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https://openalex.org/W4250111297	https://europepmc.org/articles/pmc4857056?pdf=render	English	null	Retracted: An Improved Differential Evolution Solution for Software Project Scheduling Problem	The scientific world journal/TheScientificWorldjournal	2,016	cc-by	333	[1] A. C. Biju, T. Aruldoss Albert Victoire, and K. Mohanasun- daram, “An improved differential evolution solution for soft- ware project scheduling problem,” The Scientific World Journal, vol. 2015, Article ID 232193, 9 pages, 2015. Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2016, Article ID 4835617, 1 page http://dx.doi.org/10.1155/2016/4835617 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2016, Article ID 4835617, 1 page http://dx.doi.org/10.1155/2016/4835617 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2016, Article ID 4835617, 1 page http://dx.doi.org/10.1155/2016/4835617 Retraction Retracted: An Improved Differential Evolution Solution for Software Project Scheduling Problem The Scientific World Journal Received 20 April 2016; Accepted 20 April 2016 The Scientific World Journal Received 20 April 2016; Accepted 20 April 2016 Copyright © 2016 The Scientific World Journal. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Scientific World Journal has retracted the article titled “An Improved Differential Evolution Solution for Software Project Scheduling Problem” [1]. After conducting a thorough investigation, we have strong reason to believe that the peer review process was compromised.h This article was originally submitted to a Special Issue titled “Recent Advances in Metaheuristics and its Hybrids.” In late 2015, Dr. Xavier Delorme, the lead guest editor on the Special Issue, alerted us that his identity had been compromised. After further investigation, we discovered that several peer review reports in this issue had been submitted from similarly compromised email accounts. In late 2015, Dr. Xavier Delorme, the lead guest editor on the Special Issue, alerted us that his identity had been compromised. After further investigation, we discovered that several peer review reports in this issue had been submitted from similarly compromised email accounts. We are retracting the articles in keeping with the “COPE statement on inappropriate manipulation of the peer review process.” There is no evidence that any of the authors or edi- tors, including Dr. Delorme, were aware of this misconduct.
https://openalex.org/W2614133581	https://publications.goettingen-research-online.de/bitstream/2/58880/2/foods-06-00038-v2.pdf	English	null	Rapid Prediction of Moisture Content in Intact Green Coffee Beans Using Near Infrared Spectroscopy	Foods	2,017	cc-by	7,714	Rapid Prediction of Moisture Content in Intact Green Coffee Beans Using Near Infrared Spectroscopy Adnan Adnan 1, Dieter von Hörsten 2, Elke Pawelzik 1 and Daniel Mörlein 3,* 1 Division Quality of Plant Products, Department of Crop Sciences, University of Goettingen, Carl Sprengel Weg 1 37075 Goettingen Germany; adnan adnan@stud uni goettingen de (A A ); Adnan Adnan 1, Dieter von Hörsten 2, Elke Pawelzik 1 and Daniel Mörlein 3,* Adnan Adnan 1, Dieter von Hörsten 2, Elke Pawelzik 1 and Daniel Mörlein 3,* 1 Division Quality of Plant Products, Department of Crop Sciences, University of Goettingen, Carl-Sprengel-Weg 1, 37075 Goettingen, Germany; adnan.adnan@stud.uni-goettingen.de (A.A.); epawelz@gwdg.de (E.P.) 1 Division Quality of Plant Products, Department of Crop Sciences, University of Goettingen, Carl-Sprengel-Weg 1, 37075 Goettingen, Germany; adnan.adnan@stud.uni-goettingen.de (A.A.); epawelz@gwdg.de (E.P.) 1 Division Quality of Plant Products, Department of Crop Sciences, University of Goettingen, Carl-Sprengel-Weg 1, 37075 Goettingen, Germany; adnan.adnan@stud.uni-goettingen.de (A.A.); epawelz@gwdg.de (E.P.) p g g 2 Institute for Application Techniques in Plant Protection, Julius Kühn Institute, Messeweg 11/12, 38140 Braunschweig, Germany; dieter.von-hoersten@jki.bund.de g y j 3 Department of Animal Sciences, University of Goettingen, Albrecht-Thaer-Weg 3, D-37075 Goettingen, Germany * Correspondence: daniel.moerlein@agr.uni-goettingen.de; Tel.: +49-551 * Correspondence: daniel.moerlein@agr.uni-goettingen.de; Tel.: +49-551-39-5611; Fax: +49-551-39-558 Academic Editors: Theo H. Varzakas and Charalampos Proestos Received 10 March 2017 Accepted 17 May 2017 Published 19 May 2017 Academic Editors: Theo H. Varzakas and Charalampos Proestos Received: 10 March 2017; Accepted: 17 May 2017; Published: 19 May 2017 Academic Editors: Theo H. Varzakas and Charalampos Proestos Received: 10 March 2017; Accepted: 17 May 2017; Published: 19 May 2017 Academic Editors: Theo H. Varzakas and Charalampos Proestos Received: 10 March 2017; Accepted: 17 May 2017; Published: 19 May 2017 Abstract: Moisture content (MC) is one of the most important quality parameters of green coffee beans. Therefore, its fast and reliable measurement is necessary. This study evaluated the feasibility of near infrared (NIR) spectroscopy and chemometrics for rapid and non-destructive prediction of MC in intact green coffee beans of both Coffea arabica (Arabica) and Coffea canephora (Robusta) species. Diffuse reﬂectance (log 1/R) spectra of intact beans were acquired using a bench top Fourier transform NIR instrument. MC was determined gravimetrically according to The International Organization for Standardization (ISO) 6673. Samples were split into subsets for calibration (n = 64) and independent validation (n = 44). A three-component partial least squares regression (PLSR) model using raw NIR spectra yielded a root mean square error of prediction (RMSEP) of 0.80% MC; a four component PLSR model using scatter corrected spectra yielded a RMSEP of 0.57% MC. A simpliﬁed PLS model using seven selected wavelengths (1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm) yielded a similar accuracy (RMSEP: 0.77% MC) which opens the possibility of creating cheaper NIR instruments. Rapid Prediction of Moisture Content in Intact Green Coffee Beans Using Near Infrared Spectroscopy Adnan Adnan 1, Dieter von Hörsten 2, Elke Pawelzik 1 and Daniel Mörlein 3,* 1 Division Quality of Plant Products, Department of Crop Sciences, University of Goettingen, Carl Sprengel Weg 1 37075 Goettingen Germany; adnan adnan@stud uni goettingen de (A A ); In conclusion, NIR diffuse reﬂectance spectroscopy appears to be suitable for rapid and reliable MC prediction in intact green coffee; no separate model for Arabica and Robusta species is needed. Keywords: quality; rapid methods; infrared spectroscopy; Coffea arabica (Arabica); Coffea canephora (Robusta); chemometrics Keywords: quality; rapid methods; infrared spectroscopy; Coffea arabica (Arabica); Coffea canephora (Robusta); chemometrics foods foods foods foods foods 1. Introduction International standards for MC measurement of green coffee beans are The International Organization for Standardization (ISO) 1446, 1447, and 6673 [3,14]. Thereof, ISO 6673 which requires less preparation and the shortest drying time (105 ◦C for 16 h) is widely accepted as a reference method among importing and exporting countries. Apparently these gravimetric methods do not sufﬁce when the information on MC is needed instantly [5] which is why we researched alternative methods. Near infrared spectroscopy (NIRS) has been widely investigated for rapid, often non-destructive, determination of the compositional and quality traits of agricultural products. For example, previous work in our group predicted rapid and non-destructive analysis of mango quality attributes using NIRS and chemometrics [15]. NIRS makes use of the fact that near infrared (NIR) radiation in the range of 780–2500 nm predominantly interacts with hydrogen bonds—e.g., O–H, C–H, N–H, S–H. NIR radiation that hits a sample may be transmitted, absorbed, or reﬂected, this depends on the chemical composition and physical factors of the sample. The intensity of transmitted, absorbed, or reﬂected radiation is then recorded by NIRS [16,17]. Speciﬁc wavelengths (1450 and 1940 nm) were identiﬁed to be highly correlated with water content [3,18,19]. Predicting MC using NIRS in any agricultural product is more complex and should not be based on wavelengths limited to 1450 and 1940 nm. MC does not only reﬂect water, but also loss of volatile compounds during drying [3]. In fact, NIR has some disadvantages, e.g., overlapping of wavelengths that correspond to speciﬁc organic compounds, and scattering problems [16,20]. It is therefore necessary to carefully develop calibration models for NIR based predictions [18,19]. Prediction of MC by NIRS has been developed over years for many agricultural products [21]. A regression model was developed to predict MC in (ground) green coffee bean (Coffea arabica from Brazil) based on NIR diffuse reﬂectance (log 1/R) spectra [22]. To the best of our knowledge, this is the ﬁrst study investigating the prediction of moisture content in intact green coffee beans of both Coffea arabica (Arabica) and Coffea canephora (Robusta) species by near infrared spectroscopy (NIRS) and chemometrics. The main goal of this paper was to study the feasibility of near infrared spectroscopy (NIRS) to predict moisture content (MC) in intact green coffee beans. We developed and validated calibration models based on diffuse reﬂectance spectra which were obtained using a benchtop near infrared instrument. 1. Introduction Moisture content (MC) is one of the most important quality parameters of green coffee beans. Most importing and exporting countries regulate MC as one of the quality standards for green coffee beans. The safety range for MC is 8.0–12.5%, based on fresh matter [1–3]. MC outside the safety range impairs the bean quality and safety. Beans with a MC above 12.5% are not allowed to be shipped and traded [4]. MC below 8% causes shrunken beans and an unwanted appearance [5], whereas MC above 12.5% facilitates fungal growth and mycotoxin production (e.g., ochratoxin A) that are risks to human health [6,7]. Coffee is harvested in the form of ripe berries and has a MC of more than 60% [8]. These ripe berries are processed through several steps of (wet or dry) postharvest treatments resulting in green coffee beans. Farmers generally dry the beans under the sun. The dried beans often do not meet the standard requirements for MC, resulting in a lower price [9]. For example, green beans obtained in Foods 2017, 6, 38; doi:10.3390/foods6050038 www.mdpi.com/journal/foods Foods 2017, 6, 38 2 of 11 the Bengkulu Province of Indonesia had a MC of 10.1–18.6% [10] and those in West Nusa Tenggara Province had a MC of 11.0–14.1% [11]. the Bengkulu Province of Indonesia had a MC of 10.1–18.6% [10] and those in West Nusa Tenggara Province had a MC of 11.0–14.1% [11]. MC control is also important for the storability of the beans. An inappropriate storage environment (e.g., non-aerated silos and bag storage) affects MC ﬂuctuation. The MC of green coffee beans stored in non-aerated silos increased up to 15.4% during rainy season. This moisture increase leads to the accumulation of glucose and an unpleasant taste in the beverage [12]. Furthermore, MC is crucial before the roasting process. The same roasting temperature and time with different MCs can result in different quality attributes—like color, density, and aroma—of the end product [13]. Consequently, an identical MC of green coffee beans is important for the roasting procedure in order to produce a consistent quality of roasted beans. Therefore, a fast and accurate determination of MC in green coffee beans is vital. Up to date, the standard method for determining MC is the gravimetric method, where a drying chamber with a certain temperature and time is used to dry the beans and afterwards the mass loss is calculated. 1. Introduction Our decision to involve both Arabica and Robusta species stems from the fact that both species are commercially important but vary in their chemical composition. Furthermore, we used intact green beans such as no sample preparation would be needed—yet such an approach has not been documented. The results are especially relevant for those involved in coffee trading, production, and quality control. We also demonstrate the possibility of creating a simple NIR instrument which only uses a few important wavelengths to predict MC, rather than employing the full NIR spectrum. 2. Materials and Methods A schematic representation of the experimental set up is given in the supplementary information (Figure S1). 3 of 11 Foods 2017, 6, 38 Foods 2017, 6, 38 2.2. Near-Infrared Spectroscopy A bench top Fourier transform near infrared (FT-NIR) instrument with sample cup rotation (Thermo Nicolet Antaris MDS, Thermo Fisher, Waltham, MA, USA) was used to acquire diffuse reﬂectance spectra (log 1/R) of bulk samples of green coffee beans (40 g) on a Petri dish with a diameter of 7 cm. Spectra were collected according to a workﬂow developed using the software Result Integration Software (ResultTM version 3.0, Thermo Fisher, Waltham, MA, USA). Internal background spectra were collected once every hour. High resolution diffuse reﬂectance (log 1/R) spectra at a wavelength range of 1000 to 2500 nm with 2 nm intervals were recorded as the averages of 64 scans. Thus, the spectra consisted of 1557 data points. Three replicates were acquired per sample and the spectra were averaged before further calculations. In total, this resulted in 108 spectra of 12 samples differing in moisture content, species, and origin. 2.1. Materials Green Arabica and Robusta coffee beans that were harvested in 2013 were obtained from a local trading company in Indonesia. The materials were divided into separate sample sets for calibration and validation purposes (Table 1). The beans were placed in an open plastic box with the size of 15.5 × 11 × 6 cm, and were stored in a climatic chamber (Rumed® type 1301, Rubarth Apparate GmbH, Laatzen, Germany) at 25 ◦C and a relative humidity range of 30–85%, in order to obtain a broad range of MC within 6–22%. Upon equilibration, samples were removed from the climatic chamber to record diffuse reﬂectance (log 1/R) data. Immediately thereafter, MC of the beans was determined. Table 1. Characteristics of the coffee samples including species and origin. No. Purpose Species Origin 1 Calibration Arabica West Nusa Tenggara 2 South Sulawesi 3 Aceh 4 Robusta South Sumatera 5 Bali 6 East Java 7 North Sumatera 8 Validation Arabica West Java 9 North Sumatera 10 Robusta South Sumatera 11 East Java 12 Bengkulu Table 1. Characteristics of the coffee samples including species and origin. No. Purpose Species Origin 1 Calibration Arabica West Nusa Tenggara 2 South Sulawesi 3 Aceh 4 Robusta South Sumatera 5 Bali 6 East Java 7 North Sumatera 8 Validation Arabica West Java 9 North Sumatera 10 Robusta South Sumatera 11 East Java 12 Bengkulu Table 1. Characteristics of the coffee samples including species and origin. 2.2. Near-Infrared Spectroscopy 2.2. Near-Infrared Spectroscopy 2.4. Data Processing The statistical software (The Unscrambler® X version 10.2 Network Client, CAMO software AS, Oslo, Norway) was used for further processing of the spectral data. Regression models to predict MC in green coffee beans were developed with a subset of calibration samples (n = 64), and then the models were tested using the subset of validation samples (n = 44) to evaluate the accuracy. Firstly, spectral outliers were identiﬁed using Principal Component Analysis (PCA) and Hotelling’s T2 ellipse 5% plot, based on all samples’ (n = 108) raw spectra. Afterwards, several pre-processing methods were applied to compensate the disadvantages of NIR, e.g., the scattering and material size [16,23]. In detail, smoothing (moving average, Gaussian ﬁlter, median ﬁlter) window size of 3, 7, 11, 15, 19; Savitsky–Golay derivative (First derivative, two polynomial order; second derivative, two polynomial order; third derivative, three polynomial order) window size of 3, 7, 11, 15, 19; normalization (area, mean); baseline correction (baseline offset, linear baseline correction); standard normal variate (SNV); orthogonal signal correction (OSC) (non-linear iterative partial least squares algorithm, number of component 1); multiplicative scatter correction (MSC) (full MSC model); and extended multiplicative scatter correction (EMSC) were applied. Subsequently, the models were compared in terms of prediction accuracy and model robustness (number of latent variables). MSC and EMSC were applied to the calibration data. Upon model validation, the processing was also applied to the validation data set prior to prediction. Calibration models were developed using both partial least squares regression (PLSR) and multiple linear regression (MLR). For PLSR, the full spectra (1557 wave numbers, mean centered) were used. Full cross validation was applied to estimate calibration errors. Regression coefﬁcients were obtained from PLSR to determine the important wavelengths, i.e., those that correlated most to MC. A subset of selected wavelengths was then used as an input for full rank MLR and PLS regression to identify the most parsimonious yet robust model. Leverage correction was applied with MLR to estimate calibration errors. The calibration models derived from PLSR and MLR were evaluated by the number of latent variables (LVs), R2 of calibration, R2 of cross validation, root mean square error of calibration (RMSEC), and root mean square error of cross validation (RMSECV). Finally, all models were validated in terms of their prediction accuracy using a separate validation data set. 2.3. Moisture Content Determination MC (% wet basis) was determined was based on ISO 6673 [3]. A forced air electrical oven (Thermicon P® type UT6120, Heraeus Instruments GmbH, Hanau, Germany) was used to dry approximately 10 g whole green coffee beans in open glass petri dishes (diameter: 14 cm, height: 2.3 cm) at 105 ± 1 ◦C for 16 h. Samples were limited to six origins with two replications per drying cycle in order to maintain an equal amount of mass loss during drying. The petri dishes were closed with glass lids immediately after drying had completed, and then they were stored in desiccators for 1 h in order to cool down the samples to ambient temperature. Finally, samples were weighted (Type LP 620 S, Sartorius AG, Göttingen, Germany) to calculate MC based on weight loss; data are given as the average from two replications (Equation (1)). Across all samples, average standard deviation of replicate MC determinations was 0.21% MC (Median: 0.08% MC). MC = Ww −Wd Ww (1) MC = Ww −Wd Ww MC = Ww −Wd Ww (1) 4 of 11 Foods 2017, 6, 38 where MC is the moisture content (%) of green coffee beans (wet basis), WW is the wet weight of the sample, and Wd is the weight of the sample after drying. 2.4. Data Processing Parameters used were R2 of prediction, root mean square error of prediction (RMSEP), standard error of prediction (SEP), bias, and residual predictive deviation (RPD) [22,24]. 3.1. Spectral Properties, Outliers, and Effect of Pre-Processing According to an initial PCA using all raw spectra and projection of the Hotelling’s T2 ellipse, four samples were suspected as spectral outliers (Figure 1). Subsequent modeling with and without these potential outliers, respectively revealed that model accuracy was not signiﬁcantly affected. Thus, the suspected outliers were not excluded. Inspection of the raw data also revealed that the NIR diffuse reﬂectance spectra of intact green coffee beans are inﬂuenced by scatter (Figure 2a). Several pre-processing methods were applied to eliminate the scatter. Application of EMSC proved to improve the prediction accuracy; the EMSC corrected spectra are shown in Figure 2b. Inspection of EMSC corrected spectra indicated that several wavelength regions reﬂect the chemical information regarding moisture content. 5 of 11 of 11 5 of 11 Foods 2017, 6, 38 Foods 2017, 6, 3 Foods 2017, 6, 3 ds 2017, 6, 38 5 o Foods 2017, 6, 38 5 of 11 Figure 1. Score plot of principal component analysis (PCA) using raw infrared spectra (log 1/R) with Hotelling’s T2 ellipse for outlier inspection. Calibration samples (squares) and validation samples (circles) are marked accordingly. PC: principal component. Figure 1. Score plot of principal component analysis (PCA) using raw infrared spectra (log 1/R) with Hotelling’s T2 ellipse for outlier inspection. Calibration samples (squares) and validation samples (circles) are marked accordingly. PC: principal component. , , Figure 1. Score plot of principal component analysis (PCA) using raw infrared spectra (log 1/R) with Hotelling’s T2 ellipse for outlier inspection. Calibration samples (squares) and validation samples (circles) are marked accordingly. PC: principal component. Figure 1. Score plot of principal component analysis (PCA) using raw infrared spectra (log 1/R) with Hotelling’s T2 ellipse for outlier inspection. Calibration samples (squares) and validation samples (circles) are marked accordingly. PC: principal component. Figure 1. Score plot of principal component analysis (PCA) using raw infrared spectra (log 1/R) with Hotelling’s T2 ellipse for outlier inspection. Calibration samples (squares) and validation samples (circles) are marked accordingly. PC: principal component. Figure 1. Score plot of principal component analysis (PCA) using raw infrared spectra (log 1/R) with Hotelling’s T2 ellipse for outlier inspection. Calibration samples (squares) and validation samples (circles) are marked accordingly. PC: principal component. Figure 2. Diffuse reflectance spectra (log 1/R) of calibration model. Raw spectra (a); EMSC (extended lti li ti tt ) t d t (b) Figure 2. Diffuse reflectance spectra (log 1/R) of calibration model. 3.1. Spectral Properties, Outliers, and Effect of Pre-Processing Raw spectra (a); EMSC (extended multiplicative scatter) corrected spectra (b). Figure 2. Diffuse reﬂectance spectra (log 1/R) of calibration model. Raw spectra (a); EMSC (extended multiplicative scatter) corrected spectra (b). Figure 2. Diffuse reflectance spectra (log 1/R) of calibration model. Raw spectra (a); EMSC (extended lti li ti tt ) t d t (b) Figure 2. Diffuse reflectance spectra (log 1/R) of calibration model. Raw spectra (a); EMSC (extended multiplicative scatter) corrected spectra (b). Figure 2. Diffuse reﬂectance spectra (log 1/R) of calibration model. Raw spectra (a); EMSC (extended multiplicative scatter) corrected spectra (b). 3 2 Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reﬂectance Spectra PC 2 and 3 together explain 94% of MC variance (Figure 3b). Principal components (PCs) 1 and 2 of the PLSR model based on raw spectra explain 99% of spectral data variance and 51% of MC variance; a clear separation of Arabica and Robusta species is to be seen (Figure 3a). PC 2 and 3 together explain 94% of MC variance (Figure 3b). Principal components (PCs) 1 and 2 of the PLSR model based on raw spectra explain 99% of spectral data variance and 51% of MC variance; a clear separation of Arabica and Robusta species is to be seen (Figure 3a). PC 2 and 3 together explain 94% of MC variance (Figure 3b). Foods 2017, 6, 38 7 of 11 Figure 3. Score plots of PLSR for moisture content prediction based on raw diffuse reflectance (log 1/R) near infrared spectra. A distinct clustering of Arabica and Robusta coffee samples is observed when displaying PC 1 vs. PC2 (herein: factor‐1 and factor‐2) (a); Sample allocation is following moisture content indicating the importance of PC 2 and 3 for moisture prediction (b); Weighted regression coefficients obtained from PLSR using raw spectra (c). Weighted regression coefficients obtained from PLSR on raw data (Figure 3c) were then used to Figure 3. Score plots of PLSR for moisture content prediction based on raw diffuse reﬂectance (log 1/R) near infrared spectra. A distinct clustering of Arabica and Robusta coffee samples is observed when displaying PC 1 vs. PC2 (herein: factor-1 and factor-2) (a); Sample allocation is following moisture content indicating the importance of PC 2 and 3 for moisture prediction (b); Weighted regression coefﬁcients obtained from PLSR using raw spectra (c). 7 o ods 2017, 6, 38 Figure 3. Score plots of PLSR for moisture content prediction based on raw diffuse reflectance (log 1/R) near infrared spectra. A distinct clustering of Arabica and Robusta coffee samples is observed when displaying PC 1 vs. PC2 (herein: factor‐1 and factor‐2) (a); Sample allocation is following moisture content indicating the importance of PC 2 and 3 for moisture prediction (b); Weighted regression coefficients obtained from PLSR using raw spectra (c). Figure 3. Score plots of PLSR for moisture content prediction based on raw diffuse reﬂectance (log 1/R) near infrared spectra. A distinct clustering of Arabica and Robusta coffee samples is observed when displaying PC 1 vs. 3 2 Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reﬂectance Spectra 3.2. Prediction of Moisture Content from NIR Reflectance Spectra Several preprocessing methods were applied to build the model (see Section 2.4). Nevertheless, none of the preprocessing methods yielded a better accuracy than models using raw data. Selected results of the various chemometric approaches to predict MC from NIR reflectance spectra are given in Table 2. The most parsimonious PLSR model on the full spectral range was achieved using raw spectra and three latent variables. Its prediction accuracy was, however, somewhat compromised when using the independent validation data set. Using the EMSC corrected spectra instead of the raw spectra yielded a similar R² while the prediction errors were comparably low both for the calibration and the validation data set. Yet, this model used four latent variables, e.g., it was less parsimonious compared to the model based on raw data. Several preprocessing methods were applied to build the model (see Section 2.4). Nevertheless, none of the preprocessing methods yielded a better accuracy than models using raw data. Selected results of the various chemometric approaches to predict MC from NIR reflectance spectra are given in Table 2. The most parsimonious PLSR model on the full spectral range was achieved using raw spectra and three latent variables. Its prediction accuracy was, however, somewhat compromised when using the independent validation data set. Using the EMSC corrected spectra instead of the raw spectra yielded a similar R² while the prediction errors were comparably low both for the calibration and the validation data set. Yet, this model used four latent variables, e.g., it was less parsimonious compared to the model based on raw data. Several preprocessing methods were applied to build the model (see Section 2.4). Nevertheless, none of the preprocessing methods yielded a better accuracy than models using raw data. Selected results of the various chemometric approaches to predict MC from NIR reﬂectance spectra are given in Table 2. The most parsimonious PLSR model on the full spectral range was achieved using raw spectra and three latent variables. Its prediction accuracy was, however, somewhat compromised when using the independent validation data set. Using the EMSC corrected spectra instead of the raw spectra yielded a similar R2 while the prediction errors were comparably low both for the calibration and the validation data set. Yet, this model used four latent variables, e.g., it was less parsimonious compared to the model based on raw data. 3 2 Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reﬂectance Spectra 6 of 11 Foods 2017, 6, 38 Ta Table 2. Statistical parameters of the developed prediction models for moisture content (MC) in green coffee beans using near infrared spectra. Model Parameter Full Spectral Range PLSR Spectral Subset R EMSC R (MLR) R (PLS) Model Parameter Full Spectral Range PLSR Spectral Subset Raw EMSC Raw (MLR) Raw (PLS) Calibration LVs 3 4 n/a 3 R2 calibration 0.9834 0.9850 0.9839 0.9743 R2 cross validation 0.9802 0.9811 0.9779 0.9698 RMSEC (% MC) 0.52 0.49 0.51 0.65 RMSECV (% MC) 0.58 0.56 0.60 0.71 Prediction R2 prediction 0.9641 0.9817 0.9632 0.9669 RMSEP (% MC) 0.80 0.57 0.93 0.77 Bias (% MC) 0.42 0.28 0.45 0.39 RPD 6.21 8.53 3.47 6.39 PLSR: partial least squares regression using full spectral range (1000 to 2500 nm, 1557 data points); MLR/PLS: multiple linear and partial least squares regression using selected wavenumbers (1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm); LVs: Latent variables (for PLS only); R2: the coefﬁcient of determination; RMSEC: root mean square error of valibration; RMSECV: root mean square error of cross validation; RMSEP: root mean square error of prediction; SEP: standard error of prediction; RPD: residual predictive deviation; n/a: not applicable; MC: moisture content. 3 2 Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reﬂectance Spectra Raw EMSC Raw (MLR) Raw (PLS) Calibration LVs 3 4 n/a 3 R2 calibration 0.9834 0.9850 0.9839 0.9743 R2 cross validation 0.9802 0.9811 0.9779 0.9698 RMSEC (% MC) 0.52 0.49 0.51 0.65 RMSECV (% MC) 0.58 0.56 0.60 0.71 Prediction R2 prediction 0.9641 0.9817 0.9632 0.9669 RMSEP (% MC) 0.80 0.57 0.93 0.77 Bias (% MC) 0.42 0.28 0.45 0.39 RPD 6.21 8.53 3.47 6.39 PLSR: partial least squares regression using full spectral range (1000 to 2500 nm, 1557 data points); MLR/PLS: multiple linear and partial least squares regression using selected wavenumbers (1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm); LVs: Latent variables (for PLS only); R2: the coefficient of determination; RMSEC: root mean square error of valibration; RMSECV: root mean square error of cross validation; RMSEP: root mean square error of prediction; SEP: standard error of prediction; d l d d / l bl C PLSR: partial least squares regression using full spectral range (1000 to 2500 nm, 1557 data points); MLR/PLS: multiple linear and partial least squares regression using selected wavenumbers (1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm); LVs: Latent variables (for PLS only); R2: the coefﬁcient of determination; RMSEC: root mean square error of valibration; RMSECV: root mean square error of cross validation; RMSEP: root mean square error of prediction; SEP: standard error of prediction; RPD: residual predictive deviation; n/a: not applicable; MC: moisture content. PLSR: partial least squares regression using full spectral range (1000 to 2500 nm, 1557 data points); MLR/PLS: multiple linear and partial least squares regression using selected wavenumbers (1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm); LVs: Latent variables (for PLS only); R2: the coefficient of determination; RMSEC: root mean square error of valibration; RMSECV: root mean square error of cross validation; RMSEP: root mean square error of prediction; SEP: standard error of prediction; Principal components (PCs) 1 and 2 of the PLSR model based on raw spectra explain 99% of spectral data variance and 51% of MC variance; a clear separation of Arabica and Robusta species is to be seen (Figure 3a). PC 2 and 3 together explain 94% of MC variance (Figure 3b). p pp Principal components (PCs) 1 and 2 of the PLSR model based on raw spectra explain 99% of spectral data variance and 51% of MC variance; a clear separation of Arabica and Robusta species is to be seen (Figure 3a). 3 2 Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reﬂectance Spectra PC2 (herein: factor-1 and factor-2) (a); Sample allocation is following moisture content indicating the importance of PC 2 and 3 for moisture prediction (b); Weighted regression coefﬁcients obtained from PLSR using raw spectra (c). Foods 2017, 6, 38 7 of 11 Weighted regression coefﬁcients obtained from PLSR on raw data (Figure 3c) were then used to study whether the model could be even simpliﬁed. Note that weighted and raw regression coefﬁcients are the same as long as spectral data are not scaled but only mean centered; this was applied here. Seven wavelengths were selected due to their regression weights. That is, the intensities of 1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm were used as input data to develop a MLR calibration model. Thus, a similarly accurate model was obtained (Table 2); the prediction error for the validation test set was signiﬁcantly lower (p < 0.05) for the MLR model (0.93% MC) as compared to the EMSC model using raw data (0.57% MC). The resulting MLR model is given in Equation (2). MC (%) = −4.20 + 115.02 (V1) + 0.40 (V2)– 116.18 (V3) + 76.16 (V4) −97.72 (V5) + 63.76 (V6) −17.59 (V7) (2) (2) where, V1 to V7 are the intensities of the wavelengths 1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm, respectively. When subjecting this spectral subset to PLS, the predictive ability of a three LV model was even improved as compared to the full-rank MLR model (Table 2); its prediction error (0.77% MC) was signiﬁcantly lower than the MLR model (p = 0.015). It is, however, not signiﬁcantly different from the PLSR model using raw data (p > 0.05). PLSR and MLR using raw spectral data yielded a good correlation of reference versus predicted MC (Figure 4a,b). Also, the model’s bias is close to the error of the reference method (0.21% MC, see 2.3.). Foods 2017, 6, 38 8 of 11 Figure 4. Predicted vs. measured moisture content of green coffee beans based on raw diffuse reflectance (log 1/R) near infrared spectra. (a) PLSR; (b) MLR. Figure 4. Predicted vs. measured moisture content of green coffee beans based on raw diffuse reﬂectance (log 1/R) near infrared spectra. (a) PLSR; (b) MLR. Figure 4. Predicted vs. measured moisture content of green coffee beans based on raw diffuse reflectance (log 1/R) near infrared spectra. (a) PLSR; (b) MLR. Figure 4. Predicted vs. 3 2 Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reflectance Spectra 3.2. Prediction of Moisture Content from NIR Reﬂectance Spectra measured moisture content of green coffee beans based on raw diffuse reﬂectance (log 1/R) near infrared spectra. (a) PLSR; (b) MLR. 4.2. Prediction of Moisture Content Using NIR Infrared Spectra Raw spectra were selected as an input to build the ﬁnal PLSR model because this resulted in the lowest number of latent variables, the highest R2 and lowest root mean square error compared to other pre-processing methods (Table 2). A model with these criteria is preferable. Kamruzzaman et al. [29] also considered the number of latent variables together with R2 and prediction errors to select the most appropriate model for prediction of water, fat, and protein content in lamb meat. Both the robustness and the predictive ability of a given model are of importance. If one considers only R2, RMSEP, or RPD, which reﬂect the predictive ability, likely models using more latent variables would be preferred over models using less latent variables. In terms of robustness, however, a model using less latent variables is less prone to overﬁtting than a model using more latent variables. Further examination of the PLSR score plots (based on raw spectra) revealed a distinct clustering of Arabica and Robusta samples on the ﬁrst latent variable, explaining 98% in the spectral data variance but only 4% of moisture variance (Figure 3a). To understand this clustering, the loading weights of the ﬁrst LV were inspected. As a result, important wavelengths are related to several chemical compounds, e.g., caffeine, chlorogenic acid, lipids, protein and amino acids, sucrose, carbohydrates, trigonelline and, of course, water [30]. These compounds were shown to vary between species which is why their spectral contributions can be used to discriminate between species [31,32] Using PC 2 and 3 which together explain 94% of moisture variance, samples are allocated according to moisture content levels (Figure 3b). Thus, a three component PLSR model allows prediction of moisture content on both Arabica and Robusta species. The advantages of inputting raw spectra rather than pre-processed spectra ﬁrstly reduces the complexity of calculations and therefore secondly reduces the computation time. These advantages will be useful for online and real time prediction in the future. The statistical parameters of calibration and prediction accuracy were similar for the developed PLSR models, especially for the model based on EMSC corrected spectra. This indicates that the PLSR model is robust in terms of predicting unknown samples accurately. We also investigated PLSR models based on raw spectra within individual species. However, the results were not better than the PLSR model which was developed across species. 4.1. Outliers and Effect of Pre‐Processing 4.1. Outliers and Effect of Pre-Processing For outlier detection, PCA and subjection of the Hotelling’s T2 ellipse along with residuals and influence plot, and Q‐residuals plot, were used which are common approaches in multivariate analysis. Identifying true outliers is important to prevent false inferences [25]. In this experiment, four samples were suspected to be outliers (Figure 1). Explained spectral variance (PC1 + PC2) based on diffuse raw data reflectance (log 1/R) was 99%. Elimination of suspected outliers did not increase the explained variance. Further comparisons of PLSR with and without the suspected outliers yielded only very slight improvement in R2 which indicates that the suspected outliers were no real outliers. Similarly, Morales‐Medina and Guzmán [26] examined multivariate data using Hotelling’s T2 ellipse. They also decided to not exclude the suspected outliers because they did not significantly affect the explained variance found through PCA. Various pre‐processing methods were applied to the raw spectra. This aims at reducing noise For outlier detection, PCA and subjection of the Hotelling’s T2 ellipse along with residuals and inﬂuence plot, and Q-residuals plot, were used which are common approaches in multivariate analysis. Identifying true outliers is important to prevent false inferences [25]. In this experiment, four samples were suspected to be outliers (Figure 1). Explained spectral variance (PC1 + PC2) based on diffuse raw data reﬂectance (log 1/R) was 99%. Elimination of suspected outliers did not increase the explained variance. Further comparisons of PLSR with and without the suspected outliers yielded only very slight improvement in R2 which indicates that the suspected outliers were no real outliers. Similarly, Morales-Medina and Guzmán [26] examined multivariate data using Hotelling’s T2 ellipse. They also decided to not exclude the suspected outliers because they did not signiﬁcantly affect the explained variance found through PCA. Foods 2017, 6, 38 8 of 11 Various pre-processing methods were applied to the raw spectra. This aims at reducing noise and improving the accuracy of the prediction model [27]. EMSC was effective to remove scatter which was shown also in other studies [28]. Accordingly, the prediction errors were the lowest when using EMSC corrected data for PLSR (Table 2). The resulting model, however, was surprisingly less parsimonious, i.e., it needed one more latent variable. Pizarro et al. 4.1. Outliers and Effect of Pre‐Processing 4.1. Outliers and Effect of Pre-Processing [27] also reported that none of the pre-processing methods studied (ﬁrst and second derivation, MSC, standard normal variate) improved the prediction for ash and lipid content in roasted coffee signiﬁcantly as compared to using raw data; only OSC and direct orthogonal signal correction (DOSC) enhanced the model performance remarkably. 4.2. Prediction of Moisture Content Using NIR Infrared Spectra The PLSR model obtained in this experiment resulted in a similar accuracy compared to what was reported by Morgano et al. [22]. That study predicted the MC of green Arabica coffee beans, based on smoothed spectra, which yielded an R2 of calibration = 0.507, R2 of validation = 0.669, and RMSEV of 0.55% MC (R2 recalculated from r). Even simpliﬁed MLR and PLS models were built using selected wavelengths based on their relative importance in the PLSR model. This experiment showed that near infrared diffuse reﬂectance intensities at 1155, 1212, 1340, 1409, 1724, 1908, and 2249 nm highly correspond to MC (Figure 3c). According to Ribeiro et al. [30], these wavelengths are related to the absorbance of the second overtone of C–H, ﬁrst combination overtone of C–H, ﬁrst overtone of O–H and N–H, second overtone of C=O, and combination of O–H and N–H, respectively. Obviously, these wavelengths are not exactly located at the water bands which indicate that it may well be useful to apply indirect relationships in prediction models. Plus, it was shown that the degradation of organic components during drying for MC determination needs to be considered. Reh et al. [3] proved that, using ISO 6673, the beans lose 0.39% of their mass besides water. Thus, MC is calculated as a sum of extracted water and mass Foods 2017, 6, 38 9 of 11 losses of other compounds. Similarly, Pan et al. [33] found that MC in beet slices highly corresponded to spectral intensities at 968, 1078, and 1272 nm, i.e., not exactly located at the water bands. The MLR model, as well as the PLS model based on the spectral subset, yielded a good accuracy both for calibration and validation thus proving their robustness (Figure 4b). The biases measured by PLSR and MLR were close to the method error of determining moisture content based on ISO 6673. Moreover, the ratio of the standard deviation of the target variable and the SEP of a given model, commonly referred to as RPD (residual predictive deviation), is often used to assess the performance of prediction models; higher RPD values indicate a better predictive performance [24]. Here, the models yielded RPD values of about 3 to 8 (Table 2) which is considered good [34]. 4.2. Prediction of Moisture Content Using NIR Infrared Spectra This shows the potential of near infrared spectroscopy to replace the reference method when a fast and non-destructive prediction is needed, e.g., when trading or for in-line process control. Finally, the remarkable reduction of variables without a relevant loss of accuracy opens the possibility of creating a simple NIR instrument which only uses a few important wavelengths to predict MC, rather than employing the full NIR spectrum. Speciﬁc LED light sources emitting only selected wavelengths can potentially reduce the costs of an NIR instrument. 5. Conclusions The results indicate that a fast, non-destructive prediction of moisture content in intact green coffee beans is feasible using near infrared diffuse reﬂectance spectroscopy. EMSC effectively reduces scatter apparent in raw spectra. Thus, the prediction accuracy using EMSC corrected spectra is improved at the cost of a somewhat less parsimonious model. A simpliﬁed model based on only seven selected wavelengths points to the possibility of a cheaper instrumentation. The calibration model can be applied for both Arabica and Robusta species. In conclusion, NIR is deemed feasible to replace gravimetric methods for routine applications where a timely result may outweigh the loss of accuracy as compared to the drying methods. Supplementary Materials: The following are available online at www.mdpi.com/2304-8158/6/38/s1, Figur chematic representation of the experimental set up. Acknowledgments: We would like to thank the Ministry of Agriculture of the Republic of Indonesia for gran he scholarship to Adnan. Author Contributions: Adnan and Dieter von Hörsten conceived and designed the experiments; Adnan performed the experiments; Adnan and Daniel Mörlein analyzed the data; Daniel Mörlein contributed analysis tools; Adnan and Daniel Mörlein wrote the paper. Elke Pawelzik and Daniel Mörlein supervised the study. All authors read and approved the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. International Coffee Organization (ICO). National quality standards. In Proceedings of the Promotion and Market Development Committee 6th Meeting, Belo Horizonte, Brazil, 9 September 2013. Available online: http://www.ico.org/documents/cy2012-13/pm-29e-quality-standards.pdf (accessed on 1 February 2017). 2. Pittia, P.; Nicoli, M.C.; Sacchetti, G. Effect of moisture and water activity on textural properties of raw and roasted coffee beans. J. Texture Stud. 2007, 38, 116–134. [CrossRef] 3. Reh, C.; Gerber, A.; Prodolliet, J.; Vuataz, G. Water content determination in green coffee—Method comparison to study speciﬁcity and accuracy. Food Chem. 2006, 96, 423–430. 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https://openalex.org/W4388503101	https://ejournal.iaida.ac.id/index.php/Tarbiyatuna/article/download/2388/1317	Arabic	null	ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN	Tarbiyatuna	2,023	cc-by-sa	9,617	53 53 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) مستخلص البحث مستخلص البحث هتدف هذه الدراسة لرتقية كفاءة الطالبات يف مهارة القراءة. رأت الباحثة يف الفصل األول اإلبتدائي مبعهد دار اللغة والدعوة باغل افتقارهن إىل كفاءة يف مهارة القراءة. حىت بعضهن مل يستطعن على قراءة النصوص العربية جيّدا و مل يفهمن مبا قرأن . حدثت هذه احلالة بسبب عدم اهتمامهن مبهارة القراءة وتبدو األمر ممال. وكانت أسال يب التعليم املستخدمة غري فعالة ثقيلة للغاية. لذالك أرادات الباحث أن جتر ب إحدى األساليب املفرحة يف عملية التعليم وهي اللعبة اللغوية األوراق املمزقة. هي إحدى األلعاب لتحسني قدرة الطالبات على قراءة اللغة ال عربية، يف هذه اللعبة متكن زيادة كفاءة الطالبات يف إتقان املفردات أيضا. هذه اللعبة مناسبة هبذا البحث ألن هتد ف إىل تدريب الطالبات على قراءة و .تأليف القصص العربية بناءا على هذه املشكلة، إن األهداف من هذا البحث هي ملعرفة كيفية تطبيق و ملعرفة فعلية استخدام الل عبة اللغوية لرتقية كفاءة الطالبات يف مهارة القراءة يف الفصل األول اإلبتدائي مبعهد دار اللغو والدعوة بانقيل. منهج هذا البحث هو جتريب . وأسلوب البحث املستخدمة هي اإلختبار واملالحظة واملقابلة والوثائق. ونتائج من هذ البحث هي أن استخدام اللعبة اللغوية األوراق ا ملمزقة قي تعليم اللغة العربية فعّالة لرتقية كفاءة الطالبات يف مهارة القراءة لدى الطالبات الفصل األوّل .اإلبندائي مبعهد دار اللغة والدعوة بانقيل الكلمات المفتاحية: اللعبة اللغوية، األوراق الممزقة، مهارة القراءة الكلمات المفتاحية: اللعبة اللغوية، األوراق الممزقة، مهارة القراءة ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL- MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah1, Muthmainnah2 Rizka.irtadhoni@gmail.com1, Muthmainnah@gmail.com2 Universitas Islam Internasional Darullughah Wadda’wah Bangil Pasuruan Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 54 berat diterima oleh siswa. Oleh karena itu peneliti ingin mencoba salah satu metode yang menyenangkan dalam proses pembelajaran yaitu permainan bahasa sobekan kertas. Ini adalah salah satu permainan untuk meningkatkan kemampuan siswa dalam membaca bahasa arab. Dalam permainan ini juga memungkingkan untuk meningkatkan kemampuan siswa dalam penguasaan kosa kata. Permainan ini cocok untuk penelitian ini karena bertujuan untuk melatih siswa membaca dan mengarang cerita berbahasa arab. Berdasarkan permasalahan tersebut tujuan dari penelitian ini adalah untuk mengetahui bagaimana penerapan dan keefektifitasan penggunaan permainan bahasa untuk meningkatkan kompetensi keterampilam membaca pada siswa kelas ibtida’dipondok Darullughah wadda’wah bangil pasuruan. Metode yang digunakan dalam penelitian ini adalah eksperimen. Instrumen yang digunakan adalah tes, observasi, wawancara dan dokumentasi. Hasil penelitian ini adalah penggunaan permainan bahasa sobekan kertas dalam pembelajaran bahasa arab efektif dalam meningkatkan kompetensi keterampilan membaca siswa siswa kelas ibtida’dipondok Darullughah wadda’wah bangil pasuruan. berat diterima oleh siswa. Oleh karena itu peneliti ingin mencoba salah satu metode yang menyenangkan dalam proses pembelajaran yaitu permainan bahasa sobekan kertas. Ini adalah salah satu permainan untuk meningkatkan kemampuan siswa dalam membaca bahasa arab. Dalam permainan ini juga memungkingkan untuk meningkatkan kemampuan siswa dalam penguasaan kosa kata. Permainan ini cocok untuk penelitian ini karena bertujuan untuk melatih siswa membaca dan mengarang cerita berbahasa arab. Berdasarkan permasalahan tersebut tujuan dari penelitian ini adalah untuk mengetahui bagaimana penerapan dan keefektifitasan penggunaan permainan bahasa untuk meningkatkan kompetensi keterampilam membaca pada siswa kelas ibtida’dipondok Darullughah wadda’wah bangil pasuruan. Metode yang digunakan dalam penelitian ini adalah eksperimen. Instrumen yang digunakan adalah tes, observasi, wawancara dan dokumentasi. Hasil penelitian ini adalah penggunaan permainan bahasa sobekan kertas dalam pembelajaran bahasa arab efektif dalam meningkatkan kompetensi keterampilan membaca siswa siswa kelas ibtida’dipondok Darullughah wadda’wah bangil pasuruan. Kata Kunci: Permainan linguistik, sobekan kertas, keterampilan membaca ABSTRAK Penelitian ini bertujuan untuk meningkatkan kemampuan sisawa dalam keterampilan membaca. Peneliti melihat pada siswa kelas satu ibtida’ dipondok Darullughah Wadda’wah Bangil bahwa mereka kurang memiliki kompetensi dalam keterampilan membaca. Sehingga hanya sedikit dari mereka yang dapat membaca bahasa arab dan memahami apa yang mereka baca. Kasus ini terkadi karena mereka kurang tertarik dengan keterampila membaca yang terkesan membosankan. Metode belajar yang digunakan guru sangatlah tidak efektif dan ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah Muthmainnah Rizka Anugrah, Muthmainnah Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 1. المقدمة كا نت اللغة العربية لغة خالدة غنية ، مبفرداهتا وتركيبها وأوزاهنا فإهنا تنمو وتتطور باستمرار وهي أدق اللغات نظاما و .أوسعها اشتقاقا وأمجلها أدقا وسعت حضارة األ مم املختلفة وصارت لغة العلوم واآلداب والفنون قرونا طويلة، وهي من أشهر اللغات العامة وأقواها على حتدي الصعوبات غري العصور. فقلبت األزمنة وتوالت اخلطوب واحملن واألحد اث احلسام وهى ثابتة ناصرة رائعة، وما زالت منذ مخسة عشر قرنا لغة حية مشرفة متطورة ىف حني تالشت اللغة من اللغة األ خرى وانقرضت ( ،أمني الكخن1992 : 9 .) كا نت اللغة العربية لغة خالدة غنية ، مبفرداهتا وتركيبها وأوزاهنا فإهنا تنمو وتتطور باستمرار وهي أدق اللغات نظاما و .أوسعها اشتقاقا وأمجلها أدقا وسعت حضارة األ مم املختلفة وصارت لغة العلوم واآلداب والفنون قرونا طويلة، وهي من أشهر اللغات العامة وأقواها على حتدي الصعوبات غري العصور. فقلبت األزمنة وتوالت اخلطوب واحملن واألحد اث احلسام وهى ثابتة ناصرة رائعة، وما زالت منذ مخسة عشر قرنا لغة حية مشرفة متطورة ىف حني تالشت اللغة من اللغة األ خرى وانقرضت ( ،أمني الكخن1992 : 9 .) كانت اللغة العربية من اللغات اليت استعملت للتفاهم بني مجيع الشعوب كما أهنا لغة التعليم يف مجيع املدارس واملعاهد وأكثر الكلية اجلامعية. وهي من اللغة املهمة اليت تكلم هبا أكثر من مائتني مليونا شخصا يف العامل واستخدمها حوايل كانت اللغة العربية من اللغات اليت استعملت للتفاهم بني مجيع الشعوب كما أهنا لغة التعليم يف مجيع املدارس واملعاهد وأكثر الكلية اجلامعية. وهي من اللغة املهمة اليت تكلم هبا أكثر من مائتني مليونا شخصا يف العامل واستخدمها حوايل ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 55 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) .عشرين دولة رمسية1 وهي إحدى اللغات الدولية جبانب اإلجنلزية والفرنسية. ومن املمكنات االخرى ، أن هذه اللغة ستكون لغة مهمة ىف الناحية التجارية وعالقة دولية ( Azhar Arsyad, 2022:11 ). يف تعلم اللغة العربية ، هناك أربع مهارات جيب أن حيققها الطالب أو ،لألشخاص الذين يرغبون يف تعلم اللغة العربية وفهمها، وهي: مهارة االستماع ومهارة الكالم، ومهارة القراءة ومهارة الكتابة. وإحدى املهارات املطلوبة لتعلم اللغة العربية هي مهارات .القراءة مهارات القراءة باللغة العربية هي مهارات جيب أن متتلكها الطالبات من أجل تدريب الطالبات على إتقان الفهم القرائي وتنمية قدرهتن على قراءة اللغة العربية ( Azhar Arsyad, 2022:11 ) . كانت اللغة العربية يف بلدنا اإلندونيسي مل تتطور مثل اللغات األجنبية األخرى كاإلجنلزية والفرنسية والصينية واليابانية. Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) عرفنا أن اللغة األجنبية مل تكن أسهل من لغة األم يف تدريسها. بل، هى أصعب إن كانت طريقة تدريسها ال تعطي حرية يف التفكري واخليال. وتكون عملية التعليم والتعلّم يف الفصل إال لتناول املواد الدراسية ووصول مقدار التقومي من مناهج الدراسي املقرر. فيكون التالميذ فراغا من حامسي يف التعلم اجل يدة. فتحتاج هذه الظاهرة إىل عال جها بتطوير طريقة التعليم نفسها ، فإن التعليم بطريقة جيدة تصر جيداة يف حص هلا و ،( أجيف هرموان2011 : 129 .) جيادل موليانتو سوماردي أنه يف تدريس اللغة ، فإن أحد اجلوانب اليت غالبًا ما يربزها الناس هو جانب األسلوب. غالبًا ما حيكم النجاح أو فشل الربنامج عل ى تعليم اللغة من حيث الطريقة املستخدمة ، ألن الطريقة حتدد حمتوى وكيفية تدريس اللغة ،( موليانتو سوماردي1973 : 70 .) الطريقة عبارة عن تصميم الشام ل لعرض املادة اللغوية باإلنتظام ، وال توجد أجزاء املتضاربة وكلها تستند إىل افرتاض ات هنج املعني. مبعىن آخر ، ال طريقة هي خطة شاملة فيما يتعلق ب غ رض اللغة بشكل منهجي بناءً على هنج احملدد ،( مشسدين أشراف2006 : 82 .) ، استنادًا إىل الوصف أعاله هت دف الباحثة إلجراء البحوث يف املعهد دار اللغة والدعوة با ل نقي، يعىن يف الفصل األول اإلبتدائي "أ". يريد أن يعرف استخدام األلعاب ال لغوية يف تدريس اللغة العربية لرتقية مهارة ال قراءة اليت يتم تطبيقها يف الفصل ، أل ت ن عتقد الباحثة أن ت عترب الط البات يف ذلك الفصل يستطيع قراءة اللغة .العربية جيدا يف األنشطة اليومية ويف عملية التعلم حتو اول الباحثة أن تطبيق يف إحدى طر ي قة التعلم وهي اللعبة اللغ وية يعىن "ا ألوراق املمزقة". يف هذه اللعبة تخ تار املعلم ة قص ة القصرية من الكتب "العربية "للناشئني جزء الثاين . مث تقطع إىل عدة قطع الكلمة، الليت مكتوب على عدة أوراق، ثم تح كي املعلم ة قصة، تو قوم املعلم ة بتوزيع األوراق حتتوي على عدة ا جلمل، بعد ذالك، يُطلب من الط البات لرتكيب األوراق بناء على ا .لقصة اليت متت قراءهتا باستخدام هذه اللعبة اللغوية، ميكن لط ل البات ترقية مهارة القراءة لديه ن يف تعلم عرفنا أن اللغة األجنبية مل تكن أسهل من لغة األم يف تدريسها. بل، هى أصعب إن كانت طريقة تدريسها ال تعطي حرية يف التفكري واخليال. وتكون عملية التعليم والتعلّم يف الفصل إال لتناول املواد الدراسية ووصول مقدار التقومي من مناهج الدراسي املقرر. فيكون التالميذ فراغا من حامسي يف التعلم اجل يدة. 1. المقدمة 1, Juni 2023 ISSN: 2774-5724 (media Online) 1. المقدمة وهذا يدل أن هذه الظاهرة شيئ حمزن ألن سكان اإلندونيسية أكثرهم مسلمون، وكان القرآن الكرمي بلسان عريب مبني، ولكنهم اليفهمون به إال قليال منهم. فال عجب إذا كانوا املتأخرين من البالد األخرى أخصها يف إتصا ل دولني. ولذالك حيتاجون هبا الوسعة الشديدة والطرائق العديدة يف .عالج هذه املشكلة من املهارة القراءة يف اللغة العربية بناءً على جتربة الباحثة ، ضعف ت قدرة الطالبات على ال تكلم باللغة العربية يف املتوسط ، فإن عددًا من الطالبات أقل قدرة على قراء ة اللغة العربية. كما حدث يف "الفصل األول اإلبتدائي "أ يف املعهد دار اللغة والدعوة با نقيل . ألن املعلم يف التدريس وا لتعلم يستخدم الطريقة القدمية فقط، يعىن حيث يركز املعلم على وجود كتاب إرشادي دون استخدام الوسائل التعلم اليت ميكن لرتقية مهارة القراءة للط البات ، حىت جتعل الط ال بات أن يشعر بالنعاس وامللل يف ال تعلم. أو باستخدام طريقة الرتمجة ، يعىن يقرأ املعلم قراءة النص يف اجلملة بعد ترمجتها على الفور إىل لغة اإلندونيسية ، كما أ نه جيعل الط البات .غري مستقلني ويعتمدون دائمًا على املعلم كانت اللغة العربية يف بلدنا اإلندونيسي مل تتطور مثل اللغات األجنبية األخرى كاإلجنلزية والفرنسية والصينية واليابانية. وهذا يدل أن هذه الظاهرة شيئ حمزن ألن سكان اإلندونيسية أكثرهم مسلمون، وكان القرآن الكرمي بلسان عريب مبني، ولكنهم اليفهمون به إال قليال منهم. فال عجب إذا كانوا املتأخرين من البالد األخرى أخصها يف إتصا ل دولني. ولذالك حيتاجون هبا الوسعة الشديدة والطرائق العديدة يف .عالج هذه املشكلة من املهارة القراءة يف اللغة العربية .عالج هذه املشكلة من املهارة القراءة يف اللغة العربية بناءً على جتربة الباحثة ، ضعف ت قدرة الطالبات على ال تكلم باللغة العربية يف املتوسط ، فإن عددًا من الطالبات أقل قدرة على قراء ة اللغة العربية. كما حدث يف "الفصل األول اإلبتدائي "أ يف املعهد دار اللغة والدعوة با نقيل . ألن املعلم يف التدريس وا لتعلم يستخدم الطريقة القدمية فقط، يعىن حيث يركز املعلم على وجود كتاب إرشادي دون استخدام الوسائل التعلم اليت ميكن لرتقية مهارة القراءة للط البات ، حىت جتعل الط ال بات أن يشعر بالنعاس وامللل يف ال تعلم. أو باستخدام طريقة الرتمجة ، يعىن يقرأ املعلم قراءة النص يف اجلملة بعد ترمجتها على الفور إىل لغة اإلندونيسية ، كما أ نه جيعل الط البات .غري مستقلني ويعتمدون دائمًا على املعلم ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 56 Jurnal Tarbiyatuna, Vol. 4, No. Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) فتحتاج هذه الظاهرة إىل عال جها بتطوير طريقة التعليم نفسها ، فإن التعليم بطريقة جيدة تصر جيداة يف حص هلا و ،( أجيف هرموان2011 : 129 .) فيه () جيادل موليانتو سوماردي أنه يف تدريس اللغة ، فإن أحد اجلوانب اليت غالبًا ما يربزها الناس هو جانب األسلوب. غالبًا ما حيكم النجاح أو فشل الربنامج عل ى تعليم اللغة من حيث الطريقة املستخدمة ، ألن الطريقة حتدد حمتوى وكيفية تدريس اللغة ،( موليانتو سوماردي1973 : 70 .) الطريقة عبارة عن تصميم الشام ل لعرض املادة اللغوية باإلنتظام ، وال توجد أجزاء املتضاربة وكلها تستند إىل افرتاض ات هنج املعني. مبعىن آخر ، ال طريقة هي خطة شاملة فيما يتعلق ب غ رض اللغة بشكل منهجي بناءً على هنج احملدد،( مشسدين أشراف2006 : 82 .) حتو اول الباحثة أن تطبيق يف إحدى طر ي قة التعلم وهي اللعبة اللغ وية يعىن "ا ألوراق املمزقة". يف هذه اللعبة تخ تار املعلم ة قص ة القصرية من الكتب "العربية "للناشئني جزء الثاين . مث تقطع إىل عدة قطع الكلمة، الليت مكتوب على عدة أوراق، ثم تح كي املعلم ة قصة، تو قوم املعلم ة بتوزيع األوراق حتتوي على عدة ا جلمل، بعد ذالك، يُطلب من الط البات لرتكيب األوراق بناء على ا .لقصة اليت متت قراءهتا باستخدام هذه اللعبة اللغوية، ميكن لط ل البات ترقية مهارة القراءة لديه ن يف تعلم ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN 57 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) اللغة العربية. اختار ت الباحثة هذه اللعبة اللغوية من أجل تكييف الط ا لبات عل ى التعود قراءة اللغة العربية و .قدرة التعبري عنه مرة أخرى من خالل اللغة شفهيًا وكتابيًا والفرق بني هذه الدراسة والدراسات ال سابقة انطالقا من نوع اللعبة املستخدمة : إن الدرسات اليت قامت هبا حممد رضا السقاف ة زكية السكينة و بشري مصطفى إقتصرت على استخدام اللعبة اللغوية األخرى يف تعليم مهارة القراءة وأما هذه الدراسة إقتصرت على استخدام اللعبة اللغوية األورق املمزقة لرتقية كفاءة الطالبات .فصل األول اإلبتدائي يف معهد دار اللغة والدعوة باغل يرجى أن تكون نتائج هذا البحث مسهمة يف إثراء املعلومات عن كيفية تعليم ا للغة اللغة العربية بالفرح ولرتقية كفاءة الطالبات يف مهارة القراءة. فيمكن استخدام .النتائج هذا البحث كالنظري يف تعليم اللغة العربية خاصة يف مهارة القراءة ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 2. اإلطار النظري أ) مفهوم اللعبة اللغوية األ لعاب اللغوية نوع من أنواع الوسائل التعليمية، ونشاط مهم عن أنشطة التعليم االتصايل ،( عبد الرمحان إبراهيم الفوزان1933 : 130 ) . األلعاب اللغوية وسيلة جديدة استفادات منهابرامج تعليم اللغات يف السنوات األحرية، وأثبت تطبيقاته نتائج إجيابية يف كثري من البالد اليت هتتم بتطوير .نظم تعليم لغاهتا2 يستخدم اصطالح (أللعاب) يف تعليم اللغة، لكي يعطي جماال واسعا يف األنشطة الفصيلة، لتزويد املعلم والدراس بوسيلة ممتاعة ومشوقة للتدريب على عناصر اللغة، وتوفري احلوافر لتنمية املهارت اللغة املختلفة، وهي أيضا توظف بعض العمليات العقلية مثل (التخمني) إلضفاء أبعاد اتصالية على ت لك ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 2. اإلطار النظري أ) مفهوم اللعبة اللغوية األ لعاب اللغوية نوع من أنواع الوسائل التعليمية، ونشاط مهم عن أنشطة التعليم االتصايل ،( عبد الرمحان إبراهيم الفوزان1933 : 130 ) . 1) فوائد اللعبة اللغوية 1) فوائد اللعبة اللغوية تصبح لعبة متكن فعال أن جتعل التعلم متعة ومثرية، وتشجيع التعلم، ميكن حىت تكون مبثابة اختبار. ألعاب يف التعلم اليت قادرة على خلق جو من الفرحة وحترير املعلومات االستخباراتية الكاملة وغري احملظورة، ميكن أن تعطي الكثري من ( التربعات مصطفى عبد العزيز ، 1403 : 7) . ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah رممحرامح ( التربعات مصطفى عبد العزيز ، 1403 : 7) . 2) مزايا اللعبة اللغوية يف عملي ة األلعاب اللغوية ، سيشارك الط بلا ات بشكل امل باشر ة يف التعلم النشط من خالل اإل شراك مجيع احلواس واملشاعر حبماس ال كبري ، ألن فيها عناصر ال رتفيهية ومنافسة ال صحية، وأما بالنسبة لبعض من املزايا يف لعبة اللغوية فهو كما يلي : أ) .ميكن أن تقلل من امللل للطالبات يف عملية التعلم يف الفصل الدراسة ب) مع النافسة بني الطالبات، ميكن أن تعزز روح طالبات إىل. أكثر تقدما ج) لعبة اللغة ميكن بناء عالقة وتطوير وجمموعة من الكفاءات االجتماعية .للطالبات د) املواد اليت يتم إبالغها ميكن أن ترتك انطباعا يف قلوب الطالبات حىت .جتربة تدريب املهارات صعوبات نسيان 3) عيوب اللعبة اللغوية من خالل األلعاب ، سيتعلم الطالب ات الكثري عن احلياة ، مثل الدراسة الذاتية ، واجلرأة على املنافسة ، والتواصل االجتماعي ، والتحلي بروح ، القيادة Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) األلعاب اللغوية وسيلة جديدة استفادات منهابرامج تعليم اللغات يف السنوات األحرية، وأثبت تطبيقاته نتائج إجيابية يف كثري من البالد اليت هتتم بتطوير .نظم تعليم لغاهتا2 يستخدم اصطالح (أللعاب) يف تعليم اللغة، لكي يعطي جماال واسعا يف األنشطة الفصيلة، لتزويد املعلم والدراس بوسيلة ممتاعة ومشوقة للتدريب على عناصر اللغة، وتوفري احلوافر لتنمية املهارت اللغة املختلفة، وهي أيضا توظف بعض العمليات العقلية مثل (التخمني) إلضفاء أبعاد اتصالية على ت لك يستخدم اصطالح (أللعاب) يف تعليم اللغة، لكي يعطي جماال واسعا يف األنشطة الفصيلة، لتزويد املعلم والدراس بوسيلة ممتاعة ومشوقة للتدريب على عناصر اللغة، وتوفري احلوافر لتنمية املهارت اللغة املختلفة، وهي أيضا توظف بعض العمليات العقلية مثل (التخمني) إلضفاء أبعاد اتصالية على ت لك ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah Muthmainnah 58 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) ( األنشيطة، وتتيح للطالبات نوعا من اإلختبار للغة اليت يستخدم هنا مصطفى عبد العزيز ، 1403 : 7) . 2) مزايا اللعبة اللغوية يف عملي ة األلعاب اللغوية ، سيشارك الط بلا ات بشكل امل باشر ة يف التعلم النشط من خالل اإل شراك مجيع احلواس واملشاعر حبماس ال كبري ، ألن فيها عناصر ال رتفيهية ومنافسة ال صحية، وأما بالنسبة لبعض من املزايا يف لعبة اللغوية فهو كما يلي : أ) .ميكن أن تقلل من امللل للطالبات يف عملية التعلم يف الفصل الدراسة ب) مع النافسة بني الطالبات، ميكن أن تعزز روح طالبات إىل. أكثر تقدما ج) لعبة اللغة ميكن بناء عالقة وتطوير وجمموعة من الكفاءات االجتماعية .للطالبات د) املواد اليت يتم إبالغها ميكن أن ترتك انطباعا يف قلوب الطالبات حىت .جتربة تدريب املهارات صعوبات نسيان من خالل األلعاب ، سيتعلم الطالب ات الكثري عن احلياة ، مثل الدراسة الذاتية ، واجلرأة على املنافسة ، والتواصل االجتماعي ، والتحلي بروح، القيادة ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 59 59 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 59 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) وزيادة الثقة، وما سوى .ذلك ولكن وراء كل ذلك البد أن هناك عيوب فيه. وفيما يلي بعض من العيوب يف تنفيذ اللعبة اللغوية( fatul Mujib dan Nailur Rahmawati, 2002:38 ) : أ) عدد الطالبات كبرية جدا مما تسبب يف صعوبات إلشراك مجيع الطالبات .يف اللعبة ب) لعبة اللغة عادة ما يتبع بالضحك واهلتاف حبفاوة حيث أن الطالبات يف .اللعبة .يف اللعبة ج) لعبة اللغة عادة ما يتبع بالضحك واهلتاف حبفاوة حيث أن الطالبات ميكن أن تتدخل من تنفيد.التعليمات يف البئة األخرى د) .ميكن أن ترسل املوضوع ليست كلها من خالل لعبة اللغوية ه) .لعبة اللغة عموما ال تعترب لغة التعلم يف الربامج, ولكن فقط كإهلاء ج) لعبة اللغة عادة ما يتبع بالضحك واهلتاف حبفاوة حيث أن الطالبات ميكن أن تتدخل من تنفيد.التعليمات يف البئة األخرى د) .ميكن أن ترسل املوضوع ليست كلها من خالل لعبة اللغوية ه) .لعبة اللغة عموما ال تعترب لغة التعلم يف الربامج, ولكن فقط كإهلاء 4) أهداف اللعبة اللغوية 4) أهداف اللعبة اللغوية لعبة اللغوية يستطيع أن دعم حتقيق أهداف الت .عليم الستعاب املعلم جيانب ذالك، يستطيع اللعبة أن تنمية موقفا إجيايب للطالبات كاتضامن، روح رياضية ، .ابتكار وثقة النفس وهتداف اللعبة اللغوية لنيل اإلرتاح و ترتيب املهارة ،اللغوية (االستماع، الكالم، القراءة، الكتابة وعناصر اللغة (املفردات و )القواعد اللعبة اللغوية اليت تتكامل يف التعليم أن تكون :األهداف التالية أ) تثري التفاعل اللفظي للطالبات ب) تزيد الطالقة والتقة نفس يف الطالبات ج) توفر حمتوى التعليم د) أداة لتقليل اململ(Fathul Mujib dan Nailur Rahmawati,2002: 38) ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN i k h h i h Rizka Anugrah, Muthmainnah 60 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 1) مفهوم اللعبة األوراق الممزقة لعبة األوراق املمزقة هي لعبة لرت قية مهارات الط البات يف قراءة اللغة العربي ة، ويف هذه اللعبة يستطيع الط البات ل.فهم الدرس لعبة األوراق املمزقة هي هتدف إىل مترين ال طالبات يف القراءة و ترتيب القص.ة األدوات املستخدمة يف هذه اللعبة : األوراق املمز قة (ال قصة القصرية أ و القص)ة املزاحة. ( ناصف مصطفى عبد العزيز، 2012 : . 12 ( . ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah ى 2) خطوات لعبة األوراق الممزقة التعلم املمتع ة يعين التعلم وفقًا لألجواء اليت حتدث يف الط بلا ات . من املأمول أن يصبح ال تعلم مهارات القراءة عرضًا ماديًا ما يكون ممتعًا وينتظره بلا الط ات .بفارغ الصرب وأما خطوات القراءة الستخدام طريقة القراءة بلعبة األوراق املمزقة كما يلى ( Ulin Nuha,1992: 911 ).:1) تسأل املدرسة عن استعداد ال طالبات يف التعل م . 2 ) تعد املدرسة السبورة: تكتب التاريخ وموضوع املادة . 3 )تشرح املدرسة عن املادة اليت ستعلمه ا . 4 ) تعطي املدرسة املفردات اجلديدة . 5 )تقسم املدرسة ال طالبات إىل بعض اجملموعات . 5 ) تقسم املدرسة األوراق املمز قة إىل ثالث اجملموعات . 6 ) تقرأ املدرسة بعض القصة يف اللغة العربية إىل اجملموعات . 7 ) يطلب اجملموعات لرتتيب القصة بواسطةاألوراق املمز قة اليت جيد ال طالبات . 8 )يعرض اجملموعات قصته أمام الفصل.9 ) تلخص املدرسة مادة التع لم. ب. 4) أهداف اللعبة اللغوية مهارة القراءة 1) مفهوم مهارة القراءة 2) خطوات لعبة األوراق الممزقة التعلم املمتع ة يعين التعلم وفقًا لألجواء اليت حتدث يف الط بلا ات . من املأمول أن يصبح ال تعلم مهارات القراءة عرضًا ماديًا ما يكون ممتعًا وينتظره بلا الط ات .بفارغ الصرب وأما خطوات القراءة الستخدام طريقة القراءة بلعبة األوراق املمزقة كما يلى ( Ulin Nuha,1992: 911 ).:1) تسأل املدرسة عن استعداد ال طالبات يف التعل م . 2 ) تعد املدرسة السبورة: تكتب التاريخ وموضوع املادة . 3 )تشرح املدرسة عن املادة اليت ستعلمه ا . 4 ) تعطي املدرسة املفردات اجلديدة . 5 )تقسم املدرسة ال طالبات إىل بعض اجملموعات . 5 ) تقسم املدرسة األوراق املمز قة إىل ثالث اجملموعات . 6 ) تقرأ املدرسة بعض القصة يف اللغة العربية إىل اجملموعات . 7 ) يطلب اجملموعات لرتتيب القصة بواسطةاألوراق املمز قة اليت جيد ال طالبات . 8 )يعرض اجملموعات قصته أمام الفصل.9 ) تلخص املدرسة مادة التع لم. املدرسة مادة التع لم. ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 61 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) مهارة القراءة هي مهارات اليت ميكن أن تدرب الطالبات على .مهارات القراءة والفهم وميكنهن تنمية قدرهتن على قراءة اللغة العربية )مهارة القراءة هي قدرة تعريف الشيئ املكتوب (الرموز املكتوبة وفهمه ا بالنطق والفهم.مهارة القراءة يشتمل على الناحيتني، األوىل : تغري .الكتابة إىل األصوات والثانية : استعاب معاىن كل األحوال اليت تعرب بالكتابة و األصوات.القراءة هي فهم احملتويات املكتوبة بنطق ما يك تبه املدرس. إذان، كانت القراءة حتتوى على مهارتان وهي تعريف ا أللفاظ وفهمها suharsimi arikunto,1891: 15 ) ). وفهمها suharsimi arikunto,1891: 15 ) ). 2) أهداف مهارة القراءة أن نعرف األهداف من تعلم مهارة القراءة لزيادة قدرة الطالبات على .النطق الكلمات نطقا سليما وفهم املقروء وغري ذلك ، فإن مهارات القراءة هلا العديد من األهداف األخرى :منها إكساب الطالبات القدرة على نطق الكلمات نطقا سليما ، إقدار الطالبات على إخراج احلروف من خمارجها ومتييز أصواهتا، إكساب الطالبات رصيدا من املفردات والرتكيب، تنمية الطالبات حنو القراءة واإلصطالح، مساعدة الطالبات على تكوين عادت التعريف البصري على الكلمات وفهم معناها أو معىن اجلمل والرتكيب، إقدار الطالبات على القراءة السريعة الواعية املصحوبة بفهم املادة املقروءة يف القراءتني الصامتة واجلهرية ( نور هادي، 2011 : 77 ) . 3) أنواع مهارة القراءة ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 62 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 62 62 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 62 عندما نقرأ نصًا ، لدينا سببان رئيسيا ن ألنه من الضروري قراءة النص, أوال هو املتعة و ثاني ا هو احلصول على املعلومات.عندما نقرأ ، منيل إىل استخدام ة مخس ة طرق لفهم النص :يعين الغرض األساسي من القراءة متكني املتعلم من اختزان صور الكلمات يف ذهنه ليصبح قادرا على القرءة السريعة املقرونة بالفهم، والقراءة السريعة ال تعين يف البداية الوقت وأمنا املراد هبا أن يظهر على نطق املتعلمني االرتباح لدى التاعمل مع النص، دون تفكري كثر، فين تقبل من كلمة إىل أخرى، ومن سطر إىل آخر بسهولة ويسر وال يبدو عليه العناء واالجهاد ( لدكتور يوسف الصميلي1885 م– 1405 : 69 .) )ب القراءة الصامتة القراءة الصامتة هي القراءة الطبيعية املستعملة يف احلياة لكسب املعرفة وحتتيق املتعة، وإليها يصري القارئ فيما يقرأ غالبا. وعليها يعتمد يف حياته العملية، وهبا تتاح الفرصة الواسعة لرتقية الفهم وتوسيع حم .اله والقراءة الصامتة تستخدم يف مجيع مراحل التعليم، ولكن بنس ب متفاوتة، فهي تناسب منو ال طالبات تناسبا طرديا مبعىن أنه كلما منا ال طالبات زاد وقت القراءة الصامتة ( ، جودت الركابي1988 : 38 .) ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 63 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) تعترب الق راءة اجلهرية مهارة خاصة ميكن أنيك ون تعلمها غاية يف حد ذاته، و ميكن أيضا أن يكون وسيلة أو مرحلة من مراحل تعلم املهارة الكلية للقراءة، فالقراءة اجلهرية ينظر إليها كخطوة أوىل وضرورية للقراءة الصامتة والكتابة أيضا. إذا قبل أن يتمكن املتعلم من أداء هذ ين النشاطني البد أن تتأكد لديه بشكل تام الغالق ة بني الصوت ورمزة الكتايب ( ، حممود كامل الناقة1885 م– 1405 : 191 .) يقصد بالقراءة البليغة أو املعربة، إتقان فن األداء اللغوي نطقا وفصال ووصال ووقفا وهلجة، فيعطي القارئ للعبارات واجلمل معنها املراد تعجبا أو استفهاما أو تقريرا أو غري ذلك، لتحقيق جزء أهداف القراءة أي التأثري يف املستمع وجذب انتباهه إىل املقروء (الدكتور يوسف ا لصميلي 1885 م– 1405 هـ: 75 .) ه )القراءة المكثفة يقصد هبا ذلك النوع من القراءة الذي جيري داخل الفصل هبدف تنمية مهارات القراءة عند الدارسني. وزيادة رصيهم اللغوية. وختتار هلذا مواد على مستوى من الصعوبة يدرب الدارس عل اكتساب مهارات التعرف والفهم والنقد والتفاعل. ويدور النشاط يف هذا النوع من القراءة حتت إشراف املعلم ويف الفصل الدراسي ( pangadilan Rambe, 2015: 124 ). من أنواع القراءة أعاله ، يف هذه الدراسة أخذ الباحث ة نوع القراءة اجلهرية , ألن يتوافق مع حالة بلاط ال اتفي ال فصل أول اإل بتداء "أ" الذي بعضهمناقصب.القراءة ف أخذ الباح ثة هذا النوع من القراءة لرتقية كفاءة الطالبات يف مهارة .القراءة ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 64 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 3. منهجية البحث يستخدم هذا البحث مدخل الكمي اليت حتتاج اىل الرموز االرقامية الجاد صورة املعلومات البيانية والتفصيلية وختتار الباحثة هذا املدخل، الهنا تريد أن تعرف عم لية التدريس وتريد أن جترب وختترب فعالية استخدام اللعبة اللغوية .""األوراق املمزقة ويسمي بالكمي، ألن الباحثة حتتاج اىل البيانات تكون على صورة االرقام من العينة الطالبات الاليت يطلنب منهن اإلجابة عن أسئلة حول هذا البحث، أو تعطي وصفا رقيما يشري إىل حجم هذه الظاهرة أو حجمها ودرجة اليت تتعلق هبا الظواهر املختلفة األخري. ونوعه هو البحث التجريب، يعين اجلهد البحثي أن يأخذ تأثريا من املتغري املعني على .املتغري اآلخر مبراقبة جيدة ونوع البحث هو حبث التجريب Experiment Research بطرق اجملموعة الواحدةOne Group Method يعين أن يكون الفرق يف نتائج االختبارين القبلي والبعدي بواحدة . اجملموعة التجريب ناجتاً عن تأثرها باملتغري التجريب واألسلوب جلمع البيانات يف .هذا البحث هي اإلختبار واملالحظة واملقابلة والوثائق في هذه ال بحث استخدم ت الباحثة حساب معادلةSPSS والذي يف حتليل البيانات الكمية تستخدم الباحثة حتليال إحصائها وصفيا. وهذا الرموز لتقومي :إنتاج التصميم x  3. منهجية البحث يستخدم هذا البحث مدخل الكمي اليت حتتاج اىل الرموز االرقامية الجاد صورة املعلومات البيانية والتفصيلية وختتار الباحثة هذا املدخل، الهنا تريد أن تعرف عم لية التدريس وتريد أن جترب وختترب فعالية استخدام اللعبة اللغوية .""األوراق املمزقة ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah صورة املعلومات البيانية والتفصيلية وختتار الباحثة هذا املدخل، الهنا تريد أن تعرف عم لية التدريس وتريد أن جترب وختترب فعالية استخدام اللعبة اللغوية .""األوراق املمزقة ويسمي بالكمي، ألن الباحثة حتتاج اىل البيانات تكون على صورة االرقام من العينة الطالبات الاليت يطلنب منهن اإلجابة عن أسئلة حول هذا البحث، أو تعطي وصفا رقيما يشري إىل حجم هذه الظاهرة أو حجمها ودرجة اليت تتعلق هبا الظواهر املختلفة األخري. ونوعه هو البحث التجريب، يعين اجلهد البحثي أن يأخذ تأثريا من املتغري املعني على .املتغري اآلخر مبراقبة جيدة ونوع البحث هو حبث التجريب Experiment Research بطرق اجملموعة الواحدةOne Group Method يعين أن يكون الفرق يف نتائج االختبارين القبلي والبعدي بواحدة . اجملموعة التجريب ناجتاً عن تأثرها باملتغري التجريب واألسلوب جلمع البيانات يف .هذا البحث هي اإلختبار واملالحظة واملقابلة والوثائق في هذه ال بحث استخدم ت الباحثة حساب معادلةSPSS والذي يف حتليل البيانات الكمية تستخدم الباحثة حتليال إحصائها وصفيا. وهذا الرموز لتقومي :إنتاج التصميم N x X  = :البيان .األوراق املمزقة ويسمي بالكمي، ألن الباحثة حتتاج اىل البيانات تكون على صورة االرقام من العينة الطالبات الاليت يطلنب منهن اإلجابة عن أسئلة حول هذا البحث، أو تعطي وصفا رقيما يشري إىل حجم هذه الظاهرة أو حجمها ودرجة اليت تتعلق هبا الظواهر املختلفة األخري. ونوعه هو البحث التجريب، يعين اجلهد البحثي أن يأخذ تأثريا من املتغري املعني على .املتغري اآلخر مبراقبة جيدة ونوع البحث هو حبث التجريب Experiment Research بطرق اجملموعة الواحدةOne Group Method يعين أن يكون الفرق يف نتائج االختبارين القبلي والبعدي بواحدة . اجملموعة التجريب ناجتاً عن تأثرها باملتغري التجريب واألسلوب جلمع البيانات يف .هذا البحث هي اإلختبار واملالحظة واملقابلة والوثائق في هذه ال بحث استخدم ت الباحثة حساب معادلةSPSS والذي يف حتليل البيانات الكمية تستخدم الباحثة حتليال إحصائها وصفيا. وهذا الرموز لتقومي :إنتاج التصميم Rizka Anugrah, Muthmainnah 65 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 65 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 65 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) X= املعدل x= مجلة النتائج احملصولة من جيمع الط البات N= عدد الط البات تو ش حر الباحثة النتيجة احملصولة يف االختبار القبلي باستخدام: 1 x ، والنتيجة يف االختبار البعدي باستخدام: 2 x . لتحليل البيانات يف البحث التجريي ( Experiment Research ) بطرق اجملموعة الواحدة( One Group Method ) لتقومي فعالية أحد الطريقة املستخدمة: t= t= :البيان t = قيمةt.test ( t )احلسايب d =التفريق بني الن تيجة كل الطالبات يف اإلختبار القبلي والنتيجة يف اإلختبار البعدي أو احملصولة من2 x - 1 x . d M = معدل مجلةd والرموز :املستخدم ∑𝑑 𝑁 t قيمةt.test ( t )احلسايب d =التفريق بني الن تيجة كل الطالبات يف اإلختبار القبلي والنتيجة يف اإلختبار البعدي أو احملصولة من2 x - 1 x . d M = معدل مجلةd والرموز :املستخدم ∑𝑑 𝑁 d =التفريق بني الن تيجة كل الطالبات يف اإلختبار القبلي والنتيجة يف اإلختبار البعدي أو احملصولة من2 x - 1 x . d M = معدل مجلةd والرموز :املستخدم ∑𝑑 𝑁 𝐗2 d = احنراف ال نتيجة كل الفاعل أو الطالبات، والرموز املستخدم :d - d M ∑𝐗2 d = ا جلملة املربعة من احنراف نتيجة كل الفاعل أو الطالبات ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Ri k A h M th i h Rizka Anugrah, Muthmainnah Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 66 66 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) N )= عدد الطالبات (العينة وهناك القانون يف معرفة فعالية التجربة، إذا كانت النتيجة احملصولة من الرموزt.test ( .t احلسايب) أكثر من النتيجة املوجودة يف جدوال درجة الصدق( Significance and Confidence Level ،) فكانت الطريقة املستخدمة يف التجربة هي فعالية. والعكس منه يدل على فشيلة الطريقة. والنتيجة املوجودة يف جدوال درجة الصدق تسمىt. table ( t .)اجلدوايل t.test (t < ).احلسايبt.table (t ..اجلدوايل) = حصلت التجربة t.test (t )احلسايبt.table (> t .اجلدوايل) = فشلت التجربة وألنّ هذا البحث هو كالبحث اإلج تماعي فكانت الدرجة املستخدمة هي 1 % - 5 % وجدوالt. table (t اجلدوايل) يذكر يف قائمة املالحق . ج ال حث t.test (t < ).احلسايبt.table (t ..اجلدوايل) = حصلت التجربة t.test (t )احلسايبt.table (> t .اجلدوايل) = فشلت التجربة قامت الباحثة باملالحظة ثالثة االيا م من يوم اجلمعة29 يوىل إىل يوم األحد31 يوىل2022. ويف تاريخ 1 حىت20 أ غوستوس2022 بدأت الباحثة التطبيق يف س تة :لقاءات.أما تفصيل ذلك كما يلي الرقم المادة الخطوات 1. هي املالحق . 4. نتائج البحث ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Ri k A h M th i h التعارف - القاء السالم - ت قرأ الدعاء قبل الدرس - سألت الطالبات عن أحواهلن ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 67 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) ISSN: 2774-5724 (media Online) 2 . املقدمة -إعطاءاملوضوع اليت يف كتاب العربي ة للناشئني يف تعليم مهارة ال قراءة ، مث يقرأن .معا -فشرحت الباحثةيف .املادة حتت املوضوع .وحبثتنا عن املفردات الصعوبة 3 . التطبيق -ت عرّف ا الط لبات على الطريقة اليت سيتم استخدامها. - بينت كيفية استخدام اللعبة اللغوية ."األوراق املمزقة" يف املادة - السؤال واجلواب. - عملية التعليم باستخدام اللعبة اللغوية .""األوراق املمزقة - تقسم ال باحثة ال طالبات إىل بعض اجملموعات. - تقسم ال باحثة األوراق املمز قة إىل بعض اجملموعات. - تقرأ ال باحثة بعض القصة يف اللغة العربية إىل اجملموعات. - يطلب اجملموعات لرتتيب القصة بواسطةاألوراق املمز قة اليت جيد ال طالبات. - يعرض اجملموعات قصته أمام الفصل. - تلخص ال باحثة مادة التع لم. ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KA AT TOLIBAT FI MAHARAH AL QIRO’AH FI AL FASL AL IBTIDA’I BIMA’HAD DARU AL LUGH 2 . املقدمة -إعطاءاملوضوع اليت يف كتاب العربي ة للناشئني يف تعليم مهارة ال قراءة ، مث يقرأن .معا -فشرحت الباحثةيف .املادة حتت املوضوع .وحبثتنا عن املفردات الصعوبة 3 . التطبيق -ت عرّف ا الط لبات على الطريقة اليت سيتم استخدامها. - بينت كيفية استخدام اللعبة اللغوية ."األوراق املمزقة" يف املادة - السؤال واجلواب. - عملية التعليم باستخدام اللعبة اللغوية .""األوراق املمزقة - تقسم ال باحثة ال طالبات إىل بعض اجملموعات. - تقسم ال باحثة األوراق املمز قة إىل بعض اجملموعات. - تقرأ ال باحثة بعض القصة يف اللغة العربية إىل اجملموعات. - يطلب اجملموعات لرتتيب القصة بواسطةاألوراق املمز قة اليت جيد ال طالبات. - يعرض اجملموعات قصته أمام الفصل. - تلخص ال باحثة مادة التع لم. ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Ri k A h M th i h 68 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) ISSN: 2774-5724 (media Online) - والتدريبات . 4 . اإلختتام -ارشادات واملواعظ - دعاء اإلختتام بعد الدرس -اإلختتام بالسالم ISSN: 2774-5724 (media Online) - والتدريبات . 4 . اإلختتام -ارشادات واملواعظ - دعاء اإلختتام بعد الدرس -اإلختتام بالسالم ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH AD-DA’WAH BANGIL PASURUAN اللّقاء األول، عندما دخلت الباحثة يف الفصل ، كانت حالة الفصل غ ري مستقرة للغاية ألن بعض ال طالبات قد استيقظ ن من النوم وبعضه ن كان يتحدث مع بعضه ن ًالبعض ، وبالتايل كان جو الفصل صاخبًا جد ا وخرج عن السيطرة. يف ذلك الوقت ، استعان الباحث ة بقسم الرت بية إليقاظ ال طالبات والتحكم هبم حىت يكونوا مستعدين وجلسوا بشكل منظم للمشاركة يف الدرس ( مالحظة يف التارخ1 اغستوس2022 ) قبل أن تنفذ الباحثة إختبار قبلي ، عقدت الباحثة جلسة السؤال واجلواب مع الط بلا ات ح ول رأيه ن يف تعلم اللغة العربية ، وشعر ن معظمه ن أن التعلم اللغة العربية مم ل للغاية وغري ممتع. من هنا أعط ت الباحث ة بعض األشياء اإلجيابية لتغيري األفكار غري املرحية يف تعلم اللغة العربية. عندما اللّقاء األول، عندما دخلت الباحثة يف الفصل ، كانت حالة الفصل غ ري مستقرة للغاية ألن بعض ال طالبات قد استيقظ ن من النوم وبعضه ن كان يتحدث مع بعضه ن ًالبعض ، وبالتايل كان جو الفصل صاخبًا جد ا وخرج عن السيطرة. يف ذلك الوقت ، استعان الباحث ة بقسم الرت بية إليقاظ ال طالبات والتحكم هبم حىت يكونوا مستعدين وجلسوا بشكل منظم للمشاركة يف الدرس ( مالحظة يف التارخ1 اغستوس2022 ) قبل أن تنفذ الباحثة إختبار قبلي ، عقدت الباحثة جلسة السؤال واجلواب مع الط بلا ات ح ول رأيه ن يف تعلم اللغة العربية ، وشعر ن معظمه ن أن التعلم اللغة العربية مم ل للغاية وغري ممتع. من هنا أعط ت الباحث ة بعض األشياء اإلجيابية لتغيري األفكار غري املرحية يف تعلم اللغة العربية. عندما ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN k h h h Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) بدأت إختبار قبلي، مل ت ستجيب بعضه ن بشكل جيد وكانوا أقل محاسًا لإلجابة على األسئ لة. ويرتبط هذا الشرط برأي أحد الطالبات من قسم الرتبية امسها نور حليمة شريف يف مقاب ال لة سابقة قال ت "يصعب إدارة ال طالبات "والكثري منهم ينامون أثناء التعلم( مقابلة مع قسم الرتبية نور حليمة شريف يف التا رخ30 يويل2022 ) في اللقاء االخري ، ذكّرت الباحثة على الطالبات بأنه يف ال لقاء التايل ، جيب أن تكون ظروف الفصل عند دخول الباحث ة .جاهزة ومل يعد أحد يشعر بالنعاس اللقاء الثانية،مقارنةً بال لقاء السابق ، يف هذا ال لقاء ت مل عد الباحث ة يساعده قسم الرتبية أل ن الظروف الصفية يف هذا ال لقاء كانت أكثر استقرارًا من سابقتها. ال يوجد سوى عدد قليل م ن ال طالبات الذين يبدون نعسانًا جدًا ولكنه ن ن حياول إيقاظ أنفسه ن ملتابعة الدرس( مالحظة يف التارخ6 اغستوس2022 .) ،عندما يكونون مستعدين بدأ ت الباحث ة في تعريف الطريقة التعليم ب استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية ،كفاءة الطالبات يف مهارة القراءة تو شرح كيفي ته باستخدام الكتاب للناشئني يف جزء الثاين باملوضوع"البيت " . واحدا تلو االخر استجاب بلا الط ات جيد ايف ه هذ ،الدرس وكانوا متحمسني جدًا لطرح األسئلة. يف هذ ه اللقاء ،ت قسم الباحث ة على بلا الط ات إىل عدة اجمل موعات ، وذكره ن ن أهن في اللقاء التايل جيب أن جيلس ن .يف كل جمموعة مت تقسيمها اللقاء الثالث ة، يف هذه اللّقاء مت الرتحيب بالباحثني بأجواء مفعمة باحليوية والسعادة من الط بلا ات ، على الرغم من أهن ن كان ال يزال هناك 69 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) بدأت إختبار قبلي، مل ت ستجيب بعضه ن بشكل جيد وكانوا أقل محاسًا لإلجابة على األسئ لة. ويرتبط هذا الشرط برأي أحد الطالبات من قسم الرتبية امسها نور حليمة شريف يف مقاب ال لة سابقة قال ت "يصعب إدارة ال طالبات "والكثري منهم ينامون أثناء التعلم( مقابلة مع قسم الرتبية نور حليمة شريف يف التا رخ30 يويل2022 ) في اللقاء االخري ، ذكّرت الباحثة على الطالبات بأنه يف ال لقاء التايل ، جيب أن تكون ظروف الفصل عند دخول الباحث ة .جاهزة ومل يعد أحد يشعر بالنعاس ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah الفصل عند دخول الباحث ة .جاهزة ومل يعد أحد يشعر بالنعاس اللقاء الثانية،مقارنةً بال لقاء السابق ، يف هذا ال لقاء ت مل عد الباحث ة يساعده قسم الرتبية أل ن الظروف الصفية يف هذا ال لقاء كانت أكثر استقرارًا من سابقتها. ال يوجد سوى عدد قليل م ن ال طالبات الذين يبدون نعسانًا جدًا ولكنه ن ن حياول إيقاظ أنفسه ن ملتابعة الدرس( مالحظة يف التارخ6 اغستوس2022 .) ،عندما يكونون مستعدين بدأ ت الباحث ة في تعريف الطريقة التعليم ب استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية ،كفاءة الطالبات يف مهارة القراءة تو شرح كيفي ته باستخدام الكتاب للناشئني يف جزء الثاين باملوضوع"البيت " . واحدا تلو االخر استجاب بلا الط ات جيد ايف ه هذ ،الدرس وكانوا متحمسني جدًا لطرح األسئلة. يف هذ ه اللقاء ،ت قسم الباحث ة على بلا الط ات إىل عدة اجمل موعات ، وذكره ن ن أهن في اللقاء التايل جيب أن جيلس ن .يف كل جمموعة مت تقسيمها اللقاء الثالث ة، يف هذه اللّقاء مت الرتحيب بالباحثني بأجواء مفعمة باحليوية والسعادة من الط بلا ات ، على الرغم من أهن ن كان ال يزال هناك 9 - 10 بلاط ات شبه ت ا ني للقاء السابق. ولكن كان هناك اختالف ول ب ل س ر ب ي ز ومل ي اللقاء الثانية،مقارنةً بال لقاء السابق ، يف هذا ال لقاء ت مل عد الباحث ة يساعده قسم الرتبية أل ن الظروف الصفية يف هذا ال لقاء كانت أكثر استقرارًا من سابقتها. ال يوجد سوى عدد قليل م ن ال طالبات الذين يبدون نعسانًا جدًا ولكنه ن ن حياول إيقاظ أنفسه ن ملتابعة الدرس( مالحظة يف التارخ6 اغستوس2022 .) ،عندما يكونون مستعدين بدأ ت الباحث ة في تعريف الطريقة التعليم ب استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية ،كفاءة الطالبات يف مهارة القراءة تو شرح كيفي ته باستخدام الكتاب للناشئني يف جزء الثاين باملوضوع"البيت " . واحدا تلو االخر استجاب بلا الط ات جيد ايف ه هذ ،الدرس وكانوا متحمسني جدًا لطرح األسئلة. يف هذ ه اللقاء ،ت قسم الباحث ة على بلا الط ات إىل عدة اجمل موعات ، وذكره ن ن أهن في اللقاء التايل جيب أن جيلس ن .يف كل جمموعة مت تقسيمها اللقاء الثالث ة، يف هذه اللّقاء مت الرتحيب بالباحثني بأجواء مفعمة باحليوية والسعادة من الط بلا ات ، على الرغم من أهن ن كان ال يزال هناك 9 - 10 بلاط ات شبه ت ا ني للقاء السابق. ولكن كان هناك اختالف ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Ri k A h M th i h Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 70 70 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) ال واضح عن ال لقاء األول ألهن ن ن جلس بدقة يف كل جمموعة، مما يشري إىل ن أهن مستعد ات للتعلم ( مالحظة يف التارخ8 اغستوس2022 .) ،اللقاء الرابعة و صلت الباحثة طريقة التعلم وكانت اإلجابة كل جمموعة يف ذلك الوقت متحمسة للغاية ، وسأل ت الباحث ة مرة أخرى برأيه ن حول تعلم اللغة العربية وأجاب معظمهم "كان ممتعًا وممتعًا وشيقًا وأمورًا إجيابية أخرى " . ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah الفصل عند دخول الباحث ة .جاهزة ومل يعد أحد يشعر بالنعاس اللقاء الثانية،مقارنةً بال لقاء السابق ، يف هذا ال لقاء ت مل عد الباحث ة يساعده قسم الرتبية أل ن الظروف الصفية يف هذا ال لقاء كانت أكثر استقرارًا من سابقتها. ال يوجد سوى عدد قليل م ن ال طالبات الذين يبدون نعسانًا جدًا ولكنه ن ن حياول إيقاظ أنفسه ن ملتابعة الدرس( مالحظة يف التارخ6 اغستوس2022 .) ،عندما يكونون مستعدين بدأ ت الباحث ة في تعريف الطريقة التعليم ب استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية ،كفاءة الطالبات يف مهارة القراءة تو شرح كيفي ته باستخدام الكتاب للناشئني يف جزء الثاين باملوضوع"البيت " . واحدا تلو االخر استجاب بلا الط ات جيد ايف ه هذ ،الدرس وكانوا متحمسني جدًا لطرح األسئلة. يف هذ ه اللقاء ،ت قسم الباحث ة على بلا الط ات إىل عدة اجمل موعات ، وذكره ن ن أهن في اللقاء التايل جيب أن جيلس ن .يف كل جمموعة مت تقسيمها اللقاء الثالث ة، يف هذه اللّقاء مت الرتحيب بالباحثني بأجواء مفعمة باحليوية والسعادة من الط بلا ات ، على الرغم من أهن ن كان ال يزال هناك 9 - 10 بلاط ات شبه ت ا ني للقاء السابق. ولكن كان هناك اختالف ،اللقاء الخامسة يف هذا ال لقاء ت قام الباحث ة بتذكري الط بلا ات باالستعداد يف ال لقاء اآليت سي كون هناك اإل ختبار عب ال دي ، ويف هذا ال لقاء اخلامس ة شعر ت الباحث ة أن الطالبات يف هذه الفصل استجاب ن بشكل جيد للغاية وكان ت متحمس ات دائمًا للمشاركة يف األنشطة .التعليمية ،اللقاء السادسة أجرت الباحثة اإل ختبار البعدي مبدة يف كتاب "العربية للناشئني" جزء الثاين يف امل ."وضوع "البيت و أجواء الفصل مرحية ، وهادئة ن أهن متحمس ات إلجابة على األسئلة. ويف هناية هذا اللقاء قدمت الباحثة جوائز للمجموعة املتحمسة وثالثة الطالبات نشطني وأعلى الدرجات يف الفصل( مالخظة يف التارخ20 أغستوس2022 .) بعد كل اللقاء، شعرت الباحثة بالرضا واالمتنان الشديد بسبب االست جابة اجليدة واملشاركة من ال طالبات على الرغم ، أنه مل يكن من .املمكن يف السابق التحكم يف الفصل بشكل صحيح ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah Muthmainnah 71 71 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) من خالل هذه ال لقاء ، تعلم الباحث ة حقًا حت لي بالصرب يف التعامل مع ال طالبات ألنه ليس بالسهولة اليت قد يتخيلها املرء ولكن أيضًا ليس بالصعوبة اليت قاهلا بعض معلم ة الفصل الليل، أحده ن امسها فاطمة الزهراء قالت"غالبًا ما يكون ال طالبات صا خبني يف الفصل، ناهيك عن الذي تنمن و تتحدثن هنا وهناك. جيعلنا مضطرين إىل التحلي بالصرب اإلضايف يف التع امل معها" ( مقابلة مع املعل م ة فاطمة الزهراءيف التارخ31 يويل 2022 .) حدث% 97 يف الواقع يف ال لقاء األول و لكن بالنسبة اللقاء التايل ، شعر ت الباحث ة أنه مت ختفيضه إىل% 30. ألن الباحث ة تشعر أن التعلم حيدث ان يعتمد على قدرة املعلم ة ب التحكم يف الفصل وإتقانه جيدًا أم ال ، كما جيب أن يكون لدى املعلم ة لا طريقة حىت ت كون ال طالبات هتتمن تردن و .متابعة الدرس جيدًا تردن و .متابعة الدرس جيدًا ولضعفهن يف ال قراءة باللغة العربية حتاول يف ابتداء دراستهن بتقدمي االختبار القبلي يف أول اللّقاء قبل عملية البحث،وهذا ملعرفة كفاءة ا الط لبات يف ال قراءة باللغة .العربية قبل التطبيق وكان هذا البحث هو الدراسة التجربية تصميم جمموعة واحدة وهنا فصل األول اإلبتدائي "أ". وتستخدم الباحثة هلذا البحث بتصميم اجملموعة الواحدة . ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah الفصل عند دخول الباحث ة .جاهزة ومل يعد أحد يشعر بالنعاس اللقاء الثانية،مقارنةً بال لقاء السابق ، يف هذا ال لقاء ت مل عد الباحث ة يساعده قسم الرتبية أل ن الظروف الصفية يف هذا ال لقاء كانت أكثر استقرارًا من سابقتها. ال يوجد سوى عدد قليل م ن ال طالبات الذين يبدون نعسانًا جدًا ولكنه ن ن حياول إيقاظ أنفسه ن ملتابعة الدرس( مالحظة يف التارخ6 اغستوس2022 .) ،عندما يكونون مستعدين بدأ ت الباحث ة في تعريف الطريقة التعليم ب استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية ،كفاءة الطالبات يف مهارة القراءة تو شرح كيفي ته باستخدام الكتاب للناشئني يف جزء الثاين باملوضوع"البيت " . واحدا تلو االخر استجاب بلا الط ات جيد ايف ه هذ ،الدرس وكانوا متحمسني جدًا لطرح األسئلة. يف هذ ه اللقاء ،ت قسم الباحث ة على بلا الط ات إىل عدة اجمل موعات ، وذكره ن ن أهن في اللقاء التايل جيب أن جيلس ن .يف كل جمموعة مت تقسيمها اللقاء الثالث ة، يف هذه اللّقاء مت الرتحيب بالباحثني بأجواء مفعمة باحليوية والسعادة من الط بلا ات ، على الرغم من أهن ن كان ال يزال هناك 9 - 10 بلاط ات شبه ت ا ني للقاء السابق. ولكن كان هناك اختالف وجتعل الباحثة نفس الطالبات يف جتريبة، وهو يقارن حتصيلهن يف الظرف املختلفة، وهو حصيلتهن قبل تطبيق األنشطة املستخدمة وحصيلتهن بعد تطبيقها. وحصيلتهن حمصولة من االختبار يعين .االختبار القبلي واالختبار بعدي ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN k h h h ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah Muthmainnah Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 72 72 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) ودرجة كل نقطة من العناصر األربع ملهارة ال قراءة من ال طالقة و مفهوم القراءة و خمارج احلرف ترتاوح بني واحد وأربع. فكان الطالقة درجته = 1 - 4، = القواعد4 - 1،و = مفهوم القراءة1 - 4 ،وخمارج احلرف = درجاهتا1 - 4 ، إذن كان أعلى النتيجة16 وهو مأخذ من4 × 4 = 16 ( .صالح العريب، عبداجمليد1981 : 174 - 173 ) . :وملعرفة النتيجة لكل من الطالبات، استخدام الباحثة الرمز األيت ودرجة كل نقطة من العناصر األربع ملهارة ال قراءة من ال طالقة و مفهوم القراءة و خمارج احلرف ترتاوح بني واحد وأربع. فكان الطالقة درجته = 1 - 4، = القواعد4 - 1،و = مفهوم القراءة1 - 4 ،وخمارج احلرف = درجاهتا1 - 4 ، إذن كان أعلى النتيجة16 وهو مأخذ من4 × 4 = 16 ( .صالح العريب، عبداجمليد1981 : 174 - 173 ) . :وملعرفة النتيجة لكل من الطالبات، استخدام الباحثة الرمز األيت ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH K AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGH AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 16 ( .صالح العريب، عبداجمليد1981 : 174 - 173 ) . :وملعرفة النتيجة لكل من الطالبات، استخدام الباحثة الرمز األيت .. 1 × م د =ن البيان: - ن = النتيجة. - م = جمموع النتيجة الطالبات احملصولة. - د = الدّرجة األعلى وهي4 × 4 = 16 وهذا الجدول لمعرفة مستوى النتائج وتقديرها لكل من الطالبات. مستوى النتائج الطالبات الرقم مسافة تحديد النتيجة الدرجة 1 . 90 - 100 ممتاز ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 73 73 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 2 . 80 - 89 جيد 3 . 70 - 79 كافة 4 . 60 - 69 ناقص 5 . أقل من 59 ضعيف Jurnal Tarbiyatuna, Vol. 4, No. قائمة المراجع قائمة المراجع ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 1, Juni 2023 ISSN: 2774-5724 (media Online) من اإلختبار القبلي نالت الباحثة النتائج ب أن 4،0 % من الطالبات حصلن على درجة "جيد "، وتكون4،0 % ،" منهن يف مستوى "كافة وحصلت 24،0 % منهن على نتيجة "ناقص "، وتكون 68،0 % في " مستوى "ضعيف ، ."وال أحد حصلت على نتيجة "جيد جدا نظرا إىل أهداف هذا البحث، يعين ملعرفة فعالية إستخدام اللعبة "األ وراق املمزقة " لرتقية كفاءة الطالبات يف مهارة القراءة يف الفصل األول اإلبتدائي "أ" مبعهد دار اللغة والدعوة بانقيلمن ناحية الطالقة و مفهوم القراءة و خمارج احلرف فقدمت هلن الباحثة اإل خ تبار البعدي ، وهذا اإلختبار معقود بعد إجراء التجربة وهي تعليمهن باستخدام تلك املادة وهتدف به معرفة تأثري استخدامها يف تنمية كفاءة الطالبات يف مهارة ال قراءة. ومن الإلختبار البعدي نالت الباحثة النتائج ب أن 28،0 % من الطالبات حصلن على درجة "جيد "، وتكون20،0 % منهن يف مستوى "كافة "، وحصلت 36،0 % منهن على نتيجة "ناقص "، وتكون 16،0 % ."يف مستوى "ضعيف " وال أحد حصلت على نتيجة "جيد جدا ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah Muthmainnah 74 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) بعد أن عقدت اإلختبار القبلي واإلختبار البعدي قارنت الباخثة .النتائج من ذلك إختبارين او عتماد على الرمز املوجودة وجدت الباحثة أن مجيع نتائجt. test (t )احلسايب 6،97 أكرب من قيمةt.table t) اجلدويل( سواء كانت يف الدرجة5 % 2،06 أو يف الدرجة1 % . 2،79 و أن عدد نتائج االختبار = القبلي 1233,7 = باملعدل49,35 وبعد إجراء االختبار البعدي كانت = النتائج1757,75 = باملعدل70,75 ، وهذه تدل على أن هناك تأثريا بيّنا بعد استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية كفاءة الطالبات يف مهارة القراءة يف الفصل األول اإلبتدائي "أ" مبعهد دار اللغة والدعوة بانقيل ، ألن نتائج االختبار البعدي أكرب من نتائج اال ختبار القبلي. ولذلك أن بعد استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية كفاءة الطالبات فعّاال لتنمية يف مهارة القراءة يف الفصل األول اإلبتدائي "أ" مبعهد دار اللغة والدعوة بانقيل . 5. اإلختتام 5. اإلختتام اعتمادا على البيانات السابقة رأت الباحثة بأن التعليم اجلذّاب يستط يع أن يرتقي محّاسة الطالبات يف تعلم اللغة العربية خاصة يف مهارة القراءة. باستخدام اللعبة اللغوية جتعل الفصل أكثر متعة وهذا احلال يأثّر كثريا على ارتقاء نتائج .الطالبات. نظرا من نتيجة اإلختبار البعدي أكرب من اإلختبار القبلي ولذلك خلصت الباحثة أن استخدام اللعبة اللغوية "األوراق املمزقة" لرتقية كفاءة الطالبات فعّاال لتنمية يف مهارة القراءة يف الفصل األول اإلبتدائي "أ" مبعهد دار اللغة والدعوة بانقيل. قائمة المراجع ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah 75 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) أجيف هرموان،منهجية تعلم اللغة العربية ، (باندون ،غ: املراهقة روزدا كاريا2011 ) أمني الكخن،دليل أحباثة مدنية يف تعليم الغة العربية يف مرحلة التعليم األساسي :(تونس ,جمهول املطبع1992 ) ( ،جودت الركايب، طرق تدريس اللغة العربية،دمشق، سورية: دار الفكر1988 )م ،الدكتور يوسف الصميلي اللغة العربية وطرق تدرسها نظرية وتطبيقا ، (مكة املكرمة:جامعة أم القرى1885 م– 1405 )هـ ذوفان عبيدات و صاحبه, البحث العلمي: مفهومه أدواته, واسالبه, دار أسامة, الرياض ,مبكة املكرمة1996 م ، رشدي أحمد طعيمة تعليم العربية لغري الناطقني هبا مناهجه و أساليبه :،(مصر ،إيسيسكو1989 ) ,مشسدين أشراف منهجية تعلم ال لغة العربية ،، (يوجياكارتا: األكادميية بوجا2006 ) ،صاحل ذياب هندي و هشام عامر عليان دراسات يف املناهج و األساليب العامة ، ،(عمان: دارالفكر1987 ) ،صاحل عبد اجمليد العرىب تعلم اللغات احلية و تعليمها بني النظرية و التطبيق (لبنان : ،مكتبة لبنان1981 ) عبد الرمح( .ان إبراهيم الفوزان, إضاءات ملعلمي اللغة العربية لغري الناطقني هبا :الرياض ,جامعة امللك سعود1932 ) العريب، صالح عبداجمليد. تعلّم اللغات احلية وتعليمها، بني النظرية التطبيق، الطبعة األوىل ،(لبنان: مكتبة لبنان1981 )م حممد على اجلويل، أساليب تدريس اللغة العربية،( الرياض: امل ملكة العربية السعودية 1986 )م ،حممد علي اخلويل االختبارات اللغوية ،، (األردن: دارالفالح2000 ) حممود كامل الناقة، تعليم اللغة للناطقني بلغات أخرى، (مكة املكرمة: جا معة أم القرى 1885 م– 1405 )هـ ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 76 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 76 Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 76 76 مصطفى عبد العزيز, األ لعاب اللغوية تعل :يم اللغات األجنبية, (رياض1403 ) ،موليانتو سوماردي تدريس اللغات األجنبية استعراض من حيث املنهجية ,(جاكرتا: جنم ،القمر1974 ) ، ناصف مصطفى عبد العزيز األلعب اللغوي يف تعليم اللغات األجنبية ، (الرياض : دار ,املريح2012 ) نور هادي ، املواجه لتعليم املهارات اللغوية لغري الناطقني هبا,(ماالنق: مطبعة جامعة .موالنا مالك إبراهيم اإلسالميةاحلكومية2011 ) Azhar Arsyad, bahasa arab dan metode pengajaran (yoqyakarta: pustaka pelajar, 2002) Azhar Arsyad, bahasa arab dan metode pengajaran (yoqyakarta: pustaka pelajar, 2002) Azhar Arsyad, bahasa arab dan metode pengajaran (yoqyakarta: pustaka pelajar, 2002) Bisri Mustofa dkk, Metode dan Strategi Pembelajaran Bahasa Arab, (Malang: UIN-Maliki Press, 2012) Bungin, Burhan. Metodelogi Penelitian Kuantitatif: Komunikasi, Ekonomi, Dan Kebijakan Publik Serta Ilmu-Ilmu Sosial Lainnya, Cet. Ke-3 (Jakarta: Prenada Media Group, 2008 ) Fathul Mujib dan Nailur Rahmawati. Metode Permainan-permainan Edukatif dalam Belajar BahasaArab.(Jogjakarta: Diva Press.2012) Khaeruddin, et all, Kurikulum Tingkat Satuan Pendidikan, Konsep Dan Implementasinya Di Madrasah, (Jogjakarta, Pilar Media, 2007) MelSilberman, active learning101 Strategies to Teach My Subject, Sebuah pengantarDr. Moh Ainin, Metodologi Penelitian Bahasa Arab (Hilal Pustaka, 2010 M) Nurgiyantoro, Birhan. Penilaian pembelajaran bahasa berbasis kompetensi. )Yogyakarta: BPFE), 2010 ( Pangadilan Rambe, Pembelajaran Bahasa Arab Tingkat Dasar, (Pekanbaru: Adefa Grafika, 2115( Prof.Dr.Sugiyono, Metode Penelitian Kuantitatif & Kualitatif dan R&D. Suharsimi Arikunto, Dasar-Dasar Evaluasi Pendidikan, (Jakarta: Bumi Aksara, 1891) Suharsimi Arikunto, Prosedur Penelitian Suatu Pendekatan Praktek,(Jakarta: Rineka Cipta) Ulin Nuha, Metodologi Super Efektif Pembelajaran Bahasa Arab, (Jogjakarta : Diva Press, 2192) ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Jurnal Tarbiyatuna, Vol. 4, No. 1, Juni 2023 ISSN: 2774-5724 (media Online) 76 ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN ISTIKHDAM AL-LU’BAH AL-LUGHOWIYAH AL-AUROQ AL-MUMAZZAQOH LITARQIYAH KAFA’AH AT-TOLIBAT FI MAHARAH AL-QIRO’AH FI AL-FASL AL-IBTIDA’I BIMA’HAD DARU AL-LUGHAH WA AD-DA’WAH BANGIL PASURUAN Rizka Anugrah, Muthmainnah
W4376640992.txt	https://bmcmedgenomics.biomedcentral.com/counter/pdf/10.1186/s12920-023-01536-5	en		The identification of a two-gene prognostic model based on cisplatin resistance-related ceRNA network in small cell lung cancer	BMC medical genomics	2,023	cc-by	6,744	(2023) 16:103 Zhang et al. BMC Medical Genomics https://doi.org/10.1186/s12920-023-01536-5 BMC Medical Genomics Open Access RESEARCH The identification of a two‑gene prognostic model based on cisplatin resistance‑related ceRNA network in small cell lung cancer Yani Zhang1,2, Qizhi Zhu1,2, Jian Qi1,2, Meng Fu1,2, Ao Xu4,5, Wei Wang4,5, Hongzhi Wang1,2,3, Jinfu Nie1,2,3 and Bo Hong1,2,3* Abstract Background Small cell lung cancer (SCLC) is a very malignant tumor with rapid growth and early metastasis. Platinum-based chemo-resistance is the major issue for SCLC treatment failure. Identifying a new prognostic model will help to make an accurate treatment decision for SCLC patients. Methods Using the genomics of drug sensitivity in cancer (GDSC) database, we identified cisplatin resistance-related lncRNAs in SCLC cells. Based on the competing endogenous RNA (ceRNA) network, we identified the mRNAs correlated with the lncRNAs. Using Cox and LASSO regression analysis, a prognostic model was established. The survival prediction accuracy was evaluated by receiver operating characteristic (ROC) curve and Kaplan–Meier analysis. GSEA, GO, KEGG and CIBERSORT tools were used for functional enrichment and immune cells infiltration analysis. Results We first screened out 10 differentially expressed lncRNAs between cisplatin resistant and sensitive SCLC cells from GDSC database. Based on ceRNA network, 31 mRNAs were identified with a correlation with the 10 lncRNAs. Furthermore, two genes (LIMK2 and PI4K2B) were identified by Cox and LASSO regression analysis to construct a prognostic model. Kaplan–Meier analysis indicated that the high-risk group had a poor overall survival compared with the low-risk group. The predicted area under the ROC curve (AUC) was 0.853 in the training set, and the AUC was 0.671 in the validation set. In the meanwhile, the low expression of LIMK2 or the high expression of PI4K2B in SCLC tumors was also significantly associated with poor overall survival in both training and validation sets. Functional enrichment analysis showed that the low-risk group was enriched in the apoptosis pathway and high immune infiltration of T cells. Finally, an apoptosis-related gene Cathepsin D (CTSD) was identified to be up-regulated in the low-risk group, and its higher expression correlated with better overall survival in SCLC. Conclusion We established a prognostic model and potential biomarkers (LIMK2, PI4K2B and CTSD), which could help to improve the risk stratification of SCLC patients. Keywords Small cell lung cancer, Cisplatin, lncRNA, ceRNA, Prognostic model Correspondence: Bo Hong bhong@hmfl.ac.cn Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. BMC Medical Genomics (2023) 16:103 Introduction Lung cancer is a growing global problem and the most common cause of cancer-related death [1, 2]. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for 85% of lung cancers as the most common type of lung cancer [3]. SCLC accounts for only 15% of lung cancer cases, but it is a highly metastatic and recalcitrant neuroendocrine carcinoma [4]. Rapid growth and early metastasis lead to high mortality in SCLC. As a highly aggressive disease, ~ 70% of SCLC cases have been disseminated in the initial clinical presentation, and surgery is only appropriate for a small number of patients with localized disease [5]. The standard first-line treatment for SCLC is platinum-based chemotherapy [6]. Although platinum-based chemo-therapy in SCLC patients with an initial remission rate is as high as 78%, most of the patients recur after 6 months. The median overall survival (OS) of SCLC is only about 10 months [7]. Due to the primary resistance to chemotherapy in a subset of SCLC patients and the acquired resistance to chemotherapy after treatment in almost all SCLC patients, it is desirable to be able to identify SCLC patients with chemo-resistant response. Once these SCLC patients are identified with chemo-resistance and poor survival, immune therapy may be added to improve their survival. Therefore, identifying new biomarkers for chemotherapy and constructing a prognostic model can help to make an accurate treatment decision for SCLC patients. Discovering new diagnostic and prognostic biomarkers can be useful to improve risk prediction and therapeutic strategies for cancer patients. The recent study by Giannos et al. identified ten prognostic biomarkers by analyzing TGF-β induced EMT-related genes in NSCLC. Most of these biomarkers were involved in protein ubiquitination, indicating that deregulation of ubiquitination may contribute to the EMT-associated NSCLC progression [8]. In a bioinformatics study of cervical cancer, PCNA gene has been identified as a potential biomarker of cervical intraepithelial neoplasia progression [9]. In esophageal cancer, Feng et al. identified 152 differentially expressed genes between tumors and normal tissues, and construct a 7-gene prognosis model [10]. Long non-coding RNAs (lncRNAs), the non-coding RNAs with a length of more than 200 bp and without protein-coding potential, are tightly linked to regulating cisplatin resistance in various cancers [11]. Many studies have shown that lncRNAs regulate chemo-resistance and cancer progression through sponging miRNAs, which relieves the inhibitory effect of miRNAs on mRNA targets [12]. In SCLC, Sun et al. have reported that lncRNA HOTTIP sponged miR-216a that targeted BCL-2 to increase the expression of BCL-2 and chemoresistance Page 2 of 12 [13]. Zeng et al. have indicated that Linc00173 upregulated Etk by sponging miRNA-218 and caused chemoresistance and poor survival in SCLC [14]. Sun et al. have demonstrated that lncRNA MEG3 sponged miR-15a-5p to mediate CCNE1 expression in cancer-associated fibroblast derived exosomes to promote chemoresistance and cancer progression in SCLC [15]. Therefore, lncRNAs have been demonstrated to play a key role in SCLC chemoresistance by functioning as a competing endogenous RNA (ceRNA). In the study, through developing a cisplatin resistancerelated ceRNA network, we constructed a SCLC prognostic model based on the expression of two mRNAs, LIM domain kinase 2 (LIMK2) and Phosphatidylinositol 4-kinase IIβ (PI4K2B). This model and the two genes (LIMK2 and PI4K2B) have the potential to predict patients’ survival and chemo-resistance in SCLC. Methods Data acquisition IC50 values and RNAs expression data of SCLC cell lines were collected from genomics of drug sensitivity in cancer (GDSC, version 8.3, updated in June 2020). The gene expression data based on RNA-seq and clinical information from 77 SCLC patients was obtained from George et al. [16]. The gene expression data and clinical information of 48 SCLC patients from GSE60052 [17] were downloaded from the Gene Expression Omnibus database (GEO). The gene expression data of 43 normal tissues and 15 SCLC tumors from GSE40275 [18] was obtained from GEO. The building of cisplatin resistance‑related ceRNA network To build the cisplatin resistance-related ceRNA network, we first identified cisplatin resistance-related lncRNAs. In the GDSC database, with logFC > 0.1 and P < 0.05 thresholds, differential expressed lncRNAs were identified between cisplatin-sensitive and cisplatin-resistant group. Subsequently, the miRNAs targeted by lncRNAs were identified in the miRDB database with a target score greater than 80. The mRNAs targeted by miRNAs were identified in miRWalk and NPInterv4 databases, and then the mRNAs obtained by the two databases were intersected. Finally, the correlation of lncRNA-mRNA pairs were examined by Pearson correlation analysis with an absolute value of correlation coefficient > 0.4 and P < 0.05. Construction of the prognostic model Using 31 mRNAs identified by ceRNA network, univariate Cox regression analysis was performed to determine the correlation between mRNA expression level and OS in SCLC. The mRNAs with P < 0.05 were selected as prognostic genes. LASSO regression analysis was then Zhang et al. BMC Medical Genomics (2023) 16:103 used to further screen mRNAs and eliminate collinearity between mRNAs. Finally, multivariate Cox regression analysis was used to build a model to obtain the regression coefficient of each mRNA. The risk score was calculated according to the formula: n risk score = Coefi ∗ xi i=1 “i” is the number of genes, “Coef ” is the regression coefficient of this gene, and “x” is the expression value of this gene. The specific risk score = − 1.07LIMK2 + 0.42* PI4K2B. Based on the risk scores of patients, the “SurvMiner” package in R was used to calculate the optimal cut-off point and then patients were divided into high-risk and low-risk groups. Kaplan–Meier (KM) survival analysis was performed to assess differences in OS between highrisk and low-risk patients. The “survivalROC” package was used to build the receiver operating characteristic curve (ROC) and calculate the area under ROC curve (AUC). Risk assessment scatter plot showed survival status of patients in high-risk and low-risk groups. Functional enrichment analysis Functional enrichment analysis was performed using R packages including “clusterProfiler”, “org.hs.eg.db”, “GOplot”, “ReactomePA” and “enrichplot”. The George dataset [16] and GSE60052 dataset [17] were analyzed by Gene Set Enrichment Analysis (GSEA) to reveal the biological processes between the high-risk and low-risk group. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis [19] were performed on the differentially expressed genes between the high-risk and low-risk group. The pathways with P < 0.05 and false discovery rate (q value) < 0.05 were considered to be significant. Immune cells infiltration analysis We use the R package “CIBERSORT” to calculate the different expression levels of immune cells between the high-risk group and low-risk group, and analyze the correlation between the risk score and the level of immune infiltration. Immunohistochemical (IHC) staining The IHC staining was used to examine the expression levels of LIMK2 and PI4K2B in SCLC tumors and normal tissues. Paraffin-embedded sections were incubated in the oven for 3 h. Subsequently, the sections were dewaxed in xylene and ethanol (100%, 95%, 85% and 75%, each for 5 min), and placed in citric acid buffer for antigen repair. After blocked with peroxide blocking Page 3 of 12 solution for 30 min, slides were incubated overnight in a wet chamber at 4 °C with primary antibodies in a concentration of 1:100 (anti-LIMK2, 12350-1-AP, Proteintech, Wuhan, China), and 1:100 (anti-PI4K2B, 15074-1-AP, Proteintech, Wuhan, China). Then, the slides were incubated for 30 min using a secondary antibody (KIT-5020, MaxVision-HRP mouse/rabbit, Maxim, Fuzhou, China). DAB kit (ZLI-9018, Zhongshan Golden Bridge, Beijing, China) was used to stain the sections, and then the slides were counterstained with hematoxylin, dehydrated, and mounted. Statistic analysis All analyses were performed with R version 4.0.2 and corresponding packages. Wilcoxon test and t test were used to compare the difference. P < 0.05 was considered to be significant. Result The establishment of a prognostic model based on the cisplatin resistance‑related ceRNA network in SCLC In order to obtain the lncRNA signature related to cisplatin resistance in SCLC, we collected the expression data of lncRNAs in SCLC cell lines from the GDSC database. 53 SCLC cell lines were divided into two groups, 13 cisplatin-sensitive cell lines (cisplatin IC50 < 10) and 40 cisplatin-resistant cell lines (cisplatin IC50 > 10). By comparing the two groups at the thresholds of logFC > 0.1 and P < 0.05, 10 differential expressed lncRNAs were identified, including 3 down-regulated lncRNAs (GUSBP11, AC016747 and LA16c-358B7), and 7 up-regulated lncRNAs (RP4-593H12, DIRC1, LINC02872, LINC02875, LINC01098, CYP51A1-AS1 and LINC02853) in cisplatin-sensitive cell lines (Fig. 1A). Then, we identified 120 miRNAs targeted by the 10 lncRNAs using the miRDB database. Subsequently, mRNAs targeted by the 120 miRNAs were searched in miRWalk and NPInterv4 databases respectively, and 2637 common mRNAs were obtained in both databases (Fig. 1B and Additional file 1: Table S1). Furthermore, Pearson correlation analysis was performed to examine the correlation of lncRNAs and mRNAs expression. Finally, 31 pairs lncRNA-mRNAs were identified with the absolute value of correlation coefficient > 0.4 and P < 0.05 (Additional file 2: Table S2). To establish a prognostic risk model, 31 identified mRNAs were analyzed by univariate Cox regression, LASSO regression and multivariate Cox regression using the SCLC patients’ dataset by George et al. [16]. In univariate Cox regression analysis, five mRNAs (IRAK1, SCAF1, LIMK2, PI4K2B and HMGA2) were significantly associated with OS (Fig. 1C). LASSO regression analysis further screened out 4 mRNAs, including Zhang et al. BMC Medical Genomics (2023) 16:103 Page 4 of 12 Fig. 1 The establishment of a two-gene prognostic model based on cisplatin resistance-related ceRNA network in SCLC. A Volcanic map shows differential expression of lncRNAs in cisplatin-resistant and cisplatin-sensitive SCLC cell lines. The red dots represent significantly up-regulated lncRNAs in cisplatin-sensitive cell lines, the blue dots represent significantly down-regulated lncRNAs in cisplatin-sensitive cell lines, and the gray dots represent lncRNAs with no difference (logFC > 0.1 and P < 0.05). B The flowchart describes the construction of ceRNA network. 31 pairs of lncRNA-mRNA with significant correlation were identified in the network. C Univariate Cox regression analysis shows the 5 of 31 cisplatin resistance-related mRNAs associated with OS in George dataset. D LASSO regression analysis screened out 4 mRNAs (IRAK1, SCAF1, LIMK2 and PI4K2B). E Multivariate Cox regression analysis indentified 2 mRNAs (LIMK2 and PI4K2B) as independent prognostic factors in George dataset IRAK1, SCAF1, LIMK2 and PI4K2B (Fig. 1D). Then, we performed multivariate Cox regression analysis on the 4 mRNAs and identified the two mRNAs (LIMK2 and PI4K2B), which were independent prognostic factors in SCLC (Fig. 1E). Finally, we used the coefficients of multivariate Cox regression analysis to construct a SCLC prognostic model as the following formula: Risk Score = − 1.07Exp LIMK2 + 0.42Exp PI4K2B. The validation of the prognostic model in SCLC KM survival analysis and ROC curve analysis were used to evaluate the prognostic value of the model in SCLC. We calculated the risk score of each patient in the George dataset [16] and used the “survminer” package to determine the optimal cut-off score (− 2.36), which divided the patients into a high-risk group (n = 26) and a low-risk group (n = 51). The KM survival analysis showed that the OS in the high-risk group was significantly shorter than that in the low-risk group (Fig. 2A). We then evaluated the predictive power and accuracy of the prognostic risk model according to ROC curve analysis, and the predicted AUC was 0.853 (Fig. 2B). The risk score and survival status distribution of each patient were shown in Fig. 2C, indicating that the patients with higher risk scores were more likely to have a poorer prognosis compared to patients with the lower risk scores. The expression heatmap showed that LIMK2 expression was higher in the low-risk group, while PI4K2B expression was lower in the low-risk group (Fig. 2D). To further validate the prognostic value of the model in SCLC patients, we analyzed an independent dataset (GSE60052) with 48 SCLC patients. Similarly, the patients from the dataset were divided into the highrisk group (n = 7) and a low-risk group (n = 41) by the optimal cut-off of risk scores (− 4.23). The results of KM survival analysis showed that the OS of high-risk group was significantly shorter than that of the low-risk group (Fig. 2E). The predicted AUC value was 0.671 (Fig. 2F). The risk score and survival status distribution of each patient were shown in Fig. 2G. Similar to the George dataset [16], LIMK2 expression was higher and PI4K2B expression was lower in the low-risk group (Fig. 2H). Thus, these data indicate that the model based on the expressions of LIMK2 and PI4K2B performs a good prognostic value in SCLC. Zhang et al. BMC Medical Genomics (2023) 16:103 Page 5 of 12 Fig. 2 Performance evaluation of the two-gene prognostic model in SCLC. A KM survival curve analysis of SCLC patients in George dataset. SCLC patients were divided into low-risk (n = 51) and high-risk (n = 26) groups based on the optimal cut-off point. B ROC curve shows AUC in George dataset (n = 77). C In George dataset, the risk scores and survival status distribution of SCLC patients. D In George dataset, heat map shows the expression of LIMK2 and PI4K2B in SCLC. E KM survival curve analysis of SCLC patients in GSE60052 dataset. SCLC patients were divided into low-risk (n = 41) and high-risk (n = 7) groups based on the optimal cut-off point. F ROC curve shows AUC in GSE60052 dataset (n = 48). G In GSE60052 dataset, the risk scores and survival status distribution of SCLC patients. H In GSE60052 dataset, heat map shows the expression of LIMK2 and PI4K2B in SCLC Zhang et al. BMC Medical Genomics (2023) 16:103 The assessment of the prognostic value of the single LIMK2 or PI4K2B gene in SCLC Furthermore, we evaluated the prognostic value of the single LIMK2 or PI4K2B gene in SCLC. KM analysis showed that low expression of LIMK2 was significantly associated with poor survival in the both George [16] (Fig. 3A) and GSE60052 data sets (Fig. 3C). High expression of PI4K2B was significantly associated with poor survival in the both George [16] (Fig. 3B) and GSE60052 data sets (Fig. 3D). These results were consistent with the hazard ratio (HR) of Cox regression analysis on the two genes (LIMK2 and PI4K2B). Next, we evaluated the expression of LIMK2 and PI4K2B in SCLC tumor and normal tissues by using Page 6 of 12 GSE40275 and GSE60052 datasets. The data indicated that mRNA level of LIMK2 was significantly higher in normal tissues in both datasets (Fig. 3E, G). The mRNA level of PI4K2B was significantly higher in SCLC tumors in GSE40275 dataset (Fig. 3F). In GSE60052 dataset, mRNA level of PI4K2B was higher in SCLC tumors, but the difference did not reach statistic significance (Fig. 3H). Furthermore, the expression of LIMK2 and PI4K2B was verified in our collected SCLC specimens by IHC. The IHC data showed that the expression of LIMK2 was down-regulated in SCLC tumors, compared with normal tissues. LINK2 was primarily expressed in the nucleus of normal cells (Fig. 3I). However, the expression of PI4K2B was up-regulated in SCLC tumors, compared Fig. 3 Prognostic value of single LIMK2 or PI4K2B gene in SCLC. A KM survival analysis of LIMK2 in George dataset (n = 77). SCLC patients were divided into low-expression (n = 13) and high-expression (n = 64) groups based on the optimal cut-off point. B KM survival analysis of PI4K2B in George dataset (n = 77). SCLC patients were divided into low-expression (n = 25) and high-expression (n = 52) groups based on the optimal cut-off point. C KM survival analysis of LIMK2 in GSE60052 dataset (n = 48). SCLC patients were divided into low-expression (n = 9) and high-expression (n = 39) groups based on the optimal cut-off point. D KM survival analysis of PI4K2B in GSE60052 dataset (n = 48). SCLC patients were divided into low-expression (n = 12) and high-expression (n = 36) groups based on the optimal cut-off point. E In GSE40275 dataset, boxplot of LIMK2 mRNA expression in SCLC tumors and normal tissues. F In GSE40275 dataset, boxplot of PI4K2B mRNA expression in SCLC tumors and normal tissues. G In GSE60052 dataset, boxplot of LIMK2 mRNA expression in SCLC tumors and normal tissues. H In GSE60052 dataset, boxplot of PI4K2B mRNA expression in SCLC tumors and normal tissues. I IHC detection of LIMK2 expression in two SCLC clinical specimens (T: Tumor, N: Normal tissue). J IHC detection of PI4K2B expression in two SCLC clinical specimens (T: Tumor, N: Normal tissue) Zhang et al. BMC Medical Genomics (2023) 16:103 with normal tissues. PI4K2B was primarily expressed in the cytoplasm of SCLC cells (Fig. 3J). Therefore, the mRNA levels of LIMK2 and PI4K2B in SCLC tumors and normal tissues were consistent with the IHC data. Functional enrichment analysis of the model To elucidate the biological functions and potential pathways related to the risk model, we conducted GO and KEGG enrichment analyses based on differentially expressed genes between the high-risk and lowrisk group. In the George dataset [16], according to the threshold of logFC > 1 and P < 0.05, 2266 differentially expressed genes were identified between the high-risk and low-risk group (Fig. 4A). The GO term analysis of Page 7 of 12 these differentially expressed genes revealed the functional enrichment of mitochondrial and ribosome activities, including electron transfer activity, cytochrome C oxidase activity and translational initiation et al. (Fig. 4B). The KEGG analysis of these genes also enriched the terms of mitochondrial oxidative phosphorylation and ribosome terms (Fig. 4C). Furthermore, we employed GSEA to analyze the biological process and signaling pathway enriched in the high-risk and low-risk groups. All genes were sorted in ascending order according to the logFC between the two groups, and then they were subjected to GSEA enrichment analysis. In both George [16] and GSE60052 dataset, the apoptosis pathway was enriched Fig. 4 Functional enrichment analysis of the prognostic model. A Heat map of differentially expressed genes between low-risk group and high-risk group in George dataset. B Functional enrichment analysis of GO terms for the differentially expressed genes between low-risk group and high-risk group in George dataset. Yellow stars indicate the terms associated with ribosomes, and red stars indicate the terms associated with mitochondria. C Functional enrichment analysis of KEGG for the differentially expressed genes between low-risk group and high-risk group in George dataset. Yellow stars indicate the terms associated with ribosomes, and red stars indicate the terms associated with mitochondria. D GSEA enrichment analysis of both George dataset and GSE60052 dataset shows that the apoptotic pathway is enriched in the low risk group Zhang et al. BMC Medical Genomics (2023) 16:103 in the low-risk group (Fig. 4D). The mitochondrial is an important organelle to regulate apoptosis. Therefore, the results of functional enrichment analyses suggest that the prognostic model may be related to mitochondrial-regulated cellular apoptosis. Cathepsin D (CTSD) is an important apoptosis‑related gene enriched in the model In order to explore the key proteins of the apoptosis pathway enriched in the model, we analyzed the core genes in the apoptosis pathway between the highrisk and low-risk group. According to the threshold of LogFC > 0.1 and P < 0.05, 19 key apoptosis-related genes were identified in the George dataset [16], and 39 key apoptosis-related genes were identified in the GSE60052 dataset (Fig. 5A, B). Among these genes, CTSD was down-regulated in high-risk group in both datasets (Fig. 5C, D). Moreover, we evaluated the prognostic value of CTSD in SCLC. KM analysis indicated that the low expression of CTSD was associated with poor OS in both George [16] and GSE60052 datasets (Fig. 5E, F). Therefore, these results suggest that CTSD could be an important apoptotic modulator enriched in the model. Page 8 of 12 The immune infiltrating analysis of the risk groups in SCLC The infiltrating levels of 22 immune cells in the risk group of SCLC were explored using the CIBERSORT algorithm. We constructed the landscapes of different immune cell subtypes in each sample (Fig. 6A). The data demonstrated that the levels of macrophages M0 and T follicular helper cells were significantly higher in the low-risk group compared with the high-risk group (Fig. 6B–D). Moreover, the correlation analysis between the risk scores and 22 immune infiltrating cells demonstrated that the risk score was significantly related to the infiltration level of T follicular helper cells. With the risk score increased, the infiltrating level of T follicular helper cells decreased (Fig. 6E). The data suggest that the high risk group of the model is related to low immune cells infiltration. The ceRNA network of the SCLC prognostic model Taken together, these results identified cisplatin resistance-related ceRNA network (AC016747.3/hsa-miR195-3p/LIMK2 and LINC02875/hsa-miR-4266/PI4K2B) in SCLC. In the meanwhile, based on the ceRNA network, we established a two-gene prognostic model, which had a good ability to predict the survival of SCLC patients. Furthermore, the identified ceRNA network may affect cisplatin resistance and patients’ survival Fig. 5 CTSD is identified as an important apoptotic regulator in both George dataset and GSE60052 dataset. A In George dataset, heat map of 19 differential expressed core genes of apoptotic pathway between the low-risk and high-risk group. B In GSE60052 dataset, heat map of 39 differential expressed core genes of apoptotic pathway between the low-risk and high-risk group. C In George dataset, boxplot of CTSD expression in the low-risk and high-risk groups D In GSE60052 dataset, boxplot of CTSD expression in the low-risk and high-risk groups. E KM survival analysis of CTSD in George dataset. SCLC patients were divided into low-expression (n = 44) and high-expression (n = 33) groups based on the optimal cut-off point. F KM survival analysis of CTSD in GSE60052 dataset. SCLC patients were divided into low-expression (n = 23) and high-expression (n = 25) groups based on the optimal cut-off point Zhang et al. BMC Medical Genomics (2023) 16:103 Page 9 of 12 Fig. 6 The immune infiltrating analysis of the risk groups in SCLC. A In George dataset, landscape of different immune cell subtypes for each sample (n = 77). B In George dataset, the differences in the level of 22 immune cell infiltration between high-risk group (n = 26) and low-risk group (n = 51). C Macrophages M0 cell infiltration with significantly different levels between the high risk group and low risk group. D T follicular helper cells infiltration with significantly different levels between the high risk group and low risk group. E Pearson correlation analysis of T follicular helper cells infiltration and risk score of each SCLC sample through mitochondrial-regulated apoptosis and immune infiltration (Fig. 7). Discussion In this study, we first identified 10 cisplatin resistancerelated lncRNAs in SCLC cell lines. Through the ceRNA network and correlation analysis, we found 31 mRNAs correlated with the 10 lncRNAs. Then, using Cox regression analysis, we established a prognostic model based on the expression of LIMK2 and PI4K2B genes in SCLC. Furthermore, functional enrichment analysis found that the risk groups in the model could be related to mitochondrial-regulated cellular apoptosis pathway and T cells infiltration. In our model, the higher expression of LIMK2 gene correlated with better survival in SCLC. As previously reported, LIMK2 was a serine/threonine protein kinase that positively associated with OS in lung squamous cell carcinoma (LUSC), which was consistent with our findings. The previous study indicated that LIMK2 expression negatively correlated with immune checkpoints and immune cell infiltration in LUSC, while our study showed that the low risk group (high LIMK2 expression) exhibited high immune cell infiltration. The study Zhang et al. BMC Medical Genomics (2023) 16:103 Fig. 7 The cisplatin resistance-related ceRNA network and possible functional mechanism. The cisplatin resistance-related ceRNA network AC016747.3/hsa-miR-195-3p/LIMK2 and LINC02875/ hsa-miR-4266/PI4K2B could affect mitochondrial-regulated cellular apoptosis and T cell immune infiltration, thereby leading to cisplatin resistance and poor survival in SCLC also found a DHRS4-AS1/miR-423-5p/LIMK2 ceRNA axis in LUSC, while our study found an AC016747.3/ hsa-miR-195-3p/LIMK2 ceRNA axis in SCLC [20, 21]. Our model suggests that LIMK2 acts as a tumor suppressor gene. Previous studies have demonstrated that low expression of LIMK2 in colorectal cancer enhanced the accumulation of β-catenin in the nucleus to activate Wnt signaling pathway and promote tumor progression, while high expression of LIMK2 inhibited the proliferation and migration of tumor cells [22]. However, some studies also reported that LIMK2 acted as an oncogene. For instance, LIMK2 downregulated NKX3.1 to increase the oncogenicity of castration-resistant prostate cancer [23], and overexpressed LIMK2 exhibited as a facilitator of triple-negative breast cancer metastasis by regulating SRPK1 [24]. In our model, the lower expression of PI4K2B gene correlated with better survival in SCLC, which suggests that PI4K2B may function as an oncogene in SCLC. Mazzocca et al. have indicated that PI4K2B and CD81 could synergistically inhibit the migration of liver cancer cells, and low expression of PI4K2B increased the migration of liver cancer cells [25]. Page 10 of 12 Alli-Balogun et al. also demonstrated the effect of PI4K2B on the movement of Hela cells, and they found that depletion of PI4K2B induced the formation of invadopodia containing matrix metalloproteinase, to increase cellular invasion [26]. On the contrary, these previous studies have indicated that PI4K2B may function as a tumor suppressor to inhibit tumor invasion. In order to explore the mechanistic role of the model in SCLC, GO and KEGG enrichment analysis indicated that the differentially expressed genes between highrisk group and low-risk group were enriched in the mitochondrial function. GSEA analysis showed that the apoptotic pathway was enriched in the low-risk group. Cisplatin-based chemotherapy works by forming platinum–DNA adducts, which prevent rapidly dividing cells from duplicating their DNA, thereby leading to cellular apoptosis. The inhibition of apoptosis is an important mechanism for cancer cells to evade cisplatin toxicity and lead to drug resistance [27, 28]. The construction of the model is based on cisplatin resistance-related ceRNA network. Consistently, the functional mechanism of the prognostic model could be related to mitochondrial-regulated apoptotic pathway. In this study, we identified CTSD as a key regulator of apoptosis in SCLC. CTSD is an aspartic endopeptidase [29, 30]. Previous studies have shown that CTSD responds to apoptosis stimulation. CTSD is located upstream of cytochrome C release and caspase-3 activation in chemotherapy-induced apoptosis pathway [31–33]. Secomandi et al. have indicated that the overexpression of CTSD in neuroblastoma reduced the proliferation of tumor cells by down-regulating oncogenic MAPK signaling pathway, and the high expression of CTSD correlated with better prognosis [34]. In our study, the higher expression of CTSD was also associated with a better prognosis of SCLC. Therefore, our study suggests that CTSD plays a critical role in the response to the chemotherapy of SCLC. The study has also some limitations. Firstly, the prognostic model was built based on the bioinformatics analysis of publicly available datasets, and the results need to be further validated in prospective studies with a large sample size. Secondly, the study only focused on two genes (LIMK2 and PI4K2B) and their relationship with apoptosis pathway and immune infiltration. Other identified genes (such as IRAK1, SCAF1 and HMGA2) and pathways (such as protein translation in ribosome) may be also important in the chemo-resistance and progression of SCLC. Thirdly, the biological function and mechanism of the identified ceRNA network and biomarkers need to be validated using in vitro and in vivo experiments in the future. Zhang et al. BMC Medical Genomics (2023) 16:103 Conclusion Based on cisplatin resistance-related ceRNA network, our study established a two-gene (LIMK2 and PI4K2B) prognostic model in SCLC. The functional role of the model was related to mitochondrial-regulated apoptosis and immune cell infiltration. Furthermore, we identified an important apoptosis-related protein CTSD, whose expression positively associated with OS in SCLC. Therefore, the two-gene prognostic model and these new identified biomarkers (LIMK2, PI4K2B and CTSD) could provide an accurate treatment decision for SCLC patients. Abbreviations SCLC Small cell lung cancer NSCLC Non-small cell lung cancer OS Overall survival LncRNA Long non-coding RNA ceRNA Competing endogenous RNA LIMK2 LIM domain kinase 2 PI4K2B Phosphatidylinositol 4-kinase IIβ GDSC Genomics of drug sensitivity in cancer GEO Gene expression omnibus database KM Kaplan–Meier ROC Receiver operating characteristic AUC Areas under the ROC curves GSEA Gene set enrichment analysis GO Gene ontology KEGG Kyoto encyclopedia of genes and genomes HR Hazard ratio CTSD Cathepsin D Page 11 of 12 2022HSC-CIP015), and the Fundamental Research Funds for the Central Universities (Grant Number: WK9110000125). Availability of data and materials This study analyzed publicly available datasets. These data can be found here: https://www.cancerrxgene.org and https://www.ncbi.nlm.nih.gov/geo. Declarations Ethics approval and consent to participate The study was carried out in accordance with the guidelines of the Declarations of Helsinki. This study was approved by the Ethics Committee of Hefei Institutes of Physical Science (No. SWYX-Y-2021-12). SCLC paraffin-embedded sections were obtained with written informed consent of the patients. Consent for publication Not applicable. Competing interests The authors have no financial/commercial conflicts of interest regarding the study. Author details 1 Anhui Province Key Laboratory of Medical Physics and Technology, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, People’s Republic of China. 2 University of Science and Technology of China, Hefei, Anhui, People’s Republic of China. 3 Hefei Cancer Hospital, Chinese Academy of Sciences, Hefei, Anhui, People’s Republic of China. 4 Department of Pathology, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China. 5 Division of Life Sciences and Medicine, Intelligent Pathology Institute, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China. Received: 12 December 2022 Accepted: 9 May 2023 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12920-023-01536-5. Additional file 1. Table S1: 4465 lncRNAs-miRNA-mRNA axes in the cisplatin resistance-related ceRNA network. Additional file 2. Table S2: 31 pairs lncRNA-mRNAs. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant Number: 81872438), the Program of Research and Development of Key Common Technologies and Engineering of Major Scientific and Technological Achievements in Hefei (Grant Number: 2021YL007), the Collaborative Innovation Program of Hefei Science Center, CAS (Grant Number: 2022HSC-CIP015), and the Fundamental Research Funds for the Central Universities (Grant Number: WK9110000125). Author contributions BH and YZ designed the study. YZ, QZ and JQ performed the bioinformatics analysis and interpreted the results. MF, AX and WW collected SCLC clinical samples and performed the IHC analysis. YZ wrote the first draft of the manuscript. BH, HW and JN revised the manuscript. All authors read and approved the final manuscript. Funding This study was supported by the National Natural Science Foundation of China (Grant Number: 81872438), the Program of Research and Development of Key Common Technologies and Engineering of Major Scientific and Technological Achievements in Hefei (Grant Number: 2021YL007), the Collaborative Innovation Program of Hefei Science Center, CAS (Grant Number: References 1. Woodman C, Vundu G, George A, Wilson CM. Applications and strategies in nanodiagnosis and nanotherapy in lung cancer. Semin Cancer Biol. 2021;69:349–64. https://doi.org/10.1016/j.semcancer.2020.02.009. 2. Stratmann JA, Timalsina R, Atmaca A, Rosery V, Frost N, Alt J, et al. Clinical predictors of survival in patients with relapsed/refractory small-cell lung cancer treated with checkpoint inhibitors: a German multicentric real-world analysis. Ther Adv Med Oncol. 2022;14:17588359221097192. https://doi.org/10.1177/17588359221097191. 3. Chen Z, Fillmore CM, Hammerman PS, Kim CF, Wong KK. 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https://openalex.org/W4232656184	https://research.rug.nl/files/64849114/1_s2.0_S002210311630230X_main.pdf	English	null	When power analyses based on pilot data are biased: Inaccurate effect size estimators and follow-up bias	null	2,017	cc-by	11,282	When power analyses based on pilot data are biased Albers Casper; Lakens Daniël Published in: Journal of Experimental Social Psychology DOI: 10.1016/j.jesp.2017.09.004 DOI: 10.1016/j.jesp.2017.09.004 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Publication date: 2018 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Albers, C., & Lakens, D. (2018). When power analyses based on pilot data are biased: Inaccurate effect size estimators and follow-up bias. Journal of Experimental Social Psychology, 74, 187-195. https://doi.org/10.1016/j.jesp.2017.09.004 Citation for published version (APA): Albers, C., & Lakens, D. (2018). When power analyses based on pilot data are biased: Inaccurate effect size estimators and follow-up bias. Journal of Experimental Social Psychology, 74, 187-195. https://doi.org/10.1016/j.jesp.2017.09.004 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). University of Groningen University of Groningen Citation for published version (APA): Albers, C., & Lakens, D. (2018). When power analyses based on pilot data are biased: Inaccurate effect size estimators and follow-up bias. Journal of Experimental Social Psychology, 74, 187-195. https://doi.org/10.1016/j.jesp.2017.09.004 A R T I C L E I N F O Keywords: Eﬀect size Power analysis Follow-up bias Eta-squared Omega-squared Epsilon-squared When designing a study, the planned sample size is often based on power analyses. One way to choose an eﬀect size for power analyses is by relying on pilot data. A-priori power analyses are only accurate when the eﬀect size estimate is accurate. In this paper we highlight two sources of bias when performing a-priori power analyses for between-subject designs based on pilot data. First, we examine how the choice of the eﬀect size index (η2, ω2 and ε2) aﬀects the sample size and power of the main study. Based on our observations, we recommend against the use of η2 in a-priori power analyses. Second, we examine how the maximum sample size researchers are willing to collect in a main study (e.g. due to time or ﬁnancial constraints) leads to overestimated eﬀect size estimates in the studies that are performed. Determining the required sample size exclusively based on the eﬀect size esti- mates from pilot data, and following up on pilot studies only when the sample size estimate for the main study is considered feasible, creates what we term follow-up bias. We explain how follow-up bias leads to underpowered main studies. Our simulations show that designing main studies based on eﬀect sizes estimated from small pilot studies does not yield desired levels of power due to accuracy bias and follow-up bias, even when publication bias is not an issue. We urge researchers to consider alternative approaches to determining the sample size of their studies, and discuss several options. in reported eﬀect sizes is a challenge when performing a-priori power analyses based on published research. When power analyses based on pilot data are biased: Inaccurate eﬀect size estimators and follow-up bias☆ Casper Albersa,⁎,1,2, Daniël Lakensb,2, Casper Albersa,⁎,1,2, Daniël Lakensb,2 Casper Albersa,⁎,1,2, Daniël Lakensb,2 a University of Groningen, The Netherlands b Eindhoven University, The Netherlands a University of Groningen, The Netherlands b Eindhoven University, The Netherlands a University of Groningen, The Netherlands b Eindhoven University, The Netherlands http://dx.doi.org/10.1016/j.jesp.2017.09.004 Received 18 April 2016; Received in revised form 1 September 2017; Accepted 10 September 2017 ☆The Supplementary Material, including the full R code for the simulations and plots can be obtained from the Open Science Framework https://osf.io/zq9mg/. ⁎ Corresponding author: Grote Kruisstraat 2/1, 9712, TS, Groningen, The Netherlands. 1 Department of Psychology, University of Groningen, The Netherlands. 2 Both authors contributed equally to this manuscript. 3 School of Innovation Sciences, Eindhoven University of Technology, The Netherlands. E-mail address: c.j.albers@rug.nl (C. Albers). Available online 16 October 2017 0022-1031/ © 2017 Elsevier Inc. All rights reserved. ☆The Supplementary Material, including the full R code for the simulations and plots can be obtained from the Open Science Framework https://osf.io/zq9mg/. ⁎ Corresponding author: Grote Kruisstraat 2/1, 9712, TS, Groningen, The Netherlands. 1 Department of Psychology, University of Groningen, The Netherlands. 2 Both authors contributed equally to this manuscript. 3 School of Innovation Sciences, Eindhoven University of Technology, The Netherlands. E-mail address: c.j.albers@rug.nl (C. Albers). ☆The Supplementary Material, including the full R code for the simulations and plots can be obtained from the Open Science Framewor ⁎ Corresponding author: Grote Kruisstraat 2/1, 9712, TS, Groningen, The Netherlands. E-mail address: c.j.albers@rug.nl (C. Albers). http://dx.doi.org/10.1016/j.jesp.2017.09.004 Received 18 April 2016; Received in revised form 1 September 2017; Accepted 10 September 2017 Available online 16 October 2017 0022-1031/ © 2017 Elsevier Inc. All rights reserved. Copyright The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. down policy believe that this document breaches copyright please contact us providing details, and we will remove access to the wo vestigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Journal of Experim ental Social Psychology 74 (2018) 187–195 Contents lists available at ScienceDirect 3. Bias in power analyses The sampling distributions of η2, ω2 and ε2 are considerably skewed (shown in Fig. 1 for η2). Furthermore, the smaller the sample size, the more variable the eﬀect size estimate is. Statisticians have warned against using eﬀect size estimates from small samples in power analyses (Leon, Davis, & Kraemer, 2011). The two main reasons researchers should be careful when using eﬀect sizes from the published literature in power analyses is that eﬀect size estimates from small studies are inaccurate, and that publication bias inﬂates eﬀect sizes. At the same time, many applied statistics texts recommend using eﬀect sizes from related studies reported in the literature to perform a power analysis (e.g., Fritz, Morris, & Richler, 2012; Polit & Beck, 2004; Sawyer & Ball, 1981). In many cases, this is the only information researchers have about the possible size of the eﬀect they are interested in. For example, the Reproducibility Project (Open Science Collaboration, 2015) relied on the eﬀect sizes observed in the original studies to perform power analyses for replication studies. = + σ σ σ . T B W 2 2 2 The subscripts W, B, and T indicate ‘within’ samples, ‘between’ samples, and ‘total’. Equivalently, one can decompose the so-called sums of squares: = + SS SS SS . T B W One of the most common eﬀect size indices in one-way ANOVA is eta-squared (η2), which describes the proportion of variance that is explained by group membership. It dates back to {Citation}Pearson (1911), who introduced it in a regression context, and to Fisher (1928), who used it in the ANOVA context. In statistical packages such as SPSS, eta-squared is the default eﬀect size measure. Eta-squared is an up- wardly biased estimate of the true population eﬀect size, and two al- ternative eﬀect size indices have been suggested that are less biased, namely epsilon-squared (ε2, Kelley, 1935) and omega-squared (ω2, Hays, 1963). For background reading on these (and other) indices, we refer to Levine and Hullett (2002), Okada (2013) and McGrath and Meyer (2006), and the references therein. Eta-squared, epsilon-squared, and omega-squared are deﬁned as follows4: The statistical power of a test depends on the true eﬀect size, the sample size, and the alpha level that is used. 1. Introduction Lakens Journal of Experim ental Social Psychology 74 (2018) 187–195 Journal of Experim ental Social Psychology 74 (2018) 187–195 Journal of Experim ental Social Psychology 74 (2018) 187–195 C. Albers, D. Lakens C. Albers, D. Lakens C. Albers, D. Lakens = = η σ σ SS SS , B T B T 2 2 2 = − × ε SS df MS SS , B B W T 2 = − × + ω SS df MS SS MS , B B W T W 2 = − × + ω SS df MS SS MS , B B W T W 2 where, using standard ANOVA-notation, SS, MS and df denote the sum- of-squares, mean sum-of-squares, and degrees of freedom. From these eﬀect size estimates, the well-known Cohen's d and Cohen's f can be estimated (Cohen, 1988). For population eﬀect sizes Cohen (1988, p. 276) states that d = 2f with f2=η2/(1−η2). An unbiased estimate of Cohen's d is called Hedges' g (see Lakens, 2013), and recommendations in this article concerning the use of ω2 and ε2 instead of η2 extend to the use of Hedges' g instead of Cohen's d. Fig. 1. Distribution of η2 for a between-subjects t-test with a sample size (per group) of n = 50, and a medium true population eﬀect size (η2 = 0.0588). The lower x-axis in- dicates the n per group required to achieve .80 power based on the observed eﬀect size indicated by the upper x-axis. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare. 4877414, CC-BY4.0. Alternative formulas for these eﬀect sizes, where the computation is based only on the F-value and the degrees of freedom, are given in Appendix A. These indices are estimators of the unknown true population eﬀect size and, as such, contain possible bias and variability. It is well-known that η2 has more bias than the other two indices, but the other two indices have more variability (cf. Albers, 2015; Lakens, 2015; Okada, 2013). The amount of bias and variability of these indices depends on the size of the sample and the true population eﬀect size. When looking at performance measures that take both bias and variability into ac- count, such as the (root) mean squared error, none of the three indices is uniformly optimal and very little is known on in which situations one method outperforms another. 2. Eta-squared, Epsilon-squared, and Omega-squared In experimental psychology, it is extremely common to perform studies where participants are randomly assigned to diﬀerent condi- tions, and analyze the results using analysis of variance (ANOVA) or (unpaired) t-tests (where a t-test is mathematically identical to a one- way ANOVA with two groups). We will illustrate our main points using ANOVA and the related eﬀect sizes, but our conclusions generalize to other eﬀect sizes and statistical tests. In one-way ANOVA, all analyses are based on the following decomposition of the variance. The total variance of all measurements together, σT 2, is split into a part that can be attributed to group-membership (σB 2) and a part that can not (σW 2): 2 2 2 1. Introduction The ﬁrst goal of the current manuscript is to provide practical guidelines on how to deal with these diﬀerent eﬀect size estimates when used in a-priori power analysis based on the eﬀect size estimate in a previous study. yielded lower eﬀect size estimates (cf. Greenwald, 1975), simply be- cause these studies require less resources to observe a statistically sig- niﬁcant result in the expected direction. We examine how this under- standable behavior leads to an overestimation of the true eﬀect size, on average, when performing a-priori power analyses, and thus leads to follow-up studies that are underpowered. Based on these observations, we argue against recent recommendations (Sakaluk, 2016) to use small pilot studies to explore eﬀects. In the discussion, we oﬀer some general recommendations to design well-powered studies. 4 Note that although these three indices are estimators, we adopt the usual convention to denote them without a ‘hat’. 1. Introduction It is common practice in psychological and behavioral research to express the results of a quantitative study in at least two numbers: One expressing the probability or likelihood of data under speciﬁed statis- tical models, usually through a p-value or Bayes factor, and one ex- pressing the magnitude of the eﬀect, often through a (standardized) eﬀect size (ES). Reporting eﬀect size estimates serves various purposes, one of which is facilitating cumulative science by allowing other re- searchers to use the eﬀect size estimate in a-priori power analyses (Cohen, 1988). Power analyses can be used to design studies that have a desired probability of observing a statistically signiﬁcant eﬀect, as- suming there is a true eﬀect of a speciﬁed size. However, a-priori power analyses are only accurate when the eﬀect size estimate is accurate. It has been pointed out that eﬀect sizes reported in the literature are known to be inﬂated due to publication bias, and this widespread bias In this manuscript, we focus on two other sources of bias in power analyses that play an important role in power analysis even when publication bias and researchers' degrees of freedom do not inﬂuence eﬀect size estimates (e.g., when researchers perform their own pilot study). These sources of bias point out clear limitations of the common practice to use the eﬀect size from a pilot study to determine the sample size of a follow-up study through an a-priori power analysis. First, we will discuss the relatively straightforward matter of the impact of a biased eﬀect size estimator (η2), compared to less biased eﬀect size estimators (ε2 and ω2) on the sample size estimate in power analyses. Second, we examine a source of bias which we refer to as follow-up bias. Eﬀect size estimates vary around the true eﬀect size. Even without publication bias, researchers are more likely to follow-up on initial studies that yielded higher eﬀect size estimates than initial studies that Fig. 1. Distribution of η2 for a between-subjects t-test with a sample size (per group) of n = 50, and a medium true population eﬀect size (η2 = 0.0588). The lower x-axis in- dicates the n per group required to achieve .80 power based on the observed eﬀect size indicated by the upper x-axis. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare. 4877414, CC-BY4.0. C. Albers, D. 4. Follow-up bias It is not uncommon that researchers perform a pilot study and use the information from the pilot study to decide whether or not to carry out a large scale follow-up study. The eﬀect size in a pilot study is often used to determine what the sample size in a follow-up study should be based on an a-priori power analysis. This practice has been observed (and criticized) by statisticians (e.g., Kraemer, Mintz, Noda, Tinklenberg, & Yesavage, 2006), but as members of ethical review boards and local research funding committees, both authors often see that eﬀect sizes used in power analyses are exclusively based on pilot data. According to Wald (1945), this double sampling inspection pro- cedure dates back to Dodge and Romig (1929). In the ﬁrst simulation study, where we aim to quantify the bias in power analysis due to the choice of the eﬀect size index, we follow the procedure outlined in Appendix C. Power analysis for an ANOVA re- quires an iterative approach (outlined in Appendix B). In this paper, we apply this computational procedure as programmed in the R (R Core Team, 2017) package pwr (version 1.1–3, Champely, 2015) to examine how well the desired power is achieved as a function of the chosen eﬀect size index and the maximum sample size a researcher is willing to collect, denoted by n⁎. g g The likelihood that researchers will perform a follow-up study after a pilot study depends on how large the observed eﬀect is, either ex- pressed as a p-value, or a standardized eﬀect size (for any given sample size, the eﬀect size and the p-value for a statistical test are directly related). Researchers might decide to follow up on a study when the p- value is small (e.g., p < 0.15) or equivalently, when the eﬀect size is large enough (e.g., η2 > 0.05). When a pilot study yields a very small eﬀect size estimate (or a very large p-value), power analysis will suggest a sample size is needed that is so large that the study is unfeasible, given limited resources. It could even be, when calculating ω2 or ε2, that the estimated eﬀect size is negative, in which case a power analysis cannot be performed based on the observed eﬀect size. Although researchers might in principle be interested in the presence vs. 5. Simulation design For the simulation design, we varied the ﬁve parameters. First, in line with Okada (2013), the number of groups (K) in the ANOVA was 2, 3, or 4. This implies the simulations include the t-test and One-Way ANOVA with 3 and 4 groups. Second, the number of observations per group in the pilot study (npilot) was either 10, 25 or 50. A minimum of 50 participants in each condition for the pilot study has been re- commended (Harris, 2001; Simmons, Nelson, & Simonsohn, 2013), but smaller sample sizes are still common (Fraley & Vazire, 2014). Third, the true population eﬀect size (ESp) was small (.0099), medium (.0588), or large (.1379), in line with Cohen's (1988) rules of thumb and cor- responding to using d = .2, .5, or .8, respectively. These values are also consistent with Okada's (2013) simulation design. Fourth, the desired power in the follow-up study, (0.9 or 0.8 power, i.e. β = 0.1 or 0.2). Finally, for the pilot and follow-up studies we calculated three eﬀect size indices from the sample (ESs) used to estimate ESp, namely η2, ω2 or ε2. For any true eﬀect size the variation in the eﬀect size estimate from a pilot study can lead to follow-up bias: The tendency of researchers to not follow up on pilot studies where eﬀect size estimates are small (e.g., those estimates close to 0 in Fig. 1), whereas the researcher would have followed-up on the pilot study if the true eﬀect size had been known and accurately estimated. Suppose that a researcher has to decide be- tween studying the eﬀects of intervention A or B, but does not have the means to study both. Based on two pilot studies the researcher will conclude that the eﬀect of one intervention, e.g. A, is larger than in- tervention B, and follow-up on Intervention A. This approach has re- cently been advocated by Sakaluk (2016). However, in small pilot studies, diﬀerences between eﬀect sizes will themselves often not be statistically signiﬁcant, and eﬀect size estimates have high variability. For all simulations, the signiﬁcance level was set at α = 0.05. We decided not to vary this parameter of the design as people almost ex- clusively work with α = 0.05, and this would thus unnecessarily in- crease the complexity of the simulation design. 3. Bias in power analyses The goal of a power analysis is to control Type II error rates, or to limit the probability of observing a non-signiﬁcant result, assuming there is an eﬀect of a speciﬁc size. In the presence of bias, researchers might unknowingly increase the Type II error rate of their studies. Alternatives to a-priori power analysis exists, such as deciding upon a smallest eﬀect size of interest and using this to determine the required sample size in a power analysis (e.g. Lakens & Evers, 2014, Lang & Sesic, 2006, denoted the ‘minimal clinically important diﬀerence’ in medical research, Jäschke, Singer, & Guyatt, 1989). Other researchers have suggested to perform conservative power analyses (Perugini, Gallucci, & Costantini, 2014), or to model and correct for bias (Taylor & Muller, 1996). Nevertheless, researchers might believe that building on eﬀect size 188 Journal of Experim ental Social Psychology 74 (2018) 187–195 C. Albers, D. Lakens introduced through the judgement of others (other researchers, re- viewers, or editors). Researchers themselves are more likely to continue research lines where pilot studies estimate a larger compared to smaller eﬀect size estimate, because the a-priori power analysis will indicate less resources are required to examine the eﬀect with suﬃcient power. Therefore, whenever researchers choose to perform a study after per- forming a power analysis based on an observed eﬀect size, an implicit selection process has already taken place, in that the follow-up study would not have been performed, had the pilot study revealed a tiny or even negative eﬀect size estimate. By only performing follow-up studies when the observed eﬀect size estimate falls on the right side of the distribution in Fig. 1, the eﬀect size estimate that is used in power analyses is upwardly biased. Statistically, both publication bias and follow-up bias mean the power analysis is based on a truncated eﬀect size distribution (Taylor & Muller, 1996). estimates from their own pilot studies is a valid approach, given that these eﬀect size estimates are not inﬂuenced by publication bias. In this article, we show that even without problematic research practices such as publication bias or p-hacking, designing studies by relying on the eﬀect size from a pilot study when performing an a-priori power ana- lysis will, on average, lead to underpowered designs. 4. Follow-up bias absence of an eﬀect for purely theoretical reasons, in scientiﬁc practice they often have a maximum number of participants they are willing to collect for a study, either due to monetary or time constraints. Given any maximum sample size, there is a corresponding smallest eﬀect size of interest (SESOI) that can be investigated with a decent level of power. Whenever eﬀect size estimates in pilot studies are smaller than the SESOI, researchers might not to follow up on a line of research because they either suspect there is no true eﬀect, or because examining this eﬀect would require too much resources, if the eﬀect size estimate in the pilot study is accurate. If researchers only follow-up on studies with a high η2 estimate (e.g., η2 > 0.05, which requires 76 participants in each condition) the eﬀect sizes used in follow-up studies are on average upwardly biased, which leads to underpowered studies. In practical scenario's, it is impossible to distinguish the bias due to choice of eﬀect size index from the follow-up bias; only the combined bias will be observed. Therefore, we start with a somewhat unpractical scenario, where the follow-up bias is practically zero. Thus, all observed bias will be due to the choice of eﬀect size index. Once this ﬁrst si- mulation study provides insight in how this type of bias operates, we will consider realistic scenario's and study the added bias due to follow- up bias in the second simulation study. We ﬁrst report the results of the simulation when using n⁎ ≥100,000 as ‘unpractically large to follow up on’. This large value means the follow-up bias in the simulation will be minimal, and allows us to focus on the consequences of diﬀerent eﬀect size calculations η2, ω2 and ε2. Subsequently, we examine the consequences for follow-up bias given that most researchers have a maximum sample size per condition they are willing to collect that is substantially lower than 100,000. There, we employ values for n⁎ that are more in line with realistic situations in psychology. 3. Bias in power analyses As our simulations reveal, the deviation from the desired Type II error rate can be quite substantial, showing that the use of eﬀect sizes from pilot studies will generally not be a good approach to designing future studies. 6. Results Consider a study designed to test a diﬀerence between two groups (so, K = 2, a t-test). We will ﬁrst focus on a true population eﬀect size that is large (ESp = .1379), before investigating small and medium ef- fects. A pilot study, consisting of npilot measurements for each group, is performed and the data from this sample is used to estimate the po- pulation eﬀect size (as long as the eﬀect size estimate in the pilot study is larger than 0 for ω2 and ε2). Power analyses are performed to com- pute the sample size of the main study to reach a power of 0.8, as- suming the eﬀect size estimate from the pilot study is the true popu- lation eﬀect size. Subsequently, a data set of this size suggested by the power analysis is generated, and the true power of the main study is calculated. Note that in practice, the true population eﬀect size and the true power of studies is unknown, and these values are only known in simulation studies. sizes estimates from the samples will vary around the true eﬀect size. A higher estimate for the eﬀect size will yield a lower value for nmain in a power analysis, and a lower estimate for the eﬀect size will yield a higher value for nmain. The median of the eﬀect size distribution lies roughly at η2 = 0.05, which corresponds to nmain = 76 measurements per group in the main study. An estimated eﬀect size 0.025 above this median, yields nmain = 50, whereas an estimated eﬀect size of 0.025 below this median yields nmain = 155 Clearly, 50 is much closer to the accurate sample size required to achieve 0.8 power of 76 observations per group than 155 is. Due to the non-linear relation between sample size and power, the probability that nmain is severely overestimated is much larger than the probability that nmain is severely underestimated. In mathematical statistics, this phenom- enon is known as Jensen's inequality (Jensen, 1906). Fig. 2 shows box plots and violin plots for eﬀect sizes calculated from the results of the main study, for all three eﬀect size indices, based on pilot studies with npilot 10, 25, or 50 participants (left facet, middle facet, and right facet) in each condition. The simulation conﬁrms that the mean estimates (indicated by the black dots in Fig. 6. Results 2) are very close to the true population eﬀect size of .1379 (indicated by the dashed horizontal lines), with η2 having a (relatively small) positive bias. In line with previous work (Okada, 2013), ω2 and ε2 give mean eﬀect size estimates closer to the true eﬀect size. The average bias (or deviation from the true eﬀect size) is provided in detail in Table 1. Furthermore, it can be seen in Fig. 2 that that when npilot increases (e.g., right facet), the variation in the estimates decreases. This is not surprising: With a larger sample, estimates will be, on average, more accurate. Especially with small pilot samples, it is likely that the eﬀect size is either severely under- or overestimated. This, in turn, leads to sample sizes for the main study that are either too low or too high. Thus, small pilot studies lead to large variation in eﬀect size estimates both in the pilot study itself, as in the follow-up study when the sample size is based on the eﬀect size observed in the pilot study. This is demonstrated in Fig. 3. The distribution of nmain based on an a- priori power analysis (assuming the eﬀect size in the pilot study is the true eﬀect size) is very skewed, showing that, on average, the sample size for the main study is considerably overestimated. Especially with very small sample sizes, this leads to follow-up studies where the median sample size falls below the required sample size to reach a power of 0.8. As a ﬁnal and most important step, we can see the consequences of the too small sample sizes in the main study when we analyze the power of the main study. Fig. 4 shows that with only npilot = 10 measurements per There is another complication in determining the sample size of the main study, nmain. As Fig. 1 shows, the relation between estimated eﬀect size in the pilot and nmain following a power analysis is clearly non-linear. Be- cause of the uncertainty in estimating the eﬀect sizes in small samples, eﬀect Fig. 3. Box plots and violin plots for the estimated sample size per condition required to reach 0.8 power based on the eﬀect size estimate from a pilot study with npilot (left: 10, middle: 25, right: 50) participants per condition for the t-test with a large population eﬀect size (0.1379). Table 1 Population eﬀect size npilot Small Medium Large Average η2 10 +0.0153 +0.0232 +0.0349 +0.0245 25 +0.0068 +0.0150 +0.0270 +0.0163 50 +0.0042 +0.0124 +0.0245 +0.0137 Average +0.0088 +0.0169 +0.0288 +0.0182 ε2 10 −0.0001 −0.0005 −0.0016 −0.0007 25 −0.0000 −0.0004 −0.0013 −0.0006 50 −0.0000 −0.0003 −0.0012 −0.0005 Average −0.0000 −0.0004 −0.0014 −0.0006 ω2 10 −0.0001 −0.0012 −0.0034 −0.0016 25 −0.0001 −0.0007 −0.0029 −0.0012 50 −0.0000 −0.0006 −0.0028 −0.0012 Average −0.0001 −0.0008 −0.0030 −0.0013 5. Simulation design All ﬁve parameters were fully crossed, yielding 3 × 3 × 3 × 3 × 2 = 162 combinations. For each combination, R = 1,000,000 replications were drawn (the Both follow-up bias and publication bias lead to a preference for inﬂated eﬀect sizes, but are not the same: Whereas follow-up bias is created by the boundaries a researcher sets for her/himself – most notably the maximum sample size they can collect – publication bias is 189 Journal of Experim ental Social Psychology 74 (2018) 187–195 C. Albers, D. Lakens Table 1 Mean bias in eﬀect size estimate for the 3 × 3 combinations of population eﬀect size and pilot sample size, for the three eﬀect size measures. supplementary material shows this number to be suﬃcient); yielding a total of 162 million simulated samples. A detailed breakdown of the steps in the simulation design is provided in Appendix C and the cor- responding R script is available at https://osf.io/zq9mg/. Table 1 Mean bias in eﬀect size estimate for the 3 × 3 combinations of population eﬀect size and pilot sample size, for the three eﬀect size measures. 6. Results The dashed horizontal line indicates the required sample size (n = 26) to achieve 0.8 power for the true eﬀect size. The vertical axis is capped at n = 250. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198053, CC-BY4.0. Fig. 2. Box plots (marking the median and the 1st and 3rd quartiles of the distribution) and violin plots for eﬀect size estimates for t-tests with a large true population eﬀect size. The dashed line marks the true eﬀect size, and the dots mark the mean eﬀect size estimate in the simulation. The panels denote, from left to right, npilot values of 10, 25, and 50. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198050, CC-BY4.0. Fig. 3. Box plots and violin plots for the estimated sample size per condition required to reach 0.8 power based on the eﬀect size estimate from a pilot study with npilot (left: 10, middle: 25, right: 50) participants per condition for the t-test with a large population eﬀect size (0.1379). The dashed horizontal line indicates the required sample size (n = 26) to achieve 0.8 power for the true eﬀect size. The vertical axis is capped at n = 250. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198053, CC-BY4.0. Fig. 2. Box plots (marking the median and the 1st and 3rd quartiles of the distribution) and violin plots for eﬀect size estimates for t-tests with a large true population eﬀect size. The dashed line marks the true eﬀect size, and the dots mark the mean eﬀect size estimate in the simulation. The panels denote, from left to right, npilot values of 10, 25, and 50. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198050, CC-BY4.0. 190 Journal of Experim ental Social Psychology 74 (2018) 187–195 C. Albers, D. Lakens C. Albers, D. Lakens Fig. 4. Mean power for a t-test, when the sample size for the main study is based on an a- priori power analysis to achieve a power of 0.8 (dotted line) based on the eﬀect size estimate observed in a pilot study with npilot (left: 10, middle: 25, right: 50), when the true eﬀect size is large (.1379). Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare. 5198056, CC-BY4.0. sizes are small, using eﬀect sizes estimates from small pilot studies to perform power analyses for a main study is not a useful approach to design well-powered main studies. Main studies remain underpowered, but to a lesser extent, when npilot is set to 25, or with npilot = 50. The observed power is closer to the true power in these studies. 6.1. Results for lower population eﬀect sizes So far, we have looked at simulations where the population eﬀect size is large. Power analyses based on relatively small pilot studies become more inaccurate when the eﬀect size is medium or small. Fig. 5a displays the true power for follow-up studies when examining small eﬀect sizes, and Fig. 5b displays the true power for medium eﬀect sizes, complementing Fig. 4. The smaller the sample size of the pilot study, the larger the var- iance of the eﬀect size estimate, and thus the wider its distribution. For ε2 and ω2, this means that a relatively larger percentage of eﬀect size estimates falls below 0, and cannot be used for a power analysis. When power analyses are performed using ε2 and ω2, the eﬀect sizes calcu- lated from the pilot studies overestimate the true eﬀect size as ex- plained above, leading to relatively underpowered studies. Fig. 5a shows that for small eﬀect sizes and npilot = 10, the main study is ex- tremely underpowered with power less than half of what it should be. Perhaps situations like these won't occur often in practice: even with an alpha-level of 0.10 or 0.15, pilot studies are likely to be non-signiﬁcant and interest in the line of research would diminish. When true eﬀect The power when follow-up studies have a maximum sample size of 100,000 is 0.58, 0.72, and 0.76 (the middle bar in Figs. 4 and 5a,b, and the dashed lines in Fig. 6). When the maximum sample size a researcher is willing to collect lies below 250 participants per group, the true power in follow-up studies is even lower. For example, when examining a medium true eﬀect size (the middle red line in Fig. 6), and relying on a pilot study with 25 participants in each sample, researchers who are willing to collect a maximum of 100 participants in each of two groups (so 200 in total) will achieve at most a power of 0.6, in the long run. For Fig. 5. a (left) and b (right). Mean power for a t-test, when the sample size for the main study is based on an a-priori power analysis to achieve a power of 0.8 (dotted line) based on the eﬀect size estimate observed in a pilot study npilot (left: 10, middle: 25, right: 50), when the true eﬀect size is small (.0099; panel a) and medium (0.0588; panel b). 7. Follow-up bias In the simulations reported above, we have set the maximum sample size for a follow-up study to 100,000 participants to be able to illustrate the eﬀects of accuracy bias. With extremely rare exceptions, collecting 100,000 participants will not be feasible in practice. Fraley and Vazire (2014) examined the total sample sizes in six psychology journals between 2006 and 2010, and found that median total sample sizes range from 211 to as low as 51.5. It is therefore important to examine the consequences of follow-up bias, or the tendency to only perform follow-up studies when sample sizes in pilot studies are suﬃ- ciently large. Fig. 4. Mean power for a t-test, when the sample size for the main study is based on an a- priori power analysis to achieve a power of 0.8 (dotted line) based on the eﬀect size estimate observed in a pilot study with npilot (left: 10, middle: 25, right: 50), when the true eﬀect size is large (.1379). Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare. 5198056, CC-BY4.0. group, the main study is seriously underpowered, on average. The reason for this is mainly the accuracy bias due to the skewness introduced by Jensen's inequality. We can simulate the consequences of follow-up bias as a function of the maximum sample size a researcher is willing, or has the resources, to collect. Remember that for a given study, the choice to perform a follow-up study based on a maximum sample size is directly related to the smallest eﬀect size or a largest p-value a researcher will decide to follow up on (see Fig. 1). By re-analyzing our simulation results, we can examine what happens when the maximum sample size in a follow-up study is lowered from 100,000 to more realistic values. In Fig. 6 we present these re-analyses for small, medium, and large true eﬀect sizes. We only look at η2 for sake of simplicity (the eﬀects of follow up bias on ω2 and ε2 being similar to η2), and examine follow-up studies designed to have a power of 0.8 and a pilot study with npilot = 25. 6. Results Further- more, it can be seen that the diﬀerences between the three eﬀect sizes, η2, ω2 and ε2, are substantial. Even though η2 is positively biased, the fact that it can't be negative yields follow-up studies with better power (because more power analyses yield sample size estimates below 100,000), but only when eﬀect sizes are small, and/or when eﬀect sizes are medium, and pilot studies are small (situations that might not often occur in practice). 6.1. Results for lower population eﬀect sizes Reprinted from http:// dx.doi.org/10.6084/m9.ﬁgshare.5198059 and http://dx.doi.org/10.6084/m9.ﬁgshare.5198062, CC-BY4.0. Fig. 5. a (left) and b (right). Mean power for a t-test, when the sample size for the main study is based on an a-priori power analysis to achieve a power of 0.8 (dotted line) based on the eﬀect size estimate observed in a pilot study npilot (left: 10, middle: 25, right: 50), when the true eﬀect size is small (.0099; panel a) and medium (0.0588; panel b). Reprinted from http:// dx.doi.org/10.6084/m9.ﬁgshare.5198059 and http://dx.doi.org/10.6084/m9.ﬁgshare.5198062, CC-BY4.0. 191 Fig. 6. Power for main studies (where the sample size is based on a power analysis with an eﬀect size estimated from a pilot study with npilot = 25), as a function of the true eﬀect size and the maximum sample size per group a researcher is willing to collect in the main study. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198065, CC-BY4.0. C. Albers, D. Lakens Journal of Experim ental Social Psychology 74 (2018) 187–195 C. Albers, D. Lakens in the simulation, with respect to K, npilot, ESs and ESp. Overall, η2 yields considerably less powerful main studies than ω2 and ε2. Furthermore, in all situations, the average power lies well below the desired level. On average, the achieved power is about 80% of the desired power. Out of all 162 simulated conditions, only nine conditions have an average power level that reaches (or exceeds by 1%) the desired level of power. These conditions aimed for a desired power of 0.8, examined a large true eﬀect, with two conditions having npilot = 25, and the other six have npilot = 50. None of the seven conditions relied on η2. Ironically, these are exactly the situations least representative of current practices in psychology, where eﬀect sizes are often not large (Richard, Bond, & Stokes-Zoota, 2003), studies have low sample sizes (Fraley & Vazire, 2014), and η2 is used more often than ε2 or ω2 (Open Science Collaboration, 2015). Fig. 6. Power for main studies (where the sample size is based on a power analysis with an eﬀect size estimated from a pilot study with npilot = 25), as a function of the true eﬀect size and the maximum sample size per group a researcher is willing to collect in the main study. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198065, CC-BY4.0. 7.1. More than two groups First, one can determine the smallest eﬀect size of interest (SESOI), based on either utility or theoretical ar- guments, and use the SESOI in an a-priori power analysis. This leads to main studies that have a pre-determined statistical power to detect or reject the smallest eﬀect size that is deemed worthwhile to study. For example, if researchers decide their SESOI is a medium eﬀect size of η2 = .0588 a study with 87 participants in each of two groups will in the long run have a power of 0.9 to detect the SESOI, or reject it in an equivalence test (Lakens, 2017). Choosing a SESOI allows researchers to control their Type II error rate exactly for eﬀect sizes they care about. Alternatively, researchers might simply decide upon the maximum sample size they are willing to collect. Such a maximum number can and should be motivated, e.g. based on theoretical constraints or the amount of means available. Fig. 7 shows the statistical power as a function of the sample size for a two-group ANOVA for small, medium, and large eﬀects. Simply collecting a maximum number of observations will lead to considerably higher power than aiming for a power of 0.8, given a maximum sample size you are willing to collect (see Fig. 6), but Alternative approaches exist. First, one can determine the smallest eﬀect size of interest (SESOI), based on either utility or theoretical ar- guments, and use the SESOI in an a-priori power analysis. This leads to main studies that have a pre-determined statistical power to detect or reject the smallest eﬀect size that is deemed worthwhile to study. For example, if researchers decide their SESOI is a medium eﬀect size of η2 = .0588 a study with 87 participants in each of two groups will in the long run have a power of 0.9 to detect the SESOI, or reject it in an equivalence test (Lakens, 2017). Choosing a SESOI allows researchers to control their Type II error rate exactly for eﬀect sizes they care about. 8. Discussion We have shown that the practice of conducting a pilot study, esti- mating the eﬀect size from the data, and using the eﬀect size estimate in an a-priori power analysis to decide upon the sample size of the follow- up study leads to substantially underpowered main studies in most realistic situations. Although researchers are often reminded that eﬀect size estimates from small studies can be unreliable (e.g., Lakens & Evers, 2014), researchers are rarely informed about the consequences of using biased eﬀect size estimates in power analyses. Researchers who design studies based on eﬀect size estimates observed in pilot studies will unknowingly design on average underpowered studies, as long as they don't take bias in the estimated eﬀect sizes and follow-up bias into account. The diﬀerence between the desired and achieved power can be especially worrying when the sample size of the pilot study and/or the population eﬀect size is small, or when researchers are not willing to collect large sample sizes in the main study. It is important that re- searchers are aware of these pitfalls when designing studies. large true eﬀects, the additional bias in the true power due to follow-up bias is only pronounced when the maximum sample size a researcher is willing to collect is small (see the upper curve in Fig. 6). For small true eﬀects, follow-up bias leads to main studies that are not even close the desired 0.8 power. Fig. 6 clearly shows that designing main studies based on eﬀect sizes estimated from small pilot studies does not yield desired levels of power due to accuracy bias and follow-up bias. Tables S4 to S6 in the Supplementary Material provide the observed power for each of the 162 conditions for various levels of n⁎. 7.2. Change in power When the power is set to 0.9 rather than 0.8, the impact of negative or small eﬀect size estimates is roughly similar. Averaged over all conditions with 0.8 desired power, the achieved power is 0.647, which is 80.9% of 0.8 power. Averaged over all conditions with 0.9 desired power, the achieved power is 0.717, which is 79.7% of 0.9 power. When the true population eﬀect is small, and/or the pilot study is small, the observed power is slightly better when studies were designed to have 0.9, compared to 0.8 power. In the other conditions, the opposite holds. (see Tables S1, S2, and S3). Alternatively, researchers might simply decide upon the maximum sample size they are willing to collect. Such a maximum number can and should be motivated, e.g. based on theoretical constraints or the amount of means available. Fig. 7 shows the statistical power as a function of the sample size for a two-group ANOVA for small, medium, and large eﬀects. Simply collecting a maximum number of observations will lead to considerably higher power than aiming for a power of 0.8, given a maximum sample size you are willing to collect (see Fig. 6), but 7.1. More than two groups When the number of groups increases, the diﬀerences in power and the proposed sample size for the main study due to diﬀerences in npilot become somewhat smaller: The biases we discuss in this paper are more severe for the t-test than for a four group ANOVA. This, however, is largely due to the set-up of the simulation study, where an increase in the number of groups also mean an increase in the total sample size. For example, npilot = 25 means that the total sample size equals K × 25. For a t-test this is 2 × 25 = 50, whereas for a one-way ANOVA with four groups it is 4 × 25 = 100. For instance, with two groups and a large eﬀect size, a pilot study with a total sample size of 50 has a power of 0.79, but with three groups and a large eﬀect, the pilot study has a total sample size 75, and 0.87 power. The Supplementary Material provides similar information for each of the 1628 conditions. Tables S1 to S3 provide, for the three types of eﬀect size studied in this paper, the median nmain, the average and median bias, the root mean-squared error, the percentage of simulations where the power analysis yielded a sample size estimate above n⁎ and the average achieved power. Based on the results of the simulations in this manuscript, we oﬀer the following recommendations for researchers who want to decide upon a sample size when designing their study. First and foremost, we recommend against performing a pilot study to estimate an eﬀect size, and subsequently using this eﬀect size estimate in a power analysis to design a follow-up study. This can lead to seriously underpowered study designs, especially when the sample size of the pilot and/or the true eﬀect size is small to medium. Not only is this approach inaccurate, it is ineﬃcient. The data from the pilot study is either completely ignored, or at least not included in the main study. We don't see how this waste of pilot data can be justiﬁed, when the end result is a procedure that yields underpowered main studies. Pilot studies can be performed be- cause they have other uses, such as determining the feasibility of per- forming the designed study in practice, which is especially useful when trying out new methods or procedures (Leon et al., 2011). Alternative approaches exist. 7.3. Summary Tables 2 summarizes the ﬁndings of Tables S1, S2 and S3 for the diﬀerent values of one parameter, averaging across all other parameters 192 Journal of Experim ental Social Psychology 74 (2018) 187–195 C. Albers, D. Lakens Fig. 7. Power for a two-group ANOVA as a function of the true eﬀect size and the sample size per group. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198068, CC- BY4.0. C. Albers, D. Lakens Average estimates of the observed power in main studies for a desired power level of 0.8 and 0.9 for (i) number of groups, (ii) the size of pilot study, (iii) eﬀect size index, (iv), and true population eﬀect size. 0.8 0.9 K = 2 .659 .726 K = 3 .644 .715 K = 4 .638 .711 npilot = 10 .553 .617 npilot = 25 .665 .736 npilot = 50 .723 .799 ε2 .655 .724 η2 .626 .699 ω2 .660 .728 ESp small .472 .534 ESp medium .705 .777 ESp large .764 .840 Fig. 7. Power for a two-group ANOVA as a function of the true eﬀect size and the sample size per group. Reprinted from http://dx.doi.org/10.6084/m9.ﬁgshare.5198068, CC- BY4.0. Maxwell (2017). Researchers can choose the level of truncation (e.g., making the assumption that only studies with p < 0.05 appear in the literature), and perform a power analysis based on this truncated F- distribution. When sequential analyses are not possible, the use of safeguard power or truncated F-distributions are good approaches to compensate for the bias in traditional power analyses where the eﬀect size is derived from a pilot study or the published literature. Maxwell (2017). Researchers can choose the level of truncation (e.g., making the assumption that only studies with p < 0.05 appear in the literature), and perform a power analysis based on this truncated F- distribution. When sequential analyses are not possible, the use of safeguard power or truncated F-distributions are good approaches to compensate for the bias in traditional power analyses where the eﬀect size is derived from a pilot study or the published literature. it is less eﬃcient (you will end up collecting more participants than when you had performed an a-priori power analysis). A more eﬃcient alternative approach to designing studies is to use se- quential analyses, which allows researchers to analyze the data multiple times (e.g., after 50, 100, 150, and 200 participants have been collected) whilst controlling Type I error rates. 8.1. Suggestions for future research This manuscript focused on the use of eﬀect sizes to determine the sample size in follow-up studies using balanced one-way ANOVA's. For unbalanced designs (designs with substantially diﬀerent sample sizes per cell) the bias in power analysis might be diﬀerent (Kline, 2013). Furthermore, in other experimental designs, such as regressions, more- way factorial ANOVAs and within-subject designs, follow-up bias will also distort power analyses. The extent of the bias in these designs could be quantiﬁed in future simulation studies. Researchers interested in preventing bias in those situations are recommended to adapt our si- mulations to their situation of interest, or use sequential designs in- stead. Furthermore, because the strength of the bias in power analysis is most severe if the maximum sample size researchers are willing to collect is relatively small, and the SESOI is thus relatively large, it is interesting to empirically examine what the distribution of the SESOI and maximum sample size researchers are willing to collect is in dif- ferent research domains, and how this aﬀects follow-up bias. We believe recent recommendations such as “Explore small, conﬁrm big” (Sakaluk, 2016) are not useful. Small pilot studies only provide useful information to design follow-up studies when eﬀect sizes are large. When the true eﬀect size is large (η2 = 0.1379) even relatively small follow-up studies (e.g., n = 34 in each group for a t-test) have suﬃcient (i.e., 0.9) power. When the true eﬀect size is small or medium, small pilot studies will have low power and eﬀect size estimates have high variability, which make it diﬃcult to decide whether, or how, a follow-up study should be designed. Do not use extremely small sample sizes (e.g., npilot = 10) to estimate the true eﬀect size. These are too small to get even remotely accurate estimates for nmain in a power analysis. Researchers become increasingly aware of the importance of de- signing well-powered studies. Several journals now require authors to justify their sample sizes, and although power analyses are only one possible justiﬁcation, it seems likely power analyses will become more widely used. Power analysis can be one of the factors informing a study design, but it should not be mechanistically used to determine the sample size that will be collected. Open practices The study in this article earned the Open Materials badge for transparent practices. Materials for this study are available at https://osf.io/zq9mg/. 7.3. Summary For a frequentist introduction of se- quential analysis, see Lakens (2014), for a Bayesian introduction, see Schönbrodt, Wagenmakers, Zehetleitner, and Perugini (2017), for the mathematical background, see Wald (1945), and Siegmund (2013). It can be shown mathematically that sequential analyses are more eﬃcient than the double sampling scheme with a pilot study, and sequential analyses are especially appropriate whenever the true eﬀect size is relatively uncertain. However, sequential designs are not always feasible. For example, in a 5- year longitudinal study, one can only look at the data after ﬁve years, and each subsequent look at the data adds another 5 years to the research project. In such designs, power analyses will still be an important aspect of the study design. In such situations, and alternative approach is to perform a conditional power analysis, where an initial internal pilot study is collected, based on which a power analysis is performed (if the eﬀect is not yet sig- niﬁcant based on the available data), after which the remainder of the re- quired sample is collected, and all data is combined in the ﬁnal analysis. 8.1. Suggestions for future research We feel it is important that re- searchers realize possible sources of bias in the estimated sample sizes they need for a desired level of power, and are advised to attempt to correct for these biases when designing a study, or use other approaches to determine the sample size of a study. Do not use η2 in power analyses as this leads to the lowest power, on average (Table 2) – use ε2 or ω2 instead. Note that 56% of the studies in the Reproducibility Project used η2 or ηp 2 as the eﬀect size index for the a-priori power analysis. Power analyses based on pilot studies almost always yield estimates of the sample size of the main study nmain that are too low. Whenever power analyses are based on eﬀect size estimates from previous re- search (either pilot studies, or published studies) we recommend re- searchers take measures to compensate for this bias. A possible solution is to perform power analyses with ∼η 2 rather than η2, where ∼η 2 is the lower bound of a 80% conﬁdence interval for η2. This recommendation, known as safeguard power analysis, was proposed by Perugini et al. (2014). Alternatively, one can model the bias, and calculate nmain based on a truncated F-distribution. This approach, building on work by Taylor and Muller (1996), was recently recommended by Anderson and Appendix B. Computational power analysis Power analyses are computationally tricky and therefore usually performed using software as GPower (Faul, Erdfelder, Lang, & Buchner, 2007) or the pwr R package (Champely, 2015). For a One-Way ANOVA, it requires an iterative approach, which is as follows (cf. Champely, 2015; Cohen, 1988; Faul et al., 2007). First, the so-called non-centrality parameter λ is speciﬁed via denotes the required sample size per group and δ2 the eﬀect size in the population. Next, n is obtained by equ = ′ − − − − − F F α k n k β k n k λ , 1, ( 1) 1 , 1, ( 1), = ′ − − − − − F F α k n k β k n k λ , 1, ( 1) 1 , 1, ( 1), (1) Here, α denotes the level of signiﬁcance and 1 – β denotes the desired power (cf. Cohen, 1988). A solution for n is found through the bisection method: (i) Specify a lower bound n⁎ and upper bound n⁎ for n, + ∗ p y ⁎ p (ii) Compute Eq. (1) for ′ = + ∗ ∗ n n n 2 , (ii) Compute Eq. (1) for ′ = + ∗ ∗ n n n 2 , 2 (iii) When the left-hand-side of () exceeds the right-hand-side set n⁎ = n′, else set n⁎ = n’, Repeat steps (i)–(iii) until the absolute diﬀerence between the left-hand side and right-hand side of Eq. (1) is smaller than some pre-speciﬁed tolerance level. Acknowledgements We wish to thank the reviewers, Dr. Marco Perugini, Michèle Nuijten, MSc., and an anonymous reviewer, for their comments that helped to improve the manuscript considerably. 193 Journal of Experim ental Social Psychology 74 (2018) 187–195 C. Albers, D. Lakens Appendix C. Overview of the six steps in the simulation study For all performed power analyses, a study identical to the pilot study but with a sample size based on the power analysis was performed. For these studies, we calculated three eﬀect size estimates (η2, ε2, and ω2), performed the statistical test and stored the p-value and the sample size of the study. When the power analysis in step 5 yielded NA, all values for the eﬀect size, p-value, and sample size were set to NA as well. Appendix A. Computation of eﬀect sizes based on reported F-statistics The formulas on page 5 seem to suggest that full details of the ANOVA table are required to compute the eﬀect sizes. This is not the case: with some algebra (Carroll & Nordholm, 1975; Cohen, 1988) it can be shown that, in the between-subject designs studied in this paper, all eﬀect sizes can be computed on basis of reported F values and degrees of freedom only. (Note that for the t-test, F = t2 and dfB = 1). 2 2 2 2 2 2 In a one-way ANOVA η2 equals ηp 2 (and ω2 = ωp 2, and ε2 = εp 2). In more complex designs than One-Way ANOVA's partial eﬀect sizes are used in a-priori power analysis. Researchers often don't report ωp 2 or εp 2. Fortunately, for designs where all factors are manipulated (but not for studies with measured factors or ANCOVA's) these (partial) eﬀect size indices, as well as Cohen's f, can be calculated from the F-value and both degrees of freedom: = × × + η F df F df df , p B B W 2 = − + + ω F F 1 , p df df 2 1 W B = − + ε F F 1 , p df df 2 W B = × f F df df . B W A spreadsheet document to calculate ηp 2, ωp 2 and εp 2 from the F-value and degrees of freedom is available fr Appendix C. Overview of the six steps in the simulation study 1. Input: K, npilot, ESp, ESs, β, and α. 2. Compute f = √(ESp/1 – ESp) 2. Compute f = √(ESp/1 – ESp) p f ( p/ p) 3. Generate pilot data: npilot observations per group, from normal distributions with means: –f and +f (K = 2), −f, 0 and +f (K = 3), −f, −f, +f and +f (K = 4) and a standard deviation of 1. 4 Based on the pilot data estimate either 2 ω2 or 2 3. Generate pilot data: npilot observations per group, from normal distributions with means: –f and +f (K = 2), −f, 0 and +f (K = 3), −f, −f, +f and +f (K = 4) and a standard deviation of 1. 2 2 2 4. Based on the pilot data, estimate either η2, ω2 or ε2. 4. Based on the pilot data, estimate either η , ω or ε . 5. Perform an a-priori power analysis using the pwr package to determine the sample size per group of the main experiment. When this exceeds n⁎ = 100,000 (e.g. when ω2 = 0), the sample size is coded as ‘not available’. 4. Based on the pilot data, estimate either η , ω or ε . 5. Perform an a-priori power analysis using the pwr package to determine the sample size per group of the main experiment. When this exceeds n⁎ = 100,000 (e.g. when ω2 = 0), the sample size is coded as ‘not available’. η , lysis using the pwr package to determine the sample size per group of the main experiment. When this exceeds 0), the sample size is coded as ‘not available’. 5. 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https://openalex.org/W2176538015	https://ejournals.bc.edu/index.php/ihe/article/download/6891/6108	English	null	Widening Access and Raising Fees in Britain	International higher education	2,015	cc-by	2,555	Michael Shattock Michael Shattock Michael Shattock was registrar of Warwick University. He is currently visiting professor at the Center for Higher Education at the University of London Institute of Education. Address: Centre for Higher Education Studies, Institute of Education, University of London, Bedford Place, London U.K. E-mail: <shattock@he.u-net.com>. had come from state schools went on television to defend the college’s selection policy, and the vice-chancellor who had in the past been congratulated by the secretary of state for education, David Blunkett, for the university’s efforts to broaden its intake, accused Gordon Brown of setting back the university’s plans for widening access by reinforc- ing a stereotyped image it was trying to lose. The univer- sity went into a successful media overdrive to show that offers to candidates from state schools had increased from 48 percent to 53 percent over the past five years at the ex- pense of the independent schools, that it had recently com- pleted a major review of its admissions arrangements designed precisely to broaden the entry, and that it had more than 30 schemes already targeted on attracting can- didates from disadvantaged backgrounds. “Oxford is com- mitted,” said the vice-chancellor, “to recruiting the best students it can identify whatever their background” but he wanted Oxford to continue to “have a reputation for being fiercely meritocratic.” T T he internal contradictions of U.K. higher education policy have recently been paraded for all to see in two separate but connected events. The first, in May, was when the chancellor of the exchequer, Gordon Brown, an Edinburgh graduate, accused Oxford University of elitism in denying an undergraduate place to study medicine to a candidate from a state comprehensive school in the North East, an impoverished part of the country. The candidate concerned, who was excellently qualified, subsequently turned down offers of entrance to a number of other well- known universities, including Edinburgh, in favor of a place at Harvard. The accusation of elitism in admissions poli- cies was then leveled at a group of “top” universities by a succession of government ministers, including the prime minister, and the Parliamentary Select Committee launched an inquiry into the whole question of access to higher edu- cation. 14 14 INTERNATIONAL HIGHER EDUCATION INTERNATIONAL HIGHER EDUCATION old ones, establishing links with Western universities, in- viting lecturers from abroad, etc. These efforts face many objective and subjective obstacles and restrictions caused by rigid state educational laws. tion address specific goals—examples are “Textbooks for University Students,” “Foreign Languages,” and “Teach- ers.” Although there are no reliable data as yet concerning the effectiveness of such programs, their success already seems doubtful. Most respondents to a survey conducted for this report were unaware of the existence of any active national programs in these areas. This demonstrates that the programs were developed in the traditional “secluded” Soviet bureaucratic manner without the involvement of the academic community in either the development or imple- mentation. This, in turn, implies that the programs most likely will remain on paper only. To summarize, the goals of Belarusian authorities and their policies for higher education reform remain complex and con- tradictory. There are some signs that the authorities understand the need for reform in the context of the political, social, and economic changes in Belarus and in neighboring countries. The officially proclaimed goals of reform, however, have been strongly affected by the anti-Western stance of the current Belarusian au- thorities and have drifted away from those accepted soon after Belarus’s independence. The international dimension of higher education reform priorities has almost completely disappeared. The state is increasing its pressure on universities and exercises strict control over virtually all aspects of university policies and practices. In general, the situation is much more encouraging at the university level. Many deans, department chairs, and faculty members are reform-minded and hope to introduce curriculum changes for their faculties. This may be facili- tated by introducing new courses, updating the content of Widening Access and Raising Fees: Can These Policies Be Reconciled in the UK? Michael Shattock Michael Shattock On examination the case that provoked the accusation turned out to be a particularly bad example in that the col- lege concerned (selection is by colleges not by the univer- sity, at Oxford) had interviewed 23 candidates, all very well qualified, for five places, and the candidates admitted in- cluded two candidates from state schools and three who were from ethnic minorities. Students at the college who The internal contradictions of U.K. higher education policy have recently been paraded for all to see in two separate but connected events. The internal contradictions of U.K. higher education policy have recently been paraded for all to see in two separate but connected events. 15 Blunkett has called it an “access challenge to both govern- ment and universities.” On taking office, he introduced fees of £1,000 but means-tested them so that students from dis- advantaged backgrounds continued to enter free (the fees were abolished in Scotland following a revolt in the Scot- tish Parliament). At the same time, however, he removed student maintenance grants, and this represented a serious disincentive to mature students who, in general, are reluc- tant to utilize the student loan scheme, so that mature num- bers have fallen. In May he announced a £10m fund to pay £1,000 “opportunity bursaries” from 2001–2002 together with support for university summer schools where children still at school can experience university life. More support has just been announced in the government spending re- view. Universities are themselves beginning to create schol- arship schemes of one kind or another but it is increasingly evident that the real difficulty starts further back in per- suading children from disadvantaged backgrounds to stay on at school beyond age 16 and if they do obtain good A- level qualifications to enter higher education at a time when, according to Barclays Bank a typical student owed £5,286 on graduation, a figure that has risen by 17 percent in one year. The second event was the publication in July of a re- port by professor David Greenaway, an economist at Nottingham University, entitled Funding Universities to Meet National and International Challenges, which argued that charging students much higher fees was the only way that U.K. Michael Shattock universities could maintain their international stand- ing and fulfill the national role envisaged for them in the face of a 50 percent reduction in the unit of funding from the state over the past 20 years. The report offered several scenarios but sought to protect access by the introduction of an improved income-contingent loan scheme and by using some of the additional revenue raised from fees to pay bursaries to students from disadvantaged backgrounds. The report, commissioned by Russell Group of leading uni- versities, has attracted widespread publicity and some tan- gible support in the quality press. g pp q y p The difficulty in which the United Kingdom finds it- self is that the government believes that competition through market mechanisms will drive up standards, im- prove efficiency, and reduce costs—a policy it inherited from the Tories—but it also believes in social inclusion and a significant widening of access to higher education. How- ever, while only 7 percent of the school population is in private education, these pupils make up 20 percent of the numbers taking “A” levels and 30 percent of those achiev- ing the top grades in three subjects (the normal selection requirement of entry to Oxford, for example). In the sciences, independent schools are even more dominant— providing 42 percent of A-level candidates in physics, 45 percent in chemistry, and 47 percent in mathematics in the top grades. Research for the National Inquiry into Higher Education (the Dearing Committee) showed that, while 4 out of 5 18-year-olds from senior managerial and professional backgrounds entered higher education, no more that 1 in 10 did so from unskilled and partly skilled backgrounds. Dearing demonstrated that the breakthrough into mass higher education after 1988 did nothing to change the social mix of entering students. Naturally the “best” universities (that is, in the United Kingdom, those that are also the most research intensive) attract the best-qualified candidates and, since entry is highly competitive, they find that their entry is socially skewed. Research presented at the Royal Economic Society’s conference in July has also showed that social class has a significant impact on gradu- ate earnings: students from advantaged family backgrounds earn 3 percent more than the less advantaged, students who went to independent schools have a similar advantage after graduation compared to students from state schools. The Foreign Invasion of Israeli Higher Education they don’t mind the lack of intellectual dialogue that is sup- posed to characterize any meaningful education at this level. It is important to stress that more than responding to an existing demand, these institutions have themselves cre- ated a new demand. The issue of accountability has not received the attention it deserves. Roxana G. Reichman Roxana G. Reichman is assistant professor at Gordon Teachers College, Haifa, Israel. E-mail: <reichmanr@hotmail.com>. Roxana G. Reichman Roxana G. Reichman is assistant professor at Gordon Teachers College, Haifa, Israel. E-mail: <reichmanr@hotmail.com>. The Council of Higher Education (CHE) decided to open the gates of higher education to foreign institutions because of public pressure at the beginning of the 1990s. Since these institutions belong to the private sector, for which the financial bottom line is the most important cri- teria and where self-regulation can sometimes be almost nonexistent, the CHE realizes that developments in this arena might threaten the reputation of any degree and of any university. Five main guidelines have to be met by any foreign university in order to be recognized in Israel. These guidelines need to be analyzed in the light of the main goal, which is quality control and accountability. T T here is an entirely new trend in Israeli higher educa- tion—a new diversification in the nation’s system of postsecondary education. Currently, only 56 percent of the 199,000 Israeli students study at one of the country’s seven main universities—20 percent at one of the colleges (in- cluding teachers education colleges), 16 percent at the Open University, and 8 percent at one of the branches of foreign universities that have opened during the last decade (most of which are British or American). There are various ways of looking at this new dynamism in Israeli higher educa- tion. It is, of course, encouraging to see that the system has almost doubled in size within 15 years. That means greater access to postsecondary education, especially for those stu- dents who have historically been underprivileged. The bad news is that some of the branch campuses of foreign aca- demic institutions offer quick degrees, with no attention whatsoever to academic standards, no basic facilities such as libraries, computers, etc., and a teaching staff whose qualifications are sometimes questionable. Other branches make a significant effort to meet standards while at the same time answering the needs of the population they serve. Michael Shattock There was also an “earning premium” from graduating from a top university—that is, a university that did well in the league tables. The difficulty in which the United King- dom finds itself is that the government believes that competition through mar- ket mechanisms will drive up standards, improve efficiency, and reduce costs—a policy it inherited from the Tories It is against this background that the debate about the need to generate more university funding from stu- dent fees is taking place. In addition to the general dis- tress in universities about the decline in state support for higher education and the resulting relative fall by 30 percent in salary levels, there is deep concern, first, that the fee income stream that the government introduced post-Dearing was being matched by a further reduction in state support rather than being the top-up intended by Dearing; and, second, that the funding system has, in the words of the rector of Imperial College, “echoes of the command economy of the Eastern bloc” with gov- ernment controlling the number of students as well as all the funding mechanisms. The suggestion that uni- versities might break out of this stranglehold by charg- ing fees at serious levels is therefore doubly attractive, particularly in a university that is highly attractive to students. But there are counterarguments. Greenaway sets out the evidence on the rate of return for graduate qualifications and sees this as justifying high fees that g Although recent research has updated the story, the general position about the impact of social class on the en- try to universities has been known for many years. David 16 INTERNATIONAL HIGHER EDUCATION INTERNATIONAL HIGHER EDUCATION can be paid for out of future earnings, but the econo- mists who addressed the Royal Economics Society were convinced that their evidence pointed to increased fees being a considerable disincentive to less-advantaged students even with a more favorable loan scheme. Moreover, while the report is notably balanced in the way it presents its arguments and in particular in the way it advocates the redirection of fee income into bur- saries for the disadvantaged, it does not address the prob- able plight of those universities that are at the bottom of the league tables but that are addressing the government’s access agenda as vigorously as those at the top are addressing its research agenda. Michael Shattock Their position offers the sharpest conflict between the government’s twin policies of market orientation and social inclusion. The introduction of higher fees would certainly favor the top universities but at the expense of the bottom; the bottom are delivering social inclusivity, but the top are not for the reasons described above and, once freed from government constraints, might be even less likely to do so. y The debate will no doubt continue until after the next general election, but the fear must be that attitudes will polarize either because one or the other of the main politi- cal parties endorses some elements of the debate or because the argument becomes increasingly institutionally self-interested. The dilemmas it exposes are not, of course, limited to the United Kingdom. The Foreign Invasion of Israeli Higher Education The 15,000 students who could not gain access to any of the “traditional” institutions of higher education are willing to pay a tuition almost twice as high as that charged by public higher education institutions because they want to get a degree without having to give up their full-time jobs or, in some cases, without having to devote themselves to hard intellectual work. They see the degree as a means for social mobility or simply as a way to further their careers, and The Council of Higher Education (CHE) decided to open the gates of higher education to foreign institutions because of public pressure at the beginning of the 1990s. First, any branch of a foreign university will have to prove that the time needed to complete the degree is simi- lar to that required by Israeli universities. The CHE does not oppose creative measures—such as, three semesters a year instead of two semesters at the traditional universi- ties—but it wants to prevent a situation in which a degree is awarded to persons who do not have the necessary knowl- edge in their respective fields. Second, all students who are accepted will either have passed the matriculation exams that are prerequisites for
https://openalex.org/W2972656459	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1007721&type=printable	English	null	A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	21,031	PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries Cedoljub Bundalovic-TormaID1,2¤a, Gregory B. Whitfield1,2¤b, Lindsey S. Marmont1,2¤c, P. Lynne HowellID1,2, John ParkinsonID1,2,3* 1 Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada, 2 Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, 3 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤a Current address: Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada ¤b Current address: De´partement de Microbiologie, Infectiologie et Immunologie, Universite´ de Montre´al, Montre´al, QC, Canada ¤c Current address: Department of Microbiology, Harvard Medical School, Boston, Massachusetts, United States of America * john.parkinson@utoronto.ca ¤a Current address: Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada ¤b Current address: De´partement de Microbiologie, Infectiologie et Immunologie, Universite´ de Montre´al, Montre´al, QC, Canada C t dd D t t f Mi bi l H d M di l S h l B t M h tt U it d ¤a Current address: Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada ¤b Current address: De´partement de Microbiologie, Infectiologie et Immunologie, Universite´ de Montre´al, Montre´al, QC, Canada ¤c Current address: Department of Microbiology, Harvard Medical School, Boston, Massachusetts, United States of America * john.parkinson@utoronto.ca ¤c Current address: Department of Microbiology, Harvard Medical School, Boston, Massachusetts, United States of America * john parkinson@utoronto ca OPEN ACCESS Citation: Bundalovic-Torma C, Whitfield GB, Marmont LS, Howell PL, Parkinson J (2020) A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries. PLoS Comput Biol 16(4): e1007721. https://doi.org/10.1371/journal. pcbi.1007721 In bacteria functionally related genes comprising metabolic pathways and protein com- plexes are frequently encoded in operons and are widely conserved across phylogenetically diverse species. The evolution of these operon-encoded processes is affected by diverse mechanisms such as gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification, increasing the potential evolution of novel biological pathways, and enabling pre-existing pathways to adapt to the requirements of particular environments. Despite the fundamental importance that these mechanisms play in bacterial environmental adaptation, a systematic approach for studying the evolution of operon organization is lacking. Herein, we present a novel method to study the evolution of operons based on phylogenetic clustering of operon-encoded protein families and geno- mic-proximity network visualizations of operon architectures. We applied this approach to study the evolution of the synthase dependent exopolysaccharide (EPS) biosynthetic sys- tems: cellulose, acetylated cellulose, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), Pel, and alginate. These polymers have important roles in biofilm formation, antibiotic tolerance, and as virulence factors in opportunistic pathogens. Our approach revealed the complex evolu- tionary landscape of EPS machineries, and enabled operons to be classified into evolution- arily distinct lineages. Cellulose operons show phyla-specific operon lineages resulting from gene loss, rearrangement, and the acquisition of accessory loci, and the occurrence of whole-operon duplications arising through horizonal gene transfer. Our evolution-based classification also distinguishes between PNAG production from Gram-negative and Gram- positive bacteria on the basis of structural and functional evolution of the acetylation modifi- cation domains shared by PgaB and IcaB loci, respectively. We also predict several pel-like operon lineages in Gram-positive bacteria and demonstrate in our companion paper Author summary In bacterial genomes, biological processes are frequently encoded by neighbouring co- transcribed genes, termed operons. In addition, operon-associated genes often belong to distinct evolutionary families with diverse biological functions. Studying the evolution of bacterial operons provides valuable insight into understanding the biological significance of genes involved in environmental adaptation. To date, no systematic approach has been devised to examine both the complex evolutionary relationships of operon encoded genes and the evolution of operon organization as a whole. To address this challenge, we devel- oped an integrative method to study operon evolution by combining phylogenetic tree based clustering and genomic-context networks. We applied this method to perform the first systematic survey of all known synthase-dependent exopolysaccharide biosynthetic machineries, demonstrating the generalizability of our approach for operons of diverse size, protein family composition, and species distribution. Our method identified distinct biofilm operon clades across phylogenetically diverse bacteria, that result from gene rear- rangement, duplication, loss, fusion, and horizontal gene transfer. We found different evolutionary trajectories for Gram-negative and Gram-positive PNAG biofilm production machineries, and in a companion paper (Whitfield et al PLoS Pathogens, in press) present experimental validation that the Pel polysaccharide is produced by a Gram-positive bacterium. StudentandFellowResources/RTC/Training- Programs/restracomp/index.html). Computing resources were provided by the SciNet HPC Consortium; SciNet is funded by: the Canada Foundation for Innovation (https://www.innovation. ca/) under the auspices of Compute Canada (https://www.computecanada.ca/); Ontario Research Fund–Research Excellence (https://www. ontario.ca/page/ontario-research-fund) and the University of Toronto (https://www.utoronto.ca/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3’,5’-dimeric guanosine monophosphate (c-di-GMP). (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3’,5’-dimeric guanosine monophosphate (c-di-GMP). (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3’,5’-dimeric guanosine monophosphate (c-di-GMP). Funding: JP and CB-T were supported by grants from the Natural Sciences and Engineering Research Council (RGPIN-2014-06664 & RGPIN- 2019-06852; http://www.nserc-crsng.gc.ca/) and the National Institutes of Health (NSERC; R21AI126466; https://www.nih.gov/). This work was also supported in part by grants from the Canadian Institutes of Health Research (CIHR; http://www.cihr-irsc.gc.ca/) (MOP 43998 and FDN154327 to PLH). PLH is a recipient of a Canada Research Chair (http://www.chairs-chaires.gc.ca/ home-accueil-eng.aspx). GBW and LSM have been supported by graduate scholarships from NSERC. GBW has been supported by a graduate scholarship from Cystic Fibrosis Canada (https:// www.cysticfibrosis.ca/). LSM has been supported by graduate scholarships from the Ontario Graduate Scholarship Program (https://osap.gov. on.ca/OSAPPortal/en/A-ZListofAid/PRDR019245. html), and The Hospital for Sick Children Foundation Student Scholarship Program (http:// www.sickkids.ca/Research/ Funding: JP and CB-T were supported by grants from the Natural Sciences and Engineering Research Council (RGPIN-2014-06664 & RGPIN- 2019-06852; http://www.nserc-crsng.gc.ca/) and the National Institutes of Health (NSERC; R21AI126466; https://www.nih.gov/). This work was also supported in part by grants from the Canadian Institutes of Health Research (CIHR; http://www.cihr-irsc.gc.ca/) (MOP 43998 and FDN154327 to PLH). PLH is a recipient of a Canada Research Chair (http://www.chairs-chaires.gc.ca/ home-accueil-eng.aspx). GBW and LSM have been supported by graduate scholarships from NSERC. GBW has been supported by a graduate scholarship from Cystic Fibrosis Canada (https:// www.cysticfibrosis.ca/). LSM has been supported by graduate scholarships from the Ontario Graduate Scholarship Program (https://osap.gov. on.ca/OSAPPortal/en/A-ZListofAid/PRDR019245. html), and The Hospital for Sick Children Foundation Student Scholarship Program (http:// www.sickkids.ca/Research/ StudentandFellowResources/RTC/Training- Programs/restracomp/index.html). Computing resources were provided by the SciNet HPC Consortium; SciNet is funded by: the Canada Foundation for Innovation (https://www.innovation. ca/) under the auspices of Compute Canada (https://www.computecanada.ca/); Ontario Research Fund–Research Excellence (https://www. ontario.ca/page/ontario-research-fund) and the University of Toronto (https://www.utoronto.ca/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Editor: Mark M. Tanaka, University of New South Wales, AUSTRALIA Received: September 18, 2019 Accepted: February 11, 2020 Published: April 1, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pcbi.1007721 Copyright: © 2020 Bundalovic-Torma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. 1 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 Introduction The generation of novel genomes through next generation sequencing is creating a wealth of opportunities for understanding the evolution of biological systems. A key challenge is the development of robust and systematic approaches that allow genes to be classified into func- tional categories and which are also capable of inferring evolutionary relationships. In bacterial genomes, functionally-related genes corresponding to metabolic pathways or protein com- plexes are often encoded by neighbouring co-regulated and co-transcribed loci, termed an operon. Computational prediction of operons based on the conservation of short inter-genetic distances found between homologous genes across phylogenetically diverse bacteria has been frequently used to predict the biological roles of neighbouring uncharacterized genes [1–6]. Such approaches are valuable for computational inference of gene function in experimentally uncharacterized organisms and facilitate comparative genomics of adaptive traits across phylo- genetically diverse bacteria. Competing interests: The authors have declared that no competing interests exist. Analyzing patterns of sequence divergence within each gene yields insights into species- specific functionalities. However, genes in an operon do not function in isolation but typically form parts of higher-order, biological modules (e.g. protein complexes or metabolic pathways). Consequently, analysing evolutionary events in an operonic context provides additional opportunities to better infer functional relationships. For example, while sequence divergence has the potential to impact the function of a single gene, evolutionary events that alter operon structure (e.g. rearrangements, duplications, gains and losses) have the potential to dramati- cally alter the overall function of the operon [7,8]. Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 2 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries Due to the lack of a systematic framework, very few studies have attempted to examine the influence of evolutionary events on operon structure [9,10]. Phylogenetic-tree based classifica- tion of 197 ATP binding motif sequences associated with operon-encoded bacterial ATP-bind- ing cassette (ABC) transporters was successful in resolving two evolutionarily distinct transporter clades associated with import and export functions [11]. Gene duplications have been shown to play an important role in driving protein superfamily expansion and are posi- tively correlated with bacterial genome size [12]. Duplications have been found to be associ- ated with biological processes associated with environmental adaptation of species clusters [13], such as outer membrane polysaccharide export proteins involved in capsule biosynthesis [14,15], and amplification of beta-lactamase enzymes associated with increased antibiotic resistance [16]. Introduction The study of co-localized “gene blocks” across bacteria has also shown that gene duplication, loss, and rearrangement play important roles in shaping the large-scale orga- nization of bacterial genomes [10]. Key to these analyses is the use of a rigorous and systematic approach for assigning genes into evolutionarily related ‘families’ that are likely to share simi- lar functions. However, the inference of biological function based on sequence similarities of genes or proteins is often complicated by functional divergence arising through recent gene duplication events. A variety of metrics have been employed for determining the relatedness of genes and their protein products from which groups (i.e. clustering) can be defined. These metrics include: evolutionary distances derived through the construction of phylogenetic trees [17–19]; global protein sequence similarities [20–22]; and shared sequence features such as conserved amino acids at specific sites or shared amino acid subsequences, which define motifs or structural domains [23,24]. The aim of these approaches is to automatically resolve large protein families comprising potentially thousands of genes into a smaller number of clusters defining evolutionarily related subfamilies with similar biological roles. An additional challenge faced by clustering methodologies is defining which set of clusters result in an optimal partitioning of the underlying data. To help guide such partitioning, a variety of cluster validation approaches have been devised. These are broadly divided into two categories: external-validation and internal-validation, based on whether previous information is available for the data being clustered [25]. For example, methods developed for classifying orthologous genes (i.e. those related by common ancestry) and paralogous genes (i.e. those emerging from duplication after a speciation event) rely on internal-validation tests. In one such approach, internal branch lengths between one-to-many homologous gene relationships are compared between two species [26]. In an alternative approach, clustering is employed to define “triangles” of proteins with significant sequence similarity occurring between three dis- tinct species [20,27]. However, to distinguish the finer-scale evolutionary relationships occur- ring within an orthologous group or gene family, phylogenetic tree construction is required. Such methods have typically focused on well-characterized biological systems, e.g. homologs of the bacterial flagellar and type III secretion system subunits [28] and diverse systems associ- ated with the type IV filament superfamily [29], which have utilized an external-validation approach for defining functionally distinct phylogenetic clades. Introduction Here we build on these methods and present a framework for the systematic classification and analysis of diverse gene families in the context of operons. Focusing on synthase-depen- dent exopolysaccharide (EPS) biosynthetic machineries, we use our framework to explore how gene divergence in combination with duplication, loss, and rearrangement events have shaped the evolution of EPS operons, and may have influenced the biofilm producing capabilities of evolutionarily diverse bacteria. EPS are an important component of bacterial biofilms that not only ensure survival in response to limited nutrient availability, but are also involved in antibiotic tolerance, immune evasion and serve as virulence factors in many clinically relevant pathogens [30–32]. Distinct PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 3 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries mechanisms have been identified in the production of bacterial EPS, including the well-char- acterized Wzx/Wzy and ABC transporter-dependent pathways [33], and synthase-dependent systems [34]. Typically, Gram-negative synthase-dependent EPS systems are organized as discrete oper- ons comprised of genes encoding: 1) an inner membrane associated polysaccharide synthase; 2) a regulatory domain or co-polymerase subunit responsible for binding the intracellular sig- naling molecule cyclic-3’,5’-dimeric guanosine monophosphate (c-di-GMP); 3) periplasmic polysaccharide modification enzymes; and 4) a periplasmic tetratricopeptide repeat (TPR) domain coupled with an outer membrane pore [34]. This operonic organization allows bacte- ria to acquire complete EPS functionality through discrete lateral gene transfer events and may act as a key driver in niche adaptation [35]. To date five synthase-dependent EPS have been identified: cellulose, acetylated cellulose, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), alginate and the Pel polysaccharide. While much interest has focused on the molecular basis of biofilm formation, these systems have been characterized for only a relatively limited set of bacterial species. Consequently, little is known concerning how these systems have evolved. Of interest is how mechanisms such as gene divergence, duplication, acquisition, loss, and rearrangement of EPS operons have contributed to bacterial adaptation to diverse environments, and from a human health perspective, contributed to a pathogen’s ability to infect and cause disease. While a previous survey of cellulose EPS machineries has been reported [36], a comprehensive systematic analysis of all EPS machineries is lacking. In this study, we describe a phylogenetic tree-based clustering method for defining protein sequence subfamilies and apply it to study the evolutionary relationships of operons. Introduction This method was employed for the systematic classification of EPS operons predicted from a survey of over a thousand bacterial genomes. Applying a graphical visualization approach, we demon- strate that phylogenetic clustering enables the resolution of discrete EPS operon clades which differ in their organization from experimentally characterized operons, providing valuable insights toward further understanding the roles of gene duplication, rearrangement, and loss/ absence in the evolution of biofilm production between phylogenetically diverse species from distinct environmental niches. For example, we demonstrate the biological implications of operon evolution that has been shaped by horizontal gene transfer (HGT) and subsequent divergence, for two cellulose operon clades among Proteobacteria which correspond to the production of cellulose polymers with different structural organizations. Furthermore, we note that most of our operon predictions are novel and demonstrate the value of applying computa- tional predictions to guide the discovery of EPS production in previously uncharacterized species. We highlight an example for Pel production, which was initially identified and charac- terized in Pseudomonas aeruginosa [37] and other Gram-negative bacteria. Our approach identified several pel-like operons in some Bacillus spp. and other Gram-positive bacteria, which appeared to be regulated by the intracellular signaling molecule c-di-GMP. In our com- panion paper (Whitfield et al PLoS Pathogens, in press) we experimentally validate these find- ings by demonstrating the production of Pel by the Gram-positive Bacillus cereus ATCC 10987 and its regulation by c-di-GMP. A systematic survey of bacterial EPS operons reveals EPS systems across bacteria of diverse lifestyles and environmental niches SpN 1 1 1 1 1 1 1 1 2 2 2 2 ... ... ... ... Curation of EPS Operon Loci Protein Sequences from known EPS producing bacteria (A) Operon Prediction Pipeline Overview 1. Construct EPS specific HMMs 2. Identify putative EPS loci Sp1 Sp2 SpN 3. Reconstruct EPS operons based on genome proximity 4. Define clusters for each EPS locus 5. Visualize operon networks Total Species Genomes 4 60 367 140 288 65 51 60 43 44 26 56 65 41 67 29 (B) Predicted Operons - Summary Lifestyle: % Non-Pathogen Lifestyle: % Pathogen Lifestyle: % Unknown Niche: % Host-Associated Niche: % Environmental / Other Niche: % Unknown 50 35 50 5 14 1 0 0 53 75 47 25 32 45 5 0 0 1 14 Acetylated cellulose Alginate Cellulose Pel PNAG Curation of EPS Operon Loci Protein Sequences from known EPS producing bacteria (A) Operon Prediction Pipeline Overview 1. Construct EPS specific HMM 4. Define clusters for each EPS locus 5. Visualize operon networks Total Species Genomes 4 60 367 140 288 65 51 60 43 44 26 56 65 41 67 29 (B) Predicted Operons - Summary Lifestyle: % Non-Pathogen Lifestyle: % Pathogen Lifestyle: % Unknown Niche: % Host-Associated Niche: % Environmental / Other Niche: % Unknown 50 35 50 5 14 1 0 0 53 75 47 25 32 45 5 0 0 1 14 Acetylated cellulose Alginate Cellulose Pel PNAG (B) Predicted Operons - Summary (B) Predicted Operons - Summary (A) Operon Prediction Pipeline Overview NCBI 1733 Completely Sequenced Bacterial Genomes Sp1 Sp2 ... SpN 1 1 1 1 1 1 1 1 2 2 2 2 ... ... ... ... s 2. Identify putative EPS loci Sp1 Sp2 SpN 3. Reconstruct EPS operons based on genome proximity 1. Construct EPS specific HMMs 1. Construct EPS specific HMMs 3. Reconstruct EPS operons based on genome proximity 3. A systematic survey of bacterial EPS operons reveals EPS systems across bacteria of diverse lifestyles and environmental niches Visualize operon networks Total Species Genomes 4 60 367 140 288 65 51 60 43 44 26 56 65 41 67 29 (B) Predicted Operons - Summary Lifestyle: % Non-Pathogen Lifestyle: % Pathogen Lifestyle: % Unknown Niche: % Host-Associated Niche: % Environmental / Other Niche: % Unknown 50 35 50 5 14 1 0 0 53 75 47 25 32 45 5 0 0 1 14 Locus Rearrangement Locus Loss Locus Duplication Locus Fusion Acetylated cellulose Alginate Cellulose Pel PNAG Operon Duplication (C) Predicted Operons - Evolutionary Events 16 34 66 60 0 13 80 14 4 0 3 3 11 5 75 13 14 3 12 0 0 3 0 2 0 % Operons by Event Deinococcales (1) Caulobacterales (1) Alteromonadales (2) Lactobacillales (1) Rhodospirillales (1) Petrotogales (1) Streptomycetales (3) Corynebacteriales (6) ) 4 ( s ela r e tc a b o d o h R Thermotogales (2) Neisseriales (1) Campylobacterales (1) Xanthomonadales (16) Rhodospirillales (2) NA (1) Bacillales (1) Bifidobacteriales (6) Chroococcales (1) Chloroflexales (2) Rubrobacterales (1) Thermotogales (5) Rhodospirillales (2) Thermales (2) Lactobacillales (6) Burkholderiales (11) Bacillales (4) Desulfuromonadales (2) Enterobacterales (118) Chlorobiales (2) Fusobacteriales (3) Burkholderiales (1) ) 1 ( s elaidirts ol C Enterobacterales (58) Rhizobiales (11) Desulfuromonadales (2) Bacillales (1) Clostridiales (2) Clostridiales (3) Aquificales (3) Deinococcales (1) Bacillales (49) Flavobacteriales (2) Burkholderiales (23) Nautiliales (1) Thermoanaerobacterales (2) Rhizobiales (8) Bacillales (19) Lactobacillales (25) Lactobacillales (8) Tissierellales (1) Chloroflexales (1) Clostridiales (1) Syntrophobacterales (1) Nitrospirales (2) Flavobacteriales (1) Methylococcales (1) Pseudomonadales (56) Selenomonadales (1) Burkholderiales (44) Acidobacteriales (5) Alteromonadales (3) Oscillatoriales (3) Magnetococcales (1) Bacillales (2) Clostridiales (1) Lactobacillales (10) Myxococcales (5) Clostridiales (18) Acidithiobacillales (2) Nitrosomonadales (1) Pasteurellales (11) Desulfobacterales (2) Gammaproteobacteria (1) Candidatus Methylomirabilis (1) Alteromonadales (12) Fusobacteriales (1) Desulfovibrionales (2) Desulfurobacteriales (1) Pseudomonadales (17) Thermoanaerobacterales (1) Burkholderiales (2) Lactobacillales (1) Cytophagia (1) Sphingomonadales (10) Solirubrobacterales (1) Oceanospirillales (1) Campylobacterales (2) Branch Colours (Class) Gamma-proteobacteria Beta-proteobacteria Epsilon-proteobacteria Alpha-proteobacteria Delta-proteobacteria Deinococci Flavobacteria Actinobacteria Thermotogae Aquificae Clostridia Bacilli Pie Chart Colours (EPS Operon) Acetylated Cellulose Alginate Cellulose Pel PNAG (D) Operon Phylogenetic Distribution Acetylated Cellulose Alginate Pel PNAG Cellulose Operon Combinations (Number of Genomes) 0 0 2 0 0 1 0 300 200 100 1 199 105 87 20 10 7 5 1 2 2 30 1 (E) Co-Occuring Operons 280 300 Acetylated cellulose Alginate Cellulose Pel PNAG NCBI 1733 Completely Sequenced Bacterial Genomes Sp1 Sp2 ... A systematic survey of bacterial EPS operons reveals EPS systems across bacteria of diverse lifestyles and environmental niches A schematic overview of the pipeline we used for classifying bacterial operons is provided in Fig 1A. The process begins with a systematic survey of all five previously characterized synthase-dependent EPS systems (cellulose, acetylated cellulose, PNAG, Pel, and alginate) (S1 4 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries NCBI 1733 Completely Sequenced Bacterial Genomes Sp1 Sp2 ... SpN 1 1 1 1 1 1 1 1 2 2 2 2 ... ... ... ... Curation of EPS Operon Loci Protein Sequences from known EPS producing bacteria (A) Operon Prediction Pipeline Overview 1. Construct EPS specific HMMs 2. Identify putative EPS loci Sp1 Sp2 SpN 3. Reconstruct EPS operons based on genome proximity 4. Define clusters for each EPS locus 5. A systematic survey of bacterial EPS operons reveals EPS systems across bacteria of diverse lifestyles and environmental niches Reconstruct EPS operons based on genome proximity Define clusters for each EPS locus Locus Rearrangement Locus Loss Locus Duplication Locus Fusion Acetylated cellulose Alginate Cellulose Pel PNAG Operon Duplication (C) Predicted Operons - Evolutionary Events 16 34 66 60 0 13 80 14 4 0 3 3 11 5 75 13 14 3 12 0 0 3 0 2 0 % Operons by Event Deinococcales (1) Caulobacterales (1) Alteromonadales (2) Lactobacillales (1) Rhodospirillales (1) Petrotogales (1) Streptomycetales (3) Corynebacteriales (6) ) 4 ( s ela r e tc a b o d o h R Thermotogales (2) Neisseriales (1) Campylobacterales (1) Xanthomonadales (16) Rhodospirillales (2) NA (1) Bacillales (1) Bifidobacteriales (6) Chroococcales (1) Chloroflexales (2) Rubrobacterales (1) Thermotogales (5) Rhodospirillales (2) Thermales (2) Lactobacillales (6) Burkholderiales (11) Bacillales (4) Desulfuromonadales (2) Enterobacterales (118) Chlorobiales (2) Fusobacteriales (3) Burkholderiales (1) ) 1 ( s elaidirts ol C Enterobacterales (58) Rhizobiales (11) Desulfuromonadales (2) Bacillales (1) Clostridiales (2) Clostridiales (3) Aquificales (3) Deinococcales (1) Bacillales (49) Flavobacteriales (2) Burkholderiales (23) Nautiliales (1) Thermoanaerobacterales (2) Rhizobiales (8) Bacillales (19) Lactobacillales (25) Lactobacillales (8) Tissierellales (1) Chloroflexales (1) Clostridiales (1) Syntrophobacterales (1) Nitrospirales (2) Flavobacteriales (1) Methylococcales (1) Pseudomonadales (56) Selenomonadales (1) Burkholderiales (44) Acidobacteriales (5) Alteromonadales (3) Oscillatoriales (3) Magnetococcales (1) Bacillales (2) Clostridiales (1) Lactobacillales (10) Myxococcales (5) Clostridiales (18) Acidithiobacillales (2) Nitrosomonadales (1) Pasteurellales (11) Desulfobacterales (2) Gammaproteobacteria (1) Candidatus Methylomirabilis (1) Alteromonadales (12) Fusobacteriales (1) Desulfovibrionales (2) Desulfurobacteriales (1) Pseudomonadales (17) Thermoanaerobacterales (1) Burkholderiales (2) Lactobacillales (1) Cytophagia (1) Sphingomonadales (10) Solirubrobacterales (1) Oceanospirillales (1) Campylobacterales (2) Branch Colours (Class) Gamma-proteobacteria Beta-proteobacteria Epsilon-proteobacteria Alpha-proteobacteria Delta-proteobacteria Deinococci Chloroflexia Flavobacteria Actinobacteria Thermotogae Aquificae Clostridia Bacilli Pie Chart Colours (EPS Operon) Acetylated Cellulose Alginate Cellulose Pel PNAG (D) Operon Phylogenetic Distribution Acetylated Cellulose Alginate Pel PNAG Cellulose Operon Combinations (Number of Genomes) 0 0 2 0 0 1 0 300 200 100 1 199 105 87 20 10 7 5 1 2 2 30 1 (E) Co-Occuring Operons 280 300 Locus Rearrangement Locus Loss Locus Duplication Locus Fusion Acetylated cellulose Alginate Cellulose Pel PNAG Operon Duplication (C) Predicted Operons - Evolutionary Events 16 34 66 60 0 13 80 14 4 0 3 3 11 5 75 13 14 3 12 0 0 3 0 2 0 % Operons by Event (C) Predicted Operons - Evolutionary Events Locus Rearrangement Locus Loss Locus Duplication Locus Fusion Acetylated cellulose Alginate Cellulose Pel PNAG Operon Duplication (C) Predicted Operons - Evolutionary Events 16 34 66 60 0 13 80 14 4 0 3 3 11 5 75 13 14 3 12 0 0 3 0 2 0 % Operons by Event Deinococcales (1) Caulobacterales (1) Alteromonadales (2) Lactobacillales (1) Rhodospirillales (1) Petrotogales (1) Streptomycetales (3) Corynebacteriales (6) ) 4 ( s ela r e tc a b o d o h R Thermotogales (2) Neisseriales (1) Campylobacterales (1) Xanthomonadales (16) Rhodospirillales (2) NA (1) Bacillales (1) Bifidobacteriales (6) Chroococcales (1) Chloroflexales (2) Rubrobacterales (1) Thermotogales (5) Rhodospirillales (2) Thermales (2) Lactobacillales (6) Burkholderiales (11) Bacillales (4) Desulfuromonadales (2) Enterobacterales (118) Chlorobiales (2) Fusobacteriales (3) Burkholderiales (1) ) 1 ( s elaidirts ol C Enterobacterales (58) Rhizobiales (11) Desulfuromonadales (2) Bacillales (1) Clostridiales (2) Clostridiales (3) Aquificales (3) Deinococcales (1) Bacillales (49) Flavobacteriales (2) Burkholderiales (23) Nautiliales (1) Thermoanaerobacterales (2) Rhizobiales (8) Bacillales (19) Lactobacillales (25) Lactobacillales (8) Tissierellales (1) Chloroflexales (1) Clostridiales (1) Syntrophobacterales (1) Nitrospirales (2) Flavobacteriales (1) Methylococcales (1) Pseudomonadales (56) Selenomonadales (1) Burkholderiales (44) Acidobacteriales (5) Alteromonadales (3) Oscillatoriales (3) Magnetococcales (1) Bacillales (2) Clostridiales (1) Lactobacillales (10) Myxococcales (5) Clostridiales (18) Acidithiobacillales (2) Nitrosomonadales (1) Pasteurellales (11) Desulfobacterales (2) Gammaproteobacteria (1) Candidatus Methylomirabilis (1) Alteromonadales (12) Fusobacteriales (1) Desulfovibrionales (2) Desulfurobacteriales (1) Pseudomonadales (17) Thermoanaerobacterales (1) Burkholderiales (2) Lactobacillales (1) Cytophagia (1) Sphingomonadales (10) Solirubrobacterales (1) Oceanospirillales (1) Campylobacterales (2) (D) Operon Phylogenetic Distribution Operon Combinations Number of Genomes) 200 100 199 105 87 30 (E) Co-Occuring Operons 280 300 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries Fig 1. Summary of predicted bacterial EPS operons. (A) Overview of the main steps of the EPS operon prediction pipeline: 1) De novo construction of reference operon locus HMM models; 2) Sequence homology searches against 1733 fully sequenced bacterial genomes retrieved from NCBI; 3) Prediction of putative EPS operons through genomic-proximity reconstruction of significant locus hits; 4) Definition of clusters defining evolutionary relationships for each EPS locus; 5) Integration of operon and cluster predictions to visualize operon networks. (B) Number of predicted EPS operons and percentages of species summarized by bacterial lifestyle (pathogen, non-pathogen, unknown) and corresponding niche (host-associated, environmental/other, unknown). (C) Percentage of evolutionary events associated with EPS operons: Locus loss (core EPS operon loci not detected by HMM searches); Locus rearrangement (EPS operons featuring locus orderings that differ from the canonical operon for that type–S1 Table); Locus duplication (defined by two loci possessing a significant match to the same EPS HMM within the same operon); Operon duplication, defined as a genome encoding two copies of the same type of EPS system, separated by greater than 10 kb; Locus fusion, loci possessing significant matches to multiple EPS HMMs. (D) A cladogram based on 16S rRNA sequences illustrating the phylogenetic distribution of predicted EPS operons. Branches are coloured according to taxonomic class and piecharts represent the proportion of EPS operon types identified for each clade. Leaf labels represent the major taxonomic families found in each clade, along with the number of genomes represented. 16S rRNA sequences were retrieved from full genome sequences using Barrnap (https://github.com/tseemann/barrnap), clustered using CD-HIT [94] at 95% sequence identity and aligned using MUSCLE [92] for phylogenetic tree construction with Fasttree [103] and subsequent visualization with iTOL [104]. (E) An upset plot (generated using the R (https://www.r-project.org) package UpSetR [105]) representing the number of different EPS operon combinations identified across bacterial genomes. https://doi.org/10.1371/journal.pcbi.1007721.g001 https://doi.org/10.1371/journal.pcbi.1007721.g001 & S2 Tables) through an iterative hidden Markov-model (HMM) -based search strategy and subsequent genomic-proximity based reconstruction of 1733 complete reference and repre- sentative bacterial genomes (downloaded April 20, 2015—see Methods). We identified 407 cellulose, 321 PNAG, 146 Pel, 64 alginate, and 4 acetylated cellulose EPS “operons” defined as comprising at least: 1) a polysaccharide synthase subunit; and 2) one additional locus involved in EPS modification or transport as defined previously [30] (S3 Table). PLOS COMPUTATIONAL BIOLOGY These could be allo- cated to 367, 288, 140, 60 and 4 different bacterial species, respectively (Fig 1). We note that for all previously characterized EPS producing species included in this set of fully sequenced genomes, we successfully detected an operon corresponding to the type of EPS produced (S4 Table). Furthermore, for experimentally characterized species lacking a fully sequenced genome, we also identified several closely related strains possessing an EPS operon identical to the characterized strain, providing a valuable resource for further experimental validation. PNAG was significantly enriched in pathogen genomes (161/288–56%; Chi-squared test p- value = 2.05e-09). Conversely, Pel (84/140–60%; Chi-squared test p-value ~ 1.83e-4), alginate (39/60–65%; Chi-squared test p-value ~ 3.5e-2) and cellulose (187/367–51%; Chi-squared test p-value = 2.05e-14) were significantly enriched in non-pathogen genomes (Fig 1C and S1 Fig). Interestingly, both cellulose and PNAG operons were significantly associated with genomes with host-associated lifestyles (Chi-squared p-values ~ 1.05e-9 and ~1.48e-12, respec- tively). From a phylogenetic perspective, each EPS system was well represented by Proteobac- teria, with cellulose, PNAG and Pel additionally featuring operons from Bacilli and Clostridia, which to our knowledge have not been previously reported (Fig 1D). Further, we note that Pel operons exhibited the greatest diversity of bacterial families (Shannon index of bacterial fami- lies– 2.74) with representation in Thermotogae, Actinobacteria and Rubrobacteria, among others. While most genomes contain only a single synthase-dependent EPS system, we observed many instances of co-occurrence (Fig 1E), with cellulose and PNAG systems being the most common combination (83 genomes), followed by alginate and Pel (20 genomes). Notably, all species possessing three systems were Pseudomonas spp., e.g.: Pseudomonas prote- gens strains Pf-5 and CHA0 (alginate, Pel and PNAG); Pseudomonas fluorescens SBW25 and Pseudomonas sp. TKP (acetylated cellulose, alginate, and PNAG). (D) Operon Phylogenetic Distribution (D) Operon Phylogenetic Distribution Chlorobiales (2) s (2) cterales (1) Nitrospirales (2) s (5) bacterales (2) atus Methylomirabilis (1) ulfovibrionales (2) Acetylated Cellulose Alginate Pel PNAG Cellulose Operon Combinations (Number of Genomes) 0 0 2 0 0 1 0 300 200 100 1 199 105 87 20 10 7 5 1 2 2 30 1 (E) Co-Occuring Operons 280 300 5 / 32 Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY Evolution of EPS operons is driven by gene duplication, loss and rearrangements The processes underlying EPS operon evolution across diverse bacterial phyla are poorly understood. We examined how operon organization is influenced by the following evolution- ary events that are likely to affect EPS production capabilities among bacteria: 1) single locus or whole operon duplications, which could lead to dosage effects and alter the level of EPS PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 6 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries modification or export; 2) locus losses, that may indicate a reduction or loss in EPS production or modification, or may suggest supplementation of the lost function with a novel gene; 3) operon rearrangements which may affect the regulation of EPS production through the order of expression of individual EPS system components; and, 4) gene-fusions, resulting in enhanced co-expression of interacting subunits. For each set of predicted EPS operons, the resulting number of operon evolutionary events was defined relative to the locus composition and order of reference Gram-negative experi- mentally characterized operons [30,38–41]. In contrast to previous studies of operon evolution [28,29], we use the term evolutionary events to refer to key changes that define distinct organi- zations between evolutionarily distinct operon clades and not divergence of operons from an ancestral state. With the exception of acetylated cellulose, locus losses were found to be the most frequent event (~46% of predicted operons lacked one or more reference loci), and occurred with the greatest frequency for Pel which exhibited an average loss of 2.6 loci lost per operon (S5 Table). Among all EPS systems the majority of locus losses were associated with the outer membrane pore encoding loci (441 / 993–44% of all locus loss events identified) among Gram-positive species (S5 Table), consistent with the lack of an outer membrane bilayer in Gram-positive cell envelope architectures. Operon rearrangements were the next most frequent evolutionary events (~ 39%), largely associated with cellulose operons [36] (327 / 407–80%). Focusing on duplication events, within-operon loci duplications tended to be more common than whole-operon duplications (2 or more core EPS loci identified > = 1 kb apart), with the exception of cellulose operons (29 whole operon duplications compared to 24 loci duplications). All duplicated operons were found to be separated by at least 10 kb, suggest- ing they may have been acquired through HGT rather than tandem duplication of a pre-exist- ing operon [42,43]. Systematic phylogenetic distance-based clustering of EPS operon loci and genomic-proximity networks identifies evolutionarily distinct operon clades To better understand how these evolutionary events may have altered operon function, we next devised an agnostic, systematic classification strategy to cluster each family of EPS operon loci on the basis of phylogenetic distance (Fig 2A; see Methods). In brief, for each EPS operon locus, multiple sequence alignments were generated and used to construct phylogenetic trees. From these trees, we defined sets of clusters through an iterative scan of the tree structure that captures an increasing sequence distance between family members, starting at the leaves and ending at the root. During this scan, sequences are grouped into a cluster if they share a com- mon node (i.e. are within a specified evolutionary distance). To define the optimal set of clus- ters for each locus, we then applied three cluster quality scoring schemes (Q1, Q2 and Q3) based on the following metrics: proportion of sequences clustered (to maximize the number of sequences clustered); average silhouette score (to minimize the occurrence of clusters contain- ing highly divergent sequences); and the Dunn index (to maximize the separation of closely related sequences from divergent sequences). For each scoring scheme, we defined the optimal pattern of clustering based on the evolutionary distance (expected number of substitutions per site) derived from a maximum-likelihood constructed phylogenetic tree (see Methods for more details) that maximizes the quality score. Comparisons across scoring schemes (see below) for cellulose operon loci identified Q2 as providing the most informative sets of clus- ters. Applying this scoring scheme to all EPS loci revealed the average number of sequence clusters generated correlated with the total number of operons predicted for each type of EPS system (Fig 2B), which further corresponded to the underlying differences in species PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 7 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 8 / 32 Classification of bacterial biofilm machineries Classification of bacterial biofilm machineries Fig 2. Clustering of EPS loci. (A) Schematic illustrating the process of scanning through a phylogentic tree and identifying sets of clusters associated at different evolutionary distance cutoffs. Here evolutionary distance is defined as the number of expected amino-acid substitutions normalized over the multiple sequence alignment length. To identify optimal patterns of clusters, we examined three scoring schemes (Q1, Q2 and Q3). Q1 is defined as the sum of the average silhouette score for all clusters: μ(s(i)) and the Dunn index (DI). Q2 is defined as the sum of the proportion of sequences identified in clusters (Sc/©m), μ(s(i)) and DI. Q3 is defined as the product of (Sc/Sm) and the sum of μ(s(i)) and DI. For the family of genes related to the bcsA locus, each scoring scheme identifies a different optimal evolutionary distance cutoff resulting in defining different sets of clusters. (B) Graph illustrating the average number of sequence clusters predicted (sum of # of clusters over all loci / total number of EPS loci) for each type of EPS operon. (C) Graph illustrating the average evolutionary distance of EPS loci cluster members with other members of the same cluster. (D) Cellulose operon networks generated using the different types of scoring scheme cutoffs used in (A). For each network, nodes indicate clusters of sequences representing individual cellulose loci, edges indicate genome proximity between the two linked loci. Nodes are organized into sets of four, ordered from top to bottom as bcsA, bcsB, bcsZ and bcsC. Node size indicates the number of family members associated with that locus cluster. Node colour indicates phylogenetic representation of cluster members. Edge colour indicates genomic proximity of phylogenetic clusters. At higher evolutionary distances (as defined by Q2 and Q3), networks yield more informative patterns of evolutionary relationships as illustrated by larger clusters of loci featuring larger number of interconnections. https://doi.org/10.1371/journal.pcbi.1007721.g002 https://doi.org/10.1371/journal.pcbi.1007721.g002 distributions of EPS systems (Fig 1D). For example, the cellulose system was predicted to have the largest average number of sequence clusters overall (30 clusters) and also had the greatest species diversity (Shannon index 2.16 –S2 Fig) compared to all other systems. Furthermore, for each EPS system the variability of the number of sequence clusters predicted per locus (Fig 2C) suggests differing degrees of locus evolution that are likely to be the result of different structural and functional constraints. Classification of bacterial biofilm machineries For example, a higher degree of conservation would be expected for glycosyl transferase (GT) subunits to maintain efficient co-ordination between polymerization and inner membrane transport of EPS, while increased variability of periplas- mic modification enzymes suggests that only a subset of highly conserved motifs are required to carry out polysaccharide modification reactions. y p y To compare patterns of clusters identified by each scoring scheme, we applied the three scoring schemes to each set of genomically-neighbouring protein sequences assigned by HMM searches cellulose EPS machinery. Here we focused on four core cellulose genes: bcsA, bcsB, bcsZ, and bcsC encoding the inner membrane GT, co-polymerase, periplasmic hydrolase and outer membrane export pore, respectively [36]. While previous studies have identified additional genes associated with the production of cellulose biofilms (e.g. bcsD, bcsE, bcsF, bcsG and bcsQ; [36]), to simplify our analyses we limited our query to bcsA, bcsB, bcsC, and bcsZ based on the observation that these are the most abundant genes when comparing all identified cellulose biosynthesis operons. Although this may bias the diversity of operons iden- tified, given that a functional cellulose biosynthesis locus should contain the synthase genes bcsA and bcsB, we expect our analysis should result in few false negative predictions. From the resulting clusters we generated operon genomic-proximity networks (Fig 2D). These networks provide a visual display of the conservation of individual loci, together with their respective genomic proximity to yield patterns of sequence divergence associated with the emergence of distinct forms of operon organization. In the absence of any clustering (Q0), the network trivi- ally resolves into individual operons featuring up to four loci. Applying the Q1 scoring scheme to each locus, the network reveals a variable number of clusters across operon loci, with each cluster generally comprising sequences belonging to the same bacterial genus. Application of the Q2 scoring scheme results in the generation of clusters of increased size, encompassing species featuring distinct operon organizations and compositions. For example, two distinct lineages of alpha-proteobacterial cellulose operons can be easily distinguished, one of which is more closely related in sequence and composition to gamma-proteobacterial operons, and a second which lacks two loci and appears evolutionarily divergent from gamma-proteobacterial operons [25]. However, these distinctions were more difficult to resolve using the Q3 scoring scheme due to clustering of highly divergent sequences. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 8 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 8 / 32 Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 https://doi.org/10.1371/journal.pcbi.1007721.g002 Phylogenetic clustering and genomic proximity networks reveal evolutionary events driving EPS operon divergence Having generated clustering patterns for each EPS locus, we next used the sets of cellulose operon associated loci, bcsA, bcsB, bcsZ, and bcsC, to examine how these patterns might inform on the evolution of this EPS system. Relative to BcsA, the three other subunits (BcsB, BcsZ and BcsC) display greater sequence diversity as indicated by a larger number of sequence clusters (Fig 3). Detailed structure-function studies of the BcsA-BcsB inner membrane cellulose synthase complex, outlined below, illustrate how these findings are consistent with their known functional roles. Further inspection of the cellulose operon network identifies a num- ber of sub-networks comprised of taxon-specific loci clusters associated with distinct patterns of operon organization as illustrated through the following examples: 1) a subnetwork com- prised of loci from several beta-proteobacteria, represented here by Burkholderia cenocepacia and Pandoraea promenusa (Fig 3(i)), which feature a rearrangement of the bcsA locus and novel locus gains (also supported by inspection of corresponding Genbank genomic annota- tions) as indicated by a genomic distance of > 0.1 kb between bcsA and the neighbouring locus bcsC; 2) a subnetwork composed of loci from several species of the alpha-proteobacterial Zymomonas show rearrangement of bcsZ and/or the loss of bcsB or bcsZ (Fig 3(ii)). Further inspection reveal such losses were due to gene fusion events; 3) a subnetwork composed of loci from a separate group of alpha-proteobacteria which reveals a diverse set of bcsB loci that addi- tionally lack the bcsC outer membrane pore (Fig 3(iii)); 4) a subnetwork of loci from a group of gamma-proteobacteria reveal instances of HGT and divergence (Fig 4A). In this latter example, our network identifies two distinct clades of operons, sharing a common group of bcsA loci, but featuring two evolutionarily divergent sets of bcsB, bcsZ and bcsC loci which co- occur in several genomes separated by inter-genic distances greater than 10 kb. Detailed inves- tigation of the operonic arrangements of species possessing single copies of either of these clades of operons reveal two distinct loci organizations: the first representing the canonical cel- lulose locus order (clade A1), bcsABZC, found among E. coli and Salmonella enterica strains; while the second represents a non-canonical locus ordering (clade B1), in which the periplas- mic glycoside hydrolase, BcsZ, has undergone a rearrangement, bcsABCZ. This clade is found among Dickeya, Erwinia and Pantoea spp. (Fig 4B). Of note, we found that several species (e.g. Classification of bacterial biofilm machineries Given the trade-off between clustering highly divergent sequences (Q3) with the depiction of individual operons (Q1), we applied the Q2 scoring scheme to generate clusters for all EPS loci (S6 Table). 9 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries Using this locus-specific phylogenetic clustering approach, we were able to devise a classifi- cation scheme to define EPS locus clades based on the average evolutionary distance of a group of clustered locus sequences to a reference operon sequence (S3 Table). For example, the cellulose polysaccharide synthase locus, bcsA, from Escherichia coli is assigned to clade 1, while divergent alpha-proteobacterial species including Rhodobacter sphaeroides are assigned to clade 2. We further resolved operons into distinct groups based on the genomic co-occur- rence patterns of locus clades; e.g. for the cellulose operon (bcsABZC) we identify clade combi- nations of 1:1:1:1, 1:2:2:2 and 1:3:5:3, which correspond to operons identified in Escherichia spp. and other closely related enterobacteria, Klebsiella spp., and Burkholderia spp., respectively. Phylogenetic clustering and genomic proximity networks reveal evolutionary events driving EPS operon divergence Phylogenetically clustered operon loci are arranged vertically with respect to the canonical ordering of the cellulose operon (indicated by grey side bar). Inset boxes depict selected examples of cellulose operon clades, illustrating how the network can inform on evolutionary events: (i) Rearrangement of bcsA among betaproteobacteria–Here, bcsA appears in closer proximity to bcsC than to bcsB or bcsZ (as indicated by a cyan coloured edge for the former and a grey coloured edge for the latter). Further the cyan edge indicates a relatively large intergenic distance, suggesting a locus gain between bcsA and bcsC, confirmed upon inspection of the genome of Burkholderia cenocepacia; (ii) Rearrangement and gene fusions in alpha-proteobacteria–in examples 1 and 2 the red edge indicates operons in which bcsB is closer to bcsC than bcsZ the cyan edges suggest that bcsZ is present but appears after bcsC (example 1) roximity network of phylogenetically clustered cellulose operons. Phylogenetically clustered operon loci are arranged vertic Fig 3. Genomic-Proximity network of phylogenetically clustered cellulose operons. Phylogenetically clustered operon loci are arranged vertically with respect to the Fig 3. Genomic-Proximity network of phylogenetically clustered cellulose operons. Phylogenetically clustered opero Fig 3. Genomic-Proximity network of phylogenetically clustered cellulose operons. Phylogenetically clustered operon loci are arranged vertically with respect to the canonical ordering of the cellulose operon (indicated by grey side bar). Inset boxes depict selected examples of cellulose operon clades, illustrating how the network can inform on evolutionary events: (i) Rearrangement of bcsA among betaproteobacteria–Here, bcsA appears in closer proximity to bcsC than to bcsB or bcsZ (as indicated by a cyan coloured edge for the former and a grey coloured edge for the latter). Further the cyan edge indicates a relatively large intergenic distance, suggesting a locus gain between bcsA and bcsC, confirmed upon inspection of the genome of Burkholderia cenocepacia; (ii) Rearrangement and gene fusions in alpha-proteobacteria–in examples 1 and 2, the red edge indicates operons in which bcsB is closer to bcsC than bcsZ, the cyan edges suggest that bcsZ is present, but appears after bcsC (example 1), while in other operons, bcsZ appears missing (example 2). Detailed inspection of example operons (e.g. Zymomonas spp.) reveals the fusion of the periplasmic hydrolase and outer membrane pore (BcsZC), in example 3, the apparent loss of bcsB in another Zymomonas spp. Phylogenetic clustering and genomic proximity networks reveal evolutionary events driving EPS operon divergence Enterobacter and Klebsiella spp.) possess both operon clades. The clades have been suggested to have originated by HGT [19]; a hypothesis further supported by our phylogenetic clustering assignments (Fig 4C). Furthermore, we identified two additional divergent BcsB sequences associated with a novel organization of operon clade B1 and include several loci with other roles in cellulose production (designated operon clade B2; Fig 4D). The divergence of BcsB sequences associated with clade B2 were also found to distinguish bacterial genomes possess- ing multiple cellulose operons of distinct evolutionary lineages: Proteus mirabilis (2 cellulose PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 10 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries Fig 3. Genomic-Proximity network of phylogenetically clustered cellulose operons. Phylogenetically clustered operon loci are arranged vertically with respect to the canonical ordering of the cellulose operon (indicated by grey side bar). Inset boxes depict selected examples of cellulose operon clades, illustrating how the network can inform on evolutionary events: (i) Rearrangement of bcsA among betaproteobacteria–Here, bcsA appears in closer proximity to bcsC than to bcsB or bcsZ (as indicated by a cyan coloured edge for the former and a grey coloured edge for the latter). Further the cyan edge indicates a relatively large intergenic distance, suggesting a locus gain between bcsA and bcsC, confirmed upon inspection of the genome of Burkholderia cenocepacia; (ii) Rearrangement and gene fusions in alpha-proteobacteria–in examples 1 and 2, the red edge indicates operons in which bcsB is closer to bcsC than bcsZ, the cyan edges suggest that bcsZ is present, but appears after bcsC (example 1), while in other operons, bcsZ appears missing (example 2). Detailed inspection of example operons (e.g. Zymomonas spp.) reveals the fusion of the periplasmic hydrolase and outer membrane pore (BcsZC), in example 3, the apparent loss of bcsB in another Zymomonas spp. is explained by a fusion between the inner membrane cellulose synthase complex subunits (BcsAB); (iii) Loss of outer membrane pore, BcsC, and divergence of the inner membrane cellulose co-polymerase, BcsB, in alpha- Fig 3. Genomic-Proximity network of phylogenetically clustered cellulose operons. Phylogenetically clustered operon loci are arranged vertically with respect to the canonical ordering of the cellulose operon (indicated by grey side bar). Inset boxes depict selected examples of cellulose operon clades, illustrating how the network can Fig 3. Genomic-Proximity network of phylogenetically clustered cellulose operons. Genomic-proximity networks of pel operons reveal a novel pel locus in the gram positive bacterium, bacillus cereus that is regulated by c-di-GMP Examination of the genomic-proximity networks of pel loci also reveal novel operon organizations across phylogenetically divergent bacteria (Fig 5). As with cellulose loci bcsA and bcsZ, we identify examples of operon rearrangements involving pelB (outer membrane transport pore and TPR domain) loci and pelA (periplasmic modification hydrolase) (Fig 5(ii), 5(iiib) and 5(iv)), across several species associated with diverse environments. Again, consistent with our findings for cellu- lose, we noted loci losses and acquisitions. Although it has not been demonstrated that the pel operon forms a trans-envelope biosynthetic complex, the ordering of operon loci has been shown to play an important role in the assembly of macromolecular complexes [46] and optimizing bio- synthetic pathways [47], suggesting that there exists a functional coupling between Pel outer membrane transport and periplasmic modification [48]. However, the effects of these rearrange- ment events on Pel production still remain to be experimentally investigated. We also observed a high degree of overall conservation among components which are known to play key roles in Pel biogenesis, such as the putative polysaccharide synthase (PelF), putative inner membrane protein (PelG), hydrolase/deacetylase (PelA) and cyclic-di-GMP receptor (PelD) [32]. In contrast, a greater degree of divergence can be seen among inner (PelE) and outer membrane (PelB, PelC) transport associated loci. In these loci, there appears a consis- tent pattern of clustering across bacterial phyla suggesting co-evolution of potentially physically interacting components, however no evidence of interaction has been shown to date. Our genomic proximity network revealed two distinct clades in several Gram-positive spe- cies (Fig 5(v)). Of the synthase dependent EPS operons known to date, only PNAG production has been genetically and structurally characterized in Gram-positive Staphylococci [49]. Oper- ons reconstructed from initial HMM searches identified putative pel operons in several Gram- positive bacteria, comprised of the GT encoding PelF and the PelG putative transport protein (Fig 5). To determine whether these were bona-fide pel operons with additional loci, iterative HMM searches were performed including additional protein sequences from predicted pel operons. These searches revealed additional loci including a homolog of PelD (S3 Fig). C-di- GMP signaling in Gram-positive bacteria is less well characterized [50] and this finding sug- gests a role for this secondary metabolite in regulating biofilm formation in these species. In our companion paper, we have experimentally validated our predictions by showing that single gene deletions within the predicted B. PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries proteobacteria–in these taxa, BcsB appears highly divergent (as indicated by their identification through more sensitive HMM searches–grey nodes) and no BcsC was identified (confirmed through inspection of representative operons). Further interpretation of the operons identified in the box denoted with an asterisk ‘’, which represent HGT events, are illustrated in Fig 4. Node size indicates the relative number of sequences per phylogenetic cluster; node colouring represents the taxonomic distribution of loci for a given cluster; edges connect clusters which co-occur in the same genome(s); edge colour indicates the genomic-proximity of loci clusters. https://doi.org/10.1371/journal.pcbi.1007721.g003 https://doi.org/10.1371/journal.pcbi.1007721.g003 operons: Clades A1 and B2) and Enterobacter spp. (3 cellulose operons: Clades A1, B1 and B3) (Fig 4E). Additional sequence database searches revealed that the non-core loci associated with operon clades B2 and B3 share functionally homologous loci to the cellulose accessory protein D (AxcesD), which has been characterized as increasing the efficiency of cellulose pro- duction in the Acetobacter xylinus cellulose synthase complex [44]; GalU an uridine triphos- phate (UTP)-glucose-1-phosphate uridylyltransferase involved in cellulose precursor biosynthesis; and an additional uncharacterized locus predicted to possess both PAS_9 and GGDEF signaling domains, indicating the potential adaptation in Proteus and Enterobacter spp. to produce varied forms of cellulose upon different environmental stimuli [45]. Phylogenetic clustering and genomic proximity networks reveal evolutionary events driving EPS operon divergence is explained by a fusion between the inner membrane cellulose synthase complex subunits (BcsAB); (iii) Loss of outer membrane pore, BcsC, and divergence of the inner membrane cellulose co-polymerase, BcsB, in alpha- PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 11 / 32 PLOS COMPUTATIONAL BIOLOGY Genomic-proximity networks of pel operons reveal a novel pel locus in the gram positive bacterium, bacillus cereus that is regulated by c-di-GMP cereus pel operon result in a loss of EPS production, and that PelD regulates EPS production through binding of c-di-GMP (Whitfield et al PLoS PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 12 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries BcsC_G5-1 BcsB_G5-2 BcsC_G1-1 BcsB_G5-3 BcsB_G5-1 BcsZ_G19 BcsA_G1 BcsB_G1-1 BcsZ_G1-1 Non-Duplicated Operon Clades Duplication / HGT of Operon Clades Duplication / HGT of Operon Clades + Divergence Two HGT Events of Operon Clades + Divergence A1:”Cannonical” Cellulose Operon B1: Divergent bcsBZC + Operon Rearrangement of bcsZC A1 + B1 A2: Loss of bcsZ X Loss of bcsZ Locus bcsB Divergence Locus Gain bcsZ Rearrangement (A) B1 A1 B1 B1 + B2 A1 A1 A1 A2 + B2 B2: Divergent B1 + Locus Gain Between bcsC and bcsZ A2 B2 + A1 + B1 + B3 bcsB Divergence B1 + A1 B3 + B1 B3 (B) (C) (D) (E) B3: bcsB Divergence of B2 Operon bcsA bcsB bcsZ bcsC bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 Dickeya dadantii 3937 Klebsiella pneumoniae 342 Proteus mirabilis BB2000 Enterobacter sp. R4-368 Non-Duplicated Operon Clades A1:”Cannonical” Cellulose Operon B1: Divergent bcsBZC + Operon Rearrangement of bcsZC bcsZ Rearrangement B1 A1 B1 A1 bcsA bcsB bcsZ bcsC bcsA bcsB bcsC bcsZ Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 Dickeya dadantii 3937 Non-Duplicated Operon Clades Non-Duplicated Operon Clades A1:”Cannonical” Cellulose Operon B1 A1 BcsC_G5-1 BcsB_G5-2 BcsC_G1-1 BcsB_G5-3 BcsB_G5-1 BcsZ_G19 BcsA_G1 BcsB_G1-1 BcsZ_G1-1 (A) (B) (C) (A) (B) Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 A1 Dickeya dadantii 3937 bcsB Duplication / HGT of Operon Clades Duplication / HGT of Operon Clades + Divergence Two HGT Events of Operon Clades + Divergence A1:”Cannonical” Cellulose Operon B1: Divergent bcsBZC + Operon Rearrangement of bcsZC A1 + B1 A2: Loss of bcsZ X Loss of bcsZ Locus bcsB Divergence Locus Gain B1 B1 + B2 A1 A1 A2 + B2 B2: Divergent B1 + Locus Gain Between bcsC and bcsZ A2 B2 + A1 + B1 + B3 bcsB Divergence B1 + A1 B3 + B1 B3 (C) (D) (E) B3: bcsB Divergence of B2 Operon bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ Klebsiella pneumoniae 342 Proteus mirabilis BB2000 Enterobacter sp. Genomic-proximity networks of PNAG uncover locus loss and duplication events in pathogenic and environmental bacteria To examine how locus duplication, loss, and rearrangement events have contributed to the evolution of PNAG operons across bacterial phyla, selected examples of pga operon clusters were identified and compared (S4 Fig). For example, within a group of enterobacteria possess- ing related pgaD loci, there exist a number of closely related pathogenic enterobacteria that have lost pgaA (E. coli ETEC H10407), as well as pgaB (Shigella flexneri 5 str. 8401). These losses suggest these taxa may no longer be able to produce PNAG (S4(i.a) Fig). Interestingly, we also observed a lack of pga operons among pathogenic Salmonella spp. genomes surveyed in this study. Previous work has shown that loss of PNAG production is associated with adap- tation of Salmonella spp. to an intracellular pathogenic lifestyle [52]. Although PNAG produc- tion in E. coli H10407 has not been examined, our findings are also consistent with the adaptation of E. coli H10407 from a commensal to a pathogenic lifestyle [53], where enterotox- ins and colonization factors serve a crucial role for attachment to the intestinal epithelium and enhanced toxicity [54,55]. Furthermore, it has been previously shown that biofilm formation in S. flexneri impairs invasive ability and virulence [56], which suggests that the loss of PNAG production in S. flexneri [57] is the result of adaptation to an intracellular mode of infection. These results shed light on the relationship between biofilm production capability and adapta- tion of enteric bacteria toward a pathogenic lifestyle. Based on the divergence of pgaB loci, we also identified pga operon clades corresponding to partial and whole operon duplications in aquatic bacteria, including a partial duplication of the pga operon specific to the important pathogen Acinetobacter baumannii spp. and Methylo- vora versatilis 301, respectively (S4(ii) Fig). Also, in environmental bacteria we discovered a novel pga organization resulting from rearrangement of pgaC, and a lack of pgaB and pgaD loci, which may have been too divergent to detect from initial HMM searches (S4(iii) Fig). Although our HMM models only used Gram-negative pga operon protein sequences, we also identified a number of Gram-positive pga operons consisting of pgaB and pgaC (S4(i.b) and S4(i.c) Fig). Upon closer inspection these loci were found to correspond to Staphylococcus polysaccharide intercellular adhesion (PIA) loci icaB and icaA, respectively. This suggests a potential common evolutionary origin of synthase-dependent PNAG production between Gram-positive and -negative organisms. PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries Fig 4. Horizontal gene transfer of cellulose operons identified from analysis of the genomic-proximity network. Here we show how a subgraph (A) from the global cellulose EPS operon genomic-proximity network (Fig 3()), may be interpreted to reveal HGT events involving two distinct gamma proteobacterial operon clades, A (canonical bcsABZC) and B (bcsABC-Z). (B) Examples of operons in two species which possess either a single A1 (“canonical”) or B1 (rearrangement of bcsZC) operon clade. (C) Example from Klebsiella pneumoniae in which a single genome contains both A1 and B1 operons, indicating a HGT event. (D) Example from Proteus mirabilis featuring two copies (designated A2 and B2 respectively) of the cellulose EPS operon, which appear to be divergent forms of A1 and B1: A2 features an apparent loss of the bcsZ locus from A1; B2 features a locus gain between bcsC and bcsZ from B1. Example from Enterobacter spp. in which the genome carries three copies of the cellulose EPS operon. In addition to clade A1 and B1 operon arrangements, a further operon (designated B3) appears in which bcsB has diverged from a B2 clade operon. Arrows within the network schematics depict the order of loci within the operon and are coloured according to intergenic distance: red < 100bp; cyan >100bp & <5 kb; grey >5 kb. https://doi.org/10.1371/journal.pcbi.1007721.g004 Pathogens, in press). This work provides the first and crucial piece of evidence which suggests that divergent Gram-positive pel operons, particularly those belonging to the same phyloge- netic clade as B. cereus, likely possess the ability to produce a Pel-dependent biofilm. However, it is also possible that significantly divergent operon loci may not result in the production of EPS of identical composition to that characterized in Gram-negative bacteria, as in the case of PNAG modification between Gram-negative (pga) and Gram-positive (ica) operons [51]. Non-Duplicated Operon Clades R4-368 A1:”Cannonical” Cellulose Operon A1:”Cannonical” Cellulose Operon B1: Divergent bcsBZC + Operon Rearrangement of bcsZC p B1: Divergent bcsBZC + Operon Rearrangement of bcsZC p B1: Divergent bcsBZC + Operon Rearrangement of bcsZC B1: Divergent bcsBZC + Operon Rearrangement of bcsZC Duplication / HGT of Operon Clades A1 + B1 B1 + A1 A1 C) bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ Klebsiella pneumoniae 342 A1 + B1 A1 Duplication / HGT of Operon Clades Duplication / HGT of Operon Clades (C) bcsZ bcsC bcsB A2 L f b Z B2 A2 + B2 (D) Duplication / HGT of Operon Clades + Divergence A2: Loss of bcsZ X Loss of bcsZ Locus bcsB Divergence Locus Gain B2 A2 + B2 B2: Divergent B1 + Locus Gain Between bcsC and bcsZ A2 B2 + (D) bcsA bcsB bcsC bcsA bcsB bcsC bcsZ Proteus mirabilis BB2000 (D) Duplication / HGT of Operon Clades + Divergence Duplication / HGT of Operon Clades + Divergence Duplication / HGT of Operon Clades + Divergence X Loss of bcsZ Locus bcsB Divergence Locus Gain A2 B2 + bcsA bcsB bcsC bcsA bcsB bcsC bcsZ Proteus mirabilis BB2000 Proteus mirabilis BB2000 Loss of bcsZ Locus bcsC bcsB bcsC A2: Loss of bcsZ B2: Divergent B1 + Locus Gain Between (E) Two HGT Events of Operon Clades + Divergence A1 + B1 + B3 bcsB Divergence B1 + A1 B3 + B1 B3 (E) B3: bcsB Divergence of B2 Operon bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ Enterobacter sp. R4-368 A1 + B1 + B3 B1 B3 (E) Two HGT Events of Operon Clades + Divergence A1 + B1 + B3 bcsB Divergence B1 + A1 B3 + B1 B3 (E) B3: bcsB Divergence of B2 Operon bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ Enterobacter sp. R4-368 Two HGT Events of Operon Clades + Divergence Two HGT Events of Operon Clades + Divergence bcsB Divergence B1 + A1 B3 + bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ bcsA bcsB bcsC bcsZ Enterobacter sp. R4-368 Enterobacter sp. R4-368 A1 + B1 + B3 B3: bcsB Divergence of B2 Operon B3: bcsB Divergence of B2 Operon PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 13 / 32 PLOS COMPUTATIONAL BIOLOGY Genomic proximity networks of alginate uncover distinct operon clades in pseudomonas spp. and atypical operon architectures in environmental bacteria Although the majority of alginate operons were predicted largely among Pseudomonas spp. genomes (S5 Fig), phylogenetic clustering and genomic-proximity network reconstruction revealed an array of events influencing alginate operon evolution. For example, two distinct alginate operon clades were identified among Pseudomonas spp., defined by whole operon duplication and rearrangement of alginate polysaccharide modification loci (S5(i) and S5(ii) Fig). Also identified were divergent, “atypical”, alginate operons (S5(iii) Fig) comprising extensive rearrangements and also losses of functionally related subsets of alginate loci, e.g. outer membrane transport loci (algKE), and polysaccharide modification machinery (algGX- LIJF). Closer examination of the alginate genomic-proximity network also indicated a greater number of clusters for alg44 and algX loci, which were reflective of increased divergence among distinct alginate operon clades. Given that both loci play related roles in the regulation, polymer-modification, and assembly of the alginate EPS secretion machinery [60], these results provide an avenue for future research toward elucidating how species may modify algi- nate production to adapt to diverse environmental niches. PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries Fig 5. Genomic-proximity network of phylogenetically clustered pel operons. Phylogenetically clustered operon loci are arranged vertically with respect to the canonical ordering of the pel operon (indicated by grey side bar). As for Fig 4, inset boxes depict selected examples of pel operon clades, illustrating how the network can inform on evolutionary events: (i) Canonical organization of the pel operon, as defined in the Pseudomonas aeruginosa genome.; (ii) Duplication of the pel operon in Nitrosospira multiformis with subsequent evolution through locus gain and loss, as well as rearrangement of pelA; (iii) pelB fission, locus gain and rearrangement in aquatic thermophilic species; (iv) A potentially novel duplicated pel operon identified in Leptospirillum ferrooxidans comprised of divergent pelA and pelF loci; (v) pel operons identified in Gram-positive species including divergent pelD loci involved in regulation through c-di-GMP. Node size indicates the relative number of sequences per phylogenetic cluster; node border colouring represents the taxonomic distribution of loci for a given cluster; grey filled nodes indicate loci predicted by iterative HMM searches; edges connect clusters which co-occur in the same genome(s); edge colour indicates the genomic-proximity of loci clusters. https://doi.org/10.1371/journal.pcbi.1007721.g005 detectable pgaA locus, a possible role of these gene clusters in EPS production was investigated. One member of this operon clade, Thauera sp. MZ1T, inhabits a wide range of environments, and is an abundant producer of EPS responsible for viscous bulking in activated sludge waste- water treatment processes [58]. Furthermore, a recent mutagenesis study [59] demonstrated that biofilm-formation defective Thaurea mutants could be rescued by the complementation of the predicted pgaB deacetylase locus identified in the present study. Together, these findings suggest that the divergence of deacetylase and duplication of GT related loci in PNAG biosyn- thesis have resulted in the emergence of a distinct operon lineage. Genomic-proximity networks of PNAG uncover locus loss and duplication events in pathogenic and environmental bacteria A clade of pga operons were also identified possessing varying numbers of divergent pgaC loci resulting from repeated tandem duplication events (S4(v) Fig). Despite lacking a Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 14 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 15 / 32 Sequence variability of phylogenetic clusters reveals different degrees of structural conservation of cellulose biosynthesis machinery With the availability of a crystal structure for the BcsA-BcsB inner membrane complex respon- sible for cellulose biosynthesis and transport [61], we examined the potential structural and functional consequences of the sequence variability of the BcsA and BcsB phylogenetic clusters highlighted above (Fig 3). In brief, we generated multiple sequence alignments of eight BcsA and BcsB sequences summarizing the evolutionary diversity of cellulose operon clades identi- fied in Fig 3. Residue conservation information from this alignment were subsequently mapped onto the structure of the BcsA-BcsB complex (PDB ID:4HG6 [62]; S7 Fig). The results of the following analysis are also consistent when including all predicted BcsA and BcsB sequences. We identified a high degree of sequence conservation among BcsA sequences cor- responding to the GT domain responsible for cellulose polymerization. Conserved residues mapped specifically to a cleft in the GT domain where a uridine diphosphate (UDP) carrier moiety is bound and oriented through a conserved QxxRW motif to enable polymerization of glucose monomers of the growing cellulose chain [61]. Conversely, the PilZ domain of BcsA, involved in regulation of the GT function in response to c-di-GMP levels shows low conserva- tion overall, except for the subset of residues required for c-di-GMP binding. Further, the peri- plasmic region of BcsB shows low sequence conservation overall, aside from a number of highly conserved residues in the carbohydrate binding and ferredoxin domains. These residues include one (L193 of the Rhodobacter sphaeroides ATCC 17025 reference sequence) represent- ing a putative cellulose binding residue oriented in close proximity to the growing cellulose chain near the exit of the BcsA inner membrane translocation channel. From phylogenetic sequence clustering, structurally relevant conservation features of the cellulose synthase com- plex were identified which should facilitate further investigation of cellulose EPS production across phylogenetically diverse species. For example, c-di-GMP binding residues of the PilZ domain of BcsA vary in conservation across phylogenetic clusters, which could impact the ability of the protein to bind the nucleotide. This may in turn limit access of activated glucose monomers to the GT domain, thus altering the rate of cellulose polymerization. Insertion/ deletion events are also observed across BcsB phylogenetic clusters that may facilitate the recruitment of additional periplasmic processing proteins [63], or macromolecular assembly of the BcsA-BcsB complex [64]. This might result in differences in the higher-ordered structur- ing of cellulose microfibres as a consequence of adaptation to diverse environmental niches. Sequence variability of phylogenetic clusters reveals different degrees of structural conservation of cellulose biosynthesis machinery These results demonstrate how the application of our phylogenetic clustering methodology can be extended to provide biologically informative insights into the function of components of EPS secretion machineries. Genomic proximity networks of acetylated cellulose operons reveals duplication of co-polymerase subunits and sequence homology of loci with alginate acetylation machinery From the genome sequences surveyed, only four species were identified as possessing acety- lated cellulose operons, comprising two distinct operon clusters with differing operon consti- tutions among three Pseudomonas spp. and Bordetella avium 197N (S6 Fig). Contrary to cellulose phylogenetic clusters, the polysaccharide synthase, wssB, was divided into distinct Gamma- and Beta- proteobacterial clusters. We also found a distinct phylogenetic cluster iden- tifying a unique tandem duplication of wssC in Bordetella avium 197N, which was not observed among orthologous cellulose bcsB co-polymerase loci (S6(ii) Fig). This observation might suggest a divergent mechanism of action of cellulose inner membrane transport. As we previously observed (Fig 1E), 3 out of 4 of the predicted acetylated cellulose operons were also found to co-occur with alginate operons. Additional HMM-searches identified significant PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 16 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries sequence similarity between acetylated cellulose wssBCDE operon sequences to those previ- ously identified as bcsABZC, as well as between acetylated cellulose acetylation-machinery and their functional homologs in alginate operons (WssH–AlgI; WssI–AlgJ/AlgX). Taken together, these findings suggest that acetylated cellulose production has likely evolved through the dupli- cation and operonic acquisition of the alginate acetylation machinery loci. Phylogenetic clustering elucidates the structural and functional divergence of the pgaB locus, revealing the evolution of PNAG production across gram-negative and gram-positive bacteria Unique to pga operons is a locus encoding an outer membrane export pore, pgaA [72] and, in ica operons, an additional integral membrane protein, icaC, which has been pro- posed to be involved in PNAG O-succinylation [67]. Using Gram-negative pga loci as seed sequences for the reconstruction of synthase-dependent PNAG operons, we were also able to identify Gram-positive ica operons based on significant sequence similarities to pgaB and pgaC loci. Our phylogenetic clustering approach also revealed that pgaC/icaA sequences clus- tered into a single clade, while pgaB/icaB were associated with distinct sequence clades (S4 Fig). To explore the evolution of Gram-negative and Gram-positive pga and ica operons, we generated multiple sequence alignments for representative sequences of 18 PgaB clades. Our phylogenetic clustering results confirm previous observations [67] that the glycoside hydrolase domain is exclusively associated with Gram-negative pga operons (PgaB_G1) and is absent in a clade of Staphylococcus Gram-positive ica sequences (PgaB_G3; S8A Fig). We also identified additional Gram-positive icaB clades among non-Staphylococcus spp., e.g. Bacillus and Lacto- coccus (S1 Table), which possess operons lacking the icaC locus [67]. Interestingly, we also identified a number of divergent Gram-negative pgaB clades resembling icaB clade sequences. Members of these clades lacked the canonical N-terminal glycosyl hydrolase domain, and were distinguished by possessing N-terminal fusions, primarily of GT domains. Furthermore, these pgaB clades are associated with operons lacking detectable pgaA outer membrane pore locus and pgaD (S4(v) Fig). Although PNAG production in these species has not been experimen- tally confirmed, these findings suggest that if the polymer is produced, it may be regulated through a novel mechanism, that glycoside hydrolase activity might not be essential for PNAG export across all Gram-negative species, and that other modes of export may exist. The N-ter- minal fusion of GT with PgaB de-acetylase domains would also suggest that the de-acetylase activity of PgaB in these organisms may be associated with the periplasmic face of the inner membrane, in contrast to dual domain PgaB clades where the protein is predicted to function at the periplasmic face of the outer membrane [72]. In addition to these novel domain fusion events, PgaB phylogenetic clustering enabled us to l di i ff i h l i f h d l d i diff In addition to these novel domain fusion events, PgaB phylogenetic clustering enabled us to resolve distinct events affecting the evolution of the deacetylase domain across different operon clades. Phylogenetic clustering elucidates the structural and functional divergence of the pgaB locus, revealing the evolution of PNAG production across gram-negative and gram-positive bacteria We also identified additional Gram-positive icaB clades among non-Staphylococcus spp., e.g. Bacillus and Lacto- coccus (S1 Table), which possess operons lacking the icaC locus [67]. Interestingly, we also identified a number of divergent Gram-negative pgaB clades resembling icaB clade sequences. Members of these clades lacked the canonical N-terminal glycosyl hydrolase domain, and were distinguished by possessing N-terminal fusions, primarily of GT domains. Furthermore, these pgaB clades are associated with operons lacking detectable pgaA outer membrane pore locus and pgaD (S4(v) Fig). Although PNAG production in these species has not been experimen- tally confirmed, these findings suggest that if the polymer is produced, it may be regulated through a novel mechanism, that glycoside hydrolase activity might not be essential for PNAG export across all Gram-negative species, and that other modes of export may exist. The N-ter- minal fusion of GT with PgaB de-acetylase domains would also suggest that the de-acetylase activity of PgaB in these organisms may be associated with the periplasmic face of the inner membrane, in contrast to dual domain PgaB clades where the protein is predicted to function at the periplasmic face of the outer membrane [72]. pga and ica loci appear to be similar [66], there are important differences between the roles of pga and ica operon loci [67]. Common to both operons is the presence of an integral mem- brane GT locus, pgaC and icaA, which are both members of the GT-2 family and share sequence homology [67]. In addition, non-homologous loci encoding integral membrane pro- teins, pgaD and icaD, are also present and required for the full function of their respective GTs [66,68]. In Gram-negative bacteria, PNAG production is regulated through physical interac- tions between PgaD and PgaC which are stabilized by the allosteric binding of c-di-GMP [56]. In Staphylococci, PNAG production does not depend on c-di-GMP and is likely regulated by an alternate signaling pathway [69]. Deacetylation of PNAG is carried out by pgaB and icaB loci and has been shown to play a crucial role in biofilm formation and immune evasion [51,70]. pgaB also possesses an additional C-terminal glycoside hydrolase domain which cleaves the PNAG polymer following its partial deacetylation [71], although the mechanism of how these activities are coordinated and the biological role of the hydrolase activity is unknown. Phylogenetic clustering elucidates the structural and functional divergence of the pgaB locus, revealing the evolution of PNAG production across gram-negative and gram-positive bacteria PNAG production is found across phylogenetically diverse species and is carried out by the pgaABCD operon of Gram-negative bacteria [39] and icaADBC operon of Gram-positive bac- teria [65]. Although the functional and immunological properties of PNAG produced by the 17 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries pga and ica loci appear to be similar [66], there are important differences between the roles of pga and ica operon loci [67]. Common to both operons is the presence of an integral mem- brane GT locus, pgaC and icaA, which are both members of the GT-2 family and share sequence homology [67]. In addition, non-homologous loci encoding integral membrane pro- teins, pgaD and icaD, are also present and required for the full function of their respective GTs [66,68]. In Gram-negative bacteria, PNAG production is regulated through physical interac- tions between PgaD and PgaC which are stabilized by the allosteric binding of c-di-GMP [56]. In Staphylococci, PNAG production does not depend on c-di-GMP and is likely regulated by an alternate signaling pathway [69]. Deacetylation of PNAG is carried out by pgaB and icaB loci and has been shown to play a crucial role in biofilm formation and immune evasion [51,70]. pgaB also possesses an additional C-terminal glycoside hydrolase domain which cleaves the PNAG polymer following its partial deacetylation [71], although the mechanism of how these activities are coordinated and the biological role of the hydrolase activity is unknown. Unique to pga operons is a locus encoding an outer membrane export pore, pgaA [72] and, in ica operons, an additional integral membrane protein, icaC, which has been pro- posed to be involved in PNAG O-succinylation [67]. Using Gram-negative pga loci as seed sequences for the reconstruction of synthase-dependent PNAG operons, we were also able to identify Gram-positive ica operons based on significant sequence similarities to pgaB and pgaC loci. Our phylogenetic clustering approach also revealed that pgaC/icaA sequences clus- tered into a single clade, while pgaB/icaB were associated with distinct sequence clades (S4 Fig). To explore the evolution of Gram-negative and Gram-positive pga and ica operons, we generated multiple sequence alignments for representative sequences of 18 PgaB clades. Our phylogenetic clustering results confirm previous observations [67] that the glycoside hydrolase domain is exclusively associated with Gram-negative pga operons (PgaB_G1) and is absent in a clade of Staphylococcus Gram-positive ica sequences (PgaB_G3; S8A Fig). Phylogenetic clustering elucidates the structural and functional divergence of the pgaB locus, revealing the evolution of PNAG production across gram-negative and gram-positive bacteria Using the E. coli K12 MG1655 sequence of the largest PgaB clade (PgaB_G1) as a reference, multiple sequence alignments against other representative PgaB clade sequences identified several regions of insertion/deletion events (S8A Fig). When these regions were mapped to the published crystal structure of PgaB (PDB ID: 4F9D [73]), they were found to correspond to distinct structural elements surrounding the conserved deacetylase core (S8B and S8C Fig). We assigned insertion/deletion regions a number according to their order of appearance in the multiple sequence alignment of PgaB deacetylase domains, and divided them into two categories (S8D Fig). The first two indel regions, 1 and 2, resided in the N- PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 18 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries terminal region of the reference E. coli sequence, and corresponded to beta-strands flanking the conserved active site residues involved in deacetylation, His55, Asp114, and Asp115. Region 1 was associated with Gram-positive icaB and comprised insertions of ~10aa in Staphy- lococcus aureus VC40 (PgaB_G3), as well as Bacillus infantis NRL B-14911 (PgaB_G7), Lacto- bacillus plantarum 16 (PgaB_G9), Leptospirillum ferriphilum ML-04 (PgaB_11). Structural characterization of Ammonifex degensii IcaB (PgaB_G3) identified residues overlapping with Region 1 as encoding a hydrophobic loop responsible for membrane localization in this species [74]. Region 2 was found to be exclusive to Gram-negative pgaB loci and comprised a much larger insert of ~77aa in Geobacter metallireducens GS-15 (PgaB_G2), Crinalium epipsammum PCC 9333 (PgaB_G5), and Colwellia psychrerythraea 34H (PgaB_6). The functional role of this insertion is unknown. The last three insertion/deletion regions, 3–5, occurred in a region oriented away from the deacetylase active site, and correspond to two beta-turn motifs and an alpha-helix cap, respec- tively. To further elucidate the biological import of identified PgaB indel regions, we examined regions 3, and 5 in the context of Gram-negative PNAG modification. In the E. coli K12 MG1655 PgaB_G1 sequence, region 3 encompasses a beta-turn with an elongated loop, which is spatially proximal to a disordered loop and alpha helix (pos. 367–392) on the N-terminal region of the PgaB glycoside hydrolase domain. Region 3 also encodes a histidine (E. coli PgaB —H189) which is part of the nickel binding pocket of Gram-negative PgaB deacetylases. Both regions contain polar and electrostatically charged residues which are highly conserved across PgaB_G1 sequences (S8E Fig). Region 5 corresponds to an 8 amino acid elongation of an alpha-helix (pos. Phylogenetic clustering elucidates the structural and functional divergence of the pgaB locus, revealing the evolution of PNAG production across gram-negative and gram-positive bacteria 219–226), which also appears to provide an additional point of contact between the deacetylase and hydrolase domains. Although region 5 is also shared with icaB associated sequences (PgaB_G3), region 3 appears only in other dual deacetylase-hydrolase Gram-positive pgaB sequences identified in the sporulating bacteria Lachnoclostridium phyto- fermentans ISDg and Kitasatospora setae KM-5043. Although initial PFAM searches failed to identify the additional Gram-positive C-terminal domains, subsequent BLAST searches revealed them to be homologous to glycoside hydrolases. In region 4 a unique 29 amino acid insertion was also identified in Lachnoclostridium phytofermentans ISDg (PgaB_G16), which may play a compensatory role for the absence of 9aa in region 3. These insertion regions sug- gest an overall functional importance in ensuring stability between each domain and could play a role in coordinating their activities. These findings in combination with our identifica- tion of ica-like operon organizations among environmental Gram-negative species (S4(v) Fig) suggest that Gram-negative pga operons may share a common evolutionary origin with Gram- positive ica operons. Recent research is providing growing evidence for the emergence of the di-derm Gram-negative architecture from sporulating monodermal Gram-positives [75], which provides a plausible evolutionary context for the insertion/deletion events observed among pgaB/icaB deacetylase domains. Through the loss of inner membrane localization [74] (Region 1), the compensatory gain of an N-terminal palmitoylation site [66], along with a C- terminal fusion of a hydrolase domain (Regions 3–5), an ancestral deacetylase locus may have been adapted to regulate the export of PNAG [66] at the outer-membrane of Gram-negative pga operon lineage. The last three insertion/deletion regions, 3–5, occurred in a region oriented away from the deacetylase active site, and correspond to two beta-turn motifs and an alpha-helix cap, respec- tively. To further elucidate the biological import of identified PgaB indel regions, we examined regions 3, and 5 in the context of Gram-negative PNAG modification. In the E. coli K12 MG1655 PgaB_G1 sequence, region 3 encompasses a beta-turn with an elongated loop, which is spatially proximal to a disordered loop and alpha helix (pos. 367–392) on the N-terminal region of the PgaB glycoside hydrolase domain. Region 3 also encodes a histidine (E. coli PgaB ) h h f h k l b d k f d l h Discussion In this work we describe a novel and generalizable approach for the systematic classification and presentation of bacterial protein families in the context of their host operon. Protein fami- lies are defined as sets of homologs (groups of related sequences having a common evolution- ary ancestor) sharing a particular set of sequence motifs or structural domains that can be 19 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries utilized to determine their biological roles. For example, the PFAM database utilizes curated sets of protein family sequences in the generation of profile HMMs [76]. A key challenge that complicates the definition of these relationships are evolutionary events such as duplication, gene fusion, and HGT. In attempts to account for such events, a variety of computational approaches have been developed for refining functional assignments. These operate either by graphical clustering of pair-wise protein sequence similarities (e,g, COG [27], OrthoMCL [19] and EggNOG [20]), or through the generation of hierarchical evolutionary relationships and construction of phylogenetic trees (e.g. TreeFAM [77] and TreeCL [78]). However, these methods are limited in their ability to provide further resolution of sequence diversity within a family that might otherwise offer additional insights into evolutionary events that allow taxa to adapt to specific environments. Agnostic approaches to define sub-clusters of evolutionarily related protein families have ranged from phylogenetic tree reconstructions [79] to hierarchical clustering of pairwise global sequence alignments [80]. Here we present an extension of previous efforts and introduce a novel systematic approach for defining protein sub-family relationships through the clustering of phylogenetic trees. Key to this approach is defining a scoring function that allows a phyloge- netic tree to be resolved into optimal clusters that best capture the similarities between cluster members, as well as the dissimilarities between clusters. Combining two clustering quality met- rics (Silhouette and Dunn index) and proportion of sequences clustered, we demonstrate that our approach classifies a diverse array of operon-associated protein families into taxonomically consistent and functionally informative sub-clusters. Genomic-proximity networks were also constructed to provide an intuitive means of utilizing phylogenetic clusters to examine diverse mechanisms of operon evolution across taxonomically diverse bacterial genomes. Genomic- proximity networks have previously been utilized for inferring functional relationships [81], understanding mechanisms underlying bacterial genomic organization into functionally related gene clusters [82], and transcriptional regulation of bacterial operons [83]. Discussion In this study we extend the application of genomic-proximity networks as a tool for the systematic exploration of operon evolution resulting from locus divergence, loss, duplication, and rearrangement events. To demonstrate the effectiveness of our approach, we applied our methods to classify the synthase-dependent bacterial EPS operon machineries for 5 different polymers: cellulose, acety- lated cellulose, alginate, Pel and PNAG. There has been only one previous attempt to classify synthase-dependent EPS operons and this focused specifically on the cellulose system [36]. In that study, cellulose operons were categorized into four major types, based on the presence or absence of experimentally validated accessory loci involved in cellulose production. Here, we based our analysis on the four core operon loci, bcsABZC, deemed essential for cellulose pro- duction. Cellulose operon clades identified in this study showed little consistency with the previ- ously defined four major cellulose operon types [36], suggesting that the conservation of accessory loci is more variable across bacterial species compared to loci encoding core EPS functionalities. However, one operon type was identified in this analysis, representing the loss of the BcsC outer membrane transporter identified among a subset of alpha-proteobacterial genomes, which include several known cellulose producing species [62,84] suggesting a novel mechanism of cellulose export (Fig 3(iii)) [36]. We also found that the loss of BcsC has resulted in an increased divergence of BcsB loci in these genomes, which highlights the key role of BcsB as an intermediary between cellulose biogenesis and periplasmic transport (S7 Fig). In general, inner membrane components involved in EPS polymerization were found to be relatively conserved across all systems examined, while periplasmic and outer membrane com- ponents showed a relatively increased degree of evolution, which are likely to have important functional implications. For example, in the cellulose and Pel operon networks (Figs 3 and 5 and S5 Table), rearrangement events involving the periplasmic glycosyl hydrolase (BcsZ) and PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 20 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries glycosyl hydrolase/deacetylase (PelA) were found to be a defining feature of several operon clades. It is interesting to note that these rearrangements have resulted in a change in the ordering of bcsZ and pelA relative to their respective outer membrane transport pore loci, which highlights the important role of polysaccharide modification in both the biogenesis and regulation of extracelluar EPS transport [48,85,86]. Discussion Similarly, the rearrangement of alginate modification machinery loci (algIJF) was observed as a distinguishing feature of Pseudomonas spp. operon clades. These findings suggest that rearrangement and locus ordering may serve as an important means of regulating EPS production by modifying the timing of translation of modification enzymes, which could affect the assembly of EPS complexes or the structural properties of EPS produced [47,64,87]. Furthermore, identifying operon clades through a phylogenetic approach elucidated numerous instances of cellulose whole operon duplications arising from HGT of two evolution- ary distinct operon clades (Fig 4). Such large-scale duplications, if they are functional, may either serve as a dosage response to given environmental stressors, as observed in the duplica- tion of bacterial multiple-drug transporter operons [88], or could be under the regulation of dif- ferent environmental stimuli. Interestingly, representative species of the two cellulose operon lineages identified in HGT events, e.g. the plant and human pathogens, D. dadantii and S. enter- ica, respectively, are known to produce structurally distinct forms of cellulose with different properties and roles in pathogenesis [89,90]. In addition, we identified that BcsB divergence was also seen to accompany the rearrangement or horizontal transfer of these operons, which fur- ther suggests that it may play a key role in the fine-tuning of cellulose production by coordinat- ing the export of growing cellulose polymers through the periplasm. Furthermore, our analyses of acetylated cellulose, alginate and PNAG operons suggest a dynamic evolutionary scenario for the evolution of EPS biofilm production through the acquisition of novel polysaccharide modi- fication loci. The limited number of acetylated cellulose operons identified, their frequent co- occurrence in alginate possessing species, and significant sequence similarities between acetyla- tion machinery loci, suggests that the cellulose acetylation machinery is likely to have originated from previously existing alginate operons in Pseudomonas spp. The evolutionary trajectories of Gram-positive and Gram-negative PNAG operon lineages appears to have resulted through the fusion of glycosyl hydrolase and deacetylase domains in Gram-negative pgaB loci. A further key finding from this study was the identification of homologous pel operons in the genomes of several Gram-positive bacteria. With the additional identification of homologs of PelD through iterative HMM searches, our analyses have uncovered a novel example of c- di-GMP regulation of biofilm machinery in Gram-positive bacteria. In the accompanying paper we experimentally validate that a predicted pel-like operon in B. Discussion cereus ATCC 10987 is responsible for biofilm production and is regulated by the binding of c-di-GMP to PelD (Whitfield et al PLoS Pathogens, in press). Together this work demonstrates a novel integrative approach combining phylogenomics and genomic-context approaches to systematically explore the adaptive implications of sequence divergence of protein families associated with operon associated EPS secretion machineries. Further extension of this work holds great potential as a general approach for elu- cidating how bacterial operon encoded biological pathways and complexes have contributed to bacterial adaptation to and survival in diverse environmental niches and lifestyles. Prediction of EPS operons To identify putative EPS operons, we applied an iterative HMM-based sequence similarity pro- filing strategy. For each set of EPS loci, we first constructed a HMM from previously character- ized EPS producing bacteria (S3 Table); alignments were constructed using MUSCLE v.3.8.1551 [92], with default settings, from which HMM-models were built using HMMER v.3.1b2 [93], with default settings. As the number of characterized EPS producing species var- ies greatly by system, each HMM was then used to identify additional EPS loci within the set of 1733 bacterial genomes and build an expanded HMM model for downstream operon predic- tion. First a protein-BLAST search of reference sequence EPS protein coding sequences was performed against the set of reference+representative completely sequenced bacterial genomes downloaded from NCBI (e-value threshold of < = 1e-5). Next, these putative EPS sequences were subject to a second-round of all-vs-all protein-BLAST searches. These results were then processed using an in-house custom perl script to select non-redundant sequences (percent- age-identity of < 97%) in an incremental fashion: starting with the reference EPS sequence used in the first step, its highest scoring non-redundant match was selected and subsequently used to identify the next non-redundant sequence with the highest scoring match, and repeated until a pre-defined number of sequences were selected. To capture a consistent degree of locus sequence diversity and HMM sensitivity for each EPS system, we selected 20 sequences to represent each locus with the expectation that this would provide an adequate lower-bound on sampling potential amino acid substitutions for sites undergoing random mutation, i.e. not-under functional constraints. Using these new sets of HMMs, sets of EPS loci for the reconstruction of EPS operons (see below) were predicted through sequence simi- larity searches of the 1733 genomes using HMMER, with default settings. Significant sequence matches were defined as those with E-values < = 1e-5. HMM models as well as the table detail- ing lifestyles and environmental niches of bacteria used in this study (S8 Table) have been made available online (https://github.com/ParkinsonLab/eps_biofilms). To reconstruct putative EPS operons from the sets of loci retrieved from our searches, we first retrieved locus start and stop positions for each locus from their RefSeq entry. Sources of data Sequences corresponding to experimentally characterized EPS operon loci were obtained from the National Centre for Biotechnology Information (NCBI) reference sequence database [91] 21 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries (S3 Table). Fully sequenced genomes and associated protein sequences were obtained for 1758 bacteria from the NCBI (Retrieved April 20th 2015) (S7 Table). In this set of genomes the major phyla represented are largely Gram-negative Proteobacteria (47%), followed by Gram- positive Firmicutes (~20%) and Actinobacteria (~10%). For each bacterial strain predicted to possess an EPS operon, metadata corresponding to niche (host-associated or environmental) and lifestyle (pathogenic or non-pathogenic) were collated from literature searches (S8 Table– and is also made available for download from https://github.com/ParkinsonLab/eps_biofilms). Prediction of EPS operons We then define putative operons using the following two rules: first only loci that occur within a distance of twice the size of a reference EPS operon to other loci are considered; second intergenic distances of indi- vidual loci must be < = 5 kb; third putative operons must consist of at least one locus encoding a putative polysaccharide synthase, together with at least one other locus. To detect previously undiscovered loci that may have been missed in the first rounds of HMM searches, predicted loci of reconstructed operons were used to generate expanded locus-specific HMM models and were subjected to an additional round of HMM searches. This process was performed using custom Perl scripts and results in a list of predicted EPS operons identified in each of the 1733 genomes. Classification of EPS loci Systematic classification of each EPS operon family starts with first merging closely related sequences using CD-HIT v.4.6.3 [94] with default settings (using global sequence identity threshold 0.9; word length 5) to generate a non-redundant set of sequences for each family. Multiple sequence alignments (MSAs) were then generated using MUSCLE and trimmed using trimal v.1.2rev59 [95] (using -automated1 setting). The resulting alignment was then used to construct a consensus phylogenetic tree using PhyML v.3 [96], with default parameters (LG substitution model, with 1000 bootstrap replicates). For each consensus tree, pairwise evo- lutionary distances (defined as the number of expected average number of amino-acid substi- tutions per site) for all locus protein sequences were extracted using a custom perl script. These evolutionary distances were subsequently used to iteratively generate sets of clusters, with proteins sharing an evolutionary distance less than a defined cutoff (starting at 0 and incrementing by 0.01) placed in the same cluster. This results in the generation of increasingly coarse clusters of sequences with increasing sequence dissimilarity, such that in the final step all sequences are assigned to a single cluster. At this stage, for all possible clusterings three met- rics are calculated and summed together to calculate a clustering quality score: (1) proportion of sequences clustered (p) number of sequences clustered / total number of sequences); (2) the average silhouette score (s_avg) [97]: For each sequence, i, its silhouette score, s(i), is defined as: s ið Þ ¼ bðiÞ aðiÞ maxðaðiÞ; bðiÞÞ Where a(i) = average evolutionary distance (expected number of substitutions per site) i) is the lowest average evolutionary distance to any other cluster of which i is not a member; and (3) Dunn index (DI)[98], for a set of m clusters, its Dunn index, DI, is defined as: DI ¼ min1ijm dðCi; CjÞ max1km Dk DI ¼ min1ijm dðCi; CjÞ max1km Dk Where DI is the evolutionary distance between clusters i and j and Δc is the size of cluster c. Note that a higher s(i) indicates that a sequence is well matched to other members of its cluster and not well matched to neighbouring clusters. Furthermore, a higher DI indicates clusters that are compact (smaller cluster sizes) and well differentiated (larger inter-cluster distances). Thus, the evolutionary distance cutoff which maximizes p + s_avg + DI is chosen as the opti- mal phylogenetic clustering for a given set of EPS locus sequences. Classification of evolutionary events For each EPS system (cellulose, acetylated cellulose, PNAG, Pel, and alginate), the locus assign- ments of each reconstructed operon was compared to a defined reference EPS operon 22 / 32 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries compositions and locus ordering (S5 Table) and were classified into the following evolution- ary events; 1) locus losses—the total number of reference loci missing or not detected by HMM searches; 2) locus duplications–number of distinct loci appearing as multiple significant hits to the same HMM model < 10 kb apart; 3) locus fusions–the number of loci that were sig- nificant hits to two or more reference EPS locus HMM models; 4) operon rearrangements–the number of predicted operons with locus ordering (accounting for transcriptional direction) different from the reference operon; 5) operon duplications–number of predicted operons (as defined above) present in the same genome > = 10 kb apart. Classification of EPS loci In these analyses we have chosen not to incorporate bootstrap support parameters. Boot- strap values have previously been used in phylogenetic-based clustering approaches (particu- larly for epidemiological studies of pathogen transmission and evolution [99,100]) and provide an important metric in assessing the reliability of phylogenetic tree topologies [101]. In contrast, we define clusters in a topology-agnostic manner by clustering sequences based PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 23 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries only on pairwise evolutionary distances. This provides a readily automated approach for defin- ing evolutionary relationships, particularly for sets of sequences that exhibit diverse phyloge- netic distributions that are subject to varying evolutionary selection pressures and feature a variety of sequence lengths. Construction of EPS operon genomic-proximity networks To visualize evolutionary and genomic organization relationships of predicted EPS operons, genomic proximity networks were generated in which each node represents an individual EPS locus cluster (as defined above), and an edge connecting a pair of nodes represents the average genomic distance (base pairs) between loci represented by each node found in the same genome. Further, nodes are represented as pie-charts indicating phylogenetic distribution of each EPS locus, as defined by NCBI taxonomic classification scheme. Networks were visual- ized using Cytoscape (version 3.5) [102]. Supporting information S1 Fig. Lifestyle and niche distribution of predicted EPS operons. The number of bacterial genomes with different combinations of predicted EPS operons, further represented with their distribution (% bacterial genomes) across different lifestyles and environmental niches. (PDF) S2 Fig. Species diversity of predicted synthase dependent EPS systems (shannon diversity). (PDF) Inset boxes depict selected examples of alginate operon clades distinguished by evolutionary events: Inset boxes depict selected examples of alginate operon clades distinguished by evolutionary events: i) Canonical alginate operon organization with a partial operon duplication event identified in Pseudomonas resinovorans 136 resulting in the loss of alginate acetylation machinery (ib–indicated by A); ii) A distinct alginate operon clade (ii.a-c) identified by rearrangement of acetylation machinery (indicated by B) as well as HGT events with canonical alginate operon possessing species; iii) Atypical alginate operons involving loss of outer membrane transport loci or portions of acetylation machinery in deep sea dwelling bacteria. Node size indicates the relative number of sequences per phylogenetic cluster; node colouring represents the taxonomic distribution of loci for a given cluster; edges connect clusters which co-occur in the same genome(s); edge colour indicates the genomic- proximity of loci clusters. (PDF) S6 Fig. Genomic-proximity network of phylogenetically clustered acetylated cellulose operons. Phylogenetically clustered operon loci are arranged according to the canonical acety- lated cellulose operon ordering indicated by the grey sidebar. Inset panels identify three acety- lated cellulose operons identified in Pseudomonas spp. (i) and a single Bordetella avium genome possessing a duplicated polysaccharide co-polymerase wssC locus (ii—indicated by red asterisk). Node size indicates the relative number of sequences per phylogenetic cluster; node colouring represents the taxonomic distribution of loci for a given cluster; edges connect clusters which co-occur in the same genome(s); edge colour indicates the genomic-proximity of loci clusters. (PDF) S7 Fig. Phylogenetic sequence clustering reflect differences in structural conservation between cellulose synthase complex subunits BcsA and BcsB. Top panel—Sequence conser- vation was mapped onto the cellulose synthase complex, BcsA-BcsB (4HG6 –Rhodobacter sphaeroides ATCC 17025) comprising sequences from eight species representing distinct cellu- lose operon clades (Fig 4(i)–4(iv)). Lower panels—structural and multiple sequence align- ments indicate a high degree of conservation corresponding to BcsA glycosyl hydrolase catalytic core domain and regions of the cellulose translocation channel (i) and UDP binding sites of the BcsA PilZ domain (ii). In Contrast, low overall sequence conservation is found among the carbohydrate binding and ferredoxin domains (CBD1-2, and FD1-2) of BcsB sequences, except the highly conserved cellulose binding site residing in CBD-2 (iii). The translocated cellulose polymer is indicated in green. BcsA domains identified using PFAM predictions for the R. sphaeroides reference sequence, BcsB domains were assigned according to [45]. S2 Fig. Species diversity of predicted synthase dependent EPS systems (shannon diversity). (PDF) S3 Fig. Identification of gram-positive pel operons. (A) Subnetwork depicting Gram-posi- tive pel operon clades with varying numbers of loci identified as significant matches (e- value < 1e-5) in first-pass (unfilled nodes) and iterative HMM searches (grey nodes). Selected examples shown: (i) PelA-PelFG sequences identified by first-pass HMM hits; (i.b) Iterative HMM searches identifying additional pelA loci in B. cereus ATCC 10987, a known pellicle pro- ducing Gram-positive; (ii) Additional pelD loci identified by iterative HMM; (iii) Gram-posi- tive pel operons with only pelF and pelG loci identified. (B) Operon organizations of selected examples of Gram-positive pel operons (corresponding highlighted in panel A) with additional highly divergent loci identified (red boxes: hits above HMM e-value threshold of 1e-5). (PDF) S4 Fig. Genomic-proximity network of phylogenetically clustered pga operons. Phylogenet- ically clustered operon loci are arranged according to the canonical pga operon ordering indi- cated by the grey sidebar. Inset boxes depict selected examples of pga operon clades distinguished by evolutionary events: i) Divergence of pgaD corresponding to related entero- bacterial species including pathogen-specific losses of pgaA and pgaB loci critical for PNAG export; ii) Operon duplications occurring in aquatic niche dwelling bacteria, including a par- tial duplication of the pga operon specific to the opportunistic pathogen Acinetobacter bau- mannii spp. and a whole operon duplication identified in Methylovora versatilis; iii) A unique pga operon organization among environmental bacteria lacking a pgaD locus; iv) Gram-posi- tive ica operons (annotated by their HMM hits to corresponding Gram-negative pga loci) with divergent icaB loci, resulting from novel domain acquisitions (iv.b and iv.c); v) A novel pga derived operon resulting from multiple tandem duplications of the pgaC polysaccharide synthase and lack of detectable pgaA outer membrane pore and pgaD. Node size indicates the relative number of sequences per phylogenetic cluster; node colouring represents the taxo- nomic distribution of loci for a given cluster; edges connect clusters which co-occur in the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 24 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries same genome(s); edge colour indicates the genomic-proximity of loci clusters. (PDF) S5 Fig. Genomic-proximity network of phylogenetically clustered alginate operons. Phylo- genetically clustered operon loci are arranged according to the canonical alginate operon ordering indicated by the grey sidebar. S2 Fig. Species diversity of predicted synthase dependent EPS systems (shannon diversity). (PDF) Multiple sequence alignment was visualized generated using Geneious 10.2.2 (http:// www.geneious.com), protein structure was visualized using Chimera 1.11.2 [106]. (PDF) S8 Fig. Phylogenetic clustering reveals structural evolution of PNAG PgaB periplasmic modifying enzyme distinguishing gram-negative and gram-postive PNAG operon clades. A)—Multiple sequence alignment of representative sequences comprising all PgaB phyloge- netic clusters. Global sequence conservation compared against E. coli MG1655 K12 PgaB, phy- logenetic cluster PgaB_G1, indicates presence of polysaccharide deacetylase domain (blue box) PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007721 April 1, 2020 25 / 32 PLOS COMPUTATIONAL BIOLOGY Classification of bacterial biofilm machineries but an absence of glycosyl-hydrolase domain in non-PgaB_G1 sequences. Red arrows indicate phylogenetic group specific N-terminal domain fusions predicted by PFAM searches; C-termi- nal domain fusions identified (red box) as putative hydrolase domains from BLAST searches. B)—A close up view of sequence conservation of PgaB polysaccharide deacetylase domains with indel events highlighted: green boxes indicate insertions identified in non PgaB_G1 sequences; teal boxes indicate insertions in PgaB_G1 sequence residing in the C-terminal alpha-helix cap (yellow box). C–Crystal structure of E. coli PgaB (4F9D) indicating conserva- tion of the deacetylase domain catalytic core. D–Deacetylase domain with indel regions indi- cated according to the colour scheme described for panel B. E–C-terminal alpha helical cap region of the PgaB deacetylase domain indicating insertions of the PgaB_G1 region that are spatially proximal to an N-terminal region of the hydrolase domain (light purple); comparison of the same regions with PgaB_G1 sequence conservation indicated. Multiple sequence align- ment was visualized generated using Geneious 10.2.2 (http://www.geneious.com), protein structure was visualized using Chimera 1.11.2 [106]. (PDF) S2 Table. Phylogenetic sequence clade assignments of reconstructed synthase-dependent EPS operon loci. (XLSX) S2 Table. Phylogenetic sequence clade assignments of reconstructed synthase-dependent EPS operon loci. (XLSX) S1 Table. Reconstructed synthase-dependent EPS operons. (XLSX) S2 Table. Phylogenetic sequence clade assignments of reconstructed synthase-dependent EPS operon loci. (XLSX) S3 Table. EPS reference operon loci by species and RefSeq accession number. (XLSX) S4 Table. Previously experimentally characterized EPS producing species; successful iden- tification of a predicted EPS operon by the present study, and additional novel EPS operon possessing strains identified. (XLSX) S5 Table. Summary of synthase-dependent EPS operon evolutionary events. (XLSX) S6 Table. Synthase-dependent EPS operon locus clade associations. (XLSX) S7 Table. NCBI reference complete bacterial genomes and genbank accessions used for synthase-dependent EPS operon reconstruction. (XLSX) S8 Table. Associated lifestyle and environmental niche metadata for bacterial reference genomes. (XLSX) Author Contributions Conceptualization: P. Lynne Howell, John Parkinson. Data curation: Cedoljub Bundalovic-Torma, Gregory B. Whitfield, Lindsey S. Marmont. Formal analysis: Cedoljub Bundalovic-Torma, Gregory B. Whitfield, Lindsey S. Marmont. Funding acquisition: P. Lynne Howell, John Parkinson. Investigation: Cedoljub Bundalovic-Torma, Gregory B. Whitfield, Lindsey S. Marmont. / / References 1. Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D. Detecting protein function and protein-protein interactions from genome sequences. Science. 1999 Jul 30; 285(5428):751–3. https:// doi.org/10.1126/science.285.5428.751 PMID: 10427000 2. Mao X, Ma Q, Zhou C, Chen X, Zhang H, Yang J, et al. DOOR 2.0: presenting operons and their func- tions through dynamic and integrated views. Nucleic Acids Res. 2014 Jan; 42(D1):D654–9. 3. Ermolaeva MD, White O, Salzberg SL. 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https://openalex.org/W2805223788	https://europepmc.org/articles/pmc6037818?pdf=render	English	null	Genome Plasticity in Cultured Leishmania donovani: Comparison of Early and Late Passages	Frontiers in microbiology	2,018	cc-by	16,603	Correspondence: Sucheta Tripathy tsucheta@iicb.res.in; tsucheta@gmail.com Nahid Ali nali@iicb.res.in Correspondence: Sucheta Tripathy tsucheta@iicb.res.in; tsucheta@gmail.com Nahid Ali nali@iicb.res.in nali@iicb.res.in †Present address: Roma Sinha, Mater Medical Research Institute, Translational Research Institute, Woolloongabba, Brisbane, QLD, Australia Raghwan, Division of Bacteriology, National Institute of Cholera and Enteric Diseases, Kolkata, India Mohammad Shadab, Department of Dermatology, School of Medicine, The University of Alabama, Birmingham, AL, United States ‡These authors have contributed equally to this work. †Present address: Roma Sinha, Mater Medical Research Institute, Translational Research Institute, Woolloongabba, Brisbane, QLD, Australia Raghwan, Specialty section: This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology Keywords: Leishmania donovani, genomics, in vitro passage, promastigotes, genome plasticity INTRODUCTION Received: 19 March 2018 Accepted: 25 May 2018 Published: 03 July 2018 Leishmania donovani is an intracellular obligate parasite of mammalian macrophages and causes visceral leishmaniasis or kala-azar, which is a fatal disease if not treated on time. The disease severity varies from host to host, region to region as well as between parasite strains. Extensive research by diﬀerent groups exploiting genomic, proteomic, metabolomic, immunologic, and animal models have pointed toward tripartite determinants mediating the development of this disease viz. the vector, host and pathogen. Although vector and host characteristics are important for symptomatic disease, parasite characteristics majorly determine the fate of the disease (McCall et al., 2013). Promastigotes are the culturable form of Leishmania. Stationary phase culture of Leishmania is expected to contain a large number of metacyclic parasites and have been routinely used for experimental infections. Metacyclic promastigotes are injected into the host during blood Edited by: Renato A. Mortara, Federal University of São Paulo, Brazil Roma Sinha1†‡, Mathu Malar C2,3‡, Raghwan1†‡, Subhadeep Das2,3, Sonali Das1, Mohammad Shadab1†, Rukhsana Chowdhury1, Sucheta Tripathy2,3* and Nahid Ali1* Reviewed by: Nilmar Silvio Moretti, Federal University of São Paulo, Brazil Marcelo R. S. Briones, Federal University of São Paulo, Brazil Lucile Maria Floeter-Winter, Universidade de São Paulo, Brazil Reviewed by: Nilmar Silvio Moretti, Federal University of São Paulo, Brazil Marcelo R. S. Briones, Federal University of São Paulo, Brazil Lucile Maria Floeter-Winter, Universidade de São Paulo, Brazil 1 Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India, 2 Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India, 3 Academy of Scientiﬁc and Innovative Research (AcSIR), New Delhi, India Leishmania donovani possesses a complex heteroxenic life cycle where infective metacyclic promastigotes are pre-adapted to infect their host and cope up with intracellular stress. Exploiting the similarities between cultured and sandﬂy derived promastigotes, we used early and late passage cultured promastigotes to show speciﬁc changes at genome level which compromise pathogen ﬁtness reﬂected in gene expression and infection studies. The pathogen loses virulence mostly via transcriptional and translational regulations and long-time cultivation makes them struggle to convert to virulent metacyclics. At the genomic level very subtle plasticity was observed between the early and the late passages mostly in defense-related, nutrient acquisition and signal transduction genes. Chromosome Copy number variation is seen in the early and late passages involving several genes that may be playing a role in pathogenicity. Our study highlights the importance of ABC transporters and calpain like cysteine proteases in parasite virulence in cultured promastigotes. Interestingly, these proteins are emerging as important patho-adaptive factors in clinical isolates of Leishmania. We found that the currently available genome of Leishmania in the NCBI database are from late passages. Our early passage genome can act as a reference for future studies on virulent isolates of Leishmania. The annotated leads from this study can be used for virulence surveillance and therapeutic studies in the Indian subcontinent. ORIGINAL RESEARCH published: 03 July 2018 doi: 10.3389/fmicb.2018.01279 Keywords: Leishmania donovani, genomics, in vitro passage, promastigotes, genome plasticity Citation: This study also throws light on the pathologically signiﬁcant genes common to clinical and laboratory strains which must be considered for virulence surveillance at least in the Indian subcontinent. meal and are the infective stage of the parasite. We and others have observed that continuous axenic cultivation of Leishmania leads to loss of virulence over a period of time (Dey et al., 2002; Ali et al., 2013). The relative virulence of stationary phase promastigotes is proportional to peanut agglutinin negative promastigotes contained within these populations (da Silva and Sacks, 1987) which is attributable to exposed surface carbohydrates. It has also been observed that the in vitro maintenance of Leishmania promastigotes by cultivation over longer periods may reduce their ability to diﬀerentiate into amastigote forms (Moreira et al., 2012; Ali et al., 2013). Studies at the mRNA and proteomic levels indicated diﬀerential expression of virulence related genes in both promastigotes and amastigotes cultured axenically for long periods compared to freshly isolated/transformed or early passage parasites (Lei et al., 2010; Pescher et al., 2011; Ali et al., 2013; Magalhaes et al., 2014). These studies were done with the aim of standardizing protocols for the use of axenic Leishmania cultures in infection studies and to identify virulence factors. Studies at genomic level mostly identiﬁed parasite evolution in the Indian subcontinent under drug pressure or disease phenotype (Zhang et al., 2014; Imamura et al., 2016). As promastigotes are the infective form of Leishmania, and cultured stationary phase promastigotes show remarkable consistency with sandﬂy derived metacyclic promastigotes (Inbar et al., 2017), we chose the former system to unravel the genetic mechanism of parasite pre-adaptation to host environment and how it is lost with continuous in vitro passages. We report here the global changes in the genome and transcriptome of serially passaged L. donovani AG83 strain which cause speciﬁc alterations in few genes and their expression that lead to loss of virulence reﬂected in the infection studies. We also try to link these adaptive features with parasite’s nutritional environment and complex life cycle, and how closely they relate to the global evolution of L. donovani clinical isolates as available through published work. This study also throws light on the pathologically signiﬁcant genes common to clinical and laboratory strains which must be considered for virulence surveillance at least in the Indian subcontinent. FCS (Sigma-Aldrich) (Banerjee et al., 2008). Macrophage Infection in Vitro Macrophages isolated from the peritoneal cavity of hamsters (12–16 weeks old) by injecting chilled RPMI-1640-10% FCS were pooled and cultured overnight on glass cover slips as described elsewhere (Bhowmick et al., 2010). AG83 promastigotes of diﬀerent passages were allowed to infect peritoneal macrophages (MoI of 10:1) for 3 h, washed with warm 20 mM PBS, pH 7.2 and further incubated in fresh medium till 72 h. At 24 and 72 h post- infection, cells were ﬁxed in methanol and stained with Giemsa for microscopic determination of intracellular parasite numbers per 100 host cells. Data were analyzed using GraphPad Prism 5 software. Determination of Splenic and Hepatic Parasite Burden Hamsters (5–6 weeks) were infected by injecting 2 × 107 stationary phase promastigotes by intracardiac injection suspended in 20 mM PBS. Animals were sacriﬁced 8 weeks post-infection for determination of parasite burden by Leishman Donovan Units (LDU) in Giemsa stained impression smears and limiting dilution assay in ﬁvefold serial dilutions of homogenized organs as described previously (Banerjee et al., 2008). Data analysis was performed on GraphPad Prism 5 software. High Throughput Genome Sequencing High quality genomic DNA was extracted from stationary phase promastigotes of the early (HTI4) and late (HTI5) passage of L. donovani AG83 using a commercial procedure as recommended by the manufacturer (Qiagen, Germany). Size check, integrity and presence of contaminants in the DNA samples were assessed through gel electrophoresis. DNA purity was measured using a Nano Drop 2000 spectrophotometer (Thermo Scientiﬁc, Waltham, MA, United States). Two separate libraries of L. donovani AG83 were prepared one each for early and late passages. De novo paired end sequencing was done on Illumina HiSeq 2500 with the read length of 125 base pairs with an insert size of 250 bp. A total of 66 and 44 million raw reads were generated from early and late passages, respectively. Transcriptome Sequencing For each passage, parasites were harvested by chilling on ice, spun down, and washed once in cold PBS solution, pH 7.4, and suspended in RNA Later. Total RNA was isolated from promastigotes of early (2nd), intermediate (11th), and late (25th) passage (referred to as q1, q2, and q3, respectively, in the ﬁgures) by using “Roche High Pure RNA Isolation Kit,” (Product no.11828665001) according to manufacturer’s protocol. The purity and concentration of each RNA sample was checked by using the Agilent 2100 bioanalyzer (Agilent Technologies, CA, United States) before proceeding for further downstream analyses. Paired end transcriptome sequencing was carried out Citation: Parasites were sub- cultured till 25th passage for some experiments. Golden Syrian Hamsters, 5–6 weeks old, reared in institute facilities were used for the purpose of parasite maintenance. Ethics Statement All animal experiment protocols adhered to the guidelines of the Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA), Ministry of Environment and Forest, Government of India, and were approved by the Animal Ethics Committee (147/1999/CPSCEA) of CSIR-IICB. Citation: Sinha R, C MM, Raghwan, Das S, Das S, Shadab M, Chowdhury R, Tripathy S and Ali N (2018) Genome Plasticity in Cultured Leishmania donovani: Comparison of Early and Late Passages. F t Mi bi l 9 1279 July 2018 \| Volume 9 \| Article 1279 1 Frontiers in Microbiology \| www.frontiersin.org Gene Expression in Cultured Leishmania donovani Sinha et al. meal and are the infective stage of the parasite. We and others have observed that continuous axenic cultivation of Leishmania leads to loss of virulence over a period of time (Dey et al., 2002; Ali et al., 2013). The relative virulence of stationary phase promastigotes is proportional to peanut agglutinin negative promastigotes contained within these populations (da Silva and Sacks, 1987) which is attributable to exposed surface carbohydrates. It has also been observed that the in vitro maintenance of Leishmania promastigotes by cultivation over longer periods may reduce their ability to diﬀerentiate into amastigote forms (Moreira et al., 2012; Ali et al., 2013). Studies at the mRNA and proteomic levels indicated diﬀerential expression of virulence related genes in both promastigotes and amastigotes cultured axenically for long periods compared to freshly isolated/transformed or early passage parasites (Lei et al., 2010; Pescher et al., 2011; Ali et al., 2013; Magalhaes et al., 2014). These studies were done with the aim of standardizing protocols for the use of axenic Leishmania cultures in infection studies and to identify virulence factors. Studies at genomic level mostly identiﬁed parasite evolution in the Indian subcontinent under drug pressure or disease phenotype (Zhang et al., 2014; Imamura et al., 2016). As promastigotes are the infective form of Leishmania, and cultured stationary phase promastigotes show remarkable consistency with sandﬂy derived metacyclic promastigotes (Inbar et al., 2017), we chose the former system to unravel the genetic mechanism of parasite pre-adaptation to host environment and how it is lost with continuous in vitro passages. We report here the global changes in the genome and transcriptome of serially passaged L. donovani AG83 strain which cause speciﬁc alterations in few genes and their expression that lead to loss of virulence reﬂected in the infection studies. We also try to link these adaptive features with parasite’s nutritional environment and complex life cycle, and how closely they relate to the global evolution of L. donovani clinical isolates as available through published work. Parasite Culture and Maintenance in Animals Leishmania donovani (MHOM/IN/1983/AG83; ATCC repository number PRA R⃝-413TM) amastigotes from infected hamster spleen were transformed to promastigotes in Schneider’s Drosophila medium (Sigma-Aldrich) at 22◦C and sub-cultured as promastigotes in M 199 (Sigma-Aldrich) both supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 2 Gene Expression in Cultured Leishmania donovani Sinha et al. on Illumina HiSeq platform, with the read length of 125 base pairs with an insert size of 250 bp generating 37–42 million raw reads (approximately). The library has been sequenced following manufacturer’s instructions using the HiSeq SBS Kit v4 (Part # 15034097Rev.B) to generate paired-end reads. Additional quality control of raw data using FastQC (11) was performed. The reads were preprocessed using Trimmomatic and the poor quality sequences, contaminated sequences were removed using the blast and blat based searches using the reference (LdBPK82A1; Bioproject: PRJNA171503) from Genbank. The high quality ﬁltered reads were used for downstream analyses. Trapnell et al., 2012; Kim et al., 2013) by aligning the RNAseq reads of three passages with the reference genome of LdBPK282A1. The pattern of expression across samples was calculated by using the normalized Fragments per Kilo base of transcript per Million (FPKM) values. R scripts were used to construct box plots and perform hierarchical clustering (Supplementary Figure S1). Putative secretary functions were assigned to the DEGs by using Secretome tool (Bendtsen et al., 2004). Total RNA was isolated from 4 × 107 parasite promastigotes of diﬀerent passages, using TriZOL-chloroform method. Pure RNA pellet was suspended in 50 µl DEPC treated RNase free water and concentration was measured using Nanodrop 2000 (Thermo Fisher Scientiﬁc). Almost 2 µg pure RNA was used for cDNA synthesis following manufacturer’s protocol using Revert Aid First Strand cDNA synthesis kit (Thermo Fisher, MA, United States). Diﬀerential expression analysis of parasite genes was performed using SYBR green master mix (Roche). Ld GAPDH was used as reference gene. Reaction was run in Roche light cycler 96 (Roche) and fold change of diﬀerent genes was expressed as 2−11Ct, where 1Ct = gene Ct- reference gene Ct and 1(1Ct) = 1Ct test – 1Ct control. RNA was collected from n = 3 hamster and proceeded for further long passage preparation. Also data per group (A2, A11, and A25th passage) represents n = 3 experiments performed for each group. 1https://github.com/madhubioinfo/STLab-assembler Copy Number Variant Detection py The reads mapped by using BWA were used to estimate the read depth for each sample at each chromosome using the coverage values from Samtools v0.1.18 (Li et al., 2009). After removing the duplicates using Picard the CNVnator (Abyzov et al., 2011) was used to identify copy number variants. We used 100 bp as the window size for creating the histogram bins of read depth in CNVnator. For CNVnator, we removed calls which were less than the cutoﬀof t-test calculated e-value of 1e-5. Additionally, we used companion server (Steinbiss et al., 2016) for predicting orthologous genes from the two culture passages using Genbank Leishmania major as the reference. This server predicted many interesting features including the pseudogenes present in the genomes that were not predicted by Augustus. Genomes of two passages were compared using MUMmer, NUCmer, and PROmer (Kurtz et al., 2004). Cell Viability and Apoptotic Cell Death Assay Promastigotes of early (2nd) and late (25th) passages were treated with graduated doses of miltefosine for 24 h and the percent viability was determined by enzymatic reduction of 3-[4,5- dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan. In parallel experiments the miltefosine treated promastigotes were analyzed for apoptotic cell death by AnnexinV-FITC and propidium iodide staining acquired in FITC and PE channels, respectively, on FACS LSR Fortessa and Genome and Transcriptome Assembly and Functional Annotation The early and late passage genomes were assembled using Allpaths-LG assembler (Ribeiro et al., 2012). We simulated reads with 6 K and 20 K insert sizes from the existing genomes of LdBPK282A1 retrieved from Genbank (PRJNA171503) with WGsim (Li et al., 2008). The assembly from Allpaths was resolved up to chromosome level reference based genome assembly by nucleotide alignment using Nucmer and further using our in-house scripts1. RNAseq reads from all passages were aligned using blat (Kent, 2002) with the Genbank data of LdBPK282A1 for removal of contaminants. The cleaned reads were used for transcriptome assembly using Trinity transcriptome assembler (Grabherr et al., 2011; Haas et al., 2013) (Supplementary Table S1). We used BUSCO (Simão et al., 2015) for predicting the genome completeness and the core ortholog analysis was computed using eukaryotic genomes (Supplementary Table S2). SNP Identiﬁcation Sequenced illumina genomic reads of early and late passages were aligned with the reference genome of LdBPK282A1 using Burrows-Wheeler Aligner (Li and Durbin, 2009), After marking the duplicates using PICARD tools the variants were called using the Genome Analysis toolkit (GATK) (Van der Auwera et al., 2013), obtained variants were ﬁltered with the cutoﬀof QD < 2.0 \|\|MQ < 40\|\|FS > 60.0\|\| ReadPosRankSum < −8.0. We predicted protein coding genes of the assembled genomes using Augustus (Stanke et al., 2008) and Scipio pipeline with LdBPK282A1 coding sequences as training material (Bioproject: PRJNA171503). Predicted genes were annotated using BLAST (Altschul et al., 2009) as well as InterProScan (Quevillon et al., 2005) searches. Whole genome protein sequences were submitted to the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. GO ids were mapped back to their ontology functions using in house scripts. GO enrichment was analyzed in the categories of molecular function, cellular component and biological process. Metabolic pathways analysis was done using the KASS server (Moriya et al., 2007). Differential Expression Analysis of Transcripts We carried out diﬀerential expression analysis of transcripts using Tuxedo suit tools (Langmead et al., 2009; July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 3 Gene Expression in Cultured Leishmania donovani Sinha et al. protein spots within diﬀerent gels. In this study, we have applied a cut-oﬀof at least 1.5-fold up-regulation/down-regulation of protein for the study. Experimental molecular weights and isoelectric points were calibrated according to Bio-Rad standard 2-DE PAGE marker. Spots were manually excised and processed according to the In-Gel Tryptic Digestion Kit (Thermo Fisher, MA, United States). The trypsin digested samples were concentrated using speed-vac before loading the samples for MALDI-TOF Mass Spectrometry analysis on 4800 MALDI ToF/ToF analyzer (Applied Biosystems, CA, United States). Zip Tip protein concentrator (Millipore, MA, United States) was used for concentrating and de-salting of the low abundance proteins. The samples were dissolved in a solvent consisting of 0.1% triﬂuoroacetate and 50% acetonitrile in MilliQ Water. Then, 0.5 µl of sample solution was mixed with 0.5 µl of matrix solution (1 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in the aforementioned solvent), applied to a 384-MALDI sample target plate, and dried in air. Peptides were evaporated with a ND: YAG laser at 355 nm, using a delayed extraction approach. They were accelerated with 25 kV injection pulse for ToF analysis. Each spectrum was the cumulative average of 1000 laser shots. The MS/MS spectrum was collected in MS/MS 1 kV positive reﬂectron mode with fragments generated by post source decay. The MS/MS mass tolerance was set to ±20 ppm. After processing, 10 MS/MS precursors were selected (Minimum signal to noise ratio-50). Before each analysis, the instrument was calibrated with the Applied Biosystems 4700 Proteomics Analyzer Calibration Mixture. Data interpretation was performed using the GPS Explorer Software (Applied Biosystems, CA, United States), and an automated database search was performed using the MASCOT program (Matrix Science Ltd., London, United Kingdom). analyzed for percent FITC positive and FITC-PI double positive cells on FACS Diva software. Preparation of Leishmanial Antigen The promastigotes (2nd–5th and 22nd–25th passage) were washed three times in cold 20 mM PBS, pH 7.2 and re-suspended at a concentration of 1.0 g cell pellet in 50 ml of cold 5 mMTris– HCl buﬀer, pH 7.6 and Leishmanial antigen (Lag) was prepared by ultrasonication as described earlier (Bhowmick et al., 2010). LAg was precipitated in 90% acetone-10% TCA, solubilised in rehydration buﬀer (7 M urea, 2 M thiourea, 2% Nonidet P-40, 2% dithiothreitol, and 2% pharmalyte, all from GE Healthcare LifeSciences, Little Chalfont, United Kingdom except NP-40 from Sigma) overnight and estimated by Bradford method (Bradford, 1976). Two independent batches were prepared from each passage for analysis. Second Dimension-SDS-PAGE Before the second dimension, proteins in the IEF strips were reduced in equilibration buﬀer (6 M urea, 2% SDS, 300 mM Tris–HCl pH 8.8, 20% glycerol) containing 20 mg/ml DTT and alkylated in dark with 25 mg/ml iodoacetamide, again dissolved in equilibration buﬀer, for 15–20 min each. Strips were washed with SDS-PAGE running buﬀer and separated across 12% SDS-PAGE gels (30% acrylamide, 0.8% bis-acrylamide) using a vertical system (Bio-Rad) and standard Tris/glycine/SDS buﬀer. Gels were run at 30 mA/gel until the tracking dye left the gel. The protein standard was purchased from Bio-Rad (pre- stained SDS-PAGE broad range). For western blot analysis, total cell lysates of early and late passage promastigotes were run on SDS-PAGE followed by incubation with antibodies for gp63 and HDAC. The images were developed using Luminata Forte (Thermo Fisher, MA, United States) on Gel Doc XR (Bio-Rad). The anti-gp63 antibody raised in rabbit (in house) and anti- mouse HDAC antibody (Cell Signaling Technology) were used for the assay. RESULTS AND DISCUSSION Continuous in Vitro Passaging Leads to Loss of Virulence of Promastigotes Data Availability The Genome sequences of Early and Late passages of L. donovani MHOM/IN/1983/AG83 is available at NCBI with submission numbers GCA_001989955.1 and GCA_001989975.1, respectively. Isoelectric Focusing The isoelectric focusing (IEF) was performed using the PROTEAN IEF Cell system (Bio-Rad). 500–600 µg of whole cell protein was added to 300 µl of rehydration buﬀer for 11 cm IPG strip (pH 4–7 non-linear, Bio-Rad). Protein sample was applied to the IPG strips and kept at room temperature (16–18 h) for passive rehydration. IEF was performed at 250 V for 20 min; 10,000 V for 2 h 50 min; 10,000–40,000 V/h and an optional step of 500–1000 V 10–15 h. Continuous in Vitro Passaging Leads to Loss of Virulence of Promastigotes g We ﬁrst compared the infection causing capacity of promastigotes from diﬀerent passages in vitro and in vivo. Stationary promastigotes recovered from diﬀerent passages were quantiﬁed and employed in the experiments. Hamsters and hamster derived macrophages were highly susceptible to infection, with infection persisting up to 25th passage (Figures 1, 2) and displayed a decline in infectivity with more time spent in the culture. The average number of amastigotes per macrophage was 14.4 ± 3.2 at 24 h, which increased to 21.6 ± 3.9 at 72 h (Figure 1A) post-infection with early passage. There was over a 50-fold reduction in the number of intra-macrophagic amastigotes compared to the 2nd passage when parasites of 25th passage were used for infection (Figure 1, p < 0.0001, One way Anova). Parasites from the 2nd passage could infect Spot Handling and Tryptic Digestion for Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS/MS) Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS/MS) The 2-DE gels were stained with Coomassie Brilliant Blue G-250. Gel images were taken on the Gel Doc XR+ (Bio-Rad) and analysis was done using the PD Quest software version 8.0.1 (Bio-Rad Laboratories) and manual checking. The gel having a higher number of spots was used to locate the corresponding July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 4 Gene Expression in Cultured Leishmania donovani Sinha et al. FIGURE 1 \| In vitro hamster derived macrophage infection with different passages. Hamster macrophages were infected with stationary promastigotes of Leishmania donovani AG83, as described in Section “Materials and Methods” and the cells were incubated for 24 h and 72 h at 37◦C, 5% CO2. (A) The number of amastigotes in each passage and (B) the percentage of infected cells were analyzed by counting 100 cells. Data shown are mean ± SE of experiment performed in quadruplet. FIGURE 1 \| In vitro hamster derived macrophage infection with different passages. Hamster macrophages were infected with stationary promastigotes of Leishmania donovani AG83, as described in Section “Materials and Methods” and the cells were incubated for 24 h and 72 h at 37◦C, 5% CO2. (A) The number of amastigotes in each passage and (B) the percentage of infected cells were analyzed by counting 100 cells. Data shown are mean ± SE of experiment performed in quadruplet. FIGURE 1 \| In vitro hamster derived macrophage infection with different passages. Hamster macrophages were infected with stationary promastigotes of Leishmania donovani AG83, as described in Section “Materials and Methods” and the cells were incubated for 24 h and 72 h at 37◦C, 5% CO2. (A) The number of amastigotes in each passage and (B) the percentage of infected cells were analyzed by counting 100 cells. Data shown are mean ± SE of experiment performed in quadruplet. FIGURE 2 \| Infection in hamsters. Hamsters were infected by intracardiac injection of 2 × 107 L. donovani AG83 promastigotes. Animals were sacriﬁced at 8 week post-infection and (A,C) liver and (B,D) spleen parasite burden was determined by (A,B) LDU and (C,D) LDA.Data represent mean ± SE of two independent experiments (n = 4–6). (E) The mean weights of liver and spleen were compared with respective organ weights of uninfected animals. FIGURE 2 \| Infection in hamsters. Hamsters were infected by intracardiac injection of 2 × 107 L. donovani AG83 promastigotes. Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS/MS) Animals were sacriﬁced at 8 week post-infection and (A,C) liver and (B,D) spleen parasite burden was determined by (A,B) LDU and (C,D) LDA.Data represent mean ± SE of two independent experiments (n = 4–6). (E) The mean weights of liver and spleen were compared with respective organ weights of uninfected animals. July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 5 Gene Expression in Cultured Leishmania donovani Sinha et al. 96.83 ± 1.4% of macrophages at 72 h while only 8 ± 1.6% cells were infected with promastigotes from 25th passage (Figure 1B). From these experiments it seemed the infectivity is aﬀected at two stages, ﬁrst at the level of host-parasite interaction evident from lower infection at 24 h, and second, parasite sustenance inside macrophages, as seen in clearance of parasites after 72 h. Hamsters infected with 25th passage promastigotes presented over 6.5-fold less parasite burden in both liver (LDU of 515.6 ± 82.44) and spleen (LDU of 211.5 ± 42.21) as compared to those infected with the 2nd passage (LDU of 3358 ± 132.9 and 1416 ± 126.6, respectively, in liver and spleen) (Figures 2A,B, p < 0.0001, One Way Anova) at 8 week post-infection. As the number of live amastigotes inside the organs gives an idea of the disease severity, we additionally assessed the parasite burden in the infected liver and spleen of diﬀerentially infected hamsters by serial dilution method. The number of parasites showed a gradual decline with passage number thus reinstating the fact that continuous axenic culture of promastigotes leads to loss of virulence of the parasite. As shown in Figure 2C, the number of live amastigotes came down from 1016 in the liver of hamsters infected with virulent parasites (2nd passage) to as low as 100 parasites in the animals with attenuated infection (25th passage) (p < 0.0001, One Way Anova). The splenic infection level was very high in hamsters infected with early passage promastigotes, and reached levels as high as 1021 (Figure 2D, p < 0.0001, One Way Anova) whereas the parasites failed to survive and multiply in the animals infected with late passage, and only about 100 parasites could be detected in the piece of organ examined by us. The hamsters with mild infection also demonstrated reduced liver and spleen size (Figure 2E) unlike the severely infected groups. Chromosome Copy Number Variation Drives Gene Expression in Leishmania The previous studies on L. donovani (Leprohon et al., 2009; Dumetz et al., 2017) reported that aneuploidy and Copy number variants regulate the gene expression. In our study we identiﬁed SNPs from early and late passages. Total 4390 heterozygous loci were found in early passage and 4356 heterozygous loci were found in late passage of the genome. The local copy number variant detection was performed on the basis of read depth binning ratio approach. It was already known that deletion and duplication event plays an important role in these genomes to maintain the virulence in the diﬀerent environmental conditions (Dumetz et al., 2017). Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS/MS) Thus early passage promastigotes displayed increased ﬁtness inside host cells reﬂected in higher parasite burden while late passage promastigotes were successfully cleared by macrophages. in early and late passages, compared to 7967 protein coding genes in LdBPK282A1 (Table 1B). The less number of protein coding genes from our cultures are mostly attributed to the presence of assembly gaps. The numbers of predicted pseudogenes in early and late passages were 73 and 82, respectively, and this number is signiﬁcantly higher than 54 pseudogenes predicted in LdBPK282A1 strain, but similar to what is reported in the Srilankan L. donovani (70) strains (Zhang et al., 2014). Presence of large protein families such as calpain-like proteases and amastins are found to occur in large directional clusters in Genbank strain as well as our strains. We noticed a large amastin cluster in chromosome 8 in Genbank that are present in chromosome 10 in our strain (Table 2). The GO Biological Process annotation of early and late passages indicates there are no changes in the gene numbers of processes in early and late passages. However, genes responsible for defense system (GO: 0006952) and positive regulation of cell proliferation (GO: 0008284) are missing in later passages (Figure 3, lower panel and Supplementary Data Sheet S1). Thus repeated ex host passages have made the late passage parasites suited to extracellular life where selection pressure against host defense system is not needed. Further, the cellular machinery needed for transformation to infective form is also perturbed which may impair their ability to infect the host. Chromosome Copy Number Variation Drives Gene Expression in Leishmania The previous studies on L. donovani (Leprohon et al., 2009; Dumetz et al., 2017) reported that aneuploidy and Copy number variants regulate the gene expression. In our study we identiﬁed SNPs from early and late passages. Total 4390 heterozygous loci were found in early passage and 4356 heterozygous loci were found in late passage of the genome. Late Passage Genome Has Higher Number of Pseudogenes Assembly name Number of contigs Number of chromosomes Total assembly size (in bases) Largest chromosome (in bases) Smallest chromosome (in bases) Gaps (in bases) N50 and GC% HTI4 2382 36 32196393 2743999 284264 3663498 1058081, 59% HTI5 2445 36 32148377 2714535 283355 3653324 1058043, 58% LdBPK282A1 (GCA_000227135.2) 2152 36 32444968 2713248 283432 1192833 1024085, 59% TABLE 1B \| Gene prediction statistics for early (HTI4) and late (HTI5) passages using companion server. Genome name Gene Protein coding ncRNA Pseudo snRNA SnoRNA tRNA HTI-4 Early passage (2nd) 7656 7563 14 73 2 1 67 HTI-5 Late passage (25th) 7643 7552 15 82 2 1 66 LdBPK282A1 (GCA_000227135.2) 8079 7967 37 54 0 0 64 mbly statistics for early passage (HTI4), late passage (HTI5) and Genbank Leishmania donovani LdBPK282A1 genomes. TABLE 1A \| Summary of assembly statistics for early passage (HTI4), late passage (HTI5) and Genbank Leishmania donovani LdBPK282A1 genomes. Assembly name Number of contigs Number of chromosomes Total assembly size (in bases) Largest chromosome (in bases) Smallest chromosome (in bases) Gaps (in bases) N50 and GC% HTI4 2382 36 32196393 2743999 284264 3663498 1058081, 59% HTI5 2445 36 32148377 2714535 283355 3653324 1058043, 58% LdBPK282A1 (GCA_000227135.2) 2152 36 32444968 2713248 283432 1192833 1024085, 59% TABLE 1B \| Gene prediction statistics for early (HTI4) and late (HTI5) passages using companion server. Genome name Gene Protein coding ncRNA Pseudo snRNA SnoRNA tRNA HTI-4 Early passage (2nd) 7656 7563 14 73 2 1 67 HTI-5 Late passage (25th) 7643 7552 15 82 2 1 66 LdBPK282A1 (GCA_000227135.2) 8079 7967 37 54 0 0 64 FIGURE 3 \| Mummerplot comparisons and Gene Ontology analysis of early and late passage genomes to the NCBI reference genomes. (Upper) The y-axis represents HTI4 (left) or HTI5 (middle) whole genome assemblies compared to x-axis representingLdBPK282A1 assembly. The HTI4 (x-axis) versus HTI5 (y-axis) chromosomal assembly comparison is given in the right panel. The dots represent the positions of conserved DNA sequences on the genomes. (Lower) REVIGO was used to visualize the summary of signiﬁcantly enriched GO terms. The scatterplots show the cluster representatives in a two dimensional space derived by applying multidimensional scaling to a matrix of the GO terms’ semantic similarities for early (left panel) and late (right panel) passages. Clustal differences are represented in boxes. Late Passage Genome Has Higher Number of Pseudogenes Genome plasticity in Leishmania has been linked to its ability to adapt to diﬀerent environments (Sterkers et al., 2012). This prompted us to check for the genetic adaptations which may account for adaptability to growth in culture medium and the associated loss of infectivity as a result of continuous passaging. The genomes of early and late passages were assembled into complete 36 chromosomes with a genome size of 32.2 and 32.1 MB, respectively (Table 1A). The genomes of these two passages, however, had about 11.3% gaps compared to 3.67% gaps in LdBPK282A1 from Genbank [Accession number: GCA_000227135.2]. A chromosome wise comparison of early and late passages with LdBPK282A1 (Supplementary Figure S2) didn’t reveal much diﬀerence, whereas the whole genome comparisons clearly pointed out the small but prominent changes in the genome of the late passage, which was comparable to the NCBI reference LdBPK282A1 strain (Figure 3, upper panel). We used RATT and companion for transferring annotation from LdBPK282A1 strain (Downing et al., 2011) that resulted in 7563 and 7552 predicted protein coding genes, respectively, The copy Number variation on our passages was done using LdBPK282A1 as reference, with an e-value cut-oﬀof 1e−5 for ﬁltering. Three hundred and eighty CNV events were qualiﬁed in late passage and 365 CNV events were qualiﬁed in early passage (Supplementary Data Sheet S2). The size of CNV events ranged from 0.7 to 271 kb. We checked for the regions which were overlapping with the protein coding genes. A total of 230 deletion events and 135 duplication events were identiﬁed in the early passage while 234 deletions events and 146 duplication events were identiﬁed in the late passage genome (Supplementary Data Sheet S2). Uniq gene lists that had undergone changes are listed in Supplementary Data Sheet S2: uniq_CNV. The genes undergoing structural changes in later passages compared to earlier passages comprised of several ABC Transporters (10), Amino acid transporters (7), Amastins (3), GP63, calpain like cysteine proteases to name a few. Interestingly, we have also reported changes in gene July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 6 Gene Expression in Cultured Leishmania donovani Sinha et al. TABLE 1A \| Summary of assembly statistics for early passage (HTI4), late passage (HTI5) and Genbank Leishmania donovani LdBPK282A1 genomes. Late Passage Genome Has Higher Number of Pseudogenes Bubble color indicates the user-provided p-value; size indicates the frequency of the GO term in the underlying GOA database (bubbles of more general terms are larger). TABLE 1B \| Gene prediction statistics for early (HTI4) and late (HTI5) passages using companion server. FIGURE 3 \| Mummerplot comparisons and Gene Ontology analysis of early and late passage genomes to the NCBI reference genomes. (Upper) The y-axis represents HTI4 (left) or HTI5 (middle) whole genome assemblies compared to x-axis representingLdBPK282A1 assembly. The HTI4 (x-axis) versus HTI5 (y-axis) chromosomal assembly comparison is given in the right panel. The dots represent the positions of conserved DNA sequences on the genomes. (Lower) REVIGO was used to visualize the summary of signiﬁcantly enriched GO terms. The scatterplots show the cluster representatives in a two dimensional space derived by applying multidimensional scaling to a matrix of the GO terms’ semantic similarities for early (left panel) and late (right panel) passages. Clustal differences are represented in boxes. Bubble color indicates the user-provided p-value; size indicates the frequency of the GO term in the underlying GOA database (bubbles of more general terms are larger). July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org Gene Expression in Cultured Leishmania donovani Sinha et al. TABLE 2 \| Gene Copy number in early, late passages and Genbank strain LdBPK282A1. Late Passage Genome Has Higher Number of Pseudogenes This gene was reported to be over expressed in Srilankan L. donovani strains causing visceral disease versus those causing cutaneous lesions (Zhang et al., 2014). Noticeable protein coding genes falling in the variant regions on chromosome 13 of late passage genome were some acetyl transferases including histone acetyltrasferase, N-acetyl transferase subunit ARD1, RAS-related protein RAB5, mitogen activated protein kinase 2, etc. which may have a role in cell cycle expression proﬁling of GP63 and Calpain like cysteine proteases between the late and early passages. We found more CNV events in chromosomes 5, 6, 8, 15, and 31 in the genome of early passage as per earlier reports (Laﬃtte et al., 2016; Iantorno et al., 2017). High number of CNV events play a major part in the gene expression regulation of in vitro cultured promastigotes (Dumetz et al., 2017). Interestingly we observed an increased frequency of CNVs in the protein coding regions of the late passage particularly in chromosome 3, 4, 13, 16, and 20. Protein coding regions corresponding to calpain like cysteine protease genes on chromosome 20 displayed multiple duplication events. Phosphoglycerate kinase B, cytosolic fragment on chromosome 20 showed an insertion event in the late passage which was not found in the earlier passage. This gene was reported to be over expressed in Srilankan L. donovani strains causing visceral disease versus those causing cutaneous lesions (Zhang et al., 2014). Noticeable protein coding genes falling in the variant regions on chromosome 13 of late passage genome were some acetyl transferases including histone acetyltrasferase, N-acetyl transferase subunit ARD1, RAS-related protein RAB5, mitogen activated protein kinase 2, etc. which may have a role in cell cycle Late Passage Genome Has Higher Number of Pseudogenes Gene product Family size Distribution on chromosomes HTI4 HTI5 LdBPK282A1 HTI4 HTI5 LDBPK282A1 Kinesins 51 50 49 Scattered Scattered∗ Scattered∗ Protein kinases 259 258 255 Scattered Scattered∗ Scattered∗ MAP kinases 17 17 19 Scattered∗ Scattered∗ Scattered Amastins 14 15 26 10,24,28,30,34,36 10,24,28,30,34,36 8,24,28,29,30,34 PSA2 (GP46) metalloproteases 28 29 29 Scattered∗ Scattered Scattered Serine peptidases 17 18 13 Scattered Scattered Scattered Protein phosphatase 120 118 86 Scattered Scattered Scattered Tuzins 4 4 6 8, 29∗,34 8, 29∗,34 8,29,34 Amino acid permeases 15 15 18 Scattered∗ Scattered∗ Scattered HSP 11 12 10 Scattered∗ Scattered∗ Scattered Calpain-like cysteine peptidase 29 31 26 4,17,18,20(7),21,25, 27(5),31(6),32,33,36 4,17,18,20(8),21,25, 27(4),31(7),32,33,34, 36 4,14,18,20(8),21, 25,27,30,31(5),32,33,34 Phosphoglycan β 1,3 galactosyltransferases 3 4 10 2∗,14,31∗,36∗# 2∗,14,31∗,36∗# 2,14,31,36 Dynein heavy and light chain 44 44 44 Scattered Scattered Scattered Helicases 84 84 72 Scattered Scattered Scattered Pteridine transporters 1 1 2 6,10∗# 6,10∗# 6,10 Microtubule-associated proteins 72 72 72 Scattered Scattered Scattered ABC transporters 39 42 39 Scattered∗ Scattered Scattered∗ Vesicle transporters 4 4 4 11,23,31,32 11.23,31,32 11,23,31,32 DNAJ protein/chaperone 61 61 29∗∗ Scattered Scattered Scattered∗∗ Long-chain fatty acid CoA ligases 9 9 9 1,3,13,19∗,28,36∗# 1,3,13,19∗,28,36∗# 1,3∗∗,13,19∗,28,36∗# Cyclophilins 1 15 13∗∗ 1,6,16,18,22,23,24,25, 30,31,33,35,36 1,6,16,18,22,23,24,25, 30,31,33,35,36 1,6,16,22,23,25,30,31, 33,35,36 Histone acetyl transferase/histone deacetylase 8 8 7∗∗ 8,14,16,21,24,26,28 8,14,16,21,24,26,28 8,14,16,21,24,28 Nucleoside hydrolase 4 4 3∗∗ 14,18,26,29 14,18,26,29 14,18,29 ∗One or more genes not present due to assembly gaps. ∗∗Gene prediction error. #Gene absent in the scaffold due to assembly gaps. TABLE 2 \| Gene Copy number in early, late passages and Genbank strain LdBPK282A1. progression and morphogenesis of Leishmania (Wiese, 1998; Yadav et al., 2016). expression proﬁling of GP63 and Calpain like cysteine proteases between the late and early passages. We found more CNV events in chromosomes 5, 6, 8, 15, and 31 in the genome of early passage as per earlier reports (Laﬃtte et al., 2016; Iantorno et al., 2017). High number of CNV events play a major part in the gene expression regulation of in vitro cultured promastigotes (Dumetz et al., 2017). Interestingly we observed an increased frequency of CNVs in the protein coding regions of the late passage particularly in chromosome 3, 4, 13, 16, and 20. Protein coding regions corresponding to calpain like cysteine protease genes on chromosome 20 displayed multiple duplication events. Phosphoglycerate kinase B, cytosolic fragment on chromosome 20 showed an insertion event in the late passage which was not found in the earlier passage. Frontiers in Microbiology \| www.frontiersin.org Single Nucleotide Polymorphisms in the ABC Transporter Coding Genes The evolution of pathogenicity in microbes has been attributed to ordered changes in the functionality of the genes as a result of physiological constraints encountered by the organism in their immediate environment. A KEGG pathway analysis didn’t show major diﬀerences in the number of genes involved (Supplementary Data Sheet S3) although detailed mutational analysis revealed interesting changes linked directly or indirectly to loss of virulence in the later passage. Among the defense gene products, ABC transporters are important proteins involved in drug resistance, nutrient acquisition and pathogen virulence (Glavinas et al., 2004) and have expanded in the pathogenic protists as a parasitic adaptation (Zhang et al., 2014; Jackson et al., 2016). Genomes of L. donovani AG83 in early and late passages contain 42 copies of the genes spread over the genome. However, there are at least two instances where the ABC transporters are July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 8 Gene Expression in Cultured Leishmania donovani Sinha et al. in the early passage promastigotes (HTI4) indicate enhanced intracellular survival strategy. undergoing polymorphism that could result in reduced pathogen ﬁtness. ABC transporter at LDON_230007700.1; HTI5:chr23: 91219–96174 in late passage has undergone several changes at the nucleotide level leading to an inactive ABC transporter in the late passages. Our Copy Number variant analysis also detected CNV duplication event of 12000 bp in the region of 86801–98800 in chromosome 23 in late passage. In earlier passages, a functional ABC transporter [HTI4:Chr23:91908–92025] resided inside a larger gene locus at HTI4:chr23:89807–101617 (Figure 4). We found a CNV duplication event as well in chromosome 23: 86801–98700 in early passage that corroborates this ﬁnding. Calpain like proteases play a very important role in infection process. A single insertion in 172519th position in HTI5 on chromosome 27 (Chr27: 172458–188744) leads to frameshift mutation causing this gene to become a pseudogene. The corresponding functional gene in early passage is present at Chr27: 172465–188750 (Figure 8). In case of Genbank L. donovani genome, the protein sequence is more identical to the HTI5 protein sequence. Recent reports have pointed toward the regulatory role played by expressed pseudogenes in cancer cells and parasites (Wen et al., 2011). To check the downstream impact of mutations in ABC transporters and CALPs, we checked the relative sensitivity of early and late passage promastigotes to the drug miltefosine. Single Nucleotide Polymorphisms in the ABC Transporter Coding Genes Interestingly, these transporter proteins have links to the MFS superfamily (Huynh et al., 2006) and insight into genetic structure, mechanism and regulation via domain duplication in this class of proteins appears intriguing in designing targeted therapies. Single Nucleotide Polymorphisms in the ABC Transporter Coding Genes Early passage parasites displayed an IC50 of 30 µM whereas the late passage promastigotes showed increased apoptotic death under drug pressure and an IC50 of 18 µM (Supplementary Figure S3). Interestingly, parasite CALPs may serve other functions in the intracellular form which determine disease outcome and host responses (Branquinha et al., 2013) and are thus potent drug targets. This may open up new avenues in understanding Leishmania biology. The implications of these modiﬁcations need further investigation. Interestingly, there is another ABC transporter gene in chromosome 31 with gene id LDON_310017500.1 in HTI4 and gene id LDON_310017000.1 in HTI5 where a substitution at 5012th position (GGG ->GCA a non-synonymous substitution which leads to a change from G->A amino acid at 1671st position in HTI5 (Figure 5A). The same gene undergoes two substitutions at the 5010th position (from AGC ->AGT) (Figure 5B) leading to synonymous substitution. The Genbank L. donovani strain LdBPK282A1 gene corresponding to this gene has the ‘A’ variant in this locus (Figure 5C). This observation is also supported by our CNV analysis in chromosome 31 as well as by Imamura et al. (2016). This gene codes for pentamidine resistance transporter protein. Major facilitator Protein (MFS class) is responsible for solute transport via membrane. Recently their role in stress responses and virulence has been proposed by various groups (Shah et al., 2014; Zhang et al., 2014). One such gene in HTI4 has been pseudogenized and this gene is absent in HTI5 (assembly gaps can’t be ruled out as one of the plausible causes though). A small segment of the gene (297–360 bp) is copied at two places both in HTI5 and HTI4 with one point non-synonymous mutation (AAC->AGC) (gene id: LDON_290021200 in HTI4) resulting in N ->S (Figure 6). The presence of this duplicated domain and its role in gene regulation and pathogenicity may be intriguing. Functional members of this family is present in duplicates in HTI5 as well as HTI4 (HTI5: Chr29: 711070– 711429; HTI5: Chr29: 710625–710970 and HTI4: chr29: 711414– 711772; HTI4:chr29: 710661–711007). This indicates selection pressure is working on MFS classes of proteins. Studies on pathogenic organisms demonstrate that the acquisition of iron is very important in the intracellular pathogenesis process and Leishmania possesses molecular machinery for iron regulation (Huynh and Andrews, 2008). Frontiers in Microbiology \| www.frontiersin.org Comparative Transcriptome Proﬁles Between Passages Reveal Calpain Proteins Play an Important Role in Maintaining Virulence g The information from genomic studies done in this work as well as previous studies on axenic promastigotes indicated a strong link between parasite pre-adaptation and virulence. We hypothesized that continuous axenic cultivation may lead to altered transcript expression. To check for speciﬁc changes we analyzed the diﬀerential levels of transcript expression in three diﬀerent culture stages (early, intermediate, and late passages) using RNAseq. Overall, there was no massive transcript expression switch between the early and late passages (Figure 9 and Supplementary Figure S1) although speciﬁc genes presented diﬀerential expression (Supplementary Data Sheet S4) which correlated to certain extent with the CNV. The relative gene expression of a few genes was validated by RT-PCR (Supplementary Figure S4). Among the genes coding for surface active proteins, there was down regulation in membrane bound acid phosphatase and surface antigen like protein transcript levels (Supplementary Data Sheet S4) in the later passages. Acid phosphatase activity is needed for virulence and is also involved in endosome sorting (Katakura and Kobayashi, 1988). This may also be an adaptive response to the hydrolytic environment encountered inside sandﬂy gut and mammalian cells (Papadaki et al., 2015). Cyclin-dependent kinase pho85- like protein is a morphogenesis related protein having strong link to virulence in Ustilago (Castillo-Lluva et al., 2007) although its function in Leishmania has not been elucidated to date; it is implicated in environmental signaling in yeast (Carroll and O’Shea, 2002). This gene was under-expressed in the later passages. Among the cytoskeletal and ﬂagellar proteins, a A fully functional acetyl-CoA synthatase gene in HTI4 has undergone modiﬁcation [chr23: 199275–199598 in HTI5 and chr23: 198391–19851 in HTI4] leading to loss of function (Figure 7). There is also domain duplication in HTI4: LDON_230010500.1 from 1 to 118th position. The stringency in nutrient acquisition is key to preparedness of the parasite to intracellular environment (Carman et al., 2008) and loss of function of acetyl-CoA synthetase may restrict the parasite’s capacity to thrive in nutrient poor conditions using alternative carbon sources (McConville et al., 2015). Domain duplication July 2018 \| Volume 9 \| Article 1279 9 Sinha et al. Gene Expression in Cultured Leishmania donovani FIGURE 4 \| Nucleotide and protein sequence comparison in L. donovani ABC transporter gene in Chr 23 of early and late passages. (Upper) Shows multiple nucleotide polymorphisms in HTI4 and HTI5 including indels and nucleotide substitutions. Comparative Transcriptome Proﬁles Between Passages Reveal Calpain Proteins Play an Important Role in Maintaining Virulence (Lower) Describes the protein sequence polymorphism as a result of sequence changes. The differences leading to functional loss are marked as boxes. FIGURE 5 \| Synonymous and non-synonymous substitution in early and late passages leading to altered ABC transporter genes in chromosome 31. (A) A pair wise comparison between the protein coding genes in HTI4 and HTI5 showing single amino acid mismatches. (B) Nucleotide comparison in two genes indicates few substitutions, out of which the ﬁrst one is a synonymous substitution and the second one is a non-synonymous substitution. A comparison between HTI4, HTI5 and Genbank strain (C) clearly indicates change of G–A in late passage same as the reference genome. FIGURE 4 \| Nucleotide and protein sequence comparison in L donovani ABC transporter gene in Chr 23 of early and late passages (Upper) Shows multiple FIGURE 4 \| Nucleotide and protein sequence comparison in L. donovani ABC transporter gene in Chr 23 of early and late passages. (Upper) Shows multiple nucleotide polymorphisms in HTI4 and HTI5 including indels and nucleotide substitutions. (Lower) Describes the protein sequence polymorphism as a result of sequence changes. The differences leading to functional loss are marked as boxes. protein sequence comparison in L. donovani ABC transporter gene in Chr 23 of early and late passages. (Upper) Shows multiple HTI4 and HTI5 including indels and nucleotide substitutions. (Lower) Describes the protein sequence polymorphism as a result o rences leading to functional loss are marked as boxes. FIGURE 5 \| Synonymous and non-synonymous substitution in early and late passages leading to altered ABC transporter genes in chromosome 31. (A) A pair wise comparison between the protein coding genes in HTI4 and HTI5 showing single amino acid mismatches. (B) Nucleotide comparison in two genes indicates few substitutions, out of which the ﬁrst one is a synonymous substitution and the second one is a non-synonymous substitution. A comparison between HTI4, HTI5 and Genbank strain (C) clearly indicates change of G–A in late passage same as the reference genome. FIGURE 5 \| Synonymous and non-synonymous substitution in early and late passages leading to altered ABC transporter genes in chromosome 31. (A) A pair wise comparison between the protein coding genes in HTI4 and HTI5 showing single amino acid mismatches. (B) Nucleotide comparison in two genes indicates few substitutions, out of which the ﬁrst one is a synonymous substitution and the second one is a non-synonymous substitution. Comparative Transcriptome Proﬁles Between Passages Reveal Calpain Proteins Play an Important Role in Maintaining Virulence A comparison between HTI4, HTI5 and Genbank strain (C) clearly indicates change of G–A in late passage same as the reference genome. paraﬂagellar rod protein 1D (PFR 1D) was over expressed in early passage compared to the intermediate passage consistent with previous proteomic data (Magalhaes et al., 2014) on diﬀerentially passaged promastigotes. PFR 1D is essential for proper ﬂagellar motility, a marker of promastigote virulence (Ginger et al., 2008) and it seems that ﬂagellar morphology gets aﬀected after few July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 10 Sinha et al. Gene Expression in Cultured Leishmania donovani FIGURE 6 \| Schematic showing domain copy of MFS gene in Chromosome 29. A pseudo gene of MFS class of proteins present in HTI4 is missing in HTI5. However, a small domain from region 297–360 is being copied at two different locations in chromosome 29 with a non-synonymous substitution (AAC->AGC). FIGURE 6 \| Schematic showing domain copy of MFS gene in Chromosome 29. A pseudo gene of MFS class of proteins present in HTI4 is missing in HTI5. However, a small domain from region 297–360 is being copied at two different locations in chromosome 29 with a non-synonymous substitution (AAC->AGC). FIGURE 7 \| Comparison of Acetyl Co-A synthetase genes in early and late passage. Gene coding for Acetyl CoA Synthetase undergoes several substitutions in t late passage which makes it non-functional. FIGURE 7 \| Comparison of Acetyl Co-A synthetase genes in early and late passage. Gene coding for Acetyl CoA Synthetase undergoes several substitutions in the late passage which makes it non-functional. Data Sheet 4) presented a typical pattern of expression where they were down-regulated between early and intermediate passages and ultimately had increased expression in the ﬁnal Data Sheet 4) presented a typical pattern of expression where they were down-regulated between early and intermediate passages and ultimately had increased expression in the ﬁnal passages and ultimately leads to loss of virulence. Interestingly, multiple members of calpain like cysteine proteases (CALPs) (XLOC_001409, XLOC_000608, XLOC_001023; Supplementary July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 11 Sinha et al. Gene Expression in Cultured Leishmania donovani FIGURE 8 \| Frameshift mutation in Calpain like cysteine protease gene due to a single insertion in late passage. A single insertion in HTI5 (as well as Genbank LDBPK 282 A1) completely changes the coding frame of the gene (Upper panel) making it inactive. The changes in late passage and Genbank strain is consistent. FIGURE 8 \| Frameshift mutation in Calpain like cysteine protease gene due to a single insertion in late passage. A single insertion in HTI5 (as well as Genbank LDBPK 282 A1) completely changes the coding frame of the gene (Upper panel) making it inactive. The changes in late passage and Genbank strain is consistent. FIGURE 8 \| Frameshift mutation in Calpain like cysteine protease gene due to a single insertion in late passage. A single insertion in HTI5 (as well as Genbank LDBPK 282 A1) completely changes the coding frame of the gene (Upper panel) making it inactive. The changes in late passage and Genbank strain is consistent. FIGURE 8 \| Frameshift mutation in Calpain like cysteine protease gene due to a single insertion in late passage. A single insertion in HTI5 (as well as Genbank LDBPK 282 A1) completely changes the coding frame of the gene (Upper panel) making it inactive. The changes in late passage and Genbank strain is consistent. beta-oxidation of fatty acids, as observed by others (Alcolea et al., 2009) (Supplementary Data Sheet S4). Frontiers in Microbiology \| www.frontiersin.org July 2018 \| Volume 9 \| Article 1279 As opposed to the earlier observations (Moreno et al., 2014), level of tyrosine amino transferase presented a reverse trend, as it was slightly downregulated in the early passage, although protein expression may reveal diﬀerent results as observed previously for Trypanosoma cruzi (dos Santos et al., 2012). Purine acquisition and inter-conversion is an important determinant of environmental sensing and morphogenesis in Leishmania. We observed a decrease in expression of a putative ribonucleoside- diphosphate reductase small chain, involved in this pathway in the late passage promastigotes in sync with earlier observation in L. donovani under purine stressed condition (Martin et al., 2014). On the other hand an ATP diphosphohydrolase was upregulated in the virulent parasites indicating preparedness for host- cell invasion and immunomodulation (Figueiredo et al., 2016) reﬂected in the diﬀerential host response in our infection studies. Amino acid transporter (aATP11) which has also been associated with purine starved condition (Martin et al., 2014) triggering parasite diﬀerentiation to metacyclic form (Inbar et al., 2017) passages. Some of the calpains have lost protease domain to gain microtubule organization function like SMP-1 (Branquinha et al., 2013). SMP-1 is required in all promastigote stages and also for transformation of amastigotes to promastigotes for the development of ﬂagella (Tull et al., 2010). Thus over expression of these atypical CALPs in the later passages indicate struggle of the promastigotes to convert to infective forms. The role of calpains in signal transduction and cytoskeletal remodeling is well documented in trypanosomatids (Galetovic et al., 2011), and structural deformities may aﬀect the virulence function of the parasite. Metabolic reprogramming is a hallmark of Leishmania life-cycle where the parasite switches between two extreme environmental conditions. In the sandﬂy gut glucose and amino acids are the primary source of carbon while once inside the host, fatty acids and amino acids provide the necessary carbon for metabolism. Increased transcript abundance of 3-hydroxyisobutyryl-coenzyme a hydrolase-like protein in the early passages relative to the late is in consistency with the preparedness of the infection ready parasites to use July 2018 \| Volume 9 \| Article 1279 July 2018 \| Volume 9 \| Article 1279 12 Gene Expression in Cultured Leishmania donovani Sinha et al. was upregulated in the late passage, again reinstating the fact that these parasites lag in morphological transformation and try hard to attain infective status when growing in culture for a long time. Prostaglandin f2-alpha synthase involved in prostaglandin biosynthetic pathway is reduced in expression in the more infective promastigotes while the gene expression is more pronounced in the later passages indicating non-completion of metacyclogenesis as reported earlier (Araujo-Santos et al., 2014). Leishmania salvage folate and pteridines from their host or vector as they cannot synthesize them. The expression of these genes is higher in infective promastigotes and is known to aid in survival under extreme intra macrophagic conditions (Papadopoulou et al., 2002; Alcolea et al., 2010). As expected a gene for this protein on chromosome 6 was upregulated till 11th passage followed by a signiﬁcant under expression in the late passage (Figure 9 and Supplementary Data Sheet S4). Altogether it signiﬁes that nutrient availability is strongly linked to gene expression in Leishmania and that many metabolites and classical metabolic enzymes are capable of altering the state of cellular diﬀerentiation and development as has been extensively studied in cancer cells (van der Knaap and Verrijzer, 2016). Metallopeptidases are important virulence factors in Leishmania and we already observed diﬀerential regulation of the major surface peptidase gp63 at protein level. Here, we observed slightly upregulated expression of two peptidases a metallopeptidase and a thimet oligopeptidase in the late passage. Transcriptomic data from late logarithmic phase promastigotes of Trypanosoma brucei growing in normal and purine supplemented medium displayed under expression of thimet oligopeptidase A in the former system linking it to purine stressed condition when approaching metacyclic state (Fernandez-Moya et al., 2014) corroborating our ﬁnding. FIGURE 9 \| Hierarchical Clustering Heatmap of differentially expressed genes in early, intermediate and late passages. Heat maps were generated to show the comparative log 2 abundance ratios between early, intermediate, and late passages using RNAseq data. Upregulated proteins are depicted by blue bars, downregulated proteins by red bars. FIGURE 9 \| Hierarchical Clustering Heatmap of differentially expressed genes in early, intermediate and late passages. Heat maps were generated to show the comparative log 2 abundance ratios between early, intermediate, and late passages using RNAseq data. Upregulated proteins are depicted by blue bars, downregulated proteins by red bars. FIGURE 9 \| Hierarchical Clustering Heatmap of differentially expressed genes in early, intermediate and late passages. Heat maps were generated to show the comparative log 2 abundance ratios between early, intermediate, and late passages using RNAseq data. Upregulated proteins are depicted by blue bars, downregulated proteins by red bars. July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 13 Gene Expression in Cultured Leishmania donovani Sinha et al. and Supplementary Figure S6). There was very little overlap between transcriptomic and proteomic data which reinstates the fact that leishmanial gene expression is majorly controlled at post-transcriptional level (Lahav et al., 2011). A total of 35 diﬀerentially expressed spots were identiﬁed, out of which 31 spots presented higher expression in the early passage while four spots were highly expressed in the late passage. For example, gp63 (Olivier et al., 2012), HSP 70 (Ramirez et al., 2013a), elongation factor 1 alpha (Nandan et al., 2003), etc. have all been reported as virulence factors and were over expressed in the early passage. Western blot analysis on whole cell lysates of early and late passage promastigotes conﬁrmed increased expression of gp63 in the virulent parasites (Supplementary Figure S5). The pyruvate dehydrogenase complex and its component dihydrolipoamide acetyltransferase has been associated with multiple functions including oxidative defense and regulation of gene expression in various pathogenic microbes (Spalding and Prigge, 2010), and presented an increased expression in the early passage. Apart from these, many hypothetical proteins also displayed diﬀerential protein expression and may determine the invasive capacity of the parasite. Transcript expression of each gene is also presented in Table 3 and presented a similar trend. Most of the proteins which displayed diﬀerential expression in our study form a part of the infectious exosomal proteins (Silverman et al., 2010). The changes in exosomal milieu probably changes the modulatory capacity of the parasites of late passage inside macrophages, which in turn make them more susceptible to microbicidal attack. The ultimate protein repertoire required for successful host– parasite interaction is the result of ordered changes in the genetic make-up and/or gene expression in the promastigotes. As pointed out above, genetic and epigenetic control of gene expression is central to adaptation of the parasite to its immediate and prospective environment. Consistent with that we observed an increased abundance of a histone-like transcription factor (CBF/NF-Y) and archaeal histone transcript in early passage relative to the late passage. The histone modifying enzyme histone deacetylase was also over expressed in the early passage. This protein has been associated with the disease causing stage of the parasite (Vergnes et al., 2005) and its higher activity may be linked to more invasiveness in the parasite. The higher protein level expression of HDAC in early passage promastigotes was also conﬁrmed by western blot on whole cell lysates (Supplementary Figure S5). Together with this, protein modifying enzyme farnesyltransferase presented a downward trend in the late passage (Figure 9 and Supplementary Data Sheet S4). This gene product has been associated with severe growth impairment in trypanosomatids (Gillespie et al., 2007). A putative pre-mRNA splicing factor ATP-dependent RNA helicase, member of DEAD box RNA helicase family presented increased expression till 11th passage. These proteins were shown to be strongly associated with mRNA of diﬀerentiating promastigotes as well as interactome of in vitro cultured amastigotes (Saxena et al., 2007). These proteins are mostly transiently upregulated or downregulated controlling the translational machinery. One of them has been associated with growth defects in L. infantum (Barhoumi et al., 2006). DNA repair related MutS-like proteins play important role in preventing genotoxicity and oxidative damage, a survival strategy adapted by many prokaryotic and eukaryotic pathogens (Genois et al., 2014), was upregulated in the virulent passage. Among the other diﬀerentially expressed transcripts are non-protein coding rRNA genes and conserved proteins with unknown functions. Non-coding RNAs along with proteins in the secreted exosomal milieu of Leishmania are known to modulate host–parasite interaction and changes in the composition of these exosomes may aﬀect infectivity (Lambertz et al., 2015). An upregulated calpain like protein related to small myristoylated protein-1 (SMP-1) may also signify the struggle of late passage promastigotes to transform into infective metacyclic forms and as already mentioned in the previous section. It is also interesting to note that some calpain-like proteins, particularly those lacking the protease domain, are upregulated in the late passages while some others with protease activity are downregulated or psedogenized in the avirulent late passage parasites. Another consistently upregulated protein in the late passage was a putative ATPase beta subunit. Although there is no report in protozoans, a report linked increased acid sensitivity of bacterial pathogen to ATPase overexpression (McEntire et al., 2004). This may explain partly the non-viability of late passage parasites inside acidic host phagolysosomes. Beta tubulin was signiﬁcantly up regulated in the non-infectious stage of the pathogen (Coulson et al., 1996). On the contrary alpha tubulin was mostly down regulated in the later passages. Tubulin plays a very important role in cytoskeleton formation and its expression is post-transcriptionally controlled and tightly linked to parasite transformation (Ramirez et al., 2013b). During the infection cycle, the pathogen utilizes hosts cytoskeletal machinery for pathogenicity (Gruenheid and Finlay, 2003) probably leading to less expression of these proteins. The results indicate that subtle genomic and transcriptomic variations drive the adaptation of promastigotes to culture condition and these changes mostly lead to loss of virulence in addition to morphological and metabolic modiﬁcations. Of particular interest is the role played by peptidases of diﬀerent classes mainly metallopeptidases and calpain-like cysteine proteases in host–parasite interaction. Another important aspect is the importance of purine stress and modes of purine acquisition which modulate parasite and host responses to infection. Signiﬁcant Regulation of Virulence Proteins in Comparative Protein Expression Studies Till date the loss of infectivity in late passage culture has been explained by reduced expression of virulence factors, particularly those which can interact with and modulate the host cells. Proteomic comparison of crude membrane extracts of L. donovani AG83 promastigotes (LAg) revealed altered protein expression, particularly a decrease in expression of some known virulence factors after continuous passaging (Table 3 Our study indicates that pathogenicity in L. donovani is a complex mechanism wherein the parasites are pre-adapted to a pathogenic lifestyle where nutritional stress and environmental sensing lead to global changes in both genes and gene expressions, July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 14 Gene Expression in Cultured Leishmania donovani Sinha et al. TABLE 3 \| List of proteins differentially expressed (≥1.5-folds) between early and late passages identiﬁed by MALDI-ToF MS/MS. Spot Number Assembly gap? Signiﬁcant Regulation of Virulence Proteins in Comparative Protein Expression Studies location∗ Protein annotation Accession number of protein Protein score Exp/Thr M.W Exp/ Thr pI aFold change (MS/MS) bFold change Values using RNAseq 1 Yes Constitutive major surface protease CAC37962.1 434 68/63.8 5.9/6.84 0.66 −0.67 2 Yes Constitutive major surface protease CAC37962.1 393 68.5/63.8 5.7/6.84 0.45 −0.42 3 Yes GP63, leishmanolysin XP_001463701.2 75 69/63.8 5.6/6.84 0.05 −0.42 4 Yes GP63, leishmanolysin XP_001463701.2 114 40/63.8 5.4/6.84 0.1 −0.42 5 Yes Beta tubulin XP_003878331.1 347 55.8/49.7 5.2/4.71 6.38 0.22 6 Yes Putative heat shock 70-related protein 1, mitochondrial precursor XP_003877392.1 271 68.5/63.8 5.8/6.84 0.23 −0.46 8 Chr36: 1099755–1098367 Putative dihydrolipoamide acetyltransferase precursor XP_001469441.1 382 62.4/48.62 6.4/7.02 0.44 −0.76 9 Yes ∗∗Activated C kinase protein, partial ABS82039.1 103 31.2/30.63 6.7/6.63 0.66 −0.20 10 Chr32: 766497–767468 Putative heat shock protein-like protein XP_001467884.1 308 22.5/35.49 5/4.94 0.32 −0.24 11 chr36: 2665342–2666118 Conserved hypothetical protein XP_001469385.1 703 24/29.12 5.4/5.82 0.24 0.54 12 chr25: 350814–352157 ∗∗Gamma tubulin XP_003860740.1 274 22.9/49.7 5.2/4.71 0.28 −0.16 13 chr23: 9640–10320 ∗∗Mitochondrial peroxiredoxin AAX73294.1 314 20.6/25.34 5.4/6.43 0.27 −0.82 14 chr22: 569013–569636 i/6 autoantigen-like protein XP_001465646.1 156 26.2/23.01 6/5.68 0.37 −0.03 15 chr36: 1493409–1494356 Hypothetical protein, conserved (MORN repeat) XP_003865511.1 142 18.1/35.67 4.8/5.90 0.22 −0.16 16 chr35: 896773–897597 Putative RNA-binding protein XP_003392812.1 344 22.6/30.25 6.5/7.85 0.61 −0.05 17 chr20: 601988–602383 Putative small myristoylated protein-1 XP_001465265.1 56 17.4/12.94 6.4/4.70 1.96 0.56 18 chr35: 552681–553127 ∗∗Putative ubiquitin-conjugating enzyme E2 XP_001568232.1 46 13.1/16.65 6.3/6.08 0.43 −0.03 19 chr23: 133149–134204 Putative NADP-dependent alcohol dehydrogenase XP_001465717.1 388 43.2/38.45 6.5/5.96 0.54 −1.48 20 chr25: 458908–460485 Putative ATPase beta subunit XP_001466152.1 211 60.5/56.32 5.5/5.14 2.06 0.62 21 chr28: 488208–489566 ∗∗Phenylalanine-4-hydroxylase XP_001684410.1 71 48.8/51.48 6.2/5.93 0.37 −0.21 22 chr36: 2726362–2727795 ∗∗Protein disulﬁde isomerase XP_001469404.1 206 55.5/52.34 5.5/5.42 0.34 −0.23 23 chr25: 671470–672522 Putative pyruvate dehydrogenase E1 beta subunit XP_001466210.1 72 45.8/37.84 5.5/5.64 0.27 −2.02 24 Yes Alpha tubulin XP_003873239.1 113 14.9/49.75 4.8/4.89 0.35 −0.04 25 chr21: 9660–11141 Conserved hypothetical protein XP_001564720.1 55 23.4/52.12 5.6/6.45 0.43 −0.13 26 chr21: 413285–414697 Alpha tubulin XP_003873239.1 68 11.3/49.75 5.6/4.89 0.36 −0.04 27 chr7: 303267–303773 Conserved hypothetical protein/putative Qa-SNARE protein XP_003872214.1/ XP_001463282.1 59 11.5/14.35 5.2/9.15 0.52 −1.24 28 Yes Conserved hypothetical protein XP_001470159.1 82 33.5/24.26 5.6/5.71 0.61 −0.62 29 chr28: 460266–462242 ∗∗Putative glucose-regulated protein 78 XP_001470161.1 207 75.2/71.94 5.5/5.05 1.55 0.04 30 Yes ∗∗Putative calmodulin XP_001463554.1 50 55/70.47 5.6/4.41 0.45 −0.54 31 Yes ∗∗S-adenosylmethionine synthetase XP_003392704.1 73 48.5/43.12 6.1/5.49 0.45 −0.08 (Continued) TABLE 3 \| List of proteins differentially expressed (≥1.5-folds) between early and late passages iden Spot Number Assembly gap? Signiﬁcant Regulation of Virulence Proteins in Comparative Protein Expression Studies location∗ Protein annotation Accession number of protein Pro sco 1 Yes Constitutive major surface protease CAC37962.1 43 2 Yes Constitutive major surface protease CAC37962.1 39 3 Yes GP63, leishmanolysin XP_001463701.2 7 4 Yes GP63, leishmanolysin XP_001463701.2 11 5 Yes Beta tubulin XP_003878331.1 34 6 Yes Putative heat shock 70-related protein 1, mitochondrial precursor XP_003877392.1 27 8 Chr36: 1099755–1098367 Putative dihydrolipoamide acetyltransferase precursor XP_001469441.1 38 9 Yes ∗∗Activated C kinase protein, partial ABS82039.1 10 10 Chr32: 766497–767468 Putative heat shock protein-like protein XP_001467884.1 30 11 chr36: 2665342–2666118 Conserved hypothetical protein XP_001469385.1 70 12 chr25: 350814–352157 ∗∗Gamma tubulin XP_003860740.1 27 13 chr23: 9640–10320 ∗∗Mitochondrial peroxiredoxin AAX73294.1 31 14 chr22: 569013–569636 i/6 autoantigen-like protein XP_001465646.1 15 15 chr36: 1493409–1494356 Hypothetical protein, conserved (MORN repeat) XP_003865511.1 14 16 chr35: 896773–897597 Putative RNA-binding protein XP_003392812.1 34 17 chr20: 601988–602383 Putative small myristoylated protein-1 XP_001465265.1 5 18 chr35: 552681–553127 ∗∗Putative ubiquitin-conjugating enzyme E2 XP_001568232.1 4 19 chr23: 133149–134204 Putative NADP-dependent alcohol dehydrogenase XP_001465717.1 38 20 chr25: 458908–460485 Putative ATPase beta subunit XP_001466152.1 21 21 chr28: 488208–489566 ∗∗Phenylalanine-4-hydroxylase XP_001684410.1 7 22 chr36: 2726362–2727795 ∗∗Protein disulﬁde isomerase XP_001469404.1 20 23 chr25: 671470–672522 Putative pyruvate dehydrogenase E1 beta subunit XP_001466210.1 7 24 Yes Alpha tubulin XP_003873239.1 11 25 chr21: 9660–11141 Conserved hypothetical protein XP_001564720.1 5 26 chr21: 413285–414697 Alpha tubulin XP_003873239.1 6 27 chr7: 303267–303773 Conserved hypothetical protein/putative Qa-SNARE protein XP_003872214.1/ XP_001463282.1 5 28 Yes Conserved hypothetical protein XP_001470159.1 8 29 chr28: 460266–462242 ∗∗Putative glucose-regulated protein 78 XP_001470161.1 20 30 Yes ∗∗Putative calmodulin XP_001463554.1 5 31 Yes ∗∗S-adenosylmethionine synthetase XP_003392704.1 7 s identiﬁed by MALDI-ToF MS/MS. Signiﬁcant Regulation of Virulence Proteins in Comparative Protein Expression Studies aA value greater than 1.5 in column ‘a’ indicates up-regulation and less than 1/1.5 = 0.66 indicates down regulation. bNegative values indicates down regulation and positive values indicate upregulation. parasites. The most important factor responsible for adaptive changes is the nutritional status of the immediate environment, sensing which the promastigotes undergo morphological and metabolic switch. This phenomenon is also strongly connected with transporter system in parasitic protists (Dean et al., 2014). In our genetic studies, we found domain duplication in acetyl CoA synthetase gene in the early passage promastigotes. Recent works have highlighted the importance of this enzyme in L. donovani infectivity owing to lipid and ergosterol biosynthesis (Soumya et al., 2017). On a broader note, this enzyme and its product acetyl CoA has been linked to global gene expression and chromatin regulation (van der Knaap and Verrijzer, 2016). This supports the theory of diﬀerential gene expression, and epigenetic changes in the virulent versus avirulent parasites, and reiterates the fact that nutrition and transcription are tightly linked. Another facet of the study relates to the importance of DNA repair and recombination activity prevalent in these parasites which promote antigenic variation suitable for intracellular adaptability and pathogenicity and needs further delving. This was indicated by diﬀerential regulation of MutS like protein at transcriptional level which has been implicated in quorum sensing independent developmental regulation in Trypanosoma recently (Zimmermann et al., 2017). Nevertheless, this in vitro cultured parasite model of gene regulation and virulence provides a basis for understanding not merely parasite adaptation to culture conditions and its genetic basis but also underlines the fact that common pathways are involved in intraspeciﬁc and intrastrain variations observed in Leishmania. Most of the analysis done so far in literature is based on comparison of several clinical isolates in Leishmania. However, comparison of various cultured passages of promastigotes are reported here. ultimately leading to the production of eﬀector molecules inside the host culminating in infection. The heteroxenic lifecycle and pleomorphic nature of the parasite adds another dimension to this process as successful transformation to metacyclic promastigotes determines virulence phenotype both in in vivo (other studies) in sandﬂy and in vitro (others and our study) in culture medium. It is re-established from these studies that long term cultivation of Leishmania promastigotes in axenic environment diminishes their adaptability toward intracellular life, and many of the factors involved in this process are markers of natural virulence attenuation. Frontiers in Microbiology \| www.frontiersin.org Signiﬁcant Regulation of Virulence Proteins in Comparative Protein Expression Studies Protein score Exp/Thr M.W Exp/ Thr pI aFold change (MS/MS) bFold change Values using RNAseq 434 68/63.8 5.9/6.84 0.66 −0.67 393 68.5/63.8 5.7/6.84 0.45 −0.42 75 69/63.8 5.6/6.84 0.05 −0.42 114 40/63.8 5.4/6.84 0.1 −0.42 347 55.8/49.7 5.2/4.71 6.38 0.22 271 68.5/63.8 5.8/6.84 0.23 −0.46 382 62.4/48.62 6.4/7.02 0.44 −0.76 103 31.2/30.63 6.7/6.63 0.66 −0.20 308 22.5/35.49 5/4.94 0.32 −0.24 703 24/29.12 5.4/5.82 0.24 0.54 274 22.9/49.7 5.2/4.71 0.28 −0.16 314 20.6/25.34 5.4/6.43 0.27 −0.82 156 26.2/23.01 6/5.68 0.37 −0.03 142 18.1/35.67 4.8/5.90 0.22 −0.16 344 22.6/30.25 6.5/7.85 0.61 −0.05 56 17.4/12.94 6.4/4.70 1.96 0.56 46 13.1/16.65 6.3/6.08 0.43 −0.03 388 43.2/38.45 6.5/5.96 0.54 −1.48 211 60.5/56.32 5.5/5.14 2.06 0.62 71 48.8/51.48 6.2/5.93 0.37 −0.21 206 55.5/52.34 5.5/5.42 0.34 −0.23 72 45.8/37.84 5.5/5.64 0.27 −2.02 113 14.9/49.75 4.8/4.89 0.35 −0.04 55 23.4/52.12 5.6/6.45 0.43 −0.13 68 11.3/49.75 5.6/4.89 0.36 −0.04 59 11.5/14.35 5.2/9.15 0.52 −1.24 82 33.5/24.26 5.6/5.71 0.61 −0.62 207 75.2/71.94 5.5/5.05 1.55 0.04 50 55/70.47 5.6/4.41 0.45 −0.54 73 48.5/43.12 6.1/5.49 0.45 −0.08 TABLE 3 \| List of proteins differentially expressed (≥1.5-folds) between early and late passages identiﬁed by MALDI-ToF MS/MS. July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 15 Gene Expression in Cultured Leishmania donovani Sinha et al. TABLE 3 \| Continued Spot Number Assembly gap? location∗ Protein annotation Accession number of protein Protein score Exp/Thr M.W Exp/ Thr pI aFold change (MS/MS) bFold change Values using RNAseq 32 Chr30:122: 5029–1226102 Conserved hypothetical protein XP_001467184.1 115 30.2/40.83 5.9/5.32 0.11 −0.16 33 chr36: 1115280–1116995 ∗∗Mitochondrial ATP-dependent zinc metallopeptidase, putative, metallo-peptidase, Clan MA(E), Family M41 CCM19617.1 52 19.47/72.38 5.7/8.49 0.57 −0.99 34 chr11: 473373–474023 Elongation factor 1-alpha XP_003392396.1 112 52.36/49.12 6.8/9.03 0.61 −1.31 35 chr25: 548571–549221 Putative GTPase XP_001463009.1 223 12.4/24.24 5.9/6.09 0.29 −0.31 ∗Assembly location was calculated in reference to early passages. ∗∗These spots were located on both gels but identiﬁed once. Exp/Thr M.W., Experimental/Theoretical molecular weight. Exp/Thr pI, Experimental/Theoretical isoelectric point. Fold change, mean upregulation or downregulation with respect to early passage. aA value greater than 1.5 in column ‘a’ indicates up-regulation and less than 1/1.5 = 0.66 indicates down regulation. bNegative values indicates down regulation and positive values indicate upregulation. ∗Assembly location was calculated in reference to early passages. ∗∗These spots were located on both gels but identiﬁed once. Exp/Thr M.W., Experimental/Theoretical molecular weight. Exp/Thr pI, Experimental/Theoretical isoelectric point. Fold change, mean upregulation or downregulation with respect to early passage. July 2018 \| Volume 9 \| Article 1279 Signiﬁcant Regulation of Virulence Proteins in Comparative Protein Expression Studies Leishmania have evolved several strategies to sense their immediate environment individually as well as collectively and respond to the same by controlling gene expression, and ultimately cell division and transformation. Transporter proteins are known to be involved in this pathway in other pathogens (Hlavacek et al., 2009). Continuous axenic cultivation possibly reverses this pathoadaptive response by inducing mutations in these genes which leads to loss of virulence. Further, pseudogenization of calpain like proteins involved in signal transduction and parasite transformation results in growth arrest at non-infective stages. Studies on in vitro cultured drug resistant and sensitive clinical isolates of L. donovani provided evidence that the former system has added advantage of increased metacyclogenesis in culture and higher infectivity index in host cells (Vanaerschot et al., 2010), which is also reﬂected in the miltefosine sensitivity assay done on the early and late passage promastigotes. Moreover, early and late passage promastigotes display diﬀerential infectivity (this work) because interaction of virulent L. donovani with host macrophages triggers diﬀerential transcriptional modulation in the latter as compared to avirulent strains (manuscript communicated elsewhere). A parallel gene expression study in the intracellular amastigotes also pointed toward higher expression of transporter and calcium-dependent cysteine-type endo peptidase activity genes (GO: 0005215; GO: 0004198) in more virulent forms (manuscript communicated elsewhere), strengthening the relevance of pre-adaptation in virulent Interestingly, in this study, we report a large number of ABC transporters undergoing structural changes in later passages, thus their inactivation may be a possible cause of loss of virulence. We have also found that in addition to several important genes such as GP63 and heat shock proteins which are already known to play major role in virulence, calpain like cysteine proteases may July 2018 \| Volume 9 \| Article 1279 Frontiers in Microbiology \| www.frontiersin.org 16 Gene Expression in Cultured Leishmania donovani Sinha et al. have signiﬁcant role in loss of virulence since the genes undergo alteration and the expression levels change. of early and late passage promastigotes can act as references for future genomic studies on Leishmania, particularly related to virulence and drug resistance. Annotated leads from this study and further annotation and functional characterization of hypothetical proteins can provide novel drug/vaccine targets for disease management. CONCLUSION The work presented here highlights the importance of newly emerging pathoadaptive factors like transporters and calpain-like proteins in Leishmania virulence. Our work can add to the pool of information on adaptive genomics in L. donovani and careful mining of these genes can be used for virulence surveillance at least throughout the Indian subcontinent. The genome sequences SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.01279/full#supplementary-material Signiﬁcant Regulation of Virulence Proteins in Comparative Protein Expression Studies This work re-establishes the ﬁndings of other studies at transcriptomic and proteomic level to exercise caution while using serially passaged promastigotes for infectivity and therapeutic studies but also adds genetic analysis to this list. The revelation from our study that the NCBI reference strain was derived from clinical isolate which was cultured in vitro for multiple passages reiterates that subtle changes in the genome due to axenic cultivation may have important implications on the virulence phenotype. Moreover, similar genomic modiﬁcations may come into play when Leishmania infects diﬀerent hosts, diﬀerent vectors or even diﬀerent sites of the same host which leads to diﬀerential host responses (Elso et al., 2004). Thus within-host or even within-vector selection pressure appears to be the major factor driving the modiﬁcations in the parasite which make them adaptable to their environment (Shaw, 1997), and is accompanied by no major gene losses but rather subtle polymorphisms that possibly alter the functionality of the genes. It is further evidenced from the higher expression of transporter and calpain-like proteins in visceralizing L. donovani strains from Sri Lanka (Zhang et al., 2014) compared to strains causing cutaneous lesions. Virulence is a function of nutritional status of the parasites and more time spent in the nutrient rich culture medium diminishes their pathoadaptive characteristics. Signiﬁcant is the fact that changes in genes and gene expression occur in same/similar set of genes both in culture and in nature for virulence attenuation and cultured promastigotes can well mimic their sandﬂy counterparts but long-term cultures should be avoided. Changes in transporters and certain house- keeping genes can be markers of virulence. Additionally the new class of calpain peptidases are emerging as important virulence determinants along with more established metallopeptidases and cathepsin- like cysteine peptidases. ACKNOWLEDGMENTS We acknowledge Mr. Sandip Chakraborty for Mass Spectrometry support and Mr. Janmenjay Midya for assistance in animal experiments from CSIR-IICB. We are thankful to Mr. Samrat Ghosh and Mr. Arijit Panda from Computational Biology and Genomics Lab, Structural Biology and Bioinformatics Division, CSIR-IICB for setting up the internal Blast Page. The assistance of Ms. Shreya Dev during experiments is duly acknowledged. AUTHOR CONTRIBUTIONS RS and NA conceived the study. RS, R, NA, and ST designed the experiments. RS, ST, MC, SdD, and R analyzed the data. RS, ST, MC, R, and NA wrote the manuscript. RS, R, SD, and MS performed and optimized the experiments. RS, MC, SD, and ST prepared ﬁgures. RC contributed equipment and reagents. All authors critically revised and approved the manuscript. FUNDING This work was funded by Council of Scientiﬁc and Industrial Research (CSIR), Government of India Network Project BSC0114. RS, R, MS, and SD received fellowships from CSIR. MC received fellowship from Department of Science and Technology and SdD from Department of Biotechnology, Government of India. This work was funded by Council of Scientiﬁc and Industrial Research (CSIR), Government of India Network Project BSC0114. RS, R, MS, and SD received fellowships from CSIR. MC received fellowship from Department of Science and Technology and SdD from Department of Biotechnology, Government of India. This work was funded by Council of Scientiﬁc and Industrial Research (CSIR), Government of India Network Project BSC0114. RS, R, MS, and SD received fellowships from CSIR. MC received fellowship from Department of Science and Technology and SdD from Department of Biotechnology, Government of India. promastigotes of Leishmania infantum. Genomics 93, 551–564. doi: 10.1016/j. ygeno.2009.01.007 REFERENCES Sustained cell polarity and virulence in the phytopathogenic fungus Ustilago maydis depends on an essential cyclin-dependent kinase from the Cdk5/Pho85 family. J. 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https://openalex.org/W1980439589	https://www.scielo.br/j/hcsm/a/vzxGR7tSvqgG3kSWG9DpsQL/?lang=pt&format=pdf	Portuguese	null	As atividades do naturalista José Bonifácio de Andrada e Silva em sua 'fase portuguesa' (1780-1819)	História, ciências, saúde-Manguinhos	2,004	cc-by	13,152	Alex Gonçalves Varela Mestre em geociências na área de educação aplicada às geociências, IGE/UNICAMP Maria Margaret Lopes Professora da Pós-Graduação em Educação Aplicada às Geociências, IGE/UNICAMP Maria Rachel Fróes da Fonseca Pesquisadora do Departamento de Pesquisa da Casa de Oswaldo Cruz/Fiocruz VARELA, A. G.; LOPES, M. M. e FONSECA, M. R. F. da: As atividades do filósofo natural José Bonifácio de Andrada e Silva em sua fase portuguesa (1780-1819). História, Ciências, Saúde Manguinhos, vol. 11(3): 685-711, set.-dez. 2004. VARELA, A. G.; LOPES, M. M. e FONSECA, M. R. F. da: As atividades do filósofo natural José Bonifácio de Andrada e Silva em sua fase portuguesa (1780-1819). História, Ciências, Saúde Manguinhos, vol. 11(3): 685-711, set.-dez. 2004. José Bonifácio de Andrada e Silva tem presença marcada na historiografia, de forma quase consensual, como o Patriarca da Independência, primado concedido a seu perfil de estadista e parlamentar. Contudo, ele notabilizou-se também como estudioso e pesquisador do mundo natural. Dentre outras atividades nesta área, administrou espaços governamentais portugueses ligados diretamente à mineração e à agricultura. Um destes loci institucionais foi a Intendência Geral das Minas e Metais do Reino, órgão que formulava a política de exploração mineral em Portugal no início do século XIX e realizava pesquisas no campo da mineralogia. A Intendência foi um espaço de importante produção científica no campo da mineralogia e contribuiu para a difusão das modernas idéias científicas pela sociedade portuguesa, o que ratifica a tese de que a Academia não era a única instituição produtora de ciência em Portugal. As atividades do naturalista José Bonifácio de Andrada e Silva em sua fase portuguesa (1780-1819) As atividades do naturalista José Bonifácio de Andrada e Silva em sua fase portuguesa (1780-1819) The work of naturalist José Bonifácio de Andrada e Silva during his Portuguese phase (1780-1819) PALAVRAS-CHAVE: história das ciências; história das geociências; história do Brasil; história das ciências naturais. VARELA, A. G.; LOPES, M. M. and FONSECA, M. R. F. da: The work of naturalist José Bonifácio de Andrada e Silva during his Portuguese phase (1780-1819). História, Ciências, Saúde Manguinhos, vol. 11(3): 685-711, Sept.-Dec. 2004. José Bonifácio de Andrada e Silva has his presence noticed into History, towards his identification as the Patriarch of the Independence, which corresponds to his profile of Statesman and Parliamentary. However, he also became noted as a studious man and a researcher of the natural world. He managed Portuguese governmental spaces connected directly to mineralogy and agriculture. One of these institutional loci was the General Departament of Mines and Metals of the Kingdom, institution that formulates the politics of mineral exploitation in Portugal at the beginning of the 19th century and carried on researches in the mineralogy field. The Departament became an important space of scientific production in the mineralogy field, contributing to the spread of the modern scientific ideas to the Portuguese society. Maria Rachel Fróes da Fonseca KEYWORDS: Science history, Geo-sciences, History of Brazil, Natural Science History. 685 4 vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA O O estudo da trajetória de José Bonifácio de Andrada e Silva apresenta um campo apropriado e perspectivas fecundas de trabalho em história das ciências. Isso porque, em primeiro lugar, sua presença na bibliografia especializada se faz, de forma quase que consensual, principalmente em torno de sua identificação como o Patriarca da Independência, o que, grosso modo, corres- ponde ao primado concedido a seu perfil de estadista e parlamentar. Mas estas são análises que enfatizam o viés político de sua trajetória histórica, deixando de incorporar sua dimensão de naturalista. José Bonifácio notabilizou-se não apenas como homem público, mas também como estudioso e pesquisador do mundo natural. Ele participou de viagens científicas, foi sócio de inúmeras sociedades científicas européias, publicou diversas memórias no âmbito da história natural e administrou espaços governamentais portugueses ligados diretamente à mineração e à agricultura. Desse modo, em que pese a densidade da bibliografia a seu respeito, há lacunas que estimulam o caminho da reflexão em novas direções. As atividades do naturalista José Bonifácio de Andrada e Silva em sua fase portuguesa (1780-1819) O objetivo deste trabalho é resgatar o viés naturalista de José Bonifácio durante o período em que viveu em Portugal, tendo como premissa fundamental o fato de que seu perfil como filósofo natural e homem público não pode ser estudado de forma isolada. Como homem típico da Ilustração que ele era, cabe cruzar e entrelaçar os dois aspectos mencionados. História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA juntaram-se às elites cultas da metrópole que também ali estudavam; juntos leriam as mesmas obras e receberiam a mesma formação (Nizza da Silva, 1999). José Bonifácio ingressou na Faculdade de Filosofia criada no conjunto das reformas pombalinas com o objetivo de ensinar ciências naturais e ciências físico-químicas , cujo curso regular tinha duração de quatro anos. Não havia qualquer curso preparatório, e no ensino sobressaíam os compêndios de Antonio Genovese, Carl von Linné, Petrus von Musschenbroek e a História natural de Plínio. Durante o período em que esteve na universidade, José Bonifácio recebeu uma ampla formação. Na Faculdade de Direito cursou as cadeiras de português, direito natural, história do direito civil romano, elementos de direito romano, elementos de direito canônico, direito civil pátrio e jurisprudência. Na Faculdade de Filosofia, por sua vez, fez as cadeiras de história natural, física experimental, química teórica e prática. E na Faculdade de Matemática freqüentou o curso de geometria. No período em que estudou em Coimbra, pôde observar o desleixo com a aplicação das medidas reformistas empreendidas por Pombal. Este fato levou-o a escrever, em 1785, juntamente com Francisco de Melo Franco, outro português natural do Brasil e que lá estudava, um poema satírico intitulado No reino da estupidez, em que mestres e cursos recebiam pesadas críticas. Foi, portanto, nesse espaço institucional, de onde a reforma pombalina não conseguira varrer de uma vez só os modelos tradicionais, que José Bonifácio recebeu o título de bacharel em filosofia e leis, no dia 16 de julho de 1787 embora o diploma só lhe fosse concedido em julho do ano seguinte. Em virtude de sua titulação, optaremos por denominá-lo filósofo natural. Isso porque a palavra cientista ainda não era usada naquela época, evitando-se assim anacronismos históricos (Barnes, 1987, p. 8). Além disso, cabe registrar que foi como filósofo que ele próprio se autodefiniu em uma de suas notas: Eu não sou partidarista da mitosofia ou da teosofia. Sou filósofo, isto é, constante indagador da verdadeira e útil sabedoria. Deixo aos Platônicos velhos e novos o seu Autoagathon; e procuro somente conhecer os homens e as coisas pelo lado do seu uso prático, para deles adquirir o conhecimento útil (IHGB, 192, 59 H). José Bonifácio na Universidade de Coimbra José Bonifácio de Andrada e Silva nasceu em Santos, em 1763. Era filho de dona Maria Bárbara da Silva e Bonifácio José de Andrada. O pai era alto funcionário da Coroa, embora também tivesse outras atividades, como o comércio, e possuía a segunda maior fortuna de Santos. Tinha outros irmãos, dentre os quais destacaram-se Martim Francisco e Antônio Carlos. A instrução primária do menino foi dada pela própria família, destacando-se nessa tarefa seus tios padres, uma vez que as escolas primárias de Santos não tinham um ensino de boa qualidade. Juntamente com os dois irmãos, José Bonifácio foi para São Paulo com o intuito de receber uma formação que complementasse aquela recebida no âmbito familiar. Nesta cidade, freqüentou o curso preparatório mantido por frei Manuel da Ressurreição, o que lhe possibilitou os primeiros contatos com a cultura clássica. Também assistiu a aulas de gramática, retórica e filosofia, matérias indispensáveis para quem desejava estudar em Coimbra (Sousa, 1957). Em 1780 viajou para Portugal, matriculando-se na Universidade de Coimbra, nos cursos de direito canônico e filosofia natural. Naquele espaço, ele e seus dois irmãos, membros da elite colonial, 686 História, Ciências, Saúde Manguinhos, Rio de Janeiro ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Cabe registrar que seguiremos a análise de Ferrone (1997) sobre o estudioso das ciências do século XVIII, para analisarmos a atuação de José Bonifácio de Andrada e Silva. Da mesma forma que a carreira dos estudiosos que viviam nas sociedades do Ancien Régime, a de José Bonifácio como filósofo foi caracterizada por encerrar-se completamente na fidelidade a uma espécie de dupla identidade. Primeiro, observa-se sua adesão ao modelo do homem de ciência organicamente ligado ao Estado, que aceitava inteiramente a lógica e os valores de uma sociedade hierarquizada, estabelecida, organizada por ordens, classes e corpos diferenciados segundo dignidades, honras, onipresença do privilégio e categorias. O Estado atribuía ao estudioso das ciências honras e privilégios, conforme o costume e a lógica do Ancien Régime, que iam desde uma isenção parcial dos rendimentos à dispensa do serviço militar, à honrosa possibilidade de ser levado à presença do rei, ao recebimento de bolsas de estudo, à participação no cerimonial da Corte e nas manifestações públicas. O compromisso com o monarca e com o sistema de organização da vida intelectual assente no patronage permitia, aliás, desenvolver a fundo as potencialidades do método científico e aumentar o número dos protagonistas, em virtude dos financiamentos, das pensões, dos privilégios ampliados pelo soberano. O homem de ciência do século XVIII, no contexto do Antigo Regime, era basicamente um funcionário do Estado, cujas atividades eram financiadas pelos monarcas, revelando assim o pacto tácito com o poder. Por outro lado, observa-se na prática científica do filósofo estudado a adesão e difusão do enciclopedismo, a ideologia científica do progresso, o utilitarismo e o pragmatismo, assim como a vontade e o desejo de classificar os elementos do mundo natural, traços que caracterizam o moderno pensamento científico. Ademais, cabe assinalar o fato de ele ser membro da República das Letras, com os seus valores cosmopolitas, uma vez que participou ativamente de inúmeras sociedades científicas e publicou trabalhos sobre suas pesquisas, que seguiam o método moderno da observação e da experimentação. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Embora assinalando esses dois aspectos que caracterizavam o moderno pensamento científico, o pragmatismo e o utilitarismo, José Bonifácio não mencionava uma terceira atitude que também estaria presente em suas ações como estudioso e que complementa as duas já citadas, reforçando assim a modernidade do seu pensamento: a atitude de identificar e classificar os elementos do mundo da natureza, sobretudo os minerais. 687 vol. 11(3):685-711, set.-dez. 2004 AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA científica por diversos países europeus, juntamente com Manuel Ferreira da Câmara Bethencourt e Sá e Joaquim Pedro Fragoso. Para a realização deste empreendimento, o ministro Luiz Pinto de Souza baixou uma minuciosa Instrução para a realização da viagem de aperfeiçoamento técnico através da Europa (31.5.1790). Nesta foi determinado que Manuel Ferreira da Câmara seria o chefe de Brigada, sendo responsável pela decisão do tempo dos estudos e das viagens, do destino de cada um dos sócios, e dos sítios aonde deviam empregar-se (Falcão, III, 1963, p. 169). O recebimento da bolsa de estudos para a realização da viagem oferecida pelo governo português nos permite afirmar que José Bonifácio acabava por inserir-se na lógica do prestígio, uma vez que vivia sob a proteção do Estado (por meio de cargos, pensões, mesadas etc.). Ao passar a viver literalmente às custas da Coroa portuguesa, passava a ter uma posição privilegiada em sua sociedade porque isso significava a proximidade com a Coroa, a participação em sua vida e o recebimento de pensões. Portanto, privilegiada porque dependente (Elias, 1995). Na Instrução estavam listados determinados locais por onde os filósofos deveriam passar. O percurso, longe de ser delineado arbitra- riamente, era estipulado pelo poder administrativo. Em segundo lugar, os participantes contariam com uma ampla rede de diplomatas em todos os locais que visitassem, facilitando a entrada e permanência nos países determinados pelo poder régio. Primeiramente deveriam ir à França, país expoente da Ilustração européia e onde ocorreu a chamada revolução química, liderada por Antoine Laurent Lavoisier, assim como importante centro em que se desenvolveu a Escola de Mineralogia Cristalográfica, que teve como expoentes Romé de LIsle e o abade René-Just Haüy. Em Paris deveriam fazer um curso completo de química com M. Fourcroy (Antoine François de Fourcroy, 1755-1809) e outro de mineralogia docimástica com M. Le Sage (Balthazar-Georges Sage, 1740-1824). José Bonifácio fez o curso de Fourcroy, pois recebeu um certificado que atesta a sua presença em um curso particular de Mineralogia e Química em meu laboratório [ Fourcroy ] (Falcão, III, op. cit., p. 170). Ao freqüentar as aulas deste importante químico francês, o filósofo natural entrava em contato com as principais idéias da revolução química, uma vez que Fourcroy colaborou para a formulação da nova nomenclatura química, baseada na teoria da oxidação e da combustão e que negava a existência do flogisto. Adquirindo os modernos conhecimentos mineralógicos: a viagem científica pela Europa Central e do Norte No dia 4 de março de 1789 José Bonifácio, conduzido pelas mãos do duque de Lafões, ingressou na Academia Real das Ciências de Lisboa. Este espaço foi, por excelência, o centro de apropriação das idéias da Ilustração em Portugal no período mariano e de sua adequação à realidade da sociedade lusa. Logo que entrou para a Academia, sob a proteção do duque de Lafões, José Bonifácio foi nomeado para a realização de uma viagem 688 História, Ciências, Saúde Manguinhos, Rio de Janeiro vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Paris (Falcão, III, op. cit., p. 172). Se tivesse feito o curso de Le Sage, José Bonifácio teria estudado a mineralogia docimástica, área de especialização do estudioso francês. O curso que Duhamel oferecia na Escola de Minas estava relacionado à arte do minerador, à arte do metalurgista, à geometria elementar subterrânea, teórica e prática, ou ao tratado dos filões ou veios mineralógicos e sua disposição pelo seio da terra (Arlet, 1991, p. 97). Na França, foi admitido como membro correspondente da Sociedade Filomática de Paris, em sessão de 29 de janeiro de 1791, da qual era presidente Alexandre Brogniart (1770-1847). Por sua vez, a 4 de março de 1791, foi admitido como membro da Sociedade de História Natural de Paris, onde apresentou a Memória sobre os diamantes do Brasil, publicada pela primeira vez no ano de 1792, nos Annales de Chimie da mesma sociedade. Uma versão inglesa do mesmo artigo foi publicada no ano de 1797, no Journal of Natural, Philosophy, Chemistry and Arts de Londres. Passando para a descrição cristalográfica, comentou as várias formas de diamantes existentes no Brasil. Ao identificar cristalo- graficamente as produções diamantíferas presentes em Serro do Frio, Bonifácio fez uso de dois sistemas de classificação de minerais, o de Johann Gottschalk Wallerius e o de Romé de lIsle. O primeiro sistema baseava-se no uso do critério químico para a divisão dos minerais e distinguia as características minerais internas das externas. Os caracteres externos que permitiriam a classificação eram cor, forma, gosto, cheiro (propriedades físicas), usos e ocorrência. Caso estas características fornecessem um quadro incompleto e incerto, utilizavam-se então as análises químicas (Guntau, 1997, p. 212). Por sua vez, o sistema de classificação de Romé de lIsle baseava- se nos aspectos formais do sistema de classificação proposto por Lineu, ou seja, o uso da forma do cristal para classificação e a insis- tência na hierarquia das classes minerais. Em seu Ensaio de cristalografia, de 1772, Romé de LIsle argumentava que os cristais eram ordenados de acordo com a forma e encadeados juntos em grupos de formas secundárias derivadas de algumas primárias, por meio de partições imaginárias. Ele afirmava que os cristais eram compostos por pequenas moléculas integrantes salinas, elas próprias compostas por moléculas constituintes ácidas e alcalinas. Cada mineral possuía uma estrutura e uma composição fixadas. Portanto, mantinham-se as classes minerais então necessárias para a taxonomia lineana (Laudan, 1987, p. 76; Hooykaas, 1994, p. 56). AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Aceitar a nova nomenclatura significava, assim, aderir às novas idéias (Bensaude-Vincent, Stengers, 1996). Por sua vez, o curso de mineralogia programado para ser feito com o professor Le Sage não foi realizado sob a responsabilidade deste, mas do professor Guillot Duhamel, na Escola de Minas de 689 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA ambos os personagens na Universidade de Coimbra, espaço que se caracterizava por apresentar um enfoque eclético e pragmático. Da mesma forma que Couto, também um estudioso da mineralogia, José Bonifácio foi aluno do paduano Domenico Vandelli, primeiro lente de química da Universidade de Coimbra e também professor de história natural, assim como principal expoente do subgrupo de naturalistas da Academia Real das Ciências de Lisboa, de cujos quadros participava o filósofo natural. Na Universidade de Coimbra, Vandelli seguia o método de Lineu nas cadeiras em que lecionava. Após o período na França, os filósofos dirigiram-se, seguindo a Instrução, para Freiberg, na Saxônia, centro mais avançado em mineração e estudos correlatos da Europa, além de sediar a primeira academia de minas do mundo, a Bergakademie Freiberg. Deveriam freqüentar o curso de minas daqueles distritos, assim como assentar naquele local praça de mineiros, para adquirirem todos os conhecimentos práticos. Ambos receberam autorização da Superintendência das Minas para seguir pelas obras de mineração e as instalações de depuração e lavagem a elas pertencentes (Falcão, III, op. cit., p. 173). José Bonifácio assistiu ao curso de orictognosia e geognosia dado pelo professor Abraham Gottlob Werner, de quem se tornaria discípulo (idem, ibidem, p. 176). A geognosia (literalmente, conhecimento da terra) era o campo da mineralogia relativo à classificação das massas das rochas e suas relações espaciais. Os geognostas, como eles próprios se chamavam, tentavam definir e descrever as formações que seriam reconhecidas para além de uma simples região, alcançando escalas globais. A tarefa de reconhecer formações em diferentes locais e, assim, fazer a classificação tão aplicável quanto fosse possível foi tentada empiricamente por diferentes critérios (Rudwick, 1997). p p Abraham Gottlob Werner foi sem dúvida o responsável pela institucionalização da geognosia. Ele não foi o criador desta ciência, uma vez que ela era o resultado científico de um saber muito mais antigo, originário da Europa Central e da Suécia, sendo que outros autores já haviam usado a expressão em suas publicações (Ellemberger, 1994, p. 246). Para o saxão, a geognosia era uma subdivisão da mineralogia. Ela distinguia-se da mineralogia geográfica, que estudava a distribuição das rochas e dos minerais pela superfície, e da orictognosia, o conhecimento das substâncias fósseis do subsolo. Uma vez formado em Freiberg, José Bonifácio partiu em direção a outras regiões de minas da Saxônia e Boêmia, e a outras localizadas na Hungria e na Áustria. ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA O uso de sistemas de classificação tão distintos na prática científica de José Bonifácio de Andrada e Silva não era apanágio apenas deste filósofo natural. José Vieira Couto, outro estudioso da filosofia natural daquela época, em sua prática científica de classificação dos minerais, também utilizava diversos sistemas de classificação (Silva, 2002, pp. 72-4). Tal tendência pode ser explicada pela formação de 690 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA A seguir foram em direção à Hungria, pois receberam autoriza- ção para visitar as minas e usinas metalúrgicas locais (Falcão, III, op. cit., p. 176). Na Áustria estiveram, na Caríntia e Estíria e visitaram as salinas de Gmünden. Partiram depois para várias regiões da Itália. Neste país José Bonifácio fez importantes contatos científicos, como, por exem- plo, o físico Alexandre Volta, em Pávia, na província de Turim. Ao visitar as montanhas Euganei, no sul de Pádua, escreveu uma memória lida na Academia Real das Ciências de Lisboa, dissertação que se encontra atualmente perdida, sobre a sua viagem geognós- tica aos montes Eugâneos, no território de Pádua. Nesta memória, Andrada seguia as idéias netunistas do geognosta alemão Werner, que afirmava que a água dos mares era o agente principal da formação da crosta terrestre, em contraposição ao escocês James Hutton, defensor da corrente de pensamento denominada plutonista, que enfatizava a ação interna como a responsável pela formação das rochas. A opção do filósofo pelas idéias netunistas aparece nessa memória, em que defendia uma gênese sedimentar para as rochas da região: ... fundado em observações mineralógicas, diversifico da opinião de Strange, Ferber, Fortis e Spallanzani, que atribuem origem vulcânica às rochas que formam estes outeiros (Falcão, I, op. cit., p. 145). Após a vista à Itália, os filósofos deveriam ir às minas de Ekatharinemburgo, na Rússia, o que não ocorreu, assim como não foram à Inglaterra, onde deveriam visitar as minas da Escócia e do País de Gales. Também não foram à Espanha, visitar sobretu-do as minas de Almadén. Contudo estiveram nos países nórdicos. Na Suécia, Bonifácio recebeu autorização para penetrar as usinas de ferro e de prata, bem como ter ingresso nas minas desses metais, além de ter visitado Svenska Bergslagen, região da Suécia Central rica em minas e minérios (Falcão, III, op. cit., p. 172). Cabe registrar que Bonifácio foi admitido como membro da Real Academia de Ciências de Estocolmo, no dia 25 de outubro de 1797 (idem, ibidem, p. 183). Já na Dinamarca, na cidade de Kungsberg, Bonifácio visitou as usinas de ferro e de prata, bem como as minas desses metais. Em relação à Noruega, sabemos que ali esteve, pois recebeu autorização para ir da Suécia àquele país. Contudo não obtivemos informações sobre suas atividades científicas no local. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Em sua visita à cidade de Berlim, na Prússia, foi admitido como membro da Sociedade dos Amigos da Natureza de Berlim, no dia 17 de janeiro de 1797. 691 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA todo descrevia 12 espécies minerais, sendo quatro até então desconhecidas e as demais, oito variedades de minerais. Para tais pesquisas, como o próprio Andrada afirmou, foi de extrema impor- tância a ajuda do professor Abilgaard (Peter Christian Abilgaard, 1740-1801), que o auxiliou nas análises em Copenhague. A descrição dos minerais foi feita seguindo a sua própria maneira, assim como os resultados das análises que já havia feito de alguns deles, junto com os outros, que no momento são o objeto da minha ocupação, e daqueles que o professor Abilgaard havia se comprometido em fazer as análises em Copenhague (Falcão, I, op. cit, p. 87). Embora Bonifácio tenha anunciado que as descrições dos minerais estavam baseadas em seu próprio método, este diferia pouco dos utilizados pelas escolas cristalográficas da época e por Werner, que descreveu os minerais com base em suas propriedades e caracte- rísticas externas. Abraham Gottlob Werner também foi o grande responsável pela classificação do reino mineral. Rejeitando a aplicação ao reino mineral do sistema de classificação proposto por Lineu para o vegetal, baseado no sistema sexual das plantas, o mineralogista saxão afirmou que os minerais deveriam ser classificados de acordo com sua composição, uma vez que nela residia a característica essencial. Os minerais seriam classificados levando-se em conta suas características externas e sua composição química (Laudan, op. cit., p. 81). No ano de 1774, Werner publicou uma obra intitulada Sobre as características externas dos minerais, em que apresentava uma técnica para identificar os minerais por meio dos sentidos humanos. Entre elas estavam forma, superfície, brilho externo, fratura, forma dos fragmentos, transparência, traços, cor, dureza, flexibilidade, adesão à língua e som. Descreveu as características individuais dos minerais de forma detalhada e subdividiu-as de uma maneira que as maxi- mizava segundo a utilidade da identificação. Apenas para a cor vermelha, Werner distinguiu 13 variedades diferentes. Werner estava convencido da importância das características externas, não apenas para a identificação dos minerais, mas tam- bém para o estudo da sua composição. Alegava que, se a aparência de um mineral muda quando sua composição química é alterada, devia haver uma correlação entre esta química e as características externas. Por outro lado, reconhecia que as características externas não podiam formar a base de um sistema natural. Ele estava con- vencido, em definitivo, de que os sistemas minerais deveriam ser baseados na composição química e nas propriedades e características externas. ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Sobre a viagem pelos países nórdicos, Bonifácio escreveu a memória Exposé succinte des caractéres et des propriétés de plusieurs nouveaux minéraux de Suéde et de Norwége, avec quelques observations chimiques faites sur ces substances. O documento consiste na descrição das espécies minerais pesquisadas pelo autor durante suas viagens pela Suécia e Noruega, enviada a Monsieur Beyer, inspetor de minas em Schneeberg. Ao 692 AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Por meio do diagnóstico de combinações específicas de qualidades e baseado em características externas, os tipos minerais poderiam ser reconhecidos rapidamente e por métodos relativa- mente simples. Werner tornou-se muito famoso e foi considerado, 693 vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA por algum tempo, o supremo mestre de um método de identificação incomparável na mineralogia. Com seu trabalho estabeleceu uma perfeita versão do método histórico natural de identificação mineral e, simultaneamente, uma metodologia para a mineralogia como disciplina, que começou a emergir como ciência distinta da história natural (Laudan, op. cit.). Além de Werner, o cristalografista francês Romé de lIsle (1736- 1790) também dispôs um conjunto de características externas como determinantes das espécies minerais. Entre elas estavam a forma cristalina, a dureza e o peso específico. Contudo o trabalho deste homem de ciência francês difere do trabalho do saxão pelo fato de aplicar o sistema de classificação de Lineu ao reino mineral. Bonifácio, ao descrever os minerais, baseou-se em propriedades e características externas como cor, peso específico, forma dos fragmentos, textura, transparência, brilho, presença de formas cristais, clivagem e local de ocorrência, ou seja, as mesmas utilizadas por Werner e Romé de lIsle em seus diferentes sistemas de classificação. Os quatro minerais descritos pela primeira vez na memória,de acordo com as suas características e propriedades externas foram: Espodumênio (Li Al [ Si2 O6 ]) Cor: branco esverdeado; Peso específico: 3,218; Dureza: risca o vidro e é riscado pelo quartzo; Formas cristais: [não menciona]; Clivagem: sólidos de clivagem romboidal; Transparência: pouco transparente; Textura: lamelar; Brilho: do tipo madrepérola; Local de ocorrência: encontrado na formação de ferro da Ilha de Utö (ao sul de Estocolmo). Dureza: risca o vidro e é riscado pelo quartzo; Formas cristais: [não menciona]; Clivagem: sólidos de clivagem romboidal; Transparência: pouco transparente; Textura: lamelar; Brilho: do tipo madrepérola; Local de ocorrência: encontrado na formação de ferro da Ilha de Utö (ao sul de Estocolmo). Petalita (Li [ Al Si4 O10 ]) Petalita (Li [ Al Si4 O10 ]) Cor: rosa. Peso específico: 2,620; Dureza: risca o vidro; Formas cristais: {não menciona]; Clivagem: [não menciona]; Transparência: bordas pouco transparentes; Textura: foliada; Brilho: comum e brilhante, com um pouco de esplendor Local de ocorrência: encontrado próximo à Ilha de Utö, Sala e Fingruvan, próximo a Koppaberg, na Suécia. Petalita (Li [ Al Si4 O10 ]) Cor: rosa. Peso específico: 2,620; Dureza: risca o vidro; Formas cristais: {não menciona]; Clivagem: [não menciona]; Transparência: bordas pouco transparentes; Textura: foliada; Brilho: comum e brilhante, com um pouco de esplendor Local de ocorrência: encontrado próximo à Ilha de Utö, Sala e Fingruvan, próximo a Koppaberg, na Suécia. Criolita (Na3 AlF) Cor: branca como a neve; Peso específico: 2,9698; História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Dureza: risca a calcita, riscada pela fluorita; Formas cristais: [não menciona]; Clivagem: sólidos de clivagem cúbico; Transparência: [não menciona]; Textura: espesso e bastante folheado; Brilho: pouco brilhante; Local de ocorrência: Groenlândia. Dureza: risca a calcita, riscada pela fluorita; Formas cristais: [não menciona]; Clivagem: sólidos de clivagem cúbico; Transparência: [não menciona]; Textura: espesso e bastante folheado; Brilho: pouco brilhante; Local de ocorrência: Groenlândia. Textura: espesso e bastante folheado; Local de ocorrência: Groenlândia. Escapolita (um grupo de minerais semelhante ao grupo de feldspatos da classe dos plagioclásios, constituindo uma série de cristais mistos); Cor: branco amarelado ou branco acizentado; Peso específico: 3,680-3,708; Dureza: risca o vidro; Formas cristais: forma colunas quadriláteras quase retangulares; Clivagem: sólidos de clivagem romboidal; Transparência: pouco transparente; Textura: lamelar; Brilho: pouco brilhante; brilho externo do tipo vítreo; Local de ocorrência: encontrado nas minas de ferro próximo a Arendal, na Noruega. Com a descoberta dos quatro novos minerais e sua descrição escapolita, criolita, espodumênio e petalita , Bonifácio passou a pertencer, em 1800, a um grupo de mineralogistas reconhecidos, como I. Born, A.G. Ekeberg, R. J. Haüy, A.G. Werner, por ter descoberto toda uma série de novas espécies, em um período em que a mineralogia estava especialmente em ascensão. O reconhecimento do trabalho de José Bonifácio ocorreu no ano de 1868, quando o mineralogista americano J. Dana, em sua homenagem, deu o nome de Andradita à granada de ferro e cálcio (Ca3 Fe2 (SiO4)3) (Guntau, 2000, p. 269). vol. 11(3):685-711, set.-dez. 2004 Criolita (Na3 AlF) Criolita (Na3 AlF) Cor: branca como a neve; Peso específico: 2,9698; 694 História, Ciências, Saúde Manguinhos, Rio de Janeiro História, Ciências, Saúde Manguinhos, Rio de Janeiro ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA havia se ausentado da nação lusa durante o governo pombalino e assim pôde observar o estado da ciência em outros países europeus, como a França e a Inglaterra. Ao regressar, considerou de extrema importância a necessidade de se criar uma academia de ciências como as existentes naqueles países a Royal Society (1662), em Londres, e a Académie Royale des Sciences (1666), em Paris , para que se fomentasse a cultura científica em Portugal. O plano de criação da academia e seus estatutos, foram elaborados em conjunto pelo duque de Lafões e pelo naturalista abade José Correia da Serra, outro que estivera fora durante o período pombalino, e apre- sentado à dona Maria I, que deu parecer favorável a 24 de dezembro de 1779. A rainha tornar-se-ia, em 1783, a protetora da academia. A instituição estava dividida em três classes, duas de ciências, ciências da observação meteorologia, química, anatomia, botânica e história natural; e ciências do cálculo aritmética, álgebra, geometria, mecânica e astronomia) e uma de belas-letras, que se deveria dedicar ao estudo dos vários ramos da literatura portuguesa. Cada uma das classes tinha oito sócios efetivos, além dos sócios supranumerários, honorários e correspondentes. Possuíam um observatório matemático, um laboratório químico e dois museus de história natural, ou seja, espaços voltados para as pesquisas no campo da história natural e baseadas na observação e experi- mentação. A academia publicava as Memórias, estimulando e promovendo a produção intelectual nos mais variados campos, como mineralogia, agricultura e economia, assim como estudos que tratavam de produtos naturais como algodão, oliveira, vinha, castanheiras e carvalhos, entre outros. Por meio das Memórias, realizou-se um verdadeiro levantamento da natureza do Reino e da Colônia. Os autores refletiam sobre os obstáculos que impediam a nação lusa de se igualar aos países europeus de além-Pirineus e remédios sugeriam para superar esta defasagem econômica. O corpo acadêmico era constituído por um grande número de associados, de orientações e ofícios, como reis, clérigos, naturalistas, proprietários de terras, ministros, professores e colonos de várias posses ultramarinas, devendo assim a academia ser compreendida não de uma maneira uniforme e coesa, mas como uma agremiação que resultou de diversas correntes ou estilos de pensamento. Contudo um elemento lhes era comum: o projeto de esclarecimento da socie- dade portuguesa. A Academia Real das Ciências de Lisboa A viagem científica foi sem dúvida de extrema importância para o reconhecimento da trajetória de José Bonifácio como homem de ciência, não só em Portugal como nos meios científicos e universitários dos principais países europeus. Mas, seria o espaço da Academia Real das Ciências de Lisboa que, como afirmou um dos seus biógrafos (Sousa, op. cit., p. 66), lhe abriria os caminhos de uma carreira de filósofo e lhe traria a glória e muitas decepções, o puro gozo intelectual e todas as matérias reservadas aos que excedem a carreira comum. A academia foi criada por iniciativa de dom João Carlos de Bragança, segundo duque de Lafões e, tio de dona Maria I, que 695 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA naturalista-utilitarista (Munteal Filho, 1993). Ele e os componentes deste subgrupo composto por figuras de expressão junto aos mecanismos decisórios do Estado português e com formação básica em medicina, química e história natural esboçaram uma visão de mundo que centrava no domínio da natureza a alternativa para o processo de superação, por Portugal, da defasagem econômica com relação à Europa das Luzes. Vandelli e esses naturalistas partilhavam do princípio de que se deveria realizar um profundo inventário da natureza nas colônias, natureza que seria estudada em estabelecimentos científicos como os jardins botânicos e museus de história natural, por meio dos métodos de classificação e dissecação. Por outro lado, o conheci- mento da natureza estava diretamente relacionado à política fomentista do governo mariano e joanino, pois acreditava-se que as produções naturais da colônia ajudariam na recuperação econômica do Reino e valorizava-se a agricultura baseada nas práticas científicas de orientação pragmática, que viam na natureza tropical uma fonte geradora de riqueza. A natureza colonial deveria ser cientificamente conhecida e explorada, de modo a contribuir com a industrialização portuguesa. Nesse espaço de discussão científica e sociabilidade intelectual, o filósofo natural José Bonifácio publicou diversas memórias científicas. Em seus escritos,colocava a ciência como algo que podia ser útil para a sociedade do império colonial português. Por ciência útil devemos entender o conjunto de matérias que possibilitariam a solução ou a transformação da realidade vivida até então. José Bonifácio acreditava que o papel da ciência não se restringia ao processo de conhecimento, mas transcendia-o, pois tinha o poder de transformar a sociedade. Ele procurava tornar público os conhecimentos que produzissem meios de combate às doenças, possibilitassem a introdução de novos cultivos, permitissem baratear certos produtos e, contribuíssem para a preservação da natureza, entre outros. Em seus trabalhos científicos, a ciência tinha como função social resolver problemas. A utilidade era a vértebra de sua concepção de ciência. Esta encontra-se a serviço do homem, da sociedade. Para ele, a ciência era prática, aplicada, devia aju-dar a resolver os males que imperavam na sua época. Sua função era semear idéias úteis pela sociedade. Como ele próprio afirmava, se das minhas idéias se quiser tirar proveito, folgarei infinito de ser útil (Falcão, I, op. cit., p. 48). ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Nesse espaço ganhou destaque o paduano Domenico Vandelli, que abraçou o ecletismo do reformismo ilustrado, pelo qual se posicionou em favor de algumas idéias do mercantilismo, adotando tanto os princípios fisiocráticos italianos e franceses como os princípios da economia clássica inglesa (Novais, 1984, p. 108). Na academia, Vandelli era como o principal expoente do subgrupo da vertente 696 História, Ciências, Saúde Manguinhos, Rio de Janeiro vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA estatal para superação da crise, o que mostra a tomada de consciência, pelo autor, da situação em que Portugal se encontrava no momento. E o que justifica a análise particular de cada uma das Memórias da sua fase portuguesa José Bonifácio observava como o econômico, o político e o científico são indissociáveis. Em uma nota reafirmou a necessidade da aplicação do conhe- cimento científico em prol da sociedade: Desde que eu começei a pensar que as ciências eram um emérito fútil quando não se aplicavam ao bem público, não pude deixar de espantar-me vendo o desleixo dos sábios e o pouco caso que faziam do bem público (IHGB, 192, 36, fl. 4). Essa preocupação com a utilidade da ciência, ou melhor, com o conhecimento científico destinado ao uso e aperfeiçoamento da humanidade, mostra a presença das idéias baconianas nas memórias escritas por Bonifácio. Mostrava-se ele, assim, amplamente conectado ao pensamento científico moderno, uma vez que buscava tornar o conhecimento científico algo prático e útil. Ao propor que a ciência devia gerar utilidades para a sociedade, contribuindo para solucionar os problemas que nela existissem, acreditamos que ele partilhava da utopia do pensamento ilustrado, a concepção de que o conhecimento científico contribuiria para o aperfeiçoamento das sociedades, tornando-as melhores e perfeitas (Manuel, Manuel, 1979). A divulgação das pesquisas científicas de José Bonifácio em memórias, anais, revistas, periódicos, boletins etc. demonstra clara- mente a presença no seu pensamento do ideal ilustrado de esclare- cimento, a função educadora que os sábios e letrados deveriam cumprir na sociedade. Em suas memórias científicas, o principal objeto de estudo era a natureza. Para conhecê-la, Bonifácio submetia-a à observação e à experimentação. A natureza era a sua grande aliada na luta pelo conhecimento revelado. Ele buscava encontrar no reino natural os princípios que regiam o mundo e procurava arrancar o seu segredo, submetê-lo à luz do entendimento e penetrá-lo com os poderes do espírito. A natureza seria o locus perfeito para o exercício da sensibilidade e da razão. José Bonifácio estudava os três reinos do mundo natural animal, vegetal e mineral por meio de suas características intrín- secas, identificando, classificando, ordenando e fazendo uma sistematização taxonômica de cada espécie natural. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA A intensa difusão de conhecimentos científicos que há na obra de José Bonifácio deve ser entendida como um verdadeiro planejamento racional de ações voltadas para o futuro e para projetos prospectivos baseados em análises históricas sistematizadas e atualizadas. As memórias publicadas eram parte de um planejamento 697 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA como fonte de conhecimento, e portanto deveriam ser conhecidas cientificamente. Mas também as entendia como fonte de riquezas, porque seriam capazes de gerar lucros para que a Coroa portuguesa fomentasse sua economia e industrialização. Por ele a natureza era encarada de forma quase divina, como produtora de riquezas e como mestra da própria vida (Munteal Filho, Kury, 1995, p. 116). ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Partia da obser- vação detalhada dos fatos para estabelecer a classificação, para encontrar assim na natureza as suas próprias leis, e seguia, como já mencionamos, uma variedade de sistemas de classificação, como os de Lineu, Werner, Wahlerius, Lamarck e Brotero. Em suas memórias transparece uma perspectiva bastante otimista dos elementos do mundo natural. As produções deste eram vistas 698 vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA empresa, o dr. Barradas deveria remeter para a universidade, mais precisamente para o Gabinete de História Natural e do Jardim Botânico, as coleções que recolhesse de produtos e plantas, com suas descrições competentes. O responsável pelas instruções dessa viagem filosófica foi o dr. José Bonifácio, que as elaborou em 10 de dezembro de 1806 (Loc.: BN Manus. 5,4, 11, folha 1). Recordemos que o envio de expedições filosóficas para os domínios ultramarinos, sobretudo para o Brasil, fazia parte das iniciativas do governo mariano no sentido de promover um maior conhecimento sobre as produções naturais das colônias. Os naturalistas responsáveis por essas viagens deveriam ir ao local estipulado, recolher as espécies naturais que encontrassem e, depois, enviá-las aos estabelecimentos científicos lisboetas, onde seriam experimentadas, aclimatadas e tornadas úteis ao Reino português. Neste caso, as instituições científicas que deveriam receber as remessas dos produtos da viagem do dr. Barradas seriam o Gabinete de História Natural e o Horto Botânico, ambos da Universidade de Coimbra, espaços por excelência de pesquisa e classificação dos produtos do mundo natural. Além de ter elaborado as instruções, o lente de metalurgia mostrou todo o seu conhecimento sobre os produtos do mundo natural da colônia americana ao elaborar uma lista imensa com nomes de peixes, aves, mamíferos, anfíbios, madeiras, frutos, raízes, entre outros, que o dr. Barradas deveria tentar coletar e enviar ao Gabinete de História Natural e ao Horto Botânico. Durante o tempo em que esteve na universidade, Bonifácio pôde observar como andava o funcionamento da instituição. Suas observações não diagnosticaram um bom desenvolvimento das ciências naquele espaço institucional. Tendo estudado e conhecido as principais escolas de minas da época, como Freiberg e Paris, pôde observar a defasagem que havia entre aquelas e a reformada Universidade de Coimbra, sobretudo no campo das ciências naturais. Em notas pessoais e cartas a importantes homens da viradeira, como dom Rodrigo de Souza Coutinho, expressou toda a sua insatisfação em relação ao ensino praticado pelo corpo docente conimbricense e a administração universitária. Para além das queixas, Bonifácio teve sempre um reduzido número de alunos, cerca de cinco por ano, e as verbas para a compra de equipamentos para a realização das aulas práticas e das pesquisas foram sempre bastante minguadas. Também não tinha o museu científico da universidade uma boa coleção mineralógica, que, como ele próprio afirmou, servisse e valesse coisa alguma. A cadeira de metalurgia na Universidade de Coimbra Além do espaço institucional da Academia Real das Ciências de Lisboa, José Bonifácio também desenvolveu atividades de pesquisa no campo da história natural em outras instituições portuguesas. Pela carta régia de 21 de janeiro de 1801, o príncipe regente dom João determinou que a cadeira de agricultura da Universidade de Coimbra fosse separada da cadeira de botânica do curso de filosofia. Esta última voltava a ser incorporada à zoologia e à mineralogia na cadeira de história natural. Pela mesma carta régia, dom João criou a cadeira de metalurgia, que deveria ser ensinada no quarto ano do curso de Filosofia, juntamente com a de agricultura. Para lente da cadeira de metalurgia foi nomeado o filósofo José Bonifácio de Andrada e Silva, pela Carta Régia de 15 de abril de 1801. A justificava para tanto encontrava-se no fato de ele ter viajado como pensionário meu [dom João] por espaço de dez anos, com conhecido aproveitamento, por países em que esta ciência [metalurgia] principalmente se cultiva, observado a natureza em grande, e estudado todas as práticas que lhe são relativas. Na mesma carta, dom João nomeava-o quinto lente da Faculdade de Filosofia, conferia-lhe gratuitamente o grau de doutor na mesma faculdade e, além do ordenado de quinhentos mil-réis próprio do quinto lente proprietário, receberia a quantia de trezentos mil-réis a cada ano, pelos penosos trabalhos das viagens que fez continuadas pelo longo espaço dos referidos anos a fim de se habilitar para o meu Real Serviço (Ata de 15.5.1801, 1978, p. 264). Não encontramos qualquer regulamento ou programa da cadeira de metalurgia em nas coleções de manuscritos que estão nos arquivos e bibliotecas pesquisados. Por sua vez, pelo que está registrado nas atas das reuniões da Congregação da Faculdade de Filosofia, sua participação foi bem pequena. Em uma das que esteve presente, a reunião de 25 de maio de 1808, foi um dos que propôs a adoção do Traité elementaire de minéralogie, de Alexandre Brogniart, para ser o compêndio de mineralogia (ibidem, p. 321). José Bonifácio também foi encarregado de elaborar as instruções para uma expedição filosófica da Universidade de Coimbra. O príncipe regente encarregou o dr. Luiz Antonio da Costa Barradas de realizar uma viagem pela capitania de Pernambuco. Nessa 699 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA como aqueles relativos a ausência de verbas e apoio governamental foram fatores importantes para a falta de sucesso de tal empresa. Mesmo assim entendemos a atitude de criar a cadeira de metalurgia como um esforço das autoridades portuguesas em tentar colocar a universidade em pé de igualdade com aquelas de Além-Pirineus, uma vez que arregimentou para esse fim mr. DAndrada, filósofo que havia viajado pelas nações ilustradas da Europa e adquirido os principais conhecimentos metalúrgicos da época. José Bonifácio foi jubilado da cadeira de metalurgia no dia 29 de julho de 1813 e, por decreto de 12 de outubro de 1822, foi desligado da universidade, três anos após o seu regresso para o Brasil. Pode ter sido após o insucesso da cadeira de metalurgia, apenas uma mera hipótese, que José Bonifácio redigiu um manuscrito no qual apresentava uma série de fatores que impediam o desenvolvimento das ciências naturais em Portugal. Entre essas causas estavam: a falta de museus, gabinetes de física e laboratórios; a ausência do estudo das ciências naturais no plano de educação dos jovens; a falta de sociedades econômicas e patrióticas para espalhar as luzes; o péssimo estado das ciências naturais na Universidade de Coimbra; a não-extração, ou má mineração dos metais; e o pequeno número de imprensas e de governadores hábeis para abrir estampas (Dolhnikoff, 1998, pp. 340-1). Todos esses fatores, na visão do autor, contribuíam para a não-prosperidade das ciências naturais em Portugal, impedindo assim que o país superasse a situação de defasagem cultural-científica no qual se encontrava em relação aos países de Além-Pirineus. ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Portanto, pelo que podemos observar nos documentos da época, parece que Bonifácio não foi feliz no seu empreendimento de tentar criar e institucionalizar a cadeira de metalurgia na Universidade de Coimbra. Os empecilhos postos pela própria universidade, assim 700 vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA filósofo natural e o de homem público passaram a caminhar lado a lado na história de vida do personagem, não podendo ser dissociados. Não são duas carreiras diferentes ou sucessivas, mas, durante toda a sua estada em Portugal, ele levou simultaneamente uma vida de funcionário do Reino e uma vida de naturalista. José Bonifácio era um típico representante dos laços estreitos que se criavam durante o reformismo ilustrado português mariano e joanino entre os sábios e o governo, como já assinalamos. Essa cooptação dos naturalistas pelo Estado, sobretudo no final do século XVIII, permite observar a valorização daqueles que detinham o conhecimento científico e técnico, sobretudo para dar parecer sobre os mais variados assuntos econômico-administrativos. Em síntese, isso demonstra o reconhecimento do poder da ciência pelo Estado (Matos, 1998). p No período da viradeira, ocorreu uma forte identificação entre ciência e política, ou melhor, entre aqueles que produziam o conhecimento científico e os que eram capazes de arregimentar apoio e recursos financeiros necessários ao desenvolvimento das ciências. O Estado burocrático português arregimentou os naturalistas da Academia Real das Ciências de Lisboa com o intuito de acumular várias tarefas, entre as quais se podem destacar o mapeamento, o diagnóstico, o conhecimento e a orientação de políticas direcionadas ao levantamento das riquezas naturais, ou melhor, das produções naturais do território português e de todo o seu império ultramarino. Esse fato permite observar o quanto a academia, por meio das suas propostas de caráter científico, estava extremamente conectada ao Estado português (Munteal Filho, 2001, pp. 48-9 ). Exemplo disso é o caso de arregimentação do naturalista José Bonifácio de Andrada e Silva pelo ministro da Marinha e Ultramar, dom Rodrigo de Sousa Coutinho, para ocupar uma série de cargos públicos estatais no campo da esfera administrativa. Dom Rodrigo não pouparia esforços para gastar os recursos necessários à pesquisa das produções naturais do Reino de Portugal, sobretudo os minerais, e para a preservação de matas e bosques. p p q José Bonifácio foi nomeado intendente das Minas do Reino de Portugal pela Carta Régia de 18 de maio de 1801. Pela mesma carta foi encarregado de dirigir e administrar as Minas e Fundições de Ferro de Figueiró dos Vinhos. Para tal, seria condecorado com uma beca honorária com o predicamento de primeiro banco, e ficaria mantida a pensão de oitocentos mil-réis de que havia gozado durante o tempo das suas viagens pela Europa. Associando os estudos científicos à administração pública das minas e bosques: a associação do perfil de filósofo natural e homem público na trajetória do ilustrado José Bonifácio de Andrada e Silva A grande atuação que o filósofo natural José Bonifácio vinha tendo no âmbito da Academia Real das Ciências de Lisboa despertou a atenção de alguns de seus membros, como dom Rodrigo de Sousa Coutinho, ministro da Marinha e Ultramar, que admirava o trabalho do Andrada e via nele o homem indicado para a realização de seus projetos. Assim é que ele foi chamado para criar a cadeira de metalurgia da Universidade de Coimbra, participando ativamente da Ilustração portuguesa. Juntamente com esse cargo, assumiu outros na vida pública portuguesa, como o de intendente geral das Minas e Metais do Reino, membro do Tribunal das Minas, administrador das antigas minas de carvão de Buarcos, entre outros. A partir desse momento, passou a ter de dividir seu tempo entre os estudos científicos e os cargos estatais, sobretudo aqueles relativos à esfera administrativa portuguesa. Em outras palavras, o perfil de 701 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Por sua vez, pela carta Régia do príncipe regente de 1º de julho de 1802, o naturalista José Bonifácio foi arregimentado para assumir a direção da administração das sementeiras e plantações nos areais das costas portuguesas, que começou por Couto de Lavos. Essa carta vinha associar a administração das matas e bosques à das minas, na pessoa do naturalista José Bonifácio, homem que viajara por diversos países europeus e havia feito contato com os modernos conhecimentos científicos relacionados a esses ramos da administração pública. Esse fato mostra a importância que o Estado burocrático português dava aos filósofos naturais, e, no caso em questão, a necessidade de minas e bosques serem regulados por princípios científicos, com o objetivo de promover a utilidade pública. Um novo regimento para o funcionamento das minas e estabelecimentos metálicos do Reino foi mandado criar pelo príncipe regente, em função da criação da intendência e da nomeação de José Bonifácio. O alvará de 30 de janeiro de 1802 definiu a competência do intendente geral das Minas e Metais do Reino e sua respectiva área de atuação. O intendente geral seria o diretor e administrador das Minas e Ferrarias de Portugal, estando a ele subordinadas todas as pessoas e oficiais que nela prestassem assistência e trabalhassem, assim como todos os indivíduos empregados nas minas e estabelecimentos minerais portugueses, fossem eles funcionários do rei, fossem de companhias particulares de mineração e apuração. Além de administrar as minas, também ficava encarregado da direção e administração de bosques e matas. Contudo, no ano de 1804, por Decreto de 4 de maio, a administração das minas e estabelecimentos mineiros do Reino foi entregue à direção da Fábrica das Sedas e Obras das Águas Livres. Esse decreto anulava as amplas atribuições e poderes concedidos ao Intendente para superintender em tudo que dissesse respeito as minas, ferrarias, bosques e matas, concedidos pelo Alvará supracitado de 1802. Diniz (1939: 31) justificou tal ato em função da demissão de D. Rodrigo de Sousa Coutinho, organizador da Intendência Geral das Minas e Metais do Reino, e inspetor geral das mesmas, do cargo de ministro da Fazenda, presidente do Real Erário, e amigo pessoal de José Bonifácio. Luiz de Vasconcelos e Sousa lhe sucedeu na presidência do Real Erário e entregou a administração mineira à direção da Fábrica das Sedas. ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Ainda na mesma carta, ficava encarregado de estabelecer e firmar o ensino da cadeira de metalurgia na Universidade de Coimbra durante o período de seis anos. Após esse tempo, ficaria unicamente ocupado da Intendência das Minas e Metais, assim como das de Figueiró dos Vinhos, e da abertura das minas de carvão de pedra. 702 História, Ciências, Saúde Manguinhos, Rio de Janeiro vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA e por eliminar as crises periódicas de combustível que, no passado, sempre haviam obrigado as nações a moverem-se em busca de florestas por cortar. Os principais centros industriais deslocaram-se para a vizinhança de centros carboníferos, e houve uma melhoria no processo de exploração do carvão mineral, com a utilização de máquinas a vapor para retirar a água acumulada nas minas. Por sua vez, o uso do ferro, sobretudo o do ferro fundido, veio substituir a produção de ferro por redução do minério, processo que deixava o produto cheio de impurezas e difícil de refinar. A falta do carvão vegetal necessário para fundir grandes quantidades de metal também foi um dos fatores da mudança. O ferro fundido era utilizado principalmente na manufatura de armas, em especial na de canhões. José Bonifácio apresentou à Academia de Ciências algumas memórias mineralógicas. Nesses textos ele apresentava as atividades práticas de mineração nas regiões que pesquisava, assim como descrevia minuciosamente os minerais, o local onde estes eram encontrados e sua importância para o desenvolvimento da nação portuguesa. Essas memórias relacionavam-se ao cargo estatal que Andrada exercia como intendente geral das Minas e Metais do Reino. Até então, as artes mineiras ou seja, desenvolvimento, lavra, trata- mento e fiscalização das explorações das minas haviam estado sob a alçada do Corpo de Oficiais de Artilharia (Ferreira, 1988, p. 30). Pela primeira vez o cargo era ocupado por um filósofo natural dedicado à pesquisa e investigação da natureza mineral. Nas memórias evidencia-se a tentativa de seu autor de inventariar o estado da arte da mineração em Portugal. Nelas Bonifácio mapeava os problemas existentes na atividade mineradora e apresentava propostas para superar os entraves existentes ao seu desenvolvimento. O filósofo tentava fazer um levantamento extenso e pormenorizado das riquezas minerais presentes no solo português e destacava suas potencialidades para a nação. A mineração, ao lado da agricultura, constituía a base fundamental das riquezas permanentes do Estado luso. Por meio das suas memórias científicas, destacadamente as mineralógicas, José Bonifácio ajudou a criar e a sustentar uma rede de informação (Domingues, 2000) que permitiu ao Estado do período da viradeira conhecer de forma mais aprofundada e precisa todo o território português, ou seja, reconhecer os limites físicos da sobe- rania, bem como as potencialidades econômicas do território administrado. Todas as informações fornecidas pelo naturalista e recebidas pelos dirigentes do Estado deveriam contribuir para o conhecimento global do espaço luso. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA No âmbito da intendência, José Bonifácio esforçou-se por tentar encontrar no subsolo português dois elementos de extrema importância, sobretudo com o advento da Revolução Industrial: o ferro e o carvão. O carvão mineral sob a forma de coque veio substituir a madeira, combustível e material estrutural básico de todas as civilizações anteriores. Esse novo elemento caracterizava-se por ser mais barato 703 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA alimentaria uma ciência especulativa ou teórica. O saber científico tinha um caráter eminentemente prático, pois a ciência que ele praticava tinha como fim ser útil. As descrições e amostras de produtos, sobretudo os minerais, recolhidos durante suas viagens de campo por diversos pontos do território português destinavam- se não só à inventariação, catalogação e classificação das espécies, ou ao reconhecimento das potencialidades naturais. Deveriam contribuir para o desenvolvimento econômico do Reino, para o incremento das indústrias, das manufaturas e do comércio, entre outros fatores. As informações científicas contidas nas memórias de Bonifácio estavam baseadas na observação e na experimentação. O conhe- cimento científico, para ele, devia ser prático e experimental. A ciência que o entusiasmava era aquela de matriz baconiana, que tinha como função resolver problemas práticos. A essa característica juntava-se o fato de ele sempre fazer análises prospectivas em seus estudos e propor a necessidade de utilizar os recursos naturais de forma planejada e racional, pois eles apresentavam grandes poten- cialidades econômicas para o Estado português. Dessa forma, pode- se afirmar que o conhecimento científico estava integrado a um programa que, desenvolvido na Intendência das Minas e Metais do Reino e publicado em memórias na Academia Real das Ciências, tinha repercussões na ciência, na economia e na política. p , p As memórias elaboradas por Bonifácio se referiam a trabalhos práticos concretos, descritos nos menores detalhes. Elas explici- tavam como essa política portuguesa de aproveitamento racional dos recursos naturais, sobretudo os minerais, foi efetivada e posta em prática pela Intendência das Minas, locus de produção científica e que ajudava a criar e sustentar as redes de informação. As memórias mineralógicas constituíram verdadeiros estudos analíticos das potencialidades minerais do país, por meio de exames cuidadosos de detalhes, de trabalhos de campo, de mapeamentos acoplados às informações históricas obtidas tanto de documentos de arquivos como de ruínas arqueológicas que muitas vezes datavam da ocupação romana do território português ou dos antigos reinados. Originavam-se também do conhecimento empírico acumulado pelos lavradores, habitantes rústicos do local. Isso representa que a política da intendência parecia priorizar as regiões de algum modo já conhecidas, com potencialidades minerais a serem checadas, confir- madas e, mais uma vez, exploradas racional e cientificamente. A quantidade de minerais identificados por José Bonifácio em seu trabalho na intendência vinha ao encontro de uma política estatal que tinha como objetivo a produção mineral. ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA As informações presentes nas memórias do naturalista José Bonifácio não se destinavam a fins meramente administrativos, nem 704 vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Quanto à prática científica de José Bonifácio, observamos que, no campo da mineralogia, ele seguiu o common sense dessa ciência, no período do final do século XVIII e início do século XIX, inserindo- se em suas correntes principais tanto pelos termos que empregava como pela metodologia de trabalho. Ele preocupava-se em descrever, identificar e classificar os materiais minerais em seu local de ocorrência, dando ao seu trabalho um caráter geográfico no qual o trabalho de campo adquiria papel essencial. Uma outra característica de sua prática científica foi a ênfase na observação das regularidades permanentes. A prática científica de José Bonifácio, analisada pelo exame das memórias, insere-se em uma tradição de pesquisa que buscava relatar o que Kenneth Taylor (1988, p. 2) chamou de regularidades permanentes. O estudo de tais regularidades, também denominadas condições gerais ou constantes ou regularidades de disposição, era uma prática domi- nante nos trabalhos geológicos do século XVIII, estando presente em Buffon, Louis Bourguet, Nicolas Desmarest, Horace Benedict de Saussure, Jean-André Deluc, entre outros. O interesse em identificar e estudar as regularidades refletia o empirismo habitual da época, assim como o desejo de generalizações, de se criarem leis no domínio da geologia. Os autores citados estavam preocupados em estudar os grandes traços dos continentes e dos mares: altura, localização, orientação e espessura das montanhas, o movimento das águas dos mares e dos rios, a disposição das camadas estratigráficas, os minerais presentes em tais camadas, entre outras regularidades. Cabe ressaltar ainda que, nos trabalhos desses autores, imperava o estudo das regularidades estáticas, entendidas como conseqüência de um processo, e não como suas causas, a explicação de como um deter- minado fenômeno ocorreu. José Bonifácio enfatizou, em suas memórias, as regularidades estáticas, buscando sempre apontar o local das minas, fazer a descrição do terreno, identificar os materiais que o formavam, a quantidade de minerais, como estavam contidos nas camadas estra- tigráficas, cor, forma, tamanho, peso e dureza, se estavam em profundidade ou superfície. Estas são as principais regularidades observadas pelo filósofo em suas dissertações. Embora não tenha se dedicado enfaticamente às reflexões teóricas sobre a formação da crosta terrestre, o que mais lhe interessava era conhecer as potencialidades econômicas dos minerais, para, assim, ajudar a resolver os graves problemas econômicos que Portugal enfrentava naquele momento. José Bonifácio foi um naturalista que se caracterizou pelo ecle- tismo e pragmatismo, características do pensamento ilustrado do século XVIII. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Em função disso, ele examinou as ocorrências de diversos minerais, como ouro, chumbo, ferro e prata, entre outros. 705 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA não considerava de utilidade imediata. Um exemplo claro desse ecletismo era a utilização, pelo autor, de diferentes sistemas de classificação dos minerais, como os de Lineu, Wallerius e Werner, o que lhe permitiu classificar inclusive quatro novos minerais, como já comentamos. A recorrência a diversos sistemas era necessária para que ele pudesse conhecer e identificar os produtos minerais úteis aos interesses da Coroa portuguesa. ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA O próprio Voltaire afirmava: Meu amigo, sempre fui eclético. E assim também agia Bonifácio, que bebia em todas as fontes e tirava delas sempre o melhor, deixando de lado aquilo que 706 vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA império português centralizado na América. Estadistas como dom Rodrigo tinham como missão precípua a fundação de um novo império que teria como sede o Rio de Janeiro e que deveria impor- se sobre as demais capitanias (Dias, 1986). Para esse trabalho contaram com a colaboração e o empenho dos ilustrados coloniais, ganhando destaque, entre eles, José Bonifácio, imbuído de idéias reformadoras, mas sempre no intuito de orientar a Coroa e não de romper com ela (Maxwell, 1999). A vinculação do letrado com a idéia de um Império centrado nos trópicos é registrada nesse discurso de 1819, no qual José Bonifácio deixou clara a sua opção política pelo projeto de dom Rodrigo. Primeiramente afirmou jamais ter desonrado o nome de Português e acadêmico, mostrando um sentimento de nacionalidade que apontava para uma nação luso-brasleira. Em um segundo momento, quando buscou explicitar a referência à emancipação do Reino do Brasil após o ano de 1808, com- preendeu-a como realidade concreta e conseqüente de sua conversão em sede do Império. José Bonifácio reconheceu a condição de emancipação do Reino do Brasil em relação ao sistema de dominação colonial. Tal condição só poderia ter sido adquirida a partir da transferência da Corte e de sua instalação na cidade do Rio de Janeiro, a abertura dos portos e a elevação do Brasil a Reino Unido de Portugal e Algarves. Assim comentou o naturalista: Consola-me igualmente a lembrança de que da vossa parte pagareis a obrigação em que está todo o Portugal para com a sua filha emancipada, que precisa pôr em casa, repartindo com ela das vossas luzes, conselhos, e instruções (Falcão, I, op. cit., p. 472). Cabe também ressaltar que essa idéia de emancipação não implicava separação da metrópole, mas estreita união com Portugal. Esse modelo de emancipação sem solução de continuidade mantinha os laços de compromisso entre as duas partes constitutivas da monarquia portuguesa e, ao mesmo tempo, a autodeterminação na gestão dos interesses do Brasil. A antiga colônia, como afirmou Lyra, mantinha os laços de amizade e de afeto natural para com a mãe pátria (Lyra, 1994, p. 143). A seguir José Bonifácio afirmava as potencialidades do Brasil para o desenvolvimento do novo Império lusitano centralizado nos trópicos, deixando clara a opção pelo projeto político de dom Rodrigo: E que país, esse Senhores, para uma nova civilização e para novo assento das ciências! Que terra para um grande e vasto império! A despedida Na sessão de 24 de junho de 1819 da Academia Real das Ciências de Lisboa, Bonifácio apresentou um discurso que teve um tom de despedida, pois naquele momento ele deixava o antigo Portugal, que lhe havia adotado como filho, para o novo Portugal, onde havia nascido. Portanto, passados 36 anos da sua chegada ao Reino, Bonifácio voltava aos seus pátrios lares da montanhosa, mas amena Província de São Paulo (Falcão, I, op. cit., p. 445). A referência ao Brasil como um novo Portugal permite afirmar que o país não era mais visto pelo autor como uma mera extensão do Reino, mas como a sede do novo Império lusitano: a partir da transferência da Corte no ano de 1808, o Brasil tornara-se a nova sede da Coroa portuguesa. No discurso, Bonifácio tentou deslembrar-se das almas dege- neradas que procuraram às vezes atrapalhar o seu patriotismo e seus bons desejos, e descreveu sua história durante o tempo em que esteve no Reino, construindo a sua própria memória. Ele a iniciou pelos estudos jurídicos e filosóficos na Universidade de Coimbra e relatou sua entrada para Academia Real das Ciências de Lisboa, logo após a formatura, dando início à carreira nas letras. No ano de 1790, teve de se ausentar da academia, por ter sido designado pela rainha dona Maria I para viajar pela Europa com o intuito de aprofundar seus conhecimentos nos ramos da química, mineralogia e geologia. Dessa viagem relatou ter honrado, entre as nações e sábios da Europa, o nome de Acadêmico e Português (idem, ibidem, p. 446). O retorno a Portugal coincidiu com sua nomeação, no ano de 1801, para o cargo de intendente geral das Minas e Metais do Reino, o que acabou por afastá-lo da corporação por alguns anos. A esse cargo se juntariam outros, mostrando a valorização desse ilustrado sábio colonial pelos homens dirigentes lusos e sua participação de forma ativa na Ilustração portuguesa. Sua volta à academia ocorreu no ano de 1809. Em junho de 1812 tomou posse como vice-secretário da instituição e, com a morte do primeiro-secretário, assumiu este posto. O discurso, proferido após a vinda da família real para o Brasil, permite observar a identificação do autor com a idéia de um grande 707 vol. 11(3):685-711, set.-dez. 2004 História, Ciências, Saúde Manguinhos, Rio de Janeiro ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. DA FONSECA Banhadas suas costas em triângulo pelas ondas do Atlântico; com um sem-número de rios caudais, e de ribeiras empoladas, que o retalham em todos os sentidos, não há parte alguma do sertão, que não participe mais ou menos do proveito que o mar lhe pode dar para o trato mercantil, e para 708 História, Ciências, Saúde Manguinhos, Rio de Janeiro AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA REFERÊNCIAS BIBLIOGRÁFICAS E FONTES DOCUMENTAIS I - Manuscritos de José Bonifácio Manuscritos do Instituto Histórico e Geográfico Brasileiro (IHGB): Notas sobre o alambique. S / D., S / L. Loc.: IHGB L. 192 Doc. 36. Notas sobre economia política. S/D., S/L. Loc.: IHGB L. 191 Doc. 65. REFERÊNCIAS BIBLIOGRÁFICAS E FONTES DOCUMENTAIS I - Manuscritos de José Bonifácio Manuscritos do Instituto Histórico e Geográfico Brasileiro (IHGB): Notas sobre o alambique. S / D., S / L. Loc.: IHGB L. 192 Doc. 36. Notas sobre economia política. S/D., S/L. Loc.: IHGB L. 191 Doc. 65. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA o estabelecimento das grandes pescarias. A grande cordilheira que o corta de norte a sul, o divide por ambas as vastas fraldas e pendores em dois mundos diferentes, capazes de criar todas as produções da terra inteira. Seu assento central quase no meio do globo, defronte e à porta com a África, que deve senhorear, com a Ásia à direita, e com a Europa à esquerda, qual outra região se lhe pode igualar? Riquíssimo nos três reinos da natureza, com o andar dos tempos nenhum outro país poderá correr parelhas com a nova Lusitânia (Falcão, I, op. cit., p. 144). O ano de 1808 sem dúvida representou uma profunda mudança no quadro da dominação colonial portuguesa. O Rio de Janeiro se tornou efetivamente a nova sede da metrópole. Com isso, as antigas relações entre Brasil e Portugal foram invertidas. O primeiro tornava- se a sede da monarquia portuguesa, enquanto o segundo perdia a função de pólo dinamizador do sistema e passava a depender cada vez mais do primeiro, uma vez que aqui seria o local de edificação do grande e vasto Império. Como afirmou Souza (1999, p. 57), Portugal tornou-se colônia do Brasil. Na parte final do discurso, o filósofo comentava o que o Brasil necessitava para ser o centro do novo Império e utilizava a expressão monarquia brasílica (Falcão, I, op. cit., pp. 472-3), e não Império lusitano, utilizado por Souza Coutinho. Sugeria assim uma certa naturalização da Corte na nova sede da monarquia, já que ali ela se fixava e não pretendia mais sair. José Bonifácio, naturalista ligado aos interesses do Estado, despedia-se da nação portuguesa com a consciência de ter des- pendido um grande esforço na contribuição para o processo de ins- titucionalização das ciências naturais em Portugal, ao atuar em instituições de pesquisa e universitárias particularmente voltadas para a mineração. Suas memórias científicas, fruto de seus trabalhos práticos na intendência, foram o exemplo maior dessa contribuição. Por outro lado, ele tentou modernizar a administração das minas e de matas e bosques, buscando tornar a Intendência das Minas do Reino de Portugal uma empresa competitiva e capaz de operar como as instaladas em regiões da Saxônia, Freiberg, França, Itália, entre outras. Tudo isso foi feito tendo sempre em mente ser o mais humilde e fiel súdito português. REFERÊNCIAS BIBLIOGRÁFICAS E FONTES DOCUMENTAIS I - Manuscritos de José Bonifácio 709 vol. 11(3):685-711, set.-dez. 2004 ALEX G. VARELA, MARIA M. LOPES E MARIA RACHEL F. 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Munteal Filho, Oswaldo Uma sinfonia para o novo mundo: a Academia Real das Ciências de Lisboa e os 1998 caminhos da Ilustração luso-brasileira na crise do antigo sistema colonial. Tese de doutoramento, Departamento de História, UFRJ, Rio de Janeiro. (mimeo.) Munteal Filho, Oswaldo, Cultura científica e sociabilidade intelectual no Brasil setecentista: um estudo Kury, Lorelai Brilhante acerca da Sociedade Literária do Rio de Janeiro. 1995 Acervo: Revista do Arquivo Nacional, Rio de Janeiro, vol. 8. Munteal Filho, Oswaldo Domenico Vandelli no anfiteatro da natureza: a cultura científica do 1993 reformismo ilustrado português na crise do antigo sistema colonial (1779-1808). Dissertação de mestrado, PUC-Rio, Rio de Janeiro. (mimeo.) Nizza da Silva, Maria A cultura luso-brasileira: da reforma da universidade à Independência do Beatriz Brasil. Lisboa, Editorial Estampa. 1999 Novais, Fernando A. O reformismo ilustrado luso-brasileiro: alguns aspectos. 1984 Revista Brasileira de História, nº 7. Rudwick, Martin Minerals, strata and fossils. Em N. Jardine, J. A. Secord e E. C. Spray (org.). 1997 Cultures of natural history. Cambridge, Cambridge University Press. Silva, Clarete O desvendar do grande livro da natureza: um estudo da obra do mineralogista Paranhos da José Vieira Couto, 1798-1805. 2002 São Paulo/Campinas, Annablume/Fapesp/Unicamp. Sousa, Octavio História dos fundadores do Império do Brasil: José Bonifácio de Andrada e Tarquínio de Silva, vol. I. Rio de Janeiro, José Olympio. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA 1957 Souza, Iara Lis Carvalho Pátria coroada: o Brasil como corpo político autônomo (1780-1831). 1999 São Paulo, Unesp. Taylor, Kenneth L. Les lois naturelles dans la géologie du XVIII éme siècle: recherches 1988 préliminaires. Em Travaux du Comite Français dHistoire de la Géologie Troisième série, t. II, Paris. Recebido para publicação em maio de 2003 From mineralogy to geology: the foudations of a science, 1650-1830. Chicago, The University of Chicago Press. A utopia do poderoso Império Portugal e Brasil: bastidores da política, 1798-1822. Rio de Janeiro, Sete Letras. Utopian thought in the Western world. Cambridge, Cambridge University Press. A ciência a serviço da reforma do Estado: a química em Portugal no final do século XVIII, início do século XIX. Em Ana Luísa Janeira et alii (org.). Divórcio entre cabeças e mãos? Laboratórios de química em Portugal (1772-1955). Lisboa, Livraria Escolar Editora. A geração de 1790 e a idéia do Império luso-brasileiro. Em Kenneth Maxwell Chocolate, piratas e outros malandros. Ensaios tropicais. Rio de Janeiro, Paz e Terra. Munteal Filho, Oswaldo 2001 O liberalismo num outro Ocidente: política colonial, idéias fisiocratas e reformismo mercantilista. Em Lucia Maria Paschoal Guimarães, Maria Emília Prado (org.). O liberalismo no Brasil Imperial: origens, conceitos e prática. Rio de Janeiro, Revan/Uerj. Munteal Filho, Oswaldo 1998 Uma sinfonia para o novo mundo: a Academia Real das Ciências de Lisboa e os caminhos da Ilustração luso-brasileira na crise do antigo sistema colonial. Tese de doutoramento, Departamento de História, UFRJ, Rio de Janeiro. (mimeo.) Munteal Filho, Oswaldo, Kury, Lorelai Brilhante 1995 Cultura científica e sociabilidade intelectual no Brasil setecentista: um estudo acerca da Sociedade Literária do Rio de Janeiro. Acervo: Revista do Arquivo Nacional, Rio de Janeiro, vol. 8. Munteal Filho, Oswaldo 1993 Domenico Vandelli no anfiteatro da natureza: a cultura científica do reformismo ilustrado português na crise do antigo sistema colonial (1779-1808). Dissertação de mestrado, PUC-Rio, Rio de Janeiro. (mimeo.) Nizza da Silva, Maria Beatriz 1999 Novais, Fernando A. 1984 Rudwick, Martin 1997 Silva, Clarete Paranhos da 2002 Sousa, Octavio Tarquínio de 1957 Souza, Iara Lis Carvalho 1999 Taylor, Kenneth L. 1988 Nizza da Silva, Maria Beatriz A cultura luso-brasileira: da reforma da universidade à Independência do Brasil. Lisboa, Editorial Estampa. A cultura luso-brasileira: da reforma da universidade à Independência do Brasil. Lisboa, Editorial Estampa. Novais, Fernando A. 1984 O reformismo ilustrado luso-brasileiro: alguns aspectos. Revista Brasileira de História, nº 7. O reformismo ilustrado luso-brasileiro: alguns aspectos. AS ATIVIDADES DO NATURALISTA JOSÉ BONIFÁCIO DE ANDRADA E SILVA Revista Brasileira de História, nº 7. Minerals, strata and fossils. Em N. Jardine, J. A. Secord e E. C. Spray (org.). Cultures of natural history. Cambridge, Cambridge University Press. Minerals, strata and fossils. Em N. Jardine, J. A. Secord e E. C. Spray (org.). Cultures of natural history. Cambridge, Cambridge University Press. O desvendar do grande livro da natureza: um estudo da obra do mineralogista José Vieira Couto, 1798-1805. São Paulo/Campinas, Annablume/Fapesp/Unicamp. História dos fundadores do Império do Brasil: José Bonifácio de Andrada e Silva, vol. I. Rio de Janeiro, José Olympio. Sousa, Octavio Tarquínio de 1957 Pátria coroada: o Brasil como corpo político autônomo (1780-1831). São Paulo, Unesp. Les lois naturelles dans la géologie du XVIII éme siècle: recherches préliminaires. Em Travaux du Comite Français dHistoire de la Géologie Troisième série, t. II, Paris. Recebido para publicação em maio de 2003. Aprovado para publicação em janeiro de 2004. Pátria coroada: o Brasil como corpo político autônomo (1780-1831). São Paulo, Unesp. Les lois naturelles dans la géologie du XVIII éme siècle: recherches préliminaires. Em Travaux du Comite Français dHistoire de la Géologie Troisième série, t. II, Paris. Recebido para publicação em maio de 2003. Aprovado para publicação em janeiro de 2004. 711 vol. 11(3):685-711, set.-dez. 2004
https://openalex.org/W2415567469	http://publications.lib.chalmers.se/records/fulltext/237194/local_237194.pdf	English	null	Quality and legal aspects in public care procurement	The TQM journal	2,016	cc-by	10,135	http://dx.doi.org/10.1108/TQM-09-2014-0075 Downloaded from: http://publications.lib.chalmers.se/publication/237194 Notice: Changes introduced as a result of publishing processes such as copy-editing and formatting may not be reflected in this document. For a definitive version of this work, please refer to the published source. Please note that access to the published version might require a subscription. Chalmers Publication Library (CPL) offers the possibility of retrieving research publications produced at Chalmers University of Technology. It covers all types of publications: articles, dissertations, licentiate theses, masters theses, conference papers, reports etc. Since 2006 it is the official tool for Chalmers official publication statistics. 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Downloaded by CHALMERS UNIVERSITY OF TECHNOLOGY At 00:56 29 September 2016 (PT Downloaded by CHALMERS UNIVERSITY OF TECHNOLOGY At 00:56 (2016),"The “5S” approach to improve a working environment can reduce waiting time: Findings from hospitals in Northern Tanzania", The TQM Journal, Vol. 28 Iss 4 pp. 664-680 http://dx.doi.org/10.1108/ TQM-11-2014-0099 (2016),"The “5S” approach to improve a working environment can reduce waiting time: Findings from hospitals in Northern Tanzania", The TQM Journal, Vol. 28 Iss 4 pp. 664-680 http://dx.doi.org/10.1108/ TQM-11-2014-0099 (2016),"The order and level of management standards implementation: Changes during the time", The TQM Journal, Vol. 28 Iss 4 pp. 636-647 http://dx.doi.org/10.1108/TQM-02-2015-0027 (2016),"The order and level of management standards implementation: Changes during the time", The TQM Journal, Vol. 28 Iss 4 pp. 636-647 http://dx.doi.org/10.1108/TQM-02-2015-0027 (2016),"TQM application by engineering consultants in Hong Kong", The TQM Journal, Vol. 28 Iss 4 pp. 561-587 http://dx.doi.org/10.1108/TQM-06-2014-0049 Quality and legal aspects in public care procurement The TQM Journal Vol. 28 No. 4, 2016 pp. 648-663 Emerald Group Publishing Limite 1754-2731 DOI 10.1108/TQM-09-2014-0075 Received 23 September 20 Revised 19 December 2014 10 March 2015 Accepted 20 May 2015 648 Downloaded by CHALMERS UNIVERSITY OF TECHNOLOGY At 00:56 29 September 2016 (PT) Carolina Camén Service Research Center (CTF), Karlstad University, Karlstad, Sweden Henrik Eriksson Department of Technology Management and Organization, Chalmers University of Technology, Göteborg, Sweden, and Received 23 September 2014 Revised 19 December 2014 10 March 2015 Accepted 20 May 2015 Henrik Eriksson Department of Technology Management and Organization, Chalmers University of Technology, Göteborg, Sweden, and Rickard Garvare Department of Business Administration, Technology and Social Sciences, Luleå University of Technology, Luleå, Sweden Rickard Garvare g Funding for this investigation has been provided by Forte, the Swedish Research Council for Health, Working Life and Welfare, under Grant No. 2013-0462. Abstract Purpose – The purpose of this paper is to assess the applicability of care quality concepts as contract award criteria for public procurement of health and social care, using the case of Sweden. Purpose – The purpose of this paper is to assess the applicability of care quality concepts as contr award criteria for public procurement of health and social care using the case of Sweden pose – The purpose of this paper is to assess the applicability of care quality concepts as contract d criteria for public procurement of health and social care, using the case of Sweden. p p p p p pp y q y p award criteria for public procurement of health and social care, using the case of Sweden. Design/methodology/approach – Based on a literature review, European and Swedish legal texts, government regulations as well as 26 Swedish court review cases concerning care procurement have been analysed. Findings – Methods used for assessing care quality are seldom useful for predicting the quality to be delivered by a potential contractor. Legal principles of transparency and equal treatment of tenderers make it necessary to apply strict requirements for verification. Research limitations/implications – Results refer primarily to a Swedish context but could be applicable throughout the EU. Further studies of relations between award criteria and public/private collaborative practices for improving care quality during contractual periods are desirable. Practical implications – Local and regional procurement officials should benefit from a better understanding of how quality criteria should be designed and applied to the award procedures for care contracts. Care providers in the private sector would also be able to develop their quality strategies and present their abilities more efficiently when tendering for public contracts. p y g p Social implications – Issues of quality of health and social care are of obvious importance for social sustainability. Public awareness of care quality problems is evident and often a cause of media concern. Originality/value – This investigation pinpoints the difference between traditional care quality thinking and the legal principles underlying contract award in public procurement of care services. Keywords Sweden, Performance appraisal, Health care, Privatization, Government policy, Patient care, Local government, Municipal, Quality concepts, Public procurement, Older people, Court cases l government, Municipal, Quality concepts, Public procurement, Older people, Paper type Research paper © Bröchner, Camén, Eriksson, Garvare. Published by Emerald Group Publishing Limited. About Emerald www.emeraldinsight.com Emerald is a global publisher linking research and practice to the benefit of society. The company manages a portfolio of more than 290 journals and over 2,350 books and book series volumes, as well as providing an extensive range of online products and additional customer resources and services. Emerald is both COUNTER 4 and TRANSFER compliant. The organization is a partner of the Committee on Publication Ethics (COPE) and also works with Portico and the LOCKSS initiative for digital archive preservation. *Related content and download information correct at time of download. The current issue and full text archive of this journal is available on Emerald Insight at: www.emeraldinsight.com/1754-2731.htm The current issue and full text archive of this journal is available on Emerald Insight at: www.emeraldinsight.com/1754-2731.htm Quality and legal aspects in public care procurement Jan Bröchner Department of Technology Management and Organization, Chalmers University of Technology, Göteborg, Sweden Carolina Camén Service Research Center (CTF), Karlstad University, Karlstad, Sweden Henrik Eriksson Department of Technology Management and Organization, Chalmers University of Technology, Göteborg, Sweden, and © Bröchner, Camén, Eriksson, Garvare. Published by Emerald Group Publishing Limited. This paper is published under the Creative Commons Attribution (CC BY 3.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this paper (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at: http://creativecommons.org/licences/by/ 3.0/legalcode 1. Introduction Global demographic change implies rising numbers of elderly people needing care services. Sweden is one of the countries where the age pyramid changed its shape early on, and the expansion of care has been accompanied by outsourcing to private sector providers, with the public sector typically acting as buyer of care services. When public authorities procure services from private providers, it is often obvious that contracts should not be awarded with lowest price as the only criterion for provider selection. Frequently, public procurement, which is in the focus of the present investigation, involves also one or more quality criteria, as for construction projects (Waara and Bröchner, 2006), public transport (Camén, 2010; Rönnbäck, 2012) and other services, including care (Bergman and Lundberg, 2013). For health and social care procurement (or commissioning: Murray, 2009), we may expect the quality issues to be particularly difficult to manage, although a huge number of health quality studies have been produced over many decades. However, in the context of selecting a private provider, there are two major obstacles to using earlier research on care quality. Th fi b l li i l i h d d f i 649 The first obstacle lies in translating methods and measures from retrospective studies of care quality into predictive tools for the context of public procurement: will a particular provider deliver the desired quality level after having gained the contract? Darby and Karni (1973) distinguish between search, experience and credence qualities of a good. Search qualities are those that can be ascertained prior to purchase, thus relevant to a context of service procurement, be it private or public; credence qualities are expensive to judge even after purchase. Most health care services are associated with credence qualities, and difficulties related to measurement of credence qualities make the monitoring of care contracts costly. g y Second, will quality criteria derived from earlier care quality research stand up to judicial scrutiny in case of a conflict arising from a contract award decision? Few authors have noticed the growing importance of how the legal system treats health care quality. McHale (2002) discusses how English courts have dealt with review cases concerning issues of professional practice. Such review cases concern the illegality, irrationality and procedural impropriety of decisions. However, it is both costly and time-consuming to litigate. Abstract This paper is published under the Creative Commons Attribution (CC BY 3.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this paper (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at: http://creativecommons.org/licences/by/ 3.0/legalcode g Funding for this investigation has been provided by Forte, the Swedish Research Council for Health, Working Life and Welfare, under Grant No. 2013-0462. Quality and legal aspects 2. Methodology 650 After an initial literature review of the care quality literature, we investigate how the “quality care” concept is defined and understood in documents regulating the service of elderly care, including court cases with public procurement conflicts. This elderly care context was chosen for our content analysis of texts because of: first, its particular challenges when defining and assessing quality, as quality often is thought to be a largely subjective matter determined by users or customers; in this context the elderly themselves might suffer from cognitive impairment, so that relatives or friends are relevant stakeholders; and second, the important volume of care services to elderly delivered by private firms; and third, public concern with controversial media reports of mistreatment of elderly by procured care firms in Sweden. We relied on a straightforward application of systematic content analysis (Hall and Wright, 2008), where pre-selected terms were located in the texts. Additionally, simple tabulation of term frequencies in court verdicts was performed. Our analysis should be applicable to most national sets of regulative frameworks. It departs from how care quality is conceptualized in national legislation. Next, we look at how requirements, explicit and implicit, on procedures and criteria have been formulated in regulations concerning public procurement. Ultimately, the objective here is to identify acceptable approaches and concepts from quality theories and practices in general. Our illustration is the case of public procurement of care services for elderly in Sweden. To address the issue identified and to get a deeper understanding of applicability of care quality concepts as contract award criteria for public procurement, our data consist of: first, all relevant European public procurement directives since 1971; second, Swedish statues concerning health care and social care for the 1970-2013 period; third, Swedish 2009-2014 verdicts in administrative appeal cases, based on keyword searches in the InfoTorg legal database; and fourth, an overview of care quality literature. We relied on the InfoTorg legal database with full text verdicts from administrative courts. Here, we searched the January 2009-June 2014 period for review decisions where the three keywords procurement (upphandling), care (vård) and quality (kvalitet) occurred jointly. The results were screened to ensure that only care services contracts were in focus, which was the case for a total of 26 verdicts. 1. Introduction While these cases are post-event (scrutiny of alleged clinical negligence), “legislation may also facilitate this process/determining the appropriate standard of care/ in a proactive manner”. In the USA, it is competition law, including antitrust and consumer protection, that can be said to raise issues of health care quality (Sage et al., 2003). The two Institute of Medicine reports To Err is Human (IOM, 1999) and Crossing the Quality Chasm (IOM, 2001) underscored how law is related to quality of care. Over time, courts started taking consumer preferences and not only professional standards into account when identifying quality. Predictive assessments of care quality are implicit when allowing health insurers to select providers and purchase care services based on both price and quality. Also pointed out by Sage et al. (2003) is the potential for public purchasing to develop and implement market-oriented solutions to quality problems, mentioning standardized consumer information as an example. Highly pertinent is the identification by Hyman (2004) of barriers (relevance, complexity, framing, scepticism, inadequate demand) to legal use of health care quality research in the context of US competition law. Within EU, the relation between health care quality and competition law, as interpreted by the courts, is less clear and less important (cf. Fornaciari, 2009). p y p ( ) Nevertheless, while unknown before 1989 in European procurement directives as well as in most national legislation concerning public services, “quality” as a legal term has now spread widely. The purpose of this investigation is to assess the applicability of care quality concepts as contract award criteria for public procurement of health and social care. We use the case of Sweden as our point of departure since the national emphasis on care quality measurement is obvious in international comparisons (Kollberg et al., 2005), in addition to the fact that the share of health and social care procurement is high, also seen in an international perspective (Anell et al., 2012). TQM 28,4 Downloaded by CHALMERS UNIVERSITY OF TECHNOLOGY At 00:56 29 September 2016 (PT) In principle, satisfaction survey results from tenderers’ prior contracts could be required by a procuring entity as part of the documentation for assessing the degree of “quality” when awarding a contract. The entity could require that tenderers submit proof of a certified quality assurance system that includes the use of satisfaction surveys. Furthermore, it could be specified that the contracting authority is to receive results from satisfaction surveys of a particular type and at stated intervals. Measurement of patient and personnel satisfaction with care is obviously related to the care quality issue. A pioneer study as that of Abdellah and Levine (1957) has been followed by numerous later investigations; Abusalem et al. (2013) have reviewed 23 studies of patient satisfaction in home health care, listing the dimensions and scale types used in these studies, which show great variety of approaches, unlike what has emerged as standard practices for customer satisfaction measurement in consumer services. Obviously, the applicability to (health) care of the SERVQUAL scale (Parasuraman et al., 1985, 1988) depends on the validity of the original US executive interviews and customer focus groups, considering that the four services chosen were retail banking, credit card, securities brokerage, and product repair and maintenance. Three of these services have been transformed fundamentally in the last 30 years by ICT applications; only the fourth has had and retains a slight resemblance to health care services. Thus the five “scale dimensions” of tangibles, reliability, responsiveness, assurance and empathy may be partly irrelevant to a care context. The five SERVQUAL dimensions can be compared with five “quality characteristics” or “quality attributes” of home services for the elderly (continuity, availability/time, influence, personal relation and “the suitability of home helpers in terms of competence and personal manners”) chosen by Edebalk et al. (1995). Lee et al. (2000) proceeded by adding two dimensions (core medical service, professionalism/skills) to the five SERVQUAL dimensions when assessing health care service quality. More ambitiously, Koerner (2000) retraced much of the method used for developing SERVQUAL and did so in an inpatient nursing context; factor analysis led to five mostly different service “quality dimensions”: uncertainty reduction, reliability, compassion, relationships and individualized care. Furthermore a USA-UK comparison of health care quality in nursing homes has relied on a version of the SERVQUAL scale itself, although eliminating “assurance”; the statistical results were slightly weak (Kilbourne et al., 2004). Quality and legal aspects 3. Literature review Our review of the relevant literature begins with the topic of quality of care, followed how quality is related to public procurement and how quality is treated in the European procurement directives and in European case law. Finally, earlier studies of care quality in the context of public procurement are brought up. 3.1 Quality of care The literature on quality of care is huge and also predates the stream of generic service quality studies that began in the 1980s (Martínez and Martínez, 2010). Currently, the US Institute of Medicine (IOM, 2006) recognizes six quality-related goals: safety, effectiveness, patient-centeredness, timeliness, efficiency and equity. Already Sheps (1955) distinguished between three purposes of evaluations of hospital quality: regulatory, stimulus for the improvement of quality, and studying the effects of specific programmes or procedures on the quality of care. It is the third purpose of evaluation which comes closest to the use of quality criteria in procurement/ commissioning of care services, but then it takes the form of a predictive evaluation. Donabedian (1966) outlined three approaches (outcome, process, structure) to assessing quality of medical care; it is structure and process that are more relevant for procurement/commissioning of care services. Outcome would refer to documented outcomes from earlier contracts with other clients. 651 Downloaded by CHALMERS UNIVERSITY OF TECHNOLOGY At 00:56 29 September 2016 (PT) Several types of criticism have been levied against the original SERVQUAL measures (Ladhari, 2008, 2009); among critical comments that are of interest when analysing care satisfaction, validity of items has been questioned as well as the fact that SERVQUAL focuses on the process of service delivery rather than outcomes. p y If involvement of those who receive care is a policy objective, a quality focus on outcomes may imply that “outcomes for people” are more important than the achievement of a predetermined level of service quality (Willis and Bovaird, 2012). Wreder et al. (2009) identified several groups of stakeholders in public eldercare, starting with consensus among nurses who all mentioned the patient’s relatives, doctors and their colleagues. Examples of studies of caregiver’s work satisfaction in care for older people are given by Suhonen et al. (2013). Service provision is an interactive process (Grönroos and Voima, 2013), which is brought out clearly in the study by Gill et al. (2011) on service co-creation in community-based aged health care. identified several groups of stakeholders in public eldercare, starting with consensus among nurses who all mentioned the patient’s relatives, doctors and their colleagues. Examples of studies of caregiver’s work satisfaction in care for older people are given by Suhonen et al. (2013). Service provision is an interactive process (Grönroos and Voima, 2013), which is brought out clearly in the study by Gill et al. (2011) on service co-creation in community-based aged health care. 652 y g The problems of comparing results from care satisfaction surveys have several sources: many: satisfaction measures may primarily reflect interpersonal care experiences, including the effect of language barriers, patient experiences of their health status regardless of care they have received, feelings of fulfilment of patients’ a priori desires; furthermore, the timing of satisfaction measurement (Manary et al., 2013). Ambiguities of satisfaction survey interpretation increase when patients may suffer from dementia (Zank and Leipold, 2001). A recent Swedish study has compared care satisfaction among old people receiving public care at home and in other accommodation (Karlsson et al., 2013), finding that functional impairment and health complaints overshadowed differences in where they lived. In order to assess and predict the quality of service in advance, the quality level or quality maturity of the organization can be measured. The existence of certified management systems against the ISO 9001:2008 standard might be a potential indicator. TQM 28,4 Downloaded by CHALMERS UNIVERSITY OF TECHNOLOGY At 00:56 29 September 2016 (PT) Fulfilling requirements according to this standard does show that there is a well-documented methodology within the organization, rather than proving an ability to meet customer needs, requirements and expectations (Poksinska, 2010). A more direct way to assess the quality maturity of an organization is to use quality award models such as the Malcolm Baldrige National Quality Award, the European Excellence Award and the Swedish Quality Award. Although this goes beyond the immediate needs for contracting, it is recognized that relying on award models can provide benefits such as increased awareness of overall issues and customer orientation, and furthermore that quality improvement initiatives are supported (Hendricks and Singhal, 1997; Corredor and Goñi, 2011). A drawback of these award schemes is that they require considerable resources for organizational assessment, especially in the phase of description of activities when employees engage in self-assessment, and that evaluations require specific skills (Eriksson, 2003). Looking back at the preceding decade of market-related change in the provision of social services in England, Knapp et al. (2001) identified the issues of high transaction costs and of to what extent price competition would damage quality of care. 3.2 Public procurement P i i i i 3.2 Public procurement Procuring entities can influence quality by prequalification requirements (Eadie et al., 2012), by specifying quality standards (Kuypers and Gruppen, 2008; Enquist et al., 2011) in the tendering documentation and by applying quality criteria when awarding contracts. How a contract is formulated influences the ability and willingness of the service provider to manage quality (Camén, 2010). Contractors can be provided with a variety of contractual incentives for both cost reducing activities and quality (Albano et al., 2006). The tendering documents also provide the fundament for client monitoring of services actually delivered, which can be essential for quality development (Dean and Kiu, 2002). However, detailed and static contracts reduce the scope for flexibility in developing the contractual relationship (Camén, 2010) and can thus impact quality management. This lack of flexibility is stronger under public procurement legislation than in long-term private industry relationships (Camén et al., 2012). A contract awarded under the Act has a fixed expiry date, which means that the relationship between the contracting authority and the provider ends when the contract expires. It is virtually impossible to award a well-known incumbent additional contract periods, referring to favourable experiences of working together during the current contractual period; instead a new procurement process must be initiated in order to award a new contract to a provider. Legally, the relationships between contracting authorities and their service providers have a clear beginning and end. fixed expiry date, which means that the relationship between the contracting authority and the provider ends when the contract expires. It is virtually impossible to award a well-known incumbent additional contract periods, referring to favourable experiences of working together during the current contractual period; instead a new procurement process must be initiated in order to award a new contract to a provider. Legally, the relationships between contracting authorities and their service providers have a clear beginning and end. 653 Hitherto, two award principles have been possible: first, choosing the economically most advantageous tender; or second, lowest price. The procuring entity has to declare in advance how tenders will be evaluated. If the economically most advantageous tender is chosen, the criteria are usually a predefined combination of price and quality. In principle, several alternative award mechanisms can be identified, and it can be shown, relying on microeconomic theory, that price-to-quality scoring has a number of shortcomings (Bergman and Lundberg, 2013). Quality and legal aspects 3.2 Public procurement P i i i i A fundamental approach to the relevant theory of scoring auctions has been developed by Asker and Cantillon (2008; 2010; see also Dini et al., 2006). A Portuguese study reveals alternatives that are consistent with the European 2004 procurement directive (Mateus et al., 2010). 3.3 Quality in European procurement directives 3.3 Quality in European procurement directives 3.3 Quality in European procurement directives Quality as a legal term has successively gained prominence in the European procurement directives. When the first Council Directive (71/305/EEC, Article 29) gave five examples of criteria when the award of contracts was made to the most economically advantageous tender, quality was not explicitly among them. Instead, the examples were “price, period for completion, running costs, profitability, technical merit”, where at least the last example appears to have a strong relation to quality. Later, in Directive 89/440/EEC, Annex III mentioned “quality” and “quality assurance” in the definition of technical specifications, and in Directive 92/50/EEC (Article 36), the examples of criteria under most economically advantageous tender had been reshaped: “quality, technical merit, aesthetic and functional characteristics, technical assistance and after-sales service, delivery date, delivery period or period of completion, price”. As already in the 1971 directive, the contracting authority was required to state the award criteria “where possible in descending order of importance” in the contract documents or in the contract notice. Again, in directive 2004/18/EC (Article 53), the list of examples of criteria was reordered and a few additions were inserted: “quality, price, technical merit, aesthetic and functional characteristics, environmental characteristics, running costs, cost-effectiveness, after-sale service and technical assistance, delivery date, delivery period or period of completion, price”. Furthermore, the contracting authority was now required to specify in advance also the relative weighting which it gives to each of the criteria. With the new 2014/24/EU directive (Article 67), the identification of the most economically advantageous tender may include “the best price-quality ratio”, to be assessed on the basis of criteria “including qualitative, environmental and/or social aspects”. Next, the directive mentions that such criteria may comprise (examples are then given): first, “quality, including technical merit, aesthetic and functional characteristics”; and under second, “organisation, qualification and experience of staff […] where the quality of the staff […] can have a significant impact on the level of performance […]”. As to the legal semantics, this means that the quality concept has been moved to a higher hierarchical level instead of being listed in parallel with other concepts, which was the case beginning with the 1992 directive. 3.4 European court rulings on award criteria National interpretation of European procurement directives is influenced strongly by court rulings at the European level. Four important cases are relevant here. The European Court of Justice (ECJ) Case C-87/94 Commission v. Belgium (25 April 1996) highlighted that directive text requiring contracting entities to state “in the contract documents or in the tender notice all the criteria they intend to apply to the award, where possible in descending order of importance” is intended to inform tenderers of the features to be taken into account in identifying the economically most advantageous offer. “All the tenderers are thus aware of the award criteria to be satisfied by their tenders and the relative importance of those criteria. Moreover, that requirement ensures the observance of the principles of equal treatment of tenderers and of transparency”. p y Again, according to the ECJ SIAC Construction Case C-19/00 (18 October 2001), the principle of equal treatment implies an obligation of transparency in order to enable compliance with it to be verified. More specifically, this means that “the award criteria must be formulated, in the contract documents or the contract notice, in such a way as to allow all reasonably well-informed and normally diligent tenderers to interpret them in the same way”. Finally, when tenders are being assessed, the award criteria must be applied objectively and uniformly to all tenderers. pp j y y The Wienstrom Case C-448/01 (4 December 2003) showed that criteria must be accompanied by requirements which permit the accuracy of the information contained in tenders to be effectively verified. Award criteria must be sufficiently clearly formulated to satisfy the requirements of equal treatment and transparency of award procedures. Or, as stated by the Court in the “Max Havelaar” Netherlands Case C-368/10 (10 May 2012), “compliance with the principles of equality, non-discrimination and transparency requires that the award criteria are objective, ensuring that tenders are compared and assessed objectively and thus in conditions of effective competition”. In the same case, the court points to “both the principle of the equal treatment of potential tenderers and the principle of transparency of the award criteria, the formulation of the award criteria being such as to allow all reasonably well-informed tenderers exercising ordinary care to know the exact scope thereof and thus to interpret them in the same way”, which is wording only slightly different from the 2001 verdict. TQM 28,4 3.3 Quality in European procurement directives In addition, the new directive allows for weightings to be expressed by “providing for a range with an appropriate maximum spread” and it also recognizes the situation where a weighting is not possible “for objective reasons”, and then (similar to the 1992 directive) “indicate the criteria in descending order of importance”. g p However, it is certainly not only in the award criteria that a contracting authority may take quality into account. As an extreme, unlikely to occur in practice, if all requirements for quality could be entered in the specifications for a contract, then identifying the best tender and the award might be done on the basis of price alone. 654 Quality and legal aspects 3.4 European court rulings on award criteria 3.5 Quality in public procurement of care A global overview on quality-based purchasing in health care has been made by Waters et al. (2004). In the UK, quality criteria, premia, incentives are now common in local authority social care commissioning (Hardy and Wistow, 1998; Knapp et al., 2001; Rubery et al., 2013). The Swedish policy orientation and change towards an increased reliance on care outsourcing has been described in a European perspective by Pavolini and Ranci (2008), as well in a Nordic perspective by Rauch (2008) and Brennan et al. (2012); Sobis (2013) has compared Sweden to Poland. and Ranci (2008), as well in a Nordic perspective by Rauch (2008) and Brennan et al. (2012); Sobis (2013) has compared Sweden to Poland. ( ); ( ) p There are few studies of quality and non-contractible quality under public care contracting. Eggleston and Zeckhauser (2002) applied transaction cost analysis to the quality problem. Empirical studies based on such approaches have failed to give clear patterns of quality reduction or increase upon outsourcing from the public sector. Stolt et al. (2011) concluded from Swedish statistics that private care providers seem to emphasize service aspects rather than structural quality factors; Brennan et al. (2012) reviewed a number of studies that indicate lower quality associated with marketisation of care. Mutiganda (2014) takes a pessimistic view based on a case study in Finland of procured public care of the elderly, whereas Bergman et al. (2014), comparing mortality rates, have found Swedish evidence for good effects of competition and private provision of care under contract. There is little agreement among these authors on how care quality outcomes should be measured, and there are no simple answers to the question of how to take into account the prior condition of older individuals when a private provider begins to operate under a particular contract. 655 4. The Swedish context As a legal term, kvalitet has a long history in Swedish statutes, but then referring to the quality only of tangible goods. The first instance of kvalitet of public services was introduced in the late 1970s as a change to the Local Government Act, then limiting local authority powers to devolve responsibility to bodies with representatives of labour market organizations for the quality of local government services (Bill 1978/ 79:188). Sweden has separate legislative frameworks for health and social care, currently and primarily the 1982 Health and Medical Services Act and the 2001 Social Services Act. The county councils are responsible for health-related services, while after a 1992 reform, municipalities are responsible for social care. In the 1982 Act, Section 2a centres on “good care”, which means that services must “(1) be of good quality and cater to the patient’s need of security in care and treatment; (2) be readily available; (3) be founded on respect for the self-determination and privacy of the patient; (4) promote good contacts between the patient and health and medical personnel”. Given this enumeration, it is obvious that “good quality” must be interpreted as subordinated to “good care”. 656 Kvalitet as a term entered the Social Services Act (as Section 7a) in the late 1990s (Bill 1996/97:124, pp. 52-54). The government then stated that quality could not be viewed only in a client/user perspective but should also be assessed in a staff, management and citizen perspective. Furthermore, attaining good quality in social services was seen as requiring a number of ingredients, such as rule of law, individual influence and easily available care and services. The encounter between social workers and clients was seen as core to social work; trustworthy collaboration between the individual and social services staff as well as respect for individual integrity were said to be of great importance for quality. Moreover, it was said in the Bill to be essential that the social services show responsiveness and empathy. Monitoring and evaluating of services, including a voice for those who take part of or use the service efforts, were emphasized; it was also said that the attempts to define good quality in social services led to the conclusion that quality development should focus on all parts of the activities: the organizational structure, the work process and the result obtained. 4. The Swedish context In this study we have chosen to focus on care services in Sweden and procurement of these services. Especially, we are interested in “quality of care” as formulated concept in documents regulating such services. The 290 municipalities are legally responsible for care for the elderly; each municipality can chose to deliver care services in-house (by municipal employees) or to outsource them. Local authorities in Sweden increasingly buy care and the scope has gradually increased throughout the 1990s (Nutek, 2007). In 2005 the purchase of care was estimated to SEK33 billion, corresponding to about EUR3.5 billion (Nutek, 2007). According to data from 2004, all counties put out any part of the care to private providers and only 12 municipalities out of 290 handled care of the elderly entirely in-house (Nutek, 2007). The number of employees in commercial nursing homes increased from less than 1 per cent of the workforce in 1990 to 16 per cent in 2010 (Stolt et al., 2011). 4.1 National legislation on care provider selection If the care service is outsourced, the supplier is selected by means of the public tendering process regulated by the Swedish Act on Public Procurement (LOU), revised in 2007 and implementing the European procurement directives. More recently, the 2008 Act on System of Choice in the Public Sector (LOV) has been introduced, regulating what applies when authorities allow individuals themselves to choose a supplier from among approved suppliers in a system of choice. In systems of choice, prices are established in advance. LOV applies to municipalities and counties when they introduce systems of choice for health and medical care and for social services. Counties must have systems of choice in primary care. For municipalities, and for the counties’ other activities, introducing systems of choice is voluntary. Since quality criteria are applied in LOV systems mostly in local, retrospective evaluations of service deliveries, and the Act itself fails to mention quality, our analysis here is confined to LOU contracting, where quality is treated explicitly during the selection process. 4.2 Quality in Swedish care legislation While it is not within current competence of the EU to regulate health care quality (Hervey and McHale, 2005), Swedish care legislation has developed on its own, successively increasing its reliance on quality terminology. However, the development is not fully consistent, as will be seen: is “patient safety” included in “care quality” or not? TQM 28,4 4. The Swedish context In general, it emerges that the government distinguished between antecedents or preconditions of quality and quality itself. In the same 1996/1997 Bill, the government developed further an analysis of quality in care for older people, underlining as a quality aspect that relatives should be brought into the planning of care and be supported in various ways. In addition, it was stated that well-functioning collaboration within social services and together with other affected municipal entities was of great importance for quality in the care of older people. However, in the revised and current 2001 Social Services Act (Chapter 3, Section 3), it is only stated that “Measures within social services shall be of good quality. […] The quality of activities shall be systematically and continuously developed and assured”, thus a much shorter text than in the corresponding article of the Health and Medical Services Act. 5. Review cases involving “quality” in care services procurement There is no thorough overview available, but a sampling of quality criteria used in 5. Review cases involving “quality” in care services procurement 5. Review cases involving “quality” in care services procurement There is no thorough overview available, but a sampling of quality criteria used in local and regional public procurement of care services indicates great variety. Certain municipalities tend to specify quality and associated processes as numerous detailed requirements, relying on assigning points to just a few quality subcriteria when identifying the economically most advantageous tender. In general, between two and five quality subcriteria is the usual range, and these often include staff numbers according to educational levels and also processes for quality assurance. There are examples of tenderers having to describe how they are to achieve “meaningful everyday experience” or similar vague concepts, a text that will then be graded by officials or expert readers. p An analysis of contested care procurement decisions in Swedish local and regional government during January 2009-June 2014, where the cases have involved “quality”, reveals the principles used by administrative courts. The time period chosen reflects that the revised Swedish national legislation, implementing the 2004/18/EC procurement directive, came into force for new procurement processes on 1 January 2008. 657 Most of the contested award decisions concern procurement of institutional care services for older people. Some have concerned youth care homes. Degree of detail in the criteria as formulated in the tendering documentation can be contentious, given the consequent risk of arbitrary decisions by the procuring entity. How scale points for quality are assigned during evaluation is often contentious. We find “transparency” and “equal treatment” to be prominent as principles. “Transparency” is interpreted more than once as including “openness and predictability”, or as being “clearly formulated”, or as a combination of “clarity and predictability”. Occasionally, issues of “proportionality” and “non-discrimination” arise, as well as “objective requirements”. Reliance by procuring entities on the outcome of earlier treatment might impair “openness and predictability” of an evaluation. Proportionality is discussed typically when compulsory requirements, e.g. for a specified type or item of documentation of bidders’ quality systems, have not been fulfilled. Proportionality is often questioned by tendering firms, but the courts then find mostly that their tenders had not contained information that the tendering documents had stated as compulsory. Quality and legal aspects 5. Review cases involving “quality” in care services procurement A frequent context for referring to transparency is when the evaluation criteria have been imprecise; sometimes, the procuring entity is found to have failed to apply its own predefined criteria during tender evaluation, which in its turn could be seen as in conflict with the principle of equal treatment. The total number of cases is given in Table I. There were 21 cases in administrative courts of first instance, and five cases went further to appeal courts during the period in question. During the second half of the period, the volume of cases in lower courts increased. The recent European Directive 2014/24/EU on public procurement derives (Article 1) the five principles of equal treatment, non-discrimination, mutual recognition, proportionality and transparency from the free movement of goods, freedom of establishment and the freedom to provide services. However, analysis of the EU principles is complicated by the uncertain or inconsistent relation between them, in particular how transparency is related to the other principles (Arrowsmith et al., 2000, p. 73ff.; Prechal and De Leeuw, 2007; Bovis, 2009). Year Cases in courts of first instance Cases in appeal courts 2009 2 1 2010 1 2011 2012 4 2013 9 2 2014 ( January-June) 6 1 Table I. Cases in administrative courts involving care quality criteria, January 2009-June 2014 Table II shows how the administrative courts of first instance have invoked transparency, equal treatment and other fundamental principles in their 21 review cases. Of the five principles invoked by these courts, two occur more frequently: equal treatment and transparency. It is difficult to identify trends during the period. p y y g p The administrative court of appeal is the court of second instance. There are four such courts, and during the period, they have reviewed five cases involving quality, care and procurement. The administrative courts of appeal invoked equal treatment and transparency in a few out of five review cases, but they often avoided stating the principles explicitly in their decisions, although – or perhaps because – the court of first instance had done so. 658 TQM 28,4 6. Conclusion Th l i strictly national standards for use in public care procurement creates unacceptable barriers to cross-border service trade, given that there is little international consensus on how to measure care quality. q y The concept of “improvement science” has recently emerged to provide a comprehensive framework for research focused on health care quality (Marshall et al., 2013). Improvement science is dominated by concepts and models from quality management and is guided by a patient-oriented perspective. This field of research coincides with an increased emphasis on evidence-based medicine and practice as well as a need to develop and spread knowledge more effectively, thereby contributing to quality improvement in health care. The role of commissioning for care quality improvement has been identified in an English NHS setting by Gillam and Siriwardena (2013), but the ability of procuring entities and providers jointly to improve quality during a contractual period is limited because of the restricted scope for modifying a public contract, once awarded to an external provider. Our results refer primarily to the Swedish context but are applicable throughout the European Union. Further studies of the relation between award criteria and public/private collaborative practices for improving care quality during contractual periods are desirable. Although the state-of-the-art of quality management and of public procurement on the other hand is well advanced in each of their fields, a number of questions remain on how quality criteria can be understood in contexts where services are provided by means of a contractual relationship between two parties, while it is a third party, here the care recipient, who is the primary beneficiary. In line with Balbastre-Benavent and Canet-Giner (2011), there emerges a clear need to examine how quality is operationalized, measured, evaluated and acted upon in such contexts, in particular regarding the use of various quality models. 659 Downloaded by CHALMERS UNIVERSITY OF TECHNOL There are at least three important implications for policy makers from this study. First, it appears fruitful to promote contracts of longer duration, also making it possible to reward care providers for a history of quality improvements, or to penalize them for absence of improvement practices. Second, it is urgent that quality concepts and tools are developed further to fit the context of public procurement of care services. Although there are many different concepts and tools available, few seem to be adequate in this context. 6. Conclusion Th l i Third, one way forward may be to benchmark procurement procedures in which a wider range of quality concepts and tools have been used successfully. Downloaded by CHALMERS UNIVERSIT Quality and legal aspects Anell, A., Glenngård, A.H. and Merkur, S. (2012), “Sweden: health system review”, Health Systems in Transition, Vol. 14 No. 5, pp. 1-159. Arrowsmith, S., Linarelli, J. and Wallace, D. (2000), Regulating Public Procurement: National and International Perspectives, Kluwer Law International, London. Abusalem, S., Myers, J.A. and Aljeesh, Y. (2013), “Patient satisfaction in home health care”, Journal of Clinical Nursing, Vol. 22 Nos 17-18, pp. 2426-2435. Albano, G.L., Calzolari, G., Dini, F., Iossa, E. and Spagnolo, G. (2006), “Procurement contracting strategies”, in Dimitri, N., Piga, G. and Spagnolo, G. (Eds), Handbook of Procurement, Cambridge University Press, Cambridge, pp. 82-120. 6. Conclusion Th l i The analysis presented here shows that the use of quality specifications and quality criteria for the award of care service contracts, as well as the scope for quality improvement, is limited by requirements for transparency and verification in the context of public procurement. Thus it is paradoxical that “quality” as a term has spread widely through legislation during the last decades, while there is little room for exploiting the findings of quality research when public authorities choose to outsource care services. There are many obstacles to inclusion of a wider range of quality concepts in the procurement of care. Two major concerns identified in this paper are transfer of methods and measures from retrospective studies of care quality into predictive tools suitable for the procurement context, and whether quality criteria derived from earlier care quality research will stand up to judicial scrutiny. Moreover, the legally required expiry dates for awarded contracts reduces the scope for long-term perspectives on quality improvement. Quality concepts and tools appropriate for public procurement criteria are few. Procurement officials may hesitate to include innovative criteria from the care quality literature because of perceived risk of judicial review, preferring to rely on tried and tested simpler practices. When awarding a contract for care services, the contracting authority has to prescribe and also to predict the quality of the services to be delivered by a successful tenderer. Although much work has been carried out on retrospective measurement of care quality concerning health outcomes, many important issues remain unresolved. When service providers submit tenders, it is not always possible to rely on surveys of outcomes and patient satisfaction during earlier contracts, since lack of standardization and sources of potential bias give rise to uncertainty that makes objective verification difficult. It is a matter of future research to investigate whether the development of EU principle (number of cases) Year Equal treatment Non- discrimination Mutual recognition Proportionality Transparency 2009 1 1 1 1 2 2010 2011 2012 3 1 2013 5 1 1 8 2014 ( January-June) 3 1 3 1 Table II. 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ERS UNIVERSITY OF TECHNOLOGY At 00:56 29 September 2016 (PT) About the authors Jan Bröchner, PhD, is a Professor of Organization of Construction and currently heading the Division of Service Management in the Department of Technology Management and Organization at the Chalmers University of Technology, Göteborg, Sweden. His research interests include construction procurement, productivity, innovation support policy and facilities management. Jan Bröchner is the corresponding author and can be contacted at: jan.brochner@chalmers.se Carolina Camén is a Senior Lecturer in Business Administration at the Service Research Center (CTF) at the Karlstad University, Sweden. Her main research interest is in the areas of contracts and service management – especially the use of contracts and their role in service management. Downloaded by CHALMERS UNIVERSITY OF TECH Henrik Eriksson, PhD, is an Associate Professor at the Division of Quality Sciences and the Centre for Healthcare Improvement (CHI) at the Chalmers University of Technology, Göteborg, Sweden. His research interests include quality improvement in healthcare, quality management in theory and practice, process management and its applications, business excellence and quality awards, as well as quality management in small- and medium-sized organizations. Rickard Garvare, PhD, is a Professor of Quality Management at the Department of Business Administration, Technology and Social Sciences at the Luleå University of Technology, Sweden. His present research efforts are focused on learning and implementation of quality-related methodologies, exploring gaps between knowledge and practice. 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https://openalex.org/W4214829762	https://hal.univ-lille.fr/hal-03871945/document	English	null	Cleaning of Wastewater Using Crosslinked Poly(Acrylamide-co-Acrylic Acid) Hydrogels: Analysis of Rotatable Bonds, Binding Energy and Hydrogen Bonding	Gels	2,022	cc-by	14,599	To cite this version: Salah Hamri, Tewfik Bouchaour, Djahida Lerari, Zohra Bouberka, Philippe Supiot, et al.. Cleaning of Wastewater Using Crosslinked Poly(Acrylamide-co-Acrylic Acid) Hydrogels: Analysis of Rotatable Bonds, Binding Energy and Hydrogen Bonding. Gels, 2022, Gels, 8 (3), pp.156. 10.3390/gels8030156. hal-03871945 HAL Id: hal-03871945 https://hal.univ-lille.fr/hal-03871945v1 Submitted on 25 Nov 2022 HAL is a multi-disciplinary open access rchive for the deposit and dissemination of sci- ntific research documents, whether they are pub- shed or not. The documents may come from eaching and research institutions in France or L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires Cleaning of Wastewater Using Crosslinked Poly(Acrylamide-co-Acrylic Acid) Hydrogels: Analysis of Rotatable Bonds, Binding Energy and Hydrogen Bonding Salah Hamri, Tewfik Bouchaour, Djahida Lerari, Zohra Bouberka, Philippe Supiot, Ulrich Maschke To cite this version: Salah Hamri, Tewfik Bouchaour, Djahida Lerari, Zohra Bouberka, Philippe Supiot, et al.. Cleaning of Wastewater Using Crosslinked Poly(Acrylamide-co-Acrylic Acid) Hydrogels: Analysis of Rotatable Bonds, Binding Energy and Hydrogen Bonding. Gels, 2022, Gels, 8 (3), pp.156. 10.3390/gels8030156. hal-03871945 Hamri, Tewfik Bouchaour, Djahida Lerari, Zohra Bouberka, Philippe Supiot, Ulrich Maschke Distributed under a Creative Commons Attribution 4.0 International License Salah Hamri 1,2, Tewﬁk Bouchaour 2, Djahida Lerari 1, Zohra Bouberka 3, Philippe Supiot 4 and Ulrich Maschke 4,* 1 Center for Scientiﬁc and Technical Research in Physico-Chemical Analysis (CRAPC), BP 384, Industrial Zone, BouIsmaïl 42004, Algeria; salah_hamri@yahoo.fr (S.H.); lerari_zinai@yahoo.fr (D.L.) 1 Center for Scientiﬁc and Technical Research in Physico-Chemical Analysis (CRAPC), BP 384, Industrial Zone, BouIsmaïl 42004, Algeria; salah_hamri@yahoo.fr (S.H.); lerari_zinai@yahoo.fr (D.L.) 2 Macromolecular Research Laboratory (LRM), Faculty of Sciences, Abou Bekr Belkaid University, BP 119, Tlemcen 13000 Algeria; bouchaour@yahoo fr g y ( ) y ( ) 2 Macromolecular Research Laboratory (LRM), Faculty of Sciences, Abou Bekr Belkaid University, BP 119, Tlemcen 13000, Algeria; bouchaour@yahoo.fr g y ( ) y ( ) 2 Macromolecular Research Laboratory (LRM), Faculty of Sciences, Abou Bekr Belkaid Unive Tlemcen 13000, Algeria; bouchaour@yahoo.fr g y 3 Laboratoire Physico-Chimie des Matériaux-Catalyse et Environnement (LPCMCE), Université des Sciences et de la Technologie d’Oran Mohamed Boudiaf (USTOMB), Oran 31000, Algeria; bouberkazohra@yahoo.fr 4 CNRS, INRAE, Centrale Lille, UMR 8207—UMET—Unité Matériaux et Transformations, Université de Lille, 59000 Lille, France; philippe.supiot@univ-lille.fr g y 3 Laboratoire Physico-Chimie des Matériaux-Catalyse et Environnement (LPCMCE), Université des Sciences et de la Technologie d’Oran Mohamed Boudiaf (USTOMB), Oran 31000, Algeria; bouberkazohra@yahoo.fr 4 CNRS, INRAE, Centrale Lille, UMR 8207—UMET—Unité Matériaux et Transformations, Université de Lille, 59000 Lille, France; philippe.supiot@univ-lille.fr * Correspondence: ulrich.maschke@univ-lille.fr de la Technologie d’Oran Mohamed Boudiaf (USTOMB), Oran 31000, Algeria; bouberkazohra@yahoo.fr 4 CNRS, INRAE, Centrale Lille, UMR 8207—UMET—Unité Matériaux et Transformations, Université de Lille, 59000 Lille, France; philippe.supiot@univ-lille.fr * Correspondence: ulrich.maschke@univ-lille.fr p pp p * Correspondence: ulrich.maschke@univ-lille.fr Abstract: The discharge of untreated wastewater, often contaminated by harmful substances, such as industrially used dyes, can provoke environmental and health risks. Among various techniques, the adsorption of dyes, using three-dimensional (3D) networks consisting of hydrophilic polymers (hy- drogels), represents a low-cost, clean, and efﬁcient remediation method. Three industrially used dyes, Methylene Blue, Eosin, and Rose Bengal, were selected as models of pollutants. Poly(acrylamide) (poly(AM)) and poly(acrylamide-co-acrylic acid) (poly(AM-co-AA)) networks were chosen as adsor- bent materials (hydrogels). These polymers were synthesized by crosslinking the photopolymeriza- tion of their respective monomer(s) in an aqueous medium under exposure to UV light. Experimental adsorption measurements revealed substantially higher dye uptakes for poly(AM-co-AA) compared to poly(AM) hydrogels. In this report, a theoretical model based on docking simulations was applied to analyze the conformation of polymers and pollutants in order to investigate some aspects of the adsorption process. In particular, hydrogen and halogen interactions were studied. Citation: Hamri, S.; Bouchaour, T.; Lerari, D.; Bouberka, Z.; Supiot, P.; Maschke, U. Cleaning of Wastewater Using Crosslinked Poly(Acrylamide- co-Acrylic Acid) Hydrogels: Analysis of Rotatable Bonds, Binding Energy and Hydrogen Bonding. Gels 2022, 8, 156. https://doi.org/10.3390/ gels8030156 Keywords: wastewater; pollutant; dye; hydrogel; modeling; docking simulation Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. HAL Id: hal-03871945 https://hal.univ-lille.fr/hal-03871945v1 Submitted on 25 Nov 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License gels Salah Hamri 1,2, Tewﬁk Bouchaour 2, Djahida Lerari 1, Zohra Bouberka 3, Philippe Supiot 4 and Ulrich Maschke 4,* The presence of strong hydrogen bonding plays a crucial role in the retention of dyes, whereas halogen bonding has a small or negligible effect on adsorption. An evaluation of binding energies allowed us to obtain information about the degree of afﬁnity between polymers and dyes. The number of rotatable bonds in the copolymer exceeds those of poly(AM),meaning that poly(AM-co-AA) is revealed to be more suitable for obtaining a high retention rate for pollutants. 1. Introduction Water is an important liquid for human beings, the universe, and all life existing on earth [1,2]. This liquid can be easily polluted by different dyes [3,4]. Both water and dyes are still largely used in the textile industry, meaning that the wastewater after production is a mixture of dyes and water. Unfortunately, the elimination of the wastewater often occurs through discharge into rivers and other efﬂuents [5–12]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). This negative situation has motivated many researchers to publish many reports in the ﬁeld of the treatment of water polluted with dyes [13–15]. Several physical and chemical techniques have been developed to purify water from these compounds, including photocatalysis, oxidation, ﬁltration, coagulation/ﬂocculation, and adsorption [16–20]. In particular, adsorption processes have been studied intensively because of their low cost, https://www.mdpi.com/journal/gels Gels 2022, 8, 156. https://doi.org/10.3390/gels8030156 Gels 2022, 8, 156 2 of 17 easy access, and effective dye removal, in which the dissolved dye compounds adsorb on the surface of suitable adsorbents [21]. easy access, and effective dye removal, in which the dissolved dye compounds adsorb on the surface of suitable adsorbents [21]. Many biodegradable materials and effective adsorbents obtained from natural re- sources have been used to remove dyes from aqueous solutions. Hydrogels were frequently applied for this purpose, consisting of a three-dimensional polymeric material with the capacity to uptake an important amount of water due to the presence of hydrophilic groups in their structure, such as –OH, –CONH, and –SO3H [22]. Copolymers based on acrylamide (AM) and acrylic acid (AA) have been applied to remove dyes. AM monomer is soluble in water, and linear poly(AM) ﬁnds many uses as water-soluble thickeners and ﬂocculation agents [23–25], whereas AA represents the simplest unsaturated carboxylic acid. This colorless liquid is miscible with water, alco- hols, ethers, and chloroform [26–28]. Solpan et al. [29] used (poly(AM-co-AA) hydrogels for the uptake of the cationic dyes, safranin-O and magenta. The diffusion of water and cationic dyes within hydrogels showed non-Fickian behavior. Corona-Rivera et al. [30] applied poly(AM-co-AA) crosslinked with N,N′-methylene bisacrylamide (NMBAM) for the removal of Remazol red dye from aqueous solutions, ﬁnding the maximum dye adsorp- tion capacity for peculiar experimental conditions, with an adsorption mechanism well represented by the Langmuir model. 1. Introduction p y g The diffusion of colored water inside the hydrogel depends on many factors, such as the dye structure and the functional groups on the polymer chains. The dye can generate an attraction through electrostatic interaction, which also represents an important key to removing dye from an aqueous medium, whereby a dye molecule and a receptor behave similarly to a ship and a harbor. In the ﬁeld of biochemistry, the theory of docking was largely applied to study the interaction between ligand and protein, allowing us to explain the afﬁnity between these components [31–37]. In this study, the interactions between the dyes and polymer networks were investigated. The docking method was applied to analyze different interactions, with the receptor and ligand representing the polymer matrix and dye, respectively. This simulation method has the advantage of enabling us to predict the preferred orientation of one molecule to a second one when bound to each other to form a stable complex [38–40]. Interestingly, this helps us to economize cost and time of research work. In a previous paper [41], the interaction between a polymer based on HEMA monomer and Eosin Y (EY) as a pollutant was discussed. It was found that the theoretical prediction correlates well with experimental results. In the literature, some authors report on poly(AM- co-AA) crosslinked with NMBAM [42–46]. In this work, AM and AA were copolymerized and crosslinked with HDDA since it contributes to the high level of conversion of acrylic double bonds [47,48]. Under the UV-visible light exposure in the presence of a suitable photoinitiator (Darocur 1173), a chemically crosslinked three-dimensional copolymer was successfully obtained. In contrast to thermal polymerization, which often requires ele- vated temperatures, photopolymerization can be performed at room temperature [49]. In most reports on poly(AA), thermal polymerization was applied using a source of free radicals, together with a chemical stabilizer, such as ammonium persulfate and tetram- ethylethylenediamine [50–52]. This method has disadvantages such as long polymerization times, unstable and toxic reagents, and tedious preparation steps. On the other hand, photopolymerization using an initiator sensitive to light represents a quicker method that is less tedious and less toxic. The ﬁeld of the exploitation of these polymeric materials is, thus, enlarged to applications in which the elevation of temperature is not advised. The ﬁnal properties of UV-polymerized gels depend on the UV-visible spectrum of the source, light intensity and uniformity, and exposure times [53]. 2. Results and Discussion 2. Results and Discussion 2.1. Effect of Crosslinker Content on Equilibrium Swelling 2.1. Effect of Crosslinker Content on Equilibrium Swelling To ﬁnd out the optimal dye concentration for the retention study, the UV-visible spectra of the dyes were screened in the concentration range from 32 × 10−3 mg·mL−1 to 64 × 10−3 mg·mL−1. According to the obtained results (Figure S1), the spectra correspond- ing to 64 × 10−3 mg·mL−1 reveal saturation effects for all absorbance bands except those from RB. Electronic spectra associated with the lower concentration of 32 × 10−3 mg·mL−1 were acceptable; therefore, this concentration was chosen for the retention study. To find out the optimal dye concentration for the retention study, the UV-visible spectra of the dyes were screened in the concentration range from 32 × 10−3 mg·mL−1 to 64 × 10−3 mg·mL−1. According to the obtained results (Figure S1), the spectra corresponding to 64 × 10−3 mg·mL−1reveal saturation effects for all absorbance bands except those from RB. Electronic spectra associated with the lower concentration of 32 × 10−3 mg·mL−1 were acceptable; therefore, this concentration was chosen for the retention study. Figure 1 presents the evolution of poly(AM) swelling equilibrium versus the composi- tion of a crosslinking agent (HDDA) for each dye solution. Equilibrium swelling data were remarkably increased by decreasing the crosslinker content. The best results of maximum equilibrium swelling were obtained with 1 wt% of HDDA. In this case, swelling values in solutions of RB, BM, and EY were found at around 870%, 850%, and 900%, respectively. The crosslinking density essentially governs the diffusion of the dyes in the polymer networks, as well as the swelling behavior. p y Figure 1 presents the evolution of poly(AM) swelling equilibrium versus the com- position of a crosslinking agent (HDDA) for each dye solution. Equilibrium swelling data were remarkably increased by decreasing the crosslinker content. The best results of maximum equilibrium swelling were obtained with 1 wt% of HDDA. In this case, swelling values in solutions of RB, BM, and EY were found at around 870%, 850%, and 900%, respectively. The crosslinking density essentially governs the diffusion of the dyes in the polymer networks, as well as the swelling behavior. Figure 1. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM) hydrogel in dye solutions. Figure 2 presents the evolution of the swelling equilibria of poly(AM-co-AA) versus Figure 1. 1. Introduction The model dyes studied in this report were Rose Bengal (RB), EY, and Methylene Blue (MB), presenting anionic and cationic natures. These dyes are widely used in many applications, thus increasing the probability that they contribute to water pollution, since even small quantities can easily affect the water quality [54–56]. Gels 2022, 8, 156 3 of 17 n since To understand the different interatomic interactions between dyes and polymers, the docking simulation method was exploited. Two model systems, crosslinked poly(AM)/dye and poly(AM-co-AA)/dye, were considered using Avogadro software. These model sys- tems were all energy-minimized, and the conformation of polymer/dye systems was simulated using Auto-Dock Vina software [57,58], and then visualized and analyzed using UCSF Chimera. To understand the different interatomic interactions between dyes and polymers, the docking simulation method was exploited. Two model systems, crosslinked poly(AM)/dye and poly(AM-co-AA)/dye, were considered using Avogadro software. These model systems were all energy-minimized, and the conformation of polymer/dye systems was simulated using Auto-Dock Vina software [57,58], and then visualized and analyzed using UCSF Chimera. 2. Results and Discussion 2. Results and Discussion Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM) hydrogel in dye solutions. Figure 2 presents the evolution of the swelling equilibria of poly(AM-co-AA) versus Figure 1. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM) hydrogel in dye solutions. Figure 1. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM) hydrogel in dye solutions. Figure 2 presents the evolution of the swelling equilibria of poly(AM-co-AA) versus the composition of HDDA for each dye solution. The copolymer prepared with 1 wt% HDDA shows the highest equilibrium swelling with 70%, 72%, and 71% in solutions of BM, RB, and EY, respectively. In comparison with poly(AM), poly(AM-co-AA) presents a much lower equilibrium swelling due to the addition of AA units, thus increasing the crosslinking density. Swelling equilibrium values of 34%, 35%, and 29% were obtained for 4 wt% HDDA in solutions of BM, RB, and EY, respectively. For 7 wt% HDDA, the corresponding swelling data were 18%, 19%, and 19%. Figure 2 presents the evolution of the swelling equilibria of poly(AM-co-AA) versus the composition of HDDA for each dye solution. The copolymer prepared with 1 wt% HDDA shows the highest equilibrium swelling with 70%, 72%, and 71% in solutions of BM, RB, and EY, respectively. In comparison with poly(AM), poly(AM-co-AA) presents a much lower equilibrium swelling due to the addition of AA units, thus increasing the crosslinking density. Swelling equilibrium values of 34%, 35%, and 29% were obtained for 4 wt% HDDA in solutions of BM, RB, and EY, respectively. For 7 wt% HDDA, the corresponding swelling data were 18%, 19%, and 19%. Figure 2 presents the evolution of the swelling equilibria of poly(AM-co-AA) versus the composition of HDDA for each dye solution. The copolymer prepared with 1 wt% HDDA shows the highest equilibrium swelling with 70%, 72%, and 71% in solutions of BM, RB, and EY, respectively. In comparison with poly(AM), poly(AM-co-AA) presents a much lower equilibrium swelling due to the addition of AA units, thus increasing the crosslinking density. Swelling equilibrium values of 34%, 35%, and 29% were obtained for 4 wt% HDDA in solutions of BM, RB, and EY, respectively. For 7 wt% HDDA, the corresponding swelling data were 18%, 19%, and 19%. Figure 2 presents the evolution of the swelling equilibria of poly(AM-co-AA) versus the composition of HDDA for each dye solution. ff f 2.2.1. Rose Bengal Dye 2.2.1. Rose Bengal Dye poly(AM-co-AA). A rete whereas 97% of RB was r ff f 2.2.1. Rose Bengal Dye 2.2.1. Rose Bengal Dye poly(AM co AA). A reten whereas 97% of RB was re g y Figure 3 illustrates the retention behavior of RB using poly(AM) and poly(AM-co-AA). A retention rate of about 7% for RB by poly(AM) was obtained, whereas 97% of RB was removed in the case of poly (AM-co-AA) (Figure S2). Figure 3 illustrates the retention behavior of RB using poly(AM) and poly(AM-co-AA). A retention rate of about 7% for RB by poly(AM) was obtained, whereas 97% of RB was removed in the case of poly (AM-co-AA) (Figure S2). w e eas 9 % o was e oved i t e case o po y (AM co AA) ( igu e S ). The hydrogen bonding between chains of the neutral poly(AM) provokes physical crosslinking effects; the poly(AM) network is brittle and its glass transition was reported to be around 450 K [59,60]. The hydrogen bonding between chains of the neutral poly(AM) provokes physical crosslinking effects; the poly(AM) network is brittle and its glass transition was reported to be around 450 K [59,60]. Figure 3. Retention behavior of RB in presence of crosslinked poly(AM) and poly(AM-co-AA) (1 wt% HDDA, after 24 h contact time). Figure 3. Retention behavior of RB in presence of crosslinked poly(AM) and poly(AM-co-AA) (1 wt% HDDA, after 24 h contact time). Figure 3. Retention behavior of RB in presence of crosslinked poly(AM) and poly(AM-co-AA) (1 wt% HDDA, after 24 h contact time). Figure 3. Retention behavior of RB in presence of crosslinked poly(AM) and poly(AM-co-AA) (1 wt% HDDA, after 24 h contact time). Figure 3. Retention behavior of RB in presence of crosslinked poly(AM) and poly(AM-co-AA) (1 wt% HDDA, after 24 h contact time). From the Morse curve [61], considering large distances, the energy is zero (no in- teraction). This means that two atoms placed infinitely far away do not interact with each other, or they are not bonded to each other. At inter-nuclear distances in the order of the The hydrogen bonding between chains of the neutral poly(AM) provokes physical crosslinking effects; the poly(AM) network is brittle and its glass transition was reported to be around 450 K [59,60]. From the Morse curve [61], considering large distances, the energy is zero (no in- teraction). 2. Results and Discussion 2. Results and Discussion The copolymer prepared with 1 wt% HDDA shows the highest equilibrium swelling with 70%, 72%, and 71% in solutions of BM, RB, and EY, respectively. In comparison with poly(AM), poly(AM-co-AA) presents a much lower equilibrium swelling due to the addition of AA units, thus increasing the crosslinking density. Swelling equilibrium values of 34%, 35%, and 29% were obtained for 4 wt% HDDA in solutions of BM, RB, and EY, respectively. For 7 wt% HDDA, the corresponding swelling data were 18%, 19%, and 19%. Gels 2022, 8, 156 Gels 2022, 7, x FO 4 of 17 4 of 17 Figure 2. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM-co-AA) hydrogel in dye solutions. Figure 2. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM-co-AA) hydrogel in dye solutions. Figure 2. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM-co-AA) hydrogel in dye solutions. 2.2. Effect of Crosslinker Content on Absorbance 2.2.1. Rose Bengal Dye Figure 2. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM-co-AA) hydrogel in dye solutions. Figure 2. Effect of the composition of HDDA (wt%) on equilibrium swelling of poly(AM-co-AA) hydrogel in dye solutions. 2.2. Effect of Crosslinker Content on Absorbance 2.2.1. Rose Bengal Dye 2.2. Effect of Crosslinker Content on Absorbance 2.2. Effect of Crosslinker Content on Absorbance Figure 3 illustrates the retention b poly(AM-co-AA) A retention rate of about ff f 2.2.1. Rose Bengal Dye 2.2.1. Rose Bengal Dye poly(AM-co-AA). A rete whereas 97% of RB was r This means that two atoms placed infinitely far away do not interact with each y From the Morse curve [61], considering large distances, the energy is zero (no interac- tion). This means that two atoms placed inﬁnitely far away do not interact with each other, Gels 2022, 8, 156 5 of 17 5 of 17 or they are not bonded to each other. At inter-nuclear distances in the order of the atomic diameter, attractive forces dominate. At smaller distances between two atoms, the force is repulsive and the energy of the two atoms is high. p gy g The distance between atoms has, thus, an important effect on the interactions of the system. It was found that interactions can be classiﬁed as strong and medium for a distance interval of [2.5, 3.1] Å and [3.1, 3.55] Å, respectively, whereas distances between selected atoms greater than 3.55Å correspond to weak or non-existing interactions [41]. In the case of crosslinked poly(AM) hydrogel, the distances between chlorine and oxygen atoms are greater than 3.55 Å, which shows that the interactions are weak. The hydrogen bond with 2.48 Å represents a strong interaction, but the neutrality of the hydrogel cannot allow this bond to be constructed (Table 1 and Figure 4). 5 of 17 Table 1. Interatomic distances obtained from interactions of the two polymers with RB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/RB I...O 4.721 C...O 6.197 O...H 2.486 Poly(AM-co-AA)/RB I...O 3.823 Cl...O 3.752 O...H 2.246 atomic diameter, attractive forces dominate. At smaller distances between two atoms, the force is repulsive and the energy of the two atoms is high. The distance between atoms has, thus, an important effect on the interactions of the system. It was found that interactions can be classified as strong and medium for a dis- tance interval of [2.5, 3.1] Å and [3.1, 3.55] Å, respectively, whereas distances between selected atoms greater than 3.55Å correspond to weak or non-existing interactions [41]. In the case of crosslinked poly(AM) hydrogel, the distances between chlorine and oxygen atoms are greater than 3.55 Å, which shows that the interactions are weak. The hydrogen bond with 2.48 Å represents a strong interaction, but the neutrality of the hy- drogel cannot allow this bond to be constructed (Table 1 and Figure 4). Figure 4. ff f 2.2.1. Rose Bengal Dye 2.2.1. Rose Bengal Dye poly(AM-co-AA). A rete whereas 97% of RB was r Crosslinked poly(AM)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 4. Crosslinked poly(AM)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Table 1. Interatomic distances obtained from interactions of the two polymers with RB using the docking simulation method. atomic diameter, attractive forces dominate. At smaller distances between two atoms, the force is repulsive and the energy of the two atoms is high. Table 1. Interatomic distances obtained from interactions of the two polymers with RB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/RB I...O 4.721 C...O 6.197 O...H 2.486 Poly(AM-co-AA)/RB I...O 3.823 Cl...O 3.752 O...H 2.246 atomic diameter, attractive forces dominate. At smaller distances between two atoms, the force is repulsive and the energy of the two atoms is high. The distance between atoms has, thus, an important effect on the interactions of the system. It was found that interactions can be classified as strong and medium for a dis- tance interval of [2.5, 3.1] Å and [3.1, 3.55] Å, respectively, whereas distances between selected atoms greater than 3.55Å correspond to weak or non-existing interactions [41]. In the case of crosslinked poly(AM) hydrogel, the distances between chlorine and oxygen atoms are greater than 3.55 Å, which shows that the interactions are weak. The hydrogen bond with 2.48 Å represents a strong interaction, but the neutrality of the hy- d o el a ot allo thi bo d to be o t u ted (Table 1 a d Fi u e 4) Figure 4. Crosslinked poly(AM)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 4. Crosslinked poly(AM)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 4. Crosslinked poly(AM)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 4. Crosslinked poly(AM)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Table 1. Interatomic distances obtained from interactions of the two polymers with RB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/RB I…O 4.721 C...O 6.197 O...H 2.486 Poly(AM-co-AA)/RB I...O 3.823 Cl...O 3.752 O...H 2.246 In the crosslinked poly(AM-co-AA)/RB system, there are weak and average electro- static interactions between the AA fraction and RB. 2.2.2. Methylene Blue Dye 2.2.2. Methylene Blue Dye y y Figure 6 shows that y y Figure 6 shows that crosslinked poly(AM) presents a negligible retention of MB (about 1%), whereas poly(AM-co-AA) removes MB at rates of 45% for a contact time of 24 Figure 6 shows that crosslinked poly(AM) presents a negligible retention of MB (about 1%), whereas poly(AM-co-AA) removes MB at rates of 45% for a contact time of 24 h. Figure 6 shows that crosslinked poly(AM) presents a negligible retention of MB (about 1%), whereas poly(AM-co-AA) removes MB at rates of 45% for a contact time of 24 h. Figure 6. Retention of MB in presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% wt HDDA, after 24 h contact time). In the neutral hydrogel, the interatomic distance between nitrogen and oxygen is 5.37 Å, which means that there is a weak attraction between these two atoms. A similar situation occurs between sulfur and hydrogen atoms (Table 2 and Figure 7). Initial and Figure 6. Retention of MB in presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% wt HDDA, after 24 h contact time). In the neutral hydrogel, the interatomic distance between nitrogen and oxygen is 5.37 Å, which means that there is a weak attraction between these two atoms. A similar situation occurs between sulfur and hydrogen atoms (Table 2 and Figure 7). Initial and final spectra of the dye are shown in Figure S3. Figure 6. Retention of MB in presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% wt HDDA, after 24 h contact time). In the neutral hydrogel, the interatomic distance between nitrogen and oxygen is 5.37 Å, which means that there is a weak attraction between these two atoms. A similar situation occurs between sulfur and hydrogen atoms (Table 2 and Figure 7). Initial and ﬁnal spectra of the dye are shown in Figure S3. Figure 6. Retention of MB in presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% t HDDA afte 24 h o ta t ti e) Figure 6. Retention of MB in presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% wt HDDA, after 24 h contact time). Figure 6. Retention of MB in presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% wt HDDA, after 24 h contact time). (1% wt HDDA, after 24 h contact time). In the neutral hydrogel, the interatomic distance between nitrogen and oxygen is 5.37 Å, which means that there is a weak attraction between these two atoms. ff f 2.2.1. Rose Bengal Dye 2.2.1. Rose Bengal Dye poly(AM-co-AA). A rete whereas 97% of RB was r The interaction between chlorine and iodine with oxygen is of a halogen type. The corresponding interatomic distance is higher than 3.55 Å, thus resulting in a weak interaction. Furthermore, the hydrogen bond with 2.24 Å represents a strong interaction, because the copolymer is charged in the aqueous medium and becomes a polyelectrolyte. Repulsion occurs between the negative charges of the AA parts and, consequently, the dye was retained by the strong hydrogen bond (O...H). The AA fraction of poly(AM-co-AA) has increased the retention percentage from 7% to 97%; therefore, it can be concluded that this copolymer effectively retains RB in an aqueous medium (Table 1 and Figure 5). Gels 2022, 8, 156 Gels 2022, 7, x FOR 6 of 17 6 of 17 Figure 5. Crosslinked poly(AM-co-AA)/RB system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. Figure 5. Crosslinked poly(AM-co-AA)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 5. Crosslinked poly(AM-co-AA)/RB system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. 2 2 2 M th l Bl D Figure 5. Crosslinked poly(AM-co-AA)/RB system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. Figure 5. Crosslinked poly(AM-co-AA)/RB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. tion; (b) enlargement of the hydrogen bonding interaction. 2 2 2 Methyle e Blue Dye 2.2.2. Methylene Blue Dye 2.2.2. Methylene Blue Dye . . . Met y e e ue ye Figure 6 shows that c 2.2.2. Methylene Blue Dye 2.2.2. Methylene Blue Dye y y Figure 6 shows that A similar situation occurs between sulfur and hydrogen atoms (Table 2 and Figure 7). Initial and In the neutral hydrogel, the interatomic distance between nitrogen and oxygen is 5.37 Å, which means that there is a weak attraction between these two atoms. A similar situation occurs between sulfur and hydrogen atoms (Table 2 and Figure 7). Initial and final spectra of the dye are shown in Figure S3. In the neutral hydrogel, the interatomic distance between nitrogen and oxygen is 5.37 Å, which means that there is a weak attraction between these two atoms. A similar situation occurs between sulfur and hydrogen atoms (Table 2 and Figure 7). Initial and ﬁnal spectra of the dye are shown in Figure S3. Gels 2022, 8, 156 Gels 2022 7 x FOR 7 of 17 7 of 17 7 of 17 7 of 17 Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/MB S...H 4.229 N...O 5.373 Poly(AM-co-AA)/MB S...H 2.670 N...O 3.924 7 of 17 Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/MB S…H 4.229 N…O 5.373 Poly(AM-co-AA)/MB S…H 2.670 N…O 3.924 Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/MB S...H 4.229 N...O 5.373 Poly(AM-co-AA)/MB S...H 2.670 N...O 3.924 7 of 17 Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/MB S...H 4.229 N...O 5.373 Poly(AM-co-AA)/MB S...H 2.670 N...O 3.924 7 of 17 Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 7. 2.2.2. Methylene Blue Dye 2.2.2. Methylene Blue Dye y y Figure 6 shows that Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/MB S…H 4.229 N…O 5.373 Poly(AM-co-AA)/MB S…H 2.670 N…O 3.924 Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/MB S…H 4.229 N…O 5.373 Poly(AM-co-AA)/MB S…H 2.670 N…O 3.924 Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 7. Crosslinked poly(AM)/MB system: (a)3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Poly(AM-co-AA)/MB S…H 2.670 N…O 3.924 Table 2. Interatomic distances obtained from interactions of the two polymers with MB using the docking simulation method. System Bonds Distance (Å) Poly(AM)/MB S…H 4.229 N O 5 373 Sulfur atoms have been known to participate in hydrogen bonds. It has been shown that the sulfur atom is a poor H-bond acceptor, but a moderately good H-bond donor [62]. In the copolymeric hydrogel, there is a hydrogen bond with an interatomic distance of 2.67 Å, which is considered to be a strong bond, facilitating an increase in the retention percentage from 1% to 45% (Table 2 and Figure 8). Sulfur atoms have been known to participate in hydrogen bonds. It has been shown that the sulfur atom is a poor H-bond acceptor, but a moderately good H-bond donor [62]. In the copolymeric hydrogel, there is a hydrogen bond with an interatomic distance of 2.67 Å, which is considered to be a strong bond, facilitating an increase in the retention percentage from 1% to 45% (Table 2 and Figure 8). Poly(AM-co-AA)/MB S…H 2.670 N…O 3.924 Sulfur atoms have been known to participate in hydrogen bonds. It has been shown that the sulfur atom is a poor H-bond acceptor, but a moderately good H-bond donor [62]. 2.2.2. Methylene Blue Dye 2.2.2. Methylene Blue Dye y y Figure 6 shows that In the copolymeric hydrogel, there is a hydrogen bond with an interatomic distance of 2.67 Å, which is considered to be a strong bond, facilitating an increase in the retention percentage from 1% to 45% (Table 2 and Figure 8). Figure 8. Crosslinked poly(AM-co-AA)/MB system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. Figure 8. Crosslinked poly(AM-co-AA)/MB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 8. Crosslinked poly(AM-co-AA)/MB system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. Figure 8. Crosslinked poly(AM-co-AA)/MB system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Gels 2022, 8, 156 Gels 2022, 7, x FO 8 of 17 8 of 17 2.2.3. Eosin Y Dye 2.2.3. Eosin Y Dye Figure 9 reveals a very small adsorption effect of EY by poly(AM) (0.4%). Halogens participating in the halogen bonding of the investigated dyes include iodine (I) (present in RB), bromine (Br) (present in EY), and chlorine (Cl) (present in RB). These halogens are able to act as donors and follow the general trend of Cl < Br < I, with iodine normally forming the strongest interactions [63,64]. y Figure 9 reveals a very small adsorption effect of EY by poly(AM) (0.4%). Halogens participating in the halogen bonding of the investigated dyes include iodine (I) (present in RB), bromine (Br) (present in EY), and chlorine (Cl) (present in RB). These halogens are able to act as donors and follow the general trend of Cl < Br < I, with iodine normally forming the strongest interactions [63,64]. Figure 9 reveals a very small adsorption effect of EY by poly(AM) (0.4%). Halogens participating in the halogen bonding of the investigated dyes include iodine (I) (present in RB), bromine (Br) (present in EY), and chlorine (Cl) (present in RB). These halogens are able to act as donors and follow the general trend of Cl < Br < I, with iodine normally forming the strongest interactions [63,64]. y Figure 9 reveals a very small adsorption effect of EY by poly(AM) (0.4%). Halogens participating in the halogen bonding of the investigated dyes include iodine (I) (present in RB), bromine (Br) (present in EY), and chlorine (Cl) (present in RB). These halogens are able to act as donors and follow the general trend of Cl < Br < I, with iodine normally forming the strongest interactions [63,64]. Figure 9 reveals a very small adsorption effect of EY by poly(AM) (0.4%). Halogens participating in the halogen bonding of the investigated dyes include iodine (I) (present in RB), bromine (Br) (present in EY), and chlorine (Cl) (present in RB). These halogens are able to act as donors and follow the general trend of Cl < Br < I, with iodine normally forming the strongest interactions [63,64]. y Figure 9 reveals a very small adsorption effect of EY by poly(AM) (0.4%). Halogens participating in the halogen bonding of the investigated dyes include iodine (I) (present in RB), bromine (Br) (present in EY), and chlorine (Cl) (present in RB). 2.2.3. Eosin Y Dye 2.2.3. Eosin Y Dye These halogens are able to act as donors and follow the general trend of Cl < Br < I, with iodine normally forming the strongest interactions [63,64]. Figure 9. Retention of EY in the presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% wt HDDA, after 24 h contact time). Figure 9. Retention of EY in the presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1 % wt HDDA, after 24 h contact time). Figure 9. Retention of EY in the presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1% wt HDDA, after 24 h contact time). Figure 9. Retention of EY in the presence of crosslinked poly(AM) and poly(AM-co-AA) hydrogels (1 % wt HDDA, after 24 h contact time). For this neutral system (poly(AM)), there is a Br…O bond with an interatomic dis- tance higher than 3.07 Å and a hydrogen bond with 6.53 Å (Table 3 and Figure 10) that qualify these bonds as weak bonds. Initial and final spectra are presented in Figure S4. A strong hydrogen bonding of poly(AM-co-AA) exists with 1.872 Å, which improves the retention of the MB dye (Figure 11). For this neutral system (poly(AM)), there is a Br...O bond with an interatomic distance higher than 3.07 Å and a hydrogen bond with 6.53 Å (Table 3 and Figure 10) that qualify these bonds as weak bonds. Initial and ﬁnal spectra are presented in Figure S4. A strong hydrogen bonding of poly(AM-co-AA) exists with 1.872 Å, which improves the retention of the MB dye (Figure 11). Table 3. Interatomic distances obtained from interactions of the two polymers with EY using the docking simulation method. System Bonds Distance (Å) Poly(AM)/EY O...H 6.536 Br...O 3.072 Poly(AM-co-AA)/EY O...H 1.872 Br...O 3.618 Table 3. Interatomic distances obtained from interactions of the two polymers with EY using the docking simulation method. Figure 12 represents a summary of the adsorption results for poly(AM-co-AA) hydro- gel, showing retention percentages of 45%, 50%, and 97% for MB, EY, and RB, respectively. Poly(AM-co-AA) functions, thus, with considerable efﬁciency, removing a high percentage of RB, though it is less effective for EY and MB. This difference can be explained by the different molecular structures and architectures of these dyes. Moreover, RB possesses more functional groups compared to EY and MB. Gels 2022, 8, 156 9 of 17 e S4. A ves the 9 of 17 e S4. A ves the y ( g ) Figure 10. 2.2.3. Eosin Y Dye 2.2.3. Eosin Y Dye Crosslinked poly(AM)/EY system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 10. Crosslinked poly(AM)/EY system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. 9 of 17 Table 3. Interatomic distances obtained from interactions of the two polymers with EY using the docking simulation method. System Bonds Distance (Å) Poly(AM)/EY O…H 6.536 Br…O 3.072 Poly(AM-co-AA)/EY O…H 1.872 Br…O 3.618 Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the inter- action; (b) enlargement of the hydrogen bonding interaction. Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. 2 3 Bi di E d N b f R t t bl B d A l i Figure 10. Crosslinked poly(AM)/EY system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 10. Crosslinked poly(AM)/EY system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. 9 of 17 Table 3. Interatomic distances obtained from interactions of the two polymers with EY using the docking simulation method. System Bonds Distance (Å) Poly(AM)/EY O…H 6.536 Br…O 3.072 Poly(AM-co-AA)/EY O…H 1.872 Br…O 3.618 Figure 10. Crosslinked poly(AM)/EY system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Figure 10. Crosslinked poly(AM)/EY system: (a) 3-D representation of results of the interaction; (b) enlargement of the hydrogen bonding interaction. Poly(AM-co-AA)/EY O…H 1.872 Br…O 3.618 Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the inter- action; (b) enlargement of the hydrogen bonding interaction. Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the inter- action; (b) enlargement of the hydrogen bonding interaction. Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the inter- action; (b) enlargement of the hydrogen bonding interaction. Figure 11. Crosslinked poly(AM-co-AA)/EY system: (a) 3-D representation of results of the interac- tion; (b) enlargement of the hydrogen bonding interaction. Figure 12 represents a summary of the adsorption res 2.3. Binding Energy and Number of Rotatable Bonds Analysis Figure 12 represents a summary of the adsorption re 2.3. Binding Energy and Number of Rotatable Bonds Analysis Figure 14a presents 104 rotatable bonds of the crosslinked poly(AM-co-AA) model, us- ing Autodock tools. Figure 14b shows the AA repetition unit of poly(AM-co-AA), exhibiting two rotatable bonds, C201–C203 and C203–C204. Figure 12. Illustration of the uptake efficiency for all dyes in presence of poly(AM-co-AA) hydrogel. Figure 12. Illustration of the uptake efﬁciency for all dyes in presence of poly(AM-co-AA) hydrogel. Table 4. Binding energies of polymer/dye systems. Poly(AM-co-AA)/MB −4.4 A rotatable bond is defined as any single non-ring bond attached to a non-terminal, non hydrogen atom In Figure 13a presenting the crosslinked poly(AM) model using Figure 12. Illustration of the uptake efficiency for all dyes in presence of poly(AM-co-AA) hydrogel. Figure 12. Illustration of the uptake efﬁciency for all dyes in presence of poly(AM-co-AA) hydrogel. Table 4. Binding energies of polymer/dye systems. Polymer/Dye Binding Energy (kcal/mol) Poly(AM)/RB −7.0 Poly(AM)/EY −5.2 Poly(AM)/MB −4.1 Poly(AM-co-AA)/RB −7.7 Poly(AM-co-AA)/EY −5.1 Poly(AM-co-AA)/MB −4.4 Poly(AM-co-AA)/MB −4.4 A rotatable bond is defined as any single non-ring bond attached to a non-terminal, non-hydrogen atom. In Figure 13a, presenting the crosslinked poly(AM) model using Autodock tools, most bonds were nonrotatable. When two HDDA units were very close, the crosslinker creates rigidity in the polymer network. The presence of a single cross- linking unit leads to more rotatable bonds. In Figure 13b, representing the AM repetition unit, we can see that C214–N215 bonds are nonrotatable; the same situation applies for C219–N220: amide C-N bonds present a high energy barrier for rotation [65,66]. C212–C214 and C223–C219 bonds are rotatable, i.e., the AM repetition unit possesses one rotatable bond. Binding Energy (kcal/mol) n two HDDA units were v (a) (b) Figure 13. Rotatable bonds of the crosslinked poly(AM) model using Autodock tools: (a) 92 rotat- able bonds, (b) one rotatable bond of the AM repetition unit. Green: rotatable, magenta: nonrotat- able, red: unrotatable bond. Figure 13. Rotatable bonds of the crosslinked poly(AM) model using Autodock tools: (a) 92 rotatable bonds, (b) one rotatable bond of the AM repetition unit. Green: rotatable, magenta: nonrotatable, red: unrotatable bond. (b) (a) (b) (a) Figure 13. Rotatable bonds of the crosslinked poly(AM) model using Autodock tools: (a) 92 rotat- able bonds, (b) one rotatable bond of the AM repetition unit. Green: rotatable, magenta: nonrotat- able, red: unrotatable bond. Figure 13. Rotatable bonds of the crosslinked poly(AM) model using Autodock tools: (a) 92 rotatable bonds, (b) one rotatable bond of the AM repetition unit. Figure 12 represents a summary of the adsorption re 2.3. Binding Energy and Number of Rotatable Bonds Analysis drogel, showing retention percentages of 45%, 50%, and 97% for MB, EY, and RB, re- spectively. Poly(AM-co-AA) functions, thus, with considerable efficiency, removing a high percentage of RB, though it is less effective for EY and MB. This difference can be explained by the different molecular structures and architectures of these dyes. Moreo- ver, RB possesses more functional groups compared to EY and MB. AutodockVina software allows us to determine the binding energies, which were used to evaluate if dyes could have stable complex interactions with polymeric hydrogels. The negative sign of the binding energy means that the dye was bound spontaneously without consuming energy. If the sign is positive, the binding occurs only if the required energy is available. Lower values of binding afﬁnity correspond to a higher stability of polymer/dye complexes. Consequently, hydrogel/dye systems with the highest and lowest stability in Table 4 were poly(AM-co-AA)/RB and poly(AM)/MB, respectively. A rotatable bond is deﬁned as any single non-ring bond attached to a non-terminal, non-hydrogen atom. In Figure 13a, presenting the crosslinked poly(AM) model using Autodock tools, most bonds were nonrotatable. When two HDDA units were very close, the crosslinker creates rigidity in the polymer network. The presence of a single crosslinking unit leads to more rotatable bonds. In Figure 13b, representing the AM repetition unit, we can see that C214–N215 bonds are nonrotatable; the same situation applies for C219–N220: amide C-N bonds present a high energy barrier for rotation [65,66]. C212–C214 and C223–C219 bonds are rotatable, i.e., the AM repetition unit possesses one rotatable bond. Gels 2022, 8, 156 Gels 2022, 7, x FOR 10 of 17 e can be Moreo- 10 of 17 Figure 12. Illustration of the uptake efficiency for all dyes in presence of poly(AM-co-AA) hydrogel. Figure 12. Illustration of the uptake efﬁciency for all dyes in presence of poly(AM-co-AA) hydrogel. Table 4. Binding energies of polymer/dye systems. 2.3. Binding Energy and Number of Rotatable Bonds Analysis AutodockVina software allows us to determine the binding energies, which were used to evaluate if dyes could have stable complex interactions with polymeric hydro- gels. The negative sign of the binding energy means that the dye was bound spontane- ously without consuming energy. If the sign is positive, the binding occurs only if the required energy is available. Lower values of binding affinity correspond to a higher stability of polymer/dye complexes. Figure 12 represents a summary of the adsorption re 2.3. Binding Energy and Number of Rotatable Bonds Analysis Consequently, hydrogel/dye systems with the highest and lowest stability in Table 4 were poly(AM-co-AA)/RB and poly(AM)/MB, re- spectively. Table 4. Binding energies of polymer/dye systems. Polymer/Dye Binding Energy (kcal/mol) Poly(AM)/RB −7.0 Poly(AM)/EY −5.2 Poly(AM)/MB −4.1 Poly(AM-co-AA)/RB −7.7 Poly(AM-co-AA)/EY −5.1 Poly(AM-co-AA)/MB −4.4 A rotatable bond is defined as any single non-ring bond attached to a non-terminal, 2.3. Binding Energy and Number of Rotatable Bonds Analysis AutodockVina software allows us to determine the used to evaluate if dyes could have stable complex intera gels. The negative sign of the binding energy means that ously without consuming energy. If the sign is positive, required energy is available. Lower values of binding a stability of polymer/dye complexes. Consequently, hy highest and lowest stability in Table 4 were poly(AM-co-A spectively. Table 4. Binding energies of polymer/dye systems. Polymer/Dye Bind Poly(AM)/RB Poly(AM)/EY Poly(AM)/MB Poly(AM-co-AA)/RB Poly(AM-co-AA)/EY Figure 12. Illustration of the uptake efficiency for all dyes in presence of poly(AM-co-AA) hydrogel. Figure 12. Illustration of the uptake efﬁciency for all dyes in presence of poly(AM-co-AA) hydrogel. Table 4. Binding energies of polymer/dye systems. Polymer/Dye Binding Energy (kcal/mol) Poly(AM)/RB −7.0 Poly(AM)/EY −5.2 Poly(AM)/MB −4.1 Poly(AM-co-AA)/RB −7.7 Poly(AM-co-AA)/EY −5.1 Poly(AM-co-AA)/MB −4.4 Poly(AM-co-AA)/MB −4.4 A rotatable bond is defined as any single non-ring bond attached to a non-terminal, non-hydrogen atom. In Figure 13a, presenting the crosslinked poly(AM) model using Autodock tools, most bonds were nonrotatable. When two HDDA units were very close, the crosslinker creates rigidity in the polymer network. The presence of a single cross- linking unit leads to more rotatable bonds. In Figure 13b, representing the AM repetition unit, we can see that C214–N215 bonds are nonrotatable; the same situation applies for C219–N220: amide C-N bonds present a high energy barrier for rotation [65,66]. C212–C214 and C223–C219 bonds are rotatable, i.e., the AM repetition unit possesses one rotatable bond. (a) (b) Figure 13. Rotatable bonds of the crosslinked poly(AM) model using Autodock tools: (a) 92 rotat- able bonds, (b) one rotatable bond of the AM repetition unit. Green: rotatable, magenta: nonrotat- able, red: unrotatable bond. Figure 14a presents 104 rotatable bonds of the crosslinked poly(AM-co-AA) model, using Autodock tools. Figure 14b shows the AA repetition unit of poly(AM-co-AA), ex- hibiting two rotatable bonds, C201–C203 and C203–C204. Figure 13. Rotatable bonds of the crosslinked poly(AM) model using Autodock tools: (a) 92 rotatable bonds, (b) one rotatable bond of the AM repetition unit. Green: rotatable, magenta: nonrotatable, red: unrotatable bond. 3 Conclusions 3. Conclusions Conclusions Conclusions 3. Conclusions The UV photopolymerization technique in an aqueous medium was chosen to elaborate chemically crosslinked poly(AM) and poly(AM-co-AA) as dye adsorbent hy- drogels. Experimental parameters, such as the optimal percentage of HDDA as a cross- linking agent, as well as the suitable dye concentration for analysis, were found to be1 wt% and 32 × 10−3 mg·mL−1, respectively. All dyes show negligible retention effects using the neutral poly(AM), and significant adsorption for the polyelectrolyte poly(AM-co-AA). It was found that 97% of RB was removed efficiently by the copolymer (MB: 45%, EY: 50%), which can be related to the presence of one more functional group compared to the other dyes, and also due to strong hydrogen bonding (O…H) with an interatomic distance of 2.24 Å, which plays a key role in interaction. As a consequence, this copolymer could be considered to be an efficient hydrogel with which to remove the The UV photopolymerization technique in an aqueous medium was chosen to elabo- rate chemically crosslinked poly(AM) and poly(AM-co-AA) as dye adsorbent hydrogels. Experimental parameters, such as the optimal percentage of HDDA as a crosslinking agent, as well as the suitable dye concentration for analysis, were found to be1 wt% and 32 × 10−3 mg·mL−1, respectively. All dyes show negligible retention effects using the neutral poly(AM), and signiﬁcant adsorption for the polyelectrolyte poly(AM-co-AA). It was found that 97% of RB was removed efﬁciently by the copolymer (MB: 45%, EY: 50%), which can be related to the presence of one more functional group compared to the other dyes, and also due to strong hydrogen bonding (O...H) with an interatomic distance of 2.24 Å, which plays a key role in interaction. As a consequence, this copolymer could be considered to be an efﬁcient hydrogel with which to remove the considered dyes from a water medium. considered dyes from a water medium. The conformation of polymers and pollutants were analyzed via a docking simula- tion. Interestingly, halogen bonding could be neglected, whereas hydrogen bonding plays a key role for dye retention. The system composed of poly(AM-co-AA)/RB has a binding energy of −7.7 kcal/mol, which means that this system has the highest stability compared to the other investigated polymer/dye systems. An analysis of rotatable bonds shows that the AA repetition unit presents two rotatable bonds, whereas that of AM has The conformation of polymers and pollutants were analyzed via a docking simulation. Figure 12 represents a summary of the adsorption re 2.3. Binding Energy and Number of Rotatable Bonds Analysis Green: rotatable, magenta: nonrotatable, red: unrotatable bond. Figure 14a presents 104 rotatable bonds of the crosslinked poly(AM-co-AA) model, using Autodock tools. Figure 14b shows the AA repetition unit of poly(AM-co-AA), ex- hibiting two rotatable bonds, C201–C203 and C203–C204. Figure 14a presents 104 rotatable bonds of the crosslinked poly(AM-co-AA) model, us- ing Autodock tools. Figure 14b shows the AA repetition unit of poly(AM-co-AA), exhibiting two rotatable bonds, C201–C203 and C203–C204. Gels 2022, 8, 156 Gels 2022, 7, x FO 11 of 17 11 of 17 11 of 17 11 of 17 (a) (b) Figure 14. Rotatable bonds of the crosslinked poly(AM-co-AA) model using Autodock tools: (a) 104 rotatable bonds, (b) two rotatable bonds of the AA repetition unit. Green: rotatable, magenta: nonrotatable, red: unrotatable bond. Figure 14. Rotatable bonds of the crosslinked poly(AM-co-AA) model using Autodock tools: (a) 104 rotatable bonds, (b) two rotatable bonds of the AA repetition unit. Green: rotatable, magenta: nonrotatable, red: unrotatable bond. (a) (b) (a) (b) Figure 14. Rotatable bonds of the crosslinked poly(AM-co-AA) model using Autodock tools: (a) 104 rotatable bonds, (b) two rotatable bonds of the AA repetition unit. Green: rotatable, magenta: nonrotatable, red: unrotatable bond. Figure 14. Rotatable bonds of the crosslinked poly(AM-co-AA) model using Autodock tools: (a) 104 rotatable bonds, (b) two rotatable bonds of the AA repetition unit. Green: rotatable, magenta: nonrotatable, red: unrotatable bond. The crosslinked poly(AM) model presents 92 rotatable bonds whereas the cross- linked poly(AM-co-AA) presents 104 rotatable bonds (Table 5). This difference creates more conformations for the copolymer compared to the homopolymer, so that poly- mer–dye interactions are favored for crosslinked poly(AM-co-AA). The crosslinked poly(AM) model presents 92 rotatable bonds whereas the crosslinked poly(AM-co-AA) presents 104 rotatable bonds (Table 5). This difference creates more conformations for the copolymer compared to the homopolymer, so that polymer–dye interactions are favored for crosslinked poly(AM-co-AA). Table 5. Number of rotatable bonds of poly(AM)/HDDA, poly(AM-co-AA)/HDDA and dyes, ob- tained by Autodock software. Table 5. Number of rotatable bonds of poly(AM)/HDDA, poly(AM-co-AA)/HDDA and dyes, obtained by Autodock software. Table 5. Number of rotatable bonds of poly(AM)/HDDA, poly(AM-co-AA)/HDDA and dyes, ob- tained by Autodock software. Table 5. Number of rotatable bonds of poly(AM)/HDDA, poly(AM-co-AA)/HDDA and dyes, obtained by Autodock software. Table 5. Number of rotatable bonds of poly(AM)/HDDA, poly(AM-co-AA)/HDDA and dyes, ob- tained by Autodock software. Product oly(AM Product poly(AM/HDDA) poly(AM-co-AA)/HDDA AM repeat unit AA repeat unit RB EY MB poly(AM/HDDA) poly(AM-co-AA)/HDDA AM repeat unit AA repeat unit RB EY MB Figure 12 represents a summary of the adsorption re 2.3. Binding Energy and Number of Rotatable Bonds Analysis Product Number of Rotatable Bonds poly(AM/HDDA) 92 poly(AM-co-AA)/HDDA 104 AM repeat unit 01 AA repeat unit 02 RB 2 EY 2 MB 2 Table 5. Number of rotatable bonds of poly(AM)/HDDA, poly(AM-co-AA)/HDDA and dyes, obtained by Autodock software. Product Number of Rotatable Bonds poly(AM/HDDA) 92 poly(AM-co-AA)/HDDA 104 AM repeat unit 01 AA repeat unit 02 RB 2 EY 2 MB 2 4.1. Materials The m (S i t Q The mo (Saint-Quent Cray Valley 2 h d 2 Cray Valley, 2 h d 2 2-hydroxy-2-m The monomers used in this study were AM and AA (both from Sigma-Aldrich (Saint- Quentin-Fallavier, France), purity: 99%), the crosslinking agent was HDDA (from Cray Valley, Courbevoie, France), purity: 98%), and the photoinitiator was 2-hydroxy-2-methyl-1- phenyl-propane-1 (commercial designation: Darocur 1173) (from Ciba-Geigy, purity: 97%). RB (purity: 95%), EY (purity: 99%), and MB (purity: 70%) (all from Sigma-Aldrich) were applied as dyes. All products were used as received without puriﬁcation. The chemical structures of the reagents are illustrated in Table 6. Abbreviations are given in Table S1. (Saint-Quentin-Fallavier, France), purity: 99%), the crosslinking agent was HDDA (from Cray Valley, Courbevoie, France), purity: 98%), and the photoinitiator wa 2-hydroxy-2-methyl-1-phenyl-propane-1 (commercial designation: Darocur 1173) (from Ciba-Geigy, purity: 97%). RB (purity: 95%), EY (purity: 99%), and MB (purity: 70%) (al from Sigma-Aldrich) were applied as dyes. All products were used as received withou purification. The chemical structures of the reagents are illustrated in Table 6. Abbrevia tions are given in Table S1. ( Q , ), p y ), g g ( Cray Valley, Courbevoie, France), purity: 98%), and the photoinitiator was 2-hydroxy-2-methyl-1-phenyl-propane-1 (commercial designation: Darocur 1173) (from Ciba-Geigy, purity: 97%). RB (purity: 95%), EY (purity: 99%), and MB (purity: 70%) (all from Sigma-Aldrich) were applied as dyes. All products were used as received without purification. The chemical structures of the reagents are illustrated in Table 6. Abbrevia- tions are given in Table S1. 2-hydroxy-2-methyl-1-phenyl-propane-1 (commercial designation: Darocur 1173) (from Ciba-Geigy, purity: 97%). RB (purity: 95%), EY (purity: 99%), and MB (purity: 70%) (all from Sigma-Aldrich) were applied as dyes. All products were used as received without purification. The chemical structures of the reagents are illustrated in Table 6. Abbrevia- tions are given in Table S1. Table 6. Chemical structure of monomers, crosslinker, and dyes. 2-hydroxy-2-methyl-1-phenyl-propane-1 (commercial designation: Darocur 1173) (from Ciba-Geigy, purity: 97%). RB (purity: 95%), EY (purity: 99%), and MB (purity: 70%) (all from Sigma-Aldrich) were applied as dyes. All products were used as received without purification. The chemical structures of the reagents are illustrated in Table 6. Abbrevia- tions are given in Table S1. Table 6. Chemical structure of monomers, crosslinker, and dyes. Ciba-Geigy, purity: 97%). RB (purity: 95%), EY (purity: 99%), and MB (purity: 70%) (all from Sigma-Aldrich) were applied as dyes. All products were used as received without purification. The chemical structures of the reagents are illustrated in Table 6. Abbrevia- tions are given in Table S1. Table 6. 4.1. Materials The m (S i t Q The mo (Saint-Quent Cray Valley 2 h d 2 Cray Valley, 2 h d 2 2-hydroxy-2-m Chemical structure of monomers, crosslinker, and dyes. Chemical Structure Table 6. Chemical structure of monomers, crosslinker, and dyes. Table 6. Chemical structure of monomers, crosslinker, and dye Table 6. Chemical structure of monomers, crosslinker, and dyes Chemical Stru Chemical Struc Chemical Struc Table 6. Chemical structure of monomers, crosslinker, and dyes. Table 6. Chemical structure of monomers, crosslinker, and dyes. Table 6. Chemical structure of monomers, crosslinker, and dyes. Chemical Structure Chemical Structure Chemical Structure Table 6. Chemical structure of monomers, crosslinker, and dyes. Table 6. Chemical structure of monomers, crosslinker, and dye Table 6. Chemical structure of monomers, crosslinker, and dyes Chemical Stru Chemical Struc Chemical Struc Table 6. Chemical structure of monomers, crosslinker, and dyes. Name Chemical Structure Acrylamide (AM) Table 6. Chemical structure of monomers, crosslinker, and dyes. Name Chemical Structure Acrylamide (AM) Acrylic acid (AA) 1,6-Hexanedioldiacrylate (HDDA) Rose Bengal sodium salt (RB) Eosin Y (EY) Acrylic acid (AA) Table 6. Chemical structure of monomers, crosslinker, and dyes. Name Chemical Structure Acrylamide (AM) Acrylic acid (AA) 1,6-Hexanedioldiacrylate (HDDA) Rose Bengal sodium salt (RB) Eosin Y (EY) 1,6-Hexanedioldiacrylate (HDDA) Name Chemical Structure Acrylamide (AM) Acrylic acid (AA) 1,6-Hexanedioldiacrylate (HDDA) Rose Bengal sodium salt (RB) Eosin Y (EY) Rose Bengal sodium salt (RB) Name Chemical Structure Acrylamide (AM) Acrylic acid (AA) 1,6-Hexanedioldiacrylate (HDDA) Rose Bengal sodium salt (RB) Eosin Y (EY) Eosin Y (EY) Acrylamide (AM) Acrylic acid (AA) 1,6-Hexanedioldiacrylate (HDDA) Rose Bengal sodium salt (RB) Eosin Y (EY) Methylene Blue (MB) Gels 2022, 7, x FOR PEER REVIEW 13 of 17 Methylene Blue (MB) 4.2. Hydrogel Synthesis First, the AM monomer was dissolved in distilled water. Then, 0.5 wt% of Darocur 1173 was added. To underline the crosslinker effect on the water uptake capacity of the obtained hydrogels, a set of three solutions with different percentages of HDDA (1 wt%, 4 wt%, and 7 wt%) was prepared. After steering for 24 h, the solutions were exposed to 4.2. Hydrogel Synthesis First, the AM monomer was dissolved in distilled water. Then, 0.5 wt% of Darocur 1173 was added. To underline the crosslinker effect on the water uptake capacity of the obtained hydrogels, a set of three solutions with different percentages of HDDA (1 wt%, 4 wt%, and 7 wt%) was prepared. 4.1. Materials The m (S i t Q The mo (Saint-Quent Cray Valley 2 h d 2 Cray Valley, 2 h d 2 2-hydroxy-2-m After steering for 24 h, the solutions were exposed to UV irradiation for 30 min, using a TL08 UV lamp, with a characteristic wavelength of λ = 365 nm and Chemical Structure Chemical Structur 3 Conclusions 3. Conclusions Interestingly, halogen bonding could be neglected, whereas hydrogen bonding plays a key role for dye retention. The system composed of poly(AM-co-AA)/RB has a binding energy of −7.7 kcal/mol, which means that this system has the highest stability compared to the other investigated polymer/dye systems. An analysis of rotatable bonds shows that the AA repetition unit presents two rotatable bonds, whereas that of AM has one; therefore, poly(AM-co-AA) has more conformations than poly(AM), thus increasing dye retention. Gels 2022, 8, 156 12 of 17 12 of 17 4.3. Dye Retention Experiments The selected cylindrical hydrogel (1.5 g), as an adsorbent, was immersed in 32 × 10−3 mg·mL−1 of dye solution at room temperature (T = 23 ◦C) for 24 h. Then, the hydrogel was separated by ﬁltration, and the residual concentration of the considered dye solution was introduced in a glass ﬂask and evaluated using a dual-beam ultraviolet–visible spectrometer (Specord 200 plus, Analytik Jena, Jena, Germany). 4.4. Equilibrium Swelling Measurements 4 2 H d l S th i 4.2. Hydrogel Synthesis 4 2 H d l S th i 4.2. Hydrogel Synthesis 4.2. Hydrogel Synthesis First, the AM monomer was dissolved in distilled water. Then, 0.5 wt% of Darocur 1173 was added. To underline the crosslinker effect on the water uptake capacity of the obtained hydrogels, a set of three solutions with different percentages of HDDA (1 wt%, 4 wt%, and 7 wt%) was prepared. After steering for 24 h, the solutions were exposed to UV irradiation for 30 min using a TL08 UV lamp with a characteristic wavelength of λ = First, the AM monomer was dissolved in distilled water. Then, 0.5 wt% of Darocur 1173 was added. To underline the crosslinker effect on the water uptake capacity of the obtained hydrogels, a set of three solutions with different percentages of HDDA (1 wt%, 4 wt%, and 7 wt%) was prepared. After steering for 24 h, the solutions were exposed to UV irradiation for 30 min, using a TL08 UV lamp, with a characteristic wavelength of λ = 365 nm and Gels 2022, 8, 156 13 of 17 an intensity of 1.5 mW/cm2. For the sake of comparison, a hydrogel copolymer was generated. A stock solution of 50 wt%/50 wt% AM/AA was ﬁrstly prepared. In the second step, three solutions were prepared with different percentages of HDDA: 1 wt%, 4 wt%, and 7 wt%, with 98.5 wt%, 95.5 wt%, and 92.5 wt% of the AM/AA solution, respectively. Finally, Darocur 1173 as a photoinitiator was added to each of these solutions (0.5 wt%). All solutions were prepared at room temperature. After the polymerization/crosslinking process (see also Figure S5), all samples were obtained in pellet form (sample thickness: 3 mm, diameter: 2.5 cm) and washed in distilled water to remove all remaining residue. 4.4. Equilibrium Swelling Measurements In order to underline the equilibrium swelling of the elaborated hydrogels, the sample was weighed in the dry state and then immersed in a dye solution for 24 h under stirring. Then, the sample was wiped with a ﬁlter paper to remove free liquid on the surface before being weighed. The degree of swelling was calculated according to Equation (1). τ(%) = 100 mt −m0 m0 (1) (1) where τ(%) represents the degree of swelling, mt stands for the weight of the swollen network at time t, and m0 is the weight of the initially dried network. 4.5. Model Proposition Two model systems were proposed. The ﬁrst one represents the crosslinked poly(AM)/ HDDA system, based on three chains of poly(AM) containing ten units each. Chains were connected by three HDDA crosslinking nodes. The second model system, the crosslinked poly(AM-co-AA), was created similar to the ﬁrst model. RB, EY, and MB were all presented in 3-D. All models were energy-minimized using auto-optimization with force ﬁeld UFF and the steepest descent algorithm of the Avogadro software. The output simulation implies eight conformations; the best conformation of each hydrogel/dye system was illustrated based on their energy. 4.6. Software Author Contributions: S.H.: conceptualization T B : software validation simulation calculati Informed Consent Statement: Not applicable. T.B.: software, validation, simulation calculat viewing, editing. P.S.: reviewing, editing. U.M d d d h bl h d f h Data Availability Statement: Not applicable. , , viewing, editing. P.S.: reviewing, editing. U.M Data Availability Statement: Not applicable. read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors gratefully acknowledge the support of the Research Center CRAPC Tipaza-Algeria and LRM Laboratory of Tlemcen University-Algeria. read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors gratefully acknowledge the support of the Research Center CRAPC Tipaza-Algeria and LRM Laboratory of Tlemcen University-Algeria. read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors gratefully acknowledge the support of the Research Center CRAPC Tipaza-Algeria and LRM Laboratory of Tlemcen University-Algeria. Institutional Review Board Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Institutional Review Board Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.6. Software Avogadro version 4.8.6 was used to visualize and optimize the model systems. The ﬁles were saved in the molecule ﬁle format pdb. AutoDock version 1.5.6, a molecular modeling simulation software, represents a suite of automated docking tools. It is designed to predict how dyes bind to polymer networks; the grid box allows the user to limit the space of interaction analysis (Figure 15). The simulation was conducted in dimensions of grid box points (x = y = z = 126 Å), and the grid box center dimensions were set as mentioned in Table S2. The other parameters were maintained and were used as defaults. Finally, the output ﬁle (log.txt) was analyzed, and the best docking results regarding binding energies were selected and investigated with other software programs. The Chimera calculation software UCSF version 1.5.3 was used to analyze interatomic distances. Gels 2022, 8, 156 14 of 17 It is de- user to 14 of 17 It is de- user to Figure 15. Grid box containing dye and polymer network using Autodock tools. Figure 15. Grid box containing dye and polymer network using Autodock tools. Figure 15. Grid box containing dye and polymer network using Autodock tools. Figure 15. Grid box containing dye and polymer network using Autodock tools. The simulation was conducted in dimensions of grid box points (x = y = z = 126 Å), and the grid box center dimensions were set as mentioned in Table S2. The other param- eters were maintained and were used as defaults. Finally, the output file (log.txt) was analyzed, and the best docking results regarding binding energies were selected and in- vestigated with other software programs. The Chimera calculation software UCSF ver- sion 1.5.3 was used to analyze interatomic distances. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/gels8030156/s1, Figure S1: UV-visible absorption spectra of the three dyes at different concentrations (solid line: C = 0.064 mg/mL, dashed line: C = 0.032 mg/mL); Figure S2: UV-visible absorption spectra of RB solutions with a contact time of 24 h; Figure S3: UV absorption spectra of MB solutions with a contact time of 24 h; Figure S4: UV-visible absorption spectra of EY solutions with a contact time of 24 h; Figure S5: Representation of crosslinked poly(acrylamide-co-acrylic acid)/HDDA; Table S1: Deﬁnition of Abbreviation; Table S2: Grid box center dimension for all systems. 4.6. Software The simulation was conducted in dimensions of grid box points (x = y = z = 126 Å), and the grid box center dimensions were set as mentioned in Table S2. The other param- eters were maintained and were used as defaults. Finally, the output file (log.txt) was analyzed, and the best docking results regarding binding energies were selected and in- vestigated with other software programs. The Chimera calculation software UCSF ver- sion 1.5.3 was used to analyze interatomic distances. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/gels8030156/s1, Figure S1: UV-visible absorption spectra of the three dyes at different concentrations (solid line: C = 0.064 mg/mL, dashed line: C = 0.032 mg/mL); Figure S2: UV-visible absorption spectra of RB solutions with a contact time of 24 h; Figure S3: UV absorption spectra of MB solutions with a contact time of 24 h; Figure S4: UV-visible absorption spectra of EY solutions with a contact time of 24 h; Figure S5: Representation of crosslinked poly(acrylamide-co-acrylic acid)/HDDA; Table S1: Deﬁnition of Abbreviation; Table S2: Grid box center dimension for all systems. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: UV-visible absorption spectra of the three dyes at different concentrations (solid line: C = 0.064 mg/mL, dashed line: C = 0.032mg/mL); Figure S2: UV-visible absorption spectra of RB solutions with a contact time of 24 h; Figure S3: UV absorption spectra of MB solutions with a contact time of 24 h; Figure S4: UV visible absorption spectra of EY solutions with a contact time of 24 h; Figure S5: Author Contributions: S.H.: conceptualization, methodology, writing—original draft preparation. T.B.: software, validation, simulation calculations. D.L.: supervision, reviewing, editing. Z.B.: reviewing, editing. P.S.: reviewing, editing. U.M.: editing, reviewing, submission. All authors have read and agreed to the published version of the manuscript. 24 h; Figure S4: UV visible absorption spectra of EY so Representation of crosslinked poly(acrylamide-co-acry Funding: This research received no external funding. 24 h; Figure S4: UV visible absorption spectra of EY so Representation of crosslinked poly(acrylamide-co-acr Funding: This research received no external funding. breviation; Table S2: Grid box center dimension for all sy Institutional Review Board Statement: Not applicable. breviation; Table S2: Grid box center dimension for all sy Institutional Review Board Statement: Not applicable. Author Contributions: S.H.: conceptualization T B : software validation simulation calculati Informed Consent Statement: Not applicable. 5. Ranganathan, K.; Jeyapaul, S.; Sharma, D.C. Assessment of water pollution in different bleaching based paper manufacturing and textile dyeing industries in India. Environ. Monit. Assess. 2007, 134, 363. [CrossRef] g g 4. 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https://openalex.org/W4281757996	https://europeanjournaloftaxonomy.eu/index.php/ejt/article/download/1807/6943	English	null	The genus Milnesium (Eutardigrada, Apochela, Milnesiidae) in Argentina: description of three new species and key to the species of South America	European Journal of Taxonomy	2,022	cc-by	25,250	M o n o g r a p h urn:lsid:zoobank.org:pub:522FD009-B4C9-4A80-871E-883E2EBE09C8 The genus Milnesium (Eutardigrada, Apochela, Milnesiidae) in Argentina: description of three new species and key to the species of South America Alejandra M. ROCHA 1,, Andrea X. GONZÁLEZ-REYES 2, Belén OSTERTAG 3 & Oscar LISI 4 Keywords. Tardigrades, taxonomy, Neotropical region, Salta, Santa Rosa, ontogenetic change. Alejandra M. ROCHA 1,, Andrea X. GONZÁLEZ-REYES 2, Belén OSTERTAG 3 & Oscar LISI 4 1 National University of La Pampa, Faculty of Exact and Natural Sciences, Uruguay 151, CP 6300, Santa Rosa, La Pampa, Argentina. 2 National University of Salta, Faculty of Natural Sciences, Bolivia 5150, CP 4400, Salta, Argentina. 3 National Scientific and Technical Research Council (CONICET), Uruguay 151, CP 6300, Santa Rosa, La Pampa, Argentina. 4 University of Catania, Department of Biological, Geological and Environmental Sciences, Section of Animal Biology, Androne 81, 95124 Catania, Italy. * Corresponding author: rochaale64@hotmail.com 2 Email: axgonzalezr@yahoo.com.ar 3 Email: belenostertag@gmail.com 4 Email: olisi@unict.it 2 Email: axgonzalezr@yahoo.com.ar 3 Email: belenostertag@gmail.com 4 Email: olisi@unict.it European Journal of Taxonomy 822: 1–54 ISSN 2118-9773 https://doi.org/10.5852/ejt.2022.822.1807 www.europeanjournaloftaxonomy.eu 2022 · Rocha A.M. et al. This work is licensed under a Creative Commons Attribution License (CC BY 4.0). European Journal of Taxonomy 822: 1–54 ISSN 2118-9773 https://doi.org/10.5852/ejt.2022.822.1807 www.europeanjournaloftaxonomy.eu 2022 · Rocha A.M. et al. This work is licensed under a Creative Commons Attribution License (CC BY 4.0). ISSN 2118-9773 www.europeanjournaloftaxonomy.eu 2022 · Rocha A.M. et al. European Journal of Taxonomy 822: 1–54 https://doi.org/10.5852/ejt.2022.822.1807 Introduction The genus Milnesium Doyère, 1840 had been considered monospecific for 62 years (from 1928 to 1990), and its only species Milnesium tardigradum Doyère, 1840 was regarded as rather variable and cosmopolitan. Starting in 1990 (Binda & Pilato 1990), authors started describing new species of the genus, each one less variable and not cosmopolitan. Since 2000, the rate of description of new species of the genus has increased remarkably, and Michalczyk et al. (2012a, 2012b) redescribed the nominal species questioning its presumed cosmopolitism. Descriptions of new species continued, so that today 45 species are attributed to the genus (excluding the subspecies Milnesium tardigradum trispinosa Rahm, 1931 and the species M. dujiangensis Yang, 2003, according to Morek et al. 2016 and Suzuki 2016). Based on the superficial morphology of the dorso-lateral cuticle, two species groups were established: the tardigradum group and the granulatum group, with smooth and sculptured cuticles, respectively (Michalczyk et al. 2012a, 2012b). Such groups, from the phylogenetic point of view, have been recently questioned (Morek et al. 2016), and even invalidated (Morek & Michalczyk 2020), but they can still be practical for purely morphological comparison between species. Of the 45 known species of Milnesium, 8 (17.8 %) are South American. Claps & Rossi (1984) made the first record of Milnesium in Argentina, for Salta Province. To date, Argentina has 5 recorded species of Milnesium (11.1% of the global total) but the South American tardigrade fauna is still poorly known (e.g., González Reyes et al. 2020). In this paper, we describe three new species of Milnesium from two regions of Argentina, one region in the North (Salta) and the other in the South-central area (La Pampa). 1 urn:lsid:zoobank.org:author:37D7AFFE-F588-478D-91AE-6E8BEA8754F9 2 urn:lsid:zoobank.org:author:4688D9CD-0D48-4928-A0DA-EF4B4C13A31F 3 urn:lsid:zoobank.org:author:7E8091DC-BFEA-42FE-AAE6-F5A2DB49958F 4 urn:lsid:zoobank.org:author:1EE34EFB-1742-49A8-BC11-109B67E2BEA3 1 urn:lsid:zoobank.org:author:37D7AFFE-F588-478D-91AE-6E8BEA8754F9 2 urn:lsid:zoobank.org:author:4688D9CD-0D48-4928-A0DA-EF4B4C13A31F 3 urn:lsid:zoobank.org:author:7E8091DC-BFEA-42FE-AAE6-F5A2DB49958F 4 urn:lsid:zoobank.org:author:1EE34EFB-1742-49A8-BC11-109B67E2BEA3 Abstract. A revision of microscope slides deposited between 2014 and 2017 in the collection of Rocha and Doma (National University of La Pampa, Argentina) revealed three new species of the genus Milnesium Doyère, 1840: M. pelufforum sp. nov., M. irenae sp. nov. and M. quiranae sp. nov. Milnesium pelufforum sp. nov. is mostly characterized by ten transverse bands of sculptured cuticle and pseudoplates (the first band, until now, never detected in the genus), six peribuccal lamellae and claw configuration [2-2]-[2-2] in young or [2-3]-[3-2] in senior specimens. Milnesium irenae sp. nov. is mostly characterized by complex cuticular ornamentation including a fine reticulation different from the typical one in the genus; it also has pseudoplates, six peribuccal lamellae, medioventral peribuccal papilla reduced, stylets, their furcae and supports very developed, and claw configuration [2-3]-[2-2]. Milnesium quiranae sp. nov. is mostly characterized by smooth cuticle, six peribuccal lamellae, and claw configuration [3-3]-[3-3]; with growing, the medioventral peribuccal papilla reduces and the buccal tube becomes wider. With the present contribution the genus Milnesium now has 48 valid species, and the number of described limno-terrestrial tardigrade species from South America has risen to 11, including 8 from Argentina with 5 from Salta and La Pampa province. 1 European Journal of Taxonomy 822: 1–54 (2022) Rocha A.M., González-Reyes A.X., Ostertag B. & Lisi O. 2022. The genus Milnesium (Eutardigrada, Apochela, Milnesiidae) in Argentina: description of three new species and key to the species of South America. European Journal of Taxonomy 822: 1–54. https://doi.org/10.5852/ejt.2022.822.1807 Material and methods Morphometric data were obtained using the AxioVision software SE64, given in micrometers (μm) and handled with the Excel template “Apochela” (ver. 1.4) from the tardigrade Register (Michalczyk & Kaczmarek 2013); the Excel files with the complete datasets of measurements are provided as supplementary material. Student t-tests (one-side tests) for statistical significance of differences between species morphometry (only when ranges of the given characters of the two compared species overlapped) were perfomed through Microsoft Office Excel software and the results are reported in Tables 4–5, 7, 11 (relative to the various differential diagnoses); when ranges did not overlap, they are reported directly in the text of the differential diagnoses. The buccal tube was measured according to Michalczyk et al. (2012a, 2012b) and claws according to Tumanov (2006). Percentage ratios between the length of the structure considered and that of the buccal tube (pt) were calculated following Pilato (1981) and pt ranges are indicated in italics within square brackets. Claw configuration is given according to Michalczyk et al. (2012a, 2012b). Species diagnoses were performed using original species descriptions, taxonomic keys and other useful pieces of literature: Binda & Pilato 1990; Maucci 1991; Pilato et al. 2002, 2016; Kaczmarek et al. 2004, 2012; Tumanov 2006; Kaczmarek & Michalczyk 2007; Meyer & Hinton 2010, 2012; Michalczyk et al. 2012a, 2012b; Meyer et al. 2013; Bartels et al. 2014; Ciobanu et al. 2015; Meyer 2015; Morek et al. 2016, 2019a, 2019b, 2020a; Pilato & Lisi 2016; Moreno-Talamantes et al. 2019, 2020; Surmacz et al. 2019 and Sugiura et al. 2020. For comparison with our material, the following type specimens from the Pilato and Binda Collection (Museum of the Section of Animal Biology, Department of Biological, Geological and Environmental Sciences, University of Catania) were examined: holotype and paratypes of Milnesium brachyungue Binda & Pilato, 1990 (slides Nos 3940–49), M. minutum Pilato & Lisi, 2016 (slide No. 4127), M. reticulatum Pilato, Binda & Lisi, 2002 (slides Nos 4851–4860), M. sandrae Pilato & Lisi, 2016 (slide No. 4290) and M. tumanovi Pilato, Sabella & Lisi, 2016 (slides Nos 3904, 3916). Paratypes of M. almatyense Tumanov, 2006 (slide No. 5106), M. antarcticum Tumanov, 2006 (slide No. 5104), M. asiaticum Tumanov, 2006 (slide No. 5105) M. beasleyi Kaczmarek, Jakubowska & Michalczyk, 2012 (slide No. 5518), and M. longiungue Tumanov, 2006 (slide No. 5103) were also examined. Material and methods This contribution is part of an ongoing review work on the Rocha and Doma tardigradological collection (Department of Natural Sciences of the National University of La Pampa, Argentina, deposited between 2014 and 2017). About 85 slides were examined, of which 26 were selected because containing three new species of the genus Milnesium described in the present paper. The material had been extracted from lichen and moss samples growing on sidewalk trees, taken from the cities of Salta and Santa Rosa. Samples were taken from trees of similar size (about 5 m tall) at 1.3 m height from the trunk surface facing the road. The samples were collected in May 2014 (Salta) and September 2017 (Santa Rosa), and were stored in paper bags at room temperature. For processing, they were hydrated in a plastic sieve (1.1 mm mesh), then transferred 24 h later to Petri dishes filled with mineral water for examination, using a stereoscopic microscope. Tardigrades and exuviae were separated with a micropipette. Active tardigrades were killed by placing them in a heater at about 60°C, or else using hot water (80°C). The material was mounted on slides with polyvinyl-lactophenol medium. Tardigrades were identified using phase contrast microscopy (PCM; Leica DM 500), equipped with a digital camera (ICC 50 HD). Detection of pseudoplates, in all three new species described in the present paper, and assessment of their configuration was performed using a fluorescence microscope (FM; Carl Zeiss Palm MicroBean realease 4.8 and digital camera AxioCam 506 color). Pseudoplate arrangement formulas are given following Moreno-Talamantes et al. (2019) (whose system, in turn, was based on that proposed by Michalczyk & Kaczmarek (2010) for the numbering of gibbosities in species of Doryphoribius and Isohypsibius); such formulas consist of a sequence of 2 ROCHA A.M. et al., Three new species of Milnesium from Argentina Roman numbers, indicating the pseudoplate rows from the most anterior (I) to the most posterior (IX in species of Milnesium according to Moreno-Talamantes et al. 2019), and each Roman number (i.e., row of pseudoplates) is followed by an Arabic number indicating the number of pseudoplates of the indicated row. However, in the present work it was necessary to add a pseudoplate row (the most cephalic, never detected until now) with respect to the nine indicated by Moreno-Talamantes et al. (2019), as explained in the Discussion and shown in Table 3. Diagnosis Ten transverse bands, better defined in senior specimens, of sculptured cuticle consisting of dimples forming a reticular pattern. Dimples, larger in young than in senior specimens, often showing some internal structure; ten rows of pseudoplates also present, better outlined in senior specimens, formula CP: I:1; II:4; III:6; IV:6; V:10; VI:10; VII:10; VIII:12; IX:12; X:4. Pseudoplate rows coincide with sculptured bands but sculpturing is not limited only on pseudoplates. Six peribuccal lamellae, six peribuccal papillae equal in size, two lateral cephalic papillae. Buccal tube nearly cylindrical, wider in senior specimens; claw configuration [2-2]-[2-2] in young, [2-3]-[3-2] in senior specimens (but with very small basal spurs, especially on legs IV). Material and methods Having worked with already mounted slides, determination of exact life stage was not possible; we separated, when ontogenetic change was observed (i.e., in Milnesium pelufforum sp. nov. and Milnesium quiranae sp. nov.), smaller from larger specimens according to the morphological and morphometric differences between the differentiable groups. For confirmation, we applied the method proposed by Surmacz et al. (2020), and the results perfectly corresponded to our grouping of specimens: in Milnesium pelufforum, two groups were identified, with the smaller specimens called ‘young’ (hatchlings or hatchlings plus second instar), and the larger called ‘senior’ (from second or third instar on); in Milnesium quiranae, three groups were identified, called ‘young’ (hatchlings or hatchlings plus second instar), ‘intermediate’ (second or third instar) and ‘senior’ (from third or fourth instar on) (Supp. file 7). Regarding Milnesium irenae sp. nov., instead, none of the approaches gave the possibility to separate specimen groups. The three graphs obtained through said analysis, representing the clustering or its absence for each species, are provided in supplementary materials. 3 European Journal of Taxonomy 822: 1–54 (2022) In preparing differential diagnoses we made the following decisions: 1) referring to the classical two morphological groups of the genus Milnesium (tardigradum and granulatum) for morphological convenience; very recently (Morek et al. 2016, 2021; Morek & Michalczyk 2020), the phylogenetic non-significance of those two groups was pointed out, but Morek & Michalczyk (2020) proposed new phylogenetic groups each with a variable morphology, thus not usable for the aims of the present paper. 2) For species with ontogenetic change, we prepared separate differential diagnoses for young and senior specimens. 3) In morphometric comparisons, when only a single value of a metric character, instead of a range, is reported for a compared species, this reflects the only information available from the literature. 4) When the original description of a compared species made no distinction between the measurements of external and internal claws of legs I–III, or of anterior and posterior claws of legs IV, we provide equivalent measurement indication for our new species for more direct comparison. Institutional acronyms Specimens from the following institutions and collections were examined (curator in parenthes MCNS = Museum of Natural Sciences, National University of Salta, Argentina (Ivanna Cruz) UNICT = Università degli Studi di Catania, Italy, Museum of the Department of Animal Biology ‘Marcello La Greca’, Italy, Binda and Pilato collection (Giovanni Pilato) UNLPam = National University of La Pampa, Faculty of Exact and Natural Sciences, Argentina, (Rocha Alejandra Mariana) MCNS = Museum of Natural Sciences, National University of Salta, Argentina (Ivanna C MCNS = Museum of Natural Sciences, National University of Salta, Argentina (Ivanna Cruz) UNICT = Università degli Studi di Catania, Italy, Museum of the Department of Animal Biology ‘Marcello La Greca’, Italy, Binda and Pilato collection (Giovanni Pilato) UNLPam = National University of La Pampa, Faculty of Exact and Natural Sciences, Argentina, (Rocha Alejandra Mariana) Results Taxonomic account Phylum Tardigrada Doyère, 1840 Class Eutardigrada Richters, 1926 Order Apochela Schuster, Nelson, Grigarick & Christenberry, 1980 Family Milnesiidae Ramazzotti, 1962 Genus Milnesium Doyère, 1840 Milnesium pelufforum sp. nov. urn:lsid:zoobank.org:act:0DA71F31-0FA3-4F27-91EC-CC028C5AC808 Figs 1–12, Tables 1–5 Phylum Tardigrada Doyère, 1840 Class Eutardigrada Richters, 1926 Order Apochela Schuster, Nelson, Grigarick & Christenberry, 1980 Family Milnesiidae Ramazzotti, 1962 Genus Milnesium Doyère, 1840 Milnesium pelufforum sp. nov. urn:lsid:zoobank.org:act:0DA71F31-0FA3-4F27-91EC-CC028C5AC808 Figs 1–12, Tables 1–5 Morphological description Body length from 216 µm to 620 µm, reddish colour before mounting, eyes present (habitus in Figs 1, 3, 8). Dorsal and dorsolateral cuticle with variously shaped dimples (depressions) (Figs 1–3, 6, 11) forming a reticular pattern, arranged in ten transverse bands (as a tendency in young, as a rule in senior specimens), one band on each of the ten subsegments of the body (Figs 1, 3, 11) including the very first, cephalic one; dimples often showing internal structures of variable appearance (Figs 2, 6, 11), but further investigations are required to ascertain whether apparent presence or absence, and shape, of such internal structures is just due to matter of focus under the microscope and orientation of the structures in the preparation; dimples vary in size but not in shape or details of internal structure, between young and senior specimens. Pseudoplates, better outlined in senior specimens, present (Figs 4–5, 9–10), also arranged in ten transverse bands (again, including the very first, cephalic subsegment); pseudoplate rows correspond with the sculptured bands but the sculpturing is not limited only on pseudoplates; pseudoplate formula is CP: I:1; II:4; III:6; IV:6; V:10; VI:10; VII:10; VIII:12; IX:12; X:4 (based on senior specimens). Row I is situated at the level of the buccal tube and has only one medial pseudoplate, difficult to see, more or less rectangular, laying transversally, with rounded angles. Row II, situated just anteriorly to legs I, has four pseudoplates: two medial, about trapezoidal, touching in the central line along their longer side, and two separate lateral, about rectangular, laying obliquely and difficult to see. Row III, situated in line with legs I, has six pseudoplates: four central arranged in two pairs, all four connected, with the two anterior, difficult to see, about rectangular, and the two caudal, triangular, pointing backwards; two lateral pseudoplates, about ellyptical, laying obliquely and difficult to see. Row IV, situated between legs I and II, has six pseudoplates: the four medial arranged in two pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these four pseudoplates vaguely rectangular but with the transverse line dividing the anterior and posterior couple not straight; two rounded lateral pseudoplates, difficult to see. Etymology The new species is dedicated to Maria Cristina Moly de Peluffo and Julio Ricardo Peluffo, the first researchers of tardigrades from the National University of La Pampa, Argentina. 4 ROCHA A.M. et al., Three new species of Milnesium from Argentina Paratypes Paratypes ARGENTINA • 2 ♀♀; same collection data as for holotype; MCNS Tar.000021(2), Tar.000021(4) • 4 ♀♀; same collection data as for holotype; UNICT 5898(1) to 5898(4) • 17 ♀♀; same collection data as for holotype; UNLPam 389(1) to 391(3), 454(1) to 454(5), 465(1) to 465(4), 456(4) to 456(5), 503(1) to 503(3). Material examined Holotype ARGENTINA • ♀; Salta Province, Salta City; 24°47′18″ S, 65°24′38″ W, 1150 m a.s.l.; 2 May 2014; Rocha-Doma leg.; moss and lichens from trees; MCNS Tar.000021(3). Morphological description Row V, situated in line with legs II, has ten pseudoplates: the six medial arranged in three pairs, transversally elongated, connected, forming a unique about rectangular/trapezoidal structure; each of these six pseudoplates vaguely rectangular but with the two transverse lines dividing the adjacent anterior/posterior pairs of pseudoplates not straight; lateral to that central complex, on each side, two separate pseudoplates, difficult to see, with the mid- lateral less developed and vaguely in the shape of a curved trapezium, while the most lateral is more developed and vaguely quadrangular. Row VI, situated between legs II and III, has ten pseudoplates: two medial rectangular, sided laterally and very elongated transversally; lateral to them, on each side, four separate pseudoplates difficult to see, arranged in a quadrangle. Row VII, situated in line with legs III, has ten pseudoplates: the six medial arranged in three pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these six pseudoplates vaguely rectangular but with the two transverse lines dividing the adjacent anterior/posterior pairs of pseudoplates not straight; lateral to that central complex, on each side, two separate pseudoplates, with the mid-lateral less developed and vaguely in the shape of a curved trapezium, while the most lateral is more developed and vaguely quadrangular. Row VIII, situated just posterior to legs III, has twelve pseudoplates with the four medial p g p p yp y g q yi Row IV, situated between legs I and II, has six pseudoplates: the four medial arranged in two pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these four pseudoplates vaguely rectangular but with the transverse line dividing the anterior and posterior couple not straight; two rounded lateral pseudoplates, difficult to see. Row V, situated in line with legs II, has ten pseudoplates: the six medial arranged in three pairs, transversally elongated, connected, forming a unique about rectangular/trapezoidal structure; each of these six pseudoplates vaguely rectangular but with the two transverse lines dividing the adjacent anterior/posterior pairs of pseudoplates not straight; lateral to that central complex, on each side, two separate pseudoplates, difficult to see, with the mid- lateral less developed and vaguely in the shape of a curved trapezium, while the most lateral is more developed and vaguely quadrangular. Morphological description Row VI, situated between legs II and III, has ten pseudoplates: two medial rectangular, sided laterally and very elongated transversally; lateral to them, on each side, four separate pseudoplates difficult to see, arranged in a quadrangle. Row VII, situated in line with legs III, has ten pseudoplates: the six medial arranged in three pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these six pseudoplates vaguely rectangular but with the two transverse lines dividing the adjacent anterior/posterior pairs of pseudoplates not straight; lateral to that central complex, on each side, two separate pseudoplates, with the mid-lateral less developed and vaguely in the shape of a curved trapezium, while the most lateral is more developed and vaguely quadrangular. Row VIII, situated just posterior to legs III, has twelve pseudoplates with the four medial 5 European Journal of Taxonomy 822: 1–54 (2022) arranged in two pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these four pseudoplates vaguely rectangular but with the two anterior well visible and not thin, while the two posterior difficult to see and very thin; lateral to that central complex, on each side, four separate pseudoplates arranged in a quadrangle: the two mid-lateral elongated transversally, the two very lateral vaguely quadrangular; besides, the two anterior pseudoplates of the tetrad difficult to see, while the two posterior better visible. Row IX, situated between legs III and IV, has twelve pseudoplates with a central aggregation of 10 pseudoplates forming a complex pattern (see Fig. 10), and two lateral single pseudoplates about quadrangular, aligned mid-posteriorly. Row X, situated just anterior to legs IV, has four about quadrangular pseudoplates, sided and aligned in a single row transversally. arranged in two pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these four pseudoplates vaguely rectangular but with the two anterior well visible and not thin, while the two posterior difficult to see and very thin; lateral to that central complex, on each side, four separate pseudoplates arranged in a quadrangle: the two mid-lateral elongated transversally, the two very lateral vaguely quadrangular; besides, the two anterior pseudoplates of the tetrad difficult to see, while the two posterior better visible. Row IX, situated between legs III and IV, has twelve pseudoplates with a central aggregation of 10 pseudoplates forming a complex pattern (see Fig. 10), and two lateral single pseudoplates about quadrangular, aligned mid-posteriorly. Fig. 1. Milnesium pelufforum sp. nov. Schematic drawing of a young and a senior specimen showing the ten sculptured bands. Morphological description Row X, situated just anterior to legs IV, has four about quadrangular pseudoplates, sided and aligned in a single row transversally. Cuticular grooves present dorsally, but this and other details of the cuticular ornamentation vary depending on life stage (see below). I II III IV V VI VII VIII IX X I II III IV V VI VII VIII IX X Young Senior Fig. 1. Milnesium pelufforum sp. nov. Schematic drawing of a young and a senior specimen showing the ten sculptured bands. Senior Fig. 1. Milnesium pelufforum sp. nov. Schematic drawing of a young and a senior specimen showing the ten sculptured bands. Fig. 1. Milnesium pelufforum sp. nov. Schematic drawing of a young and a senior specimen showing the ten sculptured bands. 6 ROCHA A.M. et al., Three new species of Milnesium from Argentina Six peribuccal lamellae and six peribuccal papillae plus two lateral papillae present. Buccal tube (Figs 7A, 12A) nearly cylindrical (posterior/anterior width ratio 82–100%), more slender in young specimens; pt of the stylet support insertion point on the buccal tube [65.0–73.8]. Six peribuccal lamellae and six peribuccal papillae plus two lateral papillae present. Buccal tube (Figs 7A, 12A) nearly cylindrical (posterior/anterior width ratio 82–100%), more slender in young specimens; pt of the stylet support insertion point on the buccal tube [65.0–73.8]. Claws of the Milnesium type with configuration [2-2]-[2-2] in young (Fig. 7B–C), [2-3]-[3-2] in senior specimens (Fig. 12B–D) but with very small basal spurs where present, in particular on legs IV where they are just a little spine (Fig. 12B–D, white arrows); claws stout, secondary branches with basal thickenings (‘lunulae’; Figs 7C and 12C–D, white arrowheads), primary branches with small accessory points (Figs 7B and 12B–C, black arrows); cuticular bars present on legs I–III (Figs 7B and 12B–C, black arrowheads); percentual ratio of secondary branches with respect to primary branches for each claw couple higher for legs I, slightly lower for legs II–III and more significantly lower for legs IV (Tables 1 and 2). More detailed description is given in the following paragraphs separating young and senior specimens. Young specimens (probably hatchlings: 216–305 µm; Figs 1, 3–7, Table 1, Supp. file 1) Cuticular dimples larger, especially in proportion to the body size (about 2–3.5 µm); dimples tend to form ten transverse bands almost touching one another and connected, at least in some areas, by dimples appearing less evident (Fig. 1). Morphological description Pseudoplates less developed and less distinct from one another in each row than is senior specimens (Figs 4–5). Few simple, short, cuticular depressions can be present on some segments, tending to lay transversally, the most developed is constantly present at the level of legs III in the shape of a transverse groove (Figs 1, 3, 6, white arrowheads). Buccal tube (Fig. 7A) more slender than in senior specimens (pt of standard width [43.7–52.1]) Claw configuration [2-2]-[2-2] (Fig. 7B–C); percentual ratio of secondary branches with respect to primary branches for each claw couple with a less marked difference (than in senior specimens) between legs I and legs IV (Table 1): 71–90% for claws I, 66–86% for claws IV, difference is 4–5%. Senior specimens (probably from second instar on: 390–620 µm; Figs 1, 8–12, Table 2, Supp. file 2) Cuticular dimples smaller than in young specimens (about 1–2 µm), especially in proportion to the body size; ten clear transverse bands of dimples, more spaced from one another than in young specimens and separated by smooth cuticle (Figs 1, 11). Starting with legs II in about 50% of the specimens, with legs III in the rest of them, irregular, usually branched cuticular depressions are present, tending to lay Fig. 2. Milnesium pelufforum sp. nov. Schematic drawing of cuticular dimples showing their shapes and different appearances of internal structures. Fig. 2. Milnesium pelufforum sp. nov. Schematic drawing of cuticular dimples showing their shapes and different appearances of internal structures. Fig. 2. Milnesium pelufforum sp. nov. Schematic drawing of cuticular dimples showing their shapes and different appearances of internal structures. 7 European Journal of Taxonomy 822: 1–54 (2022) Fig. 3. Milnesium pelufforum sp. nov., paratype, young (slide No. 5898 (Pilato and Binda Collection)). Habitus. The arrowhead indicates the dorsal cuticular groove constantly present in young specimens at the level of legs III. Scale bar = 30 µm. Fig. 3. Milnesium pelufforum sp. nov., paratype, young (slide No. 5898 (Pilato and Binda Collection)). Habitus. The arrowhead indicates the dorsal cuticular groove constantly present in young specimens at the level of legs III. Scale bar = 30 µm. Fig. 4. Milnesium pelufforum sp. nov., paratype, young (slide No. UNLPam 389-1) under UV fluorescence microscope, showing various pseudoplates. Scale bar = 50 µm. Fig. 4. Milnesium pelufforum sp. nov., paratype, young (slide No. UNLPam 389-1) under UV fluorescence microscope, showing various pseudoplates. Scale bar = 50 µm. 8 ROCHA A.M. et al., Three new species of Milnesium from Argentina Fig. 5. Semi-schematic drawing of pseudoplate configuration in the young specimens of Milnesium pelufforum sp. nov. Fig. 5. Semi-schematic drawing of pseudoplate configuration in the young specimens of Milnesium pelufforum sp. nov. Fig. 6. Milnesium pelufforum sp. nov., paratype, young (slide No. 5898 (Pilato and Binda Collection)). Details of the cuticular ornamentation. A. General view of the dorsal cuticle showing several bands of sculptured cuticle and the cuticular groove at the level of legs III (arrow). B. Magnification of a portion of dorsal cuticle shown in A, better showing the cuticular dimples, some with evident internal structure (arrowheads), and others with more or less out of focus internal structure (the internal of the dimples is not bright, e.g., the dimples in the centre of the photo). Scale bars: A = 20 µm; B = 10 µm. Fig. 6. Milnesium pelufforum sp. nov., paratype, young (slide No. 5898 (Pilato and Binda Collection)). Details of the cuticular ornamentation. A. General view of the dorsal cuticle showing several bands of sculptured cuticle and the cuticular groove at the level of legs III (arrow). B. Magnification of a portion of dorsal cuticle shown in A, better showing the cuticular dimples, some with evident internal structure (arrowheads), and others with more or less out of focus internal structure (the internal of the dimples is not bright, e.g., the dimples in the centre of the photo). Fig. 2. Milnesium pelufforum sp. nov. Schematic drawing of cuticular dimples showing their shapes and different appearances of internal structures. Scale bars: A = 20 µm; B = 10 µm. 9 European Journal of Taxonomy 822: 1–54 (2022) transversally or obliquely, more conspicuous on the caudal segments and all in general more developed and complex than in young specimens (Figs 1, 8, 11). Buccal tube (Fig. 12A) stouter than in young specimens (pt of standard width [55.2–64.0]). 10 Fig. 7. Milnesium pelufforum sp. nov., paratype, young (slide No. UNLPam 503-1). Buccal tube and claws. A. Buccal tube and related structures. B. Claws of legs I; the black arrow indicates the accessory points, the black arrowhead indicates the leg cuticular bar. C. Claws of legs IV; the white arrowhead indicates a ‘lunule’. Scale bars = 10 µm. Fig. 7. Milnesium pelufforum sp. nov., paratype, young (slide No. UNLPam 503-1). Buccal tube and claws. A. Buccal tube and related structures. B. Claws of legs I; the black arrow indicates the accessory points, the black arrowhead indicates the leg cuticular bar. C. Claws of legs IV; the white arrowhead indicates a ‘lunule’. Scale bars = 10 µm. 10 ROCHA A.M. et al., Three new species of Milnesium from Argentina Claw configuration [2-3]-[3-2] (Fig. 12B–D); if these senior specimens include already the second instar, this would indicate early claw configuration change. Basal spurs of internal secondary branches I–III very small, and those of anterior secondary branches IV reduced to a little spine (Fig. 12B–D, white arrows); secondary branches with basal thickenings (‘lunulae’) which are larger on legs IV (Fig. 12C–D, white arrowheads); percentual ratio of secondary branches with respect to primary branches for each claw couple with a more marked difference between legs I and legs IV (Table2): 87–99% for claws I, 72–86% for claws IV, difference is 13–15%. Remarks It was not possible to examine under SEM the (already mounted) studied material, therefore we considered the bright spots forming the reticular pattern visible under PCM as ‘dimples’ (or depressions), and not true pseudopores (as defined by Morek et al. 2020a) as a consequence of an interpretation, also suggested by one anonymous reviewer. Such structures perfectly correspond in size, spatial distribution, and general appearance, to what can be seen in PCM images of many other species descriptions, in which the structures have been determined using SEM; true pseudopores are, instead, far smaller and usually more scattered and less visible. Milnesium pelufforum sp. nov. is the first species of the genus described with cuticular dimples that show some internal structure and form the reticulation arranged in ten transverse bands, as well as pseudoplates. The presence of ten transverse rows/bands of both structures, instead of a maximum of nine (the rule until now in Milnesium) is the result of their presence in the new species also on the very first, cephalic, subsegment, where they were not found (or noticed) in the other species until now; this requires an update in the indication of the pseudoplate formula of all species of Milnesium with pseudoplates (see Discussion and Table 3). Morphometric data are given in Tables 1 (young specimens) and 2 (senior specimens); in Tables 4 and 5 (for senior and young specimens respectively) the statistically significant differences (through Student t-tests) of overlapping pt ranges of claw heights between the new species and the similar ones. Fig. 8. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3). Habitus. Scale bar = 50 µm. Fig. 8. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3). Habitus. Scale bar = 50 µm. 11 11 European Journal of Taxonomy 822: 1–54 (2022) Table 1 (continued on next page). Measurements (in μm) and pt values of selected morphological structures of young specimens of Milnesium pelufforum sp. nov. mounted in polyvinyl-lactophenol medium. Range refers to the lowest and the highest values among all measured specimens. Pt values are provided in italics. Abbreviations: N = number of specimens or structures measured; SD = standard deviation. Remarks Character N Range Mean SD µm pt µm pt µm pt Body length 12 216–305 1080 – 1615 259 1262 28 160 Peribuccal papillae length 2 3.1–3.2 14.7 – 15.1 3.1 14.9 0.1 0.3 Lateral papillae length 8 2.9–3.3 14.6 – 15.9 3.1 15.1 0.1 0.6 Buccal tube Length 12 17.9–21.7 – 20.5 – 1.0 – Stylet support insertion point 12 13.0–15.7 65.3 – 73.8 14.7 71.6 0.7 2.3 Anterior width 12 9.6–12.1 46.6 – 55.9 10.8 52.6 0.7 2.5 Standard width 12 9.2–10.7 43.7 – 52.1 9.7 47.5 0.5 2.5 Posterior width 12 9.1–10.2 45.4 – 53.7 9.7 47.5 0.3 2.3 Standard width/length ratio 12 44%–52% – 47% – 3% – Posterior/anterior width ratio 12 82%–99% – 91% – 5% – Claw 1 lengths External primary branch 11 9.5–11.2 45.0 – 53.7 10.2 49.9 0.5 2.7 External base + secondary branch 11 7.9–9.1 37.9 – 47.4 8.4 41.2 0.4 2.4 External branches length ratio 10 77%–90% _ 83% 5% Internal primary branch 10 9.1–10.3 43.8 – 52.5 9.8 48.1 0.4 2.9 Internal base + secondary branch 11 7.3–9.1 35.9 – 44.7 8.0 38.9 0.5 2.5 Internal branches length ratio 9 71%–89% – 81% 5% Claw 2 lengths External primary branch 11 9.8–11.6 46.1 – 55.9 10.6 51.6 0.5 3.2 External base + secondary branch 12 7.9–9.2 38.2 – 45.6 8.4 40.7 0.4 2.2 External branches length ratio 11 74%–92% – 80% 5% Internal primary branch 11 9.7–11.0 46.0 – 56.5 10.3 50.3 0.4 3.1 Internal base + secondary branch 12 7.3–9.0 34.8 – 42.0 7.9 38.3 0.6 2.2 Internal branches length ratio 11 69%–86% – 77% 5% Claw 3 lengths External primary branch 11 9.8–11.4 46.4 – 57.0 10.6 52.0 0.5 3.2 External base + secondary branch 10 7.5–9.4 37.7 – 45.0 8.4 41.1 0.6 2.3 External branches length ratio 10 73%–85% – 80% 4% Internal primary branch 12 9.9–10.7 47.9 – 57.9 10.3 50.4 0.3 2.7 Internal base + secondary branch 9 7.2–8.7 35.7 – 40.6 7.9 38.3 0.6 2.1 Internal branches length ratio 9 69%–82% – 76% 4% 12 ROCHA A.M. et al., Three new species of Milnesium from Argentina Table 1 (continued). Measurements (in μm) and pt values of selected morphological structures of young specimens of Milnesium pelufforum sp. nov. mounted in polyvinyl-lactophenol medium. Differential diagnosis Based on the presence of dimples on the dorsal cuticle, Milnesium pelufforum sp. nov. can be similar to many species of the old granulatum group (Michalczyk et al. 2012a, 2012b), including both species with true dimples ascertained through SEM, and others in which the value of the bright spots visible under PCM (whether dimples or pseudopores) on the cuticle is still to be verified, but the appearance under PCM can be similar to the cuticular sculpturing of Milnesium pelufforum. The new species differs from all of them (indeed, from all congeneric species, according to their descriptions/redescriptions) by the presence of ten transverse bands of sculptured cuticle and rows of pseudoplates, and the presence of dimple internal structures. We here provide separate differential diagnoses for young and senior specimens of the new species, due to the ontogenetic change, limiting the comparisons to species with the same claw configurations. Senior specimens of Milnesium pelufforum sp. nov. can be similar to those of M. beasleyi Kaczmarek, Jakubowska & Michalczyk, 2012, M. cassandrae Moreno-Talamantes, Roszkowska, García-Aranda, Flores-Maldonado & Kaczmarek, 2019, M. krzysztofi Kaczmarek & Michalczyk, 2007, M. lagniappe Meyer, Hinton & Dupré, 2013, M. reticulatum Pilato, Binda & Lisi, 2002 and M. pacificum Sugiura, Minato, Matsumoto & Suzuki, 2020 by having a more or less similar cuticular ornamentation (seen under PCM) and claw configuration [2-3]-[3-2], but the senior specimens of the new species differ from them as follows: 1. Milnesium beasleyi only known from the type locality in Turkey, by different body colour: reddish in M. pelufforum sp. nov. vs yellow in M. beasleyi; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs no band arrangement described in M. beasleyi; dimple diameter of about 1–2 µm in M. pelufforum vs 0.1–0.4 µm in M. beasleyi; presence of pseudoplates in M. pelufforum vs not reported in M. beasleyi; statistically significant lower pt of the lateral papillae, [13.8–19.9, mean 16.0] in M. pelufforum vs [19.6–23.7, mean 21.5] in M. beasleyi (t11 = -10.06, p < 0.001); different buccal tube width: higher pt of standard width [55.2–64.0] in M. pelufforum vs [31.2–39.8] in M. beasleyi; statistically significant differences about pt of several claw heights (Table 4). 2. Milnesium cassandrae (adults) only known from the terra typica (Mexico; Moreno-Talamantes et al. 2019, 2020), by different body colour: reddish in M. pelufforum sp. nov. Remarks Character N Range Mean SD µm pt µm pt µm pt Claw 3 lengths Anterior primary branch 9 10.8– 12.4 50.9 – 60.5 11.7 56.2 0.6 3.3 Anterior base + secondary branch 12 7.8– 9.7 37.5 – 45.8 8.5 41.3 0.6 2.6 Anterior branches length ratio 9 66%– 84% – 75% 5% Posterior primary branch 9 11.4– 12.7 54.2 – 63.0 12.3 59.0 0.4 2.9 Posterior base + secondary branch 11 7.9– 10.3 37.5 – 47.6 9.1 43.7 0.7 3.0 Posterior branches length ratio 9 67%– 86% – 75% 5% Differential diagnosis vs white or transparent with light yellow brownish tones before fixation in M. cassandrae; sculptured cuticle with dimples 13 European Journal of Taxonomy 822: 1–54 (2022) Table 2 (continued on next page). Measurements (in μm) and pt values of selected morphological structures of senior specimens of Milnesium pelufforum sp. nov. mounted in polyvinyl-lactophenol medium. Range refers to the lowest and the highest values among all measured specimens. Pt values are provided in italics. Abbreviations: N = number of specimens or structures measured; SD = standard deviation. Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Body length 12 390–620 1334–2087 526 1629 78 206 576 1624 Peribuccal papillae length 8 6.1–8.8 19.5–25.9 7.5 22.9 1.0 2.6 8.4 23.7 Lateral papillae length 11 4.0–6.0 13.8–19.9 5.2 16.0 0.7 1.7 5.3 14.8 Buccal tube Length 12 26.1–36.6 – 32.3 – 3.2 – 35.5 – Stylet support insertion point 12 18.1–23.8 65.0–69.4 21.6 67.0 1.8 1.9 23.4 66.1 Anterior width 12 16.0–23.2 58.1–66.3 20.1 62.0 2.3 2.9 22.5 63.3 Standard width 12 15.2–22.8 55.2–64.0 19.1 59.0 2.2 3.3 20.6 58.2 Posterior width 12 15.4–22.8 56.0–65.0 19.6 60.5 2.3 3.6 22.2 62.5 Standard width/length ratio 12 55%–64% – 59% – 3% – 58% – Posterior/anterior width ratio 12 91%–100% – 97% – 2% – 99% – Claw 1 lengths External primary branch 11 12.1–15.8 41.8–48.9 14.5 45.1 1.4 2.7 15.4 43.3 External base + secondary branch 10 11.0–15.7 37.4–46.2 13.5 42.0 1.5 3.5 13.5 38.2 External branches length ratio 9 87%–98% – 92% 3% 88% Internal primary branch 9 11.6–15.0 39.1–46.8 13.6 42.9 1.3 3.1 ? ? Internal base + secondary branch 11 11.0–15.3 36.0–43.0 12.8 39.8 1.4 2.8 13.2 37.1 Internal spur 7 2.6–4.0 7.6–12.8 3.4 10.1 0.5 1.9 2.8 7.9 Internal branches length ratio 8 90%–99% – 94% 3% _ Claw 2 lengths External primary branch 10 12.2–18.0 46.7–55.2 16.1 50.0 1.9 3.5 17.3 48.8 External base + secondary branch 12 10.9–16.2 37.7–47.6 14.1 43.7 1.6 3.2 14.7 41.5 External branches length ratio 10 81%–95% – 88% 4% 85% Internal primary branch 9 12.1–17.3 43.6–52.2 15.0 47.9 1.7 3.3 16.4 46.4 Internal base + secondary branch 9 11.4–15.3 37.6–45.0 13.7 41.4 1.2 3.0 15.1 42.6 Internal spur 7 2.8–4.9 10.7–15.9 4.0 12.6 0.6 1.9 ? ? Differential diagnosis Internal branches length ratio 7 82%–92% – 86% 3% 92% Claw 3 lengths External primary branch 9 12.4–18.4 46.6–56.0 15.8 50.0 2.0 3.8 16.9 47.7 External base + secondary branch 8 11.4–14.9 38.6–47.8 13.4 43.0 1.3 2.8 14.9 42.0 External branches length ratio 8 79%–92% – 86% 4% 88% Internal primary branch 9 11.7–16.8 42.8–52.8 14.8 46.6 1.8 3.9 15.4 43.4 Internal base + secondary branch 8 11.9–14.5 35.3–44.0 13.0 39.7 0.8 3.3 13.1 36.9 Internal spur 8 3.0–5.0 10.4–16.2 3.9 12.4 0.7 2.0 4.0 11.3 Internal branches length ratio 7 80%–86% _ 83% 2% 85% 14 ROCHA A.M. et al., Three new species of Milnesium from Argentina Table 2 (continued). Measurements (in μm) and pt values of selected morphological structures of senior specimens of Milnesium pelufforum sp. nov. mounted in polyvinyl-lactophenol medium. Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Claw 4 lengths Anterior primary branch 11 16.3–22.8 58.1 – 67.3 19.8 61.6 2.0 3.2 22.8 64.3 Anterior base + secondary branch 11 11.8–18.0 42.4 – 52.4 15.0 46.7 1.9 3.2 16.3 46.0 Anterior spur 8 2.0–3.9 6.1 – 13.3 2.7 8.3 0.8 2.7 2.2 6.2 Anterior branches length ratio 11 72%–86% – 76% 4% 72% Posterior primary branch 10 17.7–23.0 61.1 – 70.0 20.8 64.7 2.0 3.5 21.9 61.8 Posterior base + secondary branch 12 12.8–18.1 44.2 – 53.4 15.9 49.2 1.8 3.2 17.5 49.3 Posterior branches length ratio 10 72%–80% 75% 3% 80% arranged in ten bands in M. pelufforum vs sparsely distributed, not forming bands or reticular design, in M. cassandrae; presence of ten pseudoplate rows in M. pelufforum vs nine rows in M. cassandrae, and with different number in each row (Table 3); statistically significant larger dimple diameter, 1–2 µm in M. pelufforum vs 0.6–1.4 µm in M.cassandrae (t13 = -18.02, p < 0.001); statistically significant higher pt of stylet support insertion point, [65.0–69.4, mean 67.0] in M.pelufforum vs [58.7–67.6, mean 63.5] in M. cassandrae (t12 = 3.33, p < 0.01); statistically significant higher buccal tube posterior/anterior width ratio, 91–100%, mean 97% in M. pelufforum vs 81–96%, mean 89% in M. cassandrae (t12 = 4.39, p < 0.001); statistically significant differences about pt of some claw heights (Table 4). 3. Milnesium krzysztofi known from Costa Rica (type locality), Perú (Kaczmarek et al. 2014) and Colombia (Lisi et al. 2014; Londoño et al. 2015; Melo et al. Differential diagnosis 2015), by different body colour: reddish in M. pelufforum sp. nov. vs white or transparent in M. krzysztofi; eyes present in M. pelufforum vs absent in M. krzysztofi; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs not forming bands in M. krysztofi; different number of pseudoplates, not reported in M. krzysztofi, but visible in Kaczmarek & Michalczyk (2007: figs 2–7) in were it is possible to see at least six rows with different number/arrangement of pseudoplates; buccal tube nearly cylindrical in M. pelufforum vs funnel- shaped in M.krzysztofi: anterior and posterior width of the buccal tube of this species not available from description but the difference is visually evident comparing Fig. 12A of the present paper with Kaczmarek & Michalczyk (2007: fig. 12); different buccal tube width: higher pt of standard width [55.2–64.0] in M. pelufforum vs [33.1–38.4], in M.krzysztofi; statistically significant differences about pt of several claw heights (Table 4). 4. Milnesium lagniappe only known from the type locality in USA, by different body colour: reddish in M. pelufforum sp. nov. vs white or transparent in M. lagniappe; eyes visible in M. pelufforum vs not visible in M. lagniappe; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs arranged in nine bands in M. lagniappe; different number/arrangement of pseudoplates, not reported in M. lagniappe, but visible in Meyer et al. (2013: fig. 1a-b) where it is possible to see at least seven rows different from the correspondent 10 rows of M. pelufforum; statistically significant larger dimple diameter, 1–2 µm in M. pelufforum vs 0.7–1.3 µm in M. lagniappe (t13 = 9,01, p < 0.001); statistically significant lower pt of the peribuccal papillae, [19.5–25.9, mean 22.9] in M. pelufforum vs [22.7– 34.7, mean 28.0] in M. lagniappe (t8 = 4.32, p < 0.001); statistically significant lower pt of the lateral papillae: [13.8–19.9, mean 16.0] in M. pelufforum vs [16.9–30.5, mean 23.2] in M. lagniappe (t11 = 3.79, p < 0.001); lower pt of stylet support insertion point [65.0–69.4] in M. pelufforum vs [69.7–73.4] in 15 European Journal of Taxonomy 822: 1–54 (2022) Fig. 9. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3), under UV fluorescence microscope, showing various pseudoplates. Scale bar = 50 µm. Fig. 9. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3), under UV fluorescence microscope, showing various pseudoplates. Scale bar = 50 µm. Fig. Differential diagnosis 10. Semi-schematic drawing of pseudoplate configuration in the senior specimens of Milnesium pelufforum sp. nov. Fig. 10. Semi-schematic drawing of pseudoplate configuration in the senior specimens of Milnesium pelufforum sp. nov. 16 ROCHA A.M. et al., Three new species of Milnesium from Argentina Fig. 11. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3). Overview of the ten bands of cuticular dimples from the head (above) to the caudal end of the body (below) A. First and second bands (roman numbers). B. Third and fourth bands (the fourth also shows in the centre several dimples with internal structures). C. Fifth and sixth bands. D. Seventh and eighth bands (the seventh also shows in the centre some dimples with internal structures). E. Ninth band. F. Tenth band. In all pictures (A–F) cuticular grooves are also visible. Scale bars = 10 µm. Fig. 11. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3). Overview of the ten bands of cuticular dimples from the head (above) to the caudal end of the body (below) A. First and second bands (roman numbers). B. Third and fourth bands (the fourth also shows in the centre several dimples with internal structures). C. Fifth and sixth bands. D. Seventh and eighth bands (the seventh also shows in the centre some dimples with internal structures). E. Ninth band. F. Tenth band. In all pictures (A–F) cuticular grooves are also visible. Scale bars = 10 µm. 17 European Journal of Taxonomy 822: 1–54 (2022) M. lagniappe; different buccal tube width: lower pt of standard width [55.2–64.0] in M. pelufforum vs [63.4–77.9] in M. lagniappe; statistically significant differences about pt of several claw heights (Table 4). 5. Milnesium reticulatum only known from the type locality in the Seychelles islands, by different body colour: reddish in M. pelufforum sp. nov. vs transparent in M. reticulatum; different number of peribuccal lamellae: six in M. pelufforum vs four in M. reticulatum; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs nine in M. reticulatum; presence of pseudoplates in M. pelufforum vs not reported in M. reticulatum; higher pt buccal tube standard width, [55.2–64.0] in M. pelufforum vs [30.4–37.4] in M. reticulatum; higher pt of many claw lengths: external primary and secondary branches of leg II, [46.7–55.2] and [37.7–47.6] in M. pelufforum vs [35.6–38.8] and [26.8–29.6] respectively in M. Differential diagnosis pacificum; dimple diameter about 1–2 µm in M. pelufforum vs about 0.50–0.65 µm in M. pacificum; presence of ten rows of pseudoplates in M. pelufforum vs nine rows in M. pacificum; statistically significant higher pt of the peribuccal papillae [19.5–25.9, mean 22.9] in M. pelufforum vs [16.3–22.4, mean 18.7] in M. pacificum (t8 = 5.09, p < 0.001); different buccal tube width: higher pt of standard width [55.2–64.0] in M. pelufforum vs [33.0–40.8] in M. pacificum; statistically significant higher pt of stylet support insertion point [65.0–69.4, mean 67.0] in M. pelufforum vs [57.1–67.8, mean 62.2] in M. pacificum (t12 = 4.54, p < 0.001); statistically significant differences about pt of most claw heights (Table 4). Young specimens of Milnesium pelufforum sp. nov. can be similar to four species, M. cassandrae Moreno- Talamantes, Roszkowska, García-Aranda, Flores-Maldonado & Kaczmarek, 2019, M. katarzynae Kaczmarek, Michalczyk & Beasley, 2004, M. pacificum Sugiura, Minato, Matsumoto & Suzuki, 2020 and M. variefidum Morek, Gąsiorek, Stec, Blagden & Michalczyk, 2016, due to a more or less similar cuticular ornamentation and claw configuration [2-2]-[2-2]. Young specimens of M. pelufforum differ from them as follows: 1. Milnesium cassandrae (hatchlings and youngs) only known from the terra typica (Mexico; Moreno- Talamantes et al. 2019, 2020), by different body colour: reddish in M. pelufforum nov. sp. vs white or transparent with light yellow brownish tones before fixation in M. cassandrae; presence of ten rows of pseudoplates, better developed, in M. pelufforum vs nine rows poorly developed in M. cassandrae; eyes present in M. pelufforum vs absent in hatchling and youngs of M. cassandrae; different buccal tube width: higher pt of standard width [43.7–52.1] in M. pelufforum vs [28.2–39.4] in M. cassandrae; statistically significant higher pt of stylet support insertion point [65.3–73.8, mean 71.6] in M. pelufforum vs [56.5–70.5, mean 66.3] in M. cassandrae (t12 = 4.38, p < 0.005); statistically significant differences about pt of almost all claw heights (Table 5). 2. Milnesium katarzynae known from China, Sichuan Province (type locality), Costa Rica (Kaczmarek et al. 2014) and Colombia (Caicedo et al. 2014; Londoño et al. 2015; Melo et al. 2015), by different body colour: reddish in M. pelufforum sp. nov. vs white in M. katarzynae; eyes present in M. pelufforum vs absent of M. katarzynae; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs no band arrangement in M. Differential diagnosis reticulatum; internal primary and secondary branches II [43.6–52.2] and [37.6–45.0] in M. pelufforum vs [33.2–36.6] and [26.0–27.1] respectively in M. reticulatum; external primary and secondary branches III [46.6–56.0] and [38.6–47.8] in M. pelufforum vs [35.6] and [26.8– Fig. 12. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3). Cephalic region and claws. A. Cephalic region with the buccal tube and related structures in focus. B. Claws of legs I. C. Claws of legs III. D. Claws of legs IV. In B–D the black arrows indicate the accessory points, the white arrows indicate the basal spurs, the white arrowheads indicate the ‘lunulae’, the black arrowheads indicate the leg cuticular bars. Scale bars = 10 µm. Fig. 12. Milnesium pelufforum sp. nov., holotype, ♀ (slide No. MCNS tar. 000021-3). Cephalic region and claws. A. Cephalic region with the buccal tube and related structures in focus. B. Claws of legs I. C. Claws of legs III. D. Claws of legs IV. In B–D the black arrows indicate the accessory points, the white arrows indicate the basal spurs, the white arrowheads indicate the ‘lunulae’, the black arrowheads indicate the leg cuticular bars. Scale bars = 10 µm. 18 ROCHA A.M. et al., Three new species of Milnesium from Argentina 29.6] in respectively M. reticulatum; internal primary and secondary branches of leg III [42.8–52.8] and [35.3–44.0] in M. pelufforum vs [33.2] and [26.0–27.1] respectively in M. reticulatum; anterior primary and secondary branch IV [58.1–67.3] and [42.4–52.4] in M. pelufforum vs [37.9–39.7] and [29.2–33.0] respectively in M. reticulatum; posterior primary and secondary branches branch IV [61.1–70.0] and [44.2–53.4] in M. pelufforum vs [41.7–44.3] and [29.2–35.0] respectively in M. reticulatum. 29.6] in respectively M. reticulatum; internal primary and secondary branches of leg III [42.8–52.8] and [35.3–44.0] in M. pelufforum vs [33.2] and [26.0–27.1] respectively in M. reticulatum; anterior primary and secondary branch IV [58.1–67.3] and [42.4–52.4] in M. pelufforum vs [37.9–39.7] and [29.2–33.0] respectively in M. reticulatum; posterior primary and secondary branches branch IV [61.1–70.0] and [44.2–53.4] in M. pelufforum vs [41.7–44.3] and [29.2–35.0] respectively in M. reticulatum. 6. Milnesium pacificum only known from the terra typica in Japan, by different body colour: reddish in M. pelufforum sp. nov. vs creamy withe, transparent or with three brownish longitudinal stripes in M. pacificum; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs no band arrangement in M. Differential diagnosis katarzynae; dimple diameter of about 2–3.5 µm in young specimens of M. pelufforum vs about 0.5–1.5 µm in M. katarzynae; pseudoplates present in M. pelufforum vs not reported in M. katarzynae; different buccal tube width: higher pt of standard width [43.7–52.1] in M. pelufforum vs [21.7– 26.6] in M. katarzynae; statistically significant higher pt of stylet support insertion point [65.3–73.8, mean 71.6] in M. pelufforum vs [73.3–78.3, mean 75.8] in M. katarzynae (t12 = -6.13, p < 0.001); different pt of external primary and secondary branches I, [41.8–48.9] and [37.4– 46.2] in M. pelufforum vs [40.0–40.7] and [26.6–26.7] respectively in M. katarzynae; external primary and secondary branches II [46.7–55.2] and [37.7–47.6] vs [40.0–40.7] and [26.7–28.3] respectively in M. katarzynae; external primary and secondary branches III [46.6–56.0] and [38.6–47.8] vs [40.7–41.8] and [28.3] respectively in M. katarzynae, anterior primary and secondary branches IV [58.1–67.3] and [42.4–52.4] vs [43.5–43.8] and [26.7–28.3] respectively in M. katarzynae. 19 European Journal of Taxonomy 822: 1–54 (2022) Table 3 (continued on next page). Information about pseudoplates for all species that possess them (including some new reports). The correspondence of the new row indication (present paper) and the previous by Moreno-Talamantes et al. (2019) is furnished. “-” indicates no pseudoplate reported for that row; “?” indicates unclear state. Level along the body Anterior Leg I Between I–II Leg II Between II–III Leg III Behind leg III Posterior Reference Rows (present paper) Rows (Moreno-Talamantes et al. 2019) I II (I) III (II) IV (III) V (IV) VI (V) VII (VI) VIII (VII) IX (VIII) X (IX) M. pelufforum sp. nov. 1 4 6 6 10 10 10 12 12 4 Present paper: formula Figs 5, 10 (schematic drawings) Figs 4, 9 (UV fluorescence) M. irenae sp. nov. – 4 4 8 10 8 10 8 12 4 Present paper: formula Fig. 15 (schematic drawing) Fig. 14 (UV fluorescence) M almatyense Morek et al. 2020b – ? ? 4 4 2 4 4 10 1 Morek et al. 2020b: Fig. 2a (PCM) Fig. 2b (schematic drawing) Fig. 3a–d, e (SEM) M. berladnicorum Morek et al. 2016 – 1? 1? 2? 2 2 6 4 8 2 Morek et al. 2016: Fig. 6a (schematic drawing) Description M. cassandrae Moreno-Talamantes et al. 2019 – 4 2 4 10 6 10 8 10 2 Moreno-Talamantes et al. 2019: formula Fig. 1a (PCM) Fig. 3 (UV fluorescence) Fig. Differential diagnosis 4 (schematic drawing) M eurystomun Morek et al. 2020a – 2 2 2 4 2 4 4 10 4 Morek et al. 2020a: Fig. 8 (PCM) Fig. 9 (schematic drawing) Description M variefidum Morek et al. 2016 – – – 2 6 2 6 4 10 2 Morek et al. 2016: Fig. 2a (PCM) Fig. 2b–c (SEM) Fig. 2d (schematic drawing) Description 20 ROCHA A.M. et al., Three new species of Milnesium from Argentina Table 3 (continued). Information about pseudoplates for all species that possess them (including some new reports). The correspondence of the new row indication (present paper) and the previous by Moreno-Talamantes et al. (2019) is furnished. “-” indicates no pseudoplate reported for that row; “?” indicates unclear state. Level along the body Anterior Leg I Between I–II Leg II Between II–III Leg III Behind leg III Posterior Reference M. beatae Tibbs et al. 2016 Pseudoplates reported but without formula/drawing Tibbs et al. 2016: Fig. 1b–d (UV fluorescence) M. pacificum Sugiura et al. 2020 Pseudoplates reported but without formula/drawing Sugiura et al. 2020: Fig. 6a–b (PCM) Fig. 6a’–b’(UV fluorescence) M. alpigenum Morek et al. 2019b Pseudoplates reported but without formula/drawing Morek et al. 2019b: Fig. 1b (SEM), 1D (PCM) M. inceptum Morek et al. 2019b Pseudoplates reported but without formula/drawing Morek et al. 2019b: Fig. 2d (PCM), 2E (SEM) M. krysztofi Kaczmarek & Michalczyk 2007 Pseudoplates not reported but visible in figures Kaczmarek & Michalczyk 2007: Figs 2–7 (SEM, PCM) M. reductum Morek et al. 2020b Pseudoplates reported but without formula/drawing Morek et al. 2020b: Only row IX– Fig. 4d (SEM) M. dornensis Ciobanu et al. 2015 Pseudoplates reported but without formula/drawing Ciobanu et al. 2015: Only row IX – Fig. 4c, e (PCM, SEM) M. lagniappe Meyer et al. 2013 Pseudoplates not reported but visible in one figure Meyer et al. 2013: Fig. 1a (PCM) 21 European Journal of Taxonomy 822: 1–54 (2022) 22 Table 4 (continued on next page). Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between senior specimens of Milnesium pelufforum sp. nov. and the similar species. Species Character M. pelufforum sp. nov. M. beasleayi M. cassandrae M. krzysztofi M. lagniappe M. Differential diagnosis pacificum Claw/Leg I External primary branch 41.8–48.9 (45.1) 47.2–59.7 (53.3) t11 = -4.45, p < 0.001 External secondary branch 37.4–46.2 (42.0) 33.2–38.7 (35.7) t10 = 5.20, p < 0.001 Internal primary branch 39.1–46.8 (42.9) 39.2–44.0 (41.9) t9 = 2.59, p < 0.05 46.3–56.3 (50.5) t9 = -4.68, p < 0.001 34.4–48.1 (40.4) t9 = 2.13, p < 0.05 Internal secondary branch 36.0–43.0 (39.8) 34.2–36.4 (35.5) t11 = 4.41, p < 0.001 Claw/Leg II External primary branch 46.7–55.2 (50.0) 51.7–63.5 (57.9) t10 = -3.92, p < 0.001 36.8–52.7 (46.9) t10 = 2.10, p < 0.05 External secondary branch 37.7–47.6 (43.7) 32.1–39.8 (36.8) t12 = 5.25, p < 0.001 35.5–43.6 (39.5) t12 = 2.05, p < 0.05 Internal primary branch 43.6–52.2 (47.9) 37.8–55.6 (44.0) t9 = 2.10, p < 0.05 37.4–47.2 (43.7) t9 = 2.73, p < 0.05 48.8–62.1 (54.3) t9 = -4.87, p < 0.001 Internal secondary branch 37.6–45.0 (41.4) 34.3–38.0 (35.9) t7 = 4.75, p < 0.001 32.4–42.1 (36.8) t7 = 5.56, p < 0.001 Claw/Leg III External primary branch 46.6–56.0 (50.0) 49.7–63.0 (56.1) t9 = -4.69, p < 0.001 22 ROCHA A.M. et al., Three new species of Milnesium from Argentina 23 Table 4 (continued). Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between senior specimens of Milnesium pelufforum sp. nov. and the similar species. Species Character M. pelufforum sp. nov. M. beasleayi M. cassandrae M. krzysztofi M. lagniappe M. Differential diagnosis pacificum External secondary branch 38.6–47.8 (43.0) 33.8–39.7 (37.4) t8 = 6.47, p < 0.001 33.2–42.3 (38.6) t8 = 2.40, p < 0.05 Internal primary branch 42.8–52.8 (46.6) 45.5–51.5 (48.0) t9 = 13.59, p < 0.001 50.0–60.1 (55.2) t9 = -8.23, p < 0.001 Internal secondary branch 35.3–44.0 (39.7) 32.7–36.9 (35.2) t8 = 5.11, p < 0.001 31.9–35.8 (43.3) t8 = 4.96, p < 0.001 39.1–48.3 (42.6) t8 = -2.71, p < 0.01 37.6–51.3 (43.0) t8 = –2.80, p < 0.01 Claw/Leg IV Anterior primary branch 58.1–67.3 (61.6) 62.9–74.0 (69.0) t11 = 5.20, p < 0.001 41.6–60.3 (50.6) t11 = 4.43, p < 0.001 Anterior secondary branch 42.4–52.4 (46.7) 34.8–51.8 (41.3) t11 = 2.11, p < 0.05 32.8–47.1 (42.1) t11 = 3.29, p < 0.01 Posterior primary branch 61.1–70.0 (64.7) 59.0–64.2 (60.8) t10 = 3.33, p < 0.01 66.1–76.6 (70.9) t10 = -5.19, p < 0.001 41.1–61.1 (53.1) t10 = 4.37, p < 0.001 Posterior secondary branch 44.2–53.4 (49.2) 39.7–46.7 (42.8) t12 = 4.99 p < 0.001 37.2–53.2 (43.6) t12 = 2.45, p < 0.05 39.0–43.7–(40.7) t12 = 6.74, p < 0.001 23 European Journal of Taxonomy 822: 1–54 (2022) Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges heights between young specimens of Milnesium pelufforum sp. nov. and the similar species. Table 5. Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between young specimens of Milnesium pelufforum sp. nov. and the similar species. Table 5. Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between young specimens of Milnesium pelufforum sp. nov. and the similar species. Species Character M. pelufforum sp. nov. M. cassandrae M. pacificum M. Differential diagnosis variefidum Claw/Leg I External primary branch 45.0–53.7 (49.9) 35.1–52.8 (42.9) T9 = 3.44, p < 0.01 External secondary branch 37.9–47.4 (41.2) 30.1–39.7 (35.1) T10 = 4.8, p < 0.001 Internal primary branch 43.8–52.5 (48.1) 35.8–48.4 (42.1) T9 = 3.91, p < 0.005 32.8–45.1 (38.9) T10 = 4.69, p < 0.001 Internal secondary branch 35.9–44.7 (38.9) 30.2–39.6 (34.3) T10 = 3.81, p < 0.005 23.7–37.4 (32.5) T10 = 3.48, p < 0.005 Claw/Leg II External primary branch 46.1–55.9 (51.6) 29.6–52.1 (41.4) T10 = 3.45, p < 0.005 38.0–50.9 (43.7) T10 = 4.25 p < 0.001 External secondary branch 38.2–45.6 (40.7) 30.9–40.9 (36.3) T11 = 3.94, p < 0.001 27.6–39.7 (33.8) T11 = 4.65, p < 0.001 Internal primary branch 46.0–56.5 (50.3) 37.4–53.5 (43.3) T10 = 3.14, p < 0.005 Internal secondary branch 34.8–42.0 (38.3) 29.9–40.9 (34.7) T11 = 1.91, p < 0.05 25.9–38.1 (32.0) T11 = 2.93, p < 0.01 Claw/Leg III External primary branch 46.4–57.0 (52.0) 39.4–48.9 (44.7) T10 = 5.78, p < 0.001 38.8–50.3 (44.4) T10 = 3.87 p < 0.001 External secondary branch 37.7–44.0 (41.1) 31.3–38.5 (35.1) T9 = 6.93, p < 0.001 25.9–39.0 (33.3) T9 = 3.39, p < 0.005 Internal primary branch 47.9–57.9 (50.4) 39.0–47.2 (44.4) T11 = 4.48, p < 0.001 31.0–50.9 (44.7) T11 = -2.93, p < 0.01 Internal secondary branch 35.7–40.6 (38.3) 30.4–40.0 (34.2) T18 = 3.48, p < 0.001 25.5–37.2 (32.0) T8 = 3.81, p < 0.005 Claw/Leg IV Anterior primary branch 50.9–60.5 (56.2) 41.5–54.9 (49.7) T8 = 2.73, p < 0.01 40.0–56.2 (48.7) T8 = 2.77, p < 0.001 Anterior secondary branch 37.5–45.8 (41.3) 26.1–40.7 (34.4) T10 = 3.37, p < 0.005 30.6–42.2 (35.2) T10 = 2.54, p < 0.01 Posterior primary branch 54.2–63.0 (59.0) 45.8–56.7 (52.3) T11 = 3.99, p < 0.001 Posterior secondary branch 37.5–47.6 (43.7) 30.6–40.9 (36.1) T10 = 5.65, p < 0.001 27.5–41.0 (36.2) T10 = 3.63, p < 0.005 3. Milnesium pacificum (hatchlings) only known from the terra typica in Japan, by different body colour: reddish in M. pelufforum sp. nov. vs creamy white, transparent or with three brownish longitudinal stripes in M. pacificum; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs no band arrangement in M. pacificum; dimple diameter about 2–3.5 µm in M. pelufforum vs about 0.61– 0.82 µm in M. pacificum; shorter peribuccal papillae, pt [14.7–15.1] in M. pelufforum vs [15.8–20.7] in M. Paratypes Paratypes ARGENTINA • 1 ♀; same collection data as for holotype; MCNS Tar.000023(2) • 4 ♀♀; same collection data as for holotype; UNICT 5899(1) to 5899(4) • 13 ♀♀; same collection data as for holotype; UNLPam 1367(1), 1430(1) to 1430(3), 1599(1), 1599(3), 1603(1), 1603(3), 1603(4), 1656(1) to 1656(4). Etymology We dedicate the new species to the researcher Irene Luisa Doma. Differential diagnosis pacificum; longer lateral papillae, pt [14.6–15.9] in M. pelufforum sp. nov. vs [12.1–14.2] in 24 ROCHA A.M. et al., Three new species of Milnesium from Argentina M. pacificum; different buccal tube width: higher pt of standard width [43.7–52.1] in M. pelufforum vs [30.1–39.4] in M. pacificum; statistically significant higher pt of stylet support insertion point [65.3– 73.8, mean 71.6] in M. pelufforum vs [58.7–69.2, mean 63.9] in M. pacificum (t12 = 5.18 p < 0.001); statistically significant differences about pt of many claw heights (Table 5). M. pacificum; different buccal tube width: higher pt of standard width [43.7–52.1] in M. pelufforum vs [30.1–39.4] in M. pacificum; statistically significant higher pt of stylet support insertion point [65.3– 73.8, mean 71.6] in M. pelufforum vs [58.7–69.2, mean 63.9] in M. pacificum (t12 = 5.18 p < 0.001); statistically significant differences about pt of many claw heights (Table 5). 4. Milnesium variefidum (hatchlings and youngs) only known from the type locality in Scotland, by the body colour: reddish in M. pelufforum sp. nov. vs yellowish before fixation and transparent afterwards in M. variefidum; sculptured cuticle with dimples arranged in ten bands in M. pelufforum vs cuticle appearing smooth but with faint pseudopores, not arranged in bands, in M. variefidum; presence of ten pseudoplate rows better visible and outlined in M. pelufforum vs seven rows of pseudoplates only occasionally visible or poorly developed in M. variefidum (Table 3); different buccal tube width: pt of standard width [43.7–52.1] in M. pelufforum vs [22.1–33.8] in M. variefidum; different pt ranges of claw heights: external primary and secondary branch I [45.0–53.7] and [37.9–47.4] in M. pelufforum vs [33.7–44.7] and [27.7–35.8] in M. variefidum respectively; internal secondary branch I [35.9–44.7] in M. pelufforum vs [28.2–35.4] in M. variefidum; internal primary branch II and III [46.0–56.5] and [47.9–57.9] in M. pelufforum vs [33.9–45.6] and [34.2–47.2] in M. variefidum respectively; statistically significant differences about pt of most of the other claw heights (Table 5). Milnesium irenae sp. nov. urn:lsid:zoobank.org:act:E52817A9-9080-427A-B4DF-AD4C58C52352 Figs 13–18, Tables 3, 6–7 Diagnosis Peculiar, complex cuticular ornamentation including cuticular grooves, branched rugosity and a true, very fine, reticular design appearing different from that of the congeneric species with reticulated cuticle. Nine rows of pseudoplates present, formula: CP: I:0; II:4; III:4; IV:8; V:10; VI:8; VII:10; VIII:8; IX:12; X:4. Six peribuccal lamellae present, six peribuccal papillae with the medioventral reduced, two lateral cephalic papillae; buccal tube slightly funnel-shaped; stylets, their furcae and supports very developed. Claw configuration [2-3]-[2-2]; internal secondary branches of legs I–III with long basal spurs directed towards, almost touching the claw base. Ontogenetic change not observed in the available material, but hatchlings are probably lacking. Holotype Holotype ARGENTINA • ♀; La Pampa Province, Santa Rosa City; 36º37′13″ S, 64º17′26″ W; about 177 m a.s.l.; 17 Sep. 2017; Rocha-Doma leg; moss and lichens from trees; UNLPam 1657(1). Morphological description Body reddish up to 664 µm long (habitus in Fig. 13); large eyes present. Cuticle with complex sculpture (Fig. 16): on the dorsal surface of trunk segments (starting with legs I in very few specimens, with legs II in some, and with legs III in the majority of them), clearly visible cuticular grooves (Fig. 16A–B, D) 25 European Journal of Taxonomy 822: 1–54 (2022) Fig. 13. Milnesium irenae sp. nov., holotype, ♀ (slide No. UNLPam 1657-1). Habitus. Scale bar = 50 µm. Fig. 13. Milnesium irenae sp. nov., holotype, ♀ (slide No. UNLPam 1657-1). Habitus. Scale bar = 50 µm. Fig. 14. Milnesium irenae sp. nov., paratype, ♀ (slide No. UNLPam 1430-1). View under UV fluorescence microscope, showing various pseudoplates. Scale bar = 50 µm. Fig. 14. Milnesium irenae sp. nov., paratype, ♀ (slide No. UNLPam 1430-1). View under UV fluorescence microscope, showing various pseudoplates. Scale bar = 50 µm. 26 ROCHA A.M. et al., Three new species of Milnesium from Argentina are present, some developing from cuticular invaginations for muscle attachments (Fig. 16B arrows); the grooves form, more peripherally, a rugosity made of wrinkles that sometimes outline on some areas a network (Fig. 16A–B arrowheads) visible as bright crossing lines delimiting dark elongated ‘dots’. The wrinkles gradually become interrupted and disappear, giving way to a reticular design (Fig. 16) consisting of a fine-scale mesh, elongated in a few areas, isodiametric in the rest with a mesh diameter of about 0.5–1.0 µm. This reticule made of isodiametric mesh, though difficult to see, is quite spread on the cuticle, also on more cephalic segments where the other elements of the cuticular sculpture are absent: on all specimens it is visible at least starting with legs II, but in some it starts with legs I. This reticule, made of a delicate mesh, has a rather different appearance from the more common reticular pattern, resulting from close dimples, of many other species of Milnesium; in M. irenae sp. nov. it looks instead more similar to the more irregular reticulations present in Parachela Schuster, Nelson, Grigarick & Christenberry, 1980, such as, for example, several species of Isohypsibiidae Sands, McInnes, Marley, Goodall-Copestake, Convey & Linse, 2008 (e.g., Dianea sattleri (Richters, 1902) and Ursulinius pappi (Iharos, 1966)) and Doryphoribiidae Gąsiorek, Stec, Morek & Michalczyk, 2019 (e.g., Doryphoribius bindae Lisi, 2011 and Grevenius kotovae (Tumanov, 2003)). Morphological description Row VII, situated in line with legs III, has ten pseudoplates: the six medial arranged in three pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these six pseudoplates vaguely rectangular but with the two transverse lines dividing the three pairs not straight; laterally, on each side, a longitudinally elongated pseudoplate, and a more lateral, bigger, about bean-shaped. Row a unique about rectangular structure; each these six pseudoplates vaguely rectangular but with the two transverse lines dividing the adjacent anterior/posterior pairs of pseudoplates not straight; two comma- shaped pseudoplates, just lateral to the central complex and aligned posteriorly, plus two more lateral pseudoplates, laying more anteriorly, bigger and about oval but difficult to see. Row VI, situated between legs II and III, has eight pseudoplates aligned in a single transverse row, with the four medial ones connected laterally and thus forming a unique wide rectangle (but with the longitudinal lines dividing the four pseudoplates weakly outlined); lateral to that central complex, two separate pseudoplates on each side, with the mid-lateral about oval, and the most lateral, bigger, more or less rounded/oval. Row VII, situated in line with legs III, has ten pseudoplates: the six medial arranged in three pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these six pseudoplates vaguely rectangular but with the two transverse lines dividing the three pairs not straight; laterally, on each side, a longitudinally elongated pseudoplate, and a more lateral, bigger, about bean-shaped. Row Fig. 16. Milnesium irenae sp. nov. A–B. Paratype, ♀ (slide No. UNLPam 1367-1). C. Paratype, ♀ (slide No. MCNS tar. 000023-2). D. Paratype, ♀ (slide No. UNICT 5899). Details of the cuticular ornamentation of the caudal segments (where it is more evident). A. Muscular attachments, grooves and rugosity arrangement, and, partially, the reticular pattern, are visible. B. Schematic drawing based on A, showing all components of the ornamentation: muscular attachments (arrows), grooves and rugosity system (in light grey) which forms crossings in some areas (arrowheads), reticular pattern. C. The reticular pattern is well visible. D. All cuticular sculpture components as in A are shown in another paratype. Scale bars = 10 µm. Fig. 16. Milnesium irenae sp. nov. A–B. Paratype, ♀ (slide No. UNLPam 1367-1). C. Paratype, ♀ (slide No. MCNS tar. 000023-2). D. Paratype, ♀ (slide No. UNICT 5899). Details of the cuticular ornamentation of the caudal segments (where it is more evident). Morphological description Pseudoplates present, arranged in 9 rows, formula: CP: I:0; II:4; III:4; IV:8; V:10; VI:8; VII:10; VIII:8; IX:12; X:4 (Figs 14–15). Row I is absent; row II, situated anteriorly to legs I, has four pseudoplates, all difficult to see: two medial, about quadrangular, touching in the central line, and two lateral, about trapezoidal, laying obliquely. Row III, situated in line with legs I, has four central pseudoplates arranged in two pairs, connected, with two anterior about rectangular, and two caudal, vaguely triangular pseudoplates. Row IV, situated between legs I and II, has eight pseudoplates: the four medial arranged in two pairs, transversally elongated, connected, forming a unique about rectangular structure; each of these four pseudoplates is also about rectangular but with the transverse line dividing the two anterior from the two posterior pseudoplates weakly outlined; two about rectangular pseudoplates, just lateral to the central complex, longitudinally elongated, plus two more lateral, bigger but difficult to see, about trapezoidal/ellyptical laying obliquely and slightly more anteriorly. Row V, situated in line with legs II, has ten pseudoplates: the six medial arranged in three pairs (the most cephalic difficult to see), transversally elongated, connected, forming Fig. 15. Semi-schematic drawing of pseudoplate configuration in Milnesium irenae sp. nov. Fig. 15. Semi-schematic drawing of pseudoplate configuration in Milnesium irenae sp. nov. 27 European Journal of Taxonomy 822: 1–54 (2022) a unique about rectangular structure; each these six pseudoplates vaguely rectangular but with the two transverse lines dividing the adjacent anterior/posterior pairs of pseudoplates not straight; two comma- shaped pseudoplates, just lateral to the central complex and aligned posteriorly, plus two more lateral pseudoplates, laying more anteriorly, bigger and about oval but difficult to see. Row VI, situated between legs II and III, has eight pseudoplates aligned in a single transverse row, with the four medial ones connected laterally and thus forming a unique wide rectangle (but with the longitudinal lines dividing the four pseudoplates weakly outlined); lateral to that central complex, two separate pseudoplates on each side, with the mid-lateral about oval, and the most lateral, bigger, more or less rounded/oval. Morphological description A. Muscular attachments, grooves and rugosity arrangement, and, partially, the reticular pattern, are visible. B. Schematic drawing based on A, showing all components of the ornamentation: muscular attachments (arrows), grooves and rugosity system (in light grey) which forms crossings in some areas (arrowheads), reticular pattern. C. The reticular pattern is well visible. D. All cuticular sculpture components as in A are shown in another paratype. Scale bars = 10 µm. 28 ROCHA A.M. et al., Three new species of Milnesium from Argentina VIII, situated just caudally to legs III, has eight pseudoplates aligned in a single transverse row, with the four medial connected laterally and thus forming a unique wide rectangle (but with the longitudinal lines dividing the four pseudoplates weakly outlined); lateral to that central complex, aligned with its posterior margin, two separate pseudoplates on each side, with the mid-lateral one about oval, and the most lateral one, bigger, more or less rounded/oval. Row IX, situated between legs III and IV, has twelve pseudoplates with a median aggregation of 10 pseudoplates (the more cephalic pair difficult to see) forming a complex pattern (see Fig. 15), and two lateral single pseudoplates about quadrangular/ rounded, aligned posteriorly. Row X, situated just anterior to legs IV, has four pseudoplates, sided and aligned in a single row transversally, with the medial ones about quadrangular, the lateral ones about rounded. Six peribuccal lamellae present, and six peribuccal papillae plus two lateral papillae present; medio-ventral peribuccal papilla reduced, (Fig. 17B arrow; pt of such papilla [7.3–10.4] vs [14.6–20.8] of the other peribuccal papillae). Buccal tube (Fig. 17) not perfectly cylindrical, slightly wider anteriorly (posterior/ Fig. 17. Milnesium irenae sp. nov. A. Holotype, ♀ (slide No. UNLPam 1657-1). B. Paratype, ♀ (slide No. UNLPam 1599-3). C. Paratype, ♀ (slide No. UNLPam 1656-3). Cephalic region. A. Buccal tube and the noticeable development of the stylets, their furcae and supports. B. The reduced medio-ventral peribuccal papilla (arrow) in a paratype. C. Another paratype showing the same structures as in A, illustrating clearly the whale-tail shaped stylet furcae; the peribuccal lamellae are partially visible. Scale bars = 10 µm. Fig. 17. Milnesium irenae sp. nov. A. Holotype, ♀ (slide No. UNLPam 1657-1). B. Paratype, ♀ (slide No. UNLPam 1599-3). C. Paratype, ♀ (slide No. UNLPam 1656-3). Cephalic region. A. Buccal tube and the noticeable development of the stylets, their furcae and supports. B. Morphological description The reduced medio-ventral peribuccal papilla (arrow) in a paratype. C. Another paratype showing the same structures as in A, illustrating clearly the whale-tail shaped stylet furcae; the peribuccal lamellae are partially visible. Scale bars = 10 µm. 29 European Journal of Taxonomy 822: 1–54 (2022) Table 6 (continued on next page). Measurements (in μm) and pt values of selected morphological structures of specimens of Milnesium irenae sp. nov. mounted in polyvinyl-lactophenol medium. Range refers to the lowest and the highest values among all measured specimens. Pt values are provided in italics. Abbreviations: N = number of specimens or structures measured; SD = standard deviation. Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Body length 24 349–664 1190 – 1614 524 1417 83 135 638 1571 Peribuccal papillae length 9 5.3–8.3 14.6 – 20.8 6.6 17.9 1.0 1.8 ? ? Ventral peribuccal papillae length 6 2.9–4.2 7.3 – 10.4 3.3 8.7 0.5 1.3 ? ? Morphological description et al., Three new species of Milnesium from Argentina Table 6 (continued). Measurements (in μm) and pt values of selected morphological structures of specimens of Milnesium irenae sp. nov. mounted in polyvinyl-lactophenol medium. Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Claw 4 lengths Anterior primary branch 22 17.5–25.4 55.2 – 64.2 22.4 60.0 2.2 3.2 22.4 55.2 Anterior base + secondary branch 24 11.0–19.2 39.8 – 49.0 16.0 43.4 2.0 2.7 18.6 45.8 Anterior branches length ratio 21 63%–83% – 72% – 5% – 83% – Posterior primary branch 23 17.0–27.2 59.4 – 68.4 23.8 63.6 2.5 2.9 27.2 67.0 Posterior base + secondary branch 24 12.0–19.7 40.6 – 50.4 16.8 45.5 2.2 2.8 19.4 47.8 Posterior branches length ratio 22 65%–78% – 71% – 3% – 71% – anterior width ratio 78–88%); stylets very robust, with very developed furcae, whale-tail shaped, and with supports which after their insertion on the buccal tube becoming gradually wider, assuming overall a triangular shape (Fig. 17A, C); pt of stylet support insertion point on the buccal tube length [68.9–74.9]. anterior width ratio 78–88%); stylets very robust, with very developed furcae, whale-tail shaped, and with supports which after their insertion on the buccal tube becoming gradually wider, assuming overall a triangular shape (Fig. 17A, C); pt of stylet support insertion point on the buccal tube length [68.9–74.9]. anterior width ratio 78–88%); stylets very robust, with very developed furcae, whale-tail shaped, and with supports which after their insertion on the buccal tube becoming gradually wider, assuming overall a triangular shape (Fig. 17A, C); pt of stylet support insertion point on the buccal tube length [68.9–74.9]. Claws of the Milnesium type with configuration [2-3]-[2-2] (Fig. 18), rather robust, in particular the secondary branches; primary branches of legs IV more slender than on legs I–III (compare in Fig. 18A– B with C–D). Percentual ratio of secondary branches with respect to primary branches for each couple higher on legs I (79–96%), slightly lower on legs II–III (72–95%) and definitely lower on legs IV (63– 83%). Internal secondary branches of legs I–III with long basal spurs which form an acute angle inferiorly and have tip nearly reaching the claw base (Fig. 18A). Secondary branches with basal thickenings (‘lunulae’, Fig. 18A, C white arrowheads), primary branches with very thin, short, accessory points (Fig. Morphological description Lateral papillae length 21 4.0–7.0 11.6 – 17.6 5.5 15.0 0.9 1.3 6.5 16.0 Buccal tube Length 24 25.5–42.4 – 36.9 – 4.1 – 40.6 – Stylet support insertion point 24 18.1–30.5 68.9 – 74.9 26.7 72.2 3.2 1.4 30.4 74.9 Anterior width 24 13.3–24.6 50.7 – 60.6 20.4 55.0 2.9 2.9 24.6 60.6 Standard width 24 12.0–23.1 46.1 – 56.9 18.8 50.6 2.9 3.3 23.1 56.9 Posterior width 24 10.7–20.9 41.4 – 51.5 16.8 45.2 2.5 2.7 20.9 51.5 Standard width/length ratio 24 46%–57% – 50% – 3% – 57% – Posterior/anterior width ratio 24 78%–88% – 82% – 3% – 85% – Claw 1 lengths External primary branch 22 11.0–18.3 38.7 – 47.1 15.9 43.0 2.0 2.3 18.3 45.1 External base + secondary branch 22 10.3–16.0 34.1 – 42.6 14.0 38.1 1.6 2.6 15.9 39.2 External branches length ratio 19 82%–94% – 89% – 3% – 87% – Internal primary branch 22 11.0–17.8 35.3 – 44.7 15.1 40.6 2.0 3.0 17.8 43.8 Internal base + secondary branch 22 9.5–15.5 32.7 – 41.3 13.5 36.2 1.5 2.6 14.6 36.0 Internal spur 14 4.1–6.5 11.4 – 16.2 5.0 13.3 0.6 1.2 5.4 13.3 Internal branches length ratio 20 79%–96% – 90% – 5% – 82% – Claw 2 lengths External primary branch 24 13.5–20.7 43.0 – 52.9 17.8 48.3 2.0 2.8 20.3 50.0 External base + secondary branch 23 10.6–16.8 35.4 – 44.7 14.6 39.6 1.8 2.6 16.3 40.1 External branches length ratio 22 73%–95% – 83% – 5% – 80% – Internal primary branch 22 12.8–19.9 40.7 – 50.8 17.2 45.9 1.9 2.9 19.2 47.3 Internal base + secondary branch 22 10.1–16.6 34.2 – 43.0 14.2 38.0 1.6 2.6 14.9 36.7 Internal spur 19 4.2–6.9 12.5 – 18.3 5.8 15.3 0.8 1.7 6.8 16.7 Internal branches length ratio 20 77%–93% – 83% – 4% – 78% – Claw 3 lengths External primary branch 24 13.5–22.0 44.8 – 55.9 18.6 50.3 2.3 2.9 22.0 54.2 External base + secondary branch 23 10.5–18.2 35.7 – 44.9 14.9 40.4 1.9 2.5 18.2 44.8 External branches length ratio 22 75%–89% – 81% – 4% – 83% – Internal primary branch 23 13.2–20.5 42.2 – 52.3 17.5 47.6 2.0 3.3 20.1 49.5 Internal base + secondary branch 23 10.3–16.7 34.2 – 44.4 14.4 38.4 1.6 2.6 15.4 37.9 Internal spur 20 4.4–6.8 12.0 – 17.8 5.7 15.2 0.7 1.6 6.0 14.8 Internal branches length ratio 21 72%–93% – 81% – 5% – 77% – 30 ROCHA A.M. Morphological description 18B black arrow); long and thick cuticular bars present under the claws I–III (Fig. 18A black arrowhead). Tiny cuticular tubercles, often difficult to see, present on all legs, more visible on legs IV (Fig. 18C–D white arrows). Remarks Milnesium irenae sp. nov. is the only described species of the genus with this form of complex cuticular sculpture, and with reticular pattern more similar to some Parachela than to other congeneric species with ‘reticulated’ cuticle. According to the literature, other congeneric species have a ‘reticule’ actually made of dimples close to one another, while in M. irenae the reticule mesh is not given by dimples. The medioventral peribuccal papilla is reduced (comments in Discussion). We found specimens with body length from 349 µm up to 664 µm (presumably, from second instar on) without remarkable differences between the smallest and the largest; therefore, possible ontogenetic change is unknown in the passage from hatchlings to second instar, while seems to be absent from second to third instar. Morphometric data are given in Table 6 and Supp. file 3; in Table 7 the statistically significant differences (through Student t-tests) of overlapping pt ranges of claw heights between the new species and the similar ones. Differential diagnosis Milnesium irenae sp. nov., due to its unusual cuticular sculpture, differs from all known species of the genus, though morphologically it has more affinity with some species of the old granulatum group. In addition, the characters of the stylets, their furcae and supports have peculiarities which differentiate it from many, if not all, congeneric species (the characters of those structures have not been described in detail in all past species descriptions). Here, we differentiate the new species from those having the same claw configuration [2-3]- [2-2] at least in some life stages, plus some kind of cuticular sculpture. These species are: M. almatyense 31 European Journal of Taxonomy 822: 1–54 (2022) Tumanov, 2006 (youngs and seniors), M. berladnicorum Ciobanu, Zawierucha, Moglan & Kaczmarek, 2014, and adults of M. variefidum Morek, Gąsiorek, Stec, Blagden & Michalczyk, 2016. Milnesium irenae sp. nov. differs from them as follows: 1. Milnesium almatyense known from Kazakhstan (type locality), Kyrgyz Republic (Morek et al. 2020b), by different body colour: reddish in M.irenae sp. nov. vs white in M. almatyense; eyes present in M. irenae vs absent of M. almatyense; different and more complex cuticular sculpture in M. irenae (with tiny mesh, long, branched grooves, and tubercles) equal in specimens of all sizes found vs simpler (with delicate reticulation only in hatchlings, while only pseudopores in juveniles and adults) in M. almatyense; nine rows of pseudoplates in M. irenae vs eight in M. almatyense with different number of pseudoplates in each correspondent row (Table 3); higher pt of buccal tube standard width, [46.1–56.9] in M. irenae vs [26.2–33.1] in M. almatyense; higher buccal tube standard width/length ratio, 46–57% in M. irenae vs 26–33% in M. almatyense. Fig. 18. Milnesium irenae sp. nov. A, C. Holotype, ♀ (slide No. UNLPam 1657-1). B. Paratype, ♀ (UNLPam 1430-3). D. Paratype, ♀ (slide No. UNLPam 1603-3). Claws and leg characters. A. Leg II; the white arrowhead indicates a lunule, the black arrowhead indicates the leg cuticular bar. B. Legs II; the black arrow indicates primary branch accessory points. C. Legs IV; the white arrowhead indicates a lunule. D. Legs IV, magnified; the white arrows indicate some tubercles. Scale bars = 10 µm. Fig. 18. Milnesium irenae sp. nov. A, C. Holotype, ♀ (slide No. UNLPam 1657-1). B. Paratype, ♀ (UNLPam 1430-3). D. Paratype, ♀ (slide No. UNLPam 1603-3). Claws and leg characters. A. Tumanov, 2006 (youngs and seniors), M. berladnicorum Ciobanu, Zawierucha, Moglan & Kaczmarek, 2014, and adults of M. variefidum Morek, Gąsiorek, Stec, Blagden & Michalczyk, 2016. 2. Milnesium berladnicorum only known from the type locality in Romania, by different body colour: reddish in M. irenae sp. nov. vs brownish in M. berladnicorum; different and more complex cuticular sculpture in M. irenae (with tiny mesh, long, branched grooves, and tubercles) vs simpler (with just bright spots, not ascertained whether either dimples or pseudopores) in M. berladnicorum; different number of pseudoplates in each correspondent row (Table 3); statistically significant higher buccal tube posterior/anterior width ratio, 78–88%, mean 82%, in M. irenae vs 69–79%, mean 73%, in adults of M. berladnicorum; (t25 = 8.09, p < 0.001). Differential diagnosis variefidum with different number of pseudoplates in each correspondent row (Table 3); statistically significant differences about pt of almost all claw heights (Table 7). 3. Milnesium variefidum only known from the type locality in Scotland, by different body colour: reddish in M. irenae sp. nov. vs white or transparent in M. variefidum; eyes present in M. irenae vs absent in M. variefidum; different cuticular ornamentation, with tiny mesh, long, branched grooves, and tubercles in M. irenae vs with scattered pseudopores, minute wrinkles and only some short grooves in M. variefidum; presence of nine rows of pseudoplates in M. irenae vs seven rows in M. variefidum with different number of pseudoplates in each correspondent row (Table 3); statistically significant differences about pt of almost all claw heights (Table 7). Milnesium quiranae sp. nov. urn:lsid:zoobank.org:act:DDC4D209-B7E0-411A-8D83-0E086CB44D04 Figs 19–21, Tables 8–11 Diagnosis Smooth cuticle, six peribuccal lamellae, six peribuccal papillae with the medioventral reducing with growing, two lateral cephalic papillae. Buccal tube nearly cylindrical in all life stages, becoming wider with growing. Claws with configuration [3-3]-[3-3] in all life stages. Differential diagnosis Leg II; the white arrowhead indicates a lunule, the black arrowhead indicates the leg cuticular bar. B. Legs II; the black arrow indicates primary branch accessory points. C. Legs IV; the white arrowhead indicates a lunule. D. Legs IV, magnified; the white arrows indicate some tubercles. Scale bars = 10 µm. 32 ROCHA A.M. et al., Three new species of Milnesium from Argentina Table 7. Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between Milnesium irenae sp. nov. and the similar species. Species Character M. irenae sp. nov. M. variefidum Claw/Leg I External primary branch 38.7–47.1 (43.1) 33.7–44.7 (40.0) t21 = 2.20, p < 0.05 External secondary branch 34.1–42.6 (38.0) 27.7–35.8 (31.8) t23 = 6.00, p < 0.001 Internal secondary branch 32.7–41.3 (36.2) 28.2–35.4 (31.7) t22 = 4.50, p < 0.001 Claw/Leg II External primary branch 43.0–52.9 (48.2) 38.0–50.9 (43.7) t25 = 3.56, p < 0.001 External secondary branch 35.4–44.7 (39.5) 27.6–39.7 (33.8) t23 = 4.29, p < 0.001 Internal primary branch 40.7–50.8 (45.9) 33.9–45.6 (40.6) t21 = 3.93, p < 0.001 Internal secondary branch 33.0–43.0 (37.8) 25.9–38.1 (32.0) t22 = 4.64, p < 0.001 Claw/Leg III External primary branch 44.8–55.9 (50.9) 38.8–50.3 (44.4) t24 = 4.60, p < 0.001 External secondary branch 35.7–44.9 (40.3) 25.9–39.0 (33.3) t23 = 5.68, p < 0.001 Internal primary branch 42.2–52.3 (47.7) 34.2–47.2 (41.0) t23 = 4.39, p < 0.001 Internal secondary branch 34.2–44.4 (38.3) 25.5–37.2 (32.0) t23 = 4.93, p < 0.001 Claw/Leg IV Anterior primary branch 55.1–64.2 (59.8) 46.6–61.0 (52.1) t22 = 3.28, p < 0,01 Anterior secondary branch 39.8–49.0 (43.3) 30.6–42.2 (35.2) t24 = 4.69, p < 0.001 Posterior primary branch 59.4–68.4 (63.6) 35.9–62.4 (53.1) t22 = 4.53, p < 0.001 Posterior secondary branch 40.6–50.4 (45.5) 27.5–41.0 (36.2) t23 = 7.51, p < 0.001 33 European Journal of Taxonomy 822: 1–54 (2022) 3. Milnesium variefidum only known from the type locality in Scotland, by different body colour: reddish in M. irenae sp. nov. vs white or transparent in M. variefidum; eyes present in M. irenae vs absent in M. variefidum; different cuticular ornamentation, with tiny mesh, long, branched grooves, and tubercles in M. irenae vs with scattered pseudopores, minute wrinkles and only some short grooves in M. variefidum; presence of nine rows of pseudoplates in M. irenae vs seven rows in M. Etymology The new species is dedicated to the researcher Estela Maris Quirán, who published important contributions on the scientific knowledge of invertebrates in La Pampa, Argentina. Fig. 19. Milnesium quiranae sp. nov., paratype, ♀ (slide No. UNLPam 566-1). Buccal tube and claws of a young. A. Buccal tube and related structures. B. Legs I and II; the black arrow indicates the accessory points, the black arrowhead indicates the leg cuticular bar, the white arrowhead indicates a lunule. C. Leg III; the black and the white arrowheads indicate the same as in B. D. Legs IV with their claws. Scale bars = 10 µm. Fig. 19. Milnesium quiranae sp. nov., paratype, ♀ (slide No. UNLPam 566-1). Buccal tube and claws of a young. A. Buccal tube and related structures. B. Legs I and II; the black arrow indicates the accessory points, the black arrowhead indicates the leg cuticular bar, the white arrowhead indicates a lunule. C. Leg III; the black and the white arrowheads indicate the same as in B. D. Legs IV with their claws. Scale bars = 10 µm. 34 ROCHA A.M. et al., Three new species of Milnesium from Argentina Paratypes Paratypes ARGENTINA • 2 ♀♀; same collection data as for holotype; MCNS Tar.000025(3), Tar.000025(4) • 4 ♀♀; same collection data as for holotype; UNICT 5900(1) to 5900(4) • 21 ♀♀; same collection data as for holotype; UNLPam 558(1) to 558(3), 558(5), 560(1), 560(3), 560(4), 564(4), 565(1), 565(4), 565(5), 566(1) to 566(4), 570(1), 573(5), 576(4), 576(5), 586(1), 586(2). Holotype yp ENTINA • ♀; Salta Province, Salta City; 24°47′18″ S, 65°24′38″ W; 1150 m a.s.l.; 2 May 201 a-Doma leg.; moss and lichens from trees; MCNS Tar.000024(3). Material examined Holotype ARGENTINA • ♀; Salta Province, Salta City; 24°47′18″ S, 65°24′38″ W; 1150 m a.s.l.; 2 May 2014; Rocha-Doma leg.; moss and lichens from trees; MCNS Tar.000024(3). Morphological description Body length up to 770 µm (habitus in Fig. 20A), reddish colour before mounting, eyes present. Cuticle smooth (Fig. 20B–C) without dimples, wrinkles, pseudopores, reticulations, pseudoplates or gibbosities. Fig. 20. Milnesium quiranae sp. nov., holotype, ♀ (slide No. MCNS Tar.000024-3). A. Habitus. B. Smooth cuticle at the level of legs I. C. Smooth cuticle between legs III and IV. D. Smooth cuticle of the caudal end of the body. Scale bars: A = 50 µm; B–D = 10 µm. Fig. 20. Milnesium quiranae sp. nov., holotype, ♀ (slide No. MCNS Tar.000024-3). A. Habitus. B. Smooth cuticle at the level of legs I. C. Smooth cuticle between legs III and IV. D. Smooth cuticle of the caudal end of the body. Scale bars: A = 50 µm; B–D = 10 µm. Fig. 20. Milnesium quiranae sp. nov., holotype, ♀ (slide No. MCNS Tar.000024-3). A. Habitus. B. Smooth cuticle at the level of legs I. C. Smooth cuticle between legs III and IV. D. Smooth cuticle of the caudal end of the body. Scale bars: A = 50 µm; B–D = 10 µm. 35 European Journal of Taxonomy 822: 1–54 (2022) Table 8 (continued on next page). Measurements (in μm) and pt values of selected morphological structures of young specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Range refers to the lowest and the highest values among all measured specimens. Pt values are provided in italics. Abbreviations: N = number of specimens or structures measured; SD = standard deviation. Morphological description et al., Three new species of Milnesium from Argentina Table 8 (continued). Measurements (in μm) and pt values of selected morphological structures of young specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Table 8 (continued). Measurements (in μm) and pt values of selected morphological structures of young specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Character N Range Mean SD µm pt µm pt µm pt Internal spur 12 3.7–7.1 12.8 – 19.6 5.3 17.8 1.1 1.9 Internal branches length ratio 18 66%–81% – 75% – 4% – Claw 4 heights Anterior primary branch 19 13.0–19.0 47.4 – 58.1 15.4 53.7 2.0 3.6 Anterior base + secondary branch 19 9.7–13.6 35.8 – 45.5 11.6 40.4 1.4 2.8 Anterior spur 15 4.0–6.8 13.4 – 21.3 5.3 17.6 1.1 2.8 Anterior branches length ratio 19 69%–82% – 75% – 4% – Posterior primary branch 19 12.6–20.0 50.3 – 61.5 16.2 56.4 2.4 3.6 Posterior base + secondary branch 19 10.0–14.3 37.4 – 47.2 12.2 42.6 1.5 3.1 Posterior spur 12 3.2–7.0 12.1 – 19.2 4.4 15.7 1.1 2.4 Posterior branches length ratio 19 68%–86% – 76% – 5% – Six peribuccal lamellae and six peribuccal plus two lateral papillae present; medio-ventral peribuccal papilla reduced in senior specimens (Fig. 21B black arrowhead). Buccal tube (Figs 19A, 21A) from slightly funnel-shaped to almost cylindrical (posterior/anterior width ratio 80–98%), wider in senior specimens; pt of stylet support insertion point on the buccal tube [65.5–75.1]. Stylet furcae relatively large (Figs 19A, 21A). Claws (Figs 19B–D, 21C–E) of the Milnesium type with configuration [3-3]-[3-3]. Secondary branches of legs IV more robust than on legs I–III (this difference is less marked in senior specimens; compare Fig. 19B–C with 19D, and Fig. 21C–D with 21E); basal spurs of internal claws of legs I–III and anterior claws of legs IV more developed than external I–III and posterior IV (Tables 8 and 9), but this difference may not be visible depending on claw position. Secondary branches with basal thickenings (‘lunulae’, Figs 19B–C, 21E, white arrowheads), primary branches with very small accessory points (Figs 19B, 21C, black arrows); cuticular bars present on legs I–III (Figs 19C, 21C, black arrowheads). Morphological description Character N Range Mean SD µm pt µm pt µm pt Body length 19 270–460 1112 – 1499 380 1322 61 108 Peribuccal papillae length 13 4.6–7.4 15.5 – 23.9 5.6 20.6 0.9 2.0 Lateral papillae length 16 4.0–6.6 12.6 – 20.6 5.1 17.9 0.9 2.0 Buccal tube Length 19 23.0–36.5 – 28.8 – 4.7 – Stylet support insertion point 19 16.3–25.4 67.4 – 75.1 20.2 70.2 2.9 2.1 Anterior width 19 10.4–18.2 44.0 – 52.6 13.8 47.7 2.7 2.5 Standard width 19 10.1–18.8 43.2 – 51.5 13.6 46.9 2.9 3.0 Posterior width 19 9.0–16.6 36.5 – 45.5 12.1 41.8 2.4 3.0 Standard width/length ratio 19 43%–52% – 47% – 3% – Posterior/anterior width ratio 19 80%–96% – 88% – 4% – Claw 1 heights External primary branch 19 10.8–15.0 39.8 – 49.8 12.7 44.4 1.4 3.5 External base + secondary branch 17 8.6–12.9 31.2 – 40.6 10.5 36.4 1.4 2.6 External spur 7 2.9–4.0 12.5 – 16.6 3.6 13.6 0.5 1.5 External branches length ratio 17 75%–91% – 82% – 5% – Internal primary branch 19 10.0–14.0 37.9 – 47.0 11.9 41.8 1.3 3.2 Internal base + secondary branch 18 8.4–12.5 30.1 – 37.8 9.9 35.0 1.2 2.7 Internal spur 11 3.7–6.1 13.8 – 19.4 4.8 16.3 0.9 1.6 Internal branches length ratio 18 76%–95% – 83% – 5% – Claw 2 heights External primary branch 19 11.1–16.8 45.0 – 54.5 14.1 49.3 1.5 3.6 External base + secondary branch 16 9.0–12.7 33.5 – 43.3 10.9 37.4 1.3 2.8 External spur 6 3.3–5.0 10.0 – 16.4 4.1 14.1 0.8 2.4 External branches length ratio 16 68%–81% – 76% – 4% – Internal primary branch 19 10.6–16.2 43.3 – 52.8 13.6 47.6 1.6 3.5 Internal base + secondary branch 19 8.4–12.2 32.0 – 41.3 10.3 36.0 1.2 2.7 Internal spur 12 3.4–6.4 14.9 – 19.9 5.3 17.4 0.9 1.4 Internal branches length ratio 19 70%–82% – 76% – 4% – Claw 3 heights External primary branch 17 12.1–17.6 43.3 – 53.1 14.1 48.4 1.6 3.5 External base + secondary branch 18 9.0–12.2 32.9 – 43.3 10.9 37.8 1.1 3.3 External spur 8 3.0–5.0 12.6 – 16.4 4.1 14.7 0.8 1.5 External branches length ratio 16 68%–83% – 78% – 4% – Internal primary branch 18 11.7–16.2 43.0 – 52.6 13.6 47.3 1.5 3.5 Internal base + secondary branch 19 8.6–12.0 30.1 – 40.8 10.2 35.8 1.1 3.4 36 ROCHA A.M. Morphological description Percentual ratio of secondary branches with respect to primary branches for each couple slightly higher for legs I than for all other legs (for all specimens collectively, legs I have 66–95% vs legs II–IV 62–86%; Tables 8–10); the ratio for all legs is on average slightly lower in senior than in young specimens (for all legs collectively, young specimens have 66–95% vs 63–87% in senior; compare Table 8 with Table 9). Young specimens (hatchlings or hatchlings plus second instar: 270–460 µm; Fig. 19, Table 8, Supp. file 4) Medioventral peribuccal papilla similar in size to the others; buccal tube (Fig. 19A) more slender (pt of buccal tube standard width [43.2–51.5]). Intermediate specimens (probably second or third instar: 476–568 µm; Table 10, Supp. file 6) These specimens show intermediate metric characters between young and senior. Medio-ventral peribuccal papilla reduced (Fig. 21B black arrowhead; pt of such papilla [9.8–12.5] vs [22.6–31.6] of the other peribuccal papillae); buccal tube wider (pt of buccal tube standard width [59.1–67.9]; Fig. 21A). 37 European Journal of Taxonomy 822: 1–54 (2022) Differential diagnosis Based on having smooth cuticle, M. quiranae sp. nov. belongs to the old tardigradum group (Michalczyk et al. 2012a, 2012b). The new species, lacking pseudopores and pseudoplates, and having three points on the secondary branches of all claws [3-3]-[3-3] and six peribuccal lamellae, is similar to M. antarcticum Tumanov, 2006; M. asiaticum Tumanov, 2006; M. barbadosense Meyer & Hinton, 2012; M. bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014; M. brachyungue Binda & Pilato, 1990; M. burgessi Schlabach, Donaldson, Hobelman, Miller & Lowman, 2018; M. eurystomum Maucci, 1991 (emended by Morek et al. 2020a); M. longiungue Tumanov, 2006; M. minutum Pilato & Lisi, 2016; M. pseudotardigradum Surmacz, Morek & Michalczyk, 2019 M. sandrae Pilato & Lisi, 2016; M. shilohae Meyer, 2015; M. swansoni Young, Chappell, Miller & Lowman, 2016; M. tumanovi Pilato, Sabella & Lisi, 2016; M. validum Pilato, Sabella, D’Urso & Lisi, 2017; M. zsalakoae Meyer & Hinton, 2010. For more precise comparisons, we first compare only senior specimens of the new species with all mentioned similar species, apart from M. minutum, which was very probably described based on young specimens and is therefore compared with the young of M. quiranae sp. nov.; senior specimens of M. quiranae differ from: 1. Milnesium antarcticum, only known from the type locality in Antarctica, by different body colour: reddish in M. quiranae sp. nov. vs reddish-brown in M. antarcticum; different buccal tube width: higher pt of standard width [59.1–67.9] in M. quiranae vs [35.4–43.9] in M. antarcticum; different pt of many claw heights: external primary and secondary branches I [45.6–54.9] and [34.2–41.8] in M. quiranae vs [34.0–43.2] and [22.9–28.4] respectively in M. antarcticum; external primary and secondary branches III [49.3–58.3] and [36.2–43.1] in M. quiranae vs [38.5–45.7] and [18.9–21.1] respectively in M. antarcticum; posterior primary and secondary branches IV [60.1–69.7] and [44.0– 51.7] in M. quiranae vs [49.8–55-9] and [28.0–34.3] respectively in M. antarcticum. 2. Milnesium asiaticum only known from the type locality in Kyrgyz Republic (Central Asia), by different body colour: reddish in M. quiranae sp. nov. vs slightly reddish or white in M. asiaticum; higher pt of buccal tube standard width [59.1–67.9] in M. quiranae vs [30.0–41.6] in M. asiaticum; higher pt of stylet supports insertion point [67.5–73.6] in M. quiranae vs [63.9–66.9] in M. asiaticum. Statistically significant differences about pt of several claw heights (Table 11). 3. Remarks The medioventral peribuccal papilla is reduced in senior specimens (comments in Discussion). Morphometric data are given in Table 8 for young, Table 9 for senior, Table 10 for intermediate specimens; in Table 11 the statistically significant differences (through Student t-test) of overlapping pt ranges of claw heights between the new species and the similar ones. Fig. 21. Buccal tube, peribuccal papillae and claws of senior specimens of Milnesium quiranae sp. nov. A, C–E. Holotype, ♀ (slide No. MCNS Tar.000024-3). B. Paratype, ♀ (slide No. UNLPam 560-1). A. Buccal tube and related structures. B. Reduced medioventral peribuccal papilla (black arrowhead). C. Leg II with its claws; the black arrow indicates the accessory points, the black arrowhead indicates the leg cuticular bar. D. Leg III with its claws. E. Legs IV with their claws, the white arrowhead indicates a lunule. Scale bars = 10 µm. Fig. 21. Buccal tube, peribuccal papillae and claws of senior specimens of Milnesium quiranae sp. nov. A, C–E. Holotype, ♀ (slide No. MCNS Tar.000024-3). B. Paratype, ♀ (slide No. UNLPam 560-1). A. Buccal tube and related structures. B. Reduced medioventral peribuccal papilla (black arrowhead). C. Leg II with its claws; the black arrow indicates the accessory points, the black arrowhead indicates the leg cuticular bar. D. Leg III with its claws. E. Legs IV with their claws, the white arrowhead indicates a lunule. Scale bars = 10 µm. 38 ROCHA A.M. et al., Three new species of Milnesium from Argentina Differential diagnosis Milnesium barbadosense known from the type locality in Barbados Islands (Caribbean Sea) and from Mexico (Moreno-Talamantes et al. 2019, 2020); by different body colour: reddish in M. quiranae sp. nov. vs white or transparent in M. barbadosense; eyes present in M. quiranae vs absent in M. barbadosense; different buccal tube width: higher pt of standard width [59.1–67.9] in M. quiranae vs [27.2–49.7] in M. barbadosense; statistically significant differences about pt of many claw heights (Table 11). 4. Milnesium bohleberi, only known from the type locality in USA, by different body colour: reddish in M. quiranae sp. nov. vs white or transparent in M. bohleberi; eyes present in M. quiranae vs absent in M. bohleberi; statistically significant higher pt of the peribuccal papillae [22.6–31.6, mean 25.8] in M. quiranae vs [27.2–32.3, mean 30.1] in M. bohleberi (t7 = -4.42, p < 0.005); statistically significant differences about pt of buccal tube anterior width [57.9–66.1, mean 62.5] in M. quiranae vs [63.4– 74.7, mean 68.1] in M. bohleberi (t15 = -5.12, p < 0.001); statistically significant differences about pt of several claw heights (Table 11). 5. Milnesium brachyungue, known from Chile (type locality) and Colombia (Londoño et al. 2015); by different body colour: reddish in M. quiranae sp. nov. vs transparent in M. brachyungue; different buccal tube width: higher pt of standard width [59.1–67.9] in M. quiranae vs [37.0] in M. brachyungue; 39 European Journal of Taxonomy 822: 1–54 (2022) Table 9 (continued on next page). Measurements (in μm) and pt values of selected morphological structures of senior specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Range refers to the lowest and the highest values among all measured specimens. Pt values are provided in italics. Abbreviations: N = number of specimens or structures measured; SD = standard deviation. Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Body length 14 585 – 770 1453 – 1871 675 1628 55 119 585 1453 Peribuccal papillae length 6 9.2 – 13.0 22.6 – 31.6 10.8 26.1 1.6 3.6 ? ? Ventral peribuccal papillae length 4 4.2 – 5.0 9.8 – 12.5 4.8 11.5 0.4 1.2 ? ? Differential diagnosis Lateral papillae length 14 7.2 – 11.2 18.8 – 27.2 8.9 21.5 1.0 2.3 8.3 20.6 Buccal tube Length 14 38.3 – 43.2 – 41.4 – 1.5 – 40.3 – Stylet support insertion point 14 26.6 – 30.5 67.5 – 73.6 29.2 70.4 1.1 1.6 29.0 72.0 Anterior width 14 23.8 – 28.1 57.9 – 66.1 25.9 62.4 1.2 2.9 26.6 66.1 Standard width 14 24.3 – 29.1 59.1 – 67.9 26.8 64.7 1.5 3.2 24.7 61.4 Posterior width 14 21.2 – 26.0 52.3 – 61.6 23.9 57.6 1.5 3.3 21.2 52.7 Standard width/length ratio 14 59% – 68% – 65% – 3% – 61% – Posterior/anterior width ratio 14 80% – 97% – 92% – 4% – 80% – Claw 1 heights External primary branch 14 18.6 – 22.6 45.6 – 54.9 20.4 49.2 1.1 2.9 18.6 46.2 External base + secondary branch 14 13.8 – 17.5 34.2 – 41.8 16.3 39.4 1.1 2.1 13.8 34.2 External spur 11 4.6 – 6.8 10.6 – 16.5 5.4 12.9 0.7 1.7 5.0 12.4 External branches length ratio 14 74% – 87% – 80% – 4% – 74% – Internal primary branch 14 17.6 – 21.4 43.2 – 52.0 19.3 46.6 1.1 2.8 17.6 43.7 Internal base + secondary branch 14 12.3 – 16.4 30.6 – 38.9 15.2 36.7 1.2 2.3 12.3 30.6 Internal spur 12 6.4 – 8.5 15.0 – 20.7 7.3 17.7 0.5 1.5 7.6 18.9 Internal branches length ratio 14 70% – 86% – 79% – 4% – 70% – Claw 2 heights External primary branch 14 19.0 – 23.4 47.2 – 56.6 21.8 52.6 1.5 3.1 20.7 51.4 External base + secondary branch 14 14.5 – 17.6 36.4 – 42.5 16.3 39.4 1.0 1.9 14.8 36.8 External spur 10 5.4 – 8.0 12.6 – 19.4 6.8 16.5 0.8 2.1 ? ? External branches length ratio 14 71% – 81% – 75% – 3% – 72% – Internal primary branch 14 18.9 – 23.0 46.3 – 54.9 21.1 51.0 1.4 2.8 20.0 49.7 Internal base + secondary branch 14 14.6 – 16.5 35.5 – 40.2 15.6 37.8 0.6 1.5 14.6 36.2 Internal spur 12 6.6 – 9.7 16.2 – 23.5 8.2 19.8 1.1 2.4 7.6 18.9 Internal branches length ratio 14 68% – 80% – 74% – 4% – 73% – Claw 3 heights External primary branch 13 20.5 – 24.8 49.3 – 58.3 22.5 54.2 1.4 3.3 ? Differential diagnosis ? External base + secondary branch 14 15.0 – 18.0 36.2 – 43.1 16.4 39.6 1.1 2.3 15.3 38.0 External spur 12 5.0 – 8.0 11.6 – 19.4 6.5 15.6 0.9 2.3 5.8 14.3 External branches length ratio 13 63% – 81% – 74% – 4% – ? – Internal primary branch 14 19.5 – 23.1 46.0 – 56.0 21.3 51.4 1.2 2.9 20.4 50.5 40 ROCHA A.M. et al., Three new species of Milnesium from Argentina Table 9 (continued). Measurements (in μm) and pt values of selected morphological structures of senior specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Table 9 (continued). Measurements (in μm) and pt values of selected morphological structures of senior specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Internal base + secondary branch 13 14.0 – 16.5 34.8 – 40.2 15.3 37.1 0.8 1.7 14.0 34.8 Internal spur 14 7.5 – 9.8 17.9 – 23.3 8.5 20.5 0.8 1.9 9.4 23.3 Internal branches length ratio 13 66% – 77% – 72% – 4% – 69% – Claw 4 heights Anterior primary branch 14 23.5 – 27.5 56.4 – 65.6 25.7 61.9 1.5 2.7 23.6 58.7 Anterior base + secondary branch 14 17.1 – 20.4 42.4 – 49.6 18.9 45.5 0.9 2.2 17.1 42.5 Anterior spur 13 7.5 – 9.8 17.8 – 23.7 8.6 20.7 0.8 1.9 8.2 20.4 Anterior branches length ratio 14 67% – 79% – 74% – 3% – 72% – Posterior primary branch 14 26.0 – 28.9 60.1 – 69.7 27.3 65.9 1.1 2.8 26.0 64.6 Posterior base + secondary branch 14 18.0 – 21.3 44.0 – 51.7 19.5 47.0 1.1 2.4 18.0 44.6 Posterior spur 11 6.4 – 8.1 15.4 – 20.7 7.0 16.9 0.6 1.6 ? ? Posterior branches length ratio 14 68% – 76% – 71% – 2% – 69% – different pt of many claw heights: external primary and secondary branch I [45.6–54.9] and [34.2–41.8] in M. quiranae vs [22.8] and [22.8] respectively in M. brachyungue; external primary and secondary branch II [47.6–56.6] and [36.4–42.5] in M. quiranae vs [24.5] and [23.9] respectively in M. brachyungue; external primary and secondary branch III [49.3–58.3] and [36.2–43.1] in M. quiranae vs [27.1] and [23.7] respectively in M. brachyungue; posterior primary and secondary branch IV [60.1–69.7] and [44.0–51.7] in M. Differential diagnosis Measurements (in μm) and pt values of selected morphological structures of intermediate specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Range refers to the lowest and the highest values among all measured specimens. Pt values are provided in italics. Abbreviations: N = number of specimens or structures measured; SD = standard deviation. Differential diagnosis quiranae vs [33.1] and [24.6] respectively in M. brachyungue. different pt of many claw heights: external primary and secondary branch I [45.6–54.9] and [34.2–41.8] in M. quiranae vs [22.8] and [22.8] respectively in M. brachyungue; external primary and secondary branch II [47.6–56.6] and [36.4–42.5] in M. quiranae vs [24.5] and [23.9] respectively in M. brachyungue; external primary and secondary branch III [49.3–58.3] and [36.2–43.1] in M. quiranae vs [27.1] and [23.7] respectively in M. brachyungue; posterior primary and secondary branch IV [60.1–69.7] and [44.0–51.7] in M. quiranae vs [33.1] and [24.6] respectively in M. brachyungue. 6. Milnesium burgessi only known from the type locality in USA; by different body colour: reddish in M. quiranae sp. nov. vs transparent to yellow in M. burgessi; pseudoplates absent in the new species vs present in M. burgessi; statistically significant differences of pt of all branches I–IV in M. quiranae (Table 11) including anterior primary branch IV [56.4–65.6] in M. quiranae vs [70.7–89–6] in M. burgessi (statistical significance not calculated due to completely separate ranges). 7. Milnesium eurystomum known from Greenland (type locality), from Argentina and Chile (Maucci 1996), Mongolia (Kaczmarek & Michalczyk 2006), Arkansas, USA (Land et al. 2012), Alaska, USA (Johansson et al. 2013), Norway, United Kindong (Scotland) (Morek et al. 2020a), by different body colour: reddish in M. quiranae vs brownish in M. eurystomum; no evident ontogenetic shape change of the buccal tube in M. quiranae (which remains more or less cylindrical) vs marked ontogenetic shape change in M. eurystomum (the tube becomes definitely funnel-shaped); statistically significant higher buccal tube posterior/anterior width ratio, 80–97%, mean 92% in M. quiranae vs 53–92%, mean 75% in M. eurystomum (t15 = 4.72, p < 0.001); statistically significant higher pt of stylet supports insertion point [67.5–73.6, mean 70.5] in M. quiranae vs [60.3–69.8, mean 65.1] in M. eurystomum (t15 = 4.51, p < 0.001); different pt of external primary branch I and III [45.6–54.9] and [49.3–58.3] in M. quiranae vs [30.3–43.3] and [31.8–48.1] respectively in M. eurystomum; internal primary branch II [46.3–54.9] in M. quiranae vs [30.5–45.6] in M. eurystomum posterior primary branch IV [60.1–69.7] in M. quiranae vs [35.4–58.9] in M. eurystomum; statistically significant differences of pt of many other claw heights (Table 11). 41 European Journal of Taxonomy 822: 1–54 (2022) Table 10 (continued on next page). Differential diagnosis Character N Range Mean SD µm pt µm pt µm pt Body length 10 476 – 568 1240 – 1511 539 1415 32 86 Peribuccal papillae length 5 7.7 – 8.3 20.1 – 21.9 8.0 21.1 0.2 0.8 Lateral papillae length 8 6.0 – 8.0 15.0 – 21.1 6.6 17.3 0.7 1.9 Buccal tube Length 10 37.0 – 40.0 – 38.1 – 0.8 – Stylet support insertion point 10 25.8 – 27.0 65.5 – 71.1 26.2 68.8 0.3 1.5 Anterior width 10 18.4 – 21.5 49.0 – 56.1 20.3 53.4 1.0 2.6 Standard width 10 19.3 – 23.3 52.2 – 60.7 21.4 56.1 1.2 2.6 Posterior width 10 17.7 – 19.8 44.3 – 52.1 18.5 48.6 0.8 2.5 Standard width/length ratio 10 52% – 61% – 56% – 3% – Posterior/anterior width ratio 10 85% – 98% – 91% – 4% – Claw 1 lengths External primary branch 10 15.4 – 18.3 41.6 – 48.1 17.5 45.9 0.8 1.9 External base + secondary branch 9 12.6 – 13.6 33.3 – 35.8 13.2 34.7 0.3 0.9 External spur 5 4.2 – 6.3 11.1 – 16.4 4.9 12.8 0.8 2.1 External branches length ratio 9 72% – 82% – 76% – 3% – Internal primary branch 9 14.4 – 18.1 38.8 – 47.6 16.8 44.2 1.1 2.5 Internal base + secondary branch 10 12.0 – 15.1 31.0 – 39.7 12.7 33.3 0.9 2.4 Internal spur 8 5.3 – 6.5 14.1 – 17.0 6.0 15.7 0.4 1.0 Internal branches length ratio 9 66% – 87% – 74% – 6% – Claw 2 lengths External primary branch 10 17.0 – 19.8 45.9 – 51.1 18.6 48.9 0.9 1.8 External base + secondary branch 10 12.5 – 14.4 33.2 – 37.8 13.5 35.4 0.7 1.4 External spur 9 4.6 – 7.0 12.2 – 18.4 5.3 13.9 0.8 2.0 External branches length ratio 10 65% – 75% – 72% – 3% – Internal primary branch 10 16.7 – 19.0 45.2 – 50.0 18.2 47.8 0.7 1.8 Internal base + secondary branch 10 12.0 – 14.4 31.9 – 37.9 13.6 35.7 0.7 1.6 Internal spur 9 5.2 – 7.0 13.7 – 18.6 6.3 16.6 0.5 1.5 Internal branches length ratio 10 66% – 79% – 75% – 4% – Claw 3 lengths External primary branch 10 17.2 – 20.8 46.5 – 52.6 19.2 50.4 1.0 2.0 External base + secondary branch 10 12.5 – 14.8 33.8 – 39.0 13.9 36.4 0.8 1.8 External spur 8 5.0 – 7.8 13.0 – 19.5 5.8 15.1 1.0 2.3 External branches length ratio 10 67% – 79% – 72% – 4% – Internal primary branch 10 16.2 – 20.0 43.9 – 52.6 18.4 48.4 1.2 2.7 Internal base + secondary branch 9 12.5 – 14.5 33.8 – 38.2 13.5 35.5 0.6 1.5 42 ROCHA A.M. Differential diagnosis et al., Three new species of Milnesium from Argentina Table 10 (continued). Measurements (in μm) and pt values of selected morphological structures of intermediate specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Table 10 (continued). Measurements (in μm) and pt values of selected morphological structures of intermediate specimens of Milnesium quiranae sp. nov. mounted in polyvinyl-lactophenol medium. Character N Range Mean SD µm pt µm pt µm pt Internal spur 10 6.4 –7.4 16.7 – 19.5 7.1 18.7 0.3 0.9 Internal branches length ratio 9 67% –79% – 74% – 4% – Claw 4 lengths Anterior primary branch 9 20.9 –23.1 55.7 – 60.7 22.3 58.5 0.9 1.9 Anterior base + secondary branch 10 13.9 –17.2 37.2 – 45.3 15.5 40.7 1.2 2.6 Anterior spur 8 5.6 –9.0 15.1 – 23.5 7.0 18.3 1.1 2.7 Anterior branches length ratio 9 62% –79% – 70% – 5% – Posterior primary branch 10 22.2 –26.0 60.1 – 67.9 24.6 64.7 1.2 2.6 Posterior base + secondary branch 10 14.7 –17.9 39.7 – 47.1 16.4 43.0 1.0 2.2 Posterior spur 5 5.0 –7.6 13.2 – 20.2 6.2 16.4 1.2 3.2 Posterior branches length ratio 10 64% –71% – 66% – 2% – 8. Milnesium longiungue, only known from the type locality in India, by different body colour: reddish in M. quiranae sp. nov. vs white in M. longiungue; different buccal tube width: higher pt of standard width [59.1–67.9] in M. quiranae vs [33.8–59.1] in M. longiungue; accessory points present in M. quiranae vs absent in M. longiungue; higher pt of the stylet support insertion point: [67.5–73.6] in M. quiranae vs [59.1–66.7] in M. longiungue; statistically significant differences about pt of external primary branch III (Table 11); lower pt of posterior primary branch IV [60.1–69.7] in M. quiranae vs [81.8–92.4] in M. longiungue. 8. Milnesium longiungue, only known from the type locality in India, by different body colour: reddish in M. quiranae sp. nov. vs white in M. longiungue; different buccal tube width: higher pt of standard width [59.1–67.9] in M. quiranae vs [33.8–59.1] in M. longiungue; accessory points present in M. quiranae vs absent in M. longiungue; higher pt of the stylet support insertion point: [67.5–73.6] in M. quiranae vs [59.1–66.7] in M. longiungue; statistically significant differences about pt of external primary branch III (Table 11); lower pt of posterior primary branch IV [60.1–69.7] in M. quiranae vs [81.8–92.4] in M. Differential diagnosis shillohe Claw/LegI External primary branch 45.6–54.9 (49.3) 30.2–50.1 (41.0) t15 = 3.42, p < 0.01 52.2–79.5 (62.7) t15 = -4.92, p < 0.001 External secondary branch 34.2–41.8 (39.5) 29.2–36.0 (32.4) t15 = 6.12, p < 0.001 23.9–36.9 (30.7) t15 = 5.85, p < 0.001 38.8–55.2 (45.2) t15 = -3.54, p < 0.001 26.4–38.7 (31.7) t15 = -4.85, p < 0.001 32.8–35.4 (34.1) t15 = -8.69, p < 0.001 29.5–35.5 (32.7) t15 = -8.01, p < 0.001 Internal primary branch 43.2–52.0 (46.6) 47.9–75.6 (60.0) t15 = -4.83, p < 0.001 28.5–44.9 (35.0) t15 = -5.65, p < 0.001 Internal secondary branch 30.6–38.9 (36.8) 36.7–53.7 (44.2) t15 = -4.37, p < 0.001 Claw/Leg II External primary branch 47.2–56.6 (52.7) 29.8–59.6 (42.4) t15 = 2.68, p < 0.01 46.1–70.2 (62.2) t15 = -2.69, p < 0.01 32.9–47.9 (38.4) t15 = -7.37, p < 0.001 External secondary branch 36.4–42.5 (39.3) 22.3–47.4 (29.6) t15 = 2.19, p < 0.05 40.8–51.5 (45.4) t15 = -5.38, p < 0.001 33.3–37.8 (35.5) t15 = -6.01, p < 0.001 Internal primary branch 46.3–54.9 (51.2) 42.6–76.6 (59.6) t15 = -2.26, p < 0.05 Internal secondary branch 35.5–40.2 (37.8) 28.4–33.5 (32.4) t15 = 7.68, p < 0.001 35.2–52.3 (42.8) t14 = -2.98, p < 0.01 Claw/Leg III External primary branch 49.3–58.3 (54.2) 30.0–54.9 (43.7) t14 = 3.71, p < 0.001 49.7–76.1 (62.9) t14 = -2.84, p < 0.01 57.1–73.5 (65.4) t14 = 5.68, p < 0.001 44 Table 11 (continued on next page). Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between senior specimens of Milnesium quiranae sp. nov. and the similar species. Species Character M. quiranae sp. nov. M. asiaticum M. barbadosense M. bohleberi M. burgessi M. eurystomum M. longiungue M. sandrae M. Differential diagnosis longiungue. 9. Milnesium pseudotardigradum, only known from the type locality in Iceland, by different body colour: reddish in M. quiranae sp. nov. vs yellowish in M. pseudotardigradum; statistically significant higher pt of the peribuccal papillae [22.6–31.6, mean 25.8] in M. quiranae vs [13.7–24.8, mean 19.9] in M. pseudotardigradum (t7 = 2.56, p < 0.05); statistically significant higher pt of the lateral papillae [18.8–27.2, mean 21.5] in M. quiranae vs [11.0–21.8, mean 15.6] in M. pseudotardigradum; t15 = 3.83, p < 0.001; different buccal tube width: pt of standard width [59.1–67.9] in M. quiranae vs [25.5–50.8] in M. pseudotardigradum; claw configuration: [3-3]-[3-3] in all the specimens (young and senior specimens) in M. quiranae vs [3-3]-[3-3] only in hatchlings in M. pseudotardigradum; changes during the ontogeny of claw configuration are absent in M. quiranae vs with double change in M. pseudotardigradum. 10. Milnesium sandrae, only known from the type locality in Hawaii (USA), by different body colour, reddish in M. quiranae vs transparent in M. sandrae; different buccal tube width: pt of standard width [59.1–67.9] in M. quiranae vs [44.9–48.0] in M. sandrae; pt of the stylet support insertion point: [67.5– 73.6] in M. quiranae vs [58.0–60.5] in M. sandrae; by different pt of many claw heights: external primary branches I–III [45.6–54.9]-[47.2–56.6]-[49.3–58.3] in M. quiranae vs [38.8–43.5]-[42.4–46.6]- [43.4–46.1] respectively in M. sandrae; posterior primary and secondary branch IV [60.1–69.7] and [44.0–51.7] in M. quiranae vs [54.0–57.1] and [38.0–40.2] respectively in M. sandrae, and statistically significant differences of pt of external secondary branches I–III (Table 11). 11. Milnesium shilohae, only known from the type locality in Hawaii (USA), by different body colour: reddish in M. quiranae sp. nov. vs white or transparent in M. shilohae; statistically significant difference of pt of lateral papillae [18.8–27.2, mean 21.5] in M. quiranae vs [12.8–21.8, mean 17.2] in 43 European Journal of Taxonomy 822: 1–54 (2022) 44 Table 11 (continued on next page). Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between senior specimens of Milnesium quiranae sp. nov. and the similar species. Species Character M. quiranae sp. nov. M. asiaticum M. barbadosense M. bohleberi M. burgessi M. eurystomum M. longiungue M. sandrae M. Differential diagnosis shillohe External secondary branch 36.2–43.1 (39.5) 30.9–37.3 (34.1) t15 = 5.90, p < 0.001 23.6–41.1 (30.4) t15 = 3.70, p < 0.001 40.4–58.2 (48.2) t15 = –4.49, p < 0.001 34.7–36.7 (35.5) t15 = -5.77, p < 0.001 29.7–37.1 (32.6) t13=4.01, p < 0.001 Internal primary branch 46.0–56.0 (51.5) 49.9–74.4 (61.7) t15 = –4.10, p < 0.001 30.3–48.7 (38.1) t15 = –5.91, p < 0.001 Internal secondary branch 34.8–40.2 (37.1) 30.7–35.8 (34.0) t13 = 4.84, p < 0.001 36.5–51.3 (44.7) t13 = –3.43, p < 0.01 Claw/LegIV Anterior primary branch 56.4–65.6 (62.0) 32.2–59.7 (43.7) t14 = 4.77, p < 0.001 Anterior secondary branch 41.4–49.6 (45.3) 35.7–41.4 (37.5) t15 = 8.93, p < 0.001 41.1–61.6 (49.6) t15 = –2.06, p < 0.05 29.8–43.8 (36.7) t15 = –4.26, p < 0.001 35.2–42.7 (39.0) t15 = 4.88, p < 0.001 Posterior primary branch 60.1–69.7 (65.7) 63.9–76.0 (69.7) t15 = –3.05, p < 0.01 35.5–66.3 (48.7) t15 = 4.38, p < 0.001 66.6–96.2 (84.3) t15 = –4.82, p < 0.001 Posterior secondary branch 44.0–51.7 (46.8) 36.8–46.7 (41.1) t15 = 4.42, p < 0.001 24.3–42.5 (34.0) t15 = 6.56, p < 0.001 44.5–60.5 (52.6) t15 = –3.26, p < 0.01 30.4–47.7 (37.2) t15 = –4.18, p < 0.001 35.9–44.3 (39.7) t15 = 6.28, p < 0.001 45 Table 11 (continued on next page). Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between senior specimens of Milnesium quiranae sp. nov. and the similar species. Species Character M. quiranae sp. nov. M. asiaticum M. barbadosense M. bohleberi M. burgessi M. eurystomum M. longiungue M. sandrae M. Differential diagnosis shillohe Claw/LegI External primary branch 45.6–54.9 (49.3) 30.2–50.1 (41.0) t15 = 3.42, p < 0.01 52.2–79.5 (62.7) t15 = -4.92, p < 0.001 External secondary branch 34.2–41.8 (39.5) 29.2–36.0 (32.4) t15 = 6.12, p < 0.001 23.9–36.9 (30.7) t15 = 5.85, p < 0.001 38.8–55.2 (45.2) t15 = -3.54, p < 0.001 26.4–38.7 (31.7) t15 = -4.85, p < 0.001 32.8–35.4 (34.1) t15 = -8.69, p < 0.001 29.5–35.5 (32.7) t15 = -8.01, p < 0.001 Internal primary branch 43.2–52.0 (46.6) 47.9–75.6 (60.0) t15 = -4.83, p < 0.001 28.5–44.9 (35.0) t15 = -5.65, p < 0.001 Internal secondary branch 30.6–38.9 (36.8) 36.7–53.7 (44.2) t15 = -4.37, p < 0.001 Claw/Leg II External primary branch 47.2–56.6 (52.7) 29.8–59.6 (42.4) t15 = 2.68, p < 0.01 46.1–70.2 (62.2) t15 = -2.69, p < 0.01 32.9–47.9 (38.4) t15 = -7.37, p < 0.001 External secondary branch 36.4–42.5 (39.3) 22.3–47.4 (29.6) t15 = 2.19, p < 0.05 40.8–51.5 (45.4) t15 = -5.38, p < 0.001 33.3–37.8 (35.5) t15 = -6.01, p < 0.001 Internal primary branch 46.3–54.9 (51.2) 42.6–76.6 (59.6) t15 = -2.26, p < 0.05 Internal secondary branch 35.5–40.2 (37.8) 28.4–33.5 (32.4) t15 = 7.68, p < 0.001 35.2–52.3 (42.8) t14 = -2.98, p < 0.01 Claw/Leg III External primary branch 49.3–58.3 (54.2) 30.0–54.9 (43.7) t14 = 3.71, p < 0.001 49.7–76.1 (62.9) t14 = -2.84, p < 0.01 57.1–73.5 (65.4) t14 = 5.68, p < 0.001 44 ROCHA A.M. et al., Three new species of Milnesium from Argentina 45 Table 11 (continued on next page). Statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of claw heights between senior specimens of Milnesium quiranae sp. nov. and the similar species. Species Character M. quiranae sp. nov. M. asiaticum M. barbadosense M. bohleberi M. burgessi M. eurystomum M. longiungue M. sandrae M. Differential diagnosis Milnesium swansoni only known from the type locality in USA; by different body colour: reddish in M. quiranae sp. nov. vs transparent to yellow in M. swansoni; higher pt of buccal tube standard width [59.1–67.9] in M. quiranae vs [39.2–42.2] in M. swansoni, statistically significant higher pt of the stylet support insertion point: [67.5–73.6] in M. quiranae vs [66.6–68.2] in M. swansoni (t15 = 7.39, p < 0.001). 13. Milnesium tumanovi only known from the type locality in Yalta (Crimea) by body colour: reddish in M. quiranae sp. nov. vs transparent in M. tumanovi; different buccal tube width: higher pt of standard width [59.1–67.9], in M. quiranae vs [55.1] in M. tumanovi; higher pt of the stylet support insertion point: [67.5–73.6] in M. quiranae vs [52.3] in M. tumanovi; different pt of the external primary branches I–III [45.6–58.3], [47.2–56.6] and [49.3–58.3] respectively in M. quiranae vs [43.0], [43.4] and [32.6] in M. tumanovi, external secondary branches I and III [34.2–41.8], [36.2–43.1] respectively in M. quiranae vs [32.6] and [33.0] respectively in M. tumanovi, posterior primary and secondary branch IV [60.1–69.7] and [44.0–51.7] in M. quiranae vs [55.3] and [42.2] respectively in M. tumanovi. 14. Milnesium validum, only known from the type locality in Antarctica, by different body colour, reddish in M. quiranae sp. nov. vs colourless in M. validum; different buccal tube width: higher pt of standard width [59.1–67.9] in M. quiranae vs [29.9–43.9] in M. validum; higher pt of stylet support insertion point: [67.5–73.6] in M. quiranae vs [62.0–65.1] in M. validum; higher pt of several claw heights: external primary and secondary branches I [45.6–54.9] and [34.2–41.8] in M. quiranae vs [36.0–38.3] and [25.4–28.6] respectively in M. validum; external primary and secondary branches II [47.2.-56.6] and [36.4–42.5] in M. quiranae vs [37.2–42.1] and [27.0–30.3] respectively in M. validum; external primary and secondary branches III [49.4–58.3] and [36.2–43.1] in M. quiranae vs [38.5–42.5] and [30.4] respectively in M. validum, and posterior primary and secondary branches IV [60.1–69.7] and [41.4–49.6] in M. quiranae vs [47.9–49.3] and [28.8–32.9] respectively in M. validum. 15. Milnesium zsalakoae only known from the type locality in USA by different body colour: reddish in M. quiranae sp. nov. vs white or transparent in M. zsalakoae; different buccal tube width: pt of standard width [59.1–67.9] in M. quiranae vs [36.8–41.9] in M. zsalakoae; accessory points present in M. quiranae vs absent in M. Differential diagnosis shillohe External secondary branch 36.2–43.1 (39.5) 30.9–37.3 (34.1) t15 = 5.90, p < 0.001 23.6–41.1 (30.4) t15 = 3.70, p < 0.001 40.4–58.2 (48.2) t15 = –4.49, p < 0.001 34.7–36.7 (35.5) t15 = -5.77, p < 0.001 29.7–37.1 (32.6) t13=4.01, p < 0.001 Internal primary branch 46.0–56.0 (51.5) 49.9–74.4 (61.7) t15 = –4.10, p < 0.001 30.3–48.7 (38.1) t15 = –5.91, p < 0.001 Internal secondary branch 34.8–40.2 (37.1) 30.7–35.8 (34.0) t13 = 4.84, p < 0.001 36.5–51.3 (44.7) t13 = –3.43, p < 0.01 Claw/LegIV Anterior primary branch 56.4–65.6 (62.0) 32.2–59.7 (43.7) t14 = 4.77, p < 0.001 Anterior secondary branch 41.4–49.6 (45.3) 35.7–41.4 (37.5) t15 = 8.93, p < 0.001 41.1–61.6 (49.6) t15 = –2.06, p < 0.05 29.8–43.8 (36.7) t15 = –4.26, p < 0.001 35.2–42.7 (39.0) t15 = 4.88, p < 0.001 Posterior primary branch 60.1–69.7 (65.7) 63.9–76.0 (69.7) t15 = –3.05, p < 0.01 35.5–66.3 (48.7) t15 = 4.38, p < 0.001 66.6–96.2 (84.3) t15 = –4.82, p < 0.001 Posterior secondary branch 44.0–51.7 (46.8) 36.8–46.7 (41.1) t15 = 4.42, p < 0.001 24.3–42.5 (34.0) t15 = 6.56, p < 0.001 44.5–60.5 (52.6) t15 = –3.26, p < 0.01 30.4–47.7 (37.2) t15 = –4.18, p < 0.001 35.9–44.3 (39.7) t15 = 6.28, p < 0.001 45 European Journal of Taxonomy 822: 1–54 (2022) M. shilohae (t15 = 3.73, p < 0.001); different buccal tube width: lower pt of standard width, [59.1–67.9] in M. quiranae vs [47.1–55.9] in M. shilohae; lower pt of stylet support insertion point: [67.5–73.6] in M. quiranae vs [75.5–77.5] in M. shilohae; different pt of many claw heights: external primary branch I [45.6–54.9] in M. quiranae vs [34.2–40.3] in M. shilohae, external primary and secondary branches II [47.2–56.6] and [36.4–42.5] in M. quiranae vs [37.4–44.1] and [28.2–35.9] respectively in M. shilohae; internal primary and secondary branches II [46.3–54.9] and [35.5–40.2] in M. quiranae vs [35.7–42.0] and [28.8–34.5] respectively in M. shilohae; external primary branch III [49.3–58.3] in M. quiranae vs [35.2–46.8] in M. shilahoe; statistically significant differences of pt of external secondary branch III (Table 11); different pt anterior and posterior primary branches IV [56.4–65.6] and [60.1–69.7] in M. quiranae vs [42.6–51.1] and [48.3–55.5] respectively in M. shilohae and statistically significant differences about pt of external secondary branch I, external secondary branch III and anterior and posterior secondary branches IV (Table 11). 12. Differential diagnosis zsalakoae; different pt of several claw heights: internal primary branches I–III [43.2– 52.0], [46.3–54.9], [46.0–56.0] in M. quiranae. vs [64.4–68.6], [64.7–80.4], [80.5–88.6], respectively in M. zsalakoae; internal secondary branch I [30.6–38.9] in M. quiranae vs [45.4–64.7] in M. zsalakoae and anterior primary branch IV [56.4–65.6] in M. quiranae vs [94.8–102.9] in M. zsalakoae. With regard to young and intermediate specimens of M. quiranae sp. nov., as already indicated, differences of senior specimens with the above mentioned species tend to remain valid, because the 46 ROCHA A.M. et al., Three new species of Milnesium from Argentina only ontogenetic changes in the new species pertain to the buccal tube width and the medioventral peribuccal papilla reduction; thus, apart from those two characters, the already provided differential diagnosis is valid also for young and intermediate specimens of M. quiranae (e.g., considering body colour, presence/absence of eyes, pt of stylet support insertion point and of claw heights). It is only worth mentioning that for M. burgessi, M. longiungue, M. pseudotardigradum, M. sandrae, M. shilohae and M. tumanovi, the buccal tube width is comparable with with young and/or intermediate specimens of M. quiranae, but in all cases there are the other differences that keep the species well differentiated. We here provide detailed comparison of young specimens of M. quiranae only with M. minutum, for the reasons expressed above. Young specimens of M. quiranae sp. nov. differ from M. minutum (only known from the type locality in Italy) by different body colour: reddish in M. quiranae sp. nov. vs transparent in M. minutum; different buccal tube width: higher pt of buccal tube standard width [43.2–51.5] in M. quiranae vs [38.6–42.4] in M. minutum; higher pt of the stylet support insertion point: [67.4–75.1] in M. quiranae vs [63.0–65.9] in M. minutum; different pt of many claw heights: external primary and secondary branches I, [39.8–49.8] and [31.2–40.6] in M. quiranae vs [39.1] and [28.3] in M. minutum; external primary and secondary branches II [45.0–54.5] and [33.5–43.3] in M. quiranae vs [42.2–44.3] and [29.5–31.4] respectively in M. minutum; posterior secondary branches IV [37.4–47.2] in M. quiranae vs [33.5–34.5] in M. minutum. Dichotomous key of the species of Milnesium Doyère, 1840 from South America 1. Dorsal cuticle smooth ........................................................................................................................ 2 – Dorsal cuticle ornamented (pseudopores-reticulum-dimples) ........................................................... 5 2. Claw configuration [2-2]-[2-2] ...........................................................M. kogui Londoño et al., 2015 – Claw configuration [3-3]-[3-3] .......................................................................................................... 3 3. Discussion The discovery of the three new species of Milnesium described in the present paper provides an opportunity to comment on the species of this genus. In the past, ontogenetic changes were not known in the genus Milnesium, until the scientific contribution of Morek et al. (2016) describing M. variefidum, followed by Moreno-Talamantes et al. (2019, 2020), Surmacz et al. (2019), Morek et al. (2020a) and Sugiura et al. (2020). It is worth mentioning that two of the three species described in the present paper show ontogenetic changes: M. pelufforum sp. nov. shows changes in the dorsal cuticle characteristics, buccal tube width and claw configuration, while M. quiranae sp. nov. shows changes in buccal tube width and the medioventral buccal papilla reduction. All these recent discoveries lead to the suspicion that ontogenetic changes in the genus Milnesium may be rather common, but have not yet been reported for species already described. For example, about the ontogenetic change affecting the buccal tube width, young specimens with a more slender buccal tube and senior specimens with a wider one, were reported for M. eurystomum, M. pseudotardigradum, and here for M. pelufforum and M. quiranae We believe this could be a common situation, because for several species, the range of pt of the buccal tube width in the species description appears quite wide, possibly hiding the ontogenetic change under discussion. Those species are: M. alpigenum Ehrenberg, 1853 (pt of the buccal tube standard width 25.5–42.3, specimen size 367–877 µm; Morek et al. 2016, 2019b), M. barbadosense (pt of the buccal tube standard width 27.2–49.7, specimen size 283–686 µm; Meyer & Hinton 2012), M. burgessi Schlabach, Donaldson, Hobelman, Miller & Lowman, 2018 (pt of the buccal tube standard width 52.9–68.5, specimen size 343–1030 µm; Schlabach et al. 2018), M. reductum Pilato, Binda & Lisi, 2002 (pt of the buccal tube standard width 20.0–39.8, specimen size 363–730 µm; Morek et al. 2020b) and M. tardigradum (pt of the buccal tube standard width 18.4–51.8, specimen size 278–789 µm; Morek et al. 2019a). Even in some recent species descriptions/redescriptions that report ontogenetic change, metric characters of specimens of all sizes are reported together. This could hide important morphometric differences between young and senior specimens, e.g., for buccal tube width. Therefore, we strongly recommend that future species descriptions/redescriptions should always report separate morphometric characters for young and senior specimens. Differential diagnosis BT funnel-shaped in adults (posterior/anterior width ratio lower than 65%) ...................................... ...............................................M. eurystomum Maucci, 1991 (amended by Michalczyk et al. 2012b; amended by Morek et al. 2020a) – BT about cylindrical in adults (posterior/anterior width ratio higher than 75%) .............................. 4 4. Pt of BT standard width lower than 52 in adults ...................M. brachyungue Binda & Pilato, 1990 – Pt of BT standard width higher than 52 in adults ...............................................M. quiranae sp. nov. 5. Claw configuration [3-3]-[3-3] .......................................................................................................... 6 – Claw configuration different .............................................................................................................. 7 6. Nine rows of pseudoplates, pt of SS [63.8–66.7]; pt of BT standard width [58.1–65.6] .................... ......................................................................................................M. beatae Roszkowska et al., 2015 – Without pseudoplates, pt of SS [70.0–73.7], pt of BT standard width [24.2–32.3] ............................ ............................................................................................. M. argentinum Roszkowska et al., 2015 7. Claw configuration [2-2]-[2-2] ...............................................M. katarzynae Kaczmarek et al., 2004 – Claw configuration different .............................................................................................................. 8 8. Claw configuration [2-3]-[2-2], pt of BT standard width [46.1–56.9], with 9 rows of pseudoplates .... .................................................................................................................................... M. irenae sp. nov. – Claw configuration [2-3]-[3-2] ..............................................................................................................9 Dichotomous key of the species of Milnesium Doyère, 1840 from South America 47 European Journal of Taxonomy 822: 1–54 (2022) 9. Cuticular dimples arranged in 10 bands, small tubercles around all claw bases, pt of BT standard width [55.2–64.0], young specimens with claw configuration [2-2]-[2-2] .....M. pelufforum sp. nov. – Cuticular dimples present not forming bands, pt of BT standard width [33.0–38.0], no tubercles around claw bases ...................................................... M. krzysztofi Kaczmarek & Michalczyk, 2007 9. Cuticular dimples arranged in 10 bands, small tubercles around all claw bases, pt of BT standard width [55.2–64.0], young specimens with claw configuration [2-2]-[2-2] .....M. pelufforum sp. nov. – Cuticular dimples present not forming bands, pt of BT standard width [33.0–38.0], no tubercles around claw bases ...................................................... M. krzysztofi Kaczmarek & Michalczyk, 2007 y g pi g p ff p – Cuticular dimples present not forming bands, pt of BT standard width [33.0–38.0], no tubercles around claw bases ...................................................... M. krzysztofi Kaczmarek & Michalczyk, 2007 Abbreviations: BT = buccal tube; SS = stylet support insertion point on the buccal tube. Discussion We also suggest, for species in which ontogenetic change is verified, that the differential diagnosis treats young and senior specimens separately if possible and useful, with comparisons to similar species, in order to provide more precise, complete differential diagnosis which facilitates species distinction. Another, more recent achievement (firstly thanks to Michalczyk et al. 2012a) regards the discovery of reduction of the medio-ventral peribuccal papilla. We found this in M. irenae sp. nov. and M. quiranae sp. nov. Maybe this trait might prove to be, with further investigations, less rare than it appears now. For the moment it has been reported (additionally to the two new species mentioned above) for M. alpigenum, M. beasleyi, M. bohleberi, M. eurystomum, M. granulatum, M. inceptum Morek, Suzuki, Schill, Georgiev, Yankova, Marley & Michalczyk, 2019b, M. pacificum, M. tardigradum and M. variefidum. In addition, we noticed that such papilla looks to be reduced also in M. almatyense (Morek et al. 2020b: fig. 2c), M. berladnicorum (Morek et al. 2016: fig. 6b) and M. reductum (Morek et al. 2020b: fig. 4b). It is also important to ascertain in the various species if ontogenetic reduction of such papilla may occur, like we found in M. quiranae. 48 ROCHA A.M. et al., Three new species of Milnesium from Argentina About more detailed characters of the species described in the present paper, we find it interesting to discuss some features of the cuticular ornamentation of M. pelufforum sp. nov. and M. irenae sp. nov. About more detailed characters of the species described in the present paper, we find it interesting to discuss some features of the cuticular ornamentation of M. pelufforum sp. nov. and M. irenae sp. nov. Milnesium pelufforum sp. nov. has ten bands of sculptured cuticle coinciding with ten rows of pseudoplates, while, until now, the maximum number of those bands/rows was nine. This because the new species has the first band and row in the first cephalic subsegment, where, until now, no band of sculpture or row of pseudoplates has been reported. This raises the need of updating the way to indicate the pseudoplate formula which was provided by Moreno-Talamantes et al. (2019). Discussion As a matter of fact, it is necessary from now on to deal with ten possible rows/bands, which is the maximum number possible considering there are ten subsegments on the entire body (head plus four trunk segment, each one divided into two subsegments at least regarding the cuticle). We therefore propose to always indicate all ten rows for correct comparison between species, according to Table 3. Milnesium pelufforum sp. nov. also shows cuticular dimples provided with internal structures of variable appearance. Based on the literature, no other species of the genus has been reported having these characters until now, but it is not possible to exclude that some species described in the past might have these characters which passed unnoticed. Further investigations are required to ascertain whether presence and shape of the dimple internal structures are constant. Under PCM, we could not spot those internal structures inside all dimples, and their shape appeared variable; however, we cannot exclude that this is just due to matter of focus under the microscope and position of the structures in the preparation, so that those internal structures might be present in all dimples and always with the same morphology. Regarding the cuticular sculpture of M. irenae sp. nov., with the today available data it looks one of the most complex ones within the genus. It also includes a reticulation which is different from all other ‘reticular’ sculptures of the other congeneric species. We mean that the latter ones actually have dimples close to one another (we ascertained this detail from the species descriptions and related photos) which gives the appearance of a reticulation. In M. irenae, instead, the reticulation does not look as consisting of dimples but of a delicate mesh appearing more similar to reticulations present in Parachela (e.g., several species of Isohypsibiidae and Doryphoribiidae). Lisi (2011) described the reticulation of some species of Doryphoribiidae as consisting of cuticular ridges delimiting a mesh, and this looks exactly the case for M. irenae. Until now, the only similar case could be the delicate reticulation of the hatchlings of M. almatyense (see Morek et al. 2020b). It is clear that deeper investigations, especially using SEM, are needed (including for the new species described in the present paper) to clarify the still pending problems. Conclusion Valuable data were obtained from the review of a limited number of slides of the tardigrade collection deposited at the National University of La Pampa, containing 10 870 specimens in total. This shows how important it is to investigate and describe in detail biological archives that represent the biodiversity of the natural heritage of a given place and time; such work, based on descriptive morphology of the species, can also provide contributions for deciphering both intra- and interspecific variability. Thanks to the analysis of specimens of the genus Milnesium deposited in the above mentioned collection, three previously unknown species were described, so that the number of species in this genus in South America increases from 8 to 11 and in Argentina in particular from 5 to 8. The results of this research also contribute to gain knowledge about the family Milnesiidae (e.g., about morphological details and ontogenetic aspects), and provide information about faunistics of tardigrades of the southern hemisphere, as yet insufficiently known. 49 49 European Journal of Taxonomy 822: 1–54 (2022) Acknowledgements We are grateful to the associate Editor and the anonymous reviewers who improved the quality of the article, to Prof. Giovanni Pilato for making his collection available for our study, and to Irene Luisa Doma for her valuable collaboration in collecting and analysing the samples we used. We are also grateful to Cecilia Bobillo from the Forensic Genetics Laboratory, Public Ministry of the Judicial Power of La Pampa, for her valuable help and collaboration in taking the fluorescence microscopy images, and to Prof. Paul M. Ramsay who provided a thorough linguistic review of the manuscript. 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Zoological Journal of the Linnean Society 188 (3): 681–693. https://doi.org/10.1093/zoolinnean/zlz040 51 European Journal of Taxonomy 822: 1–54 (2022) Morek W., Gąsiorek P., Stec D., Blagden B. & Michalczyk Ł. 2016. Experimental taxonomy exposes ontogenetic variability and elucidates the taxonomic value of claw configuration in Milnesium Doyère, 1840 (Tardigrada: Eutardigrada: Apochela). Contributions to Zoology 85 (2): 173–200. https://doi.org/10.1163/18759866-08502003 Morek W., Stec D., Gąsiorek P., Surmacz B. & Michalczyk Ł. 2019a. Milnesium tardigradum Doyère, 1840: the first integrative study of interpopulation variability in a tardigrade species. Journal of Zoological Systematics and Evolutionary Research 57 (1): 1–23. https://doi.org/10.1111/jzs.12233 Morek W., Suzuki A., Schill R., Georgiev D., Yankova M., Marley N. & Michalczyk L. 2019b. 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References https://doi.org/10.11646/zootaxa.1122.1.1 Manuscript received: 2 July 2021 Manuscript accepted: 15 March 2022 Published on: 3 June 2022 Topic editor: Tony Robillard Section editor: Daniel Stec Desk editor: Pepe Fernández Manuscript received: 2 July 2021 Printed versions of all papers are also deposited in the libraries of the institutes that are members of the EJT consortium: Muséum national d’histoire naturelle, Paris, France; Meise Botanic Garden, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of Denmark, Copenhagen, Denmark; Naturalis Biodiversity Center, Leiden, the Netherlands; Museo Nacional de Ciencias Naturales-CSIC, Madrid, Spain; Real Jardín Botánico de Madrid CSIC, Spain; Zoological Research Museum Alexander Koenig, Bonn, Germany; National Museum, Prague, Czech Republic. Supplementary material For all the following Excel files (1–6), the Excel template “Apochela” (ver. 1.4) from the tardigrade Register (Michalczyk & Kaczmarek 2013) was used. Supp. file 1. Measurements of Milnesium pelufforum young. Complete morphometric dataset of the young specimens of Milnesium pelufforum sp. nov. (from which Table 1 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6929 Supp. file 1. Measurements of Milnesium pelufforum young. Complete morphometric dataset of the young specimens of Milnesium pelufforum sp. nov. (from which Table 1 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6929 Supp. file 2. Measurements of Milnesium pelufforum senior. Complete morphometric dataset of the senior specimens of Milnesium pelufforum sp. nov. (from which Table 2 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6931 Supp. file 3. Measurements of Milnesium irenae. Complete morphometric dataset of all specimens of Milnesium irenae sp. nov. (from which Table 6 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6933 Supp. file 4. Measurements of Milnesium quiranae young. Complete morphometric dataset of the young specimens of Milnesium quiranae sp. nov. (from which Table 8 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6935 53 European Journal of Taxonomy 822: 1–54 (2022) Supp. file 5. Measurements of Milnesium quiranae senior. Complete morphometric dataset of the senior specimens of Milnesium quirane sp. nov. (from which Table 9 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6937 Supp. file 6. Measurements of Milnesium quiranae intermediate. Complete morphometric dataset of the intermediate specimens of Milnesium quiranae sp. nov. (from which Table 10 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6939 Supp. file 6. Measurements of Milnesium quiranae intermediate. Complete morphometric dataset of the intermediate specimens of Milnesium quiranae sp. nov. (from which Table 10 in the paper derives) (Excel file). https://doi.org/10.5852/ejt.2022.822.1807.6939 Supp. file 7. Supplementary figure – statistics for life stages. Figure illustrating the results of the analysis of Surmacz et al. (2020) for detection of different life stages (through correlation between growth in body length and buccal tube length) applied to the entire population of each new species of the present paper. A. Milnesium pelufforum sp. nov.. Two evident clusters corresponding to young and senior specimens were outlined. B. Milnesium quiranae sp. nov. Three evident clusters corresponding to young, intermediate and senior specimens were outlined. C. Milnesium irenae sp. nov. No cluster was distinguishable with the available dataset (png file). https://doi.org/10.5852/ejt.2022.822.1807.6941 54 54
https://openalex.org/W2146808843	https://europepmc.org/articles/pmc4450469?pdf=render	English	null	Early biomarkers of joint damage in rheumatoid and psoriatic arthritis	Arthritis research & therapy	2,015	cc-by	11,710	© 2015 Mc Ardle et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. REVIEW Open Access Abstract Joint destruction, as evidenced by radiographic findings, is a significant problem for patients suffering from rheumatoid arthritis and psoriatic arthritis. Inherently irreversible and frequently progressive, the process of joint damage begins at and even before the clinical onset of disease. However, rheumatoid and psoriatic arthropathies are heterogeneous in nature and not all patients progress to joint damage. It is therefore important to identify patients susceptible to joint destruction in order to initiate more aggressive treatment as soon as possible and thereby potentially prevent irreversible joint damage. At the same time, the high cost and potential side effects associated with aggressive treatment mean it is also important not to over treat patients and especially those who, even if left untreated, would not progress to joint destruction. It is therefore clear that a protein biomarker signature that could predict joint damage at an early stage would support more informed clinical decisions on the most appropriate treatment regimens for individual patients. Although many candidate biomarkers for rheumatoid and psoriatic arthritis have been reported in the literature, relatively few have reached clinical use and as a consequence the number of prognostic biomarkers used in rheumatology has remained relatively static for several years. It has become evident that a significant challenge in the transition of biomarker candidates to clinical diagnostic assays lies in the development of suitably robust biomarker assays, especially multiplexed assays, and their clinical validation in appropriate patient sample cohorts. Recent developments in mass spectrometry-based targeted quantitative protein measurements have transformed our ability to rapidly develop multiplexed protein biomarker assays. These advances are likely to have a significant impact on the validation of biomarkers in the future. In this review, we have comprehensively compiled a list of candidate biomarkers in rheumatoid and psoriatic arthritis, evaluated the evidence for their potential as biomarkers of bone (joint) damage, and outlined how mass spectrometry-based targeted and multiplexed measurement of candidate biomarker proteins is likely to accelerate their clinical validation and the development of clinical diagnostic tests. Early biomarkers of joint damage in rheumatoid and psoriatic arthritis Angela Mc Ardle1, Brian Flatley1, Stephen R. Pennington1† and Oliver FitzGerald1,2† Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 DOI 10.1186/s13075-015-0652-z * Correspondence: Stephen.Pennington@ucd.ie; Oliver.Fitzgerald@ucd.ie †Equal contributors 1Conway Institute of Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland 2Department of Rheumatology, St Vincent’s University Hospital, Elm Park, Dublin 4, Ireland Introduction the Classification for Psoriatic Arthritis CASPAR criteria [4, 5]. However, it is recognised that disease onset may occur much earlier, even prior to symptom onset [6, 7]. Several disease-specific characteristics differentiate RA from PsA. For example, rheumatoid factor (RF) is present often at high titre in 80 % of RA patients whereas it is present at low titre in only 13 % of PsA patients. PsA is included among the spondylarthopathies because it shares both clinical features and association with HLAB27 with other spondylarthopathy members. The presence of psoriasis is a hallmark of PsA, although joint involvement may precede skin manifestations in ~10 % of patients. Asymmetric joint involvement is seen commonly in PsA whereas joint involvement in RA follows a symmetrical pattern. Dactylitis, enthesitis, sacroiliitis and interphalan- geal joint involvement are also more common in PsA [3]. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are the most prevalent forms of inflammatory arthritis affect- ing ~1 % and ~ 0.3 to 1 % of the population, respectively [1, 2]. Disease aetiology is unknown but it is thought that both genetic and environmental factors trigger the onset of these arthropathies [3]. The onset of RA and PsA is clini- cally recognised when a patient presents with symptoms fulfilling disease classification criteria, importantly the American College of Rheumatology criteria for RA and Page 2 of 12 Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 At the cellular level, histological studies have revealed important differences between synovial tissue in RA and PsA [8]. Angiogenesis is dysregulated in both conditions and abnormal vessel morphology and function has been reported. Increased straight, branching vascularisation is a prominent feature observed in RA joints, whereas the formation of elongated, bushy, torturous blood vessels is a more marked feature of the PsA joint [8, 9]. In the RA joint there is increased macrophage infiltration and sub- sequent synovial invasion compared with that observed in PsA. As a result, lining layer hyperplasia observed in RA is more striking than that observed in PsA [3]. Con- versely, PsA is characterised by more extensive infil- tration of polymorphonuclear cells [8]. It has been reported that the extent of T-cell and B-cell infiltration is comparable in both conditions and the formation of germinal centres (zones of T-cell and B-cell prolifera- tion) are observed in both PsA and RA joints [8, 10, 11]. Introduction The differences in synovitis in RA and PsA are illus- trated in Fig. 1. Radiographic progression Radiographic progression is considered a consequence of synovial inflammation. However, at least for RA, the observations that bone loss can occur before clinical onset and at very early stages of disease have been widely ac- knowledged [6, 7]. These observations are surprising since synovitis requires some time to destroy bone to an extent that is clinically detectable. Synovitis might thus not be the exclusive cause of joint damage. An alternative Fig. 1 Synovitis in rheumatoid arthritis and psoriatic arthritis. Synovitis in rheumatoid arthritis (RA) and psoriatic arthritis (PsA) is triggered by unknown event(s). It is thought that a genetic predisposition and/or environmental cues trigger inappropriate activation of synoviocytes, thereby promoting an autoimmune inflammatory response. Once activated, synoviocytes produce proinflammatory cytokines that in turn activate proximal cells, including endothelial cells that line the blood vessels supplying the joint. This results in dysregulated angiogenesis and the increased infiltration of leukocytes, including monocytes, macrophages, neutrophils, mast cells, eosinophils, B cells and T cells. Infiltrating cells produce cytokines that act in synergy to propagate the inflammatory response. Importantly, tumour necrosis factor alpha (TNFα) and interleukin (IL)-17 are cytokines with major implied roles in PsA and RA pathogenesis and represent important therapeutic targets. With the development of a chronic inflammatory response, the synovial lining becomes hyperplastic. Fibroblasts and macrophages form an invasive matrix (pannus) that promotes the destruction of cartilage and bone. Activation of osteoclast cells promotes bone resorption whereas activation of osteoblasts promotes bone proliferation Fig. 1 Synovitis in rheumatoid arthritis and psoriatic arthritis. Synovitis in rheumatoid arthritis (RA) and psoriatic arthritis (PsA) is triggered by unknown event(s). It is thought that a genetic predisposition and/or environmental cues trigger inappropriate activation of synoviocytes, thereby promoting an autoimmune inflammatory response. Once activated, synoviocytes produce proinflammatory cytokines that in turn activate proximal cells, including endothelial cells that line the blood vessels supplying the joint. This results in dysregulated angiogenesis and the increased infiltration of leukocytes, including monocytes, macrophages, neutrophils, mast cells, eosinophils, B cells and T cells. Infiltrating cells produce cytokines that act in synergy to propagate the inflammatory response. Importantly, tumour necrosis factor alpha (TNFα) and interleukin (IL)-17 are cytokines with major implied roles in PsA and RA pathogenesis and represent important therapeutic targets. With the development of a chronic inflammatory response, the synovial lining becomes hyperplastic. Fibroblasts and macrophages form an invasive matrix (pannus) that promotes the destruction of cartilage and bone. Radiographic progression Activation of osteoclast cells promotes bone resorption whereas activation of osteoblasts promotes bone proliferation Fig. 1 Synovitis in rheumatoid arthritis and psoriatic arthritis. Synovitis in rheumatoid arthritis (RA) and psoriatic arthritis (PsA) is triggered by unknown event(s). It is thought that a genetic predisposition and/or environmental cues trigger inappropriate activation of synoviocytes, thereby promoting an autoimmune inflammatory response. Once activated, synoviocytes produce proinflammatory cytokines that in turn activate proximal cells, including endothelial cells that line the blood vessels supplying the joint. This results in dysregulated angiogenesis and the increased infiltration of leukocytes, including monocytes, macrophages, neutrophils, mast cells, eosinophils, B cells and T cells. Infiltrating cells produce cytokines that act in synergy to propagate the inflammatory response. Importantly, tumour necrosis factor alpha (TNFα) and interleukin (IL)-17 are cytokines with major implied roles in PsA and RA pathogenesis and represent important therapeutic targets. With the development of a chronic inflammatory response, the synovial lining becomes hyperplastic. Fibroblasts and macrophages form an invasive matrix (pannus) that promotes the destruction of cartilage and bone. Activation of osteoclast cells promotes bone resorption whereas activation of osteoblasts promotes bone proliferation Page 3 of 12 Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 measure joint damage in RA. This method provides sep- arate scores for erosion and joint space narrowing in the hands, wrists and feet. A modified Sharp–van der Heijde score is used to measure radiographic progression in PsA where the distal interphalangeal joints of the hands are also included [13, 14]. concept suggests that autoimmune processes begin years before the clinical onset of disease and that these pro- cesses promote joint destruction. Indeed, levels of anti- citrullinated protein antibodies (ACPA) can be detected in RA years before the clinical onset of disease. A compara- tive imaging study used micro computerised tomography to assess bone densities in healthy individuals that were ei- ther positive or negative for ACPA. The ACPA-positive in- dividuals exhibited significant alterations in cortical bone architecture as compared with the ACPA-negative individ- uals. These findings support the theory that bone damage is not exclusively a consequence of synovitis [7]. While X-ray imaging is the best validated technique for detecting bone erosion, it is limited by its two-dimensional character. Additional imaging modalities such as magnetic resonance imaging, micro computerised tomography and ultrasound are utilised to identify distinguishing features between RA and PsA. Radiographic progression in RA and PsA is remarkably different. Biomarkers Molecular events that drive joint damage may precede dis- ease onset and can cause detrimental long-term effects such as disability [6, 7, 10, 15, 16]. It is therefore essential to begin therapy as soon as possible in order to prevent ir- reversible damage. Some of the therapies currently avail- able are associated with high cost and potential for side effects. Additionally, up to 40 % of patients will not meet the primary outcome measure and, in those who do re- spond, levels of disease activity can remain significant. To add to the complexity, not all patients will develop a de- structive form of disease. Aggressive treatment should thus be reserved for patients that will develop a more se- vere form of disease [17]. In light of this, the need to de- velop biomarker signatures predictive of joint damage in RA and PsA is of critical importance. Currently there is a major international effort driven by the Outcome Mea- sures in Rheumatology Clinical Trials and the Group for Research and Assessment in Psoriasis and Psoriatic Arth- ritis well underway in RA and to start soon in PsA whereby biomarker samples are being prospectively col- lected in an effort to identify predictors of radiographic damage. Fig. 2 X-ray image of changes in bones observed in psoriatic arthritis. Bone changes in psoriatic arthritis (PsA) patients may differ between patients and may also differ within the same patient. The heterogeneity observed within a PsA patient is illustrated. Left-hand radiograph from a PsA patient showing severe erosive disease and subluxation at the first distal interphalangeal (DIP) with fluffy periosteal new bone formation on the terminal phalange. Ankylosis of the second DIP joint is also demonstrated Fig. 2 X-ray image of changes in bones observed in psoriatic arthritis. Bone changes in psoriatic arthritis (PsA) patients may differ between patients and may also differ within the same patient. The heterogeneity observed within a PsA patient is illustrated. Left-hand radiograph from a PsA patient showing severe erosive disease and subluxation at the first distal interphalangeal (DIP) with fluffy periosteal new bone formation on the terminal phalange. Ankylosis of the second DIP joint is also demonstrated Biomarkers serve as objective molecular indicators of pathological processes such as the development of joint destruction. Owing to the heterogeneous nature of RA and PsA, identifying a single biomarker predictive of joint damage would be an onerous task. Radiographic progression RA is a bone-resorbing, ero- sive disease whereas the pattern of radiographic progres- sion in PsA is more complex. As in RA, bone resorption and erosion may be evident in PsA, but radiographic pro- gression may also be marked by more severe resorption or osteolysis and commonly bony proliferation is prominent. To the best of our knowledge early protein biomarkers of bone proliferation have not yet been reported in the litera- ture, but these markers will probably differ from bio- markers of erosion. Distinguishing patterns of disease progression in RA and PsA have been captured by differ- ent imaging modalities and are summarised in Table 1. Previously, PsA was believed to be a mild nonprogres- sive form of arthritis. However, it is now well understood that 47 % of PsA patients will develop erosions within 2 years of symptom onset, and that of the patients suffer- ing from polyarticular PsA at least 20 % are at risk of progressing to a severe destructive phenotype (mutilans) comparable with that observed in RA [12]. What is more, bone changes observed in PsA are particularly heterogeneous both between and also within individual sufferers (Fig. 2). X-ray images are used in the clinic to follow radiographic progression and may be scored to measure the extent of joint damage. The Sharp–van der Heijde scoring method is most commonly used to Biomarkers In PsA it is the enthesis that are major sites of inflammation, whereas in RA the synovium becomes chronically inflamed. Inflammation of the tendons is also prevalent in both disorders although more severe in RA. Distinct features of PsA include bony proliferation, and dactylitis. DIP, distal interphalangeal; μCT, micro computational tomography; MRI, magnetic resonance imaging; PsA, psoriatic arthritis; RA, rheumatoid arthritis; US, ultrasound Disease features present in RA and PsA and the radiological imaging technique used to measure the feature. The number of erosions observed in RA appears to be greater than that in PsA. However, more sophisticated higher resolution techniques reveal this is not accurate because erosions in PsA are generally smaller and their detection requires these more sensitive techniques. Hence, μCT reveals a comparable extent of bone erosion in RA and PsA. MRI and US capture differences in sites affected by inflammation in these disorders. In PsA it is the enthesis that are major sites of inflammation, whereas in RA the synovium becomes chronically inflamed. Inflammation of the tendons is also prevalent in both disorders although more severe in RA. Distinct features of PsA include bony proliferation, and dactylitis. DIP, distal interphalangeal; μCT, micro computational tomography; MRI, magnetic resonance imaging; PsA, psoriatic arthritis; RA, rheumatoid arthritis; US, ultrasound Disease features present in RA and PsA and the radiological imaging technique used to measure the feature. The number of erosions observed in RA appears to be greater than that in PsA. However, more sophisticated higher resolution techniques reveal this is not accurate because erosions in PsA are generally smaller and their detection requires these more sensitive techniques. Hence, μCT reveals a comparable extent of bone erosion in RA and PsA. MRI and US capture differences in sites affected by inflammation in these disorders. In PsA it is the enthesis that are major sites of inflammation, whereas in RA the synovium becomes chronically inflamed. Inflammation of the tendons is also prevalent in both disorders although more severe in RA. Distinct features of PsA include bony proliferation, and dactylitis. DIP, distal interphalangeal; μCT, micro computational tomography; MRI, magnetic resonance imaging; PsA, psoriatic arthritis; RA, rheumatoid arthritis; US, ultrasound of damage in RA [18–20]. Consequently there is great interest in identifying a panel of biomarkers that could be incorporated into a signature predictive of disease progression. Biomarkers Indeed, no sin- gle biomarker has so far emerged as a reliable predictor Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 Page 4 of 12 Table 1 Radiological features that distinguish between rheumatoid arthritis and psoriatic arthritis Disease feature Rheumatoid arthritis Psoriatic arthritis Imaging technique Reference Number of erosions +++ + X-ray [18] +++ +++ μCT [82] Severity of erosions +++ ++ μCT [83] Shape of erosions Ʊ + +++ μCT [83] Tubule + +++ μCT U +++ + μCT Erosion distribution Preponderance for radial sites Evenly distributed μCT [83] DIP joint erosion – +++ US, MRI, X-ray [84] Number of osteophytes + +++ μCT [82] Severity of osteophytes (size) + +++ μCT [82] Bone proliferation + +++ US, MRI, X-ray [84] Inflammatory changes Synovitis +++ ++ MRI, US [84] Tenosynovitis +++ ++ MRI, US [84] Enthesitis + +++ MRI, US [85] Dactylitis – +++ US, MRI [86] Mutilans (erosions on both sides of joints) + X-ray [87] Disease features present in RA and PsA and the radiological imaging technique used to measure the feature. The number of erosions observed in RA appears to be greater than that in PsA. However, more sophisticated higher resolution techniques reveal this is not accurate because erosions in PsA are generally smaller and their detection requires these more sensitive techniques. Hence, μCT reveals a comparable extent of bone erosion in RA and PsA. MRI and US capture differences in sites affected by inflammation in these disorders. In PsA it is the enthesis that are major sites of inflammation, whereas in RA the synovium becomes chronically inflamed. Inflammation of the tendons is also prevalent in both disorders although more severe in RA. Distinct features of PsA include bony proliferation, and dactylitis. DIP, distal interphalangeal; μCT, micro computational tomography; MRI, magnetic resonance imaging; PsA, psoriatic arthritis; RA, rheumatoid arthritis; US, ultrasound Mutilans (erosions on both sides of joints) Disease features present in RA and PsA and the radiological imaging technique used to measure the feature. The number of erosions observed in RA appears to be greater than that in PsA. However, more sophisticated higher resolution techniques reveal this is not accurate because erosions in PsA are generally smaller and their detection requires these more sensitive techniques. Hence, μCT reveals a comparable extent of bone erosion in RA and PsA. MRI and US capture differences in sites affected by inflammation in these disorders. Biomarkers Such a signature could be used as an indi- cator of joint destruction and thus facilitate clinicians in making more informed clinical decisions and help guide personalised medical approaches [18, 20]. It is important to recognise that joint destruction contributes signifi- cantly to disability in patients suffering from inflamma- tory arthritis, but it is not the sole cause of disability. Joint inflammation, swelling, pain and disability occur in the absence of joint destruction [21]. In PsA, for ex- ample, there are many debilitating phenotypes of disease but not all PsA phenotypes include bone erosion [22]. It would be useful to have a biomarker that distinguishes between different disease phenotypes (for example, pro- gressors from nonprogressors). Such a biomarker would help inform the clinical decision of whether to treat symptoms only or to adopt a more aggressive treatment strategy in order to prevent radiographic progression. is limited to observational prospective cohort studies that used radiographic progression as a measure of joint de- struction and included patients with early-stage disease at baseline. Observational studies are considered inferior to randomised control trials due to the fact that results can be influenced by confounding factors such as a patient’s response to therapy. Biomarkers predictive of joint dam- age at baseline in a treatment-naïve patient may not be predictive after initiation of therapy due to the suppressive effect of the pharmacological intervention on inflamma- tory mediators. However, evidence derived from well- designed prospective cohort studies that follow patients longitudinally and correct for confounding factors is con- sidered important in the clinical decision-making process and is comparable with that provided by randomised con- trol trials [23, 24]. Limiting this approach, there is a pau- city of early cohort studies in PsA in which radiographic progression is measured. As disease activity and changes in tissue organisation may serve as surrogate measures of joint damage, we have included soluble biomarkers that correlated with these disease parameters in the PsA stud- ies reviewed. In addition, the potential opportunities afforded by new mass spectrometry-based protein meas- urement approaches will be highlighted. These methods The aim of this review is to identify previously published reports that include proteins predictive of joint damage at early disease time points. Joint damage is most definitively measured by radiographic progression. Hence this review Mc Ardle et al. Biomarkers Arthritis Research & Therapy (2015) 17:141 Page 5 of 12 was reported recently that a distinct family of auto- antibodies which recognise carbamylated antigens, the anti-carbamylated protein antibodies, are predictive of a severe disease course in early RA patients even after correction for RF and ACPA. It was demonstrated that patients who were positive for immunoglobulin G anti- carbamylated protein antibodies but negative for ACPA developed a more severe disease course compared with patients who were ACPA-positive only. Those who were positive for both anti-carbamylated protein anti- bodies and ACPA were comparable in severity [30]. Anti-carbamylated protein antibodies might thus prove a useful biomarker in RA, and their ability to predict joint damage warrants further research. are reaching clinical diagnostics application and have the potential to change dramatically the landscape of protein biomarkers. Cytokines and chemokines Cytokines play a major role in promoting joint damage and their association with radiographic progression has been investigated in several early RA cohort studies. It has been reported separately that elevated levels of inter- leukin (IL)-6, IL-16, IL-22, IL-33, chemokine ligand (CCL)11 and chemokine (C-X-C) motif ligand (CXCL)13 are predictive of radiographic progression [31–35]. The finding that IL-6 was associated with joint damage is controversial, however, and contradictory findings have been reported. Klein-Wieringa and colleagues demon- strated that elevated levels of IL-6 were not significantly correlated with radiographic progression in an early RA cohort [36]. This is in disagreement with Knudsen and colleagues, who demonstrated that IL-6 was a strong in- dependent predictor of radiographic progression [31]. These controversial findings might be explained in part by differences in study design. Klein-Wieringa and colleagues measured serum levels of IL-6 in a large RA cohort consisting of 253 patients. Patients enrolled in this study received three different treatment strategies. Radiographic progression was assessed over 4 years using the Sharp–van der Heijde scoring method. Multivariate regression analysis was used to find an association be- tween levels of IL-6 and disease progression. This model corrected for confounding factors including therapeutic intervention. In contrast, Knudsen and colleagues mea- sured plasma levels of IL-6 in a smaller RA cohort consist- ing of 51 patients and treatment change was permitted over the duration of the study. Radiographic progression was measured using the Larsen method. Levels of IL-6 were measured 13 times over 2 years and the mean con- centration (area under the curve) of IL-6 measurements over 24 months were used to associate levels of IL-6 with bone erosions observed at 12 and 24 months [31, 36]. Conversely, a study by van Leeuwen and colleagues dem- onstrated that elevated levels of plasma IL-6 were not pre- dictive of damage [37]. However, the IL-6 assay used during this study was less sensitive than that used by Markers of the acute phase response p p Inflammation in the synovium is reflected by a systemic inflammatory response. Increased erythrocyte sedimen- tation rate (ESR) and hepatocyte production of acute phase proteins including C-reactive protein (CRP) and acute phase serum amyloid A (A-SAA) are surrogate markers of this process. The acute phase response has been considered important in the context of RA as it mirrors synovial inflammation. CRP and the ESR have been shown to correlate with radiographic progression and these indices have been incorporated into composite scores typically used to predict damage. However, these are general markers of inflammation; they are not dis- ease specific. Furthermore, CRP and the ESR remain normal in a proportion of RA patients that may progress – and thus additional and more sensitive predictors are re- quired. One RA cohort study which incorporated patients with a range of disease durations showed that A-SAA cor- related with radiographic progression. It would be interest- ing to investigate the correlation of A-SAA with joint damage in an early cohort as this protein reflects not only systemic but also local inflammation [25]. Auto-antibodies A b d Auto-antibodies such as RF and ACPA are very useful prognostic markers in RA. It has previously been re- ported that patients positive for ACPA or RF are more likely to develop erosions compared with those who are negative for both. Additionally it was reported that those who were positive for ACPA and negative for RF were more likely to progress to a severe disease state com- pared with those who were RF-positive but ACPA- negative. Furthermore it was noted that patients positive for both ACPA and RF were at greatest risk of disease progression [26–28]. ACPA emerge as the most useful indicator of joint damage. Indeed, RF is quite limited in its ability to predict damage because this antibody is not disease specific. Moreover RF is considered useful only at early disease time points, and its predictive power is lost as disease progresses. In contrast, in addition to being more sensitive, ACPA are more disease specific and useful at both early and later disease time points [26, 29]. There exist, however, subgroups of patients who are susceptible to joint damage but who test nega- tive for both RF and ACPA. Since RF and ACPA have been proven very useful indicators of damage, the identi- fication of novel auto-antibodies may prove advanta- geous especially in patients who lack RF and ACPA. It Page 6 of 12 Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 year [41]. Clavel and colleagues confirmed the finding that VEGF was predictive of joint damage in early RA patients. Additional analysis revealed that VEGF levels at inclusion were correlated with initial Sharp scores and Sharp scores measured after 1 year. It was also demonstrated that levels of angiopoeitin-1 (a marker of angiogenesis) were predict- ive of damage at inclusion and after 1 year. This finding suggests that VEGF and angiopotietin-1 remain predica- tive at later disease stages after initiation of therapy [40]. Knudsen and colleagues [31]. The differential findings in relation to IL-6 highlight the impact of study design on reported findings in the literature. Single reports provide evidence that IL-16, IL-22, IL-33, CCL11 and CXCL13 retain prognostic capacity in early RA. It was interest- ing to note that CCL11 was associated with reduced radiographic progression and thus may have a protect- ive role during pathogenesis [33]. Calprotectin l Calprotectin is released from activated leukocytes that derive mainly from the inflamed synovium in RA pa- tients. A recent cohort study by Hammer and colleagues demonstrated that RA patients with higher baseline levels of calprotectin developed more severe radio- graphic damage after 10 years compared with patients with lower levels of calprotectin. Furthermore, calprotec- tin levels measured at baseline and at 10 years signifi- cantly correlated with radiographic damage after 10 years. The correlation observed for calprotectin was similar to that observed for CRP and ESR [39]. Taken together these results can lead to the conclusion that calprotectin is more specific and just as sensitive at pre- dicting joint damage as CRP and may provide valuable information in the context of a biomarker signature pre- dictive of joint damage. Adipokines Ad k Adipokines are cytokines produced by fat cells. These proteins are elevated in patients with RA, where they are thought to have potent immunomodulatory effects. A study by Rho and colleagues demonstrated that adipo- nectin, visfatin and leptin correlated with measures of radiographic progression. Adiponectin and visfatin were shown to correlate with increased radiographic progres- sion, whereas leptin was associated with reduced pro- gression [38]. Klein-Wieringa and colleagues confirmed the finding that adiponectin is predictive of radiographic progression in patients with early RA [36]. Auto-antibodies A b d Additional studies should be carried out to validate the finding that the aforementioned molecules independently correlate with radiographic progression. Products of collagen degradation Fragments released as a result of type I and type II colla- gen degradation – including C-terminal telopeptide of col- lagen (CTX)I and CTXII, collagen type II degradation product epitopes C2C and C1,2C as well as matrix pro- teins such as cartilage oligomatrix protein (COMP) – are reflective of bone and cartilage damage. Several studies have investigated the association of these molecules with radiographic progression in early RA patients. Three au- thors reported that elevated levels of CTXI and CTXII are associated with long-term radiographic progression in early RA patients [42–44]. What is more, Garnero and colleagues reported that CTXI and CTXII were even more predictive of joint damage compared with CRP and ESR [42]. Bakker and colleagues found that elevated baseline levels of C1,2C were associated with radiographic progres- sion after 1 year of treatment [45]. Additionally Verstappen and colleagues demonstrated that C2C significantly corre- lated with radiographic progression after 1 year and im- portantly remained predictive the following year [46]. Andersson and colleagues demonstrated that increments in COMP levels during a 3-month period following diag- nosis were predictive of joint damage at 1-year, 2-year and 5-year follow-up [47]. The culmination of positive findings makes these degradation products attractive candidates for a biomarker panel predictive of joint damage. Enzyme mediators of destruction The association of matrix metalloproteinase (MMPs), pro- teases that promote cartilage breakdown, with joint dam- age has also been investigated. Previous early RA cohort studies demonstrated that elevated levels of MMP-3 at baseline correlate significantly with radiographic progres- sion [48–50]. However, another study documented that serial, longitudinal measurements of MMP-3 fail to correl- ate with measures of joint damage [51]. It can thus be speculated that MMP-3 may be a useful predictive marker of joint destruction at disease onset prior to treatment. MMP-1 might also have prognostic utility and its associ- ation with joint damage has been assessed. Previously, ele- vated baseline levels of MMP-1 were demonstrated to significantly correlate with radiographic progression ob- served at 12 months [49]. It has also been reported that serial measurements of MMP-1 over a period of 18 Markers of angiogenesis h b l f The ability of angiogenic markers to predict joint dam- age in RA has been reported [41, 57]. Abnormalities in angiogenesis are more pronounced in PsA compared with RA and levels of VEGF and angiopotietin-2 have been reported to be higher during PsA relative to RA [56]. It is thus logical to conclude that these molecules could act as early markers of radiographic progression, but there is a lack of data in the literature to validate this hypothesis. Interestingly, the association of VEGF with active versus inactive disease and with changes in syn- ovial vascular morphology has been described [56–58]. Molecules that regulate bone turnover g Dalbeth and colleagues examined the association between soluble mediators of bone remodelling (receptor activator of nuclear factor-κB ligand, osteoprotegerin, wnt signalling pathway inhibitor-1, macrophage colony-stimulating fac- tor) with radiographic progression in PsA patients with established disease duration. A positive correlation be- tween macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand concentrations with radiographic progression was described [59]. Connolly and colleagues found that baseline levels of A-SAA were independently associated with 1-year radiographic pro- gression in PsA patients with long disease duration. A- SAA promotes the production of MMPs by fibroblast-like synoviocytes. A-SAA levels were demonstrated to correl- ate with MMP-1, MMP-3, MMP-13 and MMP/tissue in- hibitor of matrix metalloproteinases [25]. Since it has been demonstrated previously that MMP-1 and MMP-3 are as- sociated with radiographic progression in early RA, it could be speculated that an association between A-SAA and MMPs might correlate with radiographic progression in early PsA – this warrants further research. There are very few studies reporting the association of soluble cytokines with joint damage in PsA. However, cytokines that segregate patients with polyarthritis from those with oligoarticular disease have been identified. In an early PsA cohort, elevated levels of IL-1 were detected in the synovial fluid of polyarthritic patients compared with those with monoarthritis, suggesting that this protein is a marker of disease progression [53]. A Norwegian co- hort found that elevated levels of IL-12p40, interferon alpha, IL-15 and CCL3 could segregate PsA patients with polyarticular disease from those with oligoarticular disease [54]. Since patients with polyarthritis have more severe joint involvement, these molecules might drive progres- sion and would be interesting to investigate in the context of early PsA. Biomarkers of joint damage in psoriatic arthritis Cytokines The cytokine expression profile in the PsA synovium is relatively similar to that observed in RA [8]. IL-17 has emerged as an important cytokine in autoimmune dis- eases and this protein in synergy with tumour necrosis factor alpha contributes to pathogenesis in RA and PsA. It has previously been demonstrated that, compared with osteoarthritic controls, levels of IL-17 are elevated in the synovial tissue of patients with PsA and RA (no signifi- cant difference between the two). It was also demon- strated that in vitro stimulation of synovial tissue with IL-17 induced proteins involved in matrix turnover and cartilage destruction [52]. Markers of angiogenesis Angiogenesis is central to the development of RA syno- vitis and thus assessing levels of angiogenic markers in RA patients may prove useful for predicting joint damage. Vascular endothelial growth factor (VEGF) is thought to be the most important mediator of angiogenesis and there is good evidence to suggest that this molecule should be considered a candidate marker of joint damage in RA [40, 41]. A study by Ballara and colleagues demonstrated in early RA that elevated VEGF levels at inclusion signi- ficantly correlated with radiographic progression after 1 Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 Page 7 of 12 compared with levels in those who had less than 10 af- fected joints [56]. These results together suggest that S100A8/S100A9 may be associated with joint damage and that this marker might provide additional information in the context of a biomarker signature. months correlate significantly with measures of joint dam- age [51]. In contrast, Young-Min and colleagues found no correlation between levels of MMP-1 and joint damage in early RA patients [50]. It is certainly possible that differ- ences in study design and data analysis gave rise to these discrepancies, and the relationship between MMP-1 and joint damage warrants further investigation. Calgranulin (S100A8/S100A9) A study by Kane and colleagues demonstrated that ele- vated levels of calgranulin (S100A8/S100A9) correlated with measures of disease activity and markers of intra- articular inflammation (white blood cell counts). The study further demonstrated that treatment with metho- trexate resulted in a significant decrease in S100A8/ S100A9 levels. A significant reduction in swollen joint count, Richie articular index and Disease Activity Score was also observed after treatment, suggesting that S100A8/ S100A9 may also correlate with joint damage in PsA [55]. Additional evidence reporting an association between S100A8/S100A9 with progressive disease exists. A study by Aochi and colleagues demonstrated that S100A8/ S100A9 levels in PsA patients (disease duration not speci- fied) with more than 10 affected joints were higher An emerging mass spectrometry technology An emerging mass spectrometry technology The development of a protein biomarker identified in discovery experiments for clinical assay is both long and challenging [60]. The clinical validation of candidate biomarkers has traditionally relied on the development of antibody-based assays. Enzyme-linked immunosorbent assays (ELISAs) are one of the most widely used methods for clinical diagnostic protein biomarker mea- surements [61, 62]. Large numbers of candidate biomarkers are emerging and it has become apparent that an alternative technology is required to evaluate them in a time- and cost-effective Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 Page 8 of 12 manner [63, 64]. The relatively recent introduction of a multiple reaction monitoring (MRM) mass spectrometry platform for the measurement of peptides has provided the opportunity to develop multiplexed assays for simul- taneously measuring multiple candidate biomarkers and to progress them through the biomarker development pipeline [65]. There are a number of key drivers for the adoption of MRM as a viable alternative to the traditional antibody-based approach. The economics of MRM ana- lysis far outweigh methods requiring antibodies (western blot or ELISA). Good antibodies are relatively expensive and in some cases not always available for the proteins of interest. Even for proteins for which antibodies are avail- able, the length of time needed to optimise an assay using MRM mass spectrometry is much shorter relative to that using antibody-based protein detection [63, 64]. well to therapy compared with those who did not re- spond [75]. Luminex technology is another example of a multiplex platform for biomarker validation. This technology is based on polystyrene beads that are coated with specific capture antibodies and impregnated with dyes of differ- ent intensities. Interrogation of the beads with lasers re- sults in the identification of a bead and hence an analyte due to its unique spectral properties [76]. The luminex is cable of measuring proteins that span a low dynamic range (<103) which would not be detected by MRM. In addition to being a high-throughput assay, the sensitivity and specificity of the luminex technique is comparable with the ELISA [77, 78]. This technology is limited, how- ever, by availability of antibodies against proteins of interest, high cost and the quantity of sample required for analysis [78]. using antibody-based protein detection [63, 64]. MRM assays are developed for peptides released from proteolytically cleaved proteins. An emerging mass spectrometry technology A peptide that is unique to the protein of interest (a proteotypic peptide, gener- ally ranging from 7 to 25 amino acids in length) and that is routinely observed by the mass spectrometer is crucial to guaranteeing accurate detection of specific proteins [66]. By measuring only selected proteotypic peptides, the abundance of selected proteins can be definitively established [65, 67, 68]. In MRM mode, only those pep- tides of interest pass through the mass spectrometer by setting the first quadrupole to filter based on the known mass/charge ratio of the peptide. In the second quadru- pole the peptides are then fragmented and will produce fragments of known size. The third quadrupole is then used to filter these fragments, allowing them to pass to a detector. The prior knowledge of peptide sequences is used to direct the mass spectrometry in MRM mode. The triple-quadrupole mass spectrometer can be set to filter hundreds of peptides in the first quadrupole and thousands of peptide fragments (transitions) in the third quadrupole, thereby enabling many proteins to be mea- sured. Therefore, for example, in a typical analytical run (<30 minutes) it is possible to measure hundreds of peptides [69, 70], which is analogous to performing hundreds of western blot analyses or ELISAs within a 30-minute timeframe. Indeed the MRM approach has been implemented in several large-scale biomarker studies over the past couple of years. These assays have so far proved particularly useful in the field of toxicol- ogy and oncology [71–73]. This technique has not yet been exploited to its full potential in rheumatology, but evidence supporting its potential utility does exist. For example, an MRM assay was developed for CRP and this assay was then used to distinguish progressive from nonprogressive arthritic patients [74]. More recently, Ademowo and colleagues developed an MRM assay for a panel of 57 synovial tissue proteins. This assay was then used to predict PsA patients who responded Conclusions In relation to what has been shown to date, considerably more candidate biomarkers have been identified in RA compared with PsA (Table 2). No single biomarker has been validated as a potent predictor of joint damage and it is well recognised that a multi-biomarker panel is needed to compensate for the heterogeneity between in- dividuals. A multi-biomarker panel incorporating 12 serum proteins has been shown to accurately reflect dis- ease activity in RA [18]. This panel of proteins may be measured using a blood-based test referred to as a multi-biomarker disease activity (MBDA) test. Concen- trations of the 12 biomarkers are incorporated into an algorithm that provides a low, moderate or high disease activity score as an output [18]. A recent study has shown that the MBDA test correlates well with measures of CRP, but there may also be discordance between levels of CRP and the MBDA test. In patients with both low levels of CRP and a low MBDA score, radiographic progression is infrequent. In contrast, in patients with low levels of CRP but a high MBDA score, a significant proportion developed radiographic progression during 1 year of follow-up. Levels of the multi-biomarker panel (which include CRP) thus better predict radiographic progression than CRP alone [79]. In a further study the MBDA test has been shown to predict risk of radio- graphic progression and outperform CRP as a predictive biomarker in the SWEFOT cohort trial. In this trial, only patients with a high MBDA score were at risk of devel- oping radiographic progression. In contrast, a substantial proportion of patients with low, moderate and high levels of CRP were at risk of developing joint damage [80]. Finally, the ability of the MBDA score to predict joint damage has been demonstrated in an additional trial that included 163 patients from the Leiden Early Arthritis Cohort. The MBDA score and the Disease Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 Page 9 of 12 Table 2 Candidate biomarkers of joint damage in rheumatoid arthritis and psoriatic arthritis Candidate biomarker Evidence for role in inflammatory arthritis Use Inflammatory proteins C-reactive protein Opsonisation and compliment activation RA Calprotectin (S100A12) Ca2+ binding protein released upon phagocyte activation, important intracellular and extracellular roles RA/PsA Calgranulin (S100A8/S100A9) Ca2+ binding protein with pleotropic effects. Conclusions Regulates myeloid derived cells PsA A-SAA Promotes the production of MMPs RA/PsA Cytokines IL-1 Promotes activation of keratocytes, endothelial cells, chondrocytes and osteoclasts. Promotes the production of proinflammatory cytokines PsA IL-6 Promotes neutrophil chemotaxis and production of proinflammatory cytokines, induces an acute phase response RA IL-13 Promotes antibody production by B cells RA IL-15 Induces T cell proliferation and B cell differentiation. Recruits memory T cells to the synovium and induces TNFα production PsA IL-16 Promotes chemotaxis of CD4+ T cells, monocytes and eosinophils. Modulates T-cell activation RA IL-22 Induces proliferation of fibroblasts and production of MCP-1 (monocyte chemokine) RA IL-33 Promotes chronic inflammatory response RA Chemokines CCL3 Lymphocyte, monocyte, basophil, eosinophil chemoattractant PsA CCL11 Eosinophil chemoattractant PsA CXCL13 B-cell chemoattractant RA Adipokines Adiponectin Induces IL-6 and MMP-1 production by SLFs. Promotes IL-6, TNFα and MCP-1 production in chondrocytes RA Visfatin Role unclear, thought to modulate inflammation RA Markers of angiogenesis VEGF Potent inducer of angiogenesis and vascular permeability RA/PsA Angiopotietin-1 Promotes angiogenesis (growth of new blood vessels) RA Angiopotietin-2 Promotes angiogenesis PsA Auto-antibodies Rheumatoid factor Forms immune complexes, promotes complement activation and formation of rheumatoid nodules RA Anti-CCP Promotes complement activation RA Anti-Carp Bind homocitrulline containing proteins RA Enzyme mediators of destruction MMP-1 Degrades collagen RA MMP-3 Degrades collagen RA Regulators of bone remodelling RANKL Induces osteoclast bone destruction PsA M-CSF Induces aggressive phenotype in macrophages PsA Products of collagen degradation COMP Cartilage oligomatrix protein RA CTXI C-terminal telopeptide of collagen type I RA CTXII C-terminal telopeptide of collagen type I RA C1,2C Collagen type II degradation product RA C2C Collagen type II degradation product RA Candidate biomarkers predictive of joint damage in RA and PsA have been identified in the literature. These include inflammatory proteins, cytokines, chemokines, adipokines, markers of angiogenesis, auto-antibodies, enzyme mediators of destruction, molecules that regulate bone turnover and products of collagen degradation. For references see text. Candidate biomarkers predictive of joint damage in RA and PsA have been identified in the literature. These include inflammatory proteins, cytokines, chemokines, adipokines, markers of angiogenesis, auto-antibodies, enzyme mediators of destruction, molecules that regulate bone turnover and products of collagen degradation. For references see text. A-SAA acute-phase serum amyloid A; anti-Carp, anti-carbamylated protein antibodies; CCL, chemokine ligand; CCP, cyclic citrullinated peptide; CXCL, chemokine (C-X-C) motif ligand; IL, interleukin; M-CSF, macrophage colony stimulating factor; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; PsA, psoriatic arthritis; RANKL, receptor activator of nuclear factor-κB ligand; SLF, synovium-like fibroblasts; TNFα, tumour necrosis factor alpha; VEGF, vascular endothelial growth factor Abbreviations ACPA: Anti-citrullinated protein antibodies; A-SAA: Acute phase serum amyloid A; CCL: Chemokine ligand; COMP: Cartilage oligomatrix protein; CRP: C-reactive protein; CTX: C-terminal telopeptide of collagen; CXCL: Chemokine (C-X-C) motif ligand; ELISA: Enzyme-linked immunosorbent assay; ESR: Erythrocyte sedimentation rate; IL: Interleukin; MBDA: Multi-biomarker disease activity; MMP: Matrix metalloproteinase; MRM: Multiple reaction monitoring; PsA: Psoriatic arthritis; RA: Rheumatoid arthritis; RF: Rheumatoid factor; VEGF: Vascular endothelial growth factor. Conclusions A-SAA acute-phase serum amyloid A; anti-Carp, anti-carbamylated protein antibodies; CCL, chemokine ligand; CCP, cyclic citrullinated peptide; CXCL, chemokine (C-X-C) motif ligand; IL, interleukin; M-CSF, macrophage colony stimulating factor; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; PsA, psoriatic arthritis; RANKL, receptor activator of nuclear factor-κB ligand; SLF, synovium-like fibroblasts; TNFα, tumour Table 2 Candidate biomarkers of joint damage in rheumatoid arthritis and psoriatic arthritis Candidate biomarker Evidence for role in inflammatory arthritis Candidate biomarkers predictive of joint damage in RA and PsA have been identified in the literature. These include inflammatory proteins, cytokines, chemokines, adipokines, markers of angiogenesis, auto-antibodies, enzyme mediators of destruction, molecules that regulate bone turnover and products of collagen degradation. For references see text. A-SAA acute-phase serum amyloid A; anti-Carp, anti-carbamylated protein antibodies; CCL, chemokine ligand; CCP, cyclic citrullinated peptide; CXCL, chemokine (C-X-C) motif ligand; IL, interleukin; M-CSF, macrophage colony stimulating factor; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; PsA, psoriatic arthritis; RANKL, receptor activator of nuclear factor-κB ligand; SLF, synovium-like fibroblasts; TNFα, tumour necrosis factor alpha; VEGF, vascular endothelial growth factor Candidate biomarkers predictive of joint damage in RA and PsA have been identified in the literature. These include inflammatory proteins, cytokines, chemokines, adipokines, markers of angiogenesis, auto-antibodies, enzyme mediators of destruction, molecules that regulate bone turnover and products of collagen degradation. For references see text. A-SAA acute-phase serum amyloid A; anti-Carp, anti-carbamylated protein antibodies; CCL, chemokine ligand; CCP, cyclic citrullinated peptide; CXCL, chemokine (C-X-C) motif ligand; IL, interleukin; M-CSF, macrophage colony stimulating factor; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; PsA, psoriatic arthritis; RANKL, receptor activator of nuclear factor-κB ligand; SLF, synovium-like fibroblasts; TNFα, tumour necrosis factor alpha; VEGF, vascular endothelial growth factor Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 Page 10 of 12 Page 10 of 12 Mc Ardle et al. Arthritis Research & Therapy (2015) 17:141 Activity Score in 28 joints–CRP score were assessed as predictors of radiographic progression. The study found that patients with low MBDA had significantly less radiographic progression than patients who meet Dis- ease Activity Score in 28 joints–CRP European League Against Rheumatism defined remission. It was also shown that patients with a high MBDA score were six times more likely to develop radiographic progression compared with patients with a high Disease Activity Score in 28 joints–CRP score, who were only twice as likely to progress [81]. Conclusions Research Centre is funded by the Programme for Research in Third level Institutions, as administered by the Higher Education Authority of Ireland. The authors appreciate the technical support from Aisha Q Butt while the manuscript was being completed. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 21. Scott DL, Smith C, Kingsley G. Joint damage and disability in rheumatoid arthritis: an updated systematic review. Clin Exp Rheumatol. 2003;21:S20–7. 21. Scott DL, Smith C, Kingsley G. Joint damage and disability in rheumatoid arthritis: an updated systematic review. Clin Exp Rheumatol. 2003;21:S20–7. 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Ultrasonography, magnetic resonance imaging, radiography, and
https://openalex.org/W3037462407	https://hal.archives-ouvertes.fr/hal-02879916/file/PMP.BMCMCB_2020.pdf	English	null	Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease	BMC molecular and cell biology	2,020	cc-by	12,850	Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease Pierre Laville, Sandrine Fartek, Natacha Cerisier, Delphine Flatters, Michel Petitjean, Leslie Regad To cite this version: Pierre Laville, Sandrine Fartek, Natacha Cerisier, Delphine Flatters, Michel Petitjean, et al.. Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease. BMC Molecular and Cell Biology, 2020, 21, pp.46. 10.1186/s12860-020-00290-1. hal-02879916 Laville, Sandrine Fartek, Natacha Cerisier, Delphine Flatters, Michel Petitjean, Leslie Regad To cite this version: Pierre Laville, Sandrine Fartek, Natacha Cerisier, Delphine Flatters, Michel Petitjean, et al.. Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease. BMC Molecular and Cell Biology, 2020, 21, pp.46. 10.1186/s12860-020-00290-1. hal-02879916 Distributed under a Creative Commons Attribution 4.0 International License © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. HAL Id: hal-02879916 https://hal.science/hal-02879916v1 Submitted on 10 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License (2020) 21:46 Laville et al. BMC Molecular and Cell Biology https://doi.org/10.1186/s12860-020-00290-1 BMC Molecular and Cell Biology Open Access Abstract Background: Drug resistance is a severe problem in HIV treatment. HIV protease is a common target for the design of new drugs for treating HIV infection. Previous studies have shown that the crystallographic structures of the HIV- 2 protease (PR2) in bound and unbound forms exhibit structural asymmetry that is important for ligand recognition and binding. Here, we investigated the effects of resistance mutations on the structural asymmetry of PR2. Due to the lack of structural data on PR2 mutants, the 3D structures of 30 PR2 mutants of interest have been modeled using an in silico protocol. Structural asymmetry analysis was carried out with an in-house structural-alphabet-based approach. Results: The systematic comparison of the asymmetry of the wild-type structure and a large number of mutants highlighted crucial residues for PR2 structure and function. In addition, our results revealed structural changes induced by PR2 flexibility or resistance mutations. The analysis of the highlighted structural changes showed that some mutations alter protein stability or inhibitor binding. Conclusions: This work consists of a structural analysis of the impact of a large number of PR2 resistant mutants based on modeled structures. It suggests three possible resistance mechanisms of PR2, in which structural changes induced by resistance mutations lead to modifications in the dimerization interface, ligand recognition or inhibitor binding. Keywords: HIV-2 protease, Drug-resistance mutations, Structural asymmetry, Structural alphabet studies have shown that the natural resistance of HIV-2 to protease inhibitors (PIs) can be partially explained by sub- stitutions located within the PR2 pocket. These substitu- tions induce structural changes in pocket residues, modifying pocket properties and the internal interactions between PR2 and PIs, thus altering PI binding [3–10]. Other studies have suggested that substitutions between PR1 and PR2 could modify PI binding by altering the tran- sition between the open and closed forms involved in lig- and binding [11, 12]. Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease Pierre Laville, Sandrine Fartek, Natacha Cerisier, Delphine Flatters, Michel Petitjean and Leslie Regad* Background Proteases (PRs) are an important therapeutic target in the treatment of HIV-1 and HIV-2 infections because of their indispensable role in Gag and Gag-Pol polyprotein hy- drolysis during the viral maturation process [1]. Currently, nine drugs targeting PR for the treatment of HIV-1 have been approved for clinical use by the Food and Drug Administration (FDA), only three of which (darunavir (DRV), saquinavir (SQV), and lopinavir (LPV)) are effect- ive in the treatment of HIV-2 infection [1, 2]. Some In addition to the natural resistance of HIV-2 to most HIV-1 PIs, HIV-2 can evolve to achieve drug resistance through the accumulation of mutations on its PR. How- ever, few studies conducted to date provide information * Correspondence: leslie.regad@u-paris.fr Université de Paris, BFA, UMR 8251, CNRS, ERL U1133, Inserm, F-75013 Paris, France * Correspondence: leslie.regad@u-paris.fr Université de Paris, BFA, UMR 8251, CNRS, ERL U1133, Inserm, F-75013 Paris, France * Correspondence: leslie.regad@u-paris.fr Université de Paris, BFA, UMR 8251, CNRS, ERL U1133, Inserm, F-75013 Paris, France For example, the I82F mutation has been observed in the presence of ritonavir (RTV) and indinavir (IDV) in some studies [14, 16, 19], while the I54M mutation has appeared under treatment with amprenavir (APV) [16], nelfinavir (NFV) [14], and IDV [19]. Phenotypic susceptibility assays confirmed that the I82F mutation confers resistance to IDV as well as NFV and LPV [16]. In contrast, Raugi et al. [19] found that this mutation does not increase resistance to LPV but causes hypersus- ceptibility to both DRV and SQV. In addition, some mu- tations can appear together to confer high resistance to several PIs [7, 15, 17, 19, 24, 25]. For example, the I54M and I82F mutations confer cross-resistance to all PIs. The V62A and L99F mutations confer cross-resistance to three PIs (IDV, NFV, and LPV) [16]. As no tridimen- sional (3D) structure of the PR2 mutant is available in the Protein Data Bank (PDB [26]), no structural analysis was performed to study the mechanisms explaining HIV-2 resistance induced by these mutations. However, knowledge of these mechanisms leading to PR2 resist- ance is important for designing new PR2 inhibitors. PR2 is a homodimer of 99-residue monomers. The interaction between the two monomers occurs through the Nter, catalytic, and Cter regions. Diverse ligands (pep- tide substrates with different amino acid sequences and structurally different inhibitors) bind to the PR2 central binding site. The comparison of the unbound and PI- bound PR2 structures showed that the flap region under- goes large structural changes upon ligand binding. In the unbound form, the flap region adopts an open conform- ation allowing ligand entry. Upon ligand binding, the flap region recloses over the central binding pocket [1]. In this work, we explored the structural effects of some drug resistance mutations of PR2 by comparing the structural asymmetry of the wild-type and drug- resistant mutants of PR2. The studied PR2 drug- resistant mutants harbored one, two or three mutations. As no structural data are available for these PR2 mu- tants, we constructed their 3D structures using molecu- lar modeling as in [10]. We then detected structural asymmetry (i.e., positions exhibiting different local con- formations in the two PR2 chains) in the wild-type and mutant structures using our structural-alphabet-based approach [31]. The comparison of the structural asym- metry of wild-type and mutant structures highlighted three possible mechanisms that could explain PR2 resist- ance to PIs. The analyses of the crystallographic structures of PR2 have shown that the two monomers do not exhibit the same global and local conformations, indicating that in crystallographic structures, PR2 exhibits structural asym- metry [27–32]. This structural asymmetry is translated by slightly different orientation of its two monomers, quanti- fied by a two-fold axis of 178.20° to 179.80° and a root mean square deviation (RMSD) of 0.35 to 1.02 Å [27–30, 33]. The largest deviations between the two monomers are located in the tail, elbow, and flap regions [27–31, 33]. Using a structural-alphabet-based approach, 31% of PR2 positions were identified as structurally asymmetric (i.e., Page 2 of 15 Page 2 of 15 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 exhibiting different local conformations in their two chains), among which 75% were located outside of the PI- binding pocket [31, 33]. In a recent study, we explored the structural asymmetry of unbound PR [33]. Its two mono- mers exhibit a Cα-RMSD value of 0.53 Å, which is smaller than that computed on the bound PR2 [27–30]. Our structural-alphabet-based approach highlighted that 35% of unbound PR2 positions are asymmetric. In the un- bound and bound structures, the asymmetric positions are distributed throughout the structure, particularly in the interface region and in the flap, fulcrum, elbow, and α- helix regions and the binding site [31, 33]. Thus, the crys- tallographic PR2 structure exhibits structural asymmetry in its backbone, and this property is also found in the un- bound structure. These results highlighted the asymmetric properties of the crystallographic structures of PR2, which are not caused by ligand binding alone. Indeed, proteins are dynamic objects that adjust the positions of their atoms to respond to different events, such as partner bind- ing. In the case of the crystallographic PR2 structures, the structural asymmetry results from crystal packing [27, 28, 30, 33], protein dimerization [31], and ligand binding [28, 29, 31]. Different studies have differentiated the PR2 asymmetry induced by ligand binding that is important for ligand recognition and binding [28, 29, 31] to struc- tural asymmetry corresponding to an intrinsic factor allowing the structural deformation of the target [34–36]. For example, Mulichak et al. [28] showed that the binding of a peptidic inhibitor in the PR2 specifically induces a move of the region 79–82 of chain B allowing inhibitor binding. about mutations conferring resistance to PIs. Genome sequencing studies of HIV-2 from different HIV-2- infected patients have described the selection of some mutations that confer drug resistance in HIV-2 [7, 13– 22]. These resistance mutations were subsequently con- firmed using phenotypic susceptibility assays [7, 15–17, 19, 23]. Several studies have shown that a single muta- tion can confer resistance to one or several PIs, such as V47A, I50V, I54M, I82F, I84V, and L90M [7, 15, 17, 19, 24]. However, some studies have led to contradictory re- sults. Quantification of PR2 structural deformation induced by drug resistance mutations We focused on a set of 30 drug-resistant mutants containing from one to three mutations (Fig. 1a). The 3D structure of each mutant was built using FoldX software [39] with five replications (see Methods), Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Page 3 of 15 Fig. 1 Presentation of drug-resistant mutants. a List of the 30 drug-resistant mutants with their different mutations. b Localization of the mutations observed in the 30 drug-resistant mutants onto the 3D structure of wild-type PR2. PR2 is displayed in cartoon mode and colored according to the 13 extracted PR2 regions defined in [32, 37, 38]. Each PR2 region is colored as follows: the Nter and Cter regions in grey, the R1 region in dark blue, the fulcrum region in green, the catalytic region in purple, the R2 region in orange, the elbow region in blue, the flap region in magenta, the cantilever region in yellow, the R3 region in pink, the wall region in cyan, the R4 region in brown, and the α-helix region in red. Positions, where drug-resistance mutations occur, are displayed in stick mode. c Limits of the 13 structural and functional regions extracted from PR2. The 20 pocket residues were highlighted in red Fig. 1 Presentation of drug-resistant mutants. a List of the 30 drug-resistant mutants with their different mutations. b Localization of the mutations observed in the 30 drug-resistant mutants onto the 3D structure of wild-type PR2. PR2 is displayed in cartoon mode and colored according to the 13 extracted PR2 regions defined in [32, 37, 38]. Each PR2 region is colored as follows: the Nter and Cter regions in grey, the R1 region in dark blue, the fulcrum region in green, the catalytic region in purple, the R2 region in orange, the elbow region in blue, the flap region in magenta, the cantilever region in yellow, the R3 region in pink, the wall region in cyan, the R4 region in brown, and the α-helix region in red. Positions, where drug-resistance mutations occur, are displayed in stick mode. c Limits of the 13 structural and functional regions extracted from PR2. The 20 pocket residues were highlighted in red RMSDaa (Bartlett-test p-value = 2.10−11): triple mu- tants showed more conserved structures than the other types of mutants. Quantification of PR2 structural deformation induced by drug resistance mutations Thus, compared to single or double mutants, the insertion of three mutations in- duced more structural diversity relative to the wild- type structure, but the five modeled mutant structures were more similar to each other. resulting in a set of 150 mutant models. To compare the mutant and wild-type structures, we computed the all-atom RMSD, denoted as RMSDaa, between the mutant structure and the three minimized wild-type structures (3EBZmini). The five structures of each mu- tant could exhibit large RMSDaa (i.e., different confor- mations) (Additional file 1: Figure S1). As expected, single mutants (i.e., those with only one mutation) ex- hibited a smaller average RMSDaa than the other mu- tants, indicating that introducing a single mutation induces less structural change than introducing sev- eral mutations (Additional file 1: Figure S1). In addition, there was a link between the number of mu- tations and structural diversity in terms of RMSDaa. Indeed, the three types of mutants (single, double and triple) did not exhibit the same variability in terms of Characterization of structural asymmetry in the PR2 mutant set On average, a mutant structure contains 31.8 ± 4.4 asym- metric positions, which is close to the number of asym- metric positions in the three 3EBZmini structures. The five structures of a given mutant do not always have the same number of asymmetric positions and can exhibit few com- mon asymmetric positions, such as the G48V mutation (m5); see Additional file 5: Figure S5. The variability of the number of asymmetric positions per mutant does not de- pend on the number of mutations (Bartlett test p-value = 0.14). As expected, a link was found between variability in terms of RMSDaa and both (i) the variability in terms of the number of asymmetric positions (Pearson correlation coefficient = 0.73) and (ii) the number of common asymmetric positions between the five mutant structures (Pearson coefficient between the standard deviation of the RMSDaa value for the 5 mutant conformations and the number of common asymmetric positions = −0.65). Thus, the mutants exhibiting greater diversity in terms of RMSDaa corresponded to the mutants showing five struc- tures exhibiting different numbers of asymmetric positions with few common asymmetric positions. A total of 18 positions did not exhibit the same asym- metry status in the three 3EBZmini structures (Fig. 2a). These 18 positions were not more flexible (in terms of B-factor value) or more accessible (in terms of accessible surface area (ASA) values) residues relative to other po- sitions (Kruskal-Wallis test p-values = 0.25 and 0.46, Additional files 3 and 4: Figures S3 and S4). This sug- gests that the asymmetric variability of these positions Fig. 2 Localization of asymmetric and symmetric positions in the wild-type and mutant structure. a 3EBZmini structure colored according to residue asymmetric behavior in the three wild-type structures: the 51 residues that are symmetric in the three 3EBZmini structures are colored blue, the 27 residues asymmetric in the three 3EBZmini structures are colored red, and the 18 residues exhibiting different asymmetric statuses in the three 3EBZmini structures are colored yellow. Characterization of structural asymmetry in the three 3EBZmini structures does not result from the intra-flexibility of PR2. How- ever, we have previously shown that these 18 positions exhibit different conformations in the 18 available struc- tures of PR2 complexed with different ligands [32]. Thus, these positions could modify their local conform- ation to adapt to different ligands. We determined the structural symmetric and asymmet- ric positions in the three 3EBZmini structures and their location in the 13 PR2 regions defined in [32, 37, 38] (Additional file 2: Figure S2). Half of the positions (53% of positions) were detected as symmetric (i.e., showing similar local conformations in the two chains in the three 3EBZmini structures). The entire PR2 structure was sampled, particularly the fulcrum, flap, and canti- lever regions (Fig. 2a). Thus, the conserved local con- formation in both monomers at these positions is important for PR2, particularly for pocket residues 28, 30, 53, 81 and 82, which could be important for ligand binding. The three 3EBZmini structures contained be- tween 34 and 38 asymmetric positions, with 28% of the positions showing asymmetry in the three 3EBZmini structures. These positions were located throughout the structure, particularly in the α-helix and cantilever re- gions (Fig. 2a). This asymmetry conservation suggests an important role of these positions, particularly for the elbow and flap positions (positions 40–42, 50, 51, 58), which could be important for PR2 deformation. Detection of structural asymmetry in the wild-type and mutant structures of PR2 To explore the structural effects of the studied drug resistance mutations, we compared the structural asymmetry in the three 3EBZmini structures and the 150 modeled mutant structures using an approach based on a structural alphabet [31]. Page 4 of 15 Page 4 of 15 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Laville et al. BMC Molecular and Cell Biology Characterization of structural asymmetry in the PR2 mutant set b 3EBZmini structure colored according to residue asymmetric status in the mutant set: the 25 residues that are symmetric in the 150 mutants are blue, the 25 residues that are overrepresented in terms of asymmetry in the mutant set and are asymmetric in the three 3EBZmini structures are red, the 11 residues that are overrepresented in terms of asymmetry in the mutant set and are symmetric in the three 3EBZmini structures are orange, and the 2 residues that are asymmetric in the three 3EBZmini structures and are not overrepresented in the mutant set are yellow. Additional file 2: Figure S2 lists the positions of each type in the wild-type and mutant sets and their distributions in the 13 regions Fig. 2 Localization of asymmetric and symmetric positions in the wild-type and mutant structure. a 3EBZmini structure colored according to residue asymmetric behavior in the three wild-type structures: the 51 residues that are symmetric in the three 3EBZmini structures are colored blue, the 27 residues asymmetric in the three 3EBZmini structures are colored red, and the 18 residues exhibiting different asymmetric statuses in the three 3EBZmini structures are colored yellow. b 3EBZmini structure colored according to residue asymmetric status in the mutant set: the 25 residues that are symmetric in the 150 mutants are blue, the 25 residues that are overrepresented in terms of asymmetry in the mutant set and are asymmetric in the three 3EBZmini structures are red, the 11 residues that are overrepresented in terms of asymmetry in the mutant set and are symmetric in the three 3EBZmini structures are orange, and the 2 residues that are asymmetric in the three 3EBZmini structures and are not overrepresented in the mutant set are yellow. Additional file 2: Figure S2 lists the positions of each type in the wild-type and mutant sets and their distributions in the 13 regions Fig. 2 Localization of asymmetric and symmetric positions in the wild-type and mutant structure. a 3EBZmini structure colored according to residue asymmetric behavior in the three wild-type structures: the 51 residues that are symmetric in the three 3EBZmini structures are colored blue, the 27 residues asymmetric in the three 3EBZmini structures are colored red, and the 18 residues exhibiting different asymmetric statuses in the three 3EBZmini structures are colored yellow. Characterization of structural asymmetry in the PR2 mutant set This indicates that the resistance mutations do not affect the structural symmetry of these positions, particularly the mutations occurring at some of these symmetric positions (K7R, V10I, V71I, I82F, and I82L). These positions were frequent in the fulcrum, flap, and cantilever regions and absent in the Nter, Cter, elbow, R3, and R4 regions (Fig. 2b). Five of them (28, 30, 53, 81, 82) were located in the binding site, confirming the important role of these positions in ligand binding. In the mutant set, 36 positions correspond to overrep- resented asymmetric positions and were denoted as ORa- sym positions. Their asymmetry does not arise at random. These ORasym positions are located throughout the structure except in the R1 and catalytic regions (Fig. 2b). These positions did not consist of more flexible (in terms of B-factor) or exposed (in terms of ASA) residues on average than other asymmetric positions (T-test p- values = 0.40 and 0.53, respectively, additional files 3 and 4: Figures S3 and S4). Seventy percent of the ORasym po- sitions were also asymmetric in the three 3EBZmini struc- tures. These positions were particularly common in the α-helix, flap, cantilever, and fulcrum regions (Fig. 2b). Thus, the studied drug resistance mutations do not modify the structural asymmetry of these positions, re- inforcing the important role of the structural asymmetry of these positions. These overrepresented asymmetric positions could be important for PR2 structure or activ- ity, particularly the four residues belonging to the dimerization region (4, 5, 97, and 98) and the five pocket residues (23, 32, 47, 50, 80). To highlight the changes in asymmetry that are puta- tively induced by resistance mutations, we studied the conservation and location of structural changes in all structures containing at least one of the nine mutations observed in multiple mutants (K7R, I46V, I54M, V62A, V71I, I82F, I84V, L90M, and L99F); see Fig. 4. We noted that some changes in asymmetry occurred in all mutants exhibiting a given mutation. For example, a structural change at position 83 was observed in all mutants exhi- biting the I82F or I84V mutation. The location of con- served structural changes in the PR2 structures allowed us to differentiate two types of changes in asymmetry: those occurring far from a mutated residue and those occurring close to a mutated residue, which were puta- tively induced by mutation. The remaining 11 ORasym positions were symmetric in the three 3EBZmini structures. Characterization of structural asymmetry in the PR2 mutant set b 3EBZmini structure colored according to residue asymmetric status in the mutant set: the 25 residues that are symmetric in the 150 mutants are blue, the 25 residues that are overrepresented in terms of asymmetry in the mutant set and are asymmetric in the three 3EBZmini structures are red, the 11 residues that are overrepresented in terms of asymmetry in the mutant set and are symmetric in the three 3EBZmini structures are orange, and the 2 residues that are asymmetric in the three 3EBZmini structures and are not overrepresented in the mutant set are yellow. Additional file 2: Figure S2 lists the positions of each type in the wild-type and mutant sets and their distributions in the 13 regions Page 5 of 15 Page 5 of 15 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 mutant varied from 6 (mutant m2) to 36 (mutant m5) and did not depend on the number of mutations (P-value of the Kruskal-Wallis test = 0.55). Figure 3 presents the network connecting a mutant with its asymmetric positions. We observed that some changes in asymmetry occurred at positions connected with many mutations (which correspond to central nodes in the network), while others were connected to few mutations (which correspond to external nodes in the network, Fig. 3). For example, changes in asymmetry at positions 40, 41, 33, 18, and 98 were observed in more than 20 mutants (Fig. 3). These changes in asymmetry were not specific to certain mutations, suggesting that they were not induced by mutations. In contrast, structural backbone asymmetry at posi- tions 6 and 78 was observed only in mutants I54M/ I84V (m17) and K7R/I46V/L99F (m26), respectively, while such asymmetry at position 62 was observed in mutants I84V (m12), G48V (m5), and I84V/L90M (m23). The loss of structural asymmetry at positions 12, 64, and 75 was only observed in mutant G48V (m5), but the five structures of these mutants did not exhibit this loss. To characterize the structural asymmetry in the mu- tant set, we then computed the asymmetry occurrence (AO) for each position (i.e., the number of mutant struc- tures exhibiting asymmetry for a considered position). A total of 26% of the positions that were symmetric in all mutant structures were also symmetric in the three 3EBZmini structures. Asymmetric changes occurring far from mutated residues and, thus, putatively not related to mutations and, thus, putatively not related to mutations Figure 4 shows that the nonspecific changes in asymmetry occurred at positions 40 and 41 in most mutants, but they were located far from mutated residues. As previously assumed, the high frequency of these two changes in asymmetry suggests that they are not induced by a mutation. This was con- firmed by the fact that they occurred at two exposed residues located among the elbow residues (Add- itional file 4: Figure S4). Thus, the loss of asymmetry observed at positions 40 and 41 could be induced by residue flexibility. In the mutants with K7R, I46V, I54M, I84V, or L99F mutations, changes in Characterization of structural asymmetry in the PR2 mutant set In addition, two positions (40 and 41) were asymmetric in the three 3EBZmini structures and were not overrepresented in the mutant set. Among these 13 positions, four were close (44 and 80) or corresponded (47 and 90) to mutated positions. Thus, these drug resistance mutations could be respon- sible for the changes in asymmetry at these mutated po- sitions and their nearby residues, and they could modify the asymmetry of more distant residues. Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Link between drug resistance mutations and changes in asymmetry occurring in mutants The edges are colored according to whether the change in asymmetry occurred at a position located in the binding pocket (in red) or outside of the binding pocket (in gray) Fig. 3 Network summarizing the link between the 30 drug-resistant mutants and changes in asymmetry. In this network, white square nodes correspond to mutants, and colored nodes (square and circle) correspond to positions where a change in asymmetry occurs. Positions are colored according to the 13 regions extracted from the PR2 structure. See Fig. 1b for the region color legend. The shape of position nodes indicates the type of change in asymmetry occurring at the studied position: the circular nodes indicate asymmetry and the square nodes indicate the loss of asymmetry. This network connects a mutant to a position if the position presents a change in asymmetry in at least one of its five structures. The edge thickness is proportional to the number of mutant structures exhibiting the change (ranging from 1 to 5). The edges are colored according to whether the change in asymmetry occurred at a position located in the binding pocket (in red) or outside of the binding pocket (in gray) changes in asymmetry that occurred at position 33 or 8 and mutations I54M and L90M. asymmetry at positions 40 and 41 were accompanied by the loss of asymmetry at positions 18, 21, 58, 59, and 60 (Fig. 4). As these positions are located close to flexible residues 40 and 41, we suggest that these changes in asymmetry could be induced by the changes in asymmetry at positions 40 and 41. The nine mutants with the I54M mutation also presented a loss of asymmetry at position 33, which was also observed in all mutants exhibiting either the I84V or L90M mutation. Although this change in asymmetry was specific to certain mutations, it was not located close to the mutated residues (Fig. 4). The seven mutants containing the L90M mutation exhibited asymmetry at position 8, which was located far from the mutated residue 90 (Fig. 4). Thus, it is difficult to reach a conclusion regarding the link between the Link between drug resistance mutations and changes in asymmetry occurring in mutants For each mutant, we determined how many of its five structures exhibited a change in asymmetry at each position relative to the three wild-type structures. The number of changes in asymmetry observed for each Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Page 6 of 15 (2020) 21:46 Fig. 3 Network summarizing the link between the 30 drug-resistant mutants and changes in asymmetry. In this network, white square nodes correspond to mutants, and colored nodes (square and circle) correspond to positions where a change in asymmetry occurs. Positions are colored according to the 13 regions extracted from the PR2 structure. See Fig. 1b for the region color legend. The shape of position nodes indicates the type of change in asymmetry occurring at the studied position: the circular nodes indicate asymmetry and the square nodes indicate the loss of asymmetry. This network connects a mutant to a position if the position presents a change in asymmetry in at least one of its five structures. The edge thickness is proportional to the number of mutant structures exhibiting the change (ranging from 1 to 5). The edges are colored according to whether the change in asymmetry occurred at a position located in the binding pocket (in red) or outside of the binding pocket (in gray) Fi 3 N k i i h li k b h 30 d i d h i I hi k hi d d Fig. 3 Network summarizing the link between the 30 drug-resistant mutants and changes in asymmetry. In this network, white square nodes correspond to mutants, and colored nodes (square and circle) correspond to positions where a change in asymmetry occurs. Positions are colored according to the 13 regions extracted from the PR2 structure. See Fig. 1b for the region color legend. The shape of position nodes indicates the type of change in asymmetry occurring at the studied position: the circular nodes indicate asymmetry and the square nodes indicate the loss of asymmetry. This network connects a mutant to a position if the position presents a change in asymmetry in at least one of its five structures. The edge thickness is proportional to the number of mutant structures exhibiting the change (ranging from 1 to 5). Asymmetric changes close to mutated residues putatively related to mutations The comparison between the wild-type and mutant structures confirmed the import- ance of generating several structures per mutant. Some changes in asymmetry occurred at residues in- volved in the PI-binding pocket. For example, all mu- tants containing the I46V (or I84V) mutation exhibited asymmetry at pocket residue 48 (or 31 and 84). As resi- dues 48 and 84 establish hydrophobic contacts with PIs (Additional file 6: Figure S6), we concluded that the I46V and I84V mutations could induce structural back- bone changes at positions 31, 48, and 84 that could modify structural asymmetry and PI interactions. g g p In the first step of this study, we analyzed the struc- tural asymmetry in three minimized wild-type structures of PR2 and a set of 150 modeled mutant structures. Considering a large set of PR2 structures (wild-type and mutants) at the same time allowed us to extract infor- mation about the role of particular residues. Our results highlighted 25 residues that are symmetric in the wild- type and mutant structures i.e. presenting the same local conformation in the two monomers. Eight of these resi- dues (17, 28, 30, 53, 68, 81, 82, 87) were previously de- tected as symmetric in a set of 19 structures of wild-type PR2 available in the PDB [31], in which they exhibited the same conformation [32]. This conservation of sym- metric status highlights the important role of the sym- metry of these residues for PR2. As residues 28, 30, 53, 81, and 82 are located in the PR2 pocket and residue 87 establishes interactions with pocket residues 28–29 [10, 30, 32], we concluded that the conserved conformation of these residues in the two chains is important for the binding site conformation and ligand binding. In con- trast, our results identified 25 residues that were charac- terized as asymmetric in the three minimized wild-type structures and overrepresented in terms of asymmetry in the mutant structures. Twelve of these 25 asymmetric positions (18, 42, 50, 51, 59, 60, 64, 75, 77, 83, 92, and 93) were previously detected as overrepresented in terms of asymmetry in the 19 PR2 structures available in the PDB [31]. These results confirm that the backbone asymmetry of these residues is important for PR2 struc- ture and function [31]. Asymmetric changes close to mutated residues putatively related to mutations Figure 4 shows that the three mutants with the V62A mu- tation exhibited asymmetry at positions 38 and 39 and loss of asymmetry at positions 40 and 41. Mutated residue 62 was close to residue 38, which was close to residues 39–41 (Fig. 4). Thus, the V62A mutation could be responsible for the changes in asymmetry at positions 38–41. How- ever, the loss of asymmetry at positions 40 and 41 was highly recurrent in the mutant set, whereas the asymmetry at positions 38 and 39 was specific to V62A. Thus, it is dif- ficult to conclude that the V62A mutation induces changes in asymmetry at positions 38 and 39. Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Page 7 of 15 Laville et al. BMC Molecular and Cell Biology (See legend on next page.) Fig. 4 (See legend on next page.) Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Page 8 of 15 (See figure on previous page.) Fig. 4 Detection of changes in asymmetry in mutant structures. For this analysis, we selected all structures exhibiting the K7R, I46V, I54M, V62A, V71I, I82F, I84V, L90M, or L99F mutation. a presents matrices summarizing the positions exhibiting changes in asymmetry for the nine selected mutations. The matrix rows present mutant and matrix columns corresponding to PR2 positions. A matrix cell indicates the number of mutant structures (from 0 to 5) exhibiting changes in asymmetry at a given position. Positions in blue exhibit changes in asymmetry in all mutants. PR2 regions are presented at the top of the figure and are colored according to the color code of Fig. 1. b Localization of positions exhibiting changes in asymmetry in the PR2 structures. Proteins are displayed in cartoon mode and colored blue. Mutations are displayed with sticks and colored red. Positions exhibiting changes in asymmetry in the mutant structures relative to the wild-type structure are displayed in stick mode. Only positions exhibiting changes in asymmetry in at least one structure of all mutants harboring the studied mutations are indicated in the 3D structure of PR2 previous study [10] based on FoldX software [39] and an energy minimization step. As FoldX software is based on a side-chain rotamer library, we built five structures per mutant to consider the different rotamers associated with each amino acid. Asymmetric changes close to mutated residues putatively related to mutations In addition, residues 18, 42, 59, and 75 are involved in the H-bond network with resi- dues of the elbow, which is an important region for the flexibility of PR2 [32]. Thus, the structural asymmetry of these residues seems to be important for the transition between the PR2 open and closed forms. Residues 50 and 51 are involved in the interface of the two mono- mers, suggesting that the backbone asymmetry of these Other changes in asymmetry occurred at residues that were located outside of the binding site but were im- portant for its conformation, such as residues 33, 83, and 89 [10, 32]. Figure 4 shows that the structures of all mutants with I82F, I82L, or I84V mutations exhibited a loss of asymmetry at these important positions. Thus, these changes in asymmetry could be a consequence of structural changes induced by mutations at positions 82 and 84, which could modify the conformation of the binding pocket and, thus, indirectly alter PI binding. Concerning the L99F mutation, we observed that the four mutants containing this mutation exhibited changes in asymmetry at positions 91, 92, 93, and 98. Residues 91, 92, 93, and 98 are involved in the interface between the two monomers: residues 91 and 92 establish non bonded contacts at the interface, and residue 98 estab- lishes hydrogen bonds with residues 2 and 96 of the sec- ond monomer (Additional file 7: Figure S7). Thus, the L99F mutation could induce structural asymmetry at interface residues that could have an impact on the PR2 interface and modify the stability of the dimer. Discussion In this study, we explored the impact of resistance muta- tions arising in the PR2 target on its backbone asym- metry to obtain information about the structural effects of these mutations. Several studies have shown that al- though PR2 is a homodimer, its crystallographic struc- tures exhibit structural asymmetry that is important in the mechanism of PI recognition and binding [10, 27– 30, 33]. In this work, we compared the local structural asymmetry of a wild-type PR2 structure and 30 resistant mutants. As no structure of the PR2 mutant was avail- able in the PDB, we modeled the structure of the 30 studied mutants using the protocol developed in our Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Page 9 of 15 Page 9 of 15 Laville et al. BMC Molecular and Cell Biology within residues important for the binding site con- formation. Indeed, we showed that the I84V mutation could induce changes in asymmetry at positions 33, 83, and 89 that are involved in the hydrogen-bond network with pocket residues [32]. Thus, the I84V mutation could also alter the PI-binding pocket by modifying pocket properties through structural changes at positions 33, 83, and 89. residues is induced by dimerization. Residue 83 has been previously shown to be important for the binding site conformation [28, 30, 32]. Thus, the structural asym- metry of residue 83 could be important for PI recogni- tion and binding. All these results confirm that backbone asymmetry is an intrinsic property of PR2 that is involved in its flexibility and ligand recognition and binding. They also confirm the interest in mining several structures associated with a target to offer valuable insight into target structure, flexibility and interaction mechanisms [40–42]. One possible explanation for the observed structural asymmetry that we did not consider in this study is the error or packing in the crystallographic structure. We previously showed that 24 positions were involved in crystal packing in the PR2 structure complexed with DRV (PDB code 3EBZ) [37]. In addition, we previously highlighted some structural asymmetry linked to crystal packing in unbound PR2, for example, at positions 3 and 18 [33]. This suggests that the detected conservation of the asymmetry of some positions results from crystal packing. However, in this study, structural asymmetry was extracted from structures (crystallographic struc- tures and models) that were energetically minimized. Discussion This process enables the removal of crystal packing and contact with no biological relevance in the PR2 dimer. Indeed, we have previously shown that minimized struc- tures exhibit fewer structural asymmetric positions than crystallographic structures [33]. Thus, using energetically minimized structures decreases the impact of crystal er- rors and packing on structural asymmetry. One solution to remove this source of asymmetry could be to extract structural asymmetry from structure models generated during molecular dynamics simulations. In addition, the analysis of asymmetry during molecular dynamics simu- lations could provide information about the link between structural asymmetry and PR2 deformations induced by these substitutions and facilitate a detailed understand- ing of the role of these substitutions. One limitation of our study is that we focused on the structural changes occurring in the backbone of PR2 without considering side-chain deformations. Even though our results sug- gest the existence of some structural deformations in- duced by mutations that could lead to PR2 resistance to PIs, we may have missed some mechanisms. For ex- ample, we observed that all mutants with the L90M mu- tation exhibited a change in asymmetry at position 8. As positions 8 and 90 were distant from each other in the PR2 structure, we concluded that this structural change was not induced by this mutation. However, one possi- bility is that the L90M mutation could have induced structural changes in some side chains that were not de- tected by our approach. These structural changes could spread within the structure to lead to the deformation of the backbone of pocket residue 8. Thus, to better under- stand the resistance of PR2 induced by some mutations, it would be interesting to consider the deformations We showed that wild-type and mutant PR2 structures exhibit different asymmetric statuses at some positions. We distinguished changes in asymmetry between wild- type and mutant structures occurring at the same posi- tions in many mutants. Some of these changes in asym- metry occurred at flexible residues and sites located far from mutated residues, such as residues at 18, 40, and 41. Thus, these changes in asymmetry were putatively induced by PR2 intra-flexibility. On the other hand, some changes in asymmetry occurred in some mutants at positions close to the mutated residues, suggesting that these changes in asymmetry resulted from structural changes induced by mutations. Discussion By analyzing the loca- tions of these changes in backbone asymmetry, our re- sults suggested different putative mechanisms of resistance, as observed in PR1 [43]. First, we proposed that resistance mutations could induce structural changes at the interface of the two monomers. For ex- ample, our results suggest that the L99F mutation might induce changes in asymmetry at interface positions 91, 92, 93, and 98, which could modify the PR2 interface and alter its stability. This deformation of the dimer interface induced by resistance mutation was previously observed in PR1 mutants with an L24I, F53L or I50V mutation [44, 45]. Liu et al. [44] showed that the L24I mutation induces structural changes in PR1 that modify the contacts between the two Cter regions formed by residues 95–99, which explains the reduced stability in urea and the increased dissociation of the dimer. Second, our results suggest that some resistance mutations could directly modify PI binding. For example, we showed that mutations I46V and I84V could induce structural changes at pocket residues 31, 48, and 84, where the last two residues interact with PIs through hydrophobic con- tacts. Thus, these structural changes located in the PI- binding pocket induced by mutations could directly alter PI binding. This resistance mechanism has been previ- ously observed in some PR1 mutants. For example, Tie et al. [6] showed that mutation I84V induces structural changes in PR1 resulting in the loss of two van der Waals contacts between residue 84 and DRV. The third putative resistance mechanism corresponds to induced structural changes outside of the binding pocket but Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Page 10 of 15 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 occurring in side chains in the future. Another limitation of our study is that we considered only one template during the mutant structure-modeling step: wild-type PR2 complexed with DRV (PDB code 3EBZ [46]). How- ever, not all studied mutants are resistant to DRV. For example, Raugi et al. [19] showed that the L90M mutant is resistant to SQV and exhibits only a weak decrease in susceptibility to DRV. Thus, the use of a single template complexed with only one PI could explain why, for some mutants, we did not identify significant structural changes. Discussion It would be interesting to consider the complete resistance profile of each mutant. This will re- quire the construction of mutant structures complexed with all PIs and knowledge of the resistance profile of each mutant. However, the resistance profile is particu- larly difficult to obtain for all mutants because there are few studies that have tested the effect of PIs against HIV-2 mutants using enzymatic or phenotypic suscepti- bility assays [9, 16, 17, 19, 23] and some of these studies have led to opposing results [1]. In conclusion, this study was a structural analysis of the impact of a large number of PR2 resistance muta- tions using modeled mutant structures. Our results pro- vide a better understanding of the structural effects of mutations of PR2 and, thus, of PR2 resistance to PIs. Extraction of structural and functional regions of PR2 Extraction of structural and functional regions of PR2 We considered thirteen regions extracted from each PR2 monomer as in [32, 37, 38] (Fig. 1b and c). The binding pocket (20 residues per chain) was determined using the limits presented in [32] (Fig. 1c). Location of flexible and rigid residues of PR2 In this paper, we studied the structural impact of resist- ance mutations occurring in PR2, an important thera- peutic target in HIV-2 infection treatment. More specifically, we explored the effect of resistance mutations on structural backbone asymmetry, a property involved in dimerization, ligand recognition and binding. To do so, we detected the differences in terms of structural asym- metry between the wild-type and 30 modeled mutant structures. Studying a large set of mutant structures at the same time allowed us to confirm the functional and struc- tural roles of some PR2 residues, such as residues 28, 30, 53, 81, 82, and 87, that have been identified as important for the binding site conformation and ligand binding. Location of flexible and rigid residues of PR2 The flexibility of PR2 residues was quantified using B- factor values found in the PDB file that measure the atomic displacement factor of residues [47]. From the 3EBZ structure, we extracted the B-factor values of each atom. For each residue, the average B-factor value of its atoms was then calculated. The higher the average B- factor value of a residue is, the more flexible the residue is. Location of accessible and buried residues of PR2 The ASA value of protein residues is usually used to dif- ferentiate surface-exposed, important for protein inter- actions, and buried residues, important for protein stability [48]. According to [49], the ASA value is de- fined as the area of the surface swept out by the center of a probe sphere rolling over a molecule. To compute the ASA value of PR2 residues, NACCESS software [49] was run using 3EBZ structure and a radius probe sphere of 1.4 Å. The higher the ASA value of a residue is, the more accessible the residue is. In addition, the comparison between the structural asym- metry of wild-type and mutant structures allowed the detec- tion of some structural changes that could be induced by either PR2 flexibility or the studied resistance mutations. The analysis of the latter type of structural changes revealed three possible resistance mechanisms of PR2 that could occur to- gether. First, we observed that resistance mutations could modify the PR2 interface and its stability, such as mutation L99F that induces structural changes at interface positions 91, 92, 93 and 98. The second mechanism involves the alter- ation of the PI interaction directly induced by mutations, such as the I46V and I84V mutations that induce structural changes at pocket residues. The third resistance mechanism corresponds to indirect modifications of PI binding. We noted that the I84V mutation could modify the PI-binding site by inducing structural changes at residues important for the maintenance of the conformation of the PI-pocket bind- ing site. Methods Presentation of the wild-type PR2 structure To model the PR2 mutant structures, we used the crys- tallographic structure of PR2 in complex with DRV (PDB code: 3EBZ [46]). We chose this structure because it is the only available structure of PR2 complexed with one of the three FDA-recommended drugs for the treat- ment of HIV-2 infection (DRV, LPV, and SQV). This structure shows a good resolution of 1.20 Å. Modeling of mutant PR2 structures We selected 30 drug-resistant mutants of PR2 contain- ing one (14 single mutants), two (9 double mutants), or three mutations (7 triple mutants, Fig. 1a) from the lit- erature. Figure 1b indicates the location of the mutations in the selected PR2-drug resistant mutants according to the wild-type structure (PDB code: 3EBZ). Six of them (46, 47, 48, 50, 82, and 84) were located in the binding pocket. Page 11 of 15 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 3EBZmini structures using PyMOL software [55]. The all- atom RMSD values, denoted as RMSDaa, were computed between the superimposed structures using PyMOL soft- ware [55]. 3EBZmini structures using PyMOL software [55]. The all- atom RMSD values, denoted as RMSDaa, were computed between the superimposed structures using PyMOL soft- ware [55]. As the 3D structure of these mutants was not available in the PDB, we modeled their 3D structure using an in silico protocol based on the FoldX suite [39], as we de- scribed previously [10]. To do so, we applied the follow- ing protocol to the PR2 crystallographic structure in complex with DRV (PDB code: 3EBZ). First, we prepared the protein structure by removing the DRV ligand, metal atoms, and water molecules. Second, we applied the RepairPDB command of the FoldX suite to reduce the energy content of the structure. Third, we performed in silico mutagenesis using the BuildModel command of the FoldX suite. This command first introduces one or several mutations in the two chains of the wild-type structure using a side-chain rotamer library. Second, it performs an optimization of side chain of amino acids in the vicinity of the mutated residue(s). Each model was generated with five replications, and other options were set to the defaults. At the end of the mutagenesis step, five structures were modeled per mutant. This resulted in a set of 150 mutant structures for the 30 selected drug-resistant mutants. Fourth, we applied an energetic minimization protocol to the 150 modeled mutant struc- tures using the protocol developed in [12]. The mono- protonated state was assigned to the oxygen atom OD2 of Asp25 in chain B using PROPKA software [50]. The system was solvated in a truncated octahedron box of explicit solvent (TIP3P water model) with a 12.0 Å buffer in each dimension. Modeling of mutant PR2 structures An appropriate number of chloride ions were added to produce a neutral charge in the sys- tem. Protein and water molecules were described using the force field AMBER ff99SB [51]. A two-step energy minimization was carried out in GROMACS [52] using a combination of steepest descent and conjugate gradi- ent algorithms of roughly 1000 and 2000 iterations. Water molecules and counterions were relaxed through a first step energy minimization, using a pos- ition harmonic restraining force of 100 kcal.mol-1 Å-2 on the heavy atoms of the protein. A second step of energy minimization was performed by removing re- straints on protein atoms. The particle mesh Ewald (PME) method was adopted to treat the long-range electrostatic interactions [53, 54]. The cutoff distances for the long-range electrostatic and van der Waals in- teractions were set to 10.0 Å. l l d h l h To analyze the impact of the number of mutations in- troduced in the mutants on the structural variability be- tween the mutants and the wild-type structures, we compared the average RMSDaa values of the single, double, and triple mutants using a Kruskal-Wallis test and pairwise Wilcoxon tests. We also compared the variability in terms of the RMSDaa values of single, double, and triple mutants using a Bartlett test and pair- wise Fisher tests. Quantification of structural asymmetry We also applied the minimization protocol to the 3EBZ crystallographic structure with three replications, resulting in three minimized structures of wild-type PR2 referred to as the 3EBZmini1, 3EBZmini2, and 3EBZmini3 structures. y y We first counted the number of asymmetric positions in the three minimized wild-type and 150 mutant structures, as described in [31]. To quantify the struc- tural asymmetry in the mutant structure set, we de- termined the occurrence of asymmetry (AO) at each position, i (i.e., the number of mutant structures exhi- biting asymmetry at position i). The statistical signifi- cance of the AO value of position i was determined using the overrepresentation p-value, pAO. The pAO of Analysis of the structural asymmetry of PR2 Detection of structural asymmetry in PR2 structures. f y y The detection of structural asymmetry in a dimer cor- responded to the identification of positions that exhibit different backbone conformations in the two chains. In this study, we extracted structural asymmetry from the three minimized wild-type structures of PR2 and the 150 modeled mutant structures using the structural alphabet-based approach that we previously developed [31]. This protocol, presented in Fig. 5, is based on the HMM-SA structural alphabet (Hidden Markov Model - Structural Alphabet), which is a library of 27 prototypes of 4-Cα residues classified according to their geometry, assigned structural letters and labeled [a, A-Z] [56]. First, HMM-SA was used to simplify the 3D structure of the two chains of p residues in two sequences of (p-3) struc- tural letters overlapping by 3 residues, where each struc- tural letter corresponded to the geometry of a 4-Cα fragment. Each structural letter was assigned to the third residue of the 4-residue fragment. As the fragments overlapped by 3 residues, no structural letter was assigned to the first, second, and last residues. Second, the structural letters of each position in both chains were compared. A position has two possible asymmetry profiles: either the position is asymmetric (i.e., exhibiting different local conformations) (=structural letters) in the two chains, or it is symmetric (i.e., exhibiting the same local conformation) (=structural letter) in the two chains. Comparison of wild-type and mutant structures p yp We computed the RMSD values between the 150 mu- tant structures and the three 3EBZmini structures. The mutant structures were optionally superposed onto the Laville et al. BMC Molecular and Cell Biology (2020) 21:46 Page 12 of 15 Fig. 5 Protocol used to compare the structural asymmetry of the wild-type and mutant structures of PR2. Step 1: Modeling of the 3D structure of PR2 mutants. First, the mutation is introduced into the wild-type structure of PR2 (PDB code: 3EBZ) using FoldX software [39] with five replications. Then, an energetic minimization step is applied to the wild-type and modeled mutant structures using Gromacs software [52]. Wild-type and mutant structures are presented in cartoon mode and colored according to their two chains: chain A is colored magenta, and chain B is colored cyan. Mutated residues are presented in stick mode and are indicated with black arrows. Step 2: Detection of structural asymmetry in the wild-type and mutant structures of PR2 using a protocol based on the HMM-SA structural alphabet. The HMM-SA structural alphabet [56] is a library of 27 protein prototypes of 4 residues classified according to their geometry. First, HMM-SA is used to simplify each PR2 chain of 99 residues into a sequence of 96 structural letters, in which each structural letter corresponds to the geometry of a 4-Cα fragment (i.e., representing the local conformation of each residue). Then, the structural letter for each position in the two sequences is compared to localize (i) symmetric positions that correspond to positions exhibiting the same local conformation (=structural letter) in the two chains of the dimer and (ii) asymmetric positions that correspond to positions exhibiting different local conformations (=structural letters) in the two chains of the dimer. To quantify the structural asymmetry in PR2 structures, the number of asymmetric positions in the dimer is finally counted Fig. 5 Protocol used to compare the structural asymmetry of the wild-type and mutant structures of PR2. Step 1: Modeling of the 3D structure of PR2 mutants. First, the mutation is introduced into the wild-type structure of PR2 (PDB code: 3EBZ) using FoldX software [39] with five replications. Then, an energetic minimization step is applied to the wild-type and modeled mutant structures using Gromacs software [52]. Wild-type and mutant structures are presented in cartoon mode and colored according to their two chains: chain A is colored magenta, and chain B is colored cyan. Funding h k This work was supported by an ANRS Grant. NC and PL are supported by a fellowship from the Ministère de l’Education Nationale de la Recherche et de Technologie (MENRT). Quantification of the changes in asymmetry per mutant For a given mutant, we computed the number of its five structures exhibiting changes in asymmetry for the 78 selected positions. This number was ranked from 0 to 5. A value of 0 for a position means that the position ex- hibits the same asymmetry status in the three 3EBZmini structures and the five structures of the mutant. In con- trast, a position with a value of 5 means that the position exhibits a different asymmetry status in the three 3EBZ- mini structures and the five structures of the mutant. For all mutants, these data were summarized in a network that connects mutants with a position when at least one structure of the mutant exhibits a change in asymmetry relative to the wild-type structure at the position. The Acknowledgements Not applicable. Not applicable. Supplementary information Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12860-020-00290-1. Additional file 1. Structural comparison between mutant structures and the three 3EBZmini structures. Additional file 2. Summary of structural asymmetry in the three wild- type structures 3EBZmini and the 150 mutant structures. Additional file 3. Flexibility of PR2 residues quantified using B-factor values. Additional file 4. Exposure of PR2 residues quantified using ASA values. Additional file 5. Quantification of structural asymmetry in the 150 structures of the PR2 mutants. Additional file 6. Interaction between PR2 and three drugs (APV, DRV, and IDV). Additional file 7. Residues involved in the interface of the wild-type PR2 dimer. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12860-020-00290-1. pAO ið Þ ¼ prob AOrandom ið Þ > AO ið Þ ¼ n AOrandom ið Þ > AO ið Þ nsimu ðEq:1Þ ðEq:1Þ ðEq:1Þ where [n {AOrandom}(i) > AO(i)] is the number of simu- lations in which AOrandom(i) is higher than AO(i), and nsimu is the total number of simulations. An asymmetric position was considered statistically overrepresented if its pAO was smaller than a threshold of 0.0005, as determined using the Bonferroni adjust- ment to consider multiple tests (0.05/96 positions). Additional file 7. Residues involved in the interface of the wild-type PR2 dimer. Additional file 7. Residues involved in the interface of the wild-type PR2 dimer. Authors’ contributions LR d d d LR conceived and designed the work. MP and LR supervised the project. NC and LR collected data. SF and DF generated the 150 mutant models. PL and LR analyzed and interpreted the data. LR wrote the manuscript. All authors reviewed the manuscript. All authors approved the version of the manuscript to be published. Abbreviations 3D: Tridimensional; AO: Asymmetry occurrence; APV: Amprenavir; ASA: Accessible surface area; Cter: C-terminal end; DRV: Darunavir; FDA: Food and Drug Administration; HIV: Human immunodeficiency virus; HIV-1: Human immunodeficiency virus type 1; HIV-2: Human immunodeficiency virus type 2; HMM-SA: Hidden Markov Model - Structural Alphabet; IDV: Indinavir; LPV: Lopinavir; NFV: Nelfinavir; Nter: N-terminal end; ORasym: Over-represented asymmetric; PDB: Protein Data Bank; PI: Protease inhibitor; PME: Particle Mesh Ewald; PR1: HIV-1 protease; PR2: HIV-2 protease; RMSD: Root mean square deviation; RMSDaa: RMSD computed using all common atoms; SQV: Saquinavir Study of the link between mutations and changes in asymmetry Location of changes in asymmetry in mutant structures relative to wild-type structure Consent for publication Not applicable. Consent for publication Not applicable. Location of changes in asymmetry in mutant structures relative to wild-type structure A change in asymmetry corresponds to a position that exhibits a difference in asymmetry status (asymmetric or symmetric) in both a given mutant structure and the three 3EBZmini structures. Two types of changes in asymmetry were defined: asymmetry (or loss of asym- metry) occurs at a given position of a mutant structure if the position is asymmetric (or symmetric) in the mu- tant structure, whereas it is symmetric (or asymmetric) in the 3EBZmini structures. To locate the positions exhi- biting changes in asymmetry in each mutant structure, we compared the asymmetry status in the mutant and 3EBZmini structures. To do so, we focused on only the 78 positions exhibiting the same status (asymmetric or symmetric) in the three 3EBZmini structures. Comparison of wild-type and mutant structures Mutated residues are presented in stick mode and are indicated with black arrows. Step 2: Detection of structural asymmetry in the wild-type and mutant structures of PR2 using a protocol based on the HMM-SA structural alphabet. The HMM-SA structural alphabet [56] is a library of 27 protein prototypes of 4 residues classified according to their geometry. First, HMM-SA is used to simplify each PR2 chain of 99 residues into a sequence of 96 structural letters, in which each structural letter corresponds to the geometry of a 4-Cα fragment (i.e., representing the local conformation of each residue). Then, the structural letter for each position in the two sequences is compared to localize (i) symmetric positions that correspond to positions exhibiting the same local conformation (=structural letter) in the two chains of the dimer and (ii) asymmetric positions that correspond to positions exhibiting different local conformations (=structural letters) in the two chains of the dimer. To quantify the structural asymmetry in PR2 structures, the number of asymmetric positions in the dimer is finally counted was associated with a mutant structure. In each ran- dom binary sequence, the 0 and 1 values represent the symmetric and asymmetric positions, respectively. The number of asymmetric positions in the random sequence, j, corresponds to the number of asymmetric position i was computed by comparing its observed (AO(i)) and expected (AOrandom(i)) occurrences. The AOrandom occurrence of position i was computed in a random set generated with 150 random binary se- quences of 96 positions, where each random sequence Page 13 of 15 Laville et al. BMC Molecular and Cell Biology (2020) 21:46 positions in the jth mutant structure. The AOrandom value of position i corresponds to the number of ran- dom sequences in which that position is considered an asymmetric position (i.e., showing a value of 1 at the position). pAO is estimated as the probability that AOrandom is higher than AO(i) using Eq. 1 and 2000 random sets. network was drawn using the igraph library of R soft- ware [57]. network was drawn using the igraph library of R soft- ware [57]. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. pp Not applicable. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Page 14 of 15 Page 14 of 15 Laville et al. 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W4231524308.txt	https://zenodo.org/records/1517808/files/article.pdf	de		Ueber das Zusammenwirken von Stäbchendoppelbrechung und Eigendoppelbrechung, III	Colloid and polymer science/Colloid & polymer science	1,917	public-domain	8,512	173 dings aber zun~iciast nicht mehr als ein bloBer Anzeiger. Es ist ja heute ein eindeutiger SchluB aus Viskositfits~inderungen auf das Wesen der durch diese verratenen Zustands~inderungen der Kolloide meist noch nicht ziehbar. So kann die Viskositiit von Kolloiden zunehmen mit zunehmendem, aber auch mit abnehmendem Dispersitiitsgrade. GewiB haben nur jene Messungsangaben in der Biologie einen tiefen wissenschaftlichen Wert, ,bei welchen zu tibersehen ist, was fiir Aenderungen des Systems energetisch in der Viskositiit zum Ausdruck kommen" [ S c h i b i g ] 2 9 ) . Immer noch istdie ~) J. S c h i b i g , loc. cir. auf der Versammlung der F~iraday-Society im Jahre 1913 aufgeworfene Frage often: ,Was bedeutp.n, was meinen denn nur eigentlich alle diese Viskosittitsmessungen, was zeigen sie eigentlich? ~ Doch halten wir es vorerst lieber mit der trGstenden Bemerkung Wo. O s t w a l d ' s [S. 262]3~ ,Der phtinomenologische Teil unserer Kenntnisse ist keineswegs weniger wicht!g." Dem ist wohl ganz besonders dann so, wenn - - wie bei der Viskosittttsmessung lebenden Plasmas m der ph~nomenologische Teil der Hauptsache nach erst geschaffen werden muB. a0) Wo. O s t w a I d, Fuflnote 9. Ueber das Zusammenwirken yon Stlibchendoppelbrechung und Eigendoppelbrechun9, i11.'t (Mittr Von H. Am b ro n n (Jena), (v,i~gegangeoa m 17.Vebnmr1917.) aus dem Institut fiir Mikroskopie an der Universit/lt Jena.) VIII. In der zweiten Mitteilung habe ich das optische Verhalten des bleib,md deformierten Zelloidins eingehend geschildert. Im Verlauf der Gangunterschiede, wie er durch das Veriindern des Brechungsexponenten der Imbibitionsfliissigkeit bei denoum etwa 100 Proz. veriiingerten Streifen hervorgerufen wird, sind vor allem zwei Eigenttimlichkeiten bemerkenswert. Bei der allmiihliehen Steigerung der Werte yon 1,33 bis 1,72 tritt fiir alle Farben des sichtbaren Spektrums zweimal optische lsotropie und damit in Verbindung eine zweimalige Umkehr des Vorzeichens de," Oesamtdoppelbrechung ein. Schon durch den sehr einfachen Versuch, der auf S. 274 und 275 in Mitteilung II beschrieben worden ist, kann man sich leicht von diesem merkwfirdigen Verhal'ten des Zelloidins iiberzeugen; je nach der HShe des Brechungsexponenten der eingelagerten Fliissigkeit ist die sog. akzidentelle Doppelbrechung der gedehnten Streifen positiv, Null oder negativ. In dieser Beziehung weicht also das Zelloidin im imbibierten Zustande von allen anderen bisher auf ihre akzidentellte Doppelbrechung untersuchten KSrper ganz wesentlich ab. Man kannte zwar auch bisher schon KSrper, bei denen d i e akzidentelle Doppelbrechung im Verlauf einer liinger andauernden Dehnung ihr Vorzeichen t) Vgl. Koll.-Zeitschr. 18, 90--97 und 273--281 (1916). einmal umkehrt, so z. B. das Kirschgummi 2), die Guttapercha 3), das mit echtem Kampfer hergeste!lte Zelluloid 4), aber diese Umkehr erfolgt stets nut bei einem bestimmten Deformationsgrad und ist g~inzlich unabhiingig yon einer Aenderung des Brechungsexponenten des KSrpers oder eines seiner Bestandteile. Die in Fig. 3 in Mitteilung II dargestellten Kurven lassen deutlich erkennen, in weleher Weise die H6he des Ganguntersehieds yon der Differenz der Brechungsexponenten der Grundsubstanz und der eingelagerten Fliissigkeit abhiingt. Erreicht diese Differenz den Wert Null, so zeigt aueh die Kurve des Gangunterseltieds ein Minimum. Dieses Minimum tritt natiirlich nicht bei allen Wellenliingen zugleich auf, sondern seine Lage ist abh~ingig vom Gange der Dispersion, da jene Differenz der Brechungsexponenten nicht fiir alle Wellenl~ingen zugleieh verschwinden kann. Die betr~chtlichen Schwankungen in der H6he der Gangunterschiede und die noch st~irkere Verschiedenheit in der Dispersion der Doppelbrechung sind dabei be.o) Ueber das optische Verhalten una die Stmktur des Kirschgummis (Ber. d. Deutsch. Bot. Oes. 7, 103 bis 114, 1889). s) Ueber Anomalien bei der akzidentellen Doppelbrechung (Ber. d. Kgl. S/ichs. Ges. d. Wiss. math.-phys. Kl. Naturw. Tell 50, 1--31, 1898). 4) Ueber anomale Doppelbrechung beim Zelluloid (Ber. d. Kgl. Sachs. Oes. d. Wiss. math.-physik. Kl. Naturw. Teil 63, 249--257 und 402---4,06, 1911); vgi. auch Koll.-Zeitschr. 9, t47--153 (1911). 174 sonders bemerkenswert. Wie bereits in der ersten Mitteilung gezeigt werden konnte, linden sich dieselben Eigenschaften bei der St~ibchendoppelbrechung im Sinne O. Wiener's (vgl. Mitteilung I). Diese ist aber, wenigstens soweit es sich um farblose KOrper handelt, stets positiv; denn ein System parallel gestellter St~ibchen, die von einem Medium umgeben sind, dessen Brechungsexponent yon dem der St~ichensubstanz abweicht, verh~ilt sich nach den Wie n e r'schen Untersuchungen wie ein einachsiger, positiver Kristall, dessen optische Achse den L~ingsachsen der St[ibchen parallel steht. Die Beobachtungen am Zelloidin haben nun zwar mit Sicherheit ergeben, dab die St~irke der Doppelbrechung mit dem Werte yon n~ (Brechungsexponent der Imbibitionsflfissigkeit) ver~inderlich ist, zugleich aber auch, dab die Doppelbrechung nur innerhalb zweier ganz bestimmten Bezirke der Werte yon n2 positivist, w[ihrend sie bei anderen Werten negativ wird oder sogar vOllig verschwindet. Es kann deshalb die beobachtete Anisotropie nicht allein auf die Stiibchendoppelbrechung zur/ickgefiihrt werden, es mull noch eine andere Komponente hinzukommen, die das entgegengesetzte Vorzeichen besitzt. Nut auf diese Weise kommt man zu einer befriedigenden Erkliirung des so eigentfimlichen Verlaufs der Gangunterschiede. Diese zweite Komponente mull aber rein zur Oeltung kommen, wenn n~ gleich nl (Brechungsexponent d~s Zelloidins) wird, denn dann verschwindet nach der Theorie die St~ibchendoppelbrechung vollst~indig und die noch iibrig bleibende Anisotropie mug also mit einer Eigenschaft der Stabchen seibst zusammenheingen. Ich habe diese Komponente deshalb als E i g e n d o p p e l b r e c h u n g bezeichnet; sie wird w~ihrend des ganzen Verlaufs der Beobachtungen sowohl in ihrer St[irke, wie in ihrem Charakter unveriindert bleiben, wenn die Deformation dieselbe bleibt und die Sfiibchensubstanz durch die imbibierenden F1/issigkeiten weder chemische noch physikalische Veriinderungen erf~ihrt. Wir h~itten es also mit dem Zusammenwirken zweier Komponenten zu tun, yon denen die eine konstant, die andere aber mit dem Werte yon n~ veranderlich ist. Beide Komponenten haben jedoch entgegengesetztes Vorzeichen, demnach mull auch die Resultierende in zwei ganz bestimmten F[illen Null werden, autlerdem mull eine zweimalige Umkehr des Vorzeichens eintreten. Hat dagegen auch die Eigendoppelbrechung den positiven Charakter, so wird zwar der Verlauf der Kurven der Gangunterschiede im wesentlichen ahnlich sein, die Umkehr des Vorzeichens jedoch mull dann ebenso unterbleiben wie das Eintreten der Isotropie. Wie schon friiher mehrfach erw[ihnt wurdeS), zeigt die durch Denitrierung aus dem Zelloidin gewonnene Zellulose ein solches Verhalten. Da aber auch in diesem Falle die Stiibchendoppelbrechung mitwirkt, so mug sich ebenfalls eine deuflich bemerkbare Ver~inderung in der Dispersion der Gesamtdoppelbrechung ergeben, wenn sie auch nicht eine so starke Abweiehung in den Interferenzfarben hervorrufen kann. Die in dem n~ichsten Abschnitt zusammengestellten Beobachtungen werden, zeigen, dab das optische Verhalten der Zellulose in der Tat dem eben Oesagten entspricht und dall nur untergeordnete Abweichungen auft.reten, die auf andere Weise ihre Er-kl~irung linden kOnnen. lX. Die Vorbereitung der zu den Beobachtungen benutzten Objekte geschah stets in der Weise, dab die um 100 Proz. ihrer urspr/inglichen L~inge bleibend deformierten Zelloidinstreifen etwa acht Tage hindurch unter mehrmaliger Erneuerung der Fl/issigkeit in Ammoniumsulfid verblieben und danach ebenfalls mehrere Tage in destilliertem Wasser geniigend ausgewaschen wurden. DaB auf diese .Weise eine vollsteindige Denitrierung erzielt wird, davon kann man sich leicht auf zweifache Art iiberzeugen. Zunichst IM~t sich feststellen, dab Schnitte durch solche Streifen in ihrer ganzen Ausdehnung die Zellulosereaktion mit Chlorzinkjod ergeben und dabei denselben sehr starken Dichroismus zeigen, den gew0hnliche Zellulosemembranen mit diesem Reagens erhalten. Perner kann man sich dutch liingere Behandlung der denitrierten Streifen mit einem Alkohol-Aethergemisch /iberzeugen, dab nichts mehr in L0sung geht, dab also alles Zelloidin wirklich in Zellulose umgewandelt ist. Dutch die Denitrierung wird, wie ich fr/iher schon erwiihnt habe, eine geringe Formver[inderung verursacht; aber diese finder nicht etwa in der Weise statt, dab die durch Dehnung hervorgerufene VerI[ingerung in erster Linie verkiirzt wird, sondern es ergibt sich eine fast gleichmaBige Ver~inderung in allen Richtungen. Ein Beispiel mOge dies erliutern: Ein Streifen, der vor der Denitrierung 20 mm lang, 1,8 mm breit und 1,25 mm dick war, 6) Zeitschr. f. wiss. biikroskopie 82, 56 (1915); ferner Koll.-Zeitschr.18, 280 (1916). 175 zeigte nach vollstiindiger Denitrierung die Mat3e : 18,2 mm, 1,6 mm und 1,1 ram, er hatte also in jeder Richtung eine Verkfirzung um etwa 9 - - 1 0 Proz. erfahren, dabei aber seine prismatische Gestalt w~llst~indig beibehalten. Es ist nun beachtenswext, dat3 die Formveriinderung eines Streifens von denselben Dimensionen aus frischem, nicht deformiertem Zelloidin bei derselben Behandlung fast genau dieselbe war. Obwohl bei dem ganzen Vorgang der Denitrierung eine sehr weitgehende chemisehe Ver~inderung der Zelloidinteilchen herbeigeffihrt wird, so ist doch die damit verbundene Formveriinderung eine recht geringe. Dies ist jedoeh durchaus nicht so fiberraschend, wie es auf den ersten Blick scheint, denn man well3, dat3 auch bei dem umgekehrten Vorgang, n~imlich bei der Nitrierung der ZellulosefasernS), die iiul~ere Form fast vollstiindig beibehalten wird. Ganz anders verhiilt es sich aber mit tier St.~irke der Doppelbrechung, die vor und nach der Denitrierung feslzustellen ist. Wie aus der Vergleichung der Tabelle V in Mitt. I1 (Koll.-geitschr. 18, 270) und der folgenden Tabelle VII sofort zu ersehen ist, wird die Doppelbrechung nach der Umwandlung um mehr als das Doppelte erh6ht, denn die Zahlen ffir die Gangunterschiede in der Tabelle V gelten ffir einen 3,7 ram dicken Zelloidinstreifen, die in der Tabelle VII dagegen ffir einen nur 1 mm dicken Zelluiosestreffen. Diese sehr starke Ver~inderung im optischen Verhalten ist ebenfalls nicht iiberrasehend, wenn man bedenkt, daf~ bei der Nitrierung der ZeUulosefasern ein ganz /ihnlicher Vorgang sich abspielt. Es m6ge genfigen, auf diese grofien Verschiedenheiten im Verhalten der reinen 7ellulose und der Nitrozellulose hier nur kurz ninzuCveisen; ich werde spiiter eingehender darauf zurfickkommen. Als Imbibitionsflfissigkeiten ffir die Zellulosestreifen dienten: Wasser, Aethylalkohol, Benzylalkohol und Monobromnaphthalin. Man kann jede dieser Flfissigkeiten dutch die im Brechungsexponenten niichst h6here ailmiihlich verdriingen und erh~ilt somit Mischungen, deren Exponenten zwischen 1,33 und 1,66 liegen. Beim Zelloidin konnte ich im wesentlichen mit L6sungen yon Kaliumquecksilberjodid in verschiedenen Verdfinnungen auskommen, wodurch die Beobachtungen sehr vereinfacht wurden. Leider liel~ sich dieses Salz bei tier Zellulose ~) Hans A m b r o n n , Ueber die Aendemng des optischen Verhaltens der Zellulose bei der Nitriemng (Diss., Jena 1914) Vgl. auch Koll -Zeitschr. 13, 200 his 207 (1913). nicht verwenden, da seine L6sungen eine betr~ichtliche Quellung verursachten. Nach mehreren Versuehen mit anderen Flfissigkeiten w~ihlte ich die vier bereits genannten, weft die Streifen in ihnen keinerlei bemerkbare Formver~inderungen zeigten. Wenn aueh der Spielraum der Brechungsexponenten etwas kleiner war, als bei den Versuchen am Zelloidin, so genfigte er doch v611ig, um einen Ueberblick fiber den Verlauf der Gesamtdoppelbrechung und damit auch der dabei wirksamen St~ibchendoppelbrechung zu erhalten. Die Messungen der Ganguntersehiede wurden wie friiher beim Zelloidin an zahlreichen Streifen yon verschiedener Dicke sowohl in aufsteigender wie in absteigender Reihenfolge durchgeffihrt. Die Mittelwerte aus den gewonnenen Zahlen wurden dann auf eine Streifendicke yon 1 mm umgerechnct. Neben dem Kompensatorokular nach H. S i e d e n t o p f benutzte ich diesmal auch noch zur bequemeren Orientierung eine Anzahl Glimmerpliittchen yon allm~ihlich steigenden Ganguntersehieden, die ffir die in Betracht kommenden Wellenliingen genau geeicht waren. Als Lichtquellen dienten wieder die Quecksilberbogenlampe mit den K 6 h le r'schen Lichtfiltern ffir die Wellenliingen 436 ut~ und 546 ~ und eine MikroskopierNernstlampe nach H. S i e d e n t o p f in Verbindung mit zwei Farbglasplatten; davon war die eine die dunkle Rotglasplatte, die ich frfiher schon benutzte, und die andere ein Blauglas. Bei den hohen Gangunterschieden, "wie sie die dickeren Streifen zeigten, erwies sieh die Rotglasplatte als nicht gentigend, da sie ein zu breites Gebiet aus dem Rot durchliet~, w~ihrend beim Zufiigen des Blauglases nur eine schmale Zone zwischen 670 t~a und 680 ta~a wirksam wurde, bei der aueh recht hohe Gangunterschiede noch geniigend schwarze Interferenzstreifen erg~ben, Im folgenden ist dieses Gebiet stets mit 675 ~aa bezeichnet. Nach den ffir das Verhalten des Zelloidins bereits gegebenen Darlegungen sind die Zahlen in Tabelle VII und die Kurven in Fig. 5 ohne weiteres verstiindlich. Die Ordinaten bedeuten die beobachteten Gangunterschiede 7o f ii r e i n e n S t r e i f e n y o n 1 m m D i c k e , und die Abszissen sind die Brechungsexponenten der Imbibitionsflfissigkeiten fiir die D-Linie. Es ist also auch bei der Zellulose eine betr~ichtliche Ver~inderung der Gesamtdoppelbrechung mit der Steigerung des Brechungsexponenten der Imbibitionsfliissigkeit verknfipft; die Gangunterschiede gehen aber hier ffir keine 176 Tabelle'VlI. (n2)n 436 ~/* 7'0 546 ~/~ 675 tff~ 1,33 10,95 8,50 6,55 1,08 1,35 10,70 8,35 6,45 1,08 1,37 10,48 8,24 6,33 1,07 1,39 lff,03 7,85 6,02 1,08 1,41 9, 44 7,38 5,70 1,071,43 8,90 6,94 5,35 1,08 1,45 8,48 6,59 5,08 1,08 1,47 8,14 6,30 4,85 1,09 1,49 7,87 6,10 4,68 1,09 1,51 7,77 1,09 6,02 4,60 1,53 7,72 5,96 4,55 I,I0 1,55 7,75 5,99 4,53 l,lO 1,57 7,8.6 6,04 4,55 1,11 1,59 8,07 6,17 4,65 1,12 1,61 8,46 6,32 4,81 1.13 1,63 8,83 6,65 4,98 1,14 1,65 9,47 7,14 5,28 1,16 1,661 9,95 7,47 5,48 1,17 In der letzten Reihe der Tal elle sind Lie Zahlen fiir die Dispersion der Oesamtdoppeibrechung wie frtiher in der Form (na-no)~ t~u wiedergegeben, o (na-no)6v5 p# Der Brechungsexponent 1,661 ist der des yon mir benutzten Monobromnaphthalins bei Zimmertemperatur. o~,, Wellenltinge auf Null herab, sm behalten stets dasselbe Vorzeichen. Dort, wo die Kurven die tiefsten Stellen erreichen, wird, wie auch beim Zelloidin, die Eigendoppelbrechung der Zellulose allein zur Geltung kommen. Wiirde man die Werte v o n (n2)l) , die jenen Stellen entsprechen, mit hinreichender Genauigkeit bestimmen kOnnen, so kSnnte man daraus auch, unter Berficksichtigung der Dispersion der angewandten-Fltissigkeiten, die Dispersion der Zellulose berechnen. Wie schon frtiher hervorgehoben wurde, ist aber die Feststellung der Lage der Minima, wegen des an diesen Stellen sehr flachen Vedaufes der Kurven, mit Schwierigkeiten verkniipft. Es handelte sich ja hier auch gar nicht darum, die Dispersion der Zellulose zu ermitteln, aber es hatte doch ein gewisses Interesse, zu prtifen, ob der sich ergebende Wert mit einem gr6fleren Fehler behaftet sei. Bei der durchgeftihrten Ntiherungsreehnung, auf die ich hier nicht weiter eingehen will, ergab sich ftir das in Betracht kommende Gebiet zwischen 436 ~u~ und 6 7 5 / ~ der Wert yon etwa 0,02, der ftir einen festen KOrper yore mitfleren Brechungsexponenten 1,54. durchaus wahrscheinlich ist. Eine tihnliche Unsicherh e r besteht, wie gleichfails friiher schon ausgeftihrt wurde, bei der Berechnung der Eigendoppelbrechung aus den Minimalwerten ftir die Gangunterschiede: 7,72, 5,95, 4,53; dazu kommt noch, daft der Betrag fiir 81, d. h. der Anteil an der Gesamtdicke des Streifens ebenso, wie beim Zelloidin, nur mit einiger Wahrscheinlichkeit zu bestimmen ist. Wtihlt man unter Berticksichtigung der Angaben auf S. 279 in Mitteilung II 81 -- 0,25, so erhtilt man ftir die Eigendoppelbrechung a I folgende Werte: (al)43~ ~t~ : 0,01346, (al)546 ~ = 0,01301, (al)~76 ~9 = 0,01223, und daraus ~q-----1,10. Fig. 5 F/Jr die Berechnung der Werte a2, der Sttlrke der Stabchendoppelbrechung, ftillt dagegen diese Unsicherheit weg, da hierbei ja die Gesamtdicke des Streifens, fiir den in Tabelle VII dargestellten Fall also 1 ram, in Betracht kommt; man hat nut die Differenzen 7o - - 71 zu bilden und daraus fiir die verschiedenen Wellenltingen die zugeh6rig e Sttirke der Sttibehendoppelbrechung a 2 zu berechnen. In. derTabelle VIII sind die gefundenen Zahlen zusammengestellt und auflerdem in der letzten Reihe die Werte ffir ~2 angegeben, wie sie sich direkt aus den Zahlen ffir 72 = 7o - - 71 ergeben. Perner ist in Fig. 6 wieder wie frfiher der Verlauf der Werte a~ ftir die Wellenltinge yon 546 ~tx dar- 177 gestellt. Man sieht daraus, daft dasselbe, ~as auf S. 279 zur Fig. 4 gesagt worden ist, auch hier im wesentlichen seine Giltigkeit beh[ilt. dal~ bei der Zellulose der Verlauf der Kurve1~ nicht so gleichm~iBig ist wie beim Zelloidin. Es macht sich hier sowohl beim Sinken der Werte, wie auch beim Ansteigen eine St6rung T a b o l l e VIII. bemerkbar, die auf dem absteigenden Ast starker als auf dem ansteigenden hervortritt. Ich vermutete zun~chst irgend einen versteckten (n2)D 436 t u ~ 546/~ 675 ~/~ ~2 Beobachtungsfehter, doch ergab sich bei den sehr zahlreichen Versuchsreihen, die den an1,39 1,36 1,03 1,33 1,40 geffihrten Zahlen zu Grmlde liegen, sowohl in 1,30 1,00 1,35 1,31 1,31 aufsteigender wie in absteigender Reihenfolge 1,22 0,99 1,37 1,20 1,25 1,00 1,00 1,04 fast immer dasselbe Resultat. Die Versuche 1,39 1,01 0,79 0,95 0,80 1,41 0,75 wurden stets in der Weise angestellt, dal~ die 0,54 0,55 0,92~ 1,43 0,51 0,37 0,90~B" Streifen zuerst in reinem Wasser, darauf in 1,45 0,33 0,35 verschiedenen Oemischen yon Aethylalkohol 0,22 0,85] 1,47 0,18 0,19 0,10 0,66 L 0,08 und Wasser, dann in absolutem Alkohol und 1,49 0,06 0,047 0,48 A 0,03, 1,51 O,g2 von da ab in Gemlschen yon Alkohol und 0,00; 1,53 0,02 ~ Ch Benzylalkohol bis zum reinen Benzylalkohol und 1,55 0,013 schlie$1ich in Oemischen yon Benzylalkohol und 0,014 4,5 0,05 1,57 0,06 0,08 1,9 0,12 Monobromnaphthalin bis zum reinen Monobrom1,59 0,15 0,19 1,7 1,61 0,32 0,25 naphthalin untersucht wurden. In ]eder diesel" 0,30 1,6 0,40 1,63 0,48 Flfissigkeiten blieben die Streifen mindestens 0,51 1,6. 0,65 1,65 0,76 24 Stunden, oft auch noch D.nger, ehe die 0,64 1,5 0,83 1,661 0,97 Messung der Gangunterschiede vorgenommen ) Die Buchstaben B, L, A und Ch ~edeuten hier wurde. Man daft somit wohl annehmen, dat~ wieder dasselbe wie in Tabelle V. innerhalb dieser Zeit jeweils eine vollst~indige Durchdringung der verhaltnismafiig kleinen (n,3-no) a7 Objekte -- es handelte sich doch stets nur um kurze Streifen yon 3 - . 4 mm L~inge und 1--1,5 mm Dicke - - stattgefunden hatte und das Brechungsverm6gen der umgebenden Flfissigkeit auch zur Wirkung kam. Das ist aber doch an zwei ganz deutlich sich abhebenden Stellen offenbar nicht der Fall gewesen; denn sonst mfiSten diese StOrungen wegfallen, zumal sie auch beim Zeltoidin gar nicht zu beobachten Fig. 6 waren. Worauf nun diese Ungleichmfil~igkeiten, Vergleicht man die Tabellen V und VII deren Betrag ja durchaus nicht betrfichtlich ist, sowie die zugeh6rigen Kurventafeln Fig. 3 und beruhen, vermag ich zur Zeit nicht mit SicherFig. 5 genauer mit einander, so erkennt man heir anzugeben. Meine Vermutung geht dahin, sofort, daft das Verhalten der Zellulose mit daft das Adsorptionsverm6gen der Zelluloseteilchen sich gegenfiber den vier Flfissigkeiten dem des Zelloidins in einem wichtigen Punkte fast ganz fibereinstimmt. In beiden Fallen verschieden verh~ih, was wohl auch yon vornsinkt zun~ichst die Kurve der Gangunterschiede herein durchaus nicht unwahrscheinlich ist. Bestehen aber solche Verschiedenheiten, so w~re der Gesamtdoppelbrechung mit wachsenden Werten yon n~, bei den Werten zwischen 1,52 es auch ohne weiteres verst~.ndlich, wenn z. B. und 1,56 wird ffir die verschiedenen Wellen- beim Uebergang yore Wasser zum Benzylalkolangen nacheinander ein Minimum erreicht, und hol dutch den absoluten Alkohol hindu'rch und yon da ab steigen die Kurven wieder mil ebenso beim Verdr/ingen des Benzylalkohols weiter wachsendem n~. Ganz dasselbe zeigt durch das Monobromnaphthalin zeitweise geringe sich natfirlich auch bei den Werten ffir die Unterschiede im BrechungsvermSgen der ImSt~irke der St~ibchendoppelbrechung, die aus bibitionsflfissigkeit und der umgebenden Mischden beobachteten Gangumerschieden und der ung, deren Brechungsexponenten man direkt Streifendicke berechnet wurden. Zugleich fiillt bestimmt, erhalten bleiben. Daft das Adsorpaber auch bei dem Betrachten der Kurven auf, tionsverm6gen der Zellulose gegen Wasser o,o. o1 12 178 starker als gegen Alkohol ist, h~itte gar nichts Ueberraschendes; und ein ganz ~hnliches Verh~.ltnis k6nnte zwischen dem Benzylalkohol und dem Monobromnaphthalin bestehen. Wird z. B. das Wasser den Zellulosest~ibchen durch den absoluten Alkohol nicht vollst~,ndig entzogen und erst durch den hinzukommenden Benzylalkohol v611ig verdr~ingt, so mfiBte sich etwas Aehnliches im Vedauf der Kurven zeigen, wie w i r e s tats~chlich beobachlen. Und ganz entsprechend wfirden die Dinge liegen, wenn die Zellulose gegenfiber dem Benzylalkohol ein st~irkeres Adsorptionsverm6gen als gegen das Monobromnaphthalin bes~iBe. Ich m6chte ]edoch ausdrticklich betonen, dab diese Bemerkungen nicht mehr als eine Vermutung enthalten sollen ; denn zur Entscheidung dieser Frage bedfirfte es weiterer systematisch durchgeffihrter Untersuchungen. Dat~ bei dem Verhalten des Zelloidins derartige St6rungen nicht beobachtet wurden, lieBe sich dann auf Grund ]ener Vermutung wohl einfach damit erkl~iren, dab hier eigentlich nur eine Fltissigkeit, n~mlich das Kaliumqueeksilberiodid in w/isseriger L6sung in sehr verschiedenen Konzentrationen in Betracht kava. Hier d/.irften deshalb kaum gr6Bere Verschiedenheiten im Adsorptionsverm6gen, die solehe St6rungen hervorrufen wfirden, zur Geltung kommen. Abgeselien yon den beiden Ungleichm~iBigkeiten im Verlauf der Gangunterschiede, die fibrigens, wie aus der Tabelle und der Kurventafel hervorgeht, ffir alle untersuchten WellenIfingen an denselben Stellen !iegen, n~mlich ill der N/ihe der Werte 1,37 find 1,61 fiir n,~, zeigt das Verhalten der Zellulose und des Zelloidins in dem wichtigsten Punkte gute Uebereinstimmung. Die Gesamtdoppelbrechung ist in beiden F~illen yon d'em Brechungsexponenten der eingelagerten Fltissigl~eit abh~ingig und zwar in derselben Weise, wie dies nach der Theorie der Stfibchendoppelbrechung zu erwarten war. Berechnet man nach Berticksichtigung der Eigendoppelbreehung den Gang der St~rke der Stabchendoppelbrechung, so ergib[ sich auch ffir die Zellulose der charakteristische Verlauf in der Dispersion der St~bchendoppelbrechung, wie aus den ~-Werten in der Tabelle VIII deutlich hervorgeht. Auch das allm~hliche Ansteigen der %-Werte in Tabelle VII stimmt damit gut fiberein, denn hier f/illt natfirlicb eine zweimalige Umkehr im Vorzeichen der Doppelbrechung weg, da ia beide Komponenten, aus denen sich die Gesamtdoppelbrechung zusammensetzt, dasselbe Vorzeichen besitzen. Es kann sich deshalb, wie schon erw~ihnt wurde, keine so starke Abweichung i:l der Reihenfolge der Interferenzfarben zeigen, wie sie ffir das Zelloidin charakteristisch ist. Ganz besonders bemerkenswert ist es, dab die Zahlen ffir die Stiirke der reinen St~ibehendoppelbrechung in den beiden Fiillen so gut fibereinstimmen, w~ihrend doch die Eigendoppelbrechung, wie sie sich aus den Minima der Kurven ffir die Gangunterschiede ergibt, so betfiichtliche Unterschiede nicht bloB in ihrer St/irke, sondern auch in ihrem Vorzeichen aufweist. Allerdings ist dutch die Denitrierung eine ganz wesentliche ehemische und auch physikalische Vefiinderung eingetreten, aber man darf nach frfiheren Untersuchungen annehmen, dab der Aufbau der Systeme aus Stiibchen, die yon Flfissigkeit umhfillt sind, im wesentlichen derselbe geblieben ist ; auch die Beibehaltung der Form nach einem so starken Eingriff spricht entschieden ffir die Berechtigung dieser Annahme. Denn so, wie nicht nur bei der Nitrierung der Zellulosefasern, sondern auch bei der nachfolgenden Denitrierung die Faserform vollst~ndig erhalten bleibt, die Anordnung der die Fasern aufbauenden Teilchen also unvefiindert fortbesteht,, so darf man auch hier annehmen, dab die Anordnung der stfibchenf6rmigen Teilchen in den Zelloidinund in den Zellulosestreifen im wesentliehen dieselbe bleibt. 1st dies aber in der Tat so, dann muB aueh die Stiirke der Stfibchendoppelbreehung in diesen beiden F/illen nahezu gleich groi~ sein; denn die Werte f/ir nl, die Brechungsexponenten der Stfibchen, sind bei dem Zelloidin und der Zellulose offenbar nut wenig yon einander versehieden, und damit mfissen die Differenzen nl--n2, die ftir die St/irke der St~bchendoppelbreehung maflgebend sind, ebenfalls nur geringe Untersehiede zeigen, leh werde sp~iter auf diesen wiehtigen Punkt noehmats zurfickkommen. Am Sehlusse dieses Abschnittes m6ge noeh kurz auf die schon erw~ihnten geringen Dimensions~inderungen der Streifen bei der Denitrierung hingewiesen werden. Es wurde auch schon angedeutet, dab dieselben Dimensions~inderungen eintreten, wenn man nicht gedehnte, sondern frische optiseh vollstiindig isotrope Streifen denitriert. Solche Streifen zeigen nun nach der Umwandlung, auch ohne dab sie irgend eine kfinstliche Spannung erfahren haben, eine zwar schwache aber doeh immerhin deutliche Doppelbrechung; die l~ingere Achse des Indexellipsoids liegt auch hier parallel zur L~ingsrichtung der Streifen. Diese Doppel- 179 brechung hat also, wenigstens bei jener pnsmatischen Form der Streifen, denselben Charakter, wie d~e durch starke Dehnung hervorgerufene; sie zeigt aber auch dieselbe Abhangigkeit yon dem Brechungsexponenten der eingelagerten F1/issigkeit. Bringt man derartige Streifen nacheinander in Wasser, Aethylalkohol, Benzylalkohol und Monobromnaphthalin, so ist die Doppelbrechung" im Benzylalkohol am schw~ichsten und bei d/innen Streifen kaum bemerkbar, in Wasser erreicht sie den h6chsten Betrag, in Monobromnaphthalin ist sie etwas geringer. Dazwischen treten nat/.irlich alle Uebergange auf. [)as ist also ganz dasselbe Verhalten, das auch die vorher stark gedehnten Streifen aufweisen, und man darf daher wohl mit Recht annehmen, dab hier ebenfalls schon ein Zusammenwirken yon Stabchen- und Eigendoppelbrechung vorliegt. Halt man an der Annahme fest, dag die optische Isotropie des ungedehnten Zelloidins auf die regellose Lagerung der stabchenf~rmigen Teilchen zur/ickzuf/.ihren sei, so ist es auch verstandlich, wenn allein dutch die Dirnensions~inderungen bei der Denitrierung zu 'Zellulose eine gewisse Umlagerung dieser Stabchen eintritt, und die Summe der Einzelwirkungen ietzt yon Null verschieden ist. Liegt, wie in unserem Falle, ein prismafischer Streifen vor, in dem die Dimension in einer Richtung die in den andern bedeutend /iberwiegt, so wird die bei der Denitrierung eintretende Querkontraktion eine optische Anisotropie hervorrufen, vorausgesetzt, dat3 die Stabchen an sich schon optisch anisotrop sind. Es wirkt eben in diesem Falle die Querkontraktion so, als ob der Streifen dutch die Denitrierung einen allseitigen Druek senkrecht zur Langsrichtung und dadurch eine bleibende Deformation erfahren hatte. Bedenkt man augerdem noch, daf, wie ebenfalls schon fr/iher hervorgehoben wurde, bei der Denitrierung eine Vermehrung der Trockensubstanz, also eine Verringerung der Imbibitionsfl/issigkeit eintritt, und damit dieselbe Anzahl der Stabchen in einem in der Querrichtung engeren Raum Platz finden muf, so hat clie entstehende Anisotropie gar nichts Ueberraschendes. Eine genauere quantitative Untersuchung hiertiber wtirde sehr erwtinscht sein; da aber die eben geschilderten Ver~inderungen f/Jr (tie zun~ichst zu er6rternden Beziehungen zwischen Stabchen- und Eigendoppelbrechung nur yon nebensachlicher Bedeutung sind, so wurden jene Fragen nicht welter verfolgt, als es f/.ir den Hauptzweck n6tig erschien. Der Verlauf der Gangunter- schiede, wie er oben ausf/ihrlich geschildert wurde, wird namlich dadurch nicht weiter beeinfluflt, daf durch die Denitrierung an sich schon eine schwache Eigendoppelbrechung entsteht; diese addiert sich eben zu der welt betrachtlicheren, die durch die starke bleibende Deformation hervorgerufen ist. Es wird also an dem Gesamtergebnis, soweit es die Feststellung der St~ibchendoppelbrechung und ihres Zusammenwirkens mit der Eigendoppelbrechung betrifft, gar nichts geiindert. Nur wenn man den Betrag der Eigendoppelbrechung f/Jr eine beslimmte bleibende Verlangerung genau berechnen will, muf man die durch die Denitrierung allein schon entstandene beriicksichtigen. Da abet die genaue quantitative Bestimmung der Starke der Eigendoppelbrechung aus verschiedenen fr/.iher schon angedeuteten Gr/inden auf Schwierigkeiten st6t~t, so hat es f/Jr unsere Fragen ke;nen Zweck, neben dem welt iiberwiegenden Einfluf der wirklich vollzogenen Spannungsdeformation jenen geringen dutch die Denitrierung allein eingetretenen durch die Rechnung scharf hervorzuheben. Dagegen hat es yon anderen Gesichtspunkten aus ein gewisses Interesse, zu untersuchen, in welcher Weise die urspr/-ingliche Form der Zelloidinsttick~e auf die Art der Doppelbrechung nach der Denitrierung einen Einfluf hat. Es ist von vornherein zu erwarten, dab ein solcher Einflug besteht, daft z. B. ein Wtirfel oder eine kreisf6rmige Scheibe sich anders verhalten, als ein prismatischer Streifen, dessen Lange den Querdurchmesser bedeutend fibertrifft. DaB in dieser Beziehung ein ganz charakteristischer Unterschied besteht, haben die zur vorl~iufigen Orientierung angestellten Versuche schon mit Sicherheit ergeben und ic h hoffe, in einer spateren Mitteilung dar/iber genauer berichten zu kSnnen. X. Das besonders merkw/irdige Verhalten des Zelloidins hatte die Veranlassung zu der hier vertretenen Annahme gegeben, dab die akzidentelle Doppelbrechung dieses K6rpers im imbibierten Zustand auf das Zusammenwirken zweier Komponenten von entgegengesetztem Vorzeichen zur/ickzuf/ihren sei. Auf Orund dieser Annahme liet~ sich auch sofort der iiberraschende zweimalige" Durchgang durch den isotropen Zustand erkl~iren. Allerdings konnte dabei die Starke der 'Doppelbrechung in beiden Komponenten nicht zugleich konstant sein, wie dies bei den Apophylliten oder bei den ganz 12" 180 ahnlich sich verhaltenden Mischkristallen aus dem optisch positiv einachsigen Bleidithionat und dem isomorphen abet negativen Strontiumdithionat der Fall istT). Denn hierbei kann nur ein einmaliger Durchgang dutch den isotropen Zustand bei einer ganz bestimmten Zusammensetzung der Mischung auftreten, wie es auch die Erfahrung bestatigt hatte. Soil dagegen eine zweimalige Nullwirkung statffinden, so muff die eine Komponente eine Doppelbrechung helvorrufen, deren Vorzeichen zwar stets dasselbe bleibt, deren St~irke abet in einer ganz bestimmten Weise ver~inderlich ist. Al's eine solche Art yon Doppelbrechung konnte nun in dem Zwei-Stoff-System des imbibierten Zelloidins nur die St~ibchen- oder die Schichtendoppelbrechung im Sinne O. W i e n e r's in Betracht kommen. Besteht das System aus einer festen und einer fliassigen Komponente, so mut3 in beiden F~illen durch Veriinderung des BrechungsvermSgens der Fl/issigkeit auch eine Veriinderung in der Starke der (3esamtdoppelbrechung eintreten. Mit Rficksicht auf die zweimalige Nullwirkung bei allm~ihlicher ErhShung des Brechungsexponenten no, waren zwei M6glichkeiten denkbar: entweder lag Stiibchendoppelbrechung vor, dann mu~te die Doppelbrechung der anderen Komponente negativen Charakter besitzen; oder es handelte sich um Schichtendoppelbrechung, dann muBte die Doppelbrechung~ der anderen Komponente positiv sein. Bei den bleibend verl~ingerten Zelloidinstreifen kann, wie sp~iter noch ausffihrlicher zu begrfinden sein wird, als die ver~inderliche Komponente nur die St~ibchendoppelbrechung in Frage kommen ; also muff die Doppelbrechung der anderen Komponente, d.h. in diesem Falle die der St~bchen selbst, negativ sein. Hieraus ergeben sich zwei wichtige Folgerungen: erstens mfissen die Teilchen, aus denen die Streifen aufgebaut sind, rgtumlich anisotrop sein, d. h. sie m/issen eine der St~tbchenform sich nahernde GestaI.' haben; und zweitens mfissen diese Teilchen nicht bloB r~iumlich, sondern auch optiseh anisotrop sein. Dutch die starke bleibende Verl~ingerung werden die St~ibchen so orientiert, dat~ ihre L~ingsachsen einer Parallelstellung zustreben und somit ihre Gesamtwirkung optisch als eine yon Nuli verschiedene Summe der Einzeiwirkungen erscheint, wahrend im nicht deformierten Zelloidin wegen der v611ig regellosen Lagerung de~ St~ibchen diese Summe der Einzelwirkungen gleich Null werden muff, well sie sich immer paarweise gegenseitig aufheben. 7) Zeitschr. f. Krystallogr. 52, 48--57 (1913). Diese Folgerungen sind aber nur berechtig\|, wenn der Verlauf der Gesamtdoppelbrechung und der aus ihr errechneten St~ibchendoppelbrechung mit den Forderungen /ibereinstimmt, die sich aus den W i e n e r'schen Gleichungen ergeben. Wie die Untersuchungen gezeigt haben, ist dies in der Tat in weitgehender Weise der Fall. Wenn auch die Kurve f/Jr die St~ibchendoppelbrechung nicht ganz mit derienigen fibereinstimmt, die ffir einen ldealfall gefunden wurde, so zeigt sie doch im wesentlichen ganz ~ihnliche Eigenschaften, wie aus den Darlegungen auf S. 280 in Mitteilung II hervorgeht. Besonders wichtig erscheint mir auch, daI~ ganz dieselben starken Verschiedenheiten in der Dispersion der Doppelbrechung auftreten, wie ein Vergleich der Tabellen VI und VIII sofort ergibt. Ebenso wie das Verhalten des Zelloidins l~iBt auch das der Zellulose die gezogenen Folgerungen bereehtigt erscheinen. Hier wie dort ist die gesetzm~iffige Ver~inderung der Gesamtdoppelbrechung und der daraus zu bestimmenden Stabchendoppelbrechung mit dem allm~ihlichen Ansteigen der Werte von n2 unverkennbar. Wenn auch der Verlauf der Kurven nicht ganz so gleichmaffig wie beim Zelloidin ist, indem an zwei Stellen geringe StSrungen bemerkbar werden, so sind diese doch so geringf/igig, dab sie die wesentliehen Uebereinstimmungen fiberhaupt nicht beeintriichtigen kSnnen. Das Auftreten der Minima an den Stellen, wo die Differenz nl m n g . = 0 wird, und das Bestehen der gr6Bten Gangunterschiede in den FlfJssigkeiten, fiJr die die Differenzen n l - n~ am h6chsten sind, sowie die dazwischen liegenden stetigen Uebergange lassen die groBe Aehnlichkeit in beiden F~illen mit gen/igender Sicherheit hervortreten; zumal jene gerirrgen StSrungen in anderer Weise eine ganz befriedigende Erkliirung finden kSnnen. Auch dag in der Dispersion der Doppelbrechung dieselben starken Verschiedenheiten auftreten, wie sie sich theoretisch f/it ein solches Zwei-Stoff-System ergeben m/issen, spricht entschieden ffir die Berechtigung ]ener Folgerungen. Es kOnnte mir nun vielleicht eingewendet werden, dab die aus den Beobachtungen gezogenen Schl/Jsse gewissermaffen schon in den gemachten Voraussetzungen fiber die Mitwirkung yon St~ibchendoppelbrechung versteckt seien. Solchen etwaigen Einw/irfen mSchte ich gleich ietzt in einigen Bemerkungen entgegentreten. DaB wir es in den untersuchten Kt~rpern mit S y s t e m e n zu tun haben, die sich 181 aus einer festen und einer fl~ssigen Ko m p o n e n t e zusammensetzen, unterliegt jedenfails keinem Zweifel. Es erscheint mir ferner als ganz sicher, dab die zur lmbibition verwendeten Flfissigkeiten weder beim Zelloidin noch bei der Zellulose irgend eine chemische Ver/inderung der festen Teilchen hervorrufen. Das Zelloidin verhiilll sich gegenf.iber dem Wasser und ebenso auch den verschieden konzentrierten L6sungen yon Kaliumquecksilberjodid v611ig indifferent, wie vielfache Vorversuche sowohl mit Zelloidinstreifen, wie auch mit unver~inderten Nitrozellulosefa,~ern yon hohem Stickstoffgehalt bewiesen haben. Ebenso rul~en weder Wasser, noch Alkohol, noch "13enzylalkohol oder Monobromnaphthalin irgend eine chemische Aenderung der Zellulose hervor. Alle Fl~ssigkeiten, die eine Q~tellung oder eine merkbare Formveranderung an den Streifen bewirkt hatten, waren bei der Auswahl sorgf/iltig ausgeschieden worden. Auch dafll die ganzen Aenderungen in der Gesamtdoppelbrechung beim Wechseln der Pl~ssigkeiten vollstandig reversibel waren, spricht entschieden dafiir, dab die chemischen und auch physikalischen Eigenschaften der festen Teilchen unver~indert blieben. Es w/ire ferner auch kaum zu erkl~iren, warum beim Zelloidin nur bei einer ganz bestimmten Konzentration der Quecksilbersalzl6sung das Minimum der Kurve erreicht wird, w~ihrend sowohl bei einem nur wenig geringeren oder nur wenig h6heren Wassergehalt der LOshng sogleich wieder ein Ansteigen des Ganguntersehieds in demselben Sinne eintritt. Dasselbe gilt f/.ir das Verhalten der Zellulose; auch hier ist mit Sicherheit zu beobachten, wie nur. bei einer ganz bestimmten Mischung das Minimum der Kurven erreicht wird. Ein nur geringer Zusatz yon der hOher oder der niedriger brechenden Fl/.issigkeit bewirkt sofort wieder ein Ansteigen des Gangunterschieds in dem gleichen Sinne. Atle diese Ueberlegungen fiihren, wie ich glaube, unbedingt zu dem Schlusse, dab es sich hier um eine mit dem BrechungsvermSgen der flfissigen Komponente veriinderliche Doppelbrechung handelt. Wiiren nun die festen Teilchen an sich optisch isotrop, so mfiBte bei Anwendung e i n e r Fliissigkeit, deren Brechungsexponent mit dem der Teilchen /ibereinstimmt, das System ebenfalls isotrop bleiben. Allerdings tritt beim Zelloidin, wie wir wissen, z w e i m al Isotropie auf, aber nur dann, wenn die Differenz nl - - n~ nicht Null, sondern das eine Mal einen-positiven und das andere Mal einen negativen Wert hat. Bei der Zellulose dagegen wird die Gesamtdoppelbrechung ~berhaupt bei keiner F1/.issigkeit Null. Wird also n 1 - - n2 = 0, so lal~t sich bei beiden K6rpern nicht Isotropie, sondern eine bestimmte Starke der Doppelbrechung beobachten, die beim Zelloidin das negative, bei der Zellulose das positive Vorzeichen besitzt. Diese Doppelbrechung kann aber nur eine Eigenschaft der festen Komponente sein, und da die festen Teilchen durch die verschiedenen Fl~issiglteiten keine Ver~inderungen erfahren, so muB sie bei allen Versuchen konstant bleiben. Ich habe sie deshalb yon vornherein als Eigendoppelbrechung bezeichnet. Noch auf eine weitere Beobachtung m6chte ich kurz hinweisen, die zwar nichts Neues lehrt, die aber in bequemer Weise den entgegengesetzten Charakter der Eigendoppelbrechung in beiden K6rpern bezeugt. Stellt man aus den stark gedehnten Streifen nicht zu dfinne Querschnitte her, so kann man an diesen auch das Verhalten im sog. konvergenten Licht pr/.ifen. W~ihlt man zunachst diejenigen Fl~ssigkeiten, bei denen n l - n~ = 0 ist, so erkennt man sofort, dat~ die Schnitte aus dem Zelloidin das Achsenbild eines negativen einachsigen und die aus Zellulose das eines positiven Kristalls zeigen. Beim Zelloidin kann man nun das negative Achsenbild leicht in ein positives umwandeln, wenn man z. B. die Schnitte in Wasser legt. Bei der Zellulose dagegen zeigen die Schnitte in alien F1/.issigkeiten das positive Achsenbild. In den Zelloidinschnitten sieht man auch beim allmahlichen Steigen des Wertes yon n2 recht gut dieselben Erscheinungen, wie sie ffir die Achsenbilder verschiedener VarietY.ten yon Apophylliten seit langem bekannt sind. Xl. In jedem System, das aus einer flrassigen und einer festen Komponente in stark dispersere Zustande besteht, kann nur dann optische lsotropie eintreten, wenn die Differenz n1 - - n 2 verschwindet und die Teilchen v611ig regellos gelagert sind. Dabei ist es gleichgiltig, ob die Teilchen selbst optisch isotrop oder anisotrop sind; denn auch im zweiten Falle m/JBte die Summe der Einzelwirkungen Null sein. Bleibt n 1 - - n 2 = 0 und wird durch irgend einen EinfluB die Lagerung der Teilchen in der Weise geiindert, dab eine Ungleichwertigkeit der Richtungen in dem System zustande kommt, dann kann Doppelbrechung auftreten, wenn die Teilchert selhst anisotrop sind. Mag die Aenderung der Lagerung nun durch mechanische Defor- 182 matlon, dureh ein elektrisches oder magnetisches Peld, oder such durch irgend eine gerichtete Str6mung erzielt werden; stets muB dann die Summe der Einzelwirkungen yon Null verschieden sein. Das ist im wesentlichen auch der Sinn der in den letzten-Jabren oft besproebenen L a n g e v i n ' s c h e n Orientierungshypothese, die besonders zur Erkl~irung der elektro- urrd magnetooptischen Doppelbrechung gewisser kolloider LSsungen aufgestellt worden ist. Auch die merkwfirdigen optischen Eigenschaften der sog. flfissigen Kristalle, sowie manche Erscheinungen bei geriehte~en Str/3mungen in gewissen kolloiden L6sungen sind auf eine Orientierung an sich schon optisch anisotroper Teilchen zurfickgefiihrt wordenS). Auf alle diese Dinge kann jedoch in dieser Mitteilung nicht niihe~r eingegangen werden, es soil vielmehr nur darauf hingewiesen werden, dab derartige Anschauungen keineswegs neu sind. Sehon vor mehr als 50 Jahren haben E. B r f i c k e und C. N ~ i g e l i die Anisotropie der Fasern des Tier- und Pflanzenk6rpers auf eine gleichsinnige, oder doch anniihernd gleichsinnige Orientierung anisotroper Molekfilkomp'exe zuriickgefiihrt, und besonders N ~ig e I i hat diese Anschauung in mehreren sehr exakten Arbeiten genauer zu begrfinden versucht; e r nannte spiiter diese Teilchen ~Mizelle ~ und danach hat man seine Anschauung such wohl als die Hypothese der kristallinischen Mizelle, oder kurz als Mizellarhypothese bezeichnetg). Sie behandelt keineswegs nut die optischen Eigenschaften der Zellhiiute u. dergl., sondern such die Quellbarkeit, die Wachstumserscheinungen, die Koh~sionsverh~ltnisse usw. Auch auf diese Dinge niiher einzugehen konnte hier nicht meine Aufgabe sein; aber ich wollte doch auf jene Ansichten fiber den Aufbau der Zellh~iute, besonders der Zellulosefasem hinweisen, well ja die beiden K6rper, die ich bei meinen Untersuchungen vorzugsweise benutzte, als Derivate der in der Natur vorkommenden Zellulose betrachtet werden miissen. Hiilt man an der N iig e I i'schen Ansicht fest und berficksichaj Vgl. hierzu: W. V o i g t , Elektrooptik und Magnetooptik (HandwOrterbuch d. Naturw. 3, 483, u. 6, 712), ferner: Ders., Fliissige Kristalle und anisotrope Fltissii~keiten (Physik. Zeitschr. 17, 76--87, 128--135, 152--161); H. D i e s s e l h o r s t und H. F r e u n d I i c h, Ueber Sehlierenbildung in kolloiden LOsungen und ein Verfahren, die Gestalt der Kolloidteilehen festzustellen (Physik. Zeitschr. 17, 117--128, 1916). 9) Eine Zusammenstellung der Literatur hiertiber finder sich in : V.v. E b n e r, Untersuehungen fiber die Anisotropie organisierter Substanzen (Leipzig 1882). figt man aui~erdem die Vorg~inge bei der Entstehung der Nitrozellulose, die fast vollkommene Erhaltung der Faserform und den dabei eintretenden Wechsel im Charakter der Doppelbreehung, so liegt die weitere Annahme nahe, dab die Form der Zellulosemizelle trotz dem starken chemischen Eingriff gar keine wesentliche Aenderung ihrer Gestalt erf~.hrt. Auch bei der starken Auflockerung, die die SchieBbaumwolle bei der Herstellung des Zelloidins erflihrt, kann wohl nur eine v611ige Desorientierung der Teilchen eintreten, ihre Einzelgestalt wird aber sicher beibehalten. Und wandelt man nun wieder das Zelloidin durch Dethitrierung in Zellulose urn, so wird, wie schon mehrfach angedeutet worden ist, auch hierbei keine irgendwie wesentliche Aenderung in der Form der Mizelle stattfinden. Wir kommen so auch auf einem ganz anderen Wege zu demselben SchluB, den wir bereits aus den Ergebnissen der Untersuchungen fiber den Verlauf. der Doppelbrechung in den bleibend verliingerten Streifen aus Zelloidin und Zellutose gezogen hatten, dab niimlich die festen Teilchen in diesen Systemen nicht nur r~iumlich, sondern auch optisch anisotrop seien. Die Teilchen haben eben st~ibchenf6rmige Gestalt, und bei der starken bleibenden Deformation wird ihre frfibere regellose Lagerung so vedlndert, dab sie nunmehr einer Orientierung zustreben, in der ihre Liingsachsen mehr und mehr eine der Dehnungsrichtung sich niihernde Lage einnehmen. Aus dem optischen Verhalten der Nitrozellulosefasern, wie es sich aus den Untersuchungen H a n s A m b r o n n ' s t~ ergeben hat, dfirfen wir ferner schlieisen, dab die st~ibchenf6rmige MizeUe mit ihren Liingsachsen parallel der Liingsachse der Fasern gestellt ist; besonders die Eigenschaften der gefiirbten Fasern machen dies sehr wahrscheinlich. Wie ich frfiher schon gezeigt habe, ergeben sich bei der Einlagerung yon Farbstoffen in die verliingerten Zelloidinund Zellulosestreifen ganz dieselben Verschiedenheiten in der Absorption nach verschiedenen Richtungen. Es erseheint mir deshalb ganz berechtigt, wenn ich auch hieraus den Schlug ziehe, dab die festen Teilchen in diesen K6rpern im wesentlichen dieselbe Gestalt haben, m6gen sie nun in bezug auf ihre Liingsachse positive oder negative Doppelbrechung besitzen. Aus allen diesen auf verschiedenen Wegen gewonnenen Ergebnissen daft man nun wohl mit groi3er Sicherheit. annehmen, daiS zu der stets positiven Stiibchendoppelbrechung beim ~0) Hans A m b r o n n , a. a. O. 30 flgde, 183 Zelloidin negative und bei der Zellulose positive Eigendoppelbrechung hinzukommt und dab das Zusammenwirken dieser beiden Komponenten den charakteristischen Verlauf der Gesamtdoppelbrechung, wie er sich bei meinen genauen Messungen darstellte, in ganz befriedigender Weise zu erkl~ren vermag. Die Eigendoppelbrechung darf man wohl auch trotz den eingetretenen Spannungsdeformationen als konstant annehmen, denn es sind zum Erzeugen der bleibenden Verl~ingerungen nur schwache Spannungen n6tig; das Zelloidin im imbibierten Zustande ist eine sehr plastische Masse, die schon durch einen schwachen Fingerdruck eine Veranderung der Form erf/ihrt. Es ist deshalb kaum anzunehmen, dab in einem solchen K6rper, der nahezu 80 Proz. Fl/~issigkeit enthalt, eine Deformation der St/ibchen selbst eintritt, es ist ohne Zweifel viel wahrscheinlicher, dab nur eine Aenderung in ihrer Lage herbeigeffihrt wird. Wir haben es also mit einem ganz ahnlichen Vorgang zu tun, wie ich ihn fr/.iher bei einigen pflanzlichen Gummiarten angenommen habe und wie man ihn k/instlich erzeugen kann, wenn man Faden aus einer Mischung yon Kolophonium und Wachs zieht. Auch auf diese Untersuchungen kann ich hier nicht n~iher eingehen, sondern mub auf die hier/iber ver6ffentlichten Arbeiten verweisen ~1). XII. Die Werte f/it die Eigendoppelbrechung, wie sie aus den Gangunterschieden berechnet wurden, bei denen die Stiibchendoppelbrechung als ausgeschaltet betrachtet werden konnte, ergeben nun nicht etwa die St~irke der Doppelbrechung der Stabchen selbst, aus denen die Systeme aufgebaut sind, sondern nur diejenige Differenz der Brechungsexponenten in zwei senkrecht aufeinander stehenden Richtungen, die aus der Summe der Einzelwirkungen entsteht. Erst wenn die Deformation soweit getrieben werden k6nnte, dab alle St/ibchen wirklich parallel orientiert w~iren, dann w/irde die Eigendoppelbrechung des Systems mit der der Einzelst/ibchen /.ibereinstimmen. Je starker die bleibende Verlangerung ist, desto mehr mut~ die Richtung der Langsachsen der einzelnen St/ibchen yon der regellosen Lage abweichen. Die Gesamtgeirkung des Systems wird deshalb, zun/ichst ganz abgesehen yon der hinzukommenden Stab1~) Ueber das opltische Verhalten der Kutikul~l und der verkorkten Membranen (Ber. d. Deutsch. Bot. ~es. 6, 226--230~ 1888), sowie die schon unter ~)__4) genannten Mitteilungen. chendoppelbrechung, von dem Grade der Deformation abh~ingen. Ein Streifen, d e r n u r um 25 Proz. seiner urspr/inglichen L~inge gedehnt worden ist, zeigt natfirlich eit~e geringere Doppelbrechung, als ein sotcher, der eine bleibende Verl/ingerung um 100 Proz. erfahren hat Um vergleichbare Ergebnisse bei den Messungen der Gangunterschiede zu erhalten, sind deshalb stets, wie schon anfangs erw/ihnt worden ist, in demselben Grade verl/ingerte Streifen verwendet worden; denn nur in diesem Falle darf man annehmen, dab die Sdirke der Doppelbrechung der einen Komponente, also der Eigendoppelbrechung, konstant bleibt, wenn nach einander verschieden brechende Imbibitionsflfissigkeiten in das System eingeffihrt werden Abet nicht bloB die Eigendoppelbrechung ist yon d'em Grade der Deformation abh~.ngig sondern auch die St~ibchendoppelbrechung denn diese mub gleichfalls um so h6her werden je mehr die Orientierung der St~ibchen sich der v611igen Parallelstellung niihert. Sowohl die Eigendoppelbrechung wie die St~ibchendoppelbrechung mub deshalb stets niedriger sein, als in einem System, in dem eine vollkommen parallele Anordnung der St~.bchen vorhanden ist. Da dieser Grenzfall aber durch mechanische Deformation wohl kaum erreicht werden kann, so wird sich ffir jeden Grad der Vell~.ngerung auch eine bestimmte St~irke der Eigendoppelbrechun~ und f/ir den Wert yon n2 eir~e bestimmte St~irke der Stiibchendoppelbrechung ergeben. Besitzen beide entgegengesetztes Vorzeichen, so muB die Gesamtdoppelbrechung dann Null werden, wenn die aus der Sttirke der beiden Anisotropien sich ergebenden Oangunterschiede absolut gleich werden. Dab dieser Fall bei demselben Wert yon n2 ffir verschiedene Grade der Verlangerung eintritt, ist yon vornherein unwahrscheinlich; denn die Ver~inderung der Eigendoppelbrechung und die der St~ibchendoppelbrechung bei zunehmender Deformation stehen wohl sicher nicht in so einfacher Beziehung zu einander. Es mub also, wie fibrigens die bereits angestellten Versuche bestatigt haben, der Nullwert f/Jr den Gangunterschied des ganzen Systems bei Verschiedenheit im Grad der Deformation auch bei verschiedenen Werten yon n2 auftreten. Es ist nun yon besonderem Interesse, dieses Wandern der Nullwerte durch genaue Messungen fiir verschiedene Wellenl~ingen zu verfolgen. Man kann dabei entweder in der Weise verfahren, dab man an demselben Streifen, der mit einer Fliissigkeit yon bestimmtem Brech- 184 ungsexponenten durchtrankt ist, bei fortschreitender Dehnung beobachtet, oder indem man Streifen yon bestimmter Verlangerung nacheinander mit verschiedenen F1/jssigkeiten imbibiert und dann feststellt, bei welehen Werten yon ne der Gangunterschied auf Null herabgeht. Auf diese Weise den Einflut~ des Deformationsgrades auf die St~irke der Gesamtdoppelbreehung zu pr/jfen, ist nat/jvl~eh n ur beim Zelloidin mSglich; denn nur hier kann eine solche Nullwirkung erwartet werden. Aber auch bei der Zellulose kann man die Unterschiede untersuchen, die sich bei verschiedener Starke der Deformation geltend maehen. Wie die bisher angestellten Versuche ergeben haben, wird mit zunehmender Deformation sowohl die Starke der Stabchendoppelbrechung wie die der Eigendoppelbrechung gesteigert. Die genaueren Messungen dieser Veranderungen sind noch keineswegs abgeschlossen, aber es dart schon ietzt hervorgehoben werden, dab die Beziehungen zwischen dem Deformationsgrad und der Gesamtdoppelbrechung sowohl wie der Eigenund St~ibehendoppelbrechung im besonderen manchen interessanten AufschluB zu geben versprechen. Hat man einmal die genauen Zahlen ~/ir die Stiirke der Doppelbrechung bei 25, 50, 75, 100 Proz. bleibender Verlangerung, so kann man daraus vielleicht auch durch Extrapolation die Werte f/Jr noeh betrachtlich starkere Deformationen wenigstens annaherungsweise erhalten, und aus diesen werden sich dann eher Schl/jsse auf das Verhalten bei vollstandiger Parallelstellung der St$ibehen ziehen lassen. Hierfiber hoffe ich in einer weiteren Mitteilung eingehender berichten zu k6nnen. Eine viel gr6Bere Ann~iherung ari vOllig parallele Orientierung der Stabchen oder Mizelle ist iedenfalls in vielen Zellh~iuten von Pflanzen verwirklicht, besonders in den sehr langgestreckten Bastfasern, wie sie manche Nesselarten besitzen. Solche Zellen sind oft fiber 20 cm lang, und ihre W~inde zeigen eine St~irke der Doppelbrechung, die die des Quarzes mehr als sechsmal /jbertrifft. Hieraus kOnnte man eher auf die Starke der Doppelbrechung der Zellulosestabchen schlieBen; denn iene Membranen verhalten sich innerhalb kleiner Flachengebiete fast wie einheitliche Krist~ilie. lhr gesamter Aufbau stellt allerdings wegen der radialen und konzentrischen Anordnung der optischen Symmetrieachsen ein zylinderf6rmiges Kristallaggregat dar. Vergleicht man nun. mit der Doppelbrechung dieser Zellulose die aus den vorstehenden Messungen ermittelte, so fallt der groBe Unterschied auf; denn das System der Stabchen in den bleibend verlangerten Zellulosestreifen besitzt bei mittlerer Wellenl~inge f/jr die Differenz n a ~ no nur den Wert 0,013, die Starke der Doppelbrechung ist also nut wenig gr6Ber als die des Quarzes, die 0,009 f/jr dieselbe Wellenlange betr~igt. Diese groBe Verschiedenheit erscheint wohl etwas ~berraschend, sie erklart sich aber leicht daraus, dab in den untersuchten Zellulosestreifen der Idealfall der v61ligen Parallelstellung der Stabchen sicher bei weitem noch nicht erreicht ist. AuBerdem ist wohl auch anzunehmen, dat~ die Doppelbrechung der durch Denitrierung gewonnenen Zellulosemizelle etwas geringer ist, als die der Zellw~nde in den Nesselfasern. Auch f/Jr die Nitrozellulose besteht ein ahnlicher Unterschied. Wie aus fr/jheren Beobachtungenl~) hervorgeht, besitzen die nitrierten Ramiefasern yon 13,16 Proz. Stickstoffgehalt f/jr die Wellenlange 546/~/~ einen Gangunterschied yon 0,32; daraus berechnet sich bei einer Dicke yon 20 die Starke der Doppelbrechung no - - na : 0,008. Nach den angenaherten Bestimmungen der entsprechenden Zahl f/ir die gedehnten Zelloidinstreifen ergibt sich dagegen f/jr dieselbe Wellenlange nur 0,001. Erst wenn man in der Lage w~ire, nach der vorhin angedeuteten Merhode die Starke der Doppelbrechung ffir v611ige Parallelstellung der Stiibchen zu ermitteln, k6nnte man auch einen genaueren quantitativen Vergleich dieser Zahlen ansteUen. Am Schlusse dieser Mitteilung m6chte ieh nochmals auf diejenigen Ergebnisse hinweisen, denen meines Erat:htens eine allgemeinere Bedeutung zukommt. Die Messungen haben bewiesen, dab die Doppelbrechung der untersuchten K6rper in einer bestimmten Weise yon dem Brechungsverm6gen der eingelagerten Fl/jssigkeit abh~ingt. Die Kurve der Gangunterschiede zeigt in beiden Fallen ein Minimum, wenn das Brechungsverm6gen der festen Teilchen und das der F1/jssigkeit /jberelnstimmt. Es wurde daraus der SchluB gezogen, dab die beobachtete Doppelbrechung dutch das Zusammelawirken zweier Komponenten zustande kommt. Davon bleibt die eine.bei der Einlagerung verschieden brechender F1/jssigkeiten konstant; sie muB als eine Eigenschaft des Systems der festen Teilchen betrachtet werden, und ich habe sie deshalb als Eigendoppelbrechung bezeichnet. ,Die andere dagegen ist mit dem Brechungsverm6gen der Fl/jssigkeit veranderlich, und auf 1~) Hans Ambronn a.a.O. 24, 185 Grund verschiedener Ueberlegungen kam ich zu dem weiteren SchluB, dab diese zweite Komponente als St/ibchendoppelbrechung im Sinne O. W i e n e r's aufzufassen sei. Ist abet diese Folgerung berechtigt, dann mfissen die festen Teilchen in den untersuchten Systemen st/ibchenf6rmige Gestalt besitzen und durch die bleibende Verl/ingerung so orientiert werden, dab ihre L~ingsachsen einer Parallelstellung mit der Dehnungsrichtung zustreben. Je st/irker die Verhingerung ist, desto mehr wird die Anordnung der vorher regellos gelagerten Teilchen sich diesem Zustand n~ihern, und desto st[irker muB sowohl die Eigendoppelbrechung wie die St~ibchendoppelbrechung des Systems werden. Besitzt die Eigendoppelbrechung, wie dies beim Zelloidin der Fall ist, das negative Vorzeichen, so muff in zwei bestimmten F/illen als Resur tierende optische Isotropie eintreten, was die Versuche best~itigt haben. Gerade dieser Urnstand scheint mir besondere Beweiskraft ffir die vorgetragene Ansicht zu haben. Es w/ire sehr erw/inscht, wenn noch auf anderem Wege die r/iumliche Anisotropie der Teilchen und der Uebergang aus der regellosen Lagerung in eine n~tehr orientierte Anordnung bewiesen werden k6nnte. Es liegt nahe, an die Durchstrahlung der Streifen mit R6ntgenstrahlen zu denken. Jedoch glaube ich, dab dabei kein befriedigendes Ergebnis zu erhalten w~ire, well die gleichsinnige Orientierung selbst bei sehr ~tarker Verltingerung keineswegs so welt vorgeschritten ist, dab sich wie bei einem einheitlichen Kristall ein charakteristisches L a u e Diagramm ergeben w/irde. Auch F. R i n n e la) hat an stark gedehnten Zelluloidstreifen keine Wirkung in diesem Sinne erhalten. Mehr Aussicht auf Erfolg dtirften dagegen Versuche haben, die nach der Methode yon P. D e b y e und P. S c h e r r e r 14) angestellt wtirden und zwar nicht la) F. Rin n e, Beitrtige zur Kenntnis der KristallR6ntgenogramme (Ber. der Kgl. Sachs. Oes. der Wiss. Math.- phys. KI. 67, 312, 1915~. 14) p. D e b y e und P. S c h e r r e r , Interferenzen aus regellos orientierten Teilchen im ROntgenlicht, I an den gedehnten, sondern an den noch isotropen Streifen. Nach den theoretischen Darlegungen und den Versuchen dieser beiden Forscher kann man bei Durchstrahlung sog. amorpher K6rper mit R6ntgenstrahlen aus den Interferenzerscheinungen feststellen, ob ein kristallinischer oder amorpher Zustand vorliegt. Die Untersuchungen wurden mit feinen Pulvern verschiedener K6rper, wie amorphem Bor, amorphem Silizium, Lithiumfluorid u . a . angestellt, und ergaben mit Sicherheit den kristallinischen Zustand dieter K6rper. D e b y e und S c h e r r e r weisen auch darauf hin, dab die Interferenzen, die W F r i e d r i c haS) bei der Durchstrahlung yon Wachs und Paraffin erhalten hatte, fiir das Vorhandensein regellos gelagerter sehr kleiner Kristtiilchen in diesen K6rpern sprechen. DaB eine solche mikrokristallinische Struktur bei Wachs und Paraffin sowie bei vielen Fetten u. dergl, in der Tat besteht, darauf haben schon Versuche von D. Brewster aus der Mitre des vorigen Jahrhunderts schliet~en lassen16). Man kann fibrigens die zahllosen sehr kleinen Krist~illchen z.B. in einer dfinnen Wachsschicht im Polarisationsmikroskop bei stiirkerer Vergr6Berung sofort erkennen. Wfirde man nun bei der Durehstrahlung frischer Zelloidinstficke [ihnliche Interferenzwirkungen erhalten, so wtire damit ein sehr erwfinschter weiterer Beweis ffir die r/iumliche und optische Anisotropie der darin enthaltenen festen Teilchen erbracht. Leider bin ich zur Zeit nicht in der Lage, derartige Versuche anzustellen. (Nachr. d. Kgl. Oes. d. Wiss. in OOttingen Math.-phys. Ki. 47, 1--15, 1916, und Physik. Zeitschr. 17, 277 bis 283, 1916). 15) W. F r i e d r i c h , Physik. Zeitschr. 14, 317 (1913). t6) D. B r e w s t e r , On the production of crystalline structure in crystallised powders by compression and traction (Trans. of the Roy. Soc. ol Edinburgh 20, 558, 1853). Vgl. auch die oben schon unter 2), ) und u) zitierten Arbeiten, sowie S. S c h w e n d e n e r , ~3esammelte botan. Abhandl. 1, 375 0898).
https://openalex.org/W2734455281	https://ro.uow.edu.au/cgi/viewcontent.cgi?article=3699&context=aiimpapers	English	null	Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair	Scientific reports	2,017	cc-by	11,740	Disciplines Disciplines Engineering \| Physical Sciences and Mathematics Abstract Abstract Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called "Biopen". To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co- axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10seconds of exposure to 700mW/cm2 of 365nm UV-A, containing >90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering. Publication Details Publication Details Duchi, S., Onofrillo, C., O'Connell, C. D., Blanchard, R., Augustine, C., Quigley, A. F., Kapsa, R. M. I., Pivonka, P., Wallace, G., Di Bella, C. & Choong, P. F. M. (2017). Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair. Scientific Reports, 7 5837-1-5837-12. University of Wollongong University of Wollongong Research Online Research Online Australian Institute for Innovative Materials - Papers Australian Institute for Innovative Materials 1-1-2017 Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair repair Serena Duchi University of Wollongong, University of Melbourne Carmine Onofrillo University of Wollongong, carmine@uow.edu.au Cathal D. O'Connell University of Wollongong, cathal@uow.edu.au Romane Blanchard University of Melbourne Cheryl Augustine University of Melbourne See next page for additional authors Follow this and additional works at: https://ro.uow.edu.au/aiimpapers Part of the Engineering Commons, and the Physical Sciences and Mathematics Commons Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Australian Institute for Innovative Materials Follow this and additional works at: https://ro.uow.edu.au/aiimpapers Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Duchi, S., Onofrillo, C., O'Connell, C. D., Blanchard, R., Augustine, C., Quigley, A. F., Kapsa, R. M. I., Pivonka, P., Wallace, G., Di Bella, C. & Choong, P. F. M. (2017). Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair. Scientific Reports, 7 5837-1-5837-12. Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair Received: 14 December 2016 Accepted: 14 June 2017 Published: xx xx xxxx Received: 14 December 2016 Accepted: 14 June 2017 Published: xx xx xxxx Serena Duchi1,2, Carmine Onofrillo2, Cathal D. O’Connell2, Romane Blanchard1, Cheryl Augustine1, Anita F. Quigley2,3,4, Robert M. I. Kapsa2,3,4, Peter Pivonka1, Gordon Wallace2, Claudia Di Bella1,2,5 & Peter F. M. Choong1,2,5 Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called “Biopen”. To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co-axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10 seconds of exposure to 700 mW/cm2 of 365 nm UV-A, containing >90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering. Three-dimensional (3D) bioprinting epitomizes the fusion of biology and engineering.h h p g p gy g g The ability to design and fabricate complex structures by printing living cells and biomaterials functional- ized with biological molecules is revolutionizing tissue engineering and regenerative medicine1, while enabling new possibilities in drug screening and toxicology2–4. The generation of organized 3D tissue constructs via a layer-by-layer deposition process that combines cells and biomaterials in an ordered and predetermined way, allows the fabrication of multi-cellular constructs where cell-cell and cell-material interactions can mimic the physiological environment and where cellular responses to stimuli are more reflective of those found in vivo5. In recent years this technology has inspired application for in vitro biofabrication of cartilage tissues6, 7. Authors Authors Serena Duchi, Carmine Onofrillo, Cathal D. O'Connell, Romane Blanchard, Cheryl Augustine, Anita F. Quigley, Robert M. I Kapsa, Peter Pivonka, Gordon G. Wallace, Claudia Di Bella, and Peter F. M Choong This journal article is available at Research Online: https://ro.uow.edu.au/aiimpapers/2651 www.nature.com/scientificreports www.nature.com/scientificreports Received: 14 December 2016 Accepted: 14 June 2017 Published: xx xx xxxx www.nature.com/scientificreports/ To achieve this, the bioprinting parameters of the Biopen system requires a bio-ink that: (i) sets rapidly enough to allow handheld application to the lesion by the surgeon; (ii) generates a bio-synthetic cartilage construct of sufficient stiffness to immediately withstand the forces within the intra-articular environment and (iii) delivers viable cells with the ability to form functional chondral tissue.f To overcome the challenges stated and accommodate the requirements above-mentioned for effective forma- tion of functional synthetic cartilage, we applied a co-axial extrusion strategy to our prototyped Biopen device. Our co-axial bioprinting is designed to deliver a hydrogel of uniform chemistry (GelMa/HAMa), with mechanical stiffness sufficient to withstand compressive force at the site of chondral lesion whilst segregating cells from the PI chemistry. By its use of a single polymer chemistry, this approach expands on other current approaches that advocate use of a combination of cross-linkable materials with different base polymer chemistries to try to achieve a balance between structure and functional integrity of the printed construct. In addition, our approach links for the first time the Core/Shell principle of deposition with in situ clinical (at the time of surgery) application for repair of musculoskeletal tissue such as cartilage, thereby bypassing the time-consuming step of pre-surgical laboratory-based biofabrication. y In our Biopen system, the Core component of the co-axial hydrogel contains infrapatellar Adipose-derived Mesenchymal Stem/Stromal Cells (ADSCs) encapsulated within a naturally derived hydrogel (Gelatin methacry- loyl/Hyaluronic acid methacrylate, GelMa/HAMa, 10%/2%)7. The outer Shell component of this co-axial system contains the same hydrogel (GelMa/HAMa, 10%/2%), which becomes photo-polymerizable due to the addition of the PI. Hardening of the Shell provides the structural properties that allow 3D printing, while the ADSCs are preserved in a relatively soft, cell-friendly environment inside the Core. p yt y In this work we derive the balance of material, cells and fabrication process parameters that facilitate the process of in situ deposition of chondrogenic cells using our Biopen device. We evaluate in the first instance the ideal PI based on desired/ideal mechanical (stiffness) properties and in situ photo-rheology of photo-crosslinked GelMa/HAMa hydrogels. We then show that our co-axial Biopen deposition process enables the use of other- wise cytotoxic photo-polymerization chemistries, by which to make viable 3D cell-containing constructs with mechanical (stiffness) properties suitable for cartilage regeneration. www.nature.com/scientificreports/ such as gelatin and hyaluronic acid facilitates chemical cross linking that further expands the application scope of these materials11. Crosslinking can be achieved by physical crosslinking (reversible), chemical crosslinking (irreversible) or a combination of both12 and promotes a robust state change of hydrogels from (viscous) liquid to semi-solid. This provides otherwise-absent structural stability in 3D hydrogel material configurations that retain native cell adhesion properties and otherwise mimic extracellular matrix. In turn, this facilitates cell encapsulation and deposition in 3D for additive biofabrication technologies such as 3D bioprinting13–15. In current practise, chem- ical crosslinking is largely accepted as the most effective, efficient and controllable method by which to generate cross-linked hydrogels with handling and mechanical stiffness properties most appropriate to their intended use16. The crosslinking reaction can be initiated by irradiation of a photo-initiator chemical within the hydro- gel by light of a specific wavelength. This irradiation initiates a free-radical mediated polymerization reaction between the methacrylate and photo-initiator that cross-links the bio-polymer chains to form a hydrogel. The major challenge facing chemical photo-cross-linking of cell-containing hydrogels is compromised cell viability due to cytotoxic by-products generated in-process by the cross-linking chemistry17. y y p g p y g y Photo-crosslinking chemistry engenders three possible sources of cytotoxicity: (i) exposure to the photo-initiator (PI) chemical itself, (ii) exposure to UV light, (iii) exposure to free radicals created through light degradation of the PI. The most deleterious effects have been shown to occur upon exposure to the PI and UV light together, suggesting that in-process evolution of free-radicals is the most damaging step of the crosslink- ing process18. Minimizing the PI concentrations and light intensity can alleviate cell toxicity but comes at the expense of longer crosslinking times (10–30 minutes), necessary to achieve adequate biomechanical properties. Prolonging the crosslinking increases time required to print cell-containing constructs, and thereby places lim- itations on the clinical applicability of bioprinting. Some recently reported strategies propose “pre-setting” the structure19, but the additional steps could make again the procedure impractical for a direct surgical application. p g p p g pp In our previous work we developed a novel handheld 3D printer device called “Biopen”20 with the aim of pro- moting intra-surgery in situ bioprinting for cartilage biofabrication. www.nature.com/scientificreports/ These results demonstrate that co-axial extru- sion facilitates rapid photo-cross-linking under high intensity UV-A to deposit a viable cell-containing GelMa/ HAMa hydrogel and makes possible the in situ surgeon-mediated deposition of viable biosynthetic cartilage repair structures. Extending from this, the co-axial approach opens the scope for use of fabrication materials that are precluded on the basis of their inherent toxicity issues, from additive biofabrication of functional tissue constructs. Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair However, challenges still exist in the development of a fully functional tissue construct that can replicate its natural coun- terpart8, 9.f p An important factor in chondral tissue engineering is the choice of biomaterial for scaffolds. Early work using materials such as chitosan have given way to more tissue-compliant hydrogels based on natural polymers, such as gelatine mainly due to their cytocompatibility and constitutional relevance to mammalian tissue10. Furthermore, such hydrogels’ hydrophilic nature, chemical stability and biodegradability lend favourably towards their use as versatile scaffolds for 3D printing of bio-synthetic tissue constructs using appropriate cells. Addition of chemi- cally cross-linkable side-groups such as methacrylate/methacrylamide groups to biologically-derived hydrogels 1University of Melbourne, Department of Surgery, St Vincent’s Hospital Melbourne, 29 Regent Street-Clinical Science Building, 3065, Fitzroy, VIC, Australia. 2ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, Innovation Campus, University of Wollongong, Northfields Ave, 2522, Wollongong, NSW, Australia. 3Department of Clinical Neurosciences, 5th Floor Daly Wing, St. Vincent’s Hospital, 3065, Fitzroy, VIC, Australia. 4Department of Medicine, St Vincent’s Hospital Melbourne, 3065, Fitzroy, VIC, Australia. 5Department of Orthopaedics, St Vincent’s Hospital Melbourne, 3065, Fitzroy, VIC, Australia. Serena Duchi and Carmine Onofrillo contributed equally to this work. Correspondence and requests for materials should be addressed to C.D. (email: claudia.dibella@unimelb.edu.au) Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 1 www.nature.com/scientificreports/ Results E bli In order to obtain a GelMa/HAMa hydrogel stiff enough to use for cartilage repair upon rapid photo-crosslinking, we first focused our study on the screening of different photocur- ing conditions by scoring three different photo-initiators (PIs) and photocuring time. g y gf p ( ) p g For this purpose, we measured Young’s Modulus, by mechanical compression testing, as a function of GelMa/HAMa hydrogel exposure time to 365 nm (UV light) at high intensity (700 mW/cm2) using lithium-acylphosphinate (LAP), IRGACURE2959 (IRGA) and VA086 at the same concentration (0.1% w/v). g y gf p p g For this purpose, we measured Young’s Modulus, by mechanical compression testing, as a function of GelMa/HAMa hydrogel exposure time to 365 nm (UV light) at high intensity (700 mW/cm2) using lithium-acylphosphinate (LAP), IRGACURE2959 (IRGA) and VA086 at the same concentration (0.1% w/v). y p p Data obtained showed that the compression modulus of GelMa/HAMa hydrogels increase as a function of UV photocuring time (Fig. 1). However, the rates of reaction and the final modulus achieved dramatically dif- fer between the three PIs. VA086 resulted in hydrogels with the lowest modulus, with its maximum of 60 kPa achieved at 120 seconds of light exposure. At shorter exposure times (10 s), the VA086 achieved a modulus of only 9 kPa while even shorter exposures did not result in stable crosslinking (i.e. the hydrogel remained in a liquid state). These poor mechanical properties are most likely due to the evolution of nitrogen species creating bubbling within the hydrogel17. IRGA resulted in considerably stronger hydrogels, achieving a compressive modulus of 190 kPa at 120 s exposure. At short exposure time (10 s), IRGA did not result in stable crosslinking, most likely due to oxygen inhibition of the photo-initiation reaction22. LAP achieved by far the highest modulus values (380 kPa at 20 s exposure), even at much shorter crosslinking time. IRGA and VA086 photo-activation do not lead to hydrogel hardening with short exposure times (0.5, 1, 2 and 5 s), while longer exposure times never equal the performance of LAP, which start to reach a plateau after only 5 seconds. Based on these data, and pending toxicity evaluation, we selected LAP as the potential PI of choice for use with the co-axial Biopen system. p p y In situ photo-rheology was then used to further characterize the rate of the photo-crosslinking reaction using the selected photo-initiator (LAP). Results E bli Establishment of the optimal photo-initiator molecule for GelMa/HAMa bioprint- ing. Chondrocytes within native articular hyaline cartilage exist in a highly compressive environment. The knee joint of a human of the global average 60 kg body mass experiences some 6 to 20 MPa of force, depending on activity21. For this reason, the modulus of GelMa/HAMa in which the ADSCs-derived chondrogenic cells are to be delivered to the osteochondral lesion via the Biopen needs to be sufficient to withstand compressive forces within the joint so as to sufficiently protect chondrocyte development in situ and prevent the collapse of the implanted scaffold. Furthermore, in light of the surgical scaffold application to the lesion, the structure needs to attain maximal modulus in the shortest period of time possible. This thus becomes the first priority for choice of photo-initiator with which to induce cross-linking of the GelMa/HAMa. In conjunction with this major require- ment, the methodology needs to be minimally toxic to the cells and to allow delivery of viable proliferative cells that can undergo chondrogenesis sufficiently to form functional hyaline cartilage. Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 2 www.nature.com/scientificreports/ Figure 1. The LAP photo-initiator is able to generate crosslinked hydrogels with the highest modulus values at shortest exposure time (10 s). Compressive modulus (kPa) relative to mechanical properties of GelMa/HAMa, where crosslinking was obtained with three different PIs: Lithium-acylphosphinate (LAP); IRGACURE2959 (IRGA), and VA086 with different photocuring times. The moduli reached by LAP dependent crosslinking are several fold higher respect to the other PIs tested. IRGACURE2959 and VA086 do not achieve the moduli of LAP, even with longer exposure times. Error bars represent standard error of the mean between three replicates. The calculated statistical significance (p < 0.05) was obtained by unpaired t test. Figure 1. The LAP photo-initiator is able to generate crosslinked hydrogels with the highest modulus values at shortest exposure time (10 s). Compressive modulus (kPa) relative to mechanical properties of GelMa/HAMa, where crosslinking was obtained with three different PIs: Lithium-acylphosphinate (LAP); IRGACURE2959 (IRGA), and VA086 with different photocuring times. The moduli reached by LAP dependent crosslinking are several fold higher respect to the other PIs tested. IRGACURE2959 and VA086 do not achieve the moduli of LAP, even with longer exposure times. Error bars represent standard error of the mean between three replicates. The calculated statistical significance (p < 0.05) was obtained by unpaired t test. Structure: GelMa/HAMa hydrogel stiffness. Results E bli This measurement records the storage modulus (which is proportional to the degree of crosslinking) as a function of time after the sample has been illuminated with 365 nm UV light. Figure 2A shows typical data obtained for four LAP concentrations (0.005%, 0.01%, 0.05% and 0.1%). All sam- ples showed an increase in storage modulus as a function of time, though the rate of crosslinking increases with increasing LAP concentration. LAP 0.1% achieves close to its maximum storage modulus after approximately 60 s exposure, while LAP 0.05% requires more than 250 s to achieve maximum storage modulus. These data highlight the kinetic advantages of incorporating a higher concentration of PI (Note: these measurements were performed at an intensity of 100 mW/cm2). Figure 2B shows in situ photo-rheometry for LAP 0.1% at various exposure times. After 1 s of light exposure, the GelMa/HAMa material continues to crosslink via dark polymerization, achieving a storage modulus of 30 kPa at 1000 s exposure time. Meanwhile a light exposure of 10 s achieves a final storage modulus very close to that achieved for a similar sample exposed to continuous light exposure. Thus, the production of a 300kPa GelMa/HaMa hydrogel functionalized with 0.1% LAP can be achieved with a light exposure of only 10 s at 365 nm and 700 mW/cm2, thereby fulfilling the modulus requirement for in situ Biopen mediated deposition of co-axial cells-GelMa/HAMa during surgery. Cell Viability: Photo-initiated crosslinking under long-wave UV (UV-A) irradiation. Having established LAP-mediated photo-initiation of GelMa/HAMa crosslinking as the method of choice for our intended appli- cation, we undertook cytotoxicity evaluation of the methodology, with particular focus on the photo-initiation process. The free-radical photo-crosslinking reaction involves three possible sources of cytotoxicity: (i) exposure to the PI, (ii) exposure to UV light, (iii) exposure to free radicals generated by UV-mediated photocuring of the PI. These were assessed for our system by 7 days’ growth of Adipose derived Stromal/Stem Cells (ADSCs) in the presence of: (i) LAP (0.1% w/v) on its own; (ii) UV exposure at 700 mW/cm2 and (iii) LAP with UV irradiation to activate the full photo-initiation process and its generation of free radical transients.if UV light exposure at 700 mW/cm2 alone (without the presence of PI) did not significantly affect cell viabil- ity, with cell viability in irradiated cultures comparable to those of untreated (CNTRL) cells throughout the 7 days (Fig. 3). Results E bli However, the possibility that in remaining viable, the cells had endured UV-irradiation damage and mutation cannot be excluded by our data. Cell populations exposed to LAP in absence of UV irradiation Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 3 www.nature.com/scientificreports/ Figure 2. Higher concentration of LAP photoinitiator favour higher storage modulus. In situ photo-rheometry of GelMa/HAMa hydrogels incorporating LAP during the crosslinking reaction. Each plot shows storage modulus as a function of time with the UV light (365 nm, 100 mW/cm2) switched on at 100 s. (A) Storage modulus as a function of continuous UV exposure time for four concentrations of LAP (0.005% = open squares, 0.01% = light grey squares, 0.05% = grey squares and 0.1% = black squares). Both the reaction rate and the final storage modulus are impacted by LAP concentration. Low concentrations produce slow reactions resulting in low modulus hydrogels. The two highest LAP concentrations (0.05% and 0.1%) achieve comparable final modulus (~70 kPa), although the 0.05% concentration requires much longer exposure time to achieve this. (B) Effect of dark polymerization. GelMa/HAMa hydrogels incorporating 0.1% LAP were exposed to continuous UV exposure (thick black line) or single bursts of short times (10 s, 2 s and 1 s). The arrows indicate the point at which the UV light was switched off for the continuous exposure (100 s). During light exposure, all hydrogels follow similar crosslinking kinetics, however after the light turns off dark polymerization can continue for many minutes. In the case of the 10 s exposed hydrogel, the dark polymerization is sufficient to match the material crosslinked with a (much longer) continuous exposure. igure 2. Higher concentration of LAP photoinitiator favour higher storage modulus. In situ photo-rheometry Figure 2. Higher concentration of LAP photoinitiator favour higher storage modulus. In situ photo-rheometry of GelMa/HAMa hydrogels incorporating LAP during the crosslinking reaction. Each plot shows storage modulus as a function of time with the UV light (365 nm, 100 mW/cm2) switched on at 100 s. (A) Storage modulus as a function of continuous UV exposure time for four concentrations of LAP (0.005% = open squares, 0.01% = light grey squares, 0.05% = grey squares and 0.1% = black squares). Both the reaction rate and the final storage modulus are impacted by LAP concentration. Low concentrations produce slow reactions resulting in low modulus hydrogels. Results E bli The two highest LAP concentrations (0.05% and 0.1%) achieve comparable final modulus (~70 kPa), although the 0.05% concentration requires much longer exposure time to achieve this. (B) Effect of dark polymerization. GelMa/HAMa hydrogels incorporating 0.1% LAP were exposed to continuous UV exposure (thick black line) or single bursts of short times (10 s, 2 s and 1 s). The arrows indicate the point at which the UV light was switched off for the continuous exposure (100 s). During light exposure, all hydrogels follow similar crosslinking kinetics, however after the light turns off dark polymerization can continue for many minutes. In the case of the 10 s exposed hydrogel, the dark polymerization is sufficient to match the material crosslinked with a (much longer) continuous exposure. Figure 3. Cell cytotoxicity induced by PI and UV irradiated PI. ADSCs cultured in 2D and assayed along 7 days in culture with a metabolic test (Cell Titer-Blue®) to measure the cytotoxicity induced by cell exposure to UV light alone (UV), LAP on its own (LAP) and UV exposed LAP (LAP-UV) compared to untreated cells (CNTRL). Error bars represent standard error of the mean between three replicates. The calculated statistical significance was obtained by unpaired t test and calculated versus CNTRL. At day 7 statistics is calculated also for LAP-UV versus LAP. Figure 3. Cell cytotoxicity induced by PI and UV irradiated PI. ADSCs cultured in 2D and assayed along 7 days in culture with a metabolic test (Cell Titer-Blue®) to measure the cytotoxicity induced by cell exposure to UV light alone (UV), LAP on its own (LAP) and UV exposed LAP (LAP-UV) compared to untreated cells (CNTRL). Error bars represent standard error of the mean between three replicates. The calculated statistical significance was obtained by unpaired t test and calculated versus CNTRL. At day 7 statistics is calculated also for LAP-UV versus LAP. displayed a significant reduction in percentage viability (even at day 1), indicating that in itself, LAP was toxic to the cells. Further complexity of the LAP toxicity dynamic was evident upon cell exposure to LAP in the presence of UV light, which delivered significantly greater reduction in viable cell number compared to cells exposed to UV and LAP on their own even 1 day after exposure commenced. Results E bli z-stacks were acquired every 10 µm and 3D rendering was performed with NIS elements software using the Alpha-blending algorithm. A Nikon Plan Fluor 10x DIC L N1 NA0.3 objective lens was used. The panel shows the same image representative of 3D rendered of superimposed green and red channels in three different orientations. Despite evidence that the sub-population of cells left viable at day 1 post LAP/UV irradiation retained some proliferative ability, the live cell count was significantly lower than the LAP alone throughout the growth period, indicating that the initial LAP toxicity was exacerbated by photo-activation, most likely due to free radicals gen- erated by LAP photo-activation-mediated degradation. This rationale gained further support by observation of eventual entry of cells exposed to photo-activated LAP to non-proliferative G0 cell cycle phase over the subse- quent week post 7 days (day 15; Supp. Fig. 1B). q p y y pp g Under the same UV exposure conditions, the cell viability was also affected by the photoactivation of the two other PIs considered, IRGA and VA086 (Supp. Fig. 1B,C). pp g These data represent an “itemized” analysis of each component of our photo-polymerization system’s “in-process” potential to adversely affect ADSCs viability and thus provide a baseline “maximal” possible toxicity level. This is based on the premise that LAP diffusion (and therefore its bio-availability to the cells) will be signif- icantly less in denser structures such as the cross-linked GelMa/HAMa hydrogels proposed for use in this work than in the liquid culture media used for this analysis. These data lay a comparative platform for evaluating if and, if so, how much of the toxicity of LAP photoactivation is eradicated by i) mono-axial and ii) co-axial encapsula- tion of ADSCs in GelMa/HAMa using our rapid Biopen-mediated process. The Core/Shell solution. Collectively, the photo-polymerization protocol defined above satisfy the modu- lus (at least to some degree) and viability criteria necessary for our application of ADSCs encapsulated in GelMa/ HAMa hydrogel to repair osteochondral lesions in situ as part of our Biopen technology. y g p p p gy However, having established the significant (additive) toxicity of the LAP and photoactivation process on the ADSCs that we wished to use for chondrogenic differentiation, it remained to establish if and to what extent this PI toxicity translated to the full process. Results E bli Exposure to UV light in presence of LAP, caused an eminent decrease in cell number respect to the control, which was not significatively different between all the concentrations tested (0.005%, 0.01%, 0.05% and 0.1%; Supp. Fig. 1A) so that the observed cytotoxicity was not reduced by exposing the cells to lower concentrations of LAP. displayed a significant reduction in percentage viability (even at day 1), indicating that in itself, LAP was toxic to the cells. Further complexity of the LAP toxicity dynamic was evident upon cell exposure to LAP in the presence of UV light, which delivered significantly greater reduction in viable cell number compared to cells exposed to UV and LAP on their own even 1 day after exposure commenced. Exposure to UV light in presence of LAP, caused an eminent decrease in cell number respect to the control, which was not significatively different between all the concentrations tested (0.005%, 0.01%, 0.05% and 0.1%; Supp. Fig. 1A) so that the observed cytotoxicity was not reduced by exposing the cells to lower concentrations of LAP. Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 4 www.nature.com/scientificreports/ Figure 4. Core/Shell 3D printing by co-axial extrusion. (A) Schematic representation of the 3D co-axial handheld printer. (B) Schematic representation of the co-axial nozzle. (C) Picture of the cartridges dedicated to Core and Shell loading in the printer, with relative magnification of the nozzle during co-axial deposition. (D) Representative 3D rendered confocal images of Core/Shell printed sample labeled with fluorescent beads. The Shell (GelMa/HAMa plus LAP 0.1%) is shown in red channel, while the Core (GelMa/HAMa) is shown in green channel. z-stacks were acquired every 10 µm and 3D rendering was performed with NIS elements software using the Alpha-blending algorithm. A Nikon Plan Fluor 10x DIC L N1 NA0.3 objective lens was used. The panel shows the same image representative of 3D rendered of superimposed green and red channels in three different orientations. Figure 4. Core/Shell 3D printing by co-axial extrusion. (A) Schematic representation of the 3D co-axial handheld printer. (B) Schematic representation of the co-axial nozzle. (C) Picture of the cartridges dedicated to Core and Shell loading in the printer, with relative magnification of the nozzle during co-axial deposition. (D) Representative 3D rendered confocal images of Core/Shell printed sample labeled with fluorescent beads. The Shell (GelMa/HAMa plus LAP 0.1%) is shown in red channel, while the Core (GelMa/HAMa) is shown in green channel. Results E bli y p Our approach postulated that the compound toxicity of LAP and photoactivation process should i) be consid- erably lessened by actual polymerization of the GelMa/HAMa in the first instance and ii) be further lessened by compartmentalization of the ADSCs in an inner, non-cross-linked GelMa/HAMa Core surrounded by a photo cross-linked Shell as part of a co-axial gelation process. Towards this end, we adapted our prototyped handheld Biopen printer (Fig. 4A)20 to eject an organized Core/Shell bioink in which the inner soft core, containing cells laden in non-cross-linked GelMa/HAMa, is surrounded by a robust shell made of cross-linked-GelMa/HAMa. This co-axial approach avoids the deposition of cells together with the photo-initiator, which will only be pres- ent in the shell. A dual concentric co-axial nozzle, characterized by an inner and outer orifice, enables the two different compartments (core and shell) to be dispensed (Fig. 4B,C), with a maximum resolution being 500 μm (Supp. Fig. 2A). On the basis of the mechanical and photo-rheology data obtained previously, we used a 0.1% w/v LAP concentration and crosslinking at 365 nm, 700 mW/cm2, at 10 s exposure time for shell hardening. In order to verify the Core/Shell printing capability of our device in the selected conditions, two GelMa/HAMa solutions Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 5 www.nature.com/scientificreports/ Figure 5. The co-axial printing produces 3D printed samples with high cell viability. (A,B) Representative 3D rendered confocal images of ADSCs Core/Shell (co-axial) and mono-axial bioprinted samples stained with Calcein-AM (live cells, green channel). The samples were labeled with fluorescent beads for identification of the Shell (GelMa/HAMa plus LAP 0.1%, cyan channel). Mono-axial samples were also labelled with fluorescent beads for analysis (GelMa/HAMa plus LAP 0.1% and ADSCs, cyan channel). z-stacks were acquired every 10 μm and 3D rendering was performed with NIS elements software using the Alpha-blending algorithm. Images show representative 3D rendered of single and superimposed (merge) cyan and green channels of confocal single 2D z-stacks. (C) The graph shows the quantification of Calcein-AM stained ADSCs to measure cytotoxicity over time in culture right after UV irradiation in co-axial (Core/Shell) and mono-axial 3D printed samples, and in 2D monolayer. The co-axial approach demonstrates higher cell viability and proliferation compared to mono-axial samples and 2D at 7 days post irradiation exposure. Error bars represent standard error of the mean between three replicates. Results E bli Statistical significance (p < 0.05) was determined by One way Anova test with Dennett’s correction. (D) The graph shows the quantification of live and dead staining performed with Calcein-AM (live cells, green lines) and SYTOX (dead cells, red lines) to measure cytotoxicity over time in culture in both Core/Shell (co-axial, solid line) printed and mono-axial (dotted line) samples. The Core/Shell approach demonstrates significantly higher cell viability and proliferation compared to mono-axial samples at 10 days. Error bars represent standard error of the mean between three replicates. Statistical significance (p<0.05) was determined by unpaired t test. Figure 5. The co-axial printing produces 3D printed samples with high cell viability. (A,B) Representative 3D rendered confocal images of ADSCs Core/Shell (co-axial) and mono-axial bioprinted samples stained with Calcein-AM (live cells, green channel). The samples were labeled with fluorescent beads for identification of the Shell (GelMa/HAMa plus LAP 0.1%, cyan channel). Mono-axial samples were also labelled with fluorescent beads for analysis (GelMa/HAMa plus LAP 0.1% and ADSCs, cyan channel). z-stacks were acquired every 10 μm and 3D rendering was performed with NIS elements software using the Alpha-blending algorithm. Images show representative 3D rendered of single and superimposed (merge) cyan and green channels of confocal single 2D z-stacks. (C) The graph shows the quantification of Calcein-AM stained ADSCs to measure cytotoxicity over time in culture right after UV irradiation in co-axial (Core/Shell) and mono-axial 3D printed samples, and in 2D monolayer. The co-axial approach demonstrates higher cell viability and proliferation compared to mono-axial samples and 2D at 7 days post irradiation exposure. Error bars represent standard error of the mean between three replicates. Statistical significance (p < 0.05) was determined by One way Anova test with Dennett’s correction. (D) The graph shows the quantification of live and dead staining performed with Calcein-AM (live cells, green lines) and SYTOX (dead cells, red lines) to measure cytotoxicity over time in culture in both Core/Shell (co-axial, solid line) printed and mono-axial (dotted line) samples. The Core/Shell approach demonstrates significantly higher cell viability and proliferation compared to mono-axial samples at 10 days. Error bars represent standard error of the mean between three replicates. Statistical significance (p < 0.05) was determined by unpaired t test. were prepared with different coloured fluorescent beads before the 3D printing procedure. Confocal imaging analysis showed the core completely surrounded by the shell compartment with minimal apparent mixing of the two compartments (Fig. Results E bli 4D) (the core is shown in green, the shell in red). Mechanical compression testing showed that the Core/Shell scaffold structure reached a Young’s Modulus of 195 KPa (±66.138). Thus, the gen- erated Core/Shell scaffold presented “as-close-as-possible-to-optimal” mechanical characteristics for cartilage regeneration applications, giving rise to the possibility of building 3D cartilage-repair structures in situ during surgery (Supp. Fig. 2B,C). Cell behavior in the Core/Shell structure. ADSCs were printed out in (i) mono-axial and (ii) co-axial photo-cross-linked GelMa/HAMa using the Biopen. The distribution and viability of cells within the respective structures were evaluated by Calcein-AM and SYTOX staining and fluorescence imaging (Fig. 5 and Supp. Fig. 3). Three-dimensionally rendered single z stacks obtained with confocal microscopy showed that after co-axial bio- printing, live and dead cells are mainly distributed in the Core (Fig. 5A and Supp. Fig. 3A,B). This confirmed our Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 6 www.nature.com/scientificreports/ Figure 6. The Core/Shell design allows cells to proliferate. (A) Representative 3D rendered confocal images of ADSCs bioprinted samples stained with Calcein-AM (live cells, green channel) at day 1 and day 15 after printing. The Core/Shell samples were generated by labeling with fluorescent beads the Shell (GelMa/HAMa plus LAP 0.1%, cyan channel). z-stacks were acquired every 10 μm and 3D rendering was performed with NIS elements software using the Alpha-blending algorithm. A Nikon Plan Fluor 10x DIC L N1 NA0.3 objective lens was used. Images show representative 3D rendered of superimposed (merge) cyan and green channels of confocal single 2D z-stacks. (B) The graph shows the representation of the percentages of the same area occupied by cells (Cells percentage) and Shell (Shell percentage). (C) Fluorescence images of 10 μm cryosections obtained from the cryopreserved bioprinted samples. The cyan channel represents the labeled Shell, while cells have been stained with DAPI before imaging. A fluorescent Olympus IX70 inverted microscope with a SPOT Diagnostic RT-Slider camera and SPOT Diagnostic software was used. Olimpus 4X UPlanFL NA0.13, 10X CPlanFL RC NA0.3 and 20X LCPlanFL RC2 NA0.4 objective lenses were used. Figure 6. The Core/Shell design allows cells to proliferate. (A) Representative 3D rendered confocal images of ADSCs bioprinted samples stained with Calcein-AM (live cells, green channel) at day 1 and day 15 after printing. The Core/Shell samples were generated by labeling with fluorescent beads the Shell (GelMa/HAMa plus LAP 0.1%, cyan channel). Results E bli z-stacks were acquired every 10 μm and 3D rendering was performed with NIS elements software using the Alpha-blending algorithm. A Nikon Plan Fluor 10x DIC L N1 NA0.3 objective lens was used. Images show representative 3D rendered of superimposed (merge) cyan and green channels of confocal single 2D z-stacks. (B) The graph shows the representation of the percentages of the same area occupied by cells (Cells percentage) and Shell (Shell percentage). (C) Fluorescence images of 10 μm cryosections obtained from the cryopreserved bioprinted samples. The cyan channel represents the labeled Shell, while cells have been stained with DAPI before imaging. A fluorescent Olympus IX70 inverted microscope with a SPOT Diagnostic RT-Slider camera and SPOT Diagnostic software was used. Olimpus 4X UPlanFL NA0.13, 10X CPlanFL RC NA0.3 and 20X LCPlanFL RC2 NA0.4 objective lenses were used. system’s ability to print a Core/Shell structure in which the ADSCs are compartmentalized to the Core, away from the LAP and photo-activation by-products. On the other hand, cell distribution in the mono-axial printed hydro- gel showed both live and dead cells throughout the structure without any overt compartmentalisation (Fig. 5B and Supp. Fig. 3C,D).i g Quantitative evaluation of live cells in these configurations over 7 days, demonstrated that the photopolym- erization of GelMa/HAMa is able to shield the cytotoxic effect that stem from the direct contact with the pho- toinitiator and the generation of free radicals (Fig. 3C). Despite the shielding effect, a drop in viable cell numbers beyond the 7 days in the ADSCs was evident within the mono-axial construct (Fig. 3C,D). This suggests that the mono-axial photo-cross-linking configuration does not adequately protect the cells in the system from the tox- icity generated by this printing process. In contrast to this, in the co-axial configuration the cells were observed to retain proliferative capability throughout the test period, indicating a protective effect imparted on the cells through the co-axial printing process (Fig. 3C). Further cell viability analysis along time in culture shows that compared to the mono-axial configuration the co-axial displays a steeper increase in cell number by day 10, reaching a 30% increase from the initial number (Fig. 3D). The mono-axial bioprinting configuration shows instead a viability decrease by 30% along with an increase in the number of dead cells (Fig. 3D). Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x Discussion d Our Biopen device was designed to print stem cells in 3D constructs directly into damaged cartilage during sur- gery in situ. To translate this freeform biofabrication tool into the clinical setting, in this work we aimed to define a bioprinting process that delivered a cell-laden structure with adequate structural integrity to support viable cell delivery to the highly compressive osteochondral lesion environment. Assuring high rates of cell survival remains an important challenge in 3D bioprinting applications since it is critically related to the bioprinting process, the materials used and the intra- and post-printing chemical reac- tions/by-products. Hydrogel-based materials have been widely used as cell carriers and scaffolds in tissue engi- neering due to their structural compliance with natural extracellular matrix23. The extrusion bioprinting process necessitates a rapid phase transition from liquid during extrusion, to semi-solid to form a robust bioscaffold24. Matching the mechanical properties of the resulting bioscaffold to the target tissue is therefore important, since contained cellular behavior is, in part, and particularly for cartilage/chondrocytes, mediated by their mechanical microenvironment25. Light-activated free-radical crosslinking (photo-polymerization) provides rapid reaction rates and gener- ates uniform hydrogels with excellent temporal and spatial control of important properties such as mechanical stiffness. The degree of crosslinking, and hence mechanical strength of the printed scaffold, is a function of the reaction conditions (such as light intensity and exposure time) and the properties of the PI22. The most preva- lent PI used in putative tissue engineering applications is 2-hydroxy-1-[4-(2- hydroxyethyl) phenyl]-2-methyl- 1-propanone (IRGACURE® 2959), owing to its water solubility and moderate cytotoxicity relative to other PIs26. However this PI was found to be toxic (in 2D culture, 30 minutes’ exposure to the PI in media and then between 3 and 7 minutes’ direct exposure to UV-A @ 4 mW/cm2) to different degrees in the various cell types evaluated and would require much longer UV exposure to photo-initiate cross-linking to the extent required for our proposed intra-surgical in situ application. g pp In recent years, other PIs have emerged as promising candidates for photo-crosslinking in the presence of cells. These include 2,20-Azobis[2-methyl-N-(2-hydroxyethyl) propionamide] (VA086), vitaminB227 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Whilst photo cross-linking with very high cell viability has indeed been achieved using VA08628, this again required very low-intensity exposure to 365 nm UV-A at 4 mW/ cm2 (again, unacceptably long exposure time requirements for our purposes). Results E bli y y g ( g ) Given the nature of the temporal cell viability dynamics observed within the co-axial structures, it is likely that this geometry results in a protective effect that emanates from removing the cells from exposure to the free radicals generated by the photo-activation process.l g y p p Taken together, our data clearly reflects the ability of co-axial Core/Shell bioprinting to better maintain the urvival of stem cell niche. An additional feature of the Core/Shell bioprinting is that as time in culture progresses, the relative construct volume occupied by the cells considerably increase compared to the volume occupied by the shell (Fig. 6A, Supp. Fig. 4). Evident in Fig. 6B, the initial percentage of the space occupied by the shell decreases conversely to that occupied by the cells, providing good rationale for the expectation that likewise, if used surgically in situ to repair an osteochondral lesion, the co-axial structure would provide structural protection to the ADSCs within, allow- ing their expansion and chondrogenic differentiation. This lends support towards the further expectation that by eventually overcoming the physical constraint imposed by the GelMa/HAMa vehicle, the cells will be able to ori- entate into the structural layering required for the formation of functional hyaline cartilage. Fluorescence imag- ing analysis performed on single sections obtained from cryopreserved multilayered core/shell-printed samples, 7 www.nature.com/scientificreports/ Figure 7. Core/Shell printing facilitates a layer by layer deposition of cell-laden hydrogels with structural integrity. Representative epifluorescent image of Calciein-AM (green channel) stained cells after 7 days from printing. The Shell and the Core has been differently labeled with fluorescent beads (cyan channel for Shell, violet channel for Core) to estimate the compartmentalization. An epifluorescent Olympus IX70 inverted microscope with a SPOT Diagnostic RT-Slider camera and SPOT Diagnostic software was used with an Olimpus 4X UPlanFL NA0.13 objective lens. Figure 7. Core/Shell printing facilitates a layer by layer deposition of cell-laden hydrogels with structural integrity. Representative epifluorescent image of Calciein-AM (green channel) stained cells after 7 days from printing. The Shell and the Core has been differently labeled with fluorescent beads (cyan channel for Shell, violet channel for Core) to estimate the compartmentalization. An epifluorescent Olympus IX70 inverted microscope with a SPOT Diagnostic RT-Slider camera and SPOT Diagnostic software was used with an Olimpus 4X UPlanFL NA0.13 objective lens. Results E bli shows that the compartmentalization of the core is maintained for at least 15 days, since fluorescent beads are not internalized by cells despite the measured reduction of the shell (Fig. 6C). Thus, the increase in cell volume can be ascribed to the proliferation capability of the cells bioprinted within the Core. Layer by layer deposition using co-axial Core/Shell bioprinting allows building of composite 3D bioscaffolds if/when required. Epifluorescence imaging of layered “criss-cross” core/shell fibres shows live cells embedded in the core compartments along two distinctive layers, on the top of each other (Fig. 7, Live cells in green channel). Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x www.nature.com/scientificreports/ By this process, we were able to extrude a soft non-cross-linked GelMa/HAMa core that contained ADSCs with an outer Shell component containing LAP PI that was photo cross-linked and provided structural support adequate for potential use within an osteochondral repair environment.h The co-axial cell/hydrogel structure that we generated successfully separated the ADSCs from the damaging effects of the LAP photo-activation of cross-linking. This was evident by high numbers (>95%) of viable cells in the co-axial structures immediately after the printing and photocuring steps and throughout all subsequent time in culture (Fig. 5). This was not observed in the mono-axial construct where the direct exposure of cells to the photo-activation process led to significant down-turn in cell viability. Furthermore, the co-axial process facili- tated post-polymerization maintenance of cell proliferation within the structure, with progressive expansion of the relative volume occupied by the cells inside the overall structure (Fig. 6).f p y g Various studies have reported co-axial 3D bioprinting19, 30, 31 to achieve structural integrity of the bioscaffold for deposition of hydrogels with low viscosity. Compared to those strategies, our approach is based on a new concept of co-axial Core/Shell geometry by which to protect printed cells from the fabrication process, thereby increasing the scope of materials that can be used for printing tissue constructs without compromising structural or cellular integrity. As such, our co-axial strategy geometrically compartmentalizes a solid phase that facilitates appropriate stiffness requirements and a viscous liquid phase that preserves cell viability by separating cells from PIs and the cytotoxic chemical by-products coming from the crosslinking reaction going on within the solid phase. In addition, the co-axial approach presented here uses a single hydrogel chemistry, which makes the sys- tem more readily translatable as a tool in the surgical field.h y gi The results of the current study and of our previous work with this innovative Biopen printing device20 demon- strate that co-axial bioprinting has great potential for in vivo application directly at the surgical point of cartilage repair. The handheld 3D bioprinter’s ability to deposit co-axial cell-containing scaffolds lends favorably towards the potential for its eventual clinical translation, particularly in the field of musculoskeletal tissue regeneration and repair32. www.nature.com/scientificreports/ of UV-cured polymers for in situ musculoskeletal tissue regeneration if associated in-process toxicity issues are not considered and accommodated.h of UV-cured polymers for in situ musculoskeletal tissue regeneration if associated in-process toxicity issues are not considered and accommodated.h Therefore given our intent to address the highly compressive osteochondral environment, the aim of our study was to develop a reliable method that allows rapid and efficient hydrogel hardening, while facilitating optimal cell survival. We reasoned that high cell viability and efficient hardening could be achieved by devoting different physical compartments to different structural components during the 3D printing process: one for cell deposition and one for structural integrity. g y Consequently, we evaluated the crosslinking capacity of three PIs and selected 0.1% w/v LAP as the ideal PI for cross-linking GelMa/HAMa hydrogel to a modulus of ~300 kPa. This configuration allowed minimization of cell exposure to UV by application of a photocuring time of just 10 s with long-wave UV-A (365 nm) irradiation at 700 mW/cm2 UV (Figs 1 and 2). g Fairbanks and coauthors29 achieved high levels of post activation cell viability by crosslinking PEG-diacrylate using LAP with very low light intensity irradiation (10 mW/cm2), thereby reducing exposure to PI degradation-mediated free radical production. However, using the same conditions in our GelMa/HAMa hydro- gel, would require a very long exposure time to reach a stiffness comparable to the 300kPa achieved in our system. Our approach sought an alternative means by which to preserve in-process cell viability without (excessively) compromising on the important cross-linkage-dependent stiffness requirement for in situ osteochondral repair application using our Biopen technology20.ffi pp g p gy We sought to achieve this protective effect by segregating the PI away from cells to maintain the most efficien hotocuring conditions under a short time exposure (700 mW/cm2 for 10 s).h The selected photocuring settings used in this study allowed us to print cell-laden GelMa/HAMa hydrogel using our 3D Biopen device modified by incorporation of an extrusion nozzle designed for the co-axial deposi- tion of materials (Fig. 4). This facilitated the geometric separation of two compartments of differing constitution in a Core/Shell configuration. www.nature.com/scientificreports/ Specifically, with consideration of the nature of the GelMa/HAMa hydrogel and its mechanical properties, the Biopen-mediated bioprinting approach presented here has direct relevance to cartilage regenera- tion and repair, but will undoubtedly have applications in other areas when fully developed12. Further studies in this area will address the development and application of the co-axial Biopen approach described here to repair of cartilage in animal models as immediate precursor studies to clinical translation of this exciting technology. Discussion d Vitamin B2 has been proposed as a novel natural biocompatible photocrosslinking agents, nevertheless to obtain an ECM based material with 10/20kPa stiffness, a two-step photo/thermal process is required. The approach is thus not compatible with our settings that required a fast “real time” 3D bioprinting compatible with time for surgical procedures, and high stiffness for chondral based defect repair. Meanwhile, LAP has been advocated for its relative cytocompatibility and its ability to initiate crosslinking when exposed to higher (and therefore safer) wavelengths (>400 nm) of light29. Nevertheless, the photo cross-linking reaction remains the major potential source of cytotoxicity, and can therefore affect the final outcome of the designed application. Exposure to PIs, to UV light or to free radicals gen- erated through light degradation of the PI, can all be adverse to cell viability (Fig. 3)26 and consequently, the (bio) functionality of the printed construct and the need for structural integrity (via degree of cross-linking) and cell viability need to be finely balanced in the adopted approach. Minimizing the PI concentrations and light inten- sity can alleviate cell toxicity but may reduce the efficiency of the crosslinking reaction, thus compromising the mechanical properties of the printed product. From this viewpoint, achieving adequate biomechanical properties thus requires longer crosslinking/UV exposure times (10–30 minutes), thereby limiting the clinical translatability Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 8 www.nature.com/scientificreports/ Methods The contact area between the sample with the compressing plate was measured using a Dino Lite 4111T microscope at 20X magnification and calibrated using a reference slide of known dimensions. Additionally, the point of inflexion of load versus time serves as contact point between the sample surface and the compression plate and gives the sample height. Subsequently, load and displacement are converted into stress (σ) and strain (ε) using the sample surface area and height. The compressive modulus was computed using stress data between 10 and 15% strain as follows: Ec = (σ15 − σ10)/(ε15 − ε10). This procedure was repeated for each of the 55 samples. In situ photo-rheology. All rheology experiments were performed on a Physica MCR 301 Rheometer (Anton Paar) in a parallel plate geometry (15 mm disk, 0.5 mm measuring distance) at room temperature (21– 23 °C). For the flow experiments, the shear rate was ramped up from 0.1 to 100 s−1 over 5 minutes. A pre-shear of 5 s−1 for 2 minutes was introduced before the flow experiment to eliminate rheological history. Oscillatory meas- urements were performed at 1% strain and 1.5 Hz frequency. For in situ UV curing, light from the UV light source (Omnicure 1000, Lumen Dynamix LDGI) was routed through a 5mm optical fibre to illuminate the underside of the sample through a quartz crystal stage. The UV intensity (100 mW/cm2) was measured at the sample using a UV meter before and after each experiment. (Note: these rheology measurements were performed at an inten- sity of 100 mW/cm2 which was the maximum intensity achievable through the optical fibers on this system. The intensity used for other measurements in this study was generally 700 mW/cm2). Cell culture. Sheep Adipose Derived Stromal/Stem cells (ADSCs) were isolated from sheep infrapatellar fat pad (IPFP) as previously described33. After isolation, cells were maintained in culture media containing low glu- cose DMEM (St. Louis, LA, USA) supplemented with 10% FBS (GIBCO, Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 U ml−1 Penicillin and 100 μg ml−1 Streptomycin solution (GIBCO), 2mM L-Glutamine (GIBCO), and 15 mM HEPES (GIBCO), 20 ng ml−1 epidermal growth factor (EGF) and 1 ng ml−1 fibroblast growth factor (FGF) (R&D Systems Inc., Minneapolis, MN, USA). Co-axial Core/Shell and unstructured mono-axial bioprinting. Methods Bioink preparation. Gelatin-methacryloyl/hyaluronic acid methacryloyl (GelMa/HAMa) was synthe- sized as previously described20. The materials was dissolved to a final concentration of 100 mg ml−1 GelMa and 20 mg ml−1 HAMa (10%GelMa-2%HAMa) in sterile PBS containing 100 U ml−1 penicillin and 100 μg ml−1 of streptomycin (GIBCO). Stem cells (ADSCs, see below for details of cells isolation and expansion) were mixed through the GelMa/HAMa to a final concentration of 5 × 106 cells ml−1 and carefully loaded in the Core printing chamber. The Shell printing chamber was loaded with GelMa/HAMa and the indicated PIs. Photoinitiators and UV photocuring. The following three photoinitiators (PIs) were used to initiate photocrosslinking of the GelMa/HAMa hydrogel: VA086 (2, 2′-Azobis [2-methyl-N-(2 hydroxyethyl) propiona- mide]) from Sigma Aldrich; IRGACURE® 2959 (IRGA2959) from Sigma Aldrich and lithium-acylphosphinate (LAP) from Tokyo Chemical Industry Co. (Tokyo, Japan).i y y y p Light irradiation was achieved using a 365 nm UV source (Omnicure LX400+, Lumen DynamixLDGI) fitted with a 12 mm lens (25 mm focal distance) at maximum intensity. The light source was placed directly on the bottom of the plastic wells where cells or bioprinted samples were deposited. Under these conditions, the light intensity was measured at 700 mW/cm2 through the plastic. Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 9 www.nature.com/scientificreports/ Mechanical test. Flat discs (1 cm diameter, 1 mm thickness) of cured hydrogel were prepared by irradiating the hydrogel inside a Polydimethylsiloxane (PDMS) mold, covered with a thin glass coverslip. The tests were performed at room temperature using a TA Electroforce 5500 mechanical loading device (TA Instruments, New Castle, USA) fitted with a calibrated 5 lbf load cell. A 4.2 cm diameter compression plate was mounted on the mover. The protocol follows a procedure proposed by Loessner et al.14. The contact point with the bottom of a glass dish is recorded before the sample is placed in the same dish (in an unconfined environment) and com- pressed by a plate much larger than the nominal sample diameter (1 cm). Samples were hydrated by deposing droplets of PBS solution on their surface before testing. The displacement was controlled by a ramp function, lowering the compression plate at a rate of 0.01 mm/s, until a total displacement that is much larger than 15% of the sample height is achieved. Load, displacement and time are recorded from the test. Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x Methods For co-axial Core/Shell 3D bioprint- ing, both chambers was loaded with GelMa/HAMa, but the LAP was added only to the Shell chamber diluted at 0.1% v/w. The extruded samples were then UV irradiated at room temperature, washed three times in PBS 1X and new complete culture medium was added in each well of a 24 were plates. To generate the unstructured mono-axial samples (no Core/Shell compartmentalization) the 3D extrusion was performed by using only 1 car- tridge containing GelMa/HAMa, LAP 0.1% w/v and ADSCs. The samples were then UV irradiated at room tem- perature as described, washed three times in PBS 1X and new complete culture medium was added in each well. Cell viability assays in monolayer. In the 2D ADSCs cultures to determine the cytotoxicity, cells were plated at 2000 cell cm−1 in 48 well plates and let adhere O/N at 37 °C/5%CO2. Then for each well the three differ- ent PIs (LAP, VA086 and IRGA) were added at 0.1% in PBS v/w in a final volume of 0.5 mL/well, and for the LAP also at 0.005%, 0.01%, 0.05% and 0.1% v/w concentrations. After 10 minutes incubation at room temperature, the cells were UV irradiated as described in the previous paragraph, washed three times in PBS 1X and new complete culture medium was added in each well. To measure cell cytotoxicity during time in culture, the following cell viability tests were used: Calcein-AM staining (Thermo Fisher Scientific Inc.) for LAP, VA086 and IRGA compar- ison; CyQUANT® (Thermo Fisher Scientific Inc.) for the different concentration of LAP; Cell Titer-Blue® Cell viability assay (Promega, Madison, WI, USA) for the LAP by itself in comparison with UV light exposure and UV exposure alone. For CyQUANT and Cell Titer-Blue tests, cell viability was assessed using the manufacturer protocols in triplicate by acquiring fluorescent signal at each time point with a CLARIOSTAR microplate reader (BMG LABTECH, Ortenberg, Germany).l g y Live cell counts in cells monolayer experiments were performed in triplicate by acquiring epifluorescence images with Olympus IX70 inverted microscope with a SPOT camera and software using the indicated objective lenses. References 1. Guillotin, B. & Guillemot, F. Cell patterning technologies for organotypic tissue fabrication. Trends Biotechnol. 29, 183–190 (2011). 2. Peng, W., Derya, U. & Ozbolat, I. T. Bioprinting Towards Physiologically-relevant Tissue Models for Pharmaceutics. Trends Biotechnol. 34, 722–732 (2016). p g g g yp ( ) 2. Peng, W., Derya, U. & Ozbolat, I. T. Bioprinting Towards Physiologically-relevant Tissue Models for Pharmaceutics. Trends Biotechnol. 34, 722–732 (2016). 2. Peng, W., Derya, U. & Ozbolat, I. T. Bioprinting Towards Physiologically-relevant Tissue Models Biotechnol. 34, 722–732 (2016). 3. Zhang, Y. S. et al. Bioprinting the Cancer Microenvironment. ACS Biomater. Sci. Eng 2, 1710–1721 (2016). g p g g 4. Vanderburgh, J., Sterling, J. A. & Guelcher, S. A. 3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening. Ann. Biomed. Eng. 1–16, doi:10.1007/s10439-016-1640-4 (2016). g g g 4. Vanderburgh, J., Sterling, J. A. & Guelcher, S. A. 3D Printing of Tissue Engineered Constructs for In Vitro Modeli P i d D S i A Bi d E 1 16 d i 10 1007/ 10439 016 1640 4 (2016) 4. Vanderburgh, J., Sterling, J. A. & Guelcher, S. A. 3D Printing of Tissue Engineered Constructs for In Vitro Mode Progression and Drug Screening. Ann. Biomed. Eng. 1–16, doi:10.1007/s10439-016-1640-4 (2016). 4. Vanderburgh, J., Sterling, J. A. & Guelcher, S. A. 3D Printing of Tissue Engineered Constructs for In Vitro Progression and Drug Screening. Ann. Biomed. Eng. 1–16, doi:10.1007/s10439-016-1640-4 (2016). 5. Ferris, C. J. et al. Bio-ink for on-demand printing of living cells. Biomater. Sci. 1, 224 (2013). 6. Fedorovich, N. E. et al. Biofabrication of osteochondral tissue equivalents by printing topologically defined, cell-laden hydroge scaffolds. Tissue Eng. Part C. Methods 18, 33–44 (2012). 6. Fedorovich, N. E. et al. Biofabrication of osteochondral tissue equivalen scaffolds. Tissue Eng. Part C. Methods 18, 33–44 (2012). 6. Fedorovich, N. E. et al. Biofabrication of osteochondral tisf f g 7. Schuurman, W. et al. Gelatin-Methacrylamide Hydrogels as Potential Biomaterials for Fabrication of Tissue-Engineered Cartilage Constructs. Macromol. Biosci. 13, 551–561 (2013). 8. Murphy, S. V. & Atala, A. 3D bioprinting of tissues and organs. Nat Biotech 32, 773–785 (2014). b l d f b h ll d f d 9. Ozbolat, I. T. & Yu, Y. Bioprinting toward organ fabrication: Challenges and future trends. IEEE Trans. Biomed. Eng. 6 (2013). 10. Tibbitt, M. W. & Anseth, K. S. References Hydrogels as Extracellular Matrix Mimics for 3D Cell. Culture. 103, 655–663 (2009). 11. Khetan, S. & Burdick, J. Cellular encapsulation in 3D hydrogels & Burdick, J. Cellular encapsulation in 3D hydrogels for tissue engin 11. Khetan, S. & Burdick, J. Cellular encapsulation in 3D hydrogels for tissue engineering. J. Vis. Exp. 3 7, doi:10.3791/1590 (2009 12. Bartnikowski, M., Wellard, R., Woodruff, M. & Klein, T. Tailoring Hydrogel Viscoelasticity with Physical and Chemical Crosslin Polymers (Basel). 7, 2650–2669 (2015). 3. Yue, K. et al. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials 73 254–271 (2015). 254–271 (2015). 14. Loessner, D. et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue 254–271 (2015). 14. Loessner, D. et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms Nat Protoc 11 727 746 (2016) ( ) 14. Loessner, D. et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat. Protoc. 11, 727–746 (2016). p 15. Klotz, B. J., Gawlitta, D., Rosenberg, A. J. W. P., Malda, J. & Melchels, F. P. W. Gelatin-Methacryloyl Hydrogels: To Biofabrication-Based Tissue Repair. Trends Biotechnol. 34, 394–407 (2016). 5. Klotz, B. J., Gawlitta, D., Rosenberg, A. J. W. P., Malda, J. & Melchels, F. P. W. Gelatin Methacryloyl Hydrogels: Toward Biofabrication-Based Tissue Repair. Trends Biotechnol. 34, 394–407 (2016). . Malda, J. et al. 25th Anniversary Article: Engineering Hydrogels f 17. Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S. & Dubruel, P. A review of trends and limitations in hydr f l ( ) lliet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S. & Dubruel, P. A review of trends and limitations in hydrogel-rapid rototyping for tissue engineering Biomaterials 33 6020 6041 (2012) 17. Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S. & Dubruel, P. A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. Biomaterials 33, 6020–6041 (2012).hf 17. Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S. & Dubruel, P. A review of trends and limitations in hydrogel rapid prototyping for tissue engineering. Biomaterials 33, 6020–6041 (2012).hf h prototyping for tissue engineering. Biomaterials 33, 6020–6041 (2012) h prototyping for tissue engineering. Biomaterials 33, 6020–6041 (2012).hf . Fedorovich, N. E. et al. The effect of photopolymerization on stem on stem cells embedded in hydrogels. Core/Shell distribution and cell viability assays in mono and co-axial printed scaffolds. h imaging of Core/Shell fluorescent compartments were performed by incorporating fluorescent beads (Sphero Core/Shell distribution and cell viability assays in mono and co-axial printed scaffolds. The imaging of Core/Shell fluorescent compartments were performed by incorporating fluorescent beads (Spherotech Inc., Lake Forest, IL, USA) into the Core (Blue beads) and Shell (Nile Red beads). Each bead type was 1.7–2.2 µm in diameter and was used at 0.1% w/v. Printed samples were then transferred onto 35 mm glass bottom dishes (MatTek Corporation, Ashland, MA, USA) for imaging. The mono-axial samples were printed containing Nile Red fluorescent beads only. l Cell viability in co-axial and mono-axial 3D bioprinting, was assessed using Calcein-AM and SYTOXTM Green (Thermo Fisher Scientific Inc.), respectively to stain live and dead cells, according to the manufacturer protocols. Confocal imaging was performed with NikonA1R confocal microscope using a Nikon Plan Fluor 10x DIC L N1 N.A. 0.3 objective lens. Digital images were processed using NIS-Elements software (Nikon, Amsterdam, Netherlands) without biased manipulations. 3D rendering was performed with NIS elements software using the (Thermo Fisher Scientific Inc.), respectively to stain live and dead cells, according to the manufacturer protocols. Confocal imaging was performed with NikonA1R confocal microscope using a Nikon Plan Fluor 10x DIC L N1 N.A. 0.3 objective lens. Digital images were processed using NIS-Elements software (Nikon, Amsterdam, Netherlands) without biased manipulations. 3D rendering was performed with NIS elements software using the Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 10 www.nature.com/scientificreports/ Histological analysis. Statistical Analysis. All statistical analyses were performed using GraphPad Prism software© with a statis- tical significance level of 0.05 indicated as p < 0.05. Differences between cytotoxicity of the PIs were determined using Unpaired t test or one-way Anova tests with Bonferroni or Dunnet corrections.i g p y In all graphs stars represents * is p ≤ 0.05; is p ≤ 0.01; * is p ≤ 0.001; not significant (n.s.) is p > 0.05. In all graphs stars represents * is p ≤ 0.05; is p ≤ 0.01; * is p ≤ 0.001; not significant (n.s.) is p > 0.05. Ethical statement. Use of all animals and procedures (isolation of ADSCc from sheep infrapatellar fat pad) in this study was approved by the University of Melbourne Animal Ethics Committee [ID 1513586] and all the experiments were performed in accordance with relevant guidelines and regulations. Ethical statement. Use of all animals and procedures (isolation of ADSCc from sheep infrapatellar fat pad) in this study was approved by the University of Melbourne Animal Ethics Committee [ID 1513586] and all the experiments were performed in accordance with relevant guidelines and regulations. www.nature.com/scientificreports/ Alpha-blending algorithm. All the images shown in this study are representative of at least three independent experiments. Live and dead in the 3D a NikonTiE microscope equipped with a fully automated A1 confocal laser (A1R, Nikon, Amsterdam, Netherlands) and NIS-Elements software. The percentage of live and dead cells was calcu- lated as follows: % of Live or Dead cells = 100 × n. Live or 100 n.Dead/TOT (n.Live + n.Dead) and 100% was normalized to day 0. An average of three different fields was counted per sample from at least three independent experiments. p Orthogonal projections obtained with the NIS-Elements software on the single z-stacks images were used for the representation of the volume occupied by Calcein-AM positive cells and fluorescent beads present in the Shell. The fluorescence measured from the two compartments at different time points was used to estimate their relative percentage over the same total area. Histological analysis. Samples were fixed in 1% paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA) for 4 hr at room temperature, embedded in O.C.T. TM Compound (Tissue-Tek, Sakura, Leiden, Netherlands) and flash frozen in liquid nitrogen. Cryosections of 10 μm thickness were mounted onto glass slides and stained with Safranin O (Sigma-Aldrich) for 10 minutes, dipped in 95% and 100% EtOH, cleared three times for 1 minute each in Xylene (Chem-Supply, GILLMAN, SA, Australia) and then mounted in Pertex medium (Grale HDS, Ringwood, VIC, Australia). For fluorescence analysis, 10 μm thickness slices were washed 2 times in PBS, permeabilized for 10 minutes in PBS-0.25%TritonX-100 (PBT) and then nuclei were stained by incu- bation with 5 µg/mL DAPI (Thermo Fisher Scientific Inc.) for 10 min at room temperature. The sections were washed in PBS, mounted with Fluoromount-G (Southern Biotech, Birmingham, AL, USA) onto glass slides and imaged using an epifluorescent Olympus IX70 inverted microscope using a SPOT Diagnostic RT-Slider camera and SPOT Diagnostic software using the indicated objective lenses. Images were processed using Photoshop software (Adobe). Acknowledgements g Funding from (1) Arthritis Australia – Zimmer Australia Grant, (2) Victorian Orthopaedic Research Trust, (3) Foundation for Surgery John Loewenthal Research Grants (RACS) and (4) The Australian Research Council Centre of Excellence Scheme (Project Number CE 140100012) is gratefully acknowledged. This work was carried out with the support from the St Vincent’s Hospital (Melbourne) Research Endowment Fund. The authors would like to thank the Australian National Nanofabrication Facility – Materials node for equipment use. References & Gelinsky, M. A versatile method for combining different biopolymers in a core/shell fashion by 3D plotting to achieve mechanically robust constructs. Biofabrication 8, 45001 (2016). phenyl 2,4,6 trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials 30, 6702 6707 (2009). 30. Akkineni, A. R., Ahlfeld, T., Lode, A. & Gelinsky, M. A versatile method for combining different biopolymers in a core/shell fashion by 3D plotting to achieve mechanically robust constructs. Biofabrication 8, 45001 (2016). 31. Costantini, M. et al. 3D bioprinting of BM-MSCs-loaded ECM biomimetic hydrogels for in vitro neocartilage form Biofabrication 8, 35002 (2016).f f , ( ) 2. Maruthappu, M. & Keogh, B. How might 3D printing affect clinical practice? BMJ 30, 349 (2014). pp g g p gf p 33. Ye, K. et al. Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold. PLoS One 9 (20 Author Contributions S.D., C.O., and C.D.O. performed the experiments and data analyses; S.D., C.O. prepared the Figures 1, 2, 4, 5, 6 and 7, the Supplementary file material and the manuscript; C.D.O. prepared Fig. 3; R.B. and P.P. performed compression tests and data analyses, and revised the manuscript; C.A. provided technical assistance; C.D.B., G.W., P.C. conceived the study, discussed the results and revised the manuscript; R.K. and A.Q. provided technical support and analytically revised the manuscript. Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x References Biomaterials 30, 344–353 (20 ,hf p p y y g , ( ) 19. Colosi, C. et al. Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low-Viscosity Bioink. Adv. Mater. 28, 677–684 (2016). 0. O’Connell, C. D. et al. Development of the Biopen: a handheld device for surgical printing of adipose stem cells at a chondral wound site. Biofabrication 8, 15019 (2016).h f 21. Adams, M. A. The mechanical environment of chondrocytes in articular cartilage. Biorheology 43, 537–545 (2006). . & Grellmann, W. In Principles of Polymerization 79, 9–20 (2001).f p f y 3. Zhu, J. & Marchant, R. E. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev. Med. Devices 8, 607–26 (2011). l ll h f b f h h d f 23. Zhu, J. & Marchant, R. E. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev. M 24. Ferris, C. J., Gilmore, K. G., Wallace, G. G. & In Het Panhuis, M. Biofabrication: An overview of the approaches used for printing of living cells. Appl. Microbiol. Biotechnol. 97, 4243–4258 (2013). g pp 25. Rehfeldt, F., Engler, A. J., Eckhardt, A., Ahmed, F. & Discher, D. E. Cell responses to the mechanochemical microenvironment–implications for regenerative medicine and drug delivery. Adv. Drug Deliv. Rev. 59, 1329–39 (2007). Scientific Reports \| 7: 5837 \| DOI:10.1038/s41598-017-05699-x 11 www.nature.com/scientificreports/ 6. Williams, C. G., Malik, A. N., Kim, T. K., Manson, P. N. & Elisseeff, J. H. Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing hydrogels and cell encapsulation. Biomaterials 26, 1211–1218 (2005). 7. Jang, J. et al. Tailoring mechanical properties of decellularized extracellular matrix bioink by vitamin B2-induced photo-crosslinking Acta Biomater. 33, 88–95 (2016).h ( ) 28. Billiet, T., Gevaert, E., De Schryver, T., Cornelissen, M. & Dubruel, P. The 3D printing of gelatin methacrylamide cell-laden tissue- engineered constructs with high cell viability. Biomaterials 35, 49–62 (2014). g g y 9. Fairbanks, B. D., Schwartz, M. P., Bowman, C. N. & Anseth, K. S. Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials 30, 6702–6707 (2009). 0 Akkineni A R Ahlfeld T Lode A & Gelinsk M A ersatile method for combining different biopol mers in a core/shell fashion 29. Fairbanks, B. D., Schwartz, M. P., Bowman, C. N. & Anseth, K. S. Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials 30, 6702–6707 (2009). 30. Akkineni, A. R., Ahlfeld, T., Lode, A. © The Author(s) 2017 Additional Information Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-05699-x Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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W4211020873.txt	https://ejop.psychopen.eu/index.php/ejop/article/download/262/262.pdf	en		Research that makes a difference	PsycEXTRA Dataset	2,009	cc-by	602	Europe’s Journal of Psychology 2/2009, pp.1-2 www.ejop.org Research that makes a difference By Cary L. Cooper, CBE Lancaster University Given the dramatic economic times we are all living in today, it is important, more than ever before, that the research we undertake in the field of psychology is of relevance to society. The economic recession is one of the worst in recent history, and will have many consequences for the cultures that we live and work in. It will not only affect the field of occupational psychology but also clinical, developmental and neuro-cognitive psychology as well. The impact of this downturn is having consequences for individual’s health and well-being, family relationships, the roles of men and women at work and in the family, and for old age as well as work opportunities begin to dry up for older workers. It is incumbent of research psychologists that our work is relevant and applied to the contemporary problems we currently face. We will always engage of blue-sky or basic psychological research, but many of the problems people face in Europe during these difficult times require apposite and effective solutions. The time has come for applied psychologists to work on the issues that really matter in understanding human behaviour in times of distress. This is a golden opportunity for us, as many of the changes that are taking place across Europe provide us with a natural laboratory for our science, and the practice of applied psychology. There are likely to be cutbacks in a variety of the public services, whether in education or health or social services, so the focus of our work should be to identify what is happening, what we can do about it and how we can pinpoint and synchronize our interventions (and, of course, assess their impact). By being instrumental in our focus on current issues, it is likely to make a difference in our respective countries, and show the usefulness of the science of psychology. We should take this opportunity of the changing economic order to explore new areas of research for psychology, as the old adage goes “if you always do what you always did—you’ll always get what you always got”. I would like to encourage the Europe’s Journal of Psychology new generation of psychological researchers and practitioners to explore the dynamics of our times, of the current turmoil that many are experiencing and develop new paradigms for research and practice. This is our challenge, or as the old Chinese proverb goes, about the importance of understanding human behaviour, “if you are planning for one year, plant rice. If you are planning for ten years, plant trees. If you are planning for a hundred years, plant people”. I hope you will enjoy this issue of the journal and look forward to seeing very timely and innovative articles in the future. About the author: Cary Cooper is a Director and founder of Robertson Cooper Ltd, Professor of Organisational Psychology and Health and Pro Vice Chancellor at Lancaster University. Recently awarded the Lifetime Practitioner Award from the British Psychology Society in recognition of his services to the profession, he is recognised as a world-leading expert on stress and is the media’s first choice for comment on workplace issues. He is a Fellow of the British Psychological Society, The Royal Society of Arts, The Royal Society of Medicine, The Royal Society of Health and an Honorary Fellow of the Royal College of Physicians. He is also the President of the British Association for Counselling and Psychotherapy, Editor-in-Chief of the Blackwell Encyclopedia of Management and the author /editor of over 100 books. 2
https://openalex.org/W2020787734	http://www.scirp.org/journal/PaperDownload.aspx?paperID=2004	English	null	A Multi-objective QoS Optimization with Fuzzy Based Parameter Setting for Real-Time Multicasting	International journal of communications, network and system sciences/International journal of communications, network, and system sciences	2,010	cc-by	7,787	Int. J. Communications, Network and System Sciences, 2010, 3, 530-539 doi:10.4236/ijcns.2010.36071 Published Online June 2010 (http://www.SciRP.org/journal/ijcns/). Abstract We propose a multi-objective Pareto-optimal technique using Genetic Algorithm (GA) for group communi- cation, which determines a min-cost multicast tree satisfying end-to-end delay, jitter, packet loss rate and blocking probability constraints. The model incorporates a fuzzy-based selection technique for initialization of Quality of Service (QoS) parameter values at each instance of multicasting. The simulation results show that the proposed algorithm satisfies on-demand QoS requirements (like high availability, good load balanc- ing and fault-tolerance) made by the hosts in varying topology and bursty data traffic in multimedia commu- nication networks. Keywords: QoS, Fuzzy, Multicast, Real-Time, Multi-Objective Keywords: QoS, Fuzzy, Multicast, Real-Time, Multi-Objective Keywords: QoS, Fuzzy, Multicast, Real-Time, Multi-Objective Copyright © 2010 SciRes. 3.1. Network Model Multi-objective optimization is used to solve optimiza- tion problems that have two or more number of conflict- ing objectives, where there may not exist an unique op- timal solution. Discovering and fixing optimal solutions in such scenarios is an open problem [6,11]. In general, almost all real-world problems are of multiple objectives, where each objective needs to be satisfied. For such type of problems a single best solution does not exist with simultaneous satisfaction of all objectives. In fact, we may have a set of optimal solutions in the entire search space, when all objectives are considered. These solu- tions are known as Pareto-optimal solutions. None of the solutions in this set is absolutely better then any other. So, any one of the solution can be an acceptable solution. In this context, mathematically we can define our problem more precisely, and introduce some related definitions in order to explain our GA based proposed model. It is noted that all of the objectives are in a minimized form in the following discussion. The network consists of n number of similar mobile hosts moving within a three-dimensional space. The nodes can communicate among themselves either di- rectly or indirectly via intermediate nodes. All nodes are capable of transreceiving as well as forwarding the re- quest of their neighbors to its near by nodes with broad- casting. For any instant of time the nodes along with their links can be represented as G = (V, E), where V and, E represent the set of nodes and edges in the network respectively. A multicast tree T = (VT, ET) in the graph G is rooted at source node s with destination nodes {d1, d2, …, dt} ( ) t n  act as group members for the source node. A tree is having k number of branches and each branch is of r number of edges. The QoS parame- ters considered for multi-objective optimization during multicasting from a single source to its group members are as follows: (k t ) ) ) ) Bandwidth (BW): The bandwidth of a tree is the av- erage bandwidth of all its branches; whereas bandwidth of any branch of a tree is the minimum of all its edge bandwidths from source to the corresponding destination node. 1. Introduction Our proposed model, its en- vironment setup procedure and implementation with GA are presented in Section 3. The performance evaluation and analysis of the model are elaborated in Section 4. Section 5 concludes the paper and discusses potential future work. Pareto-optimality concepts. Our proposed model, its en- vironment setup procedure and implementation with GA are presented in Section 3. The performance evaluation and analysis of the model are elaborated in Section 4. Section 5 concludes the paper and discusses potential future work. Pareto Front: It is defined as . { ( )\| } PF Z X X P   3. Proposed Model In this section we introduce the multi-objective model with its attributes, and their mapping to GA with Pare- to-optimal verification. 1. Introduction To fulfill the on-demand request of the users we use Pareto optimal GA, which guarantees for achieving bet- ter QoS from a large search space. When the size of the network is large the situation demands optimization of QoS parameters such as: delay, jitter, path length (hops), packet loss rate and variation of load among the nodes involved, with high fault-tolerance from the network within acceptable cost. Most of the research works focus on multicasting to a group for near optimal, fast solution with multi-tree backups using GA [9-11]. Multicast services have been used for real-time multime- dia applications to transport audio-video frames, among a group of users. During real-time communication the related audio-video frames are required to be delivered at the end nodes in a synchronous manner [1]. Further, the frequent change of service types, session timings with QoS requirements by the group members increases the communication complexity of the network [2,3]. In a wireless medium the situation further deteriorates than fixed network due to unpredictable mobility of the host nodes as well as their variations in resource requirements. It is also important to keep the network live with all pos- sible satisfactions to the users during that period. Such a scenario requires multi-objective optimizations with con- straints satisfactions [4,5]. The situation becomes critical when the destination nodes require multi-rate multicast sessions [6]. Hence development of multi-objective op- timization algorithm for multi-rate traffic during multi- casting is a challenge for efficient allocation of resources in a dynamically changing network [7,8]. However, a Pareto optimal algorithm can provide better results by fulfilling users’ requirement, irrespective of irrelevant transformation of parameters [4]. However, real-time multimedia applications require multi-rate data flow to the served nodes in an on-demand basis with optimized resource allocation and cost in- curred. For addressing multi-objective optimization is- sues GAs are suitable [9]. In this paper, we propose a multi-objective evolution- ary algorithm supporting multi-rate data flow across the network using GA. The algorithm approximates Pareto front by generating a set of non-dominated solutions. Multiple services can be formulated as a multi-objective model [12]. Our model assumes that complete knowl- edge about the network is available to all the nodes pre- sent inside the region. The paper is organized as follows. In Section 2, we define the multi-objective optimization (MOO) and IJCNS S. C. RAI ET AL. 531 Pareto-optimality concepts. 3.1. Network Model Both branch bandwidth and tree bandwidth are represented in Equations (2) and (3) as follows: ( l BW (BW ( b BW T Multi-Objective Optimization (MOO): Let Z1(X), Z2(X), …, Zn(X) are n number of objectives to be opti- mized with gi(X) ≤ 0, i = 1, 2,…, k1 as inequality con- straints and hi(X) = 0, i = 1, 2, …, k2, as equality con- straints for the m-dimensional vector X = (x1, x2, …, xm) then MOO [13] can be defined as follows: 1 2 1 2 1 2 1 2 ( ) ( ( ), ( ), , ( )) ( ) 0, 1, 2, , ( ) 0, 1, 2, ..., ( , , , ) , 1, 2, , n i i m m i i Minimize Z X Z X Z X Z X subject to g X i k h X i k where X x x x and x i m                    min , 1,2, , b l BW BW l r    (2) { , 1,2,..., } T b BW mean BW b k   (3) (2) (3) (2) (1) (3) Delay (DL): Delay between two corresponding node (branch delay: ) is the sum of the delay at the in- termediate nodes (node delay: ) as well as the propagation delay through the links (link delay: ) from source to destination. For a multicast tree the tree delay is the maximum delay among all branch delays ( ). The two different delays can be shown as: b DL v DL e DL ( T DL b DL ) 1, 2, , i m    Copyright © 2010 SciRes. 3.2. Formulation of Problem where, : current mutation probability, : Maxi- m P max m P mum mutation probability, : Minimum mutation probability, : current generation, and : maximum generation min m P curGen maxGen The Bandwidth (BW), end-to-end delay (DL), jitter (JT), packet loss rate (PLR) and blocking probability (BP) are the five QoS parameters as defined in Subsection 3.1 are considered for our model. All the five objectives are to be satisfied simultaneously within their desired accept- able range for multi-objective optimization. Except BW all other objectives will be minimized, where as the for- mer is to be maximized for optimal utilization. As de- fined in Equation (1) our MOO problem can be formu- lated as: Fitness Test: The fitness of a tree is evaluated in two steps. In the first step, we evaluate Delay, Jitter, PLR and BP using Equations (5), (7), (9) and (10) respectively. Instead of bandwidth we evaluate deficiency from max- imum bandwidth ( ) s BW . So, and repre- sented in Equations (2) and (3) are modified as follows. b BW T BW 1 2 3 ( , , , , ) , , , , 0, 1,2,3,4 i Optimize Z Z BW DL JT PLR BP subject to DL JT PLR BP and BW where i and is some positivethreshold value 4               (11 1 2 3 ( , , , , ) , , , , 0, 1,2,3,4 i Optimize Z Z BW DL JT PLR BP subject to DL JT PLR BP and BW where i and is some positivethreshold value 4               ( max{ , 1,2,..., } s s b l BW BW l r   (13) { , s s T b BW mean BW b E  } T  (14) (13) (14) (11) , and BW  0, 1,2,3,4 i where i   At the time of evaluation, if we find an invalid node, which is not an intermediate node in the respective path, then a penalty value is added to the delay. If the BW, JT, PLR and BP values lie outside the desired range, for each case we add a small penalty to the delay. 3.2. Formulation of Problem The values of DL, s BW and JT are rationalized within the range of 0 and 1. Then in the second step the fitness value is evalu- and is some positivethreshold value  and is some positivethreshold value  T number of destinations not reached BP total number of destinations  (10) (10) (max min ) max max m m m m P P curGen P P Gen     (12) (max min ) max max m m m m P P curGen P P Gen     (12) (12) The Genetic Operators: 1) Selection: We have used an elitist model for selec- tion of the individuals for the next generation of popula- tion. 30 percent of the best fit individuals are selected at the first step for the next generation. Rest 70 percent of the population are selected using Roulette Wheel selec- tion method. 1) Selection: We have used an elitist model for selec- tion of the individuals for the next generation of popula- tion. 30 percent of the best fit individuals are selected at the first step for the next generation. Rest 70 percent of the population are selected using Roulette Wheel selec- tion method. ) e 1 (1 T b e E PLR PLR     (8) { , 1,2,..., } T b PLR mean PLR b k   (9) (8) 2) Crossover: Standard uniform crossover technique is used for the crossover of two parents selected randomly from the matting pool. (9) Blocking Probability (BP): The ratio between num- ber of blocked calls to the number of offered calls in a tree is considered as the blocking probability for that tree. In other words it is the ratio of unreached nodes to the total number of destination nodes available in a multicast tree. Mathematically it is represented as: 3) Mutation: We observe that using the standard mutation technique we do not obtain the requisite level of performance. When the mutation probability is bit low it does not yield any result. In our model we have kept the mutation probability very high, i.e., 0.2 and slowly decrease it with the progress of the generations using the following method. 3) Mutation: We observe that using the standard mutation technique we do not obtain the requisite level of performance. When the mutation probability is bit low it does not yield any result. In our model we have kept the mutation probability very high, i.e., 0.2 and slowly decrease it with the progress of the generations using the following method. Pareto Dominance: If and 1 2 ( , , , ) m U u u u   The different phases of GA for natural evolution through encoding, selection, crossover and mutation operations are discussed as below. Wheel Selection approach. The fitness/objective function is the multi-objective function defined in Equation (11). The different phases of GA for natural evolution through encoding, selection, crossover and mutation operations are discussed as below. Encoding: We use tree encoding for each chromo- some to represent a multicast tree due to the 5 properties such as 1) all feasible trees can be represented; 2) the probability of representing all feasible tree is same; 3) only trees are represented; 4) low time complexity for encoding and decoding, and 5) no global effect on the tree, due to small change in representation, as proposed in [14]. Encoding: We use tree encoding for each chromo- some to represent a multicast tree due to the 5 properties such as 1) all feasible trees can be represented; 2) the probability of representing all feasible tree is same; 3) only trees are represented; 4) low time complexity for encoding and decoding, and 5) no global effect on the tree, due to small change in representation, as proposed in [14]. T T b e e E v V v JT JT J      T (6) max{ , 1,2,..., } T b JT JT b   k (7) T T b e e E v V v JT JT J      T (6) max{ , 1,2,..., } T b JT JT b   k (7) (6) (7) ) ) Packet Loss Rate (PLR): It is defined as a ratio of the number of lost packets to the total number of transmitted packets. PLR for a tree branch is the cumulative along each edge of the path from root to the leaf node; where as the PLR for a multicast tree is the average loss rate of all branches present in the tree. Both can be represented mathematically as: ( b PLR e (PLR ( ) T PLR Pareto Dominance: If and 1 2 ( , , , ) m U u u u   Pareto Dominance: If and are two characteristic functions then dominates V(u v) if and only if the condition is satisfied for  and 1 2 ( , , , ) m U u u u   i i m u v  {1, i 1 2 ( , , , ) m V v v v   U {1,2 i , , }:  2,   . , }: i i m u v  v T T b e e E v V DL DL DL       (4) max{ , 1,2,..., } T b DL DL b k   (5) (4) (4) Pareto Optimality: A solution is said to be Pareto Optimal if and only if X  : ( ) ( ) X Z X  Z X   (5) Jitter (JT): It is defined as the average delay variation between any source and destination nodes during trans- mission of data packets. It is due to processing delay variation at the intermediate nodes as well as the propa- Pareto Optimality Set: A Pareto Optimality set P is defined as { \| : ( ) ( P X X Z X Z X     )} where 1 2 ( , , , ) m X x x x   is any other element of. Pareto Optimality Set: A Pareto Optimality set P is defined as { \| : ( ) ( P X X Z X Z X     )} where 1 2 ( , , , ) m X x x x   is any other element of. Copyright © 2010 SciRes. IJCNS S. C. RAI ET AL. 532 gation delay variation during communication through the links. In a multicast tree the jitter for any branch ( ) b JT is the sum of the jitter at the intermediate vertices ( v) JT and in each edge ( e) JT ; whereas the jitter for a multicast tree is the maximum among all branches and are given in the following equations. Wheel Selection approach. The fitness/objective function is the multi-objective function defined in Equation (11). The different phases of GA for natural evolution through encoding, selection, crossover and mutation operations are discussed as below. Wheel Selection approach. The fitness/objective function is the multi-objective function defined in Equation (11). Copyright © 2010 SciRes. 3.4. Simulation Procedure 1) Scenario Generation: For our simulation purpose, we have considered a three dimensional (3D) space to simu- late real life wireless scenarios. The 3D coordinates of a node are randomly generated. The Euclidean distance between each pair of nodes is measured. If the distance between any two nodes is found to be less then 250 me- ters then a link is established between these two nodes and accordingly the adjacency matrix is formed. The scenario represented in Figure 1 is one of our network topology formed during simulation. good result. For the subsequent simulations, we take a high value such as 0.2 for the mutation probability to ensure a diversified search and slowly reduces as the network converges to an optimal solution to narrow down the search in the close proximity. Once the network is formed, the source and destina- tions are generated randomly and GA is applied to find out the optimal multicast tree over a set of evolutions. Also we define different cut-off ranges for various net- work parameters to fulfill the demands of multimedia applications. These cut-off values were considered in the algorithm while a tree is selected for optimality. In our model we have considered the nodes are of ho- mogeneous with respect to their transmission range, proc- essing capability and buffer length [15,16]. The wireless bandwidth is equally shared among the mobile entities. At any instance of the simulation the available bandwidth is optimized in consideration with the QoS required by the end nodes. To emulate the scenario the available band- width for each node can be obtained randomly. However, random initialization of other QoS parameters like de- 2) Simulation Parameters: The Network and GA parameter values considered for our simulation are given in the Table 1 and Table 2 respectively. During initial stage of our simulation, we have given a constant low value to the mutation probability, which did not yield 2) Simulation Parameters: The Network and GA parameter values considered for our simulation are given in the Table 1 and Table 2 respectively. During initial stage of our simulation, we have given a constant low value to the mutation probability, which did not yield 0 1000 2000 3000 4000 5000 0 2000 4000 6000 0 50 100 150 200 250 300 Breadth (meter) Length (meter) Height (meter) Figure 1. 3.3. Simulation Model Using GA In our proposed model, we choose a heuristic based Ge- netic Algorithm with binary crossover and Roulette Copyright © 2010 SciRes. IJCNS IJCNS S. C. RAI ET AL. 533 Table 1. Network Parameters. Table 1. Network Parameters. Network Parameters Min Max Bandwidth 0 25 Delay 200 1000 Jitter 0 650 Packet Loss Rate 0 1 Table 2. GA Parameters. GA Parameter Value Population Size 100 Crossover Probability 0.6 Mutation Probability 0.2 ated as follows ated as follows ated as follows 1 2 3 4 5 s f w DL w BW w JT w PLR w BP      (15) where , , , , and are user defined con- stants in the range [0, 1]. The fit value i.e. 1 w 2 w 3 w 4 w 5 w 1 (1 ) fit f   in the range [0, 1] is maximized by using GA. Network Parameters Min Max Bandwidth 0 25 Delay 200 1000 Jitter 0 650 Packet Loss Rate 0 1 Table 2. GA Parameters. Copyright © 2010 SciRes. 4.1. A Study of QoS Parameters Hop Count (HC) represents the maximum number of hops required to reach any destination in the multicast tree. where the triplets (xD, nD, Di), (xJ, nJ, Ji), (xL, nL, Li) and (xP, nP, Pi) represent maximum, minimum and cur- rent values of DL, JT, PLR, and BP of the ith node re- spectively. xW and xi represent maximum and allocated BW for the ith node respectively. The parame- ters ( , ) D D a c , ( , ) J J a c , ( , ) L L a c , and ( , ) P P a c used in evaluation of sigmoidal function are suitably selected constants which are obtained after a maximum number of testing. Delay (DL): is the maximum delay along any branch of the tree. A penalty is added if no link found between two nodes. Packet Loss Rate (PLR): is the average packet loss along the tree branches. The cut-off for PLR is set at 10%. If a branch having more than 10% packet loss then it is assigned a penalty, such that the model tries to search a path less then10% PLR. Jitter (JT): is the maximum delay variation along any branch of the tree. The cut-off is set at 20% otherwise a penalty is added. 3.4. Simulation Procedure 3-D Network topology generation with 100 nodes (volume: 6000 × 5000 × 300 cubic meters). Breadth (meter) Figure 1. 3-D Network topology generation with 100 nodes (volume: 6000 × 5000 × 300 cubic meters). Copyright © 2010 SciRes. IJCNS S. C. RAI ET AL. 534 Table 3. Average results of QoS parameters with different group size. lay(DL), jitter (JT) and packet loss rate (PLR) and blocking probability (BP) may results unpredictable net- work performance. Although there is no established rela- tionship among the parameters [7,17], we propose a sigmoidal fuzzy-logic based parameter initialization with error model for our GA approach in (17). Table 3. Average results of QoS parameters with different group size. Table 3. Average results of QoS parameters with different group size. Tot Gr HC DL PLR JT BW BP PP 10 3 2.16 442.43 0.012 40.14 21.99 0 3.01E+07 20 5 2.76 413.36 0.013 46.39 22.52 0 2.88E+10 30 8 8.13 1545.4 0.033 53.9 20.44 0 6.13E+11 40 10 5.34 726.61 0.02 57.86 21.05 0 5.61E+12 50 13 7.9 3123.7 0.032 58.39 21.59 0 3.81E+13 60 15 9.48 3581.3 0.035 60.01 21.31 0 1.81E+14 70 18 5.93 1835.1 0.025 51.86 21.23 0 2.26E+14 80 20 6.81 1902.3 0.026 52.59 21.57 0 3.66E+14 90 23 7.6 2240.4 0.02 48.59 20.02 0 7.28E+14 100 25 8.44 2190.1 0.023 42.66 20.42 0 6.08E+14 1 ( ; , ) 1 exp( ( )) sig x a c a x c    (16) 0.2 fs sig rand sig     (17) (16) (17) We establish the relationship between available band- width and other parameters with the following formulae as given in Equations (18) to (21). ( ) (( ); , ) i i D D D nD XD nD fs xW x a c      (18) ( ) (( ); , ) i i J J J nJ xJ nJ fs xW x a c      (19) ( ) (( ); , i i L nL xL nL fs xW x a c      ) L L i P P (20) ( ) (( ); , ) iP nP xP nP fs xW x a c      (21) Copyright © 2010 SciRes. 4. Simulation Results and Performance Analysis Bandwidth (BW): of the multicast tree is the average bandwidth requirement of all the tree branches. The cut-off BW is taken as 2 Mbps. Blocking Probability (BP): is the ratio of not getting a node to the total number of destination nodes in the mul- ticast tree. In this simulation our objective is to test if the proposed model is producing an optimal solution with multi-ob- jective constraints. During simulation, the total number of nodes were varied from 10 to 100 by keeping density of the nodes in the space same by changing the space volume according to total number of nodes such that the maximum number of neighbors present for a node is lim- ited to 6. The multicast group size was kept as one fourth of the total nodes. The maximum numbers of generations for evolution were fixed at 250. As the objectives are conflicting in nature, to obtain the Pareto-optimal solu- tion we have considered different sets of randomly se- lected weights in Equation (15) for each simulation. However, one may consider meta-heuristic approach for dynamic selection of those weights. The range of net- work parameters and GA parameters considered for evaluating QoS parameters are represented in Table 1 and Table 2 respectively. The simulation results of QoS parameters are shown in Table 3. Possible Paths (PP): in the tree are the average possi- ble paths available for any destinations in the tree. The PP field gives an estimation of the search space and also proves the efficiency of our algorithm to find an optimal tree from such a huge search space. Also the BP field proves the definiteness of this algorithm to find a path from source node to destination node (if exists). The ‘Tot’ represents the total number of nodes con- sidered for the simulation and ‘Gr’ represents the multi- cast group size. In order to maintain node density, we ha- ve increased the volume of the search space proportion- ately as the total number of nodes. The QoS parameter values were obtained by changing the total number of nodes and the respective multicast group size. The QoS parameter values presented in the table are the average values of 25 simulations. The multicast tar- gets are randomly chosen for each simulation and QoS Copyright © 2010 SciRes. IJCNS IJCNS S. C. RAI ET AL. 535 work conditions is assumed to be constant, i.e. 4.2. A Simulation Study The robustness of our GA based approach is the inher- ent capability of obtaining optimal solution after search- ing from a large search space, which is shown in Figure 2. The robustness of our GA based approach is the inher- ent capability of obtaining optimal solution after search- ing from a large search space, which is shown in Figure 2. Further the increase in group size of a multicast envi- ronment results in marginal elevation in the node density. The node density of a multicast tree is considered as the ratio between numbers of nodes in the tree to the size of the group. i.e. represented as The overall results from our simulation studies have been presented in Table 3. We now present the details of a sin- gle simulation study to show the working of the algo- rithm and the performance results that were obtained. In this simulation, we have considered 10 nodes as a net- work from the environment consisting of 100 nodes. In the selected group, the node with index number 14 was taken as the source node and nodes with indices 5, 11, 15, 17, 20 were considered as the target nodes. g g p Further the increase in group size of a multicast envi- ronment results in marginal elevation in the node density. The node density of a multicast tree is considered as the ratio between numbers of nodes in the tree to the size of the group. i.e. represented as number of nodesinthetree nodedensity sizeof the group  (22) (22) While measuring intermediate node density, the net- Table 4 presents the status of a typical simulation. The 10 20 30 40 50 60 70 80 90 100 0 0.5 1 1.5 2 2.5 3 x 10 6 Number of Nodes Average number of valid paths Figure 2. A subset of total search space representing average number of valid paths with 5 intermediate nodes. Average number of valid paths ure 2. A subset of total search space representing average number of valid paths with 5 intermediate nodes Table 4. Multicast Tree with QoS Parameter Values. Table 4. Multicast Tree with QoS Parameter Values. 4. Simulation Results and Performance Analysis the adja- cency matrix representing the links among the nodes is not altered. parameters are also reinitialized for each simulation. We have not considered HC as a parameter for optimization; as a result we obtained different results for different tar- get size depending upon the availability connectivity. As we have considered a minimum delay of 200 ms per node, the delay affects with increase in the number of hops. However, this algorithm has ensured to find a path where the delay gets minimized. The results of QoS parameter values obtained from simulation show that our proposed model can adapt and scale well to large group members and capable of ob- taining near optimal solution from a large search space. Copyright © 2010 SciRes. IJCNS 4.3. Performance Analysis In the following, we compare our work with some of the other related work. Gomathy et al. [11] have evaluated the packet delivery ratio and end-to-end delay of ODMRP, CAMP, and NTPMR multicast routing protocols after incorporation of a fuzzy based priority scheduler. Pinto et al. [21] has considered maximum end-to-end delay, average delay, maximum link utilization and cost of multicast tree as the four multi objective parameters for For the purpose of comparison, we have simulated the approach presented in [18] and the performance results have been represented as Model 1 results. The simulation results of the current study have been presented as Model For the purpose of comparison, we have simulated the approach presented in [18] and the performance results have been represented as Model 1 results. The simulation results of the current study have been presented as Model 2 results. In a typical simulation we have taken the total number of nodes in the network as 20, one source and 5 destinations, with a population size 10, the GA was al- lowed to run for 250 generations. The QoS metric values of the population are recorded after every 25 generations. 2 results. In a typical simulation we have taken the total number of nodes in the network as 20, one source and 5 destinations, with a population size 10, the GA was al- lowed to run for 250 generations. The QoS metric values of the population are recorded after every 25 generations. IJCNS 0 50 100 150 200 250 300 4 6 8 10 12 14 16 18 20 22 Bandwidth Number of Iterations (Kpbs) Figure 3. Pareto front of bandwidth allocation in Model 1. 0 50 100 150 200 250 300 8 10 12 14 16 18 20 22 Number of Iteration Bandwith allocated (Kpbs) Figure 4. Pareto front of bandwidth allocation in Model 2. 0 50 100 150 200 250 300 4 6 8 10 12 14 16 18 20 22 Bandwidth Number of Iterations (Kpbs) Figure 3. Pareto front of bandwidth allocation in Model 1. 0 50 100 150 200 250 300 4 6 8 10 12 14 16 18 20 22 Bandwidth Number of Iterations (Kpbs) The results of Model 1 are presented in Figures 3, 5, 7, 9, 11 and that of Model 2 in Figures 4, 6, 8, 10 and 12. Copyright © 2010 SciRes. 4.2. A Simulation Study Result obtained after generation 25 50 75 100 125 150 Multicast Tree 14→11 14→15 14→5 14→17 14→6→20 14→11 14→15 14→5 14→12→17 14→6→20 14→11 14→15 14→5 14→12→17 14→6→20 14→11 14→15 14→5 14→12→17 14→6→20 14→11 14→15 14→5 14→12→17 14→6→20 14→11 14→15 14→5 14→12→17 14→6→20 HC 1.2 1.4 1.4 1.4 1.4 1.4 DL 240.52 280.35 280.35 280.35 280.35 280.25 PLR 0.097278 0.09291 0.09291 0.09291 0.09291 0.09016 JIT 48.462 46.835 46.835 46.835 46.835 43.37 BW 21.521 21.672 21.672 21.672 21.672 22.006 BP 0.0 0.0 0.0 0.0 0.0 0.0 S. C. RAI ET AL. 536 one parameter, resulting multi-objective for two parame- ters only as against 5 distinct parameters considered in our study. results obtained after every 25 simulations were recorded, the multicast tree obtained and the QoS parameter values at that instance have been represented in this table. From the table it can be observed that the multicast tree pro- duced by this method does not change after 50 iterations and stabilizes. Please note that the values given in Table 4 for HC, DL, PLR, JIT, BW, and BP are the mean val- ues of the different multicast trees represented in the population for the respective generation. These values converge after 150 generations. The non-dominated solution of bandwidth utilization and end-to-end delay converges almost in the same num- ber of generations as pointed by Roy et al. [20]. The call blocking (BP) percentage was found to be 10% with 10 requests per second in [20]. However in our study, we have obtained 0.0 BP with network size in the range of [10, 100] where the respective maximal group size is one fourth of the network size. 4.3. Performance Analysis The objective of this experimentation (given in Equa- tion 11) is to maximize the bandwidth (BW) and to mi- nimize all other parameters. From Figures 3 and 4, we can see that optimal bandwidth allocation in the current study is 22.01 Kbps as against 5.98 Kbps in Model 1, giving performance improvement of 267.99%. One of the objectives is to minimize the delay while optimizing all other parameters. The Pareto fronts for delay in Model 1 and Model 2 have been presented in Figures 5 and 6 respectively. The optimal delay was rounded to be 794.47 ms in Model 1 as against 280.25 ms in Model 2, which shows a performance improvement of 64.72 % in delay parameter. Figure 3. Pareto front of bandwidth allocation in Model 1. 0 50 100 150 200 250 300 8 10 12 14 16 18 20 22 Number of Iteration Bandwith allocated (Kpbs) Figure 4. Pareto front of bandwidth allocation in Model 2. Figure 3. Pareto front of bandwidth allocation in Model 1. The Pareto fronts for HC have been presented in Fig- ures 7 and 8 respectively for Model 1 and Model 2. The Pareto fronts for jitter have been presented in Figures 9 and 10 respectively. The optimal level of jitter was 149.87 ms for Model 1 and 43.37 ms for Model 2, giving an improvement in performance of 71.06 %. The Pareto fronts for PLR have been presented as Figures 11 and 12. 0 50 100 150 200 250 300 8 10 12 14 16 18 20 22 Number of Iteration Bandwith allocated (Kpbs) The optimal level of PLR for Model 1 is 0.4368 and for Model 2 is 0.09016, giving an improvement of perform- ance of 79.36%. The performance of this work is compared with the performance of the models suggested by other research- ers. On comparing our results with that of Roy et al. [19], we find that our approach outperforms the approach pre- sented in [19]. However, Roy et al. has considered only three parameters such as bandwidth requirement, band- width utilization and end-to-end delay. The bandwidth requirement and bandwidth utilization possibly relates to Figure 4. Pareto front of bandwidth allocation in Model 2. S. C. RAI ET AL. 537 0 50 100 150 200 250 300 200 400 600 800 1000 1200 1400 Number of Iterations Delay Figure 5. Pareto front of delay in Model 1. 4.3. Performance Analysis RAI ET AL. 538 0 50 100 150 200 250 300 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Number of Iterations Packet Loss rate Figure 11. Pareto front of PLR in Model 1. 0 50 100 150 200 250 300 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Number of Iterations Packet Loss Rate Figure 12. Pareto front of PLR in Model 2. 0 50 100 150 200 250 300 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Number of Iterations Packet Loss rate tion environments considered by these researchers are not compatible to ours to make a meaningful performance comparison. 5. Conclusions We have proposed a multi-objective multicast model for wireless ad-hoc network using GA. In this model we have considered five QoS parameters such as bandwidth, end-to-end delay, jitter, packet loss rate and blocking probability for multi-objective optimization. Simulation studies were carried out with a network of 100 nodes moving in a 3D space of volume: 5000 × 6000 × 300 cubic meters with the number of multicast destination nodes varying in the range 5 to 30. Our model estab- lished near optimal relationships among the QoS pa- rameters to satisfy multi-objective optimization. Irre- spective of increase in group size our model could opti- mize path length between any sources to its group mem- bers. Selection of min-cost paths from source to multiple destinations for multicasting is a NP-hard problem. Our approach could effectively obtain near optimal solution for QoS multicast applications in varying network condi- tions. As per our knowledge no relationships among the QoS parameters have so far been established. We pro- pose a fuzzy-based parameter setting approach which establishes a Sigmoidal relationship among the parame- ters. Due to the dynamism of the network there is a pos- sibility of sudden change of the network type and status of resources. The empirical results show superiority over the randomly selected resource values. As a future work, we plan to consider biological-inspired optimized tech- niques for estimation of multicast cost arising from queuing delays and propagation error. Figure 11. Pareto front of PLR in Model 1. 0 50 100 150 200 250 300 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Number of Iterations Packet Loss Rate Figure 12. Pareto front of PLR in Model 2. 4.3. Performance Analysis 0 50 100 150 200 250 300 200 300 400 500 600 700 800 900 1000 1100 Number of Iterations Average Delay Figure 6. Pareto front of delay in Model 2. 0 50 100 150 200 250 300 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 Number of Iterations Number of Intermediate nodes Figure 7. Pareto front of HC in Model 1. 0 50 100 150 200 250 300 1.2 1.4 1.6 1.8 2 2.2 Number of Iterations Number of Intermediate nodes from Source to Destination (ms) Figure 8. Pareto front of HC in Model 2. 0 50 100 150 200 250 300 100 150 200 250 300 350 400 450 500 550 600 Number of Iterations Jitter (ms) (ms) Figure 9. Pareto front of jitter in Model 1. 0 50 100 150 200 250 300 0 50 100 150 200 250 300 350 400 450 500 Number of Iteration Jitter (ms) Figure 10. Pareto front of jitter in Model 2. Copyright © 2010 SciRes. IJCNS S. C. RAI ET AL. 537 0 50 100 150 200 250 300 1.2 1.4 1.6 1.8 2 2.2 Number of Iterations Number of Intermediate nodes from Source to Destination 0 50 100 150 200 250 300 200 400 600 800 1000 1200 1400 Number of Iterations Delay (ms) Figure 5. Pareto front of delay in Model 1. Figure 8. Pareto front of HC in Model 2. Figure 8. Pareto front of HC in Model 2. g 0 50 100 150 200 250 300 100 150 200 250 300 350 400 450 500 550 600 Number of Iterations Jitter (ms) 0 50 100 150 200 250 300 200 300 400 500 600 700 800 900 1000 1100 Number of Iterations Average Delay (ms) Figure 6. Pareto front of delay in Model 2. Figure 9. Pareto front of jitter in Model 1. Figure 9. Pareto front of jitter in Model 1. Figure 9. Pareto front of jitter in Model 1. 0 50 100 150 200 250 300 0 50 100 150 200 250 300 350 400 450 500 Number of Iteration Jitter (ms) 0 50 100 150 200 250 300 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 Number of Iterations Number of Intermediate nodes Number of Intermediate nodes Figure 7. Pareto front of HC in Model 1. Figure 10. Pareto front of jitter in Model 2. Copyright © 2010 SciRes. S. C. Copyright © 2010 SciRes. 6. References Dong, “A Fuzzy Genetic Algorithm for QoS Multicast Routing,” Journal of Computer Comm- unication, Vol. 26, No. 6, 2003, pp. 506-512. [17] G. M. B. Oliveira and P. T. Araujo, “Determining Multicast Routes with QoS and Traffic Engineering Requirements Based on Genetic Algorithm,” Proceeding of IEEE Conference on Cybernetics and Intelligent Systems, Singapore, 1-3 December 2004, pp. 666-670. [7] A. Khisti, U. Erez and G. W. Wornell, “Fundamental Limits and Scaling Behavior of Cooperative Multicasting in Wireless Networks,” IEEE Transactions on Infor- mation Theory, Vol. 52, No. 6, June 2006, pp. 2762-2770. [18] S. C. Rai, B. B. Mishra, A. K. Nayak, R. Mall and S. K. Pradhan, “A Multi-Objective Pareto Optimal Genetic Algorithm for QoS Multicasting,” Proceeding of IEEE International Advance Computing Conference, Patiala, 2009, pp. 1303-1307. 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Guan, “Optimized Video Multicasting Over Wireless Ad Hoc Networks Using Distributed Algorithm,” IEEE Transactions on Circuits and Systems for Video Technology, Vol. 19, No. 6, June 2009, pp. 796-807. optimization, where the maximum end-to-end delay and average delay may be considered as related features. Maximum delay, average delay, maximum link utilization, cost of the tree are the four multi objective features con- sidered by Crichigno et al. [22]; where the first two ob- jectives belong to one feature. Fabregat et al. [4] consid- ered a static network and they have optimized maximal link utilization, hop count, average delay, and bandwidth consumption parameters. Marwaha et al. [23] have con- sidered end-to-end delay, packet delivery ratio, packet loss ratio, routing load and route failure as the optimiza- tion parameters for a mobile ad-hoc network with 900 seconds as the network pause time, where the packet de- livery ratio and the packet loss ratio are related features. 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Kang, “A New QoS Multicast Routing Model and its Immune Optimization Algorithm,” Lecture Notes in Computer Science, Vol. 4159, 2006, pp. 369-378. [14] C. C. Palmer and A. Keshenbaum, “Representing Trees in Genetic Algorithms,” Proceeding of IEEE Inter- national Conference on Evolutionary Computation, Orlan- do, Vol. 1, 1994, pp. 379-384. [23] S. Marwaha, D. Srinivasan, C. K. Tham and A. Vasilakos, “Evolutionary Fuzzy Multi-Objective Routing for Wireless Mobile Ad Hoc Networks,” Proceeding of Evolutionary Computation (CEC’04), San Diego, Vol. 2, 2004, pp. 1964-1971. [15] S. Sivavakeesar, G. Pavlou and A. Liotta “Stable Clustering through Mobility Prediction for Large-Scale IJCNS Copyright © 2010 SciRes.
https://openalex.org/W2111941367	https://europepmc.org/articles/pmc4114648?pdf=render	English	null	Polymorphism of the p38β gene in patients with colorectal cancer	Oncology Letters	2,014	cc-by	3,116	Polymorphism of the p38β gene in patients with colorectal cancer JAN DIMBERG1, RENATE SLIND OLSEN2,3, MARITA SKARSTEDT4, STURE LÖFGREN2, NIKLAS ZAR5 and ANDREAS MATUSSEK2 1Department of Natural Science and Biomedicine, University College of Health Sciences, Jönköping, SE‑551 11; 2Department of Laboratory Services, Ryhov County Hospital, Jönköping, SE‑551 85; 3Division of Drug Research, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping SE‑581 85; Departments of 4Clinical Microbiology and 5Surgery, Ryhov County Hospital, Jönköping, SE‑551 85, Sweden Received January 24, 2014; Accepted June 12, 2014 Received January 24, 2014; Accepted June 12, 2014 DOI: 10.3892/ol.2014.2315 Abstract. The p38 mitogen‑activated protein kinase (MAPK) signaling pathways have been proposed to participate in the pathological process of cancer by affecting inflammation, proliferation, metastasis and cell survival. A single nucleotide polymorphism (SNP; rs2235356, ‑1628A→G) in the promoter region of the p38β gene has been proposed as a genetic modi fier for colorectal cancer (CRC) in a Chinese population. The present study evaluated the susceptibility of patients possessing this SNP to CRC, in addition to determining its association with clinical parameters in Swedish patients with CRC. Using the LightSNiP genotyping assay, this SNP was screened in 389 patients with CRC and 517 control subjects. No significant difference in the genotype distribution or in the allelic frequencies was identified between the two groups nor was any association identified with the clinical parameters. These findings indicate that the ‑1628A→G polymorphism of the p38β gene is not significantly associated with a suscepti bility to CRC in a Swedish population. Genetic variation, such as single nucleotide polymorphisms (SNPs), is hypothesized to contribute to individual variations in CRC susceptibility (5). Polymorphic variants of genes are significant factors that mediate inflammatory responses and facilitate CRC progression (6). The prognosis of CRC is depen dent on the extent of local and metastatic tumor spread and the degradation of the extracellular matrix (ECM) that surrounds cancerous tissue. Matrix metalloproteinases (MMPs) have fundamental roles in the degradation of basal membranes and the ECM, as well as being associated with tumor invasion and a poor prognosis (7). Our previous study demonstrated that the gene polymorphism of the MMP12 gene is associated with a higher risk of disseminated CRC (8). Mitogen‑activated protein kinase (MAPK) signaling path ways (2,9) consist of a large family of serine‑threonine kinases, which respond to extracellular signals, such as cellular stress and growth factors. These signals control fundamental cellular processes, such as cell proliferation, differentiation, cell survival and are considered to be significant in the progression of different types of cancer (10), including CRC (2,9,10). p38 kinases are members of the MAPK family and consist of four isoforms (α, β, γ and δ) (9‑11). An increased expression level of p38 has been observed in CRC (12) and breast cancer (13) patients. Introduction There are numerous genetic pathways, which affect colorectal cancer (CRC) initiation and progression. These include the chromosomal instability pathways, which are associated with accumulation of activating mutations in oncogenes, such as KRAS, BRAF and PI3KCA or inactivation of tumor suppressor genes, such as APC and p53 (1,2). Furthermore, aberrant meth ylation of gene promoter regions has been widely investigated and these epigenetic events within certain signaling pathways result in gene suppression and contribute to the development of CRC (3,4). Recently, it was shown that an SNP, rs2235356, ‑1628A→G, in the promoter region of the p38β gene was correlated with an increased risk of CRC in a Chinese population (14). In the present study, this SNP was analyzed to assess its value as a risk factor and as a predictor of disease outcome in Swedish CRC patients. ONCOLOGY LETTERS 8: 1093-1095, 2014 ONCOLOGY LETTERS 8: 1093-1095, 2014 Patients and methods The tumors were located in the colon in 218 cases and in the rectum in 171 cases, and were classified by Dukes' classification system (15): Stage A, n=65; stage B, n=144; stage C, n=121; and stage D, n=59. Blood donors (n=517) with no known history of CRC, who were from the same geographical region as the patients with CRC were selected as control subjects. The control group consisted of 255 males and 262 females, with a mean age of 60 years (range, 33‑79 years). Blood samples were centrifuged using Hettich Universal 320R Centrifuge (Andreas Hettich GmbH & Co. KG, Tuttligen, Germany) at 1500 x g for 10 min to separate the plasma and blood cells and were stored at ‑78˚C. software (version 19; IBM, Armonk, NY, USA) and P<0.05 w considered to indicate a statistically significant difference. Results Genotype distribution. The frequencies of the gene polymo phisms in the patients and the control group subjects indicat no significant difference in the genotype distribution or alle Table I. Genotypic and allelic distribution of the p38β gene polymorphism (-1628A→G) in patients with CRC and in healthy control subjects. A, Genotype distribution CRC, % Controls, % A→G (n=389) (n=517) A/A 28.3 (110) 24.9 (129) A/G 45.5 (177) 51.3 (265) G/G 26.2 (102) 23.8 (123) B, Allele distribution CRC, % Controls, % Variant (n=778) (n=1034) A 51.0 (397) 50.6 (523) G 49.0 (381) 49.4 (511) CRC patients vs. control subjects: Genotype and allele distribution, no significant differences (P>0.05). CRC, colorectal cancer. Table II. Genotypic and allelic distributions of the p38β ge polymorphism (-1628A→G) regarding tumour location a disease stage in patients with CRC. A, Genotype Location Stage ---------------------------------------- ------------------------------------------------- Colon Rectum Dukes' A + B Dukes' C + A→G % (n) % (n) % (n) % (n) A/A 28.4 (62) 28.1 (48) 28.2 (59) 28.3 (51) A/G 45.0 (98) 46.2 (79) 46.0 (96) 45.0 (81) G/G 26.6 (58) 25.7 (44) 25.8 (54) 26.7 (48) Total (218) (171) (209) (180) B, Allele Location Stage ------------------------------------------ ------------------------------------------------ Colon Rectum Dukes' A + B Dukes' C + Variant % (n) % (n) % (n) % (n) A 50.9 (222) 51.2 (175) 51.2 (214) 50.8 (183 G 49.1 (214) 48.8 (167) 48.8 (204) 49.1 (177 Total (436) (342) (418) (360) Colon vs. rectum and Dukes' A + B vs. Dukes' C + B: Genoty and allele distribution, no significant differences (P>0.05). CR colorectal cancer. Table II. Results Genotype distribution. The frequencies of the gene polymor phisms in the patients and the control group subjects indicated no significant difference in the genotype distribution or allelic frequencies (Table I). The genotypes in the CRC patients and the control group subjects were not associated with clinical characteristics, such as age and gender (data not shown). When subdividing the patients into groups of colon and rectal cancer, or localized Dukes' A and B and disseminated Dukes' C and D disease, no significant difference was identified between location and disease with regard to the genotypes and alleles (Table II). In addition, analysis using the Kaplan‑Meier method demonstrated that the p38β genotypes were not associated with survival rate (data not shown). DNA extraction and genotype determination. Genomic DNA was isolated from the blood samples using the QIAamp DNA Blood kit (Qiagen, Valencia, CA, USA). DNA samples were genotyped using the LightSNiP rs2235356 genotyping assay (TIB Molbiol, Berlin, Germany). DNA (10 ng) was mixed with Reagent Mix, LightCycler® FastStart DNA Master HybProbe (Roche Diagnostics GmbH, Mannheim, Germany), 5.7 µl H2O and 0.8 µl MgCl2 and amplified using the LightCycler® 480 Real‑Time PCR system (Roche Diagnostics GmbH) according to the manufacturer's instructions. Neither the patient nor the control group showed a significant deviation in genotypic frequency as assessed by the Hardy‑Weinberg equilibrium (data not shown). Statistical analysis. Differences in the frequencies of the p38β gene polymorphism between patients and the control group, as well as between clinical characteristics within the CRC subgroup were analyzed using the χ2 test and the Hardy‑Weinberg equilibrium was assessed with regard to the genotypes. Survival analysis was performed using Cox's regres sion and Kaplan‑Meier analysis was conducted with the log‑rank test. Statistical analysis was performed using SPSS statistical Patients and methods Dukes' C + B: Genotype and allele distribution, no significant differences (P>0.05). CRC, colorectal cancer. software (version 19; IBM, Armonk, NY, USA) and P<0.05 was considered to indicate a statistically significant difference. software (version 19; IBM, Armonk, NY, USA) and P<0.05 was considered to indicate a statistically significant difference. Patients and methods Genotypic and allelic distributions of the p38β gene polymorphism (-1628A→G) regarding tumour location and disease stage in patients with CRC. Table I. Genotypic and allelic distribution of the p38β gene polymorphism (-1628A→G) in patients with CRC and in healthy control subjects. polymorphism (-1628A→G) in patients with CRC and in healthy control subjects. A, Genotype distribution CRC, % Controls, % A→G (n=389) (n=517) A/A 28.3 (110) 24.9 (129) A/G 45.5 (177) 51.3 (265) G/G 26.2 (102) 23.8 (123) B, Allele distribution CRC, % Controls, % Variant (n=778) (n=1034) A 51.0 (397) 50.6 (523) G 49.0 (381) 49.4 (511) CRC patients vs. control subjects: Genotype and allele distribution, no significant differences (P>0.05). CRC, colorectal cancer. A, Genotype Location Stage ---------------------------------------- ----------------------------------------------------- Colon Rectum Dukes' A + B Dukes' C + D A→G % (n) % (n) % (n) % (n) A/A 28.4 (62) 28.1 (48) 28.2 (59) 28.3 (51) A/G 45.0 (98) 46.2 (79) 46.0 (96) 45.0 (81) G/G 26.6 (58) 25.7 (44) 25.8 (54) 26.7 (48) Total (218) (171) (209) (180) B, Allele Location Stage ------------------------------------------ ---------------------------------------------------- Colon Rectum Dukes' A + B Dukes' C + D Variant % (n) % (n) % (n) % (n) A 50.9 (222) 51.2 (175) 51.2 (214) 50.8 (183) G 49.1 (214) 48.8 (167) 48.8 (204) 49.1 (177) Total (436) (342) (418) (360) Colon vs. rectum and Dukes' A + B vs. Dukes' C + B: Genotype and allele distribution, no significant differences (P>0.05). CRC, colorectal cancer. Patient groups. The patients comprised 212 males and 177 females with a mean age of 70 years (range, 25‑93 years). The tumors were located in the colon in 218 cases and in the rectum in 171 cases, and were classified by Dukes' classification system (15): Stage A, n=65; stage B, n=144; stage C, n=121; and stage D, n=59. Blood donors (n=517) with no known history of CRC, who were from the same geographical region as the patients with CRC were selected as control subjects. The control group consisted of 255 males and 262 females, with a mean age of 60 years (range, 33‑79 years). Blood samples were centrifuged using Hettich Universal 320R Centrifuge (Andreas Hettich GmbH & Co. KG, Tuttligen, Germany) at 1500 x g for 10 min to separate the plasma and blood cells and were stored at ‑78˚C. Colon vs. rectum and Dukes' A + B vs. Patients and methods Patients, controls and ethical procedures. The present study collected blood samples from 389 consecutive patients from southeastern Sweden who underwent surgical resection for primary colorectal adenocarcinomas at the Department of Surgery, Ryhov County Hospital (Jönköping, Sweden) between 1996 and 2013. Clinicopathological characteristics of the patients were obtained from surgical and pathological records. The investigation was approved by the local Ethical Committee of the Faculty of Health Sciences (Dnr. 2013/271‑31; Linköping, Sweden) and informed consent was obtained from the participants. Correspondence to: Dr Andreas Matussek, Department of Laboratory Services, Ryhov County Hospital, Sjukhusvägen 1, Jönköping SE‑551 85, Sweden E‑mail: andreas.matussek@lj.se Key words: p38β, promoter region, single nucleotide polymorphism, colorectal cancer DIMBERG et al: p38β GENE POLYMORPHISM IN CRC 1094 DIMBERG et al: p38β GENE 1094 Patient groups. The patients comprised 212 males and 177 females with a mean age of 70 years (range, 25‑93 years). The tumors were located in the colon in 218 cases and in the rectum in 171 cases, and were classified by Dukes' classification system (15): Stage A, n=65; stage B, n=144; stage C, n=121; and stage D, n=59. Blood donors (n=517) with no known history of CRC, who were from the same geographical region as the patients with CRC were selected as control subjects. The control group consisted of 255 males and 262 females, with a mean age of 60 years (range, 33‑79 years). Blood samples were centrifuged using Hettich Universal 320R Centrifuge (Andreas Hettich GmbH & Co. KG, Tuttligen, Germany) at 1500 x g for 10 min to separate the plasma and blood cells and were stored at ‑78˚C. Table I. Genotypic and allelic distribution of the p38β gene polymorphism (-1628A→G) in patients with CRC and in healthy control subjects. A, Genotype distribution CRC, % Controls, % A→G (n=389) (n=517) A/A 28.3 (110) 24.9 (129) A/G 45.5 (177) 51.3 (265) G/G 26.2 (102) 23.8 (123) B, Allele distribution CRC, % Controls, % Variant (n=778) (n=1034) A 51.0 (397) 50.6 (523) G 49.0 (381) 49.4 (511) CRC patients vs. control subjects: Genotype and allele distribution, no significant differences (P>0.05). CRC, colorectal cancer. p β 1094 Patient groups. The patients comprised 212 males and 177 females with a mean age of 70 years (range, 25‑93 years). Discussion The p38 MAPKs are involved in inflammation, prolif eration, differentiation, and cell death (2,9‑11) and increased levels have been observed in CRC patients (12). MMPs are ONCOLOGY LETTERS 8: 1093-1095, 2014 1095 2. Markowitz SD and Bertagnolli MM: Molecular origins of cancer: Molecular basis of colorectal cancer. N Engl J Med 361: 2449‑2460, 2009. fundamental in the degradation of the ECM and are associ ated with tumor invasion and a poor prognosis (7). The p38 MAPK signaling pathways have been proposed to promote metastasis (16) by activating different transcription factors. One of these transcription factors, activator protein‑1 (AP1) (11,16) appears to be important, as it controls MMP expression (16‑19) and may impact CRC progression via tumor invasion (20). , 3. Kondo Y and Issa JP: Epigenetic changes in colorectal cancer. Cancer Metastasis Rev 23: 29‑39, 2004. 4. Naghibalhossaini F, Zamani M, Mokarram P, Khalili I, Rasti M and Mostafavi‑Pour Z: Epigenetic and genetic analysis of WNT signaling pathway in sporadic colorectal cancer patients from Iran. Mol Biol Rep 39: 6171‑6178, 2012. p , 5. Mammano E, Belluco C, Bonafé M, Olivieri F, Mugianesi E, Barbi C, Mishto M, Cosci M, Franceschi C, Lise M and Nitti D: Association of p53 polymorphisms and colorectal cancer: modu lation of risk and progression. Eur J Surg Oncol 35: 415‑419, 2009. To the best of our knowledge, studies are limited regarding the association between p38 genetic polymorphisms and the risk of CRC. However, it was recently demonstrated that an SNP (rs2235356, ‑1628A→G) in the promoter region of the p38β gene was associated with an increased risk of CRC in a Chinese population (14). To elucidate whether this SNP is correlated with the risk of CRC in other ethnic groups, this particular SNP was analyzed in Swedish patients with CRC. In the present study, clinicopathological variables, such as tumor localization, stage and survival were evaluated, which is similar to a previous study by Huang et al (14).i 6. Theodoropoulos G, Papaconstantinou I, Felekouras E, Niketeas N, Karakitsos P, Panoussopoulos D, Lazaris ACh, Patsouris E, Bramis J and Gazouli M: Relation between common polymorphisms in genes related to inflammatory response and colorectal cancer. World J Gastroenterol 12: 5037‑5043, 2006. 7. Egeblad M and Werb Z: New functions for the matrix metal loproteinases in cancer progression. Nat Rev Cancer 2: 161‑174, 2002. 8. 1. Al‑Sohaily S, Biankin A, Leong R, Kohonen‑Corish M and Warusavitarne J: Molecular pathways in colorectal cancer. J Gastroenterol Hepatol 27: 1423‑1431, 2012. Discussion Van Nguyen S, Skarstedt M, Löfgren S, Zar N, Andersson RE, Lindh M, Matussek A and Dimberg J: Gene polymorphism of matrix metalloproteinase‑12 and ‑13 and association with colorectal cancer in Swedish patients. Anticancer Res 33: 3247‑3250, 2013. In the current study, it was identified that the genotype distribution and allelic frequencies of patients were not significantly associated with CRC when compared with the control subjects. Functionally, the ‑1628A→G p38β gene poly morphism may influence CRC progression via the p38 MAPK signaling pathways as a regulator of the proliferation, survival and metastasis. In addition, no significant correlation between the genotypes and survival rate or disseminated disease was detected. Furthermore, the genotypes were not identified to be associated with other clinical parameters. , 9. Fang JY and Richardson BC: The MAPK signaling pathways and colorectal cancer. Lancet Oncol 6: 322‑327, 2005. 10. Dhillon AS, Hagan S, Rath O and Kolch W: MAP kinase signaling pathways in cancer. Oncogene 26: 3279‑3290, 2007. g g p y g 11. Zarubin T and Han J: Activation and signaling of the p38 MAP kinase pathway. Cell Res 15: 11‑18, 2005. p y 12. Pailas S, Boissière F, Bibeau F, Denouel A, Mollevi C, Causse A, Denis V, Vezzio‑Vié N, Marzi L, Cortijo C, et al: Targeting the p38 MAPK pathway inhibits irinotecan resistance in colon adenocarcinoma. Cancer Res 71: 1041‑1049, 2011. p p y adenocarcinoma. Cancer Res 71: 1041‑1049, 2011. 13. Chen L, Mayer JA, Krisko TI, Speers CW, Wang T, Hilsenbeck SG and Brown PH: Inhibition of the p38 kinase suppresses the prolif eration of human ER‑negative breast cancer cells. Cancer Res 69: 8853‑8861, 2009. In conclusion, the present study indicated that the ‑1628A→G polymorphism in the promoter region of the p38β gene does not appear to be a useful tumor marker that reflects the clinical outcome in Swedish patients with CRC. In the future it would be of interest to evaluate this SNP in a large cohort of various ethnicities. 14. Huang Q, Chen D, Song S, Fu X, Wei Y, Lu J, Wang L and Wang J: A genetic variation of the p38β promoter region is correlated with an increased risk of sporadic colorectal cancer. Oncol Lett 6: 3‑8, 2013. 15. Wu JS: Rectal cancer staging. Clin Colon Rectal Surg 20: 148‑157, 2007. 16. del Barco Barrantes I and Nebreda AR: Roles of p38 MAPKs in invasion and metastasis. Acknowledgements 17. Jormsjö S, Ye S, Moritz J, Walter DH, Dimmeler S, Zeiher AM, Henney A, Hamsten A and Eriksson P: Allele‑specific regulation of matrix metalloproteinase‑12 gene activity is associated with coronary artery luminal dimensions in diabetic patients with manifest coronary artery disease. Circ Res 86: 998‑1003, 2000. The present study was supported by grants from Futurum, the Academy for Healthcare, County Council (grant no. 105891; Jönköping, Sweden), the Foundation of Clinical Cancer Research (grant no. 110426-1; Jönköping, Sweden) and the University College of Health Sciences (Jönköping, Sweden). y y 18. Benbow U and Brinckerhoff CE: The AP‑1 site and MMP gene regulation: what is all the fuss about? Matrix Biol 15: 519‑526, 1997. 19. Simon C, Simon M, Vucelic G, Hicks MJ, Plinkert PK, Koitschev A and Zenner HP: The p38 SAPK pathway regulates the expression of the MMP‑9 collagenase via AP‑1‑dependent promoter activation. Exp Cell Res 271: 344‑345, 2001. Discussion Biochem Soc Trans 40: 79‑84, 2012. References p p 20. Ashida R, Tominaga K, Sasaki E, Watanabe T, Fujiwana Y, Oshitani N, Higuchi K, Mitsuyama S, Iwao H and Arakawa T: AP‑1 and colorectal cancer. Inflammopharmacology 13: 113‑125, 2005.
https://openalex.org/W2953322712	https://hal.inria.fr/hal-02365736/file/485999_1_En_1_Chapter.pdf	English	null	An Educational Intervention for Teaching Secure Coding Practices	IFIP advances in information and communication technology	2,019	cc-by	5,943	To cite this version: Vuyolwethu Mdunyelwa, Lynn Futcher, Johan Van Niekerk. An Educational Intervention for Teaching Secure Coding Practices. 12th IFIP World Conference on Information Security Education (WISE), Jun 2019, Lisbon, Portugal. pp.3-15, 10.1007/978-3-030-23451-5_1. hal-02365736 Distributed under a Creative Commons Attribution 4.0 International License ⋆The ﬁnancial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the authors, and are not necessarily to be attributed to the NRF. 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Distributed under a Creative Commons Attribution 4.0 International License An Educational Intervention for Teaching Secure Coding Practices⋆ Vuyolwethu Mdunyelwa1[0000−0001−9625−0925], Lynn Futcher1[0000−0003−0406−8718], and Johan van Niekerk1,2[0000−0003−1739−4563] 1 Nelson Mandela University, Port Elizabeth, South Africa {vuyolwethu.mdunyelwa, lynn.futcher, johan.vanniekerk}@mandela.ac.za 2 NoroﬀUniversity College, Kristiansand, Norway johan.vanniekerk@noroff.no Abstract. Cybersecurity vulnerabilities are typically addressed through the implementation of various cybersecurity controls. These controls can be operational, technical or physical in nature. The focus of this paper is on technical controls with a speciﬁc focus on securing web applications. The secure coding practices used in this research are based on OWASP. An initial investigation found that there was a general lack of adherence to these secure coding practices by third year software development stu- dents doing their capstone project at a South African University. This research therefore focused on addressing this problem by developing an educational intervention to teach secure coding practices, speciﬁcally fo- cusing on the data access layer of web applications developed in the .NET environment. Pre-tests and post-tests were conducted in order to deter- mine the eﬀectiveness of the intervention. Results indicated an increase in both knowledge and behaviour regarding the identiﬁed secure coding practices after exposure to the intervention. Keywords: Educational Intervention · Secure Coding Practices · OWASP · Web Application Security. 1 Introduction With the recent increase in cyber-related attacks, cybersecurity is becoming a key area of concern for many organisations. Web applications often handle very sensitive data, used for carrying out critical tasks such as banking, online shopping and online tax ﬁling [9]. These applications are trusted by billions of users for performing such daily activities. However, 75% of all attacks on the internet are executed through the application layer of the OSI model [6], and more than 76% of web applications have vulnerabilities [2]. Handling risks related to the security of web applications is a major challenge for many organizations. Not surprisingly, Web applications have recently received attention from academia and industry to initiate some defence mechanisms to protect them from security threats [9]. Many of these Web applications have common vulnerabilities which can be easily corrected [18] through introducing secure coding practices. The secure coding practices used in this research are based on OWASP. An initial investigation found that there was a general lack of adherence to these secure coding practices by third year software development students doing their capstone project at a South African University. This research therefore focused on addressing this problem by developing an educational intervention to teach secure coding practices, speciﬁcally focusing on the data access layer of web applications developed in the .NET environment. The following section highlights the related literature, while Section 3 provides the research design. Section 4 presents the educational intervention including both the knowledge and behavioural components. This is followed by Section 5 which provides the results of the veriﬁcation of the educational intervention before concluding in Section 6. 2 Related Literature More than 90,000 vulnerabilities have been recorded in the Symantec compre- hensive vulnerability database over the past two decades, from 24,560 vendors representing over 78,900 products. On average, over 340,000 web attacks were blocked from web applications per day in 2014 [2]. Although this improved to 229,000 in 2016 [2], it still remains a serious concern since most attacks are no longer on the networks, but more on the software applications that run on the application layer. If 76% of web applications contain known vulnerabilities, it means that 24% of the scanned web applications do not contain known vulnera- bilities. Therefore, it is possible for web applications to avoid known vulnerabil- ities. Those web applications without known vulnerabilities probably adhere to some form of best practice for secure software development. This is true as some researchers suggest that applying such practices and methodologies can improve security in software application [1, 7]. There are various organisations and institutions responsible for developing stan- dards and best practices. These include the National Institute of Standards and Technology (NIST), the International Organizations for Standardization (ISO) and the International Electro-Technical Commission (IEC), the Microsoft Devel- oper Network (MSDN) and the Open Web Application Project (OWASP) which provides best practices for improving security in web applications. The best practices provided by these organisations were evaluated and OWASP was considered the most relevant for identifying fundamental secure coding prac- tices to be taught to software developers. OWASP is known by many organisa- tions for its Top 10 Vulnerability List (Table 1) that it publishes and updates periodically [4, 6, 11]. This list focusses on identifying the most serious web ap- plication security vulnerabilities for many organisations [16]. The Top 10 list changes according to which vulnerability is most dominant at any given time. Table 1: OWASP Top 10 Vulnerability List 2017 [15]. Vulnerability Description SQL Injection Injection ﬂaws occur when untrusted data is sent to an inter- preter as part of a command or query. Broken Authentica- tion This relates to authentication and session management that are often implemented incorrectly. It allows attacks to compromise information, and session tokens or exploit other implementation ﬂaws. Sensitive Data Expo- sure Many web applications and Application Programming Interfaces (APIs) do not properly protect data allowing attackers to steal or modify data. 2 Related Literature SP7 Encrypt connection strings. SP8 Connection strings should be based in a conﬁguration ﬁle. SP9 Encrypt sensitive data using acceptable encryption algorithms. Table 2: Secure Coding Practices. Adapted from [16]. Table 2: Secure Coding Practices. Adapted from [16]. SP Secure Coding Practices SP1 Use Parameterised SQL commands for all data access, without exception. SP2 Do not use SQL command with a string made up of a concatenated SQL strings. SP3 Properly validate input ﬁelds. SP4 Apply the Principle of Least Privilege when setting up the database of your choice. SP5 When using SQL Server, prefer integrated authentication over SQL authenti- cation. SP6 Using stored procedures is the most eﬀective way to counter the SQL injection vulnerability. SP7 Encrypt connection strings. SP8 Connection strings should be based in a conﬁguration ﬁle. SP9 Encrypt sensitive data using acceptable encryption algorithms. The secure coding practices shown in Table 2 are referred to using the codes SP1 to SP9 throughout this paper. If one of them is not properly handled, it can be easy for an attacker to access and modify information that is in the database. For example, if the connection string is found in other parts of the application code and not locked in the conﬁguration ﬁle, it can be easy for an attacker to access the information using the same connection string to connect to the database. Or, if the expected values in an input ﬁeld are not whitelisted in a system with concatenated SQL strings, attackers can use characters to manipulate the SQL string in the database and the information would be at risk. These vulnerabilities cannot be prevented by programmers unless they know the types of ﬂaws that exist in their code [1, 3]. Similarly, they cannot implement these security controls unless they are taught how they work [8]. Once software developers have been taught about secure coding practices, it is more likely that they will have the requisite knowledge [5]. However, there has to be some form of compliance instrument to monitor their adherence, since it is known that people with the requisite knowledge do not always behave accordingly. Therefore, an educational intervention that focuses on both knowledge and behaviour was developed and provided to software development students to improve the security of their web applications. 2 Related Literature XML External Enti- ties External entities can be used to disclose ﬁles using the ﬁle Uni- form Resource Identiﬁers (URIs) handler, internal ﬁle shares, internal port scanning, remote code execution, and denial of service attacks. Broken Access Con- trol Restrictions on what authenticated users are allowed to do are often not properly enforced, allowing attackers to exploit ﬂaws to access unauthorized functionality, or data, such as other users’ accounts or change access rights. Secure Misconﬁgura- tion This is mostly a result of insecure default conﬁgurations, incom- plete or ad hoc conﬁgurations. Cross-Site Scripting Also know as XSS, it allows attackers to execute scripts in the victim’s browser which can hijack user sessions, deface web sites, or redirect users to malicious sites. Insecure Deserialisa- tion Insecure deserialisation leads to remote code execution, deserial- isation ﬂaws can also be used to perform tasks such as injection attacks, and privilege escalation attacks. Using Components with Known Vulner- abilities Components including libraries, frameworks and other software modules, run with the same privileges as the application. If a vulnerable component is exploited, such attacks can facilitate serious data loss or server take over. Insuﬃcient Logging and Monitoring This allows attacks to further attack systems, maintain persis- tence, pivot to more systems, and temper, extract, or destroy data. Table 1: OWASP Top 10 Vulnerability List 2017 [15]. The risk posed by each of these vulnerabilities can be reduced by more than one type of control. For the data access layer within .NET, OWASP recommends speciﬁc secure coding practices. Table 2 presents the nine secure coding practices (SP1 to SP9) for data access based on OWASP and used in this study. These relate to some of the vulnerabilities shown in Table 1. As an example, parame- terised SQL commands (SP1), or the use of stored procedures (SP6), can block SQL injections. Therefore more than one control can reduce a vulnerability. Table 2: Secure Coding Practices. Adapted from [16]. SP Secure Coding Practices SP1 Use Parameterised SQL commands for all data access, without exception. SP2 Do not use SQL command with a string made up of a concatenated SQL strings. SP3 Properly validate input ﬁelds. SP4 Apply the Principle of Least Privilege when setting up the database of your choice. SP5 When using SQL Server, prefer integrated authentication over SQL authenti- cation. SP6 Using stored procedures is the most eﬀective way to counter the SQL injection vulnerability. 3 Research Design This research was conducted in the School of Information and Communication Technology at a comprehensive institution in South Africa, oﬀering both degrees and vocational qualiﬁcations. In this case, the sample was drawn from students registered for their third year in the National Diploma: Software Development. In South Africa, there are no locally recognised curricular guidelines for comput- ing. Many universities therefore rely on the recommendations provided in global computing curricular publications. The Association of Computing Machinery (ACM) Information Technology curricular guidelines have been used to model IT qualiﬁcations. The IT2008 and the more recent IT2017 curricular guidelines require students in computing and engineering disciplines to engage in a cap- stone project during their ﬁnal year of study [13, 12]. Since the diploma is a three year qualiﬁcation, students are required to complete a capstone project in their third year of study. These capstone projects take place over a full year of study. According to the ACM IT curricular guidelines, capstone projects should typically adhere to the following [13, 12]: – Project groups of 3 to 5 students; – Based on a real-world problem; – Must be integrative; – Students should have completed most of the curriculum before attempting the project. – Students should have completed most of the curriculum before attempting the project. Students registered for the diploma are introduced to programming and busi- ness application systems development. Therefore, most of their capstone projects focus on developing applications for solving real world problems using business applications. When students choose the capstone projects, many of them focus on web, mobile or gaming applications, while a few develop desktop applications. Although students are taught speciﬁcally to develop software in a Windows environment using the .NET framework, students may develop their capstone projects in the programming language of their choice. Most project students choose web applications in the .NET development environment as this is where their skills lie. This research focused on two aspects relating to secure coding practices, namely knowledge and behavioural compliance of the students and involved four main phases: – Phase 1 was the ﬁrst phase for this research which started oﬀby analysing students’ behaviour relating to secure coding practices. This was done by conducting a code review on previously completed third year capstone projects, which were developed in the .NET environment. The results for this be- havioural analysis indicated low levels of compliance to the identiﬁed secure coding practices. 4 Educational Intervention The educational intervention was split into two parts, where the ﬁrst part focused on the knowledge, and the second part focused on the behaviour of students relating to secure coding practices. Owing to the lack of knowledge on the part of the students, the researcher realised the need to create a knowledge component that could assist students in acquiring the requisite knowledge regarding secure coding practices. The need to address behavioural compliance was also realised since it is known that having knowledge does not necessarily ensure that people would behave accordingly [17]. Both the knowledge and behavioural components of this research were designed using the identiﬁed secure coding practices in Table 2. 3 Research Design – Phase 2 addressed the knowledge assessment phase for this research, which assessed students’ knowledge relating to secure coding practices. This was achieved using a questionnaire, which served as a pre-test for this study. Results from the pre-test indicated that students lacked knowledge relating to secure coding practices. Therefore, students lacked in both the knowledge and behavioural aspects. – Phase 3 comprised of an educational intervention for addressing both the knowledge and behavioural aspects, which students lacked in Phase 1 and 2. In terms of the knowledge aspect, students were provided with online lessons relating to secure coding practices to work through; in terms of the behavioural aspect, students were given a checklist to check their application code against the listed secure coding practices. behavioural aspect, students were given a checklist to check their application code against the listed secure coding practices. – Phase 4 involved the veriﬁcation of the educational intervention for this research. The ﬁrst part of this phase was the knowledge veriﬁcation (Phase 4A), and the second part was the behavioural veriﬁcation (Phase 4B). – Phase 4 involved the veriﬁcation of the educational intervention for this research. The ﬁrst part of this phase was the knowledge veriﬁcation (Phase 4A), and the second part was the behavioural veriﬁcation (Phase 4B). The results for Phase 1 and 2 were published in the 2017 Human Aspects in Information Security and Assurance (HAISA) conference [14]. The focus of this paper is therefore on Phases 3 and 4. The following section describes the educa- tional intervention (Phase 3), while Phase 4 (A and B) are discussed in Section 5. 4.1 Knowledge Component The knowledge component for this research took the form of a blended learn- ing course, called the Web Application Security Course, that students worked through to improve their knowledge regarding secure coding practices. Design of the Knowledge Component The knowledge component for this research included online lessons that the researcher designed using the identiﬁed secure coding practices. For each of the secure coding practices, their impor- tance and the security implications if they were ignored were explained. The online learning platform that was used to design the lessons was the Moodle Learning Management System that runs on the university’s website. Moodle is a learning management system used by educators to create eﬀective blended learning material for students in various higher educational institutions. Moodle has been adopted by many institutions for its cost eﬀectiveness, its ability to expand with increased student populations, and its ability to meet the needs of institutions, students and educators [10]. Figure 1 provides an overview of the process followed by the students when completing the online lessons on Moodle. Lesson 1 SP1 Lesson 1 SP1 Lesson 1 SP1 Lesson 1 SP1 Next Next Quiz Lesson 2 SP2 Lesson 2 SP2 Lesson 2 SP2 Lesson 2 SP2 Next Next Quiz Quiz Question 1 ______________________________ ____ A__________ B_________ C___________ Lesson Lesson 5 SP5 Lesson 8 SP8 Lesson 9 SP9 Lesson 9 SP9 Lesson 9 SP 9 Lesson 9 SP9 Next Fig. 1: Lesson Content Process Flow. SP SP2 Fig. 1: Lesson Content Process Flow. The lessons took the form of interactive Microsoft PowerPoint slides, which were converted to videos, for students to work through. Each secure coding prac- tice was addressed in a single lesson. After completing each lesson, the students were required to take a quiz, which allowed them to reﬂect on the content of the lesson. The quiz had four questions assigned to each lesson. The students had to answer only one randomly generated quiz question before continuing to the next lesson. If the student selected the incorrect answer, they were required to work through the lesson again, and if they selected an incorrect answer once again, a diﬀerent question would be randomly generated. Alternatively, if they selected the correct answer, they were allowed to continue to the next lesson. 4.1 Knowledge Component A brief overview of each secure coding practice (SP1 to SP9) as listed in Table 2, within the knowledge component follows: – SP1 (Using Parameterised SQL commands): The content for this secure cod- ing practice ﬁrstly provides the background relating to parameterised SQL commands in order to equip students with the necessary information relat- ing to this secure coding practice. The remainder of the lesson shows the students how parameterised SQL commands can be implemented in their code, and why it is necessary to use them. – SP2 (Concatenated SQL strings): Content for this secure coding practice begins by introducing what is meant by concatenated SQL strings. The les- son proceeds by showing how programmers make use of concatenated SQL strings and the negative implications of using them. This lesson also provides a way in which to avoid using concatenated SQL strings, which is by means of parameterised SQL commands. – SP3 (Input validation): The content for this secure coding practice begins by discussing validation in general. It also highlights the various types of validation, such as blacklisting and whitelisting, and why they are important. The content also provides suggestions on what to use when dealing with validation, for example, ASP.NET Regular Expressions to tell input ﬁelds which values to accept. – SP3 (Input validation): The content for this secure coding practice begins by discussing validation in general. It also highlights the various types of validation, such as blacklisting and whitelisting, and why they are important. The content also provides suggestions on what to use when dealing with validation, for example, ASP.NET Regular Expressions to tell input ﬁelds which values to accept. – SP4 (Principle of Least Privilege): This secure coding practice content ex- plains what the Principle of Least Privilege is and why it is important when developing web applications. This content also provides a scenario where the use of this secure coding practice is shown and how it can be implemented. – SP5 (Authentication): The content of this secure coding practice was ad- dressed by means of a video adapted from YouTube. The video was embed- ded in the slides and distributed as a single lesson to the students to listen to and to watch. 4.1 Knowledge Component – SP6 (Using Stored Procedures): The content for this secure coding practice focusses on how stored procedures are used and why they are important, providing examples on how they should be implemented in a web application. – SP7 + SP8 (Connection strings): These two secure coding practices both deal with connection strings, and have been addressed collectively in the same lesson. The content ﬁrst provides detail about the importance of connection strings, and how they should be handled when developing web applications, idi d t il h t i l t b th th di ti with connection strings, and have been addressed collectively in the same lesson. The content ﬁrst provides detail about the importance of connection strings, and how they should be handled when developing web applications, providing detail on how to implement both the secure coding practices. – SP9 (Encryption): For this secure coding practice, an analogy is used to explain the concept of encryption to the students. The lesson further ex- plains the analogy to clarify the concepts for the students. Since OWASP provides recommendations relating to acceptable encryption algorithms, the content for this lesson also emphasises the use of the encryption algorithms recommended by OWASP when developing web applications. All the lessons were followed by a quiz question to check students’ understanding of the content contained in the lesson they had worked on. The results for the short content quizzes were not recorded, since answers were simply used to ensure that students do not move to the next lesson without understanding the content in the previous lesson. Administering the Knowledge Component The Web Application Security lessons were prepared by the researcher and distributed to the students on Moo- dle. The students were permitted to work through the lessons as often as they wanted. During a lecture, the researcher explained the process that the students needed to follow when completing the online content. Most students worked through the content in the computer laboratories at the university as soon as it was made active and available to them. A total of 120 students completed the online lessons. The students had to work through all the lessons, since they were required to take a quiz which served as a Post-test (Phase 4A) for which marks were recorded. 4.2 Behavioural Compliance Monitoring Instrument Although it is possible for a student to have the requisite knowledge and not perform accordingly when developing their web applications, it is most unlikely for them to behave accordingly when they do not have the requisite knowledge. Therefore, it was deemed necessary to ﬁrstly educate the students on secure coding practices and then to monitor their adherence to these practices. This section provides details on how the behaviour of students was monitored when developing their web applications as part of their third year capstone projects. Design of Behavioural Compliance Instrument The behavioural compli- ance instrument took the form of a checklist as seen in Table 3. The code review checklist for this research was adapted from the secure coding practices in Table 2 and was provided to the students electronically via Moodle. Table 3: Code Review Checklist. SP Questions SP1 Do they make use of parameterised SQL commands for all data access? Yes / No (Number of Instances) SP2 Do they make use of concatenated strings in the queries? Yes (Number of Instances) / No SP3 Are all input ﬁelds validated? Input Properly Validated / Input not Properly Validated / No Validation SP4 Do they make use of the Principle of Least Privilege when setting up their databases? Yes / No SP5 Do they use integrated authentication or do they use SQL authentication? Integrated Authentication / SQL Authentication SP6 Do they use stored procedures for their queries? Yes / No / Inconsistent Use of Stored Procedures and Queries SP7 Do they encrypt their connection strings? Yes / No SP8 Does the connection string only appear once in the web.conﬁg ﬁle? Yes / No SP9 Is all the sensitive data being encrypted using the OWASP recommended methods? Encrypted Using Acceptable Method / No Encryption / Encrypted Not using Acceptable Method Conducting the Behavioural Compliance Instrument During a lecture the researcher explained to the students how they should go about using the checklist to review their capstone projects. They were required to check all web forms accessing the data access layer against the secure coding practices for SP1 to SP9 using the checklist provided in Table 3. Having worked through the knowledge component, as discussed in Section 4.1, the students should have Table 3: Code Review Checklist. 4.2 Behavioural Compliance Monitoring Instrument Conducting the Behavioural Compliance Instrument During a lecture the researcher explained to the students how they should go about using the checklist to review their capstone projects. They were required to check all web forms accessing the data access layer against the secure coding practices for SP1 to SP9 using the checklist provided in Table 3. Having worked through the knowledge component, as discussed in Section 4.1, the students should have Conducting the Behavioural Compliance Instrument During a lecture the researcher explained to the students how they should go about using the checklist to review their capstone projects. They were required to check all web forms accessing the data access layer against the secure coding practices for SP1 to SP9 using the checklist provided in Table 3. Having worked through the knowledge component, as discussed in Section 4.1, the students should have acquired the requisite knowledge relating to the secure coding practices that should be implemented in their web applications. p pp Since most students worked in groups when developing their web applications, they were also required to conduct a peer code review on each other’s web forms using the checklist provided. The peer code review helped the students to double check whether they had really adhered to the secure coding practices as indicated in their own code reviews. Feedback from the students was positive and most students found the checklist helpful for their code and to ensure compliance to the secure coding practices. Since most students worked in groups when developing their web applications, they were also required to conduct a peer code review on each other’s web forms using the checklist provided. The peer code review helped the students to double check whether they had really adhered to the secure coding practices as indicated in their own code reviews. Feedback from the students was positive and most students found the checklist helpful for their code and to ensure compliance to the secure coding practices. 5 Eﬀectiveness of the Educational Intervention Once students had completed the educational intervention, it was necessary to determine its eﬀectiveness. The knowledge component of the educational inter- vention was responsible for providing students with knowledge regarding secure coding practices. Having completed the online course, the students were expected to implement the learnt secure coding practices in their capstone projects, show- ing behavioural compliance. The veriﬁcation of the knowledge component was achieved through an online quiz distributed to the students through the Moodle site as discussed in Section 5.1. Veriﬁcation of the behavioural compliance component took the form of a code review by the researcher on the students’ capstone projects as discussed in Section 5.2. 5.1 Knowledge Veriﬁcation The setup for the post-test questionnaire was such that students were only al- lowed to work through the post-test after they had completed the lessons in the knowledge component of the educational intervention, referred to as the Web Application Security Course. The 113 students who completed the post- test were only allowed to work through the post-test once. The results for the post-test questionnaire were automatically recorded on the Moodle site, where the researcher was able to export the data to an Excel spreadsheet for analysis. A comparison of the knowledge pre- and post-test is shown in Table 4. Table 4: Knowledge Assessment and Veriﬁcation Results (Pre-Test vs Post-Test). Table 4: Knowledge Assessment and Veriﬁcation Results (Pre-Test vs Post-Test). Phases SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 Phase 2 74% 36% 30% 58% 26% 39% 20% 3% 1% Phase 4A 95% 95% 89% 91% 91% 77% 93% 88% 83% Variance 21% 59% 59% 33% 65% 38% 73% 85% 82% Table 4 shows the results for Phase 2, Knowledge Assessment (pre-test), and Phase 4A, Knowledge Veriﬁcation (post-test). There was a substantial im- provement in the students’ knowledge as indicated in the second row, Phase 4A. Students’ knowledge has improved in all of the secure coding practices (SP1 to SP9), as seen in the variances. SP2, SP3, SP5 and SP7 showed reasonable im- provements, while SP8 and SP9 showed the highest improvements with variances above 80%. As mentioned previously, knowledge acquisition does not guarantee a change in behaviour. In order to monitor the adherence of the students to these secure coding practices when developing their web applications behavioural com- pliance monitoring was required. 6 Conclusion The results of this study indicate that students’ adherence to secure coding practices can be positively impacted through a formal educational intervention. However, it is important that such an intervention addresses both the knowledge and behaviour of students since having the requisite knowledge does not ensure compliance. It is for this reason that a behavioural compliance monitoring in- strument formed part of the study. This is a step towards educating students in secure application development which is essential in addressing the many secu- rity vulnerabilities existing in Web applications today. Limitations of this study do exist. Firstly, this study addressed only the iden- tiﬁed secure coding practices which were determined from OWASP. Secondly, the identiﬁed secure coding practices only focused on the data access layer of Web applications developed in the .NET environment. Future research could investigate similar interventions within various other application development contexts. 5.2 Behavioural Veriﬁcation The behavioural veriﬁcation instrument used was the same checklist used in Phases 1 and 3 as shown in Table 3. The checklist was used by the researcher to conduct a code review on the third year capstone projects. The code review was conducted by the researcher before the ﬁnal submission of the software development projects. The researcher ﬁrst informed the students about the code review process scheduled to take place during a session in the computer laboratory. Students ﬁlled in their group names and were required to be in the computer laboratory in order for their projects to be reviewed. The code review was conducted during the students’ practical sessions. For each of the capstone projects, the researcher reviewed ﬁve web forms per project, which connected to the database and were related to the capstone projects’ main functionality. 17 groups were present for the code review, and they were all reviewed successfully, in the presence of the students who belonged to the group being reviewed. Table 5 shows the results from the behavioural analysis for the students before and after exposure to the educational intervention. Table 5: Behavioural Veriﬁcation Results (Phase 1 and 4B). Phases SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 Phase 1 86% 84% 77% 60% N/A 38% N/A 68% 31% Phase 4B 96% 96% 100% 91% N/A 96% N/A 100% 100% Variance 10% 12% 23% 31% N/A 58% N/A 32% 69% Table 5: Behavioural Veriﬁcation Results (Phase 1 and 4B). As can be seen from the results in Table 5, there was an improvement in students’ adherence to secure coding practices after the educational interven- tion, with most capstone project groups having adhered to all the secure coding practices. Although SP5 and SP7 were recommended by OWASP, they were not required by the capstone projects from which the sample for this research was drawn. All averages per secure coding practice were between 90% and 100%, with SP3, SP8, and SP9 showing 100% compliance. SP6 and SP9 showed the largest improvements of 58%(SP6) and 69%(SP9) respectively, while SP3, SP4 and SP8 showed good improvements of between 20% and 35%. 7 Ethical Considerations This research project adhered to all ethical requirements of the Nelson Mandela University and obtained ethics approval from the university research committee (REF H15-ENG-ITE-009). References 1. Bishop, M., Dai, J., Dark, M., Ngambeki, I., Nico, P., Zhu, M.: Evaluating se- cure programming knowledge. In: IFIP World Conference on Information Security Education. pp. 51–62. Springer (2017) 2. Chandrasekar, K., Cleary, G., Cox, O., O Gorman, B.: Internet Security Threat Report. Tech. Rep. April, Symantec (2017), https://www.symantec.com/security- center/threat-report 2. Chandrasekar, K., Cleary, G., Cox, O., O Gorman, B.: Internet Security Threat Report. Tech. Rep. April, Symantec (2017), https://www.symantec.com/security- center/threat-report 3. Chi, H., Jones, E.L., Brown, J.: Teaching Secure Coding Practices to STEM Students. Proceedings of the 2013 on InfoSecCD ’13 In- formation Security Curriculum Development Conference - InfoS- ecCD ’13 pp. 42–48 (2013). https://doi.org/10.1145/2528908.2528911, http://dl.acm.org/citation.cfm?doid=2528908.2528911 3. Chi, H., Jones, E.L., Brown, J.: Teaching Secure Coding Practices to STEM Students. Proceedings of the 2013 on InfoSecCD ’13 In- formation Security Curriculum Development Conference - InfoS- ecCD ’13 pp. 42–48 (2013). https://doi.org/10.1145/2528908.2528911, http://dl.acm.org/citation.cfm?doid=2528908.2528911 4. Chung, S., Hansel, L., Bai, Y., Moore, E., Taylor, C., Crosby, M., Heller, R., Popovsky, V., Endicott-Popovsky, B.: What approaches work best for teaching secure coding practices? 2014 HUIC Education & STEM Conference (2014) 4. Chung, S., Hansel, L., Bai, Y., Moore, E., Taylor, C., Crosby, M., Heller, R., Popovsky, V., Endicott-Popovsky, B.: What approaches work best for teaching secure coding practices? 2014 HUIC Education & STEM Conference (2014) 5. Conklin, A., White, G.: A graduate level assessment course: A model for safe vulnerability assessments. In: Proceedings of the 9th Colloquium for Information Systems Security Education (2005) 6. Customs Solutions Group: A CISO ’ s Guide to Applica- tion Security. Tech. rep., Customs Solutions Group (2012), http://h30528.www3.hp.com/Security/CISOGuideToApplicationSecurity.pdf 7. Dark, M., Ngambeki, I., Bishop, M., Belcher, S.: Teach the Hands, Train the Mind . . . A Secure Programming Clinic! Proceeding of the 19th Colloquium for Infor- mation System Security Education (2015) 8. Dark, M., Stuart, L., Ngambeki, I., Bishop, M.: Eﬀect of the secure programming clinic on learners’ secure programming practices. In: Journal of The Colloquium for Information System Security Education. vol. 4, pp. 18–18 (2016) 9. Deepa, G., Thilagam, P.S.: Securing web applications from injection and logic vulnerabilities: Approaches and challenges. Information and Software Technology 74, 160–180 (2016), http://dx.doi.org/10.1016/j.infsof.2016.02.005 ( ) // / / 10. Florian, T.P., Zimmerman, J.P.: Understanding by Design , Moodle , and Blended Learning : A Secondary School Case Study. MERLOT Journal of Online Learning and Teaching 11(1), 120–128 (2015) 11. References Li, X., Xue, Y.: A survey on server-side approaches to securing web applications. ACM Computing Surveys (CSUR) 46(4), 54 (2014) 12. Lunt, B., Sabin, M., Hala, A., Impagliazzo, J., Zhang, M.: Information Technology Curricula 2017. Tech. rep., Association for Computing Machinery (ACM) IEEE Computer Society (2017) 13. Lunt, B.M., Ekstrom, J.J., Lawson, E.: Curriculum guidelines for undergraduate degree programs in Information Technology. Tech. rep., Association for Computing Machinery (ACM) IEEE Computer Society (2008) 14. Mdunyelwa, V.S., Niekerk, J.F.V., Futcher, L.A.: Secure coding practices in the software development capstone projects. In: Eleventh International Sympo- sium on Human Aspects of Information Security & Assurance, HAISA 2017, Adelaide, Australia, November 28-30, 2017, Proceedings. pp. 282–291 (2017), http://www.cscan.org/openaccess/?paperid=353 15. OWASP: OWASP Top 10 (2017), https://www.owasp.org/index.php/Top 10- 2017 Top 10 16. OWASP: The OWASP Foundation (2017), https://www.owasp.org/index.php/Main Page 16. OWASP: The OWASP Foundation (2017), https://www.owasp.org/index.php/Main Pag 17. Vroom, C., Von Solms, R.: Towards information security behavioural compliance. Computers and Security 23(3), 191–198 (2004) 18. Zhu, J., Xie, J., Lipford, H.R., Chu, B.: Supporting secure programming in web ap- plications through interactive static analysis. Journal of Advanced Research 5(4), 449–462 (2014), http://dx.doi.org/10.1016/j.jare.2013.11.006
https://openalex.org/W4286624726	https://zenodo.org/records/6885157/files/To%E2%80%98ychiboev%20Abbosjon%20Erali%20o%E2%80%98g%E2%80%98li5.pdf	Kirghiz, Kyrgyz	null	KORXONA BOSHQARUVINI AVTOMATLASHTIRISH TIZIMLARINING XARAKTERISTIKALARI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,066	Muhammad Al-Xorazmiy nomidagi TATU Farg‘ona filiali talabalari Annotatsiya: Axborotni boshqarish tizimi - bu axborotni qayta ishlash va boshqaruv qarorlarini qabul qilish uchun mo'ljallangan axborot, texnik, dasturiy ta'minot va boshqa texnologik vositalar va mutaxassislar yig'indisidir. Ushbu maqolada, korxona boshqaruvini avtomatlashtirish haqida ma’lumotlar keltirib o‘tilgan. Kalit so‘zlar: Avtomatlash, axborot, texnologiya, soha, kompaniya, biznes, iqtisodiy ob'ekt. Kalit so‘zlar: Avtomatlash, axborot, texnologiya, soha, iqtisodiy ob'ekt. Avtomatlashtirilgan axborot tizimining asosiy komponenti axborot texnologiyalari (IT) hisoblanadi., uning rivojlanishi IP ning rivojlanishi va faoliyati bilan chambarchas bog'liq. KONFERENSIYA \| 1 ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR info@bestarticle.uz BEST ARTICLE RESPUBLIKA ILMIY – ONLAYN KONFERENSIYASI 2022 https://sites.google.com/view/imxu/ Axborot texnologiyalari (AT) - bu iqtisodiy ob'ektni boshqarish muammolarini hal qilish uchun dasturiy va texnik vositalarga asoslangan axborotni ro'yxatga olish, uzatish, to'plash va qayta ishlash jarayoni. Avtomatlashtirishning asosiy maqsadi axborot texnologiyalari- birlamchi ma'lumotlarni qayta ishlash orqali yangi sifatdagi axborotni olish, ular asosida optimal boshqaruv qarorlari ishlab chiqiladi. Axborot texnologiyalari uchun avtomatlashtirilgan axborot tizimlari asosiy muhit bo'lib, uning tarkibiy elementlari ma'lumotlarni o'zgartirish vositalari va usullari hisoblanadi. 1. Turli faoliyat sohalaridagi kichik korxonalarda axborot texnologiyalari, qoida tariqasida, buxgalteriya hisobi muammolarini hal qilish, biznes jarayonlarining ayrim turlari bo'yicha ma'lumotlarni to'plash, kompaniya faoliyatining yo'nalishlari bo'yicha axborot ma'lumotlar bazalarini yaratish va telekommunikatsiya muhitini tashkil etish bilan bog'liq. foydalanuvchilarni bir-biri bilan va boshqa korxona va tashkilotlar bilan bog'lash. 2. O'rta tashkilotlarda (korxonalarda) elektron hujjat aylanishining ishlashi va uning muayyan biznes jarayonlari bilan bog'lanishi boshqaruv darajasi uchun katta ahamiyatga ega. Bunday tashkilotlar (korxonalar, firmalar) kompaniya faoliyati bilan bog'liq hal qilinishi kerak bo'lgan funktsional vazifalar doirasining kengayishi, avtomatlashtirilgan ma'lumotlar ombori va arxivlarini tashkil etish bilan tavsiflanadi, bu esa hujjatlarni to'plash imkonini beradi. turli formatlar, ularning tuzilishi, qidirish imkoniyatlari, axborotni ruxsatsiz kirishdan himoya qilish va boshqalar mavjudligini nazarda tutadi. KONFERENSIYA \| 2 ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR info@bestarticle.uz BEST ARTICLE RESPUBLIKA ILMIY – ONLAYN KONFERENSIYASI 2022 BEST ARTICLE RESPUBLIKA ILMIY – ONLAYN KONFERENSIYASI 2022 https://sites.google.com/view/imxu/ 3. Yirik tashkilotlarda (korxonalarda) axborot texnologiyalari zamonaviy dasturiy- texnika majmuasi, jumladan telekommunikatsiya vositalari, ko‘p mashinali komplekslar, rivojlangan mijoz-server arxitekturasi, yuqori tezlikdagi korporativ kompyuterlardan foydalanish asosida quriladi. tarmoqlar. Avtomatlashtirilmagan (qog'ozga asoslangan) tizimlarning afzalliklari: mavjud echimlarni amalga oshirish qulayligi; Avtomatlashtirilmagan (qog'ozga asoslangan) tizimlarning afzalliklari: 2. Ikkinchi doira biznesni rivojlantirish uchun strategik boshqaruv qarorlarini ishlab chiqish uchun mo'ljallangan tahlil vazifalari bilan bog'liq. 2. Ikkinchi doira biznesni rivojlantirish uchun strategik boshqaruv qarorlarini ishlab chiqish uchun mo'ljallangan tahlil vazifalari bilan bog'liq. 3. Tahlil vazifalarining uchinchi doirasi taktik qarorlarni ishlab chiqishga qaratilgan. 4. Vazifalarning to'rtinchi doirasi iqtisodiy ob'ektning funktsional quyi tizimlariga muvofiq iqtisodiy ob'ektni operativ boshqarish vazifalari bilan bog'liq. Odatda, boshqaruv tizimlari uch darajaga bo'linadi: strategik, taktik va operatsion. a, boshqaruv tizimlari uch darajaga bo'linadi: strategik, taktik va operatsion. I. Strategik daraja top-menejerlarga qaratilgan. Strategik boshqaruv darajasining asosiy maqsadlari quyidagilardan iborat: · Tashkilotni rivojlantirishning ustuvor yo'nalishlari tizimini aniqlash; · Tashkilot rivojlanishining istiqbolli yo'nalishlarini baholash; II. Qarorlarni qabul qilishning taktik darajasi ma'lumotlarni avtomatlashtirilgan qayta ishlash va individual, asosan zaif tuzilgan vazifalarni hal qilishga yordam II. Qarorlarni qabul qilishning taktik darajasi ma'lumotlarni avtomatlashtirilgan qayta ishlash va individual, asosan zaif tuzilgan vazifalarni hal qilishga yordam KONFERENSIYA \| 3 ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR info@bestarticle.uz beradigan modellarni amalga oshirishga asoslangan. Taktik etakchilik darajasining asosiy maqsadlari: · butun tashkilotning barqaror ishlashini ta'minlash; · Tashkilotning rivojlanishi uchun salohiyatni oshirish; · Tashkilotni rivojlantirish jarayonida to'plangan salohiyat asosida buyurtmalarni bajarish uchun asosiy ish rejalari va jadvallarini yaratish va tuzatish. Qarorlarni qabul qilishning operativ (operativ) darajasi barcha avtomatlashtirilgan axborot texnologiyalarining asosidir. Ushbu darajada iqtisodiy ob'ektning turli funktsional vazifalarini hal qilish uchun juda ko'p sonli joriy operatsiyalar amalga oshiriladi. Shu bilan birga, operativ boshqaruvning eng muhim ustuvor yo'nalishlari qatoriga quyidagilar kiradi: · To‘plangan salohiyatdan foydalangan holda oldindan rejalashtirilgan tadbirlarni amalga oshirish orqali foyda olish; · To‘plangan salohiyatdan foydalangan holda oldindan rejalashtirilgan tadbirlarni amalga oshirish orqali foyda olish; rejalashtirilgan ishlab chiqarish jarayonidan chetlanishlarni hisobga olish, jamlash va tahlil qilish; · Keraksiz og'ishlarni bartaraf etish yoki minimallashtirish uchun echimlarni ishlab chiqish va amalga oshirish. Korxona boshqaruvini avtomatlashtirish tizimlarining xarakteristikalari. . Kirish darajasidagi tizimlar. https://sites.google.com/view/imxu/ Kichkina korxonalar orasida boshlang'ich darajadagi tizimlar keng tarqalgan bo'lib, ulardan kundalik faoliyatida muvaffaqiyatli foydalanadi. Bunday axborot tizimlarining o'ziga xos xususiyati korxona biznes-jarayonlarining cheklanganligidir. Ushbu toifadagi dasturiy mahsulotlar o'z maqsadiga ko'ra bir-biridan sezilarli darajada farq qilishi mumkin: bu ham buxgalteriya, ham ombor va savdo tizimlarini o'z ichiga olishi mumkin. Ammo, shunga qaramay, bu tizimlarning umumiy jihatlari ko'p: ajratilgan resurslarga past talablar. Ushbu toifadagi tizimlar zamonaviy sanoat ma'lumotlar bazasi boshqaruvi ostida ishlashi mumkin, ammo ular kichik korxonalarda ham ishlatilishi mumkin. Bunday tizimning mumkin bo'lgan foydalanuvchilari soni 1 dan bir necha o'nlabgacha. Ma'lumki, foydalanuvchi o'z-o'zidan sotib olishi, o'rnatishi va ishlashni boshlashi mumkin, ammo ishlab chiquvchilar juda ko'p dasturlarni yaratishga harakat qilishadi. keng imkoniyatlar, bu esa bunday tizimlarni ushbu va undan yuqori sinflarning boshqa tizimlari bilan birlashtirish imkonini beradi. ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR info@bestarticle.uz BEST ARTICLE RESPUBLIKA ILMIY – ONLAYN KONFERENSIYASI 2022 ADABIYOTLAR RO‘YXATI Акбаров Д. Е. и др. Исследования Вопросов Необходимых Условий Крипто Стойкости Алгоритмов Блочного Шифрования С Симметричным Ключом //CENTRAL ASIAN JOURNAL OF MATHEMATICAL THEORY AND COMPUTER SCIENCES. – 2021. – Т. 2. – №. 11. – С. 71-79. Акбаров Д. Е., Умаров Ш. А. Анализ приложения логических операций к криптографическим преобразованиям средств обеспечения информационной ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR info@bestarticle.uz https://sites.google.com/view/imxu/ BEST ARTICLE RESPUBLIKA ILMIY – ONLAYN KONFERENSIYASI 2022 безопасности //Universum: технические науки. – 2020. – №. 2-1 (71). – С. 14- ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR info@bestarticle.uz BEST ARTICLE RESPUBLIKA ILMIY – ONLAYN KONFERENSIYASI 2022 https://sites.google.com/view/imxu/ безопасности //Universum: технические науки. – 2020. – №. 2-1 (71). – С. 14- 19. Тожибоев И. Т. Краевые задачи в специальной области для уравнения смешанного типа //Вестник Томского государственного университета. Математика и механика. – 2018. – №. 56. – С. 17-28. Mirzapolatovich E. O., Eralievich T. A., Mavlonzhonovich M. M. Analysis of Static Characteristics Optoelectronic Level Converters Liquids and Gases Based on Hollow Light Guides //EUROPEAN JOURNAL OF INNOVATION IN NONFORMAL EDUCATION. – 2022. – Т. 2. – №. 6. – С. 29-31. 19. Тожибоев И. Т. Краевые задачи в специальной области для уравнения смешанного типа //Вестник Томского государственного университета. Математика и механика. – 2018. – №. 56. – С. 17-28. Mirzapolatovich E. O., Eralievich T. A., Mavlonzhonovich M. M. Analysis of Static Characteristics Optoelectronic Level Converters Liquids and Gases Based on Hollow Light Guides //EUROPEAN JOURNAL OF INNOVATION IN NONFORMAL EDUCATION. – 2022. – Т. 2. – №. 6. – С. 29-31. KONFERENSIYA \| 6 6 KONFERENSIYA \|
https://openalex.org/W2019790268	https://europepmc.org/articles/pmc4244563?pdf=render	English	null	MTA1—a stress response protein: a master regulator of gene expression and cancer cell behavior	Cancer and metastasis reviews/Cancer metastasis reviews	2,014	cc-by	8,332	MTA1—a stress response protein: a master regulator of gene expression and cancer cell behavior Rui-An Wang Published online: 21 October 2014 Published online: 21 October 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com drugs that can block metastasis and thus reduce cancer- caused death. Hundreds of molecules are closely related to metastasis. MTA1 is one that attracts widespread attention for its close relationship with cancer progression, metasta- sis, and its fantastic role in many other cellular processes. While MTA1 research and review articles are mounting, they still lack insight about what stimulates MTA1 expres- sion and why its overexpression drives metastasis, such as a biological meaning behind all these phenomena. I here present a new carcinogenesis theory viewpoint, stem cell misplacement theory (SCMT) [1], which explains why cancer occurs and metastasizes. We may have a glimpse of MTA1’s role in carcinogenesis and cancer metastasis, not from a mechanistic but a biological point of view. Abstract Gene mutation’s role in initiating carcinogenesis has been controversial, but it is consensually accepted that both carcinogenesis and cancer metastasis are gene-regulated processes. MTA1, a metastasis-associated protein, has been extensively researched, especially regarding its role in cancer metastasis. In this review, I try to elucidate MTA1’s role in both carcinogenesis and metastasis from a different angle. I propose that MTA1 is a stress response protein that is upreg- ulated in various stress-related situations such as heat shock, hypoxia, and ironic radiation. Cancer cells are mostly living in a stressful environment of hypoxia, lack of nutrition, and immune reaction attacks. To cope with all these stresses, MTA1 expression is upregulated, plays a role of master reg- ulator of gene expression, and helps cancer cells to survive and migrate out of their original dwelling. Keywords MTA1 . Stress protein . Carcinogenesis . Metastasis . Hypoxia . Immune stress . Epithelial stem cell misplacement . Apoptosis Cancer Metastasis Rev (2014) 33:1001–1009 DOI 10.1007/s10555-014-9525-1 Cancer Metastasis Rev (2014) 33:1001–1009 DOI 10.1007/s10555-014-9525-1 2 Carcinogenesis by stem cell misplacement—carcinoma cells are strayed epithelial cells in the stroma The traditional view of carcinogenesis as a result of accumu- lated gene mutation faces increasing challenges [1–5] and evidence falsifying the somatic mutation theory (SMT) is emerging. First, intensive cancer genome studies failed to reveal any specific gene mutation combinations as the cause of cancer. Second, most chemical carcinogens are not genotoxic [6], and those which are genotoxic are not neces- sarily carcinogenic, such as the famous anti-TB drug isoniazide. Third, increasing evidence shows that most high occurrence gene mutations in cancer cells are associated with better clinical outcomes, which means gene mutations lower cancer malignancy. For example, IDH1 and IDH2 mutations are associated with better glioma patient prognosis [7–9], and Braf mutations are associated with better prognosis in acral lentiginous melanoma [10]. R.<A. Wang () Department of Pathology and Pathophysiology, The Fourth Military Medical University Xi’an, Xi’an 710032, China e-mail: wangra@fmmu.edu.cn R.<A. Wang State Key Lab for Cancer Biology, Department of Pathology, Xijing Hospital, Xi’an, China 1 Introduction Metastasis is the primary cause of cancer-related death. In the past half century, paramount efforts have been made to elucidate the mechanisms involved in cancer metastasis, especially molecular mechanisms, with an aim to design R.<A. Wang State Key Lab for Cancer Biology, Department of Pathology, Xijing Hospital, Xi’an, China R.<A. Wang () Department of Pathology and Pathophysiology, The Fourth Military Medical University Xi’an, Xi’an 710032, China e-mail: wangra@fmmu.edu.cn Cancer Metastasis Rev (2014) 33:1001–1009 1002 2.1 The possible path from normal epithelial cells to invasive cancer 2.1 The possible path from normal epithelial cells to invasive cancer 2.1 The possible path from normal epithelial cells to invasive cancer invasive lobular carcinoma (ILC) are both characterized by e- cadherin expression loss, and LCIS is thought to be the precursor lesion of ILC. Paradoxically, if simple LCIS was diagnosed, no specific treatment was needed, since it has been proven that LCIS did not necessarily further progress [16]. Where ILC comes from remains unclear. The third level of evidence includes clinical epidemiology. Evidence has shown that if DCIS was left untreated, only 20 % of patients would develop invasive breast cancer in 10 years [17]. By this speed, if all invasive breast cancer derived from DCIS, its incidence should be many times that of invasive breast cancer, but the opposite is true. Invasive breast cancer incidence is four times that of DCIS [18]. In humans, around 80–90 % of malignant tumors are epithelially derived carcinomas. Ever since Dr. Broaders first systemically described the in situ carcinoma lesion in 1932 [11], the lesion has been seen as the earliest form of cancer. With further morphological observations, the stepwise carci- nogenesis model was gradually accepted by the scientific field. This model asserts that an epithelial cell is malignantly transformed due to gene mutation, further proliferates to form atypical hyperplasia, progresses to in situ carcinoma, and with gene mutation accumulation, it breaks down the basement membrane separating the epithelium from the connective stro- ma. It becomes invasive cancer in the stroma, where it can metastasize to distant sites by lymphatics or blood vessels [12]. The model was widely accepted and was taken as fact. With this evidence, we concluded that not all invasive breast cancer is derived from in situ carcinoma [1]. There must be an alternative carcinogenesis path that creates epithelial-derived invasive cancer. 2.3 Carcinogenesis by stem cell misplacement The above described evidence strongly suggests that the step- wise carcinogenesis model of in situ carcinoma to invasive breast cancer is logically impossible [19]. This implies that carcinoma must be grown out de novo from the stroma, i.e., developed from the displaced epithelial cells. The SCMTwe proposed solved the above puzzle [1]. SCMT posits that carcinoma originates from normal/non-transformed epithelial stem cells displaced in the stroma by the damaged basement membrane (BM) [1]. All known carcinogenic factors, such as inflammation and chronic injury, can damage the BM. Historically, German pathologist Julius Cohnheim suggested carcinogenesis by displaced embry- onic stem cells some 150 years ago [20]. 2.2 Paradoxes in the classic model of in situ carcinoma to invasive carcinoma The current question is not whether the epithelial stem cells can be displaced to the stroma, but regards the fate of the misplaced epithelial cells in the wrong environment. In most cases, we would expect that the misplaced cells die out. How- ever, some could manage to survive. Since they are epithelial cells by nature, they will form epithelial structure types. Usu- ally, if they can differentiate and form BM, they are benign structures like a cyst, a benign tumor, or even normal glandular tissues. However, if they failed to differentiate and form BM, they are carcinoma, i.e., cancer (Fig. 1). The MCF-DCIS cell line is an interesting example that proves the above hypothesis principle. This cell line was derived from the benign MCF10A cell line. When injected into the mammary fat pad of nude mice, it formed DCIS, meaning there is myoepithelial cell differentiation and basement membrane formation [21]. Unfortunately, this exclusive study approach has never been applied to test the classic carcinogenic model. Morphological observations provide support but not evidence for the model per se. Since the alternative model has never been studied, we cannot say it is wrong. Interestingly, the classic carcinogenesis model has been studied for many decades, so we should be able to falsify it if it was wrong. In fact, paradoxes falsifying the in situ carcinoma to invasive carcinoma model are accu- mulating and urging us to take a different stance. The paradoxical evidence comes from different levels. The first evidence level is of molecular pathology [1]. HER2 is a well-known oncogene often amplified and overexpressed in breast cancer. Intriguingly, ductal carcinoma in situ (DCIS), which is deemed to be the precursor lesion of invasive ductal cancer, has a much higher rate (50–60 %) of HER2 amplifi- cation and overexpression than that of invasive breast cancer, which is about 25 % positive for HER2 [13–15]. Yet, we cannot say that HER2 inhibits DCIS progression to invasive ductal carcinoma. The second level of evidence came from histological pathology. Lobular carcinoma in situ (LCIS) and 1 Introduction However, this model has never been extensively tested, and its dominance hinders researchers from thinking otherwise, i.e., normal epithelial cells displaced to the connective tissue stroma sites and developed into cancer in the wrong environ- ment. The basic difference between these two models is that the classic model posits that epithelial cells malignantly trans- form first and then enter the stroma by a process called epithelial-mesenchymal transition (EMT), while the alterna- tive model states that the epithelial cells enter the stroma first and then transform to cancer cells in the wrong environment [1]. With these two possible choices, logically, we cannot prove one model is right unless we prove the other is wrong. 2.5 EMT as a camouflage Epithelial mesenchymal transition has been extensively stud- ied in cancer metastasis research over the past decade. Most studies focused on the mechanisms and signaling pathways involved in EMT with the aim of targeting therapy. However, people rarely asked why cancer cells would ever start EMT. Obviously, the notion of in situ carcinoma progressing to invasive cancer by EMT does not hold up, as the invasive cancer does not derive from the in situ carcinoma. LCIS is characterized by e-cadherin expression loss [16], an EMT hallmark. Ironically, it has been clinically proven that LCIS lesions do not further develop and do not need special treat- ment [16]. Fig. 1 Carcinogenesis by stem cell misplacement. The displaced epithe- lial cells by the damage of basement membrane have a potential to develop into different benign or malignant lesions as shown in the figure. Dysplasia, in situ carcinoma such as DCIS, and invasive carcinoma are distinct lesion entities, instead of different developmental stages of the same lesion epithelial cells in the stroma are in a stressful state. Compared to the epithelium microenvironment, the stroma is a place of hyperoxia and rich in immune cells, antibodies, and other immune-related cytokines. Moreover, the situations introduc- ing epithelial cell displacement to the stroma are often asso- ciated with inflammatory reactions. The misplaced epithelial cells must fight for their existence. Interestingly, increasing population is an effective way of maintaining existence. Therefore, the higher the environmental pressure and greater the cell death, the faster the misplaced epithelial cells must grow [19, 22]. Also, pathologists saw that increased apoptosis was associated with higher cancer malignancy and poor clin- ical outcome [22–28]. I propose that EMT is a way of immune escape. We know that by nature, carcinoma consists of epithelial cells trapped in mesenchymal tissue. The mesenchymal tissue is not the home of epithelial cells. These epithelial cancer cells thus become the easy target of the immune system. Interestingly, although immune response against cancer has been found for almost six decades [57], there have been no cancer-specific antigens identified for most cancer types. Though the issue has not been explored before by immunologists, I believe that epithe- lial cell invasion to mesenchyme would provoke an immune response, and the antigen might be the epithelial marker that discriminates cancer cells from the surrounding mesenchymal cells. 2.4 Survival pressure drives cancer cells to proliferate and metastasize The primary cause for epithelial cells to transform to cancer cells in the stroma is survival pressure (Fig. 2). The misplaced Cancer Metastasis Rev (2014) 33:1001–1009 1003 Fig. 1 Carcinogenesis by stem cell misplacement. The displaced epithe- lial cells by the damage of basement membrane have a potential to develop into different benign or malignant lesions as shown in the figure. Dysplasia, in situ carcinoma such as DCIS, and invasive carcinoma are distinct lesion entities, instead of different developmental stages of the same lesion cell lung cancer, Bcl-2 overexpression is a predicting factor of favorable clinical outcomes [45–51]. Inducing apoptosis as a therapeutic strategy has been touted for the past two decades in both academics and indus- trial labs without much success. A recent study showed that the IAP inhibitor, which was developed to treat cancer, pro- motes breast cancer metastasis to bone [52]. Similarly, anti- angiogenic agents and radiotherapy were all found to stimu- late cancer metastasis [53–55]. Therefore, metastasis is a basic response of cancer cells to stress [56]. 2.5 EMT as a camouflage Pay atten- tion that apoptosis also plays a positive role in the process of carcinogen- esis instead of being a barrier Displaced by chronic injury Environmental stress Cancer cells Released growth factors Apoptosis Fig. 2 The role of MTA1 in carcinogenesis. By the SCMT model, epithelial cells are displaced to the stromal tissue. The environmental stresses such as inflammation, immunosurveillance, reactive oxygen species (ROS), reactive nitrogen intermediates (RNI), stimulate the expression of MTA1, which in turn promotes the malignant transforma- tion, proliferation, and EMT of the misplaced epithelial cells. Pay atten- tion that apoptosis also plays a positive role in the process of carcinogen- esis instead of being a barrier ironic radiation, inflammation, as well as heat shock all strongly upregulated MTA1 expression [65–73]. Since hyp- oxia, ironic radiation, and heat shock are all stress agents, we may conclude that MTA1 is a stress response protein. Its expression in the adverse and fluctuating immediate cancer cell surroundings may help survival in harsh conditions and escape from danger. In many stress conditions, such as trauma and inflammation, growth factors are released. Therefore, heregulin-stimulated MTA1 expression also falls in this stress response category (Figs. 2 and 3). esophagus would turn to columnar epithelium, which is more resistant to gastric acid. This is termed metaplasia, a form of pathological adaptation. Similarly, we proposed the concept of molecular adaptation [58]. The molecular adaptations include adaptive mutations and adaptive epigenetic modifications. The former includes point mutations, amplifications, and deletions. The concept of adaptive mutation was proposed by Cairns three decades ago [59] and has been a controversial issue since then. Most contemporary molecular geneticists are New Dar- winists and hold that gene mutations are stochastic in nature. They do not believe in adaptive mutation. It is true that we do not know the adaptive mutation mechanism, but that does not mean it does not exist. We can use the Braf V600E mutation in nevus cells as an example. Around 80 % of nevus cells have this point mutation [60]. Obviously, we cannot explain this phenomenon as a random mutation. Cell cycle regulators are also good examples of molecular adaptions. It is well known that cyclins are often overexpressed and cyclin-dependent kinases (CDKs) are over-activated in cancer, yet the cancer cell proliferation cycle duration is not shorter than the corre- sponding normal cells but prolonged or showing no change [61]. 3.1 Stress response proteins are upregulated in cancer As described above, carcinoma cells live in a stressful envi- ronment quite different from the epithelium. Initially, when A B C D Fig. 3 MTA1 expression in a dimethylnitrosamine-induced mouse liver carcinogenesis model. Dimethynitrosamine was given at a dose of 100 mg/Kg body weight by gavage, and 0.1 ml of 20 % of CCl4 in olive oil was given after 3 days by gavage, twice per week. MTA1 expression was upregulated, and more obviously seen in the cytoplasm. This sug- gests that MTA1 also functions in the cytoplasm in stress. A control, B 60 days, C 150 days after treatment. D negative staining control of a tissue slide from a mouse of 150 days after treatment. Bar=30 μm B D A C B 2.5 EMT as a camouflage This paradox is explained by molecular adaptation. The prolonged cell cycle means there is increased resistance and thus requires more cyclins and more active CDKs. Otherwise, cells cannot divide. 2.5 EMT as a camouflage Thus, to lower the risk of being targeted, the cancer cells would reduce epithelial marker expressions, and, as an adap- tation response, express some mesenchymal cell-type pro- teins. This is in accordance with the biosphere law. We see that jungle animals exhibit colors and patterns similar to their environment to lower their chances of being targeted. There- fore, EMT is a way of immune escape by the strategy of camouflage (Fig. 2). Although it is still widely believed that resistance to apo- ptosis is a hallmark of cancer [29, 30], the evidence favors the opposite view [19, 22, 23]. So far, there are no documented carcinogenic agents that can promote cell survival. Instead, they are mostly cytotoxic and induce cell death. For example, aflatoxin and various viruses whose infection induces liver cancer all induce liver cell death. The HBV virus X protein is the most potent factor of the HBV virus’s carcinogenic effects and is an apoptosis-inducing protein [31–38]. Of the other known apoptosis-inducing genes, such as the cell death recep- tor CD95 and death executor protein Caspase-3, all are known to promote tumor growth [39, 40]. Conversely, anti-apoptosis factors inhibit carcinogenesis and cancer growth. Autophagy inhibits apoptosis and carcinogenesis [41]. Bcl-2, the anti- apoptotic protein prototype, inhibits carcinogenesis and can- cer cell growth both in vitro and in vivo [42–44]. In various cancers, including breast cancer, colon cancer, and non-small 2.6 Molecular adaptations during carcinogenesis progression 2.6 Molecular adaptations during carcinogenesis progression Adaptation is an important pathology concept and a general biosphere phenomenon. The esophageal epithelium is strati- fied squamous epithelium, which is resistance to wear and tear but not resistant to acid. Therefore, when gastric acid reflux happens often, the epithelium of the lower part of the 1004 Cancer Metastasis Rev (2014) 33:1001–1009 Environmental stress Displaced by chronic injury Malignant evoluon Proliferaon, EMT MTA1 BM Apoptosis Cancer cells Released growth factors Inﬂammaon Immunosurveillance ROS, RNI Fig. 2 The role of MTA1 in carcinogenesis. By the SCMT model, epithelial cells are displaced to the stromal tissue. The environmental stresses such as inflammation, immunosurveillance, reactive oxygen species (ROS), reactive nitrogen intermediates (RNI), stimulate the expression of MTA1, which in turn promotes the malignant transforma- tion, proliferation, and EMT of the misplaced epithelial cells. 3 MTA1 is a stress response protein D C MTA1 was initially isolated from highly invasive breast can- cer cell lines, and its expression was associated with cancer progression and metastasis in a variety of human cancers [62–64] However, the factor responsible for upregulating MTA1 in cancer was unknown until Mazumdar et al. found that heregulin, a ligand for HER3, was capable of inducing MTA1 expression [65]. It was later discovered that hypoxia, Fig. 3 MTA1 expression in a dimethylnitrosamine-induced mouse liver carcinogenesis model. Dimethynitrosamine was given at a dose of 100 mg/Kg body weight by gavage, and 0.1 ml of 20 % of CCl4 in olive oil was given after 3 days by gavage, twice per week. MTA1 expression was upregulated, and more obviously seen in the cytoplasm. This sug- gests that MTA1 also functions in the cytoplasm in stress. A control, B 60 days, C 150 days after treatment. D negative staining control of a tissue slide from a mouse of 150 days after treatment. Bar=30 μm Cancer Metastasis Rev (2014) 33:1001–1009 1005 prognosis [10]. More interestingly, although apoptosis is taken as a barrier to carcinogenesis and resistance to apoptosis is regarded as a hallmark of cancer, the overexpression of the antiapoptotic oncogene Bcl-2 was an indicator of favorable prognosis in breast cancer, colon cancer, and non-small cell lung cancers. However, MTA1 overexpression was unani- mously associated with more advanced cancer stages, in- creased metastasis tendency, and unfavorable outcomes [78, 79]. So far, the reported correlation between MTA1 overex- pression and cancer progression and prognosis includes breast cancer [80–83], colon cancer [63, 84], esophageal cancer [64, 85], lung cancer [86, 87], liver cancer [88, 89], gastric cancer [63], thymoma [90], ovarian cancer [91, 92], nasopharyngeal cancer [93, 94], pancreatic cancer [95, 96], prostate cancer [97, 98], and chorionic carcinoma [99]. epithelial cells just land to the stroma, it is hyperoxic, with ample reactive oxygen species and reactive nitrogen interme- diates, immune cells, and cytokines. When they proliferate and grow, it is hypoxic due to lack of blood supply. Therefore, most, if not all, stress response proteins are highly expressed in cancer cells. For example, heat shock proteins, hypoxia inducible factors, and MAPK kinases such as p38MAPK, MAPK13, p53, and MTA1 are all stress-related proteins and proposed therapeutic cancer targets. 5 Biological functions of MTA1 Though MTA1 is described as a stress response protein, how it helps cells in environmental stress remains unclear. Though its functional roles are still elusive, many targeting genes and collaboration partners have been identified at MTA1 downstream. 5.1 Functions at the molecular level—regulation of gene expression by both affecting protein transcription and stabilization After MTA proteins were found to be a component of nucle- osome remodeling and the deacetylation (NuRD) complex, many downstream targets were discovered. Mazumdar et al. first found that MTA1 inhibits ER transactivation activity by recruiting HDAC2 to the promoters of ER targeting genes [74]. It was later found that MTA1 binds transcription factor Six3 [100] and in a negative feedback fashion inhibits Six3 expression and its downstream targets [101]. Paradoxically, MTA1 was also found to be a coactivator protein [102]. By binding and recruiting Pol II and C-Jun to the FosB promoter, MTA1 stimulates FosB expression [102]. Further, MTA1 binds FosB in the E-cadherin promoter region and recruits HDAC2 to downregulate E-cadherin expression, a hallmark of the epithelial mesenchymal transition [102]. Since ER, Six3, FosB, and possibly many other transcription factors regulate the expression of a broad spectrum of genes, MTA1 may thus exert a wide range of regulatory functions. 3 MTA1 is a stress response protein 3.2 Induction of MTA1 expression by heat shock, hypoxia, irradiation, and X protein of hepatitis B virus and dimethylnitrosamine The first factor known to be able to stimulate MTA1 expres- sion in breast cancer cells is the growth factor heregulin [74]. We know that growth factors are often released during trauma, so heregulin can be regarded as a stress-related factor. Korean scientists later found that hypoxia induced MTA1 expression, which helped stabilize HIF1α by recruiting histone deacetylase complex 1(HDAC1) [69]. Since HIF1a plays an important role in angiogenesis and promotion of cancer me- tastasis, MTA1 also has a part to play in both the normal wound healing and cancer. Li et al. further found that ionic radiation induced marked elevation of MTA1 protein expres- sion in U2OS osteosarcoma cells, mammary glands, thymus, and skin of mice [70]. The increased amount of MTA1 helps stabilize p53 and thus plays a role in repairing damaged DNA [71]. Moreover, MTA1 protein levels were elevated in a germ cell tumor cell line after heat shock and protected the cell from heat shock-induced apoptosis [68]. The X protein of hepatitis B virus is generally believed to be responsible for the virus’s carcinogenic effect [75]. It promotes both liver cell apoptosis and proliferation. Interestingly, X protein strongly induced MTA1 protein expression [66, 76]. Dimethynitrosamine (DEN) has a strong toxicity to the liver. Long-term treatment of rodents with DEN can induce liver cancer. We found that along with the increased liver cell damage, the MTA1 expres- sion level was also increased not only in the nuclei but in the cytoplasm (Fig. 3) [77]. References 1. Wang, R. A., Li, Z. S., Zhang, H. Z., Zheng, P. J., Li, Q. L., Shi, J. G., et al. (2013). Invasive cancers are not necessarily from preformed in situ tumours—an alternative way of carcinogenesis from misplaced stem cells. Journal of Cellular and Molecular Medicine, 17, 921–926. 4 MTA1 overexpression is associated with unfavorable prognosis There are numerous paradoxes in our current knowledge about cancer. Although it is widely believed that cancer is the result of accumulated gene mutations, many of these mutations are associated with better clinical outcomes. For example, IDH1 and IDH2 mutations are associated with much better glioma patient prognosis, and the BRAF mutation was associated with more favorable acral lentiginous melanoma Except for functioning as a transcriptional coregulator, MTA1 was also found to stabilize proteins by directly binding to them and inhibiting their break down through ubiquitination inhibition. For example, the expressions of both MTA1 and p53 were upregulated in cells exposed to ionizing radiation [71]. MTA1 was found to bind and stabilize p53, which is notoriously known to have a very short half-life [71]. Cancer Metastasis Rev (2014) 33:1001–1009 1006 Similarly, in cells cultured under hypoxic conditions, MTA1 and HIF1α expression were both upregulated, and MTA1 bonded to and stabilized HIF1α. They collaborate to promote angiogenesis and may improve the condition of nourishment and oxygen supply [68]. p53. MTA1 overexpression helps cells cope with environmen- tal stressors like hypoxia or hyperoxia, hyperthermia, immune reactions, and possibly radiation and chemotherapies, which would increase their chances of survival in the adverse envi- ronment. MTA1 overexpression stabilizes both HIF1α and p53, which both play important roles in carcinogenesis. 5.2 Functions at the cellular level Both carcinogenesis and cancer metastasis are rather com- plex issues, though, and so is MTA1’s role in these processes. Metastasis is a cancer cell stress response, and MTA1 as a stress protein is a stress level indicator. Therefore, it is no surprise that MTA1 overexpression correlates well with can- cer metastasis and is often an indicator of poor prognosis, no matter it has a role in metastasis or not. Conversely, as many studies have shown, MTA1 does play a role in helping cancer cells coping with stress by increasing their survival, angio- genesis, migration and invasion abilities, and epithelial mes- enchymal transition in collaboration with other stress proteins such as HIF1α, p53, and TGFR. Though it appears to be an attractive target for blocking cancer metastasis, it may not be that promising. Of the molecules involved in cancer metasta- sis, MTA1 is an important one but certainly not the only. The force driving cancer metastasis is stress, and the struggle for existence and MTA1 overexpression is a sign of these stresses. MTA1 was found to block p53-induced apoptosis [103], induce cell proliferation [101], and promote epithelial mesen- chymal transition in different studies [102]. However, promo- tion of cell survival is not positively linked with cell prolifer- ation. In fact, in most cases, or by principle, apoptosis reduc- tion and cell proliferation are negatively correlated [19]. The less apoptosis, the slower the cell grows [19]. Bcl-2, the typical anti-apoptotic protein, inhibits cell growth [43] as well as p202, an interferon-induced antiapoptotic protein [104, 105]. Conversely, CD95, caspase 3, and HBVx, all induce apoptosis and promote tumor cell growth [39, 40, 75]. The case of MTA1 is more complex. Environmental stress stimu- lates its expression, which may protect cells from apoptosis in a certain context, but is still not enough to make cells live long in an adverse environment. Therefore, cells may still show increased proliferation. As for EMT and metastasis, it is quite natural that MTA1 mediates these processes, but it cannot be the only molecule. Without MTA1, cells would still be able to migrate and invade, though perhaps be compromised. Acknowledgments The author wants to thank Professor Rakesh Kumar for his support and valuable discussions and suggestions in the preparation of this manuscript. Grant support: from NSFC grant No. 30971535 Conflicts of interest None. Studies from MTA1 gene-modified mice have revealed a large range of functions MTA1 may play. MTA1 was found to play roles in circadian rhythm maintenance [106], embryonic de- velopment regulation, and visual performance by regulating rhodopsin expression [102]. A reduced rate of breast cancer metastasis to lung was observed in the MTA1 null genetic background [107]. More functions of MTA1 at body level are expected to be revealed by gene-modified animal studies. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 6 Conclusion Oncology Reports, 26, 1479– 1485. 27. Lipponen, P. 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Overexpression of the c-erbB- 2 oncoprotein: why does this occur more frequently in ductal carcinoma in situ than in invasive mammary carcinoma and is this of prognostic significance? European Journal of Cancer, 28, 644– 648. 33. Hu, L., Chen, L., Yang, G., Li, L., Sun, H., Chang, Y., et al. (2011). HBx sensitizes cells to oxidative stress-induced apoptosis by accel- erating the loss of Mcl-1 protein via caspase-3 cascade. Molecular Cancer, 10, 43. 34. Kim, J. Y., Song, E. H., Lee, H. 6 Conclusion MTA1 is a key factor in cancer metastasis, and its overexpres- sion was consistently found to be associated with cancer’s advanced stages, higher malignancy degree, and poorer pa- tient prognosis. The biological meaning behind these phenom- ena remains unknown. By using stem cell misplacement the- ory, I interpreted that carcinoma are developed from misplaced epithelial stem cells in the stressful wrong stroma environment, which is often affected by subtle, chronic in- flammation. 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https://openalex.org/W3113483433	https://revistaseletronicas.pucrs.br/ojs/index.php/oficinadohistoriador/article/download/37845/26352	Portuguese	null	As Humanidades Ambientais	Oficina do Historiador	2,020	cc-by	2,899	OPEN ACCESS OPEN ACCESS http://dx.doi.org/10.15448/2178-3748.2020.2.37845 As Humanidades Ambientais: emergência, características e sua contribuição para a historiografia brasileira As Humanidades Ambientais: emergência, características e sua contribuição para a historiografia brasileira The Environmental Humanities: emergence, features and contribution to Brazilian historiography Recebido em: 27/4/2020. Aprovado em: 23/7/2020. Publicado em: 21/12/2020. OFICINA DO HISTORIADOR Oficina do historiador, Porto Alegre, v. 13, n. 2, p. 1-5, jul.-dez. 2020 e-ISSN: 2178-3748 1 Universidade Federal de São Paulo (Unifesp), Guarulhos, SP, Brasil. Bianca Letícia de Almeida1 id / 86 Resumo: O objetivo desta resenha é analisar The Environmental Humanities: A Critical Introduction (2017), de Robert S. Emmett e David E. Nye, ainda sem tra dução para o português. Por meio do que é discutido no livro e de informações externas, essa resenha tem a intenção de apresentar as Humanidades Ambientais para pesquisadores brasileiros, sobretudo historiadores, e incentivar discussões e buscas de soluções para problemas ambientais locais. Isso pode ser alcan çado por intermédio de pesquisas científicas interdisciplinares em diálogo com movimentos sociais, artistas, opinião pública e outras esferas da sociedade. orcid.org/0000-0002-8660-9559 b.almeida@unifesp.br Recebido em: 27/4/2020. Aprovado em: 23/7/2020. Publicado em: 21/12/2020. Palavras-chaves: Humanidades Ambientais. História ambiental. Movimento ambiental. Abstract: The aim of this review is to analyze The Environmental Humanities: A Critical Introduction (2017), by Robert S. Emmett and David E. Nye, not trans lated to Portuguese yet. Considering the book’s discussion and other external information, this review’s intention is to present the Environmental Humanities to Brazilian scholars, especially historians, and to motivate discussions and solutions to local environmental problem. This can be reach by interdisciplinary scientific researches in dialog with social movements, artists, public opinion and other social organizations. Artigo está licenciado sob forma de uma licença Creative Commons Atribuição 4.0 Internacional. Keywords: Environmental Humanities. Environmental history. Environmental movement. EMMETT, Robert S.; NYE, David E. The Environmental Humanities: A Critical Introduction. Cambridge: MIT Press, 2017. 216 p. A obra Enviromental Humanities: a critical introduction, publicada pela MIT Press e escrita por Robert S. Emmett e David E. Nye, tem como objeto as Humanidades Ambientais, um campo com características interdisciplinar e internacional, que começou a criar forma no início do século XXI. Esta resenha tem o objetivo de apresentar e de analisar os principais pontos do livro, trazendo as discussões traçadas para o Brasil e, especialmente, para os historiadores. As experiências diversas dos autores, além de enriqueceram as discus sões do livro, mostram como é possível trabalhar em parceria com diversas áreas. David E. Nye é pesquisador de História da Ciência e Tecnologia, na Universidade de Minnesota, e professor de Estudos Americanos, na Uni versidade da Dinamarca do Sul. Entre os seus temas principais de pesquisa 1 Universidade Federal de São Paulo (Unifesp), Guarulhos, SP, Brasil. Oficina do historiador, Porto Alegre, v. 13, n. 2, p. 1-5, jul.-dez. Bianca Letícia de Almeida1 id / 86 2020 \| e-37845 2/5 estão: trabalho, consumo e história da tecnologia e energia elétrica.2 É autor de diversos livros, entre eles Electrifying America (1990), America’s Assembly Line (2013) e American Illuminations (2018). Já Robert S. Emmett, é diretor do programa acadêmico do internacional Rachel Carson Center for Environment and Society e professor assistente visitante de Es tudos Ambientais em Roanoke College (Virgínia, EUA). Suas pesquisas giram em torno dos seguintes assuntos: memória, mídia digital, novela de protes to, ecologia urbana e zonas de extração.3 Publicou Cultivating Environmental Justice: A Literary History of U.S. Garden Writing, em 2016. danças acontecem, sendo o real desafio propor alterações em planos políticos, diplomáticos e no cotidiano das pessoas, porque não basta pos suir aparato tecnológico se ele não for aplicado. Algumas sugestões de pesquisa, neste sentido, são entender por que o uso de energia per ca pita dos EUA é quase o dobro da Europa inteira; o que faz os cidadãos escolherem o seu meio de transporte principal; e como é problemático cidades se dizerem “verdes” quando descartam o seu lixo em países emergentes. A tese é desenvolvida no decorrer das 216 páginas, divididas em oito capítulos. No pri meiro, como uma espécie de introdução, Nye e Emmett discorrem sobre a emergência do campo; e, no segundo, trazem discussões sobre o meio ambiente no que se refere mais à pers pectiva geográfica. Assim, discutiu-se o conceito de place, que traz um senso de pertencimento com o espaço ao invés de só vê-lo como fonte de economia, os perigos do ecoturismo e uma discussão sobre New Wilds. O capítulo três apre senta o consumo de energia e de experiências de cidades sustentáveis. O papel da ciência e as possibilidades de contribuir ou causar danos na natureza foi explorado no quarto capítulo; no seguinte, discutiu-se as visões negativas sobre o Antropoceno; e, no sexto, as práticas alternativas que preservam a natureza. No sétimo capítulo, foram abordados como as humanidades ambien tais podem desconstruir a noção de “humano”, pautadas em discussões do novo materialismo, estudos feministas, críticas pós-coloniais, eco logia queer e animal studies. No capítulo oito, como conclusão, os autores apontaram algumas condições para que as humanidades ambientais sejam bem recebidas e praticadas, bem como possíveis críticas e limitações. 2 THE MIT PRESS (Cambridge). David E. Nye. [201-?]. Disponível em: https://mitpress.mit.edu/contributors/david-e-nye. Acesso em: 4 nov. 2019. 3 RACHEL CARSON CENTER FOR ENVIRONMENT AND SOCIETY (Munich). Dr. Robert Emmett. 2019. Disponível em: https://www.car soncenter.uni-muenchen.de/staff_fellows/archive/robert_emmett/index.html. Acesso em: 4 nov. 2019. Bianca Letícia de Almeida1 id / 86 O objetivo dos pesquisadores foi de apresentar as Humanidades Ambientais no que se refere à sua origem, temas, conceitos principais, iniciati vas vindas principalmente da Europa Ocidental, Austrália e EUA, e a relação com outros estudos – como o pós-colonial e o feminismo. Conforme os autores indicaram, não tiveram a intenção de defender algum método ou ideologia específica, nem apresentar soluções prontas, mas mostrar o trabalho que os humanistas têm feito (EMMETT; NYE, 2017, p. 1-2). Ainda que, por diversas vezes, haja pronunciamentos em tom de manifesto, indicando que há premissas em comum dentro da área, como a afirmação que o aquecimento global é real (2017, p. 1); que os humanos não possuem direitos especiais em relação às outras espécies (2017, p. 11); e que as culturas ocidentais não são superiores as outras (2017, p. 11). O argumento principal do livro, e que funda menta o campo científico, é de que as huma nidades têm um papel crucial para investigar e solucionar problemas ambientais (EMMETT; NYE, 2017, p. 2). Atuam desde a projeção de novas comunidades e a revitalização de cidades mais antigas, até em análises culturais, já que se en tende que a solução para o desgaste ambiental não está meramente na criação de novas tec nologias, mas em entender questões culturais e políticas. Dessa forma, é menos no conhecimento científico e mais na realidade social que as mu Conforme bem observou Roman Bartosch, em uma resenha em inglês sobre o livro, Nye e Emmett abordaram diversos temas introdutórios, não seguindo ordem cronológica ou metodoló gica, e superaram o desafio de quais assuntos Bianca Letícia de Almeida As Humanidades Ambientais: emergência, características e sua contribuição para a historiografia brasileira 3/5 Bianca Letícia de Almeida As Humanidades Ambientais: emergência, características e sua contribuição para a historiografia brasileira 3 3/5 apontam que as Humanidades Ambientais surgi ram da confluência de departamentos, como o de literatura, filosofia, história, geografia, estudos de gênero e antropologia (2017, p. 3). Duas iniciativas simultâneas colocaram pela primeira vez esses estudos em diálogo entre si e com discussões ambientais: uma veio de pesquisadores australia nos que usaram o nome “ecological humanities” e a outra partiu do Massachusetts Institute of Technology (MIT), que promoveu o Workshop on Humanistic Studies of the Environment, entre 1991 e 1995 (2017, p. 3). Desde 2010, o consenso girou em torno do nome Environmental Humani ties. Bianca Letícia de Almeida1 id / 86 Inicialmente, seus pesquisadores eram da Austrália, dos EUA e da Europa Ocidental, porém atualmente, já existem acadêmicos em todos os continentes (2017, p. 4-5). No momento de escrita desta resenha, foram encontradas poucas inicia tivas brasileiras em uma busca pela internet. Uma delas foi a Conferência Humanidades Ambientais e Ambientes Humanizados, realizada na Univer sidade Federal de Minas Gerais (UFMG), em 2018. escolher e com qual profundidade de duas formas. Primeiramente, apontaram como entendimento básico, que as ciências ambientais têm como denominador conceitual comum a mudança climática do Antropoceno e, em segundo lugar, reivindicaram a contribuição das humanidades em debates epistemológicos, éticos e políticos sobre a mudança ambiental (BARTOSCH, 2019, p. 1). Ademais, além de discussões teóricas que podem priorizar visões sobre o ambiente no que tange à perspectiva geográfica de conservação do mundo natural e na interação entre humanos e natureza, durante todo o livro houve a preocupa ção em trazer exemplos concretos de questões, iniciativas e estudos de diversas partes do mundo. Em relação ao Brasil, foi citado, por exemplo, o trabalho Genesis de Sebastião Salgado como uma iniciativa que lida com a questão “wilderness and wilds” de forma não centrada em uma genealogia euro-americana (EMMETT; NYE, 2017, p. 42-44); o movimento Sem Terra (MST) como exemplo de grupo que luta pela reforma agrária (2017, p. 120); e o programa de reciclagem de Curitiba, onde os seus residentes podem trocar 1kg de lixo orgânico por vegetais frescos (2017, p. 122). É interessante mencionar que diversas uni versidades nos EUA e na Austrália possuem programas de pós-graduação em Humanidades Ambientais. O da Universidade de Nova Gales do Sul, em Sidney, por exemplo, inclui diversas áreas, como filosofia e ética ambiental, história ambien tal e política ambiental. De caráter internacional, foi fundada em 2012 a revista Environmental Hu manities, coordenada de maneira colaborativa por diversas universidades do Canadá, da Suécia, da Austrália e dos EUA – um indício do crescimento do novo campo. Em relação ao Brasil, em 2002 foi criada a Associação Nacional de Pós-Graduação e Pesquisa em Ambiente e Sociedade (Anppas), que congrega cerca de 40 centros de pesquisa em meio ambiente e sustentabilidade e promove encontros a cada dois anos (O’GORMAN et al., 2019, p. 436). A ANPPAS, desde 2011, assumiu a responsabilidade editorial da revista Ambiente & Sociedade, fundada em 1997 com o suporte administrativo do Núcleo de Estudos e Pesquisas Ambientais (Nepam). Bianca Letícia de Almeida1 id / 86 Por outro lado, é necessário que as discussões ambientais ocorram também em tons globais. É importante problematizar, por exemplo, que enquanto alguns países e cidades se autodenominam “verdes”, na verdade exportam lixo para economias em emer gência ou se valem de matérias-primas extraídas e de tecnologia produzida em locais que poluem. Esse tipo de “sustentabilidade” e crescimento é, na verdade, dentro de uma perspectiva global, insustentável (2017, p. 124-125). A obra, em contrapartida, traz diversos con ceitos que podem contribuir para pesquisas históricas em geral e não só para as que lidam com questões ambientais. São exemplos: justiça ambiental (environmental justice), ecoracismo (ecoracism), decrescimento (degrowth) e loca lização (localization). Os dois primeiros são im portantes aos estudos sobre minorias étnicas e de classe, que são altamente impactados pela degradação ambiental. No contexto americano, mas não só, grupos que dependem do ecossiste ma para sua subsistência se viram afetados pela poluição e pelos resíduos perigosos. Nas cidades, geralmente, os descartes de lixo são localizados em comunidades de afro-americanos, pobres, e outros grupos em desvantagem (2017, p. 17). O livro, assim, traz aos historiadores que se interessam pelo meio ambiente uma discussão epistemológica a respeito das mudanças nas áreas de humanidades para a compreensão do meio ambiente, o que é importante porque muitos pesquisadores de humanidades, muitas vezes, precisam se legitimar diante daqueles que não veem espaço do campo nas discussões ambientais. Outra contribuição é a atualização da agenda ambiental, ou seja, o historiador am biental pode se atualizar sobre os grandes temas que têm movimentado a discussão sobre meio ambiente. Assim, partir do presente, pode-se formular questões sobre o passado. Já degrowth remete à consciência de que o progresso e o crescimento econômico não se desvinculam da poluição e do desperdício. As sim, deve-se buscar a diminuição do que antes entendíamos como avanço, como o consumo de energia e de produtos industriais, lixo, intensidade de carbono e, até mesmo, a taxa de nascimentos. Isso não significa, necessariamente, perda na qualidade de vida, mas em uma mudança de paradigma. Finalmente, o termo localização é apresentado como oposto à globalização e uma Ademais, atualmente, cresce cada vez mais a cobrança de se explicitar o papel social de cada pesquisa científica para a sociedade contempo rânea. Ora, no que tange à História Ambiental, os resultados de suas pesquisas somados a outras áreas que estudam o meio ambiente só tendem a se fortalecer. Bianca Letícia de Almeida1 id / 86 Vinculado à Universidade Estadual de Campinas (Unicamp), o Nepam pos sui um programa de doutorado que aproxima as Por justamente se tratar de um livro introdu tório, ele é recomendado, conforme apontaram os próprios autores, para qualquer pessoa in teressada no ecocriticismo, mesmo que não esteja de alguma forma vinculada à academia, já que as humanidades ambientais lidam com o cotidiano e ações práticas, não concernentes somente para pesquisadores. Ademais, é preci so ressaltar que a crítica ambiental, que tomou folego a partir dos anos 1970, veio das ruas, isto é, de movimentos sociais (FERRI, 2017). O grande impulso foi o perigo iminente de que a vida hu mana poderia acabar, seja pelo esgotamento de recursos naturais, poluição e suas consequências, envenenamento por substâncias químicas, entre outros fatores. Desta forma, a pauta ambiental entrou em discussões na opinião pública, em produções artísticas, em movimentos sociais e na academia. Todos descrevendo e buscando meios de solucionar o problema. A respeito da origem do campo, Nye e Emmett Oficina do historiador, Porto Alegre, v. 13, n. 2, p. 1-5, jul.-dez. 2020 \| e-37845 4/5 áreas das ciências biológicas e sociais em busca da reflexão e da pesquisa ambiental. resposta ao Antropoceno. A noção é baseada em modos de viver com menos desperdícios e con sumindo produtos e alimentos locais (2017, p. 118). Dentre as mais variadas disciplinas que con tribuem com as Humanidades Ambientais, o papel da História, conforme se pode observar na obra, não está em só fornecer exemplos, mas em contribuir com análises sobre temas-chave, como industrialização e imperialismo. A partir desses momentos, acelerou-se a extração de recursos naturais, o consumo, a poluição, o cres cimento populacional, a extinção de espécies e o aquecimento global (2017, p. 2, 3). Embora Nye e Emmett tenham enfatizado esses momentos históricos, relacionado com a degradação am biental, é importante salientar que é possível rea lizar pesquisa histórica sobre qualquer momento do passado em que houve impacto humano na natureza, tenha ele degradado ou conservado (COLACIOS, 2017, p. 15). p p Conforme os autores sublinharam, todas as possíveis soluções não se aplicam para todos os lugares e precisam ser pensadas considerando contextos específicos. Isso também serve para a utilização de conceitos, até porque partes dos problemas sociais e ambientais da contempora neidade vieram a partir de violentas supressões de outros modos e saberes de vida. Bianca Letícia de Almeida e no uso dessas substâncias aliadas a dados; experiências e conhecimentos que provam que a agroecologia é possível, mesmo em dimen sões nacionais, só potencializam a luta contra a utilização desses produtos. Mestranda em História pela Universidade Federal de São Paulo (Unifesp), em Guarulhos, SP, Brasil. Endereço para correspondência Bianca Letícia de Almeida Universidade Federal de São Paulo (UNIFESP) Estrada do Caminho Velho, 333, Prédio Arco, sala 33 Jd. Nova Cidade, 07252312 Guarulhos, SP, Brasil Endereço para correspondência Bianca Letícia de Almeida Universidade Federal de São Paulo (UNIFESP) Estrada do Caminho Velho, 333, Prédio Arco, sala 33 Jd. Nova Cidade, 07252312 Guarulhos, SP, Brasil Por fim, ao terminar a obra, o leitor brasileiro entrará em contato com algumas temáticas dis cutidas em outros países e poderá se perguntar se é válido investir no campo interdisciplinar sobre questões ambientais no País, não ignorando como ele impactaria as identidades de cada disciplina que busca congregar. De todo modo, o livro expressa bem os seus argumentos e cumpre o objetivo de apresentar as principais temáticas e contribuições das Humanidades Ambientais. Bianca Letícia de Almeida1 id / 86 Um exemplo a se citar é o combate aos agrotóxicos: iniciativas que tragam questões como o histórico desses insumos, a literatura crítica a eles, a questão do poder político e econô mico envolvido na produção, na comercialização Bianca Letícia de Almeida As Humanidades Ambientais: emergência, características e sua contribuição para a historiografia brasileira 5/5 Bianca Letícia de Almeida As Humanidades Ambientais: emergência, características e sua contribuição para a historiografia brasileira 5/5 Bianca Letícia de Almeida Referências BARTOSCH, Roman. The environmental humanities. A critical introduction, Green Letters, London, v. 23, p. 1-3, mar. 2019. Disponível em: https://www.tandfonline. com/doi/full/10.1080/14688417.2019.1586150. Acesso em: 27 jul. 2020. https://doi.org/10.1080/14688417.20 19.1586150. COLACIOS, Roger Domenech. “Os meios ambientes da História Ambiental brasileira: pela abertura da cai xa-preta.” História Revista, Goiânia, v. 22, p. 6-22, 2017. https://doi.org/10.5216/hr.v22i2.47142. FERRI, Gil Karlos. “História Ambiental: historiografia comprometida com a vida”. Café História, [s. l.], p. 1-8, 04 abr. 2017. Disponível em: https://www.cafehistoria. com.br/historia-ambiental-historiografia-comprometi da-com-a-vida/#_ftn6. Acesso em: 26 jul. 2020. O’GORMAN, Emily; VAN DOOREN, Thom; MÜNSTER, Ursula; ADAMSON, Joni; MAUCH, Christof ; SÖRLIN, Sverker; ARMIERO, Marco; LINDSTRÖM, Kati; HOUSTON, Donna; PÁDUA, José Augusto; RIGBY, Kate; JONES, Owain; MOTION, Judy; MUECKE, Stephen; CHANG, Chia-Ju; LU, Shuyuan; JONES, Christopher; GREEN, Lesley; MATOSE, Frank; TWIDLE, Hedley; SCHNEIDER -MAYERSON, Matthew; WIGGIN, Bethany; JØRGENSEN, Dolly. Teaching the Environmental Humanities. Environ mental Humanities, v. 11, n. 2, p. 427-460, 2019. https:// doi.org/10.1215/22011919-7754545.
https://openalex.org/W3018455884	https://eprints.whiterose.ac.uk/161818/1/2020.01.15.907808v1.full.pdf	English	null	Reconstructing Genotypes in Private Genomic Databases from Genetic Risk Scores	Journal of computational biology	2,021	cc-by	14,940	Proceedings Paper: Proceedings Paper: Paige, Brooks, Bell, James, Bellet, Aurelien et al. (2 more authors) (2020) Reconstructing Genotypes in Private Genomic Databases from Genetic Risk Scores. In: Schwartz, Russell, (ed.) Lecture Notes in Computer Science:Research in Computational Molecular Biology: 24th Annual International Conference, RECOMB 2020, Padua, Italy, May 10–13, 2020, Proceedings. Lecture Notes in Bioinformatics (LNBI) . Springer Nature Switzerland , pp. 266-268. https://doi.org/10.1007/978-3-030-45257-5_32 https://doi.org/10.1007/978-3-030-45257-5_32 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/161818/ White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/161818/ Version: Accepted Version Version: Accepted Version This is a repository copy of Reconstructing Genotypes in Private Genomic Databases from Genetic Risk Scores. https://doi.org/10.1007/978-3-030-45257-5_32 ⋆This project was funded by the Alan Turing Institute Research Fellowship under EPSRC Research grant (TU/A/000017); EPSRC/BBSRC Innovation Fellowship (EP/S001360/1), and under the EPSRC grant EP/N510129/1. It was also partly funded by a grant from CPER Nord-Pas de Calais/FEDER DATA Advanced data science and technologies 2015-20 Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: preprint 1 The Alan Turing Institute, London, UK 4 University of Warwick, Coventry, UK 4 University of Warwick, Coventry, UK 5 Department of Biology, University of York, York, UK {daphne.ezer}@york.ac.u 5 Department of Biology, University of York, York, UK {daphne.ezer}@york.ac.uk Abstract. Some organisations like 23andMe and the UK Biobank have large genomic databases that they re-use for multiple diﬀerent genome- wide association studies (GWAS). Even research studies that compile smaller genomic databases often utilise these databases to investigate many related traits. It is common for the study to report a genetic risk score (GRS) model for each trait within the publication. Here we show that under some circumstances, these GRS models can be used to re- cover the genetic variants of individuals in these genomic databases—a reconstruction attack. In particular, if two GRS models are trained using a largely overlapping set of participants, then it is often possible to de- termine the genotype for each of the individuals who were used to train one GRS model, but not the other. We demonstrate this theoretically and experimentally by analysing the Cornell Dog Genome database. The accuracy of our reconstruction attack depends on how accurately we can estimate the rate of co-occurrence of pairs of SNPs within the private database, so if this aggregate information is ever released, it would dras- tically reduce the security of a private genomic database. Caution should be applied when using the same database for multiple analysis, especially when a small number of individuals are included or excluded from one part of the study. Keywords: Genomic privacy · Genetic risk scores · GWAS Keywords: Genomic privacy · Genetic risk scores · GWAS Reconstructing Genotypes in Private Genomic Databases from Genetic Risk Scores⋆ Brooks Paige1,2[0000−0002−4797−1563], James Bell1[0000−0003−4493−4297], Aur´elien Bellet3[0000−0003−3440−1251], Adri`a Gasc´on1,4, and Daphne Ezer1,4,5[0000−0002−1685−6909] Brooks Paige1,2[0000−0002−4797−1563], James Bell1[0000−0003−4493−4297], Aur´elien Bellet3[0000−0003−3440−1251], Adri`a Gasc´on1,4, and Daphne Ezer1,4,5[0000−0002−1685−6909] Brooks Paige1,2[0000−0002−4797−1563], James Bell1[0000−0003−4493−4297], Aur´elien Bellet3[0000−0003−3440−1251], Adri`a Gasc´on1,4, and Daphne Ezer1,4,5[0000−0002−1685−6909] 1 The Alan Turing Institute, London, UK 2 Department of Computer Science, UCL, London, UK 3 Inria, France 4 University of Warwick, Coventry, UK 1 Introduction In a survey of genomic privacy experts, the long-term privacy of genomic infor- mation was deemed both the most important and the most challenging problem . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B. Paige et al. 2 to overcome [11]. If an individual’s password or ID number gets leaked, then it is always possible to change it. However, it is impossible for a person to change their genetic code and they will pass part of it onto their children, so any in- formation leaks can have long-term impacts on both the individual and their descendants. While much of the research focus on long-term privacy of genomic databases rests on the longevity of the encryption scheme [7], it is also important to remember that these genomic databases are not just sitting on a server some- where, but are being continually utilised for making new scientiﬁc discoveries. Each time these databases are accessed and the scientiﬁc results are published, there is a risk that information will be leaked and that eventually this would enable an attacker to reconstruct private information held in the database. Genomic researchers are already aware that some forms of aggregate data from their databases should not be released publicly, because there is a risk that an attacker may be able to determine whether a particular individual is a member of the database (a membership inference attack). For instance, such attacks have already been developed for summary statistics about the frequency of single nucleotide polymorphisms (SNPs) [2, 5, 15]. Membership inference attacks have also been developed for the case where a person is allowed to repeatedly query a database to learn if at least one individual contains a particular SNP [13, 14, 16]. These kinds of aggregate statistics about the frequency or presence/absence of a particular SNP might be useful to release to the broader research community, but it is not an essential output of the research process. However, the main research ﬁndings — i.e. the SNPs associated with the trait of interest and their strength of association — are essential to publish since the entire purpose of these genomic research projects is to uncover the relationship between genetic variants and phenotypic traits. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 1 Introduction Moreover, knowledge of these SNPs can lead to new diagnosis procedures or new potential drug targets, so their release is important for the public interest [18]. Yet, even this information can potentially leak private information about individuals in the database. For instance, [8] found that information about individuals in a genomic database is leaked when studies publish whether each SNP is correlated or anti-correlated to the trait of interest. It is important to quantify how much information is leaked by publishing these research ﬁndings, so that scientists can make informed decisions about when to publish their results and whether it is worth risking the privacy of the participants. In this manuscript, we demonstrate that the kind of research output that is published from genome-wide association studies (GWAS) has the potential to leak enough information to recover the SNPs of individuals in the database (a reconstruction attack), under speciﬁc circumstances. In particular, we focus on the release of Genetic Risk Scores (GRS), a common research output for ﬁnding genetic associations with continuous traits [1, 3, 4, 10, 12, 20]. We also focus on cases where a database is repeatedly used to perform a GWAS analysis, but not all the individuals are part of all the analyses. This could be the case because some individuals drop out of the study or skip speciﬁc survey questions. Al- ternatively, some databases, such as 23andMe, may grow in size over time and . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint Reconstruction attacks from GRS 3 Fig. 1. We investigate the case where two GWAS stud- ies are performed on two data sets that mostly con- tain the same individuals. 1 Introduction We reconstruct the genotype of those individuals added to the second study, using the GRS from each study and an estimate of SNP frequencies. Private data set 1 + Private data set 2 Public data set Publicly known: Genetic Risk Scores Genetic Risk Scores Frequencies of SNPs and frequencies of pairs of SNPs co-occurring in the same individual Attacker reconstructs genotype of additional individuals in data set 2 GWAS GWAS ( "#!) ( "#!"#) (%) (&#) Private data set 1 + Private data set 2 Public data set Publicly known: Genetic Risk Scores Genetic Risk Scores Frequencies of SNPs and frequencies of pairs of SNPs co-occurring in the same individual Attacker reconstructs genotype of additional individuals in data set 2 GWAS GWAS ( "#!) ( "#!"#) (%) (&#) Fig. 1. We investigate the case where two GWAS stud- ies are performed on two data sets that mostly con- tain the same individuals. We reconstruct the genotype of those individuals added to the second study, using the GRS from each study and an estimate of SNP frequencies. Fig. 1. We investigate the case where two GWAS stud- ies are performed on two data sets that mostly con- tain the same individuals. We reconstruct the genotype of those individuals added to the second study, using the GRS from each study and an estimate of SNP frequencies. allow several GWAS to be performed within a short period of time. Under these circumstances, we demonstrate that it is possible to completely reconstruct the SNPs of an individual using a custom Expectation-Maximisation (EM) algo- rithm. We also provide suggestions for avoiding this kind of attack. To be clear, this manuscript focuses on the simpler case in which the exact same trait is investigated in multiple GWAS studies; however, we expect that some version of this attack may be developed in the near future for the case of multiple highly correlated traits. B. Paige et al. B. Paige et al. 4 We also brieﬂy discuss how loosening additional restrictions would impact our ability to predict individual genotypes. In particular, we analyse the case where the two sets of SNPs that are used by the two studies are not identical. These results imply that if two sets of GRS are released on two genetic data sets with largely overlapping populations, it may be possible to reconstruct the genotypes of those individuals who participated in one study but not the other (Figure 1). 2 Methods Genetic risk score (GRS) models describe the relationship between a particular phenotype of interest and particular SNPs. These models are ﬁt in a two-stage process: ﬁrst, a reduced set of SNPs is selected from a potentially very large pool of candidates; then, this reduced set is used as the independent variables in a linear regression analysis. The set of SNPs is selected by ﬁrst ﬁltering for those that signiﬁcantly correlate to the trait of interest, after controlling for other covariates. These SNPs are then further ﬁltered to ensure that they are far apart from one another, in order to decrease the correlation between them. In this setting, we suppose M individuals have taken part in a study, and N SNPs have passed the ﬁltration steps to be used in a linear model. Let yM be the vector of M real-valued phenotypes, and XM be an M × N binary matrix, where XM[i, j] = 1 if individual i has SNP j. To include an intercept term in the linear model, we deﬁne a design matrix ΦM to be the M × (N + 1) matrix ΦM = XM 1M . (1) (1) The GRS model parameter βM is just the coeﬃcient vector of the linear model The GRS model parameter βM is just the coeﬃcient vector of the linear model yM = ΦMβM + ǫ, (2) (2) where ǫ is independent Gaussian noise. Given ΦM and phenotypes yM, the max- imum likelihood estimate of this parameter has a closed form where ǫ is independent Gaussian noise. Given ΦM and phenotypes yM, the max- imum likelihood estimate of this parameter has a closed form ˆβM ≜1 M K−1(Φ⊤ MyM), (3) (3) where we have deﬁned the symmetric (N + 1) × (N + 1) matrix K as where we have deﬁned the symmetric (N + 1) × (N + 1) matrix K as K = 1 M Φ⊤ MΦM. (4) (4) Now, suppose a second study is run, targeting the same phenotype, which adds a single extra individual with SNPs represented by the N length vector x0. This corresponds to adding the row φ⊤ 0 = [x⊤ 0 1] to the design matrix, and extending y with the additional phenotypic value y0 for the new individual. The updated estimator (i.e. 1.1 Overview of scenarios that will be investigated We demonstrate a series of reconstruction attacks that enable us to infer the genotypes of individuals in private genomic databases, based on publicly released GRS. These attacks will initially be deployed on a very favorable scenario, but the scope of the attack will be subsequently expanded, building up to the scenario shown in Figure 1. It is worth noting that the reconstruction attacks that we will describe do not depend on (i) how the SNPs were initially ﬁltered or (ii) how strongly they associate with the trait of interest. We will begin by investigating a simple scenario: two GWAS studies are performed to identify SNPs associated with the same trait, and the two studies use the same set of participants, except that the second study includes one extra individual. In addition, we will assume that we know the frequencies of each SNP and the frequencies that pairs of SNPs co-occur in the same individual. We assume that both studies publish the coeﬃcients associated with the GRS models that they infer as part of the analysis. Next, we will consider the case in which the second study includes more than one additional participant and demonstrate that in many circumstances this still allows us to easily reconstruct the individual genotypes of all the individuals that are found in the second study but not the ﬁrst (see Section 3.2). Afterwards, we will demonstrate that we do not need to know the precise frequencies of SNPs and frequencies of co-occurring SNPs, as long as we have a reasonable estimate of these values from public databases (see Section 3.3). . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B. Paige et al. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint – For i = 1, . . . , N: Kii estimates the probability that SNP i has value 1 (i.e. the frequency of the SNP in the population). 2 Methods the GRS values for the second study) is given by ˆβM+1 = (Φ⊤ MΦM + φ0φ⊤ 0 )−1(Φ⊤ MyM + y0φ0). (5) (5) . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 5 Reconstruction attacks from GRS We assume that both GRS models ˆβM and ˆβM+1 are released publicly. An attacker aims to use this knowledge to reconstruct φ0 (the genotype of the added individual). Through algebraic re-arrangement we ﬁnd that: φ0 = 1 C K(ˆβM+1 −ˆβM) (6) (6) where C is a scalar, speciﬁcally C = 1 M (y0 −φ⊤ 0 ˆβM+1). This means that φ0 is a scalar multiple of K(ˆβM+1 −ˆβM). where C is a scalar, speciﬁcally C = 1 M (y0 −φ⊤ 0 ˆβM+1). This means that φ0 is a scalar multiple of K(ˆβM+1 −ˆβM). Our approach thus centers on the use of the vector we deﬁne as d1, Our approach thus centers on the use of the vector we deﬁne as d1, d1 ≜K(ˆβM+1 −ˆβM) = Cφ0, (7) (7) corresponding to a rescaled copy of the input SNP data in the design matrix φ0, which can be easily computed from the two parameter vectors if the matrix K is known. As we will see in Section 3.1, we can use d1 to exactly reconstruct the added individual with 100% accuracy. We additionally consider the case where m additional individuals have been included in the second study, yielding a new GRS model ˆβM+m including these M + m participants. The extra rows of the design matrix now form a matrix Φm of size m × (N + 1), where each row is an individual that was added to the second study and each column is a SNP (and the last column contains only 1). The corresponding analog to Eq. (7) for multiple individuals, which we derive in Appendix B, is dm ≜K(ˆβM+m −ˆβM) = Φ⊤ mCm, (8) (8) where Cm is a vector of length m. 2 Methods For suﬃciently small m (relative to N), exact reconstruction of all m added individual genomes is also possible in this setting, following the algorithm we will introduce in Section 3.2. The previous examples have focused on cases in which the participants in the ﬁrst study are a subset of the individuals in the second study. In Appendix C we consider the case in which the ﬁrst study has some participants that are not found in the second study and vice versa. We show that the same strategies for reconstructing the genome can be used as in the previous scenario that we discussed, in which multiple participants are added to the second study. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 2.1 Estimation of K As it turns out, the entries of matrix K correspond to simple population-level statistics of the SNPs, which could either be inadvertently released (under the assumption they would be safe to share), or could be estimated from another sample from the same population. In fact, the entries of K depend only on the SNP frequencies and SNP co-occurrence frequencies in the dataset: . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B. Paige et al. 6 – For i = 1, . . . , N −1 and j > i: Kij = Kji estimates the probability that SNP i and SNP j are both 1 simultaneously (i.e. the frequency of SNP i and SNP j co-occurring in the same individual). – For i = 1, . . . , N −1 and j > i: Kij = Kji estimates the probability that SNP i and SNP j are both 1 simultaneously (i.e. the frequency of SNP i and SNP j co-occurring in the same individual). – For i = 1, . . . , N −1 and j > i: Kij = Kji estimates the probability that SNP i and SNP j are both 1 simultaneously (i.e. the frequency of SNP i and SNP j co-occurring in the same individual). – For i = 1, . . . , N and j = N + 1: Kij = Kji also estimates probability that SNP i has value 1, i.e. Ki,N+1 = KN+1,i = Kii. – For i = 1, . . . , N and j = N + 1: Kij = Kji also estimates probability that SNP i has value 1, i.e. Ki,N+1 = KN+1,i = Kii. – Finally, KN+1,N+1 = 1. Thus, knowledge of SNP frequencies and pairwise co-frequencies from the orig- inal study are all that is required in order to compute K. In the following Sec- tions 3.1 and 3.2, we consider adding one and multiple individuals at once, respectively, in the setting where this matrix K can be estimated exactly. However, while ˆβM, ˆβM+1 and M are likely to be published along with the study, an attacker would often need to estimate K from other publicly available data. 3 Results The key observation from the previous section is that the vectors d1 and dm, derived from the change in parameter vectors ˆβ from a ﬁrst study to a second study, take only a ﬁnite number of values thanks to the fact that the design matrices Φ contain only zeros and ones. In particular, when m new individuals are added to the second study, each entry of the vector dm can only take at most 2m values, and a zero value corresponds to the setting where all individuals have the most common variant for that SNP. This section describes algorithmically how these vectors can be used to re- cover the genomes of the additional individuals, as well as empirical tests which use the Cornell Dog Genome dataset as a case study [6]. More details on the experimental setup can be found in Appendix A. 2.1 Estimation of K Most studies will report some information about the study population (such as whether the study focused on individuals from a speciﬁc continent), which can help with estimating K. From this information, we can estimate the value of K in similar populations as those used in the study using publicly available data, e.g. from the HapMap project. Our additional experiments in Section 3.3 use a custom EM algorithm to ﬁnd maximum likelihood estimates of φ0 when the matrix ˆK ≈K is estimated from independent public data. The derivation of this EM algorithm is given in Appendix D.3, and a formal analysis of the reconstruction error of φ0 given the error in ˆK is found in Appendix D.1. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 3.1 Complete reconstruction of one individual’s genotype when SNP frequency information is known For now, we also assume we are in the setting where the matrix K is known, e.g. because the SNP frequency information has been publicly released. ˆ ˆ Given K, ˆβM+1 and ˆβM, we can use d1 (a vector of length N +1) to precisely determine the genotype of the individual who was added to the database. For each i = 1, . . . , N, the ith entry of d1 is either equal to 0 if φ0 contains a 0 (i.e. the individual does not have the SNP at that index) or to C if φ0 contains a 1 (i.e. the individual has the SNP at that index). In other words, it is possible to exactly read oﬀthe SNPs of the added individual in this setting. Indeed, we tested this strategy on the Cornell Dog Database and found that we were able to reconstruct the genotype of the dog that was added to the second study with 100% accuracy, both on common and uncommon SNPs (see Figure 2(A)). 3.1 Complete reconstruction of one individual’s genotype when SNP frequency information is known The ﬁrst, most straightforward case is when only one participant is added be- tween the ﬁrst and second studies, i.e. where ˆβM is the GRS for the ﬁrst study (containing M participants), and ˆβM+1 is the GRS for the second study as de- scribed in Eq. (3) and (5). Both of these are vectors of length N + 1, where the ﬁrst N indices correspond to the relationship between each SNP and the . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint Reconstruction attacks from GRS 7 0 0 0 0 1 0 0 A 0 1 0 B 0 0 1 C 1 1 0 A+B 1 0 1 A+C 0 1 1 B+C 1 1 1 A+B+C !( #$!"# −#$!)[(] Possible combination of SNPs at index i A B !( #$!"# −#$!)[(] C Fig. 2. (A) We have perfect accuracy in reconstructing the genotype when K is known (using 200 random SNPs to estimate average breed weight in the Cornell Dog Database). (B) We can reconstruct all the genotypes of multiple dogs that are added to the second study and (C) this works in practice using the data from the Cornell Dog Database, as in (A). 0 0 0 0 1 0 0 A 0 1 0 B 0 0 1 C 1 1 0 A+B 1 0 1 A+C 0 1 1 B+C 1 1 1 A+B+C !( #$!"# −#$!)[(] Possible combination of SNPs at index i B !( #$!"# −#$!)[(] C Fig. 2. (A) We have perfect accuracy in reconstructing the genotype when K is known (using 200 random SNPs to estimate average breed weight in the Cornell Dog Database). (B) We can reconstruct all the genotypes of multiple dogs that are added to the second study and (C) this works in practice using the data from the Cornell Dog Database, as in (A). trait and the last element is the intercept of the linear model. 3.3 Accurate estimation of an individual’s genotype when SNP frequency information is estimated from a public database Previously, we assumed that the attacker had access to the matrix K, which consists of population-level statistics on frequencies and co-occurrence frequencies of SNPs. While this could be released volun- tarily by organisations which are not aware of the risk, we now consider the case where K is not directly available to the attacker but is instead es- timated from a separate pub- lic database assumed to corre- spond to individuals from the same population. 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 true x0 0.0004 0.0002 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 Value of d0 ^ 000 001 010 011 100 101 110 111 True SNP for three additional dogs 0.004 0.003 0.002 0.001 0.000 0.001 0.002 0.003 0.004 Value of dm Fig. 3. Example values taken by the noisy vector ˆd, given the true value of the corresponding SNP in the genome. (Left) adding one new participant; (right) adding three new participants. These ﬁgures are analogous to those in Figure 2, albeit in the case where K is not known and instead estimated from an independent public database. 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 true x0 0.0004 0.0002 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 Value of d0 ^ 000 001 010 011 100 101 110 111 True SNP for three additional dogs 0.004 0.003 0.002 0.001 0.000 0.001 0.002 0.003 0.004 Value of dm 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 true x0 0.0004 0.0002 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 Value of d0 ^ 000 001 010 011 100 101 110 111 True SNP for three additional dogs 0.004 0.003 0.002 0.001 0.000 0.001 0.002 0.003 0.004 Value of dm Fig. 3. Example values taken by the noisy vector ˆd, given the true value of the corresponding SNP in the genome. (Left) adding one new participant; (right) adding three new participants. These ﬁgures are analogous to those in Figure 2, albeit in the case where K is not known and instead estimated from an independent public database. We simulated this scenario using the Cornell Dog Database by taking one random set of dogs for building the GRS model, and a second non-overlapping set of dogs for estimating ˆK. We compared the value of ˆd1 = ˆK(ˆβM+1 −ˆβM) with the known value of φ0. 3.2 Complete reconstruction of multiple individuals’ genotype when SNP frequency information is known Each value in Cm corresponds to a speciﬁc individual who was added to the second study. Each value in dm can be described as a sum of a unique combination of values in Cm. For instance, if dm[i] = Cm[j] + Cm[k], this means that the SNP at position i is found in individual j and k, but no one else. We tested this approach using the Cornell Dog Database, in a test scenario where the second study added three diﬀerent dogs. We were able to uniquely identify the genotypes of all three dogs with 100% accuracy, both with common and uncommon SNPs (Figure 2(C)). . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 3.2 Complete reconstruction of multiple individuals’ genotype when SNP frequency information is known We now consider the case where m additional individuals have been included in the second study, yielding a new GRS model ˆβM+m including these M + m participants. Consider again Eq. (8) above. The ith row of Φm is a binary vector that represents the combination of the m individuals who have SNP i. This means that, for a ﬁxed value of Cm, the value of the vector dm at index i is uniquely determined by the combination of individuals who have SNP i (Figure 2(B)). In other words, there will be at most 2m unique values taken by entries of dm, each corresponding to a combination of the values in vector Cm (see Figure 2(C)). If we were to learn which values of dm are also found in Cm, then we could infer the complete genotypes of all the m individuals added to the second study. We would be able to reconstruct m complete genotype vectors, although it would be impossible to know which of the genotypes corresponded to which of the m individuals. In fact, in many cases it is extremely straightforward to determine . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: preprint B. Paige et al. 8 which values in dm correspond to values in Cm. Here we describe a simple algo- rithm for ﬁnding Cm when there are exactly 2m unique values in dm. If this is not the case, please see the more complete algorithm in Section G. 1. First, extract all unique, non-zero values from dm. 2. Find the sum of all pairs of values in this vector. 3. Find all values that are in (1), but not in (2). The values of Cm appear in this list. There is no way to know which value of Cm corresponds to which index, so for simplicity we can randomly assign them indices. 4. Each value in Cm corresponds to a speciﬁc individual who was added to the second study. Each value in dm can be described as a sum of a unique combination of values in Cm. For instance, if dm[i] = Cm[j] + Cm[k], this means that the SNP at position i is found in individual j and k, but no one else. 4. 3.3 Accurate estimation of an individual’s genotype when SNP frequency information is estimated from a public database baseline estimated 0.0 0.2 0.4 0.6 0.8 1.0 Public set: 400 dogs baseline estimated 0.0 0.2 0.4 0.6 0.8 1.0 Public set: 200 dogs baseline estimated 0.0 0.2 0.4 0.6 0.8 1.0 Public set: 800 dogs Accuracy baseline estimated 0.0 baseline estimated 0.0 baseline estimated 0.0 Fig. 4. Accuracy at reconstruction of genomes x0 using EM estimation and a noisy estimate ˆK, as compared to a natural baseline which always predicts the most common variant at each SNP locus. We use this as a baseline, because without any additional information about βM and βM+1, the most accurate prediction of the dog’s genotype would be to predict the most common variant at each locus. Here we deﬁne accuracy as the proportion of SNPs that are correctly identiﬁed in the dog that was found in the second GWAS study, but not the ﬁrst. Each distribution is constructed from 500 experimental test points, in which we (i) took 10 random splits of the full dog data set, assigning dogs to either the public and private data set (ii) for each split, we tested the reconstruction 50 times, each time adding a diﬀerent randomly sampled dog to the second GWAS study. The private dataset always has 1000 individuals; the public test dataset is of increasing size, improving performance. Fig. 4. Accuracy at reconstruction of genomes x0 using EM estimation and a noisy estimate ˆK, as compared to a natural baseline which always predicts the most common variant at each SNP locus. We use this as a baseline, because without any additional information about βM and βM+1, the most accurate prediction of the dog’s genotype would be to predict the most common variant at each locus. Here we deﬁne accuracy as the proportion of SNPs that are correctly identiﬁed in the dog that was found in the second GWAS study, but not the ﬁrst. Each distribution is constructed from 500 experimental test points, in which we (i) took 10 random splits of the full dog data set, assigning dogs to either the public and private data set (ii) for each split, we tested the reconstruction 50 times, each time adding a diﬀerent randomly sampled dog to the second GWAS study. The private dataset always has 1000 individuals; the public test dataset is of increasing size, improving performance. 3.3 Accurate estimation of an individual’s genotype when SNP frequency information is estimated from a public database We observe that ˆd1 has signiﬁcantly diﬀerent values at indices where φ0[i] = 0 and φ0[i] = 1; examples for the cases where one and three dogs are added can be seen in Figure 3. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint Reconstruction attacks from GRS 9 baseline estimated 0.0 0.2 0.4 0.6 0.8 1.0 Public set: 400 dogs baseline estimated 0.0 0.2 0.4 0.6 0.8 1.0 Public set: 200 dogs baseline estimated 0.0 0.2 0.4 0.6 0.8 1.0 Public set: 800 dogs Accuracy Fig. 4. Accuracy at reconstruction of genomes x0 using EM estimation and a noisy estimate ˆK, as compared to a natural baseline which always predicts the most common variant at each SNP locus. We use this as a baseline, because without any additional information about βM and βM+1, the most accurate prediction of the dog’s genotype would be to predict the most common variant at each locus. Here we deﬁne accuracy as the proportion of SNPs that are correctly identiﬁed in the dog that was found in the second GWAS study, but not the ﬁrst. Each distribution is constructed from 500 experimental test points, in which we (i) took 10 random splits of the full dog data set, assigning dogs to either the public and private data set (ii) for each split, we tested the reconstruction 50 times, each time adding a diﬀerent randomly sampled dog to the second GWAS study. The private dataset always has 1000 individuals; the public test dataset is of increasing size, improving performance. 3.3 Accurate estimation of an individual’s genotype when SNP frequency information is estimated from a public database 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 atypicality 0.0 0.2 0.4 0.6 0.8 1.0 accuracy Public set: 200 dogs baseline estimated 0.34 0.36 0.38 0.40 0.42 0.44 0.46 atypicality 0.0 0.2 0.4 0.6 0.8 1.0 accuracy Public set: 400 dogs baseline estimated 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 atypicality 0.0 0.2 0.4 0.6 0.8 1.0 accuracy Public set: 800 dogs baseline estimated Fig. 5. Results of Figure 4 broken down by individual dogs. Here each point represents a dog and we deﬁne atypicality as the proportion of uncommon variants that the dog has compared to the public database– for instance, if 51% or more of dogs in the public database have a G in a speciﬁc locus, but this dog has a T, then this would count towards the dog’s atypicality. In other words, dogs further to the right are less and less similar to average dog present in the public dataset (measured by percentage of diﬀerent variants). In contrast to the most-common-variant baseline, our method generalizes well even to dogs which are highly dissimilar to those in the public dataset. Larger public databases (right) provide more accurate population estimates ˆK, leading to more accurate reconstructions overall. B. Paige et al. 10 Fig. 5. Results of Figure 4 broken down by individual dogs. Here each point represents a dog and we deﬁne atypicality as the proportion of uncommon variants that the dog has compared to the public database– for instance, if 51% or more of dogs in the public database have a G in a speciﬁc locus, but this dog has a T, then this would count towards the dog’s atypicality. In other words, dogs further to the right are less and less similar to average dog present in the public dataset (measured by percentage of diﬀerent variants). In contrast to the most-common-variant baseline, our method generalizes well even to dogs which are highly dissimilar to those in the public dataset. Larger public databases (right) provide more accurate population estimates ˆK, leading to more accurate reconstructions overall. 3.3 Accurate estimation of an individual’s genotype when SNP frequency information is estimated from a public database The main challenge is that the vector ˆd1 now includes additional noise, so we cannot simply use its entry at index N + 1 to estimate C, nor do the entries i with φ0[i] = 0 also correspond directly to ˆd1[i] = 0. Instead, we develop a custom expectation-maximisation algorithm to ﬁnd a maximum likelihood estimate of the constant C and recover φ0, i.e. to determine the probability that each φ0[i] = 0 or φ0[i] = 1, based on the value of ˆd1 (see Section D.3 for details). We ﬁnd that this method can successfully reconstruct the correct value of φ0[i] much better than a baseline which uses the public dataset to independently estimate the most common variant for each SNPs (see Figure 4). Crucially, we show that our approach is able to reconstruct, with relatively high accuracy, the genotypes of dogs even when they diﬀer signiﬁcantly from those in the public dataset (see Figure 5). This shows that our attack is able to extract information about the particular individuals that diﬀer across the two studies, not merely about the general population as in the most-common-variant baseline. By deﬁnition, dogs that have genotypes that diﬀer signiﬁcantly from the general population have a higher proportion of uncommon SNPs, and the ability to recover these uncommon SNPs is particularly important from a privacy perspective. Indeed, uncommon SNPs can be used to identify a particular individual and are also more likely to be associated with disease phenotypes, which is sensitive information. In general, we ﬁnd that the larger the public dataset available, and the more similar the dataset is to the unknown private dataset, the better we are able to reconstruct the genome of the added individual. Full details and description of the experimental setting are given in Section A. We also derive theoretical error bounds for our estimate of φ0 based on the error in ˆK in Section D.1. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 10 B. Paige et al. 3.4 Accurate estimation of an individuals’ genotype when diﬀerent SNPs are used in each study When GRS models are constructed, the ﬁrst step is to ﬁlter the set of SNPs down to a small set of SNPs that are (i) signiﬁcantly correlated to the trait after covariates are considered and (ii) far apart from one another along the genome. If the two studies use two diﬀerent sets of SNPs to construct the GRS model, it is still possible to recover whether or not each of the SNPs in the overlap is present in the new individual. This process is highly analogous to the previous cases and is detailed in Appendix F. 4 Discussion In this manuscript, we demonstrate that private information is leaked when GRS models are published, speciﬁcally in the case where two sets of largely overlap- ping individuals are used for multiple studies. In particular, we show that we can recover SNPs from an individual in a private database—a reconstruction attack. Even though we would not have a name associated with this genotype, it may be possible to identify the individual once the genotypic data is available to the attacker. For instance, the attacker may have access to partial genotypic infor- mation of the individual and then be able to identify them. Alternatively, they could use the genotype information to predict ethnicity and other phenotypic traits that could then be used to uniquely identify the individual. We also note that even an incomplete reconstruction attack (in which only a proportion of the SNPs are correctly identiﬁed) is likely to be suﬃcient to perform a member- ship inference attack. Investigating the relationship between the reconstruction . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint Reconstruction attacks from GRS 11 attack and the membership attack will be a subject of future research. Impor- tantly, if the attackers were unable to link the genomic data with a particular individual, the reconstruction attack would still be a breach in privacy that could have serious consequences. For instance, the patient may have only consented to have their genomic data used in particular kinds of research studies, while the attacker may use the reconstructed genomic data for a diﬀerent (potentially unethical) purpose. Suggestions for good practice. We provide a number of simple suggestions for good practice that would help limit this attack. 1. 4 Discussion Aggregate statistics about the frequency of SNPs in the database or the fre- quency of co-occurrence of SNPs should never be released. We have shown that this information, combined with GRS, allows to precisely reconstruct individual genomes in various settings. It may be possible to release noisy versions of SNP frequency data, but this would be equivalent to releasing ˆK (our estimated K from the public database). With our EM algorithm, we have demonstrated that it is still possible to do some genotypic reconstruc- tion with a noisy ˆK, but this becomes harder as the noise in ˆK increases. However, providing a very noisy ˆK may be of limited utility to the scientiﬁc community. 2. If a genetic data set is intended to serve for multiple complementary anal- yses, it is important that all study participants are used in every analysis performed. If there is missing phenotypic data from a few individuals, they should not be included in any of the analyses performed, or their privacy may be compromised. 3. When multiple individuals are added in between two studies, then the ability to reconstruct the genomes depends on the number of SNPs being large relative to the number of individuals. In particular, if m new dogs are added, exact reconstruction is only possible using the approach in Section 3.2 if the number of SNPs N > 2m. Thus, we suggest to avoid releasing multiple studies which diﬀer by fewer than log2 N individuals. Extensions and future work. While we have analyzed the case where the genome is represented by binary values of 0 or 1, often studies instead count the number of times each allele is present, which would lead to a design matrix Φ containing values 0, 1, or 2. In this scenario, K no longer contains the frequencies of SNPs and their co-occurrences, but something slightly more complicated that we de- scribe in Appendix H. This does not dramatically change the approach in this paper, except in that the vector dm can take 3m possible values, rather than 2m. In practice, then, studies which use allele counts are somewhat more robust to attacks; the multiple dog reconstruction attack would likely be ambiguous if 3m > N, rather than 2m > N. A possible countermeasure to our reconstruction attack could consist in ran- domly perturbing the GRS models before releasing them, as done in diﬀerentially private linear regression [19]. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 4 Discussion However, a naive application of this strategy could . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B. Paige et al. 12 destroy the utility of the models. A formal and empirical analysis of the eﬀective- ness of such protection against reconstruction attacks, as well as of the usefulness of the resulting GRS models to genomic researchers, is beyond the scope of this paper and left for future work. Another countermeasure could consist in refraining from releasing precise information about the population structure of the study population to prevent the attacker from estimating K eﬀectively. This would however limit the utility of the research study, because the researchers would not know to what popula- tions the research applies to. An investigation of how diﬀerences in population structures impact Kest will be undertaken in public human genomic data sets in future work, but is also beyond the scope of this paper. Our work has a number of limitations. For instance, we only test our EM algorithm on dog data. Dog populations may have diﬀerent population structures than human populations due to selective breeding, so in the future we aim to test how properties of population structure will impact our ability to estimate K and the accuracy of our reconstruction attack. In addition, the expectation- maximisation algorithm described here is only explicitly described for the case in which one participant is added to the database, and the matrix K is estimated from a public database. We are currently extending the algorithm to the broader case in which multiple participants are added at once. It may seem on the surface unlikely that two GWAS analyses will include nearly the same participants. One potentially common setting where this could arise is when a single study collects both genotype and phenotype data from a single set of participants, and releases multiple models to predict multiple traits. 4 Discussion In this case, there may be a small number of individuals who are used in one analysis, but not the other; for instance, there may be a small subset of participants who skip a particular survey question that was used to collect phenotype information, and this is indeed evident in a recent study ([9]). In such settings, it could be very possible for multiple released GRS models to be computed on sets of individuals which diﬀer by only a few participants. In future work, we aim to extend our analysis and attack to settings where multiple GRS models are released, each predicting diﬀerent but highly correlated traits. // / / 2. Cai, R., Hao, Z., Winslett, M., Xiao, X., Yang, Y., Zhang, Z., Zhou, S.: Determin- istic identiﬁcation of speciﬁc individuals from GWAS results. In: Bioinformatics (2015). https://doi.org/10.1093/bioinformatics/btv018 . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 3. Chouraki, V., Reitz, C., Maury, F., Bis, J.C., Bellenguez, C., Yu, L., Jakobsdottir, J., Mukherjee, S., Adams, H.H., Choi, S.H., Larson, E.B., Fitzpatrick, A., Uitter- linden, A.G., De Jager, P.L., Hofman, A., Gudnason, V., Vardarajan, B., Ibrahim- Verbaas, C., Van Der Lee, S.J., Lopez, O., Dartigues, J.F., Berr, C., Amouyel, P., References 1. Belsky, D.W., Moﬃtt, T.E., Sugden, K., Williams, B., Houts, R., McCarthy, J., Caspi, A.: Development and evaluation of a genetic risk score for obesity. Biodemography and Social Biology (2013). https://doi.org/10.1080/19485565.2013.774628 2. Cai, R., Hao, Z., Winslett, M., Xiao, X., Yang, Y., Zhang, Z., Zhou, S.: Determin- istic identiﬁcation of speciﬁc individuals from GWAS results. In: Bioinformatics (2015). https://doi.org/10.1093/bioinformatics/btv018 3. Chouraki, V., Reitz, C., Maury, F., Bis, J.C., Bellenguez, C., Yu, L., Jakobsdottir, J., Mukherjee, S., Adams, H.H., Choi, S.H., Larson, E.B., Fitzpatrick, A., Uitter- linden, A.G., De Jager, P.L., Hofman, A., Gudnason, V., Vardarajan, B., Ibrahim- Verbaas, C., Van Der Lee, S.J., Lopez, O., Dartigues, J.F., Berr, C., Amouyel, P., . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 13 Reconstruction attacks from GRS Bennett, D.A., Van Duijn, C., Destefano, A.L., Launer, L.J., Ikram, M.A., Crane, P.K., Lambert, J.C., Mayeux, R., Seshadri, S.: Evaluation of a Genetic Risk Score to Improve Risk Prediction for Alzheimer’s Disease. Journal of Alzheimer’s Disease (2016). https://doi.org/10.3233/JAD-150749 Bennett, D.A., Van Duijn, C., Destefano, A.L., Launer, L.J., Ikram, M.A., Crane, P.K., Lambert, J.C., Mayeux, R., Seshadri, S.: Evaluation of a Genetic Risk Score to Improve Risk Prediction for Alzheimer’s Disease. Journal of Alzheimer’s Disease (2016). https://doi.org/10.3233/JAD-150749 ( ) p // g/ / 4. 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It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 15 Reconstruction attacks from GRS A Experimental details Cornell Dog Database: To experimentally test the reconstruction attacks, we used data from the Cornell Dog Genome Database, which contains data about SNPs from a wide range of dog breeds and a number of associated phenotypic traits. The two traits we focused on were average breed weight and average breed height, because these two phenotypes had the fewest number of missing values. For the initial investigation, we binarised the genotype matrix—considering all heterogenous alleles to have a value of 1. (We also repeated the analysis with the original genotype matrix.) Only common SNPs (i.e. SNPs that were found in 25% to 75% of the dogs) were used, leaving 23,497 SNPs. For each linear model built, M = 1000 dogs were randomly sampled as the “private” dataset and N = 200 SNPs were randomly selected. To ensure that the SNPs that were sampled were spatially distributed, the SNPs were randomly sampled in a stratiﬁed way, so one SNP was selected in every 23,497 200 -sized bin. Experiment with imprecise K: First, two linear models were constructed to predict average breed weights: one with the M = 1000 randomly sampled dogs and another that contained 1 additional randomly sampled dog. This gives ˆβM and ˆβM+1. To mimic the process of estimating K from a public database, we randomly sampled an additional 200, 400, or 800 dogs that were not included as part of the original set and used this to estimate K, which we refer to as ˆK. Now we could calculate ˆK(ˆβM+1 −ˆβM) and compare this to the known φ0 for the additional dog from the second study. These additional dogs are taken from a third “test” dataset, disjoint from both the public and private data. The plots in Figures 4 and 5 are produced by re-running the algorithms across 10 random public / private / test splits, where the “test” dataset has 50 dogs which are each individually considered as candidates for the (M + 1)th dog added to the private dataset. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B Adding multiple dogs Here we explain Equations (7) and (8). Note that the former is a special case of the latter so we will only explain the latter in detail. First note that by deﬁnition ˆβM = (Φ⊤ MΦM)−1Φ⊤ MyM = (MKM)−1ΦT MyM, ˆβM+m = (Φ⊤ M+mΦM+m)−1Φ⊤ M+myM+m = (MKM + mKm)−1Φ⊤ M+myM+m. Substituting these into the left hand side of the following equation gives the right hand side: Substituting these into the left hand side of the following equation gives the right hand side: (MKM + mKm)ˆβM+m −MKM ˆβM = ΦT mym. (9) can be rearranged to give (MKM + mKm)ˆβM+m −MKM ˆβM = ΦT mym. (9) (9) This equation can be rearranged to give KM(ˆβM+m −ˆβM) = 1 M ΦT mym −m M Km ˆβM+m = 1 M ΦT m(ym −Φm ˆβM+m). . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B. Paige et al. 16 Deﬁning the length m vector Cm = 1 M (ym −Φm ˆβM+m) yields the form used in Equation (8). For the special case of m = 1, Cm is a scalar and we recover Eq. (7). C Case in which each GWAS study adds two new sets of participants This manuscript mostly explores the case in which one study’s participants are a subset of the other study’s participants. Here we demonstrate that this is equivalent to the case where each of the two studies contain a small number of participants that are not found in the other study. In particular, let us say that the ﬁrst study has M + a participants and the second study has M + b participants, where the ﬁrst M participants are shared between the studies, but there are a participants that are found in the ﬁrst study but not the second, and b participants that are found in the second study but not the ﬁrst. Following on from Equation 9, we see that: KM(ˆβM+a −ˆβM+b) = KM(ˆβM+a −ˆβM) −KM(ˆβM+b −ˆβM) = 1 M h ΦT a (ya −Φa ˆβM+a) −ΦT b (yb −Φb ˆβM+b) i . Let us deﬁne the following (N +1)×(a+b) matrix obtained by concatenating the two genotype matrices: Φa+b = [Φa, Φb] (10) (10) and the following a + b length vector: ra+b = h (ya −Φa ˆβM+a), −(yb −Φb ˆβM+b) i (11) (11) Then this gives us: KM(ˆβM+a −ˆβM+b) = 1 M Φa+bra+b (12) (12) This means that having two non-overlapping participant sets is equivalent to the setting in which the ﬁrst study is a subset of the second (only m is now a + b). . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint D Estimating K If the true matrix K is unknown, it can be estimated with public data. We denote this estimator by ˆK. In order for ˆK to be an accurate estimate the data that it is generated from must be drawn from the same (or a suﬃciently similar) population as that used in the private study. We will model this assuming no discrepancy between population distributions, however when we discuss how to . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint 17 Reconstruction attacks from GRS evaluate whether the estimate is good that assessment should account for this systematic error as well. In the following we are primarily concerned with the error due to the subsam- pling in both the private and public data sets. For convenience we only consider the case of adding a single individual, though the generalization is quite straight- forward. D.1 Analytic bound on ∥φ0 −ˆφ0∥ we can show that E[∥K −ˆK∥] = O(1/ q min( ˆ M, M)). This shows that the error in estimating K is small as long as the private and public databases are large enough. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint D.1 Analytic bound on ∥φ0 −ˆφ0∥ If ˆK is substituted for K in our reconstruction equation (7) we get an approx- imation of φ0 which we denote ˆφ0. We would like to bound the (relative) error between φ0 and ˆφ0. In the following, we ignore the constant factors C and ˆC for simplicity, noting that these scaling factors are estimated from the resulting φ0 or ˆφ0 anyway. We thus consider ϕ0 = K(ˆβM+1 −ˆβM) and ˆϕ0 = ˆK(ˆβM+1 −ˆβM). Using ∥·∥on vectors, and also on matrices to denote the corresponding operator norm. The relative error between ϕ0 and ˆϕ0 is given by: ∥ϕ0 −ˆϕ0∥ ∥ϕ0∥ = ∥( ˆK ˆK−1 −ˆKK−1)ϕ0∥ ∥ϕ0∥ ≤∥ˆK ˆK−1 −ˆKK−1∥= ∥ˆK( ˆK−1 −K−1)∥. Note that ˆK−1 −K−1 = ˆK−1(K −ˆK)K−1 and hence ∥ˆϕ0 −ϕ0∥ ∥ϕ0∥ ≤∥K−1∥∥K −ˆK∥, (13) (13) This means we can bound the error by two quantities. The term ∥K−1∥is bounded above by 1/ min(eig(K)), which is ﬁnite as soon as K is non-singular. This is not a strong requirement as in the case of linear regression it is required for ˆβM+1 and ˆβM to exist. Note that in the case of L2-regularized linear regression (i.e., ridge regression), K is replaced by K + λI where λ is the regularization parameter, and we can directly bound this term by λ. ˆ ˆ p , y y The key term in (13) is ∥K −ˆK∥, the error in estimating K by ˆK. Let us assume that the public database used to obtain ˆK follows the same distribution as the private database used to ﬁt the GRS models. Denote by ˆ M the number of individuals used to estimate ˆK. Then, under classic boundedness assumptions and leveraging matrix concentration inequalities such as matrix Bernstein [17] q p y y The key term in (13) is ∥K −ˆK∥, the error in estimating K by ˆK. Let us assume that the public database used to obtain ˆK follows the same distribution as the private database used to ﬁt the GRS models. Denote by ˆ M the number of individuals used to estimate ˆK. Then, under classic boundedness assumptions and leveraging matrix concentration inequalities such as matrix Bernstein [17] we can show that E[∥K −ˆK∥] = O(1/ q min( ˆ M, M)). This shows that the error in estimating K is small as long as the private and public databases are large enough. D.2 Modelling the error in ˆ K In this section we deﬁne a model to capture the error in ˆK, which leads to the expectation maximization algorithm for estimating φ0 which is used in the experiments. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B. Paige et al. 18 As our estimated ˆK drifts from the true K, this expression ˆK(ˆβM+1 −ˆβM) would produce a wider range of values than just 0 and C. Let ǫij ∼N(0, σ2) be independent noise, which we assume corrupts each element of Kij; i.e. given the estimated matrix ˆK, suppose Kij ∼N( ˆKij, σ2), (14) (14) for some small σ2. This is clearly an oversimpliﬁcation (as we know K is e.g. bounded and symmetric), but is a useful starting point that allows derivation of a simple estimation algorithm. For notational brevity, in this and the following section we deﬁne the vector ∆= ˆβM+1 −ˆβM (15) (15) which corresponds to the diﬀerence between the two GRS model parameter vectors. Given the true value of K, the system of equations Cφ0 = K∆ Cφ0 = K∆ relates the known quantity ∆and the Gaussian-distributed K with the unknown value of C and of the vector φ0. If K is Gaussian (following Eq. (14)), then the linear transformation K∆is Gaussian as well. We denote each of the rows of K as a vector ki, i = 1, . . . , N; then for each row, the scalar value k⊤ i ∆∼N(ˆk⊤ i ∆, σ2∆⊤∆), meaning overall the vector K∆is distributed N( ˆK∆, σ2∆⊤∆I). With some algebraic re-arrangement, and since for the true underlying value of K we have K∆= Cφ0, we can write this as meaning overall the vector K∆is distributed N( ˆK∆, σ2∆⊤∆I). With some algebraic re-arrangement, and since for the true underlying value of K we have K∆= Cφ0, we can write this as ˆK∆∼N(Cφ0, σ2∆⊤∆I) (16) (16) where C and σ are parameters we need to estimate. The vector ˆK∆is observed “data”, computed from the public SNP database and the two released parameter vectors. We can model each of the entries of φ0, which are zeros and ones, as Bernoulli distributions, whose prior probabilities correspond to the public dataset estimated frequencies. D.2 Modelling the error in ˆ K This suggests a model for ˆK∆which is akin to a constrained mixture of Gaussians. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint D.3 Derivation of EM algorithm for reconstructing an individual’s genotype Given the model in Eq. (16), we can deﬁne an EM algorithm for maximum likelihood estimation of C and σ2, which then permits easy inference for each entry of φ0. Denote the entries of φ0 as z1, . . . , zN+1; denote their prior probabilities as α1, . . . , αN+1, where α1, . . . , αN are the (public) population frequencies for each . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint Reconstruction attacks from GRS 19 SNP, and αN+1 = 1.0. Let x1, . . . , xM denote the entries of the ﬁxed (observed) vector x = ˆK∆, which is distributed p(x\|C, z, σ2) = N(x\|Cz, σ2∆⊤∆I). Supposing we know values of C, σ2, to estimate the entries of φ0 we want to ﬁnd p(z\|x, C, σ2), Supposing we know values of C, σ2, to estimate the entries of φ0 we want to ﬁnd p(z\|x, C, σ2), p(z\|x, C, σ2) ∝p(x\|C, z, σ2)p(z) For each zi, i = 1, . . . , N, we deﬁne the quantity For each zi, i = 1, . . . , N, we deﬁne the quantity πi = p(zi = 1\|x, C, σ2) = αiN(xi\|C, σ2∆⊤∆) αiN(xi\|C, σ2∆⊤∆) + (1 −αi)N(xi\|0, σ2∆⊤∆), (17) ) (17) ) (17) ( 7) the posterior probability of each particular entry taking a value of 1, rather than 0. A maximum likelihood algorithm to estimate C, σ2 proceeds by alternately: ( 7) the posterior probability of each particular entry taking a value of 1, rather than 0. A maximum likelihood algorithm to estimate C, σ2 proceeds by alternately: 1. Given C, σ2, estimate the posterior distribution π = p(z\|x, C, σ2); 1. Given C, σ2, estimate the posterior distribution π = p(z\|x, C, σ2); 2. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint D.3 Derivation of EM algorithm for reconstructing an individual’s genotype ˆσ2 ← 1 N∆⊤∆ PN i=1 πi(xi −ˆC)2 + (1 −πi)x2 i . To initialize the algorithm, we can set πi to some initial probabilities, and ﬁnd initial values for ˆC, ˆσ2; we experimented with both setting to the prior probabil- ities per-SNP estimated from the public data, as well as to the vector of all zeros (corresponding to a “hard” initialization at the value of the baseline estimate), and found no qualitative diﬀerence in performance. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint D.3 Derivation of EM algorithm for reconstructing an individual’s genotype Given the posterior π, maximize L = Eπ[log p(x\|C, z, σ2)] with respect to C and σ2. 1. Given C, σ2, estimate the posterior distribution π = p(z\|x, C, σ2); 2. Given the posterior π, maximize L = Eπ[log p(x\|C, z, σ2)] with respect to C and σ2. 2. Given the posterior π, maximize L = Eπ[log p(x\|C, z, σ2)] with respect to and σ2. To maximize C and σ2, we ﬁrst compute the derivatives of To maximize C and σ2, we ﬁrst compute the derivatives of L = X i X zi p(zi\| . . . ) log p(xi\|C, zi, σ2) = N X i=1 πi log N(xi\|C, σ2∆⊤∆) + (1 −πi) log N(xi\|0, σ2∆⊤∆). We take derivative w.r.t. C, We take derivative w.r.t. C, We take derivative w.r.t. C, ∂L ∂C = X i πi σ2∆⊤∆(xi −C) and set equal to zero to ﬁnd and set equal to zero to ﬁnd ˆC = P i πixi P i πi . (18) (18) For the noise term σ2, we have For the noise term σ2, we have ∂L ∂σ2 = N X i=1 πi ∂ ∂σ2 log N(xi\|C, σ2∆⊤∆) + (1 −πi) ∂ ∂σ2 log N(xi\|0, σ2∆⊤∆) which with some algebraic rearrangement becomes which with some algebraic rearrangement becomes ˆσ2 = 1 N∆⊤∆ N X i=1 πi(xi −C)2 + (1 −πi)x2 i . (19) (19) . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint B. Paige et al. 20 These updates taken together can be used to deﬁne an EM algorithm which optimizes the values of C and σ2, despite the fact that the entries of φ0 are unknown; once C and σ2 are then known, the vector π will give probability estimates for each entry of φ0. The overall EM algorithm can be summarized by the following iterative up- dates: 1. πi ≡p(zi = 1\|x, ˆC, ˆσ2) = αiN(xi\| ˆC, ˆσ2∆⊤∆) αiN(xi\| ˆC, ˆσ2∆⊤∆) + (1 −αi)N(xi\|0, ˆσ2∆⊤∆) , 2. ˆC ← P i πixi P i πi , 3. ˆσ2 ← 1 N∆⊤∆ PN i=1 πi(xi −ˆC)2 + (1 −πi)x2 i . 1. πi ≡p(zi = 1\|x, ˆC, ˆσ2) = αiN(xi\| ˆC, ˆσ2∆⊤∆) αiN(xi\| ˆC, ˆσ2∆⊤∆) + (1 −αi)N(xi\|0, ˆσ2∆⊤∆) , P P i i 3. F Estimating φ0 with diﬀerent SNP sets Here we analyse what can still be said in the event that the two studies do not use exactly the same set of SNPs. We will still assume the sets of SNPs considered to have a signiﬁcant overlap. For this purpose we will need a greater variety of notation. A primed variable denotes that it corresponds to the second set of SNPs, e.g. K′ is the co-occurrence matrix from the original M users for the second experiment. If a vector or matrix is surrounded by square brackets this denotes that object but with any rows or columns corresponding to SNPs not in the overlap removed, e.g. [K] denotes the co-occurrence matrix from the ﬁrst experiment restricted to the overlapping SNPs. As before, from the ﬁrst experiment, we have As before, from the ﬁrst experiment, we have K ˆβ = ΦT y (20) (20) and now, from the second experiment, we have and now, from the second experiment, we have (K′ + φ′T 0 φ′ 0)ˆβ′ = Φ′T y′. (21) (21) Taking the diﬀerence between these expressions, as before, gives Taking the diﬀerence between these expressions, as before, gives K′ ˆβ′ −K ˆβ = Φ′T y′ −ΦT y −φ′T 0 φ′ 0 ˆβ′. (22) (22) Restricting to the overlapping set gives that Restricting to the overlapping set gives that [K′ ˆβ′] −[K ˆβ] = [Φ′T y′ −ΦT y −φ′T 0 φ′ 0 ˆβ′]. (23) Noting that [K] = [K′] and that [Φ′T y′] −[ΦT y] = [φT 0 y0] we get that [K]([ˆβ′] −[ˆβ]) = [φT 0 ](y′ 0 −φ0 ˆβ′). (24) [K′ ˆβ′] −[K ˆβ] = [Φ′T y′ −ΦT y −φ′T 0 φ′ 0 ˆβ′]. (23) (23) [K]([ˆβ′] −[ˆβ]) = [φT 0 ](y′ 0 −φ0 ˆβ′). (24) (24) Analogously to the previous cases (y′ 0 −φ0 ˆβ′) is a scalar which we can label C and we get [φT 0 ] = 1 C [K]([ˆβ′] −[ˆβ]). (25) (25) Thus if K is known it can be used to deduce whether the additional individual has each of the SNPs in the overlapping set. If K is not known exactly it can be estimated from public data just as in the same SNP case. E Scaling of EM algorithm with size of private dataset Figure 6 demonstrates the change in accuracy of the EM algorithm over a range of diﬀerent private database sizes. For this test, a synthetic dataset with 100 SNPs and 1,000,000 individuals is generated; 10,000 are held out as a public database, and 30 individuals are taken as a ﬁxed test dataset of new dogs to add and are used to estimate EM algorithm accuracy, across increasingly large private database sizes. The algorithm has stable performance for increasingly large private databases. 103 104 105 106 Private dataset size 0.70 0.75 0.80 0.85 0.90 0.95 1.00 Per-SNP accuracy Fig. 6. Accuracy at reconstruction of the genome of one additional individual, using EM estimation and a noisy estimate ˆK, measured as the size of the initial private database increases. For very small private databases, accuracy is very high, as changes in entries of β are clearly attributable to the new individual. Beyond a certain threshold, overall accuracy is quite stable. Error bars show mean and two standard deviations. 103 104 105 106 Private dataset size 0.70 0.75 0.80 0.85 0.90 0.95 1.00 Per-SNP accuracy Fig. 6. Accuracy at reconstruction of the genome of one additional individual, using EM estimation and a noisy estimate ˆK, measured as the size of the initial private database increases. For very small private databases, accuracy is very high, as changes in entries of β are clearly attributable to the new individual. Beyond a certain threshold, overall accuracy is quite stable. Error bars show mean and two standard deviations. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint Reconstruction attacks from GRS 21 . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint G Algorithm for identifying unique genotypes of multiple dogs, when a precise K is known While the simple approach described in the main manuscript will work in many cases, there are a few special circumstances where a more complex algorithm may be required. In particular, it would not work if there are combinations of SNPs that are not observed among the individuals added to the database. For B. Paige et al. 22 instance, if there is not a single SNP location where the ﬁrst individual has a SNP variant and the others do not, then we would miss the corresponding value in Cm. However, it is still possible to identify all the values in Cm through a more complex algorithm: 1. First, extract all unique, non-zero values from dm. 2. Find the sum of all pairs of values in (1). . Find all values that are in (1), but not in (2 4. If there are exactly m values in (3) and the sum of these values equal the last value of dm (corresponding to the intercept term), then you have found the correct values of Cm. 5. Otherwise, this suggests that there are one or more elements of Cm that are missing from (3) and possibly a few values in (3) that are not in Cm. 6. Begin by subtracting every pair of values in (3). These are now also potential values of Cm 7. Search for a set of m values from (3) and (6) that sum to the last element of dm. There may be more than one set of values for which this is true. 8. If this search is unsuccessful, repeat steps 6-7. Eventually, a set of m values summing to dm should be found. 9. If more than one possible set of values is found for Cm in (7), it is still pos- sible to compare these sets and identify which is the most likely to contain the true values of Cm. For each possible Cm vector, a set of genotypes can be constructed for the m additional individuals. Using the frequencies of each SNP, it is possible to calculate the probability of observing each geno- type. The set of values that produces the most likely genotypes for the m individuals is most likely to be the correct one. G Algorithm for identifying unique genotypes of multiple dogs, when a precise K is known Additionally, this algorithm depends on the fact that it is extremely unlikely that if someone were to sample three random continuous numbers i, j and k, it would just so happen that i + j = k. There is an extremely small chance that a value of Cm would be un-discoverable because of a coincidence of this nature. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint H Description of K when the genotypes are non-binary In many cases, GRS are calculated on genotype matrices that are non-binary. In particular, they may take on three discrete values 0, 1 and 2, where 0 indicates that the most common variant is homozygous, 1 indicates that the individual is heterozygous for the uncommon variant, and 2 indicates that the individual is homozygous for the uncommon variant. If this is the case, the description of K will change. However, it is still the case that the entries of K depend only on the SNP frequencies and SNP co- occurrence frequencies in the dataset, and that knowledge of SNP frequencies and pairwise co-frequencies from the original study, are all that is required in order to compute K. – For i = 1, . . . , N: Kii = pAa +4pAA where paa is the frequency of individuals being heterozygous for the uncommon variant and pAA is the frequency of individuals being homozygous for the uncommon variant. . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted January 15, 2020. . https://doi.org/10.1101/2020.01.15.907808 doi: bioRxiv preprint Reconstruction attacks from GRS 23 – For i = 1, . . . , N −1 and j > i: Kij = Kji = pAa/Bb + 2pAA/Bb + 4pAA/BB where pAa/Bb is the frequency that both SNPs are simultaneously hetero- gygous, pAA/Bb is the frequency that one SNP is homozygous for the rare variant and the other is heterogygous simultaneously, and pAA/BB is the frequency that that uncommon variants are found to be homozygous simul- taneously. – For i = 1, . . . , N −1 and j > i: Kij = Kji = pAa/Bb + 2pAA/Bb + 4pAA/BB where pAa/Bb is the frequency that both SNPs are simultaneously hetero- gygous, pAA/Bb is the frequency that one SNP is homozygous for the rare variant and the other is heterogygous simultaneously, and pAA/BB is the frequency that that uncommon variants are found to be homozygous simul- taneously. – For i = 1, . . . H Description of K when the genotypes are non-binary , N −1 and j > i: Kij = Kji = pAa/Bb + 2pAA/Bb + 4pAA/BB where pAa/Bb is the frequency that both SNPs are simultaneously hetero- gygous, pAA/Bb is the frequency that one SNP is homozygous for the rare variant and the other is heterogygous simultaneously, and pAA/BB is the frequency that that uncommon variants are found to be homozygous simul- taneously. – For i = 1, . . . , N and j = N + 1: Kij = Kji = pAa + 2pAA. – Finally, KN+1 N+1 = 1. – For i = 1, . . . , N and j = N + 1: Kij = Kji = pAa + 2pAA. Fi ll K 1 – For i = 1, . . . , N and j = N + 1: Kij = Kji = pAa + 2pAA. – Finally, KN+1,N+1 = 1. – Finally, KN+1,N+1 = 1. – Finally, KN+1,N+1 = 1.
https://openalex.org/W2121158664	https://journalofinequalitiesandapplications.springeropen.com/counter/pdf/10.1186/1029-242X-2012-14	English	null	An introduction to 2-fuzzy n-normed linear spaces and a new perspective to the Mazur-Ulam problem	Journal of inequalities and applications	2,012	cc-by	11,238	© 2012 Park and Alaca; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. An introduction to 2-fuzzy n-normed linear spaces and a new perspective to the Mazur-Ulam problem Choonkil Park1 and Cihangir Alaca2* * Correspondence: cihangiralaca@yahoo.com.tr 2Department of Mathematics, Faculty of Science and Arts, Celal Bayar University, 45140 Manisa, Turkey * Correspondence: cihangiralaca@yahoo.com.tr 2Department of Mathematics, Faculty of Science and Arts, Celal Bayar University, 45140 Manisa, Turkey Mathematics Subject Classification (2010): 03E72; 46B20; 51M25; 46B04; 46S40. Keywords: Mazur-Ulam theorem, α-n-norm, 2-fuzzy n-normed linear spaces, n-isometry, n-Lipschitz mapping. RESEARCH Open Access Open Access An introduction to 2-fuzzy n-normed linear spaces and a new perspective to the Mazur-Ulam problem Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Abstract The purpose of this article is to introduce the concept of 2-fuzzy n-normed linear space or fuzzy n-normed linear space of the set of all fuzzy sets of a non-empty set. We define the concepts of n-isometry, n-collinearity n-Lipschitz mapping in this space. Also, we generalize the Mazur-Ulam theorem, that is, when X is a 2-fuzzy n- normed linear space or ℑ(X) is a fuzzy n-normed linear space, the Mazur-Ulam theorem holds. Moreover, it is shown that each n-isometry in 2-fuzzy n-normed linear spaces is affine. Full list of author information is available at the end of the article p Mathematics Subject Classification (2010): 03E72; 46B20; 51M25; 46B04; 46S40. 1. Introduction A satisfactory theory of 2-norms and n-norms on a linear space has been introduced and developed by Gähler [1,2]. Following Misiak [3], Kim and Cho [4], and Malčeski [5] developed the theory of n-normed space. In [6], Gunawan and Mashadi gave a sim- ple way to derive an (n - 1)-norm from the n-norms and realized that any n-normed space is an (n - 1)-normed space. Different authors introduced the definitions of fuzzy norms on a linear space. Cheng and Mordeson [7] and Bag and Samanta [8] intro- duced a concept of fuzzy norm on a linear space. The concept of fuzzy n-normed lin- ear spaces has been studied by many authors (see [4,9]). Recently, Somasundaram and Beaula [10] introduced the concept of 2-fuzzy 2- normed linear space or fuzzy 2-normed linear space of the set of all fuzzy sets of a set. The authors gave the notion of a-2-norm on a linear space corresponding to the 2- fuzzy 2-norm by using some ideas of Bag and Samanta [8] and also gave some funda- mental properties of this space. In 1932, Mazur and Ulam [11] proved the following theorem. Mazur-Ulam Theorem. Every isometry of a real normed linear space onto a real normed linear space is a linear mapping up to translation. Baker [12] showed an isometry from a real normed linear space into a strictly convex real normed linear space is affine. Also, Jian [13] investigated the generalizations of the Mazur-Ulam theorem in F*-spaces. Rassias and Wagner [14] described all volume pre- serving mappings from a real finite dimensional vector space into itself and Väisälä Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 2 of 17 [15] gave a short and simple proof of the Mazur-Ulam theorem. Chu [16] proved that the Mazur-Ulam theorem holds when X is a linear 2-normed space. Chu et al. [17] generalized the Mazur-Ulam theorem when X is a linear n-normed space, that is, the Mazur-Ulam theorem holds, when the n-isometry mapped to a linear n-normed space is affine. They also obtain extensions of Rassias and Šemrl’s theorem [18]. Moslehian and Sadeghi [19] investigated the Mazur-Ulam theorem in non-archimedean spaces. Choy et al. [20] proved the Mazur-Ulam theorem for the interior preserving mappings in linear 2-normed spaces. 1. Introduction They also proved the theorem on non-Archimedean 2- normed spaces over a linear ordered non-Archimedean field without the strict convex- ity assumption. Choy and Ku [21] proved that the barycenter of triangle carries the barycenter of corresponding triangle. They showed the Mazur-Ulam problem on non- Archimedean 2-normed spaces using the above statement. Xiaoyun and Meimei [22] introduced the concept of weak n-isometry and then they got under some conditions, a weak n-isometry is also an n-isometry. Cobzaş [23] gave some results of the Mazur- Ulam theorem for the probabilistic normed spaces as defined by Alsina et al. [24]. Cho et al. [25] investigated the Mazur-Ulam theorem on probabilistic 2-normed spaces. Alaca [26] introduced the concepts of 2-isometry, collinearity, 2-Lipschitz mapping in 2-fuzzy 2-normed linear spaces. Also, he gave a new generalization of the Mazur-Ulam theorem when X is a 2-fuzzy 2-normed linear space or ℑ(X) is a fuzzy 2-normed linear space. Kang et al. [27] proved that the Mazur-Ulam theorem holds under some condi- tions in non-Archimedean fuzzy normed space. Kubzdela [28] gave some new results for isometries, Mazur-Ulam theorem and Aleksandrov problem in the framework of non-Archimedean normed spaces. The Mazur-Ulam theorem has been extensively stu- died by many authors (see [29,30]). In the present article, we introduce the concept of 2-fuzzy n-normed linear space or fuzzy n-normed linear space of the set of all fuzzy sets of a non-empty set. We define the concepts of n-isometry, n-collinearity, n-Lipschitz mapping in this space. Also, we generalize the Mazur-Ulam theorem, that is, when X is a 2-fuzzy n-normed linear space or ℑ(X) is a fuzzy n-normed linear space, the Mazur-Ulam theorem holds. It is moreover shown that each n-isometry in 2-fuzzy n-normed linear spaces is affine. 2. Preliminaries , x′ n, t)}, (N1) For all t Î ℝwith t ≤0, N(x1, x2, ..., xn, t) = 0, (N2) For all t Î ℝwith t > 0, N(x1, x2, ..., xn, t) = 1 if and only if x1, x2, ..., xn are linearly dependent, (N6) N(x1, x2, ..., xn, t) is a non-decreasing function of t Î ℝand lim t→∞N(x1, x2, . . . , xn, t) = 1. Then (X, N) is called a fuzzy n-normed linear space or in short f-n-NLS. Theorem 2.1 [9] Let (X, N) be an f-n-NLS. Assume that (N7) N(x1, x2, ..., xn,t) > 0 for all t > 0 implies that x1, x2, ..., xn are linearly dependent. 2. Preliminaries Definition 2.1([31]) Let n Î N and let X be a real vector space of dimension d ≥n. (Here we allow d to be infinite.) A real-valued function ∥●, ..., ●∥on X × · · · × X n satis- fying the following properties (1) ∥x1, x2, ..., xn∥= 0 if and only if x1, x2, ..., xn are linearly dependent, (1) ∥x1, x2, ..., xn∥= 0 if and only if x1, x2, ..., xn are linearly dependent, (2) ∥x1, x2, ..., xn∥is invariant under any permutation, (3) ∥x1, x2, ..., axn∥= \|a\| ∥x1, x2, ..., xn∥for any a Î ℝ, (4) ∥x1, x2, ..., xn-1, y + z∥≤∥x1, x2, ..., xn-1, y∥+ ∥x1, x2, ..., xn-1, z∥, is called an n- (4) ∥x1, x2, ..., xn-1, y + z∥≤∥x1, x2, ..., xn-1, y∥+ ∥x1, x2, ..., xn-1, z∥, is called an n- norm on X and the pair (X, ∥●, ..., ●∥) is called an n-normed linear space. norm on X and the pair (X, ∥●, ..., ●∥) is called an n-normed linear space. Definition 2.2 [9] Let X be a linear space over S (field of real or complex numbers). A fuzzy subset N of Xn × ℝ(ℝ, the set of real numbers) is called a fuzzy n-norm on X Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 3 of 17 y (N1) For all t Î ℝwith t ≤0, N(x1, x2, ..., xn, t) = 0, (N2) For all t Î ℝwith t > 0, N(x1, x2, ..., xn, t) = 1 if and only if x1, x2, ..., xn are linearly dependent, (N3) N(x1, x2, ..., xn, t) is invariant under any permutation of x1, x2, ..., xn, (N4) For all t Î ℝwith t > 0, N(x1, x2, . . . , λxn, t) = N x1, x2, . . . , xn, t λ , if l ≠0, l Î S, (N5) For all s, t Î ℝ N(x1, x2, . . . , xn + x′ n, s + t) ≥min{N(x1, x2, . . . , xn, s), N(x1, x2, . . . (iv) ∥(x, ∨tνt)∥= ∧t ∥(x, νt)∥for all νt Î (0, 1]. (1) ∥f1, f2, ..., fn∥= 0 if and only if f1, f2, ..., fn are linearly dependent, (2) ∥f1, f2, ..., fn∥is invariant under any permutation, (3) ∥f1, f2, ..., lfn∥= \|l\| ∥f1, f2, ..., fn∥for any l Î S, 3. 2-fuzzy n-normed linear spaces In this section, we define the concepts of 2-fuzzy n-normed linear spaces and a-n- norms on the set of all fuzzy sets of a non-empty set. Definition 3.1 Let X be a non-empty and ℑ(X) be the set of all fuzzy sets in X. If f Î ℑ(X) then f = {(x, μ): x Î X and μ Î (0, 1]}. Clearly f is bounded function for \|f(x)\| ≤1. Let S be the space of real numbers, then ℑ(X) is a linear space over the field S where the addition and scalar multiplication are defined by f + g = {(x, μ) + (y, η)} = {(x + y, μ ∧η) : (x, μ) ∈f and (y, η) ∈g} f + g = {(x, μ) + (y, η)} = {(x + y, μ ∧η) : (x, μ) ∈f and (y, η) ∈g} and λf = {(λx, μ) : (x, μ) ∈f} λf = {(λx, μ) : (x, μ) ∈f} where l Î S. where l Î S. where l Î S. The linear space ℑ(X) is said to be normed linear space if, for every f Î ℑ(X), there exists an associated non-negative real number ∥f∥(called the norm of f) which satisfies (i) ∥f∥= 0 if and only if f = 0. For \|\|f\|\| = 0 ⇔{\|\|(x, μ)\|\| : (x, μ) ∈f} = 0 ⇔x = 0, μ ∈(0, 1] ⇔f = 0. \|\|f\|\| = 0 ⇔{\|\|(x, μ)\|\| : (x, μ) ∈f} = 0 ⇔x = 0, μ ∈(0, 1] ⇔f = 0. (ii) ∥lf∥= \|l\| ∥f∥, l Î S. For \|\|λf\|\| = {\|\|λ(x, μ)\|\| : (x, μ) ∈f, λ ∈S} = {\|λ\|\|\|(x, μ)\|\| : (x, μ) ∈f} = \|λ\|\|\|f\|\|. (ii) ∥lf∥= \|l\| ∥f∥, l Î S. For \|\|λf\|\| = {\|\|λ(x, μ)\|\| : (x, μ) ∈f, λ ∈S} = {\|λ\|\|\|(x, μ)\|\| : (x, μ) ∈f} = \|λ\|\|\|f\|\|. (iii) ∥f + g∥≤∥f∥+ ∥g∥for every f, g Î ℑ(X). For \|\|f + g\|\| = {\|\|(x, μ) + (y, η)\|\| : x, y ∈X, μ, η ∈(0, 1]} = {\|\|(x + y), (μ ∧η)\|\| : x, y ∈X, μ, η ∈(0, 1]} = {\|\|(x, μ ∧η)\|\| + \|\|(y, μ ∧η)\|\| : (x, μ) ∈f, (y, η) ∈g} = \|\|f\|\| + \|\|g\|\|. (iii) ∥f + g∥≤∥f∥+ ∥g∥for every f, g Î ℑ(X). 3. 2-fuzzy n-normed linear spaces For \|\|f + g\|\| = {\|\|(x, μ) + (y, η)\|\| : x, y ∈X, μ, η ∈(0, 1]} = {\|\|(x + y), (μ ∧η)\|\| : x, y ∈X, μ, η ∈(0, 1]} = {\|\|(x, μ ∧η)\|\| + \|\|(y, μ ∧η)\|\| : (x, μ) ∈f, (y, η) ∈g} = \|\|f\|\| + \|\|g\|\|. = \|\|f\|\| + \|\|g\|\|. Then (ℑ(X),∥●∥) is a normed linear space. Then (ℑ(X),∥●∥) is a normed linear space. Then (ℑ(X),∥●∥) is a normed linear space. Define \|\|x1, x2, . . . , xn\|\|α = inf{t : N(x1, x2, . . . xn, t) ≥α, α ∈(0, 1)}. x2, . . . , xn\|\|α = inf{t : N(x1, x2, . . . xn, t) ≥α, α ∈(0, 1)}. Then {∥●, ●, ..., ●∥a : a Î (0, 1)} is an ascending family of n-norms on X. We call these n-norms as a-n-norms on X corresponding to the fuzzy n-norm on X Then {∥●, ●, ..., ●∥a : a Î (0, 1)} is an ascending family of n-norms on X. We call these n-norms as a-n-norms on X corresponding to the fuzzy n-norm on X. Definition 2.3 Let X be any non-empty set and ℑ(X) the set of all fuzzy sets on X. For U, V Î ℑ(X) and l Î S the field of real numbers, define Definition 2.3 Let X be any non-empty set and ℑ(X) the set of all fuzzy sets on X. For U, V Î ℑ(X) and l Î S the field of real numbers, define Definition 2.3 Let X be any non-empty set and ℑ(X) the set of all fuzzy sets on X. For U, V Î ℑ(X) and l Î S the field of real numbers, define and lU = {(lx, ν): (x, ν) Î U}. Definition 2.4 A fuzzy linear space X = X × (0, 1] over the number field S, where the addition and scalar multiplication operation on X are defined by (x, ν) + (y, μ) = (x + y, ν∧μ), l(x, ν) = (lx, ν) is a fuzzy normed space if to every (x, ν) ∈X there is asso- ciated a non-negative real number, ∥(x, ν)∥, called the fuzzy norm of (x, ν), in such away that (i) ∥(x, ν)∥= 0 iff x = 0 the zero element of X, ν Î (0, 1], (ii) ∥l(x, ν)∥= \|l\| ∥(x, ν)∥for all (x, ν) ∈X and all l Î S, (ii) ∥l(x, ν)∥= \|l\| ∥(x, ν)∥for all (x, ν) ∈X and all l Î S, (iii) ∥(x, ν) + (y, μ) \|\| ≤∥(x, ν ∧μ)∥+ ∥(y, ν ∧μ)∥for all (x, ν), (y, μ) ∈X , (iv) ∥(x, ∨tνt)∥= ∧t ∥(x, νt)∥for all νt Î (0, 1]. (iv) ∥(x, ∨tνt)∥= ∧t ∥(x, νt)∥for all νt Î (0, 1]. Define Page 4 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Then (ℑ(X),∥●∥) is a normed linear space. Definition 3.2 A 2-fuzzy set on X is a fuzzy set on ℑ(X). Definition 3.2 A 2-fuzzy set on X is a fuzzy set on ℑ(X). Definition 3.3 Let X be a real vector space of dimension d ≥n (n Î N) and ℑ(X) be the set of all fuzzy sets in X. Here we allow d to be infinite. Assume that a [0, 1]- ℑ(X) × · · · × ℑ(X) Definition 3.3 Let X be a real vector space of dimension d ≥n (n Î N) and ℑ(X) be the set of all fuzzy sets in X. Here we allow d to be infinite. Assume that a [0, 1]- valued function ∥●, ..., ●∥on ℑ(X) × · · · × ℑ(X) n satisfies the following properties valued function ∥●, ..., ●∥on ℑ(X) × · · · × ℑ(X) n satisfies the following properties (1) ∥f1, f2, ..., fn∥= 0 if and only if f1, f2, ..., fn are linearly dependent, (2) ∥f1, f2, ..., fn∥is invariant under any permutation, (3) ∥f1, f2, ..., lfn∥= \|l\| ∥f1, f2, ..., fn∥for any l Î S, Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 5 of 17 (4) ∥f1, f2, ..., fn-1, y + z∥≤∥f1, f2, ..., fn-1, y∥+ ∥f1, f2, ..., fn-1, z∥. Then (ℑ(X),∥●,...,●∥) is an n-normed linear space or (X, ∥●, ..., ●∥) is a 2-n-normed linear space. Definition 3.4 Let ℑ(X) be a linear space over the real field S. A fuzzy subset N of ℑ(X) × · · · × ℑ(X) n ×R is called a 2-fuzzy n-norm on X (or fuzzy n-norm on ℑ(X)) if and only if Definition 3.4 Let ℑ(X) be a linear space over the real field S. Then (ℑ(X),∥●∥) is a normed linear space. A fuzzy subset N of ℑ(X) × · · · × ℑ(X) n ×R is called a 2-fuzzy n-norm on X (or fuzzy n-norm on ℑ(X)) if and only if and only if and only if (2-N1) for all t Î ℝwith t ≤0, N(f1, f2, ..., fn, t) = 0, (2-N1) for all t Î ℝwith t ≤0, N(f1, f2, ..., fn, t) = 0, (2-N2) for all t Î ℝwith t > 0, N(f1, f2, ..., fn, t) = 1 if and only if f1, f2, ..., fn are line- arly dependent, (2-N3) N(f1, f2, ..., fn, t) is invariant under any permutation of f1, f2, ..., fn, (2-N4) for all t Î ℝwith t > 0, N(f1, f2, ..., lfn, t) = N(f1, f2, ..., fn, t/\|l\|), if l ≠0, l Î S, (2-N5) for all s, t Î ℝ, N(f1, f2, . . . , fn + f ′ n, s + t) ≥min{N(f1, f2, . . . , fn, s), N(f1, f2, . . . , f ′ n, t)}, N(f1, f2, . . . , fn + f ′ n, s + t) ≥min{N(f1, f2, . . . , fn, s), N(f1, f2, . . . , f ′ n, t)}, (2-N6) N(f1, f2, ..., fn, ·): (0, ∞) ® [0, 1] is continuous, (2-N6) N(f1, f2, ..., fn, ·): (0, ∞) ® [0, 1] is continuous, (2-N7) lim t→∞N(f1, f2, . . . , fn,t) = 1. Then ℑ(X), N) is a fuzzy n-normed linear space or (X, N) is a 2-fuzzy n-normed lin- ear space. Remark 3.1 In a 2-fuzzy n-normed linear space (X, N), N(f1, f2, ..., fn, ·) is a non- decreasing function of ℝfor all f1, f2,...,fn Î ℑ(X). Remark 3.2 From (2-N4) and (2-N5), it follows that in a 2-fuzzy n-normed linear space, (2-N4) for all t Î ℝ with t > 0, N(f1, f2, . . . , λfi, . . . , fn, t) = N f1, f2, . . . , fi, . . . , fn, t \|λ\| , if l ≠0, l Î S, (2-N5) for all s, t Î ℝ, N(f1, f2, . . . , fi + f ′ i, . . . , fn, s + t) ≥min{N(f1, f2, . . . , fi, . . . , fn, s), N(f1, f2, . . . Then (ℑ(X),∥●∥) is a normed linear space. , f ′ n\|\| t + \|\|f1, f2, . . . , f ′ n\|\| s ⇒\|\|f1, f2, . . . , fn + f ′ n\|\| s ≤ s + t s · t \|\|f1, f2, . . . , f ′ n\|\| ⇒\|\|f1, f2, . . . , fn + f ′ n\|\| s + t ≤\|\|f1, f2, . . . , f ′ n\|\| t ⇒s + t + \|\|f1, f2, . . . , fn + f ′ n\|\| s + t ≤t + \|\|f1, f2, . . . , f ′ n\|\| t ⇒ s + t s + t + \|\|f1, f2, . . . , fn + f ′n\|\| ≥ t t + \|\|f1, f2, . . . , f ′n\|\| ⇒N(f1, f2, . . . , fn + f ′ n, s + t) ≥N(f1, f2, . . . , f ′ n, t). (i) s + t < 0, (ii) s = t = 0, (iii) s + t > 0; s > 0, t < 0; s < 0, t > 0, then the above relation is obvious. If (iv) s > 0, t > 0, s + t > 0, then N(f1, f2, . . . , fn + f ′ n, s + t = s + t s + t + \|\|f1, f2, . . . , fn + f ′n\|\|. If s s + \|\|f1, f2, . . . , fn\|\| ≥ t t + \|\|f1, f2, . . . , f ′n\|\| ⇒\|\|f1, f2, . . . , fn\|\| s ≤\|\|x1, x2, . . . , x′n\|\| t ⇒\|\|f1, f2, . . . , fn\|\| s + \|\|f1, f2, . . . , f ′ n\|\| s ≤\|\|f1, f2, . . . , f ′ n\|\| t + \|\|f1, f2, . . . , f ′ n\|\| s ⇒\|\|f1, f2, . . . , fn + f ′ n\|\| s ≤ s + t s · t \|\|f1, f2, . . . , f ′ n\|\| ⇒\|\|f1, f2, . . . , fn + f ′ n\|\| s + t ≤\|\|f1, f2, . . . , f ′ n\|\| t ⇒s + t + \|\|f1, f2, . . . , fn + f ′ n\|\| s + t ≤t + \|\|f1, f2, . . . Then (ℑ(X),∥●∥) is a normed linear space. , fn\|\| = 1 ⇔t = t + \|\|f1, f2, . . . , fn\|\| ⇔\|\|f1, f2, . . . , fn\|\| = 0 ⇔f1, f2, . . . , fn are linearly dependent. (2-N3) For all t Î ℝwith t > 0, N(f1, f2, . . . , fn, t) = t t + \|\|f1, f2, . . . , fn\|\| = t t + \|\|f1, f2, . . . , fn, fn−1\|\| = N(f1, f2, . . . , fn, fn−1, t) = · · · . (2-N4) For all t Î ℝwith t > 0 and l Î F, l ≠0, (2-N4) For all t Î ℝwith t > 0 and l Î F, l ≠0, N(f1, f2, . . . , fn, t/\|λ\|) = t/\|λ\| t/\|λ\| + \|\|f1, f2, . . . , fn\|\| = t/\|λ\| (t + \|λ\| \|\|f1, f2, . . . , fn\|\|)/\|λ\| = t t + \|λ\| \|\|f1, f2, . . . , fn\|\| = t (t + \|\|f1, f2, . . . , λfn\|\| = N(f1, f2, . . . , λfn, t). (2-N5) We have to prove N(f1, f2, . . . , fn + f ′ n, s + t) ≥min{f(x1, f2, . . . , fn, s), N(f1, f2, . . . , f ′ n, t)}. N(f1, f2, . . . , fn + f ′ n, s + t) ≥min{f(x1, f2, . . . , fn, s), N(f1, f2, . . . , f ′ n, t)}. (i) s + t < 0, (ii) s = t = 0, (iii) s + t > 0; s > 0, t < 0; s < 0, t > 0, then the above relation is obvious. If (iv) s > 0, t > 0, s + t > 0, then N(f1, f2, . . . , fn + f ′ n, s + t = s + t s + t + \|\|f1, f2, . . . , fn + f ′n\|\|. If s s + \|\|f1, f2, . . . , fn\|\| ≥ t t + \|\|f1, f2, . . . , f ′n\|\| ⇒\|\|f1, f2, . . . , fn\|\| s ≤\|\|x1, x2, . . . , x′n\|\| t ⇒\|\|f1, f2, . . . , fn\|\| s + \|\|f1, f2, . . . , f ′ n\|\| s ≤\|\|f1, f2, . . . Then (ℑ(X),∥●∥) is a normed linear space. , f ′ i, . . . , fn, t)}. (2-N4) for all t Î ℝ with t > 0, N(f1, f2, . . . , λfi, . . . , fn, t) = N f1, f2, . . . , fi, . . . , fn, t \|λ\| , if l ≠0, l Î S, (2-N5) for all s, t Î ℝ, ≥min{N(f1, f2, . . . , fi, . . . , fn, s), N(f1, f2, . . . , f ′ i, . . . , fn, t)}. The following example agrees with our notion of 2-fuzzy n-normed linear space. The following example agrees with our notion of 2-fuzzy n-normed linear space. Example 3.1 Let (ℑ(X),∥●,●,...,●∥) be an n-normed linear space as in Definition 3.3. Define N(f1, f2, . . . , fn,t) = ⎧ ⎨ ⎩ t t + \|\|f1, f2, . . . , fn\|\|if t > 0, t ∈R, 0if t ≤0 for all (f1, f2, . . . , fn) ∈ℑ(X) × · · · × ℑ(X) n . Then (X, N) is a 2-fuzzy n-normed linear pace. N(f1, f2, . . . , fn,t) = ⎧ ⎨ ⎩ t t + \|\|f1, f2, . . . , fn\|\|if t > 0, t ∈R, 0if t ≤0 space. space. p Solution. (2-N1) For all t Î ℝwith t ≤0, by definition, we have N(f1, f2, ..., fn, t) = 0. Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 6 of 17 (2-N2) For all t Î ℝwith t > 0, N(f1, f2, . . . , fn, t) = 1 ⇔ t t + \|\|f1, f2, . . . , fn\|\| = 1 ⇔t = t + \|\|f1, f2, . . . , fn\|\| ⇔\|\|f1, f2, . . . , fn\|\| = 0 ⇔f1, f2, . . . , fn are linearly dependent. (2-N2) For all t Î ℝwith t > 0, N(f1, f2, . . . , fn, t) = 1 ⇔ t t + \|\|f1, f2, . . . , fn\|\| = 1 ⇔t = t + \|\|f1, f2, . . . , fn\|\| ⇔\|\|f1, f2, . . . , fn\|\| = 0 ⇔f1, f2, . . . , fn are linearly dependent. N(f1, f2, . . . , fn, t) = 1 ⇔ t t + \|\|f1, f2, . . . Then (ℑ(X),∥●∥) is a normed linear space. , f ′ n\|\| t ⇒ s + t s + t + \|\|f1, f2, . . . , fn + f ′n\|\| ≥ t t + \|\|f1, f2, . . . , f ′n\|\| ⇒N(f1, f2, . . . , fn + f ′ n, s + t) ≥N(f1, f2, . . . , f ′ n, t). If Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 7 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Similarly, if Thus N(f1, f2, . . . , fn + f ′ n, s + t) ≥min{N(f1, f2, . . . , fn, s), N(f1, f2, . . . , f ′ n, t) (2-N6) It is clear that N(f1, f2, ..., fn, ·): (0, ∞) ® [0, 1] is continuous. (2-N7) For all t Î ℝwith t > 0, lim t→∞N(f1, f2, . . . , fn, t) = lim t→∞ t t + \|\|f1, f2, . . . , fn\|\| = lim t→∞ t t(1 + (1/t)\|\|f1, f2, . . . , fn\|\|) = 1, as desired. As a consequence of Theorem 3.2 in [10], we introduce an interesting notion of ascending family of a-n-norms corresponding to the fuzzy n-norms in the following theorem. Theorem 3.1 Let (ℑ(X), N) is a fuzzy n-normed linear space. Assume that (2-N8) N(f1, f2, ..., fn, t) > 0 for all t > 0 implies f1, f2, ..., fn are linearly dependent. Define \|\|f1, f2, . . . , fn\|\|α = inf(t : N(f1, f2, . . . , fn, t) ≥α, α ∈(0, 1)}. Then {∥●, ●, ..., ●∥a : a Î (0, 1)} is an ascending family of n-norms on ℑ(X). Then {∥●, ●, ..., ●∥a : a Î (0, 1)} is an ascending family of n-norms on ℑ(X). These n-norms are called a-n-norms on ℑ(X) corresponding to the 2-fuzzy n-norm on X. These n-norms are called a-n-norms on ℑ(X) corresponding to the 2-fuzzy n-norm on X. Proof. (i) Let ∥f1, ..., fn∥a = 0. This implies that inf {t : N(f1, ..., fn, t) ≥a}. Then, N(f1, f2, ..., fn, t) ≥a > 0, for all t > 0, a Î (0, 1), which implies that f1, f2, ..., fn are linearly dependent, by (2-N8). Conversely, assume f1, f2, ..., fn are linearly dependent. Then (ℑ(X),∥●∥) is a normed linear space. . . , fn α + f1, f2, . . . , f ′ n α = inf{t : N(f1, f2, . . . , fn, t) ≥α} + inf(s : N(f1, f2, . . . , f ′ n, s) ≥α} = inf {t + s : N(f1, f2, . . . , fn, t) ≥α, N(f1, f2, . . . , f ′ n, s) ≥α} ≥inf {t + s : N(f1, f2, . . . , fn + f ′ n, t + s) ≥α}, ≥inf {r : N(f1, f2, . . . , fn + f ′ n, r) ≥α}, r = t + s = f1, f2, . . . , fn + f ′ n α. Hence Hence \|\|f1, f2, . . . , fn + f ′ n\|\|α ≤\|\|f1, f2, . . . , fn\|\|α + \|\|f1, f2, . . . , f ′ n\|\|α. Thus {∥●, ●, ..., ●∥a : a Î (0, 1)} is an a-n-norm on X. Let 0 <a1 <a2. Then, f1, f2, . . . , fn α1 = inf{t : N(f1, f2, . . . , fn, t) ≥α1}, f1, f2, . . . , fn α2 = inf{t : N(f1, f2, . . . , fn, t) ≥α2}. As a1 <a2, {t : N(f1, f2, . . . , fn, t) ≥α2} ⊂{t : N(f1, f2, . . . , fn, t) ≥α1} implies that inf{t : N(f1, f2, . . . , fn, t) ≥α2} ≥inf{t : N(f1, f2, . . . , fn, t) ≥α1} which implies that \|\|f1, f2, . . . , fn\|\|α2 ≥\|\|f1, f2, . . . , fn\|\|α1. Hence {∥●, ●, ..., ●∥a : a Î (0, 1)} is an ascending family of a-n-norms on x corre- sponding to the 2-fuzzy n-norm on X. Then (ℑ(X),∥●∥) is a normed linear space. This implies that N(f1, f2, ..., fn, t) = 1 for all t > 0. For all a Î (0, 1), inf {t : N(f1, f2, ..., fn, t) ≥a}, which implies that ∥f1, f2, ..., fn∥a = 0. (ii) Since N(f1, f2, ..., fn, t) is invariant under any permutation, ∥f1, f2, ..., fn∥a = 0 under any permutation. (iii) If l ≠0, then \|\|f1, f2, . . . , λfn\|\|α = inf{s : N(f1, f2, . . . , fn, s) ≥α} = inf{s : N(f1, f2, . . . , fn, s \|λ\| ≥α}. \|\|f1, f2, . . . , λfn\|\|α = inf{\|λ\|t : N(f1, f2, . . . , fn, t) ≥α} = \|λ\| inf{t : N(f1, f2, . . . , fn, t) ≥α} = \|λ\| \|\|f1, f2, . . . , fn\|\|α. Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 8 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 8 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 If l = 0, then \|\|f1, f2, . . . , λfn\|\|α = \|\|f1, f2, . . . , 0\|\|α = 0 = 0\|\|f1, f2, . . . , fn\|\|α = \|λ\| \|\|f1, f2, . . . , fn\|\|α, ∀λ ∈S(ﬁeld). (iv) (iv) (iv) f1, f2, . . . , fn α + f1, f2, . . . , f ′ n α = inf{t : N(f1, f2, . . . , fn, t) ≥α} + inf(s : N(f1, f2, . . . , f ′ n, s) ≥α} = inf {t + s : N(f1, f2, . . . , fn, t) ≥α, N(f1, f2, . . . , f ′ n, s) ≥α} ≥inf {t + s : N(f1, f2, . . . , fn + f ′ n, t + s) ≥α}, ≥inf {r : N(f1, f2, . . . , fn + f ′ n, r) ≥α}, r = t + s = f1, f2, . . . , fn + f ′ n α. Hence \|\|f1, f2, . . . , fn + f ′ n\|\|α ≤\|\|f1, f2, . . . , fn\|\|α + \|\|f1, f2, . . . , f ′ n\|\|α. Thus {∥●, ●, ..., ●∥a : a Î (0, 1)} is an a-n-norm on X. Let 0 <a1 <a2. Then, f1, f2, . 4. On the Mazur-Ulam problem , fn\|\|α + \|\|f ′ 0, f1, . . . , fn\|\|α \|\|f0 + f ′ 0, f1, . . . , fn\|\|α = \|\|f0, f1, . . . , fn\|\|α + \|\|f ′ 0, f1, . . . , fn\|\|α for all f1, f2,...,fn Î ℑ(X) and a Î (0, 1). for all f1, f2,...,fn Î ℑ(X) and a Î (0, 1). Proof. Let f ′ 0 = tf0 for some t > 0. Then we have \|\|f0 + f ′ 0, f1, . . . , fn\|\|α = \|\|f0 + tf0, f1, . . . , fn\|\|α \|\|f0 + f ′ 0, f1, . . . , fn\|\|α = \|\|f0 + tf0, f1, . . . , fn\|\|α = (1 + t)\|\|f0, f1, . . . , fn\|\|α = \|\|f0, f1, . . . , fn\|\|α + t\|\|f0, f1, . . . , fn\|\|α = \|\|f0, f1, . . . , fn\|\|α + \|\|f ′ 0, f1, . . . , fn\|\|α for all f1, f2,...,fn Î ℑ(X) and a Î (0, 1). Definition 4.2 The elements f0, f1, f2, ..., fn of ℑ(X) are said to be n-collinear if for every i, {fj - fi : 0 ≤j ≠i ≤n} is linearly dependent. Definition 4.2 The elements f0, f1, f2, ..., fn of ℑ(X) are said to be n-collinear if for every i, {fj - fi : 0 ≤j ≠i ≤n} is linearly dependent. Remark 4.1 The elements f0, f1, and f2 are said to be 2-collinear if and only if f2 - f0 = r(f1 - f0) for some real number r. Remark 4.1 The elements f0, f1, and f2 are said to be 2-collinear if and only if f2 - f0 = r(f1 - f0) for some real number r. Now we define the concept of n-Lipschitz mapping. Now we define the concept of n-Lipschitz mapping. Now we define the concept of n-Lipschitz mapping. Definition 4.3 We call Ψ an n-Lipschitz mapping if there is a ≥0 such that Definition 4.3 We call Ψ an n-Lipschitz mapping if there is a ≥0 such that \|\|(f1) −(f0), . . . , (fn) −(f0)\|\|β ≤k\|\|f1 −f0, . . . , fn −f0\|\|α for all f0, f1, f2,...,fn Î ℑ(X) and a, b Î (0, 1). The smallest such is called the n- Lipschitz constant. 4. On the Mazur-Ulam problem In this section, we give a new generalization of the Mazur-Ulam theorem when X is a 2-fuzzy n-normed linear space or ℑ(X) is a fuzzy n-normed linear space. Hereafter, we use the notion of fuzzy n-normed linear space on ℑ(X) instead of 2-fuzzy n-normed linear space on X. Definition 4.1 Let ℑ(X) and ℑ(X) be fuzzy n-normed linear spaces and Ψ : ℑ(X) ® ℑ (Y) a mapping. We call Ψ an n-isometry if \|\|f1 −f0, . . . , fn −f0\|\|α = \|\|(f1) −(f0), . . . , (fn) −(f0)\|\|β for all f0, f1, f2,...,fn Î ℑ(X) and a, b Î (0, 1). For a mapping Ψ, consider the following condition which is called the n-distance one preserving property (nDOPP). Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 9 of 17 (nDOPP) Let f0, f1, f2, . . . , fn ∈ℑ(X) with f1 −f0, . . . , fn −f0 α = 1. (nDOPP) Let f0, f1, f2, . . . , fn ∈ℑ(X) with f1 −f0, . . . , fn −f0 α = 1. Then ∥Ψ(f1) - Ψ(f0), ..., Ψ(fn) - Ψ(f0)∥b = 1. Then ∥Ψ(f1) - Ψ(f0), ..., Ψ(fn) - Ψ(f0)∥b = 1. Then ∥Ψ(f1) - Ψ(f0), ..., Ψ(fn) - Ψ(f0)∥b = 1. Lemma 4.1 Let f1, f2,...,fn Î ℑ(X), a Î (0, 1) and ħ Î ℝ. Then, \|\|f1, . . . , fi, . . . , fj, . . . , fn\|\|α = \|\|f1, . . . , fi, . . . , fj + ¯hfi, . . . , fn\|\|α Lemma 4.1 Let f1, f2,...,fn Î ℑ(X), a Î (0, 1) and ħ Î ℝ. Then, \|\|f1, . . . , fi, . . . , fj, . . . , fn\|\|α = \|\|f1, . . . , fi, . . . , fj + ¯hfi, . . . , fn\|\|α for all 1 ≤i ≠j ≤n. for all 1 ≤i ≠j ≤n. Proof. It is obviously true. Proof. It is obviously true. Lemma 4.2 For f0, f ′ 0 ∈ℑ(X), if f0 and f ′ 0 are linearly dependent with some direc- tion, that is, f ′ 0 = tf0 for some t > 0, then \|\|f0 + f ′ 0, f1, . . . , fn\|\|α = \|\|f0, f1, . . . 4. On the Mazur-Ulam problem Lemma 4.3 Assume that if f0, f1, and f2 are 2 -collinear then Ψ(f0), Ψ(f1) and Ψ(f2) are 2-collinear, and that Ψ satisfies (nDOPP). Then Ψ preserves the n-distance k for each k Î N. Proof. Suppose that there exist f0, f1 Î ℑ(X) with f0 ≠f1 such that Ψ(f0) = Ψ(f1). Since dimℑ(X) ≥n, there are f2,...,fn Î ℑ(X) such that f1 - f0, f2 - f0, ..., fn - f0 are linearly inde- pendent. Since ∥f1 - f0, f2 - f0, ..., fn - f0∥a ≠0, we can set z2 = f0 + f2 −f0 \|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α . Then we have Then we have \|\|f1 −f0, z2 −f0, f3 −f0, . . . , fn −f0\|\|α = f1 −f0, f2 −f0 \|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α , f3 −f0, . . . , fn −f0 α = 1. Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 10 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Since Ψ preserves the unit n-distance, Since Ψ preserves the unit n-distance, \|\|(f1) −(f0), (z2) −(f0), . . . , (fn) −(f0)\|\|β = 1. But it follows from Ψ (f0) = Ψ (f1) that \|\|(f1) −(f0), (z2) −(f0), . . . , (fn) −(f0)\|\|β = 0, But it follows from Ψ (f0) = Ψ (f1) that But it follows from Ψ (f0) = Ψ (f1) that \|\|(f1) −(f0), (z2) −(f0), . . . , (fn) −(f0)\|\|β = 0, which is a contradiction. Hence, Ψ is injective. Let f0, f1, f2, ..., fn be elements of ℑ(X), k Î N and which is a contradiction. Hence, Ψ is injective. which is a contradiction. Hence, Ψ is injective. Let f0, f1, f2, ..., fn be elements of ℑ(X), k Î N and which is a contradiction. Hence, Ψ is injective. Let f0, f1, f2, ..., fn be elements of ℑ(X), k Î N and \|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α = k. We put We put gi = f0 + i k(f1 −f0), i = 0, 1, . . . , k. Then \|\|gi+1 −gi, f2 −f0, . . . , fn −f0\|\|α = f0 + i + 1 k (f1 −f0) − f0 + i k(f1 −f0) , f2 −f0, . . . 4. On the Mazur-Ulam problem . , fn −h\|\|α = \|\|f1 −h, f2 −f0, . . . , fn −f0\|\|α. Lemma 4.4 Let h, f0, f1, ..., fn be elements of ℑ(X) and let h, f0, f1 be 2-collinear. Then \|\|f1 −h, f2 −h, . . . , fn −h\|\|α = \|\|f1 −h, f2 −f0, . . . , fn −f0\|\|α. Proof. Since h, f0, f1 are 2-collinear, there exists a real number r such that f1 - h = r(f0 - h). It follows from Lemma 4.1 that \|\|f1 −h, f2 −f0, . . . , fn −f0\|\|α = \|\|r(f0 −h), f2 −f0, . . . , fn −f0\|\|α = \|r\| \|\|f0 −h, f2 −f0, . . . , fn −f0\|\|α = \|r\| \|\|f0 −h, f2 −h, . . . , fn −h\|\|α = \|\|r(f0 −h), f2 −h, . . . , fn −h\|\|α = \|\|f1 −h, f2 −h, . . . , fn −h\|\|α. This completes the proof. Theorem 4.1 Let Ψ be an n-Lipschitz mapping with the n-Lipschitz constant ≤1. Assume that if f0, f1, ..., fn are m-collinear then Ψ(f0), Ψ(f1), ..., Ψ(fm) are m-collinear, m = 2, n, and that Ψ satisfies (nDOPP), then Ψ is an n-isometry. Proof. It follows from Lemma 4.3 that Ψ preserves n-distance k for all k Î N. For f0, f1, ..., fn Î X, there are two cases depending upon whether ∥f1 - f0, ..., fn - f0∥a = 0 or not. In the case ∥f1 - f0, ..., fn - f0∥a = 0, f1 - f0, ..., fn - f0 are linearly dependent, that is, n-collinear. Thus f1 - f0, ..., fn - f0 are linearly dependent. Thus ∥Ψ(f1) - Ψ(f0), ..., Ψ(fn) - Ψ(f0)∥b = 0. In the case ∥f1 - f0, ..., fn - f0∥a > 0, there exists an n0 Î N such that n0 > \|\|f1 −f0, . . . , fn −f0\|\|α. Assume that \|\|(f1) −(f0), . . . , (fn) −(f0)\|\|β < \|\|f1 −f0, . . . , fn −f0\|\|α. We can set h = f0 + n0 \|\|f1 −f0, . . . , fn −f0\|\|α (f1 −f0). Then we get Then we get \|\|h −f0, . . . , fn −f0\|\|α \|\|h −f0, . . . , fn −f0\|\|α = f0 + n0 \|\|f1 −f0, . . . , fn −f0\|\|α (f1 −f0) −f0, . . . 4. On the Mazur-Ulam problem , fn −f0 α = 1 k (f1 −f0), f2 −f0, . . . , fn −f0 α = i k\|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α = k k = 1 for all i = 0, 1, ..., k - 1. Since Ψ satisfies (nDOPP), \|\|(gi+1) −(gi), (f2) −(f0) . . . , (fn) −(f0)\|\|β = 1 (4:1) (4:1) for all i = 0, 1, ..., k - 1. Since g0, g1, and g2 are 2-collinear, Ψ (g0), Ψ(g1) and Ψ(g2) are also 2-collinear. Thus there is a real number r0 such that Ψ(g2) - Ψ(g1) = r0 (Ψ(g1) - Ψ(g0)). It follows from (4.1) that \|\|(g1) −(g0), (f2) −(f0), . . . , (fn) −(f0)\|\|β = \|\|(g2) −(g1), (f2) −(f0), . . . , (fn) −(f0)\|\|β = \|\|r0((g1) −(g2)), (f2) −(f0), . . . , (fn) −(f0)\|\|β = \|r0\| \|\|((g1) −(g0)), (f2) −(f0), . . . , (fn) −(f0)\|\|β. Thus, we have r0 = 1 or -1. If r0 = -1, Ψ(g2) - Ψ(g1) = -Ψ(g1) + Ψ(g0), that is, Ψ(g2) = Ψ(g0). Since Ψ is injective, g2 = g0, which is a contradiction. Thus r0 = 1. Then we have Ψ(g2) - Ψ(g1) = Ψ(g1) - Ψ(g0). Similarly, one can obtain that Ψ(gi+1) - Ψ(gi) = Ψ(gi) - Ψ(gi-1) for all i = 0, 1, ..., k - 1. Thus Ψ(gi+1) - Ψ(gi) = Ψ(g1) - Ψ(g0) for all i = 0, 1, ..., k - 1. Hence (f1) −(f0) = (gk) −(g0) = (gk) −(gk−1) + (gk−1) −(gk−2) + · · · + (g1) −(g0) = k((g1) −(g0)). Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 11 of 17 Hence \|\|(f1) −(f0), (f2) −(f0), . . . , (fn) −(f0)\|\|β = \|\|k((g1) −(g0)), (f2) −(f0), . . . , (fn) −(f0)\|\|β = \|\|k(g1) −(g0), (f2) −(f0), . . . , (fn) −(f0)\|\|β = k. This completes the proof. This completes the proof. This completes the proof. This completes the proof. Lemma 4 4 Let h f f f be elements of ℑ(X) and let h f f be 2 collinear Then Lemma 4.4 Let h, f0, f1, ..., fn be elements of ℑ(X) and let h, f0, f1 be 2-collinear. Then \|\|f1 −h, f2 −h, . . 4. On the Mazur-Ulam problem , fn −f0 α = n0 \|\|f1 −f0, . . . , fn −f0\|\|α \|\|f1 −f0, . . . , fn −f0\|\|α = n0. It follows from Lemma 4.3 that \|\|(h) −(f0), . . . , (fn) −(f0)\|\|β = n0. Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 12 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 12 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 12 of 17 By the definition of h, h −f1 = n0 \|\|f1 −f0, . . . , fn −f0\|\|α −1 (f1 −f0). Since n0 \|\|f1 −f0, . . . , fn −f0\|\|α > 1, n0 \|\|f1 −f0, . . . , fn −f0\|\|α > 1, h - f1 and f1 - f0 have the same direction. It follows from Lemma 4.2 that h - f1 and f1 - f0 have the same direction. It follows from Lemma 4.2 that \|\|h −f0, f2 −f0, . . . , fn −f0\|\|α = \|\|h −f1, f2 −f0, . . . , fn −f0\|\|α + \|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α. Since Ψ(h), Ψ(f1), Ψ(f2) are 2-collinear, we have Since Ψ(h), Ψ(f1), Ψ(f2) are 2-collinear, we have \|\|(h) −(f1), (f2) −(f0), . . . , (fn) −(f0)\|\|β = \|\|(f1) −(h), (f2) −(h), . . . , (fn) −(h)\|\|β ≤\|\|f1 −h, f2 −h, . . . , fn −h\|\|α = \|\|f1 −h, f2 −f0, . . . , fn −f0\|\|α = n0 −\|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α ≤\|\|f1 −h, f2 −h, . . . , fn −h\|\|α = \|\|f1 −h, f2 −f0, . . . , fn −f0\|\|α = n0 −\|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α by Lemma 4.4. By the assumption, by Lemma 4.4. By the assumption, by Lemma 4.4. By the assumption, by Lemma 4.4. By the assumption, n0 = \|\|(h) −(f0), (f2) −(f0), . . . , (fn) −(f0)\|\|β ≤\|\|(h) −(f1), (f2) −(f0), . . . , (fn) −(f0)\|\|β + \|\|(f1) −(f0), (f2) −(f0), . . . , (fn) −(f0)\|\|β < n0 −\|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α + \|\|f1 −f0, f2 −f0, . . . , fn −f0\|\|α which is a contradiction. Hence Ψ is an n-isometry. 4. On the Mazur-Ulam problem Lemma 4.5 Let g0, g1 be elements of ℑ(X). Then v = g0 + g1 2 is the unique element of ℑ(X) satisfying 1 2\|\|g0 −gn, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = \|\|g1 −v, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = \|\|g0 −gn, g0 −v, g2 −gn, . . . , gn−1 −gn\|\|α for some g2,...,gn Î ℑ(X) with ∥g0 - gn, g1 - gn, g2 - gn, ..., gn-1 - gn∥a ≠0 and v, g0, g1 2- collinear. Proof. Let ∥g0 - gn, g1 - gn, g2 - gn, ..., gn-1 - gn∥a ≠ 0 and \|\|f0 −fn, f1 −fn, f2 −fn, . . . , fn−1 −fn\|\|α ̸= 0.. Then v, g0, g1 are 2-collinear. It follows from Lemma 4.1 and gn - g0 = g1 - g0 - (g1 - gn) that Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 13 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 13 of 17 \|\|g1 −v, g1 −gn, g2 −gn, . . . gn−1 −gn\|\|α = g1 g0 + g1 2 , g1 −gn, g2 −gn, . . . , gn−1 −gn α = 1 2\|\|g1 −g0, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = 1 2\|\|g0 −gn, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α and similarly and similarly \|\|g0 −gn, g0 −v, g2 −gn, . . . , gn−1 −gn\|\|α = 1 2\|\|g0 −gn, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α. Now we prove the uniqueness. Let u be an element of ℑ(X) satisfying the above properties. Since u, g0, g1 are 2-colli- near, there exists a real number t such that u = tg0 + (1 - t)g1. It follows from Lemma 4.1 that 1 2\|\|g0 −gn, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = \|\|g1 −u, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = \|\|g1 −(tg0 + (1 −t)g1), g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = \|t\| \|\|g1 −g0, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = \|t\| \|\|g0 −gn, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α and 1 2\|\|g0 −gn, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α = \|\|g0 −gn, g0 −u, g2 −gn, . . . 4. On the Mazur-Ulam problem , gn−1 −gn\|\|α = \|\|g0 −gn, g0 −(tg0 + (1 −t)g1), g2 −gn, . . . , gn−1 −gn\|\|α = \|1 −t\| \|\|g0 −gn, g0 −g1, g2 −gn, . . . , gn−1 −gn\|\|α = \|1 −t\| \|\|g0 −gn, g1 −gn, g2 −gn, . . . , gn−1 −gn\|\|α. Since ∥g0 - gn, g1 - gn, g2 - gn, ..., gn-1 - gn∥a ≠0, we have 1 2 = \|1 −t\| = \|t\|. Therefore, we get t = 1 2 and hence v = u. Lemma 4.6 If Ψ is an n-isometry and f0, f1, f2 are 2-collinear then Ψ(f0), Ψ(f1), Ψ(f2) are 2-collinear. Proof. Since dimℑ(X) ≥n, for any f0 Î ℑ(X), there exist g1,...,gn Î ℑ(X) such that g1 - f0, ..., gn - f0 are linearly independent. Then \|\|g1 −f0, . . . , gn −f0\|\|α = \|\|(g1) −(f0), . . . , (gn) −(f0)\|\|β ̸= 0 and hence, the set A = {Ψ(f) -Ψ(f0) : f Î ℑ(X)} contains n linearly independent vectors. Assume that f0, f1, f2 are 2-collinear. Then, for any f3,...,fn Î ℑ(X), \|\|f1 −f0, . . . , fn −f0\|\|α = \|\|(f1) −(f0), . . . , (fn) −(f0)\|\|β = 0, i.e. Ψ(f1) - Ψ(f0), ..., Ψ(fn) - Ψ(f0) are linearly dependent. Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 14 of 17 If there exist f3, ..., fn-1 such that Ψ(f1) - Ψ(f0), ..., Ψ(fn-1) - Ψ(f0) are linearly indepen- dent, then A = {(fn) −(f0) : fn ∈ℑ(X)} ⊂span {(f1) −(f0), . . . , (fn−1) −(f0)}, A = {(fn) −(f0) : fn ∈ℑ(X)} ⊂span {(f1) −(f0), . . . , (fn−1) −(f0)}, which contradicts the fact that A contains n linearly independent vectors. which contradicts the fact that A contains n linearly independent vectors. Then, for any f3, ..., fn-1, Ψ(f1) - Ψ(f0), ..., Ψ(fn-1) - Ψ(f0) are linearly dependent. If there exist f3, ..., fn-2 such that Ψ(f1) - Ψ(f0), ..., Ψ(fn-2) - Ψ(f0) are linearly indepen- dent, then which contradicts the fact that A contains n linearly independent vectors. Then, for any f3, ..., fn-1, Ψ(f1) - Ψ(f0), ..., Ψ(fn-1) - Ψ(f0) are linearly dependent. Then, for any f3, ..., fn-1, Ψ(f1) - Ψ(f0), ..., Ψ(fn-1) - Ψ(f0) are linearly dependent. 4. On the Mazur-Ulam problem If there exist f3, ..., fn-2 such that Ψ(f1) - Ψ(f0), ..., Ψ(fn-2) - Ψ(f0) are linearly indepen- dent, then A = {(fn−1) −(f0) : fn−1 ∈ℑ(X)} ⊂span {(f1) −(f0), . . . , (fn−2) −(f0)}, which contradicts the fact that A contains n linearly independent vectors. And so on, Ψ(f1) - Ψ(f0), Ψ(f2) - Ψ(f0) are linearly dependent. Thus Ψ(f0), Ψ(f1), and Ψ(f2) are 2-collinear. hich contradicts the fact that A contains n linearly independent vectors. which contradicts the fact that A contains n linearly independent vectors. which contradicts the fact that A contains n linearly independent vectors. And so on, Ψ(f1) - Ψ(f0), Ψ(f2) - Ψ(f0) are linearly dependent. Thus Ψ(f0), Ψ(f1), and Ψ(f2) are 2-collinear. And so on, Ψ(f1) - Ψ(f0), Ψ(f2) - Ψ(f0) are linearly dependent. Thus Ψ(f0), Ψ(f1), and Ψ(f2) are 2-collinear. Theorem 4.2 Every n-isometry mapping is affine. Theorem 4.2 Every n-isometry mapping is affine. Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 15 o Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 15 of 17 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 15 of 17 By Lemma 4.6, we obtain that f0 + f1 2 , Ψ(f0), and Ψ(f1) are 2-collinear. By Lemma 4.5, we get f0 + f1 2 = (f0) + (f1) 2 for all f, g Î ℑ(X) and a, b Î (0, 1). By Lemma 4.6, we obtain that f0 + f1 2 , Ψ(f0), and Ψ(f1) are 2-collinear. By Lemma 4.5, we get f0 + f1 2 = (f0) + (f1) 2 for all f, g Î ℑ(X) and a, b Î (0, 1). Since Ψ(0) = 0, we can easily show that Ψ is additive. It follows that Ψ is Q −linear-linear. Let r Î ℝ+ with r ≠1 and f Î ℑ(X). By Lemma 4.6, Ψ(0), Ψ(f) and Ψ(rf) are also 2- collinear. It follows from Ψ(0) = 0 that there exists a real number k such that Ψ(rf) = kΨ(f). Since dimℑ(X) ≥n, there exist f1, ..., fn-1 Î ℑ(X) such that ∥f, f1, f2, ..., fn-1∥a ≠0. Since Ψ(0) = 0, for every f0, f1, f2, ..., fn-1 Î ℑ(X), \|\|f0, f1, f2, . . . , fn−1\|\|α = \|\|f0 −0, f1 −0, f2 −0, . . . , fn−1 −0\|\|α = \|\|(f0) −(0), (f1) −(0), (f2) −(0), . . . , (fn−1) −(0)\|\|β = \|\|(f0), (f1), (f2), . . . , (fn−1)\|\|β. Thus we have r\|\|f, f1, f2, . . . , fn−1\|\|α = \|\|rf, f1, f2, . . . , fn−1\|\|α = \|\|(rf), (f1), (f2), . . . , (fn−1)\|\|β = \|\|k(f), (f1), (f2), . . . , (fn−1)\|\|β = \|k\| \|\|(f), (f1), (f2), . . . , (fn−1)\|\|β = \|k\| \|\|f, f1, f2, . . . , fn−1\|\|α. r\|\|f, f1, f2, . . . , fn−1\|\|α = \|\|rf, f1, f2, . . . , fn−1\|\|α = \|\|(rf), (f1), (f2), . . . , (fn−1)\|\|β = \|\|k(f), (f1), (f2), . . . , (fn−1)\|\|β = \|k\| \|\|(f), (f1), (f2), . . . , (fn−1)\|\|β = \|k\| \|\|f, f1, f2, . Theorem 4.2 Every n-isometry mapping is affine. Proof. Let Ψ be an n-isometry and F(f) = Ψ(f) - Ψ(0). Then F is an n-isometry and F (0) = 0. Thus we may assume that Ψ(0) = 0. Hence it suffices to show that Ψ is linear. Let f0, f1 Î ℑ(X) with f0 ≠f1. Since dimℑ(X) ≥n, there exist f2,...,fn Î ℑ(X) such that \|\|f0 −fn, f1 −fn, f2 −fn, . . . , fn−1 −fn\|\|α ̸= 0. Since Ψ is an n-isometry, we have \|\|(f0) −(fn), (f1) −(fn), (f2) −(fn), . . . , (fn−1) −(fn)\|\|β ̸= 0. It follows from Lemma 4.1 that It follows from Lemma 4.1 that (f0) −(fn), (f0) − f0 + f1 2 , (f2) −(fn), . . . , (fn−1) −(fn) β = (fn) −(f0), f0 + f1 2 −(f0), (f2) −(f0), . . . , (fn−1) −(f0) β = fn −f0, f0 + f1 2 −f0, f2 −f0, . . . , fn−1 −f0 α = 1 2\|\|fn −f0, f1 −f0, f2 −f0, . . . , fn−1 −f0\|\|α = 1 2\|\|(fn) −(f0), (f1) −(f0), (f2) −(f0), . . . , (fn−1) −(f0)\|\|β = 1 2\|\|(f0) −(fn), (f1) −(fn), (f2) −(fn), . . . , (fn−1) −(fn)\|\|β. And we get (f1) − f0 + f1 2 , (f1) −(fn), (f2) −(fn), . . . , (fn−1) −(fn) β = f0 + f1 2 −(f1), (fn) −(f1), (f2) −(f1), . . . , (fn−1) −(f1) β = f0 + f1 2 −f1, fn −f1, f2 −f1, . . . , fn−1 −f1 α = 1 2\|\|f0 −f1, fn −f1, f2 −f1, . . . , fn−1 −f1\|\|α = 1 2\|\|(f0) −(f1), (fn) −(f1), (f2) −(f1), . . . , (fn−1) −(f1)\|\|β = 1 2\|\|(f0) −(fn), (f1) −(fn), (f2) −(fn), . . . , (fn−1) −(fn)\|\|β. (f1) − f0 + f1 2 , (f1) −(fn), (f2) −(fn), . . . , (fn−1) −(fn) β = f0 + f1 2 −(f1), (fn) −(f1), (f2) −(f1), . . . , (fn−1) −(f1) β = f0 + f1 2 −f1, fn −f1, f2 −f1, . . . , fn−1 −f1 α 1 f f f f f f f f = 1 2\|\|(f0) −(f1), (fn) −(f1), (f2) −(f1), . . . , (fn−1) −(f1)\|\|β = 1 2\|\|(f0) −(fn), (f1) −(fn), (f2) −(fn), . . . , (fn−1) −(fn)\|\|β. Theorem 4.2 Every n-isometry mapping is affine. Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Park and Alaca Journal of Inequalities and Applications 2012, 2012:14 http://www.journalofinequalitiesandapplications.com/content/2012/1/14 Page 16 of 17 Since ∥rf - q2f, h1 - q2f, h2 - q2f, ..., hn-1 - q2f∥a ≠0, \|\|(f), (h1) −(q2f), (h2) −(q2f), . . . , (hn−1) −(q2f)\|\|β ̸= 0. Thus we have r + q2 <q2 - q1, which is a contradiction. Hence k = r, that is, Ψ(rf) = rΨ(f) for all positive real numbers r. Therefore Ψ is ℝ-linear, as desired. We get the following corollary from Theorems 4.1 and 4.2. Corollary 4.1 Let Ψ be an n-Lipschitz mapping with the n-Lipschitz constant ≤1. Suppose that if f, g, h are 2-collinear, then Ψ(f), Ψ(g), Ψ(h) are 2-collinear. If Ψ satisfies (nDOPP), then Ψ is an affine n-isometry. Authors’ contributions All h i d f All authors conceived of the study, participated in its design and coordination, drafted the manuscript, participated in the sequence alignment, and read and approved the final manuscript. 5. Conclusion In this article, the concept of 2-fuzzy n-normed linear space is defined and the con- cepts of n-isometry, n-collinearity, n-Lipschitz mapping are given. Also, the Mazur- Ulam theorem is generalized into 2-fuzzy n-normed linear spaces. Author details 1 Author details 1Department of Mathematics, Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Korea 2Department of Mathematics, Faculty of Science and Arts, Celal Bayar University, 45140 Manisa, Turkey Received: 24 May 2011 Accepted: 19 January 2012 Published: 19 January 2012 Received: 24 May 2011 Accepted: 19 January 2012 Published: 19 January 2012 Acknowledgements Acknowledgements The authors would like to thank the referees and area editor Professor Mohamed A. El-Gebeily for giving useful suggestions and comments for the improvement of this article. Theorem 4.2 Every n-isometry mapping is affine. . . , fn−1\|\|α. r\|\|f, f1, f2, . . . , fn−1\|\|α = \|\|rf, f1, f2, . . . , fn−1\|\|α = \|\|(rf), (f1), (f2), . . . , (fn−1)\|\|β = \|\|k(f), (f1), (f2), . . . , (fn−1)\|\|β = \|k\| \|\|(f), (f1), (f2), . . . , (fn−1)\|\|β = \|k\| \|\|f, f1, f2, . . . , fn−1\|\|α. = \|k\| \|\|f, f1, f2, . . . , fn−1\|\|α. Since ∥f, f1, f2, ..., fn-1∥a ≠0, \|k\| = r. Then Ψ(rf) = rΨ(f) or Ψ(rf) = -rΨ(f). First of all, assume that k = -r, that is, Ψ(rf) = -rΨ(f). Then there exist positive rational numbers q1, q2 such that o <q1 <r <q2. Since dimℑ(X) ≥n, there exist h1, ..., hn-1 Î ℑ(X) such that \|\|rf −q2f, h1 −q2f, h2 −q2f, . . . , hn−1 −q2f\|\|α ̸= 0. Then we have Then we have (q2 + r)\|\|(f), (h1) −(q2f), (h2) −(q2f), . . . , (hn−1) −(q2f)\|\|β (q2 + r)\|\|(f), (h1) −(q2f), (h2) −(q2f), . . . , (hn−1) −(q2f)\|\|β \|\|(q2 + r)(f), (h1) (q2f), (h2) (q2f), . . . , (hn−1) (q2f)\|\|β = \|\|q2(f) −(−r(f)), (h1) −(q2f), (h2) −(q2f), . . . , (hn−1) −(q2f)\|\|β = \|\|(rf) −(q2f), (h1) −(q2f), (h2) −(q2f), . . . , (hn−1) −(q2f)\|\|β = \|\|rf −q2f, h1 −q2f, h2 −q2f, . . . , hn−1 −q2f\|\|α = (q2 −r)\|\|f, h1 −q2f, h2 −q2f, . . . , hn−1 −q2f\|\|α ≤(q2 −q1)\|\|f, h1 −q2f, h2 −q2f, . . . , hn−1 −q2f\|\|α = \|\|q1f −q2f, h1 −q2f, h2 −q2f, . . . , hn−1 −q2f\|\|α = \|\|(q1f) −(q2f), (h1) −(q2f), (h2) −(q2f), . . . , (hn−1) −(q2f)\|\|β. 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https://openalex.org/W2764038501	https://zenodo.org/records/1138205/files/PK_article_14674.pdf	English	null	The Mosses of Crocker Range Park, Malaysian Borneo	PhytoKeys	2,017	cc-by	17,165	Keywords Bryophytes, CRP, Crocker Range, East Malaysia, Sabah Abstracth This paper reports the mosses from Crocker Range Park (CRP) in Sabah, Malaysian Borneo. In total, 293 species, three subspecies and eight varieties belonging to 118 genera and 36 families are reported. This represents about 40% and 47% of the species and infra-specific taxa reported from Borneo and Sabah, re spectively. Out of these, six species are new records for Borneo, namely Barbella horridula, Chaetomitrium lancifolium, Distichophyllum leiopogon, Rhaphidostichum luzonense, Rosulabryum capillare and Taxiphyllum taxirameum and 12 species and one variety are new to Sabah. With these additions, the current number of mosses in Sabah and Borneo are 651 and 766, respectively. The largest family of mosses is Calymperaceae with 35 species and one subspecies, followed by Sematophyllaceae with 32 species and two varieties and Pylaisiadelphaceae with 21 species and one variety. In conclusion, CRP has a very high species richness of mosses which is the second highest in Borneo, after Mount Kinabalu. rneo 71 Launched to accelerate biodiversity research A peer-reviewed open-access journal rneo 71 Launched to accelerate biodiversity research A peer-reviewed open-access journal The PhytoKeys 88: 71–107 (2017) doi: 10.3897/phytokeys.88.14674 http://phytokeys.pensoft.net The PhytoKeys 88: 71–107 (2017) doi: 10.3897/phytokeys.88.14674 http://phytokeys.pensoft.net Monica Suleiman1, Dunstan Polus Masundang1, Hiroyuki Akiyama2 1 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kina balu, Sabah, Malaysia 2 Museum of Nature and Human Activities & Phylogenetic Division, Institute of Na tural and Environmental Science, Hyogo Prefectural University, Yayoigaoka-6, Sanda, Hyogo 669-1546, Japan Corresponding author: Monica Suleiman (monicas@ums.edu.my) Academic editor: Y. Mutafchiev \| Received 23 June 2017 \| Accepted 20 September 2017 \| Published 11 October 2017 Citation: Suleiman M, Masundang DP, Akiyama H (2017) The Mosses of Crocker Range Park, Malaysian Borneo. PhytoKeys 88: 71–107. https://doi.org/10.3897/phytokeys.88.14674 Citation: Suleiman M, Masundang DP, Akiyama H (2017) The Mosses of Crocker Range Park, Malaysian Borneo. PhytoKeys 88: 71–107. https://doi.org/10.3897/phytokeys.88.14674 Copyright Monica Suleiman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The Mosses of Crocker Range Park, Malaysian Borneo Monica Suleiman1, Dunstan Polus Masundang1, Hiroyuki Akiyama2 Introduction Crocker Range Park (CRP) is located in the west coast of Sabah, East Malaysia in Bor neo (latitude 5°07' to 5°56'N and longitude 115°50' to 116°28'E). This park is about 110 km long and 15 km wide, covering an area of 139,919 ha, making it the largest terrestrial park and protected area in Sabah. This park was first designated as a For Copyright Monica Suleiman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) 72 est Reserve under the Forest Ordinance in 1969 but was subsequently converted to a State Park in 1984 for the conservation of natural resources and ecosystems, under the jurisdiction of Sabah Parks Trustees (Usui et al. 2006). In June 2014, Crocker Range was designated as a UNESCO Biosphere Reserve consisting of the whole area of CRP and the three forest reserves within the range. CRP, in the past, had received less attention from bryologists when compared to Kinabalu Park. These two parks are both on the Crocker Range which is the longest range in Sabah, extending from Kudat (northern tip of Borneo) to Sipitang (southern part of Sabah). CRP has become more accessible after the establishment of seven sub stations within the park between the years 2003 and 2005 and the opening of a new road system from Ulu Kimanis (western part) to Keningau Town (eastern part), cutting through the central part of the park. Another factor which may have contributed to the lesser attention received by CRP is the fact that its highest peak is only 2,076 m a.s.l., just half of that of Mount Kinabalu (4,059 m a.s.l.). Nevertheless, 27% of the total area of CRP is more than 1,000 m a.s.l., with 16 peaks above this height (Usui et al. 2006). To date, only two studies on mosses from this park have been published. Suleiman and Akiyama (2004) reported 126 species of mosses belonging to 74 genera and 27 fami lies, collected during the CRP Scientific Expedition in 2002 at Ulu Kimanis and adjacent areas within the elevations of 500–1,400 m a.s.l. Introduction Recently, Suleiman and Jotan (2015) reported 38 species and three varieties of mosses belonging to 17 genera and 11 families collected during a diversity study of epiphytic mosses along the Minduk Sirung Trail, a new 12 km trail connecting Mount Alab and Mahua substations (north-eastern part). In their study, mosses were collected from only three sampling areas of 20 m × 20 m. y y p g There are two other unpublished studies on mosses in CRP. The first one was by Kong (2006), who conducted a study on the diversity of mosses in Keningau Research Permanent Plot which is only 50 m × 50 m. She collected 40 species belonging to 26 genera and 14 families. The second one was by Chin (2008), who has studied the di versity of epiphytic mosses within 0–2 m of tree trunks, in the Mount Alab Permanent Research Plot (50 m × 50 m). She collected 20 species in 10 genera and seven families in this mossy forest (1,700–1,800 m a.s.l.). The present report attempts to produce a com prehensive checklist of mosses found in CRP based on collections from the year 2002 to 2008 and herbarium specimens deposited in the BORNEENSIS Herbarium of the Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah (BORH) and Herbarium of the Museum of Nature and Human Activities, Hyogo (HYO). Methods All specimens of mosses from the following 12 localities within the park were examined and identified. Areas covered are Inobong Visitor and Research Station, Mount Alab, Mile 32-Longkogungan Village, Longkogungan-Kuyungon Village, Salt Trail, Mahua Substa tion, Mount Minduk Sirung, CRP Headquaters, Ulu Senagang Substation, Melalap Sub station, Ulu Membakut Substation and Ulu Kimanis Substation (Figure 1). These locali The Mosses of Crocker Range Park, Malaysian Borneo 73 Figure 1. Map of Crocker Range Park showing the localities of collections from the year 2002 to 2008. Inset is map of Sabah, Malaysian Borneo. Figure 1. Map of Crocker Range Park showing the localities of collections from the year 2002 to 2008. Inset is map of Sabah, Malaysian Borneo. ties range from lowland to upper montane forests, covering secondary to primary forests, from 50 m to 2,000 m a.s.l. Details of the collection localities are listed in Table 1. Iden tified specimens were deposited at BORH and a set of duplicates were sent to the Her barium of Sabah Park (SNP). Some duplicates were also deposited at HYO, Herbarium of University of Malaya (KLU) and Herbarium of Royal Botanic Gardens Victoria (MEL). Results and discussion 2002. HA-Cr 1–467 Ulu Kimanis, CRP Headquaters and Mt. Alab pass, 27 Aug.–15 Sept. 2002. MS 1182–1244 Mahua Waterfall, 5°49.60'N, 116°23.11'E, 8-23 July 2003. MS 1245–1263 Salt Trail, Tikolod to Inobong, 5°39.62'N, 116°15.49'E to 5°51.51'N, 116°8.33'E, 23–28 Aug. 2003. MS 1386–1391 Mahua Waterfall, Nature Trail and trail to Minduk Sirung, 5°49.60'N, 116°23.11'E, 12–13 Dec. 2003. MS 1406–1407 Mt. Alab, above Gunung Emas Restaurant, 14 Dec. 2003. MS 1430–1461 Tenom, Melalap, trail to Tarangtali Hill, along Mesisilad River and Kallang Waterfall, 27–29 Jan. 2004. KWL 1–96 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116°06.16'E, 12 Oct. 2005. MS 1488–1489 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 5 July 2006. CMK 1–163 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 24–25 May 2007. Cr 1–467 Ulu Kimanis, CRP Headquaters and Mt. Alab pass, 27 Aug.–15 Sept. 2002. 1182–1244 Mahua Waterfall, 5°49.60'N, 116°23.11'E, 8-23 July 2003. 1263 Salt Trail, Tikolod to Inobong, 5°39.62'N, 116°15.49'E to 5°51.51'N, 116°8.33'E, 23–28 Aug. 2003. 391 Mahua Waterfall, Nature Trail and trail to Minduk Sirung, 5°49.60'N, 116°23.11'E, 12–13 Dec. 2003. 1407 Mt. Alab, above Gunung Emas Restaurant, 14 Dec. 2003. 1461 Tenom, Melalap, trail to Tarangtali Hill, along Mesisilad River and Kallang Waterfall, 27–29 Jan. 2004. KWL 1–96 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116°06.16'E, 12 Oct. 2005. MS 1488–1489 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 5 July 2006. KWL 1–96 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116°06.16'E, 12 Oct. 2005. MS 1488–1489 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 5 July 2006. –163 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 24–25 May 2007. DPM CMK 1–163 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 24–25 May 2007. MS & DPM MS & DPM 2357–2434 Mt. Alab, around Mt. Alab Garden, 5°49.31'E, 116°20.49'E, 13 Dec. 2007 DPM 2–112 Mt. Alab, vicinity of Mt. Alab Substation and Permanent Research Plot, 5°49.31'N, 116°20.49'E, 19–20 Jan. 2008. DPM 2–112 Mt. Alab, vicinity of Mt. Alab Substation and Permanent Research Plot, 5°49.31'N, 116°20.49'E, 19–20 Jan. 2008. Mahua, Mt. Minduk Sirung, 5°49.60'N, 116°23.11'E, 1–3 Apr. 2008. MS & DPM 2533–2712 Mahua, Mt. Minduk Sirung, 5°49.60'N, 116°23.11'E, 1–3 Apr. 2008. DPM 126–180 Ulu Senagang, trail to waterfall and along park boundary, 5°21.78'N, 116°1.72'E, 28–29 Aug. 2008. DPM 126–180 Ulu Senagang, trail to waterfall and along park boundary, 5°21.78'N, 116°1.72'E, 28–29 Aug. 2008. DPM 181–258 Inobong Visitor and Research Station, trail to waterfall and Buayan Village, 5°51.51'N, 116°8.33'E, 1–2 Sept. 2008. Results and discussion A total of 1,403 specimens of mosses from CRP were examined during this study. Amongst these, 293 species, three subspecies and eight varieties belonging to 118 gen era and 36 families were identified (Table 2 and Appendix 1). The five dominant fami lies of mosses in CRP are Calymperaceae with 35 species and one subspecies (11.8%), followed by Sematophyllaceae with 32 species and two varieties (11.2%), Pylaisiadel phaceae with 21 species and one variety (7.2%), Dicranaceae with 21 species (6.9%) and Daltoniaceae with 20 species (6.6 %). All of these families, except for Dicranaceae, are lowland families as ca. 70% of CRP land area is below 1,000m a.s.l. Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) 74 Table 1. Locality and collection details of mosses from Crocker Range Park from the year 2002 to 2008. CMK - Chin Mui Ken; DPM - Dunstan Polus Masundang; HA-Cr - Hiroyuki Akiyama-Crocker; KWL - Kong Wai Ling; MS - Monica Suleiman. Collection numbers Locality MS 877–1006 Ulu Kimanis, Mt. Rinangisan and surrounding areas, 5°28.15'N, 116°03.53'E, 27–30 Aug. 2002. HA-Cr 1–467 Ulu Kimanis, CRP Headquaters and Mt. Alab pass, 27 Aug.–15 Sept. 2002. MS 1182–1244 Mahua Waterfall, 5°49.60'N, 116°23.11'E, 8-23 July 2003. MS 1245–1263 Salt Trail, Tikolod to Inobong, 5°39.62'N, 116°15.49'E to 5°51.51'N, 116°8.33'E, 23–28 Aug. 2003. MS 1386–1391 Mahua Waterfall, Nature Trail and trail to Minduk Sirung, 5°49.60'N, 116°23.11'E, 12–13 Dec. 2003. MS 1406–1407 Mt. Alab, above Gunung Emas Restaurant, 14 Dec. 2003. MS 1430–1461 Tenom, Melalap, trail to Tarangtali Hill, along Mesisilad River and Kallang Waterfall, 27–29 Jan. 2004. KWL 1–96 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116°06.16'E, 12 Oct. 2005. MS 1488–1489 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 5 July 2006. CMK 1–163 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 24–25 May 2007. MS & DPM 2357–2434 Mt. Alab, around Mt. Alab Garden, 5°49.31'E, 116°20.49'E, 13 Dec. 2007. DPM 2–112 Mt. Alab, vicinity of Mt. Alab Substation and Permanent Research Plot, 5°49.31'N, 116°20.49'E, 19–20 Jan. 2008. MS & DPM 2533–2712 Mahua, Mt. Minduk Sirung, 5°49.60'N, 116°23.11'E, 1–3 Apr. 2008. DPM 126–180 Ulu Senagang, trail to waterfall and along park boundary, 5°21.78'N, 116°1.72'E, 28–29 Aug. 2008. DPM 181–258 Inobong Visitor and Research Station, trail to waterfall and Buayan Village, 5°51.51'N, 116°8.33'E, 1–2 Sept. 2008. MS & DPM 3776–3877 Mt. Alab, vicinity of Mt. Alab Substation, 5°49.31'N, 116°20.49'E, 8–10 Sept. Results and discussion 2008 MS & DPM 3878–3936 CRP Headquaters, Permanent Research Plot & Crocker Trail, 5°23.97'N, 116°06.16'E, 11 Sept. 2008. DPM 259–318 Ulu Membakut, along Membakut River and park boundary adjacent to Inantul Village, 5°20.97'N, 115°54.06'E, 18–20 Sept. 2008. MS & DPM 3937–4057 Longkogungan and Kuyungon Village, 5°49.97'N, 116°19.33'E to 5°42.56'N, 116°19.33'E, 22–23 Sept. 2008. MS & DPM 4058–4095 Bolotikon Village to Melungung Camp, 25 Sept. 2008. MS 4123–4130 Ulu Kimanis, Mt. Rinangisan and Permanent Research Plot, 5°28.15'N, 116°03.53'E, 13–14 Nov. 2008. MS 4131–4136 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116° 06.16'E, 19 Dec. 2008. Table 1. Locality and collection details of mosses from Crocker Range Park from the year 2002 to 2008. CMK - Chin Mui Ken; DPM - Dunstan Polus Masundang; HA-Cr - Hiroyuki Akiyama-Crocker; KWL - Kong Wai Ling; MS - Monica Suleiman. Collection numbers Locality MS 877–1006 Ulu Kimanis, Mt. Rinangisan and surrounding areas, 5°28.15'N, 116°03.53'E, 27–30 Aug. 2002. HA-Cr 1–467 Ulu Kimanis, CRP Headquaters and Mt. Alab pass, 27 Aug.–15 Sept. 2002. MS 1182–1244 Mahua Waterfall, 5°49.60'N, 116°23.11'E, 8-23 July 2003. MS 1245–1263 Salt Trail, Tikolod to Inobong, 5°39.62'N, 116°15.49'E to 5°51.51'N, 116°8.33'E, 23–28 Aug. 2003. MS 1386–1391 Mahua Waterfall, Nature Trail and trail to Minduk Sirung, 5°49.60'N, 116°23.11'E, 12–13 Dec. 2003. MS 1406–1407 Mt. Alab, above Gunung Emas Restaurant, 14 Dec. 2003. MS 1430–1461 Tenom, Melalap, trail to Tarangtali Hill, along Mesisilad River and Kallang Waterfall, 27–29 Jan. 2004. KWL 1–96 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116°06.16'E, 12 Oct. 2005. MS 1488–1489 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 5 July 2006. CMK 1–163 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 24–25 May 2007. MS & DPM 2357–2434 Mt. Alab, around Mt. Alab Garden, 5°49.31'E, 116°20.49'E, 13 Dec. 2007. DPM 2–112 Mt. Alab, vicinity of Mt. Alab Substation and Permanent Research Plot, 5°49.31'N, 116°20.49'E, 19–20 Jan. 2008. MS & DPM 2533–2712 Mahua, Mt. Minduk Sirung, 5°49.60'N, 116°23.11'E, 1–3 Apr. 2008. DPM 126–180 Ulu Senagang, trail to waterfall and along park boundary, 5°21.78'N, 116°1.72'E, 28–29 Aug. 2008. DPM 181–258 Inobong Visitor and Research Station, trail to waterfall and Buayan Village, 5°51.51'N, 116°8.33'E, 1–2 Sept. 2008. MS & DPM 3776–3877 Mt. Alab, vicinity of Mt. Alab Substation, 5°49.31'N, 116°20.49'E, 8–10 Sept. 2008 MS & DPM 3878–3936 CRP Headquaters, Permanent Research Plot & Crocker Trail, 5°23.97'N, 116°06.16'E, 11 Sept. 2008. DPM 259–318 Ulu Membakut, along Membakut River and park boundary adjacent to Inantul Village, 5°20.97'N, 115°54.06'E, 18–20 Sept. Results and discussion 2008. MS & DPM 3937–4057 Longkogungan and Kuyungon Village, 5°49.97'N, 116°19.33'E to 5°42.56'N, 116°19.33'E, 22–23 Sept. 2008. MS & DPM 4058–4095 Bolotikon Village to Melungung Camp, 25 Sept. 2008. MS 4123–4130 Ulu Kimanis, Mt. Rinangisan and Permanent Research Plot, 5°28.15'N, 116°03.53'E, 13–14 Nov. 2008. MS 4131–4136 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116° 06.16'E, 19 Dec. 2008. The species richness of mosses in the study area is very high; 40% of the total of 66 d f fi d f d 4 f h l f 6 Table 1. Locality and collection details of mosses from Crocker Range Park from the year 2002 to 2008. CMK - Chin Mui Ken; DPM - Dunstan Polus Masundang; HA-Cr - Hiroyuki Akiyama-Crocker; KWL - Kong Wai Ling; MS - Monica Suleiman. Collection numbers Locality MS 877–1006 Ulu Kimanis, Mt. Rinangisan and surrounding areas, 5°28.15'N, 116°03.53'E, 27–30 Aug. 2002. HA-Cr 1–467 Ulu Kimanis, CRP Headquaters and Mt. Alab pass, 27 Aug.–15 Sept. 2002. MS 1182–1244 Mahua Waterfall, 5°49.60'N, 116°23.11'E, 8-23 July 2003. MS 1245–1263 Salt Trail, Tikolod to Inobong, 5°39.62'N, 116°15.49'E to 5°51.51'N, 116°8.33'E, 23–28 Aug. 2003. MS 1386–1391 Mahua Waterfall, Nature Trail and trail to Minduk Sirung, 5°49.60'N, 116°23.11'E, 12–13 Dec. 2003. MS 1406–1407 Mt. Alab, above Gunung Emas Restaurant, 14 Dec. 2003. MS 1430–1461 Tenom, Melalap, trail to Tarangtali Hill, along Mesisilad River and Kallang Waterfall, 27–29 Jan. 2004. KWL 1–96 CRP Headquaters, Permanent Research Plot, 5°23.97'N, 116°06.16'E, 12 Oct. 2005. MS 1488–1489 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 5 July 2006. CMK 1–163 Mt. Alab, Permanent Research Plot, 5°49.31'N, 116°20.49'E, 24–25 May 2007. MS & DPM 2357–2434 Mt. Alab, around Mt. Alab Garden, 5°49.31'E, 116°20.49'E, 13 Dec. 2007. DPM 2–112 Mt. Alab, vicinity of Mt. Alab Substation and Permanent Research Plot, 5°49.31'N, 116°20.49'E, 19–20 Jan. 2008. MS & DPM 2533–2712 Mahua, Mt. Minduk Sirung, 5°49.60'N, 116°23.11'E, 1–3 Apr. 2008. DPM 126–180 Ulu Senagang, trail to waterfall and along park boundary, 5°21.78'N, 116°1.72'E, 28–29 Aug. 2008. DPM 181–258 Inobong Visitor and Research Station, trail to waterfall and Buayan Village, 5°51.51'N, 116°8.33'E, 1–2 Sept. 2008. Table 1. Locality and collection details of mosses from Crocker Range Park from the year 2002 to 2008. CMK - Chin Mui Ken; DPM - Dunstan Polus Masundang; HA-Cr - Hiroyuki Akiyama-Crocker; KWL - Kong Wai Ling; MS - Monica Suleiman. Collection numbers Locality MS 877–1006 Ulu Kimanis, Mt. Rinangisan and surrounding areas, 5°28.15'N, 116°03.53'E, 27–30 Aug. Results and discussion DPM 181–258 Inobong Visitor and Research Station, trail to waterfall and Buayan Village, 5°51.51'N, 116°8.33'E, 1–2 Sept. 2008. MS & DPM 3776–3877 Mt. Alab, vicinity of Mt. Alab Substation, 5°49.31'N, 116°20.49'E, 8–10 Sept. 2008 MS & DPM 3878–3936 CRP Headquaters, Permanent Research Plot & Crocker Trail, 5°23.97'N, 116°06.16'E, 11 Sept. 2008. M 259–318 Ulu Membakut, along Membakut River and park boundary adjacent to Inantul Village, 5°20.97'N, 115°54.06'E, 18–20 Sept. 2008. The species richness of mosses in the study area is very high; 40% of the total of 766 species and infra-specific taxa reported from Borneo and 47% of the total of 651 species and infra-specific taxa reported from Sabah (Andi and Suleiman 2005, Sulei man et al. 2006, 2009, 2011a, 2011b, 2017, Suleiman and Akiyama 2007, Higuchi et al. 2008, Akiyama 2010, Ho et al. 2010, Ellis et al. 2010, 2015, 2016a, 2016b, Andi et al. 2015, Chua and Suleiman 2015, Mohamed et al. 2010, Suleiman and Rimi 2016). The Mosses of Crocker Range Park, Malaysian Borneo 75 Table 2. Mosses reported from Crocker Range Park (See Appendix 1 for species checklist). No. Families Genera Species, subspecies and varieties 1. Bartramiaceae 1 3 spp. 2. Brachytheciaceae 3 4 spp. 3. Bryaceae 4 7 spp. 4. Calymperaceae 7 35 spp. and 1 subsp. 5. Cryphaeaceae 1 1 sp. 6. Daltoniaceae 6 20 spp. 7. Dicranaceae 8 21 spp. 8. Diphysciaceae 1 3 spp. 9. Ditrichaceae 1 1 sp. 10. Entodontaceae 3 3 spp. 11. Fissidentaceae 1 13 spp. and 1 var. 12. Garovagliaceae 1 4 spp. and 1 var. 13. Hookeriaceae 1 1 sp. 14. Hypnaceae 6 12 spp. 15. Hypnodendraceae 3 6 spp. 16. Hypopterygiaceae 3 4 spp. 17. Leskeaceae 2 2 spp. 18. Leucobryaceae 6 16 spp. and 2 var. 19. Leucomiaceae 1 1 sp. 20. Meteoriaceae 7 11 spp. 21. Mniaceae 1 3 spp. 22. Myuriaceae 1 1 sp. 23. Neckeraceae 7 14 spp. 24. Orthotrichaceae 2 7 spp. 25. Pilotrichaceae 4 5 spp. 26. Polytrichaceae 2 8 spp. and 2 subsp. 27. Pottiaceae 3 4 spp. 28. Pterobryaceae 7 9 spp. 29. Pylaisiadelphaceae 6 21 spp. and 1 var. 30. Racopilaceae 1 3 spp. and 1 var. 31. Regmatodontaceae 1 1 sp. 32. Rhizogoniaceae 2 4 spp. 33. Sematophyllaceae 10 32 spp. and 2 var. 34. Sphagnaceae 1 3 spp. 35. Symphyodontaceae 2 4 spp. 36. Thuidiaceae 2 6 spp. Total 118 293 spp., 3 subsp. Results and discussion Chaetomitrium lancifolium + + 5. Clastobryum scalare + 6. Distichophyllum leiopogon + + 7. Leucobryum javense var. cyathifolium + 8. Leucobryum juniperoideum + 9. Papillidiopsis malayana + 10. Rhaphidostichum luzonense + + 11. Rosulabryum capillare + + 12. Schoenobryum concavifolium + 13. Taxiphyllum taxirameum + + Total 6 13 Table 4. Moss species and infra-specific taxa reported from mountainous areas in Borneo. Geographical area Elevation Range (m a.s.l.) Number of moss species and infra-specific taxa % of moss species and infra-specific taxa Kinabalu Park 600-4,095 386 51 Crocker Range Park 50-2,076 304 40 Mount Trus Madi 600-2,642 194 26 Mount Lumaku 700-1,966 130 17 Table 3. New records of mosses to Borneo and Sabah. No. Moss species and variety New records Borneo Sabah 1. Acroporium macroturgidum + 2. Acroporium ramicola + 3. Barbella horridula + + 4. Chaetomitrium lancifolium + + 5. Clastobryum scalare + 6. Distichophyllum leiopogon + + 7. Leucobryum javense var. cyathifolium + 8. Leucobryum juniperoideum + 9. Papillidiopsis malayana + 10. Rhaphidostichum luzonense + + 11. Rosulabryum capillare + + 12. Schoenobryum concavifolium + 13. Taxiphyllum taxirameum + + Total 6 13 Table 3. New records of mosses to Borneo and Sabah. Table 4. Moss species and infra-specific taxa reported from mountainous areas in Borneo. Geographical area Elevation Range (m a.s.l.) Number of moss species and infra-specific taxa % of moss species and infra-specific taxa Kinabalu Park 600-4,095 386 51 Crocker Range Park 50-2,076 304 40 Mount Trus Madi 600-2,642 194 26 Mount Lumaku 700-1,966 130 17 Table 4. Moss species and infra-specific taxa reported from mountainous areas in Borneo. Several of the mosses found in CRP are of temperate entities and rarely reported in Borneo, namely Claopodium prionophyllum, Elmeriobryum philippinense, Entodon plicatus, Erythrodontium squarrosum, Leucomium strumosum, Mesonodon flavescens, Oxyrrhynchium vagans, Pseudoleskeopsis zippelii, Regmatodon declinatus and Schoenobryum concavifolium. Five of these species, namely Claopodium prionophyllum, Entodon plicatus, Erythrodontium squarrosum, Mesonodon flavescens and Oxyrrhynchium vagans, have only been collected once in Borneo (Dixon 1916, Iwatsuki and Noguchi 1975, Akiyama et al. 2001). Elmerio bryum philippinense was collected during the study and reported as new to Borneo by Ellis et al. (2016a). In addition, three species endemic to Borneo were also found in this park: Benitotania elimbata, Ectropothecium ptychofolium and Acroporium ramicola (Appendix 1). Crocker Range Park ranks the second highest (cf. Results and discussion and 8 var. Table 2. Mosses reported from Crocker Range Park (See Appendix 1 for species checklist). No. Families Genera Species, subspecies and varieties 1. Bartramiaceae 1 3 spp. 2. Brachytheciaceae 3 4 spp. 3. Bryaceae 4 7 spp. 4. Calymperaceae 7 35 spp. and 1 subsp. 5. Cryphaeaceae 1 1 sp. 6. Daltoniaceae 6 20 spp. 7. Dicranaceae 8 21 spp. 8. Diphysciaceae 1 3 spp. 9. Ditrichaceae 1 1 sp. 10. Entodontaceae 3 3 spp. 11. Fissidentaceae 1 13 spp. and 1 var. 12. Garovagliaceae 1 4 spp. and 1 var. 13. Hookeriaceae 1 1 sp. 14. Hypnaceae 6 12 spp. 15. Hypnodendraceae 3 6 spp. 16. Hypopterygiaceae 3 4 spp. 17. Leskeaceae 2 2 spp. 18. Leucobryaceae 6 16 spp. and 2 var. 19. Leucomiaceae 1 1 sp. 20. Meteoriaceae 7 11 spp. 21. Mniaceae 1 3 spp. 22. Myuriaceae 1 1 sp. 23. Neckeraceae 7 14 spp. 24. Orthotrichaceae 2 7 spp. 25. Pilotrichaceae 4 5 spp. 26. Polytrichaceae 2 8 spp. and 2 subsp. 27. Pottiaceae 3 4 spp. 28. Pterobryaceae 7 9 spp. 29. Pylaisiadelphaceae 6 21 spp. and 1 var. 30. Racopilaceae 1 3 spp. and 1 var. 31. Regmatodontaceae 1 1 sp. 32. Rhizogoniaceae 2 4 spp. 33. Sematophyllaceae 10 32 spp. and 2 var. 34. Sphagnaceae 1 3 spp. 35. Symphyodontaceae 2 4 spp. 36. Thuidiaceae 2 6 spp. Total 118 293 spp., 3 subsp. and 8 var. Table 2. Mosses reported from Crocker Range Park (See Appendix 1 for species checklist). Out of the 293 species, three subspecies and eight varieties of mosses in CRP, six are new to Borneo and 13 are new to Sabah (Table 3). Amongst the six species new to Borneo, four were found in the lowland areas between 70 m and 680 m a.s.l. Lowland areas in Borneo have not been given enough bryological attention, probably due to the miscon ception that the lowland rainforest has poor species richness of bryophytes. For instance, Chaetomitrium lancifolium, which was collected at 70 m a.s.l. in CRP, represents a second known record after its type collection from the Maluku Islands (see Appendix 1 for details). Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) 76 Table 3. New records of mosses to Borneo and Sabah. No. Moss species and variety New records Borneo Sabah 1. Acroporium macroturgidum + 2. Acroporium ramicola + 3. Barbella horridula + + 4. Results and discussion Table 4) in terms of number of mosses reported from mountainous areas in Borneo (Frahm et al. 1990, Suleiman and Edwards 2002, Suleiman and Akiyama 2004, Higuchi et al. 2008, Akiyama et al. 2001, Andi et al. 2015, Suleiman et al. 2011b). CRP recorded about 40% of the mosses re ported from Borneo although the highest point in CRP is only 2,076 m a.s.l. This indi cates that CRP has high species richness of mosses, second to that of Mount Kinabalu. Meanwhile, the number of mosses on Mount Trus Madi and Mount Lumaku were much lower, with 26% and 17%, respectively. Although Mount Trus Madi is much higher in terms of elevation, the number of mosses reported from the mountain was far lower than Several of the mosses found in CRP are of temperate entities and rarely reported in Borneo, namely Claopodium prionophyllum, Elmeriobryum philippinense, Entodon plicatus, Erythrodontium squarrosum, Leucomium strumosum, Mesonodon flavescens, Oxyrrhynchium vagans, Pseudoleskeopsis zippelii, Regmatodon declinatus and Schoenobryum concavifolium. Five of these species, namely Claopodium prionophyllum, Entodon plicatus, Erythrodontium squarrosum, Mesonodon flavescens and Oxyrrhynchium vagans, have only been collected once in Borneo (Dixon 1916, Iwatsuki and Noguchi 1975, Akiyama et al. 2001). Elmerio bryum philippinense was collected during the study and reported as new to Borneo by Ellis et al. (2016a). In addition, three species endemic to Borneo were also found in this park: Benitotania elimbata, Ectropothecium ptychofolium and Acroporium ramicola (Appendix 1). Crocker Range Park ranks the second highest (cf. Table 4) in terms of number of mosses reported from mountainous areas in Borneo (Frahm et al. 1990, Suleiman and Edwards 2002, Suleiman and Akiyama 2004, Higuchi et al. 2008, Akiyama et al. 2001, Andi et al. 2015, Suleiman et al. 2011b). CRP recorded about 40% of the mosses re ported from Borneo although the highest point in CRP is only 2,076 m a.s.l. This indi cates that CRP has high species richness of mosses, second to that of Mount Kinabalu. Meanwhile, the number of mosses on Mount Trus Madi and Mount Lumaku were much lower, with 26% and 17%, respectively. Although Mount Trus Madi is much higher in terms of elevation, the number of mosses reported from the mountain was far lower than The Mosses of Crocker Range Park, Malaysian Borneo 77 from CRP. Results and discussion Mount Lumaku, on the other hand, has a similar height to the highest peak of CRP but its species richness is only about half that of CRP. Two of the contributing factors are that CRP receives a high annual rainfall and it has a relatively larger area of pristine primary lowland forests than Mount Trus Madi and Mount Lumaku. Nonethe less, a diversity study should be carried out to determine the true diversity of these areas. Acknowledgements We would like to thank Sabah Parks for granting research permits to MS (2001–2008) and assistance given during field surveys. We are indebted to the late Dr. Benito C. Tan, Dr. Yong Kien Thai, Dr. Ho Boon Chuan and Dr. Niel Klazenga for helping to identify and verify some of the specimens. MS would like to thank Universiti Malaysia Sabah for funding her to visit Japan to study some specimens of mosses deposited in HYO. Conclusion CRP is a huge protected area and large parts of this park have not been surveyed during the present study. Thus, additional explorations in less accessible areas will definitely in crease the number of mosses in this park and provide a better understanding of the dis tribution of species within the park. The large area of lowland forests in CRP is an asset to this protected area as it harbours important species of mosses and other plants. Large areas of lowland forest in other parts of Borneo have been cleared for agriculture and development, adding to the importance of conservation of this UNESCO Biosphere Reserve. This study identifies CRP as one of the hotspots of moss diversity in Borneo. References Akiyama H (2010) Taxonomic revision of the genus Trismegistia (Pylaisiadelphaceae, Musci). Humans and Nature 21: 1–77. Akiyama H (2012) Contributions to the moss flora of Borneo, 2. Schoenobryum concavifolium (Cryphaeaceae, Musci), new to Borneo. Tropical Bryology 34: 12–14. 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Academy of Sciences Malaysia, Kuala Lumpur and Sabah Foundation, Kota Kinabalu, 135–156. Noguchi A (1976) A taxonomic revision of the family Meteoriaceae of Asia. Journal of the Hat tori Botanical Laboratory 41: 231–357. Noguchi A, Iwatsuki Z, Yamaguchi Y (1994) Illustrated moss flora of Japan. Part 5. Nichinan: Journal of the Hattori Botanical Laboratory, 1013–1253. Usui S, Sato H, Lee-Agama A, Chua R (2006) Crocker Range Management Plan. Sabah Parks, Kota Kinabalu. Suleiman M, Edward SR (2002) Mosses of Mt. Trus Madi, Sabah, Malaysia. Tropical Bryology 21: 57–64. Suleiman M, Akiyama H (2004) A preminary checklist of the mosses of Crocker Range Park. References In: Maryati M, Zulhazman H, Tachi T, Nais J (Eds) Crocker Range Scientific Expedition 2002. Universiti Malaysia Sabah, Kota Kinabalu, 1–15. Suleiman M, Akiyama H (2007) Checklist of mosses from Southern Part of Maliau Basin Conservation Area, Sabah, East Malaysia. Journal of Tropical Biology and Conservation 3: 67–75. Suleiman M, Akiyama H (2014) Malesian Chaetomitrium (Symphyodontaceae, Musci): Type illustrations, taxonomical notes and key to the species. Human and Nature 25: 1–62. http://www.hitohaku.jp/publication/r-bulletin/No25_01-1.pdf Suleiman M, Jotan P (2015) Diversity of epiphytic mosses along an altitudinal gradient at Min duk Sirung Trail in Crocker Range Park, Sabah, Malaysia. Sepilok Bulletin 21, 22: 49–58. Suleiman M, Rimi R (2016) The mosses of Gaya Island with two new records for Borneo. Sabah Parks Nature Journal 10: 1–8. Suleiman M, Akiyama H, Tan BC (2006) A revised catalogue of mosses reported from Borneo. Journal of the Hattori Botanical Laboratory 99: 107–183. https://www.researchgate.net/ publication/232732051_A_revised_catalogue_of_Mosses_reported_from_Borneo Suleiman M, Masundang DP, Tan BC (2009) A Checklist of mosses from Golden Hope Oil Palm Plantation and surrounding areas, Tawau, Sabah, East Malaysia. Journal of Tropical Biology and Conservation 5: 45–52. http://www.ums.edu.my/ibtpv2/images/publication/ JTBC/JTBC-VOL-5/5-monica%2024%20dec.pdf Suleiman M, Chua M-S, Fadzilah A-K (2011a) Mosses from the southern part of Imbak Can yon Conservation area. In: Latiff A, Sinun W (Eds) Imbak canyon Conservation Area, Sa Suleiman M, Chua M-S, Fadzilah A-K (2011a) Mosses from the southern part of Imbak Can yon Conservation area. In: Latiff A, Sinun W (Eds) Imbak canyon Conservation Area, Sa Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) 80 bah — Geology, Biodiversity and Socio-economic Environment. Akademi Sains Malaysia, Kuala Lumpur and Sabah Foundation, Kota Kinabalu, 269–281. bah — Geology, Biodiversity and Socio-economic Environment. Akademi Sains Malaysia, Kuala Lumpur and Sabah Foundation, Kota Kinabalu, 269–281. p Suleiman M, Fadzilah A-K, Masundang DP (2011b) The mosses of Mount Lumaku, Sipitang, Sabah, Malaysia. Tropical Bryology 33: 23–30. Suleiman M, Masundang DP, Akiyama A (2017) Thamnobryum negrosense (E.B. Bartram) Z. Iwats. & B. C. Tan (Neckeraceae, Musci), a new record for Borneo. Bryological Research 11(8): 229–231. Tan BC, Iwatsuki Z (1991) A new annotated Philippine moss checklist. Harvard Papers in Botany 3: 1–64. Tixier P (1977) Clastobryoïdées et taxa apparentés. Revue Bryologique et Lichénologique 43: 397–464. Yamaguchi T (1993) A revision of the genus Leucobryum (Musci) in Asia. Journal of the Hattori Botanical Laboratory 73: 1–123. Botanical Laboratory 73: 1–123. Bartramiaceae Philonotis bartramioides (Griff.) D.G. Griffin & W.R. Buck On boulders by river banks and road sides, 500–1580 m, DPM 128; MS & DPM 3939. Philonotis bartramioides (Griff.) D.G. Griffin & W.R. Buck On boulders by river banks and road sides, 500–1580 m, DPM 128; MS & DPM 3939. Philonotis bartramioides (Griff.) D.G. Griffin & W.R. Buck On boulders by river banks and road sides, 500–1580 m, DPM 128; MS & DPM 3939. Philonotis hastata (Duby) Wijk & Margad. On boulders, 385 m, MS 1447, 1448, 1458. Philonotis secunda (Dozy & Molk.) Bosch & Sande Lac. On soil by road sides and along trails in partially shaded and open areas, 680–1800 m, HA-Cr 140; MS 927; MS & DPM 3814, 3873 Appendix 1 Checklist of mosses from Crocker Range Park. ecklist of mosses from Crocker Range Park. The families, genera and species were arranged in alphabetical order. Species re ported for the first time for Sabah and Borneo are marked with ‘’ and ‘’, respec tively. CMK - Chin Mui Ken; DPM - Dunstan Polus Masundang; HA-Cr - Hiroyuki Akiyama-Crocker; KWL - Kong Wai Ling; MS - Monica Suleiman. Calymperes afzelii Sw. On boulder by river, open area, 550 m, MS & DPM 3996. Rhynchostegium javanicum (Bél.) Besch. On a wet rock beside waterfall, 980–1100 m, HA-Cr 292. On a wet rock beside waterfall, 980–1100 m, HA-Cr 292. y p Arthrocormus schimperi (Dozy & Molk.) Dozy & Molk. On humus, rotten logs, tree trunks and tree bases, 80–1145 m, DPM 200, 278, 311; KWL 51; MS 1445; MS & DPM 4064. Rhodobryum aubertii (Schwägr.) Thér. h On rock by a river, 1030 m, MS 1215, 1219. h On rock by a river, 1030 m, MS 1215, 1219. Rosulabryum rubens (Mitt.) J.R. Spence On soil in an open area, 50 m, DPM 295. Rosulabryum capillare (Hedw.) J.R. Spence Rosulabryum capillare (Hedw.) J.R. Spence On rotten log by a stream, 1600 m, MS & DPM 2695. On rotten log by a stream, 1600 m, MS & DPM 2695. Plants yellowish-red, forming lax tufts, 1.5 cm tall, matted with rhizoids at base. Leaves large, flaccid, spatulate, 2.0–2.7 mm × 0.5–0.7 mm; apex broad, rounded with an abruptly long piliform apiculus, arista 0.4–0.6 mm long, coloured; costa reddish, very strong at base, attenuate towards apex; margins revolute, plane 1/3 above, den ticulate in apical region, strongly bordered throughout by 1–4 rows of elongated cells, strongly thick-walled, reddish, Mid lamina cells rhomboidal, 49–54 μm × 17–25 μm, thin-walled, rectangular towards leaf base. Sporophyte not seen. This species is almost cosmopolitan in distribution but is not common in Malesia where it has been recorded previously only from New Guinea, the Philippines and Malaya. It is easily identified by the spatulate leaves with broadly and rounded apex and an abruptly long piliform apiculus as illustrated by Eddy (1996). Bryaceae Bryaceae Brachymenium nepalense Hook. On fallen logs, and tree and shrub trunks, 1150–1400 m, HA-Cr 191, 359; MS 946; MS & DPM 4033. Bryum coronatum Schwägr. Bryum coronatum Schwägr. On crevice by a road-side in open area, 1370 m, MS 925. On crevice by a road-side in open area, 1370 m, MS 925. Rhodobryum aubertii (Schwägr.) Thér. On rock by a river, 1030 m, MS 1215, 1219. Bryum apiculatum Schwägr. ryum apiculatum Sc wäg . On soil and boulders, 650–1800 m, MS & DPM 3875, 3991; HA-Cr 210. Bryum clavatum (Schimp.) Müll. Hal. On concrete in open area by road-side, 900 m, MS & DPM 3912. Bryum coronatum Schwägr. On crevice by a road-side in open area, 1370 m, MS 925. Brachytheciaceae Oxyrrhynchium vagans (A. Jaeger) Ignatov & Huttunen On a rock by a river, 1020 m, MS 1199. Rhynchostegiella vriesei (Dozy & Molk.) Broth. On a tree trunk, 940–1120 m, HA-Cr 263. Rhynchostegium celebicum (Sande Lac.) A. Jaeger On rotten logs and rocks, 400–1030 m, DPM 184; MS 1210. 81 81 The Mosses of Crocker Range Park, Malaysian Borneo Rhynchostegium javanicum (Bél.) Besch. Calymperaceae y p Arthrocormus schimperi (Dozy & Molk.) Dozy & Molk. On humus, rotten logs, tree trunks and tree bases, 80–1145 m, DPM 200, 278, 311; KWL 51; MS 1445; MS & DPM 4064. 82 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Calymperes fasciculatum Dozy & Molk. On a tree trunk, 1280 m, MS & DPM 3956. Calymperes lonchophyllum subsp. beccarii (Hampe) M. Menzel On rocks, roots and tree bases by streams, 70–100 m, DPM 259, 292, 308. Calymperes lonchophyllum subsp. beccarii (Hampe) M. Menzel On rocks, roots and tree bases by streams, 70–100 m, DPM 259, 292, 308. y p p y p p On rocks, roots and tree bases by streams, 70–100 m, DPM 259, 292, 308. Calymperes porrectum Mitt. On boulder and tree bases by streams, 100–680 m, DPM 135, 268; HA-Cr 154. Calymperes robinsonii B.C. Tan & W.D. Reese Calymperes robinsonii B.C. Tan & W.D. Reese On boulders and stone-wall by rivers, 410 m, MS 1457. On boulders and stone-wall by rivers, 410 m, MS 1457. Calymperes serratum A. Braun ex Müll. Hal. On shrub trunks, 680–1600 m, HA-Cr 141; MS & DPM 2675. Calymperes strictifolium (Mitt.) G. Roth Calymperes strictifolium (Mitt.) G. Roth Calymperes strictifolium (Mitt.) G. Roth On tree trunks and tree bases, 680 m, HA-Cr 139. On tree trunks and tree bases, 680 m, HA-Cr 139. Calymperes taitense (Sull.) Mitt. On tree root, 500 m, DPM 139. Calymperes taitense (Sull.) Mitt. On tree root, 500 m, DPM 139. Exostratum blumii (Nees ex Hampe) L.T. Ellis On boulders, roots, tree trunks and tree bases, 400–1400 m, DPM 194, 198, 202; HA-Cr 61, 257, 351, 395.; KWL 126a, 22b, 23, 25a, 26, 34, 43, 52a, 53a, 56, 60, 62, 84, 90, 93, 100b, 101, 114. Exostratum sullivantii (Dozy & Molk.) L.T. Ellis On a tree trunk, 1310 m, MS 981. Leucophanes angustifolium Renauld & Cardot On stone-walls, boulders, roots, tree trunks and bases, 100–1025 m, DPM 131, 155, 160, 162, 271; MS 1209; MS & DPM 3926, 3982. Leucophanes candidum (Schwägr.) Lindb. On rotten logs and tree trunks by river banks, 100 m, DPM 272, 313, 318. Leucophanes candidum (Schwägr.) Lindb. On rotten logs and tree trunks by river banks, 100 m, DPM 272, 313, 318. Leucophanes octoblepharioides Brid. On boulders, rotten logs, tree trunk, roots and tree stump, 80–1230 m, DPM 163, 165, 179, 180, 207, 267, 310; KWL 22a, 23, 25c, 93; 126b; HA-Cr 138, 389; MS 1435; MS & DPM 2533. The Mosses of Crocker Range Park, Malaysian Borneo 83 Mitthyridium fasciculatum (Hook. & Grev.) H. Rob. subsp. fasciculatum On rotten logs, treelet trunks and river bank, 100–1220 m, DPM 274; HA-Cr 181; MS 997. Mitthyridium fasciculatum subsp. cordotii (M. Fleisch.) B.C. Tan & L.T. Ellis On rotten and fallen logs, and tree trunks, 550–1145 m, DPM 147; HA-Cr 427; MS & DPM 3883. Mitthyridium repens (Harv.) H. Rob. Mitthyridium repens (Harv.) H. Rob. On decaying logs, tree trunks and tree bases, 400–800 m, DPM 205, 221; HA-Cr 121. Mitthyridium subluteum (Müll. Hal.) H.K. Nowak On climber, 1220 m, MS 996. Mitthyridium subluteum (Müll. Hal.) H.K. Nowak On climber, 1220 m, MS 996. Mitthyridium undulatum (Dozy & Molk.) H. Rob. On tree trunk beside streams, 950–1050 m, KWL 20a; MS 1212; MS & DPM 3932. Octoblepharum albidum Hedw. Growing on rotten logs, tree trunks and tree bases, 50–900 m, DPM 204, 222, 249, 281, 304; HA-Cr 317; MS 1431, 1434; MS & DPM 4013, 4014. Syrrhopodon albo-vaginatus Schwägr. On tree trunks and bases, and rotten logs, 50–1145 m, DPM 181, 156, 305; HA-Cr 422; KWL 95. Syrrhopodon aristifolius Mitt. On tree trunks and rotten logs, 650–1145 m, DPM 244, 257; KWL 91. Syrrhopodon ciliatus (Hook.) Schwägr. On rotten logs by river, 80–100 m, DPM 273, 309, 315, 316. Syrrhopodon confertus Sande Lac. On tree ferns, palm trees, tree trunks, tree bases and roots 610–1145 m, HA-Cr 423; KWL 2, 25b, 122; MS & DPM 3878. Syrrhopodon croceus Mitt. On rotten logs, 50–900 m, DPM 299; HA-Cr 202. Syrrhopodon gardneri (Hook.) Schwägr. On rotten logs, decaying logs, tree trunks and tree bases, 1100–1800 m, DPM 79; HA-Cr 326; MS 885; MS & DPM 3816, 3881. Syrrhopodon involutus Schwägr. On rotten logs, 705 m, MS 1432. 84 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Syrrhopodon japonicus (Besch.) Broth. On soil, climbers, tree trunks, bases and buttress, tree stumps and rotten logs, 1100–1800 m, HA-Cr 83, 91, 198, 321; MS 899, 924; MS & DPM 2547, 2615, 3830, 3950, 4030. Syrrhopodon laevis (Dixon) Mohamed & W.D. Reese Growing on rotten logs and tree trunks, 1700–1800 m, CMK 58, 52; DPM 25; MS & DPM 2616, 3791, 3817. Syrrhopodon loreus (Sande Lac.) W.D. Reese On roots, buttress, tree trunks and bases, 100–750 m, DPM 201, 266, 270; MS 4129; MS & DPM 4071. Syrrhopodon muelleri (Dozy & Molk.) Sande Lac. On tree trunks and bases, 1100–1300 m, HA-Cr 96; KWL 104; MS & DPM 2538, 3908. Syrrhopodon prolifer Schwägr. On soil, tree trunk and tree bases, 600–1800 m, DPM 229; CMK 84, 158; HA-Cr 363; MS & DPM 2706. Syrrhopodon spiculosus Hook. & Grev. On rotten logs, 600–1200 m, DPM 225; HA-Cr 87. Syrrhopodon spiculosus Hook. & Grev. On rotten logs, 600–1200 m, DPM 225; HA-Cr 87. Syrrhopodon tjibodensis M. Fleisch. Cryphaeaceae Schoenobryum concavifolium (Griff.) Gangulee Schoenobryum concavifolium (Griff.) Gangulee f On concrete in an open area, 800 m, MS & DPM 4054.h This species has been reported as new to Borneo based on a collection from Kaliman tan (Akiyama, 2012). This species has been reported as new to Borneo based on a collection from Kaliman tan (Akiyama, 2012). Mitthyridium repens (Harv.) H. Rob. On decaying logs, climbers and tree trunks, 1350–1800 m, MS 908, 919, 948; MS & DPM 3781, 4046. Syrrhopodon tristichus Nees ex Schwägr. On humus, tree stumps, rotten logs, shrub trunks and branches, tree branches and roots, 1370–1810 m, DPM 5, 67; HA-Cr 15, 199; MS 886, 923; MS & DPM 2541, 2624, 3855, 3942, 4043. Distichophyllum cuspidatum (Dozy & Molk.) Dozy & Molk. Distichophyllum cuspidatum (Dozy & Molk.) Dozy & Molk. On tree trunk and branches, shrub trunks and decaying logs, 1150–1800 m, CMK 163; DPM 58, 96a, 98, 108; HA-Cr 30b, 33, 74, 196, 336; MS 976; MS & DPM 2597, 2625, 3826. Daltoniaceae Achrophyllum javense (Dixon ex J. Froehl.) Z. Iwats., B.C. Tan & Touw On a wet boulder at streambed, 1600 m, MS & DPM 2693. Achrophyllum javense (Dixon ex J. Froehl.) Z. Iwats., B.C. Tan & Touw On a wet boulder at streambed, 1600 m, MS & DPM 2693. p y j ( J ) , u On a wet boulder at streambed, 1600 m, MS & DPM 2693. Benitotania elimbata H. Akiyama, T. Yamag. & Suleiman On tree trunks, 1800 m, MS & DPM 3825. Benitotania elimbata H. Akiyama, T. Yamag. & Suleiman On tree trunks, 1800 m, MS & DPM 3825. The Mosses of Crocker Range Park, Malaysian Borneo 8 85 Calyptrochaeta parviretis (M. Fleisch.) Z. Iwats., B.C. Tan & Touw Calyptrochaeta parviretis (M. Fleisch.) Z. Iwats., B.C. Tan & Touw On tree trunks, rotten logs, boulders and shrub branches, 680–1425 m, HA-Cr 150, 399, 408, 410; MS 961; MS & DPM 3898. Calyptrochaeta cf. ramosa (M. Fleisch.) B.C. Tan & H. Rob. On the base of tree trunk, 1300 m, HA-Cr 63, det. B.C. Ho. I h ll h h i i f h i b h l f b d h 3 4 f l Calyptrochaeta cf. ramosa (M. Fleisch.) B.C. Tan & H. Rob. It has all the characteristics of the species but the leaf border has 3–4 rows of elongated cells instead of 2–3 rows. Calyptrochaeta remotifolia (Müll. Hal.) Z. Iwats., B.C. Tan & Touw On fallen logs and boulders, 770–1800 m, HA-Cr 280; MS & DPM 3864, 4084. Daltonia armata E.B. Bartram On rotten logs, bamboo stump and tree trunks, 750–1350 m, HA-Cr 111; MS & DPM 3897, 3907, 4031, 4095. On rotten logs, bamboo stump and tree trunks, 750–1350 m, HA-Cr 111; MS & DPM 3897, 3907, 4031, 4095. Daltonia contorta Müll. Hal. Daltonia contorta Müll. Hal. On shrub trunks and branches, 1150–1400 m, HA-Cr 14, 192, 358. On shrub trunks and branches, 1150–1400 m, HA-Cr 14, 192, 358. Distichophyllum acuminatum Bosch & Sande Lac On shrubs, 1240–1360 m, HA-Cr 30a, 277, 346. Distichophyllum catinifolium J. Froehl. Distichophyllum catinifolium J. Froehl. On tree trunk and bases beside a stream, 1160 m, HA-Cr 396. On tree trunk and bases beside a stream, 1160 m, HA-Cr 396. Distichophyllum cirratum Renauld & Cardot On rocks, rotten logs and soil, 1100–1700 m, HA-Cr 29, 31, 32, 354, 355, 356, 375; MS & DPM 3854, 3892. Distichophyllum subcuspidatum Nog. & Z. Iwats. On trunk of a shrub, 1512 m, MS 921. Distichophyllum subcuspidatum Nog. & Z. Iwats. On trunk of a shrub, 1512 m, MS 921. Distichophyllum spathulatum (Dozy & Molk.) Dozy & Molk. On a rotten log, 1127 m, MS 1392. Distichophyllum cf. tortile Dozy & Molk. ex Bosch & Sande Lac. On a rotten log, 750 m, MS & DPM 4081. This specimen has all the characteristics of the species except for its leaf border which consists only of 1–2 rows of cells. Commonly, the species has 2–3 rows of cells. Distichophyllum cf. tortile Dozy & Molk. ex Bosch & Sande Lac. On a rotten log, 750 m, MS & DPM 4081. This specimen has all the characteristics of the species except for its leaf border which consists only of 1–2 rows of cells. Commonly, the species has 2–3 rows of cells. Ephemeropsis tjibodensis K.I. Goebel On palm, tree and shrub leaves by rivers, 750–1700 m, HA-Cr 20; MS & DPM 2666, 3843, 3887, 3976, 4074. Dicranaceae Braunfelsia dicranoides (Dozy & Molk.) Broth Braunfelsia dicranoides (Dozy & Molk.) Broth f ( y ) On tree trunk, tree base and humus, 1100–1200 m alt, HA-Cr 84, 182, 222. Distichophyllum leiopogon Dixon Growing on soil in partially shaded area, 1700 m, MS & DPM 3853, det. B.C. Ho. Leaves crisped when dry, spathulate, 3.0 mm × 1.2–1.3 mm, apex rounded to obtuse, with a small mucro, costa reaching 3/4 of leaf length, margin entire, border with 1–3 of cell. Lamina cells rectangular to hexagonal, 40–50 μm × 15–32 μm, thin walled. Calyptra smooth, fringed at base. Seta 7 mm, papillose throughout.h This species was previously recorded in the Philippines and New Guinea (Ho et al. 2010). Its cells near the leaf margin are only slightly smaller than the paracostal regions, distinguishing it from other species with spathulate or obovate leaf shapes. 86 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Distichophyllum malayense Damanhuri & Mohamed On fallen decaying tree trunks and rotten logs, 750–1800 m, HA-Cr 381, 357; MS & DPM 3858, 4040, 4073, 4075, 4077, 4080. Distichophyllum mittenii Bosch & Sande Lac. On rotten logs and tree roots, 750–1880 m, HA-Cr 7, 285, 353; MS 986; MS & DPM 2652, 2681, 2696, 3844, 4037, 4072, 4076, 4087. Distichophyllum nigricaule Mitt. ex Bosch & Sande Lac. On moist to wet rocks by streams 560 m, HA-Cr 304, 313. Distichophyllum osterwaldii M. Fleisch. On moist to wet rocks and boulders, and rotten logs, 750–1800 m, HA-Cr 100, 286, 377, 385; MS & DPM 3862, 4079. Dicranoloma assimile (Hampe) Paris On tree buttress, trunks and roots, rotten logs and soil, 1160–1760 m, MS 889, 1257; MS & DPM 2554, 2566; 2635, 2636, 2638, 3948, 3951, 4028. Campylopus serratus Sande Lac. Campylopus serratus Sande Lac. On soil and rotten logs, 500–700 m, DPM 217; MS & DPM 4062. Campylopus umbellatus (Schwägr. & Gaudich. ex Arn.) Paris On soil, crevice, gravel, concrete and humus, 900–1800 m, HA-Cr 22, 56, 208; MS 926, 952; MS & DPM 3801, 3813, 3938. Cryptodicranum armittii (Müll. Hal.) E.B. Bartram On tree trunk, 1700–1800 m, MS & DPM 2603, 3828. yp ( ) On tree trunk, 1700–1800 m, MS & DPM 2603, 3828. Dicranella setifera (Mitt.) A. Jaeger On wet soil in open areas, 680–1800 m, HA-Cr 113, 426; MS & DPM 3874. Dicranella setifera (Mitt.) A. Jaeger On wet soil in open areas, 680–1800 m, HA-Cr 113, 426; MS & DPM 3874. Dicranoloma assimile (Hampe) Paris On tree buttress, trunks and roots, rotten logs and soil, 1160–1760 m, MS 889, 1257; Dicranoloma assimile (Hampe) Paris O b k d l d l 6 6 MS Dicranoloma assimile (Hampe) Paris Dicranoloma billardierei (Brid.) Paris Dicranoloma billardierei (Brid.) Paris On humus and rotten logs, 1720–1800 m, MS & DPM 2610, 2640, 3821, 3792. Braunfelsia edentula (Mitt.) Wijk & Margad. Braunfelsia edentula (Mitt.) Wijk & Margad. On humus and shrub trunks, 1730–1800 m, MS & DPM 2606, 3803. On humus and shrub trunks, 1730–1800 m, MS & DPM 2606, 3803 On humus and shrub trunks, 1730–1800 m, MS & DPM 2606, 3803. Braunfelsia plicata (Sande Lac.) Broth. On fallen log, 1200–1730 m, MS & DPM 3836, 4025. Campylopus ericoides (Griff.) A. Jaeger On boulders and soil, 500–1000 m, DPM 212, 213, 216; MS & DPM 3993, 4053. 87 The Mosses of Crocker Range Park, Malaysian Borneo 87 Campylopus exasperatus (Nees & Blume) Brid. var. exasperatus On soil, 1800 m, DPM 110. Campylopus fragilis subsp. zollingerianus (Müll. Hal.) J.-P. Frahm On soil in an open area, 1150 m, HA-Cr 178. Campylopus laxitextus Sande Lac. On rotten branches and logs, and humus on tree bases, 1080–1800 m, MS & DPM 3815, 3900, 4049; HA-Cr 51, 187, 360. Dicranoloma blumii (Nees) Paris Dicranoloma blumii (Nees) Paris On trunks of shrubs and trees, 1100–1800 m, CMK 42, 151; HA-Cr 92; MS & DPM 2565, 2569, 3787. Dicranoloma braunii (Müll. Hal.) Paris On shrub and tree trunks, tree bases, roots and rotten stumps, 1080–1800 m, CMK 33, 123; KWL 59; MS & DPM 2536, 2560, 2605, 2621, 2622, 2671, 3905, 3937. Dicranoloma brevisetum (Dozy & Molk.) Paris On tree and shrub trunks, rotten logs and climbers, 1150–1870 m, CMK 33; CMK 131; DPM 3, 6, 81; HA-Cr 215, 320, 369; MS & DPM 2564, 2580, 2585, 2593, 2598, 2649, 2687, 3789, 3798, 3856, 4039. Dicranoloma brevisetum (Dozy & Molk.) Paris On tree and shrub trunks, rotten logs and climbers, 1150–1870 m, CMK 33; CMK 131; DPM 3, 6, 81; HA-Cr 215, 320, 369; MS & DPM 2564, 2580, 2585, 2593, 2598, 2649, 2687, 3789, 3798, 3856, 4039. 88 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Diphysciaceae h f l Diphysciaceae Diphyscium foliosum (Hedw.) D. Mohr On wet rock, 1400 m, HA-Cr 349. Diphyscium longifolium Griff. On rocks, 1200–1340 m, MS 974, 985, 1393. Diphyscium mucronifolium Mitt. On rocks and wet boulders, 560–1700 m, HA-Cr 42, 160, 161, 171, 308, 347; MS 938, 939; MS & DPM 3848, 3891. Ditrichaceae Garckea phascoides Müll. Hal. On road banks in sunny and open areas, 680–800 m, HA-Cr 116 Entodontaceae Entodon plicatus Müll. Hal. On soils, boulders, rocks, tree branches and rotten logs, 600–1130 m, MS 1194, 1221, 1243; MS & DPM 3994, 4006. Holomitrium cylindraceum (P. Beauv.) Wijk & Margad. On fallen log, 1090 m, MS 1388. Holomitrium cylindraceum (P. Beauv.) Wijk & Margad. On fallen log, 1090 m, MS 1388. Leucoloma molle (Müll. Hal.) Mitt. On boulders, tree and shrub trunks, 680–1600 m, HA-Cr 48, 155, 240; KWL 110; MS 1239, 1240, 2673, 3888, 4023. Leptotrichella brasiliensis (Duby) Ochyra. On rock by road side, 1340 m, MS 956. Leptotrichella brasiliensis (Duby) Ochyra. On rock by road side, 1340 m, MS 956. Leptotrichella miqueliana (Mont.) Lindb. ex Broth. On soil of trail banks, 680–950 m, HA-Cr 425; MS & DPM 3929. Entodontaceae Erythrodontium squarrosum (Hampe) Paris On a shrub trunk by a road side, 900 m, MS & DPM 3911. Erythrodontium squarrosum (Hampe) Paris Erythrodontium squarrosum (Hampe) Paris On a shrub trunk by a road side, 900 m, MS & DPM 3911. Mesonodon flavescens (Hook.) W.R. Buck On concrete, boulders and tree trunks in open areas, 600–800 m, MS & DPM 4003, 4005, 4055, 4057. 89 The Mosses of Crocker Range Park, Malaysian Borneo 89 Fissidentaceae Fissidens ceylonensis Dozy & Molk. On rocks, wet boulders and soil, 900–1700 m, HA-Cr 411; MS 975a; MS & DPM 3847, 3849, 3916. Garovaglia brachythecioides Nog. & Z. Iwats. Garovaglia brachythecioides Nog. & Z. Iwats. On fallen logs and tree branches, 650–1100 m, MS 3896; MS & DPM 4009. Garovaglia elegans (Dozy & Molk.) Hampe ex Bosch & Sande Lac. On shrub branches, tree trunks and fallen branches, 1320–1700 m, HA-Cr 391; MS 942, 958; MS & DPM 2591, 3850. Garovaglia plicata (Brid.) Bosch & Sande Lac. subsp. plicata On climber, shrub and tree trunks, and fallen branches, 1090–1750 m, MS 1389; MS & DPM 2631, 3824, 3852. Fissidens crassinervis Sande Lac. On rocks, 1370 m, MS 884. Fissidens crassinervis Sande Lac. On rocks, 1370 m, MS 884. Fissidens crenulatus var. elmeri (Broth.) Z. Iwats. & Tad. Suzuki Fissidens crenulatus var. elmeri (Broth.) Z. Iwats. & Tad. Suzuki On termite mount and rocks, 1080–1145 m, KWL 111, 124; MS & DPM 3902. Fissidens crenulatus var. elmeri (Broth.) Z. Iwats. & Tad. Suzuki On termite mount and rocks, 1080–1145 m, KWL 111, 124; MS & DPM 3902. ( ) termite mount and rocks, 1080–1145 m, KWL 111, 124; MS & DPM 3902 idens crispulus Brid. var. crispulus Fissidens crispulus Brid. var. crispulus Growing on rocks, roots, boulders, stone-walls, soils and rotten logs, 100–1120 m, DPM 117, 149, 182, 264, 282, 269; MS 1441; HA-Cr 125, 256, HA-Cr 429; MS 1450, 1460, 3978, 4059, 4069. Fissidens crispulus var. robinsonii (Broth.) Z. Iwats. & Z.H. Li On rock, soil and stone-wall by streams and road side, 100–680 m, DPM 133, 286, 287; HA-Cr 126. Fissidens geppii M. Fleisch. On wet boulders and rocks by waterfall, and on soil in disturbed area, 900–1120 m, HA-Cr 247, 250; MS & DPM 3921, 3925. Fissidens hollianus Dozy & Molk. On tree branches, rotten logs, boulders and tree bases, 400–1600 m, DPM 132, 203; MS & DPM 2698, 2700, 3917. Fissidens hyalinus Wilson & Hook. Growing on wet rocky cliff and rocks by waterfall, 900–1100 m, HA-Cr 293; MS & DPM 3924. Fissidens javanicus Dozy & Molk. On soil, roots, rocks and boulders at streambed and by rivers, 560–1020 m, HA-Cr 143, 316; MS 1197; MS & DPM 3998, 3999, 4068. Fissidens kinabaluensis Z. Iwats. On soil and termite mount 1100 m, MS & DPM 3886. Fissidens nobilis Griff. On soil and rocks along trail and river banks, 410–1250 m, DPM 134; HA-Cr 156; MS 1397; MS & DPM 3927, 3966, 4060. 90 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Fissidens pallidus Hook. f. & Wilson On soil and rock, 700–1300 m, DPM 253; HA-Cr 86; MS 989, 1002, 1252; MS & DPM 3952, 4020. Fissidens pallidus Hook. f. & Wilson On soil and rock, 700–1300 m, DPM 253; HA-Cr 86; MS 989, 1002, 1252; MS & DPM 3952, 4020. Fissidens taxifolius Hedw. On stream bank, 680 m, HA-Cr 153. Garovagliaceae Garovagliaceae Garovaglia angustifolia Mitt. var. angustifolia On tree trunks and branches and rotten branches, 770–1770 m, KWL 98; MS 1489; MS & DPM 3975, 4083. Garovaglia angustifolia var. bogorensis (M. Fleisch.) During On tree branches, and fallen and rotten logs, 650–1145 m, DPM 255; KWL 11, 67; MS 1205; MS & DPM 3977, 4008. Hookeriaceae Hookeria acutifolia Hook. & Grev. Hookeria acutifolia Hook. & Grev. On moist to wet boulders beside streams, 1160–1230 m, HA-Cr 383, HA-Cr 398. f On moist to wet boulders beside streams, 1160–1230 m, HA-Cr 383, HA-Cr 398 Ectropothecium ptychofolium N. Nishim. Ectropothecium ptychofolium N. Nishim. On tree trunk and base, and shrub branches, 1220–1800 m, DPM 74; HA-Cr 272, 468; MS 983; MS & DPM 2704. p p y f On tree trunk and base, and shrub branches, 1220–1800 m, DPM 74; HA-Cr 272, 468; MS 983; MS & DPM 2704. Ectropothecium striatulum Dixon ex E.B. Bartram On a rotten log and stone-wall, 500–1240 m, DPM 176; MS 1396. Ectropothecium cf. falciforme (Dozy & Molk.) A. Jaeger On tree trunks, 1800 m, CMK 40, 69; MS & DPM 3802. It has all the gametoph tic characters of the species but sporoph tes are not present to It has all the gametophytic characters of the species but sporophytes are not present to confirm its identity. Hypnodendraceae Dendro-hypnum beccarii Hampe On tree branches, tree trunks and shrub stems, 1370–1800 m, DPM 35, 80, 94; MS 929; MS & DPM 2658, 2583, 3822. Hypnaceae Hypnaceae Ectropothecium eleganti-pinnatum (Müll. Hal.) A. Jaeger On a rock, 400 m, DPM 197. Ectropothecium ichnotocladum (Müll. Hal.) A. Jaeger On shrub leaves, 1440–1600 m, HA-Cr 25; MS & DPM 2676, 2688. Ectropothecium moritzii A. Jaeger On a decaying log, 1800 m, DPM 60. Ectropothecium moritzii A. Jaeger On a decaying log, 1800 m, DPM 60. 91 The Mosses of Crocker Range Park, Malaysian Borneo Elmeriobryum philippinense Broth. On concrete and rocks by road sides in open areas, 1800 m, MS & DPM 3805, 3808, 3809. Pseudotaxiphyllum pohliaecarpum (Sull. & Lesq.) Z. Iwats. On soil and stone-wall, 600–1400 m, HA-Cr 23, 80; DPM 167, 227. Pseudotaxiphyllum pohliaecarpum (Sull. & Lesq.) Z. Iwats. Pseudotaxiphyllum pohliaecarpum (Sull. & Lesq.) Z. Iwats. Pseudotaxiphyllum pohliaecarpum (Sull. & Lesq.) Z. Iwats. p y p p On soil and stone-wall, 600–1400 m, HA-Cr 23, 80; DPM 167, 227. Taxiphyllum taxirameum (Mitt.) M. Fleisch. Taxiphyllum taxirameum (Mitt.) M. Fleisch. Taxiphyllum taxirameum (Mitt.) M. Fleisch. On rocks beside stream, 400 m, DPM 188, det. B.C. Tan & B.C. Ho.l Plant small, stems long-creeping, flattened, yellowish-green. Leaves ovate-lanceolate, 1.1–1.2 mm × 0.4 mm, apex gradually finely acuminate, costa short, double and indis tinct, inflexed on basal part, margin denticulate. Lamina cells long-rhomboidal, 40–52 μm × 5–7 μm, thin-walled. Sporophyte not seen.h This species has a wide distribution and has been recorded in Java, Malaya, Singa pore, Sumatra and the Philippines. It is characterised by its complanate and spreading leaves, with elongated stems as illustrated by Noguchi et al. (1994). Trachythecium verrucosum (A. Jaeger) M. Fleisch. On soil, 940–1120 m, HA-Cr 246; MS 1226. Trachythecium verrucosum (A. Jaeger) M. Fleisch. On soil, 940–1120 m, HA-Cr 246; MS 1226. Vesicularia dubyana (Müll. Hal.) Broth. Vesicularia dubyana (Müll. Hal.) Broth. On rocks beside stream, 130 m, DPM 284. On rocks beside stream, 130 m, DPM 284. Vesicularia reticulata (Dozy & Molk.) Broth. On rocks and stone-wall, 400–450 m, DPM 211, 183, 190. Hypnodendraceae Dendro-hypnum beccarii Hampe On tree branches, tree trunks and shrub stems, 1370–1800 m, DPM 35, 80, 94; MS 929; MS & DPM 2658, 2583, 3822. Mniodendron dendroides (Brid.) Wijk & Margad. Mniodendron dendroides (Brid.) Wijk & Margad. On shrub and tree branches, roots and rotten logs, 1240–1800 m, DPM 59, 62, 63, 73, 84; HA-Cr 18, 76, 281; MS & DPM 2685, 3947. Touwiodendron diversifolium (Broth. & Geh.) N.E. Bell, A.E. Newton & D. Quandt On rotten logs and soils, 1160–1750 m, HA-Cr 380, 402; MS 934, 936, 1404; MS & DPM 2563, 3834, 3945, 4041. Hypnodendraceae 92 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Dendro-hypnum fuscomucronatum (Müll. Hal.) N.E. Bell, A.E. Newton & D. Quandt On boulders and rocks by streams and rivers, 1000–1030 m, G.Gunsalam s.n.; MS 1183, 1184, 1186, 1236. Dendro-hypnum milnei (Mitt.) N.E. Bell, A.E. Newton & D. Quandt On rocks and boulders by rivers, 680–1230 m, HA-Cr 132, 384; MS 1234, 1227, 1250; MS & DPM 3963, 4092. Dendro-hypnum subspininervium subsp. arborescens (Mitt.) N.E. Bell, A.E. Newton & D. Quandt Dendro-hypnum subspininervium subsp. arborescens (Mitt.) N.E. Bell, A.E. Newton & D. Quandt On roots, stumps, tree trunks and boulders, 100–1360 m, DPM 289; HA-Cr 26, 168; MS & DPM 4070. Q On roots, stumps, tree trunks and boulders, 100–1360 m, DPM 289; HA-Cr 26, 168; MS & DPM 4070. Leucobryum javense var. cyathifolium (Dixon) T. Yamag. y j y f g On humus, 1800 m, MS & DPM 3786. This is the second report of this variety in Borneo; the first one was from Mount Mulu, Sarawak (Yamaguchi, 1993). This is the second report of this variety in Borneo; the first one was from Mount Mulu, Sarawak (Yamaguchi, 1993). This is the second report of this variety in Borneo; the first one was from Mount Mulu, Sarawak (Yamaguchi, 1993). Leucobryum aduncum Dozy & Molk. var. aduncum Leucobryum aduncum Dozy & Molk. var. aduncum On soil, roots, rotten logs, tree trunks and bases, 50–1400 m, DPM 164, 218, 294, 296; KWL 15a; MS & DPM 2545, 2710, 3946, 4050. Leucobryum aduncum var. scalare (Müll. Hal. ex M. Fleisch.) A. Eddy On soil, root, rotten logs and tree trunks, 550–1450 m, DPM 175, 154, 220, 231, 232, 243; HA-Cr 135, 372; KWL 81; MS 998, 1430; MS & DPM 2555, 4012. Leucobryum aduncum var. scalare (Müll. Hal. ex M. Fleisch.) A. Eddy On soil, root, rotten logs and tree trunks, 550–1450 m, DPM 175, 154, 220, 231, 232, 243; HA-Cr 135, 372; KWL 81; MS 998, 1430; MS & DPM 2555, 4012. Leucobryum arfakianum Müll. Hal. ex Geh. On soil, tree trunks and tree bases, 750–1700 m, MS 902, 903, 913, 918, 920, 1245; MS & DPM 2618. Leucobryum bowringii Mitt. Hypopterygiaceae Hypopterygiaceae Cyathophorum spinosum (Müll. Hal.) M. Fleisch. On rotten logs, boulders and shrub trunks, 830–1100 m, MS 1230, 1391; MS & DPM 3988. Hypopterygium tamarisci (Sw.) Brid. ex Müll. Hal. On rocks, boulders and rotten logs, 500–1600 m, DPM 161; HA-Cr 130, 268; MS 1202; MS & DPM 2686, 3919, 3961. Hypopterygium vriesei Bosch & Sande Lac. On boulders, 650–830 m, DPM 170; MS & DPM 3986. Lopidium struthiopteris (Brid.) M. Fleisch. On shrub and tree trunks, roots and rotten logs, 560–1600 m, HA-Cr 244, 299; KWL 86; MS & DPM 2539, 1229, 1394, 2692, 3933. y Bryohumbertia subcomosa (Dixon) J.-P. Frahm On rotten logs, stumps, humus and soil, 1350–1850 m, DPM 4; HA-Cr 361; MS & DPM 2647b, 3796, 4048. On rotten logs, stumps, humus and soil, 1350–1850 m, DPM 4; HA-Cr 361; MS & DPM 2647b, 3796, 4048. Campylopus comosus (Schwägr.) Bosch & Sande Lac. On soil, 600–1150 m, DPM 230; HA-Cr 188. Campylopus comosus (Schwägr.) Bosch & Sande Lac. On soil, 600–1150 m, DPM 230; HA-Cr 188. The Mosses of Crocker Range Park, Malaysian Borneo 93 Leucobryum bowringii Mitt. On humus and rotten logs, 1280–1600 m, MS & DPM 2669, 3954, 4045. Dicranodontium uncinatum (Harv.) A. Jaeger On humus and tree trunks, 1800–1900 m, MS & DPM 2642, 2647a, 2653, 3869. Leucobryum chlorophyllosum Müll. Hal. Leucobryum chlorophyllosum Müll. Hal. On soil, rotten logs, tree trunks and stump, 400–1150 m, DPM 208, 219, 226, 233, 234, 251; KWL 100a, 53b; MS 1439, 3880; MS & DPM 4015, 4052. Leucobryum javense (Brid.) Mitt. var. javense On soil, humus, rotten logs, tree trunks, roots and climbers, 600–1800 m, CMK 46, 70; DPM 27, 31, 224, 228, 236, 246, 250, 258; HA-Cr 400; MS 880, 892, 911; MS & DPM 1253, 2617, 2627, 3867,4091, 4011. Leucobryum javense (Brid.) Mitt. var. javense On soil, humus, rotten logs, tree trunks, roots and climbers, 600–1800 m, CMK 46, 70; DPM 27, 31, 224, 228, 236, 246, 250, 258; HA-Cr 400; MS 880, 892, 911; MS & DPM 1253, 2617, 2627, 3867,4091, 4011. Leucobryum javense (Brid.) Mitt. var. javense On soil, humus, rotten logs, tree trunks, roots and climbers, 600–1800 m, CMK 46, 70; DPM 27, 31, 224, 228, 236, 246, 250, 258; HA-Cr 400; MS 880, 892, 911; MS & DPM 1253, 2617, 2627, 3867,4091, 4011. Leucobryum javense (Brid.) Mitt. var. javense Leucobryum javense var. cyathifolium (Dixon) T. Yamag. Leucobryum javense var. cyathifolium (Dixon) T. Yamag. Pseudoleskeopsis zippelii (Dozy & Molk.) Broth. Pseudoleskeopsis zippelii (Dozy & Molk.) Broth. On wet boulders in river beds, 1010–1030 m, MS 1193, 1213. Leucomiaceae Leucomium strumosum (Hornsch.) Mitt. On moist boulder, 1120 m, HA-Cr 258. On moist boulder, 1120 m, HA-Cr 258. Schistomitrium apiculatum (Dozy & Molk.) Dozy & Molk. Schistomitrium apiculatum (Dozy & Molk.) Dozy & Molk. On tree branch, 1150–1770 m, DPM 102; HA-Cr 233. Schistomitrium apiculatum (Dozy & Molk.) Dozy & Molk. On tree branch, 1150–1770 m, DPM 102; HA-Cr 233. Schistomitrium mucronifolium (A. Braun in Müll. Hal.) M. Fleisch. On climbers, branches, tree trunks and rotten logs, 1100–1800 m, DPM 21, 32, 34; HA-Cr 79, 183; MS 995; MS & DPM 2578, 2705, 3795, 3835. Schistomitrium robustum Dozy & Molk. On treelet trunk, 1700 m, MS & DPM 2600, 2604. Leucobryum juniperoideum (Brid.) Müll. Hal. Leucobryum juniperoideum (Brid.) Müll. Hal. On humus and tree bases, 1150–1800 m, MS & DPM 2534, 3870. In Borneo, this species has been previously reported from Kalimantan and Sarawak. It is widespread in Europe, Macronesia, Madagasca, Turkey, Caucasus, Asia and Malesia (Yamaguchi, 1993). In Borneo, this species has been previously reported from Kalimantan and Sarawak. It is widespread in Europe, Macronesia, Madagasca, Turkey, Caucasus, Asia and Malesia (Yamaguchi, 1993). Leucobryum sanctum (Nees ex Schwägr.) Hampe On humus, soil, root and rotten logs, 50–1400 m, DPM 152, 254, 261, 298; HA-Cr 6; MS 969, 1249, 1263, 1433, 1437, 1440, 1444; MS & DPM 4044, 4047, 4067. Leucobryum sanctum (Nees ex Schwägr.) Hampe On humus, soil, root and rotten logs, 50–1400 m, DPM 152, 254, 261, 298; HA-Cr 6; MS 969, 1249, 1263, 1433, 1437, 1440, 1444; MS & DPM 4044, 4047, 4067. 94 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Leucobryum scabrum Sande Lac. On tree trunks, 1400–1800 m, CMK 60, 115; HA-Cr 371. y On tree trunks, 1400–1800 m, CMK 60, 115; HA-Cr 371. Leucobryum sumatranum Broth. ex M. Fleisch. Meteoriaceae Aerobryopsis crispifolia (Broth. & Geh.) M. Menzel O k d f ll l d b h 850 1425 MS 945 957 1248 Aerobryopsis crispifolia (Broth. & Geh.) M. Menzel Aerobryopsis longissima (Dozy & Molk.) M. Fleisch. On twigs, shrub branches, rotten logs and tree trunks, 100–1400 m, DPM 275, 277; HA-Cr 50, 77; MS 949; MS & DPM 3893, 3957. Barbella flagellifera (Cardot) Nog. Barbella horridula Broth. On a tree trunk, 550 m, DPM 146. Plants yellowish-green, laxly branched, branches strongly complanate, 1–3 cm long and 5 mm wide, sparsely leaved, often with long flagellae at tips. Branch leaves spread Leskeaceae Claopodium prionophyllum (Müll. Hal.) Broth. On boulders and soils, 940–1260 m, HA-Cr 245, 269; MS 1191, 1399; MS & DPM 3960. Leucobryum sumatranum Broth. ex M. Fleisch. On humus, roots and rotten logs, 1100–1620 m, HA-Cr 93; MS 992, 1254; MS & DPM 2586, 3955, 4029. Schistomitrium apiculatum (Dozy & Molk.) Dozy & Molk. On tree branch, 1150–1770 m, DPM 102; HA-Cr 233. Pseudobarbella ancistrodes (Renauld & Cardot) Manuel On a shrub branch by rivers, 850 m, MS & DPM 3984b, 3973. On a shrub branch by rivers, 850 m, MS & DPM 3984b, 3973. Barbella horridula Broth. The Mosses of Crocker Range Park, Malaysian Borneo 95 ing, narrowly linear-lanceolate, 3.2–3.3 mm × 0.4–0.5 mm, slightly plicate, apex gradually acuminate; margin serulate throughout, recurved on one side at the base; costa single, faint and reaching half of midleaf. Mid lamina cells linear, 108–113 μm × 10–12, rather thin-walled, sometimes with minute 1 (–2) papillae adaxially, long- rhomboidal and thick-walled across insertion. Sporophyte not seen.h This species was previously reported from the Philippines and Sumatra. It can be recognised by its strongly complanate foliation, laxly branched, and linear-lanceolate leaves which are gradually attenuate. The minute papillae are difficult to observe and absent in some leaves. Barbella horridula can be distinguished from B. stevensii (Renauld et Cardot) M. Fleisch. in Broth. from the former hyaline lamina cells (No guchi, 1976). Cryptopapillaria fuscescens (Hook.) M. Menzel On shrub and tree branches and treelet trunks, 1070–1700 m, HA-Cr 415; MS 971, 1006, 1233; MS & DPM 3840, 3941. Floribundaria floribunda (Dozy & Molk.) M. Fleisch. On termite mount and shrub branches, 850–1080 m, HA-Cr 207; MS 928; MS & DPM 3903, 3968. Floribundaria pseudofloribunda M. Fleisch. Floribundaria pseudofloribunda M. Fleisch. On tree trunks, treelet stumps, rocks, boulders, stone-wall, climbers and soil, 550–1090 m, DPM 129, 158, 159, 169; MS 1189, 1231, 1232, 1237; MS & DPM 3915, 3990, 3997. Meteoriopsis reclinata (Müll. Hal.) M. Fleisch. On a shrub trunk by road side, 900 m, MS & DPM 3913. Meteoriopsis reclinata (Müll. Hal.) M. Fleisch. On a shrub trunk by road side 900 m MS & DPM 3913 Meteoriopsis reclinata (Müll. Hal.) M. Fleisch. On a shrub trunk by road side, 900 m, MS & DPM 3913. Meteorium polytrichum Dozy & Molk. On tree and shrub branches, and fallen logs, 750–1600 m, MS 1192, 1211, 1390; MS & DPM 2665, 2679, 3972, 3984a, 3985, 4094. Pseudobarbella ancistrodes (Renauld & Cardot) Manuel On a shrub branch by rivers, 850 m, MS & DPM 3984b, 3973. Floribundaria intermedia Thér. h On shrub trunks and leaves, tree branches and buttress, 580–1200 m, MS & DPM 2535, 3987, 4000, 4086. Plagiomnium succulentum (Mitt.) T.J. Kop. Plagiomnium succulentum (Mitt.) T.J. Kop. On wet boulders beside waterfall, 900–1030 m, HA-Cr 254; MS 1207, 1216; MS & DPM 3922. Myuriaceae Oedicladium rufescens (Reinw. & Hornsch.) Mitt. On a decaying log, 1500 m, MS 1403. Mniaceae Plagiomnium integrum (Bosch & Sande Lac.) T.J. Kop. Plagiomnium integrum (Bosch & Sande Lac.) T.J. Kop. Plagiomnium integrum (Bosch & Sande Lac.) T.J. Kop. On rocks and tree roots, 640–1240 m, HA-Cr 163, 283. On rocks and tree roots, 640–1240 m, HA-Cr 163, 283. Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) 96 Neckeraceae Circulifolium exiguum (Bosch & Sande Lac.) S. Olsson, Enroth & D. Quandt On tree trunks, shrub trunks and bases, roots and rotten logs, 500–1021 m, DPM 157; HA-Cr 122; MS 1200, 1201; MS & DPM 4078. Circulifolium microdendron (Mont.) S. Olsson, Enroth & D. Quandt On stone-walls, boulders, rocks, tree trunks and stumps, 550–1160 m, DPM 143; HA-Cr 129; MS 1244; MS & DPM 3931, 3958. Himantocladium cyclophyllum (Müll. Hal.) M. Fleisch. On soil, stone-walls, tree trunks, shrub trunks and boulders, 100–1600 m, DPM 171, 178, 263, 312; MS 1442; MS & DPM 2659, 3959. Himantocladium plumula (Nees) M. Fleisch. On boulders, rocks, and tree trunks, branches, twigs, bases and roots, 100–1145 m, DPM 136, 166, 168, 193, 283, 290; HA-Cr 146, 159, 267; KWL 16, 41, 63; MS 1190; MS & DPM 3910, 3928, 3970, 4085. Himantocladium warburgii (Broth.) M. Fleisch. On tree trunks, 1300–1650 m, HA-Cr 58, 461. Himantocladium warburgii (Broth.) M. Fleisch. On tree trunks, 1300–1650 m, HA-Cr 58, 461. Homaliodendron flabellatum (Sm.) M. Fleisch. Homaliodendron flabellatum (Sm.) M. Fleisch. On tree and shrub trunks, and rotten logs, 1100–1700 m, HA-Cr 403; MS 967,1241; MS & DPM 2664, 3846, 4038. Neckeropsis gracilenta (Bosch & Sande Lac.) M. Fleisch. Neckeropsis gracilenta (Bosch & Sande Lac.) M. Fleisch. p g On climbers, tree trunks, twigs, and decaying logs, 100–1100 m, DPM 280; HA-Cr 169; MS 1228; MS & DPM 3934, 4090. Neckeropsis lepineana (Mont.) M. Fleisch. On fallen logs and tree trunks, 800–1030 m, MS 1188; MS & DPM 3983, 4089. Pinnatella kuehliana (Bosch & Sande Lac.) M. Fleisch. Pinnatella kuehliana (Bosch & Sande Lac.) M. Fleisch. On tree roots and stone-walls, 550–1120 m, DPM 150; HA-Cr 249; MS & DPM 3979. On tree roots and stone-walls, 550–1120 m, DPM 150; HA-Cr 249; MS & DPM 39 The Mosses of Crocker Range Park, Malaysian Borneo 97 Pinnatella mucronata (Bosch & Sande Lac.) M. Fleisch. Pinnatella mucronata (Bosch & Sande Lac.) M. Fleisch. On tree trunks and bases, 100–1145 m, DPM 144, 291; HA-Cr 137; KWL 45. On tree trunks and bases, 100–1145 m, DPM 144, 291; HA-Cr 137; KWL 45. Thamnobryum incurvum (Nog.) Nog. & Z. Iwats. h On moist to wet boulders by streams and waterfall, 940–1120 m, HA-Cr 251, 264. Thamnobryum subserratum (Hook. ex Harv.) Nog. & Z. Iwats. Thamnobryum subserratum (Hook. ex Harv.) Nog. & Z. Iwats. O b ld d k b f ll d i 1040 1100 MS 1 h y g On boulders and wet rocks by waterfall and rivers, 1040–1100 m, MS 1225, 1235, 1238. Touwia elliptica (Bosch & Sande Lac.) S. Olsson, Enroth & D. Quandt On rocks and boulders, 400–1230 m, DPM 185; HA-Cr 158, 265, 291, 379; MS & DPM 4088. Touwia negrosensis (E.B. Bartram) S. Olsson, Enroth & D. Quandt On wet boulders, 900 m, MS & DPM 3920. Macromitrium longicaule Müll. Hal. Macromitrium longicaule Müll. Hal. On boulders, fallen branches, shrub branches and tree trunks, 600–1710 m, MS 944b; MS & DPM 2623, 2711, 3980, 4002, 4007. Macromitrium longipilum A. Braun ex Müll. Hal. On tree trunks, rotten logs and fallen logs, 1150–1800 m, DPM 106; HA-Cr 227, 228; MS & DPM 3793, 3837, 3800. Macromitrium ochraceum (Dozy & Molk.) Müll. Hal. On fallen tree branches and tree trunks, 1340–1800 m, MS 944a; MS & DPM 2587, 2691, 3784. Macromitrium orthostichum Nees ex Schwägr. On boulders and fallen branches, 650–1150 m, HA-Cr 193; MS & DPM 3992, 4093. Orthotrichaceae Orthotrichaceae Macromitrium cuspidatum Hampe On tree trunk, 1150 m, HA-Cr 176. Orthotrichaceae Macromitrium cuspidatum Hampe On tree trunk, 1150 m, HA-Cr 176. Macromitrium cuspidatum Hampe On tree trunk, 1150 m, HA-Cr 176. On tree trunk, 1150 m, HA-Cr 176. Macromitrium orthostichum Nees ex Schwägr. Macromitrium orthostichum Nees ex Schwägr. Macromitrium orthostichum Nees ex Schwägr. On boulders and fallen branches, 650–1150 m, HA-Cr 193; MS & DPM 3992, 4093. Macromitrium salakanum Müll. Hal. On fallen branches, leaves and logs, 400–1380 m, HA-Cr 147; KWL 68, 102; MS 970, 1259, 1260. Schlotheimia wallisii Müll. Hal. On climber and treelet trunk, 1720–1770 m, MS 1488; MS & DPM 3839. Schlotheimia wallisii Müll. Hal. Hookeriopsis utacamundiana (Mont.) Broth. p On a wet rock by a streamlet, 1240 m, HA-Cr 284. On a wet rock by a streamlet, 1240 m, HA-Cr 284. Polytrichaceae Dawsonia beccarii Broth. & Geh.f On rocky cliff in open area, 1780 m, MS & DPM 3877. On rocky cliff in open area, 1780 m, MS & DPM 3877. Dawsonia longifolia (Bruch & Schimp.) Zanten On soil, 1600–1800 m, DPM 65, 69; MS & DPM 2590, 3940. Cyclodictyon blumeanum (Müll. Hal.) Kuntze Cyclodictyon blumeanum (Müll. Hal.) Kuntze On moist to wet rocks, 940–1120 m, HA-Cr 243, 294. Hookeriopsis utacamundiana (Mont.) Broth. On a wet rock by a streamlet, 1240 m, HA-Cr 284. Schlotheimia wallisii Müll. Hal. On climber and treelet trunk, 1720–1770 m, MS 1488; MS & DPM 3839. Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) 98 Pilotrichaceae Pilotrichaceae Actinodontium rhaphidostegum (Müll. Hal.) Bosch & Sande Lac. Growing on rotten logs, shrub branches and fallen branches, 1080–1400 m, HA-Cr 21, 184; MS & DPM 3906. Callicostella papillata (Mont.) Mitt. var. papillata On rotten logs, 400–1250 m, DPM 192, 209; HA-Cr 148; MS & DPM 3989 4058. Callicostella papillata (Mont.) Mitt. var. papillata On rotten logs, 400–1250 m, DPM 192, 209; HA-Cr 148; MS & DPM 3989, 4051, 4058. Callicostella papillata var. prabaktiana (Müll. Hal.) Streimann On rotten logs, boulders and on rocky cliffs, 100–1360 m, DPM 285; HA-Cr 41, 259, 282, 314. Dawsonia longifolia (Bruch & Schimp.) Zanten Pogonatum cirratum (Sw.) Brid. subsp. cirratum On soil, 1350–1490 m, MS 1402; HA-Cr 53, 55. Pogonatum cirratum subsp. fuscatum (Mitt.), Hyvönen On soil and humus, 750–1340 m, MS 1255, 1256; MS & DPM 4066; MS & DPM 3909; HA-Cr 201; MS 955. Pogonatum cirratum subsp. macrophyllum (Dozy & Molk.) Hyvönen On soil, 1220–1800 m, DPM 95; MS & DPM 2561, 3811, 3876, 3944, 4042; MS 991, 954. Pogonatum iwatsukii Touw On boulders and rocks, 1320–1400 m, MS 930, 940; HA-Cr 350. Pogonatum neesii (Müll. Hal.) Dozy On soil and boulders in open areas, 680–1800 m, MS & DPM 3871, 3995; MS 951; HA-Cr 54, 120, 424. The Mosses of Crocker Range Park, Malaysian Borneo 99 Pogonatum piliferum (Dozy & Molk.) Touw On rocks and soil, 400–1360 m, DPM 199; MS & DPM 4018, 4065; HA-Cr 27, 134, 306, 311, 428; MS 990. Pogonatum rutteri (Thér. & Dixon) Dixon On soil, 1070–1360 m, HA-Cr 28, 394; MS 1003, 1004. Pogonatum rutteri (Thér. & Dixon) Dixon On soil, 1070–1360 m, HA-Cr 28, 394; MS 1003, 1004. Pogonatum subtortile (Müll. Hal.) A. Jaeger On soil in an open area, 900–1160 m, HA-Cr 204, 406. Pogonatum subtortile (Müll. Hal.) A. Jaeger On soil in an open area, 900–1160 m, HA-Cr 204, 406. Pottiaceae Chionoloma bombayense (Müll. Hal.) P. Sollman (1996) On concrete and stone-wall, 1040–1820 m, DPM 112; MS 1224. Chionoloma bombayense (Müll. Hal.) P. Sollman (1996) y On concrete and stone-wall, 1040–1820 m, DPM 112; MS 1224. Barbula javanica Dozy & Molk. On thin soil covering wet rocks along stream, 410–1800 m, MS 1461; MS & DPM 3872. Barbula consanguinea (Thwaites & Mitt.) A. Jaeger On moist stone-wall along river, 1040 m, MS 1223. Barbula javanica Dozy & Molk. On thin soil covering wet rocks along stream, 410–1800 m, MS 1461; MS & DPM 3872. Barbula consanguinea (Thwaites & Mitt.) A. Jaeger On moist stone wall along river 1040 m MS 1223 Barbula javanica Dozy & Molk. Hyophila involuta (Hook.) A. Jaeger Growing on boulders, concrete and stone-walls by waterfall and rivers in open and partially shaded areas, 100–1820 m, DPM 111; DPM 127, 148; DPM 279; HA-Cr 253; MS 1446; MS 1217, 1218; MS & DPM 3923, 4001, 4056. Pterobryaceae Calyptothecium recurvulum (Broth.) Broth. Hanging on tree trunks by rivers or waterfall, 850–1030 m, MS 1187; MS & DPM 3969, 3974. Symphysodontella cylindracea (Mont.) M. Fleisch. Symphysodontella cylindracea (Mont.) M. Fleisch. On shrub trunks, rotten logs, tree trunks and branches, and climbers, 560–1650 m, HA-Cr 300, 439; MS 962, 968, 1405; MS & DPM 2589, 2674, 3894. Trachyloma indicum Mitt. y On rotten logs and tree trunks, 1425–1650 m, HA-Cr 459; MS 960. On rotten logs and tree trunks, 1425–1650 m, HA-Cr 459; MS 960. Cryptogonium phyllogonioides (Sull.) Isov. On a tree trunk, 940–1120 m, HA-Cr 248. On a tree trunk, 940–1120 m, HA-Cr 248. On a tree trunk, 940–1120 m, HA-Cr 248. Neolindbergia rigida (Bosch & Sande Lac.) M. Fleisch. On fallen trees, 1040–1100 m, MS 1195; MS & DPM 3884. Neolindbergia rugosa (Lindb.) M. Fleisch. Neolindbergia rugosa (Lindb.) M. Fleisch. Neolindbergia rugosa (Lindb.) M. Fleisch. On tree trunks and fallen logs, 530–1120 m, HA-Cr 260; MS 1443. On tree trunks and fallen logs, 530–1120 m, HA-Cr 260; MS 1443. Pterobryopsis crassicaulis (Müll. Hal.) M. Fleisch. On rotten logs and fallen branches, 680–1460 m, HA-Cr 145; MS 1401; MS & DPM 4027. 100 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Pylaisiadelphaceae Brotherella falcata (Dozy & Molk.) M. Fleisch. Brotherella falcata (Dozy & Molk.) M. Fleisch. Brotherella falcata (Dozy & Molk.) M. Fleisch. On a shrub branch, 1400 m, HA-Cr 339, det. B.C. Tan. On a shrub branch, 1400 m, HA-Cr 339, det. B.C. Tan. Clastobryum cf. asperrimum (Dixon) B.C. Tan On a decaying log, 1765 m, DPM 90.h Clastobryum cf. asperrimum (Dixon) B.C. Tan On a decaying log, 1765 m, DPM 90. Clastobryum cf. asperrimum (Dixon) B.C. Tan On a decaying log, 1765 m, DPM 90.h The specimen is similar to the species except for its leaves that are much larger, reaching 1.6 mm long. The specimen is similar to the species except for its leaves that are much larger, reaching 1.6 mm long. Clastobryum cuculligerum (Sande Lac.) Tixier On a fallen branch, 1150 m, HA-Cr 180. Clastobryum epiphyllum (Renauld & Cardot) B.C. Tan & Touw On rotten twigs and tree trunks, 500–1080 m, DPM 126; MS & DPM 3899a. Clastobryum epiphyllum (Renauld & Cardot) B.C. Tan & Touw On rotten twigs and tree trunks, 500–1080 m, DPM 126; MS & DPM 3899a Clastobryum epiphyllum (Renauld & Cardot) B.C. Tan & Touw On rotten twigs and tree trunks, 500–1080 m, DPM 126; MS & DPM 3899a Isopterygium minutirameum (Müll. Hal.) A. Jaeger On a tree base, 500 m, DPM 214. On a tree base, 500 m, DPM 214. Mastopoma armitii (Broth. & Geh.) Broth. On shrub and tree trunks and leaves, and rotten logs, 900–1800 m, CMK 44, 47; DPM 39; HA-Cr 11, 203. Mastopoma armitii (Broth. & Geh.) Broth. On shrub and tree trunks and leaves, and rotten logs, 900–1800 m, CMK 44, 47; DPM 39; HA-Cr 11, 203. Mastopoma brauniana (Bosch & Sande Lac.) H. Akiyama On a tree trunk, 1400 m, HA-Cr 3. Clastobryum scalare (Müll. Hal.) Tixier Clastobryum scalare (Müll. Hal.) Tixier On tree trunks and branches, leaves and shrub branches, 1800 m, MS & DPM 3790. In Borneo, this species has been reported from Sarawak and Kalimantan (Dixon 1935; Tixier 1977). Isocladiella surcularis (Dixon) B.C. Tan & Mohamed On shrub and tree trunks, and roots, 1100–1800 m, CMK 12; HA-Cr 81; KWL 17, 54, 71, 112, 121; MS 993; MS & DPM 2558, 3882, 4019. Isopterygium albescens (Hook.) A. Jaeger On rotten logs and decaying stump, 1100–1145 m, KWL 8, 12, 70, 85, 105a, 106, 109; MS & DPM 3885. Isopterygium bancanum (Sande Lac.) A. Jaeger On lianas and fallen trees, 1145 m, KWL 7, 72. 101 The Mosses of Crocker Range Park, Malaysian Borneo Isopterygium minutirameum (Müll. Hal.) A. Jaeger On a tree base, 500 m, DPM 214. Mastopoma papillosum Broth. Mastopoma papillosum Broth. Growing on shrub branches, 1730 m, MS & DPM 3838. Growing on shrub branches, 1730 m, MS & DPM 3838. Mastopoma uncinifolium (Broth.) Broth. Mastopoma uncinifolium (Broth.) Broth. On shrub branches, rotten logs, roots, tree trunks and humus, 1150–1770 m, DPM 82, 101a, 103; HA-Cr 13, 221; MS & DPM 2709, 3833. Taxithelium lindbergii (A. Jaeger) Renauld & Cardot On tree trunk, branches of shrubs and leaves, 1160–1800 m, HA-Cr 273, 401; MS & DPM 2662, 3866. Taxithelium instratum (Brid.) Broth. Taxithelium instratum (Brid.) Broth. Taxithelium instratum (Brid.) Broth. On rotten logs and rocks, 50–800 m, DPM 187, 300, 306; HA-Cr 119, 420. Taxithelium instratum (Brid.) Broth. On rotten logs and rocks, 50–800 m, DPM 187, 300, 306; HA-Cr 119, 420. On rotten logs and rocks, 50–800 m, DPM 187, 300, 306; HA-Cr 119, 420. Taxithelium isocladum (Bosch & Sande Lac.) Renauld & Cardot On a small shrub branch, 1600–1650 m, HA-Cr 455. Taxithelium vernieri (Duby) Besch. Growing on shrub branches, twigs and fallen tree, 1100–1400 m, KWL 87, 125; HA- Cr 78, 236, 337. Racopilaceae Racopilum cuspidigerum (Schwägr.) Ångström On moist rock beside a stream, 680 m, HA-Cr 131. Racopilum laxirete Broth. On tree root, 950 m, MS & DPM 3930. Racopilum laxirete Broth. On tree root, 950 m, MS & DPM 3930. Racopilum laxirete Broth. On tree root, 950 m, MS & DPM 3930. Racopilum spectabile Reinw. & Hornsch. var. spectabile On shrub leaves and trunks, rotten logs, soils and boulders by rivers, 550–1600 m, DPM 151; HA-Cr 72, HA-Cr 151, 386; MS & DPM 2701; MS 931, 1206, 1398; MS & DPM 3967, 3981. Racopilum spectabile Reinw. & Hornsch. var. spectabile On shrub leaves and trunks, rotten logs, soils and boulders by rivers, 550–1600 m, DPM 151; HA-Cr 72, HA-Cr 151, 386; MS & DPM 2701; MS 931, 1206, 1398; MS & DPM 3967, 3981. Trismegistia brachyphylla M. Fleisch. Trismegistia brachyphylla M. Fleisch. On shrub branches and trunks, tree trunks and bases, and rotten logs, 1100–1190 m, HA-Cr 101, 102, 105, 110, 223; MS 999. Trismegistia calderensis (Sull.) Broth. var. calderensis On tree branches, shrub trunks and tree stump, 1220–1800 m, MS 937; MS & DPM 1258, 2582, 3797, 3819. Trismegistia calderensis var. subintegrifolia (Broth.) H. Akiyama On tree trunks and bases, rotten logs, rotten wood, fallen log, tree branches, soil and rocks, 560–1810 m, HA-Cr 1, 5, 26, 47, 66, 67, 70, 103, 106, 107, 109, 194, 213, 214, 229, 230, 322, 324, 390, 392, 393. Trismegistia calderensis var. subintegrifolia (Broth.) H. Akiyama On tree trunks and bases, rotten logs, rotten wood, fallen log, tree branches, soil and rocks, 560–1810 m, HA-Cr 1, 5, 26, 47, 66, 67, 70, 103, 106, 107, 109, 194, 213, 214, 229, 230, 322, 324, 390, 392, 393. 102 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Trismegistia lancifolia var. valetonii (M. Fleisch. ex Dixon) H. Akiyama On tree trunks and roots, shrub branches, fallen branches, decaying tree, rotten logs and boulders, 610–1150 m, HA-Cr 107, 127, 128, 166, 167, 194, 212, 419, 421; KWL 10, 21, 24, 30, 32, 49, 50, 52b, 57, 61, 65, 74, 82, 88, 92, 96, 99, 103; 113; MS 1438. Trismegistia lancifolia var. valetonii (M. Fleisch. ex Dixon) H. Akiyama On tree trunks and roots, shrub branches, fallen branches, decaying tree, rotten logs and boulders, 610–1150 m, HA-Cr 107, 127, 128, 166, 167, 194, 212, 419, 421; KWL 10, 21, 24, 30, 32, 49, 50, 52b, 57, 61, 65, 74, 82, 88, 92, 96, 99, 103; 113; MS 1438. Trismegistia panduriformis var. prionodontella (Broth.) H. Akiyama On shrub branches, tree trunks and bases, rotten logs, decaying wood and boulders, 1100–1770 m, DPM 83, 86, 92, 2672, 4022; HA-Cr 45, 104; MS 935. Racopilum spectabile var. subisophyllum Herzog Racopilum spectabile var. subisophyllum Herzog On rotten branches and roots, 1600–1800 m, DPM 85; MS & DPM 2678, 3857. Regmatodontaceae Regmatodontaceae Regmatodon declinatsus (Hook.) Brid. On a boulder beside a stream, 1350 m, MS & DPM 4034. On a boulder beside a stream, 1350 m, MS & DPM 4034. On a boulder beside a stream, 1350 m, MS & DPM 4034 Rhizogoniaceae Pyrrhobryum latifolium (Bosch & Sande Lac.) Mitt. On tree trunks and bases, and rotten logs, 940–1280 m, HA-Cr 90, 220, 241; MS & DPM 3949. Pyrrhobryum spiniforme (Hedw.) Mitt. On tree trunks, rotten logs, humus, roots and stone-walls, 400–1780 m, DPM 49, 89, 97, 142, 206, 237, 252; HA-Cr 275, 378; KWL 13, 28, 58, 66, 77, 94, 97, 107; MS 882, 1185; MS & DPM 3832, 3879, 4016, 4017. Pyrrhobryum spiniforme (Hedw.) Mitt. On tree trunks, rotten logs, humus, roots and stone-walls, 400–1780 m, DPM 49, 89, 97, 142, 206, 237, 252; HA-Cr 275, 378; KWL 13, 28, 58, 66, 77, 94, 97, 107; MS 882, 1185; MS & DPM 3832, 3879, 4016, 4017. Rhizogonium graeffeanum (Müll. Hal.) A. Jaeger On tree trunks and bases, and rotten stumps, 1400–1800 m, HA-Cr 12, 319; MS 901; MS & DPM 3831. Rhizogonium graeffeanum (Müll. Hal.) A. Jaeger On tree trunks and bases, and rotten stumps, 1400–1800 m, HA-Cr 12, 319; MS 901; MS & DPM 3831. 103 The Mosses of Crocker Range Park, Malaysian Borneo Rhizogonium lamii Reimers g On tree buttress and trunks, 1600–1800 m, MS & DPM 2599, 2626, 2668, 3827. On tree buttress and trunks, 1600–1800 m, MS & DPM 2599, 2626, 2668, 3827. Sematophyllaceae Sematophyllaceae Acanthorrhynchium papillatum (Harv.) M. Fleisch. On tree trunks and tree bases, 560–1145 m, HA-Cr 309; KWL 117; MS & DPM 1436. Acanthorrhynchium papillatum (Harv.) M. Fleisch. y p p e trunks and tree bases, 560–1145 m, HA-Cr 309; KWL 117; MS & DPM 1436. Acroporium adspersum (Hampe) Broth. On a tree trunk, 1800 m, CMK 125. p p On a tree trunk, 1800 m, CMK 125. On a tree trunk, 1800 m, CMK 125. Acroporium convolutum (Sande Lac.) M. Fleisch. var. convolutum On a tree trunk, 1425 m, MS 963, det. B.C. Tan. Acroporium convolutum var. elatum (Dixon) B.C. Tan On rotten logs, 550–1400 m, DPM 153, 239; HA-Cr 296, 310, 368. Acroporium diminutum (Brid.) M. Fleisch. On tree trunks and branches, decaying logs, climber trunks and shrub branches, 1150–1810 m, CMK 28; DPM 71; HA-Cr 190, 231, 327, 335, 342, 370; MS 877, 893, 959; MS & DPM 2581, 2619, 2629, 2663. Acroporium downii (Dixon) Broth. On rotten logs and bamboo stumps, 50–1770 m, DPM 101b, 301, 314; KWL 15b; MS & DPM 4063. Acroporium johannis-winkleri Broth. Acroporium johannis-winkleri Broth. On tree trunks and branches, roots, rotten logs, fallen logs and shrub branches, 1100– 1880 m, CMK 103, 148; DPM 7, 10, 12, 13, 18, 26, 28, 29, 42, 50, 52, 55, 57, 66, 72, 77, 78, 99, 105, 107; HA-Cr 82; MS 987; MS & DPM 2601, 2650, 4021. Acroporium lamprophyllum Mitt. On leaves, tree trunks, treelet trunks, fallen logs, rotten logs, 1145–1850 m, DPM 9, 24, 38, 44; KWL 18; MS 915; MS & DPM 2648. Acroporium macroturgidum Dixon On humus, 1800 m, MS & DPM 3818, confirmed by B.C. Tan. In Borneo, this species has been previously reported from Kalimantan and Sarawak (Suleiman et al. 2006). Acroporium praelongum var. aciphylloides B.C. Tan On tree trunks, rotten logs and fallen tree, 1340–1800 m, MS 977; MS & DPM 2607, 3799. 104 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) Acroporium ramicola (Hampe) Broth. On tree shrub branches, 1400–1800 m, CMK 6, 90; HA-Cr 367, det. B.C. Tan. In Borneo, this species has been known only from the type collection from Sarawak (Suleiman et al. 2006). On tree shrub branches, 1400 1800 m, CMK 6, 90; HA Cr 367, det. B.C. Tan. In Borneo, this species has been known only from the type collection from Sarawak (Suleiman et al. 2006). Acroporium rigens (Broth. ex Dixon) Dixon. Acroporium rigens (Broth. ex Dixon) Dixon. On rocks and on rotten logs 600–1370 m, DPM 238; MS 883. Acroporium rufum (Reinw. & Hornsch.) M. Fleisch. On tree tree trunks and branches, and decaying logs, 1400–1800 m, CMK 76, 133; DPM 8, 33, 51; HA-Cr 73; MS & DPM 2595, 3794. Acroporium secundum (Reinw. & Hornsch.) M. Fleisch. On shrubs and branches, decaying logs and rotten climbers, 1300–1780 m, DPM 47; HA-Cr 276; MS & DPM 2549, 2609. Acroporium stramineum (Reinw. & Hornsch.) M. Fleisch. var. stramineum On tree trunks, decaying fallen log, climbers, shrub branches and fallen branches, 1150–1850 m, DPM 14, 37, 61; CMK 22, 5; HA-Cr 185, 189, 362; MS 912, 953; MS & DPM 2646, 3785. Acroporium stramineum var. hamulatum (M. Fleisch.) B.C. Tan On tree trunks, bases and branches, and decaying fallen branches, 1150–1520 m, HA- Cr 177, 186; MS 890, 900, 914; MS & DPM 2553. Acroporium strepsiphyllum (Mont.) B.C. Tan var. strepsiphyllum On a fallen log in an open area, 1790 m, DPM 43. Chionostomum hainanense B.C. Tan & Y. Jia On a tree trunk beside a small pond, 1800 m, MS & DPM 3812, det. K.T. Yong & B.C. Tan. Meiothecium hamatum (Müll. Hal.) Broth. On tree trunks on shrub branches in open areas, 1150–1800 m, HA-Cr 172, 418; MS & DPM 3778, 3782. Meiothecium microcarpum (Harv.) Mitt. On boulders in an open area, 600 m, MS & DPM 4004. Papillidiopsis malayana (Dixon) BC. Tan On a tree branch, 1800 m, MS & DPM 3863, det. B.C. Tan. In Borneo, this species has been previously reported only from Kalimantan (Dixon, 1935). 105 The Mosses of Crocker Range Park, Malaysian Borneo Papillidiopsis ramulina (Thwaites & Mitt.) W.R. Buck & B.C. Tan On moist soil in steep galley, 900 m, HA-Cr 205, det. B.C. Tan. Papillidiopsis ramulina (Thwaites & Mitt.) W.R. Buck & B.C. Tan On moist soil in steep galley, 900 m, HA-Cr 205, det. B.C. Tan. Papillidiopsis stissophylla (Hampe & Müll. Hal.) B.C. Tan & Y. Jia On Melastoma branch, shrub branches and trunks, 1600–1800 m, MS & DPM 2690, 3841, 3842, 3865, det. B.C. Tan & K.T. Yong. Radulina barbonica var. barbonica (Bél.) W.R. Buck On palm leaves, 385–1400 m, MS 1452; HA-Cr 343. Rhaphidostichum piliferum (Broth.) Broth. Rhaphidostichum piliferum (Broth.) Broth. On rotten shrub branch and trunk, 1720 m, MS & DPM 2608, 2612. Rhaphidostichum luzonense (Broth.) Broth. Rhaphidostichum luzonense (Broth.) Broth. On boulder s in streambed, 680 m, HA-Cr 149, det. B.C. Tan. On boulder s in streambed, 680 m, HA-Cr 149, det. B.C. Tan. Plants large, yellowish-green, glossy, stems elongate, prostrate, densely branched. Leaves broadly ovate to oblong-ovate, 2.7–3.0 mm × 0.6–0.7 mm, semitubulose, concave, abruptly contracted to a long acuminate apex, ecostate, margin entire below and denticulate at extreme apex. Lamina cells vermicular, 66–98 μm × 5–7 μm, thin- walled, smooth; alar cells large, oval, coloured, inflated. Sporophyte not seen. l This species is characterised by the broadly ovate to oblong-ovate leaves and abruptly contracted into long acuminate apex with denticulate acumen. It was previ ously only reported from the Philippines (Bartram, 1939; Tan & Iwatsuki, 1991). ematophyllum subpinnatum (Brid.) E. Britton On tree and shrub trunks, 900–950 m, MS & DPM 3914, 3935, det. B.C. Tan. Sematophyllum subpinnatum (Brid.) E. Britton On tree and shrub trunks, 900–950 m, MS & DPM 3914, 3935, det. B.C. Tan. Sematophyllum subpinnatum (Brid.) E. Britton On tree and shrub trunks, 900–950 m, MS & DPM 3914, 3935, det. B.C. Tan. Sematophyllum subpinnatum (Brid.) E. Britton On tree and shrub trunks, 900–950 m, MS & DPM 3914, 3935, det. B.C. Tan. Trichosteleum boschii (Dozy & Molk.) A. Jaeger On shrub trunks and leaves, twigs, tree trunks and branches, rotten logs and rock, 600– 1800 m, DPM 87, 100, 242; HA-Cr 35, 39, 142, 407; MS & DPM 2614, 2655, 3829. Trichosteleum pseudomammosum M. Fleisch. On a tree trunk, 1800 m, CMK 99. Trichosteleum pseudomammosum M. Fleisch. On a tree trunk, 1800 m, CMK 99. Trichosteleum cf. saproxylophilum (Müll. Hal.) B.C. Tan, W.B. Schofield & H.P. Ramsay On a tree trunk, 370 m, MS 1261, det. B.C. Tan. This specimen has all the characteristics of T. saproxylophilum except for the larger size of the perichaetial leaves (1.7 mm × 0.4 mm) and branch leaves (2.5 mm–2.9 mm × 0.4 mm–0.5 mm). Trichosteleum cf. saproxylophilum (Müll. Hal.) B.C. Tan, W.B. Schofield & H.P. Ramsay On a tree trunk, 370 m, MS 1261, det. B.C. Tan. Thi i h ll th h t i ti f T p l phil t f th l i Trichosteleum cf. saproxylophilum (Müll. Hal.) B.C. Tan, W.B. Schofield & H.P. Ramsay On a tree trunk, 370 m, MS 1261, det. B.C. Tan. This specimen has all the characteristics of T. Rhaphidostichum luzonense (Broth.) Broth. saproxylophilum except for the larger size of the perichaetial leaves (1.7 mm × 0.4 mm) and branch leaves (2.5 mm–2.9 mm × 0.4 mm–0.5 mm). On a tree trunk, 370 m, MS 1261, det. B.C. Tan. This specimen has all the characteristics of T. saproxylophilum except for the larger size of the perichaetial leaves (1.7 mm × 0.4 mm) and branch leaves (2.5 mm–2.9 mm × 0.4 mm–0.5 mm). Trichosteleum stigmosum Mitt. On tree trunks and base, rotten logs and soil, 50–730 m, DPM 215, 240, 248; DPM 297; HA-Cr 318, 307. Trichosteleum stigmosum Mitt. On tree trunks and base, rotten logs and soil, 50–730 m, DPM 215, 240, 248; DPM 297; HA-Cr 318, 307. Symphyodontaceae y p y Chaetomitrium orthorrhynchum (Dozy & Molk.) Bosch & Sande Lac. On shrub leaves and branches, tree branches and climber, 310–1600 m, HA-Cr 239; MS 1203, 1262; MS & DPM 2689, 4036. Warburgiella cupressinoides Müll. Hal. ex Broth. On a rotten log, 1800 m, MS & DPM 3820. Warburgiella cupressinoides Müll. Hal. ex Broth. On a rotten log, 1800 m, MS & DPM 3820. Warburgiella cupressinoides Müll. Hal. ex Broth. On a rotten log, 1800 m, MS & DPM 3820. Warburgiella circinata Dixon On humus, 1400 m, HA-Cr 364, det. B.C. Tan. Warburgiella circinata Dixon On humus, 1400 m, HA-Cr 364, det. B.C. Tan. Warburgiella cf. breviseta (Broth.) Broth. The specimens have all the characteristics of the species but the leaf cells are smooth throughout. Sphagnaceae Sphagnum cuspidatulum Müll. Hal. On humus, 1800 m, MS & DPM 3776, 3788. Sphagnum cuspidatulum Müll. Hal. On humus, 1800 m, MS & DPM 3776, 3788. Sphagnum junghuhnianum Dozy & Molk. On humus and tree trunk, 1800 m, MS & DPM 3807, 3780, 3804; DPM 19, 54; CMK 78. Sphagnum perichaetiale Hampe On humus, 1800 m, MS & DPM 3806. Trichosteleum stigmosum Mitt. 106 Monica Suleiman et al. / PhytoKeys 8: 71–107 (2017) *Chaetomitrium lancifolium Mitt. On a tree branch by a river, partially shaded in secondary lowland forest, 70 m, DPM 3 On a tree branch by a river, partially shaded in secondary lowland forest, 70 m, DPM 307. Plants medium size for the genus, stems to 3 cm long, irregularly and rather laxly branched. Branches 6–10 mm long, sometimes cuspidate at tips, with clusters of fila mentous propagules at tips, sometimes extending to the mid of branches, propagules 1/3 of leaf length. Branch leaves erect to erect-spreading when dry, slightly homomal lous, leaves often twisted half above; little altered when wet, except not twisted above; oblong-lanceolate to ovate-lanceolate, concave, 1.3–1.4 mm × 0.4–0.5 mm, apex gradually long-acuminate ending in a narrow point, strongly constricted below apices, costa distinct but short, margin sometimes slightly undulate in upper 1/3, strong ly regularly serrate to denticulate to the base, teeth strongly bifid, trifid or multifid. Lamina cells linear, 60 μm × 5 μm in mid-lamina, thick-walled, strongly prorate to spiculose-prorate to the base in adaxial and abaxial sides; alar cells small forming 5–6 short-rectangular cells. Sporophyte not seen.hh This species has only been recorded from the Maluku Islands, its type locality. The distinguishing features of this species have been reported recently by Suleiman and The Mosses of Crocker Range Park, Malaysian Borneo 107 Akiyama (2014). It is closely related to C. papillifolium Bosch & Sande Lac., differing only in seta length and leaf apex. Based on the type material, this species has a very short seta measuring only 4.4–4.5 cm. Chaetomitrium leptopoma (Schwägr.) Bosch & Sande Lac. On shrub leaves and branches, 850–1600 m, HA-Cr 234, 325, 334; MS & DPM 2702, 3964. Dimorphocladon borneense Dixon Dimorphocladon borneense Dixon On ginger leaves by rivers, 748–770 m, MS 1246; MS & DPM 4082, det. B.C. Ho Thuidiaceae Pelekium velatum Mitt. Growing on rocks, tree trunks, rotten logs and stone-walls, 400–900 m, HA-Cr 164; DPM 130; 172, 173, 191; MS & DPM 3918. Pelekium versicolor (Hornsch. ex Müll. Hal.) Touw On a decaying log, 1050 m, MS 1386. Thuidium cymbifolium (Dozy & Molk.) Dozy & Molk. On boulders, rocks, stone-wall, rotten logs, stumps, climber and soils, 500–1800 m, DPM 93, 138; HA-Cr 404; MS 1208, 1214, 1220; MS & DPM 2677, 3851, 3860, 3971. Thuidium plumulosum (Dozy & Molk.) Dozy & Molk. On tree bases, rotten logs, stone-wall, rocks and boulders, 100–680 m, DPM 141, 186, 189, 260; HA-Cr 144, 162; MS 1449, 1451. Thuidium plumulosum (Dozy & Molk.) Dozy & Molk. On tree bases, rotten logs, stone-wall, rocks and boulders, 100–680 m, DPM 141, 186, 189, 260; HA-Cr 144, 162; MS 1449, 1451. Thuidium plumulosum (Dozy & Molk.) Dozy & Molk. On tree bases, rotten logs, stone-wall, rocks and boulders, 100–680 m, DPM 141, 186, 189, 260; HA-Cr 144, 162; MS 1449, 1451. Thuidium pristocalyx (Müll.Hal.) A. Jaeger var. pristocalyx On boulders, tree trunks and buttress, climbers, roots and lianas, 850–1600 m, KWL 29, 37, 38, 39, 40; HA-Cr 88, 344; MS 984; MS & DPM 2670, 3904, 3965. Thuidium pristocalyx (Müll.Hal.) A. Jaeger var. pristocalyx On boulders, tree trunks and buttress, climbers, roots and lianas, 850–1600 m, KWL 29, 37, 38, 39, 40; HA-Cr 88, 344; MS 984; MS & DPM 2670, 3904, 3965.
https://openalex.org/W4297994739	https://www.frontiersin.org/articles/10.3389/fphys.2022.1007692/pdf	English	null	Genipin modified lyophilized platelet-rich fibrin scaffold for sustained release of growth factors to promote bone regeneration	Frontiers in physiology	2,022	cc-by	9,342	TYPE Original Research PUBLISHED 30 September 2022 DOI 10.3389/fphys.2022.1007692 TYPE Original Research PUBLISHED 30 September 2022 DOI 10.3389/fphys.2022.1007692 TYPE Original Research PUBLISHED 30 September 2022 DOI 10.3389/fphys.2022.1007692 OPEN ACCESS EDITED BY Ce Shi, Jilin University, China REVIEWED BY Chao Liu, Dalian Medical University, China Yanyan Li, Northeast Forestry University, China CORRESPONDENCE Han Jin, jinhan@hrbmu.edu.cn Bin Zhang, zhangbin@hrbmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Skeletal Physiology, a section of the journal Frontiers in Physiology RECEIVED 30 July 2022 ACCEPTED 01 September 2022 PUBLISHED 30 September 2022 CITATION Liu X, Yin M, Li Y, Wang J, Da J, Liu Z, Zhang K, Liu L, Zhang W, Wang P, Jin H and Zhang B (2022), Genipin modiﬁed lyophilized platelet-rich ﬁbrin scaffold for sustained release of growth factors to promote bone regeneration. Front. Physiol. 13:1007692. doi: 10.3389/fphys.2022.1007692 OPEN ACCESS EDITED BY Ce Shi, Jilin University, China REVIEWED BY Chao Liu, Dalian Medical University, China Yanyan Li, Northeast Forestry University, China CORRESPONDENCE Han Jin, jinhan@hrbmu.edu.cn Bin Zhang, zhangbin@hrbmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Skeletal Physiology, a section of the journal Frontiers in Physiology RECEIVED 30 July 2022 ACCEPTED 01 September 2022 PUBLISHED 30 September 2022 CITATION Liu X, Yin M, Li Y, Wang J, Da J, Liu Z, Zhang K, Liu L, Zhang W, Wang P, Jin H and Zhang B (2022), Genipin modiﬁed lyophilized platelet-rich ﬁbrin scaffold for sustained release of growth factors to promote bone regeneration. Front. Physiol. 13:1007692. doi: 10.3389/fphys.2022.1007692 Xiaoyao Liu1,2†, Mingjing Yin1,2†, Ying Li 1†, Jianqun Wang1, Junlong Da1, Zhongshuang Liu3, Kai Zhang1, Lixue Liu1, Wenxuan Zhang1, Peijun Wang2, Han Jin1* and Bin Zhang1,4* 1Heilongjiang Provincial Key Laboratory of Hard Tissue Development and Regeneration, The Second Afﬁliated Hospital of Harbin Medical University, Harbin, China, 2Department of Stomatology, The First Afﬁliated Hospital of Harbin Medical University, Harbin, China, 3Department of Stomatology, Shenzhen University General Hospital, Shenzhen University, Shenzhen, China, 4Heilongjiang Academy of Medical Sciences, Harbin, China Lyophilized platelet-rich ﬁbrin (L-PRF) was shown to further activate resident platelets in platelet-rich ﬁbrin causing a higher amount of growth factors release. However, it still required further experimental studies to resolve the uncontrolled degradation and burst release problem. In this study, the nature crosslinker genipin is introduced to improve the performance of L-PRF scaffold. We used a series of gradient concentration genipin solutions to react with L-PRF. The crosslinking degree, micro morphology, mean pore size, water absorption and mechanical properties of the crosslinked scaffold were evaluated. In order to study the effect of genipin modiﬁcation on the release kinetics of growth factors from L-PRF, we detected the release of platelet- derived growth factor, vascular endothelial growth factor and transforming growth factor in vitro by ELISA. To investigate the biodegradability of the crosslinked L-PRF in vivo, the scaffolds were transplanted subcutaneously into backs of rats, and the materials were recovered at 1, 2 and 4 weeks after implantation. The biodegradation, inﬂammatory reaction and biocompatibility of the scaffolds were examined by histological staining. Introduction 2012). GP can cross-link with amino acids, and the gardenia blue produced by the reaction is a safe, non-toxic natural food pigment with good anti-inﬂammatory effect. As one of the effective components of Chinese herbal medicine, genipin can be also used for anti-inﬂammatory, antibacterial, antithrombotic, anti-tumor drugs and treatment of diabetes, jaundice and other diseases (Koo et al., 2006; Hou et al., 2008; Lelono and Itoh, 2009). Therefore, in comparision with other chemical crosslinking agents, genipin is more biosafety, which eliminates the step of repeated rinse to remove the free crosslinking agent after crosslinking reaction is completed. In recent years, GP has been more and more applied in the ﬁeld of tissue engineering, which can crosslink with a variety of natural scaffolds to improve the mechanical properties, regulate the degradation rate and control the release rate of drug (Yao et al., 2005; Yoo et al., 2011; Zhang et al., 2016). However, there is no study on appling GP to modify L-PRF yet. In our study, a variety of crosslinking schemes are designed to explore the feasibility of crosslinking L-PRF with genipin. We try to determin an simple and convenient crosslinking scheme and prepare a new type of GP crosslinked L-PRF scaffold. The physical and biological properties of the scaffold and the release kinetics of growth factors are investigated. The effect and application of genipin-modiﬁed L-PRF (GP/L-PRF) in bone regeneration are also explored. Bone tissue defects caused by periodontal disease, periapical disease, trauma and tumor that typically lead to inadequate bone volume are still challenges for dentists (Stumbras et al., 2019). Bone healing is a complicated and well-orchestrated physiological process in which blood clot formation is the initial and foremost phase to prevent excessive bleeding (Kolar et al., 2010; Claes et al., 2012). Beyond the hemostatic property, blood clots have proven to be critical to tissue healing, serving as a nature scaffold to deliver various growth factors and interact biologically with cells for tissue regeneration (Wang et al., 2017). Inspired by the natural healing blood clot, blood- derived products have attracted signiﬁcant interests in recent years (Yang and Xiao, 2020). Platelet-rich ﬁbrin (PRF) prepared by centrifuging autologous blood is described as a natural ﬁbrin scaffold containing all the constituents of blood that are favorable to tissue healing (Dohan Ehrenfest et al., 2012; Kumar and Shubhashini, 2013). Preparation of L-PRF Ten milliliters of autologous blood in 10 ml coated glass tubes without anticoagulants was obtained from the forearm vein of volunteers. The whole blood was immediately centrifuged at 3,000 rpm for 10 min (Labofuge 400Rcentrifuge, Heraeus, Hanau, Germany) according to the PRF protocol (Choukroun et al., 2006). Then the PRF clots were gently removed from the centrifuge tube, and the red blood cells at the bottom of PRF were cut off carefully. For the preparation of lyophilized PRF, PRF clots were frozen and stored at −80°C for 30 min and then freeze- dried overnight using a Labconco lyophilizer at −51+C (Free Zone, Labconco, Kansas City, MO, United State). OPEN ACCESS EDITED BY Ce Shi, Jilin University, China REVIEWED BY Chao Liu, Dalian Medical University, China Yanyan Li, Northeast Forestry University, China CORRESPONDENCE Han Jin, jinhan@hrbmu.edu.cn Bin Zhang, zhangbin@hrbmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Skeletal Physiology, a section of the journal Frontiers in Physiology RECEIVED 30 July 2022 ACCEPTED 01 September 2022 PUBLISHED 30 September 2022 CITATION Liu X, Yin M, Li Y, Wang J, Da J, Liu Z, Zhang K, Liu L, Zhang W, Wang P, Jin H and Zhang B (2022), Genipin modiﬁed lyophilized platelet-rich ﬁbrin scaffold for sustained release of growth factors to promote bone regeneration. Front. Physiol. 13:1007692. doi: 10.3389/fphys.2022.1007692 Finally, the genipin crosslinked/uncrosslinked L- Platelet-rich ﬁbrin scaffolds were implanted with freshly prepared SHED cell sheets into rat critical size calvarial defects and the skull samples were recovered to examine the treatment efﬁcacy of genipin crosslinked L-PRF by histologic and radiographic approaches. Results of this study indicated that genipin can be used to modify L-PRF at room temperature at a very low concentration. Genipin-modiﬁed L-PRF shows better biomechanical performance, slower biodegradation, good bioavailable and sustained release of growth factors. The 0.01% w/v and 0.1% w/v genipin crosslinked L-PRF have good porous structure and signiﬁcantly promote cell proliferation and enhance the expression of key genes in osteogenesis in vitro, and work best in promoting bone regeneration in vivo. CITATION Liu X, Yin M, Li Y, Wang J, Da J, Liu Z, Zhang K, Liu L, Zhang W, Wang P, Jin H and Zhang B (2022), Genipin modiﬁed lyophilized platelet-rich ﬁbrin scaffold for sustained release of growth factors to promote bone regeneration. Front. Physiol. 13:1007692. doi: 10.3389/fphys.2022.1007692 COPYRIGHT © 2022 Liu, Yin, Li, Wang, Da, Liu, Zhang, Liu, Zhang, Wang, Jin and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. COPYRIGHT © 2022 Liu, Yin, Li, Wang, Da, Liu, Zhang, Liu, Zhang, Wang, Jin and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Physiology frontiersin.org 01 Liu et al. 10.3389/fphys.2022.1007692 KEYWORDS lyophilized PRF, genipin, sustained release, growth factor, bone tissue engineering KEYWORDS lyophilized PRF, genipin, sustained release, growth factor, bone tissue engineering Introduction For the past few years, PRF has been widely used in the treatment of bone deﬁciency in the ﬁeld of stomatology, and has achieved remarkable curative effect owe to its ﬁbrin network and growth factor release (Mazor et al., 2009; Oncu and Alaaddinoglu, 2015; Pradeep et al., 2015). However, freshly prepared PRF must be used immediately in order to retain the bioactivity of growth factors. In addition, like all natural scaffold materials, the bio-degradation rate of PRF is fast and irregular, along with the rapid release of growth factors, and then enzymatically hydrolyzed. Lyophilized PRF (L-PRF) is more conducive to preservation and transportation, that will contribute to its clinical application and promotion (Roseti et al., 2017). The pores of ﬁbrin network in L-PRF are more abundant and larger, which would be in favour of cell migration and vascular invasion (Ma et al., 2018; Wang et al., 2019). What’s more, the freeze-thaw process further promotes platelet activation. However, accompanied by the change of network structure, the burst release of L-PRF after rehydration is more obvious (Roseti et al., 2017). Moreover, neither fresh nor lyophilized PRF has insufﬁcient mechanical properties. Due to these deﬁciencies, their applications are mostly limited to combined use with other scaffold materials. In our previous study, we explored a controlled release strategy based on the combination of fresh and lyophilized PRF with different ratios, which increased bone formation in vivo (Liu et al., 2019). We consider that it is clinical signiﬁcance to explore a simple and safe method to improve the performance of L-PRF for better applications. Proliferation assay Each lyophilized sample was weighed as M0 and then saturated with PBS (pH = 7.4) for 24 h at 37°C. The surface moisture was removed by blotting gently every half hour with ﬁlter paper and turgid weight M1 was recorded. Water absorption (Wa) was calculated as follows: 0.1 g of scaffolds were placed into 24-well plates with 1 ml of complete α-MEM medium and incubated in incubator for 14 days. The conditioned medium was collected every day and an equal volume of medium was added. All collected media were stored at 4°C. Wa M1 −M0 M0 × 100% SHEDs were seeded at a density of 3,000 cells per well in 96- well plates. The incubation medium was discarded after 48 h culture, and the conditioned media mentioned above was added to each well following experimental grouping. Cell proliferation was measured by Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan). The cross-sectional microstructure of scaffolds freeze-dried again was visualized using scanning electron microscopy (SEM; SM-5800LV, JEOL, Tokyo, Japan). The average pore diameter of crosslinked or non- crosslinked scaffold was calculated by ImageJ software. The scaffold was cut into 2 mm thickness, from which standard disk sample of 2 mm diameter × 1 mm thickness was made using a tissue puncher. Mechanical properties were determined using a universal testing machine Intron5569. Crosslinking L-PRF with different concentrations of genipin Crosslinking has been a common method to improve the properties of biomaterials. Genipin (GP) extracted from gardenia jasminoides ellis fruit is a natural crosslinker with good biocompatibility and low cytotoxicity compared with other crosslinking agents such as glutaraldehyde (Frohbergh et al., For each sample, 1 ml of GP was slowly added into L-PRF prepared from 10 ml of whole blood and mixed thoroughly. After 1 min, the excess solution was discarded. Then, samples were Frontiers in Physiology frontiersin.org 02 Liu et al. 10.3389/fphys.2022.1007692 wavelength for ELISA measurement was 450 nm. All assays were tested in triplicate. incubated for 24 h at room temperature to allow crosslinking reactions to proceed. wavelength for ELISA measurement was 450 nm. All assays were tested in triplicate. A series of gradient concentration GP solutions, including 0 w/v, 0.01% w/v, 0.1% w/v, 0.5% w/v and 1% w/v were applied to crosslink L-PRF. According to the concentration of genipin, the samples were grouped as 0 w/v L-PRF (non-crosslinked), 0.01% w/v GP/L-PRF, 0.1% w/v GP/L-PRF, 0.5% w/v GP/L-PRF and 1% w/v GP/L-PRF. All the experiments were performed in triplicates with three specimens in each group. Characterization of GP/L-PRF scaffold Scaffolds of each group described above were then lyophilized again and 3 mg of the sample was weighed into EP tube respectively incubated with 100 μL of deionized water for 1 h. Then the ninhydrin (NHN) colorimetric assay was used to determine the degree of crosslinking as previously described (Sanchez et al., 2017). To prepare cell membrane sheets, SHEDs were planted into 12-well plates with a density of 5 × 105 cells/mL. Cells were then grown continually without passaging for 14 days. Medium was changed every 48 h, and 50 mg/L ascorbic acid was added. Cell culture Culture of pulp stem cells from human exfoliated deciduous teeth (SHEDs) was carried out by adherent culture methods. Cells were cultured in α-MEM, supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientiﬁc, Inc.), 100 U/mL penicillin and 100 mg/ml streptomycin (PS; Thermo Fisher Scientiﬁc, Inc.). They were incubated in a 5% CO2 atmosphere at 37°C. Quantiﬁcation of growth factors derived from GP/L-PRF To induce osteogenic differentiation, extracts were prepared as explained before, expecting that the α-MEM was replaced with osteogenic differentiation medium containing 10 nM of dexamethasone, 10 mM of β-glycerophosphate, and 100 μM of ascorbic acid (Sigma, Sigma Chemical Co., St. Louis, MO, United States). First, extracts of all the groups of scaffolds were prepared. Brieﬂy, either crosslinked or non-crosslinked lyophilized PRF scaffolds (0.1 g) were placed in 5 ml centrifuge tubes containing 1 ml of α-Minimum Essential Medium (α-MEM; Thermo Fisher Scientiﬁc, Inc. Waltham, MA, United States) without fetal bovine serum. The tubes were placed in a shaker incubator at 37°C at 100 rpm. The conditioned medium was collected at the times of 1, 3, 7, 14, 21, and 28 days of culture, and an equal volume of medium was added to the tubes. All collected mediums were stored at -80°C and analyzed at the same time to reduce bias. All ELISA kits were purchased from Boster Biological Technology and used according to the manufacturer’s protocol, and the SHEDs were seeded into 24-well cell culture plates at a concentration of 3 × 104 cells/well. After cell attachment, different conditioned media were added to different wells following experimental grouping. After 14 days of co-culture, cells were ﬁxed and stained using Alizarin Red S (ARS, Sigma, Sigma Chemical Co., St. Louis, MO, United States) for detecting mineralization. Then ARS staining was released by cetylpyridinium chloride (Sigma-Aldrich) and quantiﬁed by spectrophotometry at 560 nm. Frontiers in Physiology frontiersin.org 03 Liu et al. 10.3389/fphys.2022.1007692 Animal surgical procedure First, the implants were prepared as described below. Samples of each group were prepared in the form of circular discs of 5 mm diameter and 2 mm thickness with a 5 mm diameter skin biopsy puncher. Results A total of 22 healthy male Sprague-Dawley (SD) rats, weighing around 200 g, were employed to construct the 5 mm critical calvarial bone defect model. After exposure of the cranium via skin incision, two 5 mm symmetrical full- thickness bone defects were created using an annular bone dill. The pre-prepared scaffolds and SHEDs membrane sheets were then implanted in the calvarial defects. The calvarial defects were randomly divided into seven groups: blank group (non- implant), Cell sheet (CS) group (only SHEDs membrane sheets), 0 w/v + CS group, 0.01% w/v + CS group, 0.1%w/v + CS group, 0.5% w/v + CS group and 1% w/v + CS group. The day of surgery was assigned as day 0. Rats were sacriﬁced with an overdose of 200 mg/ml pentobarbital sodium at 4 and 8 weeks after surgery. The entire cranium samples were extracted and stored in 4% paraformaldehyde for subsequent analysis. Statistical analyses For in vivo degradation study, 1 cm long incisions were performed on the backs of rats with a distance of 2 cm from the midline, 2 cm intervals in the same side, and subcutaneous pockets created by blunt dissection. Scaffold samples were implanted in subcutaneous pockets on the backs of rats randomly. Animals were sacriﬁced after 1 week, 2 and 4 weeks of implantation. The implants were removed along with the surrounding skin and subcutaneous tissue, and perfused with 4% PFA immediately. All statistical analyses were conducted using GraphPad Prism (v8.4.0). The data was statistically analyzed with one- way or two-way analysis of variance (ANOVA). All experiments were repeated at least three times, and all data were presented as mean ± standard deviation (SD). Differences with p ≤0.05 were taken as statistically signiﬁcant. Hematoxylin and eosin staining After radiographic studies, the calvarial specimens were decalciﬁed in 10% ethylenediaminetetracetic acid (EDTA) for about 4 weeks, sectioned by bisecting the 5 mm diameter defects, and then embedded in parafﬁn. Serial sections were prepared in the thickness of 4 μm from the middle part for hematoxylin and eosin staining (H&E). Quantitative PCR Gene transcription and expression of RUNX2, COL-1 and OCN in SHEDs induced by conditioned media for 14 days were studied by qPCR analysis. Total RNA was extracted with the TRIzol reagent on the 14th day of osteogenic induction. QPCR was performed by an Mx3005P system using SYBR® Premix Ex TaqTM (Takara Biotechnology Co., Ltd.), according to the manufacturer’s instructions. The RUNX2 primers were 5′- TGGTTACTGTCATGGCGGGTA-3′ (forward) and 5′-TCT CAGATCGTTGAACCTTGCTA-3′ (reverse); the COL-1 primers were 5′- GAGGGCCAAGACGAAGACATC -3′ (forward) and 5′-CAGATCACGTCATCGCACAAC-3′ (reverse); the OCN primers were 5′-CACTCCTCGCCCTAT TGGC-3′ (forward) and 5′- CCCTCCTGCTTGGACACAAAG-3′(reverse); the β-actin primers were 5′-CATGTACGTTGCTATCCAGGC-3′ (forward) and 5′-CTCCTTAATGTCACGCACGAT-3′ (reverse). Radiographs of the cranium samples were taken by a Faxitron Specimen Radiography System (Model MX-20; Faxitron X-ray Corporation, Wheeling, IL) at 26 kVp and exposure time of 11 s. The degree of bone regeneration was examined by a micro-CT scanner (μCT35, Scanco Medical AG, Bassersdorf, Switzerland) with a 10 μm voxel size using the following parameters: 114 mA, 70 kVp, and exposure time of 300 ms. The new bone formation was calculated as the percentage fraction of new bone area to the total defect area using the methods described in previous study (Liu et al., 2019). L-PRF could crosslink with genipin L-PRF could react with genipin to produce blue compound, and the blue color was deepened as time extended. The crosslinking time of 24 h was observed to be sufﬁcient for complete crosslinking by prior experimentation. For the same duration of crosslinking, the greater the GP concentration used, the darker the blue color (Figures 1A,B). The ninhydrin (NHN) colorimetric assay was used to determine the degree of crosslinking. The result of the experiment shows the degree of crosslinking increased along with the increasing concentration of genipin (Figure 1C). Frontiers in Physiology frontiersin.org 04 Liu et al. Liu et al. 10.3389/fphys.2022.1007692 FIGURE 1 (A) Scaffolds crosslinked with genipin for 30 min (B) Scaffolds crosslinked with genipin for 24h, (C) The degree of crosslinking after 24 h (p < 0.05, p < 0.01,p < 0.001,**p < 0.0001) FIGURE 1 (A) Scaffolds crosslinked with genipin for 30 min (B) Scaffolds crosslinked with genipin for 24h, (C) The degree of crosslinking after 24 h (p < 0.05, p < 0.01,p < 0.001,**p < 0.0001) Crosslinking improved the physical properties of L-PRF The effect of genipin modiﬁcation on the release kinetics of growth factors from L-PRF was studied by ELISA in vitro. The experiment was illustrated in Figures 2I–K and Supplementary Tables S1–S3. Although all groups had burst release within 24 h, the amount of GFs release from GP/L-PRF decreased with increasing crosslinking degree. After an initial burst release within the ﬁrst day, GP/L-PRF showed slow release for the following days. The rate of GFs release decreased with the degree of crosslinking increased. The cumulative amount of PDGF-AB, TGF-β1 and VEGF in crosslinked groups were signiﬁcantly lower than that in the control at all time points. The total release of three growth factors was different, with the highest release of TGF-β1, and the lowest release of VEGF. Scanning electron microscopy image showed either crosslinked or non-crosslinked L-PRF scaffold had highly interconnected porous structures (Figures 2A–E). The number of macropores between 100–200 μm in diameter was increased in 0.01% w/v group and 0.1% w/v group compared to other groups, and adjacent macropores were interconnected by micropores. The average pore diameter was measured from SEM images. The mean diameter was increased in each genipin crosslinked group compared with the non-crosslinked control group, and the differences were signiﬁcant (Figure 2F). Of these, both 0.01% w/v GP/L-PRF and 0.1% w/v GP/L-PRF exhibited increasing average pore size larger than 100 μm (0.01% w/v vs control, and 0.1% w/v vs control, p < 0.0001). The water absorption rates of the scaffolds were illustrated in Figure 2G. After 1 h immersion, the water absorption rate of all the samples tended to plateau, and reached the saturation limit after 24 h of immersion. Compared with the control group, all the GP crosslinked L-PRF showed increased water uptake, and 0.01% w/v GP/L-PRF and 0.1% w/v GP/L-PRF produced a signiﬁcant difference. Crosslinking slowed the rate of degradation of L-PRF The degradation of L-PRF was accompanied by the release of GFs. We then conducted subcutaneous implantation experiment to explore the biodegradability of GP/L-PRF in vivo, and observe its biocompatibility in the meanwhile. One week after surgery, the HE results showed that the scaffold material in each group was not completely biodegradable. Non-crosslinked L-PRF had a loose and thin ﬁber network, and contained a large number of inﬁltration cells (Figure 3A). With the crosslinking degree of L-PRF increased, the ﬁber network structure became increasingly compact, and the number of cells inﬁltrated within scaffolds markedly decreased. Scaffolds of 1% w/v GP/L-PRF showed compactness microstructure with few pores visible (Figures 3B–E). The mechanical test results indicated that elastic modulus increased as the degree of crosslinking increased (Figure 2H). Elastic modulus of crosslinked L-PRF was higher than that of non-crosslinked L-PRF. The average modulus of elasticity of 1% w/v GP/L-PRF was signiﬁcantly higher than control (p < 0.05). Although increasing values of elastic modulus were also shown in all other crosslinked groups, the differences were not signiﬁcant compared with the control group. Frontiers in Physiology frontiersin.org 05 Liu et al. 10.3389/fphys.2022.1007692 FIGURE 2 Scanning electron microscope (SEM) images of (A) 0w/v group (B) 0.01%w/v group (C) 0.1%w/v group (D) 0.5%w/v group and (E) 1%w/v group (F) The mean pore size of scaffolds, (G) Water absorption of scaffold (H) Elastic modulus of uncrosslinked and crosslinked group; (I–K) The cumulative release of growth factors of scaffolds. (The 0w/v group was used as control: p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, the release ofT GF-β1 at day1, 0.01%w/v VS control group, p > 0.05; other groups at all time points VS control group, p < 0.05). FIGURE 2 Scanning electron microscope (SEM) images of (A) 0w/v group (B) 0.01%w/v group (C) 0.1%w/v group (D) 0.5%w/v group and (E) 1%w/v group (F) The mean pore size of scaffolds, (G) Water absorption of scaffold (H) Elastic modulus of uncrosslinked and crosslinked group; (I–K) The cumulative release of growth factors of scaffolds. (The 0w/v group was used as control: p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, the release ofT GF-β1 at day1, 0.01%w/v VS control group, p > 0.05; other groups at all time points VS control group, p < 0.05). GP/L-PRF scaffold had No cell toxicity corresponding extracts from scaffolds. The 0.01% w/v group had the highest expression of all three genes compared to others. The expression of RUNX2 of cells with extracts treatment was higher compared with that of the control group (Figure 4H). COL-1 expression of 0w/v group and 0.5% w/v group did not show a signiﬁcant expression difference when compared to the control, while the expression of 0.01% w/v group and 0.1% w/v group was signiﬁcantly higher (Figure 4I). All groups except 0.5% w/v group signiﬁcantly increased OCN expression on day 14 compared with control (Figure 4J). To evaluate biocompatibility of crosslinked scaffolds, cytotoxicity test was performed on the extracts of scaffolds, and α-MEM complete medium was used as negative control group. As shown in Figure 4A, either non-crosslinked or crosslinked L-PRF showed no signiﬁcant inhibitory effect on SHEDs proliferation at any of the time points examined. The extracts from 0.01% w/v GP/L-PRF could signiﬁcantly promote the proliferation of SHEDs until day 7. The extracts from 0.5% w/ v group reduced cell proliferation slightly. Crosslinking slowed the rate of degradation of L-PRF FIGURE 3 Photomicrographs of hematoxylin and eosin of (A) 0w/v group (B) 0.01%w/v group, (C) 0.1%w/v group (D) 0.5%w/v group, (E) 1%w/v group retrieved at 1 w and (F) 0.01%w/v group (G) 0.1%w/v group, (H) 0.5%w/v group (I) 1%w/v group retrieved at 2 w postoperatively. (S: scaffold, CT: connective tissue, Black triangular arrows indicate scattered scaffold debris). FIGURE 3 Photomicrographs of hematoxylin and eosin of (A) 0w/v group (B) 0.01%w/v group, (C) 0.1%w/v group (D) 0.5%w/v group, (E) 1%w/v group retrieved at 1 w and (F) 0.01%w/v group (G) 0.1%w/v group, (H) 0.5%w/v group (I) 1%w/v group retrieved at 2 w postoperatively. (S: scaffold, CT: connective tissue, Black triangular arrows indicate scattered scaffold debris). Crosslinking slowed the rate of degradation of L-PRF Scanning electron microscope (SEM) images of (A) 0w/v group (B) 0.01%w/v group (C) 0.1%w/v group (D) 0.5%w/v group and (E) 1%w/v group (F) The mean pore size of scaffolds, (G) Water absorption of scaffold (H) Elastic modulus of uncrosslinked and crosslinked group; (I–K) The cumulative release of growth factors of scaffolds. (The 0w/v group was used as control: p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, the release ofT GF-β1 at day1, 0.01%w/v VS control group, p > 0.05; other groups at all time points VS control group, p < 0.05). The L-PRF of the non-crosslinked control group underwent complete biodegradation at 2 weeks after surgery (not shown). Only a small amount of scattered scaffold debris surrounded by inﬂammatory cells was presented in 0.01% w/v GP/L-PRF group (Figure 3F). Obvious degradation was also observed in the 0.1% w/v GP/L-PRF group. Most of the crosslinked L-PRF is replaced by ﬁbrous connective tissue and cells. The boundaries between crosslinked scaffold and surrounding tissues were blurred, with new tissues growing inside the scaffold while the scaffold structure collapsed (Figure 3G). Many cells had inﬁltrated from the scaffold margin of 0.5% w/ v group and ﬁber arrangement became looser (Figure 3H). There was no apparent degradation seen in 1% w/v group (Figure 3I). Inﬂammatory responses were not observed in any group. Abundant neovascularization was found around the scaffolds in all groups. At 4 weeks, the scaffolds in all groups were completely degraded except 1% w/v group (Supplementary Figure S1B). Some granules with irregular shape could be seen in 0.5% w/v group (Supplementary Figure S1A). Neovascularisation was visible in the connective tissue. As the results mentioned above, including average pore diameter and biodegradability showed the performance of 1% w/v GP/L-PRF was not satisfactory, the group was no longer shown in the following experiments. Frontiers in Physiology frontiersin.org 06 Liu et al. 10.3389/fphys.2022.1007692 FIGURE 3 Photomicrographs of hematoxylin and eosin of (A) 0w/v group (B) 0.01%w/v group, (C) 0.1%w/v group (D) 0.5%w/v group, (E) 1%w/v group retrieved at 1 w and (F) 0.01%w/v group (G) 0.1%w/v group, (H) 0.5%w/v group (I) 1%w/v group retrieved at 2 w postoperatively. (S: scaffold, CT: connective tissue, Black triangular arrows indicate scattered scaffold debris). The extracts from GP/L-PRF enhance osteogenic gene expression Mineralization nodules were detected through Alizarin red staining after 14 days of induction using an osteogenic differentiation medium (control) or corresponding extracts from 0w/v group, 0.01% w/v group, 0.1% w/v group and 0.5% w/v group. The mineralized nodules were more intense in the 0.01% w/v group and 0.1% w/v group, compared with those in the other groups. The 0.5% w/v group had the least mineralized nodule formation (Figures 4B–F). The result of semi-quantitative analysis of ARS showed that the OD values of 0.01% w/v group and 0.1% w/v group were signiﬁcantly higher compared to the control group, at which the OD values of 0.01% w/v group were highest. 0w/v group and 0.5% w/v group represent no signiﬁcant difference compared to control (Figure 4G). Finally, the in vivo repair efﬁcacy of bone defects was evaluated in a rat defect model by implant GP/L-PRF combined with cell sheets. After implantation for 4 weeks, the blank group presented only a small amount of new bone tissues around the edge of defects. Various degrees of bone repair were observed on the edges and extended towards the center of the defect in all groups except blank control group (Figure 5A). Quantitative analysis of percentage of new bone area in calvarial defects revealed the area of newly formed bone in the 0w/v + CS group and 0.1% w/v + CS group were signiﬁcantly greater than that in the CS group (Figure 5B). After 8 weeks, the radiological ﬁndings showed an obvious increase in bone mass except for blank group. Continuous growth of new bone was still not observed in the blank group. The new bone of 0.01% w/v + CS group and 0.1% w/v + CS group ﬁlled almost the entire defect area and integrated with The expression of osteogenesis related genes Runx2, Col-I and OCN were determined by qRT-PCR after 14 days of culture with osteogenic differentiation medium (control) or Frontiers in Physiology frontiersin.org 07 Liu et al. 10.3389/fphys.2022.1007692 FIGURE 4 (A) Cell proliferation and cytotoxicity assay of SHEDs by CCK-8; Alizarin red staining of (B) Control group (C) 0w/v group (D) 0.01w/v group (E) 0.1%w/v group and (F) 0.5%w/v group (G) Semi quantitative results of alizarin red staining; (H–J) Real time PCR results of osteogenic–speciﬁc markers. (p < 0.05, p < 0.01, p < 0.001. RUNX2: Runt related transcription factor 2, COL-I: Collagen I,OCN: Osteocalcin). The extracts from GP/L-PRF enhance osteogenic gene expression FIGURE 4 (A) Cell proliferation and cytotoxicity assay of SHEDs by CCK-8; Alizarin red staining of (B) Control group (C) 0w/v group (D) 0.01w/v group (E) 0.1%w/v group and (F) 0.5%w/v group (G) Semi quantitative results of alizarin red staining; (H–J) Real time PCR results of osteogenic–speciﬁc markers. (p < 0.05, p < 0.01, p < 0.001. RUNX2: Runt related transcription factor 2, COL-I: Collagen I,OCN: Osteocalcin). Frontiers in Physiology frontiersin.org 08 Liu et al. Liu et al. 10.3389/fphys.2022.1007692 FIGURE 5 Radiographs and micro-CT analysis of bone regeneration in rat critical size calvarial defects at 4 and 8 weeks postoperatively (A) and quantiﬁcation analysis of the regenerated tissue covering the calvarial defect at 4 w (B) and 8 w (C). (The CS group was used as control: p < 0.05, *p < 0.001,*p < 0.0001. CS: cell sheet). FIGURE 5 Radiographs and micro-CT analysis of bone regeneration in rat critical size calvarial defects at 4 and 8 weeks postoperatively (A) and quantiﬁcation analysis of the regenerated tissue covering the calvarial defect at 4 w (B) and 8 w (C). (The CS group was used as control: p < 0.05, *p < 0.001,**p < 0.0001. CS: cell sheet). arrangement was more regular. The regenerated tissue completely fused with the host bone in the 0.01% w/v + CS group. the original bone at the defect margins (Figure 5A). The percentage of new bone formation was (86.6 ± 0.2)% in 0.01% w/v + CS group and (85.1 ± 0.1)% in 0.1% w/v + CS group, signiﬁcantly higher than that of the 0w/v + CS group. Compared with CS group, the area of newly formed bone tissues was signiﬁcantly larger in each L-PRF combined with CS group (Figure 5C). Discussion Blood clot plays a very important role during osteogenesis thanks to the structure characteristics and growth factors release (Lai et al., 2010; Shiu et al., 2018; Yang and Xiao, 2020). Inspired by the impact of natural blood clot, scholars have explored the innovation and application of blood products over the years (Yang and Xiao, 2020). Platelet-rich ﬁbrin (PRF), a second generation of platelet concentrates, has already been widely used in modern medicine (Gassling et al., 2010; Sindel et al., 2017; Pripatnanont et al., 2013). Lyophilization was shown to activate resident platelets in PRF causing the further release of GFs thus promoting tissue healing (Roseti et al., 2017). However, the more obvious burst release of GFs of L-PRF cannot meet the requirement for bone tissue engineering (Li et al., 2014). In previous studies, we explored a biomimetic strategy to promote bone regeneration based on the combination of fresh and lyophilized PRF with different ratios (Sanchez et al., 2017). This study aims for tuning the release of GFs, decreasing the rate of degradation and improving the performance of L-PRF by crosslinking with genipin. The histological ﬁndings were consistent with imaging ﬁndings (Figure 6). In the blank control groups, almost no new bone was found in the defect 4 weeks postoperatively and only ﬁbrous connective tissue covered the empty defects. A large area of residual materials could been seen in group of 0.5% w/v + CS. Histological sections of CS group, 0w/v + CS group, 0.01% w/ v + CS group and 0.1% w/v + CS group presented abundant new island of bone and neovascularization. Osteoblasts were visibly arrayed on the surfaces of newly formed bone tissues in the 0w/v + CS group, 0.01% w/v + CS group and 0.1% w/v + CS group. The defect area of the blank group still only had thin ﬁbrous connective tissues at 8 weeks. Compared with 4 weeks, the trabecular bones were thickened, the number was increased and the connections were compact in CS group and scaffolds combined CS groups. The nascent bone in combination groups was thicker than that in CS alone group. It was obvious that trabecular bone in the 0.01% w/v + CS group and 0.1% w/v + CS group appeared to be thicker and more integrated, and the The histological ﬁndings were consistent with imaging ﬁndings (Figure 6). Discussion In the blank control groups, almost no new bone was found in the defect 4 weeks postoperatively and only ﬁbrous connective tissue covered the empty defects. A large area of residual materials could been seen in group of 0.5% w/v + CS. Histological sections of CS group, 0w/v + CS group, 0.01% w/ v + CS group and 0.1% w/v + CS group presented abundant new island of bone and neovascularization. Osteoblasts were visibly arrayed on the surfaces of newly formed bone tissues in the 0w/v + CS group, 0.01% w/v + CS group and 0.1% w/v + CS group. The defect area of the blank group still only had thin ﬁbrous connective tissues at 8 weeks. Compared with 4 weeks, the trabecular bones were thickened, the number was increased and the connections were compact in CS group and scaffolds combined CS groups. The nascent bone in combination groups was thicker than that in CS alone group. It was obvious that trabecular bone in the 0.01% w/v + CS group and 0.1% w/v + CS group appeared to be thicker and more integrated, and the Frontiers in Physiology frontiersin.org 09 Liu et al. 10.3389/fphys.2022.1007692 FIGURE 6 Photomicrographs of hematoxylin and eosin retrieved at 4 w and 8 w postoperatively. (S: scaffold, CT, connective tissue; NB, new bone; CS, cell sheet. The blue arrows indicate osteoblasts, Blue triangular arrows indicate the irregular particle residue). FIGURE 6 Photomicrographs of hematoxylin and eosin retrieved at 4 w and 8 w postoperatively. (S: scaffold, CT, connective tissue; NB, new bone; CS, cell sheet. The blue arrows indicate osteoblasts, Blue triangular arrows indicate the irregular particle residue). naturally rich in growth factors (Dohan et al., 2006; Kumar et al., 2015). The soluble ﬁbrinogen polymerizes slowly into ﬁbrin network under the action of a physiological concentration of thrombin, during which growth factors are trapped in the ﬁber network (Dohan et al., 2006). Growth factors, which are not bound to ﬁbrin, are rapidly released from PRF, while the bound growth factors are gradually released with the degradation of network (Atienza-Roca et al., 2018). Lyophilization promotes the further release of GFs derived from PRF, and meanwhile along with the change of network structure leads to a faster degradation and release rate (Li et al., 2014; Roseti et al., 2017). Previous studies have shown that the covalent bonding of GFs into scaffold can reduce the burst release (Masters, 2011). Discussion The extracts of non-crosslinked L-PRF enhance the proliferative capability but not the osteogenic differentiation ability, that may be related to the high concentration of GFs. We ﬁnd that new bone formation in 0.5% w/v GP/L-PRF group is slower because the GFs release is slower than other groups. From the above results, it is suggested that proper local concentration of GFs is important for bone tissue regeneration. ability of stem cell sheets. These ﬁndings are consistent with our studies. In vivo experiments in the present study also conﬁrmed the new bone formation ratio of GP/L-PRF combined with SHEDs is higher than that of non- crosslinked L-PRF. By 8 weeks after the operation, 0.01% w/v and 0.1% w/v GP/L-PRF were able to almost completely heal the bony defect. The extracts of non-crosslinked L-PRF enhance the proliferative capability but not the osteogenic differentiation ability, that may be related to the high concentration of GFs. We ﬁnd that new bone formation in 0.5% w/v GP/L-PRF group is slower because the GFs release is slower than other groups. From the above results, it is suggested that proper local concentration of GFs is important for bone tissue regeneration. also owes to the change of ﬁbrin network structure. The structure characteristic of L-PRF includes a dense surface layer and loose inner structure. The surface pore architecture denser due to crosslinking is also contributed to controlled release. The decreased rate of degradation is also in favor of the sustained release of GFs. The subcutaneous degradation experiment in rat shows that the higher the crosslinking degree is, the slower the degradation rate is. The non- crosslinked L-PRF completely degraded after 2 weeks of in vivo implantation, while 0.01% w/v and 0.1% w/v GP/L-PRF degraded to debris, 0.5% w/v GP/L-PRF degraded relatively slowly and 1% w/v GP/L-PRF showed no noticeable degradation. Hence, a signiﬁcant amount of GFs would have been released at 1 week when referring to non-crosslinked L-PRF. The sustained release of 0.01% w/v and 0.1% w/v GP/ L-PRF continues over a period of 2 weeks. The release rate is slower in 0.5% w/v GP/L-PRF and the slowest in 1% w/v GP/ L-PRF. It can be found that the pattern of biomaterials degrading in vivo is in line with the pattern of GFs release in vitro. Degrading too slowly also has adverse effects of tending to be recognised as foreign and eliciting host immune responses against the antibody. Discussion It is suggested that the 1% w/v GP/ L-PRF is not available for tissue healing. It is worth noting that in our subcutaneous degradation experiment, no obvious inﬂammatory reaction occurred in all groups, which may be attributed to the biocompatibility of blood derivatives, the modiﬁcation of antigenic sites by crosslinking and lyophilization (Koo et al., 2006; Zhang et al., 2017; Yip et al., 2019). This may also be related to the anti-inﬂammatory effect of the crosslinked product geniposide, which needs further experiments. The cells cultured with conditioned medium from 0.5% w/v GP/L-PRF exhibited decreased proliferation from day 5, and decreased mineralized nodules and critical osteogenesis-related gene expression. We speculate the high concentration of genipin may be the reason. The dark blue particles observed in the in vivo experiment may provide evidence for excess GP. This study is the ﬁrst to present an approach to improve L-PRF by crosslinking with genipin. The present results show that reaction can be conducted at room temperature by very low concentrations of genipin. The obtained 0.01% w/v and 0.1% w/v GP/L-PRF has suitable pore structure and ability of sustained GFs release. Moreover, the crosslinked material presents good in vitro cell compatibility and in vivo biocompatibility. They can stimulate proliferation and osteogenic differentiation of SHEDs in vitro and increase bone formation on the bone defect area in vivo. GP/L-PRF combined with stem cell sheets accelerates bone defect healing. The present study provides a new way of thinking to improve the performance of L-PRF and an economical, safe and convenient method for sustained growth factors release. Bone healing is regulated by various growth factors. It has been reported that bone repairing process involved revascularization, osteogenesis, bone remodeling, and the proliferation and differentiation of osteoblasts occurred during the initial 14 days (Uggeri et al., 2007). PDGF favors division and proliferation of osteoblasts within the damage site (Hollinger et al., 2008; Rao et al., 2009). TGF-β, the most abundant GFs derived from PRF, recruits circulated stem cells to the site of injury and promotes the deposition and mineralization of the bone matrix (Fennen et al., 2016; Perez et al., 2018). VEGF is a key component responsible for angiogenesis, which beneﬁts stem cell recruitment and transportation of nutrients (Roseti et al., 2017; Iaquinta et al., 2019). Our research has shown that 0.01% w/v and 0.1% w/v GP/L-PRF can continuously release growth factors within 14 days. Discussion The in vitro studies ﬁnd that the proliferation and osteogenic differentiation of SHEDs are stimulated by extracts derived from 0.01% w/v and 0.1% w/v GP/L-PRF. David et al. found PRF could both promote the proliferation of hBMSCs and enhance ALP activity and mineralized nodule formation. Sindel and others described that PRF enhanced the osteogenic Data availability statement The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. Ethics statement The animal study and clinical ethics were reviewed and approved by the Ethics Committee at The Second Afﬁliated Hospital of Harbin Medical University (SYD2020-068, KY2021-154). Discussion Muiznieks in his study stated that genipin could be used to crosslink elastin or elastin polypeptides by formed covalent crosslinks (Muiznieks, 2018). In this study, genipin is ﬁrst used to crosslink with L-PRF. From the results, it can be seen that the burst release of GP/L-PRF is evidently decreased compared with L-PRF, suggesting the GFs may be conjugated onto ﬁber network which is necessary to further verify by further experiments. After burst release, GP/L-PRF releases GFs slowly and the cumulative release is always lower than non-crosslinked L-PRF during 28 days. The 0.1% w/v GP/L-PRF sustainably releases GFs at an almost constant rate. This slow-release effect In order to accelerate bone reconstruction, an ideal implant requires both a porous structure and optimal water uptake properties, which is favorable for cell inﬁltration, adhesion and proliferation (Thavornyutikarn et al., 2014; Wang et al., 2016; Anitua et al., 2018). The average pore size shows a tendency to increase ﬁrst and then decrease with the increase of the crosslinking degree. It could be observed that both macropores with a mean diameter larger than 100 μm and highly interconnected small pores coexisted in the 0.01% w/v group and 0.1% w/v group, meets the requirement for bone regeneration. The saturated water absorption of the 0.01% w/v group and 0.1% w/v group are signiﬁcantly higher than that of non-crosslinked L-PRF, suggesting there is a higher porosity. The number of macropores is obviously reduced and the pore wall is nearly smooth almost without pores in the 1% w/v group, which is not adapted to bone reconstruction. After rehydration, non-crosslinked L-PRF lacks the required mechanical properties and maneuverability. As is shown in Figure 2G, the elastic modulus of non-crosslinked L-PRF is the lowest, whereas that of L-PRF crosslinked by 1% w/v genipin is the highest, illustrating crosslinking improves the mechanical properties of L-PRF. Growth factors also play important roles in bone tissue engineering (Kneser et al., 2006; Iaquinta et al., 2019). PRF is Frontiers in Physiology frontiersin.org 10 Liu et al. 10.3389/fphys.2022.1007692 10.3389/fphys.2022.1007692 ability of stem cell sheets. These ﬁndings are consistent with our studies. In vivo experiments in the present study also conﬁrmed the new bone formation ratio of GP/L-PRF combined with SHEDs is higher than that of non- crosslinked L-PRF. By 8 weeks after the operation, 0.01% w/v and 0.1% w/v GP/L-PRF were able to almost completely heal the bony defect. References Hou, Y. C., Tsai, S. Y., Lai, P. Y., Chen, Y. S., and Chao, P. D. L. (2008). Metabolism and pharmacokinetics of genipin and geniposide in rats. Food Chem. Toxicol. 46 (8), 2764–2769. doi:10.1016/j.fct.2008.04.033 Hou, Y. C., Tsai, S. Y., Lai, P. Y., Chen, Y. S., and Chao, P. D. L. (2008). Metabolism and pharmacokinetics of genipin and geniposide in rats. Food Chem. Toxicol. 46 (8), 2764–2769. doi:10.1016/j.fct.2008.04.033 Anitua, E., Troya, M., and Zalduendo, M. (2018). Progress in the use of dental pulp stem cells in regenerative medicine. Cytotherapy 20 (4), 479–498. doi:10.1016/ j.jcyt.2017.12.011 Iaquinta, M. R., Mazzoni, E., Manfrini, M., D’Agostino, A., Trevisiol, L., Nocini, R., et al. (2019). Innovative biomaterials for bone regrowth. Int. J. Mol. Sci. 20 (3), E618. doi:10.3390/ijms20030618 Atienza-Roca, P., Cui, X., Hooper, G. J., Woodﬁeld, T. B. 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Author contributions XL, MY and YL conceived and designed the research, performed analysis and wrote the majority of the Frontiers in Physiology 11 frontiersin.org Liu et al. 10.3389/fphys.2022.1007692 Acknowledgments The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2022.1007692/full#supplementary-material We express our sincere gratitude to all the volunteers who donated blood for this study. Funding All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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https://openalex.org/W4236610321	https://zenodo.org/record/4061617/files/readmefile.pdf	English	null	Experimental simulation of Titan's stratospheric photochemistry: benzene (C6H6) ices	null	2,020	cc-by	208	C6H6_dp=0,5mbar_130K.CSV : solid benzene infrared spectrum recorded at 130k C6H6_dp=0,5mbar_16K.CSV : solid benzene infrared spectrum recorded at 16K C6H6_dp=0,5mbar_50K.CSV : solid benzene infrared spectrum recorded at 50K C6H6_dp=0,5mbar_70K.CSV : solid benzene infrared spectrum recorded at 70K C6H6_dp=0,5mbar_90K.CSV : solid benzene infrared spectrum recorded at 90K glace C6H6 irradiée.CSV : Infrared spectrum of benzene ice obtained after 2880 minutes of irradiation at >230 nm performed at 70K C6H6_dp=0,5mbar_130K.CSV : solid benzene infrared spectrum recorded at 130k C6H6_dp=0,5mbar_16K.CSV : solid benzene infrared spectrum recorded at 16K C6H6_dp=0,5mbar_50K.CSV : solid benzene infrared spectrum recorded at 50K C6H6_dp=0,5mbar_70K.CSV : solid benzene infrared spectrum recorded at 70K C6H6_dp=0,5mbar_90K.CSV : solid benzene infrared spectrum recorded at 90K glace C6H6 irradiée.CSV : Infrared spectrum of benzene ice obtained after 2880 minutes of irradiation at >230 nm performed at 70K glace C6H6 irradiée.CSV : Infrared spectrum of benzene ice obtained after 2880 minutes of irradiation at >230 nm performed at 70K. glace C6H6.CSV : Infrared spectrum of benzene ice before irradiation at >230 nm performed at 70K. résidu C6H6_300K.CSV : Infrared spectrum obtained at room temperature after benzene ice irradiation. résidu C6H6_300K.CSV : Infrared spectrum obtained at room temperature after benzene ice irradiation. résidu C6H6_300K.CSV : Infrared spectrum obtained at room temperature after benzene ice irradiation.
https://openalex.org/W4280583695	https://pure.ulster.ac.uk/ws/files/101328673/sensors-22-03612.pdf	English	null	Image Sensing and Processing with Convolutional Neural Networks	Sensors	2,022	cc-by	2,082	General rights Th i ht General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the docum Unless otherwise stated, users are permitted to download a copy of the output for personal study or non-commercial research and are permitted to freely distribute the URL of the output. They are not permitted to alter, reproduce, distribute or make any commercial use of the output without obtaining the permission of the author(s). 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Advance online publication. https://doi.org/10.3390/s22103612 Link to publication record in Ulster University Research Portal Link to publication record in Ulster University Research Portal Document Version Publisher's PDF, also known as Version of record Take down policy Take down policy The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 sensors sensors Editorial Image Sensing and Processing with Convolutional Neural Networks Sonya Coleman 1,* , Dermot Kerr 1 and Yunzhou Zhang 2 1 School of Computing, Engineering and Intelligent Systems, Ulster University, Londonderry BT48 7JL, UK; d.kerr@ulster.ac.uk 2 College of Information Science and Engineering, Northeastern University, Shenyang 110819, China; zhangyunzhou@ise.neu.edu.cn * C d l @ l k Convolutional neural networks are a class of deep neural networks that leverage spatial information, and they are therefore well suited to classifying images for a range of applications. These networks use an ad hoc architecture inspired by our understanding of processing within the visual cortex. Convolutional neural networks (or CNNs) provide an interesting method for representing and processing image information and form a link between general feed-forward neural networks and adaptive ﬁlters. Two-dimensional CNNs are formed by one or more layers of two-dimensional ﬁlters, with possible non-linear activation functions and/or down-sampling. CNNs possess key properties of translation invariance and spatially local connections (receptive ﬁelds). Given this, deep learning using convolutional neural networks (CNNs) has quickly become the state of the art for challenging computer vision applications. Image quality is critical for many applications. CNNs have a key role to play in directly dealing with low-quality images or in image enhancement applications. Tchendjou et al. [1] presented a new objective method incorporating a CNN for the estimation of visual per- ceiving quality without the need for a reference image or assumptions on the image quality. Wang et al. [2] explored the effect of geometric disturbance corresponding to attitude jitter using a GAN to explore the usefulness for jitter detection, revealing the enormous poten- tial of GAN-based methods for the analysis of attitude jitter from remote sensing images. Han et al. [3] proposed a deep supervised residual dense network, which uses residual dense blocks to enhance features along with an encoder and decoder to reduce the differences be- tween the features for underwater degraded images. Xiao et al. [4] focused on blur detection as an image segmentation problem where a multi-scale dilated convolutional neural network (MSDU-net) extracts features with dilated convolutions and a U-shape architecture fuses the different-scale features, supporting the image segmentation task. Yang et al. [5] proposed a novel deeply recursive low- and high-frequency fusing network for single-image super- resolution (SISR) tasks which adopts the structure of parallel branches with a focus on reducing computational and memory resources. Citation: Coleman, S.; Kerr, D.; Zhang, Y. Image Sensing and Processing with Convolutional Neural Networks. Sensors 2022, 22, 3612. https://doi.org/10.3390/ s22103612 Received: 11 April 2022 Accepted: 6 May 2022 Published: 10 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. CNNs can play a leading role in environmental applications. For example, pollution in the form of litter in the natural environment is one of the great challenges of our times. Cordova et al. [6] developed an automated litter detection system that can help assess waste occurrences in the environment. A comparative study involving state-of- the-art CNN architectures highlights the role CNNs can play to support this. Similarly, Wei et al. [7] developed models for predicting the wind speed and wave height near the coasts of ports during typhoon periods, where gated recurrent unit (GRU) neural networks and convolutional neural networks (CNNs) were combined and adopted to formulate the typhoon-induced wind and wave height prediction models. Wu et al. [8] targeted the detection of speciﬁc crop types from crowdsourced road-view photos and clearly demonstrated the superior accuracy of this approach. Xu et al. [9] presented an accurate Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sensors Sensors 2022, 22, 3612. https://doi.org/10.3390/s22103612 Sensors 2022, 22, 3612 2 of 3 and robust detection of road damage that is essential for public transportation safety, and Chou et al. [10] developed a smart dredging construction site system using automated techniques to automate the audit work at the control point, which manages trucks in river dredging areas. g g Healthcare is an important application area that AI and CNNs, in particular, can have an impact on. Speciﬁcally, the role of 5G-IoT plays a crucial part in e-health applications, and to this end, Anand et al. [11] proposed a new deep learning model to detect malware attacks based on a CNN. In contrast, Barros et al. [12] presented a hybrid model to classify lung ultrasound videos captured by convex transducers to diagnose COVID-19 with an average accuracy of 93% and sensitivity of 97%. The Clock Drawing Test (CDT) is a rapid, inexpensive, and popular screening tool for cognitive functions. Park et al. [13] presented a mobile phone application, mCDT, and suggested a novel, automatic, and qualitative scoring method and deep learning that provides the ability to differentiate dementia disease. Alsamadony et al. [14] applied DCNNs to improve the quality of rock CT images and reduce exposure times by more than 60% simultaneously. The approach is applicable to any computed tomography technology. Ankita et al. [15] presented an approach in which convolutional layers are combined with long short-term memory (LSTM) for human activity recognition (HAR); providing an accuracy of 97.89%, this has applications in assistive living and healthcare. Robotics is an important application area for CNNs, and to help robots grasp specific objects in multi-object scenes, the powerful feature extraction capabilities of CNNs have been proposed. Different from anchor-based grasp detection algorithms, Li et al. [16] successfully developed a keypoint-based scheme demonstrating that a robot can grasp the target in single- object and multi-object scenes with overall success rates of 94% and 87%, respectively. This Special Issue provides a forum for high-quality peer-reviewed papers that broaden the awareness and understanding of recent CNN developments, applications of CNNs for computer vision tasks, and associated developments in CNN architectures, processing components, connective structures, and learning mechanisms, and in dealing with CNN constraints in respect to data preparation and training. References Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Tchendjou, G.T.; Simeu, E. 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Forecasting of Typhoon-Induced Wind-Wave by Using Convolutional Deep Learning on Fused Data of Remote Sensing and Ground Measurements. Sensors 2021, 21, 5234. [CrossRef] [PubMed] g 8. Wu, F.; Wu, B.; Zhang, M.; Zeng, H.; Tian, F. Identiﬁcation of Crop Type in Crowdsourced Road View Photos with Deep Convolutional Neural Network. Sensors 2021, 21, 1165. [CrossRef] [PubMed] 9. Xu, H.; Chen, B.; Qin, J. A CNN-Based Length-Aware Cascade Road Damage Detection Approach. Sensors 2021, 21, 689. [CrossRef] [PubMed] 10. Chou, J.-S.; Liu, C.-H. Automated Sensing System for Real-Time Recognition of Trucks in River Dredging Areas Using Computer Vision and Convolutional Deep Learning Sensors 2021 21 555 [CrossRef] [PubMed] [ ] [ ] 10. Chou, J.-S.; Liu, C.-H. Automated Sensing System for Real-Time Recognition of Trucks in River Dredging Areas Using Computer Vision and Convolutional Deep Learning. Sensors 2021, 21, 555. 11. Anand, A.; Rani, S.; Anand, D.; Aljahdali, H.M.; Kerr, D. 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https://openalex.org/W4361973927	https://figshare.com/articles/journal_contribution/Supplementary_Table_S4_from_An_Epigenetic_Reprogramming_Strategy_to_Resensitize_Radioresistant_Prostate_Cancer_Cells/22411400/1/files/39857315.pdf	English	null	Supplementary Table S3 from An Epigenetic Reprogramming Strategy to Resensitize Radioresistant Prostate Cancer Cells	null	2,023	cc-by	349	n n n n us ALDH- population of DU145 P cells. 5 P ALDH+ 3 Mean DU145 P ALDH- Mean DU145 P ALDH+ t-test Fold Change Regulation 1229 1643 1028 0.032 1.60 down 872 291 1401 0.034 4.82 up 3586 2540 3750 0.002 1.48 up 2739 1415 3967 0.111 2.80 11020 3478 12000 0.003 3.45 up 260 116 393 0.017 3.39 up 401 401 380 0.464 1.05 91 77 72 0.742 1.06 1507 1522 1244 0.339 1.22 41 33 36 0.446 1.09 44 33 42 0.208 1.29 576 574 549 0.835 1.05 19 18 19 0.643 1.09 173 174 162 0.668 1.07 45 60 46 0.179 1.30 31 38 36 0.715 1.07 21 27 20 0.022 1.41 down 320 426 303 0.118 1.41 330 460 337 0.045 1.36 down 308 334 324 0.503 1.03 61 54 57 0.804 1.06 19 30 22 0.074 1.34 37 25 27 0.842 1.05 73 41 68 0.013 1.66 up 56 39 43 0.711 1.10 2295 2005 2047 0.779 1.02 1126 757 1239 0.006 1.64 up 448 418 525 0.272 1.26 74 48 75 0.000 1.56 up 39 51 33 0.008 1.52 down 121 73 127 0.038 1.74 up 21 19 25 0.054 1.35 ALDH- population of DU145 P cells. LDH+ 3 Mean DU145 P ALDH- Mean DU145 P ALDH+ t-test Fold Change Regulation 1229 1643 1028 0.032 1.60 down 872 291 1401 0.034 4.82 up 3586 2540 3750 0.002 1.48 up 2739 1415 3967 0.111 2.80 11020 3478 12000 0.003 3.45 up 260 116 393 0.017 3.39 up 401 401 380 0.464 1.05 91 77 72 0.742 1.06 1507 1522 1244 0.339 1.22 41 33 36 0.446 1.09 44 33 42 0.208 1.29 576 574 549 0.835 1.05 19 18 19 0.643 1.09 173 174 162 0.668 1.07 45 60 46 0.179 1.30 31 38 36 0.715 1.07 21 27 20 0.022 1.41 down 320 426 303 0.118 1.41 330 460 337 0.045 1.36 down 308 334 324 0.503 1.03 61 54 57 0.804 1.06 19 30 22 0.074 1.34 37 25 27 0.842 1.05 73 41 68 0 013 1 66 up
https://openalex.org/W4328097162	https://bcpublication.org/index.php/BM/article/download/4391/4279	English	null	The Nexus between Population Structure, Debt Ratio, Unemployment, and Leadership Election in Aging Countries	BCP business & management	2,023	cc-by	5,208	The Nexus between Population Structure, Debt Ratio, Unemployment, and Leadership Election in Aging Countries Ziyou Deng* College of Business and Economics, Australian National University, Canberra, 2601 Corresponding author: u6214237@alumni.anu.edu.au Ziyou Deng ess and Economics, Australian National University, Canberra, 2601, Australia Corresponding author: u6214237@alumni.anu.edu.au Abstract. For the past decades, aging of population has been a growing concern for many countries, imposing downward pressure on consumption, production, and population growth. Meanwhile, voters in aging countries, influenced by socioeconomic conditions like higher age dependency ratio, rising unemployment rate, constant inflow of immigration and lower fertility rate, might have specific preferences for the corresponding policy targets purposed by the leadership election candidates and might further affect the economic and immigration policies implemented by the new governments after elections. With the investigation of how the preferences of the voters for economic and immigration policies, sharped by the socioeconomic conditions, affect the implementation of the corresponding policies after leadership elections, candidates of these elections might be capable of gaining more support from voters by modifying their election platform, accordingly. In this study, three multinomial logistic models are conducted with government expenditure, taxation, and immigration policies set as dummy dependent variables, separately. And the results imply that, in aging countries, a high level of unemployment tends to induce the new governments to impose more stringent immigration policy and reduce taxation, while the possibility of a decrease of government expenditure would be higher. However, for countries with higher population growth, the new governments would be more likely to implement a tax increase. Keywords: Aging; election; population structure; debt ratio; unemployment rate; multinomial logistic model BCP Business & Management Volume 40 (2023) BCP Business & Management Volume 40 (2023) ECRM 2022 1. Introduction And since the aging population has not been improved by most aging countries for the past decades, in despite of tremendous effort in solving it, it is reasonable to predict this problem would exist for the next few decades. trying to boost the economy. And since the aging population has not been improved by most aging countries for the past decades, in despite of tremendous effort in solving it, it is reasonable to predict this problem would exist for the next few decades. Generally, there are three major ways of stimulating the economy, confronting population, with respect to government expenditure, taxation, and immigration policy, p p p g p g p y p y For government expenditure, some countries would choose to increase government expenditure to create jobs and encourage investments while the other countries might try to avoid deficit and relieve the pressure in paying the increasing pension through cutting down the government budget. For taxation, although rising taxation to increase government revenue and stimulate consumption by income redistribution seems reasonable for some countries, a tax relief to encourage investment and consumption for maximizing the total tax revenue in the end would be impose by the other countries. For immigration policy, despite promoting immigration to fill the vacancies in working age population sounds perfectly reasonable, in many aging countries, on the contrary, the criteria and restriction toward immigration have been risen caused by the waves of opposition from those who believed their jobs and welfare have been comprised by immigration. Besides, it is worthwhile to notice that, in this context, refugee problem is in line with immigration to a great extent for the similar reasons. In general, governments should try to keep policies stable and continued since in most cases it takes time to take effect. However, through election of the leader, the policies about government expenditure, taxation, and immigration change frequently. And since research showed a relationship between economic condition and election result, especially economic condition in the year primary to the election year, it is reasonable to assume that the candidates would adjust and emphasize their statements in election platform accordingly [10-11]. Thus, it would be meaningful to investigate the nexus of the policies stated and implemented by those who won the election and the corresponding economic conditions before election. This paper is organized as follows. 1. Introduction For the past decades, the concern for aging of population have been growing, and it is predicted that by 2050, on a global basis, the number of old would be the same as the number of young, and people under age 15 and over age 60 would account for 42% of the global population in total [1]. In terms of size and reach with respect to global economy, according to World Atlas, 21 countries of the top 25 countries with the largest aging population proportion, defined as percentage of population over 65 years old, are members of Organization for Economic Co-operation and Development (OECD), and OECD accounted for 60.27 percent of the global GDP in 2021 according to World Development Indicator from the World Bank [2]. Clearly, the influence of aging is widespread and might be inestimable. As for the specific effects of an aging population structure to a society, studies showed that a fall in productivity and consumption should be a major one in economic prospect, while pension pressures should not be neglected, either [3-5]. Moreover, Yoshino and Taghizadeh-Hesary claimed that aging of population could be a structural problem causing recession [6]. More fundamentally, in many aging countries, the aging of population is caused by the combination of growing life expectancy and declining fertility rate [7]. Thus, for one thing, if the fertility rate is below replacement level, in long run, the competitiveness of the overall economy might be weakened with a shrinking population. For another, if the retirement age stays the same, the age dependency ratio, calculated by dividing population of non-working group by the working age population, would gradually rise with the aging of population, which increase the burden of the working age population, especially for working class. Besides, Although there are arguments that the economy of the countries aging fast have grown more in the past decades, which is possibly caused by the adoption of automation technology, it is more widely accepted that adverse demographic developments have exerted downward pressure on economic growth [8-9]. Therefore, aging of population would be a major challenge for many governments 274 BCP Business & Management Volume 40 (2023) ECRM 2022 trying to boost the economy. 1. Introduction Section 2 would include literature reviews about government expenditure, taxation, and immigration policy against the backdrop of aging population, while in section 3 would introduce methodology. Then empirical analysis would be in section 4. Lastly, section 5 would discuss about conclusion and implication. 2. Literature Review Overall, the effect of population aging could be included in four aspects. The first one would be the potential downward pressure on current consumption and production. The second one would be the extra burden on pension and welfare system. And the third one would be the increasing stress on the working age with an increasing age dependency ratio and the prospective rise of tax from government while the last one is about the sustainability of the society with fertility rate lower than the replacement level and the latent national debt crisis due to expansionary government spending. Correspondingly, candidates of the leadership election would set and modify their policy objectives to get more support from voters under given socioeconomic conditions. 2.1 Government Expenditure and Taxation Policy For many governments, the tax increase and expansionary government expenditure are usually conducted in the same period to stimulate economy, acting as the opposite of the combination of tax relief and government expenditure decrease conducted by the other governments. For instance, it is typical for the United States government to swing back and forth from increasing tax and government expenditure when Democratic is in charge to tax relief and cutting down government expenditure when Republican in charge, which could be regarded as the continuing fight between the ideas of the “freshwater” economists and the “saltwater” economists [12]. Generally, governments seeking for sustainable economic growth would try to avoid a large amount of fiscal surplus or deficit since the former one leaves too many funds out of production and the latter one might cause massive debt problems, despite with a proper amount of deficit used for 275 BCP Business & Management Volume 40 (2023) ECRM 2022 investing in productive capacity would benefit economy [13]. And in practice, rising national debts or taxation is the frequently used method to make up for the deficit caused by the expansionary expenditure while tax relief usually force government to limit their expenditure to avoid massive deficit. Therefore, although for some other countries, taxation policy is not necessarily related to government expenditure, it would be more practical to discuss government expenditure and taxation policy in the same section, instead of analyzing them separately. investing in productive capacity would benefit economy [13]. And in practice, rising national debts or taxation is the frequently used method to make up for the deficit caused by the expansionary expenditure while tax relief usually force government to limit their expenditure to avoid massive deficit. Therefore, although for some other countries, taxation policy is not necessarily related to government expenditure, it would be more practical to discuss government expenditure and taxation policy in the same section, instead of analyzing them separately. For the successors of the Neo-Keynesian, they prefer stimulating economic by increasing government expenditure and deficit financing, as they believe with more job opportunities and demand created, employment and general equilibrium would rise to meet the effective demand. This is how the supporters of expansionary government expenditures convince the voters of the effectiveness of the economic policies in their election platform. 2.1 Government Expenditure and Taxation Policy As more job opportunities would be created by increasing government expenditure, it is reasonable to assume that countries with high er unemployment rates in year primary to the election year would be more likely to support candidates with plans for expansionary government expenditure. Nonetheless, an increase of government expenditure would cause great burden on national finance and might even cause massive deficit. Thus, as discussed above, many candidates might propose increasing to make up for the deficit through the direct increase of the tax revenue. Whereas some other leaders believe in the theory of Laffer curve, which shows that existence of the optimal level of average tax rate and indicates that a proper tax relief would increase the tax revenue in the end [14]. Thus, for stimulating economic growth, instead of rising government expenditure and deficit financing, they are more likely to conduct a tax cut and reduce government expenditure. Besides, the typical shape of Laffer curve is only available under the assumption of complete market and a closed economy, which is almost unrealistic in the background of globalization and the prosperity of neo liberalism [14]. However, most private companies and capitalists would still embrace the tax relief as the direct beneficiaries, which keeps the competitiveness of tax relief and reduction of government expenditure in election platforms. Nevertheless, there are arguments that against the background of aging population, the effect of government expenditure policy might not be expected [15]. Similarly, the effect of monetary policy on consumption would also be less [16]. In addition, although in some countries, tax relief was assumed to positively affect employment, Ferraro and Fiori stated that the effect of tax relief on employment would be weakened due to population aging [17-18]. Plus, considering that the effects of population on marginal propensities to consume are unclear and vary for different income levels, whether, in practice, changing fiscal and monetary policies would bring back the consumption under an older population structure is not clear [3]. However, in this study, the discussion about monetary policy would not be involved, because in some countries monetary policy is independent from the governments, which makes it less likely to be affected through elections. 2.2 Immigration Policy Apart from the economic policies, many countries attend to solve the aging population problem via a more direct way, which is improving the population structure by promoting immigration, despite a study shows that the impact of the post-1990 immigration on the share of the working-age population of the United States is minimal, as it added those outside of the working-age population in a similar proportion [19]. In practice, the attitude toward immigration would be different from countries to countries, for countries with serious problem of aging population, to attract new immigrants, theoretically, the criteria of immigration should be relatively low, and the corresponding welfare should be relatively high. However, some of the nativists attribute their unemployment to the immigrants since the unemployment rate of natives kept declining with a constant inflow of immigrants [20]. Thus, even though there are arguments that the effect of the immigrants on the employments and wages of the natives is positive as the relationship of immigrants and natives in the job markets is complementary 276 BCP Business & Management Volume 40 (2023) ECRM 2022 rather than competitive, the immigration policies in these countries might even be tightened, corresponding to the public opinion [21]. In contrast, for countries with less concern about the aging population problem and higher attractiveness to immigrants, the criteria would be set relatively high, to improve economic competitiveness at the same time by introducing immigrant investors or skilled workers. Besides, in this sense, an expansionary government expenditure might be welcomed since more job opportunities would be created to solve the unemployment problem. rather than competitive, the immigration policies in these countries might even be tightened, corresponding to the public opinion [21]. In contrast, for countries with less concern about the aging population problem and higher attractiveness to immigrants, the criteria would be set relatively high, to improve economic competitiveness at the same time by introducing immigrant investors or skilled workers. Besides, in this sense, an expansionary government expenditure might be welcomed since more job opportunities would be created to solve the unemployment problem. 2.2 Immigration Policy Nonetheless, although some advocates of immigrant believe that immigrant could fix aging population fundamentally with higher fertility rate, for some aging countries, the fertility rates of immigrants are so close to the fertility rates of natives that the influence of the immigrants on the overall fertility rate is minimal, not to mention that in the United States, the fertility rates of immigrants have even declined faster than the fertility rates of natives since 2008 [22]. Then again, the fertility rate in the Global North, which includes most developed countries, is much lower than the Global South, where most countries are developing [23]. With stringent immigrant criteria, immigrants would be more likely from the North, which further lowers the overall fertility of immigrants. 3.1 Ecometric Model Three Multinomial logistic regression would be conducted with immigration policy, government expenditure policy, and taxation policy as dependent variables, respectively. Immigration policy, government expenditure policy, and taxation policy conducted by the winners are set as dummy variables, with 0 for not mentioning at all, 1 for promoting immigration, increasing government expenditure, and increasing tax, respectively, and -1 for restricting immigration, reducing government expenditure, and decreasing tax, respectively. The model formula is as follows, 𝑌∗ = 𝐶𝑜𝑛𝑠𝑡𝑎𝑛𝑡+ 𝑋𝛽 + 𝜀 (1) (1) where Y is the policy dummy variable, X is the set of independent variables and control variables, β is the corresponding coefficients and ε is the error term subject to the logistic distribution. And the independent variables include population growth, fertility rate, debt to GDP ratio, age dependency, and unemployment rate, denoted as Population_G, Fertility, Debt, Dependency, and Unemployment, respectively. Besides, net migration rate, GDP per capita, or GDP per capita growth are considered as control variables (see Table 1). 277 ECRM 2022 Table 1. Details of variables Variable Symbol Meaning Measurement Dependent variable GE T IM Government expenditure policy Taxation policy Immigration policy Dummy variable 0 for not changing 1 for positive -1 for negative Independent variables Population_G Population growth percentage Fertility Fertility rate percentage Debt Debt to GDP ratio percentage of GDP Dependency Age dependency ratio Non-working age population divided by working age population Unemployment Unemployment rate Unemployed population divided by the sum of unemployed population and employed population Control variables Migration Net migration rate Net migration population divided by total population PGDP PGDP_G GDP per capita GDP per capita growth In USD percentage Note: data are drawn from World Development Indicators of World Bank Database (WDI), United Nation Department of Economic and Social Affairs, tradingeconomics, and macrotrend. Note: data are drawn from World Development Indicators of World Bank Database (WDI), United Nation Department of Economic and Social Affairs, tradingeconomics, and macrotrend. 3.2 Data Description The data are drawn across 13 aging countries, including Finland, France, Germany, Greece, Italy, Japan, South Korea, Portugal, Spain, Sweden, Switzerland, the United Kingdom, and the United States, for the past 3 elections, excluding transitional governments. p g g Table 2 shows the descriptive statistics of variables across the chosen countries. For debt to GDP ratio, the minimum value is 32.2 while the maximum value is 270, and the corresponding standard deviation is 57.477, which is extremely high, illustrating that debt issues might be highly different from countries to countries. For population growth and fertility rate, they are generally low, and their standard deviations are small too, matching the intuition for aging countries. The standard deviations for age dependency ratio, unemployment rate, GDP per capita growth, and population growth are medium, while net migration rate is negative for some countries, demonstrating an outflow of population. Besides, the minimum GDP per capita growth is -24.7, suggesting that some countries might have experienced severe recession. And the average age dependency ratio is 53.027, which means, for aging countries in past decades, approximately two people in working age need to support the living of one person out of working age, which might seem to be acceptable. But the maximum value is 69.483. In other words, ten people in working age might need to support the living of seven people out working age, not to mention that people in working age does not necessarily have jobs. Table 2. Descriptive statistics of variables Obs Min Max Average Std Dev Median Population_G 39 -0.659 1.851 0.389 0.524 0.397 Fertility 39 1.089 1.979 1.551 0.247 1.457 Debt 39 32.2 270 93.674 57.477 78.4 Dependency 39 36.327 69.483 53.027 6.761 53.426 Unemployment 39 2.8 24.9 8.287 5.591 7.78 PGDP_G 39 -24.7 18.33 1.561 9.331 3.03 Migration 39 -5.966 10.742 2.738 3.371 2.857 278 BCP Business & Management Volume 40 (2023) ECRM 2022 3.3 Model Likelihood Ratio Test To test the effectiveness of multinomial logistic models with immigration, government expenditure, and taxation set as dependent variables, separately, model likelihood ratio tests are done. And the results indicates that, all multinomial logistic models are effective (shown in Table 3). Table 3. Multinomial logistic model likelihood ratio test χ2 df p value AIC value BIC value IM model 23.902 14 0.047 93.171 119.788 GE model 31.159 14 0.001 81.379 107.996 T model 35.882 14 0.001 80.039 106.656 4. Empirical Analysis 4.1 Empirical Result The results of these three multinomial logistic regressions are shown in Table 4. Table 4. Results of multinomial logit/logistic regression result of multinomial logit regression for immigration policy result of multinomial logistic regression for government expenditure policy result of multinomial logistic regression for taxation policy 0 1 0 1 0 1 Population_G 1.285 1.763 Population_G -2.982 -8.639* Population_G 0.716 6.572* (0.574) (0.872) (-1.472) (-1.975) (0.23) (2.09) Fertility -4.479 -1.079 Fertility 5.659 16.591* Fertility -3.197 -1.351 (-1.037) (-0.287) (1.49) (1.993) (-0.583) (-0.315) Debt -0.02 -0.011 Debt -0.036 0.09 Debt -0.051* 0.035 (-0.985) (-0.627) (-1.399) (1.70) (-2.035) (1.31) Dependency 0.203 0.159 Dependency -0.284 -0.677 Dependency 0.123 -0.273 (1.07) (0.99) (-1.762) (-1.875) (0.596) (-1.218) Unemployment -0.291* -0.355* Unemployment -0.072 -0.804* Unemployment -0.631* -0.319 (-2.178) (-2.053) (-0.582) (-2.175) (-1.993) (-1.415) PGDP 0 0 PGDP_G -0.112 -0.101 PGDP_G -0.066 -0.019 (-1.481) (-1.269) (-1.166) (-0.986) (-0.650) (-0.232) Migration -0.411 -0.324 Migration 0.328 0.396 Migration -0.796 -0.788 (-1.094) (-0.887) (1.04) (1.16) (-1.664) (-1.649) Intercept 5.724 0.314 Intercept 9.859 9.569 Intercept 9.393 14.8 (0.763) (0.042) (1.60) (1.19) (1.09) (1.57) Likelihood test χ2(14)=23.902 p=0.047 Likelihood test χ2(14)=36.159 p=0.001 Likelihood test χ2(14)=35.882 p=0.001 Dependent variable: IM Dependent variable: GE Dependent variable: T McFadden R2: 0.281 McFadden R2: 0.423 McFadden R2: 0.428 Cox & Snell R2: 0.458 Cox & Snell R2 : 0.604 Cox & Snell R2 : 0.602 Nagelkerke R2: 0.517 Nagelkerke R2 : 0.680 Nagelkerke R2 : 0.681 * p<0.05 ** p<0.01, z value in parentheses * p<0.05 ** p<0.01, Z value in parentheses * p<0.05 ** p<0.01, Z value in parentheses The result with respect to immigration policy indicates that an increase of unemployment level would rise the possibilities of implementation of more stringent immigration policy. And compared i h i i i li hi h l l f l ld l h ibili i f Table 3. 3.3 Model Likelihood Ratio Test Multinomial logistic model likelihood ratio test χ2 df p value AIC value BIC value IM model 23.902 14 0.047 93.171 119.788 GE model 31.159 14 0.001 81.379 107.996 T model 35.882 14 0.001 80.039 106.656 Table 3. Multinomial logistic model likelihood ratio test χ2 df p value AIC value BIC value p he results of these three multinomial logistic regressions are shown in Table 4. Table 4. Results of multinomial logit/logistic regression result of multinomial logit regression for immigration policy result of multinomial logistic regression for government expenditure policy result of multinomial logistic regression for taxation policy 0 1 0 1 0 1 Population_G 1.285 1.763 Population_G -2.982 -8.639* Population_G 0.716 6.572* (0.574) (0.872) (-1.472) (-1.975) (0.23) (2.09) Fertility -4.479 -1.079 Fertility 5.659 16.591* Fertility -3.197 -1.351 (-1.037) (-0.287) (1.49) (1.993) (-0.583) (-0.315) Debt -0.02 -0.011 Debt -0.036 0.09 Debt -0.051* 0.035 (-0.985) (-0.627) (-1.399) (1.70) (-2.035) (1.31) Dependency 0.203 0.159 Dependency -0.284 -0.677 Dependency 0.123 -0.273 (1.07) (0.99) (-1.762) (-1.875) (0.596) (-1.218) Unemployment -0.291* -0.355* Unemployment -0.072 -0.804* Unemployment -0.631* -0.319 (-2.178) (-2.053) (-0.582) (-2.175) (-1.993) (-1.415) PGDP 0 0 PGDP_G -0.112 -0.101 PGDP_G -0.066 -0.019 (-1.481) (-1.269) (-1.166) (-0.986) (-0.650) (-0.232) Migration -0.411 -0.324 Migration 0.328 0.396 Migration -0.796 -0.788 (-1.094) (-0.887) (1.04) (1.16) (-1.664) (-1.649) Intercept 5.724 0.314 Intercept 9.859 9.569 Intercept 9.393 14.8 (0.763) (0.042) (1.60) (1.19) (1.09) (1.57) Likelihood test χ2(14)=23.902 p=0.047 Likelihood test χ2(14)=36.159 p=0.001 Likelihood test χ2(14)=35.882 p=0.001 Dependent variable: IM Dependent variable: GE Dependent variable: T McFadden R2: 0.281 McFadden R2: 0.423 McFadden R2: 0.428 Cox & Snell R2: 0.458 Cox & Snell R2 : 0.604 Cox & Snell R2 : 0.602 Nagelkerke R2: 0.517 Nagelkerke R2 : 0.680 Nagelkerke R2 : 0.681 * p<0.05 ** p<0.01, z value in parentheses * p<0.05 ** p<0.01, Z value in parentheses * p<0.05 ** p<0.01, Z value in parentheses The result with respect to immigration policy indicates that an increase of unemployment level would rise the possibilities of implementation of more stringent immigration policy. And compared to a tight immigration policy, higher level of unemployment rate would lower the possibilities of not The result with respect to immigration policy indicates that an increase of unemployment level would rise the possibilities of implementation of more stringent immigration policy. And compared to a tight immigration policy, higher level of unemployment rate would lower the possibilities of not changing immigration policy or applying a looser immigration policy. 3.3 Model Likelihood Ratio Test Apart from these, the effects of other variables are not statistically significant. The result with respect to immigration policy indicates that an increase of unemployment level would rise the possibilities of implementation of more stringent immigration policy. And compared to a tight immigration policy, higher level of unemployment rate would lower the possibilities of not changing immigration policy or applying a looser immigration policy. Apart from these, the effects of other variables are not statistically significant. 279 BCP Business & Management Volume 40 (2023) BCP Business & Management Volume 40 (2023) ECRM 2022 Then, for government expenditure policy, population growth, fertility rate, and unemployment rate all have statistically significant effects on expenditure policy. Higher fertility rate would impose a positive and large effect on expansionary expenditure, while population growth and unemployment rate would have negative influence on rising government expenditure. Whereas for taxation policy, higher level of population growth would lead to increase of taxation, and the effect would be noteworthy. Nonetheless, rather than keeping the taxation constant, higher debt to GDP ratio and unemployment rate would be more likely to conduct a tax cut, though the effect of debt seems to be minimal. 4.2 Robustness Check Robustness checks are conducted by replacing the fertility rate with the fertility rate growth, as a measurement of the trend of birth, and the results are shown in Table 5 for immigration, government expenditure, and taxation policy. Table 5. Results of robustness check of multinomial logistic regression result of robustness check of multinomial logistic regression for immigration policy result of robustness check of multinomial logistic regression for government expenditure policy result of robustness check of multinomial logistic regression for taxation policy 0 1 0 1 0 1 Population_G -0.278 1.315 Population_G -0.983 -1.467 Population_G -0.883 6.004* (-0.156) (0.854) (-0.66) (-0.87) (-0.41) (2.334) Fertility_G 0.59 0.041 Fertility_G -0.079 -1.007 Fertility_G -0.345 -0.248 (1.042) (0.078) (-0.16) (-1.72) (-0.67) (-0.43) Debt -0.016 -0.009 Debt -0.04 0.053 Debt -0.04 0.042 (-0.832) (-0.598) (-1.50) (1.656) (-1.89) (1.84) Dependency 0.111 0.13 Dependency -0.13 -0.131 Dependency 0.025 -0.325 (0.767) (1.121) (-1.16) (-0.94) (0.176) (-1.85) Unemployment -0.316* -0.346* Unemployment -0.094 -0.57* Unemployment -0.67* -0.3 (-2.413) (-2.057) (-0.74) (-1.98) (-2.19) (-1.40) PGDP 0 0 PGDP_G -0.07 0.03 PGDP_G -0.09 -0.018 (-1.687) (-1.365) (-0.84) (0.352) (-0.98) (-0.21) Migration -0.374 -0.243 Migration 0.075 0.027 Migration -0.579 -0.637 (-1.098) (-0.772) (0.283) (0.095) (-1.41) (-1.72) Intercept 4.569 -0.068 Intercept 10.931 6.316 Intercept 9.045 14.34 (1.092) (1.567) (1.609) (0.852) (1.113) (1.575) Likelihood test χ2 (14)=24.344 p=0.042 Likelihood test χ2 (14)=31.426 p=0.005 Likelihood test χ2 (14)=35.990 p=0.001 Dependent variable: IM Dependent variable: GE Dependent variable: T McFadden R2: 0.286 McFadden R2: 0.367 McFadden R2: 0.429 Cox & Snell R2 : 0.464 Cox & Snell R2 : 0.553 Cox & Snell R2 : 0.603 Obviously, for immigration policy, the result is the same even though the trend of birth measured by fertility rate growth instead of fertility rate, as unemployment rate still have statistically significant effect on immigration policy. However, for government expenditure policy, the effects of population growth and fertility are not statistically significnat anymore, while unemployment rate still shows a statistically sinificant effect on expansionary government expenditure. Similarly for taxation policy, the effect of debt is not statistically significant while the robustness of the results with respect to population growth and unemlpoyment is verified. Obviously, for immigration policy, the result is the same even though the trend of birth measured by fertility rate growth instead of fertility rate, as unemployment rate still have statistically significant effect on immigration policy. 4.2 Robustness Check However, for government expenditure policy, the effects of population growth and fertility are not statistically significnat anymore, while unemployment rate still shows a statistically sinificant effect on expansionary government expenditure. Similarly for taxation policy, the effect of debt is not statistically significant while the robustness of the results with respect to population growth and unemlpoyment is verified. 280 BCP Business & Management Volume 40 (2023) ECRM 2022 5. Conclusion and Implication In sum, in aging countries, aging dependency, debt to GDP ratio, fertility rate, and net migration do not have statistically significant effect on immigration, government expenditure, and taxation policy, while population growth would be more likely to result in an expansionary government expenditure. And unemployment has statistically significant influence on immigration, government expenditure, and taxation policy. Aging countries with high level of unemployment would prefer to tight the immigration policies and increase their government expenditures while implement a tax relief. The corresponding implication would be for the candidates of the leadership election in aging countries with high level of unemployment, which is about including expansionary expenditure and stringent immigration policies into their election platform. In the meantime, to make up the deficit due to expansionary expenditure and pension burden from aging population, tax reform might be a better idea than increasing national debt. Although, the debt to GDP ratio does not seem to induce a preference expenditure and taxation policy, too much debt might be unsustainable and even cause debt crisis. References per Sarah. “Economic and social implications of aging societies.” Science 346 (2014): 587 - 591 [2] Haider Faraz “Countries with the largest aging population in the world”. World Atlas, 2017, drawn from https://www.worldatlas.com/articles/countries-with-the-largest-aging-population-in-the-world.html [3] Dynan Karen E., Edelberg Wendy, Palumbo Michael G.. “The Effects of Population Aging on the Relationship among Aggregate Consumption, Saving, and Income.” The American Economic Review 99 (2009): 380-386. [4] Loser Claudio, fajgenbaum Jose, Kohli Harpaul Alberto, Vikelyte Ieva. “How Aging Societies May Affect Global Growth Prospects.” Global Journal of Emerging Market Economies 9 (2017): 38 - 74. [5] Wang Huan, Huang Jianyuan, Yang Qi. “Assessing the Financial Sustainability of the Pension Plan in China: The Role of Fertility Policy Adjustment and Retirement Delay.” Sustainability, 2019. [6] Yoshino Naoyuki, Taghizadeh‐Hesary Farhad. “Causes and Remedies of the Japan's Long‐Lasting Recession: Lessons for China.” Political Economy - Development: Domestic Development Strategies eJournal, 2016. [7] Acemoglu, Daron, Pascual Restrepo. “Secular Stagnation? The Effect of Aging on Economic Growth in the Age of Automation.” ERN: Other Macroeconomics: Consumption, 2017. [8] Bloom David, Canning David, Finlay Jocelyn. “Population Aging and Economic Growth in Asia.” (2010). [9] Ferrero, Giuseppe Maria, Marco Gross, Stefano Neri. “On Secular Stagnation and Low Interest Rates: Demography Matters.” Labor: Demographics & Economics of the Family eJournal, 2019. [10] Aidt Toke S., Francisco José Veiga, Linda Veiga. “Election results and opportunistic policies: A new test of the rational political business cycle model.” Public Choice 148 (2011): 21-44. [11] Healy Andrew, Lenz Gabriel. “Substituting the End for the Whole: Why Voters Respond Primarily to the Election‐Year Economy.” American Journal of Political Science 58 (2014): 31-47. [12] Myatt Tony, Maclean Brian K.. “Is Freshwater Skepticism on Fiscal Multipliers Rooted in Theory?” International Journal of Political Economy 43 (2014): 107 - 94. [13] Lane Chrie, Harris Elliott. “Mounting Debt Threatens Sustainable Development Goals” IMF, Sustainable Development Goals, 2018. [14] Fève Patrick, Matheron Julien, Sahuc Jean-Guillaume. “The Laffer curve in an incomplete-markets economy.” Working Papers, 2, 1-26, 2013. [15] Miyamoto Hiroaki, Naoyuki Yoshino. “A note on population aging and effectiveness of fisal policy.” Macroeconomic Dynamics 26 (2020): 1679 - 1689. [16] Wong Arlene. “Transmission of Monetary Policy to Consumption and Population Aging.” [17] Skedinger Per. “Effects of Payroll Tax Cuts for Young Workers.” ERN: Labor Policy & Regulation (Topic), 2014. 281 BCP Business & Management Volume 40 (2023) ECRM 2022 [18] Ferraro Domenico, Fiori Giuseppe. References “The Aging of the Baby Boomers: Demographics and Propagation of Tax Shocks.” Labor: Demographics & Economics of the Family eJournal, 2020. [19] Camarota Steven A, Zeigler Karen. “Projecting the Impact of Immigration on the U.S. Population.” Center for Immigration Studies. 2019. [20] Camarota Steven A.. “Immigrant Gains and Native Losses in the U.S. Job Market, 2000 to 2010.” Center for Immigration Studies. 2011. [21] Jaynes Gerald D.. “The Impact of Illegal Immigration on the Wages and Employment Opportunities of Black Workers”. 2010 [22] Camarota Steven A, Zeigler Karen. “Immigrant and Native Fertility 2008 to 2017.” Center for Immigration StudiesLow-immigration, 2019. [23] Hozmann Robert. “Demographic Alternatives for Aging Industrial Countries: Increased Total Fertility Rate, Labor Force Participation, or Immigration.” Employment Law eJournal (2005) 282
https://openalex.org/W4200037719	https://nursing.jmir.org/2021/4/e31038/PDF	English	null	Exploring Reasons for Delayed Start-of-Care Nursing Visits in Home Health Care: Algorithm Development and Data Science Study (Preprint)	null	2,021	cc-by	8,030	Abstract Background: Delayed start-of-care nursing visits in home health care (HHC) can result in negative outcomes, such as hospitalization. No previous studies have investigated why start-of-care HHC nursing visits are delayed, in part because most reasons for delayed visits are documented in free-text HHC nursing notes. Objective: The aims of this study were to (1) develop and test a natural language processing (NLP) algorithm that automatically identifies reasons for delayed visits in HHC free-text clinical notes and (2) describe reasons for delayed visits in a large patient sample. Methods: This study was conducted at the Visiting Nurse Service of New York (VNSNY). We examined data available at the VNSNY on all new episodes of care started in 2019 (N=48,497). An NLP algorithm was developed and tested to automatically identify and classify reasons for delayed visits. Results: The performance of the NLP algorithm was 0.8, 0.75, and 0.77 for precision, recall, and F-score, respectively. A total of one-third of HHC episodes (n=16,244) had delayed start-of-care HHC nursing visits. The most prevalent identified category of reasons for delayed start-of-care nursing visits was no answer at the door or phone (3728/8051, 46.3%), followed by patient/family request to postpone or refuse some HHC services (n=2858, 35.5%), and administrative or scheduling issues (n=1465, 18.2%). In 40% (n=16,244) of HHC episodes, 2 or more reasons were documented. Conclusions: To avoid critical delays in start-of-care nursing visits, HHC organizations might examine and improve ways to effectively address the reasons for delayed visits, using effective interventions, such as educating patients or caregivers on the importance of a timely nursing visit and improving patients’ intake procedures. (JMIR Nursing 2021;4(4):e31038) doi: 10.2196/31038 Exploring Reasons for Delayed Start-of-Care Nursing Visits in Home Health Care: Algorithm Development and Data Science Study Exploring Reasons for Delayed Start-of-Care Nursing Visits in Home Health Care: Algorithm Development and Data Science Study Maryam Zolnoori1, PhD; Jiyoun Song1, PhD; Margaret V McDonald2, MSW; Yolanda Barrón2, MS; Kenrick Cato1, PhD; Paulina Sockolow3, DrPH; Sridevi Sridharan2, MSc; Nicole Onorato2, BSc; Kathryn H Bowles2,4, PhD; Maxim Topaz1, PhD 1School of Nursing, Columbia University, New York, NY, United States 2Center for Home Care Policy & Research, Visiting Nurse Service of New York, New York, NY, United States 3College of Nursing and Health Professions, Drexel University, Philadelphia, PA, United States 4Center for Transitions and Health, School of Nursing, University of Pennsylvania, Philadelphia, PA, United States Corresponding Author: Maxim Topaz, PhD School of Nursing Columbia University 560 W 168th St New York, NY, 10032 United States Phone: 1 (212) 609 1774 Email: mt3315@cumc.columbia.edu 1School of Nursing, Columbia University, New York, NY, United States 2Center for Home Care Policy & Research, Visiting Nurse Service of New York, New York, NY, United States 3College of Nursing and Health Professions, Drexel University, Philadelphia, PA, United States 4Center for Transitions and Health, School of Nursing, University of Pennsylvania, Philadelphia, PA, United States JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 1 (page number not for citation purposes) Zolnoori et al Zolnoori et al JMIR NURSING Original Paper Introduction Over the last several decades in the United States, recognition has been growing of the importance of post–acute care settings, including long-term care hospitals, inpatient rehabilitation facilities, and skilled nursing facilities [1,2]. One specific type of post–acute care setting, home health care (HHC), has been growing rapidly; according to the Centers for Medicare & Medicaid Services (CMS), the expenditure on HHC was over US $102 billion in 2018, which indicated a more than 30% increase compared to 5 years ago.[3] Currently, more than 12,000 HHC agencies in the United States serve more than 6 million patients annually [4]. In addition, the United States and global populations are becoming older and suffering from more complex health conditions that require care in the community [5,6]; hence, the demand for HHC services is projected to increase incrementally in the following decades. Fortunately, recent data science analytical techniques can extract insights from free-text documentation. Specifically, natural language processing (NLP) algorithms can be applied to large collections of clinical documents to identify a diverse range of concepts and extract meaning from documents [24]. Often, NLP algorithms rely on manually curated, rule-based vocabularies generated by subject matter experts [24]. An example of NLP algorithm use is the identification of social risk factors among patients discharged from hospitals using discharge summaries [24]. The researchers (including physicians, nurses, and pharmacologists) first developed comprehensive vocabularies of words and expressions that describe several key social risk factor categories including tobacco use, drug abuse, depression, and housing instability. Words and expressions representing the concept of drug abuse include amphetamine abuse, cocaine abuse, and intravenous drug use. NLP was used to search discharge summaries (n=~100,000) to find the manually curated, rule-based terms related to 1 or more social risk factor categories. HHC consists of intermittent home visits conducted by skilled health care providers (eg, registered nurses, physical therapists, social workers). Currently, over 6 million patients are admitted to more than 12,000 HHC agencies across the United States annually [7-9]. Despite national and local efforts for quality improvement, approximately 1 in 5 HHC patients are hospitalized or visit the emergency department during their HHC episode [7,10]. Up to two-thirds of these hospitalizations occur within the first 2 weeks of HHC services [11-15]. A significant portion of hospitalizations and emergency department visits from HHC may be prevented by timely and appropriately targeted home care services [13,15-17]. KEYWORDS delayed start-of-care nursing visit; home healthcare services; natural language processing; nursing note; NLP; nursing; eHealth; home care; clinical notes; classification; clinical informatics JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 1 (page number not for citation purposes) https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX JMIR NURSING Zolnoori et al notes. Given a large volume of clinical notes (~100,000), manual review is not feasible, calling for the use of data science methods. Study Setting The CMS requires agencies to conduct a start-of-care visit within 48 hours of HHC referral or within 48 hours of the patient’s return home [21]. However, we found about one-third of patients admitted to a large HHC agency were seen later than 48 hours post–hospital discharge date in our ongoing study focused on identifying patients at risk for poor outcomes during the hospital to HHC transition (R01-NR018831). Delays in the initiation of HHC services may have a profound negative impact on patients’ outcomes. The majority of patients who are admitted to HHC are vulnerable older adults who are discharged from hospitals with chronic and acute medical conditions such as advanced heart failure, acute coronary syndrome, and diabetes [4]. Many of these patients need immediate access to care to avoid negative outcomes; studies have shown that avoiding delays in the start of care among HHC patients can improve functional and physiological outcomes [22] and reduce the risk of hospitalization and emergency department visits by up to 50% [23]. This study was conducted at Visiting Nurse Service of New York (VNSNY), the largest not-for-profit urban home care agency in the United States. The study research protocol was approved by the institutional review board of the VNSNY in October 2019. Introduction In HHC settings, previous applications of NLP focused on identifying patients who experienced falls [25], dementia symptoms [26], and hospitalization risk factors [27]. This study demonstrates the application of NLP to analyze reasons for delayed start-of-care HHC nursing visits. Specifically, we aimed to (1) develop and test an NLP algorithm that automatically (without human involvement) identifies reasons for potential delay in timing of the start-of-care HHC nursing visit in free-text clinical notes and (2) use descriptive statistics to describe the identified reasons for delayed start-of-care HHC nursing visits in a large patient sample. The start-of-care nursing visit is one of the most critical steps of the HHC episode [18,19]. This visit includes medication reconciliation, patient self-care capability assessment, home environment examination, and discussion regarding caregiver availability and ability to help. Based on this evaluation, a unique care plan is created [20]. Hence, appropriate timing of the first visit is crucial. https://nursing.jmir.org/2021/4/e31038 JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 2 (page number not for citation purposes) Structure of Clinical Notes With Delayed Start-of-Care We extracted 118,767 clinical notes documented for HHC episodes with delayed visits. More than 1 note could be documented for an episode. There were 22 note types with the most common being narrative notes that included nursing and other HHC admission staff comments (23,753/118,767, 20%), initial welcome call notes that described the initial welcome telephone call to introduce HHC services to the patient (n=20,190, 17%), intake clinical comment notes that provided clinical details of the admitted patients (n=14,252, 12%), insurance and other additional information notes with information on patient insurance and additional clinical factors (eg, list of medications; n=9501, 8%), telephone communication notes describing any phone contact between HHC staff and patients or family (n=4750, 4%), and 17 other less frequent note categories (n<3563, <3% each). As the second step of developing the NLP algorithm, we created regular expression rules to capture language describing patient or family requests to postpone HHC or refuse some HHC services. This included the following phrases: “pt declined VNS visit today,” “patient refused visit and asks for visit tomorrow,” “daughter asks to reschedule SOC for Friday.” These phrases have some word patterns, such as “patient/pt/family refused,” “asks for visit tomorrow,” and “reschedule SOC for Friday,” that were used to develop regular expression rules. Table 1 provides more examples of regular expressions applied to identify reasons for delayed start-of-care HHC nursing visits in clinical notes. Multimedia Appendix 1 provides examples of regular expression syntaxes used for identifying patterns of delayed start-of-care for the specified categories. Study Sample The study sample comprises all HHC patients admitted during the calendar year of 2019 following a hospitalization. The patients’ status of hospitalization prior to HHC was extracted from the M1000_3 variable on the Outcome and Assessment Information Set (OASIS), a patient-specific, standardized assessment required under Medicare HHC rules [28]. The study sample comprises 45,390 admitted patients, some of whom were admitted more than once, resulting in 48,497 HHC episodes. CMS reimbursement for an HHC episode in 2019 was for 60 days of care. As we were interested in understanding all reasons for delayed start-of-care nursing visits, we conducted this analysis at the HHC episode level. No previous study has investigated why start-of-care HHC nursing visits are delayed. One explanation may be that most delayed visit reasons are documented in free-text HHC nursing JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 2 (page number not for citation purposes) XSL•FO RenderX Zolnoori et al JMIR NURSING certain language patterns describing reasons for delayed visits. Regular expression is a powerful text search technique that uses alphabetic characters, numeric expressions, and nonalphanumeric expressions. The regular expression approach can help find certain predefined lists of keywords in the texts [29]. The goal is to capture as much lexical variation as possible using keywords and language patterns. Dates of hospital discharge and start-of-care HHC nursing visit were extracted from the OASIS. We calculated the difference in days between the following OASIS items: M1005: Inpatient Discharge Date and M0090: Date Assessment Completed. This study defined delayed start-of-care HHC nursing visits as visits conducted more than 2 days after hospital discharge. Approximately one-third (16,244/48,497, 33.5%) of episodes met this criterion. As the first step of developing the NLP algorithm, the data preprocessing steps involved lowercasing, stripping (removing extra spaces), and removing special characters (eg, [, \, ^, $, \|, ?, *, +, (, ), ]). We did not remove stop words because they were important for identifying the reasons for delayed start-of-care. For example, negation indicators (such as no and not) or auxiliary words (such as do, have, and has) were important for identifying if a patient requested to postpone the HHC services. We also did not perform lemmatization or stemming because keeping the original form of words, particularly the verbs, was important for identifying a pattern of late visit. Documentation Categories for Documenting Delayed Start-of-Care The electronic health record (EHR) had a standardized option that allowed HHC staff to document reasons for delayed visits. The standardized categories included no answer at the door or phone, administrative or scheduling issues, and patient/family request to postpone or refuse some HHC services. JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 3 (page number not for citation purposes) Visiting nurse not available Visiting nurse not available aHHC: home health care. bItalicized text denotes the identified language in the clinical notes that indicates reasons for delayed start-of-care HHC nursing visits. cVM: voice message. dMSG: message. eSOC: start-of-care. aHHC: home health care. bItalicized text denotes the identified language in the clinical notes that indicates reasons for delayed start-of-care HHC nursing visits. cVM: voice message. dMSG: message. eSOC: start-of-care. dMSG: message. eSOC: start-of-care. of the precision and recall). These metrics vary between 0 and 100%, with higher values indicating better NLP algorithm concept identification performance; values above 75% indicate high performing NLP algorithms. Evaluation of the NLP algorithm To evaluate the accuracy of our NLP algorithm, we extracted 100 clinical notes from each of the 22 note types, resulting in 2200 clinical notes. Each note was reviewed independently by 3 HHC experts, who labeled the note with 1 or more of the 3 delayed start-of-care HHC nursing visit categories, where applicable. The experts included a PhD-prepared registered nurse, a social worker, and an experienced research analyst. There was a high level of initial interrater agreement between the experts (κ>0.8) [30]. Disagreements were resolved via expert team discussion. Natural Language Processing for Analysis of Clinical Notes with Delayed Start-of-Care We developed an NLP algorithm using a regular expression technique [24] where algorithmic rules were crafted to match JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 3 (page number not for citation purposes) https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX Zolnoori et al JMIR NURSING Table 1. Examples of regular expressions applied to identify reasons for delayed start-of-care HHCa nursing visits in clinical notes. Example from a clinical noteb Category and examples of regular expressions No answer at the door or phone “SOC attempted. Unable to leave VM to confirm visit, mailbox full, unable to leave MSG” Unable to leave VMc; unable to leave MSGd “Unable to reach patient, contact numbers listed are incorrect.” Unable to reach; contact numbers are incorrect “Multiple phone calls and walk by attempts and no response, patient not found” No response; patient not found Patient/family request to postpone or refuse some HHC services “Patient declined SOC today” Patient declined “Caregiver and daughter informed VN that pt will not be available today and made the request to reschedule SOC for tomorrow” Request to schedule SOCe for tomorrow Administrative or scheduling issues “Request for VN visit for wound care clinical triage needed, case is pending admin approval.” Case is pending “Intake referral inquiry, subject: FW: missing information, patient is missing attachment or referral source.” Missing information “SOC visit rescheduled for tomorrow. VN unable to make scheduled appt. time. Rescheduled” Visiting nurse not available aHHC: home health care. bItalicized text denotes the identified language in the clinical notes that indicates reasons for delayed start-of-care HHC nursing visits. cVM: voice message. dMSG: message. eSOC: start-of-care. Administrative or scheduling issues “Request for VN visit for wound care clinical triage needed, case is pending admin approval.” “Intake referral inquiry, subject: FW: missing information, patient is missing attachment or referral source.” Case is pending Missing information “SOC visit rescheduled for tomorrow. VN unable to make scheduled appt. time. Rescheduled” JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 4 (page number not for citation purposes) Cohort Demographic Table 2 provides a summary of the demographics of patients admitted to VNSNY. Table 2 provides a summary of the demographics of patients admitted to VNSNY. Table 2 provides a summary of the demographics of patients admitted to VNSNY. The majority of patients admitted to VNSNY were aged over 65 years (29,985/48,497, 61.8%). Females comprised a larger fraction of the patient population compared to males. The proportion of non-Hispanic White patients was significantly higher than that of other races. Hypertension (28,804/48,497, 59.4%) was the most common disease among patients, followed by diabetes (n=15,204, 31.4%) and cancer (n=7591, 15.7%). The majority of the patients (36,106/48,497, 74.5%) could receive support from their family members or their caregivers. We applied our NLP algorithm on the testing set and calculated the accuracy of NLP in identifying reasons for delayed visits. For each category of delayed visit reasons, we calculated the precision (defined as the number of true positives out of the total number of predicted positives returned by the NLP algorithm), recall (the number of true positives out of the actual number of positives), and F-score (the weighted harmonic mean JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 4 (page number not for citation purposes) https://nursing.jmir.org/2021/4/e31038 https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX Zolnoori et al JMIR NURSING Table 2. Demographics of patients admitted to the Visiting Nurse Service of New York (VNSNY). Patients (N=48,497), n (%) Characteristic Demographics Age (years) 18,512 (38.2) <65 29,985 (61.8) ≥65 Sex 28,094 (57.9) Female 20,403 (42.1) Male Race 21,070 (43.5) Non-Hispanic White 11,574 (23.9) Non-Hispanic Black 11,416 (23.5) Hispanic 4437 (9.2) Other Type of insurance 5777 (11.9) Dual eligibility 13,147 (27.1) Medicare FFSa only 420 (0.9) Medicaid FFS only 15,954 (32.9) Any HMOb 13,199 (27.2) Other (eg, private) Living situation 36,106 (74.5) Living with others (eg, congregate living) 12,391 (25.6) Living alone Current condition 6682 (13.8) Congestive heart failure 4882 (10.1) Cardiac arrhythmias 28,804 (59.4) Hypertension 6678 (13.8) Chronic pulmonary disease 15,204 (31.4) Diabetes 5244 (10.8) Renal failure 7591 (15.7) Cancer a Patients (N=48,497), n (%) aFFS: fee-for-service. bHMO: health maintenance organization. Although the total F-score of the NLP algorithm for identifying the reason of delayed start-of-care is promising, it also implies that the algorithm could not capture all variations for delayed start-of-care in clinical notes. Cohort Demographic For example, it did not identify the following notes indicating that the patient postponed the start-of-care: (1) “Pt will return call from [nurse], he prefers the soc after 3 (PM),” (2) “Spoke with patient’s caregivers, stated that they are moving to a new apartment and the patient would like to have soc in another time.” JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 5 (page number not for citation purposes) Measuring Performance of the NLP algorithm The overall accuracy of NLP in detecting language related to reasons of delayed visits was high, as reflected by the total F-score of 0.77 (Table 3). When applied to all clinical notes in the study (n=118,767), the NLP algorithm identified 21,433 reasons for delayed visits documented in 20,536 clinical notes for 10,644 unique HHC episodes. https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX XSL•FO RenderX JMIR NURSING Zolnoori et al Table 3. Precision, recall, and F-score of the natural language processing algorithm to identify categories of delayed visit reasons. Scores above 0.75 indicate good performance. F-score Recall Precision Delayed visit reason category 0.76 0.81 0.73 No answer at the door or phone 0.77 0.71 0.85 Administrative or scheduling issues 0.77 0.73 0.81 Patient or family request to postpone or refuse some home health care services 0.77 0.75 0.8 Mean Reasons for Delayed Start of Care Within Clinical of the natural language processing algorithm to identify categories of delayed visit reasons. Scores above 0.75 Reasons for Delayed Start-of-Care Within Clinical Prevalence of Delayed Start-of-Care HHC Visits in Standardized Documentation Overall, 72.2% of the delayed visit reasons (8051/11,129, Figure 1) were documented in clinical notes. When the NLP algorithm results and the standardized documentation categories were combined, reasons for delayed visits were identified for 11,148 of the 16,244 HHC episodes (68.6%). We found that 23.1% (2572/11,129) of delayed visit reasons were documented in both documentation sources (Figure 1), while only 4.5% (n=506) of the reasons were documented in standardized documentation only. For the remaining 31.4% HHC episodes (5096/16,244), no delayed visit reasons were identified. In total, 16,244 HHC episodes had a delayed start-of-care HHC visit. Among all 16,244 episodes with a delayed start-of-care nursing visit, 3097 (19.1%) had reasons for delayed start-of-care documented using standardized documentation. Of the 3097 cases with documentation, 5484 unique reasons for delayed start-of-care HHC nursing visits were found (a single HHC episode could have more than 1 reason documented). Figure 1. Sources of documented reasons for delayed start-of-care HHC nursing visits. Figure 1. Sources of documented reasons for delayed start-of-care HHC nursing visits. The most prevalent categories of reasons for delayed start-of-care HHC nursing visits identified by the NLP algorithm were “no answer at the door or phone” (3728/8051, 46.3%), followed by “patient/family request to postpone or refuse some HHC services” (n=2858, 35.5%) and “administrative or scheduling issues” (n=1465, 18.2%). Further analysis showed that in more than 40% of episodes with delayed start-of-care HHC nursing visits, 2 or more reasons were documented. Figure 2 shows that 17.5% (1409/8051) of the HHC episodes had 2 reasons for delayed start-of-care, 9.9% (794/8051) of HHC episodes had 3 reasons, and 5.7% (459/8051) of the HHC episodes had 4 reasons. The analysis of cooccurrence of reasons for delayed start-of-care HHC nursing visits (Figure 3) indicated that 19% (1529/8051) of HHC episodes with documented delayed visit reasons had both “no answer at the door or phone” and “patient/family request to postpone or refuse some HHC services”; 8% (644/8051) of HHC episodes had both “no answer at the door or phone” and “administrative or scheduling issues.” All 3 reasons were documented for 7% (563/8051) of HHC episodes. Figure 1. Sources of documented reasons for delayed start-of-care HHC nursing visits. The most prevalent categories of reasons for delayed start-of-care HHC nursing visits identified by the NLP algorithm (794/8051) of HHC episodes had 3 reasons, and 5.7% (459/8051) of the HHC episodes had 4 reasons. Prevalence of Delayed Start-of-Care HHC Visits in Standardized Documentation The most prevalent categories of reasons for delayed start-of-care HHC nursing visits identified by the NLP algorithm were “no answer at the door or phone” (3728/8051, 46.3%), followed by “patient/family request to postpone or refuse some HHC services” (n=2858, 35.5%) and “administrative or scheduling issues” (n=1465, 18.2%). The most prevalent categories of reasons for delayed start-of-care HHC nursing visits identified by the NLP algorithm were “no answer at the door or phone” (3728/8051, 46.3%), followed by “patient/family request to postpone or refuse some HHC services” (n=2858, 35.5%) and “administrative or scheduling issues” (n=1465, 18.2%). (794/8051) of HHC episodes had 3 reasons, and 5.7% (459/8051) of the HHC episodes had 4 reasons. (794/8051) of HHC episodes had 3 reasons, and 5.7% (459/8051) of the HHC episodes had 4 reasons. The analysis of cooccurrence of reasons for delayed start-of-care HHC nursing visits (Figure 3) indicated that 19% (1529/8051) of HHC episodes with documented delayed visit reasons had both “no answer at the door or phone” and “patient/family request to postpone or refuse some HHC services”; 8% (644/8051) of HHC episodes had both “no answer at the door or phone” and “administrative or scheduling issues.” All 3 reasons were documented for 7% (563/8051) of HHC episodes. Further analysis showed that in more than 40% of episodes with delayed start-of-care HHC nursing visits, 2 or more reasons were documented. Figure 2 shows that 17.5% (1409/8051) of the HHC episodes had 2 reasons for delayed start-of-care, 9.9% JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 6 (page number not for citation purposes) https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX XSL•FO RenderX Zolnoori et al JMIR NURSING gure 2. Number of reasons for delayed start-of-care home health care (HHC) nursing visits per home health care episode. Figure 2. Number of reasons for delayed start-of-care home health care (HHC) nursing visits per home health care episode. Figure 3. Among cooccurring reasons for delayed start-of-care, overlap of reasons for delayed start-of-care home health care (HHC) nursing visits. Discussion Principal Findings This is the first study we are aware of that examines reasons for delayed start-of-care HHC nursing visits in a large population of HHC patients. Identifying reasons for delayed start-of-care is critical due to the association between delayed care and negative outcomes. Prevalence of Delayed Start-of-Care HHC Visits in Standardized Documentation The majority of patients requiring HHC are clinically complex and vulnerable older adults who were discharged from hospitals with a need for immediate post–acute care [31]. Delayed start-of-care may increase the risk of JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 7 https://nursing.jmir.org/2021/4/e31038 (page number not for citation purposes) FO ng reasons for delayed start-of-care, overlap of reasons for delayed start-of-care home health care (HHC) nursing visits. Figure 3. Among cooccurring reasons for delayed start-of-care, overlap of reasons for delayed start-of-care home health c of HHC patients. Identifying reasons for delayed start-of-care is critical due to the association between delayed care and negative outcomes. The majority of patients requiring HHC are clinically complex and vulnerable older adults who were discharged from hospitals with a need for immediate post–acute care [31]. Delayed start-of-care may increase the risk of JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 7 (page number not for citation purposes) https://nursing.jmir.org/2021/4/e31038 Figure 2. Number of reasons for delayed start-of-care home health care (HHC) nursing visits per home health care episode. Principal Findings To resolve these issues, HHC organizations may need to develop better and more streamlined patient intake procedures and identify several ways to adjust schedules when a nurse calls in sick or is otherwise unavailable to take on new cases. Our results indicate that in more than 40% of HHC episodes, there were 2 or more reasons for delayed start-of-care HHC nursing visits. The most common cooccurring reasons indicated in about 20% of HHC episodes were “no answer at the door or phone” and “patient/family request to postpone or refuse some HHC services.” In a relatively small number of cases (7%), patients had a combination of all 3 reasons for delayed start-of-care HHC nursing visits. We hypothesize that patients with cooccurring reasons might be more likely to have a delayed start-of-care HHC nursing visit due to the challenges of addressing all reasons of delays in this group of patients. Clinicians may decide to prioritize patients with cooccurring reasons for appropriate interventions to reduce the likelihood of delayed start-of-care. The patient’s demographic and clinical factors might affect their risk for a delayed start-of-care HHC nursing visit. Identifying the relationship between these factors and reasons for delayed visits may help clinicians target patients at high risk for delayed visits with specific interventions. This is an area that requires further investigation. Our findings indicate that the most prevalent category of delayed start-of-care HHC nursing visits was “no answer at the door or phone.” Potential explanations include having incorrect phone or address information transmitted to the HHC organization, last minute decisions by patients to recover in a location other than their own home, and a lack of communication between the HHC organization and the patient about the patient’s availability. To address this issue, referring hospital clinicians may need to discuss the timing and importance of the first visit with the patient and family and obtain accurate contact information. In addition, it may be necessary to develop a clear communication plan with the HHC agency in the process of transferring the patient from the hospital to the HHC setting. Previous studies [34-42] and our anecdotal experience show that in reality, very little information is available to HHC nurses about newly admitted patients. Usually, HHC referrals from hospitals or primary care providers include administrative information, such as patients’ insurance status and billing address. Principal Findings This is the first study we are aware of that examines reasons for delayed start-of-care HHC nursing visits in a large population This is the first study we are aware of that examines reasons for delayed start-of-care HHC nursing visits in a large population https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX JMIR NURSING Zolnoori et al complications, emergency department visits, hospitalizations, and even death [32]. many patients may not understand the value of HHC and refuse care. A recent national study found that more than half of the patients referred to HHC from hospitals never receive HHC services [44]. Another study found that about 1 in 3 patients referred to post–acute health services, including HHC, refuse care [18]. The patients who refused post–acute care had twice higher odds of hospitalization or emergency department visits compared to patients with timely visits. Increased patient understanding of the importance of HHC services might help to resolve some of the issues related to this prevalent reason for delayed start-of-care HHC nursing visits. The findings of this study indicate that when a reason is provided, most reasons for delayed start-of-care HHC nursing visits (8051/11,129, 72.2%) are documented exclusively in narrative clinical notes. Large-scale analysis of thousands of clinical notes can now be accomplished via innovative data science methods, including NLP. We developed and tested an NLP algorithm to identify patterns of documented delayed start-of-care HHC nursing visit reasons in clinical notes. The NLP algorithm achieved high accuracy in identifying documented reasons when tested on an expert-reviewed sample of clinical notes. NLP algorithms have a wide range of potential applications in extracting meaningful patterns from EHR clinical notes [33]. We believe that NLP algorithms will be integrated into HHC EHRs in the future to process ongoing clinical documentation. Such integration can help HHC managers and practicing nurses to identify and address reasons for delayed start-of-care HHC nursing visits, potentially reducing delays in HHC services and improving patient outcomes. In about 20% of HHC episodes, delayed start-of-care was due to “administrative or scheduling issues.” Some of the specific issues were inadequate or incomplete insurance coverage, lack of forms and documents, and a lack of available HHC nurses to complete a start-of-care visit on time. Principal Findings It may be that patient contact information indicated in referrals is not always accurate, hence the delays in care due to phone calls placed to incorrect or outdated phone numbers or visits to outdated addresses. A recent report explored the availability of address information in a large hospital system and found that for 99% of patients (~2.1 million patients), a billing address was documented [43]. However, if patients were located at another address, the documentation of an alternative address was missing for 99.7% of these patients. Further investigation of the accuracy of the referral address and phone information might shed light on some of the interventions that can help reduce delayed care. https://nursing.jmir.org/2021/4/e31038 JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 8 (page number not for citation purposes) Conflicts of Interest None declared. Implications for Health Care Administrators The findings of this study provide insight into the application of NLP techniques to identify reasons for delayed start-of-care in HHC agencies and potential interventions to address the delays and avoid subsequent negative outcomes. HHC administrators may begin considering the use of NLP techniques to analyze the HHC nursing visits for patients that were seen later than 48 hours post–hospital discharge date to identify the reasons for delays and potential interventions. Obtaining and maintaining accurate patient contact information, educating patients about the importance of timely HHC services, and improving the procedures of patient intake and the scheduling system are among the interventions we suggested to reduce the risk of delayed start-of-care due to the following reasons, respectively: “no answer at the door or phone,” “patient/family request to postpone or refuse some home health care services,” and “administrative or scheduling issues.” The second most prevalent reason for delayed start-of-care HHC nursing visits was “patient/family request to postpone or refuse some HHC services.” Although some related care delays are probably unavoidable, referring physicians and HHC clinicians may need to spend more time articulating the importance of start-of-care HHC nursing visits. Several recent studies indicate https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX JMIR NURSING Zolnoori et al identified by NLP were incorrect due to algorithm errors. It is unclear if additional reasons would have been identified if more information on these episodes was provided. Limitations The findings of this study should be considered in light of several limitations. First, the data set was from a single HHC agency located in the northeast United States. Although the HHC agency is large, its services are limited to individuals residing in a particular catchment area. Therefore, the reasons for delayed visits may not be generalizable to HHC agencies in other locations. Second, the available data did not have information on the hour of discharge from the hospital, so the analysis was based on days, not hours as in the CMS regulations. Third, the NLP algorithms developed in this study had a total precision of 0.8 and recall of 0.75. These findings indicate that some reasons for delayed start-of-care HHC nursing visits Conclusions To avoid delays in critical start-of-care nursing visits, HHC agencies and hospitals should consider examining and improving accurate patient or family contact information collection. In addition, HHC agencies should consider providing targeted education about the importance of the early initiation of HHC services to reduce patient and family refusals and/or requests to postpone care. Finally, HHC agencies should make efforts to reduce administrative and scheduling issues to enable timely and effective care. Acknowledgments Research reported in this publication was supported by the National Institute of Nursing Research (NINR) under Award Number R01NR018831. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NINR. 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JAMA Netw Open 2020 Sep 01;3(9):e2015470 [FREE Full text] [doi: 10.1001/jamanetworkopen.2020.15470] [Medline: 32876682] https://nursing.jmir.org/2021/4/e31038 Abbreviations Abbreviations CMS: Centers for Medicare & Medicaid Services EHR: electronic health record HHC: home health care NINR: National Institute of Nursing Research NLP: natural language processing OASIS: Outcome and Assessment Information Set VNSNY: Visiting Nurse Service of New York Edited by E Borycki; submitted 07.06.21; peer-reviewed by W Zhang, M Afzal, E Gjevjon; comments to author 23.07.21; revised version received 31.08.21; accepted 28.10.21; published 30.12.21 Please cite as: Zolnoori M, Song J, McDonald MV, Barrón Y, Cato K, Sockolow P, Sridharan S, Onorato N, Bowles KH, Topaz M Exploring Reasons for Delayed Start-of-Care Nursing Visits in Home Health Care: Algorithm Development and Data Science Study JMIR Nursing 2021;4(4):e31038 URL: https://nursing.jmir.org/2021/4/e31038 doi: 10.2196/31038 PMID: 34967749 ©Maryam Zolnoori, Jiyoun Song, Margaret V McDonald, Yolanda Barrón, Kenrick Cato, Paulina Sockolow, Sridevi Sridharan, Nicole Onorato, Kathryn H Bowles, Maxim Topaz. Originally published in JMIR Nursing (https://nursing.jmir.org), 30.12.2021. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Nursing, is properly cited. The complete bibliographic information, a link to the original publication on https://nursing.jmir.org/, as well as this copyright and license information must be included. JMIR Nursing 2021 \| vol. 4 \| iss. 4 \| e31038 \| p. 11 (page number not for citation purposes) https://nursing.jmir.org/2021/4/e31038 XSL•FO RenderX
https://openalex.org/W2033710332	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1002408&type=printable	English	null	Impact of Network Structure and Cellular Response on Spike Time Correlations	PLOS computational biology/PLoS computational biology	2,012	cc-by	15,397	Introduction empirical data from one class of stimuli will generalize to other stimulus classes and recording sites. Moreover, a mechanistic understanding of the origin of correlations, and knowledge of the patterns we can expect to see under different assumptions about the underlying networks, will help resolve recent controversies about the strength and pattern of correlations in mammalian cortex [1,20,21]. Finally, understanding the origin of correlations will inform the more ambitious aim of inferring properties of network architecture from observed patterns of activity [22–24]. New multielectrode and imaging techniques are revealing the simultaneous activity of neural ensembles and, in some cases, entire neural populations [1–4]. This has thrust upon the computational biology community the challenge of characterizing a potentially complex set of interactions – or correlations – among pairs and groups of neurons. Beyond important and rich challenges for statistical modeling [5], the emerging data promises new perspectives on the neural encoding of information [6]. The structure of correlations in the activity of neuronal populations is of central importance in understanding the neural code [7–13]. However, theoretical [9– 11,14–16], and empirical studies [17–19] do not provide a consistent set of general principles about the impact of correlated activity. This is largely because the presence of correlations can either strongly increase or decrease the fidelity of encoded information depending on both the structure of correlations across a population and how their impact is assessed. Here, we examine the link between network properties and correlated activity. We develop a theoretical framework that accurately predicts the structure of correlated spiking that emerges in a widely used model – recurrent networks of general integrate and fire cells. The theory naturally captures the role of single cell and synaptic dynamics in shaping the magnitude and timescale of spiking correlations. We focus on the exponential integrate and fire model, which has been shown to capture membrane and spike responses of cortical neurons [25]; however, the general approach we take can be applied to a much broader class of neurons, a point we return to in the Discussion. A basic mechanistic question underlies the investigation of the role of collective activity in coding and signal transmission: How do single-cell dynamics, connection architecture, and synaptic dynamics combine to determine patterns of network activity? Systematic answers to this question would allow us to predict how Our approach is based on an extension of linear response theory to networks [24,26]. Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NSF grants DMS-0817649, DMS-1122094, and a Texas ARP/ATP award to KJ, as well as NSF Grants DMS-1122106, DMS- 0818153, and a Burroughs Wellcome Career Award at the Scientic Interface to ESB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jrtrousd@math.uh.edu James Trousdale1*, Yu Hu2, Eric Shea-Brown2, Kresˇimir Josic´1,3 1 Department of Mathematics, University of Houston, Houston, Texas, United States of America, 2 Department of Applied Mathematics, Program in Neurobiology and Behavior, University of Washington, Seattle, Washington, United States of America, 3 Department of Biology and Biochemistry, University of Houston, Houston, Texas, United States of America PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 Abstract Novel experimental techniques reveal the simultaneous activity of larger and larger numbers of neurons. As a result there is increasing interest in the structure of cooperative – or correlated – activity in neural populations, and in the possible impact of such correlations on the neural code. A fundamental theoretical challenge is to understand how the architecture of network connectivity along with the dynamical properties of single cells shape the magnitude and timescale of correlations. We provide a general approach to this problem by extending prior techniques based on linear response theory. We consider networks of general integrate-and-fire cells with arbitrary architecture, and provide explicit expressions for the approximate cross-correlation between constituent cells. These correlations depend strongly on the operating point (input mean and variance) of the neurons, even when connectivity is fixed. Moreover, the approximations admit an expansion in powers of the matrices that describe the network architecture. This expansion can be readily interpreted in terms of paths between different cells. We apply our results to large excitatory-inhibitory networks, and demonstrate first how precise balance – or lack thereof – between the strengths and timescales of excitatory and inhibitory synapses is reflected in the overall correlation structure of the network. We then derive explicit expressions for the average correlation structure in randomly connected networks. These expressions help to identify the important factors that shape coordinated neural activity in such networks. Citation: Trousdale J, Hu Y, Shea-Brown E, Josic´ K (2012) Impact of Network Structure and Cellular Response on Spike Time Correlations. PLoS Comput Biol 8(3): e1002408. doi:10.1371/journal.pcbi.1002408 Editor: Olaf Sporns, Indiana University, United States of America Citation: Trousdale J, Hu Y, Shea-Brown E, Josic´ K (2012) Impact of Network Structure and Cellular Response on Spike Time Correlations. PLoS Comput Biol 8(3): e1002408. doi:10.1371/journal.pcbi.1002408 Editor: Olaf Sporns, Indiana University, United States of America Editor: Olaf Sporns, Indiana University, United States of America Received October 21, 2011; Accepted January 11, 2012; Published March 22, 2012 Received October 21, 2011; Accepted January 11, 2012; Published March 22, 2012 Copyright: 2012 Trousdale et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. usdale et al. Author Summary Is neural activity more than the sum of its individual parts? What is the impact of cooperative, or correlated, spiking among multiple cells? We can start addressing these questions, as rapid advances in experimental techniques allow simultaneous recordings from ever-increasing pop- ulations. However, we still lack a general understanding of the origin and consequences of the joint activity that is revealed. The challenge is compounded by the fact that both the intrinsic dynamics of single cells and the correlations among then vary depending on the overall state of the network. Here, we develop a toolbox that addresses this issue. Specifically, we show how linear response theory allows for the expression of correlations explicitly in terms of the underlying network connectivity and known single-cell properties – and that the predic- tions of this theory accurately match simulations of a touchstone, nonlinear model in computational neurosci- ence, the general integrate-and-fire cell. Thus, our theory should help unlock the relationship between network architecture, single-cell dynamics, and correlated activity in diverse neural circuits. Upon reaching a threshold vth, an action potential is generated, and the membrane potential is reset to vr, where it is held constant for an absolute refractory period tref. The output of cell i is characterized by the times, ti,k, at which its membrane potential reaches threshold, resulting in an output spike train yi(t)~ P k d(t{ti,k). Synaptic interactions are modeled by delayed a-functions fi(t)~ X j (Jij yj)(t), where Jij(t)~ Wij t{tD,j t2 S,j ! exp { t{tD,j tS,j t§tD,j 0 tvtD,j 8 > > < > > : : ð2Þ fi(t)~ X j (Jij yj)(t), where Jij(t)~ Wij t{tD,j t2 S,j ! exp { t{tD,j tS,j t§tD,j 0 tvtD,j 8 > > < > > : : ð2Þ ð2Þ 0 The N\|N matrix J contains the synaptic kernels, while the matrix W contains the synaptic weights, and hence defines the network architecture. In particular, if gL is the membrane conductance, gLWij is the area under a post-synaptic current evoked in cell j by a spike in the presynaptic cell i, and along with the membrane and synaptic time constants, determines the area under a post-synaptic potential. Wij~0 represents the absence of a synaptic connection from cell j to cell i. directly related to the spike triggered average [30]. The correlation structure of the network is then estimated using an iterative approach. Impact of Network Structure on Spike Correlations emulate the rapid onset of action potentials. Unless otherwise specified, we utilize the exponential IF model (EIF), so that y(v):DT exp½(v{vT)=DT [25]. Cells are subject to internally induced fluctuations due to channel noise [36], and externally induced fluctuations due to inputs not explicitly modelled [37]. We model both by independent, Gaussian, white noise processes, ﬃﬃﬃﬃﬃﬃﬃﬃ s2 i ti q ji(t) [38]. An external signal to cell i is represented by gi(t). Results The membrane potential of an IF neuron receiving input EX(t), with vanishing temporal average, SX(t)T~0, evolves according to Our goal is to understand how the architecture of a network shapes the statistics of its activity. We show how correlations between spike trains of cells can be approximated using response characteristics of individual cells along with information about synaptic dynamics, and the structure of the network. We start by briefly reviewing linear response theory of neuronal responses [28,34,35], and then use it to approximate the correlation structure of a network. t_v~{(v{EL)zy(v)zEz ﬃﬃﬃﬃﬃﬃﬃ s2t p j(t)zEX(t): ð3Þ ð3Þ The time-dependent firing rate, r(t), is determined by averaging the resulting spike train, y(t)~ P j d(t{tj), across different realizations of noise, j(t), for fixed X(t). Using linear response theory, we can approximate the firing rate by Linear response of individual cells Neuronal network models are typically described by a complex system of coupled nonlinear stochastic differential equations. Their behavior is therefore difficult to analyze directly. We will use linear response theory [28,34,35,39] to approximate the cross-correlations between the outputs of neurons in a network. We first review the linear approximation to the response of a single cell. We illustrate the approach using current-based IF neurons, and explain how it can be generalized to other models in the Discussion. Author Summary As in prior work [31–33], the resulting expressions admit an expansion in terms of paths through the network. We apply this theory to networks with precisely balanced inhibition and excitation in the inputs to individual cells. In this state individual cells receive a combination of excitatory and inhibitory inputs with mean values that largely cancel. We show that, when timescales and strengths of excitatory and inhibitory connections are matched, only local interactions between cells contribute to correlations. Moreover, our theory allows us to explain how correlations are altered when precise tuning balance is broken. In particular, we show how strengthening inhibition may synchronize the spiking activity in the network. Finally, we derive results which allow us to gain an intuitive understanding of the factors shaping average correlation structure in randomly connected networks of neurons. Table 1 provides an overview of all parameters and variables. Introduction We start with a linear approximation of a neuron’s response to an input. This approximation can be obtained explicitly for many neuron models [27–29], and is 1 March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 Impact of Network Structure on Spike Correlations PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 Network model r(t)~r0z(A EX)(t), ð4Þ ð4Þ To illustrate the results we consider a network of N nonlinear integrate-and-fire (IF) neurons with membrane potentials modeled by where r0 is the (stationary) firing rate when E~0. The linear response kernel, A(t), characterizes the firing rate response to first order in E. A rescaling of the function A(t) gives the spike-triggered average of the cell, to first order in input strength, and is hence equivalent to the optimal Weiner kernel in the presence of the signal j(t). [39,40]. In Figure 1, we compare the approximate firing rate obtained from Eq. (4) to that obtained numerically from Monte Carlo simulations. ti _vi~{(vi{EL,i)zy(vi)zEiz ﬃﬃﬃﬃﬃﬃﬃﬃ s2 i ti q ji(t)zfi(t)zgi(t): ð1Þ Here EL,i is the leak reversal potential, and Ei represents the mean synaptic input current from parts of the system not explicitly modeled. A spike-generating current y(vi) may be included to PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 2 Impact of Network Structure on Spike Correlations Table 1. Notation used in the text. Symbol Description vi,ti,EL,i,si Membrane potential, membrane time constant, leak reversal potential, and noise intensity of cell i. Ei,si Mean and standard deviation of the background noise for cell i. vth,vr,tref Membrane potential threshold, reset, and absolute refractory period for cells. y(v),VT,DT Spike generating current, soft threshold and spike shape parameters for the IF model [25]. fi(t),gi(t) Synaptic input from other cells in the network, and external input to cell i. tS,i,tD,i Synaptic time constant and delay for outputs of cell i. yi(t) Spike train of cell i. Wij The j?i synaptic weight, proportional to the area under a single post-synaptic current for current-based synapses. Jij(t) The j?i synaptic kernel - equals the product of the synaptic weight Wij and the synaptic filter for outputs of cell j. Cij(t) The cross-correlation function between cells i,j defined by Cij(t)~cov(yi(tzt),yj(t)). Nyi(t,tzt),rij(t) Spike count for cell i, and spike count correlation coefficient for cells i,j over windows of length t. ri,Ai(t),C0 ii Stationary rate, linear response kernel and uncoupled auto-correlation function for cell ij. Kij(t) The j?i interaction kernel - describes how the firing activity of cell i is perturbed by an input spike from cell j. It is defined by Kij(t)~(Ai Jij)(t). Network model yn i (t),Cn ij(t) The nth order approximation of the activity of cell i in a network which accounts for directed paths through the network graph up to length n ending at cell i, and the cross-correlation between the nth order approximations of the activity of cells i,j. g(t),~g(v) ~g(v) is the Fourier transform of g(t) with the convention ~g(v)~F½g(v): Ð ? {? e{2pivtg(t)dt doi:10.1371/journal.pcbi.1002408.t001 doi:10.1371/journal.pcbi.1002408.t001 The linear response kernel A(t) depends implicitly on model parameters, but is independent of the input signal, EX(t), when E is small relative to the noise ﬃﬃﬃﬃﬃﬃﬃ s2t p j(t). In particular, A(t) is sensitive to the value of the mean input current, E. We emphasize that the presence of the background noise, j, in Eq. (3) is essential to the theory, as noise linearizes the transfer function that maps input to output. In addition, when applying linear response methods, there is an implicit assumption that the fluctuations of the input X(t) do not have a significant effect on the response properties of the cell. Linear response in recurrent networks The linear response kernel can be used to approximate the response of a cell to an external input. However, the situation is more complicated in a network where a neuron can affect its own activity through recurrent connections. To extend the linear response approximation to networks we follow the approach introduced by Lindner et al. [26]. Instead of using the linear response kernel to approximate the firing rate of a cell, we use it to approximate a realization of its output Figure 1. Illustrating Eq. (4). (A) The input to the post-synaptic cell is a fixed spike train which is convolved with a synaptic kernel. (B) A sample voltage path for the post-synaptic cell receiving the input shown in A) in the presence of background noise. (C) Raster plot of 100 realizations of output spike trains of the post-synaptic cell. (D) The output firing rate, r(t), obtained by averaging over realizations of the output spike trains in C). The rate obtained using Monte Carlo simulations (shaded in gray) matches predictions of linear response theory obtained using Eq. (4) (black). doi:10.1371/journal.pcbi.1002408.g001 y(t)&y0(t)z(A X)(t): ð5Þ ð5Þ Here y0(t) represents a realization of the spike train generated by an integrate-and-fire neuron obeying Eq. (3) with X(t)~0. Our central assumption is that a cell acts approximately as a linear filter of its inputs. Note that Eq. (5) defines a mixed point and continuous process, but averaging y(t) in Eq. (5) over realizations of y0 leads to the approximation in Eq. (4). Hence, Eq. (5) is a natural generalization of Eq. (4) with the unperturbed output of the cell represented by the point process, y0(t), instead of the firing rate, r0. We first use Eq. (5) to describe spontaneously evolving networks where gi(t)~0. Equation (1) can then be rewritten as Figure 1. Illustrating Eq. (4). (A) The input to the post-synaptic cell is a fixed spike train which is convolved with a synaptic kernel. (B) A sample voltage path for the post-synaptic cell receiving the input shown in A) in the presence of background noise. (C) Raster plot of 100 realizations of output spike trains of the post-synaptic cell. (D) The output firing rate, r(t), obtained by averaging over realizations of the output spike trains in C). The rate obtained using Monte Carlo simulations (shaded in gray) matches predictions of linear response theory obtained using Eq. (4) (black). doi:10.1371/journal.pcbi.1002408.g001 Figure 1. Illustrating Eq. (4). PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 ~y(v)~(I{~K(v)){1~y0(v), ~y(v)~(I{~K(v)){1~y0(v), which can be simplified to give which can be simplified to give which can be simplified to give where the interaction matrix ~K has entries defined by Kij(t):(Ai Jij)(t). When averaged against its conjugate trans- pose, this expression yields an approximation to the full array of cross-spectra in the recurrent network: Cij(t)&C1 ij(t)~dijC0 ii(t)z(Kij C0 jj)(t)z (K{ ji C0 ii)(t)z X k (Kik K{ jk C0 kk)(t) ð9Þ ð9Þ S~y(v)~y(v)T~(I{~K(v)){1S~y0(v)~y0(v)T(I{~K(v)){1: ð7Þ S~y(v)~y(v)T~(I{~K(v)){1S~y0(v)~y0(v)T(I{~K(v)){1: ð7Þ ð7Þ where we used f {(t)~f ({t). Ostojic et al. obtained an approximation closely related to Eq. (9). [24] They first obtained the cross-correlation between a pair of neurons which either receive a common input or share a monosynaptic connection. This can be done using Eq. (4), without the need to introduce the mixed process given in Eq. (5). Ostojic et al. then implicitly assumed that the correlations not due to one of these two submotifs could be disregarded. The correlation between pairs of cells which were mutually coupled (or were unidirectionally coupled with common input) was approximated by the sum of correlations introduced by each submotif individually. We next present a distinct derivation of this approximation which allows for a different interpretation of the ansatz given by Eq. (5). We iteratively build to the approximation in Eq. (7), showing how this expression for the correlation structure in a recurrent network can be obtained by taking into account the paths through the network of increasing length. We start with realizations of spike trains, y0 i , generated by IF neurons obeying Eq. (6) with fi(t)~E fi½ . This is equivalent to considering neurons isolated from the network, with adjusted DC inputs (due to mean network interactions). Following the approximation given by Eq. (5), we use a frozen realization of all y0 i to find a correction to the output of each cell, with X(t) set to the mean-adjusted synaptic input, Equation (9) provides a first approximation to the joint spiking statistics of cells in a recurrent network. However, it captures only the effects of direct synaptic connections, represented by the second and third terms, and common input, represented by the last term in Eq. (9). The impact of larger network structures, such as loops and chains are not captured, although they may significantly impact cross-correlations [41–43]. Experimental studies have also shown that local cortical connectivity may not be fully random [44–46]. Linear response in recurrent networks (A) The input to the post-synaptic cell is a fixed spike train which is convolved with a synaptic kernel. (B) A sample voltage path for the post-synaptic cell receiving the input shown in A) in the presence of background noise. (C) Raster plot of 100 realizations of output spike trains of the post-synaptic cell. (D) The output firing rate, r(t), obtained by averaging over realizations of the output spike trains in C). The rate obtained using Monte Carlo simulations (shaded in gray) matches predictions of linear response theory obtained using Eq. (4) (black). doi:10 1371/journal pcbi 1002408 g001 ti _vi~{(vi{EL,i)zy(vi)zEi ’z ﬃﬃﬃﬃﬃﬃﬃﬃ s2 i ti q ji(t)z(fi(t){E fi½ ), ð6Þ where Ei’~EizE fi½ and E :½ represents the temporal average. Ei’~EizE fi½ and E :½ represents the temporal averag Lindner et al. used Eq. (5) as an ansatz to study the response of an all–to–all inhibitory network. They postulated that the spiking output yi(t) of cell i in the network, can be approximated in the doi:10.1371/journal.pcbi.1002408.g001 PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 3 Impact of Network Structure on Spike Correlations Impact of Network Structure on Spike Correlations The cross-correlation between the processes y1 i (t) in Eq. (8) gives a first approximation to the cross-correlation function between the cells, frequency domain by ~yi(v)~~y0 i (v)z~Ai(v) X j ~Jij(v)~yj(v) ! , Cij(t)&C1 ij(t)~E (y1 i (tzt){ri)(y1 j (t){rj) h i ~E (y0 i (tzt){ri)(y0 j (t){rj) h i z X k E (Kik ½y0 k{rk)(tzt)(y0 j (t){rj) h i z X k E (y0 i (tzt){ri)(Kjk ½y0 k{rk)(t) z X k,l E (Kik ½y0 k{rk)(tzt)(Kjl ½y0 l {rl)(t) where ~yi~F½yi{ri are the zero-mean Fourier transforms of the processes yi, and ~f ~F(f ) for all other quantities (see Table 1 for the Fourier transform convention). The term in parentheses is the Fourier transform of the zero-mean synaptic input, (fi(t){E fi½ ), in Eq. (6), and ~y0 i (v) represents a realization of the spiking output of cell i in the absence of synaptic fluctuations from the recurrent network (i.e assuming fi~E fi½ ). In matrix form this ansatz yields a simple self-consistent approximation for the firing activities ~yi which can be solved to give Impact of Network Structure on Spike Correlations Impact of Network Structure on Spike Correlations Figure 2. Iterative construction of the linear approximation to network activity. (A) An example recurrent network. (B)–(D) A sequence of graphs determines the successive approximations to the output of neuron 1. Processes defined by the same iteration of Eq. (11) have equal color. (B) In the first iteration of Eq. (11), the output of neuron 1 is approximated using the unperturbed outputs of its neighbors. (C) In the second iteration the results of the first iteration are used to define the inputs to the neuron. For instance, the process y1 2 depends on the base process y0 1 which represents the unperturbed output of neuron 1. Neuron 4 receives no inputs from the rest of the network, and all approximations involve only its unperturbed output, y0 4. (D) Cells 3 and 4 are not part of recurrent paths, and their contributions to the approximation are fixed after the second iteration. However, the recurrent connection between cells 1 and 2 implies that subsequent approximations involve contributions of directed chains of increasing length. y0 4. (D) Cells 3 and 4 are not part of recurrent paths, and their contributions to the approximation are fixed after the second iteration. However, the recurrent connection between cells 1 and 2 implies that subsequent approximations involve contributions of directed chains of increasing length. g g doi:10.1371/journal.pcbi.1002408.g002 impact of next nearest neighbors. Successive iterations include the impact of directed chains of increasing length: The isolated output from an independent collection of neurons is filtered through n stages to produce the corrected response (See Figure 2.) impact of next nearest neighbors. Successive iterations include the impact of directed chains of increasing length: The isolated output from an independent collection of neurons is filtered through n stages to produce the corrected response (See Figure 2.) Notation is simplified when this iterative construction is recast in matrix form to obtain ynz1(t) ~y0(t)z(K ½yn{r)(t) ~y0(t)z P nz1 k~1 (K(k) ½y0{r)(t), n§0, ð11Þ ð11Þ where yn(t)~½yn i (t) and r~½ri are length N column vectors, and K(k) represents a k-fold matrix convolution of K with itself. We define the convolution of matrices in the Methods. ~y(v)~(I{~K(v)){1~y0(v), It is therefore important to understand the effects on network architecture on correlations. X(t)~fi(t){E fi½ : As noted previously, the linear response kernel is sensitive to changes in the mean input current. It is therefore important to include the average synaptic input E fi½ in the definition of the effective mean input, E’i. We therefore propose an iterative approach which accounts for successively larger connectivity patterns in the network [32,33]. We again start with y0 i (t), a realization of a single spike train in isolation. Successive approximations to the output of cells in a recurrent network are defined by The input from cell j to cell i is filtered by the synaptic kernel Jij(t). The linear response of cell i to a spike in cell j is therefore captured by the interaction kernel Kij, defined above as Kij(t):(Ai Jij)(t): ynz1 i (t)~y0 i (t)z X j (Kij ½yn j {rj)(t), n§0: ð10Þ The output of cell i in response to mean-adjusted input, y0 j (t){rj, from cell j can be approximated to first order in input strength using the linear response correction The output of cell i in response to mean-adjusted input, y0 j (t){rj, from cell j can be approximated to first order in input strength using the linear response correction To compute the correction to the output of a neuron, in the first iteration we assume that its inputs come from a collection of isolated cells: When n~1, Eq. (10) takes into account only inputs from immediate neighbors, treating each as disconnected from the rest of the network. The corrections in the second iteration are computed using the approximate cell responses obtained from the first iteration. Thus, with n~2, Eq. (10) also accounts for the y1 i (t)~y0 i (t)z X j (Kij ½y0 j {rj)(t): ð8Þ ð8Þ We explain how to approximate the stationary rates, rj, in the Methods. PLoS Computational Biology \| www.ploscompbiol.org PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 4 Impact of Network Structure on Spike Correlations response, cf. Eq. (4). However, Eq. (11) does not capture nonlinear corrections to the response of individual cells, as the output of each cell is determined linearly from its input. It is the input that can contain terms of any order in connection strength stemming from directed paths of different lengths through the network. ~C n(v)~E½~yn(v)~yn(v) ~ P n k,l~0 ~K k(v)E½~y0(v)~y0(v)(~K )l(v) ~ X n k~0 ~K k(v) ! E ~y0(v)~y0(v) X n l~0 (~K )l(v) ! , ð14Þ We use the theoretical framework developed above to analyze the statistical structure of the spiking activity in a network of IF neurons described by Eq. (1). We first show that the cross- correlation functions between cells in two small networks can be studied in terms of contributions from directed paths through the network. We use a similar approach to understand the structure of correlations in larger all–to–all and random networks. We show that in networks where inhibition and excitation are tuned to exactly balance, only local interactions contribute to correlations. When such balance is broken by a relative elevation of inhibition, the result may be increased synchrony in the network. The theory also allows us to obtain averages of cross-correlation functions conditioned on connectivity between pairs of cells in random networks. Such averages can provide a tractable yet accurate description of the joint statistics of spiking in these networks. where X denotes the conjugate transpose of the matrix X. As before, the zero-mean Fourier transforms ~yn i of the processes yn i are defined by ~yn i ~F½yn i {ri, and ~f ~F(f ) for all other quantities. Defining Y(X) to be the spectral radius of the matrix X, when Y(~K)v1, we can take the limit n?? in Eq. (14) [47,48], to obtain an approximation to the full array of cross-spectra ~C(v)&~C ?(v)~ lim n?? ~C n(v) ~(I{~K(v)){1~C 0(v)(I{~K (v)){1: ð15Þ ð15Þ The correlation structure is determined by the response properties of cells together with synaptic dynamics and network architecture. Network interactions are described by the matrix of synaptic filters, J, given in Eq. (2), while the response of cell i to an input is approximated using its linear response kernel Ai. Synaptic dynamics, architecture, and cell responses are all combined in the matrix K, where Kij describes the response of cell i to an input from cell j (See Eq. (1)). Impact of Network Structure on Spike Correlations The correlation structure of network activity is approxi- mated in Eq. (15) using the Fourier transforms of the interaction matrix, K, and the matrix of unperturbed autocorrelations C0. As noted previously, this generalizes the approach of Lindner et al. [26] (also see [13]). In the limit n??, directed paths of arbitrary length contribute to the approximation. Equation (15) therefore takes into account the full recurrent structure of the network. Note that Eq. (15) may be valid even when Y(~K)w1. However, in this case the series in Eq. (14) do not converge, and hence the expansion of the correlations in terms of paths through the network is invalid. We confirmed numerically that Y(~K)v1 for all of the networks and parameters we considered. Statistics of the response of microcircuits Finally, consider the network response to external signals, gi(t), with zero mean and finite variance. The response of the neurons in the recurrent network can be approximated iteratively by We first consider a pair of simple microcircuits to highlight some of the features of the theory. We start with the three cell model of feed-forward inhibition (FFI) shown in Figure 3A [51]. The interaction matrix, ~K(v), has the form ynz1~y0zK ½yn{rzA g, ~K(v)~ 0 0 0 ~KE2E1(v) 0 ~KE2I(v) ~KIE1(v) 0 0 0 B @ 1 C A, where A~diag(Ai) and g(t)~½gi(t). External signals and recurrent synaptic inputs are both linearly filtered to approximate a cell’s response, consistent with a generalization of Eq. (4). As in Eq. (12), the nth approximation to the matrix of correlations is where cells are indexed in the order E1,E2,I. To simplify notation, we omit the dependence of ~K(v) and other spectral quantities on v. C(t)&Cn(t)~ X n k,l~0 (K(k) C0 (K{)(lT))(t)z Note that ~K is nilpotent of degree 3 (that is, ~K3:0), and the inverse of (I{~K) may be expressed as X n{1 k,l~0 (K(k) A Cg (A{) (K{)(lT))(t), (I{~K){1~(Iz~Kz~K2)~ 1 0 0 ~KE2E1z ~KE2I ~KIE1 1 ~KE2I ~KIE1 0 1 0 B @ 1 C A:ð17Þ :ð17Þ where Ch(t)~E g(tzt)g(t)T is the covariance matrix of the external signals. We can again take the Fourier transform and the limit n??, and solve for ~C(v). If Y(~K)v1, where Ch(t)~E g(tzt)g(t)T is the covariance matrix of the external signals. We can again take the Fourier transform and the limit n??, and solve for ~C(v). If Y(~K)v1, Substituting Eq. (17) into Eq. (15) (and noting that a similar equation as Eq. (17) holds for (I{~K){1) yields an approximation to the matrix of cross-spectra. For instance, Substituting Eq. (17) into Eq. (15) (and noting that a similar equation as Eq. (17) holds for (I{~K){1) yields an approximation to the matrix of cross-spectra. For instance, PLoS Computational Biology \| www.ploscompbiol.org Impact of Network Structure on Spike Correlations The nth approximation to the matrix of cross-correlations can be written in terms of the interaction kernels, Kij, and the autocorrelations of the base processes y0 as Cij(t)&Cn(t) ~E (yn(tzt){r)(yn(t){r)T ~ P n k,l~0 (K(k) C0 (K{)(lT))(t), n§0, ð12Þ ð12Þ where K{(t)~K({t), X(kT)~(X(k))T, and X(k) is the k-fold matrix convolution of X with itself. Eq. (12) can be verified by a simple calculation. First, Eq. (11) directly implies that yn(t)~y0(t)z X n k~1 (Kk ½y0{r)(t), n§0, which we may use to find, for each n§0, which we may use to find, for each n§0, Cn(t) :E (yn(tzt){r)(yn(t){r)T ~E (y0(tzt){r)(y0(t){r)T z P n k~1 E (Kk ½y0{r)(tzt)(y0(t){r)T z P n k~1 E (y0(tzt){r)(Kk ½y0{r)T(t) z P n k,l~1 E (Kk ½y0{r)(tzt)(Kl ½y0{r)T(t) ~C0(t)z P n k~1 (Kk C0)(t)z P n k~1 (C0 (K{)kT)(t) z P n k,l~1 (Kk C0 (K{)lT)(t): ð13Þ ð13Þ Figure 2. Iterative construction of the linear approximation to network activity. (A) An example recurrent network. (B)–(D) A sequence of graphs determines the successive approximations to the output of neuron 1. Processes defined by the same iteration of Eq. (11) have equal color. (B) In the first iteration of Eq. (11), the output of neuron 1 is approximated using the unperturbed outputs of its neighbors. (C) In the second iteration the results of the first iteration are used to define the inputs to the neuron. For instance, the process y1 2 depends on the base process y0 1 which represents the unperturbed output of neuron 1. Neuron 4 receives no inputs from the rest of the network, and all approximations involve only its unperturbed output, Since K0 ij(t)~dijd(t), Eq. (13) is equivalent to Eq. (12). If we apply the Fourier transform, to Eq. (12), we find that for each v, PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 5 Impact of Network Structure on Spike Correlations ~C?(v)~(I{~K(v)){1(~C0(v)z~A(v)~Cg(v)~A(v))(I{~K(v)){1:ð16Þ When the signals comprising g are white (and possibly correlated) corrections must be made to account for the change in spectrum and response properties of the isolated cells [26,49,50] (See Methods). ~C? E2I~ ~KE2I ~C0 I z ~KE2E1 ~K IE1 ~C0 E1z ~KE2ID~KIE1D2 ~C0 E1 ~ (~AE2~JE2I)~C0 I \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄ} I z (~AE2~JE2E1)(~AI ~JIE1) ~C0 E1 \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} II z (~AE2~JE2E1)D~AI ~JIE1D2 ~C0 E1 \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} III : ð18Þ ð18Þ We note that Eq. (11), which is the basis of our iterative approach, provides an approximation to the network’s output which is of higher than first order in connection strength. This may seem at odds with a theory that provides a linear correction to a cell’s March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 PLoS Computational Biology \| www.ploscompbiol.org 6 Impact of Network Structure on Spike Correlations Figure 3. The relation between correlation structure and response statistics in a feed-forward inhibitory microcircuit. (A) The FFI circuit (left) can be decomposed into three submotifs. Equation (18) shows that each submotif provides a specific contribution to the cross- correlation between cells E2 and I. (B) Comparison of the theoretical prediction with the numerically computed cross-correlation between cells E2 and I. Results are shown for two different values of the inhibitory time constant, tI (tI~5 ms, solid line, tI~10 ms, dashed line). (C) The contributions of the different submotifs in panel A are shown for both tI~5 ms (solid) and tI~10 ms (dashed). Inset shows the corresponding change in the inhibitory synaptic filter. The present color scheme is used in subsequent figures. Connection strengths were +40 mV:ms for excitatory and inhibitory connections. In each case, the long window correlation coefficient r(?) between the two cells was &{0:18. doi:10.1371/journal.pcbi.1002408.g003 Figure 3. The relation between correlation structure and response statistics in a feed-forward inhibitory microcircuit. (A) The FFI circuit (left) can be decomposed into three submotifs. Equation (18) shows that each submotif provides a specific contribution to the cross- correlation between cells E2 and I. (B) Comparison of the theoretical prediction with the numerically computed cross-correlation between cells E2 and I. Results are shown for two different values of the inhibitory time constant, tI (tI~5 ms, solid line, tI~10 ms, dashed line). (C) The contributions of the different submotifs in panel A are shown for both tI~5 ms (solid) and tI~10 ms (dashed). ~C?(v)~(I{~K(v)){1(~C0(v)z~A(v)~Cg(v)~A(v))(I{~K(v)){1:ð16Þ Inset shows the corresponding change in the inhibitory synaptic filter. The present color scheme is used in subsequent figures. Connection strengths were +40 mV:ms for excitatory and inhibitory connections. In each case, the long window correlation coefficient r(?) between the two cells was &{0:18. doi:10.1371/journal.pcbi.1002408.g003 Figure 3B shows that these approximations closely match numerically obtained cross-correlations. ~C0 X is the uncoupled power spectrum for cell X. ~K~ 0 ~KE1E2 ~KE2E1 0 ! so that (I{~K){1~ 1 1{ ~KE1E2 ~KE2E1 (Iz~K): Equation (15) gives the following approximation to the matrix of cross-spectra ~K~ 0 ~KE1E2 ~KE2E1 0 ! (I{~K){1~ 1 1{ ~KE1E2 ~KE2E1 (Iz~K): Equation (18) gives insight into how the joint response of cells in this circuit is shaped by the features of the network. The three terms in Eq. (18) are directly related to the architecture of the microcircuit: Term I represents the correlating effect of the direct input to cell E2 from cell I. Term II captures the effect of the common input from cell E1. Finally, term III represents the interaction of the indirect input from E1 to E2 through I with the input from E1 to I (See Figure 3C). A change in any single parameter may affect multiple terms. However, the individual contributions of all three terms are apparent. so that Equation (15) gives the following approximation to the matrix of cross-spectra Equation (15) gives the following approximation to the matrix of cross-spectra To illustrate the impact of synaptic properties on the cross- correlation between cells E2 and I we varied the inhibitory time constant, tI (See Figure 3B and C). Such a change is primarily reflected in the shape of the first order term, I: Multiplication by ~JE2I is equivalent to convolution with the inhibitory synaptic filter, JE2I. The shape of this filter is determined by tI (See Eq. (2)), and a shorter time constant leads to a tighter timing dependency between the spikes of the two cells [24,52–55]. In particular, Ostojic et al. made similar observations using a related approximation. In the FFI circuit, the first and second order terms, I and II, are dominant (red and dark orange, Figure 3B). The relative magnitude of the third order term, III (light orange, Figure 3B), is small. The next example shows that even in a simple recurrent circuit, terms of order higher than two may be significant. ~C ? PLoS Computational Biology \| www.ploscompbiol.org ~C?(v)~(I{~K(v)){1(~C0(v)z~A(v)~Cg(v)~A(v))(I{~K(v)){1:ð16Þ The relation between correlation structure and response statistics for two bidirectionally cou Figure 4. The relation between correlation structure and response statistics for two bidirectionally coupled, excitatory cells. (A) The cross-correlation between the two cells can be represented in terms of contributions from an infinite sequence of submotifs (See Eq. (20)). Though we show only a few ‘‘chain’’ motifs in one direction, one should note that there will also be contributions to the cross-correlation from chain motifs in the reverse direction in addition to indirect common input motifs (See the discussion of Figure 5). (B), (E) Linear response kernels in the excitable (B) and oscillatory (E) regimes. (C), (F) The cross-correlation function computed from simulations and theoretical predictions with first and third order contributions computed using Eq. (19) in the excitable (C) and oscillatory (F) regimes. (D), (G) The auto-correlation function computed from simulations and theoretical predictions with zeroth and second order contributions computed using Eq. (19) in the excitable (D) and oscillatory (G) regimes. In the oscillatory regime, higher order contributions were small relative to first order contributions and are therefore not shown. The network’s symmetry implies that cross-correlations are symmetric, and we only show them for positive times. Connection strengths were 40 mV:ms. The long window correlation coefficient r(?) between the two cells was &0:8 in the excitable regime and &0:5 in the oscillatory regime. The ISI CV was approximately 0.98 for neurons in the excitable regime and 0.31 for neurons in the oscillatory regime. doi:10.1371/journal.pcbi.1002408.g004 Figure 4. The relation between correlation structure and response statistics for two bidirectionally coupled, excitatory cells. (A) The cross-correlation between the two cells can be represented in terms of contributions from an infinite sequence of submotifs (See Eq. (20)). Though we show only a few ‘‘chain’’ motifs in one direction, one should note that there will also be contributions to the cross-correlation from chain motifs in the reverse direction in addition to indirect common input motifs (See the discussion of Figure 5). (B), (E) Linear response kernels in the excitable (B) and oscillatory (E) regimes. (C), (F) The cross-correlation function computed from simulations and theoretical predictions with first and third order contributions computed using Eq. (19) in the excitable (C) and oscillatory (F) regimes. (D), (G) The auto-correlation function computed from simulations and theoretical predictions with zeroth and second order contributions computed using Eq. March 2012 \| Volume 8 \| Issue 3 \| e1002408 ~C?(v)~(I{~K(v)){1(~C0(v)z~A(v)~Cg(v)~A(v))(I{~K(v)){1:ð16Þ ~ 1 j1{ ~KE1E2 ~KE2E1j2 (Iz~K) ~C0 E1 0 0 ~C0 E2 0 @ 1 A(Iz~K ) ~ 1 j1{ ~KE1E2 ~KE2E1j2 ~C0 E1zj ~KE1E2j2 ~C0 E2 ~K E2E1 ~C0 E1z ~KE1E2 ~C0 E2 KE2E1 ~C0 E1zK E1E2 ~C0 E2 ~C0 E2zjKE2E1j2 ~C0 E1 0 @ 1 A: ð19Þ ð19Þ In contrast to the previous example, this approximation does not terminate at finite order in interaction strength. After expanding, the cross-spectrum between cells E1 and E2 is approximated by More generally, the interaction matrices, K, of recurrent networks are not nilpotent. Consider two reciprocally coupled excitatory cells, E1 and E2 (See Figure 4A, left). In this case, PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 PLoS Computational Biology \| www.ploscompbiol.org 7 Impact of Network Structure on Spike Correlations Figure 4. The relation between correlation structure and response statistics for two bidirectionally coupled, excitatory cells. (A) The cross-correlation between the two cells can be represented in terms of contributions from an infinite sequence of submotifs (See Eq. (20)). Though we show only a few ‘‘chain’’ motifs in one direction, one should note that there will also be contributions to the cross-correlation from chain motifs in the reverse direction in addition to indirect common input motifs (See the discussion of Figure 5). (B), (E) Linear response kernels in the excitable (B) and oscillatory (E) regimes. (C), (F) The cross-correlation function computed from simulations and theoretical predictions with first and third order contributions computed using Eq. (19) in the excitable (C) and oscillatory (F) regimes. (D), (G) The auto-correlation function computed from simulations and theoretical predictions with zeroth and second order contributions computed using Eq. (19) in the excitable (D) and oscillatory (G) regimes. In the oscillatory regime, higher order contributions were small relative to first order contributions and are therefore not shown. The network’s symmetry implies that cross-correlations are symmetric, and we only show them for positive times. Connection strengths were 40 mV:ms. The long window correlation coefficient r(?) between the two cells was &0:8 in the excitable regime and &0:5 in the oscillatory regime. The ISI CV was approximately 0.98 for neurons in the excitable regime and 0.31 for neurons in the oscillatory regime. doi:10.1371/journal.pcbi.1002408.g004 p p Figure 4. ~C?(v)~(I{~K(v)){1(~C0(v)z~A(v)~Cg(v)~A(v))(I{~K(v)){1:ð16Þ After the cells are coupled, this oscillatory behavior is reflected in the cross- and auto-correlations where the dominant contributions are due to first and zeroth order terms, respectively (See Figures 4F,G). that only local interactions contribute to correlations. When this balance is broken, terms corresponding to longer paths through the network shape the cross-correlation functions. One conse- quence is that a relative increase in inhibition can lead to elevated network synchrony. We also show how to obtain tractable and accurate approximation of the average correlation structure in random networks. Orders of coupling interactions. It is often useful to expand Eq. (15) in terms of powers of ~K [31]. The term ~Kn ~C0(~K)m in the expansion is said to be of order nzm. Equivalently, in the expansion of ~C? ij , the order of a term refers to the sum of the powers of all constituent interaction kernels ~Kab. We can also associate a particular connectivity submotif with each term. In particular, nth order terms of the form A symmetric, all–to–all network of excitatory and inhibitory neurons. We begin with an all–to–all coupled network of N identical cells. Of these cells, NE make excitatory, and NI make inhibitory synaptic connections. The excitatory cells are assigned indices 1, . . . ,NE, and the inhibitory cells indices NEz1, . . . ,N. All excitatory (inhibitory) synapses have weight WE~ GE NE (WI~ GI NI ), and timescale tE (tI). The interaction matrix ~K may then be written in block form, ~Kian{1 ~Kan{1an{2 ~Ka1j ~C0 jj matrix ~K may then be written in block form, ~K~~A~J, where ~J~ ~JE1NENE ~JI1NENI ~JE1NI NE ~JI1NI NI ! : are associated with a directed path j?a1? ?an{2?an{1?i from cell j to cell i. Similarly, the term ~C0 ii ~K ia1 ~K an{2an{1 ~K an{1j corresponds to a n-step path from cell i to cell j. An (nzm)th order term of the form Here 1N1N2 is the N1\|N2 matrix of ones, ~JX is the weighted synaptic kernel for cells of class X (assumed identical within each ~Kian{1 ~Kan{1an{2 ~Ka1a0 ~C0 a0a0 ~K a0b1 ~K bm{2bm{1 ~K bm{1j class), and ~A is the susceptibility function for each cell in the network. ~C?(v)~(I{~K(v)){1(~C0(v)z~A(v)~Cg(v)~A(v))(I{~K(v)){1:ð16Þ (19) in the excitable (D) and oscillatory (G) regimes. In the oscillatory regime, higher order contributions were small relative to first order contributions and are therefore not shown. The network’s symmetry implies that cross-correlations are symmetric, and we only show them for positive times. Connection strengths were 40 mV:ms. The long window correlation coefficient r(?) between the two cells was &0:8 in the excitable regime and &0:5 in the oscillatory regime. The ISI CV was approximately 0.98 for neurons in the excitable regime and 0.31 for neurons in the oscillatory regime. doi:10.1371/journal.pcbi.1002408.g004 The contributions of different sub-motifs to the cross- and auto- correlations are shown in Figures 4C, D when the isolated cells are in a near-threshold excitable state (CV&0:98). The auto- correlations are significantly affected by network interactions. We also note that chains of length two and three (the second and third submotifs in Figure 4A) provide significant contributions. Earlier approximations do not capture such corrections [24]. ~C? E1E2~ X ? k,l~0 (~KE1E2 ~KE2E1)k(~K E1E2 ~K E2E1)l ( ~K E2E1 ~C0 E1z ~KE1E2 ~C0 E2): ð20Þ ð20Þ Directed paths beginning at E1 and ending at E2 (or vice-versa) are of odd length. Hence, this approximation contains only odd powers of the kernels ~KEiEj, each corresponding to a directed path from one cell to the other. Likewise, the approximate power spectra contain only even powers of the kernels corresponding to directed paths that connect a cell to itself (See Figure 4A). The operating point of a cell is set by its parameters (ti,EL,i, etc.) and the statistics of its input (Ei,si). A change in operating point can significantly change a cell’s response to an input. Using linear response theory, these changes are reflected in the response functions Ai, and the power spectra of the isolated cells, ~C0. To highlight the role that the operating point plays in the approximation of the correlation structure given by Eq. (15), we PLoS Computational Biology \| www.ploscompbiol.org PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 8 Impact of Network Structure on Spike Correlations elevated the mean and decreased the variance of background noise by increasing Ei and decreasing si in Eq. (1). With the chosen parameters the isolated cells are in a super-threshold, low noise regime and fire nearly periodically (CV&0:31). ~C?(v)~(I{~K(v)){1(~C0(v)z~A(v)~Cg(v)~A(v))(I{~K(v)){1:ð16Þ Although the effect of autaptic connections (those from a cell to itself) is negligible (See Figure S2 in Text S1), their inclusion significantly simplifies the resulting expressions. represents the effects of an indirect common input n steps removed from cell i and m steps removed from cell j. This corresponds to a submotif of the form i/an{1/ /a0?b1? ?bn{1?j consisting of two branches originating at cell a0. (See Figure 5, and also Figure 6A and the discussion around Eqs. (18,20).) We define ~mE~NE~JE,~mI~NI ~JI, and ~m~~mEz~mI. Using induction, we can show that ~Kk~~Ak~mk{1~J: Statistics of the response of large networks doi:10 1371/journal pcbi 1002408 g006 which allows us to calculate the powers ~Kk ~Kl when k,l=0, which allows us to calculate the powers ~Kk ~Kl when k,l=0, which allows us to calculate the powers ~Kk ~Kl when k,l=0, appear at any order. In other words, in the precisely balanced case only local interactions contribute to correlations. ~Kk ~Kl~~Ak(~A)l~mk{1(~m)l{1~mc1NN: To understand this cancelation intuitively, consider the contribution of directed chains originating at a given excitatory neuron, j. For tw0, the cross-correlation function, Cij(t), is determined by the change in firing rate of cell i at time t given a spike in cell j at time 0. By the symmetry of the all–to–all connectivity and stationarity, the firing of cell j has an equal probability of eliciting a spike in any excitatory or inhibitory cell in the network. Due to the precise synaptic balance, the postsynaptic current generated by the elicited spikes in the excitatory population will cancel the postsynaptic current due to elicited spikes in the inhibitory population on average. The contribution of other motifs cancel in a similar way. An application of Eq. (15) then gives an approximation to the matrix of cross-spectra: ~C ?~~C0 X ? k,l~0 ~K k~K l~ ~C0 ~A 1{~A~m ! ~Jz ~A 1{~A~m ! ~J z ~A 1{~A~m 2 ~mc1NNzIN " # ð21Þ ð21Þ In Figure 6B, we show the impact of breaking this excitatory- inhibitory balance on cross-correlation functions. We increased the strength and speed of the inhibitory synapses relative to excitatory synapses, while holding constant, for sake of compar- ison, the long window correlation coefficients r(?) between excitatory pairs (note that, by symmetry, all excitatory pairs should have the same correlation coefficient). Moreover, the degree of network synchrony, characterized by the short window correlation coefficients, is increased (See Figure 6B inset). Intuitively, a spike in one of the excitatory cells transiently increases the likelihood of spiking in all other cells in the network. Since inhibition in the network is stronger and faster than excitation, these additional spikes will transiently decrease the likelihood of spiking in twice removed cells. The cross-spectrum between two cells in the network is therefore given by The cross-spectrum between two cells in the network is therefore given by ½~C ? ij i[X,j[Y~ ~C0 ~A 1{~A~m ! PLoS Computational Biology \| www.ploscompbiol.org Statistics of the response of large networks The full power of the present approach becomes evident when analyzing the activity of larger networks. We again illustrate the theory using several examples. In networks where inhibition and excitation are tuned to be precisely balanced, the theory shows Direct matrix multiplication yields ~J~J~~mc1NN where ~mc~NED~JED2zNID~JID2, Figure 5. The motifs giving rise to terms in the expansion of Eq. (15). (A) Terms containing only unconjugated (or only conjugated) interaction kernels ~Kab correspond to directed chains. (B) Terms containing both unconjugated and conjugated interaction kernels ~Kab correspond to direct or indirect common input motifs. doi:10.1371/journal.pcbi.1002408.g005 Figure 5. The motifs giving rise to terms in the expansion of Eq. (15). (A) Terms containing only unconjugated (or only conjugated) interaction kernels ~Kab correspond to directed chains. (B) Terms containing both unconjugated and conjugated interaction kernels ~Kab correspond to direct or indirect common input motifs. doi:10.1371/journal.pcbi.1002408.g005 PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 9 Impact of Network Structure on Spike Correlations Figure 6. All–to–all networks and the importance of higher order motifs. (A) Some of the submotifs contributing to correlations in the all– to–all network. (B) Cross-correlations between two excitatory cells in an all–to-all network (NE~80,NI~20) obtained using Eq. (21) (Solid – precisely tuned network with ~m:0 [GE~{GI~140mVms,tE~tI~10ms], dashed – non-precisely tuned network with ~m=0 [GE~168mVms,GI~{210mVms, tE~10ms,tI~5ms]). (C) Comparison of first and second order contributions to the cross-correlation function in panel A in the precisely tuned (left) and non-precisely tuned (right) network. In both cases, the long window correlation coefficient r(?) was 0.05. doi:10.1371/journal.pcbi.1002408.g006 Figure 6. All–to–all networks and the importance of higher order motifs. (A) Some of the submotifs contributing to correlations in the all– to–all network. (B) Cross-correlations between two excitatory cells in an all–to-all network (NE~80,NI~20) obtained using Eq. (21) (Solid – precisely tuned network with ~m:0 [GE~{GI~140mVms,tE~tI~10ms], dashed – non-precisely tuned network with ~m=0 [GE~168mVms,GI~{210mVms, tE~10ms,tI~5ms]). (C) Comparison of first and second order contributions to the cross-correlation function in panel A in the precisely tuned (left) and non-precisely tuned (right) network. In both cases, the long window correlation coefficient r(?) was 0.05. Statistics of the response of large networks Additionally, the impact of direct common input to cells Ei and Ej on correlations is both larger in magnitude (because we increased the strength of both connection types) and sharper (the faster inhibitory time constant means common inhibitory inputs induce sharper correlations). These changes are reflected in the shape of the second order, common input term D~AD2~mc in Eq. (24) (see dotted orange lines in Figure 6C). neuronal networks is typically sparse, and connection probabilities can follow distinct rules depending on area and layer [57]. The present theory allows us to consider arbitrary architectures, as we now illustrate. We consider a randomly connected network of NE excitatory and NI inhibitory cells coupled with probability p. To simplify the analysis, every cell receives exactly pNE excitatory and pNI inhibitory inputs. Thus, having fixed in-degree (that is, the number of inputs is fixed and constant across cells), each cell receives an identical level of mean synaptic input. In addition, we continue to assume that cells are identical. Therefore, the response of each cell in the network is described by the same linear response kernel. The excitatory and inhibitory connection strengths are GE=(pNE) and GI=(pNI), respectively. The timescales of excitation and inhibition may differ, but are again identical for cells within each class. In sum, unbalancing excitatory and inhibitory connections via stronger, faster inhibitory synapses enhances synchrony, moving a greater proportion of the covariance mass closer to t~0 (See Figure 6B). To illustrate this effect in terms of underlying connectivity motifs, we show the contributions of length two chains and common input in both the precisely tuned and non- precisely tuned cases in Figure 6C. A similar approach would allow us to understand the impact of a wide range of changes in cellular or synaptic dynamics on the structure of correlations across networks. The approximation of network correlations (Eq. (15)) depends on the realization of the connectivity matrix. For a fixed realization, the underlying equations can be solved numerically to approximate the correlation structure (See Figure 7A). How- ever, the cross-correlation between a pair of cells of given types has a form which is easy to analyze when only leading order terms in 1=N are retained. Random, fixed in-degree networks of homogeneous excitatory and inhibitory neurons. Connectivity in cortical Specifically, the average cross-spectrum for two cells of given types is (See Section 1 in Text S1) Figure 7. Statistics of the response of large networks ~mY NY z ~A 1{~A~m ! ~m X NX z ~A 1{~A~m 2 ~mczdij " # , ð22Þ ~C0 ~A 1{~A~m ! ~mY NY z ~A 1{~A~m ! ~m X NX z ~A 1{~A~m 2 ~mczdij " # , ð22Þ ð22Þ where X,Y[fE,Ig. In Eq. (22) the first two terms represent the effects of all unidirectional chains originating at cell j and terminating at cell i, and vice versa. To see this, one should expand the denominators as power series in ~A~m. The third term represents the effects of direct and indirect common inputs to the two neurons, which can be seen by expanding this denominator as a product of power series in ~A~m and (~A~m). In Figure 6A, we highlight a few of these contributing motifs. Linear response theory allows us to confirm this heuristic observation, and quantify the impact of the imbalance on second order statistics. Expanding Eq. (22) for two excitatory cells to second order in coupling strength, we find Interestingly, when excitation and inhibition are tuned for precise balance (so that the mean excitatory and inhibitory synaptic currents cancel, and ~m~~mEz~mI~0). Using ~m~0 in Eq. (22) yields ~C? EiEj~~C0 ~A ~mE NE z~A ~m E NE zj~Aj2~mcz~A2~m ~mE NE z(~A)2~m ~m E NE zdij zO(jj~Kjj3): ð24Þ ½~C?i[X,j[Y~~C0 ~A ~mY NY z~A ~m X NX zD~AD2~mczdij : ð23Þ ð23Þ Compared to the balanced case, there is no longer a complete cancellation between contributions of chains involving excitatory and inhibitory cells, and the two underlined terms appear as a result (compare with Eq. (23)). These terms capture the effects of Effects of direct connections between the cells are captured by the first two terms, while those of direct common inputs to the pair are captured by the third term. Contributions from other paths do not PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 10 Impact of Network Structure on Spike Correlations all length two chains between cells Ei or Ej, starting at one and terminating at the other. The relative strengthening of inhibition implies that chains of length two provide a negative contribution to the cross-correlation function at short times (cf. [56], see the dashed orange lines in Figure 6C). PLoS Computational Biology \| www.ploscompbiol.org Discussion We have extended and further developed a general theoretical framework that can be used to describe the correlation structure in a network of spiking cells. The application of linear response theory allows us to find tractable approximations of cross- correlation functions in terms of the network architecture and single cell response properties. The approach was originally used to derive analytical approximations to auto- and cross-spectra in an all–to–all inhibitory network in order to study the population response of the electrosensory lateral line lobe of weakly electric fish [26]. The key approximation relies on the assumption that the activity of cells in the network can be represented by a mixed point and continuous stochastic process, as given in Eq. (9). This approximation may be viewed as a generalization of classic Linear-Poisson models of neural spiking: the crucial difference is the replacement of the stationary firing rate by a realization of an integrate-and-fire spiking process. This allows for the retention of the underlying IF spiking activity while additionally posing that neurons act as perfect linear filters of their inputs. An iterative construction then leads to the expressions for approximate cross- correlations between pairs of cells given by Eq. (15). Average correlations between cells in the random network conditioned on first order connectivity. As Figure 7B shows there is large variability around the mean excitatory-inhibitory cross-correlation function given by the leading order term of Eq. (25). Therefore, understanding the average cross-correlation between cells of given types does not necessarily provide much insight into the mechanisms that shape correlations on the level of individual cell pairs. Instead, we examine the average correlation between a pair of cells conditioned on their first order (direct) connectivity. We derive expressions for first order conditional averages correct to O(1=N2) (See Section 2 in Text S1). The average cross- spectrum for a pair of cells with indices i=j, conditioned on the value of the direct connections between them is The linear response framework of Lindner et al. [26] was extended by Marinazzo et al. [60] to somewhat more complex networks, and compared with other studies in which networks exhibit collective oscillations. In addition, other works [13,61,62] used linear response techniques to study information in the collective response of cells in a network. More recently, Ostojic et al. [24] obtained formulas for cross-correlations given in Eq. (9), which correspond to the first step in the iterative construction. Discussion Their approach captures corrections due to direct coupling (first order terms) and direct common input (second order terms involving second powers of interaction kernels; see also [49,63]). Our approach can be viewed as a generalization that also accounts for length two directed chains, along with all higher order corrections. As Figure 4 illustrates, these additional terms can be significant. The present approach also allows us to calculate corrected auto-correlations, in contrast with that of Ostojic et al. E ~C ? ij j~Jij,~Jji n o i[X,j[Y~ ~C0 ~A~Jijz~A~J jiz ~A2~m 1{~A~m ! ~mY NY z ~A2~m 1{~A~m ! ~m X NX z ~A 1{~A~m 2 ~mc " # zO(1=N2): ð26Þ E ~C ? ij j~Jij,~Jji n o i[X,j[Y~ ~C0 ~A~Jijz~A~J jiz ~A2~m 1{~A~m ! ~mY NY z ~A2~m 1{~A~m ! ~m X NX z ~A 1{~A~m 2 ~mc " # zO(1=N2): ð26Þ Here we set ~Jij~0 if we condition on the absence of a connection j?i, and ~Jij~~JY=p if we condition on its presence. The term ~Jji is set similarly. Although Eq. (26) appears significantly more complicated than the cell-type averages given in Eq. (25), they only differ in the underlined, first order terms. The magnitude of expected contributions from all higher order motifs is unchanged and coincides with those in the all–to–all network. Our work is also closely related to that of Pernice et al. [31], who analyzed the correlation structure in networks of interacting Hawkes processes [58,59]. Both studies represent correlations between cell pairs in terms of contributions of different connectivity motifs. However, our methods also differ: while their expressions are exact for Hawkes processes, Pernice et al. did not compare their results to those obtained using physiological models, and did not account for the response properties of individual cells (though it is possible that both can be achieved approximately by using appropriate kernels for the Hawkes processes). Moreover, for simplicity Pernice et al. examined only ‘‘total’’ spike count covariances, which are the integrals of the cross-correlation Figure 7C shows the mean cross-correlation function for mutually coupled excitatory-inhibitory pairs. Taking into account the mutual coupling significantly reduces variability (Compare with Figure 7B). To quantify this reduction, we calculate the mean reduction in variability when correlation functions are computed conditioned on the connectivity between the cells. Impact of Network Structure on Spike Correlations where T represents pairs of cells of a given type and connection (in the present example these are reciprocally coupled excitatory- inhibitory pairs), NT is the number of pairs of that type in the network, CCT T (t) is the leading order approximation of average correlations given only the type of cells in T (as in Eq. (25)), and CFOC T (t) the leading order approximation to average correlations conditioned on the first order connectivity of class T (as in Eq. (26)). We make use of the norm DD:DD2 defined by DDf DD2~ Ð Df D2 1=2. Figure 7D shows merror averaged over twenty networks. In particular, compare the reduction in variability when conditioning on bidirectional coupling between excitatory-inhibitory pairs shown in Figures 7B,C, with the corresponding relative error in Figure 7D (circled in red). E ~C ? ij n o i[X,j[Y~~C0 ~A 1{~A~m ! " ~mY NY z ~A 1{~A~m ! ~m X NX z ~A 1{~A~m 2 ~mc 3 5zO 1=N2 , ð25Þ ð25Þ when i=j. This shows that, to leading order in 1=N, the mean cross-spectrum between two cells in given classes equals that in the all–to–all network (see Eq. (22)). Therefore our previous discussion relating network architecture to the shape of cross-correlations in the all–to–all network extends to the average correlation structure in the random network for large N. Pernice et al. [31] derived similar expressions for the correlation functions in networks of interacting Hawkes processes [58,59], which are linear, self-exciting point processes with history-dependent intensities. They assumed that either the network is regular (i.e., both in- and out-degrees are fixed) or has a sufficiently narrow degree distribution. Our analysis depends on having fixed in- degrees, and we do not assume that networks are fully regular. Both approaches lead to results that hold approximately (for large enough N) when the in-degree is not fixed. Statistics of the response of large networks Correlations in random, fixed in-degree networks. (A) A comparison of numerically obtained excitatory-inhibitory cross-correlations to the approximation given by Eq. (26). (B) Mean and standard deviation for the distribution of correlation functions for excitatory-inhibitory pairs of cells. (Solid line – mean cross-correlation, shaded area – one standard deviation from the mean, calculated using bootstrapping in a single network realization). (C) Mean and standard deviation for the distribution of cross-correlation functions conditioned on cell type and first order connectivity for a reciprocally coupled excitatory-inhibitory pair of cells. (Solid line – mean cross-correlation function, shaded area – one standard deviation from the mean found by bootstrapping). (D) Average reduction in L2 error between cross-correlation functions and their respective first-order conditioned averages, relative to the error between the cross-correlations and their cell-type averages. Blue circles give results for a precisely tuned network, and red squares for a network with stronger, faster inhibition. Error bars indicate two standard errors above and below the mean. GE,GI,tE,tI for panels A-C are as in the precisely tuned network of Figure 6, and the two networks of panel D are as in the networks of the same figure. doi:10.1371/journal.pcbi.1002408.g007 Figure 7. Correlations in random, fixed in-degree networks. (A) A comparison of numerically obtained excitatory-inhibitory cross-correlations to the approximation given by Eq. (26). (B) Mean and standard deviation for the distribution of correlation functions for excitatory-inhibitory pairs of cells. (Solid line – mean cross-correlation, shaded area – one standard deviation from the mean, calculated using bootstrapping in a single network realization). (C) Mean and standard deviation for the distribution of cross-correlation functions conditioned on cell type and first order connectivity for a reciprocally coupled excitatory-inhibitory pair of cells. (Solid line – mean cross-correlation function, shaded area – one standard deviation from the mean found by bootstrapping). (D) Average reduction in L2 error between cross-correlation functions and their respective first-order conditioned averages, relative to the error between the cross-correlations and their cell-type averages. Blue circles give results for a precisely tuned network, and red squares for a network with stronger, faster inhibition. Error bars indicate two standard errors above and below the mean. GE,GI,tE,tI for panels A-C are as in the precisely tuned network of Figure 6, and the two networks of panel D are as in the networks of the same figure. PLoS Computational Biology \| www.ploscompbiol.org Statistics of the response of large networks doi:10.1371/journal.pcbi.1002408.g007 March 2012 \| Volume 8 \| Issue 3 \| e1002408 PLoS Computational Biology \| www.ploscompbiol.org 11 Impact of Network Structure on Spike Correlations Impact of Network Structure on Spike Correlations The linear response kernel and power spectrum for a general integrate and fire neuron model can be easily obtained [29]. In addition, it is also possible to obtain the rate, spectrum, and susceptibility for modulation of the mean conductance in the case of conductance-based (rather than current-based) synapses (See [67] and Section 3 in Text S1). As the linear response kernel is directly related to the spike triggered average [24,30], the proposed theoretical framework should be applicable even to actual neurons whose responses are characterized experimentally. The possibilities for future applications are numerous. For example, one open question is how well the theory can predict correlations in the presence of adaptive currents [67]. In addition, the description of correlations in terms of architecture and response properties suggests the possibility of addressing the difficult inverse problem of inferring architectural properties from correlations [22–24,64]. Ostojic et al. applied linear response methods to the latter problem. It is our hope that the present approach will prove a valuable tool in moving the computational neuroscience community towards a more complete understanding of the origin and impact of correlated activity in neuronal populations. It may be surprising that linear response theory can be used to provide corrections to cross-correlations of arbitrary order in network connectivity. The key to why this works lies in the accuracy of the linearization. A more accurate approximation could be obtained by including second and higher order corrections to the approximate response of a single cell, as well as corrections to the joint response. While including such terms is formally necessary to capture all contributions of a given order in network connectivity [32,33], the success of of linear response theory suggests that they are small for the cases at hand. In short, the present approximation neglects higher-order corrections to the approximate response of individual cells, along with all corrections involving joint responses, but accounts for paths through the network of arbitrary length. The possibilities for future applications are numerous. For example, one open question is how well the theory can predict correlations in the presence of adaptive currents [67]. In addition, the description of correlations in terms of architecture and response properties suggests the possibility of addressing the difficult inverse problem of inferring architectural properties from correlations [22–24,64]. Ostojic et al. applied linear response methods to the latter problem. Impact of Network Structure on Spike Correlations It is our hope that the present approach will prove a valuable tool in moving the computational neuroscience community towards a more complete understanding of the origin and impact of correlated activity in neuronal populations. As expected from the preceding discussion, simulations suggest that, for IF neurons, our approximations become less accurate as cells receive progressively stronger inputs. The physical reasons for this loss of accuracy could be related to interactions between the ‘‘hard threshold’’ and incoming synaptic inputs with short timescales. Additionally, while the theory will work for short synaptic timescales, it will improve for slower synaptic dynamics, limiting towards being essentially exact in the limit of arbitrarily long synaptic time constants (note the improvement in the approximation for the FFI circuit for the slower timescale exhibited in Figure 3). Another important factor is background noise, which is known to improve the accuracy of the linear description of single cell responses. We assume the presence of a white noise background, although it is possible to extend the present methods to colored background noise [25,65]. Discussion For a single network, the relative decrease in variability can be quantified using merror~ 1 NT X (i,j)[T i wj DDCij(t){CFOC T (t)DD2 DDCij(t){CCT T (t)DD2 , PLoS Computational Biology \| www.ploscompbiol.org March 2012 \| Volume 8 \| Issue 3 \| e1002408 12 Impact of Network Structure on Spike Correlations PLoS Computational Biology \| www.ploscompbiol.org Measures of spike time correlation We quantify dependencies between the responses of cells in the network using the spike train auto- and cross-correlation functions [39]. For a pair of spike trains, yi(t),yj(t), the cross-correlation function Cij(t) is defined as Impact of Network Structure on Spike Correlations functions. However, as they note, their approach can be extended to obtain the temporal structure of cross-correlations. Similarly, Toyoizumi et al. [64] derive expressions for cross-correlations in networks of interacting point process models in the Generalized Linear Model (GLM) class. These are very similar to Hawkes processes, but feature a static nonlinearity that shapes the spike emission rate. other studies. Ostojic and Brunel [66] examined this accuracy in the relatively simple case of a neuron receiving filtered Gaussian noise in addition to a white background. Chacron et al. [61] noted that linear response approaches applied to networks of perfect integrators begin to display significant errors at larger connection strengths. Marinazzo et al. [60] remarked on the errors induced by network effects in linear response approximations to correlations in a delayed feedback loop. In particular, these errors were attributed to network effects such as synchrony in the excitatory population. The authors noted that such activity can not be correctly modeled by a linear approach. To illustrate the power of the present linear response theory in analyzing the factors that shape correlations, we considered a number of simple examples for which the approximation given by Eq. (15) is tractable. We showed how the theory can be used both to gain intuition about the network and cell properties that shape correlations, and to quantify their impact. In particular, we explained how only local connections affect correlations in a precisely tuned all–to–all network, and how strengthening inhibition may synchronize spiking activity. In each case, we use comparisons with integrate-and-fire simulations to show that linear response theory makes highly accurate predictions. Although we have demonstrated the theory using networks of integrate–and–fire neurons, the approach is widely applicable. The linear response kernel and power spectrum for a general integrate and fire neuron model can be easily obtained [29]. In addition, it is also possible to obtain the rate, spectrum, and susceptibility for modulation of the mean conductance in the case of conductance-based (rather than current-based) synapses (See [67] and Section 3 in Text S1). As the linear response kernel is directly related to the spike triggered average [24,30], the proposed theoretical framework should be applicable even to actual neurons whose responses are characterized experimentally. Although we have demonstrated the theory using networks of integrate–and–fire neurons, the approach is widely applicable. 1. Cohen M, Kohn A (2011) Measuring and interpreting neuronal correlations. Nat Neurosci 14: 811–819. Acknowledgments The authors thank Robert Rosenbaum, Brent Doiron and Srdjan Ostojic for many helpful discussions. ~C0 ii(v; s2 i )z(se i )2D~Ai(v)D2 ? ~C0 ii(v; s2 i z(se i )2) ~C0 ii(v; s2 i )z(se i )2D~Ai(v)D2 ? ~C0 ii(v; s2 i z(se i )2) The response function A should be adjusted likewise. Convolution of matrices. Let X(t)~½Xij(t) and Y(t)~½Yij(t) be n1\|n2 and n2\|n3 matrices of functions, respectively. We define the convolution of matrices (X Y)(t) to be the n1\|n3 matrix of functions with entries defined by (X Y)ij(t)~ X k (Xik Ykj)(t): Calculation of stationary rates in a recurrent network. The stationary firing rate of an IF neuron can be computed as a function of the mean and intensity of internal noise (Ei,si) and other cellular parameters (ti,ELi, etc…) [69]. Denote the stationary firing rate of cell i in the network by ri, and by ri,0(E,s) the stationary firing rate in the presence of white noise with mean E and variance s2. We keep the dependencies on other parameters are implicit. The stationary rates, ri, in the recurrent network without external input are determined self-consistently by Expectations and convolutions commute for matrix convolutions as matrix expectations are taken entry-wise. Each entry of a matrix convolution is a linear combination of scalar convolutions which commute with expectations. Additionally, we adopt the convention that the zeroth power of the interaction matrix, K0 ij(t), is the diagonal matrix with K0 ij(t)~d(t) when i~j. Hence K0 ij(t) acts as the identity matrix under matrix convolution. Supporting Information ri~ri,0(Ei ’,si)~ri,0(Eiz X j Wijrj,si) i~1, . . . ,N , Text S1 Supplementary information file containing derivations and additional content, such as an exploration of the error of the theory. Supporting information figures were included in this file (and not separately). where we used E fi½ ~ P j WijE yj ~ P j Wijrj. This equality holds because the synaptic kernels, Jij, were normalized to have area Wij. These equations can typically be solved by fixed-point iteration. (PDF) Note that this provides an effective mean input, Ei’, to each cell, but does not give adjustments to the variance, si. We assume that the major impact of recurrent input is reflected in Ei’, and ignore corrections to the cell response involving higher order statistics of the input. This approach is valid as long as fluctuations in the recurrent input to each cell are small compared to si, and may break down otherwise [27]. Impact of Network Structure on Spike Correlations the statistics of the network response to inputs gi(t) of finite variance. As noted by [26], when inputs have infinite variance additional corrections are necessary. As a particular example, consider the case where the processes are correlated white noise, i.e., when gi(t)~ ﬃﬃﬃc p xc(t)z ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{c p xi(t), where xc,xi are independent white noise processes with variance se. Then each gi is also a white noise process with intensity se i , but E gi(tzt)gj(t) ~½dijd(t)z(1{dij)cd(t)se i . The firing rate of cell i in response to this input is ri~r0(Ei’, ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ (si)2z(se i )2 q ), and the point around which the response of the cell is linearized needs to be adjusted. cov Nyi(t,tzT),Nyj(t,tzT) ~ ðT {T Cij(t)(T{DtD)dt: We can interpret the cross-correlation as the conditional probability that cell i spikes at time tzt given that cell j spiked at time t. The conditional firing rate, Hij(t)~ lim Dt?0 1 Dt Pr Nyi(tzt,tztzDt)w0DNyj(t,tzDt)w0 , is the firing rate of cell i conditioned on a spike in cell j at t units of time in the past, and Cij(t)~rj(Hij(t){ri): Define the Fourier transform of a function f (t) as ~f (v)~F½f (t)(v): Ð ? {? f (t)e{2pivtdt: We will often make use of the cross-spectrum between the output of cells i,j, given by ~Cij(v)~E ~yi(v)~y j (v) h i , which is the Fourier transform of the cross-correlation function of cells i,j. The power spectrum ~Cii(v) is the cross-spectrum between a cell and itself, and is the Fourier transform of the auto-correlation function. is the firing rate of cell i conditioned on a spike in cell j at t units of time in the past, and Cij(t)~rj(Hij(t){ri): Finally, we may apply an additional correction to the linear response approximation of autocorrelations. For simplicity, we ignore coupling in Eq. (16) (so that ~K~0). Linear response predicts that ~Cii(v)~~C0 ii(v; s2 i )z(se i )2D~Ai(v)D2, where we have introduced explicit dependence on s2 i , the variance of white noise being received by an IF neuron with power spectrum ~C0 ii(v; s2 i ), in the absence of the external signal. The approximation may be improved in this case by making the following substitution in Eq. (16) [26,50]: Define the Fourier transform of a function f (t) as ~f (v)~F½f (t)(v): Ð ? {? Author Contributions Conceived and designed the experiments: JT YH ESB KJ. Performed the experiments: JT. Analyzed the data: JT. Contributed reagents/materials/ analysis tools: JT YH ESB KJ. Wrote the paper: JT YH ESB KJ. Conceived and designed the experiments: JT YH ESB KJ. Performed the experiments: JT. Analyzed the data: JT. Contributed reagents/materials/ analysis tools: JT YH ESB KJ. Wrote the paper: JT YH ESB KJ. Correction to statistics in the presence of an external white noise signals. Expression (16) can be used to compute 2. Ohki K, Chung S, Ch’ng Y, Kara P, Reid R (2005) Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433: 597–603. Cij(t)~cov yi(tzt),yj(t) : Cij(t)~cov yi(tzt),yj(t) : The auto-correlation function Cii(t) is the cross-correlation between a spike train and itself, and C(t) is the matrix of cross- correlation functions. Denoting by Nyi(t1,t2)~ Ð t2 t1 yi(s)ds the number of spikes over a time window ½t1,t2, the spike count correlation, rij(T), over windows of length t is defined as, We found that linear response theory remains applicable in a wide range of dynamical regimes, including relatively low noise, superthreshold regimes where cells exhibit strong oscillatory behavior. Moreover, the theory can yield accurate approximations of strong correlations due to coupling: for the bidirectionally coupled excitatory circuit of Figure 4, the approximate cross- correlations match numerically obtained results even when correlation coefficients are large (rE1E2(?)&0:8 in the excitable regime, &0:5 in the oscillatory regime). Additional discussion of the limits of applicability of linear response to the computation of correlations in networks can be found in the Supplementary Information. There, we show that the approximation is valid over a range of physiological values in the case of the all-to-all network, and that the theory gives accurate predictions in the presence of low firing rates (see Figures S3, S4 in Text S1). rij(T)~ cov Nyi(t,tzT),Nyj(t,tzT) ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ var Nyi(t,tzT) var Nyj(t,tzT) r : We assume stationarity of the spiking processes (that is, the network has reached a steady state) so that rij(T) does not depend on t. We also use the total correlation coefficient rij(?)~ limT?? rij(T) to characterize dependencies between the processes yi and yj over arbitrarily long timescales. The limits of linear response approximations of time-dependent firing activity and correlations have been tested in a number of The spike count covariance is related to the cross-correlation function by [7,68] March 2012 \| Volume 8 \| Issue 3 \| e1002408 March 2012 \| Volume 8 \| Issue 3 \| e1002408 13 Impact of Network Structure on Spike Correlations Impact of Network Structure on Spike Correlations f (t)e{2pivtdt: We will often make use of the cross-spectrum between the output of cells i,j, given by ~Cij(v)~E ~yi(v)~y j (v) h i , which is the Fourier transform of the cross-correlation function of cells i,j. The power spectrum ~Cii(v) is the cross-spectrum between a cell and itself, and is the Fourier transform of the auto-correlation function. Define the Fourier transform of a function f (t) as ~f (v)~F½f (t)(v): Ð ? {? f (t)e{2pivtdt: We will often make use of the cross-spectrum between the output of cells i,j, given by ~Cij(v)~E ~yi(v)~y j (v) h i , which is the Fourier transform of the cross-correlation function of cells i,j. The power spectrum ~Cii(v) is the cross-spectrum between a cell and itself, and is the Fourier transform of the auto-correlation function. Numerical methods. Simulations were run in C++, and the stochastic differential equations were integrated with a standard Euler method with a time-step of 0.01 ms. General parameter values were as follows: ti~20ms, EL,izEi~{54mV, si~ ﬃﬃﬃﬃﬃ 12 p mV, vth~ 20mV, vr~{54mV, tref ~2ms, VT~{52:5mV, DT~1:4mV, tE~10ms, tI~5ms, tD,i~1ms. Marginal statistics (firing rates, uncoupled power spectra and response functions) were obtained using the threshold integration method of [29] in MATLAB. 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https://openalex.org/W3087871339	https://www.research.ed.ac.uk/files/166993954/Global_assessment_of_C_reactive_protein_and_health_related_outcomes.pdf	English	null	Global assessment of C-reactive protein and health-related outcomes: an umbrella review of evidence from observational studies and Mendelian randomization studies	European journal of epidemiology	2,020	cc-by	16,833	an umbrella review of evidence from observational studies and Mendelian randomization studies Citation for published version: Markozannes, G, Koutsioumpa, C, Cividini, S, Monori, G, Tsilidis, KK, Kretsavos, N, Theodoratou, E, Gill, D, Ioannidis, JP & Tzoulaki, I 2020, 'Global assessment of C-reactive protein and health-related outcomes: an umbrella review of evidence from observational studies and Mendelian randomization studies', European Journal of Epidemiology. https://doi.org/10.1007/s10654-020-00681-w Citation for published version: Markozannes, G, Koutsioumpa, C, Cividini, S, Monori, G, Tsilidis, KK, Kretsavos, N, Theodoratou, E, Gill, D, Ioannidis, JP & Tzoulaki, I 2020, 'Global assessment of C-reactive protein and health-related outcomes: an umbrella review of evidence from observational studies and Mendelian randomization studies', European Journal of Epidemiology. https://doi.org/10.1007/s10654-020-00681-w Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: European Journal of Epidemiology General rights g Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Edinburgh Research Explorer Global assessment of C-reactive protein and health-related outcomes an umbrella review of evidence from observational studies and Mendelian randomization studies Global assessment of C‑reactive protein and health‑related outcomes: an umbrella review of evidence from observational studies and Mendelian randomization studies Georgios Markozannes1 · Charalampia Koutsioumpa1,2,3 · Sofia Cividini4 · Grace Monori5 · Konstantinos K. Tsilidis1,5 · Nikolaos Kretsavos1 · Evropi Theodoratou6,7 · Dipender Gill5 · John PA Ioannidis8,9,10,11,12 · Ioanna Tzoulaki1,5 Georgios Markozannes1 · Charalampia Koutsioumpa1,2,3 · Sofia Cividini4 · Grace Monori5 · Konstantinos K. Tsilidis1,5 · Nikolaos Kretsavos1 · Evropi Theodoratou6,7 · Dipender Gill5 · John PA Ioannidis8,9,10,11,12 · Ioanna Tzoulaki1,5 Received: 3 December 2019 / Accepted: 25 August 2020 © The Author(s) 2020 Take down policy Th U i i f Ed p y The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 European Journal of Epidemiology https://doi.org/10.1007/s10654-020-00681-w REVIEW REVIEW * Ioanna Tzoulaki i.tzoulaki@imperial.ac.uk Abstract C-reactive protein (CRP) has been studied extensively for association with a large number of non-infectious diseases and outcomes. We aimed to evaluate the breadth and validity of associations between CRP and non-infectious, chronic health outcomes and biomarkers. We conducted an umbrella review of systematic reviews and meta-analyses and a systematic review of Mendelian randomization (MR) studies. PubMed, Scopus, and Cochrane Database of Systematic Reviews were systematically searched from inception up to March 2019. Meta-analyses of observational studies and MR studies examin- ing associations between CRP and health outcomes were identified, excluding studies on the diagnostic value of CRP for infections. We found 113 meta-analytic comparisons of observational studies and 196 MR analyses, covering a wide range of outcomes. The overwhelming majority of the meta-analyses of observational studies reported a nominally statistically significant result (95/113, 84.1%); however, the majority of the meta-analyses displayed substantial heterogeneity (47.8%), small study effects (39.8%) or excess significance (41.6%). Only two outcomes, cardiovascular mortality and venous throm- boembolism, showed convincing evidence of association with CRP levels. When examining the MR literature, we found MR studies for 53/113 outcomes examined in the observational study meta-analyses but substantial support for a causal association with CRP was not observed for any phenotype. Despite the striking amount of research on CRP, convincing evidence for associations and causal effects is remarkably limited. Keywords Umbrella review · Meta-analysis · Systematic review · C-reactive protein · CRP · Mendelian randomization · Bias Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10654-020-00681-w) contains supplementary material, which is available to authorized users. 12 Meta-Research Innovation Center at Stanford (METRICS), Stanford, CA 94305, USA Abstract 7 Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 8 Department of Medicine, Stanford Prevention Research Center, Stanford University School of Medicine, Stanford, CA 94305, USA 9 Department of Health Research and Policy, Stanford University School of Medicine, Stanford, CA 94305, USA 10 Department of Biomedical Data Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA 11 Department of Statistics, Stanford University School of Humanities and Sciences, Stanford, CA 94305, USA 12 Meta-Research Innovation Center at Stanford (METRICS), Stanford, CA 94305, USA 7 Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 7 Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK * Ioanna Tzoulaki i.tzoulaki@imperial.ac.uk 1 Department of Hygiene and Epidemiology, University of Ioannina Medical School, 45110 Ioannina, Greece 2 Department of Neurobiology, Harvard Medical School, Boston, MA, USA 3 BBS Program, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA 4 Department of Biostatistics, University of Liverpool, Liverpool, UK 5 Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, UK 6 Centre for Global Health, Usher Institute, University of Edinburgh, Edinburgh, UK 1 Department of Hygiene and Epidemiology, University of Ioannina Medical School, 45110 Ioannina, Greece 8 Department of Medicine, Stanford Prevention Research Center, Stanford University School of Medicine, Stanford, CA 94305, USA 2 Department of Neurobiology, Harvard Medical School, Boston, MA, USA 9 Department of Health Research and Policy, Stanford University School of Medicine, Stanford, CA 94305, USA 3 BBS Program, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA 10 Department of Biomedical Data Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA 4 Department of Biostatistics, University of Liverpool, Liverpool, UK 11 Department of Statistics, Stanford University School of Humanities and Sciences, Stanford, CA 94305, USA 5 Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, UK 12 Meta-Research Innovation Center at Stanford (METRICS), Stanford, CA 94305, USA 6 Centre for Global Health, Usher Institute, University of Edinburgh, Edinburgh, UK (0123 1 3456789) 3 G. Markozannes et al. meta-analyses of observational studies), without quantitative synthesis of effect sizes, and studies where CRP concentra- tions were the outcome. Also, due to the well-known role of CRP in infectious disease diagnosis, articles which investi- gated infections as the outcome of interest were excluded. Data sources and searches of observational studies We systematically searched PubMed, Scopus, and Cochrane Database of Systematic Reviews, from inception to 31 March 2019, for meta-analyses of observational studies examining the association of CRP with any health outcome (see search algorithms in Additional file 1: Appendix Table 1). All iden- tified publications went through a three-step parallel review of title, abstract, and full text (performed by CK, GMa, SC, NK) based on predefined inclusion and exclusion criteria. For meta-analyses of observational studies, we estimated the summary effects obtained from the random-effects method [11, 12] for which we also estimated the 95% prediction intervals to indicate the possible interval that could include the effect size of a new study examining the same associa- tion and describe the uncertainty of the summary effect size [13]. The heterogeneity between studies was assessed using the I2 metric, which has a range between 0 and 100%. It is calculated as the ratio of the variance between-studies over the sum of the variances between and within studies [14]. Values exceeding 50% or 75% are considered to represent large or very large heterogeneity, respectively. Small study effects were assessed with the use of the Egger’s regression asymmetry test [15]. A P ≤ 0.10 combined with a more con- servative effect in the largest study than in random-effects meta-analysis was judged to provide evidence for small- study effects. Abstract We also excluded meta-analyses using only cross-sectional assessments, meta-analyses of only crude (unadjusted) esti- mates, and associations reported as correlation coefficients. Where more than one article with overlapping outcomes was retrieved, the article with the meta-analysis of only prospec- tive studies, the most comprehensive meta-analysis (the one including the largest number of studies), or the more recently published one was included in the final analysis (in order of preference). Introduction C-reactive protein (CRP) is one of the most widely used biomarkers in clinical practice. First identified in 1930 [1], this acute phase reactant was initially used as a biomarker for infection [2]. The advent of high-sensitivity CRP measure- ment in the 1990s, alongside experimental and clinical evi- dence suggesting a potential role of inflammation in cardio- vascular disease a few years later [3, 4], increased research interest in CRP. It has since been examined as a potential risk factor for an ever-expanding list of diseases including different cardiovascular outcomes, cancers, metabolic and skeletal diseases and autoimmune diseases [5–9]. Today, despite intensive research efforts, the role of CRP in the etiology of common diseases remains unclear. Three independent investigators (CK, GMa and SC) extracted the data, which were checked by a second inves- tigator (IT, ET) and in case of discrepancies consensus was reached. From each eligible meta-analysis, we extracted information on the first author, journal and year of pub- lication, examined risk factors and the number of studies considered, type of metric reported (hazard ratio, risk ratio, odds ratio [OR], in order of preference), maximally adjusted effect sizes and 95% confidence intervals (CIs), number of total studies included, design of the original studies, unit of comparison, number of cases and population. When the number of cases or controls for individual studies was not reported, we abstracted them from the original studies when possible. When CRP was examined in more than one level of comparison (e.g. as a continuous biomarker and by tertiles), we extracted the data for the comparison having the largest number of component studies. Umbrella review is a systematic overview of systematic reviews and meta-analyses that assesses the evidence from the current literature in a field of research [10]. We aimed to systematically summarize and evaluate the breadth and validity of associations between CRP and health outcomes using the umbrella review methodology. We summarized meta-analyses of observational studies, examined the extent of phenotypic associations with CRP, and evaluated the strength of associations and bias in these identified associa- tions. At the same time, we performed a systematic review of Mendelian randomization (MR) studies considering CRP levels as the exposure, to assess the evidence for causality stemming from this literature. Study selection and data extraction of observational studies We included systematic reviews and meta-analyses of observational studies that examined associations between CRP levels and health outcomes that had identified at least three studies per outcome examined, keeping only articles that were full publications and in the English language. We excluded studies without systematic literature searches (for 1 3 3 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… We further applied the excess statistical significance test, which evaluates whether there is a relative excess of formally significant findings in the published literature due to any reason (e.g., publication bias, selective reporting of outcomes or analyses) [16]. It is a Chi square-based test that assesses whether the observed number of studies with nominally significant results is larger than their expected number. We used the effect size of the largest study (smallest standard error) in each meta-analysis to calculate the power of each study using a non-central t distribution. Excess sta- tistical significance was claimed at two-sided P ≤ 0.10 with observed > expected as previously proposed [16, 17]. excluding infections. The titles, abstracts, and full texts of the resulting papers were examined in detail by two authors (GMa and IT), and discrepancies were resolved by consen- sus. From each eligible MR study, two authors (GMo and GMa) extracted data in relation to first author, journal and year of publication, the study cohort/s, sample size, num- ber of cases (as applicable), type of data used (individual participant or summary level), the instrumental variables (single-nucleotide polymorphisms [SNPs]), the instrument selection approach, population ancestry, SNP exclusion cri- teria, % variance explained by the instruments, the outcome phenotypes, the MR effect estimate and the corresponding CIs. When we observed a nominally significant association (P < 0.05) in the main MR analysis, we further extracted and evaluated all information on sensitivity MR analyses. Evidence grading of Mendelian randomization studies We classified the evidence of the associations that had P < 0.05 as strong, highly suggestive, suggestive, and weak based on a set of previously used criteria whose rationale has been described elsewhere in detail [10, 18–20]. In brief, these criteria try to consider the level of statistical signifi- cance, amount of evidence, consistency, and lack of signals of bias. Thus, we classified as strong evidence those associa- tions that had significance P < 1×10−6 based on the random effects model, more than 1000 cases, the I2 metric was less than 50%, there was no evidence of small study effects, the prediction interval did not include the null value, and there was no evidence for excess significance bias. Associations were classified as highly suggestive when P < 1×10−6 based on the random-effects model, more than 1000 cases, and the P value of the largest study in the meta-analysis was < 0.05. The associations with P < 0.001, and more than 1000 cases were considered as suggestive. Finally, associations were considered as weak when P < 0.05 on the random effects model. We stratified MR analyses into those using instrumental variables which included only variants located in the CRP gene and those using instrumental variables with SNPs that were significantly associated with CRP levels from through- out the genome (i.e., not restricted to the CRP gene). The latter approach for selecting instruments is more likely to incorporate invalid instruments that have pleiotropic effects [22]. Indeed, a genome-wide association study (GWAS) of CRP has revealed a large number of genetic variants, which were not specific to CRP, but influence other inflammatory cytokines including interleukin-6 receptor (IL-6R) and inter- leukin 1 family member 10 (ILF10) [23]. For MR analyses restricted to variants located in the CRP gene, we consid- ered MR evidence as ‘potentially supportive’ when the main analysis reported a P < 0.01 [20] and there was consistent evidence from sensitivity analyses; ‘limited/inconsistent evi- dence’ when there was 0.01 < P < 0.05 or P < 0.01 without further support from sensitivity analysis, and ‘not present’ when P > 0.05. For MR analyses with variants throughout the genome for CRP, we considered as ‘limited/inconsistent evidence’ when there was P < 0.05 and further support from sensitivity analysis, and ‘not present’ otherwise. Some meta-analyses used estimates from studies with different study designs. Evidence grading of Mendelian randomization studies Due to the inherent limitations of cross-sectional and case–control studies to examine temporal associations, we performed a sensitivity analysis by exclud- ing cross-sectional and case–control studies. Finally, for each association in the strong and highly sug- gestive category, we reassessed the evidence after examin- ing each meta-analysis in depth by assessing the eligibility of the included studies as well as verifying the data used in the meta-analysis using AMSTAR (A MeaSurement Tool to Assess systematic Reviews) [21]. Data sources and searches, study selection and data extraction of Mendelian randomization studies Our literature search yielded 4100 eligible articles. Fol- lowing title review, 863 articles were considered eligible (Fig. 1), and after abstract screening, 552 articles were potentially eligible for full text review. Finally, 55 studies [5, 24–77] including 113 comparisons of different outcomes We used the search algorithm (See Additional file 1: Appen- dix Table 1) to identify MR studies evaluating potential causal association between CRP levels and health outcomes, 1 3 G. Markozannes et al. were included in the umbrella review of observational studies, consisting of 952 primary estimates. To facilitate interpretation, the different outcomes were classified into the following groups: cancer-related (52 outcomes), car- diovascular-related (31 outcomes), kidney-related (7 out- ) k l t l (6 t ) l i l (3 t ) pregnancy-related (2 outcomes), respiratory-related (2 out- comes), and other (10 outcomes). The majority of the primary studies were cohorts (N = 823; 86.5%, of which 497 were prospective, 264 retro- spective, and 62 of unclear design), followed by case–control t di (N 115 12 1%) Oth t d d i i t d f Fig. 1 Flowchart of study selection for a umbrella review and b Mendelian Randomization review G. Markozannes et al. Fig. 1 Flowchart of study selection for a umbrella review and b Mendelian Randomization review Fig. 1 Flowchart of study selection for a umbrella review and b Mendelian Randomization review pregnancy-related (2 outcomes), respiratory-related (2 out- comes), and other (10 outcomes). were included in the umbrella review of observational studies, consisting of 952 primary estimates. To facilitate interpretation, the different outcomes were classified into the following groups: cancer-related (52 outcomes), car- diovascular-related (31 outcomes), kidney-related (7 out- comes), skeletal (6 outcomes), neurological (3 outcomes), The majority of the primary studies were cohorts (N = 823; 86.5%, of which 497 were prospective, 264 retro- spective, and 62 of unclear design), followed by case–control studies (N = 115; 12.1%). Other study designs consisted of 1 3 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… cross-sectional studies (N = 6; 0.6%), case-cohorts (N = 7; 0.7%), and one case-crossover study (0.1%). study quality (9 of the 11 papers) based on an AMSTAR score between 4 and 7, and only one had a score of 8. Finally, one study [41] was a pooled analysis and therefore it could not be evaluated based on the AMSTAR tool (Additional file 1: Appendix Table 3). Data sources and searches, study selection and data extraction of Mendelian randomization studies Ninety-five out of 113 associations (84.1%) presented a statistically significant effect at P < 0.05 under the ran- dom-effects model, 67 remained significant at P < 0.001, whereas 34 associations had a statistically significant effect at P < 1×10−6 (Table 1). However, only 24 (21.2%) associa- tions had a 95% prediction interval that excluded the null. The largest study was statistically significant in 71 of the 113 comparisons (62.8%) and was more conservative than the meta-analysis estimate in 87 of 113 comparisons (77%) (Table 1). Twenty-three associations (20.4%) presented very large between-study heterogeneity (I2 > 75%), and 31 (27.4%) associations had large heterogeneity estimates (I2 > 50% and I2 < 75%). In 45 (39.8%) of the 113 associa- tions the Egger’s test was statistically significant (P < 0.1) and the random effects estimate was inflated compared to the largest study (Table 1). Forty-seven associations (41.6%) showed evidence of excess significance, meaning that the number of observed studies with statistically significant results exceeded the number of expected ones (Table 1). CRP levels and health outcomes reported in Mendelian randomization studies A total of 196 primary MR analyses were identified from 37 studies [79–115] covering 82 distinct phenotypes (Table 2 and Additional file 1: Appendix Tables 4, 5). The majority of associations were investigated through two-sample MR methodologies (130 out of 196; 66%). The median number of participants included in MR studies was 26 405 (range 134 to 184 305). The most frequently examined phenotypes included cardiovascular diseases (coronary heart disease and stroke) (n = 19; 9.7%), type 2 diabetes (n = 8; 4.1%), schizo- phrenia (n = 8; 4.1%), and body mass index (BMI) (n = 6; 3.1%). Eighty-four MR analyses (60 unique outcomes, Table 2) used instrument variants at the CRP gene locus, and 112 used instruments from throughout the genome The SNPs used as instruments varied vastly among studies. The four most commonly used SNPs among the 196 MR associa- tions were rs1130864 (n = 78; 39.8% of the comparisons), rs1205 (n = 74; 37.8%), rs2794520 (n = 74; 37.8%), and rs3093077 (n = 65; 33.2%); all these variants fall within or close the CRP gene region. Assessment of epidemiological credibility Of the 113 associations, only two cardiovascular outcomes (cardiovascular mortality and venous thromboembolism) fulfilled the necessary criteria to be categorized in the strong level of evidence (Table 1). Ten (8.9%) associations were supported by highly suggestive evidence, 6 of which were on cardiometabolic outcomes. The highly suggestive associations were all-cause mortality in general population, all-cause mortality in patients with chronic kidney disease, long-term mortality in chronic obstructive pulmonary dis- ease (COPD) patients, long-term mortality or CVD in acute coronary syndrome (ACS)/unstable coronary heart disease (CHD)/angina patients, mortality or CVD in stable coronary artery disease (CAD) patients, CHD in general population, overall survival in hepatocellular carcinoma patients, Bath Ankylosing Spondylitis Disease Activity Index-50 (BAS- DAI50) in ankylosing spondylitis patients, ovarian cancer in general population, and type 2 diabetes in general popula- tion. There were 16 comparisons that were categorized in the suggestive level of evidence and 67 in the weak level. Finally, 18 comparisons did not present a statistically sig- nificant association. When we excluded the case–control or cross-sectional studies, only seven comparisons were affected. Only six of those comparisons had at least 3 remaining studies in order to be re-evaluated and for all six the evidence categorization remained the same (Additional file 1: Appendix Table 2). Overall, 12 distinct phenotypes presented significant associations at a P < 0.01, of which four (Crohn’s disease, ischemic heart disease, systolic and diastolic blood pres- sure) presented significant associations (P < 0.01) when the instruments were restricted to CRP gene locus (Appendix Tables 4 and 5). However, independent MR analyses did not show consistent evidence for Crohn’s disease and ischemic heart disease, and none of the aforementioned phenotypes had support from sensitivity analyses.i Nine phenotypes presented significant (P < 0.01) causal effect estimates when instruments from throughout the genome were considered and of those, only schizophrenia and bipolar disorder presented consistent evidence in sen- sitivity analyses and in analysis restricted to SNPs within CRP locus, but only at P < 0.05. Nonetheless, the result on bipolar disorder [113] was not confirmed by an earlier study [107] where MR using only CRP gene SNPs did not reach statistical significance at P < 0.05. Schizophrenia had evidence from independent studies and sensitivity analysis (weighted median and inverse variant weighted estimate), but this was not supported by MR Egger analysis and the sensitivity analysis using only CRP gene SNPs (P = 0.04). Assessment of epidemiological credibility Overall, only 14 outcomes had evidence available from both MR analyses and meta-analyses of observational studies (Table 3). The evidence between the observational i When we assessed the meta-analyses in either the strong or the highly suggestive evidence category, we observed that the majority of the meta-analysis papers were on moderate 1 3 1 3 G. Markozannes et al. G. Markozannes et al. 1 3 Table 1 Health outcomes and assessment of evidence in meta-analyses of observational studies References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Cancer-related outcomes Zheng et al. [69] High vs Low Hepatocel- lular carcinoma Overall survival HR 11 1071/1885 2.15 (1.76, 2.63) 1.0E − 13 1.80 (1.30, 2.30) 1.36, 3.39 27 (0, 64) 0.171 9/4.49 0.010 Highly sugges- tive Zeng et al. [67] High vs Low General population (women) Ovarian cancer RR 7 2011/33288 1.91 (1.51, 2.41) 5.0E − 08 1.67 (1.03, 2.70) 1.41, 2.59 0 (0, 58) 0.015d 4/6.14 NP Highly sugges- tive Liao et al. [48] High vs Low Non-small cell lung carcinoma Overall survival HR 14 1342/2491 1.63 (1.36, 1.94) 1.0E − 07 1.03 (1.00, 1.06) 0.88, 3.01 90 (85, 93) 0.002d 11/2.61 2.0E − 06 Suggestive Guo et al. [32] per unit lnCRP Breast cancer Overall survival HR 13 3180/15112 1.28 (1.13, 1.44) 5.9E − 05 1.03 (1.00, 1.06) 0.89, 1.83 77 (58, 85) 0.004d 6/0.71 3.3E − 05 Suggestive Li et al. [20] High vs Low General population Cancer mortality RR 8 4748/55720 1.26 (1.11, 1.42) 1.9E − 04 1.28 (1.11, 1.48) 1.00, 1.58 17 (0, 63) 0.505 3/5.33 NP Suggestive Guo et al. [32] per unit lnCRP General population Lung cancer HR 7 1045/127867 1.34 (1.15, 1.57) 2.3E − 04 1.51 (1.21, 1.88) 0.89, 2.03 45 (0, 75) 0.600 4/3.80 1.000 Suggestive Guo et al. [33] per unit lnCRP Breast cancer Cancer- specific survival HR 7 1320/12932 1.38 (1.15, 1.66) 6.5E − 04 1.16 (1.02, 1.32) 0.86, 2.20 51 (0, 77) 0.095d 4/1.22 0.021 Suggestive Wang et al. [59] High vs Low Renal cell carcinoma, receiving tyrosine kinase inhibitors Overall survival HR 8 490/1158 2.83 (2.26, 3.56) 2.5E − 19 3.17 (2.20, 4.68) 1.90, 4.22 12 (0, 61) 0.226 6/7.11 NP Weak Yu et al. Assessment of epidemiological credibility [66] High vs Low Gastric cancer Overall survival HR 12 771/2597 1.77 (1.56, 2.00) 3.9E − 19 1.54 (1.25, 1.92) 1.38, 2.27 19 (0, 59) 0.207 9/4.54 0.013 Weak Hu et al. [38] High vs Low Metastatic renal cell carcinoma Overall survival HR 5 487/729 2.56 (2.05, 3.19) 1.2E − 16 2.10 (1.50, 3.00) 1.78, 3.67 0 (0, 64) 0.795 5/3.08 0.164 Weak Fang et al. [30] High vs Low Nasopharyn- geal carci- noma Overall survival HR 5 439/3691 1.84 (1.57, 2.17) 1.9E − 13 1.82 (1.47, 2.25) 1.41, 2.40 0 (0, 64) 0.168 5/3.11 0.164 Weak Dai et al. [29] High vs Low Renal cell carcinoma Overall survival HR 12 865/2305 2.51 (1.93, 3.26) 4.7E − 12 1.20 (1.15, 1.26) 1.06, 5.95 93 (90, 95) 0.002d 11/3.85 3.1E − 05 Weak Fang et al. [30] High vs Low Nasopharyn- geal carci- noma Distant metastasis- free survival HR 3 449/3513 1.81 (1.52, 2.14) 1.0E − 11 1.71 (1.38, 2.13) 0.60, 5.47 0 (0, 73) 0.061d 3/2.35 1.000 Weak 1 3 Table 1 Health outcomes and assessment of evidence in meta-analyses of observational studies References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Cancer-related outcomes Zheng et al. [69] High vs Low Hepatocel- lular carcinoma Overall survival HR 11 1071/1885 2.15 (1.76, 2.63) 1.0E − 13 1.80 (1.30, 2.30) 1.36, 3.39 27 (0, 64) 0.171 9/4.49 0.010 Highly sugges- tive Zeng et al. [67] High vs Low General population (women) Ovarian cancer RR 7 2011/33288 1.91 (1.51, 2.41) 5.0E − 08 1.67 (1.03, 2.70) 1.41, 2.59 0 (0, 58) 0.015d 4/6.14 NP Highly sugges- tive Liao et al. [48] High vs Low Non-small cell lung carcinoma Overall survival HR 14 1342/2491 1.63 (1.36, 1.94) 1.0E − 07 1.03 (1.00, 1.06) 0.88, 3.01 90 (85, 93) 0.002d 11/2.61 2.0E − 06 Suggestive Guo et al. [32] per unit lnCRP Breast cancer Overall survival HR 13 3180/15112 1.28 (1.13, 1.44) 5.9E − 05 1.03 (1.00, 1.06) 0.89, 1.83 77 (58, 85) 0.004d 6/0.71 3.3E − 05 Suggestive Li et al. [20] High vs Low General population Cancer mortality RR 8 4748/55720 1.26 (1.11, 1.42) 1.9E − 04 1.28 (1.11, 1.48) 1.00, 1.58 17 (0, 63) 0.505 3/5.33 NP Suggestive Guo et al. Assessment of epidemiological credibility [32] per unit lnCRP General population Lung cancer HR 7 1045/127867 1.34 (1.15, 1.57) 2.3E − 04 1.51 (1.21, 1.88) 0.89, 2.03 45 (0, 75) 0.600 4/3.80 1.000 Suggestive Guo et al. [33] per unit lnCRP Breast cancer Cancer- specific survival HR 7 1320/12932 1.38 (1.15, 1.66) 6.5E − 04 1.16 (1.02, 1.32) 0.86, 2.20 51 (0, 77) 0.095d 4/1.22 0.021 Suggestive Wang et al. [59] High vs Low Renal cell carcinoma, receiving tyrosine kinase Overall survival HR 8 490/1158 2.83 (2.26, 3.56) 2.5E − 19 3.17 (2.20, 4.68) 1.90, 4.22 12 (0, 61) 0.226 6/7.11 NP Weak 1 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… p References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Zheng et al. [69] High vs Low Hepatocel- lular carcinoma TNM stage HR 3 185/689 3.23 (2.29, 4.56) 2.7E − 11 3.29 (2.22, 4.88) 0.34, 30.25 0 (0, 73) 0.808 2/2.39 NP Weak Hu et al. [38] High vs Low Localised renal cell carcinoma Progression- free survival HR 4 233/881 3.27 (2.25, 4.77) 6.9E − 10 3.26 (1.79, 6.53) 1.43, 7.49 0 (0, 68) 0.721 4/3.58 1.000 Weak Woo et al. [61] High vs Low Colorectal cancer Cancer- specific survival HR 3 126/579 4.37 (2.63, 7.26) 1.3E − 08 4.90 (2.33, 10.31) 0.16, 117.79 0 (0, 73) 0.594 3/2.99 1.000 Weak Dai et al. [29] High vs Low Renal cell carcinoma Cancer- specific survival HR 12 783/2843 3.52 (2.18, 5.69) 2.7E − 07 1.23 (1.17, 1.30) 0.63, 19.74 92 (88, 94) 1.4E − 04d 12/3.19 1.3E − 07 Weak Dai et al. [29] High vs Low Upper uri- nary tract and bladder cancer Overall survival HR 3 278/408 1.63 (1.33, 1.99) 2.7E − 06 1.56 (1.18, 2.06) 0.44, 6.08 0 (0, 73) 0.043d 3/0.60 0.008 Weak Luo et al. [50] High vs Low Urothelial bladder cancer Cancer- specific survival HR 4 373/1495 1.64 (1.32, 2.03) 7.3E − 06 1.96 (1.42, 2.69) 0.86, 3.12 21 (0, 74) 0.937 2/3.21 NP Weak Dai et al. Assessment of epidemiological credibility [29] High vs Low Upper uri- nary tract and bladder cancer Cancer- specific survival HR 8 411/1384 1.81 (1.39, 2.36) 1.3E − 05 1.20 (1.10, 1.30) 0.83, 3.97 73 (31, 85) 3.0E − 04d 8/0.72 4.5E − 09 Weak Hu et al. [38] High vs Low Localised renal cell carcinoma Cancer- specific survival HR 3 102/759 3.40 (1.95, 5.92) 1.6E − 05 3.87 (1.70, 8.82) 0.09, 124.54 0 (0, 73) 0.571 2/2.69 NP Weak Liu et al. [40] per unit lnCRP Prostate cancer Progression- free survival HR 3 54/316 1.50 (1.25, 1.81) 1.9E − 05 1.44 (1.17, 1.77) 0.45, 5.07 0 (0, 73) 0.568 2/2.18 NP Weak Woo et al. [61] High vs Low Colorectal cancer Overall survival HR 4 184/778 2.04 (1.45, 2.86) 4.0E − 05 1.88 (1.10, 3.20) 0.97, 4.29 0 (0, 68) 0.290 3/1.99 0.372 Weak Zheng [69] High vs Low Hepatocel- lular carcinoma Tumor vascular invasion HR 5 256/915 3.05 (1.78, 5.23) 4.7E − 05 4.11 (2.58, 6.53) 0.66, 14.22 44 (0, 78) 0.494 3/4.32 NP Weak Wang et al. [114] High vs Low Renal cell carcinoma, receiving tyrosine kinase Progression- free survival HR 4 81/449 2.35 (1.53, 3.63) 1.0E − 04 2.48 (1.74, 3.59) 0.60, 9.18 23 (0, 75) 0.999 3/1.91 0.355 Weak 1 3 G. Markozannes et al. 3 References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Liu et al. [49] High vs Low Prostate cancer Cancer- specific survival HR 4 162/822 1.91 (1.36, 2.69) 2.2E − 04 1.48 (0.83, 2.66) 0.89, 4.09 1 (0, 68) 0.020d 2/1.75 1.000 Weak Rocha et al. [54] High vs Low Metastatic prostate cancer Overall survival HR 6 432/659 1.42 (1.18, 1.72) 2.8E − 04 1.11 (1.02, 1.20) 0.81, 2.51 72 (8, 86) 0.006d 5/0.35 3.6E − 06 Weak Dai et al. [29] High vs Low Renal cell carcinoma Recurrence- free survival HR 8 189/1485 3.09 (1.66, 5.74) 3.8E − 04 1.23 (1.14, 1.33) 0.39, 24.50 89 (82, 93) 0.012d 7/2.52 0.002 Weak Huang et al. [39] High vs Low Esophageal cancer Overall survival HR 8 683/1329 2.00 (1.36, 2.94) 4.0E − 04 1.18 (1.03, 1.36) 0.59, 6.83 81 (60, 89) 0.027d 6/0.64 6.1E − 06 Weak Zheng et al. Assessment of epidemiological credibility [69] High vs Low Hepatocel- lular carcinoma Recurrence- free survival HR 3 245/445 2.66 (1.54, 4.58) 4.3E − 04 3.05 (1.68, 5.52) 0.02, 410.95 34 (0, 81) 0.799 2/2.44 NP Weak Zhou et al. [70] per unit lnCRP General population Colorectal cancer RR 18 4779/152418 1.12 (1.05, 1.21) 1.3E − 03 1.06 (0.99, 1.13) 0.90, 1.41 52 (4, 71) 0.069d 6/1.28 0.001 Weak Zhou et al. [70] per unit lnCRP General population Colon cancer RR 13 9715/153763 1.12 (1.05, 1.21) 1.4E − 03 1.00 (0.92, 1.07) 0.94, 1.34 38 (0, 66) 0.052d 4/0.65 0.003 Weak Liu et al. [49] High vs Low Prostate cancer Overall survival HR 4 273/471 1.38 (1.13, 1.68) 0.002 1.11 (1.02, 1.20) 0.57, 3.31 82 (35, 91) 0.038d 4/0.24 1.2E − 05 Weak Dai et al. [29] High vs Low Upper uri- nary tract and bladder cancer Recurrence- free survival HR 3 266/727 1.62 (1.20, 2.19) 0.002 1.45 (1.06, 1.99) 0.10, 27.49 36 (0, 81) 0.133 3/0.88 0.025 Weak Guo et al. [33] per unit lnCRP General population Any cancer HR 11 11459/194796 1.11 (1.04, 1.18) 0.002 1.00 (0.94, 1.07) 0.89, 1.37 70 (34, 82) 0.717 5/0.55 1.1E − 04 Weak Hu et al. [38] High vs Low Clear cell renal cell carcinoma Cancer- specific survival HR 3 269/522 2.98 (1.48, 6.00) 0.002 2.64 (1.04, 6.70) 0.01, 1243.25 25 (0, 79) 0.619 2/2.55 NP Weak Zheng et al. [69] High vs Low Hepatocel- lular carcinoma Tumor number HR 5 448/935 2.36 (1.36, 4.10) 0.002 1.81 (1.23, 2.66) 0.40, 14.08 62 (0, 84) 0.568 4/2.59 0.377 Weak Leuzzi et al. [44] High vs Low Early stage non-small cell lung carcinoma Mortality HR 10 2106/3165 1.42 (1.11, 1.81) 0.005 1.06 (0.95, 1.18) 0.64, 3.16 81 (63, 88) 0.162 8/0.58 5.0E − 09 Weak 1 3 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… obal assessment of C reactive protein and health related outcomes: an umbrella review of… 1 3 References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Wang et al. [58] High vs Low General population (women) Breast cancer RR 11 5371/69157 1.26 (1.06, 1.49) 0.007 0.89 (0.76, 1.06) 0.79, 2.02 50 (0, 73) 0.006d 2/2.15 NP Weak Guo et al. Assessment of epidemiological credibility [32] per unit lnCRP Breast cancer Disease-free survival HR 9 1790/8350 1.18 (1.04, 1.34) 0.009 1.03 (1.00, 1.07) 0.83, 1.69 76 (47, 86) 0.080d 3/0.48 0.010 Weak Godos et al. [31] High vs Low Patients who underwent sigmoidos- copy/colo- noscopy Advanced adenoma OR 4 1092/2330 1.59 (1.09, 2.32) 0.016 1.10 (0.76, 1.59) 0.41, 6.14 44 (0, 80) 0.431 2/0.39 0.050 Weak Qin et al. [73] High vs Low Diffuse large B-cell lymphoma patients Overall survival HR 11 579/2681 2.67 (1.95, 3.64) 6.7E − 10 1.51 (1.04, 2.20) 1.06, 6.67 60 (2, 78) 0.029d 11/5.76 0.001 Weak Qin et al. [112] High vs Low Diffuse large B-cell lymphoma patients Progression- free survival HR 5 353/1269 2.19 (1.68, 2.86) 7.4E − 09 1.91 (1.28, 2.85) 1.22, 3.92 16 (0, 70) 0.961 4/3.78 1.000 Weak Li et al. [20] High vs Low Patients with bone neo- plasms Overall survival HR 5 315/816 1.87 (1.28, 2.75) 0.001 1.40 (1.00, 1.80) 0.54, 6.45 62 (0, 84) 0.473 4/0.97 0.006 Weak Chen et al. [77] High vs Low Pancreatic cancer patients Overall survival HR 5 266/551 2.28 (1.38, 3.79) 0.001 1.36 (0.99, 1.88) 0.43, 12.06 71 (0, 87) 0.009d 3/1.29 0.112 Weak Godos et al. [31] High vs Low Patients who underwent sigmoidos- copy/colo- noscopy Colorectal adenoma (total) OR 12 3350/8308 1.23 (0.98, 1.54) 0.077 1.10 (0.76, 1.59) 0.61, 2.46 54 (0, 75) 0.322 3/1.23 0.117 NS Guo et al. [33] per unit lnCRP General population (men) Prostate cancer HR 5 1586/48450 1.07 (0.98, 1.17) 0.156 1.12 (0.97, 1.30) 0.92, 1.24 0 (0, 64) 0.482 0/0.94 NP NS Zheng et al. [69] High vs Low Hepatocel- lular carcinoma Tumor differ- entiation HR 3 46/364 1.58 (0.74, 3.40) 0.237 2.26 (0.85, 6.01) 0.01, 223.37 0 (0, 73) 0.324 0/1.01 NP NS Zhang et al. [68] High vs Low General population Colorectal adenoma RR 11 6303/14925 1.11 (0.89, 1.38) 0.347 1.32 (1.45, 0.57) 0.58, 2.13 64 (2, 80) 0.574 4/6.52 NP NS Hu et al. [38] High vs Low Clear cell renal cell carcinoma Overall survival HR 3 220/607 1.32 (0.66, 2.65) 0.426 1.43 (0.86, 2.39) 0.00, 1537.81 48 (0, 84) 0.699 0/0.68 NP NS 1 3 G. Markozannes et al. G. Assessment of epidemiological credibility Markozannes 1 3 ( ) References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Zhou 2014 [70] per unit lnCRP General population Rectal cancer RR 12 1170/48209 1.03 (0.90, 1.17) 0.705 0.99 (0.88, 1.10) 0.72, 1.46 43 (0, 69) 0.923 3/0.60 0.020 NS Godos et al. [31] High vs Low Patients who underwent sigmoidos- copy/colo- noscopy Non- advanced adenoma OR 5 536/1625 1.06 (0.57, 1.98) 0.843 0.77 (0.46, 1.29) 0.12, 9.44 77 (20, 89) 0.972 3/1.13 0.080 NS Cardiovascular-related outcomes Li et al. [47] High vs Low General population CVD mortal- ity RR 6 1612/35727 2.05 (1.64, 2.57) 3.6E − 10 1.49 (1.00, 2.21) 1.34, 3.13 13 (0, 66) 0.115 5/5.05 NP Strong Kunutsot et al. [43] per 1 SD lnCRP General population Venous thrombo- embolism HR 9 2225/81625 1.14 (1.08, 1.19) 2.9E − 07 1.18 (1.06, 1.32) 1.07, 1.21 0 (0, 54) 0.743 3/2.48 0.714 Strong Heming et al. [35] High vs Low Stable CAD Mortality or CVD RR 53 5244/50519 1.94 (1.71, 2.20) 5.2E − 25 1.14 (1.06, 1.23) 0.97, 3.88 77 (70, 82) 4.8E − 11d 38/7.21 9.1E − 22 Highly sugges- tive ERFC [41] per 1 SD lnCRP General population CHD HR 31 5373/111899 1.38 (1.27, 1.49) 6.6E − 16 1.27 (1.11, 1.44) 1.09, 1.73 26 (0, 52) 0.724 16/10.59 0.056 Highly sugges- tive He et al. [78] High vs Low ACS/unsta- ble CHD/ angina Mortality or CVD (long-term) RR 11 1276/9011 2.18 (1.78, 2.68) 8.6E − 14 1.70 (1.30, 2.60) 1.21, 3.93 50 (0, 73) 0.024d 9/8.95 1.000 Highly sugges- tive Bibek et al. [26] High vs Low Patients undergoing PCI MACE RR 33 4120/34367 1.96 (1.65, 2.34) 2.8E − 14 1.10 (1.00, 1.20) 0.86, 4.50 84 (79, 88) 1.5E − 05d 24/2.72 1.7E − 19 Suggestive Bibek et al. [26] High vs Low Patients undergoing PCI Mortality RR 26 1358/33068 3.00 (2.18, 4.12) 1.4E − 11 1.08 (0.93, 1.24) 0.84, 10.69 78 (68, 84) 1.4E − 04d 15/1.57 2.1E − 12 Suggestive Xu et al. [63] per 1 mg/L CRP General population Ischemic stroke RR 10 3071/125260 1.15 (1.09, 1.22) 1.2E − 06 1.09 (1.04, 1.14) 1.01, 1.30 37 (0, 69) 0.006d 6/1.18 3.8E − 04 Suggestive Saito et al. Assessment of epidemiological credibility [55] High vs Low East Asians CHD RR 3 1319/310964 1.76 (1.29, 2.40) 3.4E − 04 1.39 (1.04, 1.86) 0.08, 40.61 49 (0, 84) 0.547 3/2.38 1.000 Suggestive Correia et al. [28] High vs Low ACS Mortality or CVD (long-term) OR 6 424/3270 4.58 (2.78, 7.53) 2.1E − 09 2.80 (1.81, 4.32) 1.00, 20.86 69 (0, 85) 0.016d 6/5.49 1.000 Weak Yo et al. [65] High vs Low AF AF recur- rence OR 9 333/632 4.05 (2.51, 6.54) 9.3E − 09 1.60 (1.00, 2.50) 0.95, 17.34 66 (12, 82) 3.6E − 04d 9/1.61 1.9E − 07 Weak 1 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… obal assessment of C reactive protein and health related outcomes: an umbrella review of… 1 3 References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Bibek et al. [26] High vs Low Patients undergoing PCI MI RR 24 974/23271 1.80 (1.47, 2.21) 1.0E − 08 1.42 (1.14, 1.76) 1.00, 3.25 42 (0, 63) 0.003d 7/4.71 0.299 Weak Singh et al. [56] High vs Low Peripheral artery disease Major CVD HR 4 194/752 2.26 (1.65, 3.09) 3.5E − 07 1.89 (1.18, 3.02) 1.14, 4.49 0 (0, 68) 0.185 4/2.08 0.126 Weak Mincu et al. [52] High vs Low Patients with STEMI All-cause mortality RR 6 142/2721 2.68 (1.78, 4.04) 2.2E − 06 2.62 (1.94, 3.50) 0.96, 7.53 49 (0, 78) 0.136 4/3.04 0.688 Weak Mincu et al. [52] High vs Low Patients with STEMI Recurrent MI RR 4 28/1480 3.51 (1.90, 6.48) 5.8E − 05 2.84 (1.27, 6.35) 0.92, 13.47 0 (0, 68) 0.422 2/1.23 0.591 Weak Singh et al. [56] per unit lnCRP Peripheral artery disease Major CVD HR 5 179/1184 1.38 (1.16, 1.63) 2.1E − 04 1.47 (1.13, 1.98) 0.97, 1.95 12 (0, 68) 0.449 2/1.03 0.276 Weak Bibek et al. [26] High vs Low Patients undergoing PCI Coronary revascu- larization RR 21 2115/21694 1.31 (1.11, 1.56) 0.002 0.91 (0.81, 1.02) 0.71, 2.43 69 (47, 79) 0.001d 5/1.63 0.020 Weak Correia et al. [28] High vs Low ACS Mortality or CVD (short- term) OR 12 1203/13256 1.65 (1.20, 2.27) 0.002 1.45 (1.20, 1.74) 0.68, 3.98 62 (14, 78) 0.546 6/5.29 0.775 Weak Padayachee et al. Assessment of epidemiological credibility [53] High vs Low Vascular surgery MACE OR 3 67/386 2.74 (1.36, 5.51) 0.005 2.55 (1.12, 5.83) 0.03, 252.05 0 (0, 73) 0.916 1/1.49 NP Weak Saito et al. [55] High vs Low East Asians Stroke RR 6 2292/91852 1.40 (1.10, 1.77) 0.006 0.93 (0.64, 1.35) 0.78, 2.49 33 (0, 73) 0.116 2/0.73 0.158 Weak Saito et al. [55] High vs Low East Asians Ischemic stroke RR 4 1226/85331 1.40 (1.08, 1.81) 0.010 1.19 (0.82, 1.73) 0.80, 2.46 0 (0, 68) 0.018d 0/1.46 NP Weak Mincu et al. [52] High vs Low Patients with STEMI In-hospital target revascu- larization RR 3 13/1222 3.17 (1.30, 7.72) 0.011 4.53 (1.44, 14.23) 0.00, 7582.37 27 (0, 79) 0.234 2/1.19 0.567 Weak Bibek et al. 2014 [26] High vs Low Patients undergoing PCI Restenosis RR 9 511/2765 1.45 (1.07, 1.96) 0.016 1.10 (0.83, 1.45) 0.63, 3.37 59 (0, 79) 0.431 4/0.58 0.002 Weak Padayachee et al. [53] High vs Low Vascular surgery Cardiac death OR 4 34/477 4.15 (1.18, 14.52) 0.026 5.38 (0.62, 46.50) 0.26, 64.96 0 (0, 68) 0.552 1/2.50 NP Weak 1 3 G. Markozannes et al. 3 References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Barron et al. [25] per 1 SD CRP Adults (mean age: 50–75) CVD mortal- ity HR 3 569/7269 1.31 (1.02, 1.69) 0.033 1.28 (1.14, 1.44) 0.07, 23.53 81 (0, 92) 0.582 2/1.28 0.579 Weak Padayachee et al. [53] High vs Low Vascular surgery All-cause mortality (long-term) OR 4 53/530 2.19 (1.02, 4.67) 0.043 3.43 (1.15, 10.28) 0.40, 11.83 1 (0, 68) 0.889 1/2.16 NP Weak Yu et al. [76] High vs Low Patients with acute ischemic stroke All-cause mortality HR 6 663/3035 2.45 (1.47, 4.06) 5.4E − 04 2.00 (1.70, 1.30) 0.48, 12.52 29 (0, 76) 0.912 5/5.07 NP Weak Saito et al. [55] High vs Low East Asians CHD RR 4 625/74626 1.75 (0.96, 3.19) 0.068 1.13 (0.70, 1.82) 0.13, 22.80 72 (0, 88) 0.159 2/0.53 0.089 NS Barron et al. [25] per 1 SD CRP Adults (mean age: 50–75) CHD mortal- ity HR 3 333/7269 1.20 (0.93, 1.56) 0.160 1.27 (1.09, 1.48) 0.07, 21.28 71 (0, 89) 0.760 2/0.81 0.181 NS Padayachee et al. Assessment of epidemiological credibility [53] High vs Low Vascular surgery MI (nonfatal) OR 3 36/386 1.37 (0.62, 3.00) 0.436 1.24 (0.52, 2.97) 0.01, 222.04 0 (0, 73) 0.319 0/0.20 NP NS Saito et al. [55] High vs Low East Asians Hemorrhagic stroke RR 4 863/85331 1.04 (0.66, 1.65) 0.850 0.70 (0.46, 1.07) 0.21, 5.16 39 (0, 79) 0.061 0/2.62 NP NS Kidney-related outcomes Li et al. [46] High vs Low Chronic kidney disease All-cause mortality HR 17 2327/9022 1.21 (1.14, 1.29) 5.6E − 10 1.02 (1.01, 1.03) 1.01, 1.46 89 (84, 92) 3.9E − 05d 14/1.82 1.3E − 11 Highly sugges- tive Li et al. [46] High vs Low Chronic kidney disease CVD mortal- ity HR 14 7685.966/14498 1.19 (1.10, 1.28) 2.3E − 05 1.02 (1.01, 1.03) 0.95, 1.49 76 (57, 84) 1.1E − 04d 7/0.75 3.1E − 06 Suggestive Herselman et al. [36] per 1 mg/L CRP Dialysis All-cause mortality HR 9 503/1608 1.03 (1.02, 1.05) 1.9E − 04 1.02 (1.01, 1.03) 0.99, 1.08 74 (40, 85) 0.001d 8/0.45 3.5E − 10 Weak Avram et al. [24] High vs Low Peritoneal dialysis All-cause mortality HR 15 619/3333 1.04 (1.02, 1.06) 3.0E − 04 1.02 (1.01, 1.03) 0.98, 1.10 80 (67, 87) 1.5E − 04d 11/0.76 6.0E − 12 Weak Herselman et al. [36] per 1 mg/L CRP Dialysis CVD mortal- ity HR 4 137/1047 1.06 (0.98, 1.15) 0.133 1.00 (0.98, 1.02) 0.77, 1.46 86 (59, 93) 0.075d 2/0.20 0.014 NS Chan et al. [27] High vs Low Children with HSP HSP nephri- tis OR 5 380/955 1.33 (0.78, 2.28) 0.298 1.20 (0.78, 1.83) 0.26, 6.91 56 (0, 82) 0.907 1/0.52 0.420 NS 1 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… obal assessment of C reactive protein and health related outcomes: an umbrella review of… References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Shen (2016) High vs Low Peritoneal dialysis CVD mortal- ity HR 5 134/832 1.69 (0.50, 5.74) 0.403 1.03 (1.01, 1.05) 0.02, 146.16 91 (83, 95) 0.794 4/0.25 3.1E − 05 NS Skeletal-related outcomes Maneiro et al. Assessment of epidemiological credibility [51] High vs Low AS on anti- TNF BASDAI50 OR 6 1384/2570 2.14 (1.71, 2.68) 2.5E − 11 1.94 (1.53, 2.45) 1.32, 3.48 22 (0, 69) 0.015d 6/3.99 0.188 Highly sugges- tive Wu et al. [62] High vs Low General population Fracture RR 6 2421/14382 2.14 (1.51, 3.05) 2.2E − 05 1.78 (1.27, 2.46) 0.75, 6.11 62 (0, 82) 0.047d 5/5.09 NP Suggestive Maneiro et al. [51] High vs Low AS on anti- TNF ASAS20 OR 6 865/1262 2.53 (2.00, 3.21) 1.7E − 14 2.18 (1.34, 3.53) 1.81, 3.54 0 (0, 61) 0.508 5/4.58 1.000 Weak Maneiro et al. [51] High vs Low AS on anti- TNF ASAS40 OR 3 758/1524 2.03 (1.49, 2.76) 7.0E − 06 2.02 (1.60, 2.55) 0.12, 33.85 28 (0, 80) 0.559 3/2.11 0.560 Weak Maneiro et al. [51] High vs Low AS on anti- TNF BASDAI OR 5 940/1617 1.04 (1.01, 1.08) 0.004 1.02 (1.01, 1.04) 0.94, 1.16 86 (64, 92) 0.092d 4/0.26 3.3E − 05 Weak Jin et al. [40] High vs Low Osteoarthritis Disease pro- gression OR 4 2469/10619 0.97 (0.71, 1.33) 0.855 1.12 (0.81, 1.54) 0.28, 3.40 57 (0, 84) 0.598 1/1.08 NP NS Neurological-related outcomes Koyama et al. [42] High vs Low General population Dementia HR 5 746/4392 1.45 (1.10, 1.91) 0.008 1.21 (0.85, 1.73) 0.68, 3.11 39 (0, 76) 0.358 1/1.06 NP Weak Koyama et al. [42] High vs Low General population Alzheimer’s disease HR 7 565/5401 1.21 (1.03, 1.42) 0.021 1.23 (1.00, 1.52) 0.98, 1.49 0 (0, 58) 0.913 0/1.12 NP Weak Yang et al. [64] High vs Low Non- demented adults Cognitive decline RR 4 1001/5170 1.29 (0.95, 1.75) 0.101 1.24 (0.96, 1.63) 0.49, 3.39 25 (0, 75) 0.939 1/1.46 NP NS Respiratory-related outcomes Leuzzi et al. [45] High vs Low COPD Mortality (late) HR 15 2287/11728 1.53 (1.32, 1.77) 1.5E − 08 1.48 (1.28, 1.71) 0.93, 2.52 69 (40, 80) 0.010d 12/7.49 0.021 Highly sugges- tive Leuzzi et al. [45] High vs Low COPD Mortality (early) RR 11 802/6688 1.15 (0.93, 1.42) 0.183 1.22 (1.11, 1.34) 0.60, 2.21 87 (78, 91) 0.517 8/1.44 9.6E − 06 NS 1 G. Markozannes et al. G. Assessment of epidemiological credibility Markoz 1 3 Table 1 (continued) References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Pregnancy-related outcomes Wei et al. [60] High vs Low (plasma CRP) Pregnant women Spontaneous preterm birth OR 5 934/3543 1.61 (1.22, 2.11) 6.6E − 04 1.17 (0.84, 1.63) 0.81, 3.18 27 (0, 73) 0.040d 3/0.83 0.035 Weak Wei et al. [60] High vs Low (amniotic fluid CRP) Pregnant women Spontaneous preterm birth OR 3 165/647 8.75 (1.86, 41.12) 0.006 2.80 (0.99, 7.94) 0.00, 3.11E + 08 68 (0, 89) 0.068d 2/2.69 NP Weak Other outcomes Li et al. [47] High vs Low General population All-cause mortality RR 14 9285/71016 1.75 (1.55, 1.98) 8.3E − 19 1.49 (1.24, 1.78) 1.16, 2.64 60 (16, 77) 0.192 12/12.97 NP Highly sugges- tive Wang et al. [5] per unit lnCRP General population Type 2 diabetes RR 22 5836/40435 1.26 (1.16, 1.37) 5.8E − 08 1.17 (1.06, 1.29) 0.92, 1.71 64 (38, 76) 0.204 11/4.59 0.002 Highly sugges- tive Hong et al. [37] High vs Low Adults ≥ 40 yeas Age-related macular degenera- tion OR 11 3232/41690 1.69 (1.28, 2.23) 2.2E − 04 1.24 (0.87, 1.78) 0.78, 3.63 51 (0, 74) 0.004d 4/3.56 0.755 Suggestive Wu et al. [75] High vs Low Patients receiving allogeneic stem cell transplant Overall survival HR 14 1275/3216 1.63 (1.34, 1.98) 8.8E − 07 0.96 (0.91, 1.13) 0.85, 3.12 77 (59, 85) 3.7E − 04d 8/2.64 0.002 Suggestive Tian et al. [74] High vs Low Type 2 diabetic patients All-cause mortality RR 6 1121/9843 2.03 (1.49, 2.75) 6.5E − 06 1.77 (1.29, 2.42) 0.82, 5.00 60 (0, 82) 0.167 4/5.27 NP Suggestive Jayedi et al. [71] High vs Low General population Hypertension RR 12 18877/137918 1.26 (1.13, 1.39) 1.5E − 05 1.09 (1.03, 1.16) 0.94, 1.68 65 (23, 80) 0.153 7/3.78 0.060 Suggestive Tian et al. [80] High vs Low Type 2 diabetic patients Cardio- vascular mortality RR 6 1451/21148 1.74 (1.35, 2.23) 1.7E − 05 2.09 (1.57, 2.77) 0.98, 3.08 28 (0, 71) 0.944 3/5.35 NP Suggestive Wu et al. Assessment of epidemiological credibility [75] High vs Low Patients receiving allogeneic stem cell transplant non-relapse mortality HR 14 513/3128 2.06 (1.62, 2.62) 4.4E − 09 1.50 (1.24, 1.82) 1.03, 4.12 52 (0, 72) 0.007d 8/4.87 0.094 Weak 3 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… ( ) References Contrast Population Outcome Meta- analysis metric N Stud- ies N cases/N popula- tion Random effects (95% CI)a Random effects P Largest study (95% CI)b Prediction interval I2 Egger’s Pc Excess Significance Evidence Grade O/E Pd Wu et al. [75] High vs Low Patients receiving allogeneic stem cell transplant acute graft versus host disease HR 7 104/1133 1.35 (1.07, 1.71) 0.013 1.00 (0.98, 1.01) 0.71, 2.57 77 (40, 87) 0.002d 4/2.25 0.222 Weak Soysal et al. [57] High vs Low Elderly Frailty OR 3 1045/2939 1.06 (0.78, 1.44) 0.694 1.05 (0.72, 1.54) 0.15, 7.68 0 (0, 73) 0.678 0/0.20 NP NS All statistical tests were two-sided ACS Acute coronary syndrome; AF Atrial fibrillation; anti-TNF anti-tumor necrosis factor; AS Ankylosing spondylitis; ASAS Assessment in Ankylosing Spondylitis response criteria; BASDAI Bath Ankylosing Spondylitis Disease Activity Index; CAD Coronary artery disease; CHD Coronary heart disease; CI confidence interval; COPD Chronic obstructive pulmonary disease; CRP C-reactive protein; CVD Cardiovascular disease; HR Hazard ratio; HSP Henoch-Schönlein purpura; MACE Major Adverse Cardiac Events; MI Myocardial infarction; NP Not pertinent; NS Not significant; OR Odds ratio; RR Relative risk; STEMI ST-elevation myocardial infarction a Random-effects refers to summary relative risk (95% CI) using the meta-analysis random-effects model b Largest study (smallest standard error) c P-value from the Egger’s regression asymmetry test d Denotes doth a P-value < 0.1 and that the largest study is more conservative that the summary random effects estimate e P-value of the excess statistical significance test. Expected number of statistically significant studies is estimated using the point estimate of the largest study (smallest standard error) as the plausible effect size 1 3 G. Markozannes et al. 1 3 Table 2 Health outcome and characteristics of Mendelian randomization studies. Only studies with instruments from the CRP gene are presented. One study is selected per outcome based on the largest sample sizea Reference Phenotype N cases Total N SNPs used in the GRS instrument Level of exposure Metric Causal effect estimateb Pc MR method Wium-Andersen et al. Assessment of epidemiological credibility [101, 102] All-cause mortality 4778 78809 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 1.08 (0.86, 1.34) NR 1SMR, IPD, IV regres- sion Prins et al. [107] Alzheimer disease 4663 13020 rs1130864, rs3093077 Per unit of lnCRP OR 1.26 (0.89, 1.78) 0.2 2SMR, PSD, IVW meta- analysis Prins et al. [107] Amyotrophic lateral sclerosis 4133 12263 rs1130864, rs1205 Per unit of lnCRP OR 0.79 (0.60, 1.04) 0.09 2SMR, PSD, IVW meta- analysis Wium-Andersen et al. [101, 102] Any cancer 12343 78809 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.94 (0.81, 1.08) NR 1SMR, IPD, IV regres- sion Marott et al. [96] Atrial fibrillation 2111 46876 rs1205, rs1130864, rs3091244, rs3093077 Per doubling of CRP OR 0.76 (0.62, 0.93) NR 1SMR, IPD, GLSR Prins et al. [107] Autism 90 1566 rs1130864, rs1205, rs3093077 Per unit of lnCRP OR 1.02 (0.97, 1.07) 0.38 2SMR, PSD, IVW meta- analysis Prins et al. [107] Bipolar disorder 7481 16731 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.17 (0.97, 1.42) 0.11 2SMR, PSD, IVW meta- analysis Allin et al. [91] Bladder and urinary tract cancer 531 46618 rs1205, rs1130864, rs3091244, rs3093077 Per doubling of CRP OR 0.73 (0.42, 1.25) NR 1SMR, IPD, GLSR Prins et al. [107] BMI (in SD) NA 123864 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP MD − 0.017 (− 0.06, 0.02) 0.41 2SMR, PSD, IVW meta- analysis Allin et al. [91] Breast cancer 1402 46618 rs1205, rs1130864, rs3091244, rs3093077 Per doubling of CRP OR 1.05 (0.77, 1.43) NR 1SMR, IPD, GLSR Prins et al. [107] CAD 60801 184305 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.00 (0.93, 1.07) 0.965 2SMR, PSD, IVW meta- analysis Kivimäki et al. [86] Carotid intima-media thickness (mm) NA 3016 rs1130864, rs1205, rs3093077 Per doubling of CRP (mean age of 49.2) MD − 0.001 (− 0.025, 0.023) NR 1SMR, IPD, IV regres- sion Prins et al. [107] Celiac disease 4533 15283 rs1130864, rs1205, rs3093077 Per unit of lnCRP OR 0.96 (0.77, 1.21) 0.75 2SMR, PSD, IVW meta- analysis Prins et al. [107] Chronic kidney disease 6271 74354 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.04 (0.88, 1.22) 0.67 2SMR, PSD, IVW meta- analysis Allin et al. [91] Colorectal cancer 858 46618 rs1205, rs1130864, rs3091244, rs3093077 Per doubling of CRP OR 1.10 (0.74, 1.64) NR 1SMR, IPD, GLSR Wium-Andersen et al. Assessment of epidemiological credibility [101, 102] COPD 3853 78809 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.87 (0.69, 1.11) NR 1SMR, IPD, IV regres- sion Dahl et al. [97] COPD hospitalization 2285 40109 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.82 (0.59, 1.13) NR 1SMR, IPD, GLSR Prins et al. [107] Crohn disease 6333 21389 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 0.78 (0.65, 0.94) 0.009 2SMR, PSD, IVW meta- analysis Prins et al. [107] Cutaneous psoriasis 1363 4880 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.10 (0.76, 1.59) 0.62 2SMR, PSD, IVW meta- analysis Prins et al. [107] DBP (mmHg) NA 69368 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP MD 0.70 (0.20, 1.19) 0.006 2SMR, PSD, IVW meta- analysis 1 3 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… p 1 3 Table 2 (continued) Reference Phenotype N cases Total N SNPs used in the GRS instrument Level of exposure Metric Causal effect estimateb Pc MR method Wium-Andersen et al. [101, 102] Depression 1183 78809 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.79 (0.51, 1.22) NR 1SMR, IPD, IV regres- sion Prins et al. [107] eGFRcr (in mm/ min/1.73 m2) NA 74354 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP MD 0.004 (− 0.01, 0.02) 0.4 2SMR, PSD, IVW meta- analysis Sunyer et al. [84] FEF25-75% (ml) NA 134 rs1205 Per doubling of CRP MD − 1283.5 (− 2792.7, 225.7) NR 1SMR, IPD, IV regres- sion Bolton et al. [98] FEV1 NA 1224 rs1800947 CG/GG compared with CC MD 0.01 (− 0.08, 0.11) 0.82 1SMR, IPD, Genotype used as a proxy for exposure, without fur- ther estimation Sunyer et al. [84] FVC (ml) NA 134 rs1205 Per doubling of CRP MD − 628.0 (− 1402.8, 146.8) NR 1SMR, IPD, IV regres- sion Brunner et al. [85] HbA1c (%) NA 4678 rs1130864, rs1205, rs3093077 Per doubling of CRP (mean age of 49) GMR 0.996 (0.981, 1.011) NR 1SMR, IPD, IV regres- sion Timpson N, 2005 HDL cholesterol (mmol/L) NA 3206 rs2794521, rs1800947, rs1130864, rs1205 Per doubling of CRP MD 0.006 (-0.072, 0.084) NR 1SMR, IPD, IV regres- sion Brunner et al. Assessment of epidemiological credibility [85] HOMA-IR NA 3912 rs1130864, rs1205, rs3093077 Per doubling of CRP (mean age of 49) GMR 1.035 (0.934, 1.145) NR 1SMR, IPD, IV regres- sion Wium-Andersen [101, 102] Hospitalization or death with depression 1145 76479 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.79 (0.51, 1.22) NR 1SMR, IPD, IV regres- sion Davey Smith et al. [79] Hypertension NR 3529 rs1800947 Per doubling of CRP OR 1.03 (0.61, 1.73) NR 1SMR, IPD, IV regres- sion Prins et al. [107] IBD (all types) 13020 47794 rs1130864, rs1205, rs3093077 Per unit of lnCRP OR 0.97 (0.84, 1.13) 0.7 2SMR, PSD, IVW meta- analysis Prins et al. [107] Ischemic stroke (all types) 3548 9520 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.19 (0.93, 1.53) 0.16 2SMR, PSD, IVW meta- analysis Prins et al. [107] Knee osteoarthritis 5755 24260 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 0.94 (0.78, 1.13) 0.5 2SMR, PSD, IVW meta- analysis Viikari et al. [83] Leptin (ng/ml) NA 1655 rs2794521, rs3091244, rs1800947, rs1130864, rs1205 Per doubling of CRP MD 0.02 ± 0.06 0.76 1SMR, IPD, IV regres- sion Allin et al. [91] Lung cancer 678 46618 rs1205, rs1130864, rs3091244, rs3093077 Per doubling of CRP OR 1.15 (0.67, 1.98) NR 1SMR, IPD, GLSR Prins et al. [107] Major depressive disorder 9240 18759 rs1130864, rs1205, rs3093077 Per unit of lnCRP OR 0.98 (0.81, 1.18) 0.81 2SMR, PSD, IVW meta- analysis Casas et al. [81] Non-fatal MI 985 5216 rs1130864 TT compared with CT/ CC OR 1.01 (0.74 – 1.38) 0.95 1SMR, IPD, multivariate logistic regression Wium-Andersen et al. [101, 102] Not accomplishing 16001 75504 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 1.09 (0.96, 1.23) NR 1SMR, IPD, IV regres- sion 3 G. Markozannes et al. 1 3 Reference Phenotype N cases Total N SNPs used in the GRS instrument Level of exposure Metric Causal effect estimateb Pc MR method Prins et al. [107] Parkinson disease 5333 17352 rs1130864, rs1205, rs3093077 Per unit of lnCRP OR 1.00 (0.85, 1.17) 0.96 2SMR, PSD, IVW meta- analysis Wium-Andersen et al. [101, 102] Prescription antidepres- sant medication use 8641 76539 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.98 (0.83, 1.15) NR 1SMR, IPD, IV regres- sion Allin et al. [91] Prostate cancer 560 46618 rs1205, rs1130864, rs3091244, rs3093077 Per doubling of CRP OR 1.02 (0.62, 1.69) NR 1SMR, IPD, GLSR Prins et al. Assessment of epidemiological credibility [107] Psoriasis vulgaris 4007 8941 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.23 (0.96, 1.57) 0.11 2SMR, PSD, IVW meta- analysis Prins et al. [107] Psoriatic arthritis 1946 6880 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.45 (1.04, 2.04) 0.03 2SMR, PSD, IVW meta- analysis Davey Smith et al. [79] Pulse pressure (mm Hg) NA 3529 rs1800947 Per doubling of CRP MD − 0.40 (− 5.38, 4.57) NR 1SMR, IPD, IV regres- sion Prins et al. [107] Rheumatoid arthritis 5538 25702 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 0.94 (0.77, 1.15) 0.55 2SMR, PSD, IVW meta- analysis Prins et al. [107] SBP (mmHg) NA 69372 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP MD 1.23 (0.45, 2.01) 0.002 2SMR, PSD, IVW meta- analysis Hartwig et al. [109] Schizophrenia 35476 82315 rs1130864, rs1205, rs1800947, rs3093077 Per 2-fold of lnCRP OR 0.93 (0.86, 1.00) 0.04 2SMR, PSD, weighted generalized linear regression Wium-Andersen et al. [101, 102] Self-reported antide- pressants 5002 75169 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 1.16 (0.95, 1.43) NR 1SMR, IPD, IV regres- sion Prins et al. [107] Serum albumin level (gr/dl) NA 53189 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP MD − 0.002 (− 0.02, 0.01) 0.77 2SMR, PSD, IVW meta- analysis Prins et al. [107] Serum protein level (gr/dl) NA 25537 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP MD 0.008 (− 0.02, 0.04) 0.64 2SMR, PSD, IVW meta- analysis Prins et al. [107] Systemic lupus erythe- matous 1311 4651 rs1130864, rs1205, rs3093077 Per unit of lnCRP OR 1.20 (0.80, 1.81) 0.38 2SMR, PSD, IVW meta- analysis Prins et al. [107] Systemic sclerosis 2356 7518 rs1130864, rs1205, rs3093077 Per unit of lnCRP OR 1.07 (0.78, 1.45) 0.68 2SMR, PSD, IVW meta- analysis Rode et al. [104] Telomere length in base pairs NA 45069 rs3091244 Per doubling of CRP MD − 66 (− 124, − 7) NR 1SMR, IPD, IV regres- sion Timpson et al. [80] Triglycerides (mmol/L) NA 3206 rs2794521, rs1800947, rs1130864, rs1205 Per doubling of CRP GMR 0.99 (0.92, 1.08) NR 1SMR, IPD, IV regres- sion Prins et al. [107] Type 1 diabetes 9934 26890 rs1130864, rs1205 Per unit of lnCRP OR 1.15 (0.90, 1.47) 0.26 2SMR, PSD, IVW meta- analysis Prins et al. Assessment of epidemiological credibility [92, 93] Venous Thromboem- bolism 1370 46470 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.80 (0.56, 1.12) NR 1SMR, IPD, GLSR Timpson et al. [80] Waist-to-hip ratio NA 3206 rs2794521, rs1800947, rs1130864, rs1205 Per doubling of CRP MD 0.005 (− 0.007, 0.016) NR 1SMR, IPD, IV regres- sion Wium-Andersen et al. [101, 102] Wanting to give up 4846 75694 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 1.02 (0.83, 1.26) NR 1SMR, IPD, IV regres- sion 1SMR one-sample Mendelian randomization; 2SMR two-sample Mendelian randomization; BMI body mass index; CAD Coronary artery disease; COPD chronic obstructive pulmonary disease CRP c-reactive protein; DBP diastolic blood pressure; FEF forced expiratory flow; FEV1 Forced expiratory volume in 1 s; FVC Forced vital capacity; GLSR Generalized least squares regres sion; GMR Geometric Means Ratio; HDL high density lipoprotein; HOMA-IR Homeostatic Model Assessment for Insulin Resistance; HR Hazard ratio; IBD irritable bowel syndrome; IPD indi vidual participant data; IV Instrumental variable; IVW Inverse variance weighted; MD mean difference; MI Myocardial infarction; NR not reported; OR odds ratio; PSD published summary data RR Relative risk; SBP systolic blood pressure; SNP single nucleotide polymorphism Assessment of epidemiological credibility [107] Type 2 diabetes 6698 22570 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.11 (0.94, 1.32) 0.23 2SMR, PSD, IVW meta- analysis Prins et al. [107] Ulcerative colitis 6687 26405 rs1130864, rs1205, rs1800947, rs3093077 Per unit of lnCRP OR 1.10 (0.92, 1.31) 0.29 2SMR, PSD, IVW meta- analysis 1 3 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… studies and MR analyses was concordant for three out- comes where both meta-analyses of observational studies and MR analyses were not statistically significant (P ≥ 0.05). The remaining studies showed various degree of evidence (weak, suggestive, highly suggestive) with meta-analyses of observational studies and no evidence or limited inconsistent evidence from MR. Finally, MR did not support causality for venous thromboembolism whose evidence was graded as strong in the observational meta-analysis evidence. ( ) Reference Phenotype N cases Total N SNPs used in the GRS instrument Level of exposure Metric Causal effect estimateb Pc MR method Zacho et al. [92, 93] Venous Thromboem- bolism 1370 46470 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 0.80 (0.56, 1.12) NR 1SMR, IPD, GLSR Timpson et al. [80] Waist-to-hip ratio NA 3206 rs2794521, rs1800947, rs1130864, rs1205 Per doubling of CRP MD 0.005 (− 0.007, 0.016) NR 1SMR, IPD, IV regres- sion Wium-Andersen et al. [101, 102] Wanting to give up 4846 75694 rs3091244, rs1130864, rs1205, rs3093077 Per doubling of CRP OR 1.02 (0.83, 1.26) NR 1SMR, IPD, IV regres- sion 1SMR one-sample Mendelian randomization; 2SMR two-sample Mendelian randomization; BMI body mass index; CAD Coronary artery disease; COPD chronic obstructive pulmonary disease CRP c-reactive protein; DBP diastolic blood pressure; FEF forced expiratory flow; FEV1 Forced expiratory volume in 1 s; FVC Forced vital capacity; GLSR Generalized least squares regres sion; GMR Geometric Means Ratio; HDL high density lipoprotein; HOMA-IR Homeostatic Model Assessment for Insulin Resistance; HR Hazard ratio; IBD irritable bowel syndrome; IPD indi vidual participant data; IV Instrumental variable; IVW Inverse variance weighted; MD mean difference; MI Myocardial infarction; NR not reported; OR odds ratio; PSD published summary data RR Relative risk; SBP systolic blood pressure; SNP single nucleotide polymorphism Reference Phenotype N cases Total N SNPs used in the GRS instrument Level of exposure Metric Causal effect estimateb Pc MR method Zacho et al. Conclusions Our umbrella review showed an impressive body of litera- ture on CRP including 113 comparisons from 55 studies for separate phenotypes and 196 MR analyses to assess causality of epidemiologic associations. Only 14 phenotypes had evi- dence from meta-analysis of observational studies and MR analyses. Most summary meta-analytic estimates of obser- vational studies yielded nominally statistically significant results for a direct association between CRP and different phenotypes. Nonetheless, only two of these associations had strong results with no suggestions of biases (cardiovascular mortality and venous thromboembolism in general popula- tion) and none of these had supporting evidence of a causal role for CRP in MR investigations. Low-grade inflammation has been suggested to be involved in many chronic diseases, which may explain the breadth and depth of phenotypes examined in relation to CRP, a general marker of inflammation that can be inexpen- sively measured in epidemiological and clinical settings. A search of “C-reactive protein or CRP” yields 74,622 items as of March 05, 2019, and the vast number of meta-analy- ses that we identified are efforts to summarize this huge, expanding literature. A large proportion of studies examined CRP as a prognos- tic marker of cancer incidence but also of cancer survival. Out of those 52 comparisons, there was highly suggestive evidence for only two associations (ovarian cancer incidence and overall survival in hepatocellular carcinoma). The evi- dence from the remaining literature was classified as sugges- tive or weak. MR efforts, including one on lung cancer, did not highlight any evidence of causality either, although their sample sizes were modest for less common cancers. Chronic inflammation may still be linked to cancer development and progression, as other lines of evidence suggest a higher risk of cancer amongst individuals with inflammatory conditions (e.g., inflammatory bowel diseases and risk of colon cancer), or higher risk of cancer in relation to infections (e.g. human papillomaviruses and cervix cancer) [115–119]. However, CRP, as a general marker of inflammation, is unlikely to capture the specific inflammatory mediating pathways link- ing inflammation to cancer development and progression. 1 3 G. Markozannes et al. Conclusions Table 3 Comparison of evidence from observational studies meta-analysis and Mendelian randomization (MR) studies taking into account both CRP gene-only and genome-wide significant instruments Population (observational) Outcome (observational) Grade (observational) Outcome (MR) Grade (MR) General population Venous thromboembolism Strong Venous Thromboembolism No evidence General population All-cause mortality Highly suggestive All-cause mortality No evidence General population Coronary Heart Disease Highly suggestive Coronary Heart Disease No evidence General population Type 2 diabetes Highly suggestive Type 2 diabetes Limited/inconsistent evidence General population Hypertension Suggestive Hypertension No evidence General population Ischemic stroke Suggestive Ischemic stroke (all types) No evidence AF patients Atrial fibrillation (recur- rence) Weak Atrial fibrillation No evidence General population Alzheimer’s disease Weak Alzheimer’s disease Limited/inconsistent evidence General population (women) Breast cancer Weak Breast cancer No evidence General population Colon cancer Weak Colon cancer No evidence General population Colorectal cancer Weak Colorectal cancer Limited/inconsistent evidence Vascular surgery patients Non-fatal Myocardial Infarction No evidence Non-fatal Myocardial Infarction Limited/inconsistent evidence General population (men) Prostate cancer No evidence Prostate cancer No evidence General population Rectal cancer No evidence Rectal cancer No evidence CRP and cardiovascular diseases have been subject to an increasing body of research and debate. Our review found that the associations of CRP with cardiovascular mortal- ity and venous thromboembolism were supported by strong evidence. Furthermore, we found highly suggestive evidence between higher CRP and risk of CHD, type 2 diabetes and mortality or CVD on stable CAD patients and on unstable CHD/ACS/angina patients. Nonetheless, MR studies have repeatedly failed to provide evidence for causal association with CHD; an observation further supported from rand- omized controlled trials [120]. The observational literature of CRP is likely to suffer from diverse biases and the effect size of the associations may be inflated [121, 122]. Beyond causality, even efforts to show that CRP could at least be used in risk prediction have also not demonstrated convinc- ing results [123, 124]. Accordingly, the relative risks that we noted for cardiovascular mortality (2.05, in fact just 1.49 in the largest study) and venous thromboembolism (only 1.14) do not suggest a substantial predictive potential. The role of inflammation in atherosclerotic plaque initiation, progres- sion and rupture has been supported by various other lines of evidence [125], but this may not necessarily prove that CRP should have clinical utility. a causal association. Conclusions A large proportion (48.2%) of the examined observational meta- analyses displayed substantial heterogeneity (I2 > 50%), small study effects (39.5%), and excess significance bias (41.2%), which, in addition to the small effect estimates increase the probability of false-positive findings. MR approaches use genetic variants as instrumental variables to establish whether an exposure is causally related to a dis- ease or trait. The genetic variants are unrelated to confound- ing factors, and therefore, this approach is not as prone to confounding and reverse causation bias. At the same time, genetic association estimates in MR represent the average lifetime association of the variants with the outcome for all those in the considered population, and are therefore less vulnerable to measurement error [132]. Nonetheless, MR also shares some of the limitations of observational epi- demiology literature including small sample sizes, instru- ment bias and low power, and poor reporting has further additional limitations [22]. For example, we observed that at least half of the MR studies on CRP used instruments derived from genome-wide association studies including genetic variants on genes of other inflammatory cytokines such as IL-6. These approaches may introduce potential plei- otropy and can thus bias MR estimates. There are several methodologies to account for the violation of the pleiotropy assumption of MR, but these cannot always identify pleio- tropic effects, and therefore, can only partly disentangle the complex pleiotropy previously shown between CRP and lipid and metabolic pathways [133]. describe the possibility that the results are susceptible to bias and uncertainty. In this extensive systematic review of meta-analyses of observational studies on CRP and disease outcomes and of the evidence stemming from MR studies, we could not find strong evidence supported by both study designs in rela- tion to CRP and the most frequently studied non-infection phenotypes in the literature. Observational studies presented robust evidence of association between higher CRP levels and cardiovascular mortality and venous thromboembolism, but without causality support from MR studies. Following claims that CRP maybe be a novel CVD risk factor [134], it has been extensively studied in relation to an ever-increas- ing list of phenotypes and diseases, but it does not seem to be crucially relevant to any of them. Despite intensive research efforts, our study shows that there is little evidence that CRP may constitute a priority interventional target for any diseases. Conclusions CRP might be elevated in COPD patients due to reverse causality as the disease is associated with triggering an inflammatory response. Reverse causality is likely to explain other associations such as mortality in patients with chronic kidney disease or overall survival in hepatocellular carcinoma patients. In these instances CRP could serve as a predictive factor for disease severity, but studies assessing its value over and above validated exist- ing risk prediction algorithms are essential to support any prediction claim [123]. Some particular mention needs to be made on schizo- phrenia, where, among the tentative MR findings described in this review we found several studies of CRP and schizo- phrenia onset. Yet, there is a distinctive lack of observa- tional data on this association, and those that exist [129, 130], mainly focus on the reverse pathway of the associa- tion (how schizophrenia affects CPR levels) than what is the focus of this review. In our MR review we found multiple studies and sensitiv- ity analyses show evidence for causal effect, but with very modest P-values, when only CPR SNPs were used in the genetic instruments. One recent analysis (published after the search date of our review [131]) found even lower P-values with inverse variance weights and generalized summary MR modeling. The putative causal association with schizophre- nia is even more interesting because it suggests a protective effect of CRP on schizophrenia, while observational data had suggested an association of CRP with higher schizo- phrenia risk [130]. COPD is associated with an abnormal inflammatory response beyond the lungs with evidence of low-grade systemic inflammation which causes systemic manifesta- tions such as weight loss, skeletal muscle dysfunction, an increased risk of cardiovascular disease, osteoporosis and depression [125–128]. We found highly suggestive evidence that CRP is associated with late (but not with early) mortal- ity in COPD patients. However, MR studies did not support Overall, the overwhelming majority of the meta-analyses of observational studies reported a nominally statistically 1 3 1 3 Global assessment of C-reactive protein and health-related outcomes: an umbrella review of… significant result (84%) in contrast to MR studies where only 37 of the 196 (19%) analyses presented nominally statisti- cally significant results. These two study designs may be subject to different biases in the biomedical field. Conclusions Authors’ contributions IT had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. IT had the original idea for the manuscript and all authors contributed to design the study. CK, GM performed the analyses and all authors interpreted the results. CK, GM and IT wrote the first draft of the manuscript and all authors commented on previous versions of the manuscript. All authors critically reviewed, wrote and approved the final version. Funding ET is supported by a CRUK Career Development Fellowship (C31250/A22804). DG is funded by the Wellcome Trust. Funding ET is supported by a CRUK Career Development Fellowship (C31250/A22804). DG is funded by the Wellcome Trust. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. Limitations of our approach need to be acknowledged. Our review focused on existing meta-analyses, and there- fore, outcomes that were not assessed in a meta-analysis are not included in this review. Furthermore, we did not appraise the quality of the individual studies but the quality of the actual meta-analyses. We refer interested readers to the quality assessments already made by the authors of each original meta-analysis and we did not wish to change the eligibility criteria based on quality since this would add our own subjective in study selection. We did not include evi- dence from randomised control trial meta-analyses as these examine a wide range of anti-inflammatory treatments which are not specific to CRP lowering effects. Statistical tests for small-study effects and excess significance bias should also be interpreted with caution in case of large between-study heterogeneity and both tests have limited power in the pres- ence of few studies or sparse studies with significant results. Finally, we adopted credibility assessment criteria, which were based on established tools for observational evidence; however, none of the components of these criteria provides firm proof of credibility of evidence, but they cumulatively Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W2097500752	https://ijmhs.biomedcentral.com/counter/pdf/10.1186/1752-4458-5-22	English	null	Determinants of parents' experiences with outpatient child and adolescent mental health services	International journal of mental health systems	2,011	cc-by	6,391	© 2011 Holmboe et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Open Access Olaf Holmboe1, Hilde H Iversen1 and Ketil Hanssen-Bauer2,3 Olaf Holmboe1, Hilde H Iversen1 and Ketil Hanssen-Bauer2,3 * Correspondence: olh@nokc.no 1Norwegian Knowledge Centre for the Health Services, P.O. Box 7004 St Olavs plass, 0130 Oslo, Norway Full list of author information is available at the end of the article Abstract Background: Few studies have investigated how demographic, clinical and organizational characteristics influence parents’ experiences with child and adolescent mental health services (CAMHS). The objective of this study was to determine the effects of these characteristics on parents’ experiences using data from a large national postal survey. Method: A questionnaire was mailed to 17,871 parents or other primary caregivers whose children were attending 1 of the 86 outpatient CAMHS in Norway in 2006. Multiple regression analysis was used to explore the associations between demographic, clinical and organizational characteristics, and three scales of parents’ experiences. Results: The questionnaire was completed by 7906 parents (46%). Organizational characteristics such as involvement of the parents in treatment and accessibility to the clinic explained most of the variation in all three scales of parents’ experiences. Although the effects of demographic and clinical characteristics of the children in some instances were statistically significant, they only accounted for a small amount of the total explained variance. Conclusion: Accessibility to the clinic and involvement of the parents in treatment are much stronger predictors of parental experiences with outpatient CAMHS than are demographic and clinical variables. Accessibility and involvement are at least partly influenced by the clinics themselves, and hence parental satisfaction may be enhanced by making the clinics more accessible and by involving the parents/caregivers in the treatment. experiences, Parent satisfaction, Child and adolescent mental health services, National survey Keywords: User experiences, Parent satisfaction, Child and adolescent mental health services, Natio Determinants of parents’ experiences with outpatient child and adolescent mental health services Olaf Holmboe1, Hilde H Iversen1 and Ketil Hanssen-Bauer2,3 Background satisfaction with CAMHS [4,5]. Most studies have iden- tified one or more variables that are significantly asso- ciated with parental satisfaction with aspects of CAMHS, but we are not aware of any variables that are significantly related to parental satisfaction with CAMHS across all studies, or of studies that have pro- vided information on the explanatory power of such variables beyond their statistical significance. Patient satisfaction and experiences are increasingly used as indicators of quality in health care. Parents are often an integral part of the treatment within the child and adolescent mental health services (CAMHS), and their opinion may be crucial to the engagement and continuation of treatment [1]. Few studies have investi- gated the associations between background variables and parents’ reported experiences with CAMHS [2,3]. It has been suggested that the demographic and clinical characteristics of the patients–in addition to organiza- tional data–are needed when investigating parental The findings regarding parent satisfaction with CAMHS in the published literature is often contradic- tory. For example, some studies have found no relation- ship between demographic variables and parental satisfaction [4,6], while others have identified some sta- tistically significant relationships. One study showed that fathers tended to be less positive than mothers [7], and two other studies found that the parents of older Correspondence: olh@nokc.no 1Norwegian Knowledge Centre for the Health Services, P.O. Box 7004 St Olavs plass, 0130 Oslo, Norway Full list of author information is available at the end of the article Page 2 of 9 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 children were less satisfied than those of younger chil- dren [8,9]. The parents’ age has been shown to be posi- tively associated with their satisfaction with CAMHS [7,10]. Family composition has rarely been examined in studies of parent satisfaction with CAMHS. Single and remarried mothers reported greater changes in their child’s behaviour and improved coping with their child’s problems than did married mothers in one study [11], while parents of children living at home reported statis- tically significant higher levels of satisfaction than those with children living away from home in another [12]. Ethnic background has been shown to be only weakly correlated with parental satisfaction. Several studies found no statistically significant association between satisfaction and ethnic background [4,7,12-14], while Heflinger et al. [10] found that parents of black children were more satisfied with one aspect of the treatment. Background of demographic, clinical and organizational variables on parents’ experiences in Norway, as measured using a national postal survey. Materials and methods Norwegian CAMHS are applied to those aged < 18 years, are a part of the publicly funded National Health Service and are available regardless of parental income. Private services of this kind are very few in Norway and their influence is negligible. In 2006, CAMHS in Norway treated a total of 47,280 children and adolescents (4.3% of the population aged 0-17 years), mostly (46,214) as outpatients. By the end of 2006, 3507 full-time equiva- lent employees worked in the Norwegian CAMHS, of whom 1773 (51%) worked at outpatient units [17]. At the time of the present study there were 86 outpatient CAMHS units in Norway. There is some evidence that health status can influ- ence patients’ satisfaction with health services in general [15]; however, it is unknown whether this relationship holds true regarding parents’ experiences with CAMHS. The type or severity of the child’s mental problems seems to have little effect on the parent satisfaction with CAMHS [4,6,10], but one study found that the satisfac- tion with the treatment outcome was less among par- ents of children referred with externalizing symptoms than for those with internalizing symptoms [8]. There is also some evidence that the severity of the child’s symp- toms and the stress felt by the parents while caregiving is negatively associated with their satisfaction with the services [2,4,14], and Godley et al. [2] found this to be the best predictor of parent satisfaction with CAMHS. But again, another study found no such relationship [16]. Some studies have found a statistically significant correlation between improvement in the child’s mental problems and the parent satisfaction with the services [1,6]. Finally, the length of treatment was found to be positively associated with satisfaction in some studies [7,8] but not in others [4,12]. The project was approved by The Regional Committee for Medical Research Ethics. Dispensation from patient confidentiality was given by The Directorate for Health and Social Affairs. Sample The study sample consisted of parents or primary care- givers in 17,871 families. To be included in the study, the child had to be less than 16 years old with at least one appointment at an outpatient CAMHS during the final 4 months of 2006. The sample comprised a maxi- mum of 400 patients per clinic; participants were chosen randomly if the clinic had more than 400 patients dur- ing the study period. They received a questionnaire by mail, and were asked to return it in a prestamped envel- ope. Two reminders were sent to non-respondents. We compensated for non-responses by dividing the sample into seven response homogeneity groups [18]. These groups were based on diagnosis, length of treat- ment, ethnic background and group of inclusion, and used as a basis for weighting for non-responses. Tele- phone interviews were conducted with a sample of nearly 400 randomly selected non-respondents using a short version of the questionnaire. In addition, administrative and organizational aspects of the clinic may affect patients’ satisfaction with CAMHS. One study of outpatient CAMHS found that a longer waiting time from referral to the start of exami- nation or treatment was associated with a lower level of satisfaction [8]. Furthermore, one study found that a higher frequency of consultations at the clinic increased the satisfaction scores [4], while the findings of another study did not support this [6]. Questionnaire and register data The questionnaire was developed following a review of the international literature, interviews with parents attending two outpatient CAMHS units, discussions within an expert panel and pilot testing of the question- naire [19]. The clinics transferred data regarding the child’s name, address, gender, age, date of referral, date of treatment start and end, reason for referral, diagnoses, and mother’s and father’s ethnic backgrounds. From these data we computed the waiting time and length of treatment (in units of days), and grouped ethnic In summary, the results from published studies that have explored the relationship between demographic, clinical and organizational variables on the one hand and the parents’ experiences with outpatient CAMHS on the other are not consistent. The aim of this study was to determine the influence and explanatory power Page 3 of 9 Page 3 of 9 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 variables. The coefficients for all of the correlations between the independent variables were r < 0.4. We first conducted linear regressions to assess the relationship between the scales and each of the independent vari- ables separately. Independent variables with p < 0.1 in the bivariate regression analyses were included in multi- variate models, where the independent variables were grouped and entered in four blocks. This enabled us to assess the explained variation attributable to each group of variables in addition to the statistical significance of the independent variables’ relationship to the scales. The first block included the child’s age and gender. The sec- ond block included clinical data about the child, such as the grouped diagnosis variable, the Global Assessment of Psychosocial Disability score on axis 6 and whether the child had previously been in contact with CAMHS. Variables on the parents’ background were entered in the third block, and organizational variables related to the clinics’ accessibility, involvement of the parents and the parents’ knowledge of the services were entered in the forth block. background and diagnosis into appropriate categories. The diagnoses at CAMHS are registered in a multiaxial system of six axes based on the International Classifica- tion of Diseases (ICD)-10 [20,21]. Axis 4 refers to somatic diseases and was excluded. Questionnaire and register data Axis 6 refers to the child’s level of social functioning as measured by the Global Assessment of Psychosocial Disability scale, rated from 0 (superior/good social functioning) to 8 (profound and pervasive social disability), and was kept as a sepa- rate variable. The main diagnoses on the remaining axes were grouped into one variable. Statistical analyses l l Statistical analyses were carried out using SPSS 15. Exploratory factor analyses identified three scales in the questionnaire, all of which had satisfactory psychometric properties [19]. The first scale, “Relationship with health personnel”, comprised eight items, including care, understanding, respectfulness, cooperation and enough time. The second scale, “Information and participation”, comprised four items addressing information about the child’s condition and treatment alternatives, and the parents’ influence on treatment. The third scale, “Out- come”, comprised three items regarding changes in the child’s condition and social functioning. The items included in each scale and their response categories are shown in table 1. Results The questionnaire was returned by 7906 parents or pri- mary caregivers (46% of those sent the questionnaire), and a further 226 out of 395 parents (57%) responded during the telephone follow-up study. An analysis of this material revealed that the level of satisfaction was the same for the telephone and mail respondents [19]. These results, together with weighting for response Regression analyses were conducted with each of the three scales as dependent variables, and demographic, clinical and organizational variables as independent Table 1 The scales and their respective single items Scale Items Relationship with health personnela Were the health personnel thoughtful and considerate towards you? Did the health personnel understand your concerns as a parent/guardian? Were the health personnel thoughtful and considerate towards your child? Were the health personnel polite and respectful towards you? Did the health personnel speak to you in a way that was understandable? Did the health personnel take your views seriously? Did you get enough time for contact and conversation with the health personnel? Did the health personnel cooperate well with you? Information and participationa Were you asked to give your views about the choice of treatment program? Did you have an influence in the choice of treatment program? Did you receive information on the different types of treatment available to your child? Did you receive information on your child’s psychological condition? Outcomeb Compared with before treatment started at the outpatient clinic, how is your child’s well-being now? Compared with before treatment started at the clinic, how does your child function in your family now? Compared with before treatment started at the clinic, how does your child function outside of your family now (at school, at nursery, among friends and other social situations)? Response categories for single items included in the scales: a Not at all, To a small extent, To a moderate extent, To a large extent, To a very large extent b Much worse A little worse Neither better nor worse A little better Much better Table 1 The scales and their respective single items Holmboe et al. Results International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Page 4 of 9 The organizational variables accounted for most of the variation explained by our models, increasing the adjusted R2 values from 2% to 43% for the “Relationship with health personnel” scale, from 2% to 41% for the “Information and participation” scale and from 4% to 20% for the “Outcome” scale. Most of these independent variables exerted statistically significant effects on all three scales. The exceptions were the following four variables: 1) “Length of treatment” had no statistically significant effect on the “Information and participation” scale, 2) “Recorded waiting time” was included only on the “Outcome” scale and did not show a statistically sig- nificant effect on this scale, 3) “Perceived waiting time” had no statistically significant effect on the “Outcome” scale, and 4) “Parents participate in consultations” had no statistically significant effect on the “Information and participation” or “Outcome” scale. propensity, led to the conclusion that the low response rate had not induced serious bias, and hence that gener- alization to the entire population was justified [19]. The characteristics of the respondents and the children are presented in table 2. Table 3 gives the distribution of the variables related to the clinics’ accessibility and involvement of the par- ents. These include length of treatment period and the recorded waiting time, taken from the clinics’ registers, and the following six items from the questionnaire: 1) Perceived waiting time, 2) number of consultations in the previous 3 months, 3) having received a suitable number of consultations in the previous 3 months, 4) ease of contacting the therapist outside appointments, 5) parents’ participation in consultations and 6) parents’ understanding of the services. The results of the multiple regression analyses are pre- sented in table 4. The adjusted R2 values for the full model were 0.43 for the “Relationship with health per- sonnel” scale, 0.41 for the “Information and participa- tion” scale and 0.20 for the “Outcome” scale. Altogether the demographic and clinical variables explained 2-4% of the total variance of each of the three scales. Thus, the organizational variables accounted for most of the explained variance. On all scales, three variables had considerably higher b values than the others, two of which were common to all three scales. Results Firstly, the variable “Parents’ under- standing of the services” had the highest b value on all the three scales: 0.326, 0.429 and 0.248 for “Relationship with health personnel”, “Information and participation” and “Outcome”, respectively. Secondly, parents who answered that they had a “Suitable number of consulta- tions” in the previous 3 months were more positive than parents who answered that they had less than a suitable number of consultations. The b values were 0.191, 0.154 and 0.186 for “Relationship with health personnel”, “Information and participation” and “Outcome”, respec- tively. For the “Relationship with health personnel” and “Information and participation” scales, “Ease of contact- ing the therapist outside appointments” had the second highest b values (0.263 and 0.193, respectively), indicat- ing a higher score as a result of easier access. “Number of consultations in the previous 3 months” had the third highest b value on the “Outcome” scale (-0.132), pre- dicting that a lower number of consultations is asso- ciated with a better perceived outcome. The child’s age was significantly negatively correlated with the “Relationship with health personnel” scale. On the “Information and participation” scale, parents of boys were significantly more satisfied than parents of girls. However, these variables explained only a small amount of the variation. Both of these variables were left out of the multivariate analysis for the “Outcome” scale because none of them were significantly related with this scale in the bivariate linear regression analysis. None of the clinical variables significantly affected the “Relationship with health personnel” scale. For the other two scales, parents of children with hyperkinetic or con- duct disorders tended to be more positive than the others. Parents of children previously treated by CAMHS were statistically significantly more negative on the “Outcome” scale. The clinical data explained slightly more than 3% of the explained variation on “Outcome” scale, and had even less explanatory power on the other two scales. Discussion In this study we evaluated the predictors of parents’ experiences with three aspects of outpatient CAMHS. Organizational aspects of the clinic, such as the parents’ perceived accessibility and involvement, were much stronger predictors than were demographic and clinical variables. This is in accordance with another study that found that parental satisfaction is predicted by the degree to which clinics are able to meet the parents’ desires and expectations [22]. The parents’ background explained only a small amount of the variation on the “Relationship with health personnel” scale. Older and married parents and parents with a Western or mixed background were more posi- tive. These variables did not add to the explained var- iance on the “Information and participation” scale and only marginally to that on the “Outcome” scale, even though higher education predicted less satisfaction on both, and non-Nordic Europeans were significantly more positive than Norwegians on the “Information and participation” scale. “Parents’ understanding of the services” had the high- est b value on the three scales. The “Information and participation” scale addressed the parents’ experience with the information about the child’s condition, Page 5 of 9 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. aWorld Health Organization [20,21] Discussion International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Table 2 Characteristics of parents/primary caregivers and their children n % Mean (SD) Median Child’s age in years 7906 11.3 (3.22) 12 Parents’ age in years 7693 40.3 (6.62) 40 Child’s gender 7903 Female 37 Male 63 Diagnosis axes 1, 2, 3 and 5a 7906 Hyperkinetic or conduct disorders (F90, F91, F92) 30 Emotional disorders (F3, F4, F5, F93) 18 Developmental disorders (F7, F8) 8 Psychosocial problems only (Axis 5) 9 No mental or behavioural disorders or psychosocial problems 8 Other mental or behavioural disorders (F0, F1, F2, F6, F94-F99) 7 Not coded or invalid code used 21 Axis 6 (Global Assessment of Psychosocial Disability scale)a 4945 0 Superior/good social functioning 2 1 Moderate social functioning 17 2 Slight social disability 27 3 Moderate social disability 34 4 Serious social disability 16 5 Serious and pervasive social disability 3 6 Unable to function in most areas 1 7 Gross and pervasive social disability 0 8 Profound and pervasive social disability 0 Previously treated by CAMHS 7651 Never 74 Once 11 More than once 14 Parents’ gender 7639 Female 85 Male 15 Marital status 7700 Married 54 Cohabitant 19 Neither married nor cohabitant 27 Household’s highest education level 7787 Primary school 7 High school 41 University graduate 30 University postgraduate 22 Number of parents in paid work 7906 0 17 1 41 2 42 Parents’ ethnic background 6352 Norwegian 89 Western or mixed 8 Non-Western 2 Native language 7718 Norwegian 95 Sami 0 Other Nordic 1 Other European 2 Non-European 2 aWorld Health Organization [20,21] Table 2 Characteristics of parents/primary caregivers and their children Page 6 of 9 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Table 3 Descriptive statistics for the clinics’ accessibility and involvement of the parents n % Mean (SD) Median Length of treatment (days) 7903 436.5 (531.7) 256 Recorded waiting time (days) 7902 83.1 (82.7) 55 Perceived waiting time 7700 None 13 Not for long 44 Fairly long 22 Very long 21 Number of consultations in the previous 3 months? Discussion 7173 Just one 35 2-5 46 6-12 17 More than 12 2 Suitable number of consultations in the previous 3 months 7361 No, far too few 14 No, too few 21 Yes, a suitable number 65 Ease of contacting the therapists outside appointments 5966 Not at all 9 To a small extent 16 To a moderate extent 35 To a large extent 29 To a very large extent 11 Parents’ participation in consultations 7580 No, never 13 Yes, occasionally 49 Yes, often 39 Parents’ understanding of the services 7547 Very poor 4 Quite poor 7 Neither poor nor good 21 Quite good 42 Very good 26 Table 3 Descriptive statistics for the clinics’ accessibility and involvement of the parents variable left out did not change the overall results, and the major part of the explained variance was still attri- butable to the organizational data. treatment and the parents’ influence on the choice of treatment. It seems reasonable that a high score on these variables implies that parents feel they have a good understanding of the services. The “Relationship with health personnel” scale included a question about the cooperation between health personnel and parents, and whether health personnel speak in a way that is understandable. A high score on these questions is likely to lead to a high score on the understanding of the ser- vices. The parents understanding of the services also predicted a high score on the “Outcome” scale, which is about changes in the child’s condition and social func- tioning. The time it takes to gain a good understanding of the services might also be associated with the time needed to see a positive outcome. A good understanding of the services might also imply a more realistic expec- tation about the degree and speed of improvement. Having experienced a “Suitable number of consulta- tions in the previous 3 months” was a stronger predictor on all three scales than the actual number of consulta- tions. It seems intuitively correct that a less-than-suita- ble number of consultations in the previous 3 months would predict a lower score on all three scales. On the other hand, a higher number of consultations in the pre- vious 3 months also predicted lower scores on the “Out- come” and “Information and participation” scales. This apparently contradictory finding may be explained by considering the severity of the child’s problem. Discussion It seems reasonable that a more severe condition elicits a greater effort from the clinic. It is also known from studies of patient satisfaction in general that a poorer health status is associated with a lower satisfaction score [10,15]. Our data also suggest that the frequency of consultations is highest at the onset of treatment. Parents in the early stage of treatment may not yet have reached the point where a notable outcome may be observed, or they may have not yet received sufficient information. It has also It can be argued that the “Parents’ understanding of the services” itself is an outcome of the “Relationship with health personnel” and “Information and participa- tion” scales, and that it should not be included as an independent variable. This does not apply to the “Out- come” scale. Initial analyses with this independent Page 7 of 9 Page 7 of 9 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. in the bivariate regression analysis. b Blank spaces in the p column indicate that the variable was not statistically significant (p > 0.05). cient column indicate that the variable was not included in the final model because it had no statistically significant effect (p > 0.1) alysis Discussion International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Table 4 Regression results: b coefficienta, pb and cumulative adjusted R2 (Adj R2) for each block entered Relationship with health personnel Information and participation Outcome Independent variables b p Adj R2 b p Adj R2 b p Adj R2 (Intercept) 46.76 0.000 27.36 0.000 78.02 0.000 CHILD’S DEMOGRAPHIC DATA 0.004 0.010 Child’s gender (ref Female) Male 0.050 0.001 Child’s age -0.063 0.000 -0.014 CHILD’S CLINICAL DATA AND PREVIOUS CONTACT WITH CAMHS 0.009 0.021 0.034 Diagnosis axes 1,2,3 and 5 (ref hyperkinetic or conduct disorders) Emotional disorders -0.029 -0.106 0.000 -0.033 Developmental disorders 0.015 -0.014 -0.103 0.000 Psychosocial problems only 0.013 -0.043 0.005 -0.080 0.000 No mental or behavioural disorders or psychosocial problems 0.009 -0.023 -0.077 0.000 Other mental or behavioural disorders 0.008 -0.021 -0.059 0.001 Not coded or invalid code used -0.018 -0.023 -0.044 0.013 Axis 6 (Global Assessment of Psychosocial Disability scale) -0.029 0.007 -0.030 Previously treated by CAMHS -0.030 -0.074 0.000 PARENTS’ DEMOGRAPHIC DATA 0.019 0.021 0.036 Parents’ gender (ref Female) Male -0.005 0.007 Parents’ age 0.047 0.006 -0.017 Marital status (ref Married) Cohabiting -0.010 -0.018 Neither married nor cohabiting -0.040 0.035 -0.032 Household’s highest education -0.052 0.000 -0.087 0.000 Number of parents in paid work -0.015 -0.015 0.021 Parents’ ethnic background (ref Norwegian) Western or mixed 0.038 0.012 0.009 Non-Western 0.002 0.022 Native language (ref Norwegian) Other Nordic 0.018 Other European 0.032 0.020 Non-European 0.021 ACCESSIBILITY AND INVOLVEMENT OF THE PARENTS 0.425 0.407 0.199 Length of treatment -0.034 0.044 0.026 0.062 0.001 Recorded waiting time -0.022 Perceived waiting time -0.073 0.000 -0.043 0.004 -0.026 Number of consultations in the previous 3 months 0.059 0.000 -0.031 0.040 -0.132 0.000 Suitable number of consultations in the previous 3 months 0.191 0.000 0.154 0.000 0.186 0.000 Ease of contacting the therapist outside appointments 0.263 0.000 0.193 0.000 0.086 0.000 Parents’ participation in consultations 0.040 0.013 0.027 -0.036 Parents’ understanding of services 0.326 0.000 0.429 0.000 0.248 0.000 a Blank spaces in the b-coefficient column indicate that the variable was not included in the final model because it had no statistically significant effect (p > 0.1) in the bivariate regression analysis. b Blank spaces in the p column indicate that the variable was not statistically significant (p > 0.05). Discussion Regression results: b coefficienta, pb and cumulative adjusted R2 (Adj R2) for each block entered coefficienta, pb and cumulative adjusted R2 (Adj R2) for each block entered Page 8 of 9 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 material, and there is little support for ethnic back- ground as a predictor for parental satisfaction with out- patient CAMHS in the literature. been suggested that dissatisfied patients drop out of treatment more quickly than others [23], and will thus not be among the long-term users with a lower fre- quency of consultations. Clearly this is a complex ques- tion that needs further investigation. Strengths of the study Neither recorded nor perceived waiting time signifi- cantly affected the “Outcome” scale, but a longer per- ceived waiting time predicted lower scores on the other two scales. This suggests that parents who have experi- enced a long waiting time develop a negative attitude that is difficult to change. One strength of this study is its comprehensiveness. All 86 outpatient CAMHS units in Norway participated, with a total number of 7906 respondents. Various back- ground variables were collected from the clinics’ regis- ters and the parents’ self-reports. The parents’ evaluations of the services were collected with a ques- tionnaire with satisfactory psychometric properties [19]. The “Outcome” scale addresses changes in the child’s condition and social functioning, while the other two scales have more of a relational and communicative character. This makes it more plausible that the child’s diagnosis, “Previous contact with CAMHS” and “Length of treatment” have a stronger effect on the “Outcome” scale than on the other two scales. The observation that previous contact with CAMHS predicts a lower score on the “Outcome” scale may indicate that the child’s condition is severe or chronic, with little perceived improvement. The response rates in patient experience surveys in CAMHS have generally been low [3], but it has been shown that reminders are effective at increasing response rates [15]; in this study the non-respondents received two reminders. A response rate of 46% may seem low, but the follow-up study of the non-respon- ders revealed that the results are representative of the entire population. Author details 1Norwegian Kno Author details 1Norwegian Knowledge Centre for the Health Services, P.O. Box 7004 St Olavs plass, 0130 Oslo, Norway. 2Centre for Child and Adolescent Mental Health, Eastern and Southern Norway, P.O. Box 4623 Nydalen, 0405 Oslo, Norway. 3Department of Research and Development, Division of Mental Health Services, Akershus University Hospital, 1478 Lørenskog, Norway. The results may have been influenced by non-Norwe- gian respondents being underrepresented in the sample; although they represent a small group at the national level, they may constitute a relatively large proportion of the patients in certain areas. The questionnaire was dis- tributed only in Norwegian, and so parents experiencing the greatest linguistic barriers in living in Norway were unable to respond to this survey. Nevertheless, to the extent that the ethnic background or native language had an effect, Norwegians were less satisfied. But again, this explains only a minor part of the variation in this Conclusions The parents’ assessment of their experiences with CAMHS is strongly predicted by variables related to the clinics’ accessibility and the involvement of the parents. The demographic and clinical characteristics have less explanatory power. The most important single item for all three scales is the parents’ understanding of the ser- vices. In addition, the perception of being offered a sui- table number of consultations and having easy access to the therapist outside appointments has a strong predic- tive effect on the parents’ experiences on the three aspects of the services assessed in this study. These vari- ables are at least partly controlled by the clinics them- selves, and clinics could therefore enhance parental satisfaction by reducing the waiting time, being accessi- ble during treatment, involving the parents and being attentive to their concerns. Authors’ contributions OH led the development of the questionnaire and was involved in data acquisition. All authors contributed to the design of the study. OH conducted the analyses and drafted the manuscript. All authors have made significant contributions by critically reviewing the manuscript and have read and approved the final version. Limitations of the study This study considered only the experiences of the par- ents, and not those of the children. However, there is evidence that the parents’ perception of the services is correlated to their children’s perception only to a lim- ited degree [2,14], and that children are more negative than their parents [2,16]. Other studies have shown that the correlation between parents’ and child’s satisfaction increases with the age of the child [24,25]. However, the parents’ perspective represents more than a proxy for the children’s experiences, since parents often form an integral part of the treatment process. Clinical data beyond the main diagnosis for each of the six axes were not available for this study. Informa- tion about comorbidity and multi-informant assessment of the child’s condition could have increased the appar- ent importance of the child’s condition as a predictor of parental experiences. Assessments before and after treat- ment might have provided a more valid measure of treatment outcome. References 1. Rey JM, Plapp JM, Simpson PL: Parental satisfaction and outcome: a 4- year study in a child and adolescent mental health service. Aust N Z J Psychiatry 1999, 33:22-28. y y 2. Godley SH, Fielder EM, Funk RR: Consumer satisfaction of parents and their children with child/adolescent mental health services. Eval Program Plann 1998, 21:31-45. 3. Young SC, Nicholson J, Davis M: An Overview of Issues in Research on Consumer Satisfaction with Child and Adolescent Mental Health Services. J Child Fam Stud 1995, 4:219-238. 3. Young SC, Nicholson J, Davis M: An Overview of Issues in Research on Consumer Satisfaction with Child and Adolescent Mental Health Services. J Child Fam Stud 1995, 4:219-238. doi:10.1186/1752-4458-5-22 Cite this article as: Holmboe et al.: Determinants of parents’ experiences with outpatient child and adolescent mental health services. International Journal of Mental Health Systems 2011 5:22. 4. Measelle JR, Weinstein RS, Martinez M: Parent satisfaction with case managed systems of care for children and youth with severe emotional disturbance. J Child Fam Stud 1998, 7:451-467. 5. Riley SE, Stromberg AJ, Clark J: Assessing parental satisfaction with children’s mental health services with the youth services survey for families. J Child Fam Stud 2005, 14:87-99. 6. Steinhausen HC: Parental satisfaction with achievements and experiences within the scope of a child and adolescent psychiatric polyclinic (in German). Prax Kinderpsychol Kinderpsychiatr 1983, 32:286-292. 6. Steinhausen HC: Parental satisfaction with achievements and experiences within the scope of a child and adolescent psychiatric polyclinic (in German). Prax Kinderpsychol Kinderpsychiatr 1983, 32:286-292. y y 7. Brannan AM, Sonnichsen SE, Heflinger CA: Measuring satisfaction with children’s mental health services: validity and reliability of the satisfaction scales. Eval Program Plann 1996, 19:131-141. 7. Brannan AM, Sonnichsen SE, Heflinger CA: Measuring satisfaction with children’s mental health services: validity and reliability of the satisfaction scales. Eval Program Plann 1996, 19:131-141. 8. Bjorngaard JH, Wessel AH, Osborg OS, Hanssen-Bauer K: User satisfaction with child and adolescent mental health services: impact of the service unit level. Soc Psychiatry Psychiatr Epidemiol 2008, 43:635-641. 8. Bjorngaard JH, Wessel AH, Osborg OS, Hanssen-Bauer K: User satisfaction with child and adolescent mental health services: impact of the service unit level. Soc Psychiatry Psychiatr Epidemiol 2008, 43:635-641. 9. Gerkensmeyer JE: Examining parent satisfaction with services for children and adolescents with mental health problems. Dissertation Abstracts International: Section B: The Sciences and Engineering 2000, 60(8-B):3850. Competing interests h h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 24 June 2011 Accepted: 15 September 2011 Published: 15 September 2011 Received: 24 June 2011 Accepted: 15 September 2011 Published: 15 September 2011 Page 9 of 9 Page 9 of 9 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 References 10 H fli CA Si ki CG S h ll SH K ll h KJ P t/ i 10. Heflinger CA, Simpkins CG, Scholle SH, Kelleher KJ: Parent/caregiver satisfaction with their child’s Medicaid plan and behavioral health providers. Mental Health Services Research 2004, 6:23-32. 11. Kotsopoulos S, Elwood S, Oke L: Parent satisfaction in a child psychiatric service. Canadian Journal of Psychiatry/Revue Canadienne de Psychiatrie 1989, 34:530-533. 12. Gerkensmeyer JE, Austin JK: Development and testing of a scale measuring parent satisfaction with staff interactions. J Behav Health Serv Res 2005, 32:61-73. 13. Copeland VC, Koeske G, Greeno CG: Child and mother client satisfaction questionnaire scores regarding mental health services: race, age, and gender correlates. Res Soc Work Pract 2004, 14:434-442. 14. Garland AF, Haine RA, Boxmeyer CL: Determinates of youth and parent satisfaction in usual care psychotherapy. Eval Program Plann 2007, 30:45-54. 15. Crow R, Gage H, Hampson S, Hart J, Kimber A, Storey L, Thomas H: In The measurement of satisfaction with healthcare: implications for practice from a systematic review of the literature. Volume 6. Health Technology Assessment; 2002(32). 16. Barber AJ, Tischler VA, Healy E: Consumer satisfaction and child behaviour problems in child and adolescent mental health services. Journal of Child Health Care 2006, 10:9-21. 17. Pedersen PB, Bjerkan AM, Bjørngaard JH, Bremnes R, Halsteinli V, Hatling T, Kaspersen S, Lilleeng SE, Sitter M, Venner B, Waagan T: SAMDATA sectorial report for psychiatric health care services 2006 [in Norwegian]. Trondheim, SINTEF helse; 2007. 18. Särndal CE, Swensson B, Wretman J: Model Assisted Survey Sampling New York: Springer; 2003. 19. Garratt A, Bjertnaes OA, Holmboe O, Hanssen-Bauer K: Parent Experiences Questionnaire for Outpatient Child and Adolescent Mental Health Services (PEQ-CAMHS Outpatients): reliability and validity following a national survey. Child and Adolescent Psychiatry and Mental Health 2011, 5. Holmboe et al. International Journal of Mental Health Systems 2011, 5:22 http://www.ijmhs.com/content/5/1/22 References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 20. World Health Organization: The ICD-10 classification of mental and behavioural disorders, clinical description and diagnostic guidelines Geneva; 1992. 21. World Health Organization: Multiaxial classification of child and adolescent psychiatric disorders Cambridge, Cambridge University Press; 1996. 22. Gerkensmeyer JE, Austin JK, Miller TK: Model testing: examining parent satisfaction. Arch Psychiatr Nurs 2006, 20:65-75. 23. Byalin K: Assessing parental satisfaction with children’s mental health services: A pilot study. Eval Program Plann 1993, 16:69-72. 23. Byalin K: Assessing parental satisfaction with children’s mental health services: A pilot study. Eval Program Plann 1993, 16:69-72.
https://openalex.org/W3217126537	https://zenodo.org/records/5762943/files/ZK_article_75539.pdf	English	null	Songs and morphology in three species of the Chorthippus biguttulus group (Orthoptera, Acrididae, Gomphocerinae) in Russia and adjacent countries	ZooKeys	2,021	cc-by	18,458	ZooKeys 1073: 21–53 (2021) doi: 10.3897/zookeys.1073.75539 https://zookeys.pensoft.net ZooKeys 1073: 21–53 (2021) doi: 10.3897/zookeys.1073.75539 https://zookeys.pensoft.net ZooKeys 1073: 21–53 (2021) doi: 10.3897/zookeys.1073.75539 https://zookeys.pensoft.net http://zoobank.org/A991F9BF-945B-4491-9123-6222298863EA http://zoobank.org/A991F9BF-945B-4491-9123-6222298863EA Citation: Tarasova T, Tishechkin D, Vedenina V (2021) Songs and morphology in three species of the Chorthippus biguttulus group (Orthoptera, Acrididae, Gomphocerinae) in Russia and adjacent countries. ZooKeys 1073: 21–53. https://doi.org/10.3897/zookeys.1073.75539 Abstract Songs and morphology are compared between Chorthippus miramae (Vorontsovsky, 1928) that was previ ously named as C. porphyropterus and two other closely related species, C. brunneus (Thunberg, 1815) and C. maritimus Mistshenko, 1951. We compare them because the calling song of C. miramae was previously shown to have song elements similar to those of other two species. One morphological character, the length of stridulatory file, appeared to be the best character to distinguish between all three species. For C. maritimus and C. miramae, we present the morphological descriptions since they are absent in the li terature. We also establish the synonymy C. maritimus = C. bornhalmi Harz, 1971, syn. n. = C. biguttulus eximius Mistshenko, 1951, syn. n. In the song analysis, we analyse not only the sound but also the leg- movement pattern, which is very helpful to find a homology between various song elements. We show that the calling song of C. miramae usually contains two elements, one element being similar to the C. brun- neus calling song, and another – to the C. maritimus calling song. Despite some similarities, the calling song elements in C. miramae have some peculiarities. The courtship song of C. miramae is similar to the C. brunneus song, whereas the rivalry songs of C. miramae comprise both the maritimus-like elements and the unique ones. C. miramae generally demonstrates a richer song repertoire than the other two species. Songs and morphology in three species of the Chorthippus biguttulus group (Orthoptera, Acrididae, Gomphocerinae) in Russia and adjacent countries Tatiana Tarasova1, Dmitry Tishechkin2, Varvara Vedenina1 1 Institute for Information Transmission Problems, Russian Academy of Sciences, Bolshoy Karetny per.19, Mos- cow 127051 Russia 2 Department of Entomology, Faculty of Biology, Moscow State University, Leninskie Gory, Moscow 119234 Russia Corresponding author: Varvara Vedenina (vedenin@iitp.ru) Academic editor: T. Robillard \| Received 20 September 2021 \| Accepted 30 October 2021 \| Published 29 November 2021 Copyright Tatiana Tarasova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction In singing Orthoptera, the song is an important component of reproductive isolation. Acoustic signals are often used in taxonomy, when sibling species are similar in mor phology, but different in songs. In grasshoppers of subfamily Gomphocerinae, the song is produced by stroking the stridulatory file of each hind femur across a raised vein on the fore wing. It is noteworthy that using both hind legs, the grasshoppers have two separate sound-producing devices, which must be coordinated with one another. The stridulatory movements of the two legs often differ in amplitude and pattern, and the legs can exchange roles from time to time, which leads to an increase of song comple xity (e.g., Elsner 1974; Helversen and Elsner 1977; Helversen and Helversen 1994). To distinguish cryptic grasshopper species, not only the sound recordings but also the recordings of the leg movements are used by various authors (Helversen 1986; Gotts berger and Mayer 2007; Vedenina and Helversen 2003, 2009; Willemse et al. 2009; Vedenina et al. 2012; Tarasova et al. 2021). Closely related grasshopper species belonging to the Chorthippus biguttulus group offer an excellent example of the cryptic species complex that can only be reliably identified by the male calling songs (Ragge and Reynolds 1988, 1998; Helversen 1989; Ragge et al. 1990; Bukhvalova 1993, 1998; Ingrisch 1995; Willemse et al. 2009; Sirin et al. 2010). This group includes four species with large ranges across Europe and Asia: C. biguttulus (Linnaeus, 1758), C. brunneus (Thunberg, 1815), C. mollis (Charpen tier, 1825), and C. maritimus Mistshenko, 1951. Other species of this group with smaller ranges occur in southern Europe, namely, C. jacobsi Harz, 1975 and C. yersini Harz, 1975 in the Iberian Peninsula, C. rubratibialis Schmidt, 1978 in Italy and C. bornhalmi Harz, 1971 in the Balkans. Two additional species are endemic to Greece (Willemse et al. 2009) and two more to Anatolia (Sirin et al. 2010). Several species and subspecies only occur in Russia and adjacent territories, in particular, C. porphyropterus (Vorontsovsky 1928) (Benediktov 1999, 2005).h The main subject of the current study is one species of the biguttulus group, C. por- phyropterus, which we name as C. miramae (Vorontsovsky, 1928 nec Ramme, 1936, 1951), and two closely related species, C. brunneus and C. maritimus, whose songs resemble song elements of C. miramae. Since in Russia and adjacent countries C. brun- neus, C. maritimus and C. Introduction miramae often occur with two other species of the biguttulus group, C. biguttulus and C. mollis, we describe the main morphological differences from the latter two species as well. Keywords Calling song, courtship song, grasshoppers, leg-movement pattern, rivalry song, stridulatory file Copyright Tatiana Tarasova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 22 Materials and methods Localities where material was collected are shown in Fig. 1. All localities were num bered and all numbers are listed in Results, in the paragraph “Material examined”. On the map, however, only localities with song recordings are numbered. Songs and morphology in Chorthippus biguttulus group 23 Figure 1. Map of localities where the specimens of Chorthippus brunneus (green triangles), C. maritimus (red squares) and C. miramae (blue circles) were collected. The localities with song recordings are num bered and marked by filled icons. Figure 1. Map of localities where the specimens of Chorthippus brunneus (green triangles), C. maritimus (red squares) and C. miramae (blue circles) were collected. The localities with song recordings are num bered and marked by filled icons. Morphological analysis In all specimens studied, we measured the following morphological characters: the lengths of pronotum, forewing and hind femur, the width of costal and subcostal (C & Sc) areas of fore wing, the distance from the center of stigma to the tip of fore wing, the length of stridulatory file and the distance from the most distal stridulatory peg to the tip of knee (Table 1, Fig. 2). In 10 specimens of each sex and species, the body length, the width of fore wing and the number of stridulatory pegs were measured. These morphological features have been chosen on the basis of the literature (Ragge et al. 1988; Bukhvalova 1993; Benediktov 1999; Willemse et al. 2009).The length of pronotum was measured along the midline. The length of forewing was measured from the humeral plate to the tip of the wing; the widths of the C & Sc areas were measured at the point where costal area was of the greatest width (Fig. 2B). The length of hind femur was measured from the anterior margin of the upper basal lobe to the hind margin of the upper knee-lobe; the length of stridulatory file was measured from the most proximal peg to the most distal peg; the distance between stridulatory file to the tip of the knee was measured from the most distal peg to the hind margin of the upper knee-lobe (Fig 2C). Morphological studies were carried out with an MBS-9 light microscope at 8–56× magnification using an ocular micrometer. Material for the morphological analysis was taken from the Zoological Museum of Moscow State University (ZMMU) and the personal collections of V. Vedenina (CV). All statistical analyses were performed using Excel 2016 and STATISTICA 12.0.0. To visualize and clarify the differences in morphology between the three species, a principal component analysis (PCA) was applied to 6 morphological cha racters (Fig. 3E). Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 24 Figure 2. Morphology of fore wing and hind leg in Chorthippus miramae (Vorontsovsky) from Orenburg region A fore wing with complete venation B fore wing with main veins; C hind leg. The measured mor phological characters are indicated with arrows and brackets. Songs and morphology in Chorthippus biguttulus group 25 Table 1. Morphological measurements in three species of the Chorthippus biguttulus group. For each character, mean, standard deviation, min and max are shown. Abbreviations in brackets see in Fig. 2. Table 1. Morphological measurements in three species of the Chorthippus biguttulus group. For each character, mean, standard deviation, min and max are shown. Abbreviations in brackets see in Fig. 2. Number of specimens Males Females C. miramae C. maritimus C. brunneus C. miramae C. maritimus C. brunneus 133 122 53 50 28 35 Length of pronotum, mm 3.14±0.25 2.60–3.70 3.25±0.23 2.80–3.60 3.06±0.15 2.80–3.50 4.17±0.36 3.50–4.90 4.26 ±0.25 3.80–4.80 3.89±0.27 3.40–4.40 Length of fore wing, mm 14.06±0.89 12.10–16.30 14.87±1.09 12.50–16.60 14.30±0.82 12.40–15.70 17.49±1.46 12.40–19.90 17.78±1.18 15.20–20.70 17.12±1.29 14.30–19.20 Length from stigma to tip of fore wing, mm 6.07±0.64 4.30–7.70 5.90±0.69 4.50–7.30 5.61±0.38 4.60–6.20 8.38±0.73 6.60–10.80 7.47±0.97 5.80–9.90 6.75±0.99 4.10–8.30 Width of C & Sc areas, mm 10.53±0.96 7.00–13.00 9.52±0.84 7.50–11.00 9.05±0.61 7.50–10.00 7.58±0.65 6.00–9.00 7.20±0.75 6.00–9.00 7.10±0.64 6.00–9.00 Length of hind femur, mm 10.09±0.58 8.90–11.7 10.21±0.61 9.20–12.80 9.57±0.41 8.70–10.70 13.21±1.09 9.30–15.20 13.48±0.99 11.90–15.40 12.21±0.99 10.20–14.40 Length of stridulatory file, mm 5.78±0.87 3.10–7.45 4.41±0.55 3.40–6.30 3.13±0.25 2.70–3.90 7.35±0.76 5.70–9.20 5.45±1.04 3.50–8.50 4.29±0.99 3.10–7.70 Length from last distal peg to tip of knee, mm 2.73±0.69 1.60–5.05 4.11±0.55 2.20–5.50 4.74±0.29 4.20–5.40 3.63±0.57 2.60–4.90 5.63±0.69 4.10–7.00 5.75±1.00 2.30–7.40 converted with a PC card L-305 (L-Card Ltd., Russia). The ambient temperature near a singing male in the field was 20–40°C. converted with a PC card L-305 (L-Card Ltd., Russia). The ambient temperature near a singing male in the field was 20–40°C. i During stridulation of the males studied in laboratory, both the sound and the hind leg movements were recorded with a custom-built opto-electronic device (Helversen and Elsner 1977; Hedwig 2000). A piece of reflecting foil was glued to the distal part of each hind leg femur of a male and two opto-electronic cameras were focused on the illuminated reflecting dots. Each camera was equipped with a position-sensitive photodiode that converted the upward and downward movements of the hind legs into voltage signals. These signals, together with the recordings of the sounds (a micro phone type 4191, ½ inch; a conditioning amplifier type 2690; Brüel & Kjaer, Nærum, Denmark), were A/D-converted with a custom-built PC card. The sampling rate was 1325 Hz for recording the stridulatory movements and 100 kHz for sound recordings. In the laboratory, the ambient temperature near a singing male was 30–32°C. Song recordings and analysis The calling song was recorded from an isolated male; the courtship song was recorded when a male was sitting near a female; the rivalry song was recorded from males sitting near each other. Recordings of the calling and rivalry songs in the field were carried out with a MD-382 microphone (upper frequency limit 12.5 kHz; before 2008), or a Spirit IM-01 microphone (upper frequency limit 20 kHz), and an Elektronika-302-1 cassette recorder (upper frequency limit 10 kHz; before 2005), or a Sony Walkman MZ-NH900 minidisk recorder (sampling frequency 44.1 kHz). The signals were A/D Songs and morphology in Chorthippus biguttulus group Nomenclatural notes The names Stauroderus mollis porphyroptera and S. miramae (both currently included in the subgenus Glyptobothrus Chopard, 1951) were described by Vorontsovsky (1928a, b) in two papers on grasshoppers from Orenburg published in the same issue. S. mollis porphyroptera was described as a new variation and designated as a var. nov.; therefore, the authorship of Vorontsovsky in this case is beyond doubt (Vorontsovsky 1928a, p. 12). Vorontsovsky attributed the authorship of the S. miramae to Ramme, with the following comment: “For the identification of this species, as well as the form, I identi fied as a variety of the species Stauroderus mollis, I take the opportunity to express here my deep gratitude to E.F. Miram, who informed me that S. miramae has just been described from Crimea by Dr. Ramme as a new species.” (Vorontsovsky 1928a: 12, footnote). Actually, Ramme mentioned Chorthippus miramae for the first time only in 1939 without a description, specifying that this species “will be described in the near future” (Ramme 1939: 131). Therefore, the name C. miramae Ramme, 1939 is sug gested to be a nomen nudum. Only in 1951, Ramme described this species based on material from Ukraine, Crimea, Southern and South-eastern European Russia, Cauca sus, and Transcaucasia, with the type locality in Southern Crimea (Ramme 1951). On the other hand, Vorontsovsky (1928b) presented a short description of C. miramae. For this reason, he is the author of the taxon from Orenburg in spite of the fact that he attributed the authorship to Ramme. Further, the study of signals showed that C. miramae Vorontsovsky and C. miramae Ramme represent the different species (see below). Summarizing the following three taxa were described in the papers mentioned above: C. mollis porphyroptera (Vorontsovsky 1928) from the type locality in Oren burg, C. miramae (Vorontsovsky, 1928) from the type locality in Orenburg, and C. mi- ramae Ramme, 1951 from the type locality in Southern Crimea. According to the study of Bukhvalova (1993) based on investigation of the male songs, the Chorthippus biguttulus group includes 5 species in Russia: C. biguttulus (Lin naeus, 1758), C. brunneus (Thunberg, 1815), C. mollis (Charpentier, 1825), C. mi- ramae Ramme, 1939 and C. yersini Harz, 1975. The study of the songs of specimens from Crimea, Southern European Russia, North Caucasus, Central Asia, and the Rus sian Far East showed that C. Songs and morphology in Chorthippus biguttulus group y p g g All recordings were analyzed with COOLEDIT 2.0 (Syntrillium, Seattle, WA) and TURBOLAB 4.0 (Bressner Technology, Gröbenzell, Germany). All statistical analyses were performed using Excel 2016 and STATISTICA 12.0.0. For the song description we used the following terminology (Figs. 4, 6): pulse – the sound produced by one stroke of a hind leg (the shortest measurable unit or the first- order unit); syllable – the sound produced by one complete up and down movement of the hind legs, starting when the legs leave their initial position and ending when the legs return to their original position and representing the repeated unit of a stable structure (the second-order unit); echeme – series of consistent syllables separated by pauses (the third-order unit). We measured three characters in C. brunneus (echeme rate, echeme duration and pulse rate), four characters in C. maritimus (echeme rate and duration and syllable rate and duration) and seven characters in C. miramae (echeme rate, echeme duration and pulse rate for the brunneus-like echeme and echeme rate and duration Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 26 and syllable rate and duration for the maritimus-like echeme). To visualize and clarify the differences in calling song between the three species, a PCA was applied to 5 song characters (Fig. 5E). We did not use echeme rate for both types of echemes because not all recorded males produced several echemes. When a character was equal to 0, we changed it to 0.01 by convention because we only used the logarithmic values for PCA. Nomenclatural notes miramae Ramme, 1939 sensu Bukhvalova (1993) is a good species, which is widespread throughout the southern part of Russia and adjacent terri tories. It was described as C. biguttulus meridionalis Mistshenko, 1950 from mountains Songs and morphology in Chorthippus biguttulus group 27 of Central Asia (Mistshenko 1950), as C. miramae Ramme, 1951 from Crimea, and as C. maritimus Mistshenko, 1951 from the Russian Far East (Bey-Bienko and Mist shenko 1951). However, it differs from the taxa described by Vorontsovsky (1928a, b) from Orenburg (Bukhvalova 1998). The name C. biguttulus meridionalis Mistshenko, 1950 is invalid, since it is a junior homonym of C. bicolor var. meridionalis (Fruhstor fer, 1921). The name C. miramae Ramme, 1951 is a junior homonym of C. miramae (Vorontsovsky, 1928). As a result, the valid name of this taxon should be C. maritimus Mistshenko, 1951. It should be also noted that some authors improperly considered the date of publication of the name C. miramae Ramme to be 1939 (Bey-Bienko and Mistshenko 1951; Harz 1975; Bukhvalova 1993; Wosnessenskij 1996) and treated this taxon as a subspecies of C. brunneus (Bey-Bienko and Mistshenko 1951; Harz 1975). p C. bornhalmi Harz, 1971 was described from Croatia in the Balkans and has been shown to occur from Italy to Turkey (Willemse et al. 2009; Sirin et al. 2010). The range of C. maritimus extends from southern Ukraine to the Russian Far East. In the current study, we compare the morphology and songs in C. bornhalmi (from Bulgaria and Greece) and C. maritimus, and establish the synonymy C. maritimus Mistshenko, 1951 = C. bornhalmi Harz, 1971, syn. n. y C. biguttulus eximius Mistshenko, 1951 was described from Sukhumi, Abkhazia (Mistshenko 1951). A study of songs from the environs of the type locality (loc. 34 in Fig. 1) showed that this subspecies also is identical to C. maritimus. Since C. maritimus (as C. biguttulus maritimus) and C. biguttulus eximius were described in the same pa per, we choose a valid name C. maritimus for this species and establish the synonymy C. maritimus = C. biguttulus eximius syn.n. g y C. miramaellus Wosnessenskij, 1996 and C. sinuatus Mistshenko and Wosnessen skij, 1996 proposed by Wosnessenskij (1996) to replace C. miramae Ramme, 1951 and C. biguttulus meridionalis Mistshenko, 1950 respectively, are the junior synonyms of C. maritimus (Bukhvalova 1998). We suggest that C. Nomenclatural notes maritimus tsejensis Bukhvalova, 1993 from North Ossetia, North Caucasus (Bukhvalova 1993) and C. meridionalis karakalen- sis Sytshev and Woznesenskij, 1995 from South-western Turkmenistan (Sychov and Voznesensky 1995) also belong to C. maritimus; however, additional studies are needed to clarify their status. It should be noted that M.M. Sychov and A.Yu. Voznesensky transliterated their own names in different ways in different papers, both in English and Latin; here we present their original spellings from the corresponding papers. p g p g p g p p Benediktov (1999) reinvestigated material from Orenburg used by Vorontsovsky and concluded that C. mollis porphyroptera (Vorontsovsky, 1928) and C. miramae (Vo rontsovsky, 1928) are synonyms. Benediktov (1999) compared the lengths of stridu latory files (the most characteristic feature of this species) in the type specimens of Vorontsovsky and found them to be identical. He proposed C. porphyropterus as the valid name, raising its rank, and changing its gender ending. However, according to chapter 24 of the International Code of Zoological Nomenclature (1999), when syno nyms are established simultaneously, but are proposed at different ranks, the name proposed at a higher rank takes precedence. Consequently, the valid name of the taxon from Orenburg should be C. miramae (Vorontsovsky, 1928). Also, Benediktov (1999) established the synonymy C. porphyropterus = C. biguttulus forma tomensis Berezhkov, Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 28 1956, proposed the new combination C. porphyropterus euchedickei Helversen, 1989 for C. biguttulus euchedickei Helversen, 1989, and pointed out that C. yersini Harz, 1975 sensu Bukhvalova, 1993 is conspecific with C. miramae (Vorontsovsky, 1928). The true identity of C. biguttulus forma tomensis described known only from the bank of the Tom’ River near Ust’-Iskitim, ca. 85 km south of Tomsk, Western Siberia (Be rezhkov 1956), requires confirmation from song recordings from the type locality. The combination C. biguttulus euchedickei was restored by Willemse et al. (2009). The conspecificity of C. yersini sensu Bukhvalova, 1993 nec Harz, 1975 and C. miramae (Vorontsovsky, 1928) are absolutely correct. Later on, Benediktov (2005) established the synonymy C. porphyropterus = C. brunneus mistshenkoellus Oliger, 1974 on the basis of investigation of the types of C. brunneus mistshenkoellus Oliger, 1974 from Tolyatti, Samara region. However oscil lograms of the song of C. maritimus from Tolyatti (Benediktov and Mikhailenko 2017) cast doubt on this synonymy. The status of C. brunneus (Thunberg, 1815) is unambiguous. Nomenclatural notes In addition to the nominotypical subspecies, this species includes C. brunneus mistshenkoellus mentioned above and C. brunneus brevis Klingstedt, 1939 from Southern Finland (Klingstedt 1939), the statuses of which require further clarification.h i In the current paper, we consider the following three taxa: C. brunneus (Thunberg, 1815), C. maritimus Mistshenko, 1951, and C. miramae (Vorontsovsky, 1928). C. maritimus tsejensis Bukhvalova, 1993, C. meridionalis karakalensis Sytshev et Woznesen skij, 1995, C. brunneus mistshenkoellus Oliger, 1974, and C. brunneus brevis Klingstedt, 1939 are excluded from the consideration since their statuses are unclear. Chorthippus brunneus (Thunberg) Gryllus brunneus Thunberg, 1815: 256. Gryllus brunneus Thunberg, 1815: 256. Material examined. Bulgaria: 4 Sofia region, lake Iskyr, 29.VI.2002, 1 ♂ 5 ♀, leg. V. Vedenina, song recordings in 1 ♂ (CV); Moldova: 10 Vinnitza region, Volchinetz, ab. 5 km W Mogilev-Podol’sky, 17.VII.1997, 1 ♂, leg. V. Vedenina (CV); Roma- nia: 11 Dobrudzha region, 14 km S Constantza, Ephoria-Nord, 09.IX.1997, 2 ♂ 3 ♀, leg. A. Loginov (ZMMU); Ukraine: 8 Ivano-Frankovsk region, environs of Mikulichin, 09–14.VIII.1996, 6 ♂ 1 ♀, leg. V. Vedenina (CV); 9 Khmelnitsky re gion, 28 km NNW of Kamenetz-Podolsky, near Beloe, 25.VI.2010, 1 ♂ 1 ♀, leg. V. Vedenina, song recordings in 1 ♂ (CV); 12 Odessa region, Kiliya district, environs of Vilkovo, 30.VI.1997, 2 ♂, leg. V. Vedenina (CV); 13 Odessa region, ab. 30 km NW of Belgorod-Dnestrovsky, near Krasnaya Kosa village, 29.VI.1997, 1 ♂, leg. V. Vedenina (CV); 16 Nikolaev region, Pervomaisk district, surr. of Kuripchino vil lage, 27.06.1997, 1♂, leg. V. Vedenina (CV); 18 Cherkassy region, Kanev district, Kanev reserve, 12–18.VI.1996, 12 ♂ 5 ♀, leg. V. Vedenina (ZMMU); 19 Kirovo grad region, environs of Aleksandriya, 04.VII.1997, 2 ♂ 2 ♀, leg. V. Vedenina (CV); 20 Nikolaev region, Pervomaisk district, environs of Kuripchino village, beach of Songs and morphology in Chorthippus biguttulus group 29 Yuzhny Bug river, 27.VI.1997, 1 ♂ 1 ♀, leg. V. Vedenina, song recordings in 2 ♂ (CV); 22 Poltava region, Mirgorod district., V.Sorochintzy, 27–28.VI.1985, 4 ♂ 5 ♀, 25–28.VII.1993, 3 ♂ 5 ♀, 24.VII–26.VIII.1994, 5 ♂, leg. V. Vedenina, song re cordings in 6 ♂ (ZMMU, CV); 25 Dnipro region, Pavlograd district, Samara reserve, 12–15.VII.1996, 4 ♂ 4 ♀, leg. V. Vedenina (CV); Russia: 1 Kaliningrad region, environs of Svetlogorsk, forest road, 16.VIII.2005, 3 ♂ 1 ♀, leg. N. Kulygina, song recordings in 1 ♂ (CV); 14 St-Peterburg, 27.08.1997, 1 ♂, leg. V. Vedenina (CV); 32 Voronezh region, Novaja Usman’ district, near Maklok village, 29.VI.2006, 3 ♂, leg. N. Kulygina (CV); 35 Kostroma region, Manturovo district, environs of. Ano sovo, 07–08.VIII.2009, 2 ♂ 1 ♀, leg. V. Vedenina, song recordings in 2 ♂ (CV); 40 Saratov region, Krasny Kut district, near D‘yakovka, 17.VII.2004, 3 ♂, leg. D. Tishechkin, song recordings in 2 ♂ (ZMMU). Yuzhny Bug river, 27.VI.1997, 1 ♂ 1 ♀, leg. V. Vedenina, song recordings in 2 ♂ (CV); 22 Poltava region, Mirgorod district., V.Sorochintzy, 27–28.VI.1985, 4 ♂ 5 ♀, 25–28.VII.1993, 3 ♂ 5 ♀, 24.VII–26.VIII.1994, 5 ♂, leg. V. Gryllus brunneus Thunberg, 1815: 256. Vedenina, song re cordings in 6 ♂ (ZMMU, CV); 25 Dnipro region, Pavlograd district, Samara reserve, 12–15.VII.1996, 4 ♂ 4 ♀, leg. V. Vedenina (CV); Russia: 1 Kaliningrad region, environs of Svetlogorsk, forest road, 16.VIII.2005, 3 ♂ 1 ♀, leg. N. Kulygina, song recordings in 1 ♂ (CV); 14 St-Peterburg, 27.08.1997, 1 ♂, leg. V. Vedenina (CV); 32 Voronezh region, Novaja Usman’ district, near Maklok village, 29.VI.2006, 3 ♂, leg. N. Kulygina (CV); 35 Kostroma region, Manturovo district, environs of. Ano sovo, 07–08.VIII.2009, 2 ♂ 1 ♀, leg. V. Vedenina, song recordings in 2 ♂ (CV); 40 Saratov region, Krasny Kut district, near D‘yakovka, 17.VII.2004, 3 ♂, leg. D. Tishechkin, song recordings in 2 ♂ (ZMMU). Distribution. (Fig. 1). The range of this species extends from Europe to the south-western part of European Russia. In Europe this species occurs over a wide range, excluding the central and southern part of the Iberian Peninsula and Greece (Ragge and Reynolds 1988, Sirin et al. 2010). Further to the east, it occurs in the Baltic republics, Belarus, Moldova, and Ukraine. The eastern border of the range lies on the longitude of the Saratov and Kostroma regions of Russia. The species tends to be mesophilic. The range of C. brunneus overlaps with that of C. maritimus in south-eastern Europe, Moldova, Ukraine, and the south-eastern part of Euro pean Russia. Recognition. (Table 1, Fig. 3). The males of C. brunneus can be distinguished from the males of C. miramae and C. maritimus by a short stridulatory file (Fig. 3A). This, however, is not applicable to the females (Fig. 3B). Both sexes of C. brunneus are characterized by the lowest number of stridulatory pegs (58–93 in ♂, 51–95 in ♀.). In comparison with C. miramae and C. maritimus, both sexes of C. brunneus tend to have the shortest pronotum, the narrowest C & Sc areas of fore wing, and the stigma closest to the wing tip (Table 1). The PCA applied to 6 characters shows a substantial overlap between C. brunneus and C. maritimus (Fig. 3C, D). In PCA, however, we do not use the number of stridulatory pegs, since this value was measured for a small number of males. Meanwhile, it was previously shown that C. brunneus can be easily distinguished from all other species of the C. Gryllus brunneus Thunberg, 1815: 256. biguttulus group by the lowest number of stridulatory pegs, especially in nominate subspecies (Oliger 1974; Ragge and Reynolds 1988; Bukhvalova 1993; Willemse et al. 2009). Recognition. (Table 1, Fig. 3). The males of C. brunneus can be distinguished from the males of C. miramae and C. maritimus by a short stridulatory file (Fig. 3A). This, however, is not applicable to the females (Fig. 3B). Both sexes of C. brunneus are characterized by the lowest number of stridulatory pegs (58–93 in ♂, 51–95 in ♀.). In comparison with C. miramae and C. maritimus, both sexes of C. brunneus tend to have the shortest pronotum, the narrowest C & Sc areas of fore wing, and the stigma closest to the wing tip (Table 1). The PCA applied to 6 characters shows a substantial overlap between C. brunneus and C. maritimus (Fig. 3C, D). In PCA, however, we do not use the number of stridulatory pegs, since this value was measured for a small number of males. Meanwhile, it was previously shown that C. brunneus can be easily distinguished from all other species of the C. biguttulus group by the lowest number of stridulatory pegs, especially in nominate subspecies (Oliger 1974; Ragge and Reynolds 1988; Bukhvalova 1993; Willemse et al. 2009). Calling song (Table 2, Figs. 4, 5). The calling song of C. brunneus consists of seve ral short echemes repeated at the rate of about 0.3–2.1 /s. Each echeme lasts on ave rage 0.1–0.4 s and has a relatively stable temporal structure. It consists of short pulses, which are grouped into 4–7 syllables (Fig. 4C). The gaps between the subsequent syl lables can’t be traced by the sound analysis, but they can be distinguished by the analy sis of the leg movements. The two legs are moved with a large phase shift, and some times almost alternately (Fig. 4E). Each leg generates one short pulse during a straight upstroke, whereas two short pulses are produced during a two-step downstroke. The pulse duration and the pulse rate vary in the ranges of 7–8 ms and 91–111/s, respec tively (at the temperature 29–30°C). The population from loc. 40, shows an extremely Tatiana Tarasova et al. Gryllus brunneus Thunberg, 1815: 256. Morphological differences between Chorthippus brunneus (green dots), C. maritimus (red dots), and C. miramae (blue dots). A,B length of stridulatory file vs. distance from the last stridulatory peg to the tip of knee in males (A) and females (B) C,D results of Principal Component Analysis based on 6 characters are shown for PC 1 and PC 2 in males (C) and females (D) E loadings of different characters to PC 1 and PC 2. long echeme duration and low echeme and pulse rate (Table 2). Notably, the values are relatively stable within the same population.h long echeme duration and low echeme and pulse rate (Table 2). Notably, the values are relatively stable within the same population.h Courtship and rivalry songs. The courtship and rivalry (Fig. 4F, G) songs of C. brunneus are similar to the calling song. Gryllus brunneus Thunberg, 1815: 256. / ZooKeys 1073: 21–53 (2021) 30 A length of stridulatory f ile, mm f o pit d n a g e p la t sid n e e w t e b e c n a t sid m m , e e n k 2 3 4 5 6 7 8 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 B length of stridulatory f ile, mm f o pit d n a g e p la t sid n e e w t e b e c n a t sid m m , e e n k 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 C PC 1 2 C P -5 -4 -3 -2 -1 0 1 2 3 4 -5 -4 -3 -2 -1 0 1 2 3 4 5 D PC 1 2 C P -6 -5 -4 -3 -2 -1 0 1 2 3 4 -3 -2 -1 0 1 2 3 4 5 Morphological characters males females PC 1 PC 2 PC 1 PC 2 Length of pronotum 0,37 0,74 0,70 -0,43 Length of fore wing 0,28 0,90 0,58 -0,70 Distance from stigma to tip of fore wing 0,68 0,30 0,82 -0,13 Width of C & Sc areas 0,73 0,00 0,43 0,21 Length of stridulatory file 0,89 -0,31 0,86 0,35 Distance from last peg to end of knee -0,76 0,61 -0,57 -0,77 E A length of stridulatory f ile, mm f o pit d n a g e p la t sid n e e w t e b e c n a t sid m m , e e n k 2 3 4 5 6 7 8 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 B length of stridulatory f ile, mm f o pit d n a g e p la t sid n e e w t e b e c n a t sid m m , e e n k 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 8 B length of stridulatory f ile, mm f o pit d n a g e p la t sid n e e w t e b e c n a t sid m m , e e n k 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 A length of stridulatory f ile, mm f o pit d n a g e p la t sid n e e w t e b e c n a t sid m m , e e n k 2 3 4 5 6 7 8 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 B length of stridulatory f ile, mm A B C PC 1 2 C P -5 -4 -3 -2 -1 0 1 2 3 4 -5 -4 -3 -2 -1 0 1 2 3 4 5 D D PC 1 2 C P -6 -5 -4 -3 -2 -1 0 1 2 3 4 -3 -2 -1 0 1 2 3 4 5 C D C PC 1 D PC 1 Morphological characters males females PC 1 PC 2 PC 1 PC 2 Length of pronotum 0,37 0,74 0,70 -0,43 Length of fore wing 0,28 0,90 0,58 -0,70 Distance from stigma to tip of fore wing 0,68 0,30 0,82 -0,13 Width of C & Sc areas 0,73 0,00 0,43 0,21 Length of stridulatory file 0,89 -0,31 0,86 0,35 Distance from last peg to end of knee -0,76 0,61 -0,57 -0,77 E E Figure 3 Figure 3. Chorthippus maritimus Mistshenko Chorthippus miramae Ramme, 1939: 131, nomen nudum. Chorthippus miramae Ramme, 1939: 131, nomen nudum. Songs and morphology in Chorthippus biguttulus group 31 Table 2. Calling songs parameters of Chorthippus brunneus. For each parameter, medians, the lower and upper quartiles are shown. Locality Number of recorded males (measurements) Temperature, ˚ C echeme duration, s echeme rate, /s pulse rate, /s 1 1 (10) 32 0.2 0.7 125 0.1; 0.2 0.6; 0.9 115; 161 4 1 (9) 31–35 0.2 1.1 100 0.2; 0.2 0.7; 1.9 100; 122 9 1 (10) 24–25 0.2 0.7 143 0.2; 0.2 0.6; 0.8 143; 167 20 2 (16) 30 0.2 2.1 100 0.2; 0.2 1.0; 2.5 83; 111 22 6 (51) 29 0.2 1.1 91 0.2; 0.2 0.4; 1.3 83; 111 35 2 (20) 29–30 0.1 1.1 111 0.1; 0.1 0.7; 2.5 91; 129 40 2 (24) 28; 32–33 0.4 0.3 57 0.3; 0.5 0.2; 0.4 51; 66 Table 2. Calling songs parameters of Chorthippus brunneus. For each parameter, medians, the lower and upper quartiles are shown. Chorthippus meridionalis Mistshenko, 1950: 790. Chorthippus biguttulus maritimus Mistshenko, 1951: 514. Chorthippus miramae Ramme, 1951: 389. Chorthippus biguttulus eximius Mistshenko, 1951: 515, syn. n. Chorthippus bornhalmi Harz, 1971: 336, syn. n. Chorthippus miramaellus Woznessenskij, 1996: 204. Chorthippus sinuatus Mistshenko et Woznessenskij, 1996: 204. Chorthippus meridionalis Mistshenko, 1950: 790. Chorthippus biguttulus maritimus Mistshenko, 1951: 514. Chorthippus miramae Ramme, 1951: 389. Chorthippus biguttulus eximius Mistshenko, 1951: 515, syn. n. Chorthippus bornhalmi Harz, 1971: 336, syn. n. Chorthippus miramaellus Woznessenskij, 1996: 204. Chorthippus sinuatus Mistshenko et Woznessenskij, 1996: 204. Chorthippus miramaellus Woznessenskij, 1996: 204. Material examined. Bulgaria: 4 Sofia region, lake Iskyr, 29.VI.2002, 6 ♂ 5 ♀, leg. V. Vedenina (ZMMU); 5 Vraca region, ab. 3 km S of Vraca, Vracniki Balekan National Park, Memorial Botev, 30.VI.2002, 2 ♂, leg. V. Vedenina (CV); Greece: 2 Phthiotis, environs of Timfristos, NE slope, 27.V.1998, 1 ♂, leg. V. Vedenina (CV); 3 Phthiotis, ab 40 km NW Lamia environs of Lautra Kaitsas, 26.V.1998, 3 ♂ 1 ♀, leg. V. Vedenina (CV); 6 Macedonia, Drama, Mt Falakro above Volakas, 5 km NE Elatia, 24.VII.2004, 1 ♂, leg. V. Vedenina, song recordings in 2 ♂ (CV); 7 Macedonia, Drama, W. Rodopi, 5 km NE Elatia, 23.VII.2004, 1 ♂ 1 ♀, leg. V. Vedenina (CV); Ukraine: 15 Odessa region, near Sychavka, 03.VII.1997, 5 ♂, leg. V. Chorthippus maritimus Mistshenko Vedenina (ZMMU); 17 Kirovograd region, Novoukrainka district, environs of Pomoshnaya, 26.VI.1997, 2 ♂, leg. V. Ve denina, song recordings in 2 ♂ (CV); 21 Kherson region, Chernomorsky nature re serve, Solyonoozerny area, 25.VII–05.VIII.1995, 2 ♂ 1 ♀, leg. V. Vedenina (CV); 23 Crimea, Bakhchisaray district, 3–4 km E of Gluboky Yar, 11.VI.1997, 4 ♂, leg. D. Tishechkin, song recordings in 4 ♂ (ZMMU); 24 Crimea, Simferopol’ district, en virons of Pereval’noe, 20.VI.1997, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); 25 Dnipro region, Pavlograd district, Samara reserve, 12–15.VII.1996, 6 ♂, leg. V. Vedenina (CV); 26 Crimea, Kerch peninsula, E shore of Kazantip bay, en virons of cape Chagany, 26.VI.1997, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 27 Kharkov region, Izjum district, Kamyshevacha, 15.VII.1996, 5 ♂ 1 ♀, leg. V. Vedenina (ZMMU); 28 Kharkov region, Izjum, Kremenetz hill, 15.VII.1996, 1 Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 32 Figure 4. Oscillograms of calling songs A–E and rivalry songs F,G in Chorthippus brunneus from K troma region (A) Poltava region (B) and Saratov region (F). Song recordings are presented at four di ent speeds (faster oscillograms of the indicated parts of the songs shown in C,D,E,G). In all oscillogr the two upper lines are recordings of hind leg movements and the lower line is the sound record Different song parameters are indicated by brackets and arrows. The ambient temperature near a sin male was 29 – 32°C. Figure 4. Oscillograms of calling songs A–E and rivalry songs F,G in Chorthippus brunneus from Kos troma region (A) Poltava region (B) and Saratov region (F). Song recordings are presented at four differ ent speeds (faster oscillograms of the indicated parts of the songs shown in C,D,E,G). In all oscillograms the two upper lines are recordings of hind leg movements and the lower line is the sound recording. Different song parameters are indicated by brackets and arrows. The ambient temperature near a singing male was 29 – 32°C. ♂, leg. V. Vedenina (CV); Abkhazia: 34 Sukhumi region, slopes near highway Sukhumi – Gagra, 21–22.X.2005, 5 ♂ 5 ♀, leg. V. Vedenina, song recordings in 3 ♂ (ZMMU); Russia: 33 Krasnodarsky krai, near highway Krasnaya Poljana – Adler, 22.X.2005, 4 ♂ 3 ♀, leg. V. Vedenina, song recordings in 4 ♂ (CV); 39 Saratov, slopes near Polivanovka, 28.VI.2020, 2 ♂, leg. V. Chorthippus maritimus Mistshenko Vedenina, song recordings in 2 ♂ (CV); 41 Saratov region, Krasnokutsk district, near D’yakovka, 28.VI.2020, 6 ♂ 1 ♀, leg., song recordings in 5 ♂ (CV); 43 Saratov region, SW from Khvalynsk, environs of Ul’yanino village, 19.VII.2005, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); 44 Saratov Songs and morphology in Chorthippus biguttulus group 33 region, ab. 6 km NW of Ershov, 22.VI.2018, 3 ♂, leg. V. Vedenina (CV); 45 Saratov region, 15 km NE Ozinki, 23.VI.1996, 4 ♂, leg. D. Tishechkin, song recordings in 4 ♂ (ZMMU); 42 Krasnoyarsk region, Astrakhan‘ district, environs of Dosang railway sta tion, 03.VII.2000, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 75 Irkutsk region, Olkhon district, 20 km from Jelantsy to strait Olkhonskie vorota, 15.VII.2003, 4 ♂, leg. D. Tishechkin, song recordings in 4 ♂ (ZMMU); 77 Buryatia, Barguzin val ley, Ina river, 4 – 5 km downstream from Ina, 17.VII.2007, 3 ♂, leg. D. Tishechkin, song recordings in 2 ♂ (ZMMU); 78 Chita region, Klichka range, ab. 15 km W Kli chka, 22.VII.2003, 2 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 79 Amur region, 15 km S Svobodny, environs of Malaya Sazanka, 05.VII.1995, 4 ♂, leg. D. Tishechkin, song recordings in 4 ♂ (ZMMU); 80 Primorskiy kray, Pogranichny dis trict, environs of Barabash-Levada, 20.VII.1995, 3 ♂, leg. D. Tishechkin, song record ings in 3 ♂ (ZMMU); 81 Primorskiy kray, Pogranichny district, Khanka lake, 15 km S Turiy Rog, 21.VII.2006, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); 82 Southern Sakhalin, environs of Sokol, 02.VIII.2015, 4 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); Kazakhstan: 62 Almaty region, 40 km N from Almaty, environs of Kara-Oi village, 12.VI.2017, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 63 Almaty, botanical garden, 07.VII.1994, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); 65 Almaty region, ab. 20 km NE of Taldykorgan, 02.VII.2016, 4 ♂, 1 ♀, leg. V. Vedenina & T. Pushkar, song recordings in 1 ♂ (CV); 66 Kazakhstan, Almaty region, near Kapal, 01.VII.2016, 1 ♂, leg. V. Vedenina & T. Pushkar, song recordings in 1 ♂ (CV); 67 Kazakhstan, Almaty region, ab. 2.5 km W of Kapal, 02.VII.2016, 4 ♂ 4 ♀, leg. V. Vedenina & T. Chorthippus maritimus Mistshenko maritimus and C. brunneus overlap in Eastern Europe, Ukraine and the south-eastern part of European Russia. Moreover, C. maritimus and C. brunneus often occur syntopically. The range of C. maritimus also overlaps with the range of C. miramae in the south-eastern part of European Russia and in surroundings of the Baikal Lake, however, they do not occur in the same biotopes.h Recognition. (Table 1, Fig. 3). The males of C. maritimus can be distinguished from the males of C. brunneus by the longer stridulatory file (Fig. 3A) and the higher number of stridulatory pegs (see Description). These characters are also mentioned as the dis tinguishing features between C. brunneus and C. bornhalmi by other authors (Willemse et al. 2009; Skejo and Ivcovic 2015). The length of stridulatory file in C. maritimus is intermediate between those in C. miramae and C. brunneus. Both sexes of C. maritimus also tend to have the longest fore wings and pronotum in comparison with C. miramae and C. brunneus (Table 1). C. maritimus can be also distinguished from other species of the biguttulus group by the narrower costal area of fore wing. By contrast, C. maritimus differs from C. mollis by the wider costal area of fore wing and by the lower density of stridulatory pegs (Bukhvalova 1993; Oliger 1974). C. bornhalmi and C. biguttulus eximius are not different in morphology from C. maritimus from Ukraine and Russia.h f Description. (Table 1, Fig. 3). The head structure as in genus. Ratio length of verti cal diameter of eye to maximum length of foveolae 2.8–3.4 in ♂, 3.0–3.2 in ♀; ratio minimum interocular distance to length of subocular groove 0.6–0.8 in ♂, 0.7–0.9 in ♀. Antennae filiform. Prozona is slightly shorter than metazona; median carina is distinct and continuous. Lateral pronotal keels are distinctly incurved, ratio between minimum and maximum widths 2.3–2.6 in ♂, 2.3–2.9 in ♀. In western populations keels are more angled, min/max width ratio up to 3.0. Tympanal aperture slit-like, 2.3–2.8 times in ♂, 2.6–2.8 in ♀ as long as broad. Fore and hind wings well developed in both sexes, wings far surpassing the apices of the hind knee. Costal area of fore wing has maximum width in the middle part or in the last third of the wing. Subcostal area narrow, its width 0.25–0.3 mm in ♂, 0.15–0.2 mm in ♀ (measured on the line of maximal width of costal area). Chorthippus maritimus Mistshenko Pushkar, song recordings in 2 ♂ (ZMMU); 68 Urzhar region, 27 km SSE Taskesken, 5.5 km NW Karakol, 24.VI.2019, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); Turkmenistan: 49 Ahal region, Kaka district, 6–7 km S of Dushak, 14.V.2014, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); Kyrgyzstan: 51 Batken region, Leilek district, Turkestan range, 12 km S from Katran village, 11.VII.2014, 1 ♂, leg. D. Tishechkin, song record ings in 1 ♂ (ZMMU); 53 Batken region, N shore of Tortkul’skoye reservoir, 12 km WSW Batken, 09.VII.2014, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 54 Jalal-Abad region, Chatkal range, Sary-Chelek nature reserve, environs of Arkyt, 22.VII.2008, 2 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 57 Chuy region, Jayyl district, Karakol river, 10 km upstream from confluence with Suusamyr, 07.VII.2016, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 58 Chuy re gion, Djumgal river, between Baizak and Chaek, 30.VI.2014, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU); 64 Issyk-Kul‘ region, Tossor river, 18 km E from Kadji-Sai, 15.VII.2013, 1 ♂, leg. D. Tishechkin, song recordings in 1 ♂ (ZMMU). Distribution. (Fig. 1). C. maritimus is a widespread trans-Palearctic species. It includes C bornhalmi from the Balkans and Anatolia (Willemse et al 2009; Sirin et al Distribution. (Fig. 1). C. maritimus is a widespread trans-Palearctic species. It includes C. bornhalmi from the Balkans and Anatolia (Willemse et al. 2009; Sirin et al. 2010; Skejo et al. 2018) and as C. biguttulus eximius from Sukhumi, Abkhazia (Mist shenko 1951). It also occurs in Moldova and southern Ukraine (Heller et al. 1998). In the territory of Russia, its range stretches from Krasnodarsky krai to Sakhalin along the southern border. This species also occurs in Caucasus, southern Kazakhstan, Turk menistan, very likely Uzbekistan, Kyrgyzstan, Mongolia, northern-east China, Korea Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 34 and Japan (Storozhenko 2002). The ranges of C. maritimus and C. brunneus overlap in Eastern Europe, Ukraine and the south-eastern part of European Russia. Moreover, C. maritimus and C. brunneus often occur syntopically. The range of C. maritimus also overlaps with the range of C. miramae in the south-eastern part of European Russia and in surroundings of the Baikal Lake, however, they do not occur in the same biotopes.h and Japan (Storozhenko 2002). The ranges of C. Songs and morphology in Chorthippus biguttulus group 35 Calling song (Table 3, Figs. 5, 6). The calling song of C. maritimus usually contains one to several echemes of median duration ranged from 1 to 4 s. In some populations (49, 62, 63), however, the median echeme duration is higher, ranging between 5–11.1 s (Table 3, Fig. 5C). The echeme rate also greatly varies between different populations (0.05–0.42 / s). The number of syllables per echeme varies in the range of 15 to 40, in populations with prolonged echemes – in the range from 40 to 70. The syllable duration is relatively stable within the same population; however, its median duration can vary between the populations in the range of 86–162 ms (Fig. 5D). At the beginning of each echeme, the sound is very soft, but then it reaches maximum loudness after the first third of the echeme duration, being constant until the echeme end (Fig. 6D). The syllables are gener ated by the leg movements with a small phase shift, which comprise the straight upstroke and stepwise downstroke (Fig. 6E, F). Both upstroke and downstroke have the similar duration. The leg upstroke generates a noisy sound with unclear structure and slightly increasing amplitude; the stepwise downstroke generates 4–5 distinct pulses. The pulses, Table 3. Calling songs parameters of Chorthippus maritimus. For each parameter, medians, the lower and upper quartiles are shown. Chorthippus maritimus Mistshenko Ratio width of fore wing to C & Sc areas 3.1–3.5 in ♂, 4.4–4.7 in ♀. Apical constriction (distance from C and Sc confluence to the wing tip) prolonged, ratio length of apical constriction to the wing length 3.3–3.8 in ♂, 3.5–3.8 in ♀. Stigma far from the wing tip, ratio length between stigma center and the wing tip to the wing length 2.4–2.7 in ♂, 2.3–2.5 in ♀. Hind femur gracile, ratio femur length to maximum width 4.4–4.6 in ♂, 4.4–4.7 in ♀. Stridulatory file consists of one row, its length nearly equal to the distance between last peg and tip of hind knee. The number of stridulatory pegs 100–168 in ♂, 104–157 in ♀. Body coloration varies from light straw to dark brown, sometimes with a red tone. The ventral side of the body lighter than dorsal side, and densely pubescent. Fore wings smoky, with a few dark spots in M area. Hind wings transparent at the base and slightly smoky in apical part, distal half of C area smoky or brownish. Hind femur in the inner side with black lengthwise line. Hind knees dark brown or blackish, particularly on upper lobe. Hind tibiae orange or reddish. Measurements in mm. Body length: 15–18 in ♂, 19–26 in ♀, pronotum length: 3.1– 3.4 in ♂, 4.1–4.4 in ♀, fore wing length: 14.1–15.5 in ♂, in 17.2–18.5 in ♀, fore wing id h 3 1 3 4 i ♂3 2 3 5 i ♀hi d f l h 9 8 10 6 i ♂12 8 14 1 i ♀ Measurements in mm. Body length: 15–18 in ♂, 19–26 in ♀, pronotum length: 3.1– 3.4 in ♂, 4.1–4.4 in ♀, fore wing length: 14.1–15.5 in ♂, in 17.2–18.5 in ♀, fore wing width 3.1–3.4 in ♂, 3.2–3.5 in ♀, hind femur length: 9.8–10.6 in ♂, 12.8–14.1 in ♀. Songs and morphology in Chorthippus biguttulus group Songs and morphology in Chorthippus biguttulus group Locality Number of recorded males (measurements) Temperature, ˚ C echeme duration, s echeme rate, /s syllable duration, ms syllable rate, /s 6 2 (10) 30 4.0 0.19 103 8.5 3.6; 4.6 0.18; 0.19 99; 105 8.3; 9.1 17 2 (10) 32 1.0 0.20 129.5 9.4 0.9; 1.1 0.18; 0.25 127; 132 8.6; 10.0 23 4 (40) 31–35 1.7 0.25 102 9.3 1.5; 1.9 0.20; 4.5 96; 106 8.9; 9.5 24 3 (18) 24–25 1.4 0.3 104 8.8 1.2; 1.6 0.29; 0.34 102; 112 8.5; 9.0 34 3 (14) 30 2.8 0.42 136 7.0 2.0; 4.4 0.21; 0.46 119; 159 5.6; 8.1 39 2 (13) 29 2.1 0.24 103 9.2 1.4; 2.7 0.22; 0.27 100; 106 9.0; 9.4 41 5 (15) 29–30 2.4 0.20 100 9.3 2.0; 2.9 0.16; 0.22 95; 108 8.7; 9.9 43 3 (12) 28; 32–33 1.3 0.25 86 10.1 1.2; 1.7 0.22; 0.28 81; 124 7.2; 10.8 45 4 (12) 32–36 2.0 0.23 119 7.8 1.6; 2.3 0.19; 0.24 110; 127 7.2; 8.1 49 3 (25) 34–35 10.7 0.07 159 6.4 4.9; 12.2 0.06; 0.08 157; 165 6.3; 6.5 54 1 (10) 35–39 3.2 0.16 135 7.0 2.0; 5.2 0.13; 0.26 133; 136 6.9; 7.0 62, 63 4 (11) 30–32; 35 11.1 0.05 162 5.9 7.8; 11.5 0.04; 0.05 134; 164 5.7; 6.1 75 4 (12) 31 2.2 0.14 86 10.1 1.7; 5.2 0.13; 0.18 83; 94 9.7; 10.5 77 2 (15) 20; 27–30 2.5 0.13 133 7.0 1.7; 5.2 0.12; 0.23 124; 147 6.3; 7.7 79 4 (13) 31 2.0 0.14 90 10.3 1.7; 2.9 0.09; 0.18 87; 104 9.2; 10.7 80 3 (18) 38–40 2.1 0.13 87 10.9 1.9; 2.5 0.07; 0.16 85; 90 9.6; 11.1 82 3 (20) 35–40 2.3 0.15 88 10.5 1.8; 3.5 0.12; 0.18 85; 90 10.3; 11.2 e 3. Calling songs parameters of Chorthippus maritimus. For each parameter, medians, the lower an r quartiles are shown. Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 36 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 C. miramae C. maritimus C. brunneus 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0 20 40 60 80 100 120 140 160 180 200 220 C. miramae C. Songs and morphology in Chorthippus biguttulus group brunneus echeme duration, s C 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0 20 40 60 80 100 120 140 160 180 200 220 C. miramae C. maritimus C. brunneus syllable duration, ms D syllable duration, ms echeme duration, s D C PC 1 -5 -4 -3 -2 -1 0 1 2 -2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 PC 2 Calling song characters PC 1 PC 2 maritimus-like echeme duration 0,94 -0,30 maritimus-like syllable duration 0,94 -0,30 maritimus-like syllable rate 0,85 -0,46 brunneus-like echeme duration -0,72 -0,69 brunneus-like pulse rate -0,82 -0,56 E F Figure 5. Differences in calling songs between Chorthippus brunneus, C. maritimus, and C. miramae A–D box plots for the brunneus-like echeme duration (A) for the brunneus-like pulse rate (B) for the maritimus-like echeme duration (C) and the maritimus-like syllable duration (D) medians (dots), first and third quartiles (boxes), the 10th and 90th percentiles (whiskers), and outliers (dots beyond whiskers) are shown E results of Principal Component Analysis based on 5 song characters are shown for PC 1 and PC 2 in C. brunneus (green PC 1 -5 -4 -3 -2 -1 0 1 2 -2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 PC 2 Calling song characters PC 1 PC 2 maritimus-like echeme duration 0,94 -0,30 maritimus-like syllable duration 0,94 -0,30 maritimus-like syllable rate 0,85 -0,46 brunneus-like echeme duration -0,72 -0,69 brunneus-like pulse rate -0,82 -0,56 E F PC 1 -5 -4 -3 -2 -1 0 1 2 -2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 PC 2 E E E Figure 5. Differences in calling songs between Chorthippus brunneus, C. maritimus, and C. miramae A–D box plots for the brunneus-like echeme duration (A) for the brunneus-like pulse rate (B) for the maritimus-like echeme duration (C) and the maritimus-like syllable duration (D) medians (dots), first and third quartiles (boxes), the 10th and 90th percentiles (whiskers), and outliers (dots beyond whiskers) are shown E results of Principal Component Analysis based on 5 song characters are shown for PC 1 and PC 2 in C. brunneus (green dots), C. maritimus (red dots), and C. miramae (blue dots) F loadings of different characters to PC 1 and PC 2. however, can be sometimes fuzzy. The durations and rates of echeme and syllable in C. bornhalmi (from loc. Songs and morphology in Chorthippus biguttulus group maritimus C. brunneus echeme duration, s pulse rate /s 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0 2 4 6 8 10 12 14 16 C. miramae C. maritimus C. brunneus echeme duration, s 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0 20 40 60 80 100 120 140 160 180 200 220 C. miramae C. maritimus C. brunneus syllable duration, ms PC 1 -5 -4 -3 -2 -1 0 1 2 -2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 PC 2 Calling song characters PC 1 PC 2 maritimus-like echeme duration 0,94 -0,30 maritimus-like syllable duration 0,94 -0,30 maritimus-like syllable rate 0,85 -0,46 brunneus-like echeme duration -0,72 -0,69 brunneus-like pulse rate -0,82 -0,56 A B C D E F Figure 5. Differences in calling songs between Chorthippus brunneus, C. maritimus, and C. miramae A–D box plots for the brunneus-like echeme duration (A) for the brunneus-like pulse rate (B) for the maritimus-lik echeme duration (C) and the maritimus-like syllable duration (D) medians (dots), first and third quartile (boxes), the 10th and 90th percentiles (whiskers), and outliers (dots beyond whiskers) are shown E results o Principal Component Analysis based on 5 song characters are shown for PC 1 and PC 2 in C. brunneus (green dots), C. maritimus (red dots), and C. miramae (blue dots) F loadings of different characters to PC 1 and PC 2 h b f Th d d f h d ll bl C 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 C. miramae C. maritimus C. brunneus echeme duration, s l / A 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0 20 40 60 80 100 120 140 160 180 200 220 C. miramae C. maritimus C. brunneus pulse rate /s B B A 30 36 48 73 74 76 6 23 41 45 49 63 75 79 1 9 20 22 35 40 Locality 0 2 4 6 8 10 12 14 16 C. miramae C. maritimus C. Songs and morphology in Chorthippus biguttulus group 6) and in C. biguttulus eximius (from loc. 34) fall into the range of values in C. maritimus from several localities (Table 3, Fig. 5C, D). The syllable structure is also quite similar in C. bornhalmi (Fig. 6E) and C. biguttulus eximius (Fig. 6F). Songs and morphology in Chorthippus biguttulus group 37 ure 6. Oscillograms of calling songs A–F and rivalry songs G,H in Chorthippus maritimus from Pri skiy kray (A) Macedonia (B) Sukhumi region (C) and Saratov region (G). Song recordings are present three different speeds (faster oscillograms of the indicated parts of the songs shown in D,E,F,H). In a lograms the two upper lines are recordings of hind leg movements and the lower line is the sound re ing. The ambient temperature near a singing male was 33 – 34°C in (A) and 29 – 30°C in other cases 6. Oscillograms of calling songs A–F and rivalry songs G,H in Chorthippus maritimu Figure 6. Oscillograms of calling songs A–F and rivalry songs G,H in Chorthippus maritimus f morskiy kray (A) Macedonia (B) Sukhumi region (C) and Saratov region (G). Song recordings are ed at three different speeds (faster oscillograms of the indicated parts of the songs shown in D,E,F,H oscillograms the two upper lines are recordings of hind leg movements and the lower line is the s cording. The ambient temperature near a singing male was 33 – 34°C in (A) and 29 – 30°C in oth Figure 6. Oscillograms of calling songs A–F and rivalry songs G,H in Chorthippus maritimus from Pri morskiy kray (A) Macedonia (B) Sukhumi region (C) and Saratov region (G). Song recordings are present ed at three different speeds (faster oscillograms of the indicated parts of the songs shown in D,E,F,H). In all oscillograms the two upper lines are recordings of hind leg movements and the lower line is the sound re cording. The ambient temperature near a singing male was 33 – 34°C in (A) and 29 – 30°C in other cases. p gh p g g g Rivalry song (Fig. 6G, H). The rivalry song of C. maritimus contains echemes of a shorter duration than the calling song. In some males the first syllable of the rivalry echeme lasts 1.5–2 times as long as the subsequent syllables, which results from the prolonged first downstroke (Fig. 6H). Songs and morphology in Chorthippus biguttulus group The pulses produced during the first downstroke are repeated twice as slowly as the pulses of the subsequent syl lables. The subsequent 2–8 syllables are of the same structure as the syllables in the calling song. Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 38 Stauroderus miramae Vorontsovsky, 1928a: 12. Vedenina & N. Sevastianov (ZMMU); 50 Kostanay region, Naurzum nature reserve, 04–11.VIII.1938, 13 ♂ 6 ♀, leg. Derevitskaya, 11.VIII–25.IX.1939, 3 ♂ leg. Pokrovskyi, 24.VII.1947, 1 ♂ A. Formozov (ZMMU); 52 Akmola region, Tselinograd district, ab. 4 km SWW from Zhaynak, 09.VII.2019, 3 ♂, leg. V. Vedenina, N. Sevas tianov & T. Tarasova, song recordings in 1 ♂ (CV); 55 Akmola region, Arshaly district, 7 km N Vishnevka, Ishym river floodplain, 11.VII.2019, 3 ♂, leg. V. Vedenina, N. Sev astianov & T. Tarasova, song recordings in 2 ♂ (CV); 56 Akmola region, Jerementau district, 4.5 km NE from Baysary, 03.VII.2019, 2 ♂, leg. V. Vedenina, N. Sevastianov & T. Tarasova, song recordings in 2 ♂ (CV); 59 Pavlodar region, Ekibastuz district, ab. 3 km W of Schidert, 04.VII.2019, 6 ♂ 1 ♀, leg. V. Vedenina, N. Sevastianov & T. Material examined. Russia: 29 Krasnodarsky kray, environs of Gelendzhik, 06.X.2011, 8 ♂ 4 ♀, leg. V. Vedenina & L. Shestakov, song recordings in 3 ♂(ZMMU); 30 Kras nodarsky kray, Gelendzhik district, environs of Aderbievka, 07.VII.1997, 8 ♂ 8 ♀, leg. D. Tishechkin, song recordings in 4 ♂ (ZMMU); 31 Krasnodarsky kray, Gelendzhik district, environs of Praskoveevka; 12.VII.1997, 2 ♂, leg. D. Tishechkin (ZMMU); 36 N. Caucasus, N. Ossetia, environs of Alagir, Ardon river floodplain, 09.VIII.1990, 2 ♂ 2 ♀, leg. M. Bukhvalova, song recordings in 2 ♂ (ZMMU); 37 N. Caucasus, N. Ossetia, Sunzhensky range, environs of Elkhotovo, 10–12.VIII.1990, 2 ♂ 1 ♀, leg. M. Bukhvalova (ZMMU); 38 N. Caucasus, N. Ossetia, Sunzhensky range, environs of Bekan lake, 14.VIII.1985, 3 ♂ 3 ♀, leg. D. Tishechkin (ZMMU); 47 Orenburg re gion, environs of Studentzy, 14.VII.2012, 1 ♂, leg. V. Vedenina & L. Shestakov, song recordings in 1 ♂ (CV); 48 Orenburg region, environs of Guberlya railway station, 07–09.VII.1996, 37 ♂ 13 ♀, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU), Material examined. Russia: 29 Krasnodarsky kray, environs of Gelendzhik, 06.X.2011, 8 ♂ 4 ♀, leg. V. Vedenina & L. Shestakov, song recordings in 3 ♂(ZMMU); 30 Kras nodarsky kray, Gelendzhik district, environs of Aderbievka, 07.VII.1997, 8 ♂ 8 ♀, leg. D. Tishechkin, song recordings in 4 ♂ (ZMMU); 31 Krasnodarsky kray, Gelendzhik district, environs of Praskoveevka; 12.VII.1997, 2 ♂, leg. D. Tishechkin (ZMMU); 36 N. Caucasus, N. Ossetia, environs of Alagir, Ardon river floodplain, 09.VIII.1990, 2 ♂ 2 ♀, leg. M. Bukhvalova, song recordings in 2 ♂ (ZMMU); 37 N. Stauroderus miramae Vorontsovsky, 1928a: 12. Caucasus, N. Ossetia, Sunzhensky range, environs of Elkhotovo, 10–12.VIII.1990, 2 ♂ 1 ♀, leg. M. Bukhvalova (ZMMU); 38 N. Caucasus, N. Ossetia, Sunzhensky range, environs of Bekan lake, 14.VIII.1985, 3 ♂ 3 ♀, leg. D. Tishechkin (ZMMU); 47 Orenburg re gion, environs of Studentzy, 14.VII.2012, 1 ♂, leg. V. Vedenina & L. Shestakov, song recordings in 1 ♂ (CV); 48 Orenburg region, environs of Guberlya railway station, 07–09.VII.1996, 37 ♂ 13 ♀, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU), g g g 69 Altai Republic, ab. 26 km SE of Ongudai, environs of Kupchegen’, 08.VIII.2017, 5 ♂ 3 ♀, leg. V. Vedenina & N. Sevastianov, song recordings in 1 ♂ (ZMMU); 70 Tyva republic, environs of Erzin, Tore-Kchan’ lake, 31.VII.1989, 1 ♂ 1 ♀, leg. S. Byzov (ZMMU); 71 Tyva republic, environs of Erzin, Erzin river floodplain, 20.VII–06. VIII.1989, 3 ♂ 3 ♀, leg. M. Bukhvalova, song recordings in 3 ♂ (ZMMU); 72 Tyva republic, environs of Erzin, Tes-Kchem river floodplain, 03–06.VIII.1989, 3 ♂, leg. M. Bukhvalova (ZMMU); 73 Irkutsk region, Nizhneudinsk district, Uk river estuary, confluence with Uda, 02.VII.2003, 5 ♂, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU); 74 Buryatia, Selenginsk district, 5 km N from Novoselenginsk, Selenga river valley, 07.VII.2007, 5 ♂, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU); 76 Buryatia, Zaigrayevo district, 10 km Onokhoy, Bryanka river valley, 21.VII.2007, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); Kazakhstan: 46 West- Kazakhstan region, ab. 50 km W of Ural’sk, environs of Kamenka, 23.VI.2018, 5 ♂, leg. V. Vedenina & N. Sevastianov (ZMMU); 50 Kostanay region, Naurzum nature reserve, 04–11.VIII.1938, 13 ♂ 6 ♀, leg. Derevitskaya, 11.VIII–25.IX.1939, 3 ♂ leg. Pokrovskyi, 24.VII.1947, 1 ♂ A. Formozov (ZMMU); 52 Akmola region, Tselinograd district, ab. 4 km SWW from Zhaynak, 09.VII.2019, 3 ♂, leg. V. Vedenina, N. Sevas tianov & T. Tarasova, song recordings in 1 ♂ (CV); 55 Akmola region, Arshaly district, 7 km N Vishnevka, Ishym river floodplain, 11.VII.2019, 3 ♂, leg. V. Vedenina, N. Sev astianov & T. Tarasova, song recordings in 2 ♂ (CV); 56 Akmola region, Jerementau district, 4.5 km NE from Baysary, 03.VII.2019, 2 ♂, leg. V. Vedenina, N. Sevastianov & T. Tarasova, song recordings in 2 ♂ (CV); 59 Pavlodar region, Ekibastuz district, ab. 3 km W of Schidert, 04.VII.2019, 6 ♂ 1 ♀, leg. V. Vedenina, N. Sevastianov & T. 69 Altai Republic, ab. Stauroderus miramae Vorontsovsky, 1928a: 12. Stauroderus miramae Vorontsovsky, 1928a: 12. y Stauroderus mollis porphyroptera Vorontsovsky, 1928b: 31, 34 y Stauroderus mollis porphyroptera Vorontsovsky, 1928b: 31, 34. p p y p y Chorthippus porphyropterus (Vorontsovsky, 1928): Benediktov, 1999: 42. Chorthippus porphyropterus (Vorontsovsky, 1928): Benediktov, 1999: 42. Material examined. Russia: 29 Krasnodarsky kray, environs of Gelendzhik, 06.X.2011, 8 ♂ 4 ♀, leg. V. Vedenina & L. Shestakov, song recordings in 3 ♂(ZMMU); 30 Kras nodarsky kray, Gelendzhik district, environs of Aderbievka, 07.VII.1997, 8 ♂ 8 ♀, leg. D. Tishechkin, song recordings in 4 ♂ (ZMMU); 31 Krasnodarsky kray, Gelendzhik district, environs of Praskoveevka; 12.VII.1997, 2 ♂, leg. D. Tishechkin (ZMMU); 36 N. Caucasus, N. Ossetia, environs of Alagir, Ardon river floodplain, 09.VIII.1990, 2 ♂ 2 ♀, leg. M. Bukhvalova, song recordings in 2 ♂ (ZMMU); 37 N. Caucasus, N. Ossetia, Sunzhensky range, environs of Elkhotovo, 10–12.VIII.1990, 2 ♂ 1 ♀, leg. M. Bukhvalova (ZMMU); 38 N. Caucasus, N. Ossetia, Sunzhensky range, environs of Bekan lake, 14.VIII.1985, 3 ♂ 3 ♀, leg. D. Tishechkin (ZMMU); 47 Orenburg re gion, environs of Studentzy, 14.VII.2012, 1 ♂, leg. V. Vedenina & L. Shestakov, song recordings in 1 ♂ (CV); 48 Orenburg region, environs of Guberlya railway station, 07–09.VII.1996, 37 ♂ 13 ♀, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU), 29.VI.2018, 1 ♂, leg. V. Vedenina & N. Sevastianov, song recordings in 1 ♂ (CV); 69 Altai Republic, ab. 26 km SE of Ongudai, environs of Kupchegen’, 08.VIII.2017, 5 ♂ 3 ♀, leg. V. Vedenina & N. Sevastianov, song recordings in 1 ♂ (ZMMU); 70 Tyva republic, environs of Erzin, Tore-Kchan’ lake, 31.VII.1989, 1 ♂ 1 ♀, leg. S. Byzov (ZMMU); 71 Tyva republic, environs of Erzin, Erzin river floodplain, 20.VII–06. VIII.1989, 3 ♂ 3 ♀, leg. M. Bukhvalova, song recordings in 3 ♂ (ZMMU); 72 Tyva republic, environs of Erzin, Tes-Kchem river floodplain, 03–06.VIII.1989, 3 ♂, leg. M. Bukhvalova (ZMMU); 73 Irkutsk region, Nizhneudinsk district, Uk river estuary, confluence with Uda, 02.VII.2003, 5 ♂, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU); 74 Buryatia, Selenginsk district, 5 km N from Novoselenginsk, Selenga river valley, 07.VII.2007, 5 ♂, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU); 76 Buryatia, Zaigrayevo district, 10 km Onokhoy, Bryanka river valley, 21.VII.2007, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); Kazakhstan: 46 West- Kazakhstan region, ab. 50 km W of Ural’sk, environs of Kamenka, 23.VI.2018, 5 ♂, leg. V. Stauroderus miramae Vorontsovsky, 1928a: 12. 26 km SE of Ongudai, environs of Kupchegen’, 08.VIII.2017, 5 ♂ 3 ♀, leg. V. Vedenina & N. Sevastianov, song recordings in 1 ♂ (ZMMU); 70 Tyva republic, environs of Erzin, Tore-Kchan’ lake, 31.VII.1989, 1 ♂ 1 ♀, leg. S. Byzov (ZMMU); 71 Tyva republic, environs of Erzin, Erzin river floodplain, 20.VII–06. VIII.1989, 3 ♂ 3 ♀, leg. M. Bukhvalova, song recordings in 3 ♂ (ZMMU); 72 Tyva republic, environs of Erzin, Tes-Kchem river floodplain, 03–06.VIII.1989, 3 ♂, leg. M. Bukhvalova (ZMMU); 73 Irkutsk region, Nizhneudinsk district, Uk river estuary, confluence with Uda, 02.VII.2003, 5 ♂, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU); 74 Buryatia, Selenginsk district, 5 km N from Novoselenginsk, Selenga river valley, 07.VII.2007, 5 ♂, leg. D. Tishechkin, song recordings in 5 ♂ (ZMMU); 76 Buryatia, Zaigrayevo district, 10 km Onokhoy, Bryanka river valley, 21.VII.2007, 3 ♂, leg. D. Tishechkin, song recordings in 3 ♂ (ZMMU); Kazakhstan: 46 West- Kazakhstan region, ab. 50 km W of Ural’sk, environs of Kamenka, 23.VI.2018, 5 ♂, leg. V. Vedenina & N. Sevastianov (ZMMU); 50 Kostanay region, Naurzum nature reserve, 04–11.VIII.1938, 13 ♂ 6 ♀, leg. Derevitskaya, 11.VIII–25.IX.1939, 3 ♂ leg. Pokrovskyi, 24.VII.1947, 1 ♂ A. Formozov (ZMMU); 52 Akmola region, Tselinograd district, ab. 4 km SWW from Zhaynak, 09.VII.2019, 3 ♂, leg. V. Vedenina, N. Sevas tianov & T. Tarasova, song recordings in 1 ♂ (CV); 55 Akmola region, Arshaly district, 7 km N Vishnevka, Ishym river floodplain, 11.VII.2019, 3 ♂, leg. V. Vedenina, N. Sev astianov & T. Tarasova, song recordings in 2 ♂ (CV); 56 Akmola region, Jerementau district, 4.5 km NE from Baysary, 03.VII.2019, 2 ♂, leg. V. Vedenina, N. Sevastianov & T. Tarasova, song recordings in 2 ♂ (CV); 59 Pavlodar region, Ekibastuz district, ab. 3 km W of Schidert, 04.VII.2019, 6 ♂ 1 ♀, leg. V. Vedenina, N. Sevastianov & T. Songs and morphology in Chorthippus biguttulus group 39 Tarasova, song recordings in 3 ♂ (ZMMU); 60 Pavlodar region, Zhelezinsky district, near Pyatiryzhsk, 22.VII 1 ♂ 1 ♀ leg. Ingenitskyi (ZMMU), 05.VII.2019, 2 ♂, leg. V. Vedenina, N. Sevastianov & T. Tarasova, song recordings in 2 ♂ (CV); 61 Pavlodar region, Terenkol’ district, bank of the Irtysh river, 05.VII.2019, 6 ♂, leg. V. Vedenina, N. Sevastianov & T. Tarasova, song recordings in 1 ♂ (CV).h Distribution. (Fig. 1). Stauroderus miramae Vorontsovsky, 1928a: 12. The range of this species stretches in the form of a ribbon from the Black Sea coast eastwards to Transbaikalia. C. miramae occurs in Krasno darsky krai and Caucasus, Orenburg region, northern Kazakhstan, Altai, Tyva, Irkutsk region and Transbaikalia. The ranges of C. miramae and C. maritimus overlap in the south-eastern part of European Russia and in surroundings of Baikal Lake. Recognition. (Table 1, Figs. 2, 3). C. miramae can be distinguished from most species of the biguttulus group by remarkably long stridulatory file (Fig. 2C). This feature was previously shown by Benediktov (1999), who described the last distal stridulatory peg to be situated at least at a level of the second tibial spine when tibia is attached to femur. Within the biguttulus group, a similarly long file is only shown in C. biguttulus euhedickei von Helversen, 1989, that occurs in the southern Balkans and Anatolia and in C. maroccanus Nadig, 1986, that occurs in North Africa (Ragge and Reynolds 1988; Willemse et al. 2009). The latter two taxa, however, are quite different from C. miramae in other morphological characters and songs. In other species of the biguttulus group, the length of stridulatory file is noticeably shorter, and the last distal stridulatory peg is situated at least at the level of the 4th tibial spine when the legs are bent (Benediktov 1999). Notably, in C. miramae, the number of stridulatory pegs is only slightly higher than in C. maritimus, and can’t be considered as a good character. C. miramae tends to have the longest distance between stigma and the wing tip, and the broadest width of C & Sc areas in comparison to C. maritimus and C. brunneus. The PCA based on 6 morphological characters shows that C. miramae represents a separate cluster from C. maritimus and C. brunneus, but it is stronger in males than in females (Fig. 3C, D). Description. (Table 1, Figs. 2, 3). The head structure as in genus. Ratio length of vertical diameter of eye to maximum length of foveolae 3.2–3.6 in ♂, 2.8–3.2 in ♀; ratio minimum interocular distance to length of subocular groove 0.6–0.8 in ♂, 0.7– 1.0 in ♀. Antennae filiform. Median carina distinct and continuous. Prozona slightly shorter than metazona. Lateral pronotal keels distinctly incurved, ratio minimum to maximum widths 2.1–2.6 in ♂, 2.4–2.6 in ♀. Tympanal aperture 2.8–3.3 times in ♂, 2.8–3.4 in ♀ as long as broad. Stauroderus miramae Vorontsovsky, 1928a: 12. Fore and hind wings well developed in both sexes, wings far surpassing the apices of the hind knee. Width of costal area of fore wing reaches its maximum in the middle or in the last third part (Fig. 2A, B). Width of subcostal area 0.3–0.35 mm in ♂, 0.2–0.23 mm in ♀ (measured along the line of maximal width of costal area). Ratio width of fore wing to width of C & Sc areas 3.0–3.2 in ♂, 4.3–4.5 in ♀. Length of apical constriction (distance from C and Sc confluence to the wing tip) is a quarter of the wing length. Ratio length between stigma center and the wing tip to the wing length 2.1–2.8 in ♂, 1.8–1.9 in ♀. Hind femur gracile, ratio femur length to maximum width 4.5–4.9 in ♂, 4.6–4.9 in ♀. Stridulatory file remarkably long in both Description. (Table 1, Figs. 2, 3). The head structure as in genus. Ratio length of vertical diameter of eye to maximum length of foveolae 3.2–3.6 in ♂, 2.8–3.2 in ♀; ratio minimum interocular distance to length of subocular groove 0.6–0.8 in ♂, 0.7– 1.0 in ♀. Antennae filiform. Median carina distinct and continuous. Prozona slightly shorter than metazona. Lateral pronotal keels distinctly incurved, ratio minimum to maximum widths 2.1–2.6 in ♂, 2.4–2.6 in ♀. Tympanal aperture 2.8–3.3 times in ♂, 2.8–3.4 in ♀ as long as broad. Fore and hind wings well developed in both sexes, wings far surpassing the apices of the hind knee. Width of costal area of fore wing reaches its maximum in the middle or in the last third part (Fig. 2A, B). Width of subcostal area 0.3–0.35 mm in ♂, 0.2–0.23 mm in ♀ (measured along the line of maximal width of costal area). Ratio width of fore wing to width of C & Sc areas 3.0–3.2 in ♂, 4.3–4.5 in ♀. Length of apical constriction (distance from C and Sc confluence to the wing tip) is a quarter of the wing length. Ratio length between stigma center and the wing tip to the wing length 2.1–2.8 in ♂, 1.8–1.9 in ♀. Hind femur gracile, ratio femur length to maximum width 4.5–4.9 in ♂, 4.6–4.9 in ♀. Stridulatory file remarkably long in both Tatiana Tarasova et al. Stauroderus miramae Vorontsovsky, 1928a: 12. / ZooKeys 1073: 21–53 (2021) 40 sexes: distance between the last peg and the knee tip 2–2.7 times in ♂, 1.7–2.4 in ♀ as large as length of stridulatory file. In males, stridulatory pegs form one row and have different density along the file (Fig. 2C). Most proximal part of stridulatory file starts with several rare and dispersed pegs that are followed by more densely disposed pegs. The second part of stridulatory file more prolonged, consisting of more rare pegs with stable inter-peg intervals. In the third, most distal part the peg density decreases pro portionally to the length of stridulatory file, and the pegs often do not lay in one raw. In females, stridulatory pegs arranged in one row and distributed rarer than in males. The peg density decreases from the proximal towards the distal parts. The number of stridulatory pegs 118–182 in ♂, 98–157 in ♀. Body coloration similar to coloration of C. maritimus. Measurements in mm. Body length: 14–18 in ♂, 18–24 in ♀, pronotum length: 2.9– 3.3 in ♂, 3.8–4.4 in ♀, fore wing length: 13.3–14.6 in ♂, in 16.4–18.3 in ♀, fore wing width 3.1–3.6 in ♂, 3.2–3.5 in ♀, hind femur length: 9.7–10.4 in ♂, 12.6–14.0 in ♀. g Calling song. (Table 4, Figs. 5, 7). The calling song of C. miramae includes the two types of randomly alternating echemes, typical maritimus-like and optional brunneus- like echemes. The first echeme type was present in the songs of all 34 males recorded, the second echeme type – in the songs of 28 males. The song usually starts with the maritimus-like echeme, which is similar to the C. maritimus calling song, but lasting shorter (the median duration varies in the range of 0.3–2.9 s). The number of syllables per echeme varies in the range of 5 to 35. Each echeme starts with the low-amplitude syllables. In short echemes, the amplitude reaches its maximum in about the echeme middle (Fig. 7F). In long echemes, the amplitude gradually increases, and keeps a constant level after about one quarter of an echeme (Fig. 7G). The syllables are about 1.5 times as short as the syllables in C. maritimus, lasting in the range of about 66–114 ms (Table 4). The syllable duration is rather stable within one population; however, it is more variable between populations. Stauroderus miramae Vorontsovsky, 1928a: 12. Oscillographic analysis shows no distinct pulses within the syllables in some populations, whereas distinct pulses are visible on the oscillograms of the songs from other populations. The shift between the two legs is greater in C. miramae than in C. maritimus (Fig. 7I, J).h The brunneus-like echemes are more often produced by the males from the Sibe rian and the east-european Russian populations, but they are rare in the songs from northern Kazakhstan. The echeme duration in C. miramae is almost three times as high as in C. brunneus (Fig. 5A). Similarly to C. brunneus, the C. miramae echeme consists of the short pulses, the amplitude of which gradually increases, reaching maximum intensity at about half of its duration, and then gradually decreases towards the end. The pulse duration and the pulse rate in C. miramae are almost the same as in C. brun- neus (9–13 ms and 77–96 /s respectively, data are given for 29–30°C). However, the leg movement patterns are different in two species. In C. miramae, the brunneus-like echeme is produced by simple up and down leg-movements that vary in amplitude and duration (Fig. 7J). In C. brunneus, each leg generates a simple upstroke but a two-step downstroke (Fig. 4D). The oscillographic analysis of the C. miramae song shows that the pulses highly vary in amplitude and duration, whereas the pulses in the C. brunneus Songs and morphology in Chorthippus biguttulus group e 7. Oscillograms of calling songs of Chorthippus miramae from Orenburg region A, West-K gion C and Buryatia D,E. Song recordings are presented at three different speeds (faster o of the indicated parts of the songs shown in F–J. At small scales (A–D) the maritimus-like ec distinguished from the brunneus-like echemes by the higher amplitude. In all oscillograms t lines are recordings of hind leg movements and the lower line is the sound recording. The am Songs and morphology in Chorthippus biguttulus group Songs and morphology in Chorthippus biguttulus group Songs and morphology in Chorthippus biguttulus group 41 Figure 7. Oscillograms of calling songs of Chorthippus miramae from Orenburg region A, West-Kazakh stan region C and Buryatia D,E. Song recordings are presented at three different speeds (faster oscillo grams of the indicated parts of the songs shown in F–J. At small scales (A–D) the maritimus-like echemes can be distinguished from the brunneus-like echemes by the higher amplitude. Stauroderus miramae Vorontsovsky, 1928a: 12. In all oscillograms the two upper lines are recordings of hind leg movements and the lower line is the sound recording. The ambient temperature near a singing male was 34 – 35 °C in (A,E) and 29 – 31°C in other cases. song are much more stable in these parameters. In some males of C. miramae, the pulses are tended to group into syllables; the pulse number per syllable is unstable (Fig. 7H).h The order of the two echeme types in the C. miramae song is erratic, though there are some common variants in different populations. For example, several maritimus- like echemes are followed by one brunneus-like echeme (Fig. 7D). Another variant implies alternation of the two echeme types. A rarer case is when one maritimus-like echeme is followed by several echemes of the second type (Fig. 7A, E). The intervals between echemes of the same type may exceed the echeme duration 1.5–3 times for Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 42 Table 4. Calling songs parameters of Chorthippus miramae. For each parameter, medians, the lower and upper quartiles are shown. Stauroderus miramae Vorontsovsky, 1928a: 12. Locality Number of recorded males (measurements) Temperature, ˚ C maritimus-like part brunneus-like part echeme duration, s echeme rate, /s syllable duration, ms syllable rate, /s echeme duration, s echeme rate, /s pulse rate, /s 29–30 7 (40) 25–28; 2.9 0.28 114 7.81 1.4 0.19 76.9 30–32 1.8; 3.8 0.16; 0.29 99;135 7.16;8.53 1.0; 1.7 0.14; 0.20 66.7; 90.1 36 2 (20) 30 2.4 0.07 86 9.2 0.8 0.21 95.5 2.2; 2.7 0.07; 0.07 78; 98 9.7; 11.3 0.8; 0.9 0.17; 0.22 81.7;102.8 48 6 (60) 28–30; 0.9 0.47 66 13.8 0.4 0.31 83.3 34 0.8; 0.9 0.40; 0.59 60; 74 12.5; 14.9 0.3; 0.8 0.29; 0.37 71.4; 100 56 2 (15) 31 0.3 1.4 69 14.6 n/a n/a n/a 0.3; 0.4 1.2; 1.8 61; 76 13.2; 16.3 71 3 (13) 25–26; 0.8 0.40 76 10.5 1.0 n/a 47.6 30 0.8; 1.1 0.36; 0.45 64; 91 9.6; 13.0 0.9; 1.2 23.4; 62.5 73 5 (26) 0.8 0.36 68 13.0 0.3 0.31 83.3 0.7; 0.8 0.34; 0.44 64; 75 12.4; 14.3 0.3; 0.7 0.30; 0.33 70.2; 93.2 74 5 (20) 29–30 2.0 0.16 85 10.7 1.2 0.26 76.9 2.0; 2.8 0.15; 0.17 81; 91 10.1; 11.2 1.0; 1.3 0.22; 0.28 71.4; 83.3 76 3 (10) 35 2.6 0.20 80 11.0 1.3 0.23 83.3 1.9; 3.0 0.19; 0.21 75; 85 10.7; 11.6 1.0; 1.5 0.20; 0.30 71.4; 90.9 *n/a – non-applicable Table 4. Calling songs parameters of Chorthippus miramae. For each parameter, medians, the lower and upper quartiles are shown. Table 4. Calling songs parameters of Chorthippus miramae. For each parameter, medians, the lower and upper quartiles are shown. the maritimus-like echemes, and 3–5 times for the brunneus-like echemes. An interval between the maritimus-like and the subsequent brunneus-like echemes can be very short (Fig. 7F, J), or can exceed the echeme duration 3–5 times.h Courtship song and female response song. (Fig. 8). The courtship song of C. mi- ramae consists of the brunneus-like echemes. However, the courtship sound is much soft er than in the calling song. The courtship echemes are shorter than in the calling song, not reaching 1 s (the median duration is about 0.4 s). The echemes are usually repeated at the rate of about 0.2–0.6/s, and their duration varies from 0.7 to 1.0 s. Pulses are short (6–9 ms), frequent (repeated at the rate of 61–95/s), and of a low amplitude (Fig. 8F). What is the function of the long stridulatory file? The morphological analysis conducted in the current study shows that one character, the length of stridulatory file, appears to be the most reliable character to distinguish C. miramae, C. maritimus and C. brunneus. The difference in the file length between C. maritimus and C. brunneus can be explained by the difference in the peg number. By contrast, the extremely long file in C. miramae is not due to the significant increase in the peg number, but due to the more widely spaced pegs in the distal part of the file.hi The long stridulatory files are known in some other species of the biguttulus group. C. biguttulus euchedickei from the southern Balkans and north-western Anatolia (Wil lemse et al. 2009) and C. maroccanus Nadig, 1976 from north-western Africa (Ragge and Reynolds 1988), are also characterized by extraordinary long stridulatory files and the widely spaced distal pegs. In C. brunneus brevis Klingstedt 1939 from Southern Finland and north-east Russia (Ragge and Reynolds 1998; Benediktov 2017), the file length is much greater than in the nominate subspecies. In C. brunneus brevis, how ever, the increased length of the stridulatory file can be explained by the increase in the peg number. In one endemic of the biguttulus group in Anatolia, C. relicticus Sirin, Helversen & Ciplak, 2010, the peg number was shown to be extremely high (175–225 in male, 194–245 in female; Sirin et al. 2010). Unfortunately, the authors did not measure the length of stridulatory file in C. relicticus, but we assume that the file could be also long.ihf What could be a function of the long stridulatory file? The different parts of the long file can be used during stridulation to produce various song elements (Vedenina et al. 2007; Vedenina and Helversen 2009). This, however, is only evident in C. bi- guttulus euchedickei (Helversen 1989; Willemse et al. 2009). The calling song of this species consists of 1–3 typical loud echemes, similar to those in the nominate form, that are followed by 1–5 softer aftersongs (quiet parts of the song produced at the end of singing). Aftersongs are produced at a low position of the legs, and presumably the distal pegs are used for sound generation. In C. miramae, however, the long stridula tory file does not seem to be specifically involved in sound generation: at least, no song elements were found to be generated by distal pegs only. Stauroderus miramae Vorontsovsky, 1928a: 12. / ZooKeys 1073: 21–53 (2021) 44 strokes. Rarely, the males produce the maritimus-like echeme without the first syllable of distinct pulses (Fig. 9H). strokes. Rarely, the males produce the maritimus-like echeme without the first syllable of distinct pulses (Fig. 9H). The same male may produce echemes of different structure in the rivalry situ ations. Some females are actively responding to the male rivalry songs. Stauroderus miramae Vorontsovsky, 1928a: 12. In some cases, the leg movements do not produce any sound at all (Fig. 8A, D). A female produces the brunneus-like song in response to the male courtship or rivalry song (Fig. 8A, B). The female alternates her response echemes with the male echemes (Fig. 8D). The duration of the female echeme is similar to that in the male courtship, or 1.5–2 times longer than in the male courtship. The leg movement pattern in the female response song is similar to that in the male courtship song, but less regu lar (Fig. 8E, F). The pulses are longer (10–21 ms) and repeated at the rate of 43–77/s, especially in the first third of the echeme (Fig. 8D, E). i Rivalry song. (Fig. 9). Several males of C. miramae sitting close to each other pro duce a diversity of echemes of different duration, structure and leg movement pattern. For example, one can find a rivalry song similar to that of C. maritimus, which starts with the prolonged first syllable, which results from the prolonged first downstroke Songs and morphology in Chorthippus biguttulus group Songs and morphology in Chorthippus biguttulus group Songs and morphology in Chorthippus biguttulus group 43 Figure 8. Oscillograms of courtship songs and female response songs in Chorthippus miramae from Pav lodar region A West-Kazakhstan region B and Altai republic C. Song recordings are presented at three different speeds (faster oscillograms of the indicated parts of the songs shown in D–F). During courtship, a male can produce audible (C,F) or silent (A,D) variants of song. Female responses with leg movements recordings (B, E) and without them (A,D) are shown. In all oscillograms the two upper lines are record ings of hind leg movements and the lower line is the sound recording. The ambient temperature near a singing specimen was 29 – 31°C. (Fig. 9D, E). The pulses produced during the first downstroke follow twice as slowly as the pulses of the subsequent syllables. The subsequent syllables are of the same struc ture as in the maritimus-like echeme of the calling song.i Most often, the males produce single syllables similar to the first one with distinct pulses described above. These syllables are repeated at the rate of about 2–2.5 /s (Fig. 9F, G). Notably, the two legs may produce different number of the up and down Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) Tatiana Tarasova et al. What is the function of the long stridulatory file? The leg movements in other species of the biguttulus group with a long file or high peg number have not been stu died or studied only for certain song types. It is noteworthy that stridulatory pegs function not only as a mechanic part of the stridulatory apparatus, but also as the mechanoreceptors (Hustert et al. 1999). It was Songs and morphology in Chorthippus biguttulus group 45 wn in two distantly related species of Gomphocerinae, C. biguttulus and re 9. Oscillograms of rivalry songs in Chorthippus miramae from Altai republic A and n of Kazakhstan B,C. Song recordings are presented at three different speeds (faster oscillo ndicated parts of the songs shown in D–H). In all oscillograms the two upper lines are reco leg movements and the lower line is the sound recording. The ambient temperature near was 29 – 31°C. Figure 9. Oscillograms of rivalry songs in Chorthippus miramae from Altai republic A and Pavlodar region of Kazakhstan B,C. Song recordings are presented at three different speeds (faster oscillograms of the indicated parts of the songs shown in D–H). In all oscillograms the two upper lines are recordings of hind leg movements and the lower line is the sound recording. The ambient temperature near a singing male was 29 – 31°C. shown in two distantly related species of Gomphocerinae, C. biguttulus and Syrbula montezuma (Saussure, 1861), that two sensory cells innervate each peg in themale and each tubercle in the female. These mechanoreceptors can deliver specific propriocep tive information about the contact between the stridulatory file and the vein of the fore wing. A subtle sensory control is required for measuring the pressure of the leg against the wing. The current study of the C. miramae songs shows that the loud maritimus-like echeme is apparently produced by legs being more pressed to the wings than during Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) 46 the softer brunneus-like echeme (Fig. 7). The latter echeme may be also produced with different leg pressure depending on the calling or courtship behavior; during courtship, the sound can be even absent despite the appropriate leg movements (Fig. 8). p pp p g g In species of the C. albomarginatus group, the peg number and density differ only at the proximal parts of the stridulatory files (Vedenina and Helversen 2009). Peculiarities of the Chorthippus miramae song The calling song of C. miramae is conspicuously different from the songs of C. brun- neus and C. maritimus, by the presence of two types of echemes, which were recorded in 82% of males. In the calling songs of 18% of C. miramae males, however, only the maritimus-like echemes were recorded. The latter specimens, however, clearly belong to C. miramae based on morphology and courtship and rivalry songs. p gy p y g Until now, the calling songs of C. miramae were only presented under the name C. ye- rsini by Bukhvalova (1993) and C. porphyropterus by Benediktov (2005). Both authors claim the presence of the two echeme types. From the oscillograms presented, one could see many similarities with the songs of C. maritimus and C. brunneus. The current song analysis that includes not only the sound but also the leg-movement analysis indicates that both maritimus-like and brunneus-like elements have some peculiarities in the C. miramae song. The maritimus-like echemes rarely show the distinct pulses within syllable, whereas such pulses in the calling song of C. maritimus are typically present. This may be determined by the larger shift between the two legs in C. miramae than in C. maritimus. The brunneus- like echeme in the C. miramae song is produced by simple up and down leg-movements, whereas each leg generates a simple upstroke but a two-step downstroke in the C. brunneus song. The sound pulses, however, are of a similar temporal structure in both species. The similarities between the calling songs could explain why C. miramae is not found together with C. maritimus and C. brunneus in the same biotopes (despite the latter two species often occur syntopically). According to the concept of ‘acoustic niches’ (Bukhvalova 2006; Tishechkin and Bukhvalova 2009), the combination of the syllable rate and syllable tem poral pattern determines the species ‘place’ in the acoustic environment of the grasshopper community. Since these song parameters overlap within the species pairs C. miramae / C. maritimus and C. miramae / C. brunneus, the absence of each pair in the same biotope is not surprising. C. miramae generally demonstrates a richer song repertoire than the other two spe cies. The courtship song of C. miramae is similar to the brunneus-like echeme, but the sound is very soft. In some cases, leg movements of C. What is the function of the long stridulatory file? The vari ous species of this group produce different and very conspicuous visual displays in a particular part of the courtship: during the stroke with the hind tibiae, the femora are kept at the extra-high, almost vertical, position. At this moment, the proximal pegs may participate in producing sound. Therefore, the divergence in visual display and the changes in the peg morphology in the albomarginatus group could strengthen each other. A similar assumption can be made for the evolution in song and the stridulatory file structure in the biguttulus group. Peculiarities of the Chorthippus miramae song miramae do not produce any sound at all, which may be interpreted by a female as a visual display. Notably, there is Songs and morphology in Chorthippus biguttulus group 47 no specific courtship song in both C. brunneus and C. maritimus. As for a rivalry song, this is present in C. maritimus and C. miramae but not in C. brunneus. The rivalry song of C. miramae is similar to that in C. maritimus. It comprises the first syllable with dis tinct pulses lasting longer than the subsequent syllables with fuzzy pulses. More often, however, the rivalry repertoire in C. miramae includes short syllables similar to the first one in the maritimus-like echeme but repeated at the rate of 2–2.5/s. In most species of the biguttulus group, the rivalry song is similar to the calling song (Ragge and Reynolds 1998). The rivalry song may be shorter than the calling song, but similar in temporal structure and usually does not contain any new elements. Only C. maroccanus produces a characteristic rivalry song containing two elements, one element similar to the calling song and the second unique element. Thus, C. mi- ramae is another species of this group, in which the rivalry song is principally different from the calling song. The relationship of Chorthippus miramae with other members of the biguttulus group It has been suggested that the biguttulus group comprises many young, closely related species, some of which may be of hybrid origin. Some species of this group were found to hybridize in nature (e.g., Ragge 1976; Bridle and Butlin 2002; Kleukers et al. 2004; Nolen et al. 2020), whereas some of them were hybridized in laboratory in no-choice conditions and produced viable and fertile offspring (Helversen and Helversen 1975; Gottsberger and Mayer 2007). The similarity of the C. miramae song with the songs of C. brunneus and C. maritimus might suggest a hybrid origin of C. miramae. g gg y g One of the most well studied hybrid zones within the biguttulus group is a hybrid zone between C. jacobsi and C. brunneus in northern Spain (e.g., Bridle and Butlin 2002; Saldamando et al. 2005; Bridle et al. 2006). The calling song of C. jacobsi is similar to the song of C. maritimus, but of a shorter duration. Songs of F1, F2 and backcross hybrids between C. jacobsi and C. brunneus were intermediate between the songs of both parental species in all song parameters (Saldamando et al. 2005). At the same time, no combination of the parental song elements was found in the hybrid songs. Similarly, natural hybrids between C. maritimus (named as C. bornhalmi) and C. brunneus from north-eastern Italy were shown to sing intermediate songs (Kleukers et al. 2004). In European Russia and Ukraine, these two species often occur in the same biotope allowing them to hybridize. We suggest that the C. brunneus song with unusually long echeme duration and low echeme and pulse rate recorded from loc. 40 (Table 2) may be also attributed to the hybrid. F1 hybrids between C. maritimus and C. brunneus bred in our laboratory only revealed intermediate songs (unpublished data). It is therefore unlikely that C. miramae could evolve from the hybrids between C. maritimus and C. brunneus.h The number of stridulatory pegs in hybrids between C. jacobsi and C. brunneus (Sal damando et al. 2005) and C. brunneus and C. bornhalmi (Kleukers et al. 2004) were shown to be also intermediate between those in parental species. In C. miramae, the peg number is similar to that of C. maritimus, but significantly larger than that in C. brunneus. 48 Tatiana Tarasova et al. / ZooKeys 1073: 21–53 (2021) Other results were obtained for hybrids between C. biguttulus and C. The relationship of Chorthippus miramae with other members of the biguttulus group mollis (Helversen and Helversen 1975) and C. brunneus and C. biguttulus (Gottsberger and Mayer 2007). A combination of the parental song elements and even novel song ele ments were found in hybrids between C. biguttulus and C. mollis. Thus, the hybrid song may be considered as more complex in comparison with the parental songs. In hybrids between C. albomarginatus and C. oschei (unrelated species to the biguttulus group), the values of several song parameters were significantly larger or smaller than those in the parental songs (Vedenina et al. 2007). Notably, the leg-movement patterns appeared to be simpler in hybrids than these in both parentals. In hybrids between C. brunneus and C. biguttulus, the species-specific syllable structure was largely lost, because the leg-movement patterns were also simplified in comparison to the parental patterns (Gottsberger and Mayer 2007). These divergences in inheritance of different song parameters are likely the result from incompatibility of neuronal networks that control stridulatory leg movements in hybrids. This hypothesis was offered by Helvers en and Helversen (1975). They suggested the two pattern-generating neuronal net works to be formed in the central nervous system of hybrids because of nonhomology of the parental elements. The outputs of the two networks converge in a common final pathway, probably at the level of the motoneurons, and may lead to the superimposed pattern of the hybrid song. In C. brunneus, C. biguttulus, and C. mollis, the song ele ments in terms syllable structure are suggested to be nonhomological. In C. biguttulus, for example, the first and the loudest pulse in each syllable is generated by an accentu ated downstroke of the legs; each syllable is usually produced by three up-and-down leg movements; the two legs moving in slightly different patterns (e.g., Elsner 1974; Helversen and Helversen 1983, 1994). It is no coincidence that some authors attribute these three species to different subgroups of the biguttulus group (Willemse et al. 2009; Sirin et al. 2010). By contrast, syllable of the calling song in C. brunneus, C. jacobsi and C. maritimus ( = brunneus subgroup) is produced by similar leg movements (simple upstroke and stepwise downstroke) and may be considered as homological element. It is therefore not surprising why hybrids between the species within the brunneus sub group generate purely intermediate songs without novel elements or combination of the parental elements. Considering all the aforesaid, what can we say about the origin of C. The relationship of Chorthippus miramae with other members of the biguttulus group miramae? We hypothesize that this species could have evolved as a result of hybridization between other species of the biguttulus group, for example, between C. biguttulus and C. mariti- mus. The two species are vicariant: the first one occurs in the north, the second one – in the south. For example, in the Ukraine C. biguttulus is found more in the north, where as C. maritimus more in the south. Eastwards, this border is shifting, the ranges overlap, and the species may occur syntopically. In the latter case, however, C. maritimus can be found in the first half of summer, whereas C. biguttulus – in the second half of summer. This indicates that the species tend not to meet, probably because the syllable rate in calling songs is quite similar; the syllable structure, however, is very different. Mean while, we do not exclude that hybridization may occur between these species when one of them is rare and another is abundant. To date, no laboratory hybrids were bred Songs and morphology in Chorthippus biguttulus group 49 between them, and nothing is known about biguttulus × maritimus hybrid song. The hybridization experiments between these species could be a subject of future studies. We also hypothesize that C. miramae could diverge from C. maritimus. The latter species is widespread in Anatolia, where it occurs in highlands, thus forming iso lated populations. In Anatolia, there is also another species of the biguttulus group, C. relicticus, occurring very locally in the Southern Anatolian Taurus (Sirin et al. 2010). Its calling song is similar to the brunneus-like echeme of C. miramae, which is produced by simple up and down strokes of the legs moving in antiphase. Sirin et al. (2010) suggest that this species could have radiated from a C. maritimus (named as C. bornhalmi in the paper) like ancestor in an interglacial refugium. In southern territories, the members of the biguttulus group, being the cold-resistant species, are suggested to be isolated during interglacial periods and spread down and expanded their ranges during glacial periods. If we suggest the divergence of C. miramae from C. maritimus, the spreading of the former to the north could occur, on the contrary, during interglacial periods. To test both hypotheses (hybrid origin of C. miramae or its divergence from a C. maritimus-like ancestor in a glacial refugium), it is necessary to conduct genomic studies. The relationship of Chorthippus miramae with other members of the biguttulus group A recent analysis of mitochondrial and nuclear genomes in the biguttulus group in Western Europe (Nolen et al. 2020) shows that four species, C. brunneus, C. biguttulus, C. rubratibialis and C. mollis, experienced a long period of geographic isolation, followed by secondary contact and extensive introgression. According to Nolen et al. (2020), C. mollis was the first species to split, C. bigut- tulus was the next, followed by C. rubratibialis and C. brunneus. Mitochondrial ge nomes suggest that the radiation is relatively recent, dating to the mid-Pleistocene. Thus, the species of the biguttulus group must have experienced multiple episodes of contraction and expansion during the multiple glacial periods that affected the European continent. Taking this into account, it would be especially interesting to sample other species of the biguttulus group, especially those at or near the de scribed refugia in Eurasia. References Benediktov AA (1999) Little-known taxa of grasshoppers of the Chorthippus biguttulus group (Orthoptera, Acrididae, Gomphocerinae). Moscow University Biological Sciences Bulletin 54 (1): 41–45. Benediktov AA (1999) Little-known taxa of grasshoppers of the Chorthippus biguttulus group (Orthoptera, Acrididae, Gomphocerinae). Moscow University Biological Sciences Bulletin 54 (1): 41–45. Benediktov AA (2005) Fauna and acoustic signals of the genus Chorthippus Fieb. (Orthoptera, Acrididae) from southern Siberia. Proceedings of the Russian Entomological Society 76: 118–130. [in Russian with English summary] Benediktov AA (2005) Fauna and acoustic signals of the genus Chorthippus Fieb. (Orthoptera, Acrididae) from southern Siberia. Proceedings of the Russian Entomological Society 76: 118–130. [in Russian with English summary] Benediktov AA (2017) To the knowledge of rare species of Acrididae from protected areas in north-western Russia: Chorthippus brunneus brevis (Orthoptera, Acrididae). Nature Conservation Research 2(2): 84–89. [in Russian with English summary] https://doi. org/10.24189/ncr.2017.003 Benediktov AA, Mikhailenko AP (2017) On the northern border of the range of Chorthippus maritimus in European Russia. Works of the Stavropol Department of Russian Entomo logical Society 13: 34–36. [in Russian] Berezhkov RP (1956) Grasshoppers of Western Siberia. Tomsk University Publisher, Tomsk, 175 pp. Bey-Bienko GJa, Mistshenko LL (1951) Locusts and grasshoppers of the USSR and adjacent countries. Part II. Academy of Sciences of USSR Publisher, Moscow-Leningrad, 287 pp. [pp. 381–667, in Russian, English translation of Russian original: Bey-Bienko GJa, Mist shenko LL (1964) Locusts and Grasshoppers of the U.S.S.R and Adjacent Countries. Part II. 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https://openalex.org/W4385728014	https://www.researchsquare.com/article/rs-3165099/latest.pdf	English	null	Addition of Green and Black Liquor in Kraft Pulping of Eucalyptus dunnii wood: Possible Solutions for the Problems with Kraft Pulping Caused by High Calcium Content	Research Square (Research Square)	2,023	cc-by	7,230	Vijaya Vegunta Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Raghu Deshpande Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Pär A. Lindén Wallenberg Wood Science Center, WWSC, Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Andres Garcia Montes del Plata Colonia Department Maria Björk Stora Enso, Biomaterials division Ulla Jansson Stora Enso, Biomaterials division Gunnar Henriksson (  ghenrik@kth.se ) Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Mikael E. Lindström Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Raghu Deshpande Department of Fiber and Polymer Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, KTH Research Article Keywords: Kraft pulping, green liquor, black liquor, Eucalyptus dunnii, Calcium, Delignification Posted Date: August 7th 2023 Keywords: Kraft pulping, green liquor, black liquor, Eucalyptus dunnii, Calcium, Delignification eywords: Kraft pulping, green liquor, black liquor, Eucalyptus dunnii, Calcium Posted Date: August 7th, 2023 Posted Date: August 7th, 2023 Page 1/28 Page 1/28 DOI: https://doi.org/10.21203/rs.3.rs-3165099/v1 DOI: https://doi.org/10.21203/rs.3.rs-3165099/v1 DOI: https://doi.org/10.21203/rs.3.rs-3165099/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Cellulose on December 20th, 2023. See the published version at https://doi.org/10.1007/s10570-023-05603-z. Page 2/28 Abstract In our previous study, we demonstrated that Eucalyptus dunnii samples containing high calcium content show inferior pulping properties concerning delignification and polysaccharide degradation. This led us to investigate alternative methods for improving the pulping process of these samples. In the present work, we evaluated the effects of incorporating black and green liquors into the Eucalyptus dunnii chips before kraft pulping, aiming to enhance the pulping process and overcome the negative impact of high calcium content. The addition of both black and green liquors resulted in specific enhancements, with the green liquor having a more significant impact on the pulping process. Even wood samples with the highest calcium content demonstrated satisfactory pulping results when treated with green liquor. Delignification occurred more rapidly, and selectivity was higher for samples pre-treated with green liquor before kraft pulping. Moreover, calcium tended to follow the fiber under these conditions rather than being released into the black liquor, which may contribute to the improved pulping performance. Subsequent bleaching tests revealed that the bleachability of green liquor-treated pulp was nearly identical to that of a control pulp, while maintaining a higher viscosity. This suggests that incorporating green liquor into the pre-treatment process not only improves the pulping performance of Eucalyptus dunnii samples with high calcium content but also maintains desirable bleachability characteristics. To better understand the underlying mechanisms of these findings, we discuss the potential chemical explanations behind the observed improvements. Introduction For many years, kraft pulping has maintained its status as the most crucial method for processing wood, primarily due to its low operating costs, which can be attributed in part to efficient chemical recovery systems. Other factors contributing to its success include its relatively high selectivity of delignification and its broad tolerance for various raw materials (Ragnar et al. 2013). Despite this wide range of acceptable raw materials, not all wood types are pulped at the same rate. The lignin structure is a key factor, with softwood, and particularly compression wood, delignifying at a slower rate than hardwood. This slower rate is a result of a higher content of stable condensed bonds in the lignin (Gonzalo Epelde et al. 1998). In addition to lignin structure, covalent bonds between lignin and polysaccharides, known as "lignin carbohydrate complexes" (LCC), significantly influence delignification rates. These complexes, which exhibit variations between different tree species, serve as rate-limiting factors for delignification (Lawoko et al. 2004). One less studied aspect in the field of kraft pulping is the impact of inorganic content of wood on the kinetics of the process. An interesting example of this is the calcium oxalate content present in Page 3/28 Eucalyptus dunnii wood; recent research has revealed that wood samples of this species with a higher calcium content exhibit suboptimal performance during kraft pulping. Specifically, these samples display slower delignification rates and a more significant degree of cellulose degradation (Vegunta et al. 2022). In laboratory-scale experiments, pulping samples with the highest calcium content, which reached levels of 4.7 grams of calcium per kilogram of wood, proved to be a challenge. This finding highlights the importance of considering the inorganic content of wood when optimizing kraft pulping processes. Interestingly, this issue was less pronounced, yet still significant, at the industrial scale. One possible explanation is that industrial white liquor may be "less pure" due to incomplete causticizing, meaning it contains carbonates. Alternatively, the addition of black liquor to fresh chips before pulping could partially alleviate the calcium-related challenges in some manner (Vegunta et al. 2022). Kraft pulping of wood chips using black or green liquor is a potential solution to address challenges associated with high calcium content in wood. Since green liquor is an intermediate in the chemical recovery system, it is readily available in the kraft mill alongside black liquor (Ragnar et al. 2013). Materials Wood chips of standard kraft pulping size Eucalyptus dunnii with a calcium content of 3366 mg/kg were from Soriano farm at Bequilo district, Uruguay. E. dunnii, with a calcium content of 3756 mg/kg, was from Grecco farm and Los Cercos, District, Uruguay. E. dunnii, with a calcium content of 4668 mg/kg, was from Grecco farm and Los Cercos, District, Uruguay Sodium carbonate (99.5%) was from Sigma Aldrich, St. Louis, USA, Nitric acid (65%) is from Sigma Aldrich, Burlington, USA, hydrogen peroxide (30 at%H2O2) was from Sigma Aldrich St. Louis, USA, Sulphuric acid (72%), was from Alfa Aesar, Kendel, Germany). Introduction Previous experiments with green liquor kraft pulping have demonstrated encouraging results, such as increased delignification rate, higher pulp yield, enhanced strength, improved selectivity, and reduced chemical consumption (Andrews and Chang H 1985; Ban et al. 2004; Klevinska and Treimanis 1997; Svedman and Tikka 1998). Nevertheless, the exact mechanisms behind these improvements remain elusive. In this study, we examine the impact of adding black and green liquor prior to kraft pulping on the delignification kinetics of E. dunnii wood with varying calcium contents including as high as of 4.7 g/kg – a quality that is difficult to pulp under normal conditions in laboratory (Vegunta et al. 2022). Storage of the Samples: The black liquor obtained after pulping was stored in the refrigerator. Prior to analysis, the samples were left on the table to reach room temperature and thoroughly mixed. Page 4/28 Page 4/28 Preparation of Black Liquor for impregnation experiment: Black liquor production was conducted in the laboratory for impregnation experiments, utilizing E. dunnii wood chips containing 3366 mg/kg of calcium. The kraft cooking conditions for producing black liquor included an impregnation temperature of 110°C for 90 minutes, 35% sulfide content, a 4:1 liquid-to-wood ratio, 18% effective alkali (EA), and kraft cooking at 145°C for 1 hour. Consequently, the laboratory- produced black liquor, containing hydroxyl and sulfide ions, closely resembles industrial black liquor. Kraft Cooking Three sets of kraft pulping experiments were conducted, using E. dunnii wood chips with varying calcium content: the first with 3366 mg/kg, the second with 4668 mg/kg, and the third with 3756 mg/kg. In the first set of experiments, different kraft pulping process conditions were assessed, and the effects of black or green liquor additions on pulping efficiency were evaluated. The second set of experiments involved E. dunnii wood chips with a very high calcium content of 4668 mg/kg, which were challenging to pulp in the laboratory using a conventional protocol. Green liquor was used as both impregnation and cooking liquor in this case. The objective was to further examine and confirm the role of green liquor on the pulping performance of wood with very high calcium content. The third set of experiments utilized E. dunnii wood chips with a calcium content of 3756 mg/kg, similar to the wood chips in the first set. This study aimed to assess the effects of green liquor on the bleachability of the produced kraft pulp. Both batches had calcium content too high to achieve reasonable defibration in our laboratory using standard pulping techniques (Vegunta et al. 2022). The kraft pulping experiments were conducted using a 1000 ml steel autoclave multiunit digester system, with the process controlled by a computer system and an electric heater. Impregnation took place in steel autoclaves, where wood chips were degassed under vacuum for 30 minutes. Following this, the liquor was drawn into the autoclaves and positioned in a stream-heated glycol bath. It took 10 minutes for the required temperature to be reached, so the actual impregnation time commenced 10 minutes after placing the autoclaves in the electric heater. The impregnation and cooking liquor conditions were consistent across all kraft cooks in this study. Impregnation conditions included a temperature of 110°C and a duration of 90 minutes. As for the kraft cooking liquor conditions, they involved a liquid-to-wood ratio of 4:1, with effective alkali and sulfide charges set at 18% and 35%, respectively. Kraft Cook Series 2: E. dunnii wood chips containing 4668 mg/kg of calcium were used. Kraft cooking was performed using synthetic green liquor. The impregnation and cooking chemical conditions remained the same as previously described, followed by the kraft cook. The kraft cooking conditions were set at 145°C for 210 minutes. Preparation of Green Liquor: Green liquor preparation involved adding sodium carbonate with a concentration of 0.66M to the water, after adjusting the effective alkali (18%) and sulfide (35%) content in the white liquor. The liquid-to-wood ratio was maintained at 4:1. White liquor, a precursor to green liquor, is created from sodium hydroxide and sodium sulfide stock solutions. Kraft Cook Series 1: Page 5/28 E. dunnii wood chips with a calcium content of 3366 mg/kg were used in this study. Kraft cooks were conducted using reference white liquor, a combination of black and white liquor, and green liquor. The impregnation and cooking chemicals were the same as previously described. Kraft cooking was performed at various H-factors, with a constant time of 210 minutes and temperatures ranging from 138°C to 170°C. Kraft Cook Series 3: E. dunnii wood chips with a calcium content of 3768 mg/kg were used. Kraft cooking was conducted using reference white liquor, a combination of black and white liquor, and synthetic green liquor, aiming to achieve similar kappa numbers (13–14). The impregnation and cooking chemical conditions were consistent with prior descriptions, followed by the kraft cook. The kraft cooking conditions for the three cooking liquors were as follows: white liquor (170°C, 210 minutes), black and white liquor (170°C, 210 minutes), and green liquor (145°C, 210 minutes). Bleaching: A DEDED bleaching sequence was conducted on kraft pulp (with similar kappa numbers) obtained from E. dunnii wood using reference white liquor, a combination of black and white liquor, and synthetic green liquor. The drainability (ISO 5267-1), water retention value (WRV) for 100 mesh (ISO 23714), tensile index (ISO 1924-2), tensile stiffness (ISO 1924-2), and tear index (ISO 1974) were evaluated for both unbleached and bleached pulps. Page 6/28 Table 1 Chemical composition details for DEDED bleaching sequence on kraft pulps. D0 E1 D1 E2 D2 Acid Reaction temperature (°C) 60 70 70 70 70 20–25 Reaction time (min) 60 90 120 90 120 15 pH target at the end 2-2.5 10.5–11 4-4.5 10.5–11 4-4.5 4.5-0 ClO2 (%) 4.76 - 2.00 - 0.90 - NaOH (%) - 1.90 0.50 0.50 0.10 - Lignin Isolation: Lignin Isolation: Page 6/28 Concentrated sulfuric acid (97 to 98% H2SO4) was added to 20 ml of impregnation liquor until the pH of the solution reached 2–3. The mixture was then centrifuged at 4800 rpm for ten-minute intervals, with 5- minute breaks in between. After centrifugation, the supernatant was separated from the precipitate, which was subsequently washed twice with a sulfuric acid solution at pH 2 and centrifuged again. The resulting pellet was dried in an oven overnight at 80°C, and the weight was recorded. The precipitation process was performed in duplicate. Concentrated sulfuric acid (97 to 98% H2SO4) was added to 20 ml of impregnation liquor until the pH of the solution reached 2–3. The mixture was then centrifuged at 4800 rpm for ten-minute intervals, with 5- minute breaks in between. After centrifugation, the supernatant was separated from the precipitate, which was subsequently washed twice with a sulfuric acid solution at pH 2 and centrifuged again. The resulting pellet was dried in an oven overnight at 80°C, and the weight was recorded. The precipitation process was performed in duplicate. Determination of Dry Solid Content and Ash Content: Determination of Dry Solid Content and Ash Content: Approximately 5 ml of black liquor was placed in a ceramic crucible and kept in an oven at a temperature of 105°C until a constant mass was achieved (typically within 12 to 15 hours). The mass was then recorded. Following this, the residue from the 105°C drying process was heated to 600°C for 6 hours in the oven, and the resulting weight, representing the ash content, was documented. Determination of Oxalic Acid Using HPLC: Approximately 100 mg of dried black liquor samples were diluted with a dilution factor of 1000 using Milli-Q water. The diluted samples were filtered through a 0.25 µm nylon filter and transferred to HPLC vials. Oxalic acid content was determined using high-performance liquid chromatography (HPLC) with a Thermo Fischer Scientific system (USA) equipped with a ROA-organic acid column (Thermo Fischer Scientific, USA) and a refractive index detector. The mobile phase consisted of a sulfuric acid solution with a pH between 2 and 8 (0.45 M) at a flow rate of 0.5 ml/min. The oven temperature was set at 50°C. Klason Lignin and Sugar Composition Analysis: All pulp samples were Wiley milled using a 40 mesh screen and then subjected to acid hydrolysis to determine lignin and sugar content. Initially, 3 ml of 72% H2SO4 was added to each sample, followed by placement in a vacuum desiccator for 1 hour and 20 minutes with occasional stirring. The mixtures were then diluted with 84 ml of Milli-Q water and autoclaved at 125°C for 1 hour. Subsequently, the samples were filtered through a glass fiber filter using a 3-piece filtration setup. The filtrates were diluted at a 1:10 ratio for sugar analysis and acid-soluble lignin determination. The insoluble (Klason lignin) fraction was dried in an oven at 105°C and weighed. Acid-soluble lignin was measured using a Shimadzu UV-2550 UV-VIS spectrophotometer at an absorbance of 205 nm. Carbohydrate content was determined using a Dionex ICS-3000 high-performance anion-exchange chromatography system with pulsed amperometric detection (HPAEC-PAD), featuring a CarboPac PA1 column (Thermo Scientific, USA), an injection volume of 25 µl, and a flow rate of 1 ml/min. Page 7/28 Page 7/28 External sugar standards based on the sample were used for calibration. The results were reported as anhydrous sugars and performed in duplicate. Metal Ion Content Analysis: The total metal ion content in the pulp samples was measured using ICP-OES (Thermo Scientific iCAP 7000 series). Before the ICP-OES measurements, approximately 100 mg of each sample was taken for analysis. Initially, 7 ml of aqua regia solution (2 ml H2O2 + 5 ml HNO3) was added to the samples. The tubes were sealed and then placed in an ultrasonic bath for several minutes. After this, the samples were left overnight for acid digestion. The samples were then filtered using filter paper and diluted to 50 ml with Milli-Q water. This solution was further diluted to a 1:50 ratio using 5% HNO3. Finally, 10 ml of the diluted sample was subjected to metal analysis using ICP-OES. Residual alkali determination The residual alkali of black liquor samples was determined according to the SCAN-N 33:94 standard. he residual alkali of black liquor samples was determined according to the S Kappa number determination Kappa number of pulp samples was determined according to the ISO 302:2004 standard. Results and discussion Previous research has demonstrated that higher calcium content results in slower delignification and more severe cellulose degradation in E. dunnii with calcium contents of 705, 870, and 1500 mg/kg, and has proposed possible mechanisms for these effects (Vegunta et al. 2022). Even under harsh conditions, kraft pulping with lab-made white liquor proved to be practically challenging for samples containing more than 3000 mg/kg calcium. However, high calcium content had a lesser impact on industrial-scale pulping, although severe problems did arise. The difference between laboratory and industrial-scale pulping lies in the fact that black liquor is typically added to wood chips in a pulp mill (Ragnar et al. 2013), whereas white liquor may become contaminated with green liquor due to insufficient causticization. As a result, the composition of the liquors used for impregnation and kraft cooking in the pulp mill and a lab varies significantly. In this study, we conducted kraft pulping on a laboratory scale using white liquor, a combination of black and white liquor, and synthetic green liquor (prepared in our lab from pure chemicals). Further details on the composition can be found in the materials and methods section. Page 8/28 Table 2 Details of kraft pulping experiments using different cooking liquors. E. dunnii wood chips with a calcium content of 3366 mg/kg were used. H- factor Total pulp yield Screened pulp yield (%) Rejects (%) Kappa number unbleached pulp White liquor (reference) 387.8 64 17 48 24 948.8 55 43 11.8 21 2237.3 54 50 3.3 20 3389.6 51 47 2 13 Black + white liquor 387.8 51 28 23.1 25 948.8 58 47 11.5 22 2237.3 57 53 4.2 20 3389.6 52 51 1.4 14 Green liquor (synthetic) 247.6 57 37 20.4 19 278.9 56 52 5 18 387.8 55 55 0 14 Table 1 shows the characteristics of kraft pulps produced using various cooking liquors at different H- factors. Kraft pulping was carried out with the aim of achieving a similar kappa number in the pulp. To achieve the desired kappa number, the cooking temperature was selected as a variable factor (H-factor). In this study, kraft pulping of E. dunnii wood chips with a calcium content of 3366 mg/kg was performed at temperatures ranging from 138°C to 140°C, 145°C, 155°C, 165°C, and 170°C. Table 1 shows the characteristics of kraft pulps produced using various cooking liquors at different H- factors. Results and discussion Kraft pulping was carried out with the aim of achieving a similar kappa number in the pulp. To achieve the desired kappa number, the cooking temperature was selected as a variable factor (H-factor). In this study, kraft pulping of E. dunnii wood chips with a calcium content of 3366 mg/kg was performed at temperatures ranging from 138°C to 140°C, 145°C, 155°C, 165°C, and 170°C. The rate of delignification of high calcium-content wood chips is significantly influenced by green liquor. Despite a lower H-factor for green liquor pulping, high calcium-containing wood chips could be pulped with fewer rejects to achieve a lower kappa (as shown in Figs. 1). Additionally, the kappa numbers were considerably lower for the pulps pulped with green liquor. In contrast, the addition of black liquor did not produce any significant effects (as illustrated in Figs. 1 and 2). E. dunnii wood chips with a calcium content of 3366 mg/kg were employed in this study. Additionally, the viscosity of the pulps generated using green liquor was higher at a given Kappa number (as depicted in Fig. 3). This indicates that green liquor, in some manner, protects cellulose from degradation during kraft pulping. Page 9/28 Page 9/28 Page 9/28 These findings are consistent with previous studies demonstrating that green liquor has a significant impact on the delignification rate and pulp quality in kraft pulping (Andrews and Chang 1985; Ban et al. 2004; Klevinska and Treimanis 1997; Svedman and Tikka 1998). While earlier research suggested that green liquor could be used to increase the sulfur content in pulping, our experiments did not support this explanation since the sulfur content was not increased by the addition of green liquor. Instead, we propose that it is the carbonate ions in green liquor that are responsible for the positive effects by forming inert calcium carbonate crystals, thereby preventing the negative effects of calcium ions during pulping. Figure 5 provides a schematic representation of this hypothesis. However, if this hypothesis is correct, calcium must remain in the fiber even during kraft cooking and not be significantly transferred to the pulping liquor. The data in Fig. 6 support the hypothesis in Fig. 5, indicating that green liquor-treated pulp contains considerably more calcium than white and black liquor- treated pulp. indicating that green liquor-treated pulp contains considerably more calcium than white and black liquor- treated pulp. Results and discussion Table 3 presents the data on the residual alkali, oxalic acid, inorganic, organic, and lignin content of the black liquor collected after each kraft cook. The data indicate that black liquor always contains total dry solids (%) and lignin content (%), as well as inorganics. Additionally, a substantial amount of residual alkali was detected in the black liquor obtained using green liquor at a low kappa number, suggesting that kraft pulping selectivity is more favorable for this kraft cook at a low kappa number, particularly when compared to white liquor and black combined with white liquor. Page 10/28 Page 10/28 Table 3 Characterization of black liquor collected from kraft cooks performed using different cooking liquors. H- factor Dry solids (%) Ash content (%) Residual alkali (mol/l) Precipitated lignin (%) Oxalic acid White liquor (reference) 387.8 19 ± 0.09 62 ± 0.03 0.5 5.5 ± 0.02 1.7 948.8 15 ± 0.05 56 ± 0.04 0.2 3.9 ± 0.3 1.9 2237.3 14 ± 0.06 48 ± 0.2 0.3 3.3 ± 0.2 0.9 3389.6 16 ± 0.04 47 ± 0.03 0.2 3.4 ± 0.1 3.4 Black + white liquor 387.8 22 ± 0.5 57 ± 0.02 0.8 7.5 ± 0.06 1.6 948.8 22 ± 0.05 55 ± 0.04 0.3 8.2 ± 0.3 1.9 2237.3 21 ± 0.8 54 ± 0.03 0.4 4.8 ± 0.3 1.8 3389.6 22 ± 0.06 59 ± 0.02 0.2 5.5 ± 0.4 1.3 Green liquor (synthetic) 247.6 17 ± 0.9 56 ± 0.03 0.4 2.4 ± 0.2 1.7 278.9 18 ± 1 56 ± 0.05 0.5 3.3 ± 0.2 1.9 387.8 17 ± 1 47 ± 0.05 0.6 4.5 ± 0.3 1.9 Figure 5 illustrates a hypothetical interpretation of the chemistry of cooking liquors with regard to calcium oxalate ions in wood chips. Previous research has identified calcium oxalate in the lumen of wood, and during kraft pulping with strongly alkaline white liquor, oxalate and calcium ions can deteriorate into the cell wall. According to our previous study (Vegunta et al. 2022), these calcium ions can accelerate polysaccharide degradation while delaying delignification. If this hypothesis is correct, calcium must remain in the fiber during kraft cooking and should not be significantly transferred to the pulping liquor. The data presented in Fig. 6 support this hypothesis, as green liquor-treated pulp contains considerably more calcium than white and black liquor-treated pulp. Results and discussion Figure 5 illustrates a hypothetical interpretation of the chemistry of cooking liquors with regard to calcium oxalate ions in wood chips. Previous research has identified calcium oxalate in the lumen of wood, and during kraft pulping with strongly alkaline white liquor, oxalate and calcium ions can deteriorate into the cell wall. According to our previous study (Vegunta et al. 2022), these calcium ions can accelerate polysaccharide degradation while delaying delignification. If this hypothesis is correct, calcium must remain in the fiber during kraft cooking and should not be significantly transferred to the pulping liquor. The data presented in Fig. 6 support this hypothesis, as green liquor-treated pulp contains considerably more calcium than white and black liquor-treated pulp. Given the success of kraft pulping with high calcium content E. dunni wood, we tested pulping with wood containing even higher calcium content – 4668 mg/kg wood – a quality that had been virtually impossible to pulp in the laboratory with conventional white liquor pulping. As expected, pure white liquor pulping did not result in defibrillation under the conditions used (see materials and methods), but adding green liquor allowed for pulping to generate pulp with low rejects and acceptable kappa numbers (Table 3) Page 11/28 Page 11/28 Table 4 Details regarding pulp produced using high calcium- containing wood chips (4668mg/kg) using green liquor. Green liquor (synthetic) 100% (0.66M) Rejects (%) 0.1 Total yield (%) 52 Screened pulp yield (%) 52 Kappa number 18.6 Dry solids In Black liquor (%) 18 Ash content in black liquor (%) 59 Precipitated lignin (%) 4.7 Glucose (% on wood) 31.8 Xylose (% on wood) 9.4 Klason lignin (%) 3.97 Viscosity (ml/g) 1369 Calcium content in pulp sample (mg/kg) 4138 HEXA Unbleached pulp (µmol/g) 11.5 HEXA Bleached pulp(µmol/g) 11 en Liquor on Bleaching of Pulp Table 4 containing wood chips (4668mg/kg) using green liquor. Results and discussion Green liquor (synthetic) 100% (0.66M) Rejects (%) 0.1 Total yield (%) 52 Screened pulp yield (%) 52 Kappa number 18.6 Dry solids In Black liquor (%) 18 Ash content in black liquor (%) 59 Precipitated lignin (%) 4.7 Glucose (% on wood) 31.8 Xylose (% on wood) 9.4 Klason lignin (%) 3.97 Viscosity (ml/g) 1369 Calcium content in pulp sample (mg/kg) 4138 HEXA Unbleached pulp (µmol/g) 11.5 HEXA Bleached pulp(µmol/g) 11 Impact of Green Liquor on Bleaching of Pulp Impact of Green Liquor on Bleaching of Pulp In order to assess the impact of green liquor on the bleaching of high calcium content wood chips, additional batches of pulp were prepared as controls and subjected to bleaching experiments. The pulp was produced at a specific kappa number (13–14) to investigate the effects on pulp quality and strength. Wood chips with a calcium content of 3758 mg/kg were used for these experiments, and three different impregnation liquors were employed in the kraft cooking process. These experiments were conducted to determine whether the enhanced mineral content from green liquor would have any adverse effects on pulp bleaching, due to coprecipitated transition metal ions, among other factors. Page 12/28 Table 5 Characterization of kraft cooks subjected to bleaching experiments. White liquor (reference) Black + white liquor Green liquor (synthetic) Rejects (%) 1.3 0 0 Total yield (%) 45.7 46.3 51.5 Screened pulp yield (%) 44.4 46.3 51.5 Kappa number 17 16 16.1 Dry solids In Black liquor (%) 14 23 16 Ash content in black liquor (%) 59 60 59 Precipitated lignin (%) 5.1 9.2 5.7 Viscosity (ml/g) 882 896 1364 Calcium content in pulp sample (mg/kg) 1925 2362 2580 HexA Unbleached pulp (µmol/g) 22.5 31.3 13.5 Glucose (% on wood) 24.5 27.7 31 Xylose (% on wood) 5.5 7.2 7.7 Klason lignin (%) 2.5 2.3 4.9 Table 5 Table 5 summarizes the properties of the pulp obtained through kraft pulping with different impregnation liquors. The green liquor impregnation resulted in higher pulp yield, faster delignification, and higher viscosity at the same kappa number, consistent with previous experiments using wood chips with a calcium content of 3366 mg/kg. All pulps were kraft pulped under identical chemical conditions with different H-factors to achieve a similar kappa number. The total yield increased by 5% when green liquor impregnation was used, compared to white and black + white liquor. Results and discussion 7–11, suggests that calcium carbonate crystals in pulps should not significantly affect the bleaching process. Results and discussion The hexeneuronic acid content was slightly lower in the pulp obtained using green liquor impregnation. Table 5 displays the total inorganic metal content in unbleached pulp samples produced using different impregnation liquors. The unbleached pulp obtained from green liquor impregnation has a slightly higher calcium content compared to those obtained from black liquor + white liquor and white liquor (Table 5). This suggests that the calcium content follows the fiber line instead of being washed out with black liquor. However, the presence of calcium carbonate crystals in the pulp may trap transition metal ions and hinder the bleaching process. Hence, bleaching experiments with the sequence DEDED were conducted on pulps cooked with white and green liquors to comparable kappa numbers to assess the impact of green liquor on bleaching efficiency. The green liquor impregnated pulp samples had significantly higher intrinsic viscosity compared to those impregnated with white liquor and black + white liquor. This Page 13/28 Page 13/28 indicates an improvement in the mechanical properties of the pulp. The findings are consistent with the tensile strength and tear index results obtained from unbleached and bleached pulp samples (see Figs. 8–9). The viscosity (Fig. 7) of unbleached and bleached pulp generated with white liquor and black liquor + white liquor is lower at a given kappa number than that of green liquor impregnated pulp. The use of higher H-factor in pulp obtained from white liquor and black liquor + white liquor could be a reason for the lower viscosity. The benefits of the green liquor treatment in achieving high viscosity were mostly retained even after bleaching, as depicted in Fig. 7. This is evident in the improved tensile index (Fig. 8) and tear index (Fig. 9) for the bleached pulps previously treated with green liquor. Refining of pulp fibers through PFI refining enhances the paper-making properties of pulp fibers, improving their binding ability and increasing their strength. As shown in Fig. 9, both unbleached and bleached pulps processed with green liquor impregnation have higher mechanical and tear indices. The higher hemicellulose content on the fiber surfaces in green liquor-treated pulps may contribute to their improved strength properties. However, this increase in strength appears to cause an increase in drainage resistance (Fig. 10). The bleaching of green liquor-treated pulps was found to be equally effective in developing brightness as it was for the control samples. This, together with the data in Figs. Acknowledgements Monica Heberling, Montes del Plata Colonia Department, is gratefully acknowledged for her role in the sampling of the wood chips samples. Consent for publication Not applicable. Authors' contributions All authors contributed to the planning of the experiments. Most of the experimental work was performed by Vegunta with help from Lindén and Deshpande, except for the bleaching experiments and part of the pulp characterization which was performed by Björk with instructions from Jansson. Garcia and Jansson selected and procured the wood chips samples which were used in the experiments. Vegunta, Henriksson and Sevastyanova wrote the major part of the text, but all authors participated in reading and commenting on the text. Availability of data and materials All data generated or analysed during this work are included in this publication. Raw data for the figures are available from the corresponding author on request. Funding For Vegunta, Garcia, Björk and Jansson, financial support from Stora Enso is gratefully acknowledged. For Lindén, support from the Knut and Alice Wallenberg Foundation (KAW) through the Wallenberg Wood Science Center, KAW 2015.0390, is gratefully acknowledged. Ethics approval and consent to participate Not applicable. Conclusions In this study, we have conducted a series of experiments researching high calcium-content wood chips that cannot pulp under conventional kraft cooking conditions using white liquor on a lab scale. Instead, we could pulp these highly impossible wood chips using green liquor kraft pulping at a lower H-factor. The bleaching of green-liquor cooked pulps was not affected. The following conclusions can be drawn from this work: Exceptionally low reject content and low kappa number at similar H-factor using green liquor impregnation can be achieved compared to reference white liquor and black + white liquor. Kappa number was lower at given H-factor for pulps of calcium rich wood when green liquor was added to the white liquor. The bleached pulps produced using green liquor impregnation have increased tensile, and tear index. The bleached pulps produced using green liquor impregnation have incre The addition of green liquor to kraft pulping directs the calcium to stay with the fiber rather than to go with the black liquor. Kraft cooking with green liquor results in improved selectivity of the kraft pulping. Green liquor usage makes pulping of Eucalypts with even a very high ca Green liquor-treated pulps could be bleached with good results, mainly keeping the advantages from the unbleached pulp. Green liquor-treated pulps could be bleached with good results, mainly keeping the advantages from the unbleached pulp. Page 14/28 Page 14/28 A hypothesis for the effects of green liquor based on the formation of calcium carbonate (Fig. 5) has been presented. A hypothesis for the effects of green liquor based on the formation of calcium carbonate (Fig. 5) has been presented. Competing Interests The authors declare that they have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper. References Page 15/28 Page 15/28 Page 15/28 1. Andrews EK, Chang HM (1985) Extended delignification kraft pulping of softwoods effect of treatment on chips and pulp with sulfide containing liquors. J Wood Chem Technol 5:431-450. 2. Ban W, Song J, Lucia LA (2004) Insight into the Chemical Behaviour of Softwood Carbohydrates during High-Sulfidity Green Liquor Pre-treatment. Ind Eng Chem Res 43:1366-1372. 2. Ban W, Song J, Lucia LA (2004) Insight into the Chemical Behaviour of Softwood Carbohydrates during High-Sulfidity Green Liquor Pre-treatment. Ind Eng Chem Res 43:1366-1372. 3. Ban W, Wang S, Lucia LA (2003) The relationship of pretreatment pulping parameters with respect to pulp qualities: optimization of green liquor pretreatment conditions for improved kraft pulping. 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Vegunta V, Sethikumar ER, Lindén P, Sevastyanova O, Vilaplana F, Garcia A, Björk M, Jansson U, Henriksson G, Lindström ME (2022) High calcium content of Eucalyptus dunnii wood affects delignification and polysaccharide degradation in kraft pulping. Nordic Pulp Paper Res J 37(2):338- 348. Figures Figures Page 17/28 Figure 1 Reject (%) as a function of H-factor. Page 18/28 Figure 1 Reject (%) as a function of H-factor. Reject (%) as a function of H-factor. Reject (%) as a function of H-factor. Page 18/28 Figure 2 Kappa number as a function of H-factor. Page 19/28 Figure 2 Kappa number as a function of H-factor. Figure 2 Kappa number as a function of H-factor. Kappa number as a function of H-factor. Page 19/28 Figure 3 Intrinsic viscosity (ml/g) as a function of H-factor. Page 20/28 Figure 3 Intrinsic viscosity (ml/g) as a function of H-factor. Figure 3 Figure 3 Intrinsic viscosity (ml/g) as a function of H-factor. Intrinsic viscosity (ml/g) as a function of H-factor. Page 20/28 Page 20/28 Figure 4 Intrinsic viscosity (ml/g) as a function of kappa number. Figure 4 Intrinsic viscosity (ml/g) as a function of kappa number. Page 21/28 Page 21/28 Page 21/28 Figure 5 Hypothetical explanation for the strong positive effect of green liquor impregnation. Page 22/28 Figure 6 Calcium content in pulp (mg/kg) as a function of kappa number Page 23/28 Figure 6 Calcium content in pulp (mg/kg) as a function of kappa number Figure 6 Calcium content in pulp (mg/kg) as a function of kappa number Page 23/28 Page 23/28 Page 23/28 Figure 7 Intrinsic viscosity (ml/g) after kraft cooking and after bleaching. Page 24/28 Figure 7 Intrinsic viscosity (ml/g) after kraft cooking and after bleaching. Figure 7 Intrinsic viscosity (ml/g) after kraft cooking and after bleaching. Intrinsic viscosity (ml/g) after kraft cooking and after bleaching. Page 24/28 Page 24/28 Figure 8 Tensile index (Nm/g) of pulps produced at targeted kappa number using different cooking liquor. Figure 8 Tensile index (Nm/g) of pulps produced at targeted kappa number using different cooking liquor. Tensile index (Nm/g) of pulps produced at targeted kappa number using different cooking liquor. Page 25/28 Figure 9 Tear index (mNm2/g) of pulp pulps produced at targeted kappa number using different cooking liquor. Figure 9 Figure 9 Tear index (mNm2/g) of pulp pulps produced at targeted kappa number using different cooking liquor. Page 26/28 Figure 10 PCC brightness of unbleached and bleached pulp Figure 10 PCC brightness of unbleached and bleached pulp Figure 10 PCC brightness of unbleached and bleached pulp Figure 10 PCC brightness of unbleached and bleached pulp Page 27/28 Figure 11 Drainage resistance of pulps produced at targeted kappa number using different cooking liquor. Figure 11 Drainage resistance of pulps produced at targeted kappa number using different cooking liquor. Drainage resistance of pulps produced at targeted kappa number using different cooking liquor. 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https://openalex.org/W3030030005	https://ro.uow.edu.au/cgi/viewcontent.cgi?article=5909&context=sspapers	English	null	Augmenting cancer registry data with health survey data with no cases in common: the relationship between pre-diagnosis health behaviour and post-diagnosis survival in oesophageal cancer	BMC cancer	2,020	cc-by	9,370	University of Wollongong University of Wollongong Research Online Research Online Faculty of Social Sciences - Papers (Archive) Faculty of Arts, Social Sciences & Humanities 1-1-2020 Augmenting cancer registry data with health survey data with no cases in Augmenting cancer registry data with health survey data with no cases in common: The relationship between pre-diagnosis health behaviour and common: The relationship between pre-diagnosis health behaviour and post-diagnosis survival in oesophageal cancer post-diagnosis survival in oesophageal cancer Paul Fahey Andrew Page Glenn Stone Thomas E. Astell-Burt University of Wollongong, thomasab@uow.edu.au University of Wollongong University of Wollongong Research Online Research Online Sciences - Papers (Archive) Faculty of Arts, Social Sciences & Humanities Faculty of Social Sciences - Papers (Archive) Faculty of Arts, Social Sciences & Humanities Augmenting cancer registry data with health survey data with no cases in Augmenting cancer registry data with health survey data with no cases in common: The relationship between pre-diagnosis health behaviour and common: The relationship between pre-diagnosis health behaviour and post-diagnosis survival in oesophageal cancer post-diagnosis survival in oesophageal cancer Thomas E. Astell-Burt University of Wollongong, thomasab@uow.edu.au Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Disciplines Disciplines Education \| Social and Behavioral Sciences Abstract Abstract 2020 The Author(s). Background: For epidemiological research, cancer registry datasets often need to be augmented with additional data. Data linkage is not feasible when there are no cases in common between data sets. We present a novel approach to augmenting cancer registry data by imputing pre- diagnosis health behaviour and estimating its relationship with post-diagnosis survival time. Methods: Six measures of pre-diagnosis health behaviours (focussing on tobacco smoking, 'at risk' alcohol consumption, overweight and exercise) were imputed for 28,000 cancer registry data records of US oesophageal cancers using cold deck imputation from an unrelated health behaviour dataset. Each data point was imputed twice. This calibration allowed us to estimate the misclassification rate. We applied statistical correction for the misclassification to estimate the relative risk of dying within 1 year of diagnosis for each of the imputed behaviour variables. Subgroup analyses were conducted for adenocarcinoma and squamous cell carcinoma separately. Results: Simulated survival data confirmed that accurate estimates of true relative risks could be retrieved for health behaviours with greater than 5% prevalence, although confidence intervals were wide. Applied to real datasets, the estimated relative risks were largely consistent with current knowledge. For example, tobacco smoking status 5 years prior to diagnosis was associated with an increased age-adjusted risk of all cause death within 1 year of diagnosis for oesophageal squamous cell carcinoma (RR = 1.99 95% CI 1.24,3.12) but not oesophageal adenocarcinoma RR = 1.61, 95% CI 0.79,2.57). Conclusions: We have demonstrated a novel imputation- based algorithm for augmenting cancer registry data for epidemiological research which can be used when there are no cases in common between data sets. The algorithm allows investigation of research questions which could not be addressed through direct data linkage. Publication Details Publication Details Fahey, P., Page, A., Stone, G. & Astell-Burt, T. (2020). Augmenting cancer registry data with health survey data with no cases in common: The relationship between pre-diagnosis health behaviour and post- diagnosis survival in oesophageal cancer. BMC Cancer, 20 (1), This journal article is available at Research Online: https://ro.uow.edu.au/sspapers/4833 Fahey et al. BMC Cancer (2020) 20:496 https://doi.org/10.1186/s12885-020-06990-3 Open Access Augmenting cancer registry data with health survey data with no cases in common: the relationship between pre- diagnosis health behaviour and post- diagnosis survival in oesophageal cancer Paul P. Fahey1* , Andrew Page2, Glenn Stone3 and Thomas Astell-Burt4 Paul P. Fahey1* , Andrew Page2, Glenn Stone3 and Thomas Astell-Burt4 © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: For epidemiological research, cancer registry datasets often need to be augmented with additional data. Data linkage is not feasible when there are no cases in common between data sets. We present a novel approach to augmenting cancer registry data by imputing pre-diagnosis health behaviour and estimating its relationship with post-diagnosis survival time. Methods: Six measures of pre-diagnosis health behaviours (focussing on tobacco smoking, ‘at risk’ alcohol consumption, overweight and exercise) were imputed for 28,000 cancer registry data records of US oesophageal cancers using cold deck imputation from an unrelated health behaviour dataset. Each data point was imputed twice. This calibration allowed us to estimate the misclassification rate. We applied statistical correction for the misclassification to estimate the relative risk of dying within 1 year of diagnosis for each of the imputed behaviour variables. Subgroup analyses were conducted for adenocarcinoma and squamous cell carcinoma separately. Results: Simulated survival data confirmed that accurate estimates of true relative risks could be retrieved for health behaviours with greater than 5% prevalence, although confidence intervals were wide. Applied to real datasets, the estimated relative risks were largely consistent with current knowledge. For example, tobacco smoking status 5 years prior to diagnosis was associated with an increased age-adjusted risk of all cause death within 1 year of diagnosis for oesophageal squamous cell carcinoma (RR = 1.99 95% CI 1.24,3.12) but not oesophageal adenocarcinoma RR = 1.61, 95% CI 0.79,2.57). Conclusions: We have demonstrated a novel imputation-based algorithm for augmenting cancer registry data for epidemiological research which can be used when there are no cases in common between data sets. The algorithm allows investigation of research questions which could not be addressed through direct data linkage. Keywords: Cancer registries, Alcohol drinking, Oesophageal neoplasms, Exercise, Obesity, Tobacco smoking * Correspondence: p.fahey@westernsydney.edu.au 1School of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia Full list of author information is available at the end of the article * Correspondence: p.fahey@westernsydney.edu.au 1School of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia Full list of author information is available at the end of the article Data sources Oesophageal cancer cases were extracted from the Sur- veillance, Epidemiology, and End Results Program (SEER) cancer registries database, which combines data from can- cer registries in up to 13 US States covering up to 28% of the US population [19]. Available data included patient demographics and outcomes (including survival time). Oesophageal cancer is the seventh most common can- cer by site [5], has low survival [6], and caused an esti- mated 1 in 20 cancer deaths worldwide in 2018 [5]. It has been estimated that 71% of male and 59% of female oesophageal cancer deaths in the US arise from modifiable health behaviours: including smoking (50%), alcohol con- sumption (17%) and excess body weight (27%) [7]. The impact of pre-diagnosis health behaviour on oesophageal cancer survival is uncertain. As survival times are short, the carry-over effect of pre-diagnosis behaviour may be important, and potentially impact treatment choices [8]. Further, as health behaviours in populations change over time [9, 10], predicting the impact of behaviour on cancer survival would assist in forecasting future disease burden and health service requirements. All records of primary oesophageal cancers diagnosed between 2006 to 2014 were downloaded using the SEERStat utility [20]. After excluding 112 cases < 35 years of age as atypical, the dataset contained 34,972 oesophageal cancer cases. Health behaviour data of US residents were extracted from the Behavioural Risk Factor Surveillance System (BRFSS) [21]. This telephone survey of the adult popula- tion of US residents (all States) has been conducted an- nually since 1984. All 3,018,830 records from 2001 to 2009 were included. Given that health behaviour can change after diagnosis [22, 23] the BRFSS health behaviour best represented the health behaviour of oesophageal cancer cases pre- diagnosis. We added a 5-year lag to minimise the risk of early symptoms influencing behaviour. The initial year was the earliest year in which BRFSS used a consistent definition for health behaviours selected for the present study. The end year was the most recently available SEER cancer registry data which allowed at least 12- months follow-up. Associations between oesophageal cancer incidence and health behaviour (including tobacco smoking, alco- hol consumption, body mass index and physical activity) differ by histological sub-type [11, 12] with oesophageal squamous cell carcinoma (ESCC) and oesophageal adenocarcinoma (EAC) usually examined separately. Similar differences may exist for survival time [13, 14]. Data sources Nowadays, cancer survival data is generally available through cancer registries [15], but not data on pre- diagnosis health behaviour. Registry data needs to be augmented with additional data collection or linkage to external data sources. Additional data collection can be time consuming, expensive and subject to survivor bias [16] and data linkage needs the same individuals to be present and identifiable in both data collections and is less feasible for rare disease like oesophageal cancer. Methods The role of cancer registries has changed considerably over time [4]. Since the 1990s, for example, the develop- ment of specialised data linkage infrastructure has open wide new research applications [4]. However, data link- age may not be feasible in all circumstances. There are still research questions which are waiting for a suitable method of analysis. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Fahey et al. BMC Cancer (2020) 20:496 Page 2 of 11 Page 2 of 11 Background with large datasets and careful calibration, imputing a completely missing variable could return valid results. We describe and evaluate an algorithm for assessing the relationship between pre-diagnosis health behaviours and survival at one-year post-diagnosis for oesophageal cancer where survival is derived from cancer registry data and key health behaviours are fully imputed using unrelated health survey data. In 2011 it was estimated that that the cost of maintain- ing the United States’ National Program of Cancer Registries was $US60.77 per case [1]. The estimated number of new United States cancer cases in 1999 was 1,291,451 [2] and 1,762,450 in 2019 [3] an increase of 36% in 20 years. As in any public investment, there is al- ways a need to maintain, and indeed increase, benefits of cancer registries relative to costs. Outcomes, predictors and subgroups The dichotomous outcome was all-cause mortality within 1 year of diagnosis. Six self-reported measures of health behaviour were selected based on previous associations with oesophageal cancer [11, 24] and availability in the BRFSS dataset: Current tobacco smoking (yes or no), defined as daily or less than daily smoking; When faced with missing data, researchers sometimes use imputation [17]. Imputing data is likely to lead to misclassification of health behaviours (such as smokers classified as non-smokers and vice-versa). However, re- peated observations of the same behaviour can be used to quantify, and subsequently correct for misclassifica- tion [18]. In this paper we investigate the possibility that, When faced with missing data, researchers sometimes use imputation [17]. Imputing data is likely to lead to misclassification of health behaviours (such as smokers classified as non-smokers and vice-versa). However, re- peated observations of the same behaviour can be used to quantify, and subsequently correct for misclassifica- tion [18]. In this paper we investigate the possibility that, Alcohol consumption – possible binge drinking (yes or no), defined as ≥5 standard drinks for males or ≥ 4 standard drinks for females on at least one occasion in the month prior to survey; Alcohol consumption – possible heavy drinking (yes or no), defined as > 2 standard drinks per day for Fahey et al. BMC Cancer (2020) 20:496 Page 3 of 11 Page 3 of 11 Page 3 of 11 men and > 1 standard drink per day for women in the month prior to survey; men and > 1 standard drink per day for women in the month prior to survey; 5 years earlier and one age-group younger than the cor- responding SEER cancer case. men and > 1 standard drink per day for women in the month prior to survey; There were 37,440 possible combinations of the auxil- iary variable categories, 7397 of which occurred within the SEER oesophageal cancer cases. Of these, 6986 (94.4%) contained at least one eligible BRFSS donor record. Physical activity (yes or no), defined as any physical activity or exercise in the past 30 days other than for regular job; g j Obese (yes/no), defined as body mass index ≥30 kg/ m2; and Current tobacco smoking with regular alcohol (yes or no), defined as current tobacco smoking with ≥1 standard drink of alcohol per day on average in the previous month. Imputation method and covariates The complete absence of data on health behaviour meant that regression-based imputation and multiple imputation could not be used [25]. Random cold deck imputation [17] based on demographic strata was appro- priate, as there were demographic variables in common between the two datasets and individuals from the same demographic group have a greater likelihood of engaging in similar health behaviours [26]. Only 458,780 of the BRFSS health behaviour records matched the SEER cases on the auxiliary variables. The number with missing health behaviour ranged from 564 (0.1%) for physical activity to 17,624 (3.9%) for obesity. To avoid imputing a missing value into a missing value, these records were excluded. To avoid cumulative ef- fects, we created six separate donor datasets (each con- taining complete cases for one of the six health behaviours) and imputed each health behaviour independently. In random cold deck imputation individuals are allo- cated into strata according to auxiliary variables and then, within each stratum, one ‘donor’ record is ran- domly selected for each ‘recipient’ record. The BRFSS health behaviour data were the donor records and the SEER cancer registry data were the recipients. The re- cipient record is assigned the behaviour of the donor record. The more, and the more informative, the auxil- iary variables the greater the chance the imputed behav- iour will be correct. Missing data, exclusions and the final dataset Approximately 80% of the 35,084 eligible oesophageal cancer cases were included in the analyses. (Additional file 2). SEER cases were excluded for missing survival time or auxiliary variables (n = 2784, 8.0%) or failing to find two donor records (from 4353 to 4453 (12.4 to 12.7%) varying between health behaviours). Cases with- out two donor records were more likely to be older, from earlier study years and California residents (Add- itional file 3). Outcomes, predictors and subgroups To allow calibration, we randomly selected two BRFSS donor records for each SEER case (without replace- ment), such that each cancer case had two imputed values for each lifestyle variable. Where donor records were exhausted before cancer cases, the cancer case was omitted from the analysis (see Additional file 1). Histological subgroups were defined using Inter- national Classification of Diseases for Oncology, third edition (ICD-O-3) with 805–808 indicating ESCC (n = 10,454) and 814–838 indicating EAC (n = 17,950). Checking the algorithm with simulated data Checking the algorithm with simulated data For each behaviour, we cross-tabulated the first set of imputed values against 1 year survival status and calcu- lated the relative risk of death within 1 year, RRi. The subscript i signifies that the imputed data were used in the calculations. In the absence of a cohort showing the true relationship between pre-diagnosis health behaviour and post- diagnosis survival time, we used simulated data to test the algorithm. The first set of imputed behaviour was designated to be the ‘true’ health behaviour of each cancer case. For each health behaviour we separately simulated seven survival status variables (repeated 100 times): to produce relative risks of 0.50, 0.66, 0.80, 1.00, 1.25, 1.50 and 2.00 while maintaining the overall rate of the health behav- iour pi and 1 year death rate (Additional file 7). Other potential predictors of survival times were investi- gated using log-binary regression with associated log likeli- hood ratio statistics and area under the receiver operator curves (Additional file 5). Age was identified as a confounder as both post-diagnosis survival and proportion recording each health behaviours were lower among older age groups (Additional file 5). To adjust for this, age-adjusted relative risks, adjRRi, were estimated using the Cochrane-Mantel- Haenzel method [28]. Other potential demographic predic- tors of survival were found to be of lesser impact or confounded with age (see Additional file 5). The imputed relative risks were obtained using the second set of imputed health behaviours. As the second set of imputed values were selected independently and without replacement, they had a similar relationship with the first set of simulated data as with the actual cancer cases. The main difference is that the simulated survival data, being based only on the behaviour of inter- est, have no relationship with (confounding from) any other variables. The true data were likely to display more complex relationships. Beyond the demographic variables, cancer stage at diag- nosis (coded by SEER according to the AJCC Cancer Sta- ging Manual 6th Edition [29]) was confirmed as a stronger predictor of survival (Additional file 5) but, occurring after health behaviour exposure, may partially lie on the disease pathway. That is, smokers may have more advanced dis- ease at diagnosis due to their smoking and so correcting for cancer stage at diagnosis may falsely attenuate the as- sociation between pre-diagnosis smoking and survival post diagnosis [30]. Calibrating the effectiveness of imputation Six auxiliary variables were used: We used the paired imputed values to calibrate the im- putation process (see Additional file 4). In brief, let pi represent the proportion of imputed values where the behaviour is present. If the imputation process retained no information on behaviour, the expected proportion of behaviour present to behaviour present matches is p2 i - the agreement arising through chance alone. If the imputation process is informative, the proportion of be- haviour present to behaviour present matches is greater than chance. We modelled these excess matches as pi(1 −pi) ρ where ρ is a measure of correlation [27]. – Age category at diagnosis (5-year groups from 35- 39y to 75-79y then >80y); – Gender (male; female); – Marital status (married, including common law; single or never married; widowed; divorced); – Race (white; black; Asian or Pacific Islander; American Indian or Alaska Native); – State of residence (Alaska; California; Connecticut; Georgia; Hawaii; Iowa; Kentucky; Louisiana; Michigan; New Jersey; New Mexico; Utah; Washington); We estimated pi as the proportion imputed to have the behaviour (averaged across the two imputed values) and estimated ρ using the phi coefficient (the correlation coefficient for dichotomous variables) between the pairs of imputed values. All analyses were conducted separ- ately for each health behaviour. – Year of diagnosis (2006 to 2014). To produce the 5-year lag, we defined the donor re- cords to be BRFSS health behaviour records which were Page 4 of 11 Fahey et al. BMC Cancer (2020) 20:496 Fahey et al. BMC Cancer (2020) 20:496 Checking the algorithm with simulated data Subgroup analyses for cancer stage at diag- nosis are provided in Additional file 8. Calibrating the imputation The estimated proportion of cancer cases with a given health behaviour, pi, ranged from a median of 0.737 for physical activity to 0.034 for current smoking with regu- lar drinking (Table 1). The phi coefficients, φ, show that there is usually a positive correlation between the two imputed values, albeit weak (medians between 0.008 and 0.077). This confirms that some information about health behaviour is being conveyed through the random cold deck imputation. The value npi(1 −pi)ρ, the number of correct matches greater than would be expected through chance, quantifies the information conveyed through the imputation. ‘Heavy drinking’, and ‘current smoking with regular drinking’, had the lowest preva- lence (median of 0.05 or less), the lowest correlations be- tween imputed observations (median less than 0.025) and hence lowest information (medians below 20 matches beyond chance). Non-differential misclassification errors will, barring random error and confounding, attenuate the estimated relative risk toward the null [31]. The mathematical rela- tionship between the relative risk using the imputed data, RRi, and the true relative risk for the cancer cases, RRT, is derived in Additional file 6. In brief, if the preva- lence of behaviour is the same between the donor re- cords and cancer cases in each stratum, the true relative risk can be estimated using RRT ¼ 1− RRi−1 ð Þ RRi−1 ð Þpi 1−ρ ð Þ−ρ Extreme values of pi and/or ρ can be problematic. For example, when ρ = 0, RRT is negative: an impossible value for a relative risk. Analyses using simulated survival status Random cold deck imputation was repeated 100 times, separately for each of the six health behaviours. As donor records were selected at random within strata, each statistic varied between repetitions. Results were re- ported as the median value from the 100 repetitions with the associated 2.5 and 97.5 percentiles as empirical 95% confidence intervals. We report subgroup analyses for ESCC and EAC. Where more than 5% of the estimates of the true relative risk RRT were impossible, the imput- ation process was labelled as ‘failed’. The simulated relative risks of survival were accurate to two-decimal places and precise (with a maximum mar- gin of error of 0.07) (Table 2). The relative risks ob- tained by using the (second) imputed behaviour (RRi) were substantially attenuated toward the null differing from 1.0 only in the second decimal place. Estimation of the true relative risk from the imputed relative risk failed for the two least common health be- haviours: ‘heavy drinking’ and ‘current smoking with regular drinking’. For the other four behaviours, the Fahey et al. Analyses using simulated survival status BMC Cancer (2020) 20:496 Page 5 of 11 Table 1 The estimated proportions with each health behaviour, the phi coefficient between imputed values and the estimated excess matches for each analysis Behaviour 5 years before diagnosis N Estimated proportion with behaviour, bpi Estimated phi coefficient, ^ρ = φ Estimated excess matches, nbpið1−bpiÞ^ρ Median 95% CI Median 95% CI Median 95% CI Current smoking overall 27,835 0.159 0.157,0.162 0.071 0.059,0.084 262.2 220.1312.2 ESCC 8914 0.166 0.162,0.170 0.077 0.061,0.097 94.8 74.5120.7 EAC 15,726 0.157 0.153,0.159 0.066 0.052,0.081 137.0 107.4169.5 Binge drinking Overall 27,750 0.100 0.098, 0.102 0.060 0.049,0.077 150.5 121.5192.1 ESCC 8891 0.086 0.082,0.089 0.060 0.042,0.086 42.2 29.8,61.1 EAC 15,673 0.109 0.106,0.111 0.058 0.042,0.079 88.6 63.6120.3 Heavy drinking Overall 27,749 0.048 0.047,0.050 0.011 0.002,0.025 14.3 2.7,32.0 ESCC 8888 0.046 0.043,0.049 0.015 −0.002,0.036 5.7 −0.7,14.2 EAC 15,676 0.050 0.048,0.052 0.008 −0.004,0.028 6.0 −3.0,20.8 Physical activity Overall 27,830 0.737 0.734,0.740 0.034 0.026,0.046 185.1 139.4247.4 ESCC 8912 0.716 0.709,0.721 0.036 0.016,0.056 64.7 29.6100.2 EAC 15,724 0.750 0.746,0.754 0.031 0.013,0.047 91.4 40.0,138.4 Obese Overall 27,796 0.257 0.254,0.261 0.030 0.020,0.042 160.2 108.4226.8 ESCC 8898 0.262 0.255,0.268 0.045 0.024,0.061 77.0 41.4104.6 EAC 15,709 0.256 0.251,0.261 0.023 0.012,0.041 67.8 35.0,122.4 Current smoking with regular drinking Overall 27,735 0.034 0.033,0.035 0.022 0.009,0.038 19.8 8.0,34.2 ESCC 8883 0.031 0.029,0.033 0.024 −0.000,0.049 6.2 −0.0,13.5 EAC 15,670 0.035 0.034,0.037 0.021 0.004,0.042 11.5 2.1,22.4 bpi proportion of imputed values where the health behaviour is present ^ρ = φ the correlation between the pairs of imputed values (calculated as the phi coefficient) nbpið1−bpiÞ^ρ= the excess number of correct matches greater than would be expected through chance alone Median median of 100 repetitions of the imputation algorithm, 95% CI = empirical 95% confidence interval created from the 2.5 and 97.5 percentiles obtained from 100 repetitions of the imputation algorithm, N number of SEER oesophageal cancer cases receiving data from two donor records from the BRFSS health behaviour datasets ESCC oesophageal squamous cell carcinoma, EAC oesophageal adenocarcinoma Table 1 The estimated proportions with each health behaviour, the phi coefficient between imputed values and the estimated excess matches for each analysis imputed relative risks had the opposite direction of asso- ciation confirming the potential for confounding by age. Current tobacco smoking 5 years prior to diagnosis was detrimental to one-year survival after diagnosis following adjustment for age, particularly in ESCC where the esti- mated relative risk was 2.0 (95%CI 1.24, 3.12). Analyses using simulated survival status For ESCC, the median relative risk for binge drinking 5 years prior to diagnosis was 1.52 although the range of possible rela- tive risks was wide (95% CI 0.44,2.75). Similar results were seen for obesity (ESCC estimated RR 1.73, 95%CI 0.83,4.17). Physical activity 5-years prior to diagnosis was protective for survival with median estimated median of the estimated true relative risk was accurate to one, and often two, decimal places. However, the con- fidence intervals were wide and few excluded no association. Analyses using true survival status When imputing the health behaviours onto SEER cancer cases, the median imputed relative risks (RRi) are attenu- ated to close to 1.0 (Table 3). Less expectedly, most of the median risks are less than 1.0; suggesting that most behaviours were associated with a lower rate of death within one year of diagnosis. Many of the age-adjusted Fahey et al. Analyses using true survival status BMC Cancer (2020) 20:496 Page 6 of 11 Table 2 Result of simulation-based testing of whether or not the imputation can Target RR Simulated data RR Imputed RR (RRi) Imp Median 95% CI Median 95% CI Fre Current smoking RR = 0.5 0.501 0.475,0.521 0.964 0.934,0.993a 0 RR = 0.66 0.660 0.635,0.683 0.973 0.944,0.999a 0 RR = 0.80 0.799 0.771,0.823 0.983 0.952,1.017 0 RR = 1.00 1.001 0.976,1.026 0.997 0.967,1.027 0 RR = 1.25 1.249 1.220,1.287 1.017 0.989,1.048 0 RR = 1.50 1.499 1.465,1.528 1.032 1.005,1.059a 0 RR = 2.00 2.000 1.974,2.034 1.064 1.034,1.092a 0 Binge drinking RR = 0.5 0.501 0.474,0.526 0.967 0.940,0.996a 0 RR = 0.66 0.659 0.624,0.692 0.976 0.945,1.015 1 RR = 0.80 0.798 0.758,0.830 0.988 0.959,1.025 0 RR = 1.00 0.997 0.963,1.033 0.999 0.971,1.032 0 RR = 1.25 1.245 1.213,1.278 1.016 0.984,1.054 0 RR = 1.50 1.499 1.463,1.534 1.030 0.990,1.068 0 RR = 2.00 1.999 1.978,2.028 1.058 1.021,1.093a 0 Heavy Drinking RR = 0.5 0.500 0.450,0.548 0.995 0.945,1.046 40 RR = 0.66 0.661 0.606,0.697 0.995 0.946,1.046 34 RR = 0.80 0.799 0.746,0.847 0.997 0.944,1.053 43 RR = 1.00 0.997 0.949,1.045 0.998 0.940,1.041 32 RR = 1.25 1.251 1.210,1.300 1.003 0.959,1.053 22 RR = 1.50 1.497 1.459,1.535 1.012 0.956,1.059 24 RR = 2.00 Not possible Not possible Physical activity RR = 0.5 0.500 0.491,0.509 0.974 0.951,0.997a 0 RR = 0.66 0.659 0.645,0.671 0.983 0.959,1.006 0 RR = 0.80 0.800 0.782,0.818 0.993 0.971,1.017 0 RR = 1.00 1.002 0.976,1.022 1.001 0.978,1.021 0 RR = 1.25 1.250 1.219,1.276 1.006 0.977,1.030 0 RR = 1.50 1.499 1.455,1.549 1.013 0.987,1.037 2 RR = 2.00 2.003 1.939,2.083 1.021 1.002,1.047 3 Obese RR = 0.5 0.499 0.485,0.517 0.983 0.960,1.008 1 RR = 0.66 0.660 0.634,0.680 0.989 0.962,1.016 2 RR = 0.80 0.802 0.777,0.823 0.995 0.967,1.015 1 RR = 1.00 1.002 0.981,1.024 0.999 0.980,1.024 0 RR = 1.25 1.250 1.222,1.274 1.009 0.989,1.030 0 RR = 1.50 1.500 1.468,1.534 1.014 0.987,1.039 0 RR = 2.00 2.002 1.961,2.041 1.025 0.997,1.044 0 Current smoking with regular drinking RR = 0.5 0.504 0.441,0.550 0.988 0.931,1.034 37 RR = 0.66 0.660 0.600,0.713 0.997 0.932,1.066 31 Table 2 Result of simulation-based testing of whether or not the imputation can be used to predict relative risk Target RR Simulated data RR Imputed RR (RRi) Impossible Result (RRT < 0) Estimated true RR (RRT) Median 95% CI Median 95% CI Frequency Median 95% CIb Current smoking RR = 0.5 0.501 0.475,0.521 0.964 0.934,0.993a 0 0.519 0.163,0.904a RR = 0.66 0.660 0.635,0.683 0.973 0.944,0.999a 0 0.638 0.300,0.985a RR = 0.80 0.799 0.771,0.823 0.983 0.952,1.017 0 0.753 0.375,1.226 RR = 1.00 1.001 0.976,1.026 0.997 0.967,1.027 0 0.957 0.577,1.444 RR = 1.25 1.249 1.220,1.287 1.017 0.989,1.048 0 1.254 0.856,1.793 RR = 1.50 1.499 1.465,1.528 1.032 1.005,1.059a 0 1.486 1.069,1.947a RR = 2.00 2.000 1.974,2.034 1.064 1.034,1.092a 0 2.047 1.542,2.532a Binge drinking RR = 0.5 0.501 0.474,0.526 0.967 0.940,0.996a 0 0.478 0.087,0.927a RR = 0.66 0.659 0.624,0.692 0.976 0.945,1.015 1 0.629 0.173,1.316 RR = 0.80 0.798 0.758,0.830 0.988 0.959,1.025 0 0.805 0.341,1.448 RR = 1.00 0.997 0.963,1.033 0.999 0.971,1.032 0 0.981 0.518,1.492 RR = 1.25 1.245 1.213,1.278 1.016 0.984,1.054 0 1.271 0.739,2.029 RR = 1.50 1.499 1.463,1.534 1.030 0.990,1.068 0 1.517 0.831,2.246 RR = 2.00 1.999 1.978,2.028 1.058 1.021,1.093a 0 2.014 1.352,2.717a Heavy Drinking RR = 0.5 0.500 0.450,0.548 0.995 0.945,1.046 40 failed failed RR = 0.66 0.661 0.606,0.697 0.995 0.946,1.046 34 failed failed RR = 0.80 0.799 0.746,0.847 0.997 0.944,1.053 43 failed failed RR = 1.00 0.997 0.949,1.045 0.998 0.940,1.041 32 failed failed RR = 1.25 1.251 1.210,1.300 1.003 0.959,1.053 22 failed failed RR = 1.50 1.497 1.459,1.535 1.012 0.956,1.059 24 failed failed RR = 2.00 Not possible Not possible Physical activity RR = 0.5 0.500 0.491,0.509 0.974 0.951,0.997a 0 0.504 0.319,0.901a RR = 0.66 0.659 0.645,0.671 0.983 0.959,1.006 0 0.632 0.367,1.231 RR = 0.80 0.800 0.782,0.818 0.993 0.971,1.017 0 0.833 0.449,1.907 RR = 1.00 1.002 0.976,1.022 1.001 0.978,1.021 0 1.025 0.488,2.092 RR = 1.25 1.250 1.219,1.276 1.006 0.977,1.030 0 1.206 0.541,2.961 RR = 1.50 1.499 1.455,1.549 1.013 0.987,1.037 2 1.514 0.722,4.078 RR = 2.00 2.003 1.939,2.083 1.021 1.002,1.047* 3 2.127 1.055,10.987a Obese RR = 0.5 0.499 0.485,0.517 0.983 0.960,1.008 1 0.550 0.028,1.322 RR = 0.66 0.660 0.634,0.680 0.989 0.962,1.016 2 0.665 0.114,1.772 RR = 0.80 0.802 0.777,0.823 0.995 0.967,1.015 1 0.846 0.316,1.676 RR = 1.00 1.002 0.981,1.024 0.999 0.980,1.024 0 0.962 0.461,2.067 RR = 1.25 1.250 1.222,1.274 1.009 0.989,1.030 0 1.335 0.601,2.300 RR = 1.50 1.500 1.468,1.534 1.014 0.987,1.039 0 1.440 0.606,2.796 RR = 2.00 2.002 1.961,2.041 1.025 0.997,1.044 0 1.995 0.886,3.234 Current smoking with regular drinking RR = 0.5 0.504 0.441,0.550 0.988 0.931,1.034 37 failed failed RR = 0.66 0.660 0.600,0.713 0.997 0.932,1.066 31 failed failed Fahey et al. Analyses using true survival status exclude relative risk equals 1) b excludes impossible result Target RR – the relative risk we attempted to achieve in the simulated data Simulated data RR – the relative risk which was actually achieved between the first imputed value and the simulated one-year survival status Imputed RR (RRi) – the relative risk calculated using the second imputed data point as the imputed behaviour Impossible result – instances where the estimated true relative risk was impossible (a negative value) Estimated True RR (RRT) – the estimated true relative risk derived from the imputed relative risk and calibration parameters bpi and ^ρ Median median of 100 repetitions of the imputation algorithm, 95% CI = empirical 95% confidence interval created from the 2.5 and 97.5 percentiles obtained from 100 repetitions of the imputation algorithm, a 95% confidence intervals exclude no association (i.e. exclude relative risk equals 1) b excludes impossible result relative risks of approximately 0.50 (95%CI 0.31, 1.03) for oesophageal cancer overall. evidence of association between smoking and survival in EAC [24, 33]. The unadjusted protective effects of smoking has also been reported [34, 35] as has the change in the direction of the association following age adjustment [35]. Estimates of the relative risks could not be retrieved for the less common behaviours ‘heavy drinking’ and ‘current smoking with regular drinking’. The one relative risk which was retrieved - a median RR of 3.35 for current smoking with regular drinking in all oesophageal cancer - was accompanied by wide uncertainty (95% CI 0.77,11.84). g j A previous meta-analysis found that ever drinking alcohol had a detrimental association with survival in ESCC (HR 1.36, 95% CI 1.15, 1.61) but not in EAC (HR = 1.08 95% CI 0.85, 1.37) [24]. More recent re- sults from China (HR = 1.58, 95% CI 1.21,2.07 [36, 37], HR = 1.45 95% CI 1.13,1.87 [37]) and Japan (HR = 2.37 95% CI 1.24,4.53 [38]) also support the detrimental impact of pre-diagnosis alcohol con- sumption on survival in ESCC. We could not esti- mate the association between heavy drinking and survival. However, for binge drinking five years prior to diagnosis, the median relative risk was 1.52 in ESCC, although the confidence interval (95% CI 0.44, 2.75) allows no association. Subgroup analyses on cancer stage at diagnosis (Additional file 8), suggests that pre-diagnosis health behaviours have stronger relationships with one-year survival in those who are not metastatic at diagnosis. Analyses using true survival status BMC Cancer (2020) 20:496 Page 7 of 11 tion-based testing of whether or not the imputation can be used to predict relative risk (Continued) Table 2 Result of simulation-based testing of whether or not the imputation can be used to predict relative risk (Continued) Target RR Simulated data RR Imputed RR (RRi) Impossible Result (RRT < 0) Estimated true RR (RRT) Median 95% CI Median 95% CI Frequency Median 95% CIb RR 0 80 0 797 0 744 0 863 0 991 0 928 1 052 34 f il d f il d Table 2 Result of simulation-based testing of whether or not the imputation can be used to predict relative risk (Continued) Target RR Simulated data RR Imputed RR (RRi) Impossible Result (RRT < 0) Estimated true RR (RRT) Median 95% CI Median 95% CI Frequency Median 95% CIb RR = 0.80 0.797 0.744,0.863 0.991 0.928,1.052 34 failed failed RR = 1.00 0.996 0.943,1.049 1.001 0.940,1.059 25 failed failed RR = 1.25 1.250 1.183,1.298 1.009 0.954,1.059 16 failed failed RR = 1.50 1.497 1.454,1.545 1.000 0.958,1.065 19 failed failed RR = 2.00 Not possible Not possible Target RR – the relative risk we attempted to achieve in the simulated data Simulated data RR – the relative risk which was actually achieved between the first imputed value and the simulated one-year survival status Imputed RR (RRi) – the relative risk calculated using the second imputed data point as the imputed behaviour Impossible result – instances where the estimated true relative risk was impossible (a negative value) Estimated True RR (RRT) – the estimated true relative risk derived from the imputed relative risk and calibration parameters bpi and ^ρ Median median of 100 repetitions of the imputation algorithm, 95% CI = empirical 95% confidence interval created from the 2.5 and 97.5 percentiles obtained from 100 repetitions of the imputation algorithm, a 95% confidence intervals exclude no association (i.e. Discussion This study shows that an entirely missing variable can be imputed and return accurate estimates of relative risks. Nearly all correlation coefficients were positive, in- dicating that the imputation conveyed some information about health behaviour, although confidence intervals were wide. However, for the less common behaviours (heavy drinking and current smoking with regular drink- ing), no interpretable information could be retrieved. Previous studies have reported that pre-diagnosis smoking with regular alcohol consumption produced a disproportionately high risk to post-diagnosis survival in ESCC (HR 3.84, 95% CI 2.02,7.32 [13]). We observed a similar association (RR = 3.25, 95% CI 0.77,11.84) with wider confidence intervals. The choice of health behaviour variables was re- stricted to measures available through the BRFSS health survey. However, the results are consistent with the literature. We found that tobacco smoking 5 years prior to diagnosis was associated with increased risk of death 1 year after diagnosis in ESCC (RR = 1.99, 95% CI 1.24,3.12) and, with less certainty, EAC (RR = 1.61, 95% CI 0.79,2.57). Recent meta analyses estimated hazard ratios (HRs) of 1.41 (95% CI 1.22, 1.64) and 1.41 (95% CI 0.96,2.09) for current smoking relative to never smoked in mainly ESCC populations [32, 33] and 1.19 (95% CI 1.04,1.36) for ever smoking compared to never smoked in ESCC [24] with no In relation to obesity, a recent North American study [39] found self-reported obesity was associated with lower survival times in EAC compared to normal weight (HR 1.77, 95% CI 1.25, 2.51) and a 27 year follow-up of 29,446 participants in China [40] found higher body mass index protective of death from ESCC (HR = 0.97 per unit increase, 95% CI 0.95,0.99). We found, in con- trast, that obesity 5 years pre-diagnosis may be detri- mental to one-year post diagnosis survival for ESCC (median RR = 1.73) although confidence intervals were wide (95% CI 0.83,4.17). Fahey et al. Discussion BMC Cancer (2020) 20:496 Page 8 of 11 Table 3 Estimated relative risks of 1-year survival derived from imputed pre-diagnosis behaviours for SEER oesophageal cancer cases, 2006–2014; unadjusted and age adjusted Imputed RR (RRi) Impossible Result (RRi < 0) Estimated True RR (RRT) Age-adjusted Imputed RR (adjRRi) Impossible Result (adjRRi < 0) Age-adjusted Estimated True RR (adjRRT) Median 95% CI Frequency Median 95% CI Median 95% CI Frequency Median 95% CI Current smoking All 0.986 0.954,1.009 0 0.806 0.380,1.130 1.051 1.014, 1.078 0 1.794 1.215,2.357a ESCC 1.025 0.981,1.067 0 1.349 0.733,2.142 1.064 1.016, 1.111 0 1.990 1.240,3.117 a EAC 0.959 0.914,1.000 a 5 0.478 0.039,1.003 b 1.038 0.985, 1.085 0 1.613 0.785,2.571 Binge drinking All 0.933 0.900,0.964 49 failed failed 0.997 0.961, 1.032 1 0.951 0.445,1.539 b ESCC 0.998 0.936,1.059 4 0.991 0.167,1.995 b 1.033 0.968, 1.101 0 1.515 0.440,2.754 EAC 0.914 0.863,0.961 a 72 failed failed 0.989 0.935, 1.046 3 0.818 0.181,1.890 b Heavy drinking All 0.981 0.932,1.028 61 failed failed 1.010 0.963, 1.060 23 failed failed ESCC 0.995 0.912,1.066 48 failed failed 1.012 0.929, 1.088 36 failed failed EAC 0.974 0.907,1.039 66 failed failed 1.011 0.938, 1.077 35 failed failed Physical activity All 0.954 0.934,0.978 a 0 0.319 0.165,0.564 a 0.974 0.956, 1.001 0 0.507 0.307,1.030 ESCC 0.959 0.925,0.991 2 0.345 0.073,0.811 a,b 0.971 0.933, 1.003 1 0.452 0.102,1.071b EAC 0.957 0.929,0.986 a 1 0.311 0.109,0.675 a,b 0.984 0.954, 1.013 0 0.627 0.285,2.180 Obese All 0.969 0.946,0.993 a 24 failed failed 1.008 0.983, 1.036 0 1.262 0.559,2.931 ESCC 1.000 0.968,1.039 0 1.004 0.134,2.378 1.027 0.992, 1.068 0 1.733 0.834,4.167 EAC 0.949 0.917,0.987 a 76 failed failed 0.996 0.960, 1.035 8 failed failed Current smoking with regular drinking All 0.987 0.930,1.058 40 failed failed 1.044 0.986, 1.120 2 3.254 0.771,11.843 b ESCC 1.044 0.946,1.146 12 failed failed 1.076 0.973, 1.180 11 failed failed EAC 0.963 0.861,1.052 60 failed failed 1.032 0.919, 1.123 13 failed failed Imputed RR (RRi) – the relative risk calculated using the imputed behaviour Impossible result – instances where the estimated true relative risk was impossible (a negative value) Estimated True RR (RRT) – the estimated true relative risk derived from the imputed relative risk and calibration parameters bpi and ^ρ Median median of 100 repetitions of the imputation algorithm, 95% CI = empirical 95% confidence interval created from the 2.5 and 97.5 percentiles obtained from 100 repetitions of the imputation algorithm, a 95% confidence intervals exclude no association (i.e. g p esult – instances where the estimated true relative risk was impossible (a negative value) Availability of data and materials The SEER Research Data used in this study are made available to the public at no cost, subject to data-use agreement (https://seer.cancer.gov/data/). The BRFSS data sets used in this study are freely available from https:// www.cdc.gov/brfss/index.html. Authors’ contributions In this paper we have demonstrated a novel imputation- based algorithm for augmenting cancer registry data for epidemiological research and established its face-validity. The algorithm adds information obtained from an exter- nal data set with (presumed) no cases in common, to the cancer registry data via demographic variables in com- mon. The algorithm is subject to much higher random error than direct data linkage (depending on how inform- ative the demographic variables are), and requires larger sample sizes to compensate. However, it does avoid the aggravation of confidentiality issues (and associated data security costs) arising from direct data linkage. PF conducted all analyses and writing. AP, GS and TA-B provided regular and substantial input in the conception, methods of analysis and interpretation of results, and reviewed and improved a number of drafts of this paper. All authors have read and approved the final manuscript. Supplementary information Shows the derivation of the mathematical relationship between the imputed relative risk and the true relative risk and thus introduces the formula used to correct for misclassification errors within the imputed health behaviours. Additional file 6. Shows the derivation of the mathematical relationship between the imputed relative risk and the true relative risk and thus introduces the formula used to correct for misclassification errors within the imputed health behaviours. Additional file 7. Describes how health behaviour and survival status were assigned to cancer cases so as to produce the target relative risk in the simulated data sets. Additional file 7. Describes how health behaviour and survival status were assigned to cancer cases so as to produce the target relative risk in the simulated data sets. Additional file 8. Tabulates the results of sub-group analyses on cancer stage I, II and III combined and for cancer stage IV. Additional file 8. Tabulates the results of sub-group analyses on cancer stage I, II and III combined and for cancer stage IV. Additional file 8. Tabulates the results of sub-group analyses on cancer stage I, II and III combined and for cancer stage IV. Discussion exclude relative risk equals 1) b excludes impossible result Table 3 Estimated relative risks of 1-year survival derived from imputed pre-diagnosis behaviours for SEER oesophageal cancer cases, 2006–2014; unadjusted and age adjusted Fahey et al. BMC Cancer (2020) 20:496 Fahey et al. BMC Cancer (2020) 20:496 Page 9 of 11 Page 9 of 11 One benefit of the algorithm is that it does not add any additional information about individuals to the can- cer registry data and so, unlike direct data linkage, does not exacerbate the issues of confidentiality and data se- curity. (The imputed behaviours are only slightly more likely to be correct than an uninformed guess.) The algo- rithm also provides protection against biases. Data were obtained from the SEER cancer registries which are cen- suses with good population coverage. Many sampling and non-response biases in the BRFSS health behaviour data [41] are eliminated when using a census as the ref- erence. However, we used rigid matching criteria and failed to match 20% of cases. Further investigation of the trade-off between exact matching and biases arising from failure to match is required. questions which cannot be addressed through direct data linkage; due to insufficient individuals in common, insuf- ficient matching variables and/or costs associated with data confidentiality and security. By increasing the range of research question which can be addressed with cancer registry data, the algorithm further augments the bene- fits of cancer registries. Abbreviations BRFSS B h i BRFSS: Behavioral Risk Factor Surveillance System; CI: Confidence interval; EAC: Esophageal adenocarcinoma; ESCC: Esophageal squamous cell carcinoma; HR: Hazard ratio; RR: Relative risk; SEER: Surveillance, Epidemiology, and End Results Program We do not have access to any true gold standard for validity testing. A gold standard would be an oesophageal cancer dataset where behaviour was mea- sured 5 years prior to diagnosis. Supplementary information y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12885-020-06990-3. Additional file 1. Provides a conceptual map of the steps in the imputation process. Additional file 2. Charts the inclusion and exclusion of data records from both data sources. Additional file 3. Shows the proportions of eligible SEER cancer cases that were unable to be matched with two donor records with non- missing smoking status. As with direct data linkage, our investigations were lim- ited to available health behaviour measures, rather than all clinically important risk factors. Potentially important health behaviours such as diet [11, 42] and hot beverages [42] were unavailable. The number and variety of auxiliary variables available for matching donor to recipient records was also limited. Our only investigation of clustering in health behaviours [43] was for the combination of current smoking and regular alcohol consumption. Additional file 4. Details the mathematical model used to quantify the agreement between the pairs or imputed values assigned to each cancer case. Additional file 4. Details the mathematical model used to quantify the agreement between the pairs or imputed values assigned to each cancer case. Additional file 5. Shows the strength of associations between candidate confounding variables and one-year survival. Shows why age group is an important potential confounder as both the proportion sur- viving and proportion with the health behaviour present decrease in older age groups. Additional file 5. Shows the strength of associations between candidate confounding variables and one-year survival. Shows why age group is an important potential confounder as both the proportion sur- viving and proportion with the health behaviour present decrease in older age groups. The results display considerable uncertainty with few instances where the empirical confidence intervals ex- cluded the null. 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Author details 1 1School of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia. 2Translational Health Research Institute, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia. 3School of Computing, Engineering and Mathematics, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia. 4Population Wellbeing and Environment Research Lab (PowerLab), School of Health and Society, Faculty of Social Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. 1School of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia. 2Translational Health Research Institute, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia. 3School of Computing, Engineering and Mathematics, Western Sydney University, 4 19. Surveillance, Epidemiology, and End Results (SEER) Program. Research Data (1973–2013). 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BMC Med Res Methodol. 2016;16(1):155. 42. Abnet CC, Arnold M, Wei W-Q. Epidemiology of esophageal squamous cell carcinoma. Gastroenterology. 2018;154(2):360–73. 43. Meader N, King K, Moe-Byrne T, Wright K, Graham H, Petticrew M, et al. A systematic review on the clustering and co-occurrence of multiple risk behaviours. BMC Public Health. 2016;16(1):657. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2199349590	https://figshare.com/articles/journal_contribution/Occurrence_of_pharmaceuticals_in_surface_waters_analytical_method_development_and_environmental_risk_assessment/1575734/2/files/2360159.pdf	English	null	Occurrence of pharmaceuticals in surface waters: analytical method development and environmental risk assessment	International journal of environmental analytical chemistry	2,015	cc-by	3,891	1 1 Legends of figures Figure SD1. Matrix effect evaluation. The percentage of signal suppression is depicted for river (a) and lake water (b) Figure SD1. Matrix effect evaluation. The percentage of signal suppression is depicted for river (a) and lake water (b) Occurrence of pharmaceuticals in surface waters: Analytical method development and environmental risk assessment List of tables Table SD1. Levels of pharmaceuticals worldwide. Table SD2. Physicochemical properties of the target pharmaceuticals. Table SD3 Instrumental parameters for target pharmaceuticals using LC-ESI-MS in SIM mode. Table SD4. Limits of detection (LODs), limits of quantification (LOQs), precision in the same day (RSDr) and in different days (RSDR) in distilled, river and lake water. Table SD5. Mean recoveries (%) and RSD (%) in distilled, river and lake water, after spiking with 0.2 and 2 μg/L (n=3). Table SD6. Ecotoxicological data of the analysed pharmaceuticals, for acute toxicity. Table SD7. Ecotoxicological data of the analysed pharmaceuticals, for chronic toxicity. Supplementary Data (SD) Supplementary Data (SD) 1 Table SD1. Levels of pharmaceuticals in surface waters worldwide 2 Table SD1. Levels of pharmaceuticals in surface waters worldwide Therapeutic group Compound Concentration (ng/L) Reference Analgesics/ Non-steroidal anti- inflammatory drugs (NSDAIDs)] Paracetamol 250 (max) [1] 10000 (max) [2] 154 - 634 [3] n.d. - 872 [4] Phenazone 950 (max) [5] bql-13 [6] 27 (max) [7] Salicylic acid 8800 (max) [8] 4100 (max) [5] 7.8 - 9.6 [9] <1.6-85.8 [10] <0.3-302 [11] n.d. [12] Diclofenac 1200 (max) [5] 156±6.0 [7] 45.5 (mean) [13] Ibuprofen 144-2370 [14] 280-530 [5] 29.4- 490.4 [15] 29.1 (mean) [13] 2796 (max) [16] Ketoprofen 7 (mean), 9808 (max) [17] n.d. - 22 [18] 0.4 - 39.5 [19] 10 - 190 [20] 0.5 - 14  [21] n.d. [12] Indomethacin n.d. - 33.5±8  [22] n.d. - 62.5 [4] <7 (mean), 2323(max)  [17] Mefenamic acid n.d. - 39.8 [23] 0–326 [22] Lipid regulators 0.3 - 169  [21] Gemfibrozil 29.1-59.2 [23] 41 (mean) [17] 1.9 - 3.5 [20] 26-1014 [24] Fenofibrate 9 (max) [6] 1200 (max) [5] n.d. [3] Bezafibrate 29±2.5 [7] bql - 16 [25] 2 16 - 43 [3] Anibiotics Sulfamethazine <LOQ - 52 [3] n.d. - <LOQ [4] <4(mean), 1626 (max) [17] Sulfamethoxazole 93±4.4 [7] 10 - 79 [25] 11 (mean), 56 (max) [17] n.d. - 5.1 [18] 0.3 - 60 [20] 0.5 - 4 [21] Ciprofloxacin <31 (mean), 740 (max) [17] <LOQ - 822, 30.4 (mean) [26] n.d. - 13.6 [27] Erythromycin n.d. - 137±15 [22] 16 - 23 [3] 75 - 100 [28] 1.1 - 20.7 [29] 16 - 23 [3] Antiepileptic Carbamazepine n.d. - 595±14 [22] 6300 (max) [30] 1100 [5] 1238 [16] 1.3 - 2.6 [7] 0.5 - 684 [21] <4 - 595 [22] n.d. - 31.6 [18] Antipsychotic Risperidone 0.088 ± 0.066 / 0.017 ± 0.008 [31] 0.34 [32] Psychomotor stimulant Caffeine 29.1 - 659 [29] 6000 (max) [2] 4275 [16] 24 (mean) [17] 1 - 1813 [20] 220 (mean) [33] Disinfectant Triclosan 35 - 1023 [34] 11, 263 (max) [17] 3-39 [19] 5-95 [21] n.d. [3] Estrogen Estriol n.d. - 1 [34] <LOD - 16 [26] Beta- blocker Atenolol n.d. - 690±26 [22] 0 -34 [18] 1 - 560 [21] n.d. - <1237 [4] H2 receptor antagonist Cimetidine n.d. - <LOQ [4] Anibiotics 3 3 [17] <6 (mean), 63 (max) Table SD2. Physicochemical properties of the target pharmaceuticals. Quantitation ions in bold 1 Therapeutic group Compound Molecular formula MW pKa[35] LogKow[35] Analgesics/ Non-steroidal anti- inflammatory drugs (NSDAIDs) Paracetamol C8H9NO2 151.17 9.4 0.46 Phenazone C11H12N2O 188.23 1.5 0.38 Salicylic acid C7H6O3 138.12 2.3/3.5[36] 1.13/2.26/- 2.42[36] Diclofenac C14H11Cl2NO2 296.15 4.2 4.51/0.7[36] Ibuprofen C13H18O2 206.28 4.9 3.97/0.45[36] Ketoprofen C16H14O3 254.28 4.45 3.12 Indomethacin C19H16ClNO4 357.79 4.5 4.27 Mefenamic acid C15H15NO2 241.28 4.3 5.12 Lipid regulators Gemfibrozil C15H22O3 250.34 4.7 4.77 Fenofibrate C20H21ClO4 360.83 4.5 5.19 Bezafibrate C19H20ClNO4 361.82 3.6[36] 4.25[36] Anibiotics Sulfamethazine C12H14N4O2S 278.33 7.59 0.89 Sulfamethoxazole C10H11N3O3S 253.28 5.7[36] 0.89[36] Ciprofloxacin C17H18FN3O3 331.37 6.09[37] 0.00 [37] Erythromycin C37H67NO13 733.93 8.88 [37] 2.47 [37] Antiepileptic Carbamazepine C15H12N2O 236.27 7/13.9[36] 2.47 Antipsychotic Risperidone C23H27FN4O2 410.45 8.76 2.5 Psychomotor stimulant Caffeine C8H10N4O2 194.20 10.4 -0.007 Glucocorticoid steroid Budesonide C25H34O6 430.53 7.9 2.18 Disinfectant Triclosan C12H7Cl3O2 289.54 4.5/8.1[36] 4.80/5.34[36] Estrogen Estriol C18H24O3 288.38 10.4 2.45 Beta- blocker Atenolol C14H22N2O3 266.34 9.6 0.16 H2 receptor antagonist Cimetidine C10H16N6S 252.34 6.8 0.4 Literature data from: Kosma et al. [36], Verlicchi et al. [37] and Rivera et al. [38]. Table SD2. Physicochemical properties of the target pharmaceuticals. 4 Table SD3. Instrumental parameters for target pharmaceuticals using LC-ESI-MS in SIM mode. Compound Polarity (ESI) Retention time (min) m/z ions Relative ion intensity (%) Cimetidine + 6.47 253, 159 100.0, 60.1 Paracetamol + 6.52 152, 110 100.0, 40.0 Atenolol + 6.75 267, 190, 145 100.0, 21.0, 19.3 Caffeine + 8.20 195, 138 100.0, 60.2 Sulfamethazine + 10.83 279, 156, 186 100.0, 84.3, 60.1 Phenazone + 11.48 189, 147, 56 100.0, 86.5, 12.3 Ciprofloxacin + 12.30 332, 288 100.0, 44.5 Sulfamethoxazole + 13.78 254, 156, 92 100.0, 40.2, 12.3 Carbamazepine + 17.77 237,194, 192 100.0, 80.4, 48.6 Bezafibrate + 22.74 362, 276, 316 100.0, 25.9, 14.9 Erytrhomycin + 22.85 734, 576 100.0 , 20.6 Budesonide + 23.39 431, 413, 323 100.0, 98.3, 29.8 Fenofibrate + 26.05 319 100.0 Risperidone + 30.28 411, 110 100.0, 81.9 Estriol - 3.12 287, 171 100.0, 92.4 Salicylic acid - 12.21 137, 93 100.0, 97.8 Ibuprofen - 12.58 205, 160, 161 100.0, 90.2, 71.1 Ketoprofen - 18.89 253, 209 100.0, 81.9 Indomethacin - 23.67 356, 312, 314 100.0, 75.3, 68.8 Diclofenac - 23.84 294, 295, 250 100.0, 40.9, 12.7 Gemfibrozil - 24.04 249, 121 100.0, 82.63 Mefenamic acid - 27.08 240, 196, 180 100.0, 94.0, 90.8 Triclosan - 29.64 288, 287 100.0, 90.9 *Quantitation ions in bold Table SD3. Instrumental parameters for target pharmaceuticals using LC-ESI-MS in SIM mode. 1 5 5 Table SD4. Limits of detection (LODs), limits of quantification (LOQs), precision in the same day (RSDr) and in different days (RSDR) in distilled, river and lake water. Compound IDL (ng/L) LOD (ng/L) LOQ (ng/L) RSDr (%) (n=5) RSDR (%) (n=5) DW RW LW DW RW LW DW RW LW DW RW LW Cimetidine 4.9 3.5 3.7 5.1 11.6 14.4 19.9 4.5 6.7 5.8 5.2 7.1 7.4 Paracetamol 12.9 12.3 12.8 12.7 40.6 42.2 41.9 9.8 10.0 11.3 13.0 14.1 14.5 Atenolol 15.1 17.0 18.1 20.3 56.1 56.1 62.9 6.1 5.7 6.1 6.8 7.1 6.9 Caffeine 14.1 9.4 9.8 13.1 30.6 32.3 43.2 5.1 9.0 10.1 9.2 9.0 8.8 Sulfamethazine 5.2 4.7 5.2 8.9 15.5 15.6 26.7 2.9 2.1 3.2 5.1 6.9 8.5 Phenazone 2.8 2.7 2.9 5.6 8.9 11.3 21.8 1.9 5.5 5.9 4.3 5.2 6.1 Ciprofloxacin 8.9 9.9 10.8 27.1 32.7 33.5 84.0 7.5 8.9 9.0 7.9 8.3 10.1 Sulfamethoxazole 5.7 3.8 4.7 6.8 12.5 17.4 25.2 2.5 6.1 6.8 3.7 5.1 5.1 Carbamazepine 5.2 4.8 5.2 7.3 17.8 16.6 23.4 2.1 3.6 4.1 4.2 6.7 7.2 Bezafibrate 4.2 5.9 6.2 6.1 18.3 23.6 23.2 3.5 3.5 4.1 7.9 8.5 9.2 Erytrhomycin 9.8 9.3 10.1 9.8 32.6 30.3 29.4 4.3 7.1 8.8 5.1 5.9 6.7 Budesonide 20.1 26.1 28.1 29.9 96.6 95.5 101.7 4.6 7.8 9.4 10 11.1 13.1 Fenofibrate 9.0 9.3 9.7 10.3 30.7 32.0 34.0 5.3 6.9 6.4 16.1 18.5 21.5 Risperidone 65.1 73.5 87.0 101.0 220.5 304.5 353.5 7.5 8.9 10.4 8.1 9.7 9.9 Estriol 29.4 29.7 31.6 40.0 98.0 101.1 128.0 6.0 9.0 10.8 7.1 8.9 9.1 Salicylic acid 40.0 46.0 47.1 47.6 147.2 141.3 142.8 3.0 5.1 7.4 6.1 6.5 6.9 Ibuprofen 76.9 81.7 83.0 91.0 318.6 307.1 336.7 4.1 7.3 7.4 4.7 6.1 6.9 Ketoprofen 14.1 10.0 12.5 12.9 37.0 41.3 42.6 3.9 4.5 4.2 5.7 8.9 8.1 Indomethacin 22.3 25.0 25.8 24.9 82.5 85.1 82.2 4.6 4.9 5.7 5.1 5.8 5.8 Diclofenac 15.4 15.9 17.1 18.5 52.5 56.4 61.1 5.4 7.9 7.1 11.2 13.2 15.2 Gemfibrozil 37.9 38.1 40.0 45.7 125.7 124.0 141.7 9.7 10.7 10.4 10.9 10.1 10.9 Mefenamic acid 7.9 6.2 7.1 8.2 21.7 23.4 27.1 4.9 5.7 5.2 6.1 8.3 9.4 Triclosan 28.7 34.5 37.0 41.3 113.9 111.0 123.9 4.8 7.1 8.6 5.0 9.1 11.7 6 (%) in distilled, river and lake water, after spiking with 0.2 and 2 μg/L (n=3). Table SD5. 1 Mean recoveries (%) and RSD (%) in distilled, river and lake water, after spiking with 0.2 and 2 μg/L (n=3). Compound % Recoveries after spiking with 0.2 μg/L (RSD %) % Recoveries after spiking with 2 μg/L (RSD %) DW RW LW DW RW LW Cimetidine 86.1 (5.8) 82.7 (4.1) 79.1 (4.4) 89.5 (4.7) 87.3 (4.1) 85.1 (4.8) Paracetamol 76.7 (5.1) 78.2 (5.2) 74.9 (5.6) 83.8 (5.7) 81.7 (6.1) 80.4 (5.7) Atenolol 70.1 (7.8) 69.6 (7.2) 67.2 (8.1) 71.3 (6.5) 74.8 (8.9) 73.1 (8.6) Caffeine 89.1 (5.8) 88.1 (5.2) 86.2 (3.2) 98.1 (2.7) 95.3 (2.9) 95.3 (2.9) Sulfamethazine 78.4 (6.8) 75.4 (6.5) 73.2 (6.1) 81.1 (4.3) 81.9 (5.1) 81.9 (5.1) Phenazone 89.1 (3.7) 89.1 (3.5) 86.7 (3.9) 98.7 (11.2) 96.7 (10.5) 94.3 (9.3) Ciprofloxacin 80.1 (7.6) 78.1 (7.3) 75.4 (7.1) 83.1 (6.4) 81.1 (4.4) 82.5 (4.9) Sulfamethoxazole 93.1 (4.1) 90.4(4.7) 91.5 (3.8) 100.1 (2.9) 91.1 (3.9) 89.7 (3.2) Carbamazepine 93.7 (9.4) 91.9 (9.2) 93.4 (9.1) 99.1 (10.5) 95.2 (8.7) 91.4 (8.9) Bezafibrate 77.7 (7.9) 75.3 (8.1) 75.1(7.8) 97.1 (8.1) 79.9 (7.2) 78.7 (7.4) Erytrhomycin 95.0 (15.1) 92.1(13.2) 90.8(11.1) 97.4 (12.1) 94.2 (12.9) 92.2 (13.1) Budesonide 69.7 (9.0) 64.9 (9.3) 61.3(9.5) 74.8 (8.9) 69.1 (8.9) 70.8 (8.2) Fenofibrate 52.1 (3.5) 51.0 (3.2) 53.0 (3.8) 60.8 (8.9) 55.3 (3.9) 52.1 (3.7) Risperidone 30.0 (19.1) 27.5 (17.1) 25.2(16.3) 34.9 (13.9) 32.1 (11.8) 30.6 (10.6) Estriol 43.4(9.1) 47.8 (9.9) 46.8 (9.7) 50.2 (3.9) 50.1 (3.0) 49.3 (3.2) Salicylic acid 57.9 (3.1) 56.7 (3.6) 52.4 (3.1) 60.0 (2.7) 60.1 (3.3) 68.3 (4.1) Ibuprofen 67.3 (4.2) 64.1 (3.8) 60.8 (3.2) 81.4 (2.9) 69.2 (4.0) 67.1 (4.9) Ketoprofen 52.1 (2.4) 57.7 (2.9) 54.2 (2.3) 61.7 (4.2) 57.2 (5.0) 52.7 (5.1) Indomethacin 74.2 (5.1) 70.1 (5.3) 70.3 (5.0) 75.1 (5.9) 74.1 (5.1) 72.1 (5.9) Diclofenac 100.4 (9.1) 100.0 (8.5) 98.2 (7.9) 111.1 (3.4) 104.6 (8.3) 102.1 (7.1) Gemfibrozil 58.3 (9.7) 56.0 (8.7) 53.1 (8.1) 59.1 (4.0) 58.0 (8.1) 56.1 (8.6) Mefenamic acid 89.1 (8.0) 78.3 (7.2) 81.2 (7.1) 94.4 (3.2) 92.1 (3.1) 91.8 (3.0) Triclosan 44.2 (11.1) 48.1 (11.3) 47.7 (10.2) 57.4 (6.0) 57.4 (6.0) 55.5 (6.7) 7 7 (lowest values in bold) Compounds Species Test Toxicity (mg/L) Reference Cimetidine Fish EC50 571 [38] Invertebrates (Daphnid) EC50 35 [38] Invertebrates (D. magna) EC50 271.3 [39] Algae EC50 40 [38] Paracetamol Fish EC50 40 [38] Fish (B. rerio) EC50 378 [40] Invertebrates (Daphnid) EC50 41 [38] Invertebrates (D. magna) EC50 136 (24 h) [41] Invertebrates (D. 1 magna) EC50 9.2 (48 h) [41] Invertebrates (D. magna) EC50 50 [40] Algae EC50 2549 [38] Algae (S. subcapitata) EC50 134 [40] Atenolol Invertebrates EC50 30 [36] Invertebrates (Daphnid) LC50 33.4 (48h) [42] Algae LC50 620 (24h) [42] Caffeine Fish EC50 805 [38] Fish LC50 87 (<96h) [42] Invertebrates (Daphnid) EC50 46 [38] Fish LC50 182 (48h) [42] Algae EC50 46 [38] Sulfamethazine Fish EC50 517 [38] Invertebrates (Daphnid) EC50 4 [38] Algae EC50 38 [38] Phenazone Fish EC50 3 [43] Invertebrates (D. magna) EC50 6.7 [43] Algae EC50 1.1 [43] Ciprofloxacin Fish EC50 246000 [38] Fish (B. rerio) EC50 >100 [40] Invertebrates (Daphnid) EC50 991 [38] Invertebrates (D. magna) EC50 >60 [40] Algae EC50 938 [38] Algae (P. subcapitata) EC50 2.97 [40] Sulfamethoxazole Fish EC50 562.5 [44] Fish EC50 890 [38] Invertebrates (D. magna) EC50 25.2 [44] Invertebrates (B. Calyciflorus) EC50 26 [40] Invertebrates (C. dubia) EC50 15.5 [40] Invertebrates (D. magna) EC50 25 [40] Invertebrates (Daphnid) EC50 4.5 [38] Invertebrates (Daphnid) LC50 10 (48h) [42] Algae (S. leopoliensis) EC50 0.027 [44] Algae (P. subcapitata) EC50 0.146 [40] Algae (C. meneghiniana) EC50 2.4 [40] Algae (P. subcapitata) EC50 0.57 [40] Algae EC50 51 [38] 8 8 9 Carbamazepine Fish EC50 101 [38] Fish EC50 35.4 [43] Invertebrates (Daphnid) EC50 111 [38] Invertebrates (D. magna) EC50 76.3 [43] Algae EC50 70 [38] Algae EC50 85 [43] Bezafibrate Fish EC50 5.3 [45] Fish LC50 >100 (<96h) [42] Invertebrates (D. magna) EC50 30 [45] Invertebrates (Daphnid) EC50 25 [38] Invertebrates (Daphnid) LC50 >100 (48h) [42] Algae EC50 18 [45] Algae EC50 >100 (24h) [42] Erytrhomycin Fish EC50 900 [36] Invertebrates EC50 15 [36] Algae EC50 0.02 [36] Budesonide Fish LC50 19 (<96h) [42] Invertebrates (Daphnid) LC50 20 (48h) [42] Fenofibrate Algae EC50 0.8 [45] Invertebrates (D. magna) EC50 50 [45] Invertebrates (Daphnid) EC50 0.35 [38] Algae EC50 0.1 [45] Risperidone Fish LC50 6 (<96h) [42] Invertebrates (Daphnid) LC50 6 (48h) [42] Algae LC50 N.A. [42] Salicylic acid Fish EC50 1.29 [43] Fish LC50 37 (<96h) [42] Invertebrates (D. magna) EC50 59 [43] Invertebrates (Daphnid) LC50 118 (48h) [42] Algae EC50 48 [43] Algae LC50 >100 (24h) [42] Ibuprofen Fish EC50 5 [43] Fish LC50 173 (<96h) [42] Invertebrates (D. magna) EC50 9.02 [43] Invertebrates (D. magna) EC50 9.06 (48h) [41] Invertebrates (D. 1 magna) EC50 101 [40] Invertebrates (Daphnid) LC50 9.1 (48h) [42] Invertebrates (Daphnid) EC50 38 [38] Algae EC50 4 [43] Algae (S.costatum) EC50 7.1 (96h) [41] Algae (D. subspicatus) EC50 342 [40] Algae LC50 7.1 (24h) [42] Algae EC50 26 [38] Ketoprofen Fish EC50 32 [38] Fish EC50 157.7 [46] Invertebrates (Daphnid) EC50 248 [38] Invertebrates EC50 9.05 [46] Algae EC50 164 [38] Algae EC50 7.87 [46] Indomethacin Fish EC50 3.9 [38] 9 9 Invertebrates (Daphnid) EC50 26 [38] Algae EC50 18 [38] Diclofenac Fish EC50 531 [43] Invertebrates (D. magna) EC50 22 [43] Invertebrates (Daphnid) EC50 5057 [38] Invertebrates (D. magna) EC50 68 [40] Invertebrates (T. Platyurus) EC50 41 [40] Invertebrates (D. magna) EC50 80 [40] Diclofenac Invertebrates (C. dubia) EC50 23 [40] Algae EC50 14.5 [40] Algae EC50 2911 [38] Algae (D. subspicatus) EC50 72 [40] Algae (P. subcapitata) EC50 16 [40] Algae (C. meneghiniana) EC50 19 [40] Gemfibrozil Fish EC50 0.9 [43] Invertebrates (D. magna) EC50 10.4 [43] Invertebrates (Daphnid) EC50 6 [38] Algae EC50 4 [43] Triclosan Fish (P. Promelas) EC50 0.26 (96h) [47] Fish (L. macrochirus) EC50 0.37 (96h) [47] Invertebrates (D. magna) EC50 0.39 (48 h) [47] Invertebrates (Daphnid) LC50 0.13 (48h) [42] Algae (Scenedesmus) EC50 0.0014 [47] Algae LC50 0.0045 (48h) [42] Table SD7. Ecotoxicological data of the analyzed pharmaceuticals, for chronic toxicity (lowest values in bold) Compounds Species Test Toxicity (mg/L) Reference Ciprofloxacin Algae (P. subcapitata) NOEC 1.09 [48] Sulfamethoxazole Fish (D. Rerio) NOEC >8 [49] Invertebrates (B. calyciflorus) NOEC 0.25 [49] Invertebrates (C. dubia) NOEC 0.25 [49] Invertebrates (C. dubia) NOEC 0.21 [40] Invertebrates (B. calyciflorus) NOEC 9.63 [40] Algae (P. subcapitata) NOEC 0.09 [49] Algae (C. meneghiniana) NOEC 0.25 [49] Algae (L. gibba) NOEC 0.01 [40] Carbamazepine Fish (D. Rerio) NOEC 25 [49] Invertebrates (B. calyciflorus) NOEC 0.377 [49] Invertebrates (C. dubia) NOEC 0.025 [49] Algae (P. subcapitata) NOEC >100 [49] Algae (C. meneghiniana) NOEC 10 [49] Algae (S. leopoliensis) NOEC 17.5 [49] Bezafibrate Fish NOEC 3.8 [45] Table SD7. Ecotoxicological data of the analyzed pharmaceuticals, for chronic toxicity (lowest values in bold) 10 Erytrhomycin Algae (P. Subcapitata) NOEC 0.0103 (144h) [50] Algae (P. Subcapitata) NOEC 0.0103 (72h) [50] Budesonide Invertebrates (Daphnid) NOEC 10 [42] Fenofibrate Fish NOEC 0.048 [45] Ibuprofen Algae (Synechocystissp) NOEC >1 [40] Algae (D. subspicatus) NOEC 103 [40] Diclofenac Fish (D. Rerio) NOEC 4 [49] Fish (D. Rerio) NOEC 0.5 [40] Fish (S. 1 trutta) NOEC 0.0015 [40] Fish (O. mykiss) NOEC 0.005 [40] Invertebrates (B. calyciflorus) NOEC 12.5 [49] Invertebrates (C. dubia) NOEC 1 [49] Invertebrates (D. magna) NOEC 10 [40] Algae (P. subcapitata) NOEC 10 [49] Algae (C. meneghiniana) NOEC 10 [49] Algae (S. leopoliensis) NOEC 10 [49] Algae (D. subspicatus) NOEC 49 [40] Gemfibrozil Fish NOEC 0.93 [45] Triclosan Fish (O. mykiss) NOEC 0.034 [51] Algae (S. subspicatus) NOEC 0.0007 [51] Algae (Scenedesmus) NOEC 0.00069 [47] Figure SD1 (a) 0 10 20 30 40 50 60 70 80CimetidineParacetamolAtenololCaffeineSulfamethazinePhenazoneCiprofloxacinSulfamethoxazoleCarbamazepineBezafibrateErytrhomycinBudesonideFenofibrateRisperidoneEstriolSalicylic acidIbuprofenKetoprofenIndomethacinDiclofenacGemfibrozilMefenamic acidTriclosan (%) Signal Suppression Pharmaceutical Erytrhomycin Algae (P. Subcapitata) NOEC 0.0103 (144h) [50] Algae (P. Subcapitata) NOEC 0.0103 (72h) [50] Budesonide Invertebrates (Daphnid) NOEC 10 [42] Fenofibrate Fish NOEC 0.048 [45] Ibuprofen Algae (Synechocystissp) NOEC >1 [40] Algae (D. subspicatus) NOEC 103 [40] Diclofenac Fish (D. Rerio) NOEC 4 [49] Fish (D. Rerio) NOEC 0.5 [40] Fish (S. trutta) NOEC 0.0015 [40] Fish (O. mykiss) NOEC 0.005 [40] Invertebrates (B. calyciflorus) NOEC 12.5 [49] Invertebrates (C. dubia) NOEC 1 [49] Invertebrates (D. magna) NOEC 10 [40] Algae (P. subcapitata) NOEC 10 [49] Algae (C. meneghiniana) NOEC 10 [49] Algae (S. leopoliensis) NOEC 10 [49] Algae (D. subspicatus) NOEC 49 [40] Gemfibrozil Fish NOEC 0.93 [45] Triclosan Fish (O. mykiss) NOEC 0.034 [51] Algae (S. subspicatus) NOEC 0.0007 [51] Algae (Scenedesmus) NOEC 0.00069 [47] Figure SD1 (a) 0 10 20 30 40 50 60 70 80CimetidineParacetamolAtenololCaffeineSulfamethazinePhenazoneCiprofloxacinSulfamethoxazoleCarbamazepineBezafibrateErytrhomycinBudesonideFenofibrateRisperidoneEstriolSalicylic acidIbuprofenKetoprofenIndomethacinDiclofenacGemfibrozilMefenamic acidTriclosan (%) Signal Suppression Pharmaceutical Figure SD1 (a) 11 Figure SD1 (b) 0 10 20 30 40 50 60 70 80CimetidineParacetamolAtenololCaffeineSulfamethazinePhenazoneCiprofloxacinSulfamethoxazoleCarbamazepineBezafibrateErytrhomycinBudesonideFenofibrateRisperidoneEstriolSalicylic acidIbuprofenKetoprofenIndomethacinDiclofenacGemfibrozilMefenamic acidTriclosan (%) Signal Suppression Pharmaceutical Figure SD1 (b) 12 12 References [1] M. 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https://openalex.org/W4255017409	https://www.biogeosciences.net/13/5259/2016/bg-13-5259-2016.pdf	English	null	High resolution neodymium characterization along the Mediterranean margins and modeling of ε<sub>Nd</sub> distribution in the Mediterranean basins	null	2,016	cc-by	14,196	Biogeosciences, 13, 5259–5276, 2016 www.biogeosciences.net/13/5259/2016/ doi:10.5194/bg-13-5259-2016 © Author(s) 2016. CC Attribution 3.0 License. Biogeosciences, 13, 5259–5276, 2016 www.biogeosciences.net/13/5259/2016/ doi:10.5194/bg-13-5259-2016 © Author(s) 2016. CC Attribution 3.0 License. Correspondence to: Mohamed Ayache (mohamed.ayache@lsce.ipsl.fr) Received: 24 March 2016 – Published in Biogeosciences Discuss.: 5 April 2016 Revised: 12 August 2016 – Accepted: 8 September 2016 – Published: 22 September 2016 Received: 24 March 2016 – Published in Biogeosciences Discuss.: 5 April 2016 Revised: 12 August 2016 – Accepted: 8 September 2016 – Published: 22 September 2016 Revised: 12 August 2016 – Accepted: 8 September 2016 – Published: 22 September 2 change (BE) as an important process in the Nd oceanic cycle. Nevertheless this approach simulates a too-radiogenic value in the Mediterranean Sea; this bias will likely be corrected once the dust and river inputs will be included in the model. Abstract. An extensive compilation of published neodymium (Nd) concentrations and isotopic composi- tions (Nd IC) was realized in order to establish a new database and a map (using a high-resolution geological map of the area) of the distribution of these parameters for all the Mediterranean margins. Data were extracted from different kinds of samples: river solid discharge deposited on the shelf, sedimentary material collected on the margin or geological material outcropping above or close to a margin. Additional analyses of surface sediments were done in order to improve this data set in key areas (e.g. Sicilian strait). This work highlights that a signiﬁcant interannual vari- ability of εNd distribution in seawater could occur. In par- ticular, important hydrological events such as the Eastern Mediterranean Transient (EMT), associated with deep water formed in the Aegean sub-basin, could induce a shift in εNd at deep/intermediate depths that could be noticeable in the eastern part of the basin. This underlines that the temporal and geographical variations of εNd could represent an inter- esting insight of Nd as tracer of the Mediterranean Sea cir- culation, in particular in the context of palaeo-oceanographic applications. The Mediterranean margin Nd isotopic signatures vary from non-radiogenic values around the Gulf of Lion, (εNd values ∼−11) to radiogenic values around the Aegean and the Levantine sub-basins up to +6. Using a high-resolution regional oceanic model (1/12◦of horizontal-resolution), εNd distribution was simulated for the ﬁrst time in the Mediterranean Sea. 1 Introduction The high resolution of the model provides a unique op- portunity to represent a realistic thermohaline circulation in the basin and thus apprehend the processes governing the Nd isotope distribution in the marine environment. Results are consistent with the preceding conclusions on boundary ex- The Mediterranean Sea is a semi-enclosed sea of great in- terest because it is submitted to large range of dynami- cal processes and interactions, such as strong air–sea ex- changes leading to open-sea deep-water convection feeding a 1Laboratoire des Sciences du Climat et de l’Environnement LSCE/IPSL, CEA-CNRS-UVSQ, Université Paris-Saclay, 91191 Gif Y tt F 1Laboratoire des Sciences du Climat et de l’Environnement LSCE/IPSL, CEA-CNRS-UVSQ, Université 91191 Gif-sur-Yvette, France 2ENSTA ParisTech, Université Paris-Saclay, 828 bd des Maréchaux, 91762 Palaiseau CEDEX, France 3Laboratoire de Météorologie Dynamique, École Polytechnique, Palaiseau, France 4SEDISOR/UMR6538 “Laboratoire Domaines Océaniques”, IUEM, CNRS-UBO, Plouzané, France 5Mercator-Océan, Ramonville Saint-Agne, France 6Météo-France, Toulouse, France 7LEGOS, Université de Toulouse, CNRS, CNES, IRD, UPS, Toulouse, France 7LEGOS, Université de Toulouse, CNRS, CNES, IRD, UPS, Toulouse, France orrespondence to: Mohamed Ayache (mohamed.ayache@lsce.ipsl.fr) M. Ayache et al.: High-resolution neodymium characterization Frost et al. (1986) and Spivack and Wasserburg (1988) pro- posed that the additional Nd source might be the partial dis- solution of river particles and/or aeolian particles. Greaves et al. (1991) argued that the missing source might rather be of marine origin. Schijf et al. (1991) suggested that the Black Sea was a net source to the Mediterranean Sea. Based on a two-box model Henry et al. (1994) suggested that the εNd in the Ligurian sub-basin deep waters required an exchange involving 30 ± 20 % of the sinking particles of atmospheric origin. Finally, Tachikawa et al. (2004) proposed that the missing term could be sediments deposited on the margins. In other words, the origin of this radiogenic input remains unclear. The present study aims to compile data and develop modelling tools for clarifying this issue. Nd residence time ranges from 700 to 1500 years in the global ocean (e.g. Lacan et al., 2012), long enough to be transported within the global thermohaline circula- tion system and short enough to avoid complete homoge- nization. Therefore, εNd is often considered to be a “quasi- conservative” tracer. In other words, εNd values of the wa- ter masses could be conserved up to long distances from the source of lithogenic inputs. In such a context, it could be used to tag water masses with distinct isotopic compositions in or- der to constrain water mass mixing and pathways, as well as the thermohaline circulation in modern and palaeo ocean cir- culation (e.g. Lacan and Jeandel, 2005; Jeandel, 1993; Jean- del et al., 1998; Frank, 2002; Goldstein and Hemming, 2003; Piotrowski et al., 2004, 2012; Stichel et al., 2012; Martin et al., 2012; Pena et al., 2013; Molina-Kescher et al., 2014; Arsouze et al., 2008). However, because Nd is particle reac- tive, Nd parameters are also successfully used to study Nd exchange between dissolved and particulate phases (Bertram and Elderﬁeld, 1993; Henry et al., 1994; Jeandel et al., 1995; Tachikawa et al., 1999, 2003; Garcia-Solsona et al., 2014; Rousseau et al., 2015; Haley et al., 2014). The circulation of the Mediterranean Sea is driven by the fact that the mean evaporation exceeds the mean pre- cipitation, leading to a density increase along surface wa- ter mass paths and subsequent strong convective events in winter. M. Ayache et al.: High-resolution neodymium characterization 5260 thermohaline circulation cell (Malanotte-Rizzoli and Robin- son, 1988), strait transports and dynamics or cross-shore ex- changes. From a biogeochemical perspective, it is a region receiving the highest aerosol loads owing to air masses car- rying numerous and various aerosol types (Lelieveld et al., 2002; Nabat et al., 2014), where oligotrophy occurs and with a characteristic dynamic of the deep chlorophyll maximum (Mignot et al., 2014). Under the stress of the global change and anthropogenic forcing, understanding the functioning of the Mediterranean Sea and quantifying the biogeochemical cycles is a priority (MerMex-Group, 2011). the waters ﬂowing along these margins, called the “bound- ary exchange” (BE) was the missing Nd source that could balance both the concentration and isotopic distributions of Nd on regional and world scales. Since these pioneer works, many studies have conﬁrmed this hypothesis (Rickli et al., 2009, 2010; Stichel et al., 2012; Wilson et al., 2012; Grenier et al., 2013; Carter et al., 2012). The modelling studies have reached the same conclusions on the relative importance of the BE on the Nd oceanic cycle on the global scale, although dust and river inputs could locally affect the surface waters, such as off the Sahara.(Arsouze et al., 2007; Siddall et al., 2008; Arsouze et al., 2009; Rempfer et al., 2011). Neodymium (Nd) is a Rare Earth Element (REE) with seven naturally occurring isotopes. The radiogenic iso- tope 143Nd is produced by the radioactive α-decay of 147Sm. At the continent surface, the Nd isotopic composition (usu- ally expressed as εNd 1 of a given material) is a function of the Sm / Nd ratio characterizing this material, which is pri- marily a function of its age and lithology. On a global scale, it is higher in the Earth’s mantle compared to its crust. As a consequence, the εNd of the continents presents a heteroge- neous distribution (Goldstein and Hemming, 2003; Jeandel et al., 2007). The Nd inﬂux brought by the Atlantic inﬂow in the Strait of Gibraltar is smaller than the Nd outﬂux exiting with the Mediterranean outﬂow (Tachikawa et al., 2004; Greaves et al., 1991; Henry et al., 1994). The εNd value of the Mediterranean outﬂow was estimated to be −9.4 (Henry et al., 1994; Tachikawa et al., 2004), which is higher than that of the Atlantic inﬂow (εNd = −11.8; Spivack and Wasser- burg, 1988). Thus, a source of radiogenic Nd is required to balance these ﬂuxes. 1εNd = [(143Nd/144Nd)sample/(143Nd/144Nd)CHUR −1] × 104, where (143Nd/144Nd)CHUR = 0512638 is the averaged earth value (Jacobsen and Wasserburg, 1980). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. M. Ayache et al.: High-resolution neodymium characterization The main deep-water sources are located in the Gulf of Lion (south of France) for the western Mediter- ranean Sea (WMed), and the Adriatic sub-basin for the east- ern Mediterranean Sea (EMed; Millot and Taupier-Letage, 2005). In the mid-1990s a shift in the deep-water forma- tion site occurred during the Eastern Mediterranean Tran- sient (EMT) events. The EMT describes a temporary change in the Eastern Mediterranean Deep Water (EMDW) for- mation site that switched from the Adriatic to the Aegean sub-basin (Roether et al., 1996, 2007; Lascaratos et al., 1999; Malanotte-Rizzoli et al., 1999; Theocharis et al., 1992, 1999). The new source has produced large quantities of very dense water masses, in particular the Cretan Deep Wa- ter (CDW) that overﬂowed through the Cretan Arc straits and subsequently ﬁlled the eastern Mediterranean with wa- ters denser than the previously existing deep and bottom wa- Nd sources to the ocean are lithogenic, and the mean εNd of an oceanic basin is representative of the surrounding continents (Jeandel et al., 2007). During the last few years, signiﬁcant progress has been made in understanding how dif- ferent water masses acquire their Nd IC (Isotopic Composi- tion). In the early 2000s, Tachikawa et al. (2003) and La- can and Jeandel (2005) suggested that exchange of Nd be- tween the sediments deposited on the oceanic margins and Biogeosciences, 13, 5259–5276, 2016 www.biogeosciences.net/13/5259/2016/ M. Ayache et al.: High-resolution neodymium characterization M. Ayache et al.: High-resolution neodymium characterization 5261 Figure 1. The ﬁlled contours indicate the geological province limit based on geological age (i.e. each colour represents an age) from a high- resolution digital geological map (http://www.geologie.ens.fr/spiplabocnrs/spip.php?rubrique67) while the circles ﬁlled in blue represent the location of the discrete data compiled from the EarthChem database (see Supplement 1), and in red the location of the stations correspond to the sediments analysed as part of the present work. Figure 1. The ﬁlled contours indicate the geological province limit based on geological age (i.e. each colour represents an age) from a high- resolution digital geological map (http://www.geologie.ens.fr/spiplabocnrs/spip.php?rubrique67) while the circles ﬁlled in blue represent the location of the discrete data compiled from the EarthChem database (see Supplement 1), and in red the location of the stations correspond to the sediments analysed as part of the present work. constitute the Mediterranean margins, which are expected to interact with the water masses, are presented. M. Ayache et al.: High-resolution neodymium characterization This high- resolution mapping was established using a detailed geolog- ical map, providing the most realistic representation of the Mediterranean geology existing so far (see Figs. 1 and 2, and Supplement 1). ter. However, EMT was revealed using hydrographic and an- thropogenic tracers such as CFC and 3H. Both are transients and are not imprinted in the sediments. Establishing the oc- currence of a similar EMT event in the past would require the identiﬁcation of proxies that clearly identify the distribu- tion and circulation of the different water masses, which is memorized in the sediments. The approach by Arsouze et al. (2007) to simulate the BE was evaluated and generally accepted by the scientiﬁc community (e.g. Arsouze et al., 2009), following this pro- tocol made up for the global scale, we implemented the neodymium in a high-resolution regional model (NEMO- MED12) developed for the Mediterranean Sea. We used dis- solved εNd data compiled by Tachikawa et al. (2004), Vance et al. (2004), Henry et al. (1994) to evaluate the ability of this model to reproduce the main features of the circulation and mixing of the Mediterranean Sea water masses for which Nd signatures are known. These tools provided perspectives on (i) the εNd distribution in the whole Mediterranean Sea, (ii) the impact of the interannual variability of the thermoha- line circulation (e.g. EMT event) on the modelled εNd distri- bution, and (iii) high resolution of the geological ﬁeld on the one hand and the model on the other hand can reveal possible local heterogeneities which could reﬂect local BE effects. Tachikawa et al. (2004) demonstrated that the Nd iso- topic signature is more conservative than the salinity in the Mediterranean Sea, the latter being strongly affected by the evaporation. In addition, these authors revealed that the Mediterranean water masses are well distinguished by their Nd isotopic signatures. The Mediterranean Sea makes an ex- cellent “laboratory test” basin for studying the potential εNd distribution variations as it is a semi-enclosed basin with a quite short residence time of the waters (50–100 years; Mil- lot and Taupier-Letage, 2005). This paper also aims to in- vestigate how EMT events affect the Nd distribution in the Mediterranean basin in order to estimate the potential of this tracer to characterize from palaeo archive (e.g. corals, foraminifera) the occurrence of such event in the past. Mod- elling represents an appropriate tool to address this question. M. Ayache et al.: High-resolution neodymium characterization In this study, we developed a new modelling platform for simulating Nd isotopic composition at high resolution in the Mediterranean basin. This study is part of the work carried out to assess the robustness of the NEMO-MED12 model, used to study the thermohaline circulation and the biogeochemical cycles in the Mediterranean Sea, and it improves our ability to predict First, the results of a dense compilation of the concentra- tions and isotopic compositions of the different materials that www.biogeosciences.net/13/5259/2016/ 2.1 Sediment core tops and erodible material data the future evolution of this basin under increasing anthro- pogenic pressure (Drobinski et al., 2012). Surface sediments collected on the shelf or the slope were our ﬁrst choice because they provide direct information on the geochemical and isotopic characteristics of the mate- rial in contact with the water masses. Sediments deposited during the recent Holocene were taken into consideration. When surface sediment data were missing, we took into ac- count the Nd parameters of erodible material deposited along the coasts (Jeandel et al., 2007). The EarthChem database provides a good spatial covering in the northern coastline (Fig. 1), in contrast to the southern coastline (e.g. Algerian coast), while there are no published data for the Tunisian, Libyan and Egyptian coasts. We therefore distinguished be- tween the two cases Sects. 2.1.1 and 2.1.2. 2 Data compilation and representation on the Mediterranean margins Here we present the data compilation procedure and result- ing map (Fig. 2) allowing us to characterize the Nd isotopic signatures and concentrations of all the margins surrounding the Mediterranean Sea. Our approach was to prioritize the use of data directly measured in the outcropping sediments or geological ﬁelds, as proposed by Jeandel et al. (2007). An extensive compi- lation of all published Nd concentrations and isotopic val- ues was made using the EarthChem database (http://www. earthchem.org) with a zoomed-in image of the Mediter- ranean region (latitude between 28 to 48◦N, and between 10◦W and 40◦E in longitude). This yielded assets of more than 14 200 discrete data reported in Supplement 1 and lo- cated in Fig. 1. When data were missing in crucial areas (e.g. Strait of Sicily), we directly measured them on core-top sediments. www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 M. Ayache et al.: High-resolution neodymium characterization M. Ayache et al.: High-resolution neodymium characterization 5262 Longitudes (°E) Latitudes (°N) Figure 2. Extrapolated map providing a picture of the Nd signature of all the margins surrounding the Mediterranean Sea. (a) Low-resolution conﬁguration ORCA2 (reproduced from Jeandel et al., 2007) and (b) high-resolution conﬁguration NEMO-MED12 (this work). Hatched areas correspond to uncharacterized areas in the published literature (before 2007) as done by Jeandel et al. (2007). Longitudes (°E) Figure 2. Extrapolated map providing a picture of the Nd signature of all the margins surrounding the Mediterranean Sea. (a) Low-resolution conﬁguration ORCA2 (reproduced from Jeandel et al., 2007) and (b) high-resolution conﬁguration NEMO-MED12 (this work). Hatched areas correspond to uncharacterized areas in the published literature (before 2007) as done by Jeandel et al. (2007). 2.1.2 Areas with low spatial resolution of available data For the southern coast, we gathered sediment samples from miscellaneous origins. Those were collected during the cruises of ETNA80, DEDALE and NOE near the Tunisian, Libyan and the Egyptian coasts respectively (see Table 1). In the Strait of Sicily samples were collected along two tran- sects, Sciacca–Pantelleria (SP) and Pozzallo–Malta (PM), perpendicular to the southern coast of Sicily (Tranchida et al., 2011). The sampling site identiﬁcations, depths, collection dates and positions are compiled in Table 1. establishing the fraction dissolved at the air–sea interface is also challenging. Sediment samples were analysed for Nd radiogenic iso- topes. About 100 mg of samples were weighted and dis- solved in teﬂon beakers in a mixture of ultrapure quartex HF (24N), HNO3 (14N) and HClO4 (12N) for 4 days at 160 ◦C on a hot plate. After evaporation to dryness sam- ples were dissolved in aqua regia and heated for 24 h at 130 ◦C. Nd fractions were chemically separated follow- ing conventional column chemistry procedures described in Révillon et al. (2011). Nd isotope compositions were mea- sured in static mode on a Thermo TRITON at the PSO (Pole de Spectrométrie Océan) in Brest, France. All mea- sured Nd ratios were normalized to 146Nd / 144Nd = 0.7219. During the course of analysis, Nd standard solution La Jolla gave 0.511854 ± 0.000008 (2α; n = 28, recommended value 0.511850) and JNdi gave 0.512099 ± 0.000010 (2α; n = 6, recommended value 0.512100). Procedural blanks were all below 200 pg and therefore negligible in all cases. The main river systems of the Mediterranean basin are the Nile, Po and Rhone. River plume extensions were established using maps and satellite images from data banks provided by Ludwig et al. (2009). Nd input derived from the Nile river water and/or particles could be transported eastward and northward to the Rhodes Gyre where the LIW is formed. Tachikawa et al. (2004) sug- gest that the most signiﬁcant radiogenic Nd source to the EMed is partially dissolved Nile River particles, radiogenic Nd supplies to the eastern basin being formed by dissolved and particulate loads (εNd of about ∼−4). Henry et al. (1994) have studied the potential impact of river inputs on the Nd isotopic composition of the WMed. The Rhone transports 80 % of the solid riverine discharge into the north-western Mediterranean Sea (Leveau and Coste, 1987). According to Henry et al. 2.1.2 Areas with low spatial resolution of available data (1994) the Rhone dissolved water and superﬁcial sediments display an average εNd value of −10.2 ± 0.5. M. Ayache et al.: High-resolution neodymium characterization 5263 Table 1. Coordinates of the studied cores together with water depth. Cruise Years Longitude Latitude Depth εNd (◦E) (◦N) (m) ETNA80 1980 11.48 36.30 263 −10.09 13.44 33,23 736 −10.92 DEDALE 1987 25.59 33.51 3020 −8.15 NOE 1984 30.01 32.19 1465 −4.49 30.1 31.53 495 −3.92 Strait of Sicily 2003 12.57 37.30 29.5 −11.67 14.37 36.36 87.4 −11.05 14.22 36.16 488.2 −11.22 12.32 36.56 117 −8.06 Table 1. Coordinates of the studied cores together with water depth. treatment, results and uncertainties). This approach was to generate relatively robust Nd isotopic signatures character- izing the whole of the northern Mediterranean Sea margins, with a standard deviation less than 2εNd units in most cases. treatment, results and uncertainties). This approach was to generate relatively robust Nd isotopic signatures character- izing the whole of the northern Mediterranean Sea margins, with a standard deviation less than 2εNd units in most cases. 2.1.1 Areas with high spatial resolution of available data For this kind of region (i.e. Italian coast) and for each geolog- ical province, we carefully established the extent to which we could extrapolate the measured values to the whole province. The average εNd values for each geological province were calculated, taking into account the number of cores collected in a given area, and the geochemical characteristics of the analysed sediments. We neglected data for speciﬁc areas like very small volcanoes which were not representative of the geochemical and isotopic characteristics of the region, such as in the Strait of Sicily (see Supplement 1 reports the full Finally, we used a high-resolution numerical geological map to interpolate/extrapolate data from similar geological areas. Below we brieﬂy discuss the pros and cons of this lat- ter approach. Biogeosciences, 13, 5259–5276, 2016 www.biogeosciences.net/13/5259/2016/ 2.3 Extrapolation and extrapolation air–sea ﬂuxes (Herrmann and Somot, 2008; Herrmann et al., 2010). The heat ﬂux is applied with a retroaction term using the ERA-40 sea surface temperature (SST). Extrapolating the collected discrete data is required to allo- cate the margins with continuous Nd concentration and iso- topic compositions. In other words, we attribute an isotopic signature and Nd concentration to any margin area liable to be in contact with the waters ﬂowing through. To this end, the tools developed by Jeandel et al. (2007) for the world margins were adapted for the Mediterranean Sea. The initial state (temperature, salinity) for the Mediter- ranean Sea came from the MedAtlas-II (Rixen et al., 2005; MEDAR-MedAtlas-group, 2002) climatology weighted by a low-pass ﬁlter with a time window of 10 years using the Me- dAtlas data covering the 1955–1965 period, following Beu- vier et al. (2012b). For the temperature and salinity in the buffer zone (west of the Strait of Gibraltar), the initial state is prescribed from the 2005 World Ocean Atlas (Antonov et al., 2006; Locarnini et al., 2006). River run-off is pre- scribed from the interannual data set of Ludwig et al. (2009). The Black Sea is not explicitly included in the models, but is rather treated as one of the major freshwater sources of the Mediterranean Sea located at the Dardanelles strait, with a ﬂux corresponding to the Dardanelles net budget estimates of Stanev and Peneva (2002). Because the Nd isotopic composition of any ﬁeld is closely related to its geological nature and age (O’Nions et al., 1979; Goldstein et al., 1984, 1997; Allegre, 2005) we used a high-resolution digital geological map which provides the contours of the ﬁelds (Fig. 1) of a given geological age and type (http://www.geologie.ens.fr/spiplabocnrs/spip.php? rubrique67). This allowed us to estimate the size of the coastal segments that could provide material with the same Nd characteristics. In poorly documented areas, we ﬁrst considered the age and geochemical nature of the ﬁeld (Jeandel et al., 2007). Then we applied the same isotopic signature as similar ﬁelds of the same age based on the Nd model-age relationships (Allegre, 2005; Goldstein et al., 1984, 1997; O’Nions et al., 1979). This was thoroughly done by checking the geology, geochemistry and Nd signatures of the ﬁelds identiﬁed as the source of deposited material. 2.3 Extrapolation and extrapolation Note that the resolution of the available data in the Mediterranean basin is relatively high, reducing the uncertainties of the approach described above (see Sect. 2, Supplement 1), Fig. 2b reveals the im- provement allowed by this approach by comparing the high- resolution patchwork of ﬁeld documented to an extraction of the Mediterranean Sea basin (Fig. 2a) from the global low- resolution distribution of Jeandel et al. (2007) (see Sect. 4.1). NEMO-MED12 model simulates the main features of the thermohaline circulation and mixing of the Mediterranean Sea water masses and their interannual variability. In par- ticular, the propagation of the Levantine Intermediate Wa- ter (LIW) from the eastern to the western basin is produced with realistic timescale compared to the observations (Ay- ache et al., 2015a). However, some aspects of the model still need to be improved. For example, the formation of Adri- atic Deep Water (AdDW) is too weak, leading to a too-low contribution to the EMDW in the Ionian sub-basin (Palmiéri et al., 2015; Ayache et al., 2015a, b). The atmospheric forc- ing used by Beuvier et al. (2012b) includes some modiﬁ- cations to improve dense water ﬂuxes through the Cretan Arc during the EMT. As established in the previous ver- sion of NEMO-MED8 (1/8◦of horizontal resolution, Beu- vier et al., 2010), the model is able to reproduce a transient deep-water formation as observed for the EMT, but the sim- ulated transient produced less Eastern Mediterranean Deep Water (EMDW). Beuvier et al. (2012b) later performed a sensitivity test with modiﬁed forcing. The ARPERA forc- ings were modiﬁed over the Aegean sub-basin, by increas- ing daily water loss by 1.5 mm, daily surface heat loss by 40 W m−2, and the daily wind stress modulus by 0.02 N m−2 during November to March in the winters of 1991–1992 and 1992–1993, as done by Herrmann and Somot (2008) to study deep convection in the Gulf of Lion. This resulted in aver- age wintertime increases in heat loss (+18 %), water loss (+41 %) and wind intensity (+17 %) over the Aegean sub- basin. These changes generate an improved circulation that satisfyingly reproduced the formation and renewal of the deep water in the eastern basin during the EMT event (Ay- ache et al., 2015a). These performances of the dynamical model have to be kept in mind when analysing the Nd simu- lations. M. Ayache et al.: High-resolution neodymium characterization 5264 2.2 River and dust inputs In contrast, the Po river is less documented. The geograph- ical extension of Po river drainage basin was deﬁned using the digital geological map referenced above, and Ludwig et al. (2009) database. These information allowed us to ex- tract the Nd isotopic signatures from the work of Conticelli et al. (2009), Prelevi´c et al. (2008) and Owen (2007). These studies are the most representative of the Po drainage basin so far. Dissolved Nd in river water is efﬁciently removed from solu- tion by coagulation of colloids during the estuarine mixing. The recent compilation of Rousseau et al. (2015) conﬁrmed that on a global scale 71.8 ± 16 % of dissolved riverine Nd is removed by this process, in agreement with preceding works (e.g. Elderﬁeld et al., 1990). In addition, these authors evi- denced that lithogenic Nd is released at higher salinities by the suspended particulate material discharged by the river. Globally, this could represent 5700 ± 2600 Mg of dissolved Nd annually brought to the ocean by this mechanism, a ﬂux 6–17 times larger than the dissolved one, and 8 to 21 times larger than the atmospheric ﬂux, assuming 2 % dust dissolu- tion. Note that the mechanism evidenced in the Amazon es- tuary by Rousseau et al. (2015) does not describe all the pro- cesses likely to affect the sediments deposited on the shelves and margins. The hypothesis was modelled by Arsouze et al. (2009) and Rempfer et al. (2011). Dust input is also difﬁcult to constrain: while ﬂux is sporadic and hard to characterize, Finally, we included aeolian inputs in our compilation. To this extent, the review of Scheuvens et al. (2013) was of great help. Indeed, this work presents a review of bulk compo- sition data of northern dust inputs, their potential sediment sources and their elemental, isotope and mineralogical char- acteristics. Actually, these aeolian data will not be immedi- ately used in our modelling approach. However, we consid- ered relevant to present them with the remaining data, which allowed us to propose the most comprehensive data set. All the discrete data extracted from the literature are re- ported in Supplement 1 and Fig. 1. www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 Biogeosciences, 13, 5259–5276, 2016 M. Ayache et al.: High-resolution neodymium characterization www.biogeosciences.net/13/5259/2016/ 3.2 The tracer model Using different modelling approaches, Arsouze et al. (2007, 2010) and Rempfer et al. (2011) have shown that the ex- change between the continental margins and seawater, the boundary exchange (BE) represents the major source of Nd on the global scale. This source represents more than 90 % of the total input, whereas dissolved riverine and dust inputs could only be signiﬁcant in the upper 500 m. However, so far only little is known about the importance of the BE in semi- enclosed and/or interiors basins like the Mediterranean Sea, where atmospheric and river ﬂuxes could also have signiﬁ- cant impacts on the εNd distribution. As a ﬁrst approach, we chose to simulate only the Nd iso- topic composition (εNd) in order to test the BE hypothesis in the Mediterranean Sea (Arsouze et al., 2007). This approach does not require explicitly simulating the Nd concentration, allowing us to focus on the timescale of the process stud- ied. As in Arsouze et al. (2007, 2010), εNd is implemented in the model as a passive conservative tracer which does not affect ocean circulation. It is transported into the Mediter- ranean Sea by NEMO-MED12 physical ﬁelds using a classi- cal advection–diffusion equation, including the sources and sinks (SMS term, eq1). The rate of change of oceanic Nd isotopic composition is as follows: Figure 3. Map of the εNdmargin used in the model simulation, made by interpolation of Fig. 1b on the oceanic margins of the Mediter- ranean Sea (see Sect. 2). maskmargin is the percentage of continental margin in the grid box which represents the proportion of the surface in the grid where the BE process occurs. This quantity is estimated from the high-resolution bathymetry of the 10th version of the Mercator-LEGOS bathymetry at a resolution of 30′′ × 30′′. δεNd δt = S (εNd) −U · ∇εNd + ∇· (K∇εNd), (1) (1) The topographic extension of the oceanic margins of the Mediterranean Sea has been chosen to the ∼540 m (Fig. 3) following the margin deﬁnition used to model the iron cycle in the Mediterranean Sea by Palmiéri (2014). where S(εNd) represents the SMS term, U · ∇εNd is the three-dimensional advection and ∇· (K ∇εNd) is the lateral and vertical diffusion of εNd. The exchanges of the Nd with the Atlantic Ocean are spec- iﬁed through a buffer zone between 11◦W and the Strait of Gibraltar. 3.2 The tracer model εNd values in the buffer zone are prescribed from observation using NE Atl. MED-15 vertical proﬁle from Spi- vack and Wasserburg (1988). Since εNd is a passive tracer, simulations could be run in ofﬂine mode using the pre-computed transport ﬁelds (U, V , W) from the NEMO-MED12 dynamical model (Beuvier et al., 2012b). Physical forcing ﬁelds are read daily and inter- polated at each time step of 20 min. Ofﬂine simulations are performed for computational efﬁciency, allowing many sen- sitivity tests on the SMS term parameterization. The same approach was used by Ayache et al. (2015b) to simulate the mantle and crustal helium isotope signature, by Ayache et al. (2015a) to model the anthropogenic tritium invasion, and by Palmiéri et al. (2015) to simulate CFCs and anthropogenic carbon storage. We established some sensitivity experiments regarding the optimal value of τ in the Mediterranean basin (Arsouze et al., 2007, 2010). In this aim, six tests were performed, referred to as EXP1, EXP2, EXP3 and EXP4 with τ = 1, 3 months, 6 months and 1 year respectively. As surface ocean cur- rents are generally more dynamic than deep ones, providing more energy for sediment–seawater interactions, we realized an additional simulation (EXP5) wherein τ increases expo- nentially with depth from 1 month at the surface to 1 year at 600 m depth. We also explored the possibility that our BE parameterization might be dependent on the mineralogi- cal maturity of margin sediments (e.g. granitic vs. basaltic). Hence, relaxing time τ in EXP6 is varying linearly on a timescale of 1 month for the most radiogenic isotopic sig- nature (i.e. εNd = +6 on the extreme east of the Mediter- ranean margin) to 1 years for the most non-radiogenic values (i.e. εNd = −12 along the Spanish coast). The only SMS term taken into account in the present study is BE (Arsouze et al., 2007, 2010). It is parameterized by a relaxing equation between the ocean and the continental margin: S (εNd) = 1/τ εNdmargin −εNd · maskmargin, (2) (2) where τ is the characteristic relaxing time (i.e. the charac- teristic time needed to transfer isotopic properties from the continental margin to the ocean), εNd is the Nd isotopic com- position of seawater, εNdmargin is the value of the material deposited along the continental margin (see Sect. M. Ayache et al.: High-resolution neodymium characterization 5265 Figure 3. Map of the εNdmargin used in the model simulation, made by interpolation of Fig. 1b on the oceanic margins of the Mediter- ranean Sea (see Sect. 2). 3.1 Description of the model We use the free-surface ocean circulation model NEMO (Nucleus for European Modelling of the Ocean) (Madec and NEMO-Team., 2008) in a regional conﬁguration called NEMO-MED12 (Beuvier et al., 2012a), already used for biogeochemical studies (Ayache et al., 2015a, b; Guyennon et al., 2015; Palmiéri et al., 2015). This model uses the stan- dard ORCA grid of NEMO at 1/12◦resolution. This corre- sponds to a grid cell size in the Mediterranean area varying with latitude between 6 and 8 km, from 46 to 30◦N and it ex- tends into the Atlantic Ocean to 11◦W (buffer zone). Vertical resolution varies with depth from 1Z = 1 m at the surface to 1Z = 450 m at the bottom with 35 levels in the ﬁrst 1000 m (50 levels in total). Daily mean ﬁelds of momentum, freshwater ﬂux (evapo- ration minus precipitation) and net heat ﬂux from the high- resolution atmospheric data set (ARPERA) are used for the www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 3.2 The tracer model 2), and The simulations were initialized with uniform isotopic composition of εNd = −7 and integrated to steady state; M. Ayache et al.: High-resolution neodymium characterization 5266 Figure 4. Model–data comparison for the six simulations performed with different relaxing time at the steady state (see Table 3) and the in situ data from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994): (a) model–data correlation, red line is the linear regression from EXP2. Diagonal dashed lines are lines εNd (modelled) = εNd (data), εNd (modelled) = εNd (data) + 3εNd and εNd (modelled) = εNd (data) −3. (b) Model–data comparison as a function of depth, dashed solid line represents the data from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994). Figure 4. Model–data comparison for the six simulations performed with different relaxing time at the steady state (see Table 3) and the in situ data from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994): (a) model–data correlation, red line is the linear regression from EXP2. Diagonal dashed lines are lines εNd (modelled) = εNd (data), εNd (modelled) = εNd (data) + 3εNd and εNd (modelled) = εNd (data) −3. (b) Model–data comparison as a function of depth, dashed solid line represents the data from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994). i.e. the global averaged drift was less than 10−3 εNd per thou- sand years, for more than 75 years of spin-up run. nature (values around −12). The Algerian, Tunisian, French and Spanish coasts display relatively homogeneous values between −11.5 and −10. Such east–west gradient of Nd iso- topic signature is also observed in the seawater data, where poorly radiogenic waters from the Atlantic are progressively shifted toward more radiogenic values in the Levantine basin (Tachikawa et al., 2004). 4.1 Map of the outcropping Nd values All the details revealed by this new high-resolution map will be used to set boundary conditions in the regional simu- lation (see Sect. 3, Fig. 3). Results of the Nd parameter mapping are represented in Fig. 2, cold colours represent the old non-radiogenic rocks whereas the warm colours correspond to the recent radio- genic ones. www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 Biogeosciences, 13, 5259–5276, 2016 4.2 The characteristic margin-to-ocean exchange time Tectonic and associated volcanic activities led to the very complex morphology and geology in the Mediterranean re- gion, comprising small islands (e.g. Corsica, Cyprus), sub- basins (e.g. Adriatic, Aegean and Tyrrhenian), and many nar- row straits (e.g. Strait of Sicily, Otranto Passage). This partic- ular context prevents the use of low-resolution grid to repre- sent this region properly. This motivated the realization of the high-resolution (1/12◦× 1/12◦) version of the Nd isotopic signature (Fig. 2b) and Nd concentration (see Supplement 5) for this basin. We ﬁrst explored the impact of changing the value of the re- laxing time on the εNd distribution in the Mediterranean Sea. This was made following the strategy adopted by Arsouze et al. (2010) in the North Atlantic basin, although NEMO- MED12 model has higher horizontal and vertical resolutions (1/12◦in this study compared to 1/4◦in Arsouze et al., 2010). The results of these different sensitivity tests are com- pared with in situ observations collected by Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994) using cor- relation plots, coloured maps and sections (Figs. 4 and 5). The general trend is that the margin Nd isotopic signa- tures vary from non-radiogenic values in the WMed, to ra- diogenic values when reaching the Aegean and Egyptians coasts, the most radiogenic ﬁelds (εNd up to +6) being lo- cated around the eastern border of the Levantine sub-basins, and in the volcanic region of the south of Italy (Fig. 2b). In contrast, the southern Sicilian ﬁelds and the northern Alboran sub-basin are characterized by the most negative isotopic sig- The simulated εNd distributions in EXP2 and EXP3 (re- laxing time of 3 and 6 months respectively) present a better correlation with in situ data relative to the other experiments, with correlation coefﬁcients close to 0.75 and 0.60 respec- tively (Table 3). The difference between in situ data (dashed line) and the different sensitivity experiments as a function of depth (Fig. 4b) reveals that EXP2 provides the best agree- www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 M. Ayache et al.: High-resolution neodymium characterization 5267 Figure 5. Output of model from EXP2 (t = 3 months) at the steady state. Upper panel: horizontal maps for surface waters (a), interme- diate waters (b), and deep waters (c). Lower panel E–W section in WMed (d), and EMed (e), whereas colour-ﬁlled dots represent in situ observations from Tachikawa et al. 4.2 The characteristic margin-to-ocean exchange time (2004), Vance et al. (2004) and Henry et al. (1994). Both use the same colour scale. Figure 5. Output of model from EXP2 (t = 3 months) at the steady state. Upper panel: horizontal maps for surface waters (a), interme- diate waters (b), and deep waters (c). Lower panel E–W section in WMed (d), and EMed (e), whereas colour-ﬁlled dots represent in situ observations from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994). Both use the same colour scale. 4.3 The εNd distribution ment with observations, despite a slight overestimation of εNd between 0.3 and 2εNd units. The data–model differences are more important for the other experiments which produced too-radiogenic simulations (of more than 2εNd units). The horizontal distribution of εNd (Supplement 2–4) conﬁrms this statistical correlation, showing that only EXP2 and EXP3 produced reasonable east–west gradients of εNd. EXP1 gen- erated too-pronounced εNd geographical gradients, particu- larly in surface waters along the continental margins, sug- gesting an overestimation of the exchange compared to the transport. On the opposite a simulation with a relaxing time of 1 year (EXP4) leads to a homogeneous εNd distribution in surface and deep waters with a low data-model correlation, indicating an underestimation of the boundary exchange pro- cess. EXP5 (τ increases with depth) conducted to a strong gradient over the entire water column in WMed, showing that surface-to-deep variation of the BE rate was likely overesti- mated in this simulation. EXP6 displayed a realistic E–W gradient in the surface waters, but a too-homogeneous εNd signal in the intermediate and deep waters, suggesting that the BE rate seems weakly affected by the lithology of the margin sediments. Finally, we consider that the characteris- tic exchange time providing the best agreement with obser- vations is close to 3 months. This value is consistent with the results obtained by Arsouze et al. (2010) with its simulation of the North Atlantic area. Therefore we will only consider EXP2 for the rest of our analysis. ment with observations, despite a slight overestimation of εNd between 0.3 and 2εNd units. The data–model differences are more important for the other experiments which produced too-radiogenic simulations (of more than 2εNd units). The horizontal distribution of εNd (Supplement 2–4) conﬁrms this statistical correlation, showing that only EXP2 and EXP3 produced reasonable east–west gradients of εNd. EXP1 gen- erated too-pronounced εNd geographical gradients, particu- larly in surface waters along the continental margins, sug- gesting an overestimation of the exchange compared to the transport. On the opposite a simulation with a relaxing time of 1 year (EXP4) leads to a homogeneous εNd distribution in surface and deep waters with a low data-model correlation, indicating an underestimation of the boundary exchange pro- cess. EXP5 (τ increases with depth) conducted to a strong gradient over the entire water column in WMed, showing that surface-to-deep variation of the BE rate was likely overesti- mated in this simulation. 4.3 The εNd distribution EXP6 displayed a realistic E–W gradient in the surface waters, but a too-homogeneous εNd signal in the intermediate and deep waters, suggesting that the BE rate seems weakly affected by the lithology of the margin sediments. Finally, we consider that the characteris- tic exchange time providing the best agreement with obser- vations is close to 3 months. This value is consistent with the results obtained by Arsouze et al. (2010) with its simulation of the North Atlantic area. Therefore we will only consider EXP2 for the rest of our analysis. The monthly-averaged εNd horizontal distributions resulting from EXP2 for the surface waters (0–200 m), the intermedi- ate waters (200–600 m) and the deep waters (600–3500 m), are represented in Fig. 5a–c respectively, together with the data from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994). The model results are extracted after the steady state (in 1987) of the simulation and considered rep- resentative of a pre-EMT situation (i.e. EMT, Roether et al., 2007). The model correctly simulates the pronounced εNd E–W gradient characterizing the surface waters (Fig. 5a). The val- ues simulated in the WMed and eastern Levantine basin are consistent with the observations while the simulated values in the Aegean and central Levantine basin tend to be too radiogenic. At intermediate depths, both modelled and ob- served E–W gradients are less pronounced than at the sur- face (Fig. 5b). However averaged simulated values are rel- atively too radiogenic at the intermediate level (−5.8 com- pared to −9.4 ± 0.69, Table 2). Especially high εNd signa- tures are simulated in the Aegean sub-basin, over the Strait of Sicily and in the Tyrrhenian sub-basins (Fig. 5b). A sig- niﬁcant model–data disagreement was found in the Alboran sub-basin which largely overestimates the observations. The εNd distribution in the deep waters is relatively homogeneous over the whole basin except in the Aegean sub-basin and Strait of Sicily (Fig. 5c). www.biogeosciences.net/13/5259/2016/ www.biogeosciences.net/13/5259/2016/ M. Ayache et al.: High-resolution neodymium characterization 5268 Table 2. Mean εNd for the Mediterranean Sea, from EXP3 and for the in situ data from Tachikawa et al. (2004). Model In situ data Intermediate Average Intermediate Average waters all depths waters all depths Mediterranean Sea −5.8 −6.2 −7.6 ± 1.37 −7.8 ± 1.54 Eastern basin (EMed) −4.7 −5.1 −7 ± 0.85 −7.1 ± 1.08 Western basin (WMed) −5.8 −6.3 −9.4 ± 0.69 −9.6 ± 0.48 Figure 6. Comparison of average vertical proﬁles of Nd isotopic signature (Nd-IC) from EXP2 (t = 3 months) in the whole Mediterranean Sea. Model results are in blue, while red indicates the in situ data from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994). n εNd for the Mediterranean Sea, from EXP3 and for the in situ data from Tachikawa et al. (2004). Table 2. Mean εNd for the Mediterranean Sea, from EXP3 and for the in situ data from Tachikawa et al. (2004). Figure 6. Comparison of average vertical proﬁles of Nd isotopic signature (Nd-IC) from EXP2 (t = 3 months) in the whole Mediterranean Sea. Model results are in blue, while red indicates the in situ data from Tachikawa et al. (2004), Vance et al. (2004) and Henry et al. (1994). The Levantine Intermediate Water mass (LIW) is well identiﬁed by its marked radiogenic signature. LIW is pro- duced in the Levantine sub-basin before passing Crete at 28◦E, where measured εNd values reach −5 (Fig. 5). The εNd isotopic signature is well identiﬁed over the entire LIW trajectory at the intermediate level (between 200 and 600 m depth), with values around −4.8 in the Algerian sub-basin and up to −5.7 in the Alboran sub-basin (Fig. 5d). The res- olution of the available data hardly allows us to evaluate the model performance for this water mass; nevertheless, station 74 (33◦7′ N, 33◦5′ E) in the eastern Levantine basin exhibits a discernible radiogenic signal associated to LIW (more pronounced than the modelled one), while station 51 (33.5◦N, 27◦E) in the western Levantine basin exposes a rel- atively homogeneous vertical isotopic signature. The surface waters originating from the Atlantic Ocean (Atlantic Waters, AW) are characterized by the most negative signature (value around −9) and are transported over all the Mediterranean Sea, allowing them to be clearly identiﬁed. M. Ayache et al.: High-resolution neodymium characterization The εNd signa- tures of the deep-water mass display values around −6.5, consistent with the observations available in the eastern basin (Fig. 5e). Except in the Alboran sub-basin, where pronounced mis- matches are simulated between the model and the observa- tions, the model captures the general features of the vertical proﬁles of Nd isotopic signatures, especially in the Levantine sub-basin (averaged over the entire water column), produc- ing a realistic and signiﬁcant radiogenic signature associated to LIW at the intermediate level (Fig. 6), although the εNd values can be overestimated in some places by almost 2εNd units. www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 4.4 The interannual variability After 1992, which is referred to as the beginning of the EMT event, an important change of εNd dis- tributions is simulated over all the Mediterranean Sea, with regional values shifted by almost 0.5εNd units. Mediterranean Sea. After 1992, which is referred to as the beginning of the EMT event, an important change of εNd dis- tributions is simulated over all the Mediterranean Sea, with regional values shifted by almost 0.5εNd units. In comparison with the steady-state situation for the Mediterranean Sea circulation (pre-EMT), the surface wa- ters are relatively less radiogenic in the Levantine sub-basin, the Algerian, and the Alboran sub-basins between 1995 and 1999 (Fig. 8a and b). After 2001 these surface waters be- came more radiogenic over the whole basin. At the interme- diate level only the EMed presents a less radiogenic signature in 1995; indeed the εNd are more radiogenic over the entire basin after 2001 (Fig. 8f–h). The deep waters are globally more radiogenic between 1987 and 2010, especially in the EMed where increase of 1 units of εNd are simulated around the Aegean sub-basin. The vertical section illustrates the im- portant penetration of the surface and intermediate waters characterized with radiogenic εNd into the deep waters near The drastic change caused by the EMT event at the begin- ning of the 1990s is further illustrated by showing the differ- ences of εNd distributions between the pre-EMT situations in 1987 and the subsequent years up to 2010. The analysis on different horizontal levels (Fig. 8), as well as along the E– W sections (Fig. 9), provides a better understanding of the source of ventilation for the interior of the Mediterranean Sea, and the connection between the surface, intermediate and deep-water redistribution. 4.4 The interannual variability In this section, we analyse inter annual variations on the re- distribution of εNd over the Mediterranean basin, with a spe- cial focus on the possible impact of the EMT events. The evo- lution of the monthly averaged εNd at the intermediate level (between 200 and 600 m) in different “boxes” following the LIW trajectory from the Levantine sub-basin to the Algerian sub-basin (including Ionian, Strait of Sicily, Tyrrhenian, and Gulf of Lion) is represented in Fig. 7 for the 40 years of the simulation. It shows that εNd signatures vary seasonally with maximum amplitude of 0.2εNd units. The EMT event signif- icantly impacts the εNd signature at the global scale of the Biogeosciences, 13, 5259–5276, 2016 www.biogeosciences.net/13/5259/2016/ 5269 M. Ayache et al.: High-resolution neodymium characterization M. Ayache et al.: High-resolution neodymium characterization Table 3. Summary of the main characteristics for each experience. Experience Relaxing time τ Regression coefﬁcient for data/model points EXP1 1 month 0.32 EXP2 3 months 0.75 EXP3 6 months 0.60 EXP4 1 year 0.34 EXP5 τ varying vertically from 1 month at the surface to 10 months at 540 m 0.27 EXP6 τ 1 month (max εNdmargin) to 1 year (min εNdmargin) 0.39 Figure 7. The εNd evolution from EXP2 (τ = 3 months) at the intermediate level in (a) (average depth between 200 and 600 m), deep levels in (b) (average depth between 600 and 3500 m). In the Gulf of Lion (yellow), Algerian sub-basin (blue), Levantine Ionian s-b (green), Strait of Sicily (red), Tyrrhenian s-b (black). Mediterranean Sea After 1992 which is referred to as the In comparison with the steady state situatio e 7. The εNd evolution from EXP2 (τ = 3 months) at the intermediate level in (a) (average depth between 200 and 600 m), a levels in (b) (average depth between 600 and 3500 m). In the Gulf of Lion (yellow), Algerian sub-basin (blue), Levantine s b ( ) S i f Si il ( d) T h i b (bl k) Figure 7. The εNd evolution from EXP2 (τ = 3 months) at the intermediate level in (a) (average depth between 200 and 600 m), and for the deep levels in (b) (average depth between 600 and 3500 m). In the Gulf of Lion (yellow), Algerian sub-basin (blue), Levantine s-b (cyan), Ionian s-b (green), Strait of Sicily (red), Tyrrhenian s-b (black). Mediterranean Sea. www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 M. Ayache et al.: High-resolution neodymium characterization M. Ayache et al.: High-resolution neodymium characterization 5270 Figure 8. Horizontal maps showing the difference relative to 1987 from EXP2 (τ = 3 month), in the left column for the surface level (0– 200 m), in the middle column for the intermediate layer (between 250–600 m, and for the deep layer in the right column (600–3500 m). Figure 8. Horizontal maps showing the difference relative to 1987 from EXP2 (τ = 3 month), in the left column for the surface level (0– 200 m), in the middle column for the intermediate layer (between 250–600 m, and for the deep layer in the right column (600–3500 m). www.biogeosciences.net/13/5259/2016/ 5 Discussion the Cretan Arc as a consequence of the EMT that shifted the Nd isotopic signature by almost +1.3εNd units in the bottom waters (Fig. 7b). This radiogenic signal is maximum in 1995 at the bottom water around the Cretan Arc near 26◦E, and for the following years (i.e. 1997, 1999, 2005 and 2010) propa- gates in the deep waters of the whole Levantine sub-basin, which typically becomes more radiogenic of +0.5 of εNd (Fig. 9). The high-resolution simulation presented here provides a too-radiogenic signature of Nd isotopic signature in the Mediterranean Sea; nevertheless this approach conﬁrms the primordial role of the BE as the major source of Nd in the marine environment, similar to what has been previously demonstrated for the global ocean (Arsouze et al., 2007) and the Atlantic basin (Arsouze et al., 2010). This reinforces the preceding conclusions of BE as a major process in the Nd oceanic cycle, even at regional scale and in a semi-enclosed basin such as the Mediterranean basin. Although the pro- cesses leading to BE are still not fully understood (Jeandel and Oelkers, 2015), the resulting timescale is of the order of few months, in agreement with Arsouze et al. (2010). This timescale is also consistent with the kinetic rates of Nd re- lease from basaltic material during the batch experiments conducted by Pearce et al. (2013). It is also consistent with the ﬁeld data and their Lagrangian modelling developed by Grenier et al. (2013) in the highly dynamic south-western Paciﬁc. Taking into account the lithology of the margin sed- The amplifying tracer penetration caused by the EMT event generates less radiogenic values at the LIW layer in the EMed in 1995 and 1999, because this water is mixed with upwelled pre-EMT less radiogenic water masses. In con- trast the simulated values become globally more radiogenic in the WMed. The radiogenic transient signal enters inside the western basin through the LIW outﬂow (up to +0.6εNd unit) and gradually penetrates into the deep water through time. However the most εNd shift was simulates in the Lev- antine sub-basin deep water with more than 1.3 unit change (Figs. 7b and 9). Biogeosciences, 13, 5259–5276, 2016 www.biogeosciences.net/13/5259/2016/ M. Ayache et al.: High-resolution neodymium characterization 5271 Figure 9. Colour-ﬁlled contours represent simulated Nd isotopic composition for the WMed in the left column, and the EMed sections are shown in the right column. 5 Discussion The ﬁrst line show the situation in 1987 (pre-EMT), the others sections show the difference in the Nd-IC between 1995 and 2010 (post-EMT period) corrected to the 1987 situation. M. Ayache et al.: High-resolution neodymium characterization 5271 Figure 9. Colour-ﬁlled contours represent simulated Nd isotopic composition for the WMed in the left column, and the EMed sections are shown in the right column. The ﬁrst line show the situation in 1987 (pre-EMT), the others sections show the difference in the Nd-IC between 1995 and 2010 (post-EMT period) corrected to the 1987 situation. Mediterranean basin (i.e. the Nile, Po and Rhone) are char- acterized by a wide range of Nd isotopic signature, with an average εNd value of −10.2 for the Rhone, and rather radio- genic Nd isotopic ratios for the Nile (εNd ∼−4). The input of Saharan dust has important effects on the Mediterranean region (Guerzoni et al., 1997), where the Nd isotopic com- positions of aerosols range from −9.2 in the eastern part of northern Africa (e.g. Egypt) to −16 in the central and west- ern parts of northern Africa (Grousset and Biscaye, 2005; Scheuvens et al., 2013). Previous studies suggest that the εNd distribution at the near surface, for the most part, reﬂects river and aerosol inputs (Piepgras and Wasserburg, 1987; Arsouze et al., 2009; Jones et al., 2008; Siddall et al., 2008). Hence, it is clear that taking into account dust and river input in future work could improve the simulation of Nd isotopic distribu- tion in the Mediterranean Sea. Mediterranean basin (i.e. the Nile, Po and Rhone) are char- acterized by a wide range of Nd isotopic signature, with an average εNd value of −10.2 for the Rhone, and rather radio- genic Nd isotopic ratios for the Nile (εNd ∼−4). The input of Saharan dust has important effects on the Mediterranean region (Guerzoni et al., 1997), where the Nd isotopic com- positions of aerosols range from −9.2 in the eastern part of northern Africa (e.g. Egypt) to −16 in the central and west- ern parts of northern Africa (Grousset and Biscaye, 2005; Scheuvens et al., 2013). Previous studies suggest that the εNd distribution at the near surface, for the most part, reﬂects river and aerosol inputs (Piepgras and Wasserburg, 1987; Arsouze et al., 2009; Jones et al., 2008; Siddall et al., 2008). 5 Discussion Hence, it is clear that taking into account dust and river input in future work could improve the simulation of Nd isotopic distribu- tion in the Mediterranean Sea. iments did not improve our simulations. This requires more laboratory experiments, targeted on the issue of the nature of the sediments. Nevertheless the comparison with the avail- able data in the Mediterranean Sea reveals that this approach simulates a slightly too-radiogenic value in the surface and intermediate waters, especially in the Aegean and the Alb- oran sub-basins. The uniquely available observation in the Alboran sub-basin is located close to the Strait of Gibraltar, and shows εNd values characteristic of the outﬂow from the Atlantic sector. The model fails to reproduce this signal asso- ciated to the advection of water mass of Atlantic origin (see Fig. 5a), due to a simulated net water ﬂux input from the At- lantic that stands in the lower range compared to observations (Beuvier et al., 2012a). However, the global radiogenic bias will likely be corrected once the dust and river inputs are sim- ulated. Indeed, those could locally affect the surface waters with less radiogenic values. The main river systems of the 6 Conclusions This study proposes a new map compiled from in situ data with a sufﬁcient resolution to cover the very complex mor- phology and geology of the Mediterranean Sea. This map shows Nd isotopic signatures for all the Mediterranean Sea margins. The quality of this interpolated map means it can be used as a continuous source of εNd to make a link between an ocean circulation model and the tracer inputs from the margins in order to better understand the thermohaline cir- culation in modern and palaeo ocean circulation. This com- pilation provides a complete picture of the εNd of the whole Mediterranean margins which could interest other earth sci- ence ﬁelds (e.g. solid earth, weathering, tectonic, etc.). The sequence of the EMT events occurring in the EMed at the beginning of the 1990s has completely changed the deep-water mass structure. Different hypotheses concerning the preconditioning of the EMT and its timing have been pro- posed in the literature (Roether et al., 1996, 2007; Malanotte- Rizzoli et al., 1999; Lascaratos et al., 1999; Theocharis et al., 1992, 1999; Theocharis and Kontoyiannis, 1999; Samuel et al., 1999; Zervakis et al., 2000; Stanev and Peneva, 2002; Klein et al., 1999; Gertman et al., 2006; Josey, 2003). In our simulation, the εNd distribution between 1995 and 2001 also revealed that LIW and deep-water signatures were very dif- ferent from the pre-EMT picture (see Fig. 7). This ampliﬁca- tion of mixing caused by the EMT generates accumulation of radiogenic water at the bottom. The 1995 section emphasizes the severe impact of the EMT on water mass distribution, which transfers massive volumes of surface/intermediate wa- ters into the deep layers, with the highest contributions to- ward the bottom and south of Crete (Fig. 8), causing a tem- porary change in the EMDW origin, from the Adriatic to Aegean sub-basin in 1992–1993. The renewal of the deep- water masses is very similar to the tritium/helium-3 redis- tribution observed by Roether et al. (2013), which is satisfy- ingly simulated by our regional model (Ayache et al., 2015a). This gives some more reliability to the evolution of εNd dis- tribution simulated after the EMT event (Fig. 9). The εNd distribution was simulated using a high-resolution regional model at 1/12◦of horizontal resolution (6–8 km). The boundary exchange (BE) parameterization was per- formed via a relaxing term toward the isotopic composition of the margin from this new geological map. M. Ayache et al.: High-resolution neodymium characterization 5272 The LIW layer is particularly characterized by the most ra- diogenic signature in the intermediate level between 200 and 600 m, which is in good agreement with in situ observations from Tachikawa et al. (2004) especially with the highest εNd value of −4.8 found at about 200 m in the easternmost Lev- antine basin. The LIW represents the principal movement of water mass from the EMed into the WMed. This LIW signature is conserved in the WMed, allowing us to study the impact of interannual variability, including the excep- tional events observed in the ventilation of the deep waters (e.g. EMT) in the whole basin. The too-radiogenic isotopic signature simulated in the LIW layer at 25◦E can be ex- plained by the fact that the LIW are formed NW of Lev- antine sub-basin near the Cretan Arc, where the margin IC are about −4, leading to a relatively radiogenic signature as we consider only the margin Nd source. Also, tritium/helium (Ayache et al., 2015a) and CFC (Palmiéri et al., 2015) simu- lations have shown that the model overestimates the mixing near the Cretan Arc and, as a consequence, the Levantine sub-basin isotopic signature is overrepresented in this water mass. Our results suggest that the shift is more important in the Levantine deep water, compared to intermediate water where the EMT impact is lower. This sensitivity test gives a use- ful diagnostic on the long-term variability of Mediterranean Sea circulation and demonstrates the potential of Nd to detect a EMT-like event. However the weak formation of AdDW could affect the simulated sift of seawater Nd IC in the Io- nian deep water. www.biogeosciences.net/13/5259/2016/ www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 www.biogeosciences.net/13/5259/2016/ 7 Data availability The data used in this study was obtained from an extensive compilation of all published Nd concentrations and isotopic values using the EarthChem database (accessible from http:// www.earthchem.org). All supplemental ﬁgures and data ref- erences (Sect. 2) can be found in the Supplement. Bertram, C. and Elderﬁeld, H.: The geochemical balance of the rare earth elements and neodymium isotopes in the oceans, Geochim. Cosmochim. Ac., 57, 1957–1986, doi:10.1016/0016- 7037(93)90087-D, 1993. Beuvier, J., Sevault, F., Herrmann, M., Kontoyiannis, H., Ludwig, W., Rixen, M., Stanev, E., Béranger, K., and Somot, S.: Modeling the Mediterranean Sea interannual variability during 1961–2000: Focus on the Eastern Mediterranean Transient, J. Geophys. Res., 115, C08017, doi:10.1029/2009JC005950, 2010. M. Ayache et al.: High-resolution neodymium characterization whole Mediterranean basin. It conﬁrms that εNd represents an appropriate proxy to improve our knowledge on the long- term trend in the Mediterranean Sea circulation, especially to explore whether EMT-type events occurred in the past (Roether et al., 2014; Gaˇci´c et al., 2011). New Nd palaeo data (e.g. Jiménez-Espejo et al., 2015) or recent Nd observa- tions collected on corals or foraminifera in the context of the PaleoMeX (Palaeo Mediterranean Experiment) programme should give an opportunity to address this question. Arsouze, T., Treguier, A. M., Peronne, S., Dutay, J. C., Lacan, F., and Jeandel, C.: Modeling the Nd isotopic composition in the North Atlantic basin using an eddy-permitting model, Ocean Sci- ence, 6, 789–797, doi:10.5194/os-6-789-2010, 2010. Ayache, M., Dutay, J.-C., Jean-Baptiste, P., Beranger, K., Arsouze, T., Beuvier, J., Palmieri, J., Le-vu, B., and Roether, W.: Mod- elling of the anthropogenic tritium transient and its decay prod- uct helium-3 in the Mediterranean Sea using a high-resolution regional model, Ocean Science, 11, 323–342, doi:10.5194/os-11- 323-2015, 2015a. Ayache, M., Dutay, J.-C., Jean-Baptiste, P., and Fourré, E.: Sim- ulation of the mantle and crustal helium isotope signature in the Mediterranean Sea using a high-resolution regional circu- lation model, Ocean Science, 11, 965–978, doi:10.5194/os-11- 965-2015, 2015b. The Supplement related to this article is available online at doi:10.5194/bg-13-5259-2016-supplement. Beuvier, J., Béranger, K., Lebeaupin Brossier, C., Somot, S., Se- vault, F., Drillet, Y., Bourdallé-Badie, R., Ferry, N., and Lyard, F.: Spreading of the Western Mediterranean Deep Water after win- ter 2005: Time scales and deep cyclone transport, J. Geophys. Res., 117, C07022, doi:10.1029/2011JC007679, 2012a. Acknowledgements. We would like to thank Koji Suzuki and an anonymous reviewer for their careful reading of the manuscript and helpful remarks. The authors wish to acknowledge F. Bassinot, G. Tranchida and P. Censi for sediment samples. We thank S. Conticelli who kindly answered our questions on geological features of the Italian coast, which greatly helped for the interpo- lation. Hugo Pradalier is acknowledged for his contribution at the beginning of this work. Beuvier, J., Lebeaupin Brossier, C., Béranger, K., Arsouze, T., Bourdallé-Badie, R., Deltel, C., Drillet, Y., Drobinski, P., Lyard, F., Ferry, N., Sevault, F., and Somot, S.: MED12, Oceanic component for the modelling of the regional Mediterranean Earth System, Mercator Ocean Quarterly Newsletter, 46, 60–66, 2012b. Edited by: K. Suzuki Reviewed by: two anonymous referees Carter, P., Vance, D., Hillenbrand, C., Smith, J., and Shoosmith, D.: The neodymium isotopic composition of waters masses in the eastern Paciﬁc sector of the Southern Ocean, Geochim. Cos- mochim. Ac., 79, 41–59, doi:10.1016/j.gca.2011.11.034, 2012. Conticelli, S., Guarnieri, L., Farinelli, A., Mattei, M., Avanzinelli, R., Bianchini, G., Boari, E., Tommasini, S., Tiepolo, M., Prele- vi´c, D., and Venturelli, G.: Trace elements and Sr-Nd-Pb iso- topes of K-rich, shoshonitic, and calc-alkaline magmatism of the Western Mediterranean Region: Genesis of ultrapotassic to calc- alkaline magmatic associations in a post-collisional geodynamic setting, Lithos, 107, 68–92, doi:10.1016/j.lithos.2008.07.016, 2009. 6 Conclusions The charac- teristic margin-to-ocean exchange time is about 3 months in the Mediterranean Sea, in good agreement with the previous estimation of Arsouze et al. (2010) in the northern Atlantic basin. The high resolution of the geological ﬁeld on the one hand and the model on the other hand has revealed local het- erogeneities attributed to local BE effects that would not be detected using a coarse-resolution model. This is not con- ﬁrmed by the data yet but they could be improved when all the measurements done in the framework of MedBlack Geo- traces cruise are available. Our next step is therefore to use a fully prognostic coupled dynamical/biogeochemical model with an explicit represen- tation of all Nd sources (i.e. atmospheric dusts, dissolved river ﬂuxes, and margin sediment re-dissolution) and sinks (i.e. scavenging) to simulate the Nd oceanic cycle in another dedicated study. More in situ data should help to improve knowledge of Nd and its isotope cycles in the Mediterranean Sea, which will better constrain the ﬂuxes of solid mate- rial and exchange between the continental margin and open ocean. The EMT modiﬁes the characteristics of EMDW in the Levantine sub-basin by increasing the εNd signature over the entire eastern basin (Fig. 9). Hence the LIW layer is also af- fected by this εNd shift, which is next transferred rapidly in the WMed by the overﬂow of the Strait of Sicily. The LIW signal then propagates at depth in the western basin, illustrat- ing how the EMT event modiﬁes water mass characteristics and potentially affects the formation of deep and bottom wa- ter masses in this sub-basin. The boundary sources with εNd implemented as a pas- sive conservative has represented an interesting opportunity to explore the interannual variability on the εNd distribu- tion. Indeed, the Eastern Mediterranean Transient (EMT) sig- nal from the Aegean sub-basin was simulated, conducting a signiﬁcant and measurable evolution of εNd signal over the www.biogeosciences.net/13/5259/2016/ Biogeosciences, 13, 5259–5276, 2016 5273 References Allegre, C.: Géologie Isotopique, Belin, Paris, 2005. Allegre, C.: Géologie Isotopique, Belin, Paris, 2005. Allegre, C.: Géologie Isotopique, Belin, Paris, 2005. Antonov, J. I., Locarnini, R. A., Boyer, T. P., Mishonov, A. V., and Garcia, H. 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https://openalex.org/W2861254206	https://www.ocean-sci.net/14/617/2018/os-14-617-2018.pdf	English	null	100 years of atmospheric and marine observations at the Finnish Utö Island in the Baltic Sea	Ocean science	2,018	cc-by	10,595	Correspondence: Lauri Laakso (lauri.laakso@fmi.ﬁ) Correspondence: Lauri Laakso (lauri.laakso@fmi.ﬁ) Correspondence: Lauri Laakso (lauri.laakso@fmi.ﬁ) Received: 29 December 2017 – Discussion started: 25 January 2018 Revised: 20 June 2018 – Accepted: 26 June 2018 – Published: 11 July 2018 Abstract. The Utö Atmospheric and Marine Research Station introduced in this paper is located on Utö Is- land (59◦46.84′ N, 21◦22.13′ E) at the outer edge of the Archipelago Sea, by the Baltic Sea towards the Baltic Proper. Meteorological observations at the island started in 1881 and vertical proﬁling of seawater temperature and salinity in 1900. Since 1980, the number of observations at Utö has rapidly increased, with a large number of new meteorolog- ical, air quality, aerosol, optical and greenhouse gas param- eters, and recently, a variety of marine observations. In this study, we analyze long-term changes of atmospheric temper- ature, cloudiness, sea salinity, temperature and ice cover. Our main dataset consists of 248 367 atmospheric temperature observations, 1632 quality-assured vertical seawater temper- ature and salinity proﬁles and 8565 ice maps, partly digi- tized for this project. We also use North Atlantic Oscillation (NAO), major Baltic inﬂow (MBI) and Baltic Sea river runoff data from the literature as reference variables to our data. Our analysis is based on a statistical method utilizing a dynamic linear model. The results show an increase in the atmospheric temperature at Utö, but the increase is signiﬁcantly smaller than on land areas and has taken place only since the early 1980s, with a rate of 0.4 ◦C decade−1 during the last 35 years. We also see an increase in seawater temperatures, especially on the surface, with an increase of 0.3 ◦C decade−1 for the last 100 years. In deeper water layers, the increase is smaller and inﬂuenced by vertical mixing, which is modulated by in- ﬂow of saline water from the North Sea and freshwater inﬂow from rivers and by wind-driven processes inﬂuenced by the local bathymetry. The date when air temperature in the spring exceeds +5 ◦C became 5 days earlier from the period 1951– 1980 to the period 1981–2010 and the date when sea surface water temperature exceeds +4 ◦C changed to 9 days earlier. Sea ice cover duration at Utö shows a decrease of approxi- mately 50 % during the last 35 years. Correspondence: Lauri Laakso (lauri.laakso@fmi.ﬁ) Based on the combined results, it is possible that the climate at Utö has changed into a new phase, in which the sea ice no longer reduces the local temperature increase caused by the global warming. 1 Introduction Recently, average atmospheric concentration of carbon diox- ide has exceeded 400 ppm (Kilkki et al., 2015) and the effects of climate change have become continuously more visible throughout the Earth (IPCC, 2013; Mikkonen et al., 2015; Iles and Hegerl, 2017). The Baltic Sea, with shallow waters and variable ice cover, rapidly responds to both annual and long-term changes (Lehmann et al., 2011; HELCOM, 2013). However, the responses are still slower than those observed over land areas, due to thermal inertia of the water body. Previous studies from the Baltic Sea area show that dur- ing the 20th century, air temperatures have increased until 1930, decreased until the 1960s and started to increase again since the 1980s (HELCOM, 2013). Simultaneously, the sea surface temperatures have followed the atmospheric temper- atures, with a clear increase due to a recent decrease in dura- tion of ice cover. Seawater salinities in the Baltic Sea follow both changes in freshwater inﬂow and major Baltic inﬂows (MBIs). The MBIs increase stratiﬁcation, leading to reduced vertical mixing. 100 years of atmospheric and marine observations at the Finnish Utö Island in the Baltic Sea Lauri Laakso1,2, Santtu Mikkonen3, Achim Drebs1, Anu Karjalainen1, Pentti Pirinen1, and Pekka Alenius1 1Finnish Meteorological Institute, Erik Palménin aukio 1, Helsinki, Finland 2Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa Lauri Laakso1,2, Santtu Mikkonen3, Achim Drebs1, Anu Karjalainen1, Pentti Pirinen1, and Pekka Alenius1 1Finnish Meteorological Institute, Erik Palménin aukio 1, Helsinki, Finland 2Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa 3Department of Applied Physics, University of Eastern Finland, Kuopio, Finland Lauri Laakso1,2, Santtu Mikkonen3, Achim Drebs1, Anu Karjalainen1, Pentti Pirinen1, and Pekka Alenius1 1Finnish Meteorological Institute, Erik Palménin aukio 1, Helsinki, Finland 2Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa 3Department of Applied Physics, University of Eastern Finland, Kuopio, Finland Ocean Sci., 14, 617–632, 2018 https://doi.org/10.5194/os-14-617-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Ocean Sci., 14, 617–632, 2018 https://doi.org/10.5194/os-14-617-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island In Finland, the longest observed data series containing sea temperatures and salinities together with meteorological variables are from the Island of Utö at the outer edge of the Archipelago Sea (Ahlnäs, 1961). Recently, Utö station was selected as one of the World Meteorological Organization (WMO) long-term observing stations in the recognition of its irreplaceable cultural and scientiﬁc heritage (World Me- teorological Organization, 2017). However, despite observa- tions having started already in 1881, a limited number of meteorological studies (e.g., Riihelä et al., 2015; Laapas and Venäläinen, 2017) and only few studies focusing on sea ice and hydrography (Ahlnäs, 1961; Haapala and Alenius, 1994; Haapala and Leppäranta, 1997; Jevrejeva et al., 2004) have been published. One reason for this is that a signiﬁcant part of the observation data has not been digitized or quality as- sured until the current study. In our study, we use the meteo- rological, hydrographic and sea ice observations carried out in Utö, Baltic Sea, during the period 1881–2016. Occasional saline water inﬂows (major Baltic inﬂows) from the North Sea (Matthäus et al., 2008) and continuous inﬂow of freshwater from the rivers (Ahlnäs, 1961; Hansson et al., 2011; Väli et al., 2013) keep the stratiﬁcation strong. Occasional saline water inﬂows (major Baltic inﬂows) from the North Sea (Matthäus et al., 2008) and continuous inﬂow of freshwater from the rivers (Ahlnäs, 1961; Hansson et al., 2011; Väli et al., 2013) keep the stratiﬁcation strong. ) p g Utö Island (59◦46.84′ N, 21◦22.13′ E), where the observa- tions of this study are made, is located at the outer edge of the Archipelago Sea towards the Baltic Sea (Fig. 1). Utö is the southernmost permanently inhabited island in the Finnish archipelago. Its land area is approximately 0.8 km2 and the average permanent population between 20 and 30 people. Utö Island has had a lighthouse and a pilot station since the 18th century, with several generations of pilots living on the island. Due to its location and permanent population, local pilots (and during the 20th century, military ofﬁcers) have carried out observations on a daily basis, with the exception of some short breaks during WWI. The sea area 1 km west of Utö is relatively deep (104 m) and is connected to the open sea through a deep channel (see Fig. A1). L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island Due to the regional bottom topography, depth struc- ture and prevailing wind direction from southwest, currents at the Utö deep may be relatively strong in comparison to typ- ical values in the Baltic Sea. This is visible in, e.g., seabed erosion (unpublished data). During the last decades, sea ice is observed at Utö every few years (Jevrejeva et al., 2004) while summertime cyanobacterial algae blooms are observed almost annually, in July or August (e.g., Kahru et al., 1994; Seppälä et al., 2007). The paper has three aims: (1) to bring the data series dig- itized for this project into the scientiﬁc domain and make them available for other scientists; (2) to describe the obser- vations and typical atmospheric and marine conditions and ranges of variability at Utö Atmospheric and Marine Re- search Station; (3) to study the long-term changes of seawater properties, ice cover and meteorology at Utö. The paper has the following structure. First, we describe the general environmental characteristics of the measurement place. Next, we continue by describing the observations and supporting datasets, data quality-assurance methods and the tools used for statistical analysis of the data. The paper con- tinues with time series of air temperature, cloudiness and seawater temperatures, salinities and densities. The paper is closed with conclusions, some future plans and an appendix with a short description of the current observations and the local bathymetry. pp The Finnish Meteorological Institute started meteorologi- cal observations at Utö already in 1881 and a ﬁxed oceano- graphic station has been in operation since 1900. Atmo- spheric trace gas and aerosol measurements were started on the island in 1980 in the framework of European Monitoring and Evaluation Programme (EMEP) (Ruoho-Airola et al., 2003; Laurila and Hakola, 1996). In 2012, the Finnish Mete- orological Institute (FMI) and Finnish Environment Institute (SYKE) started the construction of a marine research station on the island, leading to a combined Utö Atmospheric and Marine Research Station (Finnish Meteorological Institute, 2017). The list of current observations and the site descrip- tion are given in Appendix A (Table A1 and Fig. A2). Published by Copernicus Publications on behalf of the European Geosciences Union. 618 2 Measurement site and general characteristics The Baltic Sea is a shallow semi-enclosed seasonally ice- covered sea with average depth of 55 m and maximum depth of 459 m (Leppäranta and Myrberg, 2009). In spite of the shallow depth, the vertical stratiﬁcation is strong in summer in shallow areas and throughout the year in areas that are deeper than the mean depth. The upper layer of the sea has a strong seasonal cycle, which is also reﬂected partly to the deeper water. Parts of the Baltic Sea are ice covered every winter, so that the extent of the annual maximum ice cover varies between 50 × 103 and 340 × 103 km2 (Seinä and Palo- suo, 1996; Vainio, 2001) of the total area of 420 × 103 km2. The salinity varies from more than 20 ‰ in Kattegat down to less than 2 ‰ at the ends of the large bays in the north- ern part of the Gulf of Bothnia and Gulf of Finland (Feistel et al., 2010). In deep areas of the Baltic Proper, there is a permanent halocline somewhere between 60 and 80 m depth. Observations at Utö are part of the HELCOM marine monitoring network, Integrated Carbon Observing System (ICOS), Finnish Marine Research Infrastructure (FINMARI) and Joint European Research Infrastructure network for Coastal Observatory – Novel European eXpertise for coastal observaTories (JERICO-NEXT) (Puillat et al., 2016). It is also planned to become a part of European Aerosols, Clouds, and Trace gases Research Infrastructure (ACTRIS). www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 619 Figure 1. Map of the Baltic Sea. The Utö measurement site is in- dicated with a red star. The local bathymetry around Utö Island is shown in Fig. A1 in Appendix A. est measurement has varied but was always more than 80 m. The temperature observations started in 1900 and salinities have been measured since 1911. There were also some salin- ity records from the period 1900–1911, but due to poor data quality, they are excluded from our analysis. The routine observations (until 2003) were done in ﬁxed oceanographic stations in principle with 10-day intervals, on every month’s 1st, 11th and 21st days. Because the obser- vations from small boats are weather dependent, the exact observation days sometimes differ from the scheduled, and also the number of winter observations is smaller than that of the summer observations. 3.1 Observations and other data y Sea ice data were obtained from ice maps done during the ice season for the Gulf of Finland. The ice charts are based on ice observations done in a large number of locations, one of the observing sites being Utö. We used these generalized ice data instead of direct local ice observations from Utö, as there were few periods when we were not able to deter- mine whether the sea ice observations at Utö were missing, or there was simply no ice. For the same reason, we also ex- cluded from our analysis the ice thickness observations made at Utö during the period 1897–2015. The use of ice charts also provided us with better general understanding of the ice situation in the vicinity of Utö. The ice data used in this study are based on 8564 manually analyzed ice maps from the pe- riod 1914–2016. In this study, we focus on long-term changes at Utö during the period 1881–2016. As we do not have all data available for the whole period, and there are gaps in the data, the best coverage for combined data is for the period 1911–2016. Meteorological observations including air temperature, wind speed and direction, cloudiness and sea surface temper- ature have been carried out with developing methods since 1881, initially three times a day and later on with increas- ing time resolution, currently with 10 min logging interval. As the methodology and exact observation locations have changed during this long period, and there are limited meta- data from early measurements, especially the older data con- tain uncertainties. Meteorological data for the period 1881– 1959 used in this study were manually digitized from an- nual written records; data since 1959 are taken from FMI electronic archives. These data were automatically checked from clear outliers and bad data, in addition to the original quality assurance done for all FMI operational meteorologi- cal observations. Our study focuses on air temperatures, but cloudiness and winds are also shortly discussed. NAO data were taken from Jones et al. (1997) with updates from Osborn (2004, 2006, 2011). MBI data were taken from Matthäus et al. (2008), with the latest updates from Mohrholz et al. (2015) and Nauman et al. (2018). River runoff data for the period 1900–2016 are a combi- nation of observations for the period 1900–1995 (Hansson et al., 2011) and modeling for the period 1996–2016 (Jo- hansson, 2018). 2 Measurement site and general characteristics Thus, the results for winter months include higher uncertainties than for the other seasons. There was a gap in the marine observations in 2003– 2012, because of lack of observers at that time period. Ob- servations were started again in 2013, with an RBR XR- 620 conductivity–temperature–depth (CTD) with a RINKO- III dissolved oxygen sensor, measuring temperature, salin- ity and pressure with 0.5 db (∼0.5 m) intervals. The proﬁles have been measured once every 10 days when weather and ice conditions have permitted. These new data are combined with the earlier ﬁxed depth data in order to obtain as long time series as possible. Figure 1. Map of the Baltic Sea. The Utö measurement site is in- dicated with a red star. The local bathymetry around Utö Island is shown in Fig. A1 in Appendix A. Oceanographic proﬁles were visually inspected, speciﬁ- cally for this study, using a code written in MATLAB. All suspicious proﬁles, like those with clearly wrong salinities and/or temperatures or impossible density proﬁles, were re- jected. After the quality check, we had 1520 good-quality full vertical proﬁles of temperature and salinity from the pe- riod 1911–2002 and 112 more proﬁles from the period April 2013–July 2017. www.ocean-sci.net/14/617/2018/ 3.2 Time series analysis methods A trend is a change in the statistical properties of the back- ground state of a system (Chandler and Scott, 2011). The simplest case is a linear trend, in which, when applicable, we need to specify only the trend coefﬁcient and its uncer- tainty. Natural systems evolve continuously over time, and often it is not appropriate to approximate the background evolution with a constant trend. Furthermore, the time se- ries can include multiple time-dependent cycles, and they are typically non-stationary; i.e., their distributional properties change over time. In this work, we applied a dynamic linear model (DLM) approach to time series analysis of multiple meteorological variables measured at Utö Island. Dynamic linear models are regression models whose regression coefﬁcients can depend on time. DLM is a state-space model capable of modeling univariate or multivariate time series also in the presence of non-stationarity, structural changes and irregular patterns. With a properly set up and estimated DLM model, we can detect signiﬁcant changes in the background states and esti- mate the trends. The magnitude of the trend in an individual model is not prescribed by the modeling formulation. This dynamic approach is well known and documented in time series literature (Chatﬁeld, 1989; Harvey, 1991; Hamilton, 1994; Migon et al., 2005). The method is the same one that was already applied in Mikkonen et al. (2015) for the Finnish mean temperature time series. DLM is used to statistically describe the underlying processes that generate variability in the observations. The method effectively decomposes the se- ries into basic components, such as level, trend, seasonality and noise. The components can be allowed to change over time, and the magnitude of this change can be modeled and estimated. The part of the variability that is not explained by the chosen model is assumed to be uncorrelated noise and we can evaluate the validity of this assumption by statistical model residual diagnostics. Figure 2. Average annual air temperatures at Utö during 1881– 2016. The solid line represents temperatures calculated with DLM, and the gray area shows the 95 % conﬁdence range calculated using bootstrap method. The blue line over the DLM curve is a 5-year run- ning mean of atmospheric temperature at Utö. The thin black line with diamonds shows the decadal average temperatures calculated for all of Finland (Mikkonen et al., 2015). variation is included in the model, and separately in different seasons of the year. 3.1 Observations and other data The offset between the two datasets was cor- rected by calculating averages for both datasets for the over- Vertical proﬁles of seawater salinity and temperature have been measured at the Utö deep (59◦46.96′ N, 21◦20.96′ E) at standard depths down to 100 m. The depth of the deep- www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 620 p Figure 2. Average annual air temperatures at Utö during 1881– 2016. The solid line represents temperatures calculated with DLM, and the gray area shows the 95 % conﬁdence range calculated using bootstrap method. The blue line over the DLM curve is a 5-year run- ning mean of atmospheric temperature at Utö. The thin black line with diamonds shows the decadal average temperatures calculated for all of Finland (Mikkonen et al., 2015). lapping period (1950–1995) and correcting the modeled data with the difference. lapping period (1950–1995) and correcting the modeled data with the difference. www.ocean-sci.net/14/617/2018/ 4.1 Long-term changes in atmospheric temperatures Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 00 years of atmospheric and marine observations at Utö Island 621 Figure 3. North Atlantic Oscillation (NAO) during 1880–2016. Panel (a) shows yearly NAO values and panel (b) shows the trend of the NAO, together with 5-year running mean values. Figure 3. North Atlantic Oscillation (NAO) during 1880–2016. Panel (a) shows yearly NAO values and panel (b) shows the trend of the NAO, together with 5-year running mean values. Figure 4. Average seasonal air temperatures at Utö during 1881– 2016. Winter: December, January, February; spring: March, April, May; summer: June, July, August; autumn: September, October, November. As in Fig. 2, the black line represents DLM-calculated Figure 4. Average seasonal air temperatures at Utö during 1881– 2016. Winter: December, January, February; spring: March, April, May; summer: June, July, August; autumn: September, October, November. As in Fig. 2, the black line represents DLM-calculated trend, the gray area represents error for the trend, and the blue line represents the 5-year running average. Figure 4. Average seasonal air temperatures at Utö during 1881– 2016. Winter: December, January, February; spring: March, April, May; summer: June, July, August; autumn: September, October, November. As in Fig. 2, the black line represents DLM-calculated trend, the gray area represents error for the trend, and the blue line represents the 5-year running average. Figure 3. North Atlantic Oscillation (NAO) during 1880–2016. Panel (a) shows yearly NAO values and panel (b) shows the trend of the NAO, together with 5-year running mean values. We also investigated the annual average temperatures against the NAO indices (Fig. 3) (Hänninen et al., 2000; Os- born, 2004, 2006, 2011) and found that on average, lower temperatures are connected to highest negative NAO values, and vice versa (visible also in the 5-year running mean shown in Figs. 2 and 3) (Lehmann et al., 2011). However, we were not able to explain the temperature trend or the longer (> 10- year) periods with higher and lower temperatures with the NAO cycle. Figure 5. Cloud fraction during 1881–2005. In addition to overall temperature trend, it is of interest to look the changes in different seasons (Fig. 4). Using sim- ply 3 calendar months as seasons, we see similar trends in each season as in the annual temperature (Fig. 2). Looking at individual seasons, we notice that the long-term increase in annual temperatures (Fig. 4.1 Long-term changes in atmospheric temperatures 2) results especially from the increase of temperatures during the winter and spring. 4.1 Long-term changes in atmospheric temperatures Figure 2 represents the annual average atmospheric temper- ature at Utö during the period 1881–2016, together with the mean values for all of Finland. For illustrative purposes, we also included 5-year running mean (requiring at least 40 % data coverage) in this and subsequent ﬁgures; however, quan- titative results are based on DLM analyses only. According to DLM analysis, annual average temperature at Utö has in- creased from 6.0 ◦C in 1881 to 7.5 ◦C in 2015. The total in- crease would have been 0.11 ◦C decade−1 if it were linear, which is lower than the average increase (0.14 ◦C decade−1) observed in Finland (Mikkonen et al., 2015). The model provides a method to detect and quantify trends, but it does not directly provide explanations for the observed changes, i.e., whether, for example, natural vari- ability could explain the changes in the background levels. The model construction procedure and equational formula- tion follow closely the ones described in Mikkonen et al. (2015), and the results were calculated with software pack- age DLM for R statistical language described in Petris et al. (2009) and Petris (2010). Conﬁdence limits for the trend es- timates were calculated with the maximum entropy bootstrap for time series method (Hrishikesh and López-de Lacalle, 2009). The variables of interest in this study were air temper- ature, cloudiness, seawater temperature in different depths, water salinity and density. Each variable was inspected in both manners: as total measurement series, where monthly While in Mikkonen et al. (2015) the temperature in- crease follows the pattern in global temperature time se- ries (NASA, 2017), where the warming has taken place in two periods, before the 1940s and after the 1960s, in Utö the temperature increase has taken place only since 1980 without observable trends before that. This leads to an in- crease of 0.4 ◦C decade−1 during the last 35-year period, in line with results reported by Lehmann et al. (2011) and Almén et al. (2017), and the concluding remark of Mikko- nen et al. (2015) stating that within the last 40 years the rate of temperature change in Finland has varied between 0.2 and 0.4 ◦C decade−1. Ocean Sci., 14, 617–632, 2018 www.ocean-sci.net/14/617/2018/ L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island L. www.ocean-sci.net/14/617/2018/ 4.3 Long-term changes in seawater temperatures, salinities and sea ice ﬁler 2 km south of Utö (Fig. A1, location 6); (2) studying the publicly available bathymetric maps (see Fig. A1); and (3) investigating individual vertical salinity proﬁles. Figure 6 shows the monthly median seawater temperatures, salinities and densities in the Utö deep during the period 1911–2016. From the ﬁgure, we see the annual cycle of the water body: strong vertical stratiﬁcation in the summer, with mixed layer depths around 20 m; vertical mixing throughout the whole water body in October; and the seasonal variation of salinity in all depths. Generally, at our site, average sea surface temperature varies between 18 ◦C during the summer and 0 ◦C during the winter while bottom temperature range is from 2 to 5 ◦C. Surface salinities vary between 6 and 7 ‰, water being less saline during the summer and more saline in winter. At the bottom, the situation is opposite, with up to 8 ‰ in summer and around 7 ‰ in winter. Density fol- lows the cycle of salinity. The values for individual years and months may be signiﬁcantly different from these me- dians due to MBIs bringing large amounts of saline water to the Baltic Sea, variation of annual temperatures, wind- driven water transport (Liblik and Lips, 2017), upwelling (Myrberg and Andrejev, 2003), river runoff and existence of sea ice cover. In contrast to the Baltic Proper, we do not see a permanent halocline between 60 and 80 m depth (Lep- päranta and Myrberg, 2009) in the average ﬁgure shown here. We investigated the reasons for this behavior by (1) com- paring vertical salinity proﬁles at Utö with those observed at point LL15 (59◦10.99′ N, 021◦44.80′ E; approximately 70 km south–southeast (SSE) of Utö, max depth 130 m) and a few CTD proﬁles taken next to the new cabled bottom pro- Based on this analysis, we observed the following: (1) salinity proﬁles at Utö and LL15 correlate between the surface and approximately 60 m depth, but the salinities be- low 60 m are higher at point LL15; (2) the bathymetric map (see Fig. A1) shows two potential sills with depth of approx- imately 60 m in the channel between the observing site and open sea; (3) while the halocline between 60 and 80 m is not seen continuously, it is sometimes observed for shorter, few- month periods. 4.2 Cloudiness and wind The quality assurance for atmospheric temperatures is rela- tively easy. For cloudiness and especially wind, the situation is more complicated. We analyzed the changes in cloudiness for the period 1881–2005 for which we had visual (manual) observations available. After October 2005, the cloud obser- vations have been done with a ceilometer, and the results are not comparable with the previous data. Figure 5 shows the time series of cloudiness on a scale from 0 to 8. We see in- crease until 1990 and after that a decrease until the end of our visual (manual) observations, 2005. Automated observa- tions (not shown) since 2005 show again an increasing trend in cloudiness. Further investigations focusing on reasons be- hind the changes in cloudiness are, however, out of the scope of this paper. Figure 5. Cloud fraction during 1881–2005. We also looked at wind time series (period 1959–2016) and found no signiﬁcant changes in wind direction nor wind speed, in line with a recent study for the period 1979–2008 (Laapas and Venäläinen, 2017). However, because the wind observations are very sensitive to inhomogeneities in meth- ods and location (Pryor et al., 2009; Wan et al., 2010; Feser et al., 2015; Laapas and Venäläinen, 2017), more analyses for observations done prior to 1959 are needed before further use of this part of the dataset. Ocean Sci., 14, 617–632, 2018 www.ocean-sci.net/14/617/2018/ www.ocean-sci.net/14/617/2018/ L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 622 Figure 6. Monthly median seawater temperature, salinity and density in Utö during 1911–2016. Figure 6. Monthly median seawater temperature, salinity and density in Utö during 1911–2016. Figure 6. Monthly median seawater temperature, salinity and density in Utö during 1911–2016. www.ocean-sci.net/14/617/2018/ L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 623 Figure 7. Average seawater temperatures in Utö at depths of −5, −50 and −90 m. For better visualization, we have used a combina- tion of lines in black and blue for −5 m, red and magenta for −50 m, and blue and black for −90 m, as shown in the legend. Figure 8. Average seawater salinities in Utö at −5, −50 and −90 m depths. For details, see Fig. 7. Figure 7. Average seawater temperatures in Utö at depths of −5, −50 and −90 m. For better visualization, we have used a combina- tion of lines in black and blue for −5 m, red and magenta for −50 m, and blue and black for −90 m, as shown in the legend. Figure 8. Average seawater salinities in Utö at −5, −50 and −90 m depths. For details, see Fig. 7. Figure 8. Average seawater salinities in Utö at −5, −50 and −90 m depths. For details, see Fig. 7. Figure 7. Average seawater temperatures in Utö at depths of −5, −50 and −90 m. For better visualization, we have used a combina- tion of lines in black and blue for −5 m, red and magenta for −50 m, and blue and black for −90 m, as shown in the legend. Figure 8. Average seawater salinities in Utö at −5, −50 and −90 m depths. For details, see Fig. 7. Figure 9. Average seawater densities in Utö at −5, −50 and −90 m depths. For details, see Fig. 7. vestigate 5 and 90 m depths was clear, selection of 50 m was also supported by visual analysis of the temperature, salinity and density proﬁles: we inspected all proﬁles visually and found that during the summer months (June, July, August) thermocline was almost always above the 50 m depth. Figure 7 represents the trends in water temperatures at these three depths. We see that the surface temperature fol- lows the behavior of atmospheric temperatures (Fig. 2), with a rapid increase since the 1980s and a warmer period from the 1930s until the 1960s. The overall increase has been ap- proximately 0.3 ◦C decade−1 during the last 100 years. For deeper layers, we observe partly different trends, with a faster increase in temperatures in the 1980s and a drop or hiatus during the last few years. 4.3 Long-term changes in seawater temperatures, salinities and sea ice Based on these observations, we may conclude that the statement by Ahlnäs (1961), “The trench-like gully opens into the open sea in the south and the deep samples may be taken to reﬂect the characteristics of the corresponding wa- ter layers in the Northern Baltic” may not fully capture all dynamic aspects of the observing site. Exact measurement depths and the number of different ob- servation depths have varied during the last 100 years. We decided to focus on three different depths where we have the most data, while the depths have also physical meaning: 5 m represents the sea surface layer which is quite directly inﬂu- enced by the atmosphere but is most probably not inﬂuenced by measurement errors, 50 m depth which is at the old winter water layer that is not directly inﬂuenced by the surface pro- cesses in summer and that is also the middle point between the surface and bottom, and 90 m which is the closest point to the bottom, with high data coverage. While the decision to in- www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island As the main heating to the seawa- ter comes from the surface of the sea, the higher increase of deep water temperatures during the 1980s and 1990s, and re- cent decrease have to be inﬂuenced by other phenomena than simply the increasing atmospheric temperatures. Figure 9. Average seawater densities in Utö at −5, −50 and −90 m depths. For details, see Fig. 7. The seawater temperatures and salinities combine in Fig. 9 as seawater density. As the salinity is a key factor (in layers where temperature variations are small) inﬂuencing seawater density, the density curves follow the changes in salinity. Fig- ure 9 shows smaller vertical density gradients in the 1980s and 1990s than earlier and increase of density stratiﬁcation during the last few years. Figure 8 shows the changes in salinity at different depths. We see that the salinity has varied signiﬁcantly during the observing period but there is no general trend in the data. If we compare our salinity data (50 and 90 m depths) with those reported for 200 m depth in the Gotland Deep (BY15) by Fonselius and Valderrama (2003), we see that the periods of maximum and minimum salinities are correlated between the two sites. These changes, we assume, are responsible for the in- creased water temperatures at 50 and 90 m depths during the period 1980–2000 and the recent decrease since 2012, and are the explanation for why there is a difference between the behavior (slope) of surface water being directly in contact with the atmosphere and the deeper water layers during this period (1980–2000). In the salinity stratiﬁcation, we see the following changes: the stratiﬁcation was strongest in the 1950s and weakest in the 1980s and 1990s during the stagnation period when no major Baltic inﬂows occurred. The stratiﬁcation increased again since 2013. The observations at Utö are insufﬁcient to explain directly the reasons for changes in the salinity stratiﬁcation. However, www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 624 624 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island Figure 10. MBIs during 1880–2016 (a, b). There are no data available for the World War periods of 1915–1920 (WWI) and 1940–1946 (WWII). L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island Panel (c) shows the total river runoff to the Baltic Sea during the period 1900–2016. The unit is percent relative to the period average. Please see the text for details on data sources. Figure 10. MBIs during 1880–2016 (a, b). There are no data available for the World War periods of 1915–1920 (WWI) and 1940–1946 (WWII). Panel (c) shows the total river runoff to the Baltic Sea during the period 1900–2016. The unit is percent relative to the period average. Please see the text for details on data sources. Figure 11. Wintertime sea ice cover at Utö. Panel (a) shows the yearly duration of the ice cover and panel (b) shows the DLM trend and 5-year moving averages of the length of the ice season. the frequency and strength of major Baltic inﬂows (Feistel et al., 2008, update based on Nauman et al., 2018), shown in Fig. 10, clearly explain that the changes are related to major Baltic inﬂows since the 1980s. There have not been similar recent changes in the river runoff (Fig. 10c) able to explain the observed salinity increase. We also see a rapid increase in salinities in the 1940s. While there are no MBI data due to WWII (Matthäus et al., 2008), the study by Ahlnäs (1961) supports strong MBIs during that period, combined with reduced river discharge (Hansson et al., 2011). Utö is at the border between the open Baltic Proper and the Archipelago Sea. The sea ice cover (Fig. 11) has thus varied greatly from year to year. We see that the average duration of sea ice cover has decreased by 50 % since 1980, in line with the increased average temperature (Fig. 2) and previous studies (Jevrejeva et al., 2004; Merkouriadi and Leppäranta, 2014). This decrease in ice cover may have enhanced the re- cent rapid increase in air temperatures at Utö since the open sea is a large source of latent heat, which leads to higher at- mospheric temperatures than when the sea is ice covered. Ice cover also increases albedo, which may have inﬂuenced the surface temperatures during the spring. Figure 11. Wintertime sea ice cover at Utö. Panel (a) shows the yearly duration of the ice cover and panel (b) shows the DLM trend and 5-year moving averages of the length of the ice season. ceeds 5 ◦C has changed to 5.4 days earlier from the previous periods to 1981–2010. 5 Conclusions In this study, we used an approximately 100-year long time series of atmospheric and marine observations carried out at Utö. The focus was on long-term changes and potential im- pacts of warming climate to the Baltic Sea hydrography. In an earlier study by Mikkonen et al. (2015), a clear increase of at- mospheric temperatures was observed throughout continen- tal Finland. In the sea areas, however, changes are dampened by the large heat capacity of the sea. In winter, the sea ice inﬂuences albedo, along with sensible and latent heat ﬂuxes between the sea and the atmosphere. In our study, we saw an increase in the atmospheric and sea (surface) water temperatures only since the 1980s, which is different from the Finnish average air temperature increase observed throughout the 20th century. As the increase ob- served at Utö is mostly due to the warmer spring and winter months, we assume that the impact of warming climate is visible especially after the reduction of wintertime sea ice cover. Figure 12. Monthly average air temperatures (a) and sea surface water temperatures (b) at Utö during the four 30-year reference pe- riods. We also found that there was a clear reduction in the verti- cal stability of the water column during the so-called stagna- tion period (1980–2010), when there were less major Baltic inﬂows than before. This enhanced mixing, together with in- creased air temperature may have been responsible for the increased deep water temperatures during this period. The latest observations since 2013 show again an increase in ver- tical stratiﬁcation due to recent MBIs, which have increased the bottom salinities and decreased the temperatures, we as- sume, due to reduced vertical mixing. riods are given in Table 1. The air temperature averages for all periods are calculated based on three observations per day, which was the initial observing frequency. Based on later high time resolution data, we estimated that the average air temperature values given in Table 1 are approximately 0.17 ◦C larger than the true average values using high time resolution data, and correspondingly, the cloud fraction val- ues are 0.14 larger than the true averages. The data show clearly the high natural variability of en- vironmental variables. However, these averages calculated stepwise for ﬁxed 30-year climate periods also hide the rapid change which has taken place especially in temperatures since 1980, which is clearly visible in ﬁgures showing the trends. L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island As the increase ob- served at Utö is mostly due to the warmer spring and winter months, we assume that the impact of warming climate is visible especially after the reduction of wintertime sea ice cover. 1891–1920 1921–1950 1951–1980 1981–2010 Air temperature (◦C) 5.77 (7.56) 6.15 (7.91) 5.99 (7.73) 6.72 (7.46) Cloud fraction (0 to 8) 4.70 (5.75) 5.20 (3.25) 5.32 (2.97) 5.46 (2.70) Seawater temperature (◦C) −5 m – 7.95 (5.67) 7.73 (5.52) 8.12 (5.49) −50 m – 3.76 (2.04) 3.63 (1.90) 4.31 (2.52) −90 m – 3.36 (1.61) 3.18 (1.39) 3.95 (2.06) Seawater salinity (‰) −5 m – 6.40 (0.34) 6.72 (0.29) 6.46 (0.30) −50 m – 7.01 (0.50) 7.44 (0.54) 6.87 (0.37) −90 m – 7.31 (0.69) 7.75 (0.66) 6.97 (0.38) Sea ice cover duration Days – 31.8 (36.5) 37.5 (34.7) 27.8 (33.0) 1891–1920 1 Air temperature (◦C) 5.77 (7.56) 6 Cloud fraction (0 to 8) 4.70 (5.75) 5 Seawater temperature (◦C) −5 m – 7 −50 m – 3 −90 m – 3 Seawater salinity (‰) −5 m – 6 −50 m – 7 −90 m – 7 Sea ice cover duration Days – 3 Figure 12. Monthly average air temperatures (a) and sea surface water temperatures (b) at Utö during the four 30-year reference pe- riods. 5 C In th serie Utö. pacts earlie mosp tal Fi by th inﬂu betw In (surf diffe obser serve mont visib cove Figure 12. Monthly average air temperatures (a) and sea surface water temperatures (b) at Utö during the four 30-year reference pe- riods. L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 625 Table 1. Average values with standard deviations for the 30-year periods of 1891–1920, 1921–1950, 1951–1980 and 1981–2010. As there have been gaps in observations, the uncertainties between variables and periods vary. Due to the limited amount of hydrographic and sea ice data during the ﬁrst period (1891–1920), only values for air temperature and cloud fraction are shown. Table 1. Average values with standard deviations for the 30-year periods of 1891–1920, 1921–1950, 1951–1980 and 1981–2010. As there have been gaps in observations, the uncertainties between variables and periods vary. Due to the limited amount of hydrographic and sea ice data during the ﬁrst period (1891–1920), only values for air temperature and cloud fraction are shown. 1891–1920 1921–1950 1951–1980 1981–2010 Air temperature (◦C) 5.77 (7.56) 6.15 (7.91) 5.99 (7.73) 6.72 (7.46) Cloud fraction (0 to 8) 4.70 (5.75) 5.20 (3.25) 5.32 (2.97) 5.46 (2.70) Seawater temperature (◦C) −5 m – 7.95 (5.67) 7.73 (5.52) 8.12 (5.49) −50 m – 3.76 (2.04) 3.63 (1.90) 4.31 (2.52) −90 m – 3.36 (1.61) 3.18 (1.39) 3.95 (2.06) Seawater salinity (‰) −5 m – 6.40 (0.34) 6.72 (0.29) 6.46 (0.30) −50 m – 7.01 (0.50) 7.44 (0.54) 6.87 (0.37) −90 m – 7.31 (0.69) 7.75 (0.66) 6.97 (0.38) Sea ice cover duration Days – 31.8 (36.5) 37.5 (34.7) 27.8 (33.0) Figure 12. Monthly average air temperatures (a) and sea surface water temperatures (b) at Utö during the four 30-year reference pe- riods. 5 Conclusions In this study, we used an approximately 100-year long time series of atmospheric and marine observations carried out at Utö. The focus was on long-term changes and potential im- pacts of warming climate to the Baltic Sea hydrography. In an earlier study by Mikkonen et al. (2015), a clear increase of at- mospheric temperatures was observed throughout continen- tal Finland. In the sea areas, however, changes are dampened by the large heat capacity of the sea. In winter, the sea ice inﬂuences albedo, along with sensible and latent heat ﬂuxes between the sea and the atmosphere. In our study, we saw an increase in the atmospheric and sea (surface) water temperatures only since the 1980s, which is different from the Finnish average air temperature increase observed throughout the 20th century. L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island The date when seawater temperature at 5 m depth exceeds 4 ◦C has changed even to 8.8 days ear- lier from the period 1951–1980 (and 1921–1950) to 1981– 2010. Finally, we calculated average monthly air and sea surface temperatures at Utö for four different 30-year reference pe- riods, 1891–1920, 1921–1950, 1951–1980 and 1981–2010 (Fig. 12). The averages show the recent warming of winters and springs with a small increase in springtime seawater tem- perature. In time, the date when average air temperature ex- Average air temperatures, cloud fractions, seawater tem- peratures and salinities and durations of sea ice cover, to- gether with standard deviations for the different 30-year pe- Ocean Sci., 14, 617–632, 2018 www.ocean-sci.net/14/617/2018/ www.ocean-sci.net/14/617/2018/ www.ocean-sci.net/14/617/2018/ 5 Conclusions Our results are in line with previous studies on climate and hydrographic changes on the northern Baltic Sea region. In the future, our aim is to continue the analyses of this dataset with other methods, and studies focusing more on individual changes and processes. The data and analysis represented in this study also form a solid base for detailed process and biogeochemical stud- www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 626 Together with our new observations, the long data series represented in this paper will support better understanding of both the earlier observations and current, ongoing physical, chemical and biological changes in the Baltic Sea. ies which are an integral part of the JERICO-NEXT concept of integrated coastal observatories (Puillat et al., 2016). An interesting study utilizing the time series presented in this paper together with the new observations will be to use the new cabled bottom proﬁler together with an acoustic Doppler current proﬁler (ADCP) (Figs. A1 and A2) to study the occa- sional inﬂows of saline bottom water which may have signif- icant impacts on the Archipelago Sea ecosystem (Vuorinen et al., 2015). Another planned study combining hydrographic observations with biogeochemistry and climate change is to use the proﬁler together with the ﬂow-through system to an- alyze the thickness of biologically active layer and its con- nection to the marine carbon cycle. Data availability. Meteorological data digitized and used in this article are available through the Finnish Meteorological Institute open data portal (https://en.ilmatieteenlaitos.ﬁ/open-data, last ac- cess: 9 July 2018). Hydrographic data used in this study are avail- able through SeaDataNet (https://www.seadatanet.org/, last access: 9 July 2018). Please cite this article when using the data. Ocean Sci., 14, 617–632, 2018 www.ocean-sci.net/14/617/2018/ L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 627 Appendix A: Observations at Utö Atmospheric and Marine Research Station Table A1. Continuous atmospheric and marine observation at Utö. Site refers to numbers and coordinates in Fig. A1. Variable Site Start (year) Reference Meteorological T , p, WS, WD, RH 5 1881 Laapas and Venäläinen (2017) observations Precipitation, cloudiness 5 1881 Global, diffuse and UV radiation 3 1998 Riihelä et al. (2015) Visibility 5 2002 Cloud cover and height 5 2006 3-D wind proﬁle (Doppler lidar) 5 2012 Hirsikko et al. (2014), Tuononen et al. (2017) Weather camera 4 2014 Aerosol and trace Aerosol mass (PM10) 5 1980 Ruoho-Airola et al. (2003) SO2 5 1980 Ruoho-Airola and Salmi (2001) Aerosol chemical composition (PM10) 5 1980 Ruoho-Airola et al. (2003) NOx, O3 5 1986 Engler et al. (2007) Aerosol mass (PM2.5) 5 2003 Aerosol size distribution 5 2004 Engler et al. (2007), Hyvärinen et al. (2008) Aerosol absorption 5 2007 Hyvärinen et al. (2011) Aerosol scattering 5 2010 Hyvärinen et al. L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island (2011) Aerosol chemical composition (PM2.5) 5 2011 Phosphorus deposition 5 2014 Makkonen et al. (2015) Radon 5 2015 Vesterbacka (2017) Atmospheric greenhouse CO2, CH2 and CO gas concentrations 3 2012 Kilkki et al. (2015) measurements CO2 ﬂux 2 2012 Honkanen et al. (2018) Marine observations Sea ice observations 1 1897 Seinä and Palosuo (1996) Temperature and salinity proﬁles (0 to −90 m) 1 1900 Ahlnäs (1961) Nutrient and chlorophyll proﬁles (0 to −70 m) 1 2001 Suomela (2003) Sea ice radar 4 2013 Temperature, salinity, O2, turbidity, 2 2014 chlorophyll (−5 m) Currents (0 to −23 m) and surface waves 2 2014 Haavisto (2015) Automatic Identiﬁcation System (AIS) 2 2015 Bottle sampler 2 2015 Spectrometric observations of phytoplankton 2 2016 pCO2 2 2016 Honkanen et al. (2018) pH, DIC 2 2016 Cabled bottom proﬁler (−5 to −70 m) 6 2018 Temperature, salinity, O2, turbidity, ﬂuorescence (three wavelengths) Currents (0 to −75 m) and surface waves 6 2018 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island Appendix A: Observations at Utö Atmospheric and Marine Research Station t al.: 100 years of atmospheric and marine observations at Utö Island 627 Table A1. Continuous atmospheric and marine observation at Utö. Site refers to numbers and coordinates in Fig. A1. Ocean Sci., 14, 617–632, 2018 www.ocean-sci.net/14/617/2018/ www.ocean-sci.net/14/617/2018/ L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 628 lose to Utö Island. 1: hydrographic observations (59◦46.96′ N, 21◦20.96′ E until 200 research station (59◦46.90′ N, 21◦21.45′ E); 3: greenhouse gases (GHGs) and sola ◦46.84′ N, 21◦22.13′ E); 5: meteorological and air quality station (59◦46.76′ N, 21◦22 .41′ N, 21◦22.13′ E); red line: 50 m contour; black arrows: possible sills (60 m depth). M 2, 2018 www.ocea Figure A1. Bathymetry close to Utö Island. 1: hydrographic observations (59◦46.96′ N, 21◦20.96′ E until 2002; currently 59◦46.97′ N, 21◦20.78′ E); 2: marine research station (59◦46.90′ N, 21◦21.45′ E); 3: greenhouse gases (GHGs) and solar radiation (59◦47.12′ N, 21◦22.04′ E); 4: radar (59◦46.84′ N, 21◦22.13′ E); 5: meteorological and air quality station (59◦46.76′ N, 21◦22.51′ E); 6: cabled bottom proﬁler and ADCP (59◦45.41′ N, 21◦22.13′ E); red line: 50 m contour; black arrows: possible sills (60 m depth). Map source: National Land Survey of Finland (2018). www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 L. www.ocean-sci.net/14/617/2018/ Ocean Sci., 14, 617–632, 2018 L. Laakso et al.: 100 years of atmospheric and marine observations at Utö Island 630 Author contributions. LL was behind the idea of the paper, did most of the writing, drew all the ﬁgures except the map (Fig. 1) and did most of the data QC and analyses. SM did all statistical analy- ses, wrote Sect. 3.2 and participated in writing throughout the paper. PA provided the hydrographic data, supported in the QC process and contributed signiﬁcantly to the introduction and interpretation of the results. AD and PP were responsible for the meteorological data used in the paper and their quality assurance. AK analyzed and digitized the ice map data used in the paper. All authors commented and improved the paper during the writing process. Engler, C., Lihavainen, H., Komppula, M., Kerminen, V.-M., Kul- mala, M., and Viisanen, Y.: Continuous measurements of aerosol properties at the Baltic Sea, Tellus, 59B, 728–741, 2007. Feistel, R., Nausch, G., and Wasmund, N. (Eds.): State and evolu- tion of the Baltic Sea, 1952–2005: a detailed 50-year survey of meteorology and climate, physics, chemistry, biology, and ma- rine environment, John Wiley & Sons, Hoboken, USA, 703 pp. + Digital supplement 1 cd-rom, 2008. 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https://openalex.org/W2999487987	https://concept.mgimo.ru/jour/article/download/299/265	Russian	null	SECTION «INTERCULTURAL COMMUNICATION» AT THE RAMI MGIMO-75 CONVENTION	Koncept: filosofiâ, religiâ, kulʹtura	2,020	cc-by	772	* В.И. Коннов – к.социол.н., доцент кафедры философии МГИМО МИД России. ** М.В. Силантьева – д.философ.н., заведующая кафедрой философии МГИМО МИД Рос сии. НАУЧНАЯ ЖИЗНЬ НАУЧНАЯ ЖИЗНЬ DOI: 10.24833/2541-8831-2019-4-12-204-205 СЕКЦИЯ «МЕЖКУЛЬТУРНАЯ КОММУНИКАЦИЯ» НА КОНВЕНТЕ РАМИ МГИМО-75 В.И. Коннов, М.В. Силантьева* Московский государственный институт международных отношений (университет) МИД России. 119454, Москва, проспект Вернадского, 76. 22 октября 2019 г. в рамках XII Кон вента РАМИ, проводимо го в год 75-летия МГИМО работала организованная кафедрой философии им. А.Ф. Шишкина секция «Межкультурная комму никация». Она представ лена на мероприятиях РАМИ с 2010 г. и неизмен но пользуется интересом как со стороны предста вителей МГИМО, так и учёных других вузов. это удалось: охватывая культурное про странство от обеих Америк до Японии и от Испании до Ирана, Турции и Китая, российские и зарубежные исследовате ли поделились своими наработками в изучении проблемы культуры, так или иначе связанных с международными от ношениями. это удалось: охватывая культурное про странство от обеих Америк до Японии и от Испании до Ирана, Турции и Китая, российские и зарубежные исследовате ли поделились своими наработками в изучении проблемы культуры, так или иначе связанных с международными от ношениями. 22 В этом году лидерство было явно за регионоведческой тематикой, причём на первый план вышли исследования, посвящённые Китаю и Центральноази атскому региону, иберийским и ибероа мериканским культурам, Ирану (здесь особый интерес вызвало выступление Лейлы Хоссейни), Скандинавии (отдель ного внимания заслуживают выступле ния С.Ю. Дианиной и Д.А. Талагаевой). Коммуникационные процессы в Европе (Италия, Португалия, Британия, Ирлан дия, Польша и т.д.) затрагивали в основ ном двусторонние и трёхсторонние про цессы межкультурного взаимодействия. Культура в определён ном смысле – это «обо всём и сразу». Не случайно современные дискуссии о ней заострены на вопросе о «культурном раз нообразии». Именно разнообразие куль туры и есть её специфика, тем ценнее возможность услышать и обсудить раз ные аспекты этого жизненного явления, в первую очередь коммуникативные. И нам кажется, что на площадке МГИМО Как всегда, в структуре проблематики, представленной на обсуждение в рамках 204 В.И. Коннов, М.В. Силантьева Самары и ряда других городов России, а также Ирана и Турции. МГИМО был представлен специалистами кафедры мировой литературы и культуры, язы ковых кафедр, кафедры сравнитель ной политологии, кафедры филосо фии и др. От кафедры философии были представлены доклады В.С. Глаголева, М.В. Силантьевой, А.В. Шестопала и Н.Ф. Желудовой, Н.И. Бирюкова, В.И. Кон нова, В.В. Печатнова, Р.Ф. Додельцева, Н.В. Литвака, С.Н. Лютовой, С.М. Мед ведевой, Т.В. Панфиловой, В.П. Терина, Д.Н. Беловой и И.А. Чупровой, а также Д.Г. Горина и В.В. Сухомлиновой. СЕКЦИЯ «МЕЖКУЛЬТУРНАЯ КОММУНИКАЦИЯ» НА КОНВЕНТЕ РАМИ МГИМО-75 секции, можно выделить различные бло ки, от исследований в области искусства (где анализировались актуальные тен денции и культурные контексты музы ки, живописи (включая книжную иллю страцию), поэзии, кинематографа и даже экспериментального театра) до лингво культурологических исследований. секции, можно выделить различные бло ки, от исследований в области искусства (где анализировались актуальные тен денции и культурные контексты музы ки, живописи (включая книжную иллю страцию), поэзии, кинематографа и даже экспериментального театра) до лингво культурологических исследований. Стоит подчеркнуть, что последова тельный рост числа докладов в области пересечения лингвистики, филологии и культурологии, выполненных на вы соком теоретическом уровне (то есть с привлечением глубоко философских идей и концептов) показывает востребо ванность секции со стороны расширяю щих свою научно-исследовательскую работу сотрудников языковых кафедр МГИМО, как, впрочем, и специалистов- лингвокультурологов других вузов. Ценной особенностью ставшего еже годным конвента РАМИ является воз можность обмена исследовательским опытом в широком кругу специалистов. В качестве отдельного пункта, делающе го секцию привлекательной для боль шого количества участников (их прибы ло более 70 человек), следует выделить возможность посмотреть на мир глазами другого, создав фундамент не только для борьбы интерпретаций, но и для глубо кого и безусловно продуктивного меж культурного диалога, формирующего поле совместного продвижения в изуче нии актуальной, теоретически значимой и практически востребованной пробле матики. Среди наиболее обсуждаемых тем, вызвавших оживленный интерес ауди тории, стали: экспансия лингвокультуры Испании; эволюция элементов русского, английского, французского, голландско го, абхазского и других языков, особен ности политического и экономического дискурса в сравнительной методологии и многое другое. Тематическое поле дис куссий, разумеется, не могло обойтись без обсуждения вопросов философии культуры, науковедения и религиоведе ния, медийного и в целом виртуального пространства и специфики коммуника ции в данных областях. И благодаря высокой квалифика ции участников Конвента этот диалог к тому же позволяет уточнять и коррек тировать методологические и теорети ческие подходы в рамках конкретных исследований, открывает новые науч ные горизонты как в изучении «старых» наболевших проблем межкультурной коммуникации, так и в освоении прин ципиально новых тем – таких например, как цифровизация и искусственный ин теллект. В рамках секции прозвучали сообще ния, посвящённые анализу теоретиче ских моделей межкультурной комму никации, проблем научной политики и научной дипломатии, вопросам форми рования и развития религиозных кон фессий. В работе секции приняли участие го сти из вузов Москвы, Санкт-Петербурга, 205
https://openalex.org/W220503443	https://europepmc.org/articles/pmc3793720?pdf=render	English	null	5-Fluoro-<i>N</i>′-(4-methylcyclohexylidene)-3-phenyl-1<i>H</i>-indole-2-carbohydrazide	Acta crystallographica. Section E	2,013	cc-by	6,145	Table 1 Hydrogen-bond geometry (A˚ , ). Received 1 July 2013; accepted 3 July 2013 Cg1, Cg2, Cg3, Cg6 and Cg8 are the centroids of the 1H-pyrrole and benzene rings of the 1H-indole ring system of molecule A, the phenyl ring of molecule A, the 1H-pyrrole ring of the 1H-indole ring system of molecule B and the phenyl ring of molecule B, respectively. Key indicators: single-crystal X-ray study; T = 296 K; mean (C–C) = 0.003 A˚; R factor = 0.057; wR factor = 0.144; data-to-parameter ratio = 17.5. D—H A D—H H A D A D—H A N1—H1 O2 0.86 2.15 2.895 (2) 145 N4—H4 O1 0.86 2.04 2.811 (2) 149 C17—H17A Cg1i 0.97 2.66 3.594 (3) 163 C17—H17B Cg3 0.97 2.74 3.685 (3) 164 C31—H31 Cg6ii 0.93 2.87 3.658 (2) 144 C35—H35 Cg2iii 0.93 2.96 3.627 (3) 130 C39—H39B Cg8 0.97 2.72 3.667 (3) 164 C42—H42A Cg1iv 0.97 2.99 3.848 (3) 148 Symmetry codes: (i) x þ 1; y þ 1; z; (ii) x þ 1; y þ 2; z þ 1; (iii) x þ 1; y þ 1; z þ 1; (iv) x 1; y; z. The title compound, C22H22FN3O, crystallized with two independent molecules (A and B) in the asymmetric unit; these are linked by a pair of N—H O hydrogen bonds, forming a pseudo-centrosymmetric dimer with an R2 2(10) motif. In addition, a number of C—H interactions are also observed. The 1H-indole ring systems in molecules A and B are essentially planar [maximum deviations of 0.019 (2) and 0.014 (2) A˚ , respectively] and make dihedral angles of 77.64 (10) and 69.50 (9), respectively, with thephenyl rings. Data collection: X-AREA (Stoe & Cie, 2002); cell reﬁnement: X- AREA; data reduction: X-RED32 (Stoe & Cie, 2002); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 2012); software used to prepare material for publication: WinGX (Farrugia, 2012). 5-Fluoro-N000-(4-methylcyclohexylidene)- 3-phenyl-1H-indole-2-carbohydrazide Reﬁnement R[F 2 > 2(F 2)] = 0.057 wR(F 2) = 0.144 S = 1.03 8702 reﬂections 496 parameters 2 restraints Related literature For the synthesis and characterization of some bioactive indole derivatives, see: Akkurt et al. (2010, 2013); Cihan- U¨ stu¨ndag˘ & C¸ apan (2012); Zhang et al. (2004). For puckering analysis, see: Cremer & Pople (1975). For the graph-set analysis of hydrogen bonding, see: Bernstein et al. (1995). The authors acknowledge the Faculty of Arts and Sciences, Ondokuz Mayıs University, Turkey, for the use of the Stoe IPDS 2 diffractometer (purchased under grant F.279 of the University Research Fund). This work was supported by the Scientiﬁc Research Projects Coordination Unit of I˙stanbul University (project No. T-471/25062004) Experimental Crystal data C22H22FN3O Mr = 363.43 Triclinic, P1 a = 11.6630 (6) A˚ b = 13.5320 (7) A˚ c = 14.7754 (8) A˚ = 112.967 (4) = 95.936 (4) Acta Cryst. (2013). E69, o1211–o1212 doi:10 Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: SJ5341). organic compounds = 0.09 mm1 T = 296 K 0.68 0.52 0.33 mm = 111.385 (4) V = 1915.4 (2) A˚ 3 Z = 4 Mo K radiation Data collection Stoe IPDS 2 diffractometer Absorption correction: integration (X-RED32; Stoe & Cie, 2002) Tmin = 0.948, Tmax = 0.972 Acta Crystallographica Section E Acta Crystallographica Section E Structure Reports Online Acta Crystallographica Section E St t R t Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 ISSN 1600-5368 Data collection Stoe IPDS 2 diffractometer Absorption correction: integration (X-RED32; Stoe & Cie, 2002) Tmin = 0.948, Tmax = 0.972 26097 measured reﬂections 8702 independent reﬂections 5714 reﬂections with I > 2(I) Rint = 0.058 26097 measured reﬂections 8702 independent reﬂections 5714 reﬂections with I > 2(I) Rint = 0.058 Sevim Tu¨rktekin C¸elikesir,a Mehmet Akkurt,a* Go¨kc¸e Cihan U¨stu¨ndag˘,b Gu¨ltaze C¸apanb and Orhan Bu¨yu¨kgu¨ngo¨rc H atoms treated by a mixture of independent and constrained reﬁnement max = 0.42 e A˚ 3 min = 0.37 e A˚ 3 aDepartment of Physics, Faculty of Sciences, Erciyes University, 38039 Kayseri, Turkey, bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, 34116 Beyazit, Istanbul, Turkey, and cDepartment of Physics, Faculty of Arts and Sciences, Ondokuz Mayıs University, 55139 Samsun, Turkey Correspondence e-mail: akkurt@erciyes.edu.tr Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: SJ5341). Zhang, H. Z., Drewe, J., Tseng, B., Kasibhatla, S. & Cai, S. X. (2004). Bioorg. Med. Chem. 12, 3649–3655. References Akkurt, M., C¸ elik, I´., Cihan, G., C¸ apan, G. & Bu¨yu¨kgu¨ngo¨r, O. (2010). Acta Cryst. E66, o830. Akkurt, M., Zopun, M., C¸ apan, G. & Bu¨yu¨kgu¨ngo¨r, O. (2013). Acta Cryst. E69, o1137. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. Cihan-U¨ stu¨ndag˘, G. & C¸ apan, G. (2012). Mol. Divers. 16, 525–539. Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1358. Akkurt, M., C¸ elik, I´., Cihan, G., C¸ apan, G. & Bu¨yu¨kgu¨ngo¨r, O. (2010). Acta Cryst. E66, o830. Akkurt, M., Zopun, M., C¸ apan, G. & Bu¨yu¨kgu¨ngo¨r, O. (2013). Acta Cryst. E69, o1137. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. Cihan-U¨ stu¨ndag˘, G. & C¸ apan, G. (2012). Mol. Divers. 16, 525–539. Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1358. Experimental Crystal data Crystal data b = 13.5320 (7) A˚ c = 14.7754 (8) A˚ = 112.967 (4) = 95.936 (4) g Cihan-U¨ stu¨ndag˘, G. & C¸ apan, G. (2012). Mol. Divers. 16, 525–539. Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1358. g Cihan-U¨ stu¨ndag˘, G. & C¸ apan, G. (2012). Mol. Divers. 16, 525–539. Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1358. C¸elikesir et al. o1211 Acta Cryst. (2013). E69, o1211–o1212 o1211 o1211 C¸elikesir et al. o1 doi:10.1107/S1600536813018436 Farrugia, L. J. (2012). J. Appl. Cryst. 45, 849–854. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Stoe & Cie (2002). X-AREA and X-RED32. Stoe & Cie, Darmstadt, Germany. Acta Cryst. (2013). E69, o1211–o1212 , ( ) y , Stoe & Cie (2002). X-AREA and X-RED32. Stoe & Cie, Darmstadt, Germany. Experimental A mixture of 5-fluoro-3-phenyl-1H-indole-2-carbohydrazide (0.005 mol) and 4-methyl cyclohexanone (0.007 mol) was refluxed in 15 ml ABS. ethanol for 5 h. The precipitate obtained was purified by recrystallization from an ethanol-water mixture. Yield:73%, mp.: 466.5–468.5 K. IR(KBr): υmax 3348, 3240 (N—H), 1652 (C=O) cm-1. 1H-NMR (DMSO-d6/500 MHz): δ 0.87 (d, 3H, J=5.3 Hz, 4-CH3-cyc.), 1.00–1.14 (m, 1H, CH2-cyc.), 1.44–1.64 (br. m, 4H, CH, CH2-cyc.), 1.66–1.84 (m, 2H, CH2-cyc.), 2.15 (s, 1H, CH2-cyc.), 2.29 (s, 1H, CH2-cyc.), 7.12 (br. t, 2H, J=8.5 Hz, H4,H6-ind.), 7.33–7.50 (m, 6H, H7, 3-C6H5-ind.), 9.44 (s, 1H, CONH), 12.02 (s, 1H, NH) p.p.m.. Analysis calculated for C22H22FN3O: C 72.71, H 6.10, N 11.56%. Found: C 72.67, H 6.39, N 11.57%. (cyc.=cyclohexylidene, ind.=indole). organic compounds o1212 C¸elikesir et al. C22H22FN3O Acta Cryst. (2013). E69, o1211–o1212 supplementary materials Comment Indole-2-carbohydrazides are attractive targets in organic synthesis because of the biological potential of the indole scaffold and the synthetic utility of the carbohydrazide function (Zhang et al., 2004; Akkurt et al., 2010; 2013). The title compound has been synthesized as a member of a series of indolylhydrazones with antituberculosis properties (Cihan- Üstündağ & Çapan, 2012). To fully characterize the structure, we now report on the X-ray diffraction analysis of the title compound. In the title compound, (I), (Fig. 1), the asymmetric unit contains two crystallographically independent molecules, A and B, whose cyclohexane rings adopt distorted chair conformations [the puckering parameters (Cremer & Pople, 1975) are QT = 0.520 (3) Å, θ = 168.2 (3)°, φ = 31.9 (15)° for molecule A (with N1), and QT = 0.520 (3) Å, θ = 168.2 (3)°, φ = 31.9 (15)° for molecule B (with N4)]. In the title compound, (I), (Fig. 1), the asymmetric unit contains two crystallographically independent molecules, A and B, whose cyclohexane rings adopt distorted chair conformations [the puckering parameters (Cremer & Pople, 1975) are QT = 0.520 (3) Å, θ = 168.2 (3)°, φ = 31.9 (15)° for molecule A (with N1), and QT = 0.520 (3) Å, θ = 168.2 (3)°, φ = 31.9 (15)° for molecule B (with N4)]. The 1H-indole ring systems of both molecules A and B are essentially planar [maximum deviations are 0.019 (2) Å for C1 in molecule A and 0.014 (2) Å for C26 in molecule B]. The 1H-indole ring systems of molecules A and B make dihedral angles of 77.64 (10) and 69.50 (9)° with their phenyl rings, respectively. The C14–C15–N2–N3, C15–N2–N3– C16, C36–C37–N5–N6, C37–N5–N6–C38 torsion angles are 174.92 (18), -175.2 (2), -179.95 (17) and 178.4 (2)°, respectively. In the crystal, the two molecules in the asymmetric unit are connected to each other, forming N—H···O dimers (Table 1, Fig. 2), giving rise to R22(10) ring patterns (Bernstein et al., 1995). Furthermore, C—H···π interactions (Table 1) contribute to the stability of the crystal packing in (I). supplementary materials Acta Cryst. (2013). E69, o1211–o1212 [doi:10.1107/S1600536813018436] Acta Cryst. (2013). E69, o1211–o1212 [doi:10.1107/S1600536813018436] supplementary materials 1.5Ueq(C,N). The H atoms (N2)H2N and (N5)H5N of the two amide groups were found in a difference Fourier map and were refined freely. The crystal studied was a non-merohedral twin (twin law 0.24 0.00 - 0.75 - 0.09 - 1.00 0.05 - 1.26 0.00 - 0.24), with the minor twin component refining to 0.00116 (8). 1.5Ueq(C,N). The H atoms (N2)H2N and (N5)H5N of the two amide groups were found in a difference Fourier map and were refined freely. The crystal studied was a non-merohedral twin (twin law 0.24 0.00 - 0.75 - 0.09 - 1.00 0.05 - 1.26 0.00 - 0.24), with the minor twin component refining to 0.00116 (8). Computing details Data collection: X-AREA (Stoe & Cie, 2002); cell refinement: X-AREA (Stoe & Cie, 2002); data reduction: X-RED32 (Stoe & Cie, 2002); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 2012); software used to prepare material for publication: WinGX (Farrugia, 2012). Figure 1 Acta Cryst. (2013). E69, o1211–o1212 Refinement H atoms bonded to C atoms and the H atoms (N1)H1 and (N4)H4 of the two of the four amide groups were positioned geometrically with C—H = 0.93 - 0.98 Å, and N—H =0.86 Å and refined using a riding model with Uiso(H) = 1.2 or Acta Cryst. (2013). E69, o1211–o1212 sup-1 supplementary materials Figure 1 The molecular structure of (I), showing the atom labelling scheme. Displacement ellipsoids for non-H atoms are drawn at the 30% probability level. sup-2 Acta Cryst. (2013). E69, o1211–o1212 supplementary materials Figure 2 View of the N—H···O dimer in the unit cell. H atoms not participating in hydrogen bonding have been omitted for clarity and hydrogen bonds are drawn as dashed lines. supplementary materials Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.057 wR(F2) = 0.144 S = 1.03 8702 reflections 496 parameters 2 restraints Hydrogen site location: mixed H atoms treated by a mixture of independent and constrained refinement W = 1/[Σ2(FO2) + (0.0744P)2 + 0.1246P] WHERE P = (FO2 + 2FC2)/3 (Δ/σ)max < 0.001 Δρmax = 0.42 e Å−3 Δρmin = −0.37 e Å−3 Special details Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.057 wR(F2) = 0.144 S = 1.03 8702 reflections 496 parameters 2 restraints Hydrogen site location: mixed H atoms treated by a mixture of independent and constrained refinement W = 1/[Σ2(FO2) + (0.0744P)2 + 0.1246P] WHERE P = (FO2 + 2FC2)/3 (Δ/σ)max < 0.001 Δρmax = 0.42 e Å−3 Δρmin = −0.37 e Å−3 Special details Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.057 wR(F2) = 0.144 S = 1.03 8702 reflections 496 parameters 2 restraints Special details Hydrogen site location: mixed H atoms treated by a mixture of independent and constrained refinement W = 1/[Σ2(FO2) + (0.0744P)2 + 0.1246P] WHERE P = (FO2 + 2FC2)/3 (Δ/σ)max < 0.001 Δρmax = 0.42 e Å−3 Δρmin = −0.37 e Å−3 Geometry. Bond distances, angles etc. have been calculated using the rounded fractional coordinates. All su's are estimated from the variances of the (full) variance-covariance matrix. The cell e.s.d.'s are taken into account in the estimation of distances, angles and torsion angles Refinement. Refinement on F2 for ALL reflections except those flagged by the user for potential systematic errors. Weighted R-factors wR and all goodnesses of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The observed criterion of F2 > σ(F2) is used only for calculating -R-factor-obs etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. Figure 2 g View of the N—H···O dimer in the unit cell. H atoms not participating in hydrogen bonding have been omitted for clarity and hydrogen bonds are drawn as dashed lines. View of the N—H···O dimer in the unit cell. H atoms not participating in hydrogen bonding have been omitted for clarity and hydrogen bonds are drawn as dashed lines. 5-Fluoro-N′-(4-methylcyclohexylidene)-3-phenyl-1H-indole-2-carbohydrazide Crystal data C22H22FN3O Mr = 363.43 Triclinic, P1 Hall symbol: -P 1 a = 11.6630 (6) Å b = 13.5320 (7) Å c = 14.7754 (8) Å α = 112.967 (4)° β = 95.936 (4)° γ = 111.385 (4)° V = 1915.4 (2) Å3 Z = 4 F(000) = 768 Dx = 1.260 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 35686 reflections θ = 2.0–28.0° µ = 0.09 mm−1 T = 296 K Prism, colourless 0.68 × 0.52 × 0.33 mm Data collection Stoe IPDS 2 diffractometer Radiation source: sealed X-ray tube, 12 x 0.4 mm long-fine focus Plane graphite monochromator Detector resolution: 6.67 pixels mm-1 ω scans Absorption correction: integration (X-RED32; Stoe & Cie, 2002) Tmin = 0.948, Tmax = 0.972 26097 measured reflections 8702 independent reflections 5714 reflections with I > 2σ(I) Rint = 0.058 θmax = 27.6°, θmin = 2.0° h = −15→15 k = −17→17 l = −19→19 sup-3 Acta Cryst. (2013). E69, o1211–o1212 supplementary materials Acta Cryst. (2013). E69, o1211–o1212 supplementary materials Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq F1 0.33494 (17) −0.02996 (12) 0.06230 (14) 0.1113 (7) O1 0.68124 (14) 0.68746 (12) 0.28626 (9) 0.0679 (5) N1 0.52435 (14) 0.45358 (13) 0.24287 (11) 0.0543 (5) N2 0.70784 (16) 0.62496 (14) 0.12738 (12) 0.0547 (5) N3 0.78349 (15) 0.73959 (13) 0.14306 (11) 0.0564 (5) C1 0.46778 (18) 0.33259 (17) 0.20839 (13) 0.0553 (6) C2 0.3836 (2) 0.2633 (2) 0.24439 (16) 0.0669 (7) C3 0.3400 (2) 0.1422 (2) 0.19316 (18) 0.0771 (9) C4 0.3792 (2) 0.09106 (19) 0.10798 (19) 0.0761 (8) C5 0.4602 (2) 0.15512 (18) 0.06997 (16) 0.0678 (7) C6 0.50520 (18) 0.27986 (16) 0.12087 (13) 0.0547 (6) C7 0.58613 (17) 0.37471 (16) 0.10276 (12) 0.0510 (5) C8 0.64165 (17) 0.35989 (15) 0.01471 (12) 0.0495 (5) C9 0.56439 (19) 0.32276 (19) −0.08081 (14) 0.0640 (7) C10 0.6149 (2) 0.3174 (2) −0.16231 (14) 0.0724 (8) C11 0.7423 (2) 0.34579 (18) −0.15024 (14) 0.0685 (7) C12 0.8187 (2) 0.3774 (2) −0.05808 (16) 0.0773 (8) C13 0.7693 (2) 0.3848 (2) 0.02451 (14) 0.0689 (7) C14 0.59506 (17) 0.47922 (16) 0.17903 (12) 0.0501 (5) C15 0.66499 (16) 0.60585 (16) 0.20329 (12) 0.0499 (5) C16 0.81127 (17) 0.74851 (16) 0.06423 (14) 0.0546 (6) C17 0.7672 (2) 0.6516 (2) −0.04384 (15) 0.0716 (7) C18 0.8715 (3) 0.66082 (19) −0.09684 (16) 0.0813 (8) C19 0.9486 (3) 0.7848 (2) −0.08349 (18) 0.0837 (9) C20 1.0026 (2) 0.87295 (18) 0.02981 (17) 0.0725 (7) C21 0.8962 (2) 0.87190 (18) 0.08106 (17) 0.0705 (8) C22 1.0472 (3) 0.7915 (3) −0.1398 (3) 0.1207 (14) F2 0.95169 (12) 1.23386 (12) 0.77835 (10) 0.0893 (5) l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Acta Cryst. (2013). Acta Cryst. (2013). E69, o1211–o1212 supplementary materials pp y sup-6 Acta Cryst. (2013). Acta Cryst. (2013). E69, o1211–o1212 supplementary materials E69, o1211–o1212 sup-4 supplementary materials O2 0.39600 (13) 0.58695 (12) 0.35468 (10) 0.0678 (5) N4 0.62682 (15) 0.79327 (14) 0.47138 (11) 0.0576 (5) N5 0.28375 (16) 0.68606 (16) 0.42914 (13) 0.0627 (6) N6 0.16405 (16) 0.59508 (15) 0.36881 (11) 0.0648 (5) C23 0.72321 (18) 0.90083 (16) 0.54295 (13) 0.0535 (6) C24 0.85522 (19) 0.95099 (19) 0.55570 (15) 0.0632 (7) C25 0.9302 (2) 1.0619 (2) 0.63591 (16) 0.0674 (7) C26 0.8732 (2) 1.12177 (18) 0.70176 (15) 0.0645 (7) C27 0.74541 (18) 1.07482 (17) 0.69316 (14) 0.0589 (6) C28 0.66725 (17) 0.96119 (15) 0.61105 (12) 0.0506 (6) C29 0.53239 (17) 0.88540 (15) 0.57767 (12) 0.0501 (6) C30 0.43961 (16) 0.91430 (15) 0.63207 (12) 0.0498 (5) C31 0.4100 (2) 1.00510 (18) 0.63397 (15) 0.0653 (7) C32 0.3272 (2) 1.0345 (2) 0.68837 (18) 0.0777 (8) C33 0.2746 (2) 0.9750 (2) 0.74062 (18) 0.0849 (9) C34 0.3030 (2) 0.8843 (2) 0.73919 (18) 0.0847 (10) C35 0.3847 (2) 0.85356 (19) 0.68489 (15) 0.0659 (7) C36 0.51112 (17) 0.78338 (16) 0.49088 (12) 0.0513 (6) C37 0.39356 (18) 0.67621 (16) 0.41905 (13) 0.0541 (6) C38 0.06794 (19) 0.61469 (18) 0.38764 (14) 0.0626 (6) C39 0.0688 (2) 0.7241 (2) 0.46963 (18) 0.0765 (8) C40 −0.0321 (2) 0.6933 (2) 0.52374 (18) 0.0854 (9) C41 −0.1642 (2) 0.6026 (2) 0.45358 (19) 0.0816 (10) C42 −0.1578 (2) 0.4916 (2) 0.3791 (2) 0.0907 (10) C43 −0.0627 (2) 0.5196 (2) 0.31960 (18) 0.0865 (9) C44 −0.2606 (3) 0.5777 (4) 0.5126 (3) 0.1196 (16) H1 0.51700 0.50530 0.29580 0.0650* H2 0.35810 0.29860 0.30130 0.0800* H2N 0.688 (2) 0.563 (2) 0.0781 (14) 0.068 (6)* H3 0.28380 0.09360 0.21520 0.0920* H5 0.48450 0.11810 0.01300 0.0810* H9 0.47700 0.30100 −0.09040 0.0770* H10 0.56190 0.29450 −0.22550 0.0870* H11 0.77660 0.34350 −0.20470 0.0820* H12 0.90490 0.39420 −0.05030 0.0930* H13 0.82280 0.40670 0.08710 0.0830* H17A 0.69710 0.65430 −0.08270 0.0860* H17B 0.73410 0.57470 −0.04360 0.0860* H18A 0.92920 0.63560 −0.07030 0.0980* H18B 0.83270 0.60620 −0.16950 0.0980* H19 0.88800 0.80630 −0.11310 0.1000* H20A 1.05170 0.95280 0.03810 0.0870* H20B 1.06030 0.85250 0.06290 0.0870* H21A 0.93390 0.92490 0.15410 0.0850* H21B 0.84470 0.90180 0.05370 0.0850* H22A 1.10930 0.77190 −0.11260 0.1450* H22B 1.08940 0.87120 −0.13210 0.1450* H22C 1.00660 0.73590 −0.21130 0.1450* H4 0.63670 0.73980 0.42170 0.0690* Acta Cryst. (2013). E69, o1211–o1212 sup-5 supplementary materials supplementary materials E69, o1211–o1212 H5N 0.2992 (17) 0.750 (2) 0.4704 (16) 0.067 (6)* H24 0.89120 0.91000 0.51080 0.0760* H25 1.01860 1.09750 0.64670 0.0810* H27 0.71140 1.11640 0.73980 0.0710* H31 0.44570 1.04670 0.59870 0.0780* H32 0.30760 1.09540 0.68900 0.0930* H33 0.21960 0.99550 0.77720 0.1020* H34 0.26700 0.84340 0.77490 0.1020* H35 0.40290 0.79170 0.68390 0.0790* H39A 0.05350 0.77160 0.43910 0.0920* H39B 0.15300 0.77220 0.51960 0.0920* H40A −0.00490 0.66310 0.56740 0.1020* H40B −0.03670 0.76600 0.56770 0.1020* H41 −0.19180 0.63690 0.41320 0.0980* H42A −0.24240 0.43520 0.33140 0.1090* H42B −0.13290 0.45420 0.41660 0.1090* H43A −0.05610 0.44730 0.27760 0.1040* H43B −0.09490 0.54540 0.27410 0.1040* H44A −0.26420 0.65070 0.55500 0.1440* H44B −0.23490 0.54640 0.55490 0.1440* H44C −0.34390 0.52030 0.46520 0.1440* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 F1 0.1200 (13) 0.0613 (8) 0.1456 (14) 0.0303 (9) 0.0534 (11) 0.0465 (9) O1 0.0915 (10) 0.0593 (8) 0.0553 (7) 0.0372 (8) 0.0418 (7) 0.0201 (6) N1 0.0650 (10) 0.0590 (9) 0.0493 (7) 0.0337 (8) 0.0316 (7) 0.0251 (7) N2 0.0649 (10) 0.0471 (8) 0.0495 (8) 0.0234 (8) 0.0289 (7) 0.0182 (7) N3 0.0590 (9) 0.0512 (8) 0.0618 (8) 0.0266 (8) 0.0326 (7) 0.0230 (7) C1 0.0599 (11) 0.0630 (11) 0.0557 (9) 0.0329 (10) 0.0258 (8) 0.0317 (9) C2 0.0704 (13) 0.0824 (14) 0.0676 (11) 0.0383 (12) 0.0347 (10) 0.0452 (11) C3 0.0762 (15) 0.0790 (15) 0.0914 (15) 0.0298 (13) 0.0344 (12) 0.0554 (13) C4 0.0800 (15) 0.0568 (12) 0.0924 (15) 0.0273 (12) 0.0287 (12) 0.0370 (11) C5 0.0749 (14) 0.0607 (12) 0.0730 (12) 0.0350 (11) 0.0308 (11) 0.0288 (10) C6 0.0586 (11) 0.0575 (10) 0.0565 (9) 0.0314 (9) 0.0247 (8) 0.0270 (8) C7 0.0552 (10) 0.0563 (10) 0.0493 (8) 0.0309 (9) 0.0241 (8) 0.0237 (8) C8 0.0573 (10) 0.0481 (9) 0.0465 (8) 0.0288 (8) 0.0238 (8) 0.0177 (7) C9 0.0580 (11) 0.0761 (13) 0.0534 (10) 0.0312 (11) 0.0201 (9) 0.0232 (9) C10 0.0820 (15) 0.0837 (15) 0.0470 (9) 0.0384 (13) 0.0228 (10) 0.0234 (10) C11 0.0844 (15) 0.0646 (12) 0.0539 (10) 0.0337 (11) 0.0386 (10) 0.0196 (9) C12 0.0593 (12) 0.0933 (16) 0.0709 (13) 0.0368 (12) 0.0342 (11) 0.0236 (11) C13 0.0608 (12) 0.0920 (15) 0.0512 (9) 0.0410 (12) 0.0212 (9) 0.0226 (10) C14 0.0550 (10) 0.0583 (10) 0.0462 (8) 0.0304 (9) 0.0254 (7) 0.0252 (8) C15 0.0528 (10) 0.0581 (10) 0.0469 (8) 0.0320 (9) 0.0251 (7) 0.0224 (8) C16 0.0539 (10) 0.0545 (10) 0.0623 (10) 0.0273 (9) 0.0282 (8) 0.0275 (8) C17 0.0747 (14) 0.0708 (13) 0.0549 (10) 0.0169 (11) 0.0197 (10) 0.0295 (10) C18 0.1014 (18) 0.0663 (13) 0.0631 (12) 0.0262 (13) 0.0425 (12) 0.0231 (10) C19 0.1068 (18) 0.0744 (14) 0.0788 (14) 0.0367 (14) 0.0553 (14) 0.0397 (12) C20 0.0766 (14) 0.0552 (11) 0.0834 (13) 0.0224 (11) 0.0436 (12) 0.0310 (10) Atomic displacement parameters (Å2) Acta Cryst. supplementary materials (2013). Acta Cryst. (2013). E69, o1211–o1212 supplementary materials E69, o1211–o1212 sup-6 supplementary materials C21 0.0832 (15) 0.0560 (11) 0.0819 (13) 0.0344 (11) 0.0445 (12) 0.0323 (10) C22 0.166 (3) 0.0908 (19) 0.130 (2) 0.058 (2) 0.108 (2) 0.0568 (18) F2 0.0637 (8) 0.0726 (8) 0.0934 (9) 0.0224 (7) 0.0195 (7) 0.0107 (7) O2 0.0712 (9) 0.0612 (8) 0.0633 (7) 0.0334 (7) 0.0345 (7) 0.0141 (6) N4 0.0643 (10) 0.0611 (9) 0.0542 (8) 0.0353 (8) 0.0347 (7) 0.0217 (7) N5 0.0600 (10) 0.0581 (10) 0.0574 (9) 0.0282 (9) 0.0224 (8) 0.0115 (8) N6 0.0615 (10) 0.0671 (10) 0.0511 (8) 0.0295 (9) 0.0185 (7) 0.0121 (7) C23 0.0591 (11) 0.0580 (10) 0.0523 (9) 0.0303 (9) 0.0301 (8) 0.0262 (8) C24 0.0627 (12) 0.0732 (13) 0.0670 (11) 0.0391 (11) 0.0382 (10) 0.0316 (10) C25 0.0560 (12) 0.0743 (13) 0.0743 (12) 0.0296 (11) 0.0304 (10) 0.0330 (11) C26 0.0613 (12) 0.0613 (12) 0.0624 (11) 0.0250 (10) 0.0218 (9) 0.0217 (9) C27 0.0628 (12) 0.0591 (11) 0.0564 (9) 0.0312 (10) 0.0289 (9) 0.0215 (9) C28 0.0562 (10) 0.0550 (10) 0.0517 (9) 0.0301 (9) 0.0297 (8) 0.0263 (8) C29 0.0571 (11) 0.0533 (10) 0.0488 (8) 0.0282 (9) 0.0280 (8) 0.0252 (8) C30 0.0526 (10) 0.0512 (9) 0.0438 (8) 0.0249 (8) 0.0232 (7) 0.0161 (7) C31 0.0738 (13) 0.0611 (11) 0.0727 (12) 0.0370 (11) 0.0381 (10) 0.0310 (10) C32 0.0784 (15) 0.0675 (13) 0.0924 (15) 0.0461 (12) 0.0401 (13) 0.0251 (12) C33 0.0807 (15) 0.0965 (17) 0.0811 (14) 0.0510 (14) 0.0527 (13) 0.0262 (13) C34 0.0865 (16) 0.1159 (19) 0.0835 (14) 0.0558 (15) 0.0595 (13) 0.0568 (14) C35 0.0705 (13) 0.0786 (13) 0.0714 (12) 0.0408 (11) 0.0435 (10) 0.0429 (11) C36 0.0591 (11) 0.0551 (10) 0.0486 (8) 0.0303 (9) 0.0306 (8) 0.0239 (8) C37 0.0636 (11) 0.0583 (10) 0.0485 (8) 0.0317 (9) 0.0310 (8) 0.0241 (8) C38 0.0644 (12) 0.0696 (12) 0.0514 (9) 0.0327 (11) 0.0206 (9) 0.0218 (9) C39 0.0735 (14) 0.0701 (14) 0.0823 (14) 0.0390 (12) 0.0305 (12) 0.0231 (11) C40 0.0867 (17) 0.1039 (18) 0.0774 (14) 0.0603 (16) 0.0352 (13) 0.0333 (13) C41 0.0775 (16) 0.1055 (19) 0.0903 (15) 0.0522 (15) 0.0395 (13) 0.0573 (14) C42 0.0626 (14) 0.0847 (17) 0.1170 (19) 0.0308 (13) 0.0188 (13) 0.0428 (15) C43 0.0684 (15) 0.0888 (17) 0.0716 (13) 0.0378 (13) 0.0089 (11) 0.0084 (12) C44 0.104 (2) 0.174 (3) 0.144 (3) 0.077 (2) 0.073 (2) 0.109 (3) sup-7 Acta Cryst (2013) E69 o1211 o1212 C21 0.0832 (15) 0.0560 (11) 0.0819 (13) 0.0344 (11) 0.0445 (12) 0.0323 (10) C22 0.166 (3) 0.0908 (19) 0.130 (2) 0.058 (2) 0.108 (2) 0.0568 (18) F2 0.0637 (8) 0.0726 (8) 0.0934 (9) 0.0224 (7) 0.0195 (7) 0.0107 (7) O2 0.0712 (9) 0.0612 (8) 0.0633 (7) 0.0334 (7) 0.0345 (7) 0.0141 (6) N4 0.0643 (10) 0.0611 (9) 0.0542 (8) 0.0353 (8) 0.0347 (7) 0.0217 (7) N5 0.0600 (10) 0.0581 (10) 0.0574 (9) 0.0282 (9) 0.0224 (8) 0.0115 (8) N6 0.0615 (10) 0.0671 (10) 0.0511 (8) 0.0295 (9) 0.0185 (7) 0.0121 (7) C23 0.0591 (11) 0.0580 (10) 0.0523 (9) 0.0303 (9) 0.0301 (8) 0.0262 (8) C24 0.0627 (12) 0.0732 (13) 0.0670 (11) 0.0391 (11) 0.0382 (10) 0.0316 (10) C25 0.0560 (12) 0.0743 (13) 0.0743 (12) 0.0296 (11) 0.0304 (10) 0.0330 (11) C26 0.0613 (12) 0.0613 (12) 0.0624 (11) 0.0250 (10) 0.0218 (9) 0.0217 (9) C27 0.0628 (12) 0.0591 (11) 0.0564 (9) 0.0312 (10) 0.0289 (9) 0.0215 (9) C28 0.0562 (10) 0.0550 (10) 0.0517 (9) 0.0301 (9) 0.0297 (8) 0.0263 (8) C29 0.0571 (11) 0.0533 (10) 0.0488 (8) 0.0282 (9) 0.0280 (8) 0.0252 (8) C30 0.0526 (10) 0.0512 (9) 0.0438 (8) 0.0249 (8) 0.0232 (7) 0.0161 (7) C31 0.0738 (13) 0.0611 (11) 0.0727 (12) 0.0370 (11) 0.0381 (10) 0.0310 (10) C32 0.0784 (15) 0.0675 (13) 0.0924 (15) 0.0461 (12) 0.0401 (13) 0.0251 (12) C33 0.0807 (15) 0.0965 (17) 0.0811 (14) 0.0510 (14) 0.0527 (13) 0.0262 (13) C34 0.0865 (16) 0.1159 (19) 0.0835 (14) 0.0558 (15) 0.0595 (13) 0.0568 (14) C35 0.0705 (13) 0.0786 (13) 0.0714 (12) 0.0408 (11) 0.0435 (10) 0.0429 (11) C36 0.0591 (11) 0.0551 (10) 0.0486 (8) 0.0303 (9) 0.0306 (8) 0.0239 (8) C37 0.0636 (11) 0.0583 (10) 0.0485 (8) 0.0317 (9) 0.0310 (8) 0.0241 (8) C38 0.0644 (12) 0.0696 (12) 0.0514 (9) 0.0327 (11) 0.0206 (9) 0.0218 (9) C39 0.0735 (14) 0.0701 (14) 0.0823 (14) 0.0390 (12) 0.0305 (12) 0.0231 (11) C40 0.0867 (17) 0.1039 (18) 0.0774 (14) 0.0603 (16) 0.0352 (13) 0.0333 (13) C41 0.0775 (16) 0.1055 (19) 0.0903 (15) 0.0522 (15) 0.0395 (13) 0.0573 (14) C42 0.0626 (14) 0.0847 (17) 0.1170 (19) 0.0308 (13) 0.0188 (13) 0.0428 (15) C43 0.0684 (15) 0.0888 (17) 0.0716 (13) 0.0378 (13) 0.0089 (11) 0.0084 (12) C44 0.104 (2) 0.174 (3) 0.144 (3) 0.077 (2) 0.073 (2) 0.109 (3) Geometric parameters (Å, º) F1—C4 1.361 (3) C18—H18B 0.9700 F2—C26 1.361 (3) C19—H19 0.9800 O1—C15 1.225 (2) C20—H20B 0.9700 O2—C37 1.224 (2) C20—H20A 0.9700 N1—C1 1.366 (3) C21—H21B 0.9700 N1—C14 1.378 (3) C21—H21A 0.9700 N2—C15 1.350 (3) C22—H22A 0.9600 N2—N3 1.385 (3) C22—H22B 0.9600 N3—C16 1.279 (3) C22—H22C 0.9600 N1—H1 0.8600 C23—C24 1.395 (3) N2—H2N 0.80 (2) C23—C28 1.410 (3) N4—C36 1.379 (3) C24—C25 1.365 (3) N4—C23 1.364 (3) C25—C26 1.399 (3) N5—N6 1.376 (3) C26—C27 1.360 (3) N5—C37 1.352 (3) C27—C28 1.400 (3) N6—C38 1.276 (3) C28—C29 1.427 (3) N4—H4 0.8600 C29—C30 1.488 (3) N5—H5N 0.78 (2) C29—C36 1.384 (3) Acta Cryst. supplementary materials (2013). E69, o1211–o1212 sup-7 supplementary materials Acta Cryst. (2013). E69, o1211–o1212 C1—C6 1.413 (3) C30—C31 1.384 (3) C1—C2 1.397 (3) C30—C35 1.384 (3) C2—C3 1.364 (4) C31—C32 1.389 (3) C3—C4 1.394 (3) C32—C33 1.358 (4) C4—C5 1.361 (4) C33—C34 1.376 (4) C5—C6 1.404 (3) C34—C35 1.383 (3) C6—C7 1.431 (3) C36—C37 1.469 (3) C7—C8 1.489 (3) C38—C43 1.494 (3) C7—C14 1.380 (3) C38—C39 1.499 (3) C8—C9 1.382 (3) C39—C40 1.515 (4) C8—C13 1.379 (3) C40—C41 1.501 (4) C9—C10 1.383 (3) C41—C42 1.511 (4) C10—C11 1.365 (4) C41—C44 1.515 (5) C11—C12 1.359 (3) C42—C43 1.517 (4) C12—C13 1.387 (3) C24—H24 0.9300 C14—C15 1.473 (3) C25—H25 0.9300 C16—C21 1.500 (3) C27—H27 0.9300 C16—C17 1.496 (3) C31—H31 0.9300 C17—C18 1.507 (4) C32—H32 0.9300 C18—C19 1.506 (4) C33—H33 0.9300 C19—C22 1.485 (5) C34—H34 0.9300 C19—C20 1.516 (3) C35—H35 0.9300 C20—C21 1.518 (3) C39—H39A 0.9700 C2—H2 0.9300 C39—H39B 0.9700 C3—H3 0.9300 C40—H40A 0.9700 C5—H5 0.9300 C40—H40B 0.9700 C9—H9 0.9300 C41—H41 0.9800 C10—H10 0.9300 C42—H42A 0.9700 C11—H11 0.9300 C42—H42B 0.9700 C12—H12 0.9300 C43—H43A 0.9700 C13—H13 0.9300 C43—H43B 0.9700 C17—H17B 0.9700 C44—H44A 0.9600 C17—H17A 0.9700 C44—H44B 0.9600 C18—H18A 0.9700 C44—H44C 0.9600 C1—N1—C14 108.92 (16) H21A—C21—H21B 108.00 N3—N2—C15 120.98 (16) C19—C22—H22A 110.00 N2—N3—C16 115.91 (16) C19—C22—H22B 109.00 C1—N1—H1 126.00 H22A—C22—H22C 110.00 C14—N1—H1 126.00 C19—C22—H22C 109.00 N3—N2—H2N 128.4 (19) H22A—C22—H22B 109.00 C15—N2—H2N 110.5 (18) H22B—C22—H22C 109.00 C23—N4—C36 109.59 (16) N4—C23—C24 130.90 (19) N6—N5—C37 122.72 (18) N4—C23—C28 107.45 (19) N5—N6—C38 116.56 (18) C24—C23—C28 121.65 (18) C36—N4—H4 125.00 C23—C24—C25 118.2 (2) C23—N4—H4 125.00 C24—C25—C26 119.6 (2) C37—N5—H5N 110.4 (17) F2—C26—C27 118.9 (2) N6—N5—H5N 126.8 (17) C25—C26—C27 123.9 (2) sup-8 Acta Cryst. (2013). E69, o1211–o1212 supplementary materials s Acta Cryst. (2013). supplementary materials E69, o1211–o1212 N1—C1—C6 107.95 (18) F2—C26—C25 117.2 (2) N1—C1—C2 130.44 (19) C26—C27—C28 117.09 (19) C2—C1—C6 121.6 (2) C27—C28—C29 132.93 (19) C1—C2—C3 117.7 (2) C23—C28—C29 107.56 (16) C2—C3—C4 120.3 (2) C23—C28—C27 119.52 (19) F1—C4—C3 117.2 (2) C28—C29—C36 106.29 (18) F1—C4—C5 118.9 (2) C30—C29—C36 129.37 (19) C3—C4—C5 123.9 (2) C28—C29—C30 124.30 (16) C4—C5—C6 116.8 (2) C29—C30—C31 120.77 (18) C5—C6—C7 133.18 (19) C29—C30—C35 120.51 (19) C1—C6—C5 119.7 (2) C31—C30—C35 118.7 (2) C1—C6—C7 107.12 (18) C30—C31—C32 120.3 (2) C6—C7—C8 125.71 (17) C31—C32—C33 120.6 (2) C8—C7—C14 127.95 (19) C32—C33—C34 119.7 (2) C6—C7—C14 106.25 (17) C33—C34—C35 120.4 (2) C7—C8—C13 122.73 (16) C30—C35—C34 120.4 (2) C9—C8—C13 117.73 (18) N4—C36—C29 109.11 (17) C7—C8—C9 119.52 (19) N4—C36—C37 118.25 (16) C8—C9—C10 121.1 (2) C29—C36—C37 132.6 (2) C9—C10—C11 120.20 (19) N5—C37—C36 114.19 (18) C10—C11—C12 119.6 (2) O2—C37—N5 123.4 (2) C11—C12—C13 120.6 (2) O2—C37—C36 122.4 (2) C8—C13—C12 120.69 (19) N6—C38—C43 117.47 (19) N1—C14—C7 109.76 (18) C39—C38—C43 114.5 (2) N1—C14—C15 117.64 (16) N6—C38—C39 128.0 (2) C7—C14—C15 132.59 (18) C38—C39—C40 112.2 (2) O1—C15—C14 121.94 (17) C39—C40—C41 114.7 (2) N2—C15—C14 115.28 (16) C40—C41—C42 109.5 (2) O1—C15—N2 122.8 (2) C42—C41—C44 113.5 (3) N3—C16—C21 116.31 (18) C40—C41—C44 111.9 (2) C17—C16—C21 115.47 (19) C41—C42—C43 112.0 (2) N3—C16—C17 128.2 (2) C38—C43—C42 112.8 (2) C16—C17—C18 113.6 (2) C23—C24—H24 121.00 C17—C18—C19 113.8 (2) C25—C24—H24 121.00 C18—C19—C22 113.2 (3) C24—C25—H25 120.00 C20—C19—C22 113.5 (3) C26—C25—H25 120.00 C18—C19—C20 109.6 (2) C26—C27—H27 121.00 C19—C20—C21 111.3 (2) C28—C27—H27 121.00 C16—C21—C20 111.7 (2) C30—C31—H31 120.00 C3—C2—H2 121.00 C32—C31—H31 120.00 C1—C2—H2 121.00 C31—C32—H32 120.00 C4—C3—H3 120.00 C33—C32—H32 120.00 C2—C3—H3 120.00 C32—C33—H33 120.00 C6—C5—H5 122.00 C34—C33—H33 120.00 C4—C5—H5 122.00 C33—C34—H34 120.00 C8—C9—H9 119.00 C35—C34—H34 120.00 C10—C9—H9 119.00 C30—C35—H35 120.00 C9—C10—H10 120.00 C34—C35—H35 120.00 C11—C10—H10 120.00 C38—C39—H39A 109.00 Acta Cryst (2013) E69 o1211–o1212 N1—C1—C6 107.95 (18) F2—C26—C25 N1—C1—C2 130.44 (19) C26—C27—C28 C2—C1—C6 121.6 (2) C27—C28—C29 C1—C2—C3 117.7 (2) C23—C28—C29 C2—C3—C4 120.3 (2) C23—C28—C27 F1—C4—C3 117.2 (2) C28—C29—C36 F1—C4—C5 118.9 (2) C30—C29—C36 C3—C4—C5 123.9 (2) C28—C29—C30 C4—C5—C6 116.8 (2) C29—C30—C31 C5—C6—C7 133.18 (19) C29—C30—C35 C1—C6—C5 119.7 (2) C31—C30—C35 C1—C6—C7 107.12 (18) C30—C31—C32 C6—C7—C8 125.71 (17) C31—C32—C33 C8—C7—C14 127.95 (19) C32—C33—C34 C6—C7—C14 106.25 (17) C33—C34—C35 C7—C8—C13 122.73 (16) C30—C35—C34 C9—C8—C13 117.73 (18) N4—C36—C29 C7—C8—C9 119.52 (19) N4—C36—C37 C8—C9—C10 121.1 (2) C29—C36—C37 C9—C10—C11 120.20 (19) N5—C37—C36 C10—C11—C12 119.6 (2) O2—C37—N5 C11—C12—C13 120.6 (2) O2—C37—C36 C8—C13—C12 120.69 (19) N6—C38—C43 N1—C14—C7 109.76 (18) C39—C38—C43 N1—C14—C15 117.64 (16) N6—C38—C39 C7—C14—C15 132.59 (18) C38—C39—C40 O1—C15—C14 121.94 (17) C39—C40—C41 N2—C15—C14 115.28 (16) C40—C41—C42 O1—C15—N2 122.8 (2) C42—C41—C44 N3—C16—C21 116.31 (18) C40—C41—C44 C17—C16—C21 115.47 (19) C41—C42—C43 N3—C16—C17 128.2 (2) C38—C43—C42 C16—C17—C18 113.6 (2) C23—C24—H24 C17—C18—C19 113.8 (2) C25—C24—H24 C18—C19—C22 113.2 (3) C24—C25—H25 C20—C19—C22 113.5 (3) C26—C25—H25 C18—C19—C20 109.6 (2) C26—C27—H27 C19—C20—C21 111.3 (2) C28—C27—H27 C16—C21—C20 111.7 (2) C30—C31—H31 C3—C2—H2 121.00 C32—C31—H31 C1—C2—H2 121.00 C31—C32—H32 C4—C3—H3 120.00 C33—C32—H32 C2—C3—H3 120.00 C32—C33—H33 C6—C5—H5 122.00 C34—C33—H33 C4—C5—H5 122.00 C33—C34—H34 C8—C9—H9 119.00 C35—C34—H34 C10—C9—H9 119.00 C30—C35—H35 C9—C10—H10 120.00 C34—C35—H35 C11—C10—H10 120.00 C38—C39—H39A 107.95 (18) F2—C26—C25 117.2 (2) 130.44 (19) C26—C27—C28 117.09 (19) 121.6 (2) C27—C28—C29 132.93 (19) 117.7 (2) C23—C28—C29 107.56 (16) 120.3 (2) C23—C28—C27 119.52 (19) 117.2 (2) C28—C29—C36 106.29 (18) 118.9 (2) C30—C29—C36 129.37 (19) 123.9 (2) C28—C29—C30 124.30 (16) 116.8 (2) C29—C30—C31 120.77 (18) 133.18 (19) C29—C30—C35 120.51 (19) 119.7 (2) C31—C30—C35 118.7 (2) 107.12 (18) C30—C31—C32 120.3 (2) 125.71 (17) C31—C32—C33 120.6 (2) 127.95 (19) C32—C33—C34 119.7 (2) 106.25 (17) C33—C34—C35 120.4 (2) 122.73 (16) C30—C35—C34 120.4 (2) 117.73 (18) N4—C36—C29 109.11 (17) 119.52 (19) N4—C36—C37 118.25 (16) 121.1 (2) C29—C36—C37 132.6 (2) 120.20 (19) N5—C37—C36 114.19 (18) 119.6 (2) O2—C37—N5 123.4 (2) 120.6 (2) O2—C37—C36 122.4 (2) 120.69 (19) N6—C38—C43 117.47 (19) 109.76 (18) C39—C38—C43 114.5 (2) 117.64 (16) N6—C38—C39 128.0 (2) 132.59 (18) C38—C39—C40 112.2 (2) 121.94 (17) C39—C40—C41 114.7 (2) 115.28 (16) C40—C41—C42 109.5 (2) 122.8 (2) C42—C41—C44 113.5 (3) 116.31 (18) C40—C41—C44 111.9 (2) 115.47 (19) C41—C42—C43 112.0 (2) 128.2 (2) C38—C43—C42 112.8 (2) 113.6 (2) C23—C24—H24 121.00 113.8 (2) C25—C24—H24 121.00 113.2 (3) C24—C25—H25 120.00 113.5 (3) C26—C25—H25 120.00 109.6 (2) C26—C27—H27 121.00 111.3 (2) C28—C27—H27 121.00 111.7 (2) C30—C31—H31 120.00 121.00 C32—C31—H31 120.00 121.00 C31—C32—H32 120.00 120.00 C33—C32—H32 120.00 120.00 C32—C33—H33 120.00 122.00 C34—C33—H33 120.00 122.00 C33—C34—H34 120.00 119.00 C35—C34—H34 120.00 119.00 C30—C35—H35 120.00 120.00 C34—C35—H35 120.00 120.00 C38—C39—H39A 109.00 Acta Cryst. Acta Cryst. (2013). E69, o1211–o1212 supplementary materials N1—C1—C2—C3 178.9 (2) C26—C27—C28—C29 −179.6 (2) C6—C1—C2—C3 1.2 (3) C26—C27—C28—C23 0.8 (3) C2—C1—C6—C7 177.4 (2) C27—C28—C29—C30 −2.5 (4) N1—C1—C6—C7 −0.8 (2) C23—C28—C29—C36 −0.6 (2) C1—C2—C3—C4 0.0 (4) C27—C28—C29—C36 179.8 (2) C2—C3—C4—C5 −0.5 (4) C23—C28—C29—C30 177.10 (18) C2—C3—C4—F1 178.2 (2) C28—C29—C36—N4 1.0 (2) C3—C4—C5—C6 −0.2 (4) C28—C29—C30—C31 69.5 (3) F1—C4—C5—C6 −178.8 (2) C28—C29—C36—C37 −176.4 (2) C4—C5—C6—C1 1.3 (3) C30—C29—C36—N4 −176.60 (19) C4—C5—C6—C7 −177.8 (2) C36—C29—C30—C35 69.0 (3) C5—C6—C7—C8 2.9 (4) C36—C29—C30—C31 −113.3 (2) C5—C6—C7—C14 179.6 (2) C30—C29—C36—C37 6.1 (4) C1—C6—C7—C8 −176.27 (19) C28—C29—C30—C35 −108.2 (2) C1—C6—C7—C14 0.4 (2) C31—C30—C35—C34 −0.7 (3) C6—C7—C8—C13 −105.2 (3) C29—C30—C35—C34 177.08 (19) C14—C7—C8—C13 78.9 (3) C29—C30—C31—C32 −177.44 (19) C6—C7—C14—C15 178.9 (2) C35—C30—C31—C32 0.3 (3) C6—C7—C14—N1 0.1 (2) C30—C31—C32—C33 0.2 (3) C6—C7—C8—C9 76.6 (3) C31—C32—C33—C34 −0.4 (4) C8—C7—C14—C15 −4.5 (4) C32—C33—C34—C35 0.0 (4) C14—C7—C8—C9 −99.4 (3) C33—C34—C35—C30 0.5 (3) C8—C7—C14—N1 176.70 (19) N4—C36—C37—N5 −164.94 (19) C9—C8—C13—C12 2.6 (4) C29—C36—C37—O2 −169.0 (2) C7—C8—C13—C12 −175.6 (2) C29—C36—C37—N5 12.2 (3) C7—C8—C9—C10 174.7 (2) N4—C36—C37—O2 13.8 (3) C13—C8—C9—C10 −3.7 (4) N6—C38—C39—C40 −136.0 (2) C8—C9—C10—C11 1.8 (4) C43—C38—C39—C40 45.4 (3) C9—C10—C11—C12 1.1 (4) N6—C38—C43—C42 132.9 (2) C10—C11—C12—C13 −2.1 (4) C39—C38—C43—C42 −48.3 (3) C11—C12—C13—C8 0.2 (4) C38—C39—C40—C41 −49.3 (3) N1—C14—C15—O1 13.7 (3) C39—C40—C41—C42 54.3 (3) C7—C14—C15—N2 15.9 (3) C39—C40—C41—C44 −179.0 (3) C7—C14—C15—O1 −165.0 (2) C40—C41—C42—C43 −55.5 (3) N1—C14—C15—N2 −165.41 (18) C44—C41—C42—C43 178.6 (2) N3—C16—C21—C20 134.3 (2) C41—C42—C43—C38 53.5 (3) Hydrogen-bond geometry (Å, º) Hydrogen-bond geometry (Å, º) d h id f h l d b i f h i d l i f l l h h l i f Hydrogen-bond geometry (Å, ) Cg1 Cg2 Cg3 Cg6 and Cg8 are the centroids of the 1H-pyrrole and benzene rings of the 1H-indole ring system of molecule A the phenyl ring of sup 11 A t C t (2013) E69 1211 1212 Hydrogen-bond geometry (Å, ) Cg1, Cg2, Cg3, Cg6 and Cg8 are the centroids of the 1H-pyrrole and benzene rings of the 1H-indole ring system of molecule A, the phenyl ring of molecule A, the 1H-pyrrole ring of the 1H-indole ring system of molecule B and the phenyl ring of molecule B, respectively. supplementary materials (2013). E69, o1211–o1212 sup-9 supplementary materials s Acta Cryst (2013) E69 o1211 o1212 C12—C11—H11 120.00 C38—C39—H39B 109.00 C10—C11—H11 120.00 C40—C39—H39A 109.00 C13—C12—H12 120.00 C40—C39—H39B 109.00 C11—C12—H12 120.00 H39A—C39—H39B 108.00 C12—C13—H13 120.00 C39—C40—H40A 109.00 C8—C13—H13 120.00 C39—C40—H40B 109.00 C18—C17—H17A 109.00 C41—C40—H40A 109.00 H17A—C17—H17B 108.00 C41—C40—H40B 109.00 C16—C17—H17B 109.00 H40A—C40—H40B 108.00 C18—C17—H17B 109.00 C40—C41—H41 107.00 C16—C17—H17A 109.00 C42—C41—H41 107.00 C17—C18—H18A 109.00 C44—C41—H41 107.00 C17—C18—H18B 109.00 C41—C42—H42A 109.00 C19—C18—H18A 109.00 C41—C42—H42B 109.00 H18A—C18—H18B 108.00 C43—C42—H42A 109.00 C19—C18—H18B 109.00 C43—C42—H42B 109.00 C20—C19—H19 107.00 H42A—C42—H42B 108.00 C18—C19—H19 107.00 C38—C43—H43A 109.00 C22—C19—H19 107.00 C38—C43—H43B 109.00 H20A—C20—H20B 108.00 C42—C43—H43A 109.00 C19—C20—H20A 109.00 C42—C43—H43B 109.00 C19—C20—H20B 109.00 H43A—C43—H43B 108.00 C21—C20—H20A 109.00 C41—C44—H44A 109.00 C21—C20—H20B 109.00 C41—C44—H44B 110.00 C16—C21—H21A 109.00 C41—C44—H44C 109.00 C16—C21—H21B 109.00 H44A—C44—H44B 110.00 C20—C21—H21A 109.00 H44A—C44—H44C 109.00 C20—C21—H21B 109.00 H44B—C44—H44C 109.00 C1—N1—C14—C15 −179.61 (17) C17—C16—C21—C20 −47.1 (3) C14—N1—C1—C2 −177.1 (2) N3—C16—C17—C18 −138.7 (3) C14—N1—C1—C6 0.9 (2) C21—C16—C17—C18 42.9 (3) C1—N1—C14—C7 −0.6 (2) C16—C17—C18—C19 −46.9 (3) C15—N2—N3—C16 −175.2 (2) C17—C18—C19—C22 −177.5 (2) N3—N2—C15—O1 6.0 (3) C17—C18—C19—C20 54.7 (3) N3—N2—C15—C14 −174.92 (18) C18—C19—C20—C21 −58.6 (3) N2—N3—C16—C21 −178.36 (19) C22—C19—C20—C21 173.7 (3) N2—N3—C16—C17 3.3 (3) C19—C20—C21—C16 55.0 (3) C23—N4—C36—C37 176.82 (18) C24—C23—C28—C29 −179.1 (2) C23—N4—C36—C29 −1.0 (2) N4—C23—C24—C25 −179.6 (2) C36—N4—C23—C28 0.6 (2) C28—C23—C24—C25 −0.7 (3) C36—N4—C23—C24 179.6 (2) N4—C23—C28—C29 0.0 (2) C37—N5—N6—C38 178.4 (2) C24—C23—C28—C27 0.6 (3) N6—N5—C37—O2 1.3 (3) N4—C23—C28—C27 179.71 (18) N6—N5—C37—C36 −179.95 (17) C23—C24—C25—C26 −0.5 (4) N5—N6—C38—C39 −1.7 (3) C24—C25—C26—F2 −177.7 (2) N5—N6—C38—C43 176.9 (2) C24—C25—C26—C27 2.0 (4) C2—C1—C6—C5 −1.9 (3) C25—C26—C27—C28 −2.1 (3) N1—C1—C6—C5 179.92 (19) F2—C26—C27—C28 177.62 (19) sup-10 Acta Cryst. (2013). E69, o1211–o1212 supplementary materials Acta Cryst. (2013). E69, o1211–o1212 Symmetry codes: (i) −x+1, −y+1, −z; (ii) −x+1, −y+2, −z+1; (iii) −x+1, −y+1, −z+1; (iv) x−1, y, z. supplementary materials D—H···A D—H H···A D···A D—H···A N1—H1···O2 0.86 2.15 2.895 (2) 145 N4—H4···O1 0.86 2.04 2.811 (2) 149 C17—H17B···N2 0.97 2.43 2.811 (3) 103 C39—H39B···N5 0.97 2.44 2.814 (3) 102 C17—H17A···Cg1i 0.97 2.66 3.594 (3) 163 C17—H17B···Cg3 0.97 2.74 3.685 (3) 164 C31—H31···Cg6ii 0.93 2.87 3.658 (2) 144 Cg8 are the centroids of the 1H-pyrrole and benzene rings of the 1H-indole ring system of molecule A, the phenyl ring of ole ring of the 1H-indole ring system of molecule B and the phenyl ring of molecule B, respectively. sup-11 supplementary materials C35—H35···Cg2iii 0.93 2.96 3.627 (3) 130 C39—H39B···Cg8 0.97 2.72 3.667 (3) 164 C42—H42A···Cg1iv 0.97 2.99 3.848 (3) 148 Symmetry codes: (i) −x+1, −y+1, −z; (ii) −x+1, −y+2, −z+1; (iii) −x+1, −y+1, −z+1; (iv) x−1, y, z. Symmetry codes: (i) −x+1, −y+1, −z; (ii) −x+1, −y+2, −z+1; (iii) −x+1, −y+1, −z+1; (iv) x−1, y, z. sup-12 Acta Cryst. (2013). E69, o1211–o1212
https://openalex.org/W4313299640	https://zenodo.org/record/7496386/files/61.pdf	English	null	Eco-Tourism: Concepts and Application by Smart Rural Areas: Case Study in Trenggalek, East Java	Journal of economics, finance and management studies	2,022	cc-by	4,677	Journal of Economics, Finance and Management Studies ISSN (print): 2644-0490, ISSN (online): 2644-0504 Volume 5 Issue 12 December 2022 Article DOI: 10.47191/jefms/v5-i12-61, Impact Factor: 6.274 Page No. 4000-4005 Journal of Economics, Finance and Management Studies ISSN (print): 2644-0490, ISSN (online): 2644-0504 Volume 5 Issue 12 December 2022 Article DOI: 10.47191/jefms/v5-i12-61, Impact Factor: 6.274 Page No. 4000-4005 Ni Wayan Sri Aryani1, Sherlinda Octa Yuniarsa2 1Faculty of Engineering, University of Udayana, Bali Province 2 Faculty of Economic and Business, University of Brawijaya Ni Wayan Sri Aryani1, Sherlinda Octa Yuniarsa2 1Faculty of Engineering, University of Udayana, Bali Province 2 Faculty of Economic and Business, University of Brawijaya ABSTRACT: Indonesia has a good tourist market, East Java is an island that has a very magnificent of tourism potential, then Trenggalek is particular as a tourist-magnet in South East Java. Several southern regions such as Trenggalek appear to play a huge role in sustaining economic growth, especially in the southern coastal areas. This several factors have supported by environment, knowledge, and expertise of the local population through the economy. Particularly in Trenggalek and generally in East Java is to apply the concept of international education with a package tour for tourism development in the short and long term as well. In this case, how to make local people economically profit in the sustaining tourism progress, how to develop eco-tourism programs and products meets the needs of tourist market and how to promote the concepts of cultural education. The methodology use a site visits with capability to quickly act and growth some innovation. The results of this study provide some several choices of answers a concepts and applications that be able to show entrepreneur potential in Trenggalek, East Java. Some concepts have 4 components, such as tourism destinations, tourism communication between local people and local government, tourism good services, and also value change. For the over time, local people in Trenggalek area will be more confident displaying their expertise. They can introduce about how to develop culture through creative learning programs with domestic tourists. There is a flow chart for some tourists to get a location using a smart village technology, it learn together about tourism for business, such as making business processed by making batik (culture), and learning for java language (education). So, some concepts and applications it can joined by local citizens, government, and stakeholders to change for ecotourism. Because a survival for ecotourism is depend on earth. Hopefully, an expectation can be applied in East Java by using a smart village, specifically for tourists to increase a economic growth. KEYWORDS: Eco-Tourism, Development, Smart Village, East Java KEYWORDS: Eco-Tourism, Development, Smart Village, East Java INTRODUCTION But, these sections are dealt within descriptive and weakly theory ways. (1991: 451) So, this problem is of fundamental importance as it has led to an absence of an adequate theoretical critique for understanding the dynamics of tourism and the social activities it involves. The first is concerned primarily with auditing, categorizing, listing, and grouping the outputs or consequent of tourism; the second approach is concerned primarily with conceptualizing the forces which is impact on tourism and, through an analysis of these forces, providing a broader context for understanding tourism. In this case, the crucial difference in the latter approach is that tourism concept and application in Indonesia. It seen as a focal lens through which is broader considerations can be taken into account, and it confirms the multidisciplinary foundation upon which tourism research is built as the only way in which tourism can be comprehended. As a personal social activity, tourism is practiced by a diverse range of the population; as an industry, it is multi-sectoral; and as a means of economic and cultural exchange, it has also been many facets and forms. Any comprehensive analysis of the field must therefore be multidisciplinary; and of necessity a study of tourism must be a net importer of ideas, themes, and concepts from the broader social sciences. Accordingly, this research discuss about how to draws on economics-tourism, development theory, environmental theory, social theory, and international relations, for example. Inevitably, this breadth of consideration will mean that a number of relevant aspects are not examined in depth, and do not necessarily cover the complexity of the matters under discussion. However, it will serve as a stimulant to further thinking, discussion, research, and study too. At the same time, it using the concepts of a range of academic and intellectual fields in order much better to understand about economics-tourism. The study of economic-tourism helps to illuminate more general economic, social, and environmental processes. Then, it just try not to know and see tourism as a discrete field of study. For both points stress the increasing significance of tourists with the second factor highlighting the importance of social class. This is not to say that class is only factor in study a tourism. INTRODUCTION But, it is a significant factor and is especially important for the analysis of new forms of tourism, in that the world of tourism is rife with the class distinction in everyday world (Crick, 1989: 334). Yet an analysis of the significance of class and tourism is only weakly developed. Naturally, the analysis of tourist has centre with around either classifying tourists or carrying out motivation and attitude surveys. Although, such approaches are interest in the case they have tended to limit the scope of tourism analysis. This research also has two points of host communities as objects of economic tourism or as controllers of economic tourism. So, it could be consideration for some peoples about the different between conventional mass tourism or new forms of economic tourism. Indeed, there are may be something happen to clear an idea between local authorities and local services providers for mass tourism clientele have been a greater degree to control and power over their activity too. Because, tourism is not only a form of trade, not of goods perhaps, although some commodities or local product of tourist destinations can talk about tourist local product is now firmly can established and accepted also (Rio Summit, 1992). Arden-Clarke argued that the whole dealing with trade amounted to an evasion of the key trade and environment issues, rather than a basis for their solution (1992: 13). TEORITICAL FRAMEWORK Tourism And Geographical Imagination Theory INTRODUCTION Globalization is a concept that is increasingly invoked for the analysis of tourism. The seemingly limitless spread of tourism to the four corners of the world, for the embracing of virtually any form for activities and the general ubiquity for tourists and tourism too. The temptation to reference globalization in research of tourism has been irresistible, often through casual and uncritical statements. Globalization has been an especially appealing concept for geographers because it can emphasizes for the way in which economic, social, cultural, and environmental relationship have been stretched and interwoven across the globe as well. Increasingly potential of tourism argues that tourism growth offers a means for Third World countries to escape the confines of underdevelopment and that new forms of tourism in particular allow this transition to be achieved sustainably and equitably. Building upon this fundamental precept, this research also explores and challenges the notions of sustainability, globalization, and development and their relationship to contemporary of economics tourism in the Trenggalek. Adopting a broad geography and conceptual perspective, the authors contend that a clear to understanding of the tourism process and output, then it is has also been relationship to development can be achieved by an interdisciplinary approach touching on environmentalism, sociocultural studies, human geography, economics, and development studies. Although over-simplifying, we could characterize the ‘geography of tourism’ as being primarily concerned with: the description of travel flows; micro-scale spatial structure and land use of tourist places and facilities; economics; social, culture, and environmental impacts of tourist activity; impacts of tourism in third world countries; geographic patterns of recreation and leisure pastimes; and the planning implications JEFMS, Volume 5 Issue 12 December 2022 www.ijefm.co.in Page 4000 Page 4000 Eco-Tourism: Concepts and Application by Smart Rural Areas: Case Study in Trenggalek, East Java of all these topics…These are vital elements of the study of travel and tourism. But, these sections are dealt within descriptive and weakly theory ways. (1991: 451) Eco-Tourism: Concepts and Application by Smart Rural Areas: Case Study in Trenggalek, East Java of all these topics…These are vital elements of the study of travel and tourism. But, these sections are dealt within descriptive and weakly theory ways. (1991: 451) Eco-Tourism: Concepts and Application by Smart Rural Areas: Case Study in Trenggalek, East Java of all these topics…These are vital elements of the study of travel and tourism. Eco-Tourism: Concepts and Application by Smart Rural Areas: Case Study in Trenggalek, East Java Finally, geographical imagination also makes a very distinctive contribution to our understanding of globalization and its impacts (Allen and Massey, 1995). There is a sense that we are living in a smaller, more compressed and interconnected world, and tourism is often invoked in this process of globalization, a process perceived differently, by different people in different places. On the one hand, places are drawn into the sphere of global tourism and the feeling of a smaller world encourages consumption of further places. On the other hand, some places deemed unattractive to tourism are marginalized from the processes of global interde- pendence. The relationship is rather a complex and symbiotic one. Tourism is both cause and consequence within globalization. Tourism in a shrinking world Globalization is a concept that is increasingly invoked in the analysis of tourism. With the seemingly limitless spread of tourism to the four corners of the world, the embracing or virtually any form of activity and the general ubiquity of tourists and tourism, the temptation to reference globalization in discussions of tourism has been irresistible, often through casual and uncritical state- ments. Globalization is much more than an abstract concept and represents a fact of our everyday lives that the world, in some crucial respects, has shrunk (Bauman, 1998). Globalization has been an especially appealing concept for geographers because it emphasizes the way in which economic, social, cultural, and environmental relationships have been stretched and interwoven across the globe. Economic globalization conveys the manner in which economic relationships and flows have been stretched across the globe. In the context of tourism, many point to the phenomenal growth of the industry in a global sense (it is now reputed to be the largest single industry) and the rapidity with which new places are continuously drawn into the tourism process. Take for example an average travel agent and consider the range of destinations on offer. Not only has the number of holiday destinations increased, but also the distances between destinations and markets has increased markedly, and we will be examining how new tourism practices have helped to accelerate this process. This also suggests that globalization is about capitalizing on the revolutions in telecommunications, finance, and transport, all of which have been instrumental in the globalization of tourism. In addition, tour- ism for an increasing number of Third World countries is big business. It has been suggested that it is not just capital and com- modities that can be transported and transferred easily across the world, but tourists too. It is necessary, therefore to consider how changes in contemporary global capitalism have impacted upon the development of tourism, a point we take up later. The Educational Element It is often stated that an important difference between the new forms of tourism and conventional tourism is found in an element of educational input into the activity. This does not mean that it is necessary to reach high academic levels in order to be a sus- tainable tourist; but a greater understanding of how our natural and human environment works is often a goal, if not always stated, of the activity as well, then it is stated as a goal without being practiced. One notable exception to this is Krippendorf (1987), who encourages the dissemination of information about the tourist to those they are visiting: By supplying the host population with comprehensive information about tourists and tourism, many misunderstanding could be eliminated, feelings of aggression prevented, more sympathetic attitudes developed and a better basis for hospitality and contact with tourists created…Such information should aim at introducing the host population…to the tourists’ background: their country, their daily life (working and housing conditions, etc.), their reasons for traveling and their behavior patterns. (1987: 143). Tourism And Geographical Imagination Theory Tourism is one of the principal ways through which our world-views are shaped. This not only results from our holidays, but also from the way destinations are represented by travel reviews, travel programs, and documentaries, travel brochures, and guides, advertising, then the way in which we exchange our holiday experiences. Some geographers have adopted the term geographical imagination as shorthand for these processes: the way we understand the geographical world, and the way in which we repre- sented it, to ourselves and to others (Massey, 1995c:41). It is also shorthand for emphasizing that activities, issues, places, and so on, are subject to competing interpretations. This involves the way in which we represent both our own activities (how we define ourselves as, for example, tourists, travelers, visitors, and what each of these categorizations entails) and the places in which we holiday (for example, built-up beach resorts or remote regions). This example of once divergent imaginations helps to emphasize two other points. First, some individuals, companies, institutions, and countries will be better able to diffuse their particular imaginations to others. When we think of Bali, Goa, or Hawaii, for example, the images and representations that are called forth are less likely to be of local people struggling to maintain cultural identity in the wake of mass tourism development and more likely to be of palm-fringed beaches and crystal blue waters (often the products of travel brochures, travel reviews, and holiday programs). In short, some imaginations are more powerful than others (Allen and Massey, 1995). JEFMS, Volume 5 Issue 12 December 2022 www.ijefm.co.in Page 4001 Page 4001 RESULTS The degree of tourism development differs considerably in each of the three settlement. Variables are describing income, house- hold economic strategies and demographic were analyzed for each of the above categories. The findings for this research is to increase social differentiation as a results of tourism developments, to assignment of the majority of beach to low status, low- paid, temporary jobs, can reduce access for local people to the natural resources on which they depend on their livelihoods, escalating for prices, land and agriculture product speculation, increased outside ownership of local resources and local commu- nity, then deterioration of the biophysical environment too. In Trenggalek, can pressure of business may render this, but cynicism may also explain it for the flimsiest pamphlet of any infor- mation for the tourist thatch it can be used as evidence an educational input, then genuine motives for the operators and the real desire to aim for sustainable economic tourism. For smart tourism card, economic sustainability, we can argue that it was not a condition which competes with other social as- pects. So, it was not the only condition of economic sustainability for tourism, but as might appear to be a case from the thought of numerous active peoples in the industry areas. Because, the real condition of this as an element of economic sustainability in no way reduces an significance to acceptance the villages conditions as well. Nor does it could very important for the contextual social issues of power over tourist activities. The question is still about who gains financially by stakeholder and who loses finan- cially often sets a power and control economic issues in sharper and more immediate focus to access transportation from every areas in Indonesia. METHODOLOGY This research is used qualitative data by secondary data and primary data. The techniques of this research is used by tools of sustainability to economic tourism such as area protection, industry regulation, visitor management techniques, Environmental Impact Assessment (EIA), carrying capacity calculations, consultation techniques, codes of conduct, and also sustainability indica- tors. Population in this research is a domestic tourist who visited the coastal tourist sites in Prigi. Where the retrieval is non- probability sampling for all research objects do not have the same opportunities to be selected as research samples, because in implementation used consideration by some certain things that are imposed to the sub group. Sampling that has been selected randomly for this research, that is by purposive sampling method. Purposive sampling means taking respondents based on certain considerations (Juanda, 2009). The number of selected respondents is 90 people based on the demographic aspect, the arrival, the purpose for the tour, and the benefits gained during the tour. Respondents were selected on terms of adulthood aged 17 years and over, physically and spiritually healthy, able to communicate well, and quite like to love traveling. Some tourists visit the crowd, ranging from two people to 7 people or more. Sampling is done during holidays, Saturday and Sunday. JEFMS, Volume 5 Issue 12 December 2022 Page 4002 www.ijefm.co.in Page 4002 Factors Affecting Demand (Frequency of Travel) Calculating Travel Costs is to estimate an economic value of coastal areas in Pr case, using the approach individually and can be used the following formula: C BT + BK + B + B + B + B (1) Calculating Travel Costs is to estimate an economic value of coastal areas in Prigi using the travel cost method approach. In this case, using the approach individually and can be used the following formula: C = BT + BK + BTM + BP + BW + B1…..(1) C = Traveling Towards Tour Area BT = Transportation Cost BK = Cost of Consumption BTM = Admission Fee BP = Parking Fee BW = Time Charge B1 = Other Costs Characteristics of Tourists Based on the characteristics of age, it turns out most of the respondents who make natural tourist visits to Watulimo district, which is 50% range of age around 17-25 years. Most of the domestic tourists in this young age group are not married and love to travel nature tours. Intensity visit about 3-5 times on Saturday or Sunday with friends. Some of them are student or college stu- dents, who really love nature with the feel of the beach and enjoy the beautiful sunset in the south of East Java. The second group is 38% of respondents with an age range of about 26-40 years. The third age group is 12% with respondents aged about 40 and above. In this case, the second and third age groups usually visit with family for nature and culture tours. Based on the level of education, the respondents of domestic tourists are dominated by the beach area tourists with high school that is equal to 50%. Then, the respondents of domestic tourists with undergraduate which is 38% mostly unmarried and love nature and cultural tourism. Meanwhile, the domestic tourist group with the last education level S2 12%, which is married and work both from Treng- galek and from out of town. Thus, as a whole can be seen from the characteristics of domestic tourists in Trenggalek. The respondents of domestic tourists who visit the coastal areas mostly work as private employees, entrepreneurs, and civil serv- ants who have income per month around Rp 1,500,000. - Rp 3,000,000. When viewed from the level of income, it can be concluded that tourism activities to the Prigi area is very affordable by domestic tourists from various levels of income even by tourists who have no income per month. Based on the frequency of domestic tourist visits for one year, it turns out that most of the approximately 10% of respondents of domestic tourists visit 1-3 times. They are domestic tourists who have known the tourist location for less than a year. Then by approximately 90%, domestic tourists visit more than 3 times because a lot of activities and they wanna refreshing with their JEFMS, Volume 5 Issue 12 December 2022 www.ijefm.co.in Page 4003 Page 4003 Eco-Tourism: Concepts and Application by Smart Rural Areas: Case Study in Trenggalek, East Java community to enjoy a magnificent of beach in Trenggalek. Total Economic Value The existence of nature tourism in Prigi area turned out to provide benefits of environmental and human resources better, either in the form of goods (local product) or services. One of the economically valuable environmental services is nature tourism ser- vices. Total economic value is known in the resources and environment as a value trying to describe the overall value of resources and environment in a particular region. The value is the sum of the use value and the non-use value. The value of environmental services can be utilized for tourism activities that are grouped as direct benefit or direct use value. This value is also reflected by the price of the use of a tourist site. This price is not only the entrance fee that has been paid, but by travel cost, cost efficiency time needed to do the tour. Because a number of individual tourist assessments of a tourist visit has been based on the hope of getting the tour (Wijayanti, 2009). Rating of Domestic Tourist To Karanggongso Beach Tourist Site As much as 20% argue that the mileage to go far enough with the road conditions are twisted. Around more than 50% of respond- ents from domestic tourists say that it is not difficult to reach the natural and cultural tourist sites in Prigi area. This is due to the road is quite good and not too narrow. In addition, beautiful natural scenery and cultural learning with the hospitality of officers make them increasingly get many benefits from these tourist destinations. Where, the location of Prigi tourist area has a fairly high level of natural beauty surrounded by several mountains and there are also mangrove forests. This beautiful location turned out to be away from the crowds of the city, congestion, and free of air pollution, so it is still quite beautiful. Some domestic tourists are less amenable to the lack of awareness of officials or local government of public facilities that are less well organized and less clean. Of the various characteristics above shows that the level of natural scenery and culture to be the highest value among others. Characteristics of Tourists Thus, it can be concluded that each tourist has a different frequency level of travel. As much as 75% of the respondents of domestic tourists in the area Prigi travel distance to the tourist attractions approximately 30 minutes. Unlike the case with respondents who have to travel a distance of more than 30 minutes, which is turned out a place to stay close with the tour. Visitor Management Techniques A range of visitor management techniques exist for use by those who cater for and control the movements of tourists. There are several texts which outline these in depth (Ceballos-Lascurain, 2001); Elkington and Hailes, 1992; Lavery, 1971; Lindberg and Haw- kins, 1993; Witt and Moutinho, 1994). So, worthy of particularly note is the current trend towards for the restriction of motorized vehicles in areas normally attractive to lovers of nature. In this case, on the premise that the motor car as currently run is inherently unsustainable. Then, this trend would seem like a move which the scientific community, the host, and also the planner could all agree works towards the goal of sustainability. The key of visitor management technique in Trenggalek is that of differential charg- ing for foreign and national visitors, which is a policy is not always understood by the visiting tourists from the North, but it promotes any condition of local tourists as an inherent aspect of sustainability. CONCLUSIONS In tourist-speak, suitable destinations are just as likely to be countries as they are to be specific small-scale resorts, towns or settlements. In fact a browse through the brochures of new forms of tourism shows that most are organized by country or even by groups of countries rather that by resort for community. But, the countries that tour operators speak of are nation states and are run by governments which often represent different interest and have different priorities from those of local communities. For local governments are more concerned with national planning strategies that it require to explore the potential of their natural environments. In other case, some ministers who speak radically, convincingly, and frequently about protection of the nation environmental and cultural treasures are the same people who sign some agreement which allow transnational companies to build a hotel or tourism complex whose development pays no heed to the environmental, social, and cultural impact caused. Governments are urged to improve and reorientate pricing and subsidy policies in issues related to tourism, to diversity beach economies by creating and strengthening tourism, to provide mechanism to preserve threaten areas that could protect wildlife, converse biological diversity or serve as national parks, to promote environmental sound leisure and tourism activities, building on beach areas, agricultural areas, and also culture areas with one tourism card. JEFMS, Volume 5 Issue 12 December 2022 www.ijefm.co.in Page 4004 Page 4004 There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. Eco-Tourism: Concepts and Application by Smart Rural Areas: Case Study in Trenggalek, East Java REFERENCES 1) Adams, W. 2001. Green Development: Environment and Sustainability In The Third World. Lon 1) Adams, W. 2001. Green Development: Environment and Sustainability In The Third World. London: Routledge, 2nd edn. 2) Briassoulis, H. 1992. 'Environmental impact of tourism: framework for analysis and evaluation', in H. Briassoulis and J. van der Straaten (eds), Tourism and Environment. London: Kluwer Academy. 2) Briassoulis, H. 1992. 'Environmental impact of tourism: framework for analysis and evaluation', in H. Briassoulis and J. van der Straaten (eds), Tourism and Environment. London: Kluwer Academy. 3) Cater, E. 1994. 'Ecotourism in the Third World: Issues And Prospects For Sustainability', At E. Carter and G. Lowman (eds). Ecotourism: Sustainable Choice?. Chichester: Wiley. 3) Cater, E. 1994. 'Ecotourism in the Third World: Issues And Prospects For Sustainability', At E. Carter and G. Lowman (eds). Ecotourism: Sustainable Choice?. Chichester: Wiley. 4) Fauzi A. 2014. Valuasi Ekonomi Dan Penilaian Kerusakan Sumber Daya Alam Dan Lingkungan. PT. Gramedia Pustaka Utama, Jakarta. 4) Fauzi A. 2014. Valuasi Ekonomi Dan Penilaian Kerusakan Sumber Daya Alam Dan Lingkungan. PT. Gramedia Pustaka Utama, Jakarta. 5) Forsyth, T. 1996. Sustainable Tourism: Moving from Theory To Practice, London: Tourism Concern. 6) Juanda B. 2009. Metode Penelitian Ekonomi Dan Bisnis. IPB Press, Bogor. 6) Juanda B. 2009. Metode Penelitian Ekonomi Dan Bisnis. IPB Press, Bogor. 7) Mason, P. and Mowforth, M. 1995. Codes Of Conduct In Tourism. Progress in Tourism and Hospitality Research, 2: 151- 67. 7) Mason, P. and Mowforth, M. 1995. Codes Of Conduct In Tourism. Progress in Tourism and Hospitality Research, 2: 151- 67. 8) Miller, G. 2001. Development Of Indicators For Sustainable Tourism: Delphi Survey Results Of Tourism Researchers. Tour- ism Management, 22: 351-62. 8) Miller, G. 2001. Development Of Indicators For Sustainable Tourism: Delphi Survey Results Of Tourism Researchers. Tour- ism Management, 22: 351-62. 9) Mowforth, M. and Munt, I. 2003. Tourism and sustainability: New development and tourism in the third world. London: Routledge, 2nd edn. 9) Mowforth, M. and Munt, I. 2003. Tourism and sustainability: New development and tourism in the third world. London: Routledge, 2nd edn. 11) Steward, J. and Hams, T. 1991. Local Government For Sustainability. Luton: Local Government Management Board. 11) Steward, J. and Hams, T. 1991. Local Government For Sustainability. Luton: Local Government Management Board. JEFMS, Volume 5 Issue 12 December 2022 Page 4005 www.ijefm.co.in
https://openalex.org/W2751175723	https://www.nature.com/articles/s41467-017-00462-2.pdf	English	null	Misalignment with the external light environment drives metabolic and cardiac dysfunction	Nature communications	2,017	cc-by	11,130	1 Division of Diabetes, Endocrinology and Gastroenterology, School of Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PL, UK. Correspondence and requests for materials should be addressed to D.A.B. (email: david.bechtold@manchester.ac.uk) ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 A further, more prevalent circadian insult occurs when the phase of our internal timing (chronotype) does not match with patterns of societal-driven activity (commonly referred to as social jet-lag). Indeed, large population and targeted cohort studies are now linking chronotype with a variety of health, psychiatric and life history variables10–13. Given that modern life has disturbed the natural temporal structure of our environment, it is critical that we understand the mechanisms which link clock desynchrony to pathophysiological outcomes. T In mammals, circadian timekeeping is centred on the feedback coupling of the transcriptional activators CLOCK and BMAL1, and repressors PERIOD, CRYPTOCHROME and REVERB. Much of our understanding of clock function has been deﬁned through genetic ablation of these and additional constituent factors14, 15. Animal studies demonstrate that genetic disruption of individual core clock genes can have a severe impact on heath, ranging from disturbances in metabolism and inﬂammatory response, to altered bone formation and neu- rodegeneration14, 16, 17. However, due to pleotropic and/or developmental activity of the targeted clock genes, it is rarely possible to isolate the inﬂuence of clock timing per se from that of the ablated factor18, 19. Moreover, in the context of human health, misalignment with the environment is overwhelmingly the principal source of circadian disruption, rather than the relatively minor contributions of genetic disruption in clock gene function. Despite human circadian desynchrony being widely acknowledged as deleterious to health, the reasons for increased risk remain unclear, and the pathways to disease undeﬁned. Here, we recapitulate in mice the circadian misalignment that occurs during shift work and in human subjects with extremechronotype20–22. We show that long-term housing of mice under light–dark (LD) cycles that do not match a normal 24 h cycle leads to pronounced physiological disturbance, including altered metabolic efﬁciency and substrate utilisation, and a profound depression of cardiac function, including signiﬁcant prolongation of PR and QT intervals. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 In mammals, circadian timekeeping is centred on the feedback coupling of the transcriptional activators CLOCK and BMAL1, and repressors PERIOD, CRYPTOCHROME and REVERB. Much of our understanding of clock function has been deﬁned through genetic ablation of these and additional constituent factors14, 15. Animal studies demonstrate that genetic disruption of individual core clock genes can have a severe impact on heath, ranging from disturbances in metabolism and inﬂammatory response, to altered bone formation and neu- rodegeneration14, 16, 17. However, due to pleotropic and/or developmental activity of the targeted clock genes, it is rarely possible to isolate the inﬂuence of clock timing per se from that of the ablated factor18, 19. Moreover, in the context of human health, misalignment with the environment is overwhelmingly the principal source of circadian disruption, rather than the relatively minor contributions of genetic disruption in clock gene function. Despite human circadian desynchrony being widely acknowledged as deleterious to health, the reasons for increased risk remain unclear, and the pathways to disease undeﬁned. Here, we recapitulate in mice the circadian misalignment that occurs during shift work and in human subjects with extremechronotype20–22. We show that long-term housing of mice under light–dark (LD) cycles that do not match a normal 24 h cycle leads to pronounced physiological disturbance, including altered metabolic efﬁciency and substrate utilisation, and a profound depression of cardiac function, including signiﬁcant prolongation of PR and QT intervals. Our study T he periodic succession of night and day inﬂuences nearly all forms of life on earth. As a result, organisms have evolved internal circadian clocks capable of keeping near precise 24 h time. By tracking time internally, organisms adapt their biology to match cyclical ﬂuctuations in the environment (e.g. light, food availability, predation) and thus respond optimally. In mammals, the circadian system consists of a network of tissue clocks housed across the body and coordinated by a central pacemaker located within the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Within all of these sites, the molecular clock machinery drives rhythmic transcriptional and metabolic pathways in a tissue-speciﬁc manner, a process critical to proper tissue function1–3. Altered circadian function, be it through genetic variation, lifestyle factors (e.g. chronic shift work, sleep restriction, nocturnal light exposure) or experimental perturbation (e.g. forced desynchrony) are linked to a wide range of pathogenic states from metabolic disease to cancer4–9. Misalignment with the external light environment drives metabolic and cardiac dysfunction DOI: 10.1038/s41467-017-00462-2 OPEN Alexander C. West1, Laura Smith1, David W. Ray 1, Andrew S.I. Loudon1, Timothy M. Brown1 & David A. Bechtold 1 Alexander C. West1, Laura Smith1, David W. Ray 1, Andrew S.I. Loudon1, Timoth & David A. Bechtold 1 & David A. Bechtold 1 Most organisms use internal biological clocks to match behavioural and physiological processes to speciﬁc phases of the day–night cycle. Central to this is the synchronisation of internal processes across multiple organ systems. Environmental desynchrony (e.g. shift work) profoundly impacts human health, increasing cardiovascular disease and diabetes risk, yet the underlying mechanisms remain unclear. Here, we characterise the impact of desynchrony between the internal clock and the external light–dark (LD) cycle on mammalian physiology. We reveal that even under stable LD environments, phase misalignment has a profound effect, with decreased metabolic efﬁciency and disrupted cardiac function including prolonged QT interval duration. Importantly, physiological dysfunction is not driven by disrupted core clock function, nor by an internal desynchrony between organs, but rather the altered phase relationship between the internal clockwork and the external environment. We suggest phase misalignment as a major driver of pathologies associated with shift work, chronotype and social jetlag. 1 Division of Diabetes, Endocrinology and Gastroenterology, School of Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PL, UK. Correspondence and requests for materials should be addressed to D.A.B. (email: david.bechtold@manchester.ac.uk) 1 NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 Diurnal proﬁles c–g reﬂect mean ± 95% CI. a, c–h ∗p < 0.05, ∗∗p < 0.01 one-way ANOVA ett’s post hoc test (vs. 24 h control group); b ∗∗p < 0.01 Students t-test; i ∗p < 0.05 vs. 24 h LD two-way ANOVA with Tukey’s post hoc test URE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 ARTIC 50 45 40 35 30 25 22.5 24 27 22.5 24 27 22.5 24 22.5 24 22.5 24 34 32 30 28 26 Fat weight (g) Lean weight (g) Body weight (g) Body weight (g) epWAT (mg/g BW) 25 20 15 10 5 0 30 0 10 20 60 40 20 0 * a b 7 22.5 24 22.5 24 22.5 24 34 32 30 28 26 Fat weight (g) Lean weight (g) Body weight (g) 25 20 15 10 5 0 60 40 20 0 b a 120 100 80 60 120 140 100 80 60 22.5 24 27 VO2 (ml/h) VO2 (ml/h) * c 0.60 0.55 0.50 0.45 0.40 0.35 0.7 0.6 0.5 0.4 0.3 22.5 24 27 EE (kcal/h) EE (kcal/h) d d c 1.10 1.05 1.00 0.95 0.90 0.85 0.80 1.1 1.0 0.9 0.8 0.7 22.5 24 27 RER RER (VCO2/VO2) e ** 22.5 24 27 COx (mg/min) COx (mg/min) 3 2 1 0 3 4 2 1 0 f f f e 0.4 0.3 0.2 0.1 0 27 h LD 24 h LD 22.5 h LD Food consumption (g) g 100 80 60 40 20 22.5 24 27 Food intake (mg/h) * * h 160 120 80 40 0 22.5 24 27 Food intake (mg/h) * * * Light Dark i i i g h Fig. 2 Phase misalignment drives reduced energy efﬁciency and metabolic disturbance. a Body weight and epididymal adipose tissue mass (epWAT) of mice maintained in 22.5, 24 or 27 h LD conditions for 17 week. epWAT was signiﬁcantly lower in 22.5 h LD mice compared with age-matched 24 h controls (n > 7/group). b By 1 year of LD exposure, both body weight and whole-body fat mass were signiﬁcantly lower in 22.5 h LD housed mice compared with matched 24 h LD mice (n = 9–10/group). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 lights the importance of phase alignment of the circadian 30 h symmetrical LD cycles for 17 week (from 8 week of 50 45 40 35 30 25 0.60 0.55 0.50 0.45 0.40 0.35 0.7 0.6 0.5 0.4 0.3 120 100 80 60 120 140 100 80 60 0.4 0.3 0.2 0.1 0 1.10 1.05 1.00 0.95 0.90 0.85 0.80 27 h LD 24 h LD 22.5 h LD 160 120 80 40 0 100 80 60 40 20 1.1 1.0 0.9 0.8 0.7 22.5 24 27 22.5 24 27 22.5 24 27 22.5 24 27 22.5 24 22.5 24 22.5 24 22.5 24 27 22.5 24 27 22.5 24 27 22.5 24 27 34 32 30 28 26 Fat weight (g) EE (kcal/h) COx (mg/min) COx (mg/min) EE (kcal/h) VO2 (ml/h) VO2 (ml/h) Food intake (mg/h) Food intake (mg/h) Food consumption (g) RER RER (VCO2/VO2) Lean weight (g) Body weight (g) Body weight (g) epWAT (mg/g BW) 25 20 15 10 5 0 30 0 10 20 60 40 20 0 ** 3 2 1 0 3 4 2 1 0 * * * * * * * Light Dark a b d c f e g h i 2 Phase misalignment drives reduced energy efﬁciency and metabolic disturbance. a Body weight and epididymal adipose tissue mass (epWAT maintained in 22.5, 24 or 27 h LD conditions for 17 week. epWAT was signiﬁcantly lower in 22.5 h LD mice compared with age-matched 24 h con 7/group). b By 1 year of LD exposure, both body weight and whole-body fat mass were signiﬁcantly lower in 22.5 h LD housed mice compared hed 24 h LD mice (n = 9–10/group). c–f Diurnal rhythms and mean values for oxygen consumption (VO2; c), energy expenditure (EE; d), respira ange rate (RER; e) and carbohydrate oxidation (COx; f) for mice maintained under 22.5, 24 and 27 h LD cycles (n = 7–16/group). A signiﬁcant incr R and COx was evident in non-24 h LD housed mice. g–i Diurnal proﬁles of food intake (g) and mean intake/h (h) reveal signiﬁcantly increased umption in mice maintained under either 22.5 or 27 h LD cycles compared with 24 h LD control. Mice housed under non-24 h LD cycles also umed more food during the light phase of the cycle (i). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 c–f Diurnal rhythms and mean values for oxygen consumption (VO2; c), energy expenditure (EE; d), respiratory exchange rate (RER; e) and carbohydrate oxidation (COx; f) for mice maintained under 22.5, 24 and 27 h LD cycles (n = 7–16/group). A signiﬁcant increase in RER and COx was evident in non-24 h LD housed mice. g–i Diurnal proﬁles of food intake (g) and mean intake/h (h) reveal signiﬁcantly increased food consumption in mice maintained under either 22.5 or 27 h LD cycles compared with 24 h LD control. Mice housed under non-24 h LD cycles also consumed more food during the light phase of the cycle (i). Diurnal proﬁles c–g reﬂect mean ± 95% CI. a, c–h ∗p < 0.05, ∗∗p < 0.01 one-way ANOVA with Dunnett’s post hoc test (vs. 24 h control group); b ∗∗p < 0.01 Students t-test; i ∗p < 0.05 vs. 24 h LD two-way ANOVA with Tukey’s post hoc test highlights the importance of phase alignment of the circadian clock to the environment and implicates disrupted entrainment, common place in the modern world, as being a major driver of pathology. 30 h symmetrical LD cycles for 17 week (from 8 week of age, n > 8/group). Mice maintained under 20 h or 30 h LD cycles were unable to entrain, and exhibited a free-running rhythm with a high degree of phase dispersion between individual animals (Fig. 1; Supplementary Fig. 1). In contrast, mice maintained in 22.5 h, 24 h and 27 h conditions achieved stable entrainment in locomotor activity and core body temperature (Tb). However, in comparison to 24 h LD housed mice, the phase of behavioural or physiological rhythms (as deﬁned by the acrophase of Tb and activity) were respectively delayed or advanced relative to the onset of night under 22.5 h and 27 h NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 Our study Tb (°C) 38 37 36 35 0 10 20 0 11.25 22.5 0 Time (h) 0 24 h 48 h 0 24 h 48 h 0 24 h 48 h 0 24 h 48 h 0 24 h 48 h 0 20 h 40 h 0 22.5 h 45 h 0 24 h 48 h 0 27 h 54 h 0 30 h LD 27 h LD 24 h LD 22.5 h LD 20 h LD Duration Duration 30 h 60 h 12 24 0 13.5 27 0 15 30 (Plotted on LD cycle) (Plotted on 24 h cycle) a b Fig. 1 Long-term housing under non-24 h LD cycles drives phase desynchrony between physiological rhythms and the LD cycle. a Representative body temperature (Tb) rhythms from mice maintained on 20, 22.5, 24, 27 and 30 h LD cycles. Top panels are double plotted relative to LD cycle time; bottom panels are plotted on 24 h frequency. b Group Tb proﬁles across the LD cycle (derived from 10 consecutive cycles, after 40 d LD exposure) highlight th delayed (22.5 h) and advanced (27 h) phase of entrainment in non-24 h LD housed mice. Data in b reﬂect mean ± SEM, n = 7/group for 20 and 30 h LD n = 10/group for 22.5, 24 and 27 h LD a 24 h 24 h LD 30 h LD 30 h 20 h 20 h LD 27 h 27 h LD 30 h LD 30 h 22.5 h 22.5 h LD b Fig. 1 Long-term housing under non-24 h LD cycles drives phase desynchrony between physiological rhythms and the LD cycle. a Representative body temperature (Tb) rhythms from mice maintained on 20, 22.5, 24, 27 and 30 h LD cycles. Top panels are double plotted relative to LD cycle time; bottom panels are plotted on 24 h frequency. b Group Tb proﬁles across the LD cycle (derived from 10 consecutive cycles, after 40 d LD exposure) highlight the delayed (22.5 h) and advanced (27 h) phase of entrainment in non-24 h LD housed mice. Data in b reﬂect mean ± SEM, n = 7/group for 20 and 30 h LD; n = 10/group for 22.5, 24 and 27 h LD NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications 2 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 conditions (Fig. 1a, b; Supplementary Fig. 1). Thus, the high degree of phase coordination among individual mice and the long-term stability of their phase alignment relative to the LD cycle make the 22.5 h and 27 h LD cycle conditions ideal to test the physiological consequence of environmental desynchrony. energy expenditure was examined. In comparison to matched 24 h LD housed mice, a signiﬁcant reduction in adiposity was observed in 22.5 h LD conditions (Fig. 2a). No signiﬁcant differences were observed in comparable analyses of mice housed under 27 h LD conditions. Reduced fat mass in 22.5 h LD mice was exacerbated by12 months of 22.5 h LD exposure, by which time a signiﬁcantly lower body weight was also evident (Fig. 2b). Results Entrainment to non-24 h light cycles leads to reduced energy efﬁciency. To establish an altered phase relationship between the environmental LD cycle and the internal circadian clock, male C57Bl/6J mice were placed under 20 h, 22.5 h, 24 h, 27 h and 3 NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 5b; analyses limited to periods of >20 min of inactivity before HR measure).Critically, longitudinal analyses of ECG recordings in wild-type C57B6J mice revealed that non-resonant LD conditions not only increased inter-beat (RR) interval (Fig. 5d), but also slowed cardiac conduction parameters, with signiﬁcantly lengthening of PR, QT and RR-adjusted QT (QTc) intervals evident in mice housed under either 22.5 or 27 h LD cycles (Fig. 5e–h). Entrainment to non-24 h LD cycles does not disrupt clock function. Despite the clear phase misalignment relative to the light cycle, mice entrained to non-resonant LD cycles exhibited a robust amplitude and consistent phase across different physiological parameters (Supplementary Fig. 1), suggesting that internal circadian timing is maintained in the animals. Therefore, we next sought to identify how central and peripheral oscillators respond to non-24 h LD cycles, with a focus on the 22.5 h LD condition. Quantiﬁcation of Per1 in the master SCN clock revealed a peak in expression during the early light phase within the 24 h LD housed mice (Fig. 3a, b). In line with the altered timing of activity and Tb in 22.5 h LD housed mice, the rhythm in Per1 expression in the SCN of these mice was signiﬁcantly delayed in phase and dampened in amplitude relative to the 24 h LD group (Fig. 3a, b). This was reﬂected in circulating corticos- terone, an important synchronising agent for peripheral tissue clocks whose secretion is strongly inﬂuenced by the SCN26. Corticosterone rhythms were signiﬁcantly delayed in phase in mice entrained to 22.5 h LD cycles, when compared with 24 h LD housed animals (Fig. 3c, d). Rhythms in both SCN gene expression and corticosterone were signiﬁcantly dampened in the 22.5 h LD housed mice, suggesting that the phase misalignment with the external LD cycle weakens the central oscillator. y g Prolonged inter-beat intervals can result from changes in repolarising potassium channels35. However, we did not observe aberrant ion channel expression in the 22.5 h LD mice (Supplementary Fig. 2). In our studies, HR slowing occurred rapidly upon transition from 24 h to non-resonant LD cycles, and was evident in mice switched from 24 h LD to constant light (Supplementary Fig. 3). A similar LD cycle-dependent slowing of HR was observed in the accelerated period CK1εtau mutant mice (free-running period of 20 h), when housed under 22.5 h LD cycles (Supplementary Fig. 3). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 Therefore, we next proﬁled regulatory factors and pathways known to be under circadian control (Fig. 4; Supplementary Fig. 2). The expression proﬁle of manyfactors (e.g. wee1) matched that of the core clock, remaining robustly rhythmic but delayed in phase under 22.5 h LD conditions. However, many other factors (e.g. nrf2, pparg, ezh2, RelA) exhibited signiﬁcant transcriptional dysregulation,with alterations in amplitude and/or mesor. Dysregulation of affectedpathways (e.g. antioxident response, histone/DNA methylation) was highly tissue speciﬁc, demonstrating the tissue selective impact of phase misalignment (Supplementary Fig. 2). Thus,despite maintenance of robust core circadian clock function in non-resonant mice, disruption of timing was evident in a number of key clock-output pathways. mice exhibited higher rates of both oxygen consumption (VO2; Fig. 2c) and energy expenditure (EE; Fig. 2d). Interestingly, both the 22.5 h and 27 h LD conditions caused a signiﬁcant increase in respiratory exchange rate (RER) and carbohydrate oxidation (COx) rate (Fig. 2e, f), indicating an increased reliance on carbohydrate substrates for energy generation in these mice. To assess feeding behaviour, mice were temporarily housed in automated feeding cages, and food intake monitored continuously for 5 days. In line with the increase in RER and COx, mice housed under non-24 h LD conditions exhibited a signiﬁcant increase in food intake when compared with matched 24 h LD controls (Fig. 2g, h), which was due in large part to an increased intake during the light phase of the cycle (Fig. 2i). Together these ﬁndings show that entrainment to non-24 h LD cycles has a pronounced effect on energy efﬁciency (higher food intake without increased body weight) and substrate utilisation. Exposure to non-resonant LD cycles impairs cardiac function. The heart houses a robust circadian clock (Fig. 3e)32, 33, and cardiovascular health is adversely affected by shift work and forced desynchrony protocols9, 34. We therefore examined the impact of 22.5 h and 27 h LD cycles on heart rate (HR) and electrocardiogram (ECG) parameters. Mice exhibited a robust diurnal rhythm in HR under 24 h LD; however both 22.5 h and 27 h LD cycles disrupted the HR rhythms, and signiﬁcantly slowed HR across the LD cycle (Fig. 5a; Supplementary Fig. 1d–f). The reduction in HR was not related to changes in locomotor activity (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 Consistent with reduced adiposity, 22.5 h LD housed p y g q y y Energy metabolism is tightly coupled to the circadian clock23–25; therefore, the impact of 22.5 h and 27 h LD environment on body weight, adiposity, feeding behaviour and 24 h 22.5 h a a 24 h 22.5 h 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 3 2 1 0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 1.5 1.5 1.0 1.0 2.0 0.5 0.5 0.0 0.0 3 2 1 0 Liver Lung Adrenal Heart Kidney EpFat 80 60 40 20 0 2.0 1.5 1.0 0.5 0.0 1.5 1.0 2.5 2.0 0.5 0.0 4 3 2 1 0 30 20 10 0 300 200 100 0 15 10 5 0 15 10 5 0 15 10 5 0 1.5 1.0 0.5 0.0 1.5 2.5 1.0 2.0 0.5 0.0 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * a a 4 3 2 1 0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 80 100 60 40 20 0 80 100 60 40 20 0 60 40 20 0 30 20 10 0 30 40 20 10 0 30 40 20 10 0 30 40 20 10 0 120 80 40 0 25 20 15 10 5 0 120 80 40 0 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 6 4 2 0 8 6 4 2 0 8 6 4 2 0 Corticosterone (ng/ml) Relative OD (a.u.) 80 100 60 40 20 0 a a Per1 Cort 24 h LD 22.5 h LD 24 h 22.5 h Liver Bmal1 Per2 Cry1 Nr1D1 Dbp Lung Adrenal gland Heart Relative expression (vs 24 h LD ZT0) Kidney epWAT scWAT Pancreas 24 h a b c d e f 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 3 2 1 0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 1.5 1.5 1.0 1.0 2.0 0.5 0.5 0.0 0.0 3 2 1 0 Liver Lung Adrenal Heart Kidney EpFat ScFat Pancreas 80 60 40 20 0 2.0 1.5 1.0 0.5 0.0 1.5 1.0 2.5 2.0 0.5 0.0 4 3 2 1 0 30 20 10 0 300 200 100 0 15 10 5 0 15 10 5 0 15 10 5 0 1.5 1.0 0.5 0.0 1.5 2.5 1.0 2.0 0.5 0.0 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * a a 4 3 2 1 0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 80 100 60 40 20 0 80 100 60 40 20 0 60 40 20 0 30 20 10 0 30 40 20 10 0 30 40 20 10 0 30 40 20 10 0 120 80 40 0 25 20 15 10 5 0 120 80 40 0 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 6 4 2 0 8 6 4 2 0 8 6 4 2 0 Corticosterone (ng/ml) Relative OD (a.u.) 80 100 60 40 20 0 a a Per1 Cort 24 h LD 22.5 h LD 24 h 22.5 h Liver Bmal1 Per2 Cry1 Nr1D1 Dbp Lung Adrenal gland Heart Relative expression (vs 24 h LD ZT0) Kidney epWAT scWAT Pancreas 24 h 22.5 h b c d e f NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecomm Corticosterone (ng/ml) 80 100 60 40 20 0 a c 2.0 1.5 1.0 0.5 0.0 Relative OD (a.u.) a b Per1 Cort 24 h LD 22.5 h LD d b d c 2.0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 3 2 1 0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 80 60 40 20 0 2.0 1.5 1.0 0.5 0.0 1.5 1.0 2.5 2.0 0.5 0.0 4 3 2 1 0 30 20 10 0 300 200 100 0 15 10 5 0 15 10 5 0 15 10 5 1.5 1.0 0.5 0.0 1.5 2.5 1.0 2.0 0 5 * * * * * * * * * * * * * * * * * * * * * * * a a 30 40 20 10 0 120 80 40 0 25 20 15 10 5 0 120 80 40 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 6 4 2 24 h 22.5 h Liver Bmal1 Per2 Cry1 Nr1D1 Dbp Lung Adrenal gland Heart expression (vs 24 h LD ZT0) Kidney e 2.0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 3 2 1 0 1.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 1.5 1.5 1.0 1.0 2.0 0.5 0.5 0.0 0.0 3 2 1 0 Liver Lung Adrenal 80 60 40 20 0 2.0 1.5 1.0 0.5 0.0 1.5 1.0 2.5 2.0 0.5 0.0 4 3 2 1 0 30 20 10 0 300 200 100 0 15 10 5 0 15 10 5 0 15 10 5 0 1.5 1.0 0.5 0.0 1.5 2.5 1.0 2.0 0.5 0.0 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * a a 4 3 2 1 0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 80 100 60 40 20 0 80 100 60 40 20 0 60 40 20 0 30 20 10 0 30 40 20 10 0 30 40 20 10 0 30 40 20 10 0 120 80 40 0 25 20 15 10 5 0 120 80 40 0 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 8 6 4 2 0 6 4 2 0 8 6 4 2 0 8 6 4 2 0 24 h 22.5 h Liver Bmal1 Per2 Cry1 Nr1D1 Dbp Lung Adrenal gland Heart Relative expression (vs 24 h LD ZT0) Kidney epWAT scWAT Pancreas e f e Adrenal gland NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 clock. Fig. 3 Robust circadian rhythms in clock gene expression are maintained in non-24 h LD housed mice. Representative brain sections a and quantiﬁcation b of radioactive in situ hybridisation for Per1 expression in the SCN from mice housed in 24 h (black) or 22.5 h (red) conditions for 17 week. c Circulating corticosterone proﬁles were delayed relative to the LD cycle in mice housed under or 22.5 h conditions. d Acrophase analysis of Per1 and corticosterone rhythms in 24 and 22.5 h housed mice reveals consistent phase delay. e Proﬁling of clock gene expression in peripheral tissues of mice housed in 24 or 22.5 h LD conditions for 17 weeks. f Acrophase analyses across genes and tissues (plotted relative to respective light cycle) demonstrates that synchronisation both within and across tissue clocks is maintained in non-24 h LD conditions. All data plotted mean ± SEM relative to the respective light cycle (24 or 22.5 h) and normalised to ZT0 of the 24 h LD group (n = 4/time-point/group). ∗Signiﬁcant (p < 0.05) difference in phase between 24 and 22.5 h proﬁles; a signiﬁcant difference in amplitude or mesor between 24 and 22.5 h LD conditions (sinusoidal waveform ﬁts with F tests for shared characteristics). Scale bar in a = 3 mm NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 Our studies suggest that suppression of cardiac function and lengthening of QT, results from discordance between the phase of the local clockwork and extrinsic signals such as altered autonomic input to the heart. We tested this directly in 22.5 and 24 h LD housed mice by blocking vagal and sympathetic input to the heart using atropine/propranolol administration38–40. The impact of autonomic block on HR was assessed in both the active (2 h after lights on; ~ZT2) and inactive (2 h after lights off, ~ZT14) phases of the cycle under both lighting conditions. Indeed, at both time points, complete autonomic blockade caused a signiﬁcant increase in HR under 22.5 h LD, but not in 24 h LD housed mice and resulted in comparable HR in both groups (Fig. 5k–m). Thus, our ﬁndings clearly demonstrate that discordance between environmental light/dark cycles and the internal clockwork cause a rapid and profound depression of cardiac function, including prolonged PR and QT interval. This effect is independent of locomotor activity, and is mediated by altered autonomic signalling. indicate that it is the altered phase relationship between the external light cycle and internal circadian timing that drives the aberrant physiology. Many animal studies have inferred the importance of circadian timing for maintaining a healthy physiological state through genetic disruption of one or more components of the underlying molecular clock. However, this approach rarely allows the importance of altered timing per se to be determined due to constitutive loss of the factor and its pleiotropic actions19. In contrast, resonance studies, as we use here, exploit the altered interaction between an intact biological clock and its external environment. The adaptive beneﬁt of matching internal circadian time to environmental cycles has been robustly demonstrated in lower organisms18, and a few studies have demonstrated that altered circadian timing can reduce survival ﬁtness in natural settings41, 42. Yet few studies have used circadian resonance to reveal the impact of circadian timing to internal physiology and health in mammalian models. Here we show that under non-24 h LD cycles, mice exhibited markedly reduced energy efﬁciency and increased reliance on carbohydrate energy substrates. This ﬁnding reinforces the long held assertion that circadian timing serves to optimise response to the environment, including optimising cycles of energy storage and mobilisation. Decreased energy efﬁciency was at least in part due to increased energy expenditure in the 22.5 h LD housed mice. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 3 2 1 0 3 1.4 1.2 1.0 0.8 0.6 1.4 1.3 1.2 1.1 1.0 0.9 0.8 1.50 1.25 1.00 0.75 0.50 2 1 0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 2.0 1.5 2.5 1.0 0.5 0.0 2.0 1.5 2.5 1.0 0.5 0.0 1.5 1.0 0.5 0.0 5 4 3 2 1 0 4 3 2 1 0 3 2 1 0 4 5 3 2 1 0 10 8 6 4 2 0 Wee1 Adrenal gland Heart epWAT Liver Relative expression (vs 24 h LD ZT0) * * * * a a a a a a a a a a a a Pparγ Ezh2 Nrf2 RelA 2.0 1.5 1.0 0.5 0.0 4 5 3 2 1 0 24 h LD 22.5 h LD Fig. 4 Phase misalignment disrupts rhythmic gene expression in a pathways and tissue-speciﬁc manner. Transcript proﬁles of major clock-controlled regulator genes of cell cycle (Wee1), epigenetic regulation (Ezh2), metabolism (Pparγ), oxidative stress (Nrf2) and inﬂammation (RelA) from mice housed in 24 or 22.5 h conditions for 17 weeks. All data plotted mean ± SEM relative to the respective light cycle (24 or 22.5 h) and normalised to ZT0 of the 24 h LD group (n = 4/time-point/group). ∗Signiﬁcant (p < 0.05) difference in phase between 24 and 22.5 h proﬁles; a = signiﬁcant difference in amplitude or mesor between 24 and 22.5 h LD conditions (sinusoidal waveform ﬁts with F tests for shared characteristics) Fig. 4 Phase misalignment disrupts rhythmic gene expression in a pathways and tissue-speciﬁc manner. Transcript proﬁles of major clock-controlled regulator genes of cell cycle (Wee1), epigenetic regulation (Ezh2), metabolism (Pparγ), oxidative stress (Nrf2) and inﬂammation (RelA) from mice housed in 24 or 22.5 h conditions for 17 weeks. All data plotted mean ± SEM relative to the respective light cycle (24 or 22.5 h) and normalised to ZT0 of the 24 h LD group (n = 4/time-point/group). ∗Signiﬁcant (p < 0.05) difference in phase between 24 and 22.5 h proﬁles; a = signiﬁcant difference in amplitude or mesor between 24 and 22.5 h LD conditions (sinusoidal waveform ﬁts with F tests for shared characteristics) HR can be acutely affected by environmental light via autonomic signalling36, 37. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 This suggests that aberrant light exposure drives altered cardiac function. Due to the altered phase of entrainment, both 22.5 and 27 h LD housed mice are exposed to light at inappropriate times (relative to their internal rhythm). Therefore, we assessed the effect of acute mistimed light exposure on HR by exposing control 24 h LD housed mice to a 2 h light pulse in the early active (dark) phase (ZT14-16). As expected, light exposure caused a signiﬁcant reduction in activity, Tb and HR. However, unlike Tb and activity, mistimed light caused a profound decrease in HR that remained depressed in light-pulsed mice for a further two LD cycles as the mice regained normal phase alignment to the LD cycle (Fig. 5i, j; Supplementary Fig. 4). Contrary to expectation, transcript proﬁles in peripheral tissues remained strongly rhythmic in both 24 h and 22.5 h LD housed groups (Fig. 3e). Strikingly, acrophase analyses revealed a consistent phase-delay relative to the LD cycle across all of the peripheral tissues examined (Fig. 3f), similar to that observed in the SCN. There was also a remarkable preservation of the phase relationship of clock components, both within and between tissue sets. A few exceptions were evident (e.g. Bmal1 expression in the lung), but these were limited to single gene proﬁles in individual tissues. Thus, despite chronic entrainment to a short-running LD cycle, the circadian system remains intact and faithfully retains its amplitude and internal synchrony. y y Many cellular processes including metabolism27, cell cycle28, oxidative stress29, inﬂammation30 and epigenetic modiﬁcation31 are subject todirect transcriptional control bycomponents of the 5 5 NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 Phase misalignment may also lead to reduced nutrient absorption and/or utilisation as both are inﬂuenced by the circadian system43. In a notable study, Martino and colleagues reported that short period CK1εtau/+ mutant hamsters (endogenous period of ~22 h) were prone to cardio-renal dysfunction when housed under 24 h LD cycles, which did not manifest if the animals were maintained on a 22 h LD cycle44. In agreement with our studies, this indicates that an altered phase of entrainment is sufﬁcient to drive pathology, and highlights a particular vulnerability of the cardiovascular system. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 In pursuit of further consequences of environmental desynchrony, we proﬁled clock output genes across tissues. These studies showed tissue speciﬁc, and gene speciﬁc patterns of dysregulation, with enrichment for speciﬁc pathways (notably oxidative stress response in the heart and adrenal gland). Disruption of rhythmic transcription despite robust circadian clock gene oscillation has been demonstrated previously in both mice and humans45–47, indicating a de-coupling possibly due to mistimed external signals. Moreover, it is becoming clear that rhythmic processes within a tissue can be remodelled and detached from the circadian clock under environmental or pathological challenge48, 49. The altered expression of epigenetic regulators (e.g. EZH2, TET enzymes) in response to non-24 LD schedules, is strikingly similar to that observed in the SCN of 22 h LD housed mice reported by Brown and colleagues45, and suggests that epigenetic mechanisms drive reprogramming of clock-controlled processes during phase misalignment. Discussion h d These studies provide direct evidence that chronic desynchrony between the internal circadian clock and environmental light cycles profoundly impacts mammalian physiology. Housing mice under non-24 h LD cycles created an experimental paradigm, which mimics the phase misalignment experienced by humans with extreme chronotype and those engaged in shift work. Despite achieving stable entrainment, phase misalignment had a rapid impact on energy metabolism and cardiac rate and conduction, which did not diminish over many months. Remarkably, dysregulated metabolic and cardiac proﬁles were not driven by disruption of the molecular clockwork, nor by a loss of phase synchrony between different tissue oscillators (internal desynchrony). Instead, our studies clearly NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 ARTICLE Methods Animals. All animal experiments were licenced under the Animals (Scientiﬁc Procedures) Act of 1986 (UK), and conducted in accordance with University of Manchester animal welfare committee guidelines. Male C57Bl/6J mice were purchased from Charles River (UK), and Ck1εtau mice67 were bred at the Uni- versity of Manchester. At 8 weeks of age, male mice were assigned randomly to each LD condition and transferred into light-tight housing cabinets and main- tained in 20, 22.5, 24, 27 or 30 h LD cycles for 17–52 weeks. Light levels were maintained at 463 lux during the light phase and during light pulse experiments. Ambient temperature was 22 ± 2 °C, with food and water available ad libitum. Mice remained group housed throughout, except where single housing was necessitated by physiological monitoring (e.g. CLAMS, ECG telemetry, food intake recording). Due to different lighting schedules, blinding of the experimenter to experimental conditions was not possible. Behavioural and physiological monitoring. To assess body composition, whole body lean and fat mass was assessed using the EchoMRI system (Echo Medical Systems). For long-term (>10 week) recording of body temperature, mice were implanted with iButton temperature loggers (Maxim, DS1922L-F5). Implants were de-housed, programmed and encapsulated in a 20% Poly(ethylene-co-vinyl acetate) and 80% parafﬁn mixture68 before implantation into the peritoneal cavity (ip). Temperature recordings were calibrated by immersion of the iButtons into set temperature water baths before implantation. In separate experiments, Tb and locomotor activity rhythms were recorded using indwelling radio-telemetry devices (TA-F10, DSI international) implanted ip. Continuous measures of food intake were recorded from singly housed animals using PhenoMaster behavioural cages (TSE Systems). Mice (>8 week LD cycle exposure) were acclimatised to the cages for two cycles, after which food consumption was measured in 5 min bins for 3–4 LD cycles. To assess metabolic gas exchange, mice were individually housed in indirect calorimetry cages (CLAMS, Columbus Instruments). As above, mice (>8 week LD cycle exposure) were acclimatised to the cages for two cycles, fol- lowing which O2 consumption and CO2 production were recorded every 10 min for >3 LD cycles. RER was derived from these measures (VCO2/VO2), as were protein oxidation independent COx (4.55∗VCO2−3.21∗VO2)69 and energy expenditure (3.815∗VO2 + 1.232∗VCO2). Remarkably, depression of HR was observed in response to acute mistimed light exposure, which persisted over several days. Methods The detrimental impact of mistimed light is clear, and is supported by reports of depressed HR and prolonged QT in mice housed under constant light or subject to light-restricted feeding57. Cardiomyocyte-speciﬁc deletion of Bmal1 leads to a similar lengthening of QRS and QTc intervals, which was ascribed to aberrant expression of cardiac ion channels Scn5a and Kcnh258, 59. We did not observe altered expression of Scn5a, Kcnh2, or a number of other ion channels in cardiac tissue of 22.5 h housed mice. We show that autonomic block can normalise HR in non-resonant LD housed mice, demonstrating an underlying role for inappropriate autonomic signalling during phase misalignment with the LD cycle. In contrast to the mice, acute light exposure increases HR in humans. Nevertheless, important parallels exist between rodent and human HR/ECG responses to altered lighting/circadian desynchrony. Rats and humans exhibit a similar diurnal variation in the fractal structure of HR (a measure of HR variability), despite the opposite nature of diurnal/nocturnal activity in the species60. Most importantly, altered autonomic signalling and long QTc interval have been consistently reported in studies of shift work in humans (e.g. refs 61–63). Thus, in both humans and mice, the strong inﬂuence of both extrinsic (autonomic) and intrinsic (local car- diac clock) timing signal to cardiac pacing is likely to increase the vulnerability of the cardiovascular system to dysfunction under conditions of circadian disruption. Heart rate and ECG recording. Mice were implanted with ETA-F10 radio-tele- metry devices (Data sciences international) for recording of locomotor activity, Tb and ECG. Devices were implanted ip with ECG recording leads brought through the abdominal wall and negative lead secured ~1 cm right of midline at upper chest, positive lead secured ~1 cm to the left of midline at the xiphoid plexus. Mice recovered for 7–10 days before the start of recording the experiment. Mice were excluded from the study if lead placement was not maintained throughout the experiment. For longitudinal studies (i.e. >7 days), activity, Tb and 10 s ECG data sweeps were collected every 10 min, and a minimum of three consecutive days used for each analyses period. ‘Inactive heart rate’ was deﬁned as periods where no activity had been recorded for >20 min before the ECG sweep recording. Following recording, ECG waveforms (10 s sweeps at 1 sweep/10 min) were processed and analysed using bespoke software programme written in Matlab (Mathworks). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 Cardiac physiology exhibits pronounced circadian rhythmicity, driven both by the local cardiac clock and through circadian variation in autonomic input to the heart32, 33, 50–53. In our studies, control mice exhibited robust rhythms in ECG parameters including RR, PR and QTc. All of these parameters were signiﬁcantly prolonged when mice were placed under non-24 LD cycles. Altered cardiac conduction is associated with severe pathology and sudden cardiac death, and is therefore of profound interest to human health. The incidence of sudden cardiac death follows a strong diurnal rhythm, with peak incidence around the transition from sleep to wake54, 55; and increased incidence of heart disease and cardiac events is associated with nightshift workers56. Our results indicate that mistimed light drives inappropriate autonomic input to the heart, and suggests that circadian misalignment increases vulnerability to conduction block and ventricular arrhythmia (due to signiﬁcant lengthening of PR and QTc). The dampening of the central SCN circadian oscillator observed in the non-resonant housed mice may weaken normal circadian control over autonomic output to the heart and other tissues, and thereby exacerbate phase misalignment between the local tissue clocks and autonomic drive. on circadian phase in humans22, 64–66, our ﬁndings provide important new insight into human pathologies associated with shift work, chronotype and social jetlag. Fig. 5 Misalignment with environmental LD cycles disrupts cardiac rate and leads to long QT interval. a Incomparison to matched 24 h LD cycle housed mice, mean heart rate (HR) was signiﬁcantly reduced in mice maintained in 22.5 and 27 h LD cycles, which was accompanied by a signiﬁcant reduction in the amplitude of HR rhythms in the 27 h LD mice. b HR remained signiﬁcantly reduced in non-24 h LD housed mice even when analyses were limited to periods of inactive (no recorded movement for 20 min preceding HR measure). c Murine ECG beat waveform with deﬁning features labelled. d–h Diurnal proﬁles and mean interval durations for RR (d), PR (e), QRS (f), QT (g) and RR-adjusted QT (QTc; h) of mice maintained under 24 h, 22.5 h or 27 h LD conditions. All parameters were signiﬁcantly lengthened under non-24 h LD cycles. i, j A 2 h light pulse (LP, red) from ZT14-16 caused a signiﬁcant reduction in night-time HR when compared with the average HR proﬁle recorded over three preceding cycles (blue, NLP). k–m Complete autonomic blockade (AB) increased HR at ZT2 (~2 h after lights on; k) and ZT14 (~2 h after lights off; l) in 22.5 h LD housed mice, and normalised it to 24 h LD conditions. m Change in HR (vs. baseline) during autonomic blockade at ZT2 and ZT14. Diurnal proﬁles depict mean ± 95%CI. ∗p < 0.05, ∗∗p < 0.01. a–h One-way ANOVA with Dunnett’s post hoc test (vs. 24 h control group); j paired t-test; k–m repeated measures two-way ANOVA with Tukey’s post hoc test NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 750 650 550 450 PR RR R R P P T T J J Q Q S S QT QRS HR (BPM) Inactive HR (BPM) HR (BPM) 22.5 24 27 22.5 24 27 22.5 h 24 h 27 h 700 600 500 400 300 700 650 600 550 500 450 a b c HR (BPM) 22.5 24 27 700 650 600 550 500 450 750 650 550 450 HR (BPM) Inactive HR (BPM) HR (BPM) 22.5 24 27 22.5 h 24 h 27 h 700 650 600 550 500 450 a b Inactive HR (BPM) 22.5 24 27 700 600 500 400 300 b b 750 650 550 450 HR (BPM) 22.5 h 24 h 27 h a PR RR R R P P T T J J Q Q S S QT QRS c a c 750 650 550 450 PR RR R R P P T T J J Q Q S S QT QRS HR (BPM) Inactive HR (BPM) HR (BPM) RR interval (ms) QRS interval (ms) QRS interval (ms) QTc QT interval (ms) QT interval (ms) RR interval (ms) PR interval (ms) PR interval (ms) 9 8 7 6 9 10 60 55 50 45 40 35 ** No pulse Pulse 700 600 500 400 NLP LP 700 600 500 400 300 60 50 40 30 60 50 40 30 QTc 60 50 40 30 8 7 6 5 160 140 120 100 80 130 120 110 100 90 22.5 24 27 22.5 24 27 22.5 24 27 22.5 24 27 22.5 24 27 22.5 24 27 22.5 24 27 22.5 h 24 h 27 h 700 600 500 400 300 40 35 30 25 * * 40 35 45 30 25 700 650 600 550 500 450 HR (BPM) HR (BPM) 650 600 550 450 500 400 150 100 50 0 –50 650 600 550 450 500 400 HR (BPM) ΔHR (BPM) ZT14 ** * * * ZT2 22.5 24 22.5 24 AB Baseline 22.5 24 22.5 24 AB Baseline 22.5 24 22.5 24 ZT14 ZT2 * a b d e h i f g c j k l m E COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications RR interval (ms) RR interval (ms) 160 140 120 100 80 130 120 110 100 90 22.5 24 27 d PR interval (ms) PR interval (ms) 22.5 24 27 40 35 30 25 * * 40 35 45 30 25 e d e QRS interval (ms) QRS interval (ms) 9 8 7 6 9 10 8 7 6 5 22.5 24 27 * f QT interval (ms) QT interval (ms) 60 55 50 45 40 35 60 50 40 30 22.5 24 27 g g f QTc No pulse Pulse 700 600 500 400 NLP LP 700 600 500 400 300 60 50 40 30 QTc 60 50 40 30 22.5 24 27 HR (BPM) HR (BPM) h i j QTc 60 50 40 30 QTc 60 50 40 30 22.5 24 27 h No pulse Pulse 700 600 500 400 300 HR (BPM) i 700 600 500 400 NLP LP HR (BPM) j j i h 650 600 550 450 500 400 HR (BPM) * ZT2 22.5 24 22.5 24 AB Baseline k l 650 600 550 450 500 400 ZT14 ** * 22.5 24 22.5 24 AB Baseline l 150 100 50 0 –50 ΔHR (BPM) * 22.5 24 22.5 24 ZT14 ZT2 m l l k m AB Baseline NATURE COMMUNICATIONS\| 8: 417 \| DOI: 10.1038/s41467-017-00462-2\| www.nature.com/naturecommunications 7 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-00462-2 QRS, QT) were then collected for each beat by automated identiﬁcation and recording of appropriate deﬂection points between each valid R–R interval in the trace. Within our data sets >80% of analysed traces passed this quality control step and, as a matter of routine, we manually inspected randomly chosen subsets of valid and excluded traces to conﬁrm appropriate categorisation and analysis. Importantly, we found that using more or less stringent quality control parameters did not affect the overall results of our subsequent analysis, nor did we ﬁnd any bias with respect to the proportion of traces excluded from analysis during active vs. inactive portions of the daily cycle. For autonomic blockade, conscious free-moving 22.5 h and 24 h LD housed mice were injected either 2 h after lights on (~ZT2) or 2 h after lights off (~ZT14) with atropine (0.5 mg/kg, ip) followed by propranolol (1 mg/kg, ip). During autonomic blockade studies, radio-telemetry data (activity, Tb and HR) was collected every minute. Baseline HR was derived from 1 h pre-injection, with HR during complete autonomic blockade collected 20–30 min post-administration. 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This work was supported by the Biotechnology and Biological We thank the Biological Services Unit at the University of Manchester for support of in vivo studies. This work was supported by the Biotechnology and Biological Sciences Research Council (UK) through grants to D.A.B. (BB/I01864/1; BB/J017744/1) and T.M.B. (BB/N007115/1). 49. Eckel-Mahan, K. L. et al. Reprogramming of the circadian clock by nutritional challenge. Cell 155, 1464–1478 (2013). in vivo studies. This work was supported by the Biotechnology and Biological Sciences Research Council (UK) through grants to D.A.B. (BB/I01864/1; BB/J017744/1) and T.M.B. (BB/N007115/1). Sciences Research Council (UK) through grants to D.A.B. (BB/I01864/1; BB/J017744/1) and T.M.B. (BB/N007115/1). 50. Scheer, F. A., Kalsbeek, A. & Buijs, R. M. Cardiovascular control by the suprachiasmatic nucleus: neural and neuroendocrine mechanisms in human and rat. Biol. Chem. 384, 697–709 (2003). Author contributions 51. Scheer, F. A. et al. Impact of the human circadian system, exercise, and their interaction on cardiovascular function. Proc. Natl Acad. Sci. USA 107, 20541–20546 (2010). All authors contributed to experimental design; A.C.W., L.S. and D.A.B. conducted experiments; A.C.W., T.M.B. and D.A.B. analyzed results; A.C.W., D.W.R., A.S.I.L., T.M. B. and D.A.B. wrote the manuscript; D.A.B. conceived the project and supervised all aspects. All authors contributed to experimental design; A.C.W., L.S. and D.A.B. conducted experiments; A.C.W., T.M.B. and D.A.B. analyzed results; A.C.W., D.W.R., A.S.I.L., T.M. B. and D.A.B. wrote the manuscript; D.A.B. conceived the project and supervised all aspects. 52. Young, M. E. et al. Cardiomyocyte-speciﬁc BMAL1 plays critical roles in metabolism, signaling, and maintenance of contractile function of the heart. J. Biol. Rhythms 29, 257–276 (2014). 53. Martino, T. A. & Young, M. E. Inﬂuence of the cardiomyocyte circadian clock on cardiac physiology and pathophysiology. J. Biol. 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Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock (vol 3, pg 29, 2014). Mol. Metab. 3, 857–857 (2014). 36. Scheer, F. A. J. L., van Doornen, L. J. P. & Buijs, R. M. Light and diurnal cycle affect autonomic cardiac balance in human; poss ible role for the biological clock. Auton. Neurosci-Basic 110, 44–48 (2004). 4. Carter, S. J. et al. A matter of time: study of circadian clocks and their role in inﬂammation. J. Leukoc. Biol. 99, 549–560 (2016). 37. Schroeder, A., Loh, D. H., Jordan, M. C., Roos, K. P. & Colwell, C. S. Circadian regulation of cardiovascular function: a role for vasoactive intestinal peptide. h l h ( ) 37. Schroeder, A., Loh, D. H., Jordan, M. C., Roos, K. P. & Colwell, C. S. Circadian regulation of cardiovascular function: a role for vasoactive intestinal peptide. Am. J. Physiol. Heart Circ. Phys. 300, H241–H250 (2011). 5. Alibhai, F. J., Tsimakouridze, E. V., Reitz, C. J., Pyle, W. G. & Martino, T. A. 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https://openalex.org/W3169522697	https://jnnp.bmj.com/content/jnnp/92/11/1186.full.pdf	English	null	Improving the efficacy of exome sequencing at a quaternary care referral centre: novel mutations, clinical presentations and diagnostic challenges in rare neurogenetic diseases	Journal of neurology, neurosurgery and psychiatry	2,021	cc-by	10,861	► ►Additional online supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/10.1136/ jnnp-2020-325437). J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325 on October 23, 2024 by guest. Prot http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Neurogenetics Original research Received 24 October 2020 Revised 10 April 2021 Accepted 5 May 2021 Published Online First 8 June 2021 ABSTRACT B k d Established pathogenic variants (n=16) presented with atypical features, such as optic neuropathy in adult polyglucosan body disease, facial dysmorphism and skeletal anomalies in cerebrotendinous xanthomatosis, steroid-responsive weakness in congenital myasthenia syndrome 10. Potentially treatable rare diseases were diagnosed, improving the quality of life in some patients.i Received 24 October 2020 Revised 10 April 2021 Accepted 5 May 2021 Published Online First 8 June 2021 on October 23, 2024 by guest. Protected by copyright. ttp://jnnp.bmj.com/ The Neurogenetics Clinic at the National Insti- tutes of Health (NIH) Clinical Centre is a quater- nary care centre for patients referred from national and international tertiary medical centres. Referrals include complex and difficult to diagnose singleton or small familial cases in which standard care eval- uations have not identified a diagnosis. In this study, we integrate gene filter lists with detailed phenotyping, biological assays and family segrega- tion analysis to diagnose patients with rare neuro- genetic disorders. This has allowed us to identify mutations in patients from diverse ethnic and racial backgrounds with a range of neurogenetic disorders including those that are rare, complex and atypical. er 23, 2024 by guest. Protected by copyright. Conclusions Integrating deep phenotyping, gene filter algorithms and biological assays increased diagnostic yield of exome sequencing, identified novel pathogenic variants and extended phenotypes of difficult to diagnose rare neurogenetic disorders in an outpatient clinic setting. guest. Protected by copyright. Improving the efficacy of exome sequencing at a quaternary care referral centre: novel mutations, clinical presentations and diagnostic challenges in rare neurogenetic diseases on October 23, 2024 by guest. Protected by copyright. http://jnnp.bmj.com/ surg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from on October 23, 2024 by guest. Protected by copyright. http://jnnp.bmj.com/ t published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Christopher Grunseich ‍ ‍ ,1 Nathan Sarkar,1 Joyce Lu,1 Mallory Owen,1 Alice Schindler,1 Peter A Calabresi ‍ ‍ ,2 Charlotte J Sumner ‍ ‍ ,2 Ricardo H Roda ‍ ‍ ,3 Vinay Chaudhry ‍ ‍ ,3 Thomas E Lloyd ‍ ‍ ,2 Thomas O Crawford ‍ ‍ ,2 S H Subramony ‍ ‍ ,4 Shin J Oh ‍ ‍ ,5 Perry Richardson,6 Kurenai Tanji ‍ ‍ ,7 Justin Y Kwan,1 Kenneth H Fischbeck,1 Ami Mankodi ‍ ‍ 1 on clinical data alone difficult. The identifica- tion of disease genes enables molecular diagnosis of patients, defines disease risk in relatives and represents the first step to a better understanding of the physiological role of the underlying protein and disease pathways, which in turn leads to ther- apeutic developments for the diseases. Whereas targeted gene panels and candidate gene testing can identify known mutations, these tests may be costly and not sensitive to unknown mutations. Whole-exome sequencing (WES) allows rapid, low cost and unbiased identification of pathogenic vari- ants in patients with suspected genetic diseases.1–5 But, the large amount of information provided by this powerful gene discovery tool can be time- consuming and often yields inconclusive results. The use of gene lists can improve diagnostic yield by filtering patient WES results to those variants identified in genes associated with disease-causing mutations.6 Yet, variants of interest identified through gene list filtering require further validation by additional methods. Correspondence to Dr Christopher Grunseich, Neurogenetics Branch, NINDS, Bethesda, Maryland, USA; christopher.grunseich@nih.gov Dr Ami Mankodi; ami.mankodi@ nih.gov ABSTRACT B k d Background We used a multimodal approach including detailed phenotyping, whole exome sequencing (WES) and candidate gene filters to diagnose rare neurological diseases in individuals referred by tertiary neurology centres. For numbered affiliations see end of article. For numbered affiliations see end of article. Methods WES was performed on 66 individuals with neurogenetic diseases using candidate gene filters and stringent algorithms for assessing sequence variants. Pathogenic or likely pathogenic missense variants were interpreted using in silico prediction tools, family segregation analysis, previous publications of disease association and relevant biological assays. Results Molecular diagnosis was achieved in 39% (n=26) including 59% of childhood-onset cases and 27% of late-onset cases. Overall, 37% (10/27) of myopathy, 41% (9/22) of neuropathy, 22% (2/9) of MND and 63% (5/8) of complex phenotypes were given genetic diagnosis. Twenty-seven disease-associated variants were identified including ten novel variants in FBXO38, LAMA2, MFN2, MYH7, PNPLA6, SH3TC2 and SPTLC1. Single-nucleotide variants (n=10) affected conserved residues within functional domains and previously identified mutation hot-spots. Established pathogenic variants (n=16) presented with atypical features, such as optic neuropathy in adult polyglucosan body disease, facial dysmorphism and skeletal anomalies in cerebrotendinous xanthomatosis, steroid-responsive weakness in congenital myasthenia syndrome 10. Potentially treatable rare diseases were diagnosed, improving the quality of life in some patients. Conclusions Integrating deep phenotyping, gene filter algorithms and biological assays increased diagnostic yield of exome sequencing, identified novel pathogenic variants and extended phenotypes of difficult to diagnose rare neurogenetic disorders in an outpatient clinic setting. Methods WES was performed on 66 individuals with neurogenetic diseases using candidate gene filters and stringent algorithms for assessing sequence variants. Pathogenic or likely pathogenic missense variants were interpreted using in silico prediction tools, family segregation analysis, previous publications of disease association and relevant biological assays. Correspondence to Dr Christopher Grunseich, Neurogenetics Branch, NINDS, Bethesda, Maryland, USA; christopher.grunseich@nih.gov Dr Ami Mankodi; ami.mankodi@ nih.gov Results Molecular diagnosis was achieved in 39% (n=26) including 59% of childhood-onset cases and 27% of late-onset cases. Overall, 37% (10/27) of myopathy, 41% (9/22) of neuropathy, 22% (2/9) of MND and 63% (5/8) of complex phenotypes were given genetic diagnosis. Twenty-seven disease-associated variants were identified including ten novel variants in FBXO38, LAMA2, MFN2, MYH7, PNPLA6, SH3TC2 and SPTLC1. Single-nucleotide variants (n=10) affected conserved residues within functional domains and previously identified mutation hot-spots. INTRODUCTION A cohort of 66 patients (age range: 13–75 years, median: 48 years) with suspected hereditary neuro- logical diseases had exome analysis as part of their diagnostic evaluation in the NIH Neurogenetics Patients with hereditary neuromuscular disorders often present with nonspecific, complex and atyp- ical phenotypes, making a precise diagnosis based Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 1186 on October 23, 2024 by guest. Protected by copy http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Neurogenetics Clinic (Bethesda, MD). These patients were referred from tertiary medical centres and had completed an evaluation by an outside neurologist. This included 50 singleton cases (76%), 9 cases from 5 sibling pairs and 7 cases from multigeneration fami- lies. Seventeen patients (26%) reported ancestry from outside of Europe including Asia and South America. A majority of patients (85%; n=56) had previous genetic testing including single and panel gene testing. All had a thorough physical examination, and relevant clinical testing such as MRI, electrophysiology studies and muscle biopsy. Genetic counselling was provided. Family members were also evaluated when feasible. Informed written consent or assent was obtained from each subject before partic- ipation in the study. gene list appropriate to their phenotype, then the other two gene lists were also applied to ensure the diagnostic yield of the neuromuscular disease gene panels. gene list appropriate to their phenotype, then the other two gene lists were also applied to ensure the diagnostic yield of the neuromuscular disease gene panels. Variants identified from gene list filtering were categorised based on the guidelines recommended by the American College of Medical Genetics and Genomics and Association for Molecular Pathology. Variants with evidence of benign impact, likely benign impact or uncertain significance were excluded from further analysis. Pathogenic or likely pathogenic missense variants with allelic frequency <0.0001 in the genome aggregation database (gnomAD) were interpreted using CADD, CDPred and ClinPred. The total of 422 candidate gene variants were identified (online supplemental data 1). Heterozygous variants with gnomAD allele frequencies between 0.0001 and 0.00003 in genes associated with an autosomal dominant pattern of inheritance were categorised as uncertain significance. Manual review of variant inspection and read sequence alignments was done using the Integrative Genomics Viewer. Diagnostic yield of exome sequencing D hi f 66 i di id l i l di Demographics of 66 individuals including disease categories, age at evaluation, gender distribution and age of symptom onset are shown in figure 1. Overall, a genetic diagnosis was made in 39% (26/66) of patients tested, with the highest yield in patients with affected family members (88%, 14/16) and those with a complex phenotype (63%, 5/8; table 1, figure 1 (online supplemental table 2). Sixteen individuals (59%) with disease onset before 20 years of age received a genetic diagnosis, whereas the yield was relatively lower (27%) in older individuals with disease onset after 40 years of age. In those families with more than one individual affected, pedigrees included autosomal dominant and recessive inheritance patterns (figure 1). Candidate genes were first evaluated following gene variant annotation as indicated in online supplemental figure 1. We then applied diagnostic gene filters to the patient’s ES vari- ants of interest to facilitate the genetic diagnosis (online supplemental table 1). Gene filters were compiled from previ- ously reported disease-associated variants listed in the Leiden Muscular Dystrophy pages (Leiden University Medical Center, NL), Neuromuscular Disease Center (Washington University, St Louis, Missouri, USA), Online Mendelian Inheritance in Man (OMIM) databases and searches of medical literature. A total of 634 gene identifiers were categorised into the following cate- gories based on their asserted association with human disease: myopathy (n=293), neuropathy (n=193) and motor neuron disease (n=148). The variants of interest were first filtered by 23, 2024 by guest. Protected by copyright. uest. Protected by copyright. INTRODUCTION We compiled further evidence to support classification of pathogenicity from additional genetic testing, family segregation analysis, previous publications of asserted disease association and relevant biological assays in patient-derived cell lines and tissues. Clinically relevant variants were validated by a Clinical Laboratory Improvement Amendment CLIA-certified laboratory. Previously reported mutations were identified through VarSome, ClinVar, OMIM and PubMed. Exome analysis f WES was performed in a research laboratory (NIH Intramural Sequencing Center) on DNA extracted from whole blood. The NimbleGen SeqCap EZ V.3.0+ UTR capture kit (Roche, Basel, Switzerland) was used to cover 96 Mb of genomic DNA (100 ug/mL). The captured DNA library was sequenced using Illu- mina HiSeq to provide coverage to call most probable genotypes (MPGs) with a score of 10 in at least 85% of targeted bases. Reads were mapped to NCBI build 37 (hg19) using the ELAND (Efficient Large-scale Alignment of Nucleotide Databases;Illu- mina, San Diego, California, USA), and Novoalign V.3.02.07 (Novocraft, Selangor, Malaysia). The aligned bam files were fed as input to bam2mpg (http://research.nhgri.nih.gov/software/ bam2mpg/index.shtml), to call MPGs at all covered positions using a probabilistic Bayesian algorithm. Genotypes with an MPG score ≥10 showed greater than 99.9% concordance with Illumina Human 1M-Quad single nucleotide polymorphism (SNP) Chip data. Sequence bases with Phred quality score >30 were included for the analysis. Where indicated whole-genome oligonucleotide array compar- ative genome hybridisation (CGH)+SNP analysis was performed by GeneDx using 180 000 oligonucleotide probes on blood DNA samples. RNAseq analysis of S.2 patient vastus lateralis muscle tissue was performed from total RNA using the Total RNA Library Prep Kit (Illumina). Sequencing of S.2 patient muscle tissue was performed on an Illumina HiSeq 2500 platform, with reads aligned to human reference (HG18). To generate ADSSL1- cDNA clones, cDNA was synthesised by reverse transcription of total cellular RNA isolated from the biopsied vastus lateralis muscle, followed by digestion with RNase H. cDNAs extending from exons 2 to 13 shared by most ADSSL1 splice isoforms were amplified through 31 cycles of PCR. Products from five PCR reactions were combined, gel-purified and cloned into pBSK for Sanger sequencing (n=30 clones). The alternate 5’UTR and coding sequence of exons 1 and 2 in the two prominent ADSSL1 isoforms were amplified from genomic DNA and analysed by Sanger sequencing. on October 23, 2024 by guest. Prot http://jnnp.bmj.com/ 7 on 8 June 2021. Downloaded from Variant identification and interpretation Th f i i i The strategy of variant annotation, interpretation, and valida- tion is highlighted in online supplemental figure 1. Exome vari- ants were filtered using VarSifter (National Human Genome Research Institute, Bethesda, USA). Variants were computation- ally annotated using ANNOVAR, presence in dbSNP (version 137), the International Genome Sample resource (n=2504 genomes), NHLBI EVS dataset of 6500 individuals, the Human Gene Mutation Database as a ‘Disease Mutation’, and ClinVar, frequency within the Exome Aggregation Consortium Database (ExAC database; n=60 706 exomes), and predicted effect on the protein using CDPred, SIFT and Polyphen-2. Chromosomal SNP microarray analysis helped to focus ES screening in relevant cases. Trio exome sequencing was used when parental DNA was available. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 Variant interpretation Ten novel pathogenic variants were identified in patients with myopathy (n=3), neuropathy (n=5) and MND (n=2; table 1). Nine amino acid substitutions were in residues otherwise conserved across species (figure 2). These variants occurred 1187 Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 on Octo http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Neurogenetics rogenetics 1 Demographics and inheritance patterns in those receiving WES. (A) The myopathy group (n=27) comprised of limb girdle muscular dystrophy distal myopathy (n=4), limb girdle congenital myasthenic syndrome (LG-CMS; n=4), congenital myopathy (n=1), and other (n=6). The neuropathy n=22) consisted of hereditary motor and sensory neuropathy (HMSN, n=13), distal hereditary motor neuropathy (dHMN, n=6) and hereditary sensory athy (HSN, n=3). These patients had a predominantly axonal (n=17), demyelinating (n=3) or mixed (n=2) patterns of nerve injury. There were nine in the motor neuron disease (MND) group and eight patients in the complex group. (B) Graph showing the distribution of ages in years at the time presentation with mean and 5/95% CIs. Mean ages at the time of presentation are 48, 46, 41 and 55 years for the myopathy, neuropathy, MND mplex phenotype groups, respectively. (C) The number of females (F) and males (M) in each disease category. (D) Distribution of age of onset in each category. The myopathy group had 11 patients with juvenile/adult onset (10–40 years), 8 patients with childhood onset (<10 y) and 8 patients with et (>40 years) disease. The neuropathy group had 11 patients with juvenile/adult onset, 6 patients with childhood onset and 5 patients with late sease. The MND group consisted of five patients with juvenile/adult onset and four patients with late-onset disease. (E) Graph showing the number on October 23, 2024 by gu http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from g Figure 1 Demographics and inheritance patterns in those receiving WES. (A) The myopathy group (n=27) comprised of limb girdle muscular dystrophy (n=12), distal myopathy (n=4), limb girdle congenital myasthenic syndrome (LG-CMS; n=4), congenital myopathy (n=1), and other (n=6). The neuropathy group (n=22) consisted of hereditary motor and sensory neuropathy (HMSN, n=13), distal hereditary motor neuropathy (dHMN, n=6) and hereditary sensory neuropathy (HSN, n=3). These patients had a predominantly axonal (n=17), demyelinating (n=3) or mixed (n=2) patterns of nerve injury. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 Variant interpretation There were nine patients in the motor neuron disease (MND) group and eight patients in the complex group. (B) Graph showing the distribution of ages in years at the time of initial presentation with mean and 5/95% CIs. Mean ages at the time of presentation are 48, 46, 41 and 55 years for the myopathy, neuropathy, MND and complex phenotype groups, respectively. (C) The number of females (F) and males (M) in each disease category. (D) Distribution of age of onset in each disease category. The myopathy group had 11 patients with juvenile/adult onset (10–40 years), 8 patients with childhood onset (<10 y) and 8 patients with late onset (>40 years) disease. The neuropathy group had 11 patients with juvenile/adult onset, 6 patients with childhood onset and 5 patients with late onset disease. The MND group consisted of five patients with juvenile/adult onset and four patients with late-onset disease. (E) Graph showing the number of diagnosed (+) and undiagnosed (−) cases in the myopathy (37%;10/27), neuropathy (41%;9/22), MND (22%;2/9) and complex (63%;5/8) phenotype groups by age group. (F) Representative pedigrees in families with multiple affected patients showing compound heterozygous mutations in GBE1 (I, F.1), compound heterozygous mutations in LAMA2 (II, F.3), heterozygous inheritance mutations in MFN2 (III, F.5), homozygous mutations in ADSSL1 (IV, S.3) and homozygous mutations in CYP271A (V, S.16). parents of proband in family F.5 not available for testing. WES, whole exome sequencing. positioned in the distal rod region of the protein where other mutations have been established to cause Laing distal myopathy and cardiomyopathies.7 in important functional domains of the proteins considered as hotspots for disease-associated variants. The MYH7 p.Le- u1533Pro variant in a distal myopathy patient (S.7) was Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 1188 Neurogenetics el pathogenic variants and phenotypes of individuals diagnosed through exome sequencing ene(Chr:position)c.DNA change p.protein change Coding effect CADD score CDPred score GnomAD freq. GnomAD Allele count / # of het/ # of hom ClinPred Score Age at diagnosis (y):Sex Phenotype (MIM#) BXO38(5:147 785 932) M_030793.4:c.843T>G p.His281Gln Het. 23.3 0 0 0/0/0 1.00 18:M Juvenile-onset upper limb distal weakness; a de novo variant. HMN2D (615575) AMA2 :129 636 905)NM_000426.3: c.3736-2A>T p.(?) Hom. 34 −30 0.00001194 3/3/0 NA 69:F Childhood-onset with milder phenotype, retained independent ambulation. LGMDR23 (618138) AMA2(6:129 204 451_129204 452)NM_000426.3:c.61_62delCA Gln21Glyfs28 Het. Neurogenetics Neurogenetics Pathogenicity was also indicated by location near other previ- ously reported mutations. The SPTLC1 and FBXO38 variants (patients S.14 and S.18) were in conserved regions containing clusters of previously published rare disease-associated muta- tions (figure 2).8 9 In two sisters (F3.1; F3.2) compound hetero- zygous loss-of-function variants were identified in the LAMA2 gene including a novel frameshift expected to truncate the protein, and a previously reported pathogenic splice site muta- tion.10 Patient S.4 had a homozygous variant that destroyed the canonical splice acceptor site in LAMA2 intron 25, and is presumed to cause loss of functional protein. A targeted exon- level oligo array CGH assay showed no complete or partial deletion and duplication of the LAMA2 gene, supporting true homozygous splice site variant. Although MFN2 mutations at p.Arg94 and p.Thr362 residues have been reported in CMT2A patients,11 the changes in our patients (S.13 and F.5.1;F5.2) with leucine and arginine substitutions, respectively, have not been previously reported. Segregation analysis for the variants was done by targeted variant testing in parents when available. To this end, we identified de novo dominant pathogenic variants in on October 23, 2024 by guest. Protected by copyrigh http://jnnp.bmj.com/ eurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from 2 Gene variant characterisation. (A) Variants resulted in amino acid substitutions that were conserved across most vertebrates. Conservatio ected in Drosophila melanogaster in five of nine variants evaluated. (B) Variants occurred in hotspot regions containing previously reported causing variants. Mutations of CACNA1S associated with congenital myopathy are shown as triangles. For MYH7, only those variants in the yosin (LMM) region are shown. Newly reported variants shown in black, ClinVar published variants indicated on top, and non-ClinVar publish b f h i V i i MYH14 i hi h i bi di d i 1190 Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 Figure 2 Gene variant characterisation. (A) Variants resulted in amino acid substitutions that were conserved across most vertebrates. Conservation was detected in Drosophila melanogaster in five of nine variants evaluated. (B) Variants occurred in hotspot regions containing previously reported disease causing variants. Mutations of CACNA1S associated with congenital myopathy are shown as triangles. For MYH7, only those variants in the light meromyosin (LMM) region are shown. Newly reported variants shown in black, ClinVar published variants indicated on top, and non-ClinVar published variants on bottom for each protein. Variant interpretation 20.2 −30 0 0/0/0 NA 34:F; 24:F Childhood-onset with milder phenotype, retained independent ambulation in sisters. LGMDR23 (618138) MFN2 :12 052 717)NM_014874.3:c.281G>T p.Arg94Leu Het. 31 −2 0 0/0/0 0.99 38:F Childhood-onset with severe disease, loss of ambulation by age 30y. CMT2A (609260) MFN2 :12 062 085) M_014874.3:c.1085C>G p.Thr362Arg Het. 24.3 −1 0 0/0/0 0.99 33:M;60:F Adult-onset with milder, later onset disease in proband and his maternal aunt. CMT2A (609260) MYH7(14:23 886 123) M_000257.2:c.4598T>C p.Leu1533Pro Het. 30 −9 0 0/0/0 0.99 65:M Childhood-onset with foot drop at age 6 years. Loss of ambulation during his late 50s. A de novo variant. Laing distal myopathy (160500) NPLA6(19:7 615 957)NM_006702.4:c.2031G>T p.Glu677Asp; NPLA6 9:7 619 463)NM_006702.4:c.2374G>C p.Gly792Arg Comp. het. 17.8;23.6 −3; −2 0.00000679;0.00006377 1/1/0;2/2/0 0.18;0.92 19:F Juvenile-onset motor neuron disease with upper >lower limb distal weakness; absent spasticity. PTLC1(19:7 619 463) M_006415.2:c.1019C>T p.Ser340Leu Het. 23.6 −2 0.00001592 4/4/0 0.92 68:M Late-onset hereditary motor sensory neuropathy with mixed sensory and motor symptoms. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 1189 on October 23, 2024 by guest. Prot http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Clinical presentations in patients with novel pathogenic variants Phenotypes of cases with novel variants are summarised in table 2 and in online supplemental data 2 section. A late-aged female (S.4), and adult sisters (F3.1, F3.2) presented with slowly progressive childhood-onset milder limb girdle dystrophy phenotype due to laminin-α2 deficiency (LGMDR23). There was no known parental consanguinity. Muscle MRI showed that the muscle periphery was primarily affected (figure 3), similar to a pattern seen in milder cases of LAMA-2 myopathies.12 Muscle biopsies were done more than 25 years previously and immunostaining was not analysed, thereby hampering the diag- nosis until WES analysis. A late-aged male (S.7) with the MYH7 variant presented with childhood-onset Laing distal myopathy.7 His exam showed distally predominant weakness with severe involvement of toe, finger and wrist extension, and ankle dorsi- flexion and a pattern consistent with Laing distal myopathy on muscle MRI (figure 3). Cardiac echo and ECG were normal. ( g ) An adult female (S.13) and F.5 family members with a MFN2 variant presented with childhood onset and late onset CMT2A, respectively. A late-aged male (S.14) with a SPTLC1 variant presented with distal weakness and loss of vibratory sensation in the lower extremities. He had symptoms of fatigue, muscle stiffness, and paresthesia. An adult male (S.18) with FBXO38 mutation had juvenile-onset HMN2D9 with distal weakness prominently in finger flexion. The phenotype of patient S.11 with homozygous SH3TC2 variants and CMT4C has been reported.13 An adult female (S.15) with a PNPLA6 variant had juvenile-onset MND with distal weakness most marked in finger flexion, ankle dorsiflexion and toe extension. Neurogenetics APBD (263570) 4.2 GSN (9:124 073 097)NM_000177.4:c.640G>T p.Asp214Tyr Het. 22.2 −10 0 0.99 70:F; 47:M Late-onset disease; bifacial weakness; lattice corneal dystrophy absent. Amyloidosis (105120) dult polyglucosan body disease; N/A, not applicable. on October 23, 2024 by guest. Protected b http://jnnp.bmj.com/ Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from two cases (S.7, MYH7; S.18, FBXO38), and compound hetero- zygous mutations in one case (S.15, PNPLA6). two cases (S.7, MYH7; S.18, FBXO38), and compound hetero- zygous mutations in one case (S.15, PNPLA6). Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 Neurogenetics Variant in MYH14 occurs within the actin binding domain. on October 23, 2024 by guest. Protected by copyright http://jnnp.bmj.com/ 21. Downloaded from Figure 2 Gene variant characterisation. (A) Variants resulted in amino acid substitutions that were conserved across most vertebrates. Conservation was detected in Drosophila melanogaster in five of nine variants evaluated. (B) Variants occurred in hotspot regions containing previously reported disease causing variants. Mutations of CACNA1S associated with congenital myopathy are shown as triangles. For MYH7, only those variants in the light meromyosin (LMM) region are shown. Newly reported variants shown in black, ClinVar published variants indicated on top, and non-ClinVar published variants on bottom for each protein. Variant in MYH14 occurs within the actin binding domain. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 1190 Neurogenetics Neurogenetics Table 2 Individuals with established mutations and phenotypic extension diagnosed through exome sequencing Patient ID Gene(Chr:position)c.DNA change p.protein change Coding effect CADD score CDPred score GnomAD ClinPred Score Age at diagnosis (y):Sex Phenotype (MIM#) S.16 CYP27A1(2:219 678 909)NM_000784.3:c1183C>T p.Arg395Cys Hom. 20.2 −14 0.0002831 0.60 33:F Childhood-onset facial dysmorphism and skeletal anomalies; juvenile-onset cataracts, cognitive decline, neuropathy. CTX (213700) F.2.1; F.2.2 DOK7(4:3 494 837_3 494 840) NM_173660.5:c.1124_1127dupTGCC p.Ala378Serfs30 Hom. N/A N/A 0 N/A 56:M;52:M Juvenile-onset steroid-responsive biceps weakness; milder limb-girdle weakness in brothers CMS10 (254300) F.1.1; F.1.2 GBE1(3:81 691 938)NM_000158.3:c.986A>C p.Tyr329Ser; GBE1(3:81 542 964_81 542 972)NM_000158.3:c.2053–3358_2053- 3350delinsTGTTTTTTACATTACAGGT p.Tyr686Serfs3 Comp. Het. 22;N/A −11;N/A 0.000318;0 0.94;N/A 46:M;44:F Adult-onset optic neuropathy, deafness, neuropathy, and white matter T2w hyperintensities in brother and sister. APBD (263570) F.4.1; F.4.2 GSN (9:124 073 097)NM_000177.4:c.640G>T p.Asp214Tyr Het. 22.2 −10 0 0.99 70:F; 47:M Late-onset disease; bifacial weakness; lattice corneal dystrophy absent. Amyloidosis (105120) APBD, adult polyglucosan body disease; N/A, not applicable. g 2 Individuals with established mutations and phenotypic extension diagnosed through exome sequencing Gene(Chr:position)c.DNA change p.protein change Coding effect CADD score CDPred score GnomAD ClinPred Score Age at diagnosis (y):Sex Phenotype (MIM#) CYP27A1(2:219 678 909)NM_000784.3:c1183C>T p.Arg395Cys Hom. 20.2 −14 0.0002831 0.60 33:F Childhood-onset facial dysmorphism and skeletal anomalies; juvenile-onset cataracts, cognitive decline, neuropathy. CTX (213700) .2 DOK7(4:3 494 837_3 494 840) NM_173660.5:c.1124_1127dupTGCC p.Ala378Serfs30 Hom. N/A N/A 0 N/A 56:M;52:M Juvenile-onset steroid-responsive biceps weakness; milder limb-girdle weakness in brothers CMS10 (254300) .2 GBE1(3:81 691 938)NM_000158.3:c.986A>C p.Tyr329Ser; GBE1(3:81 542 964_81 542 972)NM_000158.3:c.2053–3358_2053- 3350delinsTGTTTTTTACATTACAGGT p.Tyr686Serfs*3 Comp. Het. 22;N/A −11;N/A 0.000318;0 0.94;N/A 46:M;44:F Adult-onset optic neuropathy, deafness, neuropathy, and white matter T2w hyperintensities in brother and sister. yp variants Previously established pathogenic variants were identified in 17 patients (online supplemental table 2). Clinical examples with atypical presentations of rare neurological diseases are listed in table 2 and are described in online supplemental data 2 section. Two brothers (F2.1 and F2.2) with homozygous DOK7 muta- tion (CMS10)14 had a juvenile onset steroid-responsive weakness and EMG findings of small amplitude short duration motor unit potentials. The 2–5 Hz repetitive nerve stimulation test after the diagnosis of CMS10 showed 9%–17% decrement in the orbi- cularis oculi muscle. Their parents were second cousins. The initial presentation consisted of biceps muscle weakness during the teenage years, which remained predominant over the years causing disability. The effect of steroid treatment was notice- able on biceps muscle strength, and higher steroid doses were required as the disease progressed. Treatment with salbutamol/ ephedrine and adding 3,4-diaminopyridine and gradual steroid taper was recommended, although no benefit was appreciated with a short trial of albuterol.15 Two members of a family (F.4.1; F.4.2) with hereditary gelsolin amyloidosis had facial paralysis, fasciculations, and weakness of other bulbar muscles. Their first symptom was bilateral facial weakness. Lattice corneal dystrophy was absent. Mild sensory neuropathy was present, and screening for systemic complications of amyloidosis was unrevealing. An adult female (S.16) with cerebrotendinous xanthoma- tosis (CTX), a rare potentially treatable disease, presented with asymmetric hypoplasia of facial structures, short neck, Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 on Octo http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Neurogenetics g Figure 3 Examples of clinical test results aiding the genetic diagnosis by exome sequencing analysis. (A) T1-weighted axial images in F.3.1 patient laminin α2 deficient LGMDR23 show peripheral fat replacement with central sparing in the vastus lateralis, soleus and gastrocnemius muscles, but th tibialis anterior is spared. The rectus femoris muscle shows increased signal around central fascia and periphery leaving a U-shaped less affected mus (B) The tibialis anterior is mostly replaced by fat, the quadriceps and adductor muscles are prominently affected, but the rectus femoris is spared in S. patient with Laing distal myopathy. In contrast with (A), multiple muscles show internal bands of fat infiltration. (C) Fat infiltration is seen in the addu magnus, biceps femoris, semimembranosus, soleus and gastrocnemius muscles, followed by the semitendinosus, vastus and tibialis anterior muscles i patient with ADSSL1-related distal myopathy. yp variants (D) Typical symmetrical cerebellar white matter hyperintensities around dentate nucleus bilaterally in T2 MRI in S.16 patient with cerebrotendinous xanthomatosis. optic neuropathy, leukoencephalopathy, and impaired cellular GBE1 activity in a family wit adult polyglucosan body disease (APBD; E–G). (E) Axial T2-FLAIR images of brain show multiple discrete and confluent foci of high-signal in the subc white-matter, along the atria and occipital horns of the lateral ventricles, and along the white-matter tracts in the brainstem lesions. (F–G) Optical coh tomography data showing bilateral atrophy of the macular ganglion cell-inner plexiform layer (GCL-IPL) and the retinal nerve fibre layer (RNFL), indic neuronal death and axonal loss in optic nerves, respectively. Sup, superior; Inf, inferior. (H) The GBE1 enzyme activity measured in cultured skin fibrob affected siblings and unaffected relatives. Figure 3 Examples of clinical test results aiding the genetic diagnosis by exome sequencing analysis. (A) T1-weighted axial images in F.3.1 patient with laminin α2 deficient LGMDR23 show peripheral fat replacement with central sparing in the vastus lateralis, soleus and gastrocnemius muscles, but the tibialis anterior is spared. The rectus femoris muscle shows increased signal around central fascia and periphery leaving a U-shaped less affected muscle. (B) The tibialis anterior is mostly replaced by fat, the quadriceps and adductor muscles are prominently affected, but the rectus femoris is spared in S.7 patient with Laing distal myopathy. In contrast with (A), multiple muscles show internal bands of fat infiltration. (C) Fat infiltration is seen in the adductor magnus, biceps femoris, semimembranosus, soleus and gastrocnemius muscles, followed by the semitendinosus, vastus and tibialis anterior muscles in S.2 patient with ADSSL1-related distal myopathy. (D) Typical symmetrical cerebellar white matter hyperintensities around dentate nucleus bilaterally in T2-FLAIR MRI in S.16 patient with cerebrotendinous xanthomatosis. optic neuropathy, leukoencephalopathy, and impaired cellular GBE1 activity in a family with adult polyglucosan body disease (APBD; E–G). (E) Axial T2-FLAIR images of brain show multiple discrete and confluent foci of high-signal in the subcortical white-matter, along the atria and occipital horns of the lateral ventricles, and along the white-matter tracts in the brainstem lesions. (F–G) Optical coherence tomography data showing bilateral atrophy of the macular ganglion cell-inner plexiform layer (GCL-IPL) and the retinal nerve fibre layer (RNFL), indicating neuronal death and axonal loss in optic nerves, respectively. Sup, superior; Inf, inferior. (H) The GBE1 enzyme activity measured in cultured skin fibroblasts of affected siblings and unaffected relatives. yp variants Figure 3 Examples of clinical test results aiding the genetic diagnosis by exome sequencing analysis. (A) T1-weighted axial images in F.3.1 patient with laminin α2 deficient LGMDR23 show peripheral fat replacement with central sparing in the vastus lateralis, soleus and gastrocnemius muscles, but the tibialis anterior is spared. The rectus femoris muscle shows increased signal around central fascia and periphery leaving a U-shaped less affected muscle. (B) The tibialis anterior is mostly replaced by fat, the quadriceps and adductor muscles are prominently affected, but the rectus femoris is spared in S.7 patient with Laing distal myopathy. In contrast with (A), multiple muscles show internal bands of fat infiltration. (C) Fat infiltration is seen in the adductor magnus, biceps femoris, semimembranosus, soleus and gastrocnemius muscles, followed by the semitendinosus, vastus and tibialis anterior muscles in S.2 patient with ADSSL1-related distal myopathy. (D) Typical symmetrical cerebellar white matter hyperintensities around dentate nucleus bilaterally in T2-FLAIR MRI in S.16 patient with cerebrotendinous xanthomatosis. optic neuropathy, leukoencephalopathy, and impaired cellular GBE1 activity in a family with adult polyglucosan body disease (APBD; E–G). (E) Axial T2-FLAIR images of brain show multiple discrete and confluent foci of high-signal in the subcortical white-matter, along the atria and occipital horns of the lateral ventricles, and along the white-matter tracts in the brainstem lesions. (F–G) Optical coherence tomography data showing bilateral atrophy of the macular ganglion cell-inner plexiform layer (GCL-IPL) and the retinal nerve fibre layer (RNFL), indicating neuronal death and axonal loss in optic nerves, respectively. Sup, superior; Inf, inferior. (H) The GBE1 enzyme activity measured in cultured skin fibroblasts of affected siblings and unaffected relatives. Figure 3 Examples of clinical test results aiding the genetic diagnosis by exome sequencing analysis. (A) T1-weighted axial images in F.3.1 patient with laminin α2 deficient LGMDR23 show peripheral fat replacement with central sparing in the vastus lateralis, soleus and gastrocnemius muscles, but the tibialis anterior is spared. The rectus femoris muscle shows increased signal around central fascia and periphery leaving a U-shaped less affected muscle. (B) The tibialis anterior is mostly replaced by fat, the quadriceps and adductor muscles are prominently affected, but the rectus femoris is spared in S.7 patient with Laing distal myopathy. In contrast with (A), multiple muscles show internal bands of fat infiltration. yp variants (C) Fat infiltration is seen in the adductor magnus, biceps femoris, semimembranosus, soleus and gastrocnemius muscles, followed by the semitendinosus, vastus and tibialis anterior muscles in S.2 patient with ADSSL1-related distal myopathy. (D) Typical symmetrical cerebellar white matter hyperintensities around dentate nucleus bilaterally in T2-FLAIR MRI in S.16 patient with cerebrotendinous xanthomatosis. optic neuropathy, leukoencephalopathy, and impaired cellular GBE1 activity in a family with adult polyglucosan body disease (APBD; E–G). (E) Axial T2-FLAIR images of brain show multiple discrete and confluent foci of high-signal in the subcortical white-matter, along the atria and occipital horns of the lateral ventricles, and along the white-matter tracts in the brainstem lesions. (F–G) Optical coherence tomography data showing bilateral atrophy of the macular ganglion cell-inner plexiform layer (GCL-IPL) and the retinal nerve fibre layer (RNFL), indicating neuronal death and axonal loss in optic nerves, respectively. Sup, superior; Inf, inferior. (H) The GBE1 enzyme activity measured in cultured skin fibroblasts of affected siblings and unaffected relatives. Figure 3 Examples of clinical test results aiding the genetic diagnosis by exome sequencing analysis. (A) T1-weighted axial images in F.3.1 patient with laminin α2 deficient LGMDR23 show peripheral fat replacement with central sparing in the vastus lateralis, soleus and gastrocnemius muscles, but the tibialis anterior is spared. The rectus femoris muscle shows increased signal around central fascia and periphery leaving a U-shaped less affected muscle. (B) The tibialis anterior is mostly replaced by fat, the quadriceps and adductor muscles are prominently affected, but the rectus femoris is spared in S.7 patient with Laing distal myopathy. In contrast with (A), multiple muscles show internal bands of fat infiltration. (C) Fat infiltration is seen in the adductor magnus, biceps femoris, semimembranosus, soleus and gastrocnemius muscles, followed by the semitendinosus, vastus and tibialis anterior muscles in S.2 patient with ADSSL1-related distal myopathy. (D) Typical symmetrical cerebellar white matter hyperintensities around dentate nucleus bilaterally in T2-FLAIR MRI in S.16 patient with cerebrotendinous xanthomatosis. optic neuropathy, leukoencephalopathy, and impaired cellular GBE1 activity in a family with adult polyglucosan body disease (APBD; E–G). (E) Axial T2-FLAIR images of brain show multiple discrete and confluent foci of high-signal in the subcortical white-matter, along the atria and occipital horns of the lateral ventricles, and along the white-matter tracts in the brainstem lesions. Searching for a second mutation In the case of patient S.2 with a single heterozygous ADSSL1 mutation (chr14:105207568,NM_199165.2:c.910G>A, p.Asp304Asn), a second mutation in the ADSSL1 gene has not been identified. The patient had symptom onset in his 50s of mild distal myopathy predominantly involving the posterior thigh and calf muscles (figure 3). This is in contrast to a severe childhood-onset presentation in patient (S.3) with consanguineous parents (second cousins, once removed) found to have the same mutation in homozygous state and in previously reported patients with juvenile-onset disease with homozygosity20 and compound heterozygosity.21 WES anal- ysis of both parents for patient S.2 identified the mutation in his mother, suggesting germline transmission of the carrier status. A vastus lateralis muscle biopsy showed chronic myopathy with rimmed vacuoles (online supplemental figure 3). Gene panel testing was uninformative. It is possible that a second mutation exists and was not identified, although a search for another mutation in ADSSL1 with a targeted array CGH assay and evaluation of total transcript levels and Sanger sequencing of cDNA clones extending from the second to the final exons shared by most ADSSL1 splice isoforms (n=30) and of the alternate 5’UTR and exons 1 and 2 in the two most prominent ADSSL1 isoforms from genomic DNA in the biopsied vastus lateralis muscle was nonrevealing. RNAseq analysis of the biopsied muscle tissue showed an even distribution of sequencing reads across the ADSSL1 coding region with no truncation or pseudoexon inclusion and heterozygosity at codon 304 (online supple- mental figure 4). For patient S.3, an oligonucleotide array CGH+SNP showed no partial or complete deletion of the ADSSL1 gene locus, supporting true homozygous missense mutations. Her mother was a carrier for the same mutation. Her father was deceased, and his DNA was not available for testing. Other family members of S.2 and S.3 patients lived outside of the USA and were not available for segregation testing. An adult male (F1.1) with a heterozygous GBE1 p.Tyr329Ser mutation associated with adult polyglucosan body disease (APBD), presented with distal paresthesias for 6 years, followed by erectile dysfunction, fatigue and progressive deterioration in vision in both eyes for 3 years. His exam showed bilateral optic atrophy and a left superior homonymous visual field defect suggestive of a postchiasmal lesion. Features included pyramidal tract signs in the lower limbs, sensory greater than motor axonal neuropathy with pes cavus, and sensorineural deafness. Searching for a second mutation MRI showed leuko- encephalopathy with multiple FLAIR signal hyperintensities at the supratentorial white matter and additional juxtacor- tical lesions. There were also lesions involving the bilateral superior cerebellar peduncle, as well as the left medullary pyramid (figure 3). Optical coherence tomography indi- cated optic nerve axon loss (figure 3). Prior to his evaluation at the NIH, he was diagnosed with multiple sclerosis and treated with interferon beta-1a for 2 years but continued to worsen. His sister (F1.2) had a similar but milder pheno- type. The WES analysis showed a heterozygous c.986A>C, p.Tyr329Ser mutation in the GBE1 gene and subsequently, Sanger sequencing of the entire gene identified a deep intronic deletion mutation in intron 15 (online supple- mental table S2), which has been reported together with the p.Tyr329Ser mutation in APBD patients and resulted in markedly reduced GBE function.17 Family segregation studies showed that both mutations were on separate alleles. GBE activity in skin fibroblasts was 22% and 32% of the mean value in asymptomatic carriers in their family in the proband and his sister, respectively, confirming the diagnosis of APBD. on October 23, 2024 by guest. Protected by copyright. p://jnnp.bmj.com/ r 23, 2024 by guest. Protected by copyright. y guest. Protected by copyright. yp variants Twenty-two hetero- zygotes with the MYH14 variant identified in patient S.10 were present in gnomAD. The variant was located within its actin-binding domain that is highly conserved in all myosin proteins (figure 2).19 prominently affecting axial muscles. Nine heterozygotes with the CACNA1S variant identified in patient S.9 were present in gnomAD. The variant is in the S4 helix of the voltage-sensing transmembrane domain III in the Cav1.1 channel (figure 1), where arginine mutations are associ- ated with hypokalemic periodic paralysis.18 Both parents were asymptomatic and tested negative for the Asn909Ser variant. A gene panel testing for neuromuscular disorders did not reveal other mutation(s). A muscle biopsy was not available to examine the abnormalities in the T-tubules and sarcoplasmic reticulum, as reported previously in Cav1.1 myopathy. Similarly, a late-aged female (S.10) with a MYH14 variant (chr19:50760672, NM_024729.3:c.2038C>T, p.Arg680Cys) had a late onset complex disease with features of myopathy, neuropathy and deafness. Twenty-two hetero- zygotes with the MYH14 variant identified in patient S.10 were present in gnomAD. The variant was located within its actin-binding domain that is highly conserved in all myosin proteins (figure 2).19 Variants of uncertain significance I l h did i In several cases the candidate variants identified may be patho- genic, however, the allele frequency in gnomAD was higher than anticipated for autosomal dominant inheritance. An adult female (S.9) with CACNA1S variant (chr1:201035376, NM_000069.2:c.2725A>G, p.Asn909Ser) had congen- ital myopathy with severe generalised muscle weakness yp variants (F–G) Optical coherence tomography data showing bilateral atrophy of the macular ganglion cell-inner plexiform layer (GCL-IPL) and the retinal nerve fibre layer (RNFL), indicating neuronal death and axonal loss in optic nerves, respectively. Sup, superior; Inf, inferior. (H) The GBE1 enzyme activity measured in cultured skin fibroblasts of affected siblings and unaffected relatives. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 1192 on October 23, 2024 by guest. Protected http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Neurogenetics a missing rib, low average to borderline cognitive ability, and leukoencephalopathy (figure 3; online supplemental figure 2). Her major findings were abnormal facial appearance, poor scholastic performance and fatigue. Other features included sensorimotor demyelinating neuropathy and juvenile-onset cataracts. Cerebellar signs, spasticity and subcutaneous xanthomas were absent. A whole genome oligonucleotide array CGH and SNP genotyping detected several regions of extended homozygosity (>7 Mb) encompassing at least 90 Mb (3.1% or 1/32 of the genome), suggesting that the parents were first cousins, once removed. Exome sequencing identified a candidate recessive mutation within the region of homozygosity in the CYP27A1 gene associated with CTX. The potential genetic contribution of loci in addition to CYP27A1 was considered given the detection of multiple regions of homozygosity and several atypical features such as facial asymmetry, short neck and missing rib. Although the exome was analysed for genes outside of our neuromus- cular gene filters, it is conceivable that a second unidentified genetic mutation has contributed to the phenotype. Both parents were asymptomatic heterozygous carriers. Plasma cholestanol levels were seven times higher than the reference value upper limit of normal. Treatment with chenodeoxy- cholic acid was recommended.16 prominently affecting axial muscles. Nine heterozygotes with the CACNA1S variant identified in patient S.9 were present in gnomAD. The variant is in the S4 helix of the voltage-sensing transmembrane domain III in the Cav1.1 channel (figure 1), where arginine mutations are associ- ated with hypokalemic periodic paralysis.18 Both parents were asymptomatic and tested negative for the Asn909Ser variant. A gene panel testing for neuromuscular disorders did not reveal other mutation(s). A muscle biopsy was not available to examine the abnormalities in the T-tubules and sarcoplasmic reticulum, as reported previously in Cav1.1 myopathy. Similarly, a late-aged female (S.10) with a MYH14 variant (chr19:50760672, NM_024729.3:c.2038C>T, p.Arg680Cys) had a late onset complex disease with features of myopathy, neuropathy and deafness. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 Neurogenetics The presenting feature of late-onset facial weakness without corneal lattice dystrophy is atypical in patients with gelsolin amyloidosis.27 31 Similarly, skeletal anomalies and facial dysmorphism are not typically associated with CTX, whereas more common features such as cerebellar ataxia, spasticity and subcutaneous xanthomas were absent in our patient. A history of parental consanguinity, juvenile cataracts, demyelinating neuropathy and cerebellar white- matter changes on MRI supported the diagnosis. Bilateral optic neuropathy as a prominent and presenting feature together with multifocal brain white matter hyperintensi- ties led to an initial misdiagnosis of multiple sclerosis in our APBD patient, which is not unusual for this rare pleo- morphic disorder.32 Optic neuropathy has not previously been described in patients with APBD. A naturally occur- ring orthologue of human GBE1 deficiency in Norwegian forest cats caused mild to moderate storage of abnormal glycogen in retinal ganglion cells with degeneration of the optic nerve,33 suggesting that the optic nerve is vulner- able to mutation effects in APBD. The phenotype of S.14 patient with a variant in SPTLC1 is atypical and not found in in other HSAN1A cases. However, recent evidence has suggested that mutations in SPTLC1 are linked to motor phenotypes including juvenile ALS.34 Interestingly, atyp- ical phenotype of growth retardation, hypotonia, and vocal cord paralysis was reported in a patient with the p.Ser331Phe mutation in SPTLC1, which is close to the p. Ser340Leu variant identified in this study.35 Our diagnostic yield in those who presented below 20 years old was 59%, highlighting the novelty and utility of our approach. Late age of onset was a diagnostic challenge in our cohort, with a third of all patients having an age of onset over 40 years old. Within this patient group we were able to achieve a diagnostic success rate of 27%. With the availability of WES technology, the curation of gene filter sets appropriate for the disease phenotype is an important step in variant interpretation. Filtering of variants using targeted gene lists containing a total of 634 genes applied broadly across the phenotype spectrum helped in reaching the molecular diagnosis in our patients. For example, we were able to detect pathogenic variants in CYP27A1, GBE1, GSN, HEXB, and PNPLA6 genes, which have been previously associated with rare, diverse clinical presentations,25–28 and many of these genes are not typi- cally included in diagnostic gene panel testing for specific diseases. Neurogenetics Neurogenetics be better described so that more physicians can decide which next steps are appropriate in the subsequent evaluation and validation of genetic testing data. Our approach used an array of tools feasible in clinical practice ranging from predictive algorithms, bioinformatics, family segregation studies, clin- ical diagnostic tests, and biological assays, which enhanced the yield of WES testing. We were able to obtain a molecular diagnostic yield of 39% (n=26) in 66 sequential, unselected individuals with diverse presentations of rare neurogenetic disorders, which is higher than the positive rates of other studies (13%–30%).6 22 These patients were without a diag- nosis for many years despite extensive evaluations including single and panel gene testing for their conditions. Previous studies have demonstrated diagnostic success rates of 46% and 39% in cohorts receiving WES during childhood.23 24 Our diagnostic yield in those who presented below 20 years old was 59%, highlighting the novelty and utility of our approach. Late age of onset was a diagnostic challenge in our cohort, with a third of all patients having an age of onset over 40 years old. Within this patient group we were able to achieve a diagnostic success rate of 27%. variants, the reduction of SH3TC2 levels may have modi- fied the presentation of Sandhoff disease and exacerbated the patient’s neuropathy.30 When applicable and feasible, we used blood and tissue samples from patients to validate the biological consequence of the candidate pathogenic variant. For example, impaired GBE1 activity in fibroblasts of F1.1 and F1.2 patients, and elevated plasma cholestanol levels in S.16 patient confirmed the loss of enzymatic func- tion resulting from underlying mutations causing APBD and CTX, respectively. variants, the reduction of SH3TC2 levels may have modi- fied the presentation of Sandhoff disease and exacerbated the patient’s neuropathy.30 When applicable and feasible, we used blood and tissue samples from patients to validate the biological consequence of the candidate pathogenic variant. For example, impaired GBE1 activity in fibroblasts of F1.1 and F1.2 patients, and elevated plasma cholestanol levels in S.16 patient confirmed the loss of enzymatic func- tion resulting from underlying mutations causing APBD and CTX, respectively. on October 23, 2024 by guest. Protected by copyright. http://jnnp.bmj.com/ rst published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from WES analysis extended phenotypes in patients with novel as well as established disease-associated variants. Neurogenetics We believe that our approach is more successful and cost effective than panel testing. Accordingly, we were able to diagnose 11 of 23 patients with previous negative gene panel sequencing. Testing family members enhanced the diagnostic yield of WES even in singleton cases. Parental DNA testing deter- mined de novo heterozygous point mutations in FBXO38, GARS, MFN2, and MYH7 genes, and germline transmis- sion on separate alleles for recessive mutations, such as in ADSSL1, CYP27A1, GBE1, and PNPLA6 genes. The location of variants in the context of conserved functional domains and mutation ‘hot-spots’ predicted pathogenicity in previ- ously undescribed variants such as MYH7. The MYH7 gene encodes slow/β-cardiac myosin heavy chain, the motor protein of the sarcomere thick filaments which is expressed in type 1 skeletal muscle fibres and the heart ventricles. The mutated MYH7 Leu1533 residue occupied a strategic posi- tion within the light meromyosin region of the myosin rod at which the substitution of proline likely disrupted coiled-coil structure and thus affected thick filament assembly.7 on October 23, 2024 by guest. Protected by copyright. ttp://jnnp.bmj.com/ Most importantly, molecular diagnosis allows clinicians to treat and more accurately predict prognosis. Using our approach, we were able to diagnose rare and poten- tially treatable diseases such as CMS10 and CTX.14 16 Furthermore, systemic complications of disease can also be anticipated and mitigated, which have prognostic implications and impact quality of life and survival. For example, screening for systemic amyloidosis in patients with gelsolin mutation, as well as cardiac and respiratory disease in patients with mutations in MYH7, DMD and LAMA2 genes. Finally, a molecular diagnosis paves the way for disease modelling and precision medicine approaches in rare and disabling disorders with no currently available treatment. er 23, 2024 by guest. Protected by copyright. For recessive diseases, homozygosity was validated for variants in ADSSL1, CYP27A1, DOK7, LAMA2 and SH3TC2 genes through parental testing, SNP homozygosity mapping, and the absence of large gene deletions by CGH array anal- ysis. In cases with compound heterozygous mutations, the search for a second mutation involved genetic testing beyond WES analysis, including deep gene sequencing, CGH array and tissue-derived transcript-level analysis. This is exempli- fied by the individuals with variants in ADSSL1, GBE1 and HEXB genes. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 DISCUSSION h Whereas WES is well established in research and diagnostic laboratories, additional methods and approaches are needed for the interpretation and confirmation of this data, and the interface between the neurologist and the patient needs to 1193 Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 on Octo http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Correction notice This article has been corrected since it appeared Online First. Author name Chaudhury was corrected to Chaudhry. Affiliations have been updated Correction notice This article has been corrected since it appeared Online First. Author name Chaudhury was corrected to Chaudhry. Affiliations have been updated. Correction notice This article has been corrected since it appeared Online First. Author name Chaudhury was corrected to Chaudhry. Affiliations have been updated 8 Bode H, Bourquin F, Suriyanarayanan S, et al. Hsan1 mutations in serine palmitoyltransferase reveal a close structure-function-phenotype relationship. Hum Mol Genet 2016;25:853–65. Twitter Shin J Oh @shinjoh@charter.net and Ami Mankodi @ami_mankodi Twitter Shin J Oh @shinjoh@charter.net and Ami Mankodi @ami_mankodi Acknowledgements We thank the patients and their families for their participation in this study. Elizabeth Hartnett, MSW, contributed as the patient care coordinator for the NIH neurogenetics clinic. Yotam Blech-Hermoni, PhD, Sharolyn Kawakami-Schulz, PhD, and Charissa Obeng-Nyarko, BS, (NINDS) contributed to biological assays. The authors would also like to thank the NIH Intramural Sequencing Center (NISC) for whole-exome sequencing and the NIH Clinical Centre Radiology Department for assistance with MRI and x-ray imaging. Acknowledgements We thank the patients and their families for their participation in this study. Elizabeth Hartnett, MSW, contributed as the patient care coordinator for the NIH neurogenetics clinic. Yotam Blech-Hermoni, PhD, Sharolyn Acknowledgements We thank the patients and their families for their participation in this study. Elizabeth Hartnett, MSW, contributed as the patient care coordinator for the NIH neurogenetics clinic. Yotam Blech-Hermoni, PhD, Sharolyn 9 Sumner CJ, d’Ydewalle C, Wooley J, et al. A dominant mutation in FBXO38 causes distal spinal muscular atrophy with calf predominance. Am J Hum Genet 2013;93:976–83. 9 Sumner CJ, d’Ydewalle C, Wooley J, et al. A dominant mutation in FBXO38 causes distal spinal muscular atrophy with calf predominance. Am J Hum Genet 2013;93:976–83. Kawakami-Schulz, PhD, and Charissa Obeng-Nyarko, BS, (NINDS) contributed to biological assays. The authors would also like to thank the NIH Intramural Sequencing Center (NISC) for whole-exome sequencing and the NIH Clinical Centre Radiology Department for assistance with MRI and x-ray imaging. 10 Geranmayeh F, Clement E, Feng LH, et al. Genotype-Phenotype correlation in a large population of muscular dystrophy patients with LAMA2 mutations. Neuromuscul Disord 2010;20:241–50. g y biological assays. The authors would also like to thank the NIH Intram 11 Chung KW, Kim SB, Park KD, et al. Early onset severe and late-onset mild Charcot- Marie-Tooth disease with mitofusin 2 (MFN2) mutations. Brain 2006;129:2103–18.i Contributors CG, KHF and AM: conceptualisation and supervision. CONCLUSION l Peter A Calabresi http://orcid.org/0000-0002-7776-6472 Charlotte J Sumner http://orcid.org/0000-0001-5088-6012 Ricardo H Roda http://orcid.org/0000-0002-3255-7749 Vinay Chaudhry http://orcid.org/0000-0001-5625-7989 Thomas E Lloyd http://orcid.org/0000-0003-4756-3700 Thomas O Crawford http://orcid.org/0000-0003-0916-2797 S H Subramony http://orcid.org/0000-0002-8052-9240 Shin J Oh http://orcid.org/0000-0002-7989-6107 Kurenai Tanji http://orcid org/0000-0003-2230-2416 WES analysis involves convergence of human and computer-assisted analysis. In this study, we demonstrate that detailed phenotyping, family segregation studies and biological assays together with strin- gent application of algorithms and use of candidate gene filters improved the diagnostic efficiency of WES in patients with complex atypical presentations of rare neurogenetic diseases. This approach allowed us to redefine our practice, identify new disease-associated variants, expand the phenotypic spectrum of rare diseases and offer better care to our patients and at-risk family members. Shin J Oh http://orcid.org/0000 0002 7989 6107 Kurenai Tanji http://orcid.org/0000-0003-2230-2416 Ami Mankodi http://orcid.org/0000-0002-5131-7767 REFERENCES 1 Walsh M, Bell KM, Chong B, et al. Diagnostic and cost utility of whole exome sequencing in peripheral neuropathy. Ann Clin Transl Neurol 2017;4:318–25. Neurogenetics The WES analysis also led to valuable insights into the potential disease-modifying influence of additional genetic variations.29 In F.4 family with HEXB +SH3TC2 The major limitations of WES include the inability to interrogate many variants that occur in regions of genome not currently recognised as a functional or regulatory region or resulting in large genomic reorganisations such as repeat expansions, deletions or duplications. Incomplete capture and coverage of genes may result in a failure to make a molec- ular diagnosis. Gene filters may not allow for detection of mutations in genes not previously known to cause a specific disorder. The algorithms may be limited by low concordance due to inherent biases and dependency on training datasets. guest. Protected by copyright. 1194 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 o Neurogenetics Patient consent for publication Obtained. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. 24 Waldrop MA, Pastore M, Schrader R, et al. Diagnostic utility of whole exome sequencing in the neuromuscular clinic. Neuropediatrics 2019;50:96–102. 25 Guyant-Maréchal L, Verrips A, Girard C, et al. Unusual cerebrotendinous xanthomatosis with fronto-temporal dementia phenotype. Am J Med Genet A 2005;139A:114–7. 26 Lossos A, Meiner Z, Barash V, et al. Adult polyglucosan body disease in Ashkenazi Jewish patients carrying the Tyr329Ser mutation in the glycogen-branching enzyme gene. Ann Neurol 1998;44:867–72. Patient consent for publication Obtained. Ethics approval All studies were in compliance with the NIH Privacy Programme and approved by an NIH Institutional Review Board. All studies adhered to the principles specified in the Declaration of Helsinki. Ethics approval All studies were in compliance with the NIH Privacy Programme and approved by an NIH Institutional Review Board. All studies adhered to the principles specified in the Declaration of Helsinki. 17 Akman HO, Kakhlon O, Coku J, et al. Deep intronic GBE1 mutation in manifesting heterozygous patients with adult polyglucosan body disease. JAMA Neurol 2015;72:441–5. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. 18 Wu J, Yan Z, Li Z, et al. Structure of the voltage-gated calcium channel Ca(v)1.1 at 3.6 Å resolution. Nature 2016;537:191–6. Data availability statement Data are available on reasonable request. Data availability statement Data are available on reasonable request. 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Author affiliations 1 Author affiliations 1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 2Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 3Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 4Department of Neurology, University of Florida, Gainesville, Florida, USA 5Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, USA 6Department of Neurology, George Washington University, Washington, District of Columbia, USA 7Division of Neuropathology, Columbia University Medical Center, New York, New York, USA i 1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 2 Reddy HM, Cho K-A, Lek M, et al. The sensitivity of exome sequencing in identifying pathogenic mutations for LGMD in the United States. J Hum Genet 2017;62:243–52. 2Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 3 Thuriot F, Gravel E, Buote C, et al. Molecular diagnosis of muscular diseases in outpatient clinics: a Canadian perspective. Neurol Genet 2020;6:e408. y 3Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 4 Harris E, Topf A, Barresi R, et al. Exome sequences versus sequential gene testing in the UK highly specialised service for limb girdle muscular dystrophy. Orphanet J Rare Dis 2017;12:151. y , 4Department of Neurology, University of Florida, Gainesville, Florida, USA epartment of Neurology, University of Florida, Gainesville, Florida, US 5Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, USA 5 Ghaoui R, Cooper ST, Lek M, et al. Use of whole-exome sequencing for diagnosis of limb-girdle muscular dystrophy: outcomes and lessons learned. JAMA Neurol 2015;72:1424–32. 6Department of Neurology, George Washington University, Washington, District of Columbia, USA 7 6Department of Neurology, George Washington University, Washington, District of Columbia, USA 7 6 Haskell GT, Adams MC, Fan Z, et al. Diagnostic utility of exome sequencing in the evaluation of neuromuscular disorders. Neurol Genet 2018;4:e212.i 7Division of Neuropathology, Columbia University Medical Center, New York, New York, USA 7Division of Neuropathology, Columbia University Medical Center, New York, New York, USA 7 Meredith C, Herrmann R, Parry C, et al. Mutations in the slow skeletal muscle fiber myosin heavy chain gene (MYH7) cause Laing early-onset distal myopathy (MPD1). Am J Hum Genet 2004;75:703–8. Correction notice This article has been corrected since it appeared Online First. Author name Chaudhury was corrected to Chaudhry. Affiliations have been updated CG, NS, JL, MO, AS, PC, CS, RR, VC, TL, TOC, SHS, SJO, PR, KT, JK, KHF and AM: acquisition of data, editing the manuscript; investigation: CG, NS, JL, MO, AS, KHF and AM: analysis and interpretation of data; project administration: CG, KHF and AM; supervision: CG, KHF, AM; writing-original draft: CG, NS and AM. 12 Harris E, McEntagart M, Topf A, et al. Clinical and neuroimaging findings in two brothers with limb girdle muscular dystrophy due to LAMA2 mutations. Neuromuscul Disord 2017;27:170–4. 12 Harris E, McEntagart M, Topf A, et al. Clinical and neuroimaging findings in two brothers with limb girdle muscular dystrophy due to LAMA2 mutations. Neuromuscul Disord 2017;27:170–4. 13 Jerath NU, Mankodi A, Crawford TO, et al. Charcot-Marie-Tooth disease type 4C: novel mutations, clinical presentations, and diagnostic challenges. Muscle Nerve 2018;57:749–55. Funding This study was supported by Intramural Research Programme of the National Institute of Neurological Disorders and Stroke (Project # 1ZIANS002974). Funding This study was supported by Intramural Research Programme of the National Institute of Neurological Disorders and Stroke (Project # 1ZIANS002974). 14 Beeson D, Higuchi O, Palace J, et al. Dok-7 mutations underlie a neuromuscular junction synaptopathy. Science 2006;313:1975–8. Competing interests PC is principal investigator on grants to Johns Hopkins University from Annexon and Biogen, and he has received consulting fees from Disarm Therapeutics and Biogen. Competing interests PC is principal investigator on grants to Johns Hopkins University from Annexon and Biogen, and he has received consulting fees from Disarm Therapeutics and Biogen. 15 Witting N, Vissing J. Pharmacologic treatment of downstream of tyrosine kinase 7 congenital myasthenic syndrome. JAMA Neurol 2014;71:350–4. 16 Stelten BML, Huidekoper HH, van de Warrenburg BPC, et al. Long-Term treatment effect in cerebrotendinous xanthomatosis depends on age at treatment start. Neurology 2019;92:e83–95. Patient consent for publication Obtained. Patient consent for publication Obtained. Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 ORCID iDs ORCID iDs Christopher Grunseich http://orcid.org/0000-0003-4994-2472 Christopher Grunseich http://orcid.org/0000-0003-4994-2472 Christopher Grunseich http://orcid.org/0000-0003-4994-2472 1195 Grunseich C, et al. J Neurol Neurosurg Psychiatry 2021;92:1186–1196. doi:10.1136/jnnp-2020-325437 on October 23, 2024 by guest. Protected b http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-325437 on 8 June 2021. Downloaded from Neurogenetics Neurogenetics 34 Johnson JO, Chia R, Kumaran R. 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J Neurol 2015;262:1066–8. i y 31 Park KJ, Park JH, Park JH, et al. The first Korean family with hereditary gelsolin amyloidosis caused by p.D214Y mutation in the GSN gene. Ann Lab Med 2016;36:259–62. 38 Rentzsch P, Witten D, Cooper GM, et al. Cadd: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res 2019;47:D886–94. 32 Hellmann MA, Kakhlon O, Landau EH, et al. Frequent misdiagnosis of adult polyglucosan body disease. J Neurol 2015;262:2346–51. 39 Alirezaie N, Kernohan KD, Hartley T, et al. ClinPred: prediction tool to identify disease-relevant nonsynonymous single-nucleotide variants. Am J Hum Genet 2018;103:474–83. 39 Alirezaie N, Kernohan KD, Hartley T, et al. ClinPred: prediction tool to identify disease-relevant nonsynonymous single-nucleotide variants. Neurogenetics Am J Hum Genet 2018;103:474–83. 33 Fyfe JC, Kurzhals RL, Hawkins MG, et al. 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https://openalex.org/W4232174985	https://www.duo.uio.no/bitstream/10852/47072/1/12887_2012_Article_609.pdf	English	null	Erratum to: “Reduction in BMI z-score and improvement in cardiometabolic risk factors in obese children and adolescents. The Oslo adiposity intervention study - a hospital/public health nurse combined treatment.”	BMC pediatrics	2,012	cc-by	1,904	* Correspondence: Magnhild.L.P.Kolsgaard@ous-hf.no 1Department of Pediatrics, Oslo University Hospital, Ullevål, PB 4956, Nydalen 0424, Oslo, Norway Full list of author information is available at the end of the article Correction “An increase in BMI z-score was associated with a worsening of HOMA-IR, insulin, C-peptide and total/ HDL cholesterol ratio.” After publication of this work [1], it came to our atten- tion that the laboratory we use changed the method of analysis for insulin and C-peptide during our data col- lection. Before March 2007 the method used for insulin was competitive radioimmunoassay (Linco Research Inc, St Charles, MO, USA) and for C-peptide competitive luminoimmunoassay (Immulite 2000, Diagnostic Pro- ducts Corporation, CA, USA). After March 2007 non- competitive immunofluorometric assays (DELFIA kit form Wallac OY, Turku, Finland) were used. Explanation: The group with an increase in BMI z-score had a wor- sening in HOMA-IR and insulin in addition to C- peptide and total/HDL cholesterol ratio as previously reported. Corrections in the Main manuscript We have therefore recalculated the insulin and C- peptide values analyzed after March 2007, and repeated the statistical analyses involving insulin, C-peptide and HOMA-IR. In the section “Baseline characteristics” on page 4 In the section “Baseline characteristics” on page 4 The fourth-fifth sentence should have read: CORRECTION Open Access © 2012 Kolsgaard et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Explanation: Explanation: After recalculation the significant reduction in insulin in the group with a very small reduction in BMI z-score disappeared. None of the groups had significant improvements in insulin concentration (the improve- ments in group 1 and 2 were of borderline significance). Correction: “Reduction in BMI z-score and improvement in cardiometabolic risk factors in obese children and adolescents. The Oslo adiposity intervention study - a hospital/public health nurse combined treatment.” Correction: “Reduction in BMI z-score and improvement in cardiometabolic risk factors in obese children and adolescents. The Oslo adiposity intervention study - a hospital/public health nurse combined treatment.” Magnhild L Pollestad Kolsgaard1*, Geir Joner1,2, Cathrine Brunborg3, Sigmund A Anderssen4, Serena Tonstad5,6 and Lene Frost Andersen6 The seventh sentence should have read: Kolsgaard et al. BMC Pediatrics 2012, 12:77 Kolsgaard et al. BMC Pediatrics 2012, 12:77 Kolsgaard et al. BMC Pediatrics 2012, 12:77 http://www.biomedcentral.com/1471-2431/12/77 CORRECTION Open Access Corrections in the Abstract “Insulin, C-peptide, triglycerides and aerobic fitness at baseline also differed between the groups, but the other seven parameters did not differ between the groups, see corrected version of Additional file 1. The group with the largest reduction in BMI z-score (Group 1) had sig- nificantly higher aerobic fitness and significantly lower insulin and C-peptide concentration than the three other groups (data not shown).” In Results the fifth sentence should have read: “Even a very small reduction in BMI z-score (group 3) was associated with significantly lower total cholesterol, LDL cholesterol and total/HDL cholesterol ratio.” In Results the fifth sentence should have read: Explanation: Insulin should be added to the list with metabolic parameters that differed between the groups at base- line. Moreover the group with the largest reduction in BMI z-score (Group 1) had significant lower insu- lin in addition to lower C-peptide and higher aerobic fitness as previously reported. Page 2 of 3 Kolsgaard et al. BMC Pediatrics 2012, 12:77 http://www.biomedcentral.com/1471-2431/12/77 the lowest HOMA-IR value at baseline even though the difference was not statistically significant.” the lowest HOMA-IR value at baseline even though the difference was not statistically significant.” In the section “Changes in metabolic parameters and aerobic activity according to changes in BMI z-score after one year intervention” on page 4 Explanation: The second sentence should have read: We have written that the group in our study with the lowest BMI z-score initially (that is the same group that reduced their BMI z-score the most, group 1) also tended to have the lowest HOMA-IR and insulin values at the beginning of the interven- tion even though the difference was not statistically significant. The difference between group 1 and the three other groups according to insulin now become statistically significant. “We also found significant improvements in total chol- esterol, LDL cholesterol and total cholesterol/HDL chol- esterol ratio in the total group (data not shown).” Explanation: The significant reduction in HOMA-IR and insulin after one year intervention in the total group disappear. The tenth sentence (start of paragraph 3) in the same section should have read: The fifteenth sentence (start of paragraph four) on page 5 should have read: “A very small reduction in BMI z-score after one year follow-up (group 3) was associated with significantly lower total cholesterol, LDL and total cholesterol/HDL cholesterol ratio, see corrected version of Additional file 2. “We found that a reduction in BMI z-score ≥0.23 (group 4) improved insulin resistance.” Explanation: It is no longer correct that the group with a very small reduction in BMI z-score (≥0.00- < 0.10) improved insulin and insulin resistance. Insulin re- sistance only improved in the group with the largest reduction in BMI z-score, and the improvement in insulin was of borderline significance in this group. Explanation: The significant reduction in insulin in the group with a very small reduction in BMI z-score disappeared. None of the groups had significant improvements in insulin concentration (the improvements in group 1 and 2 were of borderline significance). Explanation: Explanation: This is no longer relevant for our findings since insulin did not improve in any of the groups. Explanation: h Explanation: h The improvement in HOMA-IR was no longer border- line significant in the group with the smallest reduction in BMI z-score (group 3). The group with an increase in BMI z-score now got a worsening in insulin resistance, like Reinehr earlier has reported. The last part of the sentence “though C- peptide concentrations increased, indicating increased insulin production and future risk of diabetes” is no longer relevant for our findings. The thirteenth sentence (paragraph 3) in the same sec- tion should have read: “The group with an increase in BMI z-score (group 4) had a significant increase in HOMA-IR, insulin, C-peptide and total cholesterol/HDL cholesterol ratio after the intervention.” The twenty-fifth sentence (end of paragraph four) on page 5 “No change in glucose and a simultaneous lower- ing of insulin indicates that the insulin resistance is improved, and less insulin is needed to maintain the same glucose concentration” should be deleted. Explanation: The group with an increase in BMI z-score (group 4) got a worsening in HOMA-IR and insulin in addition to C-peptide and total/HDL cholesterol ratio as we previ- ously reported. Explanation: Explanation: Explanation: The twenty-first sentence (paragraph four) on page 5 should have read: The eleventh sentence (paragraph 3) in the same section “Like Reinehr et al we found an increase in insulin re- sistance in the group with an increase in BMI z-score [3,4]”. The phrase “The improvement in HOMA-IR was of borderline significance in this group” should be deleted. Explanation: The improvement in insulin in the group with the small reduction in BMI z-score disappeared (group 3) and the group with increased BMI z-score had an wor- sening of HOMA-IR and insulin in addition to C- peptide and total/HDL cholesterol Additional files Additional file 1: Corrected Table 1. The values that changed are shown in bold. Additional file 2: Corrected Table 2. The values that changed are shown in bold. Received: 26 March 2012 Accepted: 18 June 2012 Published: 18 June 2012 Received: 26 March 2012 Accepted: 18 June 2012 Published: 18 June 2012 Author details 1D f 1Department of Pediatrics, Oslo University Hospital, Ullevål, PB 4956, Nydalen 0424, Oslo, Norway. 2Department of Health Management and Health Economics, Institute of Health and Society, University of Oslo, Oslo, Norway. 3Centre for Clinical Research, Unit of Epidemiology and Biostatistics, Oslo University Hospital, Oslo, Norway. 4Department of Sports Medicine, The Norwegian School of Sport Sciences, Oslo, Norway. 5Department of Preventive Cardiology, Oslo University Hospital, Oslo, Norway. 6Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway. Received: 26 March 2012 Accepted: 18 June 2012 Published: 18 June 2012 Kolsgaard et al. BMC Pediatrics 2012, 12:77 http://www.biomedcentral.com/1471-2431/12/77 Kolsgaard et al. BMC Pediatrics 2012, 12:77 http://www.biomedcentral.com/1471-2431/12/77 improvement in total-, LDL, and total/HDL cholesterol. An increase in BMI z-score during the one year period was associated with worsening of HOMA-IR, insulin, C- peptide and total/HDL cholesterol.” Explanation: References 1. Kolsgaard ML, Joner G, Brunborg C, Anderssen SA, Tonstad S, Andersen LF: Reduction in BMI z-score and improvement in cardiometabolic risk factors in obese children and adolescents The Oslo Adiposity Intervention Study - a hospital/public health nurse combined treatment. BMC Pediatr 2011, 11:47. 1. Kolsgaard ML, Joner G, Brunborg C, Anderssen SA, Tonstad S, Andersen LF: Reduction in BMI z-score and improvement in cardiometabolic risk factors in obese children and adolescents The Oslo Adiposity Intervention Study - a hospital/public health nurse combined treatment. BMC Pediatr 2011, 11:47. 2. Caprio S: Insulin resistance in childhood obesity. J Pediatr Endocrinol Metab 2002, 15(Suppl 1):487–92. 2. Caprio S: Insulin resistance in childhood obesity. J Pediatr Endocrinol Metab 2002, 15(Suppl 1):487–92. 3. Reinehr T, Kiess W, Kapellen T, Andler W: Insulin sensitivity among obese children and adolescents, according to degree of weight loss. Pediatrics 2004, 114(6):1569–73. 4. Reinehr T, Andler W: Changes in the atherogenic risk factor profile according to degree of weight loss. Arch Dis Child 2004, 89(5):419–22. 4. Reinehr T, Andler W: Changes in the atherogenic risk factor profile according to degree of weight loss. Arch Dis Child 2004, 89(5):419–22. doi:10.1186/1471-2431-12-77 Cite this article as: Kolsgaard et al.: Correction: “Reduction in BMI z-score and improvement in cardiometabolic risk factors in obese children and adolescents. The Oslo adiposity intervention study - a hospital/public health nurse combined treatment.”. BMC Pediatrics 2012 12:77. Conclusions The thirteenth sentence (paragraph three) on page 5 should have read: The first and second sentences in the conclusion should have read: “The group in our study with the lowest BMI z-score initially also had the lowest insulin value at the begin- ning of the intervention. This group also tended to have “In conclusion even a modest reduction in BMI z- score after one year intervention was associated with Page 3 of 3 Page 3 of 3 References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W3049614910	https://hal.archives-ouvertes.fr/hal-02919602/document	English	null	Experimental realization of dual task processing with a photonic reservoir computer	APL photonics	2,020	cc-by	7,441	To cite this version: Jeremy Vatin, Damien Rontani, Marc Sciamanna. Experimental realization of dual task processing with a photonic reservoir computer. APL Photonics, 2020, 5 (8), pp.086105. 10.1063/5.0017574. hal-02919602 Experimental realization of dual task processing with a photonic reservoir computer Cite as: APL Photonics 5, 086105 (2020); https://doi.org/10.1063/5.0017574 Submitted: 22 April 2020 . Accepted: 28 July 2020 . Published Online: 13 August 2020 Jeremy Vatin , Damien Rontani, and Marc Sciamanna HAL Id: hal-02919602 https://hal.science/hal-02919602v1 Submitted on 23 Aug 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. I. INTRODUCTION solution was proposed with time-delay reservoir computing: Instead of using physical neurons, only one physical neuron is used, and several virtual neurons are temporally spread along a delay line.8 The time separation between virtual neurons is set to be smaller than the physical-neuron response time so that the neu- rons remain in a sustained transient dynamics, which effectively translates into time-multiplexed interconnection between the virtual neurons. In that framework, adding neurons only requires length- ening the delay line. Several photonic architectures use this specific technique, with either an optoelectronic4,9,10 or an all-optical11–17 delay line. solution was proposed with time-delay reservoir computing: Instead of using physical neurons, only one physical neuron is used, and several virtual neurons are temporally spread along a delay line.8 The time separation between virtual neurons is set to be smaller than the physical-neuron response time so that the neu- rons remain in a sustained transient dynamics, which effectively translates into time-multiplexed interconnection between the virtual neurons. In that framework, adding neurons only requires length- ening the delay line. Several photonic architectures use this specific technique, with either an optoelectronic4,9,10 or an all-optical11–17 delay line. Building energy efficient systems to process data currently per- formed by computer is one of the focus problems that photonic reservoir computing is trying to address. A reservoir computing sys- tem is a specific kind of neural network with a recurrent topology, i.e., coupling signals and information are not propagating unidirec- tional in the network structure. The training, consisting of adjusting the interconnection weight between the neurons for this particular structure, is usually difficult and data intensive as it scales with the square of the network size to solve a specific task. This also implies that the physical architecture with many tunable degrees of freedom should be designed, which represents a significant technical chal- lenge for the development of efficient hardware platforms. A reser- voir computing system overcomes these hurdles by not realizing the training through internal weight adjustments but by keeping it fixed and training a readout layer unidirectionally connected to the recur- rent network. This can be achieved with a simple linear regression at the readout with simple regression algorithms.1,2 This is specif- ically interesting as it allows the use of physical components for a hardware implementation of a neural network. a)Author to whom correspondence should be addressed: jeremy.vatin@centralesupelec.fr a)Author to whom correspondence should be addressed: jeremy.vatin@centralesupelec.fr ABSTRACT We experimentally demonstrate the possibility to process two tasks in parallel with a photonic reservoir computer based on a vertical-cavity surface-emitting laser (VCSEL) as a physical node with time-delay optical feedback. The two tasks are injected optically by exploiting the polarization dynamics of the VCSEL. We test our reservoir with the very demanding task of nonlinear optical channel equalization as an illustration of the performance of the system and show the recover of two signals simultaneously with an error rate of 0.3% (3%) for a 25 km-fiber distortion (50 km-fiber distortion) at a processing speed of 51.3 Mb/s. © 2020 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0017574., s Jeremy Vatin,a) Damien Rontani, and Marc Sciamanna Jeremy Vatin,a) Damien Rontani, and Marc Sciamanna AFFILIATIONS Chair in Photonics, LMOPS EA 4423 Laboratory, CentraleSupélec and Université Lorraine, 2 rue Edouard Belin, F-57070 Metz, France ARTICLES YOU MAY BE INTERESTED IN APL Photonics 5, 081301 (2020); https://doi.org/10.1063/5.0013577 Optical parametric gain in CMOS-compatible sub-100 m photonic crystal waveguides APL Photonics 5, 066108 (2020); https://doi.org/10.1063/5.0003633 APL Photonics 5, 086105 (2020); https://doi.org/10.1063/5.0017574 5, 086105 © 2020 Author(s). APL Photonics 5, 086105 (2020); https://doi.org/10.1063/5.0017574 © 2020 Author(s). APL Photonics 5, 086105 (2020); https://doi.org/10.1063/5.0017574 © 2020 Author(s). 5, 086105 ARTICLE APL Photonics scitation.org/journal/app Experimental realization of dual task processing with a photonic reservoir computer Experimental realization of dual task processing with a photonic reservoir computer Cite as: APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 Submitted: 22 April 2020 • Accepted: 28 July 2020 • Published Online: 13 August 2020 Cite as: APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 Submitted: 22 April 2020 • Accepted: 28 July 2020 • Published Online: 13 August 2020 II. METHOD The experimental setup is depicted in Fig. 1. The reservoir itself is the same as the one we have previously studied in Ref. 20: It comprises a VCSEL (Raycan) as a physical node, which emits light at 1552.75 nm for the dominant linear polarization mode (LPx) and at 1552.89 nm for the depressed polarization mode (LPy). The bias current of the VCSEL is set at 4.5 mA, which corresponds to 1.5 times the threshold current. This choice of pumping current is based on the previous numerical analysis we conducted in Ref. 19, showing that a pumping current close to the current threshold lead to high-memory capacity and overall computing performance for the time-delay VCSEL-based reservoir computer. The feedback loop is made of a SMF-28 single mode fiber (standard telecommunication fiber) resulting in a delay line of τ = 39.4 ns. As only one calcula- tion step can be performed per round-trip, this length imposes to a i In this article, we present an experimental realization of a reser- voir computer processing two tasks simultaneously. This reservoir computer is based on the time-delay reservoir architecture, using a VCSEL as a physical node. The two tasks are injected optically in each polarization mode of the VCSEL. By carefully choosing the operating point of the reservoir computer, we show the possibility to tune the performance of the system on each processed task. As an illustration, we test our reservoir on the nonlinear optical channel equalization. This task is very demanding as signals sent in optical fiber are distorted due to several nonlinear effects, such as chromatic dispersion and Kerr effect.22 More specifically, we are able to recover FIG. 1. (a) Scheme of the experiment. The two masked signals are sent on the two modulators. Each input signal is aligned with a different polarization mode of the VCSEL. MZ: Mach–Zehnder modulator, P.C.: polarization controller, AWG: arbitrary waveform generator, Att: attenuator, Osc: Oscilloscope, ampl: amplificator, and PD: photodiode. (b) Example of two input streams generated by the AWG. The blue line corresponds to the input stream injected in the dominant polarization mode (LPx), and the red line corresponds to the stream injected in the depressed polarization mode (LPy) of the VCSEL. (c) example of signals recorded at the output of the reservoir computing system. I. INTRODUCTION Several architectures using this specific principle already exist.3–7 The vertical-cavity surface-emitting laser (VCSEL) is a good candidate to realize a time-delay reservoir computer and process data in optical networks as it is widely used in optical telecom- munication networks. One of VCSEL’s specificity is light emission along two orthogonal linear polarization modes and a faster mod- ulation frequency than an edge-emitting laser.18 We have already proven numerically19 and experimentally20 that a VCSEL-based time-delay reservoir computer is able to efficiently perform com- putation tasks, with state-of-the-art performance on various tasks such as chaotic time-series prediction and nonlinear WIFI channel equalization. i However, realizing a large physical neural network remains a technical challenge especially with photonic devices. Hence, a Parallel processing of two tasks was originally proposed in Ref. 13 using single-mode dynamics of a laser diode. Using the APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 © Author(s) 2020 5, 086105-1 ARTICLE APL Photonics scitation.org/journal/app two signals simultaneously distorted by 25 km and by 50 km of fiber and sent at 25 Gb/s with a mean error rate of 0.3% at 25 km and of 3% at 50 km, at a processing speed of 51.3 Mb/s. multimode polarization dynamics of a laser diode has also been con- sidered to perform simultaneously several tasks. It has been shown theoretically that using two longitudinal modes of an edge-emitting laser,17 the two modes of a semiconductor ring laser15 or the two polarization modes of a VCSEL21 enable parallel processing with a time-delay reservoir computing architecture. We thus experimen- tally address here the question of whether a VCSEL-based photonic reservoir, which exhibits two polarization modes, is able to perform efficiently two tasks consisting of the recovery of two optical signals being distorted by a fiber. II. METHOD This is why we set the feedback attenuation η to 17 dB to guarantee that enough power is injected to find this best operating point. FIG. 2. Optical spectra of the system under different operation conditions. LPx: dominant polarization mode, LPy: depressed polarization mode, and ML: mas- ter laser. (a) VCSEL with isotropic feedback, η = 17 dB. (b) Reservoir computer with optical injection on both polarization modes without modulation, Pinjx = Pinjy = 0.08 mW, η = 17 dB, (c) reservoir computer with optical injection on both polar- ization modes with modulation, Pinjx = Pinjy = 0.08 mW, η = 17 dB, and (d) reser- voir computer with optical injection on both polarization modes with modulation, Pinjx = 0.08 mW, Pinjy = 0.4 mW η = 17 dB. input streams is given in Fig. 1(b). Both beams are then recombined and sent in the reservoir computer. p The input layer is primarily composed of an arbitrary wave- form generator (AWG) AWG700002A from Tektronix, a tunable laser Tunics T100S from Yanista, and two Mach-Zehnder modula- tors (MZx,y) with a bandwidth of 12.5 GHz. Both modulators are working in their linear regime. The light emitted by the tunable laser is split in two different beams and sent in the two different mod- ulators. The wavelength of this laser is set to 1552.82 nm so that its wavelength is equally separated from the frequencies of the main and depressed polarization modes of the VCSEL, as presented in Fig. 2. By doing so, we ensure that having the same power in both linear polarization modes at the output of the modulators, the power is equally distributed among the two linear polarization modes of the injected VCSEL. Shifting the frequency of the master laser to one of the polarization modes of the VCSEL leads to a more efficient opti- cal injection in this mode and therefore enhances the response of this mode at the expense of the response of the other mode, for which the optical injection is reduced. The two different masked input streams, corresponding to the two tasks Tx,y to be processed, are used to drive both modulators and are generated by the AWG at a symbol rate of 25 GS/s for each stream. The output power of the modulator is controlled by an optical attenuator built inside each modulator. II. METHOD (b) Reservoir computer with optical injection on both polarization modes without modulation, Pinjx = Pinjy = 0.08 mW, η = 17 dB, (c) reservoir computer with optical injection on both polar- ization modes with modulation, Pinjx = Pinjy = 0.08 mW, η = 17 dB, and (d) reser- voir computer with optical injection on both polarization modes with modulation, Pinjx = 0.08 mW, Pinjy = 0.4 mW η = 17 dB. processing speed of 25.65 MHz per task, thus 51.3 MHz for two tasks. The speed of the system could be increased by reducing the length of the delay line, which was not possible in our case. To optimize our use of the VCSEL dynamics, we set the inter-nodes delay θ = 0.04 ns according to previous simulations19 and the frequency limitation of the experimental components (i.e., oscilloscope, arbitrary waveform generator and modulators): The optimal delay between virtual nodes that exploits the best VCSEL’s transient response is θ∗= 0.02 ns; however, the modulation bandwidth of our arbitrary waveform gen- erator (AWG) is at 25 GHz. We use for the training and testing of the reservoir only one every two nodes separated by 2θ = 0.08 ns due to the memory limitation of the computer performing the train- ing, thus leading to consider N = 492 nodes instead of N = 984. Considering an increasing number of virtual nodes while keeping the feedback delay fixed, we observed numerically an improvement of the performance up to Nth = 100. Beyond this threshold value, increasing the size of the virtual network will only lead to marginal improvement in the RC performance. In our experience, we choose N = 492 > Nth for experimental convenience rather than using all the accessible virtual nodes to speed up the training phase without compromising on the performance. There is also a polarization con- troller (P.C.) to control the optical polarization along the feedback loop. Finally, an optical attenuator Keysight 81577A (Att.) is used to control the feedback strength. In this article, the results presented are obtained with the isotropic feedback configuration, i.e., the orienta- tion of the two VCSEL’s polarization modes (LPx,y) are preserved in the external cavity prior to being fed back. Accordingly to the results obtained in Ref. 19, there is an optimum operating point for each value of the feedback strength while varying the injection power. II. METHOD The blue line corresponds to the response of the dominant polarization mode (LPx), and the red line corresponds to the response of the depressed polarization mode (LPy) of the VCSEL. (d) Scheme of the preprocessing method. The signal at the output of the fiber is averaged at twice the frequency of the input data stream, giving symbols b(1) n and b(2) n for each bit bn. This signal is temporally rescaled so that each symbol duration is τ. The ten values b(1) n−4, bn−4(2), to b(1) n and b(2) n are masked with a mask of 10 × N values and used as an input of the reservoir to reconstruct bn−2. FIG. 1. (a) Scheme of the experiment. The two masked signals are sent on the two modulators. Each input signal is aligned with a different polarization mode of the VCSEL. MZ: Mach–Zehnder modulator, P.C.: polarization controller, AWG: arbitrary waveform generator, Att: attenuator, Osc: Oscilloscope, ampl: amplificator, and PD: photodiode. (b) Example of two input streams generated by the AWG. The blue line corresponds to the input stream injected in the dominant polarization mode (LPx), and the red line corresponds to the stream injected in the depressed polarization mode (LPy) of the VCSEL. (c) example of signals recorded at the output of the reservoir computing system. The blue line corresponds to the response of the dominant polarization mode (LPx), and the red line corresponds to the response of the depressed polarization mode (LPy) of the VCSEL. (d) Scheme of the preprocessing method. The signal at the output of the fiber is averaged at twice the frequency of the input data stream, giving symbols b(1) n and b(2) n for each bit bn. This signal is temporally rescaled so that each symbol duration is τ. The ten values b(1) n−4, bn−4(2), to b(1) n and b(2) n are masked with a mask of 10 × N values and used as an input of the reservoir to reconstruct bn−2. APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 © Author(s) 2020 APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 5, 086105-2 APL Photonics FIG. 2. Optical spectra of the system under different operation conditions. LPx: dominant polarization mode, LPy: depressed polarization mode, and ML: mas- ter laser. (a) VCSEL with isotropic feedback, η = 17 dB. II. METHOD (b) Performance of the recovery of a distortion due to 50 km of optical fiber. The blue curve corresponds to the performance of the task Tx, and the red one corresponds to the performance of the task Ty. The dotted line corresponds to the choice of Pinjx reported in Figs. 4 and 5 for this specific case. APL Photonics to react to the master laser and to respond according to the mod- ulated input. This response also broadens the spectra of the two polarization modes of the VCSEL. The spectral width of the domi- nant polarization mode LPx detuned from the modulated input by 9.45 GHz. We observe also that injecting more power in the depressed mode LPy forces its emission despite not lasing when the VCSEL is free-running. We have tested the dual-tasking performance of our reservoir at solving a nonlinear optical channel equalization, which aims at reconstructing a transmitted signal only from the given distorted sig- nal at the channel’s output. We have chosen a single-mode optical fiber for the telecommunication channel. The distortion introduced by this channel is simulated using the nonlinear Schrödinger equa- tion, which models the propagation of a signal in the fiber. This equation reads as24 FIG. 3. Performance on the nonlinear channel equalization task as a function of the injection power in the main polarization mode Pinjx for a fixed ratio of injection power Pinjy Pinjx at 0.3. (a) Performance of the recovery of a distortion due to 25 km of optical fiber. (b) Performance of the recovery of a distortion due to 50 km of optical fiber. The blue curve corresponds to the performance of the task Tx, and the red one corresponds to the performance of the task Ty. The dotted line corresponds to the choice of Pinjx reported in Figs. 4 and 5 for this specific case. i∂E(z, t) ∂z = −iα 2 E(z, t) + β2 2 ∂2E(z, t) ∂t2 −γ∣E(z, t)∣2E(z, t), (1) (1) FIG. 4. Performance on the nonlinear channel equalization task after propagating 25 km in a communication channel made of optical fiber as a function of the ratio of injection Pinjy/Pinjx. (a) Example of the signal sent at the input of the fiber. (b) Corresponding received signal after 25 km of fiber. II. METHOD The blue curves corresponds to the performance of the task Tx, and the red curves corresponds to the performance of the task Ty for (c) the reservoir computer and (d) the linear classifier. The lighter area shows the standard deviation of the performance. The dotted line shows the performance of the reservoir performing the single task. where E(z, t) is the slowly varying envelop of the optical field, α is the attenuation of the fiber, β2 is the second order coefficient of disper- sion, and γ refers to the nonlinearity of the fiber. We have chosen the coefficient of the SMF-28 fiber, which is the single mode sil- ica fiber used for long haul transmission, with α = 0.2 dB km−1, β2 = −21.4 ps2 km−1, and γ = 1.2 W−1 km−1 (Ref. 25). We used the split-step Fourier method to numerically integrate the equation. The signal we use as an input of the fiber is a series of bits using pulse amplitude modulation (PAM) at 25 Gb/s. The power of the input pulse is set to 4 mW. This value is small enough to avoid signifi- cant distortion induced by the Kerr nonlinearity of the optical fiber. To compare our results with the state-of-the-art achieved with time- delay photonic reservoir computer, we perform the recovery after a 25 km and a 50 km long optical fiber.16,26 Examples of input signals are presented in Figs. 4(a) and 5(a), and their respective distorted versions after propagating in the optical fiber 25 km and 50 km are given in Figs. 4(b) and 5(b), respectively. The distortion induced by 25 km of optical fiber still allows us to identify the long lasting pulse. However, after 50 km, the distortion is more pronounced and does not allow straightforward retrieval of any section of the original signal. FIG 4 Performance on the nonlinear channel equalization task after propagating g The signal at the output of the simulated channel is a time continuous signal. Similar to the method used by Argyris et al. in Ref. 26, for each bit, we associate two features values b(1) n and b(2) n , which are the time-average values of the upper half and the lower half of the distorted signal for the duration of one bit. II. METHOD This allows the independent change of the injected power Pinjx,y of the tasks Tx,y. At the modulators output, the optical polarization of the input stream containing Tx is aligned with the main polarization mode (LPx) of the VCSEL and the one of the input stream contain- ing Ty with the depressed polarization mode (LPy). An example of The response of the reservoir is recorded at the output layer: The signal is first amplified with an erbium-doped fiber amplifier (EDFA) from Lumibird. Then, the two polarization modes of the VCSEL are separated and recorded with two photodiodes Newport 1544-B 12 GHz bandwidth, connected to an oscilloscope Tektronix DPO 71604C 16 GHz bandwidth with two channels at 50 GS/s. Examples of the experimental time series recorded for each polariza- tion mode of the VCSEL are given in Fig. 1(c). The signal-to-noise ratio (SNR) has been experimentally measured at 21 dB. With the high-resolution optical spectrum analyzer BOSA from Aragon Photonics, we can study the spectral dynamics of the system in different configurations. Figure 2(a) shows the experimental opti- cal spectrum of the reservoir computer without injection and with optical feedback. The VCSEL is lasing at 1552.72 nm, the wavelength of its dominant polarization mode. The dominant mode LPx of the VCSEL has a spectral width of 5.72 GHz with an attenuation of 17 dB in the feedback loop. The two smaller side peaks are induced by the undamped relaxation oscillations of the VCSEL,23 which frequency is measured at 3.73 GHz. Figure 2(b) presents the spectrum of the reservoir with injection but without modulating input: Under this condition, the VCSEL is emitting light only in its dominant polar- ization mode, with the wavelength of the master laser at 1552.82 nm. We notice that the slave laser exhibits wave-mixing dynamics and that it is not locked to the master laser. When modulating the mas- ter laser, its spectrum broadens and overlaps the two wavelengths of the VCSEL, as shown in Figs. 2(c) and 2(d). This allows the VCSEL APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 © Author(s) 2020 5, 086105-3 ARTICLE scitation.org/journal/app FIG. 3. Performance on the nonlinear channel equalization task as a function of the injection power in the main polarization mode Pinjx for a fixed ratio of injection power Pinjy Pinjx at 0.3. (a) Performance of the recovery of a distortion due to 25 km of optical fiber. II. METHOD For the training of the reservoir, we use 20 000 samples, i.e., sliding block of five consecutive distorted bits. Since we record optical power of LPx,y modes for the 492 nodes, the size of S is 20 000 × 984. The performance of the reservoir is tested on 5380 samples and measured using the bit error rate (BER). the value of the feedback strength fixed. This allows reducing the dimension of the space parameters to explore to find the best exper- imental operating point. By finding the best operating point, we ensure for our VCSEL-based reservoir computing system to have a combination of large memory capacity (i.e., long fading memory) and large computational ability (i.e., good aptitude for approxima- tion and generalization), as demonstrated in our previous numerical analysis.19 Furthermore, we aim at showing the tunable parameters that can control the performance of the two processed tasks Tx and Ty. Figures 4 and 5 present the influence of the ratio of injection power Pinjy Pinjx on the performance of the two processed tasks. To pro- duce these figures, we first find the best operating point for each value of this ratio: We sweep the value of Pinjx (an example is pro- vided in Fig. 3), and Pinjy is then fixed by the value of the ratio. As a result, we find the value of Pinjx that minimizes the mean BER for both Tx and Ty. This optimal value is then reported in the graph (this is why Figs. 4 and 5 do not contain any information on the effective injected power). Figure 3 shows an example of the method used to produce the performance figures.il As already stated, for each value of the feedback strength, there is a corresponding optimal injection power for the reservoir com- puter.19 That is why we vary only the injected power, while keeping FIG. 5. Performance on the nonlinear channel equalization task after propagating 50 km in a communication channel made of optical fiber as a function of the ratio of injection Pinjy/Pinjx. (a) Example of the signal sent at the input of the fiber. (b) Corresponding received signal after 50 km of fiber. The blue curves corresponds to the performance of the task Tx, and the red curves corresponds to the performance of the task Ty for (c) the reservoir computer and (d) the linear classifier. II. METHOD The input of the reservoir is realized by masking each feature value for five con- secutive bits, hence using 10 different masks (one per input value) of 985 values, which are then summed together. The masked input of the reservoir Jn−2(t) at the step n −2 reads Jn−2(t) = 4 ∑ i=0 b(1) n−i × M2i(t) + 4 ∑ i=0 b(2) n−i × M2i+1(t), (2) FIG. 4. Performance on the nonlinear channel equalization task after propagating 25 km in a communication channel made of optical fiber as a function of the ratio of injection Pinjy/Pinjx. (a) Example of the signal sent at the input of the fiber. (b) Corresponding received signal after 25 km of fiber. The blue curves corresponds to the performance of the task Tx, and the red curves corresponds to the performance of the task Ty for (c) the reservoir computer and (d) the linear classifier. The lighter area shows the standard deviation of the performance. The dotted line shows the performance of the reservoir performing the single task. (2) where Mi(t) is one of the ten different masks. A graphical illustration of the preprocessing is given in Fig. 1(d). At the output of the reser- voir, we train the system by linear regression with N = 492 nodes to recover bits bn−2. For each node, we use as a state the values of the optical power of the two orthogonal polarization modes (LPx and APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 © Author(s) 2020 5, 086105-4 5, 086105-4 ARTICLE APL Photonics scitation.org/journal/app LPy). Two different linear regressions are performed, one for each task Tx and Ty, using the whole state of the reservoir. The equa- tions of the regressions are S × ωx = bTx and S × ωy = bTy, where S is the reservoir’s state matrix containing the power associated with the dominant (LPx) and depressed (LPy) polarization mode. ωi is the vector containing the readout layer weights obtained from linear regression, and bTi is the vector containing the target output of the task Ti. Exploiting the two LP modes for each regression stems from nonlinear mixing the two input data streams in the VCSEL dynamics so that the two polarization modes contains part of the information of both processed tasks. III. RESULTS The results for the channel equalization of 25 km of propa- gation in the fiber are presented in Fig. 4(c). Figures 4(a) and 4(b) present an example of the signal at the input and output of the opti- cal fiber, respectively. We observe that the performance on tasks Tx and Ty varies with the injection ratio Pinjy/Pinjx. If this ratio is smaller than 2, task Tx is better performed than task Ty. When this ratio is higher than 2, the trend is reversed, and the task Ty is bet- ter performed. This can be explained by a polarization switching in the VCSEL output induced by optical injection (i.e., the role of the dominant and depressed polarization modes of the VCSEL are exchanged27). This phenomenon therefore increases the SNR of the task Ty injected in the depressed polarization mode. The system is able to provide a BER of 0.04% for the task Tx, while the dominant mode is strongly injected (with an injection ratio Pinjy/Pinjx of 0.2). The other task is processed with lower performance in this case, with a BER of 1.6%. When the ratio of power is greater than 0.5, the aver- age performance of the reservoir reaches a threshold of performance with a BER of 0.35%. The ratio of injected power in the polarization modes can thereby be used to easily choose the split of performance between the two performed tasks. While processing a single non- linear channel equalization task, the reservoir computer exhibits a BER of 0.08%. We notice that the performance of our VCSEL-based reservoir on a single task is comparable to the one achieved with a monomode laser diode with a more complex modulation format and similar propagation distance.26 However, processing two tasks instead of one mitigates the averaged performance of the system. FIG. 5. Performance on the nonlinear channel equalization task after propagating 50 km in a communication channel made of optical fiber as a function of the ratio of injection Pinjy/Pinjx. (a) Example of the signal sent at the input of the fiber. (b) Corresponding received signal after 50 km of fiber. The blue curves corresponds to the performance of the task Tx, and the red curves corresponds to the performance of the task Ty for (c) the reservoir computer and (d) the linear classifier. The lighter area shows the standard deviation of the performance. II. METHOD The lighter area shows the standard deviation of the performance. The dotted line shows the performance of the reservoir performing the single task. i We first present the influence of the injected power on the per- formance of both tasks Tx and Ty in Fig. 3 for the two lengths of fiber recovered: 25 km (a) and 50 km (b). On this figure, the injec- tion ratio Pinjy/Pinjx is fixed to 0.3. We can observe that there is an optimal injected power that yields the best mean performance at Pinjx = 0.09 mW for 25 km and at Pinjx = 0.2 mW for 50 km. We will only report this best value in the figures. IV. CONCLUSION Our result also shows that there is still a significant margin of improvement before considering it a viable alternative to the best DSP approaches, despite achieving level of performance compara- ble to existing photonic-based machine learning techniques on this particular task.30 Nevertheless, this result is a first step showing that analog photonic reservoir computing could be envisioned for such dual-tasking on optical channel equalization. The performance of the stand-alone linear regression (linear classifier) is presented in Fig. 5(d). The test has been realized with the same condition as the one used for the reservoir computer. The lin- ear classifier is achieving a BER of 7.5% as a best performance. When both processed signals are balanced, the linear classifier exhibits its best mean performance, with a mean BER at 8.4%. Using the non- linear effects in our VCSEL-based photonic reservoir computer in similar SNR conditions thus provides a significant benefit, allowing to improve by a factor 5 the performance on the signal-recovery task. We proved in our previous work that the bimodal dynamics of the VCSEL allows better computational performance than a single mode dynamics system. This is due to a more complex dynamics that is suitable to perform computation. Here, we proved experimentally that we can exploit the bimodal dynamics of the VCSEL to process two tasks simultaneously. This suggests that using a system exhibit- ing more dynamical modes would allow scaling up the number of tasks to be processed simultaneously. However, performing several tasks simultaneously slightly degrades the mean computational per- formance of the system. There is thus a trade-off between the num- ber of tasks to be processed and the individual performance of each task considered. Moreover, we hypothesize that the physics underly- ing the coupling mechanism between modes may also influence the performance of the reservoir computer, for instance, using longitu- dinal mode of a laser17 or the two modes of a semiconductor ring laser15 instead of using the polarization modes of the VCSEL. This may constitute an interesting frame for future studies of multimode reservoir computing. The relatively low range of power used for the input signal propagating in the fiber is consistent with the range of power use in telecommunication networks. Furthermore, it does not lead to significant trigger of the Kerr nonlinearity. IV. CONCLUSION We have realized an experimental photonic reservoir computer architecture capable of processing two tasks simultaneously. This reservoir is a time-delay reservoir computer, using a VCSEL as a physical node. The two different inputs are made by injecting two different optical signals, each being aligned with a different polar- ization mode of the VCSEL. Using this system, we have performed as an illustration two signal-recovery tasks simultaneously when the signal generated at 25 Gb/s is distorted by propagation in a 25 km or 50 km long SMF-28 optical fiber. We have been able to recover two signals with a BER of 0.3% at a processing speed of 51.3 Mb/s in total for a 25 km-distortion and with a BER of 3% at the same bit rate for a 50 km-distortion. On both tasks, the reservoir allows improving the performance by a factor 5–10, compared to processing the input signal directly under similar SNR conditions. The actual telecom- munication networks use digital signal processing (DSP) to mitigate the effects of the optical fiber29 as it allows propagating the signals along several thousands of kilometers with a BER of ∼10−3 compat- ible with forward error correction, but at the expense of important computational resources.i We also provide results on the dual channel equalization of the propagation in 50 km of single mode fiber. Since the distortion of the signal is more pronounced [Fig. 5(b)], the mean performance of the reservoir computer is expected to be lower than the one after a 25 km transmission. The performance of the reservoir computer is given in Fig. 5(c). We still observe a similar trend: The polarization switching of the VCSEL for a ratio of injection Pinjy/Pinjx ∼1, and the best achieved BER for one task is at 1.6%. The best mean performance is at 2.2%, achieved for a ratio of injection at 0.7. The system perform- ing this single task exhibits a BER of 1.9%, which is slightly below the performance previously reported.16 Contrary to the equalization of the shorter optical fiber, processing two tasks simultaneously slightly decreases the mean performance of the system, when compared to processing a single task. IV. CONCLUSION Equalizing both linear distortion and a strong Kerr effect remains a challenge in current digital signal processing (DSP)-based techniques for optical channel equalization.28 To analyze how the Kerr effect would affect the per- formance of the reservoir, we have sent in the fiber two signals with a large pulse-amplitude modulation depth of 0.5 W and recover two signals simultaneously at the output of the fiber. This power is large enough to trigger the Kerr nonlinearity (as only a few tens of mW are necessary) and make the task more complex to solve. Under these new conditions and using similar parametric and operating condi- tions, our reservoir can now recover two signals simultaneously with an optimal mean BER of 8.9% for a 25 km fiber distortion and with a mean BER of 17.9% for a 50 km distortion. A degradation of at least one order of magnitude is observed in these conditions with the level of recovery unsuitable for telecom application. However, the level of III. RESULTS The dotted line shows the performance of the reservoir performing the single task. To analyze the impact of the nonlinear transformation induced by our VCSEL-based reservoir on the task, we compare it to a APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 © Author(s) 2020 APL Photon. 5, 086105 (2020); doi: 10.1063/5.0017574 5, 086105-5 ARTICLE APL Photonics scitation.org/journal/app stand-alone linear regression (a linear classifier). 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https://openalex.org/W3000304088	https://europepmc.org/articles/pmc7013673?pdf=render	English	null	Evaluation of the Technical Performance of Football Players in the UEFA Champions League	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	10,784	Received: 18 December 2019; Accepted: 11 January 2020; Published: 17 January 2020 Abstract: This study aimed to assess the technical match performance of top-class football players in a long-term perspective. Technical performance proﬁles of players according to ﬁve playing positions (central defender, full back, wide midﬁelder, central midﬁelder, forward) and ﬁve situational variables (competition stage, match location, quality of team, quality of opponent, match outcome) were established. Technical match data of players in the UEFA Champions League from season 2009–2010 to 2016–2017 were analyzed. The true eﬀects of positional and situational variables on players’ technical performance were evaluated by the non-clinical magnitude-based inference. Results showed that the eﬀect of competition stage on player’s performance was negligible. Quality of team, quality of opponent and match outcome revealed the strongest eﬀects on player’s performance (ES: −0.42 ± 0.10–0.59 ± 0.10) while the eﬀect of match location was relatively lower (ES: −0.32 ± 0.10–0.23 ± 0.07). The number of variables that showed statistical diﬀerences under ﬁve competing contexts for wide midﬁelders and forwards were higher than those of central defenders, full backs, and central midﬁelders. Diﬀerences of players’ match performance could mainly be identiﬁed in variables related to goal scoring, passing, and organizing, these ﬁndings may provide important insights for coaches and analysts during the match preparation and training session. Keywords: technical performance proﬁle; situational variable; playing position; football; soccer; match analysis International Journal of Environmental Research and Public Health ceived: 18 December 2019; Accepted: 11 January 2020; Published: 17 January 2020 Received: 18 December 2019; Accepted: 11 January 2020; Published: 17 January 2020 Evaluation of the Technical Performance of Football Players in the UEFA Champions League Qing Yi 1,2,3,4,* , Miguel-Ángel Gómez-Ruano 3 , Hongyou Liu 5 , Shaoliang Zhang 6 Binghong Gao 1,2, Fabian Wunderlich 4 and Daniel Memmert 4 1 School of Physical Education and Sport Training, Shanghai University of Sport, Shanghai 200438, China; gaobinghong@126.com 2 Shanghai Key Lab of Human Performance, Shanghai University of Sport, Shanghai 200438, China 3 2 Shanghai Key Lab of Human Performance, Shanghai University of Sport, Shanghai 200438, China 3 Facultad de Ciencias de la Actividad Física y del Deporte (INEF), Universidad Politécnica de Madrid, 28040 Madrid, Spain; miguelangel.gomez.ruano@upm.es g y , g y p , g , 3 Facultad de Ciencias de la Actividad Física y del Deporte (INEF), Universidad Politécnica de Madrid, 28040 Madrid, Spain; miguelangel.gomez.ruano@upm.es 4 Institute of Training and Computer Science in Sport, German Sport University Cologne, 50933 Cologne, Germany; f.wunderlich@dshs-koeln.de (F.W.); d.memmert@dshs-koeln.de (D.M.) g p p , p y g , g Germany; f.wunderlich@dshs-koeln.de (F.W.); d.memmert@dshs-koeln.de (D.M.) y 5 School of Physical Education & Sports Science, South China Normal University, Guangzhou 510631, China; szu.youyou@hotmail.com 5 School of Physical Education & Sports Science, South China Normal University, Guangzhou 510631, China; szu.youyou@hotmail.com y y 6 Division of Sport Science & Physical Education, Tsinghua University, Beijing 100084, China; zsl.inef@gmail.com * Correspondence: yiqing1771@outlook.com 1. Introduction The complexity of football match performance can be reduced by using performance analysis techniques, presenting the results in systematic ways, and systematically integrating them into the coaching process [1,2]. This can be considered as valuable feedback for coaches, players, and sport researchers [3,4]. Whether performance analysis can be good feedback or an educational tool depends on the type and quality of the methods used [5]. An accurate and reliable performance proﬁle may improve the Int. J. Environ. Res. Public Health 2020, 17, 604; doi:10.3390/ijerph17020604 www.mdpi.com/journal/ijerph 2 of 12 Int. J. Environ. Res. Public Health 2020, 17, 604 eﬃciency of the analysis procedure [6,7], and can provide useful feedback that can be easily understood by sports practitioners [8]. Performance proﬁling is a descriptive analysis that brings a collection of valid and reliable psychological, physical, and technical indicators together to characterize the overall performance of players and teams [9,10]. However, the properties of match performance indicators may vary along the matches played as situational variables have an inﬂuence on them [11,12]. Therefore, the data from a single match cannot represent a player’s or a team’s typical performance [9,13]. The individual match eﬀects may generate additional variance in the data of a single match and fail to produce a signiﬁcant diﬀerence in the comparison between groups [14]. Accordingly, Hughes et al. [6] stated that the nature of the data and the performers are the two main factors that should be taken into account when considering the number of matches required. The typical performance of a player/team may only be represented by the data from a large number of matches using the technique of performance proﬁling. Due to the availability of data, the proﬁling subject has developed over time from the analysis of single players or teams to more comprehensive analysis including a larger number of players or teams [15]. Previously, median and 95% conﬁdence intervals [9,16] were used to present the typical performance of subjects and its spread of performance, while currently proﬁling techniques are commonly based on mean and 95% conﬁdence intervals [17,18]. Due to the nature of complexity and highly dynamic behaviour in football matches [18–21], the match performance of players/teams is not only inﬂuenced by technical, tactical, mental [22,23], and physiological factors [24], but also by diﬀerent situational factors [25,26]. 1. Introduction Match performance of players/teams cannot be generalized in all contexts [15] and introducing the situational variables into performance proﬁle can make it more comprehensive and systematic [17]. Match location, team quality, quality of opposition, and match outcome are examples of situational variables that have been investigated so far [17,18,27]. The quality of the opponent is considered the main factor for variation in match performance [28]. However, given the high dynamic nature of the match play highlighted previously, there is also an obvious opening for further research to take the interactions between playing positions and competing situations into deeper consideration. In the current study, suﬃcient match observations make it possible to take player positions into account to establish performance proﬁles according to diﬀerent playing positions under diﬀerent competing situations [17,27,29]. As players from diﬀerent playing positions have diﬀerent tasks in a football game, the general evaluation of all players’ match performance will result in a loss of a lot of valuable information. Therefore, a more useful way is a detailed analysis of the performance of players with proper regard to the particularity of diﬀerent playing positions and the eﬀect of situational variables. In contrast to prior studies using data from domestic leagues [17,30,31], the evaluation of data from the UEFA Champions League makes it possible to evaluate diﬀerences of performance in diﬀerent competition stages (group vs. knockout stage). Moreover, the database allows for comparisons between performances in international matches compared to domestic matches investigated in earlier studies. In order to add these aspects to the current research on performance analysis in football, the current study aimed to establish technical performance proﬁles of players in the UEFA Champions League from season 2009–2010 to 2016–2017 by comparing the between-player diﬀerences in match performance in a long-term perspective (8 full seasons) according to players’ speciﬁc ﬁeld positions and incorporating ﬁve situational variables: competition stage (group stage/knockout stage), match location, quality of team, quality of opposition and match outcome. 2.1. Data and Reliability Technical performance-related match data of players in the UEFA Champions League from season 2009–2010 to 2016–2017 (1000 matches, 768 matches from group stage, 232 matches from knockout stage) were collected for analysis. Match data were obtained from a public-accessed football statistics website “whoscored.com” (https://www.whoscored.com). Data resource of the website is the sports 3 of 12 Int. J. Environ. Res. Public Health 2020, 17, 604 analytics company OPTA Sports (London, UK). The reliability of OPTA in coding players’ match actions and events has already been successfully tested as highly reliable [32]. Because of the speciﬁcity of position for goalkeepers and a very limited applicability of technical variables to the performance of goalkeepers, match data for this position was excluded from the sample. Moreover, only the players being part of the starting line-up were included, which ﬁnally limited the database to 5136 players (3693 players from group stage, 1443 players from knockout stage) and generated 14,437 full match observations (11,095 from group stage, 3342 from knockout stage). All players in the sample were divided into 5 groups according to their playing positions [17,33]: central defender (group stage: N1 = 1012 players, n1 = 2811 full match observations, knockout stage: N2 = 380 players, n2 = 821 observations), full back (N1 = 939, n1 = 2657, N2 = 337, n2 = 772), wide midﬁelder (N1 = 711, n1 = 1214, N2 = 216, n2 = 351), central midﬁelder (N1 = 1326, n1 = 3081, N2 = 511, n2 = 963), and forward (N1 = 734, n1 = 1332, N2 = 237, n2 = 435). The current study was conducted in accordance with the Declaration of Helsinki and approved oﬃcially by the ethics committee of the Shanghai University of Sport (11DZ2261100). 2.2. Technical Variables and Situational Variables Twenty-ﬁve technical performance-related match actions or events were chosen as variables in the present study and were divided into four groups (Table 1) based on previous studies [18,34–36]. Deﬁnitions of these variables can be found in the previous studies [11,20,36]. The technical match data was analyzed under the following ﬁve situational variables: (1) competition stage: group stage and knockout stage; (2) match location: home and away; (3) quality of team: teams that qualiﬁed into the knockout stage and teams that didn’t qualify into the knockout stage; (4) quality of opponent: opponents that qualiﬁed into the knockout stage and opponents that didn’t qualify into the knockout stage; and (5) match outcome: win, draw and lose. Due to the limitation of the sample size from knockout stage, except for the situational variable of competition stage, only the match data of players in the group stage were included in the analysis of the other four situational variables. Table 1. The classiﬁcation of technical variables. Categories Variables Goal scoring Shot, Shot on target. Attacking Dispossessed, Unsuccessful touch, Fouled, Aerial won, Dribble, Oﬀside. Defending Yellow card, Total tackle, Interception, Clearance, Blocked shot, Foul. Passing and organizing Assist, Touch, Key pass, Pass accuracy (%), Pass, Cross, Accurate cross, Long ball, Accurate long ball, Through ball, Accurate through ball. 2.3. Statistical Analysis Table 1. The classiﬁcation of technical variables. Categories Variables Goal scoring Shot, Shot on target. Attacking Dispossessed, Unsuccessful touch, Fouled, Aerial won, Dribble, Oﬀside. Defending Yellow card, Total tackle, Interception, Clearance, Blocked shot, Foul. Passing and organizing Assist, Touch, Key pass, Pass accuracy (%), Pass, Cross, Accurate cross, Long ball, Accurate long ball, Through ball, Accurate through ball. 2 3 St ti ti l A l i Table 1. The classiﬁcation of technical variables. Table 1. The classiﬁcation of technical variables. 3. Results Comparisons in technical variables between players from ﬁve positions according to ﬁve situational variables are presented in Figure 1 and Table 2. Figure 1 shows a graphical representation of the performance proﬁles including the results of magnitude-based inferences. Table 2 gives a summary of all technical variables that revealed non-trivial diﬀerences. The full set of descriptive statistics of match performance proﬁles can be found in the Supplementary Materials ﬁle. There is a new ﬁnding concerning the inﬂuence of competition stage on player performance that did not appear in the previous research due to the investigation of domestic matches [17,30] or the lack of knockout matches in international tournaments [43,44]. In all variables from ﬁve positions between players from group stage and players from knockout stage no clear diﬀerences were identiﬁed, except for fouling. The comparisons regarding the other four situational variables in the group stage revealed more substantial diﬀerences in performance. The number of variables showing non-trivial diﬀerences between home games and away games were limited across all playing positions. Especially in the case of central midﬁelders, only shots showed a non-trivial diﬀerence. Central defenders and full backs made more clearances when playing away than when playing at home in the group stage. Their performance in variables related to passing and organizing (passes and crosses) in home games was better than in away games. The diﬀerences of match performance of wide midﬁelders and forwards between home and away games were mainly focused on variables related to goal scoring and variables related to passing and organizing, while the central midﬁelders only showed clear diﬀerence in shots. A similar trend was found by comparing player’s match performance when considering the eﬀect of quality of team, quality of opponent and match outcome. Across all positions from qualiﬁed teams, players playing against non-qualiﬁed teams and players of a winning team showed better performances in variables related to passing and organizing than their counterparts (players from non-qualiﬁed teams, players playing against qualiﬁed teams, and players playing in games lost/draw). Wide midﬁelders and forwards showed clear diﬀerences in variables related to goal scoring (shots and shots on target) in these three competing contexts. In addition, forwards from qualiﬁed teams and from winning teams made more dribbles when compared with their counterparts from non-qualiﬁed teams and playing in games lost/draw. Central defenders obtained more aerials won when playing with non-qualiﬁed teams than when playing with qualiﬁed teams. 2.3. Statistical Analysis According to the central limit theorem, the sampling distribution of the match performance statistics based on the large database will be normal, and the variance will be homogeneous as well. Thus, the test for data normality distribution and homogeneity of variance was not performed in the process of statistical analysis [37]. After the screening of missing values and outliers, count values of 25 technical performance-related actions or events of players were transformed into standardized scores (Z-Score) and were uniﬁed into the same scale by the formula T = 10Z + 50 [27,38]. Match performance of players from ﬁve positions were compared by means of adjusted values respectively accounting to ﬁve situational variables and were plotted into radar charts. The standardized scores (Z-Score) were then transformed and uniﬁed using the statistical software IBM SPSS Statistics 22 for Windows (IBM Corp., Armonk, NY, USA) and plotted into radar charts using the Microsoft Excel 2007 program (Microsoft, Redmond, WA, USA). The non-clinical magnitude-based inference (MBI) was used to identify the diﬀerences of match performance of players. Diﬀerences were evaluated by using the standardized smallest worthwhile change which was calculated by 0.2 times the between-subject standard deviation [39]. Comparisons between groups were conducted using the spreadsheet developed 4 of 12 Int. J. Environ. Res. Public Health 2020, 17, 604 by Hopkins and 90% conﬁdence intervals were used to make the inferences [40,41]. Magnitude of clear diﬀerences was considered as follows: trivial, 0–0.2; small, 0.2–0.6; moderate, 0.6–1.2; large, 1.2–2.0; and very large, >2.0 [39,42]. The possibility of the eﬀect to be clear was deﬁned as follows: 25–75%, possibly; 75–95%, likely; 95–99.5%, very likely; and 99.5–100%, most likely [42]. 3. Results Another important result that can be found in Table 2 is that touches and passes are the only two variables that showed clear diﬀerences for players of all positions when taking quality of team, quality of opponent, and match outcome into account. Touches and passes of wide midﬁelders showed bigger diﬀerences in these three competing situations compared with those from the other four playing positions. Moreover, match performance of players from all ﬁve playing positions in touches and passes under the situational variable of team quality showed greater diﬀerences than those under the other two situational variables. In the present study, no clear diﬀerences were detected for the match variables yellow cards, dispossessed, unsuccessful touches, total tackles, interceptions, blocked shots, fouled, and oﬀsides, neither across playing positions, nor under all ﬁve situational variables. 5 of 12 Int. J. Environ. Res. Public Health 2020, 17, 604 Table 2. Statistical diﬀerences of players’ match performance across ﬁve playing positions and ﬁve competing situations. Position Group-Knockout Home-Away Non-Qualiﬁed-Qualiﬁed Non-Qualiﬁed Opp.-Qualiﬁed Opp. 3. Results Draw/Lose-Win Variable Eﬀect Size Inference Variable Eﬀect Size Inference Variable Eﬀect Size Inference Variable Eﬀect Size Inference Variable Eﬀect Size Inference CD Clearance 0.22 ± 0.06 S * Touch 0.47 ± 0.06 S ** Touch −0.39 ± 0.06 S Touch 0.31 ± 0.07 S ** Pass −0.22 ± 0.06 S * Pass 0.50 ± 0.06 S ** Pass −0.39 ± 0.06 S Pass 0.33 ± 0.07 S PA 0.29 ± 0.06 S * AW −0.26 ± 0.06 S ** AccLB 0.21 ± 0.06 S * FB Clearance 0.23 ± 0.07 S * Touch 0.44 ± 0.07 S ** Touch −0.35 ± 0.07 S Assist 0.39 ± 0.07 S ** Cross −0.22 ± 0.07 S * PA 0.32 ± 0.07 S ** Pass −0.38 ± 0.07 S Touch 0.32 ± 0.07 S Pass 0.50 ± 0.07 S ** AccLB −0.22 ± 0.07 S * Pass 0.39 ± 0.07 S ** PA 0.27 ± 0.07 S * WM Foul 0.21± 0.11 S * Shot −0.26 ± 0.10 S Touch 0.59 ± 0.10 S Touch −0.42 ± 0.10 S Assist 0.47 ± 0.12 S KP −0.28 ± 0.10 S Pass 0.56 ± 0.10 S ** Pass −0.40 ± 0.10 S Pass 0.51 ± 0.11 S ** Cross −0.21 ± 0.10 S * ThB 0.35 ± 0.10 S * Shot −0.26 ± 0.10 S ShotOT 0.38 ± 0.11 S **** AccCross −0.21 ± 0.10 S * PA 0.30 ± 0.10 S * KP −0.24 ± 0.10 S Touch 0.55 ± 0.11 S ** AccThB 0.26 ± 0.10 S ShotOT −0.24 ± 0.10 S * Shot 0.31 ± 0.11 S * Shot 0.24 ± 0.10 S PA −0.21 ± 0.10 S * PA 0.31 ± 0.11 S * ShotOT 0.29 ± 0.10 S KP 0.29 ± 0.11 S ** Assist 0.21 ± 0.10 S * ThB 0.27 ± 0.11 S ** KP 0.21 ± 0.10 S * AccThB 0.26 ± 0.11 S ** AccLB 0.20 ± 0.10 S * AccLB 0.23 ± 0.11 S * CM Shot −0.20 ± 0.06 S * Touch 0.49 ± 0.06 S ** Touch −0.39 ± 0.06 S Assist 0.33 ± 0.07 S PA 0.35 ± 0.06 S Pass −0.37 ± 0.06 S Touch 0.39 ± 0.06 S Pass 0.49 ± 0.06 S LB −0.23 ± 0.06 S * Pass 0.38 ± 0.06 S ** ThB 0.24 ± 0.06 S PA 0.21 ± 0.06 S * Assist 0.21 ± 0.06 S * FW Shot −0.32 ± 0.10 S * Touch 0.47 ± 0.10 S Shot −0.36 ± 0.10 S Assist 0.46 ± 0.11 S KP −0.28 ± 0.10 S Pass 0.43 ± 0.10 S ** ShotOT −0.32 ± 0.10 S * Shot 0.52 ± 0.10 S **** ShotOT −0.22 ± 0.10 S * Shot 0.32 ± 0.10 S * KP −0.35 ± 0.10 S * ShotOT 0.57 ± 0.10 S **** AccCross −0.23 ± 0.10 S * ShotOT 0.32 ± 0.10 S * Assist −0.28 ± 0.10 S Touch 0.46 ± 0.10 S ** Assist 0.28 ± 0.10 S Touch −0.28 ± 0.10 S Pass 0.42 ± 0.10 S KP 0.30 ± 0.10 S Pass −0.26 ± 0.10 S ThB 0.37 ± 0.11 S PA 0.28 ± 0.10 S ThB −0.28 ± 0.10 S KP 0.34 ± 0.10 S * Dribble 0.21 ± 0.10 S * AccThB −0.27 ± 0.10 S AccThB 0.33 ± 0.11 S * Dribble 0.28 ± 0.10 S ** Note: Eﬀect sizes are presented as the magnitude of the true diﬀerence in means ± 90% conﬁdence interval, only the variables that showed clear diﬀerences were included. Note: Eﬀect sizes are presented as the magnitude of the true diﬀerence in means ± 90% conﬁdence interval, only the variables that showed clear diﬀerences were included. Positive eﬀect size indicates that the mean values of variables from group A bigger than the mean values of variables from group B, negative eﬀect size indicates that the mean values of variables from group B bigger than the mean values of variables from group A, e.g., group B-group A: group stage-knockout stage. Letters in parentheses denote the magnitude: t = trivial; s = small. Asterisks indicate the likelihood for the magnitude of the true diﬀerence in means as follows: * possible; likely; * very likely; **** most likely. Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through; AW = aerial won; CD = central defender; CM = central midﬁelder; FB = full back; FW = forward; KP = key pass; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; ball WM = wide midﬁelder. 3. Results Positive eﬀect size indicates that the mean values of variables from group A bigger than the mean values of variables from group B, negative eﬀect size indicates that the mean values of variables from group B bigger than the mean values of variables from group A, e.g., group B-group A: group stage-knockout stage. Letters in parentheses denote the magnitude: t = trivial; s = small. Asterisks indicate the likelihood for the magnitude of the true diﬀerence in means as follows: * possible; likely; * very likely; **** most likely. Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through; AW = aerial won; CD = central defender; CM = central midﬁelder; FB = full back; FW = forward; KP = key pass; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; ball WM = wide midﬁelder. Note: Eﬀect sizes are presented as the magnitude of the true diﬀerence in means ± 90% conﬁdence interval, only the variables that showed clear diﬀerences were included. Positive eﬀect size indicates that the mean values of variables from group A bigger than the mean values of variables from group B, negative eﬀect size indicates that the mean values of variables from group B bigger than the mean values of variables from group A, e.g., group B-group A: group stage-knockout stage. Letters in parentheses denote the magnitude: t = trivial; s = small. Asterisks indicate the likelihood for the magnitude of the true diﬀerence in means as follows: * possible; likely; * very likely; ** most likely. Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through; AW = aerial won; CD = central defender; CM = central midﬁelder; FB = full back; FW = forward; KP = key pass; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; ball WM = wide midﬁelder. Note: Eﬀect sizes are presented as the magnitude of the true diﬀerence in means ± 90% conﬁdence interval, only the variables that showed clear diﬀerences were included. 3. Results 40 45 50 55 Shot(T) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T*) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Full back 40 45 50 55 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T*) Clearance(S) BS(T*) Foul(T) Assist(T) Touch(T) KP(T) PA(T*) Pass(T) Cross(S) AccCross(T) LB(T**) AccLB(T) ThB(T**) AccThB(T) 40 45 50 55 Shot(T*) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) Full back 40 45 50 55 Shot(T) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T*) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Full back 40 45 50 55 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T*) Clearance(S) BS(T*) Foul(T) Assist(T) Touch(T) KP(T) PA(T*) Pass(T) Cross(S) AccCross(T) LB(T**) AccLB(T) ThB(T**) AccThB(T) 40 45 50 55 Shot(T*) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) Full back 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T**) Dribble(S) Offside(T**) YC(T) TT(T) Interception(T) Clearance(t) BS(T) Foul(T) Assist(T) Touch(T) KP(T*) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Central defender 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T*) Clearance(S) BS(T) Foul(T*) Assist(T*) Touch(T) KP(T*) PA(T) Pass(S) Cross(T*) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) 35 40 45 50 55 60 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T*) Foul(T) Assist(T) Touch(S*) KP(T) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(T*) AccThB(T) Central defender 35 40 45 50 55 60 65 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T*) TT(T) Interception(T**) Clearance(T) BS(T**) Foul(T) Assist(T) Touche(T) KP(T*) PA(T) Pass(T) Cross(T*) AccCross(T) LB(T*) AccLB(T) ThB(T) AccThB(T) Forward 40 45 50 55 60 Shot(T*) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T**) Dribble(S) Offside(T**) YC(T) TT(T) Interception(T) Clearance(t) BS(T) Foul(T) Assist(T) Touch(T) KP(T*) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Central defender 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T*) Clearance(S) BS(T) Foul(T*) Assist(T*) Touch(T) KP(T*) PA(T) Pass(S) Cross(T*) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) 35 40 45 50 55 60 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T*) Foul(T) Assist(T) Touch(S*) KP(T) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(T*) AccThB(T) Central defender 45 47 49 51 53 55 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearances(T) BS(T) Foul(T) Assist(T) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T*) Central midfielder 40 45 50 55 Shot(S) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(T) Touch(T) KP(T) PA(T**) Pass(T) Cross(T) AccCross(T**) LB(T) AccLB(T) ThB(T*) AccThB(T) 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T*) Foul(T*) Assist(S) Touch(S***) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T*) AccLB(T) ThB(S) AccThB(T) Central midfielder 40 45 50 55 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T*) Clearance(S) BS(T*) Foul(T) Assist(T) Touch(T) KP(T) PA(T*) Pass(T) Cross(S) AccCross(T) LB(T**) AccLB(T) ThB(T**) AccThB(T) 40 45 50 55 Shot(T*) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) Full back Figure 1. 3. Results Cont. 40 44 48 52 56 60 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T**) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(S) Assist(T**) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T) Group stage Knockout stage Wide midfielder 30 35 40 45 50 55 60 Shot(S) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T*) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T**) Assist(T*) Touch(T) KP(S) PA(T) Pass(T) Cross(S) AccCross(S) LB(T) AccLB(T) ThB(T) AccThB(T) Home Away 35 40 45 50 55 60 Shot(S) ShotOT(S) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T*) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S***) KP(S) PA(S*) Pass(S) Cross(T) AccCross(T*) LB(T) AccLB(S) ThB(S) AccThB(S) Non-qualified Qualified Wide midfielder Figure 1. Cont. 3. Results 40 45 50 55 60 Shot(T**) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T**) Dribble(S) Offside(T**) YC(T) TT(T) Interception(T) Clearance(t) BS(T) Foul(T) Assist(T) Touch(T) KP(T*) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Central defender 40 45 50 55 Shot(T*) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T*) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Full back 40 44 48 52 56 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T**) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(S) Assist(T**) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T) Group stage Knockout stage Wide midfielder To KP(T* PA(T**) Pass(T) Cross(T) AccCross(T*) LB(T*) AccLB( 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T*) Clearance(S) BS(T) Foul(T*) Assist(T*) Touch(T) KP(T*) PA(T) Pass(S) Cross(T*) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) 40 45 50 55 Shot(T) ShotOT(T**) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T*) Clearance(S) BS(T*) Foul(T) Assist(T) Touch(T) KP(T) PA(T*) Pass(T) Cross(S) AccCross(T) LB(T**) AccLB(T) ThB(T**) AccThB(T) 30 35 40 45 50 55 60 Shot(S) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T*) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T**) Assist(T*) Touch(T) KP(S) PA(T) Pass(T) Cross(S) AccCross(S) LB(T) AccLB(T) ThB(T) AccThB(T) Home Away Tou KP(T* PA(T**) Pass(T) Cross(T) AccCross(T**) LB(T) AccLB 35 40 45 50 55 60 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T*) Foul(T) Assist(T) Touch(S*) KP(T) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(T*) AccThB(T) Central defender 40 45 50 55 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) Full back 35 40 45 50 55 60 Shot(S) ShotOT(S) Disp(T) UnsTouch(T*) Fouled(T) AW(T) Dribble(T) Offside(T*) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S***) KP(S) PA(S*) Pass(S) Cross(T) AccCross(T*) LB(T) AccLB(S) ThB(S) AccThB(S) Non-qualified Qualified Wide midfielder Touch(S KP(T) PA(S*) Pass(S) Cross(T) AccCross(T) LB(T*) AccLB(T Figure 1. Cont. 3. Results 40 45 50 55 60 Shot(T**) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T**) Dribble(S) Offside(T**) YC(T) TT(T) Interception(T) Clearance(t) BS(T) Foul(T) Assist(T) Touch(T) KP(T*) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Central defender 40 45 50 55 Shot(T*) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T*) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Full back 40 44 48 52 56 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T**) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(S) Assist(T**) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T) Group stage Knockout stage Wide midfielder 45 47 49 51 53 55 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearances(T) BS(T) Foul(T) Assist(T) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T) Central midfielder 35 40 45 50 55 60 65 Shot(T) ShotOT(T Clea BS(T*) Foul(T) Assist(T) Touche(T) KP(T*) PA(T) Pass(T) Cross(T*) AccCross(T) LB(T*) AccLB(T) ThB(T) AccThB(T) Forward 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T*) Clearance(S) BS(T) Foul(T*) Assist(T*) Touch(T) KP(T*) PA(T) Pass(S) Cross(T*) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) 40 45 50 55 Shot(T) ShotOT(T**) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T*) Clearance(S) BS(T*) Foul(T) Assist(T) Touch(T) KP(T) PA(T*) Pass(T) Cross(S) AccCross(T) LB(T**) AccLB(T) ThB(T**) AccThB(T) 30 35 40 45 50 55 60 Shot(S) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T*) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T**) Assist(T*) Touch(T) KP(S) PA(T) Pass(T) Cross(S) AccCross(S) LB(T) AccLB(T) ThB(T) AccThB(T) Home Away 40 45 50 55 Shot(S) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(T) Touch(T) KP(T) PA(T**) Pass(T) Cross(T) AccCross(T**) LB(T) AccLB(T) ThB(T*) AccThB(T) 30 35 40 45 50 55 60 65 Shot(S) ShotOT C BS(T) Foul(T) Assist(T) Touch(T**) KP(S) PA(T*) Pass(T) Cross(T) AccCross(S) LB(T) AccLB(T) ThB(T) AccThB(T) 35 40 45 50 55 60 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T*) Foul(T) Assist(T) Touch(S*) KP(T) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(T*) AccThB(T) Central defender 40 45 50 55 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T**) AccThB(T) Full back 35 40 45 50 55 60 Shot(S) ShotOT(S) Disp(T) UnsTouch(T*) Fouled(T) AW(T) Dribble(T) Offside(T*) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S***) KP(S) PA(S*) Pass(S) Cross(T) AccCross(T*) LB(T) AccLB(S) ThB(S) AccThB(S) Non-qualified Qualified Wide midfielder 35 40 45 50 55 60 Shot(T**) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T*) Foul(T*) Assist(S) Touch(S***) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T*) AccLB(T) ThB(S) AccThB(T) Central midfielder 25 30 35 40 45 50 55 60 65 70 Shot(S*) ShotOT(S Clea BS(T) Foul(T) Assist(S) Touch(S*) KP(S) PA(S) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(S*) AccThB(S) Forward Figure 1. 3. Results Public Health 2020, 17, 604 I t J E i R P bli H lth 2020 17 FOR 6 of 12 6 f 12 *) d(T) AW(T) Dribble(T) Offside(T) (T*) ) 40 44 48 52 56 60 Shot(T*) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T**) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(S) Assist(T**) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T) Group stage Knockout stage Wide midfielder 45 47 49 51 53 55 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearances(T) BS(T) Foul(T) Assist(T) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T) Central midfielder 35 40 45 50 55 60 65 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T*) TT(T) Interception(T**) Clearance(T) BS(T**) Foul(T) Assist(T) Touche(T) KP(T*) PA(T) Pass(T) Cross(T*) AccCross(T) LB(T*) AccLB(T) ThB(T) AccThB(T) Forward ) (T*) W(T) Dribble(T) Offside(T) T) 30 35 40 45 50 55 60 Shot(S) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T*) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T**) Assist(T*) Touch(T) KP(S) PA(T) Pass(T) Cross(S) AccCross(S) LB(T) AccLB(T) ThB(T) AccThB(T) Home Away 40 45 50 55 Shot(S) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(T) Touch(T) KP(T) PA(T**) Pass(T) Cross(T) AccCross(T**) LB(T) AccLB(T) ThB(T*) AccThB(T) 30 35 40 45 50 55 60 65 Shot(S) ShotOT(S) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T**) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(T**) KP(S) PA(T*) Pass(T) Cross(T) AccCross(S) LB(T) AccLB(T) ThB(T) AccThB(T) ) d(T) W(T) Dribble(T) Offside(T) T) ) 35 40 45 50 55 60 Shot(S) ShotOT(S) Disp(T) UnsTouch(T*) Fouled(T) AW(T) Dribble(T) Offside(T*) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S***) KP(S) PA(S*) Pass(S) Cross(T) AccCross(T*) LB(T) AccLB(S) ThB(S) AccThB(S) Non-qualified Qualified Wide midfielder 35 40 45 50 55 60 Shot(T**) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T*) Foul(T*) Assist(S) Touch(S***) KP(T) PA(S**) Pass(S) Cross(T) AccCross(T) LB(T*) AccLB(T) ThB(S) AccThB(T) Central midfielder 25 30 35 40 45 50 55 60 65 70 Shot(S*) ShotOT(S) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(S) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S*) KP(S) PA(S) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(S*) AccThB(S) Forward Figure 1. Cont. 3. Results Positive eﬀect size indicates that the mean values of variables from group A bigger than the mean values of variables from group B, negative eﬀect size indicates that the mean values of variables from group B bigger than the mean values of variables from group A, e.g., group B-group A: group stage-knockout stage. Letters in parentheses denote the magnitude: t = trivial; s = small. Asterisks indicate the likelihood for the magnitude of the true diﬀerence in means as follows: possible; likely; * very likely; ** most likely. Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through; AW = aerial won; CD = central defender; CM = central midﬁelder; FB = full back; FW = forward; KP = key pass; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; ball WM = wide midﬁelder. Int. J. Environ. Res. 3. Results 40 44 48 52 56 60 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T**) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(S) Assist(T**) Touch(T) KP(T) PA(T) Pass(T) Cross(T) AccCross(T*) LB(T) AccLB(T) ThB(T) AccThB(T) Group stage Knockout stage Wide midfielder 30 35 40 45 50 55 60 Shot(S) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T*) Dribble(T) Offside(T) YC(T) TT(T**) Interception(T) Clearance(T) BS(T) Foul(T**) Assist(T*) Touch(T) KP(S) PA(T) Pass(T) Cross(S) AccCross(S) LB(T) AccLB(T) ThB(T) AccThB(T) Home Away 35 40 45 50 55 60 Shot(S) ShotOT(S) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T*) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S***) KP(S) PA(S*) Pass(S) Cross(T) AccCross(T*) LB(T) AccLB(S) ThB(S) AccThB(S) Non-qualified Qualified Wide midfielder Figure 1. Cont. 3. Results Cont. Int. J. Environ. Res. Public Health 2020, 17, 604 Int. J. Environ. Res. Public Health 2020, 17, x FO 7 of 12 7 of 12 7 of 12 7 of 12 Figure 1. Comparison of the performance profiles of different position’s players under five situational variables. Notes: letters in parentheses denote the magnitude: t = trivial; s = small. Asterisks indicate the likelihood for the magnitude of the true difference in means as follows: * possible; likely; * very likely; ** most likely. Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through ball; AW = aerial won; BS = blocked shot; Disp = player is dispossessed on the ball by an opponent-no dribble involved; KP = key pass; LB = long ball; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; TT = total tackle; UnsTouch = Unsuccessful touch; YC = yellow card. 3. Results 35 40 45 50 55 60 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(S) Dribble(T**) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S*) KP(T) PA(T) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) 40 45 50 55 Shot(T) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(T) Touch(S**) KP(T) PA(T) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(T**) AccThB(T) 35 40 45 50 55 60 Shot(S) ShotOT(S) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T**) YC(T) TT(T) Interception(T*) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(S) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Opponent non-qualified Opponent qualified 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T*) AW(T) Dribble(T**) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(T) Touch(S**) KP(T) PA(T) Pass(S) Cross(T) AccCross(T) LB(S) AccLB(T) ThB(T) AccThB(T) 25 30 35 40 45 50 55 60 65 70 Shot(S*) ShotOT(S) Disp(T*) UnsTouch(T*) Fouled(T) AW(T*) Dribble(T) Offside(T**) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(S) Touch(S) KP(S) PA(T) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(S) AccThB(S) 30 35 40 45 50 55 60 Shot(T*) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(T*) PA(T) Pass(S**) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T*) AccThB(T) Central defender 40 45 50 55 Shot(T**) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(S) Touch(S*) KP(T) PA(S*) Pass(S) Cross(T) AccCross(T) LB(T*) AccLB(T) ThB(T*) AccThB(T) Full back 35 40 45 50 55 60 Shot(S*) ShotOT(S) Disp(T) UnsTouch(T) Fouled(T*) AW(T) Dribble(T) Offside(T*) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(S) Touch(S) KP(S) PA(S*) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(S) AccThB(S) Draw/lose Win Wide midfielder 35 40 45 50 55 60 Shot(T*) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S) KP(T) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Central midfielder 25 30 35 40 45 50 55 60 65 70 Shot(S**) ShotOT(S) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(S) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S) KP(S) PA(T) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(S**) AccThB(S) Forward Figure 1. Comparison of the performance proﬁles of diﬀerent position’s players under ﬁve situational variables. Notes: letters in parentheses denote the magnitude: t = trivial; s = small. Asterisks indicate the likelihood for the magnitude of the true diﬀerence in means as follows: possible; likely; * very likely; ** most likely. 3. Results 35 40 45 50 55 60 Shot(T) ShotOT(T*) Disp(T) UnsTouch(T) Fouled(T) AW(S) Dribble(T**) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S*) KP(T) PA(T) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) 30 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(T*) PA(T) Pass(S**) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T*) AccThB(T) Central defender 40 45 50 55 Shot(T**) ShotOT(T) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(T*) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(T) Touch(S**) KP(T) PA(T) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(T**) AccThB(T) 40 45 50 55 Shot(T*) ShotOT(T) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(S) Touch(S*) KP(T) PA(S*) Pass(S) Cross(T) AccCross(T) LB(T*) AccLB(T) ThB(T*) AccThB(T) Full back 25 30 35 40 45 50 55 60 65 70 Shot(S**) ShotOT(S) Disp(T*) UnsTouch(T*) Fouled(T) AW(T*) Dribble(T) Offside(T**) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(S) Touch(S) KP(S) PA(T) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(S) AccThB(S) 25 30 35 40 45 50 55 60 65 70 Shot(S**) ShotOT(S) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(S) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S) KP(S) PA(T) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(S**) AccThB(S) Forward 35 40 45 50 55 60 Shot(T) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T*) AW(T) Dribble(T**) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(T) Touch(S**) KP(T) PA(T) Pass(S) Cross(T) AccCross(T) LB(S) AccLB(T) ThB(T) AccThB(T) 35 40 45 50 55 60 Shot(T*) ShotOT(T) Disp(T*) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S) KP(T) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Central midfielder 35 40 45 50 55 60 Shot(S*) ShotOT(S) Disp(T**) UnsTouch(T) Fouled(T) AW(T) Dribble(T) Offside(T**) YC(T) TT(T) Interception(T*) Clearance(T) BS(T) Foul(T) Assist(T) Touch(S**) KP(S) PA(S) Pass(S*) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(T) AccThB(T) Opponent non-qualified Opponent qualified 35 40 45 50 55 60 Shot(S*) ShotOT(S) Disp(T) UnsTouch(T) Fouled(T*) AW(T) Dribble(T) Offside(T*) YC(T) TT(T) Interception(T) Clearance(T) BS(T) Foul(T*) Assist(S) Touch(S) KP(S) PA(S*) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(S) ThB(S) AccThB(S) Draw/lose Win Wide midfielder 25 30 35 40 45 50 55 60 65 70 Shot(S*) ShotOT(S) Disp(T) UnsTouch(T) Fouled(T) AW(T) Dribble(S) Offside(T) YC(T) TT(T*) Interception(T) Clearance(T) BS(T) Foul(T) Assist(S) Touch(S) KP(S) PA(T) Pass(S) Cross(T) AccCross(T) LB(T) AccLB(T) ThB(S**) AccThB(S) Forward Figure 1. Comparison of the performance profiles of different position’s players under five situational variables. Notes: letters in parentheses denote the magnitude: t = trivial; s = small. 3. Results Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through ball; AW = aerial won; BS = blocked shot; Disp = player is dispossessed on the ball by an opponent-no dribble involved; KP = key pass; LB = long ball; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; TT = total tackle; UnsTouch = Unsuccessful touch; YC = yellow card. 4. Discussion The current study established technical performance proﬁles of players from the UEFA Champions League based on a large sample (N = 1000 matches from 8 seasons) to identify the diﬀerences of technical performance between players across diﬀerent situational variables and playing positions. Thus, the interaction between situational and positional variables were investigated. Generally, all variables showed small or trivial diﬀerences across ﬁve situational variables and ﬁve playing positions. The eﬀects of situational variables on the technical performance of players from diﬀerent playing positions were lower than in previous studies [31,45]. This ﬁnding may be due to the fact that the analysis of long-term data may reduce the impact of situational variables on players’ performance due to the higher stability of the performances during eight seasons under the diﬀerent contexts. The diﬀerences of player’s technical performance could mainly be identiﬁed in variables related to passing and organizing, especially touches and passes. In particular, passing and organizing abilities are time-space and task-related variables. The diﬀerent proﬁles may reﬂect that these are key determinants of performance in elite football during the last few years [45]. Therefore, the present study helps to reveal the interactions between playing positions and situational variables in these two technical variables. To the best of our knowledge, there is no research that has examined the diﬀerences of player’s performance between group stage and knockout stage of the UEFA Champions League so far. Our database provided us with the possibility to analyze the performance of players in matches from diﬀerent competition stages. Clear diﬀerences when comparing matches from group and knockout stages would be expected given the special characteristics of knockout matches such as the high importance and the presence of higher quality teams than the group stage. Surprisingly, we found that there was no statistical diﬀerence in player’s performance between group stage and knockout stage, except for fouls in the group of wide midﬁelders. This demonstrates a high consistence of player’s performance between group stage and knockout stage, although these two competition stages are characterized by diﬀerent characteristics of matches. Wide midﬁelders might have more defensive tasks when playing against strong opponents compared to when playing against weak opponents as they face higher defensive pressure [46], which could explain why wide midﬁelders from knockout stage committed more fouls than wide midﬁelders from group stage. 3. Results Asterisks indicate the likelihood for the magnitude of the true difference in means as follows: possible; likely; * very likely; **** most likely. Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through ball; AW = aerial won; BS = blocked shot; Disp = player is dispossessed on the ball by an opponent-no dribble involved; KP = key pass; LB = long ball; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; TT = total tackle; UnsTouch = Unsuccessful touch; YC = yellow card. Figure 1. Comparison of the performance proﬁles of diﬀerent position’s players under ﬁve situational variables. Notes: letters in parentheses denote the magnitude: t = trivial; s = small. Asterisks indicate the likelihood for the magnitude of the true diﬀerence in means as follows: * possible; likely; * very likely; **** most likely. Abbreviations: AccCross = accurate cross pass; AccLB = accurate long ball; AccThB = accurate through ball; AW = aerial won; BS = blocked shot; Disp = player is dispossessed on the ball by an opponent-no dribble involved; KP = key pass; LB = long ball; PA = pass accuracy in %; ShotOT = shot on target; ThB = through ball; TT = total tackle; UnsTouch = Unsuccessful touch; YC = yellow card. Int. J. Environ. Res. Public Health 2020, 17, 604 8 of 12 4. Discussion Recent research identiﬁed that match location had a signiﬁcant inﬂuence on technical variables [17], although it had limited impact on physical variables [47]. The eﬀect of match location on players’ performance in our database is lower than those of reported in prior studies. Central defenders and full backs made more clearances and less passes and crosses in away games than in home games, which may reveal that home teams tend to employ a more aggressive strategy. In home games, central defenders are involved in the organizing and attacking process, and full backs are moving forward frequently into the attacking third to make crosses for teammates. In contrast, away teams will face more defensive pressure, thus defenders have to make more clearances to block opponent’s attack. The fact that wide midﬁelders, central midﬁelders and forwards from home teams obtained more shots opportunities than their counterparts from away teams supports the theory that home teams play a more aggressive and oﬀensive strategy. Surprisingly, central midﬁelders did not show clear diﬀerences in variables related to passing and organizing between home games and away games, which may indicate that the performance of central midﬁelders in passing and organizing related variables is stable regardless of match location. Three of the situational variables (quality of team, quality of opponent, and match outcome)—although covering diﬀerent aspects—are in some way connected to teams’ strength. Consequently, similar trends in player’s technical performance were found in these three contexts. Moreover, they had a relatively greater inﬂuence on players’ technical performance than competition stage and match location. Technical performance of players from all playing positions in these three competing contexts showed clear diﬀerences in variables related to passing and organizing. Similar ﬁndings were reported in a previous study on Spanish La Liga [17], which shows that players’ performance was consistent 9 of 12 Int. J. Environ. Res. Public Health 2020, 17, 604 between a domestic league and international competition. The important role of variables related to passing and organizing can be explained as follows: Stronger teams have a higher ability to retain ball possession, to control the game, and have a higher initiative to score goals instead of preventing goals. 4. Discussion Our ﬁndings revealed that either strong teams are more likely to adopt a possession-based playing style, or that improving players’ ability in the aspect of passing and organizing can help to achieve a better match performance for football teams in the group stage. Moreover, forwards’ performance in shots, shots on target, and dribbles showed clear diﬀerences within these three contexts, which might be a result of the diﬀerences of skill level between forwards and the support they got from midﬁelders based on the advantage of possession. However, wide midﬁelders from qualiﬁed teams, playing against non-qualiﬁed teams and playing in winning games gained more shots and shots on target than wide midﬁelders from non-qualiﬁed teams, playing against qualiﬁed teams and playing in draw/losing games. This indicates that wide midﬁelders from stronger teams had more opportunities to participate in the oﬀensive phase and to invade from wide to inside, hence they are likely to get more scoring chances [17]. We also found that touches and passes were the only two variables in which match performance of players from all playing positions showed clear diﬀerences when considering the eﬀect of quality of team, quality of opponent, and match outcome. This provides us with the opportunity to explore the interaction between ﬁve playing positions and three situational variables. Wide midﬁelders showed the biggest diﬀerences in touches and passes between stronger teams and weaker teams. A possible reason is that wide midﬁelders from stronger teams played an important role in the organizing and attacking phase, while wide midﬁelders from weaker teams relatively need to take on more defensive tasks. The magnitude of diﬀerences in touches and passes for players from all ﬁve playing positions seems to be mostly inﬂuenced by the eﬀect of quality of team within these three situational variables. Most variables related to attacking and defending showed unclear diﬀerences across diﬀerent playing positions and diﬀerent situational variables, which indicates that the diﬀerences of players’ match performance does not mainly result from attacking and defending abilities, but from the ability of managing the game, keeping hold of the ball, and creating scoring opportunities [33]. 5. Conclusions The present study contributes to the current research on performance analysis in top-class football [48] by establishing more comprehensive and detailed technical proﬁles to examine the interaction of positional and situational variables on players’ technical performance. This could be an important step to provide information on players’ match performance and their interactions. Generally, the magnitudes of diﬀerences for all variables were displayed at a low level (small or trivial) based on a large dataset. Match location, quality of team, quality of opponent, and match outcome demonstrated signiﬁcant eﬀect on players’ technical performance while the eﬀect of competition stage on players’ technical performance was strongly limited. Strength-related situational variables showed similar trends and had a relatively greater inﬂuence on players’ technical performance than match location. The technical performance of each playing position also varies under diﬀerent competing contexts. Wide midﬁelders showed the biggest diﬀerences in variables related to passing and organizing within strength-related situational variables and the quality of team had a bigger impact on players’ match performance in variables related to passing and organizing compared to quality of opponent and match outcome. The diﬀerences of players’ technical performance could mainly be identiﬁed in variables related to goal scoring and variables related to passing and organizing, while there were no clear diﬀerences in most attacking and defending related variables. Technical performance of individual players can be evaluated by integrating their match data into the performance proﬁles, which may provide a valuable tool for player recruitment and talent identiﬁcation. Moreover, these technical performance proﬁles can also be used during pre-match preparation, while considering the conditions of the next match, and during post-match assessment to develop position-speciﬁc interventions in the coaching process. 10 of 12 Int. J. Environ. Res. Public Health 2020, 17, 604 However, there are still opportunities to expand the level of this research by adding relevant information in future research. There were 625 pairwise comparisons conducted to identify the diﬀerences in technical performance of players from ﬁve positions under ﬁve competing situations, which may probably result in an increase of type I errors. Thus, this issue should be addressed in further research. Previous studies have identiﬁed the inﬂuence of match status (e.g., score and time left) on the players’ match performance. 5. Conclusions All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, Q.Y., M.-Á.G.-R., and H.L.; Methodology, M.-Á.G.-R. and H.L.; Software, Q.Y. and S.Z.; Data Collection, Q.Y.; Writing—Original Draft Preparation, Q.Y.; Visualization, Q.Y. and S.Z.; Writing—Review and Editing, M.-Á.G.-R., H.L., B.G., F.W., and D.M.; Supervision, M.-Á.G.-R., H.L., F.W., and D.M.; Funding Acquisition, B.G. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Shanghai Key Lab of Human Performance (Shanghai University of Sport) under Grant No. 11DZ2261100. Funding: This work was supported by the Shanghai Key Lab of Human Performance (Shanghai University of Sport) under Grant No. 11DZ2261100. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions But as all data used within this study is aggregated data reﬂecting the whole match time, the match status hasn’t been considered as an additional situational variable in this study. Another aspect that could be valuable for future research on performance proﬁles of football players is positional data. A growing availability of positional data has led to innovations in match analysis in recent years. The investigation of advanced key performance indicators based on positional data can help to gain additional insights to performance of football players. Future research on performance proﬁles might proﬁt from this work by including variables based on positional data into the performance proﬁles and thus expanding performance analysis from technical variables to tactical variables. Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/17/2/604/s1, Table S1. Descriptive statistics of match performance proﬁles of central defenders under ﬁve competing situations. Table S2. Descriptive statistics of match performance proﬁles of full backs under ﬁve competing situations. Table S3. Descriptive statistics of match performance proﬁles of wide midﬁelders under ﬁve competing situations. Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/17/2/604/s1, Table S1 Descriptive statistics of match performance proﬁles of central defenders under ﬁve competing situations Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/17/2/604/s1, Table S1. Descriptive statistics of match performance proﬁles of central defenders under ﬁve competing situations. Table S2. Descriptive statistics of match performance proﬁles of full backs under ﬁve competing situations. Table S3. Descriptive statistics of match performance proﬁles of wide midﬁelders under ﬁve competing situations. Table S4. Descriptive statistics of match performance proﬁles of central midﬁelders under ﬁve competing situations. Table S5. Descriptive statistics of match performance proﬁles of forwards under ﬁve competing situations. Table S2. Descriptive statistics of match performance proﬁles of full backs under ﬁve competing situations. Table S3. Descriptive statistics of match performance proﬁles of wide midﬁelders under ﬁve competing situations. Table S4 Descriptive statistics of match performance proﬁles of central midﬁelders under ﬁve competing situations p p p p g Table S3. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3145148975	https://www.degruyter.com/document/doi/10.1515/opag-2021-0016/pdf	English	null	Increase in the antioxidant content in biscuits by infusions or <i>Prosopis chilensis</i> pod flour	Open Agriculture	2,021	cc-by	7,915	1 Introduction Abstract: Nowadays there is an increasing demand for healthy biscuits. The reduction in sugar and fat level, as well as the addition of bioactive compounds, is posi- tively associated with a healthy diet. In the present work, low-fat and low-sugar biscuits were prepared with infu- sions (mate, coﬀee, and tea) or with Prosopis chilensis pod ﬂour (PPF). Biscuits were made with maize starch and wheat ﬂour (gluten formulations) or with gluten- free ingredients (gluten-free). The colour, texture, and the antioxidant capacity were evaluated in dough and bis- cuits. Among the formulations prepared with infusions, the mate dough showed the lowest ﬁrmness (1.1 N (gluten)- 24.3 N (gluten-free)). However, no signiﬁcant diﬀerences were found in the fracture stress of the ﬁnal products (P > 0.05). Mate gluten biscuits and PPF gluten-free biscuits showed the highest fracture strain (16.2 and 9.4%, respec- tively) and the lowest Young’s modulus (7.3 and 13.3 MPa, respectively) in their groups. The highest antioxidant activity was found in biscuits with mate (8.7 µmol FeSO4/g (gluten)-4.3 µmol FeSO4/g (gluten-free)). These values were three times higher than the ones found in the control biscuits (2.9 µmol FeSO4/g (gluten)-3.9 µmol FeSO4/g (gluten-free)). The present results showed that the antioxidant content in biscuits could be successfully increased with infusion addition. Biscuits are one of the best-selling food products in many countries. They have a long shelf life and, because a wide variety of ingredients can be incorporated into their for- mulation, they can be a useful tool to improve the nutri- tional quality in the consumer’s diet (Guiné et al. 2020). Nonetheless, biscuits generally contain high fat levels often more than 30% and a high sugar amount rarely less than 40%; therefore, they are not considered as healthy food. The development of healthy biscuits can include the reduction in their sugar or fat levels and the incorpora- tion of diﬀerent bioactive components. Some authors made healthy biscuits through the incorporation of com- posite ﬂours (Saha et al. 2011; Chandra et al. 2015; Dhan- khar et al. 2019), ﬁbre (Brennan and Samyue 2004; Aboshora et al. 2019; Diez-Sánchez et al. 2019), resistant starch (Aparicio-Saguilán et al. 2007; Laguna et al. 2011), natural and artiﬁcial sweeteners (Mosafa et al. 2017; Nakov et al. 2019), fruits (Pathak et al. 2018), spices (Klunklin and Savage 2018; Sandhya and Waghray 2018), or fat replacers (Colla et al. 2018). Open Access. © 2021 Paula Andrea Conforti and Mariela Patrignani, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Research Article Paula Andrea Conforti, Mariela Patrignani Increase in the antioxidant content in biscuits by infusions or Prosopis chilensis pod ﬂour https://doi.org/10.1515/opag-2021-0016 received October 12, 2020; accepted February 12, 2021 https://doi.org/10.1515/opag-2021-0016 received October 12, 2020; accepted February 12, 2021 Open Agriculture 2021; 6: 243–253 Corresponding author: Paula Andrea Conforti, Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET – UNLP – CIC, Calle 47 y 116 La Plata, Buenos Aires, Argentina; Facultad de Ciencias Agrarias y Forestales-Universidad Nacional de La Plata, 60 y 119, 1900 La Plata, Argentina, e-mail: paulacon@biol.unlp.edu.ar, tel: +54-221-424-9287, fax: +54-221-424-9287 * Corresponding author: Paula Andrea Conforti, Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET – UNLP – CIC, Calle 47 y 116 La Plata, Buenos Aires, Argentina; Facultad de Ciencias Agrarias y Forestales-Universidad Nacional de La Plata, 60 y 119, 1900 La Plata, Argentina, e-mail: paulacon@biol.unlp.edu.ar, tel: +54-221-424-9287, fax: +54-221-424-9287 Mariela Patrignani: Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET – Facultad de Ciencias Exactas UNLP – CIC, Calle 47 y 116 La Plata, Buenos Aires, Argentina 2 Materials and methods Prosopis chilensis pod ﬂour (PPF) (9.0% proteins, 4.6% lipids, 74% carbohydrates, 7.2% moisture, and 4.50% ash) was prepared by grinding whole pods of Prosopis chilensis. Other ingredients were purchased in a local market, such as wheat ﬂour (ash content less than 0.65%, 10.1% protein, 14.7% moisture), rice ﬂour (6% proteins, 1.2% lipids, 80% carbohydrates, and 2.4% ﬁbre), chickpea ﬂour (16% pro- teins, 9% lipids, 45% carbohydrates, and 15% ﬁbre), cas- sava starch, maize starch, high oleic sunﬂower oil (Cañuelas, Argentina), sucrose, baking powder, vanilla, cocoa powder, black tea (La Virginia, Argentina), green tea (La Virginia, Argentina), yerba mate (Unión, Argentina), and coﬀee (La Morenita, Argentina). The chemical reagents were of ana- lytical grade. Infusions are natural aqueous extracts with high anti- oxidant content. Coﬀee (Coﬀea arabica) and tea (Camellia sinensis) are widely consumed beverages, and they are rich in bioactive phytochemicals such as chlorogenic acids, polyphenols, alkaloids, and melanoidins (Rodrigues and Bragagnolo 2013; Iriondo-DeHond et al. 2019). Mate is an ancestral beverage consumed in several South American countries made from dried leaves of Ilex paraguariensis. Mate contains high levels of chlorogenic acids (Meinhart et al. 2018), saponins, purine alkaloids (Bracesco et al. 2011) vitamins, minerals, and several amino acids (Da Silva et al. 2008). Previous research has indicated the beneﬁcial eﬀects of mate consumption (Heck and Mejia 2007; Bracesco et al. 2011; Riachi and De Maria 2017; Gómez-Juaristi et al. 2018). Moreover, because of the long history of safe usage of these infusions and their high content of antioxidant sub- stances, their addition into biscuit formulation is of parti- cular interest to increase their nutritional value. to volume, colour, tenderness, sweetness, and also acts as preservative (Mamat et al. 2010). The reduction in sugar and fat content in biscuits results in structural, textural, sensory, and hedonic consequences (Pareyt et al. 2009). Accordingly, the production of healthy low- fat and low-sugar biscuits enriched with natural antioxi- dants could be a worthwhile alternative to increase the amount of antioxidant in the diet. Besides, this may also be an attractive option for consumers who are increas- ingly concerned about the choice of healthy foods. However, the addition of new ingredients into biscuit formulations may adversely aﬀect the viscoelastic pro- perties of dough and the quality of the ﬁnal products. Therefore, any modiﬁcation of the formulation must be thoroughly evaluated. territory, and it is a great food resource for humans and animals in arid and semi-arid regions of the world. Algarrobo pod ﬂour is used for various purposes: food, wood, fodder, and also some ethnohistorical references have indicated their consumption by local indigenous people (Capparelli 2007; Capparelli and Prates 2015). Moreover, previous authors have indicated that algarrobo pod ﬂour presents a high proportion of simple sugars and ﬁbre, and also contains a signiﬁcant amount of minerals, vitamins, and polyphenols with high antioxidant activity (Cardozo et al. 2010; Sciammaro et al. 2016; Gonzales- Barron et al. 2020). It is well known that there is a positive correlation between food and health. Dietary guidelines worldwide recommend to increase the consumption of fruits and vegetables. These foodstuﬀs are rich sources of antioxi- dant and dietary ﬁbre that could reduce the risk of human diseases such as cancer, atherosclerosis, heart diseases, osteoporosis, and obesity. Biscuits are often stored for extended periods; thus, antioxidant agents are one of the most used additives in the food industry. Natural antioxidants are generally preferred to potentially toxic, synthetic substances. Thus, several authors have studied the addition of natural antioxidant in biscuits with excel- lent results (Reddy et al. 2005; Mildner‐Szkudlarz et al. 2009; Caleja et al. 2017). Furthermore, antioxidants can protect the body from oxygen radical-induced damage. Thus, the aim of this article was to verify that (1) healthy additive‐free low-fat and low-sugar biscuits with or without gluten can be made with a delicate texture by an appropriate selection of blends of diﬀerent ingredients (ﬂours and starches), and (2) the addition of infusions or algarrobo ﬂour into biscuits could improve their antioxi- dant content without aﬀecting their quality. 1 Introduction On the contrary, the increase in the number of celiac patients increases the demand of gluten-free food. Although hydrocolloids or other additives are generally used to improve the physical characteristics of these food products, they may reduce the consumer’s acceptance. Moreover, gluten-free products present a high proportion of carbohy- drates but they are deﬁcient in protein, ﬁbre, minerals, and vitamins (Saturni et al. 2010; Koidis 2016). Thus, the addition of ingredients that provide all the nutrients necessary to maintain a balanced diet and, at the same time, reduce the fat and the sugar content of gluten-free products is a matter of striking signiﬁcance. Keywords: low-fat biscuits, mate, tea, coﬀee, gluten-free * Corresponding author: Paula Andrea Conforti, Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET – UNLP – CIC, Calle 47 y 116 La Plata, Buenos Aires, Argentina; Facultad de Ciencias Agrarias y Forestales-Universidad Nacional de La Plata, 60 y 119, 1900 La Plata, Argentina, e-mail: paulacon@biol.unlp.edu.ar, tel: +54-221-424-9287, fax: +54-221-424-9287 Mariela Patrignani: Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET – Facultad de Ciencias Exactas UNLP – CIC, Calle 47 y 116 La Plata, Buenos Aires, Argentina * Corresponding author: Paula Andrea Conforti, Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET – UNLP – CIC, Calle 47 y 116 La Plata, Buenos Aires, Argentina; Facultad de Ciencias Agrarias y Forestales-Universidad Nacional de La Plata, 60 y 119, 1900 La Plata, Argentina, e-mail: paulacon@biol.unlp.edu.ar, tel: +54-221-424-9287, fax: +54-221-424-9287 Various ﬂours and starches have been studied as ingredients to replace wheat ﬂour in gluten-free foods. Algarrobo pod ﬂour or Mesquite ﬂour is a gluten-free ﬂour obtained from the grinding of the whole mature fruit (pod) of algarrobo trees (Prosopis spp.). These species are widely distributed in a large part of the South American Mariela Patrignani: Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET – Facultad de Ciencias Exactas UNLP – CIC, Calle 47 y 116 La Plata, Buenos Aires, Argentina Paula Andrea Conforti and Mariela Patrignani 244 2.3 Dough and biscuits characterization Texture proﬁle analysis (TPA) was performed with a texture analyser (TA-XT2i, Stable Micro Systems Ltd, England). The dough samples with a 2.0 cm diameter and 1 cm height were compressed and decompressed during two penetration cycles. Compression was exerted by a 7.5 cm diameter cylindrical probe with a test speed of 0.5 mm/s and with a 50 kg load cell. The strain was set at 50% and 30 s between cycles. Firmness (N), consistency (N s), adhesiveness (N s), springiness, and cohesiveness were calculated from the TPA plot (Gómez et al. 2007). The pH of dough was mea- sured with an electrode for solid samples. To evaluate whether the addition of infusions (5%) could cause aromas, colours, or ﬂavours perceived by consumers, two sensory tests were run. These assays were also used to optimize the proportion of ingredients that allowed the best texture in the low-fat and low-sugar biscuit. A total of 24 untrained panellists evaluated the colour, the texture, the aroma, the taste, and the global acceptance of four biscuits coded with random digits on a hedonic scale (1 = dislike and 10 = like very much). The ﬁrst sensory test was performed with gluten bis- cuit formulations made with four blends of wheat ﬂour and maize starch (WF:MS) (100.0 g): 20.0 g of sugar, 8.0 g of high oleic sunﬂower oil, 1.0 g of vanilla essence, 1.0 g of baking powder, and 46.0 g of tap water. In three for- mulations, 30.0 g of water was replaced by 5% infusions (mate, coﬀee, and tea). Biscuits were baked for 22 min. The fracture properties of the rectangular biscuits were studied by a three-point bending test performed with a TA.XT2 Texture Analyser (Stable Micro Systems Ltd, England), with trigger force of 25 g and load cell of 50 kg. Span length (L) was 1.7 cm, and compression speed was set at 0.1 mm/s. Samples were placed on supports with their top surface down. The large, width (d), and thickness (b) of the baked products were measured using a Vernier calliper. The force (F) needed to break the bis- cuit (N), the toughness or breaking work (N s), the defor- mation (y) before rupture (mm), and the slope (s) of force–distance curve (N mm) were determined. Texture of rectangular biscuits was expressed according to size- independent parameters (Baltsavias et al. 245 essence were added and mixed for 90 s. Finally, the mix of ﬂour, starches, and baking powder was added and mixed for 120 s. thermostatic bath during 15 min. Then, the extracts were cooled at room temperature before their use. The pH of infusions was determined using a Mettler Toledo meter (SevenMulti, China) at 25°C. Dough was set into a polypropylene bag and held at 4°C for 30 min before it was extended with a rolling pin to give a thickness of 0.3 cm. Circular pieces of dough of 2.5 cm in diameter were cut and placed on a silicon sheet. The dough pieces were baked in an oven (Ariston type F9M, Italy) at 175°C with forced convection at diﬀerent baking times. 2.3 Dough and biscuits characterization 1999), and the fracture stress (σ, equation (1)), the fracture strain (ε, equation (2)), and the Young’s modulus (E, equation (3)) were calculated as follows: The second sensory test was made with four gluten- free blends with rice ﬂour, cassava starch, maize starch, and chickpea ﬂour (RF:CS:MS:CF) (100.0 g): 5.0 g sugar and 5.0 g PPF, 1.0 g of vanilla essence, 2.0 g of baking powder, and 35.0 g of tap water. In three of them, 30.0 g water was replaced by 5% infusions (mate, coﬀee, and tea). Biscuits were baked for 18 min. Then, two new batches of biscuits were prepared using the formulations selected in the sensory tests: a con- trol formulation (with water), formulations with 5% infu- sions (mate, coﬀee, black, or green tea), and one prepared with a mix of sugar and PPF (1:1). Gluten samples (formu- lated with a WF:MS mixture) were prepared with 46 g of tap water, while gluten-free samples (prepared with the RF:CS:MS:CF mixture) with 35 g. When PPF was added into formulations, a slightly higher amount of water was necessary to add (12 mL). Six rectangular pieces (5.0 × 2.5 × 0.3 cm) of gluten or gluten-free biscuit formulations were baked at 175°C during 32 min or 22 min, respectively. (3)) = σ FL db 3 2 , 2 (1) = ε by L 6 , 2 (2) = E sL db 4 . 3 3 (3) = σ FL db 3 2 , 2 (1) = ε by L 6 , 2 (2) = E sL db 4 . 3 3 (3) (1) (2) (3) The texture was measured 24 h after baking (to mini- mize the impact of moisture gradients in the baked pro- duct during cooling) with at least ﬁve diﬀerent biscuits of each formulation. The water activity of biscuits was measured with AquaLab Serie3 (Decagon Devices, Inc., Pullman, WA; at 25°C) in duplicate. Besides, the moisture content was also determined in duplicate (AOAC 1984). 2.1 Selection of biscuit formulation by sensorial analysis It is well known that the proportion of fat and sugar strongly inﬂuences the machinability of the dough as well as the quality of the ﬁnished product (Maache- Rezzoug et al. 1998; Baltsavias et al. 1999; Rodríguez- García et al. 2013). The fat acts as lubricant and contri- butes to dough plasticity, whereas the sugar contributes Dough was prepared with a kneading machine (Phillipp Cucina, Brazil) using a dough hook attachment at medium speed (837 rpm). First, the oil and the sugar were creamed during 30 s; then, the infusions or the water and vanilla Biscuits with infusions or algarrobo pod ﬂour 2.6 Data analysis Data were statistically evaluated by analysis of variance (ANOVA) at a 0.05 signiﬁcance level. The least signiﬁcant diﬀerences (LSD) were calculated to compare the means at a level of 95% using the Fisher’s test. Non-parametric statistical tests were used when the variables did not comply with the assumption of homoge- neity of variance. As recommended by García‐Gómez et al. (2019), the diﬀerences in the sensory characteristics (colour, texture, taste, aroma, and global acceptance) were studied using Friedman’s test at 95% level of conﬁdence. = − (( − ) + + ) L a b WI 100 100 , ⁎ 2 ⁎2 ⁎2 (4) = (( − ) + ( − ) + ( − ) ) E L L a a b b Δ . ⁎ 0 ⁎ 2 ⁎ 0 ⁎ 2 ⁎ 0 ⁎ 2 (5) (4) 3 Results and discussion The extraction of antioxidants was carried out in dupli- cate on ground and sieve samples (<500 µm) of biscuits, dough, and in some ingredients with a high proportion of bioactive compounds. The extraction was performed with warm water as described by Morales et al. (2009) with slight modiﬁcations. Brieﬂy, 0.2 g of samples was weighed and extracted with 1.5 mL of distilled water (45°C) by stirring for 5 min (orbital shaker MS1, IKA, Brasil). Then, after 30 min of resting time at 4°C, samples were centrifuged for 10 min (Giumelli Z-127-D Centrifuge, Argentina). The residue was extracted one more time with 1 mL of warm water and centrifuged in the same condi- tions. Finally, the two aqueous extracts of each sample were combined and stored at −18°C before use. 3.1 Selected formulation from sensorial analysis The low-fat and low-sugar biscuits could be classiﬁed as semisweet biscuits according to Manley’s classiﬁcation (Manley 1998). The colour of the biscuits, the ﬁrst character- istic that the consumers perceive and together with texture and taste, strongly aﬀects the acceptability of the product. Data of colour, texture, and the sensory test of biscuits made with diﬀerent ingredients are presented in Tables 1 and 2. As presented in Table 1, a wide range of toughness values (196.2–625.1 N s) could be obtained from diﬀerent wheat ﬂour and maize blend composition. Biscuits made with 55:45 (WF:MS) with mate were preferred by panellist in texture, taste, and global acceptance (P < 0.05). These samples showed the highest whiteness index (69.8) and a tender texture (low toughness, 196.2 N s) dough, or dried ingredients) or µmol FeSO4 per mL in infusion samples. The surface colour of at least ﬁve samples (dough and biscuits) was measured using a Chroma meter CR- 400 (Osaka, Japan) with D65 illuminant, 10° angle of vision. The colorimeter was calibrated using a standard white plate. The Hunter parameters L* (L* = 0 [black], L* = 100 [white]), a* (−a* = green, +a* = red), and b* (−b* = blue, +b* = yellow) were determined. Moreover, the whiteness index (WI, equation (4)) (Zucco et al. 2011) and the colour diﬀerence (ΔE, equation (5)) between sam- ples and the control biscuits (note with o) were calculated as follows: 2.2 Infusions’ preparation Coﬀee, black or green tea, and yerba mate infusions were prepared (5% w/v) with warm tap water (60°C) in a 246  Paula Andrea Conforti and Mariela Patrignani 246 246  Paula Andrea Conforti and Mariela Patrignani Table 1: Colour (whiteness index), texture (toughness), and results of the sensory test of biscuits prepared with wheat ﬂour (WF) and maize starch (MS) of blends and with infusion addition WF:MS blend ratio Infusion Whiteness index Toughness (N s) Sensory test Colour Aroma Texture Taste Global acceptance 55:45 Mate 69.8c 196.2a 59.0ab 58.5a 77.0b 73.0b 74.0b 62:38 Coﬀee 64.3a 263.4a 72.0b 65.5a 57.0a 64.5ab 63.0ab 69:31 Tea 65.4ab 380.4ab 54.0a 60.5a 50.5a 50.0a 51.0a 77:23 — 66.1b 625.1b 55.0a 55.5a 55.5a 52.5a 52.0a Means with diﬀerent superscripts within the same column are signiﬁcantly diﬀerent (P < 0.05). Table 1: Colour (whiteness index), texture (toughness), and results of the sensory test of biscuits prepared with starch (MS) of blends and with infusion addition ess index), texture (toughness), and results of the sensory test of biscuits prepared with wheat ﬂour (WF) and maize and with infusion addition Means with diﬀerent superscripts within the same column are signiﬁcantly diﬀerent (P < 0.05). Means with diﬀerent superscripts within the same column are signiﬁcantly diﬀerent (P < 0.05). Generally, coﬀee and tea are prepared with very hot water (90–95°C), while mate is prepared at 60–85°C. In this study, all infusions were prepared with tap water at 60°C. Mate infusion had pH 6.30, coﬀee pH 5.62, green tea pH 5.71, and black tea pH 5.17. The pH value measured in the coﬀee infusion is within the range of 5–5.8 reported by Derossi et al. (2018). Results showed that all gluten-free biscuits showed lower WI values than gluten samples because of the colour of CF (88.60L, −0.48a, 22.86b) and PPF (74.96L, 3.87a, 28.65b) (Table 2). It should be highlighted that in both sensory tests, biscuits made with a high starch proportion were pre- ferred by the panellists. Besides, among the formulations with infusion additions, biscuits made with mate received the highest taste score. 2.5 Ferric reducing antioxidant power On the contrary, the colour, the texture, and the sen- sory characteristics of gluten-free samples were also stu- died. According to the results presented in Table 2, no signiﬁcant diﬀerences were found between samples in the sensory test (P > 0.05). The highest toughness value (96.1 N s) and the lowest whiteness index (48.1) were registered in 26:12:12:50 (RF:CS:MS:CF) blend biscuits with high content of chickpea ﬂour (P < 0.05). Thus, the formulation 25:25:25:25 (RF:MS:CS:CF) was selected because it presented the highest scores in texture and global acceptance, besides it had a low value of tough- ness according to the bending test. The ferric reducing antioxidant power (FRAP) of the sam- ples was determined as described by Benzie and Strain (1996). Brieﬂy, 0.2 mL of each extract was mixed with 1.8 mL of fresh prepared FRAP reagent and kept in the dark for 20 min. The absorbance was measured at 593 nm (UVmin-1240 spectrophotometer, Shimatdzu, Jenck S.A., Kyoto, Japan) in clear samples. A calibration curve was made with ferrous sulphate (FeSO4·7H2O) within the range of 0–1,000 μM. The FRAP of the samples was expressed as µmol FeSO4 per g of dried sample (biscuits, Biscuits with infusions or algarrobo pod ﬂour 247 3.2 Infusion preparation Results of colour and dough texture of gluten samples are presented in Tables 3 and 4. In both the tables, the highest whiteness index was found in the control sam- ples (P < 0.05), while the highest colour diﬀerence was observed in the coﬀee samples (P < 0.05). The colour diﬀerences between samples and control dough were less in the gluten-free samples because of the colour of the chickpea ﬂour. The addition of mate infusion increased the pH of the dough, whereas with other infusions or with PPF addition, the values were lower than the control. Previous authors studied the eﬀect of infusion prepara- tion conditions and its antioxidant content (Richelle et al. 2001; Sánchez-González et al. 2005; da Silveira et al. 2014). Mate extracts contain purine alkaloids (methyl xanthines), ﬂavonoids (rutin), vitamins (such as vitamin A, B complex, C, and E), tannins, chlorogenic acid and its derivatives, and numerous triterpenoid saponins derived from ursolic acid (Bracesco et al. 2011). Green tea is made by inactivating the enzymes in the fresh leaves, and it contains ﬂavonoids derivatives of catechins (monomers), whereas black tea contains more complex polyphenols (dimers and polymers) (Wang and Ho 2009). Coﬀee con- tains phenolic compounds such as chlorogenic acids, caf- feine, and diterpenic compounds (Yashin et al. 2017). No signiﬁcant diﬀerences were found between green tea, coﬀee, and control sample (Table 3). Mate sample shows the highest cohesiveness but also the lowest ﬁrm- ness and consistency values (P < 0.05). Except for the PPF dough, all samples were prepared using the same quan- tities of ingredients (water, sugar, oil, and proteins) but Table 2: Colour (whiteness index), texture (toughness), and results of the sensory test of gluten-free biscuits prepared from four blends of rice ﬂour (RF), cassava starch (CS), maize starch (MS), and chickpea ﬂour (CF) with infusions RF:CS:MS:CF blend ratio Infusion Whiteness index Toughness (N s) Sensory test Colour Aroma Texture Taste Global acceptance 25:25:25:25 Mate 57.2c 42.8a 67.0a 64.5a 64.5a 71.0a 67.0a 38:12:12:38 Tea 54.4b 53.2a 60.0a 57.0a 55.0a 56.5a 58.5a 50:12:12:26 Coﬀee 56.0bc 46.1a 65.5a 63.5a 57.0a 54.5a 59.5a 26:12:12:50 — 48.1a 96.1b 47.5a 55.0a 63.5a 58.0a 55.0a Means with diﬀerent superscripts within the same column are signiﬁcantly diﬀerent (P < 0.05). Table 4: Physical characteristics of gluten-free dough prepared from 25:25:25:25 rice ﬂour:cassava starch:maize starch:chickpea ﬂour blend with infusions or Prosopis chilensis pod ﬂour (PPF) Control Coﬀee Mate Black tea PPF Dough Firmness (N) 42.3d 30.3b 24.3a 33.8c 29.5b Consistency (N s) 212.2d 156.6b 129.6a 181.9c 162.3b Cohesiveness 0.100a 0.113c 0.105b 0.101ab 0.101ab Adhesiveness (N s) 2.2a 4.0c 3.9c 3.0b 2.3a Springiness 0.23b 0.27b 0.27b 0.21ab 0.15a Whiteness index, WI 64.7d 58.5a 61.9c 61.3bc 60.7b Colour diﬀerences, ΔE — 6.9c 3.0a 3.6a 4.8b pH 6.81 6.61 6.91 6.70 6.62 Means with diﬀerent superscripts within the same row are signiﬁ- cantly diﬀerent (P < 0.05). Table 3: Physical characteristics of dough and biscuits prepared from 55:45 wheat ﬂour:maize starch blend ﬂour with infusions or with Prosopis chilensis pod ﬂour (PPF) Table 4: Physical characteristics of gluten-free dough prepared from 25:25:25:25 rice ﬂour:cassava starch:maize starch:chickpea ﬂour blend with infusions or Prosopis chilensis pod ﬂour (PPF) Control Coﬀee Mate Green tea PPF Firmness (N) 1.5b 1.3ab 1.1a 1.4b 4.8c Consistency (N s) 8.4b 8.4b 7.0a 8.5b 32.1c Cohesiveness 1.1b 1.2b 1.4c 1.1b 0.3a Springiness 0.98a 0.98a 0.98a 0.98a 0.98a Whiteness index, WI 74.1d 59.4a 66.4b 68.9c 67.0b Colour diﬀerences, ΔE — 15.4c 11.3b 9.9a 10.2a pH 6.56 6.41 6.73 6.62 6.40 Means with diﬀerent superscripts within the same row are signiﬁ- cantly diﬀerent (P < 0.05). Control Coﬀee Mate Green tea PPF Means with diﬀerent superscripts within the same row are signiﬁ- cantly diﬀerent (P < 0.05). diﬀerent infusions; therefore, the diﬀerences in dough texture and dough pH could be related to the composition of the aqueous extracts. The texture of the sample with PPF showed the lowest cohesiveness and the highest ﬁrmness and consistency values (P < 0.05). The replace- ment of sugar by PPF increases the amount of proteins and ﬁbre in the formulation, and therefore, more water was required to obtain a homogeneous dough. Other authors reported a similar trend when replacing wheat ﬂour with algarrobo ﬂour (Bigne et al. 2016). Means with diﬀerent superscripts within the same row are signiﬁ- cantly diﬀerent (P < 0.05). higher amount of water than the other formulations. In both tables, all the aw values were lower than 0.8; there- fore, it could be considered that the pathogen growth would be inhibited (Mauer and Bradley 2017). Biscuits with lower thickness also showed lower values of aw and humidity and thus a crispy texture (high Young mod- ulus values) (P < 0.05). However, no signiﬁcant diﬀer- ences were found in the fracture stress (σ) values (P > 0.05), indicating that all biscuits had similar hardness. Finally, it was observed that the lowest colour diﬀerence was observed in the mate samples (Table 5). In Table 4, the control sample showed highest ﬁrm- ness and consistency values (P < 0.05), while the mate sample showed the lowest values. Adhesiveness of sam- ples with infusion addition was higher than control for- mulations (P < 0.05). According to the results, it could be concluded that the replacement of half of sugar by PPF produced dough with less ﬁrmness, consistency, and springiness than control sample but with similar cohe- siveness and adhesiveness values. The control biscuit (Table 5) showed the lowest values of thickness, aw and humidity, and the highest Young’s modulus values (which indicates a crispy tex- ture) (P < 0.05). On the contrary, biscuits prepared with mate showed the highest fracture strain values (ε) and the lowest Young’s modulus (P < 0.05), indicating a tender texture. Other authors also found a reduction in the ﬁrmness texture with mate addition (Faccin et al. 2015). Mate biscuits also showed a signiﬁcantly higher thickness, humidity, and aw values (P < 0.05). During baking, dough components (proteins, sugars, dietary ﬁbre, and other high water aﬃnity ingredients) are responsible for trapping water until it is released as a consequence of heating. Thus, diﬀerences in dough com- position (extracts or PPF) produce that all biscuits retain more moisture than the control formulation. Regarding colour, coﬀee and PPF biscuits presented lower WI values and higher ΔE values than other biscuits (P < 0.05). 3.2 Infusion preparation Table 2: Colour (whiteness index), texture (toughness), and results of the sensory test of gluten-free biscuits prepared from four blends of rice ﬂour (RF), cassava starch (CS), maize starch (MS), and chickpea ﬂour (CF) with infusions Means with diﬀerent superscripts within the same column are signiﬁcantly diﬀerent (P < 0.05).  Paula Andrea Conforti and Mariela Patrignani 248 Table 5: Physical characteristics biscuits prepared from the 55:45 wheat ﬂour:maize starch blend ﬂour with infusions or with Prosopis chilensis pod ﬂour (PPF) Control Coﬀee Mate Green tea PPF Fracture stress, σ (kPa) 1.7a 1.3a 0.9a 1.3a 1.5a Fracture strain, ε (%) 5.0a 8.2a 16.2b 7.9a 6.8a Young’s modulus, E (MPa) 33.9c 22.6bc 7.3a 17.8ab 18.2ab Thickness (mm) 4.5a 5.6ab 6.7b 5.6ab 5.3b Moisture content (%) 6.7a 8.7c 11.2d 7.9b 9.0c aw 0.399a 0.559b 0.672c 0.487ab 0.529b Whiteness index, WI 68.5c 57.0a 68.2c 61.8b 56.7a Colour diﬀerences, ΔE — 13.0b 8.3a 8.3a 14.3b Means with diﬀerent superscripts within the same row are signiﬁcantly diﬀerent (P < 0.05). Table 5: Physical characteristics biscuits prepared from the 55:45 wheat ﬂour:maize starch blend ﬂour with infusions or with Prosopis chilensis pod ﬂour (PPF) cs biscuits prepared from the 55:45 wheat ﬂour:maize starch blend ﬂour with infusions or with Prosopis Means with diﬀerent superscripts within the same row are signiﬁcantly diﬀerent (P < 0.05). with the dough and the biscuits samples by FRAP assay so that the results could be compared. The values found for these ingredients were (µmol FeSO4/g): 80.4 Moringa oleifera leaf powder; 1.9 dried cranberries (Vaccinium macrocarpon); 9.8 raisins (Vitis vinifera L.); and 5.5 dried plums (Prunus domestica). (P > 0.05). Coﬀee biscuits showed the highest Young’s modulus values (crispier texture) while samples with PPF showed the highest fracture strain (tender texture) (P < 0.05). The colour analysis indicated that mate and PPF biscuits presented higher WI values and lower ΔE values than coﬀee and tea biscuits (P < 0.05). Results of FRAP assay are presented in Table 7. Anti- oxidant capacity of dough depends on the amount of antioxidants added with ingredients. During baking, the outer layers of the dough are heated to 170°C, but in the inner layers, the temperature remains lower than 100°C. Decomposition of antioxidant during baking is partially compensated by the formation of Maillard products (mel- anoidins) which also possess antioxidant activity (Patri- gnani et al. 2021). Only samples made with PPF and with black tea showed a higher antioxidant activity in biscuits than dough. PPF and all infusions are shown higher FRAP values than control samples. The antioxidant activity of samples made with PPF or mate and green tea infusions was higher than samples made with coﬀee and black tea infusions. The same trend was observed between the antioxidant content of the ingredients. All It was observed that despite the diﬀerences in the texture of the dough with infusions or with PPF (Tables 3 and 4), there were no important diﬀerences in the tex- ture of biscuits (Tables 5 and 6). 3.4 Quality properties of biscuits Baking is a complex process, which includes evaporation of water, denaturation of proteins, starch gelatinization, and also Maillard reactions. Results of colour, humidity, dimensions, and fracture texture of biscuits are presented in Table 5 (gluten samples) and Table 6 (gluten-free sam- ples). Although all the samples were baked simulta- neously and had the same amount of ingredients (except for PPF), some unexpected diﬀerences were found in the moisture and water activity values. PPF samples showed a higher humidity and higher aw values than control samples, probably because they were prepared with a Results in Table 6 show that no signiﬁcant diﬀer- ences were found in the thickness of gluten-free biscuits Biscuits with infusions or algarrobo pod ﬂour  249 Means with diﬀerent superscripts within the same row are signiﬁcantly diﬀerent (P < 0.05). Table 7: FRAP results in samples prepared with 55:45 wheat ﬂour:maize starch (WF:MS) blend or with 25:25:25:25 rice ﬂour:cassava starch:maize starch:chickpea ﬂour (RF:CS:MS:CF) blend with infusions or Prosopis chilensis pod ﬂour (PPF)* Table 7: FRAP results in samples prepared with 55:45 wheat ﬂour:maize starch (WF:MS) blend or with 25:25:25:25 rice ﬂour:cassava starch:maize starch:chickpea ﬂour (RF:CS:MS:CF) blend with infusions or Prosopis chilensis pod ﬂour (PPF)* Table 7: FRAP results in samples prepared with 55:45 wheat ﬂour:maize starch (WF:MS) blend or with 25:25:25:25 rice ﬂour:cassava starch:maize starch:chickpea ﬂour (RF:CS:MS:CF) blend with infusions or Prosopis chilensis pod ﬂour (PPF)* Control Coﬀee Mate Black tea Green tea PPF Ingredients — 8.8* 12.1* 7.8* 12.8* 18.6 55:45 (WF:MS) Dough 2.0a 4.4b 8.1d — 5.7c 4.4b Biscuit 2.9a 4.5b 8.7d — 5.1bc 5.6c 25:25:25:25 (RF:CS:MS:CF) Dough 1.3a 3.3c 3.9d 2.0b — 3.0c Biscuit 1.8a 3.3b 4.3e 3.1b — 3.8c Means with diﬀerent superscripts within the same row are signiﬁcantly diﬀerent (P < 0.05). Results are expressed as µmol FeSO4/g solid samples or µmol FeSO4/mL in liquid ingredients. Means with diﬀerent superscripts within the same row are signiﬁcantly diﬀerent (P < 0.05). Results are expressed as µmol FeSO4/g solid samples or µmol FeSO4/mL in liquid ingredients on biscuit formulation should be further investigated. In addition, the results indicated that healthier low-fat and low-sugar biscuits without artiﬁcial additives and with a delicate texture could be obtained by selecting the right mix of ingredients. ingredients showed a higher antioxidant activity than dried cranberries and dried plumbs. The antioxidant capacity of mate samples (8.1 (WF:MS) – 3.9 (RF:CS:MS:CF) was three times greater than the control dough samples (2.2 (WF:MS) – 1.3 (RF:CS:MS:CF)) and almost double in biscuits samples (P < 0.05). This is in accordance with the higher antioxidant activity of mate compared to tea infusions reported by other authors (Bravo et al. 2007; Heck and Mejia 2007; da Silveira et al. 2014). No diﬀerences were found between the antioxidant activity of coﬀee and PPF dough samples, but in biscuits, PPF showed higher values than coﬀee biscuits. Acknowledgments: The authors would like to thank Mr. Fabian Conforti for providing the algarrobo pod used in the present work. Funding information: This work was ﬁnancially supported by ANPCyT (PICT 2013-0007, PICT 2016-3047), CONICET, and UNLP of Argentina. The sensorial test showed that the incorporation of the infusions at 5% level did not cause signiﬁcant changes in appearance (aroma or colour) perceived by consumers, and it could be considered a positive aspect to keep the traditional aspect of the biscuits. Author contribution: PAC: conceptualization, methodology, validation, formal analysis, investigation, visualization, data curation, funding acquisition, writing – original draft, review and editing. MP: visualization, formal analysis, writing – original draft, review and editing. 4 Conclusions Conﬂict of interest: The authors state no conﬂict of interest. Biscuits are products worldwide appreciated and con- sumed by diﬀerent categories of consumers, child, young, and adults. Therefore, the reduced sugar and fat content could have a great impact on health. Low-fat and low- sugar biscuits enriched with natural sources of anti- oxidants may be attractive for consumers who are increasingly concerned about the choice of healthy foods. Algarrobo pod ﬂour could be obtained at home, with a simple ground process, and it is a gluten-free ingredient that could be used as a partial sugar replacer. Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 3.5 Ferric reducing antioxidant power There are many diﬀerent antioxidants, and it is very dif- ﬁcult to measure each antioxidant component separately. In this study, the FRAP assay was selected because it is simple, and the reaction is reproducible and linearly related to the concentration of the antioxidant(s) present. In the present study, antioxidants were extracted under the same conditions (warm water) and analysed together Table 6: Physical characteristics of gluten-free biscuits prepared from 25:25:25:25 rice ﬂour:cassava starch:maize starch:chickpea ﬂour blend with infusions or Prosopis chilensis pod ﬂour (PPF) Control Coﬀee Mate Black tea PPF Fracture stress, σ (kPa) 0.8a 1.1a 0.9a 1.1a 1.2a Fracture strain, ε (%) 3.5a 4.6ab 6.5b 4.9ab 9.4c Young’s modulus, E (MPa) 18.8a 37.6b 17.2a 27.7ab 13.3a Thickness (mm) 5.0a 4.6a 4.9a 5.0a 5.2a Moisture content (%) 6.9ab 6.1a 9.8c 7.7b 11.1d aw 0.469a 0.453a 0.609c 0.525b 0.650d Whiteness index, WI 59.0c 52.9a 60.0c 56.4b 58.5c Colour diﬀerences, ΔE — 7.7b 3.1a 5.5ab 4.8ab Means with diﬀerent superscripts within the same row are signiﬁcantly diﬀerent (P < 0.05). Means with diﬀerent superscripts within the same row are signiﬁcantly diﬀerent (P < 0.05). 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Photo of gluten-free dough prepared from 25:25:25:25 rice ﬂour: cassava starch: maize starch: chickpea ﬂour blend ﬂour with infusions or Prosopis chilensis pod ﬂour (PPF) (1: black tea; 2: coﬀee; 3: control (prepared with water); 4: PPF; 5: mate).
https://openalex.org/W3153298919	https://www.jmir.org/2021/7/e26658/PDF	English	null	App-Based Feedback for Rehabilitation Exercise Correction in Patients With Knee or Hip Osteoarthritis: Prospective Cohort Study	JMIR. Journal of medical internet research/Journal of medical internet research	2,021	cc-by	9,283	Abstract Background: The use of digital therapeutic solutions for rehabilitation of conditions such as osteoarthritis provides scalable access to rehabilitation. Few validated technological solutions exist to ensure supervision of users while they exercise at home. Motion Coach (Kaia Health GmbH) provides audiovisual feedback on exercise execution in real time on conventional smartphones. Objective: We hypothesized that the interrater agreement between physiotherapists and Motion Coach would be noninferior to physiotherapists’ interrater agreement for exercise evaluations in a cohort with osteoarthritis. Methods: Patients diagnosed with osteoarthritis of the knee or hip were recruited at a university hospital to perform a set of 6 exercises. Agreement between Motion Coach and 2 physiotherapists’ corrections for segments of the exercises were compared using Cohen κ and percent agreement. Results: Participants (n=24) were enrolled and evaluated. There were no significant differences between interrater agreements (Motion Coach app vs physiotherapists: percent agreement 0.828; physiotherapist 1 vs physiotherapist 2: percent agreement 0.833; P<.001). Age (70 years or under, older than 70 years), gender (male, female), or BMI (30 kg/m2 or under, greater than 30 kg/m2) subgroup analysis revealed no detectable difference in interrater agreement. There was no detectable difference in levels of interrater agreement between Motion Coach vs physiotherapists and between physiotherapists in any of the 6 exercises. Conclusions: The results demonstrated that Motion Coach is noninferior to physiotherapist evaluations. Interrater agreement did not differ between 2 physiotherapists or between physiotherapists and the Motion Coach app. This finding was valid for all investigated exercises and subgroups. These results confirm the ability of Motion Coach to detect user form during exercise and provide valid feedback to users with musculoskeletal disorders. JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al Original Paper (J Med Internet Res 2021;23(7):e26658) doi: 10.2196/26658 (J Med Internet Res 2021;23(7):e26658) doi: 10.2196/26658 J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 1 (page number not for citation purposes) App-Based Feedback for Rehabilitation Exercise Correction in Patients With Knee or Hip Osteoarthritis: Prospective Cohort Study Johanna Theresia Biebl1, MD; Marzena Rykala1, BSc; Maximilian Strobel2, MSc; Pawandeep Kaur Bollinger2, PhD; Bernhard Ulm3, MSc; Eduard Kraft1, MD; Stephan Huber2, MD; Andreas Lorenz1, MD 1Department of Orthopaedics, Physical Medicine, and Rehabilitation, University Hospital, Ludwig Maximilians University of Munich, Munich, Germany 2Kaia Health GmbH, Munich, Germany 3Unabhängige statistische Beratung Bernhard Ulm, Munich, Germany these authors contributed equally Corresponding Author: Johanna Theresia Biebl, MD Department of Orthopaedics, Physical Medicine, and Rehabilitation University Hospital Ludwig Maximilians University of Munich Marchioninistr. 15 Munich, 81377 Germany Phone: 49 89440074070 Fax: 49 89440077073 Email: johanna.biebl@med.uni-muenchen.de Johanna Theresia Biebl1, MD; Marzena Rykala1, BSc; Maximilian Strobel2, MSc; Pawandeep Kaur Bollinger2, PhD; Bernhard Ulm3, MSc; Eduard Kraft1, MD; Stephan Huber2, MD; Andreas Lorenz1*, MD Corresponding Author: Johanna Theresia Biebl, MD Department of Orthopaedics, Physical Medicine, and Rehabilitation University Hospital Ludwig Maximilians University of Munich Marchioninistr. 15 Munich, 81377 Germany Phone: 49 89440074070 Fax: 49 89440077073 Email: johanna.biebl@med.uni-muenchen.de KEYWORDS mHealth; digital health; digital rehabilitation; machine learning; smartphone; osteoarthritis; exercise therapy J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 1 (page number not for citation purposes) https://www.jmir.org/2021/7/e26658 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al exercises in patients with osteoarthritis. We hypothesized that interrater agreement between physiotherapists and Motion Coach would be noninferior to that between 2 physiotherapists. Introduction Musculoskeletal conditions such as osteoarthritis and back pain result in a huge burden for patients and health care systems. Impaired mobility affects both the quality of life of the individual, for example, by increasing social isolation, and the health care system, by raising costs due to factors such as hospitalizations and secondary diseases [1-3]. Osteoarthritis can lead to pain-related fear of movement and an increased probability of further functional impairment [4]. In addition, osteoarthritis is a predictor for developing disabilities that affect activities of daily living, underlining the importance of effective interventions [5]. Ethics and Registration The study was approved by the Ethics Committee of Ludwig Maximilians University of Munich (20-162) and all participants provided informed consent before study procedures were carried out. The study was registered with the German Study Registry (Deutsches Register Klinischer Studien; DRKS00021828) prior to beginning enrollment. Several different digital solutions have been proposed to correct and optimize body pose during exercise execution to improve access to therapeutic exercises [12]. Many mobile health apps for musculoskeletal rehabilitation rely upon video instructions only and provide no means of detecting and correcting pose during exercise [9,13]. These systems, by default, leave users exposed to the risk of incorrectly performing exercise but allow for scalable access without requiring external hardware. To the best of our knowledge, there are no reports on the quality of exercise execution during the use of these systems. Other technologies, such as integrated devices containing inertial sensors, have also been validated to a limited extent, and whether they are suitable for detecting and correcting form during therapeutic exercises has not been evaluated [14,15]. Digital therapeutics that have been validated for this purpose require additional hardware such as a Microsoft Kinect device [16,17]. Procedure To evaluate the correction of osteoarthritis-specific exercises, Motion Coach provides instructions visually through an iPad’s screen and acoustically via headphones to the participants. While participants performed exercises using Motion Coach, 2 physiotherapists evaluated whether the exercises were being performed correctly. (Physiotherapists were blinded to the audiovisual feedback of Motion Coach). Furthermore, the physiotherapists evaluated the execution of an exercise set or the performance over the predefined time for static exercises as a whole on a 6-point Likert scale (0=insufficient, 5=excellent execution of movement). Participants Participants with a confirmed prior diagnosis of osteoarthritis of the hip or knee were enrolled from the outpatient population of the Department of Orthopedics, Physical Medicine and Rehabilitation, University Hospital, Ludwig Maximilians University of Munich. Inclusion criteria were (1) diagnosed hip or knee osteoarthritis and (2) age over 18 years. Exclusion criteria were (1) inability to consent (significant cognitive deficits); (2) not fluent in the German language; (3) severe medical or neurological conditions; (4) severe joint contractures that would influence the correct execution of the exercises; (5) previous hip, knee, and ankle arthrodesis; (6) osseous instabilities; or (7) severe osteoporosis. Current guidelines [6] recommend self-management programs and exercise as first-line therapies for managing osteoarthritis. The prevalence of osteoarthritis is increasing, yet cost and resource constraints limit in-person access to these therapies [7]. Digital therapeutics have emerged as an option to provide access to exercise therapy and multidisciplinary rehabilitation for patients with musculoskeletal pain conditions such as osteoarthritis and back pain [8-10]. Even though a recent survey among health professionals indicated widespread support of use of mobile health technologies in osteoarthritis treatment [11], a primary concern with using digital therapeutics for home-based exercise is the lack of supervision by health care professionals. J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 2 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Exercises performed by participants. Exercise difficulty was rated by training experts prior to study. Exercise difficulty rating Execution mode Pose Exercise name Labela High Dynamic Quadruped Hip extension bent leg a High Dynamic Standing Knee flexion (leg curl) b Medium Dynamic Standing Strengthening hip extensors c Medium Dynamic Standing Strengthen hip abductors d Medium Static Standing Strain front of thigh e Low Static Standing Elongation of the hip flexors f aLetters correspond to those in Figure 1. aLetters correspond to those in Figure 1. Figure 1. Exercises performed in this study (a) hip extension bent leg; (b) knee flexion (leg curl); (c) strengthening hip extensors; (d) strengthen hip abductors; (e) strain front of thigh; (f) elongation of the hip flexors. Figure 1. Exercises performed in this study (a) hip extension bent leg; (b) knee flexion (leg curl); (c) strengthening hip extensors; (d) strengthen hip abductors; (e) strain front of thigh; (f) elongation of the hip flexors. abductors; (e) strain front of thigh; (f) elongation of the hip flexors. Motion Coach Overview In order to give audiovisual feedback on exercise form in real time, Motion Coach uses the camera stream of a user’s mobile device and artificial intelligence–based image processing. Users place their device on the ground approximately 2 meters away, tilted slightly so they can be seen in the frame of view of the camera. The app guides the user with interactive setup instructions (Figure 2). A 2-step process is applied to each new image frame as it is captured by the camera. Motion Coach Overview In order to give audiovisual feedback on exercise form in real time, Motion Coach uses the camera stream of a user’s mobile device and artificial intelligence–based image processing. Users place their device on the ground approximately 2 meters away, tilted slightly so they can be seen in the frame of view of the camera. The app guides the user with interactive setup instructions (Figure 2). A 2-step process is applied to each new i f i i d b h Motion Coach Overview In order to give audiovisual feedback on exercise form in real time, Motion Coach uses the camera stream of a user’s mobile device and artificial intelligence–based image processing. Users place their device on the ground approximately 2 meters away, tilted slightly so they can be seen in the frame of view of the camera. J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 3 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH The app guides the user with interactive setup instructions (Figure 2). A 2-step process is applied to each new image frame as it is captured by the camera. Exercises For assessment, 6 exercises (Table 1 and Figure 1) that reflected several aspects of therapeutic exercises were chosen from the app to ensure detection by the algorithm was reliable in different circumstances. We included exercises that required a varying range of technical ability; exercises that had different modes of execution (4 dynamic and 2 static), to differentiate between exercises requiring rapid feedback in real time (due to continuous movement) and those that do not; and exercises with different levels of difficulty (low, medium, or high). Motion Coach (Kaia Health GmbH) was recently introduced to address these issues (ie, requiring that equipment be worn on the body or additional hardware) by using only smartphone front camera data and machine learning algorithms to detect the position of body segments during exercise in real time in order to provide personalized feedback. The aim of this study was to evaluate the ability of Motion Coach to detect and correct form during physiotherapeutic https://www.jmir.org/2021/7/e26658 J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 2 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX Overview In order to give audiovisual feedback on exercise form in real time, Motion Coach uses the camera stream of a user’s mobile In order to give audiovisual feedback on exercise form in real time, Motion Coach uses the camera stream of a user’s mobile https://www.jmir.org/2021/7/e26658 https://www.jmir.org/2021/7/e26658 XSL•FO RenderX Biebl et al JOURNAL OF MEDICAL INTERNET RESEARCH Figure 2. (a) User stands approximately 2 meters away from their device while the front-facing camera of the device captures user’s movements. (b) User is guided as to where to stand by a series of interactive screens. Step 1: Estimating Pose First, a Pose Estimation Machine Learning Model is applied to infer the user’s pose for each captured image frame in real time (Figure 3). This Pose Estimation Model is a convolutional neural network (typically used for image-based machine learning tasks [18]) with a proprietary architecture that runs entirely on the user’s mobile device (therefore, no raw video data leave the user’s device). The model was specifically optimized to run on a wide variety of iOS and Android devices, and the model achieves state-of-the-art performance on academic benchmarks such as the MPII Human Pose Data Set Benchmark [19]. Kaia Health trained this model using a proprietary image data set that consisted of data from people with a variety of characteristics (body shape, height, skin color, movement limitations, etc) exercising in front of their mobile device, with a wide variety of exercise movements and environmental conditions such as varying lighting and background to make the model robust. Each image in the data set had been manually labeled according to a taxonomy designed to best capture the human body in physiotherapeutic exercises. Figure 3. Examples of keypoint poses (white) inferred for various exercises by the Pose Estimation Model. Figure 2. (a) User stands approximately 2 meters away from their device while the front-facing camera of the device captures user’s movements. (b) User is guided as to where to stand by a series of interactive screens. Figure 2. (a) User stands approximately 2 meters away from their device while the front-facing camera of the device captures user’s movements. (b) User is guided as to where to stand by a series of interactive screens. ir device while the front-facing camera of the device captures user’s movements. (b ns. such as the MPII Human Pose Data Set Benchmark [19]. Data Step 2: Evaluating Geometric Expert System Step 1: Estimating Pose Step 1: Estimating Pose First, a Pose Estimation Machine Learning Model is applied to infer the user’s pose for each captured image frame in real time (Figure 3). This Pose Estimation Model is a convolutional neural network (typically used for image-based machine learning tasks [18]) with a proprietary architecture that runs entirely on the user’s mobile device (therefore, no raw video data leave the user’s device). The model was specifically optimized to run on a wide variety of iOS and Android devices, and the model achieves state-of-the-art performance on academic benchmarks First, a Pose Estimation Machine Learning Model is applied to infer the user’s pose for each captured image frame in real time (Figure 3). This Pose Estimation Model is a convolutional neural network (typically used for image-based machine learning tasks [18]) with a proprietary architecture that runs entirely on the user’s mobile device (therefore, no raw video data leave the user’s device). The model was specifically optimized to run on a wide variety of iOS and Android devices, and the model achieves state-of-the-art performance on academic benchmarks Figure 3. Examples of keypoint poses (white) inferred for various exercises by the Pose Estimation Model. Step 2: Evaluating Geometric Expert System For audiovisual feedback, spatiotemporal constraints, which were configured in advance by medical, physiotherapeutic, or sport science–trained Kaia staff, are triggered based on movement; there was no need for reconfiguration on a per-user or per-session basis. While the system was in use, constraints were checked automatically in real time, and feedback was provided if any of the configured constraints were violated. If multiple constraints were violated, the prioritization mechanism selects the feedback based on risk of injury. Data Overview Physiotherapists’ evaluations were collected on a rating sheet for each participant. Data from the app were obtained by taking a screenshot of the report of corrections after the exercises had been executed. Baseline data were collected from participants using paper-based surveys or from participants’medical reports if they were available in the system. Data from all sources were entered into a metafile in a spreadsheet (Excel; Microsoft Inc). Figure 3. Examples of keypoint poses (white) inferred for various exercises by the Pose Estimation Model. Figure 3. Examples of keypoint poses (white) inferred for various exercises by the Pose Estimation Model. Overview Kaia Health trained this model using a proprietary image data set that consisted of data from people with a variety of characteristics (body shape, height, skin color, movement limitations, etc) exercising in front of their mobile device, with a wide variety of exercise movements and environmental conditions such as varying lighting and background to make the model robust. Each image in the data set had been manually labeled according to a taxonomy designed to best capture the human body in physiotherapeutic exercises. J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 4 (page number not for citation purposes) https://www.jmir.org/2021/7/e26658 Study Endpoints The primary endpoint was overall agreement between physiotherapists’and Motion Coach’evaluations during exercise execution. For each segment, there was a dichotomous outcome (correction recommended or not). Statistical Analysis Continuous data (age, weight, height, and BMI) are described using means and standard deviations; discrete data (gender, location of osteoarthritis, WOMAC score) are described using absolute and relative numbers. Motion Coach–physiotherapist 1, Motion Coach–physiotherapist 2, Motion Coach–both physiotherapists, and physiotherapist 1–physiotherapist 2 interrater reliabilities (Cohen κ and percent agreement) were compared using z scores (α=5%). To assess whether demographic variables had any significant effect on the interrater agreement between Motion Coach and physiotherapists, subgroups for age (70 years or under, older than 70 years), gender (male, female), and BMI (30 kg/m2 or under, greater than 30 kg/m2) were formed and compared. We also assessed interrater agreement by exercise. Interrater agreement was categorized according to Cohen κ values as suggested by Landis and Koch [21]: κ < 0.00, poor agreement; κ=0.00-0.20, slight agreement; κ=0.21-0.40, fair agreement; κ=0.41-0.60, moderate agreement; κ=0.61-0.80, substantial agreement; κ=0.81-1.00, almost perfect agreement. All analyses were conducted with R software (version 4.0.2; R Foundation for Statistical Computing). Gender, age, diagnosis, location of osteoarthritis, height, weight, and the Western Ontario and McMaster Universities Arthritis Index (WOMAC) score were collected at baseline [20]. Each participant performed 6 exercises with a total of 23 rated segments (a set of repetitions of 10 for each exercise or 30 seconds of stable posing for static exercises). For each segment, each physiotherapist’s evaluation and Motion Coach’s evaluation (ie, whether correction was required or not) were collected after the participants completed each exercise. Furthermore, the overall form rating by physiotherapists was recorded on a 6-point Likert scale. Data were pooled for the primary analysis. Sample Size We calculated the sample size required for a noninferiority trial with dichotomous outcome (ie, agreement or disagreement, either between app and physiotherapists’ ratings or between the 2 physiotherapists). We used pilot data (app–physiotherapists mean ratio 0.83; physiotherapist 1–physiotherapist 2 mean ratio 0.845) from the first 16 participants of the study. We determined that 552 exercise segments would be required; therefore, given an assumption of 23 segments per participant, the number of required participants was 24 (noninferiority margin 0.05; α=5%; β=90%). A noninferiority margin of 0.05 was recently used in a comparable study [16] for evaluation of exercise correction with a digital tool. JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al Biebl et al Overview For audiovisual feedback, spatiotemporal constraints, which were configured in advance by medical, physiotherapeutic, or sport science–trained Kaia staff, are triggered based on movement; there was no need for reconfiguration on a per-user or per-session basis. While the system was in use, constraints were checked automatically in real time, and feedback was provided if any of the configured constraints were violated. If multiple constraints were violated, the prioritization mechanism selects the feedback based on risk of injury. For audiovisual feedback, spatiotemporal constraints, which were configured in advance by medical, physiotherapeutic, or sport science–trained Kaia staff, are triggered based on movement; there was no need for reconfiguration on a per-user or per-session basis. While the system was in use, constraints were checked automatically in real time, and feedback was provided if any of the configured constraints were violated. If multiple constraints were violated, the prioritization mechanism selects the feedback based on risk of injury. Physiotherapists’ evaluations were collected on a rating sheet for each participant. Data from the app were obtained by taking a screenshot of the report of corrections after the exercises had been executed. Baseline data were collected from participants using paper-based surveys or from participants’medical reports if they were available in the system. Data from all sources were entered into a metafile in a spreadsheet (Excel; Microsoft Inc). https://www.jmir.org/2021/7/e26658 J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 4 (page number not for citation purposes) XSL•FO RenderX J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 5 (page number not for citation purposes) Primary Analysis Mean agreement between the app and physiotherapists (percent agreement 0.828) was not inferior (margin 0.05; P<.001) to that between physiotherapist 1 and physiotherapist 2 (percent agreement 0.833). Participants The study population’s mean age was 67.6 (SD 8.98 years), and 20 out of the 24 participants (83%) were female. Participants (Table 2) had osteoarthritis of the knee (15/24, 62.5%), hip (6/24, 25%), or both knee and hip (3/24, 12.5%). The mean global WOMAC score was 64.9 (SD 43.3) with mean domain scores of 15.8 (SD 10.7) for Pain, 7.3 (SD 4.8) for Stiffness, and 41.9 (SD 30.5) for Physical Function. https://www.jmir.org/2021/7/e26658 XSL•FO RenderX Biebl et al JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al Table 2. Study population characteristics. Value (n=24) Characteristic Gender, n (%) 4 (17) Male 20 (83) Female Age (years) 67.6 (9.0) mean (SD) n (%) 12 (50) ≤70 12 (50 >70 years 69.5 (16.7) Weight (kg), mean (SD) 1.7 (0.1) Height (m), mean (SD) BMI (kg/m2) 24.9 (4.6) mean (SD) n (%) 20 ≤30 kg/m2 4 >30 kg/m2 Location of osteoarthritis, n (%) 6 (25) Hip 15 (63) Knee 3 (13) Both hip and knee WOMACa, n (%) 65 (43) Total score 16 (11) Pain 7 (5) Stiffness 42 (31) Physical function aWOMAC: Western Ontario and McMaster Universities Arthritis Index Biebl et al JOURNAL OF MEDICAL INTERNET RESEARCH Stiffness aWOMAC: Western Ontario and McMaster Universities Arthritis Index. percent agreement 0.815, 95% CI 0.780-0.847); Motion Coach and physiotherapist 2 (Cohen κ=0.626, 95% CI 0.474-0.697; percent agreement 0.841, 95% CI 0.807-0.870); and Motion Coach and when there was agreement between both physiotherapists (Cohen κ=0.726, 95% CI 0.654-0.798; percent agreement 0.893, 95% CI 0.862-0.920) (Figure 3). There was no detectable difference between either Motion Coach–physiotherapist 1 interrater reliability and physiotherapist 1–physiotherapist 2 interrater reliability (Cohen κ: P=.309; percent agreement: P=.46) or Motion Coach–physiotherapist interrater reliability and physiotherapist 1–physiotherapist 2 interrater reliability (Cohen κ: P=.71; percent agreement: P=.74; Figure 4). J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 6 (page number not for citation purposes) J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 7 (page number not for citation purposes) Comparison of Interrater Reliability Interrater reliability for the evaluations (Table 3) demonstrated moderate to substantial agreement between physiotherapist 1 and physiotherapist 2 (Cohen κ=0.607, 95% CI 0.535-0.679; percent agreement 0.833, 95% CI 0.800-0.864), Motion Coach and physiotherapist 1 (Cohen κ=0.551, 95% CI 0.474-0.628; J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 6 (page number not for citation purposes) https://www.jmir.org/2021/7/e26658 https://www.jmir.org/2021/7/e26658 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al Table 3. All interpretations of correct versus incorrect exercise execution. Both Individual All Assessment Disagreement Agreement Physiotherapist 2 Physiotherapist 1 Incorrect Correct Incorrect Correct Incorrect Correct 92 122 338 178 374 158 394 552 All, n Physiotherapist 1, n (%) N/A N/A N/A 56 (31.5) 338 (90.4) N/A N/Aa 394 (71.4) Correct N/A N/A N/A 122 (68.5) 36 (9.6) N/A N/A 158 (28.6) Incorrect App, n (%) 51 (55.4) 25 (20.5) 314 (92.9) 52 (29.2) 338 (90.4) 49 (31.0) 341 (86.5) 390 (70.7) Correct 41 (44.6) 97 (79.5) 24 (7.1) 126 (70.8) 36 (9.6) 109 (69.0) 53 (13.5) 162 (29.3) Incorrect aN/A: not applicable. Figure 4. Interrater reliability (percent agreement and Cohen κ, with upper and lower 95% confidence intervals). PT: physiotherapist. Subgroup Analysis No differences were found between app–physiotherapist interrater reliabilities and physiotherapist 1–physiotherapist 2 interrater reliability in any of the subgroups (Table 4 and Figure 5). Biebl et al JOURNAL OF MEDICAL INTERNET RESEARCH Table 3. All interpretations of correct versus incorrect exercise execution. Both Individual All Assessment Disagreement Agreement Physiotherapist 2 Physiotherapist 1 Incorrect Correct Incorrect Correct Incorrect Correct 92 122 338 178 374 158 394 552 All, n Physiotherapist 1, n (%) N/A N/A N/A 56 (31.5) 338 (90.4) N/A N/Aa 394 (71.4) Correct N/A N/A N/A 122 (68.5) 36 (9.6) N/A N/A 158 (28.6) Incorrect App, n (%) 51 (55.4) 25 (20.5) 314 (92.9) 52 (29.2) 338 (90.4) 49 (31.0) 341 (86.5) 390 (70.7) Correct 41 (44.6) 97 (79.5) 24 (7.1) 126 (70.8) 36 (9.6) 109 (69.0) 53 (13.5) 162 (29.3) Incorrect aN/A: not applicable. Figure 4. Interrater reliability (percent agreement and Cohen κ, with upper and lower 95% confidence intervals). PT: physiotherapist. Subgroup Analysis No differences were found between app–physiotherapist interrater reliabilities and physiotherapist 1–physiotherapist 2 interrater reliability in any of the subgroups (Table 4 and Figure 5). Biebl et al JOURNAL OF MEDICAL INTERNET RESEARCH Table 3. All interpretations of correct versus incorrect exercise execution. Comparison of Interrater Reliability Both Individual All Assessment Disagreement Agreement Physiotherapist 2 Physiotherapist 1 Incorrect Correct Incorrect Correct Incorrect Correct 92 122 338 178 374 158 394 552 All, n Physiotherapist 1, n (%) N/A N/A N/A 56 (31.5) 338 (90.4) N/A N/Aa 394 (71.4) Correct N/A N/A N/A 122 (68.5) 36 (9.6) N/A N/A 158 (28.6) Incorrect App, n (%) 51 (55.4) 25 (20.5) 314 (92.9) 52 (29.2) 338 (90.4) 49 (31.0) 341 (86.5) 390 (70.7) Correct 41 (44.6) 97 (79.5) 24 (7.1) 126 (70.8) 36 (9.6) 109 (69.0) 53 (13.5) 162 (29.3) Incorrect aN/A: not applicable. Table 3. All interpretations of correct versus incorrect exercise execution. Figure 4. Interrater reliability (percent agreement and Cohen κ, with upper and lower 95% confidence intervals). PT: physiotherapist. Subgroup Analysis No differences were found between app–physiotherapist interrater reliabilities and physiotherapist 1–physiotherapist 2 interrater reliability in any of the subgroups (Table 4 and Figure 5). Subgroup Analysis No differences were found between app–physiotherapist interrater reliabilities and physiotherapist 1 physiotherapist 2 interrater reliability in any of the subgroups (Table 4 and Figure 5). Subgroup Analysis No differences were found between app–physiotherapist interrater reliabilities and physiotherapist 1–physiotherapist 2 interrater reliability in any of the subgroups (Table 4 and Figure 5). Subgroup Analysis No differences were found between app–physiotherapist interrater reliabilities and physiotherapist 1–physiotherapist 2 J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 7 (page number not for citation purposes) https://www.jmir.org/2021/7/e26658 https://www.jmir.org/2021/7/e26658 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Table 4. Interrater agreement for age, gender, and BMI subgroups. aComparison of subrow with Physiotherapist 1 vs physiotherapist 2. J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 8 (page number not for citation purposes) Comparison of Interrater Reliability Percent agreement Cohen κ Raters P valuea Mean (95% CI) P valuea Mean (95% CI) All 0.833 (0.800-0.863) 0.607 (0.535-0.679) Physiotherapist 1 vs physiotherapist 2 .46 0.815 (0.780-0.847) .31 0.551 (0.474-0.628) App vs physiotherapist 1 .74 0.841 (0.807-0.870) .71 0.626 (0.556-0.697) App vs physiotherapist 2 0.893 (0.862-0.920) 0.726 (0.654-0.798) App vs agreement Gender Male 0.815 (0.721-0.889) 0.603 (0.436-0.770) Physiotherapist 1 vs physiotherapist 2 .34 0.761 (0.661-0.844) .26 0.456 (0.262-0.650) App vs physiotherapist 1 >.999 0.815 (0.721-0.889) >.999 0.603 (0.436-0.770) App vs physiotherapist 2 0.853 (0.753-0.924) 0.667 (0.486-0.847) App vs agreement Female 0.837 (0.800-0.870) 0.606 (0.526-0.686) Physiotherapist 1 vs physiotherapist 2 .65 0.826 (0.788-0.860) .55 0.571 (0.488-0.655) App vs physiotherapist 1 .71 0.846 (0.809-0.877) .67 0.630 (0.552-0.708) App vs physiotherapist 2 0.901 (0.867-0.929) 0.738 (0.660-0.816) App vs agreement BMI <30 kg/m2 0.848 (0.812-0.879) 0.635 (0.558-0.713) Physiotherapist 1 vs physiotherapist 2 .46 0.830 (0.793-0.864) .34 0.580 (0.497-0.663) App vs physiotherapist 1 .71 0.839 (0.802-0.872) .75 0.617 (0.539-0.696) App vs physiotherapist 2 0.895 (0.860-0.923) 0.725 (0.646-0.804) App vs agreement ≥30 kg/m2 0.761 (0.661-0.844) 0.473 (0.285-0.662) Physiotherapist 1 vs physiotherapist 2 .72 0.739 (0.637-0.825) .68 0.416 (0.220-0.612) App vs physiotherapist 1 .11 0.848 (0.758-0.914) .13 0.665 (0.504-0.825) App vs physiotherapist 2 0.886 (0.787-0.949) 0.728 (0.552-0.904) App vs agreement Age ≤70 years 0.846 (0.795-0.888) 0.578 (0.461-0.695) Physiotherapist 1 vs physiotherapist 2 .90 0.842 (0.791-0.885) .77 0.554 (0.432-0.676) App vs physiotherapist 1 .59 0.862 (0.813-0.902) .45 0.640 (0.531-0.748) App vs physiotherapist 2 0.916 (0.870-0.949) 0.743 (0.631-0.855) App vs agreement >70 years 0.823 (0.775-0.864) 0.615 (0.522-0.708) Physiotherapist 1 vs physiotherapist 2 .33 0.793 (0.742-0.837) .28 0.539 (0.438-0.640) App vs physiotherapist 1 >.999 0.823 (0.775-0.864) .95 0.611 (0.517-0.705) App vs physiotherapist 2 0.874 (0.826-0.913) 0.708 (0.613-0.803) App vs agreement aComparison of subrow with Physiotherapist 1 vs physiotherapist 2. Biebl et JOURNAL OF MEDICAL INTERNET RESEARCH Table 4. Interrater agreement for age, gender, and BMI subgroups. https://www.jmir.org/2021/7/e26658 XSL•FO RenderX Figure 5. Interrater reliability (percent agreement and Cohen κ, with upper and lower 95% confidence intervals) for (a) gender, (b) BMI, and (c) age subanalyses. PT: physiotherapist. Interrater Agreement in Different Exercises The analysis showed no detectable difference in the rates of interrater agreement in any of the exercises (Table 5 and Table 6). Table 5. Mean rating of exercise form by the physiotherapists, using a 6-point Likert scale, and interrater agreement comparisons between app–physiotherapist and physiotherapist 1–physiotherapist 2 percent agreement values for each exercise. Comparison of Interrater Reliability P value Percent agreement Rating, mean (SD) Exercisea Comparison 2c Comparison 1b App–agreement Physiotherapist 1–physiotherapist 2 App–physiotherapist 2 App–physiotherapist 1 .23 .32 0.881 (0.816-0.929) 0.851 (0.788-0.896) 0.845 (0.782-0.896) 0.804 (0.735-0.861) 2.8 (1.1) a .13 .86 0.841 (0.727-0.921) 0.875 (0.776-0.889) 0.806 (0.695-0.889) 0.792 (0.680-0.878) 3.4 (1.3) b .67 .67 0.852 (0.729-0.934) 0.750 (0.634-0.845) 0.750 (0.634-0.845) 0.778 (0.664-0.867) 4.3 (1.4) c >.999 >.999 0.938 (0.840-0.983) 0.889 (0.793-0.951) 0.889 (0.793-0.951) 0.889 (0.793-0.951) 4.5 (1.1) d .36 .48 0.946 (0.851-0.989) 0.778 (0.664-0.931) 0.861 (0.759-0.931) 0.833 (0.727-0.911) 4.5 (1.2) e .68 .12 0.912 (0.828-0.964) 0.833 (0.744-0.934) 0.875 (0.792-0.934) 0.812 (0.720-0.885) 4.8 (1.0) f aLetters correspond to those in Figure 1. bApp physiotherapist 1 vs physiotherapist 1 physiotherapist 2 Biebl et al JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al Figure 5. Interrater reliability (percent agreement and Cohen κ, with upper and lower 95% confidence intervals) for (a) gender, (b) BMI, and (c) age subanalyses. PT: physiotherapist. Figure 5. Interrater reliability (percent agreement and Cohen κ, with upper and lower 95% confidence intervals) for (a) gender, (b) BMI, and (c) age subanalyses. PT: physiotherapist. Figure 5. Interrater reliability (percent agreement and Cohen κ, with upper and lower 95% confidence intervals) for (a) gender, (b) BMI, and (c) age subanalyses. PT: physiotherapist. Interrater Agreement in Different Exercises The analysis showed no detectable difference in the rates of interrater agreement in any of the exercises (Table 5 and Table 6). Table 5. Mean rating of exercise form by the physiotherapists, using a 6-point Likert scale, and interrater agreement comparisons between app–physiotherapist and physiotherapist 1–physiotherapist 2 percent agreement values for each exercise. P value Percent agreement Rating, mean (SD) Exercisea Comparison 2c Comparison 1b App–agreement Physiotherapist 1–physiotherapist 2 App–physiotherapist 2 App–physiotherapist 1 .23 .32 0.881 (0.816-0.929) 0.851 (0.788-0.896) 0.845 (0.782-0.896) 0.804 (0.735-0.861) 2.8 (1.1) a .13 .86 0.841 (0.727-0.921) 0.875 (0.776-0.889) 0.806 (0.695-0.889) 0.792 (0.680-0.878) 3.4 (1.3) b .67 .67 0.852 (0.729-0.934) 0.750 (0.634-0.845) 0.750 (0.634-0.845) 0.778 (0.664-0.867) 4.3 (1.4) c >.999 >.999 0.938 (0.840-0.983) 0.889 (0.793-0.951) 0.889 (0.793-0.951) 0.889 (0.793-0.951) 4.5 (1.1) d .36 .48 0.946 (0.851-0.989) 0.778 (0.664-0.931) 0.861 (0.759-0.931) 0.833 (0.727-0.911) 4.5 (1.2) e .68 .12 0.912 (0.828-0.964) 0.833 (0.744-0.934) 0.875 (0.792-0.934) 0.812 (0.720-0.885) 4.8 (1.0) f aLetters correspond to those in Figure 1. bApp–physiotherapist 1 vs physiotherapist 1–physiotherapist 2. cApp–physiotherapist 2 vs physiotherapist 1–physiotherapist 2. Table 5. J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 9 (page number not for citation purposes) Discussion The purpose of this study was to compare interrater agreement of osteoarthritis knee and hip exercise assessments between Motion Coach (a novel digital tool) and trained physiotherapists; we hypothesized that assessment agreement for the Motion Coach app would not be inferior to that of physiotherapists. Our data support the hypothesis that Motion Coach is noninferior to physiotherapists in assessing whether exercise poses required correction. There was no difference between the interrater agreement of Motion Coach and physiotherapists and that among physiotherapists. This finding was also true in analyses of subgroups that consisted of men, women, participants 70 years or older, participants below 70 years, participants with BMI greater than 30 kg/m2, and participants with BMI less than 30 kg/m2 and in analyses by exercise. To the best of our knowledge, this is the first report comparing a digital software–based exercise feedback tool with conventional smartphone technology and physiotherapeutic exercise feedback for musculoskeletal conditions. Built-in smartphone inertia sensors are a viable option to deliver pose correction in rehabilitation without requiring specialized equipment or installations. Spina et al evaluated real-time smartphone motion sensor data processing as an option to assess pose in physical exercises by people with chronic obstructive pulmonary disease [25]. The system was able to provide feedback on pose and exercise feedback similar to the feedback of a trained therapist. The system required a holster to hold the smartphone and that was repositioned on the body depending on the exercise performed. While previous reports have addressed the general feasibility of exercise-related feedback using 2D RGB camera streams, the percent agreement of those systems without postprocessing limited their use [26,27]. In contrast, Motion Coach relies upon 2D camera stream postprocessing of using machine learning algorithms for valid real-time feedback for exercise correction. Previous studies [16,17] have used 3D sensors such as the Microsoft Kinect system to assess pose during exercise and give feedback to users if correction was needed. However, 3D-sensor systems are expensive and require extensive external hardware and a stationary television set, and thus have limited scalability in providing access to digital rehabilitation. Komatireddy [16] found no detectable difference in agreement between a software solution for Microsoft Kinect and a panel of physiotherapists for repetition count and the number of acceptable exercises. Comparison of Interrater Reliability Discussion Other digital rehabilitation tools for musculoskeletal pain use external inertial sensors attached to specific limbs or joints to rcise form by physiotherapists, using a 6-point Likert scale, and comparisons between app–physiotherapist and physiotherapist κ values for each exercise. aLetters correspond to those in Figure 1. Other digital rehabilitation tools for musculoskeletal pain use external inertial sensors attached to specific limbs or joints to detect exercise poses [22-24]. By nature, these systems are limited to detecting the poses of joints or body areas only where they are placed, and users must typically attach the hardware to their bodies themselves. Studies [14,15] have shown that these systems are generally capable of detecting exercise poses; however, these systems have not been systematically evaluated for their ability to provide feedback on pose during exercise execution. Comparison of Interrater Reliability However, 3D-sensor systems are expensive and require extensive external hardware and a stationary television set, and thus have limited scalability Other digital rehabilitation tools for musculoskeletal pain use external inertial sensors attached to specific limbs or joints to detect exercise poses [22-24]. By nature, these systems are limited to detecting the poses of joints or body areas only where they are placed, and users must typically attach the hardware to their bodies themselves. Studies [14,15] have shown that these systems are generally capable of detecting exercise poses; however, these systems have not been systematically evaluated for their ability to provide feedback on pose during exercise execution. Built-in smartphone inertia sensors are a viable option to deliver pose correction in rehabilitation without requiring specialized equipment or installations. Spina et al evaluated real-time smartphone motion sensor data processing as an option to assess pose in physical exercises by people with chronic obstructive pulmonary disease [25]. The system was able to provide feedback on pose and exercise feedback similar to the feedback of a trained therapist. The system required a holster to hold the smartphone and that was repositioned on the body depending on the exercise performed. While previous reports have addressed the general feasibility of exercise-related feedback using 2D RGB camera streams, the percent agreement of those systems without postprocessing limited their use [26,27]. In contrast, Motion Coach relies upon 2D camera stream postprocessing of using machine learning algorithms for valid real-time feedback for exercise correction. Table 6. Mean rating of exercise form by physiotherapists, using a 6-point Likert scale, and comparisons between app–physiotherapist and physiotherapist 1–physiotherapist 2 Cohen κ values for each exercise. P value Cohen κ Rating, mean (SD) Exercisea Comparison 2c Comparison 1b App–agreement Physiotherapist 1–physiotherapist 2 App–physiotherapist 2 App–physiotherapist 1 .20 .20 .707 (0.396-0.673) 0.656 (0.396-0.673) 0.655 (0.396-0.673) 0.534 (0.396-0.673) 2.8 (1.1) a .17 .85 .679 (0.391-0.766) 0.749 (0.391-0.766) 0.605 (0.391-0.766) 0.579 (0.391-0.766) 3.4 (1.3) b .81 .68 .596 (0.242-0.687) 0.425 (0.242-0.687) 0.397 (0.242-0.687) 0.464 (0.242-0.687) 4.3 (1.4) c .81 .92 .817 (0.476-0.884) 0.714 (0.476-0.884) 0.695 (0.476-0.884) 0.680 (0.476-0.884) 4.5 (1.1) d .44 .55 .867 (0.393-0.801) 0.478 (0.393-0.801) 0.681 (0.393-0.801) 0.597 (0.393-0.801) 4.5 (1.2) e .91 .17 .616 (0.218-0.651) 0.453 (0.218-0.651) 0.635 (0.218-0.651) 0.435 (0.218-0.651) 4.8 (1.0) f aLetters correspond to those in Figure 1. bApp–physiotherapist 1 vs physiotherapist 1–physiotherapist 2. cApp–physiotherapist 2 vs physiotherapist 1–physiotherapist 2. J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 10 (page number not for citation purposes) https://www.jmir.org/2021/7/e26658 Comparison of Interrater Reliability Mean rating of exercise form by the physiotherapists, using a 6-point Likert scale, and interrater agreement comparisons between app–physiotherapist and physiotherapist 1–physiotherapist 2 percent agreement values for each exercise. J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 9 (page number not for citation purposes) https://www.jmir.org/2021/7/e26658 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al Table 6. Mean rating of exercise form by physiotherapists, using a 6-point Likert scale, and comparisons between app–physiotherapist and physiotherapist 1–physiotherapist 2 Cohen κ values for each exercise. P value Cohen κ Rating, mean (SD) Exercisea Comparison 2c Comparison 1b App–agreement Physiotherapist 1–physiotherapist 2 App–physiotherapist 2 App–physiotherapist 1 .20 .20 .707 (0.396-0.673) 0.656 (0.396-0.673) 0.655 (0.396-0.673) 0.534 (0.396-0.673) 2.8 (1.1) a .17 .85 .679 (0.391-0.766) 0.749 (0.391-0.766) 0.605 (0.391-0.766) 0.579 (0.391-0.766) 3.4 (1.3) b .81 .68 .596 (0.242-0.687) 0.425 (0.242-0.687) 0.397 (0.242-0.687) 0.464 (0.242-0.687) 4.3 (1.4) c .81 .92 .817 (0.476-0.884) 0.714 (0.476-0.884) 0.695 (0.476-0.884) 0.680 (0.476-0.884) 4.5 (1.1) d .44 .55 .867 (0.393-0.801) 0.478 (0.393-0.801) 0.681 (0.393-0.801) 0.597 (0.393-0.801) 4.5 (1.2) e .91 .17 .616 (0.218-0.651) 0.453 (0.218-0.651) 0.635 (0.218-0.651) 0.435 (0.218-0.651) 4.8 (1.0) f aLetters correspond to those in Figure 1. bApp–physiotherapist 1 vs physiotherapist 1–physiotherapist 2. cApp–physiotherapist 2 vs physiotherapist 1–physiotherapist 2. Discussion The purpose of this study was to compare interrater agreement of osteoarthritis knee and hip exercise assessments between Motion Coach (a novel digital tool) and trained physiotherapists; we hypothesized that assessment agreement for the Motion Coach app would not be inferior to that of physiotherapists. Our data support the hypothesis that Motion Coach is noninferior to physiotherapists in assessing whether exercise poses required correction. There was no difference between the interrater agreement of Motion Coach and physiotherapists and that among physiotherapists. This finding was also true in analyses of subgroups that consisted of men, women, participants 70 years or older, participants below 70 years, participants with BMI greater than 30 kg/m2, and participants with BMI less than 30 kg/m2 and in analyses by exercise. To the best of our knowledge, this is the first report comparing a digital software–based exercise feedback tool with conventional smartphone technology and physiotherapeutic exercise feedback for musculoskeletal conditions. Previous studies [16,17] have used 3D sensors such as the Microsoft Kinect system to assess pose during exercise and give feedback to users if correction was needed. Discussion Whatman et al [29] investigated interrater agreement for lower extremity exercises in a panel of physiotherapists (segment-specific and overall agreement) with ordinal and dichotomous outcomes; interrater agreement was generally fair to good and increased with experience of the rater. The interrater agreement observed in our study, among the physiotherapists and also between the physiotherapists and Motion Coach, was high compared to those in previous studies [28,29]. This finding can be explained by the high level of experience of the physiotherapists and training of the physiotherapists on evaluation criteria prior to patient enrollment. Compared to other approaches requiring specialized hardware, the degree of agreement between both physiotherapists and Motion Coach remains high; a similar study [30] using data from the Kinect version 2 Skeleton Tracking system to assess rehabilitation exercises in 19 people with musculoskeletal and neurological limitations showed a limited correlation (r=0.60, P<.01 for the clinical subgroup) between expert’s clinical judgement and the results of various models based on sensor data. agreement during 2 exercises performed by healthy volunteers for the lower extremity with 2 distinct methods (overall rating and investigation of deviation from the neutral plane during exercise) in a panel of 3 physiotherapists and found agreement better than chance but no high levels of agreement between physiotherapists. Whatman et al [29] investigated interrater agreement for lower extremity exercises in a panel of physiotherapists (segment-specific and overall agreement) with ordinal and dichotomous outcomes; interrater agreement was generally fair to good and increased with experience of the rater. The interrater agreement observed in our study, among the physiotherapists and also between the physiotherapists and Motion Coach, was high compared to those in previous studies [28,29]. This finding can be explained by the high level of experience of the physiotherapists and training of the physiotherapists on evaluation criteria prior to patient enrollment. Compared to other approaches requiring specialized hardware, the degree of agreement between both physiotherapists and Motion Coach remains high; a similar study [30] using data from the Kinect version 2 Skeleton Tracking system to assess rehabilitation exercises in 19 people with musculoskeletal and neurological limitations showed a limited correlation (r=0.60, P<.01 for the clinical subgroup) between expert’s clinical judgement and the results of various models based on sensor data. Discussion The interrater agreement for suggesting corrections during therapeutic exercises between both physiotherapists and Motion Coach was moderate to substantial and did not differ between physiotherapists themselves and physiotherapists and Motion Coach. This finding was valid for all investigated exercises and subgroup analysis. These findings validate the ability of Motion Coach to detect form during exercise and provide audiovisual feedback to users with preexisting musculoskeletal conditions. Acknowledgments The study was part of a project (TELE-CORRECT, grant number MED-1804-0003) funded by the Bavarian Ministry of Economic Affairs, Regional Development and Energy. The authors would like to thank Luca Sonntag at Kaia Health for providing and editing images used in the manuscript. SH, PKB, and MS are employees of Kaia Health and receive salary and stock options. SH, PKB, and MS are employees of Kaia Health and receive salary and stock options. Discussion Wochartz et al [17] evaluated agreement with regard to joint angles and positions of the lower limb between a Microsoft Kinect based-system and a 3D camera-based motion system but did not evaluate its capacity to trigger corrections during therapeutic exercises; they concluded that the validity of the Kinect system to detect pose without postprocessing was restricted. To the best of our knowledge, this study is the first to evaluate the potential of a technology (Motion Coach) to trigger suitable corrections of therapeutic exercises in musculoskeletal pain rehabilitation, with the findings suggesting that Motion Coach technology triggers valid corrections as compared to trained physiotherapists. Motion Coach is a software only solution operating on off-the-shelf smartphones, without any need for additional hardware, which makes this digital therapeutic solution accessible to a broad patient population. The interrater reliability of trained physiotherapists assessments of pose during lower extremity exercises for the has been investigated: Chmielewski et al [28] investigated interrater https://www.jmir.org/2021/7/e26658 https://www.jmir.org/2021/7/e26658 J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 10 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX Biebl et al JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al The study had several limitations. First, the pool of raters was small with n=2, and a third rater was not used (in cases of disagreement between the 2 raters). In addition, the sample was heterogeneous in terms of gender distribution and localization of osteoarthritis, limiting the generalizability of the results. Other limitations arise from the fact that the assessment of pose during therapeutic exercise execution is not standardized, and thus, in this study as in comparable previous studies [28,29], no well-established standard measurement could be used to quantify exercise execution. Furthermore, dichotomous assessment of acceptable exercise is only one of several measures used in prior studies to assess form during exercise. Future studies evaluating Motion Coach will need to use more diverse outcome measures of form during exercise, for example calculations with a musculoskeletal human model. agreement during 2 exercises performed by healthy volunteers for the lower extremity with 2 distinct methods (overall rating and investigation of deviation from the neutral plane during exercise) in a panel of 3 physiotherapists and found agreement better than chance but no high levels of agreement between physiotherapists. References 1. GBD 2016 DALYsHALE Collaborators. 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[doi: 10.1016/j.ptsp.2011.07.001] [Medline: 22498149] J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 12 (page number not for citation purposes) https://www.jmir.org/2021/7/e26658 XSL•FO RenderX XSL•FO RenderX J Med Internet Res 2021 \| vol. 23 \| iss. 7 \| e26658 \| p. 13 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH Biebl et al WOMAC: Western Ontario and McMaster Universities Arthritis Index Edited by R Kukafka; submitted 20.12.20; peer-reviewed by AV Das, S Kriventsov; comments to author 19.01.2 received 03.02.21; accepted 19.04.21; published 13.07.21 ©Johanna Theresia Biebl, Marzena Rykala, Maximilian Strobel, Pawandeep Kaur Bollinger, Bernhard Ulm, Eduard Kraft, Stephan Huber, Andreas Lorenz. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 13.07.2021. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. https://www.jmir.org/2021/7/e26658 https://www.jmir.org/2021/7/e26658 XSL•FO RenderX
https://openalex.org/W2072127477	https://europepmc.org/articles/pmc3977101?pdf=render	English	null	Hamming Distance Method with Subjective and Objective Weights for Personnel Selection	The scientific world journal/TheScientificWorldjournal	2,014	cc-by	7,958	Correspondence should be addressed to M. Z. Ahmad; mzaini@unimap.edu.my Correspondence should be addressed to M. Z. Ahmad; mzaini@unimap.edu.my Received 31 August 2013; Accepted 11 November 2013; Published 17 March 2014 Academic Editors: R.-M. Chen and H. Wu Copyright © 2014 R. Md Saad et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Multicriteria decision making (MCDM) is one of the methods that popularly has been used in solving personnel selection problem. Alternatives, criteria, and weights are some of the fundamental aspects in MCDM that need to be defined clearly in order to achieve a good result. Apart from these aspects, fuzzy data has to take into consideration that it may arise from unobtainable and incomplete information. In this paper, we propose a new approach for personnel selection problem. The proposed approach is based on Hamming distance method with subjective and objective weights (HDMSOW’s). In case of vagueness situation, fuzzy set theory is then incorporated onto the HDMSOW’s. To determine the objective weight for each attribute, the fuzzy Shannon’s entropy is considered. While for the subjective weight, it is aggregated into a comparable scale. A numerical example is presented to illustrate the HDMSOW’s. Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 865495, 9 pages http://dx.doi.org/10.1155/2014/865495 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 865495, 9 pages http://dx.doi.org/10.1155/2014/865495 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 865495, 9 pages http://dx.doi.org/10.1155/2014/865495 R. Md Saad,1 M. Z. Ahmad,1 M. S. Abu,1 and M. S. Jusoh2 1 Institute of Engineering Mathematics, Universiti Malaysia Perlis, Pauh Putra Main Campus, 02600 Arau, Perlis, Malaysia 2 School of Business Innovation and Technopreneurship, Universiti Malaysia Perlis, Jalan Kangar-Alor Setar, 01000 Kangar, Perlis, Malaysia R. Md Saad,1 M. Z. Ahmad,1 M. S. Abu,1 and M. S. Jusoh2 1 Institute of Engineering Mathematics, Universiti Malaysia Perlis, Pauh Putra Main Campus, 02600 Arau, Perlis, Malaysia 2 School of Business Innovation and Technopreneurship, Universiti Malaysia Perlis, Jalan Kangar-Alor Setar, 01000 Kangar, Perlis, Malaysia 2. Preliminaries A fuzzy set 𝐴in 𝑋is defined as a set of ordered pairs (see [20]): 𝐴= {⟨𝑥, 𝜇𝐴(𝑥)⟩: 𝑥∈𝑋} , (1) (1) where 𝑋is denoted as a universe of discourse and 𝜇𝐴(𝑥) is the membership function of 𝐴defined as 𝜇𝐴: 𝑋󳨀→[0, 1] . (2) (2) A triangular fuzzy number is specified by three parameters and can be defined as triplet 𝐴= (𝑎1, 𝑎2, 𝑎3), where 𝑎1 < 𝑎2 < 𝑎3 with the 𝑥= 𝑎2 as the core of the triangle. Its membership function can be represented as [21] Literally, evaluation of certain criteria or attributes to select an appropriate alternative for specified position could become tremendous and challenging task for the decision makers. It is because some of the criteria such as leadership, personality, and creativity are referred to as qualitative criteria in which exhibits imprecise and vagueness data. In general, this uncertainty and subjective scene that occurs during the evaluation of the alternatives based on respective criteria and criteria weight may come from various sources including unquantifiable information, incomplete information, unob- tainable information, and partial ignorance [18]. For this situ- ation, commonly classical MCDM will be put aside since the alternatives rating and criteria weights for classical MCDM are usually measured in crisp numbers. Therefore, one of the best resorts to solve this problem is by applying fuzzy set theory. The fuzzy set theory is known for its flexibility in handling imprecise and uncertainty in human judgments. Bellman and Zadeh [19] had introduced the use of fuzzy set theory in MCDM and it proved to be an effective approach in dealing with uncertainty in human decision making process. Since then, it had become an important tool in constructing a decision making framework that incorporates subjective judgments that entails in the personnel selection process.h 𝜇𝐴(𝑥) = { { { { { { { { { { { { { { { { { { { 0, 𝑥< 𝑎1, (𝑥−𝑎1) (𝑎2 −𝑎1), 𝑎1 ≤𝑥≤𝑎2, (𝑎3 −𝑥) (𝑎3 −𝑎2), 𝑎2 ≤𝑥≤𝑎3, 0, 𝑥> 𝑎3. (3) (3) The 𝛼-cuts of this fuzzy number 𝐴are denoted by The 𝛼-cuts of this fuzzy number 𝐴are denoted by [𝐴]𝛼= [𝑎1 + 𝛼(𝑎2 −𝑎1) , 𝑎3 −𝛼(𝑎3 −𝑎2)] , 𝛼∈(0, 1] . (4) [𝐴]𝛼= [𝑎1 + 𝛼(𝑎2 −𝑎1) , 𝑎3 −𝛼(𝑎3 −𝑎2)] , 𝛼∈(0, 1] . 1. Introduction during the selection process. Some decision makers try to utilize rigorous and costly selection procedure and some even used the traditional method which depends on only information stated on the application forms that turn out to be quickest and inexpensive methods [1]. However, these methods actually never bring satisfaction and their final results are sometimes deniable. Thus, when multicriteria decision making (MCDM) was introduced in the early 1970’s, it had become one of favorable and important methods in this area. Some of the decision makers took a chance and grabed this opportunity to apply this method in solving personnel selection problem [3]. MCDM is known for its capabilities in evaluating, electing, or ranking a finite set of available alternatives with respect to multiple and conflicting criteria [4]. A number of methods and theories had been introduced and extended based on the utilization of this approach and the continuing study of this field had extended in a fixed rate. Preference Ranking Organization Method for Enrich- ment Evaluation (PROMETHEE) [5], linear programming techniques [6], Analytic Hierarchy Process (AHP) [7], Simple Additive Weighting (SAW) [8], and Technique for Order The rapid growth in globalization had created an intense competition between modern firms in global markets. These situations had urged the organization and firms to estab- lish a comprehensive procedure during personnel selection process. The personnel selection can be defined as a process of selecting the individuals who match the requirement and qualification to perform a particular job in an excellent way [1]. The main objective of this process is to assess the diversity among the alternatives that could pave a way of predicting the future performance [2]. Knowing the fact that personnel selection is not an easy task to be solved has awakened the conscience of decision makers to make decisive action to solve. The decision makers have to consider all aspects that are needed in this process. Hence, some of the decision makers try to solve this problem by using any kind of methods that are available and suitable for them to use. 1. Introduction Despite restructuring and reorganizing the personnel selection process, some of the firms had performed a so- called “strategic decision” to choose the best candidate 2 The Scientific World Journal 2 Preference by Similarity to Ideal Solution or TOPSIS [9] are some of the numerous examples on MCDM methods that particularly have been used by the decision makers.i changes in ranking of the alternatives when different values of 𝛼are used. The remaining of this paper is organized as follows. The next section, we briefly explain the preliminary concerning fuzzy set and Hamming distance. Section 3 will briefly explain about the Hamming distance method and subjective and objective weights. In Section 4, we propose a new algorithm for personnel selection problem. The new algorithm is called HDMSOW’s. Section 5 validates the HDMSOW’s by conducting a numerical example. The last section concludes this paper. Distance measure can be identified as one of the MCDM approaches that can be used in personnel selection process. This approach holds an important key to solve many prob- lems related to biology, science, social, and technology due to its capability of constructing some related distance measures, notably similarity, and proximity which always become a norm in various problems [10]. In recent years, the study of this method has been rapidly growing in which it resulted in proposing and improving the previous distance measure methods. Some of the well-known distance measure methods are Hamming, Euclidean, Hausdorff, and Minkowski meth- ods. Based on the existing literatures, Hamming distance is one of the methods that can be used in personnel selection process [11–13]. This method was proposed by Hamming [14] in 1950 to count the number of flipping bits in a fixed-length binary word as an estimate of error used in telecommunica- tion. Hamming distance is known for its ability in calculating the difference between two sets or elements. For example, the distance between interval-valued fuzzy sets. Consequently, apart from the decision making problem, it also has been applied in various fields such as communication [15], iris recognition [16], and engineering [17]. 2. Preliminaries Plus it is a human nature to have diverse opinion in evaluating process. Thus it is undeniable that the criteria weight plays an important role in MCDM problem as it depicted the relative weightiness of the criteria must be assigned [3]. Alternatively, numerous approaches has been generalized and introduced to solve this problem. These methods can be categorized into two groups which are subjective and objective weights.h The normalized Hamming distance for two interval-valued fuzzy numbers 𝐴and 𝐵, whose membership functions are as follows: 𝜇𝐴(𝑥𝑗) = [𝑎𝐿 𝑥𝑗, 𝑎𝑈 𝑥𝑗] , 𝜇𝐵(𝑥𝑗) = [𝑏𝐿 𝑥𝑗, 𝑏𝑈 𝑥𝑗] , 𝑗= 1, 2, . . . , 𝑛. (9) (9) 𝑗= 1, 2, . . . , 𝑛. is defined as 𝑑NHD (𝐴, 𝐵) = 1 2𝑛( 𝑛 ∑ 𝑗=1 (󵄨󵄨󵄨󵄨󵄨󵄨𝑎𝐿 𝑥𝑗−𝑏𝐿 𝑥𝑗 󵄨󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨󵄨𝑎𝑈 𝑥𝑗−𝑏𝑈 𝑥𝑗 󵄨󵄨󵄨󵄨󵄨󵄨)) . (10) (10) Definition 2 (see [25]). The weighted Hamming distance of dimension 𝑛is a mapping 𝑑WHD : [0, 1]𝑛× [0, 1]𝑛→[0, 1] that associated with weighting vector 𝑊of dimension 𝑛with 𝑊= ∑𝑛 𝑗=1, 𝑤𝑗= 1, and 𝑤𝑗∈[0, 1]. Then the weighted Hamming distance is defined as g p j j g The subjective weight are determined solely based on the preference of the decision makers [27, 28]. These evaluations are basically based on experience, perception, and knowledge [29]. In a general view, it is a process of assigning subjective preferences to the criteria [29]. AHP method, eigenvector method, and weighted least square method can be used to calculate this approach. Beside, objective weight measured the weight with the use of mathematical models such as entropy method [30] and multiple objective programming [31]. This approach solves without any consideration from the decision makers preference. The use of objective weight can overcome some of the limitations in subjective weight such as inconsistency problem in subjective weight. Furthermore, it is useful when the reliable subjective weight is not available [29]. 𝑑WHD (𝐴, 𝐵) = 𝑛 ∑ 𝑗=1 𝑤𝑗 󵄨󵄨󵄨󵄨󵄨𝜇𝐴(𝑥𝑗) −𝜇𝐵(𝑥𝑗)󵄨󵄨󵄨󵄨󵄨. (11) (11) According to [12] the weighted Hamming distance can be the normalized Hamming distance if 𝑤𝑗= 1/𝑛for 𝑗= 1, 2, . . . , 𝑛. 2. Preliminaries (4) ] (4) An interval-valued fuzzy set 𝐴in universe discourse 𝑋is denoted by (see [22, 23]) 𝐴= {(𝑥, [𝜇𝐿 𝐴(𝑥) , 𝜇𝑈 𝐴(𝑥)]) \| 𝑥∈𝑋} , (5) (5) where 𝜇𝐿 𝐴(𝑥), 𝜇𝑈 𝐴(𝑥) : 𝑋→[0, 1], 𝜇𝐿 𝐴(𝑥) is lower bound, and 𝜇𝑈 𝐴(𝑥) is upper bound of membership.h The multiplication of two interval-valued fuzzy numbers, 𝐴= [𝑎𝐿, 𝑎𝑈] and 𝐵= [𝑏𝐿, 𝑏𝑈], can be defined as (see [21]) The main objective of this paper is to propose an approach to solve personnel selection process by using Hamming distance method. Inspired by algorithm proposed by Can´os et al. [11], we extend and improve Can´os’s algorithm by adding weight in the classical Hamming distance. In our proposed method we suggest two types of weight which are subjective and objective weights. The linguistic terms correspondence to triangular fuzzy numbers are used to evaluate the performance rating values as well as the weight of the criteria, in which later will be expressed into interval valued fuzzy numbers. In this approach, we also identify the 𝐴⋅𝐵= [𝑎𝐿, 𝑎𝑈] ⋅[𝑏𝐿, 𝑏𝑈] = [𝑐𝐿, 𝑐𝑈] , (6) (6) where 𝑐𝐿= min {𝑎𝐿𝑏𝐿, 𝑎𝐿𝑏𝑈, 𝑎𝑈𝑏𝐿, 𝑎𝑈𝑏𝑈} , 𝑐𝑈= max {𝑎𝐿𝑏𝐿, 𝑎𝐿𝑏𝑈, 𝑎𝑈𝑏𝐿, 𝑎𝑈𝑏𝑈} . (7) (7) Hamming distance methods to be used in this paper are presented as follows. 3 The Scientific World Journal Definition 1 (see [24]). Given two fuzzy subsets of 𝐴and 𝐵 with a reference set, 𝑋= {𝑥1, 𝑥2, . . . , 𝑥𝑛} and memberships function 𝜇𝐴and 𝜇𝐵.hi be selected. However, when the distance values between the alternatives are the same, the decision makers will face a problem in ranking them. Thus, with the help of weights, it will help decision makers to distinguish between the criteria that valued the most for the specified job than the other criteria. 𝜇𝐴 𝜇𝐵 Then the Hamming distance is defined as 𝑑(𝐴, 𝐵) = 𝑛 ∑ 𝑗=1 󵄨󵄨󵄨󵄨󵄨𝜇𝐴(𝑥𝑗) −𝜇𝐵(𝑥𝑗)󵄨󵄨󵄨󵄨󵄨. (8) (8) 3.2. Subjective and Objective Weights. The decision makers are genuinely aware that they cannot assume that all criteria are equally important as it holds its own meaning and neediness, especially when its focus is only to one subject or position. For example, when recruiting the appropriate applicant for position credit officer, the criteria that might be valued most are experienced in credit analysis and personality assessment. Generally, the other criteria are also valuable but they are not as important as these two criteria. (iii) The interval decision matrix for criteria weight (iii) The interval decision matrix for criteria weight Step 1 (construct a decision matrix for ideal alternative). The decision matrix for ideal alternative is given as follows: 𝑊𝛼= [[[[ [ [(𝑤11) 𝐿 𝛼, (𝑤11) 𝑈 𝛼] [(𝑤12) 𝐿 𝛼, (𝑤12) 𝑈 𝛼] ⋅⋅⋅ [(𝑤1𝑛) 𝐿 𝛼, (𝑤1𝑛) 𝑈 𝛼] [(𝑤21) 𝐿 𝛼, (𝑤21) 𝑈 𝛼] [(𝑤22) 𝐿 𝛼, (𝑤22) 𝑈 𝛼] ⋅⋅⋅ [(𝑤2𝑛) 𝐿 𝛼, (𝑤2𝑛) 𝑈 𝛼] ... ... ⋅⋅⋅ ... [(𝑤𝑚1) 𝐿 𝛼, (𝑤𝑚1) 𝑈 𝛼] [(𝑤𝑚2) 𝐿 𝛼, (𝑤𝑚2) 𝑈 𝛼] ⋅⋅⋅[(𝑤𝑚𝑛) 𝐿 𝛼, (𝑤𝑚𝑛) 𝑈 𝛼] ]]]] ] , (17) 𝐼= [V1, V2, . . . , V𝑛] . (12) (12) The ideal alternative matrix represents the optimum values of 𝑛selection criteria 𝐶= {𝐶1, 𝐶2, . . . , 𝐶𝑛} that the alternatives should achieve. These values are set up by decision makers. The ideal alternative matrix represents the optimum values of 𝑛selection criteria 𝐶= {𝐶1, 𝐶2, . . . , 𝐶𝑛} that the alternatives should achieve. These values are set up by decision makers. where 0 ≤𝛼≤1. The value of 𝛼represents the degree of confidences in the decision makers’ assessment with respect to ideal alternative, alternatives rating, and criteria weights. Step 2 (construct a decision matrix for alternatives). The decision matrix for performance alternatives is given as follows: Step 5 (calculating of criteria weight). The criteria weight of 𝑛selection criteria 𝐶= {𝐶1, 𝐶2, . . . , 𝐶𝑛} evaluated by the decision makers will be calculated using two methods, which are subjective and objective weights. 𝐷= 𝐶1 𝐶2 ⋅⋅⋅𝐶𝑛 𝐴1 𝐴2 ... 𝐴𝑚 [[[[ [ 𝑥11 𝑥12 ⋅⋅⋅𝑥1𝑛 𝑥21 𝑥22 ⋅⋅⋅𝑥2𝑛 ... ... ⋅⋅⋅ ... 𝑥𝑚1 𝑥𝑚2 ⋅⋅⋅𝑥𝑚𝑛 ]]]] ] . (13) (13) (a) Subjective weight. The subjective weight of 𝑛selection criteria 𝐶= {𝐶1, 𝐶2, . . . , 𝐶𝑛} may be considered as the average weights [9] and its calculation is [9, 28] (a) Subjective weight. The subjective weight of 𝑛selection criteria 𝐶= {𝐶1, 𝐶2, . . . , 𝐶𝑛} may be considered as the average weights [9] and its calculation is [9, 28] where 𝑥𝑖𝑗represent the linguistic assessment on the utility ratings of alternative 𝐴𝑖(𝑖= 1, 2, . . . , 𝑚) with respect to 𝑛selection criteria 𝐶= {𝐶1, 𝐶2, . . . , 𝐶𝑛} evaluated by the decision makers. 𝑤𝑗= 1 𝑚( 𝑚 ∑ 𝑖=1 𝑤𝑖𝑗) , 𝑖= 1, 2, ..., 𝑚; 𝑗= 1, 2, . . . , 𝑛. 3. Hamming Distance Method and Subjective and Objective Weights 4 The Scientific World Journal The Scientific World Journal 4. Hamming Distance Method with Subjective and Objective Weights The weighting matrix represents the relative importance of 𝑛selection criteria 𝐶𝑗(𝑗= 1, 2, . . . , 𝑛) given by the decision makers. In this section, the description and algorithm for the HDM- SOWs is constructed. To our best knowledge, the study of using a weighted Hamming distance method in solving personnel selection problem has rarely been done. Merig´o and Gil-Lafuente [12] had presented a study involving the use of weighted Hamming distance method, integrated with Ordered Weighted Averaging (OWA) but without the use of fuzzy numbers. Hence, we would like to expand the use of weighted Hamming distance in personnel selection by using fuzzy data and we propose two types of weights which are subjective and objective weights. The elements of this HDMSOWs can be presented in the following descriptions. Step 4 (construct an interval-valued fuzzy number). By using 𝛼-cut of triangular fuzzy number, the interval performance matrix for alternatives, ideal alternatives, and criteria weight are derived as follows, respectively. (i) The interval decision matrix for the ideal alternative: 𝐼𝛼= [[(V1)𝐿 𝛼, (V1)𝑈 𝛼] , [(V2)𝐿 𝛼, (V2)𝑈 𝛼] , . . . , [(V𝑛)𝐿 𝛼, (V𝑛)𝑈 𝛼]] . (15) 𝐼𝛼= [[(V1)𝐿 𝛼, (V1)𝑈 𝛼] , [(V2)𝐿 𝛼, (V2)𝑈 𝛼] , . . . , [(V𝑛)𝐿 𝛼, (V𝑛)𝑈 𝛼]] . (15) ] (15) 3. Hamming Distance Method and Subjective and Objective Weights 3.1. Hamming Distance Method. Hamming distance is one of the distance measures that can be applied in personnel selection process. This is due to its ability in calculating the distance between ideal alternative and alternative. The ideal alternative is a virtual alternative in which the criteria values are expressed as close as possible to ideal values which is rationale for human thinking to achieve. There are several methods that focus on identifying and measuring the ideal alternative. However this measurement is beyond our scope of research. In this paper, the evaluation on the ideal alternative is made based on assumption of the optimum value of each criterion that alternatives should achieve for the specified job. We also disregard the usage of maximum value, for example, (1, 1, 1) in case of the triangular fuzzy number of all criteria evaluations. Rationally, it is hard for the alternatives to achieve a perfect score for some criteria especially when the evaluation of the criteria itself is made from human based judgment that mostly in subjective terms could be varied from one person to others. The ranking of alternatives is made through the comparison between the alternatives and the ideal alternative [26] such that, the alternatives with the minimum distance values are likely to One of the objective weighting measure that vastly has been used in MCDM field is Shannon’s entropy concept [32]. Shannon’s entropy concept is a general measure of uncertainty in information formulated in terms of probability theory [30]. This concept is appropriate for calculating the relative contrast intensities of criteria to represent the average intrinsic information transmitted to the decision maker [33]. It began when Shannon first introduced the application of entropy in communication theory and since then, he had contributed the most fundamental definition of the entropy measure in the information theory [34]. This concept had been applied in wide range area exemplified mathematics [35], spectral analysis [36], and economics [37]. Entropy weight is a parameter that describes how much diverse alternatives approach one another with respect to a certain criteria [3, 28]. This concept is also, relatively known in the measurement of fuzziness [38]. Hence this method is suitable to be applied in our approach as we will deal with fuzzy data. Apart from that, the total weights for all criteria values will equal to one in which satisfy the condition that need in weighted Hamming distance method. (ii) The interval decision matrix for performance alterna- tives: (ii) The interval decision matrix for performance alterna- tives: p g p Let us assume that there is a set of 𝑚possible alternatives, 𝐴= {𝐴1, 𝐴2, . . . , 𝐴𝑚} to be evaluated based on a set of 𝑛 respective criteria, 𝐶= {𝐶1, 𝐶2, . . . , 𝐶𝑛}. These evaluations are done by a set of 𝑚decision makers, 𝐸= {𝐸1, 𝐸2, . . . , 𝐸𝑚} by using linguistic variables. To capture the linguistic terms, we use triangular fuzzy numbers. The linguistic variables are divided into two categories which are the evaluation on criteria weight and the evaluation on criteria. The given algorithm is unfolded as follows. 𝐷𝛼= [[[[ [ [(𝑥11) 𝐿 𝛼, (𝑥11) 𝑈 𝛼] [(𝑥12) 𝐿 𝛼, (𝑥12) 𝑈 𝛼] ⋅⋅⋅ [(𝑥1𝑛) 𝐿 𝛼, (𝑥1𝑛) 𝑈 𝛼] [(𝑥21) 𝐿 𝛼, (𝑥21) 𝑈 𝛼] [(𝑥22) 𝐿 𝛼, (𝑥22) 𝑈 𝛼] ⋅⋅⋅ [(𝑥2𝑛) 𝐿 𝛼, (𝑥2𝑛) 𝑈 𝛼] ... ... ⋅⋅⋅ ... [(𝑥𝑚1) 𝐿 𝛼, (𝑥𝑚1) 𝑈 𝛼] [(𝑥𝑚2) 𝐿 𝛼, (𝑥𝑚2) 𝑈 𝛼] ⋅⋅⋅[(𝑥𝑚𝑛) 𝐿 𝛼, (𝑥𝑚𝑛) 𝑈 𝛼] ]]]] ] . (16) ] (16) (iii) The interval decision matrix for criteria weight (18) (18) Step 3 (construct a decision matrix for weight (criteria importance)). The weighting matrix for criteria weight; 𝑤𝑖𝑗 evaluated by the decision makers, 𝐸𝑖(𝑖= 1, 2, . . . , 𝑚) is given as follows: (b) Objective weight. The interval valued fuzzy number is transformed into crisp number before using Shan- non’s entropy concept. The crisp value of interval weight is given by [39] 𝑊= 𝐸1 𝐸2 ... 𝐸𝑚 𝐶1 𝐶2 ⋅⋅⋅𝐶𝑛 [[[[ [ 𝑤11 𝑤12 ⋅⋅⋅𝑤1𝑛 𝑤21 𝑤22 ⋅⋅⋅𝑤2𝑛 ... ... ⋅⋅⋅ ... 𝑤𝑚1 𝑤𝑚2 ⋅⋅⋅𝑤𝑚𝑛 ]]]] ] . (14) The crisp value of interval weight is given by [39] 𝑤𝑖𝑗= (𝑤𝑙 𝑖𝑗+ 𝑤𝑢 𝑖𝑗) 2 . (19 Then Shannon’s entropy concept is used to obtain the weigh (14) 𝑤𝑖𝑗= (𝑤𝑙 𝑖𝑗+ 𝑤𝑢 𝑖𝑗) 2 . (19) (19) (14) Then Shannon’s entropy concept is used to obtain the weight. The Scientific World Journal 5 (b) Objective weight. For the objective weight, the dis- tance values are calculated by using Definition 2: The details of Shannon’s entropy concept are defined as follows [27, 39]. Step 5.1. Normalized each criterion weight to obtain the projection value; 𝑝𝑖𝑗: 𝑑WHD (𝐼, 𝐷) = 𝑛 ∑ 𝑗=1 (𝑤𝑗 󵄨󵄨󵄨󵄨󵄨V𝐿 𝑗−𝑥𝐿 𝑖𝑗 󵄨󵄨󵄨󵄨󵄨+ 𝑤𝑗 󵄨󵄨󵄨󵄨󵄨V𝑈 𝑗−𝑥𝑈 𝑖𝑗 󵄨󵄨󵄨󵄨󵄨) . (29) (29) 𝑝𝑖𝑗= 𝑤𝑖𝑗 ∑𝑚 𝑖=1 𝑤𝑖𝑗 , 𝑖= 1, . . . , 𝑚, 𝑗= 1, . . . , 𝑛. (20) (20) Step 7 (ranking the candidate). The alternatives are ranked in ascending order according to the distance values for respective 𝛼values. The alternative with the less distance value is considered as the best choice. Consequently, a projection matrix representing a relative weight of each criterion from the decision maker evaluation is expressed as 𝑃= [[[[ [ 𝑝11 𝑝12 ⋅⋅⋅𝑝1𝑛 𝑝21 𝑝22 ⋅⋅⋅𝑝2𝑛 ... ... ⋅⋅⋅ ... 𝑝𝑚1 𝑝𝑚2 ⋅⋅⋅𝑝𝑚𝑛 ]]]] ] . (21) Step 8 (repeat Steps 4, 5, and 6 for different values of 𝛼). The alternatives are ranking according to the different values of 𝛼. (21) Step 9 (selection of the appropriate alternative by the decision makers). Step 5.2. Calculate entropy values 𝑒𝑗as Step 5.2. Calculate entropy values 𝑒𝑗as 5. A Numerical Example 𝑒𝑗= −𝑘 𝑚 ∑ 𝑖=1 𝑝𝑖𝑗ln 𝑝𝑖𝑗, 𝑗= 1, . . . , 𝑛, (22) (22) An example on the personnel selection in an academic institution is provided to validate the proposed algorithm. Suppose that the academic institution intends to employ a lecturer based on consideration of four main criteria which are experienced in teaching areas (𝐶1), proficiency in per- forming research (𝐶2), personality assessment (𝐶3), and past contribution (𝐶4). Assume that after preliminary selection phase, four alternatives 𝐴1, 𝐴2, 𝐴3, and 𝐴4 are qualified for final evaluation. A committee of experts (decision makers) consisting of three persons is formed, namely, 𝐷1, 𝐷2, and 𝐷3. The information of this study is given in Figures 1 and 2 and Tables 1, 2, 3, 4, and 5, while the results from the numerical examples are shown in Tables 6, 7, 8, 9, and 10. As mentioned before, TOPSIS is one of the existing MCDM methods that can be used to solve personnel selection problem. Thus it can be used to validate the proposed method and the results by using this method that is shown in Table 11. More explanation on these figures and tables are explained in the discussion section. where 𝑘is constant and let 𝑘= (ln 𝑚)−1. If 𝑝𝑖𝑗= 0, then 𝑝𝑖𝑗ln 𝑝𝑖𝑗is equal to 0. Step 5.3. Calculate the degree of diversification, 𝑑𝑗: Step 5.3. Calculate the degree of diversification, 𝑑𝑗: 𝑑𝑗= 1 −𝑒𝑗, 𝑗= 1, . . . , 𝑛. (23) (23) Step 5.4. Calculate the criteria weight, 𝑤𝑗: Step 5.4. Calculate the criteria weight, 𝑤𝑗: 𝑤𝑗= 𝑑𝑗 ∑𝑛 𝑘=1 𝑑𝑘 . (24) (24) Step 6 (calculating the distance values). (a) Subjective weight. Before calculating the distance values, calculate the overall performance evaluation of ideal alternatives and alternatives by multiplying the aggregate weight with each criterion [21]. F th id l lt ti Step 6 (calculating the distance values). (a) Subjective weight. Before calculating the distance values, calculate the overall performance evaluation of ideal alternatives and alternatives by multiplying the aggregate weight with each criterion [21]. h d l l For the ideal alternative: 𝑅𝑗= [V𝐿 𝑗, V𝑈 𝑗] ⋅[𝑤𝐿 𝑗, 𝑤𝑈 𝑗] = [𝑟𝐿 𝑗, 𝑟𝑈 𝑗] , (25) (25) and for the alternatives: and for the alternatives: 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 VP P MP M MG G VG 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 VP P MP M MG G VG Figure 2: The fuzzy linguistic variables for each alternative. Table 2: Fuzzy linguistic terms and respective fuzzy numbers for each criterion. Table 2: Fuzzy linguistic terms and respective fuzzy numbers for each criterion. Table 2: Fuzzy linguistic terms and respective fuzzy numbers for each criterion. Linguistic terms Fuzzy numbers Very poor (VP) (0, 0, 0.2) Poor (P) (0.05, 0.2, 0.35) Medium poor (MP) (0.2, 0.35, 0.5) Fair (F) (0.35, 0.5, 0.65) Medium good (MG) (0.5, 0.65, 0.8) Good (G) (0.65, 0.8, 0.95) Very good (VG) (0.8, 1, 1) Figure 2: The fuzzy linguistic variables for each alternative. Step 5. The objective and subjective weights are identified. The subjective weight is measured based on (18). Table 6 shows the subjective weight for each criterion at 𝛼= 0 and 𝛼= 0.5. While for objective weight, Shannon’s entropy concept (19)–(24) is used to obtain the weight. The projection values are shown in Table 7. Table 8 consist of entropy values (𝑒𝑗), degree of diversifications (𝑑𝑗), and the objective weight (𝑤𝑗). The use of objective weight will give an insight to the decision maker in determining which criteria is needed the most in which 𝐶3 and 𝐶4 are considered as the most important criteria based on Shannon’s entropy concept. It is known that objective weight can be obtained without consideration of decision maker’s preferences; however, since the evaluation of criteria weight exists, the objective weight is obtained based on the evaluation of criteria weight. Table 3: Decision makers’ evaluation on each criterion weight. Table 3: Decision makers’ evaluation on each criterion weight. Table 3: Decision makers’ evaluation on each criterion weight. Criteria 𝐶1 𝐶2 𝐶3 𝐶4 𝐷1 VH H H MH 𝐷2 H VH MH H 𝐷3 VH VH H H Table 4: Decision makers’ evaluation on ideal alternative. Criteria 𝐶1 𝐶2 𝐶3 𝐶4 𝐼 VG G VG MG “very good.” Table 5 illustrates each fuzzy linguistic term to its corresponding fuzzy number for each alternative. Table 3: Decision makers’ evaluation on each criterion weight. Criteria 𝐶1 𝐶2 𝐶3 𝐶4 𝐷1 VH H H MH 𝐷2 H VH MH H 𝐷3 VH VH H H Table 4: Decision makers’ evaluation on ideal alternative. 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. 𝑆𝑖𝑗= [𝑥𝐿 𝑖𝑗, 𝑥𝑈 𝑖𝑗] ⋅[𝑤𝐿 𝑗, 𝑤𝑈 𝑗] = [𝑠𝐿 𝑖𝑗, 𝑠𝑈 𝑖𝑗] , (26) (26) Step 1. Ideal alternative matrix (12) is built from the evalua- tions of the criteria based on linguistic variables taken from Wang and Lee [28] as illustrated in Figure 2 and Table 2. The linguistic terms are represented by triangular fuzzy number ranging from “very poor” to “very good.” Table 4 shows the decision makers evaluation on ideal alternative. In this paper, we assume that the 𝑚decision makers had come to an agreement in standardizing into one final value for each criterion. where where 𝑟𝐿= min {V𝐿𝑤𝐿, V𝐿𝑤𝑈, V𝑈𝑤𝐿, V𝑈𝑤𝑈} ; 𝑟𝑈= max {V𝐿𝑤𝐿, V𝐿𝑤𝑈, V𝑈𝑤𝐿, V𝑈𝑤𝑈} ; 𝑠𝐿= min {𝑥𝐿𝑤𝐿, 𝑥𝐿𝑤𝑈, 𝑥𝑈𝑤𝐿, 𝑥𝑈𝑤𝑈} ; 𝑠𝑈= max {𝑥𝐿𝑤𝐿, 𝑥𝐿𝑤𝑈, 𝑥𝑈𝑤𝐿, 𝑥𝑈𝑤𝑈} . (27) Step 2. Decision matrix for alternatives evaluation on each criterion (13) is obtained by using the same linguistic variables adopted from Wang and Lee [28] as illustrated in Figure 2 and Table 2. Similar to Step 1, these terms are captured in the form of the triangular fuzzy number. The alternatives performance evaluations are ranging from “very poor” to Then, calculate the distance values between the ideal alterna- tives with the alternatives by using Definition 1: Then, calculate the distance values between the ideal alterna- tives with the alternatives by using Definition 1: 𝑑NHD (𝑅, 𝑆) = 1 2𝑛( 𝑛 ∑ 𝑗=1 (󵄨󵄨󵄨󵄨󵄨𝑟𝐿 𝑗−𝑠𝐿 𝑖𝑗 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝑟𝑈 𝑗−𝑠𝑈 𝑖𝑗 󵄨󵄨󵄨󵄨󵄨)) . (28) (28) The Scientific World Journal The Scientific World Journal 6 6 VL L ML M MH H VH 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 Figure 1: The fuzzy linguistic variables for each criterion weight. VL L ML M MH H VH 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 Table 1: Fuzzy linguistic terms and respective fuzzy numbers for each criterion weight. Table 1: Fuzzy linguistic terms and respective fuzzy numbers for each criterion weight. each criterion weight. Linguistic terms Fuzzy numbers Very low (VL) (0, 0, 0.2) Low (L) (0.05, 0.2, 0.35) Medium low (ML) (0.2, 0.35, 0.5) Medium (M) (0.35, 0.5, 0.65) Medium high (MH) (0.5, 0.65, 0.8) High (H) (0.65, 0.8, 0.95) Very high (VH) (0.8, 1, 1) Figure 1: The fuzzy linguistic variables for each criterion weight. 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. Alternatives 𝐶1 𝐶2 𝐶3 𝐶4 𝐷1 𝐷2 𝐷3 𝐷1 𝐷2 𝐷3 𝐷1 𝐷2 𝐷3 𝐷1 𝐷2 𝐷3 𝐴1 G G F F MG F G VG VG G VG MG 𝐴2 F G G F F F G MG G MG G G 𝐴3 F VG F MG VP G VG G MG VG G G 𝐴4 G G G MG G G VG VG VG G G MG 𝐴2 F G G F F F G MG G MG G G 𝐴3 F VG F MG VP G VG G MG VG G G 𝐴4 G G G MG G G VG VG VG G G MG Table 6: Subjective weight for each criterion at 𝛼= 0 and 𝛼= 0.5. Criteria 𝛼= 0 𝛼= 0.5 𝐶1 (0.75, 0.9833) (0.84166, 0.95833) 𝐶2 (0.75, 0.9833) (0.84166, 0.95833) 𝐶3 (0.60, 0.90) (0.675, 0.825) 𝐶4 (0.60, 0.90) (0.675, 0.825) Table 7: Each criterion projection value at 𝛼= 0 and 𝛼= 0.5. Criteria 𝐷1 𝐷2 𝐷3 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 𝐶1 (0.34615) (0.35185) (0.30769) (0.29630) (0.34615) (0.35185) 𝐶2 (0.30769) (0.29630) (0.34615) (0.35185) (0.34615) (0.35185) 𝐶3 (0.35556) (0.35556) (0.28889) (0.28889) (0.35556) (0.35556) 𝐶4 (0.28889) (0.28889) (0.35556) (0.35556) (0.35556) (0.35556) Table 8: Shannon’s entropy based weight. Criteria 𝑒𝑗 𝑑𝑗 𝑤𝑗 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 𝐶1 0.99864 0.99713 0.00136 0.00287 0.12385 0.20439 𝐶2 0.99864 0.99713 0.00136 0.00287 0.12385 0.20439 𝐶3 0.99585 0.99585 0.00415 0.00415 0.37615 0.29561 𝐶4 0.99585 0.99585 0.00415 0.00415 0.37615 0.29561 Table 9: Distance value of subjective and objective weights at 𝛼= 0 and 𝛼= 0.5. Distance 𝐴1 𝐴2 𝐴3 𝐴4 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 Subjective 0.12604 0.13993 0.15181 0.17915 0.17305 0.20001 0.04979 0.06338 Objective 0.23685 0.32263 0.31193 0.40220 0.36193 0.45220 0.11239 0.14087 Table 10: Ranking of alternatives at 𝛼= 0 and 𝛼= 0.5 by using HDMSOW’s. Ranking Subjective weight Objective weight 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 1 𝐴4 𝐴4 𝐴4 𝐴4 2 𝐴1 𝐴1 𝐴1 𝐴1 3 𝐴2 𝐴2 𝐴2 𝐴2 4 𝐴3 𝐴3 𝐴3 𝐴3 Step 8. The steps are repeated by using different values of 𝛼, 𝛼 ∈ [0, 1]. Under different values of 𝛼, the decision makers may expect the different outcome in the ranking of the alternatives. 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. “very good.” Table 5 illustrates each fuzzy linguistic term to its corresponding fuzzy number for each alternative. Step 6. The distance values between the ideal alternative and the alternatives are calculated by using the Hamming distance method. For the subjective weight, the overall performance evaluation for the ideal alternative (25) and the alternatives (26) are determined beforehand the use of the normalized Hamming distance method (28). For the objective weight, the distance values are obtained from the use of the weighted Hamming distance method (29). The distance values show how much is the similarity between the alternatives and the ideal alternative. Step 3. The weighting matrix (14) for each criterion is evaluated and determined by the decision makers based on linguistic variables pictured in Figure 1 and Table 1. Like the previous step, these linguistic terms are expressed in the form of triangular fuzzy numbers and are ranging from “very low” to “very high.” Table 3 marks the evaluation of the criteria weights by the decision makers according to their own judgment in evaluating criteria’s importance for the specified job. Step 3. The weighting matrix (14) for each criterion is evaluated and determined by the decision makers based on linguistic variables pictured in Figure 1 and Table 1. Like the previous step, these linguistic terms are expressed in the form of triangular fuzzy numbers and are ranging from “very low” to “very high.” Table 3 marks the evaluation of the criteria weights by the decision makers according to their own judgment in evaluating criteria’s importance for the specified job. Step 7. The ranking of the alternatives is made based on the distance values obtained before. The alternative with the less distance value is considered as a preferable alternative to be selected. Table 9 shows the distance value for each alternative at 𝛼= 0 and 𝛼= 0.5. Table 10 shows the ranking of the alternatives based on the distance values with the use of subjective and objective weights. Step 4. By using the 𝛼-cuts of fuzzy numbers, the interval value of the fuzzy number of the performance matrics for the ideal alternative (15), the alternatives (16), and criteria weight (17) are built. The values of 𝛼show the degree of confidences for the decision makers in evaluating the criteria performance of each alternative. The Scientific World Journal 7 Table 5: Decision makers rating on alternative performance. 6. Conclusions In this paper, we have presented a novel approach of handling personnel selection process by using the Hamming distance method. Based on the fact that most of criteria assessment is in qualitative or in subjective measurement, fuzzy set theory has been applied to overcome this limitation. Furthermore, realizing the importance of weighting the criteria in deter- mining which criteria are valued the most; two types of weights have been applied in this paper which are objective and subjective weights. The objective weight is determined by the application of Shannon’s entropy concept and the subjective weight is obtained based on the preference of the decision maker. With the use of the weighted Hamming distance, the distance values between the ideal alternative and the alternatives are identified and the ranking of the alternatives based on the overall evaluation of the criteria is made. The final results showed that the criteria 𝐶3 and 𝐶4 are considered as the important criteria and 𝐴4 is considered as the best alternative to choose based on the use of sub- jective and objective weights. With emphasis on finding the distance measure between ideal alternative and alternatives with the use of subjective and objective weights, our method provides an effective way to be used. In addition, we are also incorporating fuzzy linguistic terms to express the subjective assessment that the decision makers often exhibit, while evaluating the alternatives performance in certain criteria. We also provided the numerical example to prove the validity of this approach. To verify the proposed method, the TOPSIS method is used to compare the result and we can justify that the final results are almost the same for both methods. The proposed method also can overcome some limitation in the existing methods of MCDM that are involved with the inconsistency of judgement when there are the addition of alternatives and criteria. For further research, we are going to study the appropriate methods in evaluating ideal alternatives hence improving the HDMSOWs. [4] Y. H. Chang, C. H. Yeh, and Y. W. Chang, “A new method selec- tion approach for fuzzy group multicriteria decision making,” Applied Soft Computing, vol. 13, no. 4, pp. 2179–2187, 2013. [5] T. Dereli, A. Durmusoglu, S. U. Sec¸kiner, and N. Avlanmaz, “A fuzzy approach for personnel selection process,” Turkish Journal of Fuzzy Systems, vol. 1, no. 2, pp. 126–140, 2010. [6] I. S. Fagoyinbo and I. A. 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. The decision makers also can make the decision based on the preferable 𝛼levels since the ranking may be changed at the different values of 𝛼. Apparently, the most suitable alternative for the post by using both subjective and objective weights is the alternative with the minimum distance values. From Table 10, 𝐴4 is likely to be selected by the decision makers regarding his/her distance values. Here, we also present the results by using TOPSIS method to validate the proposed approach. Consequently, the same results are recorded by using TOPSIS method in which 𝐴4 is the possible alternative to be selected. The ranking for the other alternatives also can be clarified as almost similar to the results by using the proposed method. 6. Conclusions Ajibode, “Application of linear pro- gramming techniques in the effective use of resources for staff training,” Journal of Emerging Trends in Engineering and Applied Sciences, pp. 127–132, 2010. [7] C. K. Voon, Analytic hierarchy process in academic staff selection at Faculty of Science in University Technology Malaysia [M.S. thesis], Faculty of Science, Universiti Teknologi Malaysia, Johor, Malaysia, 2009. [8] A. Afshari, M. Mojahed, and R. M. Yusuff, “Simple additive weighting approach to personnel selection problem,” Interna- tional Journal of Innovation, Management and Technology, vol. 1, no. 5, pp. 511–515, 2010. [9] P. V. Polychroniou and I. 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Liern, “Some fuzzy models for human resource management,” International Journal of Technology, Policy and Management, vol. 4, no. 4, pp. 291–308, 2004. [14] R. W. Hamming, “Error detecting and error correcting codes,” Bell System Technical Journal, vol. 29, no. 2, pp. 147–160, 1950. References [1] M. Dursun and E. E. Karsak, “A fuzzy MCDM approach for personnel selection,” Expert Systems with Applications, vol. 37, no. 6, pp. 4324–4330, 2010. [2] Z. G¨ung¨or, G. Serhadlıoˇglu, and S. E. Kesen, “A fuzzy AHP approach to personnel selection problem,” Applied Soft Comput- ing, vol. 9, pp. 641–646, 2009. [3] H. Niakan, M. Zowghi, and A. Bakhshandeh-Fard, “A fuzzy objective and subjective decision making method by non-linear normalizing and weighting operations,” in Proceedings of the International Conference on Management and Service Science (MASS ’11), pp. 1–4, Wuhan, China, August 2011. Acknowledgment Step 9. The decision makers then will select the suitable alternative to fill the vacancy based on the ranking of the alternatives. The decision makers also can make the decision based on the preferable 𝛼levels since the ranking may be changed at the different values of 𝛼. Apparently, the most suitable alternative for the post by using both subjective and objective weights is the alternative with the minimum distance values. From Table 10, 𝐴4 is likely to be selected by the decision makers regarding his/her distance values. Here, we also present the results by using TOPSIS method to validate the proposed approach. Consequently, the same results are recorded by using TOPSIS method in which 𝐴4 is the possible alternative to be selected. The ranking for the other alternatives also can be clarified as almost similar to the results by using the proposed method. This research was funded by the Ministry of Education of Malaysia under Research Acculturation Grant Scheme (RAGS): 9018-00004. 5.1. Discussion. Based on the results obtained, the proposed HDMSOWs can be summarized as follows. If there exist two or more alternatives on Table 11: Ranking of alternatives at 𝛼= 0 and 𝛼= 0.5 by using TOPSIS. Ranking Subjective weight Objective weight 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 1 𝐴4 𝐴4 𝐴4 𝐴4 2 𝐴1 𝐴1 𝐴1 𝐴1 3 𝐴3 𝐴3 𝐴3 𝐴3 4 𝐴2 𝐴2 𝐴2 𝐴2 the same ranking which indicate that they having the same distance values, the decision makers may refer to the criteria weight, which mean the alternative that perform well in the criteria that is needed the most is likely to be selected. Table 6: Subjective weight for each criterion at 𝛼= 0 and 𝛼= 0.5. Table 9: Distance value of subjective and objective weights at 𝛼= 0 and 𝛼= 0.5. Table 10: Ranking of alternatives at 𝛼= 0 and 𝛼= 0.5 by using HDMSOW’s. Table 11: Ranking of alternatives at 𝛼= 0 and 𝛼= 0.5 by using TOPSIS. Ranking Subjective weight Objective weight 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 1 𝐴4 𝐴4 𝐴4 𝐴4 2 𝐴1 𝐴1 𝐴1 𝐴1 3 𝐴3 𝐴3 𝐴3 𝐴3 4 𝐴2 𝐴2 𝐴2 𝐴2 Ranking Subjective weight Objective weight 𝛼= 0 𝛼= 0.5 𝛼= 0 𝛼= 0.5 1 𝐴4 𝐴4 𝐴4 𝐴4 2 𝐴1 𝐴1 𝐴1 𝐴1 3 𝐴2 𝐴2 𝐴2 𝐴2 4 𝐴3 𝐴3 𝐴3 𝐴3 Step 8. The steps are repeated by using different values of 𝛼, 𝛼 ∈ [0, 1]. Under different values of 𝛼, the decision makers may expect the different outcome in the ranking of the alternatives. If there exist two or more alternatives on the same ranking which indicate that they having the same distance values, the decision makers may refer to the criteria weight, which mean the alternative that perform well in the criteria that is needed the most is likely to be selected. Step 8. The steps are repeated by using different values of 𝛼, 𝛼 ∈ [0, 1]. Under different values of 𝛼, the decision makers may expect the different outcome in the ranking of the alternatives. 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https://openalex.org/W2411224281	https://europepmc.org/articles/pmc4909041?pdf=render	English	null	Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil	Memórias do Instituto Oswaldo Cruz	2,016	cc-by	3,140	Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil Humberto Fabio Boatto1,2, Sarah Desirée Barbosa Cavalcanti3, Gilda MB Del Negro3, Manoel João BC Girão1, Elaine Cristina Francisco4, Kelly Ishida5, Olga Fischman Gompertz6/+ 1Universidade Federal de São Paulo, Departamento de Ginecologia, São Paulo, SP, Brasil 2Universidade Mogi das Cruzes, Faculdade de Medicina, Departamento de Clínica Médica, Mogi das Cruzes, SP, Brasil 3Universidade de São Paulo, Hospital das Clínicas da Faculdade de Medicina, São Paulo, SP, Brasil 4Universidade Federal de São Paulo, Departamento de Infectologia, São Paulo, SP, Brasil 5Universidade de São Paulo, Instituto de Ciências Biomédicas, Departamento de Microbiologia, São Paulo, SP, Brasil 6Universidade Federal de São Paulo, Departamento de Microbiologia, Imunologia e Parasitologia, São Paulo, SP, Brasil The aim of this study was to identify Candida species isolated from women diagnosed with recurrent vulvovagi nal candidiasis (RVVC) and their partners; and to evaluate the fluconazole (FLZ) susceptibility of the isolates. In a period of six years, among 172 patients diagnosed with vulvovaginal candidiasis, 13 women that presented RVVC and their partners were selected for this investigation. The isolates were obtained using Chromagar Candida me dium, the species identification was performed by phenotypic and molecular methods and FLZ susceptibility was evaluated by E-test. Among 26 strains we identified 14 Candida albicans, six Candida duobushaemulonii, four Can dida glabrata, and two Candida tropicalis. Agreement of the isolated species occurred in 100% of the couples. FLZ low susceptibility was observed for all isolates of C. duobushaemulonii (minimal inhibitory concentration values from 8-> 64 µg/mL), two C. glabrata isolates were FLZ-resistant and all C. albicans and C. tropicalis isolates were FLZ-susceptible. This report emphasises the importance of accurate identification of the fungal agents by a reliable molecular technique in RVVC episodes besides the lower antifungal susceptibility profile of this rare pathogen C. duobushaemulonii to FLZ. Key words: Candida duobushaemulonii - recurrent vulvovaginal candidiasis - antifungal susceptibility tes Vulvovaginal candidiasis (VVC) is a very common infection that affects a great number of women at repro ductive age and the frequent cause of taking gynecologi cal medical consultation. The recurrent vulvovaginal candidiasis (RVVC) is a more severe condition that af fects 5-8% of these patients; and the reasons are poorly understood (Sobel 2007, Giraldo et al. 2013). Candida albicans is the most common causal agent but non-al bicans species have been identified (Richter et al. 2005, Sobel 2007). Among C. non-albicans, Candida glabrata and Candida tropicalis had been related with RVVC cases. Interestingly, C. glabrata isolates present lower azoles susceptibility than other species (Richter et al. 2005, Sobel 2007). cation of C. 407 407 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 111(6): 407-410, June 2016 online \| memorias.ioc.fiocruz.br doi: 10.1590/0074-02760160166 Financial support: CNPq. + Corresponding author: olga.gompertz@unifesp.br Received 22 April 2016 Accepted 12 May 2016 Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil Humberto Fabio Boatto1,2, Sarah Desirée Barbosa Cavalcanti3, Gilda MB Del Negro3, Manoel João BC Girão1, Elaine Cristina Francisco4, Kelly Ishida5, Olga Fischman Gompertz6/+ haemulonii, Candida pseudohaemulonii, Candida auris and C. duoboshaemulonii (Kim et al. 2009, Cendejas- Bueno et al. 2012). The identification at species level was achieved only after the sequencing of ITS region, which is in agreement with other authors (Cendejas-Bueno et al. 2012, Ramos et al. 2015). molecular analysis. The DNA extractions and amplifi cations by polymerase chain reaction (PCR) were per formed using Candida species-specific primers (Table) following protocols reported elsewhere (Luo & Mitchell 2002, Taira et al. 2014). Samples that were not identified by PCR were submitted to sequencing of the amplified products obtained with primers VLG/LS and ITS1/ITS4 (White et al. 1990), and the sequence similarity searches were done by BLAST (http://www.ncbi.nlm.nih.gov/blast). lonii. Classical and commercial methods of yeast iden tification are not reliable to identify rare and emerging clinical isolates of C. haemulonii complex species as C. haemulonii, Candida pseudohaemulonii, Candida auris and C. duoboshaemulonii (Kim et al. 2009, Cendejas- Bueno et al. 2012). The identification at species level was achieved only after the sequencing of ITS region, which is in agreement with other authors (Cendejas-Bueno et al. 2012, Ramos et al. 2015). y ( p g ) Twenty-six yeasts were isolated from 13 samples of women diagnosed with RVVC and from 13 samples of their partners. C. albicans (14), Candida duobushaemu lonii (6), C. glabrata (4), and C. tropicalis (2) isolates were identified by phenotypic characteristics and con firmed by molecular methods. The identification of C. albicans, C. glabrata and C. tropicalis were confirmed by PCR assays and the remaining isolates (n = 6) were accurately identified as C. duobushaemulonii by se quencing of the region ITS of rDNA, showing 100% of identity with a reference strain available in GenBank (NCBI Reference Sequence NR130694.1). The isolates of C. duobushaemulonii obtained from samples col lected from the three couples were evaluated by random amplified polymorphic DNA (RAPD) assay employing the primer OPG10 and amplification parameters previ ously described (Rocha et al. 2008); the yeast isolates demonstrated highly similar band patterns between iso lates obtained of each couple (Figure). Thus, the C. duo bushaemulonii isolated from the women with RVVC and from their partners were possibly the same isolates. The first human isolation of C. haemulonii complex species occurred in 1984 from the blood of a patient with renal failure (Lavarde et al. 1984). Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil Humberto Fabio Boatto1,2, Sarah Desirée Barbosa Cavalcanti3, Gilda MB Del Negro3, Manoel João BC Girão1, Elaine Cristina Francisco4, Kelly Ishida5, Olga Fischman Gompertz6/+ non-albicans because they had shown high est minimal inhibitory concentration (MIC) values to many antifungal agents (Richter et al. 2005, Sobel 2007, Oberoi et al. 2012, Ramos et al. 2015). The aim of this re port was to identify species of Candida clinical isolates from women diagnosed with RVVC and their partners; and to evaluate the FLZ susceptibility. p y From July 2005 to August 2011, 2,026 female patients ranging from 18-65 years old were evaluated at Gyneco logic Services of three private and two public Services in São Paulo city, São Paulo state, Brazil. Out of 172 patients who presented VVC, 13 women with RVVC and their partners were selected for this study. Secretion of the ectocervice and vagina from the women and of the foreskin and glans from their respective partners were collected with moistened swabs in sterile saline solution. In this study women with diabetes mellitus, on steroid, antibiotics or hormone therapy, in use of intrauterine de vice using vaginal douches or spermicidal, carriers of immunodeficiency virus were excluded. All procedures were previously approved by the Research Ethic Com mittee of São Paulo Hospital, Federal University of São Paulo, São Paulo, Brazil (Protocol CEP 1719/05). The VVC and RVVC therapeutics are performed by topic application of polyene and azole agents (Sobel et al. 2004, Sobel 2007). Oral fluconazole (FLZ) has also been frequently used being the first drug of choice for VVC treatment (Sobel et al. 2004), and the Public Health Service in Brazil furnishes it to the patients with VVC or RVVC. It is important to highlight the reliable identifi Samples were previously cultivated in CHROMagar Candida medium® (Becton-Dickinson, New Jersey, USA) at 37ºC for 48 h and isolated yeasts were trans ferred to Sabouraud dextrose agar (Difco, USA) for fur ther procedures. Morphological, biochemical and physi ological characterisation of the isolates were defined according to Kurtzman et al. (2011). In order to confirm phenotypic identification, the isolates were submitted to doi: 10.1590/0074-02760160166 Financial support: CNPq. + Corresponding author: olga.gompertz@unifesp.br Received 22 April 2016 Accepted 12 May 2016 doi: 10.1590/0074-02760160166 Financial support: CNPq. + Corresponding author: olga.gompertz@unifesp.br Received 22 April 2016 Accepted 12 May 2016 C. duobushaemulonii • Humberto Fabio Boatto et al. 408 lonii. Classical and commercial methods of yeast iden tification are not reliable to identify rare and emerging clinical isolates of C. haemulonii complex species as C. Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil Humberto Fabio Boatto1,2, Sarah Desirée Barbosa Cavalcanti3, Gilda MB Del Negro3, Manoel João BC Girão1, Elaine Cristina Francisco4, Kelly Ishida5, Olga Fischman Gompertz6/+ These species are op portunistic fungal pathogens that have been associated Random amplified polymorphic DNA banding pattern of Candida duobushaemulonii recovered from the three couples with the primer OPG 10. Lane M: molecular weight marker 1 Kb (arrow) (Fermentas, USA); Lines 1-2: C. duobushaemulonii isolated from the couple 1; Lines 3-4: C. duobushaemulonii isolated from the couple 2; and Lines 5-6: C. duobushaemulonii isolated from the couple 3. C. albicans, C. glabrata and C. tropicalis have been commonly identified in RVVC (Richter et al. 2005, So bel 2007). Here, we identified the three Candida spe cies and also C. duobushaemulonii from genital samples of women and their partners. The infection of Candida haemulonii complex species (including C. duobushae mulonii) is not common in humans. Lehman et al. (1993) reported variations among C. haemulonii isolates and concluded that the isolates represented a complex of C. haemulonii group I and C. haemulonii group II species. In 2012, Cendejas-Bueno et al. (2012) proposed a reclas sification of the isolates of group II as C. duobushaemu Random amplified polymorphic DNA banding pattern of Candida duobushaemulonii recovered from the three couples with the primer OPG 10. Lane M: molecular weight marker 1 Kb (arrow) (Fermentas, USA); Lines 1-2: C. duobushaemulonii isolated from the couple 1; Lines 3-4: C. duobushaemulonii isolated from the couple 2; and Lines 5-6: C. duobushaemulonii isolated from the couple 3. TABLE TABLE Primers used in polymerase chain reaction and random amplified polymorphic DNA assays: sequences, hybridisation temperature (annealing) and molecular weight of amplified products Primers Sequences Annealing Molecular weight Reference ITS1/4 F: 5’TCCGTAGGTGAACCTGCGG 3’ R: 5’TCCTCCGCTTATTGATATGC 3’ 47ºC variable 14 Candida albicans 1/2 F: 5’TTTATCAACTTGTCACACCAGA-3’ R: 5’ATCCCGCCTTACCACTACCG-3’ 55ºC 272pb 14 Candida glabrata 1/2 F: 5’TTATCACACGACTCGACACT-3’ R: 5’CCCACATACTGATATGGCCTACAA-3’ 52ºC 423pb 14 Candida tropicalis 1/2 F: 5’CAATCCTACCGCCAGAGGTTAT-3’ R: 5’TGGCCACTAGCAAAATAAGCGT-3’ 52ºC 357pb 14 F: forward; R: reverse. Primers used in polymerase chain reaction and random amplified polymorphic DNA assays: sequences, hybridisation temperature (annealing) and molecular weight of amplified products Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 111(6), June 2016 409 with onychomycosis, finger nail infections and broncho- alveolar lavage (Ramos et al. 2015), bloodstream infec tions (Ruan et al. 2010, Almeida Jr et al. 2012, Oberoi et al. 2012), fungaemia related catheter (Kim et al. 2011), and with an outbreak in neonatal care units (Khan et al. 2007). Recently, Almeida Jr et al. (2016) showed that among 14,642 positive yeast cultures from five hospitals in São Paulo (Brazil), 40 (0.3%) isolates were identified as C. haemulonii complex species. C. duobushaemulo nii was characterised in nine biological samples and the data suggested that patients with diabetes mellitus are more likely to have positive cultures for C. duobushae mulonii (Almeida Jr et al. 2016). acquisition of genital candidiasis (Sobel et al. 2004, Li et al. 2008, Giraldo et al. 2013). So, the real role of sexual transmission on RVVC has yet to be defined. In summary, in these 13 cases of RVVC we empha sise the importance of the correct identification of emer gent pathogens that had shown higher MIC values for routine antifungal drugs as FLZ. To our best knowledge, it is the first isolation of C. duobushaemulonii, a rare hu man emergent pathogen, from RVVC cases with the ac curate identification of the fungal agent. We highlight the reduced susceptibility of this species to FLZ, which rep resents a major therapeutic choice for RVVC. All these informations will be very useful to improve the manage ment of the patients with infections caused by these or ganisms and will contribute to the surveillance of RVVC. In our study, the FLZ susceptibility testing of isolates was performed by the standard kit “E-Test” (Biodisk AB, Solna, Sweden) according to manufacturer recommen dations. Reduced susceptibility to FLZ was observed for all isolates of C. REFERENCES Almeida Jr JN, Assy JGPL, Levin AS, Del Negro GMB, Giudice MC, Tringoni MP, et al. Candida haemulonii complex species, Brazil, January 2010 - March 2015. Emerg Infect Dis. 2016; 22(3): 561-3. Almeida Jr JN, Motta AL, Rossi F, Abdala E, Pierrotti LC, Kono ASG, et al. First report of clinical isolate of Candida haemulonii in Brazil. Clinics. 2012; 67(10): 1229-31. Cendejas-Bueno E, Kolecka A, Alastruey-Izquierdo A, Theelen B, Groenewald M, Kostrzewa M, et al. Reclassification of the Can dida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant hu man pathogenic yeasts. J Clin Microbiol. 2012; 50(11): 3641-51. CLSI - Clinical Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; Fourth informational supplement. CLSI document M27-S4. Wayne: Clinical and Laboratory Standards Institute; 2012. Giraldo PC, Rodrigues HM, Melo AG, do Amaral RL, Passos MRL, Eleutério Jr J, et al. Vulvovaginitis and the treatment of asymp tomatic partners: a systematic review and metanalisis. DST-J Bras Doenças Sex Transm. 2013; 25(1): 36-40. FLZ is the main antifungal agent employed in VVC; and for RVVC 10-14 days of induction therapy with a topical agent or oral FLZ, followed by FLZ, 150 mg weekly for six months, is strongly recommended (Sobel et al. 2004, Pappas et al. 2016). In our research FLZ was used for its effectiveness and because it is recommended and furnished by the Public System of Health in Brazil for RVVC treatment. In our experience, RVVC present ed clinical and mycological resolution after symptomatic and asymptomatic partner’s treatment and the use of an tifungal drugs for a long time. Khan ZU, Al-Sweih NA, Ahmad S, Al-Kazemi N, Khan S, Joseph L, et al. Outbreak of fungemia among neonates caused by Candida haemulonii resistant to amphotericin b, itraconazole , and fluco nazole. J Clin Microbiol. 2007; 45(6): 2025-7. Kim MN, Shin JH, Sung H, Lee K, Kim Ec, Ryoo N. Candida hae mulonii and closely related species at 5 university hospitals in Korea; identification, antifungal susceptibility, and clinical fea tures. Clin Infect Dis. 2009; 48: 57-61. The RVVC is a clinical condition that is characterised by three or more episodes of VVC with the isolation of the causal agent that occurs within a 12-months period. It is known that the causes of RVVC are multifactorial (Sobel 2007). TABLE duobushaemulonii (MIC ranging from 8->64 µg/mL), 50% of C. glabrata isolates exhibited re sistance (≥ 64 µg/mL), and all isolates of C. albicans and C. tropicalis were susceptible to FLZ. The antifungal susceptibility interpretation was based on the breakpoint values for FLZ recommended by the CLSI (2012). The reduced susceptibility to FLZ of C. non-albicans spe cies in RVVC cases described in this report, mainly C. glabrata and C. duobushaemulonii, is in agreement with other authors (Cendejas-Bueno et al. 2012, Almeida Jr et al. 2016). C. duobushaemulonii species complex have been isolated from several clinical sources varying from superficial to deep infections (Almeida Jr et al. 2012, Ramos et al. 2015) and they are related with lower sus ceptibility to polyenes, azoles and echinocandins (Ra mos et al. 2015, Almeida Jr et al. 2016). REFERENCES The knowledge about the relation of sexual activity with the infection is limited. The role of sexual partners, trauma of the vaginal mucosa, immunosup pressive effect of semen during sexual activity besides other factors should be taken into consideration in the cases of repetition (Li et al. 2008). Studies that collect more specimens and detailed clinical data from women with RVVC may enhance the ability to distinguish be tween recurrence and reinfection. In all cases of RVVC of our investigation, there was 100% of agreement among Candida spp. isolated from the couples as demonstrated by the RAPD assay, that may suggest transmission be tween partners. Investigations did not prove the sexual Kim S, Ko KS, Moon SY, Lee MS, Lee MY, Son JS. Catheter-related candidemia caused by Candida haemulonii in a patient in long- term care hospital. J Korean Med Sci. 2011; 26(2): 297-300. Kurtzman CP, Fell JW, Boekhout T, Robert V. Methods for isola tion, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW, Boekhout T, editors. The Yeasts, a Taxo nomic Study. Elsevier; 2011. p. 87-110. Lavarde V, Daniel F, Saez H, Arnold M, Faguer B. Peritonite mycosique a Torulopsis haemulonii. Bul Soc Fr Mycol Med. 1984; 13: 173-6. Lehmann PF, Wu LC, Pruitt WR, Meyer SA, Ahearn DG. Unrelated ness of groups of yeasts within the Candida haemulonii complex. J Clin Microbiol. 1993; 31(7): 1683-7. Li J, Fan RS, Liu XP, Li DM, Nie ZH, Li F, et al. Biased genotype dis tributions of Candida albicans strains associated with vulvovagi nal candidosis and candidal balanopostitis in China. Clin Infect Dis. 2008; 47(9): 1119-25. 410 C. duobushaemulonii • Humberto Fabio Boatto et al. Luo G, Mitchell TG. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR. J Clin Microbiol. 2002; 40(8): 2860-5. Rocha BA, Del Negro GMB, Yamamoto L, de Souza MVB, Precioso AR, Okay TS. Identification and differentiation of Candida spe cies from pediatric patients by random amplified polymorphic DNA. Rev Soc Bras Med Trop. 2008; 41(1): 1-5. Oberoi JK, Wattal C, Goel N. Non-albicans Candida species in blood stream infections in a tertiary care hospital at New Delhi, India. Indian J Med Res. 2012; 136(6): 997-1003. Ruan SY, Kuo YW, Huang CT, Hsiue HC, Hsueh PR. Infections hae mulonii due to Candida: species identification, antifungal suscep tibility and outcomes. Int J Antimicrob Agents 2010; 35(1): 85-8. REFERENCES Pappas PG, Kauffman CA, Andes DR, Clancy CJ, Marr KA, Os trosky-Zeichner L, et al. Clinical practice guideline for the man agement of candidiasis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis. 2016; 62(4): e1-50. Sobel JD, Wiesenfeld HD, Martens MGE, Danna P, Hooton TM, Rompalo A. Maintenance fluconazole therapy for recurrent vul vovaginal candidiasis. N Engl J Med. 2004; 351(9): 876-83. Sobel JD. Vulvovaginal candidosis. 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https://openalex.org/W1985377889	https://europepmc.org/articles/pmc4049582?pdf=render	English	null	Osteopontin (OPN) Is an Important Protein to Mediate Improvements in the Biocompatibility of C Ion-Implanted Silicone Rubber	PloS one	2,014	cc-by	9,149	Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: caocong@suda.edu.cn (CC); fdltmmu@sina.com (DF) * E-mail: caocong@suda.edu.cn (CC); fdltmmu@sina.com (DF) * E-mail: caocong@suda.edu.cn (CC); fdltmmu@sina.com (DF) . These authors contributed equally to this work. attached more strongly and grew faster on silicone rubber coated with carbon nanotubes (CNTs) than on uncoated rubber [11]. Among the various techniques, ion implantation is versatile and attractive because several properties such as cyto-compatibility and corrosion resistance will be enhanced, whereas the favorable attributes including biomechanical properties can usually be preserved [12–15]. Several groups have reported the use of nitrogen and carbon plasma immersion ion implantation (N-PIII and C-PIII) to modify Ti-6Al-4V [16]. Ion implantation has been also applied on biomedical polymer materials, including PMMA, polytetrafluorethylene (PTFE) and others [17]. However, few papers discussing ion implantation on silicone rubber have been found, not to mention the mechanism of its possible cyto- compatibility improvement. Osteopontin (OPN) Is an Important Protein to Mediate Improvements in the Biocompatibility of C Ion-Implanted Silicone Rubber Shao-liang Wang1., Xiao-hua Shi1., Zhi Yang1., Yi-ming Zhang1, Li-ru Shen2, Ze-yuan Lei1, Zhi-qing Zhang3, Cong Cao3, Dong-li Fan1 1 Department of Plastic and Cosmetic Surgery, Xinqiao Hospital, Third Military Medical University, Chongqing, People’s Republic of China, 2 Southwestern Institute of Physics, Chengdu, Sichuan, People’s Republic of China, 3 Institute of Neuroscience, Soochow University, Suzhou, Jiangsu, People’s Republic of China 1 Department of Plastic and Cosmetic Surgery, Xinqiao Hospital, Third Military Medical University, Chongqing, People’s Republic o Physics, Chengdu, Sichuan, People’s Republic of China, 3 Institute of Neuroscience, Soochow University, Suzhou, Jiangsu, People’s Editor: Sanjoy Bhattacharya, Bascom Palmer Eye Institute, University of Miami School of Medicine, United States of America ation: Wang S-l, Shi X-h, Yang Z, Zhang Y-m, Shen L-r, et al. (2014) Osteopontin (OPN) Is an Important Protein to Mediate compatibility of C Ion-Implanted Silicone Rubber. PLoS ONE 9(6): e98320. doi:10.1371/journal.pone.0098320 Abstract Medical device implants are drawing increasing amounts of interest from modern medical practitioners. However, this attention is not evenly spread across all such devices; most of these implantable devices can cause adverse reactions such as inflammation, fibrosis, thrombosis, and infection. In this work, the biocompatibility of silicone rubber (SR) was improved through carbon (C) ion implantation. Scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) results confirmed that these newly generated carbon- implanted silicone rubbers (C-SRs) had large, irregular peaks and deep valleys on their surfaces. The water contact angle of the SR surface decreased significantly after C ion implantation. C ion implantation also changed the surface charge distribution, silicone oxygen rate, and chemical-element distribution of SR to favor cell attachment. The dermal fibroblasts cultured on the surface C-SR grew faster and showed more typical fibroblastic shapes. The expression levels of major adhesion proteins, including talin-1, zyxin, and vinculin, were significantly higher in dermal fibroblasts cultured on C-SR coated plates than in dermal fibroblasts cultured on SR. Those same dermal fibroblasts on C-SRs showed more pronounced adhesion and migration abilities. Osteopontin (OPN), a critical extracellular matrix (ECM) protein, was up-regulated and secreted from dermal fibroblasts cultured on C-SR. Matrix metalloproteinase-9 (MMP-9) activity was also increased. These cells were highly mobile and were able to adhere to surfaces, but these abilities were inhibited by the monoclonal antibody against OPN, or by shRNA-mediated MMP-9 knockdown. Together, these results suggest that C ion implantation significantly improves SR biocompatibility, and that OPN is important to promote cell adhesion to the C-SR surface. Received December 24, 2013; Accepted May 1, 2014; Published June 9, 2014 Copyright: 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a grant from National Natural Science Foundation of China (81071574) (http://www.nsfc.gov.cn/publish/portal0/default.htm), a grant for Transformation of Scientific and Technological Achievements from Third Military Medical University (2012XZH05) (http://www.tmmu.edu.cn/), and the research start-up funds of Soochow University to Cong Cao (Q321506612)(http://www.suda.edu.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Editor: Sanjoy Bhattacharya, Bascom Palmer Eye Institute, University of Miami School of Medicine, United States of America OPN Mediate Biocompatibility Improvements of C-SR [17–19]. Depending on the physical and chemical properties of their surfaces, the implant materials absorb ECM proteins, which work as ligands to bind to integrins and other receptors on the cell surface. The ligand-receptor interaction then forms a focal adhesion complex, which activates signal transduction through focal adhesion kinase. These signals provoke recombination of the cytoskeleton, involving adhesion-associated proteins including talin-1, vinculin, zyxin and others, which facilitates cell adhesion to the implanted materials, where these cells will migrate and proliferate [17–19]. applied to observe the surface micro-morphology and the fibroblast shape. The latter was also examined by atomic force microscopy (AFM) (SAP 400, Seiko Instrument Inc.). For the fourier transform infrared spectroscopy (FTIR) analysis, the SR and C-SRs were cleaned by dehydrated alcohol. And then the materials surface ingredients were examined by FTIR microscopy (Nicolet 470 spectrometer, Thermo Scientific), with the wave number at 4 cm21, and the scan extent at 4000,400 cm21. For the X-ray Diffraction (XRD) detection, the SR and C-SRs squares were cleaned by dehydrated alcohol, non-destructive XRD (D/ MAX 1200, Rigaku Inc.) was applied to reveal information about crystallographic compounds and to determine relative abundance by comparing diffraction data to a database maintained by the International Centre for Diffraction Data. X-ray photoelectron spectroscopy (XPS) (ESCALAB 250, Thermo Scientific) was applied to characterize the adsorbed species on the surface of the above samples. The water contact angles of SR and three kinds of C-SRs were measured with a drop shape analysis system (DSA100, Kru¨ss) in the sessile mode at room temperature. The results of surface characteristic are shown in Figure 1. In the current study, we successfully modified the surface of SR by carbon (C) ion implantation. We examined the effectiveness of C ion implantation by evaluating the results of surface character- istics and the cyto-compatibility. Results indicated that when human dermal fibroblasts were cultured on carbon-implanted silicone rubber (C-SR), they tended to grow faster and showed more typical fibroblast shapes than cells cultured under SR, they also demonstrated more pronounced adhesion and migration abilities. Meanwhile, adhesion-associated proteins including talin- 1, vinculin and zyxin were up-regulated in these cells. These phenomena were found to be positively related with ion doses. In particular, we found that OPN, an ECM component and a soluble cytokine, worked as an important protein to mediate biocompat- ibility improvement of C ion-implanted silicone rubber. SR surface modification The surface of SR was modified by carbon ion implantation. Carbon atom is a compositional element of SR and is harmless to the human body. Carbon ions were implanted onto the surface of SR in a neutral and ionized alkaline bombardment-type heavy ion source, in which the ions were produced from a sintered carbon (99.99%). The silicone rubber surface was implanted with carbon ions at an ion energy of 10 keV and carbon ion doses of 161015 ions/cm2 (C-SR-1), 361015 ions/cm2 (C-SR-2) and 161016 ions/ cm2 (C-SR-3). The current density during implantation less than 600 nA/cm2 and residual gas pressure was less than 161023 Pa. All operations were carried out under sterile conditions. OPN Mediate Biocompatibility Improvements of C-SR Possible mechanisms by which OPN might improve cyto-compatibility were also investigated. We found that MMP-9 is the downstream signal molecular of OPN, playing a role in cells migration. These results suggested that C ion implantation can improve the biocompatibility of SR considerably, and that OPN works as an important protein to promote cell adhesion on the surface of C- SR. Silicone rubber (SR) preparation Silicone rubber (SR) preparation Equal amounts (10 ml) of clinical-quality liquid SR-A and SR-B (Polydimethylsiloxane, GN501, viscosity 1500–2000, Chenguang Research Institute of Chemical Engineering, China) were mixed, stirred and slowly injected into a metal plate mold (100 mm6100 mm60.5 mm). The mixture was then vacuumed under 20.1 MPa for 30 minutes. It was then allowed to solidify at room temperature for 5.5 h. Only pure silicone rubber sheets with smooth surfaces were used for further experiments. All operations were carried out under sterile conditions. Human dermal fibroblasts (16106) were collected and seeded at a density of 16103 cells/well in 96-well plate pre-coated with SR or indicated C-SRs. The cells were further cultured in growth medium in 37uC incubator for 24, 48 and 96 hours. Cell viability was then measured using a CCK-8 (Dojindo, Japan) according to manufacturer’s protocol [20]. The OD value of cells cultured on C-SR surface was normalized to that of cells cultured on SR surface. All experiments repeated six times with 6 well for each condition. Cell culture The human dermal fibroblasts (Cell Bank of Chinese Academy of Sciences, Shanghai, China) were maintained in DMEM medium (Sigma, St.Louis, MO, USA), supplemented with a 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), penicillin/Streptomycin (1:100, Sigma, St.Louis, MO, USA) and 4 mM L-glutamine (Sigma, St.Louis, MO, USA), in a CO2 incubator at 37uC. Materials and Methods Human dermal fibroblasts were collected and seeded into 24- well multi-plates with or without SR and C-SRs (sample diameter 14 mm), at a density of 56104 cells/well and cultured for 6 h. The culture medium was removed and cells were washed twice with PBS. Then adherent cells were collected by trypsinization and diluted with a trypan blue/DMEM mixture after centrifugation (1000 rpm, 200 g, 5 min) and counted with a hemacytometer. Cell adhesion rate was calculated from the proportion of adherent/seeded cells. Introduction Bio-implant materials are widely used in reconstructing and repairing human organs damaged by injuries or degradation [1]. Silicone rubber (SR)-based materials have been used for many years, as they have excellent physiology inertia, high adsorption properties, high corrosion resistance, good chemical stability and high mechanical strength [2,3]. It is also workable and convenient. However, there are still many tribulations when using this material. The intrinsically hydrophobic nature of SR surface makes cell adhesion almost impossible, causing problems like fibrous capsules, contracture formation, and displacement during long-term usage [4–6]. Poor cell adhesion on its surface allows a gap to form between the SR implant and surrounding tissues, this can lead to bacterial invasion [7]. One possible explanation for the improvements in cell attachment and retention involves protein adsorption, which is known to determine the accessibility of implanted materials to cells The surface modification to improve the biocompatibility of SR is a common way of addressing this issue. In recent years, there have been many attempts [8–10]. For example, osteoblast cells June 2014 \| Volume 9 \| Issue 6 \| e98320 1 June 2014 \| Volume 9 \| Issue 6 \| e98320 PLOS ONE \| www.plosone.org OPN Mediate Biocompatibility Improvements of C-SR Cell morphology and adhesion proteins observation by SEM and immuno-fluorescence As described previously [9], human dermal fibroblasts were cultured on SR or C-SR surface as monolayer, and then rinsed with PBS and fixed with 3% buffered glutaraldehyde for 20 min at 4uC. Then samples were dehydrated with aqueous ethanol (30– 100%) step by step. Samples were lyophilized and coated with platinum. Cell morphology was observed by SEM (AMRAY 1000- B, Amray Inc, Bedford, Mass, USA). SR and C-SR surface observation and analysis SR and C-SR surface observation and analysis For scanning electron microscope (SEM, AMRAY 1000-B, Amray Inc, Bedford, Mass, USA) observations, SR and C-SRs were cut into 10 mm610 mm squares and dried in 37uC. Afterwards, these squares were put into a vacuum pump to spray painting gold coat on the surface. SEM microscope was then As described previously [21,22], human dermal fibroblasts were cultured on SR or C-SR surface as monolayer on cover slips and were fixed in cold paraformaldehyde (4%) for 15 min at 24uC, PLOS ONE \| www.plosone.org June 2014 \| Volume 9 \| Issue 6 \| e98320 2 Figure 1. C ion implantation changes physical and chemical properties of SR surface. (A) AFM images show the micro-morphology of the surfaces of SR and three different C-SRs (C-SR-1, C-SR-2 and C-SR-3). C ion implantation changed the surface micro-morphology of SR. Note that with the ion implantation dose increased, the surface tended to be more uneven, but arranged regularly. (B and C) XPS analysis of the chemical composition of SR and three different C-SRs. C ion implantation changed the chemical composition of SR. (D) XRD analysis of SR and three different C- SRs. Results show that the XRD pattern of SR was changed after C ion implantation, indicating that new crystal structures may form. The difference is very small. (E) FTIR analysis of the formation of new bonding motifs. The spectrums for C-SRs were similar to that for pristine SR. (F) The water contact angle of SR and three different C-SRs. Note that C ion implantation significantly decreased water contact angle. p,0.05 The difference was statistically significant when C-SR-2 or C-SR-3 was compared to SR. Experiments in this figure were repeated three times, similar results were obtained each time. doi:10.1371/journal.pone.0098320.g001 OPN Mediate Biocompatibility Improvements of C-SR OPN Mediate Biocompatibility Improvements of C-SR Figure 1. C ion implantation changes physical and chemical properties of SR surface. (A) AFM images show the micro-morphology of the surfaces of SR and three different C-SRs (C-SR-1, C-SR-2 and C-SR-3). C ion implantation changed the surface micro-morphology of SR. Note that with the ion implantation dose increased, the surface tended to be more uneven, but arranged regularly. (B and C) XPS analysis of the chemical composition of SR and three different C-SRs. C ion implantation changed the chemical composition of SR. (D) XRD analysis of SR and three different C- SRs. SR and C-SR surface observation and analysis (A) FITC-labeled actin tracer was used to observe cytoskeleton by immuno-fluorescence microsco Fibroblasts cultured on C-SRs had more and larger filopodia spreading out, and their microfilament stretched longer and arranged more regularly. SEM images further demonstrated a more fibroblastic appearance of fibroblasts cultured on C-SRs. (C) Cell area depicted higher values for cells on SRs than those on SR. But the difference was not statistically significant between any two groups (p.0.05). (D) Dermal fibroblasts were cultured in well plate (at a density 16103 cells/well) for 24, 48 and 96 hours. The cell viability was analyzed by CCK-8 assay. The cell viability was also increased C ion implantation. p,0.05 The difference was statistically significant when the substrate was compared to SR at any time point. #p,0.05 T difference was statistically significant when the substrate was compared to TCP at any time point. Experiments in this figure were repeated th times, similar results were obtained each time. Bar = 20 mm. All data are expressed as mean 6 SD (error bars). doi:10.1371/journal.pone.0098320.g002 PLOS ONE \| www plosone org 4 June 2014 \| Volume 9 \| Issue 6 \| e983 Figure 2. Human dermal fibroblasts cultured on C-SR grow faster and showed a more fibroblastic appearance. Dermal fibroblasts (16106) were seeded in 6-well plate pre-coated with SR or three C-SRs (C-SR-1, C-SR-2 and C-SR-3). The cells were incubated in 37uC incubator for 48 hours for observation of cell morphology. (A) FITC-labeled actin tracer was used to observe cytoskeleton by immuno-fluorescence microscopy. Fibroblasts cultured on C-SRs had more and larger filopodia spreading out, and their microfilament stretched longer and arranged more regularly. (B) SEM images further demonstrated a more fibroblastic appearance of fibroblasts cultured on C-SRs. (C) Cell area depicted higher values for cells on C- SRs than those on SR. But the difference was not statistically significant between any two groups (p.0.05). (D) Dermal fibroblasts were cultured in 96- well plate (at a density 16103 cells/well) for 24, 48 and 96 hours. The cell viability was analyzed by CCK-8 assay. The cell viability was also increased by C ion implantation. p,0.05 The difference was statistically significant when the substrate was compared to SR at any time point. #p,0.05 The difference was statistically significant when the substrate was compared to TCP at any time point. Experiments in this figure were repeated three times, similar results were obtained each time. Bar = 20 mm. SR and C-SR surface observation and analysis Results show that the XRD pattern of SR was changed after C ion implantation, indicating that new crystal structures may form. The difference is very small. (E) FTIR analysis of the formation of new bonding motifs. The spectrums for C-SRs were similar to that for pristine SR. (F) The water contact angle of SR and three different C-SRs. Note that C ion implantation significantly decreased water contact angle. p,0.05 The difference was statistically significant when C-SR-2 or C-SR-3 was compared to SR. Experiments in this figure were repeated three times, similar results were obtained each time. doi:10 1371/journal pone 0098320 g001 doi:10.1371/journal.pone.0098320.g001 doi:10.1371/journal.pone.0098320.g001 hai, China) to zyxin were used. In Figure 4B, rabbit anti-OPN (1:100, Santa Cruz Biotech, Santa Cruz, CA, USA) was used as primary antibody and FITC-tagged secondary antibody (Invitro- gen, Shanghai, China) was used. In Figure 4C, the cytoskeleton was also stained with FITC-labeled actin Tracker probes (Beyotime, Shanghai, China). In Figure 5D, rabbit anti-a-tublin (1:1000, millipore, MA, USA) was used as primary antibody and the corresponding Cy3-tagged secondary antibody (Beyotime, Shanghai, China) was used to detect cytoskeleton. The cell nuclei were stained with 49, 69-diamidino-2-phenylindole (DAPI, 0.5 mg/ ml) (Sigma-Aldrich, St.Louis, MO, USA) in Figure 2, 3, 4 and 5. Cells were visualized through a Leica confocal microscope with washed three times with PBS. Afterwards, cells were blocked with 5% BSA in PBS (pH 7.5) for 30 min, followed by overnight incubation with the primary antibody. The corresponding secondary antibody was then added for 1 h at room temperature. In Figure 2, the cytoskeleton was stained with FITC-labeled actin Tracker probes (Beyotime, Shanghai, China). In Figure 3A, the primary antibodies were rabbit anti-talin-1 (1:500, Abcam, Cambridge, England), rabbit anti-zyxin (1:500, cell signaling, Danvers, MA, USA) or mouse anti-vinculin (1:500, Sigma- Aldrich, St.Louis, MO, USA) and the corresponding Cy3-tagged secondary antibodies to talin-1 and vinculin (Beyotime, Shanghai, China) and FITC-tagged secondary antibody (Invitrogen, Shang- PLOS ONE \| www.plosone.org June 2014 \| Volume 9 \| Issue 6 \| e98320 3 OPN Mediate Biocompatibility Improvements of C-SR Figure 2. Human dermal fibroblasts cultured on C-SR grow faster and showed a more fibroblastic appearance. Dermal fibrobla (16106) were seeded in 6-well plate pre-coated with SR or three C-SRs (C-SR-1, C-SR-2 and C-SR-3). The cells were incubated in 37uC incubator 48 hours for observation of cell morphology. SR and C-SR surface observation and analysis All data are expressed as mean 6 SD (error bars). doi:10.1371/journal.pone.0098320.g002 June 2014 \| Volume 9 \| Issue 6 \| e98320 PLOS ONE \| www.plosone.org 4 OPN Mediate Biocompatibility Improvements of C-SR Figure 3. Dermal fibroblasts adhere more strongly to the surface of C-SR with increased expression levels of adhesion proteins. Immuno-fluorescence results (A) and western blots results (B and C) show the expressions and localization of talin-1, zyxin and vinculin in dermal fibroblasts cultured on SR or different C-SRs coated 6-well plate (48 hours). Note that there were significantly more expression levels of talin-1, zyxin and vinculin in dermal fibroblasts cultured on C-SRs than in other cells. p,0.05 The difference was statistically significant when the substrate was compared to SR for detecting different adhesion protein. These experiments were repeated three times, similar results were obtained each time. Bar = 20 mm. doi:10 1371/journal pone 0098320 g003 Figure 3. Dermal fibroblasts adhere more strongly to the surface of C-SR with increased expression levels of adhesion proteins. Immuno-fluorescence results (A) and western blots results (B and C) show the expressions and localization of talin-1, zyxin and vinculin in dermal fibroblasts cultured on SR or different C-SRs coated 6-well plate (48 hours). Note that there were significantly more expression levels of talin-1, zyxin and vinculin in dermal fibroblasts cultured on C-SRs than in other cells. p,0.05 The difference was statistically significant when the substrate was compared to SR for detecting different adhesion protein. These experiments were repeated three times, similar results were obtained each time. Bar = 20 mm. d l m doi:10.1371/journal.pone.0098320.g003 PAGE) and transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). After blocking with 10% instant nonfat dry milk for 1 hour, the membrane was incubated with specific antibody overnight at 4uC, followed by incubation with secondary antibodies (HRP-conjugated) for 60 min at room temperature. The antibody binding was detected with the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Piscataway, NJ, USA). The primary antibodies used in this study were as follows: rabbit anti-OPN (1:300, Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti-zyxin (1:1000, cell signaling, Danvers, MA, USA), rabbit anti-talin-1 (1:1000, Abcam, Cambridge, England), mouse anti-vinculin (1:5000, Sigma-Al- drich, St.Louis, MO, USA) antibodies, and the anti-GAPDH (the house keeping gene, 1:1000, Sigma-Aldrich, St.Louis, MO, USA). The OPN in the supernatant of dermal fibroblast culture medium was also tested by western blot. SR and C-SR surface observation and analysis The intensity of each blot was quantified by Image J software, and was normalized to the loading the appropriate filters. All the experiments were repeated six times and 6 slides for each condition. Cell surface area for comparing cell shape was measured by Image-Pro plus 6, six cells of each sample were selected randomly for quantitative comparison. Protein isolation, western blot and data quantification q As described before [22–24], dermal fibroblasts were seeded on the 6-well plate coated with SR or C-SRs for 48 hours. Afterwards, cells were washed with ice-cold PBS and lysed using lysis buffer (pH, 7.4) containing 1% Nonidet P-40 (NP-40), 1% deoxycholate, 0.1% sodium dodecyl sulfate, 150 mmol/L NaCl and 10 mmol/L Tris-HCl. The lysates were collected and centrifuged. The concentration of the extracted protein was measured by bicinchoninic acid assay kit (catalogue B9643, Sigma- Aldrich, St.Louis, MO, USA). Aliquots of 30–40 mg of protein from each sample (treated as indicated in the legends) were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS- June 2014 \| Volume 9 \| Issue 6 \| e98320 PLOS ONE \| www.plosone.org 5 OPN Mediate Biocompatibility Improvements of C-SR Figure 4. OPN acts as an important protein to promote cell adhesion on the surface of C-SR. (A) Western blots results show the expression level of OPN secreted from dermal fibroblasts cultured on SR or C-SR coated 6-well plate (48 hours). Note that OPN was up-regulated and secreted from dermal fibroblasts cultured on C-SRs. (B) Immuno-fluorescence images show that the expression level of OPN in dermal fibroblasts cultured on C-SR coated 6-well plate was increased (48 hours). (C) FITC-labeled actin tracer results demonstrate that exogenously-added purified OPN (0.5 mg/ml) enhanced dermal fibroblasts adhesion and proliferation. On the other hand, the monoclonal antibody against OPN inhibited dermal fibroblasts adhesion and proliferation on the surface of C-SRs (48 hours). (D) Cell adhesion on different substrates. OPN promoted fibroblasts adhesion, and monoclonal antibody against OPN significantly inhibited cell adhesion. However, the difference was not statistically significant when the substrate in group (OPN2 Anti-OPN2) was respectively compared to the substrate in group (OPN+ Anti-OPN2) or group (OPN2 Anti-OPN+) (p. 0.05). The difference was significant between the substrates in group (OPN+ Anti-OPN2) and group (OPN2 Anti-OPN+) (p,0.05). These experiments were repeated three times, similar results were obtained each time (Fig. 4A, Fig. 4B and Fig. 4C). Bar = 20 mm. The cell adhesion test was performed six times, the data are expressed as mean 6 SD (Fig. 4D). doi:10.1371/journal.pone.0098320.g004 Figure 4. OPN acts as an important protein to promote cell adhesion on the surface of C-SR. (A) Western blots results show the expression level of OPN secreted from dermal fibroblasts cultured on SR or C-SR coated 6-well plate (48 hours). Protein isolation, western blot and data quantification Note that OPN was up-regulated and secreted from dermal fibroblasts cultured on C-SRs. (B) Immuno-fluorescence images show that the expression level of OPN in dermal fibroblasts cultured on C-SR coated 6-well plate was increased (48 hours). (C) FITC-labeled actin tracer results demonstrate that exogenously-added purified OPN (0.5 mg/ml) enhanced dermal fibroblasts adhesion and proliferation. On the other hand, the monoclonal antibody against OPN inhibited dermal fibroblasts adhesion and proliferation on the surface of C-SRs (48 hours). (D) Cell adhesion on different substrates. OPN promoted fibroblasts adhesion, and monoclonal antibody against OPN significantly inhibited cell adhesion. However, the difference was not statistically significant when the substrate in group (OPN2 Anti-OPN2) was respectively compared to the substrate in group (OPN+ Anti-OPN2) or group (OPN2 Anti-OPN+) (p. 0.05). The difference was significant between the substrates in group (OPN+ Anti-OPN2) and group (OPN2 Anti-OPN+) (p,0.05). These experiments were repeated three times, similar results were obtained each time (Fig. 4A, Fig. 4B and Fig. 4C). Bar = 20 mm. The cell adhesion test was performed six times, the data are expressed as mean 6 SD (Fig. 4D). d i 10 1371/j l 0098320 004 10% glycerol, 2% SDS, 0.1% bromophenol blue), and were added to the gel wells without denature (boiling). Following electropho- resis, gels were washed twice in 2.5% Triton X-100 for 30 min at 37uC to remove the SDS. Gels were further incubated at 37uC overnight in the developing buffer containing 50 mmol/L Tris- HCl, 0.2 mol/L NaCl, 5 mmo/L CaCl2 and 0.02% Triton X- 100. Gels were stained with 0.5% coomassie blue in 30% methanol, 10% glacial acetic acid for 30 min, and were then de- stained in the same solution but coomassie blue. Gelatin-degrading control (GAPDH). Each experiment was repeated at least three times, with 3 wells per replicate. Total RNA isolation and real-time reverse transcriptase polymerase chain reaction (RT-PCR) The expression of MMP-9 mRNA was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was prepared from cultured cells using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instruc- tions. The concentration and purity of RNA was measured spectrophotometrically at A260 and A280. RT-PCR was per- formed using TOYOBO ReverTra Ace RT-PCR kit according to the manufacturer’s instruction. The resulting cDNA was used as a template for PCR with specific primer pairs using Primer Premier 5.0 software (Premier Biosoft, International, Palo Alto, CA, USA). The results were analyzed using delta Ct. All real-time PCRs were performed three times at least. Zymography The MMP-9 activity was analyzed using SDS-PAGE substrate gels. Gelatin (Bloom 300, Sigma-Aldrich, St.Louis, MO, USA) was added to 10% acrylamide separating gel at a final concentration of 1 mg/ml. Samples containing equal amount proteins were mixed with non-reducing sample buffer (62.5 mM Tris-HCl, pH 6.8, PLOS ONE \| www.plosone.org June 2014 \| Volume 9 \| Issue 6 \| e98320 6 OPN Mediate Biocompatibility Improvements of C-SR Figure 5. MMP-9 is important for dermal fibroblast adhesion and migration on the surface of C-SR. (A) Transwell results show that exogenously-added OPN and MMP-9 facilitated dermal fibroblasts migration (48 hours). (B) Zymography assay shows the MMPs activity of fibroblasts cultured on SR or different C-SR coated 6-well plate (48 hours). The difference was statistically significant when any C-SR was compared to SR (p,0.05). (C) MMP-9 mRNA level in control dermal fibroblasts or in fibroblasts infected with MMP-9-shRNA or scramble- shRNA containing lentiviral particles. (D and E) Immuno-fluorescence results show that MMP-9 shRNA inhibited fibroblast adhesion and growth on the surface of C-SR and it was rescued by exogenously- added purified OPN (20 ng). p,0.05 The difference was statistically significant when the substrate in group (MMP9 shRNA or MMP9 shRNA+OPN) was compared to the substrate in group (vector). #p, 0.05 The difference was also statistically significant when the substrate in group (MMP9shRNA+OPN) was compared to the substrate in group (MMP9shRNA). These experiments were repeated three times, similar results were obtained each time. Bar = 20 mm. The cell adhesion test was performed six times, the data are expressed as mean 6 SD (Fig. 5E). doi:10.1371/journal.pone.0098320.g005 analysis, experiments were repeated three times at least. MMP-9 activity level in C-SR group was expressed in the form of fold changes vs. SR group. Transwell assay Fibroblasts were maintained for 24 h in serum-free medium, prior to treatment with OPN (0 ng/L, 5 ng/L, 10 ng/L, 20 ng/L) (Proteintech Group, Chicago, IL, USA) or MMP9 (0 ng/L, 5 ng/ L, 10 ng/L, 20 ng/L) (Proteintech Group, Chicago, IL, USA) for a further 24 h. Fibroblasts (100 ml in DMEM, 16105 cells/ml) were then plated onto upper chamber in a 24-well plate, according to the manufacturer’s instructions (Millipore, MA, USA). After migration for 24 h, penetrated cells on the filters were fixed in dried methanol, stained in 0.1% crystal violet, washed by PBS, and then photographed in each group. Figure 5. MMP-9 is important for dermal fibroblast adhesion and migration on the surface of C-SR. (A) Transwell results show that exogenously-added OPN and MMP-9 facilitated dermal fibroblasts migration (48 hours). (B) Zymography assay shows the MMPs activity of fibroblasts cultured on SR or different C-SR coated 6-well plate (48 hours). The difference was statistically significant when any C-SR was compared to SR (p,0.05). (C) MMP-9 mRNA level in control dermal fibroblasts or in fibroblasts infected with MMP-9-shRNA or scramble- shRNA containing lentiviral particles. (D and E) Immuno-fluorescence results show that MMP-9 shRNA inhibited fibroblast adhesion and growth on the surface of C-SR and it was rescued by exogenously- added purified OPN (20 ng). p,0.05 The difference was statistically significant when the substrate in group (MMP9 shRNA or MMP9 shRNA+OPN) was compared to the substrate in group (vector). #p, 0.05 The difference was also statistically significant when the substrate in group (MMP9shRNA+OPN) was compared to the substrate in group (MMP9shRNA). These experiments were repeated three times, similar results were obtained each time. Bar = 20 mm. The cell adhesion test was performed six times, the data are expressed as mean 6 SD (Fig. 5E). doi:10.1371/journal.pone.0098320.g005 Generation of MMP-9 knockdown stable dermal fibroblasts by lentiviral infection y The dermal fibroblasts were seeded in a 6-well plate with 60% confluence in growth medium with polybrene. Twenty ml/ml of lentiviral particles containing MMP-9 shRNA (Santa Cruz Biotech, Santa Cruz, CA, USA) were added to the cells for 24 h, cell culture medium was then replaced by growth medium and cells were cultured for another 24 h, stable clones expressing MMP-9 shRNA were selected using puromycin (1 mg/ml). The culture medium was replaced with fresh puromycin-containing culture medium every 2–3 days, until resistant colonies could be identified. The MMP-9 mRNA expression in the transfected cells was detected by RT-PCR in the resistant colonies. Same amount of scramble non-sense shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA, USA) was added to the control cells. Statistical analysis The data presented in this study was expressed as means 6 SD (standard deviation). Statistical difference was analyzed by one- way ANOVA followed by multiple comparisons performed with post hoc Bonferroni test (SPSS version 16.0). Value of p,0.05 was considered statistically significant. June 2014 \| Volume 9 \| Issue 6 \| e98320 OPN Mediate Biocompatibility Improvements of C-SR change the macro-scale surface of SR. However, results from the AFM images revealed that the surfaces of C-SRs were composed of larger irregular peaks and deeper valleys, while SR exhibited a relatively smooth and more homogeneous surface (Figure 1A). The surface roughness of C-SR-3, which underwent most C-ion implantation, was highest among all three C-SRs (Figure 1A). Thus, C ion implantation on the SR changed the surface microstructure. To further estimate the physical and chemical properties C-SRs, XPS analysis was employed. Results showed that C ion implantation significantly changed the surface charge distribution, silicone oxygen rate and chemical-element distribu- tion of SR, all of which could facilitate cell adhesion (Figure 1B and C). Note that with the ion implantation dose increasing, the C content in the material increased, while the Si content decreased (Figure 1C), suggesting that implanted C atom may replace the Si of SR, interrupting the original Si-O assemble, so the surface free energy increases, thereby theoretically decreasing material’s water contact angle. The XRD images exhibited characteristic peaks (black arrow) pattern with crystalline materials in both SR and C- SRs. The distinct XRD patterns among SR and C-SRs, or among three C-SRs may be attributable to different amount of C ion implantation. It is notable that the difference was very small (Figure 1D). FTIR analysis was performed to clarify the formation of new bonding motifs. The spectrums for C-SRs are similar to that for pristine SR (Figure 1E). These results of XPS, XRD and FTIR indicate that the C ion implantation may interrupt the silicon bonds, but the interaction between C ion and SR is weak. To test the hydrophilicity/hydrophobicity of these materials, water contact angle was also examined. We found that C ion implantation significantly decreased the water contact angle of SR (Figure 1F), and C-SR-3 had lowest water contact angle among all C-SRs (Figure 1F). These results together indicate C ion implantation significantly changes the physical and chemical properties of SR surface, to possibly favor an improved biocompatibility. vinculin. These proteins are involved in the formation of focal adhesion complexes, acting as a conjugation site for both cytoskeleton organization and integrin signaling transduction, which is a critical step for cell adhesion/migration initiation. Immuno-fluorescence results revealed that dermal fibroblasts cultured on C-SRs expressed higher levels of talin-1, zyxin and vinculin than cells grew on SR (Figure 3A). Discussions There are many ways to remodel the surface of SR, including microwave plasma surface modification [27], copolymerization [28] and others. By using indirect copolymerization, studies have integrated multiple drugs or related compounds onto the SR surface to improve its biocompatibility. For example, halofugi- none, an anti-fibrotic drug was attached on the surface of silicone breast implants to reduce the capsular fibrosis [29]. Oxygen/ OPN works as an important protein to promote cell adhesion on the surface of C-SR OPN originally isolated from bone, is a multifunctional acidic glycoprotein. It is secreted from cells, works as both an ECM component and a soluble cytokine [25], and is able to bind to both RGD-containing integrins (avb1, avb3, avb5, avb1, a8b1) or non-RGD integrins (a4b1, a9b1) to promote cell survival, proliferation, migration, invasion, and metastasis [26]. Western blot results in Figure 4A showed that dermal fibroblasts cultured on C-SRs had higher OPN expression and secretion than those cultured on SR. Immuno-fluorescence images in Figure 4B further confirmed higher intracellular OPN expression in those dermal fibroblasts. Importantly, exogenously-added OPN promoted dermal fibroblast adhesion on both SR and C-SR surfaces. On the other hand, the monoclonal antibody against OPN signifi- cantly inhibited cell adhesion on the C-SR surfaces (Figure 4C). However, the difference was not statistically significant when the substrate in group (OPN2 Anti-OPN2) was respectively com- pared to the substrate in group (OPN+ Anti-OPN2) or group (OPN2 Anti-OPN+) (p.0.05). The difference was significant between the substrate in group (OPN+ Anti-OPN2) and group (OPN2 Anti-OPN+) (* p,0.05). These results together suggest that dermal fibroblasts cultured on C-SR surface express and secrete higher level of OPN, which helps cell adhesion. OPN Mediate Biocompatibility Improvements of C-SR In addition, western- blot and relative intensity analysis showed that with the implanted C ion dosage increasing, the expression levels of above proteins were higher (Figure 3B and 3C). These results and results in Figure 2 suggested that the growth of dermal fibroblasts on C-SRs is better than those on SR, and the expression levels of many adhesion proteins are increased. Human dermal fibroblasts cultured on C-SR grew faster and had a more fibroblastic appearance From the cytoskeleton images in Figure 2A, we found that human dermal fibroblasts cultured on the C-SR grew faster, and showed a more fibroblastic appearance. These cells had more and larger filopodia spreading out, and their microfilament stretched longer and arranged more regularly (Figure 2A). While cells cultured on the regular SR grew slower, and had a ‘‘narrow’’ shape (Figure 2A). SEM image results further demonstrated a more fibroblastic appearance of fibroblasts cultured on C-SRs (Figure 2B). We also quantified the morphology of fibroblasts on different substrates. The cell area depicted higher values for cells on C-SRs than those on SR, and it was positively correlated with implanted C ion doses. But there was no statistically significant difference (p.0.05) (Figure 2C). Results in Figure 2D showed that dermal fibroblasts cultured on the C-SRs grew faster, and had higher cell viability. Cells cultured on C-SR-3 had the highest viability among SR and all C-SRs, but lower than tissue culture plates (TCP) (Figure 2D). These results together indicate that C ion implantation could provide a better environment for cell adhesion and growth. MMP-9 is important for dermal fibroblast adhesion and migration on the surface of C-SR MMP can promote ECM degradation during physiological and pathological tissue remodeling processes. Meanwhile, MMP-9 is also important for cell migration and skin wound healing. Transwell experiment demonstrated that both exogenously added human recombination MMP-9 and OPN promoted dermal fibroblasts migration (Figure 5A). Significantly, dermal fibroblasts cultured on the C-SR surfaces showed higher activity of MMP-9 by zymography (Figure 5B). After a stable MMP-9 knockdown dermal fibroblast line was created (Figure 5C), cell adhesion was largely inhibited on both SR and C-SR surfaces, which was rescued by OPN (Figure 5D and Figure 5E). These results together indicate that MMP-9 is important for dermal fibroblast adhesion and migration on the surface of C-SR. C ion implantation changes the physical and chemical properties of SR surface C-SR-1, C-SR-2 and C-SR-3 were generated as described. SEM and AFM were applied to observe the surface micro- morphology of SR and three different C-SRs. The SEM results failed to find any significant differences between SR and three C- SRs (data not shown), indicating that C ion implantation didn’t enzymes were identified as clear bands against the blue background of the stained gel. Images of stained gels were captured under illumination using the BIO CHEN Imagestroe 7500. The intensity of the bands was measured by densitometric June 2014 \| Volume 9 \| Issue 6 \| e98320 7 PLOS ONE \| www.plosone.org OPN Mediate Biocompatibility Improvements of C-SR June 2014 \| Volume 9 \| Issue 6 \| e98320 OPN Mediate Biocompatibility Improvements of C-SR Ion implantation affects the morphology and structure of the material, and it is dependent on the source of energy, energy flow, the beam and the composition of the target material [32,33]. When the ions are implanted into the substrate materials, the ions will collide with the target atoms. The collision processes may have three different outcomes: nuclear collision, electron collision, and charge exchange. Incident ions lose energy during every collision process and could be stopped within the materials, where they act as impurities. Most of these incident ions stay at the interstitial sites, and these interstitial impurities may migrate to substitution positions after annealing. This substitution doping gives the substrate materials better properties [34]. Sputtering effect is another important phenom- enon. This effect generally impacts the shape and morphology of substrate materials. During the implantation process, the incident ions induce collision cascades, the atoms of the target material may get enough energy to be ejected out from the substrate material [35]. On this account, the surface region of the smooth substrate will be sputtered away. This sputtering effect will be enhanced at low-lying areas, and then the substrate will become rougher. As a non-collagenic ECM protein, OPN exists both as a component of the ECM and as a soluble cytokine [45–48]. OPN contains an arginine-glycine-aspartate (RGD) domain, which promotes cell adhesion by binding integrins, and leads succedent intracellular signal transduction to promote cell proliferation and migration, etc [45,46,49]. However, the molecular mechanism behind OPN regulation of cell migration is not well understood. Several studies have reported that OPN may promote MMPs secretion to enhance cell migration. Recent studies have confirmed that OPN increases cell adhesion and migration through inducing MMP-9 or MMP-2 up-regulation and secretion in different cells [50,51]. In the current study, a high MMP-9 activity was observed in fibroblasts grew on the surface of C-SRs. Meanwhile, both recombinant OPN and MMP-9 promoted cell migration. Impor- tantly, the high mobility and adhesion abilities seen in cells were inhibited by MMP-9 RNAi, and such an effect was reversed by exogenously-added OPN. These results suggeste that MMP-9 is the downstream signal molecular of OPN, playing an important role in the migration of fibroblasts which are seeded on C-SRs. In the current study, we successfully modified SR surface through C ion implantation. It is well-known that surface characteristics play a vital role in the functions of biomaterials [36,37]. OPN Mediate Biocompatibility Improvements of C-SR behavior [37,41]. Moreover, small amounts of foreign atoms or molecules on the surface can dramatically alter the surface reactivity [42]. As ECM is crucial to mediating cell adhesion/ attachment onto the surface of implants [43]. And the cellular response to material involves a chain of complex biological reactions including protein adsorption, receptor-ligand binding and signal transduction. So the improvement of cyto-compatiblity is possible due to preliminary ECM protein adsorption on C-SRs, and subsequent signal transduction [44]. In the current study, we found a significant increased intracellular expression level of OPN, as the intracellular response. Meanwhile, we also found increased OPN in culture media, which was secreted by dermal fibroblasts cultured on the surface of C-SRs and will be absorbed on the surface of C-SRs. The pleiotropic nature of OPN allows it to promote cell adhesion, migration, survival and others, which may be due to its ability to interact with a variety of cell types via binding multiple integrins and activating many signaling pathways [45,46]. Furthermore, we found that the improved cells adhesion, migration and proliferation seen in those cells were largely inhibited by the monoclonal antibody against OPN. In a previous study, the increased expression level of OPN was observed in the early stage after skin wound, and it also displayed a possible fibrosis gene spatial profile with expression in deep dermal cell layers [47]. Based on our results and previous studies, we speculate that OPN is an important candidate among ECM proteins, will be absorbed onto the surface of C-SR when it is implanted subcutaneously, in accordance with future clinical application, and will play an important role in cell adhesion, growth and migration. ammonia plasma and poly (ethylene-alt-maleic anhydride) (PEMA) were also added to SR to improve surface functionality to permanently improve adhesion [30]. Meanwhile, scientists have used the chemical vapor deposition method to attach some biocompatible compound to the surface of SR. Poly (o-amino-p- xylylene-co-p-xylylene) (amino-PPX) and polyacrylamide (PAAm) were introduced onto SR to help improve its surface functions [2]. For all the methods mentioned above, the manufacture procedures are complicated and contain multiple steps, conditions are relatively harsh, which makes the industrial mass manufacture almost impossible. Ion implantation is often used in semiconductor device fabrication and metal finishing, as well as various applications in materials science research [31–35]. Conclusions Carbon (C) ion implantation was here used to modify silicone rubber (SR). The results of the present study suggest that C ion implantation significantly improves the cyto-compatibility of SR. The enhancement is attributable to the surface characteristics, including surface roughness, surface chemistry, and hydrophilic- ity/hydrophobicity. OPN was found to act as an important protein to promote cell adhesion to the surface of C-SR. OPN Mediate Biocompatibility Improvements of C-SR That is to say, the biological response to biomaterials is largely controlled by their surface characteristics, including chemistry, morphology, hydrophilicity/hydrophobicity, etc. So it is important to understand how C ion implantation impacts the surface characteristics of SR. C ion implantation increased the roughness of SR (Figure 1A), changed surface chemistry (Figure 1B and Figure 1C) and decreased water contact angle (Figure 1F). The AFM micrographs results showed that the surfaces of C-SRs were composed of large, irregular peaks and deep valleys, and these newly formed C-SRs had decreased water contact angle. Significantly, with more C ion implanted, the C-SR surface roughness increased and the hydrophobicity decreased. On rough surfaces, good cell adhesion/attachment and proliferation can be achieved [38], but decreased cell functions have also been reported [39]. Other factors such as hydrophilicity/hydrophobicity and surface chemistry are also considered important [40]. Compared with hydrophobic surface, hydrophilic surface is feasible for cell adhesion/attachment [40]. In our study, the dermal fibroblasts cultured on the surface of C-SRs grew faster, and were with higher mobility and better viability than those cultured under other conditions. Meanwhile, the cytoskeleton alignment was improved. All these results indicated that the cell adhesion/attachment on this modified surface was significantly improved. These results reveal that C ion implantation changes the surface roughness and water contact angle, thus the biocompatibility is improved, in accordance with previous studies [38]. Dermal fibroblasts adhere more strongly to the surface of C-SR with increased expressions of many adhesion proteins Cell adhesion process is associated with increased expressions of many adhesion-associated proteins, including talin-1, zyxin and June 2014 \| Volume 9 \| Issue 6 \| e98320 PLOS ONE \| www.plosone.org June 2014 \| Volume 9 \| Issue 6 \| e98320 8 OPN Mediate Biocompatibility Improvements of C-SR References 27. Ren TB, Weigel T, Groth T, Lendlein A (2008) Microwave plasma surface modification of silicone elastomer with allylamine for improvement of biocompatibility. J Biomed Mater Res A 86: 209–219. 1. 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Cao C, Huang XS, Han YY, Wan YS, Birnbaumer L, et al. (2009) Galpha(i1) and Galpha(i3) are required for epidermal growth factor-mediated activation of the Akt-mTORC1 pathway. Sci Signal 2:ra17 49. Rullo OJ, Woo JM, Parsa MF, Hoftman AD, Maranian P, et al. (2013) Plasma levels of osteopontin identify patients at risk for organ damage in systemic lupus erythematosus. Arthritis Res Ther 15: R18. p y g 24. Cao C, Rioult-Pedotti MS, Migani P, Yu CJ, Tiwari R, et al. (2013) Impairment of TrkB-PSD-95 Signaling in Angelman Syndrome. PLoS Biol 11:e1001478 50. Castellano G, Malaponte G, Mazzarino MC, Figini M, Marchese F, et al. (2008) Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression. Clin Cancer Res 14: 7470–7480. 25. Hunter C, Bond J, Kuo PC, Selim MA, Levinson H (2012) The role of osteopontin and osteopontin aptamer (OPN-R3) in fibroblast activity. J Surg Res 176: 348–358. 51. Mi ZY, Guo HT, Wai PY, Gao CJ, Kuo PC (2006) Integrin-linked kinase regulates osteopontin-dependent MMP-2 and uPA expression to convey metastatic function in murine mammary epithelial cancer cells. Carcinogenesis 27: 1134–1145. 26. Zhang R, Zhang Z, Pan X, Huang X, Huang Z, et al. (2011) ATX-LPA axis induces expression of OPN in hepatic cancer cell SMMC7721. Anat Rec (Hoboken) 294: 406–411. June 2014 \| Volume 9 \| Issue 6 \| e98320 June 2014 \| Volume 9 \| Issue 6 \| e98320 PLOS ONE \| www.plosone.org 10
https://openalex.org/W2808472683	https://periodicals.karazin.ua/eejp/article/download/10468/10040	Russian	null	It's Worth Remembering: "This Never Was Not in The USSR". - Ukraine, Kharkov, UPTI	East European journal of physics	2,018	cc-by	14,535	СТОИТ НАПОМНИТЬ: «ЭТОГО НИГДЕ В СССР НЕ БЫЛО». – УКРАИНА, ХАРЬКОВ, УФТИ Истоки. Аннеси (Франция) – Санкт-Петербург Мне пришлось ... зарабатывать не только на себя, но и на семью. Из автобиографии И. В. Обреимова Иван Васильевич Обреимов родился 8 марта 1894 года во Франции, в городе Аннеси. По семейным преданиям, его отец – Василий Иванович Обреимов – был высокообразованным человеком с отчаянно мятежной душой. Так, окончив с отличием математический факультет Казанского университета, он отклонил лестное предложение профессора Бальцани остаться работать на его кафедре по той лишь причине, что однажды профессор имел неосторожность в разговоре с ним перейти на повышенный тон. Поэтому последующие годы Василий Иванович Обреимов был вынужден посвятить преподаванию математики в частной гимназии. 1871 год стал роковым в его жизни: Василий Иванович был арестован и отправлен в ссылку за политическую неблагонадежность. Однако вскоре организовал удачный побег и некоторое время жил нелегально, устроившись домашним учителем. В начале 90-х годов Обреимову удалось эмигрировать за границу. Во Франции появились на свет два его сына от второго брака – Иван (1894 г.) и Александр (1896 г.). В 1896 году молодая чета Обреимовых принимает решение возвратиться в Россию. Отчасти это стало возможным потому, что многолетние хлопоты о помиловании увенчались успехом. Местом постоянного жительства был выбран Санкт-Петербург. Василию Ивановичу посчастливилось и найти неплохое место преподават восьмиклассном Коммерческом училище. Туда же он записал учениками и своих сыновей. Василию Ивановичу посчастливилось и найти неплохое место преподавателя математики в восьмиклассном Коммерческом училище. Туда же он записал учениками и своих сыновей. Об На протяжении последующих лет Василий Иванович Обреимов пытается совмещать педагогическую работу, дающую средства к существованию, с волонтёрской научно–просветительской деятельностью. Как свидетельствуют немногочисленные архивные данные, он был одним из активнейших составителей Энциклопедического словаря, а также автором двух научно–популярных книг по математике. В 1909 году семью неожиданно настигло горе – Василий Иванович скоропостижно скончался. Осиротевшая семья, оставшись без кормильца, вынуждена была оставить столицу и переехать в Гатчину. Ивану и Александру пришлось очень рано повзрослеть. К примеру, еще будучи подростком, Иван (как старший из сыновей) стал всерьез помогать матери пополнять скудный семейный бюджет. Некоторое время он подрабатывал в качестве гувернера и учителя игры на рояле. В 1910 году трудолюбивый юноша блестяще заканчивает (с золотой медалью!) Гатчинский сиротский институт и поступает в Санкт-Петербургский университет на физико-математический факультет, в группу физики. Современный читатель может прочувствовать студенческую атмосферу тех лет, ознакомившись с небезынтересной цитатой из его эпистолярного наследия: «В мое время в Петербургском университете обучалось 12000 студентов. Среди них большую часть составляли юристы. Алла Таньшинаi К 100-летию НАН Украины & 90-летнему юбилею УФТИ – ННЦ «ХФТИ» К 100-летию НАН Украины & 90-летнему юбилею УФТИ – ННЦ «ХФТИ» В канун 90-летия Национального научного центра «Харьковский физико-технический институт» (далее – ННЦ «ХФТИ») представляется уместным вспомнить как его предысторию, так и воздать дань уважения легендарным фундаторам Украинского физико-технического института (далее – УФТИ), которые ещё в 30-х годах прошлого столетия заложили краеугольный камень в основание мировой славы УФТИ – ННЦ «ХФТИ». Светлой памяти академика И. В. Обреимова, директора-организатора УФТИ i Статья написана по материалам докторской диссертации (научный куратор – академик НАН Украины Виктор Григорьевич Б ) 85 85 85 EAST EUROPEAN JOURNAL OF PHYSICS East Eur. J. Phys. Vol.5 No.1 (2018) 85-101 © Tan’shyna A , 2018 i Статья написана по материалам докторской диссертации (научный куратор – академик НАН Украины Виктор Григорьевич Барьяхтар). © Tan’shyna A., 2018 Барьяхтар). СТОИТ НАПОМНИТЬ: «ЭТОГО НИГДЕ В СССР НЕ БЫЛО». – УКРАИНА, ХАРЬКОВ, УФТИ На математическое отделение (в группы математики, астрономии, физики) поступало ежегодно около 300 человек. Сдавало государственные экзамены около 60 человек. Таким образом, курс оканчивало лишь 20% поступавших. i Статья написана по материалам докторской диссертации (научный куратор – академик НАН Украины Виктор Григорьевич Барьяхтар) © Tan’shyna A., 2018 Барьяхтар). 86 EEJP Vol.5 No.1 2018 86 EEJ 86 86 EEJP Vol.5 No.1 2018 A. Tan’shyna Весьма интересную, но немногочисленную категорию составляли великовозрастные студенты. Например, народные учителя, вышедшие на пенсию, бывшие студенты других факультетов. Они поступали в университет из любви к науке. На нас, молодых студентов, они оказывали сильное влияние. От них мы узнавали о марксизме, они помогали нам лучше понять классовую борьбу в России». Так или иначе, но самостоятельная студенческая жизнь, несмотря на тяжелейшее материальное положение, благоприятно сказалась на формировании личности Вани Обреимова: университетские годы закалили его характер и развили уверенность в своих силах. Не исключено, что именно поэтому он специально заметит в автобиографии: «Во время прохождения университетского курса зарабатывал преподаванием музыки (рояль) и демонстрированием физических опытов на публичных лекциях». Впрочем, в те времена в университете посещение занятий было не очень строгим. Как откровенно признается И. В. Обреимов, «такая свобода посещения была важной и по другой причине. С 16 лет, т. е. первого курса университета, мне пришлось, как и многим студентам, зарабатывать не только на себя, но и на семью. Это было возможно только благодаря тому, что я мог слушать не все лекции и работать в лаборатории в удобное для себя время». Примечательно, что еще на первом курсе, при выполнении лабораторного практикума по физике, Ивану Обреимову посчастливилось познакомиться с замечательным университетским профессором Дмитрием Сергеевичем Рождественским(1876–1940). Именно он–то и сумел разглядеть в любознательном и трудолюбивом студенте задатки будущего исследователя. Способного первокурсника профессор приглашает поучаствовать в работе студенческого научного кружка, который курировал тогда известный университетский профессор Орест Даниилович Хвольсон (автор первого отечественного многотомного «Курса физики», переведенного в дореволюционные годы на многие языки мира). «Физический кружок был основан в 1906 году, т. е это было дитя революции 1905 года, – уточняет в своих мемуарах И. В. Обреимов. – Официально кружком руководил профессор Орест Даниилович Хвольсон... Это было «официальное» руководство в том смысле, что защищало работу кружка от открытого полицейского надзора. До некоторой степени такой надзор осуществлял секретарь проректора: он утверждал повестку каждого заседания кружка. Средства кружка составляли членские взносы, на которые рассылались и печатались повестки. На остатки средств покупались книжки, являвшиеся собственностью кружка. СТОИТ НАПОМНИТЬ: «ЭТОГО НИГДЕ В СССР НЕ БЫЛО». – УКРАИНА, ХАРЬКОВ, УФТИ Со стороны некоторых преподавателей физики (ассистентов и лаборантов) были попытки направить работу кружка в соответствии с учебным планом, но мы их не допускали в кружок. Кружок объединял студентов всех курсов, в него входили также «оставленные при университете для подготовки к профессорскому званию» (аспиранты). Темы занятий кружка отличались разнообразием. Часть тем предлагали студенты – дипломники». Нельзя не вспомнить и тот факт, что авторитет университетского профессора физики О. Д. Хвольсона был настолько весом, что даже существовало неписанное правило: вновь назначенные преподаватели физики обязательно должны были приехать в Петербург, чтобы лично представиться Оресту Данииловичу. р р ур , р р у Д у «Совершеннейшим летописцем физики» называли современники Ореста Данииловича Хвольсона (1852– 1934). Благодаря многотомному «Курсу физики», он приобрел поистине мировую известность. «Совершеннейшим летописцем физики» называли современники Ореста Данииловича Хвольсона (1852– 1934). Благодаря многотомному «Курсу физики», он приобрел поистине мировую известность. На рубеже XIX–XX веков, по авторитетному замечанию Д. С. Рождественского, «буквально не было книг по нашей науке, где нечего было читать и где молодежь – я это испытал сам – не знала, как добраться до скудных знаний той поры». Поэтому-то по книгам О. Д. Хвольсона не одно младое поколение постигало азы физики. В те же годы даже появился методологический термин – «хвольсоновский стиль изложения». Первое издание четырехтомного «Курса физики» О. Д. Хвольсона было осуществлено издательством К. Л. Риккера в период 1897–1915 гг. В дальнейшем «Курс» выдержал семь изданий, каждое из которых было тщательно переработанным и дополненным. В 1923–1926 гг. вышло в свет последнее шеститомное издание. Известно также, что Альберт Эйнштейн отзывался об этом энциклопедическом своде физических знаний как о «превосходном». В историю же физической науки Орест Даниилович Хвольсон вошел не только как блестящий её популяризатор, но и как талантливый исследователь. В его активе было также 40 научных работ, посвященных электрофизике, магнетизму, фотометрии, актинометрии, а также изучению солнечного излучения. Со слов его современника Абрама Фёдоровича Иоффе, «Орест Даниилович хорошо читал лекции и часто выступал с публичными лекциями... В молодости он работал в лаборатории Академии наук и мечтал сделаться ее членом, для чего, однако, значение его работ было недостаточным». Кстати, Орест Даниилович и сам не стеснялся признавать, что главный его талант – это талант педагога. В 1895 году Орест Даниилович Хвольсон все же был удостоен звания члена–корреспондента Петербургской Академии наук. Рассказывают, что когда его избрали почетным членом Российской Академии наук (1920), то он остроумно заметил: «Разница между академиком и почетным академиком такая же, как между государем и милостивым государем». Гёттингенский университетii В 1913 г. ездил в г. Гёттинген, в лабораторию В 1913 г. ездил в г. Гёттинген, в лабораторию профессора Г. А. Таммана по командировке университета. Из автобиографии И. В. Обреимова р ф р В 1913 году Ивану Обреимову представился уникальный случай – пополнить свой багаж знаний за границей. По своей личной инициативе он написал письмо на имя гёттингенского профессора Г. А. Таммана, в котором запрашивал возможность поработать во время летнего семестра в его лаборатории. И ему повезло: профессор не возражал против летней стажировки. Правда, при одном условии – студент Обреимов должен был располагать средствами для оплаты обучения в Гёттингенском университете. И тут ему снова улыбнулась удача: родной университет изыскал возможность предоставить талантливому студенту научную командировку за границу. В течение четырёх месяцев пытливый паренек повышает свои знания и обучается экспериментальному мастерству в прославленном Гёттингенском университете. И надо полагать, что выбор Обреимовым именно этого университета был не случайным. СТОИТ НАПОМНИТЬ: «ЭТОГО НИГДЕ В СССР НЕ БЫЛО». – УКРАИНА, ХАРЬКОВ, УФТИ В 1895 году Орест Даниилович Хвольсон все же был удостоен звания члена–корреспондента Петербургской Академии наук. Рассказывают, что когда его избрали почетным членом Российской Академии наук (1920), то он остроумно заметил: «Разница между академиком и почетным академиком такая же, как между государем и милостивым государем». Не меньшее влияние на научное формирование студента Ивана Обреимова оказал и Павел Сигизмундович Эренфест (Ehrenfest) (1880–1933), с именем которого связано становление современной теоретической физики в дореволюционном Петербурге. 87 It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI EEJP Vol.5 No.1 2018 Родившись в Вене, «образованность ума» он получил в Австрии и Германии. Своим учителем считал выдающегося австрийского физика-теоретика Людвига Больцмана. Непродолжительное время жил в России. Последние двадцать лет своей недолгой 53–летней жизни прожил в Голландии, где возглавлял кафедру теоретической физики прославленного Лейденского университета, ранее которой руководил общепризнанный патриарх теоретической физики начала прошлого века Гендрик Антон Лоренц. Кстати, по мемуарным воспоминаниям П. С. Эренфеста, «Россия могла бы стать моей Родиной в самом глубоком значении этого слова, если бы я смог получить здесь постоянную преподавательскую работу где бы то ни было». Как ни парадоксально, но только два семестра (с 1 января 1909 года по 1 января 1910 года) он читал специальный курс дифференциальных уравнений для студентов старших курсов и лаборантов-ассистентов электромеханического факультета Политехнического института. (К слову: Эренфест был зачислен как временный преподаватель с оплатой за счет излишков институтского бюджета). Всего лишь пять лет – с 1907 по 1912 гг. – прожил Павел Сигизмундович Эренфест в Петербурге. Наиболее лаконично подытожил эти годы академик Абрам Федорович Иоффе (один из самых близких его тогдашних друзей): «Со всей решительностью он выступал против формализма университетской физики, против ее вождей. Зато он и не смог добиться права преподавать в университете, хотя даже сдал там магистерские экзамены... В годы пребывания Эренфеста в Петербурге вокруг него группировалась вся талантливая молодежь. Именно это и сыграло главную роль в развитии современной теоретической физики в Петербурге». р ур Также и Ваня Обреимов активно участвовал в заседаниях неофициального городского физического кружка, основателем и руководителем которого был П. С. Эренфест. Вот что по этому поводу он припоминает: «Осенью 1912 года я был допущен еще в одну «конспиративную» организацию – в воскресный кружок Эренфеста. Этот кружок объединял всю творческую молодую физику Петербурга. Собирались члены кружка по воскресеньям у кого-нибудь в квартире (или в Малой физической аудитории Физического Института) с 10 до 12 часов». Гёттингенский университетii Историческая справка ii ii Гёттинген (Göttingen) – один из старейших немецких городов (земля Нижняя Саксония). На рубеже XIX–XX веков Гёттингенский университет, основанный в 1734 году, был одним из авторитетнейших немецких университетов. К тому же, как заметил Генрих Гейне – один из выдающихся студентов этого университета, – Гёттинген «знаменит своими колбасами и Университетом». Не менее красноречива и следующая характеристика университетского городка: «В самом городе – ни трамваев, ни автомобилей (почти), одни велосипедисты, да прохожие, наполовину по крайней мере студенты в пестрых шапочках различных корпораций и бюргершафтов [товариществ. – Прим. А.Т.] ..., целью которых является так же, как во время оно, совместное распивание пива, распевание песен и, конечно, “Mensur” [«Дуэль» (нем.). – Прим. А.Т.], т. е. нанесение «знаков отличия» на физиономии». А по образному сравнению лауреата Нобелевской премии Макса Борна, Гёттинген был привлекателен для европейских студентов и как «Мекка немецких математиков». К тому же, по его словам, «студенты кочевали по университетским городам, проведя лето в каком-нибудь маленьком университете, чтобы насладиться природой и спортом, а зиму – в больших городах с их театрами, концертами... Еще со времен Гаусса и Вебера в Гёттингене стало традицией, что математика и физика развиваются не параллельно, а вместе. Клейн особенно энергично охранял эту традицию, расширив ее привлечением технических наук». Символичен и следующий факт: второе десятилетие прошлого столетия было ознаменовано становлением новой квантовой механики и определено как «гёттингенский период истории физики». 88 EEJP Vol.5 No.1 2018 88 EEJPVol. 88 A. Tan’shyna Окончив к осени «свои университеты» в Европе, Обреимов возвращается на родину и приступает к выполнению дипломной работы, руководителем которой по его просьбе согласился стать профессор Д. С. Рождественский. Д д В 1915 году он получает предложение остаться при alma mater для «приготовления к преподавательской и профессорской деятельности», что соответствует теперешней аспирантуре. Более того – профессор Д. С. Рождественский предлагает ему занять место своего личного ассистента. Университетская карьера в начале прошлого века предоставляла единственный путь для проведения научной работы. Правда, за свой счет. Поскольку «вузы России, как известно, были бедны. Жалованье и профессора, и преподаватели получали только за учебную работу... При университете оставляли со стипендией людей, которые подавали надежды быть хорошими преподавателями, демонстраторами, но не исследователями. Исследовательская работа, повторяю, государственными средствами не оплачивалась». Много лет спустя академик Иван Васильевич Обреимов любил с удовольствием рассказывать о своей насыщенной судьбоносными событиями молодости, и, как правило, всегда с большим уважением отзывался о своем первом наставнике – академике Дмитрии Сергеевиче Рождественском. р Д р р Кстати, не считал Иван Васильевич зазорным и откровенничать. Историческая справка ii Так, например, всегда с улыбкой вспоминал, что «Дмитрий Сергеевич был очень радушен. В квартире на Волховском по воскресеньям в час дня подавали завтрак, состоящий из пирога с капустой и чая. Всех, кто приходил в это время, угощали завтраком. Студентом я из озорства как-то придумал предлог и зашел к Рождественскому домой после воскресного кружка, меня угостили пирогом с капустой. И после я частенько у него обедал, ездил к нему на дачу и всегда бывал радушно принят». Его память сохранила и следующие факты: «Он всегда был уверен в себе. То, что ему было трудно (даже в смысле физической выносливости), он считал невозможным для других. Это его свойство иногда сильно раздражало окружающих и восстанавливало против него. В 1918 году в Ленинграде было голодно. Некоторые и сейчас не могут себе представить, что сидеть на одной картошке с солью в течение одного–двух месяцев – очень большое лишенье. ¼ или ⅛ фунта хлеба в день в этих условиях было заметным подспорьем к картошке. Мы, ученики Дмитрия Сергеевича, всячески старались помочь ему, чтобы он и его семья как можно лучше питались (например, мы выхлопотали ему у Петросовета три рабочих пайка). Впоследствии Дмитрий Сергеевич говорил: р волен, что поголодал. Я узнал, какая масса вкусных вещей существует. Например, до сих пор я ийского сала, а ведь это очень вкусная вещь». – Как я доволен, что поголодал. Я узнал, какая масса вкусных вещей существует. Наприме не ел малороссийского сала, а ведь это очень вкусная вещь». И, по-видимому, всё-таки здорово повезло студенту Ивану Обреимову. Ведь не каждому вначале жизненного пути повстречается замечательный наставник. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI Он перевел меня на должность «оставленного при университете со стипендией». р р у р Одновременно со мной Дмитрию Сергеевичу надо было устроить и других людей. При Комиссии естественных производительных сил Академии наук Д. С. Рождественскому (в то время молодому профессору – ему было тогда 42 года) удалось создать Коллегию по оптотехнике... Одновременно со мной Дмитрию Сергеевичу надо было устроить и других людей. При Комиссии естественных производительных сил Академии наук Д. С. Рождественскому (в то время молодому профессору – ему было тогда 42 года) удалось создать Коллегию по оптотехнике... было тогда 42 года) удалось создать Коллегию по ему было тогда 42 года) удалось создать Коллегию по о Через несколько месяцев мы нашли другой выход. у ) у Через несколько месяцев мы нашли другой выход. ) у есколько месяцев мы нашли другой выход. Осенью 1918 г. Советское правительство обратилось к доктору М. И. Неменову с предложением основать медицинский исследовательский рентгенологический институт. М. И. Неменов в свою очередь обратился к А. Ф. Иоффе с предложением сотрудничать с ним и основать институт, как теперь сказали бы, комплексный с тремя отделениями: медицинским, физическим и отделением радиоактивности. Осенью 1918 г. Советское правительство обратилось к доктору М. И. Неменову с предложением основать медицинский исследовательский рентгенологический институт. М. И. Неменов в свою очередь обратился к А. Ф. Иоффе с предложением сотрудничать с ним и основать институт, как теперь сказали бы, комплексный с тремя отделениями: медицинским, физическим и отделением радиоактивности. А. Ф. Иоффе просил Д. С. Рождественского присоединиться для участия в создани оптического отделения... В общем, сразу было видно, что комплексный институт не устроил бы ни одного из его организаторов, но споры об его образовании привели Д. С. Рождественского к решению о необходимости создания самостоятельного оптического института. В этом он убедил советское правительство. 15 декабря 1918 года было принято постановление Народного комиссариата просвещения об организации ГОИ». Согласно этому постановлению в задачи института входило «научное исследование производства и свойств оптического стекла», «содействие оптической промышленности в России», «научное исследование всех вопросов, касающихся лучистой энергии», «распространение оптических знаний среди специалистов и в широких массах». р В составе ГОИ было предусмотрено два отдела – научный и технический. Становление же института проходило в крайне тяжёлые годы. Первая мировая война, революция, гражданская война и интервенция породили в стране разруху, голод и холод. Оттого в те времена всё было проблемой: и приборы для экспериментов, и продукты питания, и одежда, и даже бумага. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI EEJP Vol.5 No.1 2018 Поэтому неудивительно, что «во время первой мировой войны оказалось, что Россия полностью зависит в оптике от Германии. У нас не было оптических заводов, не умели делать оптическое стекло, не умели проектировать оптические приборы... р р р р За оптическое стекло Германия получала от нас хлеб... Говорили, что за тонну оптического стекла Германия получала вагон или несколько вагонов зерна. При таких условиях началась в тогдашней России борьба за оптическое стекло. И, несомненно, главные усилия и главные успехи были связаны с именем Д. С. Рождественского. Он связался с бывшим императорским заводом; он там присутствовал при первых опытах над варкой оптического стекла, он подбирал всю ту, тогда молодую ныне почтенную ячейку, которая взялась за это дело и довела его до современного состояния». у у у, р р И надо признать, что именно недостаток военной оптики заставил тогда власть имущих поставить на государственном уровне вопрос о создании отечественной оптической базы. Роль же научного куратора выпала на долю профессора Д. С. Рождественского. И надо признать, что именно недостаток военной оптики заставил тогда власть имущих поставить на государственном уровне вопрос о создании отечественной оптической базы. Роль же научного куратора выпала на долю профессора Д. С. Рождественского. В апреле 1918 года исключительно благодаря энергии и научно-организационным хлопотам Д. С. Рождественского был создан и Отдел оптотехники на базе Подкомиссии по микроскопии КЕПС, выделившийся вскоре в самостоятельное научно-исследовательское учреждение – Государственный оптический институт (далее – ГОИ). Организация ГОИ была поручена инициатору его создания – Д. С. Рождественскому. Представляется возможным непредвзято реконструировать предысторию его создания по воспоминаниям И. В. Обреимова: «Осенью 1918 г. Дмитрий Сергеевич объявил мне, что завод стал на консервацию, платить ему мне не из чего, и я должен сам искать себе место. Это тоже было отличительной чертой Д. С. Рождественского – он не привязывался к людям. У него была одна привязанность – Ольга Антоновна, и эта привязанность исчерпывала все. р р Я настолько хорошо его знал, что не возмутился, не огорчился, а просто напомнил ему, что положение в стране не такое, чтобы можно было легко найти себе место, и что он обязан что-нибудь предпринять. Он перевел меня на должность «оставленного при университете со стипендией». Я настолько хорошо его знал, что не возмутился, не огорчился, а просто напомнил ему, что положение в стране не такое, чтобы можно было легко найти себе место, и что он обязан что-нибудь предпринять. «Было решено создать собственные оптотехнику и оптическое стекло» Я вместе с Рождественским переходили сначала Я вместе с Рождественским переходили сначала в Комиссию по изучению естественных производительных сил (КЕПС) Академии наук, а затем во вновь организованный Советской властью Государственный оптический институт. Из автобиографии И. В. Обреимова Начало научной деятельности Ивана Обреимова совпало с необратимыми изменениями внутри страны. Многих его сверстников унесла первая мировая война, революция, гражданская война. Но ему посчастливилось не только остаться в живых, но и внести свою лепту в становление и развитие отечественного оптического приборостроения. А начинался этот нелегкий путь в большую науку, с его слов, так: «Осенью 1915 г. произошло событие, сказавшееся на дальнейшей судьбе Д. С. Рождественского и моей судьбе. Было решено создать собственные оптотехнику и оптическое стекло (на Императорском фарфоровом и стекольном заводе). Генерал Струков пригласил в качестве консультанта Дмитрия Сергеевича, а Д. С. Рождественский в свою очередь меня своим ассистентом с жалованьем из личных средств 100 рублей в месяц... Дмитрий Сергеевич поручил мне наладить основную измерительную технику, создать иммерсионный метод для экспрессного измерения малых разностей показателя преломления на обломках стекла... В течение двух с половиной лет через мои руки проходили все стандартные измерения по оптическому стеклу». Уместно пояснить вкратце приведенную выше цитату. Дело в том, что во время первой мировой войны оснащение царской армии военными оптическими приборами было резко подорвано, поскольку именно Германия тогда была мировым монополистом в области производства оптического стекла. В России же отсутствовала собственная оптическая промышленность. Сборка оптических приборов осуществлялась из заграничного оптического стекла и с помощью импортного оборудования на трех заводах: на базе оптических мастерских Обуховского завода и сборочных мастерских заводов Цейсса и Герца. 89 у 3. Создать для научной работы этих специалистов жилищные условия, обеспечивающие их минимальными, безусловно для такой работы необходимыми удобствами... Постановление Центральной комиссии по улучшению быта научных специалистов при Совнаркоме об увеличении количества пайков научным специалистам Петрограда и Москвы и о выдаче им обуви и одежды 3. Выдавать научным специалистам как Москвы, так и Петрограда, кроме продовольственных пайков, по одной паре обуви, одному костюму (или материи на костюм), три пары белья (или материи на белье) и шести пар носков (или чулок) в год. Что касается теплого платья, то таковое выдавать отдельным лицам лишь в зависимости от степени нуждаемости в нем. И, по-видимому, так же не зря в числе прочих тогдашних лозунгов был и следующий: «Научиться ценить науку, отвергать “коммунистическое” чванство дилетантов и бюрократов». Понятно поэтому, что проблем у первого директора ГОИ было предостаточно. Но кроме бесчисленных научно-организационных сложностей, главные затруднения были с кадрами. Ныне имеет смысл несколько подробнее остановиться на одном из наиболее действенных его нововведений. А чтобы не погрешить против истины, процитируем по этому поводу воспоминания одного из первых сотрудников института А. И. Стожарова: «Мне было 18 лет, когда меня привлекли к работе в организовавшийся тогда Государственный оптический институт... Довелось поговорить с Дмитрием Сергеевичем. Он сказал следующее: «Я предлагаю вам поступить в организуемый нами новый Оптический институт. Вы должны будете энергично учиться, чтобы подготовиться к научной работе. От вас требуется обещание в течение двух лет не уходить из Оптического института, слушать лекции по моему указанию и обязательно вовремя сдавать экзамены. Я обеспечу вас продуктовой рабочей карточкой и буду добиваться отсрочки от призыва в армию. Думаю, что через два года вы уже сами не захотите уходить от нас». Таким образом, была организована, я бы сказал, первая аспирантура в Советском государстве... Полное название нашей должности было: младшие лаборанты при мастерских ГОИ». Безусловно, острую нужду в квалифицированных кадрах испытывали тогда многие учебные и научные учреждения. Поэтому нельзя не осознать эффективность упомянутого выше сотрудничества ГОИ с университетом. Заслуживает внимания и следующая особенность научно-организационной «кухни» ГОИ тех лет, рассказанная И. В. Обреимовым: «Возник вопрос, где размещать Оптический институт? На первых порах решили: в здании Физического Института университета. Помогла случайность. Во двор университета вклинивалось кирпичное здание конфетной фабрики Колесникова. Осмотреть фабрику отправились Архангельский и я... С помощью топора мы открыли ворота... Осмотрели фабрику и доложили Д. С. Рождественскому, что нашли пригодное для работы помещение. Здание фабрики было передано Оптическому институту... Чтобы придать зданию более красивый вид, было предложено использовать несколько плит из красного песчаника, взятых из бывшей ограды Зимнего дворца». А приведенная ниже цитата из доклада Д. С. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI р р р р р у у Приведенные ниже извлечения из правительственных документов того времени позволяют более наглядно обрисовать некоторые реалии той эпохи. Проект декрета СНК об улучшении положения научных специалистов 23 декабря 1919 г. р В целях сохранения научных сил, необходимых для социалистического строительства, для поднятия производительности народного хозяйства и культуры, а также наиболее целесообразного обеспечения нужд рабоче- крестьянской обороны Совет Народных Комиссаров постановляет: В целях сохранения научных сил, необходимых для социалистического строительства, для поднятия производительности народного хозяйства и культуры, а также наиболее целесообразного обеспечения нужд рабоче- крестьянской обороны Совет Народных Комиссаров постановляет: р р р р 1. Предоставить усиленное довольствие наиболее выдающимся специалистам тех отраслей, которые являются существенными для разрешения указанных выше задач. 1. Предоставить усиленное довольствие наиболее выдающимся специалистам тех отраслей, которые являются существенными для разрешения указанных выше задач. у р р у 2. Освободить этих специалистов от всякого рода повинностей (трудовой, военной и т. д.), не имеющих отношения к их научным занятиям. 3. Создать для научной работы этих специалистов жилищные условия, обеспечивающие их минимальными, безусловно для такой работы необходимыми удобствами... 90 90 EEJP Vol.5 No.1 2018 A. Tan’shyna 90 EEJP Vol.5 No.1 2018 Постановление Центральной комиссии по улучшению быта научных специалистов при Совнаркоме об увеличении количества пайков научным специалистам Петрограда и Москвы и о выдаче им обуви и одежды Рождественского на первом годичном собрании ГОИ (от 19 декабря 1919 г.) обнародует принципы и методы научно-организационной работы тех лет: «Оглядываясь на минувший год, мы с глубокой благодарностью вспоминаем о той поддержке, которой обязаны Петроградскому университету. В его Физическом институте зародилась наша деятельность и протекал первый год работы... Из университета мы черпаем свежие силы, молодых начинающих сотрудников, из университета черпаем ту науку, которая, как я говорил выше, нам нужна, как воздух. У нас не было в достаточном количестве необходимых приборов. Вследствие закрытия границы наш сотрудник проф. В. А. Анри не мог привести в Петроград все, что было заказано за границей... Часть приборов ревностные и настойчивые сотрудники института разыскали в России. Все это мы смогли осуществить только благодаря энергичной поддержке Комиссариата народного просвещения. Он пошел навстречу идее научно-технического учреждения не только большими, подчас выходящими из всякой нормы, средствами, но и активным содействием, в котором фактическое осуществление ставилось всегда выше формы, буква закона преступалась, если от этого выигрывало дело. Мы должны принести искреннюю благодарность Комиссариату за то, что он дал нам возможность в короткий срок увидеть воплощение дорогой для нас мысли». р Стоит также описать факты, связанные с Д. С. Рождественским, которые непосредственно освещают объективные причины резкого перелома научной судьбы его молодого соратника И. В. Обреимова. Со слов Ивана Васильевича, «Дмитрий Сергеевич был полон замыслов. Его увлекало многое, в том числе квантовая теория. Помню его прекрасный доклад о ротационном инфракрасном спектре воды, который он сделал как-то на воскресном кружке. р ру После доклада, который заинтересовал всех нас, он с жаром говорил мне: ставляете себе, если сравнить спектр поглощения паров бензола и кристалла бензола, то ведь в ола ротационной структуры не должно быть! – Вы представляете себе, если сравнить спектр поглощения паров бензола и кристалла бен кристалле бензола ротационной структуры не должно быть! Эта мысль окрылила меня. Я пришел к выводу, что спектр кристаллического бензола надо исследовать при низких температурах... Я предложил Д. С. Рождественскому поставить его эксперимент с бензолом, но получил неожиданный отпор. Дмитрий Сергеевич «забыл» свои мысли. Мало того, в самых резких тонах он запретил 91 It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI EEJP Vol.5 No.1 2018 мне заниматься экспериментом. «Спектроскописту нечего делать с конденсированной фазой. Я не могу допустить, чтобы государственные деньги тратились на глупости». Когда я напомнил ему, что мысль об эксперименте принадлежит ему, он категорически отверг это. Я убеждал его в том, что окрашенные кристаллы изменяют свою окраску при температуре жидкого воздуха. «Работать под руководством А. Ф. Иоффе было легко» В 1924 г. перешел из Оптического института в Ленинградский физико-технический институт (академика А. Ф. Иоффе), где работал до 1929 г. Из автобиографии И. В. Обреимова В 1924 г. перешел из Оптического института в Ленинградский физико-технический институт (академика А. Ф. Иоффе), где работал до 1929 г. Из автобиографии И. В. Обреимова В 20-е годы прошлого столетия ЛФТИ переживал тяжелейшую пору своего становления. В качестве показательного примера стоит процитировать весьма откровенное письмо молодого директора ЛФТИ А. Ф. Иоффе, адресованное его зарубежному другу П. С. Эренфесту. 18 июня 1920 г., Петроград Постановление Центральной комиссии по улучшению быта научных специалистов при Совнаркоме об увеличении количества пайков научным специалистам Петрограда и Москвы и о выдаче им обуви и одежды Он ответил: – Что же в этом особенного? Еще Дьюар это отметил. – Что же в этом особенного? Еще Дьюар это отметил. Беседа внешне проходившая мирно закончилась ульти – Что же в этом особенного? Еще Дьюар это отметил. Беседа, внешне проходившая мирно, закончилась ультиматумом: щ Д р Беседа, внешне проходившая мирно, закончилась ультиматумом: – Или Вы будете делать то, что я Вам скажу, или убирайтесь вон. Выбирайте! – Конечно, я выбираю второе. – Конечно, я выбираю второе. – То есть остаетесь? – Нет, ухожу. – Ну и убирайтесь вон! Я поделился с А. Ф. Иоффе идеей о спектре кристаллов при низких температурах, получил его одобрение и пригласил себе в помощницы студентку физико-математического факультета Политехникума Тосю Прихотько (ныне действительный член Академии наук УССР)... у Я обратился к своим друзьям в Оптическом институте ..., и они отчасти под влиянием энтузиазма и обаяния А. Ф. Прихотько стащили небольшой по размерам, но очень хороший спектрограф..., и мы, с А. Ф. Прихотько на санках и в трамвае доставили его в Лесной. С помощью этого спектрографа были сделаны первые наблюдения над спектрами кристаллов при температуре жидкого воздуха, и (о радость!) в спектре кристаллов азобензола в жидком воздухе появилась структура – широкие полосы». Вот так неожиданно Иван Васильевич Обреимов и стал научным сотрудником упомянутого выше Государственного физико-технического рентгенологического института (здесь и далее ЛФТИ – Ленинградский физико-технический институт), первым директором и организатором которого был Абрам Федорович Иоффе. Дорогой друг! Надеюсь, что это письмо дойдет до тебя, а поэтому спешу (сейчас 1 час ночи, а завтра в 9 утра надо сдать письмо) ска- зать главное. Мы прожили тяжелые годы и многих потеряли, но сейчас начинаем снова жить. Научная работа у нас идет все это время. Все физики сконцентрированы в 2 институтах: рентгеновский (мой) и оптический (Рождественского), а в Москве – биологической физики (Лазарев) и университет (Романов). В каждом человек по 20. Работаем много, но закончено пока немного, так как год ушел на организацию работы в новых условиях, устройство мастерских и борьбу с голодом. Сейчас главная наша беда – полное отсутствие иностранной литературы, которой мы лишились с начала 1917 г. И первая, и главная моя просьба к тебе – выслать нам журналы и главные книги по физике... Надеюсь, что это письмо дойдет до тебя, а поэтому спешу (сейчас 1 час ночи, а завтра в 9 утра надо сдать письмо) ска- зать главное. Мы прожили тяжелые годы и многих потеряли, но сейчас начинаем снова жить. Научная работа у нас идет все это время. Все физики сконцентрированы в 2 институтах: рентгеновский (мой) и оптический (Рождественского), а в Москве это время. Все физики сконцентрированы в 2 институтах: рентгеновский (мой) и оптический (Рождественского), а в Москве – биологической физики (Лазарев) и университет (Романов). В каждом человек по 20. Работаем много, но закончено пока немного, так как год ушел на организацию работы в новых условиях, устройство мастерских и борьбу с голодом. Сейчас главная наша беда – полное отсутствие иностранной литературы, которой мы лишились с начала 1917 г. И первая, и главная моя просьба к тебе – выслать нам журналы и главные книги по физике... Вторая просьба – написать о том, чем живет современная физика и ты, в частности. Мы здесь целиком поглощены строением атома и молекул... просьба к тебе – не сердись, что редко писал... Живем в Политехническом. Студентов мало, но лекции иду и в академию... Третья просьба к тебе – не сердись, что редко писал... Живем в Политехническом. Студентов ма Меня выбрали в академию... Итак, жду письма и книг. Твой далекий друг А. Иоффе Заметим, что в течение первых трех десятилетий прошлого века П. С. Эренфест пользовался исключительным авторитетом как у теоретиков, так и у экспериментаторов всего мира. В числе его самых близких друзей были такие титаны современной физики, как Альберт Эйнштейн и Нильс Бор. Явно не зря и сам А. Эйнштейн искренне признался в письме к П. Дорогой друг! Может быть, что-либо, касающееся свойства деформации сверхпроводящих монокристаллов в точке перехода». Я прошу А. Ф. продумать какую-либо красивую работу, которую можно было бы (после подготовки при комнатной температуре и в жидком воздухе) в течение двух месяцев полностью проделать в (жидком) водороде или гелии. Может быть, что-либо, касающееся свойства деформации сверхпроводящих монокристаллов в точке перехода». р И по большому счету, только благодаря хлопотам П. С. Эренфеста А. Ф. Иоффе имел возможность ежегодно с 1921-го по 1933 г. посещать передовые физические центры мира. Вот что об этом рассказывал позднее сам Абрам Федорович: «Начиная с 1921 г., я почти ежегодно бывал за границей с целью использования заграничного опыта, выяснения важнейших научных вопросов, дискуссий, консультаций и т. д. В крупнейших университетах: Париже, Берлине, Геттингене, Лейдене, Кембридже, в 15 американских университетах и во всех крупных заводских лабораториях я читал доклады и лекции. В Калифорнийском университете и Бостонском технологическом институте я читал систематические курсы. Благодаря этому я мог на деле познакомиться с организацией научной работы и преподавания в университетах и промышленных предприятиях Запада и Америки». р Вместе с тем эти годы были ознаменованы и рядом принципиально важных научно-организационных мероприятий в стенах ЛФТИ, истоки которых были известны лишь узкому кругу приближенных к А. Ф. Иоффе. фф На примере исключительно откровенных воспоминаний Ивана Васильевича Обреимова, который не стоял в стороне от ключевых институтских событий, возможно более конкретно реконструировать этот период становления ЛФТИ: «Летом 1924 г. А. Ф. Иоффе «выкинул номер». Все руководство института – А. Ф. Иоффе, его заместитель А. А. Чернышев и помощник Н. Н. Семеновiv – одновременно собралось в отпуск. Абрам Дорогой друг! Эренфесту: «Я редко встречаю людей, с которыми мне бывает хорошо. Я нуждаюсь в твоей дружбе еще больше, чем ты в моей». Но самое главное – Эренфест был необыкновенно щедр на помощь. Имеется возможность процитировать по этому поводу чрезвычайно примечательное письмо П. Эренфеста к А. Эйнштейну: «Я очень надеюсь, что ты, наконец, сможешь познакомиться с моим дорогим, дорогим Иоффе. Он очень тонкий физик и человек. Во всяком случае, ты получишь много удовольствия от этого знакомства. Наряду с моей женой, тобой и Бором он 92 A. Tan’shyna EEJP Vol.5 No.1 2018 принадлежит к числу моих ближайших друзей, и, возможно, нет другого такого человека, который высказывал бы по отношению ко мне больше любви». принадлежит к числу моих ближайших друзей, и, возможно, нет другого такого человека, который высказывал бы по отношению ко мне больше любви». Кстати, П. С. Эренфест принимал самое деятельное участие в налаживании международного научного сотрудничества. Так, спустя годы А. Ф. Иоффе в своих мемуарах специально заметит: «Когда в начале 1921 г. мы с Д. С. Рождественским и А. Н. Крыловым приехали за границу, выполняя поручение В. И. Ленина о восстановлении научных связей, решающую помощь нам оказывал Эренфест, имеющий широкие связи среди заграничных ученых. Он даже мобилизовал их на сбор для советских библиотек физических книг и журналов, вышедших во время блокады». р Добавим, что именно П. С. Эренфест содействовал и научной стажировке наиболее перспективных молодых сотрудников ЛФТИ в ведущих физических центрах мира. В частности, именно благодаря его хлопотам физтеховец Иван Васильевич Обреимов получил возможность в 1927 и 1928 гг. проводить научные исследования по спектроскопии кристаллов при низких температурах в знаменитой Лейденской криогенной лаборатории. р р По этому поводу примечательна выдержка из лейденского письма П. С. Эренфеста, адресованного А. Ф. Иоффе: «Криогенная лаборатория только что получила 100 000 долларов из фонда Рокфеллераiii на проведение работ при низких температурах [Здесь и далее курсивом выделено Эренфестом. – Прим. А.Т.] В соответствии с этим (это сообщение только для А. Ф.!) здесь очень заинтересованы в таких криогенных работах, которые должны быть проведены в наступающем году и которые бы продемонстрировали, что если только Лейден берет на себя «холод», то наиболее высококвалифицированные специалисты со всех частей света проводят свои специальные низкотемпературные исследования только в Лейдене, где они могут располагать наиболее совершенной техникой. р р Я прошу А. Ф. продумать какую-либо красивую работу, которую можно было бы (после подготовки при комнатной температуре и в жидком воздухе) в течение двух месяцев полностью проделать в (жидком) водороде или гелии. Фонд субсидировал научные стажировки молодых перспективных ученых (как правило, одногодичные), оказывал финансовую поддержку научным институтам (например, Институту теоретической физики Н. Бора в Копенгагене, Лаборатории низких температур Г. Камерлинг-Оннеса в Лейдене), оплачивал научные командировки ведущих ученых мира (в частности, их лекционную работу). iii Рокфеллеровский фонд был создан в мае 1913 г. с целью «содействия процветания человечества во всем мире». Основатель фонда – Дж. Д. Рокфеллер (Rockefeller)-старший (1839-1937), родоначальник американской династии промышленников и финансистов. Историческая справка iii Рокфеллеровский фонд был создан в мае 1913 г. с целью «содействия процветания человечества во всем мире». Основатель фонда – Дж. Д. Рокфеллер (Rockefeller)-старший (1839-1937), родоначальник американской династии промышленников и финансистов. Фонд субсидировал научные стажировки молодых перспективных ученых (как правило, одногодичные), оказывал финансовую поддержку научным институтам (например, Институту теоретической физики Н. Бора в Копенгагене, Лаборатории низких температур Г. Камерлинг-Оннеса в Лейдене), оплачивал научные командировки ведущих ученых мира (в частности, их лекционную работу). Так, к примеру, в период с 1924-го по 1928 г. было выделено 427 стипендий, из них одну треть получили физики, ⅓ - биологи, а остальная часть была поделена между химиками и представителями сельскохозяйственных наук. Стипендиатами Рокфеллеровского фонда были и сотрудники ЛФТИ: Я. И. Френкель (1925-1926 гг.), Ю. А. Крутков (1926-1927 гг.), В. А. Фок (1927-1928 гг.), К. Д. Синельников (1928-1930 гг.), Д. В. Скобельцын (1928-1930 гг.), Г. А. Гамов (1929 -1930 гг.) и Л. Д. Ландау (1930-1931 гг.). iv Семенов Николай Николаевич (1896-1986) – физикохимик, заместитель директора ЛФТИ с 1921-го по 1928 г., организатор и первый директор Института химической физики (1931), академик (1932), академик-секретарь Отделения химических наук АН СССР (1957-1963), вице-президент АН СССР (1963-1971). В 1956 г. присуждена Нобелевская премия ( ф ) Историческая справка Через несколько дней после моего назначения все уехали... Федорович предложил мне место своего помощника (вместо Н. Н. Семенова). Через несколько дней после моего назначения все уехали... у Я остался один, и. о. директора... В этом, если хотите, был тоже стиль А. Ф. Иоффе. Он давал общее направление, «первый толчок», а дальше во всем доверял. Работать у него в подчинении было морально легко. Не сказал бы, что легко и физически, ибо инициатива, которую давал Абрам Федорович требовала напряженной работы... К осени 1924 г. внутри института создалась небольшая дружная группа... Сюда определенно не входили В. Р. Бурсианv и Я. И. Френкель. В одной из частных бесед Н. Н. Семенов обратил внимание на то, что ЛФТИ «держится» на личном авторитете А. Ф. Иоффе. Но при несчастной случайности будет просто назначен новый директор, который придет со своими людьми и может аннулировать хорошие традиции института. Семенов считал правильным разбить ЛФТИ на лаборатории во главе с основными работниками, тем самым официально закрепить за ними право влиять на судьбу ЛФТИ. Когда мы пришли с этим к А. Ф. Иоффе, он страшно покраснел, но согласился. Так Физико-технический институт был разбит на несколько лабораторий... Лаборатория кристаллов – И. В. Обреимов». Ивану Васильевичу Обреимову все-таки сопутствовала удача, поскольку следующее стечение обстоятельств также сыграло знаковую роль в его научно-организационной карьере: «Наши просьбы о субсидировании, обращаемые к Научно-техническому управлению (НТУ) или к другим организациям ВСНХvi, все учащались. Иногда мы получали бюрократические ответы, например: «Переходите из Главнауки в ВСНХ, тогда денег дадим при условии, что все имущество приобретенное на эти деньги будет наше»... Подобные обстоятельства привели Н. Н. Семенова к мысли: не надо ли вообще перевести институт из Главнауки в НТУ ВСНХ... По этой причине был назначен «полный сбор» в Москве. Выехали все заместители А. Ф. Иоффе – Чернышев, Семенов, и я... В течение нескольких дней вопрос был выяснен, и только тогда приехал Абрам Федорович для разговора на высшем уровне. Мы вернулись в Ленинград с разрешением на организацию нового института, который в отличие от Физико-технического института Главнауки назывался Ленинградская физико- техническая лаборатория (ЛФТЛ). Директором обоих институтов стал А. Ф. Иоффе, его заместителем по обоим институтам – А. А. Чернышев, заместителем по ЛФТЛ – Н. Н. Семенов, заместителем по ЛФТИ – И. В. Обреимов». р Вместе с тем нельзя недооценивать и дальновидность кадровой политики первого директора ЛФТИ. Абрам Федорович Иоффе в то время открыл немало молодых талантов. Недаром, кстати, ЛФТИ в период становления называли «детским садом папы Иоффе». Мой дорогой друг! ...Позволь мне высказать некоторые возражения по поводу твоего плана забрать к себе Мандельштама и Тамма. Конечно, я прекрасно понимаю, насколько важно, во-первых, создать этим двум превосходным физикам благоприятные – по возможности – условия работы, вместо того чтобы бесполезно растрачивать их силы. Тем более, что они не только превосходные физики, но и совершенно превосходные люди. Я могу, во-вторых, очень хорошо и по достоинству оценить, что будет означать приход обоих этих людей, с их огромной ясностью мышления (!!!) и – в случае Тамма – с блестящей энергией и изобретательностью, в вашу группу для обучения лучших из молодежи. Но я все же должен тебе сказать, в чем суть моего возражения. Эта огромная концентрация внушает мне чувство глубокого страха. Французская революция сконцентрировала все в Париже; сравни теперь ситуацию там с тем, что имеет место в Германии. Одним из смертельнейших ядов такой концентрации является то, что кучка постаревших людей владеет абсолютно всем. В лучшем случае это люди, которые в молодости сделали нечто совершенно выдающееся, но зачастую и этого не бывает. Жизнь молодежи становится Историческая справка И как ни странно, а прогрессирующую уже тогда «болезнь роста» ЛФТИ усмотрел именно его далёкий лейденский друг П. С. Эренфест. П. С. Эренфест – А. Ф. Иоффе П. С. Эренфест – А. Ф. Иоффе 13 апреля 1928 г. 13 апреля 1928 г. 13 апреля 1928 г. Историческая справка iii Рокфеллеровский фонд был создан в мае 1913 г. с целью «содействия процветания человечества во всем мире». Основатель фонда – Дж. Д. Рокфеллер (Rockefeller)-старший (1839-1937), родоначальник американской династии промышленников и финансистов. Фонд субсидировал научные стажировки молодых перспективных ученых (как правило, одногодичные), оказывал финансовую поддержку научным институтам (например, Институту теоретической физики Н. Бора в Копенгагене, Лаборатории низких температур Г. Камерлинг-Оннеса в Лейдене), оплачивал научные командировки ведущих ученых мира (в частности, их лекционную работу). р ( , у р у) Так, к примеру, в период с 1924-го по 1928 г. было выделено 427 стипендий, из них одну треть получили физики, ⅓ - биологи, а остальная часть была поделена между химиками и представителями сельскохозяйственных наук. р ( у р у) Так, к примеру, в период с 1924-го по 1928 г. было выделено 427 стипендий, из них одну треть получили физики, ⅓ - биологи, а остальная часть была поделена между химиками и представителями сельскохозяйственных наук. Стипендиатами Рокфеллеровского фонда были и сотрудники ЛФТИ: Я. И. Френкель (1925-1926 гг.), Ю. А. Крутков (1926-1927 гг.), В. А. Фок (1927-1928 гг.), К. Д. Синельников (1928-1930 гг.), Д. В. Скобельцын (1928-1930 гг.), Г. А. Гамов (1929 -1930 гг.) и Л. Д. Ландау (1930-1931 гг.). Стипендиатами Рокфеллеровского фонда были и сотрудники ЛФТИ: Я. И. Френкель (1925-1926 гг.), Ю. А. Крутков (1926-1927 гг.), В. А. Фок (1927-1928 гг.), К. Д. Синельников (1928-1930 гг.), Д. В. Скобельцын (1928-1930 гг.), Г. А. Гамов (1929 -1930 гг.) и Л. Д. Ландау (1930-1931 гг.). iv Семенов Николай Николаевич (1896-1986) – физикохимик, заместитель директора ЛФТИ с 1921-го по 1928 г., организатор и первый директор Института химической физики (1931), академик (1932), академик-секретарь Отделения химических наук АН СССР (1957-1963), вице-президент АН СССР (1963-1971). В 1956 г. присуждена Нобелевская премия (совместно с английским физикохимиком С. Н. Хиншелвудом) за исследования механизма химических реакций. iv Семенов Николай Николаевич (1896-1986) – физикохимик, заместитель директора ЛФТИ с 1921-го по 1928 г., организатор и первый директор Института химической физики (1931), академик (1932), академик-секретарь Отделения химических наук АН СССР (1957-1963), вице-президент АН СССР (1963-1971). В 1956 г. присуждена Нобелевская премия (совместно с английским физикохимиком С. Н. Хиншелвудом) за исследования механизма химических реакций. 93 It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI 93 EEJP Vol.5 No.1 2018 Федорович предложил мне место своего помощника (вместо Н. Н. Семенова). Через несколько дней после моего назначения все уехали... Федорович предложил мне место своего помощника (вместо Н. Н. Семенова). EEJP Vol.5 No.1 2018 Возможно, ты позаботишься тогда и о том, чтобы кое-что из этого отрицательного последействия было уменьшено. Пожалуй, ты уже сейчас в некоторой степени сможешь провести подготовку к тому, чтобы по-настоящему хорошее пополнение тяготело бы не к Ленинграду, а смогло рассчитывать на удовлетворительные условия для развития в периферийных местах. Особенно это относится к математикам и физикам-теоретикам. Но и в их случае нужно, чтобы это происходило в дружеском контакте с тобой... р ру Прости за это, несомненно, неприятное для тебя письмо. Во всяком случае оно показывает тебе, с каким вниманием я отношусь к твоим планам... Абрам Федорович, объективно оценив искренность замечаний и пожеланий своего друга, вскоре принимает судьбоносное решение: «С 1929 г. выделил из Физико-технического института такие же институты для Томска, Харькова, Днепропетровска и Свердловска» (Из автобиографии А. Ф. Иоффе). Иван Васильевич Обреимов был направлен в Харьков. Его мемуарные заметки, являются историческим источником, откуда можно почерпнуть подробности тогдашних судьбоносных событий: «У нас в стране было всего два центра физической науки – Ленинград и Москва, и они «переманивали» многих хороших физиков из провинции... Началась пропаганда того, что научные центры, и в частности крупные физические центры, надо строить в провинции в организованном порядке. Институт получил два предложения: одно – открыть физико-технический институт в Свердловске, другое – усилить кафедру физики в Томске и создать на ее основе физико-технический институт. Институт получил два предложения: одно – открыть физико-технический институт в Свердловске, другое – усилить кафедру физики в Томске и создать на ее основе физико-технический институт. Насколько идея рассредоточения физики встретила всеобщее сочувствие, настолько конкретный вопрос – Насколько идея рассредоточения физики встретила всеобщее сочувствие, настолько конкретный вопрос – кто поедет осваивать новые места – оказался трудным... Когда я сообщил своей жене, Екатерине Александровне Пузино, о том, как проходит организация новых центров, она мне сказала: «Все твои молодые коллеги, которых ты так хвалишь, в действительности мало чего стоят. Они мастера критиковать. Но теперь, когда им дана возможность строить институты так, как они считают нужным, они дрейфят, не желают рисковать. Они предпочитают критиковать готовенькое». Я возразил: «Имей в виду, большую роль играют жены, которые не хотят уезжать из Ленинграда. Ведь если бы мне предложили уехать в провинцию, ты бы первая возразила». – «И ничего подобного. При условии, если разрешат строительство института на новом месте, я бы советовала тебе ехать». На следующий день я сказал А. Ф. Иоффе: «Если никто не желает ехать в Свердловск, то я согласен». – «Нет, – ответил он, – Вы должны оставаться здесь». «Слушали: Об организации ФТИ на Украине. Доклад акад. А. Ф. Иоффе. Постановили: Историческая справка v Бурсиан Виктор Робертович – первый ученый секретарь ЛФТИ, первый заведующий кафедрой теоретической физики физико-механического факультета Петербургского политехнического института. i Высший Совет Народного Хозяйства (ВСНХ) образован 5 декабря 1917 г. В его задачи входило проведение национализации промышленности, плановая организация народного хозяйства. После осуществления декрета о национализации – высший центральный орган по управлению народным хозяйством, главным образом промышленностью. В связи с правительственным курсом на индустриализацию страны были произведены изменения в организации и руководстве промышленностью и научно-исследовательской работой. В 1926 г. в системе ВСНХ созданы Научно- техническое управление (НТУ), комитеты и управления по отраслям промышленности. В состав НТУ вошли отраслевые научно-технические советы и институты. национализации промышленности, плановая организация народного хозяйства. После осуществления декрета о национализации – высший центральный орган по управлению народным хозяйством, главным образом промышленностью. В связи с правительственным курсом на индустриализацию страны были произведены изменения в организации и руководстве промышленностью и научно-исследовательской работой. В 1926 г. в системе ВСНХ созданы Научно- техническое управление (НТУ), комитеты и управления по отраслям промышленности. В состав НТУ вошли отраслевые научно-технические советы и институты. у у ВСНХ СССР руководил промышленными предприятиями союзного значения, ВСНХ союзных республик – остальными. Впоследствии ВСНХ СССР был реорганизован в 3 наркомата: тяжелой, легкой и лесной промышленности. ВСНХ СССР руководил промышленными предприятиями союзного значения, ВСНХ союзных республик – остальными. Впоследствии ВСНХ СССР был реорганизован в 3 наркомата: тяжелой, легкой и лесной промышленности. 94 A. Tan’shyna EEJP Vol.5 No.1 2018 тогда адом. Здоровое развитие она может получить лишь в децентрализованных местах, заполненных сотрудниками только наполовину... Это зло ужасно, потому что проявляется оно очень медленно. Но это – смертельный яд! То, что молодежь получает высшее образование в одном, двух центрах, — это очень хорошо, но затем она должна иметь возможность развернуться в процессе самостоятельной деятельности в децентрализованных местах, обеспеченных научными работниками не в полной мере. Ну, а теперь немножко о другом, что стало заметным за последнее время! Централизация приводит к возникновению «тяжелых институтов», «солидных организаций», в которые тот, кто их создает и организует, умеет на долгое время – и по всем отраслям – вдохнуть истинную жизнь. Ведь в большей или меньшей степени это плоть, которую создает его душа! Но нередко эти институты становятся для их создателей проклятием. «Тяжелые институты» наиболее правильно следовало бы – с психологической точки зрения – рассматривать как некие «гигантские игрушки», которые мальчик 50 лет позволяет себе подарить или купить. Мальчишеское упрямство мешает ему отшвырнуть их даже тогда, когда они становятся для него не- выносимыми, когда они начинают обкрадывать ядро его собственного «я»... всем отраслям – вдохнуть истинную жизнь. Ведь в большей или меньшей степени это плоть, которую создает его душа! Но нередко эти институты становятся для их создателей проклятием. «Тяжелые институты» наиболее правильно следовало бы – с психологической точки зрения – рассматривать как некие «гигантские игрушки», которые мальчик 50 лет позволяет себе подарить или купить. Мальчишеское упрямство мешает ему отшвырнуть их даже тогда, когда они становятся для него не- выносимыми, когда они начинают обкрадывать ядро его собственного «я»... р у р у р – с психологической точки зрения – рассматривать как некие «гигантские игрушки», которые мальчик 50 лет позволяет себе подарить или купить. Мальчишеское упрямство мешает ему отшвырнуть их даже тогда, когда они становятся для него не- выносимыми, когда они начинают обкрадывать ядро его собственного «я»... р р Мой дорогой Иоффе! Я очень хорошо знаю, что все это ты говорил себе сам, однако ты все же считаешь, что должен избрать путь концентрации и размаха. Но в том, что я тебе еще раз об этом написал, быть может, все-таки есть смысл. Возможно, ты позаботишься тогда и о том, чтобы кое-что из этого отрицательного последействия было уменьшено. Мой дорогой Иоффе! Я очень хорошо знаю, что все это ты говорил себе сам, однако ты все же считаешь, что должен избрать путь концентрации и размаха. Но в том, что я тебе еще раз об этом написал, быть может, все-таки есть смысл. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI р у р р ру фу ру И недаром впоследствии газета «Известия» от 11 ноября 1932 года в очерке Ф. Кандыбы «Снайперы атомного ядра» рапортовала: «Харьковский институт действительно строился темпами пятилетки и за девять месяцев был выстроен. Директор института профессор Обреимов разъезжал по Европе, закупая и заказывая самое лучшее, новейшее оборудование для лабораторий». Примечательные подробности о начальном периоде становления института можно почерпнуть из воспоминаний Ивана Васильевича Обреимова: «Группу физиков, выехавших в Харьков, провожали с помпой: на вокзале играл оркестр, слышалась дробь барабанов, плескались знамена. В эту группу входили: К. Д. Синельников, А. К. Вальтер, Н. А. Бриллиантов, А. Ф. Прихотько, В. С. Горский, В. Гей, Г. Д. Латышев, В. Волейко, П. И. Стрелков, А. И. Лейпунский (мой заместитель), Л. В. Розенкевич (теоретик), Г. Горовиц (теоретик). В становлении института, который получил название «Украинский физико-технический институт» (УФТИ), приняли участие В. А. Фок и Л. Д. Ландау, хотя они и не собирались жить в Харькове, и П. С. Эренфест, который серьезно думал о переезде в Харьков. Он приезжал туда дважды… р ф р р у р р р у А. Ф. Иоффе был назначен председателем Ученого совета УФТИ. Хотя роль его была и не велика, но значение огромно. Дело в том, что об общих принципах нам договариваться было нечего. Они были едины. Но в моменты затруднений Абрам Федорович со свойственным ему тактом всемерно поддерживал нас в правительственных органах Украины или в академических кругах. Некоторые товарищи в правительстве УССР – да и в Москве – подозревали, не избавляется ли А. Ф. Иоффе путем создания новых институтов от нежелательных ему людей или не «бегут» ли на Украину те, кто недоволен Абрамом Федоровичем. Приезды А. Ф. Иоффе в Харьков, его дружеские письма и бескорыстная поддержка во всех вопросах, которые я поднимал, рассеивали всякие подозрения. р р р р р После 1933 г., когда я был назначен председателем Ученого совета УФТИ, Абрам Федорович приезжал иногда на несколько дней к нам в гости. Ему, по-видимому, нравилась научная жизнь института, были приятны встречи с друзьями. Докладов он не делал, но подолгу беседовал с разными людьми. Не могу не отметить и прекрасное отношение к нашему институту как со стороны правительственных органов Украины, лично В. Я. Чубаря и председателя ВСНХ УССР Б. К. Сухомлина, так и со стороны харьковской интеллигенции – ученых всех факультетов университета, Электромеханического института, Математического института во главе с С. Н. Бернштейном и Н. И. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI 1.Признать организацию Физико-технического института на Украине необходимой. 2. Имея в виду, что Физико-технический институт должен вовлечь в свою работу научно-технические силы Украины и установить тесную связь с заводскими лабораториями, научно-исследовательскими учреждениями ВСНХ и Наркомпроса, считать необходимым организовать институт в Харькове... 2. Имея в виду, что Физико-технический институт должен вовлечь в свою работу научно-технические силы Украины и установить тесную связь с заводскими лабораториями, научно-исследовательскими учреждениями ВСНХ и Наркомпроса, считать необходимым организовать институт в Харькове... р р р у р 5. Просить академика Абрама Федоровича Иоффе взять на себя обязанности председателя научн нического совета института. 6. Для проведения всей подготовительной работы по организации Физико-технического института утвердить организационное бюро во главе с проф. Обреимовым, в составе профессоров Штейнберга, Желиховского, Рожанского, Перевозного, а также представителей от НТУ Украины и Укрглавнауки. 6. Для проведения всей подготовительной работы по организации Физико-технического института утвердить организационное бюро во главе с проф. Обреимовым, в составе профессоров Штейнберга, Желиховского, Рожанского, Перевозного, а также представителей от НТУ Украины и Укрглавнауки. 7. Выразить благодарность академику А. Ф. Иоффе за проявленную им инициативу в деле развития научно- исследовательской работы на Украине, в частности, отметить с удовлетворением выдвижение Ленинградским физико-техническим институтом группы высококвалифицированных научных работников для работы в Украинском физико-техническом институте». В период становления УФТИ Иван Васильевич Обреимов, будучи еще только руководителем оргбюро, входил во все детали постройки института, продумывая план строительства УФТИ с ведущими специалистами. Так, в частности, «к строительству института И. В. Обреимов привлек талантливых архитекторов П. И. Сидорова и В. И. Богомолова. Проект фундамента и нулевой цикл научного корпуса и жилого дома были выполнены за 2 недели. Пока их строили, за 4 недели был полностью готов остальной проект зданий. В качестве перекрытий были использованы конструкции с затонувшего в Севастопольской бухте и поднятого в 1928 г. корабля «Императрица Мария». Параллельно проводили испытания на огнестойкость камышита, из которого были построены стены библиотеки и конференц-зала (камышит оказался негорючим). Крыша криогенной лаборатории была установлена на рельсах. При взрыве в 1943 г. гитлеровцами здание устояло, а крыша поднялась и опустилась взрывная волна не разрушила здания функционирующего и по сей день» р у , р р ру , фу ру И недаром впоследствии газета «Известия» от 11 ноября 1932 года в очерке Ф. Кандыбы «Снайперы атомного ядра» рапортовала: «Харьковский институт действительно строился темпами пятилетки и за девять месяцев был выстроен. Директор института профессор Обреимов разъезжал по Европе, закупая и заказывая самое лучшее, новейшее оборудование для лабораторий». EEJP Vol.5 No.1 2018 Вскоре я уехал в Лейден, там окончил свою работу по спектрам кристаллов при низких температурах и стал работать на заводе Хука в Схидаме, где изготовлялась машина жидкого водорода для ЛФТИ. Неожиданно в октябре 1928 г. получаю от А. Ф. Иоффе телеграмму: «Выезжайте немедленно организовывать Харьков». Я ответил: «СОС пятьсот». Получив свои «СОС пятьсот», я вернулся в Ленинград и на следующий день выехал в Харьков, взяв с собою П. И. Стрельникова и А. И. Лейпунского». Феномен УФТИ В 1929 году приказом по ВСНХ УССР был назначен директором вновь организуемого Украинского физико-технического института в г. Харькове. Из автобиографии И. В. Обреимова Правительство Украины с признательностью и должным вниманием отнеслось к предложению вице- президента АН СССР академика А. Ф. Иоффе создать Украинский физико-технический институт в Харькове. В этой связи примечательна выдержка из Постановления заседания коллегии Научно-технического управления ВСНХ УССР от 16 мая 1928 г.: «Слушали: Об организации ФТИ на Украине. Доклад акад. А. Ф. Иоффе. Постановили: 95 95 EEJP Vol.5 No.1 2018 It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI Сотрудничество с иностранными учеными (как с носителями передового научного знания) в значительной степени способствовало приобщению уфтинцев к новейшим успехам западноевропейской науки, так как последние новости физической науки они получали, что называется, из первых уст. Сотрудничество с иностранными учеными (как с носителями передового научного знания) в значительной степени способствовало приобщению уфтинцев к новейшим успехам западноевропейской науки, так как последние новости физической науки они получали, что называется, из первых уст. Кроме того, уже на этапе становления УФТИ многим видным зарубежным ученым были посланы приглашения занять вакантные должности руководителей научных отделов. В качестве показательного примера процитируем официальное приглашение, адресованное голландскому физику-теоретику П. С. Эренфесту. Март 1929 г. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI Ахиезером, а также руководящих работников заводов (ХЭМЗ, ХПЗ), которые очень помогали УФТИ, охотно принимали на работу наших студентов, защитивших диплом, а также некоторых сотрудников УФТИ. Инженеры ХЭМЗ и Турбинного завода принимали участие в научных собраниях УФТИ и выступали у нас с докладами». Безусловно, в Харькове во всю мощь проявился как административный, так и организаторский таланты Ивана Васильевича Обреимова. И именно он – первый директор УФТИ – сумел заложить стратегические направления института, которые и позволили первенцу физической науки Украины за невероятно короткий срок выйти на передовые научные позиции. р р у Стремительнейший старт УФТИ во многом был определен именно тем, что первый директор не побоялся сделать главную ставку на фундаментальную науку и талантливую молодежь. Много лет спустя академик И. В. Обреимов с особой гордостью подчеркнет в своих мемуарах: «Если у меня есть серьезная заслуга перед 96 EEJP Vol.5 No.1 2018 96 EEJP 96 A. Tan’shyna EEJP Vol.5 No.1 2018 страной – то это одна заслуга: это то, что я культивировал теоретическую физику в Харькове и тем самым в СССР... Своих теоретиков у нас не было... р у Было сделано так, что в Харькове у нас всё время гостили приезжие учёные, так что получался центр теоретической физики... р ф Важно было, что теоретики приезжали не как гости, а длительно работали. ф было, что теоретики приезжали не как гости, а длительно работали. нигде в СССР не было» Этого нигде в СССР не было». Этого нигде в СССР не было». Этого нигде в СССР не было». Этот феномен УФТИ даже более полувека спустя будет специально отмечен на академических страницах «Вестника Российской академии наук»: «Факт приглашения ведущих иностранных ученых в Харьков можно оценить, только если учесть, что из-за ограниченных, как всегда, валютных возможностей это приглашение конкурировало с альтернативой купить новый спектрограф или какой-нибудь другой прибор. По существу, же приезд западных ученых оказал влияние на всю российскую науку того времени, поскольку в летний период в Харькове собирались и исследователи из других городов Советского Союза. Физики несомненно отметят, что Иван Васильевич приглашал наиболее активно и плодотворно работавших специалистов того времени, иными словами, приглашал “кого надо”». р Будучи дальновидным ученым, первый директор УФТИ исключительно большое значение придавал налаживанию широкого международного научного сотрудничества. На сегодняшний день имеется возможность объективно восстановить картину тогдашних уникальных международных контактов УФТИ по мемуарным заметкам И. В. Обреимова: «Было сделано так, что в На сегодняшний день имеется возможность объективно восстановить картину тогдашних уникальных международных контактов УФТИ по мемуарным заметкам И. В. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI Обреимова: «Было сделано так, что в Х ё ё й ф Харькове у нас всё время гостили приезжие учёные, так что получался центр теоретической физики. В 1929 г. приезжали П. Иордан (Гамбург), Ф. Гейтлер (Гёттинген). Ежегодно на два месяца приезжал из Ленинграда В. А. Фок... Харькове у нас всё время гостили приезжие учёные, так что получался центр теоретической физики. В 1929 г. приезжали П. Иордан (Гамбург), Ф. Гейтлер (Гёттинген). Ежегодно на два месяца приезжал из Ленинграда В. А. Фок... р у р р у у ц р р ф В 1929 г. приезжали П. Иордан (Гамбург), Ф. Гейтлер (Гёттинген). Ежегодно на два месяца приезжал из Ленинграда В. А. Фок... С весны 1930, когда мы уже въехали в своё здание, к нам приезжали: 3 раза (3 года) П. Дирак из Кембриджа, год работал Подольский из Принстона (США). Дважды приезжал П. С. Эренфест из Лейдена... Дважды приезжал Плачек и сделал с Ландау работу о строении линии рэлеевского и рамановского рассеяния. Дважды приезжал Вайскопф. Один раз приезжал Пайерлс... С весны 1930, когда мы уже въехали в своё здание, к нам приезжали: 3 раза (3 года) П. Дирак из Кембриджа, год работал Подольский из Принстона (США). Дважды приезжал П. С. Эренфест из Лейдена... Дважды приезжал Плачек и сделал с Ландау работу о строении линии рэлеевского и рамановского рассеяния. Д д В й ф Од П й Дважды приезжал Вайскопф. Один раз приезжал Пайерлс... Дважды приезжал Вайскопф. Один раз приезжал Пайерлс... В б д б Д р ф р р р Важно было, что теоретики приезжали не как гости, а длительно работали. В 1933 г. переехал окончательно Л. Д. Ландау (до сих пор он часто приезжал в качестве гостя и по хорошему свойству своего характера вмешивался во все дела УФТИ). Образовалась группа его учеников: А. И. Ахиезер, Е. М. Лифшиц, И. Я. Померанчук. В 1933 г. переехал окончательно Л. Д. Ландау (до сих пор он часто приезжал в качестве гостя и по хорошему свойству своего характера вмешивался во все дела УФТИ). Образовалась группа его учеников: А. И. Ахиезер, Е. М. Лифшиц, И. Я. Померанчук. А. И. Ахиезер, Е. М. Лифшиц, И. Я. Померанчук. В 1934 г. на три недели приезжал Нильс Бор и каждый день до обеда работал с теоретиками. Этого нигде в СССР не было». р ф ц р у В 1934 г. на три недели приезжал Нильс Бор и каждый день до обеда работал с теоретиками. Этого нигде в СССР не было». It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI 97 EEJP Vol.5 No.1 2018 Весьма показателен и следующий факт: «молодой физик-теоретик из Венгрии Л. Тисса, которому так понравилась обстановка в УФТИ, что он решил остаться в Харькове... Кстати, о его человеческих качествах говорит такой факт: ему как иностранному специалисту была назначена более высокая зарплата, чем нам. Но Тисса посчитал это несправедливым и попросил установить ему такую же зарплату, как и у других теоретиков». А вот как описывает причины Виктор Фредерик Вайскопф (кстати, в 1961-1965 гг. – генеральный директор ЦЕРНа), побудившие его приехать в Харьков: «Я не мог получить работы ни в Англии, ни во Франции .... Я почти на год уехал в Россию, в Харьков, где можно было получить работу, обеспечивающую средства к существованию». ущ Стоит процитировать по этому поводу и небезынтересное харьковское письмо П. Эренфеста: «Я пробыл в России с 14 декабря по 14 января все время в Харькове, среди моих друзей в Украинском Физико- техническом институте. Жизнь там сейчас полна трудностей. Они, может быть, не так сильно ощущаются иностранными специалистами – в смысле возможностей приобретения продуктов и других предметов. Несмотря на эти трудности, все мои друзья чувствуют себя положительно счастливыми и работают с замечательным энтузиазмом. Они очень, очень устают, в частности потому, что все здесь страшно быстро разрастается, а с этим связано много беспорядка, чертовски непроизводительно отнимающего до 80% энергии (население Харькова за несколько лет выросло с 200 тысяч человек до миллиона и продолжает увеличиваться). Но вот удивительная вещь: каждый мужчина, каждая женщина, которые учатся, чувствуют себя совершенно необходимыми обществу, и Вы представляете, что это чувство означает! Сам я точно так же немедленно почувствовал себя молодым и полным инициативы... у Все мои друзья настаивают на том, что я должен навсегда переехать в Россию и помочь им. Я начал сейчас обсуждать с моими датскими и русскими друзьями возможность некоего комбинированного плана, который позволил бы мне проводить 4 месяца в Голландии, 6 – в России, а два - странствуя между моими немецкими и скандинавскими коллегами, знакомясь с новыми работами, выполняемыми в Берлине, Лейпциге, Геттингене и Копенгагене». Научный престиж УФТИ поднимал и тот уникальный факт, что лауреат Нобелевской премии П. Дирак был избран почетным членом Ученого совета. А научными консультантами института согласились стать такие легенды физической науки тех лет, как П. Эренфест, П. Капица, Г. Гамов. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI К тому же на базе УФТИ с 1932 года начал издаваться первый советский физический журнал «Physikalische Zeitschrift der Sowjet Union» на иностранных языках. Поэтому неудивительно, что уже первые научные успехи уфтинцев были на уровне лучших работ того времени и стали достоянием широкой гласности на 17 съезде ВКП(б): «Исследовательская работа по физике в сколько-нибудь широком масштабе велась в СССР до последнего времени только в Ленинграде и Москве. Рост промышленности Союза поставил задачу создания крупных научных учреждений, работающих в области физики, и в других важнейших промышленных центрах. р ф ру р р По инициативе Украинского правительства в план первой пятилетки ВСНХ СССР была внесена организация научно- исследовательского физико-технического института в Харькове (УФТИ). Организация нового института была поручена Ленинградскому физико-техническому институту, который выделил для него большую группу (около 20 чел.) научных работников. Эта группа вместе с частью харьковских физиков и составила основное ядро института ... Развитию работ по теоретической физике институт все время уделяет особое внимание, так как эти работы, кроме их непосредственного значения, в сильной степени способствуют общему высокому уровню работ института. Тесное взаимодействие теоретических и экспериментальных работ составляет одну из самых существенных черт научного лица института ... у Большой успех работы теоретиков вызвал приток ученых из других институтов Союза и из-за границы для временной работы в институте... За истекшие три года своей работы Украинский физико-технический институт стал одним из крупн центров Союза в области физики и пользуется большим авторитетом за границей». И надо отдать должное первому директору УФТИ. Именно Иван Васильевич Обреимов не побоялся собрать в стенах вновь созданного института целую плеяду молодых и талантливых ученых, которые и вывели институт на передовые научные позиции. Причем многие прошли стажировку в крупнейших научных центрах мира. И надо отдать должное первому директору УФТИ. Именно Иван Васильевич Обреимов не побоялся собрать в стенах вновь созданного института целую плеяду молодых и талантливых ученых, которые и вывели институт на передовые научные позиции. Причем многие прошли стажировку в крупнейших научных центрах мира. Особо судьбоносным для УФТИ стало приглашение в 1932 году Льва Давидовича Ландау на должность руководителя отдела теоретической физики. Вот как этот факт был прокомментирован более полувека спустя на страницах «Вестника Российской академии наук»: «Его отношения с Иоффе обострились настолько, что Лев Давидович вынужден был уйти из института. Поскольку характеристики его оказались резко негативными, то к моменту, когда Обреимов пригласил Ландау в Харьков, он уже около года мыкался в Ленинграде без работы». На момент переезда в Харьков Ландау исполнилось всего лишь 24 года. Глубокоуважаемый и дорогой Павел Сигизмундович! Глубокоуважаемый и дорогой Павел Сигизмундович! Большой Физико-технический институт в Харькове, о котором нам пришлось несколько раз с Вами говорить, по- видимому, близок к осуществлению. Нам поручено его организовать. Одна из первых наших мыслей была привлечь Вас к организации этого института. Всем известна та роль, которую Вы уже сыграли для развития физики в России, Ваш неизменный благожелательный интерес к ней и та постоянная помощь, которую Вы оказываете. Поэтому мы просим Вас принять место консультанта Украинского физико-технического института. Мы очень просим Вас сюда приехать в этом году месяца на два. Для возмещения Ваших расходов по поездке мы приготовили 2000 рублей. Одновременно с этим мы посылаем Вам некоторые материалы по организации института. Одна из самых важных вещей в организации физики – это организация теоретической физики, и вместе с тем это для нас одна из самых трудных задач, потому что теоретиков у нас мало и теоретическая молодежь боится покидать Ленинград и Москву, чтобы не потерять руководства. Нам кажется, что если бы Вы согласились стать во главе теоретической физики Харькова, перенести туда Вашу школу, то это было бы одной из важнейших вещей не только для развития физики в нашем Союзе, но и для мировой физики. Здесь дело не только в том, что наш Союз приобрел бы в Вашем лице физика, стоящего в первом ряду физиков, но и в Вашем исключительном умении группировать вокруг себя и теоретиков и экспериментаторов, давать помощь и совет в вопросах научной организации. Нам кажется, что в смысле научной работы, быта, климата Вы могли бы иметь условия не хуже тех, которые имеете сейчас; в смысле же пользы для физики — принесли бы здесь неизмеримо большую. Мы надеемся, что во время Вашего приезда Вы разрешите поднять и подробно обсудить с Вами вопрос о полном переходе Вашем в Харьков, приезд же Ваш даст Вам возможность лично увидеть условия научной работы и обстановку. Ваши: А. Иоффе, И. Обреимов. Ваши: А. Иоффе, И. Обреимов. 97 It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI В этой связи стоит процитировать отзыв о научных трудах Л. Д. Ландау тех лет, за авторитетной подписью физика-теоретика В. Фока: «Работы Л. Д. Ландау пользуются большой известностью как у нас в Союзе, так и за границей. Так, в посвященном квантовой механике ХХIV томе (часть 1) известной германской энциклопедии физики «Handbuch der Physik» 98 98 EEJP Vol.5 No.1 2018 A. Tan’shyna 98 EEJP Vol.5 No.1 2018 имя Ландау цитируется 11 раз. Нужно заметить, что этот том издан в 1933г., а большинство работ Ландау относится к периоду после 1933 г.». имя Ландау цитируется 11 раз. Нужно заметить, что этот том издан в 1933г., а большинство работ Ландау относится к периоду после 1933 г.». имя Ландау цитируется 11 раз. Нужно заметить, что этот том издан в 1933г., а большинство работ Ландау относится к периоду после 1933 г.». р у Кроме того, Л. Д. Ландау имел личные научные контакты с ведущими физиками-теоретиками мира, так как ранее совершил европейское турне по научным центрам мира. По этому поводу приведем выдержку из его ленинградского «Отчета о научной заграничной командировке в Данию, Швейцарию и Германию в 1929- 1931гг.»: «С октября 1929 г. до апреля 1930 г. я находился в заграничной командировке за счет НКП, а затем до марта 1931 г. по рокфеллеровской стипендии. За это время я имел возможность работать в контакте с наиболее выдающимися современными теоретиками, из которых наибольшее влияние на мою работу оказали N. Воhr (Копенгаген), W. Раuli (Цюрих) и W. Неisenberg (Лейпциг)». ( ) ( р ) g ( ) Кстати, Нильс Бор впоследствии приезжал к Ландау в Харьков. Это была дань уважения и его любимому ученику, и Украинскому физико-техническому институту. До наших дней сохранился отзыв Нильса Бора о тогдашнем посещении УФТИ: «Я рад возможности выразить свои чувства высокого восхищения и удовлетворения, с которыми я увидел прекрасный новый Физико-технический институт в Харькове, где отличные условия для экспериментальной работы во всех областях современной физики сочетаются с величайшим энтузиазмом и успехами под замечательным руководством и тесным сотрудничеством с блестящим физиком-теоретиком» 22.5.1934 Нильс Бор». Нильс Бор». Харьковский период жизни Л. Д. Ландау продолжался всего лишь пять лет – с 1932-го по 1937 год. Но именно он заложил краеугольный камень в основание харьковской школы теоретической физики, ставшей впоследствии одной из самых известных украинских теоретических школ в мире. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI Много лет спустя патриарх теоретической физики Украины академик Александр Ильич Ахиезер – харьковский ученик и преемник Ландау на посту руководителя теоротдела УФТИ – имел веские основания, чтобы объективно подытожить: «Если бы меня попросили назвать всего двух физиков, в максимальной мере прославивших украинскую науку, то я бы назвал теоретика Л. Д. Ландау и экспериментатора Л. В. Шубникова». у Следует пояснить приведенную выше цитату. Дело в том, что именно Лев Васильевич Шубников возглавил в УФТИ первую(!) в СССР криогенную лабораторию. р у ( ) р у р р По воспоминаниям его жены Ольги Трапезниковой, «еще в нашу бытность в Лейдене И. В. Обреимов предложил Льву Васильевичу переехать в Харьков, во вновь созданный УФТИ, директором которого он стал». По воспоминаниям его жены Ольги Трапезниковой, «еще в нашу бытность в Лейдене И. В. Обреимов предложил Льву Васильевичу переехать в Харьков, во вновь созданный УФТИ, директором которого он стал». Лев Васильевич Шубников, будучи сотрудником ЛФТИ, обучался методике проведения эксперимента при низких температурах в прославленной Лейденской лаборатории. И выбор для научной стажировки именно Лейденского университета (1575 г. – дата основания) был тогда не случайным, так как еще в 1894 году при этом университете была организована первая в мире криогенная лаборатория, которая впоследствии стала одним из самых авторитетных мировых центров физики низких температур. Следует особо подчеркнуть: Лейденская лаборатория существенно помогла УФТИ в оснащении первой в СССР (и четвертой в мире) криогенной лаборатории, руководителем которой в период становления (1931– 1937 гг.) был Л. В. Шубников. В частности, «очень большую помощь лаборатории оказывал Э. Вирсма [куратор Лейденской лаборатории. – Прим. А. Т.]. Он каждый год, вплоть до 1935 года, приезжал в Харьков и привозил массу всяких вещей, без которых мы не могли работать...Он привозил все, что мы в Союзе не могли достать...Разумеется, все это Э. Вирсма делал с одобрения В. де Хааса [директора Лейденской лаборатории. – Прим. А. Т.] – мы получали обещанную ранее в Лейдене помощь». р ] у щ у р щ Стоит напомнить и тот факт, что именно Иван Васильевич Обреимов стоял у истоков уфтинской криогенной лаборатории. Процитируем по этому поводу его письмо к Петру Леонидовичу Капице, который в то время работал в Кавендишской лаборатории. Л. В. Каменев – П. Л. Капице Уважаемый Петр Леонидович. Сейчас мы организуем в Харькове Физико-технический институт по типу Ленинградской [физико-технической] лаборатории академика А. Ф. Иоффе. Придавая этому делу исключительное значение, я решил просить Вас принять участие в организации этого института в качестве консультанта. Если Вы соглашаетесь на мое предложение, то на Вашей обязанности будет лежать ежегодный приезд в СССР на 2-3 месяца. Вопрос о визах (въездных и проездных) для Вас и Вашей семьи не будет связан для Вас ни с какими затруднениями и может быть при Вашем первом приезде сюда урегулирован так, как это Вам будет удобно. За Вашу работу здесь Вы будете получать ежегодно 2 000 рублей, причем в эту же сумму будут входить и Ваши расходы по поездкам сюда. ру р у у у у у р Зная о ваших научных успехах, я полагаю, что Ваши приезды сюда окажут вообще существенное значение не только для Харьковского института, но и вообще для дела научно-технического развития СССР. Ознакомившись с Вашим вопросом, я вообще был удивлен, что до сего времени не велось официальных переговоров с Вами о перенесении Ваших работ, как советского ученого, в СССР. Те средства, которыми располагает НТУ, вполне позволят создать для Вас в СССР – Ленинграде, Москве или Харькове – те условия, которые необходимы для успешного развития Вашей работы. Я полагаю, что при приезде сюда мы побеседуем с Вами об этих возможностях. Я вполне гарантирую вам, однако, что никакие давления в смысле немедленного переезда сюда на Вас не будут оказаны и весь вопрос будут решен в смысле наиболее успешного и бесперебойного хода Вашей работы. Уважающий Вас, Уважающий Вас, Л. Каменев. Л. Каменев. It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI 99 EEJP Vol.5 No.1 2018 И. В. Обреимов – П. Л. Капице Кембридж, 17 ноября 1929 г. р р Дорогой Петр Леонидович. Нас всех крайне порадовало твое согласие быть консультантом у нас в Институте, и я надеюсь, что это лишь первый шаг к твоему постоянному переезду в СССР для постоянной научной работы. Ты прекрасно знаешь, как быстро научная работа развивается у нас в стране и какое громадное значение придается ей у нас. р Дорогой Петр Леонидович. Нас всех крайне порадовало твое согласие быть консультантом у нас в надеюсь, что это лишь первый шаг к твоему постоянному переезду в СССР для постоянной научной работ Я тебя могу уверить, что все необходимое, чтобы облегчить твой переезд будет предпринято и частично уже предпринято с нашей стороны. 1. Принимая во внимание твои моральные обязательства по отношению [к] Кавендишской лаборатории, мы включили в пятилетний план УФТИ на 1929/30 г. сумму в 250 тысяч рублей валютой на выкуп твоей лаборатории. Этот 5-летний план утвержден Президиумом ВСНХ УССР... 1. Принимая во внимание твои моральные обязательства по отношению [к] Кавендишской лаборатории, мы включили в пятилетний план УФТИ на 1929/30 г. сумму в 250 тысяч рублей валютой на выкуп твоей лаборатории. Этот 5-летний план утвержден Президиумом ВСНХ УССР... у р р у 3. В пятилетний план на 1929/30 год включена сумма в 300 тысяч рублей на постройку твоей Магнитной Лаборатории на участке нашего института. 3. В пятилетний план на 1929/30 год включена сумма в 300 тысяч рублей на постройку твоей Магнитной Лаборатории на участке нашего института. у у 4. Что касается твоего положения, то оно будет таким, каким ты пожелаешь, т.е. либо директором УФТИ, либо независимым Старшим Физиком, либо можешь иметь совершенно независимую лабораторию... И Об 4. Что касается твоего положения, то оно будет таким, каким ты пожелаешь, т.е. либо директором УФТИ, либо независимым Старшим Физиком, либо можешь иметь совершенно независимую лабораторию... И Об И. Обреимов И. Обреимов И. В. Обреимов – П. Л. Капице И. В. Обреимов – П. Л. Капице 12 июня 1928 г. 12 июня 1928 г. <...>. То, о чем с Вами говорили насчет провинции, осуществляется, и даже, м. б., более бурно и стремительно, чем нужно. у В наш ГФТИ поступило разом два предложения организовать 2 физических института, один в Томске, другой у Харкові. С Томском дело сделано... С Харьковом дело не только не кончено, но даже и не начато. В жертву Харькову обречен Ваш покорный слуга. В Харькове предполагается дело очень интересное – большая криогенная лаборатория с водородом и гелием. Отчасти по моей инициативе... Должен Вам сказать, что к институтам сверхдредноутам у меня влечения нет, т. ч. я мыслю себе – это очень скромный институт. Но вот когда я думаю о Вас, то мне кажется, что если бы вы там были, то это был бы допинг для нашей физики, и для физики вообще. ф Вы подумайте, что можно сделать в таком институте. А для отечества – это ведь тоже будет институт, который будет конкурировать по своему значению с Питером и будет Питер подтягивать... ф Вы подумайте, что можно сделать в таком институте. А для отечества – это ведь то конкурировать по своему значению с Питером и будет Питер подтягивать... ур р у р у р В этой связи нельзя не вспомнить и о последующей уфтинской борьбе за Петра Леонидовича Капицу, который с 1921 года находился в многолетней научной командировке в Англии. В этой связи нельзя не вспомнить и о последующей уфтинской борьбе за Петра Леонидовича Капицу, который с 1921 года находился в многолетней научной командировке в Англии. 99 It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI 10 октября высоковольтная бригада разрушила ядро лития; работы продолжаются. Директор УФТИ Обреи 10 октября высоковольтная бригада разрушила ядро лития; работы продолжаются. Директор УФТИ Обреимов. Секретарь парткома Шепелев. Местком – Федоритенко. р р д Директор УФТИ Обреимов. Секретарь парткома Шепелев. Местком – Федоритенко. Уфтинская телеграмма – беспрецедентное за всю историю СССР событие. УФТИ вскоре были выделены значительные средства для сооружения еще более мощных ядерно-физических установок. С того времени ядерная физика и техника – одно из ведущих направлений института. р ф у р у К концу 30-х годов прошлого столетия УФТИ стал одним из наиболее авторитетных институтов СССР. И первая выездная сессия физической группы АН СССР была проведена именно в Харькове, на базе УФТИ (23-24 января 1937 года). Заслуживает внимания следующая выдержка из резолюции данной сессии: «УФТИ за шесть лет своего существования превратился в один из ведущих физических институтов Советского Союза. Сессия отметила огромное научное и техническое значение создания в УФТИ криогенной лаборатории, стоящей на уровне лучших мировых лабораторий низких температур». ур у р р р р ур Этот стремительный взлет УФТИ можно по праву назвать научным феноменом. Со слов директора- организатора УФТИ Ивана Васильевича Обреимова, «мы в УФТИ правильно оценили тенденцию развития науки, что дальнейшее развитие науки пойдет по двум направлениям: физика конденсированной фазы (которую называют сейчас физикой твердого тела, или физикой кристаллов, причем оба названия меня несколько коробят) и физика атомного ядра. УФТИ был инициатором в СССР направления «атомного ядра». В других центрах этим вопросом не занимались и не предполагали заниматься, отчасти считая, что для народного хозяйства эта проблема лежит в далеком поле. Физика твердого тела. Надо помнить, что до появления Петра Капицы мы были первой и единственной лабораторией в СССР и четвертой в мире, где был жидкий водород, а с 1933 г. и жидкий гелий. Мне вспоминается встреча с О. Мейсснером в Берлинском Рейхсанштальте (аналогичен нашему Институту метрологии) в 1928 г. Он мне сказал: «Вы желаете иметь машину жидкого водорода? Вопрос в том, будете ли Вы иметь достаточно культурного механика, чтобы её обслужить. Я такого механика добыть себе не могу и сам обслуживаю эту установку. Другие на ней работают, а я обслуживаю. Немецкие механики, конечно, культурнее русских механиков. Ваша судьба такая: Вы будете механиком при машине, а другие будут с жидким водородом работать. Впрочем, я буду Вам охотно помогать». у у И он, действительно, очень помогал, не с водородом, где помощь оказывал Лейден, персонально профессор Кроммелин и главный механик Флим, а с гелием. Но пророчество Мейсснера не оправдалось. П. Л. Капица – А. А. Капице Москва, 15 апреля 1935 г. , <...>. Настроение убийственное. Представь, уже почти 8 месяцев я сижу и ничего не делаю, и это н желаю, а, конечно, потому, что я не могу... р оение убийственное. Представь, уже почти 8 месяцев я сижу и ничего не делаю, и это не потому, что не о, потому, что я не могу... Замечания: «Почему вы не поедете в Хар[ьков], работать там», так же наивны, как предложить шоферу сесть на козлы извозчика. То, что автомобиль и телега служат для езды, не значит еще, что один и тот же человек может на них ездить. Так и ученого нельзя пересаживать из одной лаборатории в другую. Первый директор УФТИ также, невзирая на авторитеты, твердо отстаивал свою точку зрения на стратегические пути развития института: «После моего окончательного переезда в Харьков, 2 апреля 1930 г., мои контакты с А. Ф. Иоффе значительно сократились. Сократились они частично и из-за того, что Абрам Федорович не во всем одобрял направление работ Харьковского института. С одной стороны, в Харькове имела место «гипертрофия теоретической физики»... С другой стороны, в Харькове успешно проводились работы по физике атомного ядра». Судьбоносен и следующий факт: именно уфтинцы впервые в СССР (и вторые в мире!) расщепили атомное ядро. Временная разница в воспроизведении знаменитого кембриджского эксперимента (апрель 1932 года) в УФТИ (октябрь 1932 года) – всего лишь полгода. На скромной экспериментальной базе института был воспроизведён фундаментальный эксперимент одной из старейших физических лабораторий мира – Кавендишской лаборатории, основанной в 1874 году при старейшем университете Европы – Кембриджском университете, который в свою очередь ведет летоисчисление с 1209 года. УФТИ тотчас же рапортовал тогдашнему советскому правительству о проведенном фундаментальном эксперименте. Ниже дословно приводится текст этой исторической телеграммы, которая была напечатана на первой полосе газеты «Правда» от 22 октября 1932 года. Разрушено ядро атома лития Крупнейшее достижение советских ученых МОСКВА, тт. СТАЛИНУ, МОЛОТОВУ, ОРДЖОНИКИДЗЕ, «ПРАВДЕ». Украинский физико-технический институт в Харькове в результате ударной работы к XV годовщине Октября добился первых успехов в разрушении ядра атома. Украинский физико-технический институт в Харькове в результате ударной работы к XV годовщине Октября добился первых успехов в разрушении ядра атома. 100 EEJP Vol.5 No.1 2018 100 A. Tan’shyna 10 октября высоковольтная бригада разрушила ядро лития; работы продолжаются. Директор УФТИ Обреи В руках механика Ивана Петровича Королева, а затем Владимира Ивановича Богатова и их воспитанников работали и две установки жидкого водорода, а впоследствии и установка жидкого гелия, полученная с помощью того же Мейсснера, работают и поныне. Мы также правильно оценили масштаб и широкий стиль работ, стиль хороших измерений. Мы не задавались целью «догнать и перегнать», а просто делали как можно лучше, как можно тщательнее те исследования, которые, мы считали, стоят в повестке дня физики. С этой стороны я должен вспомнить Льва Васильевича Шубникова, который создал в УФТИ стиль критической, тщательной, точной работы. Надо вспомнить также Вадима Сергеевича Горского. Открытие им упорядоченных и частично упорядоченных твердых растворов является гордостью УФТИ. Мы переезжали в Харьков – город, уже обладающий высокой культурой в области физики. Я имею школу Дмитрия Аполлинарьевича Рожанского, продолжателем дела которого был Александр Абрамович Слуцкин. Напомню, что до 1941 г. мировой рекорд по 30-мм-ым магнетронным колебаниям принадлежал УФТИ». Показательна также и следующая подробность: во времена директорства И. В. Обреимова широкий демократизм определял истинное лицо УФТИ. Так, в частности, «каждую неделю в УФТИ происходило заседание совета и проводился реферативный семинар... На заседаниях совета докладывались все работы, выполнявшиеся в лабораториях УФТИ, а на реферативном собрании – новые журнальные статьи по различным разделам физики. Прекрасный овальный стол, за которым сидели участники заседаний, создавал неповторимую обстановку легкости и даже интимности, чему содействовал еще подаваемый чай с пирожными. у , у р Такая обстановка не препятствовала, а, пожалуй, даже способствовала накалу бескомпромиссности критики». становка не препятствовала, а, пожалуй, даже способствовала накалу дискуссий и ности критики». Первый директор УФТИ, как отмечают его современники, имел привычку задавать докладчикам вопросы, которые, на первый взгляд, были довольно-таки просты. Но впоследствии оказывалось, что именно они вскрывают суть данной темы. «Кто не спрашивает, тот никогда не поумнеет» – любимый афоризм Ивана Васильевича Обреимова. Кстати, свой «простой» вопрос он предварял, как правило, такими словами: «Простите мне мою невежественность». К тому же Иван Васильевич всегда просил называть фамилии тех ученых, на результаты которых делается ссылка, «чтобы было ясно, из какой лаборатории вышла работа – можно ей доверять или нет». А при анализе научных результатов замечал: «Есть три вида лжи: ложь, наглая ложь и статистика, как говорил Бисмарк». Иван Васильевич Обреимов, будучи не тщеславным и не честолюбивым, ввел в институте весьма доверительный стиль руководства. 10 октября высоковольтная бригада разрушила ядро лития; работы продолжаются. Директор УФТИ Обреи Рассказывают, что на двери его служебного кабинета висела дощечка, где 101 2018 101 It is Worth Recalling: "This Was Nowhere in the USSR." - Ukraine, Kharkiv, UFTI 101 EEJP Vol.5 No.1 2018 были прибиты в ряд гвоздики, внизу которых были подписи: «Я – у Тоси», «Я – у Гарбера», «Не входить», «Я - у себя». Указателем служил металлический жетон, который вешался на соответственный гвоздик. были прибиты в ряд гвоздики, внизу которых были подписи: «Я – у Тоси», «Я – у Гарбера», «Не входить», «Я - у себя». Указателем служил металлический жетон, который вешался на соответственный гвоздик. Первый директор УФТИ, несмотря на трудности беспокойного организационного периода, не забросил и свои личные научные изыскания. По воспоминаниям Ольги Трапезниковой, у директора УФТИ «был свой отдел, куда входили В. С. Горский, Н. А. Бриллиантов, А. Ф. Прихотько и др... Я помню, как И. В. Обреимов ходил по институту с двумя ведрами на коромысле, наполненными жидким воздухом». Особой гордостью директора-организатора УФТИ была институтская библиотека, которая именно благодаря его хлопотам и была собрана. Примечательно и то, что он доверял ключи и предоставлял возможность сотрудникам УФТИ работать в любое время суток. Кроме того, по воспоминаниям академика Бориса Георгиевича Лазарева, «в институте все были увлечены спортом – альпинизмом, туризмом, теннисом, лыжами, наконец, многие занимались в школе верховой езды. Из всех этих увлечений Иван Васильевич не устоял перед последним (думаю, по примеру его ученицы Антонины Федоровны Прихотько – пятигорской казачки). Мы (Лейпунские, Хоткевич, Лазаревы) занимались в одной группе с Иваном Васильевичем... Иван Васильевич, объясняя свои физические особенности, говорил мне, что в детстве он много занимался игрой на рояле, «все время, пока другие бегали, играли, я занимался упражнениями на рояле». И надо сказать, что играл он превосходно, исполняя самые сложные произведения Бетховена, Баха». р р р В 1933 году Иван Васильевич Обреимов неожиданно для всех принимает решение: уйти с директорского поста. Официальная версия в его автобиографии изложена так: «По окончании организационного периода в 1933 г. перешел в том же институте на должность председателя НТС и заведующего лабораторией физики кристаллов». До сегодняшнего дня неизвестно, почему потребовалось отойти от дел руководства институтом именно в период стремительного взлета УФТИ.
https://openalex.org/W1492714130	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0129685&type=printable	English	null	LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1	PloS one	2,015	cc-by	10,701	LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1 Shwu-Fen Chang1, Shih-Shan Lin2, Hui-Ching Yang2, Yuan-Yi Chou2, Jhen-I Gao2, Shao- Chun Lu2* 1 Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan, 2 Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan * lsc@ntu.edu.tw Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Grant of SCL NSC96-2320-B002-022 by Ministry of Science and Technology in Taiwan (www. most.gov.tw). Grant of SFC NSC 96-2320-B-038-028 by Ministry of Science and Technology in Taiwan (www.most.gov.tw). Grant of SFC NSC99-2320- B038-009-MY3 by Ministry of Science and Technology in Taiwan (www.most.gov.tw). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differ- entiation of neutrophil progenitors which play important roles in host defense against infec- tious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expres- sion and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPβ synergistically acti- vate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPβ to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS- induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS- induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/ EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression. RESEARCH ARTICLE OPEN ACCESS Citation: Chang S-F, Lin S-S, Yang H-C, Chou Y-Y, Gao J-I, Lu S-C (2015) LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1. PLoS ONE 10(6): e0129685. doi:10.1371/journal.pone.0129685 Citation: Chang S-F, Lin S-S, Yang H-C, Chou Y-Y, Gao J-I, Lu S-C (2015) LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1. PLoS ONE 10(6): e0129685. doi:10.1371/journal.pone.0129685 Editor: Chih-Hsin Tang, China Medical University, TAIWAN Editor: Chih-Hsin Tang, China Medical University, TAIWAN TAIWAN Received: February 25, 2015 Accepted: May 12, 2015 Published: June 26, 2015 Copyright: © 2015 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. TAIWAN Received: February 25, 2015 Accepted: May 12, 2015 Published: June 26, 2015 Copyright: © 2015 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. G-CSF has therefore been suggested as a molecular target for chronic into the inflamed tissue and increased production of inflammatory mediators, such as cyto- kines, chemokines, and serum complement, which subsequently amplify the local inflamma- tory response. G-CSF has therefore been suggested as a molecular target for chronic inflammatory diseases [10–12]. Several studies have reported that G-CSF can also be produced by non-hematopoietic malignant tumors, such as hepatocellular carcinoma, pancreatic cancer, lung cancer, and gastric cancer, or cell lines derived from these [13–16]. G-CSF-producing tumors are often associated with aggressive growth and patients with this type of tumor tend to have a poor prognosis [16]. However, little is known about the pathological significance of G-CSF production by tumors and the underlying mechanisms triggering G-CSF expression. inflammatory diseases [10–12]. Several studies have reported that G-CSF can also be produced by non-hematopoietic malignant tumors, such as hepatocellular carcinoma, pancreatic cancer, lung cancer, and gastric cancer, or cell lines derived from these [13–16]. G-CSF-producing tumors are often associated with aggressive growth and patients with this type of tumor tend to have a poor prognosis [16]. However, little is known about the pathological significance of G-CSF production by tumors and the underlying mechanisms triggering G-CSF expression. It is known that LPS activates the NF-κB pathway and all three MAPK pathways (ERK, JNK/SAPK, and p38α), leading to a wide range of cellular responses, including cell differentia- tion, survival or apoptosis, and inflammatory responses [17]. We have previously reported that pretreatment with rapamycin, which blocks the activity of mTOR complex 1 (mTORC1), inhibits LPS-induced G-CSF expression by decreasing the expression of Oct-2, a crucial tran- scription factor required for this process [6]. In addition, our preliminary data showed that pre- treatment for 30 min with 10 μM U0126, a specific MAP/ERK kinase inhibitor, inhibited LPS- induced expression of G-CSF in RAW264.7 murine macrophage cells (S1 Fig). In monocytes/ macrophages, both extracellular signaling-regulated kinases, ERK1 and ERK2, are activated by LPS or cytokines, increasing proinflammatory gene expression [18, 19]. In response to stimuli, ERKs are phosphorylated at the Thr-Glu-Tyr (TEY) motif, and then activate numerous down- stream modulators, including transcription factors Elk-1, NF-AT, STAT3, and C/EBPβ [20– 22]. However, little is known about the specific involvement of ERK1 or ERK2 in LPS-induced G-CSF expression. We recently reported that ERK2 is important in G-CSF production of can- cer cells [23]. Introduction Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor, regulates the proliferation of neutrophil progenitors, and the differentiation of granulocyte lineages, and the survival and maturation of neutrophil progenitors, and their mobilization from bone marrow to peripheral tissues [1]. For decades, recombinant G-CSF has been widely used in patients receiving chemotherapy to increase the number of circulating hematopoietic progenitor cells and in certain patients with neutropenia. Endogenous G-CSF is produced by various types of PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 1 / 17 In the present study, we investigated the role of ERKs in LPS-induced G-CSF expression in macrophages and identified the essential role of ERK2 in this process. Our results demonstrated that LPS-activated ERK2 functions by remodeling local chromatin, interacting with C/EBPβ and synergizing its transactivation activity to increase G-CSF expression. This study suggests that ERK2 may be a critical therapeutic target for surplus G-CSF related diseases. ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages cells, including bone marrow stromal cells, endothelial cells, macrophages, and fibroblasts, and its production is induced by inflammatory stimuli, including cytokines, such as IL-1β and TNF-α, and pathogenic toxins, such as lipopolysaccharide (LPS), via transcriptional and post- transcriptional mechanisms [2, 3]. NF-κB, NF-IL6 (C/EBP-β), and octamer-binding factor 2 (Oct-2), are transcription factors that have been identified essential for LPS-induced G-CSF expression in macrophages [4–6], but none of these factors alone is sufficient to drive LPS’s effect on G-CSF expression. Post-transcriptionally, LPS or cytokines increases G-CSF mRNA stability, which is regulated by the AU-destabilizing element and stem-loop destabilizing ele- ment in the 3’-end untranslated region [7, 8]. Competing Interests: The authors have declared that no competing interests exist. G-CSF stimulates the proliferation and functional maturation of neutrophils and plays an important role in host defense against microbial infection. However, excessive G-CSF levels are associated with increased severity of inflammatory diseases, for example, acute lung injury and rheumatoid arthritis [9, 10]. This is primarily due to G-CSF-induced neutrophil infiltration into the inflamed tissue and increased production of inflammatory mediators, such as cyto- kines, chemokines, and serum complement, which subsequently amplify the local inflamma- tory response. G-CSF has therefore been suggested as a molecular target for chronic inflammatory diseases [10–12]. Several studies have reported that G-CSF can also be produced by non-hematopoietic malignant tumors, such as hepatocellular carcinoma, pancreatic cancer, lung cancer, and gastric cancer, or cell lines derived from these [13–16]. G-CSF-producing tumors are often associated with aggressive growth and patients with this type of tumor tend to have a poor prognosis [16]. However, little is known about the pathological significance of G-CSF production by tumors and the underlying mechanisms triggering G-CSF expression. G-CSF stimulates the proliferation and functional maturation of neutrophils and plays an important role in host defense against microbial infection. However, excessive G-CSF levels are associated with increased severity of inflammatory diseases, for example, acute lung injury and rheumatoid arthritis [9, 10]. This is primarily due to G-CSF-induced neutrophil infiltration into the inflamed tissue and increased production of inflammatory mediators, such as cyto- kines, chemokines, and serum complement, which subsequently amplify the local inflamma- tory response. Materials Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone Laboratories (Logan, UT, USA). LPS from Escherichia coli (serotype 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved as a 1 mg/mL stock PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 2 / 17 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages solution in PBS. MMLV reverse transcriptase was from Promega (Madison, WI, USA). Super- Fect Transfection reagent was purchased from Qiagen (Hilden, Germany). Antibodies against ERK, phospho-ERK, p50, p65, C/EBPβ, β-actin, or lamin B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). U0126 and PD98059 (specific MEK inhibitors) were purchased from Calbiochem (San Diego, CA) and were dissolved as stock solutions in DMSO, and were added to the culture medium 30 min before other treatments or as indicated with 0.1% DMSO in culture medium as the control. RNA isolation and RT-PCR analyses Total cellular RNA was isolated from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and its concentration was determined from the absorbance at 260 nm, then it was subjected to reverse transcription (RT)-polymerase chain reaction (PCR) analysis as described previously [6]. G-CSF, TNF-α, and GAPDH mRNA levels were determined by RT-PCR using the primers listed in S1 Table. Cell culture and LPS treatment The RAW264.7 murine macrophage cell line was cultured as described previously [6]. THP-1, a human acute monocytic leukemia cell line, was maintained and induced to differentiate into macrophages using 160 nM of phorbol 12-myristate-13-acetate (PMA) as described previously [24]. G-CSF mRNA and protein levels were compared in untreated cells and cells pretreated with the indicated concentration of inhibitor or DMSO for 30 min, then LPS was added and the cells incubated for the indicated time; unless otherwise stated, LPS treatment was at 100 ng/ ml for 6 h and the concentration of U0126 was 10 μM. Preparation of cytosolic and nuclear extracts Cytosolic and nuclear extracts were prepared as described previously [25]. Briefly, cells were washed twice with ice-cold PBS, incubated on ice for 15 min with lysis buffer (10 mM HEPES, pH 7.4, 10 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.2% Nonidet P-40, and 0.2 mM PMSF), and collected by gentle pipetting. After centrifugation at 500 xg for 5 min at 4°C, the superna- tant was collected as the cytosolic extract, and the pellet was extracted by incubation for 15 min at 4°C with intermittent vortexing in nuclear extract buffer (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, and 1x protease inhibitor cocktail), followed by centrifugation at 12,900 xg for 10 min at 4°C, after which the supernatant was collected as the nuclear extract. The Bradford method (DC Protein Assay; Bio-Rad) was used to measure the protein concentrations in the extracts, which were then stored in aliquots at—80°C. Plasmids Expression plasmids encoding mouse p50, Oct-2, or C/EBPβ were, respectively, generous gifts from Dr. Neil D. Perkins, University of Michigan, Dr. Winship Herr, Cold Spring Harbor Lab- oratory, and Dr. Gerard Elbery, Section of Nephrology, Department of Pediatrics, University of Oklahoma Health Sciences Center. Plasmids encoding the T188A or S64A mutant of C/EBPβ were generated from the wild-type C/EBPβ-expressing plasmid by PCR mutagenesis and con- firmed by sequencing analysis. A plasmid expressing the constitutively active form of ERK2 (CA-ERK2) was obtained from Upstate Co. (Charlottesville, VA, USA). All plasmids were amplified in the E. coli DH5α host strain and purified by equilibrium centrifugation in a CsCl −EtBr gradient as described previously [24]. The purity and stability of plasmid preparations were confirmed by agarose gel electrophoresis followed by ethidium bromide staining, and the DNA concentration was measured by UV absorption at 260 nm. Western blot analysis Cells were washed with ice-cold PBS, then lysed with RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 0.5% Triton X-100, 0.1% SDS, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, and 1 μg/ml of leupeptin), then samples of the cell lysate (20 μg of pro- tein/lane) were separated by 10% SDS-PAGE and transferred to a PVDF membrane, which was then blocked overnight at 4ºC with blocking buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20, and 5% fat-free milk). The blots were then incubated for 1 h at room temperature with 0.5 μg/ml of rabbit polyclonal antibody against p50, p65, ERK, phospho- ERK, or β-actin in blocking buffer, then for 40 min at room temperature with peroxidase-con- jugated anti-rabbit IgG antibodies (Amersham-Pharmacia Biotech) in blocking buffer. Bound 3 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages antibody was detected using an improved chemiluminescence detection system (NEN Life Sci- ence Products). Protein concentrations were determined by the Bradford method (DC Protein Assay, Bio-Rad). Transient transfection and reporter gene activity assay Transient transfection was carried out as described previously [25]. Briefly, RAW264.7 cells were plated and cultured in 24-well plates overnight before transfection. To determine the role of ERK in regulating the G-CSF promoter activity, 0.5 μg of the pG-CSF(-283/+35)-Luc plas- mid and 0.1 μg of the phRLTK plasmid were mixed with 0.15–0.6 μg of the expression plasmid containing p50, Oct-2, CA-ERK, or C/EBPβ cDNA or mixtures of the plasmid encoding C/ EBPβ and that encoding p50, Oct-2, or CA-ERK, and the total amount of DNA was adjusted to 1.2 μg with pcDNA3.1. The DNA mixture was transfected into RAW264.7 cells using Super- Fect transfection reagent, then 24 h later, cell lysates were prepared using the lysis buffer in the kit and Photinus and Renilla luciferase activities were measured using the Dual-Luciferase reporter assay system as described previously [25]. The light intensity produced by Photinus luciferase (test plasmid) was normalized to that produced by Renilla luciferase (control plasmid). Quantitation of G-CSF in culture medium The concentrations of human (THP-1 cells) or mouse (RAW264.7 cells) G-CSF in the culture medium were measured by enzyme-linked immunosorbent assay using human or mouse G-CSF Quantikine ELISA kits (R&D Systems) according to the manufacturer's instructions. Knockdown of ERK1 and ERK2 by RNA interference pLKO.1-shRNA plasmids encoding shRNAs targeting firefly luciferase, human ERK1 (shMAPK3-B1/ERK1i-a: 50–CTATACCAAGTCCATCGACAT–30 or MAPK3-A2/ERK1i-b: 50–CAACATGAAGGCCCGAAACTA–30), or human ERK2 (shMAPK1-F1/ERK2i-a: 50– CAAAGTTCGAGTAGCTATCAA–30 or shMAPK1-G1/ERK2i-b: TATCCATTCAGC TAACGTTCT) were obtained from the National RNAi Core Facility of the Academia Sinica, Taiwan. These plasmids were transfected, together with pMD.G and pCMV delta8.91, into a HEK293T packaging cell line using the calcium phosphate method and virus supernatants were collected from the medium 60 h after transfection [23]. For knockdown experiments, THP-1 cells were transduced for 24 h with the collected virus supernatants plus polybrene (8 μg/ml) and infected cells were selected with puromycin (10 μg/ml) for 10 days. 4 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed according to the manufacturer’s instructions (Upstate Biotech- nology Inc.) as described previously [6]. Briefly, after treatment, the cells were fixed with 1% formaldehyde for 10 min at 37°C to cross-link DNA and protein, collected by scraping, and sonicated to fragment the chromatin. Immunoprecipitation was performed using control rab- bit IgG or rabbit antibodies against ERK, C/EBPβ, p50, p65, or histone 3 (Santa Cruz), the cross-links were reversed at 65°C for 4 h and digested with proteinase K (Sigma-Aldrich) for 1 h at 45°C to remove proteins, then the immunoprecipitated DNA was recovered by phenol/ chloroform extraction and ethanol precipitation before being used as template for PCR with the following primers: G-CSF promoter (-248 to -70), forward (5'–TGGCTGGaagAGAGGAA GAG–3') and reverse (5'–CTGGGGCAACTCAGGCTTA–3') or TNF-α promoter (-270 to -4), forward (5'–CTGATTGGCCCCAGATTG–3') and reverse (5'–CTTCTGCTGGCTGGCTGT– 3') (GenBank: Y00467.1). Ten percent of the chromatin DNA used for immunoprecipitation was also subjected to PCR analysis (indicated as “input”). The number of PCR cycles was 30 or 31 for all ChIP experiments and 24 for the input samples. DNase I accessibility assay After 1% formaldehyde treatment, the cells were gently lysed with 500 μl of ice-cold Nonidet P-40 lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 0.15 mM spermine, 0.5 mM spermidine), and the lysates centrifuged at 500 xg for 5 min at 4°C to pellet the nuclei, which were washed with 200 μl of Nonidet P-40 lysis buffer and centrifuged again at 500 xg for 5 min at 4°C. After gentle rinsing with 200 μl of ice-cold buffer A (100 mM NaCl, 50 mM Tris-HCl, pH 8.0, 3 mM MgCl2, 0.15 mM spermine, and 0.5 mM spermidine), the pelleted nuclei were suspended in buffer A containing 1 μM CaCl2 for DNase I (Promega) treatment, which was performed at 37°C for 5 min for THP-1 cells or for 2 min for Raw264.7 cells, and stopped with ice-cold 10 mM EDTA, pH 8.0. After reversal of the formaldehyde cross-linking at 65°C for 4 h, proteinase K and RNase A were added to the sample, which were then incubated at 37°C overnight, then DNA was extracted using the standard phenol-chloro- form technique. Care was taken to use cut-off tips and very gentle pipetting to reduce non-spe- cific DNA sheering. After precipitation, the DNA was dissolved in H2O. Real-time PCR was used to identify the DNase-sensitive sites using the primers (final con- centration 400 nM) listed in S1 Table. DNase I-treated and non-digested DNA were tested in triplicate. All PCR reactions were performed with SYBR Green PCR Master Mix in an ABI PRISM 7300 Sequence Detection System (Applied Biosystems 7300 System Sequence Detec- tion System software version 1.3.) ΔCt values were determined by subtracting the Ct value of the control reaction. The conditions for the PCR were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 60°C for 1 min. At the end of the PCR cycles, amplifica- tion specificity was confirmed by dissociation curve analysis, and the products were separated on a 3% agarose gel and stained with ethidium bromide for visual confirmation of the PCR product. Inhibition of ERK activation prevents LPS-induced G-CSF expression in macrophages An initial experiment was designed to confirm the involvement of ERK activation in LPS-stim- ulated G-CSF production in macrophages. Phosphorylation of ERK1/2 was detected in mouse Raw264.7 macrophages after 15 to 60 min of treatment with LPS (100 ng/ml) and was blocked by 30 min pretreatment with U0126 (10 μM) (Fig 1A). To examine whether U0126 inhibited LPS-induced G-CSF production, Raw264.7 cells were incubated with or without U0126, then with LPS for 0 to 24 h. Fig 1B shows that LPS-induced G-CSF secretion in the culture medium was time-dependent and was abolished by U0126 pretreatment. In addition, the inhibitory effect of U0126 on LPS-induced G-CSF production was dose-dependent (Fig 1C). Since LPS is known to increase G-CSF mRNA expression in mouse bone marrow-derived macrophage (BMDMs), we then examined whether the ERK inhibitors U0126 or PD98059 could block this effect in primary cells. The results in S2A and S2B Fig show that G-CSF mRNA levels were sig- nificantly increased after LPS treatment for 6 h and this effect was blocked by pretreatment cells with 0.01 or 0.1 μM of U0126 or 1 or 10 μM of PD98059. Fig 1. U0126 inhibits ERK1/2 phosphorylation and G-CSF expression in LPS-stimulated RAW264.7 macrophages. (A) Cells were left untreated (lane 1) or were incubated either with LPS (100 ng/ml) for 15 to 60 min (lanes 2–4) or with DMSO or U0126 (10 μM) for 30 min, followed by addition of same concentration of LPS and incubation for 0 to 60 min (lanes 5–8), then phosphorylated ERK1/2 and total ERK1/2 were analyzed by Western blotting. The results shown are representative of those obtained in three separate experiments. (B) Cells were incubated with DMSO or U0126 (10 μM) for 30 minutes, then with LPS (100 ng/ ml) for the indicated time, then G-CSF levels in the medium were measured by ELISA. (C) Cells were incubated with DMSO or 0.01, 0.1, 1, or 10 μM of U0126 for 30 minutes, then with LPS (100 ng/ml) for 6 h and G-CSF protein levels in the medium were measured by ELISA. The results in B and C are the mean ± SD for three independent experiments (p<0.01). doi:10.1371/journal.pone.0129685.g001 Fig 1. U0126 inhibits ERK1/2 phosphorylation and G-CSF expression in LPS-stimulated RAW264.7 macrophages. Statistical analyses Results are shown as the mean + SD. Differences between mean values were evaluated using Student’s t test and were considered significant at p <0.05. 5 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages Inhibition of ERK activation prevents LPS-induced G-CSF expression in macrophages (A) Cells were left untreated (lane 1) or were incubated either with LPS (100 ng/ml) for 15 to 60 min (lanes 2–4) or with DMSO or U0126 (10 μM) for 30 min, followed by addition of same concentration of LPS and incubation for 0 to 60 min (lanes 5–8), then phosphorylated ERK1/2 and total ERK1/2 were analyzed by Western blotting. The results shown are representative of those obtained in three separate experiments. (B) Cells were incubated with DMSO or U0126 (10 μM) for 30 minutes, then with LPS (100 ng/ ml) for the indicated time, then G-CSF levels in the medium were measured by ELISA. (C) Cells were incubated with DMSO or 0.01, 0.1, 1, or 10 μM of U0126 for 30 minutes, then with LPS (100 ng/ml) for 6 h and G-CSF protein levels in the medium were measured by ELISA. The results in B and C are the mean ± SD for three independent experiments (p<0.01). doi:10.1371/journal.pone.0129685.g001 6 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages ERK2 is critical for LPS-induced G-CSF expression ERK1 and ERK2 are isoforms of the ‘classical’ MAPKs and both are activated by MAP/ERK kinase 1 (MEK1) and MEK2, members of the MAPKK family. To examine whether U0126 inhibited LPS-induced phosphorylation of ERK1/2 in THP-1 macrophages, cells were induced to differentiate into macrophages by incubation with 160 nM PMA, and then were incubated with LPS for 0.5, 1 or 2 h. Phosphorylation of ERK1/2 was detected in THP-1 macrophages after treatment with LPS (100 ng/ml) and was blocked by 30 min pretreatment with U0126 (10 μM) (S3 Fig). To determine whether ERK1 and/or ERK2 involved in LPS-induced G-CSF expression, ERK expression in THP-1 human acute monocytic leukemia cells was knocked down using shRNA clones ERK1a and ERK1b carrying two different ERK1 sequences or shRNAs ERK2a and ERK2b carrying two different ERK2 sequences. The luciferase shRNA was used as a negative control. Knockdown efficiency and specificity were confirmed by Western blotting analysis (Fig 2A and S4 Fig). After shRNA-lentiviral transduction and puromycin selection, the cells were induced to differentiate into macrophages by incubation with 160 nM PMA, and then were incubated with LPS or PBS for 4 h. As shown in Fig 2B and 2C, a Fig 2. ERK2 knockdown in THP-1 macrophages blocks LPS-induced G-CSF expression. THP-1 cells were infected with lentivirus carrying specific shRNA for ERK1 or ERK2 and induced to differentiate by incubation with PMA (160 nM) for 3 days; lentivirus carrying luciferase shRNA (Luc) was used as a control, then the following tests were performed. (A) Levels of ERK1/2 and β-actin in the cells were determined by Western blotting; the data shown are typical of the results of three experiments. (B) Cells were treated with LPS (100 ng/ml) for 8 h, then G-CSF levels in the medium were determined by ELISA; untreated Luc cells were used as the control. (C and D) Levels of G-CSF mRNA (C) and TNF-α mRNA (D) in cells were determined by RT-qPCR, normalized to the levels of GAPDH mRNA, and expressed relative to levels in the Luc control (relative value = 1). In (B-D), the results are the mean ± SD for five independent experiments. p<0.05 compared to the corresponding cells not treated with LPS. doi:10.1371/journal.pone.0129685.g002 ges blocks LPS-induced G-CSF expression. THP-1 cel Fig 2. ERK2 knockdown in THP-1 macrophages blocks LPS-induced G-CSF expression. THP-1 cells Fig 2. ERK2 knockdown in THP-1 macrophages blocks LPS-induced G-CSF expression. Cooperative regulation of G-CSF promoter activation by ERK2 and C/ EBPβ The results above suggested that ERK2 might involve in LPS-activated G-CSF expression at the transcriptional level. To further clarify whether ERK2 activation affected the G-CSF promoter activity, reporter assays were performed. Raw264.7 cells were transfected with a reporter plasmid containing the luciferase gene driven by the G-CSF promoter (-283/+35) (pG-CSF(-283/+35)- Luc), then were incubated with or without U0126, followed by LPS treatment for 6 h. As shown in S5A Fig, U0126 pretreatment reduced LPS-activated G-CSF promoter activity by 60%, sup- porting the idea that LPS-activated G-CSF promoter activity requires activation of the MEK/ERK pathway. We therefore investigated the role of ERK2 in G-CSF promoter activation and possible regulatory factors involved. Raw264.7 cells were co-transfected with the pG-CSF(-283/+35)-Luc reporter plasmid and (i) a plasmid encoding constitutively active ERK2 (CA-ERK2) or p50, Oct- 2, or C/EBPβ, all of which are involved in LPS-regulated G-CSF expression, or (ii) with pairs of plasmids consisting of the C/EBPβ-encoding plasmid and the CA-ERK2, p50, or Oct-2 plasmid. As shown in Fig 3A, co-transfection with pG-CSF(-283/+35)-Luc and the plasmid encoding p50, Oct-2, C/EBPβ, or CA-ERK2 resulted, respectively, in a 0.3-, 0.7-, 6.2-, or 2.7-fold increase in luciferase activity compared to transfection with pG-CSF(-283/+35)-Luc alone. In addition, co- transfection with the pG-CSF(-283/+35)-Luc reporter plasmid, the CA-ERK2-encoding plasmid, and the plasmid encoding C/EBPβ resulted in the highest luciferase activity, with a 28-fold increase compared to the control with the Luc reporter plasmid alone. In contrast, co-transfec- tion with the pG-CSF(-283/+35)-Luc reporter, CA-ERK2-encoding plasmid, and either the p50 or Oct-2 expression plasmid resulted in no significant increase in luciferase activity compared to that detected in the cells co-transfected with CA-ERK2-encoding plasmid alone (Fig 3A). These findings suggest the importance of the interaction between ERK2 and C/EBPβ in regulating LPS- stimulated G-CSF promoter activity. Since ERK1/2 is known to phosphorylate C/EBPβ and thereby regulate its transcriptional factor activity [26], we next examined whether LPS treatment led to ERK1/2-mediated phosphorylation of C/EBPβ. As shown in Fig 3B, LPS treatment of RAW264.7 cells for 4 h increased both the protein and phosphorylation levels of nuclear C/ EBPβ, and phosphorylation level was partially inhibited, but not the protein level, by U0126 pre- treatment. Serine 64 in the transactivation domain and threonine 188 in the regulatory domain of C/EBPβ, both have been proposed as potential target sites for phosphorylation by ERK [22, 27, 28]. ERK2 is critical for LPS-induced G-CSF expression THP-1 cells were infected with lentivirus carrying specific shRNA for ERK1 or ERK2 and induced to differentiate by incubation with PMA (160 nM) for 3 days; lentivirus carrying luciferase shRNA (Luc) was used as a control, then the following tests were performed. (A) Levels of ERK1/2 and β-actin in the cells were determined by Western blotting; the data shown are typical of the results of three experiments. (B) Cells were treated with LPS (100 ng/ml) for 8 h, then G-CSF levels in the medium were determined by ELISA; untreated Luc cells were used as the control. (C and D) Levels of G-CSF mRNA (C) and TNF-α mRNA (D) in cells were determined by RT-qPCR, normalized to the levels of GAPDH mRNA, and expressed relative to levels in the Luc control (relative value = 1). In (B-D), the results are the mean ± SD for five independent experiments. p<0.05 compared to the corresponding cells not treated with LPS. PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 7 / 17 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages significant increase at LPS-induced G-CSF protein and mRNA levels was detected in control luciferase shRNA knockdown and ERK1 knockdown cells, but not in ERK2 knockdown cells. Whereas, neither ERK1 nor ERK2 knockdown had a significant effect on LPS-induced TNF-α mRNA expression (Fig 2D), though both basal and LPS-induced TNF-α mRNA levels were slightly lower in the ERK2 knockdown cells. These results show that ERK2, but not ERK1, is essential for LPS-induced G-CSF expression, but is not a common regulator for the expression of LPS-activated genes. PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 Cooperative regulation of G-CSF promoter activation by ERK2 and C/ EBPβ To clarify the phosphorylation sites on C/EBPβ by LPS-activated ERK, these two amino acids were separately mutated to alanine by point mutation to create the C/EBPβ-S64A and C/ EBPβ-T188A mutants. In RAW264.7 cells co-transfected with the pG-CSF(-283/+35)-Luc reporter, the CA-ERK2-encoding plasmid, and either the wild-type C/EBPβ or one of the mutant C/EBPβ constructs, a 45% decrease in luciferase activity was detected in cells with C/EBPβ T188A compared to that with wild type C/EBPβ, whereas the C/EBPβ-S64A mutant had no sig- nificant effect (Fig 3C). We then mutated the consensus C/EBPβ binding site in the G-CSF pro- moter to examine its role in LPS-ERK-induced G-CSF expression. Co-transfection of Raw264.7 cells with increasing amounts of the CA-ERK2-expressing plasmid (0–0.6 μg) and the pG-CSF 8 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages Fig 3. Threonine188 of C/EBPβ is important for the interaction of ERK2 with C/EBPβ that activates G-CSF promoter. (A) RAW264.7 cells were co- transfected with a mixture of two reporter plasmids (pG-CSF(-283/+35)-Luc and phRLTK) and the CA-ERK2, p50, Oct-2, or C/EBPβ plasmid alone or the C/ EBPβ plasmid plus the CA-ERK2, p50, or Oct-2 plasmid, then luciferase activity was measured at 24 h after transfection using the Dual-luciferase reporter assay system. (B) RAW264.7 cells were pretreated for 30 min with DMSO (D) or U0126 (U) (10 μM), then were incubated with LPS (100 ng/ml) (L) for another 4 h, after which a nuclear extract (N.E.) and cytosolic extract (C.E.) were isolated and levels of total and phosphorylated C/EBPβ, phosphorylated ERK1/2, β-actin (cytosol internal control), and laminin A/C (nuclear internal control) analyzed by Western blotting. The data shown are typical of those obtained in three experiments. (C) Cells were cotransfected with reporter plasmids (0.5 μg of pG-CSF (-283/+35)-Luc mixed with 0.1 μg of phRLTK), 0.15 μg of CA-ERK expression plasmid, and 0.15 μg of the WT, T188A, or S64A C/EBP expression plasmid or 0.15 μg of pcDNA3.1 (vector control), then the reporter luciferase activities were analyzed 24 h after transfection. (D) Cells were co-transfected with pG-CSF(-283/+35)-Luc carrying the wild type or mutant C/EBPβ binding site, 0, 0.15, 0.3, 0.6 μg of CA-ERK2 expression plasmid, and the internal control phRLTK, then luciferase activities were measured 24 h after transfection. All luciferase activities are shown relative to the vector control (relative value = 1). U0126 inhibits LPS-induced binding of nuclear NF-κB and C/EBPβ to the G-CSF promoter region and decreases its accessibility to DNase To determine whether U0126 inhibited LPS-induced G-CSF expression by affecting transcrip- tion factors known to regulate G-CSF expression, we analyzed levels of the transcriptional reg- ulators including p50, p65, and C/EBPβ in the nucleus of RAW264.7 cells. As shown in Fig 4A, nuclear levels of p50, p65, and C/EBPβ were increased after 6 h of LPS treatment and this effect was unaffected by pretreatment with U0126. These results show that nuclear levels of these transcriptional factors are irrelevant for the ERK-regulated G-CSF transcription. We then used ChIP assays to investigate the effect of U0126 on the bindings of p50, p65, and C/EBPβ to the promoters of G-CSF and TNF-α in cells treated with LPS. Fig 4B shows that binding of p50, p65, or C/EBPβ to the G-CSF promoter and the TNF-α promoter was induced by LPS and that U0126 pretreatment inhibited binding of all three to the G-CSF promoter, but not the TNF-α promoter (Fig 4B). To clarify whether U0126 pretreatment affected LPS-stimulated NF-κB transcriptional activity, Raw264.7 cells were transfected with an NF-κB-driven luciferase reporter (pNF-κB-Neo-Luc), then, at 24 h after transfection, were incubated with LPS with or without U0126 pretreatment. As expected, LPS treatment for 6 h significantly upregulated NF- κB-driven luciferase activity and this effect was not affected by U0126 pretreatment (S5B Fig). These results indicate that LPS-triggered NF-κB activation, including nuclear levels of NF-κB and its transactivation activity, was not affected by U0126. Activation of the MEK-ERK signaling pathway induces histone phosphorylation, causing nucleosome remodeling at the promoters of immediate early genes [29]. DNase accessibility analysis was therefore employed to determine whether LPS activation of ERK resulted in mod- ulation of chromatin remodeling at the promoter region of the G-CSF gene. Nuclei of RAW264.7 or THP-1 macrophages pretreated with DMSO or U0126, then treated with LPS for 4 h were subjected to DNase I digestion and the amount of undigested DNA in the G-CSF and TNF-α promoter regions containing C/EBPβ binding sites was determined by quantitative real-time PCR. In Raw264.7 cells, the amount of undigested DNA in region -167/+12 of the G-CSF promoter (Fig 5A) and region -270/-4 of the TNF-α promoter (Fig 5B) was decreased after LPS treatment, while U0126 pretreatment restored the amount of undigested G-CSF Fig 4. U0126 reduces LPS-induced nuclear NF-κB and C/EBPβ binding to the G-CSF promoter in Raw264.7 macrophages. Cooperative regulation of G-CSF promoter activation by ERK2 and C/ EBPβ PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 9 / 17 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages Cooperative regulation of G-CSF promoter activation by ERK2 and C/ EBPβ In A, C, and D, values are the mean ± SD for three independent experiments. p<0.05 compared to wild-type cells. d i 10 1371/j l 0129685 003 Fig 3. Threonine188 of C/EBPβ is important for the interaction of ERK2 with C/EBPβ that activates G-CSF promoter. (A) RAW264.7 cells were co- transfected with a mixture of two reporter plasmids (pG-CSF(-283/+35)-Luc and phRLTK) and the CA-ERK2, p50, Oct-2, or C/EBPβ plasmid alone or the C/ EBPβ plasmid plus the CA-ERK2, p50, or Oct-2 plasmid, then luciferase activity was measured at 24 h after transfection using the Dual-luciferase reporter assay system. (B) RAW264.7 cells were pretreated for 30 min with DMSO (D) or U0126 (U) (10 μM), then were incubated with LPS (100 ng/ml) (L) for another 4 h, after which a nuclear extract (N.E.) and cytosolic extract (C.E.) were isolated and levels of total and phosphorylated C/EBPβ, phosphorylated ERK1/2, β-actin (cytosol internal control), and laminin A/C (nuclear internal control) analyzed by Western blotting. The data shown are typical of those obtained in three experiments. (C) Cells were cotransfected with reporter plasmids (0.5 μg of pG-CSF (-283/+35)-Luc mixed with 0.1 μg of phRLTK), 0.15 μg of CA-ERK expression plasmid, and 0.15 μg of the WT, T188A, or S64A C/EBP expression plasmid or 0.15 μg of pcDNA3.1 (vector control), then the reporter luciferase activities were analyzed 24 h after transfection. (D) Cells were co-transfected with pG-CSF(-283/+35)-Luc carrying the wild type or mutant C/EBPβ binding site, 0, 0.15, 0.3, 0.6 μg of CA-ERK2 expression plasmid, and the internal control phRLTK, then luciferase activities were measured 24 h after transfection. All luciferase activities are shown relative to the vector control (relative value = 1). In A, C, and D, values are the mean ± SD for three independent experiments. p<0.05 compared to wild-type cells. doi:10.1371/journal.pone.0129685.g003 (-283/+35)-Luc reporter carrying either the consensus C/EBPβ binding site (WT: ATTTCA- CAAA) or the mutant C/EBPβ binding site (GCGTCACGCA) resulted in a 2.8- to 16-fold increase in luciferase activity using reporter carrying the WT binding site, but only a 1.6- to 4.3-fold increase with the reporter construct carrying the mutant C/EBPβ binding site (Fig 3D). Taken together, these results demonstrate that the ERK2-mediated increase in G-CSF promoter activity, at least in part, involves phosphorylation of C/EBPβ at Thr188. U0126 inhibits LPS-induced binding of nuclear NF-κB and C/EBPβ to the G-CSF promoter region and decreases its accessibility to DNase Nuclei were then isolated and subjected to DNase I digestion for 2 min, then genomic DNA was isolated and quantitative real-time PCR was performed to measure the amount of undigested DNA in region -167/+12 of the G-CSF promoter (A) and in region -270/-4 of the TNF-α promoter (B), which is expressed as a percentage of that in the control cells. The results are the mean ± SD for three independent experiments. p<0.05 compared to the control. doi:10 1371/journal pone 0129685 g005 Fig 5. U0126 decreases the LPS-induced increased accessibility of the G-CSF promoter to DNase I. Fig 5. U0126 decreases the LPS-induced increased accessibility of the G-CSF promoter to DNase I. Raw264.7 macrophages were pretreated with DMSO or U0126 (10 μM), then incubated with LPS (100 ng/ml) or PBS for 4 h. Nuclei were then isolated and subjected to DNase I digestion for 2 min, then genomic DNA was isolated and quantitative real-time PCR was performed to measure the amount of undigested DNA in region -167/+12 of the G-CSF promoter (A) and in region -270/-4 of the TNF-α promoter (B), which is expressed as a percentage of that in the control cells. The results are the mean ± SD for three independent experiments. p<0.05 compared to the control. g y p Raw264.7 macrophages were pretreated with DMSO or U0126 (10 μM), then incubated with LPS (100 ng/ml) or PBS for 4 h. Nuclei were then isolated and subjected to DNase I digestion for 2 min, then genomic DNA was isolated and quantitative real-time PCR was performed to measure the amount of undigested DNA in region -167/+12 of the G-CSF promoter (A) and in region -270/-4 of the TNF-α promoter (B), which is expressed as a percentage of that in the control cells. The results are the mean ± SD for three independent experiments. p<0.05 compared to the control. doi:10.1371/journal.pone.0129685.g005 promoter DNA, but not TNF-α promoter DNA, to the control level. Similar results were detected in human THP-1 macrophages that LPS treatment decreased the amounts of undi- gested DNA within the promoter regions of G-CSF (Fig 6A) and TNF-α (Fig 6B) that contain a C/EBPβ binding site. Again, pretreatment with U0126 restored the amount of undigested DNA within the G-CSF promoter, but not the TNF-α promoter (Fig 6A and 6B). Furthermore, Fig 6. Knockdown of ERK2, but not ERK1, decreases LPS-induced DNase I accessibility of the G-CSF promoter. U0126 inhibits LPS-induced binding of nuclear NF-κB and C/EBPβ to the G-CSF promoter region and decreases its accessibility to DNase Raw264.7 cells were incubated with DMSO or U0126 (10 μM) for 30 min, then with LPS (100 ng/ml) or PBS for 6 h, then the following tests were carried out. (A) Nuclear levels of p50, p65, C/EBPβ, and lamin B were measured by Western blotting assay. (B) Bindings of p50, p65, or C/EBPβ to the G-CSF promoter were analyzed by ChIP assay via precipitation with antibodies against the test protein or histone H3, used as the control, removal of the antibodies with proteinase K digestion for 12 h at 45ºC, and PCR amplification of a 179-bp G-CSF promoter fragment (-248 to -70 bp) or a 274 bp TNF-α promoter fragment (-270 to -4 bp). Ten per cent of the chromatin DNA used for immunoprecipitation was subjected to PCR and is indicated as ‘input’ (bottom row). In A and B, the data are representative of the results from three independent experiments. doi:10 1371/journal pone 0129685 g004 Fig 4. U0126 reduces LPS-induced nuclear NF-κB and C/EBPβ binding to the G-CSF promoter in Raw264.7 macrophages. Raw264.7 cells were incubated with DMSO or U0126 (10 μM) for 30 min, then with LPS (100 ng/ml) or PBS for 6 h, then the following tests were carried out. (A) Nuclear levels of p50, p65, C/EBPβ, and lamin B were measured by Western blotting assay. (B) Bindings of p50, p65, or C/EBPβ to the G-CSF promoter were analyzed by ChIP assay via precipitation with antibodies against the test protein or histone H3, used as the control, removal of the antibodies with proteinase K digestion for 12 h at 45ºC, and PCR amplification of a 179-bp G-CSF promoter fragment (-248 to -70 bp) or a 274 bp TNF-α promoter fragment (-270 to -4 bp). Ten per cent of the chromatin DNA used for immunoprecipitation was subjected to PCR and is indicated as ‘input’ (bottom row). In A and B, the data are representative of the results from three independent experiments. doi:10.1371/journal.pone.0129685.g004 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 10 / 17 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages Fig 5. U0126 decreases the LPS-induced increased accessibility of the G-CSF promoter to DNase I. Raw264.7 macrophages were pretreated with DMSO or U0126 (10 μM), then incubated with LPS (100 ng/ml) or PBS for 4 h. Discussion Although recombinant G-CSF is widely used clinically, regulation of endogenous G-CSF expression has not been well characterized. In this study, we used a highly selective inhibitor of MAP/ERK kinase, U0126, and shRNA knockdown of ERK1 or ERK2 to demonstrate that LPS- induced activation of ERK2, but not ERK1, was required for LPS-induced G-CSF expression in macrophages. Moreover, our results showed that LPS-activated ERK2 interacts with C/EBPβ and increases the accessibility of the G-CSF promoter to DNase I and transcriptional factor binding to the G-CSF promoter, leading to up-regulation of G-CSF expression. ERK signaling is known associated with various cellular processes, including proliferation, differentiation, and survival. The two classical ERKs, ERK1 and ERK2, co-express in most tis- sues, but vary in relative abundance. They involve in the transcriptional regulation of multiple cellular processes by phosphorylating many substrates, including Elk1, c-Myc, and ribosomal S6 kinase, and by activating c-fos. An animal study showed that ERK2 knockout is lethal and cannot be compensated for by ERK1, while ERK1 knockout has only a mild phenotypic effect [30]. It is therefore proposed that ERK1 and ERK2 have different biological functions [31]. It is also known that ERK2 expression is more critical than ERK1 expression for survival [31]. In this study, we evidenced that ERK2, rather than ERK1, involved in LPS-induced G-CSF expres- sion in macrophages, further illustrating the different functions of these two kinases. ERK2 has been identified as a transcription regulator but the consequence of ERK2 signal- ing might be determined by the interaction of ERK2 and other proteins in either the nucleus or cytoplasm, such as nuclear factors, scaffold proteins, and anchoring proteins. For example, interaction between activated ERK2 and the IκB-NF-κB complex in the cytoplasm has been implicated in protecting myeloid leukemia cells against apoptogenic stimuli [32]. Although NF-κB is known to be a major transcriptional regulator of the expressions of inflammatory genes, NF-κB alone is not sufficient to drive transcription of G-CSF [4]. We have previously shown that, following LPS stimulation, Oct-2 binds to the promoters of the iNOS and G-CSF genes and this effect is reduced by inhibitors of PI3K, AKT, or mTOR, thus demonstrating the essential role of Oct-2 in regulating the transcription of these genes [6]. All these results suggest that both Oct-2 and NF-κB are essential for LPS-induced expression of G-CSF. U0126 inhibits LPS-induced binding of nuclear NF-κB and C/EBPβ to the G-CSF promoter region and decreases its accessibility to DNase THP-1 cells in which either ERK1 or ERK2 was knocked down using shRNA were induced to differentiate into macrophages. After incubation with DMSO or U0126 (10 μM), the cells were incubated with LPS (100 ng/ml) or PBS for 4 h, then nuclei were purified and subjected to DNase I digestion for 5 min. Genomic DNA was then isolated and the amount of undigested DNA within region -624/-450 of the G-CSF promoter (A) or within region -228/-71 of the TNF- α promoter (B) was determined by quantitative real-time PCR and expressed as a percentage of that in cells not treated with LPS and U0126. The results are the mean ± SD for three independent experiments. p<0.05 compared to the corresponding control. doi:10.1371/journal.pone.0129685.g006 Fig 6. Knockdown of ERK2, but not ERK1, decreases LPS-induced DNase I accessibility of the G-CSF promoter. THP-1 cells in which either ERK1 or ERK2 was knocked down using shRNA were induced to differentiate into macrophages. After incubation with DMSO or U0126 (10 μM), the cells were incubated with LPS (100 ng/ml) or PBS for 4 h, then nuclei were purified and subjected to DNase I digestion for 5 min. Genomic DNA was then isolated and the amount of undigested DNA within region -624/-450 of the G-CSF promoter (A) or within region -228/-71 of the TNF- α promoter (B) was determined by quantitative real-time PCR and expressed as a percentage of that in cells not treated with LPS and U0126. The results are the mean ± SD for three independent experiments. *p<0.05 compared to the corresponding control. doi:10.1371/journal.pone.0129685.g006 11 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages knockdown of ERK2 in THP-1 cells resulted in an increased amount of undigested G-CSF pro- moter DNA (Fig 6A), but had no significant effect on the TNF-α promoter (Fig 6B) in LPS- treated cells compared to LPS-treated knockdown control cells. Whereas, ERK1 knockdown had no significant effect on the amount of undigested G-CSF and TNF-α promoter DNA in LPS-treated cells compared to LPS-treated control knockdown cells. These results reveal that LPS treatment increases the accessibility of both the G-CSF and TNF-α promoters to DNase I and that U0126 pretreatment or ERK2 depletion abrogates LPS effect on DNase accessibility of the G-CSF promoter, but not the TNF-α promoter region. PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 Discussion However, as shown in Fig 4 and S6 Fig in this study, U0126 did not affect LPS-induced nuclear levels of NF- κB (p50 and p65) and Oct-2, but reduced the LPS-induced binding of NF-κB p50 and p65 to the G-CSF promoter, but not to the TNF-α promoter. In addition, LPS-induced accessibility of the G-CSF promoter to DNase I was prevented by U0126 or ERK2 knockdown, but not by ERK1 knockdown, and, again, no such effect was seen with the TNF-α promoter region (Figs 5 and 6). These results further suggest that ERK2 activation may specifically alter chromatin con- formation in the G-CSF promoter region, thus increasing its accessibility to DNase I digestion and facilitating the binding of transcription factors [33]. Carlson et al. [34] identified a poten- tial ERK2 substrate, ETV3, a transcriptional regulator that is extensively phosphorylated by activated ERK2 and loses its ability to bind to the GGAA-containing E box motif at the PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 12 / 17 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages promoters of many genes. ETV3 has been suggested to function by cooperating with SBNO2, a transcriptional corepressor, to regulate the anti-inflammatory effects of IL-10 [35]. In our study here, we provide evidences that ERK2 mediates LPS-upregulated G-CSF promoter activ- ity, at least in part, by interacting with C/EBPβ and increasing its ability to activate transcrip- tion. However, it remains unclear whether factors similar to ETV3 or other chromatin remodeling complexes involve in LPS-ERK2-regulated G-CSF expression in macrophages. NF-κB controls many genes involved in inflammation, such as G-CSF, IL-6, TNF-α and iNOS. Although we show that U0126 has no effect on LPS-increased NF-κB-dependent reporter gene expression and nuclear protein levels of p50 and p65 in RAW 264.7 macrophage. LPS-induced NF-κB activation has been reported to be inhibited by PD98059 in murine J774 macrophages [36]. The discrepancy could be due to that different cells or different methods were used. In addition, studies have shown that inhibition of ERK1/2 has no effect on the acti- vation of a stably transfected NF-κB-dependent reporter gene expression [37], or expression of NF-κB down-stream genes, such as IL-6, TNF-α and iNOS [38–40]. Furthermore, evidence showing that phosphorylation level of p65 at S468 and S536 is not inhibited by PD98059 [41, 42]. Discussion These results suggest that ERK2 may not a critical regulator of NF-κB activation, and ERK2 regulates G-CSF expression is not through inhibition of NF-κB activation. G-CSF is essential for the protective inflammatory response and for maintaining the balance between anti- and pro-inflammatory reactions in the inflammatory condition. Higher levels of G-CSF may cause an excessive inflammatory response and have been associated with morbid- ity and mortality of acute lung injury or with chronic inflammatory diseases, such as rheuma- toid arthritis [9, 10]. Knockout of G-CSF protects mice against collagen-induced arthritis, mostly due to prevention of infiltration by activated neutrophils [43]. Thus, inhibition of G-CSF activity is now considered as a new therapeutic strategy for rheumatoid arthritis and other inflammatory diseases. In this report, we provide evidences for a pivotal role of ERK2 in LPS-induced G-CSF expression in macrophages. We show that ERK2 involves in LPS-induced G-CSF expression at two distinct steps; activation of ERK2 triggers local chromatin remodeling, which may facilitate binding of transcription factors to the G-CSF promoter, and activation of ERK2 cooperates with C/EBPβ to activate G-CSF promoter activity. These effects of ERK2 on G-CSF expression are not shared by ERK1. The precise mechanism by which ERK2 causes chromatin remodeling is not known yet and further studies are needed. Supporting Information At 24 h post-transfection, the cells were pretreated with DMSO or 10 μM U0126 for 30 min, followed by addition of LPS (100 ng/ ml) for 6 h, then luciferase activities were determined using the Dual-Luciferase reporter assay system, and firefly luciferase activity was normalized to renilla luciferase activity, then the results were expressed relative to those for untreated control cells (C). The values are the mean ± SD for three independent experiments. p < 0.01 compared to the LPS-treated cells in (A) and p < 0.05 compared to the LPS-treated cells in (B). (TIF) S5 Fig. U0126 inhibits LPS-induced G-CSF promoter but not NF-κB-driven promoter activity. RAW264.7 cells were co-transfected with 1 μg of pG-CSF(−283/+35)-Luc (A) or the pTransNF-κB-Neo plasmid (B) and 0.05 μg of phRLTK. At 24 h post-transfection, the cells were pretreated with DMSO or 10 μM U0126 for 30 min, followed by addition of LPS (100 ng/ ml) for 6 h, then luciferase activities were determined using the Dual-Luciferase reporter assay system, and firefly luciferase activity was normalized to renilla luciferase activity, then the results were expressed relative to those for untreated control cells (C). The values are the mean ± SD for three independent experiments. p < 0.01 compared to the LPS-treated cells in (A) and p < 0.05 compared to the LPS-treated cells in (B). (TIF) S6 Fig. Effect of U0126 on the LPS-induced increase in nuclear Oct-2 protein. Raw264.7 cells were left untreated (lane 1) or were treated with LPS (100 ng/ml) for 6 h (lane 2) or were pretreated with U0126 (10 μM) for 30 min, then treated with LPS (100 ng/ml) for 6 h (lane 3), then nuclei were isolated and nuclear levels of Oct-2 and lamin B were determined by Western blotting. (TIF) Acknowledgments We thank the National RNAi Core Facility at the Academia Sinica, Taiwan for providing the shRNA viruses and clones. Author Contributions Conceived and designed the experiments: SFC SCL. Performed the experiments: SSL HCY YYC JIG. Analyzed the data: SSL HCY YYC JIG. Contributed reagents/materials/analysis tools: SFC SCL. Wrote the paper: SFC SCL. Supporting Information S1 Fig. Effects of inhibitors of the PI3K, MAPK, or PKC pathways on LPS-induced G-CSF protein in culture medium of RAW264.7 macrophages. RAW264.7 cells were pretreated for 30 min with DMSO, a PI3K inhibitor (50 μM LY294002), a MEK inhibitor (10 μM U0126), a JNK inhibitor (0.5 μM L-JNKi 1 trifluoroacetate), a p38 inhibitor (20 μM SB203580), or a PKC inhibitor (1 μM RO318220), then 100 ng/ml of LPS was added for 6 h, then levels of G-CSF protein in the culture medium were measured by ELISA. The values are the mean ± SD for three separate experiments. p < 0.01 compared to the DMSO-treated cells. (TIF) S2 Fig. U0126 or PD98059 inhibits LPS-induced increase of G-CSF mRNA in bone mar- row-derived macrophages. Mouse bone marrow-derived macrophages (BMDMs), cultured as described previously [Arch Biochem Biophys. 2011;508: 110–119.], were left untreated (lane 1) or were pretreated with (A) DMSO or 0.01 or 0.1 μM U0126 (lanes 2–5) or (B) DMSO or 1 or 10 μM PD98059 (lanes 2–5), then were incubated with LPS (100 ng/ml) or PBS for 6 h. Total RNA was then isolated and the levels of G-CSF and GAPDH (internal control) mRNA were PLOS ONE \| DOI:10.1371/journal.pone.0129685 June 26, 2015 13 / 17 ERK2 Mediates LPS-Stimulated G-CSF Expression in Macrophages determined by RT-PCR and analyzed by gel electrophoresis. The data shown are typical of the results obtained in two independent experiments. (TIF) S3 Fig. U0126 inhibits ERK1/2 phosphorylation in LPS-stimulated THP-1 macrophages. PMA differentiated THP-1 macrophages were left untreated (lane 1) or were incubated either with LPS (100 ng/ml) for 0.5 to 2 h (lanes 2–4) or pretreated with U0126 (10 μM) for 30 min, followed by addition of same concentration of LPS and incubation for 2 h (lane 5), then phos- phorylated ERK1/2 and total ERK1/2 were analyzed by Western blotting. (TIF) S4 Fig. Knockdown of ERK1 and ERK2 by shRNA. 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https://openalex.org/W4381952642	https://jurnalp4i.com/index.php/language/article/download/2307/2099	Malay	null	MENINGKATKAN KOMPETENSI MEMBACA DAN BERBICARA SISWA KELAS XI NAUTIKA KAPAL PENANGKAP IKAN (NKPI) DI SMK NEGERI I BIROMARU MELALUI STRATEGI PEMBELAJARAN KETERAMPILAN BERBAHASA TERPADU	Language	2,023	cc-by-sa	5,088	ABSTRAK ABSTRAK Penelitian Tindakan Kelas (PTK) ini dilaksanakan di SMK Negeri I Biromaru. Subyek penelitian adalah siswa kelas XI Program Keahlian Nautika Kapal Penangkap Ikan yang berjumlah 20 siswa. Peneliti ini menggunakan strategi pembelajaran keterampilan berbahasa terpadu untuk meningkatkan keterampilan membaca dan berbicara dalam bahasa Inggris yang digunakan dibidang kelautan dan perikanan. Dalam kegiatan pembelajaran peneliti mengintegrasikan materi-materi kejuruan (produktif) perikanan dan kelautan dalam bentuk bacaan sebagai bahan ajar dimana keterampilan membaca dan berbicara dilatih berdasarkan isi bahan bacaan. Peneliti menggunakan lembar observasi, catatan lapangan, angket, dan penilaian formatif untuk mengumpulkan data. Pada siklus 1 terdapat 8 siswa atau 40% yang memperoleh nilai 70 atau lebih pada pemahaman membaca (kriteria keberhasilan 70%), dan pada siklus 2 menjadi 14 siswa atau 70%. Pada siklus I terdapat 1 siswa atau 5% yang memperoleh nilai rata- rata keterampilan berbicara 36,00 (baik) dan 7 siswa atau 35% yang memperoleh nilai 30,00 (Cukup). Pada siklus 2 terdapat 1 atau 5% yang memperoleh nilai rata-rata 42,00 dalam keterampilan berbicara (sangat baik), 5 siswa atau 25% memperoleh nilai rata-rata 36,00 (Baik), dan 6 siswa atau 30% memperoleh nilai baik. Skor rata-rata 30,00 (Cukup). Berdasarkan data yang diperoleh strategi pembelajaran keterampilan berbahasa terpadu efektif untuk membantu siswa meningkatkan kompetensi mereka dalam keterampilan membaca dan menulis. Kata Kunci: Kompetensi Membaca dan Menulis, Strategi Pembelajaran, Keterampilan Kata Kunci: Kompetensi Membaca dan Menulis, Strategi Pembelajaran, Keterampilan Berbahasa Terpadu LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 MENINGKATKAN KOMPETENSI MEMBACA DAN BERBICARA SISWA KELAS XI NAUTIKA KAPAL PENANGKAP IKAN (NKPI) DI SMK NEGERI I BIROMARU MELALUI STRATEGI PEMBELAJARAN KETERAMPILAN BERBAHASA TERPADU LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 BADARUDIN SMK Negeri 2 Ampana Kota e-mail: qaumanmaqomi@g.mail.com PENDAHULUAN Mengacu pada penggunaan tujuan dari suatu rencana program pembelajaran, kompetensi menggambarkan hasil/kualitas pembelajaran. Kompetensi mengacu pada perilaku yang dapat diamati yang diperlukan untuk keberhasilan pemenuhan kegiatan-kegiatan dalam dunia kerja yang nyata. Kegiatan-kegiatan ini mungkin terkait dengan bidang kehidupan apa saja, meskipun biasanya dikaitkan dengan bidang pekerjaan dan kelangsungan hidup sosial di lingkungan baru (Richards, 2001:128). Menurut fungsinya, pembelajaran bahasa Inggris untuk siswa SMK di Indonesia memiliki dua jenis yaitu bahasa Inggris umum dan bahasa Inggris Untuk Keperluan Khusus (ESP). Bahasa Inggris umum adalah Bahasa Inggris sebagaimana yang kita dengarkan secara umum ditengah masyarakat dan dipelajari di SMP dan SMA. Bahasa Inggris Untuk Tujuan Tertentu (ESP) diajarkan pada siswa Sekolah Menengah Kejuruan (SMK) seperti Bahasa Inggris Maritime dan lain-lain. Dalam kaitannya Bahasa Inggris untuk tujuan tertentu, Sekolah kejuruan adalah lembaga pendidikan yang dirancang untuk menghasilkan keluarannya agar siap pakai dan terampil dalam program keahlian tertentu. Belakangan ini banyak SMK yang menyediakan berbagai program keahlian seperti perhotelan, pariwisata, farmasi, kelautan dan perikanan, ilmu komputer, dan lain sebagainya. Menurut kurikulumnya, fungsi bahasa Inggris yang diajarkan kepada siswa SMK adalah agar siswa memiliki pengetahuan dasar bahasa Inggris untuk mencapai kompetensi pada program keahlian yang menjadi jurusannya. Oleh karena itu, pendekatan yang digunakan dalam pengajaran bahasa Inggris kepada siswa program keahlian tersebut di atas adalah English For Specific Purposes (ESP). Mata pelajaran Bahasa Inggris dalam hal ini berfungsi sebagai mata pelajaran adaptif. Para siswa dilatih untuk mampu berkomunikasi dalam bahasa Inggris dalam konteks program keahliannya. Oleh karena itu, materi pembelajaran bahasa dirancang oleh guru bahasa Inggris dengan mengintegrasikan atau mengadaptasi konten bidang tertentu ke dalam kegiatan pembelajaran bahasa. Selain itu, pembelajaran bahasa Inggris mencakup dua bidang utama; pembelajaran keterampilan berbahasa dan pembelajaran komponen bahasa. Keterampilan berbahasa terdiri dari empat keterampilan, yaitu menyimak, berbicara, membaca, dan menulis. Komponen bahasa adalah kosa kata, ejaan dan pengucapan dan tata bahasa/struktur dan sebagainya . Mendengarkan dan Membaca juga dikenal sebagai keterampilan reseptif. Keterampilan ini digunakan oleh pengguna bahasa untuk menerima informasi dari bahasa lisan dan tulisan. Informasi yang diakui dengan baik yang diperoleh dari kegiatan mendengarkan dan membaca disebut sebagai pemahaman. Untuk pemahaman inilah sebenarnya pengajaran keterampilan menyimak dan membaca ditujukan kepada. Berbeda dengan menyimak dan membaca, ketermpilan berbicara dan menulis adalah keterampilan produktif yang digunakan oleh pengguna bahasa untuk mengungkapkan perasaan, pikiran, pendapat atau informasi yang telah mereka pahami dari kegiatan membaca dan menyimak. Kapasitas untuk mereproduksi informasi yang dipahami disebut sebagai kompetensi. ABSTRACT This Classroom Action Research (CAR) was conducted at SMK Negeri I Biromaru. The research subjects were class XI students of the fishing vessel nautical expertise program which consist of 20 students. Researcher employed an integrated language skills teaching strategy to increase students’ reading and speaking skills competence for marine and fisheries. In his teaching activitiesarch, the researcher integrated vocational matters in form of reading text as the teaching materials from where Reading and Writing skills are trained based on those materials. Researchers employed observation sheets, field notes, questionnaires, and formative assessments to collect data. In cycle 1 there were 8 students or 40% who scored 70 or more in reading comprehension (70% success criteria), and in cycle 2 there were 14 students or 70%. In cycle I there was 1 student or 5% who got an average speaking skill score of 36.00 (good) and 7 students or 35% who got a score of 30.00 (Enough). In cycle 2 there were 1 or 5% who got an average score of 42.00 in speaking skills (very good), 5 students or 25% got an average score of 36.00 (Good), and 6 students or 30% obtained the average score of 30.00 (Enough). Based on the above data the integrated skills teaching strategy is effective to help students improve their competency in English for marine and fisheries. Keywords: Reading and Writing Competence, Learning Strategies, Integrated Language Skills Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 66 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 PENDAHULUAN Dalam pembelajaran bahasa asing secara formal di sekolah, pemahaman dan kompetensi dapat diperoleh dengan berpartisipasi aktif dalam kegiatan belajar mengajar. Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Memiliki kompetensi keterampilan berbahasa tentu harus didukung oleh penguasaan komponen bahasa yang baik. Pengajaran komponen bahasa sangat penting untuk diintegrasikan dalam pembelajaran empat keterampilan bahasa (Listening,Speaking,Reading and Writing). Komponen bahasa yang terkandung dalam bahan ajar (Reading text) dilatihkan kepada siswa untuk mencapai kelancaran dan penguasaan (Fluency and Mastery). Dengan demikian, melalui strategi pembelajaran komponen bahasa yang terintegrasi dalam pembelajaran empat keterampilan berbahasa akan dapat melatih siswa secara langsung kegunaan dan fungsi komponen bahasa dalam mendukung pencapaian keterampilan bahasa secara nyata. 67 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 Dalam kaitannya dengan kompetensi di atas, siswa kelas XI program keahlian Nautika Kapal Perikanan (NKPI) SMKN I Biromaru masih mengalami kesulitan dalam memahami isi bacaan tentang program keahliannya. Mereka juga mengalami kesulitan untuk merekonstruksi ide-ide yang diperoleh dari bacaan ke dalam kalimat untuk menjawab pertanyaan pemahaman. Mereka sering salah melafalkan terminologi teknis bahasa Inggris yang sesuai dengan spesialisasi mereka. Selain itu, sebagian besar siswa belum terampil untuk mengonversi satu ide ke dalam struktur tata bahasa yang berbeda. Sebagai contoh: check all navigation devices before setting out to planned fishing resort (imperative sentence). Substansi kalimat ini sebenarnya dapat diungkapkan dengan menggunakan pola kalimat lain yang berbeda seperti prepositional gerund sebagai subject dan atau dengan pola kalimat Introductory “It” sebagai berikut; before setting out to planned fishing resort, check all navigation devices. Penguasaan komponen bahasa seperti di atas akan sangat membantu siswa dalam menjawab pertanyaan- pertanyaan pemahaman (comprehension questions) sekaligus bisa mereproduksi satuan-satuan ide yang ada dalam bacaan sehingga pada gilirannya siswa memiliki kompetensi berkomunikasi baik lisan maupun tertulis Munculnya kesulitan siswa sebagaimana dikemukakan di atas disebabkan oleh beberapa faktor. Pertama, bahan ajar tentang bahasa Inggris kejuruan (ESP) sangat terbatas dibandingkan dengan bahan ajar bahasa Inggris umum. Selain itu, guru bahasa Inggris umum yang ada di SMK membutuhkan waktu untuk menyediakan bahan ajar buatan guru dalam waktu yang sangat singkat karena mereka tidak menguasai mata pelajaran kejuruan (kelautan dan perikanan). Akibatnya, mereka menggunakan materi bahasa Inggris umum sebagai gantinya. PENDAHULUAN Kedua, penguasaan siswa tentang komponen bahasa utamanya aturan-aturan garamatika Bahasa Inggris dan kosa kata masih rendah sehingga merasa sulit menggunakan teknik skimming dan scanning dalam kegiatan membaca. Kesulitan belajar siswa ini bila dibiarkan jelas tidak memenuhi harapan kurikulum. Kurikulum Berbasis Sekolah (KTSP) (2007:84) menyatakan bahwa peserta didik diharapkan memiliki kompetensi untuk menguasai dasar pengetahuan dan keterampilan Bahasa Inggris untuk mendukung pencapaian program keahliannya. y Sehubungan dengan latar belakang yang telah dipaparkan di atas, peneliti terdorong untuk melakukan penelitian tindakan kelas untuk membantu siswa meningkatkan kompetensi keterampilan membaca dan menulis bahasa Inggris untuk kelautan dan perikanan. Penelitian tindakan adalah suatu pendekatan untuk memperbaiki pendidikan dengan mengubahnya dan belajar dari konsekuensi perubahan (Kemmis & McTaggart, 1992:22). Peneliti mengintegrasikan materi- materi kejuruan dalam bahan ajar lalu mengimplementasikan dalam pembelajaran keterampilan berbahasa terpadu (Reading and Speaking) dimana keterampilan membaca sebagai penekanannya. Perhatian utama peneliti dalam penelitian ini adalah untuk meningkatkan kompetensi siswa dalam memahami isi bacaan dengan asumsi bahwa jika siswa memiliki kompetensi keterampilan membaca yang baik akan memungkinkan mereka untuk menyelesaikan tugas menulis. Para siswa terlebih dahulu dilatih untuk mengumpulkan informasi dari teks bacaan dengan menggunakan strategi membaca skimming dan scanning, kemudian mereka difasilitasi untuk mereproduksi informasi yang dipahami secara tertulis melalui celah –informasi (information-gap). HASIL DAN PEMBAHASAN Guru melakukan delapan kali penilaian dalam penelitian tindakan ini; 4 kali pada siklus 1 dan 4 kali juga pada siklus 2. Guru tidak menyajikan hasil dari delapan penilaian tersebut dalam penyajian data ini melainkan hanya menyajikan dua penilaian pada setiap siklus yaitu penilaian hasil pemahaman bacaan dan penilaian hasil keterampilan menulis. Data dari kedua hasil asesmen yang dilakukan dalam dua siklus (membaca pemahaman dan keterampilan menulis) ini cukup representatif untuk menunjukkan dampak dari strategi pembelajaran keterampilan berbahasa terpadu terhadap peningkatan kompetensi siswa dalam pemahaman membaca dan keterampilan menulis dalam bahasa Inggris yang digunakan bidang kelautan dan perikanan. METODE PENELITIAN Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Penelitian ini merupakan penelitian tindakan kelas dengan strategi pembelajaran ketrampilan terpadu. Penelitian tindakan ini dilaksanakan di SMKN I Biromaru. Subyek penelitian tindakan ini adalah siswa kelas XI program keahlian Nautika kapal Penangkap Ikan 68 (NKPI) sebanyak 20 orang siswa. Instrumen yang digunakan dalam mengumpulkan data adalah lembar observasi, catatan lapangan, dan penilaian. (NKPI) sebanyak 20 orang siswa. Instrumen yang digunakan dalam mengumpulkan data adalah lembar observasi, catatan lapangan, dan penilaian. Prosedur penelitian tindakan adalah sebagai berikut1). Peneliti menyiapkan materi pembelajaran yang diramu secara kolaboratif dengan guru kejuruan.2).Peneliti menyiapkan RPP dan alat pengumpul data. 4). Peneliti mengimplementasikan perencanaan dalam kegiatan belajar mengajar yang nyata di kelas. 4).Peneliti dan kolaborator melakukan refleksi tentang pelaksanaan pembelajaran. 5). Berdasarkan hasil refleksi peneliti dapat memutuskan untuk meneruskan ke siklus berikut atau tidak. 1. Pemahaman Membaca Penilaian pemahaman bacaan yang dilakukan oleh peneliti pada setiap pertemuan ditujukan untuk mengetahui kemajuan siswa dalam menggunakan strategi membaca untuk memahami isi bacaan. Selain itu, berfungsi untuk mengetahui tingkat penguasaan siswa dalam membaca pemahaman dengan menilai kemampuan siswa dalam merumuskan ide ke dalam kalimat untuk menjawab pertanyaan pemahaman. Kriteria keberhasilan membaca pemahaman dalam penelitian tindakan ini adalah 70,00. Artinya, secara individu siswa diharapkan memperoleh skor 70.00 sebagai nilai minimal yang diperoleh dalam pemahaman bacaan. Dengan demikian, individu tersebut harus menjawab setidaknya tujuh pertanyaan dengan benar. Prestasi siswa dalam membaca pemahaman pada siklus I ditampilkan pada tabel berikut. Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 69 Tabel 1 Prestasi Siswa dalam Pemahaman Membaca pada Siklus 1 No Nama Awal Jumlah Pertanyaan Jumlah Jawaban Benar Jumlah Jawaban Salah Skor yang didapat (Berhasil/Gagal) 1. Abd 10 8 2 80,00/Sukses 2. Ari 10 7 3 70,00/Sukses 3. Adw 10 7 3 70,00/Sukses 4. Elo 10 7 3 70,00/Sukses 5. Edw 10 7 3 70,00/Sukses 6. Jauh 10 5 5 50,00/Gagal 7. Har 10 5 5 50,00/Gagal 8. Mul 10 5 5 50,00/Gagal 9. Nur 10 6 4 60,00/Gagal 10. Yud 10 8 2 80,00/Sukses 11. Khr 10 8 2 80,00/Sukses 12. Tam 10 6 4 60,00/Gagal Tabel 1 Prestasi Siswa dalam Pemahaman Membaca pada Siklus 1 J l h J l h Sk 69 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 13. Tfq 10 6 4 60,00/Gagal 14. Ton 10 9 1 90,00/Sukses 15. Sah 10 6 4 60,00/Gagal 16. Umr 10 4 6 40,00/Gagal 17. Vik 10 4 6 40,00/Gagal 18. Bur 10 6 4 60,00/Gagal 19. Zul 10 5 5 50,00/Gagal 20. Zack 10 5 5 50,00/Gagal B d k d d b l di d 8 i 40% i di k l b Berdasarkan data pada tabel di atas terdapat 8 siswa atau 40% siswa di kelas tersebut yang memperoleh nilai ≤ 70,00. Skor yang diperoleh ini setara dengan “sukses”. Siswa tersebut mampu menjawab dengan benar tujuh atau lebih dari sepuluh soal pemahaman yang diberikan kepada mereka. Mereka mampu mengkonstruksi ide-ide yang diperoleh dari bacaan untuk menjawab pertanyaan pemahaman dengan menggunakan kriteria keterampilan mikro menulis (lihat penilaian pada bab II). Artinya prestasi belajar siswa tersebut memenuhi kriteria keberhasilan 70,00. Sebaliknya, terdapat 12 atau 60% siswa yang memperoleh nilai tidak memenuhi kriteria keberhasilan 70,00. Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 1. Pemahaman Membaca Hal ini mengimplikasikan bahwa kompetensi mereka dalam membaca dengan pemahaman masih rendah. Berdasarkan data pada tabel di atas, dapat dikatakan bahwa pada siklus 1 hanya 8 siswa atau 40% siswa di kelas yang mengalami kemajuan dalam membaca dengan pemahaman karena nilai yang diperoleh dalam menjawab soal pemahaman memenuhi kriteria sukses 70.00. Sementara itu, terdapat 12 atau 60% siswa yang belum mengalami kemajuan dalam membaca dengan pemahaman karena nilai yang diperoleh dalam menjawab soal pemahaman tidak memenuhi kriteria keberhasilan 70,00. 2 Keterampilan Berbicara 2. Keterampilan Berbicara Penilaian keterampilan berbicara bertujuan untuk mengenali kompetensi siswa dalam menggunakan keterampilan berbicara untuk mereproduksi secara lisan pokok-pokok materi produktif yang digali dari bacaan melalui celah informasi (information gap) yang dipresentasikan di depan kelas secara berpasangan. Keterampilan berbicara terdiri dari dua aspek yaitu isi setara dengan pemahaman dan penyampaian setara dengan kelancaran. Penyampaian atau kelancaran mengacu pada seberapa fasih individu menyajikan komponen bahasa, pelafalan dan tata bahasa yang sesuai. Isi atau pemahaman mengacu pada seberapa baik siswa memahami pokok-pokok materi kejuruan yang disajikan. Peneliti menilai aspek-aspek tersebut dengan menggunakan speaking performance list Brown 2004. Hasil penilaian keterampilan berbicara disajikan dalam tabel berikut. Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Tabel 2. Prestasi Siswa dalam Keterampilan Berbicara pada Siklus I No Nama Awal SKOR YANG DIPEROLEH Skor rata - rata Penyampaian Isi Predikat 1. Abd 35 25 30,00/Sedang 2. Ari 35 25 30,00/Sedang 3. Adw 35 25 30,00/Sedang 4. Elo 35 25 30,00/Sedang 5. Edw 35 30 30,25/Sedang 30,00/Sedang 6. Jauh 35 25 20,20/Buruk 7. Har 28 20 20,20/Buruk 8. Abd 28 20 20,20/Buruk 9. Nur 28 20 20,20/Buruk Tabel 2. Prestasi Siswa dalam Keterampilan Berbicara pada Siklus I Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 70 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 10. Yud 28 20 36,00/Baik 11. Khr 42 30 20,20/Buruk 12. Tam 28 20 20,20/Buruk 13. Tfq 28 20 20,20/Buruk 14. Ton 28 20 20,20/Buruk 15. Sah 35 20 20,20/Buruk 16. Umr 28 20 20,20/Buruk 17. Vik 28 20 20,20/Buruk 18. Bur 28 20 20,20/Buruk 19. Zul 28 20 20,20/Buruk 20. Zack 28 20 Berdasarkan data pada tabel di atas, terdapat 1 siswa atau 5% dari total siswa di kelas tersebut yang mendapat nilai 36,00 sebagai rata-rata nilai yang diperolehnya. Menurut sistem penilaian itu dikategorikan sebagai "baik". Masih dari tabel di atas, terdapat 6 siswa atau 30% dari total siswa di kelas tersebut yang memperoleh nilai rata-rata sekitar 30.00 dan 30.25. Menurut sistem penilaian skor rata-rata ini dikategorikan sebagai “cukup”. 1. Pemahaman Membaca Penilaian pemahaman bacaan yang dilakukan oleh guru pada setiap pertemuan ditujukan untuk mengetahui kemajuan siswa dalam menggunakan strategi membaca untuk memahami isi bacaan. Selain itu, berfungsi untuk mengetahui tingkat penguasaan siswa dalam membaca pemahaman dengan menilai kemampuan siswa dalam merumuskan ide ke dalam kalimat untuk menjawab pertanyaan pemahaman. Kriteria keberhasilan membaca pemahaman dalam penelitian tindakan ini adalah 70,00. Artinya, secara individu siswa diharapkan memperoleh skor 70.00 sebagai nilai minimal yang diperoleh dalam pemahaman bacaan. Dengan demikian, individu tersebut harus menjawab setidaknya tujuh pertanyaan dengan benar. Guru menugaskan siswa untuk menjawab sepuluh soal pemahaman berdasarkan bacaan pada setiap pertemuan siklus 2. Ada dua topik yang dibahas pada siklus 2; adalah "Anak Buah Kapal” dan "Teknik Navigasi". Prestasi siswa dalam membaca dengan pemahaman disajikan pada tabel 3 sebagai berikut. Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 7 Tabel 3. Prestasi Siswa dalam Pemahaman Membaca pada Siklus 2 No Nama Awal Jumlah Pertanyaan Jumlah Jawaban Benar Jumlah Jawaban Salah Skor yang Didapat (Berhasil/Gagal) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Abd Ari Adw Elo Edw Jhr Har Mul Nur Yud Khr 10 10 10 10 10 10 10 10 10 10 10 9 9 7 9 9 6 7 7 8 8 8 1 1 3 1 1 4 3 3 2 2 2 90,00/Sukses 90,00/Sukses 70,00/Sukses 90,00/Sukses 90,00/Sukses 60,00/Gagal 70,00/Sukses 70,00/Sukses 80,00/Sukses 80/,00Sukses 80,00/Sukses Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 71 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 12. 13. 14. 15. 16. 17. 18. 19. 20. Tam Tfq Ton Sah Umr Vik Bur Zul Zack 10 10 10 10 10 10 10 10 10 9 6 9 7 6 6 6 7 5 1 4 1 3 4 4 4 3 5 90,00/Sukses 60,00/Gagal 90,00/Sukses 70,00/Sukses 60,00/Gagal 60,00/Gagal 60,00/Gagal 70,00/Sukses 50,00/Gagal LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P ISSN 2807 2316 Berdasarkan data pada tabel di atas, terdapat 14 siswa atau 70% dari total siswa di kelas tersebut yang memperoleh nilai pemahaman bacaan ≤ 70,00. Artinya terdapat 14 (70%) siswa yang memperoleh nilai membaca dengan pemahaman memenuhi kriteria keberhasilan. Terdapat 6 siswa atau 30% siswa yang nilai yang diperoleh tidak memenuhi kriteria keberhasilan karena hanya memperoleh nilai kurang dari 70,00. Siklus I Berdasarkan data yang diperoleh dari lembar kerja siswa yang tertera pada tabel 1, kesalahan yang dilakukan siswa dalam menjawab soal paling banyak adalah merekonstruksi ide menjadi kalimat yang tepat. Contoh: “Jelaskan perbedaan antara kapal kargo kering dan kapal kargo cair”. Sebagian besar siswa (13 siswa) tidak menjelaskan perbedaan kedua jenis kapal tersebut. Mereka hanya memberi contoh sebagai gantinya. Demikian pula, sebagian besar siswa membuat kesalahan tata bahasa untuk menjawab pertanyaan mengapa kapal penumpang saat ini mengalami penurunan. Tujuh siswa menjawab pertanyaan ini dengan benar dengan mengambil informasi dari kalimat terakhir paragraf dua. Siswa lain menjawab pertanyaan tersebut dengan menuliskan pertumbuhan pesat transportasi darat dan udara saja. Sebagian besar siswa juga membuat kesalahan pada tata bahasa/struktur. Para siswa tidak dapat menggunakan “Introductory It” sebagai subyek kalimat dengan kata benda tunggal dan jamak. Ada beberapa siswa (tujuh) yang mampu mengkonstruksi jawaban dengan menggunakan passive voice sesuai dengan yang diminta oleh pertanyaan, namun kebanyakan dari mereka melakukan kesalahan dalam pola kalimat ini. Singkatnya, sebagian besar siswa masih kesulitan menyusun kalimat untuk menjelaskan atau membedakan jenis-jenis kapal. Mereka mengutipnya dari paragraf. Siswa dapat menyebutkan informasi umum dari setiap paragraf (tidak ada kesalahan yang dilakukan siswa pada jenis soal ini). Berdasarkan pengamatan guru di lapangan, siswa yang memperoleh nilai memenuhi kriteria keberhasilan 70,00 (8 siswa atau 40%) mampu menggunakan strategi membaca; skimming dan scanning untuk mengetahui jawaban dari pertanyaan. Mereka juga mampu mengkonstruksi ide-ide yang mereka peroleh melalui skimming dan scanning menjadi kalimat yang benar untuk menjawab pertanyaan. Berdasarkan rata-rata yang diperoleh di atas tabel 2, dapat dikatakan bahwa ketujuh siswa di atas (35%) sudah mengalami kemajuan dalam kompetensi berbicara. Mereka dapat menggunakan item keterampilan berbicara (penyampaian dan isi) untuk mereproduksi pokok- pokok materi produktif yang mereka peroleh dari kegiatan membaca selama kelas membaca. Namun demikian, kualitas presentasi lisan mereka dalam hal penyampaian (tujuh item) berbeda (1 siswa “baik” dan 7 siswa “cukup”). Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Sebaliknya, terdapat 14 siswa atau 60% dari total siswa yang belum banyak mengalami kemajuan dalam kompetensi berbicara. Sebenarnya, para siswa ini melakukan kegiatan berbicara seperti yang ditugaskan, tetapi skor yang mereka peroleh baik dalam penyampaian maupun isi belum memenuhi kriteria keberhasilan. Rata-rata skor yang mereka peroleh hanya 20,20. Berdasarkan sistem skoring yang digunakan dalam penelitian tindakan ini, skor rata-rata ini dikategorikan “buruk”. Siswa-siswa ini selalu melihat konsep mereka dan bertindak seolah- olah mereka sedang membaca dengan suara keras. y 2. Keterampilan Berbicara Penilaian tentang keterampilan berbicara bertujuan untuk mengetahui kompetensi siswa dalam keterampilan berbicara untuk mereproduksi pokok-pokok materi kejuruan yang diperoleh dari bacaan. Hasil penilaian keterampilan berbicara dapat ditunjukan seberapa baik kemajuan siswa secara perorangan dalam menggunakan aspek-aspek keterampilan berbicara yang dinilai pada siklus 2 dibandingkan dengan siklus 1. Dengan kata lain, guru ingin mengetahui apakah penggantian pasangan dapat memberikan kontribusi terhadap peningkatan kompetensi siswa dalam keterampilan berbicara atau tidak. Hasil penilaian keterampilan berbicara disajikan pada tabel 4, sebagai berikut. Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 72 Tabel 4 Hasil Penilaian Keterampilan Berbicara pada Siklus 2 No Nama Awal SKOR YANG DIPEROLEH Skor Rata-Rata Predikat Pengiriman Isi 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Abd Ari Adw Elo Edw Jauh Har Mul Nur Yud Khr Tam Tfq Ton Sah Umr Vik Bur Zul Zack 42 42 42 42 42 35 35 28 35 28 49 28 42 28 35 35 28 28 28 28 30 30 30 30 30 25 25 20 25 20 35 20 30 20 25 25 20 20 20 20 36,00/ Bagus 36,00 Bagus 36,00/Baik 36,00/Baik 36.00/Baik 30,00/Sedang 30,00/Sedang 20,00/Buruk 30,00/Sedang 20,20/Buruk 42,00/Lumayan 20,20/Buruk 20,20/Buruk 30,00/Baik 20,20/Buruk 30,00/Sedang 30,00/Sedang 20,20/Buruk 20,20/Buruk 20,20/Buruk Tabel 4 Hasil Penilaian Keterampilan Berbicara pada Siklus 2 72 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 Berdasarkan data pada tabel di atas, pada siklus 2 terdapat 1 siswa atau 5% dari siswa di kelas ini yang memperoleh nilai rata-rata 42,00. Nilai rata-rata ini menurut sistem penilaian sama dengan “sangat baik”. Terdapat 6 siswa atau 30% yang memperoleh nilai rata-rata 36,00. Skor rata-rata ini sama dengan “baik”. Dan terdapat 5 siswa atau 25% yang memperoleh nilai 30.00 dan itu setara dengan “cukup”. Sebaliknya, ada 8 siswa atau 40% yang tidak mengalami kemajuan dalam keterampilan berbicara pada siklus 2. Ketidakmampuan siswa tersebut untuk meningkatkan kompetensi berbicaranya disebabkan rendahnya kemampuan mereka dalam aspek penyampaian. Sebagian besar dari mereka mendapatkan skor rendah pada item penyampaian (delivery). Siklus II Pada siklus 2, sebagian besar siswa sudah mampu menggunakan strategi membaca skimming dan scanning untuk mencari informasi jawaban dari pertanyaan pemahaman. Hal ini karena siswa bekerja dalam kelompok untuk menyelesaikan tugas membaca sebelum menjawab pertanyaan pemahaman secara individu. Selain itu, latihan tata bahasa juga termasuk dalam tugas membaca. Akibatnya, sebagian besar siswa (70%) memperoleh nilai yang memenuhi kriteria keberhasilan ≤ 70,00. Mereka mampu menyusun ide menjadi kalimat untuk menjawab pertanyaan pemahaman dengan benar. Sementara itu, siswa yang gagal (6 orang siswa atau 30%) sebenarnya mampu menggunakan skimming dan scanning untuk mencari jawaban tetapi mereka masih melakukan kesalahan untuk menyusun jawaban mereka dengan benar sehingga skor perolehan kurang dari 70,00. Pada siklus 2, siswa tidak banyak menghabiskan waktu untuk memahami makna soal pemahaman karena sudah disediakan oleh guru sehingga waktu yang tersedia banyak digunakan untuk melakukan kegiatan membaca untuk mencari jawaban menjawab pertanyaan pemahaman. Menurut pengamatan dan catatan lapangan, pemberian pertanyaan pemahaman beserta artinya sangat membantu siswa untuk mencari jawaban secara langsung dari bacaan. Berdasarkan data yang diperoleh dari lembar kerja siswa, kesalahan yang dilakukan siswa dalam menjawab soal pemahaman di atas adalah pada struktur dan kosa kata. Siswa yang gagal di atas (6 atau 30%) telah mencoba menguraikan jawaban mereka dalam kalimat yang cukup panjang tetapi strukturnya salah dan menggunakan banyak pengulangan. Siswa-siswa ini membutuhkan lebih banyak pelatihan dalam struktur kalimat dan kosa kata terutama sinonim agar lebih terampil menjawab pertanyaan pemahaman. Berdasarkan skor yang diperoleh dalam keterampilan berbicara pada tabel 4, dapat dikatakan bahwa 12 siswa atau 60% siswa mampu menyajikan pokok-pokok materi kejuruan yang diperoleh dari bacaan berupa information-gap secara lisan. Kemajuan ini tidak hanya dalam jumlah siswa tetapi juga dalam kualitas presentasi. Pada siklus 1 hanya 1 siswa atau 5% yang mendapat rata-rata nilai yang diperoleh setara dengan “baik” dan tidak ada siswa yang mendapat predikat 'sangat baik'. Pada siklus 2 terdapat 1 siswa atau 5% yang memperoleh rata- rata nilai setara dengan “sangat baik” (42,00). Selain itu terdapat 5 siswa atau 25% yang mendapat predikat “cukup” pada siklus 1 namun pada siklus 2 rata-rata nilai yang diperoleh meningkat setara dengan “baik” (36,00). Bahkan ada 5 siswa atau 25% yang nilai rata-ratanya sama dengan “kurang baik” pada siklus 1, menjadi “cukup” (4 siswa) dan “baik” (1 siswa) pada siklus 2. Siklus I Berdasarkan pengamatan mereka terlihat memiliki kemauan yang rendah untuk melakukan komunikasi lisan karena rendahnya rasa percaya diri yang mereka miliki. Siswa-siswa ini juga membuat banyak kesalahan dalam 73 kalimat konsep tertulis mereka. Selain itu, mereka tidak aktif mendengarkan presentasi lisan temannya. Mereka sibuk menyusun tugas berbicara mereka untuk dipresentasikan secara lisan di depan kelas. kalimat konsep tertulis mereka. Selain itu, mereka tidak aktif mendengarkan presentasi lisan temannya. Mereka sibuk menyusun tugas berbicara mereka untuk dipresentasikan secara lisan di depan kelas. Berdasarkan check list observasi, 8 atau 40% siswa di atas memiliki rasa percaya diri yang lebih besar dibandingkan dengan 12 atau 60% siswa lainnya. Mereka dengan berani tampil di depan kelas untuk mempresentasikan kesenjangan informasi mereka secara lisan. Selain itu, delapan siswa ini berpartisipasi aktif selama proses pembelajaran. Khususnya siswa yang memperoleh nilai rata-rata 36.00, ia sering mencoba mengungkapkan makna tertentu dalam bentuk tata bahasa yang berbeda dan ia tidak merasa malu menggunakan bahasa tubuh (menggerakkan bagian tubuh seperti tangan, jari, dan mata) untuk mendukung penyampaian makna. Dia juga dapat memulai pembicaraan dengan penuh perhatian, mempertahankan pembicaraan, dan menutupnya dengan menggunakan fungsi bahasa yang tepat dengan kesalahan pengucapan yang minimal dan tanpa melihat konsepnya sama sekali. Siklus II Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Berdasarkan pengamatan, peningkatan jumlah dan kualitas yang diperoleh 12 siswa atau 60% di atas disumbangkan oleh teknik berpasangan. Siswa yang lebih pandai dalam tugas 74 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 berbicara dipasangkan dengan siswa yang kurang saat menyelesaikan tugas berbicara. Selain itu, tugas-tugas berbasis struktur yang ditugaskan oleh guru untuk dikerjakan selama pembelajaran membaca juga menginspirasi pasangan untuk menciptakan celah informasi (information-gap) yang tepat untuk disajikan secara lisan. Akibatnya, sebagian besar siswa membuat banyak kemajuan dalam kompetensi keterampilan berbicara untuk mereproduksi pokok-pokok materi kejuruan dari teks bacaan. Berdasarkan pengamatan guru 5 orang siswa atau 25% yang kemampuan berbicaranya “kurang” pada siklus 2 masih lemah dalam aspek penyampaian, dan sisanya 3 siswa atau 15% lemah baik dalam penyampaian maupun isi. Gagasan utama dan pendukung dinyatakan dengan jelas meskipun beberapa gagasan dinyatakan dalam struktur kalimat yang tidak tepat. Selain itu, mereka sering membuat kesalahan pelafalan. Mereka tidak menggunakan gerak tubuh untuk mendukung penyampaian makna. Mereka hanya membaca konsep celah informasi (information-gap) dan memaparkannya secara lisan. KESIMPULAN Strategi pembelajaran keterampilan berbahasa terpadu yang diimplementasikan dalam pembelajaran membaca bahasa Inggris untuk kelautan dan perikanan kepada siswa kelas XI program keahlian Nautika Kapal Penangkap Ikan di SMK Negeri I Biromaru efektif untuk membantu siswa meningkatkan kompetensi keterampilan membaca.Berdasarkan pencapaian hasil belajar siswa dalam membaca dengan pemahaman pada siklus I terdapat 8 siswa atau 40% siswa di kelas yang memperoleh nilai ≤ 70,00. Skor yang diperoleh ini setara dengan keberhasilan atau cukup memenuhi kriteria keberhasilan 70%. Dan sisanya 12 siswa atau 60% belum memenuhi kriteria keberhasilan 70%. Pada siklus 2 persentase siswa yang memperoleh nilai membaca dengan pemahaman memenuhi kriteria keberhasilan 70,00 menjadi 14 siswa atau 70%. Persentase siswa yang belum memenuhi kriteria keberhasilan menurun dari 12 siswa (60%) pada siklus I menjadi 6 siswa atau 30% pada siklus II DAFTAR PUSTAKA Brown, Douglas H. 2001. Teaching by Principles: and Interactive Approach toLanguage Pedagogy, Second Edition. Pearson Education, Inc. New York DAFTAR PUSTAKA Brown, Douglas H. 2001. Teaching by Principles: and Interactive Approach toLanguage Pedagogy, Second Edition. Pearson Education, Inc. New York Brown, Douglas H. 2004. Language Assessment: Principles and Classroom Practices. Pearson Education, Inc. New York 10606. Emzir, 2008. Metodologi Penelitian Pendidikan Kuantitatif & Kualitatif. PT. Raja Grafindo Persada. Jakarta. Duke,Merlyn&Pearson,Jack. 2002.Sydney Micro-Skills: Redeveloped. Sydney University Press. Fiorito. Lorenzo. 2005. Teaching English for Specific Purposes (ESP). Retrieved, December 14th 2008 from : The internet TESL Journal 2005. Gatehouse. 2001. Keys issues in English specific purposes (ESP). Curriculum Development. The Internet TESL Journal, Vol. VII. No.10 October 2001. Retrieved, December, 12th 2008. From: http:// itasly.org/article/Gatehouse. ESP.html. Harmer, Jeremy,2007. How to Teach English. Pearson Education Limited, Longman. h Hortas. An. D. 2005. English for Specific Purpose. Retrieved on December, 14th 2008, from The internet TESL Journal. 2005. Kayi, Hayriye. 2007. Teaching Speaking: Activities to Promote Speaking in a Second Language. Retrieved on April23rd2009, from: http://unr.edu/homepage/hyriyekkayih[at]unr.nevada.edu.University Nevada(Nevada,USA) Kayi, Hayriye. 2007. Teaching Speaking: Activities to Promote Speaking in a Second Language. Retrieved on April23rd2009, from: htt // d /h /h i kk ih[ t] d d U i it 75 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra Vol 3. No 2. Mei 2023 E-ISSN : 2807-1670 P-ISSN : 2807-2316 Oxford. Rebecca. 2001. Integrated Skills in The EFL/ESL Classroom. An Article. University of Mary Land. 2001. Retrieved, January 15th2009 from The Internet TESL JOURNAL, Rebecca. @ Maryland. Edu. Richards Jack C 2001 Curriculum development in Language Teaching Cambridge Oxford. Rebecca. 2001. Integrated Skills in The EFL/ESL Classroom. An Article. University of Mary Land. 2001. Retrieved, January 15th2009 from The Internet TESL JOURNAL, Rebecca. @ Maryland. Edu. y Richards. Jack. C. 2001. Curriculum development in Language Teaching. Cambridge University, Press. y Soemardi, Sumantri.2007. ESP. Is it Possible? An Article. TEFLIN. Magazine. Vaughn, S et al 2001.Teaching Reading Comprehension through Collaborative Strategic Reading.Little, Brown and Company. Yildiz, Senem, 2004. Teaching English for Specific Purposes. An Article. Retrieved December 10th 2008 from The Internet TESL JOURNAL, Syldiz. @ Indiana. Edu. Copyright (c) 2023 LANGUAGE : Jurnal Inovasi Pendidikan Bahasa dan Sastra 76
https://openalex.org/W2793215231	http://www.nasemore.com/wp-content/uploads/2018/04/3_Krile_Miskovic.pdf	English	null	Optimalna uporaba kontejnerskih brodova koji opslužuju male luke	Naše more	2,018	cc-by	4,329	KEY WORDS Transportation of containers, in areas which are not interesting for large shipping operators, can pose a problem for ship cargo officer due to limited logistical assistance from ashore. In such cases, all calculations and planning have to be done on board the ship. Transportation between secondary ports to other-final ports, e.g. for servicing ports in island region, can be done by independent carriers which operate small container ships without proper logistical assistance. The efficient algorithm for optimal transport of N contingents of containers, for ship with limited capacity on the route with M ports, is being considered. The proposed algorithm has acceptable complexity and such optimization tool can be used efficiently in limited shipping surrounding. But crucial condition is well educated cargo officer, to be skilled about such management and planning tool. non-linear transportation problem multi-destination routing problem capacity management of container ships Optimal Use of Container Ships for Servicing Among Small Ports Optimalna uporaba kontejnerskih brodova koji opslužuju male luke Darijo Mišković Nautical Department University of Dubrovnik e-mail: darijo.miskovic@unidu.hr Srećko Krile Electric Engineering and Computing Department University of Dubrovnik e-mail: srecko.krile@unidu.hr Srećko Krile Electric Engineering and Computing Department University of Dubrovnik e-mail: srecko.krile@unidu.hr DOI 10.17818/NM/2018/1.3 UDK 629.56:656.615 Preliminary communication / Prethodno priopćenje Paper accepted / Rukopis primljen: 12. 2. 2018. Sažetak KLJUČNE RIJEČI nelinearni transportni problem problem usmjeravanja prometa za više odredišta upravljanje kapacitetom kontejnerskog broda Prijevoz kontejnera, posebno u područjima koja velikim prijevoznicima nisu zanimljiva, može predstavljati problem časniku palube zaduženom za organizaciju tereta zbog ograničene logističke podrške s kopna. U takvim slučajevima svi izračuni i planiranje moraju biti obavljeni na brodu. Prijevoz između malih luka prema drugim lukama odredišta, npr. u opsluživanju otočnih luka, može obavljati neovisni prijevoznik koji upravlja malim kontejnerskim brodom bez odgovarajuće logističke podrške. Učinkoviti algoritam za optimalni prijevoz N vrsta kontejnerskih kontingenata, za brod ograničenog kapaciteta, na liniji s M luka je predložen. Predloženi algoritam prihvatljive je kompleksnosti i takvo računsko sredstvo može biti uspješno primijenjeno u ograničenom okruženju broda. Jedini ključan uvjet je dobra edukacija časnika zaduženog za teret da uz pomoć ovog alata može uspješno donositi odluke o planu krcanja. S. Krile and D. Mišković: Optimal Use of Container... 1. INTRODUCTION / Uvod In addition to the above, other conditions have impact on process in whole like an efficient operator network, efficient logistical support and most important acceptable port conditions (acceptable entry time-window, weather conditions, pilotage availability etc.) Therefore, operator can adjust flexibility when set schedule is deranged and to improve plan as required. To minimize the operational cost of the venture all aspects stated must be considered and integrated into plan. This paper addresses this need by proposing a model that considers the port locations together with the operational cost. Figure 2 Potential transfer of container contingents between ports given in percentage of the total ship capacity Slika 2. Potencijalni tereti (kontingenti) kontejnera koji čekaju na prijevoz izraženi u postotku brodskog kapaciteta Figure 1 Distances between the ports Slika 1. Udaljenosti između luka Figure 2 Potential transfer of container contingents between ports given in percentage of the total ship capacity Slika 2. Potencijalni tereti (kontingenti) kontejnera koji čekaju na prijevoz izraženi u postotku brodskog kapaciteta Figure 1 Distances between the ports Slika 1. Udaljenosti između luka For test-example from fig. 2. we have optimal solution represented on fig. 4 – fig. 6. On fig. 6. it is obvious that we have almost full ship, entering in all ports, with only small amounts of idle space (5 -15 %) on the voyage from 1-2 (10%), 3-4 (5%), 4-5 (15%) and. 5-1 (10%). In that case the transport cost reduction is more important that idle (unused) capacity on board. The capacity management problem in container shipping is extended to the transportation problem of different contingents (cargo types) transported by one ship on the route with multiple sources (loading ports) and multiple destinations (ports of discharge). Loads of containers (empty and loaded) are waiting to be transported as it is shown on fig 2. The load amounts are given in percentage of total ship capacity. If all contingents have the same transportation cost it is clear that the ship will service all ports and be backed to home port. It will be happened only if total income is positive. But if it is not the case the avoidance of some port could be possible, in order to find optimal transportation cost. Amount of different cargo loads (e.g. container) is in firm correlation because the total capacity of the ship is limited in TEU (Twenty-foot Equivalent Unit). 1. INTRODUCTION / Uvod 1. INTRODUCTION / Uvod Before operator even starts planning, first task is to find ship with optimal size, draft, maneuvering characteristics and stowage capacity [4, 5]. Potential implications regarding deploying too small or big ship may have adverse effect on venture. For these stages of planning all calculations are done by ship cargo operators which are based ashore, with full logistical and computing assistance. During last 60 years container ship trade grew significantly, which forced operators to look for more efficient ways for better transportation planning [1, 2]. The main goal of all involved parties is to lower overall transportation costs and to reduce congestion of infrastructure components, by delivering containers to the destination, as soon as possible. Transportation is organized on the principle of “hub and spoke” network modeled on the aircraft industry. These are linear network between ports that meet the needs of „mega” container ships, starting on one side and ending at the other side of the world. Among them sail large ships called “mother” ships and further distribution between main and secondary ports is done with smaller container ships [3, 4]. On the other side, transportation of containers between secondary ports to other-final ports, in this scenario placed on the islands, is done by independent carriers which operate small container ships without proper logistical assistance. Route planning is usually done by ship cargo officer and can pose a great problem. When there is more than one port involved in transportation planning, important inputs like transshipment costs, fuel price for each route, sailing time, time for loading /discharge containers and the location of each port present main inputs to For container ships of great size, with set timetable for loading/discharging ports, situation is clear; cargo must be delivered as per plan. Situation is similar for container ships which serves “hub-secondary port” route. 18 In example from fig. 1 we set 5 ports with set distances. For this purpose the container ship is limited with capacity, e.g. of max. 100 TEU, in order to reach maximum cost utilization. All information about potential load can be gathered through market research or from statistics. On diagram from fig.2. we can see eight contingents waiting for transport, half of them are for empty containers (1-2,1-3,1-4-,1-5) and second half are for loaded containers ( 2-1,3-1,4-1,5-1). determine the overall efficiency of the process [4]. 1. INTRODUCTION / Uvod Taking into account the cargo waiting to be transported, we need optimal transportation plan to minimize shipping and loading/unloading expenses, transshipment cost and cost of ship’s stay in port (connected with duration of loading process). Also, it can help in definition of ship capacity arrangement or for comparison of ships with different cargo capacity. 2. MATHEMATICAL MODEL / Matematički model Transportation of empty and loaded container contingents is specific problem but it can be seen as similar transport problems [15-18]. Various types of container contingents are differentiated with i for i = 1, 2, ... , N., odd for empty and even for loaded containers, see fig.3 . The ship with defined cargo capacity is shipping from the starting port, servicing number of K potential ports. The objective is to find a loading and transshipment strategy that minimizes the total cost incurred over the whole voyage route consisting of M ports on the path (M ≤ K). Each port on the route can be for loading and for discharging. The transportation problem can be represented by a flow diagram on fig. 3. The problem can be solved with different techniques, see [3-9] but here we applied network optimization technique as the shortest path problem in the network with multiple sources/destinations. Some ports have limitation on loading capacity and most of them are secondary ports with capacity below the ship’s earning capacity. In this paper the contingents are transported from/to home port, but in general, any combination of starting/ending ports can be introduced. On figure 3. the i-th row of nodes represents the capacity state of i-th type of contingent after loading in port m. The links between nodes represent the amount of cargo transported between ports (in percentage of ship capacity). The non-linear transportation problem (NTP) with multiple (several) ports of loading (sources) and multiple destinations (sinks) is very hard (NP-hard) problem so it is still the subject of many scientific papers. The similar problem can be solved with different techniques, see [6-12]. In special circumstances the NTP can be seen as Minimum Cost Multiple Commodities Flow Problem (MCMCF); see [13] and [14]. In this paper we applied such network optimization approach. The mathematical model is formulated in section 2. Implemented algorithm is tested on some examples and results are commented in section 3. 19 “Naše more” 65(1)/2018., pp. 18-23 Figure 3 A network flow presentation of the transportation problem Slika 3. Transportni problem prikazan pomoću mrežnog prikaza tokova tereta Figure 3 A network flow presentation of the transportation problem Slika 3. Transportni problem prikazan pomoću mrežnog prikaza tokova tereta In the mathematical model form fig. the following notation is used: In the mathematical model form fig. the following notation is used: the network with former calculated weights. 2. MATHEMATICAL MODEL / Matematički model The number of all possible dm,m+1 values depends on the total number of capacity points Cp i, j and k = indices for cargo load. The N facilities are not ranked, just present different types of cargo/container contingents from 1, 2, ... , N . Odd numbers are for empty and even for loaded containers. p In this research load amount on board do not influence on voyage speed neither to oil consumption but it could be easily incorporated. m = indices the port of loading (charging) or discharging. The number of port of calls on the voyage including departure port is M (m = 1, .. , M). m = indices the port of loading (charging) or discharging. The number of port of calls on the voyage including departure port is M (m = 1, .. , M). 3. RESULTS OF BASIC HEURISTIC / Rezultati osnovne heurističke metodeii Učinkovitost brodskog prostora na putovanju (popunjenost) Figure 5 Moments of loading/discharging Slika 5. Trenutci krcanja/iskrcaja have to differentiate freight cost for each container contingent and for each transport distance between ports m and m+1. All expenses have negative polarity. It means that profit will be reduced by transportation cost dependent on distances ci,m (dm) and with transshipment (load/discharge) cost gm (xi,m) related on loading/discharging amounts. Also, the port taxes can be introduced as hm for each port specifically. The idle capacity cost can be taken in account but only as a penalty cost to force the usage of maximal capacity (prevention of unused/idle capacity). So we have adding cost: idle (W-Ii,m), where W is total capacity of the ship and Im is total cargo load on board for each contingent and for transport distance from port m to port m+1. Figure 5 Moments of loading/discharging Slika 5. Trenutci krcanja/iskrcaja Figure 5 Moments of loading/discharging Slika 5. Trenutci krcanja/iskrcaja ∑ + = = 1 1 , N i m i m I I (3.2) (3.2) Costs are often represented by the fix-charge cost or/with constant value and variable part. It should be assumed that all cost functions are concave and non-decreasing (some of them reflecting economies of scale) and they differ from one contingent to another or from one port to another. The objective function (3.1) is necessarily non-linear and exponential. The problem can be seen as looking for maximal value of profit, that is logically in relation to minimal expenses. Figure 6 Efficiency of the ship on the route (occupancy) Slika 6. Učinkovitost brodskog prostora na putovanju (popunjenost) If we differentiate freight cost, e.g. higher cost for loaded containers, that cost will be included in objective function with opposite polarity, with intention to increase the profit of transportation. Such dual min-max problem could be solved with maximization of the profit. We can use the same technique of minimization but objective function has to be with negative polarity. Definitely, introduction of different freight cost (higher cost for loaded containers) it will influence on optimal solution and we have quite different results, see fig. 7 – fig. 8. If we have demands for empty containers that exceeds the amount that we can take from home port, we can supply it from the neighboring port. This model allows such solution, so cargo manager can include such contingent in account. 3. RESULTS OF BASIC HEURISTIC / Rezultati osnovne heurističke metodeii xi,m = quantity of i-th load of cargo amounts (e.g. containers contingent) being loaded on board in port m (TEU). In route definition for example from fig. 2 we have starting and ending port 1, but any of four middle ports can also be included in the route. All distances between ports are defined in miles. From figure 2. we can see traffic demands (possible transfer of contingents) given in the percentage of the total ship capacity. In this test-example we do not have contingents between middle ports, only transport related to/from home port, but it is not limitation. Lxi,m = limitations for each port and for each cargo load. For convenience, the xi,m is assumed to be integer. , Ii,m = the amount of cargo load i at arrival in port m (or, equivalently, at the departure in port m-1). Before the first port of loading, Ii,m= 0 . After last port Ii,M+1 = 0 for i =1,…, N . Capacity values cannot be negative. step Ii = the lowest step of possible capacity loading and discharging for capacity type i. In our numerical test-examples it can be set e.g. step Ii = 5% of total capacity of the ship. Figure 4. Amount of container contingents on the ship during voyage Slika 4. Količina kontejnera za pojedini teret u tijeku plovidbe The complexity of the proposed algorithm is O(Cp 2), where Cp is the number of capacity points; see explanation in [10]. That value is in strong correlation with number of ports M and number of contingents N but also with capacity increment step Ii that can be variable from contingent to contingent. The network optimization can be divided in two steps. At first step the minimal transportation weights (cost value for transport) between all pairs of capacity points (neighbor ports on the route) are calculated (see equitation 3.1). It is clear that many values dm,m+1 that emanate two capacity points of neighbor ports. At second step we are looking for the shortest path in Figure 4. Amount of container contingents on the ship during voyage Slika 4. Količina kontejnera za pojedini teret u tijeku plovidbe Figure 4. Amount of container contingents on the ship during voyage 20 S. Krile and D. Mišković: Optimal Use of Container... Figure 5 Moments of loading/discharging Slika 5. Trenutci krcanja/iskrcaja Figure 6 Efficiency of the ship on the route (occupancy) Slika 6. 3. RESULTS OF BASIC HEURISTIC / Rezultati osnovne heurističke metodeii Popunjenost broda je bliža 100 % nego na slici 6 Figure 10 Ship’s occupancy for third example is maximal Slika 10. Popunjenost broda je ovdje maksimalna Also, the complexity is firmly dependent of number of ports on the route M and with number of contingents N. If we have very huge problem we can solve it in steps of calculation, consisting of successive iterations that can decrease the complexity to acceptable level. According to all transport costs elements (freight cost, oil consumption, transshipment cost, port taxes etc.) we can design the route which will be the most profitable. In figures 7. and 8. the resulting (the best) route is presented. Fig. 7. shows the load amounts of every contingent on board during the voyage. We can see that contingent 4 (3-1) is much higher than before. Fig. 8. shows that ship’s occupancy rises, too. For the basic algorithm option we used the same capacity increment step Ii for all contingents and it is 5%. Such capacity resolution can be satisfactorily if we have small capacity of the ship, e.g., for 40 TEU (40 x 20 foot containers), so the capacity increment is 2 TEU, that is equal to two containers (2 x 20 ft). But if we have 100 TEU ship, the capacity increment is 5 TEU, so the calculation can be far away from optimal result. In that case we have to decrease step Ii, on value of 2% or 1% (not less the value that means one container). Normally, it causes huge number of capacity points and much higher complexity, so calculation duration drastically rises. So it could be the problem for limited calculation power. In the third example we can force the maximal loading (occupancy) of the ship. In that sense we can introduce penalty cost for idle capacity, see (3.1). From fig. 9 and fig.10. it is clear that result is quite different, the idle capacity of the ship is lower. No better ship’s occupancy exists for this example, but it depends on all cost elements and their relations. In such case, where we introduce penalty cost for empty space, it can cause non-optimal transport (not maximal profit). Through many test examples it is clear that such approach functions good and calculation complexity of the optimization process is under control. 3. RESULTS OF BASIC HEURISTIC / Rezultati osnovne heurističke metodeii Such planning tool offers a lot of possibilities for modeling to final result, that is firmly connected with knowledge of cargo officers. So they have to be well trained to use such optimization tool. 4. CONCLUSIONS / Zaključci The proposed algorithm shows ability to solve very demanding transportation problem with many loading/unloading ports and with various container contingents. In distribution of loaded containers from many small ports to home port it could be very useful to optimize efficiency of cargo space and reduction of transport costs, resulting in higher profit. In the same time the transport of empty containers has to be optimized, too. With such planning tool the cargo officers (on board or in small port) is supported with capable tool for decision making, to be able to satisfy traffic demands and easily adapt to its changes. Sometimes the transportation has to be the most profitable option, but not always. Figure 9 The main part of cargo are contingents that stay on ship longer (better ship’s occupancy), but contingents closer to destination (home) port are lower in amount. Problem of voyage direction can appear, but it can be solved with different port numeration Figure 9 The main part of cargo are contingents that stay on ship longer (better ship’s occupancy), but contingents closer to destination (home) port are lower in amount. Problem of voyage direction can appear, but it can be solved with different port numeration Also, the limited calculation power in shipping surrounding doesn’t support high algorithm complexity. So this approach can solve very huge problems in steps, consisting of successive iterations that can decrease the complexity to acceptable level. In the same time it ensures the cargo officers on board a very fine optimization tool, enabling many input values and leading the optimization process in wanted direction. 3. RESULTS OF BASIC HEURISTIC / Rezultati osnovne heurističke metodeii Only condition is that surplus of empty containers are waiting in one of previous ports. All information about potential load can be gathered through market research or from statistics of port authorities. Basically we have four cost types: freight cost (income), transport cost caused with distances, port taxes and cost of loading/discharging operation (transshipment cost). As the objective function is complex function, consisting of two or more non-linear cost functions (showing effect of economy of scale), it becomes very hard problem. Complexity depends on algorithm type we use [13]. If we have no freight cost, only transport and transshipment costs (including port taxes) influence on optimal solution. In that case we are looking for minimization of equitation (3.1). For simplicity in this research, all costs elements for any contingent and any port are equal, see [17 and 18]. Figure 7 Here we prioritize the transport of loaded containers, so we can see that contingent 4 (3-1) is much higher than be- fore. Now, the fourth contingent is transported in total amount, see difference on fig. 4 The objective function dm,m+1 can be formulated as follows:         − − − − ∑ ∑ + = = 1 1 , , , , , 1 ) , ( ) ( ) ( ) ( max M m m i m m i m i m m m i m i N i I W idle h x g d c I f (3.1) for m = 1, 2, ... , M+1; i = 1, 2, ... , N . Slika 7 Ovdje se potencira prijevoz punih kontejnera, tako je kontingent 4 (3-1) mnogo veći nego u prethodnom primjeru. Sada je četvrti kontingent prevezen u punom iznosu, u usporedbi sa slikom 4 As we can see from the equation (3.1) the objective function (total cost) includes some different costs. In fact, we have dual min-max problem. Freight cost is denoted with fi,m (Ii,m) and we 21 “Naše more” 65(1)/2018., pp. 18-23 Figure 10 Ship’s occupancy for third example is maximal Slika 10. Popunjenost broda je ovdje maksimalna Figure 8 Ship’s occupancy is closer to 100 % than in previous situation from figure 6 Slika 8. Popunjenost broda je bliža 100 % nego na slici 6 Figure 8 Ship’s occupancy is closer to 100 % than in previous situation from figure 6 Slika 8. REFERENCES / Literatura [12] Zhang X., Xiea F., Jiab R. (2012). Hybrid Genetic Algorithm for Minimum Saturated Flow in Emergency Network, IJACT: International Journal of Advancements in Computing Technology, Vol. 4, No. 21, pp. 133 - 144, https:// doi.org/10.4156/ijact.vol4.issue21.17 [1] Stopford, M. (2003). Maritime Economics, 2nd Edition, Routledge, London & New York [2] The Statistical Portal. Available at: https://www.statista.com/topics/1367/ container-shipping/ [13] Ouorou, A., Mahey, P., Vial, J.Ph. (2000). A Survey of Algorithms for Convex Multi- commodity Flow Problems, Markup Languages, Vol. 46, No. 1, pp. 126-147. [3] Lun, Y.H.V. and Browne M., (2009). Fleet mix in container shipping operations, International Journal of Shipping and Transport Logistics, 1(2): 103-118. https://doi.org/10.1504/IJSTL.2009.024491 [14] Castro, J., Nabona N. (1996). An Implementation of Linear and Nonlinear Multi-commodity Network Flows, European Journal of Operational Research, Vol. 92, No.1, pp. 37-53. https://doi.org/10.1016/0377-2217(95)00137-9 [4] Sys, C., Blauwens G, Omey E., Voorde V.D.E., Witlox F. (2008). In Search of the Link between Ship Size and Operations, Transportation Planning and Technology, 31:4, 435-463, https://doi.org/10.1080/03081060802335109 [15] Krile, S. (2005). Optimal Voyage Planning in Container Shipping, Proc. of 25th International Conference of Automation in Transportation (Korema), Zagreb– Copenhagen, pp. 32-35. [5] Kendall, P. M. (1972). The theory of optimal ship size, Journal of Transport Economies and Policy, VI (2), pp. 128_146. [16] Krile, S. (2011). Logistic Support for Loading/Unloading in Shipping with Multiple Ports / Logistika za ukrcaj i iskrcaj na plovidbi s više luka, Proc of. 31st International Conference of Automation in Transportation (Korema), Pula – Milan, pp. 94–97. [6] Xiea F., Jiab R., (2012). Nonlinear fixed charge transportation problem by minimum cost flow-based genetic algorithm, Computers & Industrial Engineering, Vol. 63, No. 4, pp. 763–778. https://doi.org/10.1016/j. cie.2012.04.016 [17] Krile, S., Krile, M. (2012). Better Profitability of Multi-Stop Flight Routes, Proc. of. International Conference of Automation in Transportation (Korema), Zagreb - Wien, pp. 97-100. [7] Yan S, Chen H.C, Chen Y.H, & Lou T.C. (2007). Optimal scheduling models for ferry companies under alliances. Journal of Marine Science and Technology, Vol. 15, No. 1, pp. 53-66. [18] Krile, S. (2013). Efficient Heuristic for Non-linear Transportation Problem on the Route with Multiple Ports, Polish Maritime Research, Gdansk, Poland, Vol. 20, No 4, pp. 80-86, DOI: 10.2478/pomr-2013-0044. https://doi.org/10.2478/ pomr-2013-0044 [8] Yan S. and Chen H.L. (2002). A scheduling model and a solution algorithm for inter-city bus carriers. Transportation Research Part A: Policy and Practice, Volume 36, No. 9, pp. 805-825. https://doi.org/10.1016/S0965-8564(01)00041-6 [19] Fleisher, L. (2000). port numeration Slika 9. Glavnina tereta su kontingenti koji putuju duže (bolja po- punjenost broda), ali su zato kontingenti kojima je polazište bliže krajnjem odredištu manje zastupljeni. Može se postaviti i problem smjera obilaska luka, što je samo stvar brojčanog označavanja luka 22 S. Krile and D. Mišković: Optimal Use of Container... [11] Zenzerovic, Z., Beslic, M. (2003). Contribution to the Optimization of the Cargo Transportation Problem, Promet - Traffic - Traffico, Vol. 15, No 1, Portorož, Trieste, Zagreb, pp. 13-17. REFERENCES / Literatura Approximating multi-commodity flow independent of the number of commodities, Siam J. Discrete Math. Vol. 13, No. 4, pp. 505-520. https://doi.org/10.1137/S0895480199355754 [9] Yang S, Tang C.H, & Lee M.C. (2007). A flight scheduling model for Taiwan airlines under market competitions. The International Journal of Management and Science, Vol. 35, No. 1, pp. 61-74. [20] Fonoberova, M., Lozovanu, D. (2004). The maximum flow in dynamic networks, Computer Science Journal of Moldova 3(36), pp. 387–396, [10] Zangwill I. W. l. (1968). “Minimum Concave Cost Flows in Certain Networks”, Management Science, Vol. 14, pp. 429-450. https://doi.org/10.1287/mnsc. 14.7.429 [21] Garaix, T., Artiques C., Feillet D., Josselin D., (2009). Vehicle routing problems with alternative paths: An application to on-demand transportation, European Journal of Operational Research. Volume 204, No. 1, pp. 62-75. https://doi.org/10.1016/j.ejor.2009.10.002 [11] Zenzerovic, Z., Beslic, M. (2003). Contribution to the Optimization of the Cargo Transportation Problem, Promet - Traffic - Traffico, Vol. 15, No 1, Portorož, Trieste, Zagreb, pp. 13-17. 23 “Naše more” 65(1)/2018., pp. 18-23
https://openalex.org/W2151416708	https://europepmc.org/articles/pmc3630176?pdf=render	English	null	Primary Breast Cancer Tumours Contain High Amounts of IgA1 Immunoglobulin: An Immunohistochemical Analysis of a Possible Carrier of the Tumour-Associated Tn Antigen	PloS one	2,013	cc-by	6,954	Abstract The Tn antigen (GalNAc alpha-O-Ser/Thr) as defined by the binding of the lectin, helix pomatia agglutinin (HPA) or anti-Tn monoclonal antibodies, is known to be exposed in a majority of cancers, and it has also been shown to correlate positively with the metastatic capacity in breast carcinoma. The short O-glycan that forms the antigen is carried by a number of different proteins. One potential carrier of the Tn antigen is immunoglobulin A1 (IgA1), which we surprisingly found in tumour cells of the invasive parts of primary breast carcinoma. Conventional immunohistochemical analysis of paraffin- embedded sections from primary breast cancers showed IgA1 to be present in the cytoplasm and plasma membrane of 35 out of 36 individual primary tumours. The immunohistochemical staining of HPA and anti-Tn antibody (GOD3-2C4) did to some extent overlap with the presence of IgA1 in the tumours, but differences were seen in the percentage of stained cells and in the staining pattern in the different breast cancers analysed. Anti-Tn antibody and HPA were also shown to specifically bind to a number of possible constellations of the Tn antigen in the hinge region of IgA1. Both reagents could also detect the presence of Tn positive IgA in serum. On average 51% of the tumour cells in the individual breast cancer tumour sections showed staining for IgA1. The overall amount of staining in the invasive part of the tumour with the anti Tn antibody was 67%, and 93% with HPA. The intra-expression or uptake of IgA1 in breast cancer makes it a new potential carrier of the tumour associated and immunogenic Tn antigen. Citation: Welinder C, Baldetorp B, Blixt O, Grabau D, Jansson B (2013) Primary Breast Cancer Tumours Contain High Amounts of IgA1 Immunoglobulin: An Immunohistochemical Analysis of a Possible Carrier of the Tumour-Associated Tn Antigen. PLoS ONE 8(4): e61749. doi:10.1371/journal.pone.0061749 Editor: Yves St-Pierre, INRS, Canada Received December 21, 2012; Accepted March 13, 2013; Published April 18, 2013 Copyright: 2013 Welinder et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the Mrs. Berta Kamprad Foundation and the Gunnar Nilsson Cancer Foundation and The Danish Agency for Science Technology and Innovation (FTP), EU FP7/2007-2013-EuroGlycoArrays 215536 and EU FP7-GlycoBioM. Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bo.jansson@med.lu.se Primary Breast Cancer Tumours Contain High Amounts of IgA1 Immunoglobulin: An Immunohistochemical Analysis of a Possible Carrier of the Tumour-Associated Tn Antigen Charlotte Welinder1, Bo Baldetorp1, Ola Blixt2, Dorthe Grabau3, Bo Jansson1* Charlotte Welinder1, Bo Baldetorp1, Ola Blixt2, Dorthe Grabau3, Bo Jansson1* 1 Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden, 2 Copenhagen Center for Glycomics, Department of Cellular & Molecular Medicine, University of Copenhagen, Copenhagen, Denmark, 3 Department of Pathology, Ska˚ne University Hospital, Lund, Sweden 1 Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden, 2 Copenhagen Center for Glycomics, Department of Cellular & Molecular Medicine, University of Copenhagen, Copenhagen, Denmark, 3 Department of Pathology, Ska˚ne University Hospital, Lund, Sweden Reagents and cell lines The monoclonal M4D8 anti-human IgA1 [12] was obtained from Margaret Goodall at The Division of Immunity & Infection University of Birmingham B15 2TT United Kingdom ., the anti- human poly-Ig receptor- (pIgR] biotinylated antibody BAF2717, from R&D Systems Europe Ltd (Abingdon, United Kingdom), and the negative control mouse IgG from Jacksson ImmunoR- esearch Europe Ltd (Suffolk, United Kingdom) . The anti-Tn monoclonal antibody GOD3-2C4 was produced in-house [10]. The biotinylated lectin, HPA, was purchased from EY Labora- tories, Inc. (San Mateo, CA, USA). T47D and MCF-7 breast carcinoma cell lines were obtained from the American Type Culture Collection (ATCC). No. IgA hinge glycopeptide 1 VPSTPPTPSPSTPPTPSPSA 2 VPSTPPTPSPSTPPTPSPSA 3 VPSTPPTPSPSTPPTPSPSA 4 VPSTPPTPSPSTPPTPSPSA 5 VPSTPPTPSPSTPPTPSPSA 6 VPSTPPTPSPSTPPTPSPSA 7 VPSTPPTPSPSTPPTPSPSA 8 VPSTPPTPSPSTPPTPSPSA 9 VPSTPPTPSPSTPPTPSPSA 10 VPSTPPTPSPSTPPTPSPSA 11 VPSTPPTPSPSTPPTPSPSA 12 VPSTPPTPSPSTPPTPSPSA 13 VPSTPPTPSPSTPPTPSPSA 14 VPSTPPTPSPSTPPTPSPSA 15 VPSTPPTPSPSTPPTPSPSA 16 VPSTPPTPSPSTPPTPSPSA 17 VPSTPPTPSPSTPPTPSPSA 18 VPSTPPTPSPSTPPTPSPSA 19 VPSTPPTPSPSTPPTPSPSA 20 VPSTPPTPSPSTPPTPSPSA 21 VPSTPPTPSPSTPPTPSPSA 22 VPSTPPTPSPSTPPTPSPSA 23 VPSTPPTPSPSTPPTPSPSA 24 VPSTPPTPSPSTPPTPSPSA 25 VPSTPPTPSPSTPPTPSPSA 26 VPSTPPTPSPSTPPTPSPSA 27 VPSTPPTPSPSTPPTPSPSA 28 VPSTPPTPSPSTPPTPSPSA 29 VPSTPPTPSPSTPPTPSPSA 30 VPSTPPTPSPSTPPTPSPSA 31 VPSTPPTPSPSTPPTPSPSA 32 VPSTPPTPSPSTPPTPSPSA 33 VPSTPPTPSPSTPPTPSPSA 34 VPSTPPTPSPSTPPTPSPSA 35 VPSTPPTPSPSTPPTPSPSA 36 VPSTPPTPSPSTPPTPSPSA 37 VPSTPPTPSPSTPPTPSPSA 38 VPSTPPTPSPSTPPTPSPSA 39 VPSTPPTPSPSTPPTPSPSA 40 VPSTPPTPSPSTPPTPSPSA 41 VPSTPPTPSPSTPPTPSPSA 42 VPSTPPTPSPSTPPTPSPSA 43 VPSTPPTPSPSTPPTPSPSA 44 VPSTPPTPSPSTPPTPSPSA 45 VPSTPPTPSPSTPPTPSPSA 46 VPSTPPTPSPSTPPTPSPSA Bold and S and T indicate O-glycosylation with Ga1NAc. doi:10.1371/journal.pone.0061749.t001 Immunohistochemistry Briefly, the tissue sections (4 mm) were de-paraffinized in xylene and rehydrated stepwise in ethanol and distilled water. Before staining, the sections were treated with antigen retrieval buffer (S1699, Dako, Glostrup, Denmark) in a 2100-Retriver (PickCell Laboratories, HistoLab, Va¨stra Fro¨lunda, Sweden). The slides were then allowed to cool for at least 20 min. An automated immunostainer (TechMate 500Plus, Dako) was used for the staining procedure: 30 minutes’ staining for the primary antibody M4D8 anti-human IgA1 (dilution 1:2000), the anti-human pIgR- biotinylated antibody (dilution 10 mg/mL), the negative control mouse IgG (dilution 10 mg/mL), GOD3-2C4 (dilution 10 mg/mL), biotinylated HPA (dilution 25 mg/mL) and the secondary antibody. Staining was visualized with the EnVision TM Detection system (K5001 for the biotinylated antibodies and K5007 for the other antibodies, Dako, Denmark). The slides were counterstained with haematoxylin. The percentage of invasive tumour cells stained in each slide was evaluated on a continuous scale (0–100%). Staining intensity was assessed semi-quantitatively: 0 = completely negative slide, 1 = weak, 2 = moderate and 3 = strong intensity. Magnifications ranging from 4 to 40 times were used during scoring. The histological grade was assessed according to Elston et al. [13]. The majority of the tumours were invasive ductal breast cancers. Sample 6 was classified as mucinous and sample 35 as tubular cancer. Samples 2 and 35 were recidiv, while the remainder of the tumours were primary lesions. Introduction usefulness of an anti-Tn antibody in passive immunotherapy has been illustrated in vivo with different animal models. Treatment with the anti Tn antibody GOD3-2C4 of SCID mice grafted with a human tumour cell line significantly reduced the growth rate of the tumor and when combined with cyclophosphamide another chimeric anti Tn antibody induced complete rejection of a murine mammary tumor in immune competent animals [10,11]. The Tn antigen CD175 is generally defined as (GalNAc alpha- O-Ser/Thr) or as a cluster of the same glycan. Tn antigen is the result of an abnormal O-glycosylation. Tumour-associated chang- es such as the Tn antigen and other changes in O-glycosylation have been found to be immunogenic and present on a variety of proteins, e.g. CD43 in T-cell leukaemia cells [1], MUC-1 in colon cancer [2], CD44 in breast carcinoma [3] and nucleolin in melanoma [4]. The majority of all carcinomas, 80–90%, are positive for the Tn antigen as defined by the lectin HPA. Furthermore, up-regulation of the Tn antigen in tumours is associated with poor prognosis [3,5,6,7]. Previously HPA affinity chromatography of a number of solubilised breast cancer tumours followed by SDS-PAGE and peptide sequencing have identified a major Tn-carrying 55 kDa protein in breast cancer metastatic tissue lysate as the heavy chain of IgA1 [8]. The O-glycosylation in IgA1is normally found in the hinge region of immunoglobulin, which may theoretically carry a maximum of nine O-glycosyla- tions and it makes IgA1 a potential carrier of Tn antigen and potential target for an anti-tumour response [9]. The therapeutic We have performed a short study that demonstrates high frequency of IgA1 positive cells in primary breast tumours. IgA1 was found to be present in both the cytoplasm and plasma membrane of 35 out of 36 individual breast cancer tumours The percentage and intensity of staining correlated to some extent with the staining intensity patterns of HPA and GOD-2C4 indicating, as expected, that IgA1 is not the only protein that carries the Tn antigen in the tumour. We also demonstrate in this study that HPA and anti Tn antibody GOD3-2C4 bind different glycoforms of the GalNAc alpha-O-Ser/Thr in the hinge region of IgA. April 2013 \| Volume 8 \| Issue 4 \| e61749 1 PLOS ONE \| www.plosone.org Breast Cancer Tumours Contain IgA1 Immunoglobulin Breast Cancer Tumours Contain IgA1 Immunoglobulin Table 1. The 44 different IgA hinge glycopeptides tested with Helix Pomatia Lectin and anti-Tn antibody. Microarray analysis The fine specificity of GOD3-2C4 and HPA for different Tn antigens was analysed using a glycopeptide array. The assay was performed as described previously [14], with a synthetic screening microarray platform with O-glycosylated 20-amino-acid sequences from the hinge region of the IgA. Briefly, the construction of the array is based on chemical solid-phase glycopeptide synthesis and selective the enrichment of defined glycopeptide on a hydrogel- coated microarray glass slide. Each slide contains the glycopep- tides given in Table 1. Peptide no.1 is the non-glycosylated peptide representing the background binding. Each slide was incubated for one hour with 10 mg/mL GOD3-2C4 or HPA in PBS, pH 7.4. The GOD3-2C4 antibody was detected with Cy3-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., (Suffolk, United Kingdom) diluted 1:1000. The biotinylated HPA was detected with streptavidin-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA; diluted 1:5000). All incubation steps were separated by two washing steps in PBS containing 0.05% Tween- 20 and one washing step in PBS. After the final washing step, the slides were rinsed in water and air dried. Finally, the slides were Bold and S and T indicate O-glycosylation with Ga1NAc. doi:10.1371/journal.pone.0061749.t001 April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org 2 Breast Cancer Tumours Contain IgA1 Immunoglobulin Table 2. Summary of Histological Immunohistochemical Results. Sample Hormone receptor Histology grade IgA1, % Int. pIgR, % Int. HPA, % Int. Tn, % Int. Microarray analysis 1 2 2 40 2 40 2 100 1 80 2 2 + 3 40 3 40 3 100 2 60 2 3 + 3 30 1 1 1 100 2 90 3 4 + 1 80 3 60 1 100 2 100 2 5 + 2 60 3 0 0 50 3 30 3 6 + 1 5 2 0 0 100 2 0 0 7 2 2 80 3 20 2 100 1 10 1 8 + 3 50 3 80 3 100 3 70 3 9 + 3 20 3 10 3 90 2 60 2 10 + 1 20 3 70 3 90 1 20 2 11 2 1 30 2 10 3 100 1 10 3 12 + 3 2 1 5 3 100 3 70 2 13 + 2 70 2 60 2 100 3 90 2 14 + 3 80 3 0 0 80 1 70 1 15 + 1 90 3 50 2 100 3 100 2 16 + 1 50 2 20 1 100 2 90 2 17 + 1 30 3 1 1 90 2 30 3 18 + 2 50 2 40 1 50 1 30 2 19 + 2 10 1 30 3 100 3 80 3 20 + 3 40 2 1 1 100 3 100 3 21 2 3 1 2 0 0 60 2 20 2 22 + 1 50 2 50 2 100 3 90 3 23 + 2 80 3 60 1 100 3 100 3 24 + 1 60 2 30 2 100 2 100 2 25 + 2 80 2 10 1 100 2 30 2 26 + 1 50 1 5 1 80 1 90 1 27 + 3 10 1 20 2 100 3 70 3 28 2 2 100 3 5 1 100 3 100 3 29 2 1 70 2 60 1 100 1 50 2 30 2 3 95 2 20 2 100 3 90 2 31 + 2 60 2 80 1 100 2 100 2 32 + 3 90 2 10 1 90 1 1 1 33 + 2 0 0 0 0 100 2 100 1 34 0 3 90 2 30 3 100 2 100 2 35 + 1 50 2 20 3 80 1 90 1 36 + 3 70 3 40 2 100 3 90 3 Intensity = Int., Hormone receptor = Oestrogen and progesterone receptors. Microarray analysis doi:10 1371/journal pone 0061749 t002 Intensity = Int., Hormone receptor = Oestrogen and progesterone receptors. doi:10.1371/journal.pone.0061749.t002 96-well test plates (Lumitrac 600, Greiner-Bio One, Frickenhau- sen, Germany) were coated with 50 mL of 2 mg/mL antibody or lectin in 0.1 M sodium carbonate buffer, pH 9 at +4uC overnight. All following washing steps and dilution of reagents were performed with PBS containing 0.05% Tween 20. The plates were washed and then incubated for one hour with 50 mL of diluted (1:5) conditioned culture supernatants, medium or serum. After washing, bound IgA1 antibody was detected with HRP- conjugated rabbit anti-IgA (P-0216; DAKO). After the final washing step the bound antibody was visualized with SuperSignal Femto Maximum substrate for chemiluminescence for ELISA detection (Thermo Scientific, USA). The plates were read on a Wallac Victor II Fluorescence plate reader (PerkinElmer, USA). scanned (Pro Scan Array HT Microarray, Perkin Elmer Life and Analytical Sciences, MA, USA) and analysed using image analysis software (Scan Array Express, v 3.0, Perkin Elmer Life and Analytical Sciences). Cell culturing The human carcinoma cell lines MCF-7 and T47D were purchased from the ATCC (Rockville, MD, USA) and cultivated at 37uC in RPMI 1640 supplemented with 10% FCS. ELISA Binding to IgA was tested with a sandwich ELISA using HPA, GOD3-2C4 or polyclonal anti-IgA as a catcher antibody. White April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org 3 Breast Cancer Tumours Contain IgA1 Immunoglobulin Figure 1. Immunohistochemical staining of breast tumour specimens showing the presence of IgA1 in the invasive part of the tumour. A) In Sample 2, 40% of the tumour cells in the invasive part were stained with anti-IgA1 with a relative intensity of 3. Strong cytoplasmic and plasma membrane staining of IgA1 is observed in the invasive part of the section (INV) but only very weak staining in the in situ part (INSU). B) In Sample 7, 80% of the cells were IgA1-positive with a relative intensity of 3. doi:10.1371/journal.pone.0061749.g001 Figure 1. Immunohistochemical staining of breast tumour specimens showing the presence of IgA1 in the invasive part of the tumour. A) In Sample 2, 40% of the tumour cells in the invasive part were stained with anti-IgA1 with a relative intensity of 3. Strong cytoplasmic and plasma membrane staining of IgA1 is observed in the invasive part of the section (INV) but only very weak staining in the in situ part (INSU). B) In Sample 7, 80% of the cells were IgA1-positive with a relative intensity of 3. doi:10.1371/journal.pone.0061749.g001 IgA1, HPA and GOD3-2C4, showed overlapping staining of breast cancer tumour cells, but with clear differences in intensity, proportion and inter-cellular distribution between different breast cancer tumour sections. The sections also stained positive for pIgR, one of the receptors for IgA. The majority of the invasive tumour sections stained positive for both IgA1 and pIgR, but there was no obvious correlation between the frequency of expression. Two tumours stained negative for pIgR but still stained intensively for IgA1 (Samples 5 and 14 in Table 2). Examples of staining patterns for IgA1, HPA, GOD3-2C4 and pIgR are shown for three different breast cancer tumour samples in Figures 3, 4, 5, 6, 7, 8. The percentage staining for IgA1 was 40% in the invasive part of the tumour in Sample 1, 0% in Sample 33 and 95% in Sample 30 (Figures 3A, 5A and 7A). Forty percent of the cells were stained for IgA1 and pIgR in tumour Sample 1 (Figures 3A and 4B). The IgA1-negative tumour (Sample 33) showed no staining for pIgR (Figures 5A and 6B). Ethics statement This study was approved by the Ethics Committee of Lund University (LU 240-01). ELISA Ninety-five percent of Sample 30 was stained for IgA1, but much less for pIgR, being only 20% (Figures 7A and 8B). HPA and GOD3-2C4 staining seems to be similar to each other in the three different breast cancer tumour samples shown in Figures 3B, 4A, 5B, 6A, 7B and 8A. However, this was not the case for all breast cancer tumour samples analysed (see Table 2). A majority of the invasive tumour cells showed high intensity staining with HPA, while less intensity and frequency was seen with the monoclonal GOD3-2C4 antibody. Results Paraffin sections from 36 different breast cancer tumours were stained for IgA1, Tn antigen, pIgR and HPA. A summary of the results is presented in Table 2. The percentage and intensity of positive staining of tumour cells in the invasive part of each tissue section was analysed. Immunostaining with M4D8 (for IgA1), HPA, pIgR and GOD3-2C4 (for Tn) is compared with non- binding mouse Ig. The percentage of stained invasive tumour cells was evaluated on a continuous scale (0–100%), while the relative intensity of tissue staining was classified as 0–3. Positive staining for IgA1 was seen in the majority of the breast cancer sections. Sections morphologically classified as invasive were more intensely stained than other parts classified as cancer in situ (Figure 1A). Figures 1 and 2 illustrates four different breast cancer tumour samples stained with anti-IgA1, showing different intensity and amounts of stained cancer cells in the invasive part of the tumour. The percentage of IgA1-positive cells ranged from 0– 100%, with an overall average of 51%. Binding was frequently seen in both the cytoplasm and plasma membrane of the breast cancer tumour cells, as can be seen in Figures 1 and 2. Breast cancer cells in the invasive part also stained positive for binding of HPA and GOD3-2C4, to varying degrees. All three reagents, anti The anti-Tn antibody, GOD3-2C4, and HPA were also tested for specific binding to 46 different glycoforms of the Tn antigen Figure 2. Immunohistochemical staining of specimens from individual breast cancer tumours showing the presence of IgA1 in both lymphocytes and tumour cells. A) A section showing weak positive staining of cancer cells (Can) and intensively stained lymphocytes (lym). B) Intense anti- IgA1 staining of cancer cells in Sample 28, an ER/PGR-negative tumour in which 100% of the invasive part of the section was regarded as being maximally stained for IgA1 with an intensity of 3. doi:10.1371/journal.pone.0061749.g002 Figure 2. Immunohistochemical staining of specimens from individual breast cancer tumours showing the presence of IgA1 in both lymphocytes and tumour cells. A) A section showing weak positive staining of cancer cells (Can) and intensively stained lymphocytes (lym). B) Intense anti- IgA1 staining of cancer cells in Sample 28, an ER/PGR-negative tumour in which 100% of the invasive part of the section was regarded as being maximally stained for IgA1 with an intensity of 3. Results doi:10.1371/journal.pone.0061749.g002 April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 4 Breast Cancer Tumours Contain IgA1 Immunoglobulin Figure 3. Intermediate staining percentage of IgA1-positive tumour cells in Sample 1 compared to staining for HPA. Scale bar = 50 mm A) Anti-IgA1, B) HPA. doi:10.1371/journal.pone.0061749.g003 Figure 3. Intermediate staining percentage of IgA1-positive tumour cells in Sample 1 compared to staining for HPA. Scale bar = 50 mm A) Anti-IgA1, B) HPA. doi:10.1371/journal.pone.0061749.g003 expressed on a microarray platform. Various O-glycoforms of the 20-amino-acid IgA1 heavy-chain hinge region peptide, VPSTPPTPSPSTPPTPSPSA were tested. GOD3-2C4 shows very selective binding to peptides expressing two or more adjacent GalNAc alpha-O-Ser/Thr carbohydrate epitopes while HPA has a much broader binding pattern allowing binding to single GalNAc alpha-O-Ser/Thr carbohydrate epitopes (Table 1 and Figure 9). Using different sandwich ELISAs, we were also able to show that some healthy blood donors have Tn-positive IgA in the circulation. However, no IgA was detected in culture supernatant from two different breast cancer cell lines (Figure 10). expressed on a microarray platform. Various O-glycoforms of the 20-amino-acid IgA1 heavy-chain hinge region peptide, VPSTPPTPSPSTPPTPSPSA were tested. GOD3-2C4 shows very selective binding to peptides expressing two or more adjacent GalNAc alpha-O-Ser/Thr carbohydrate epitopes while HPA has a much broader binding pattern allowing binding to single GalNAc alpha-O-Ser/Thr carbohydrate epitopes (Table 1 and Figure 9). Using different sandwich ELISAs, we were also able to show that some healthy blood donors have Tn-positive IgA in the circulation. However, no IgA was detected in culture supernatant from two different breast cancer cell lines (Figure 10). or uptake in invasive primary breast cancer cells seems to be a frequent phenomenon. The presence of IgA in tumour sections was confirmed in a small number of tissue sections with a polyclonal anti human IgA reagents (data not shown) There may be different explanations of the enrichment of IgA1 in tumour cells, such as specific binding of the antibody to tumour cells [19] or the active uptake of IgA1. The biological functions of immunoglobulin IgA1 antibodies depend primarily on their interaction with cell surface receptors, and several cancer cell receptors are available for binding and internalization of IgA1. Fc aRI (CD89), poly-IgR, Fc a/mR, asialo-glycoprotein receptor and the transferrin receptor [20] all have the capacity to transfer IgA1 into the cell. Results For some of the breast cancer tumours studied here, a correlation was seen between the staining intensity (Figures 3A and 4B), or lack of staining (Figures 5A and 6B), between pIgR and IgA1 in the cancer cells. Some of the breast cancer tumours stained intensively for IgA1 but much weaker for pIgR (an example of such a tumour is shown in Figures 7A and 8B). This could indicate there is at least two different receptors involved or that the pIgR is down regulated. Discussion Tn antigen expression is correlated with poor prognosis regarding the metastatic potential of breast cancer [15]. CD44 and MUC-1 are already known to be Tn-positive proteins in breast cancer [3]. These proteins play a role in adhesion and/or migration, and it has been suggested that changes in their O- glycosylation might influence the function and mobility of cancer cells [16]. Streets et al. [8] found a dominating 55-kDa band in SDS-PAGE analysis after HPA affinity chromatography of lysate from metastatic breast cancer tissue. The 55-kDa band was identified as the heavy chain of IgA1. They also reported that IgA1 extracted from normal control tissues bound much less to HPA. Increased concentrations in serum of human IgG, IgM and IgA have been reported in patients with epithelial carcinomas [17], and also the occasional intra-cellular presence of secretory component and IgA in breast carcinoma [18] but no conclusive data has been presented. However, when applying a conventional immunohistochemical technique we found very high amounts of IgA1 in the majority of breast cancer tissues examined. Thirty five individual samples out of thirty six tested were positive. The breast cancer tumour cells in the invasive parts of the tumour were more frequently IgA1-positive than those in the in situ parts of the tumour (Figure 1A). Based on our observations, IgA1 expression A third explanation for the uptake of IgA1 may be the reported capacity of epithelial cancer cells to express endogenous immu- noglobulin [21]. There are a number of publications on the potential of cancer cell lines to produce immunoglobulin and other B-cell-associated proteins [21]. According to one study, cancer cell lines have the capacity to express heavy-chain IgA1 [22]. In previous work using the highly sensitive RT-nested PCR method it was shown that some cancer cell lines transcribe both immuno- globulin and T-cell receptor genes [23]. Since then, there have been reports of several cancer cell lines that express immunoglob- ulin alpha chains, both in the cytoplasm and in secreted form in the cultured supernatants of cancer cell lines [22]. Transcription of the immunoglobulin A1 heavy chain (SNC73), together with the light k and l chains, has also been detected with RT-nested PCR and immunohistochemistry in human epithelium-derived tumour cells, including the breast carcinoma cell line MCF-7 [24]. Using a Figure 4. Staining with anti-Tn and anti-pIgR in Sample 1. Scale bar = 50 mm. A) Anti-Tn, B) Anti-pIgR. doi:10.1371/journal.pone.0061749.g004 Figure 4. Discussion Alternatively the major variety of techniques such as immunohistochemical analysis, in situ hybridization and laser capture micro-dissection, Qiu et al. have demonstrated that established epithelial cancer lines including breast cancer can produce IgG in both cytoplasmic and secreted forms [25]. However, some of these results are in contrast to observations made by other researchers. When epithelial cancer cells were analysed after being sorted with FACS as EpCAM + cells from cultured cancer cell lines they were found positive for Ig mRNA but no Ig protein expression could be detected in flow cytometry, indicating very low protein expression of IgA [26]. We have obtained similar results when no IgA could be detected in any of the fixed permeabilized tumour cell line tested using flow cytometry (data not shown). We could neither detect any IgA protein in the supernatants from the cultured MCF-7 or T47D cell lines (Figure 10), as has been claimed previously [27]. Perhaps are cultivation conditions and the use of very specific sub clones of cancer cells lines very critical for the production of an efficient amounts of IgA1 in vitro and the most optimal conditions are only met in some parts of the tumour ‘‘in vivo’’. Alternatively the major p g p Human IgA1 contains both N- and O-glycosylation sites and carries nine potential O-glycosylation sites in its heavy-chain hinge region, but only a maximum of five sites are believed to be glycosylated [5,9,28]. Each attached O-glycan has a core of GalNAc alpha-O-Ser/Thr (Tn antigen) typically linked to galactose and one or two sialic acid residues shielding the Tn epitope. Abnormally glycosylated IgA1, e.g. Tn-positive IgA1, is known to play a part in autoimmune diseases such as IgA nephropathy [29]. In patients with IgA nephropathy abnormalities in O-glycan biosynthesis result in exposure of the immunogenic Tn antigen by auto-antibodies, resulting in immune complex formation and deposition in the kidneys, leading to kidney failure [29] and it is tempting to think the expression of aberrant O- glycosylation on IgA1 in a similar way could constitute the target of an anti-tumour immune response [30]. It has also been suggested by that the presence of immuno- globulins could be advantageous for a tumour cell. Li et al. [27] reported that transfecting MCF-7 cells with small interfering RNA Figure 7. High percentage of IgA1-positive tumour cells compared with staining with HPA in tumour Sample 30. Scale bar = 50 mm. Discussion Staining with anti-Tn and anti-pIgR in Sample 1. Scale bar = 50 mm. A) Anti-Tn, B) Anti-pIgR. doi:10.1371/journal.pone.0061749.g004 April 2013 \| Volume 8 \| Issue 4 \| e61749 April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Breast Cancer Tumours Contain IgA1 Immunoglobulin Figure 5. Very low percentage of IgA1-positive tumour cells in tumour Sample 33. Scale bar = 50 mm. A) Anti.IgA1, B) HPA. doi:10.1371/journal.pone.0061749.g005 Figure 5. Very low percentage of IgA1-positive tumour cells in tumour Sample 33. Scale bar = 50 mm doi:10.1371/journal.pone.0061749.g005 percentage of IgA1-positive tumour cells in tumour Sample 33. Scale bar = 50 mm. A) Anti.IgA1, B) HPA. one.0061749.g005 Figure 5. Very low percentage of IgA1-positive tumour cells in tumour Sample 33. Scale bar = 50 mm. A) Anti.IgA1, B) HPA. doi:10.1371/journal.pone.0061749.g005 Figure 6. Staining with anti-Tn and anti-pIgR in Sample 33. Scale bar = 50 mm. A) Anti-Tn, B) Anti-pIgR. doi:10.1371/journal.pone.0061749.g006 Figure 6. Staining with anti-Tn and anti-pIgR in Sample 33. Scale bar = 50 mm. A) Anti-Tn, B) Anti-pIgR. doi:10.1371/journal.pone.0061749.g006 part of the IgA1 seen in the tumour cells originates from the tumour uptake of surrounding proteins. variety of techniques such as immunohistochemical analysis, in situ hybridization and laser capture micro-dissection, Qiu et al. have demonstrated that established epithelial cancer lines including breast cancer can produce IgG in both cytoplasmic and secreted forms [25]. However, some of these results are in contrast to observations made by other researchers. When epithelial cancer cells were analysed after being sorted with FACS as EpCAM + cells from cultured cancer cell lines they were found positive for Ig mRNA but no Ig protein expression could be detected in flow cytometry, indicating very low protein expression of IgA [26]. We have obtained similar results when no IgA could be detected in any of the fixed permeabilized tumour cell line tested using flow cytometry (data not shown). We could neither detect any IgA protein in the supernatants from the cultured MCF-7 or T47D cell lines (Figure 10), as has been claimed previously [27]. Perhaps are cultivation conditions and the use of very specific sub clones of cancer cells lines very critical for the production of an efficient amounts of IgA1 in vitro and the most optimal conditions are only met in some parts of the tumour ‘‘in vivo’’. Discussion A) Anti-IgA1, B) HPA. doi:10.1371/journal.pone.0061749.g007 Figure 7. High percentage of IgA1-positive tumour cells compared with staining with HPA in tumour Sample 30. Scale bar = 50 mm. A) Anti-IgA1, B) HPA. doi:10.1371/journal.pone.0061749.g007 April 2013 \| Volume 8 \| Issue 4 \| e61749 April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Breast Cancer Tumours Contain IgA1 Immunoglobulin Figure 8. Staining with anti-Tn and anti-pIgR in Sample 30. Scale bar = 50 mm A) Anti-Tn, B) Anti-pIgR. doi:10.1371/journal.pone.0061749.g008 Figure 8. Staining with anti-Tn and anti-pIgR in Sample 30. Scale bar = 50 mm A) Anti-Tn, B) Anti-pIgR. doi:10.1371/journal.pone.0061749.g008 (siRNA) blocking the production of Ig inhibited their growth, and that the presence of cancerous Ig specifically reduced antibody- dependent cell-mediated cytotoxicity induced by an anti-human EGF receptor antibody in a dose-dependent manner, suggesting that tumour-associated Ig has a protective role. Blocking of the cancer derived IgA have been shown to suppress growth and viability of cancer cells [22]. Furthermore, the blocking of this tumour-derived IgG increased programmed cell death and inhibited tumour growth in vitro and in xeno-transplants in vivo [25].The observation that breast cancer cells contain high amounts of IgA1 in vivo needs further investigations of the origin, clonality and significance of this tumour associated immunoglob- ulin. As IgA1 is a potential carrier of the Tn antigen it may provide a target or a blocking decoy for antibody-based therapy. GOD3-2C4 is monoclonal antibody specific to the Tn antigen [10] and it preferentially binds adjacent GalNAc alpha-O-Ser/ Thr epitopes in the hinge region of IgA1, but its binding pattern in the array also indicates a preference for some amino acid sequences, indicated by its specificity for inner cluster (glycopep- tides 16, 28, 29, 30, 31, 41, 42, 44, 45) but not flanking regions (glycopeptide 23, 24 and 34), (Figure 9B). GOD3-2C4 also binds different known Tn-positive proteins from different cancer cell lines, e.g. CD44 and mucins (data not shown). The difference between the reagents is also seen in the immunohistochemistry staining patterns of HPA and GOD3-2C4 Figure 9. Helix Pomatia Lectin and GOD3-2C4 (anti-Tn antibody) glycopeptide array. Relative fluorescence signal (RFU). The mean value of five individual measurements is given for each glycopeptide. The numbers of each specific Tn peptide are listed in Table 1. A) HPA, B) GOD3-2C4. doi:10.1371/journal.pone.0061749.g009 Figure 9. Helix Pomatia Lectin and GOD3-2C4 (anti-Tn antibody) glycopeptide array. Acknowledgments Thanks to Kristina Lo¨fgren and Dr. Emiliano Clo for technical assistance 10. Welinder C, Baldetorp B, Borrebaeck C, Fredlund BM, Jansson B (2011) A new murine IgG1 anti-Tn monoclonal antibody with in vivo anti-tumor activity. Glycobiology 21: 1097–1107. Conclusions [ ] Both HPA and GOD3-2C4 bind the Tn antigen on the IgA1 hinge region and in the case of GOD3-2C4 it is clear that clustered bis-GalNAc structure are preferred. HPA has of course a broader reaction pattern. The observation that glycopeptide 20 and 29 are negative might be experimental artifact and has to be examined further. Although these peptides are glycosylated, it might very well be that the glycan structure is sterically hindered by an un-favourable conformation. A recent publication noticed a similar situation for HPA microarray experiment to IgA hinge glycopeptide (Fig. 4 in reference [31]). Both reagents also recognize a portion of the circulating IgA1 proteins in healthy blood donors. The cytosol and plasma membrane of invasive breast cancer cells frequently contain IgA1, a carrier of the immunogenic Tn antigen. The origin and possible function of the observed tumour- associated IgA1 are unknown, but its relatively high abundance makes it an interesting biomarker and potential therapeutic target 9. Mattu TS, Pleass RJ, Willis AC, Kilian M, Wormald MR, et al. (1998) The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fc alpha receptor interactions. J Biol Chem 273: 2260–2272. Author Contributions The anti-Tn antibody has in vitro and in vivo effects on the growth of tumours, and GOD3-2C4 was the first anti-Tn antibody to show an in vivo reduction of growth of a xeno-transplanted solid tumour [10]. A more dramatic and convincing therapeutic effect Conceived and designed the experiments: CW BJ OB BB. Performed the experiments: CW OB DG BJ. Analyzed the data: CW OB DG BJ BB. Contributed reagents/materials/analysis tools: CW OB DG BJ BB. Wrote the paper: CW BJ. Discussion Relative fluorescence signal (RFU). The mean value of five individual measurements is given for each glycopeptide. The numbers of each specific Tn peptide are listed in Table 1. A) HPA, B) GOD3-2C4. doi:10.1371/journal.pone.0061749.g009 April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org 7 7 Breast Cancer Tumours Contain IgA1 Immunoglobulin Figure 10. Results of sandwich IgA ELISA of human serum and cultivation supernatant from dense cultures of MCF-7 and T47D breast carcinoma cell lines. Serum or conditioned culture supernatant from two different breast carcinoma cell lines (MCF-7 sup. and T47D sup.) were incubated in test plates coated with HPA, GOD3-2C4 (Tn) or polyclonal anti-IgA antibody (IGA). A polyclonal anti-IgA HRP conjugated antibody was used for detection, and the results are presented as the quotient between a coated non-binding control and the relevant catcher reagent. Mouse serum (CTRL) and fresh RPMI 1640 cultivation medium (Medium) were used as negative controls. doi:10.1371/journal.pone.0061749.g010 Figure 10. Results of sandwich IgA ELISA of human serum and cultivation supernatant from dense cultures of MCF-7 and T47D breast carcinoma cell lines. Serum or conditioned culture supernatant from two different breast carcinoma cell lines (MCF-7 sup. and T47D sup.) were incubated in test plates coated with HPA, GOD3-2C4 (Tn) or polyclonal anti-IgA antibody (IGA). A polyclonal anti-IgA HRP conjugated antibody was used for detection, and the results are presented as the quotient between a coated non-binding control and the relevant catcher reagent. Mouse serum (CTRL) and fresh RPMI 1640 cultivation medium (Medium) were used as negative controls. doi:10.1371/journal.pone.0061749.g010 was seen with the Tn-antigen-specific chimeric monoclonal antibody (Chi-Tn, originally denoted 83D4) in a syngeneic breast cancer tumour when combined with cyclophosphamide [11]. which did not always overlap in the breast cancer samples (Table 2). This could be explained by the difference in fine specificity of the two reagents (Figure 9), but also because HPA is known to bind blood group A, while GOD3-2C4 is not cross reactive [10]. 5. Dwek MV, Ross HA, Streets AJ, Brooks SA, Adam E, et al. (2001) Helix pomatia agglutinin lectin-binding oligosaccharides of aggressive breast cancer. Int J Cancer Mar 20;95(2):79–85. 1. Ando H, Matsushita T, Wakitani Masako, Sato Takashi, Kodama-Nishida S, et al. (2008) Mouse-Human Chimeric Anti-Tn IgG1 Induced Anti-tumor Activity against Jurkat Cells in Vitro and in Vivo. Biol Pharm Bull 31:1739–44 2. Freire T, Medeiros A, Reis CA, Real FX, Osinaga E (2003) Biochemical characterization of soluble Tn glycoproteins from malignant effusions of patients with carcinomas. Oncol Rep 10:1577–85. 8. Streets AJ, Brooks SA, Dwek MV, Leathem AJ (1996) Identification, purification and analysis of a 55 kDa lectin binding glycoprotein present in breast cancer tissue. Clin Chim Acta 254: 47–61. 3. Cazet A, Julien S, Bobowski M, Burchell J, Delannoy P (2010) Tumour- associated carbohydrate antigens in breast cancer. Breast Cancer Res 12: 204. 7. Brooks SA, Hall DM, Buley I (2001) GalNAc glycoprotein expression by breast cell lines, primary breast cancer and normal breast epithelial membrane. Br J Cancer 85(7):1014–22. 6. Leathem AJ, Brooks SA (1987) Predictive value of lectin binding on breast- cancer recurrence and survival. Lancet 1(8541):1054–6 Breast Cancer Tumours Contain IgA1 Immunoglobulin Breast Cancer Tumours Contain IgA1 Immunoglobulin 11. Hubert P, Heitzmann A, Viel S, Nicolas A, Sastre-Garau X, et al. (2011) Antibody-dependent cell cytotoxicity synapses form in mice during tumor- specific antibody immunotherapy. Cancer Res 71: 5134–5143. 22. Zheng H, Li M, Ren W, Zeng L, Liu HD, et al. (2007) Expression and secretion of immunoglobulin alpha heavy chain with diverse VDJ recombinations by human epithelial cancer cells. Mol Immunol 44: 2221–2227. 12. Farris MA, Hardie D, de Lange G, Jefferis R (1985) Immunogenic and antigenic epitopes of immunoglobulins. X: Monoclonal antibodies specific for human IgA, the IgA1 and IgA2 subclasses and an nA2m(2) iso-allotypic epitope. Vox Sang 48(2):116–21. p 23. Kimoto Y (1998) Expression of heavy-chain constant region of immunoglobulin and T-cell receptor gene transcripts in human non-hematopoietic tumor cell lines. Genes Chromosomes Cancer 22: 83–86. 24. Geng LY, Shi ZZ, Dong Q, Cai XH, Zhang YM, et al. (2007) Expression of SNC73, a transcript of the immunoglobulin alpha-1 gene, in human epithelial carcinomas. World J Gastroenterol 13: 2305–2311. ( ) 13. Elston CW, Ellis IO (1991) Pathological Prognostic Factors in Breast Cancer. I. The Value of Histological Grade in Breast Cancer: Experience From a Large Study With Long-Term Follow-Up. Histopathology 19: 403–10 25. Qiu X, Zhu X, Zhang L, Mao Y, Zhang J, et al. (2003) Human epithelial cancers secrete immunoglobulin g with unidentified specificity to promote growth and survival of tumor cells. Cancer Res 63: 6488–6495. 14. Blixt O, Head S, Mondala T, Scanlan C, Huflejt ME, et al. (2004) Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci USA 101: 17033–17038. 26. Babbage G, Ottensmeier CH, Blaydes J, Stevenson FK, Sahota SS (2006) Immunoglobulin heavy chain locus events and expression of activation-induced cytidine deaminase in epithelial breast cancer cell lines. Cancer Res 66: 3996– 4000. 15. Ju T, Otto VI, Cummings RD (2011) The Tn antigen-structural simplicity and biological complexity. Angew Chem Int Ed Engl 50: 1770–1791. 16. Julien S, Adriaenssens E, Ottenberg K, Furlan A, Courtand G, et al. (2006) ST6GalNAc I expression in MDA-MB-231 breast cancer cells greatly modifies their O-glycosylation pattern and enhances their tumourigenicity. Glycobiology 16: 54–64. 27. Li M, Zheng H, Duan Z, Liu H, Hu D, et al. (2011) Promotion of cell proliferation and inhibition of ADCC by cancerous immunoglobulin expressed in cancer cell lines. Cell Mol Immunol 9:54–61 17. References 1. Ando H, Matsushita T, Wakitani Masako, Sato Takashi, Kodama-Nishida S, et al. (2008) Mouse-Human Chimeric Anti-Tn IgG1 Induced Anti-tumor Activity against Jurkat Cells in Vitro and in Vivo. Biol Pharm Bull 31:1739–44 6. Leathem AJ, Brooks SA (1987) Predictive value of lectin binding on breast- cancer recurrence and survival. Lancet 1(8541):1054–6 7. Brooks SA, Hall DM, Buley I (2001) GalNAc glycoprotein expression by breast cell lines, primary breast cancer and normal breast epithelial membrane. Br J Cancer 85(7):1014–22. 2. Freire T, Medeiros A, Reis CA, Real FX, Osinaga E (2003) Biochemical characterization of soluble Tn glycoproteins from malignant effusions of patients with carcinomas. Oncol Rep 10:1577–85. 8. Streets AJ, Brooks SA, Dwek MV, Leathem AJ (1996) Identification, purification and analysis of a 55 kDa lectin binding glycoprotein present in breast cancer tissue. Clin Chim Acta 254: 47–61. 3. Cazet A, Julien S, Bobowski M, Burchell J, Delannoy P (2010) Tumour- associated carbohydrate antigens in breast cancer. Breast Cancer Res 12: 204. 4. Hoja-Lukowicz D, Przybylo M, Pochec E, Drabik A, Silberring J, et al. (2009) The new face of nucleolin in human melanoma. Cancer Immunol Immunother 58: 1471–1480. 5. Dwek MV, Ross HA, Streets AJ, Brooks SA, Adam E, et al. (2001) Helix pomatia agglutinin lectin-binding oligosaccharides of aggressive breast cancer. Int J Cancer Mar 20;95(2):79–85. April 2013 \| Volume 8 \| Issue 4 \| e61749 April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org 8 Breast Cancer Tumours Contain IgA1 Immunoglobulin Roberts MM, Bathgate EM, Stevenson A (1975) Serum immunoglobulin levels in patients with breast cancer. Cancer 36: 221–224. 28. Takahashi K, Smith IV AD, Poulsen K, Kilian M, Julian BA, et al.(2012) Naturally Occurring Structural Isomers in Serum IgA1 O-Glycosylation. J Proteome Res 11: 692–702. 18. 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(2001) Identification of the transferrin receptor as a novel immunoglobulin (Ig) A1 receptor and its enhanced expression on mesangial cells in IgA nephropathy. J Exp Med 194: 417–425. 31. Borgert A, Heimburg-Molinaro J, Song X, Lasanajak Y, Ju T, et al. (2012) Deciphering Structural Elements of Mucin Glycoprotein Recognition. ACS Chem Biol 7:1031–1039. 21. Chen Z, Qiu X, Gu J (2009) Immunoglobulin expression in non-lymphoid lineage and neoplastic cells. Am J Pathol 174: 1139–1148. PLOS ONE \| www.plosone.org April 2013 \| Volume 8 \| Issue 4 \| e61749 PLOS ONE \| www.plosone.org 9
https://openalex.org/W2713744421	https://threatenedtaxa.org/index.php/JoTT/article/download/2730/4052	English	null	<b>Trematode infestation in coral colonies at Poshitra Reef, Gulf of Kachchh Marine National Park, Gujarat, India</b>	Journal of threatened taxa	2,017	cc-by	2,741	The Journal of Threattened Taxa fis dedficatted tto bufildfing evfidence for conservafion globally by publfishfing peer-revfiewed arficles onlfine every montth att a reasonably rapfid ratte att www .tthreattenedttaxa.org. All arficles publfished fin JoTT are regfisttered under Creafive Commons Attrfibufion 4.0 Intternafional Lficense unless ottherwfise menfioned. JoTT allows unresttrfictted use of arficles fin any medfium, reproducfion, and dfisttrfibufion by provfidfing adequatte credfitt tto tthe autthors and tthe source of publficafion. The Journal of Threattened Taxa fis dedficatted tto bufildfing evfidence for conservafion globally by publfishfing peer-revfiewed arficles onlfine every montth att a reasonably rapfid ratte att www .tthreattenedttaxa.org. All arficles publfished fin JoTT are regfisttered under Creafive Commons Attrfibufion 4.0 Intternafional Lficense unless ottherwfise menfioned. JoTT allows unresttrfictted use of arficles fin any medfium, reproducfion, and dfisttrfibufion by provfidfing adequatte credfitt tto tthe autthors and tthe source of publficafion. The Journal of Threattened Taxa fis dedficatted tto bufildfing evfidence for conservafion globally by publfishfing peer-revfiewed arficles onlfine every montth att a reasonably rapfid ratte att www .tthreattenedttaxa.org. All arficles publfished fin JoTT are regfisttered under Creafive Commons Attrfibufion 4.0 Intternafional Lficense unless ottherwfise menfioned. JoTT allows unresttrfictted use of arficles fin any medfium, reproducfion, and dfisttrfibufion by provfidfing adequatte credfitt tto tthe autthors and tthe source of publficafion. PEN ACCESS OPEN ACCESS Notte Tremattode finfesttattfion fin coral colonfies att Poshfittra Reef, Gulf of Kachchh Marfine Nattfional Park, Gujaratt , Indfia D. Adhavan, R. Chandran, S. Tfikadar & K. Sfivakumar 26 June 2017 \| Vol. 9\| No. 6 \| Pp. 10345–10346 10.11609/jott.2730.9.6.10345–10346 www .tthreattenedttaxa.org ISSN 0974-7907 (Onlfine) \| ISSN 0974-7893 (Prfintt) Bufildfing evfidence for conservafion globally Journal of Threattened Taxait For Focus, Scope, Afims, Polficfies and Gufidelfines vfisfitt http://tthreattenedttaxa.org/Aboutt_JoTT For Arficle Submfissfion Gufidelfines vfisfitt http://tthreattenedttaxa.org/Submfissfion_Gufidelfines For Polficfies agafinstt Scfienfific Mfisconductt vfisfitt http://tthreattenedttaxa.org/JoTT_Polficy_agafinstt_Scfienfific_Mfisconductt For reprfintts conttactt <finfo@tthreattenedttaxa.org> This situation indicates that if ISSN 0974-7907 (Online) ISSN 0974-7893 (Print) OPEN ACCESS environmental stress including sedimentation, freshwater influence, sewage disposal, aerial exposure, cyanide exposure and bleaching (Peters 1984). In addition to that, some colonies of Porites sp. at Poshitra Reef were spotted with pink swollen nodules (Image 1d–f). These nodules are termed as a condition called “Porites trematodiasis” which is due to an infection of digenetic trematodes (Aeby 2003; Abey 2007; Palmer et al. 2009). These trematodes are common parasites of most animal taxa occurring in the intertidal communities as they are often unnoticed by most researchers and are an integral part of intertidal ecosystems (Sousa 1991; Leung et al. 2009). It has a complex life cycle involving a molluscan first intermediate host, massive coral Porites as the second intermediate host, and coral-feeding fish as the final host (Aeby 1998). According to Palmer et al. (2009), the pink pigmentation in the swollen nodules of infected coral is due to the presence of a red fluorescent protein in compromised tissue of Scleractinian coral that plays a role in cytotoxic defense, and provides new insights into the biological mechanisms involved in immune resistance. Furthermore, the sea surface temperature (SST) data (NOAA) of the National Environmental Satellite Data and Information Service (NESDIS) showed that temperature fluctuates from 30–34 0C which is above normal in the Gulf of Kachchh region during the past six months (NOAA; 2014). The temperature fluctuation and anthropogenic pressures are perhaps the reasons for coral stress (Adhavan et al. 2014) and this may be the opportunity for the parasite to infect the stressed coral colonies at Poshitra Reef. This situation indicates that if Trematode infestation in coral colonies at Poshitra Reef, Gulf of Kachchh Marine National Park, Gujarat, India D. Adhavan 1, R. Chandran 2, S. Tikadar 3 & K. Sivakumar 4 1 Present address: Wildlife institute of India, P.O. box 18, Chandrabani, Dehradun, Uttarakhand 248001, India 1,2,3 Zoological Survey of India, Office of C.C.F. Forest Campus, “Van Sankul”, Jamnagar, Gujarat 361001, India 4 Wildlife institute of India, P.O. box 18, Chandrabani, Dehradun, Uttarakhand 248001, India 1 adhavmarine@gmail.com (corresponding author), 2 softcoralchandran@gmail.com, 3 mnp@yahoo.com, 4 ksivakumar@wii.gov.in 1 Present address: Wildlife institute of India, P.O. box 18, Chandrabani, Dehradun, Uttarakhand 248001, India 1,2,3 Zoological Survey of India, Office of C.C.F. Forest Campus, “Van Sankul”, Jamnagar, Gujarat 361001, India 4 Wildlife institute of India, P.O. box 18, Chandrabani, Dehradun, Uttarakhand 248001, India 1 adhavmarine@gmail.com (corresponding author), 2 softcoralchandran@gmail.com, 3 mnp@yahoo.com, 4 ksivakumar@wii.gov.in 1 Present address: Wildlife institute of India, P.O. box 18, Chandrabani, Dehradun, Uttarakhand 248001, India The Gulf of Kachchh (GoK) is one of the three gulfs in India, which occupies an area of 7,300km2 with 42 Islands and is enriched with various marine habitats such as coral reefs and mangroves (Nair 2002; Adhavan et al. 2014). Poshitra reef, a part of the GoK Marine National Park is located at 22.413056 N & 69.191111 E. During our regular survey for Integrated Coastal Zone Management Project to assess the intertidal diversity, between 27 and 29 March 2015, a soapy oily slick was observed on the water surface at Poshitra Reef, which indicated mass secretion of mucus from the corals (Image 1a–c). Mucus secreted by the corals creates a layer around them like a slipcover to trap the dirt (sediment) and once the coral sloughs it off, it makes a new one (Wild 2004). The mucus produced by corals has a thin-layer that supports an active community of microorganisms, which may in turn affect the health of corals through interactions with other beneficial or pathogenic bacteria (Aeby 1998). Copious mucus release by coral reefs, however, is the first obvious sign of a generalized response to DOI: http://doi.org/10.11609/jott.2730.9.6.10345-10346 Editor: M. Nithyanandan, Kuwait Institute for Scientific Research, Safat, Kuwait. Date of publication: 26 June 2017 (online & print) Manuscript details: Ms # 2730 \| Received 19 April 2016 \| Final received 30 May 2017 \| Finally accepted 06 June 2017 Citation: Adhavan, D., R. Chandran, S. Tikadar & K. Sivakumar (2017). Trematode infestation in coral colonies at Poshitra Reef, Gulf of Kachchh Marine National Park, Gujarat, India. Notte Tremattode finfesttattfion fin coral colonfies att Poshfittra Reef, Gulf of Kachchh Marfine Nattfional Park, Gujaratt , Indfia D. Adhavan, R. Chandran, S. Tfikadar & K. Sfivakumar 26 June 2017 \| Vol. 9\| No. 6 \| Pp. 10345–10346 10.11609/jott.2730.9.6.10345–10346 www .tthreattenedttaxa.org ISSN 0974-7907 (Onlfine) \| ISSN 0974-7893 (Prfintt) Bufildfing evfidence for conservafion globally Journal of Threattened Taxait For Focus, Scope, Afims, Polficfies and Gufidelfines vfisfitt http://tthreattenedttaxa.org/Aboutt_JoTT For Arficle Submfissfion Gufidelfines vfisfitt http://tthreattenedttaxa.org/Submfissfion_Gufidelfines For Polficfies agafinstt Scfienfific Mfisconductt vfisfitt http://tthreattenedttaxa.org/JoTT_Polficy_agafinstt_Scfienfific_Mfisconductt For reprfintts conttactt <finfo@tthreattenedttaxa.org> Notte Tremattode finfesttattfion fin coral colonfies att Poshfittra Reef, Gulf of Kachchh Marfine Nattfional Park, Gujaratt , Indfia D. Adhavan, R. Chandran, S. Tfikadar & K. Sfivakumar 26 June 2017 \| Vol. 9\| No. 6 \| Pp. 10345–10346 10.11609/jott.2730.9.6.10345–10346 www .tthreattenedttaxa.org ISSN 0974-7907 (Onlfine) \| ISSN 0974-7893 (Prfintt) Bufildfing evfidence for conservafion globally Journal of Threattened Taxa For Focus, Scope, Afims, Polficfies and Gufidelfines vfisfitt http://tthreattenedttaxa.org/Aboutt_JoTT For Arficle Submfissfion Gufidelfines vfisfitt http://tthreattenedttaxa.org/Submfissfion_Gufidelfines For Polficfies agafinstt Scfienfific Mfisconductt vfisfitt http://tthreattenedttaxa.org/JoTT_Polficy_agafinstt_Scf For reprfintts conttactt <finfo@tthreattenedttaxa.org> Threattened Taxa Publfisher/Hostt Parttner Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 June 2017 \| 9(6): 10345–10346 ISSN 0974-7907 (Online) ISSN 0974-7893 (Print) OPEN ACCESS Note ISSN 0974-7907 (Online) ISSN 0974-7893 (Print) OPEN ACCESS environmental stress including sedimentation, freshwater influence, sewage disposal, aerial exposure, cyanide exposure and bleaching (Peters 1984). In addition to that, some colonies of Porites sp. at Poshitra Reef were spotted with pink swollen nodules (Image 1d–f). These nodules are termed as a condition called “Porites trematodiasis” which is due to an infection of digenetic trematodes (Aeby 2003; Abey 2007; Palmer et al. 2009). These trematodes are common parasites of most animal taxa occurring in the intertidal communities as they are often unnoticed by most researchers and are an integral part of intertidal ecosystems (Sousa 1991; Leung et al. 2009). It has a complex life cycle involving a molluscan first intermediate host, massive coral Porites as the second intermediate host, and coral-feeding fish as the final host (Aeby 1998). According to Palmer et al. (2009), the pink pigmentation in the swollen nodules of infected coral is due to the presence of a red fluorescent protein in compromised tissue of Scleractinian coral that plays a role in cytotoxic defense, and provides new insights into the biological mechanisms involved in immune resistance. Furthermore, the sea surface temperature (SST) data (NOAA) of the National Environmental Satellite Data and Information Service (NESDIS) showed that temperature fluctuates from 30–34 0C which is above normal in the Gulf of Kachchh region during the past six months (NOAA; 2014). The temperature fluctuation and anthropogenic pressures are perhaps the reasons for coral stress (Adhavan et al. 2014) and this may be the opportunity for the parasite to infect the stressed coral colonies at Poshitra Reef. Trematode infestation in coral colonies at Poshitra Reef, Gulf of Kachchh Marine National Park, Gujarat, India Journal of Threatened Taxa 9(6): 10345–10346; http://doi.org/10.11609/jott.2730.9.6.10345-10346 Copyright: © Adhavan et al. 2017. Creative Commons Attribution 4.0 International License. JoTT allows unrestricted use of this article in any medium, reproduction and distribution by providing adequate credit to the authors and the source of publication. Funding: State Project Management Unit, Integrated Coastal Zone Management Project, Gujarat Ecology Commission, Gandhinagar. Competing interests: The authors declare no competing interests. Acknowledgements: We thank the ranger and other frontline staff of Marine National Park, Gujarat Forest Department, for their support during the field survey. ript details: Ms # 2730 \| Received 19 April 2016 \| Final received 30 May 2017 \| Finally accepted 06 June 2017 10345 Trematode infestation in coral colonies Adhavan et al. Image 1. a–b - on Porites colony covered by mucus secretion; c - mass mucus secretion in the intertidal region of Poshitra Reef ecosystem; d–f - Pink swollen nodules. © D. Adhavan & S. Tikadar a d b e c f b b e Image 1. a–b - on Porites colony covered by mucus secretion; c - mass mucus secretion in the intertidal region of Poshitra Reef ecosystem; d–f - Pink swollen nodules. © D. Adhavan & S. Tikadar Leung, T.L.F., K.M. Donald, D.B. Keeney, A.V. Koehler, R.C. Peoples & R. Poulin (2009). Trematode parasites of Otago Harbour (New Zealand) soft-sediment intertidal ecosystems: life cycles, ecological roles and DNA barcodes. New Zealand Journal of Marine and Freshwater Research 43: 857–865; http://doi. org/10.1080/00288330909510044 the corals fail to develop disease resistance and thermal tolerance, the reefs along the Indian subcontinent may experience a phase shift in community structure, which could impact fisheries. Nair, V.R. (2002). Status of Flora and fauna of Gulf of Kachchh, India. National Institute of Oceanography, 157pp (http://drs.nio.org/drs/ handle/2264/87) t Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 June 2017 \| 9(6): 10345–10346 Notes Four species of Commelinaceae, as additions to Andhra Pradesh, India -- S. Salamma, M. Chennakesavulu Naik, M. Anil Kumar, A. Sreenath & B. Ravi Prasad Rao, Pp. 10340–10344 Four species of Commelinaceae, as additions to Andhra Pradesh, India -- S. Salamma, M. Chennakesavulu Naik, M. Anil Kumar, A. Sreenath & B. Ravi Prasad Rao, Pp. 10340–10344 Trematode infestation in coral colonies at Poshitra Reef, Gulf of Kachchh Marine National Park, Gujarat, India -- D. Adhavan, R. Chandran, S. Tikadar & K. Sivakumar, Pp. 10345– 10346 First report of Mantibaria mantis (Dodd) (Hymenoptera: Scelionidae: Scelioninae) from India and additional descriptors for the species -- Kamalanathan Veenakumari & Prashanth Mohanraj, Pp. 10347– 10350 A new record of Tenodera fasciata (Olivier, 1792) (Insecta: Mantodea: Mantidae: Mantinae) for western India -- Gopal Ambrushi Raut & Sunil Madhukar Gaikwad, Pp. 10351–10354 First records of butterflies Anthene emolus emolus (Godart, [1924]) (Lepidoptera: Lycaenidae: Polyommatinae) and Gandaca harina assamica Moore, [1906] (Lepidoptera: Pieridae: Coliadinae) from Kumaon, Uttarakhand, India -- Sanjay Sondhi, Pp. 10355–10357 A new locality record of the rare Anomalous Nawab Polyura agrarius (Swinhoe, 1887) (Lepidoptera: Nymphalidae: Charaxinae) from central India -- Deepika Mehra, Jagatjot Singh Flora & Vivek Sharma, Pp. 10358– 10360 Taxonomic note about Willow Ermine Moth Yponomeuta rorrellus Hübner (Lepidoptera: Yponomeutidae) from Ladakh division of Jammu & Kashmir, India -- Mudasir Ahmad Dar, Shahid Ali Akbar & Govindasamy Mahendiran, Pp. 10361–10364 First record of hagfish (Cyclostomata: Myxinidae) in Indian waters Trematode infestation in coral colonies at Poshitra Reef, Gulf of Kachchh Marine National Park, Gujarat, India -- D. Adhavan, R. Chandran, S. Tikadar & K. Sivakumar, Pp. 10345– 10346 Articles Articles Co-occurrence patterns of fish communities in littorals of three floodplain lakes of the Orinoco River, Venezuela -- Gabriela E. Echevarría & Nirson González, Pp. 10249–10260 Genetic diversity of the Green Turtle (Testudines: Cheloniidae: Chelonia mydas (Linnaeus, 1758)) population nesting at Kosgoda Rookery, Sri Lanka -- E.M.L. Ekanayake, T. Kapurusinghe, M.M. Saman, D.S. Rathnakumara, P. Samaraweera & R.S. Rajakaruna, Pp. 10261– 10268 Identity of Sphaerotheca pluvialis (Jerdon, 1853) and other available names among the burrowing frogs (Anura: Dicroglossidae) of South Asia -- Neelesh Dahanukar, Shauri Sulakhe & Anand Padhye, Pp. 10269– 10285 Sphaerotheca pashchima, a new species of burrowing frog (Anura: Dicroglossidae) from western India -- Anand Padhye, Neelesh Dahanukar, Shauri Sulakhe, Nikhil Dandekar, Sunil Limaye & Kirti Jamdade, Pp. 10286–10296 Population status and species diversity of wetland birds in the Rapti and Narayani rivers and associated wetlands of Chitwan National Park, Nepal -- Bed Bahadur Khadka, Paras Mani Acharya & Sunil Lal Rajbhandari, Pp. 10297–10306 Communications Wildlife hunting by indigenous people in a Philippine protected area: a perspective from Mt. Apo National Park, Mindanao Island -- Krizler Cejuela Tanalgo, Pp. 10307–10313 Pupal shape and size dimorphism in Aedes albopictus (Skuse, 1894) (Diptera: Culicidae) -- Elvira Sánchez, Daniel Castillo & Jonathan Liria, Pp. 10314–10319 Short Communications Occurrence and conservation of the Indian Leopard (Mammalia: Carnivora: Felidae: Panthera pardus) in Cox’s Bazar District of Bangladesh -- M. Tarik Kabir, M. Farid Ahsan & Ayesha Khatoon, Pp. 10320–10324 A checklist of the avian fauna of Chittagong University campus, Bangladesh -- M. Tarik Kabir, M. Farid Ahsan, M. Mizanur Rahman & M. Manirul -- Pradip Kachhiya, Jatin Raval, Paresh Poriya & Rahul Kundu, Pp. 10334–10339 References NOAA (2014). http://www.ospo.noaa.gov/Products/ocean/sst/ anomaly/2014.htmll Adhavan, D., R.D. Kamboj, N. Marimuthu & M.M. Ballodi (2014). Seasonal variation and climate change influence coral bleaching in Pirotan Island, Gulf of Kachchh Marine National Park, Gujarat. Current Science 107(11): 1780–1781. Palmer, C.V., M.S. Roth & R.D. Gates (2009). Red fluorescent protein responsible for pigmentation in trematode-infected Porites compressa tissues. Biology Bulletin 216: 68–74. i Aeby, G.S. (1998). A digenean metacercaria from the reef coral, Porites compressa, experimentally identified as Podocotyloides stenometra. Journal of Parasitology 84: 1259–1261; http://doi. org/10.2307/3284684 Peters, E.C. (1984). “A survey of cellular reactions to environmental stress and disease in Caribbean scleractinian corals.” Helgolander Meeresunters 37: 113–137.t Peters, E.C. (1984). “A survey of cellular reactions to environmental stress and disease in Caribbean scleractinian corals.” Helgolander Meeresunters 37: 113–137. Sousa, W.P. (1991). Can models of soft-sediment community structure be complete without parasites? American Zoologist 31: 821–830. Sousa, W.P. (1991). Can models of soft-sediment community structure be complete without parasites? American Zoologist 31: 821–830.t Aeby, G.S. (2003). Corals in the genus Porites are susceptible to infection by a larval trematode. Coral Reefs 22: 216; http://doi. org/10.1007/s00338-003-0310-9 it Wild, C., M. Huettel, A. Klueter, S.G. Kremb, M.Y.M. Rasheed & B.J. Bo (2004). Coral mucus functions as an energy carrier and particle trap in the reef ecosystem. Nature 428: 66–70; http://doi.org/10.1038/ nature02344 Aeby, G.S. (2007). Spatial and temporal patterns of Porites trematodiasis on the reefs of Kaneohe Bay, Oahu, Hawaii. Bulletin of Marine Science 80 (1): 209–218. Threatened Taxa 10346 Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 June 2017 \| 9(6): 10345–10346 O O The Journal of Threatened Taxa is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows unrestricted use of articles in any medium, reproduction, and distribution by providing adequate credit to the authors and the source of publication. OPEN ACCESS ISSN 0974-7907 (Online); ISSN 0974-7893 (Print) June 2017 \| Vol. 9 \| No. 6 \| Pages: 10249–10368 Date of Publication: 26 June 2017 (Online & Print) DOI: 10.11609/jott.2017.9.6.10249-10368 www.threatenedtaxa.org Diversity and new records of intertidal hermit crabs of the genus Clibanarius (Crustacea: Decapoda: Diogenidae) from Gujarat coast off the northern Arabian Sea, with two new records for the mainland Indian coastline Diversity and new records of intertidal hermit crabs of the genus Clibanarius (Crustacea: Decapoda: Diogenidae) from Gujarat coast off the northern Arabian Sea, with two new records for the mainland Indian coastline Communications Short Communications First record of hagfish (Cyclostomata: Myxinidae) in Indian waters -- B. Fernholm, A. Biju Kumar & Michael Norén, Pp. 10365–10368 Threatened Taxa
https://openalex.org/W4317854102	https://zenodo.org/records/7562630/files/Hebraic%20Analysis%20of%20John%208-31-38.pdf	English	null	Hebraic Analysis of John 8:31-38	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	5,416	Hebraic Analysis John 8:31-38 Hebraic Analysis John 8:31-38 Michael Harvey Koplitz Michael Harvey Koplitz @2022. Copyright Rev. Dr. Michael H. Koplitz, D.Min., Ph.D. All rights reserved. 2 2 Introduction When a person is baptized as an infant and grows up in the church, different paradigms become a part of their religious DNA. The church has a message to give about Jesus Christ and His importance. Very few people study the theology and doctrines of the church to determine for themselves the accuracy of the church. The Proto-Orthodox church, which survived the pressures of the Roman Empire, decided to oppose any expression of Christianity that did not fit its dogma in its infancy. In addition, the Proto- Orthodox church would permanently destroy any writings that the rival Christians had developed. The Gnostic Christians of Northern Egypt viewed the life of Jesus of Nazareth in a completely different way than the Proto-Orthodox church did. They saw the message about the Kingdom of Heaven as the vital purpose of Jesus. His birth, death, and resurrection are not mentioned in the Gnostic Gospels. However, did the Proto- Orthodox church destroy the Gnostic Gospels when they crushed said movement? The answer is yes and no. Yes, they destroyed what they got their hands on. No, because in 1948, copies of the Gnostic religious books were discovered in Alexandria, Egypt. Once these documents were translated, the world learned what the Gnostic Christians believed. It is fascinatingly different than what the Proto-Orthodox said about these followers of Christ. Why is this understanding critical? Much research points to a different situation in the early years than what the church espouses. A lot of this information is available to anyone today. However, the Seminaries and churches will not openly discuss these 3 3 other writings about Jesus and His disciples. The scholars teaching in most Seminaries have learned their lessons from the church and closed-minded mentors who refuse to look at other possibilities. This is because the Western European world took Christianity and changed it from a Near Eastern religion to Western religion. There is a theory that Paul converted Mithras House Churches into Jesus House Churches. This is clear from the connection between the Mithras’ and Christianity’s rituals. For example, baptism was the initiation ritual of Mithras. Communion did not originate with Jesus. This ritual was a part of Mithras, where the followers would share his flesh (bread) and drink his blood (wine). There are many more rituals that Christianity picked up from Mithras. A good reference is “Christianity's Need for Mithras," which the author wrote. Introduction Did Paul create the churches in the letters he sent, which comprise the New Testament, and if so, they must have been Jewish groups who became Jewish Christians? They would have continued with their Hebraic rituals and saw Jesus of Nazareth as the Messiah that the prophets of old had promised. They would have adopted many of Jesus' teachings and tried to live by them. The letters in the New Testament are written in Greek. However, most Jews in the Roman Empire did not speak Greek; instead, they spoke Aramaic and Hebrew. These congregations would not have understood a Greek letter from Paul. Therefore, the letters in the New Testament must have been written in Aramaic and then transliterated into Greek. The same can be said for the Gospels, all of them. The church, over the centuries, decided who wrote the Gospels and their intent. The only 4 4 Gospel we can assign to a writer is Luke. The other three are up in the air about who wrote them. While in Seminary, the author was taught that the entire New Testament was originally written in Koine Greek. However, that raised the question, "Did Jesus speak Greek?" The Seminary instructors said, "no, Jesus did not speak Greek." Then the New Testament, especially the Gospels, must have been written in Aramaic. After all, Jesus spoke Aramaic and Hebrew. We know this because He was a poor tekton (a stonemason or carpenter) from an impoverished city named Nazareth. Being born to a Jewish family in Galilee, he would have learned the traditions of His people and trade. He would have learned to speak Aramaic, the language of the area. He would have learned Hebrew because that was the language of the synagogue and the Temple in Jerusalem. In other words, Hebrew was the language of God, and Jewish males learned the language. Suppose you are ready to toss this manuscript into the nearest trash can or delete it off your electronic device at this point in the introduction. In that case, the writer has your attention. This is the reaction when the writer has spoken with persons who had been indoctrinated into the church's position since birth. The author did not come into the church environment until he was 35. Therefore, the church's paradigms, dogma, and doctrine were not a part of his DNA. Instead, he questioned a lot. Introduction He found many inconsistencies between the Bible and the doctrines of the church. Seminary was an experience to learn what the church had evolved into two-thousand years after the death of Jesus. 5 There are more parts to the premise that the New Testament was originally written in Aramaic and will be explored. For the reader to grasp the subsequent phases of the proof, an open mind is critical. 6 6 Culture and Language Let us continue in the journey of examining the New Testament to determine its original language. Nothing in stone tells us that Aramaic is the Original Language of the New Testament. However, nothing says that Koine Greek was the original language of the New Testament either. Therefore, we have two theories about the original language of the New Testament. The author admits that the Seminary he attended drove home the belief that the Old Testament was written in Hebrew, except for a few spots. The New Testament was initially written in Koine Greek. The writers' research has been searching for the original meaning of Scripture for many years. The methodology for this work is called "Ancient Bible Study Methods." The method was developed by Dr. Anne Davis of the Bible Learning University in Albuquerque, New Mexico. The author studied this method with Dr. Davis as his mentor. It became clear that the search for the original meaning of the Scriptures requires that the culture and language be examined. So, the author's methodology is Dr. Davis' work, plus his Ph.D. studies combining the method, culture, and language. The language examination is easy for the Old Testament because it was written in Hebrew, and about one-half of Daniel is in Aramaic. It does not take long to realize that idioms and figures of speech in the Hebrew of the Old Testament revealed a lot about the people and situation of the day when the scrolls were written. The Targums were a valuable resource because they are the Aramaic translations the rabbis did for the people living outside of Judea. The rabbis added commentary to the Targums 7 7 because they knew that some of the idioms and speech used in the Near East would not translate well into the different areas where the Jews lived. because they knew that some of the idioms and speech used in the Near East would not translate well into the different areas where the Jews lived. The culture of the Near East has been essentially the same in many aspects since the days of Jesus. Many practices of Jesus' day are still in use today. The culture of the Jews of the Near East is built into the language. Often an Aramaic or Hebrew word has a deep meaning that is only fully understood by natives living in that culture. Culture and Language The Old Testament is filled with cultural items that do not need to be spelled out because the people knew their culture in the author's time. Suppose the New Testament in Koine Greek is a transliteration of the Aramaic. The culture, figures of speech, and idioms will be easily identified when examining the Peshitta (the Aramaic version of the New Testament). Indeed many of the so-called difficult words of Jesus are not tricky when examined in the light of the culture of Jesus' day. An example is "faith to move a mountain," Jesus said these words to His disciples. The church determined that this meant complete faith in Jesus. From the western European Greek point of view, that makes sense. What else could it possibly mean? "Faith to move a mountain" is an Aramaic idiomatic expression. What Jesus said to His followers when he said this is that his disciples needed to be faithful so that they could change the "government's view through their words." The governing body for Judaism resided on the top of a mountain. Jerusalem, with its Temple, was built on the top of Mount Zion, a very tall mountain. This idiom survived because the Aramaic Gospels were transliterated into Koine Greek. Numerous other examples support this position. 8 8 Suppose the culture and language idioms of Jesus' day can be found in the Koine Greek because it was transliterated. In that case, it supports the theory of the Aramaic versions being the original language of the Gospels and possibly even more. 9 9 1 Rocco A. Errico and George M. Lamsa, Aramaic Light on Galatians through Hebrews: A Commentary Based on Aramaic, the Language of Jesus, and Ancient near Eastern Customs (Smyma, GA: Noohra Foundation, 2005). Aramaic, the Language of Jesus, and Ancient near Eastern Customs (Smyma, GA: Noohra Found 1 Rocco A. Errico and George M. Lamsa, Aramaic Light on Galatians through Hebrews: A Commentary Based on h f d ( h d ) 1 Rocco A. Errico and George M. Lamsa, Aramaic Light on Galatians through Hebrews: A Com The Aramaic Version of the New Testament The Peshitta is the accepted Aramaic translation of the New Testament for many churches of the East. Peshitta means "simple, true, direct, and original." It is a collection of scrolls that were compiled in 150 CE. There were some revisions to the Peshitta in the fifth and sixth centuries. The Greek version of the New Testament is a transliteration of the Peshitta.1 For centuries, the Catholic church has used the Latin version of the Bible, the Vulgate, and still uses it. The Vulgate was developed around 350 CE by Jerome by order of the Pope at that time. Erasmus (1466 – 1536) was the person who put together the Greek New Testament for the Catholic church. "The New Testament, brought to light in the original Greek tongue, was compiled and made available for humanity to study and learn. Although working under and deeply associated with the Roman Catholic Church, the learned scholar declared his disagreement with those who wanted to keep the Scriptures from the common people. He said, "If only the farmer would sing something from them at his plow, the weaver moves his shuttle to their tune, the traveler lighten the boredom of his journey with Scriptural stories!" Little did he know that the work he was about to produce would change the world forever. This Greek New Testament, in printed form, would become the standard of the New Testament, launching the translations of Martin Luther and William Tyndale into the world. Thus, fulfilling his dream that all men would read the 10 Bible for themselves in their common language. His new "study Bible" had two main parts, the Greek text, and a revised Latin edition, which was more elegant and accurate than the traditional translation of Jerome's Latin Vulgate. Erasmus prefaced this monumental work of scholarship with an exhortation to Bible study. He proclaimed that the New Testament contains the "philosophy of Christ," simple and accessible teaching with the power to transform lives."2 The church recognized Erasmus' Greek New Testament in 1515 CE. The church in the Near East has been using the Peshitta as the original language of the New Testament since 150 CE. If the Greek New Testament was important to the church as an original language, then why did it adopt the Vulgate in 350 CE? The church should have adopted the Greek New Testament at the beginning. 2 “Erasmus Greek New Testament,” Insight of the King, accessed February 18, 2022, https://www.insightoftheking.com/erasmus-greek-new-testament.html. The Aramaic Version of the New Testament The Peshitta, translated into English, is used to examine Paul's letters. The rest of the methodology that the author developed for Ancient Bible Study Methods is the framework of this research. 11 The Messianic Tradition Change One problem for Peter and the Disciples was that they claimed Yeshua to be the Messiah that the prophets of the Hebrew Scriptures spoke. However, Yeshua did not do what these traditions said. The main tradition was that the Messiah would destroy oppressive Romans and reinstate the Kingdom of Israel. Yeshua would then be declared the king and sit on David's throne in Jerusalem. That did not occur. None of the messianic traditions of the day worked. So, what was the new movement going to do? They turned to the prophets and discovered Isaiah 50-53. These chapters are referred to as the Suffering Servant chapters. The Yeshua movement decided that the Suffering Servant was Yeshua. The portrayal of Yeshua's life does fit the Suffering Servant chapters. However, rabbinical interpretation then and now sees the Suffering Servant as the nation of Israel. Indeed, these chapters do describe the history of Israel. Nations have wanted to destroy the Jewish people since the time of Abraham. The diaspora from the Babylonia Exile and the Assyrian invasions looked to squelch the Jewish people. The LORD promised that a remnant of the people would always survive. That is true throughout the 4,000-year history of the Jewish people. Many nations tried to destroy them, and the LORD intervened to ensure that a remnant of the people survived. Paul must have been convinced in his encounter with Yeshua on the Damascus road that Yeshua was the Suffering Servant. It is clear from Paul's writings that he did believe this. For Paul, the Messiah was the Spiritual Messiah that the Kabbalah spoke. The 12 Kabbalah says that there will be two Messiahs. This theology is based on Zachariah 9:9. The first Messiah is Messiah ben Joseph. This Messiah was to restore the Kingdom of Heaven, a spiritual Kingdom. The second Messiah will be Messiah ben David. This Messiah was to restore the Kingdom of Israel. The Midrash from the Kabbalah did not state that the Messiah was two different souls. 13 The Kabbalah There is a large amount of material in print about the Kabbalah. The Kabbalah referred to is Moses's Secret Work from Mount Sinai. Legends say Moses received three items on Mount Sinai when he met the LORD. The first is the written law. The written law is called the Torah. The second is the oral law. The oral law was put into a written form around 200 CE called the Mishnah. The third is the secret law called the Kabbalah. The secrets of the Kabbalah are based on the Torah and were written down around 200 CE. The main books of the Kabbalah are the Zohar and the Book of Creation. Many of Yeshua's statements have Kabbalah undertones. Yeshua would have known the Kabbalah. Paul would have known the basics, at least, of the Kabbalah because of his religious education and training. There are Kabbalistic ideas in the Gospels and Paul's letters. Kabbalistic verses will be highlighted in the chapters of the letters. 14 Methodology The methodology employed is to use "Ancient Bible Study Methods" integrated with Jesus's day's customs and culture to examine the Hebrew and Christian Scriptures, thus gathering a more in-depth understanding by learning the Scriptures in the way the people of Jesus's day did. I have titled the methodology of analyzing a passage of Scripture in a Hebraic manner the "Process of Discovery." The author developed this methodology, which combines various linguistic and cultural understanding areas. There are several sections to the process, and not all the parts apply to every passage of Scripture. The overall result of developing this process is to give the reader a framework for studying the Word in more depth. The "Process of Discovery" starts with a Scripture passage. An examination of the linguistic structure of the passage is next. The linguistic structure includes parallelism, chiastic structures, and repetition. Formatting the passage in its linguistic form allows the reader to visualize what the first-century CE listener was hearing. Their corresponding sections label the chiasms, for example, A, B, C, B', A.' Not all passages of the Scriptures have a poetic form. The next step is to "question the narrative." The narrative process of questioning the narrative assumes the reader knows nothing about the passage. Therefore, the questions go from simple to complex. The next task is to identify any linguistic patterns. Linguistic patterns include, but are not limited to, irony, simile, metaphor, symbolism, idioms, hyperbole, figurative language, personification, and allegory. 15 A review of any translation inconsistencies discovered between the English NAU version and Hebrew or Greek versions is done. Sometimes, a Hebrew or Greek word is translated in more than one way. Inconsistencies also can be created by the translation committee, which may have decided to use traditional language instead of the actual translation. The decision of the translation committee is in the Preface or Introduction to the Bible. Perhaps some of the inconsistencies were intentionally added to convey some deeper meaning. An examination of every discrepancy is done. The passage is analyzed for any echoes of the Hebrew Scriptures in the Christian Scriptures. An echo occurs using a passage from the Hebrew Scriptures in the Christian Scriptures.3 Also, echoes are found when Torah (Genesis through Deuteronomy) passages are used in other Hebrew Bible books. Cross-references in the Scripture are references from one verse to another verse, which can help the reader understand the verse. 3 Mitzvot are the 613 commandments found in the Torah that please God. There are positive and negative commandments. The list was first development by Maimonides. The full list can be found at: ttp://www.jewfaq.org/613.htm. Methodology The names of persons mentioned in the passage are listed. Many Hebrew names have meaning and may be associated with places or actions. Jewish parents used to name their children based on what they felt God had in store for their children. An example is Abraham, whose original name was Abram and was changed to mean eternal father (God changed Abram's name to Abraham, indicating a function he was to perform). When the Hebrew Bible gives names, many occurrences mean something unique. The same importance can occur for the names of places. The time it takes to travel between locations can supply insight into the event. 16 Keyphrases are identified in verses when they are essential to understanding that passage. There are no rules for selecting the keywords. Searching for other occurrences of the keywords in Scripture in concordance is necessary to understand the Word's usage; this must be done in either Hebrew or Greek, not in English. A classic Hebraic approach is to find the usage of a word in the Scripture by finding other verses that contain the Word. The usage of a word in its original language is discovered by searching the Scripture in the language of the Word. Verses that contain the Word are identified, and a pattern for the usage of the Word is discovered. Each verse is examined to see what the usage of the Word is, which may reveal a model for the Word's usage. The first usage of the Word in the Scripture, primarily if used in the Torah, is essential for Hebrew words. The Christian Scriptures are used for Greek words to determine the Word usage in the Scripture. Sometimes, finding the equivalent Greek Word in the Septuagint can be beneficial as analyzing its Hebrew usage. The Rules of Hillel are used when applicable. Hillel was a Torah scholar who lived shortly before Jesus' day. Hillel developed several rules for Torah students to interpret the Scriptures, which refer to halachic Midrash. In several cases, these rules are helpful in the analysis of the Scripture. The cultural implications from the writing period are done after the linguistic analysis is completed. The culture is crucial because it is not explicitly referenced in the biblical narratives, as indicated earlier. From the linguistic analysis and the cultural understanding, it is possible to obtain a deeper meaning of the Scripture beyond the plain text's literal meaning. Methodology That is what the listeners of Jesus's time were doing. They put linguistics and culture together without even having to contemplate it. 17 The analysis will lead to findings explaining the passage's meaning in Jesus's day. Most of the time, the Hebraic analysis leads to the desire for more in-depth analysis to fully understand what Jesus was talking about or what was happening to Him. Whatever the result, a new, more in-depth understanding of the Scripture is obtained. The components of the Process of Discovery are: The components of the Process of Discovery are: Language Process of Discovery 18 New American Standard 1995 New American Standard 1995 John 8:31 So Jesus was saying to those Jews who had believed Him, “aIf you continue in My word, then you are truly bdisciples of Mine; 32 and ayou will know the truth, and bthe truth will make you free.” 33 They answered Him, “aWe are Abraham’s descendants and have never yet been enslaved to anyone; how is it that You say, ‘You will become free’?” John 8:31 So Jesus was saying to those Jews who had believed Him, “aIf you continue in My word, then you are truly bdisciples of Mine; 32 and ayou will know the truth, and bthe truth will make you free.” 33 They answered Him, “aWe are Abraham’s descendants and have never yet been enslaved to anyone; how is it that You say, ‘You will become free’?” John 8:34 Jesus answered them, “Truly, truly, I say to you, aeveryone who commits sin is the slave of sin. 35 “aThe slave does not remain in the house forever; bthe son does remain forever. 36 “So if the Son amakes you free, you will be free indeed. 37 “I know that you are aAbraham’s descendants; yet byou seek to kill Me, because My word 1has no place in you. 38 “I speak the things which I have seen 1with My Father; therefore you also do the things which you heard from ayour father.” John 8:34 Jesus answered them, “Truly, truly, I say to you, aeveryone who commits sin is the slave of sin. 35 “aThe slave does not remain in the house forever; bthe son does remain forever. 36 “So if the Son amakes you free, you will be free indeed. 37 “I know that you are aAbraham’s descendants; yet byou seek to kill Me, because My word 1has no place in you. 38 “I speak the things which I have seen 1with My Father; therefore you also do the things which you heard from ayour father.” John 8:34 Jesus answered them, “Truly, truly, I say to you, aeveryone who commits sin is the slave of sin. 35 “aThe slave does not remain in the house forever; bthe son does remain forever. 36 “So if the Son amakes you free, you will be free indeed. 37 “I know that you are aAbraham’s descendants; yet byou seek to kill Me, because My word 1has no place in you. Discussion Only the applicable sections are included in this document. 19 Chapter or Verse New American Standard 1995 38 “I speak the things which I have seen 1with My Father; therefore you also do the things which you heard from ayour father.” 20 References to the New American Standard 1995 John 8:31 aJohn 15:7; 2 John 9 bJohn 2:2 John 8:32 aJohn 1:14, 17 bJohn 8:36; Rom 8:2; 2 Cor 3:17; Gal 5:1, 13; James 2:12; 1 Pet 2:16 John 8:33 aMatt 3:9; Luke 3:8; John 8:37, 39 John 8:34 aRom 6:16; 2 Pet 2:19 John 8:35 aGen 21:10; Gal 4:30 bLuke 15:31 John 8:36 aJohn 8:32 John 8:37 1Or makes no progress aMatt 3:9; John 8:39 bJohn 7:1; 8:40 John 8:38 1Or in the presence of aJohn 8:41, 44 John 8:31 aJohn 15:7; 2 John 9 bJohn 2:2 John 8:32 aJohn 1:14, 17 bJohn 8:36; Rom 8:2; 2 Cor 3:17; Gal 5:1, 13; Jam John 8:33 aMatt 3:9; Luke 3:8; John 8:37, 39 John 8:34 aRom 6:16; 2 Pet 2:19 John 8:35 aGen 21:10; Gal 4:30 bLuke 15:31 John 8:36 aJohn 8:32 John 8:37 1Or makes no progress aMatt 3:9; John 8:39 bJohn 7:1; 8:40 John 8:38 1Or in the presence of aJohn 8:41, 44 John 8:31 aJohn 15:7; 2 John 9 bJohn 2:2 John 8:32 aJohn 1:14, 17 bJohn 8:36; Rom 8:2; 2 Cor 3:17; Gal 5:1, 13; James 2:12; 1 Pet 2:16 John 8:32 aJohn 1:14, 17 bJohn 8:36; Rom 8:2; 2 Cor 3:17; Gal 5:1, 13; James 2:12; 1 Pet 2:16 John 8:33 aMatt 3:9; Luke 3:8; John 8:37, 39 John 8:34 aRom 6:16; 2 Pet 2:19 John 8:33 aMatt 3:9; Luke 3:8; John 8:37, 39 John 8:37 1Or makes no progress aMatt 3:9; John 8:39 bJohn 7:1; 8:40 John 8:38 1Or in the presence of aJohn 8:41, 44 21 Koine Greek Koine Greek John 8:31 Ελεγεν ουν ο Ιησους προς τους πεπιστευκοτας αυτω Ιουδαιους, Εαν υμεις μεινητε εν τω λογω τω εμω, αληθως μαθηται μου εστε· 32 και γνωσεσθε την αληθειαν, και η αληθεια ελευθερωσει υμας. 33 Απεκριθησαν αυτω, Σπερμα Αβρααμ εσμεν, και ουδενι δεδουλευκαμεν πωποτε· πως συ λεγεις οτι Ελευθεροι γενησεσθε; 34 Απεκριθη αυτοις ο Ιησους, Αμην αμην λεγω υμιν, οτι πας ο ποιων την αμαρτιαν δουλος εστιν της αμαρτιας. 35 Ο δε δουλος ου μενει εν τη οικια εις τον αιωνα· ο υιος μενει εις τον αιωνα· 36 Εαν ουν ο υιος υμας ελευθερωση, οντως ελευθεροι εσεσθε. 37 Οιδα οτι σπερμα Αβρααμ εστε· αλλα ζητειτε με αποκτειναι, οτι ο λογος ο εμος ου χωρει εν υμιν. 38 Εγω ο εωρακα παρα τω πατρι μου, λαλω. και υμεις ουν ο εωρακατε παρα τω πατρι υμων, ποιειτε. 22 Process of Discovery Linguistics Section Linguistic Structure Process of Discovery Linguistics Section Linguistic Structure Process of Discovery Process of Discovery Linguistics Section Linguistic Structure The NASB 1995 was used for the chiastic structure. The NASB 1995 was used for the chiastic structure. [Yeshua’s words] 31 So Jesus was saying to those Jews who had believed Him, “aIf you continue in My word, then you are truly bdisciples of Mine; 32 and ayou will know the truth, and bthe truth will make you free.” [Response] 33 They answered Him, “aWe are Abraham’s descendants and have never yet been enslaved to anyone; how is it that You say, ‘You will become free’?” [Yeshua’s words] 34 Jesus answered them, “Truly, truly, I say to you, aeveryone who commits sin is the slave of sin. 35 “aThe slave does not remain in the house forever; bthe son does remain forever. 36 “So if the Son amakes you free, you will be free indeed. 37 “I know that you are aAbraham’s descendants; yet byou seek to kill Me, because My word 1has no place in you. 38 “I speak the things which I have seen 1with My Father; therefore you also do the things which you heard from ayour father.” Discussion This section is a question-and-answer session between Yeshua and some unknown people. This section is a question-and-answer session between Yeshua and some unknown people. Questioning the narrative 1. Why did the people respond that they were always free? (v. 33) It is puzzling why the people responded to Yeshua in this manner. Even then, the people were enslaved by the Herodians and the Romans. The people were not free when examining the materialism of the world. They were not controlled by these groups when it came to Judaism. Therefore, in a spiritual sense, the Hebrew people were never made enslaved people to a false god during that time. Yeshua said to the people that even though they believed that they were not 23 enslaved religiously, they were enslaved. The Pharisees and Sadducees did not allow the people to see the sacred writing. The people had to be made to believe they were being told the truth. Yeshua referred to them in verses thirty-one to thirty-three. The people were religious enslaved people. Their taskmasters collected taxes and offerings from the people like the Herodians and Romans did. The people were not free. 4 Rocco A. Errico and George M. Lamsa, Aramaic Light on the Gospel of John: A Commentary on the Teachings of Jesus from the Aramaic and Unchanged near Eastern Customs (Smyrna, GA: Noohra Foundation, 2002). 4 Rocco A. Errico and George M. Lamsa, Aramaic Light on the Gospel of John: A Commentary on the Teachings of Jesus from the Aramaic and Unchanged near Eastern Customs (Smyrna GA: Noohra Foundation 2002) g g p f y Jesus from the Aramaic and Unchanged near Eastern Customs (Smyrna, GA: Noohra Foundation Discussion The Aramaic word for “truth” was Shrara. This word is derived from the root word sharer, which means “to be firm, sincere, thankful.” A person who is doubtful of their faith can be easily shaken. In the ancient world, getting people to change their religious allegiance was not difficult. A prime example is the work of Saint Paul. He got Mithras House Churches to become Yeshua House churches.4 Persons who have a firm understanding of their faith understand what is truth. These persons are difficult to sway. During Yeshua’s day, sacred writings, like what became the Bible, were only available to the high priests and teachers of the Law. Torah scrolls took a lifetime to copy. The people heard about the contents of the sacred writings through the traditions of the elders who interpreted the Law. That means that the people could be told falsehoods which assisted priests in controlling the people. Pharisees and Sadducees have been accused of this practice. Servants and stewards were not hired permanently. Therefore, they often changed 24 jobs and positions for more money or better working conditions. Also, they might be hired for temporary positions. The hired person knew this when going into the job. The father’s son was an heir, meaning he was a permanent resident. The caveat can be seen in the LORD’s covenant with King David. As long as David’s descendants stayed true to the Torah, the LORD guaranteed that a son of David would stay on the throne of Israel. History demonstrates that many kings in Israel and Judah did not follow the Torah. The Assyrians destroyed the Northern Kingdom because the LORD took his protection away from them because they did not follow the Torah. Judah went into Exile, and the Babylonians murdered the royal family because the people, including the royal family, had idolatry and other Torah violations. When the Judeans were in Exile in Babylon, they reformed and returned to Torah worship. After seventy years, the LORD reinstated them into the Judean territory before the Exile. Judah regained its freedom from the Greeks for a short time, who took over vast amounts of the Persian empire. Yeshua said that if the people followed his example of how to live by the Torah, they would be freed from the slavery of outside nations. Discussion Yeshua also added that all the people would not accept his message and they would try to kill him. Why? The reason was that the Messiah was a special prophet sent by the LORD. The previous prophets sent by the LORD were all killed by the ruling authority. Yeshua knew that he was cutting his life short when he spoke as a prophet. 25 Thoughts Sometimes it sounds like a broken record because the problem of education is brought up by Yeshua often. This is another place where Yeshua says that the lack of proper education is detrimental to one’s faith. Faith and believing are action words. It is easy to say I believe or I have faith, but it is not easy to hold to it. A solid education about the sacred writings which we call the Bible today is necessary. Then the ability to be free thinkers is imperative. How can the church fix this lack of education? A question for you to answer. 26
https://openalex.org/W2062000159	https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1028&context=surgery_facpub	English	null	Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds	Healthcare	2,014	cc-by	21,562	Wound Healing: Biologics, Skin Substitutes, Biomembranes and Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds Scaffolds Krishna S. Vyas University of Kentucky, krishnavyas1@gmail.com Henry C. Vasconez University of Kentucky, hcvasc@uky.edu Follow this and additional works at: https://uknowledge.uky.edu/surgery_facpub Follow this and additional works at: https://uknowledge.uky.edu/surgery_facpub Part of the Surgery Commons Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. University of Kentucky University of Kentucky UKnowledge UKnowledge University of Kentucky University of Kentucky UKnowledge UKnowledge Surgery Faculty Publications Surgery Notes/Citation Information Notes/Citation Information Published in Healthcare, v. 2, issue 3, p. 356-400. Published in Healthcare, v. 2, issue 3, p. 356-400. Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds Digital Object Identifier (DOI) https://doi.org/10.3390/healthcare2030356 Digital Object Identifier (DOI) Digital Object Identifier (DOI) https://doi.org/10.3390/healthcare2030356 Repository Citation Repository Citation Repository Citation Repository Citation Vyas, Krishna S. and Vasconez, Henry C., "Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds" (2014). Surgery Faculty Publications. 29. https://uknowledge.uky.edu/surgery_facpub/29 Vyas, Krishna S. and Vasconez, Henry C., "Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds" (2014). Surgery Faculty Publications. 29. https://uknowledge.uky.edu/surgery_facpub/29 This Review is brought to you for free and open access by the Surgery at UKnowledge. It has been accepted for inclusion in Surgery Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. This review is available at UKnowledge: https://uknowledge.uky.edu/surgery_facpub/29 Healthcare 2014, 2, 356-400; doi:10.3390/healthcare2030356 healthcare ISSN 2227-9032 www.mdpi.com/journal/healthcare Review Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds Krishna S. Vyas and Henry C. Vasconez * Division of Plastic Surgery, Department of Surgery, University of Kentucky, Kentucky Clinic K454, 740 South Limestone, Lexington, KY 40536, USA; E-Mail: krishnavyas@uky.edu * Author to whom correspondence should be addressed; E-Mail: hcvasc@uky.edu; Tel.: +1-859-323-8082; Fax: +1-859-323-3823. Received: 26 May 2014; in revised form: 8 July 2014 / Accepted: 19 August 2014 / Published: 10 September 2014 Abstract: This review will explore the latest advancements spanning several facets of wound healing, including biologics, skin substitutes, biomembranes and scaffolds. Keywords: biologics; skin substitutes; biomembranes; scaffolds; wound healing OPEN ACCESS Healthcare 2014, 2, 356-400; doi:10.3390/healthcare2030356 Healthcare 2014, 2, 356-400; doi:10.3390/healthcare2030356 healthcare ISSN 2227-9032 www.mdpi.com/journal/healthcare Review Wound Healing: Biologics, Skin Substitutes, Biomembranes and Scaffolds Krishna S. Vyas and Henry C. Vasconez * Division of Plastic Surgery, Department of Surgery, University of Kentucky, Kentucky Clinic K454, 740 South Limestone, Lexington, KY 40536, USA; E-Mail: krishnavyas@uky.edu * Author to whom correspondence should be addressed; E-Mail: hcvasc@uky.edu; Tel.: +1-859-323-8082; Fax: +1-859-323-3823. Received: 26 May 2014; in revised form: 8 July 2014 / Accepted: 19 August 2014 / Published: 10 September 2014 Abstract: This review will explore the latest advancements spanning several facets of wound healing, including biologics, skin substitutes, biomembranes and scaffolds. Keywords: biologics; skin substitutes; biomembranes; scaffolds; wound healing OPEN ACCESS healthcare ISSN 2227-9032 www.mdpi.com/journal/healthcare OPEN ACCESS © 2014 by the authors; licensee MDPI, Basel, Switzerland. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the C This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). Commons Attribution license (http://creativecommons.org/licenses/by/3.0/) 1. Introduction The healing of wounds is a complex process that involves the activation and synchronization of intracellular, intercellular and extracellular elements, including coagulatory and inflammatory events, fibrous tissue accretion, deposition of collagen, epithelialization, wound contraction, tissue granulation and remodeling [1]. This process occurs via activation of local and systemic cells to restore tissue integrity through regeneration and scar formation, and often these cumulative processes result in satisfactory repair of damaged sites. Disruptions caused by tissue loss, inadequate blood flow, and comorbid disease states can lead to chronic wounds that are difficult to manage [2]. There are many strategies that have been applied to the treatment of wounds in the past. Early on, these were based on empirical deduction and unsubstantiated determinations. Although there was a general resistance to new concepts and modalities that impeded progress, advancements in the treatment of wounds have, nevertheless, evolved [3]. Over the past two decades, advancements in the clinical understanding of wounds and their pathophysiology have commanded significant biomedical innovations in the treatment of acute, chronic, and other types of wounds. This review will explore the latest advancements spanning several facets of wound healing, including biologics, skin substitutes, biomembranes and scaffolds. Krishna S. Vyas and Henry C. Vasconez * Division of Plastic Surgery, Department of Surgery, University of Kentucky, Kentucky Clinic K454, 740 South Limestone, Lexington, KY 40536, USA; E-Mail: krishnavyas@uky.edu * Author to whom correspondence should be addressed; E-Mail: hcvasc@uky.edu; T l 1 859 323 8082 F 1 859 323 3823 Division of Plastic Surgery, Department of Surgery, University of Kentucky, Kentucky Clinic K454, 740 South Limestone, Lexington, KY 40536, USA; E-Mail: krishnavyas@uky.edu * Author to whom correspondence should be addressed; E-Mail: hcvasc@uky.edu; Tel.: +1-859-323-8082; Fax: +1-859-323-3823. Received: 26 May 2014; in revised form: 8 July 2014 / Accepted: 19 August 2014 / Published: 10 September 2014 Abstract: This review will explore the latest advancements spanning several facets of wound healing, including biologics, skin substitutes, biomembranes and scaffolds. Abstract: This review will explore the latest advancements spanning several facets of wound healing, including biologics, skin substitutes, biomembranes and scaffolds. Abbreviations RNA Ribonucleic Acid IL-6 Interleukin 6 TNF-α Tumor Necrosis Factor Alpha LTC4 Leukotriene C4 TXB2 Thromboxane B2 UVB Ultraviolet B MIF Migration Inhibitory Factor NO Nitric Oxide RCT Randomized Controlled Trial TBSA Total Body Surface Area STSG Split-Thickness Skin Graft COX-2 Cyclooxygenase-2 IL-1β Interleukin-1 beta NF-κB Nuclear Factor kappa-light-chain-enhancer of activated B cells Healthcare 2014, 2 IL-10 Interleukin 10 ATP Adenosine Triphosphate KATP Potassium Channels CEA Cultured Epithelial Autograft HDE Humanitarian Device Exemption DFU Diabetic Foot Ulcers PMA Premarket Approval LOS Length of Stay TGF-β Transforming Growth Factor-beta CACs Circulating Angiogenic Cells OPN Osteopontin CCPE Collagen Coated Porous Polyethylene PMB Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) UPPE Uncoated Porous Polyethylene DNA Deoxyribonucleic Acid P3HT Photosensitive Polymer Poly (3-hexylthiophene) CGS Collagen/Gelatin Sponge 357 357 Healthcare 2014, 2 Healthcare 2014, 2 Healthcare 2014, 2 contamination. They also permit autolytic debridement and develop a granular wound bed. Biological skin equivalents, epidermal growth factors, stem cell therapies, and tissue engineering might also be utilized [2]. 2.1. Description Biologic wound healing therapies are those that are intended to facilitate the re-establishment of the innate repair mechanisms, and may involve the application of active biological agents, such as plant-derived active biomolecules which exhibit antioxidant, antimicrobial, or anti-inflammatory attributes. Biologic dressings prevent evaporative water loss, heat loss, protein and electrolyte loss, and 358  No clinical trials in wound healing. Table 1. Monoterpenes in wound healing. Table 1. Monoterpenes in wound healing. p g Monoterpene Company (FDA Approval) Composition Mechanism Clinical Trials Sulbogin® (SuileTM) ointment wound dressing Hedonist Biochemical Technologies Co, Taipei, Taiwan (2001, 2003) 0.7% borneol, 4.5% bismuth subgallate, Vaseline® bismuth subgallate induces macrophages to secrete growth factors to facilitate wound healing [6] decreases lesion area, enhances granulation tissue formation and re-epithelialization, initiates proliferation of collagen via the activation of fibroblasts, accelerates reestablishment of blood vessels, restricts the formation of nitric oxide [4]  Indicated for first- and second-degree burns, partial-thickness wounds, donor sites and abrasions.  In a study evaluating the effect of bismuth subgallate on biopsy punch wounds on Wistar rats, bismuth subgallate had a statistically significant improvement in the area of ulceration (day 1), distance between epithelial edges (day 4), and area of granulation tissue (day 7, 11, 18) compared to control. No significant histological differences were identified between the test and control [15].  A study of adult male rats with full-thickness wounds were evaluated using the treatment bismuth and borneol, the major components of Sulbogin® with control treatment flamazine. The experimental treatment decreased the wound lesion area, increased granulation tissue formation and re-epithelialization [6]. thymol N/A monoterpenic phenol which is usually found in thyme oil modulates prostaglandin synthesis [7]; anti-inflammatory; inhibits myeloperoxidase activity [8,9]; oxidant effects on docosahexaenoic acid [10]; prevents lipid autoxidation [11] and formation of toxic elements via the stimulation of reactive nitrogen species [12]; enhances collagen synthesis and fibroblast metabolism [9]; antimicrobial; anesthetic [16]  Wounds dressed with collagen-based containing thymol films showed significantly larger wound retraction rates at 7 and 14 days, improved granulation reaction, and better collagen density and arrangement [9].  Gelatin films impregnated with thymol have antioxidant and antimicrobial properties against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa [17]. α-terpineol N/A monoterpene alcohol derived from pine and other oils inhibits generation of prostaglandin-endoperoxide synthase [18], COX-2 [19], IL-1β [20], IL-6 [21], NF-κB [20], TNF-α and NO production [21]; increased expression of IL-10; inhibits neutrophil influx [22]; antimicrobial [23]; antifungal [24]  No clinical trials in wound healing. Clinical Trials  Indicated for first- and second-degree burns, partial-thickness wounds, donor sites and abrasions. 2.2. Mechanisms and Indications Monoterpenes represent an extensive and varied family of naturally occurring terpene-based chemical compounds that comprise the majority of essential oils. These compounds exhibit anti-inflammatory, antibacterial, and antioxidant attributes [4,5]. The primary mechanisms proposed for various monoterpenes encompass: antimicrobial activity (inhibition of microorganism ribonucleic acid (RNA) and protein biosynthesis); anti-inflammation (lowers the generation of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in mast cells, inhibition and alteration of leukotriene C4 (LTC4) release and thromboxane B2 (TXB2) release, respectively); antioxidation (inhibits the production of ultraviolet B (UVB)-induced free radicals photoprotective effects and oxidative stress); fibroblast growth and macrophage migration inhibitory factor (MIF) effects. The anti-inflammatory action of the monoterpenes is often correlated to their wound-healing effects. Monoterpenes include compounds such as borneol, thymol, α-terpineol, genipin, aucubin, d-Limonene and sericin that have either direct or indirect activities in wound healing. Although monoterpenes are poorly studied in the context of wound healing, studies suggest that they are promising for the treatment of chronic wounds (Table 1). Mai et al. [6] investigated the ointment Sulbogin® (marketed as SuileTM), comprised of borneol (a bicyclic monoterpenoid alcohol), bismuth subgallate and Vaseline®, and found it to hasten excision wound closure in adult male Sprangue-Dawley rats. Although the specific mechanism remains elusive, it is thought that bismuth subgallate may induce macrophages to secrete growth factors to facilitate wound healing. It was found to decrease the lesion area, enhance granulation tissue formation and re-epithelialization, initiate the proliferation of collagen via the activation of fibroblasts, accelerate the reestablishment of blood vessels, and restrict the formation of nitric oxide (NO) [4,6]. The monoterpenoid phenol, thymol, demonstrates multiple beneficial bioactivities toward the healing of wounds. These attributes encompass the modulation of prostaglandin synthesis [7], imparting anti-inflammatory effects in neutrophils, the inhibition of myeloperoxidase activity and a decreased influx of leukocytes [8,9], positive antioxidant effects on docosahexaenoic acid (an omega-3 fatty acid) concentrations [10], the prevention of lipid autoxidation [11] and formation of toxic elements via the stimulation of reactive nitrogen species [12], and antimicrobial activity [13,14]. The capacity of thymol for direct wound healing involves its being correlated with elevated concentrations, in the central nervous system, of macrophage MIF, as well as enhanced anti-inflammatory related tissue granulation. Furthermore, it influences collagen synthesis and fibroblast metabolism, leading to augmented fibroblast growth in vitro [9]. 359 Healthcare 2014, 2 Table 1. Monoterpenes in wound healing.  No clinical trials in wound healing. Table 1. Monoterpenes in wound healing. e  In a study evaluating the effect of bismuth subgallate on biopsy punch wounds on Wistar rats, bismuth subgallate had a statistically significant improvement in the area of ulceration (day 1), distance between epithelial edges (day 4), and area of granulation tissue (day 7, 11, 18) compared to control. No significant histological differences were identified between the test and control [15].  Wounds dressed with collagen-based containing thymol films showed significantly larger wound retraction rates at 7 and 14 days, improved granulation reaction, and better collagen density and arrangement [9].  Wounds dressed with collagen-based containing thymol films showed significantly larger wound retraction rates at 7 and 14 days, improved granulation reaction, and better collagen density and arrangement [9].  Gelatin films impregnated with thymol have antioxidant and antimicrobial properties against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa [17].  No clinical trials in wound healing. 360 Healthcare 2014, 2 Table 1. Cont. noterpene Company (FDA Approval) Composition Mechanism Clinical Trials genipin N/A fruit extract aglycone derived from iridoid glycoside crosslinking agent [25,26]; antioxidant [27]; anti-inflammatory [28]; stimulates NO production; inhibits lipid peroxidation; elevates potential of mitochondrial membranes; elevates secretion of insulin; increases ATP levels; closes KATP channels [29]  No clinical trials in wound healing.  Genipin hydrogels [30], nanogels [31], and genipin cross-linked scaffolds [32] have potential application in skin tissue engineering [33] and wound dressings [34–36] and demonstrate excellent biocompatibility and low cytotoxicity in scaffolding models [37,38]. In biomaterials studies, genipin-crosslinked gels enhance fibroblast attachment [39] and vascularization of engineered tissues [38,40] and exhibit bacterial inhibition [41].  Genipin-crosslinked gelatin-silk fibroin hydrogels have been shown to induce pluripotent cells to differentiate into epidermal lineages [42]. Genipin as a crosslinking agent is also utilized in controlling drug delivery in multiple systems [43]. aucubin N/A iridoid glycoside found in plants anti-inflammatory [44], antimicrobial, antioxidant, chemopreventive agent  No clinical trials in wound healing.  In a study of male mice with full-thickness buccal mucosal oral wounds, 0.1% aucubin-treated mice demonstrated earlier re-epithelization and matrix formation and decreased numbers of inflammatory cells compared to saline-treated controls at 1, 3, and 5 days, suggesting utility of topical aucubin in oral wound healing [45]. Table 1. Monoterpenes in wound healing. Clinical Trials  Genipin hydrogels [30], nanogels [31], and genipin cross-linked scaffolds [32] have potential application in skin tissue engineering [33] and wound dressings [34–36] and demonstrate excellent biocompatibility and low cytotoxicity in scaffolding models [37,38]. In biomaterials studies, genipin-crosslinked gels enhance fibroblast attachment [39] and vascularization of engineered tissues [38,40] and exhibit bacterial inhibition [41].  Genipin-crosslinked gelatin-silk fibroin hydrogels have been shown to induce pluripotent cells to differentiate into epidermal lineages [42]. Genipin as a crosslinking agent is also utilized in controlling drug delivery in multiple systems [43].  In a study of male mice with full-thickness buccal mucosal oral wounds, 0.1% aucubin-treated mice demonstrated earlier re-epithelization and matrix formation and decreased numbers of inflammatory cells compared to saline-treated controls at 1, 3, and 5 days, suggesting utility of topical aucubin in oral wound healing [45]. 361 Healthcare 2014, 2 Healthcare 2014, 2 Table 1. Cont. Table 1. Cont. Monoterpene Company (FDA Approval) Composition Mechanism Clinical Trials d-Limonene N/A orange-peel derived terpene d-Limonene anti-angiogenic, anti-inflammatory; decreases systemic cytokines; inhibits expression of endothelial P-selectin  No clinical trials in wound healing.  Topical d-Limonene and its metabolite perillyl alcohol were tested in murine models of chemically-induced dermatitis and mechanical skin lesions. Both significantly reduced the severity and extent of chemically-induced dermatitis. Lower levels of the inflammatory cytokines IL-6 and TNF-α, reduced neovascularization, and lower levels of P-selectin expression were observed in both models. Both d-Limonene and perillyl alcohol demonstrated anti- inflammatory effects in wound healing. Together, these effects contribute to the wound healing effects of d-Limonene [46].  Nanophyto-modified wound dressings with limonene are resistant to Staphylococcal and Pseudomonal colonization and biofilm formation compared to uncoated controls [47].  Topical limonene and other terpenes can increase permeation of silver sulphadiazine by increasing its partitioning into eschars. Burn wound antimicrobial therapy may be improved through the use of terpenes [48]. Table 1. Cont. Monoterpene Company (FDA Approval) Composition Mechanism Clinical Trials d-Limonene N/A orange-peel derived terpene d-Limonene anti-angiogenic, anti-inflammatory; decreases systemic cytokines; inhibits expression of endothelial P-selectin  No clinical trials in wound healing.  Topical d-Limonene and its metabolite perillyl alcohol were tested in murine models of chemically-induced dermatitis and mechanical skin lesions. Both significantly reduced the severity and extent of chemically-induced dermatitis. Lower levels of the inflammatory cytokines IL-6 and TNF-α, reduced neovascularization, and lower levels of P-selectin expression were observed in both models. Table 1. Monoterpenes in wound healing. No infections or adverse reactions were found in any of the wounds [49]. No infections or adverse reactions were found in any of the wounds [49].  A clinical study on silk sericin-releasing wound dressing was compared to the wound dressing Bactigras® in a clinical trial in patients with split-thickness skin graft (STSG) donor sites. The sericin dressing was less adhesive to the wound and potentially less traumatic. Wounds treated with the silk sericin dressing exhibited significantly faster rates to complete healing (12 ± 5.0 days compared to 14 ± 5.2 days) and significantly reduced pain during the first four days post- operatively [50]. In rat models, silk sericin dressing also demonstrated accelerated wound healing and greater epithelialization and type III collagen formation in full-thickness wounds [51–53].  A clinical study on silk sericin-releasing wound dressing was compared to the wound dressing Bactigras® in a clinical trial in patients with split-thickness skin graft (STSG) donor sites. The sericin dressing was less adhesive to the wound and potentially less traumatic. Wounds treated with the silk sericin dressing exhibited significantly faster rates to complete healing (12 ± 5.0 days compared to 14 ± 5.2 days) and significantly reduced pain during the first four days post- operatively [50]. In rat models, silk sericin dressing also demonstrated accelerated wound healing and greater epithelialization and type III collagen formation in full-thickness wounds [51–53]. sericin N/A protein created by silkworms (Bombyx mori) stimulates migration of fibroblasts; generates collagen in wounds, leading to activation of epithelialization; anti-inflammatory; initiates propagation and attachment of skin fibroblasts and keratinocytes sericin N/A protein created by silkworms (Bombyx mori) stimulates migration of fibroblasts; generates collagen in wounds, leading to activation of epithelialization; anti-inflammatory; initiates propagation and attachment of skin fibroblasts and keratinocytes Several animal studies conclude that sericin promotes the wound healing process without causing inflammation [54]. Sericin treated full-thickness skin wounds in rats demonstrated less inflammation, greater wound size reduction and shorter mean time to healing compared to control (betadine treated full- thickness skin wounds). Examination after 15 days of 8% sericine treatment revealed complete healing, increased collagen formation, and no ulceration compared to cream base-treated wounds which demonstrated inflammatory exudates and ulceration [55].  3D hydrogels [56] and cultured fibroblasts and keratinocytes on three- dimensional sericin matrices can potentially be used as skin equivalents in wound repair [57]. Table 1. Monoterpenes in wound healing. Both d-Limonene and perillyl alcohol demonstrated anti- inflammatory effects in wound healing. Together, these effects contribute to the wound healing effects of d-Limonene [46].  Nanophyto-modified wound dressings with limonene are resistant to Staphylococcal and Pseudomonal colonization and biofilm formation compared to uncoated controls [47].  Topical limonene and other terpenes can increase permeation of silver sulphadiazine by increasing its partitioning into eschars. Burn wound antimicrobial therapy may be improved through the use of terpenes [48]. Clinical Trials  No clinical trials in wound healing.  Topical d-Limonene and its metabolite perillyl alcohol were tested in murine models of chemically-induced dermatitis and mechanical skin lesions. Both significantly reduced the severity and extent of chemically-induced dermatitis. Lower levels of the inflammatory cytokines IL-6 and TNF-α, reduced neovascularization, and lower levels of P-selectin expression were observed in both models. Both d-Limonene and perillyl alcohol demonstrated anti- inflammatory effects in wound healing. Together, these effects contribute to the wound healing effects of d-Limonene [46].  Topical d-Limonene and its metabolite perillyl alcohol were tested in murine models of chemically-induced dermatitis and mechanical skin lesions. Both significantly reduced the severity and extent of chemically-induced dermatitis. Lower levels of the inflammatory cytokines IL-6 and TNF-α, reduced neovascularization, and lower levels of P-selectin expression were observed in both models. Both d-Limonene and perillyl alcohol demonstrated anti- inflammatory effects in wound healing. Together, these effects contribute to the wound healing effects of d-Limonene [46]. d-Limonene N/A orange-peel derived terpene d-Limonene anti-angiogenic, anti-inflammatory; decreases systemic cytokines; inhibits expression of endothelial P-selectin d-Limonene  Nanophyto-modified wound dressings with limonene are resistant to Staphylococcal and Pseudomonal colonization and biofilm formation compared to uncoated controls [47].  Topical limonene and other terpenes can increase permeation of silver sulphadiazine by increasing its partitioning into eschars. Burn wound antimicrobial therapy may be improved through the use of terpenes [48]. 362 Healthcare 2014, 2 Table 1. Cont. Clinical Trials  Double blinded randomized controlled trial (RCT) of 65 burn wounds of greater than 15% total body surface area (TBSA) were randomly assigned to either control (silver zinc sulfadiazine cream) or treatment (silver zinc sulfadiazine cream with sericin cream at a concentration of 100 μg/mL). Time to complete healing was significantly shorter for the treatment group (22.42 ± 6.33 days) compared to the control group (29.28 ± 9.27 days). No infections or adverse reactions were found in any of the wounds [49]. Healthcare 2014, 2 The monoterpenoid alcohol, α-terpineol conveys its wound healing [61] and anti-inflammatory activities via the inhibition of the generation of prostaglandin-endoperoxide synthase enzymes [18], cyclooxygenase-2 (COX-2) [19], interleukin-1 beta (IL-1β) [20] and IL-6 cytokines [21], nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [20], TNF-α and NO production [21]. Increased expression of the anti-inflammatory cytokine interleukin 10 (IL-10) is also observed. Additionally, it exhibits inhibitory effects on neutrophil influx [22], as well as robust antimicrobial [23] and antifungal activities [24]. Significant activity in tissue/scar formation is also observed with α-terpineol [61]. Cross-linkers are one of the many factors that affect the mechanical and biological properties of scaffolds used in tissue engineering. The iridoid (a secondary monoterpenoid metabolite) compound genipin may serve as a biocompatible crosslinking agent that imparts minimal cytotoxicity [25,26]. Additionally, it is an antioxidant [27] and anti-inflammatory that stimulates the generation of NO while inhibiting lipid peroxidation [28]. It also serves to elevate the potential of mitochondrial membranes, to elevate the secretion of insulin, to increase adenosine triphosphate (ATP) levels and to close potassium channels (KATP) [29], among other positive effects in wound healing [36,62]. Aucubin (an iridoid glycoside) was found to have beneficial pharmacological activities on a number of fronts, encompassing dermal wound healing [44,45,63], and capacities as an anti-inflammatory [44], antimicrobial [64], and antioxidant [65]. In addition to various specific biochemical effects, it also shows promise as a non-cytotoxic chemopreventive agent [66]. D’Alessio et al. [46] revealed that the prototype monoterpene d-Limonene in combination with its metabolite perillyl alcohol, which is derived from orange-peel, exhibited considerable anti-angiogenic, anti-inflammatory properties, epidermal repair and wound healing effects in murine models. These compounds also lowered the generation of systemic cytokines and inhibited the expression of endothelial P-selectin. Topical treatment resulted in more rapid and improved wound closure. Aramwit et al. [49] revealed that a protein derived from the silkworm cocoon called silk sericin acted to enhance the capacity for wound (second-degree burns) healing when incorporated into a common silver zinc sulfadiazine antimicrobial cream. At a concentration of 100 μg/mL, sericin was shown to stimulate the migration of fibroblasts. Siritientong et al. [35] discovered that silk sericin had the capacity to generate collagen in wounds, which led to the activation of epithelialization. Further, it served to reduce inflammation [67] and to initiate the propagation and attachment of human skin fibroblasts and keratinocytes [55,68,69]. 2.3. Contraindications Contraindications for biologics such as the monoterpenes are low. Acute toxicity of the monoterpenes is low via the oral and dermal routes of exposure in animal models [70]. Table 1. Monoterpenes in wound healing.  3D hydrogels [56] and cultured fibroblasts and keratinocytes on three- dimensional sericin matrices can potentially be used as skin equivalents in wound repair [57].  Sericin/chitosan composite nanofibers demonstrate wide spectrum bactericidal activity [58]. Sericin enriched wound dressings represent significant promise in wound healing biologics [35,59,60]. 363 3.1. Description Skin substitutes are tissue-engineered products designed to replace, either temporarily or permanently, the form and function of the skin. Skin substitutes are often used in chronic, non-healing ulcers, such as pressure ulcers, diabetic neuropathic ulcers and vascular insufficiency ulcers. Epidermal Skin Replacement duct Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages gous keratinocytes urine fibroblasts are d to form epidermal afts which are then sed into sheets and onto petroleum [71]. It is used as an nt to STSG or alone G are not available ent or severity of the Humanitarian Device Exemption (HDE) for treatment of deep dermal or full thickness burns (greater than or equal to 30% TBSA); grafting after congenital nevus removal (diabetic and venous ulcers) Burns  No RCT have been conducted to evaluate the effectiveness of this product in improving health outcomes for deep dermal/full thickness burns.  In a large, single center trial, Epicel® CEA was applied to 30 burn patients with a mean TBSA of 37% ± 17% TBSA. Epicel® achieved permanent coverage of a mean of 26% TBSA compared to conventional autografts (mean 25%). Final CEA take was a mean 69% ± 23%. Ninety percent of these severely burned patients survived [72]. Advantages  Use of autologous cells obviates rejection  Permanent large area wound coverage, especially in extensive burns [73] Disadvantages  Long culture time (3 weeks)  Variable take rate  Poor long-term results  1 day shelf life [74]  Expensive  Risk of blistering, contractures, and infection FDA Indications (Other Indications) ns ns) Clinical Trials Advantages Disadvantages ce or rmal ns l to g us Burns  No RCT have been conducted to evaluate the effectiveness of this product in improving health outcomes for deep dermal/full thickness burns.  In a large, single center trial, Epicel® CEA was applied to 30 burn patients with a mean TBSA of 37% ± 17% TBSA. Epicel® achieved permanent coverage of a mean of 26% TBSA compared to conventional autografts (mean 25%). Final CEA take was a mean 69% ± 23%. Ninety percent of these severely burned patients survived [72]. Advantages  Use of autologous cells obviates rejection  Permanent large area wound coverage, especially in extensive burns [73] Disadvantages  Long culture time (3 weeks)  Variable take rate  Poor long-term results  1 day shelf life [74]  Expensive  Risk of blistering, contractures, and infection Healthcare 2014, 2 364 These wounds contribute to substantial morbidity such as increased risk for infection, limb amputation, and death. Skin substitutes have the potential to improve rates of healing and reduce complications in a variety of other skin wounds including, but not limited to, wounds from burn injuries, ischemia, pressure, trauma, surgery and skin disorders. Skin substitutes are also used in patients whose ability to heal is compromised and in situations where skin coverage is inadequate. Goals for treating acute and chronic wounds with skin substitutes are to provide temporary coverage or permanent wound closure, to reduce healing time, to reduce post-operative contracture, to improve function, and to decrease morbidity from more invasive treatments such as skin grafting. Skin substitutes can be categorized according to whether they are acellular or cellular. Acellular products, such as cadaveric human dermis with removed cellular components, contain a scaffold or matrix of hyaluronic acid, collagen, or fibronectin. Cellular products contain living cells such as keratinocytes and fibroblasts within a matrix. These cells can be autologous, allogeneic, or from another species. Skin substitutes can be divided into three major categories: dermal replacement, epidermal replacement and dermal/epidermal replacement. They can also be used as either permanent or temporary wound coverings. A large number of skin substitutes are commercially available or in development. Table 2 details epidermal, dermal, and combined, full-thickness skin replacements that have clinical and experimental evidence of efficacy in wound healing. Information regarding type of skin replacement, regulatory status and year of United States Food and Drug Administration (U.S. FDA) approval, product description, indications, clinical and experimental trials according to wound type, and advantages and disadvantages for each product are detailed. Epidermal skin replacements require a skin biopsy from which keratinocytes are isolated and cultured on top of fibroblasts. Epicel® (Genzyme Tissue Repair Corporation, Cambridge, MA, USA) is an epidermal skin substitute composed of cultured autogeneous keratinocytes used for permanent coverage in partial or full-thickness wounds. Laserskin® (Fidia Advanced Biopolymers, Abano Terme, Italy) is composed of autologous keratinocytes and fibroblasts cultured on a laser-microperforated biodegradable matrix of benzyl esterified hyaluronic acid. Healthcare 2014, 2 365 Table 2. Skin substitutes for wound healing. Epidermal Skin Replacement Advantages Disadvantages FDA Indications (Other Indications) Diabetic Foot Ulcers (DFUs)  A multicenter RCT with unhealed (≥1 month) DFUs randomized 180 patients to receive intervention (Hyalograft-3D® autograft and then Laserskin® autograft after two weeks) or control (paraffin gauze). At 12 weeks, a 50% reduction in the intervention group was achieved significantly faster compared to control (40 versus 50 days). Complete ulcer healing was similar in both groups. The rate of ulcer reduction was greater in the treatment group. There was a significantly (3.65-fold) better chance of wound healing in a subgroup of hard-to-heal ulcers following autograft treatment of dorsal ulcers [79].  A multicenter RCT with unhealed (≥1 month) DFUs randomized 180 patients to receive intervention (Hyalograft-3D® autograft and then Laserskin® autograft after two weeks) or control (paraffin gauze). At 12 weeks, a 50% reduction in the intervention group was achieved significantly faster compared to control (40 versus 50 days). Complete ulcer healing was similar in both groups. The rate of ulcer reduction was greater in the treatment group. There was a significantly (3.65-fold) better chance of wound healing in a subgroup of hard-to-heal ulcers following autograft treatment of dorsal ulcers [79]. Advantages  Use of autologous cells obviates rejection  Can be produced in shorter period of time than confluent epidermal sheets Laserskin® Fidia Advanced Biopolymers Abano Terme, Italy Permanent skin substitute autologous keratinocytes and fibroblasts derived from a skin biopsy cultured on a laser-microperforated biodegradable matrix of benzyl esterified hyaluronic acid [75,76]. Cells proliferate and migrate through the matrix. Microperforations allow for drainage of wound exudate. (dia ven par viti  In a study of chronic (>6 months) foot ulcers over 15 cm2 in type 2 diabetic patients older than 65 years treated with Hyalograft-3D® and Laserskin® autograft, all ulcers healed at 12 months except for one, with a median healing time of 21 weeks [80].  Does not require the use of the enzyme dispase to remove the sheets from culture flasks, in contrast to CEA  In a study of 14 patients with chronic (>6 months), non-healing foot ulcers secondary to type 2 diabetes treated with Laserskin® autograft, 11/14 lesions were completely healed between 7 and 64 days post-transplantation [81]. (diabetic foot ulcers and venous leg ulcers, partial thickness burns, vitiligo) [77,78] Clinical Trials Epicel® Genzyme Tissue Repair Corporation Cambridge, MA, USA (2007) Permanent skin substitute Living Cell Therapy Cultured Epithelial Autograft (CEA) autologous keratinocytes with murine fibroblasts are cultured to form epidermal autografts which are then processed into sheets and placed onto petroleum gauze [71]. It is used as an adjuvant to STSG or alone if STSG are not available due to the extent or severity of the burns. Humanitarian Device Exemption (HDE) for treatment of deep dermal or full thickness burns (greater than or equal to 30% TBSA); grafting after congenital nevus removal (diabetic and venous ulcers) Humanitarian Device Exemption (HDE) for treatment of deep dermal or full thickness burns (greater than or equal to 30% TBSA); grafting after congenital nevus removal Epicel® Genzyme Tissue Repair Corporation Cambridge, MA, USA (2007)  Risk of blistering, contractures, and infection Healthcare 2014, 2 366 Table 2. Cont. Epidermal Skin Replacement Advantages Disadvantages Wounds  In a retrospective observational study in 30 patients with chronic wounds not responding to conventional therapy, keratinocytes on Laserskin® to treat superficial wounds or fibroblasts on Hyalograft-3D® to treat deep leg ulcers were applied; the wounds were then dressed with nanocrystalline silver dressing. A reduction in wound dimension and exudates and an increase in wound bed score was observed. The group treated with keratinocytes had a significantly greater degree of healing compared to those treated with allogenic fibroblasts [82].  Collagen matrices such as Integra® have been poor recipients of cultured keratinocytes, although some studies report successes in the use of Laserskin® on the neodermis of Integra® after the silicone membrane is removed 14–21 days post-grafting [83,84]. 367 Healthcare 2014, 2 Table 2. Cont. Dermal skin replacement Dermal skin replacement FDA Indications (Other Indications) Advantages Disadvantages Burns  33 children with partial-thickness burn wounds were randomized to receive TransCyte®, Biobrane®, or Silvazine cream. Mean time to re-epithelization was 7.5 days, 9.5 days, and 11.2 days, respectively. Wounds requiring autografting were 5%, 17%, and 24%, respectively. TransCyte® promoted faster re-epithelization, required fewer dressings, and required less autograft compared to those treated with Biobrane® or Silvazine [86].  In a randomized prospective study of 21 adults with partial-thickness burn wounds to the face, patients treated with TransCyte® had significantly decreased daily wound care time (0.35 ± 0.1 versus 1.9 ± 0.5 h), re-epithelialization time (7 ± 2 versus 13 ± 4 days), and pain (2 ± 1 vs. 4 ± 2) compared to patients treated with topical bacitracin [87]. TransCyte® Shire Regenerative Medicine, Inc. San Diego, CA, USA; Smith & Nephew, Inc., Largo, FL, USA (1997) Temporary skin substitute Composite matrix TransCyte® Shire Regenerative Medicine, Inc. San Diego, CA, USA; Smith & Nephew, Inc., Largo, FL, USA (1997) Advantages  Easy to remove compared to allograft  Widely used for partial- thickness burns  Improved healing rate  1.5 year shelf life Disadvantages  Expensive  20 pediatric patients with TBSA over 7% were treated with TransCyte® and compared to previous patients those who received standard therapy of antimicrobial ointment and hydrodebridement. Only one child required autografting in the TransCyte® group, compared to 7 children in the standard treatment group. In addition, children treated with TransCyte® had a significantly decreased length of stay (5.9 days) compared to those who received standard therapy (13.8 days) [88].  110 patients with deep partial-thickness burns were treated with dermabrasion and TransCyte® and compared with data from the American Burn Association Patient Registry. Patients with 0–19.9% TBSA burn treated with dermabrasion and TransCyte® had a significantly shorter length of stay of 6.1 days versus 9.0 days. The authors compared burns of all sizes with dermabrasion and TransCyte® and concluded that this method of managing partial-thickness burns reduced length of stay compared to standard care [89].  110 patients with deep partial-thickness burns were treated with dermabrasion and TransCyte® and compared with data from the American Burn Association Patient Registry. Patients with 0–19.9% TBSA burn treated with dermabrasion and TransCyte® had a significantly shorter length of stay of 6.1 days versus 9.0 days. Advantages  Semitransparency allows continuous observation of underlying wound surface  Cell bank fibroblasts have been tested for safety and there have been no safety issues thus far Burns The authors compared burns of all sizes with dermabrasion and TransCyte® and concluded that this method of managing partial-thickness burns reduced length of stay compared to standard care [89]. 368 Healthcare 2014, 2 Table 2. Cont. Dermal skin replacement Advantages Disadvantages FDA Indications (Other Indications) Wounds  A randomized prospective comparison study of TransCyte® and silver sulfadiazine on 11 patients with paired wound sites was performed. Wounds treated with TransCyte® had significantly quicker healing times to re-epithelialization (mean 11.14 days vs. 18.14 days). Wound evaluations at 3, 6, and 12 months revealed that wounds treated with TransCyte® healed with significantly less hypertrophic scarring than those treated with silver sulfadiazine [90].  A randomized prospective comparison study of TransCyte® and silver sulfadiazine on 11 patients with paired wound sites was performed. Wounds treated with TransCyte® had significantly quicker healing times to re-epithelialization (mean 11.14 days vs. 18.14 days). Wound evaluations at 3, 6, and 12 months revealed that wounds treated with TransCyte® healed with significantly less hypertrophic scarring than those treated with silver sulfadiazine [90]. Dermagraft® Shire Regenerative Medicine, Inc. San Diego, CA, USA (2001) Permanent or temporary skin substitute Living Cell Therapy Allogenic matrix derived from human neonatal fibroblast cryopreserved allogenic neonatal fibroblasts derived from neonatal foreskin and cultured on bioabsorbable collagen on polyglactin (Dexon) or polyglactin-910 (Vicryl) mesh for several weeks [91]. The biodegradable mesh disappears after 3–4 weeks Premarket approval (PMA) for full-thickness diabetic lower extremity ulcers present for longer than 6 weeks extending through the dermis but not to the tendon, muscle, or bone [92] (Chronic wounds, and noninfected wounds. It can be used as a temporary or permanent covering to support take of meshed STSG on excised burn wounds [93,94]) DFUs  A multicenter RCT with 314 patients with chronic DFUs to Dermagraft® or conventional therapy was performed. At 12 weeks, 30% of the Dermagraft® patients had complete wound closure compared to 18.3% of control patients. Although the incidence of adverse events was similar for both groups, the Dermagraft group (19%) experienced significantly fewer ulcer-related adverse events (infection, osteomyelitis, cellulitis) compared to the control group (32.5%) [95]. Dermagraft® Shire Regenerative Medicine, Inc. San Diego, CA, USA (2001) Permanent or temporary skin substitute Living Cell Therapy Allogenic matrix derived from human neonatal fibroblast  Easier to remove and higher patient satisfaction compared to allograft [94]  A prospective, multicenter RCT in 28 patients with chronic DFUs (>6 weeks duration) comparing intervention (Dermagraft® + saline gauze) to control (saline gauze) was performed. By week 12, significantly more DFUs healed in the intervention (71.4%) compared to the control (14.3%). Wounds closed significantly faster in patients treated with Dermagraft® and the percentage of patients with wound infection was less in the Dermagraft® group [96]. patient satisfaction compared to allograft [94]  Equivalent or better than allograft for graft take [93], wound healing time, wound exudate and infection  No adverse reactions, such as evidence of rejection [93]  A prospective, multicenter RCT in 28 patients with chronic DFUs (>6 weeks duration) comparing intervention (Dermagraft® + saline gauze) to control (saline gauze) was performed. By week 12, significantly more DFUs healed in the intervention (71.4%) compared to the control (14.3%). Wounds closed significantly faster in patients treated with Dermagraft® and the percentage of patients with wound infection was less in the Dermagraft® group [96].  Equivalent or better than allograft for graft take [93], wound healing time, wound exudate and infection  No adverse reactions, such as evidence of rejection [93] 369 Healthcare 2014, 2 Table 2. Cont. Dermal skin replacement FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages DFUs  The DOLCE trial (ID: NCT01450943) is a randomized, single-blind, comparative trial to compare the differences among acellular matrices (Oasis® (Healthpoint, Ltd Fort Worth, TX, USA), cellular matrices (Dermagraft® (Shire Regenerative Medicine, Inc.), and standard of care in the treatment of DFUs using the primary outcome of complete wound closure by 12 weeks [97].  A multicenter clinical trial of Dermagraft® in the treatment of DFUs in 62 patients after sharp debridement was performed. Patients received dressing changes with saline gauze or polyurethane foam dressings weekly. DFUs By week 12, 27/62 (44%) patients had complete wound closure, and 32/62 (52%) healed by week 20. Median time to healing was 13 weeks. Dermagraft® was safe and effective in the treatment of non-healing DFUs [98].  A prospective multicenter randomized single-blinded study to evaluate wound healing in 50 patients with DFUs was performed. Patients were randomized into one of four groups (three separate dosages of Dermagraft® and one control group). A dose response curve was observed and ulcers treated with the highest dosage of Dermagraft® healed significantly more than those treated with conventional wound closure methods. 50% (6/12) of the Dermagraft® and 8% (1/13) of the control ulcers healed completely. The percentage of ulcers to complete closure was significantly greater in the Dermagraft® group (50% or 6/12) compared to the control group (8% or 1/13) [99]. Disadvantages  Used for temporary coverage  6 month shelf life Contraindications  Clinically infected ulcers  Ulcers with sinus tracts  Hypersensitivity to bovine products Advantages Disadvantages Advantages Disadvantages FDA Indications (Other Indications) Clinical Trials Venous leg ulcers  A prospective multicenter RCT to evaluate Dermagraft® + compressive therapy versus compressive therapy alone in the treatment of venous leg ulcers was conducted. For ulcers ≤12 months duration, 49/94 (52%) patients in the Dermagraft® group versus 36/97 (37%) patients in the control group healed at 12 weeks and this was statistically significant. For ulcers ≤10 cm2, complete healing at 12 weeks was observed in 55/117 (47%) patients in the Dermagraft® group compared with 47/120 (39%) patients in the control group, and this was statistically significant. Both groups experienced similar rates of adverse events [100].  A prospective RCT in 18 patients with venous leg ulcers treated with Dermagraft® + compression therapy or compression therapy alone was performed. Healing was assessed through ulcer tracing and planimetry. The rate of healing was significantly improved in patients treated with Dermagraft® [101]. AlloDerm®/ Strattice® LifeCell Corporation Branchburg, NJ, USA (1992) Permanent skin substitute Living Cell Therapy Human skin allograft derived from donated human cadaver lyophilized human acellular cadaver dermal matrix serves as a scaffold for tissue remodeling [85] Burns, full thickness wounds [102] (breast surgery [103–105], soft tissue reconstruction [106]) DFUs  The DOLCE trial (ID: NCT01450943) is a randomized, single-blind, comparative trial to compare the differences among acellular matrices (Oasis® (Healthpoint, Ltd Fort Worth, TX, USA), cellular matrices (Dermagraft® (Shire Regenerative Medicine, Inc.), and standard of care in the treatment of DFUs using the primary outcome of complete wound closure by 12 weeks [97].  A multicenter clinical trial of Dermagraft® in the treatment of DFUs in 62 patients after sharp debridement was performed. Patients received dressing changes with saline gauze or polyurethane foam dressings weekly. By week 12, 27/62 (44%) patients had complete wound closure, and 32/62 (52%) healed by week 20. Median time to healing was 13 weeks. Dermagraft® was safe and effective in the treatment of non-healing DFUs [98].  A prospective multicenter randomized single-blinded study to evaluate wound healing in 50 patients with DFUs was performed. Patients were randomized into one of four groups (three separate dosages of Dermagraft® and one control group). A dose response curve was observed and ulcers treated with the highest dosage of Dermagraft® healed significantly more than those treated with conventional wound closure methods. 50% (6/12) of the Dermagraft® and 8% (1/13) of the control ulcers healed completely. The percentage of ulcers to complete closure was significantly greater in the Dermagraft® group (50% or 6/12) compared to the control group (8% or 1/13) [99]. 370 Healthcare 2014, 2 Table 2. Cont. Dermal skin replacement Dermal skin replacement Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Advantages Disadvantages n FDA Indications (Other Indications) Burns Dermal skin replacement Dermal skin replacement FDA Indications (Other Indications) Biologic Company A Approval) Product escription Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Wounds  36 patients with oral mucosal defects reconstructed with AlloDerm® grafts were evaluated. 34/36 cases (94.4%) were successfully replaced with mucosa and 2 grafts failed. Graft contraction occurred in 7/34 (20.6%) of patients with lip or buccal defects [109]. Advantages  Immediate permanent wound coverage  Allows grafting of ultra-thin STSG as one-stage procedure  Template for dermal regeneration  Immunologically inert since the cells responsible for immune response and graft rejection are removed during the processing  Reduced scarring  Can vascularize over exposed bone and tendon  2 year shelf life  Good aesthetic and functional outcomes (less hypertrophic scar rates, good movement)  Injectable micronized form is also available (Cymetra®) Disadvantages  Risk of transmission of infectious diseases, although no cases of viral transmission have been reported  No viral or prion screening  Collection fluid risk (seroma, hematoma, infection)  Possibility of donor rejection  Expensive  Requires two procedures  Inability to replace both dermal and epidermal components simultaneously Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Wounds  36 patients with oral mucosal defects reconstructed with AlloDerm® grafts were evaluated. 34/36 cases (94.4%) were successfully replaced with mucosa and 2 grafts failed. Graft contraction occurred in 7/34 (20.6%) of patients with lip or buccal defects [109]. Advantages  Immediate permanent wound coverage  Allows grafting of ultra-thin STSG as one-stage procedure  Template for dermal regeneration  Immunologically inert since the cells responsible for immune response and graft rejection are removed during the processing  Reduced scarring  Can vascularize over exposed bone and tendon  2 year shelf life  Good aesthetic and functional outcomes (less hypertrophic scar rates, good movement)  Injectable micronized form is also available (Cymetra®) Disadvantages  Risk of transmission of infectious diseases, although no cases of viral transmission have been reported  No viral or prion screening  Collection fluid risk (seroma, hematoma, infection)  Possibility of donor rejection  Expensive  Requires two procedures  Inability to replace both dermal and epidermal components simultaneously Advantages Burns LifeCell Corporation Branchburg, NJ, USA (1992) Permanent skin substitute Living Cell Therapy Human skin allograft derived from donated human cadaver lyophilized human acellular cadaver dermal matrix serves as a scaffold for tissue remodeling [85] Burns, full thickness wounds [102] (breast surgery [103–105], soft tissue reconstruction [106])  Three patients with full-thickness burns of the extremities were treated with AlloDerm® dermal grafts followed by thin autografts. Functional performance and aesthetics were considered good to excellent [107].  The average graft take rate in 12 patients with full-thickness burn injuries in joint areas was 91.5% at one year post AlloDerm® with ultrathin autograft. All patients had near normal range of motion at one year and aesthetic results were judged fair to good by both surgeons and patients [108]. LifeCell Corporation Branchburg, NJ, USA (1992) Permanent skin substitute Living Cell Therapy Human skin allograft derived from donated human cadaver lyophilized human acellular cadaver dermal matrix serves as a scaffold for tissue remodeling [85] Burns, full thickness wounds [102] (breast surgery [103–105], soft tissue reconstruction [106])  Three patients with full-thickness burns of the extremities were treated with AlloDerm® dermal grafts followed by thin autografts. Functional performance and aesthetics were considered good to excellent [107].  The average graft take rate in 12 patients with full-thickness burn injuries in joint areas was 91.5% at one year post AlloDerm® with ultrathin autograft. All patients had near normal range of motion at one year and aesthetic results were judged fair to good by both surgeons and patients [108].  Three patients with full-thickness burns of the extremities were treated with AlloDerm® dermal grafts followed by thin autografts. Functional performance and aesthetics were considered good to excellent [107]. 5],  The average graft take rate in 12 patients with full-thickness burn injuries in joint areas was 91.5% at one year post AlloDerm® with ultrathin autograft. All patients had near normal range of motion at one year and aesthetic results were judged fair to good by both surgeons and patients [108].  The average graft take rate in 12 patients with full-thickness burn injuries in joint areas was 91.5% at one year post AlloDerm® with ultrathin autograft. reconstruction [106]) All patients had near normal range of motion at one year and aesthetic results were judged fair to good by both surgeons and patients [108]. 371 Healthcare 2014, 2 Table 2. Cont. Table 2. Cont. Dermal skin replacement Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) FDA Indications (Other Indications) Burns  In a retrospective chart review of children aged 4 weeks to 18 years with an average of 6% TBSA partial thickness burns, patients with Biobrane® healed significantly faster compared than those treated with beta glucan collagen (9 days vs. 13 days). Patients requiring inpatient treatment had shorter length of hospital stay (2.6 vs. 4.1 days) [114] Wounds 372 Healthcare 2014, 2 Table 2. Cont. Dermal skin replacement Dermal skin replacement Advantages  Reserved for fresh wounds (<48 h) with low bacterial counts In a prospective randomized study in pediatric patients with partial thickness burns, Biobrane® was compared to topical application of 1% silver sulfadiazine. Pain, pain medication requirement, wound healing time, and length of stay (LOS) were significantly reduced in the Biobrane® group [115]. In a prospective randomized study in pediatric patients with partial thickness burns, Biobrane® was compared to topical application of 1% silver sulfadiazine. Pain, pain medication requirement, wound healing time, and length of stay (LOS) were significantly reduced in the Biobrane® group [115]. Biobrane® Smith & Nephew, St. Petersburg, FL, USA Temporary skin substitute Acellular matrix Biobrane® Smith & Nephew, St. Petersburg, FL, USA In a retrospective review, Biobrane® promoted adherence of split thickness skin grafts to the wound, allowing fluid drainage and preventing shearing. Biobrane® also facilitated healing of adjacent donor site or partial thickness burns [116]. In a controlled clinical trial of patients with partial thickness burns, compared to 1% silver sulfadiazine applied twice daily with dry gauze and elastic wraps, Biobrane® decreased healing time by 29% (10.6 days vs. 15.0 days) and reduced pain and the use for pain medication (0.6 vs. 3.0 tablets) at 24 h. There was no difference in the rate of infection [117].  In a prospective study of patients with scalp defects >5 cm requiring removal of periosteum, the biosynthetic dressing was definitive in six patients and complete closure was achieved in 3.5 months [118].  In a prospective study of patients with scalp defects >5 cm requiring removal of periosteum, the biosynthetic dressing was definitive in six patients and complete closure was achieved in 3.5 months [118].  Does not debride dead tissue [117]  In a prospective RCT of children with intermediate thickness burns with TBSA <10%, no significant difference in time to healing or pain scores were detected between use of Biobrane® or Duoderm®, although Biobrane® was more expensive [119].  373 Healthcare 2014, 2 Table 2. Cont. Dermal skin replacement Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Advantages Disadvantages FDA Indications (Other Indications) Burns  In a multicenter prospective RCT, 106 patients with life-threatening burns underwent excision and grafting. Mean burn size was 46.5% ± 15% mean TBSA. Epidermal donor sites healed 4 days sooner with Integra® compared to autograft, allograft, and xenograft. There was less hypertrophic scarring with Integra® [127].   bilayered extracellular matrix of cross-linked bovine type 1 collagen and chondroitin-6- sulfate glycosaminoglycan dermal replacement [85,126], with a thin silicone backing which acts as a temporary epidermal substitute. The product facilitates migration of macrophages and fibroblasts to initiate angiogenesis from dermal wound bed to create granulation tissue to support graft or local tissue. Once the neo- dermis is formed, the silicone layer is removed and the wound is permanently closed with a STSG on the neo-dermis [91]. Integra® was applied to surgically clean, freshly excised burn wounds in 216 burn patients at 13 burn facilities in the United States. The mean total body surface area burned was 36.5%. Once the neo-dermis was generated, a thin epidermal autograft was placed. The incidence of superficial infection at Integra® sites was 13.2% and of invasive infection was 3.1%. The mean take rate of Integra® was 76.2% with a median of 95%. The mean take rate of epidermal autograft was 87.5% with a median take rate of 98%. This study supported the evidence that Integra® is a safe and effective treatment in burn care [128]. pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, surgical wounds (donor sites/grafts, post-Moh’s surgery, post-laser surgery, podiatric, wound dehiscence), trauma wounds (abrasions, lacerations, second- degree burns, and skin tears) and draining wounds (approved through 510(k) process in 2002)    pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, surgical wounds (donor sites/grafts, post-Moh’s surgery, post-laser surgery, podiatric, wound dehiscence), trauma wounds (abrasions, lacerations, second- degree burns, and skin tears) and draining wounds (approved through 510(k) process in 2002) Integra® Dermal Regeneration Template (DRT) Integra Lifesciences Corporation Plainsboro, Plainsboro, NJ, USA (1996) In a prospective RCT comparing burn wounds treated with Integra®, STSG, and the cellulose sponge Cellonex® in 10 adult patients, all products demonstrated equal histological and immunohistological findings and equal clinical appearance after one year [129]. In a RCT of 20 children with burn size ranging from 58% to 88%, there were no significant differences between Integra® and control (autograft- allograft application) in burn size, mortality, and length of stay. Advantages Disadvantages Clinical Trials Burns  In a prospective RCT of 89 children treated within 48 hours of a superficial-thickness scald burn of 5%–25% TBSA randomized to Biobrane® or conservative treatment with topical antimicrobials and dressing changes, patients treated with Biobrane® had significantly shorter time to healing and length of stay. There was no difference in the use of systemic antibiotics or readmission for infections [124].  In a prospective RCT of 89 children treated within 48 hours of a superficial-thickness scald burn of 5%–25% TBSA randomized to Biobrane® or conservative treatment with topical antimicrobials and dressing changes, patients treated with Biobrane® had significantly shorter time to healing and length of stay. There was no difference in the use of systemic antibiotics or readmission for infections [124].  In a prospective RCT comparing Biobrane®, Duoderm®, and Xeroform for 30 skin graft donor sites in 30 patients, donor sites dressed with Xeroform had a significantly shorter time to healing of 10.5 days compared to Duoderm® (15.3 days) or Biobrane® (19.0 days). Duoderm® was reported to be the most comfortable dressing compared to Biobrane® and Xeroform. Two infections developed using Biobrane®, one using Duoderm®, and none using Xeroform. Biobrane® ($102.57 per patient) was the most expensive dressed compared to Duoderm® ($54.88 per patient) and Xeroform ($1.16 per patient) [125]. 374 Healthcare 2014, 2 Table 2. Cont. Dermal skin replacement Dermal skin replacement Biologic Company (FDA Approval) Product Description Advantages Disadvantages DFUs  Prospective study of patients with diabetic, non-infected plantar foot ulcers treated with Integra® demonstrated complete wound closure in 7/10 patients by week 12 with no recurrent ulcers at follow-up [135].  A retrospective case studies review of five patients with DFUs with extensive soft tissue deficits and exposed bone and tendon treated with Integra® followed by STSG demonstrated complete wound healing despite the failure of two grafts. No infections occured and all patients resumed ambulation [136]. Dermal skin replacement Dermal skin replacement on FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Post-excisional treatment of life threatening full thickness or deep partial thickness burn injuries [134] where autograft is not available at the time of excision or not desirable due to the condition of the patient (approved 2001); reconstruction of scar contractures when other therapies have failed or when donor sites for repair are not sufficient or desirable due to the condition of the patient; chronic lower extremity ulcers [91,92] (soft tissue defects) DFUs  Prospective study of patients with diabetic, non-infected plantar foot ulcers treated with Integra® demonstrated complete wound closure in 7/10 patients by week 12 with no recurrent ulcers at follow-up [135].  A retrospective case studies review of five patients with DFUs with extensive soft tissue deficits and exposed bone and tendon treated with Integra® followed by STSG demonstrated complete wound healing despite the failure of two grafts. No infections occured and all patients resumed ambulation [136]. Wounds  In a retrospective study of 127 contracture releases with the application of Integra® followed by epidermal autograft, 76% of the release sites, range of motion and function were rated as significantly improved or maximally improved by physicians at a mean post-operative follow-up period of 11.4 months. Patients expressed satisfaction with the results at 82% of sites. No recurrence of contracture was observed at 75% of the sites. Integra® offered functional and aesthetic benefits similar to full-thickness grafts without the associated donor site morbidity [137].  Twelve patients with large wounds were randomly divided into treatment with fibrin-glue anchored Integra® and postoperative negative-pressure therapy or conventional treatment. The take rate was significantly higher in the experimental treatment group (98% ± 2%) compared to the conventional group (78% ± 8%). The mean time from Integra® application to allograft was significantly shorter in the experimental group (10 ± 1 days) compared to the conventional treatment group (24 ± 3 days), which also resulted in shorter length of stay and potentially decreased risks of complications such as infection or thrombosis [138]. Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Advantages Disadvantages Clinical Trials Burns The Integra® group had lower resting energy expenditure and increased levels of serum constitutive proteins. The Integra® group also had increased bone mineral content and density at 24 months and improved scarring (vascularity, pigmentation, thickness) at 12 and 24 months [130]. This study supported the use of Integra® for immediate wound coverage in children with severe burns. 375 Healthcare 2014, 2 Table 2. Cont. Wounds  With the use of dressings and STSG, Integra® has been used to achieve functional and aesthetic coverage in the management of traumatic wounds of the hand with osseous, joint, or tendon exposure [139].  With the use of dressings and STSG, Integra® has been used to achieve functional and aesthetic coverage in the management of traumatic wounds of the hand with osseous, joint, or tendon exposure [139].  In a study of 31 patients who underwent Integra® grafting for reconstructive surgery, complications such as silicone detachment, failure of the graft, and hematoma were observed in nine [131]. Epidermal/Dermal Skin Replacements (Full-Thickness) Epidermal/Dermal Skin Replacements (Full-Thickness) Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Apligraf®/ Graftskin® Organogenesis, Canton, MA, USA (1998, 2001) Permanent skin substitute Living Cell Therapy Composite matrix cornified epidermal allogeneic keratinocytes derived from neonatal foreskin cultured on a type I bovine collagen gel seeded with living neonatal allogeneic human fibroblasts in dermal matrix [91] Chronic partial and full thickness venous stasis ulcers and full thickness diabetic foot ulcers [140] (epidermolysis bullosa [141], recurrent hernia repair, pressure sores, burn reconstruction) [92] Venous Leg Ulcers  A Cochrane Review concluded that a bilayer artificial skin used in conjunction with compression bandaging increases venous ulcer healing compared with a simple dressing plus compression [142].  In a prospective multicenter RCT of 240 patients with hard-to-heal chronic wounds (>1 year) receiving either intervention with Graftskin® plus compression or compression alone, treatment with Graftskin® with compression was significantly more effective than compression therapy alone in achieving complete wound closure at 8 weeks (32% vs. 10%) and significantly more effective at 24 weeks (47% vs. 19%) [143].A previously conducted prospective RCT by the same group revealed similar results [144]. Advantages  Small wounds require one application  Improved cosmetic (scar tissue, pigmentation, texture) and functional outcomes in chronic wounds [145]  Primary role in treating chronic ulcers FDA Indications (Other Indications) Wounds In a retrospective study of 127 contracture releases with the application of Integra® followed by epidermal autograft, 76% of the release sites, range of motion and function were rated as significantly improved or maximally improved by physicians at a mean post-operative follow-up period of 11.4 months. Patients expressed satisfaction with the results at 82% of sites. No recurrence of contracture was observed at 75% of the sites. Integra® offered functional and aesthetic benefits similar to full-thickness grafts without the associated donor site morbidity [137]. Twelve patients with large wounds were randomly divided into treatment with fibrin-glue anchored Integra® and postoperative negative-pressure therapy or conventional treatment. The take rate was significantly higher in the experimental treatment group (98% ± 2%) compared to the conventional group (78% ± 8%). The mean time from Integra® application to allograft was significantly shorter in the experimental group (10 ± 1 days) compared to the conventional treatment group (24 ± 3 days), which also resulted in shorter length of stay and potentially decreased risks of complications such as infection or thrombosis [138]. 376 Healthcare 2014, 2 Table 2. Cont Dermal skin replacement Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Advantages Disadvantages FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Clinical Trials Venous Leg Ulcers  A Cochrane Review concluded that a bilayer artificial skin used in conjunction with compression bandaging increases venous ulcer healing compared with a simple dressing plus compression [142].  ]  A Cochrane Review concluded that a bilayer artificial skin used in conjunction with compression bandaging increases venous ulcer healing compared with a simple dressing plus compression [142].   In a prospective multicenter RCT of 240 patients with hard-to-heal chronic wounds (>1 year) receiving either intervention with Graftskin® plus compression or compression alone, treatment with Graftskin® with compression was significantly more effective than compression therapy alone in achieving complete wound closure at 8 weeks (32% vs. 10%) and significantly more effective at 24 weeks (47% vs. 19%) [143].A previously conducted prospective RCT by the same group revealed similar results [144].  Improved cosmetic (scar tissue, pigmentation, textu and functional outcomes chronic wounds [145]  Primary role in treating chronic ulcers 377 Healthcare 2014, 2 Table 2. Cont. Table 2. Cont. Epidermal/Dermal Skin Replacements (Full-Thickness) Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages Burns  In a multicenter RCT of 38 patients with STSG wounds, Apligraf® was placed over meshed autograft while control sites were treated with meshed autograft covered with no biologic dressing or meshed allograft. There was no difference in the percent take of meshed split thickness autograft with or without Apligraf®. The Apligraf® group demonstrated significantly improved vascularity, pigmentation, wound height and Vancouver burn scar scores, demonstrating a cosmetic and functional advantage of Apligraf® compared to controls [145].      In a multicenter RCT of 38 patients with STSG wounds, Apligraf® was placed over meshed autograft while control sites were treated with meshed autograft covered with no biologic dressing or meshed allograft. There was no difference in the percent take of meshed split thickness autograft with or without Apligraf®. The Apligraf® group demonstrated significantly improved vascularity, pigmentation, wound height and Vancouver burn scar scores, demonstrating a cosmetic and functional advantage of Apligraf® compared to controls [145].     Epidermal/Dermal Skin Replacements (Full-Thickness) FDA Indications (Other Indications) Clinical Trials Donor site healing  A RCT of 60 skin donor sites treated with meshed autograft, meshed Apligraf®, or polyurethane film dressing was conducted. The healing time with Apligraf® (7.6 days) was significantly shorter than with polyurethane film dressing.   In a multicenter RCT of 10 patients treated with Apligraf®, Apligraf® dermis-only, and polyurethane film for acute STSG donor sites, there were no differences among the treatment modalities in establishing basement membrane at 4 weeks and there were no differences in other secondary outcomes [146]. foreskin (Quakers) [147] Contraindications  Infected wounds  Allergy to bovine collagen 378 Healthcare 2014, 2 Table 2. Cont. DFUs  In a multicenter RCT of 72 patients comparing Apligraf® and standard therapy versus standard therapy alone in the treatment of DFUs, there was a significantly shorter time to complete wound closure in the Apligraf® group 51.5% (17/33) compared to with standard treatment with international guidelines 26.3% (10/38) at 12 weeks [148].  In a multicenter RCT of 72 patients comparing Apligraf® and standard therapy versus standard therapy alone in the treatment of DFUs, there was a significantly shorter time to complete wound closure in the Apligraf® group 51.5% (17/33) compared to with standard treatment with international guidelines 26.3% (10/38) at 12 weeks [148].  In a prospective multicenter RCT of 208 patients randomly assigned to ulcer treatment with Graftskin® or saline-moistened gauze (control), 63/112 (56%) of Graftskin® patients achieved complete wound healing compared to 36/96 (38%) in the control at 12 weeks and this result was statistically significant. Kaplan-Meier curve to complete closure was also significantly lower for Graftskin® (65 days) compared to control (90 days). Osteomyelitis and lower- limb amputations were less frequent in the Graftskin® group [149].  Treatment with Apligraft® plus good wound care for DFUs results in 12% reduction in costs during first year of treatment compared to good wound care alone [150]. Table 2. Cont. Table 2. Cont. Epidermal/Dermal Skin Replacements (Full-Thickness) Biologic Company (FDA Approval) Product Description Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages OrCel® Forticell Bioscience, New York City, NY, USA (1998) Living Cell Therapy Composite matrix neonatal foreskin derived keratinocytes and dermal fibroblasts cultured in separate layers into a type I bovine collagen porous sponge [85]. During healing, autologous skin cells replace the cells in the product. Approved for HDE in 2001 for use in patients with dystrophic epidermolysis bullosa undergoing hand reconstruction surgery to close and heal wounds created by surgery, including donor sites; PMA approval for autograft donor sites in burn patients (overlay on split thickness skin grafts to improve cosmesis and function) [92] (chronic diabetic and venous wounds)  A randomized matched pairs study comparing treatment of split-thickness donor site wounds with OrCel® or Biobrane-L® revealed that scarring and healing times for sites treated with OrCel® were significantly shorter than for sites treated with Biobrane-L® [153]. Advantages  9 month shelf life Disadvantages  Cryopreserved  Cannot be used in infected wounds, in patients who are allergic to any animal products, or in patients allergic to penicillin, gentamycin, streptomycin, or amphotericin B GraftJacket® Wright Medical Technology, Inc., Arlington, TX, USA, licensed by KCI USA, Inc., San Antonio, TX, USA Permanent skin substitute Human skin allograft derived from donated human cadaver micronized acellular human dermis with a dermal matrix and intact basement membrane to facilitate ingrowth of blood vessels (deep and superficial wounds, sinus tract wounds, tendon repair, such as rotator cuff repair) [154] not subject to FDA pre-notification approval as it is a human cell or tissue based product DFUs  Multicenter, retrospective study in the treatment of 100 chronic, full thickness wounds of the lower extremity in 75 diabetic patients revealed a 91% healing rate and suggested its use in the treatment of complex lower extremity wounds. No significant differences were observed for matrix incorporation or complete healing. Mean time to complete healing was 13.8 weeks [155]. Advantages  2 year shelf life  Pre-meshed for clinical application  Single application  Utilized in both deep and superficial wound healing Disadvantages  Cryopreserved Wounds  In a prospective RCT of 31 patients requiring full-thickness surgical excision for non-melanoma skin cancer, patients were randomized to receive a single application of Apligraf® or to heal by secondary intention. Apligraf® reduced post-operative pain in this setting, but it was not determined whether it could decrease healing time or result in better aesthetic outcomes [151].  In a prospective controlled clinical trial, 48 deep dermal wounds were created and Apligraf®, STSG, or dressing was applied. Apligraf® demonstrated more cellular infiltrate but less vascularization compared to controls. Apligraf® demonstrated survival of allogeneic cells in acute wounds for up to six weeks and was recommended for the management of acute surgical wounds [152]. 379 Healthcare 2014, 2 Epidermal/Dermal Skin Replacements (Full-Thickness) Epidermal/Dermal Skin Replacements (Full-Thickness) OrCel® orticell Bioscience, New York City, NY, USA (1998) iving Cell Therapy omposite matrix neonatal foreskin derived keratinocytes and dermal fibroblasts cultured in separate layers into a type I bovine collagen porous sponge [85]. During healing, autologous skin cells replace the cells in the product. Approved for HDE in 2001 for use in patients with dystrophic epidermolysis bullosa undergoing hand reconstruction surgery to close and heal wounds created by surgery, including donor sites; PMA approval for autograft donor sites in burn patients (overlay on split thickness skin grafts to improve cosmesis and function) [92] (chronic diabetic and venous wounds)  A randomized matched pairs study comparing treatment of split-thickness donor site wounds with OrCel® or Biobrane-L® revealed that scarring and healing times for sites treated with OrCel® were significantly shorter than for sites treated with Biobrane-L® [153]. Advantages  9 month shelf life Disadvantages  Cryopreserved  Cannot be used in infected wounds, in patients who are allergic to any animal products, or in patients allergic to penicillin, gentamycin, streptomycin, or amphotericin B Approved for HDE in 2001 for use in patients with dystrophic epidermolysis bullosa undergoing hand reconstruction surgery to close and heal wounds created by surgery, including donor sites; PMA approval for autograft donor sites in burn patients (overlay on split thickness skin grafts to improve cosmesis and function) [92] OrCel® Forticell Bioscience, New York City, NY, USA (1998) Living Cell Therapy Composite matrix neonatal foreskin derived keratinocytes and dermal fibroblasts cultured in separate layers into a type I bovine collagen porous sponge [85]. During healing, autologous skin cells replace the cells in the product. (chronic diabetic and venous wounds) DFUs  Multicenter, retrospective study in the treatment of 100 chronic, full thickness wounds of the lower extremity in 75 diabetic patients revealed a 91% healing rate and suggested its use in the treatment of complex lower extremity wounds. No significant differences were observed for matrix incorporation or complete healing. Mean time to complete healing was 13.8 weeks [155]. (deep and superficial wounds, sinus tract wounds, tendon repair, such as rotator cuff repair) [154] (deep and superficial wounds, sinus tract wounds, tendon repair, such as rotator cuff repair) [154] not subject to FDA pre-notification approval as it is a human cell or tissue based product not subject to FDA pre-notification approval as it is a human cell or tissue based product 380 Healthcare 2014, 2 Table 2. Cont. Epidermal/Dermal Skin Replacements (Full-Thickness) p p ( ) ndications ndications) Clinical Trials Advantages Disadvantages DFUs  In a prospective multicenter RCT comparing GraftJacket® with standard of care therapies for the treatment of DFUs in 86 patients for 12 weeks, the proportion of completely healed ulcers between the groups was statistically significant. The odds of healing in the study group were 2.7 times higher than in the control group. The odds of healing were 2.0 times higher in the study group than in the control group when adjusted for ulcer size at presentation [156].  A prospective randomized study evaluating diabetic patients with lower extremity wounds demonstrated that patients treated with GraftJacket® healed significantly faster than those with conventional treatment at 1 month [157].  A prospective single center RCT comparing intervention (sharp debridement + GraftJacket® + mineral oil-soaked compression dressing) to control (wound gel with gauze dressing) for the treatment of full-thickness chronic non-healing lower extremity wounds in 28 diabetic patients revealed that at 16 weeks, 12/14 patients treated with GraftJacket® had complete wound closure compared to 4/14 patients in the control group. Significant differences were observed for wound depth, volume, and area [158].  In a prospective, randomized single blind pilot study of 40 patients with debrided diabetic lower extremity wounds, GraftJacket® was compared to the hydrogel wound dressing Curasol®. At 4 weeks, there was a significant reduction in the ulcer size in the GraftJacket® group compared to debridement only. At 12 weeks, 85% of the patients with GraftJacket® healed compared to 5% of controls [157]. Advantages Disadvantages Clinical Trials  No clinical trials available. Epidermal/Dermal Skin Replacements (Full-Thickness) Epidermal/Dermal Skin Replacements (Full-Thickness) Epidermal/Dermal Skin Replacements (Full-Thickness) Epidermal/Dermal Skin Replacements (Full Thickness) any al) tion Product Description FDA Indications (Other Indications) Clinical Trials Advantages Disadvantages DFUs  A retrospective multicenter series in 12 patients with DFUs and complex, deep, irregularly-shaped, tunneling sinus tracts treated with GraftJacket Xpress Scaffold® (a micronized, decellularized flowable soft tissue scaffold that can be delivered through a syringe into the wound cavity) demonstrated complete healing in 10/12 patients at 12 weeks [159].  In a prospective case series of 17 patients with debrided, non-infected, non-ischemic, neuropathic DFUs treated with a single application of GraftJacket® with weekly silicone dressing changes, 82.5% of wounds had complete re-epithelialization in 20 weeks, with a mean time to healing of 8.9 ± 2.7 weeks [160]. Falls, tute autologous keratinocytes and fibroblasts cultured on bovine collagen scaffold Orphan status approval as a permanent skin substitute in burns  No clinical trials available. Advantages  No risk of rejection  Permanent substitute for massive burn injury Disadvantages  No clinical trials or long- term studies available Advantages Disadvantages FDA Indications (Other Indications) Clinical Trials DFUs  In a prospective multicenter RCT comparing GraftJacket® with standard of care therapies for the treatment of DFUs in 86 patients for 12 weeks, the proportion of completely healed ulcers between the groups was statistically significant. The odds of healing in the study group were 2.7 times higher than in the control group. The odds of healing were 2.0 times higher in the study group than in the control group when adjusted for ulcer size at presentation [156].  A prospective randomized study evaluating diabetic patients with lower extremity wounds demonstrated that patients treated with GraftJacket® healed significantly faster than those with conventional treatment at 1 month [157].  A prospective single center RCT comparing intervention (sharp debridement + GraftJacket® + mineral oil-soaked compression dressing) to control (wound gel with gauze dressing) for the treatment of full-thickness chronic non-healing lower extremity wounds in 28 diabetic patients revealed that at 16 weeks, 12/14 patients treated with GraftJacket® had complete wound closure compared to 4/14 patients in the control group. Significant differences were observed for wound depth, volume, and area [158].  In a prospective, randomized single blind pilot study of 40 patients with debrided diabetic lower extremity wounds, GraftJacket® was compared to the hydrogel wound dressing Curasol®. At 4 weeks, there was a significant reduction in the ulcer size in the GraftJacket® group compared to debridement only. At 12 weeks, 85% of the patients with GraftJacket® healed compared to 5% of controls [157].  In a prospective, randomized single blind pilot study of 40 patients with debrided diabetic lower extremity wounds, GraftJacket® was compared to the hydrogel wound dressing Curasol®. At 4 weeks, there was a significant reduction in the ulcer size in the GraftJacket® group compared to debridement only. At 12 weeks, 85% of the patients with GraftJacket® healed compared to 5% of controls [157]. 381 Healthcare 2014, 2 Table 2. Cont. Table 2. Cont. Advantages  No risk of rejection  Permanent substitute for massive burn injury Disadvantages  No clinical trials or long- term studies available Healthcare 2014, 2 382 Dermal skin replacements provide greater stability to the wound and prevent the wound from contracting. Transcyte® (Shire Regenerative Medicine, Inc., San Diego, California, USA; Smith & Nephew, Inc., Largo, FL, USA) is composed of human allogeneic fibroblasts from neonatal foreskin seeded onto silicone covered bioabsorbable nylon mesh scaffold and cultured ex vivo for 4–6 weeks [85]. Transcyte® is often used as a non-living, temporary wound covering for partial- and full-thickness burns after excision [161]. A derivative of Transcyte® is Dermagraft® (Shire Regenerative Medicine, Inc., San Diego, California, USA), a skin substitute composed of living allogenic fibroblasts incorporated into a bioresorbable polyglactin mesh that secretes extracellular matrix (ECM) proteins, collagen, growth factors and cytokines into the wound site in the provision of viable living dermal substitute [162,163]. Dermagraft® has shown improvement in the treatment of chronic diabetic foot ulcers. AlloDerm®/Strattice® (LifeCell Corporation, Branchburg, NJ, USA) are lyophilized human acellular cadaver dermal matrices which serve as a scaffold for tissue remodeling. Autologous keratinocytes may be seeded and cultured on Alloderm® to form an epithelium; together; these can be utilized for wound and burn closure. Subsequent to its administration to a wound site, AlloDerm® is shown to exhibit cellular infiltration and neovascularization [164]. Biobrane® (Smith & Nephew, St. Petersburg, FL, USA) is a synthetic dermis temporary skin substitute composed of inner nylon and outer silicone with bovine collagen used for temporary coverage in partial and full-thickness burns. Integra® Dermal Regeneration Template (DRT) (Integra Lifesciences Corporation, Plainsboro, NJ, USA) is an example of a composite skin graft. It is composed of an outer layer of silicone and a cross-linked bovine type I collagen glycosaminoglycan dermal matrix. Once the dermal layer has regenerated, the silicone layer is removed and the wound is permanently closed with a split thickness skin graft (STSG) on the neo-dermis. Integra® is used for permanent coverage of full-thickness burns when combined with thin skin graft. Epidermal/Dermal skin replacements are also called as full-thickness skin substitutes and are composed of both epidermal and dermal layers. Autologous or allogeneic fibroblasts and keratinocytes are used in their preparation. The allogenically derived Apligraf® (Organogenesis, Canton, MA, USA) is a bilayered matrix construct similar to a microscopic skin layer. Specifically, it is comprised of a lower dermal layer of bovine type 1 collagen combined with human fibroblasts (extracted from postnatal foreskin) and an upper layer that consists of human keratinocytes, along with granulocyte/macrophage colony-stimulating factors. DFUs  A retrospective multicenter series in 12 patients with DFUs and complex, deep, irregularly-shaped, tunneling sinus tracts treated with GraftJacket Xpress Scaffold® (a micronized, decellularized flowable soft tissue scaffold that can be delivered through a syringe into the wound cavity) demonstrated complete healing in 10/12 patients at 12 weeks [159].  In a prospective case series of 17 patients with debrided, non-infected, non-ischemic, neuropathic DFUs treated with a single application of GraftJacket® with weekly silicone dressing changes, 82.5% of wounds had complete re-epithelialization in 20 weeks, with a mean time to healing of 8.9 ± 2.7 weeks [160]. 3.3. Clinical Trial Based Evidence Greer et al. [166] compared a number of advanced wound therapies in the treatment of ulcers in regard to the proportion of ulcers healed and time to healing. This study reviewed randomized controlled trials from the literature (MEDLINE 1995–2013, Cochrane Library, and existing systemic reviews), which involved patients who were typically middle-aged white males. The 56 trials encompassed lower extremity or foot ulcers, with 35 cases of patients with diabetic ulcers, 20 patients with venous ulcers, and one patient with arterial ulcers. The duration of therapies generally spanned from 4 to 20 weeks, with a mean ulcer duration from 2 to 94 weeks. The mean ulcer size ranged from 1.9 to 41.5 cm2. Of the advanced wound care products used in these trials, the biological skin equivalent Apligraf® demonstrated moderate-strength evidence for enhanced healing, as did negative pressure wound therapy. Low-strength evidence was shown for platelet-derived growth factors and silver cream in comparison to standard care. For arterial ulcers, there was an improvement in healing with biological skin equivalent. Although the evidence was deemed as limited, the conclusion of the authors was that several advanced wound care therapies appeared to enhance the number of ulcers healed, as well as to reduce the times for healing. A clinical randomized, double-blind, standard-controlled study was undertaken, which compared burn wounds that were treated with silver zinc sulfadiazine cream (control) against those treated with the identical cream that also contained silk sericin. The study involved 29 patients presenting with 65 burn wounds that covered at least 15% of total body surface areas. It was observed that the typical time for attaining 70% re-epithelialization in the sericin group was approximately 5–7 days shorter than the control group. The control group required 29.28 ± 9.27 days for complete burn wound healing, while the sericin group attained this condition within 22.42 ± 6.33 days with no indication of severe reaction or infection in any wound [49]. Multiple clinical trials have been conducted with the living skin equivalents Apligraf® and Dermagraft®. Healthcare 2014, 2 Healthcare 2014, 2 cover large burns. It is composed of autologous keratinocytes and fibroblasts cultured on bovine collagen scaffold [165]. 3.2. Contraindications Biological skin equivalents such as allogenically derived Apligraf® or Dermagraft® have an existing, albeit significantly low, risk of disease transmission due to their allogenicity [162]. In the case of Apligraf®, it has been verified in a number of studies that the cells it delivers are not sustained within the wound site beyond six weeks, and has inconsistent effects on the wound basement membrane, in vivo collagen composition and vascularization [2,146,152]. Healthcare 2014, 2 Apligraf® has been used for permanent coverage of non-healing chronic wounds (such as diabetic foot ulcers), surgical wounds, pressure wounds, neuropathic wounds and venous insufficiency ulcers. Apligraf® has been observed in vitro to generate extracellular matrix structural elements and modulators inclusive of tissue inhibitors of matrix metalloproteinases and glycoprotein fibronectin [2]. OrCel® (Forticell Bioscience, New York, NY, USA) is a composite matrix composed of keratinocytes and dermal fibroblasts cultured in separate layers into a type I bovine collagen porous sponge. It is used in patients with dystrophic epidermolysis bullosa undergoing hand reconstruction surgery and at autograft donor sites in burn patients [92]. GraftJacket® (Wright Medical Technology, Inc., Arlington, TX, USA, licensed by KCI USA, Inc., San Antonio, Texas, USA), is an acellular derivative of human dermis. GraftJacket® was shown to facilitate accelerated healing and initiate depth and volume reductions in wounds [156]. PermaDerm® (Regenicin, Inc., Little Falls, NJ, USA) is a newer product that acts as a permanent skin substitute to 383 4.1. Description Biocompatible vegetal biomembranes of natural rubber/latex, amniotic, polyurethane and poly-DL-lactic acid (PDLLA) comprise a class of versatile interventions for the treatment and healing of wounds. Additionally, biomembranes may be impregnated with a wide range of bioactive compounds to further facilitate and promote wound healing. Healthcare 2014, 2 Healthcare 2014, 2 384 51.5% (17 of 33) of Apligraf® patients in contrast to 26.3% (10 of 38) of control patients [148]. An additional prospective, randomized controlled trial involved 74 patients (38 autograft + Apligraf®, 36 autograft alone or + allograft) with dull and partial thickness burns. It was found at 22 months that 58% of the Apligraf® sites were deemed of better quality than the controls, with 26% as equivalent and 16% as worse. Further, Apligraf® treated patients (47%) exhibited normal vascularity in contrast to 6% of control patients [145]. A prospective, randomized controlled trial with Dermagraft® studied 314 patients (130 Dermagraft®, 115 controls) with diabetic foot ulcers. At 12 weeks, 30% of the Dermagraft® patients were healed in comparison to 8.3% of the control patients, who were treated with standard wet-to-dry dressings [95]. An additional prospective, randomized controlled trial was undertaken with 18 patients (10 Dermagraft®, eight controls) with venous ulcers, which revealed that the healing rate after 12 weeks was enhanced considerably in those patients treated with Dermagraft® + compression (five patients (50%)) as opposed to compression on its own (one patient (12.5%)). In addition, the perfusion of capillaries in the Dermagraft® group increased by 20%, in comparison to 4.9% in the compression group [101]. 3.3. Clinical Trial Based Evidence A retrospective controlled trial was undertaken that involved 2517 patients (446 Apligraf®, 1892 Regranex® (a human platelet-derived growth factor topical gel for the treatment of lower extremity diabetic neuropathic ulcers), 125 platelet releasates, 54 combined) and found that diabetic foot ulcers initially treated with Apligraf® were 31.2% more likely to heal than those administered with topical growth factor and 40% more likely to heal than those treated with platelet releasates [95]. In a prospective, randomized controlled trial involving 72 patients (33 Apligraf®, 39 with saline moistened gauze control), it was found that at 12 weeks, full wound closure was observed in 4.3. Contraindications Despite stringent preparation protocols, there might be a very low risk of bacterial or viral transmission via the use of human amniotic membranes on open wounds. 4.2. Mechanism and Indications Human amniotic membranes, such as Biomembrane® (Matrix Company, Ismailia, Egypt) are comprised of skin-like fetal ectoderm, consisting of four layers (epithelial, basement membrane, connective tissue fibroblasts, and spongy layer), which have demonstrated angiogenic properties. The membrane is freeze dried to 5% water content and then gamma irradiated (25 kgy) to ensure sterilization. These biomembranes exhibit a 1000-fold improvement in efficacy over split-thickness human skin grafts, though the specific mechanisms remain unclear [167,168]. Further, amniotic membranes are found to inhibit the alpha smooth muscle protein actin, resulting in a significant reduction in the generation of scar tissue in comparison to a moist wound dressing control [169]. Additional benefits included decreased pain, protection from infection and control of the loss of electrolytes and albumin. The polyurethane film, TegadermTM (3M, Saint Paul, MN, USA), exhibits gas semi-permeability, which acts to augment the rate of epithelialization. This may be due the retention of carbon dioxide, which translates to sustaining a low pH. The pain relief that is reportedly associated with this film may be the result of the exclusion of atmospheric oxygen, which negates the generation of prostaglandin E2, via the oxygen-reliant cyclo-oxygenase system [167,170]. An additional imparted benefit secondary to the semi-permeability of TegadermTM is the regulation of transforming growth factor beta (TGF-β) via the mediation of transepidermal water transfer [171]. It also stimulates the propagation of Healthcare 2014, 2 Healthcare 2014, 2 385 keratinocytes through the activation of integrins a5 and a6 to encourage enhanced and rapid wound healing [172]. A biocompatible vegetal biomembrane derived from the Hevea brasiliensis rubber tree exhibited the capacity to initiate angiogenesis and re-epithelialization in the chronic ulcers of diabetic patients. Its activity in the healing process appears most prominent at the inflammatory stage, where the microenvironment is transformed by robust angiogenesis followed by re-epithelialization [173]. A non-toxic, biocompatible, biodegradable, and non-carcinogenic crosslinked gelatin hydrogel biomembrane was developed for use as a wound dressing via the addition of a naturally occurring genipin crosslinking agent, and compared to a glutaraldehyde-crosslinked control. The resulting genipin infused biomembrane exhibited considerably less inflammation along with more rapid re-epithelialization and subsequent wound healing than the control, which may have been facilitated by a lower level of genipin imparted cytotoxicity [36]. 4.4. Clinical Trial Based Evidence Adly et al. [167] conducted a randomized, controlled clinical trial to compare the efficacy of an amniotic membrane (Biomembrane®) group I (23 patients) and a polyurethane membrane (TegadermTM) dressing group II (23 patients) in the treatment of burns (scald and flame). There were no notable differences between the two groups. The criteria were inclusion of both genders and all age groups with <50% total body surface area affected with either second or third degree burns. The group I patients exhibited a considerably lower infection rate (one patient (4.3%) in group I compared to three patients (13.0%) in group II) and required fewer dressing changes than group II (highest dressing change frequency was once per day in 30.4% of group I patients, in comparison to five times per day in 60.9% of group II patients). In addition, electrolyte disturbance was evident in 17.4% of patients in group I, compared with 60.9% of patients in group II. Albumin loss was indicated in 39.1% of patients in group I in contrast to 60.9% of patients in group II. In terms of pain and healing times, 43.5% of group I patients experienced pain during dressing, compared with 60.9% in group II. Healing frequency was 47.8% (11–20 days) for group I in contrast to 39.1% in group II spanning the same time period. 5.2. Mechanisms and Indications Collagen is a component of the extracellular matrix, which has found established utility as a biomaterial in cell therapies and tissue engineering via the provision of a viable substrate for the attachment and propagation of cells. In the treatment of wounds, collagen scaffolds offer a feasible platform for the topical conveyance of cells into the wound bed, increase the healing of wounds and initiate angiogenesis and neovasculogenesis. O’Loughlin et al. [174] investigated the use of type 1 collagen scaffolds for the topical delivery of autologous circulating angiogenic (CACs) cells (precursors to endothelial progenitor cells), to full thickness cutaneous ulcers. It was revealed that the CACs could also be pre-stimulated through the addition of matricellular protein osteopontin (OPN), a glycoprotein involved in immune function, neovascularization, and facilitation of cell migration and survival [175]. The inclusion of OPN served to augment wound healing. It was demonstrated that scaffolds comprised of type 1 collagen, which has been shown to sustain angiogenesis [176], when infused with CACs and enhanced with OPN, resulted in the formation of larger diameter blood vessels than untreated wounds, and thus acceleration of the wound healing process [174]. Ehashi et al. [177] compared subcutaneously implanted scaffolds for their host body reactions in order to assess their wound healing capacities. The scaffolds consisted of collagen coated porous (Ø32 μm and Ø157 μm) polyethylene (CCPE), bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) coated polyethylene, and uncoated porous polyethylene (UPPE) (control). Subsequent to their immersion in sterile solution for an appropriate period, six samples (two of each type with different pore diameters) were implanted under the skins of mouse models, and then resected after seven days. In terms of vascularization, it was observed that small vessels were induced on the UPPE, albeit contingent on the pore size (more activity seen with Ø32 μm pores than Ø157 μm pores). Interestingly, the reverse was true for the CCPE, with more activity seen with the Ø157 μm pore sample. There was no vessel growth activity associated with the PMB scaffolds. A deoxyribonucleic acid (DNA) microarray assay was then employed to conduct genetic analyses, which showed that the CCPE scaffold had a more highly distributed level of gene expression than did the PMB scaffold. The PMB scaffold showed the up-regulation of genes associated with the suppression of inflammation. The CCPE scaffold indicated up-regulation of genes related to inflammation, angiogenesis, and wound healing. Healthcare 2014, 2 Healthcare 2014, 2 386 5.1. Description Hybrid scaffolds comprised of polymeric substrates coated with bioactive materials, collagen, silk fibroin, as well as advanced tissue engineered substrates impregnated with endothelial progenitor cells, and nanomaterial-based scaffolds may be employed as advanced wound dressings to initiate and expedite wound healing. 5.2. Mechanisms and Indications The authors concluded that the up-regulation of interleukin-1b and angiogenesis associated genes within the porous scaffolds likely contributed to the mediation of tissue regeneration. A novel scaffold comprised of electrospun core-shell gelatin/poly(L-lactic acid)-co-poly-(ε-caprolactone) nanofibers, which encapsulated a photosensitive polymer poly (3-hexylthiophene) (P3HT) and epidermal growth factor (EGF) at its core, was investigated by Jin et al. [178] as a potential skin graft. It was found that fibroblast propagation was activated under exposure to light in contrast to its absence and cells akin to keratinocytes were found only on the light exposed scaffolds. The researchers propose that these light sensitive nanofibers may have utility as a unique scaffold for the healing of wounds and the reconstitution of skin. Bacterial (or microbial) cellulose has been investigated by Fu et al. [179] for its capacity to enable wound healing and skin tissue rejuvenation. Specific bacteria are involved in the biosynthesis of this natural polymer, which has unique properties in contrast to plant based cellulose, encompassing Healthcare 2014, 2 Healthcare 2014, 2 387 biocompatibility, hydrophilicity, high water retention, elasticity, transparency, conformability and the capacity for absorbing wound generated exudate during inflammation. These features position microbial cellulose to have great potential for biomedical advancements in skin tissue repair. 5.4. Clinical Trial Based Evidence The clinical performance of bacterial cellulose (BC) scaffold DermafillTM (AMD/Ritmed, Tonawanda, New York, USA) wound dressings (Acetobacter xylinum derived) was assessed by Portal et al. [181] who compared the reduction in wound size of chronic non-healing lower extremity ulcers following standard care. A total of 11 chronic wounds were evaluated for the time required to achieve 75% epithelization, by comparing non-healing ulcers prior to and following the application of DermafillTM. The median observation timeline for chronic non-healing wounds under standard care prior to the application was 315 days. When BC scaffolds were applied to these same wounds, the median time to 75% epithelization was decreased to 70 days. Thus, the authors concluded that BC scaffold-initiated wound closure for non-healing ulcers proceeded considerably more rapidly than did standard care wound dressings. Morimoto et al. [182] investigated the clinical efficacy of a unique synthetic collagen/gelatin sponge (CGS) scaffold for the treatment of chronic skin ulcers. This artificial dermal scaffold demonstrated the capacity to sustainably release basic fibroblast growth factor (bFGF) over 10 days or longer. One of the criteria for the study group was the inclusion of chronic skin ulcers that had not healed over a time period of at least four weeks. These wounds treated with CGS, which was infused with 7 or 14 μg/cm2 of bFGF following debridement, and assessed two weeks subsequent to their application. Positive improvement in the wound beds was defined by the emergence of granulated and epithelialized areas of 50% or greater. Out of a total of 17 subjects, it was observed that 16 showed wound bed improvements, with no discernable difference between the low and high dose groups. There was rapid recovery from mild adverse reactions. 5.3. Contraindications Scaffolds that are comprised of hyaluronan (an anionic polysaccharide), even though non-cytotoxic and biodegradable, may disrupt cell adhesion and the regeneration of tissues due to its hydrophilic surface properties [177]. Additional drawbacks for tissue engineered scaffolds in the case of severe burns relate to their unreliable adhesion to lesions and failure to replace dermal tissues [180]. 6. Conclusions The healing of surface and deep wounds of the epidermis is a complex multistage process, but one that may nevertheless be expedited utilizing strategies such as the application of active biologic, biomembrane or scaffold based wound dressings. Specific therapeutic compounds and cell species including epidermal stem cells may be utilized to impregnate biocompatible and/or biodegradable substrates, including membranes and scaffolds to facilitate rapid revascularization, re-epithelialization, and healing of wound beds. Healthcare 2014, 2 Healthcare 2014, 2 388 Acknowledgments This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contributions The authors’ responsibilities were as follows—Krishna S. Vyas and Henry C. 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https://openalex.org/W3208812672	https://www.intechopen.com/citation-pdf-url/78759	English	null	Staphylococcus aureus and the Veterinary Medicine	Infectious diseases	2,021	cc-by	7,934	Abstract Staphylococcus aureus has vital importance in veterinary medicine. Within the ruminants, it is one of the major causes of mastitis, the problem that was and is, with no definite solution to date. Along with that, it also affects the health of animals, pets, and poultry in several ways as the tissue tropism for this organism in poultry is the bones and the joints. This review is focused on habitat, species differentiation, differential biochemical tests, pathogenesis, clinical infections, economic importance, public health significance, immune response, the regulation of virulence in the staphylococci, and cytokines response against S. aureus. Keywords: cytokines, superantigens, tissue tropism, virulence, zinc 2. Habitat Staphylococcal species occur on humans and animals on the skin, mucosa of the upper respiratory system, lower urinary, and genital tract, and as transients in the digestive tract. They are stable in the environment, have a selective affinity for particular species. They have limited zoonotic importance [1, 2]. 1. Introduction Staphylococci are Gram-positive cocci bacteria of 1 pico-meter diameter. They are observed with gram staining under the microscope as a bunch of grapes. The word staphylococcus is originated from the Greek words staphyle and kokkos. Staphyle means the “bunch of grapes”, while the word kokkos means “the berry”. The normal habitat of staphylococci is skin and mucus membranes. There are approximately 30 species of staphylococcus. They act as commensals but some of them are opportunistic pathogens too. They a famous for their pyogenic infection- causing property. Most staphylococci are facultative anaerobes, non-motile, oxidase-negative, non-spore-forming, and catalase-positive. S. aureus subsp. aureus is the coagulase-positive that has very much importance concerning the disease sta- tus of animals. Production of coagulase is directly correlated with the pathogenicity of the staphylococcus i.e. coagulase-negative bacteria are usually non0-pathogenic to animals and humans [1]. They can be grown on non-enriched media. They are facultative anaerobes and non-motile. They are found as commensals on mucous membranes and skin. They are stable in the environment Figure 1 [99]. 1 Insights Into Drug Resistance in Staphylococcus aureus Figure 1. ‘Bunches of grapes’ appearance of Staphylococci. Modified from [1]. Insights Into Drug Resistance in Staphylococcus aureus Figure 1. Figure 1. ‘Bunches of grapes’ appearance of Staphylococci. Modified from [1]. g ‘Bunches of grapes’ appearance of Staphylococci. Modified from [1]. 3. Specie differentiation While confirming a bacterial colony to be a staphylococcus or not, it is necessary to differential differentiate it from closest resembling bacteria named micrococcus Figure 2. Growth curve of Staphylococcus aureus within bovine aortic endothelial cells. Modified from [1]. Figure 2. Figure 2. Growth curve of Staphylococcus aureus within bovine aortic endothelial cells. Modified from [1]. 2 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 and streptococcus species. The point that differentiates the Staphylococci from staphylococci is that staphylococci are mostly catalase-positive while the strep- tococci are mostly catalase-negative. Other tests of vital importance within the differentiation of the Staphylococcus species are hemolytic pattern, biochemical profiles, colonial appearance, and rRNA gene restriction patterns [2]. S. aureus and S. intermedius are often confused clinical cases of dogs and cats. Coagulase- negative staphylococci are ordinarily reserved for isolates from pure cultures. Their colonies are white, opaque and up to 4 mm in diameter, some are golden yellow and some have pigmented colonies. Sheep or ox blood agar presents alpha, beta, gamma and delta hemolysis. Strains of the staphylococcus species are differentiated based on their capability of haemolysin production [1, 2]. The growth curve of Staphylococcus aureus within bovine aortic endothelial cells under optimal conditions is presented in Figure 2. 4. Biochemical tests for differentiating Staphylococcus aureus and Staphylococcus intermedius A rapid test for the detection of acetoin has been developed [3]. Purple agar, containing bromocresol purple as a pH indicator and 1% maltose, is used to differ- entiate S. aureus from S. intermedius [4]. Purple is the color of most of the colonies of that bacteria. The energy source used by the Staphylococcus aureus in the culture medium is maltose which is utilized by that microbe and the resultant metabolic by- product is acid production. The by-product acid changes the color of the medium and colonies to yellow. Staphylococcus intermedius is a maltose fermenter so it means that it will not affect the color of the medium. There is also the commercial availability of the Biochemical tests which can be used for the confirmation of the staphylococcal species which can further be confirmed by molecular techniques like a polymerase chain reaction and multiplex PCR [5]. There are also studies on the molecular typing of the isolates of different regions of the world. The techniques that are and can be used in near future for the molecular epidemiology of the different isolates of Staphylococcus species can be but not limited to the Multilocus Sequence Typing (MLST) [6–9] and Multilocus variable number of Tandem Repeats (MLV) [10–12]. 6.2 Tick pyaemia Tick pyaemia of lambs is a disease of hill-grazing regions having the tick Ixodes ricinus. Clinical signs include septicemia and rapid death, localized abscess forma- tion, arthritis, posterior paresis, and ill-thrift. 30% of lambs between half a month old to up to three months of age can be affected. More infections are reported in spring and early summer [4–7]. 5. Pathogenesis and pathogenicity Staphylococci are pyogenic and cause suppurative lesions. Virulent factors for this gram-positive bacterium are capsule, plasmid or phage-mediated, cell wall proteins, teichoic acids, and protein A Figure 3 [2]. Figure 3. Sheep or ox blood agar with Double haemolysis of S. aureus. Modified from [1]. Figure 3. Sheep or ox blood agar with Double haemolysis of S. aureus. Modified from [1]. Figure 3. Sheep or ox blood agar with Double haemolysis of S. aureus. Modified from [1]. 3 3 Insights Into Drug Resistance in Staphylococcus aureus 6.3 Exudative epidermitis (greasy-pig disease) This is the disease pigs that are of up to 3 months old. It is contagious, with excessive sebaceous secretion, and exfoliation of the skin. Clinical signs include anorexia, depression, fever, dermatitis with an exudate. Death may be within 2–4 days with morbidity rate to be 20 to 100%, and mortality rates can be up to 90%. Isolation and identification of this bacteria can be from the vaginal mucosa and skin. Agalactia, weaning, and intercurrent infections are the predisposing factors for this disease [1]. 6.2.1 Diagnosis, treatment and control In young grazing lambs, clinical signs, microscopy of pus, and isolation and identification are required. Treatment is usually ineffective so control measures should have to be applied as tetracyclines injectables to the susceptible ones. Dipping to avoid tick-control measures should have to be practiced [2–5]. 6.1 Bovine mastitis Staphylococcal mastitis is a common form of mastitis worldwide. Most infections are subclinical, but they can be acute or chronic, per acute and gan- grenous. In gangrenous mastitis, the quarter is cold and blue-black and sloughing by the alpha-toxin causing necrosis of blood vessels and releases lysosomal enzymes [2–4]. 6. Clinical infections Staphylococci infections can be endogenous or exogenous in origin. Many infections are opportunistic and associated with other infections or the immune- compromised state of the host. Coagulase-positive staphylococci are mostly pathogenic. There are no effective vaccines against this malaise to date. Antibiotic sensitivity testing should have to be applied to check the efficacy of the drug against this bacterium. This is because many strains of this bacteria have developed resistance against many antibiotics. Common diseases of veterinary importance the Staphylococci are tick pyaemia, mastitis, botryomycosis, exudative epidermi- tis, and pyoderma [1]. 7. Staphylococcosis and poultry Along with humans and animals, poultry is also susceptible to infections by staphylococcus [13–17]. There are no definitive signs of that bacteria in the poultry and it varies from case to case and the lesions are usually dependent upon the point of entry of the bacteria within the host. Unlike the animals where the staphylococ- cus mainly targets the skin and mucosa, the skin is less likely to be infected in the poultry and the organs that are more susceptible to the infection of staphylococcus species in poultry are bones, tendons, and joints [14, 16, 18–21]. The infections are characterized by the increased heterophil count and their accumulation into the affected regions [22]. It is also responsible for the acute deaths in layers [23] within the hot climates and is required to be differentially diagnosed with the fowl cholera. Staphylococcal infections in poultry are required to have in-depth studies by future researchers as there is less knowledge about the route of entry, immunity interac- tion, pathogenesis, and the possible prognosis of that organism. It impedes chronic infections mostly in poultry having poor antibiotic response. Immunization against that pathogen also requires more in-depth studies as the currently available vaccines are not as potent as the poultry business farmers and expecting [23–25]. 7.1 Economic importance Along with the studies that they present acute infection in hot climates, they can infect almost all types of climates and target both poultry and turkey. They have very much economic importance as they decrease the feed conversion ratio, weight gain, egg production, and septicemia. They target the bones resulting in lameness and osteomalacia. Their pathological lesion may lead to the condemnation of the carcass [24, 25]. There is a study correlating the green discoloration of the liver with the staphylococcal infections and it is concluded it these studies that there is a high correlation between the green discoloration of the liver and the staphylococcal infections, and they termed that condition as the “green-liver osteomyelitis com- plex” [26, 27] of the turkeys. It should have to be remembered that this pathogen is not the only etiological agent for that correlation, other isolates within these studies were Escherichia coli and many others [26–28]. 6.5 Staphylococcal infections in dogs and cats Otitis externa and Pyoderma, endometritis, mastitis, osteomyelitis, and cystitis are reported to be due to the S. aureus in many cases [2–6]. 6.3.1 Diagnosis, treatment and control A high mortality rate in exudative, non-pruritic skin lesions. Along with the isolation and identification of the bacteria is required for confirmatory purposes. Antibiotic therapy with antiseptics is proven to be effective in many cases. Isolation of affected pigs, cleaning, and disinfection of surroundings. Antiseptic application before farrowing is also an effective way of prevention [1–3]. 4 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 6.4 Botryomycosis Botryomycosis is chronic, a suppurative granulomatous malaise of horses that is after castration infecting the stump of the scirrhous cord and mammary glands of sows [3–6]. 7.4 Clinical signs, morbidity, incubation period, and pathology Clinical signs of this disease include lameness, depression, pyrexia, and gait abnormalities, and death. Survived animals have arthritis, osteomyelitis [73, 74] unable to stand and sit on the hock and keel [25, 75]. This makes the fragility of the bones, mostly the femur and the tibiotarsus. It also leads to the congestion of the spleen, liver, lungs, and kidneys [23], gangrenous dermatitis, and ultimately the “blue wing disease” that presents the infection to the tip of wings of the birds that are infected with the chicken infectious anemia virus. Other clinical signs include enlarged yolk sac, planter abscess, discolored liver [27, 76]. Usually, the bacteria are not subjected to enough titer that may be the cause of higher mortality rates as compared to another fatal disease as the New Castle disease, etc., under optimal environmental conditions with most of the birds. But this bacterium has also been reported to have very high mortality rates that were primarily due to the immune- compromised state of the birds and the poor management conditions, and this bacteria in these conditions too is not the primary cause of the losses. The common site for the isolation and identification of that agent is the joints [18, 76–78]. 7.2 Public health significance on poultry Approximately 50% of the Staphylococcus aureus strains are responsible for human food poisoning through their enterotoxins [28–32] that are subjected to the condemnation of carcass upon their identification on food processing. Sources of the Staphylococcal infections may be the un-hygienic conditions of the processing plant and the poultry meat handling personals of the processing plant [33–36]. 5 Insights Into Drug Resistance in Staphylococcus aureus There is also a close associate of the Methicillin-resistant Staphylococcus aureus (MRSA) with the poultry meat [37–47]. MRSA has different strains each is resistant to a class of antibiotics as the commonly reported antibiotics against which the MRSA has evolved the disease tolerance includes the semi-synthetic penicillins [48, 49], Methicillin [50], fluoroquinolones [51], Vancomycin [52, 53], Sulphonamides and trimethoprim [54], tetracyclines [55–57], aminoglycosides [58–60], chloramphenicol [61], and clindamycin [62]. MecA gene is reported to be responsible for the methicillin resistance in the Staphylococcus aureus. This gene is also attributed to be transmitted from poultry to humans. The most common isolates of MRSA are CC398, ST9 [28–30]. There is also a close associate of the Methicillin-resistant Staphylococcus aureus (MRSA) with the poultry meat [37–47]. MRSA has different strains each is resistant to a class of antibiotics as the commonly reported antibiotics against which the MRSA has evolved the disease tolerance includes the semi-synthetic penicillins [48, 49], Methicillin [50], fluoroquinolones [51], Vancomycin [52, 53], [ , ], [ ], q [ ], y [ , ], Sulphonamides and trimethoprim [54], tetracyclines [55–57], aminoglycosides [58–60], chloramphenicol [61], and clindamycin [62]. MecA gene is reported to be responsible for the methicillin resistance in the Staphylococcus aureus. This gene is also attributed to be transmitted from poultry to humans. The most common isolates of MRSA are CC398, ST9 [28–30]. 7.3 History and transmission Firstly reported cases of the isolates have reported the susceptibility of bones with this pathogen and the prominent clinical signs as synovitis and arthritis [63–66]. Navel of the day-old chicks, surgery as trimming, and vaccination in un-hygienic conditions can be the trigger for the infection. Diseases that involve the predilection site to be the immune organs as being directly involved can also the root cause of this infection as the infectious bursal disease [67] and chicken infectious anemia. This is usually fatal as it leads to septicemia. Aged turkeys can have this infection with exposure to the hemorrhagic enteritis virus (HEV) [68]. Genetics of the poultry as the major MHC is also the predisposing factor for the skeletal-related problems of the poultry [69]. The incubation period of 2–3 days is a thumb rule but it is dependent upon several factors as the immune status of the host, the potency, and route of infection of the bacteria as the aerosol and tracheal routes are reported not to be the potent routes of infection [70, 71]. Infections with less than 105 organisms/kg body weight are reported to be defeated by the immune system of a healthy bird [25, 72]. 7.7 Management and control Sharp objects should not have contact with the birds of the poultry farm, Sanitation and optimal environmental conditions are key to good farming practices that will minimize the chances of infection [22, 67, 90–91]. Nutritionists are also considering the point of adding herbs and plants as Moringa oliefera [92] to boost the immune system, they also claim to have the composition of these herbs that helps the birds to cope up with the pathogens. In ovo inoculation is also advocated to boost the immune system to cope-up with the infectious agents [93]. Passive immunity against this bacterium to the susceptible population is also a rational option to cope up with the disease outbreaks [99]. 7.8 Vaccination Staphylococcal bacterins [81, 94], strain 115 [95], aerosol vaccine S. epidermidis 115 [71, 95–98] and PNSG are available with an aim to prevent the Staphylococcal infections. The capsule of live or dead cells of the Smith diffuse strain of S. aureus is most antigenic and was proved and used as the earliest potent vaccinal candidate, as the antibodies produced against the capsule can deal with the strategy of this bac- teria of dodging the phagocytosis [13–19]. The single intraperitoneal injection can protect from the challenge of a lethal dose of 108 CFU [26–27]. Anti-microcapsule vaccines are not proved to be as effective as capsular candidates. Bivalent vaccines are also been approved to be the effective ones. The capsule requires a monophos- phoryl lipid A as adjuvant and a booster dose to show an optimal antibody response [98–102]. 7.5 Immune response There are no convincing reports of the facts the active immunity or passive immunity other than that of the anti-Staphylococcus aureus antibodies may have any effect on this bacterium [79, 80]. Immunized hens can have antibodies within their 6 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 egg yolk that can be used to prevent the bacteria in vitro. Toxoids are ineffective in other species [81, 82], and vaccines have not proven to be a very effective way of controlling the disease [83–86]. egg yolk that can be used to prevent the bacteria in vitro. Toxoids are ineffective in other species [81, 82], and vaccines have not proven to be a very effective way of controlling the disease [83–86]. 7.6 Diagnosis Isolation and identification of the samples of yolk, joints, and internal organs from the infected bird should have to be practiced. Bacteria are harvested on the blood agar from the sheep or bovine and results are visible within a day of incuba- tion. Selective media for this organism can be used as mannitol salt agar [87–89]. Serology testing includes microtiter plate agglutination assay and indirect immuno- fluorescent antibody titer assay. It can be differentially diagnosed from the diseases of the joints of the poultry [79, 83]. 8. The regulation of virulence in the Staphylococci Virulence factors are the substances that aid in the pathogenesis of an organ- ism. Pathogenesis of Staphylococcus aureus does not depend on a single factor and there are a set of substances that collectively leads to the successful colonization of that bacteria into its host [98–99]. These virulence factors also diversify in their composition of proteins as exoproteins and surface proteins. To date, there are many reports of mutants, which behave differentially concerning the expres- sion of different exoproteins in different environmental conditions [100–102]. Most of the exoproteins are secreted at the post exponential phase. The polysac- charide of the capsule of Staphylococcus aureus also acts as the virulent factor. This bacterium can also be classified based on the structure of the capsule into 11 different serotypes [99]. Serotypes 1 and 2 and mucoid, while the serotypes 3 to 11 are microcapsules as which are non-mucoid and have thin capsules [96–101]. Among these 11 serotypes, 5 and 8 are the most prevalent. The capsule is vital to this bacterium as it is responsible for evading the phagocytosis by masking the C3b that is placed on the surface of these bacteria by the host immune cells. The significance of microcapsules in pathogenesis is not well established as there are many controversial studies in this regard. The genes responsible for the formation of microcapsules are cap5H, cap8J, and cap5P. The cap8B and cap5B genes are homologous to each other in several proteins, and cap8B acts as the chain length regulator of the capsule [98–100]. The chemical composition of serotypes 1, 2, 5, and 8 are presented in Figure 4. p g The agr and sar 16 loci have been extensively studied and believed to have vital importance in the virulence of this bacteria. Alpha toxin is also a virulence factor of Staphyloccocus aureus, which forms the pores to the cells resulting in cytolysis of the surrounding cells of invasion [97–100]. Not all the virulence factors are active throughout the life of the bacteria, but on the as-required basis, to overcome the metabolic burden [96–100]. Currently, the exact mechanism behind these virulence factors is not well elucidated. Staphylococcus is blessed with these virulence factors for its survival in diversified environmental conditions, and the primary purpose of these is not to cause the disease. 7.9 Treatment It is recommended to have antibiotic sensitivity testing before deciding the application of the antibiotic. The commonly used antibiotics against this bacterium are penicillin, tetracycline, streptomycin, novobiocin, sulfonamides, lincomycin, and spectinomycin. Most bacteria to date, are resistant to penicil- lin and many are resistant to other antibiotics as methicillin too. Vancomycin is considered now to be the most effective antibiotic against this bacterium. It is good to know that the cure rate of Staphylococcal infection with antibiotics does not exceed far beyond thirty percent, so vaccines should be the priority in dairy herd management [99–102]. 7 Insights Into Drug Resistance in Staphylococcus aureus 8. The regulation of virulence in the Staphylococci Passaging the bacteria to nutritive media in vitro leads to the bacteria of less virulency and the passage of bacteria to the live animal or host leads to the bacteria with more virulency [99, 100]. Microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), Sialoprotein, laminin, elastin, etc. are the proteins that are respon- sible for the adhesion of staphylococcus to its surrounding [98–100]. Figure 4. The chemical compositions of serotypes 1, 2, 5, and 8. Modified from [99]. Figure 4. The chemical compositions of serotypes 1, 2, 5, and 8. Modified from [99]. 8 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 To dodge the host immune system is a requirement of the successful colonization of each pathogen. Staphylococcus is also blessed with these factors as protein A for binding the IgG antibodies [99–101]. This bacterium has a system of coordination with environmental conditions as temperature, pH, etc. This system of coordination is named the “two-component systems” having two proteins and a single operon and upon detection of the signal these proteins active certain genes for transcription. A small colony-sized SVC subpopulation is also a potent strategy of this bacteria against the immune system of the host and antibiotic therapy [97, 98]. The bacterial secretions having mitogen properties are also called superantigens. These superantigens are pathogenic and may cause an autoimmune response. They are also responsible to activate macrophages, zinc having a vital role in that, by ini- tiating the IFN-gamma secretion from T cells. Superantigens can initiate an immune response without the increased concentration of IFN-gamma, whereas in mice it is necessary to have the increased concentration of IFN-gamma to initiate the immune response. It is not clear whether the response of MHC I and MHC II are synergistic or not, in the immunologic response against the pathogenesis of Staphylococcus Figures 5 and 6 [97–101]. Figure 5. (A) Response of IL-6 against anti-MHC-I 50 μg and MHC-II 100 μg antibodies incubated with C2D macrophages. (B) Response of TNF against anti-MHC-I 50 μg and MHC-II 100 μg antibodies incubated with C2D macrophages. Modified from [99–102]. Figure 5. Figure 5. 10. Conclusion Staphylococcus aureus has vital importance in the ruminants, as it is one of the major causes of Mastitis. Along with that, it also affects the health of animals, Pets, and Poultry in several ways as the diseases of bones in poultry. The Regulation of Virulence in the Staphylococci mainly are the exoproteins and surface proteins, and capsule, agr, sar 16 loci, and Alpha toxin. Bacteria potentiates cytokines for host resistance [97–101]. IFN-g and TNF play a protective role against Listeria monocytogenes, Mycobacterium species, Salmonella typhimurium, and Francisella tularensis. IFN-g and TNF also mediate gram-negative septic shock and endotoxin shock. Staphylococci induce TNF, interleukin-1, IFN-g, IL-2, and IL-6 in humans and animals [101, 102]. Figure 5. Figure 5. (A) Response of IL-6 against anti-MHC-I 50 μg and MHC-II 100 μg antibodies incubated with C2D macrophages. (B) Response of TNF against anti-MHC-I 50 μg and MHC-II 100 μg antibodies incubated with C2D macrophages. Modified from [99–102]. Figure 6. Response of TNF against various stimuli. (TNF: Tumor Necrosis Factor, SEA: Staphylococcal enterotoxin A, SEA-B: Staphylococcal enterotoxin A). Modified from [99]. i 6 Figure 6. Figure 6. Response of TNF against various stimuli. (TNF: Tumor Necrosis Factor, SEA: Staphylococcal enterotoxin A, SEA-B: Staphylococcal enterotoxin A). Modified from [99]. 9 Insights Into Drug Resistance in Staphylococcus aureus 9. Endogenous IFN-gamma, TNF, and IL-6 in Staphylococcus aureus infection Endogenous IFN-g plays a detrimental role in S. aureus infection. IFN-g, TNF, and IL-6 levels are elevated within 24 hours of infection even though whether the infection is lethal or non-lethal. In nonlethal cases, Bacteria is not present in the blood but in the kidneys and remains there for up to three weeks of infections. IFN-g peaks again in the spleens and kidneys. Among these three cytokines, the only cytokine that is detected in the serum is IL-6. In lethal infection, IFN-g and IL-6 in the sera and TNF in the kidneys peaked before death [98–102]. Acknowledgements I deem it utmost to express my heartiest gratitude to Dr. Shafia Tehseen Gul, Dr. Aisha Khatoon, and Dr. Muhammad Imran Arshad for scholastic guidance, ever encouraging attitude, and valuable suggestions during this project. Conflict of interest The authors declare no conflict of interest. The authors declare no conflict of interest. 10 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 Staphylococcus aureus and the Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.100202 Author details Muhammad Farhab1, Muhammad Tahir Aleem2, Shakseema Shaukat3, Ayesha Qadry3, Muhammad Zeeshan Ul Haq4, Fateh Ullah5, Muhammad Jawad6 and Amjad Islam Aqib7 1 College of Veterinary Medicine, Yangzhou University, Yangzhou, China 2 MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China 3 Veterinary Research Institute Lahore, Lahore, Pakistan 4 Department of Clinical Medicine and Surgery, University of Agriculture Faisalabad, Faisalabad, Pakistan 5 Department of Clinical Medicine, University of Veterinary and Animal Sciences Lahore, Lahore, Pakistan 6 Islamia College Peshawar, Peshawar, Pakistan 7 Department of Medicine, Faculty of Veterinary Science, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan Address all correspondence to: farhab.dvm@gamil.com © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. References [9] Saunders NA, & Holmes A. Multilocus sequence typing (MLST) of Staphylococcus aureusStaphylococcus aureus. In Methicillin-Resistant Staphylococcus Aureus (MRSA) Protocols. Humana Press. 2007. 71-85 p. [1] Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, & Fitzpatrick E. Veterinary microbiology and microbial disease. John Wiley & Sons; 2011. [1] Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, & Fitzpatrick E. Veterinary microbiology and microbial disease. John Wiley & Sons; 2011. [9] Saunders NA, & Holmes A. Multilocus sequence typing (MLST) of Staphylococcus aureusStaphylococcus aureus. In Methicillin-Resistant Staphylococcus Aureus (MRSA) Protocols. Humana Press. 2007. 71-85 p. 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https://openalex.org/W4387608434	https://www.researchsquare.com/article/rs-3425038/latest.pdf	English	null	Characterization of natural cytotoxic T lymphocytes and natural killer cells in Cuban older adults	Research Square (Research Square)	2,023	cc-by	5,046	Characterization of natural cytotoxic T lymphocytes and natural killer cells in Cuban older adults Elizabeth Hernández-Ramos (  ramos.beth94@gmail.com ) Elizabeth Hernández-Ramos (  ramos.beth94@gmail.com ) Instituto de Hematología e Inmunología Vianed Marsán-Suárez Instituto de Hematología e Inmunología Imilla Casado-Hernández Instituto de Hematología e Inmunología Mary Carmen Reyes-Zamora National Center for Biopreparations Mary Carmen Reyes-Zamora National Center for Biopreparations Luis Felipe Heredia-Guerra Research Center for the Elderly (CITED) Luis Felipe Heredia-Guerra Research Center for the Elderly (CITED) Yenisey Triana-Marrero Instituto de Hematología e Inmunología Yenisey Triana-Marrero Instituto de Hematología e Inmunología Gabriela Díaz-Domínguez Instituto de Hematología e Inmunología Yaneisy Duarte-Pérez Instituto de Hematología e Inmunología Abstract Introduction: The Cuban population has a high proportion of older adults and faces age-related changes in the immune system, known as immunosenescence. Natural killer T (NKT) cells and natural killer (NK) cells play an important role in innate inmunity and modulating adaptive immune responses. Their diminished function in older adults contributes to increased susceptibility to infectious diseases, tumors, and autoimmune diseases in this population group. Objective: To characterize distribution of NKT and NK cells in Cuban older adults. Methods: A cross-sectional study was carried out in 30 Cuban older adults, considering age, sex and malignant neoplasm comorbidity as factors of influence on the values of NK and NKT. A linear regression model was used to analyze the data, as well as a two-tailed Mann-Whitney U test to compare independent samples. In addition, odds ratios were used as measures of effect. NK cells and NKT lymphocytes were quantified in peripheral blood using flow cytometry. Results: In the studied populations of NK and NKT cells, age and sex did not show any significant differences. However, most cases exhibited values above the normal reference ranges, with the exception of one female patient. While no significant differences were found in the comparisons by sex and age, higher values of NK and NKT cells were observed in the group under 80 years old and in males. The adequate NK cell numbers in PB might be a protective factor against malignant neoplasms Conclusions: NK and NKT cells play a fundamental role in the regulation of immune response and directly influence the impairment of immune response in older adults, age and sex showed no significant impact on NKT and NK cell counts and percentages. Nevertheless, the presence of adequate NK cell percentages might be a protective factor against malignant neoplasms. Research Article Research Article Keywords: NK, NKT, malignant neoplasm, immunosenescence, innate immunity, flow cytometry Posted Date: October 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3425038/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/13 1. Introduction The innate and adaptive components of the immune system undergo significant age-related changes known as immunosenescence. These changes include the state of dysregulation and decreased immune response, contributing to morbidity and mortality due to increased incidence or reactivation of infectious diseases, autoimmunity, and cancer. The immune response of T cells is most affected by this natural process, although age-related alterations in phenotype and function have been demonstrated in other immune cell populations (1–4). Natural killer T (NKT) cells and natural killer (NK) cells are cellular subpopulations that are part of innate immunity. NKT lymphocytes recognize lipid or glycolipid antigens presented by CD1d, a non-classical MHC molecule. NKT cells represent a very small proportion of total T lymphocytes in peripheral blood (PB), although they constitute a larger number in other organs such as the liver, lungs, thymus, and spleen (5). NKT cells play an important role in regulating multiple immune responses, including microbial infection, autoimmunity, and cancer (6–8). Even in a steady state, cytokine production by NKT cells Page 2/13 Page 2/13 influences the basal state and function of other immune cells, such as dendritic cells, CD8+ T cells, and NK cells (9). influences the basal state and function of other immune cells, such as dendritic cells, CD8+ T cells, and NK cells (9). NKT cells have been found in the thymus, liver, spleen, and bone marrow. While the liver could be a potential site for extrathymic development of T cells, evidence suggests that the thymus is the main organ responsible for the development of most NKT cells (10). Increased NKT cell activity has been found in the pathogenesis of certain autoimmune diseases, allergies, and atherosclerosis (11). Additionally, research on the antitumor function of NKT cells has shown them as a therapeutic alternative against certain types of cancer (7). On the other hand, NK cells are a population of heterogeneous lymphocytes that act against a wide variety of infections and tumors. The role played by NK cells in defense against viruses is due to both their cytotoxic capacity and the cytokines they produce, particularly interferon (IFN) production. The decline in NK cell functionality, specifically their cytotoxicity, in the elderly is associated with a higher incidence of infectious diseases in this age group (12–14). NK cells are derived from pluripotent hematopoietic stem cells. T and NK cell lineages share a common precursor that expresses FcγRIII. 1. Introduction While T cell progenitors transfer to the thymus for differentiation and maturation, NK cells can develop independently of the thymus and proliferate in the bone marrow under the stimulation of growth factors. In NK cells, immunoglobulin (Ig) or T cell receptor (TCR) rearrangements do not occur, so neither Ig nor the TCR/CD3 complex are expressed on the cell surface. Mature NK and NKT cells are characterized by the expression of markers such as CD56, a neural cell adhesion molecule isoform (12, 13). The Cuban population is one of the most aged in Latin America, with approximately 20.1% being over 60 years old and a life expectancy of 78.45 years (1). Therefore, the present research aims to characterize the distribution of NKT and NK cells in older adults and their relation to age and sex. 2.1 Study Design and Subjects A cross-sectional study was conducted as part of the clinical trial “Evaluation of the Efficacy and Safety of a New Dosage Regimen of BIOMODULINA T® for the Prevention of Infections, Including COVID-19, in Older Adults in Cuba” (Public registration code: http://registroclinico.sld.cu/ensayos/RPCEC00000319- Sp). The sample consisted of 30 randomly selected subjects from the “Alfredo Gómez Gendra” Elderly Home. The study included subjects aged 60 years and older, who provided written consent to participate, and both sexes were included. Older adults receiving immunomodulatory treatments in the previous two months, with acute allergic states, a history of severe allergic reactions, or uncontrolled intercurrent disease were excluded from the study. The comorbidities existing in the patients are declared in Table 1. Page 3/13 2.2 Sample Collection and Processing Approximately 2 mL of PB was extracted from each patient using ethylenediaminetetraacetic acid (EDTA) tubes through venipuncture. All samples were processed and analyzed at the Immunology Laboratory of the IHI. Fifty microliters of each sample were taken and incubated in 15 mL tubes for 10 minutes at 4°C, protected from light, with the following fluorochrome-conjugated monoclonal antibodies: anti-CD45- fluorescein isothiocyanate (FITC), anti-CD56-phycoerythrin (PE), and anti-CD3-phycoerythrin cyanine 5 (PC5) (MACS®, Miltenyi Biotec, Germany). Subsequently, red blood cell lysis was performed using a lysing solution for 10 minutes at room temperature. The cells were washed twice with 0.9% sodium chloride and centrifuged for 10 minutes at 300g. A Beckman Coulter Gallios® 8-color flow cytometer (Beckman Coulter®, USA) was used for sample reading, with a minimum of 100,000 acquired events. The data obtained were analyzed using Kaluza Analysis® software version 1.2 for Microsoft®. The NKT lymphocyte population was defined by the immunophenotype CD45+/CD3+/CD56+, and the NK population by CD45+/CD3−/CD56+. Figure 1 presents the analysis strategy for NKT cells, where the dot plot over the total labeled cells allows for the separation of singlets; individual cells passing through the laser and presenting uniform height (FSC-H) and area (FSC-A) signals that the cytometer registers as an event, discarding cell aggregates (Fig. 1-A). Then, a dot plot was created using the pan-leukocyte antigen CD45+ expression (Fig. 1-B). Subsequently, the T lymphocyte population was defined using the CD3+ antigen for NKT cells (Fig. 1-C) and CD3− for NK cells. Finally, both subpopulations were defined using another dot plot with the CD56+ antigen (Fig. 1-D). The results were expressed as relative values (% of cells) and absolute values (cells/µL). The absolute values of both cellular subpopulations were calculated according to the following formula: Absolute count (cells/µL) = Lymphocyte count (number of cells/µL) in blood count x % of the specific cellular subpopulation of interest ÷ 100 Absolute count (cells/µL) = Lymphocyte count (number of cells/µL) in blood count x % of the specific cellular subpopulation of interest ÷ 100 Page 4/13 Page 4/13 Table 1 ab e Distribution of comorbidities in the older adults. Comorbidity Total (n = 30) Cardiovascular Diseases 25 Dementia 16 Type II Diabetes Mellitus 9 Chronic Obstructive Pulmonary Disease and Cerebrovascular Diseases 8 Malignant Neoplasms 4 Skin Infections 5 Urinary Tract Infections and Bronchial Asthma 2 Distribution of comorbidities in the older adults. 2.4 Ethics The study was approved by the Research Ethics Committees of the IHI and CITED, and the principles of the Declaration of Helsinki were applied in all processes (24). 2.3 Statistical Analysis The sample was stratified by sex and age into two groups: under 80 years old and 80 years old or older. Reference value ranges obtained from studies conducted on healthy Cuban adults were used. The Shapiro-Wilk test was used to assess the normality of the distribution of variable values. Descriptive statistics, including mean, median, standard deviation (SD), and 2.5th -97.5th percentile range were evaluated due to the variable data skewness. A linear regression model was used to assess the effect of age, and the two-tailed Mann-Whitney U test for independent samples was used to evaluate the effect of sex. The significance threshold was set at p ≤ 0.05. The data were processed using GraphPad Prism software version 9.5.0. The odds ratio (OR) and their respective confidence intervals (CI) with p ≤ 0.05 were used to determine the effect of malignant neoplasms on the relative and absolute values of NKT and NK. 3. Results No effect of age or sex on the evaluated populations was found; the p-values and r2 values were not statistically significant. Anormal values above the upper limit of the reference value ranges were found in all cases, except for one 78-year-old female patient, where only NK cells were below the normal values for her age and sex. NKT cells were anormal in 90.5% of the relative values and 81.0% of the absolute values in females (n = 21), while in males (n = 9), all values fell within the reference range. On the other hand, NK cells in females showed alterations in 42.9% of the relative values and 76.0% of the absolute values, with the latter being normal in males, although the relative values were anormal in 77.8% of males. Regarding age, NKT cells were normal in the percentage values, but 6.7% of the total patients had anormal absolute Page 5/13 Page 5/13 Page 5/13 values. In NK cells evaluated with respect to age, 3.3% of the total had both absolute and relative values outside the range of normal values. The comparisons made between the groups stratified by sex and age did not yield statistically significant results in any case. However, it is worth noting that NK cells in both distributions showed higher absolute and relative values in the group under 80 years old (Table 3) and in the male group (Table 4). The mean of the relative and absolute values of NKT cells was also higher in the group under 80 years old (Table 3), and in the male group, only the absolute values were higher (Table 4). The comparison according to neoplasia comorbidity showed that patients without neoplasia had considerably higher numbers of NK cells in PB compared to those who did have neoplasia, while the NKT cells were reduced but not significantly (Table 5). Table 2 Absolute and relative values for total NK and NKT cells (n = 30). NK (CD45+CD3−CD56+) NKT (CD45+CD3+CD56+) Median Mean (SD) 2.5th -97.5th Percentile Median Mean (SD) 2.5th -97.5th Percentile % 11.93 14.4 (7.6) 9.1–19.1 5.2 6.8 (4.3) 4.0-10.5 cells/ µL 195.6 253.6 (211.0) 136.5–292.0 80.0 126.4 (122.4) 56.9-122.7 SD: standard deviation; NKT: natural killer T lymphocyte; NK: natural killer cells; n: sample size Table 2 Absolute and relative values for total NK and NKT cells (n = 30). 3. Results Male (n = 9) 30% of Total Female (n = 21) 70% of Total Mean (SD) 2.5th -97.5th percentile Median Mean (SD) 2.5th -97.5th percentile Median p CD3+CD56+ (%) (15) 6.3 (3.5) 3.1–10.5 3.7 7.1 (4.6) 4.3–10.5 7.7 0.6567ns CD3+CD56+ (cells/µL) (15) 151.6 (160.7) 57.9-211.9 72.6 115.7 (104.9) 51.0-116.6 80.3 0.8243ns CD3−CD56+ (%) (16) 16.8 (7.1) 10.8–25.0 13.3 13.4 (7.7) 8.7–18.5 11.7 0.1635ns CD3−CD56+ (cells/µL) (16) 389.4 (321.2) 153.3-504.4 218.0 195.4 (106.7) 109.5-258.6 193.1 0.0697ns Absolute and relative values of NKT and NK cells by sex. Male (n = 9) 30% of Female (n = 21) 70% of To Absolute and relative values of NKT and NK cells by sex. Table 5 Distribution of relative and absolute values of NKT and NK according to the neoplasia comorbidity. Neoplasia (n = 4) 13.3% of Total No neoplasia (n = 26) 86.7% of Total Median CI Median CI OR [CI] p CD3+CD56+ (%) 10.6 9.0-11.6 5.1 3.9– 7.4 1.19 [1.51– 0.95]ns 0.100ns CD3+CD56+ (cells/µL) 166.2 121.8-209.8 76.0 53.9- 111.3 1.00 [1.01- 1.00]ns 0.088ns CD3−CD56+ (%) 5.5 3.1–8.3 13.5 10.3– 19.6 0.50 [1.06– 0.24]* 0.010* CD3−CD56+ (cells/µL) 95.9 70.8-114.1 208.1 165.7- 312.2 0.96 [1.00- 0.93]* 0.005* CI: confidence interval; OR: odds ratio 3. Results n; NKT: natural killer T lymphocyte; NK: natural killer cells; n: sample size SD: standard deviation; NKT: natural killer T lymphocyte; NK: natural killer c y p y p Table 3 Absolute and relative values of NKT and NK cells by age groups. < 80 years (n = 17) 56.7% of Total ≥ 80 years (n = 13) 43.3% of Total Mean (SD) 2.5th -97.5th Percentile Median Mean (SD) 2.5th -97.5th Percentile Median p CD3+CD56+ (%) (17) 7.0 (4.0) 4.2–10.5 5.9 6.649 (4.719) 3.1–10.5 5.0 0.5634ns CD3+CD56+ (cells/µL) (17) 140.0 (143.0) 54.8-127.1 87.9 108.6 (91.4) 55.4-148.4 72.7 0.4570ns CD3−CD56+ (%) (17) 15.16 (7.779) 10.28– 20.24 13.27 13.46 (7.438) 8.270– 18.97 10.44 0.3851ns CD3−CD56+ (cells/µL) (17) 281.2 (251.5) 139.1-317.6 218.0 217.5 (144.1) 120.0-274.4 173.1 0.4083ns ns: not significant Page 6/13 Page 6/13 Table 4 Absolute and relative values of NKT and NK cells by sex. Male (n = 9) 30% of Total Female (n = 21) 70% of Total Mean (SD) 2.5th -97.5th percentile Median Mean (SD) 2.5th -97.5th percentile Median p CD3+CD56+ (%) (15) 6.3 (3.5) 3.1–10.5 3.7 7.1 (4.6) 4.3–10.5 7.7 0.6567ns CD3+CD56+ (cells/µL) (15) 151.6 (160.7) 57.9-211.9 72.6 115.7 (104.9) 51.0-116.6 80.3 0.8243ns CD3−CD56+ (%) (16) 16.8 (7.1) 10.8–25.0 13.3 13.4 (7.7) 8.7–18.5 11.7 0.1635ns CD3−CD56+ (cells/µL) (16) 389.4 (321.2) 153.3-504.4 218.0 195.4 (106.7) 109.5-258.6 193.1 0.0697ns Table 5 Distribution of relative and absolute values of NKT and NK according to the neoplasia comorbidity. Neoplasia (n = 4) 13.3% of Total No neoplasia (n = 26) 86.7% of Total Median CI Median CI OR [CI] p CD3+CD56+ (%) 10.6 9.0-11.6 5.1 3.9– 7.4 1.19 [1.51– 0.95]ns 0.100ns CD3+CD56+ (cells/µL) 166.2 121.8-209.8 76.0 53.9- 111.3 1.00 [1.01- 1.00]ns 0.088ns CD3−CD56+ (%) 5.5 3.1–8.3 13.5 10.3– 19.6 0.50 [1.06– 0.24]* 0.010* CD3−CD56+ (cells/µL) 95.9 70.8-114.1 208.1 165.7- 312.2 0.96 [1.00- 0.93]* 0.005* CI: confidence interval; OR: odds ratio 4. Discussion Table 4 Table 4 Absolute and relative values of NKT and NK cells by sex. 4. Discussion Linear regression analyses to assess the effect of age did not yield r2 values close to unity, with statistically non-significant p-values for the evaluated cellular populations (data not shown). These Page 7/13 findings are consistent with the results reported by Villegas et al. regarding NKT cells and by Kokuina et al. for NK cells in two studies conducted on a Cuban adult population (15, 16). findings are consistent with the results reported by Villegas et al. regarding NKT cells and by Kokuina et al. for NK cells in two studies conducted on a Cuban adult population (15, 16). There was not influence of sex on the values of NKT cells in older adults. Rojas-Pandales et al. also did not find a sex effect on NK and NKT values in Colombian individuals, although it is worth mentioning that they only studied subjects between 18 and 58 years of age (11). However, Villegas et al. found a sex effect on the NKT population, with a slightly higher number in males. On the other hand, a study by Apoil et al. showed a sex effect on NK cells in a French adult population, with significantly higher values in males, but no age effect was observed for this subpopulation (18). Only 30% of the evaluated older adults were male, which results in an unbalanced representation of this sex, and the sample size was much smaller compared to the aforementioned studies. These factors may explain why these effects were not observed in the presented results. However, in the comparison of absolute values of NK cells in males, the p-value was close to 0.05 (p = 0.0697), and the mean was higher in both absolute and relative values, indicating that a larger sample size might reveal the sex effect. Generally, women exhibit stronger humoral and cellular immune responses than men, linked to higher antibody levels and immune mediator production. Sex chromosome genes and sex hormones like estrogen, progesterone, and androgens influence immune responses, with estrogen enhancing both cell- mediated and humoral immunity, while androgens suppress Th1 cell differentiation and reduce IFN-γ production. Nutritional and microbiome differences also impact immune function between sexes (25– 28). Taking into account that women participating in this study are over 60 years of age, the influence on the cellular immune response associated with the presence of estrogens would not be as much as significant. 4. Discussion Some sex differences in the maturation and differentiation process of NK cells persist in elderly females, where a higher NK CD56bright/CD56dim ratio has been reported compared to elderly males (17, 18, 20). The 13.33% of the patients had malignant neoplasms, and within this percentage, there was one patient with relative and absolute values below the normal reference range, but only for NK cells. Among the most studied effector immune cells with biological significance in cancer are natural killer cells (NK cells) as part of innate immunity, further research into lymphocyte immunophenotypes has led to the discovery of new subpopulations with important roles in cancer biology, such as NKT cells. The effects of immunosenescence manifest rapidly in cancer and are associated with a worse prognosis, then with less immunosurveillance support, cancer incidence considerably rises with age (4, 6, 12–14). The comparison according to neoplasia comorbidity showed that patients with neoplasia had considerably lower numbers of NK cells in PB compared to those with no neoplasia, while the NKT cells were reduced but not significantly. The OR indicates that adequate numbers of NK cells in PB might be a protective factor against malignant neoplasms. Arango et al. evaluated lymphocyte subpopulations in Cuban cancer patients and found that with increasing age, there is a tendency for a decrease in T, B, and NK lymphocyte populations, and patients with solid tumors showed the lowest values (17). Wang et al. demonstrate that age is one of the variables that most influences immunosenescence and they conclude that T cells, NK cells, and NKT cells are gradually affected in cancer (23). NK cells play important roles in the antitumor immunity in the absence of sensitization, which contribute to the prevention of cancer invasion and metastasis and finally prevented the early dissemination of cancer cells (14). It is known that terminally differentiated NK cells increase in frequency in cancer patients, whereas NK CD56bright cells are in an exhausted state, which refers to the exhaustion or dysfunction caused by immunosuppression in the tumor microenvironment and tumor evasion mechanisms that result in a decreased ability of NK cells to effectively recognize and eliminate tumor cells. In this sense, NKT cells play an important supporting role, as they not only exert immunosurveillance against cancer but also help restore effector function and the recognition and elimination capacity of cancer cells by NK cells. 4. Discussion Aging leads to decreased lymphocyte concentration and function. T lymphocyte decline is mainly due to thymic involution. Conversely, the increase of NK absolute and relative values in PB due to successive antigenic challenges is an evidence of the immunosenescence process. Subpopulation balance changes, with fewer naive lymphocytes and more memory, exhausted, and senescent phenotypes accumulate. Clonal diversity decreases, and oligoclonal populations expand, contributing to immunosenescence (4, 12–14, 22). The values outside the range of normal values were found only above the upper limit of each reference value range for both NK cells and NKT cells. The high data dispersion observed in NKT cells, could be related to previous exposures to stimulating agents. The means of the absolute values for NK and NKT cells were higher in the group under 80 years old and in the male group. However, specifically for NKT cells, the standard deviation (SD) was higher than the mean in both distributions, indicating a high dispersion among the data. NKT cells accumulate in PB as their affinity antigens are encountered, so the interindividual variability found may be directly related to the number of previous exposures to agents capable of stimulating NKT cells (15). Additionally, the expansion of this population has been reported in pathological conditions such as sarcoidosis, allergies, various types of cancer, and HIV or Human Cytomegalovirus (HCMV) infection (15, 18). Apoil et al. found in the aforementioned study that HCMV Page 8/13 seropositivity was associated with an increased frequency of CD56 + T cells (18). García et al. observed a high prevalence of HCMV infection in a group of Cuban adults aged 18 to 102 years (19). Considering this, it is possible that HCMV infection contributes to the increase in the NKT cell population in these groups. seropositivity was associated with an increased frequency of CD56 + T cells (18). García et al. observed a high prevalence of HCMV infection in a group of Cuban adults aged 18 to 102 years (19). Considering this, it is possible that HCMV infection contributes to the increase in the NKT cell population in these groups. The accumulation of terminally differentiated NK cells (CD56dim: with NKG2C+, CD57+, or FcεRIγ−) in PB can be caused by factors such as advanced age, chronic antigenic exposure following HCMV infection, or sex chromosome genes, as it has been found that males have higher values of these cell populations. 4. Discussion NKT cells reverse NK cell exhaustion through cytokine production, expression of co-stimulatory molecules, and their immunoregulatory activity, reducing immunosuppression in the tumor microenvironment (17, 20). 5. Limitations Page 9/13 Page 9/13 However, previous studies have shown certain associations between sex and these cells, although the unbalanced representation of sexes in this study may have affected the results. Further research with larger and balanced samples is recommended to obtain more solid conclusions. However, previous studies have shown certain associations between sex and these cells, although the unbalanced representation of sexes in this study may have affected the results. Further research with larger and balanced samples is recommended to obtain more solid conclusions. 6. Conclusions NK and NKT cells play a fundamental role in the regulation of immune response and directly influence the impairment of immune response in older adults. This impairment contributes to increased susceptibility to infections, tumors, and autoimmune diseases in this population group. In this study, no significant influence of age and sex was found on the values of NKT and NK cells in pheripheral blood of older adults. However, adequate numbers of circulant NK cells could be a protective factor against malignant neoplasms. Declaration of competing interests The author declares no conflict of interest, and there are no conflicts of interest between the institutions and the rest of the participating researchers. The identity of the participating patients in all processes was protected, and the ethical principles of human research were applied. References 1. Saavedra D, Fuertes SA, Suárez GM, González A, Lorenzo-Luaces P, García B et al (2019) Biomodulina T partially restores immunosenescent CD4 and CD8 T cell compartments in the elderly. Exp Gerontol 124:110633 2. Pera A, Campos C, López N, Hassouneh F, Alonso C, Tarazona R et al (2015) Immunosenescence: Implications for response to infection and vaccination in older people. Maturitas 82(1):50–55 3. Crooke SN, Ovsyannikova IG, Poland GA, Kennedy RB (2019) Immunosenescence and human vaccine immune responses. Immunity & ageing: I & A. ;16:25 3. Crooke SN, Ovsyannikova IG, Poland GA, Kennedy RB (2019) Immunosenescence and human vaccine immune responses. 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Reed RG, Al-Attar A, Presnell SR, Lutz CT, Segerstrom SC (2019) A longitudinal study of the stability, variability, and interdependencies among late-differentiated T and NK cell subsets in older adults. Exp Gerontol 121:46–54 21. Ye L, Zhang F, Li H, Yang L, Lv T, Gu W et al (2017) Circulating Tumor Cells Were Associated with the Number of T Lymphocyte Subsets and NK Cells in Peripheral Blood in Advanced Non-Small-Cell Lung Cancer. Dis Markers 2017:5727815 22. Michel JJ, Griffin P, Vallejo AN (2016) Functionally Diverse NK-Like T Cells Are Effectors and Predictors of Successful Aging. Front Immunol. ;7 22. Michel JJ, Griffin P, Vallejo AN (2016) Functionally Diverse NK-Like T Cells Are Effectors and Predictors of Successful Aging. Front Immunol. ;7 23. Wang J, Yang G, Wang D, Liu K, Ma Y, Liu H et al (2017) Changes of peripheral lymphocyte subsets and cytokine environment during aging and deteriorating gastrointestinal tract health status. Oncotarget 8(37):60764–60777 Page 11/13 24. Helsinki (2013) World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 310(20):2191–2194 25. Klein SL, Flanagan KL (2016) Sex differences in immune responses. Nat Rev Immunol 16(10):626– 638 26. Kissick HT, Sanda MG, Dunn LK, Pellegrini KL, On ST, Noel JK et al (2014) Androgens alter T-cell immunity by inhibiting T-helper 1 differentiation. Proc Natl Acad Sci USA 111(27):9887–9892 26. Kissick HT, Sanda MG, Dunn LK, Pellegrini KL, On ST, Noel JK et al (2014) Androgens alter T-cell immunity by inhibiting T-helper 1 differentiation. Proc Natl Acad Sci USA 111(27):9887–9892 27. References Edwards M, Dai R, Ahmed SA (2018) Our environment shapes us: the importance of environment and sex differences in regulation of autoantibody production. Front Immunol 9:478 27. Edwards M, Dai R, Ahmed SA (2018) Our environment shapes us: the importance of environment and sex differences in regulation of autoantibody production. Front Immunol 9:478 28. Yan J, Greer JM, Hull R, O’Sullivan JD, Henderson RD, Read SJ et al (2010) The effect of ageing on human lymphocyte subsets: comparison of males and females. Immun Ageing 7:4 28. Yan J, Greer JM, Hull R, O’Sullivan JD, Henderson RD, Read SJ et al (2010) The effect of ageing on human lymphocyte subsets: comparison of males and females. Immun Ageing 7:4 24. Helsinki (2013) World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 310(20):2191–2194 Figures Page 12/13 Figure 1 Methodology for the analysis of natural killer T (NKT) cells. Discrimination of singlets from total e (A). Segregation of the lymphocyte population using CD45 vs SSC (B). Separation of T lymphocyt using the CD3 antigen (C) and the CD56 antigen based on SSC (D). Figure 1 Methodology for the analysis of natural killer T (NKT) cells. Discrimination of singlets from total events (A). Segregation of the lymphocyte population using CD45 vs SSC (B). Separation of T lymphocytes using the CD3 antigen (C) and the CD56 antigen based on SSC (D). Page 13/13 Page 13/13
https://openalex.org/W2560573788	https://europepmc.org/articles/pmc5143613?pdf=render	English	null	Corrigendum: Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture	Frontiers in cellular and infection microbiology	2,016	cc-by	748	A corrigendum on Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture by Jiang, H., Du, H., Wang, L. M., Wang, P. Z., and Bai, X. F. (2016). Front. Cell. Infect. Microbiol. 6:1. doi: 10.3389/fcimb.2016.00001 Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture by Jiang, H., Du, H., Wang, L. M., Wang, P. Z., and Bai, X. F. (2016). Front. Cell. Infect. Microbiol. 6:1. doi: 10.3389/fcimb.2016.00001 Due to an oversight the authors did not cite the original source for Figures 1, 2. Figure 1 was adapted from Figure 5 of Schönrich et al. (2008). The revised ﬁgure caption should read: Left side: Normal endothelial cells (EC), no vascular leakage occurs. Right side: EC were infected with hantaviruses. ZO-1, VEGFR2, VE-cadherin on EC were altered. High hantavirus RNA load result in severe vascular leakage. Virus-infected ECs be cleared by virus-speciﬁc CTLs leading to vascular damage. Owing to acute thrombocytopenia, there are not suﬃcient platelets available to repair “holes” in the EC barrier, resulting in vascular leakage. In addition, cytokines produced during the innate response against pathogenic hantaviruses like TNF-α could enhance vascular permeability. Adapted from Schönrich et al. (2008). p y p Figure 2 was adapted from Figure 2 of Schönrich et al. (2015). The revised ﬁgure caption should read: Monocytes, macrophages, NK cells, and Lymphocytes produce various cytokines/ chemokines which directly or indirectly increase vascular permeability. The humoral pattern recognition receptor PTX3 and antibodies activate complement. Activated complement components induce cytoskeletal rearrangement in EC further increasing dysfunction of the EC barrier. TLRs recognize Hantavirus and mediate the innate response. Virus-infected ECs were cleared by virus-speciﬁc CTLs leading to vascular leakage. B cells produce several subclass antibodies, while only the neutralizing antibodies against G1 and G2 is beneﬁcial to decrease the viruses, then decrease vascular leakage. Adapted from Schönrich et al. (2015). Corrigendum: Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture Hong Jiang 1, Hong Du 1, Li M. Wang 2, Ping Z. Wang 1* and Xue F. Bai 1* 1 Center for Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an, China, 2 Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi’an, China Keywords: hantavirus, hemorrhagic fever with renal syndrome, Bunyavirus, hantaan virus, pathogenesis Edited and reviewed by: Shinichiro Kurosawa, Boston University School of Medicine, USA Edited and reviewed by: Shinichiro Kurosawa, Boston University School of Medicine, USA Correspondence: Ping Z. Wang wangpz63@126.com Xue F. Bai xfbai2011@163.com Correspondence: Ping Z. Wang wangpz63@126.com Xue F. Bai xfbai2011@163.com *Correspondence: Ping Z. Wang wangpz63@126.com Xue F. Bai xfbai2011@163.com g p This does not aﬀect the scientiﬁc conclusions of this article in any way. The authors apologize for this oversight. REFERENCES Schönrich, G., Krüger, D. H., and Raftery, M. J. (2015). Hantavirus-induced disruption of the endothelial barrier: neutrophils are on the payroll. Front. Microbiol. 6:222. doi: 10.3389/fmicb.2015.00222 Received: 05 April 2016 Accepted: 22 November 2016 Published: 08 December 2016 Received: 05 April 2016 Accepted: 22 November 2016 Published: 08 December 2016 Citation: Jiang H, Du H, Wang LM, Wang PZ and Bai XF (2016) Corrigendum: Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture. Front. Cell. Infect. Microbiol. 6:178. doi: 10.3389/fcimb.2016.00178 Schönrich, G., Rang, A., Lütteke, N., Raftery, M. J., Charbonnel, N., and Ulrich, R. G. (2008). Hantavirus-induced immunity in rodent reservoirs and humans. Immunol. Rev. 225, 163–189. doi: 10.1111/j.1600-065X.2008.00694.x CORRECTION published: 08 December 2016 doi: 10.3389/fcimb.2016.00178 CORRECTION published: 08 December 2016 doi: 10.3389/fcimb.2016.00178 Citation: Jiang H, Du H, Wang LM, Wang PZ and Bai XF (2016) Corrigendum: Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Copyright © 2016 Jiang, Du, Wang, Wang and Bai. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Front. Cell. Infect. Microbiol. 6:178. doi: 10.3389/fcimb.2016.00178 December 2016 \| Volume 6 \| Article 178 1 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org
https://openalex.org/W2603315599	https://europepmc.org/articles/pmc5348582?pdf=render	English	null	Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma	Clinics	2,017	cc-by	3,534	Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma Omer Bilgehan Poyrazoglu,I Ahmet Cumhur Dulger,II,* Bilge Sumbul GultepeIII I Lokman Hekim Hospital, General Surgery, Van, Turkey. II Yuzuncu Yil University, School of Medicine, Department of Gastroenterology, Van, Turkey. III Bezmialem Vakif University, School of Medicine, Microbiology, Istanbul, Turkey. OBJECTIVE: Esophageal squamous cell carcinoma is one of the most common esophageal diseases in the developing world, but the relationship between esophageal squamous cell carcinoma and Helicobacter pylori infection remains a neglected topic. The primary objective of this study was to determine the association between Helicobacter pylori infection and esophageal squamous cell carcinoma. A second purpose was to determine the incidence and factors associated with Helicobacter pylori infection following esophagectomy. METHOD: The microorganism was identiﬁed by testing the gastric biopsy materials from 95 esophageal squamous cell carcinoma patients (66 females; 39 were esophagectomized) for urease activity in a medium containing urea and a power of hydrogen detection reagent and comparing the results with those from a healthy population. Differences in patient characteristics were assessed with chi-square tests and t-tests for categorical and continuous factors, respectively. RESULTS: The patients with esophageal squamous cell carcinoma had a signiﬁcantly lower prevalence of Helicobacter pylori compared with the healthy population (po0.001). The naive and esophagectomized patients, in contrast, showed no signiﬁcant differences in Helicobacter pylori infection (p40.005). Patients with esophageal squamous cell carcinoma showed a signiﬁcant association between leukocytosis and hypoglobu- linemia and the presence of Helicobacter pylori infection (p=0.023 and p=0.045, respectively). CONCLUSION: These results suggest that Helicobacter pylori is not an etiological factor in patients with esophageal squamous cell carcinoma. We found a statistically signiﬁcant negative correlation between esoph- ageal squamous cell cancer and Helicobacter pylori infection. These ﬁndings may guide new strategies for esophageal squamous cell carcinoma therapy. KEYWORDS: Helicobacter pylori; Esophageal Squamous Cell Carcinoma; Turkey. KEYWORDS: Helicobacter pylori; Esophageal Squamous Cell Carcinoma; Turkey. Poyrazoglu OB, Dulger AC, Gultepe BS.Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma. Clinics. 2017; 72(3):150-153 Poyrazoglu OB, Dulger AC, Gultepe BS.Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma. Clinics. 2017; 72(3):150-153 Received for publication on July 12, 2016; First review completed on October 10, 2016; Accepted for publication on December 19, 2016 Corresponding author. E-mail: acdulger@gmail.com Received for publication on July 12, 2016; First review completed on October 10, 2016; Accepted for publication on December 19, 2016 Corresponding author. E-mail: acdulger@gmail.com *Corresponding author. E-mail: acdulger@gmail.com CLINICAL SCIENCE CLINICAL SCIENCE ’ INTRODUCTION cancer (in 0.1 to 3%), and gastric mucosa-associated lym- phoid-tissue (MALT) lymphoma (in o0.01%) (4). Recent studies have demonstrated a high frequency of HP infection in gastric cancer patients, suggesting that presence of HP may function as a driver of the events contributing to oncogen- esis in gastric adenocarcinomas (5,6). Furthermore, an inverse association has been established between Cag A-positive HP infection and the risk of esophageal adenocarcinoma (7). HP infection plays crucial roles in gastric carcinogenesis; however, the impact of HP on ESCC is not well understood. Esophageal squamous cell carcinoma (ESCC), one of the most aggressive digestive system tumors, is associated with numer- ous factors, including advanced age, achalasia, Plummer-Vinson syndrome, low socioeconomic status, high-starch diets lacking in fruits and vegetables, alcohol abuse, tobacco use, previous head and neck squamous cell carcinoma, and radiation ther- apy (1). It is also an important cause of mortality in the Asian esophageal cancer belt and in the eastern part of Turkey (2). p g p y Helicobacter pylori (HP), a gram-negative bacterium found on the gastric mucosa, was first isolated 30 years ago (3). Infection with HP is one cause of duodenal or gastric ulcers (reported to develop in 1 to 10% of infected patients), gastric The available data, derived from studies using serologic tests, are conflicting with respect to any association between HP and ESCC (8-10). The aim of the present study was to examine the potential correlation between ESCC and HP infection and to compare the presence of HP in naive ESCC patients and their esophagectomized counterparts. Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No potential conﬂict of interest was reported. DOI: 10.6061/clinics/2017(03)04 ’ DISCUSSION Our study revealed a strong association between HP infec- tion and a reduced risk of ESCC. In the Turkish adult popula- tion, the prevalence of HP infection is higher than reported in Western countries. A previous investigation of 4622 Turkish subjects indicated an HP infection prevalence of 82.5% (12). The prevalence of HP infection in the present study was ’ RESULTS endemic. In total, 95 ESCC patients (65 women, aged 32- 92 years) were evaluated in our clinic from July 2012 to July 2015. All the patients were diagnosed with ESCC based on established endoscopic and histopathological criteria. Of these, 39 had undergone a subtotal esophagectomy. In this esophagectomy group, esophageal reconstruction was per- formed through a subcutaneous route in 10 patients, through a retrosternal route in 25, and through a posterior mediast- inal route in 4. The reconstructed esophagus was a wide gastric tube, as described by Holscher (11). The mean age was 52.96±11.81 years in the control group (100 women), 59.53±13.93 years in the naive ESCC group (36 females), and 55.95±11.53 years in esophagectomized group (28 females). Nearly two-thirds of the ESCC patients were female. Descriptive statistics and comparison results according to the presence of HP are shown in Table 1. g p HP infection was observed in 39 (68.4%) of the 57 naive ESCC patients, 27 (69.2%) of the 39 esophagectomized patients, and 128 (85%) of the 151 dyspeptic control patients (Figure 2). We found a significantly lower rate of HP in patients with ESCC compared with the dyspeptic subjects (po0.0001), whereas no statistically significant difference was detected between the naive ESCC patients and their esopha- gectomized counterparts (p40.005). We also found no gender differences between the groups (p40.0005). Postoperatively, the patients typically underwent upper gastrointestinal endoscopy 6 weeks after surgery to deter- mine the presence or absence of HP infection. p Gastric biopsy samples were tested for urease activity using a commercial Hp-fast test kit (GI Supplys, Camp Hill, PA, USA) consisting of a urea-containing medium and a power of hydrogen (pH) detection reagent. g p Significantly higher levels of leukocytes and serum glob- ulin were also found in the ESCC patients diagnosed with HP compared with those without HP infection (p=0.023 for leukocytes and p=0.045 for serum globulin). p y g p g The control group comprised 151 dyspeptic subjects (100 women and 51 men, aged 30–85 years). Controls were also required to be medical-treatment free for at least 6 months from the time of study entry. Antrum biopsies with normal endoscopic evaluations were examined for HP using the same method. The prognostic values of the presence of HP and other clinicopathologic factors were also evaluated. ’ PATIENTS AND METHODS This retrospective trial was conducted at a university medical center in a large metropolitan area near the Iranian border of Turkey, where both ESCC and HP infection are DOI: 10.6061/clinics/2017(03)04 150 CLINICS 2017;72(3):150-153 CLINICS 2017;72(3):150-153 HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. CLINICS 2017;72(3):150-153 similar (85%). Squamous cell carcinomas are usually detected in the proximal two-thirds of the esophagus. They predomi- nantly affect elderly people and usually present as dysphagia, odynophagia, and unintentional weight loss (13). Worldwide, esophageal cancer ranks fifth in mortality among all malig- nancies, and ESCC remains the most common type. cancers are the most prevalent malignancies in both females and males in eastern part of Turkey (16). The probable culprit factors for ESCC in this region are low educational and socioeconomic status; the consumption of smoked, salted, hot, fatty foods; overconsumption of hot tea; cigarette smok- ing; poor intake of fresh fruits and vegetables, and poor hygienic conditions (17). Previous studies have shown that the eastern part of Turkey has one of the highest rates of both ESCC and HP infection. An HP infection rate of 36% was reported for gastric biopsy specimens in patients with gastric carcinoma from the Van region (18). cancers are the most prevalent malignancies in both females and males in eastern part of Turkey (16). The probable culprit factors for ESCC in this region are low educational and socioeconomic status; the consumption of smoked, salted, hot, fatty foods; overconsumption of hot tea; cigarette smok- ing; poor intake of fresh fruits and vegetables, and poor hygienic conditions (17). Previous studies have shown that the eastern part of Turkey has one of the highest rates of both ESCC and HP infection. An HP infection rate of 36% was reported for gastric biopsy specimens in patients with gastric carcinoma from the Van region (18). In Asia, upper gastrointestinal cancers constitute a major group of malignancies with high rates of morbidity and mortality. The esophageal cancer belt, originating in the Far East and extending through middle Asia and the Near East, encompasses many countries, including northern China, northern Iran and the eastern part of Turkey (14). The pre- dominant histopathological type of esophageal cancer is the squamous cell type in the endemic Asian regions, with incidence rates that may vary 200-fold among different populations within the same defined region due to cultural practices. More than 80% of ESCC patients in the rural areas of Asia also present at advanced stages that are not amenable to curative therapies; hence the need for novel preventive strategies is urgent (15). CLINICS 2017;72(3):150-153 g g ( ) Various environmental factors, including cigarette smok- ing and excessive alcohol intake, can be associated with an increased risk of developing ESCC (19). HP is considered one of the most important human carcinogens for the upper gastrointestinal tract and the stomach (20). At present, a few reports have indicated a possible relationship between HP and ESCC, but most of these were performed using non- endoscopic (serologic) techniques (7). A recent Chinese study reported an HP seropositivity of 35.3% in ESCC patients, which was lower than that of the control groups (40% and 59%) (21). A recent meta-analysis also found an association between CagA-negative HP strains and a marginally signif- icant increased risk of ESCC (7). In contrast, a prospective and serological study from China showed no association between HP and ESCC (22). Another recent meta-analysis from China showed an association between HP infection and a decreased risk of ESCC in Eastern populations and a decreased risk of esophageal adenocarcinoma (EAC) in the overall population (23). The Van region of eastern Turkey is located in the western end of the esophageal cancer belt. Esophageal and gastric Figure 1 - Traditional large oven (Tandir). p p The current study revealed no association between HP infection and ESCC among people living in the eastern part of Turkey. The low prevalence of HP in these ESCC patients was similar to that reported in the Chinese meta-analyses. We also found a predominance of ESCC in female patients, in agreement with a previous Turkish study (17). This phe- nomenon has been linked to the use of the traditional large ovens (tandirs) that use smoke to cook meals (Figure 1). The mechanisms by which these ovens induce esophageal carci- nogenesis remain undefined, but previous work suggests that smoke may play role similar to that of cigarette smoking (17). We conclude that an effective follow-up strategy for HP-negative female Asian adults will be necessary if ESCC screening is to yield public health benefits. The higher levels of leucocytes and serum globulin among ESCC patients with HP may also reflect an emerging phenomenon that requires additional investigation to determine the underlying causa- tive factors. Figure 1 - Traditional large oven (Tandir). Figure 1 - Traditional large oven (Tandir). Figure 2 - Rate of HP according to groups. The H. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. CLINICS 2017;72(3):150-153 Statistical analysis h The two groups were compared using the Mann-Whitney U-test. The differences were considered statistically signifi- cant at po0.005. Table 1 - Descriptive statistics and comparison results according to Helicobacter pylori status. HP1 n Mean Std. Dev Min. Max. p Age (years) + 65 57.02 11.514 32 92 0.239 - 30 60.43 15.939 23 87 Total 95 58.09 13.084 23 92 Hemoglobin (gr/dL) + 63 12.50 2.025 8 17 0.371 - 30 12.11 1.876 9 17 Total 93 12.38 1.976 8 17 Hematocrit + 63 37.20 5.616 24 49 0.427 - 30 36.21 5.502 28 49 Total 93 36.88 5.569 24 49 Leukocytes (/mm3) + 63 7.598 3.8817 1.8 25.0 0.023 - 30 5.803 2.4689 2.0 11.0 Total 93 7.019 3.5760 1.8 25.0 Platelets (/mm3) + 63 270.000 123.839 67.000 702.000 0.150 - 30 616.687 324.542 79.000 178.000 Total 93 443.343 184.309 67.000 178.000 ALT2 (U/L) + 63 18.94 17.766 6 142 0.284 - 30 15.27 8.026 6 39 Total 93 17.75 15.362 6 142 AST3 (U/L) + 62 33.02 48.837 2 341 0.089 - 30 17.57 6.976 9 38 Total 92 27.98 40.833 2 341 Albumin (g/dL) + 58 3.71 0.734 2 5 0.882 - 25 3.69 0.667 2 5 Total 83 3.71 0.711 2 5 Globulin (g/dL) + 47 3.07 0.533 2 4 0.045 - 19 2.73 0.772 1 4 Total 66 2.97 0.625 1 4 Calcium (mg/dL) + 57 8.92 1.362 1 13 0.370 - 27 9.17 0.639 8 11 Total 84 9.00 1.181 1 13 Std. Dev: Standard deviation 1 Helicobacter pylori, 2 Alanine transaminase, 3 Aspartate transaminase Table 1 - Descriptive statistics and comparison results according to Helicobacter pylori status. tistics and comparison results according to Helicobacter pylori status. 151 HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. infection status changed from preoperatively positive to postoperatively negative and that this changing pattern was linked to the eradication of HP via the perioperative administration of antibiotics (25). Conversely, a report from China showed a low incidence of HP infection in the gastric conduit in patients who underwent esophagectomy, pylor- oplasty, and reconstruction. The authors of that study con- cluded that this phenomenon was mostly due to the chronic reflux of bile after pyloroplasty (26). carcinoma in a Taiwanese population. Am J Gastroenterol. 2005;100 (3):588-93, http://dx.doi.org/10.1111/j.1572-0241.2005.40623.x. carcinoma in a Taiwanese population. Am J Gastroenterol. 2005;100 (3):588-93, http://dx.doi.org/10.1111/j.1572-0241.2005.40623.x. p g j 10. Henrik Simán J, Forsgren A, Berglund G, Floren CH. Helicobacter pylori infection is associated with a decreased risk of developing oesophageal neoplasms. Helicobacter. 2001;6(4):310-6, http://dx.doi.org/10.1046/j.1523- 5378.2001.00041.x. 11. Holscher AH, Voit H, Buttermann G, Siewert JR. Function of the intra- thoracic stomach as esophageal replacement. World J Surg. 1998;12(6): 835-44, http://dx.doi.org/10.1007/BF01655491. p g 12. Ozaydin N, Turkyilmaz SA, Cali S. Prevalence and risk factors of Heli- cobacter pylori in Turkey: a nationally-representative, cross-sectional, screening with the 13C-Urea breath test. BMC Public Health. 2013;13:1215, http://dx.doi.org/10.1186/1471-2458-13-1215. Our findings were in concordance with previous reports on HP status and gastric tube cancer patients. In our Turkish population, HP infection was not associated with ESCC and had a similar pattern to that reported for Asian populations. HP infection may not contribute to the development of ESCC in patients who reside in rural areas of Asia, especially not in females. Furthermore, esophageal damage could be dimin- ished by the prior presence of HP in areas with a high risk of ESCC. HP treatment has been associated with impro- ved thrombocyte levels. An increase in platelet counts was observed in only 6.7% of treated patients. In the current study, we found no relationship between HP status and thrombocyte levels (27,28). p g 13. Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med. 2003;349(23): 2241-52, http://dx.doi.org/10.1056/NEJMra035010. p g 14. Lambert R, Hainaut P. The multidisciplinary management of gastro- intestinal cancer. Epidemiology of oesophagogastric cancer. Best Pract Res Clin Gastroenterol. 2007;21(6):921-45, http://dx.doi.org/10.1016/ j.bpg.2007.10.001. j pg 15. Ke L. Mortality and incidence trends from esophagus cancer in selected geographic areas of China circa 1970-90. Int J Cancer. 2002;102(3):271-4, http://dx.doi.org/10.1002/ijc.10706. p g j 16. Koca T, Arslan D, Basaran H, Cerkesli AK, Tastekin D, Sezen D, et al. CLINICS 2017;72(3):150-153 CLINICS 2017;72(3):150-153 ’ AUTHOR CONTRIBUTIONS y J ( ) 19. Lambert R, Hainaut P. Esophageal cancer: cases and causes (part I). Endoscopy. 2007;39(6):550-5, http://dx.doi.org/10.1055/s-2007-966530. Poyrazoglu OB was involved in developing the concept and design of the study and writing the manuscript. Dulger AC was involved in imple- menting the study and collecting data. Dulger AC and Gultepe BS were involved in collecting and processing the data. All the authors read and approved the ﬁnal manuscript. py p g 20. Goodwin CS, Mendall MM, Northfield TC. Helicobacter pylori infection. Lancet. 1997;349(9047):265-9, http://dx.doi.org/10.1016/S0140-6736(96) 07023-7. 21. Wu IC, Wu DC, Yu FJ, Wang JY, Kuo CH, Yang SF, et al. Association between Helicobacter pylori seropositivity and digestive tract cancers. World J Gastroenterol. 2009;15(43):5465-71, http://dx.doi.org/10.3748/ wjg.15.5465. jg 22. Kamangar F, Qiao YL, Blaser MJ, Sun XD, Katki H, Fan JH, et al. Heli- cobacter pylori and oesophageal and gastric cancers in a prospective study in China. Br J Cancer. 2007;96(1):172-6, http://dx.doi.org/10.1038/ sj.bjc.6603517. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. Dietary and demographical risk factors for oesophageal squamous cell carcinoma in the Eastern Anatolian region of Turkey where upper gas- trointestinal cancers are endemic. Asian Pac J Cancer Prev. 2015;16(5): 1913-7, http://dx.doi.org/10.7314/APJCP.2015.16.5.1913. p g J 17. Turkdogan MK, Akman N, Tuncer I, Uygan I, Kösem M, Ozel S, et al. Epidemiological aspects of endemic upper gastrointestinal cancers in eastern Turkey. Hepatogastroenterology. 2005;52(62):496-500. y Finally, further studies are needed to address the impact of HP infection on ESCC and its natural history. y Finally, further studies are needed to address the impact of HP infection on ESCC and its natural history. y p g gy 18. Türkdog˘an K, Alici S, I˙lhan M, Dilek H, Akman E, Ayakta H, et al. Helicobacter pylori infection in gastric carcinoma in the Van region of Turkey. Turk J Gastroenterol. 1999;10(1):36-9. CLINICS 2017;72(3):150-153 pylori-fast test (Hp-fast test) is based on the detection of HP urease activity and has a high sensitivity (85%) and specificity (495%) for detecting HP infection. As this test is considered cost-effective and suitable for endoscopy units (24), we used the Hp-fast test to establish HP infection. The current data are limited regarding any changes in the prevalence of HP in patients who have undergone esopha- gectomy. We therefore performed gastroduodenoscopy with pathological examination of the biopsy specimens obtained from the gastric conduit. We observed that the rate of HP infection was lower in esophagectomized ESCC patients than in the control subjects (19% and 78%, respectively; po0.001). The rate of HP infection was similar, however, between the naive ESCC patients and the esophagectomized patients (p40.005). A Japanese study reported that the HP Figure 2 - Rate of HP according to groups. 152 ’ REFERENCES 1. Layke JC, Lopez PP. Esophageal cancer: a review and update. Am Fam Physician. 2006;73(12):2187-94. j j 23. Xie FJ, Zhang YP, Zheng QQ, Jin HC, Wang FL, Chen M, et al. Helico- bacter pylori infection and esophageal cancer risk: an updated meta- analysis. World J Gastroenterol. 2013;19(36):6098-107, http://dx.doi.org/ 10.3748/wjg.v19.i36.6098. y 2. Onuk MD, Oztopuz A, Memik F. Risk factors for esophageal cancer in eastern Anatolia. Hepatogastroenterology. 2002;49(47):1290-2. p g gy 3. 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https://openalex.org/W2912202314	https://journal.unpak.ac.id/index.php/fitofarmaka/article/download/1170/pdf	Indonesian	null	PENGARUH ANTIBIOTIKA PROFILAKSIS TERHADAP KEJADIAN INFEKSI LUKA OPERASI	Fitofarmaka	2,019	cc-by-sa	2,835	ABSTRAK Antibiotik profilaksis adalah antibiotik yang diberikan pada pasien yang akan menjalani pembedahan untuk mencegah terjadinya infeksi akibat tindakan operasi. Antibiotik profilaksis diberikan secara intravena agar dicapai konsentrasi maksimum di serum/jaringan pada saat operasi. Pemilihan antibiotika profilaksis yang sesuai pada tindakan pembedahan sangat menentukan keberhasilan dalam mencegah terjadinya infeksi luka operasi. Penelitian ini bertujuan untuk mengetahui besarnya angka kejadian infeksi luka operasi dan mengevaluasi penggunaan antibiotika profilaksis dalam pencegahan infeksi luka operasi di rumah sakit Premier Bintaro, Kota Tanggerang. Jenis penelitian ini adalah observasional dengan rancangan studi deskriptif analitik melalui penelusuran data yang dilakukan secara retrospektif pada pasien yang menjalani pembedahan di ruang operasi. Analisa dan evaluasi data berupa deskripsi pola penggunaan antibiotika profilaksis dan angka kejadian infeksi luka operasi serta hubungan antara penggunaan antibiotika profilaksis dengan kejadian infeksi luka operasi. Hasil yang diperoleh dalam penelitian ini menunjukan bahwa angka kejadian infeksi luka operasi pada tindakan pembedahan sebanyak 7 kasus (1,97%) dari jumlah total 355 kasus bedah pada periode penelitian. Antibiotika profilaksis yang paling banyak digunakan adalah sefalosporin generasi III (66,2%). Hasil Analisa dengan Fisher exact menunjukkan bahwa sifat operasi jenis antibiotika dan waktu pemberian antibiotika mempunyai hubungan bermakna dengan kejadian ILO (p<0,05). Dari penelitian terlihat pula bahwa semakin lama operasi berlangsung semakin tinggi risiko infeksi luka operasi. Antibiotika sefalosporin generasi III terbanyak yang digunakan adalah ceftriaxone injeksi. j Kata kunci: Antibiotika profilaksis, sefalosporin, luka operasi Kata kunci: Antibiotika profilaksis, sefalosporin, luka operasi Artikel Riset DOI : 10.33751/jf.v8i1.1170 Artikel Riset DOI : 10.33751/jf.v8i1.1170 Fitofarmaka Jurnal Ilmiah Farmasi Vol. 8, No.1, Juni 2018 : 43-49 p-ISSN : 2087-9164 e-ISSN : 2622-755X Oktaviana Zunnita* Program Studi Farmasi, FMIPA, Universitas Pakuan, PO Box 452 Bogor 16143, West Java, Indonesia E-mail: nadiahasna40@gmail.com Diterima : 13 Mei 2018 Disetujui : 11 Juni 2018 Disetujui : 11 Juni 2018 Direvisi : 28 Mei 2018 PENGARUH ANTIBIOTIKA PROFILAKSIS TERHADAP KEJADIAN INFEKSI LUKA OPERASI Oktaviana Zunnita Program Studi Farmasi, FMIPA, Universitas Pakuan, PO Box 452 Bogor 16143, West Java, Indonesia *E-mail: nadiahasna40@gmail.com ABSTRACT Antibiotic prophylaxis commonly given to the patients undergoing surgery to prevent infection due to surgery wound. Antibiotics prophylactic were given intravenously to achieve maximum serum/tissue concentration at the time of operation and the maximum level was maintained during the surgical procedure. Selection of appropriate antibiotic prophylaxis in surgery is crucial to prevent surgical procedure. 43 Fitofarmaka Jurnal Ilmiah Farmasi Fitofarmaka Jurnal Ilmiah Farmasi Selection of appropriate antibiotic prophylaxis in surgery is crucial to prevent surgical site infection. This study aims to determine the occurrence of surgical site infection and to evaluate the use of prophylactic antibiotics to the pationts undergoing surgery in Premier Bintaro Hospital, Kota Tanggerang hospital. The study was an observational study with descriptive analytic design using retrospective data obtained from the surgery patients. The data was analyzed and evaluated in form of the pattern of antibiotic prophylaxis usage, occurrence of surgical site infection, and relationship between antibiotic prophylaxis usage and occurrence of surgical site infection. The results obtained that during the study period, seven cases of surgical site infections occurred from the total of 355 surgical procedure (1.97%). The antibiotics prophylaxis most widely used was a third generation cephalosporin (66.2%) The results of Fisher exact analysis showed that the types of operation, type of antibiotic, and time of antibiotics administration had significant relationship with ILO (p<0,05). The research also revealed that the longer the surgery time, the higher the risk of surgical site infection. The third generation cephalosporin antibiotics used were ceftriaxone injection was the third generation cephalosporin widely used in hospital. Key words: Antibiotics prophylactic cephalosporin surgical site infection g p p y p Key words: Antibiotics prophylactic, cephalosporin, surgical site infection PENDAHULUAN antimikroba serta reaksi kolitis (Gordon, 2006). Pasien yang menjalani operasi mempunyai risiko tinggi mengalami infeksi luka operasi dan pemberian antibiotika profilaksis dapat menurunkan infeksi luka operasi. Antibiotik profilaksis diberikan sedemikian rupa sehingga dicapai konsentrasi maksimum di serum/jaringan pada saat awal sayatan bedah, dan kadar ini dijaga selama periode rentan dalam prosedur yaitu waktu antara sayatan kulit dan penutupan kulit (Burke dan Cunha, 2009). Antibiotik profilaksis harus dapat memotong aktifitas patogen terhadap luka yang terkontaminasi atau pada daerah operasi. Menurut World Health Organization (WHO, 2002), antibiotika profilaksis diberikan secara intravena satu jam sebelum pembedahan. National Surgical Infection Prevention Project menyatakan bahwa antibiotika profilaksis sebaiknya tidak digunakan lebih dari 24 jam setelah penutupan luka operasi. Penggunaan antibiotika melebihi 48 jam tidak efektif dalam menurunkan risiko infeksi, dan meningkatkan resistensi Pada umumnya, antibiotika profilaksis dianjurkan hanya untuk tindakan dengan kejadian infeksi yang tinggi dan tindakan dengan konsekuensi infeksinya sangat serius. Infeksi luka operasi atau ILO merupakan bagian dari infeksi nosokomial dan termasuk salah satu masalah kesehatan yang cukup serius di rumah sakit. Infeksi pada operasi terjadi karena masuknya kuman yang berasal dari luar atau dari permukaan tubuh ke dalam luka (infeksi eksogen) dan dapat pula karena masuknya kuman yang berasal dari dalam rongga tubuh ke dalam luka (infeksi endogen). Infeksi eksogen terjadi melalui udara, kontak langsung antara luka dengan kuman yang ada pada permukaan tubuh, atau kuman yang berasal dari alat dan tangan. Infeksi endogen umumnya berasal dari tumpahan isi rongga/ saluran sewaktu dinding rongga/ saluran itu dipotong/robek Infeksi ini terjadi pada masa setelah operasi yang ditandai dengan adanya nanah, peradangan, bengkak, rasa nyeri dan panas. Beberapa faktor penderita yang 44 Pengaruh Antibiotik…..(Oktaviana Zunnita) mempermudah terjadinya ILO ialah obesitas, diabetes, co-morbid/ penyakit penyerta, infeksi di tempat lain, kontaminasi dalam pembedahan, rawat inap pre-operatif yang panjang, karier Staphylococcus aureus, dan pertahanan tubuh yang lemah. Faktor kuman yang mempengaruhi terjadinya ILO ialah virulensi serta jumlah kuman. Faktor lokal yang dapat mempermudah infeksi luka adalah pembedahan yang lama (> 4 jam), adanya bekuan darah atau jaringan nekrotik yang tertinggal, vaskularisasi yang buruk karena jahitan yang terlalu rapat dan kuat, jenis benang, dan pemasangan drain (Sjamsuhidajat et al., 2010). Di Indonesia, angka kejadian ILO pada rumah sakit bervariasi antara 2- 18 % dari keseluruhan prosedur pembedahan. Rumah Sakit pada periode penelitian. Eklusi sampel adalah pasien yang tidak dapat diikuti perkembangan atau data rekam medik tidak terbaca serta pasien dengan daya tahan menurun (HIV/AIDS). METODE PENELITIAN Bahan Bahan pada penelitian ini adalah data dari rekam medis 355 pasien dengan kasus bedah yang menjalani operasi dan perawatan di RS Premier Bintaro, Kota Tanggerang. Parameter data rekam medik pasien yang dilihat adalah sifat operasi, lama penggunaan antibiotika, waktu operasi, jenis antibiotik dan waktu pemberian antibiotika. Kejadian luka infeksi luka paska bedah ditandai dengan adanya nanah/pus berdasarkan diagnosa dokter yang tertera dalam rekam medik pasien dilihat dalam waktu 30 hari. Kriteria Inklusi sampel adalah pasien yang menjalani operasi dan perawatan di Metode Analisa Penelitian ini merupakan penelitian deskriptif yang dilakukan secara retrospektif pada pasien yang menjalani operasi di rumas sakit Premier Bintaro, Kota Tangerang pada Mei 2011 – April 2012. Data dilihat dari beberapa definisi operasional mulai dari jenis antibiotika profilaksis yang diberikan sebelum operasi berdasarkan nama generik dilihat dari golongan antibiotika yang digunakan, seperti sefalosporin generasi 2, generasi 3, generasi 4, carbapenem dan seterusnya. Jarak pemberian antibiotika profilaksis pada pasien sebelum operasi dilihat dari ketepatan waktu pemberian profilaksis 60 menit atau lebih. Durasi penggunaan antibiotika profilaksis dilihat dari lamanya penggunaan antibiotika. Penggunaan antibiotika profilaksis digunakan dalam 24 jam atau lebih. Waktu Operasi yang diperlukan oleh pasien untuk menjalani operasi juga merupakan salah satu kategori yang dilihat dalam penelitian. Waktu operasi berlangsung < 1jam atau lebih. Sifat operasi digolongkan dalam beberapa kategori yaitu bersih, kontaminasi dan kotor. Data yang diperoleh kemudian dikelompokkan dan dianalisis. Hubungan antara kejadian infeksi luka dengan beberapa data-data yang diambil dilakukan dengan analisa fisher exact. Pada studi ini, pengumpulan data penggunaan antibitiotik profilaksis dilakukan dengan melihat penggunaan antibiotika profilaksis meliputi jenis antibiotik, rute pemberian, lama penggunaan, lama operasi, jangka waktu pemberian dan angka kejadian luka operasi. HASIL DAN PEMBAHASAN Hasil yang diperoleh dalam penelitian ini menunjukan bahwa angka kejadian infeksi luka operasi pada tindakan pembedahan sebanyak 7 45 Fitofarmaka Jurnal Ilmiah Farmasi kasus (1,97%) dari jumlah total 355 kasus bedah pada periode penelitian. Dari hasil pengamatan infeksi luka operasi dalam penelitian ini sifat operasi, jenis antibiotika, waktu pemberian dan lama operasi memperlihatkan adanya hubungan atau pengaruh dalam kejadian infeksi luka operasi. Kejadian infeksi luka operasi terjadi pada operasi bersih 1,13% yaitu pada operasi cranioplasty, koreksi scoliasis dan Stabilisasi Bone/Other Operations Bone serta ORIF (Open reduction Invasif Fraktur). Operasi kontaminasi 0,56 % yaitu pada operasi appendectomy dan laparatomi. Operasi kotor 0,28 % yaitu pada operasi Close reduction+ debridement+ internal fixasi. pada operasi cranioplasty, koreksi scoliasis dan Stabilisasi Bone/Other Operations Bone serta ORIF (Open reduction Invasif Fraktur). Operasi kontaminasi 0,56 % yaitu pada operasi appendectomy dan laparatomi. Operasi kotor 0,28 % yaitu pada operasi Close reduction+ debridement+ internal fixasi. Gambar 1. Infeksi Luka Operasi Pada Sifat Operasi, Jenis Antibiotika, Waktu Pemberian dan Lama Operasi 0 10Ber…Kon…KotorSef…Cab…Pe…La…La… ILO ILO ILO Gambar 1. Infeksi Luka Operasi Pada Sifat Operasi, Jenis Antibiotika, Waktu Pemberian dan Lama Operasi Dari gambar diatas, didapat hasil infeksi luka operasi terjadi pada sifat operasi bersih 4 pasien, kontaminasi 2 pasien dan kotor 1 pasien. Infeksi luka operasi juga terjadi pada pemberian antibiotika profilaksis tidak tepat waktu, dengan lama operasi yang berlangsung 1- 2 jam atau lebih dari 2 jam. Pada jenis antibiotika profilaksis yang digunakan, ada 5 kejadian infeksi luka yang disebabkan oleh pemakaian antibiotika golongan carbapenem (meropenem). Hal ini bisa disebabkan waktu paruh dari, antibiotika golongan ini adalah pendek yaitu 1 jam, sehingga tidak direkomendasikan dalam pembedahan, selain harganya yang mahal, antibiotika ini mempunyai resiko lebih besar untuk kejadian infeksi luka operasi apabila pemberian profilaksis tidak tepat waktu dan operasi berlangsung lama, sehingga kadar obat dalam darah sudah berkurang saat operasi berlangsung. Infeksi luka operasi terjadi pada 2 pasien dengan penggunaan antibiotika profilaksis golongan sefalosporin generasi 3, hal ini dapat disebabkan oleh pasien sudah mengalami infeksi sebelum operasi, sehingga dibutuhkan kombinasi antibiotika profilaksis, dan pemberian profilaksis harus tepat waktu dengan operasi berlangsung > 1 jam agar manfaat profilaksis optimal. Pada penelitian sebelumnya yang dilakukan oleh Amba (2007), infeksi luka operasi paling tinggi juga terjadi pada durasi operasi > 2 jam yaitu sebesar 11,3 %, pada durasi 1 – 2 jam 8,4 % dan pada durasi operasi 1 jam 0 %. HASIL DAN PEMBAHASAN Oleh karena itu dapat disimpulkan bahwa semakin lama operasi berlangsung kemungkinan terjadi infeksi luka operasi cukup besar. Dari hasil penelitian dapat dikatakan teknik aseptis sudah dilakukan dengan cermat, dan penggunaan antibiotika profilaksis dapat dilihat manfaatnya karena sebagian besar pasien yang menjalani operasi tidak mengalami infeksi luka operasi. Hal ini dapat terlihat bahwa hanya 1,97 % pasien mengalami 46 Pengaruh Antibiotik…..(Oktaviana Zunnita) infeksi luka operasi dari 355 sampel pasien yang menjalani pembedahan. Oleh karena itu antibiotik yang disarankan adalah sefalosporin generasi I dan II. Pada kasus tertentu yang dicurigai melibatkan bakteri anaerob dapat ditambahkan metronidazol. Tidak dianjurkan menggunakan sefalosporin generasi III dan IV, golongan karbapenem (dicadangkan untuk kasus yang lebih serius) dan golongan kuinolon untuk profilaksis bedah (cepat memberikan resistensi). Dalam penelitian ini infeksi luka operasi terjadi pada penggunaan antibiotika profilaksi golongan carbapenem adalah 1,41 % dan 0,56 % antibiotika golongan sefaloporin generasi 3 (ceftriaxone). Indikasi penggunaan antibiotika profilaksis lebih ditekankan pada operasi bersih dan bersih kontaminasi dan dasar pemilihan antibiotika profilaksis sesuai dengan sensitivitas dan pola bakteri patogen terbanyak pada kasus yang bersangkutan. Sebaiknya antibiotika profilaksis yang digunakan adalah spektrum sempit untuk mengurangi risiko resistensi bakteri, toksisitas rendah, tidak menimbulkan reaksi merugikan terhadap pemberian obat anestesi, bersifat bakterisidal, harga terjangkau. Tabel 1. Penggunaan Antibitotika Profilaksis Pada Periode Penelitian Tabel 1. Penggunaan Antibitotika Profilaksis Pada Periode Penelitian Antibiotika profilaksis Jumlah pemakaian Sefalosporin generasi 2 5 Sefalosporin generasi 3 235 Carbapenem 54 Aminoglikosida 4 Penicillin 41 Glikopeptida 14 Sefalosporin generasi 4 2 Tabel 1. Penggunaan Antibitotika Profilaksis Pada Periode Penelitian Antibiotika profilaksis Jumlah pemakaian dan 238 (67%) pasien menerima antibiotika tidak tepat waktu. Hal ini disebabkan oleh persiapan operasi yang meliputi kedatangan dokter dan kesiapan kamar bedah. Ada beberapa penelitian yang menunjukkan bahwa pemberian dosis tunggal antibiotika profilaksis dengan waktu paruh cukup lama, cukup memadai untuk beberapa operasi (Welmahos et al., 2002 dan Desyana et al., 2008). Pada operasi yang tidak membutuhkan waktu lama, pemberian profilaksis dosis tunggal sama efektifnya dengan pemberian profilaksis dalam waktu lebih dari 24 jam. Penggunaan antibiotika profilaksis pada penelitian ini masih banyak menggunaka antibiotika yang biasa digunakan untuk pengobatan di ruang rawat. Hal ini dapat disebabkan karena belum adanya pengaturan dalam pemberian antibiotika profilaksis dan dapat juga karena persediaan obat dipasaran untuk sefalosporin generasi 1 dan 2 dalam bentuk injeksi susah didapat atau bisa juga disebabkan oleh promosi obat antibiotika dari pihak produsen serta sensitifitas antibiotika itu sendiri masih >60 % di rumah sakit ini. KESIMPULAN Dapat disimpulkan bahwa semakin lama operasi berlangsung kemungkinan terjadi infeksi luka operasi cukup besar. Dari hasil penelitian dapat dikatakan teknik aseptis sudah dilakukan dengan cermat , dan penggunaan antibiotika yang digunakan sebagian besar menggunakan sefalosporin generasi III yaitu sebanyak 235 (66,2 %) pasien, carbapenem sebanyak 54 (15,2 %) pasien, penicillin 41 (11,5 %) pasien. HASIL DAN PEMBAHASAN Kasus ILO Selama Periode Penelitian Tabel 2. Kasus ILO Selama Periode Penelitian Jenis Operasi Tabel 3. Hasil Analisis Korelasi Fisher Exact Tabel 3. Hasil Analisis Korelasi Fisher Exact Infeksi Luka Operasi Hasil Uji Statistik Keterangan Tidak Ada Ada Sifat Operasi 348 7 p =0,010 (< 0,05) Berbeda bermakna Jenis antibiotika 348 7 p =0,05 (< 0,05) Berbeda bermakna Waktu pemberian antibiotika 348 7 p = 0,017 (< 0,05) Berbeda bermakna Lama operasi 348 7 p = 0,007 (< 0,05) Berbeda bermakna Sefalosporin generasi 3 yang banyak dipakai adalah ceftriaxone dan terdapat hubungan dan pengaruh antara sifat operasi, jenis antibiotik, waktu pemberian dan lama operasi dengan infeksi luka operasi dengan P value < 0,05. HASIL DAN PEMBAHASAN Berdasarkan hasil penelitian sebanyak 117 (33%) pasien menerima antibiotika secara intravena tepat waktu Penggunaan antibiotika profilaksis yang tidak rasional dapat meningkatkan 47 Fitofarmaka Jurnal Ilmiah Farmasi Untuk melihat hubungan antara jenis antibiotika profilaksis, waktu pemberian profilaksis dan lama penggunaan profilaksis dapat dilihat dari hasil analisis dengan fisher exact. kejadian resistensi. Rata-rata resistensi antibiotika terjadi di rumah sakit. Pada operasi tertentu, penggunaan antibiotika lebih dari 24 jam setelah operasi direkomendasikan, seperti pada appendectomy, bedah syaraf, arthroplasty dan cardiothoracic (Sign, 2008). Tabel 2. Kasus ILO Selama Periode Penelitian Jenis Operasi Sifat Operasi Jenis antibiotika Waktu pemberian antibiotika Lama operasi 1. < 1 jam tepat < 60 menit 2. 1 - 2 jam tidak tepat > 60 menit 3. > 2 jam Laparatomy & Hemicolectomy Dx kontaminasi Meropenem tidak tepat 3 Cranioplasty bersih Meropenem tidak tepat 3 Koreksi scoliosis bersih Meropenem tidak tepat 3 Appendictomy kontaminasi Ceftriaxone tidak tepat 2 Scoliosis, Stabilisasi Bone/Other Operations Bone bersih Meropenem tidak tepat 3 Orif fraktur tri melaeolar kanan bersih Ceftriaxone tidak tepat 2 Close reduction+ debridement+ internal fixasi kotor Meropenem tidak tepat 3 Tabel 3. Hasil Analisis Korelasi Fisher Exact Infeksi Luka Operasi Hasil Uji Statistik Keterangan Tidak Ada Ada Sifat Operasi 348 7 p =0,010 (< 0,05) Berbeda bermakna Jenis antibiotika 348 7 p =0,05 (< 0,05) Berbeda bermakna Waktu pemberian antibiotika 348 7 p = 0,017 (< 0,05) Berbeda bermakna Lama operasi 348 7 p = 0,007 (< 0,05) Berbeda bermakna KESIMPULAN Dapat disimpulkan bahwa semakin lama operasi berlangsung kemungkinan terjadi infeksi luka operasi cukup besar. Dari hasil penelitian dapat dikatakan teknik aseptis sudah dilakukan Sefalosporin generasi 3 yang banyak dipakai adalah ceftriaxone dan terdapat hubungan dan pengaruh antara sifat operasi, jenis antibiotik, waktu pemberian dan lama operasi dengan infeksi luka operasi dengan P value < 0 05 Tabel 2. Kasus ILO Selama Periode Penelitian Jenis Operasi Sifat Operasi Jenis antibiotika Waktu pemberian antibiotika Lama operasi 1. < 1 jam tepat < 60 menit 2. 1 - 2 jam tidak tepat > 60 menit 3. > 2 jam Laparatomy & Hemicolectomy Dx kontaminasi Meropenem tidak tepat 3 Cranioplasty bersih Meropenem tidak tepat 3 Koreksi scoliosis bersih Meropenem tidak tepat 3 Appendictomy kontaminasi Ceftriaxone tidak tepat 2 Scoliosis, Stabilisasi Bone/Other Operations Bone bersih Meropenem tidak tepat 3 Orif fraktur tri melaeolar kanan bersih Ceftriaxone tidak tepat 2 Close reduction+ debridement+ internal fixasi kotor Meropenem tidak tepat 3 Tabel 2. REFERENSI Amba, S. 2007. Hubungan penggunaan antibiotika profilaksis dengan kejadian infeksi luka operasi di ruang bedah IRNA A RSCM Amba, S. 2007. Hubungan penggunaan antibiotika profilaksis dengan kejadian infeksi luka operasi di ruang bedah IRNA A RSCM Amba, S. 2007. Hubungan penggunaan antibiotika profilaksis dengan kejadian infeksi luka operasi di ruang bedah IRNA A RSCM 48 Pengaruh Antibiotik…..(Oktaviana Zunnita) tahun 2005. Tesis. Universitas Indonesia, Jakarta. Sign. 2008. Antibiotic Prophylaxis in Surgery: A national clinical guidelines. United Kingdom. Burke, A. & M.D. Cunha. 2009. Antibiotic Essentials. 8 th ed. Physicians Press. New Delhi- India. Sjamsuhidajat, R., W. Karnadihardja, T. Prasetyono & R. Reno. 2010. Buku Ajar Ilmu Bedah. Edisi 3. Penerbit EGC. Desyana L.S., A. Soemardi & M. Radji. 2008. Evaluasi penggunaan antibiotika profilaksis di ruang bedah Rumah Sakit Kanker Dharmais Jakarta dan hubungannya dengan kejadian infeksi daerah operasi. Indonesian J. Cancer. Welmahos, G.C., K.G. Toutouzas, G. Sarkisyan, L.S. Chan & A. Jindal. 2002. Severe trauma is not an excuse for prolonged antibiotic prophylaxis. Archieves of Surgery. 137(5): 537-41. WHO. 2001. Prevention of Hospital- Acquired Infection. A Practical guide second edition. Malta. Hal. 1-8 Gordon, S.M. 2006. Antibiotics prophylaxis against postoperative wound infection. Clev. Clin. J. Med. 73: 542-545. CLEV 49
https://openalex.org/W4283276606	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0267783&type=printable	English	null	Influence of product selection criteria on clothing purchase and post-purchase behaviours: A gender and generational comparison	PloS one	2,022	cc-by	14,990	PLOS ONE PLOS ONE RESEARCH ARTICLE Influence of product selection criteria on clothing purchase and post-purchase behaviours: A gender and generational comparison Gustavo Barrera-VerdugoID, Antonio Villarroel-Villarroel Faculty of Engineering and Business, Universidad de Las Ame´ricas, Providencia, Santiago, Chile Gustavo Barrera-VerdugoID, Antonio Villarroel-Villarroel Gustavo Barrera-VerdugoID, Antonio Villarroel-Villarroel Faculty of Engineering and Business, Universidad de Las Ame´ricas, Providencia, Santiago, Chile Faculty of Engineering and Business, Universidad de Las Ame´ricas, Providencia, Santiago, Chile gbarrera@udla.cl Abstract Purchasing and consumption behaviour is a factor with an important impact on sustainable development. In this regard, the clothing category plays a key role due to the high volume of products that are manufactured in countries with poor environmental and social conditions. While some research has investigated personal, social and cultural conditions that influence these behaviours, little is currently known about the influence of the attributes of sustainable clothing selection on the frequency of sustainable purchase and post-purchase actions in this category. This research seeks to evaluate this effect by comparing the results among genders and age/generation and measuring sustainable consumption using the Young Con- sumers’ Sustainable Consumption Behaviour method, which has two dimensions: purchase choices and sufficient and frugal consumption. Responses to online surveys of 240 univer- sity students in Chile are analysed using descriptive statistics, the Mann-Whitney-Wilcoxon test and ologit regressions. The findings show significant differences between the groups analysed with respect to the influence of attributes for sustainable clothing selection and highlight the negative effect of the search for quality in men and in older people. The evi- dence highlights the need to inform the population about the characteristics of sustainable clothing that positively affect purchase and post-purchase actions such as buying second- hand clothing, repairing, exchanging and donating clothing. This study also suggests that it is important to strengthen the relationship between sustainability and clothing quality among older generations and men. OPEN ACCESS Citation: Barrera-Verdugo G, Villarroel-Villarroel A (2022) Influence of product selection criteria on clothing purchase and post-purchase behaviours: A gender and generational comparison. PLoS ONE 17(6): e0267783. https://doi.org/10.1371/journal. pone.0267783 Editor: La´szlo´ Vasa, Szechenyi Istvan University: Szechenyi Istvan Egyetem, HUNGARY Szechenyi Istvan Egyetem, HUNGARY Received: January 13, 2022 Accepted: April 17, 2022 Published: June 22, 2022 Copyright: © 2022 Barrera-Verdugo, Villarroel- Villarroel. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data files are available from the Zenodo database (https:// zenodo.org/record/6401697#.YkWhUCgn5D8). 1. Introduction The concept of sustainable development was published in 1987 in the report "Our Common Future" [1] and is defined as a form of economic and social development that satisfies people’s present needs without harming the ability of future generations to satisfy their own needs. In turn, in line with sustainable development, the United Nations (n.d.) [2] states that sustainable production and consumption implies the efficient use of resources and energy, the Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 1 / 23 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours construction of environmentally friendly infrastructure and the creation of environmentally friendly jobs with fair pay and good working conditions. From the perspective of consump- tion, these approaches condition the preference, purchase, use and disposal of products in accordance with the care of natural resources, the use of efficient energy and adequate working conditions. Another concept that favours sustainable production and consumption is fair trade, defined by the Word Fair Trade Organisation [3] as follows: "A trading system based on dialogue, transparency and respect, which seeks greater equity in international trade with spe- cial attention to social and environmental criteria. It contributes to sustainable development by offering better trading conditions and securing the rights of disadvantaged producers and workers, especially in the South" (p.6). Along these lines, in 2015, the United Nations defined the 2030 Agenda for Sustainable Development, an initiative that incorporates seventeen goals that have been included in the report "The 2030 Agenda and the Sustainable Development Goals. An opportunity for Latin America and the Caribbean", published by CEPAL [4]. Goal twelve demands ensuring sustain- able consumption and production patterns. Sustainable consumption is defined as “the use of goods and services that respond to basic needs and bring a better quality of life, while minimis- ing the use of natural resources, toxic materials and emissions of waste and pollutants over the life cycle, so as not to jeopardize the needs of future generations” [5]. Due to the recognised importance of sustainable clothing consumption, a significant amount of research has attempted to understand the consumption habits of population groups in different countries in the last ten years [6, 7] and to recognise which factors are related to or influence sustainable clothing consumption behaviours [8, 9]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 1. Introduction In the measurement of sustain- able consumption behaviours, different scales have been developed to determine their magni- tude [10, 11]. Among these methodologies, the Young Consumers’ Sustainable Consumption Behaviour (YCSCB) scale [12] is a recent measurement tool that is focused on the clothing and food category, and is accepted and valued. YCSCB presents two measurement factors in the clothing category: the first is called sufficient and frugal consumption, which is related to exchanging and borrowing clothes, buying second-hand clothes and repairing clothes, and the second is called purchase choices, which are associated with the selection of products for envi- ronmental care, social responsibility or high quality. Thus, the two YCSCB dimensions cover sustainable consumption in a global way, focusing on different and complementary areas: purchase choices are related to sustainable attributes that are valued in the choice of clothing, while sufficient and frugal consumption evaluates sus- tainable behaviours integrated with self-production, purchase and post-purchase, such as the acquisition of secondhand clothing or self-production, repair, transformation, donation or exchange of clothing. It is also possible to argue that the purchase choices factor is linked to attribute-based product selection theory, which proposes that consumers use their preferences for products based on their attributes in terms of their functional and psychological benefits or risks (consequences) to achieve their underlying values [13]; in contrast, the sufficient and fru- gal consumption factor is linked to the measurement of observable behaviours, which are sub- sequent to preference and purchase intention, considering the response hierarchy models in the field of consumer behaviour as a basis that order consumption hierarchies into three stages: cognitive, affective and behavioural [14, 15]. In addition, Sufficient and frugal con- sumption incorporates the measurement of fashion clothing disposal behaviour, a type of post-purchase behaviour that is defined as the act of getting rid of something, in other words, the end of the clothing’s life with the current owner, encompassing whether the clothing is dis- carded as scrap or given for recycling or reuse [16]. As mentioned, these two YCSCB factors address different aspects, and in view of their dis- crepancies, the following questions arise: Does people’s evaluation of attributes for selecting 2 / 23 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours clothing included in purchase choices impact the post-purchase actions embedded in sufficient and frugal consumption? 1. Introduction Does the impact differ according to people’s gender and age group? These questions are important because to date, there is little information regarding the effect of sustainable attributes in clothing selection on the frequency of sustainable purchase and post-purchase actions among different segments of the population. Most of the research pub- lished to date that has attempted to recognise determinants of sustainable purchase and post- purchase behaviour has analysed personal demographic or psychological characteristics [17, 18], the influence of the social environment [19, 20] and the impact of cultural conditions [21, 22] on behaviours such as the purchase of second-hand clothing or post-purchase behaviours such as fashion clothing disposal behaviour. Consequently, a knowledge gap that implies a research problem is recognised. It is concern- ing the lack of understanding of which attributes of sustainable clothing influence the fre- quency of behaviours in the purchase and post-purchase stages that benefit environmental care and social wellbeing. Precisely, the objective of this research is to analyse the influence of the attributes for sustainable clothing selection incorporated in the purchase choices dimen- sion on sustainable purchase and post-purchase behaviours assessed in sufficient and frugal consumption, recognising differences among genders and age/generation. This research uses the YCSCB measurement method because it has been validated and incorporated in studies about sustainable consumption [12, 23, 24]. The YCSCB scale is similar to other metrics devel- oped to assess sustainable consumption in different segments of populations [8, 25]; conse- quently, we consider YCSCB to be a valid instrument to measure sustainable consumption of clothing in this research. Searching new evidence to answer these questions and achieve the objective of this research is important, as it would help understand which attributes of clothing selection are associated with subsequent behaviours that reduce the use of natural resources and the generation of pol- lutants from clothing waste. This information could be used by governmental and nongovern- mental organisations to promote the valuation of some attributes with a favourable impact on positive purchasing and post-purchase behaviours from a sustainability perspective, such as the purchase of second-hand clothing and the repair, exchange and donation of clothing. 1. Introduction Complementarily, knowing differences by gender and age enhances the usefulness of the find- ings due to published evidence that distinguishes sustainable consumption behaviours by gen- der and age generation [25, 26] and because segmenting the results by these demographic variables facilitates the development of targeted strategies in the marketing areas of companies. Organisations could capture the preference of demographic segments, by gender or age, com- bining the attributes valued by these groups for product selection with benefits or sales promo- tions that facilitate the purchase of second-hand clothing, repair, donation, exchange or reuse of garments. 2.1. Sustainable consumption Sustainable consumption is defined as the consumption of goods and the environmental costs and economic inequality associated with the modern consumerist lifestyle. In addressing envi- ronmental issues, policy-makers should not only focus their attention on the production side of industrial companies but also encourage responsible consumer consumption behaviour [27]. Similarly, Wang and Ward [28] stated that sustainable consumption consists of the con- sumption of goods and services that are produced using techniques and materials that cause minimal damage to the environment and that satisfy the basic needs of all human beings in a socially equitable manner. Complementarily, sustainable consumption can be defined as "the PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 3 / 23 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours use of goods and services that satisfy basic needs and provide a better quality of life by mini- mising the use of natural resources, toxic materials and waste and pollutants throughout their life cycle, so as not to threaten the needs of future generations" [5]. These definitions make it clear that sustainable consumption incorporates both the production conditions of products, including the natural and human resources used in their manufacture and sale, as well as pur- chase and post-purchase behaviours related to the acquisition of second-hand products, exchange, reuse, donation or transformation; these behaviours are carried out within the life cycle of products and contribute to a lower use of natural resources, toxic materials and waste by avoiding or postponing the purchase of new products. 2.2. Sustainable clothing attributes Attribute-based product selection theory proposes that consumers prefer products according to their attributes in terms of their functional and psychological benefits or risks (conse- quences) [13]. This theory also assumes that consumers’ decision-making is a form of problem solving (rather than cognitive rationalisation) in the sense that consumers will resolve their problems by taking various actions to maximise benefits and avoid negative outcomes [29]. The attributes that are considered in product selection are defined as properties or characteris- tics of a product that are intrinsic to it, or in other words, that are linked to it and are concrete, observable, objectively measurable and relevant for choosing between alternatives [30]. Keller [31] argued that product attributes refer to a descriptive characteristic of a brand and the com- ponents of the product that customers will look for. Complementarily, Keller [32] stated that direct product-related attributes are the materials necessary for the product to function, usu- ally linked to its physical composition, and that nonproduct-related attributes are external aspects of a product, such as information about the product’s price, packaging and design, including people, groups or celebrities who use the product. In relation to attributes relevant to the selection of sustainable apparel, Patnaik et al. [33] posited that sustainable fashion products are manufactured organically and produce less wastewater and harmful chemicals; in this regard, an increasing number of apparel companies are creating quality garments and building successful brands while developing sustainable practices, such as the use of recycled and organic materials. Similarly, Rausch et al. [34] pointed out that the most important attributes of sustainable clothing are the durability of the garment, fair wages and working conditions, as well as an environmentally friendly production process. In YCSCB, Fischer et al. [12] proposed that the attributes for the selection of sustain- able apparel are: production in countries with good working conditions, compliance with fair trade in the sale of clothing, the incorporation of organic production, the absence of pollutants in the clothing, and the high quality and durability of the clothing. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 2.3. Clothing purchasing and post-purchase behaviours Recycled clothing can also be converted into new products [41]. A concept related to post-purchase behaviours is the term "disposal of unused product". In the case of clothing, Chun [42] defined this behaviour as “discontinued wear and disposal of a clothing item by giving it to others, throwing it away, using it for another purpose than wear- ing apparel, or selling it at used clothing stores or garage sale” (p. 7). More recently, Laitala [16] defined this disposal as "the act of getting rid of something, i.e., the end-of-life stage of the clothing with the present owner, regardless of whether the clothing is discarded of as waste or delivered to recycling or reuse" (p. 444). In this regard, there are several ways in which con- sumers can dispose of clothing: donating it to charity, giving it to family or friends, reselling it, or throwing it away. Donating it to charity and giving it to family or friends are common methods of reusing clothing [43, 44]. The observable post-purchase behaviour of clothing is highly relevant from the perspective of environmental and social sustainability, as it affects the volume of new clothing that is man- ufactured and distributed using natural and human resources. The clothing sector is character- ised by the generation of a high volume of waste that can be minimised with actions such as the reuse of garments, donation, exchange or recycling. Clothing production has increased sig- nificantly in the last decade, and the number of times consumers wear clothes has decreased by one-third since the beginning of the century. Complementarily, clothing sales will increase to 160 million tonnes by 2050 [45], and global consumption of clothing will reach approxi- mately 102 million tonnes by 2030 [46]. Furthermore, many of the companies in the textile and clothing industry implement production and sales strategies with a focus on massification and low prices; these strategies increasingly encourage consumers to buy, which has a negative impact on the environment and society [47]. To date, some of the research has focused on studying the habits and trends in population groups in different countries regarding secondhand clothing purchases [48, 49] and fashion clothing disposal behaviour [50, 51]. Other research has searched for factors that affect these behaviours. Gopalakrishnan and Matthews [52] argued that cheaper prices, the excitement of finding great deals, brand value and variety are the main reasons for shopping at second-hand shops. 2.3. Clothing purchasing and post-purchase behaviours Response hierarchy models published in the 20th century [35, 36], which describe the stages consumers go through in the buying process, propose three general stages. The first stage is called "cognitive" and is related to customers’ awareness and knowledge of a product and brand. The second stage is called "affective" and is associated with the emotions that products or brands generate in customers. The third stage is called "behavioural" and is related to behav- iours that can be observed and recorded, such as buying, consuming and discarding products. Examples of the use of these three stages are the AIDA model [37], which determines the atten- tion stage in the cognitive phase, interest and desire in the affective phase and action in the behavioural phase; the Hierarchy of Effects model [38], which recognises awareness and knowledge of the product in the cognitive phase, taste and preference for the product in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 4 / 23 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours affective phase, and the purchase of the product in the behavioural stage. In the last phase, which is the behavioural stage, it is possible to recognise various behavioural indicators, such as the brand purchased, the sales channel used in the purchase, the method of payment used in the purchase, and the frequency and volume of purchase. Some models also include post-pur- chase as a later stage. In this respect, Kotler and Turner [39] explain that customer satisfaction is the consequence of the customer’s experience during the distinct phases of the purchase pro- cess, which includes the stages of awakening of the need, information search, evaluation of alternatives, purchase decision and post-purchase behaviour. In the area of sustainable clothing purchasing behaviour, one widely studied conduct is the purchase of second-hand clothing, since the repurchase and reuse of clothing reduces the use of natural and human resources for the manufacture and distribution of new clothing. Regard- ing post-purchase behaviours, Bianchi and Birtwistle and Laitala [6, 16] stated that in the field of clothing, post-purchase includes actions such as the use, care, reuse, destruction or disposal of garments. Reuse of clothing may involve giving or transferring garments to friends or fam- ily, while donated clothing may be exported and sold as second-hand clothing to consumers, especially in third world countries [40]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 2.4. Clothing consumption in Chile Chile is one of the countries in Latin America with the highest per capita clothing consumption. In the last 5 years, people in Chile have increased their clothing consumption by 80%, going from 13 to 50 new garments per year on average [57]. The high access to credit in the population [58] and the fast fashion marketing strategies implemented by mass retail that are based on discounts and the distribution of credit cards for payment in instalments [59, 60] allow the middle socioeco- nomic groups to purchase clothing from fast fashion brands such as Zara or H & M. The arrival of used clothing from the United States to Chile began in 1975 under sanitary and customs regulations [61]. Thus, the purchase of secondhand clothing is accessible because in sev- eral cities, there are stores that sell used clothes. “American clothing" or "European clothing" stores that sell used clothing have been available in the country for decades [62]. The consumption of second-hand clothing is transversal among Chile’s social classes, it takes place in street sells with low prices and in stores that offer exclusivity, clothes for important occasions, costumes and very large or small special sizes. Besides, the brands positioned in the local second-hand offer: online sales channels for wholesalers, garment exchange guarantees, and application of the "recy- cled clothing" concept linked to environmental care and social responsibility [61]. In addition, some large department stores are now accepting used clothing as part payment for new clothing, this commercial strategy is mainly focused on the younger segments of the market. Moreover, Chile is a country with the highest internet penetration, with more than 14 mil- lion people connected, representing 76% of the population [63], and more than 16 million users of social media [64]. This broad digital coverage facilitates the population’s access to online shopping for clothing, both new and second-hand, and facilitates the population’s access to clothing exchange; in this sense, online platforms for buying and exchanging used clothing are currently prospering in the country [65]. With respect to the information provided to consumers, the garments include labels that identify the country of manufacture of the garment and its materials, this information have been legally defined in Chile [66]. 2.3. Clothing purchasing and post-purchase behaviours Arangdad et al. [17] stated that the physical condition of clothing and lack of awareness are the most prominent reasons preventing consumers from recycling more textile products. Pingki and Kuntala [53] recognised that creating more infrastructural facilities for recycling clothing, expanding environmental and social awareness, and implementing corporate social responsibility (CSR) activities should positively affect sustainable clothing disposal. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 5 / 23 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours Regarding clothing attributes that affect purchase and post-purchase behaviour, Pac¸o et al. [26] have argued that the main barriers to buying secondhand clothing are the perceived lower cleanliness of the garment and the fact that these clothes have already been previously used by other people. McQueen et al. [54] stated that people are less likely to donate, give away or sell malodorous clothing and are more likely to dispose of malodorous garments directly in the rubbish. Degenstein et al. [55] indicated that the severity of damage to clothing caused by its use plays a key role in how people decide to dispose of their unwanted garments and that the quality and type of garment predict the disposal method and end-of-life extension strategies. Similarly, Laitala and Klepp [56] argued that uniqueness and style are more important for those who buy second-hand clothes and that barriers to buying used clothes are lack of hygiene, health and intimacy. 2.4. Clothing consumption in Chile Consequently, it is easy for a person to recognise whether the garment is manufactured in an Asian country and whether it is made of cotton or a syn- thetic material such as polyester. In addition, due to the high internet and social media cover- age, the population has access to multiple online sources of information that facilitate the knowledge of the negative environmental and social conditions of the countries that contrib- ute to fast fashion. 3. Hypothesis Regarding of the influence of valuing sustainable attributes in clothing on sustainable purchase and post-purchase behaviours, no recent evidence has been recognised associating these 6 / 23 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours selection attributes with the frequency of these post-purchase behaviours. However, because the influence of other types of non-sustainability-related attributes on second-hand purchasing or clothing disposal behaviour has been supported, such as the smell and cleanliness of cloth- ing [56, 54], the perception that clothing is out of fashion [41] and high brand recognition of the product due to its authenticity and quality [67], this research suggests that valuing condi- tions such as organic production, fair-trade manufacturing, absence of polluting materials and quality should affect the frequency of clothing purchase and post-purchase actions that are important for social and environmental sustainability, such as exchanging, reusing, repairing, donating, disposing of clothes and buying second-hand clothes. This approach is also justified by the phases exposed in the response hierarchy models [38, 36] which support that the prefer- ence or desire for a product precedes subsequent behaviors such as its purchase and consump- tion. Under this theoretical framework, the valuation of attributes for clothing preference or selection should impact subsequent purchase and post-purchase behaviours. Consequently, the following research hypothesis is proposed: • Hypothesis 1: The valuation of sustainable attributes of clothing affects the frequency of sus- tainable purchase and post-purchase consumption behaviours. • Hypothesis 1: The valuation of sustainable attributes of clothing affects the frequency of sus- tainable purchase and post-purchase consumption behaviours. Complementarily, previous research has recognised discrepancies in sustainable consump- tion by gender. Isenhour and Ardenfors [68] concluded that women are more likely than men to consume sustainably, considering activities such as buying organic and fair-trade products, reducing travel, consuming organic food and recycling; according to their research, women express more interest in sustainable living and spend more time seeking information and alter- natives on sustainable consumption. Bulut et al. [25] noted that women show a higher level of sustainable consumption behaviour both in general behaviour and in the tendency to reuse products. More recently, Nenckova´ et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 4.1. Measurement Online surveys were distributed through the Survey Monkey platform. Respondents answered the online survey independently without the presence of an evaluator. The measurement in the survey is based on the Young Consumers’ Sustainable Consumption Behaviour (YCSCB) scale published by Fischer et al. [12]. They argued that YCSCB is based on the Sustainable Consumption Behaviour Cube (SCB-Cube) proposed by Geiger et al. [10]. The SCB-Cube pro- vides an integrative conceptual framework that includes three domains of sustainable con- sumption; the first is related to the different areas of consumption (food, housing, mobility, clothing, etc.), the second is associated with the phases of consumption (acquisition, use and disposal of consumer goods), and the third domain involves the selection of consumption impacts in two dimensions of sustainability (ecological and socioeconomic). y g Fischer et al. [12] applied this framework to develop the YCSCB in the clothing categories, addressing the stages acquisition, usage, and disposal of consumer goods. They recognised, after conducting a qualitative and quantitative study, that the sentences “I avoid buying cloth- ing items that originate in countries with poor working conditions” and “I choose clothing items from fair trade production” are related to the socioeconomic dimension in the acquisi- tion stage, and the other phrases, including "I choose high-quality and long-lasting clothing items", are linked to the ecological dimension in the acquisition stage. The choice of high-quality and long-lasting apparel can be associated with the concept of slow fashion. This is a socially conscious movement that shifts the mindset of consumers from quantity to quality, encouraging people to purchase high-quality items less frequently [78]. Hall [79] argued that slow fashion involves longer production times, the use of local materials, and a focus on quality and sustainability. In other words, slow fashion implies a longer useful life of the garment from manufacture to disposal, thus reducing the amount of clothing that needs to be manufactured and distributed and reducing the resources used (materials, trans- portation, infrastructure, etc.), affecting sustainability from an ecological perspective. The YCSCB scale is similar to other metrics developed to assess sustainable consumption. Razzaq et al. 3. Hypothesis [69] supported that men are significantly more likely to throw away their clothes if they do not use them, and Kopplin and Ro¨sch [70] have argued that women are willing to buy sustainable clothes only due to their concern for the environ- ment. Previous research has argued a higher tendency towards sustainable consumption in women with few exceptions; for example, Lang et al. [71] stated that sensitivity to fashion trends, higher income and female gender are positively correlated with frequent clothing dis- posal. Supported by the gender differences evidenced in previous publications, this research proposes that the influence of attributes for sustainable product selection on the frequency of purchase and post-purchase behaviour should be different between men and women, and con- sequently, the following research hypothesis is proposed. • Hypothesis 2: The valuation of sustainable attributes of clothing differentially affects the fre- quency of sustainable purchase and post-purchase consumption behaviours between men and women. • Hypothesis 2: The valuation of sustainable attributes of clothing differentially affects the fre- quency of sustainable purchase and post-purchase consumption behaviours between men and women. Additionally, generational differences in sustainable consumption have been argued. Bulut et al. [25] noted that older consumers of the Baby Boom generation have the highest level of unnecessary consumption of products, while younger consumers of generation Z have the lowest level of unnecessary consumption. Similarly, several studies published in the last 5 years have recognised an important trend of the centennial or "Z" generation towards environmental and social concern [72–74], which is also expressed in an important propensity towards sus- tainable clothing consumption behaviours [75, 76]. Supporting generational differences in sus- tainable consumption, Pac¸o et al. [26] stated that young consumers are more likely to buy textile products in second-hand markets due to environmental reasons, lack of money or the search for vintage fashion. Additionally, Liang and Xu [77] analysed four generational cohorts in China, differentiating between people born in the 1960s, 1970s, 1980s and 1990s, observing 7 / 23 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours that post-70s consumers avoided buying secondhand clothing, while younger generations per- ceived higher values and had higher purchase intentions for secondhand clothing than their older counterparts. 3. Hypothesis On the basis of such generational differences, this research hypothesises that the influence of sustainable clothing selection attributes on these subsequent behaviours should also be different among the centennial generation: • Hypothesis 3: The valuation of sustainable attributes of clothing differentially affects the fre- quency of sustainable purchase and post-purchase behaviours between the centennial gener- ation and older people. • Hypothesis 3: The valuation of sustainable attributes of clothing differentially affects the fre- quency of sustainable purchase and post-purchase behaviours between the centennial gener- ation and older people. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 4.1. Measurement [8] used the statements: “I will buy clothing that is made with recycled content”, “I will buy clothing that can be disposed of in an environmentally friendly manner”, “I will buy clothing that is safe for the environment”, “I will limit my use of that clothing that is made of or uses scarce resources”, “I will not buy new clothing items if I already have previous dresses in a usable state”, and “I will buy clothing that is produced in an environmentally friendly manner”. Complementarily, Bulut et al. [25] evaluated sustainable consumption behaviours among genders and generations using a four-dimensional sustainable consump- tion behaviour scale that includes coherent and similar statements to YCSCB. Consequently, we consider YCSCB to be a valid instrument to measure sustainable consumption of clothing in this research, such that it has incorporated sets of validated statements for measuring sus- tainable consumption that have also been used in previous studies related to this issue [23, 24]. 8 / 23 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours As noted previously, YCSCB incorporates two factors: purchase choices, which are associ- ated with the importance of sustainable attributes in product selection, and sufficient and fru- gal consumption, which measures purchase and post-purchase behaviours that affect environmental and social sustainability. The YCSCB items were adapted to the Chilean con- text, translated from English into Spanish, and then translated back into English for presenta- tion in this research. Before final distribution, the survey was answered by a small sample of twenty students to check the understanding of the statements and make corrections. A fre- quency scale was used in the measurement with the following levels associated with numbers: never (1), rarely (2), occasionally (3), sometimes (4), frequently (5), very frequently (6), and always (7). Seven levels were selected in this scale instead of five to allow the selection of a larger number of frequency options for the respondents. The 7-level frequency scale has been used for decades to assess the frequency of observable behaviours or the frequency of prefer- ences and intentions in research [80–82]. 4.2. Sample A total of 240 students from the Faculty of Engineering and Business at Universidad de Las Ame´ricas in Chile completed the online survey in full, 105 men, 133 women and two persons who did not respond or indicated another gender. The average age of the students was 29.41 years, and the standard deviation of their age was 9.45 years. The classification of the centen- nial generation considers the assessed persons who were born since 1996, according to the classification defined by Lanier [83]. The students surveyed are from the lower-middle, middle and upper-middle socioeco- nomic classes of Chile, which represent approximately 60% of the country’s population [84]. Students are enrolled in a private university; in addition, they pay a college fee and have access to the internet because they had to attend online classes since the beginning of the COVID-19 pandemic. Consequently, students have access to buy clothes in mass retail, in physical stores or through e-commerce, as well as to buy new and used clothes on social media. Most of the students who responded to the survey were enrolled in Commercial Engineer- ing and Business Administration Engineering. The survey was sent to approximately 1,000 stu- dents from March 2020 to October 2021, representing a response rate of nearly 24%. The 240 surveys selected for analysis in this research are those that were fully responded. All surveys included a request for informed consent that was defined by the Ethics Committee of Univer- sidad de Las Ame´ricas (ethic committee code CEC_FP_2019021). Only responses of students who accepted informed consent were considered. Table 1 details the characteristics of the stu- dent sample. 4.3. Analysis Statistical analysis of the data was performed with the statistical package STATA version 16. First, a comparative analysis of the observed variables by gender and age generation was con- ducted using the arithmetic mean and an evaluation of differences between response distribu- tions through the Mann-Whitney-Wilcoxon test. This test is a nonparametric evaluation whose null hypothesis is based on equal distributions of both populations [85] and that involves the comparison of the mode representing the 50% percentile of the data distribution. Obtaining a P value < 0.05 supports statistically significant differences. The Mann–Whitney test is used for comparison between two groups having quantitative variables that are not nor- mally distributed [86]; likewise, it is a robust competitor of the t-test in the univariate setting [87]. 9 / 23 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours PLOS ONE Table 1. Description of the sample. Average age Standard deviation of age Commercial Engineering Business Administration Engineering Industrial Engineering Other Careers Total Total centennials 21.24 1.88 68 19 8 20 115 Centennials men 21.32 1.83 32 6 5 3 46 Centennials women 21.19 1.93 36 12 3 17 68 Centennials other- gender 19.00 0.00 0 1 0 0 1 Total Older 36.94 7.05 52 42 23 8 125 Older men 37.17 7.60 22 16 19 2 59 Older women 36.89 6.49 29 26 4 6 65 Older other-gender 26.00 0.00 1 0 0 0 1 Total 29.41 9.45 120 61 31 28 240 https://doi.org/10.1371/journal.pone.0267783.t001 Table 1. Description of the sample. This research seeks to evaluate the criteria influence for selecting sustainable clothing on post-purchase actions. Therefore, a logistic regression analysis was performed for ordinal vari- ables, considering the product selection attributes incorporated in the purchase choices factor as independent variables and the purchase and post-purchase actions of clothing, associated with the sufficient and frugal consumption factor, as dependent variables. Logistic regression is typically used to evaluate the influence of a set of variables on the selection probability of a certain alternative, especially for factors that affect a given decision but cannot be directly observed [88]. Ordinal logistic regression is a regression analysis used to find and describe the relationship between ordinal scale response variables with more than two categories and a set of continuous or categorical predictor variables [89]. 4.3. Analysis It is a technical statistical technique capa- ble of processing data on an ordinal scale in situations involving several factors that can influ- ence the outcome [90]. The overall fit of the regressions is assessed by the Chi2 test, and the significance of the regression coefficients is assessed by hypothesis testing based on the param- eter P>\|z\|. Moreover, the Mann-Whitney-Wilcoxon test and logistic regressions were selected because the responses obtained for the observed variables did not show a normal distribution, consid- ering a confidence level of 99% or 95%, except for the responses obtained for the statement "I choose high quality and long-lasting clothing items". The normal distribution of the variables was evaluated with the Shapiro-Wilks test, and the results showed p-values of less than 0.05 or 0.01, which represents the absence of a normal distribution; only the measurement of the qual- ity attribute showed a higher p-value (0.05). PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE Table 2. Comparison of central tendency by group. Sufficient and frugal consumption Mean Women Mean Men Prob > \|z\| Mann-U Gender Mean Centennials Mean Olders Prob > \|z\| Mann-U Generation I buy second-hand clothing. 3.23 2.26 0.00 3.32 2.34 0.00 I wear patched and mended clothing. 3.53 2.80 0.00 3.49 2.96 0.01 I give away or swap unwanted clothing items that I no longer wear. 4.57 4.33 0.28 4.25 4.64 0.12 Instead of buying a new piece of clothing for a special occasion, I borrow something. 2.53 1.87 0.00 2.40 2.09 0.05 I look for other possible uses of unwanted clothing items. 3.84 3.42 0.11 3.96 3.35 0.02 I air my clothing items properly before deciding whether they need washing. 3.66 3.58 0.83 3.69 3.54 0.49 I throw away clothing items that I no longer wear. 3.38 3.44 0.92 3.22 3.61 0.29 Purchase choices Mean Women Mean Men Prob > \|z\| Mann-U Gender Mean Centennials Mean Olders Prob > \|z\| Mann-U Generation I avoid buying clothing items that originate in countries with poor working conditions. 2.68 2.66 0.86 2.79 2.54 0.09 I choose clothing items from fair-trade production. 3.54 3.38 0.42 3.51 3.46 0.41 I choose clothing items from organic production (e.g., made from organic cotton). 3.38 3.23 0.47 3.43 3.18 0.14 I select clothing that includes labels guaranteeing the absence of chemical pollutants. 2.97 3.22 0.32 3.20 2.95 0.10 I choose high quality and long-lasting clothing items. 4.36 4.86 0.02 4.28 4.85 0.01 Note: A p value <0.01 represents a significant difference with 99% confidence, a p value <0.05 represents a significant difference with 95% confidence, and a p value <0.10 represents a significant difference with 90% confidence. Note: A p value <0.01 represents a significant difference with 99% confidence, a p value <0.05 represents a significant difference with 95% confidence, and a p value <0.10 represents a significant difference with 90% confidence. of buying a new piece of clothing for a special occasion, I borrow something" (p<0.10), and "I look for other possible uses of unwanted clothing items" (p<0.05). In the case of the purchase choices factor, significant differences with 95% (p<0.05) confi- dence were only recognised in the statement "I choose high quality and long-lasting clothing items". PLOS ONE The arithmetic mean of the importance of this selection criterion is higher for men and older people; therefore, these results suggest that in these groups, quality is a more relevant attribute. Additionally, the centennial generation shows a higher concern than older people in avoiding clothes items that originate in countries with poor working conditions, and the dif- ference in distribution in this statement is significant only at 90% confidence (p<0.10) accord- ing to the Man-U Wilcox test. 5.1. Comparison of arithmetical means by groups Table 2 shows the arithmetic means obtained for each group evaluated. These arithmetic means suggest that the female group and centennials express a higher frequency of behaviours associated with the sufficient and frugal consumption factor. The Man-U Wilcox test also sup- ports these differences in the distribution of response frequencies between men and women and between centennials and older people. Although the arithmetic means obtained show a low frequency of behaviours in general terms, women show higher magnitudes in the state- ments "I buy second-hand clothing" (p<0.01), "I wear patched and mended clothing" (p<0.01) and "Instead of buying a new piece of clothing for a special occasion, I borrow some- thing" (p<0.01). For the centennial generation, the magnitudes are higher for the statements "I buy second-hand clothing" (p<0.01), "I wear patched and mended clothing" (p<0.05), "instead PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 10 / 23 Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE PLOS ONE Table 3. Logistic regression of women. I buy second- hand clothing I wear patched and mended clothing I give away or swap unwanted clothing items that I no longer wear Instead of buying a new piece of clothing for a special occasion, I borrow something I look for other possible uses of unwanted clothing items I air my clothing items before deciding whether they need washing I throw away clothing items that I no longer wear β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| I avoid buying clothing items that originate in countries with poor working conditions 0.43 0.00 0.27 0.02 -0.10 0.36 0.31 0.01 0.23 0.04 0.16 0.09 0.15 0.21 I choose clothing items from fair-trade production -0.03 0.82 0.00 0.99 0.46 0.00 -0.08 0.52 0.01 0.92 0.19 0.10 -0.13 0.24 I choose clothing items from organic production (e.g., made from organic cotton) 0.11 0.42 0.07 0.59 -0.02 0.85 0.17 0.19 0.12 0.35 -0.08 0.50 0.05 0.67 I select clothing that includes labels guaranteeing the absence of chemical pollutants -0.20 0.12 -0.05 0.70 -0.06 0.61 -0.10 0.44 0.10 0.43 0.19 0.13 -0.16 0.17 I choose high quality and long-lasting clothing items -0.17 0.08 0.02 0.84 0.18 0.07 -0.06 0.55 -0.07 0.45 0.03 0.76 0.13 0.17 Observations 133 133 133 133 133 133 133 Prob > chi2 0.00 0.10 0.00 0.07 0.02 0.00 0.37 Pseudo R2 0.04 0.02 0.05 0.02 0.03 0.04 0.01 https://doi.org/10.1371/journal.pone.0267783.t003 Table 3. Logistic regression of women. confidence. In particular, the statement "I avoid buying clothing items that originate in coun- tries with poor working conditions" shows a high positive regression coefficient (β = 0.43, P>\| z\| = 0.00) in the regression with the dependent variable "I buy secondhand clothing"; the state- ment "I choose clothing items from fair trade production". (β = 0.46, P>\|z\| = 0.00) in the regression with the dependent variable "I give away or swap unwanted clothing items that I no longer wear" also stands out for its high positive and significant coefficient. Mainly, it is recog- nised that among women, the concern to avoid garments that originate in countries with poor working conditions has a positive effect on the behaviour of buying second-hand clothes, bor- rowing clothes, finding new uses for clothing, and airing clothes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 5.2. Ologit regressions by gender Table 3 presents the ologit regressions in the women’s group, including the statements of suffi- cient and frugal consumption as dependent variables. The Chi2 test (prob > chi2) shows that the regressions with the dependent variables "I buy secondhand clothing", "I give away or swap unwanted clothing items that I no longer wear", "instead of buying a new piece of clothing for a special occasion, I borrow something", "I look for other possible uses of unwanted clothing items", and "I air my clothing items properly before deciding whether they need washing" have a valid goodness of fit considering a 99% (p <0.01), 95% (p <0.05) or 90% (p <0.10) confi- dence level. In contrast, the regressions associated with the action "I wear patched and mended clothing" and "I throw away clothing items that I no longer wear" as the dependent variable did not obtain good goodness of fit. In the regressions with adequate goodness-of-fit, signifi- cant regression coefficients were found at 99% (p<0.01), 95% (p<0.05) or 90% (p<0.10) PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 11 / 23 Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours PLOS ONE PLOS ONE Table 4. Logistic regression of men. I buy second- hand clothing I wear patched and mended clothing I give away or swap unwanted clothing items that I no longer wear Instead of buying a new piece of clothing for a special occasion, I borrow something I look for other possible uses of unwanted clothing items I air my clothing items before deciding whether they need washing I throw away clothing items that I no longer wear β P>\|z\| Β P>\|z\| β β P>\|z\| β P>\|z\| P>\|z\| β P>\|z\| β P>\|z\| I avoid buying clothing items that originate in countries with poor working conditions 0.28 0.08 0.06 0.65 0.22 0.13 0.26 0.10 0.16 0.27 0.00 0.99 0.29 0.05 I choose clothing items from fair-trade production 0.16 0.30 0.13 0.37 0.06 0.66 0.23 0.13 0.11 0.45 0.28 0.03 -0.37 0.01 I choose clothing items from organic production (e.g., made from organic cotton) 0.37 0.05 0.47 0.00 0.03 0.84 -0.08 0.67 0.25 0.13 0.51 0.00 0.27 0.12 I select clothing that includes labels guaranteeing the absence of chemical pollutants -0.11 0.53 -0.00 0.99 0.16 0.30 0.35 0.04 0.13 0.41 -0.20 0.17 -0.16 0.32 I choose high quality and long-lasting clothing items -0.35 0.00 -0.33 0.00 0.11 0.29 -0.28 0.03 -0.41 0.00 0.14 0.17 0.01 0.91 Observations 105 105 105 105 105 105 105 Prob > chi2 0.00 0.00 0.00 0.00 0.00 0.00 0.13 Pseudo R2 0.07 0.08 0.05 0.09 0.06 0.08 0.02 https://doi.org/10.1371/journal.pone.0267783.t004 Table 4. Logistic regression of men. items that originate in countries with poor working conditions has a positive effect on the actions "I buy second-hand clothing" (β = 0.28, P>\|z\| = 0.08) and “I throw away clothing items that I no longer wear” (β = 0.29, P>\|z\| = 0.05), and that selecting clothes that are produced under fair trade has a positive effect on post-purchase action "I air my clothing items properly before deciding whether they need washing" (β = 0.28, P>\|z\| = 0.03) and a negative effect on post-purchase action “I throw away clothing items that I no longer wear” (β = -0.37, P>\|z\| = 0.01). PLOS ONE In a complementary way, it can be seen that only quality has a negative effect on the purchase of second-hand clothes (β = -0.17, P>\|z\| = 0.08) and has a positive effect on the donation or exchange of clothes (β = 0.18, P>\|z\| = 0.07). Table 4 presents the ologit regressions in the men’s group, also incorporating the sufficient and frugal consumption statements as dependent variables. In this case, the results show that all the ologit regressions, except the regression with the dependent variable "I throw away clothing items that I no longer wear", obtained a good goodness of fit (Chi2 test with p<0.01). The main difference with respect to women is that significant negative regression coefficients were obtained, at 99% or 95% confidence, particularly these associated with the statement "I choose high quality and long-lasting clothing items". This result shows that, in the case of men, the search for quality negatively affects purchase and post-purchase behaviours that benefit environmental care and social well-being, particularly the actions "I buy second-hand clothes" (β = -0.35, P>\|z\| = 0.00), "I wear patched and mended clothing" (β = -0.33, P>\|z\| = 0.00), "instead of buying a new piece of clothing for a special occasion, I borrow something" (β = -0.28, P>\|z\| = 0.03) and "I look for other possible uses of unwanted clothing items" (β = - 0.41, P>\|z\| = 0.00). In contrast, the results show that the preference for organic production posi- tively affects the purchase of secondhand clothes (β = 0.37, P>\|z\| = 0.05), patching or mending clothes to continue using them (β = 0.47, P>\|z\| = 0.00), and airing clothes before deciding to wash them (β = 0.51, P>\|z\| = 0.00). Complementarily, it is recognised that avoiding clothing 12 / 23 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours PLOS ONE Additionally, as in the case of men, negative and significant regression coeffi- cients at 99% (P<0.01), 95% (P<0.05) or 90% (P<0.10) confidence stand out in the sentence "I select garments of high quality in their materials and elaboration, and with long duration", specifically, in the regressions with dependent variables "I buy second-hand clothing" (β = regression coefficients on regressions with dependent variables "I buy second-hand clothing" (β = 0.38, P>\|z\| = 0.01), "I wear patched and mended clothing" (β = 0.21, P>\|z\| = 0.09), "instead of buying a new piece of clothing for a special occasion, I borrow something" (β = 0.48, P>\|z\| = 0.00) and "I look for other possible uses of unwanted clothing items" (β = 0.36, P>\|z\| = 0.01). Additionally, as in the case of men, negative and significant regression coeffi- cients at 99% (P<0.01), 95% (P<0.05) or 90% (P<0.10) confidence stand out in the sentence "I select garments of high quality in their materials and elaboration, and with long duration", specifically, in the regressions with dependent variables "I buy second-hand clothing" (β = regression coefficients on regressions with dependent variables "I buy second-hand clothing" (β = 0.38, P>\|z\| = 0.01), "I wear patched and mended clothing" (β = 0.21, P>\|z\| = 0.09), Table 6. Logistic regression of older adults. PLOS ONE I buy second- hand clothing I wear patched and mended clothing I give away or swap unwanted clothing items that I no longer wear Instead of buying a new piece of clothing for a special occasion, I borrow something I look for other possible uses of unwanted clothing items I air my clothing items before deciding whether they need washing I throw away clothing items that I no longer wear β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| I avoid buying clothing items that originate in countries with poor working conditions 0.38 0.01 0.21 0.09 0.17 0.17 0.48 0.00 0.36 0.01 0.14 0.27 0.20 0.13 I choose clothing items from fair-trade production -0.04 0.75 -0.02 0.87 0.21 0.05 -0.10 0.45 -0.03 0.82 0.20 0.08 -0.20 0.08 I choose clothing items from organic production (e.g., made from organic cotton) 0.12 0.43 0.25 0.07 0.05 0.70 0.30 0.06 0.33 0.02 0.04 0.80 0.21 0.13 I select clothing that includes labels guaranteeing the absence of chemical pollutants -0.01 0.92 0.04 0.77 0.13 0.32 -0.11 0.48 0.13 0.33 0.16 0.22 -0.25 0.06 I choose high quality and long-lasting clothing items -0.29 0.01 -0.17 0.09 0.03 0.76 -0.21 0.06 -0.41 0.00 0.03 0.76 0.04 0.69 Observations 125 125 125 125 125 125 125 Prob > chi2 0.00 0.01 0.00 0.00 0.00 0.00 0.23 Pseudo R2 0.05 0.04 0.06 0.06 0.09 0.05 0.02 https://doi.org/10.1371/journal.pone.0267783.t006 Table 5. Logistic regression of the centennial generation. PLOS ONE I buy second- hand clothing I wear patched and mended clothing I give away or swap unwanted clothing items that I no longer wear Instead of buying a new piece of clothing for a special occasion, I borrow something I look for other possible uses of unwanted clothing items I air my clothing items before deciding whether they need washing I throw away clothing items that I no longer wear β P>\|z\| β P>\|z\| β P>\|z\| Β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| I avoid buying clothing items that originate in countries with poor working conditions 0.39 0.01 0.15 0.23 -0.09 0.46 0.18 0.16 0.09 0.44 0.12 0.32 0.17 0.20 I choose clothing items from fair-trade production 0.15 0.30 0.10 0.49 0.35 0.02 0.11 0.41 0.05 0.70 0.22 0.10 -0.14 0.29 I choose clothing items from organic production (e.g., made from organic cotton) 0.39 0.01 0.31 0.03 0.09 0.54 0.08 0.61 0.08 0.59 0.22 0.12 0.03 0.86 I select clothing that includes labels guaranteeing the absence of chemical pollutants -0.47 0.00 -0.17 0.21 -0.11 0.46 0.05 0.72 -0.00 0.98 -0.05 0.73 -0.01 0.92 I choose high quality and long-lasting clothing items -0.13 0.22 -0.15 0.17 0.16 0.15 -0.09 0.39 0.02 0.86 0.07 0.51 0.10 0.37 Observations 115 115 115 115 115 115 115 Prob > chi2 0.00 0.05 0.05 0.15 0.68 0.01 0.62 Pseudo R2 0.05 0.03 0.03 0.02 0.01 0.03 0.01 https://doi.org/10.1371/journal.pone.0267783.t005 Table 5. Logistic regression of the centennial generation. regression coefficients on regressions with dependent variables "I buy second-hand clothing" (β = 0.38, P>\|z\| = 0.01), "I wear patched and mended clothing" (β = 0.21, P>\|z\| = 0.09), "instead of buying a new piece of clothing for a special occasion, I borrow something" (β = 0.48, P>\|z\| = 0.00) and "I look for other possible uses of unwanted clothing items" (β = 0.36, P>\|z\| = 0.01). PLOS ONE PLOS ONE regression coefficients on regressions with dependent variables "I buy second-hand clothing" (β = 0.38, P>\|z\| = 0.01), "I wear patched and mended clothing" (β = 0.21, P>\|z\| = 0.09), "instead of buying a new piece of clothing for a special occasion, I borrow something" (β = 0.48, P>\|z\| = 0.00) and "I look for other possible uses of unwanted clothing items" (β = 0.36, P>\|z\| = 0.01). Additionally, as in the case of men, negative and significant regression coeffi- cients at 99% (P<0.01), 95% (P<0.05) or 90% (P<0.10) confidence stand out in the sentence "I select garments of high quality in their materials and elaboration, and with long duration", specifically, in the regressions with dependent variables "I buy second-hand clothing" (β = Table 5. Logistic regression of the centennial generation. I buy second- hand clothing I wear patched and mended clothing I give away or swap unwanted clothing items that I no longer wear Instead of buying a new piece of clothing for a special occasion, I borrow something I look for other possible uses of unwanted clothing items I air my clothing items before deciding whether they need washing I throw away clothing items that I no longer wear β P>\|z\| β P>\|z\| β P>\|z\| Β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| I avoid buying clothing items that originate in countries with poor working conditions 0.39 0.01 0.15 0.23 -0.09 0.46 0.18 0.16 0.09 0.44 0.12 0.32 0.17 0.20 I choose clothing items from fair-trade production 0.15 0.30 0.10 0.49 0.35 0.02 0.11 0.41 0.05 0.70 0.22 0.10 -0.14 0.29 I choose clothing items from organic production (e.g., made from organic cotton) 0.39 0.01 0.31 0.03 0.09 0.54 0.08 0.61 0.08 0.59 0.22 0.12 0.03 0.86 I select clothing that includes labels guaranteeing the absence of chemical pollutants -0.47 0.00 -0.17 0.21 -0.11 0.46 0.05 0.72 -0.00 0.98 -0.05 0.73 -0.01 0.92 I choose high quality and long-lasting clothing items -0.13 0.22 -0.15 0.17 0.16 0.15 -0.09 0.39 0.02 0.86 0.07 0.51 0.10 0.37 Observations 115 115 115 115 115 115 115 Prob > chi2 0.00 0.05 0.05 0.15 0.68 0.01 0.62 Pseudo R2 0.05 0.03 0.03 0.02 0.01 0.03 0.01 https://doi.org/10.1371/journal.pone.0267783.t005 Table 6. Logistic regression of older adults. 5.3. Ologit regressions by age generation Table 5 shows the results linked to the centennial group. According to the Chi2 test, the regres- sions with the dependent variables "I buy second-hand clothing", "I wear patched and mended clothing", "I give away or swap unwanted clothing items that I no longer wear" and "I air my clothing items properly before deciding whether they need washing" obtained adequate good- ness of fit at 99% (p <0.01), 95% (p <0.05) or 90% (p <0.10) confidence. The significant regression coefficients are mostly associated with the purchase of secondhand clothing as the dependent variable. In this regression, the statements "I avoid buying clothing items that origi- nate in countries with poor working conditions" (β = 0.39, P>\|z\| = 0.01), " I choose clothing items from organic production" (β = 0.39; P>\|z\| = 0.01) stand out for their positive influence, and the statement "I select clothes that include labels that guarantee the absence of chemical pollutants" (β = -0.47, P>\|z\| = 0.00) stands out for its negative influence. Complementarily, the selection of fair trade clothes positively affects the frequency of donating or exchanging clothes (β = 0.35, P>\|z\| = 0.02), and the selection of organic clothing positively affects the frequency of patching or mending clothes (β = 0.31, P>\|z\| = 0.03). In the case of the older persons, the results presented in Table 6 show that all ologit regres- sions, except the regression with the dependent variable "I throw away clothing items that I no longer wear", obtained adequate goodness-of-fit at 99% (p <0.01) or 95% (p <0.05) confi- dence, according to the Chi2 test. In this group, the statement "I avoid buying clothing items that originate in countries with poor working conditions" stands out because it had positive PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 13 / 23 Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours PLOS ONE I buy second- hand clothing I wear patched and mended clothing I give away or swap unwanted clothing items that I no longer wear Instead of buying a new piece of clothing for a special occasion, I borrow something I look for other possible uses of unwanted clothing items I air my clothing items before deciding whether they need washing I throw away clothing items that I no longer wear β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| β P>\|z\| I avoid buying clothing items that originate in countries with poor working conditions 0.38 0.01 0.21 0.09 0.17 0.17 0.48 0.00 0.36 0.01 0.14 0.27 0.20 0.13 I choose clothing items from fair-trade production -0.04 0.75 -0.02 0.87 0.21 0.05 -0.10 0.45 -0.03 0.82 0.20 0.08 -0.20 0.08 I choose clothing items from organic production (e.g., made from organic cotton) 0.12 0.43 0.25 0.07 0.05 0.70 0.30 0.06 0.33 0.02 0.04 0.80 0.21 0.13 I select clothing that includes labels guaranteeing the absence of chemical pollutants -0.01 0.92 0.04 0.77 0.13 0.32 -0.11 0.48 0.13 0.33 0.16 0.22 -0.25 0.06 I choose high quality and long-lasting clothing items -0.29 0.01 -0.17 0.09 0.03 0.76 -0.21 0.06 -0.41 0.00 0.03 0.76 0.04 0.69 Observations 125 125 125 125 125 125 125 Prob > chi2 0.00 0.01 0.00 0.00 0.00 0.00 0.23 Pseudo R2 0.05 0.04 0.06 0.06 0.09 0.05 0.02 https://doi.org/10.1371/journal.pone.0267783.t006 Table 6. Logistic regression of older adults. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 14 / 23 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours -0.29, P>\|z\| = 0.01), "I wear patched and mended clothing" (β = -0.17, P>\|z\| = 0.09), "instead of buying a new piece of clothing for a special occasion, I borrow something" (β = -0.21, P>\|z\| = 0.06) and "I look for other possible uses of unwanted clothing items" (β = -0.41; P>\|z\| = 0.00). PLOS ONE Complementarily, selecting clothes that are produced under fair trade guidelines affects the dependent variables "I give away or swap unwanted clothing items that I no longer wear" (β = 0.21, P>\|z\| = 0.05) and "I air my clothing items properly before deciding whether they need washing" (β = 0.20, P>\|z\| = 0.08); selecting clothes produced organically positively affects the dependent variables "I wear patched and mended clothing" (β = 0.25, P>\|z\| = 0.07), "instead of buying a new piece of clothing for a special occasion, I borrow something" (β = 0.30, P>\|z\| = 0.05) and "I look for other possible uses of unwanted clothing items" (β = 0.33, P>\|z\| = 0.02). Mainly, these results indicate that in older persons, selecting clothes manufactured in coun- tries with good working conditions and produced organically positively affects purchase or post-purchase actions that benefit environmental and social sustainability; conversely, select- ing products for quality negatively affects sustainable purchase and post-purchase actions, par- ticularly the purchase of secondhand clothes, patching or mending clothes, borrowing clothes and finding new uses for them. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 6. Discussion Regarding the centennial generation, the results mainly show that PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 15 / 23 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours sustainable attributes for clothing selection affect the frequency of secondhand clothing pur- chases. Particularly, in the centennial generation, avoiding selecting clothes that originate in countries with poor labour conditions and selecting clothes produced organically positively influences the purchase of secondhand clothes; conversely, selecting clothes that guarantee the absence of pollutants negatively influences the purchase of secondhand clothes. In the case of older persons, the clothing selection attributes "I avoid buying clothing items that originate in countries with poor working conditions" and "I choose clothing items from organic produc- tion" stand out, as they have a positive influence on buying secondhand clothes, patching or mending clothes and borrowing clothes for special occasions. Additionally, evidence that the selection of clothing for quality negatively affects buying second-hand clothes, patching or mending clothes, borrowing clothes for special occasions, and finding other uses for unwanted clothing items is relevant for older persons. This evidence allows us to validate Hypothesis 3 of this research, which states that the valuation of sustainable attributes of clothing differentially affects the frequency of sustainable purchase and post-purchase behaviours between the cen- tennial generation and older people. With the theoretical support from response hierarchy models [36, 38], this research shows that clothing preference based on attributes such as organic production, fair-trade and pollut- ant-free sales, and quality influences subsequent purchase and post-purchase behaviour, which is consistent with these models. The findings are also coherent with previous research support- ing differences in sustainability concerns, sustainable product selection and sustainable pur- chase and post-purchase actions by gender [68, 69]. They are also consistent with previous publications that recognise differences in sustainability concerns, sustainable product selection and sustainable purchase and post-purchase actions by age generation [25, 75]. This work is a new contribution to knowledge about the attributes of sustainable clothing that affect purchase and post-purchase behaviours, distinguishing this influence according to gender and age generation. The findings are involved in relevant and widely studied sustain- able clothing behaviours, such as buying secondhand clothing and repairing, reusing and donating clothes. 6. Discussion This research demonstrates that consumer valuation of sustainable clothing attributes affects the frequency of sustainable clothing purchase and post-purchase actions. The study proposes the following research questions: Does people’s evaluation of attributes for selecting clothing, included in purchase choices, impact post-purchase actions embedded in sufficient and frugal consumption? Does the impact differ according to people’s gender and age generation? The findings demonstrate that product selection based on sustainable attributes affects sustainable purchasing and post-purchase behaviours, and this effect differs by gender and age generation. The results of the logistic regressions, supported by hypothesis tests to assess the signifi- cance of the coefficients and the goodness of fit of the regression models, reveal that the fre- quency of clothing selection in terms of its sustainable attributes influences the frequency of sustainable purchase and post-purchase behaviours. The p-values of the regression coefficients, less than 0.10, 0.05 or 0.01, imply that the regression coefficients are significant at the 99%, 95% or 90% confidence level. These findings support Hypothesis 1 of this research. The ologit regressions also show that this influence is distinct between men and women. The significant regression coefficients show that in the group of women evaluated, the state- ment "I avoid buying clothing items that originate in countries with poor working conditions." is relevant, as it positively influences behaviours such as the purchase of second-hand clothes, borrowing clothes for special occasions, and finding new uses for clothes and airing clothes before deciding to wash them. In the case of men, the selection of clothing with organic mate- rials is an important attribute that positively influences buying second-hand clothes, patching or mending clothes and airing clothes before deciding to wash them. Conversely, for men, clothing selection by quality demonstrates a significant negative influence on sustainable pur- chasing and post-purchase behaviours, specifically buying second-hand clothes, patching or mending clothes, borrowing clothes for a special occasion and finding other uses for unwanted clothing items. This evidence allows us to validate Hypothesis 2 of this research, which states that the valuation of sustainable attributes of clothing differentially affects the frequency of sus- tainable purchase and post-purchase consumption behaviours between men and women. The ologit regressions also show that this influence is distinct between the centennial gener- ation and older persons. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 6. Discussion A better understanding of sustainable apparel attributes that favour or disfa- vour the frequency of sustainable purchasing and post-purchase actions is of high importance for sustainable clothing consumption due to the enormous volume of waste generated by the disposal of apparel and the poor working conditions in countries with major fast-fashion pro- duction, such as Bangladesh, Indonesia, China, Turkey and Vietnam [91]. The New York Times published the concept “Fast Fashion” to describe Zara’s goal of taking only 15 days from the design of a garment to its sale in shops [92]. Lax environmental regulations in these countries allow companies to mass-produce clothing without legal protest, causing environ- mental poverty through a volatile combination of water, chemicals and waste [93]. The results of this study have practical utility for governmental and nongovernmental orga- nisations concerned with sustainable clothing consumption. Knowledge of the attributes of clothing selection that positively or negatively affect secondhand purchasing, and actions related to fashion clothing disposal behaviour should guide policies and regulations to inform the characteristics of these products with higher detail and prominence. Governments could mandate the incorporation of labels or seals that more clearly report conditions on clothing, such as the use of organic materials, the absence of pollutants and fair-trade production, in a similar way to the seals on food packaging that warn of high sugar and saturated fat compo- nents. Previous studies have argued that increased awareness of the social and environmental impact of clothing contributes to changing consumers’ purchasing, consumption and disposal behaviours [94]; hence, governmental and nongovernmental organisations should develop communication campaigns highlighting the importance of such sustainable attributes in prod- ucts and regulate the wholesale and retail distribution of clothing. 16 / 23 PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours Information from this research can also guide the commercial strategies of apparel compa- nies seeking to capture the preference of market segments that value sustainable products. Information from this research can also guide the commercial strategies of apparel compa- nies seeking to capture the preference of market segments that value sustainable products. Because some attributes of clothing selection positively influence clothing donation, exchange or repair, fashion companies could offer benefits such as discounts on new clothing by receiv- ing used clothing in part payment and offering repair and maintenance services for clothing already purchased. 6. Discussion They could also connect these consumers with organisations, communities or other clients looking for used clothing or train consumers to transform their branded cloth- ing for new uses. Additionally, the results indicate that a preference for quality discourages sustainable pur- chase and post-purchase actions among men and older persons. These negative relationships between the preference for quality and durability and the purchase of secondhand clothing are consistent with the study by Eike et al. [95], who found that people discard clothing that they perceive to be of poor quality or poor fit. However, in the group of young people, no significant and negative regression coefficients were found between the search for quality and sustainable purchase and post-purchase behaviours, and in the women group, only one significant nega- tive regression coefficient was found. In other words, the results suggest that the centennial generation and women show fewer objections to preferring higher quality and more durable clothing—according to their perception—and to buying secondhand clothing and patching and borrowing garments. One explanation for this difference is that quality may be perceived differently among age groups and genders. Quality is defined as the consumer’s overall assessment of how well goods or services perform [96] and as the superiority of a product when compared to an alternative product from a market perspective [97]. These definitions suggest that quality is a general con- cept that depends on personal interpretation of a product. In this sense, younger people and women might relate quality mostly to slow fashion attributes such as longer production times, use of local materials, and sustainability. In contrast, older people and men may have a differ- ent perspective on quality, for example, related to exclusivity, brand tradition and sophistica- tion; these characteristics are more closely related to luxury or premium product attributes [98, 99]. Therefore, the evidence of this research suggests that public and private organisations concerned with sustainability must inform older people and men that sustainability is cur- rently an important characteristic of quality; likewise, to further promote sustainable purchas- ing and post-purchase behaviours among men and older persons who are quality seekers and who associate quality with fast fashion features. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 7. Conclusions This research provides new evidence about the importance of clothing attributes on the fre- quency of sustainable purchase and post-purchase actions, such as secondhand purchase, repair, exchange, donation and ventilation of clothing, distinguishing results by gender and age generation. The results show that for women, the manufacture of clothing in countries with good working conditions positively influences their sustainable purchasing and post-pur- chase behaviours. In the case of the centennial generation, the attributes for clothing selection mostly positively affect the purchase of secondhand clothing. In the group of men and older persons, it is remarkable that the evidence found supports the negative effect of clothing selec- tion by quality on sustainable purchase and post-purchase behaviours. Additionally, this research recognises a higher frequency of sustainable purchase and post-purchase actions incorporated in the sufficient and frugal consumption factor in women and centennial genera- tion and a higher valuation of quality, incorporated in the purchase choices factor, in men and older persons. PLOS ONE \| https://doi.org/10.1371/journal.pone.0267783 June 22, 2022 17 / 23 PLOS ONE Importance of selection criteria on sustainable clothing purchase and post-purchase behaviours 8. Limitations and future research Future research should deepen the understanding of the influence of clothing attributes on sustainable purchase and post-purchase behaviour by using qualitative techniques to obtain more meaning from the results of this research. Precisely one of the limitations of studies with a quantitative approach is the absence of motives, reasons or meanings in the results that can be found mostly in qualitative studies. In addition, this research was conducted on university students in Chile who were mostly workers enrolled in executive programmes and they have the sociodemographic conditions described in the sample section. The characteristics of the sample are a limitation for the generalisability of the results; consequently, it is pertinent to conduct further research, identifying the results by gender and age generation, in countries with different cultures in Asia, Europe and Africa. Finally, future studies should further study the discrepancies between quality and sustainable consumption among men and older persons using qualitative techniques such as interviews and focus groups to better understand the rea- sons and emotions that generate differences. Author Contributions Conceptualization: Gustavo Barrera-Verdugo. Data curation: Gustavo Barrera-Verdugo. Project administration: Gustavo Barrera-Verdugo, Antonio Villarroel-Villarroel. 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The perception of sustain- ability and circular economy: A gender/generation quantitative approach. Sustainability. 2020; 12(7), 2809. https://doi.org/10.3390/su12072809 73. Peres P, Mesquita A. Characteristics and learning needs of generation z. Academic Conferences Inter- national Limited: Proceedings of 17th European Conference on e-Learning; 2018 Nov 1–2; Greece; 2018. p. 464–473 74. Uche SC. Generation Z and corporate social responsibility [Doctoral Thesis]. Las Vegas: University of Nevada; 2018. Available from: https://search.proquest.com/openview/ 5f114c8957d722b6d91f25976e5cb9cc/1?pq-origsite=gscholar&cbl=18750 75. Jaciow M, Wolny R. New Technologies in the Ecological Behavior of Generation Z. Procedia Computer Science. 2021; 192, 4780–4789. https://doi.org/10.1016/j.procs.2021.09.256 76. Uddin SF, Khan MN. Young consumer’s green purchasing behavior: Opportunities for green marketing. 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W4312932199.txt	https://www.nomos-elibrary.de/10.5771/9783748913344-403.pdf?download_full_pdf=1&page=1	de		Autorinnen und Autoren	Nomos Verlagsgesellschaft mbH & Co. KG eBooks	2,022	cc-by	1,635	Autorinnen und Autoren Dr. Hartmut Aden ist Jurist und Politikwissenschaftler, seit 2009 Professor für Öffentliches Recht, Europarecht, Politik- und Verwaltungswissenschaft an der HWR Berlin, Fachbereich Polizei und Sicherheitsmanagement/FÖPS Berlin und seit 2020 Vizepräsident für Forschung und Transfer der HWR Berlin. Leen Al Kalla hat IT-Sicherheit an der Ruhr Universität Bochum studiert und arbeitet zur Zeit als IT-Sicherheitsanalystin bei der ConSecur. Sie hat in ihrer Bachelorarbeit die Nutzung von WhatsApp durch arabischsprachige Nut zer:innen untersucht. E-Mail: Leen.AlKallaa@ruhr-uni-bochum.de Marianne von Blomberg ist wissenschaftliche Mitarbeiterin am Projekt “The Social Credit System as a Challenge for Law and Courts in China” am Lehrstuhl für chinesi sche Rechtskultur der Universität zu Köln. Sie promoviert zu der Frage, wie Sozialkreditsysteme als alternative Mechanismen der Verhaltensregu lierung das traditionelle Recht stärken, unterwandern und verändern. Email: m.vonblomberg@uni-koeln.de Annalina Buckmann ist wissenschaftliche Mitarbeiterin am Lehrstuhl für Human-Centred Se curity an der Ruhr-Universität Bochum und forscht in Projekten des DFG-Exzellenzclusters “CASA - Cyber Security in the Age of Large-Scale Adversaries” zu Sicherheitskulturen, Digital Divide, Inklusivität und Di versität im Kontext von IT-Sicherheit und Privatsphäre in digitalen Gesell schaften. Email: Annalina.Buckmann@rub.de Dr. Niël H. Conradie ist wissenschaftlicher Mitarbeiter am Lehr- und Forschungsgebiet Ange wandte Ethik an der RWTH Aachen Universität. Derzeit liegt der Schwer punkt seiner Arbeit auf kollektiver Verantwortung und wie Fragen indivi dueller und kollektiver Verantwortung mit KI und anderen aufkommen den Technologien zusammenhängen. E-Mail: niel.conradie@humtec.rwthaachen.de 403 https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb Autorinnen und Autoren Jutta Croll ist Vorstandsvorsitzende der Stiftung Digitale Chancen und dort verant wortlich für das auf internationale Kooperation ausgerichtete Projekt Kin derschutz und Kinderrechte in der digitalen Welt. E-Mail: jcroll@digitalechancen.de Alan Dahi ist Datenschutzjurist bei beim gemeinnützigen Verein “noyb - European Center for Ditigal Right” in Wien. Er betreibt dort ua Projekte zum The ma “biometrische Daten und Datenschutz”. Email: ad@noyb.eu Dr. Martin Degeling arbeitet im Themenbereichen “usable privacy and security” und beschäf tigt sich insbesondere mit Online Tracking und der Perspektive von Nutzenden auf Datenschutzfragen. Zur Zeit ist er beider Stiftung Neue Verantwortung mit Empfehlungen zur Auditierung von Empfehlungssys temen beschäftigt. Vorher war er wissenschaftlicher Koordinator eines Graduiertenkollegs und PostDoc an der Ruhr-Universität Bochum. E-Mail: martin.degeling@ruhr-uni-bochum.de Dr. Jana Dittmann ist Professorin an der Otto-von-Guericke Universität in Magdeburg und leitet dort seit 2002 die Arbeitsgruppe “Multimedia and Security”. Sie be schäftigt sich mit verschiedenen Themenfeldern der Cybersicherheit im Zusammenspiel mit Datensparsamkeit, Souveränität und Nachhaltigkeit. Neben Security-by-Design sowie Privacy-by-Design & Default umfassen die Arbeiten Spezialthemen zu Medien-, Netzwerk- und Computer-Foren sik, Test und Evaluation von Angriffsvektoren (zum Beispiel für den Bereich Automotive, Produktionssysteme, automatisierte Steuerungen, Fi nanzwirtschaft), Verdeckte Kommunikation und Digitale Wasserzeichen, sowie den Entwurf von Mediensicherheitsprotokollen. Email: jana.ditt mann@ovgu.de Dr. Jan Fährmann ist Jurist und Kriminologe. Er hat an der HWR Berlin am Forschungsin stitut für öffentliche und private Sicherheit geforscht und unterrichtet. Mittlerweile ist er in den politischen Bereich gewechselt und arbeitet als Fraktionsreferent für die Fraktion von Bündnis 90/Die Grünen im Berliner Abgeordnetenhaus. Email: Jan.Faehrmann@hwr-berlin.de 404 https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb Autorinnen und Autoren Konstantin Fischer ist wissenschaftlicher Mitarbeiter am Lehrstuhl für Human-Centred Secu rity an der Ruhr-Universität Bochum und forscht im Projekt “Humans & Cryptography” des DFG-Exzellenzclusters “CASA - Cyber Security in the Age of Large-Scale Adversaries” zu Nutzer:innenverhalten, mit Fokus auf die Adoption von neuen Sicherheitslösungen und deren Benutzbarkeit. Dr. Michael Friedewald leitet das Geschäftsfeld “Informations- und Kommunikationstechnik” am Fraunhofer-Institut für System- und Innovationsforschung ISI in Karls ruhe. Er ist Koordinator des “Forum Privatheit und selbstbestimmtes Le ben in der digitalen Welt”. Email: michael.friedewald@iis.fraunhofer.de Clemens Gruber ist wissenschaftlicher Mitarbeiter bei der Stiftung Digitale Chancen und aktuell im BMBF-Projekt “Interaktive, visuelle Datenräume zur souverä nen, datenschutzrechtlichen Entscheidungsfindung” InviDas, tätig. E-Mail: cgruber@digitale-chancen.de PD Dr. Jessica Heesen ist Leiterin des Forschungsschwerpunkts Medienethik und Informations technik am Internationalen Zentrum für Ethik in den Wissenschaften (IZEW) der Universität Tübingen und Mitglied im Forum Privatheit. EMail: jessica.heesen@uni-tuebingen.de Franziska Herbert ist wissenschaftliche Mitarbeiterin der Arbeitsgruppe Mobile Security an der Ruhr-Universität Bochum und forscht in Projekten des DFG-Exzellenz clusters “CASA - Cyber Security in the Age of Large-Scale Adversaries” zu Wissen, Verhalten und Wahrnehmung von Nutzer:innen in den Bereichen Privatheit und IT-Sicherheit. Dr. Gerrit Hornung ist Professor für Öffentliches Recht, IT-Recht und Umweltrecht und Direk tor am Wissenschaftlichen Zentrum für Informationstechnik-Gestaltung (ITeG) an der Universität Kassel. E-Mail: gerrit.hornung@uni-kassel.de Dr. Carolin Jansen ist wissenschaftliche Mitarbeiterin im BMBF-Projekt “DYNAMO - Hoch dynamische Verbreitungsformen von Desinformation verstehen, erkennen 405 https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb Autorinnen und Autoren und bekämpfen” an der Hochschule der Medien in Stuttgart (HdM). EMail: jansenc@hdm-stuttgart.de Rita Jordan ist Vorstandsreferentin bei der Technologiestiftung Berlin. Zuvor war sie wissenschaftliche Mitarbeiterin am ScaDS.AI Dresden/Leipzig sowie assoziiertes Mitglied des Schaufler Kolleg@TU Dresden. E-Mail: rita.jor dan@ts.berlin Hannah Klöber Hannah Klöber ist Research Assistant am Projekt “The Social Credit Sys tem as a Challenge for Law and Courts in China” am Lehrstuhl für chine sische Rechtskultur der Universität zu Köln. E-Mail: hkloeber@smail.unikoeln.de. Jan Philipp Kluck ist wissenschaftlicher Mitarbeiter am Lehrstuhl Sozialpsychologie – Medi en und Kommunikation an die Universität Duisburg-Essen. Dort forscht er innerhalb des BMBF-Projekts “DYNAMO” zu den Verbreitungsformen von Falschinformationen. E-Mail: jan.kluck@uni-due.de Dr. Nicole Krämer ist Professorin für Sozialpsychologie – Medien und Kommunikation an die Universität Duisburg-Essen in der Fakultät für Ingenieurwissenschaften und Mitglied des “Form Privatheit”. E-Mail: nicole.kraemer@uni-due.de Dr. Christian Krätzer ist wissenschaftlicher Mitarbeiter an der AG “Multimedia and Security” an der Fakültät für Informatik, Otto-von-Guericke Universität Magdeburg. Er forscht und lehrt in dieser Position zu Themen der IT-Sicherheit mit Fo kus auf Mediensicherheit, Biometrie und Forensik. E-Mail: christian.kraet zer@iti.cs.uni-magdeburg.de Dr. Jörn Lamla ist Professor für Soziologische Theorie an der Universität Kassel und dort auch Direktor am Wissenschaftlichen Zentrum für InformationstechnikGestaltung (ITeG). E-Mail: lamla@uni-kassel.de Roger von Lauffenberg ist Senior Researcher am Vienna Centre for Societal Security \| VICESSE, mit dem Schwerpunkt auf eine kritische Erforschung der gesellschaftli 406 https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb Autorinnen und Autoren chen und organisatorischen Auswirkungen von KI-Systeme. E-Mail: ro ger.von.laufenberg@vicesse.eu Lena Isabell Löber ist wissenschaftliche Mitarbeiterin am Fachgebiet Öffentliches Recht mit dem Schwerpunkt Recht der Technik und des Umweltschutzes (Prof. Dr. Alexander Roßnagel) an der Universität Kassel und promoviert zum The ma digitale Desinformation und Kommunikationsgrundrechte unter be sonderer Berücksichtigung der Regulierung von Social Networks. E-Mail: l.loeber@uni-kassel.de Anna Louban ist Soziologin und Rechtsethnologin. Seit 2021 arbeitet sie als wissen schaftliche Mitarbeiterin zu KI-bezogenen Projekten im Kontext von Straf verfolgungsbehörden am Forschungsinstitut für Öffentliche und Private Sicherheit der Hochschule für Wirtschaft und Recht Berlin (FÖPS/HWR Berlin). E-Mail: Anna.Louban@hwr-berlin.de Matthias Marx ist wissenschaftlicher Mitarbeiter in der Arbeitsgruppe für Sicherheit in verteilten Systemen an der Universität Hamburg. Dort forscht er an anony mer Kommunikation und befasst sich mit der IT-Sicherheit von Kritischen Infrastrukturen. E-Mail: matthias.marx@uni-hamburg.de Dr. Rainer Mühlhoff ist Professor für Ethik der Künstlichen Intelligenz an der Universität Osna brück. Er arbeitet zu Ethik, Datenschutz und kritische Theorie im Kontext vernetzter digitaler Medien. Kontakt und weitere Informationen: https:// RainerMuehlhoff.de Dr. Saskia Nagel ist Professorin für Angewandte Ethik an der RWTH Aachen. Sie ist Mit glied des Human-Technology-Centers und des Centers für Künstliche In telligenz. E-Mail: saskia.nagel@humtec.rwth-aachen.de Dr. Lars Rinsdorf ist Professor für Journalistik an der Hochschule der Medien in Stuttgart. Seine Forschungsschwerpunkte sind journalistische Qualität und publizis tische Vielfalt, Desinformation und Innovation im Journalismus. E-Mail: rinsdorf@hdm-stuttgart.de 407 https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb Autorinnen und Autoren Dr. Alexander Roßnagel ist Seniorprofessor für öffentliches Recht mit dem Schwerpunkt Recht der Technik und des Umweltschutzes an der Universität Kassel und Sprecher des “Forum Privatheit und selbstbestimmtes Leben in der digitalen Welt”. E-Mail: a.rossnagel@uni-kassel.de Dr. Hannah Ruschemeier ist Juniorprofessorin für Öffentliches Recht mit Schwerpunkt Daten schutzrecht/Recht der Digitalisierung (Tenure) an der Fernuniversität in Hagen. E-Mail: hannah.ruschemeier@fernuni-hagen.de Dr. Stephan Schindler ist wissenschaftlicher Mitarbeiter am Fachgebiet Öffentliches Recht, ITRecht und Umweltrecht (Prof. Dr. Gerrit Hornung, LL.M.) an der Univer sität Kassel. E-Mail: stephan.schindler@uni-kassel.de Sabrina Schomberg ist wissenschaftliche Mitarbeiterin am Fachgebiet Öffentliches Recht, ITRecht und Umweltrecht (Prof. Dr. Gerrit Hornung, LL.M.) an der Univer sität Kassel. E-Mail: sabrina.schomberg@uni-kassel.de Jasmin Schreyer ist wissenschaftliche Mitarbeiterin am Lehrstuhl für Soziologie mit dem Schwerpunkt Technik - Arbeit - Gesellschaft an der Friedrich-AlexanderUniversität Erlangen-Nürnberg und arbeitet im Projekt: Digitalisierung der Arbeitswelten. Sie promoviert zu Fragen rund um digitalisierte Ar beitsbeziehungen, die durch Plattformen vermittelt und etabliert werden. Der Fokus liegt dabei auf dem Spannungsverhältnis Mensch-Technik in Bezug auf algorithmische Arbeitskoordination, das zwischen Autonomie und Kontrolle changiert. E-Mail: jasmin.schreyer@fau.de Tahireh Setz ist wissenschaftliche Mitarbeiterin am Fachgebiet Öffentliches Recht, ITRecht und Umweltrecht (Prof. Dr. Gerrit Hornung, LL.M.) an der Univer sität Kassel. E-Mail: t.setz@uni-kassel.de Dr. Martin Steinebach ist Leiter der Abteilung “Multimedia Sicherheit und IT-Forensik” am Fraunhofer SIT und Honorarprofessor zu den gleichen Themen an der TU Darmstadt. Er promovierte zu digitalen Audiowasserzeichen und forscht 408 https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb Autorinnen und Autoren heute in verschiedenen Themen der Mediensicherheit, der Sicherheit von maschinellem Lernen, der IT-Forensik und OSINT. Milan Tahraoui ist seit 2021 wissenschaftlicher Mitarbeiter am Forschungsinstitut für Öf fentliche und Private Sicherheit der Hochschule für Wirtschaft und Recht Berlin (FÖPS/HWR Berlin). Seine Forschungsgebiete umfassen internatio nales und europäisches Recht sowie Datenüberwachung (öffentlich und privat), Fragen der internationalen Sicherheit und die Regulierung von Technologie. Sabine Theis ist Senior Researcher am Institut für Arbeitswissenschaft der RWTH Aachen University. Ihr Forschungsschwerpunkt liegt auf der Bewertung und Charakterisierung von Daten- und Informationsvisualisierungssyste men und -techniken aus der Perspektive menschlicher Faktoren. Sie pro movierte zum Thema ergonomische Gestaltung Gesundheitsdatenvisuali sierungen. Inna Vogel ist wissenschaftliche Mitarbeiterin in der Abteilung von Prof. Dr.-Ing. Martin Steinebach “Multimedia Sicherheit und IT-Forensik” am Fraunho fer SIT. Sie promoviert zu der Frage wie Fake News mithilfe von ma schinellen Lernverfahren automatisiert erkannt werden können. E-Mail: inna.vogel@sit.fraunhofer.de York Yannikos ist wissenschaftlicher Mitarbeiter in der Abteilung “Media Security und IT Forensics” am Fraunhofer SIT. Seine Forschungsschwerpunkte sind das Testen IT-forensischer Tools, Darknet-Marktplätze und OSINT. E-Mail: york.yannikos@sit.fraunhofer.de 409 https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb https://doi.org/10.5771/9783748913344-403, am 14.07.2024, 16:15:13 Open Access – - https://www.nomos-elibrary.de/agb
https://openalex.org/W2588723254	https://europepmc.org/articles/pmc5331580?pdf=render	English	null	Effect of Fibre Supplementation on Body Weight and Composition, Frequency of Eating and Dietary Choice in Overweight Individuals	Nutrients	2,017	cc-by	8,928	Effect of Fibre Supplementation on Body Weight and Composition, Frequency of Eating and Dietary Choice in Overweight Individuals Vicky A. Solah 1,, Deborah A. Kerr 1, Wendy J. Hunt 1, Stuart K. Johnson 1, Carol J. Boushey 2, Edward J. Delp 3, Xingqiong Meng 4, Roland J. Gahler 5, Anthony P. James 1,6, Aqif S. Mukhtar 1,7, Haelee K. Fenton 1 and Simon Wood 1,8,9 1 School of Public Health, Faculty of Health Sciences, Curtin University, Perth WA 6845, Australia; d.kerr@curtin.edu.au (D.A.K.); w.hunt@curtin.edu.au (W.J.H.); s.johnson@curtin.edu.au (S.K.J.); T.P.James@curtin.edu.au (A.P.J.); Aqif.Mukhtar@curtin.edu.au (A.S.M.); h.fenton@curtin.edu.au (H.K.F.); simonwood@shaw.ca (S.W.) 1 School of Public Health, Faculty of Health Sciences, Curtin University, Perth WA 6845, Australia; d.kerr@curtin.edu.au (D.A.K.); w.hunt@curtin.edu.au (W.J.H.); s.johnson@curtin.edu.au (S.K.J.); T.P.James@curtin.edu.au (A.P.J.); Aqif.Mukhtar@curtin.edu.au (A.S.M.); h.fenton@curtin.edu.au (H.K.F.); simonwood@shaw.ca (S.W.) 2 Epidemiology Program, University of Hawaii Cancer Center, Honolulu, HI 96813, USA; cjboushey@cc.hawaii.edu j y 3 Video and Image Processing Laboratory, School of Electrical and Computer Engineering, Purdue University, West Lafayette, IN 47907, USA; ace@ecn.purdue.edu 3 Video and Image Processing Laboratory, School of Electrical and Computer Engineering, Purdue University, West Lafayette, IN 47907, USA; ace@ecn.purdue.edu 4 Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide 5001, Australia; rosie.meng@ﬂinders.edu.au 4 Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide 5001, Australia; rosie.meng@ﬂinders.edu.au 5 Factors Group R & D, Burnaby, BC V3N4S9, Canada; rgahler@naturalfactors.com 6 Curtin Health Innovation Research Institute, Faculty of Health Sciences, Curtin University, Perth WA 6845, Australia 7 Centre for Population Health Research, Faculty of Health Sciences, Curtin University, Perth WA 6845, Australia 8 InovoBiologic Inc., Calgary, AB Y2N4Y7, Canada 9 Food, Nutrition and Health Program, University of British Columbia, Vancouver, BC V6T1Z4, Canada Correspondence: v.solah@curtin.edu.au; Tel.: +61-8-9266-2771; Fax: +61-8-9266-2958 9 Food, Nutrition and Health Program, University of British Columbia, Vancouver, BC V6T1Z4, Canada Received: 12 December 2016; Accepted: 13 February 2017; Published: 16 February 2017 Received: 12 December 2016; Accepted: 13 February 2017; Published: 16 February 2017 Abstract: Fibre supplementation can potentially reduce energy intake and contribute to weight loss. The mechanism may be reduced frequency of eating, resulting in reduced food consumption. The objective of this research was to determine the effectiveness of ﬁbre supplementation with PolyGlycopleX® (PGX®), on body weight and composition, frequency of eating and dietary intake in 118 overweight adults. In a three-arm, parallel, blind, randomised controlled trial participants were randomised to one of three groups; 4.5 g PGX as softgels (PGXS), 5 g PGX granules (PGXG) or 5 g rice ﬂour (RF) control. nutrients nutrients nutrients nutrients 1. Introduction The burden of chronic disease in many countries is increasing concomitant with the percentage of the population with a high body mass index (BMI) [1]. Providing tools to improve the healthiness of the food environment and assisting people to make healthy food choices are important in the prevention of excessive weight gain [1]. Dietary pattern evidence has shown that higher dietary quality i.e. consumption of mainly whole grains, vegetables, fruit, nuts, legumes, seafood, plant protein and low-fat dairy leads to marked reductions in all-cause, cardiovascular disease and cancer mortality [2,3]. Similarly, a healthy dietary pattern which includes consumption of fruits, vegetables, grains, combined with a lower intake of sweets, red meat and processed meat, lowers the risk of developing colorectal cancer [4]. Evidence supports the role of dietary ﬁbre in improving metabolic health. A higher intake of ﬁbre is associated with increased satiety and reduced energy intake and therefore may be important in obesity management [5–11]. Total ﬁbre is the sum of both dietary ﬁbre (complete, intrinsic non-digestible plant carbohydrates, lignin) and functional ﬁbres (non-digestible carbohydrates that have been isolated but still have beneﬁcial physiological effects) [12]. What constitutes a ‘beneﬁcial physiological effect’ and the level of evidence required for this to be substantiated remains unspeciﬁed [13]. Solubility or the ability of a ﬁbre to dissolve in water will affect water holding capacity, viscosity and fermentability [11]. The properties of ﬁbre that are associated with appetite, energy intake and body weight include solubility [11]. Solubility remains a common classiﬁcation technique, although it has been suggested that ﬁbres should be categorised according to functional properties such as viscosity and fermentability [14], since not all ﬁbre is equal in delivering a beneﬁcial physiological effect. PolyGlycopleX (PGX) is a commercial functional ﬁbre complex, manufactured by a proprietary process (EnviroSimplex®) from three dietary ﬁbres: konjac glucomannan, sodium alginate, and xanthan gum [9]. PGX is a soluble viscous non-starch polysaccharide complex that has been identiﬁed as contributing to improved satiety, lipidaemia and glycaemia [8–10,15,16]. In the modern food environment both portion size and frequency of eating have increased in the population [17]. Mattes [17] has suggested that increased frequency of eating is a major factor in weight gain and is linked to the increased energy intake and rising BMI trends. In addition to frequency of eating, the types and amount of food and beverages consumed at an eating occasion may inﬂuence energy intake. Effect of Fibre Supplementation on Body Weight and Composition, Frequency of Eating and Dietary Choice in Overweight Individuals Prior to supplementation and at 12 weeks, participants captured before and after images of all food and beverages consumed within 4 days using a mobile food record app (mFR). The mFR images were analysed for food group serving sizes and number of eating occasions. In the PGXG group, intention-to-treat analysis showed there was a signiﬁcant reduction in waist circumference (2.5 cm; p = 0.003). Subgroup analysis showed that PGXG supplementation at the recommended dose resulted in a reduction in body weight (−1.4 ± 0.10 kg, p < 0.01), body mass index (BMI) reduction (−0.5 ± 0.10, p < 0.01), reduced number of eating occasions (−1.4 ± 1.2, p < 0.01) and a reduced intake of grain food (−1.52 ± 1.84 serves, p = 0.019). PGXG at the recommended dose resulted in a reduction in weight and BMI which was signiﬁcantly greater than that for RF (p = 0.001). These results demonstrate the potential beneﬁts of PGX ﬁbre in controlling frequency of eating and in weight loss. Keywords: ﬁbre; weight; waist circumference; frequency; eating; PolyGlycopleX® (PGX) Nutrients 2017, 9, 149; doi:10.3390/nu9020149 www.mdpi.com/journal/nutrients Nutrients 2017, 9, 149 2 of 14 2.1. Study Design 2.1. Study Design A three-arm, parallel, blind, randomised control trial was conducted to determine whether PGX supplementation would promote weight loss and reduce waist circumference and BMI, change dietary patterns and reduce the frequency of eating or number of eating occasions. Participants (118 in total) were divided between three groups, 4.5 g PGX as softgels (PGXS), 5 g PGX granules (PGXG) and 5 g rice ﬂour (RF) control, (Figure 1) and were assessed at baseline and 12 weeks. This trial was registered with the Australian New Zealand Clinical Trials Registry (reference ACTRN12614000701628). The food images sent via the mFR were stored by a participant identiﬁcation number (ID) only. No personal information was stored with the images. The research was conducted in accordance with the principles proposed by the Australian Association for Research in Education (AARE), the Australian Vice-Chancellor’s Committee (AVCC) and the National Health and Medical Research Council (NHMRC). The study was conducted in accordance with the Declaration of Helsinki and ethics approval was granted by the Human Research Ethics Committee, Curtin University (reference HR170/2014). 1. Introduction Aljuraiban et al. [18] suggest that modifying eating behaviour through more frequent meals of low energy density and high nutrient quality may be an important approach to controlling obesity. A challenge for understanding the contribution of dietary factors is our ability to measure diet. This is even more difﬁcult in overweight and obese participants who are more prone to misreporting or underreporting their energy intake [19,20]. In addition, details such as time of eating is important in examining eating frequency and are difﬁcult to accurately capture with paper-based methods. Advancement in technology has made available new image-based food recording systems such as those using a mobile food record app (mFR) [21–25]. Among the advantages of mFRs is the provision of real-time data capture which allows for the extraction of information from the images on the timing and location of the eating occasion, without relying on individuals to report these details [22,23]. Thus the mFR app allows for more accurate capture of time of eating while reducing the burden to the participant of reporting food consumption [23,24]. A unique aspect of this study was the use of the mFR to capture frequency of eating and dietary intake. The objective of this research was to determine the effectiveness of diet supplementation with the viscous and gel-forming ﬁbre, PolyGlycopleX (PGX), on body weight and composition and to determine if frequency of eating and diet can explain subsequent changes in 118 overweight adults. 3 of 14 Nutrients 2017, 9, 149 2.2. Study Participants Participants, aged 25–70 years and with BMI 25–35 kg/m2 were recruited through advertisements on Curtin University radio, as well as email communication systems. Individuals that expressed interest were screened for eligibility by completing a screening questionnaire. Participants were excluded if they were: (a) pregnant; (b) unable to complete the 12-week study; (c) undertaking extreme forms of exercise or dieting; (d) unable to attend the study centre; or (e) had an allergy to any food ingredient used in the study; (f) had previous or current renal, liver or respiratory failure; (g) had previous gastric or weight-loss surgery; (h) had any malabsorption conditions or (i) had current or recent dietary ﬁbre supplementation. The participant ﬂow diagram (Figure 1) lists the reasons for exclusion. Once assessed as eligible, further details of the study were provided and informed consent obtained. 2.3. Baseline Assessments Participants who met the selection criteria had height, weight and waist circumference anthropometric measurements taken at baseline. A portable stadiometer was used to measure height; weight was measured using a pre-calibrated digital scale and waist was measured as described by Norton and Olds [26]. To electronically record food consumption, iPods with mFR application installed were given to the participants, who were asked to keep a 4-day mFR at baseline and during the twelfth week of the study. Before beginning their baseline image-based 4-day food record, all participants received a 30-minute interactive training session by a single researcher who conducted individual or small group training sessions using PowerPoint slides on how to connect the iPod to the Wi-Fi and how to use the mFR app. Training sessions were held at Curtin University in a room with Wi-Fi access. The inclusion of a ﬁducial marker (a checkerboard pattern of known shape, size and colour) in all food record images gave a known reference of dimension and markings to assist with food identiﬁcation and portion size estimation [25]. During training, participants were able to practice taking before and after images using food models. Collected food record images were automatically uploaded from the iPod touch, when in Wi-Fi range. The iPods were coded with the participant ID ensuring each image was tagged with the participant ID, date and time of eating occasion. Participant ID was used to identify the images on the server. The server was accessible by researchers only via password. Prior to the commencement of supplementation and during the twelfth week of the study, participants took before and after images of all foods and drinks (excluding water) consumed over four consecutive days. Food consumption during the entire study was ad libitum. 4 of 14 f 14 Nutrients 2017, 9, 149 Figure 1. Participant flow diagram. RF: rice flour. Figure 1. Participant ﬂow diagram. RF: rice ﬂour. Figure 1. Participant flow diagram. RF: rice flour. Figure 1. Participant ﬂow diagram. RF: rice ﬂour. 2.4. PGX Supplementation Th PGXS PGXG 2.4. PGX Supplementation The PGXS, PGXG and RF control supplements were provided to participants in a carry bag containing a 12‐week supply labelled with a three‐digit code. PGXG and the control were provided as 5 g individual doses in identical foil sachets and PGXS was provided in plain white jars each containing one month’s supply. Research staff were blinded to the treatment allocation until all analyses were completed. In Arm 1 (PGXS) instructions were provided in writing and verbally to the participants: “Take The PGXS, PGXG and RF control supplements were provided to participants in a carry bag containing a 12-week supply labelled with a three-digit code. PGXG and the control were provided as 5 g individual doses in identical foil sachets and PGXS was provided in plain white jars each containing one month’s supply. Research staff were blinded to the treatment allocation until all analyses were completed. ( ) p g y p p 1–2 softgels three times a day in week 1, 2–4 softgels three times a day in week 2 and 4–6 softgels, three times a day in week 3 to week 12”. The recommended dose was four (4) to six (6) softgels containing 0.64 g fibre each, three times a day. This represented a supplement of dietary of between 7.6–11.4 g/day. Participants were also asked to consume 500 mL water with every softgel dose. Participants in Arm 2 were instructed to consume 5 g of PGXG containing 4.4 g fibre provided in a In Arm 1 (PGXS) instructions were provided in writing and verbally to the participants: “Take 1–2 softgels three times a day in week 1, 2–4 softgels three times a day in week 2 and 4–6 softgels, three times a day in week 3 to week 12”. The recommended dose was four (4) to six (6) softgels containing 0.64 g ﬁbre each, three times a day. This represented a supplement of dietary of between 7.6–11.4 g/day. Participants were also asked to consume 500 mL water with every softgel dose. Participants in Arm 2 were instructed to consume 5 g of PGXG containing 4.4 g ﬁbre provided in a 5 of 14 Nutrients 2017, 9, 149 single dose foil sachet taken three times a day just before or with meals over the 12-week intervention period. This represented a supplement of dietary ﬁbre of 12.2 g/day. 2.5. Post-Intervention Assessments At the end of the 12-week intervention period, measurement of participants’ height, weight and waist circumference were repeated, along with the 4-day mFR being repeated during week 12. At the end of the 12-week intervention, during the ﬁnal meeting participants reported the number of doses to the interviewer and data were recorded in participant ﬁles. 2.4. PGX Supplementation Th PGXS PGXG 2.4. PGX Supplementation Those in Arm 3 were provided with 5 g of RF containing 4 g ﬁbre in the same dose format as the PGXG, representing 12 g ﬁbre/day. RF was selected due to its neutral taste and hypoallergenicity, and it has a similar dietary ﬁbre content, energy, colour and texture to PGX [7]. The recommended dose was 1 sachet, three times per day. Directions were “Stir 1 sachet into your meal or in a drink and consume immediately. You must consume 500mL of water each time you take a sachet.” Participants were also advised that “If you have any discomfort, reduce the dose to 1 sachet a day for week 1, 2 sachets for week 2 and 3 sachets week 3 to week 12. Contact the researcher to discuss any issues.” Participants were informed of the importance of consuming water with the supplement as well as the possible gastrointestinal effects of ﬁbre such as diarrhoea, bloating and ﬂatulence as described in research by Kacinik et al. [7]. All participants were instructed to record their daily sachet or softgel intake and report in person to the researcher at the end of week 12 of the intervention. 2.6. Dietary Analysis A researcher reviewed the 4-day food record images and as needed, conﬁrmed the content of images with participants. Eating occasions were deﬁned as all food and beverages (except water) and were taken from the image metadata and any additional notes supplied by the participant. Eating occasions were categorised as beverage only, food only, single item, food and beverage or ﬁbre only. The images of ﬁbre also allowed dose of ﬁbre taken to be determined. Analysis was conducted to measure any change in eating occasions from baseline to study week 12. Images were analysed and eating occasions, types of foods and serving sizes were entered into a database speciﬁcally designed to capture the number of eating occasions and food groups according to the Australian Guide to Healthy Eating food group serves (vegetables, fruit, grain (cereal) foods, mostly wholegrain and/or high cereal ﬁbre varieties, lean meats and poultry, ﬁsh, eggs, tofu, nuts and seeds and legumes/beans, milk, yoghurt cheese and/or alternatives, mostly reduced fat) (NHMRC 2013) plus junk food (Table 1). The primary outcome variables measured at baseline and at the end of the intervention were: changes in weight, waist circumference and BMI; eating occasions and foods consumed each day classiﬁed as serves of junk food (energy-dense nutrient poor), grain (cereal), meat, dairy, fruits and vegetables for the three intervention groups. Within each intervention group, participants were further categorised into ﬁbre dose compliance level and number of eating occasions and the primary outcomes for this subgroup were also evaluated. 3.1.1. Intention-to-Treat Analysis Baseline measurements showing of all recruited participant characteristics according to treatment group are shown in Table 2. There were 92 females and 28 males, evenly distributed across the three treatment groups. In total 83 of the 118 participants (63% retention) completed the 12-week study. Greater attrition was observed in the ﬁrst two weeks of the supplementation for participants who reported stomach upsets and diarrhoea after PGXG consumption (attrition = 6); who reported diarrhoea, headaches, difﬁculty swallowing recommended PGXS dose (attrition = 6) and during the ﬁrst six weeks of supplementation in the RF group (attrition = 15) due to constipation and feeling ill. Baseline data (Table 2) shows the characteristics of study participants randomised (n = 118) and shows that at baseline there were no signiﬁcant differences (p > 0.05) in age, height, weight, waist circumference, BMI and food group servings across three groups. At baseline PGXS participants had similar baseline frequency of eating (number of eating occasions) to the PGXG and RF participants but those allocated to the PGXG intervention had a signiﬁcantly greater number of eating occasions per day than those allocated to the RF intervention (p = 0.04) (Table 2). Table 2. Characteristics of all recruited study participants randomised at baseline (n = 120) comparing groups. Table 2. Characteristics of all recruited study participants randomised at baseline (n = 120) comparing groups. All Participants PGXS (n = 40) PGXG (n = 40) RF (n = 40) Men 9 10 9 Women 31 30 31 Mean ± SD Age (years) 42.2 ± 16.0 46.5 ± 14.0 43.3 ± 16.8 Height (cm) 167.4 ± 9.1 167.3 ± 9.0 166.4 ± 7.9 Weight (kg) 82.7 ± 16.8 80.9 ± 16.6 81.3 ± 17.7 Waist (cm) 89.8 ± 12.8 90.7 ± 12.1 88.4 ± 14.3 Body Mass Index (BMI, kg/m2) 29.4 ± 4.8 28.7 ± 4.4 29.2 ± 4.8 Eating occasions per day 5.4 ± 2.8 6.3 ± 2.0 4.8 ± 2.1 Food group servings (mean daily serves ± SD) Fruit (150 g) 0.8 ± 0.8 1.0 ± 1.0 1.1 ± 1.2 Vegetable (75 g) 2.4 ± 1.5 2.6 ± 1.4 2.5 ± 1.1 Grain (cereal) (40 g bread, 75–120 g cooked rice, pasta etc. 3. Results 3.1. Baseline Characteristics 3.1.1. Intention-to-Treat Analysis or 500 kJ) 3.8 ± 1.8 4.3 ± 2.2 4 .0 ± 1.5 Dairy (250 mL milk, 40 g cheese or 500–600 kJ) 1.3 ± 0.9 1.6 ± 0.8 1.3 ± 0.8 Junk food (60 g meat pie or hot chips, 40 g donut or cake) 3.5 ± 1.9 3.1 ± 1.6 3.0 ± 1.7 Meat (65 g meat, 100 g ﬁsh or 500–699 kJ) 1.0 ± 0.7 1.6 ± 0.8 1.4 ± 0.7 Alcohol (150 mL) 0.5 ± 0.8 0.4 ± 0.5 0.1 ± 0.2 PGXS = PGX softgel, PGXG = PGX granules, RF = Rice Flour. SD = Standard Deviation. 2.7. Statistical Analysis Both intention-to-treat analysis and per-protocol analyses were performed and reported. The distribution of all outcome variables, weight, waist and BMI were checked by construction of histograms to check normality. A mixed effect model with clustering of participants’ ID and robust variance-covariance estimation were used to assess outcome variables. For the intention-to-treat analysis, all randomised participants were included and all available data at each study time point were used. For per-protocol analysis, only those who completed the study at week 12 were included and missing values were not imputed. Further analysis of subgroups by actual dose consumed was performed and reported. All tests were two tailed and a p value < 0.05 was regarded as statistically signiﬁcant. All analyses were performed using Stata MP 14.1 (Stata Corp., College Station, TX, USA). 6 of 14 6 of 14 Nutrients 2017, 9, 149 Table 1. Serve size in g or mL National Health and Medical Research Council (NHMRC), 2013 [27]. Fibre is 1 serve (g), with 2.5 serves daily minimum recommended dose. Grains Vegetable Fruit Meats Dairy Junk Food Fibre * Bread Porridge cooked Muesli/Oats raw Rice Pasta cooked Beef Lamb Pork Chicken Fish Milk Cheese Meat pie Donut or Cake PGXS PGXG 40 120 30 75–120 (1/2 cup) 75 150 65 80 100 250 mL 40 60 40 3.8 4.4 * Dose determined by study. PGXS = PGX softgel, PGXG = PGX granules. d Medical Research Council (NHMRC), 2013 [27]. Fibre is 1 serve (g), with 2.5 serves daily minimum 7 of 14 Nutrients 2017, 9, 149 3.1.2. Per-Protocol Analysis Baseline measurements according to treatment group showing participant characteristics of all who consumed the recommended dose of ﬁbre supplements are shown in Table 3. Baseline data (Table 3) shows the characteristics of study participants (n = 54) and shows that at baseline there were no signiﬁcant differences (p > 0.05) in weight, waist circumference and BMI across three groups. PGXG and RF intervention sub-group numbers of eating occasions per day were not signiﬁcantly different to each other or to the baseline group but the PGXS subgroup number of eating occasions was 7.4 times per day (Table 3). 8 of 14 Nutrients 2017, 9, 149 Table 3. Characteristics of study participants randomised at baseline (n = 54) comparing subgroups who consumed the recommended dose of ﬁbre supplements. All Participants PGXS (n = 17) PGXG (n = 18) RF (n = 17) p Value Body weight (kg) 76.5 ± 15.9 87.7 ± 20.2 78.3 ± 15.0 0.62 BMI (kg/m2) 27.2 ± 4.5 28.7 ± 5.2 28.3 ± 5.2 0.64 Waist (cm) 84.8 ± 12.2 89.2 ± 20.4 87.1 ± 13.8 0.59 Eating occasions per day 7.4 ± 2.5 6.0 ± 2.0 5.5 ± 2.6 p > 0.05 PGXS = PGX softgel, PGXG = PGX granules, RF = Rice Flour. 3.2. Effect of Intervention on Body Weight and Body Composition 3.2.1. Intention-to-Treat Analysis 3.2.1. Intention-to-Treat Analysis Intention-to-treat analysis revealed a signiﬁcant reduction in waist circumference at week 12 of minus 2.5cm (Conﬁdence Interval = −3.9, −0.8, p = 0.003) for those in the PGXG group (Table 4). No effect was seen on waist circumference for the PGXS and RF groups (p > 0.05). There was no effect of the PGXS or PGXG interventions on weight and BMI. g g 1 1 serve of fruit = 150 g, 1 serve vegetable = 75 g, 1 serve ﬁbre = 3.8 to 4.4 g. Bold values denote signiﬁcant within treatment effect. PGXS = PGX softgel, PGXG = PGX granules, RF = Rice Flour. g g , g , serve of fruit = 150 g, 1 serve vegetable = 75 g, 1 serve ﬁbre = 3.8 to 4.4 g. 3.2.2. Per-Protocol Analysis Per-protocol analyses of the subgroup who consumed the recommended dose of ﬁbre supplements showed a signiﬁcant weight loss and reduction in BMI in the PGXG intervention compared to baseline (Table 5). However, the PGXS subgroup did not show a signiﬁcant weight change during the intervention period. Compared to effect of the intervention on weight seen in the RF subgroup, that of the PGXG subgroup was signiﬁcantly greater (p = 0.001). Table 4. Change in participant characteristics from baseline to week 12 of the interventions of all participants who completed the interventions (intention-to-treat analysis). Table 4. Change in participant characteristics from baseline to week 12 of the interventions of all participants who completed the interventions (intention-to-treat analysis). All Participants PGXS (n = 32) PGXG (n = 32) RF (n = 19) Body weight (kg) 0.47 ± 1.85 −0.49 ± 0.34 −0.03 ± 0.58 BMI (kg/m2) 0.15 ± 0.65 −0.17 ± 0.13 0.01 ± 0.20 Waist (cm) −0.17 ± 2.92 −2.50 ± 0.60 p = 0.03 −1.3 ± 1.0 Eating occasions per day −0.60 ± 1.5 −0.82 ± 1.28 p = 0.01 −0.22 ± 1.72 Food group servings (mean daily serves ± SD) Fruit (150 g) 1 −0.2 ± 0.76 0.08 ± 0.7 −0.18 ± 0.75 Vegetable (75 g) 1 −0.07 ± 1.11 −0.34 ± 1.22 −0.23 ± 0.64 Grain (cereal) 0.21 ± 1.73 −0.79 ± 1.66 −0.51 ± 1.23 Dairy 0.11 ± 0.63 −0.22 ± 0.64 0.07 ± 0.35 Junk food −0.14 ± 2.00 −0.57 ± 1.29 0.28 ± 2.12 Meat 0.08 ± 0.59 0.01 ± 0.79 −0.09 ± 0.78 Alcohol −0.22 ± 0.73 0.17 ± 0.99 −0.02 ± 0.26 Fibre (3.8–4.4 g) 1 1.89 ± 0.91 2.17 ± 0.71 2.35 ± 0.58 Bold values denote signiﬁcant within treatment effect. PGXS = PGX softgel, PGXG = PGX granules, RF = Rice Flour. 1 1 serve of fruit = 150 g, 1 serve vegetable = 75 g, 1 serve ﬁbre = 3.8 to 4.4 g. 9 of 14 Nutrients 2017, 9, 149 Table 5. Change in participant characteristics from baseline to week 12 of the PGXS, PGXG and RF interventions in the subgroup analysis of those who consumed the recommended dose of ﬁbre supplements (per-protocol analysis). 3.2.2. Per-Protocol Analysis Nutrients 2017, 9, 149 9 of 14 T bl 5 Ch i i i h i i f b li k 12 f h PGXS PGXG d RF All Participants PGXS (n = 17) PGXG (n = 18) RF (n = 17) Body weight (kg) 0.22 ± 1.61 −1.4 ± 0.10 p < 0.01 −0.03 ± 0.58 BMI (kg/m2) 0.07 ± 0.59 −0.5 ± 0.10 p < 0.01 0.01 ± 0.20 Waist (cm) −1.04 ± 2.28 −1.2 ± 1.00 −1.3 ± 1.0 Eating occasions per day −1.3 ± 1.9 −1.4 ± 1.20 p < 0.01 −0.22 ± 1.72 Food group servings (mean daily serves ± SD) Fruit (150 g) −0.43 ± 0.59 −0.63 ± 0.57 p = 0.022 −0.18 ± 0.75 Vegetable (75 g) −0.35 ± 0.96 −0.82 ± 1.31 −0.23 ± 0.64 Grain (cereal) −0.93 ± 1.47 −1.52 ± 1.84 p = 0.019 −0.51 ± 1.23 Dairy −0.05 ± 0.56 −0.59 ± 0.50 p = 0.012 0.07 ± 0.35 Junk food −0.17 ± 1.48 −0.76 ± 0.85 0.28 ± 2.12 Meat −0.06 ± 0.62 0.18 ± 0.90 −0.09 ± 0.78 Alcohol −0.50 ± 0.98 0.11 ± 0.32 −0.02 ± 0.26 Fibre supplement serves (serving size 4.5–5 g) 2.6 ± 0.47 2.82 ± 0.24 2.35 ± 0.58 Bold values denote signiﬁcant within treatment effect. PGXS = PGX softgel, PGXG= PGX granules, RF = Rice Flour. SD = standard deviation. Table 5. Change in participant characteristics from baseline to week 12 of the PGXS, PGXG and RF interventions in the subgroup analysis of those who consumed the recommended dose of fibre supplements (per‐protocol analysis). 3.3. Number of Eating Occasions and Food Group Servings 3.3. Number of Eating Occasions and Food Group Servings Collection of mFR images required participants to use the iPod provided by the study to record images before and after each eating occasion during the 4-day food record (Figure 2). Collection of mFR images required participants to use the iPod provided by the study to record images before and after each eating occasion during the 4‐day food record (Figure 2). Collection of mFR images required participants to use the iPod provided by the study to record images before and after each eating occasion during the 4-day food record (Figure 2). Collection of mFR images required participants to use the iPod provided by the study to record images before and after each eating occasion during the 4‐day food record (Figure 2). Figure 2. Before and after images of an eating occasion with foil sachet containing PGXG and fiducial marker captured with the mobile food record App on an iPod. Figure 2. Before and after images of an eating occasion with foil sachet containing PGXG and ﬁducial marker captured with the mobile food record App on an iPod. Figure 2. Before and after images of an eating occasion with foil sachet containing PGXG and fiducial marker captured with the mobile food record App on an iPod. Figure 2. Before and after images of an eating occasion with foil sachet containing PGXG and ﬁducial marker captured with the mobile food record App on an iPod. 3.3.1. Intention‐to‐Treat Analysis 3.3.1. Intention-to-Treat Analysis Analysis was conducted to measure change in number of eating occasions from baseline to week 12 of the interventions. The number of eating occasions was significantly reduced in the PGXG group (p = 0.01) during the intervention (Table 4). No significant differences in number of eating occasions between baseline and week 12 were observed in the groups PGXS and RF or in food group servings in any of the intervention groups (Table 4) in the intention‐to‐treat analyses. Analysis was conducted to measure change in number of eating occasions from baseline to week 12 of the interventions. The number of eating occasions was signiﬁcantly reduced in the PGXG group (p = 0.01) during the intervention (Table 4). No signiﬁcant differences in number of eating occasions between baseline and week 12 were observed in the groups PGXS and RF or in food group servings in any of the intervention groups (Table 4) in the intention-to-treat analyses. 3.2.2. Per-Protocol Analysis All Participants PGXS (n = 17) PGXG (n = 18) RF (n = 17) Body weight (kg) 0.22 ± 1.61 −1.4 ± 0.10 p < 0.01 −0.03 ± 0.58 BMI (kg/m2) 0.07 ± 0.59 −0.5 ± 0.10 p < 0.01 0.01 ± 0.20 Waist (cm) −1.04 ± 2.28 −1.2 ± 1.00 −1.3 ± 1.0 Eating occasions per day −1.3 ± 1.9 −1.4 ± 1.20 p < 0.01 −0.22 ± 1.72 Food group servings (mean daily serves ± SD) Fruit (150 g) −0.43 ± 0.59 −0.63 ± 0.57 p = 0.022 −0.18 ± 0.75 Vegetable (75 g) −0.35 ± 0.96 −0.82 ± 1.31 −0.23 ± 0.64 Grain (cereal) −0.93 ± 1.47 −1.52 ± 1.84 p = 0.019 −0.51 ± 1.23 Dairy −0.05 ± 0.56 −0.59 ± 0.50 p = 0.012 0.07 ± 0.35 Junk food −0.17 ± 1.48 −0.76 ± 0.85 0.28 ± 2.12 Meat −0.06 ± 0.62 0.18 ± 0.90 −0.09 ± 0.78 Alcohol −0.50 ± 0.98 0.11 ± 0.32 −0.02 ± 0.26 Fibre supplement serves (serving size 4.5–5 g) 2.6 ± 0.47 2.82 ± 0.24 2.35 ± 0.58 Bold values denote significant within treatment effect. PGXS = PGX softgel, PGXG= PGX granules, RF = Rice Flour. SD = standard deviation. Number of Eating Occasions and Food Group Servings .3. Number of Eating Occasions and Food Group Servings 4. Discussion This 12 week randomised controlled study showed that when consumed at the recommended dose (per-protocol), the PGXG intervention gave a reduction in BMI and body weight, the number of eating occasions per day and consumption of servings of grain food. The weight loss and BMI reduction observed for the PGXG per-protocol intervention is most likely a result of compliance to the recommended dose, resulting in 64% of participants who consumed PGXG at the recommended dose losing weight and reducing BMI. The average 1.4-kg weight loss found in our study of overweight adults for the PGX per-protocol intervention is in agreement with previous research by Lyon et al. [28] who found a weight loss in women of 1.6 kg with 12 weeks PGX supplementation. Pal et al. [29] reported obese adults on 12-week supplementation of both artiﬁcially sweetened and ﬂavoured PGX and psyllium lost weight (1.6 kg and 1.1 kg respectively). On an individual basis, the highest weight loss in the PGXS group (intention-to-treat) was 4.4 kg, with a waist circumference decrease of 5.5 cm. However, in this group a weight gain of 6.3 kg and waist circumference increase was 9.6 cm was also recorded in one participant. This high variance in change in weight and waist circumference resulted in a non-signiﬁcant weight loss for this group. Data collected by personal communication at the 6-week visit showed ﬁve participants in the PGXS group reporting that they expected PGXS to be a “magic bullet” for weight loss, which was an unexpected issue that requires consideration in future research. Protocol in future research may involve education of participants on using PGX to control appetite. The signiﬁcant reduction in waist circumference observed in the present study for PGXG intention-to-treat group of 2.5 cm was similar in magnitude to that reported in a study by Reimer et al. [8] where PGX consumption over 14 weeks resulted in a signiﬁcant reduction of 1.96 cm in waist circumference. The signiﬁcant reduction in BMI of 0.5 observed in the present study for PGXG per-protocol sub-group was in contrast to Reimer [8] who found no signiﬁcant differences in BMI compared to baseline. The ﬁndings from our study that PGXS and PGXG consumption (intention-to-treat groups) did not affect weight is similar to research on PGX consumption by Kacinik et al. 3.3.2. Per‐Protocol Analysis F h l 3.3.2. Per-Protocol Analysis For the per‐protocol analysis, the subgroup who consumed PGXG at the recommended dose of 2.5 to 3 times per day, significantly reduced their eating occasions by 1.4 ± 1.2 (CI −2.1, −0.6, p < 0.01) during the intervention period (Table 5). Likewise, the participants who consumed PGXG at the recommended dose significantly reduced For the per-protocol analysis, the subgroup who consumed PGXG at the recommended dose of 2.5 to 3 times per day, signiﬁcantly reduced their eating occasions by 1.4 ± 1.2 (CI −2.1, −0.6, p < 0.01) during the intervention period (Table 5). Likewise, the participants who consumed PGXG at the recommended dose significantly reduced their daily intake of fruit (−0.63 ± 0.57 serves, p = 0.02), dairy (−0.59 ± 0.50 serves, p = 0.01) and grain food (−1.52 ± 1.84 serves, p = 0.02) during the intervention (Table 5). Analysis of types of foods in the mFR showed that 1.6 to 2 serves of the grain food consumed at baseline were white bread for these Likewise, the participants who consumed PGXG at the recommended dose signiﬁcantly reduced their daily intake of fruit (−0.63 ± 0.57 serves, p = 0.02), dairy (−0.59 ± 0.50 serves, p = 0.01) and grain food (−1.52 ± 1.84 serves, p = 0.02) during the intervention (Table 5). Analysis of types of foods in 10 of 14 Nutrients 2017, 9, 149 the mFR showed that 1.6 to 2 serves of the grain food consumed at baseline were white bread for these participants. This consumption of white bread for the PGXG group also dropped signiﬁcantly to 0.74 serves per day at 12 weeks of intervention. the mFR showed that 1.6 to 2 serves of the grain food consumed at baseline were white bread for these participants. This consumption of white bread for the PGXG group also dropped signiﬁcantly to 0.74 serves per day at 12 weeks of intervention. 3.4. Adverse Events Adverse effects reported by the participants were mild and agreed with common reported reactions to increased ﬁbre, for example those reported by Kacinik et al. (2011) [7] where for PGX supplementation, 30.9 % of participants reported diarrhoea, bloating and ﬂatulence whereas for rice ﬂour consumption 7.8% reported constipation. 4. Discussion While participants reported feeling full in previous PGXS and PGXG research [9,10,35,36], most participants in this study did not appear to use appetite to change their eating patterns, except in the per-protocol PGXG group who consumed the recommended dose, most likely resulting reduced appetite and reduced food intake. Dietary feedback using the mFR indicated a reduction of 1 serve white bread, each day, which may have been a contributor to the reduction of 40 g in weight daily in the PGXG per-protocol group [27]. In a previous study, a reduction of 31.5 g carbohydrate per day was recorded at 12 weeks after PGX supplementation using a 3-day food and drink diary [29]. The 3-day food and drink diary does not provided details on type carbohydrates foods, whereas the mFR reports provided detail of types of foods and serving sizes. Dietary intake records collected using the mFR can reduce the bias found in auto-assessment [24]. In the present study, PGXG was taken with 500 mL water immediately before or with a meal. The PGXG was mixed in the water, in juice or mixed into the meal. The mechanism for weight loss in the PGXG per-protocol sub-group in the present study may have been as a result of reducing dietary energy density of the meals with which it was consumed by increasing the ﬁbre and water content of meals while maintaining the volume of food eaten [37,38]. Decreasing dietary energy density has been shown to be useful in long-term weight loss [39]. The detailed dietary feedback from the mFR enabled this research to determine weight loss was possible when PGXG at the recommended dose was consumed and the reduction in the number of eating occasions may be part of the mechanism for this effect. Previous research indicates that a possible mechanism behind the appetite and body weight reduction effects of PGX may be related to circulating gut hormones [30]. Reimer et al. [30] reported increased peptide YY, which can slow gastric emptying and decreased ghrelin (an appetite stimulant) on consumption of PGX. More work is required to conﬁrm the physiological mechanisms controlling these effects of PGX. The detailed dietary feedback from the mFR enabled this research to more accurately determine food group changes after 12 weeks. 4. Discussion [7] who reported no signiﬁcant differences in weight loss between PGX and the placebo groups in a study involving a low-calorie diet of 1000 kcal/day for both treatments. There was also no weight loss recorded in a three-week study by Reimer et al. [30] where PGX was pre-mixed with breakfast cereal and consumed with yogurt. In our study participants were not on an energy restricted diet nor were the PGX and RF doses mixed with other products. The use of the mFR allowed our research to determine level of compliance to the recommended dose of the PGXG per-protocol group by reviewing the images collected. The mFR image analysis of the foods eaten, serving sizes and number of eating occasions per day enabled important information about the dietary pattern of the overweight participants to be collected. The reduced number of eating occasions found with the PGXG per-protocol subgroup, translated into reduced consumption of grain food of 1.52 serves (p = 0.019), mainly being a reduction in consumption of white bread. Research by Kerr et al. [23] reported a reduction in consumption of energy-dense nutrient-poor (EDNP) foods as a result of tailored dietary feedback using the mFR. In the present study there was 11 of 14 Nutrients 2017, 9, 149 a non-signiﬁcant reduction in junk food consumption of 0.57 in the PGXG intention-to-treat group and 0.76 serves in the PGXG per-protocol group. Pollard et al. [31] also found overweight people were more likely than those who were a healthy weight to decrease their intake grain food when trying to lose weight, supporting the reduced grain food trend found in this research. Small reductions in conscious or mindful energy intake can improve weight gain [32] and the choice to reduce grain food intake found in this research may have been the contributor to weight loss. Although we observed a reduction in daily intake of fruit and dairy, which is not desirable based on dietary guidelines [27], the magnitude of this change was minor. Data from the mFR showed participants in all groups, other than the PGXG per-protocol subgroup, appeared to make no changes to their eating pattern, as after 12 weeks of intervention, food group servings were not signiﬁcantly different to baseline. This ﬁnding is supported by previous studies [32–34] where it was reported that humans tend to consume a consistent weight or volume of food from day to day. 4. Discussion The reason the PGXS, and PGXG per-protocol groups did not reduce their weight may be because they did not reduce the number of eating occasions or change their daily intake of the foods and continued to consume the amount and type of food usually eaten. In addition, research by Polidori et al. [40] reports that weight loss leads to increased appetite, and appetite increases by approximately 100 kcal/day per kg of lost weight. The reason ﬁbre such as PGXG helps some individuals in the control of appetite and not others requires more research. References 1. Swinburn, B.; Kraak, V.; Rutter, H.; Vandevijvere, S.; Lobstein, T.; Sacks, G.; Gomes, F.; Marsh, T.; Magnusson, R. Strengthening of accountability systems to create healthy food environments and reduce global obesity. Lancet 2015, 385, 2534–2545. [CrossRef] 1. Swinburn, B.; Kraak, V.; Rutter, H.; Vandevijvere, S.; Lobstein, T.; Sacks, G.; Gomes, F.; Marsh, T.; Magnusson, R. Strengthening of accountability systems to create healthy food environments and reduce global obesity. Lancet 2015, 385, 2534–2545. [CrossRef] 2. Liese, A.D.; Krebs-Smith, S.M.; Subar, A.F.; George, S.M.; Harmon, B.E.; Neuhouser, M.L.; Boushey, C.J.; Schap, T.E.; Reedy, J. The Dietary Patterns Methods Project: Synthesis of ﬁndings across cohorts and relevance to dietary guidance. J. Nutr. 2015, 145, 393–402. [CrossRef] [PubMed] 2. Liese, A.D.; Krebs-Smith, S.M.; Subar, A.F.; George, S.M.; Harmon, B.E.; Neuhouser, M.L.; Boushey, C.J.; Schap, T.E.; Reedy, J. 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Tayyem, R.F.; Bawadi, H.A.; Shehadah, I.; Agraib, L.M.; AbuMweis, S.S.; Al-Jaberi, T.; Al-Nusairr, M.; Bani-Hani, K.E.; Heath, D.D. Dietary patterns and colorectal cancer. Clin. Nutr. 2016, in press. [CrossRef] [PubMed] 5. Delzenne, N.M.; Cani, P.D. A place for dietary ﬁber in the management of the metabolic syndrome. Curr. Opin. Clin. Nutr. Metab. Care 2005, 8, 636–640. [CrossRef] [PubMed] 6. Kristensen, M.; Jensen, M.G. Dietary ﬁbres in the regulation of appetite and food intake. Importance of viscosity. Appetite 2011, 56, 65–70. [CrossRef] [PubMed] 7. Kacinik, V.; Lyon, M.; Purnama, M.; Reimer, R.A.; Gahler, R.; Green, T.J.; Wood, S. Effect of PGX, a novel functional ﬁbre supplement, on subjective ratings of appetite in overweight and obese women consuming a 3-day structured, low-calorie diet. Nutr. Diabetes 2011, 1, 1–8. [CrossRef] [PubMed] 8. Limitation Meals and consumption of test products were self-administered; the possibility of non-compliance could not be avoided. In the current study misreporting of intake may have occurred due to participants not taking images of all food and beverages consumed. In addition, the assessment of food group serves by a trained analyst may not be sensitive enough to detect changes in dietary intake. Nutrients 2017, 9, 149 12 of 14 12 of 14 5. Conclusions Supplementation with PGX at the recommended dose resulted in a reduction in body weight (kg), BMI (kg/m2), reduced frequency of eating and reduced intake of white bread. The weight loss and BMI reduction from baseline to 12 weeks was signiﬁcantly greater for PGXG at the recommended dose than for the RF treatment. Dietary assessment using the mFR provided detailed information enabling accurate analysis of the number of eating occasions and changes to food group servings per day. Further research on reducing the frequency eating of speciﬁc foods, such as junk food is warranted. These results demonstrate the potential beneﬁts of PGX ﬁbre in controlling frequency of eating and in weight loss. Acknowledgments: PGX® and PolyGlycopleX® are registered trademarks of InovoBiologic Inc., Calgary, AB, Canada. Financial support for the submitted work was provided by Factors Group, Australia Pty Ltd. RJG owns the Factors Group of Companies, which retains an interest in PGX. SW receives consulting fees from InovoBiologic Inc. Author Contributions: V.A.S., D.A.K., W.J.H., X.M., S.K.J. and S.W. designed and conducted the research; C.J.B., D.A.K. and E.J.D. developed the mFR part of this research; R.J.G. critically reviewed the study design; X.M., V.A.S., A.S.M. and D.A.K. analysed the data; V.A.S., D.A.K., W.J.K., S.K.J., C.J.B., E.J.D., X.M., A.P.J., A.S.M., H.K.F. and S.W. wrote the paper; all authors read and approved the ﬁnal manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W3165894256	https://www.revistas.usp.br/paz/article/download/168560/172166	Latin	null	Variation in dung beetle (Coleoptera: Scarabaeidae: Scarabaeinae) assemblages in a tropical forest remnant from a Mexican National Park	Papéis Avulsos de Zoologia	2,021	cc-by	8,091	Variation in dung beetle (Coleoptera: Scarabaeidae: Scarabaeinae) assemblages in a tropical forest remnant from a Mexican National Park Gibrán Sánchez-Hernández¹⁴; Benigno Gómez¹⁵; Misraim Edivaldo Rodríguez-López¹⁶; Rolando Antonio Dávila-Sánchez²⁷ & Eduardo Rafael Chamé-Vázquez³⁸ Gibrán Sánchez-Hernández¹⁴; Benigno Gómez¹⁵; Misraim Edivaldo Rodríguez-López¹⁶; Rolando Antonio Dávila-Sánchez²⁷ & Eduardo Rafael Chamé-Vázquez³⁸ ¹ El Colegio de la Frontera Sur (ECOSUR), Departamento de Conservación de la Biodiversidad. San Cristóbal de Las Casas, Chiapas, México. ² Environmental Protection & Control (EPC), Ingeniería Ambiental. Managua, Nicaragua. ³ El Colegio de la Frontera Sur (ECOSUR), Ecología de Artrópodos y Manejo de Plagas. Tapachula, Chiapas, México. ⁴ ORCID: http://orcid.org/0000-0002-0152-1380. E‑mail: gisah16@gmail.com ⁵ ORCID: http://orcid.org/0000-0002-7260-6744. E‑mail: bgomez@ecosur.mx ⁶ ORCID: http://orcid.org/0000-0003-3192-5287. E‑mail: edivaldo.rguez@gmail.com ⁷ ORCID: http://orcid.org/0000-0003-3034-1415. E‑mail: rdavilas@hotmail.de ⁸ ORCID: http://orcid.org/0000-0002-9039-1636. E‑mail: echame@ecosur.mx Abstract. The Cañón del Sumidero National Park (PNCS) is a priority area for conservation, but there are few studies on its fauna, which evidences the need for further basic studies to produce adequate knowledge on its biodiversity. This study aimed to determine dung beetle assemblages temporal distribution, trophic preference, and daily activity patterns. We conducted samplings using baited pitfall traps in a PNCS tropical sub deciduous forest remnant, during the dry and rainy seasons between 2014 and 2015. We captured a total of 863 individuals of 20 species, 12 genera, and five tribes of Scarabaeinae. Estimators suggest that we obtained high faunistic representation (> 80%), but species richness is low compared to other regional studies. The community was characterized by a high number of rare species and few dominant species. We captured the greatest richness and abundance during rainy months, however, species composition between seasons did not differ significantly. Trophic preference was mainly generalist and we considered only four species as specialists to tapir dung. We observed a clear segregation between activity hours. Nevertheless, we determined only nine species as specialists (six nocturnal and three diurnal) and two others had generalist habits. The low diversity we found could be influenced by the constant pressure of the urban area and non-native species within the park, which alter the dung beetle assemblages. However, performing samplings for longer periods and using a wider range of resources would help us obtain more robust results and better understand species distribution patterns. Keywords. Cañón del Sumidero; Protected area; Sub deciduous forest; Temporal distribution; Trophic preference. ARTICLE ARTICLE INTRODUCTION of the three located in the state of Chiapas. Due to the hydrological and ecological processes that are developed and the biological diversity that sus tains it, this park was integrated to the wetlands of international importance or RAMSAR sites. Besides, it is part of a priority terrestrial region for the regional conservation of birds (CONANP, 2012). To create joint strategies for the conserva tion of biodiversity and sustainable development at the regional level, this National Park is consid ered along with the federal NPAs Selva El Ocote Biosphere Reserve (REBISO) and the Villa Allende Natural Resources Protection Area (APRNFVA), as well as two state protection areas (La Pera and Cerro Meyapac), as part of the natural corridor so- In Mexico, Natural Protected Areas (NPAs) are zones where the original environments have not been significantly altered by human activities or that need to be preserved and restored. The federal NPAs fall into six categories, which differ among themselves according to their manage ment objectives and the type of zoning they may be subject to. Among them, National Parks are es tablished mainly by their ecosystems scenic beau ty and their aptitude for tourism development (Íñiguez et al., 2014). The Cañón del Sumidero is one of the 67 National Parks decreed in Mexico and the largest ISSN On-Line: 1807-0205 ISSN Printed: 0031-1049 ISNI: 0000-0004-0384-1825 ISSN On-Line: 1807-0205 ISSN Printed: 0031-1049 ISNI: 0000-0004-0384-1825 ISSN On-Line: 1807-0205 ISSN Printed: 0031-1049 ISNI: 0000-0004-0384-1825 Pap. Avulsos Zool., 2021; v.61: e20216150 http://doi.org/10.11606/1807-0205/2021.61.50 http://www.revistas.usp.br/paz http://www.scielo.br/paz Edited by: Laura Rocha Prado Received: 07/04/2020 Accepted: 19/04/2021 Published: 04/06/2021 Pap. Avulsos Zool., 2021; v.61: e20216150 http://doi.org/10.11606/1807-0205/2021.61.50 http://www.revistas.usp.br/paz http://www.scielo.br/paz Edited by: Laura Rocha Prado Received: 07/04/2020 Accepted: 19/04/2021 Published: 04/06/2021 Pap. Avulsos Zool., 2021; v.61: e20216150 2/9 Sánchez-Hernández, G. et al.: Dung beetle assemblages in the Cañón del Sumidero called Complejo Selva Zoque of Natural Protected Areas (RAC, 2015). are the deciduous forest, sub deciduous forest, oak for est, xerophilous vegetation, and secondary vegetation (CONANP, 2012). According to the Köppen classification (García, 2004), the weather is warm, sub-humid with rains in summer. According to the historical data of a meteorological station located in the vicinity of the sam pling area (16°49′41″N, 93°05′42″W), the average annu al rainfall is 1,000 mm during the rainy season (May to October) and 200 mm in the dry season (November to April) (SMN, 2015). Sampling and identification In each of the two sampling areas, we delimited a transect with an approximate length of 500 m. In each transect, we installed eight baited pitfall traps with a minimum separation of 50 m from each other to pro mote their statistical independence (Larsen & Forsyth, 2005). These traps consisted of cylindrical plastic con tainers of 1 L capacity (11 cm in diameter, 18 cm deep) buried at ground level, using ethylene glycol as a preser vative liquid. The baits we used were carrion (squid and river fish), or tapir [Tapirus bairdii (Gill, 1865)] and jaguar [Panthera onca (Linnaeus, 1758)] dung. We obtained the last two attractants from specimens of a local zoo, locat ed southeast of Tuxtla Gutiérrez, Chiapas (16°43′28.8″N, 93°05′42.1″W). In addition to their utility as bioindicators, dung bee tles play a fundamental role in eliminating and recycling organic matter. They use both the carrion in different stages of decomposition and the dung of different ver tebrates, mainly mammals, to feed and nest (Halffter & Edmonds, 1982). These organisms perform a series of ecological functions, ranging from the secondary seed dispersal to the nutrient cycle and the suppression of parasites. Many of these functions result in valuable eco system services, such as biological control of pests and soil fertilization, so that habitat alteration can interrupt many of the functions in which dung beetles are in volved (Nichols et al., 2008). Therefore, this study aimed to analyze the dung beetles assemblages and to deter mine their temporal distribution, trophic preference, and daily activity patterns in a tropical sub deciduous forest remnant within the Cañón del Sumidero National Park (PNCS). This study also represents the first approach to these insects faunal knowledge in this protected area. We performed the surveys in three months during the rainy season (July and September 2014, and June 2015) and three months during the dry season (December 2014, February and April 2015), which we chose as being representative of each season. The traps were active for two days in each month of sampling and we recovered the samples every 12 h (7:00 am and 7:00 pm). Study area The Cañón del Sumidero National Park is located in the center-west of the state of Chiapas, between the limits of the physiographic regions Central Depression, Central Plateau, and Northern Mountains, with a total extension of 21,789 ha. Its extreme coordinates are be tween 16°44′N to 16°56′N and 93°00′W to 93°11′W, with an altitude that varies between 360 and 1,720 m asl. Among the predominant vegetation types in the region INTRODUCTION For this study, we selected two areas from a tropical sub deciduous forest remnant located in the core zone destined for protection within the park, 3.2 km north from the Tuxtla Gutiérrez municipality ur ban area (16°49′40″N, 93°06′09″W). The sites were sep arated by an approximate distance of 1 km (Fig. 1). We established a third sampling area, but most of the traps were looted during each sampling. Therefore, we did not consider them for analysis. called Complejo Selva Zoque of Natural Protected Areas (RAC, 2015). Following the deforestation trend of tropical areas, for almost two decades, this protected area’s original vegetation has been subjected to a series of pressures caused by human activities, which reduced it to small remnants and caused irreversible losses in the richness of their flora and fauna (CONANP, 2012). Despite this re serve’s relevance, and the importance of knowing its bio diversity, the park’s faunistic knowledge is restricted to the study of some groups of terrestrial (Ramírez-Albores, 2010; Altamirano & Ramírez-Mota, 2013; Chacón et al., 2013) and aquatic vertebrates (Velázquez-Velázquez et al., 2014), disregarding other taxa with bioindicator potential, as occurs with several invertebrate groups, mainly insects (Gerlach et al., 2013). The dung beetles of the subfamily Scarabaeinae (Coleoptera: Scarabaeidae) are abundant insects in trop ical ecosystems that come to occupy diverse types of bi omes and altitudes higher than 3,000 meters above sea level (Morón, 2003). They are also important soil entomo fauna’s components, they are well defined from the taxo nomic and functional point of view, and the methods for their sampling are standardized (Spector, 2006), which is why they are considered one of the most convenient groups for the monitoring of biological diversity (Halffter & Favila, 1993). Sampling and identification We con served the collected individuals in containers with 70% ethanol, and we identified them with a Stereoscopic microscope Zeiss Stemi 1000, using several taxonomic studies (Kohlmann, 1997; Rivera-Cervantes & Halffter, 1999; Kohlmann & Solís, 2001; Génier & Kohlmann, 2003; Kohlmann & Solís, 2006; Delgado & Kohlmann, 2007; Edmonds & Zidek, 2010, 2012; Chamé-Vázquez et al., 2020). Data analysis We determined the precipitation effect on species richness and abundance through the Pearson’s correlation coeffi cient using the R software ggpubr package (Kassambara, 2019). To visualize the dominant species in each of the used attractants, we made rank-abundance curves by transforming the relative abundances (log₁₀ + 1) of each species. We used a non-Metric Multidimensional Scaling (NMDS) to represent the grouping patterns in the spe cies composition between sampling seasons, baits, and daily activity. We also carried out an analysis of similarity (ANOSIM test) to verify the differences in the dung beetle species composition between these same comparisons. We performed these analyzes with abundance data us ing the Bray-Curtis dissimilarity index in the PAST soft ware version 3.26 (Hammer et al., 2001). Data analysis We used a species accumulation curve to verify the sampling efficiency for the entire dataset. We calculat ed the curve using the specaccum function of the Vegan package (Oksanen et al., 2019) in R software version 3.6.2. (R Core Team, 2019). We calculated the non-parametric Sánchez-Hernández, G. et al.: Dung beetle assemblages in the Cañón del Sumidero Pap. Avulsos Zool., 2021; v.61: e20216150 3/9 Pap. Avulsos Zool., 2021; v.61: e20216150 Pap. Avulsos Zool., 2021; v.61: e20216150 3/9 3/9 Figure 1. Location of the two sampling areas in a tropical forest remnant in the core zone of the Cañón del Sumidero National Park, Chiapas. Figure 1. Location of the two sampling areas in a tropical forest remnant in the core zone of the Cañón del Sumidero National Park, Chiapas by inclusion. According to their activity hours, we test ed the species’ classification with the multinomial model (CLAM‑test) developed by Chazdon et al. (2011), which is considered adequate for species classification when the sample size is small. This model allowed us to determine a statistical association of preferences (specialists and generalists) in two distinct categories (i.e., diurnal and nocturnal) based on the species’ relative abundance. We performed this analysis in the CLAM program version 1.0 (Chao & Lin, 2011) using the supermajority specialization limit (k = 0.667) and a significance level of 0.05. richness estimators Chao 1, ICE, Bootstrap, and Michaelis- Menten to determine the number of expected species, using EstimateS software version 9.1 (Colwell, 2013). We determined the precipitation effect on species richness and abundance through the Pearson’s correlation coeffi cient using the R software ggpubr package (Kassambara, 2019). To visualize the dominant species in each of the used attractants, we made rank-abundance curves by transforming the relative abundances (log₁₀ + 1) of each species. We used a non-Metric Multidimensional Scaling (NMDS) to represent the grouping patterns in the spe cies composition between sampling seasons, baits, and daily activity. We also carried out an analysis of similarity (ANOSIM test) to verify the differences in the dung beetle species composition between these same comparisons. We performed these analyzes with abundance data us ing the Bray-Curtis dissimilarity index in the PAST soft ware version 3.26 (Hammer et al., 2001). richness estimators Chao 1, ICE, Bootstrap, and Michaelis- Menten to determine the number of expected species, using EstimateS software version 9.1 (Colwell, 2013). RESULTS Species accumulation curve of the total richness captured in a trop ical forest remnant in the Cañón del Sumidero National Park, Chiapas. Table 1. Dung beetle species composition and their abundances in two sampling seasons in a forest remnant of the Cañón del Sumidero National Park, Mexico. DA = daily activity; D = diurnal; N = nocturnal; G = generalist; R = too rare. Tribes/Species Rainy season Dry season Total DA Jul14 Sep14 Jun15 Dec14 Feb15 Apr15 Tribe Ateuchini Ateuchus rodriguezi (Preudhomme De Borre) 50 125 32 5 — — 212 N Scatimus ovatus Harold 1 — 9 — — — 10 N Uroxys microcularis Howden & Young 48 34 112 2 1 1 198 N Tribe Coprini Canthidium centrale (Boucomont) 2 — — — — — 2 R Canthidium pseudoperceptibile Kohlmann & Solís — — 1 — — 1 2 R Copris laeviceps Harold 14 122 5 25 8 — 174 N Copris lugubris Boheman — 1 — — — — 1 R Dichotomius amplicollis (Harold) 2 — 3 — — — 5 N Tribe Deltochilini Canthon cyanellus LeConte — 2 — — — — 2 R Canthon femoralis (Chevrolat) 12 4 13 — — — 29 G Canthon vazquezae (Martínez, Halffter & Halffter) 6 134 18 1 — — 159 D Deltochilum sublaeve (Bates) — 1 — — — — 1 R Deltochilum scabriusculum Bates 8 4 5 — — — 17 N Tribe Onthophagini Onthophagus anthracinus Harold — — 1 — — — 1 R Onthophagus guatemalensis Bates 1 — — — 7 — 8 D Onthophagus cyanellus Bates — — 1 2 3 — 6 D Tribe Phanaeini Coprophanaeus corythus (Harold) 2 — — — — — 2 R Phanaeus demon Laporte-Castelnau — — 1 — — — 1 R Phanaeus endymion Harold 13 6 12 — — — 31 G Sulcophanaeus chryseicollis (Harold) — 2 — — — — 2 R Abundance 159 434 213 35 19 2 863 Richness 12 11 13 5 4 2 20 Seasonal abundance 807 56 Seasonal richness 20 7 Table 1. Dung beetle species composition and their abundances in two sampling seasons in a forest remnant of the Cañón del Sumidero National Park, Mexico. DA = daily activity; D = diurnal; N = nocturnal; G = generalist; R = too rare. RESULTS We captured a total of 863 individuals of 20 species and 12 genera grouped into five tribes of the subfamily Scarabaeinae (Table 1). The tribes with the most signif icant number of species were Coprini and Deltochilini, both with four species. Canthon and Onthophagus were the genera with the highest number of species registered with three species each. Ateuchus rodriguezi (Preudhomme de Borre, 1886) (n = 212), Uroxys micro cularis Howden & Young, 1981 (n = 198), Copris laevi ceps Harold, 1862 (n = 174), and Canthon vazquezae We determined the species’ preference towards any of the four baits used following the method proposed by Halffter & Arellano (2002), considering that the spe cies were specialists for a given resource when its occur rence was ≥ 80% in one of the four baits used. For this analysis, we excluded those species represented by less than five individuals (rare species) to avoid aggregation Sánchez-Hernández, G. et al.: Dung beetle assemblages in the Cañón del Sumidero Pap. Avulsos Zool., 2021; v.61: e20216150 Pap. Avulsos Zool., 2021; v.61: e20216150 4/9 (Martínez, Halffter & Halffter, 1961) (n = 159) were the most abundant species and represent 86.19% of the total abundance. On the other hand, Copris lugubris Boheman, 1858, Deltochilum sublaeve Bates, 1887, Onthophagus an thracinus Harold, 1873, and Phanaeus demon Castelnau, 1840, were represented by a single individual. The species accumulation curve does not tend to sta bility (Fig. 2), which demonstrates a high probability of encountering other dung beetle species in the area. ICE estimated the maximum number of species (S = 23.7), followed by Bootstrap (S = 22.6), Chao 1 (S = 22), and Michaelis-Menten (S = 21.5). These values suggest that the effort we invested was adequate to sample a repre sentative percentage of the dung beetle assemblages since it allowed registering between 84.4 and 93% of the species present in the landscape. Both richness and abundance varied drastically be tween sampling seasons, obtaining the highest values during the rainy season. Precipitation presents a signifi cant correlation on abundance (R = 0.99, p = 0.0004) and a high percentage of correlation but non-significant on species richness (R = 0.81, p = 0.053). We observed the highest abundance during September (n = 436), while June recorded the highest species richness (s = 13). Figure 2. RESULTS Tribes/Species Rainy season Dry season Total DA Jul14 Sep14 Jun15 Dec14 Feb15 Apr15 Tribe Ateuchini Ateuchus rodriguezi (Preudhomme De Borre) 50 125 32 5 — — 212 N Scatimus ovatus Harold 1 — 9 — — — 10 N Uroxys microcularis Howden & Young 48 34 112 2 1 1 198 N Tribe Coprini Canthidium centrale (Boucomont) 2 — — — — — 2 R Canthidium pseudoperceptibile Kohlmann & Solís — — 1 — — 1 2 R Copris laeviceps Harold 14 122 5 25 8 — 174 N Copris lugubris Boheman — 1 — — — — 1 R Dichotomius amplicollis (Harold) 2 — 3 — — — 5 N Tribe Deltochilini Canthon cyanellus LeConte — 2 — — — — 2 R Canthon femoralis (Chevrolat) 12 4 13 — — — 29 G Canthon vazquezae (Martínez, Halffter & Halffter) 6 134 18 1 — — 159 D Deltochilum sublaeve (Bates) — 1 — — — — 1 R Deltochilum scabriusculum Bates 8 4 5 — — — 17 N Tribe Onthophagini Onthophagus anthracinus Harold — — 1 — — — 1 R Onthophagus guatemalensis Bates 1 — — — 7 — 8 D Onthophagus cyanellus Bates — — 1 2 3 — 6 D Tribe Phanaeini Coprophanaeus corythus (Harold) 2 — — — — — 2 R Phanaeus demon Laporte-Castelnau — — 1 — — — 1 R Phanaeus endymion Harold 13 6 12 — — — 31 G Sulcophanaeus chryseicollis (Harold) — 2 — — — — 2 R Abundance 159 434 213 35 19 2 863 Richness 12 11 13 5 4 2 20 Seasonal abundance 807 56 Seasonal richness 20 7 Table 1. Dung beetle species composition and their abundances in two sampling seasons in a forest remnant of the Cañón del Sumidero National Park, Mexico. DA = daily activity; D = diurnal; N = nocturnal; G = generalist; R = too rare. (Martínez, Halffter & Halffter, 1961) (n = 159) were the most abundant species and represent 86.19% of the total abundance. On the other hand, Copris lugubris Boheman, 1858, Deltochilum sublaeve Bates, 1887, Onthophagus an thracinus Harold, 1873, and Phanaeus demon Castelnau, 1840, were represented by a single individual. (Martínez, Halffter & Halffter, 1961) (n = 159) were the most abundant species and represent 86.19% of the total abundance. RESULTS Pairs of baits R p Squid – Jaguar 0.005 0.49 Squid – Fish -0.06 0.59 Squid – Tapir 0.15 0.076 Jaguar – Fish 0.22 0.034* Jaguar – Tapir 0.12 0.13 Fish – Tapir 0.21 0.032* Species n Baits (%) Guild JD TD SQ RF Ateuchus rodriguezi 212 18.9 59.9 4.2 17 Generalist Canthon femoralis 29 — 86.2 10.3 3.5 Specialist Canthon vazquezae 159 39 44.6 4.4 12 Generalist Copris laeviceps 174 8 87.4 1.1 3.5 Specialist Deltochilum scabriusculum 17 11.8 70.6 — 17.6 Generalist Dichotomius amplicollis 5 — 100 — — Specialist Onthophagus cyanellus 6 66.7 — 33.3 — Generalist Onthophagus guatemalensis 8 75 12.5 — 12.5 Generalist Phanaeus endymion 31 9.7 12.9 22.6 54.8 Generalist Scatimus ovatus 10 — 100 — — Specialist Uroxys microcularis 198 6.6 79.8 2 11.6 Generalist However, the similarity between both seasons is high, and they do not present statistical differences in the spe cies composition (ANOSIM: R = 0.0787, p = 0.26) (Fig. 3A). Traps baited with tapir dung captured the greatest species richness and abundance (S = 16, n = 573), fol lowed by traps baited with jaguar dung (S = 10, n = 146), fish (S = 10, n = 109), and squid (S = 8, n = 35). the day, which presents differences in the species com position between both daily activity hours (ANOSIM: R = 0.346, p = 0.0001) (Fig. 3C). Meanwhile, 45% of the species presented a significant association with activity hours (specialists). We determined six species as noctur nal, three as diurnal, two presented generalist habits, and we considered nine too rare to classify (Table 1). We observed no species groupings in the results obtained by the NMDS diagram, which presented a high similarity percentage between baits in a group (ANOSIM: R = 0.1, p = 0.061) (Fig. 3B). However, when comparing pairs of baits, we observed statistical differ ences (Table 2). We determined only four species as tapir dung specialists and considered seven to be generalists (Table 3), while the other nine did not present associa tions due to their low abundance. Each of the baits pre sented a different hierarchical order of species, being C. laeviceps the most abundant species in tapir dung, C. vazquezae in jaguar dung, while in squid and fish was A. rodriguezi, but without presenting substantial changes between the four baits’ dominant species (Fig. 4). They emphasize C. centrale, C. RESULTS lugubris, D. amplicollis, O. anthra cinus, P. Demon, S. ovatus, and S. chryseicollis as tapir dung exclusive species, D. sublaeve jaguar dung exclusive, and C. corythus in fish. We captured five species in all the at tractants, and there were no exclusive species in squid. RESULTS On the other hand, Copris lugubris Boheman, 1858, Deltochilum sublaeve Bates, 1887, Onthophagus an thracinus Harold, 1873, and Phanaeus demon Castelnau, 1840, were represented by a single individual. The species accumulation curve does not tend to sta bility (Fig. 2), which demonstrates a high probability of encountering other dung beetle species in the area. ICE estimated the maximum number of species (S = 23.7), followed by Bootstrap (S = 22.6), Chao 1 (S = 22), and Michaelis-Menten (S = 21.5). These values suggest that the effort we invested was adequate to sample a repre sentative percentage of the dung beetle assemblages since it allowed registering between 84.4 and 93% of the species present in the landscape. Both richness and abundance varied drastically be tween sampling seasons, obtaining the highest values during the rainy season. Precipitation presents a signifi cant correlation on abundance (R = 0.99, p = 0.0004) and a high percentage of correlation but non-significant on species richness (R = 0.81, p = 0.053). We observed the highest abundance during September (n = 436), while June recorded the highest species richness (s = 13). Figure 2. Species accumulation curve of the total richness captured in a trop ical forest remnant in the Cañón del Sumidero National Park, Chiapas. Sánchez-Hernández, G. et al.: Dung beetle assemblages in the Cañón del Sumidero Pap. Avulsos Zool., 2021; v.61: e20216150 Pap. Avulsos Zool., 2021; v.61: e20216150 5/9 5/9 Figure 3. Non-Metric Multidimensional Scaling (NMDS) constructed from the Bray-Curtis index to establish the similarities between (A) seasons, (B) baits, and (C) daily activity of the dung beetle assemblages in the Cañón del Sumidero National Park, Chiapas. Figure 3. Non-Metric Multidimensional Scaling (NMDS) constructed from the Bray-Curtis index to establish the similarities between (A) seasons, (B) baits, and (C) daily activity of the dung beetle assemblages in the Cañón del Sumidero National Park, Chiapas. Table 2. Pairwise comparison of species composition between the four baits according to the analysis of similarity (ANOSIM). * Values of p < 0.05. Table 3. Trophic preference of dung beetles in a forest remnant of the Cañón del Sumidero National Park, Mexico. JD = jaguar dung; TD = tapir dung; SQ = squid; RF = fish. DISCUSSION The analysis of the dung beetle assemblages that in habit the tropical sub deciduous forest remnant studied within the Cañón del Sumidero National Park, Chiapas, indicates that there is a high local faunistic representa tion, with a sampling effort more significant than 80%, according to the four non-parametric estimators we used. However, the species richness represents only 16.13% of the dung beetles’ species diversity estimated for the state of Chiapas (Sánchez-Hernández & Gómez, 2018; Sánchez-Hernández et al., 2019a; Chamé-Vázquez et al., 2020), at the same time that it is revealing low spe cies representativeness (20 species represented by 863 Regarding daily activity patterns, we found a strong predominance of species with nocturnal habits. We cap tured 14 species and 625 individuals (72.39%) at night, and 12 species and 238 individuals (27.61%) during Sánchez-Hernández, G. et al.: Dung beetle assemblages in the Cañón del Sumidero Pap. Avulsos Zool., 2021; v.61: e20216150 Pap. Avulsos Zool., 2021; v.61: e20216150 6/9 Figure 4. Rank-abundance curves of the dung beetle assemblages collected with four different baits, pointing out the dominant species in each. U.mic = Uroxys microcularis; C.lae = Copris laeviceps; A.rod = Ateuchus rodriguezi; C.vaz. = Canthon vazquezae; P.end = Phanaeus endymion. could explain the low faunistic representation at this study’s regional scale, as well as the presence of a high proportion of species (45%) with low abundances and the clear dominance of only four species (Ateuchus rodri guezi, Uroxys microcularis, Copris laeviceps, and Canthon vazquezae) during the study since this is generally indic ative of disturbed habitats (Cajaiba et al., 2017; Silva et al., 2017; Sánchez-Hernández et al., 2018). Despite not presenting apparent differences in the species composition between sampling seasons, we recorded the lowest species richness and abundance during the low rainfall months (December, February, and April). On the contrary, the highest activity expressed with the most significant number of species and individ uals was limited to months with adequate environmen tal conditions, with higher rainfall and stable tempera tures. During this season, we captured 93.5% of the total abundance and all the species reported between the six months of sampling, including 13 exclusive species. This positive correlation between precipitation levels and the dung beetle assemblages is a typical pattern that has been previously reported in several regions of the Neotropics (Damborsky et al., 2015; Morón-Ríos & Morón, 2016; Novais et al., 2016; Rodríguez-López et al., 2019). DISCUSSION The precipitation creates favorable microclimatic condi tions for these organisms’ feeding, such as a greater forest cover and ambient humidity that avoid the rapid drying of food, as well as the soil’s humidity, which facilitates the reproduction process (Hanski & Cambefort, 1991). While during the season with low rainfall, the assemblage’s dy namics are altered, which is expressed in the decrease of richness and changes in the species structure, mainly due to the low quality and quantity of available trophic resources (Nichols et al., 2009; Damborsky et al., 2015). However, although we did not collect exclusive species during the dry season, we captured a high proportion of individuals of two Onthophagus species during this season’s months (December and February) compared to the rainy months. It suggests that performing samplings for longer periods of both seasons can help determine the temporal distribution of dung beetle assemblages adequately. Figure 4. Rank-abundance curves of the dung beetle assemblages collected with four different baits, pointing out the dominant species in each. U.mic = Uroxys microcularis; C.lae = Copris laeviceps; A.rod = Ateuchus rodriguezi; C.vaz. = Canthon vazquezae; P.end = Phanaeus endymion. individuals) in comparison with other studies conducted in protected areas nearby to the study area. For example, for the Villa Allende Natural Resources Protection Area, Arellano et al. (2008, 2013) report between 28 and 33 spe cies, while in the Selva El Ocote Biosphere Reserve, the studies of Gómez et al. (2017), and Sánchez-Hernández et al. (2018), recorded 34, and 37 species, respective ly. Differences in these results could be due to the fact that they carried out studies with higher sampling effort and analyzed a variety of habitats with greater spatial and temporal coverage. Another important aspect is that these studies included baits that are considered the most effective for monitoring Scarabaeinae communi ties (i.e., human feces and domestic pig dung), as they attract almost all of these insects’ species compared to other resources regardless of habitat type (Correa et al., 2016; Cajaiba et al., 2017). individuals) in comparison with other studies conducted in protected areas nearby to the study area. For example, for the Villa Allende Natural Resources Protection Area, Arellano et al. (2008, 2013) report between 28 and 33 spe cies, while in the Selva El Ocote Biosphere Reserve, the studies of Gómez et al. (2017), and Sánchez-Hernández et al. (2018), recorded 34, and 37 species, respective ly. CONCLUSION Therefore, these conditions would allow us to obtain more robust results and better understand the spatial and temporal distribution patterns in dung bee tle assemblages. Segregation in daily activity patterns plays a vital role in resource partition between the dung beetle species (Hanski & Cambefort, 1991). Some authors suggest that there are three daily activity schemes (diurnal, nocturnal, and crepuscular) among the beetles of the subfamily Scarabaeinae (Iannuzzi et al., 2016), while others indi cate that these patterns are narrower, with more specif ic activity ranges divided only in diurnal and nocturnal (Noriega et al., 2008). Hernández (2002) indicates that daily activity patterns may be strongly related to elytral coloration. However, other factors in local conditions such as the percentage of forest cover, relative humidity, temperature, availabili ty of resources, and competition, among others, are also determinants in the spatial and temporal distribution of beetles (Hanski & Cambefort, 1991). Therefore, the distri bution during the day can also be more heterogeneous, and there are probably more than two or three daily ac tivity patterns (Feer & Pincebourde, 2005). This information represents the first systematic fau nistic study concerning dung beetles of the subfam ily Scarabaeinae in the Cañón del Sumidero National Park. Intensifying exploration with this and other insect groups in environments characteristic of the reserve and contiguous protected areas, such as tropical deciduous forests, would provide a greater knowledge of the ento mofauna at a regional scale. It leads to decision-making tools more adequate to promote conservation, at the same time that greater connectivity can be established between the different protected areas that integrate the Complejo Selva Zoque (Sánchez-Hernández et al., 2020). Our data suggest that daily activity hours are pretty similar between species within the same genus or tribe. For example, Ateuchus, Scatimus, Uroxys (Ateuchini), Copris, and Dichotomius (Coprini) are mainly nocturnal, while the Onthophagus species (Onthophagini) are con sidered diurnal, observing a low percentage of similarity with apparent differences in the species composition between both sampling hours (p < 0.001). In this sense, it is possible that most of the Scarabaeinae species res ident in the Cañón del Sumidero exhibit these two ac tivity schemes (diurnal-nocturnal), but without being a general rule among the community, as occurs with spe cies like Canthon femoralis and Phanaeus endymion that seem to be adapted to more extreme conditions, whose patterns of distribution during the day are more het erogeneous. CONCLUSION each type of attractant to maintain different species composition and potentially induce both the number of specialists and their abundances to increase, modifying, in turn, the composition of generalist species (Sánchez- Hernández et al., 2018). On the contrary, discarding this or another similar attractant would strongly affect the survey results by completely losing a specific community component (Tocco et al., 2018). Therefore, using a great er variety of resources for the sampling of dung beetles (e.g., fruit, fungi, and including different types of carrion and dung) in subsequent studies may help to obtain a more approximate knowledge of the species that inhabit the region. At the same time, it can establish more evi dent relationships between the amplitude in the use of trophic resources with the abundance and dominance patterns in the dung beetle assemblages (Salomão et al., 2014; Tocco et al., 2018; Sánchez-Hernández et al., 2019b). In summary, we found that the dung beetles spe cies richness in the tropical forest remnant studied was low. At this study scale, the dung beetle community is homogeneous between sampling seasons and the am plitude in the use of trophic resources. The changes be tween sampling stations do not affect the composition of dung beetle assemblages, and most species seem to present generalist feeding habits. It could respond to the constant expansion of the urban zone adjacent to the re serve (Tuxtla Gutiérrez city) and non-native fauna (feral dogs and cats) within the park. However, there are some restrictions associated with our study that could limit the generalizability of the findings reported. First, we conducted samplings during short periods of each season (three rainy and three dry months). Therefore, conducting at least one annual sampling cycle will capture a greater species richness and better define the temporal distribution of the assemblages (Rodríguez-López et al., 2019). Second, our study focused only on a small region with in the PNCS. It is possible that covering a larger spatial scale and a greater variety of habitats will allow the expansion of captured dung beetle fauna. Finally, al though we used several baits for sampling, the inclu sion of other attractants would have better defined the extent of resources use that each species can present, and the configuration of guilds would have a different structure. DISCUSSION Avulsos Zool., 2021; v.61: e20216150 7/9 Pap. Avulsos Zool., 2021; v.61: e20216150 7/9 CONCLUSION These species with daily activity patterns different from most of the community members may have an advantage in occupying dung in nesting sites where their congeners are absent (Niino et al., 2014). It suggests that even at a local level, the assemblages can present more than one representation of activity, and depending on the availability of resources, some species must adapt to these conditions, mainly those that are not dominant. DISCUSSION Differences in these results could be due to the fact that they carried out studies with higher sampling effort and analyzed a variety of habitats with greater spatial and temporal coverage. Another important aspect is that these studies included baits that are considered the most effective for monitoring Scarabaeinae communi ties (i.e., human feces and domestic pig dung), as they attract almost all of these insects’ species compared to other resources regardless of habitat type (Correa et al., 2016; Cajaiba et al., 2017). The dung of Central American tapir (T. bairdii) was the bait that attracted the most significant number of species and individuals (66.34%), which indicates a solid preference for this resource, being four species (Canthon femoralis, Copris laeviceps, Dichotomius amplicollis, and Scatimus ovatus) defined as specialists in this resource. However, the high percentage of similarity between the composition and the four attractants’ species structure added to the non-significant changes among the dom inant species suggests that the diet of the dung beetle fauna studied may be more diverse. Other studies that have evaluated the food preference of beetles among a greater variety of feces have shown that the captured species diversity is always higher in the dung of mam mals that have an omnivorous feeding than in the dung of herbivores or carnivores (Filgueiras et al., 2009; Bogoni & Hernández, 2014; Correa et al., 2016). In this way, the inclusion of this type of resource (omnivorous) allows Overall, urbanization and other irregular settlements within the reserve have affected around 3,500 ha of the PNCS during the past 20 years (CONANP, 2012). Among the indirect effects of these settlements, densities of fe ral dogs and cats that have occupied territory within the PNCS drastically alter the wild communities’ dynamics, even surpassing the populations of several small and medium mammal species, besides feeding on at least nine native species of the park (Zúñiga & Sarmiento, 2014). These types of disturbances can also generate al terations in other animal groups, mainly in those that are strongly connected to the presence of mammals, as it is the dung beetles’ case (Bogoni et al., 2016), causing spe cies losses locally and imposing barriers to the displace ment of individuals, affecting richness, abundance, and the assemblage structure (Andresen & Laurance, 2007; Nichols et al., 2009; Feer & Boissier, 2015). This scenario Sánchez-Hernández, G. et al.: Dung beetle assemblages in the Cañón del Sumidero Pap. REFERENCES Edmonds, W.D. & Zídek, J. 2010. A taxonomic review of the neotropical genus Coprophanaeus Olsoufieff, 1924 (Coleoptera: Scarabaeidae, Scarabaeinae). Insecta Mundi, 0129: 1‑111. Altamirano, M.A. & Ramírez-Mota, G.M. 2013. Aves del Parque Nacional Cañón del Sumidero y sus alrededores. In: Comisión Nacional para el Conocimiento y Uso de la Biodiversidad (CONABIO) and Gobierno del Estado de Chiapas (Eds.). La biodiversidad en Chiapas: Estudio de Estado. 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https://openalex.org/W2040149203	https://hal.science/hal-00481217/document	English	null	Investigation of the variations in the water leaving polarized reflectance from the POLDER satellite data over two biogeochemical contrasted oceanic areas	Optics express	2,008	cc-by	8,773	Investigation of the variations in the water leaving polarized reflectance from the POLDER satellite data over two biogeochemical contrasted oceanic areas. 2009. Hubert Loisel, Lucile Duforêt-Gaurier, D. Dessailly, Malik Chami, P Hubert Loisel, Lucile Duforêt-Gaurier, D. Dessailly, Malik Chami, P. Dubuisson Dubuisson To cite this version: Hubert Loisel, Lucile Duforêt-Gaurier, D. Dessailly, Malik Chami, P. Dubuisson. Investigation of the variations in the water leaving polarized reflectance from the POLDER satellite data over two biogeochemical contrasted oceanic areas. 2009.. Optics Express, 2008, 16 (17), pp.12905-12918. 10.1364/OE.16.012905. hal-00481217 Distributed under a Creative Commons Attribution 4.0 International License Investigation of the variations in the water leaving polarized reflectance from the POLDER satellite data over two biogeochemical contrasted oceanic areas. Abstract: The biogeochemical characterization of marine particles suspended in sea water, is of fundamental importance in many areas of ocean science. Previous studies based on theoretical calculations and field measurements have demonstrated the importance of the use of the polarized light field in the retrieval of the suspended marine particles properties. However, because of the weakness of the water leaving polarized signal and of the limited number of appropriate spatial sensors, such measurements have never been exploited from space. Here we show that the marine polarized remote sensing reflectance, as detected from the POLarization and Directionality of the Earth’s Reflectances (POLDER) sensor, can be measured from space over bright waters and in absence of aerosols. This feasibility study is carried out over two oceanic areas characterized by different nature of the bulk particulate assemblage: the Barents sea during an intense coccolithophore bloom, and the Rio de la Plata estuary waters dominated by suspended sediments. The retrieved absolute values of the degree of polarization, P, its angular pattern, and its behavior with the scattering level are consistent with theory and field measurements. Radiative transfer simulations confirm the sensitivity of the POLDER-2 P values to the nature of the particulate assemblage. These preliminary results are very promising for the assessment of the bulk particle composition from remote sensing of the polarized signal, at least over highly scattering waters. ©2008 Optical Society of America OCIS codes: (120.0280) Remote sensing and sensors; (010.0010) Atmosphere and ocean optics; (260.5430) Polarization; (010.5620) Radiative transfer. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12905 OCIS codes: (120.0280) Remote sensing and sensors; (010.0010) Atmosphere and ocean optics; (260.5430) Polarization; (010.5620) Radiative transfer. 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Oceanogr. 52, 739-752 (2007). #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12907 References and links Nadal, G. Perry, and D. Tanré, “Remote sensing of aerosols over land surfaces from POLDER- ADEOS-1 polarized measurements,“ J. Geophys. Res. 106, 4913-4926 (2001). 23. J. Chowdhary, B. Cairns, and L. D. Travis, "Case studies of aerosol Retrievals over the ocean from multiangle, multispectral photopolarimetric remote sensing data," J. Atmos. Sci. 59, 383-397 (2002). 24. P. Goloub, M. Herman, M. Chepfer, J. Riedi, G. Brogniez, P. Couvert, and G. Sèze, “Cloud thermodynamical phase classification from the POLDER aspaceborn instrument,” J. Geophys. Res. 105, 14747-14759 (2000). 25. T. Harmel and M. 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(University of California Press, Berkley, 1958). 31. A. Morel, "Diffusion de la lumière par les eaux de mer: Resultats expérimentaux et approche théorique," in Optics of the Sea AGARD Lecture Ser. 61, (North Atlantic Treaty Org., Brussels) pp. 3.1.1–3.1.76 (1973). 32. G. F. Beardsley, “Mueller scattering matrix of sea water,” J. Opt. Soc. Am. 58, 52-57 (1968). 33. A. Lerner, E-H. Carynelisa, S. Nadav, and S. Shai, “On the Quest for the Scattering Mechanism that Determines the Polarization,” presented at the Ocean Optics Meeting XVIII, Montreal, Canada (2007). 3. A. Lerner, E-H. Carynelisa, S. Nadav, and S. Shai, “On the Quest for the Scattering Mechanism that D t i th P l i ti ” t d t th O O ti M ti XVIII M t l C d (2007) 33. A. Lerner, E H. Carynelisa, S. Nadav, and S. Shai, On the Quest for the Scattering Mechanism that Determines the Polarization,” presented at the Ocean Optics Meeting XVIII, Montreal, Canada (2007). 34. D. Rrs(λ)=Lu(λ)/Ed(λ) None of these models exploits the linear polarization of the upwelling light field from the ocean surface which can potentially be observed by an aircraft or a spacecraft. Many studies based on field measurements [12-15] as well as on theoretical or radiative transfer calculations [16-19] highlighted that the polarization of the underwater light field is sensitive to the nature of the suspended marine particles (for example phytoplankton vs. mineral). For example, Ivanoff et al. [12] stressed that the nature of particles suspended in water affects the relationships between the scattering coefficient and the degree of polarization, P. This latter parameter expresses the percentage of polarized light within the total radiation. Based on numerical radiative transfer simulations for the ocean-atmosphere system, previous investigators [18, 20] found that the polarized signal at red wavelengths may be used to discriminate phytoplankton from sediment particles. The directional and polarization information can also be used to significantly increase the efficiency of the IOPs inversion algorithms [21]. While the polarization of light is now extensively used in aerosol [22, 23] and cloud [24] remote sensing studies, it has never been exploited from space borne observations of ocean color, to our knowledge. This may partly be explained by the fact that the polarized water leaving radiation (i) only represents a small fraction of the total radiation recorded by the satellite sensor over open ocean waters and (ii) is fairly insensitive to marine constituents in open ocean waters [25]. However, over relatively bright areas, such as those encountered in coastal waters or during intense phytoplankton blooms, the polarized signal should be exploitable from remote sensing, at least at some appropriate wavelengths. In the present study, we examine the possibility of retrieving the polarized remote sensing reflectance, Rrs-p, simultaneously to the total remote sensing reflectance, Rrs, of oceanic surface layer from the POLarization and Directionality of the Earth’s Reflectances (POLDER-2) sensor [26]. Rrs-p is defined as the ratio of the polarized upwelling radiance to the downwelling irradiance. This study represents the preliminary step prior to the development of an algorithm based on space retrieval of the polarized remote sensing reflectance. Our purpose is to examine if Rrs-p can be detected from satellite remote sensing measurement of ocean color, and if the behavior of the retrieved P values are consistent with expectations. Rrs(λ)=Lu(λ)/Ed(λ) For that purpose, two different areas are selected: the Rio de la Plata estuary waters dominated by suspended sediment, and the Barents Sea north of Norway during an intense phytoplankton bloom. The consistency of the results with previous field experiments, and with theoretical calculations as obtained from radiative transfer simulations, is analyzed. 1. Introduction The absorption coefficients of phytoplankton and colored detrital matter, aphy and acdm, respectively, as well as the backscattering coefficient of suspended particulate matter, bbp, can now routinely be assessed from remote sensing measurements [1]. These coefficients provide information on new (compared to the chlorophyll concentration) biogeochemical components involved in oceanic carbon cycle studies such as particulate organic carbon (POC), etc. These space retrieved inherent optical properties (IOPs) allow to monitor the particulate and dissolved organic matter of the ocean surface. For example, the space retrieved particulate backscattering coefficient has been used to estimate the spatio-temporal variability of POC at local [2, 3, 4] and global scales [5]. In the same way, Siegel et al. [6] provided global distribution of colored detrital and dissolved material, CDM. Recent studies showed that information about the proportion between small-sized and larger particles [7], and about phytoplankton species [8, 9] and size [10, 11] may be assessed from satellite observations of ocean color over the global ocean. Whereas all these advances open new doors in oceanic carbon cycle studies, information on the nature of the bulk particulate matter (for example, mineral vs. organic) from remote sensing is still missing. Moreover, most of these current inverse methods were essentially developed for open ocean waters, and their performance generally decreases in optically complex waters such as those found in coastal areas. The inverse methods used to estimate such bio-optical information from space are based on different assumptions and mathematical developments. However, they all use the total remote sensing reflectance, Rrs(λ) which is defined as the ratio of the upwelling radiance, Lu(λ), to the downwelling irradiance, Ed(λ), at different wavelengths λ, as input parameters (or a similar radiometric quantity): Rrs(λ)=Lu(λ)/Ed(λ) (1) (1) #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12908 2. Theoretical background The scattered light is partially polarized according to the proportion defined by the degree of polarization: P= (Q2+U2+V2)0.5/I, (2) P= (Q2+U2+V2)0.5/I, (2) #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12908 where I, Q, U and V are the Stokes parameters [27]. The parameter I represents the intensity of light, whereas Q and U describe the linear polarization state, and V describes the elliptic polarization state. In the ocean, the linearly polarized radiation is generated through scattering of the penetrating sunlight. Elliptically polarized radiation can also be produced by the reflection under the sea-surface of the linearly polarized radiation [28]. However, the elliptic component is much less prominent than the linear one, and can be neglected in a good approximation [29, 30]. The degree of polarization of the ocean, P(θ), presents a bell-shaped distribution as a function of the scattering angle, θ. P(θ) generally reaches a maximum value around 90° and equals zero at the 0° and 180° scattering angles [31, 32]. Note, however, that the position of the maximum can change with the particle composition, i.e., the real part of the refractive index n and with the particle size distribution [Fig. 1(a-b)]. From in situ measurements, Ivanoff et al. [12] showed that the degree of polarization measured at a scattering angle of 90°, P(90), decreases linearly with the scattering coefficient. In clear waters, the P(90) values were found to be close to the maximum value measured for pure sea water (=0.84), whereas values as low as 0.10 were observed in eutrophic and turbid waters [12, 33]. The decrease of the degree of polarization from clear to turbid waters is mainly explained by multiple scattering events which contribute to depolarize the signal. Ivanoff et al. [12] pointed out that the dispersion observed around this decreasing trend could be related to the natural variability of the bulk suspended particle population. This assumption was confirmed by Mie theory calculations performed for homogeneous spherical particles with a size distribution approximated by a Junge-type hyperbolic model [31]. The degree of polarization increases with increasing Junge exponent ζ (i.e. with increasing the number of small particles relatively to larger ones), and with decreasing refractive index n [Fig. 1(c)]. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12909 #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12910 2. Theoretical background Therefore, the examination of the relationships between the scattering (or backscattering) coefficient and the degree of polarization may bring complementary information on the nature of the particles. (a) (b) (c) Fig. 1. Degree of polarization P (in %) as a function of the scattering angle, θ, for two extreme refractive index relative to waters, n, and for two different values of the PSD slope, ζ: a)ζ=3, and b) ζ =4. c) Contour diagrams of P(90) as a function of the parameters n (x-axis), and ζ (y-axis). Note that the P values presented in this figure are not affected by multiple scattering in contrast to what happens in the ocean. (a) (b) (b) (b) (b) (c) (c) (c) Fig. 1. Degree of polarization P (in %) as a function of the scattering angle, θ, for two extreme refractive index relative to waters, n, and for two different values of the PSD slope, ζ: a)ζ=3, and b) ζ =4. c) Contour diagrams of P(90) as a function of the parameters n (x-axis), and ζ (y-axis). Note that the P values presented in this figure are not affected by multiple scattering in contrast to what happens in the ocean. 3. POLDER-2 data processing POLDER-2 is a wide field of view imaging radiometer which flew on ADEOS-2 from April to October 2003. The POLDER-2 instrument is a camera composed of a two-dimensional CCD detector array with a rotating wheel carrying spectral (443, 490, 565, 670, 763, 765, 865, and 910 nm) and polarized (443, 670, and 865 nm) filters [26]. For each polarized channel, the Stokes parameters are derived from measurements performed by using the same three spectral filters but with the polarized filter axes turned by steps of 60° [26]. Three polarized filters are then used to rebuilt the polarized signal for each polarized channel. In addition to the classical measurements and mapping characteristics of a narrow-band imaging radiometer, POLDER-2 has the unique ability to observe each pixel from 14 different viewing directions during a single satellite pass. Among the three available polarized channels, the red one, centered at 670 nm, is a good compromise for our study. Indeed, in contrast to the blue channel at 443 nm, it is much less affected by atmospheric (Rayleigh) scattering as well as by multiple scattering process occurring in the atmosphere-ocean system which depolarizes the signal. We also disregarded the channel centered at 865 nm, due to the high sea water absorption value which considerably reduces the magnitude of the remote sensing reflectance. Fig. 2. POLDER2 selected scenes of RGB composites, over (a) the Rio de la Plata estuary (26 of May 2003), and (b) off Norway in the Barents sea (19 of July 2003). The red line in panel a corresponds to a transect specifically examined, and the numbers represent the pixel number (see text for details). Fig. 2. POLDER2 selected scenes of RGB composites, over (a) the Rio de la Plata estuary (26 of May 2003), and (b) off Norway in the Barents sea (19 of July 2003). The red line in panel a corresponds to a transect specifically examined, and the numbers represent the pixel number (see text for details). Two oceanic areas, characterized by different particulate matter composition, are selected: the Rio de la Plata estuary waters dominated by suspended sediments of terrestrial origin [Fig. 2(a)], and a phytoplankton bloom occurring offshore the Norway coasts in the Barents sea [Fig. 2(b)]. These scenes were chosen for their relatively high Rrs values at 670 nm, and their very clear sky conditions. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12911 3. POLDER-2 data processing This last point is essential since the correction for the aerosol effects on the top of atmosphere reflectance is still a very challenging task over eutrophic and turbid areas [34, 35]. Here, this correction is not necessary as the aerosol optical thickness at 670 nm of the surrounding black pixels is about 0.02 and 0.04 for the Rio de la Plata and the Barents sea images, respectively. Thus, the aerosol component can be considered as negligible in this study. For each selected scene, the top of atmosphere Stokes parameters (I, Q, U) recorded by the sensor in each viewing direction at 670 nm are corrected for Rayleigh scattering (using the measured surface pressure), for absorption by oxygen and water vapor (from ECMWF pressure and humidity profiles), and for ozone (as recorded by the Total Ozone Mapping Spectrometer sensor). The residual signal is then divided by the atmospheric diffuse transmission to obtain the above water Stokes parameters. For that purpose, the Rayleigh polarized radiance and the transmission are calculated using an atmospheric radiative transfer model developed by Duforet et al. [36]. This code accounts for the interface effects, which are modeled as a function of the sea surface wind speed value using the Cox and Munk’s model [37]. The mean wind speed value (from ECMWF) is about 2 m.s-1 and 5 m.s-1 over the Barents Sea and Rio de la Plata areas, respectively. Note that the glitter contribution on the degree of polarization is negligible as the total and polarized reflectances are measured in (the) direction(s) far from the glitter pattern. The values of P reported in this study are calculated from above water reflectance values, and are slightly different from those calculated from in water reflectance values, because of the transmission at the sea-air interface. On the basis of radiative transfer simulations, Kattawar and Adams [38], have shown that the P values calculated below the sea-air interface are generally greater than those calculated above. Over a wide range of viewing angles this difference ranges between 5% and 25% for high (75°) and low (10°) sun zenith angles, respectively. The value of the sun zenith angle is 62° for the Rio de la Plata image, and 51° for the Barents sea image, which suggest that the effect of the interface is relatively small for our study. 4. Radiative transfer simulations Radiative transfer calculations are conducted to verify the consistency of the POLDER-2 retrievals presented in this study, and especially the relationship between Rrs and P. These simulations are only performed for the Rio de la Plata image for which a reasonable assumption is that the inherent optical properties (IOPs) of the water are determined by pure sea water and sediments. In contrast, the Barents sea waters present much more variability in terms of IOPs due to the patchiness of phytoplankton biomass and the complex optical properties of the particles associated with the coccolithophores bloom (mostly due to the complex structure of the coccoliths, and of the variable concentration of detached coccoliths). Such complexity, which obviously impacts P and strongly affects the backscattering coefficient and then Rrs [39], makes the selection of input parameters for radiative transfer modeling purposes more difficult. The radiative transfer equation is solved for the ocean-atmosphere system using the OSOA model which is fully described in [18]. This model is based on the successive orders of scattering method, and is able to predict the total and polarized signals in the coupled ocean-atmosphere system. The atmospheric parameters are those used for the POLDER-2 data processing and presented in the previous section. The simulations are carryied out for a homogeneous and infinitely deep ocean, for a flat sea surface, and in the absence of inelastic process. The inherent optical properties of marine constituents at 670 nm are modeled as follows. The absorption coefficient is considered to be totally dominated by pure sea water according to Pope and Fry [40]. The total and polarized particulate phase functions are computed by means of Mie theory. For that purpose two values of refractive index typical for sediments are considered: 1.15, and 1.20 (these values are relative to water). The particulate size distribution (PSD) is assumed to follow the Junge hyperbolic function with different Junge exponent values : -3.5, -4.0, and -4.5. The concentration of sediments varies between 0.5 to 60 mg.l-1. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12912 3. POLDER-2 data processing Furthermore, Chami and McKee [15] have recently shown that P, estimated from above water radiometric field measurements performed for sun zenith angles varying from 28° to 39°, is closely related to the biogeochemical properties of the marine particles in turbid environments. 5.1 The spatial distribution of Rrs, Rrs-p, and P 5.1 The spatial distribution of Rrs, Rrs-p, and P The spatial distribution of Rrs, Rrs-p, and P over the two selected areas are displayed in Fig. 3. These maps are obtained by averaging the directional values of each parameter over the different viewing directions. The mean and standard deviation values of the scattering angle are 135.95°±12.21° and 140.98°±10.46° over the Rio de la Plata estuary and the Barents sea, respectively. The total remote sensing reflectance images clearly reveal the plume and the bloom patterns [Fig. 3(a-b)]. At 670 nm, the variability of the total remote sensing reflectance, Rrs, is proportional to that of the particulate backscattering coefficient, bbp, as the total absorption coefficient, a, is largely dominated by pure sea water absorption. Due to a much higher concentration of highly refractive suspended particles in water, the Rrs values are much higher for the Rio de la Plata Image (RPI) than for the Barents Sea Image (BSI). The concentration of suspended particulate matter, SPM, in the Rio de la Plata estuary is often greater than 50 mg.l-1, with maximum values reaching 1 g.l-1 [41]. For such SPM values, and assuming inherent optical properties values as those proposed by Doxaran et al. [42] for different turbid estuarine waters, the Rrs values should range approximately between 0.025 to 0.060 sr-1. This is consistent with the Rrs values obtained in RPI from POLDER2 [Fig. 3(a)]. The maximum Rrs values are observed at the frontal zone located at the inlet of the estuary, and are likely caused by strong resuspension effects due to the low bathymetry. For the Barents Sea Image, the Rrs values range between about 0 (the clearest waters) and 0.015 sr-1 [Fig. 3(b)]. Note that the pixels with Rrs values lower than the noise equivalent Rrs value, that is 0.0004 sr-1 [43], are not significant and only represent 3.6 % of the sea pixels. The relatively high Rrs values observed during this phytoplankton bloom are explained by the specific optical properties of the phytoplankton cells. The brightest blue color observed in Fig. 2(b) suggest the presence of coccolithophores which strongly backscatter light due to the calcium carbonate covering (coccoliths) surrounding their organic constituents. g g g Detached coccolithes of these phytoplankton cells also greatly contribute to increase the backscattering coefficient [44]. Smyth et al. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12913 #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12914 5.2 Examination of the origin of the variability of P The P variability reflects changes in the scattering angles, turbidity (i.e. contribution of the multiscattering effects), and nature of the suspended marine particles (i.e size, shape, and refractive index). Direct interpretation of the different spatial patterns of P (Fig. 3), in terms of biogeochemical properties of marine particles should then be considered with caution, even qualitatively. The impact of the different factors responsible for the variability of P are now examined separately. The evolution of Rrs, Rrs-p, and P is analyzed along a transect in the Rio de la Plata which extend from the inner part of the estuary to offshore waters (Fig. 4). Its exact location is given in Fig. 2(a). The value of the scattering angle along this transect remains roughly the same (138° ± 2°), which points out that the variations of Rrs, Rrs-p, and P are essentially governed by the turbidity and the water composition (Fig. 5). As already noticed in Fig. 3, Rrs and Rrs-p exhibit almost the same spatial evolution, however with an opposite trend between pixels 2376 and 2381 where Rrs decreases by a factor of 1.3 while Rrs-p increases by the same factor. This may be explained by a large increase of P (by a factor of 1.7), probably due to a great variability of the suspended particulate assemblage between these two locations. The degree of polarization shows slight fluctuations around the mean value of 0.18 ± 0.03 from pixel 2376 to 2411 (that is about 245 km), and then sharply increases by a factor of 2.1 up to the end of the transect (where both Rrs and Rrs-p decrease). Some interesting features can be noticed at some certain locations where P exhibits significant variations for the same Rrs value (i. e. same scattering level). For example, P varies by a factor of 1.5 between pixels 2377 and 2389, whereas Rrs is constant (0.034 sr-1). Similar observation can be done between pixels 2391 and 2397, where Rrs varies only by a factor of 1.02, whereas P increases by a factor of 1.21. This behavior can be attributed to variations in the particulate assemblage between these locations. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 2370 2380 2390 2400 2410 2420 2430 2440 2450 Rrs(x5), Rrs-p(x50), P Pixel number Rrs Rrs-p P Fig. 4. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12915 5.1 The spatial distribution of Rrs, Rrs-p, and P The P values for BSI ranges from 0.1 to 0.5, with a coefficient of variability of 35.6 (against 53.2 for RPI). P in BSI is much less organized than in RPI. The P values for BSI ranges from 0.1 to 0.5, with a coefficient of variability of 35.6 (against 53.2 for RPI). 5.1 The spatial distribution of Rrs, Rrs-p, and P [45] used satellite observations to study the occurrence of coccolithophore blooms in the Barents sea over a period of two decades. They showed from in situ measurements that the bloom occurring in July 2003 is of the coccolithophore species Emiliana huxleyi. Field radiometric measurements performed in a different area, but during a Emiliana huxleyi bloom confirm the range of remote sensing reflectance observed in our study [46]. Compared to Rrs, Rrs-p presents much lower values [Fig. 3(c-d)]. The Rrs-p values range between 0.001 and 0.012 sr-1 for RPI, and between 0 and 0.0035 sr-1 for BSI. Only Rrs-p values greater than 0.0009 sr-1 (the noise equivalent Rrs-p value) are significant [43], which represent 100% and 71.23% of the sea pixels for RPI and BSI, respectively. While the coefficient of variation (i.e. the ratio of standard deviation to the mean) of Rrs-p (40.6 %) is higher than that of Rrs (31.3%) for BSI, this is the reverse pattern for RPI (28.4 for Rrs-p, and 40.2 for Rrs). Comparison with in situ Rrs-p measurements is not discussed here as such measurements are still very rare, and generally presented in the literature in terms of degree of polarization [15]. Section 5.2 provides a comparison of the Rrs-p values estimated from POLDER-2, and those obtained from numerical simulations for the Rio de la Plata estuary. Fig. 3. Maps of Rrs, Rrs-p, and P at 670 nm for the Rio de la Plata estuary (left column), and the Barents Sea areas (right column). Fig. 3. Maps of Rrs, Rrs-p, and P at 670 nm for the Rio de la Plata estuary (left column), and the Barents Sea areas (right column). The different Rrs and Rrs-p patterns directly impact the spatial distribution of the degree of polarization [Fig. 3(e-f)]. The general spatial pattern of P is roughly inversely proportional to that of Rrs. In RPI, the highest P values (~0.4) are observed offshore, and the lowest P values (~0.05) are found at the inlet of the estuary after the frontal zone. This pattern is consistent with expectation as P should decrease from clear to more turbid waters. The spatial pattern of P in BSI is much less organized than in RPI. The P values for BSI ranges from 0.1 to 0.5, with a coefficient of variability of 35.6 (against 53.2 for RPI). P in BSI is much less organized than in RPI. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12916 5.2 Examination of the origin of the variability of P Evolution of Rrs, Rrs-p, and P along a transect in the Rio de la Plata estuary (represented by the red line in Fig. 2a). Fig. 4. Evolution of Rrs, Rrs-p, and P along a transect in the Rio de la Plata estuary (represented by the red line in Fig. 2a). The differentiation of the three main phenomena involved in the variability of P, that is its angular dependence, the turbidity, and the particulate assemblage characteristics, may be assessed by looking at the evolution of Rrs as a function of P for a restricted range of scattering angles (Fig. 5). The average value and standard deviation of the scattering angle considered in Fig. 5 is 130°±1° for both scenes. Because the Rrs variability at 670 nm is mainly driven by the particulate backscattering, this figure is somewhat similar to that presented by Ivanoff et al. [12], between the scattering coefficient at 90°, and P. 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.05 0.1 0.15 0.2 0.25 0.3 Rrs (sr-1) P r2=0.88 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.01 0.011 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Rrs (sr-1) P r2=0.89 (a) (b) Fig. 5 Scatter plot of Rrs as a function of P at 670 nm for (a) the Rio de la Plata estuary, and (b) the Barents Sea areas. The black lines in panel a and b correspond to the fits described by the equations 2 and 3, respectively. The square correlation coefficient (r2) is given. Note the different scales of the two panels. 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.01 0.011 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Rrs (sr ) P r2=0.89 (b) 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.05 0.1 0.15 0.2 0.25 0.3 Rrs (sr-1) P r2=0.88 1 (a) P (b) Fig. 5 Scatter plot of Rrs as a function of P at 670 nm for (a) the Rio de la Plata estuary, and (b) the Barents Sea areas. The black lines in panel a and b correspond to the fits described by the equations 2 and 3, respectively. The square correlation coefficient (r2) is given. Note the different scales of the two panels. The net decreasing of P with increasing Rrs, due to the multiple scattering effects, is clearly evidenced over the two different areas. 5.2 Examination of the origin of the variability of P Variation of Rrs as a function of P for the value of the scattering angle of (a) 120° and Fig. 6. Variation of Rrs as a function of P for the value of the scattering angle of (a) 120° and (b) 130° for the Rio de la Plata estuary waters. The colored symbols represent data obtained from the radiative transfer simulations performed for different values of the refractive index, n, and PSD slope, ζ, as indicated. The black dots represent the data obtained from POLDER-2 observations. Note that the P values are lower than those presented in Fig. 1 for which the calculations did not account to multiple scattering. Fig. 6. Variation of Rrs as a function of P for the value of the scattering angle of (a) 120° and (b) 130° for the Rio de la Plata estuary waters. The colored symbols represent data obtained from the radiative transfer simulations performed for different values of the refractive index, n, and PSD slope, ζ, as indicated. The black dots represent the data obtained from POLDER-2 observations. Note that the P values are lower than those presented in Fig. 1 for which the calculations did not account to multiple scattering. The relationship between Rrs and P, as revealed from the POLDER-2 observations, is evaluated for the Rio de la Plata image by the means of radiative transfer calculations carried out as described in section 4. The results of these numerical computations are superimposed on the POLDER-2 data for θ=120° [Fig. 6(a)], and θ=130° [Fig. 6(b)]. The evolution of P as a function of Rrs obtained from these radiative transfer simulations is in excellent agreement with the POLDER-2 observations. The hyperbolic trend observed between P and Rrs from POLDER-2 is well confirmed in both shape and amplitude from the radiative transfer simulations. For a given scattering angle, each variation of the refractive index and of the slope of the particle size distribution affects the P vs. Rrs relationship, likely contributing to the observed scatter in POLDER-2 data. For example, for θ=120° and Rrs = 0.06 sr-1, the increase of the degree of polarization by a factor of 1.6 observed from POLDER-2 in RPI is coherent with an increase of the PSD slope from –3.5 to –4. According to these results, it could be tempting to retrieve information about the particles assemblage (n and ζ) by examining the relationship between Rrs and P. 5.2 Examination of the origin of the variability of P Such a decreasing trend has also been reported by Chami and McKee [15] between P measured at the Brewster viewing angle and the concentration of the suspended particulate matter (which controls the value of Rrs(670), to first order). An exponential function can be fitted between P and Rrs for RPI (Eq. (3)) and BSI (Eq. (4)), in good agreement with the results of Ivanoff et al. [12]: The net decreasing of P with increasing Rrs, due to the multiple scattering effects, is clearly evidenced over the two different areas. Such a decreasing trend has also been reported by Chami and McKee [15] between P measured at the Brewster viewing angle and the concentration of the suspended particulate matter (which controls the value of Rrs(670), to first order). An exponential function can be fitted between P and Rrs for RPI (Eq. (3)) and BSI (Eq. (4)), in good agreement with the results of Ivanoff et al. [12]: P = 0.0099 Rrs (-0.768) (r2=0.88) (3) P = 0.0054 Rrs (-0.739) (r2=0.89) (4) (3) (4) (3) (4) These two relationships present very similar exponential slopes, but their offset (in linear scale) remarkably differ: for the same Rrs value, P is higher by almost a factor of 2 for the Rio del Plata estuary, than for the Barents sea. Assuming that this effect is only attributable to the nature of the suspended marine particles, this result suggests that the bulk particulate assemblage is dominated by relatively larger particles with higher refractive index in BSI than in RPI. While relatively large phytoplankton cells are expected during the bloom of Emiliana huxleyi occurring in the Barents sea, the refractive index of these cells (n=1.05, [47]) and of their associated detached coccoliths (n=1.19, [48]), have generally (depending on the concentration of detached coccoliths in waters) a lower refractive index than that of mineral particles from terrigenous origin (n=1.14-1.26, [49]). However, based on numerical Mie computations it is shown that P is much more sensitive to size than refractive index for this range of refractive index and scattering angles. Indeed, the isolines plotted in Fig. 1(c) for θ=90° tend to be much more parallel to the abscissa (i.e. to the refractive index) when the scattering angles increases [50]. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12917 5.2 Examination of the origin of the variability of P This difference could also partly be explained by a higher concentration of dissolved and particulate absorbing material in RPI than in BSI, which may make RPI waters more single scattering dominated than BSI waters. For this narrow range of scattering angle, a relatively large dispersion of the data is observed around the mean exponential trends. P can vary up to a factor of 1.5 for a given Rrs value over the two studied areas. A higher dispersion is generally observed over the Barents sea area than over the Rio de la Plata estuary. The coefficient of variability is 41% and 31% for BSI, and RPI, respectively (note that it was the reverse pattern when all scattering angles were considered in Fig. 3). Assuming that such dispersion is due to the particulate assemblage variability [12, 31], this suggests that the nature of the bulk particulate matter is more variable over the Barents Sea area than over the Rio de la Plata estuary (at least for these two images). 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 0.1 0.2 0.3 0.4 0.5 Rrs (sr-1) P Θ=120° n=1.15, ζ=-3.5 n=1.15, ζ=-4.0 n=1.15, ζ=-4.5 n=1.20, ζ=-3.5 n=1.20, ζ=-4.0 n=1.20, ζ=-4.5 Polder-2 Data 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 0.1 0.2 0.3 0.4 0.5 Rrs (sr-1) P Θ=130° n=1.15, ζ=-3.5 n=1.15, ζ=-4.0 n=1.15, ζ=-4.5 n=1.20, ζ=-3.5 n=1.20, ζ=-4.0 n=1.20, ζ=-4.5 Polder-2 Data (a) (b) Fig. 6. Variation of Rrs as a function of P for the value of the scattering angle of (a) 120° and (b) 130° for the Rio de la Plata estuary waters. The colored symbols represent data obtained from the radiative transfer simulations performed for different values of the refractive index, n, and PSD slope, ζ, as indicated. The black dots represent the data obtained from POLDER-2 observations. Note that the P values are lower than those presented in Fig. 1 for which the calculations did not account to multiple scattering. 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 0.1 0.2 0.3 0.4 0.5 Rrs (sr-1) P Θ=120° n=1.15, ζ=-3.5 n=1.15, ζ=-4.0 n=1.15, ζ=-4.5 n=1.20, ζ=-3.5 n=1.20, ζ=-4.0 n=1.20, ζ=-4.5 Polder-2 Data 1 (a) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 0.1 0.2 0.3 0.4 0.5 Rrs (sr ) P Θ=130° n=1.15, ζ=-3.5 n=1.15, ζ=-4.0 n=1.15, ζ=-4.5 n=1.20, ζ=-3.5 n=1.20, ζ=-4.0 n=1.20, ζ=-4.5 Polder-2 Data (b) P (a) P (b) Fig. 6. 5.2 Examination of the origin of the variability of P However, it should be done with caution as modifications of n for the same PSD slope does not necessarily modify the Rrs vs. P relationship (Fig. 6). This is in agreement with Mie calculations which showed that, for such scattering angles, P is much more sensitive to ζ than n [50]. Further theoretical and experimental investigations are obviously needed at this point. The effect of the particle shape on P should also be examined. 6. Concluding remarks This preliminary study shows that polarized radiations of the natural light scattered back out of the ocean can be extracted from satellite observations of ocean color at least over bright oceanic areas. The absolute values of the polarized remote sensing reflectance and of the degree of polarization are consistent with expectations, and with previous theoretical and experimental findings. Our confidence in our P retrieval from POLDER-2 is reinforced by the examination of the behavior between Rrs and P which is similar to in-situ observations made by Ivanof et al. [12] and Chami and McKee [15]. Comparisons with numerical simulations performed for the ocean-atmosphere system are also provided for the Rio de la Plata estuary. These radiative transfer simulations, performed for different physical characteristics of suspended marine particles, confirm that the scatter of the POLDER-2 data observed around the hyperbolic trend between Rrs and P may be explained by the variability of the bulk particulate assemblage composition. p g p The present study has been conducted for two idealized cases (no aerosols) to assess the feasibility of extracting water polarized radiations from remote sensing over bright areas. Prior to any generalization of these results two main studies should be conducted. First, the sensitivity of the effect of the presence of aerosols should be investigated. Atmospheric corrections over turbid waters, necessary to accurately assess the total remote sensing reflectance and a fortiori the polarized remote sensing reflectance, are still very challenging. Once good atmospheric corrections schemes are available for such waters, comparison with other bio-optical products derived from ocean color observations and related to the nature of the marine particles in suspension should be performed. Secondly, in situ measurements of the polarized remote sensing reflectance, which are still very rare, should be performed in biogeochemically contrasted waters. The behavior of P should then be compared to other bio- optical proxy usually used in situ to characterize the bulk particulate assemblage, such as the particulate backscattering to scattering ratio [51, 52]. #94370 - $15.00 USD Received 28 Mar 2008; revised 22 May 2008; accepted 12 Jun 2008; published 11 Aug 2008 (C) 2008 OSA 18 August 2008 / Vol. 16, No. 17 / OPTICS EXPRESS 12918 Acknowledgments This work was supported by Centre National d’Etude Spatiale (CNES) and Région Nord Pas- de-Calais through the doctoral fellowship of Lucile Duforet, and by Centre National de la Recherche Scientifique in the frame of the PNTS program. POLDER-2 ocean color data are provided by CNES. We thank Cyril Moulin, Jean-Marc Nicolas, and Jean-Luc Deuzé for their guidance during the POLDER-2 data processing. The authors thank two anonymous reviewers for their relevant comments.
https://openalex.org/W1942608895	https://seer.ufrgs.br/EmQuestao/article/download/50968/33977	Portuguese	null	Análise e indexação de imagens na rede Flickr	Em Questão	2,015	cc-by	7,793	Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira Doutorando; Universidade Federal da Paraíba; rraffael@gmail.com Luciane Paula Vital Doutoranda; Universidade Federal de Santa Catarina; lucianepv@yahoo.com.br Resumo: O artigo apresenta um breve panorama sobre a disseminação e democratização da internet colaborativa, e de como esse processo popularizou o uso da imagem em suporte digital. Faz uma breve análise sobre a rede social Flickr, identificando seus objetivos, suas ferramentas, e alguns dos estudos relativos à sua usabilidade no âmbito da Ciência da Informação. Delimita a abordagem para as imagens armazenadas na rede, propondo um estudo qualitativo em torno da indexação livre realizada por seus usuários. Aplica a metodologiaestabelecida em estudo anterior como a mais apropriada para indexação de imagens fotográficas em ambiente web, comparando os resultados obtidos com a indexação livre realizada pelos usuários. Palavras-chave: Imagem. Indexação de imagens. Flickr. Redes sociais. Indexação social. Indexação social. E-ISSN 1808-5245 E-ISSN 1808-5245 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q g g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015 http://dx.doi.org/10.19132/1808-5245212.7-30 \| 7 1 Introdução A década de 2001 a 2010 foi testemunha do surgimento de um novo conceito de uso da internet. A possibilidade de publicação e tratamento livre de informações por parte de seus usuários tornou-se a característica fundamental deste novo modelo, que, embora não seja um consenso, os estudiosos nomearam de Web 2.0. Em alguma medida, pode-se dizer que a criação das redes sociais foi uma consequência natural desse fato, e que a aceitação dessas por parte de uma grande parcela dos usuários da internet é um reflexo de como este modelo se estabeleceu, e tende a se aprimorar. Essas novas ferramentas disseminaram o conceito e democratizaram o uso de um suporte informacional bastante peculiar: a imagem, que migrou dos \| 7 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital álbuns de fotografias e das molduras de parede para o suporte digital, podendo agora ser acessada a qualquer instante, de qualquer lugar do planeta. [...] com as novas tecnologias de comunicação e informação uma nova fase inicia-se, e nesta, as imagens são disponibilizadas livremente pelos usuários na internet. Essa liberdade de publicação, naturalmente, impeliu os métodos de tratamento e recuperação para essa mesma ideia. Agora o usuário não só pode publicar as imagens como também categorizá-las. Esse boom de imagens disponibilizadas iniciou-se com o surgimento das redes sociais. A sociedade aderiu a esta nova forma de interagir, e as redes cresceram e popularizaram-se significativamente ao longo de poucos anos. À medida que as ferramentas destas redes se desenvolviam com o avanço dos seus servidores, as redes podiam armazenar cada vez mais informações. A publicação livre de imagens foi apenas um passo natural nesse desenvolvimento. (OLIVEIRA, 2011, p. 2). Partindo desse pressuposto, fica evidente que uma problemática tende a surgir com esse avanço, e diz respeito especificamente à maneira como estas imagens serão tratadas e recuperadas. Na literatura científica de Ciência da Informação ainda não existe um acordo sobre como as imagens devem ser tratadas, pois diversos fatores implicam nessa análise, incluindo o tipo de imagem (pinturas, fotografias, etc.) e o suporte no qual se encontra. Entretanto, alguns modelos bem construídos e eficientes podem ser encontrados, como o de Manini (2002) e Rodrigues (2007). Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Em Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 1 Introdução No trabalho intitulado Metodologias para indexação de imagens fotográficas em ambiente web (OLIVEIRA, 2011) são apresentados e analisados os elementos propostos e o comportamento de três das metodologias para indexação de imagens mais citadas na literatura científica da área da Ciência da Informação, visando identificar aquela que poderia ser considerada mais adequada à aplicação em imagens fotográficas coletadas em ambiente web. Este estudoanalisa, dentre outras, a metodologia proposta por Rodrigues (2007), na qual o autor revela que uma imagem não apenas mostra, mas também representa algo, que pode não necessariamente ter uma relação direta aos objetos apresentados. Devido a isso, entende-se que uma imagem terá dois níveis ou sentidos principais: o denotativo, que se refere “[...] àquilo que a imagem representa com ‘certa precisão’, no seu sentido real” (RODRIGUES, 2007, p.69), e o conotativo,referente àquilo “[...] que a imagem pode ‘interpretar’ em \| 8 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital um determinado contexto, em um sentido figurado e simbólico”.(RODRIGUES, 2007, p. 69). A partir dessa constatação, Rodrigues (2007) estabelece que devem ser considerados os seguintes elementos para uma análise apropriada: um determinado contexto, em um sentido figurado e simbólico”.(RODRIGUES, 2007, p. 69). A partir dessa constatação, Rodrigues (2007) estabelece que devem ser considerados os seguintes elementos para uma análise apropriada: a) descrição física, ou o “[...] formato e tamanho da imagem fotográfica, tipo de suporte, autor, transformações ocorridas a partir do original etc”. (RODRIGUES, 2007, p.75);igo de periódico NBR 6022/03; b) composição, ao considerar “[...] tipo de luz, nível de nitidez dos assuntos, ponto de vista do fotógrafo, profundidade de campo e hierarquia das figuras, enquadramento etc”.(RODRIGUES, 2007, p. 75); c) contexto arquivístico, ou os locais e fatos, históricos ou não, correspondentes àquela fotografia; d) sentidos denotativos e conotativos, correspondendo ao que imagem mostra e ao que ela representa, respectivamente; e) tematização, ao atribuir a imagem a um contexto diferente do que é mostrado, mas que possui relação direta aos elementos denotativos apresentados. Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 1 Introdução Após traçar um quadro comparativo observou-se que, para uma análise de imagens coletadas em ambiente web, a metodologia proposta por Rodrigues (2007) pode ser considerada a mais apropriada, já que enfatiza os elementos conotativos identificados numa imagem, considerando-se que uma imagem coletada na internet tem grande possibilidade de não apresentar informações técnicas referentes a esta. A partir das constatações obtidas neste estudo, buscaremos obter um breve panorama sobre a indexação realizada livremente pelos usuários de redes sociais por meio da aplicação prática da metodologia de Rodrigues (2007) e da interpretação dos dados obtidos. O banco de imagens escolhido para a pesquisa foi a rede Flickr, por se tratar de uma rede social amplamente utilizada e ter seu foco no compartilhamento de recursos visuais. Desde já delimitaremos o objeto de estudo apenas às imagens armazenadas pela rede Flickr, embora a rede atualmente permita também o compartilhamento de vídeos. \| 9 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 2 Flickr: uma breve análise Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital [...] que podem ser definidas como sistemas orgânicos baseados na atribuição livre e pessoal de [termos] à informações ou objetos visando à organização e recuperação. O neologismo folksonomia – formado pelas palavras, em inglês, folks (pessoas) e taxonomy (taxonomia) – foi cunhado [...] como forma de expressar contraposição às classificações do conhecimento tradicionais, elaboradas por especialistas e construídas baseando-se em arranjos hierárquicos. (GUEDES; DIAS, 2010, p.48). 2 Flickr: uma breve análise 2 Flickr: uma breve análise O Flickr é uma rede de compartilhamento de imagens hospedada na internet. O Flickr, segundo os próprios idealizadores,propõe novas formas para organizar fotografias, que poderão ser acessadas livremente por qualquer pessoa. Parte da solução é tornar colaborativo o processo de organizar fotos [...] No Flickr, é possível permitir que seus amigos, família e outros contatos organizem suas coisas - não apenas adicionem comentários, mas também notas e tags. [...] E, à medida que essas informações crescem como metadados, você poderá encontrar as coisas facilmente mais tarde, uma vez que toda essa informação pode ser buscada. (FLICKR, 2014c, documento eletrônico sem paginação). Embora seja considerado uma rede social, o Flickr permite que mesmo aqueles que não possuam cadastro na base tenham acesso às imagens disponibilizadas. No entanto, o usuário tem a liberdade de restringir o acesso a algumas de suas fotografias apenas àqueles usuários que lhes forem mais convenientes. O Flickr é um claro exemplo de ambienteque permite a utilização e o emprego daquilo que a literatura científica nomeou de folksonomias, [...] que podem ser definidas como sistemas orgânicos baseados na atribuição livre e pessoal de [termos] à informações ou objetos visando à organização e recuperação. O neologismo folksonomia – formado pelas palavras, em inglês, folks (pessoas) e taxonomy (taxonomia) – foi cunhado [...] como forma de expressar contraposição às classificações do conhecimento tradicionais, elaboradas por especialistas e construídas baseando-se em arranjos hierárquicos. (GUEDES; DIAS, 2010, p.48). No Flickr podemos identificar dois tipos de usuários: aqueles que possuem cadastro na base e, portanto, podem publicar fotos e se utilizar de todas as ferramentas que o Flickr oferece; e aqueles que não possuem qualquer vínculo com o sistema, apenas o utilizam para fazer pesquisas no conjunto de fotografias disponibilizadas para consulta pública. Ao primeiro tipo, chamaremos de usuário regular; ao segundo, usaremos a definição usuário- consultor. \| 10 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245 Apesar de ser descrito como um ambiente que utiliza folksonomias (HIDDERLEY; RAFFERTY, 2007), na prática o Flickr não permite que a indexação seja feita livremente pelo usuário-consultor, como acontece na maioria desses ambientes. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 As tags são como palavras-chave ou títulos que você adiciona a uma foto para facilitar encontrá-la posteriormente. Você pode criar uma tag para uma foto com frases como "Catarina caminhada trilha montanha Yosemite." Posteriormente, se você procurar por imagens de Catarina, bastará clicar nessa tag e obter todas as fotos marcadas dessa maneira. (FLICKR, 2014a, documento eletrônico sem paginação). m Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 i htt //d d i /10 19132/1808 5245212 7 30 \| 2 Flickr: uma breve análise Tags, ao contrário de termos de indexação (descritores); geralmente utilizados em unidades de informação, não possuem um mediador \| 11 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 que as controle. O Flickr diferencia-se de outros ambientes que utilizam folksonomias por estabelecer que apenas o usuário regular possua tal liberdade (embora alguns ambientes que empregam folksonomiastambém utilizem esse recurso), provavelmente como uma forma de minimizar tags ‘inúteis’. O Flickr também propõe uma lista de “hot tags”, ou “etiquetas quentes”, que consistem numa lista das tags mais utilizadas pelos usuários regulares. Essas são, possivelmente, estratégias desenvolvidas pelos administradores da base para aprimorar as formas de indexação, ou tagging, das imagens. que as controle. O Flickr diferencia-se de outros ambientes que utilizam folksonomias por estabelecer que apenas o usuário regular possua tal liberdade (embora alguns ambientes que empregam folksonomiastambém utilizem esse recurso), provavelmente como uma forma de minimizar tags ‘inúteis’. O Flickr também propõe uma lista de “hot tags”, ou “etiquetas quentes”, que consistem numa lista das tags mais utilizadas pelos usuários regulares. Essas são, possivelmente, estratégias desenvolvidas pelos administradores da base para aprimorar as formas de indexação, ou tagging, das imagens. Problemas no processo de indexação e as consequências na recuperação das imagens foram identificados por alguns autores. Rafferty e Hidderley (2007), numa pesquisa controlada, mostram como os índices de revocação podem variar imensamente de acordo com o termo atribuído, quando explicam que No exemplo mostrado, o termo WEDDING é considerado muito geral pelos autores, recuperando um número de imagens considerado absurdo. Já o termo IIJSSELMEER, segundo os autores, é específico demais para uma imagem disponibilizada numa base para uso público, recuperando apenas uma imagem. Analisar os resultados da recuperação, no entanto, não é suficiente para deduzir que a causa do problema seja a forma como os usuários indexam as imagens no Flickr. Percebemos, inclusive, que há um interesse por parte dos próprios administradores em educar os usuários a controlar os termos utilizados. 2 Flickr: uma breve análise Apenas o usuário regular pode fazê-lo e, no máximo, outros usuários regulares que receberam do proprietário das imagens uma permissão para tanto. Esses poderão indexar as imagens utilizando-se do recurso detagging (do inglês, etiquetagem), que consiste na atribuição de termos que representem os conteúdos informacionais disponibilizados na web. As tags são como palavras-chave ou títulos que você adiciona a uma foto para facilitar encontrá-la posteriormente. Você pode criar uma tag para uma foto com frases como "Catarina caminhada trilha montanha Yosemite." Posteriormente, se você procurar por imagens de Catarina, bastará clicar nessa tag e obter todas as fotos marcadas dessa maneira. (FLICKR, 2014a, documento eletrônico sem paginação). As tags são como palavras-chave ou títulos que você adiciona a uma foto para facilitar encontrá-la posteriormente. Você pode criar uma tag para uma foto com frases como "Catarina caminhada trilha montanha Yosemite." Posteriormente, se você procurar por imagens de Catarina, bastará clicar nessa tag e obter todas as fotos marcadas dessa maneira. (FLICKR, 2014a, documento eletrônico sem paginação). As tags atribuídas a determinado conteúdo servirão como uma referência conceitual aos usuários que terão acesso posterior àquela informação. No caso do Flickr, o usuário regular disponibiliza a imagem na base e atribui, a partir da sua interpretação, termos que representem a imagem. Em seguida, o usuário- consultor, ou outros usuários regulares, poderão pesquisar as imagens disponibilizadas através de palavras-chave e a base naturalmente utilizará as tags atribuídas a cada imagem como ferramenta de busca. Alguns autores preferem o termo indexação social para identificar esse processo. Cada imagem pode ter um máximo de 75 tagsatribuídas a ela. No entanto, o usuário regular tem, além das tags, outra forma de descrever uma imagem: o texto. Este pode conter, como sugere o próprio Flickr (2014a), a história da fotografia e/ou notas explicativas sobre esta. É importante ressaltar que o Flickr não se utiliza exclusivamente das tags atribuídas às imagens para fazer a recuperação, mas também de termos livres contidos nas descrições. Na hora da busca, o usuário pode escolher entre pesquisar apenas nas tags, ou por meio de termos livres, incluindo assim os textos. A […] tag […] “casamento” recuperou 795.280 imagens, quando utilizada no Flickr em 14 de fevereiro de 2006, sugerindo que este seria um exemplo de uma tag talvez ampla demais. A […] tag “Iijsselmeer” recuperou apenas uma imagem, e é um exemplo de uma tag que talvez seja específica demais para o propósito de pesquisa pública. (RAFFERTY; HIDDERLEY, 2007, p. 403, tradução nossa)1. 2 Flickr: uma breve análise É possível que, apesar de os índices de recuperação no Flickr não terem sido considerados satisfatórios pelos autores, os termos utilizados pelos usuários na atribuição de tags representem de forma efetiva os elementos apresentados na imagem. \| 12 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245 Partindo desse impasse, coletamos algumas imagens no Flickr para analisá- las com base na metodologia proposta por Rodrigues (2007), anteriormente considerada a mais adequada para o ambiente web dentre as metodologias para representação imagética encontradas, e comparar os resultados obtidos com as tags atribuídas pelos usuários. Assim, é possível perceber se a indexação de imagens que está sendo realizada por meio das ferramentas da Web 2.0 condiz qualitativamente com aquela indexação proposta pelos estudiosos da área. [...]constitui o menos rigoroso de todos os tipos de amostragem. Por isso mesmo é destituída de qualquer rigor estatístico. O pesquisador seleciona os elementos a que tem acesso, admitindo que estes possam, de alguma forma, representar o universo. Aplica-se este tipo de amostragem em estudos exploratórios ou qualitativos, onde não é requerido elevado nível de precisão. (GIL, 2008, p. 94, grifo nosso). Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 3 Procedimentos metodológicos Entendemos este estudo como sendo de caráter qualitativo, já que não buscamos uma coleta precisa de dados estatísticos, mas uma análise interpretativa, baseada essencialmente no que a literatura científica diz a respeito do tema. Através deste estudo, buscamos compreender um pouco mais sobre as modalidades de indexação imagética. O Flickr possui uma quantidade de fotos armazenadas que cresce de forma rápida e significativa. Dessa forma, percebemos de imediato que o Flickr constitui um universo de amplitude infinita. Sendo esta pesquisa de caráter qualitativo, recorremos a Gil (2008) para obter alguns esclarecimentos a esse respeito. O autor explica, dentre outras formas de coletar amostras, que a amostragem por acessibilidade ou por conveniência [...]constitui o menos rigoroso de todos os tipos de amostragem. Por isso mesmo é destituída de qualquer rigor estatístico. O pesquisador seleciona os elementos a que tem acesso, admitindo que estes possam, de alguma forma, representar o universo. Aplica-se este tipo de amostragem em estudos exploratórios ou qualitativos, onde não é requerido elevado nível de precisão. (GIL, 2008, p. 94, grifo nosso). [...]constitui o menos rigoroso de todos os tipos de amostragem. Por isso mesmo é destituída de qualquer rigor estatístico. O pesquisador seleciona os elementos a que tem acesso, admitindo que estes possam, de alguma forma, representar o universo. Aplica-se este tipo de amostragem em estudos exploratórios ou qualitativos, onde não é requerido elevado nível de precisão. (GIL, 2008, p. 94, grifo nosso). Dessa forma, buscamos tornar possível a análise da metodologia em ambiente web,mesmo admitindo a infinidade de imagens que podem ser encontradas nesse contexto.Devemos destacar, antes de explanar os \| 13 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital procedimentos, que, visando delimitar o campo de estudo, fica estabelecido que apenas as tags atribuídas na língua portuguesa serão consideradas na análise. Tags em quaisquer outras línguas serão desconsideradas, assim como aquelasque apresentem composição confusa devido a possíveis erros de grafia. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 3 Procedimentos metodológicos Em vista disso, foram realizados os seguintes procedimentos para a coleta das imagens utilizadas neste estudo: a) optamos por fazer a pesquisa como usuário consultor, uma vez que, desta forma, o cadastro na base não interfere significativamente na busca; b) na página inicial do Flickr, utilizamos os termos TAGS (para realizar a pesquisa inicial de imagens, por se tratar de um termo bastante amplo, de certa forma desprovido de qualquer conceituação, e, como já visto, muito utilizado na base); e BRASIL (para delimitar ao máximo as imagens recuperadas à imagens com tags em língua portuguesa). Dessa forma, as imagens foram recuperadas de forma aleatória no que diz respeito à sua temática; c) após a pesquisa inicial, a base dá a possibilidade de escolha entre a pesquisa em todos os textos atribuídos a uma imagem ou apenas em suas tags. Visando uma maior generalidade de imagens recuperadas, optamos por permanecer na busca realizada em todos os textos; d) tendo em vista uma maior aleatoriedade na coleta, foi selecionada uma imagem de cada página dos resultados de busca, sendo analisadas quatro imagens ao final; e) buscando uma conceituação diferenciada e equilibrada nas imagens coletadas, duas dessas foram selecionadas por conterem seres humanos, e as duas restantes por não conterem humanos representados. Coletados os dados, aplicamos a cada imagem a metodologia proposta por Rodrigues (2007) e, em seguida, comparamos os resultados com as tags atribuídas, visando analisar qualitativamente a indexação realizada pelos usuários. \| 14 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital 4 Resultados obtidos Utilizando os procedimentos metodológicos estabelecidos para a coleta e análise das imagens no Flickr, obtivemos as seguintes imagens: Figura 1 – Praia de Jurerê – Florianópolis, SC Fonte: Flickr(2014b). Figura 1 – Praia de Jurerê – Florianópolis, SC Figura 1 – Praia de Jurerê – Florianópolis, SC Fonte: Flickr(2014b). Na área reservada para o texto descritivo da imagem, o autor indica que esta imagem refere-se à Praia de Jurerê, localizada em Florianópolis, estado de Santa Catarina. As tagsatribuídas a esta imagem foram, em ordem de aparição: Quadro 1 – Tags atribuídas à Figura 1 por usuário da redeFlickr Rainbow top20travel Arco-íris A Plus Photo Jurerê The World Throgh a Lens Florianópolis Platinum Photo Santa Catarina Geo-tagged Brasil 100v+10f Brazil Novas Jurerê Fonte: elaborado pelos autores Aplicando a metodologia de Rodrigues (2007), chegamos à seguinte análise: \| 15 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 2 – Aplicação da metodologia de Rodrigues (2007) para a Figura 1 Fonte: elaborado pelos autores. DESCRIÇÃO FÍSICA COMPOSIÇÃO CONTEXTO ARQUIVÍSTICO --- --- Jurerê Florianópolis Santa Catarina SENTIDO DENOTATIVO SENTIDOS CONOTATIVOS TEMATIZAÇÃO Praia Arco-íris Chuva Tranquilidade Meteorologia Através da metodologia de Rodrigues (2007), atribuímos oito termos que poderiam recuperar essa imagem. No Contexto Arquivístico, os termos JURERÊ, FLORIANÓPOLIS e SANTA CATARINA referem-se à procedência da imagem, ou seja, ao local apresentado na fotografia, e, consequentemente, onde esta foi produzida. No Sentido Denotativo, os termos PRAIA, ARCO-ÍRIS e CHUVA representam os objetos identificados pelos autores, enquanto nos Sentidos Conotativos o termo TRANQUILIDADE expressa a sensação transmitidaaos autores no momento da observação. Entretanto, por se tratar de um elemento essencialmente subjetivo, outros termos podem ser atribuídos por observadores diferentes. Por fim, no campo Tematização, o termo METEOROLOGIA reproduz um enquadramento temático feito pelos autores diante dos elementos objetivos apresentados. Apesar desse enquadramento temático ser subjetivo, é mais fácil apontar os elementos que levaram a tal determinação, que, no caso da fotografia em questão, os elementos que levaram à atribuição das tags PRAIA e CHUVA. Entretanto, da mesma forma que os Sentidos Conotativos, a Tematização pode variar de um observador para outro. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos Como ficou claro nas tags associadas a essa imagem, alguns termos não correspondem à língua portuguesa, enquanto outros apresentam composição confusa e, portanto, deverão ser desconsiderados. Traçando um comparativo entre as tagsválidas e os termos adquiridos por meio da metodologia, temos: \| 16 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 3 – Comparação entre as tagsconsideradas válidas e os resultados obtidos na análise TAGS TERMOS Arco-íris Jurerê Jurerê Florianópolis Florianópolis Santa Catarina Santa Catarina Praia Brasil Arco-íris Novas Jurerê Chuva --- Tranquilidade --- Meteorologia Fonte: elaborado pelos autores Dentre as tags consideradas válidas para esta pesquisa, apenas quatro coincidem com os termos adquiridos através da metodologia. Termos considerados indispensáveis na indexação desta imagem, como PRAIA, não aparecem nas tags. A tag BRASIL, por sua vez, apesar de corresponder a um dos critérios de busca para a presente pesquisa e ser relevante para situar a localização do ambiente apresentado na ilustração, é excessivamente genérica para descrever, por si só, essa imagem, enquanto NOVAS JURERÊ parece ser uma indicação individual de localização para o próprio autor da fotografia, não podendo ser considerado relevante para descrevê-la. Avançando para a próxima página de busca, coletamos a seguinte imagem: \| 17 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Figura 2 – Maragogi, AL Fonte: Flickr (2014b). Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Figura 2 – Maragogi, AL Fonte: Flickr (2014b). Figura 2 – Maragogi, AL Fonte: Flickr (2014b). Nesta imagem é possível identificar um grupo de jovens observando o mar. Segundo as informações do autor da fotografia, ela foi tirada na Praia de Peroba, em Maragogi, Estado de Alagoas. Diversas tags referenciam ess imagem, como as listadas a seguir: \| 18 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 4 – Tags atribuídas à Figura 2por usuário da rede Flickr Quadro 4 – Tags atribuídas à Figura 2por usuário da rede Flickr Colors Miramar Caribe Color Boa viagem Brasileiro Maragogi Contraste O Caribe brasileiro Alagoas Cores Viagem Peroba Fortes Dicas Praia Vibrantes Ficar Beach Viva Salinas Red Paraíso Férias Yellow Paradise Piscinas Mar Geotagged Naturais Céu Beaches Maceió Azul Ondas Turismo Nuvens Rio Brasilviagem Pentax Lago Pousada Ist Vivid Hotéis D Praias Brasil azul 17mm Maragogi fotos Pousadas em Maragogi Fisheye Fotos de Maragogi Miramar Maragogi Brasil Praia de Peroba Praia de Maragogi Brazil Maragogi online Porto de Galinhas Postal Aventura Japaratinga Tamandaré Galés Catamarã Piscinas naturais de Maragogi, Alagoas Piscinas naturais de Maragogi Passeio Hotel Salinas Fotos Maragogi Referência no Turismo Fonte: elaborado pelos autores. Um olhar não muito atento é o suficiente para perceber que muitas palavras se repetem em tags diferentes; ainda, percebemos que palavras que deveriam formar uma única tag acabam tornando-se tags distintas, como, por exemplo, CARIBE e BRASILEIRO, seguidas da tag O CARIBE BRASILEIRO. Aplicando a metodologia de Rodrigues (2007) obtemos: \| 19 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 5–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 2 Fonte: elaborado pelos autores. Fonte: elaborado pelos os autores. 4 Resultados obtidos DESCRIÇÃO FÍSICA COMPOSIÇÃO CONTEXTO ARQUIVÍSTICO --- Azul Azul turquesa Peroba Maragogi Alagoas SENTIDO DENOTATIVO SENTIDOS CONOTATIVOS TEMATIZAÇÃO Praia Mar Céu azul Jovens Curiosidade Aventura Férias Turismo Pontos turísticos Na tabela comparativa entre as tags consideradas válidas e os termos obtidos teremos: Quadro 6–Comparaçãoentre as tags consideradas válidas e os resultados obtidos na análise TAGS TERMOS Maragogi Azul Alagoas Azul turquesa Peroba Peroba Praia Maragogi Mar Alagoas Céu Praia Azul Mar Nuvens Céu azul Contraste Jovens Cores Curiosidade Fortes Aventura Vibrantes Férias Viva Turismo Paraíso Pontos turísticos TAGS TERMOS Brasil --- Postal --- Ondas --- Rio --- Lago --- Praias --- Fotos --- \| 20 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245 Maragogi --- Fotos de Maragogi --- Praia --- Peroba --- Caribe --- Brasileiro --- O Caribe brasileiro --- Pousada --- Hotéis --- Viagem --- Dicas --- Ficar --- Salinas --- Boa viagem --- Brasilviagem --- Aventura --- Férias --- Piscinas --- Naturais --- Maceió --- Turismo --- Brasil azul --- Pousadas em Maragogi --- Praia de Maragogi --- Porto de Galinhas --- Japaratinga --- TAGS TERMOS Tamandaré --- Piscinas naturais de Maragogi, Alagoas --- Piscinas naturais de Maragogi --- Referência no Turismo --- Hotel Salinas --- Catamarã --- Galés --- Passeio --- Fotos Maragogi --- Fonte: elaborado pelos os autores. \| 21 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245 Com a metodologia de Rodrigues (2007) atribuímos 14 termos a essa imagem, os quais abrangem desde a sua composição, com a cor que notavelmente se destaca, até a sua tematização. Acreditamos ser possível, com esses termos, descrever eficientemente a fotografia e realizar uma recuperação eficiente. As tags consideradas válidas somam 56, dentre as quais oito coincidem com os termos atribuídos. 56poderia ser considerada uma quantidade de termos elevada, especialmente quando comparada com o resultado obtido através da metodologia de Rodrigues (2007). 4 Resultados obtidos Entretanto, é preciso levar em conta os aspectos conotativos e toda a gama de possibilidades de interpretação que a imagem pode despertar ao observador, de modo que, dependendo do contexto em que o documento seja analisado e representado, um número grande de termos pode ser considerado ideal.No entanto, é necessário estar atento a equívocos na interpretação que poderiam levar a conceituações distantes e passíveis de confusão com outras imagens.Algumas tagsatribuídas à imagem em questão podem exemplificar isso, como é o caso de PORTO DE GALINHAS ou TAMANDARÉ, que são praias localizadas no estado de Pernambuco. Ainda, a tag MACEIÓ refere-se à capital do estado de Alagoas, que não possui relação direta com a fotografia apresentada, o que poderia provocar confusão no momento da busca, visto que esse termo não corresponde corretamente à procedência da fotografia. Na terceira página de busca encontramos a seguinte imagem: Figura 3 – Ponte de madeira – Itapuã, RS Fonte: Flickr (2014b). Figura 3 – Ponte de madeira – Itapuã, RS Fonte: Flickr (2014b). \| 22 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Nesta imagem temos apenas um panorama, sem a presença de seres humanos. Segundo a descrição do autor, esta fotografia representa uma região da cidade de Itapuã, no Estado do Rio Grande do Sul. As tags atribuídas pelo autor foram: Quadro 7 – Tags atribuídas à Figura 3 por usuário da rede Flickr Itapuã PB Bem Flickr... Bem Brasil! RS Noiretblanc Gutemberg Ostemberg Interior Bianco Gutemberg Gaúcho Nero Porto Alegre Guaíba Blanco Mywinners Rio Grande do Sul Negro Bem Flickr... Bem Brasil! BW Brazil --- Fonte: elaborado pelos autores. Mais uma vez percebemos, em INTERIOR e GAÚCHO, palavras que provavelmente deveriam formar uma única tag, mas acabam tornando-se tagsdistintas. Aplicando a metodologia de Rodrigues (2007) obtemos: Quadro 8–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 3 Fonte: elaborado pelos autores. DESCRIÇÃO FÍSICA COMPOSIÇÃO CONTEXTO ARQUIVÍSTICO --- Preto e branco Itapuã Guaíba Rio Grande do Sul SENTIDO DENOTATIVO SENTIDOS CONOTATIVOS TEMATIZAÇÃO Ponte de madeira Lago Dique Imensidão Infinito Solidão Preservação ambiental Ecossistema Fonte: elaborado pelos autores. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 d i h //d d i / 0 9 32/ 808 2 2 2 30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos Adotamos 12 termos para representar esta imagem, que buscam englobar tanto o que a imagem mostra, o que ela transmiteaos autores, e os contextos em que pode estar inserida de acordo com a história do local. Itapuã é o nome de uma reserva florestal localizada no município de Guaíba, Rio Grande do Sul. Percebe-se, assim, que alguns destes termos só puderam ser atribuídos porque o autor informa através dos textos onde a fotografia foi tirada. A partir daí o indexador pode investigar o que mais pode ser relevante na sua conceituação, indo além do que a imagem revela. É importante destacar que Lancaster (2004) \| 23 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 prevê isso em seus estudos, quando aborda as questões relacionadas à indexação de imagens. No quadro comparativo teremos: Quadro 9–Comparaçãoentre as tags consideradas válidas e os resultados obtidos na análise Fonte: elaborado pelos autores. Fonte: elaborado pelos autores. TAGS TERMOS Itapuã Preto e branco Interior Itapuã Gaúcho Rio Grande do Sul Guaíba Ponte de madeira Rio Grande do Sul Lago Negro Dique Porto Alegre Imensidão --- Infinito --- Solidão --- Guaíba --- Preservação ambiental --- Ecossistema Dentre as sete tags relacionadas, apenas três coincidem com os termos atribuídos pela metodologia de Rodrigues. Identificamos umasituaçãosemelhante à encontrada na imagem analisada anteriormente, quando se adotou a tag PORTO ALEGRE, referente à capital do Estado do Rio Grande doSul, auma imagem que representa uma localidade situada no município de Guaíba, também no Rio Grande do Sul. No entanto, nesse caso é possível afirmar que, apesar de não haver uma relação direta entre a tag e a localidade apresentada, visto que são municípios diferentes, ainda assim seria admissível reconhecer uma relação relevante, já que o município de Guaíba fica localizado na Região Metropolitana de Porto Alegre. Até agora analisamos três imagens, sendo que em duas delas não aparecem seres humanos. Cumprindo os procedimentos metodológicos estabelecidos, a última imagem a ser analisada deve necessariamente conter um ser humano. Na quarta página dos resultados de busca encontramos a seguinte imagem: \| 24 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 4 Resultados obtidos 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Figura 4 – Chapada dos Veadeiros, GO Fonte: Flickr(2014b). Figura 4 – Chapada dos Veadeiros, GO Fonte: Flickr(2014b). Na imagem há apenas um rapaz com a sua câmera fotográfica em meio a uma imensa paisagem verde. Segundo os textos relacionados à fotografia, ela foi tirada na Chapada dos Veadeiros, localizada no estado de Goiás. As tags atribuídas a essa imagem, diferentemente das outras analisadas, não foram muitas, como relacionado abaixo: Quadro 10 – Tags atribuídas à Figura 4 por usuário da rede Flickr Chapada dos Veadeiros Eu Vale da Lua Borguetti Alto Paraíso Top-v1111 Me --- Fonte: elaborada pelos autores. Com a metodologia de Rodrigues (2007), obtemos os seguintes resultados: \| 25 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 11–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 4 Quadro 11–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 4 Fonte: os autores DESCRIÇÃO FÍSICA COMPOSIÇÃO CONTEXTO ARQUIVÍSTICO --- Verde Chapada dos Veadeiros Goiás SENTIDO DENOTATIVO SENTIDOS CONOTATIVOS TEMATIZAÇÃO Floresta Homem Aventura Pontos turísticos Ecossistema Na tabela comparativa, considerando os critérios determinados anteriormente para a validade das tags, teremos: Quadro 12–Comparaçãoentre as tagsconsideradas válidas e os resultados obtidos na análise TAGS TERMOS Vale da Lua Verde Alto Paraíso Chapada dos Veadeiros Eu Goiás Chapada dos Veadeiros Floresta --- Homem --- Aventura --- Liberdade --- Pontos turísticos --- Ecossistema Fonte: elaborado pelos autores. Quadro 12–Comparaçãoentre as tagsconsideradas válidas e os resultados obtidos na análise Quadro 12–Comparaçãoentre as tagsconsideradas válidas e os resultados obtidos na análise A Chapada dos Veadeiros é uma vasta região que abrange diversos municípios, dentre eles Cavalcante, Alto Paraíso de Goiás e Teresina de Goiás. Possivelmente por isso o autor atribuiu a tag ALTO PARAÍSO, referindo-se ao município em que, talvez, localizava-se no momento da fotografia. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Em Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos No entanto, sendo este termo específico demais, não foi considerado na sua análise. Da mesma forma, VALE DA LUA refere-se a uma região específica da Chapada dos Veadeiros, mas que não é possível identificar nem na descrição textual, nem na imagem em si. Assim, consideramos apenas o termo genérico CHAPADA DOS VEADEIROS. Nesse sentido, apenas uma tag coincidiu com os termos atribuídos na análise, que foram nove no total. Sobre a análise qualitativa de dados, podemos fazer uma breve comparação entre os termos e as tags, e verificar como os dados que obtivemos \| 26 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 nas análises se comportam. Todas as tagsatribuídas às quatro imagens analisadas e consideradas válidas somam o total de 83, enquanto os termos adquiridos por meio da metodologia de Rodrigues (2007) somam 43. Do total de tags, apenas 16 coincidem com o total de termos, o que resulta em aproximadamente 39,02% de compatibilidade entre as tags e os termos. Além disso, analisando as tabelas comparativas podemos perceber que, em geral, os termos que coincidiram foram apenas os que dizem respeito à localização geográfica do ponto em que a fotografia foi tirada, o que não garante uma riqueza na conceituação por meio das tags. Observamos neste estudo, também, que é essencial para o indexador identificar e considerar os diversos aspectos que uma imagem pode conter. Durante as análises, percebemos que as imagens coletadas possuem a maior parte das características propostas por Rodrigues (2007) como sendo as mais relevantes. No entanto, à exceção dos termos PASSEIO, FÉRIAS, PARAÍSO, TURISMO e REFERÊNCIAS NO TURISMO (Figura 2), que remetem à tematização da imagem, não encontramos nenhuma tag que correspondesse a elementos conotativos ou temáticos, e poucas as tags correspondentes a elementos denotativos. Entende-se, assim, que não há um equilíbrio entre os tipos de elementos descritos, sendo esses predominantemente relativos à sua composição e ao seu contexto arquivístico (RODRIGUES, 2007). Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 5 Considerações finais Apesar da baixa qualidade identificada na indexação realizada por meio das tags, o valor da indexação social não se perde. O fato de existirem ferramentas capazes de permitir esse tipo de atividade representa um avanço significativo na forma como os novos profissionais da informação devem encarar a representação da informação, seja ela imagética ou não. Alguns estudos inclusive já apontam para propostas de metodologias para a atribuição das tags (HIDDERLEY; RAFFERTY, 2007). Isso remonta, de alguma forma, aos estudos iniciais de Panofsky (1979) sobre a conceituação de imagens. Os conceitos iniciais evoluíram de tal forma que hoje somos capazes de analisar \| 27 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q g p g doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 minuciosamente imagens disponibilizadas em ambiente web, algo impensável algumas décadas atrás. É possível que, em alguma medida, estejamos no mesmo marco inicial em que se encontrava Panofsky (1979) quando iniciou o estudo sobre essas questões. Ao lado de grandes ferramentas da Web 2.0, como a Wikipedia, por exemplo, o Flickr faz cada vez mais parte do cotidiano de nossa sociedade. No entanto, as possibilidades advindas dessa evolução e a velocidade com que essa ocorreu e ainda ocorre requerem dos profissionais estudos frequentes para compreender a usabilidade e os potencializar os mecanismos dessas novas ferramentas. A indexação de imagens é uma atividade complexa e altamente passível a interpretações e questionamentos diferenciados. Devido a isso, foi necessário fazer um estudo aprofundado sobre o tema através da literatura existente, para entender realmente a problemática e buscar um embasamento científico para analisar qualitativamente as imagens. Buscamos, a partir desse estudo, criar uma reflexão em torno das problemáticas que estão surgindo no ambiente de tratamento, organização e disseminação da informação. Por mais que diversos estudos proponham formas de tratamento de imagens, é preciso estar atento à eficiência desses métodos em ambientes que não sejam restritos a bibliotecas e centros de documentação. FLICKR. Ajuda: tags. 2014a. Disponível em: FLICKR. Ajuda: tags. 2014a. Disponível em: <http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. <http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. PANOFSKY, E. Significado nas Artes Visuais. 2. ed. São Paulo: Perspectiva, 1979. RODRIGUES, Ricardo Crisafulli. Análise e tematização da imagem fotográfica. Ciência da Informação, Brasília, v. 36, n. 3, p. 67-76, set./dez. 2007. Referências Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 FLICKR. Ajuda: tags. 2014a. Disponível em: <http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. FLICKR. Busca de imagens.2014b. Disponível em: <http://www.flickr.com/search>. Acesso em: 15 out. 2014. FLICKR. Sobre o Flickr. 2014c. Disponível em: <http://www.flickr.com/about>. Acesso em: 15 out. 2014. GIL, Antônio Carlos. Métodos e técnicas de pesquisa social. 6. ed.São Paulo: Atlas, 2008. FLICKR. Ajuda: tags. 2014a. Disponível em: <http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. FLICKR. Busca de imagens.2014b. Disponível em: <http://www.flickr.com/search>. Acesso em: 15 out. 2014. FLICKR. Sobre o Flickr. 2014c. Disponível em: <http://www.flickr.com/about>. Acesso em: 15 out. 2014. GIL, Antônio Carlos. Métodos e técnicas de pesquisa social. 6. ed.São Paulo: Atlas, 2008. \| 28 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 GUEDES, Roger de Miranda; DIAS, Eduardo José Wense. Indexação social: abordagem conceitual. Revista ACB: Biblioteconomia em Santa Catarina, Florianópolis, v. 15, n. 1, p.39-53, jan./jun. 2010. HIDDERLEY, R.; RAFFERTY, P. Flickr and democratic indexing: dialogic approaches to indexing. Aslib Proceedings, London, v. 59, n. 4/5, p. 397-410, 2007. Disponível em: <www.emeraldinsight.com/0001-253X.htm>. Acesso em: 11 jan. 2012. LANCASTER, F. W. Indexação e resumos: teoria e prática. 2. ed. Brasília: Briquet de Lemos, 2004. MANINI, Miriam Paula. Análise documentária de fotografias: um referencial de leitura de imagens fotográficas Para fins documentários. São Paulo, 2002. Tese (Doutorado em Ciências da Comunicação) - Departamento de Biblioteconomia e Documentação, Escola de Comunicações e Artes, Universidade de São Paulo, São Paulo, 2002. OLIVEIRA, Rafael Alves de. Metodologias para indexação de imagens fotográficas em ambiente web. In: ENCONTRO DE ESTUDOS SOBRE TECNOLOGIA, CIENCIA E GESTÃO DA INFORMAÇÃO. Anais.... Recife: –Universidade Federal de Pernambuco, 2011. 1 CD-ROM. . OLIVEIRA, Rafael Alves de. Metodologias para indexação de imagens fotográficas em ambiente web. In: ENCONTRO DE ESTUDOS SOBRE TECNOLOGIA, CIENCIA E GESTÃO DA INFORMAÇÃO. Anais.... Recife: –Universidade Federal de Pernambuco, 2011. 1 CD-ROM. . PANOFSKY, E. Significado nas Artes Visuais. 2. ed. São Paulo: Perspectiva, 1979. Em Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Analysis and index of images on Flickr network Abstract: This article presents a brief overview on the dissemination and democratization of the collaborative web, and how this process popularized the use of the image in digital form. A brief analysis of the Flickr social network, identifying their goals, tools, and some studies on its usability in the context of information science. Sets out the approach to the images stored on the network, proposing a qualitative study about the free indexing performed by users. Applies the methodology established in a previous study as the most appropriate \| 29 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 for indexing images in a web environment, comparing the results obtained with free indexing performed by users. Keywords: Image. Indexing of images. Flickr. Social networks. Social indexing. Recebido: 16/10/2014 Aceito: 01/04/2015 Recebido: 16/10/2014 Aceito: 01/04/2015 1No original: “The [...] tag [...] “wedding” retrieved 795.280 images, when it was used on Flickr on 14 February 2006, which suggests that this an example of a tag that is perhaps too broad. The […] tag “Iijsselmeer” only retrieved one image and is an example of a tag that is perhaps too specific for public searching purposes”. 1No original: “The [...] tag [...] “wedding” retrieved 795.280 images, when it was used on Flickr on 14 February 2006, which suggests that this an example of a tag that is perhaps too broad. The […] tag “Iijsselmeer” only retrieved one image and is an example of a tag that is perhaps too specific for public searching purposes”. \| 30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30
https://openalex.org/W3100939362	http://journal.umg.ac.id/index.php/justi/article/download/2029/1247	Indonesian	null	PENINGKATAN EFISIENSI KERJA SERTA MEMINIMALISIR WASTE PADA DIVISI KAROSERI MENGGUNAKAN METODE LEAN MANUFACTURING (STUDI KASUS PT. SUMBER URIP SEJATI	JUSTI	2,020	cc-by	3,289	E-ISSN : XXXXX E-ISSN : XXXXX JUSTI (Jurnal Sistem Dan Teknik Industri) Nurul Habibi Ahmad(1), Pregiwati Pusporini(2) 1Mahasiswa Teknik Industri, Fakultas Teknik, Universitas Muhammadiya Gresik 2Dosen Teknik Industri, Fakultas Teknik, Universitas Muhammadiyah Gresik Jl. Sumatera No. 101 GKB-Gresik 61121. il ld bi bi @ il Nurul Habibi Ahmad(1), Pregiwati Pusporini(2) 1Mahasiswa Teknik Industri, Fakultas Teknik, Universitas Muhammadiya Gresik 2Dosen Teknik Industri, Fakultas Teknik, Universitas Muhammadiyah Gresik Jl. Sumatera No. 101 GKB-Gresik 61121. il ld t bi bi @ il e-mail : oldstar.biebie@gmail.com ABSTRAK PT. Sumber Urip Sejati adalah perusahaan yang bergerak dalam bidang perakitan karoseri trailer 40 feet 45 feet dan 60 feet , Kondisi yang terjadi saat ini adalah sering terjadi permasalahan yang diakibatkan karena proses perakitan terlalu lama yang tidak sesuai dengan komitmen awal dengan konsumen. Tahapan-tahapan yang diterapkan dalam penelitian ini adalah (1) penggambaran VSM current state dan future state (2) mengetahui value added dan non value added didalam proses perakitan (3) membuat skala prioritas terhadap 7 waste untuk meminimalisir pemborosan di proses perakitan trailer 40 feet. Berdasarkan hasil penelitian, didapatkan pemborosan yang paling sering terjadi adalah waiting (20,3%), motion (20,2%), Innappropriate processing (18,8%), dan inventory (18,1%). Mapping tools yang digunakan berdasarkan hasil konversi matrik adalah proses activity mapping (39,3%). Dari proses activity mapping dapat diketahui bahwa proporsi waktu inspection sebesar (6,76%). Setelah perbaikan didapatkan hasil proporsi inspeksi sebesar (4,94%). Untuk nilai value added ratio (VAR) sebelum perbaikan sebesar 88,52% setelah penerapan perbaikan nilai var menjadi (92,29%). Dalam arti perusahaan yang dulunya merakit trailer 40 feet selama 22 hari berkurang menjadi 18 hari sehingga waktu proses pengerjaan nya lebih cepat dari due date konsumen. Kata Kunci : Lean manufacturing, Value Stream Mapping (VSM), Value Stream Analysis Tools (VALSAT), Failure Measure effect analyze (FMEA) 1. Pendahuluan Karoseri (auto parts) atau body builder Karoseri (auto parts) atau body builder Truck. PT.Sumber Urip Sejati menawarkan berbagai macam jenis untuk pemasangan lantai / dek trailer juga pembuatan dump truck, load bak, box, mixer, concrate pump. Dengan berbagai jenis ukuran sesuai kebutuhan konsumen. 2.1 Lean Manufacturing Menurut James Womack dan Daniel Jones dalam Kusuma (2010) untuk menjadi lean manufacturing dibutuhkan cara berfikir yang berfokus untuk menjadikan produk mengalir melalui tahapan yang memberikan nilai tanpa adanya hambatan (one piece flow), sebuah pull system yang bersumber dari permintaan customer untuk mencapai interval proses yang pendek dan membudayakan melakukan continuous improvement dengan tekun. Berdasarkan pengamatan pada divisi karoseri di PT. Sumber Urip Sejati memungkinkan adanya pemborosan (waste) terjadi dikarenakan disetiap variable dari identifikasi awal (Tabel 1.1) kurang adanya penanganan khusus dari pihak perusahaan. Dan perusahaan juga tidak memberikan alternatif pada proses perakitan (assembling). Maka dari itu diperlukan penanganan untuk menentukan penyebab terjadinya pemborosan yang terjadi didalamnya. Salah satu pendekatan yang dapat digunakan untuk mengurangi pemborosan adalah lean manufacturing, dan Value Stream Mapping ( VALSAT). 1.3 Tujuan Penelitian Tujuan yang ingin dicapai dalam penelitian ini adalah Tujuan yang ingin dicapai dalam penelitian ini adalah 1. Mengidentifikasi waste yang terjadi pada proses perakitan (assembling). 2. Mengukur waste yang paling berpengaruh pada proses perakitan (assembling). 3. Mengidentifikasi faktor-faktor yang sering menjadi penyebab terjadinya waste pada perakitan (assembling). tersebut maka completion time proses assembling menjadi lebih lama. Untuk mendapatkan completion time yang lebih pendek maka perusahaan perlu mengurangi pemborosan (waste) yang ada. Dengan demikian completion time proses assembling pada divisi karoseri diharapkan menjadi lebih pendek dan produktifitas perusahaan meningkat. 4. Memberikan rekomendasi perbaikan beserta langkah- langkah yang perlu dilakukan untuk meminimalisir waste 1.1 Latar Belakang Pada era globalisasi ini, tingkat persaingan antar perusahaan manufaktur semakin ketat. Dengan meningkatnya persaingan antar perusahaan, pelanggan semakin tidak bersedia untuk menunggu mendapatkan pesanannya. Oleh karena itu, perusahaan yang mampu menghasilkan produk yang tepat waktu dan tepat jumlah merupakan perusahaan yang mampu bertahan dalam persaingan. PT. Sumber Urip Sejati merupakan perusahaan yang menganut “Make To Order” dan perlu untuk menyelesaikan tepat waktu pada proses assembling nya. Berdasarkan wawancara dengan pihak manajer divisi karoseri perusahaan belum mampu melaksanakan assembling nya tepat waktu sesuai dengan “due date” yang diberikan oleh konsumen. Karena pada proses karoseri terdapat permasalahan yang sering terjadi yaitu adanya pemborosan (waste) waktu tunggu (waiting), gerakan yang tidak berguna (unnecessary motion), persediaan yang tidak penting (unnecessary inventory) proses yang tidak tepat (inappropriate processing) dan menganggur (Iddle Time) yang dapat dilihat pada tabel 1.2. Dengan adanya Pemborosan (waste) Di dalam Industri rancang – bangun kendaraan, proses perencanaan hingga produksi massal dilakukan secara matang dan terukur. Kepresisian dengan tingkat efisiensi tinggi adalah hal yang mutlak saat ini, dengan tetap mempertahankan kualitas terbaiknya agar mampu bersaing secara global. Sehingga PT. Sumber Urip Sejati yang berdiri sejak tahun 1981 yang berpusat di Surabaya menawarkan kualifikasi tersebut, melalui lini produknya yang mencakup komponen suku cadang (autoparts) dan eksport import (sparepart) di industri kendaraan. Di dalam Divisi 28 E-ISSN : XXXXX JUSTI (Jurnal Sistem Dan Teknik Industri) Berdasarkan penjelasan latar belakang masalah diatas, maka permasalahan dalam penelitian ini adalah : 1. Bagaimana pendekatan lean manufacturing sebagai upaya meminimalkan waste dan meningkatkan efisiensi kerja pada divisi karoseri ? 1.3 Tujuan Penelitian 2.4 Seven Mapping tools Tujuh detail mapping tools yang memiliki kemampuan dan manfaat masing- masing untuk menemukan pemborosan (waste). Setiap alat mempunyai bobot low, medium dan high sesuai dengan ketentuan peringkatnya, dan sekaligus menunjukkan skor yang dapat mengindikasi besar kecilnya pengaruh pemborosan (waste influence) pada mapping yang dipilih. 2.2 Value Stream Mapping Value stream mapping adalah semua tindakan (value added dan non value added) saat ini diperlukan untuk menbawa produk melalui aliran utama untuk setiap produk: (1) aliran produksi dari aliran bahan baku sampai ke pelanggan dan (2) aliran design dari konsep sampai kepeluncuran (Rother & Shook, 2003). Dengan menggunakan metode Lean Manufacturing ini diharapkan PT. Sumber Urip Sejati dapat mengurangi pemborosan yang terjadi sehingga dapat meningkatkan efisiensi kerja dan memenuhi due date yang diberikan oleh konsumen. 2.3 Value Stream Analysis Tools (Valsat) Hines & Rich (1997) mengembangkan tool yaitu VALSAT 2.3 Value Stream Analysis Tools (Valsat) Hines & Rich (1997) mengembangkan tool yaitu VALSAT 1.2 Rumusan Masalah 1.2 Rumusan Masalah 29 E-ISSN : XXXXX JUSTI (Jurnal Sistem Dan Teknik Industri) 4. Hasil Penelitian Dan Pembahasan 4.1 Hasil Identifikasi pemborosan (waste) harapanya adalah untuk mempermudah pemahaman terhadap value stream yang ada dan mempermudah untuk membuat perbaikan yang berhubungan dengan pemborosan (waste) yang terdapat didalam value stream. 2.6 FMEA (Failure Mode Effect Analysis) FMEA merupakan suatu metode yang digunakan untuk mengidentifikasi dan memberikan prioritas kegagalan potensial yang terjadi pada sebuah proses atau produk (Kmenta, 2000). Process activity mapping (PAM) tool digunakan untuk memetakan setiap 2.5 Kegiatan-Kegiatan Value added Dan Non Value Added 4. Hasil Penelitian Dan Pembahasan 4. Hasil Penelitian Dan Pembahasan 4.1 Hasil Identifikasi pemborosan (waste) Proses transformasi bahan baku menjadi barang jadi adalah tujuan dari setiap perusahaan manufacturing (Davy, 2009). Proses yang membuat bahwa transformasi yang mungkin adalah hasil dari dua kegiatan yang berbeda, kegiatan yang memberi nilai tambah dan kegiatan yang tidak memberi nilai tambah. Berdasarkan hasil identifikasi pemborosan menggunakan kuisioner, sesuai hasil yang didapatkan maka jenis pemborosan yang paling dominan adalah waiting, inventory, innapropriate processing, dan unnecessary motion processing, dan unnecessary motion 4.2 VALSAT Dari hasil konversi VALSAT didapatkan Tools yang dominan untuk mengidentifikasi waste yang terjadi adalah Process Activity Mapping (39,3%) , Supply Chain Respone Matrix (20,2 %) dan Quality Filler Mapping (13,1 %), Demand Amplification mapping (12,4%), Decision point analysis (8,2%), Product variety funnel (5,1%), Phisycal structure (1,7%). Keterangan tentang pemilihan Tool : Process activity mapping (PAM) tool digunakan untuk memetakan setiap p g, y 4 2 VALSAT p g, y 4.2 VALSAT 4.2 VALSAT Dari hasil konversi VALSAT didapatkan Tools yang dominan untuk mengidentifikasi waste yang terjadi adalah Process Activity Mapping (39,3%) , Supply Chain Respone Matrix (20,2 %) dan Quality Filler Mapping (13,1 %), Demand Amplification mapping (12,4%), Decision point analysis (8,2%), Product variety funnel (5,1%), Phisycal structure (1,7%). Process activity mapping (PAM) tool digunakan untuk memetakan setiap Process activity mapping (PAM) tool digunakan untuk memetakan setiap 30 E-ISSN : XXXXX JUSTI (Jurnal Sistem Dan Teknik Industri) aktivitas didalam setiap proses perakitan . PAM sangat berguna untuk mengidentifikasi VA,NVA, & NNVA. Sedangkan SCRM (Supply chain respone matrix) digunakan untuk mendeskripsikan tentang kondisi lead time untuk setiap proses dan jumlah persediaan. Di PT. Sumber Urip Sejati ini persediaan di atur oleh pihak divisi lain. Sehingga divisi karoseri hanya akan merakit design layout yang sudah dipesan oleh konsumen. Tool Quality Filler Mapping (QFM) digunakan untuk mengevaluasi waste defect kurang cocok digunakan, dikarenakan waste defect yang kecil dan kebanyakan dari defect tersebut berupa reject. proporsi 6,76% dan ternyata terdapat delay dengan proporsi 2,45%. proporsi 6,76% dan ternyata terdapat delay dengan proporsi 2,45%. 4.4 Identifikasi Faktor- Faktor Pemborosan dengan 5 whys 4.4 Identifikasi Faktor- Faktor Pemborosan dengan 5 whys Pada bagian ini akan diidentifikasi penyebab terjadinya waste seperti, inventory, motion , waiting dan inappropriate processing sesuai hasil observasi, wawancara dan kuisioner terlihat pada tabel 4.10 Dengan mengidentifikasi seluruh waste dengan 5 whys 4.5 Identifikasi kriteria-kriteria waste 5. Analisa Dan Pembahasan 5. Analisa Dan Pembahasan 5. Analisa Dan Pembahasan 5.1 Analisa awal proses perakitan trailer dengan Value Stream Mapping 5.1 Analisa awal proses perakitan trailer dengan Value Stream Mapping 5.1 Analisa awal proses perakitan trailer dengan Value Stream Mapping Menurut metode VSM aliran fisik dan aliran informasi yang telah dibuat , dapat di identifikasi Menurut metode VSM aliran fisik dan aliran informasi yang telah dibuat , dapat di identifikasi Menurut metode VSM aliran fisik dan aliran informasi yang telah dibuat , dapat di identifikasi p 4.3 Process Activity Mapping (PAM) Pembuatan tools ini memerlukan pengamatan secara langsung terhadap proses, aktivitas tiap proses, jarak, waktu serta tenaga kerja yang terlibat. Hasilnya di inputkan kedalam tabel dimana setiap aktivitas akan dikelompokkan kedalam lima jenis aktivitas, yaitu : operasi, transportasi, inspeksi, delay dan storage. Dari tabel ini didapatkan bahwa aktivitas value added merupakan sebuah operasi, maka akan didapatkan value added activity. Operasi adalah aktivitas yang bernilai tambah (VA). Sedangkan inspeksi transportasi dan storage adalah aktivitas yang idak bernilai tambah tapi diperlukan (NNVA). Delay adalah aktivitas yang tidak bernilai tambah (NVA). 4.5 Identifikasi kriteria-kriteria waste dengan FMEA FMEA merupakan suatu metode yang digunakan untuk mengidentifikasi dan memberikan prioritas kegagalan potensial yang terjadi pada sebuah proses atau produk (Kmenta, 2000). Dalam FMEA ada 3 faktor yang dinilai terkait dengan resiko yang secara standar ditetapkan sebagai faktor yang akan dikalikan untuk mendapatkan nilai Risk Priority Number (RPN), ketiga faktor tersebut adalah : Dari tabel 4.9 dapat diketahui bahwa pada proses perakitan trailer 40 feet, proporsi waktu operation menghabiskan waktu yang paling banyak sebesar 8.640 menit atau setara 144 jam (88,52%) dari konsumsi waktu secara keseluruhan. Selanjutnya proporsi waktu terbesar kedua adalah aktivitas inspection dengan 1. Severity Uraian kriteria dari rating severity yang diperoleh dari rating standart six sigma yang telah disesuaikan dengan kondisi perusahaan. Pendefinisian untuk setiap ratting severity dapat dilihat pada halaman 28. 31 E-ISSN : XXXXX JUSTI (Jurnal Sistem Dan Teknik Industri) sebelumnya maka dapat diusulkan rekomendasi perbaikan sebelumnya maka dapat diusulkan rekomendasi perbaikan 2. Occurance Uraian kriteria dari rating severity yang diperoleh dari rating standart six sigma yang telah disesuaikan dengan kondisi perusahaan. Pendefinisian untuk setiap ratting occurance dapat dilihat pada halaman 29. sebelumnya maka dapat diusulkan rekomendasi perbaikan 3. Detection. Uraian kriteria dari rating severity yang diperoleh dari rating standart six sigma yang telah disesuaikan dengan kondisi perusahaan. Pendefinisian untuk setiap ratting detection dapat dilihat pada halaman 30. 3. Detection. Uraian kriteria dari rating severity yang diperoleh dari rating standart six sigma yang telah disesuaikan dengan kondisi perusahaan. Pendefinisian untuk setiap ratting detection dapat dilihat pada halaman 30. Nilai ranking yang muncul dalam Severity, Occurance dan Detection pada tabel FMEA berikut adalah hasil rata-rata dari pembobotan yang telah dilakukan oleh 2 responden diatas yang didapatkan dari hasil penyebaran kuisioner. 3. Detection. Uraian kriteria dari rating severity yang diperoleh dari rating standart six sigma yang telah disesuaikan dengan kondisi perusahaan. Pendefinisian untuk setiap ratting detection dapat dilihat pada halaman 30. 4.3 Process Activity Mapping (PAM) Nilai ranking yang muncul dalam Severity, Occurance dan Detection pada tabel FMEA berikut adalah hasil rata-rata dari pembobotan yang telah dilakukan oleh 2 responden diatas yang didapatkan dari hasil penyebaran kuisioner. 4.6 Risk Priority Number (RPN) 4.6 Risk Priority Number (RPN) Risk Priority Number (RPN) merupakan hasil perkalian dari Severity, Occurance dan Detection. Berdasarkan nilai kritis RPN dan atas persetujuan perusahaan maka diperoleh 3 kerusakan kritis. Dengan nilai RPN dari keempat kerusakan tersebut berada di atas 135 yang merupakan nilai paling kritis RPN. permasalahan yang terjadi dalam proses perakitan trailer 40 feet pada PT. Sumber Urip Sejati permasalahan tersebut antara lain : p g 4.6 Rekomendasi usulan perbaikan dari hasil FMEA Dimulai karna adanya penumpukan inventory pada area perakitan berupa tabung angin, spare part velg dan ban sehingga menghambat gerakan yang mempengaruhi kinerja kerja karyawan (unnecessary motion). Pemborosan sebenarnya terjadi karena banyaknya material yang menumpuk di sisi area kerja dikarenakan gudang spare part selalu penuh dan sisa dari gudang spare part di tumpuk di area sebelah gudang 4.6 Rekomendasi usulan perbaikan dari hasil FMEA Proses perakitan trailer 40 feet terdiri dari beberapa aktifitas yaitu rakit rangka body, rakit lantai, pengecatan lantai dan rangka, pemasangan spare part dan acessoris trailer. Pada proses perakitan trailer tersebut muncul waste yang dominan dari 7 waste yang ada yaitu waiting inventory motion dan innappriate processing. Berdasarkan analisis hasil FMEA yang sudah dilakukan pada tahap Kemudian ada proses yang tidak bernilai tambah dan harus dijalani seperti proses pengambilan bahan baku berupa besi mentah wf, pengambilan material (spare part), pembersihan sisa sisa plat hasil potongan dalam proses perakitan rangka body dan lantai proses terserbut akan mengakibatkan para pekerja menunggu 32 E-ISSN : XXXXX JUSTI (Jurnal Sistem Dan Teknik Industri) 2. Non-value added activity (waiting) sehingga waktu yg menunggu akan dibuat istirahat bagi para pekerja (small stop) Kurang lengkapnya peralatan peralatan penunjang bagi para pekerja (compressor, crane, mesin bubut, dll) yang mengakibatkan para pekerja akan menggunakan alat manual dan membutuhkan waktu extra ketika menggunakan nya (inappropriate processing). serta di dalam ruang lingkup perakitan, belum adanya atap yang mengakibatkan pekerja akan kepanasan (dehidrasi) dan kalau hujan pekerja akan berhenti sejenak. 2. Non-value added activity y Jenis pemborosan pada Non value added activity dari value stream mapping direkap dengan bentuk tabel agar mudah untuk dianalisis. Jenis pemborosan pada non value added ativity p p y 5.3 Analisis Process Activity Mapping (Future State) Future State Mapping 5.2 Analisis future state mapping value added activity dan non added value activity value added activity Jenis pemborosan pada value added activity dari value stream mapping direkap dengan bentuk tabel agar mudah untuk analisis. Jenis pemborosan pada value added activity p p y p p y 5.3 Analisis Process Activity Mapping (Future State) Berdasarkan hasil pengolahan data dalam tahap sebelumnya maka dapat digambarkan future process activity mapping. Dalam penggunaanya alat ini sering digunakan oleh beberapa ahli teknik industry untuk memetakan seluruh aktivitas secara detail untuk mengeliminasi waste, ketidak konsistenan, dan keirasionalan di area kerja sehingga dapat meningkatkan efisiensi kinerja melalui peningkatan 33 E-ISSN : XXXXX JUSTI (Jurnal Sistem Dan Teknik Industri) Berdasarkan pengolahan data dan analisa dalam penelitian ini, maka didapatkan kesimpulan yaitu : kualitas, mempercepat proses serta mereduksi biaya. Berdasarkan pengolahan data dan analisa dalam penelitian ini, maka didapatkan kesimpulan yaitu : Process activity mapping memberikan sebuah deskripsi tentang aliran fisik dan informasi, waktu yang diperlukan untuk setiap aktivitas jarak yang ditempuh dan pengukuran inventory dalam setiap tahap produksi. Kemudahan dalam 1. Berdasarkan hasil penelitian, didapatkan jenis pemborosan yang paling sering terjadi adalah waiting, Motion, Innappropriate processing, Inventory, Transportation, Defect, over production. 1. Berdasarkan hasil penelitian, didapatkan jenis pemborosan yang paling sering terjadi adalah waiting, Motion, Innappropriate processing, Inventory, Transportation, Defect, over production. 2. Dari hasil identifikasi berdasarkan perhitungan rata-rata urutan keseringan waste yang terjadi pada proses perakitan trailer 40 feet adalah waiting (20,3%), Motion (20,2%), Innappropriate processing (18,8%), Inventory (18,1%), mengidentifikasi sebuah aktivitas dibagi menjadi lima golongan yaitu operasi, transportasi, inventory, inspeksi dan delay. Operasi adalah aktivitas yang bernilai tambah (VA). Sedangkan inspeksi dan transportasi berjenis penting tapi tidak bernilai tambah (NNVA). Kemudian delay adalah aktivitas yang tidak bernilai tambah (NVA) yang sebaiknya dihindari untuk meningkatkan efisiensi. ● Waste waiting terjadi karena sering nya menunggu material datang, dan menunggu proses inspeksi selesai. ● Waste motion terjadi karena kondisi ruang kerja perusahaan yang sempit dan belum adanya atap g 5.4 Analisa perbandingan future state proses activity mapping dengan current state proses activity mapping ● Waste Inappropriate processing terjadi karena proses yang tidak efisien dan efektif ● Waste Inventory terjadi karena banyak nya plat plat sisa yang tidak terpakai serta banyaknya material bahan baku disekitar area kerja. ● Mapping tools yang akan digunakan berdasarkan hasil konveksi skor kuisioner kedalam matrik VALSAT adalah proses activity mapping (39,3%). Future State Mapping ● Value Added Ratio (VAR) sebelum perbaikan mempunyai prosentase nilai sebesar 88,52% sedangkan penerapan perbaikan nilai VAR menjadi 92,29% ● Dari penggunaan mapping tools, process activity mapping dapat diketahui bahwa prosentase aktivitas inspection adalah (6,76%) memiliki proporsi waktu terbesar kedua, dimana aktifitas ini termasuk dalam aktifitas necessary non value added. Setelah dilakukan perbaikan aktifitas inspection maka hasil dari nilai prosentasenya adalah (4,94%). 5.5 Analisis perbandingan value stream mapping (VSM) current state dengan future state pada proses perakitan trailer 40 feet VSM perbaikan ini digunakan untuk menggambarkan aliran nilai sistem perakitan trailer 40 feet setelah dilakukan perbaikan. Berdasarkan gambar 5.2 future state mapping, dapat diketahui bahwa perusahaan mampu mengurangi lead time proses perakitan trailer 40 feet sebesar 1.850 menit sehingga perusahaan mampu menyelesaikan perakitan trailer 40 feet selama 18 hari yang sebelumnya 22 hari 6. Kesimpulan Dan Saran 3. Berdasarkan analisis FMEA maka didapatkan Faktor faktor yang menjadi penyebab terjadi nya waste yaitu ● kurang nya peralatan yang menghambat kinerja karyawan contoh crane dan compressor pompa ban ● menunggu keputusan hasil inspeksi pada setiap proses perakitan 34 JUSTI (Jurnal Sistem Dan Teknik Industri) E-ISSN : XXXXX Manufacturing Operation and Supply Chain Management: Lean Approach, David Taylor and David Brunt. (editor). Thomas Learning. London. ● menunggu pengambilan material yang terlalu lama ● menunggu pengambilan material yang terlalu lama 4. 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https://openalex.org/W2121770040	https://europepmc.org/articles/pmc1524796?pdf=render	English	null	CCR3, CCR5, CCR8 and CXCR3 expression in memory T helper cells from allergic rhinitis patients, asymptomatically sensitized and healthy individuals	Clinical and molecular allergy	2,006	cc-by	4,877	Mille Holse, Kristian Assing and Lars K Poulsen* Address: Laboratory for Medical Allergology 7542, National University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Email: Mille Holse - mholse@yahoo.dk; Kristian Assing - kristian.assing@rh.hosp.dk; Lars K Poulsen* - lkpallgy@inet.uni2.dk * Corresponding author Received: 29 November 2005 Accepted: 19 April 2006 Clinical and Molecular Allergy 2006, 4:6 doi:10.1186/1476-7961-4-6 This article is available from: http://www.clinicalmolecularallergy.com/content/4/1/6 © 2006 Holse et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Chemokine receptors have been suggested to be preferentially expressed on CD4+ T cells with CCR3 and CCR8 linked to the T helper (Th) 2 subset and CCR5 and CXCR3 to the Th1 subset, however this remains controversial. Objective: Our aim was to compare the CCR3, CCR5, CCR8 and CXCR3 expression in memory Th cells from allergic, asymptomatically sensitized and healthy individuals. Methods: Peripheral blood mononuclear cells from 8 pollen allergic rhinitis patients, 10 asymptomatically sensitized and 10 healthy individuals were stimulated for 7 days with allergen or tetanus toxoid. CCR3, CCR5, CCR8, CXCR3, CD4 and CD45RO were detected by flow cytometry. Results: No differences in chemokine receptor expression were observed between the three groups on day 0, and seven days of unstimulated culture did not change the expression. Both antigenic stimuli increased the chemokine receptor expression, tetanus toxoid being the most potent. No differences in percentage chemokine receptor positive memory Th cells were observed between the three groups on day 7. Only a change in MFI for CCR5 was significantly different between the three groups after allergen stimulation of the Th cells. Conclusion: We conclude that even though allergen and antigen induced increased chemokine receptor expression, no differences in profiles were identified in memory Th cells from patient groups with different atopic status. BioMed Central Clinical and Molecular Allergy Open Access Research CCR3, CCR5, CCR8 and CXCR3 expression in memory T helper cells from allergic rhinitis patients, asymptomatically sensitized and healthy individuals Mille Holse, Kristian Assing and Lars K Poulsen* Address: Laboratory for Medical Allergology 7542, National University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Email: Mille Holse - mholse@yahoo.dk; Kristian Assing - kristian.assing@rh.hosp.dk; Lars K Poulsen* - lkpallgy@inet.uni2.dk * Corresponding author Published: 19 April 2006 Clinical and Molecular Allergy 2006, 4:6 doi:10.1186/1476-7961-4-6 Received: 29 November 2005 Accepted: 19 April 2006 This article is available from: http://www.clinicalmolecularallergy.com/content/4/1/6 © 2006 Holse et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central Clinical and Molecular Allergy Open Access CCR3, CCR5, CCR8 and CXCR3 expression in memory T helper cells from allergic rhinitis patients, asymptomatically sensitized and healthy individuals Mill H l K i i A i d L K P l * y Mille Holse, Kristian Assing and Lars K Poulsen* Introduction Allergic symptoms were reported in diary cards during the relevant pollen season. Symptoms were considered as pollen allergy when lasting > 7 days or when symptoms were repeatedly elicited when pollen counts exceeded a certain (individual) level [2]. The skin prick test was performed according to the guidelines of European Academy of Allergy and Clinical Immunology [18]. n = 10 for the healthy controls, n = 10 for the asymptomatically sensitized individuals and n = 8 for the allergic individuals. The chemokines and their receptors play a pivotal role in leukocyte migration and chemotaxis. It is still controver- sial whether these receptors can function as phenotypic markers on certain cell subsets. CCR3 and CCR8 have been suggested as Th2 markers whereas CXCR3 is men- tioned in the literature as a Th1 marker [4-6]. The CCR3 ligand CCL11/eotaxin is upregulated in nasal mucosa of allergic rhinitis patients during the pollen season [7]. CCL1/I-309, which is the only CCR8 ligand, is upregu- lated in patients with atopic dermatitis [8] and IL-12 inhibit its production [9]. Also, CCL1/I-309 is released by mast cells in response to IgE cross-linking [10] indicating a role in allergic inflammation. On the contrary, the IFN- γ-inducible CXCR3 ligands and some CCR5 ligands are increased in autoimmune diseases [11-14]. However, other findings show that no correlation exists between Th1/Th2 cytokine profile and chemokine receptor expres- sion on a single cell level [15] and also suggest that the chemokine receptor profile can be changed without a con- comitant change in cytokine profile [16] questioning the use of chemokine receptors as markers for T cell subsets. allergic and 3 grass pollen allergic volunteers with sea- sonal hay fever symptoms were examined during the birch or grass pollen season respectively. Skin prick test (Solu- prick, ALK-Abello, Hørsholm, Copenhagen), histamine release [17] and specific IgE against birch and grass pollen using the CAP-system (Pharmacia, Uppsala, Sweden) were determined for all volunteers (Table 1). The skin prick test was performed according to the guidelines of European Academy of Allergy and Clinical Immunology [18]. Three of the allergic patients had allergic asthma. The allergic subjects received no corticosteroid treatment for three months prior to the study. The asymptomatically sensitized and healthy control subjects took no hay fever medicine. All subjects came from the area of greater Copenhagen (Storkøbenhavn). Cell stimulation i h l bl d Peripheral blood mononuclear cells (PBMCs) were iso- lated from whole blood by gradient centrifugation on Lymphoprep (Nycomed, Roskilde, Denmark). The PBMCs were cultured (3 × 106) in 6 well plates with anti- gen in 6 ml of low endotoxin RPMI1640 medium con- taining 10% heat-inactivated autologous serum, 25 mM HEPES, 2 mM L-glutamine, 50 µM β-mercaptoethanol, 100 U/ml streptomycin/penicillin for 7 days in the pres- ence of either 15 µg/ml birch or grass allergen (ALK- Abello, Hørsholm, Copenhagen), 10 µg/ml Tetanus tox- oid (TTx) (Statens Serum Institut, Copenhagen, Den- mark) or no antigen as a control. On day 7, the cells were harvested and used for flow cytometric analysis. The lipopolysaccharide level in both allergen extracts was < 7 EU/mg and after 24 hours of stimulation of PBMCs from As the chemokine receptor profile determines the migra- tory patterns of leukocytes, we wanted to compare this profile with respect to CCR3, CCR5, CCR8 and CXCR3 in memory Th cells from allergic, asymptomatically sensi- tized and healthy individuals to obtain knowledge about their migratory potential and any differences in expres- sion patterns that might exist between these three groups. Introduction The study was approved by the local Ethical Committee and the clinical features of the patients are described in detail elsewhere [19]. Introduction some individuals seem to exhibit an IgE positive pheno- type without having any allergic symptoms. These indi- viduals have been described in the literature as asymptomatically sensitized and are phenotypically consid- ered to be a group between the allergic and the healthy individuals with an increased risk of developing allergy [2,3]. The prevalence of allergy is increasing in the westernized part of the world with estimates suggesting that 20–30% of the population is affected [1]. However, unlike the reac- tion of most IgE-sensitized individuals who upon re-expo- sure to the allergen develop symptoms due to activation and release of mediators from various immune cells, Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) Clinical and Molecular Allergy 2006, 4:6 http://www.clinicalmolecularallergy.com/content/4/1/6 Table 1: Patient characterization. Birch Grass Patients Age (range) Sex Male/Female Symptoms Spt IgE class Median(range) HR class Median(range) Symptoms Spt IgE class Median(range) HR class Median(range) Healthy 25 (22–43) 3/7 0/10 0/10 0 (0) 0 (0) 0/10 0/10 0 (0) 0 (0) AS Birch 25 (24–27) 1/4 0/5 5/5 0 (0–2) 2 (0–3) 0/5 0/5 0 (0) 0 (0) AS Grass 25 (22–31) 1/4 0/5 0/5 0 (0) 0 (0) 0/5 5/5 0 (0–2) 0 (0–3) Allergic Birch 27 (25–43) 4/1 5/5 5/5 3 (2–4) 3 (2–3) 2/5 2/5 0 (0–4) 2 (0–3) Allergic Grass 26 (24–41) 3/0 1/3 1/3 0 (0–3) 0 (0–3) 3/3 3/3 4 (2–4) 3 (0–3) AS: asymptomatically sensitized. Spt: skin prick test. HR: histamine release Skin prick tests (performed in duplicate) were considered positive when mean wheal diameter >3 mm. Allergic symptoms were reported in diary cards during the relevant pollen season. Symptoms were considered as pollen allergy when lasting > 7 days or when symptoms were repeatedly elicited when pollen counts exceeded a certain (individual) level [2]. The skin prick test was performed according to the guidelines of European Academy of Allergy and Clinical Immunology [18]. n = 10 for the healthy controls, n = 10 for the asymptomatically sensitized individuals and n = 8 for the allergic individuals. Table 1: Patient characterization. AS: asymptomatically sensitized. Spt: skin prick test. HR: histamine release Sk k ( f d d l ) d d h AS: asymptomatically sensitized. Spt: skin prick test. HR: histamine release Skin prick tests (performed in duplicate) were considered positive when mean wheal diameter >3 mm. Results Day 0 Immediate after isolation of the PBMCs, the cells were subjected to flow cytometric analysis. No significant dif- ferences in the percentage of CCR3+, CCR5+, CCR8+ and CXCR3+ memory Th cells from allergic, asymptomatically sensitized and healthy individuals were observed (Figure 1). Likewise, no differences in MFI were observed between the three donor groups (results not shown). healthy individuals no detectable amounts of TNF-α were observed. healthy individuals no detectable amounts of TNF-α were observed. Effect of stimulation No significant differences in chemokine receptors (neither expressed as the percentage of positive cells nor as MFI) were observed between day 0 and the cells having been kept in antigen-free medium for 7 days as controls. Thus the medium alone and the experimental set-up did not influence the chemokine receptor expression. After TTx stimulation a significant increase in MFI was observed for CCR3 in the allergic and asymptomatically sensitized individuals, but not in the healthy control group (Table 2). However, no increases in the percentage of CCR3+ memory Th cells were observed in any of the groups. CCR5 increased both as MFI and the percentage of CCR5+ memory Th cells in the asymptomatically sensitized and Methods Patients 10 healthy, 5 asymptomatically birch pollen sensitized, 5 asymptomatically grass pollen sensitized, 5 birch pollen Page 2 of 6 (page number not for citation purposes) Clinical and Molecular Allergy 2006, 4:6 http://www.clinicalmolecularallergy.com/content/4/1/6 Percentage chemokine receptor positive memory T helper cells day 0 Figure 1 Percentage chemokine receptor positive memory T helper cells day 0. Percentage CCR3+, CCR5+, CCR8+ and CXCR3+ memory Th cells from allergic (dots), asymptomatically sensitized (triangles) and healthy control (crosses) individuals on day 0 immediate ex vivo. - = median value. n = 10 for the healthy controls, n = 10 for the asymptomatically sensitized and n = 8 for the allergic individuals except for CCR8 where n = 6 for the asymptomatically sensitized and allergic individuals. 10.000 PBMCs were acquired for the analysis. Isotype control cut-off values were set to >98%. Samples were run in monocates. For experimental design and analysis see Methods. Percentage chemokine receptor positive memory T helper cells day 0 Figure 1 Percentage chemokine receptor positive memory T helper cells day 0. Percentage CCR3+, CCR5+, CCR8+ and CXCR3+ memory Th cells from allergic (dots), asymptomatically sensitized (triangles) and healthy control (crosses) individuals on day 0 immediate ex vivo. - = median value. n = 10 for the healthy controls, n = 10 for the asymptomatically sensitized and n = 8 for the allergic individuals except for CCR8 where n = 6 for the asymptomatically sensitized and allergic individuals. 10.000 PBMCs were acquired for the analysis. Isotype control cut-off values were set to >98%. Samples were run in monocates. For experimental design and analysis see Methods. Flow cytometry Surface markers were detected using primary labeled anti- bodies: CCR3-FITC, CCR5-FITC, CCR8-FITC, CXCR3- FITC (R&Dsystems, Abingdon, UK), CD4-PE-Cy5 and CD45RO-PE (Dakocytomation, Glostrup, Denmark). Three-color flow cytometry was performed on day 0 and day 7 on a FACScan (Becton Dickinson, Heidelberg, Ger- many) using WinList (Verity Software House, Topsham, USA) software for analysis. Isotype control cut-off values were set to > 98% and 10.000 PBMC were acquired. Gat- ing was done by firstly applying a CD4+ gate followed by determination of the percentage and mean fluorescence intensity (MFI) of the CD45RO and chemokine receptor double positive population. CD45RO is a marker of effec- tor and memory T cells, however throughout this article these CD4+ CD45RO+ T cells will be mentioned as mem- ory T helper cells for convenience. Page 3 of 6 (page number not for citation purposes) Statistical analysis Median chemokine receptor expression in memory Th cells in percentage and MFI after 7 days of stimulation with antigen (allergen (15 µg/ml) or TTx (10 µg/ml)) or no antigen as a control. n = 10 for the healthy controls, n = 10 for the asymptomatically sensitized individuals and n = 8 for the allergic individuals except for CCR8 where n = 6 for the asymptomatically sensitized and allergic individuals. 10.000 PBMCs were acquired for the analysis. Isotype control cut-off values were set to >98%. Samples were run in monocates. For experimental design and analysis see Methods. Bold text and * indicates significant differences between the antigen (allergen or TTx) stimulated samples and control samples where no antigen was added. Ag: antigen AS: asymptomatically sensitized individuals. healthy control group whereas no changes in CCR5 was observed in the allergic individuals. Also, an increase in the percentage of CCR8+ memory Th cells was observed in the healthy control group, but no significant changes in MFI were observed for this receptor. An increase in the percentage of CXCR3+ memory Th cells was observed in all three groups after TTx stimulation, however increases in MFI were only observed in the healthy control group. the MFI, the change in the CCR5 after allergen stimulation was significantly different between the three groups (P = 0.02). healthy control group whereas no changes in CCR5 was observed in the allergic individuals. Also, an increase in the percentage of CCR8+ memory Th cells was observed in the healthy control group, but no significant changes in MFI were observed for this receptor. An increase in the percentage of CXCR3+ memory Th cells was observed in all three groups after TTx stimulation, however increases in MFI were only observed in the healthy control group. Discussion Other studies have linked certain diseases with aberrant expression of one or more chemokine receptors [12,20,21]. However, very few studies have been con- ducted with regard to the phenotype of asymptomatically sensitized individuals and, to our knowledge none on chemokine receptor profiles. After stimulation with allergen, increases in the percent- age of CCR5+ memory Th cells were observed in healthy controls and in MFI in allergic individuals. Allergen stim- ulation did not induce any changes in CCR3, CCR8 and CXCR3 expression. When pooling all 28 patients in the statistical analysis, TTx was able to induce expression of all receptors both seen as a significant increase in the percent- age of chemokine receptor positive cells and as MFI. Aller- gen stimulation only induced a significant increase in the percentage of CCR5+ memory Th cells. In this study, no differences were found in receptor expression patterns immediate ex vivo for CCR3, CCR5, CCR8 and CXCR3 in memory Th cells from allergic, asymptomatically sensitized and healthy individuals despite the fact that the study was carried out in the pollen season. Our findings are in agreement with other studies report- ing equal mRNA levels of CCR3 and CCR5 in PBMCs [22] and same levels of CXCR3+ peripheral blood Th cells [21] in patients with atopic dermatitis and healthy controls, but in disagreement with other findings showing decreased percentage of CCR5+ and CXCR3+ memory Th cells in the blood from patients with atopic dermatitis compared to healthy controls [23]. Statistical analysis Samples were compared using non-parametric statistics (Kruskall-Wallis test or Wilcoxon's test for matched pairs). Values of P < 0.05 were considered significant. Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) http://www.clinicalmolecularallergy.com/content/4/1/6 Clinical and Molecular Allergy 2006, 4:6 Table 2: Chemokine receptor expression in memory T helper cells induced by 7 days of antigenic stimuli. Median chemokine receptor expression in memory Th cells in percentage and MFI after 7 days of stimulation with antigen (allergen (15 µg/ml) or TTx (10 µg/ml)) or no antigen as a control. n = 10 for the healthy controls, n = 10 for the asymptomatically sensitized individuals and n = 8 for the allergic individuals except for CCR8 where n = 6 for the asymptomatically sensitized and allergic individuals. 10.000 PBMCs were acquired for the analysis. Isotype control cut-off values were set to >98%. Samples were run in monocates. For experimental design and analysis see Methods. % median (range) MFI median (range) No Ag Allergen TTx No Ag Allergen TTx CCR3 Allergic 10.6 (4.7–32) 17.3 (9.8–29.5) 15.6 (4.4–52.2) 21 (16.2–32.3) 19.4 (16.9–37.2) 26.1* (16.6–40.5) AS 10.3 (3.3–19.7) 8.7 (2.8–23.2) 11.9 (5–28.8) 20.5 (14.7–27.2) 18.6 (16.4–32.3) 23.4 * (18.4–37.4) Healthy 8.7 (1.5–45) 11.2 (3.2–36.8) 23.4 (4.5–44.3) 19.7 (12.6–45.4) 17.8 (12.5–32.2) 23.7 (12.5–38.4) CCR5 Allergic 21.3 (7.8–31.5) 25 (4.1–53.9) 28.3 (12.3–61.1) 21.4 (16.8–27.9) 25.9 * (22.4–48.5) 26.1 (16.1–42.5) AS 15.2 (2.6–36.6) 17 (3–33.5) 16.9 * (5.2–65.9) 18.3 (15.2–24.5) 17.5 (15.4–20.5) 22.5 * (18.2–58.2) Healthy 11.7 (5.6–34.5) 13.5 * (6.7–29.7) 20.2 * (10.1–74.1) 16.7 (12.7–22) 17.3 (13.5–25.6) 25.1 * (13.4–72) CCR8 Allergic 10.4 (1.6–20.4) 12.8 (3.3–20) 12.7 (2.2–27.4) 24.3 (16.8–37.6) 27.9 (18.8–40.3) 28.2 (17.8–82.6) AS 3.7 (1.8–16.6) 3.2 (1.7–14.7) 6.3 (1.4–34.9) 21.9 (17–26.1) 22.8 (17.2–44.6) 24.9 (16.2–36.6) Healthy 5.7 (0.7–16.4) 3.9 (1.5–14) 9.3 * (3.8–35.7) 19 (12.6–49.6) 19.2 (11.7–46.6) 23.9 (12.4–48.2) CXCR3 Allergic 42.9 (22–47.5) 42.1 (19.2–67.4) 49.2 * (27–74.2) 57.4 (33.1–65.3) 54.4 (48.4–72.4) 60.6 (36.9–81.8) AS 28.1 (18–56.6) 27.8 (21–56.8) 29 * (20.8–77.4) 39.7 (29.3–59.7) 42.3 (30.6–54.5) 46.5 (33.5–115) Healthy 28.8 (19.8–46.5) 31.4 (17.3–46.9) 35.9 * (25.8–82.9) 37.8 (32–65.2) 39.3 (30.1–63.9) 57.6 * (32–162.4) Bold text and * indicates significant differences between the antigen (allergen or TTx) stimulated samples and control samples where no antigen was added. Ag: antigen AS: asymptomatically sensitized individuals. Table 2: Chemokine receptor expression in memory T helper cells induced by 7 days of antigenic stimuli. References When comparing the three groups after antigen stimula- tion, we found no differences in expression patterns between the three groups except for the change in CCR5 MFI which was significantly different between the three groups. This is the only observed difference between the three groups but as discussed above the great overlap in receptor levels would not make this finding of any clinical relevance. 1. Parronchi P, Brugnolo F, Sampognaro S, Maggi E: Genetic and envi- ronmental factors contributing to the onset of allergic disor- ders. Int Arch Allergy Immunol 2000, 121:2-9. 1. Parronchi P, Brugnolo F, Sampognaro S, Maggi E: Genetic and envi- ronmental factors contributing to the onset of allergic disor- ders. Int Arch Allergy Immunol 2000, 121:2-9. gy 2. Bodtger U, Poulsen LK, Malling HJ: Asymptomatic skin sensitiza- tion to birch predicts later development of birch pollen allergy in adults: a 3-year follow-up study. J Allergy Clin Immunol 2003, 111:149-154. 003, : 9 5 . 3. Bodtger U: Prognostic value of asymptomatic skin sensitiza- tion to aeroallergens. Curr Opin Allergy Clin Immunol 2004, 4:5-10. 4. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A: Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998, 187:875-883. 5. Zingoni A, Soto H, Hedrick JA, Stoppacciaro A, Storlazzi CT, Sini- gaglia F, D'Ambrosio D, O'Garra A, Robinson D, Rocchi M, et al.: The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J Immunol 1998, 161:547-551. 3. Bodtger U: Prognostic value of asymptomatic skin sensitiza- tion to aeroallergens. Curr Opin Allergy Clin Immunol 2004, 4:5-10. 4. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A: Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998, 187:875-883. 5. Zingoni A, Soto H, Hedrick JA, Stoppacciaro A, Storlazzi CT, Sini- gaglia F, D'Ambrosio D, O'Garra A, Robinson D, Rocchi M, et al.: The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J Immunol 1998, 161:547-551. In spite of the apparent lack of differences between the three groups with respect to chemokine receptor profile, a parallel study using a somewhat larger sample size showed that allergen stimulation induced significantly more proliferation of memory Th cells in the allergic indi- viduals compared to the asymptomatically sensitized and healthy individuals as well as a different cytokine profile [19]. J 6. http://www.clinicalmolecularallergy.com/content/4/1/6 http://www.clinicalmolecularallergy.com/content/4/1/6 http://www.clinicalmolecularallergy.com/content/4/1/6 Clinical and Molecular Allergy 2006, 4:6 asymptomatically sensitized and healthy individuals. The reason why allergic individuals do not upregulate this receptor even when stimulated with a type 1 antigen is speculative, but one reason might be due to their Th2 biased reaction pathway. However, they do show signifi- cant increases in percentage CXCR3+ memory Th cells after TTx stimulation, in accordance with this receptor's much stronger link to the Th1 phenotype [24]. the three groups after antigenic stimulation and thus we conclude that pollen allergic, asymptomatically pollen sensitized and healthy individuals cannot be distin- guished by means of chemokine receptors expression in memory Th cells and thus the migratory potentials of the memory Th cells seem to be the same between the three groups. References Sallusto F, Mackay CR, Lanzavecchia A: Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 1997, 277:2005-2007. 7. Pullerits T, Linden A, Praks L, Cardell LO, Lotvall J: Upregulation of nasal mucosal eotaxin in patients with allergic rhinitis during grass pollen season: effect of a local glucocorticoid. Clin Exp Allergy 2000, 30:1469-1475. gy 8. Gombert M, Dieu-Nosjean MC, Winterberg F, Bunemann E, Kubitza RC, Da Cunha L, Haahtela A, Lehtimaki S, Muller A, Rieker J, et al.: CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation. J Immunol 2005, 174:5082-5091. Authors' contributions All authors participated in the design of the study. KA con- ducted the patient contact and characterization, cell isola- tion and stimulation assays. MH conducted the flow cytometry and analyzed the data. All authors contributed towards the manuscript preparation with MH as the main author of the article. CCR5 appears to be the most allergen susceptible recep- tor. However, the apparent overlap in expression levels between the groups would exclude the use of this receptor as a diagnostic tool and thus is of no major clinical inter- est. Competing interests The author(s) declare that they have no competing inter- ests. Abbreviations Only when grouping all individuals, did the recall antigen TTx induce significant increases in expression of all recep- tors. The reason for the less clear effect as observed in the individual groups might be due to the great inter-individ- ual variation in chemokine receptor expression level, an observation also described by others [25]. Nevertheless, TTx induced more changes than the allergenic stimuli, an effect that is likely due to the higher frequency of TTx spe- cific T cells compared to allergen specific T cells in periph- eral blood (Glue, unpublished results). Ag: antigen AS: asymptomatically sensitized MFI: mean fluorescence intensity PBMC: peripheral blood mononu- clear cell Th: T helper TTx: Tetanus toxoid Group differences To compare the chemokine receptor expression between the three groups, the Day 7no antigen receptor level was sub- tracted from either the Day 7allergen or the Day 7TTx sample to obtain the change in receptor expression (∆Chemokine receptor). No differences in ∆Chemokine receptor for the percentage of chemokine receptor positive cells were observed between the three groups after stimulation with TTx or allergen. When comparing the ∆Chemokine receptor for Changes in chemokine receptor expression were observed after stimulation with both antigens (Table 2). CCR5 expression was induced after TTx stimulation, but only in Page 4 of 6 (page number not for citation purposes) http://www.clinicalmolecularallergy.com/content/4/1/6 Conclusion In conclusion, both antigenic stimuli were able to induce changes in chemokine receptor expression. TTx seemed to be a more potent stimulus with regard to changes in chemokine receptor expression in all three groups com- pared to the pollen allergens. No major differences in CCR3, CCR5, CCR8 and CXCR3 were found between allergic, asymptomatically sensitized and healthy individ- uals and thus chemokine receptor expression in periph- eral blood memory Th cells does not seem to be linked to patient status. No major differences were seen between 9. Iellem A, Colantonio L, Bhakta S, Sozzani S, Mantovani A, Sinigaglia F, D'Ambrosio D: Inhibition by IL-12 and IFN-alpha of I-309 and macrophage-derived chemokine production upon TCR trig- gering of human Th1 cells. Eur J Immunol 2000, 30:1030-1039. g g J 10. 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Conclusion Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 6 of 6 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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https://openalex.org/W2516900807	https://www.zora.uzh.ch/id/eprint/127145/1/BOE_manuscript_for_submission_Kopie.pdf	English	null	Optical coherence tomography endoscopic probe based on a tilted MEMS mirror	Biomedical optics express	2,016	cc-by	5,977	Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2016 g y p Duan, Can; Tanguy, Quentin; Pozzi, Antonio; Xie, Huikai (2016). Optical coherence tomography endoscopic probe based on tilted MEMS mirror. Biomedical Optics Express, 7(9):3345-3354. DOI htt //d i /10 1364/BOE 7 003345 An optical coherence tomography endoscopic probe based on a tilted MEMS mirror Can Duan1,, Quentin Tanguy1,2, Antonio Pozzi3 and Huikai Xie1, 1Department of Electrical and Computer Engineering, University of Florida, Gainesville, FL, 32611, USA 2Department of Micro Nano Science and Systems, FEMTO-ST, Besançon, 25000, France 3Small Animal Surgery Clinic, University of Zurich, Zurich 8057 Switzerland *cduan@ufl.edu; hkx@ufl.edu Abstract: This paper reports a compact microendoscopic OCT probe with an outer diameter of only 2.7 mm. The small diameter is enabled by a novel 2-axis scanning MEMS mirror with a preset 45° tilted angle. The tilted MEMS mirror is directly integrated on a silicon optical bench (SiOB). The SiOB provides mechanical support and electrical wiring to the mirror plate via a set of bimorph flexure, enabling a compact probe mount design without the requirement of a 45° slope, which is capable to dramatically reduce the probe size and ease the assembly process. Additionally, the SiOB also provides trenches with properly-designed opening widths for automatic alignment of the MEMS mirror, GRIN lens and optical fiber. The 45°-tilted MEMS mirror plate is actuated by four electrothermal bimorph actuators. The packaged 2.7 mm-diameter probe offers 2-axis side-view optical scanning with a large optical scan range of 40° at a low drive voltage of 5.5 Vdc in both axes, allowing a lateral scan area of 2.2 mm × 2.2 mm at a 3 mm working distance. High-resolution 2D and 3D OCT images of the IR card, ex vivo imaging of meniscus specimens and rat brain slices, in vivo imaging of the human finger and nail have been obtained with a TDOCT system. 2016 Optical Society of America 2016 Optical Society of America OCIS codes: (110.4500) Optical coherence tomography; (170.2150) Endoscopic imaging; (230.4685) Optical microelectromechanical devices; (170.3880) Medical and biological imaging. OCIS codes: (110.4500) Optical coherence tomography; (170.2150) Endoscopic imaging; (230.4685) Optical microelectromechanical devices; (170.3880) Medical and biological imaging. Optical coherence tomography endoscopic probe based on tilted MEMS mirro Duan, Can ; Tanguy, Quentin ; Pozzi, Antonio ; Xie, Huikai DOI: https://doi.org/10.1364/BOE.7.003345 DOI: https://doi.org/10.1364/BOE.7.003345 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-127145 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-127145 Journal Article Published Version Originally published at: Duan, Can; Tanguy, Quentin; Pozzi, Antonio; Xie, Huikai (2016). Optical coherence tomography endoscopic probe based on tilted MEMS mirror. Biomedical Optics Express, 7(9):3345-3354. DOI: https://doi.org/10.1364/BOE.7.003345 1. D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, and C. A. Puliafito, "Optical coherence tomography," Science 254(5035), 1178–1181 (1991). 2. C. W. Sun, S. Y. Lee, and K. F. Lin, "Review: Optical Scanning Probe for Optical Coherence Tomography," J. Med. Biol. Eng. 34(1), 95–100 (2014). 6. P. R. Herz, Y. Chen, A D. Aguirre, K. Schneider, P. Hsiung, J. G. Fujimoto, K. Madden, J. Schmitt, J. Goodnow, and C. Petersen, "Micromotor endoscope catheter for in vivo, ultrahigh-resolution optical coherence tomography.," Opt. Lett. 29(19), 2261–2263 (2004). 1. D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, and C. A. Puliafito, "Optical coherence tomography," Science 254(5035), 1178–1181 (1991). 2. C. W. Sun, S. Y. Lee, and K. F. Lin, "Review: Optical Scanning Probe for Optical Coherence Tomography," J. Med. Biol. Eng. 34(1), 95–100 (2014). 3. B. J. Vakoc, D. Fukumura, R. K. Jain, and B. E. Bouma, "Cancer imaging by optical coherence tomography: preclinical progress and clinical potential," Nat. Rev. Cancer 12(5), 363–368 (2012). 4. M. Atif, H. Ullah, M. Y. Hamza, and M. Ikram, "Catheters for optical coherence tomography," Laser Phys. Lett. 646(9), 629 (2011). 5. P. H. Tran, D. S. Mukai, M. Brenner, and Z. Chen, "In vivo endoscopic optical coherence tomography by use of a rotational microelectromechanical system probe.," Opt. Lett. 29(11), 1236–1238 (2004). 6. P. R. Herz, Y. Chen, A D. Aguirre, K. Schneider, P. Hsiung, J. G. Fujimoto, K. Madden, J. Schmitt, J. Goodnow, and C. Petersen, "Micromotor endoscope catheter for in vivo, ultrahigh-resolution optical coherence tomography.," Opt. Lett. 29(19), 2261–2263 (2004). 7. J. Su, J. Zhang, L. Yu, and Z. Chen, "In vivo three-dimensional microelectromechanical endoscopic swept source optical coherence tomography," Opt. Express 15(16), 10390–10396 (2007). 8. M. J. Gora, J. S. Sauk, R. W. Carruth, K. A Gallagher, M. J. Suter, N. S. Nishioka, L. E. Kava, M. Rosenberg, B. E. Bouma, and G. J. Tearney, "Tethered capsule endomicroscopy enables less invasive imaging of gastrointestinal tract microstructure.," Nat. Med. 19(2), 238–40 (2013). 9. X. Li, C. Chudoba, T. Ko, C. Pitris, and J. G. Fujimoto, "Imaging needle for optical coherence tomography.," Opt. Lett. 25(20), 1520–1522 (2000). 10. X. Liu, M. J. Cobb, Y. Chen, M. B. Kimmey, and X. Li, "Rapid-scanning forward-imaging miniature endoscope for real-time optical coherence tomography," Opt. Lett. 29(15), 1763–1765 (2004). 1. Introduction Optical coherence tomography (OCT) is a powerful biomedical imaging technique for real- time, cross-sectional imaging with 1-10 μm resolution and 1-3 mm imaging depth [1]. In recent years, OCT has been widely investigated in the area of endoscopic imaging of internal organs for cancer screening and early cancer diagnosis [2–4]. A variety of OCT probes and catheters have been developed to facilitate endoscopic OCT imaging with various optical scanning mechanisms such as using an external motor to spin an optical fiber with a prism attached [5–7], a rotary joint to rotate and linearly translate a fiber [8,9], a piezoelectric tube to tether a fiber [10–12], or a micromirror to scan the optical beam in free space at the distal end of a fiber [13–15]. Among them, MEMS micromirrors offer a more promising solution for rapid 3D endoscopic OCT imaging due to their small size, fast scanning speed and 2-axis linear scan capability. Several side-viewing and forward-viewing endoscopic OCT probes have been demonstrated in the last two decades using MEMS mirrors based on electrostatic [13], electromagnetic [14], piezoelectric [16] and electrothermal [17] actuation mechanisms. However, further minimization of MEMS micromirror enabled endoscopic OCT probes still remains a primary challenge due to the complexity of designing small probe mounts and the difficulty of assembling small probes in narrow space. y g p p Several types of planar electrothermal MEMS mirrors with 1-axis or 2-axis scan capability have been extensively employed in side-viewing endoscopic OCT probes [15,17– 19]. A typical side-viewing endoscopic OCT probe is shown in Fig. 1(a) [19], where a planar MEMS micromirror chip is mounted on a 45° sloped ferrule inside the probe body to direct an optical beam to one side of the probe. To assemble the probe, a flexible PCB (FPCB) is first glued on the slope. Then the MEMS chip is stacked on the FPCB using silver epoxy or instant glue; this step is tedious as the MEMS chip is very small and the housing space is very limited. The 45° slope takes extra space which increases both the probe size and the probe fabrication cost. The electrical connection is provided either by wire bonding [15] or flip-chip bonding [19]. Wire bonding further increases the probe size while flip-chip bonding may pose a risk of high electrical contact failure rate. References and links 1. D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, and C. A. Puliafito, "Optical coherence tomography," Science 254(5035), 1178–1181 (1991). 1. D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, and C. A. Puliafito, "Optical coherence tomography," Science 254(5035), 1178–1181 (1991). 2. C. W. Sun, S. Y. Lee, and K. F. Lin, "Review: Optical Scanning Probe for Optical Coherence Tomography," J. Med. Biol. Eng. 34(1), 95–100 (2014). 3. B. J. Vakoc, D. Fukumura, R. K. Jain, and B. E. Bouma, "Cancer imaging by optical coherence tomography: preclinical progress and clinical potential," Nat. Rev. Cancer 12(5), 363–368 (2012). 4. M. Atif, H. Ullah, M. Y. Hamza, and M. Ikram, "Catheters for optical coherence tomography," Laser Phys. Lett. 646(9), 629 (2011). 5. P. H. Tran, D. S. Mukai, M. Brenner, and Z. Chen, "In vivo endoscopic optical coherence tomography by use of a rotational microelectromechanical system probe.," Opt. Lett. 29(11), 1236–1238 (2004). 6. P. R. Herz, Y. Chen, A D. Aguirre, K. Schneider, P. Hsiung, J. G. Fujimoto, K. Madden, J. Schmitt, J. Goodnow, and C. Petersen, "Micromotor endoscope catheter for in vivo, ultrahigh-resolution optical coherence tomography.," Opt. Lett. 29(19), 2261–2263 (2004). g p y p ( ) ( ) 7. J. Su, J. Zhang, L. Yu, and Z. Chen, "In vivo three-dimensional microelectromechanical endoscopic swept source optical coherence tomography," Opt. Express 15(16), 10390–10396 (2007). 8. M. J. Gora, J. S. Sauk, R. W. Carruth, K. A Gallagher, M. J. Suter, N. S. Nishioka, L. E. Kava, M. Rosenberg, B. E. Bouma, and G. J. Tearney, "Tethered capsule endomicroscopy enables less invasive imaging of gastrointestinal tract microstructure.," Nat. Med. 19(2), 238–40 (2013). g , ( ), ( ) 9. X. Li, C. Chudoba, T. Ko, C. Pitris, and J. G. Fujimoto, "Imaging needle for optical coherence tomography.," Opt. Lett. 25(20), 1520–1522 (2000). 10. X. Liu, M. J. Cobb, Y. Chen, M. B. Kimmey, and X. Li, "Rapid-scanning forward-imaging miniature endoscope for real-time optical coherence tomography," Opt. Lett. 29(15), 1763–1765 (2004). 11. Y. Wu, Y. Leng, J. Xi, and X. Li, "Scanning all-fiber-optic endomicroscopy system for 3D nonlinear optical imaging of biological tissues.," Opt. Express 17(10), 7907–7915 (2009). 12. L. Huo, J. Xi, Y. Wu, and X. References and links JW3A.17. 22. C. Duan, Q. Tanguy, A. Pozzi, and H. Xie, "An Optical Coherence Tomography Endoscopic Probe Based on a Tilted MEMS Mirror," in Biomedical Optics (OSA, 2016), pp. JW3A.17. References and links Li, "Forward-viewing resonant fiber-optic scanning endoscope of appropriate scanning speed for 3D OCT imaging.," Opt. Express 18(14), 14375–14384 (2010). 13. W. Jung, D. T. McCormick, J. Zhang, L. Wang, N. C. Tien, and Z. Chen, "Three-dimensional endoscopic optical coherence tomography by use of a two-axis microelectromechanical scanning mirror," Appl. Phys. Lett. 88(16), 163901 (2006). 14. K. H. Kim, B. H. Park, G. N. Maguluri, T. W. Lee, F. J. Rogomentich, M. G. Bancu, B. E. Bouma, J. F. de Boer, and J. J. Bernstein, "Two-axis magnetically-driven MEMS scanning catheter for endoscopic high-speed optical coherence tomography," Opt. Express 15(26), 18130–18140 (2007). 15. J. Sun, S. Guo, L. Wu, L. Liu, S. W. Choe, B. S. Sorg, and H. Xie, "3D in vivo optical coherence tomography based on a low-voltage, large-scan-range 2D MEMS mirror," Opt. Express 18(12), 12065–12075 (2010). 16. K. H. Gilchrist, R. P. McNabb, J. A. Izatt, and S. Grego, "Piezoelectric scanning mirrors for endoscopic optical coherence tomography," J. Micromech. Microeng. 19(9), 095012 (2009). 16. K. H. Gilchrist, R. P. McNabb, J. A. Izatt, and S. Grego, "Piezoelectric sca coherence tomography," J. Micromech. Microeng. 19(9), 095012 (2009). 17. Y. Pan, H. Xie, and G. K. Fedder, "Endoscopic optical coherence tomography based on a microelectromechanical mirror," Opt. Lett. 26(24), 1966–1968 (2001). 18. C. Duan, J. Sun, S. Samuelson, and H. Xie, "Probe alignment and design issues of microelectromechanical system based optical coherence tomography endoscopic imaging.," Appl. Opt. 52(26), 6589–98 (2013). 19. S. R. Samuelson, L. Wu, J. Sun, S. W. Choe, B. S. Sorg, and H. Xie, "A 2.8-mm imaging probe based on a high-fill-factor MEMS mirror and wire-bonding-free packaging for endoscopic optical coherence tomography," J. Microelectromech. Syst. 21(6), 1291–1302 (2012). 20. Y. Xu, J. Singh, C. S. Premachandran, A Khairyanto, K. W. S. Chen, N. Chen, C. J. R. Sheppard, and M. Olivo, "Design and development of a 3D scanning MEMS OCT probe using a novel SiOB package assembly," J. Micromech. Microeng. 18(12), 125005 (2008). 21. C. Duan, W. Wang, X. Zhang, J. Ding, Q. Chen, A. Pozzi, and H. Xie, "A 45°-tilted 2-axis scanning micromirror integrated on a silicon optical bench for 3D endoscopic optical imaging," in 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS) (IEEE, 2015), pp. 948–951. 22. C. Duan, Q. Tanguy, A. Pozzi, and H. Xie, "An Optical Coherence Tomography Endoscopic Probe Based on a Tilted MEMS Mirror," in Biomedical Optics (OSA, 2016), pp. 1. Introduction The failure rate of electrical connection is high for flip-chip bonding because the pads on the small MEMS mirror chip must be aligned with the pads on the FPCB in a very small space. Silver epoxy is manually placed on the pads, so the contact areas and strengths are not uniform, easily causing broken circuits or short circuits. Another popular side-viewing probe design was demonstrated by Xu et al. [20], employing a SiOB with a pre-fabricated trench to hold a micromirror vertically. The micromirror was dropped into the trench and fixed manually, and the electrical connections between the MEMS actuators and the embedded metal paths on the SiOB were provided with solder balls [20]. But to form reliable electrical and mechanical connections is still a challenge due to significant deformation of the solder balls during reflow and curing. g g Fig. 1. (a) Illustration of the probe design with the MEMS mirror fixed on a 45° slope. (b) The new probe design with the MEMS mirror tilted 45° out of plane. Fig. 1. (a) Illustration of the probe design with the MEMS mirror fixed on a 45° slope. (b) The new probe design with the MEMS mirror tilted 45° out of plane. In this paper, we report a compact OCT probe (Fig. 1(b)) with a simplified probe mount design and simplified assembly process. This probe is enabled by a novel MEMS micromirror design with the mirror plate tilted at a preset 45° and integrated on a SiOB [21]. The tilted MEMS mirror simplifies the probe mount design by eliminating the need of a 45° slope or pocket to fix a planar MEMS mirror as in prior work [15,19]. Furthermore, the SiOB, which contains large pads connected to the actuators, enables easy and stable electrical connection. In this paper, the MEMS mirror will be introduced first, followed by the probe design, the probe assembling, and the imaging experiment results. 3. Probe design The schematic of the side-viewing OCT probe design based on the 45° tilted MEMS mirror is shown in Fig. 1(b). The side-viewing probe is defined by five main components: a MEMS chip, a GRIN lens, a single mode fiber (SMF), an FPCB and a probe mount. The MEMS chip consists of a 45° tilted MEMS mirror and a SiOB and is mounted on the FPCB for electrical connections using a wire-bonding-free packaging technique. The GRIN lens and SMF are placed in the trenches on the SiOB. The trenches are designed with proper dimensions to ensure good optical alignment of the optical components. The MEMS-FPCB module is fixed on a 25 mm-long, 2.3 mm-wide and a 0.95 mm-thick flat holder and inserted into a glass tube with an outer diameter of 2.7 mm. The flat holder is made using 3D printing, so the cost is very low compared to the prior probe design shown in Fig. 1(a) [19]. y p p p g g ( ) [ ] The optical design of the probe with light ray tracing is shown in Fig. 3. An SMF fiber (SMF-28, Corning) with an 8° cleaved fiber tip is connected to the sample arm of an OCT system to deliver a broadband light with a center wavelength of 1310 nm. The outer diameter of the SMF is 0.9 mm and the 8° cleaved fiber tip helps to reduce back-reflection light. The fiber tip is optically aligned with and physically fixed at the end of a GRIN lens. Optical UV glue (NOA 61, Norland Optical Adhesives) with a refractive index of 1.542 at 1310 nm is used to fix the fiber tip with the GRIN lens and provide refractive index matching. The distance between the fiber tip and the GRIN lens is controlled to be smaller than 0.1 mm and can be adjusted according to the requirements of the working distance and lateral resolution [18]. The GRIN lens (ILW-070, GoFoton, NJ) employed has a pitch of 0.27 with a length of 1.955 mm, a diameter of 0.7 mm and a focal length of 5 mm. A 45° tilted MEMS mirror is placed at the distal end of the flat holder, and the height of the mirror center is around 0.52 mm, as shown in Fig. 3. The mirror center is aligned with the optical axis, and the mirror plate is 45° to the incident light beam. 2. The 45° tilted MEMS mirror The 3D model of the employed 45° tilted MEMS mirror is shown in Fig. 2(a), consisting of a 2-axis scanning single-crystal-silicon (SCS) mirror plate tilted out of the surface of a SiOB. The 45° tilt of the mirror plate is achieved with the initial bending of a set of flexure bimorph cantilevers and the stopping by a stopper structure anchored on the SiOB. The flexure bimorph consists of SiO2 as the top layer and Al as the bottom layer, which bends towards the SiOB upon releasing. The tilt angle can be precisely controlled by properly choosing the flexure bimorph length and the distance from the mirror frame to the silicon sidewall [21]. The SiOB provides mechanical support and electrical wiring to the bimorph actuators as well as trenches for fixation and optical alignment of the optical components. Fig. 2. The 45° tilted MEMS mirror with long SiOB. (a) 3D model. (b) SEM of a fabricated device. (c) A close-up SEM of the mirror plate with four actuators. (d) Scan angle versus applied voltage. Fig. 2. The 45° tilted MEMS mirror with long SiOB. (a) 3D model. (b) SEM of a fabricated device. (c) A close-up SEM of the mirror plate with four actuators. (d) Scan angle versus applied voltage. Figs. 2(b) and (c) show two SEMs of a fabricated 45° tilted MEMS mirror. The fabricated device has a footprint of 2.22 mm × 6.78 mm and a height of 0.85 mm, and the mirror plate is 0.72 mm × 0.72 mm in size and tilted by 63°, which is not exactly 45° due to the silicon sidewall undercut during the device release. The maximum total optical scan angle of the mirror plate is around 40° at 5.5 Vdc for both axes, as shown in Fig. 2(d). The first mechanical resonance peak of the mirror plate for the tip-tilt scanning mode in both directions is approximately 750 Hz. More details about the design, fabrication and characterization of this MEMS mirror can be found in [21]. 3. Probe design The distance between the GRIN lens and the mirror plate center is designed as 1 mm to achieve a working distance of 3 mm with a lateral resolution around 20 μm in air. The effects of the distance between the fiber and GRIN lens, the curvature of the MEMS mirror plate and the cylindrical tubing can be found in [18]. Fig. 3. Optical design of the side-viewing endoscopic OCT probe employing the tilted MEMS mirror (not drawn to scale). Fig. 3. Optical design of the side-viewing endoscopic OCT probe employing the tilted MEMS mirror (not drawn to scale). Electrical connections to the four actuators of the MEMS mirror are required. An FPCB shown in Figs. 4(a) and (b) is used to achieve compactness while maintaining stable electrical connections. The width of the PCB is 2.25 mm, and the length is 40 mm. There are five Al- coated bonding pads on the back side of the SiOB, as shown in Fig. 4(c), and there are five exactly matched bonding pads at one end of the FPCB, as shown in Fig. 4(b). Several markers are placed on the FPCB to facilitate the alignment of the SiOB to the FPCB. The length of the SiOB is designed such that large pad size and large spacing between the pads are allowed. Thus, the MEMS chip can be directly flip-chip bonded on the FPCB without the need for wire bonding. This wire-bonding-free technique leads to reliable electrical connection and small form factor. As shown in Fig. 4(c), the size of each pad is around 1 mm × 0.6 mm and the spacing between any two pads is greater than 1 mm, which is large enough for easy assembly and to ensure reliable electrical connections as well. The other end of the FPCB has five corresponding solder pads which are soldered with five copper wires to carry four drive signals and one electrical ground. Compared to a side-viewing probe design using a planar MEMS mirror, a 45° slope or pocket or a separate SiOB with a trench for mounting the MEMS mirror is not needed in this new probe design. Only a strip with a flat surface is utilized to hold the tilted MEMS mirror, which significantly reduces the volume of the whole probe and decreases the fabrication cost of the probe mount. Fig. 4. Wire-bonding-free technique for 45° tilted MEMS mirror by using FPCB. 3. Probe design (a) and (b) Photographs of FPCB. (c) SEM of the conductive pads on the backside of a SiOB. Fig. 4. Wire-bonding-free technique for 45° tilted MEMS mirror by using FPCB. (a) and (b) Photographs of FPCB. (c) SEM of the conductive pads on the backside of a SiOB. 4. Probe assembly An endoscopic probe prototype has been assembled using the fabricated tilted MEMS mirror and other optical components. Five copper wires are first soldered on one FPCB as shown in Fig. 4(a), and then the FPCB with the copper wires is attached to a 3D printed flat strip using an instant glue. After that, a small amount of electrically conductive silver epoxy (EMS #12642-14, Electron Microscopy Sciences) is dispensed on each pad of the FPCB. Next, the MEMS micromirror chip is flipped over and pressed on the FPCB with the edges of the SiOB aligned to the corresponding markers on the FPCB to ensure proper and reliable electrical connections. The silver epoxy can be cured in 4 hours at room temperature to achieve good mechanical and electrical connections. A GRIN lens and an SMF are glued inside the trenches on the SiOB using an optical glue, and they are aligned automatically to the center of the MEMS mirror plate. The mirror plate is tilted 45° initially with respect to the incident light beam. The auto-alignment is realized by carefully designing opening widths of the trenches on SiOB in order to keep the optical axes of the fiber and the GRIN lens aligned with the center height of the mirror plate. The auto-alignment eliminates the difficulty in manual adjustment and assembly. A transparent glass tube is then slid from the distal end of the probe for protection and can be replaced by other tubes or imaging windows made of any biocompatible and transparent material. A cap can be added and fixed using glue at the end of the probe head to encapsulate the probe. Fig. 5(a) shows a fully packaged endoscopic OCT probe, composed of a probe head, an SMF fiber, and several copper wires. As shown in Fig. 5(b), the outer diameter of the probe is 2.7 mm and can be decreased by minimizing the wall thickness of the tubing. The inner diameter of the prototype is 2.3 mm and can be further reduced to 1.2 mm by modifying the SiOB design. An enlarged photograph of the probe head is shown in Fig. 5(c), indicating that the 45° tilted MEMS mirror is attached both physically and electronically on a flat holder. Fig. 5. Photographs of the side-viewing endoscopic probe. (a) Overview of the packaged probe. (b) Fully packaged probe placed beside a ruler and one dime. 4. Probe assembly (c) Close-up of the probe head. Fig. 5. Photographs of the side-viewing endoscopic probe. (a) Overview of the packaged probe. (b) Fully packaged probe placed beside a ruler and one dime. (c) Close-up of the probe head. Fig. 6(a) shows a packaged probe head with a red light delivered to the mirror plate through the SMF and GRIN lens. A semiconductor laser with a wavelength of 650 nm was used for demonstration and measurement. The longitudinal scan pattern with the drive voltage of 3 Vpp is shown in Fig. 6(b). The total optical scan angle was measured to be 18.6° in this image. In both axes, the maximum scan angle of 40° can be achieved with a maximum drive voltage of 5.5 Vdc. Fig. 6(a) shows a packaged probe head with a red light delivered to the mirror plate through the SMF and GRIN lens. A semiconductor laser with a wavelength of 650 nm was used for demonstration and measurement. The longitudinal scan pattern with the drive voltage of 3 Vpp is shown in Fig. 6(b). The total optical scan angle was measured to be 18.6° in this image. In both axes, the maximum scan angle of 40° can be achieved with a maximum drive voltage of 5.5 Vdc. Fig. 6. Photographs of the packaged probe head without tubing. (a) A packaged probe. (b) One- axis scan pattern with driven voltage of 3V for opposed actuators in the longitudinal direction. Fig. 6. Photographs of the packaged probe head without tubing. (a) A packaged probe. (b) One- axis scan pattern with driven voltage of 3V for opposed actuators in the longitudinal direction. 5.1 Imaging characterization The imaging performance of the probe is demonstrated using a TDOCT system, shown in Fig. 7. As shown in Fig. 7(a), the probe is connected to the sample arm of the TDOCT system to provide a 2D lateral scan on a sample. As shown in Fig. 7(b), the sample is placed in a glass holder which is placed on top of the probe and the light exiting the side-viewing probe scans the sample from the bottom side. The MEMS mirror inside the probe is capable of providing a raster scan with the fast scan of 1.25 Hz and the slow scan of 0.005 Hz. A series of cross- sectional 2D images can be obtained in real time, and 3D rendering can be achieved by stacking the 2D images. The lateral scan speed of the MEMS mirror can be increased, but it is limited by the axial scan speed (500 Hz) of the TDOCT system. The axial resolution is 10 μm, which is determined by the broadband light source; the lateral resolution is measured to be approximately 23 μm, which is a compromise of the 3 mm imaging depth. Fig. 7. MEMS based OCT imaging probe. (a) Schematic of a TDOCT system using an endoscopic OCT probe as the sample arm. (b) Photograph of the side-viewing probe connected to the OCT system. Fig. 7. MEMS based OCT imaging probe. (a) Schematic of a TDOCT system using an endoscopic OCT probe as the sample arm. (b) Photograph of the side-viewing probe connected to the OCT system. The lateral scan range at a working distance of 3 mm was measured by imaging a printed customized calibration pattern with a 1 inch or 2.54 mm pitch, as shown in Fig. 8(a). The ratio of the black area to the area of one unit is 95%. OCT images of the black area on the printed pattern are shown as shallow bright lines (indicated by the stars), while the imaging depth of the white lines is larger, as indicated by the arrows in Figs. 8(b) to (e). The scan range or the image width can be determined by the ratio of the total pixel number and the pixel number between the two longer bright lines (Fig. 8(b)), which indicate the space between the adjacent white patterns. 5.1 Imaging characterization If there is only one grid space shown in the image, which means the image width is smaller than 2.54 mm, the scan range can be determined by recording the displacement of the printed calibration pattern when the space moves from the left to right side of the imaging window, shown in Figs. 8(c) to (e). The lateral displacement was generated by manually moving the translational stage that held the sample. The measured width of the 2D OCT images is 1.75 mm at the working distance of 3 mm, and the height of the 2D OCT images is 2.5 mm. The corresponding optical scan angle of the probe is ±16.3° in the transverse direction when applying a 5 V drive voltage. Fig. 8. Characterization of the lateral scan range of the side-viewing probe. (a) The calibration pattern with a grid size of 2.54 mm. Cross-sectional OCT images of the printed calibration pattern: (b) driving a single actuator to scan in the x-direction; (c) to (e) driving a pair of opposing actuators in the y-direction with the translational stage at different positions. Fig. 8. Characterization of the lateral scan range of the side-viewing probe. (a) The calibration pattern with a grid size of 2.54 mm. Cross-sectional OCT images of the printed calibration pattern: (b) driving a single actuator to scan in the x-direction; (c) to (e) driving a pair of opposing actuators in the y-direction with the translational stage at different positions. 5.2 OCT imaging results OCT images of an IR card, a human finger, a human nail, rat brain tissue slices, and canine menisci samples have been obtained using the OCT probe prototype in combination with the TDOCT system. Fig. 9 shows a series of 2D OCT images obtained by the endoscopic OCT probe. Different layers of the samples are clearly observed in the real-time 2D OCT images. Fig. 9(a) shows the cross-sectional OCT image of an IR card. The top two bright lines represent the surface of the glass holder and the plastic layer of the IR card surface respectively. The penetration depth is measured to be around 0.7 mm inside the IR card. Fig. 9(b) represents both the epidermis and dermis layer as well as some sweat ducts beneath the surface of the human finger. And Fig. 9(c) shows the cross-sectional of a human nail with the imaging depth of 1.3 mm. The lunula layer was clearly delineated. 5.1 Imaging characterization In Fig. 9(c), only the right part of the image shows the presence of the lunula and the left part does not contain this layer since this cross-sectional image was taken at the interface of the normal nail area and the lunula area. The fluctuations of the nail surface were introduced by the moving finger, which can be eliminated by using an SSOCT system with a much higher imaging speed. 2D OCT images of rat brain tissue slices have also been obtained. The thickness of the rat brain tissue was measured to be 0.6 mm, as shown in Fig. 9(d). The white matter and the gray matter show different scattering properties (Fig. 9(e)) as the white matter appeared brighter than the gray matter in the OCT images. Some nerve fibers or axons were also observed as bright dots in the region of the corpus callosum, as shown in Fig. 9(f). Fig. 9. OCT imaging performance of the side-viewing probe. 2D OCT images of (a) IR card, (b) human finger, (c) human nail, and (d) to (f) rat brain tissue slice. Fig. 9. OCT imaging performance of the side-viewing probe. 2D OCT images of (a) IR card, (b) human finger, (c) human nail, and (d) to (f) rat brain tissue slice. 3D OCT renderings of the brain tissue slices were obtained by stacking a series of 2D OCT images, as shown in Figs. 10(a) to (d). The 3D images were achieved by scanning the sample in two orthogonal directions with a fast scan frequency of 1.25 Hz and a slow scan frequency of 0.005 Hz, respectively. Two stainless steel balls with different diameters were placed on top of a thin slice of a rat brain tissue. The interfaces between the balls and the brain tissue were clearly delineated by the bright curves shown in Figs. 10(a) and (c). The 3D images in Figs. 10(b) and (d) provide better views of the brain tissue slice with the ball placed on top of it. Deformation of the brain tissue generated by the weight of the stainless ball was observed. The deformation varies with the ball size, providing a potential method to study the refractive index change of the brain tissue due to mechanical deformation or stress. Figs. 6. Summary In this work, we have developed a miniaturized endoscopic side-viewing OCT probe using a 45° tilted 2-axis MEMS mirror for in vivo 3D imaging of biological specimens. The endoscopic OCT probe has an inner diameter of 2.3 mm and an outer diameter of 2.7 mm. It possesses the advantages of simplified probe mount design, reduced probe size and more stable electrical connections compared to the planar MEMS mirrors based endoscopic OCT probes. The flat strip holding the FPCB and MEMS can be created by 3D printing, resulting in low cost. The large pad spacing allowed by the SiOB makes the assembly much easier and the electrical connection much more reliable. Also, the trenches and markers on the SiOB enable quick optical alignment. High-resolution imaging capability of the probe has been demonstrated using a TDOCT system on IR cards, biological specimen and human fingers and nails. The imaging results indicate that the probe is promising for real-time in vivo imaging of the epithelial layers of internal organs with micrometer-resolution, which is attractive for the early cancer detection and the image-guided surgery. By incorporating tilted MEMS mirrors with narrower SiOB, the probe size can be further reduced to 1.5 mm, allowing even broader applications inside human body including intravascular imaging. 5.1 Imaging characterization 10(e) and (g) are 2D OCT images of a normal canine meniscus and a canine meniscus with a horizontal meniscal tear created by a single incision, respectively. The lesion was filled with a fat gel, shown as a dark gap inside the meniscus in the OCT image (Fig. 10(g)) due to the lower scattering of the gel compared to the meniscus tissue. The outline of the meniscal tear was clearly delineated in Fig. 10(g). A 3D image of the canine meniscus with the horizontal tear is shown in Fig. 10(h). It is capable of facilitating the diagnosis of meniscal tears in vivo with the MEMS-based endoscopic OCT probe integrated within the traditional arthroscope. Fig. 10. 2D and 3D OCT images obtained by the endoscopic probe. (a) 2D and (b) 3D OCT images of a rat brain tissue slice with a stainless steel ball of 2 mm in diameter. (c) 2D and (d) 3D OCT images of a rat brain tissue slice with a stainless steel ball of 1 mm in diameter. (e) 2D and (f) 3D OCT images of the normal canine meniscus. (g) 2D and (h) 3D OCT image of the canine meniscus with a simulated horizontal meniscal tear. Fig. 10. 2D and 3D OCT images obtained by the endoscopic probe. (a) 2D and (b) 3D OCT images of a rat brain tissue slice with a stainless steel ball of 2 mm in diameter. (c) 2D and (d) 3D OCT images of a rat brain tissue slice with a stainless steel ball of 1 mm in diameter. (e) 2D and (f) 3D OCT images of the normal canine meniscus. (g) 2D and (h) 3D OCT image of the canine meniscus with a simulated horizontal meniscal tear. Acknowledgements This work was supported in part by the National Science Foundation under award #1002209 and 1512531. Portions of this work were presented at the Biomedical Optics Congress in 2016, JW3A-17 [22].
https://openalex.org/W2799848229	https://www.nature.com/articles/s41467-018-04022-0.pdf	English	null	The mechanisms of crystal growth inhibition by organic and inorganic inhibitors	Nature communications	2,018	cc-by	5,623	ARTICLE OPEN ARTICLE \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications Results The microkinetic model was developed for minerals consisting of two general components in equal stoichiometry, and should therefore be generally applicable to the growth of any two components of a crystal, also at elevated temperature and pressure. This would, for example, include the most divalent metal carbonate and sulphate minerals, as long as kink nucleation is observed to be a rate-limiting step during growth. rSO4 ¼ a KIP;CaCO3 Ca2þ ½ CO2 3 1 þ KCa Ca2þ ½ ð Þ 1 þ KCO3 CO2 3 þ KSO4 SO2 4 kT h e ΔGIP;CaCO3 RT and ð1Þ rMg ¼ a KIP;CaCO3 Ca2þ ½ CO2 3 1 þ KCa Ca2þ ½ þ KMg Mg2þ ½ 1 þ KCO3 CO2 3 kT h e ΔGIP;CaCO3 RT ð2Þ rSO4 ¼ a KIP;CaCO3 Ca2þ ½ CO2 3 1 þ KCa Ca2þ ½ ð Þ 1 þ KCO3 CO2 3 þ KSO4 SO2 4 kT h e ΔGIP;CaCO3 RT and ð1Þ Calcite is an important link in the global carbon cycle, it is present in nearly all the earth’s surface environments and it plays a leading role in biomineralisation8–10, where the organic mole- cules have been shown to signiﬁcantly impact the growth rate and morphology of calcite11–13. Currently, our interest focuses on the structures and properties of biocomposites14, internal cell struc- ture in relation to the mineralising compartment15 and the role of the polymers for inducing mineralisation16–18. ð2Þ Here, the step spacing of the atomic structure, a, is 0.32 nm, and ΔGIP;CaCO3 represents the energy barrier for the rate-limiting reaction, i.e. kink nucleation. It mostly reﬂects the energy required for dehydrating the attaching unit. kT h denotes the attempt frequency, which is equal to the product of the Boltzmann constant and temperature, divided by the Planck constant. For growth inhibition by both Mg2+ and SO42−, the expressions above can be generalised assuming that the ions adsorb independently: p y g In this work, we extend the microkinetic model7 for calcite growth to accommodate adsorption of foreign species. Our goal was to understand the mechanisms that control crystal growth when ionic or organic inhibitors are present. Results Inhibition by the inorganic ions Mg2+ and SO42−. We began by modelling the effect of inorganic inhibitors, with Mg2+ and SO42 −as examples. To include these species in the microkinetic growth model7 in a thermodynamically consistent manner requires the knowledge of the solution speciation. The formation of ion pairs has been suggested as a potential growth inhibition mechanism24, signiﬁcantly affecting growth by decreasing the saturation index (SI). We therefore performed a full geochemical speciation calculation with PHREEQC25 for each experimental data point. The solution speciation results for the Mg, SO4 and MgSO4 systems were used in the microkinetic model, ﬁtted to the experimental data. The equilibrium constant for CaCO30 ion pair formation is KCaCO0 3 ¼ KIP;CaCO3 ¼ 103:22 in the PHREEQC database26,27. We have used the standard geochemistry notation for the ion pair, CaCO30. We use the IP notation to specify that it relates to the ion pair formation in the solution, which avoids confusion with adsorption equilibrium constants, which are written as KX, for adsorption of X onto the steps. Because the experimental data23 for MgSO4 inhibition were acquired at low SI (0 < SI < 1, where 0 = equilibrium), the growth from polynuclear complexes was negligible28–30, so we can use the simpler ion pair (IP) model7 to obtain microscopic growth rates (step advance- ment rates) in the presence of a single inhibitor (Mg2+ or SO42−): p y Over past decades, several models for mineral growth in the presence of inhibitors have been developed. Most require several free parameters and some empirical assumptions for deriving the model equations4,5. The classic Cabrera–Vermilyea model (CV model)4 focusses on blocking the step edge advance. A different approach, by Nielsen et al.5, expanded on the kinetic ionic model for a Kossel crystal, presented by Zhang and Nancollas6, and described inhibition as a result of either kink-site blocking, the kink-blocking model, or incorporation of the inhibitor into the crystal, the incorporation inhibition model. These models are described in more detail in Supplementary Note 1. A recently published microkinetic model for calcite growth7 uses only one physically meaningful parameter of each inhibitor, namely the adsorption energy of the inhibitor on the crystal step. The ther- modynamic base and the absence of empirical parameters makes the microkinetic model simple, yet enables reliable predictions and straightforward interpretations. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04022-0 T he ability to predict the behaviour during crystal growth would have an important scientiﬁc and economic impact. Accurate prediction of mineral growth rates in complex ﬂuids is the base for understanding a broad range of geological processes, from crystallisation of a melt in a magma chamber, through recrystallisation during diagenesis and metamorphism, to the production of secondary minerals during weathering, and the controls on biomineralisation. From an applied perspective, it would provide the ability to design biomimetic materials with predetermined properties1,2 (i.e. high mechanical strength and controlled surface area), it would aid in the systematic improve- ment of techniques to prevent or remove unwanted precipitation such as scale formation in pipes3, and it would provide input for the improved manufacture of crystalline materials. T solution, which decreases the number of growth units available for attachment on the surface. We demonstrate that the minimal set of parameters used in the microkinetic model together with geochemical speciation modelling is enough to reproduce the observed behaviour for all inorganic and organic inhibitors in our study. The mechanisms of crystal growth inhibition by organic and inorganic inhibitors S. Dobberschütz1, M.R. Nielsen1, K.K. Sand 1, R. Civioc1, N. Bovet1, S.L.S. Stipp1 & M.P. Andersson 1 S. Dobberschütz1, M.R. Nielsen1, K.K. Sand 1, R. Civioc1, N. Bovet1, S.L.S. Stipp1 & M.P. Andersson 1 Understanding mineral growth mechanism is a key to understanding biomineralisation, fos- silisation and diagenesis. The presence of trace compounds affect the growth and dissolution rates and the form of the crystals produced. Organisms use ions and organic molecules to control the growth of hard parts by inhibition and enhancement. Calcite growth in the pre- sence of Mg2+ is a good example. Its inhibiting role in biomineralisation is well known, but the controlling mechanisms are still debated. Here, we use a microkinetic model for a series of inorganic and organic inhibitors of calcite growth. With one, single, nonempirical para- meter per inhibitor, i.e. its adsorption energy, we can quantitatively reproduce the experi- mental data and unambiguously establish the inhibition mechanism(s) for each inhibitor. Our results provide molecular scale insight into the processes of crystal growth and biominer- alisation, and open the door for logical design of mineral growth inhibitors through compu- tational methods. 1 NATURE COMMUNICATIONS\| (2018) 9:1578 Results Adsorbed single ions block the sites for growth unit adsorption Fig. 1 The growth inhibition model. A generic step edge consisting of empty sites (transparent) and the sites occupied by various ions present in the solution. On calcite, kinks are formed by the attachment of a calcium carbonate ion pair (in red), which is assumed to be the rate determining reaction in the growth process. Adsorbed single ions block the sites for growth unit adsorption 0 5 10 15 0 0.2 0.4 0.6 0.8 1 MgSO4 Inhibition factor 0 5 10 15 Mg 0 5 10 15 20 25 30 35 40 0 0.2 0.4 0.6 0.8 1 SO4 This work Kink-blocking model Incorporation inhibition model Concentration of the inhibitor [mM] Fig. 2 Comparison between the model and the experimental growth inhibition data for Mg2+ and SO42−. Fits for the growth inhibition data with the extended microkinetic model (this work, black), the kink-blocking model (blue) and the incorporation inhibition model (red) for MgSO40, Mg2+ and SO42−. The microkinetic model ﬁts best and requires only two physically meaningful parameters, adsorption energy for Mg2+ and SO42− Concentration of the inhibitor [mM] Fig. 2 Comparison between the model and the experimental growth inhibition data for Mg2+ and SO42−. Fits for the growth inhibition data with the extended microkinetic model (this work, black), the kink-blocking model (blue) and the incorporation inhibition model (red) for MgSO40, Mg2+ and SO42−. The microkinetic model ﬁts best and requires only two physically meaningful parameters, adsorption energy for Mg2+ and SO42− parameters (9 and 18), the kink-blocking model (blue dashed line) underestimates the effect of MgSO40 and Mg2+. The incorporation inhibition model (red dotted line) underestimates the effect of MgSO40 and gives a ﬁt with unphysical slope change for the Mg2+ data. The ﬁt with both models is reasonable for the SO42−data. assumptions about the ratio of acute/obtuse steps in our ﬁts, because these data are not available in a macroscopic measure- ment. This means that if growth is completely inhibited on one step type, but not the other, the ﬁtted adsorption energy values only reﬂect the growing step, which would dominate the macroscopic growth rate. In the case of strong inhibition on one type of step and a weak inhibition on the other type of step, the ﬁtted adsorption energy would describe the weak inhibition. Results Only by ﬁtting the model to step-speciﬁc data, can the inhibition on each type of step be obtained. The extended microkinetic growth model is able to reproduce the inhibiting effect of Mg2+, SO42−and MgSO40. Despite using only two free parameters in total for all the three ﬁts, it performs better than the previous models5; the residual is 0.15, compared with 0.26 and 0.16. Because there are no free, empirical parameters, we can interpret the results in light of the deriving growth inhibition mechanisms. The microkinetic model indicates that the effects of Mg2+ and SO42−are additive and independent of each other. Table 1 shows the derived adsorption energies of the ions. Both Mg2+ and SO42−adsorb more weakly than Ca2+ and CO32−, but only by ~20 %. The results indicate that the dominant mechanism for inhibition by Mg2+ and SO42−is ion adsorption on steps, which blocks the growth sites, preventing attachment of CaCO30 ion pairs. This is the only mechanism that affects SO42−. For single, weakly complexing inhibitors such as SO42−, the inhibition factor can be interpreted as the fraction of step sites covered by the inhibitor (Supplementary Note 3). For Mg2+, ion pairs formation in the aqueous phase contributes an additional ~10% to total inhibition at high magnesium concen- tration, so the solution speciation must be considered for quantitative agreement (more details in Supplementary Note 5). The results indicate that it is essential to include the solution speciation for accurate growth inhibition modelling of carbonate We ﬁt the model in Equations (1)–(3) to the measured inhibition index, which is constructed in such a way that the ﬁt parameter for the energy barrier, ΔGIP;CaCO3; cancels because only the relative changes in the growth rate matter. We used KCa ¼ 103:236 and KCO3 ¼ 103:355, which were calculated to be the geometric mean of the equilibrium constants for adsorption of Ca2+ and CO32−on the acute and obtuse steps7, obtained by ﬁtting the experimental data in Sand et al.31. This leaves the adsorption equilibrium constants KMg and KSO4 as the only free parameters. As solution complexing must be taken into account simultaneously, it is important to use the concentration of free ions available for adsorption in the model, in Equations (1)–(3). A schematic diagram of the model is presented in Fig. 1. Figure 2 shows the outcome of the various ﬁtting procedures, using the parameter values listed in Supplementary Note 3 (Supplementary Tables 1-3). Results We test the extended microkinetic model on calcite growth at ambient temperature in aqueous solutions containing ionic and small organic inhibitors. Magnesium is a known poison for calcite growth, and this is relevant for biomineralisation19–21 as well as industrial materials synthesis22, so we test the model with the recently published23 experimental data for magnesium (Mg2+), sulphate (SO42−) and their ion pair (MgSO40). Organic compounds, in the form of humic and fulvic acids, are common in the earth’s surface waters, and the carboxyl functional group is also active in molecules known to control calcite growth11 and biomineralisation12, so we test the model with new experimental data for calcite growth in solutions containing acetate (CH3COO−) and benzoate (C6H5COO−). Our results show that inhibition by the inorganic species Mg2+ and SO42−is caused mainly by adsorption on calcite steps, which blocks kink nucleation. However, inhibition by the small organic acids mainly results from complexing in the rMgSO4 ¼ a KIP;CaCO3 Ca2þ ½ CO2 3 1 þ KCa Ca2þ ½ þ KMg Mg2þ ½ 1 þ KCO3 CO2 3 þ KSO4 SO2 4 kT h e ΔGIP;CaCO3 RT ð3Þ ð3Þ We can then relate the microscopic growth rate, r, from the models to the measured inhibition index, Θ, by: Θ ¼ runinhib rinhib runinhib ð4Þ ð4Þ where rinhib represents any of the rates from Equations (1)–(3) (details in Supplementary Note 2) and runinhib can be obtained by setting the inhibitor concentration to 0 in Equations (1)–(3). runinhib in this case reduces to the rate, rIP. We do not make any where rinhib represents any of the rates from Equations (1)–(3) (details in Supplementary Note 2) and runinhib can be obtained by setting the inhibitor concentration to 0 in Equations (1)–(3). runinhib in this case reduces to the rate, rIP. We do not make any NATURE COMMUNICATIONS\| (2018) 9:1578 \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04022-0 CO3 SO4 Mg Ca Ca CO3 Ca CO3 Ca CO3 Fig. 1 The growth inhibition model. A generic step edge consisting of empty sites (transparent) and the sites occupied by various ions present in the solution. On calcite, kinks are formed by the attachment of a calcium carbonate ion pair (in red), which is assumed to be the rate determining reaction in the growth process. \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications Results The data were ﬁt with the kink- blocking and incorporation inhibition models for each inhibitor separately, using 3 and 6 free parameters per curve, as in the original models5. In spite of the large number of possible ﬁtting \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:1578 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04022-0 1 2 mM Ca + benzoate 2 mM Ca + acetate 3 mM Ca + acetate 4 mM Ca + acetate 5 mM Ca + acetate 0.8 0.6 Inhibition factor 0.4 0.2 0 0 20 40 Nominal acid concentration [mM] 60 80 100 Fig. 3 Comparison between the model and the experimental growth inhibition data for acetate and benzoate. The inhibition factor for calcite growth in the presence of the small carboxylate anions, acetate (ﬁlled squares) and benzoate (open diamonds). In the legend, x mM Ca refers to a starting solution of x mM CaCl2 and x mM NaHCO3 Table 1 Adsorption energies used in and obtained by the ﬁt to experiments Ion/inhibitor Adsorption energy (kJ/mol) Ca2+ −18.5 ± 2 CO32− −19.1 ± 2 Mg2+ −14.2 ± 0.3 SO42− −16.3 ± 0.3 Acetate −1.1 ± 0.3 Benzoate −2.8 ± 2.3 Table 1 Adsorption energies used in and obtained by the ﬁt to experiments Ion/inhibitor Adsorption energy (kJ/mol) Ca2+ −18.5 ± 2 CO32− −19.1 ± 2 Mg2+ −14.2 ± 0.3 SO42− −16.3 ± 0.3 Acetate −1.1 ± 0.3 Benzoate −2.8 ± 2.3 Table 1 Adsorption energies used in and obtained by the ﬁt to experiments Ion/inhibitor Adsorption energy (kJ/mol) Ca2+ −18.5 ± 2 CO32− −19.1 ± 2 Mg2+ −14.2 ± 0.3 SO42− −16.3 ± 0.3 Acetate −1.1 ± 0.3 Benzoate −2.8 ± 2.3 calcite 1014 f g surface has been predicted to signiﬁcantly change the adsorption energy of organic molecules compared with water32,33. Because adsorption and step blocking is a potentially important inhibitory effect, changing the surface chemistry can signiﬁcantly inﬂuence growth rate, and in more complex systems, where both cations and organic molecules are present, incorpora- tion might need to be taken into account to describe growth. Inhibition by carboxylic acids acetate and benzoate. The car- boxylic acids have been shown to inﬂuence calcite growth34,35. The inhibition mechanism has been assumed to be step blocking but this has not been proven. Therefore we made growth inhi- bition experiments using acetate and benzoate, two simple organic molecules containing a single carboxylate functional group. Results We carried out PHREEQC calculations to obtain the solution speciation for each set of conditions with varying inhi- bitor concentration. In our experiments, SI varied from slightly above 1 to 0. To obtain a smooth growth rate as a function of SI, we therefore used the modiﬁed IP model, where the constant growth rate is subtracted to make the net growth rate 0 at SI = 07. The comparison of the experimental data with the model ﬁt is shown in Fig. 3. The agreement of inhibition as a function of concentration was very good for both molecules, under quite different conditions. As for the inorganic ions, only one para- meter per inhibitor was needed, the step adsorption energy. Because the deprotonated acids are anions, we used Eq. 1 with carboxylate concentration instead of sulphate. The optimised adsorption energy for acetate was −1 kJ/mol and for benzoate was −3 kJ/mol, which is signiﬁcantly weaker than the divalent inorganic ions. The difference in the adsorption energy between acetate and benzoate from the model ﬁt is consistent with DFT predictions, which predict that benzoic acid binds slightly more strongly than acetic acid36. 1 2 mM Ca + benzoate 2 mM Ca + acetate 3 mM Ca + acetate 4 mM Ca + acetate 5 mM Ca + acetate 0.8 0.6 Inhibition factor 0.4 0.2 0 0 20 40 Nominal acid concentration [mM] 60 80 100 Fig. 3 Comparison between the model and the experimental growth inhibition data for acetate and benzoate. The inhibition factor for calcite growth in the presence of the small carboxylate anions, acetate (ﬁlled squares) and benzoate (open diamonds). In the legend, x mM Ca refers to a starting solution of x mM CaCl2 and x mM NaHCO3 1 2 mM Ca + benzoate 2 mM Ca + acetate 3 mM Ca + acetate 4 mM Ca + acetate 5 mM Ca + acetate 0.8 0.6 Inhibition factor 0.4 0.2 0 0 20 40 N i l id t ti [ M] 60 80 100 Nominal acid concentration [mM] Fig. 3 Comparison between the model and the experimental growth inhibition data for acetate and benzoate. The inhibition factor for calcite growth in the presence of the small carboxylate anions, acetate (ﬁlled squares) and benzoate (open diamonds). Results In the legend, x mM Ca refers to a starting solution of x mM CaCl2 and x mM NaHCO3 For the small carboxylates studied here, the change in SI and the decreased concentration of growth units is the dominating mechanism, but step blocking accounts for ~15% of the inhibition and must be considered to describe the experimental data (Fig. 3 and Supplementary Note 5). Solution complexing as the dominant inhibition mechanism is consistent with the observa- tion that the same concentration of inhibitor is more effective for low SI conditions than for high SI. Figure 3 shows this relationship for inhibitor concentration of 100 mM. The inhibi- tion factor drops from 0.7 to 0.3 for increasing Ca concentration (i.e. increasing SI). minerals. Equations (1)–(3) should therefore be used with concentrations or activities obtained from the PHREEQC calculations. 2+ 2 The adsorption energies and uncertainties for Ca2+ and CO32− were taken from Table S1 in Andersson et al.7 and averaged for the two types of calcite steps. Adsorption energies and uncertainties for Mg2+, SO42, acetate and benzoate are from this work. They were obtained from the ﬁts to the experimental data. \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications Methods C t t Constant composition experiments. The constant composition-method setup has been used in the previous work23,38. Stock solutions of CaCl2, NaHCO3, Na2CO3 were prepared using reagent grade chemicals from Sigma-Aldrich. At the start of the experiment, 50 mL of 0.1 M NaCl solution containing equivalent aliquots of CaCl2 and NaHCO3 were added to target the required concentration, and the pH was adjusted to 8.3 by the addition of a few µL of 0.1 M NaOH. The 50 mL solution was introduced in a steered glass reactor vessel at room temperature (22 ± 1 ℃). When the pH was stable at 8.3 for about 30 min, the calcite seed (Merck calcite powder 99.95% Suprapure) was added to the system to initiate precipitation. When a constant growth rate was achieved, addition of inhibitors obtained from the stock solution was added. The change in calcite volume was less than 4% in the max- imum concentration run. The fractional inhibition was calculated as (R0−R1)/R0, where R0 is the initial growth rate and R1 is the growth rate after addition of the inhibitors. The PHREEQC calculations for the constant composition setup and carboxylate inhibition were conducted using the Minteq.v4 database. 19. Davis, K. J., Dove, P. M. & De Yoreo, J. J. The role of Mg2+ as an impurity in calcite growth. Science 290, 1134–1137 (2000). 20. Weiner, S. & Dove, P. M. in Biomineralization Vol. 54 Reviews in Mineralogy and Geochemistry (eds Dove, P. M., De Yoreo, J. J. & Weiner, S.) 1–30 (The Mineralogical Society of America, Chantilly, USA, 2013). 21. Sand, K. K., Pedersen, C. S., Matthiesen, J., Dobberschütz, S. & Stipp, S. L. S. Controlling biomineralization with cations. Nanoscale 9, 12925–12933 (2017). 22. Chen, T., Neville, A. & Yuan, M. Assessing the effect of on scale formation–bulk precipitation and surface deposition. J. Cryst. Growth 275, e1341–e1347 (2005). 23. Nielsen, M. R. et al. Inhibition of calcite growth: combined effects of Mg2+ and SO42−. Cryst. Growth Des. 16, 6199–6207 (2016). y 24. Olafson, K. N., Li, R., Alamani, B. G. & Rimer, J. D. Engineering crystal modiﬁers: Bridging classical and nonclassical crystallization. Chem. Mater. 28, 8453–8465 (2016). 25. Parkhurst, D. L. & Appelo, C. A. J. Description of Input and Examples for PHREEQC. Version 3 – A Computer Program for Speciation, Batch-Reaction, One-Dimensional Transport, and Inverse Geochemical Calculations Vol. 6 (U. S. Geological Survey Techniques and Methods, 2013). Fitting of the experimental inhibition experiments. References 1. Aizenberg, J., Muller, D. A., Grazul, J. L. & Hamann, D. R. Direct fabrication of large micropatterned single crystals. Science 299, 1205–1208 (2003). 31. Sand, K. K. et al. Calcite growth kinetics: dependence on saturation index, Ca2+:CO32−activity ratio, and surface atomic structure. Cryst. Growth Des. 16, 3602–3612 (2016). 2. Meyers, M. A., McKittrick, J. & Chen, P. Y. Structural biological materials: critical mechanics-materials connections. Science 339, 773–779 (2013). 3. Atkinson, G. & Mecik, M. The chemistry of scale prediction. J. Pet. Sci. Eng. 17, 113–121 (1997). 32. Sakuma, H., Andersson, M. P., Bechgaard, K. & Stipp, S. L. S. Surface tension alteration on calcite, induced by ion substitution. J. Phys. Chem. C 118, 3078–3087 (2014). 4. Cabrera, N. & Vermilyea, D. in Proceedings Growth and Perfection of Crystals (ed. Doremus, R. H.) 391-410 (Wiley, New York, USA, 1958). 33. Andersson, M. P., Dideriksen, K., Sakuma, H. & Stipp, S. L. S. Modelling how incorporation of divalent cations affects calcite wettability-implications for biomineralisation and oil recovery. Sci. Rep. 6, 28854 (2016). 5. Nielsen, L. C., De Yoreo, J. J. & DePaolo, D. J. General model for calcite growth kinetics in the presence of impurity ions. Geochim. Cosmochim. Acta 115, 100–114 (2013). 34. Aizenberg, J., Black, A. J. & Whitesides, G. H. Oriented growth of calcite controlled by self-assembled monolayers of functionalized alkanethiols supported on gold and silver. J. Am. Chem. Soc. 121, 4500–4509 (1999). 6. Zhang, J. W. & Nancollas, G. H. Kink density and rate of step movement during growth and dissolution of an AB crystal in a nonstoichiometric solution. J. Colloid Interface Sci. 200, 131–145 (1998). pp g 35. Nielsen, J. W. et al. Polysaccharide effects on calcite growth: the inﬂuence of composition and branching. Cryst. Growth Des. 12, 4906–4910 (2012). f 7. Andersson, M. P. et al. A microkinetic model of calcite step growth. Angew. Chem. Int. Ed. 55, 11086–11090 (2016). 36. Ataman, E., Andersson, M. P., Ceccato, M., Bovet, N. & Stipp, S. L. S. Adsorption on calcite: I. Oxygen containing and nonpolar organic molecules J. Phys. Chem. C 120, 16586–16596 (2016). 8. Mann, S. Molecular recognition in biomineralization. Nature 332, 119–124 (1988). 9. Weiner, S. & Dove, P. M. An overview of biomineralization processes and the problem of the vital effect. Rev. Mineral. Geochem. 54, 1–29 (2003). 37. Astilleros, J. M., Fernandez-Diaz, L. & Putnis, A. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04022-0 the main inhibition mechanisms8,37, is predicted to account for <5–10% of the total inhibition. 12. Borman, A. H. et al. The role in CaCO3 crystallization of an acid-Ca2+ binding polysaccharide associated with coccoliths of Emiliania-Huxleyi. Eur. J. Biochem. 129, 179–183 (1982). With the ability to predict the extent of crystal growth and recrystallisation and the effect of inhibitors, we move closer to understanding large scale (in space and time) processes that shape our earth and its climate. With this new understanding about the molecular scale mechanisms, we come closer to understanding the processes that control biomineralisation, and with the growth inhibition model, it becomes possible to systematically design improved growth inhibitors, for example, by computational screening of complexing done by the dominant ions (in the case of calcite, for Ca2+or CO32) and by determining the adsorption energies of the inhibitor species on the calcite steps. In general, strong adsorption allows for effective inhibition with lower con- centrations of inhibitor. 13. de Yoreo, J. J., Wierzbicki, A. & Dove, P. M. New insights into mechanisms of biomolecular control on growth of inorganic crystals. CrystEngComm 9, 1144–1152 (2007). 14. Gordon, L. M. & Joester, D. Nanoscale chemical tomography of buried organic-inorganic interfaces in the chiton tooth. Nature 469, 194–197 (2011). 15. Sviben, S. et al. A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga. Nat. Commun. 7, 11228 (2016). 16. Giuffre, A. J., Hamm, L. M., Han, N., De Yoreo, J. J. & Dove, P. M. Polysaccharide chemistry regulates kinetics of calcite nucleation through competition of interfacial energies. Proc. Natl Acad. Sci. USA 110, 9261–9266 (2013). 17. Hamm, L. M. et al. Reconciling disparate views of template-directed nucleation through measurement of calcite nucleation kinetics and binding energies. Proc. Natl Acad. Sci. USA 111, 1304–1309 (2014). energies. Proc. Natl Acad. Sci. USA 111, 1304–1309 (2014). 18. Smeets, P. J. M., Cho, K. R., Kempen, R. G. E., Sommerdijk, N. A. J. M. & De Yoreo, J. J. Calcium carbonate nucleation driven by ion binding in a biomimetic matrix revealed by in situ electron microscopy. Nat. Mater. 14, 394–399 (2015). Methods C t t A standard, nonlinear least squares method in the form of a trust-region reﬂective algorithm39 with a central difference scheme was used for all ﬁts, except for the incorporation inhibition model. Because of its heavy nonlinear nature, we used simulated annealing.40,41 Termination tolerances were set to 1E−15 in all cases, and numerous starting values were used to ensure that the algorithms were not trapped in local optima. 26. Plummer, L. N. & Busenberg, E. The solubilities of calcite, aragonite and vaterite in CO2-H2O solutions between 0 and 90 °C, and an evaluation of the aqueous model for the system CaCO3-CO2-H2O. Geochim. Cosmochim. Acta 46, 1011–1040 (1982). Data availability. All data are available from the authors on reasonable request 27. Sverjensky, D. A., Shock, E. L. & Helgeson, H. C. Prediction of the thermodynamic properties of aqueous metal complexes to 1000 °C and 5 kb. Geochim. Cosmochim. Acta 61, 1359–1412 (1997). Received: 27 July 2017 Accepted: 27 March 2018 Received: 27 July 2017 Accepted: 27 March 2018 Received: 27 July 2017 Accepted: 27 March 2018 28. Sillén, L. G. On equilibria in systems with polynuclear complex formation. 5. Some useful differential expressions. Acta Chem. Scand. 15, 1981–1992 (1961). 29. Demichelis, R., Raiteri, P., Gale, J. D., Quigley, D. & Gebauer, D. Stable prenucleation mineral clusters are liquid-like ionic polymers. Nat. Commun. 2, 590 (2011). 30. Habraken, W. et al. Ion-association complexes unite classical and non- classical theories for the biomimetic nucleation of calcium phosphate. Nat. Commun. 4, 1507 (2013). Discussion Only the free energy from the binding of the inhibitor to the calcite step is necessary to determine the dominant calcite growth inhibition mechanism for these small organic and inorganic inhibitors. The inhibition mechanism ranges from only step blocking (SO42−), through mainly step blocking with minor contributions from solution complexing (Mg, MgSO4), to mainly solution complexing with minor inﬂuence of step blocking (acetate, benzoate). Good agreement was obtained over a range of inhibitors and inhibition factors. There is evidence that ions are incorporated into the growing crystal23, but the good ﬁt of the model to the experimental growth data, without considering incorporation, suggests that incorpora- tion is a minor or negligible growth inhibition mechanism. Substituting ions can be incorporated to form a solid solution without signiﬁcant effect on the crystallisation rate. Incorporation without inhibition agrees with previous results7, though we observe (in Fig. 2) a small discrepancy between predictions and the experimental data. The discrepency is negligible for low inhibitor concentrations and is <5–10%, even for the highest Mg2+ and MgSO40 concentrations. This small underestimation of the inhibitory effect at high Mg2+ concentration could result from Mg2+ incorporation. Solution complexing must be considered simultaneously to adsorption in order to obtain quantitative agreement between the model and the experiment. At least for the inorganic inhibitors studied here, solution complexing was less effective than blocking by adsorption, in spite of a slightly larger equilibrium constant for solution-ion pair formation (102.98) than step adsorption energy (102.8, derived from the ﬁtted adsorption energy in Table 1). Inhibition via step blocking is, unsurprisingly, quite sensitive to the adsorption energy of the inhibitor (Supplementary Note 6). Magnesium incorporation, which has been suggested as one of g While cation incorporation into calcite does not inﬂuence the growth rate signiﬁcantly in itself, the subsequent change in surface composition would have a signiﬁcant effect on surface energy32,33 and thus impact the afﬁnity of organic inhibitors that might also be present. Divalent cation incorporation into the NATURE COMMUNICATIONS\| (2018) 9:1578 4 \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications Competing interests: The authors declare no competing interests. 41. Cerny, V. Thermodynamical approach to the traveling salesman problem - an efﬁcient simulation algorithm. J. Optim. Theory Appl. 45, 41–51 (1985). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Author contributions The ideas came from and were developed by M.P.A., S.D., M.R.N., K.K.S., N.B. and S.L.S. S. M.R.N. collected the experimental data for the inorganic inhibitors. R.C. and N.B. collected the data and performed PHREEQC calculations for the organic inhibitors. S.D. was responsible for developing the code for the previously published models and for all model ﬁtting to the experimental data for inorganic ions. M.P.A. ﬁtted the model to the organic inhibitors. M.R.N. and M.P.A. analysed and modiﬁed the growth rate equations. M.R.N. and S.D. wrote the ﬁrst draft and all authors contributed to scientiﬁc discussion and the ﬁnal paper. Acknowledgements Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. We thank K. West and L. Lakshtanov for the constant composition set up. Funding was provided by the UK Engineering and Physical Sciences Research Council [EPSRC Grant Number EP/I001514/1], through the Materials Interface with Biology (MIB) Consortium. This study sprang from work that originated under Nano-Chalk, funded by the Danish Advanced Technology Foundation (HTF) and Maersk Oil and Gas A/S and continued under W-EOR, supported by Maersk Oil. K.K.S. is grateful for funding from the Danish Council for Independent Research, Sapere Aude Program (0602-02654B). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04022-0 40. Kirkpatrick, S., Gelatt, C. D. & Vecchi, M. P. Optimization by simulated annealing. Science 220, 671–680 (1983). Competing interests: The authors declare no competing interests. References The role of magnesium in the growth of calcite: an AFM study. Chem. Geol. 271, 52–58 (2010). 10. Albeck, S., Aizenberg, J., Addadi, L. & Weiner, S. Interactions of various skeletal intracrystalline components with calcite crystals. J. Am. Chem. Soc. 115, 11691–11697 (1993). 38. Lakshtanov, L. Z., Bovet, N. & Stipp, S. L. S. Inhibition of calcite growth by alginate. Geochim. Cosmochim. Acta 75, 3945–3955 (2011). g 39. Coleman, T. F. & Li, Y. Y. An interior trust region approach for nonlinear minimization subject to bounds. SIAM J. Optim. 6, 418–445 (1996). 11. Geoffroy, C., Foissy, A., Persello, J. & Cabane, B. Surface complexation of calcite by carboxylates in water. J. Colloid Interface Sci. 211, 45–53 (1999). 5 NATURE COMMUNICATIONS\| (2018) 9:1578 5 \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications © The Author(s) 2018 \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-04022-0. pp 018-04022-0. pp 018-04022-0. NATURE COMMUNICATIONS\| (2018) 9:1578 \| DOI: 10.1038/s41467-018-04022-0\| www.nature.com/naturecommunications 6 6
https://openalex.org/W1997209371	https://www.scielo.br/j/cagro/a/GXMmhKdC6xS4DQLX9Lm6nFv/?lang=pt&format=pdf	Portuguese	null	Semeadura do híbrido Lyra de mamona (Ricinus communis L.) sob plantio direto	Ciência e Agrotecnologia	2,008	cc-by	4,203	ABSTRACT The mechanized sowing of castor bean (Ricinus communis L.) can reduce the operation time and the costs associated to labor, however the privation of information in this area have damaged the correct adoption of this technology. Thus, the research had as an aim to verify the regularity of longitudinal distribution of hybrid Lyra castor bean seeds, in a no-tillage system area, and its relationship with yield; and the viability of control charts for evaluation of sowing in this crop. The essay was carded out in a commercial area of grain production in no-tillage system. one evaluated: longitudinal distribution, percentage of normal, defective and double spaces in line, number of plants per linear meter, height of plants, height and length of primer raceme and yield. For the evaluation of the proposed parameters, one used the describable statistic, the correlation analysis and control charts. The results allowed to conclude that it is necessary to enhance the regulation of seeder-fertilizer for mechanized sowing of castor bean hybrid Lyra, in no-tillage system areas. The longitudinal distribution of castor bean affect seeds grain yield and other phytotechnic parameters and the control charts are efficient to evaluate the mechanized castor bean sowing. Index terms: Sowing, no-tillage, control charts, Ricinus communis. Seeding of castor bean (Ricinus communis L.) hybrid Lyra in no-tillage system Nilza Patrícia Ramos1, Juliana Altafin Galli 2, Edson Perito Amorim3, Marcos Roberto da Silva4, Antonio Lúcio Mello Martins2 RESUMO A semeadura mecanizada da mamona (Ricinus communis L.) pode reduzir o tempo de operação e os custos associados à mão- de-obra, porém a escassez de informações nesta área tem prejudicado a adoção correta desta tecnologia. Assim, objetivou-se com esse trabalho verificar a regularidade da distribuição longitudinal de sementes do híbrido Lyra de mamona, em área de plantio direto e sua relação com rendimento de grãos; bem como a viabilidade da aplicação de cartas de controle para a avaliação do processo de semeadura, nesta cultura. O ensaio foi conduzido em área comercial de produção de grãos de mamona sob plantio direto, sendo avaliadas: distribuição longitudinal, porcentagens de espaçamentos normais, falhos e duplos na linha, número de planta por metro linear, altura de plantas, altura e comprimento do racemo primário e rendimento de grãos. Utilizou-se a estatística descritiva, a análise de correlações e as cartas de controle para a avaliação dos parâmetros propostos. Há necessidade de refinamento nas regulagens de semeadoras-adubadoras de precisão para a semeadura do híbrido Lyra de mamona, em áreas de plantio direto; a distribuição longitudinal de sementes de mamona influencia vários parâmetros fitotécnicos, incluindo o rendimento de grãos; a aplicação da carta de controle é uma ferramenta eficiente para a avaliação do processo de semeadura da mamona. Termos para indexação: Semeadura, plantio direto, cartas de controle, Ricinus communis. Index terms: Sowing, no-tillage, control charts, Ricinus communis. MATERIAL E MÉTODOS O ensaio foi realizado em área de produção comercial de mamona do Pólo Regional de Desenvolvimento Tecnológico dos Agronegócios Centro Norte, localizado em Pindorama-SP, durante o período de safrinha (fevereiro a julho), em 2006. O solo da região é classificado como Argissolo eutrófico A moderado, textura arenosa/ média, Unidade Pindorama, e o clima caracteriza-se pela temperatura média anual de 22,8 ºC, precipitação média anual de 1.390,3 mm com altitudes variando de 498 a 594m. Os requisitos de regularidade da distribuição longitudinal de sementes para semeadoras devem estar de acordo com o mecanismo dosador empregado. Coelho et al. (1992) afirmam que os valores mínimos aceitáveis de espaçamento e máximos coeficientes de variação admissíveis para o mecanismo do tipo pneumático são 90% e 30%, respectivamente. Entretanto, esses limites de tolerância podem se alterar, em função da cultura, pois a mamona, por exemplo, pode ter valores mínimos aceitáveis de espaçamento de até 80%, pela dificuldade de distribuição de sementes, em função da classificação dos lotes em um pequeno número de peneiras e do cultivo em espaçamentos largos (0,90 e 1,00 m), que desfavorecem a uniformidade da distribuição de sementes pelas máquinas. Como material vegetal utilizou-se um lote comercial de sementes do híbrido Lyra de mamona; semeado em 24/ 02/2006 em sistema plantio direto, o qual já estava implantado na área há três anos. Anteriormente, esta mesma área foi ocupada com um campo de produção de sementes de mamona, semeado em janeiro de 2005 e colhido de maneira parcelada de junho até outubro deste referido ano. Após a colheita os restos culturais permaneceram na área, sendo roçados no mês de dezembro, para a melhor distribuição de massa seca sobre a superfície do solo. Aproximadamente 20 dias antes da semeadura, aplicou-se glifosate (4L ha-1) e 2,4D (2L ha-1) para eliminação das plantas remanescentes de mamona e outras infestantes. A verificação da qualidade de processos pode ser feita pelo controle estatístico de processo (CEP) que auxilia a detecção rápida de variações não-aleatórias envolvendo o uso de técnicas estatísticas (MONTGOMERY, 1997). Também, a carta de controle é uma técnica primária do CEP utilizada no monitoramento do mesmo, pois reflete a variabilidade existente no sistema. Entre as vantagens citadas para seu uso destacam-se: identificação de desvios resultantes de causas especiais, tornando o processo previsível em termos de atender ou não as especificações desejadas, e determinação da necessidade de alterações quando necessário (ANTUNES & ENGEL, 1999). INTRODUÇÃO FILHO, 2005). Este panorama vem se modificando com os avanços tecnológicos alcançados dentro desta cadeia produtiva, pela colaboração de técnicos de extensão e pesquisa, e do maior interesse de exploração em grandes áreas. A cultura da mamona (Ricinus communis L.), no Brasil, caracteriza-se historicamente pelo cultivo em áreas de baixo nível tecnológico, onde se inclui a semeadura manual, em covas, com espaçamentos largos e posterior operação de desbaste acarretando, muitas vezes, incrementos significativos nos custos de produção pelo uso intensivo de mão-de-obra (SANTOS et al., 2001; SAVY Como regra geral, na produção de grãos, a operação de semeadura é considerada um dos processos mais relevantes (SILVA & DANIEL, 2004), pois deve assegurar a dosagem recomendada de sementes por área, o Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 482 RAMOS, N. P. et al. acondicionamento na profundidade desejada e a distribuição uniforme das sementes ao longo da linha. Neste contexto, a mecanização desta operação visa, principalmente, maior eficiência e rapidez, além do conforto oferecido ao operador, potencializando o trabalho individual e o rendimento (SILVEIRA et al., 2005). de especificação do processo e possibilita a análise de desempenho, facilitando correções. Assim, as cartas são ferramentas que permitem análises rápidas e objetivas da operação de semeadura, podendo muitas vezes substituir a análise estatística convencional, que é mais demorada e laboriosa. Kurachi et al. (1986) relataram que as semeadoras- adubadoras, de diferentes tipos e modelos existentes no mercado brasileiro, devem ter sua eficiência avaliada por meio de dois parâmetros principais com relação à distribuição longitudinal de sementes, sendo eles, a porcentagem de espaçamentos aceitáveis e o coeficiente de variação geral dos espaçamentos aceitáveis. A uniformidade de distribuição longitudinal de sementes foi apontada entre as que mais contribuem para um estande adequado de plantas de milho e para a melhoria do seu rendimento (KURACHI et al., 1989). Como contribuição para esclarecimentos tecnológicos na cadeia produtiva da mamona, objetivou- se com este trabalho verificar a regularidade da distribuição longitudinal de sementes do híbrido Lyra de mamona, em área de plantio direto e sua relação com o rendimento de grãos, bem como a viabilidade da aplicação da carta de controle como ferramenta de avaliação e melhoria do processo de semeadura dessa cultura. Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 MATERIAL E MÉTODOS A operação de semeadura foi feita com uma semeadora-adubadora de precisão, marca Jumil, modelo 2600SH para plantio direto, com dosador de sementes do tipo pneumático e quatro unidades de semeadura espaçadas em 90 cm; a velocidade de deslocamento do conjunto trator-semeadora foi de 6 km h-1. O disco de semeadura foi utilizado na recomendação de 1,25 plantas de mamona por metro linear. O uso das cartas de controle foi eficiente para verificar a qualidade do processo de distribuição longitudinal de sementes de girassol, em ensaios conduzidos por Lino et al. (2003) trabalhando em bancadas de laboratório, e Silva et al. (2005), trabalhando com avaliações em campos comerciais, quando se conclui que sua aplicação permite a comparação imediata com os limites O uso das cartas de controle foi eficiente para verificar a qualidade do processo de distribuição longitudinal de sementes de girassol, em ensaios conduzidos por Lino et al. (2003) trabalhando em bancadas de laboratório, e Silva et al. (2005), trabalhando com avaliações em campos comerciais, quando se conclui que sua aplicação permite a comparação imediata com os limites No ponto de colheita da cultura, foram avaliados os parâmetros: distribuição longitudinal de sementes (DLS), em centímetros; porcentagem de espaçamentos aceitáveis (EN), falhos (EF) e duplos (ED) na linha Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 Semeadura do híbrido Lyra de mamona ... 483 (KURACHI et al., 1989); número de plantas por metro linear (Pl m-1); altura de plantas (AP), altura de inserção (IR) e comprimento do racemo primário (CR) em centímetros, além de rendimento de grãos (REND) em kg ha-1. As amostras foram retiradas em cinco metros lineares de cada uma das linhas de semeadura, em 20 pontos da área de produção, eqüidistantes entre si. (KURACHI et al., 1989); número de plantas por metro linear (Pl m-1); altura de plantas (AP), altura de inserção (IR) e comprimento do racemo primário (CR) em centímetros, além de rendimento de grãos (REND) em kg ha-1. As amostras foram retiradas em cinco metros lineares de cada uma das linhas de semeadura, em 20 pontos da área de produção, eqüidistantes entre si. encontravam-se elevados, sendo que para a distribuição longitudinal de sementes (DLS) o valor foi de 50,92%, bastante superior ao limite estabelecido por Coelho et al. (1992) para semeadoras pneumáticas, que foi de 30%. MATERIAL E MÉTODOS Este valor elevado sugere problemas graves na operação de semeadura, envolvendo a desuniformidade na distribuição de sementes na linha de semeadura, aspectos relacionados ao acondicionamento dessas sementes em profundidade inadequada e com a pressão exercida pelo mecanismo compactador do sulco. Utilizou-se a estatística descritiva para análise dos dados, além das correlações de Pearson; sendo a distribuição longitudinal de sementes também avaliada por carta de controle (LINO et al., 2003). A partir da recomendação de 1,25 plantas por metro linear, os limites de controle da distribuição longitudinal foram estipulados, tendo como ponto de referência central (x ref.) 80 cm. A média de DLS foi de 131,89 cm, enquanto o valor de referência seria 80 cm, indicando um desvio superior a 64 pontos percentuais, em relação ao desejado na operação; sendo classificado como ruim por Casão Júnior & Siqueira (2003). Mesmo para a mamona, que poderia tolerar um limite de desvio superior aos 10% propostos como bons por esses autores, os valores observados não foram satisfatórios e comprovam a necessidade de maiores cuidados na semeadura dessa cultura, onde deve ser incluída a maior atenção com as regulagens de rotina e também do operador responsável pela semeadura do conjunto. A denominação das classes foi proposta para girassol por Ungaro et al. (1999), enquanto os intervalos foram adaptados para a mamona a partir de intervalos anteriores propostos por Casão Júnior & Siqueira (2003). Sendo consideradas as faixas: inaceitável abaixo do ideal as distâncias entre plantas <64 cm; aceitável abaixo do ideal entre 65-71 cm; ideal entre 72-88 cm; aceitável acima do ideal entre 89-96 cm e inaceitável alto > 97 cm. Tanto a análise descritiva, quanto às estimativas das correlações de Pearson entre as características foram obtidas utilizando-se o pacote estatístico computacional SAS v. 8.1 (SAS INSTITUTE, 2000). Com relação à distribuição das porcentagens de espaçamentos aceitáveis, falhos e duplos (Tabela 1) foi verificada uma grande incidência de falhas ( = 60,88%), com valores máximos de até 95,83%, indicando a ausência quase que total de plantas em alguns pontos analisados. Por outro lado, os valores aceitáveis variaram de 4,17 a 48,43%, com coeficiente de variação de 44,86%, o que demonstra a ineficácia do processo de semeadura na área de produção. Mahl et al. (2004) também obtiveram altos coeficientes de x Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 Max valor máximo observado; Min valor mínimo observado; Ampl amplitude de variação; CV% - coeficiente de variação experimental em porcentagem; DP- desvio padrão. RESULTADOS E DISCUSSÃO Talvez, a maior quantidade de falhas se deva ao cultivo com espaçamentos maiores entre as plantas, na linha, e às características físicas e fisiológicas (mais pesadas, de tamanho grande e vigor reduzido) das sementes de mamona que reduzem as possibilidades de deposição duplas de sementes em comparação à maioria das culturas graníferas, semeadas mecanicamente. Lembrando que a distribuição e o nível de danificação das sementes dependem, principalmente, da forma, do tamanho e da uniformidade no tamanho das sementes (ROCHA et al., 1998). O híbrido Lyra de mamona, pelo seu porte baixo possui uma tendência de cultivo mais adensado, principalmente no período de safrinha, que implica em menor disponibilidade de chuvas, o que confirma a alta correlação de 0,91 observada entre PL m-1 e REND (Tabela 2). Neste caso, foram observadas populações variando de 2.780 a 23.333 pl ha-1, explicando os 85% de variação no rendimento de grãos. Assim, estudos de populações superiores a 20.000 pl ha-1 são interessantes para recomendações deste híbrido para cultivo em safrinha na região estudada. Também a necessidade de treinamento e maior intimidade dos técnicos com a cultura da mamona pode ter interferido na qualidade da operação, neste sentido Santos (2005), trabalhando com qualidade na avaliação do processo de pulverização de defensivos, demonstrou claramente a interferência do fator mão-de-obra na qualidade do processo e da necessidade de qualificação profissional. Várias outras correlações significativas entre os parâmetros analisados foram encontradas (Tabela 2), onde REND só não se correlacionou com altura de plantas (AP) e comprimento do racemos (CR). A correlação entre AP e REND varia com a espécie vegetal, sendo que no caso do híbrido Lyra de mamona, que possui caracteristicamente apenas o racemo primário e algumas vezes os secundários contribuindo para o valor final de altura, esperava-se encontrar Quando se comparou a elevada porcentagem de falhas com o rendimento de grãos, observou-se efeito altamente negativo, com variação de mais de 85% entre o valor mínimo e máximo obtidos, (208,89 e 1193,10 kg ha-1, Tabela 2 Correlações de Pearson e suas significâncias para os parâmetros: distribuição longitudinal de sementes (DLS); porcentagem de espaçamentos aceitáveis (EN), falhos (EF) e duplos (ED) na linha; número de plantas por metro linear (Pl m-1); altura de plantas (AP) e de inserção do primeiro racemo (IR); comprimento do racemo (CR) e rendimento de grãos (REND); avaliados em plantas do híbrido de mamona Lyra, conduzidas em sistema plantio direto. RESULTADOS E DISCUSSÃO Com base na estatística descritiva (Tabela 1), observou-se que os coeficientes de variação para a maioria dos caracteres relacionados ao processo de semeadura Tabela 1 Estatística descritiva da distribuição longitudinal de sementes (DLS-cm); porcentagem de espaçamentos aceitáveis (EN), falhos (EF) e duplos (ED) na linha; número de plantas por metro linear (Pl m-1); altura de plantas (AP - cm) e de inserção do racemo primário (IR- cm); comprimento do racemo (CR - cm) e rendimento de grãos (REND - kg ha-1), avaliados em plantas do híbrido de mamona Lyra, conduzidas em sistema plantio direto. Pindorama SP, 2006. Max valor máximo observado; Min valor mínimo observado; Ampl amplitude de variação; CV% - coeficiente de variação experimental em porcentagem; DP- desvio padrão. DLS EN EF ED Pl m-1 AP IR CR REND Média 131,89 28,37 60,88 12,94 0,82 85,81 42,07 34,91 674,61 Mediana 110,34 31,25 61,90 8,04 0,70 87,92 41,68 35,42 431,98 Max 295,00 48,43 95,83 42,33 2,10 100,63 51,25 41,69 1193,10 Min 51,35 4,17 12,50 0,00 0,25 70,50 36,50 26,24 208,89 Ampl. 343,65 44,26 83,33 42,33 1,85 30,13 14,75 15,45 984,22 CV% 50,92 44,86 37,02 107,34 60,16 9,89 9,86 11,74 40,74 DP 67,15 12,72 22,54 13,89 0,49 4,15 6,50 4,10 273,55 Max valor máximo observado; Min valor mínimo observado; Ampl amplitude de variação; CV% - coeficiente de variação experimental em porcentagem; DP- desvio padrão. Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 RAMOS, N. P. et al. 484 respectivamente). Esta observação se confirma na Tabela 2, onde o rendimento de grãos (REND) e DLS são inversamente correlacionados, com alto nível de significância, como já era esperado, pois com o aumento das DLS tem-se menor número de plantas por área, o que reduz rendimento de grãos. Assim, a redução de 34,4% no número de plantas por metro linear (PL m-1) (Tabela 1) em relação ao valor desejado (1,25 pl m-1) interferiu negativamente no rendimento de mamona, notando-se também correlação positiva e altamente significativa entre esses fatores (Tabela 2). variação para a porcentagem de espaçamentos falhos e duplos na cultura do milho, ao contrário de Branquinho et al. (2004), trabalhando com soja, não observaram altos valores no coeficiente de variação para esses parâmetros, mas concluíram que menos da metade das sementes foram depositadas em espaçamentos aceitáveis (= 44,80%), independente do manejo de solo adotado e da velocidade da operação. ,: significativo a 1 e 5% pelo teste t, respectivamente; ns: não significativo. RESULTADOS E DISCUSSÃO Pindorama SP 2006 ,: significativo a 1 e 5% pelo teste t, respectivamente; ns: não significativo. DLS EN EF ED Pl m-1 AP IR CR REND DLS - -0,59 -0,92 -0,66 -0,58 -0,08n.s. -0,40n.s. -0,04n.s. -0,71 EN - -0,88 0,54 0,70** 0,17n.s 0,48* -0,01n.s. 0,66 EF - -0,85 -0,92** 0,05n.s. -0,55* 0,10n.s. -0,89 ED - 0,88 -0,26n.s. 0,56* -0,17n.s. 0,91** Pl m-1 - -0,28n.s. 0,52* -0,30n.s. 0,86 AP - 0,29n.s. 0,75 -0,01n.s. IR - 0,19n.s. 0,56* CR - 0,08n.s. REND - Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 Semeadura do híbrido Lyra de mamona ... 485 ajuste de pressão do mecanismo de corte. A maior quantidade de resíduos de raízes e caule também podem prejudicar a abertura e fechamento dos sulcos de semeadura. correlação entre esses parâmetros. Também, não se observou a correlação entre CR e REND, talvez pela alta porcentagem de grãos chochos obtidos nesta área, em função da baixa precipitação do período (= 365 mm), concentrando-se menos de 65 mm durante o enchimento de grãos. Na literatura não foram encontrados relatos de semeadura mecânica de mamona, especialmente em áreas cultivadas anteriormente com essa mesma cultura, ficando difícil a comparação de resultados. Assim infere-se, com base nos dados levantados, que o uso da carta de controle de processos permite a visualização das variações pontuais da operação e pode auxiliar o ajuste na regulagem da distribuição de sementes, evitando prejuízos. A ferramenta demonstrou claramente a necessidade de maior refinamento na regulagem da máquina, associada ao treinamento da mão-de-obra para a o processo da semeadura, além da intensificação de pesquisas com esta cultura que esclareçam as melhores populações de plantas, em função da época de cultivo e das condições edafoclimáticas. A estatística descritiva permitiu uma análise numérica da operação de semeadura, refletindo a necessidade de melhor regulagem da máquina. Porém, esta análise não foi eficiente para verificar o comportamento pontual da distribuição de sementes ao longo da linha de semeadura, que possibilita identificar os erros na regulagem da máquina, os quais podem ser identificados pelas cartas de controle (MONTGOMERY, 1997). As falhas de DLS de mamona ficam bem claras ao se analisar a carta de controle do processo de semeadura do híbrido Lyra (Figura 1), onde 65 % dos pontos ficaram fora dos limites aceitáveis de distribuição. RESULTADOS E DISCUSSÃO Deste total, 92,7 % representaram distâncias superiores as aceitáveis, indicando falhas de distribuição, talvez pela não adequação de nível de vácuo do mecanismo pneumático, semeadura rasa, alta velocidade de deslocamento, fechamento ineficiente do sulco, entre outras possibilidades. CONCLUSÕES Há necessidade de refinamento e aferição nas regulagens de semeadoras-adubadoras de precisão para a semeadura mecânica do híbrido Lyra de mamona, em áreas de plantio direto. O fato da palhada anterior também ser de mamona, pode ter sido uma das causas de menor sucesso da semeadura mecânica, pois os restos vegetais gerados são maiores e mais fibrosos, exigindo mais dos discos de corte de palha, sendo necessária uma adequação especial no A distribuição longitudinal de sementes de mamona influencia vários parâmetros fitotécnicos, onde se inclui o rendimento de grãos da cultura. Figura 1 Carta de controle da distribuição longitudinal de plantas, por semeadora-adubadora tipo pneumática, observada em cultivo de comercial de mamona, híbrido Lyra, no sistema plantio direto de preparo de solo. Pindorama SP, 2006. 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 aceitável acima espaçamento observado ideal Xref ideal aceitável abaixo Figura 1 Carta de controle da distribuição longitudinal de plantas, por semeadora-adubadora tipo pneumática, observada em cultivo de comercial de mamona, híbrido Lyra, no sistema plantio direto de preparo de solo. Pindorama SP, 2006. Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 486 RAMOS, N. P. et al. velocidade e condição de solo. Engenharia Agrícola, Jaboticabal, v. 24, n. 1, p. 150-157, 2004. A aplicação da carta de controle de processos é uma ferramenta eficiente para a avaliação da semeadura de mamona. MONTGOMERY, D. C. Introduction to statistical quality control. New York: J. Wiley, 1997. 677 p. REFERÊNCIAS BIBLIOGRÁFICAS ANTUNES, L. M.; ENGEL, A. Qualidade total na agropecuária. Guariba: Agropecuária, 1999, 116 p. ROCHA, F. E. C.; CUNHA, J. P. A. R.; FRANZ, C. A. B.; FOLLE, S. M. Avaliação de três mecanismos de distribuição de sementes. Pesquisa Agropecuária Brasileira, Brasília, v. 33, n. 3, p. 331-337, 1998. BRANQUINHO, K. B.; FURLANI, C. E. A.; LOPES, A.; SILVA, R. P.; GOTTA, D. C. C.; BORSATTO, E. A. Desempenho de uma semeadora-adubadora direta, em função da velocidade de deslocamento e do tipo de manejo da biomassa da cultura de cobertura do solo. Engenharia Agrícola, Jaboticabal, v. 24, n. 2, p. 374-380, 2004. SANTOS, S. R. Proposta metodológica utilizando ferramentas de qualidade na avaliação do processo de pulverização. 2005. 59 f. (Doutorado em Engenharia Agrícola) - UNICAMP, Campinas, 2005. CASÃO JUNIOR, R.; SIQUEIRA, R. Resultados das avaliações do desempenho de semeadoras-adubadoras de plantio direto na Costa Oeste Paranaense. Londrina: IAPAR, 2003. 132 p. SANTOS, R. F.; BARROS, M. A. L.; MARQUES, F. M.; FIRMINO, P. T.; REQUIÃO, L. E. G. Análise econômica. In: AZEVEDO, D. M. P.; LIMA, E. F. (Ed.). O agronegócio da mamona no Brasil. Campina Grande: Embrapa Algodão, 2001. p. 17-35. AZEVEDO, D. M. P.; LIMA, E. F. (Ed.). O agronegócio da mamona no Brasil. Campina Grande: Embrapa Algodão, 2001. p. 17-35. COELHO, J. L. D.; MOLIN, J. P.; GADANHA JÚNIOR, C. D.; VASARHELYI, A. Avaliação do desempenho de máquinas aplicadoras a lanço na distribuição de gesso agrícola. In: CONGRESSO BRASILEIRO DE ENGENHARIA AGRÍCOLA, 21.; SIMPÓSIO DE ENGENHARIA AGRÍCOLA DO CONESUL, 1., 1992, Santa Maria. Anais... Santa Maria: Sociedade Brasileira de Engenharia Agrícola, 1992. p. 2058-2103. COELHO, J. L. D.; MOLIN, J. P.; GADANHA JÚNIOR, C. D.; VASARHELYI, A. Avaliação do desempenho de máquinas SAS Institute. SAS language and procedures: usage. Version 8.1. Cary: SAS Institute 2000. CD-ROM. SAVY FILHO, A. Mamona tecnologia agrícola. Campinas: EMOPI, 2005. 105 p. Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008 SAVY FILHO, A. Mamona tecnologia agrícola. Campinas: EMOPI, 2005. 105 p. KURACHI, S. A. H.; COSTA, J. A. S.; BERNARDI, J. A.; COELHO, J. L. D.; SILVEIRA, G. M. Avaliação tecnológica de semeadoras e/ou adubadoras: tratamento de dados de ensaio e regularidade de distribuição longitudinal de sementes. Bragantia, Campinas, v. 48, n. 2, p. 249-262, 1989. SILVA, M. R.; DANIEL, L. A. C. Cultivar máquinas, Pelotas: v. 3, p. 24-28, 2004. SILVA, M. R.; UNGARO, M. R. G.; RAMOS, N. P. Qualidade da distribuição longitudinal de cinco genótipos de girassol. In: REUNIÃO NACIONAL DE PESQUISA DE GIRASSOL, 16.; SIMPÓSIO NACIONAL SOBRE A CULTURA DO GIRASSOL, 4., 2005, Londrina. Anais... Londrina: Embrapa, 2005. p. 64-67. KURACHI, S. A. H.; SILVEIRA, G. M.; COSTA, J. A.; MORAES, R. A. D. M.; BERNARDI, J. A.; MOREIRA, C. A.; PETRONI, A. C.; SILVA, J. R.; MESQUITA, C. M. Código de avaliação de semeadoras e/ou adubadoras. Campinas: IAC, 1986. 138 p. SILVEIRA, J. C. M.; MODOLO, A. J.; SILVA, S. L.; GABRIEL FILHO, A. Força de tração e potência de uma semeadora em duas velocidades de deslocamento e duas profundidades de deposição de sementes. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, v. 9, n. 1, p. 125-128, 2005. LINO, A. C. L.; PECHE FILHO, A.; UNGARO, M. R. G.; STORINO, M. Uso de cartas de controle na avaliação do trabalho de semeadoras manuais. In: SIMPÓSIO NACIONAL DE GIRASSOL, 3.; REUNIÃO NACIONAL DE PESQUISA DE GIRASSOL, 15., 2003, Ribeirão Preto. Anais em CD-Rom. 4p. 2003. UNGARO, M. R. G.; PECHE FILHO, A.; LINO, A. C. L.; STORINO, M. Uso de SIG e de mapas temáticos de estande e produção de grãos na avaliação de lavoura de girassol (Helianthus Annuus L.). Engenharia Agrícola, Jaboticabal, v. 18, n. 3, p. 73-79, 1999. MAHL, D.; GAMERO, C. A.; BENEZ, S. H.; FURLANI, C. E. A.; SILVA, A. R. B. Demanda energética e eficiência da distribuição de sementes de milho sob variação de Ciênc. agrotec., Lavras, v. 32, n. 2, p. 481-486, mar./abr., 2008
https://openalex.org/W4307185658	https://www.researchsquare.com/article/rs-2185983/latest.pdf	English	null	The Musical Turn in Biosemiotics: An Expressivist Model of Communication	Research Square (Research Square)	2,022	cc-by	12,977	The Musical Turn in Biosemiotics: An Expressivist Model of Communication Matthew Slayton Duke University Yogi Hendlin (  hendlin@esphil.eur.nl ) Erasmus University Rotterdam Matthew Slayton Duke University Yogi Hendlin (  hendlin@esphil.eur.nl ) Erasmus University Rotterdam Research Article Keywords: Zoomusicology, synesthesia, iconicity, music, expression, interpretation Posted Date: October 24th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2185983/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License The Musical Turn in Biosemiotics 1 An Expressivist Model of Communication 2 3 Matthew Slayton, Duke University 4 Yogi Hendlin, Erasmus School of Philosophy, Erasmus University Rotterdam 5 Corresponding Author: Yogi Hendlin, hendlin@esphil.eur.nl 6 7 Abstract 8 Human music and language are two systems of communication and expression that, while 9 historically considered to overlap, have become increasingly divergent in their approach and 10 study. Music and language almost certainly co-evolved and emerged from the same semiotic 11 field, and this relationship as well as co-origin are actively researched and debated. For the 12 sake of evaluating the semiotic content of zoomusicology, we investigate music from a ‘bottom- 13 up’ biosemiotic functionalist account considering iconic, indexical, and symbolic forms of 14 meaning not in a hierarchy but according to their effects on agents. Such an approach avoids 15 overintellectualizing the representational aspects of music, and instead inverts, as it were, the 16 traditional hierarchy of semiotic categories to produce illocutionary effects. Understanding 17 aesthetics and action not as a priori separate but rather fundamentally co-arising elements of 18 the same events, the focus of musicality again returns to interpretation and how semiosis 19 precipitates expression. 20 21 Keywords 22 The Musical Turn in Biosemiotics An Expressivist Model of Communication Matthew Slayton, Duke University Yogi Hendlin, Erasmus School of Philosophy, Erasmus University Rotterdam Corresponding Author: Yogi Hendlin, hendlin@esphil.eur.nl Abstract 8 28 This article explores the different ways the life sciences, social sciences, and semiotics consider 29 music and language, and explores the consequences for the field of biosemiotics. In particular, 30 we champion expressivist models of music and push back against linguistic bias, which 31 privileges indexical and symbolic forms of meaning over the iconic. 32 Introduction 25 Human music and language are two systems of communication and expression that 26 have increasingly diverged in how they are approached and studied, which is surprising given 27 that music and language almost certainly co-evolved and emerged from the same semiotic field. 28 This article explores the different ways the life sciences, social sciences, and semiotics consider 29 music and language, and explores the consequences for the field of biosemiotics. In particular, 30 we champion expressivist models of music and push back against linguistic bias, which 31 privileges indexical and symbolic forms of meaning over the iconic. 32 It’s not surprising that we ended up emphasizing music’s symbolic rather than iconic 33 elements, as language is a referential system of communication permitting shorthand 34 extrapolation useful to passing on messages even if their quality is degraded in the passing as 35 the messages become increasingly decontextualized. Music, on the other hand, foregrounds 36 iconic, associative, and aesthetic meanings conveyed via an immediacy lost through non- 37 virtuosic repetitions.1 Both music and language employ symbols and motifs that rely on 38 foundational indexical and iconic forms of meaning, but language is something of a standard- 39 bearer for symbolic meaning, as music has become for the iconic. Our hope is that by 40 considering the origin, forms, and diverse instances of music and language in both the human 41 and non-human world, that we can place symbolic, indexical and iconic forms of meaning on 42 equal footing in terms of the depth of significance they can convey. The payoff for this work will 43 be to clarify how expression and interpretation of different kinds of meaning are defined by 44 cultural, semiotic, and ecological contexts. Abstract 8 Human music and language are two systems of communication and expression that, while 9 historically considered to overlap, have become increasingly divergent in their approach and 10 study. Music and language almost certainly co-evolved and emerged from the same semiotic 11 field, and this relationship as well as co-origin are actively researched and debated. For the 12 sake of evaluating the semiotic content of zoomusicology, we investigate music from a ‘bottom- 13 up’ biosemiotic functionalist account considering iconic, indexical, and symbolic forms of 14 meaning not in a hierarchy but according to their effects on agents. Such an approach avoids 15 overintellectualizing the representational aspects of music, and instead inverts, as it were, the 16 traditional hierarchy of semiotic categories to produce illocutionary effects. Understanding 17 aesthetics and action not as a priori separate but rather fundamentally co-arising elements of 18 the same events, the focus of musicality again returns to interpretation and how semiosis 19 precipitates expression. 20 21 Keywords 22 Zoomusicology; synesthesia; iconicity; music; expression; interpretation 23 24 Human music and language are two systems of communication and expression that, while 9 historically considered to overlap, have become increasingly divergent in their approach and 10 study. Music and language almost certainly co-evolved and emerged from the same semiotic 11 field, and this relationship as well as co-origin are actively researched and debated. For the 12 sake of evaluating the semiotic content of zoomusicology, we investigate music from a ‘bottom- 13 up’ biosemiotic functionalist account considering iconic, indexical, and symbolic forms of 14 meaning not in a hierarchy but according to their effects on agents. Such an approach avoids 15 overintellectualizing the representational aspects of music, and instead inverts, as it were, the 16 traditional hierarchy of semiotic categories to produce illocutionary effects. Understanding 17 aesthetics and action not as a priori separate but rather fundamentally co-arising elements of 18 the same events, the focus of musicality again returns to interpretation and how semiosis 19 precipitates expression. 20 21 24 1 1 Introduction 25 Human music and language are two systems of communication and expression that 26 have increasingly diverged in how they are approached and studied, which is surprising given 27 that music and language almost certainly co-evolved and emerged from the same semiotic field. Abstract 8 299) has 50 written, language is but “a special case of a more general biosemiosis.” 51 52 1. Music versus Language: a false dichotomy 53 On the surface it seems easy to differentiate language and music when it comes from 54 humans. Language is meant to communicate information and music is meant to express 55 emotion or aesthetic impressions. And yet, we know that language and music serve both 56 purposes. Synesthetes and highly trained musicians, for instance, grasp far more content in 57 music than the modern music-illiterate individual (Bragança et al. 2015). Similarly, language 58 famously contains far more (‘wet’) musicality, when spoken or even in written form than a (‘dry’) 59 mathematical, purely digital understanding of interpretation alone (Krames et al. 1974; 60 Mehrabian 1972). These false dichotomies epitomize the classic question of code duality — the 61 irreducible concomitant analog and algorithmic digital components accompanying all semiosis 62 (Hoffmeyer and Emmeche 1991). Our complaint is that there is an historical trend to privilege 63 the symbolic over the indexical, and the indexical over the iconic, as superior forms of 64 conveyance, which has led to an overemphasis on the exclusive benefits of language and 65 under-emphasis on the significance of music and musicality in semiotics (for just a recent 66 example, see Paolucci 2021).2 67 Present understandings of language coalesce around convictions that it is hierarchical 68 previous work on the subject (c.f. Barbieri 2007; Kull 2015). As Hoffmeyer (2008, p. 299) has 50 written, language is but “a special case of a more general biosemiosis.” 51 Abstract 8 45 While numerous introductions to and treatments of biosemiotics aspire to describe 46 semiosis beyond language, language remains an understandably handy and useful analogical 47 construct for describing forms of communication at numerous levels of biological organization 48 Introduction 25 Human music and language are two systems of communication and expression that 26 have increasingly diverged in how they are approached and studied, which is surprising given 27 that music and language almost certainly co-evolved and emerged from the same semiotic field. 28 This article explores the different ways the life sciences, social sciences, and semiotics consider 29 music and language, and explores the consequences for the field of biosemiotics. In particular, 30 we champion expressivist models of music and push back against linguistic bias, which 31 privileges indexical and symbolic forms of meaning over the iconic. 32 It’s not surprising that we ended up emphasizing music’s symbolic rather than iconic 33 elements, as language is a referential system of communication permitting shorthand 34 extrapolation useful to passing on messages even if their quality is degraded in the passing as 35 the messages become increasingly decontextualized. Music, on the other hand, foregrounds 36 iconic, associative, and aesthetic meanings conveyed via an immediacy lost through non- 37 virtuosic repetitions.1 Both music and language employ symbols and motifs that rely on 38 foundational indexical and iconic forms of meaning, but language is something of a standard- 39 bearer for symbolic meaning, as music has become for the iconic. Our hope is that by 40 considering the origin, forms, and diverse instances of music and language in both the human 41 and non-human world, that we can place symbolic, indexical and iconic forms of meaning on 42 equal footing in terms of the depth of significance they can convey. The payoff for this work will 43 be to clarify how expression and interpretation of different kinds of meaning are defined by 44 cultural, semiotic, and ecological contexts. 45 While numerous introductions to and treatments of biosemiotics aspire to describe 46 semiosis beyond language, language remains an understandably handy and useful analogical 47 construct for describing forms of communication at numerous levels of biological organization. 48 Our hope is to offer a remedy to some of the language-preference our field has identified in 49 2 2 previous work on the subject (c.f. Barbieri 2007; Kull 2015). As Hoffmeyer (2008, p. 1. Music versus Language: a false dichotomy 53 On the surface it seems easy to differentiate language and music when it comes from 54 humans. Language is meant to communicate information and music is meant to express 55 emotion or aesthetic impressions. And yet, we know that language and music serve both 56 purposes. Synesthetes and highly trained musicians, for instance, grasp far more content in 57 music than the modern music-illiterate individual (Bragança et al. 2015). Similarly, language 58 famously contains far more (‘wet’) musicality, when spoken or even in written form than a (‘dry’) 59 mathematical, purely digital understanding of interpretation alone (Krames et al. 1974; 60 3 Present understandings of language coalesce around convictions that it is hierarchical 68 and modular, starting from phonetics, through syntax, semantics, and pragmatics; thus it is a 69 convenient model case for talking about more abstract symbolic capacities such as cognition 70 and behavior. Language helps humans to describe our external and internal worlds and 71 construct a cohesive whole, such that language is elevated to a kind of axiom of mind and 72 experience. One way of describing this discourse is linguistic bias due to our current dominant 73 anthropic communication style, where the more decontextualized a referent can be, the more 74 flexibility it has, without regard to the concomitant loss in communicative fidelity such a 75 3 deliberately atonal move brings. This split between expression and content erroneously 76 assumes that one element can exist without the other, and that what is commonly glossed as 77 ‘content’ is superior.3 Expression is often misunderstood as a dramatization of feeling. We, 78 following Price (2013), hone in on expression as signifying being moved to act based on one’s 79 response to a change in state, internal or external. 80 deliberately atonal move brings. This split between expression and content erroneously 76 assumes that one element can exist without the other, and that what is commonly glossed as 77 ‘content’ is superior.3 Expression is often misunderstood as a dramatization of feeling. We, 78 following Price (2013), hone in on expression as signifying being moved to act based on one’s 79 response to a change in state, internal or external. 80 In order to re-discover the iconic and aesthetic aspects present in all modes of 81 communication and expression, we analyze acoustic communication in its myriad forms through 82 a musical lens. 1. Music versus Language: a false dichotomy 53 To put it more simply, we want to call any and all forms of acoustic expression 83 ‘music,’ in the way that a zoomusicologist would. Dario Martinelli (2010, p. 85), for example, 84 describes zoomusicology as the study of “the aesthetic use of sound communication among 85 animals.”4 His usage of “sound communication” may sound like a wiggle-word, not wanting to 86 call it language, but not wanting to relegate music as merely expressionist. While we see the 87 pragmatism and strategy of such a move — addressing the classic open question of whether 88 absolute music (unaccompanied by text or program) represents or expresses extra-musical 89 content — this implies an ex ante separation of aesthetic elements and semantic ones 90 (Boghossian 2020). Music has no more ability to pin down a specific interpretation than plain 91 language does. The difference, however, is the important question of how ambiguities are 92 resolved. In ordinary language, one can ask, “what do you mean by that?” With music, however, 93 we have no such luxury. Instead, we have repetition and variation on themes, call and 94 response, mimicry and silence. 95 4 Structures and processes (e.g. behaviors) are a feature of animal music. We could 96 dance around the matter and call these ‘acoustic utterances’, but such obfuscations would 97 performatively contradict our claim that various forms of animal acoustic utterance and behavior 98 can reasonably be classified as animal music — with the musical, denotative, and connotative 99 elements inextricable in their synergistic effect. These are utterances that contain musical 100 qualities for the animals themselves, and in those musical qualities, we can see aspects of 101 4 semiosis that would be lost or impoverished without them. In this discussion we err on the side 102 of behavioral, structural, and processual accounts, where expressive and communicative 103 pragmatics are sufficiently universal that we can use specific instances of musical behavior to 104 describe musical behavior in general (Marchesini and Celentano 2021; Martinelli 2005, 2009; 105 Ullrich 2018). 106 semiosis that would be lost or impoverished without them. In this discussion we err on the side 102 of behavioral, structural, and processual accounts, where expressive and communicative 103 pragmatics are sufficiently universal that we can use specific instances of musical behavior to 104 describe musical behavior in general (Marchesini and Celentano 2021; Martinelli 2005, 2009; 105 Ullrich 2018). 1. Music versus Language: a false dichotomy 53 128 We present a model of semiosonic meaning-making, discussing examples 129 from the plant and bacteria world as well as the more studied world of animal mus 130 argue which qualities render such activity musical and parse unambiguous instanc 131 musicality in alloanimal communication. We will use this frame to make an anthrop 132 for how human language lost many qualities characterized by iconicity and indexic 133 discuss how our conception of biosemiotic music, broadly conceived, can help res 134 features, leading to a more complete picture of environmentally-embedded intra- a 135 species biosemiosis. 136 137 Section 2: The case of alarm calls 138 Let’s consider a concrete example: alarm calls. Many species of monkeys, 139 make calls to communicate to conspecifics that there is danger (Zuberbühler 2009 140 animals may overhear or eavesdrop on these calls to learn information useful to th 141 1988). Yes, one function of an alarm call is to communicate vital information that c 142 upon (qua digital component); but these calls are also an excellent example of how 143 can feature in what appears to be a (quasi-)linguistic act (qua analog component). 144 of an alarm call beyond its reference to danger might include urgency or fear, or e 145 something more basic like a strong urge or need to make that call (an ‘expression 146 like to be the vocalizer in that environment, or to hear that vocalization? There we 147 qualitative, affective, or even aesthetic experience, where the category of ‘music’ c 148 clarify perhaps underappreciated aspects of the meaning-making going on. To put 149 directly, music conveys contextual aboutness. The idea that music is a language o 150 one instance of this quality, but only one piece of a larger story. An alarm call is da 151 for a “multicomponent perspective” to interpret the musical dimensions of semiosis, we agree 127 that music is an underlying adaptation, not an exaptation. 128 for a “multicomponent perspective” to interpret the musical dimensions of semiosis, we agree 127 that music is an underlying adaptation, not an exaptation. 128 We present a model of semiosonic meaning-making, discussing examples of ‘music’ 129 from the plant and bacteria world as well as the more studied world of animal music. We will 130 argue which qualities render such activity musical and parse unambiguous instances of 131 musicality in alloanimal communication. 1. Music versus Language: a false dichotomy 53 106 semiosis that would be lost or impoverished without them. In this discussion we err on the side 102 of behavioral, structural, and processual accounts, where expressive and communicative 103 pragmatics are sufficiently universal that we can use specific instances of musical behavior to 104 describe musical behavior in general (Marchesini and Celentano 2021; Martinelli 2005, 2009; 105 Ullrich 2018). 106 This move accommodating animal musicality as the null hypothesis to be defended 107 against rather than as an impositional structure to be otherwise accounted for allows 108 recuperation of lost meanings, while of course introducing the danger of overdetermining 109 utterances as music, linguistic, or meaningful, when they may be mere non-intentional 110 expressions. Yet, discussion of intentionality already bifurcates semiosis despite the fact that 111 whether an act is intentional or not, it has an effect in the world. It effects a difference that 112 makes a difference. This more expansive and permissive conception of musicality does not 113 discriminate between intentional musicality and non-intentional musicality if the overall 114 synesthetic effect is deepened/heightened/made more precise by the incorporation of musical 115 elements. This lands us in an expressivist model of communication in which no utterance, or 116 modulation of rhythm, frequency, or tone is for naught.5 Instead, these parameters are ripe with 117 affective, pictorial, and propulsive immanency. And they are not devoid of proposition or 118 argument (Rothenberg 2005, 2008). We can choose to resist the sheer illocutionary force of an 119 utterance, but that doesn’t mean that it bears no meaning for the agent or other audiences, 120 intended and unintended.6 121 Thus, our purpose here is to explore nonhuman forms of musical expression first and 122 work our way back to humans. By decentering our model focusing on nonhuman music, we 123 hope to find musical features of communication and expression that can more generally help us 124 understand how meaning arises in interactions, and the various parameters organisms have at 125 their disposal to generate and convey meaning. Following Honing et al.’s (2015, p. 3) proposal 126 5 for a “multicomponent perspective” to interpret the musical dimensions of semiosis 127 that music is an underlying adaptation, not an exaptation. 1. Music versus Language: a false dichotomy 53 We will use this frame to make an anthropological case 132 for how human language lost many qualities characterized by iconicity and indexicality, and then 133 discuss how our conception of biosemiotic music, broadly conceived, can help rescue these 134 features, leading to a more complete picture of environmentally-embedded intra- and inter- 135 species biosemiosis. 136 Let’s consider a concrete example: alarm calls. Many species of monkeys, for example, 139 make calls to communicate to conspecifics that there is danger (Zuberbühler 2009). Other 140 animals may overhear or eavesdrop on these calls to learn information useful to them (Hauser 141 1988). Yes, one function of an alarm call is to communicate vital information that can be acted 142 upon (qua digital component); but these calls are also an excellent example of how musicality 143 can feature in what appears to be a (quasi-)linguistic act (qua analog component). The qualities 144 of an alarm call beyond its reference to danger might include urgency or fear, or even 145 something more basic like a strong urge or need to make that call (an ‘expression’). What is it 146 like to be the vocalizer in that environment, or to hear that vocalization? There we might find 147 qualitative, affective, or even aesthetic experience, where the category of ‘music’ can help us 148 clarify perhaps underappreciated aspects of the meaning-making going on. To put it more 149 directly, music conveys contextual aboutness. The idea that music is a language of emotion is 150 one instance of this quality, but only one piece of a larger story. An alarm call is danger to those 151 6 who hear it: the call is iconic of that danger in its onomatopoeisis. There’s an immediate 152 recognition and reaction by tuned-in others of what the utterer is experiencing. 153 7 recognition and reaction by tuned-in others of what the utterer is experiencing. 153 Music is immediate because iconicity is immediate — an immediate aboutness that 154 connects an organism’s experienced environment, its body, and those hearing these acoustic 155 behaviors. We can think of aesthetic musical properties of communication as a synesthetic form 156 of metaphor. Based on the “the affective quality of all iconic relations” according to Peircian 157 abduction, rhemic icons (qualisigns) cannot escape their immediacy (Petrilli and Ji 2022; though 158 compare with Nöth (2014, p. 1. Music versus Language: a false dichotomy 53 16), who in a logocentric context focusing on digitality, to the 159 contrary claims that “[i]t is logically impossible for a rhematic sign to be also informative”). As 160 John Collier (2013) once phrased it, a qualisign “has an iconic relation to its object (itself) and a 161 rhemic relation to its interpretant (itself). A qualisign is an iconic rheme.” In non-virtualized 162 media, the immediacy of the iconic involves forms of synesthetic musicality, and thus include 163 affective dimensions which always accompany such signs according to their analog component. 164 The immediateness and aboutness of musical meaning can be seen in humans and 165 non-human primates where there is a direct relationship between embodiment and acoustic 166 expression. Pouw et al. (2020) demonstrated that human vocalizations contain information 167 about bodily states (such as the amount of muscle tension during upper limb movements) that 168 are detectable by listeners who hear the vocalizations but cannot see the source, so participants 169 were able to synchronize their arm movements with those of the speaker when they could not 170 see the speaker’s movements. These findings mirror those in non-human primates. For 171 example, rhesus monkeys can differentiate age-related body size based on the acoustic 172 qualities of vocalizations (Ghazanfar et al. 2007). Orangutans exploit this sonic phenomenon to 173 mislead others, cupping their mouths with their hands so they sound larger and more 174 threatening (Pisanski et al. 2016). The aesthetic dimensions of the call (e.g., timbre, tone, 175 frequency, rhythmic oscillations) provide a suite of situational clues that contextualize any 176 ‘content’, without which, misunderstanding and misinterpretation would increase. 177 7 7 It’s worth pointing out that ‘alarms’ can take many forms. There are many examples that 178 challenge the idea of what a sensible danger signal can be. The embryos of the yellow-legged 179 gull, still developing inside eggs, respond to adult alarm calls by vibrating within their shell and 180 transmitting the warning to their clutchmates (Noguera and Velando 2019). Moving beyond the 181 animal kingdom, tomato and tobacco plants emit ultrasonic sounds when stressed by lack of 182 water or if their stem is cut (Khait et al. 2019; Zweifel and Zeugin 2008). Rubber trees become 183 more productive when bombarded with ultrasonic waves on their tapping surface (Zhu et al. 184 2013). 1. Music versus Language: a false dichotomy 53 It is not possible to infer intention on the part of plants, but both cases are undeniably 185 ways in which plants produce and react to sonic expressions in their environments (Gagliano et 186 al. 2012). 187 Pauses or breaks in background acoustic signaling can be informative as well. Gray 188 squirrels have been observed to act normally when they can hear bird chatter, yet stop and hide 189 when the chatter stops. Bird chatter acts as a reassuring and indicative background sound; the 190 authors describe it as the ‘Muzak’ of the animal kingdom, of which the squirrels are always 191 peripherally aware (Lilly et al. 2019). The crackling and whooshing sounds of a healthy coral 192 reef promotes community development in fish populations whereas the sound of a damaged 193 coral reef suppresses it (Gordon et al. 2019). Many species of fish appear to derive meaning 194 from the acoustic environment about the suitability or potential of a particular location for food 195 and shelter. 196 These are emergent phenomena not just from particular, solitary organisms, but webs of 197 resonance and responding between biota and their abiotic media. The auditory, as continuous 198 membrane of reverberation, is often tapped into by species from various kingdoms to meta- 199 communicate and reach harmonies - the evolutionary stage on which all analytically identifiable 200 semiosis occurs. It doesn’t require a long conceptual leap to understand that musical valence — 201 178 These are emergent phenomena not just from particular, solitary organisms, but webs of 197 resonance and responding between biota and their abiotic media. The auditory, as continuous 198 membrane of reverberation, is often tapped into by species from various kingdoms to meta- 199 communicate and reach harmonies - the evolutionary stage on which all analytically identifiable 200 semiosis occurs. It doesn’t require a long conceptual leap to understand that musical valence — 201 whether the organism is perceiving a positive, safe, welcoming situation or a negative, 202 dangerous threatening situation is an essential part of the acoustic information we can 203 8 8 perceive. While awareness of this membrane may not be consciousness, the resonant bodies of 204 living beings always already flicker in entrainment in one way or another with the background 205 biophysics of resonance and its meta-semiotic organization. 1. Music versus Language: a false dichotomy 53 206 living beings always already flicker in entrainment in one way or another with the background 205 biophysics of resonance and its meta-semiotic organization. 206 Perhaps it is evident that these examples show how musical features like iconicity, 207 embodiment, and valence operate at sub-conscious levels in the sonic communicative 208 expressions that indicate the presence or lack of danger, health or decay; but why bother calling 209 these behaviors music? Is such a term a mere over-extension of the concept, past the point of 210 usability? Or, is there really something to be gained by framing such sounds as music? As 211 we’ve argued, musicality helps us to emphasize the depth and effect of iconicity present in all 212 expression and communication, as well as the continuity and overlaying of simultaneous and 213 displaced patterns (Nomura et al. 2018). The immediacy and inviolability of iconic 214 communication means that regardless of the other interpretations which other con- and 215 interspecifics may perceive in their overlapping Umwelten and translations, there remains a 216 mediating, moderating reverberation which physically agitates any being or medium within 217 ‘earshot’ that is equipped with body - biotic or abiotic - which can resonate with the sonic 218 emission. The general tone or mood (safety or danger), intensity of that communication (mild 219 versus severe), and technical acoustic ecological supra-compositional elements of rhythm, 220 pitch, frequency, and resonance will be picked up differentially for different sensory 221 fi ti b t f th i ht i t t th i i t l i t l 222 configurations; but for the right interpreters, these signs communicate clear environmental cues 222 to conspecifics and often interspecifics too (including other animals, other fish, other plants, etc). 223 The particular sonic expressions impart immediate sensations offering multifold meaning 224 accessible for those with the disposition to hear and feel them. 225 Section 3: Language and music overlapping in humans 227 Just as we claim that the iconicity of nonhuman music provides a full palate of 228 communicative needs, without added semantic specificity, turning to human music, we now look 229 9 at how in strictly codified musical cultures, human music’s symbolic repertoire can serve the 230 purpose of language’s semantic specificity. 231 A commonly cited example from human culture that evinces the blurred boundary 232 between language and music is the talking drums of the Yoruba people. There was great 233 anthropological interest in the use of talking drums for long-distance communication as speech 234 surrogates, sometimes across or down rivers and their roar with which vocal speech would 235 encounter sonic interference patterns. While the syntactical and semantic use of drums is not a 236 comprehensive picture of the use of the drums in Yoruba culture, it is easy to see why there was 237 so much excitement (Durojaye et al. 2021). On the surface, it’s a long-distance communication 238 system that uses elements of both language and music. Speakers of tonal languages like 239 Yoruban use sequences of two notes played on pitched drums in order to transmit information. 240 Calls and responses are deliberately chosen to minimize miscommunication on matters of 241 potential great importance. Repetition is key when receivers doubt the precision of the original 242 message. Despite their perlocutionary content, they do not lose their musicality, as one would 243 expect, if some sort of zero-sum game of mutual exclusivity were at play. Indeed, the messages 244 contain both linguistic and musical elements, and in so doing constitute a robust communication 245 system. Symbolic and iconic content here serve to reinforce each other, and convey the 246 intended message more clearly than if one or the other communication technique were used on 247 its own. 248 10 More recent scholarship expands the picture of Yoruba drumming, which is also used to 249 communicate with deities in spiritual practice and for artistic purposes (Omojola 2010). Symbolic 250 content, aesthetics, and emotion blend together in a palpable way. The talking drums can 251 communicate content that is specific and concrete like a number of some product for trade.. 252 They can also be metaphorical purposes, similar to using an aphoristic phrase to refer to 253 something, either as a short-hand or for expressive or artistic purposes. Section 3: Language and music overlapping in humans 227 In this way, the 254 combination of linguistic and musical features provides a communication system with 255 10 associations that can be both referential, as part of a codified system, and expressive, due to 256 the immediate associations hearers have to the musical features. 257 associations that can be both referential, as part of a codified system, and expressive, due to 256 the immediate associations hearers have to the musical features. 257 Another example, this time from India, looks at the Indian classical music system of 258 notation and correspondence between words and musical sounds. Svara (swara) in Sanskrit 259 connotes a specific breath, vowel, and corresponding musical note. This ancient Indian concept 260 of musical pitch is thoroughgoingly symbolic, so much so, that melodies become words, and 261 poetry can be expressed instrumentally (Datta et al. 2017). Poetry, of Tagore, say read in 262 diverse contexts must be reinterpreted, and often are instrumentally (without the lyrics) 263 interpreting the poem for the present location (Desha), circumstances (Kaala) and impetus that 264 triggered the emotion eliciting the poem (Karana). In cultures where music and language are 265 intertwined, melodies can convey to these seasoned listeners lines of well-known poetry that 266 can be backtranslated into words, similarly to how the Yoruban drum calls carry precise 267 semantic content. 268 It is a common assumption that language is informative and music is emotional; but this 269 view is incomplete (Damasio 1994). It also tends to underestimate the meaningful dimensions of 270 musicality and emotion (Nussbaum and Schweinberger 2021). Of course emotion is a central 271 component of music, but music contains much more. Music requires recognition of embodiment 272 as well as associations given by historical, cultural consensus. It cannot be ungrounded from its 273 referent (except, perhaps, in modern, experimental music). In fact, musicality and emotion are 274 both fundamental components of the meaning-making taking place in all kinds of human 275 interaction. The aesthetics, emotion, and history of an expressive utterance or action have as 276 much significance as music’s referential meaning, and recalling examples like the talking drums 277 and Indian classical music can help clarify the many facets at play. 278 Music also is malleable for different functions (e.g. performance, expression, aesthetics); 279 and not all music has all functions. Section 3: Language and music overlapping in humans 227 The expressivist model of 284 communication takes both music and language to be conveying similar intentions and distinct 285 meanings — the modes or methods are just accomplishing this according to differing degrees of 286 logocentric formality, which changes the metanarrative under which each form of understanding 287 operates. 288 Section 3: Language and music overlapping in humans 227 Different musical instantiations and purposes are explored in 280 various cultures, and exist in forms of communication and expression that might not necessarily 281 11 even be called music, despite their involving acoustic, aesthetic choices. In a way, we are 282 posing the question of whether something having musical qualities makes it music. Is bird song 283 musical but not music? The utility for such a distinction is hard to see. The expressivist model o 284 communication takes both music and language to be conveying similar intentions and distinct 285 meanings — the modes or methods are just accomplishing this according to differing degrees o 286 logocentric formality, which changes the metanarrative under which each form of understanding 287 operates. 288 289 Section 4: Felicity of Utterances and Umwelten 290 If, including language and music, all forms of semiosis are utilitarian in non-singular 291 ways – meaning that the composite semiosis is greater than any particular digital or analog 292 component – then it behooves us to stop ignoring certain aspects of semiosis as superfluous 293 extras or nonreactive backgrounds. Achieving higher-fidelity semiosis — ecologically, 294 biologically, and culturally — requires tuning into a full spectrum approach, rather than settling 295 for certain narrow bandwidths, even if the latter do provide exquisite depth. Perhaps there is a 296 zero-sum game of awareness. But then, we should at least check in once and a while to see if 297 the findings of our narrow focus correlate with the other extant forms and instantiations that are 298 sidelined in order to concentrate on particular details. Periodic matching of intensive and 299 extensive scientific study is crucial to knit together the dispersed disciplines into a reflective 300 equilibrium. Otherwise, we risk entering the hall of mirrors of ungrounded symbolic 301 communication which Terrence Deacon (2013), Iain McGilchrist (2009), and others have 302 identified and warned against. 303 In the transition to industrialization and urbanization, the relevant signal for humans 304 changed from ecological sounds (biophony and geophony) to an increased focus on 305 even be called music, despite their involving acoustic, aesthetic choices. In a way, we are 282 posing the question of whether something having musical qualities makes it music. Is bird song 283 musical but not music? The utility for such a distinction is hard to see. Section 4: Felicity of Utterances and Umwelten 290 12) — drastically reduced in contemporary speech in most cultures — 319 provide far more in terms of entrainment towards comprehension and mood attunement than we 320 have assumed. Attending to this invisible work, the latent musicality occurring in the background 321 of what we take to make the main attraction of semiosis, opens our aperture of awareness to a 322 wider range of factors and signals both endo- and exosemiotically, enabling us to more fully 323 inhabit our bodies and the places and structurings that constitute us. 324 Studies suggest that musical training not only potentiates domain specific abilities (e.g. 325 verbal memory, reasoning, visuospatial cognition, divergent thinking, etc.) associated with 326 working memory, but also benefits domain general areas such as phonological memory and 327 executive function (for a review see Ramachandra et al., 2012). Yet, with the shift to text-based 328 interfaces of language especially in the 20th century much of this musicality of language was 329 resonance with the environment became less important than communicating specific plans, 308 orders, and other directives and coordination information amongst humans only (mainly 309 speaking the same language), the exigency of the communication superseded the aesthetics of 310 the transmission, rendering the musical elements in human speech less salient. 311 Attending to the musicality of language and other allo-organism modes of semiosis not 312 only helps overcome the digital-centrism of 20th century positivist linguistics, but it also permits 313 anchoring semiosis in the analog experiences of joy and pleasure. As Kalevi Kull (2022) 314 Multirelational fitting with other aspects of one’s Umwelt suggests that the aesthetic dimensions 318 of speech (Kull 2022, p. 12) — drastically reduced in contemporary speech in most cultures — 319 provide far more in terms of entrainment towards comprehension and mood attunement than we 320 have assumed. Attending to this invisible work, the latent musicality occurring in the background 321 of what we take to make the main attraction of semiosis, opens our aperture of awareness to a 322 wider range of factors and signals both endo- and exosemiotically, enabling us to more fully 323 inhabit our bodies and the places and structurings that constitute us. 324 Studies suggest that musical training not only potentiates domain specific abilities (e.g. Section 4: Felicity of Utterances and Umwelten 290 If, including language and music, all forms of semiosis are utilitarian in non-singular 291 ways – meaning that the composite semiosis is greater than any particular digital or analog 292 component – then it behooves us to stop ignoring certain aspects of semiosis as superfluous 293 extras or nonreactive backgrounds. Achieving higher-fidelity semiosis — ecologically, 294 ways – meaning that the composite semiosis is greater than any particular digital or analog 292 component – then it behooves us to stop ignoring certain aspects of semiosis as superfluous 293 extras or nonreactive backgrounds. Achieving higher-fidelity semiosis — ecologically, 294 biologically, and culturally — requires tuning into a full spectrum approach, rather than settling 295 for certain narrow bandwidths, even if the latter do provide exquisite depth. Perhaps there is a 296 zero-sum game of awareness. But then, we should at least check in once and a while to see if 297 the findings of our narrow focus correlate with the other extant forms and instantiations that are 298 sidelined in order to concentrate on particular details. Periodic matching of intensive and 299 extensive scientific study is crucial to knit together the dispersed disciplines into a reflective 300 equilibrium. Otherwise, we risk entering the hall of mirrors of ungrounded symbolic 301 communication which Terrence Deacon (2013), Iain McGilchrist (2009), and others have 302 identified and warned against. 303 12 resonance with the environment became less important than communicating specific plans, 308 orders, and other directives and coordination information amongst humans only (mainly 309 speaking the same language), the exigency of the communication superseded the aesthetics of 310 the transmission, rendering the musical elements in human speech less salient. 311 Attending to the musicality of language and other allo-organism modes of semiosis not 312 only helps overcome the digital-centrism of 20th century positivist linguistics, but it also permits 313 anchoring semiosis in the analog experiences of joy and pleasure. As Kalevi Kull (2022) 314 describes beauty as the perfect semiotic fitting, the joining of beauty with precision is crucial to 315 overcome prejudices against ecological harmony; as if attending to the needs, demands, pleas, 316 and lives of others somehow reduced our own happiness or fitness, properly understood. 317 Multirelational fitting with other aspects of one’s Umwelt suggests that the aesthetic dimensions 318 of speech (Kull 2022, p. Section 4: Felicity of Utterances and Umwelten 290 The perceiver is in a constant state of interaction and feedback with 353 their perceptual environment, which is an idea that is central to many of the 20th century 354 cybernetic theories that came later. Particularly important for our purposes is the idea that we 355 only perceive what is useful or meaningful to us in our “evolutionary-semiotic context” (Uexküll 356 2010, p. 25). This Umwelt “can be considered as the sum total of [the] perceptual cues among 357 the stimuli in [the] environment” (Reybrouck 2015, p. 16). The environment, then, “is merely the 358 content conveys more than musicality, that it is capable of conveying more information than 334 musicality. 335 But in our current digital age dominated by short videos, often fielded by the 336 inventiveness of youth, the 21st century may witness a shift from semantic to musical features 337 becoming forefront in human semiosis. Thirty-second TikTok videos and the repetitive, theme- 338 and-variation riffing off each other (remixing), pays far more attention to the aesthetic 339 dimensions of communication, including the pleasurable or strange impressions of semiosis. 340 Unexpected and attention-getting features become increasingly salient communication features 341 in a saturated environment. The study of human semiosis needs to expand and adapt to 342 account for the variety of forms of human behavior and expression, and foregrounding music is 343 one handy way to side-step potentially limiting assumptions. 344 content conveys more than musicality, that it is capable of conveying more information than 334 musicality. 335 Mark Reybrouck is one scholar whose work is focused on reconceptualizing music 345 cognition in terms of “ecosemiotics,” meaning that rather than looking at music as a static entity, 346 Reybrouck (2012, 2015) instead views music as a process that occurs within an environmental 347 context. Specifically, listeners have a relationship with their sonic environments that is based on 348 function. All humans (and all organisms) evolved some kind of functional relationship with their 349 environments. Jakob von Uexkull first proposed a model for “perceptions, communications, and 350 purposeful behaviors” in trying to account for the experience of non-human animals (Uexküll 351 2010, p. 3). All animals (humans included) live in a perceptual environment which is significant 352 to their lives and needs. Section 4: Felicity of Utterances and Umwelten 290 325 verbal memory, reasoning, visuospatial cognition, divergent thinking, etc.) associated with 326 working memory, but also benefits domain general areas such as phonological memory and 327 executive function (for a review see Ramachandra et al., 2012). Yet, with the shift to text-based 328 interfaces of language, especially in the 20th century, much of this musicality of language was 329 lost. Especially in translation of texts, musicality is often viewed as “the most recalcitrant of all 330 features in a source-language text” (Wong 2006, p. 91), while semantic content can be 331 preserved. This discrepancy between the transferability of semantic content from aural to written 332 form and the difficulty of preserving musicality is symptomatic of the presumption that semantic 333 13 content conveys more than musicality, that it is capable of conveying more information than 334 musicality. 335 But in our current digital age dominated by short videos, often fielded by the 336 inventiveness of youth, the 21st century may witness a shift from semantic to musical features 337 becoming forefront in human semiosis. Thirty-second TikTok videos and the repetitive, theme- 338 and-variation riffing off each other (remixing), pays far more attention to the aesthetic 339 dimensions of communication, including the pleasurable or strange impressions of semiosis. 340 Unexpected and attention-getting features become increasingly salient communication features 341 in a saturated environment. The study of human semiosis needs to expand and adapt to 342 account for the variety of forms of human behavior and expression, and foregrounding music is 343 one handy way to side-step potentially limiting assumptions. 344 Mark Reybrouck is one scholar whose work is focused on reconceptualizing music 345 cognition in terms of “ecosemiotics,” meaning that rather than looking at music as a static entity, 346 Reybrouck (2012, 2015) instead views music as a process that occurs within an environmental 347 context. Specifically, listeners have a relationship with their sonic environments that is based on 348 function. All humans (and all organisms) evolved some kind of functional relationship with their 349 environments. Jakob von Uexkull first proposed a model for “perceptions, communications, and 350 purposeful behaviors” in trying to account for the experience of non-human animals (Uexküll 351 2010, p. 3). All animals (humans included) live in a perceptual environment which is significant 352 to their lives and needs. Section 4: Felicity of Utterances and Umwelten 290 360 Reybrouck also provides, drawing on James Gibson, a way of incorporating the 361 potentially ‘universal’ capacities and tendencies in music. However, because of the closed 362 feedback loop between individual and environment, these elements of music are not introduced 363 as isolated traits to be explained. Rather, “senses, in this view, do not simply function to arouse 364 sensations but pick up information, which is already structured and ordered as part of an 365 organism-environment ecosystem” (Reybrouck 2015, p. 15). The point is that organisms 366 evolved to attend to certain features of the sonic environment, which automatically shapes the 367 meanings they can perceive, but those meanings can’t be reduced to the anatomy of the 368 sensing organ or the waveform of the sound. These are all crucial components to meaning- 369 making, but they operate together as scaffolding for the evolution of future meaning (c.f. 370 projection or mapping out of the organism’s internal organization onto the outside world” 359 (Reybrouck 2015, p. 6). 360 Reybrouck also provides, drawing on James Gibson, a way of incorporating the 361 potentially ‘universal’ capacities and tendencies in music. However, because of the closed 362 feedback loop between individual and environment, these elements of music are not introduced 363 as isolated traits to be explained. Rather, “senses, in this view, do not simply function to arouse 364 sensations but pick up information, which is already structured and ordered as part of an 365 organism-environment ecosystem” (Reybrouck 2015, p. 15). The point is that organisms 366 evolved to attend to certain features of the sonic environment, which automatically shapes the 367 meanings they can perceive, but those meanings can’t be reduced to the anatomy of the 368 sensing organ or the waveform of the sound. These are all crucial components to meaning- 369 making, but they operate together as scaffolding for the evolution of future meaning (c.f. 370 Caporael et al. 2014). We are born and raised in a cultural environment that is created by other 371 people. What’s more, we don’t just perceive sonic phenomena — we structure what we perceive 372 (Reybrouck 2012, 2015). We are active participants in the production of meaning, and music 373 (including all iconic and aesthetic forms of signification) are an essential part of how physiology, 374 behavior, and culture relate over evolutionary time. Section 4: Felicity of Utterances and Umwelten 290 The perceiver is in a constant state of interaction and feedback with 353 their perceptual environment, which is an idea that is central to many of the 20th century 354 cybernetic theories that came later. Particularly important for our purposes is the idea that we 355 only perceive what is useful or meaningful to us in our “evolutionary-semiotic context” (Uexküll 356 2010, p. 25). This Umwelt “can be considered as the sum total of [the] perceptual cues among 357 the stimuli in [the] environment” (Reybrouck 2015, p. 16). The environment, then, “is merely the 358 14 projection or mapping out of the organism’s internal organization onto the outside world” 359 (Reybrouck 2015, p. 6). 360 Reybrouck also provides, drawing on James Gibson, a way of incorporating the 361 potentially ‘universal’ capacities and tendencies in music. However, because of the closed 362 feedback loop between individual and environment, these elements of music are not introduced 363 as isolated traits to be explained. Rather, “senses, in this view, do not simply function to arouse 364 sensations but pick up information, which is already structured and ordered as part of an 365 organism-environment ecosystem” (Reybrouck 2015, p. 15). The point is that organisms 366 evolved to attend to certain features of the sonic environment, which automatically shapes the 367 meanings they can perceive, but those meanings can’t be reduced to the anatomy of the 368 sensing organ or the waveform of the sound. These are all crucial components to meaning- 369 making, but they operate together as scaffolding for the evolution of future meaning (c.f. 370 Caporael et al. 2014). We are born and raised in a cultural environment that is created by other 371 people. What’s more, we don’t just perceive sonic phenomena — we structure what we perceive 372 (Reybrouck 2012, 2015). We are active participants in the production of meaning, and music 373 (including all iconic and aesthetic forms of signification) are an essential part of how physiology, 374 behavior, and culture relate over evolutionary time. 375 Musicality finds expression in dialects of semiosis, not ideolects. The biosocial meaning 376 of semiosis helps us distinguish between music, and random tonal utterances. Musicality in 377 semiosis provides many more surfaces (c f “surfaces inside surfaces” (Hoffmeyer 1998)) 378 projection or mapping out of the organism’s internal organization onto the outside world” 359 (Reybrouck 2015, p. 6). Section 4: Felicity of Utterances and Umwelten 290 375 M i lit fi d i i di l t f i i t id l t Th bi i l i 376 projection or mapping out of the organism’s internal organization onto the outside world” 359 (Reybrouck 2015, p. 6). 360 Caporael et al. 2014). We are born and raised in a cultural environment that is created by other 371 people. What’s more, we don’t just perceive sonic phenomena — we structure what we perceive 372 (Reybrouck 2012, 2015). We are active participants in the production of meaning, and music 373 (including all iconic and aesthetic forms of signification) are an essential part of how physiology, 374 behavior, and culture relate over evolutionary time. 375 Musicality finds expression in dialects of semiosis, not ideolects. The biosocial meaning 376 of semiosis helps us distinguish between music, and random tonal utterances. Musicality in 377 semiosis provides many more surfaces (c.f. “surfaces inside surfaces” (Hoffmeyer 1998)), 378 reticulations, or convolutions, for semiosis to be transmitted, creating an affective field. What 379 expressionism may lack in precision and the ability to 'force' one to a certain conclusion, it 380 makes up for in resonance — deactivating presuppositions, and rearranging prejudgments to 381 create space for novel thought and action. 382 15 Conclusion 385 By becoming more aware of the way in which musicality informs our own semiosis, we 386 can attend more deliberately to the musical qualities in nonhuman biosemiotics. Paying more 387 attention to soundscapes as cohesive structures also temper mechaphony and other intrusive 388 forms of anthrophony. Attending to our influences – say on distorting birdsong or other aural 389 forms of communication by our nonhuman cohabitants of a given territory – can also give 390 motivation to walk back the loudness which has come to represent our age. Perhaps, in a 391 quieter world, our own musicality, using the whisper or sotto voce as ways of gathering and 392 conveying our own thoughts, may introduce new modes of thought, that show our current 393 linguistic paradigm to be but one frame of reference among others, each with its own insights 394 that help contextualize the rest. Section 4: Felicity of Utterances and Umwelten 290 395 Biosemiotics as an enterprise seeks to account for the meaning-making occurring in 396 biological systems, and the truly difficult work of making this set of models as all-encompassing 397 as possible can perhaps be facilitated by tuning into the musicality inherent in semiosis. Music 398 and language stand as overlapping yet opposed modalities in human expression, and find a 399 wide range of instantiations throughout the natural world. It may be that considering both 400 modalities on equal footing can facilitate the extension of semiosis by recovering the icon from 401 among the foregrounded index and pushing on the limits of what our conceptualization of a 402 symbol can be. 403 404 405 406 407 408 409 Conclusion 385 By becoming more aware of the way in which musicality informs our own semiosis, we 386 can attend more deliberately to the musical qualities in nonhuman biosemiotics. Paying more 387 attention to soundscapes as cohesive structures also temper mechaphony and other intrusive 388 forms of anthrophony. Attending to our influences – say on distorting birdsong or other aural 389 forms of communication by our nonhuman cohabitants of a given territory – can also give 390 motivation to walk back the loudness which has come to represent our age. Perhaps, in a 391 quieter world, our own musicality, using the whisper or sotto voce as ways of gathering and 392 conveying our own thoughts, may introduce new modes of thought, that show our current 393 linguistic paradigm to be but one frame of reference among others, each with its own insights 394 that help contextualize the rest. 395 Biosemiotics as an enterprise seeks to account for the meaning-making occurring in 396 biological systems, and the truly difficult work of making this set of models as all-encompassing 397 as possible can perhaps be facilitated by tuning into the musicality inherent in semiosis. Music 398 and language stand as overlapping yet opposed modalities in human expression, and find a 399 wide range of instantiations throughout the natural world. It may be that considering both 400 modalities on equal footing can facilitate the extension of semiosis by recovering the icon from 401 among the foregrounded index and pushing on the limits of what our conceptualization of a 402 symbol can be. 403 16 411 1 411 412 References 413 Barbieri, M. (2007). Introduction to biosemiotics: the new biological synthesis. Dordrecht: 414 Springer. Section 4: Felicity of Utterances and Umwelten 290 415 Bragança, G. F. F., Fonseca, J. G. M., & Caramelli, P. (2015). Synesthesia and music 416 perception. Dementia & Neuropsychologia, 9(1), 16–23. https://doi.org/10.1590/S1980- 417 57642015DN91000004 418 Caporael, L. R., Griesemer, J. R., & Wimsatt, W. C. (2014). Developing Scaffolds in Evolution, 419 Culture, and Cognition. MIT Press. 420 Collier, J. (2013, March 25). Re: [biosemiotics:1085] Re: Neither materialism or pansemiotism. 421 Cross, G. S., & Proctor, R. N. (2014). Packaged Pleasures: How Technology and Marketing 422 Revolutionized Desire. 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New York: Oxford University Press. 438 Gagliano, M., Mancuso, S., & Robert, D. (2012). Towards understanding plant bioacoustics. 439 Trends in Plant Science, 17(6), 323–325. https://doi.org/10.1016/j.tplants.2012.03.002 440 Ghazanfar, A. A., Turesson, H. K., Maier, J. X., van Dinther, R., Patterson, R. D., & Logothetis, 441 N. K. (2007). Vocal-Tract Resonances as Indexical Cues in Rhesus Monkeys. Current 442 Biology, 17(5), 425–430. https://doi.org/10.1016/j.cub.2007.01.029 443 Gordon, T. A. C., Radford, A. N., Davidson, I. K., Barnes, K., McCloskey, K., Nedelec, S. L., et 444 al. (2019). Acoustic enrichment can enhance fish community development on degraded 445 coral reef habitat. Nature Communications, 10(1), 5414. https://doi.org/10.1038/s41467- 446 019-13186-2 447 Hauser, M. D. (1988). How Infant Vervet Monkeys Learn To Recognize Starling Alarm Calls: the 448 Role of Experience. Behaviour, 105(3–4), 187–201. 449 https://doi.org/10.1163/156853988X00016 450 Hendlin, Y. (2021). 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Taylor & Francis. 516 Pisanski, K., Cartei, V., McGettigan, C., Raine, J., & Reby, D. (2016). Voice Modulation: A 517 Window into the Origins of Human Vocal Control? Trends in Cognitive Sciences, 20(4), 518 304–318. https://doi.org/10.1016/j.tics.2016.01.002 519 Plumwood, V. (1993). Feminism and the mastery of nature. London; New York: Routledge. 520 Potvin, D. A., Parris, K. M., & Mulder, R. A. (2011). Geographically pervasive effects of urban 521 noise on frequency and syllable rate of songs and calls in silvereyes (Zosterops 522 lateralis). Proceedings of the Royal Society B: Biological Sciences, 278(1717), 2464– 523 2469. https://doi.org/10.1098/rspb.2010.2296 524 Price, H. (2013). Expressivism, pragmatism and representationalism. Burlington, VT: Ashgate 525 Publishing Company. 526 Reybrouck, M. (2012). Musical Sense-Making and the Concept of Affordance: An Ecosemiotic 527 and Experiential Approach. 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New Phytologist, 179(4), 1070–1079. 551 https://doi.org/10.1111/j.1469-8137.2008.02521.x 552 553 554 We Declare No Conflicts of Interest 555 556 Acknowledgements: We would like to thank the participants of the 22nd Gatherings in 557 Biosemiotics, in Olomouc, Czechia, for their supportive comments. 558 559 Endnotes 560 1 Barring the recent invention (ca 1850) of the technological reproduction of sound (Cross and Proctor 2014), music has always been played for specific audiences, with each performance offering an interplay between culturally-contextualized expression and audience- Methodological Framework. In A. Barcz & D. Łagodzka (Eds.), Animals and Their People 541 (pp. 3–12). BRILL. https://doi.org/10.1163/9789004386228_002 542 Methodological Framework. Endnotes 560 1 Barring the recent invention (ca 1850) of the technological reproduction of sound (Cross and Proctor 2014), music has always been played for specific audiences, with each performance offering an interplay between culturally-contextualized expression and audience- responsiveness. p 2 Such “denied dependency on a subordinated other” in the words of Val Plumwood (1993, p. 41), relates here as well as to other pairs where one aspect becomes dominating rather than complimentary or in partnership. Compare with McGilchrist (2009) and Eisler and Fry (2019). 3 This reiterates the perennial opposition in western thought between philosophy (discourse, truth) and rhetoric (style, pathos, the ability to persuade). 2 Such “denied dependency on a subordinated other” in the words of Val Plumwood (1993, p. 41), relates here as well as to other pairs where one aspect becomes dominating rather than complimentary or in partnership. Compare with McGilchrist (2009) and Eisler and Fry (2019). 3 This reiterates the perennial opposition in western thought between philosophy (discourse, truth) and rhetoric (style, pathos, the ability to persuade). 22 4 The enterprise to understand what animal music is, is related to a central question in anthropomusicology, which hold that musical universals exist (Martinelli 2008, 2009). Ethnomusicologists have identified two main possibilities for the question of musical universals: either there are musical traits present in all communities with no contrary examples, or there’s something at the pragmatic level about musical practice that is common to all communities. The question of whether music is universal among humans is less relevant for our purposes, though we are perhaps more sympathetic to the idea that the pragmatics of music (namely, establishing common ground, and expressing and communicating through repetition and variation) are sufficient universals. 5 While of course some features of utterances are accidental - artifacts of the process and physics of conveying an expression rather than intended - these are misfires. Looking into how to triangulate semiosis so as not to get derailed by reading too much into these misfires, or semiotic red herrings, is an important aspect of good semiology. But assuming that all musicality is somehow added-on rather than fundamental is erring too far in the other logocentric direction. Endnotes 560 Study of what sort of environmental and social elements cause more or less misfires in communication or speech (“distortions”) is indeed a very important part of biosemiotics, at the intersection with social design and the environmental/social/commercial determinants of health (Dowling et al. 2012; Potvin et al. 2011). Human industrial interference with natural processes lowers the fidelity of nonhuman communication (Szymkowiak and Schmidt 2022). For example, the fact that songbirds sing louder in noise-polluted urban environments, and that that loudness causes more distortions (less fidelity in interpretation from other intended listening conspecifics), should be a cause for alarm, and is an example of semiocide via humans taking up too much sonic bandwidth (Luther et al. 2016). 6 In fact, our training to resist the relevance of certain synesthetic elements of speech may render our understanding and fidelity poorer. Perhaps our lopsided focus on symbolic content broadsides us to other intuitive elements in speech which would tell us about the metastructure of discourse, power, and our willingness and desire to engage (or not) in certain milieus. 23
https://openalex.org/W3095324150	https://www.annalsofgeophysics.eu/index.php/annals/article/download/7933/7282	English	null	Energetic Particle Flux Variations around magnetic storm and huge earthquake	Annals of geophysics	2,020	cc-by	3,826	Abstract A megathrust earthquake with Mw 9.0 occurred in the North-western Pacific Ocean on March 11, 2011. From the energetic particle flux from WIND, CLUSTER and GOES in different L locations, some variation can be found around the earthquake. Among the three satellites, WIND is used to identify solar activity, and GOES is used to detect the changes from ground source. And during the same period, a magnetic storm with intensity -80nT occur. In order to validate the particle flux variation, multi-parameters relationship is compared. The results show that: (1) all energetic fluxes variation can reflect the solar activity. The far ones are connected with the F10.7 and the near ones are connected with Dst/Kp. (2) The energetic particle fluxes give a scarp change in all energy bands at the beginning coupling period and when the space recovers to be quite, the fluxes will have a long decreasing tail from high to low energy. (3) The coseismic and after effect have been detected in GOES and the pre-seismic emission should exist because the bigger decreasing fluxes in GOES are responding to the period with smaller Kp. Keywords: Energetic particle, different altitudes, earthquake. ANNALS OF GEOPHYSICS, 63, 5, PA553, doi:10.4401/ag-7933 ANNALS OF GEOPHYSICS, 63, 5, PA553, doi:10.4401/ag-7933 Energetic Particle Flux Variations around magnetic storm and huge earthquake Jianping Huang,1, 2, Xuhui Shen1, Wenjing Li1, Wei Chu1 Jianping Huang,1, 2, Xuhui Shen1, Wenjing Li1, Wei Chu1 (1) National Institute of Natural Hazards, Ministry of Emergency Management, Beijing 100085, China (2) Institute of Disaster Prevention, Sanhe 065201, China Article history: received September 19, 2018; accepted February 3, 2020 Article history: received September 19, 2018; accepted February 3, 2020 Keywords: Energetic particle, different altitudes, earthquake. 2.2 Satellite data source Many satellites have been launched into various locations to study the small-scale plasma structures in three dimensions in key plasma regions, such as the solar wind, bow shock, magnetopause, polar cusps, magnetotail and the auroral zones. Here WIND, CLUSTER, and GOES are introduced, which are located in about 200 Re, 1.2~19 Re, 5~6 Re (Earth radii, where 1 RE = 6371 km). 2.1 Space weather background Several space weather indexes are shown in Figure 1, including f 0.7, Kp, Dst, AU, AL and solar Lyman-alpha. F10.7, the solar radio flux at 10.7 cm (2800 MHz) is an excellent indicator of solar activity. From March1 to March 15, the level increased and reached maximum on March 8 and then decreased. Kp, the geomagnetic three-hourly index, is considered a proxy for the energy input from the solar wind to Earth. During the period, the high value occurred on March 10-12. Dst index represents the axially symmetric disturbance magnetic field at the dipole equator on the Earth’s surface. Major disturbances in Dst are negative, namely decreases in the geomagnetic field. These field decreases are produced mainly by the equatorial current system in the magnetosphere, usually referred to as the ring current. Only on March 11, the Dst decreased lower than -80nT. The trend of the AU and AL is similar to Dst. And the solar Lymann-alpha is almost same to F10.7. Jianping Huang et al. environment is governed principally by the interaction of energetic charged particles with electric and magnetic fields in space. In particular, near the Earth, most of these charged particles derive their energy ultimately from the Sun, directly or through the interaction of the solar wind with the Earth’s magnetosphere [Russell, et al, 2016]. Wave- particle interactions also play a fundamental role in energy exchange between plasma waves and radiation belt electrons [Walt, 1994]. Meanwhile, the similar process, but in the opposite direction, has been suggested. Before strong earthquakes, some EM waves, especially the VLF/ELF wave, can be emitted from the lithosphere [Merzer et al., 1997; Molchanov et al., 1998; Zhang X et al., 2014], penetrates into the ionosphere through atmosphere [Rozhnoi et al., 2008 and references therein], and couples with the ionospheric plasma and energetic particles [Nemec, et al., 2009]. Energetic particle variation, called precipitation or enhancement, has been taken as the pre-seismic precursor since 1980s [Galper et al., 1989]. After more and more attentions were paid on the relation between particles and earthquakes in terms of a number of parameters, including time difference, spatial shift according to case study, statistical analysis or some modeling based on wave-particle coupling theory [Voronov et al., 1990; Galper et al., 1992, 1995; Pustovetov et al., 1993; Aleksandrin et al., 2003; Sgrigna et al., 2005; Huang et al., 2010; Li et al., 2012; Fidani, 2010; Zhang et al., 2013; Wang et al., 2014]. All these studies showed that before strong earthquakes, energetic particle flux would be affected from 300 to 2000 km altitudes [Wang et al., 2014; Zhang et al., 2013]. However, when a magnetic storm and a strong earthquake happened at the same time, what does the energetic particle response? In this paper, we are devoted to exam whether the pre-seismic flux variation could be found at higher altitudes. So, the higher altitudes data from WIND, Cluster II and GOES are collected, and the time period are set around the occurrence of the M9.0 Tohoku earthquake. 1. Introduction At 05:46 (UTC) On March 11, 2011, an M9.0 earthquake occurred near Miyagi city (142.6° E, 38.1° N), off the east coast of Honshu, Tohoku area, Japan. The resultant tsunami waves damaged countless coastal communities around the Tohoku area. This earthquake is the strongest event taken place in the last 20 years. From the Japanese GPS network, the Total Electron Content (TEC) in the ionosphere above the focal region rapidly increases 40 minutes before the event [Heki, 2011, 2013; Jin, 2014]. The earthquake and the tsunami, coupling with the atmosphere, generated upward-propagating atmospheric disturbances, which reached the upper atmosphere and ionosphere, and were subsequently detected by GPS receivers [Galvan et al., 2012; Komjathy et al., 2012; Yu et al., 2015]. Those pre-seismic signals have been validated by the Lithosphere-Atmosphere- Ionosphere Coupling (LAIC) theory [Pulinets et al., 2000, 2004 and 2011; Zhao et al., 2010; Kuo et al., 2014; Zhang, et al., 2012]. According to the LAIC, the ionospheric disturbance is originated from electromagnetic field or associated with waves above strong earthquake regions [Parrot, 2012]. In addition to the TEC or electron density, the electromagnetic field and energetic particle flux can also be disturbed. It is worth noting that, the physics of the solar-terrestrial 1 Table 1. Particle Flux change onboard WIND. Table 1. Particle Flux change onboard WIND. 2.3 Wind observatory Figure 2 lists the magnetism and energetic particle records (Three-Dimensional Plasma and Energetic Particle Investigation payload, 3DP) from WIND satellite. The variation shape is similar in the three components of the magnetic field. And the amplitude of Bx is larger than that of By and Bz. The disturbance occurred on March 10 to 12. The clear sharp changes on March 10 and March 11. 2 Particle ﬂux around storm and earthquake Figure 1. Space weather index from February 20 to March 20, 2011. Figure 1. Space weather index from February 20 to March 20, 2011. For all the energetic fluxes, three “layers” can be found: the undisturbed region (blue region), the weak increased region (green region) and the core enhancement region (red region). All 4 payloads recorded the changes. The shape at the increased layer is sudden scarp and then weak slope, as shown in SOSP, SFSP and EHSP. The shape in the core enhancement region is like “Gauss shape”. The center data of Gauss shape is March 10 for all payloads and also is March 11 in EHSP and ELSP. An indentation, as shown in SOSP, ELSP and not clear in SFSP and EHSP, can also be found in Figure 2. The difference is the starting-ending date and energy range, as listed in Table 1. Table 1 listed the variation date and energy for all payloads. For ELSP, no obvious changes can be seen in the increased layer. The date in SFSP and EHSP is several hours earlier than that in SOSP and lasted longer, too. And for the enhanced layer, March 10 is the same for all payloads and March 11 is found in EHSP and ELSP. Payload SOSP SFSP EHSP ELSP Recorded Energy 0.1-4MeV 40-300keV 0.2-20keV 10-1000eV Increased Layer Response energy 0.2-4MeV 40-300keV 0.5-20keV unclear Starting Time March 8 March 7 March 7 unclear Ending Time March 12-13 March 12-16 March 12-14 unclear Core Layer Response energy 0.1 ~1MeV. 40-100keV 0.5-1keV 10-200eV Starting Time March 10 March 10 March 10 & 11 March 10 & 11 Ending Time March 11 March 11 March 10 & 12 March 10 & 11 Table 1. Particle Flux change onboard WIND. 3 Jianping Huang et al. Figure 2. Magnetism and particle ﬂux from WIND satellite. Jianping Huang et al. Figure 2. Magnetism and particle ﬂux from WIND satellite. 2.4 CLUSTER II observatory CLUSTER II is composed of four identical spacecraft flying in a tetrahedral formation, with an aim to study the impact of the Sun’s activity on the Earth’s space environment. Each orbit took approximately 57 hours to complete. Figure 3 shows the RAPID (Research with Adaptive Particle Imaging Detectors) particle flux from March 1 to March 16, 2011. Figure 3 shows that normally the flux keeps steady and only increase quickly when they pass the perigee which is close to the polar region. The flux increases from March 8 to March 10 and then decreases to normal level. CLUSTER II is composed of four identical spacecraft flying in a tetrahedral formation, with an aim to study the impact of the Sun’s activity on the Earth’s space environment. Each orbit took approximately 57 hours to complete. Figure 3 shows the RAPID (Research with Adaptive Particle Imaging Detectors) particle flux from March 1 to March 16, 2011. 3. Analysis and Discussions From Figure 1 to 4, each parameter has its variation and it is inconvenient to judge whether the variation is connected with the huge earthquake or not. Figure 5 gives some parameters with key time information. From T1 Line, it is clear that the increase in WIND from March 8 should be connected with the F10.7 high values. T2 line gives the beginning time of the main phase for this geomagnetic storm, and also explains the beginning time of enhancement in WIND and decrease in GOES. T3 and T4 give a period to show the 1st disturbance in GOES, the core enhancement in WIND and the 1st high value (> 40) in Kp. Then, T5 and T6 are for the 2nd one, and T8 and T9 are for the 3rd. T7 line gives the earthquake time which is about 14minutes later than T6. T6 is also the the lowest Dst time point for the geomagnetic storm. Figure 5. The jointly compassion of different parameters T1: the beginning time of F10.7 peak; T2: magnetic storm beginning time from Dst; T3: beginning time of the 1st GEOS ﬂux disturbance; T4: ending time of the 1st GEOS ﬂux disturbance; T5: beginning time of the 2nd GEOS ﬂux disturbance; T6: ending time of the 2nd GEOS ﬂux disturbance; T7: huge earthquake time; T8: beginning time of the 3rd GEOS ﬂux disturbance; T9: ending time of the 3rd GEOS ﬂux disturbance. Figure 5. The jointly compassion of different parameters T1: the beginning time of F10.7 peak; T2: magnetic storm beginning time from Dst; T3: beginning time of the 1st GEOS ﬂux disturbance; T4: ending time of the 1st GEOS ﬂux disturbance; T5: beginning time of the 2nd GEOS ﬂux disturbance; T6: ending time of the 2nd GEOS ﬂux disturbance; T7: huge earthquake time; T8: beginning time of the 3rd GEOS ﬂux disturbance; T9: ending time of the 3rd GEOS ﬂux disturbance. Considering the F10.7 and particle flux in WIND, they go in synchronization trend. For GOES, the situation is a little complex. For the 1st flux drop on March 10, the Kp is 4.3, and the corresponding change in particle flux is from ~6000 to ~20. For the second flux drop on March 11, the kp is 5.3, and the particle flux is from ~1000 to ~2. For the third one, the kp is 5.7 and the flux change is from ~6000 to ~100. 2.5 GOES The Geostationary Operational Environmental Satellites (GOES) all carry on the Space Environment Monitor (SEM) instrument at geosynchronous orbit around 6.6Re. The GOES Energetic Particle Sensor (EPS) measure proton, alpha-particle, and electron fluxes. GOES 13 was launched in 2006, located at 85.16° E and 0.36° S GOES 15 was launched in 2011 and located at 128.35° W and 0.03° S. 4 Particle ﬂux around storm and earthquake Figure 3. Cluster particle ﬂux from March 1 to March 16, 2011; C1, C2,C3, C4 means the 4 satellites. Particle ﬂux around storm and earthquake Figure 3. Cluster particle ﬂux from March 1 to March 16, 2011; C1, C2,C3, C4 means the 4 satellites. Figure 4. Particle ﬂux from GOES 13 and 15. Figure 4. Particle ﬂux from GOES 13 and 15. Figure 4. Particle ﬂux from GOES 13 and 15. 5 Jianping Huang et al. The electron fluxes from GOES 13 and GOES 15 approximately present the same character (Figure 4). In the lower energy, such as 40eV, 75eV, the periodic daily pump can be found, but in higher energy, it is not so obvious. As shown in Figure 4, only three decreasingly rapid fluctuations on March 10 and March 11 are found from March 7 to March 17. The first two are before the earthquake and the third one is after the earthquake. For the middle one, the change amplitude is biggest. For the after one, the amplitude is smallest. Particle ﬂux around storm and earthquake Particle ﬂux around storm and earthquake is consistent with the geomagnetic storm intensity. So generally the change amplitudes of the previous two flux should be smaller than the third one. According to GPS TEC and infrasonic record, the coseismic ionospheric effect was recorded one hour after the earthquake (Hao, et al, 2012), then there is no after-seismic effect on the 3rd flux drops in GOES because particle flux drop is 19 hours later after the earthquake. So the third drop is only the effect of high Kp. Then there should be some other source which leads to the bigger drop during the T3-T4 and T5-T6. According to the analysis above, the only source may come from the pre-seismic electromagnetic emission of the coming huge earthquake on March 11. What’s more, the duration of the lower flux values could also sustain the conclusion. For the third Kp, it is just a pulse. For the first, the duration is almost 3 hours, and for the second, the duration is almost 4.5 hours. During those durations, some local HF changes could be found which is far different from the spatial index frequency. So the possible pre-seismic effect should be considered for such two decreasing. In addition, for the T3-T4 and T8-T9, there is no more variation after the kp decreases. While after T5-T6 and T7, the fluxes in GOES occur, which should be the coseismic effect to the energetic particles. 4. Conclusions Using the various altitudes observations on WIND, CLUSTER and GOES, the high energetic particle fluxes are compared around the Tohoku huge earthquakes. The results indicate that: All satellite with particle detectors could record the solar activity. And in low orbit satellite, GOES, could also record the pre- and co-seismic particle precipitation. Particles with the different energy could response to the same source and the responding time and amplitude are different. Although the spatial background is not quite, the pre-seismic information could be detected. And with more cases, the real pre-seismic energetic particle flux changes could be quantified. Acknowledgement. Thanks to R. Lin/S. Bale at UC Berkeley, A. Szabo at NASA/GSFC for WIND and CLUSTER data and SWPC for the GOES data. Thanks SPDF, Goddard Space Flight Center, NASA for the CDAwebdata and ﬁgure support (http://cdaweb.gsfc.nasa.gov/istp_public/). Thanks for the f10.7 ﬁgure from Space Environment Prediction Center, National Space Science Center (http://www.sepc.ac.cn/F107Index_chn.php). This work is supported by the National State key R&D project (2018YFC1503501), ISSI-BJ(2019) and APSCO Earthquake Phase II. The authors would also like to thank reviewers and Editor for their valuable suggestions that have greatly improved this paper. 3. Analysis and Discussions In summary, the relative change ratio is 300, 500 and 60 but the spatial Kp is 4.3, 5.3 and 5.7. And based on previous research [He, et al, 2011], the changing amplitude 6 Jianping Huang et al. Jianping Huang et al. 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Penetration characteristics of VLF wave from atmosphere into lower ionosphere, Earthquake Science, 23, 275-282, doi:10.1007/s11589-010-0723-9 CORRESPONDING AUTHOR: Jianping HUANG, National Institute of Natural Hazards, Ministry of Emergency Management, Beijing 100085, China and Institute of Disaster Prevention, Sanhe 065201, China, e-mail: xhhjp@126.com © 2020 the Istituto Nazionale di Geofisica e Vulcanologia. All rights reserved CORRESPONDING AUTHOR: Jianping HUANG, National Institute of Natural Hazards, Ministry of Emergency Management, Beijing 100085, China and Institute of Disaster Prevention, Sanhe 065201, China, e-mail: xhhjp@126.com © 2020 the Istituto Nazionale di Geofisica e Vulcanologia. All rights reserved CORRESPONDING AUTHOR: Jianping HUANG, CORRESPONDING AUTHOR: Jianping HUANG, 9
https://openalex.org/W4200414930	https://www.researchsquare.com/article/rs-980950/latest.pdf	English	null	The Characterization of Amorphous AZO-n/Si-p Hetrojunction Diode for Solar Cell Application	Research Square (Research Square)	2,021	cc-by	5,528	The Characterization of Amorphous AZO-n/Si-p Hetrojunction Diode for Solar Cell Application Laya Dejam (  layadejam@gmail.com ) islamic azad university https://orcid.org/0000-0003-3925-1838 Abstract The aim of the present study is to verify the effect of annealing temperature variation on zinc oxide doped with aluminum (AZO) thin films deposited on p-type silicon (Si) substrates. Here, AZO/p-Si heterojunction was annealed in nitrogen environment and its structural, electrical, and optical characterizations were investigated. The results of XRD patterns showed the amorphous structure of AZO thin films. FE-SEM images illustrated the increase of grain size by increasing annealing temperature up to 500oC. The reflectance analysis showed that for this annealing temperature, that the energy band gap of AZO thin film was moved to higher energy level. The electrical properties were investigated by I–V measurement carried out in the light at room temperatures. The short circuit current (ISC), ideality factor, saturation current, and open circuit voltage (VOC) of the AZO/p-Si heterojunction strongly depended on annealing conditions due to charge carrier trapping and density of defect on interface. By considering IR and IF as reverse and forward current, the ration of IF/IR had the maximum value at 1 V which was belonged to n-AZO/p-Si heterojunction at 500oC annealing temperature. Keywords: AZO-n/Si-p hetrojunction, Amorphous, Annealing conditions, Sputtering procedure. Soghra Hosseini1, Laya Dejam* 2, Hossain Elahi 3. 1 Department of Physics, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Physics, West Tehran Branch, Islamic Azad University, Tehran, Iran 3 Assistant professor, Department of Mechanical engineering, Arak University of Technology, Arak, Iran 3 Assistant professor, Department of Mechanical engineering, Arak University of Technology, Arak, Iran * Layadejam@gmail.com Research Article Keywords: AZO-n/Si-p hetrojunction, Amorphous, Annealing conditions, Sputtering procedure Posted Date: December 23rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-980950/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Optical and Quantum Electronics on February 26th, 2022. See the published version at https://doi.org/10.1007/s11082-022-03550-w. The characterization of amorphous AZO-n/Si-p hetrojunction diode for solar cell application Soghra Hosseini1, Laya Dejam* 2, Hossain Elahi 3. 1 Department of Physics, Science and Research Branch, Islamic Azad University Tehran, Iran 2 Department of Physics, West Tehran Branch, Islamic Azad University, Tehran Iran 3 Assistant professor, Department of Mechanical engineering, Arak University o Technology, Arak, Iran Soghra Hosseini1, Laya Dejam* 2, Hossain Elahi 3. 1. Introduction Transparent conducting oxide (TCO) films have attracted a lot of attention in recent decades. Zinc oxide is a good candidate for TCO films duo to excellent electrical, structural, and optical properties like doping suitability and non-toxicity, as well as high thermal, mechanical, and chemical stability [1-2]. Therefore, ZnO can be considered as suitable choice in optoelectronic and electronic devices like, gas sensors, transparent electrodes, light emitting diodes, varistors, nanolasers heterojunctions, and etc. [3–4] Moreover, ZnO thin film deposited on p/n silicon substrate can be applied in optoelectronic devices and solar cells as a useful heterojunctions whose main advantages are the low cost of silicon and the high energy of band gap of ZnO thin films [5]. Because of high sensitivity of pure ZnO thin films to the oxidation and also its high transmittance in the visible area, ZnO thin films were doped with aluminum initially. Then, all samples were annealed in order to decrease resistivity and increase band gap energy, carrier concentration, and stability [6]. On the other hand, Al:ZnO (AZO) is a promising material for light emitting diodes, solar cells, and gas sensors along with general optoelectronic applications [7-8]. The advantages of silicon-based solar cells are like non-toxicity and low cost make them to be applied in photovoltaic systems [9]. AZO thin films as a front electrode must be conductive enough to allow lateral transfer of electron from sufficient light (80%) to the incoming light [10]. Several methods have been applied to fabricate AZO thin films including pulsed laser deposition (PLD), evaporation, chemical vapor deposition, magnetron sputtering, and spray pyrolysis [11-12]. Among these methods, thin films with high- quality are prepared by magnetron sputtering technique because of its special conditions like low temperature deposition, large-area fabrication adaptability, repeatability, and uniformity as well as working power and sputtering pressure [13]. Specific interest of ZnO/Si and AZO/p-Si heterojunctions is observed in the optoelectronic devices strong adhesion. So far, a lot of researches have been focused on AZO/p-Si hetrojunction. Bo et. al. [14] fabricated Al-doped n-ZnO/p-Si heterojunctions by the deposition of AZO film on p-Si (1 0 0) wafer and investigated their optical, microstructural, and electrical properties. They found good quality of samples and their rectifying behavior under dark condition. Urper et. al. [15] also fabricated ZnO:Al thin film by sol-gel dip coating method and studied the relationship between Al content and optical band gap of the films. 1. Introduction They also analyzed crystalline structure and electrical resistivity of samples under different annealing conditions. Baydogan et. al. [16] found hexagonal wurtzite crystal structure of ZnO:Al thin films prepared by sol-gel dip coating method. They also demonstrated that the optical band gap was broadened by increasing Al concentration. The novelty of our work is that we have focused on the optical, structural and electrical properties of amorphous AZO thin films prepared by sputtering. So far, according to our studies, amorphous AZO thin films has not been considered as a diode and making amorphous thin films is easier and cheaper. Also we have investigated the improvement of amorphous AZO/p-Si hetrojunction and find the best structural conditions for AZO/p-Si as a diode and solar cell. considered as a diode and making amorphous thin films is easier and cheaper. Also we have investigated the improvement of amorphous AZO/p-Si hetrojunction and find the best structural conditions for AZO/p-Si as a diode and solar cell. In the present work, AZO thin films were deposited on p type silicon substrate by RF magnetron sputtering method. AZO thin films were annealed with nitrogen gas at three different temperatures. Then, their electrical, optical, and structural properties as well as their morphology were investigated. For this purpose, every junction of AZO amorphous thin film and Si was taken into consideration and Si/ AZO heterojunction diode properties were determined. As can be seen, annealing in nitrogen atmosphere and Al dopant ends to the extreme enhancement of blue, violet and green emissions. Table .1 .The technical details of sputtering process for each sample Table .1 .The technical details of sputtering process for each sample Substrate Base Pressure (mbar) Work Pressure (mbar) Power (W) Time (min) Gas Thickness (±5nm) Rat (A° /S) Si 6.5 × 10−5 6 × 10−3 175 75 oxygen%𝟑𝟎 argon%𝟕𝟎 200 0.37 2. Experimental details AZO thin films were deposited by radio-frequency (RF) magnetron sputtering on p- type silicon substrate for 75 minutes. The diameter of circle-shaped target was 2.5cm with the thickness of 3 mm. The weight ratio of Al and Zn were ~10% and 90%, respectively. The working gas was constant amount of (O2+Ar) where the ratio of O2 was 30% and the power was 175 W. The initial vacuum and basic pressure were set as 6.4×10-3mbarr and 6.5×10-5mbarr, respectively. The substrates were initially cleaned and then placed in acetone and ethanol bath for 15 minutes. They were finally dried in a clean room. In order to remove any oxide residues, samples were pre-sputtered 30 minutes. Afterward, samples were annealed for 60 minutes at 400 oC, 500 oC, and 600 oC annealing temperatures at the presence of nitrogen gas and then, they were cooled down to room temperature gradually. The details of sputtering process are presented in Table 1. The thickness of prepared AZO thin film was measured 200±5 nm. To characterize the crystalline nature of samples, XRD patterns were extracted from STOE-XRD diffractometer using Cu-Kα line (l = 0.15406 nm). Also, the topography of AZO thin films was investigated by the non-contact mode atomic force microscopy (AFM) (Vecco-Autoprobcp-research) and field emission scanning microscopy (FESEM) (MIRA3 TESCAN). Energy dispersive X-ray (EDX) analysis was performed by MIRA3 TESCAN to analyze the information about the elements of samples. The optical transmittance spectra of the deposited films were recorded by the UV–VIS–NIR spectrophotometer (CARY-500 UV-VIS-NIR) in the range of 200-1600 nm. Photoluminescence (PL) was also used to reveal the luminescence characteristics of samples by Cary Eclipse spectrometer equipped with a xenon amp with 320 nm (AZO) excites wavelength. The current–voltage (I-V) measurement was finally carried out by solar simulator (SIM-1030) and Palm Sense. 3. Results and discussion 3.1 Structure properties Fig. 1 illustrates XRD patterns of AZO thin films on silicon substrates. As can be seen, there is just one peak at 68 degree related to silicon substrate and there is no peak related ZnO (hexagonal wurtzite) crystal structure. Therefore, amorphous nature of AZO thin films is demonstrated by XRD patterns. The crystalline peak of Si is of very high intensity. Therefore, the AZO thin films on the glass substrate were also grown and annealed under the same conditions to examine their XRD spectrum, which may indicate ZnO crystalline peaks. But no specific peak was observed for the crystal structure of ZnO on the glass substrates. The method of preparing the AZO thin films can be the reason for the amorphous structure of the thin films. Existence of Zn/Al target and oxygen gas enters the environment during sputtering. For thin thicknesses, it is not possible to form the crystal structure of AZO thin films. In the same manufacturing and annealing conditions with thicker thicknesses, we prepared the crystal structure of AZO thin films [10,13].Similar results have been gained by other researchers who prepared AZO thin films without annealing process [8-10]. Incidentally, the interesting point of the research is that although there is no change in thin film structure up to 600 oC annealing temperature, the electrical and optical properties of samples have been changed. Most research focus on the amorphous nature of AZO thin films. However, their crystalline structure has been studied extensively [14-16]. Fig .1 XRD patterns of AZO thin films with different annealing temperatures Fig .1 XRD patterns of AZO thin films with different annealing temperatures Fig .1 XRD patterns of AZO thin films with different annealing temperatures Fig. 2. The EDX spectra of (a) as deposited, and annealed AZO/p-Si heterojunction at (b) 400oC (c) 500oC, and (d) 600oC. Fig. 2. The EDX spectra of (a) as deposited, and annealed AZO/p-Si heterojunction at (b) 400oC (c) 500oC, and (d) 600oC. As can be seen in Fig. 2 (a-d), EDX data approved the existence of Zn and Al in as- deposited and annealed thin films and determined their stoichiometry. Moreover, any significant variation were observed in weight and atomic percentage of AZO thin films after annealing process. 3. Results and discussion 3.1 Structure properties (b) AZO/Si 400oC (a) AZO/Si As-deposited (b) AZO/Si 400oC (a) AZO/Si As-deposited (a) AZO/Si As-deposited (b) AZO/Si 400oC C o AZO/Si 600 (d) C o AZO/Si 600 (d) C o AZO/Si 500 )c( Fig.3. FESEM images (a-d) of as deposited and annealed AZO/p-Si heterojunction at different annealing temperatures C o AZO/Si 500 )c( Fig.3. FESEM images (a-d) of as deposited and annealed AZO/p-Si heterojunction at different annealing temperatures FESEM images in Fig. 3 (a-d) represent the topography of as deposited and annealed AZO thin films. As can be seen, there are irregular and non-uniform nanograins in the as deposited sample whose average size is ~55 nm. Increasing annealing temperature up to 500oC increases the average diameter because of agglomeration and the best grain adhesion is occurred at 500oC and the grain boundaries are much smaller, which is very important for electrical properties because they control the movement of electrical charges. By increasing annealing temperature to 600oC, the distribution is disrupted due to grain movements. In fact, at this temperature, the particles have not become smaller, but have dispersed, and the grain boundaries have increased. This is also confirmed in the AFM particle distribution diagram (Table 2). (a) AZO/Si As-deposited (b) AZO/Si 400oC (b) AZO/Si 400oC (a) AZO/Si As-deposited (b) AZO/Si 400oC (a) AZO/Si As-deposited (a) AZO/Si As-deposited (c) AZO/Si 500oC (d) AZO/Si 600oC Fig .4. 3D AFM images (a-d) of as deposited and annealed of AZO/p-Si heterojunction (d) AZO/Si 600oC (d) AZO/Si 600oC Fig .4. 3D AFM images (a-d) of as deposited and annealed of AZO/p-Si heterojunction Fig .5. Distribution of topology for as deposited and annealed of AZO/p-Si heterojunction Fig .5. Distribution of topology for as deposited and annealed of AZO/p-Si heterojunctio Surface morphology of AZO films was examined by their 3-D AFM images (a-d) as illustrated in Fig. 4. As can be seen, surface roughness is increased by increasing annealing temperature (Table 2) which may be due to the grains stickiness, the creation of a rough surface, and the variation of RMS roughness under nitrogen gas flow range from 0.42 to 1.61 nm. In addition, the surface of as-deposited AZO thin film is smoother and more interconnected which is formed by annealing particles at the grain boundaries, resulting in a variety of sizes. Fig. 5 shows the morphological changes of thin film surface. Particle distributions differ from the Gaussian diagram by increasing annealing temperature. 3. Results and discussion 3.1 Structure properties Especially at 600°C, the dispersion of nanoparticles is much higher which is exactly the behavior observed in FESEM images. Increasing annealing temperature increases the energy and surface mobility which results in particle size enhancement. The increase in thermal and kinetic energy has also increased the dispersion of particles on the surface which is a key factor in changing the shape of the Gaussian distribution. On the other hand, increasing the size of particles and their dispersion on the surface is because of the adhesion of nanoparticles to each other. increasing the size of particles and their dispersion on the surface is because of the adhesion of nanoparticles to each other. 3.2 Optical properties It should be noted that the results of PL analysis confirms these changes in defects, localized states, and band gap. 𝐹(𝑅) = (1−𝑅)2 2𝑅 (1) 2𝑅 where R is the reflectance of sample and depends on wavelength. Eq (2) shows the relationship between F(R) and the absorption coefficient (α) as [17]: (2) 𝑡 Where ‘t’ is the thickness of AZO thin films. To evaluate the dependency of band gap to direct allowed transition, the diagram of (αhν)2 vs. hν is illustrated in Fig. 7 and the band gap was calculated via the linear hν–intercept. The value of band gap increases gently from 3.2 eV to 3.9 eV by the increase of annealing temperature up to 500oC. But when the annealing temperature increases to 600oC, the value of band gap decreases. Hence, a critical state has occurred at 500 oC which is also confirmed in previous research [10]. In fact, annealing process decreases the defects while increasing temperature reduces the density of the localized states and decreases their band tail energy. Here, increasing annealing temperature to 500oC decrease the density of the localized states and defects and increases the band gap. 𝑡 Where ‘t’ is the thickness of AZO thin films. To evaluate the dependency of band gap to direct allowed transition, the diagram of (αhν)2 vs. hν is illustrated in Fig. 7 and the band gap was calculated via the linear hν–intercept. The value of band gap increases gently from 3.2 eV to 3.9 eV by the increase of annealing temperature up to 500oC. But when the annealing temperature increases to 600oC, the value of band gap decreases. Hence, a critical state has occurred at 500 oC which is also confirmed in previous research [10]. In fact, annealing process decreases the defects while increasing temperature reduces the density of the localized states and decreases their band tail energy. Here, increasing annealing temperature to 500oC decrease the density of the localized states and defects and increases the band gap. However, AFM and FESEM images of sample annealed at 600oC indicate surface irregularity and uneven distribution of nanoparticles and hence, the increase of defects replacement and localized states as well as decrease of band gap are expected. It should be noted that the results of PL analysis confirms these changes in defects, localized states, and band gap. Table. 2. 3.2 Optical properties Fig. 6 indicates the reflectance spectra of as deposited and annealed AZO thin films. In the reflectance spectra, a sharp band edge of about 350-400 nm in the as deposited and 400, 600 oC annealed thin films is due to strong adsorption. An obvious blue shift is only observed in AZO thin film annealed at 500 oC which is because of the suffering of its absorption edge. The reflection of AZO thin film at 550-800 nm decreases with annealing. The reduction of the reflection spectrum after annealing may be due to the nano-scaled morphology of the sample which has improved the light traps. Fig.6. Reflectance of as deposited and annealed AZO/p-Si heterojunction. Fig.6. Reflectance of as deposited and annealed AZO/p-Si heterojunction. Fig.6. Reflectance of as deposited and annealed AZO/p-Si heterojunction. Fig.6. Reflectance of as deposited and annealed AZO/p-Si heterojunctio Fig.7. Tauc’s Plot of as deposited and annealed AZO/p-Si heterojunction Fig.7. Tauc’s Plot of as deposited and annealed AZO/p-Si heterojunction The UV–Vis spectra were inquired by Kubelka- Munk theory to convert thin film reflectance to Kubelka- Munk function (F (R)), by Eq (1) [17-19]: 𝐹(𝑅) = (1−𝑅)2 2𝑅 (1) where R is the reflectance of sample and depends on wavelength. Eq (2) shows the relationship between F(R) and the absorption coefficient (α) as [17]: 𝛼= 𝐹(𝑅) 𝑡 (2) Where ‘t’ is the thickness of AZO thin films. To evaluate the dependency of band gap to direct allowed transition, the diagram of (αhν)2 vs. hν is illustrated in Fig. 7 and the band gap was calculated via the linear hν–intercept. The value of band gap increases gently from 3.2 eV to 3.9 eV by the increase of annealing temperature up to 500oC. But when the annealing temperature increases to 600oC, the value of band gap decreases. Hence, a critical state has occurred at 500 oC which is also confirmed in previous research [10]. In fact, annealing process decreases the defects while increasing temperature reduces the density of the localized states and decreases their band tail energy. Here, increasing annealing temperature to 500oC decrease the density of the localized states and defects and increases the band gap. However, AFM and FESEM images of sample annealed at 600oC indicate surface irregularity and uneven distribution of nanoparticles and hence, the increase of defects replacement and localized states as well as decrease of band gap are expected. 3.2 Optical properties Structural, optical and electrical parameters of as deposited and annealed AZO/p-Si heterojunction Sample RMS Roughne ss(nm) Eg (eV) Eg(emiss ion) (eV) Voc (V) Isc (μA) Ideality factor (n) IF/IR Is (μA) As-Dep 0.42 3.2 3.318 0.05 0.23 29.72 0.82 0.29 400oC 1.03 3.3 3.27 0.06 0.81 24.15 0.39 0.17 500oC 1.53 3.9 4.02 0.21 1.84 22.72 1.72 0.30 600oC 1.61 3.3 3.32 0.13 1.017 21.46 0.38 0.46 uctural, optical and electrical parameters of as deposited and annealed eterojunction Table. 2. Structural, optical and electrical parameters of as deposited and annealed AZO/p-Si heterojunction Fig.8. PL spectra of as deposited and annealed AZO/p-Si heterojunction Fig 8 PL spectra of as deposited and annealed AZO/p Si heterojunction Fig.8. PL spectra of as deposited and annealed AZO/p-Si heterojunction PL spectra in Fig. 8 are applied at room temperature to estimate any annihilation or defect created by annealing. PL emission proved the center of defects that act as centers of recombining charge carrier. For as-deposited and annealed AZO thin films excited at 320 nm, the PL spectra show four emission bands at UV, Violet, Blue and Green emissions. Fitting PL spectra with Gaussian diagram gives valuable information about FWHM and peak positions as summarized in Table 3. The peak at around 374 nm corresponds to UV emission or to the transition of electron from the substitution level below the conduction band to the valence band [20]. The near band-edge emission with the peak at around 393-398 nm results in UV emission because of its dependency to the recombination of free-exciton [21]. The peak around 400nm in all samples is a violet emission which appears because of the electron transition from conduction tail states to the valence states [22]. Moreover, the blue emission observed around (467-489) is revealed by the transition from shallow donor levels of oxygen vacancy to valence band which is in agreement with Xue’s report [23]. The peak at around (528-534nm) belongs to green emission which is appeared by the transition from deep donor levels of oxygen vacancies to valence band [24]. Annealing in nitrogen gas flow also affects the presence of green emission [25]. With increasing annealing temperature up to 500oC, the PL centers remarkably increase which are related to the increase in PL intensity. 3.2 Optical properties Therefore, increasing density of defects at 500°C results in the increase of recombination and hence, hence, the increase of Voc that can be seen in Table 2 where the maximum Voc is revealed in 500 oC. Table 3. PL peak positions of as-deposited and annealed AZO/p-Si heterojunction Sample Peak positions (nm) FWHM (nm) AZO/Si As-Dep 389.55 424.46 481.66 522.67 0.23784 72.416 21.105 69.589 AZO/Si 400oC 379.05 422.31 478.54 521.49 25.211 57.583 37.693 72.36 AZO/Si 500oC 307.85 414.33 488.86 510.8 1.5903 67.642 43.849 121.87 AZO/Si 600oC 372.7 415.12 439.54 491.05 113.92 64.947 652.71 79.492 3.3 The hetrojunction properties of AZO/p-Si The value of Is for n-AZO/p-Si heterojunction was also reported by for O.Urper et al.[15] and N. Baydogan et al.[16] as 1.5×10-6A and 0.3×10-6 A, respectively and they are comparable to the value of Is in the present study. As it is known, the value of n for an ideal diode is unity and for other devices is bigger than one. Here, the value of ideality factor is similar to the previous research as 38[16] and 20.1[14] which demonstrates that the present diode is not ideal and the reason is the existence of surface states and oxide layer [28]. In order to find the response of photoelectrical parameters, the values of open circuit voltage (VOC) and short circuit current (ISC) were calculated for AZO/p-Si Schottky diodes under 1000 W/m2 of light source for as deposited and annealed AZO/p-Si heterojunctions. The values of these two parameters have been measured by the results of I-V diagram in Table 2. The values of ISC and VOC are in the regions from 0.23 to 1.84 μA and 0.05 to 0.13 V, respectively. The adsorption results also show the maximum and minimum values of ISC at AZO/p-Si annealed at 500 ° C and as- deposited AZO/p-Si, respectively. In addition, VOC is related directly to the band gap so that the increase of band gap increases VOC. As can be seen in Table 2, the maximum band gap and hence, the maximum value of VOC are indicated in sample annealed at 500oC. In an ideal device, VOC is limited by radiative recombination. Because of the unique photoelectrical properties of AZO/p-Si annealed at 500 oC, it is considered as a suitable candidate in solar cell and photodiode applications. High values of n in AZO/p-Si diodes maybe be due to the presence of surface states or interfacial layers and demonstrates that the transport mechanism is no longer controlled by thermionic emission [29]. The forward bias log I vs-log V plot reveals the dominate mechanisms of transport charge in AZO/p-Si diodes along with a power law treatment of current I α Vm+1 where (m + 1) changes with the injection level and corresponds to trapping centers distribution [29]. As shown in Fig. 10, AZO/p-Si diodes follow three regimes: ohmic, space-charge-limited current (SCLC), and trap filling limit (TFL) [29]. 3.3 The hetrojunction properties of AZO/p-Si In order to find remarkable information about Schottky diode behavior, current- voltage I-V diagram were measured under illumination at room temperature and presented in Fig. 9 (a) for all samples. An almost photoelectric and rectifying behavior is illustrated in n-AZO/ p-Si heterojunctions. Fig. 9(b) also illustrates the semi-logarithmic forward and reverse bias of the experimental I–V for the n-AZO/ p-Si Schottky diodes. Fig. 9. (a) I–V diagram and (b) Semi-logarithmic I–V diagram of as-deposited and annealed AZO/p-Si heterojunction. Fig. 9. (a) I–V diagram and (b) Semi-logarithmic I–V diagram of as-deposited and annealed AZO/p-Si heterojunction. Fig. 10. Logarithmic I–V plot of as-deposited and annealed AZO/p-Si heterojunction Fig. 10. Logarithmic I–V plot of as-deposited and annealed AZO/p-Si heterojunction By considering IR and IF for reverse and forward current, the rectification IF/IR ratio at 1 V is in the range of 0.38 to 1.42. Based on p–n junction theory, the I–V relation of standard diodes is as follow [26]: By considering IR and IF for reverse and forward current, the rectification IF/IR ratio at 1 V is in the range of 0.38 to 1.42. Based on p–n junction theory, the I–V relation of standard diodes is as follow [26]: 𝐼= 𝐼𝑠exp ( 𝑞𝑉 𝑛𝑘𝐵𝑇−1 ) (4) 𝐼= 𝐼𝑠exp ( 𝑞𝑉 𝑛𝑘𝐵𝑇−1 ) 𝑞𝑉 𝑛𝑘𝐵𝑇−1 ) (4) (4) where Is and V is the saturation current and voltage bias, respectively. Is is measured from the straight line intercept of ln I at V = 0 as: where Is and V is the saturation current and voltage bias, respectively. Is is measured from the straight line intercept of ln I at V = 0 as: 𝐼𝑠= 𝐴∗𝑆𝑇2 exp (𝑞𝜑𝑏 𝑘𝐵𝑇) 𝐼𝑠= 𝐴∗𝑆𝑇2 exp (𝑞𝜑𝑏 𝑘𝐵𝑇) (5) (5) Here, q and T are the absolute value of electron charge (1.6 × 10-19 C) and temperature, respectively. Also, V, I, and S are applied voltage, net current, and the diode area, respectively. The value of Boltzmann’s constant (kB) is 1.38 × 10-23 J/K and the barrier height (eV) is labeled as φb. Also, the value of effective Richardson constant (A*) for ZNO is 32 A cm−2 K−2 [27]. The ideality factor (n) of heterojunctions is calculated in Eq. 6 from the slope of the straight line in the forward bias Ln I–V diagram [26] 𝑛= 𝑞 𝑘𝐵𝑇( 𝑑𝑉 𝑑𝐿𝑛𝐼) 𝑛= 𝑞 𝑘𝐵𝑇( 𝑑𝑉 𝑑𝐿𝑛𝐼) (6) (6) The values of Is and n are represented in table 2. 3.3 The hetrojunction properties of AZO/p-Si In the low range of voltage, for as-deposited and annealed AZO/p-Si at 400oC in region 1 (0.05 < log V < 0.08), annealed AZO/p-Si at 500oC in (0.2 < log V < 0.3), and annealed AZO/p-Si at 600oC (0.1 < log V < 0.15), an ohmic mechanism controlled the increase of current with voltage. This is because comparing to background thermal carrier density, the value of perfused effective carrier density is lower [30] and it is associated with trapping centers distribution [30]. As the intensity and density of the trap distribution centers for different annealing temperatures varied in PL analysis, the ohmic region range was also different. In the intermediate range of voltages, for as-deposited and annealed AZO/p-Si at 400oC (0.08 < log V < 0.1), annealed AZO/p-Si at 500oC (0.3 < log V < 0.4), and annealed AZO/p-Si at 600oC (0.15 < log V < 0.1) in region 2, SCLC is the dominate mechanism and the role of voltages is more than region 1. Moreover, comparing to thermal-generated free charge carrier density, the density of perfused free charge is much higher [29] which increases the current. In region 3 with high voltages, for as-deposited and annealed AZO/p-Si at 400oC (0.1 < log V < 1), annealed AZO/p-Si at 500oC (0.4 < log V < 1), and annealed AZO/p-Si at 600oC (0.2 < log V < 1), the exponential increase of current is observed. Here, the trap filling limit controlled the mechanism. Injected electrons fills the deep traps until all trap sites become fully occupied. Interestingly, comparing different regions of voltage for all samples shows that they change with almost the same slope and hence, the behavior of their occupied and trap sites are the same. Conclusion The effect of annealing process on structural, electrical, and optical properties of n- AZO/p-Si heterojunction was investigated in the preset study. The fabrication of n- AZO/p-Si heterojunction was carried out by the deposition of AZO thin films on p- Si substrate with magnetron sputtering technique. The XRD results indicated the amorphous structure of as deposited and annealed AZO thin films. FESEM images demonstrated that increasing annealing temperature up to 500oC increased the average diameter because of agglomeration so that the distribution of particles was disrupted. AFM images also illustrated that RMS roughness of samples was increased from 0.42 to 1.61 by the increase of annealing temperature. Also, by increasing temperature to 500oC, the band gap of thin films varied from 3.2 to 3.9 eV. Moreover, PL spectra showed that the PL centers and intensity were considerably increased by increasing annealing temperature. Good rectifying behavior of AZO/p-Si heterojunction annealed at 500 oC was also confirmed. Ideality factor, saturation current, rectification ratio IF/IR, open circuit voltage (VOC), and short circuit current (ISC) were the parameters optimized by the I–V measurements. The dominate mechanisms was transport charge in AZO/p-Si diodes. Since the AZO thin film is in amorphous structure, charge transfers are limited and therefore diode structures could not exhibit very good rectification and photovoltaic properties. However, since making amorphous thin films is easier and cheaper, it has been studied and the remarkable result is that by changing the surface structure of amorphous thin films and eliminating or reducing grain boundaries, electrical properties can be improved. Declaration of interest: The authors report no conflict of interests. The authors alone are responsible for the content and writing of the paper. Declaration of interest: The authors report no conflict of interests. The authors alone are responsible for the content and writing of the paper. [15] O. Urper, O. Karacasu, H. Cimenoglu, N. Baydogan, Superlattices and Microstructures 125 (2019) 81–87 References [1] P.P. Sahay, R.K. Nath, Sens Actuators B,134 (2008) 654–659 [2] L. Dejam, S. M. Elahi, M. M. Larijani and Y. S. Jalili, Bull. Mater. Sci. 38 (2015)1821-1830 [3] M.S. Wang, K.E. Lee, H.S Hahn, E.J Kim, S. Kim, J.S. Chung, E.W Shin, C. Park, Mater Lett. 61(2007) 1118–1121 [4] O. Kluth, G. Scho¨pe, J. Hu¨pkes, C. Agashe, J. 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https://openalex.org/W2769138930	https://symbiosisonlinepublishing.com/obesity-control-therapies/obesity-control-therapies19.pdf	English	null	Importance of a Blood Test Before Starting a Protein Ketogenic Diet	Obesity & control therapies	2,015	cc-by	2,002	Importance of a Blood Test Before Starting a Protein Ketogenic Diet Joaquin Perez-Guisado Rosa* Department of Genetic, University of Córdoba, Campus of Rabanales, Córdoba Received: : June 28, 2015; Accepted: July 20, 2015; Published: September 20, 2015 Corresponding author: Joaquín Pérez-Guisado Rosa, MD, Ph.D, Department of Genetic, University of Córdoba, Campus of Rabanales, Córdoba, Spain. E-mail: pv1peguj@uco.es Moreover, my Ph.D is based on this diet. For this reason, I would like to give advice based on my experience concerning this diet and specifically related to the importance of the blood test before starting this diet. As we all know, obesity is not only an aesthetic problem, but in developed countries, it is a disease that is reaching epidemic proportions and is associated with a multitude of medical conditions. Few examples of scientifically proven and its association with, are given below: Considered obesity as a disease per se and its possible association with other comorbidities, the medical consultation is very important. This checkup should include a complete medical history to consider personal and family history and physical examination that takes into account the patient’s weight, height, waist and hip circumference measurement and blood pressure. The blood test has a great importance, because on one hand it is not only important to know the external physical condition, but also we will know how our patient is regarding internal health markers, and how they evolve conducting diet and subsequent weight loss. Through the blood test, we can also determine whether the patient has any medical problem that could dissuade us from performing the ketogenic diet or could recommend us the use of any medication or additional measures. In those cases where we think that obesity could have an endocrine background, a more exhaustive blood test, also allow us to rule out or detect possible endocrine pathologies associated with it. 1. A lower life expectancy Symbiosis Symbiosis Symbiosis Symbiosis 2. A lipid profile by determining triglyceride, total cholesterol, Obesity & Control Therapies: Open Access Open Access Open Access Open Access Editorial 1. A lower life expectancy Finally, we could be facing a patient in which the values of urea and creatinine or only creatinine are exceeding normal levels without there being either associated renal insufficiency, in this case, we could find the ratio of urea and creatinine less than 10, it could be due to a liver disease, overhydration and even rhabdomyolysis. Keep in mind that urea levels depend on several factors such as the type of food, the liver protein catabolism and diuresis. While creatinine levels are more related to muscle protein catabolism and diuresis. These considerations may be of importance to us in order to know if our patient has a renal disease , which will be one in which both plasma urea and creatinine values are presented above the normal levels and compliance that, the ratio of urea and creatinine is approximately 10/1. We must be alert because it might be the case that due to a high protein diet, a high protein catabolism and even dehydration, determinations of urea and even creatinine could be above the normal levels without any renal failure associated, in this case we should find that, the urea and creatinine ratio exceeds 10 to 15. Finally, we could be facing a patient in which the values of urea and creatinine or only creatinine are exceeding normal levels without there being either associated renal insufficiency, in this case, we could find the ratio of urea and creatinine less than 10, it could be due to a liver disease, overhydration and even rhabdomyolysis. 6. Determination of uric acid, especially in patients with a history of gout or hyperuricemia. We should know that a protein ketogenic diet may increase in these patients the levels of this marker, being a relative contraindication when making this diet. If we have a patient with this problem, we could solve it through a previous normalization of uric acid by treatment with allopurinol for 2-3 weeks before starting the diet. Once the patient got such normalization, he could start the diet with the condition that he maintain the normal levels of uric acid with allopurinol treatment and we check it with regular blood sample tests (at least once a month). 3. An analysis of glucose metabolism by determining the fasting glucose. 1. A lower life expectancy In diabetic patients, if we want to know retrospectively how has been the control of their diabetes 2-3 months before the blood sample, we can determine glycosylated hemoglobin (HbA1c), which will present values below 7% if the control has been right. In general, all patients that perform the ketogenic diet improve fasting blood glucose as a result of an improvement in insulin sensitivity. 7. In those cases where we think there might be an endocrine pathology associated we should discard two diseases: hypothyroidism and Cushing syndrome. In hypothyroidism, we determine the plasma concentration of TSH, T3 and T4, and we must never forget that the best biochemical marker for the diagnosis and management of hypothyroidism is the TSH, as it should always be high. In hypothyroidism, the most frequent is having high levels of TSH with low levels of T4 and T3, however when the hypothyroidism is subclinical, the TSH remains high while the T4 is normal or low and the T3 is normal. Concerning the Cushing syndrome, the two most widely used tests when there is a suspected diagnosis are: urinary free cortisol determination in 24-hour urine test or rapid dexamethasone suppression test. Rapid dexamethasone suppression test consist of giving the patient before bedtime 1 mg of dexamethasone and determine the next morning plasma cortisol levels, which must be descended if the patient does not have Cushing Syndrome. 4. An analysis of the liver and biliary function by assessing the bilirubin, ALT, AST, GGT and alkaline phosphatase. This is important because many obese patients may have problems especially cholelithiasis and fatty liver associated with the insulin resistance. Normally, the fatty liver doesn’t have associated transaminase levels that go beyond twice the normal values and the ketogenic diet will normalize their values, although there may be an initial increase at the beginning of the diet. In those cases where we have particularly high levels of transaminases, we should do a further study because the liver problem is probably due to another entity. As for the question of what levels of maximum transaminase levels, we could include patients in the therapeutic protocol of the protein ketogenic diet, with up to three times their maximum values of transaminases, although the doctor will take the final decision and it is totally acceptable not to include a patient in the protocol if their values exceed twice than the normal values. 1. A lower life expectancy 2. A higher incidence with worse prognosis and evolution in different types of cancer such as the esophagus, stomach, colon, breast, endometrium, ovary, kidney and pancreas. 3. Cardiovascular problems or diseases associated such as dyslipidemia, hypertension, peripheral vascular disease, phlebitis and venous thromboembolic disease. 4. Endocrine problems where resistance to insulin, either directly or indirectly plays a key role, as the metabolic syndrome, impaired fasting glucose, carbohydrate intolerance, type 2 diabetes mellitus and polycystic ovary syndrome. 5. Both male and female infertility and menstrual disorders 6. Orthopedic problems such as osteoarthritis. 7. Hyperuricemia and gout. 7. Hyperuricemia and gout. Given all this, a complete blood test before conducting a protein ketogenic diet should include: 8. Chronic kidney disease due to glomerular damage. 1. The hemogram and electrolyte profile that consider the sodium, potassium, magnesium, calcium and chlorine. We all know that obesity is not synonymous of eating well or balanced, so could detect deficiencies as may be iron deficiency (microcytic anemia) of folic acid and vitamin B12 even (macrocytic anemias) in our obese patients. We should have in mind these alterations in order to focus on them appropriately so we don’t worse them with the completion of the ketogenic diet. Concerning the ions indicated, they are important to know if the patient has some sort of predisposition to electrolyte abnormalities that become apparent in the blood sample and that could exacerbate when the patient start the ketogenic diet, so we could act and prevent through the proper supplementation. 9. Gastroesophageal reflux disease and hiatal hernia. 10. Vision problems such as glaucoma, macular degeneration and cataracts 10. Vision problems such as glaucoma, macular degeneration and cataracts 11. Liver problems such as gallstones and liver steatosis, closely related to insulin resistance. 11. Liver problems such as gallstones and liver steatosis, closely related to insulin resistance. 12. Respiratory problems such as dyspnea, sleep apnea and asthma. 12. Respiratory problems such as dyspnea, sleep apnea and asthma. 13. Psychological problems such as low self-esteem, depression and anxiety. 13. Psychological problems such as low self-esteem, depression and anxiety. A protein ketogenic diet is a ketogenic diet based on proteins. I have treated successfully with this diet, mainly with the “Spanish ketogenic Mediterranean diet” to more than 100 obese patients. 2. 1. A lower life expectancy A lipid profile by determining triglyceride, total cholesterol, Corresponding author email: pv1peguj@uco.es Symbiosis Group Symbiosis Group Importance of a Blood Test Before Starting a Protein Ketogenic Diet Copyright: © 2015 Rosa LDL and HDL. In obese patients, we find very often an altered lipid profile, presenting elevated triglycerides, total cholesterol and LDL and low levels of HDL. With the ketogenic diet, we usually find in the next monthly blood test an improvement in all these parameters, this is due not only to the weight loss, but also to an improvement in insulin sensitivity. As in everything, there are exceptional cases where there could be a worsening of the lipid profile, as could be the Euthyroid Sick Syndrome (ESS), Sick Euthyroid Syndrome (SES) or Non-Thyroidal Illness Syndrome (NTIS), which is currently considered as the most common cause of alteration in plasma thyroid hormone and which could cause a typical dyslipidaemia of hypothyroidism. This alteration is a defense mechanism of our body in situations where it is necessary to consider energy savings, appearing in any patients with nonthyroidal acute or chronic diseases, in postoperative states, fasting, malnutrition or important caloric restriction, which is very typical in the anorexic disorder. The body, to lower the basal, reduces the plasma concentration of T3 by 3 ways: decreasing the binding of T3 to the thyroid hormone transport protein (thereby decreasing the reserve of these hormones and increasing their metabolism speeds ), increasing the peripheral conversion of T4 to rT3 (inactive) and decreasing the conversion of T4 to T3 (metabolically the most active). Keep in mind that urea levels depend on several factors such as the type of food, the liver protein catabolism and diuresis. While creatinine levels are more related to muscle protein catabolism and diuresis. These considerations may be of importance to us in order to know if our patient has a renal disease , which will be one in which both plasma urea and creatinine values are presented above the normal levels and compliance that, the ratio of urea and creatinine is approximately 10/1. We must be alert because it might be the case that due to a high protein diet, a high protein catabolism and even dehydration, determinations of urea and even creatinine could be above the normal levels without any renal failure associated, in this case we should find that, the urea and creatinine ratio exceeds 10 to 15. Citation: Rosa JPG (2015) Importance of a Blood Test Before Starting a Protein Ketogenic Diet. Obes Control Ther 2(2): 1-2. DOI: http://dx.doi.org/10.15226/2374-8354/2/2/00119 1. A lower life expectancy Finally, I would like to remember that we are responsible with our patients for their success in weight loss, and that we should make guidelines and follow a scientifically based protocol that produces in our patients a healthy weight loss. 5. An analysis of renal function by measuring plasma urea and creatinine. These determinations are vital because renal failure is an absolute contraindication for the protein ketogenic diet. Page 2 of 2
https://openalex.org/W1960334827	https://kclpure.kcl.ac.uk/portal/files/47945690/art_10.1007_s10803_015_2641_0.pdf	English	null	Using the Autism-Spectrum Quotient to Measure Autistic Traits in Anorexia Nervosa: A Systematic Review and Meta-Analysis	Journal of autism and developmental disorders	2,015	cc-by	11,640	Citation for published version (APA): Westwood, H., Eisler, I., Mandy, W., Leppanen, J., Treasure, J., & Tchanturia, K. (2015). Using the Autism- Spectrum Quotient to Measure Autistic Traits in Anorexia Nervosa: A Systematic Review and Meta-Analysis. Journal of Autism and Developmental Disorders, 46(3), 964-977. https://doi.org/10.1007/s10803-015-2641-0 Citation for published version (APA): Westwood, H., Eisler, I., Mandy, W., Leppanen, J., Treasure, J., & Tchanturia, K. (2015). Using the Autism- Spectrum Quotient to Measure Autistic Traits in Anorexia Nervosa: A Systematic Review and Meta-Analysis. Journal of Autism and Developmental Disorders, 46(3), 964-977. https://doi.org/10.1007/s10803-015-2641-0 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. & Kate Tchanturia Kate.Tchanturia@kcl.ac.uk Using the Autism-Spectrum Quotient to Measure Autistic Traits in Anorexia Nervosa: A Systematic Review and Meta-Analysis Heather Westwood1 • Ivan Eisler2,3 • William Mandy4 • Jenni Leppanen1 • Janet Treasure1 • Kate Tchanturia1,2,5 Published online: 5 November 2015 The Author(s) 2015. This article is published with open access at Springerlink.com Published online: 5 November 2015 Published online: 5 November 2015 The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Interest in the link between Autism Spectrum Disorder (ASD) and Anorexia Nervosa (AN) has led to estimates of the prevalence of autistic traits in AN. This systematic review and meta-analysis assessed the use of the Autism-Spectrum Quotient (AQ) or abbreviated version (AQ-10) to examine whether patients with AN have ele- vated levels of autistic traits. Seven studies were identiﬁed and subsequent meta-analysis indicated that those with AN appear to have signiﬁcant difﬁculties of a manner charac- teristic of ASD, relative to controls. Whilst this analysis supports previous indications of higher prevalence of ASD in AN, the aetiology of these traits remains unclear. Studies using more robust clinical measures of ASD within AN are needed to conﬁrm what self-report measures appear to show. 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Oct. 2024 J Autism Dev Disord (2016) 46:964–977 DOI 10.1007/s10803-015-2641-0 ORIGINAL PAPER ORIGINAL PAPER 1 Psychological Medicine, IoPPN, King’s College London (KCL), PO59, London SE5 8AF, UK 2 Child and Adolescent Eating Disorders Service, Maudsley Hospital, London SE5 8AZ, UK 3 Department of Psychology, IoPPN, King’s College London (KCL), London, UK 5 Ilia State University, Tbilisi, Georgia 4 Research Department of Clinical, Educational and Health Psychology, University College London (UCL), London, UK & Kate Tchanturia Kate.Tchanturia@kcl.ac.uk & Kate Tchanturia Kate.Tchanturia@kcl.ac.uk 1 Psychological Medicine, IoPPN, King’s College London (KCL), PO59, London SE5 8AF, UK 2 Child and Adolescent Eating Disorders Service, Maudsley Hospital, London SE5 8AZ, UK 3 Department of Psychology, IoPPN, King’s College London (KCL), London, UK 4 Research Department of Clinical, Educational and Health Psychology, University College London (UCL), London, UK 5 Ilia State University, Tbilisi, Georgia Introduction Studies with AN populations have examined the presence of both co-morbid ASD and elevated levels of autistic traits using several instruments. Studies assessing the prevalence of ASD have tended to use diagnostic cri- teria (Gillberg et al. 1995; Nilsson et al. 1998, 1999; Rastam 1992; Rastam et al. 2003) and The Asperger Syndrome Diagnostic Interview (Anckarsater et al. 2012; Nilsson et al. 1999; Rastam et al. 2003). A meta-analysis of these prevalence studies found an average ASD prevalence rate of 22.9 % (Huke et al. 2013) in eating disorder pop- ulations. The majority of these studies, however, involved the same Swedish community sample and the variety of diagnostic tools used makes it difﬁcult to compare the outcomes. The review only contained studies with adults with AN whereas a prevalence study examining ASD in adolescents with early onset-AN found that a diagnosis of ASD was no more common than in healthy controls (Pooni et al. 2012), although the AN sample did have elevated levels of ASD traits. Although there is a male bias in the diagnosis of ASD, recent evidence suggests that it may affect more females than previously thought, with diagnostic criteria and research practices leading to an over-stated gender gap (Goldman 2013; Lai et al. 2015). ASD may present dif- ferently in females than in males so that females are often under-diagnosed or mislabelled as having other impair- ments (Mandy and Tchanturia 2015). For example, females with ASD have been found to have better social skills than their male counterparts (Head et al. 2014) and females with ASD out-perform males on tests of executive functioning (Bolte et al. 2011). Within ASD, typical sex differences between males and females on empathising and systemis- ing are attenuated, with both sexes shifting towards the extreme male brain (Baron-Cohen et al. 2014). However, this study found that signiﬁcant sex differences within ASD still exist, in the same direction as Healthy Controls (HCs), highlighting the need to consider sex-differences in the diagnosis of ASD. A widely used measure of autistic traits is the Autism- Spectrum Quotient (AQ; Baron-Cohen et al. 2001) which was developed to provide a brief, self-report measure of autistic traits for use with adults, rather than for use as a diagnostic tool. Introduction When the AQ was initially developed, individuals with high functioning autism (HFA) were found to have a mean score of 35.8 (SD = 6.5) which was signif- icantly higher than the control group who had a mean score of 16.4 (SD = 6.3). Among the controls, but not in the HFA group, men were found to score signiﬁcantly higher than women. For the original validation study, a cut-off score of 32 was chosen as 80 % of those with HFA scored above this level, while only 2 % of controls did. Test–retest and inter- rater reliability of the AQ were good, and a cut-off score of 32 has acceptably high sensitivity (0.77) and speciﬁcity (0.74) (Austin 2005; Woodbury-Smith et al. 2005). Females may also display less stereotyped behaviours or restricted interests (Mandy et al. 2012) or their interests may be more subtle or in line with gender stereotypes, making them more difﬁcult to detect (Hiller et al. 2015). While AN tends to develop during adolescence, it may be that the presence of undetected ASD traits in early life interact with socio-cultural pressures to leave females in particular, sus- ceptible to the development of eating disorders. Difﬁculties with set-shifting and central coherence are consistently reported in adults with AN (for a review, see Roberts et al. 2007) but the evidence in children and adolescents is more mixed (Lang et al. 2014a, b). However, this may be due to several studies using different experimental paradigms to measure central coherence and set-shifting and under-pow- ered studies, which make the data difﬁcult to interpret or compare. It is therefore still unclear whether children with AN, who are exposed to the effects of starvation for less time, have the same cognitive inefﬁciencies as adults. Findings on ToM are also mixed, with research not supporting a speciﬁc link between ToM and AN (Tchanturia et al. 2004). Research on emotional ToM in AN suggests that problems in this area resolve with weight gain and recovery, supporting the notion that this difﬁculty is at least exacerbated by the ill state of the disorder (Oldershaw et al. 2010). The AQ is a 50-item questionnaire consisting of ﬁve domains: social skills; attention switching; attention to detail; communication and imagination. The AQ has rea- sonable face validity as only 2 % of control participants scored above the clinical cut-off during the original valida- tion study (Baron-Cohen et al. 2001). Introduction Research into the possible link between Autism Spectrum Disorders (ASD) and Anorexia Nervosa (AN) has grown considerably, with studies showing potential links both in speciﬁc characteristics of the two disorders (Oldershaw et al. 2011) and in prevalence rates of co-morbidity or elevated levels of ASD traits within AN populations (for a systematic review see Huke et al. 2013). AN is a severe eating disorder characterised by body weight signiﬁcantly lower than the normal range relative to height, fear of gaining weight and undue inﬂuence of weight and shape on self-evaluation (APA 2013). It pre- dominantly affects females, with an estimated gender ratio of 10:1 females to males, though epidemiological studies report higher variation of 3:1–12:1 (Raevuori et al. 2014). In contrast, ASD is a pervasive developmental disorder that tends to affect more males than females, with a gender ratio of 3.3:1 males to females being reported in a UK national prevalence study (Mills and Kenyon 2013). Unlike AN, which tends to have its onset in adolescence to early adulthood (Micali et al. 2013), for ASD to be diagnosed, symptoms must be present during the early developmental period. These include persistent difﬁculties with social communication and interaction and restricted, repetitive behaviours and interests (APA 2013). Keywords Autism Systematic review Meta-analysis Anorexia Nervosa Autism-Spectrum Quotient Female Despite these two disorders seeming different, several traits associated with ASD have also been found in AN populations including: difﬁculties with set-shifting (Tchanturia et al. 2012), the capacity to shift a course of thought or action according to situational demands (Lezak 1995); weak central coherence, the lack of an effect of context or difﬁculty in taking a global approach (Frith 1989; Lang et al. 2014); difﬁculties with Theory of Mind (ToM), the ability to infer the mental states of others 3 Department of Psychology, IoPPN, King’s College London (KCL), London, UK 4 Research Department of Clinical, Educational and Health Psychology, University College London (UCL), London, UK 5 Ilia State University, Tbilisi, Georgia 123 12 3 965 J Autism Dev Disord (2016) 46:964–977 (Baron-Cohen et al. 1985; Tchanturia et al. 2004); and difﬁculties with emotional processing (Davies et al. 2011; Russell et al. 2009). intermediate between typical functioning and ASD, i.e. elevated levels of ASD traits found in the general popu- lation. Introduction Items comprising each of the ﬁve domains showed moderate to high alpha coefﬁ- cients, indicating reasonable construct validity. Additional analysis also found the AQ to have moderate accuracy in diagnosis of Asperger’s Syndrome in clinical settings, i.e. correctly identifying a ‘true positive’ case as scoring higher than a ‘true negative’ case (Woodbury-Smith et al. 2005). The marked overlap in speciﬁc characteristics present in both ASD and AN has led to attempts to examine the prevalence of co-morbid ASD or elevated levels of autistic traits within AN. As ASD is considered a dimensional disorder (Bolton et al. 1994; Wiggins et al. 2012) a dis- tinction can be made between ASD as a fully diagnosed clinical disorder and the presence of sub-clinical traits, In addition to the AQ being used as a screening tool for ASD in the general population, it has been used to a examine ASD traits within clinical groups. For example, in schizophrenia (Mealey et al. 2014; Spek and Wouters 2010; 12 3 3 966 J Autism Dev Disord (2016) 46:964–977 Wouters and Spek 2011) and obsessive compulsive disorder (Cath et al. 2008; Mito et al. 2014). The criterion validity of the AQ has been found to be good, with patients with HFA scoring considerably higher than those with social anxiety disorder or obsessive compulsive disorder (Hoekstra et al. 2008). This demonstrates that the AQ can differentiate between individuals with ASD and other disorders. Information Sources and Search The electronic databases Scopus, Psychinfo, Medline and Web of Science were searched up to and including April 2015. Search terms included AQ, Autism, ASD, eating disorder and Anorexia Nervosa. Limits included English language, articles and peer-reviewed. Reference lists of eligible papers were also screened for other relevant studies and additionally, Lang et al. (2015) was included (data were available from the principal author of the study, KT). Missing data on AQ subscales were also obtained directly from the authors of one study (Huke et al. 2014). g The presence of ASD in AN represents a signiﬁcant treatment challenge (Nielsen et al. 2015) and thus identifying ASD traits within this population is important. However, disentangling the relationship between AN and ASD has proved problematic. The presence of ASD traits in AN appear to differ across different stages of the illness, sug- gesting that these traits are epiphenomena, arising as a result of the ED and only superﬁcially resembling ASD (Mandy and Tchanturia, Additionally, ASD traits in females are difﬁcult to detect and the presence of a severe eating disorder further complicates the identiﬁcation and diagnosis of ASD in this group. The presence of eating disorders in individuals with established ASD diagnoses has received less attention than ASD in AN populations. Eating disturbances, including selective eating, are known to be overrepresented in ASD (Rastam 2008) and may serve as a risk factor for the devel- opment of a clinical eating disorder, such as AN. Karlsson et al. (2013) note that eating disturbances in ASD are clini- cally acknowledged but under-researched. Thus, disentan- gling the relationship between the two disorders is difﬁcult and it is important to establish the most robust method of identifying ASD traits within AN populations. Methods Wouters and Spek 2011) and obsessive compulsive disorder (Cath et al. 2008; Mito et al. 2014). The criterion validity of the AQ has been found to be good, with patients with HFA scoring considerably higher than those with social anxiety disorder or obsessive compulsive disorder (Hoekstra et al. 2008). This demonstrates that the AQ can differentiate between individuals with ASD and other disorders. The meta-analysis was conducted according to the ‘PRISMA’ (preferred reporting items for systematic reviews and meta-analysis) statement (Moher et al. 2009). Selection The ﬁrst and principal authors (HW, KT) identiﬁed potential titles from all databases and screened the abstracts for relevance. Full-texts were then retrieved and read to determine eligibility. Texts deemed eligible were then further screened by KT in discussion with HW and any papers that did not meet inclusion criteria were excluded. Eligibility Criteria To examine whether individuals with AN have elevated levels of ASD traits, relative to the general population, several studies have utilised the AQ or the abbreviated ten- item version, the short Autism Spectrum Quotient (AQ-10; Allison et al. 2012). The AQ-10 has similar sensitivity and speciﬁcity to the full version and performed well as a screen for ASD (Booth et al. 2013), with a clinical cut-off of 6 being indicative of ASD. If validated, self-report measures such as the AQ or AQ-10 have the beneﬁt of being cost-effective and time efﬁcient and thus could provide a useful addition to more robust but time-consuming clinical assessments. Studies using the AQ or AQ-10 with both a clinical AN and HC group were included in the review. Other eligibility criteria included being published in a peer-reviewed jour- nal and being available in English. Risk of Bias in Individual Studies was assessed visually and by Egger’s test (Egger et al. 1997) to see if sample size was related to effect size. was assessed visually and by Egger’s test (Egger et al. 1997) to see if sample size was related to effect size. The risk of bias in individual studies was assessed by considering how methodology would impact on effect size in each study, for example by attending to how healthy controls were matched to clinical samples, how the AQ or AQ-10 were administered and the source of participant recruitment. Study Selection A total of seven studies were identiﬁed and included in the review, consisting of a pooled total of 328 AN patients and 1890 HCs. Of the AN sample, 90 were children or ado- lescents. Two of these studies (Baron-Cohen et al. 2013; Lang et al. 2015) had multiple data sets for adult and child/ adolescent samples resulting in nine data sets for the meta- analysis. Although the adult and child data are pooled within the Lang et al. (2015) paper, at our request the authors provided data separately for these groups. The selection process for studies is shown in Fig. 1. The main reasons for exclusion of studies were: the AQ or AQ-10 was not used to measure ASD traits, the clinical sample did not have a primary diagnosis of AN, the papers did not contain empirical data or the papers were not available in English. Synthesis of Data Synthesis of results for meta-analysis meant that only studies reporting mean and standard deviation AQ or AQ-10 scores were included in the analysis. The meta-analysis was per- formed by pooling the standardised effect sizes using a random effects model. Random effects models assume that as well as within-group variability caused by variability of scores, mean effect size is also caused by differences between studies. The model includes the between study heterogeneity, resulting in estimates with wider conﬁdence intervals than ﬁxed-effects models. Additional meta-analy- ses, again using random-effects model were conducted to compare AN patients and HCs on sub-scale scores of the AQ (social skills, attention switching, attention to detail, com- munication and imagination). Data Collection and Items The data items collected from each eligible study were: number of participants, age of participants, BMI, illness duration (for the AN group), percentage of female partic- ipants, how HCs were matched to clinical samples, use of medication, co-morbidities, IQ and the version of the AQ used i.e. full or AQ-10. In addition to demographic and experimental paradigm data, the mean and standard devi- ations of the AQ or AQ-10 score were used within the meta-analysis. In studies using the full AQ, means and standard deviations of the AQ sub-scale scores for AN and HC groups were also extracted so that these could be sta- tistically compared across studies. The aims of this systematic review and meta-analysis were to: (1) examine whether patients with AN have ele- vated levels of self-reported autistic traits relative to HCs and (2) compare the use of the AQ and AQ-10 in studies with AN populations (3) Examine whether the AQ or AQ- 10 are robust measures of ASD traits in AN populations, given the presence of other co-morbidities, the impact of the eating disorder on these traits and the stability of ASD traits during the course of AN. 123 J Autism Dev Disord (2016) 46:964–977 967 Summary Measure The principal summary measure used for analysis from all studies was the difference in means and standard deviations of AQ or AQ-10 scores between the AN sample and HCs. Additional Analysis Between studies heterogeneity was measured calculating I2 (Higgins et al. 2003) based on Cochran’s Q test: measure of heterogeneity, I2 = 100 % (Q-df)IQ. I2 ranges between 0 %, indicating no heterogeneity and 100 %, indicating high heterogeneity. Where heterogeneity was found a meta-regression was performed with age as a moderator. It was not possible to conduct a meta-regression with either BMI or gender as moderator variables due to missing data on BMI and all but one study included only female par- ticipants. Meta-regression of sub-scale scores was also not possible due to only four studies reporting this data. Statistical Analysis Analysis was carried out in STATA 11 (StataCorp, College Station, TX) with the following user contributed com- mands metan (Bradburn et al. 1998), metabias, metatrim (Steichen 1998), and metareg (Sharp 1998). For all meta- analyses Cohen’s d was used to estimate effect size and is reported for all studies together with 95 % conﬁdence intervals. The effect sizes were interpreted according to Cohen’s (1988) deﬁnitions of small (C 0.20 B 0.50), medium (C 0.50 B 0.80), large (C 0.80 B 1.30) and very large (B1.30). Positive effect size indicates that the AN group had higher scores on the AQ or AQ-10 than HC. A p value of \0.05 indicates signiﬁcant difference between groups. For sub-scale analysis, Review Manager 5.3 (Cochrane Collaboration 2014) was used. Study Characteristics Studies included in the systematic review and meta-anal- ysis are displayed in Table 1. Five of the seven included studies (Baron-Cohen et al. 2013; Calderoni et al. 2015; Courty et al. 2013; Hambrook et al. 2008; Huke et al. 2014) used the full AQ, while the remaining two studies (Lang et al. 2015; Tchanturia et al. 2013) used the AQ-10. Of the ﬁve studies using the AQ, four collected data on the sub- scale differences between the AN and HC samples (Cal- deroni et al. 2015; Courty et al. 2013; Hambrook et al. 2008; Huke et al. 2014). Risk of Bias Across Studies In all studies, groups were matched by gender and in all but one study (Courty et al. 2013), all participants were female. In three studies, the AN sample was also matched Publication bias was assessed by evaluating funnel plots of each study’s mean differences. Symmetry of the funnel plot 123 12 J Autism Dev Disord (2016) 46:964–977 968 1087 records identified through database searching 104 abstracts (23 with duplicates removed) screened for relevance 24 full texts reviewed for eligibility 80 records excluded 6 studies included from screened texts 18 records excluded 1 study included from direct contact with authors 7 studies included in data synthesis and meta-analysis Fig. 1 Systematic review search process in accordance with the PRISMA statement medication. Attempt was made by all studies to control for psychiatric illness within both the AN and HC groups. Baron-Cohen et al. (2013) excluded one participant with AN whom also had ASD and used a self-report question- naire with HCs to assess for psychiatric illness. Courty et al. (2013) ruled out ASD in the AN and HC groups and found that the AN group had higher levels of depression than the HCs. Calderoni et al. (2015) excluded anyone who displayed psychotic symptoms, had substance abuse and those with an IQ of below 80. In the AN group, 80.8 % met criteria for an anxiety or mood disorder and two patients had a co-morbid personality disorder. Huke et al. (2014) excluded participants with psychosis, drug and alcohol misuse or those with a high risk of suicide. HCs were also excluded if they had a current or previous diagnosis of a mental disorder, as was the case in Tchanturia et al. (2013). Hambrook et al. (2008) screened for psychotic disorder in the HC group. IQ was only assessed in one study (Lang et al. 2015) and years of education were recorded in two studies (Huke et al. 2013; Tchanturia et al. 2013), with no signiﬁcant differences being found between the AN and HC groups. 6 studies included from screened texts 1 study included from direct contact with authors Fig. 1 Systematic review search process in accordance with the PRISMA statement There were also differences between the studies in how the AQ or AQ-10 was administered. Risk of Bias Across Studies 2013) AN diagnosis was made in accordance with DSM-IV criteria (APA 1994) while two studies used DSM-IV-TR (APA 2000) criteria (Calderoni et al. 2015; Courty et al. 2013). One study did not state which diagnostic criteria were used, but diagnosis was checked using the Eating Disorder Examination (12th Ed, Fairburn and Cooper 1993). Calderoni et al. (2015) included only patients with Anorexia Nervosa restricting subtype (AN-R) whilst the other studies either did not specify or included a mixture of both binge/purge and restricting type AN. The studies included in the overall AQ score meta- analysis differed greatly in terms of their sample size. For example, Baron-Cohen et al. (2013) had an AN sample of 66 but a HC sample of 1609, so power calculations would only account for the smaller, AN sample. The smallest sample size was 15 in Courty et al. (2013)’s study and Lang et al. (2015)’s study had the largest, most equal sample size of 96 and 97 AN and HCs respectively. Although BMI was reported by all but one study (Baron-Cohen et al. 2013), two studies did not report AN illness duration (Baron- Cohen et al. 2013; Calderoni et al. 2015) so this could not be used as a moderator variable when examining hetero- geneity across studies. Risk of Bias Across Studies For example, due to the self-report version of the AQ only being validated for children over the age of 16, the parent version of the AQ was used for younger participants in Baron-Cohen et al. (2013)’s study. Baron-Cohen also sent the questionnaires by post, as did Courty et al. (2013). In all other studies, participants completed the questionnaires during a testing session. Whilst ﬁve of studies took place in the United Kingdom (Baron-Cohen et al. 2013; Hambrook et al. 2008; Huke et al. 2014; Lang et al. 2015; Tchanturia et al. 2013), one study was conducted in Italy (Calderoni et al. 2015) and one in France (Courty et al. 2013). AN samples were also recruited from a variety of services: three studies recruited from a mixture of outpatient, day patient and inpatient services within the same, national specialist eat- ing disorder service (Hambrook et al. 2008; Lang et al. 2015; Tchanturia et al. 2013), three only recruited from inpatient services (Calderoni et al. 2015; Courty et al. 2013; Huke et al. 2014) and one states that patients were recruited from a specialist service but does not state whether this was an inpatient setting (Baron-Cohen et al. 2013). to HCs by age (Calderoni et al. 2015; Courty et al. 2013; Huke et al. 2014) and in two studies, by level of education (Calderoni et al. 2015; Courty et al. 2013). In four of the studies (Baron-Cohen et al. 2013; Hambrook et al. 2008; Lang et al. 2015; Tchanturia et al. 2013) AN diagnosis was made in accordance with DSM-IV criteria (APA 1994) while two studies used DSM-IV-TR (APA 2000) criteria (Calderoni et al. 2015; Courty et al. 2013). One study did not state which diagnostic criteria were used, but diagnosis was checked using the Eating Disorder Examination (12th Ed, Fairburn and Cooper 1993). Calderoni et al. (2015) included only patients with Anorexia Nervosa restricting subtype (AN-R) whilst the other studies either did not specify or included a mixture of both binge/purge and restricting type AN. to HCs by age (Calderoni et al. 2015; Courty et al. 2013; Huke et al. 2014) and in two studies, by level of education (Calderoni et al. 2015; Courty et al. 2013). In four of the studies (Baron-Cohen et al. 2013; Hambrook et al. 2008; Lang et al. 2015; Tchanturia et al. Synthesis of Results The forest plotof all studies included in the overall AQ or AQ- 10 score meta-analysis is displayed in Fig. 3. The random- effects analysis, with a total sample size of 2218 (AN = 328, HC = 1890) revealed a signiﬁcant difference between AN and HC groups on the AQ or AQ-10 mean scores, d = 1.065 [95 % CI: 0.83, 1.23], z = 8.90, p = \0.001. Risk of Bias Attention to detail: MD = 0.51 [95 % CI: -0.27, 1.29], z = 1.29, p = 0.20. Communication: MD = 1.88 [95 % CI: 0.21, 3.56], z = 2.20, p = 0.03. Imagination: MD = 1.16 [95 % CI: 0.19, 2.14], z = 2.35, p = 0.02. imagination. Social skills: MD = 2.68 [95 % CI: 0.73, 4.63], z = 2.7, p = 0.007. Attention switching: MD = 2.42 [95 % CI: 1.14, 3.71], z = 3.71, p = 0.0002. Attention to detail: MD = 0.51 [95 % CI: -0.27, 1.29], z = 1.29, p = 0.20. Communication: MD = 1.88 [95 % CI: 0.21, 3.56], z = 2.20, p = 0.03. Imagination: MD = 1.16 [95 % CI: 0.19, 2.14], z = 2.35, p = 0.02. Risk of Bias There was good symmetry within the funnel plot, shown in Fig. 2, indicating no relationship between effect and study size. Additionally, Egger’s test was conducted to statisti- cally assess for publication bias, indicating no evidence of bias (p = 0.829). The use of medication by study participants was only reported in one study (Courty et al. 2013) who reported that 65.4 % of AN patients were taking psychotropic 123 J Autism Dev Disord (2016) 46:964–977 969 Table 1 Participant demographic information for included studies Author/date Group N Age M (SD) BMI M (SD) Illness duration M (SD) AQ version AQ score M (SD) Female (%) Groups matched by Adult studies Hambrook et al. (2008) AN 22 26.73 (4.77) 15.27 (1.22) 9.5 (5.0) Full 23.2 (7.3) 100 Gender HC 45 32.51 (9.93) 23.36 (3.76) 15.3 (5.5) 100 Courty et al. (2013) AN 15 23.9 (4.7) 16.4 (1.7) 4.0 (3.5) Full 20.3 (5.9) 93.3 Age, gender, education HC 15 24.0 (4.9) 21.0 (1.8) 14.8 (4.9) 93.3 Baron-Cohen et al. (2013) AN 42 17.85 (0.38)* Full 21.0 (6.8) 100 Gender HC 1038 18.56 (3.99)* 15.5 (5.6) 100 Tchanturia et al. (2013) AN 66 26.35 (8.08) 14.90 (2.13) 10.59 (6.66) AQ-10 4.53 (2.56) 100 Gender HC 66 25.68 (9.74) 21.78 (2.46) 1.85 (1.68) 100 Huke et al. (2013) AN 32 28.7 (9.65) 14.71 (1.77) 11.03 (9.33) Full 20.03 (9.65) 100 Age, gender HC 32 24.9 22.5 (3.22) 14.78 (6.23) 100 Lang et al. (2015) AN 61 26.72 (7.61) 15.44 (1.68) 8.74 (7.84) AQ-10 4.05 (2.22) 100 Gender HC 69 27.30 (9.29) 22.34 (2.42) 1.52 (1.24) 100 Child/adolescent studies Baron-Cohen et al. (2013) AN 24 Full 21.0 (6.8) 100 Gender HC 412 15.5 (5.6) 100 Lang et al. (2015) AN 41 15.07 (1.81) 80.69 % (6.57) 1.70 (1.18) AQ-10 3.88 (2.12) 100 Gender HC 43 15.11 (1.94) 100.92 % (8.37) 2.67 (1.28) 100 Calderoni et al. (2015) AN 25 14.34 (1.86) 14.63 (1.90) Full 21.04 (6.52) 100 Age, gender, education HC 170 15.01 (2.23) 19.75 (2.61) 17.46 (5.50) 100 N number of participants, BMI body mass index (kg/m2) * Overall mean age for whole sample including children and adolescents, adult mean age not available Table 1 Participant demographic information for included studies imagination. Social skills: MD = 2.68 [95 % CI: 0.73, 4.63], z = 2.7, p = 0.007. Attention switching: MD = 2.42 [95 % CI: 1.14, 3.71], z = 3.71, p = 0.0002. Additional Analysis Forest plots for the AQ sub-scale analysis are displayed in Fig. 4. The random-effects analysis, with a total sample size of 358 (94 AN, 262 HCs) revealed signiﬁcant differ- ences between AN and HCs on four of the ﬁve subscales; social skills, attention switching, communication and There was evidence of considerable heterogeneity between studies included in the total score meta-analysis (I2 = 59.8 %). A meta-regression was conducted to further 123 12 3 J Autism Dev Disord (2016) 46:964–977 970 Fig. 2 Funnel plot of studies included in the meta-analysis to assess for publication bias. Each dot represents a study included in the meta- analysis, with the Y axis representing the size of each study and the X axis, each study’s result Discussion The aim of this review was to provide a synthesis of the existing literature on the presence of autistic traits in AN, as measured by the self-report AQ or AQ-10. It also aimed to establish whether factors such as age or illness severity predict AQ scores and thus whether the AQ is a robust measure of ASD traits in AN populations. A number of methodological differences across studies were high- lighted, including the differing use of either the full AQ or shortened AQ-10, differences in sample size and the way in which confounding factors such as BMI, illness duration, medication and co-morbidities were controlled for within the studies. Data on BMI, gender and illness duration were not sufﬁcient to allow for meta-regression to explore whether these factors predict AQ scores. However, gener- ally, the studies all reported enough information to allow for synthesis and analysis of the data. Fig. 2 Funnel plot of studies included in the meta-analysis to assess for publication bias. Each dot represents a study included in the meta- analysis, with the Y axis representing the size of each study and the X axis, each study’s result The meta-analysis indicates a signiﬁcant difference between AN patients and HCs on the scores on both the AQ and AQ-10, with AN patients scoring signiﬁcantly higher, suggesting signiﬁcant difﬁculties with social skills, communication and ﬂexibility that present in a manner characteristic of autistic traits. This ﬁnding is in line with previous prevalence research in this area, for example the systematic review by Huke et al. (2013) which reported a mean ASD prevalence rate of 22.9 % in AN, higher than that prevalence of ASD within the general population which is estimated to be 1.1 % in the UK (Mills and investigate the variance, with age as a moderator. The meta-regression showed no signiﬁcant effect of age on the unexplained between-study variation. Due to incomplete data, meta-regression using other moderator variable was not possible but the potential reasons for this heterogeneity are explored within the discussion. Moderate to high heterogeneity was also found between studies included in AQ-subscale meta-analysis (I2 = 43 % to 92 %). How- ever, due to the small number of studies, this could not be examined further statistically. X axis represents the magnitude of difference between the AN and HC groups. Discussion The overall effect sizes for adult and child studies and the mean summary measure are depicted by the black diamonds X axis represents the magnitude of difference between the AN and HC groups. The overall effect sizes for adult and child studies and the mean summary measure are depicted by the black diamonds Fig. 3 Forest plot for mean total Autism-Spectrum Quotient (AQ) and short Autism-Spectrum Quotient (AQ-10), with standardised mean effect sizes for difference between Anorexia Nervosa (AN) and Healthy Controls (HC). The Y axis shows each included study and the 3 971 J Autism Dev Disord (2016) 46:964–977 Kenyon 2013). However, the ﬁndings of this meta-analysis h ld b i d i h i (2015) at 14.63, which is considered to be extrem h l h h i b h f h di Fig. 4 Mean and standard deviations sub-scale scores and forest plots for Autism-Spectrum Quotient with effect sizes for difference between Anorexia Nervosa (AN) and Healthy Controls (HC). The Y axis shows each individual study and the X axis represents the magnitude of difference between the AN and HC groups on sub-scales. The overall effect sizes for each sub-scale sc depicted by the black diamonds magnitude of difference between the AN and HC groups on the AQ sub-scales. The overall effect sizes for each sub-scale score are depicted by the black diamonds Fig. 4 Mean and standard deviations sub-scale scores and forest plots for Autism-Spectrum Quotient with effect sizes for difference between Anorexia Nervosa (AN) and Healthy Controls (HC). The Y axis shows each individual study and the X axis represents the magnitude of difference between the AN and HC groups on the AQ sub-scales. The overall effect sizes for each sub-scale score are depicted by the black diamonds ( ) y ( ) Y axis shows each individual study and the X axis represents the Kenyon 2013). However, the ﬁndings of this meta-analysis should be interpreted with some caution. Kenyon 2013). However, the ﬁndings of this meta-analysis should be interpreted with some caution. (2015) at 14.63, which is considered to be extreme AN (APA). Thus, although in both of these studies patients were recruited from inpatient settings, the severity of the AN may have been different and thus severity of AN may have account for the heterogeneous samples included in this review. Countries differ on the treatments offered to patients at different severities. Discussion Even in the UK, where ﬁve of the studies were based, there is large variation across All patients included in the studies were within the acute, ill phase of AN with BMIs considerably lower than the normal range. The highest mean BMI for the clinical group was 16.4, recorded by Courty et al. (2013); this is considered to be moderate in terms of severity (APA 2013). The lowest mean BMI was reported by Calderoni et al. 12 3 972 J Autism Dev Disord (2016) 46:964–977 AN may indicate less difﬁculties associated with ASD. This would be concordant with other research in this area, which suggests that the similarities between AN and ASD may be less pronounced in children (Lang et al. 2014a, b; Pooni et al. 2012; Rhind et al. 2014). However, research in this area is not robust enough to conﬁrm this. services in the treatment patients receive, even within the moderate to severe range of AN (Goddard et al. 2013). Severity of the eating disorder is an important consid- eration due to the possibility of ASD traits being exacer- bated by starvation or the acute state of the illness (Pellicano and Hiller 2013). With the mean duration of illness across the included studies also varying from 4 to 11 years, the possibility of illness chronicity affecting the results of the AQ cannot be ruled out. As the AQ focuses on current behavioural symptoms, rather than on ASD traits earlier on in life, it is possible that individuals with AN develop autistic-like traits as a result of their eating disorder. Lack of data meant that exploring the impact of BMI or illness duration on AQ scores was not possible within this review. While none of the included studies make claims about the aetiology of elevated autistic traits in AN, this is an important treatment consideration and warrants further investigation. Despite the self-report version of the AQ not being validated with children under the age of 16, only one of the child studies included in this review (Baron-Cohen et al. 2013) used the parental AQ version for younger partici- pants. It is therefore possible that the smaller effect found in child/adolescent studies are due to methodological dif- ferences between studies. Given the limitations of current data, interpretation of any differences in ﬁndings between adults and young people needs to be cautious. Discussion Adult sam- ples tend to consist of more chronically ill individuals and the differences in ASD ﬁndings could therefore indicate an illness effect, but equally could be maintaining factors of the eating disorder in those in whom ASD traits are observed. Illness effect could not be explored in the context of this review but could account for the heterogeneity of the included samples, or indeed whether longer illness duration elicits ASD traits. Therefore, further research is needed to determine whether the differences on the AQ and AQ-10 found between AN and HC samples extend to young people and if so, how this should be interpreted. g A study comparing the AQ in ASD and schizophrenia (Lugnega˚rd et al. 2015) found that while individuals with either ASD or schizophrenia score high on the AQ, nine individuals with schizophrenia who also met childhood criteria for ASD did not show signiﬁcantly higher scores on the AQ than the individuals without ASD. This suggests that the AQ may not be sensitive in differentiating between individuals with ASD and those with other psychiatric disorders. Additionally, it may suggest that AQ scores are not stable over time in psychiatric populations. Research on the stability of autistic traits during development (White- house et al. 2011) found that while these traits appeared to be stable in males, with scores in childhood being corre- lated with those in adulthood, this was not the case in females. Again, this indicates that autistic traits may pre- sent and develop differently in females, making it difﬁcult to interpret whether the elevated AQ scores seen in AN females are a true reﬂection of elevated autistic traits in this population. Analysis of the difference in sub-scale scores on the AQ between the AN and HC groups included only one child study (Calderoni et al. 2015) so it is not possible to draw conclusions about the difference between children and adults on sub-scale scores. The sub-scale analysis did suggest, however, that there was no signiﬁcant difference in Calderoni et al.’s (2015) study between AN and HC groups on social skills, attention to detail, communication or imagination, again highlighting the need for further studies with both adult and child samples. Whilst analysis of the AQ sub-scales was limited by fewer studies and large heterogeneity, it indicated that that there was no signiﬁcant difference between patients with AN and HCs on self-report attention to detail. Discussion This is contrary to con- sistent experimental, performance-based ﬁndings that individuals exhibit weaker central coherence, i.e. difﬁcul- ties with bigger picture thinking relative to controls (For a review, see Lopez et al. 2008). However, Lopez et al. (2008) concluded that while those with AN have global processing difﬁculties, the ﬁndings on local processing were less clear. Thus, while people with AN may struggle with bigger picture thinking, they may not necessarily favour a detail focused approach. Of the seven items assessing attention to detail on the AQ, six referred to detail focused behaviour and only one referred to bigger picture thinking (Baron-Cohen et al. 2001). This may therefore indicate subtle differences between those with Although age was used within the meta-regression in an attempt to explain heterogeneity between the studies, this was not found to be signiﬁcant. Despite this, only three studies (Baron-Cohen et al. 2013; Calderoni et al. 2015; Lang et al. 2015) included participants below the age of 18 and when child/adolescent studies were analysed sepa- rately, the effect size was smaller. There is a lack of con- sensus about whether difﬁculties such as those present in ASD, including social communication and ﬂexibility, are present prior to the onset of the AN or whether they are state dependent, resulting from starvation and an enduring illness and thus not truly autistic in origin (Pellicano and Hiller 2013). Therefore, although the results of this meta- analysis suggest elevated levels of autistic traits in AN, the aetiology of these apparent traits remains obscure. Smaller effect sizes in studies with children and adolescents with 123 973 J Autism Dev Disord (2016) 46:964–977 AN and individuals with elevated autistic traits or ASD in this domain. AN and individuals with elevated autistic traits or ASD in this domain. AN and HC groups as recent research suggests that indi- viduals with AN have lower levels of self-focused attention than HCs, possibly accounted for by differences in exec- utive function (Zucker et al. 2015). However, ﬁndings on gender differences in symptoms of ASD are not conclusive, and other research has found no gender difference in the symptoms of ASD, once IQ is controlled for (Holtmann et al. 2007). Further studies are therefore needed to address the differing presentation of ASD in males and females. Discussion Whilst these difﬁculties may not be severe enough to warrant exploration or diagnosis of ASD, they could still leave an individual vulnerable to the development of AN (Treasure and Schmidt 2013) and may impact on treatment outcome, making them clinically relevant (Nielsen et al. 2015). Whilst ﬁve of the published studies used the full version of the AQ, only two used the brief AQ-10 so separate analysis of the studies using this version could not be performed. Although the effect size of the AQ-10 studies was still signiﬁcant, it is not known whether either the AQ- 10, or indeed the full version, are speciﬁc enough to dif- ferentiate between symptoms associated with ASD and those caused by co-morbidities such as obsessive com- pulsive disorder or social anxiety disorder. Fifty-six per- cent of inpatients with eating disorders have been found to also have an anxiety disorder (Blinder et al. 2006) and certain items on the AQ may tap into symptoms of anxiety rather than ASD per se. Similarly, self-report measures such as the AQ may not be sensitive enough to detect some of the subtle traits associated with female ASD, for example because of their ability to mask the social difﬁ- culties they may experience (Lai et al. 2011). A more recent, large-scale study of 811 adults with ASD, 454 of whom were female (Baron-Cohen et al. 2014), found that AQ scores were higher in the ASD group than in HCs, with both sexes scoring above the suggested cut-off of 32. Males with ASD scored signiﬁcantly higher than their female counterparts, indicating the presence of normative sex differences within ASD, adding strength to the notion that sex differences need to be considered in the clinical diagnosis of ASD. Lai et al. (2011) found that females with ASD had higher levels of self-report autistic traits as measured by the AQ, again suggesting a gender difference in the opposite direction of Baron-Cohen et al.’s (2014) large scale study. Relying on self-report measures to estimate the preva- lence of ASD with AN may therefore lead to a false impression of the true incidence rate. A recent pilot study (Mandy and Tchanturia 2015) aimed to establish whether females with eating disorders who have social and ﬂexi- bility problems meet ASD criteria. After assessing ten women using the ADOS 2nd Edition (ADOS-2; Lord et al. Discussion Another key ﬁnding of this meta-analysis is that although AN patients were found to score signiﬁcantly higher than HCs on the AQ and AQ-10, the mean scores were not high enough to meet the indicated cut-off for ASD, or as high as those with a diagnosis of ASD. For studies using the full version of the AQ, the mean scores ranged from 20.3 to 23.2, whereas 80 % of males and 92.3 % of females with ASD scored above 32 in the originally validation study (Baron-Cohen et al. 2001). Similarly, the studies using the AQ-10 reported mean scores of 3.89 to 4.05 which are below the cut-off of 6 (Allison et al. 2012). It therefore appears that the proﬁle seen in AN represents an intermediate state between HCs and those with clinical ASD. This is not to say that scoring below 32 is not clinically signiﬁcant. It is known that females with ASD may present differently to males. For example, Lai et al. (2011) found behavioural sex differ- ences in ASD, as measured by the Autism Diagnostic Observation Schedule (ADOS), a standard clinical assess- ment tool for ASD, despite males and females not differing on childhood severity of core autistic symptoms. While the AQ was originally validated with just 13 females with Asperger’s/HFA (Baron-Cohen et al. 2001) and the scores of males and females in this group did not differ signiﬁ- cantly, evidence suggests that the diagnostic proﬁle of ASD may differ by gender, with females with HFA being less impaired in early social development (McLennan et al. 1993) and certain diagnostic items appear to be much more typical of girls than boys (Kopp and Gillberg 2011). Regardless of whether the proﬁles of males and females with ASD are comparable, the presence of elevated levels of autistic traits in AN, as measured by the AQ or AQ-10, could still indicate the presence of a neurodevelopmental disorder prior to the onset of the eating disorder. Many of the domains measured by the AQ have been found to be impaired in both children and adults with AN including social skills and communication (Doris et al. 2014; Krug et al. 2013) and despite mixed ﬁndings, there is evidence of difﬁculties with attention switching and attention to detail in young people with AN (Lang et al. 2014a, b). Discussion 2012), the standard assessment tool of ASD diagnosis, three received an ADOS-2 autism classiﬁcation, a further two met criteria for ASD and two appeared to have ASD based on clinical reports but did not score above clinical One possibility is that gender differences in self-focused attention may inﬂuence how autistic traits are reported. For example, as females are more self-focused (Ingram et al. 1988) they may be more introspective than males, reported higher levels of autistic traits despite males having equal or more symptoms. This theory has not been tested empiri- cally and is therefore an area which may warrant further research. Differences in self-focused attention may also not account for the differences in reported ASD traits between 12 3 J Autism Dev Disord (2016) 46:964–977 974 cut-off on the ADOS. Thus, whilst there is evidence that even when using thorough, clinical assessment tools such as the ADOS-2 that a subset of women with AN also have elevated levels of ASD traits, it is not known whether the AQ or AQ-10 can accurately capture this. As the AQ and AQ-10 were designed as screening instruments, rather than diagnostic tools (Baron-Cohen et al. 2001), it may still be beneﬁcial to use them in such a way within AN populations to identify individuals whom would beneﬁt from a full ASD assessment. It would, however, beneﬁt from further validity and reliability for use with this speciﬁc population. Future studies looking to address the issue of trait or state differences in levels of autistic traits in AN would beneﬁt from using thorough clinical tools such as the ADOS along with developmental history in order to ascertain whether any difﬁculties associated with ASD were present prior to the onset of the eating disorder and whether they can be detected by clinical diagnostic tools. between people with AN and ASD, the results do not allow for conclusions to be drawn regarding whether a proportion of those with AN also have an underlying ASD. The self- report nature of the AQ and AQ-10, the presence of sig- niﬁcant co-morbidities in AN and the effect of starvation on these symptoms make interpretation extremely difﬁcult. Discussion Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creative commons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creative commons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. References Allison, C., Auyeung, B., & Baron-Cohen, S. (2012). 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The sociocommunicative deﬁcit subgroup in anorexia nervosa: Autism spectrum disorders and neurocognition in a community-based, longitudinal study. Psychological Medicine, 42(9), 1957–1967. doi:10.1017/ S0033291711002881. Discussion Further research should use thorough, sensitive and vali- dated clinical assessment tools to examine ASD in AN patients and the results of these should be compared to AQ or AQ-10 scores to explore the validity of using self-report measures to assess for ASD within this population. Acknowledgments Heather Westwood would like to thank the Medical Research Council and The Institute of Psychiatry, Psychol- ogy and Neuroscience Excellence Studentship who funded her Ph.D., of which this study is part. Kate Tchanturia would like to thank the Swiss Anorexia Foundation. Both HW and KT would like to thank The Psychiatry Research for support. Author Contributions HW conducted the study and wrote the manuscript, KT designed the study, corrected drafts and proofs and supervised the project, WM corrected drafts, JL conducted meta- analysis statistics, IE and JT assisted in the write up of the study. The presence of elevated levels of autistic traits in AN may have important clinical and treatment implications. There is evidence that treatments such as Cognitive Remediation Therapy (CRT) effectively address some of the traits associated with ASD in AN including difﬁculties with set-shifting and central coherence (For a review see Tchanturia et al. 2014). More recently, Cognitive Reme- diation and Emotion Skills Training (CREST) has been developed to also target the socio-emotion difﬁculties found in ASD and AN (Tchanturia et al. 2015; Tchanturia et al. 2014). 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https://openalex.org/W4306906811	https://iopn.library.illinois.edu/journals/aliseacp/article/download/1072/843	English	null	Many Narratives	Proceedings of the Association for Library and Information Science Education Annual Conference	2,022	cc-by-sa	3,486	ABSTRACT Storytelling bridges a key epistemological divide in our field between socially constructed humanism of children’s story time and implicit positivist orientation of computational systems. Centering story and storytelling as fundamental to LIS calls for a richer variety of stories, tellers, and audiences for a future of greater inclusion. INTRODUCTION Library and information science (LIS) has a rich focus on humanistic concerns in practice, but the underlying theories of information from which we operate are almost all inflected with “an atavistic positivist perspective” (Rayward, 1994; Hjørland, 2005). We typically separate the humanist realm of story and storytelling from the empirical realm of information. Drawing on over 130 years of storytelling wisdom in youth services librarianship, this paper will define storytelling and story, describe the epistemological divide between story and information, and argue for storytelling as an epistemological bridge with implications for inclusion. AUTHOR KEYWORDS Storytelling; youth services; LIS history; innovation; DIKW ALISE RESEARCH TAXONOMY TOPICS Information ethics; research methods; children’s services; critical librarianship; social justice Many Narratives: Storytelling as Epistemological Bridge Kate McDowell University of Illinois at Urbana Champaign, USA kmcdowel@illinois.edu LITERATURE REVIEW Storytelling, which has a long tradition in LIS, has yet to inform information theory or professional practice beyond qualitative data collection methods (such as oral history). After more than a century of practice, LIS storytelling has been largely overlooked, “neglected as a source for new ways of thinking and knowing” (McDowell, 2020). Despite its long history, relatively few studies engage storytelling in LIS, with youth or otherwise. One study demonstrated the educational and social/emotional benefits of oral storytelling (Agosto, 2013). Exploring why storytelling matters, an analysis of children’s responses to oral storytelling revealed benefits in visualization, cognitive engagement, critical thinking, story sequencing abilities (Agosto, 2016). Storytelling can entrance listeners, with shared states of trance-like attention among audience members (Sturm, 1999). Similarly, neurological research finds that neural story processing involves “mirroring process of embodied subjectivity” or experiences of “narrative emotions” predicated upon story's “ability to intertwine our experience of time” (Armstrong, 2020). Specialized “mirror neurons” in the brain contribute to experiencing empathy through story (Rizzolatti, 2008), and contextual empathy cues increase the potential for empathetic experience through story (Roshanaei et al., 2019). A few qualitative studies have explored librarians’ perceptions and uses of storytelling. In libraries, youth services librarians value storytelling for its uses in motivating reading, encouraging imagination through vicarious experience, sharing culture and history, building personal relationships and emotional engagement, and more (Sturm & Nelson, 2016). Organizational storytelling among academic reference librarians conveys rich tacit knowledge, including explaining work conditions, providing warning systems, affording shared preparation for work challenges, and more (Colón-Aguirre, 2015). Qualitative studies like these draw from Elfreda Chatman’s work that explored the lived experiences of information, revealing boundaries between “life-worlds” that and are critical to understanding how people are informed and, ultimately, what they know as individuals within groups (Chatman, 1996). However, definitions of “information” typically rely on an empiricist epistemology that excludes social complexities like “opinions, intentions, desires,” and “cultural forms and social practices” (Ma, 2021). Further, cultural and social exclusions in LIS abound, ranging from microaggressions to epistemicide, so that some social complexities are more likely to be studied while others are ignored. “It is hard work teaching about racism, social justice, and other topics, especially in LIS, which is characteristically known as a white and female field, and it is even harder to teach these topics when I am typically the only person of color in the classroom” (Cooke, 2016). LITERATURE REVIEW Counter-storytelling in LIS is necessary to disrupt patterns of exclusion and bias, and it requires professionals who have heard stories in classrooms that have previously been excluded. For example, it is imperative to teach social justice stories so that students can tell those stories accurately and persuasively (McDowell & Cooke, in press). When stories are ignored, silenced, stolen, or their cultural context is betrayed, they may vanish along with the information and knowledge they contain. “Epistemicide is the killing, silencing, annihilation, or devaluing of a knowledge system,” and the harms of being told that some stories “do not count” have intergenerational impacts that information professionals must engage in “handling knowledge from every field” (Patin et al., 2021). When many stories are overwritten by only a few stories that “count,” and when information professionals are accustomed to these epistemicides as everday injustices, it may help to turn conceptually to storytelling as a way of understanding the epistemological divides by which some human activities are considered information while others are dismissed as mere stories. DEFINING STORYTELLING AND STORY Storytelling as a practice in youth services librarianship emerged in the 1890s, when libraries began offering “a regular hour for storytelling” (Dousman, 1896). The goal was “to be able to create a story, to make it live during the moment of the telling, to arouse emotions— wonder, laughter, joy, amazement” (Sawyer, 1942). Augusta Baker, the first to hold the position of Storytelling Specialist at the New York Public Library, directed new storytellers to emphasize “the story rather than upon the storyteller, who is, for the time being, simply a vehicle through which the beauty and wisdom and humor of the story comes to the listeners” (Baker & Greene, 1977). The definitions given here are also based on 15 years of teaching an LIS Storytelling course at the graduate level and 5 years of co-teaching a new Data Storytelling course that instructs students in well-evidenced and honest communication practices at the intersection of data and story. Storytelling is defined as telling a story within the dynamic triangle of teller, audience, and story. LIS storytelling is a process that co-creates a particular telling of a story. “Storytelling at its best is mutual creation” (Baker & Greene, 1977). The librarian learns the basic character, setting, and plot of the story in advance but does not memorize every word, instead committing the events to memory so that the story's specific version (adaptation, instantiation, performance, etc.) emerges in the dynamic interchange of the storytelling triangle (Agosto, 2013; Agosto, 2016; Del Negro, 2017; MacDonald, 1993; Pellowski, 1977). The three relationships of this storytelling triangle inform each other. For example, the audience's relationship to the teller depends on how they understand the teller's relationship to the story as well as the story itself (McDowell, 2020). For storytelling to occur, there must be a relationship of trust between the teller and the audience. This trust is contextual and depends on demonstrating that the teller wants this audience to know this story. The teller has a relationship to the story, whether as creator or reteller. The storyteller is not neutral as they inevitably bring a point of view. The most obvious example is that of a personal story, in which the person who lived the story is telling it. The audience has an interpretive relationship to the story informed by everything the teller says (gestures, performs, writes, records, etc.) which is not entirely controlled by the teller. DEFINING STORYTELLING AND STORY Story is both narratively patterned information and the content shared through the narrative experience of storytelling. To be a story as narratively patterned information, language must be structured by the chronology of narrative (beginning, middle, and end) and the logic of narrative (character, setting, and plot). Folklorist and acclaimed storyteller Betsy Hearne lauds the aesthetics of folkloric stories, with their “fast-moving, highly structured elemental plots” and “clearly delineated archetypal characters,” for allowing each listener “to glean different emotional, socio-cultural, intellectual, spiritual, and physical connections with a tale” (Hearne, 2011). Crossing both aesthetics and categorization, LIS storytelling ethics require respecting cultural story origins in retellings (Hearne, 1993a, 1993b). Story is also the content shared in the collective narrative experience of storytelling. Some kinds of reception can be viscerally sensed through live audience responses—laughter, applause, boos, hisses, gasps, sighs—which storytellers have categorized (Holt & Mooney, 1994). Embodiment in the “ritual storytelling situation” of story hours indicates that collective narrative experience includes “corporeal aspects,” even among pre-literate children (Hedemark & Lindberg, 2018). Most definitions of information center the individual and do not seriously consider groups, group reception, group interpretation, and other collective phenomena that shape exchanges on social media and other fast-paced “live” or “present” experiences. In short, story is both an empirical form and a socially constructed narrative experience. Story and the dynamics of storytelling constitute not merely a subset of information or of information behavior, but a fundamental information form (McDowell, 2021). Figure 1 Figure 1 Epistemological Divide between Stories and Information Stories Information -Collective (playful) meaning-making -Allowing each listener “to glean different emotional, socio-cultural, intellectual, spiritual, and physical connections with a tale” (Hearne, 2011) -Social constructionism -Individual meaning-making -“things fed into a computer” (Ma, 2021) -“atavistic positivist perspective” (Rayward in Hjørland, 2005) -Positivism g Epistemological Divide between Stories and Information EPISTEMOLOGICAL DIVIDE Most definitions of information presume that audiences are individual. Because stories are constituted through narrative experience, and audiences are partly constitutive of the stories told to and with them, storytelling offers a framework for bridging a fundamental epistemological divide in our field, between social constructionism and positivism. Figure one illustrates how story and information have constituted distinct categories in LIS, without overlap. -Social constructionism Because stories are simultaneously empirical and socially constructed, they bridge this epistemological divide. Storytelling is a way of understanding collective information processes as meaning-making, as story can convey information and storytelling centers collective audience interpretations. Social constructionism is implicit in much human-centered research within information studies (Holland, 2006). “Social constructionism” refers to understanding reality as socially constructed, rooted in American pragmatism and symbolic interactionism. Its emphasis on the “construction metaphor” allows researchers to study things “which do not have material substance,” such as stories (Berger & Luckmann, 1966; Leeds-Hurwitz, 2012). “Positivism” here refers to understandings of information and knowledge as empiricist, rationalist, and informed by the scientific method (Hjørland 2005). This way of knowing typically underlies computational information systems, presuming information to be fixed and static. In one sense, a story is static. Stories persist and the same story is retold over time, in different ways. Stories often convey information with consistency. At the same time, storytelling is a dynamic process that includes variation so that one story can become many narratives. Storytelling communicates to and with collective, social, and interpretive audiences, and audiences reshape stories when they retell them, individually and collectively. Despite a service-focused history, the power of audiences to interpret (and retell stories in their own ways) has been overlooked in LIS too frequently, eschewing humanistic perspectives for a focus on computational systems. A story can be empirically valid and variable, and this room for re-interpretation is a feature of story and storytelling. “We expect ambiguity in narrative, that is, words and events will not mean what at first they seem to” (Polletta, 2006). In other words, stories are both amenable to (empirical) structural analysis and socially constructed in the narrative experience of the audience. The epistemological implications of bridging social constructionism and positivism include opening “doors, windows, and sliding glass doors” (Sims Bishop 2003) to more narratives and, most importantly, more narrators. Storytelling dynamics and emergent stories A story that is retold, spontaneously, by those who have heard it signals a very different kind of power than an official story. If wisdom is carried by stories and accumulates in them as they are retold, then wisdom could be defined as an emergent quality of the storytelling dynamic between teller and audience. Wisdom and storytelling have long been associated, perhaps in part because story allows for accumulating wisdom, with the audience acting as editor. Being a storyteller is an action in context, not a characteristic, and many narrators are needed to enrich LIS. Just as knowledge need not be the attribute of one individual knower, storytelling may be socially constructed and enacted situationally. In this time of multi-layered global crises, breaking free of epistemologies that have engendered epistemicide is a critical move toward wisdom. Storytelling can acknowledge audiences as those who hold the power of interpretation and retelling. There is no telling without listening, and those who serve as listeners grant their attention to the teller. This cannot be forced without damaging the trust that constitutes the storytelling triangle. There is humility in the reminder that, in live oral storytelling, there is no direct line that the teller controls between the audience and the story. “The circumstance that forces you to be humble is also what makes it so miraculous when you succeed” (Lipman, 1999). STORYTELLING AS EPISTEMOLOGICAL BRIDGE Stories that last can be created with audiences, not just presented to them. LIS must make room for counter-storytelling that amplifies voices of those who have been unheard or silenced. The many narratives that would result from an LIS epistemology based on storytelling would include counternarratives, collective meaning making, and collaboration. Centering story and storytelling challenges LIS to include richer varieties of stories, tellers, and audiences whose stories are yet to be heard. This could enable practitioners and scholars alike to identify storytelling dynamics and emergent stories in action and to understand audiences as co-creators of story. CONCLUSION No matter how deeply we embrace humanism in library services, we routinely fall back to positivism in LIS concepts and theories. Storytelling bridges this longstanding divide between social constructionism and positivism and reveals exclusions of the latter. By framing story as information, our field could radically and inclusively transform where we seek information. By hearing people’s stories as not only culturally and emotionally but also informationally relevant, LIS is challenged to make visible the places where more stories are needed. As a field, we must theorize practices like storytelling that have rich potential for challenging limited epistemologies so that many narratives inform and inspire future innovations. Centering story and storytelling as conceptually fundamental to LIS challenges the field to include the richer variety of stories, tellers, and audiences whose dynamic exchanges should inform a future of greater inclusion. Audiences and tellers co-creating stories Storytellers must become listeners, to empower audiences to take up telling. Audiences should be an acknowledged part of the story-making process, not afterthoughts, receptacles, or “markets.” They should know that they are part of the story, giving reactions and feedback that change how it is told and, ultimately, what is told. Storytelling and a commitment to understanding knowledge as epistemologically diverse and comprised of many narratives would mean that, for example, indigenous knowledge takes an obvious and central place in the field (Roy, 2015). And yet changing roles alone will not solve structural inequities, biases, or power differentials. For example, between young adults and adults, changing roles does not create equality. “To disavow or deny such power differentials would rapidly erode trust” (McDowell, 2020). The triangle of teller, listener, and story is never static, and while storytelling with humility means looking for opportunities to listen, those opportunities are merely the starting point toward structural change and greater social equity. equality. “To disavow or deny such power differentials would rapidly erode trust” (McDowell, 2020). The triangle of teller, listener, and story is never static, and while storytelling with humility means looking for opportunities to listen, those opportunities are merely the starting point toward structural change and greater social equity. Finally, storytelling concepts should not be used as a substitute for rigorous definitions and assessments of cultural competence, which has not yet translated into clear action or improved diversity, equity, and inclusion outcomes in the LIS field (Poole et al., 2021). Understanding of cultural competence as a spectrum—along which one can progress or regress—requires cultural humility in order to continually re-engage necessary learning (Cooke, 2017). Storytelling can serve as a complementary tool to these approaches by showing that positivism has served as an excuse for hidden biases, so that LIS has routinely failed to hear many important stories. REFERENCES Agosto, D. E. (2013). If I Had Three Wishes: The Educational and Social/Emotional Benefits of Oral Storytelling. Storytelling, Self, Society, 9(1), 53–76. https://doi.org/10.13110/storselfsoci.9.1.0053 Agosto, D. E. (2016). Why Storytelling Matters: Unveiling the Literacy Benefits of Storytelling. Children and LIbraries, 14(2), 21–26. https://doi.org/10.5860/cal.14n2.21 Agosto, D. E. (2016). Why Storytelling Matters: Unveiling the Literacy Benefits of Storytelling. Children and LIbraries, 14(2), 21–26. https://doi.org/10.5860/cal.14n2.21 Armstrong, P. B. (2020). Stories and the brain: The neuroscience of narrative. Johns Hopkins University Press. Armstrong, P. B. (2020). Stories and the brain: The neuroscience of narrative. Johns Hopkins University Press. Baker, A., & Greene, E. (1977). Storytelling: Art and technique. Bowker. Berger, P. L., & Luckmann, T. (1966). The social construction of reality: A treatise in the sociology of knowledge. Doubleday. Bishop, K., & Kimball, M. A. (2006). Engaging Students in Storytelling. Teacher Librarian, 33(4), 28–31. Chatman, E. A. (1996). The impoverished life-world of outsiders. Journal of the American Society for Information Science, 47(3), 193–206. https://doi.org/10.1002/(SICI)1097- 4571(199603)47:3<193::AID-ASI3>3.0.CO;2-T Colón-Aguirre, M. (2015). Organizational Storytelling Among Academic Reference Librarians. Portal: Libraries and the Academy, 15(2), 233–250. Cooke, N. A. (2017). Information services to diverse populations: Developing culturally competent library professionals. Libraries Unlimited, an imprint of ABC-CLIO, LLC,. Del Negro, J. (2017). Engaging teens with story: How to inspire and educate youth with storytelling. Libraries Unlimited, An Imprint of ABC-CLIO. Dousman, M. E. (1896). Children’s Departments. Library Journal, 21(9), 406–408. Hearne, B. (1993a). Cite the Source: Reducing Cultural Chaos in Picture Books, Part One. School Library Journal, 39(7), 22–27. Hearne, B. (2011). Folklore in Children’s Literature: Contents and Discontents. In S. Wolf, K. Coats, P. Enciso, & C. Jenkins (Eds.), Handbook of Research on Children’s and Young Adult Literature2 (pp. 209–223). Routledge. Hearne, B. (1993b). Respect the Source: Reducing Cultural Chaos in Picture Books, Part Two. School Library Journal, 39(8), 33–37. Hedemark, Å., & Lindberg, J. (2018). Babies, bodies, and books—Librarians’ work for early literacy. Library Trends, 66(4), 422–441. https://doi.org/10.1353/lib.2018.0011 Hjørland, B. (2005). Empiricism, rationalism and positivism in library and information science. Journal of Documentation, 61(1), 130–155. https://doi.org/10.1108/00220410510578050 Holland, G. A. (2006). Associating social constructionism and extended cognition in information studies. Journal of Documentation, 62(1), 91–100. https://doi.org/10.1108/00220410610642066 Holt, D., & Mooney, W. (1994). How do I make a program flow? In Ready-to-tell tales: Surefire stories from America’s favorite storytellers (pp. 68–76). August House. Leeds-Hurwitz, W. (2012). Social Construction of Reality. In S. Littlejohn & K. REFERENCES Foss (Eds.), Encyclopedia of Communication Theory (pp. 891–894). SAGE Publications, Inc. https://doi.org/10.4135/9781412959384.n344 Lipman, D. (1999). Improving your storytelling: Beyond the basics for all who tell stories in work or play. August House. Ma, Y. (2021). Understanding information: Adding a non‐individualistic lens. Journal of the Association for Information Science & Technology, 72(10), 1295–1305. MacDonald, M. R. (1993). The storyteller’s start-up book: Finding, learning, performing, and using folktales including twelve tellable tales. August House. McDowell, K. (2020). Storytelling, Young Adults, and Three Paradoxes. In A. Bernier (Ed.), Transforming Young Adult Services, second edition (2nd ed., pp. 93–109). American Library Association-Neal Schuman. McDowell, K. (2021). Storytelling wisdom: Story, information, and DIKW. Journal of the Association for Information Science and Technology, 72(10 (Special Issue: Paradigm Shift in the Field of Information)), 1223–1233. https://doi.org/10.1002/asi.24466 McDowell, K., Cooke, N. Social Justice Storytelling: A Pedagogical Imperative. Library Quarterly (in press.) Overall, P. M. (2009). Cultural Competence: A Conceptual Framework for Library and Information Science Professionals. Library Quarterly, 79(2), 175–204. Library & Information Science Source. https://www.journals.uchicago.edu/doi/10.1086/597080 Patin, B., Sebastian, M., Yeon, J., Bertolini, D., & Grimm, A. (2021). Interrupting epistemicide: A practical framework for naming, identifying, and ending epistemic injustice in the information professions. Journal of the Association for Information Science and Technology, 72(10), 1306–1318. https://doi.org/10.1002/asi.24479 Pellowski, Anne. (1977). The world of storytelling. Bowker. Polletta, Francesca. (2006). It Was Like a Fever: Storytelling in Protest and Politics. University of Chicago Press. https://doi.org/10.1177/009430610803700327 Poole, A. H., Agosto, D., Greenberg, J., Xia Lin, & Erjia Yan. (2021). Where Do We Stand? Diversity, Equity, Inclusion, and Social Justice in North American Library and Information Science Education. Journal of Education for Library & Information Science, 62(3), 258–286. https://doi.org/10.3138/jelis.2020-0018 Rayward, W. B. (1994). Visions of Xanadu: Paul Otlet (1868-1944) and Hypertext. Journal of the American Society for Information Science, 45(4), 235–250. https://doi.org/10.1002/(SICI)1097-4571(199405)45:4<235::AID-ASI2>3.0.CO;2-Y Rizzolatti, Giacomo. (2008). Mirrors in the brain: How our minds share actions and emotions (C. Sinigaglia, Ed.). Oxford University Press. Roshanaei, M., Tran, C., Morelli, S., Caragea, C., & Zheleva, E. (2019). Paths to empathy: Heterogeneous effects of reading personal stories online. Proceedings - 2019 IEEE International Conference on Data Science and Advanced Analytics, DSAA 2019, 1, 570– 579. https://doi.org/10.1109/DSAA.2019.00072 Roy, L. (2015). Advancing an Indigenous Ecology within LIS Education. LIBRARY TRENDS, 64(2), 384–414. Sawyer, R. (1942). The way of the storyteller. The Viking Press. Sims Bishop, R. (2003). Reframing the Debate about Cultural Authenticity. In D. Fox & K. G. REFERENCES Short (Eds.), Stories Matter: The Complexity of Cultural Authenticity in Children’s Literature. National Council of Teachers of English. Sturm, B. W. (1999). The Enchanted Imagination: Storytelling’s Power to Entrance Listeners. School Library Media Research, 2, 1–21. https://eric.ed.gov/?id=EJ593526 Sturm, B. W., & Nelson, S. B. (2016). With Our Own Words: Librarians’ Perceptions of the Values of Storytelling in Libraries. Storytelling, Self, Society, 12(1), 4–4. https://doi.org/10.13110/storselfsoci.12.1.0004
https://openalex.org/W2077425400	https://bioone.org/journals/african-invertebrates/volume-49/issue-2/afin.049.0210/A-Review-of-Afrotropical-Trichardis-Hermann-1906-and-the-Description/10.5733/afin.049.0210.pdf	English	null	A Review of Afrotropical<i>Trichardis</i>Hermann, 1906, and the Description of the First Oriental Representative of the Genus (Diptera: Asilidae: Laphriinae)	African invertebrates	2,008	cc-by	29,182	A Review of Afrotropical Trichardis Hermann, 1906, and the Description of the First Oriental Representative of the Genus (Diptera: Asilidae: Laphriinae) Author: Londt, Jason G. H. Source: African Invertebrates, 49(2) : 171-226 Published By: KwaZulu-Natal Museum URL: https://doi.org/10.5733/afin.049.0210 Author: Londt, Jason G. H. Source: African Invertebrates, 49(2) : 171-226 Published By: KwaZulu-Natal Museum URL: https://doi.org/10.5733/afin.049.0210 Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Author: Londt, Jason G. H. Source: African Invertebrates, 49(2) : 171-226 Published By: KwaZulu-Natal Museum URL: https://doi.org/10.5733/afin.049.0210 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Complete website, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/terms-of-use. Usage of BioOne Complete content is strictly limited to personal, educational, and non - commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use African Invertebrates Vol. 49 (2) Pages 171–226 Pietermaritzburg Decem African Invertebrates December, 2008 ABSTRACT The Afrotropical species of Trichardis Hermann, 1906 are reviewed, and the first species to be recorded from the Oriental Region is described. Brief descriptions are provided for all Afrotropical species studied, and keys supplied for their identification. The following new species are described: Afrotropical T. abdelkuri (Yemen), crassipala (Burkina Faso, Mali, Niger), eburacta (Ivory Coast, Nigeria), effrena (Namibia, South Africa), glabra (Gambia), hesperia (Gambia, Senegal), lavignei (Somalia), malawi (Malawi, Tanzania, Zimbabwe), mellina (Eritrea), ornata (Chad), similis (Malawi), spicata (Mozambique), zinidi (Kenya, Tanzania); Oriental T. indica (India). The previously described species are redescribed: T. apicalis Oldroyd, 1974; cribrata (Loew, 1858); grisescens Engel, 1924; katangaensis Oldroyd, 1970; leucocoma (Wulp, 1899); nigrescens (Ricardo, 1903); picta Hermann, 1906; pohli Geller-Grimm, 2002; rueppelii (Wiedemann, 1828); terminalis Oldroyd, 1974; testacea (Macquart, 1838); turneri Oldroyd, 1974. A lectotype of Hoplistomera leucocoma Wulp, 1899 has been designated. The following new synonymies have been established: Trichardis lucifer Oldroyd, 1974 = Trichardis picta Hermann, 1906; Triclis rufescens Austen, 1914 = Trichardis leucocoma (Wulp, 1899). EY WORDS: Diptera, Asilidae, Laphriinae, Trichardis, robber flies, Afrotropical, Oriental, new species ew synonymy, identification keys. A review of Afrotropical Trichardis Hermann, 1906, and the description of the first Oriental representative of the genus (Diptera: Asilidae: Laphriinae) Jason G. H. Londt Jason G. H. Londt Natal Museum, P. Bag 9070, Pietermaritzburg, 3200 South Africa, and School of Biological & Conservation Sciences, University of KwaZulu-Natal, P. Bag X01, Scottsville, 3209 South Africa; robber4afr@telkomsa.net Jason G. H. Londt Natal Museum, P. Bag 9070, Pietermaritzburg, 3200 South Africa, and School of Biological & Conservation Sciences, University of KwaZulu-Natal, P. Bag X01, Scottsville, 3209 South Africa; robber4afr@telkomsa.net INTRODUCTION Trichardis Hermann, 1906 is primarily an Afrotropical genus. It is also recorded for the Palaearctic Region where four species, including one also found in the Afrotropics, were catalogued by Lehr (1988) (i.e. T. afanasievae Lehr, 1964; cinctella Séguy, 1934; leucocomus (Wulp, 1899); mongolica Richter, 1972). While this genus has not been previously recorded from any other region, I have found an undescribed species from India (Oriental Region), which is described at the end of this paper. The systematic position of the genus, which has been placed in both the Laphriinae and Laphystiinae, has been discussed by Londt (2007a, b) who recommends that it be treated, together with Perasis Hermann, 1905 and Hoplistomerus Macquart, 1838, within the Laphriinae. These relatively small robber flies have been confused with the generally larger species of Hoplistomerus, and it is hoped that this paper will finally resolve any confusion that may still exist. The development of our knowledge of Afrotropical Trichardis species may be summarised briefly as follows. Wiedemann (1828) – Described Dasypogon Rüppelii from ‘Abyssinia’ (= Eritrea) Macquart (1838) – Described Laphria testacea from ‘Du Cap’ (i.e. The Cape = Western Cape Province of South Africa). Loew (1858) – Described Hoplistomera cribrata from ‘Caffraria’ (i.e. the eastern parts of Southern Africa). Loew (1860) – Elaborated on his 1858 publication, providing a redescription of Hoplistomera cribrata. p Wulp (1899) – Described Hoplistomera leucocoma from Yemen, southern Arabia. Ricardo (1903) – Described Hoplistomera nigrescens from Socotra Island (Yemen). Wulp (1899) – Described Hoplistomera leucocoma from Yemen, southern Arabia. Ricardo (1903) – Described Hoplistomera nigrescens from Socotra Island (Yemen http://www.africaninvertebrates.org.za http://www.africaninvertebrates.org.za Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 172 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Hermann (1906) – Described the genus Trichardis, assigning two new species to it (picta and testacea), but neglected to designate a type species. He briefly discussed the group of genera subsequently included in the subfamily Laphystiinae. g p g q y y p y Becker (1907) – Described the genus Strobilothrix for his new species Strobilothrix albipila from Algeria, in the Palaearctic Region. Becker (1907) – Described the genus Strobilothrix for his new species Strobilothrix albipila from Algeria, in the Palaearctic Region. p g g Kertész (1909) – Catalogued two species of Trichardis (picta and testacea) from ‘Prom. bon. Sp.’ (i.e. the Cape of Good Hope). Kertész (1909) – Catalogued two species of Trichardis (picta and testacea) from ‘Prom. bon. Sp.’ (i.e. the Cape of Good Hope). p p p Hermann (1920) – Designated Laphria testacea Macquart as type species of Trichardis listing six other species—picta, cribrata, leucocoma, grisescens, erythrogaster, and ? nigrescens. T. grisescens was at the time an unpublished manuscript name, while erythrogaster, described in Hoplistomerus, has subsequently been retained in Hoplistomerus (see Londt 2007b). Engel (1924) – Provided a brief discussion of Trichardis before keying out (giving full descriptions) five species considered belonging to the genus (cribrata, grisescens, leucocoma, picta and testacea). In providing a description for grisescens he became author of the species even though he attributed the species to Hermann (see previous entry). y Efflatoun (1937) – Included T. leucocoma in an extensive review of Egyptian Asilidae (part of the Palaearctic fauna). Efflatoun (1937) – Included T. leucocoma in an extensive review of Egyptian Asilidae (part of the Palaearctic fauna). Hull (1962) – Redescribed Trichardis as part of his world revision of genera, giving Strobilothrix as a synonym and listing those species he considered to be con- generic. The Ethiopian (= Afrotropical) species listed were cribratus, grisescens (correctly attributed to Engel), nigrescens, picta, and testacea Hermann (he was apparently unaware of Macquart’s species with the same name). Two Palaearctic species were also listed, one of which was leucocoma (with albipila as a synonym). Oldroyd (1970) – In handling the Asilidae of the Congo Basin provided a brief discussion of Trichardis and the description of a new species, katangaensis. Oldroyd (1974) – Published an introduction to the Asilidae of southern Africa. MATERIAL AND METHODS Specimens studied are housed in the Natal Museum (NMSA) unless otherwise indicated. Other institutions housing material are listed below, together with the abbreviations used in the text when citing these repositories and the names of the people who kindly assisted with information and/or loans. AMGS – Albany Museum, Grahamstown, South Africa (A. Kirk-Spriggs). AMGS – Albany Museum, Grahamstown, South Africa (A. Kirk-Spriggs) BMNH – The Natural History Museum, London, UK (E. McAlister). CASC – California Academy of Sciences, San Francisco, USA (A. Carmichael HLMD – Hessisches Landesmuseum Darmstadt, Germany (W. Schneider). MCMI – Museo Civico di Storia Naturale, Milano [Milan], Italy (F. Rigato). MCMI – Museo Civico di Storia Naturale, Milano [Milan], Italy (F. Rigato MNHN – Museum National d’Histoire Naturelle, Paris, France (C. Daugeron) MRAC – Musee Royal de l’Afrique Centrale, Tervuren, Belgium (E. De Conin MRAC – Musee Royal de l’Afrique Centrale, Tervuren, Belgium (E. De Coninck MZLU – Zoological Museum, Dept. Zoology, Lund, Sweden (R. Danielsson). MZUF – Museo Zoologica de ‘La Specola’, Firenze [Florence], Italy (L. Bartoloz NHMW – Naturhistorisches Museum Wien, Wien, Austria (P. Sehnal). NHRS – Naturhistoriska Riksmuseet, Stockholm, Sweden (B. Viklund). NMNH – National Museum of Natural History, Smithsonian Institution, Washington, DC, USA (F.Ch. Thompson). OXUM – Hope Entomological Collections, Oxford University Museum of Natural History, Oxford, UK (D. Mann). SAMC – South African Museum, Cape Town, South Africa (M. Cochrane). SANC – National Collection of Insects, Pretoria, South Africa (R. Urban). SMFD – Forschungsinstitut und Naturmuseum Senckenberg, Frankfurt, Germany (P. Haase). SMFD – Forschungsinstitut und Naturmuseum Senckenberg, Frankfurt, Germany (P. Haase). ZMHB – Museum für Naturkunde, Humboldt-Universität zu Berlin, Germany (J. Ziegler). ZMHB – Museum für Naturkunde, Humboldt-Universität zu Berlin, Germany (J. Ziegler). g ZSMC – Zoologische Staatssammlung, München, Germany (B. Stock). ZSMC – Zoologische Staatssammlung, München, Germany (B. Stock). Other collections cited in this paper, but not consulted personally are: NHCY (Natural History Collection, Yemen), COGG (Collection of Fritz Geller-Grimm, Frankfurt), CWWR (Collection of Wolfgang Wranik, Rostock). A standard format has been employed in recording label information. For type specimens data is reproduced as it appears on labels, each label being demarcated by the use of single inverted commas, and each line of data separated by a spaced slash (/). Data that appear on the reverse side of a label are preceded by a ‘~’ symbol. http://www.africaninvertebrates.org.za He briefly discussed Trichardis and described four new species (apicalis, lucifer, terminalis, and turneri) in an illustrated key to seven southern African taxa that included three previously described species (cribrata, picta and testacea). Theodor (1980) – Included T. leucocoma in his review of Palestinian Asilidae focussing attention on genital morphology. Theodor (1980) – Included T. leucocoma in his review of Palestinian Asilidae focussing attention on genital morphology. Oldroyd (1980) – Catalogued (under the tribe Laphriini of subfamily Laphriinae) the then accepted Afrotropical species apicalis, cribrata, grisescens, katangaensis, leucocoma, lucifer, nigrescens, picta, terminalis, testacea, and turneri — giving grisescens Hermann as a nomen nudum, albipila as a synonym of leucocoma, and testacea Hermann as a synonym and homonym of testacea Macquart. He placed Dasypogon Rüppelii in Laphria as L. rueppelii giving Sudan as the country of origin. Geller-Grimm (1999) – Placed Dasypogon rueppelii (as rueppellii) in Trichardis. Geller-Grimm (2002) – Described Trichardis pohli from Socotra Island and provided a redescription and records of nigrescens from the island. There were, therefore, 13 valid species of Afrotropical Trichardis at the commence- ment of this study, i.e. those catalogued by Oldroyd (1980) and the species subsequently added by Geller-Grimm (1999, 2002). This study was prompted by an accumulation of There were, therefore, 13 valid species of Afrotropical Trichardis at the commence- ment of this study, i.e. those catalogued by Oldroyd (1980) and the species subsequently added by Geller-Grimm (1999, 2002). This study was prompted by an accumulation of Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 173 many specimens in the collections of the Natal Museum and other institutions. At the completion of my study a further 13 undescribed species need to be added and one new synonym established bringing the number of Afrotropical species to 25. In addition, I believe this is a good opportunity to provide the description of the first representative of the genus from the Oriental Region. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use MATERIAL AND METHODS For other specimens cited, information is provided in a briefer format, but maintaining actual information provided, and in order that facilitates electronic data capture. Square brackets are used to add useful information or comment not found on labels. In this regard, co-ordinates are usually provided when these, or a quarter-degree grid reference, do not appear on a label. When a locality could not be traced or other information is not 174 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 understood the wording may be followed by [?]. Specimens are arranged in geographical order (according to latitude) within countries, which are alphabetically ordered. An adequate generic diagnosis is provided. If a fuller description is required that of Hull (1962) may be consulted. Species descriptions are brief and confined largely to characteristics that are considered helpful in the separation of species. Final illustrations were prepared from pencil drawings and do not depict setae that are not considered to have diagnostic value. Measurements were taken as follows: antennal postpedicel length (L) includes terminal style, depth (D) is taken at maximum level; wing length is from humeral crossvein to tip, breadth is taken at its maximum level; metathoracic (hind) femur length is measured in anterior view (excluding any part of coxa or trochanter), height is taken at its maximum level. Morphological terminology generally follows McAlpine (1981). TAXONOMY Genus Trichardis Hermann, 1906 TAXONOMY Genus Trichardis Hermann, 1906 Trichardis Hermann, 1906: 137. Type species: Laphria testacea Macquart, 1838, by designation of Hermann (1920: 177). Genus Trichardis Hermann, 1906 Trichardis Hermann, 1906: 137. Type species: Laphria testacea Macquart, 1838, by designation of Hermann (1920: 177). ( ) Strobilothrix Becker, 1907: 42–43. Type species: Strobilothrix albipila Becker, 1907 [Hoplistomera leucocoma Wulp, 1899], by monotypy. Diagnosis (Figs 1–4): Laphriine asilids (wing length <7 mm) with the following combi- nation of characters. Head: Antennal postpedicel moderately elongate to clavate; proboscis small and hardly protruding beyond lower epistomal margin. Thorax: Postpronotal lobe with at least a few strongly developed macrosetae; anatergal macrosetae usually present; scutellum with weakly developed marginal macrosetae; Figs 1–4. Trichardis species: (1–3) T. testacea (Macquart, 1838): (1) dorsal view of entire male, (2) lateral view of entire male, (3) wing; (4) T. effrena sp. n., wing. See text for measurements. Figs 1–4. Trichardis species: (1–3) T. testacea (Macquart, 1838): (1) dorsal view of entire male, (2) lateral view of entire male, (3) wing; (4) T. effrena sp. n., wing. See text for measurements. LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 175 postmetacoxal area membranous; hind femora robust (length:breadth ratio <4), with a swollen appearance and usually equipped with ventral tubercles; hind leg of male usually lacking a tibial spur; vein R2+3 bent anteriorly at tip and joining R1 just before or at C; cell r5 always closed; C continues fairly strongly along wing margin to Cu+A1 before becoming much weaker along anal cell and completely absent from alula. Abdomen: Terga with discal setae beyond T1 and lacking obvious golden setation. _ genitalia: Epandrium in dorsal view hardly if at all incised (i.e. not divided into lobes); hypandrium usually absent or at best poorly developed; gonocoxites usually closely associated ventrally and rarely with a median projection (when present it is short and medially directed) and commonly with mediodistal macrosetae. Notes: Hermann's original description makes reference to ‘Hoplistomera’ and includes figs 5 (antenna) and 7 (wing), but no indication of which species was involved. Hermann (1920) separates Trichardis from Strobilothrix on the basis of femoral setation. Oldroyd (1970: 247) says the genus is not easily separated from Hoplistomerus and provides a brief discussion of the characterisation of these taxa. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use AFROTROPICAL SPECIES Trichardis abdelkuri sp. n. Figs 5, 6 Etymology: Named after the type locality, Abd el Kuri I. [also written Abd al Kuri], Yemen. Etymology: Named after the type locality, Abd el Kuri I. [also written Abd al Kuri], Yemen. Description (based on holotype in good condition): Head: Dark red-brown to black, extensively silver pruinose except for central face, frons and ocellar tubercle, setae black, pale yellow and white. Antennal scape dark red- brown, white setose except for a single pale yellow ventrally situated macroseta (right side has an additional slender black ventral macroseta); pedicel brown-yellow, setae white except for 1 or 2 longish black ones; postpedicel and style dark red-brown, postpedicel elongate spindle-shaped (L:D=4.1:1) with few pale setulae dorsally. Mystax white with some black setae along epistomal margin and below antennal sockets. Ocellar tubercle with 4 pale yellowish macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown to black, largely apruinose with gold-silver pruinose parts, fine setae whitish, more major setae pale yellow. Postpronotum largely apruinose except for narrow medial part, mesonotum largely apruinose except for margins, macrosetae shiny pale yellow, setulae shiny white. Scutellum black, entirely apruinose. Anepisternum with slender pale yellow posterior macroseta, extensively pruinose except for small area anteroventrally. Proepimeron anteriorly pruinose, posteriorly apruinose; katepi- sternum posteriorly pruinose, anteriorly apruinose; anepisternum pruinose except for anterodorsal part. Legs: Dark red-brown to black, pulvilli and empodium of similar length. Hind femur uniformly dark red-brown, length:height ratio 3.8:1, ventral tubercles poorly developed, major setae pale yellowish. Hind tibia lacking ventrodistal spur. Wing: 5.3×2.2 mm. Costal vein extends around most of wing margin, weak along anal cell, absent from alula. Membrane not extensively microtrichose—discal cell largely lacking microtrichiae (a few present centrally), cell r5 with microtrichiae limited mainly to distal half. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 176 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Abdomen: Anterior four terga dark red-brown, terminal two visible segments and hypo- pygium orange, apruinose except for narrow silver pruinose distolateral margins, setae transparent whitish. T2 dark red-brown, apruinose except for narrow silver pruinose posterior margins laterally. _ genitalia (Figs 5, 6): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without median projections distally and with mediodistally arranged macrosetae, mediodistal projection fairy slender with slightly upturned distal end. Gonostylus slender with straight distal end. Aedeagal prongs more or less straight with small terminal tubules. Variation: The paratype _ is slightly teneral and displays a few minor differences in co- loration. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Trichardis apicalis Oldroyd, 1974 Figs 7, 8, 58 Trichardis apicalis: Oldroyd 1974: 120; 1980: 355 (catalogue). Etymology: Named after the type locality, Abd el Kuri I. [also written Abd al Kuri], Yemen. Both scape and pedicel are yellowish brown and both have major setae black. Macrosetae of thorax and abdomen are transparent, lacking colour. The ^paratypes are similar to the holotype, but have the first five terga dark red-brown. Holotype: YEMEN: Abd el Kuri I.: _ ‘Abdelkuri I: [12°05'N:52°20'E] / Jebel Saleh [?], / 500–1500 ft / 7.v.1967 / K. Guichard’, ‘Brit. Mus. / 1967-455’ (BMNH). Holotype: YEMEN: Abd el Kuri I.: _ ‘Abdelkuri I: [12°05'N:52°20'E] / Jebel Saleh [?], / 500–1500 ft / 7.v.1967 / K. Guichard’, ‘Brit. Mus. / 1967-455’ (BMNH). Paratypes: 1_ 2^with same label data. Distribution and biology: Known only from the type series. The species may be confined to the island of Abd el Kuri and has so far only been collected in May. No biological information is available. Similar species: T. abdelkuri is superficially very similar to nigrescens, but the species can be reliably separated on male genital features. Although I have seen relatively few specimens of both species, all specimens of abdelkuri have mesonotal, anepisternal and ocellar setae yellowish, while these setae are mostly black in nigrescens (some variation exists). These species are also somewhat similar to pohli, but easily separated on size and male genital form. Trichardis apicalis: Oldroyd 1974: 120; 1980: 355 (catalogue). Redescription (based on _ holotype in good condition): Head: Uniformly brown-orange. Antenna uniformly brown-orange, yellowish setose; postpedicel not markedly clavate (L:D=3.2:1). Mystax uniformly yellowish on slightly and evenly convex face. Ocellar tubercle with 4 macrosetae. Proboscis brown-orange with dark red-brown distal half, palpi brown-orange. Thorax: Brown-orange, dorsal parts slightly darker; mesonotal macrosetae orange, fine setae white or pale yellow. Postpronotum entirely pruinose, mesonotum apruinose except for narrow silver pruinose lateral and posterior margins. Scutellum apruinose. Anepi- sternum with yellow posterior macroseta; dorsal half pruinose, ventral half apruinose. Proepimeron dorsally pruinose, ventrally apruinose; katepisternum dorsally pruinose, ventrally apruinose; anepisternum posteriorly pruinose, anteriorly apruinose. Legs: Brown-orange except for terminal tarsomeres and distal parts of hind tibiae; pulvilli and empodium of similar length. Hind femur brown-orange, length:height ratio 3.5:1, Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 177 ventral tubercles poorly developed. Hind tibia lacking ventrodistal spur. Wing: 5.0×2.1 mm. Costal vein strongly developed around most of wing margin, weak to absent along anal cell and alula. Membrane not extensively microtrichose—discal cell entirely lacking microtrichiae, cell r5 with microtrichiae only in distal half. Abdomen: Uniformly brown-orange, fine white setose. T2 orange, apruinose except for small areas of silver pruinescence on posterolateral corners. p p _ genitalia (Figs 7, 8): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple in structure. Gonocoxite in ventral view without median projections distally and with a few moderately developed mediodistal setae; mediodistal projection stout with upturned and darkly sclerotised distal end. Gonostylus stout with upturned, slightly clavate distal end. Aedeagal prongs more or less straight, with small, slender tips. Holotype (examined): MOZAMBIQUE: _ ‘Holo- / type’ [circular, red edge], ‘Lourenço Marques. [Maputo, 25°57'S:32°34'E] / Sept. – Dec., 1913. / H. A. Junod.’, ‘C. J. Wainwright / collection / B.M. 1948-488’, ‘Trichardis / apicalis Oldr. / det. H. Oldroyd 1972 / Holotype’ [white] (BMNH). Paratypes (examined): MOZAMBIQUE: 3_ 1^same data as holotype (BMNH); 1_ 1^same data as holotype but ‘Jan. – Mar., 1914’ (BMNH); 1_ ‘Para- / type’ [circular with yellow border], ‘Lourenço Marques / Sept.– Dec., 1913, / H.A. Junod’, ‘C. J. Wainwright / collection. / B.M. 1948–488’ (BMNH). SOUTH AFRICA: 1_ ‘South Africa, Natal Prov / Zululand. 4 mi S. Ndumu / Game Res. Camp (2632Cc) / Dec. Trichardis apicalis: Oldroyd 1974: 120; 1980: 355 (catalogue). 1, 1971; ME&BJ Irwin / dry scrub forest; 160 ft’ (NMSA); 7_ 1^1? ‘South Africa, Natal Prov / Zululand. 20 mi S. Ndumu / Game Res. Camp (2732Aa) / Nov. 29, 1971; ME&BJ Irwin / dry scrub forest; 320 ft’, ‘Trichardis / apicalis Oldr / det. H. Oldroyd 1973 / Paratypes’ (NMSA); 3_ 3^‘Ndumu Reserve [26°52'S:32°15'E], / Ingwavuma District / Tongaland, S. Africa / B. & P. Stuckenberg / 1–10 December 1963’, ‘Trichardis / apicalis Oldr / det. H. Oldroyd 1973 / Paratypes’ (NMSA); 2^‘Para- / type’ [circular with yellow border], ‘Ndumu Reserve / Ingwavuma dist. / Zululand, Natal / South Africa / 1–10.xii.63’, ‘collectors / B. & P. Stuckenberg’ (BMNH). Other material examined: BOTSWANA: 1^Ghanzi-Maun Rd, 2122AB, 19.xii.1984, Johnson; 1_ Palapye [22°33'S:27°08'E], 4.iii.1934, Ogilvie (BMNH). MOZAMBIQUE: 1^Nampula [15°07'S:39°15'E], 29.ii.1982, Feijen; 1_ Masiene [16°24'S:39°54'E], xii.1923, Lawrence (SAMC); 1_ Nyaka [16°40'S:32°42'E], ii.1924, Lawrence (SAMC); 1_ Mabote [22°02'S:34°08'E], 27–30.iii.1964, Moore (NMNH); 2_ Massangwa [?Massangera, 22°02'S:34°08'E], 1–8.ii.1964, Moore (NMNH); 1^Inhambane [23°51'S:35°29'E], i.1924, Lawrence (SAMC); 4_ 4^Chimonzo [24°58'S:33°17'E], 21–27.ii.1964, Moore (NMNH); 1? Lorenzo Marques [= Maputo, 25°57'S:32°34'E], 1909, De Azevedo (NMNH); 7_ 10^Chiqubo [?], 11–20.ii.1964, Moore (NMNH). NAMIBIA: 1_ 1^Waterberg Nat. Park Entrance, 20°32'S:17°20'E, 20.iii.1984, Stuckenberg & Londt, Acacia thornveld; 1_ Etosha-Pan, Namutoni [18°48'S:16°59'E], 23.i.1993, Koch (ZMHB). SOUTH AFRICA: 1^Messina Nat. Res., 22°24'54"S:30°05'12"E, 487 m, 14.ii.2005, Londt & Dikow, dry woodland Sand R.; 1_ 2^Soutpansberge Soutpan, 2229CD, 23–24.ii.1980, Londt & Schoeman, bushveld vegetation; 1_ 37 km N Louis Trichardt, 2229DD, i.1975, Stuckenberg, arid bushveld; 1^Wyllies Poort, 10 km N Louis Trichardt, 2229DD, 22.ii.1980, Londt & Schoeman; 1^Lapalala Nat. Res., 23°52'52"S:28°20'19"E, 1072 m, 16.ii.2005, Londt & Dikow, Acacia Combretum woodland; 4_ Mogol Nat. Res., Ellisras Dist., 23°58'S:27°45'E, 19–23.xi.1979, van Tonder, Kok, Prinsloo & Mansell (SANC); 2_ Ndumu Game Reserve, 26°55'S:32°19'E, 3.ii.1995, Koch (ZMHB); 1_ Ndumu Game Reserve Rest Camp, 2632CD, 95 m, 15.ii.1978, Brothers, Malaise trap; 1^Ndumu Reserve, Ingwavuma Dist. [27°08'S: 31°59'E], 1–10.xii.1963, Stuckenberg; 1_ 32 km N Jozini, 2732AC, 229 m, 28.xi.1971, Irwin. ZIMBABWE: 2_ Lusulu [18°04'S:27°50'E], 19.xi.1963, Phelps; 1^Hartley [18°08'S:30°09'E], xii.1930, Cuthbertson (BMNH); 1^Sawmills [19°35'S:28°01'E], 10.ii.1923, Swinburn & Stevenson; 1_ 1^Sawmills, 26.xii.1923, Stevenson (SAMC); 1^Sawmills, 14.xi.1924, Stevenson. Distribution and biology: The species, a southern African endemic, has a fairly wide distribution (Fig. 58), occurring in the northern parts of Namibia eastwards through Botswana, Zimbabwe, the northern parts of South Africa and Mozambique. Adults fly between November and March (Table 1). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Paratypes: BURKINA FASO: 2_ 1^same data as holotype (MNHN). MALI: 1_ 1^‘Coll. Mus. Tervuren / Mali: Kassarola [?] / 31.vii.1970 / G. Pierrard’ (MRAC). NIGER: 5_ 3^‘Museum Paris / Rég. O. De Zinder / Tibiri–Maradi [13°35'N:08°10'E] / (Mission Tilho) / Dr R. Gaillard 1910’, ‘Juillet’, ‘Aout’ (MNHN); 1_ 3^‘Museum Paris / Rég. O. De Zinder / Maradi / (Mission Tilho) / Dr R. Gaillard 1910’, ‘Juillet’ (MNHN). SUDAN: 3_ 3^‘11.v.55 38 / Southern Sudan / Equatoria Province / Juba [04°57'N:31°35'E] / P. Blasdale. 34–1955’ (OXUM). Holotype: BURKINA FASO: _ ‘Ouaga. [Ouagadougou, 12°25'N:01°30'W] 8.vii.69 / Haute – Volta / J.G. Pointel’ (MNHN). Trichardis apicalis: Oldroyd 1974: 120; 1980: 355 (catalogue). While little information is available concerning habitat, labels suggest that the species is found in dry to arid woodland dominated by Acacia trees. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 178 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Similar species: T. apicalis has an entirely pruinose postpronotal lobe and in this respect can be grouped with grisescens, ornata, picta, terminalis, testacea, turneri and zinidi. The species is, however, most similar to zinidi. Trichardis crassipala sp. n. Figs 9, 10 Other material examined: There are three specimens of undetermined sex from Niger (same labels as above) in MNHN Distribution and biology: The species is widely distributed in Africa north of the Equator, being found in West Africa (Mali, Burkina Faso), Central Africa (Niger) and East Africa (Sudan). Adults fly between May and August (no records for June), the northern hemisphere summer (Table 1). No information is available concerning habitat preference, but locality information suggests that this is a savannah species. Similar species: A member of what is here called the ‘cribrata species group’ which consists of crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata and indica. These species are superficially similar, but can be easily separated on characters of the male genitalia. T. crassipala is most similar to similis in that both species have well- developed hypandria. Trichardis cribrata (Loew, 1858) Figs 11, 12, 59 Hoplistomera cribrata: Loew 1958: 357; 1860: 193. Trichardis cribrata: Engel 1924: 108–109; Oldroyd 1970: 249 (fig. 28, mesopleuron); 1974: 118; 1980: 355 (catalogue). Trichardis cribratus: Hull 1962: 97 Figs 11, 12, 59 Hoplistomera cribrata: Loew 1958: 357; 1860: 193. Trichardis cribrata: Engel 1924: 108–109; Oldroyd 1970: 249 (fig. 28, mesopleuron); 1974: 118; 1980: 355 (catalogue). Trichardis cribratus: Hull 1962: 97 g , , Hoplistomera cribrata: Loew 1958: 357; 1860: 193. Trichardis cribrata: Engel 1924: 108–109; Oldroyd 1970: 249 (fig. 28, mesopleuron); 1974: 118; 1980: 355 (catalogue). T i h di ib H ll 1962 97 p ; richardis cribrata: Engel 1924: 108–109; Oldroyd 1970: 249 (fig. 28, mesopleuron); 1974: 118; 1980: 35 (catalogue). ( g ) Trichardis cribratus: Hull 1962: 97. Redescription (based on Mhlopeni Nat. Res. male in excellent condition): Head: Dark red-brown to black, finely silver pruinose except for central parts of face. Antenna orange-brown, black setose except for a few small pale yellow setae; postpedicel not markedly clavate (L:D=3.2:1). Mystax uniformly pale yellowish. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown to black, silver pruinose except for some bare areas. Postpro- notum medially strongly silver pruinose, laterally apruinose, mesonotum apruinose except for silver lateral and posterior margins, macrosetae yellow–white, fine setulae yellowish. Scutellum apruinose except for narrow anterior margin. Anepisternum with pale yellow posterior macroseta, extensively silver pruinose except for apruinose area anteroventrally. Proepimeron anteriorly pruinose, posteriorly apruinose; katepisternum posteriorly pruinose, anteriorly apruinose; anepisternum entirely pruinose. Legs: Dark red-brown, tibiae orange-brown proximally, pulvilli and empodium of similar length. Trichardis crassipala sp. n. Figs 9, 10 Etymology: From Latin crassus (thick) and palus (stake/stick). Refers to enlarged gonostyli. Description (based on holotype in excellent condition): Head: Dark red-brown to black. Antenna dark red-brown to black, black setose except for a few small white setae; postpedicel not markedly clavate (L:D=3.7:1). Mystax white, a few black macrosetae along epistomal margin, on slightly convex and mostly shiny apruinose face. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red- brown to black. Thorax: Dark red-brown to blackish, mostly apruinose, pruinose areas silvery. Post- pronotum strongly silver pruinose medially, extensively apruinose laterally, mesonotum apruinose except for narrow silver pruinose lateral and posterior margins, macrosetae pale yellow, fine setae white. Scutellum apruinose except for narrow anterior margin. Anepisternum with pale yellow posterior macroseta, dorsally pruinose, ventrally aprui- nose. Proepimeron intensively pruinose, posterior part apruinose; katepisternum ante- riorly apruinose, posteriorly pruinose; anepisternum pruinose. Legs: Dark red-brown, pulvilli and empodium of similar length. Hind femur dark red-brown, length:height ratio 3.7:1, ventral tubercles moderately developed. Hind tibia lacking ventrodistal spur. Wing: 4.2×1.6 mm. Costal vein moderately developed along entire wing margin, but weak along anal cell and absent from alula. Membrane extensively microtrichose (except for parts of some proximally situated cells)—discal and r5 cells entirely microtrichose. Abdomen: Dark red-brown anteriorly becoming progressively more brown-orange posteriorly. T2 dark red-brown, apruinose except for narrow silver pruinose posterior margin laterally. _ genitalia (Figs 9, 10): Epandrium in lateral view significantly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger fairly long and strongly dorsoventrally compressed. Hypandrium moderately developed, with characteristic bilobed shape distally. Gonocoxite in ventral view with moderately well-developed median projection distally and lacking macrosetae except for a group of small ones on median projection; mediodistal projection stout, broad, laterally flanged, strongly sclerotised with characteristic shape. Gonostylus short, slender, largely hidden by gonocoxite. Aedeagal prongs more or less straight with moderately well-developed trifurcate tip. Holotype: BURKINA FASO: _ ‘Ouaga. [Ouagadougou, 12°25'N:01°30'W] 8.vii.69 / Haute – Volta / J.G. Pointel’ (MNHN). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 179 Other material examined: There are three specimens of undetermined sex from Niger (same labels as above) in MNHN. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Trichardis crassipala sp. n. Figs 9, 10 SOUTH AFRICA: 1^Pietersburg [Polokwane, 23°54'S:29°27'E], 8.xii.1965, Hoffmann (SANC); 1^Groenfontein, 35 km E Thabazimbi, 24°34'S:27°45'E, 27.xi.1980, Kok (SANC); 1^Ben Alberts Nat Res, Thabazimbi, 24°37'S:27°23'E, 24–28.xi.1980, Kok (SANC); 1_ Sondéla Nat. Res., 24°54.127'S:28°25.191'E, 1086 m, 7–14.xi.2003, Londt, Acacia savannah; 1^Skukuza [24°59'S:31°36'E], 23.xi.1959 (BMNH); 1^Salietjie Pad, KNW [= KNP – Kruger National Park], 27 km from Skukuza, 8.xi.1960, van Schalkwyk (SANC); 1_ 1^Kruger National Park, N of Sabie R., 25.xi.1959, Munro & v. Bruggen (SANC); 1^Lydenburg Dist. [25°06'S:30°27'E], 1896, Krantz; 1^1? Wonderboom [25°36'S:29°20'E], 21.xi.1915, Roberts; 1^W. boom [Wonderboom] Pretoria, xii.1915, Munro (NMNH); 2_ 1^Swartruggens Marico [25°39'S:26°42'E], 15.i.1921, Brauns; 1_ Zwartruggens Marico, 15.i.1921, Brauns (NMNH); 1_ Rustenburg [25°40'S:27°15'E], 3.xii.1961 (BMNH); 2_ 4^Pretoria [25°44'S: 28°11'E], 28.xii.1912 (1_), 16.xii.1913 (1^), 12.xii.1914 (1^), 2.xii.1915 (1_), 18.xii.1915 (1^), xii.1929 (1^), Munro (NMNH); 1^1? Pretoria, 16.xii.1913, Munro (SANC); 1_ 2^Hennops R., 20 km W Pretoria, 25°47'S:27°55'E, 17.xii.1981, Oberprieler (SANC); 1^Brnkh.spr. [Bronkhorstspruit, 25°48'S:28°44'E], 15.xii.1906; 1_ 2^Halfway House [25°59'S:28°07'E], 22.vii.1981, Elferink; 1_ Lichtenburg [26°09'S:26°10'E] (MCMI); 3_ 2^Lichtenburg, Brauns (ZSMC); 1^‘Sammlung F. Hermann’ [no locality data, but probably from Lichtenburg] (ZSMC); 1^Kroonstad [27°40'S:27°14'E], O.R.C. [Orange River Colony = Free State Province], Eckersley (BMNH); 2_ 1^1? M’fongosi [28°42'S:30°48'E], x.1911 (1?), iii.1917 (1^), iv–xi.1934 (2_), Jones (SAMC); 2_ 3^Weenen [28°51'S:30°05'E], i.1925 (2_ 2^), iii.1925 (1^), Thomasset (BMNH); 1^Koornspruit Weenen, 2830CC, 24.xi.1981, Milton, Acacia tortilis; 1_ 1^Colenso, 2829DB, 7.x.1981, Londt; 1_ 1^20 km W Tugela Ferry, 2830CA, 26–27.ii.1977, Miller, Malaise trap; 9_ 4^c. 10 km E Estcourt [29°00'S:29°53'E], 13.xii.1995, Londt & Cradock, Acacia woodland; 1_ 1^Estcourt, 1894, Haviland (SAMC); 1_ 3^Estcourt, xii.1896 (2^in BMNH); 1_ 1^Estcourt, 1897, Marshall (BMNH); 6_ 2^Mhlopeni Nat. Res. 15 km SE Muden, 2930AB, 22.xii.1983, Londt; 2^Aliwal North [30°42'S:26°42'E], 1326 m, 1–13.i.1923, Turner (BMNH); 1^Umdala Fort Beaufort [32°48'S:26°39'E], iii.1954, S.A.Museum (SAMC); 1_ Willowmore [33°17'S:23°29'E], 10.i.1907, Brauns (ZSMC); 2_ 2^5 km NE Kenton on Sea, 3326AD, 29.xii.1985, Londt, bush & grass & stream bed; 1_ 1^Kangwane, Thomeni Res. [?], 16.i.1992, Acacia veld. ZIMBABWE: 1^Rekomitjie [16°08'S:29°24'E], i.1988, Phelps, Mopane woodland; 1^Mt Selinda [20°25'S:32°42'E], xii,1935, van Son. Distribution and biology: The species is a southern African endemic, distributed widely within the eastern half of the subregion (eastwards of about 23°E), but does not appear to occur along the subtropical and tropical eastern coast (Fig. 59). Adults fly during the summer months of October and March (there is a record for July that needs verification) (Table 1). Trichardis crassipala sp. n. Figs 9, 10 Hind femur dark red-brown, length:height ratio 3.4:1, ventral tubercles well-developed. Hind tibia lacking ventrodistal spur. Wing: 4.0×1.6 mm. Costal vein moderately developed around most of wing margin, weakening along anal cell and absent from alula. Membrane extensively microtrichose—discal and r5 cells entirely microtrichose. g g p Hind tibia lacking ventrodistal spur. Wing: 4.0×1.6 mm. Costal vein moderately developed around most of wing margin, weakening along anal cell and absent from alula. Membrane extensively microtrichose—discal and r5 cells entirely microtrichose. Abdomen: Dark red-brown with narrow brown-orange posterior margins, fine pale white setose. T2 dark red-brown, apruinose except for narrow weakly silver pruinose posterior margins laterally. g y _ genitalia (Figs 11, 12): Epandrium in lateral view significantly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without median projections distally and with a row of about 7 mediodistally arranged macrosetae; mediodistal projection long, well- developed and with slightly upturned sclerotised distal end. Gonostylus stout with 180 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 relatively straight and broadly rounded apex. Aedeagal prongs small, more or less straight and with small trifurcate tip. Type specimens: Despite an extensive search, I have not been able to trace the where- abouts of the type material. Loew’s (1858) short description, in Latin, was based on ‘_ & ^’ from ‘Caffraria (Wahlb.)’. As no holotype was designated his specimens must be considered syntypes. There is only one species from southern Africa that answers to the description, so I am confident that the material here assigned to this taxon has been correctly allocated, and that there is little need for a neotype to be designated. Type locality designation: Loew’s material, collected by Wahlberg, came from ‘Caffraria’, a term used to cover much of the eastern part of present day Southern Africa. As Wahlberg passed through the KwaZulu-Natal midlands, I hereby designate the Mhlopeni Nat. Res., SE of Muden, as type locality as a good series has been collected there. Specimens studied: LESOTHO: 5_ 3^Mamathes [29°08'S:27°51'E], 31.xii.1947 (1^), 4.i.1948 (1_), 9.i.1949 (1_), 9.xii.1949 (1_), 8.i.1950 (1_ 1^) 1.i.1952 (1_ 1^), Jacot-Guillarmod (AMGS); 1^Mahlatsa [29°13'S:28°00'E], 30.xii.1951, Jacot-Guillarmod (AMGS). MOZAMBIQUE: 1_ Maqudé [?Magude, 25°02'S:32°40'E], 29.ii.1964, Moore (NMNH); 3_ 1^Maoamba [25°36'S:32°15'E], 9–12.iii.1964, Moore (NMNH). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Holotype: IVORY COAST: _ ‘Côte D'Ivoire: 28 km / W Bouaflé. Maraoué / Nat Park 19.iv.1989 / 06°59'N:: 05°54'W / JGH Londt. Woodland / and forest margins’ (NMSA). Paratypes: IVORY COAST: 1_ 3^same data as holotype; 1_ 1^‘Côte D'Ivoire: Comoé / Nat. Park. ca. 7 km NW / Gansé. 17.iv.1989 / 08°39'N:: 03°56'W / Viewpoint 2 J Londt / riverine forest area’; 3_ ‘Côte D'Ivoire: Comoé / Nat. Park. nr where / Lolo riv meets Comoé / 08°44'N:: 03°50'W / Viewpoint 4 J Londt / 17.iv.1989 riverside’. NIGERIA: 1^‘N. Nigeria / Zaria, / Samaru. [11°10'N:07°37'E] / 18.vi.1968’, ‘J. C. Deeming / m.v. trap.’ (BMNH). LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 181 called the ‘cribrata species group’ which consists of crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata and indica. These species are superficially similar, but can be separated on characters of the male genitalia. T. cribrata appears to be a fairly distinctive species within the group. Trichardis eburacta sp. n. Figs 13, 14 Etymology: From Latin ebur (ivory) and acta (shore). Refers to the country of Ivory Coast, where most of the type specimens were collected. Description (based on holotype in excellent condition): Head: Dark red-brown to black, fine silver pruinose except for much of face and frons. Antenna dark red-brown to black, black setose; postpedicel not markedly clavate (L:D=3.7:1). Mystax white with black macrosetae along epistomal margin. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown to black. Thorax: Dark red-brown to black, silver pruinose except for some shiny apruinose areas. Postpronotum medially silver pruinose, laterally apruinose; mesonotum largely apruinose except for lateral and posterior margins. Scutellum apruinose except for narrow anterior margin. Anepisternum with pale yellow posterior macroseta, dorsally and posteriorly pruinose, anteroventrally apruinose. Proepimeron pruinose except for posterior margin, katepisternum pruinose except for anterior margin, anepisternum entirely pruinose. Legs: Dark red-brown to black, pulvilli and empodium of similar length. Hind femur dark red-brown to black, length:height ratio 3.6:1, ventral tubercles well-developed. Hind tibia lacking ventrodistal spur. Wing: 4.3×1.7 mm. Costal vein well-developed and extending along much of wing margin, weakly along anal cell, absent from alula. Membrane extensively microtrichose—discal and r5 cells entirely microtrichose. Abdomen: Dark red-brown to black anteriorly becoming progressively red-brown posteriorly. T2 dark red-brown, apruinose except for silver pruinose posterior margins laterally. _ genitalia (Figs 13, 14): Epandrium in lateral view significantly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed, lower valve long. Hypandrium greatly reduced and simple. Gonocoxite in ventral view with sharp median projection dorso- distally and without distally arranged macrosetae; mediodistal projection moderately well developed with fairly straight, broadly rounded distal end. Gonostylus fairly stout, jutting out beyond medial process of gonocoxite, with slightly upturned distal end. Aedeagal base with a short finger-like projection laterally; prongs more or less straight, stout, with small trifurcate tip. Holotype: IVORY COAST: _ ‘Côte D'Ivoire: 28 km / W Bouaflé. Maraoué / Nat Park 19.iv.1989 / 06°59'N:: 05°54'W / JGH Londt. Woodland / and forest margins’ (NMSA). Paratypes: IVORY COAST: 1_ 3^same data as holotype; 1_ 1^‘Côte D'Ivoire: Comoé / Nat. Park. ca. 7 km NW / Gansé. 17.iv.1989 / 08°39'N:: 03°56'W / Viewpoint 2 J Londt / riverine forest area’; 3_ ‘Côte D'Ivoire: Comoé / Nat. Park. Trichardis crassipala sp. n. Figs 9, 10 While little information is available concerning habitat, labels suggest that the species is found mainly in Acacia savannah and woodland. Similar species: Oldroyd (1970) compared katangaensis with cribrata, but these species are in fact quite different in many respects. T. cribrata is a member of what is here Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Trichardis effrena sp. n. Etymology: From Latin effrena (unrestrained). Refers to the absence of the costal vein along the posterior margin of the wing. Description (based on holotype in excellent condition): Head: Brown-orange, entirely fine silver pruinose. Antenna brown-orange except for distal part of postpedicel and style which are red-brown, setae orange; postpedicel clavate (L:D=2.4:1). Mystax uniformly shiny orange. Ocellar tubercle with 4 macrosetae. Proboscis proximally orange-brown distally dark red-brown, palpi orange-brown. Thorax: Brown-orange with some red-brown areas, extensively fine silver pruinose. Postpronotum medially pruinose, laterally apruinose; mesonotum brown-orange with red-brown dorsal stripe and laterally situated broad bands, apruinose except for narrow lateral and posterior margins. Scutellum entirely apruinose. Anepisternum with orange posterior macroseta, extensively pruinose except for small anteroventral area. Pro- epimeron pruinose; katepisternum red-brown pruinose posteriorly, apruinose anteriorly; anepisternum entirely pruinose. Legs: Brown-orange (femora, tarsomere 5 and hind tibiae darker), pulvilli and empodium of similar length. Hind femur orange-brown, length:height ratio 3.4:1, ventral tubercles poorly developed. Hind tibia lacking ventro- distal spur. Wing: 4.6×1.8 mm. Costal vein strongly developed only as far as wing tip, very weak or absent along entire posterior margin of wing. Membrane not extensively microtrichose—discal and r5 cell almost entirely lacking microtrichiae. Abdomen: Brown-orange, macrosetae orange, fine setulae pale yellow. T2 brown-orange, apruinose except for strong silver pruinose spot posterolaterally. p p g p p p y _ genitalia (Figs 15, 16): Epandrium in lateral view longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without median projections distally and with a distal row of about 7 macrosetae; mediodistal projection stout, with strongly upturned and scerotised distal end. Gonostylus fairly stout, with broadly rounded, fairly straight distal end. Aedeagal prongs small, more or less straight and with a small terminal end. Holotype: SOUTH AFRICA: _ ‘South Africa: N Cape / Witsand Nature Reserve / 28°33.975'S:022°29.279'E / 1150 m J Londt & T Dikow / 31.i.2004 Acacia mixed / woodland. Reception area’ (NMSA). Paratypes: NAMIBIA: 1^‘Brit. S. W. – Africa / Kalahari / L. Schultze S.’, ‘Hoplistomere / cribrata / Lw / Kalahari / ı 968. a. / Det Dr. F. Hermann [sideways]’ (ZMHB). Paratypes: NAMIBIA: 1^‘Brit. S. W. – Africa / Kalahari / L. Schultze S.’, ‘Hoplistomere / cribrata / Lw / Kalahari / ı 968. a. / Det Dr. F. Hermann [sideways]’ (ZMHB). SOUTH AFRICA: 2_ 2^‘S:Africa: NW Province / Molopo Game Reserve / Phiri Camp area / 25°46'43''S:22°55'53''E / 990 m 14.iii.2003 J Londt / Acacia Erogrostis savannah’; 1^‘S Africa: N Cape #15 / 14 km S of Hotazel / 27 19'S:22 54'E 1050 m / Trichardis eburacta sp. n. nr where / Lolo riv meets Comoé / 08°44'N:: 03°50'W / Viewpoint 4 J Londt / 17.iv.1989 riverside’. NIGERIA: 1^‘N. Nigeria / Zaria, / Samaru. [11°10'N:07°37'E] / 18.vi.1968’, ‘J. C. Deeming / m.v. trap.’ (BMNH). Distribution and biology: The species has only been found in West Africa. Adults fly during the summer months of April and June (Table 1). Little information is available Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 182 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 concerning habitat, however, I collected specimens in woodland and open areas adjacent to forests. Similar species: T. eburacta is a member of what is here called the ‘cribrata species group’ which consists of crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata, and indica. These species are superficially similar, but can be separated on characters of the male genitalia. T. eburacta appears to be a fairly distinctive species, however it is most similar to crassipala and similis in that the hypandrium is better developed than in other species and the form of the gonocoxites is very similar. Trichardis effrena sp. n. Figs 4, 15, 16, 59 Trichardis effrena sp. n. SOUTH AFRICA: 2_ 2^‘S:Africa: NW Province / Molopo Game Reserve / Phiri Camp area / 25°46'43''S:22°55'53''E / 990 m 14.iii.2003 J Londt / Acacia Erogrostis savannah’; 1^‘S Africa: N Cape #15 / 14 km S of Hotazel / 27 19'S:22 54'E 1050 m / Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 183 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Date: 14.iii.1991 / Londt & Whittington / Ga-Mogara River bed’; 1^‘South Africa: N Cape / Vaalbos National Park / Riverside Picnic site 1055 m / 28°27.470'S 024°19.994'E / 28–29.i.2004 JGH Londt & / T Dikow Acacia savannah’; 1_ ‘S Africa: N Cape / Witsand Farm, 28°32'S/ 22°30'E. 2–4.ii.1979 / B/ Lamoral, I Bampton / J. Barnley. Malaise tr’; 4_ 4^‘South Africa: N Cape / Witsand Nature Reserve / 28°33.615'S 022°29.105'E / 1160 m J Londt & T Dikow / 31.i.– 1.ii.2004 Acacia / savannah & white dune area’; 2_ 1^‘South Africa: N Cape / Witsand Nature Reserve / 28°33.673'S 022°29.656'E / 1200 m J Londt & T Dikow / 30.i.– 1.ii.2004 Acacia / savannah. Red sandy ridge’; 6_ 1^same data as holotype. Other material examined: I have seen the following specimen, identified as testacea by Engel, which appears to belong to effrena. Because the locality is so far removed from the Northern Cape records, I refrain from including the specimen in the type series. ZIMBABWE: 1_ ‘Victoria Falls [17°55'S:25°51'E], / 4.i.1920 / Rhodesia / Museum’, Pres. by / Imp. Inst. Ent. / Brit. Mus. / 1930-298.’, ‘Trichardis testacea / Merh. / Dr. E. O. Engel det.’ (BMNH). Distribution and biology: The species is a southern African endemic, being found in the Northern Cape Province of South Africa and in western Zimbabwe (Fig. 59). While I hesitate to give type status to the single Zimbabwean specimen because of its isolated position relative to the other records, I am fairly confident that the specimen is correctly labelled and identified. Botswana is generally poorly sampled and so this kind of appa- rently disjuct distributional pattern should not cause undue concern. Other asilid species have been shown to have a similar distributional pattern. For example Londt (2004) demonstrated that Laphystotes albicans (Engel, 1932) is similarly distributed. Adults of the new species are active during summer and have been collected between January and March (Table 1). This species is associated with open Acacia savannah and mixed woodland. All the specimens captured at Witsand Nat. Res. were found resting on sandy pathways. Similar species: Although sharing a number of characters with glabra and mellina, effrena is a distinctive species in that it displays a remarkable reduction in wing venation and has a distinctive male genital form. Trichardis glabra sp. n. Etymology: From Latin glabra (hairless, bald, smooth). Refers to the extensively aprui- nose thoracic pleura. Description (based on holotype in excellent condition): Head: Dark red-brown, extensively silver pruinose, but weakly on central face and ocellar tubercle, setae black, orange and white. Antennae yellow-brown except distal end of postpedicel and scape which are dark red-brown; scape with two macrosetae ventrally (1 black, 1 orange), fine setulae white and black; pedicel entirely black setose; postpedicel not markedly clavate (L:D=3.1:1), with few black setulae dorsally. Mystax entirely white. Ocellar tubercle with 2 black macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown, largely apruinose with silver pruinose parts, fine setae whitish, macrosetae brown-yellow. Postpronotum largely apruinose except for narrow medial part, mesonotum largely apruinose except for margins, macrosetae orange, setulae shiny white. Scutellum dark red-brown, entirely apruinose. Anepisternum with slender orange posterior macroseta. Pleura entirely apruinose except for the following small sections— anterior part of proepimeron, dorsal part of anepisternum, ventral part of metepisternum. Legs: Dark red-brown, pulvilli and empodium of similar length. Hind femur uniformly dark red-brown, length:height ratio 3.7:1, ventral tubercles hardly evident, major setae Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 184 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 pale yellowish. Hind tibia lacking ventrodistal spur. Wing: 4.2×1.5 mm. Costal vein extends around most of wing margin, weakly along anal cell, absent from alula. Mem- brane not extensively microtrichose—discal cell largely lacking microtrichiae (a few centrally), cell r5 with microtrichiae limited mainly to distal half. Abdomen: Terga and hypopygium dark red-brown, apruinose, setae transparent whitish. T2 dark red-brown, apruinose. , p _ genitalia (Figs 17, 18): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view with large broadly-rounded dorsomedial projection equipped with moderately developed setae; mediodistal projection sinuous at base with long slender slightly curved distal end. Gonostylus fairly broad basally with slender down- curved distal end. Aedeagal prongs more or less straight and with small terminal tubules. Holotype: GAMBIA: _ ‘Bansang [13°26'N:14°39'W], Gambia / 11.v.77 Malaise in scrub / beside river W. F. Snow Collection / pres. W. F. Snow, 1996 / OUM 02-1996’ (OXUM). Paratypes (all OXUM): GAMBIA: 1^same data as holotype; 1_ ‘Bansang, Gambia / 10.v.77 Malaise in / scrub beside river’, ‘W. F. Snow Collection / pres. W. F. Trichardis glabra sp. n. Snow, 1996 / OUM 02-1996’; 1_ same labels but ‘9.v.77’; 1^‘Bansang, Gambia / 4.iv.75 Scrub along / river bank’, ‘W. F. Snow Collection / pres. W. F. Snow, 1996 / OUM 02-1996’. Distribution and biology: Known only from the type locality in Gambia, specimens being collected in April and May (Table 1). All specimens were collected in scrub along a river bank. No other biological information is available. Distribution and biology: Known only from the type locality in Gambia, specimens being collected in April and May (Table 1). All specimens were collected in scrub along a river bank. No other biological information is available. Similar species: T. glabra is most similar to mellina, and they key out together. The male genitalia, especially the form of the gonocoxites are particularly diagnostic in this pair. T. effrena shares some characteristics with these species, but is otherwise distinctive. Trichardis grisescens Engel, 1924 Figs 19, 20 Trichardis grisescens Hermann, 1920: 178. Nomen nudum. Trichardis grisescens: Engel 1924: 108; Hull 1962: 97; Oldroyd 1980: 356 (catalogue). Trichardis grisescens Engel, 1924 Figs 19, 20 Trichardis grisescens Hermann, 1920: 178. Nomen nudum. Trichardis grisescens: Engel 1924: 108; Hull 1962: 97; Oldroyd 1980: 356 (catalogue). Trichardis grisescens Engel, 1924 Figs 19, 20 Trichardis grisescens Hermann, 1920: 178. Nomen nudum. Trichardis grisescens: Engel 1924: 108; Hull 1962: 97; Oldroyd 1980: 356 (catalogue). Trichardis grisescens Hermann, 1920: 178. Nomen nudum. Trichardis grisescens: Engel 1924: 108; Hull 1962: 97; Oldro Redescription (based on holotype in excellent condition): Head: Orange anteriorly, red-brown posteriorly; entirely silvery pruinose. Antenna brown-orange except for red-brown distal part of postpedicel and style, pale yellow setose; postpedicel clavate (L:D=2.3:1). Mystax uniformly yellow-white. Ocellar tu- bercle with 2 macrosetae. Proboscis and palpi red-brown. Thorax: Orange-brown with some darker red-brown parts, extensively silver pruinose, macrosetae pale yellow, setulae shiny pale yellow. Postpronotum entirely pruinose, mesonotum with red-brown dorsal stripe and lateral broad bands, extensively pruinose except for darker red-brown areas. Scutellum pruinose (except for narrow posterior margin). Anepisternum with pale yellow posterior macroseta, dorsally pruinose, ventrally apruinose. Proepimeron pruinose anteriorly, apruinose posteriorly; katepisternum pruinose posteriorly, apruinose anteriorly; anepisternum pruinose posteroventrally, apruinose anterodorsally. Legs: Orange-brown (but femora, tarsomere 5 and hind tibiae darker), pulvilli and empodium of similar length. Hind femur red-brown, length:height ratio 3.6:1, ventral tubercles poorly developed. Hind tibia lacking ventrodistal spur. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 185 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Wing: 5.3×1.9 mm. Costal vein extends around most of wing margin, weak along anal cell, absent from alula. Membrane extensively lacking microtrichiae—discal and r5 cells entirely lacking microtrichiae. Wing: 5.3×1.9 mm. Costal vein extends around most of wing margin, weak along anal cell, absent from alula. Membrane extensively lacking microtrichiae—discal and r5 cells entirely lacking microtrichiae. Abdomen: Red-brown, apruinose, macrosetae pale yellow, setulae shiny white. T2 red- brown, apruinose. _ genitalia (Figs 19, 20): Epandrium in lateral view longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium reduced, with somewhat pointed distal end and simple structure. Gonocoxite in ventral view without median projections and distally with a single short, stout macroseta; mediodistal projection slender with slightly upturned distal end. Gonostylus long, slender with straight distal end. Aedeagal prongs slightly sinuous, with small trifurcate tip. Holotype (examined): GAMBIA: _ ‘Essan [?Essau, 13°29'N:16°32'W] / Gambia. / J. J. Simpson. / 25.iv.1910’, ‘Sammlung / F. Hermann’, ‘Type von / Trichardis / grisescens H.i.l. / Engel’ [orange], ‘Ost-Africa / Trichardis / grisescens / Type Hrm’ [pink] (ZSMC). Note: Although Engel (1924) attributed the species to ‘Herm. In litt.’, and the specimen is labelled ‘Type Hrm’, this action has no validity and Engel himself must be credited with authorship. Trichardis grisescens Engel, 1924 Figs 19, 20 Trichardis grisescens Hermann, 1920: 178. Nomen nudum. Trichardis grisescens: Engel 1924: 108; Hull 1962: 97; Oldroyd 1980: 356 (catalogue). Other material examined: ETHIOPIA: 1^Mério Bourié Bord de la Riv Omo [04°31'N:35°59'E], 600 m, ii.1932–33 [?], Arambourg, Chappuis & Jeannel (MNHN). GAMBIA: 1_ Outside Abuko Nat. Res. at Waterworks [13°24'N:16°39'W], at light 19.00–20.00, Loc. No. 6. UTM28pk214812, 26.ii.1977, Lund Univ. Syst. Dept. Sweden (MZLU); 1^[has a holotype label placed by J.E. Chainey 1984, but is not a type], Essan [?Essau], 25.iv.1919, Simpson (BMNH); 2^Karantaba Tenda [13°33'N:14°34'W], 23.iii.[19]75, Nth Bank on stony river shore, W.F. Snow (OXUM); 1^[has a ‘typus’ label, but is not a type], Jalokunda [Jalo Kunda, 13°47'N:15°00'W], 17.iii.1911, Simpson (BMNH); 1^Gambia, 16.iii.1911, Simpson (BMNH). KENYA: 1^Archers Post [00°39'N:37°41'E], 15.i.1973, Bampton; 1^Nairobi National Park [01°16'S: 36°46'E], 16.xi.1969, Irwin & Ross (CASC); 2^Kiboko [02°11'S:37°43'E], 24.ii.1968, Hussey (BMNH); 1_ Kiboko, 28.ii.1968, Hussey (BMNH). SENEGAL: 1^Camon [?Gamon, 13°20'N:12°55'W] Oriental Re[gion]., 14.v.1966, Harvey (NMNH). Distribution and biology: A widespread species having been collected in both West Africa (Senegal, Gambia) and East Africa (Ethiopia, Kenya) and both north and south of the equator. Adults fly between November and May (no records for December) (Table 1). Label data do not provide insights into the habitat requirements of the species. Although Engel and Cuthbertson (1939) records the following for grisescens—‘In S. Rhodesia [Zimbabwe] this species is known from the Nyamandhlovu district, Matabeleland, and Urungwe, Lomagundi district. At Kariba Gorge, Zambezi River, it is found on leaf- strewn ground in September. The prey consists of leaf-hoppers and small Hymenoptera (teste W.L. Williams). Rhodesian specimens (males) are much larger than the types which came from Gambia.’—the accepted distribution indicates that these notes must refer to another species. Similar species: T. grisescens has an entirely pruinose postpronotal lobe and in this respect can be grouped with apicalis, ornata, picta, terminalis, testacea, turneri, and zinidi. The species is, however, most similar to terminalis. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Trichardis hesperia sp. n. Figs 21, 22 Etymology: From Latin hesperia (western). Refers to the West African distribution of this species. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 186 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Description (based primarily on holotype in fair condition—antennae broken off beyond pedicel, postmetacoxal area and most of anterior sterna missing presumably due to damage by dermestids—but supplemented by information from paratypes): Head: Dark red-brown to black, silver pruinose except for central part of face and frons. Antenna dark red-brown, black setose; postpedicel (_ paratype) not markedly clavate (L:D=3.6:1). Mystax white with black macrosetae along epistomal margin. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown, silver pruinose except for bare areas, macrosetae orange, setulae pale yellow. Postpronotum medially pruinose, laterally apruinose; mesonotum apruinose except for narrow lateral and posterior margins. Scutellum entirely pruinose. Anepi- sternum with orange posterior macroseta, dorsally pruinose, ventrally apruinose. Pro- epimeron pruinose; katepisternum pruinose posteriorly, apruinose anteriorly; anepi- sternum pruinose anteriorly, apruinose posteriorly. Legs: Dark red-brown, pulvilli and empodium of similar length. Hind femur dark red-brown, length:height ratio 3.6:1, ventral tubercles poorly developed. Hind tibia lacking ventrodistal spur. Wing: 4.0×1.4 mm. Costal vein extends around most of wing margin, weak along anal cell, absent from margin of alula. Membrane extensively microtrichose—discal cell microtrichose, but weakly so anteroproximally, cell r5 entirely microtrichose. Abdomen: Dark red-brown proximally rapidly becoming brown-orange more distally, macrosetae pale yellow, setulae pale white. T2 red-brown, apruinose except for narrow posterolateral margins, which have some silver pruinescence. Abdomen: Dark red-brown proximally rapidly becoming brown-orange more distally, macrosetae pale yellow, setulae pale white. T2 red-brown, apruinose except for narrow posterolateral margins, which have some silver pruinescence. _ genitalia (Figs 21, 22): Epandrium in lateral view as long as basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger long, strongly dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view with projections distally and with a few laterally positioned macrosetae; mediodistal projection stout, fairly straight. Gonostylus slender, slightly sinuous with slightly down turned tip. Aedeagal prongs more or less straight, fairly stout, with small trifurcate tip. Holotype: SENEGAL: _ ‘Museum Paris / Sénegal / Kayes [14°25'N:11°30'W] / F. De Zeltner 1905’ (MNHN). Paratypes: GAMBIA: 1^‘Keneba [13°19'44"N:16°00'54"W], Gambia / 14.viii.75 Woodland’, ‘W. F. Snow Collection / pres. W. F. Snow, 1996 / OUM 02-1996’ (OXUM); 2^‘Keneba, Gambia / 11.viii.74 Tambana / Bare ground’, ‘W. F. Trichardis hesperia sp. n. Figs 21, 22 Snow Collection / pres. W. F. Snow, 1996 / OUM 02-1996’ (OXUM); 1^‘Keneba, Gambia / 30.v.74 Tambana / dry stream bed’, ‘W. F. Snow Collection / pres. W. F. Snow, 1996 / OUM 02- 1996’ (OXUM). SENEGAL: 1_ same data as holotype (MNHN). Distribution and biology: This West African species is known from Gambia and Senegal. Adults have been collected in May and August and so the species is probably active during the northern hemisphere summer. Apart from the fact that specimens have been collected on bare ground, dry river beds and in woodland, no biological information exists. Similar species: A member of what is here called the ‘cribrata species group’ which consists of crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata, and indica. These species are superficially similar, but can be easily separated on characters of the male genitalia. T. hesperia is distinctive. Trichardis katangaensis Oldroyd, 1970 Figs 23, 24 Trichardis katangaensis: Oldroyd 1970: 248–249, fig. 29 (mesopleuron); 1980: 356 (catalogue). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 187 Redescription (based on holotype in good condition, with wings a little crumpled and with damaged hind margins): Redescription (based on holotype in good condition, with wings a little crumpled and with damaged hind margins): Head: Dark red-brown to black, silver pruinose (sparse on lower face), setae longish black, yellow and white. Antenna dark red-brown, black setose; postpedicel not markedly clavate (L:D=3.9:1). Mystax shiny yellowish with black macrosetae along epistomal margin. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown to black, postpronotal and postalar lobes orange-brown, gold- silver and silver pruinose, generally appearing more setose than many other species. Postpronotum medially narrowly pruinose, laterally extensively apruinose; mesonotum apruinose with narrow silver pruinose lateral and posterior margins, macrosetae black, setulae mixed long black and short yellow. Scutellum apruinose. Anepisternum with black posterior macroseta, pruinose except for large anteroventral area. Proepimeron entirely pruinose, katepisternum pruinose except for small central area, anepisternum entirely pruinose. Legs: Dark red-brown except for orange-brown coxae, pulvilli and empodium of similar length. Hind femur dark red-brown, length:height ratio 3.1:1 (i.e. moderately inflated), ventral tubercles well-developed. Hind tibia with well-developed ventrodistal spur. Wing: 6.1×2.2 mm. Costal vein extends along most of wing margin, weakly along anal cell, absent from margin of alula. Trichardis hesperia sp. n. Figs 21, 22 Membrane extensively micro- trichose—discal cell microtrichose but weakly so anteroproximally, cell r5 entirely microtrichose. Abdomen: Dark red-brown, macrosetae pale yellow, setulae longish white. T2 dark red- brown, apruinose except for posterolateral margins. _ genitalia (Figs 23, 24): Epandrium in lateral view longer than basal part of gono- coxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger short, only moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without median projections distally and with about four medially-directed macrosetae at about mid-length; mediodistal projection stout, with upturned forked distal end. Gonostyli stout with converging, pointed distal ends. Aede- agal prongs small, more or less straight and with small trifurcate tip. Holotype: DR CONGO: _ ‘Holotypus’ [orange], ‘Musée Du Congo / Lulua [05°56'S:25°47'E]: Kapanga / x–1932 / G. F. Overlaet’, ‘Trichardis / katangaensis Oldr / det. H. Oldroyd, 1965 / Paratype’ [white] (MRAC). Paratypes (all MRAC): DR CONGO: 1^‘Paratypus’ [orange], ‘Musée Du Congo / Elisabethville [11°40'S:27°28'E] / xi–1911 / Miss. Agric.’, ‘Trichardis / katangaensis Oldr / det. H. Oldroyd, 1965 / Holotype’ [white]. TANZANIA: 1^‘Coll. Mus Congo / Tang.: Sunkutu [?], 1140 m. / Km. 95, Rte Pepa–Moliro / H. Bomans xii–1953’, ‘Trichardis / katangaensis Oldr / det. H. Oldroyd, 1965 / Paratype’ [white]. Paratypes (all MRAC): DR CONGO: 1^‘Paratypus’ [orange], ‘Musée Du Congo / Elisabethville [11°40'S:27°28'E] / xi–1911 / Miss. Agric.’, ‘Trichardis / katangaensis Oldr / det. H. Oldroyd, 1965 / Holotype’ [white]. TANZANIA: 1^‘Coll. Mus Congo / Tang.: Sunkutu [?], 1140 m. / Km. 95, Rte Pepa–Moliro / H. Bomans xii–1953’, ‘Trichardis / katangaensis Oldr / det. H. Oldroyd, 1965 / Paratype’ [white]. Note: The two DR Congo types above were incorrectly labelled when received from MRAC. The male from Lulua, clearly designated as holotype, carried the paratype label while the female from Elizabethville was labelled as holotype. These labels have been switched. Note: The two DR Congo types above were incorrectly labelled when received from MRAC. The male from Lulua, clearly designated as holotype, carried the paratype label while the female from Elizabethville was labelled as holotype. These labels have been switched. Distribution and biology: The species is found in Central and East Africa. Adults fly between October and December (Table 1) during the southern hemisphere summer. No biological information is available. Distribution and biology: The species is found in Central and East Africa. Paratypes (all MRAC): DR CONGO: 1^‘Paratypus’ [orange], ‘Musée Du Congo / Elisabethville [11°40'S:27°28'E] / xi–1911 / Miss. Agric.’, ‘Trichardis / katangaensis Oldr / det. H. Oldroyd, 1965 / Holotype’ [white]. TANZANIA: 1^‘Coll. Mus Congo / Tang.: Sunkutu [?], 1140 m. / Km. 95, Rte Pepa–Moliro / H. Bomans xii–1953’, ‘Trichardis / katangaensis Oldr / det. H. Oldroyd, 1965 / Paratype’ [white]. Trichardis lavignei sp. n. Figs 25, 26 Figs 25, 26 Etymology: Named for Dr Robert Lavigne whose collecting activities in Somalia have added significantly to our understanding of Asilidae from this part of Africa. Description (based on holotype in good condition; the genitalia, macerated and stored in a capsule some years before this study, are intact, but somewhat squashed and inflexible, making it difficult to illustrate the structures in the standard manner used in this paper): Head: Dark red-brown to black, gold-silver pruinose except for area around antennal bases and ocellar tubercle, setae black, yellow and white. Antenna dark red-brown, mainly yellow setose (a few black); postpedicel elongate spindle-shaped (L:D=4.2:1). Mystax black, confined to lower half of face (which in profile has a slightly concave area centrally). Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown, postpronotal and postalar lobes and anterior part of scutellum orange-brown, silver pruinose except for bare areas, setae yellowish. Postpronotum apruinose except for a tiny area medially, mesonotum extensively apruinose except for narrow lateral and posterior margins, macrosetae yellow, setulae yellow and white. Scutellum entirely pruinose. Anepisternum with pale yellow posterior macroseta, dorsally pruinose, ventrally apruinose. Proepimeron pruinose; katepisternum pruinose posteriorly, apruinose anteriorly; anepisternum extensively apruinose. Legs: Dark red-brown, femora and tibiae paler proximally, pulvilli and empodium of similar length. Hind femur dark red-brown with paler proximal end, length:height ratio 3.2:1, ventral tubercles well-developed. Hind tibia with ventrodistal spur. Wing: 4.0×1.6 mm. Costal vein extends along most of wing margin, weakly along anal cell, absent from alula. Membrane extensively microtrichose—discal cell microtrichose but weakly so at proximal end, cell r5 entirely microtrichose. Abdomen: Dark red-brown proximally becoming orange-brown distally, macrosetae pale yellow, setulae white. T2 dark red-brown, entirely apruinose, tufts of white setulae posterolaterally. _ genitalia (Figs 25, 26): Epandrium significantly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view with slender projections distally and without distally arranged macrosetae; mediodistal projection unusually slender and not medially situated as in most other species. Gonostylus slender, fairly straight, with slightly hooked tip. Aedeagal prongs more or less straight, with small sinuous terminal filamentous tubules. Holotype: SOMALIA: _ ‘Somalia / Mogadishu [02°02'N:45°21'E], / v-10-86 / R. Lavigne’ (NMSA). Paratype: 1^‘Somalia / Mogadishu / vi-7-86 / R. Lavigne’ (NMSA). Distribution and biology: The species is recorded only from the type locality. Trichardis hesperia sp. n. Figs 21, 22 Adults fly between October and December (Table 1) during the southern hemisphere summer. No biological information is available. Similar species: Oldroyd (1970) compared the species to cribrata and illustrated the mesopleura of both species. Why he did this is not understood as these species do not have a great deal in common. T. katangaensis can be linked with lavignei in that both species possess hind-tibial spurs. However, both are otherwise distinctive species. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 188 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Trichardis leucocoma: Engel 1924: 107; Efflatoun 1937: 208–212, figs 150, 151 (head), 152 (wing), 153 (hind leg), 154, 155 (_ gen.), 156 (^gen.), pl. IV, fig. 34 (whole fly); Theodor 1980: 256–258, figs 433 (head), 433a (antennal postpedicel), 434 (aedeagus), 435 (epandrium and proctiger), 436 (hypandrium), 437 (gonocoxite and dististylus), 438 (spermatheca); Oldroyd 1980: 356 (catalogue). Strobilothrix albipila Becker, 1907: 43–44. Trichardis leucocoma: Engel 1924: 107; Efflatoun 1937: 208–212, figs 150, 151 (head), 152 (wing), 153 (hind leg), 154, 155 (_ gen.), 156 (^gen.), pl. IV, fig. 34 (whole fly); Theodor 1980: 256–258, figs 433 (head), 433a (antennal postpedicel), 434 (aedeagus), 435 (epandrium and proctiger), 436 (hypandrium), 437 (gonocoxite and dististylus), 438 (spermatheca); Oldroyd 1980: 356 (catalogue). Strobilothrix albipila Becker 1907: 43–44 p , Trichardis leucocomus: Hull 1962: 97; Lehr 1988: 212 (incorrect subsequent spelling). Triclis rufescens Austen, 1914: 267. Syn. n. Redescription (based on lectotype in excellent condition): Redescription (based on lectotype in excellent condition): Head: Probably orange but colour masked by strong silver pruinescence, setae white. Antenna orange except for dark red-brown style, white setose; postpedicel not markedly clavate (L:D=3.6:1). Mystax white. Ocellar tubercle with 4 macrosetae. Proboscis orange with orange-brown distal half, palpi orange. Thorax: Probably orange and red-brown but colour largely masked by strong silver pruinescence, setae white. Postpronotum entirely pruinose, mesonotum dark red-brown centrally, orange laterally, entirely silver pruinose, setae white. Scutellum pruinose except for hind margin. Anepisternum lacking posterior macroseta, entirely pruinose (weakish anteroventrally). Proepimeron, katepisternum and anepimeron pruinose. Legs: Yellowish with hind tibiae and dorsal parts of all femora red-brown, pulvilli clearly much shorter than empodium. Hind femur orange-brown with ventral parts yellow, length:height ratio 4.0:1 (i.e. slender), ventral tubercles not evident. Hind tibia lacking ventrodistal spur. Wing: 5.6×2.0 mm. Costal vein extends along most of wing margin, weakly along anal cell, absent from alula. Membrane entirely without microtrichiae. Abdomen: Brown-orange proximally becoming red-brown, hypopygium orange, silver pruinose except for small areas anterolaterally, white setose (setulae longish). T2 orange, pruinose, pruinose except for small anterolateral areas. _ genitalia (Figs 27, 28): Epandrium in lateral view longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without median projections and with about 6 well-developed medially directed macrosetae; mediodistal projection stout with slightly upturned distal end. Gonostylus stout with upturned straight distal end. Paralectotypes: YEMEN: 1^‘S. W. Arabia, / 6 m. N. of Aden / Shaik Othman. / Capt. Apr. 1. 95 / & pres. 1899 by / J. W. Yerbury.’, ‘Sammlung / F. Hermann’, ‘Type. / v.d.Wulp, / Trans. Ent. Soc., / 1899, page 90– 1.’ [white with pink edge], ‘[red square]’, ‘Trichardis / leucocoma / ^v. d. W.’, ‘Arabia / Hoplistomera / leucocoma. / Type. V. d. W.’ (ZSMC); 1^‘Type / v.d.Wulp, / Trans. Ent. Soc. / 1899, page 90–1.’ [white with red edge], ‘S. W. Arabia, / 6 m. N. of Aden, / Shaik Othman. / Capt. Feb. 24. 95 / & pres. 1899 by / J.W. Yerbury.’ ‘Hoplistomera sp nov? / nearest cribrata Loew / Dipt sud afr p 121 but / distinct’ [faint pencil handwriting] ‘1899 / 7717’, ‘Hoplistomera / leucocoma n.s.’, ‘Type Dip: 2141/4 / Hoplistomera / leucocoma / v der Wulp / Hope Dept Oxford’ (OXUM); 1_ ‘Type / v.d.Wulp, / Trans. Ent. Soc. / 1899, page 90–1.’ [white with red edge], ‘S. W. Arabia, / 6 m. N. of Aden, / Shaik Othman. / Capt. Feb. 17.95 / & pres. 1899 by / J.W. Yerbury.’, ‘1899 / 7715’, ‘Type Dip: 2142/4 / Hoplistomera / leucocoma / v der Wulp / Hope Dept Oxford’ (OXUM); 1^‘Type / v.d.Wulp, / Trans. Ent. Soc. / 1899, page 90–1.’ [white with red edge], ‘S. W. Lectotype: YEMEN: 1_ ‘Type / v.d.Wulp, / Trans. Ent. Soc. / 1899, page 90–1.’ [white with red edge], ‘S. W. Arabia, / 6 m. N. of Aden [12°50'N:45°00'E], / Shaik Othman. / Capt. Mar. 4. 95 / & pres. 1899 by / J.W. Yerbury.’, ‘1899 / 7716’, ‘Type Dip: 2143/4 / Hoplistomera / leucocoma / v der Wulp / Hope Dept Oxford’ (OXUM). Trichardis lavignei sp. n. Figs 25, 26 Adults have been recorded in May and June (Table 1). No biological information exists. Similar species: T. lavignei can be linked with katangaensis in that both species possess hind tibial spurs. However, both are otherwise distinctive species. Trichardis leucocoma (Wulp, 1899) Figs 27, 28 Hoplistomera leucocoma: Wulp 1899: 90–91. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 189 Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Mastung [29°44'N:66°56'E], 13.vi.1963, Popov (BMNH); 1_ 1? Khuzelar [?], 17.vi.1963, Popov (BMNH); 1_ 1? Guelta [?], 4.vi.1963, Popov (BMNH). MONGOLIA: 1^S.W. Mongolia, 24.vii, Söderbom, Sven Hedins Exp. Str. Asien (NHRS). PALESTINE: 1_ Rubin [31°56'N:34°42'E], 28.v.1921, Aharoni (BMNH). SAUDI ARABIA: 1_ 1^Wadi Qanuna [?Wadi Qanahu, 13°12'N:43°46'E], 30.iii.1948, Uvarov (BMNH); 1_ 1? nr Hais [13°56'N:43°29'E], 7.x.1962 (BMNH); 1_ 1^Zeidiya [15°20'N:43°01'E], 28.ix.1962 (BMNH); 1? Mahfad [?Mahfa, 15°53'N:43°15'E], 20.x.1962 (BMNH); 2_ nr Bisha [?Bitah, 16°02'N:42°59'E], 25.vi.1962 (BMNH); 1^Nejran [?], 17.vii.1962 (BMNH); 1_ [locality illegible], 20.ix.1963 (BMNH). UNKNOWN: 1^Darré Zohrab [?], Aulimesk [?], 29.v.1962 (BMNH); 1_ Lehaj [?Lahaj – may be from Yemen], 11.v.1895, Nurse (BMNH). New synonymy: I have studied the unique holotype of T. rufescens Austen, 1914 and believe it to be entirely conspecific with T. leucocoma. The species name is therefore a synonym of T. leucocoma. The genus Triclis Loew, 1851 (type species Triclis olivaceus Loew, 1851) is Palaearctic with three catalogued species (Lehr 1988) including rufescens. Although I do not claim to be familiar with the species of Triclis, in attempting to check the classification of rufescens it became clear that it is somewhat unlike other species included in Triclis. Hull (1962) uses the extent of abdominal setation to effectively isolate Triclis from Trichardis in his key. In keying rufescens the species does not agree with the condition described for Triclis. Theodor (1980) draws attention to the condition of the antennal style in keying Triclis, and rufescens does not possess a Triclis-like style. Indeed when comparing the types of rufescens and leucocoma directly, there is little doubt that these are conspecific taxa. Distribution and biology: This is primarily a Palaearctic species. Lehr (1988) summarised the distribution of the species thus—‘USSR: KZ [Kazakhstan]; Asia: Arabic States, Israel, Iran, ?Mongolia; North Africa: Morocco, Algeria, Egypt; Afrotropical Region.’ Previously recorded only from one Afrotropical location (Yemen), the new record from Niger suggests that the species may be far more widely distributed within the Afrotropics. My records show that adults have been collected between February and May as well as August and so the species probably flies during the northern hemisphere summer. Little biological information is available. However, Efflatoun (1937: 212), in his report on Egyptian asilids records: ‘T. leucocoma is very common … My records extend from end of March to end of September. LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Aedeagal prongs short, fairly stout, more or less straight, with moderately well-developed tips. Lectotype designation: Wulp (1899: 90–91) based his description on ‘Five specimens (2_ 3^) from Shaik Othman.’ A holotype was not designated and so all the specimens are syntypes. While the ZSMC female is labelled ‘Type’, I here designate one of the OXUM males as lectotype, all the other specimens are paralectotypes. Lectotype: YEMEN: 1_ ‘Type / v.d.Wulp, / Trans. Ent. Soc. / 1899, page 90–1.’ [white with red edge], ‘S. W. Arabia, / 6 m. N. of Aden [12°50'N:45°00'E], / Shaik Othman. / Capt. Mar. 4. 95 / & pres. 1899 by / J.W. Yerbury.’, ‘1899 / 7716’, ‘Type Dip: 2143/4 / Hoplistomera / leucocoma / v der Wulp / Hope Dept Oxford’ (OXUM). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 190 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Arabia, / 6 m. N. of Aden, / Shaik Othman. / Capt. Apr. 1. 95 / & pres. 1899 by / J.W. Yerbury.’, ‘1899 / 7714’, ‘Type Dip: 2144/4 / Hoplistomera / leucocoma / v der Wulp / Hope Dept Oxford’ (OXUM). Arabia, / 6 m. N. of Aden, / Shaik Othman. / Capt. Apr. 1. 95 / & pres. 1899 by / J.W. Yerbury.’, ‘1899 / 7714’, ‘Type Dip: 2144/4 / Hoplistomera / leucocoma / v der Wulp / Hope Dept Oxford’ (OXUM). Other material examined: Afrotropical: NIGER: 1_ Aïr [18°30'N:08°00'E], Tafidet Valley, North East of Agadez, viii.2004, Mamadou (OXUM). YEMEN: 2_ 1^1? Huswah, nr Aden [12°50'N:45°00'E], 14.iv.1895, Nurse (BMNH); 1_ 2^Lodar [13°56'N:45°56'E], 16.v.1967, 800 m, Guichard (BMNH). Afrotropical: NIGER: 1_ Aïr [18°30'N:08°00'E], Tafidet Valley, North East of Agadez, viii.2004, Mamadou (OXUM). YEMEN: 2_ 1^1? Huswah, nr Aden [12°50'N:45°00'E], 14.iv.1895, Nurse (BMNH); 1_ 2^Lodar [13°56'N:45°56'E], 16.v.1967, 800 m, Guichard (BMNH). Palaearctic: ALGERIA: _ (T. rufescens holotype) ‘Triclis / Type / rufescens / Austen’, ‘Algeria: / Biskra [?] / 5.vi.1897. / Rev. A.E. Eaton . 97.268.’, ‘Biskra / 5.vi.97 / _’, ‘Holotype / Triclis / rufescens Austen / det. J.E. Chainey, 1984’ (BMNH). EGYPT: 1^Wadi Husein [26°48'N:33°27'E], 1.v.1919, Adair (ZSMC); 1^Wadi Hof [29°52'N:31°19'E], 8.v.1924, H.C.E. (ZSMC); 1_ Wadi Hof, 9.vi.[19]22, Efflatoun (MCMI); 1_ Um Elek [?], 14.v.[19]26 (BMNH); 1_ W. Kakhla [?], 7.vi.[19]26, Efflatoun (BMNH). LIBYA: 5_ 1^Leptis Magna [32°59'N:14°15'E], 9.vii.1957, Guichard (BMNH). PAKISTAN: Baluchistan: 2_ 3^Turbat [26°00'N:63°06'E], 12.v.1963, Popov (BMNH); 1? Patkin [Patkin Chauki, 29°05'N:65°48'E], 2.vi.1963, Popov (BMNH); 1_ 1? Holotype: MALAWI: _ ‘Malawi 35 km SE of / Monkey Bay on road / to Mangochi 1434Aa / 12.xii.1980 500m / Londt & Stuckenberg / mixed woodland’ (NMSA). Paratypes: MALAWI: 1_ 6^same data as holotype TANZANIA: 2^‘Tanzania: Serengeti / Nat Park Trichardis malawi sp. n. Etymology: Named after Malawi, where the holotype and a number of paratypes were collected. Etymology: Named after Malawi, where the holotype and a number of paratypes were collected. Description (based on holotype in excellent condition): Head: Dark red-brown to black, extensively silver pruinose except for shiny apruinose strip centrally from ocellar tubercle to epistomal margin, setae black and white. Antenna orange-brown, distal part of postpedicel and style dark red-brown, black and white setose; postpedicel elongate spindle-shaped (L:D=4.2:1). Mystax sparse white with black macrosetae along epistomal margin. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown to black with a few small orange-brown areas (postalar lobes, anepisternum and coxae), fairly extensively silver pruinose, pale yellow-white setose. Postpronotum strongly silver pruinose medially, apruinose laterally; mesonotum aprui- nose except for margins, macrosetae pale yellowish, setulae pale white. Scutellum apruinose except for narrow silver pruinose anterior margin. Anepisternum with pale yellowish posterior macroseta, silver pruinose except for large apruinose anteroventral area. Proepimeron, katepisternum and anepisternum entirely pruinose. Legs: Orange- brown with femora slightly darker, pulvilli and empodium of similar length. Hind femur orange-brown, length:height ratio 3.5:1, ventral tubercles poorly developed. Hind tibia lacking ventrodistal spur. Wing: 4.7×1.9 mm. Costal vein extends along most of wing margin, weakly along anal cell, absent from alula. Membrane extensively microtrichose, small areas of some proximally situated cells without microtrichiae; discal and r5 cells entirely microtrichose. Abdomen: Dark red-brown anteriorly becoming brown-orange posteriorly, apruinose except for narrow silver pruinose posterolateral tergal margins, macrosetae pale yellowish, setulae whitish. T2 dark red-brown, apruinose except for hind margins laterally. _ genitalia (Figs 29, 30): Epandrium in lateral view significantly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple in structure. Gonocoxite in ventral view with weakly defined projections distally and lacking macrosetae; mediodistal projection stout with upturned darkly sclerotised distal end. Gonostylus fairly stout with slightly upturned tip. Aedeagal base well- developed, upturned mediodistally; prongs large, upwardly directed and with expanded trumpet-like openings. Holotype: MALAWI: _ ‘Malawi 35 km SE of / Monkey Bay on road / to Mangochi 1434Aa / 12.xii.1980 500m / Londt & Stuckenberg / mixed woodland’ (NMSA). Paratypes: MALAWI: 1_ 6^same data as holotype. TANZANIA: 2^‘Tanzania: Serengeti / Nat. Park. Seronera [02°16'S:34°47'E] / 23-xi-1069 / M.E. Irwin & / E.S. Ross’ (CASC). Paratypes: MALAWI: 1_ 6^same data as holotype. TANZANIA: 2^‘Tanzania: Serengeti / Nat. Park. Seronera [02°16'S:34°47'E] / 23-xi-1069 / M.E. Irwin & / E.S. Ross’ (CASC). ZIMBABWE: 1_ ‘Country Holotype: MALAWI: _ Malawi 35 km SE of / Monkey Bay on road / to Mangochi 1434Aa / 12.xii.1980 500m / Londt & Stuckenberg / mixed woodland’ (NMSA). Paratypes: MALAWI: 1_ 6^same data as holotype. TANZANIA: 2^‘Tanzania: Serengeti / Nat. Park. Seronera [02°16'S:34°47'E] / 23-xi-1069 / M.E. Irwin & / E.S. Ross’ (CASC). ZIMBABWE: 1_ ‘Country LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) The favourite hunting grounds for this Asilid … are the dried stony and sandy beds of Wadies where it sits on sand or on stones. I have never seen it sitting or settling on plants or grasses and I have caught it feeding on Musca lucidula and on two or three species of Tachinids, among which Wolfartia trina Wied.’ Similar species: T. leucocoma is very similar to rueppelii and may be a synonym of that older-named species (see discussion under rueppelii) from Algeria. Together these make Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 191 a distinctive pair not to be confused with any other Afrotropical species. The absence of anepisternal macrosetae sets them apart from all others studied by me. The fact that the species occurs in both the Afrotropical and Palaearctic regions suggests that there may be other similar species in the Palaearctic Region. Trichardis malawi sp. n. ZIMBABWE: 1_ ‘Country Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 192 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Rhodesia / Loc. Lusulu [18°04'S:27°50'E] / Date 22/xi/63 / Coll. G. Davison’ (NMSA); 1^‘Country Rhodesia / Loc. 22/xi/63 / Date Lusulu / Coll. R. J. Phelps’ (NMSA). Rhodesia / Loc. Lusulu [18°04'S:27°50'E] / Date 22/xi/63 / Coll. G. Davison’ (NMSA); 1^‘Country Rhodesia / Loc. 22/xi/63 / Date Lusulu / Coll. R. J. Phelps’ (NMSA). Distribution and biology: The species is recorded from Southern and Eastern Africa. Adults have been collected during the summer months of November and December (Table 1). I collected the Malawian specimens resting on the ground in mixed woodland. Similar species: A member of what is here called the ‘cribrata species group’ which consists of crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata and indica. These species are superficially similar, but can be easily separated on characters of the male genitalia. T. malawi has distinctive male genitalia. LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Paratypes: ERITREA: 3_ 3^same data as holotype; 1_ ‘Trichardis / erythrogaster. Herm / Typus’, ‘Pres by / Imp. Bur. Ent. / Brit Mus. / 1923–58.’, ‘Abyssinia / Nov. 1911 / R. J. Stordy’ (BMNH). Note: The specimens have poorly hand-written labels difficult to decipher. Distribution and biology: The species is known with certainty only from Ghinda in Eritrea. Apart from the fact that adults fly during June, midsummer in the northern hemisphere (Table 1), nothing is known of its biology. Similar species: T. mellina is most similar to glabra and these species key out together. The male genitalia, especially the form of the gonocoxites are particularly diagnostic in this pair. T. effrena shares some characteristics with these species, but is otherwise distinctive. Trichardis nigrescens (Ricardo, 1903) Figs 33, 34 Hoplistomera nigrescens: Ricardo 1903: 362. Trichardis nigrescens: Hull 1962: 97; Oldroyd 1980: 356 (catalogue); Geller-Grimm 2002: 470, pls 3, 17 (entire _). Redescription (based on holotype ^in good condition): Head: Dark red-brown, extensively silver pruinose except for lower face and frons (including ocellar tubercle), black and white setose. Antenna red-brown, black and pale yellow setose; postpedicel elongate spindle-shaped (L:D=5.3:1). Mystax mainly white with a few black macrosetae along epistomal margin. Ocellar tubercle with 4 yellowish macrosetae. Some black occipital macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown, silver pruinose when present, pale whitish setose. Postpronotum dark red-brown with small orange part posteriorly, largely apruinose except for medial part, mesonotum largely apruinose except for lateral and posterior margins, macrosetae black (notopleurals) and whitish, setulae shiny white. Scutellum apruinose except for narrow anterior margin. Anepisternum with slender, weakly developed posterior macroseta, extensively pruinose except for small area anteroventrally. Proepimeron anteriorly pruinose, posteriorly apruinose; katepisternum posteriorly pruinose, anteriorly apruinose. Legs: Femora dark red-brown, other segments orange-brown, pulvilli and empodium of similar length. Hind femur dark red-brown, moderately slender (length to height ratio not measured), ventral tubercles poorly developed. Hind tibia lacking ventro- distal spur. Wing: 5.5×2.1 mm. Costal vein extends along most of wing margin, weakly along anal cell, absent from alula. Membrane not extensively microtrichose—discal cell largely lacking microtrichiae (a few present), cell r5 with microtrichiae in distal half only. Abdomen: Anterior five terga dark red-brown with orange-brown hind margins, posterior terga and hypopygium mustard colour, apruinose except for silver pruinose posterolateral corners, setae whitish. T2 dark red-brown, apruinose except for silver pruinose postero- lateral corner. Trichardis mellina sp. n. Etymology: From Latin mellina (honey coloured). Refers to the orange-brown colour of this species. Description (based on holotype in good condition, with left antenna broken off beyond pedicel and mid leg broken off beyond femur): Head: Brown-orange with dark red-brown occipital area, entirely silver pruinose, white setose. Antenna brown-orange, white setose; postpedicel not markedly clavate (L:D= 3.8:1). Mystax shiny white. Ocellar tubercle with 4 macrosetae. Proboscis brown-orange proximally, red-brown distally; palpi brown-orange. Thorax: Brown-orange, gold-silver pruinose except for apruinose areas, pale whitish yellow setose. Postpronotum extensively pruinose except for narrow lateral strip, mesonotum orange-brown, extensively silver-gold pruinose (weak mediolaterally), pale yellowish setose. Scutellum gold-silver pruinose except for narrow hind margin and central part of disc. Anepisternum with pale yellowish posterior macroseta, pruinose dorsally, apruinose ventrally. Proepimeron pruinose anteriorly, apruinose posteriorly; katepisternum pruinose posteriorly, apruinose anteriorly; anepisternum pruinose pos- teriorly, apruinose anteriorly. Legs: Brown-orange, pulvilli and empodium of similar length. Hind femur brown-orange, length:height ratio 4.2:1 (slender), ventral tubercles absent. Hind tibia lacking ventrodistal spur. Wing: 4.2×1.6 mm. Costal vein extends along most of wing margin, weakly along anal cell, absent from alula. Membrane extensively microtrichose—discal and r5 cells almost entirely microtrichose (weakly proximally and adjacent to veins). Abdomen: Brown-orange, hind margins of terga yellow, entirely apruinose, pale yellowish setose. T2 brown-orange, apruinose. g p _ genitalia (Figs 31, 32): Epandrium in lateral view longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger fairly long, strongly dorsoventrally compressed. Hypandrium greatly reduced and simple. Gono- coxite in ventral view without median projections distally and with about 4 distally arranged weak macrosetae; mediodistal projection stout at base becoming slender towards sclerotised distal end. Gonostyli short, stout, with broadly-rounded converging distal ends. Aedeagal prongs slender, slightly curved, ending as small terminal filamen- tous tubules. Holotype: ERITREA: _ ‘Ghinda [15°26'N:39°07'E] / Mochi / vi-16 [1916]’ (MCMI). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 193 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) _ genitalia: Geller-Grimm (2002: figs 2–4) illustrated a male from Socotra. I here illustrate the genitalia of an already macerated _ from Homhil (Figs 33, 34). While this is probably the specimen illustrated by Geller-Grimm, I believe that my drawings more accurately depict the genital structures and the subtle differences between nigrescens and abdelkuri, the closely similar species from the nearby island of Abd el Kuri. The following is a description of the Homhil _ genitalia based on my illustrations. Epandrium Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 194 in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally com- pressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without median projections distally and with mediodistally arranged macrosetae; mediodistal projection fairly slender with slightly upturned distal end. Gonostylus slender with straight distal end. Aedeagal prongs more or less straight and with small terminal tubules. Note: Although this description is similar to that of the genitalia of abdelkuri, the genital differences in these species can easily be detected by comparing the relevant illustrations. The shape of the gonocoxite in ventral view is particularly diagnostic. Holotype (examined): YEMEN: Socotra I.: ^‘Type’ [circular, red edged], ‘Hoplistomera / nigrescens Ric. / 2^’, ‘Sokotra / Hadibu [12°40'N:53°59'E] Plains. / 11.xii.1898 / W. R. O. Grant. / 1916-75.’ ‘Holotype / Hoplistomera / nigrescens Ricardo / det. J. E. Chainey, 1984’ [has circular, red edged label stuck to top right corner of holotype label] (BMNH). Other material examined: YEMEN: Socotra I.: 1_ Homhil, 12°34'13"N:54°18'32"E, 29–30.x.2000, Pohl (HLMD); 1_ 1^Goeeh, 12°32'25"N:54°10'22"E, 240 m, 23.x.2000, Pohl (HLMD). Other material examined: YEMEN: Socotra I.: 1_ Homhil, 12°34'13"N:54°18'32"E, 29–30.x.2000, Pohl (HLMD); 1_ 1^Goeeh, 12°32'25"N:54°10'22"E, 240 m, 23.x.2000, Pohl (HLMD). Recorded specimens not studied (cited from Geller-Grimm (2002)): YEMEN: Socotra I.: 2^Goeeh, 12°32'25"N:54°10'22"E, 240 m, 23.x.2000, Pohl (NHCY 1^COGG 1^); 1^Firmihin, 12°24'41"N: 54°13'35"E, 34–25.x.2000, Pohl (HLMD); 1^Deksam, 12°32.298'N:53°56.102'E, ca 300 m, 26.x.2000, Pohl (HLMD); 2_ 1^Homhil, 12°32'N:53°56'E, 9.i.1998, Wranik (CWWR, COGG 1_). Recorded specimens not studied (cited from Geller-Grimm (2002)): YEMEN: Socotra I.: 2^Goeeh, 12°32'25"N:54°10'22"E, 240 m, 23.x.2000, Pohl (NHCY 1^COGG 1^); 1^Firmihin, 12°24'41"N: 54°13'35"E, 34–25.x.2000, Pohl (HLMD); 1^Deksam, 12°32.298'N:53°56.102'E, ca 300 m, 26.x.2000, Pohl (HLMD); 2_ 1^Homhil, 12°32'N:53°56'E, 9.i.1998, Wranik (CWWR, COGG 1_). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Distribution and biology: The species has been recorded only from four localities on the island of Socotra. Collections have been made in October, December and January (Table 1). No biological data have been recorded on specimen labels. Similar species: T. nigrescens is superficially very similar to abdelkuri, but the species can be reliably separated on male genital features. Although I have seen relatively few specimens of both species, all specimens of abdelkuri have mesonotal, anepisternal and ocellar setae yellowish while these setae are mostly but not always black in nigrescens. These two species are somewhat similar to pohli, but easily separated on size and male genital form. Trichardis ornata sp. n. Figs 35, 36 Trichardis ornata sp. n. Figs 35, 36 Etymology: From Latin ornata (handsome, splendid). Refers to the attractiveness of this species. Description (based on unique holotype in good condition, right antenna broken off beyond pedicel, genitalia slightly damaged): Head: Dark red-brown to black, entirely silver pruinose, white setose. Antennal scape and pedicel orange, postpedicel dark red-brown, setae white; postpedicel somewhat clavate (L:D=2.9:1). Mystax white. Ocellar tubercle with 4 macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown, extensively silver pruinose, pale yellow and white setose. Postpronotum entirely pruinose, mesonotum apruinose except for fairly broad silver pruinose margins, macrosetae pale yellowish, setulae white. Scutellum apruinose except for narrow silver pruinose anterior margin. Anepisternum with pale yellow posterior macroseta, entirely pruinose, but weakly so anteroventrally. Proepimeron, katepisternum and anepisternum entirely pruinose. Legs: Femora orange with dark red-brown band subapically (broad on hind legs), tibiae orange proximally red-brown distally, tarsi dark Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 195 red-brown, pulvilli and empodium of similar length. Hind femur dark red-brown distally orange proximally, length:height ratio 4.3:1 (slender), ventral tubercles poorly developed. Hind tibia lacking ventrodistal spur. Wing: 5.3×2.0 mm. Costal vein strongly developed as far as wing tip, then very weakly developed along posterior margin of wing and absent from alula. Membrane not extensively microtrichose—discal cell lacking micro- trichiae, cell r5only with weakly developed microtrichiae in distal half. Abdomen: Dark red-brown, extensively apruinose but hind margins of terga broadly pruinose and lateral parts weakly pruinose anteriorly, white setose. T2 dark red-brown, apruinose except for broad posterior bands laterally and weaker areas anterolaterally. _ genitalia (Figs 35, 36; note slight damage to tips of proctiger, aedeagal prongs, tip of mediodistal process of left gonocoxite, and tip of left gonostylus): Epandrium in lateral view longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed (tip broken off). Hypandrium greatly reduced and simple. Gonocoxite in ventral view without projections and lacking distally arranged macrosetae; mediodistal projection fairly slender with straight distal end. Gonostylus moderately slender, gently curved with broadly rounded apex. Aedeagus with fairly elongate lateral projections basally; prongs more or less straight, tip damaged. Holotype: CHAD: _ ‘Tchad, N'Djaména [12°04'N:15°08'E] / Chari – Baguirmi / 2.viii.1992 / Leg. H.R. Feijen’ (NMSA). Distribution and biology: The species is known only from the type locality. The holotype was collected in August (Table 1). Trichardis picta Hermann, 1906 Figs 37–40, 59 Trichardis picta: Hermann 1906: 139–141; Kertész 1909: 159; Engel 1924: 107; Hull 1962: 97; Oldroyd 1974: 119; 1980: 356 (catalogue). Trichardis picta: Hermann 1906: 139–141; Kertész 1909: 159; Engel 1924: 107; Hull 1962: 97; Oldroyd 1974: 119; 1980: 356 (catalogue). T i h di L if Old d 1974 120 S Trichardis picta: Hermann 1906: 139–141; Kertész 1909: 159; Engel 1924: 107; Hull 1962: 97; Oldroyd 1974: 119; 1980: 356 (catalogue). T i h di L if Old d 1974 120 S ; ( g ) Trichardis Lucifer: Oldroyd 1974: 120. Syn. n. ; ( g ) Trichardis Lucifer: Oldroyd 1974: 120. Syn. n. f y y Trichardis lucifera: Oldroyd 1980: 356 (catalogue), unjustified emendation. f y y Trichardis lucifera: Oldroyd 1980: 356 (catalogue), unjustified emendation. f y y Trichardis lucifera: Oldroyd 1980: 356 (catalogue), unjustified emendatio Redescription (based on holotype in excellent condition): Trichardis ornata sp. n. No biological information exists. Similar species: T. ornata has an entirely pruinose postpronotal lobe and in this respect can be grouped with apicalis, grisescens, picta, terminalis, testacea, turneri and zinidi. The species is, however, most similar to testacea. Redescription (based on holotype in excellent condition): Head: Dark red-brown except for brown-orange face, colours masked by strong silver pruinescence, setae white. Antenna brown-yellow except for brown distal part of postpedicel and style, setae white; postpedicel not markedly clavate (L:D=3.2:1). Mystax white. Ocellar tubercle with 6 macrosetae. Proboscis red-brown, palpi brown-orange. Thorax: Dark red-brown, colour masked by strong silver pruinescence, pale yellow- white setose. Postpronotum entirely pruinose, mesonotum entirely pruinose, but more weakly posteriorly. Scutellum pruinose except for hind margin. Anepisternum with pale yellow posterior macroseta, entirely pruinose. Proepimeron, katepisternum and anepisternum entirely pruinose. Legs: Dark red-brown, narrowly brown-orange proximally, pulvilli and empodium of similar length. Hind femur dark red-brown with brown-orange proximal parts, length:height ratio 3.9:1, ventral tubercles poorly de- veloped. Hind tibia lacking ventrodistal spur. Wing: 5.8×2.2 mm. Costal vein strongly Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 196 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 developed as far as wing tip, then very weakly developed along posterior margin of wing and absent from alula. Membrane devoid of microtrichiae, including discal and r5 cells. Abdomen: Red-brown, extensively silver pruinose except for hind margins of terga and transverse bands across each tergite at about mid-length, setae white. T2 red-brown, strongly pruinose except for apruinose hind margin and weakly pruinose central area. _ genitalia (Figs 37, 38): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger hardly dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without projections and with about 7 small, distally arranged macrosetae; mediodistal projection fairly stout with upturned sclerotised distal end. Gonostylus stout, laterally flanged, with broad laterally compressed tip. Aedeagal prongs small, straight. Notes on type material: Hermann (1906) based his description on ‘_’ (number of specimens not stated) from ‘Capland, Willowmore (Dr. Brauns)’. While Engel (1924: 107) records the following material ‘^Lichtenburg, Transvaal. – 1_^Willowmore, Kapland, Dr. Brauns leg.’, and these ZSMC specimens (listed below) carry type labels, as do a pair of ‘cotype’ specimens in the AMGS (also listed below), only one specimen was collected before the published description. The single 1905 specimen must be considered the holotype, others were presurably mislabelled as types by Engel and possibly other workers. The two BMNH specimens of lucifer (holotype and paratype) when received, were incorrectly labelled—the female from Kahn River being labelled as the holotype. Type specimens studied: SOUTH AFRICA: 1_ (holotype picta) ‘Capland / Willowmor [Willowmore: 33°17'S:23°29'E] / 20 1 1905 / Dr. Brauns’, ‘Sammlung / F. Hermann’, ‘Type von / _ Trichardis / picta Herm.’ [orange] (ZSMC); NAMIBIA: _ (holotype lucifer) ‘Holo- / type’ [circular with red border], ‘S. W. Africa: Satansplatz. [24°51'S:17°31'E] / 1300m. / 17–19.xii.1933. / K. Jordan’, ‘Brit. Mus. / 1934–288’, ‘Trichardis / lucifer Oldr. / det. H. Oldroyd 1972 / Paratype’ [white] (BMNH); 1^(paratype lucifer) ‘Para- / type’ [circular with yellow border], ‘Southern / African Exp. / B.M. 1972–1’, ‘S.W. Africa (29) / Kahn River, 5 mls. / N. Usakos [22°00'S:15°34'E] / 30–31.i.1972’, ‘Trichardis / lucifer Old. / det. H. Oldroyd 1972 / Holotype’ [white] (BMNH). Other material examined: NAMIBIA: 1^Otjitundua [18°39'S:14°14'E], iii.1926, Mus. Exped. (SAMC); 1^60 km E Otjiwarongo, 20°39'S:17°05'E, 20.iii.1984, Londt & Stuckenberg, Acacia thornveld and dry river course; 2_ 1^26 km N Windhoek, 22°20'S:17°04'E, 29.iii.1984, Londt & Stuckenberg, dry river bed Acacia riparian woodland; 2_ 1^191 km E Walvis Bay [22°57'S:14°30'E], 12.xi.1963, Moore (NMNH); 1_ 3^1? Hakas Mts [Hakos Mts, 23°10'S:16°20'E], 12.xi.1963 (1^), 13.xi.1963 (1_ 2^1?), Moore (NMNH); 4_ 3^Gobabeb [23°33'S:15°02'E], 17.xi.1963, Moore (NMNH); 3_ 1^Namib Desert Park, Kuiseb R. at Gobabeb, 2315Ca, 12.ii.1974, Irwin, riverine forest and sand; 1_ Kuiseb R., 9.xii.1976, Cunningham; 2^Keetmanshoop Dist., 17.5 km N Grünau, 2718Bc, 1350 m, 30.i.1974, Irwin, dry river bed; 2_ Fish River Canyon Park, Ai-Ais [27°55'S:17°29'E], 19–21.xi.1993, Koch (ZMHB). SOUTH Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Redescription (based on holotype in excellent condition): As Oldroyd (1974) clearly indicated that the male from Satansplatz was the holotype, these labels have been switched. While Oldroyd (1974) called the species lucifer, presumably because the type locality was Satansplatz (i.e. Satan’s Place, referring to Lucifer), the spelling was amended to read lucifera in the Afrotropical Diptera catalogue (1980) by the Editor R.W. Crosskey, a change I consider both unnecessary and inappropriate as the name lucifera has a totally different derivation (from Latin lux). f y ( ) I consider the synonymy of lucifer with picta to be entirely justified. T. picta is a widely distributed species displaying variation over its range. The paler colour of the lucifer types represents variation, and while the male genitalia do show slight differences (Figs 39, 40), these too are considered to be within acceptable limits of variation. Type specimens studied: SOUTH AFRICA: 1_ (holotype picta) ‘Capland / Willowmor [Willowmore: 33°17'S:23°29'E] / 20 1 1905 / Dr. Brauns’, ‘Sammlung / F. Hermann’, ‘Type von / _ Trichardis / picta Herm.’ [orange] (ZSMC); NAMIBIA: _ (holotype lucifer) ‘Holo- / type’ [circular with red border], ‘S. W. Africa: Satansplatz. [24°51'S:17°31'E] / 1300m. / 17–19.xii.1933. / K. Jordan’, ‘Brit. Mus. / 1934–288’, ‘Trichardis / lucifer Oldr. / det. H. Oldroyd 1972 / Paratype’ [white] (BMNH); 1^(paratype lucifer) ‘Para- / type’ [circular with yellow border], ‘Southern / African Exp. / B.M. 1972–1’, ‘S.W. Africa (29) / Kahn River, 5 mls. / N. Usakos [22°00'S:15°34'E] / 30–31.i.1972’, ‘Trichardis / lucifer Old. / det. H. Oldroyd 1972 / Holotype’ [white] (BMNH). Other material examined: NAMIBIA: 1^Otjitundua [18°39'S:14°14'E], iii.1926, Mus. Exped. (SAMC); 1^60 km E Otjiwarongo, 20°39'S:17°05'E, 20.iii.1984, Londt & Stuckenberg, Acacia thornveld and dry river course; 2_ 1^26 km N Windhoek, 22°20'S:17°04'E, 29.iii.1984, Londt & Stuckenberg, dry river bed Acacia riparian woodland; 2_ 1^191 km E Walvis Bay [22°57'S:14°30'E], 12.xi.1963, Moore (NMNH); 1_ 3^1? Hakas Mts [Hakos Mts, 23°10'S:16°20'E], 12.xi.1963 (1^), 13.xi.1963 (1_ 2^1?), Moore (NMNH); 4_ 3^Gobabeb [23°33'S:15°02'E], 17.xi.1963, Moore (NMNH); 3_ 1^Namib Desert Park, Kuiseb R. at Gobabeb, 2315Ca, 12.ii.1974, Irwin, riverine forest and sand; 1_ Kuiseb R., 9.xii.1976, Cunningham; 2^Keetmanshoop Dist., 17.5 km N Grünau, 2718Bc, 1350 m, 30.i.1974, Irwin, dry river bed; 2_ Fish River Canyon Park, Ai-Ais [27°55'S:17°29'E], 19–21.xi.1993, Koch (ZMHB). SOUTH Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 197 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Trichardis pohli: Geller-Grimm 2002: 472–474, pls 4, 18 (entire _), figs 5–7 (_ terminalia). LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) AFRICA: 1_ Baberspan, 25°07'S:26°05'E, 14–21.xii.1993, Joffe; 1^Lichtenburg [26°09'S:26°10'E], Brauns (ZSMC); 1_ 2^10 km W Bloubos Farm, 28°07'S:20°45'E, 900 m, 17.iii.1991, Londt & Whittington, red dunes [habitat] N Upington; 1_ Bloemfontein [29°10'S:26°00'E], 13.ii.1918 (SANC); 1^5 km S Laingsburg, 33°14'S:20°52'E, 700 m, 25.xi.1990, Londt & Whittington, Banks Buffels River; 1_ Gamka R. 40 km N Prince Albert, 3321BB, 500 m, 11.xi.1986, Londt & Quickelberge, sandy area/Acacias; 1_ Meiringspoort, 3322BC, 11–12.xii.1979, Londt & Stuckenberg, rocky hillside & stream edge; 8_ 2^Diepkloof ca 20 km E De Rust, 3322BD, 12.xii.1979, Londt & Stuckenberg, dry rocky hillside & stream; 1^Graaff-Reinet Urquhart Park Caravan Park, 32°15'S:24°33'E, 4–6.xii.1988, Londt, riverine vegetation, sandy ground; 1_ Middelburg [31°29'S:25°01'E], 13.ii.1925, Munro; 3_ 2^Rietvlei Nieuveld Escarpment [32°20'S:21°30'E], i.1949, Zinn & Hesse (SAMC); 1^Tankwa Karoo [32°30'E 19°45'E], i.1949, Zinn & Hesse (SAMC); 1_ Letjiesbosch Koup [32°34'S:22°16'E], iii.1937, Mus. Staff (SAMC); 2_ 7^Merweville [32°40'S:21°31'E], i–ii.1947, Zinn (SAMC); 1_ 1^Merweville Lainsburg Dist., i.1959, Zinn (SAMC); 1_ Dikbome Merweville Koup, i.1953, Zinn (SAMC); 1_ Oukloof Beaufort West [33°15'S:22°06'E], i.1949, Zinn & Hesse (SAMC); 1^Willowmore [33°17'S:23°29'E], 25.ii.1907, Brauns (ZSMC); 1_ 1^Willowmore, 5.ii.1907 (1^), 1.iii.1907 (1_), Brauns (AMGS); 2_ 3^Willowmore, 25.ii.1907 (1^), 1.xii.1909 (1_), xii.1912 (1^), 25.xii.1916 (1_), 10.xii.1920 (1^), Brauns; 2_ 1^Willowmore, 5.ii.1907 (1_), 1.xii.1920 (1^), no date (1_), Brauns (NMNH); 1_ 1^Willowmore, 20.i.1908, Brauns (BMNH); 1^Willowmore, 25.xii.1915, Brauns (MRAC); 1_ Willowmore, 15.xii.1917, Brauns (SAMC); 6_ 3^Rooinek Lainsburg Dist. [33°20'S:20°55'E], i.1949, Zinn & Hesse (SAMC); 1^Rooinek Pass, x.1952, Mus. Expd. (SAMC); 4_ 1^Tierberg Res. Stat. Prince Albert Dist., 33°07'42"S:22°16'24"E, 26.xi–5.xii.1987, Gess (AMGS); 1_ 1^De Hoek Uitenhage [33°45'S:25°24'E], 11.iii.1919, Munro (NMNH). Distribution and biology: The species is a southern African endemic being found in the western parts of the region. It ranges from northern Namibia southwards to the Western and Eastern Cape provinces (Fig. 59). Adults fly between October and March (Table 1). Personal experience and label data indicate that the species frequents Acacia woodland and is associated with sandy stream banks or dry river courses where individuals rest on the ground. Two prey records are known to me, both in AMGS: 1_ (Tierberg, Prince Albert Dist.) pinned with a pollen wasp (Hymenoptera: Masaridae), and 1^(same locality) pinned with a tachinid fly (Diptera: Tachinidae). Similar species: T. picta has an entirely pruinose postpronotal lobe and in this respect can be grouped with apicalis, grisescens, ornata, terminalis, testacea, turneri and zinidi. The species is, however, distinctive and difficult to confuse with others in this group. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Redescription (based on holotype in excellent condition): Redescription (based on holotype in excellent condition): Head: Dark red-brown to black, extensively silver pruinose, but weakly so on frons and apex of ocellar tubercle, setae black and white. Antennal scape yellow–brown, pedicel, postpedicel and style dark red-brown, setae black and white (black setae being better developed than white); postpedicel elongate spindle-shaped (L:D=4.5:1), with few black setulae dorsally. Mystax black and white (black setae better developed). Ocellar tubercle with 4 black macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown to black with orange parts, silver pruinose except for some apruinose parts, fine setae whitish, macrosetae either black (mesonotum) or white (pleura). Postpronotum largely apruinose except for narrow medial part, mesonotum dark red-brown to blackish except for orange postpronotal and postalar lobes, largely apruinose except for margins, macrosetae black, setulae shiny yellowish. Scutellum dark red-brown with orange posterior margin, anterior half silver pruinose. Anepisternum with slender black posterior macroseta, extensively pruinose except for small area antero- Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 198 ventrally. Katatergite with white macrosetae. Proepimeron anteriorly pruinose, poste- riorly apruinose; katepisternum posteriorly pruinose, anteriorly apruinose; anepisternum pruinose except for anterodorsal part. Legs: Generally dark red-brown to black anteriorly, yellowish posteriorly, pulvilli and empodium of similar length. Hind femur dark red- brown anterodorsally, yellowish posteroventrally; length:height ratio 4.2:1; ventral tubercles hardly evident, major setae pale yellowish. Hind tibia lacking ventrodistal spur. Wing: 4.0×1.6 mm. Costal vein extends along most of wing margin, weakly along anal cell, absent from alula. Membrane not extensively microtrichose—discal cell largely lacking microtrichiae (a few present centrally), cell r5with microtrichiae limited mainly to distal half. Abdomen: Terga and hypopygium dark red-brown, but with orange parts laterally, apruinose except for narrow silver pruinose distolateral margins, setae transparent whitish. T2 dark red-brown with orange parts laterally (anterior and posterior parts), apruinose except for narrow silver pruinose posterior margins laterally. _ genitalia: Holotype well illustrated by Geller-Grimm (2002: figs 5–7). Another male from Socotra (NHMW) is here illustrated (Figs 41, 42) and described: Epandrium in lateral view longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hyp- andrium greatly reduced and simple. Gonocoxite in ventral view without median pro- jections distally and with mediodistally arranged macrosetae; mediodistal projection slender with slightly upturned tip. Gonostylus long, laterally compressed, with slender slightly down turned apex. Redescription (based on holotype in excellent condition): Aedeagal prongs more or less straight and with small terminal tubules. Holotype (examined): YEMEN: Socotra I.: _ ‘Soqotra · Archipel: Soqotra, / Route von Hadibo zum Deksam- Plateau, 800 m / 12°32'N:53°56'E, 22.2.1999 / leg.: H. Pohl, SOQ35’ (HLMD). Holotype (examined): YEMEN: Socotra I.: _ ‘Soqotra · Archipel: Soqotra, / Route von Hadibo zum Deksam- Plateau, 800 m / 12°32'N:53°56'E, 22.2.1999 / leg.: H. Pohl, SOQ35’ (HLMD). Other material examined: YEMEN: Socotra I.: 1^1? Hamadara [?], 400 m, 4.iv.1967, Guichard (BMNH); 1_ 1^Socótra I., 1899, Simony (NHMW). Other material examined: YEMEN: Socotra I.: 1^1? Hamadara [?], 400 m, 4.iv.1967, Guichard (BMNH); 1_ 1^Socótra I., 1899, Simony (NHMW). Distribution and biology: A species apparently confined to Socotra I. and known with certainty from two localities. Collected in February and April (Table 1). No biological data have been recorded on labels. Similar species: A fairly distinctive species with some similarities to both abdelkuri and nigrescens. Holotype (examined): YEMEN: Socotra I.: _ ‘Soqotra · Archipel: Soqotra, / Route von Hadibo zum Deksam- Plateau, 800 m / 12°32'N:53°56'E, 22.2.1999 / leg.: H. Pohl, SOQ35’ (HLMD). Trichardis rueppelii (Wiedemann, 1828) Trichardis rueppelii (Wiedemann, 1828) Dasypogon Rüppelii: Wiedemann 1828: 569–570. Laphria rueppelii: Oldroyd 1980: 352. Trichardis rueppellii: Geller-Grimm 1999: 214. Dasypogon Rüppelii: Wiedemann 1828: 569–570. Laphria rueppelii: Oldroyd 1980: 352. Trichardis rueppellii: Geller-Grimm 1999: 214. Dasypogon Rüppelii: Wiedemann 1828: 569–570. Laphria rueppelii: Oldroyd 1980: 352. p pp y Trichardis rueppellii: Geller-Grimm 1999: 214. p pp y Trichardis rueppellii: Geller-Grimm 1999: 214. Redescription (based on holotype in fair condition: both antennae broken off beyond pedicels, hind margins of wings damaged and not showing extent of costal vein, terminal tarsomeres mostly missing, damaged or dirty): Head: Pale orange, silver pruinescence, setae white. Antennae orange, both broken off beyond pedicel, white setose. Mystax white. Ocellar tubercle with 4 macrosetae and a few fairly big setae. Proboscis orange with orange-brown distal half, palpi orange. Thorax: Brown-orange, dorsal parts red-brown, setae white. Postpronotum entirely pruinose, mesonotum dark red-brown centrally, orange laterally, extensively silver Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 199 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) pruinose except for central parts [may be worn smooth through handing?], setae white. Scutellum pruinose except for hind margin. Anepisternum lacking posterior macroseta, extensively pruinose (somewhat apruinose anteroventrally [may be worn]). Proepimeron, katepisternum and anepimeron pruinose. Legs: Uniformly orange, lengths of pulvilli and empodia difficult to study as terminal tarsomeres dirty, damaged or missing. Hind femur orange, length:height ratio 4.1:1 (i.e. slender), ventral tubercles not evident. Hind tibia lacking ventrodistal spur. Wing: 6.3×2.4 mm. Costal veins broken and missing beyond wing tips [due to damaged hind margins]. Membrane entirely without micro- trichiae. Abdomen: Uniformly brown-orange, terga silver pruinose except for small areas antero- laterally and centrally [may be worn], white setose (setulae longish). T2 orange, pruinose except for small anterolateral and central [may be worn] areas. Holotype (examined): ERITREA: ^‘Abyssinia [no locality given] / Dr Rüppell.’, ‘136’ [blue with black frame], ‘Typus’ [red with black frame], ~ ‘Dipt. / 105’ [white], ‘Trichardis / Ruppelii Wd. / det. / Dr. F. Herman’ (SMFD). Note: Wiedemann (1928) gives the provenance as ‘Aus Nubien’. This suggests the Nubian Desert which is in Eritrea and not Sudan, as listed by Oldroyd (1980). For the present it is not possible to provide a type-locality. Taxonomic status: Morphologically the holotype closely agrees with the description provided above for the leucocoma type except for a few small details as follows. Trichardis rueppelii (Wiedemann, 1828) Pruinescence of head and thorax is not as strong or extensive; the legs are uniformly orange and totally lack red-brown parts. Bearing in mind that the rueppelii holotype is a female and that some sexual dimorphism is evident in leucocoma everything points to leucocoma being a synonym of rueppelii. However, while I am reasonably sure that this will be the future taxonomic outcome, I refrain from establishing the synonymy until male specimens agreeing with leucocoma are found in Eritrea. This conservative approach also ensures retention of the well-known name, leucocoma, until further investigations of the Palaearctic Trichardis fauna have been undertaken. Trichardis similis sp. n. Figs 43, 44 Trichardis similis sp. n. Etymology: From Latin similis (similar). Refers to the similarity between the male genitalia of this species and crassipala. Description (based on holotype in excellent condition): Head: Black, silver pruinose except for strip between ocellar tubercle and epistomal margin, setae black, white and pale yellow. Antenna dark red-brown to black, black setose; postpedicel elongate spindle-shaped (L:D=4.3:1). Mystax shiny yellow-white with black macrosetae along epistomal margin. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown to black. Thorax: Dark red-brown to black, silver pruinose except where apruinose, shiny yellowish setose. Postpronotum extensively apruinose except for narrow band medially, mesonotum apruinose except for narrow lateral and posterior margins, yellowish setose. Scutellum apruinose except for narrow anterior margin. Anepisternum with pale yellowish posterior macroseta, pruinose except for large area anteroventrally. Proepi- meron pruinose anteriorly, apruinose posteriorly; katepisternum pruinose posteriorly, apruinose anteriorly; anepisternum pruinose except for small central spot. Legs: Dark Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 200 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 red-brown, pulvilli and empodium of similar length. Hind femur dark red-brown, length:height ratio 3.9:1, ventral tubercles well-developed. Hind tibia lacking ventrodistal spur. Wing: 4.3×1.7 mm. Costal vein strongly developed as far as wing tip, then very weakly developed along posterior margin of wing and absent from alula. Membrane extensively microtrichose—discal and r5cells entirely microtrichose. Abdomen: Dark red-brown proximally becoming red-brown distally, apruinose, pale yellow setose. T2 dark red-brown, apruinose. y p _ genitalia (Figs 43, 44): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger long, strongly dorsoventrally compressed. Hypandrium moderately well developed with two pairs of lobes distally. Gonocoxites in ventral view with medially directed dorsal projections and lacking macrosetae; mediodistal projection well-developed, strongly sclerotised, broad with characteristic shape. Gonostylus short, slender, poorly developed, straight. Aedeagal prongs more or less straight, small, tapering to small terminal filamentous tubules. Holotype: MALAWI: _ ‘Malawi Kasungu Nat. / Park Lifupa Camp / 1333Aa 9–10.xii.1980 / 1000 m Stuckenberg & / Londt, Brachystegia’ (NMSA). Paratypes: 1_ same data as holotype; 1^‘Malawi Chimaliro / forest reserve 1200 m / 1233Bc Stuckenberg & / Londt 9.xii.1980 / Brachystegia woodland’ (NMSA). aratypes: 1_ same data as holotype; 1^‘Malawi Chimaliro / forest reserve 1200 m / 1233Bc Stuckenbe & / Londt 9.xii.1980 / Brachystegia woodland’ (NMSA). Distribution and biology: The species is recorded from two localities in Malawi. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Trichardis similis sp. n. Adults are known to fly in December (Table 1), midsummer in the southern hemisphere. The type material was collected on the ground in Brachystegia woodland. Similar species: A member of what is here called the ‘cribrata species group’ which consists of crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata and indica. These species are superficially similar, but can be easily separated on characters of the male genitalia. T. similis is most similar to crassipala in that both species have well- developed hypandria. Trichardis spicata sp. n. Figs 45, 46 Trichardis spicata sp. n. Figs 45, 46 Etymology: From Latin spica (point, spike). Refers to the long spike-like aedeagal projections. Description (based on holotype in excellent condition): Head: Dark red-brown to black. Antenna dark red-brown to black, setae black, post- pedicel elongate spindle-shaped (L:D=5.0:1). Mystax white, with a few black macrosetae along epistomal margin, on plane and mostly shiny apruinose face (narrow pruinose strips along eye margins). Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown to black. Thorax: Dark red-brown to blackish, extensively apruinose, pruinose areas silvery. Post- pronotum strongly silver pruinose medially, extensively apruinose laterally, mesonotum apruinose except for narrow silver pruinose margins, macrosetae pale yellow, fine setae yellow-white. Scutellum apruinose except for anterior margin. Anepisternum with longish pale yellow posterior macroseta, dorsally pruinose, ventrally apruinose. Pro- epimeron pruinose except for small apruinose area posteriorly, katepisternum pruinose Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 201 except for small apruinose part anteriorly, anepisternum pruinose. Legs: Dark red-brown, tibiae paler brownish, pulvilli and empodium of similar length. Hind femur dark red- brown, length:height ratio 3.6:1, ventral tubercles moderately developed. Hind tibia lacking ventrodistal spur. Wing: 4.1×1.5 mm. Costal vein moderately developed along entire wing margin, but weak along anal cell and apparently absent from alula. Membrane extensively microtrichose (except for small parts of some proximally situated cells)— discal and r5cells entirely microtrichose. Abdomen: Dark red-brown, largely apruinose, terga weakly pruinose along hind margins. T2 dark red-brown, apruinose except for weak, narrow, silver pruinose posterior margin. Abdomen: Dark red-brown, largely apruinose, terga weakly pruinose along hind margins. T2 dark red-brown, apruinose except for weak, narrow, silver pruinose posterior margin. _ genitalia (Figs 45, 46): Epandrium in lateral view significantly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger long and moderately dorsoventrally compressed. Hypandrium highly reduced and simple. Gonocoxite in ventral view distally pointed, with moderately well-developed median hook-like projection distally and group of about 6 macrosetae laterally at mid- length; mediodistal projection short, stout, straight, strongly sclerotised. Gonostylus short, stout. Aedeagal base with pair of projections that exceptionally long, slender, strongly sclerotised, and gently downcurved to pointed tips; prongs tiny, slightly curved, poorly developed distally. Holotype: MOZAMBIQUE: _ ‘3.xii.2006 Mozambique / Sofala Prov. 30 km S Caia / 18.02S – 34.02E / P. Schüle leg.’ (MRAC). Paratypes: 1_ 1^same data and depository as holotype. Trichardis spicata sp. n. Figs 45, 46 Paratypes: 1_ 1^same data and depository as holotype. Distribution and biology: The species is known only from the type locality where it has been collected in December (Table 1). No biological data are available. Similar species: A member of what is here called the ‘cribrata species group’ which consists of crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata and indica. These species are superficially similar, but can be easily separated on characters of the male genitalia. T. spicata has distinctive male genitalia that cannot be confused with any other species. Trichardis terminalis Oldroyd, 1974 Figs 47, 48, 58 richardis terminalis: Oldroyd 1974: 118 (figs 109 _ genitalia, 110 ^genitalia); 1980: 356 (catalogue). Redescription (based on holotype in fair condition; following parts missing: right antennae beyond pedicel, left pro- and mesothoracic legs, right prothoracic tarsus, right wing): Head: Orange-brown anteriorly dark red-brown posteriorly, but colours masked by silver pruinescence, yellow and white setose. Antenna brown-orange except for red-brown distal part of postpedicel and style, yellowish setose; postpedicel clavate (L:D=2.7:1). Mystax shiny yellowish. Ocellar tubercle with 2 macrosetae. Proboscis and palpi dark red-brown. Thorax: Dark red-brown with red-brown patches, colours masked by silver pruinescence, yellowish setose. Postpronotum entirely pruinose, mesonotum red-brown with dark red-brown dorsal stripe and broad lateral bands, extensively pruinose except for central area, shiny yellowish setose. Scutellum pruinose except for posterior margin. Anepi- sternum with yellow posterior macroseta, dorsally pruinose, ventrally apruinose. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 202 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Proepimeron pruinose anteriorly, apruinose posteriorly; katepisternum pruinose poste- riorly, apruinose anteriorly; anepisternum pruinose posteriorly, apruinose anteriorly. Legs: Femora and distal half of hind tibiae red-brown, rest brown-yellow, pulvilli and empodium of similar length. Hind femur red-brown with paler apices, length:height ratio 3.6:1, ventral tubercles well-developed. Hind tibia lacking ventrodistal spur. Wing: 6.1×2.3 mm. Costal vein strongly developed as far as wing tip, then very weakly deve- loped along posterior margin of wing and absent from alula. Membrane devoid of micro- trichiae, including discal and r5cells. Abdomen: Dark red-brown, apruinose, shiny yellow setose. T2 dark red-brown, aprui- nose. _ genitalia (Figs 47, 48): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger well-developed, moderately dorsoventrally compressed. Hypandrium reduced and simple. Trichardis spicata sp. n. Figs 45, 46 Gonocoxite in ventral view without dorsomedial projections and tapering to narrowly rounded tip carrying about 4 short macrosetae; mediodistal projection mo- derately stout, tapering to slender slightly upturned tip. Gonostylus well-developed, moderately slender, jutting out beyond mediodistal projection of gonocoxite, gently downcurved distally. Aedeagal prongs slender, sinuous in lateral view, with long terminal filamentous tubules. Holotype (examined): ZIMBABWE: _ ‘S. Rhodesia / Umguza Valley [19°30'S:27°46'E] / 17.12.22 / Roy Stevenson’, ‘Trichardis / terminalis Oldr. / det. H. Oldroyd, 1972 / Holotype’ (NMSA). Paratypes (examined): BOTSWANA: 1^‘Para- / type’ [circular with yellow border], ‘S. Africa: / Bechuanaland. / Ngamiland [ca 20°30'S:22°40'E], Nov. 1930–Jan. 1931. / G.D. Hale Carpenter. / B.M. 1931–160’, ‘trichardis /terminalis Oldr. / det. H. Oldroyd 1972 / Paratype’ [white] (BMNH). ZIMBABWE: 1^‘Country Rhodesia / Loc. Chirundu [16°02'S:28°50'E] / Date 17.ii.65 / Coll. K Borthwick’, ‘Trichardis / terminalis Oldr. / det. H. Oldroyd, 1972 / Paratype’; 1^‘Country Rhodesia / Loc. Chirundu / Date 15.ii.65 / Coll. K Borthwick’, ‘Trichardis / terminalis Oldr. / det. H. Oldroyd, 1972 / Paratype’; 1^‘Para- / type’ [circular with yellow border], ‘Dovenby Farm. [19°53'S:28°29'E], / S. Rhodesia / 17.9.1922, Rhodesia / Museum’, ‘Trichardis / cribrata / Lw. / Dr. EO. Engel det.’, ‘Trichardis / terminalis Oldr. / det. H. Oldroyd 1972 / Paratype’ [white] (BMNH). Note: In describing this species Oldroyd (1974) lists his material as follows: ‘Type in Pretoria. Type-locality: RHODESIA, Umguza Valley, 17.ix.22 (Roy Stevenson)’. He then states ‘Distribution. RHODESIA: Umguza Valley; Dovenby Farm; Chivundu (Borthwick). BOTSWANA: Ngamiland, 1931 (G. D. Hale Carpenter)’. The specimens he studied are in NMSA and BMNH and carry paratype labels. So although not formally listed in his publication, I accept the above specimens as the full type series. Other material examined: BOTSWANA: 1_ 2^Maxwee [19°28'S:23°40'E], ix.1976, Russell-Smith, Mopane woodland; 1_ Kwaai [Khwai] R. 20 km W Moremi North gate [ca 19°S:23°E], 14.x.1977, Russell-Smith, Acacia giraffae woodland. NAMIBIA: 1_ E Caprivi Linyanti [17°47'S:24°23'E], 9–17.x.1970, Strydom. TANZANIA: 1_ Seranda Rd [?] K.I., 27.xi.1927, Nash (OXUM). ZIMBABWE: 2_ 2^Rekomitjie Research Station [16°08'S:29°24'E], 14.x.1973 (1_), 15.x.1973 (1_ 1^), 16.x.1973 (1^), Phelps; 1_ Sanyanti Valley [?Sanyati, 17°30'S:29°23'E], ix–x.1925, Stevenson; 12_ 8^2? Triangle [21°02'S:31°27'E], 19.ix.1963 (5_ 3^), 21.ix.1963 (5_ 4^1?), 23.ix.1963 (2_ 1^1?), Moore (NMNH). Distribution and biology: The species is known mainly from a relatively small area of southern Africa (Fig. 58), being recorded from Botswana, Namibia (the Caprivi) and Zimbabwe. A single record from Tanzania suggests a wider distribution. Trichardis testacea (Macquart, 1838) Figs 1–3, 49, 50, 60 Laphria testacea Macquart, 1838: 63. Laphria testacea Macquart, 1838: 63. Trichardis testacea Hermann, 1906: 137–139 [Junior synonym and junior secondary homonym, preocc. testacea Macquart, 1838]; Kertész 1909: 159; Engel 1924: 109–110; Hull 1962: 97. Trichardis testacea Macquart: Oldroyd 1974: 120 (figs 107 entire ^, 108 wing); 1980: 356 (catalogue). aph ia testacea acqua t, 838: 63. Trichardis testacea Hermann, 1906: 137–139 [Junior synonym and junior secondary homonym, preocc. testacea Macquart, 1838]; Kertész 1909: 159; Engel 1924: 109–110; Hull 1962: 97. q , ]; ; g ; richardis testacea Macquart: Oldroyd 1974: 120 (figs 107 entire ^, 108 wing); 1980: 356 (catalogue). Redescription (based on a _ syntype of T. testacea Hermann in good condition): Head: Orange anteriorly red-brown distally, somewhat masked by silver pruinescence, white and pale yellow setose. Antenna orange with red-brown style, pale yellow setose; postpedicel not markedly clavate (L:D=3.3:1). Mystax pale yellow. Ocellar tubercle with 4 macrosetae. Proboscis and palpi orange-brown. Thorax: Orange, fine silver pruinose, yellow and white setose. Postpronotum entirely pruinose, mesonotum extensively apruinose except for silver pruinose lateral and poste- rior margins, macrosetae shiny orange, setulae white. Scutellum apruinose except for small spots laterally. Anepisternum with orange posterior macroseta, entirely pruinose. Proepimeron, katepisternum and anepisternum entirely pruinose. Legs: Orange, pulvilli and empodium of similar length. Hind femur orange, length:height ratio 3.7:1, ventral tubercles absent. Hind tibia lacking ventrodistal spur. Wing: 5.6×2.2 mm. Costal vein strongly developed as far as wing tip, then proceeding more weakly along posterior margin of wing and absent from alula. Membrane microtrichose only in distal half— discal cell entirely microtrichose, cell r5 extensively microtrichose but weak to absent in proximal half. Wing membrane with orange stained areas. Abdomen: Orange, terga somewhat reddish laterally, each tergum with a small silver pruinose spot posterolaterally, setae pale yellow, setulae minute shiny whitish. T2 yellowish orange, laterally somewhat reddish, apruinose except for small posterolateral spot. _ genitalia (Figs 49, 50): Epandrium in lateral view longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger short, moderately dorsoventrally compressed. Hypandrium greatly reduced and simple in structure. Gonocoxite in ventral view without obvious projections distally and with about 5 medially directed distal macrosetae; mediodistal projection moderately stout with upturned sclerotised tip. Gonostylus stout with broad flange-like distal tip. Aedeagal prongs small, fairly straight, with small terminal filamentous tubules. Trichardis spicata sp. n. Figs 45, 46 Adults fly between September and February (no record for January) (Table 1). Label data indicate that the species lives in both Acacia and Mopane woodland. Similar species: T. terminalis has an entirely pruinose postpronotal lobe and in this respect can be grouped with apicalis, grisescens, ornata, picta, testacea, turneri and zinidi. The species is, however, most similar to grisescens. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 203 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 testacea / Hermann’, ‘Trichardis / testacea / Cotype / Hermann’ (MCMI); 1^‘Willowmore / Capland / Dr. Brauns’, ‘Type von / Trichardis / testacea ^/ Herm.’ [orange] (ZSMC); 1^‘Cotype’, ‘Capland / Willowmor / 12 12 1906 / Dr. Brauns’, ‘Cotype / Trichardis / testacea / Hermann’ [green] (AMGS). Notes: Macquart (1838) described his species briefly on female specimens (number not stated) from ‘Du Cap’. Unfortunately Hermann (1906), unaware of Macquart’s species, placed in another genus, described his species using the same name, thus creating a homonym. Engel (1924) subsequently provided a brief description in a key, recording for testacea Hermann ‘2_ 2^Willowmore, Kapland, Dr. Brauns leg.’ and indicated (p. 106) that ‘testacea Mcq. 1838 [Laphria] ist ein Storthyngomerus’. This unlikely identification was checked by Oldroyd (1970: footnote on p. 247) and found to be incorrect—the homonymy of Hermann’s species with that of Macquart being fully supported. p q g y pp Other material examined: BOTSWANA: 8_ 14^Farmer's Brigade Serowe, 2226BD, 21.xii.1982 (1_), 20.x.1984 (1_), 31.x.1984 (1^), xii.1985 (2_ 2^), 3.xii.1985 (1_), 9.xi.1988 (1^), 8.xii.1988 (1_), ix.1989 (1_ 3^), x.1989 (3^), x.1990 (1_ 3^), 28.xi.1990 (1^), Forchhammer; 4_ 6^same place, x.1989 (2_ 4^), xi.1989 (1_ 1^), xii.1989 (1_), ii.1990 (1^), Forchhammer, Mercury V.L. light trap (NHRS); 1_ Close to Otse [23°10'S:21°00'E] Vultury, 6.xi.1993, Viklund (NHRS); 1_ Gaberones [24°39'S:25°54'E], ii.1915, Ellenberger (MNHN); 1_ Sanitas garden Gaberones Dam, 7–19.xi.1993, Viklund (NHRS). NAMIBIA: 1^Kunene, Epupa Falls, 17°00'S:13°15'E, 22.ii.1995, Koch (ZMBH); 1_ 1^Kaoko Otavi [18°18'S:13°42'E], iii.1926, Mus. Exped. (SAMC); 1_ Etosha-Pan, Namutoni [18°48'S:16°59'E], 23.i.1993, Koch (ZMHB); 1^Brandberg Tsisab Valley [21°01'S:14°41'E], viii.1948, Strey. SOUTH AFRICA: 1_ 1^Messina Nat. Res., 22°24'54"S:30°05'12"E, 487 m, 14.ii.2005, Londt & Dikow, Mopane dry woodland Sand R. [habitat]; 1^Soutpansberge Soutpan, 2229CD, 23–24.ii.1980, Londt & Schoeman, bushveld vegetation; 1_ 37 km N Louis Trichardt, 2229DD, i.1975, Stuckenberg, arid bushveld; 1^Swartruggens Marico [25°39'S:26°42'E], 15.i.1901, Brauns; 1_ 2^Wolmaransstad [27°12'S:25°58'E], 20.x.1963 (1^), 23.x.1963 (1_ 1^), Moore (NMNH); 1_ Kroonstad [27°40'S:27°14'E], 29.xi.1965, Brothers (AMGS); 1^35 km SE Noenieput, 2720CB, 20.iii.1982, Londt & Schoeman, flowers on roadside; 1? Cape Colony, Windsorton [28°20'S:24°43'E], 20.xii.1920, Brauns; 1_ Modder R., Brandfort Dist. [2826CD], xi.1939, Mus. AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Staff (SAMC); 1_ Grootvlei [29°13'S:26°18'E], 9.xii.1913, Munro (SANC); 1_ Grootvlei, 9.xii.1914; 1^Wyk's Vlei [Vanwyksvlei] [30°21'S:21°49'E], Alston (NMNH); 1_ Wyk's Vlei, 1885, Alston (BMNH); 1_ Wyk's Vlei, 1886, Alston (SAMC); 1^Vanwyksfontein, 8 km W Norvalspont [30°38'S:25°27'E], 16.i.1985, Gess (AMGS); 1_ 2^Norval’s Pont [30°38'S:25°27'E], 9.xii.1971, Greathead (BMNH); 1_ 1^Aliwal North [30°42'S:26°42'E], 1326 m, 1–13.1923 [?], Turner (BMNH); 3_ Lady Grey [30°42'S:27°14'E], 30.xii.1924, 6.i.1925, 6.i.1926, Nel; 1_ Lady Grey, 22.xii.1925, Nel (BMNH); 1_ 13 km NW Carnarvon V. Wyksvlei Rd, 3022CC, 14.xi.1986, Londt & Quickelberge, dry riverbed Acacias; 1^Carnarvon [30°57'S:22°08'E] (SAMC); 1^Steynsburg [31°19'S:24°20'E], 1910, Ellenberger (MNHN); 1^Richmond Dist. [31°25'S:23°56'E], iii.1931, Museum Staff (SAMC); 1_ 1^Middelburg [31°29'S:25°01'E], 13.ii.1925, Munro (NMNH); 5_ 4^14 m E Middelburg, Cape Province, 8–14.xii.1960, Brown, Fürst & Haacke (SANC); 1^5 km N Nieuwoudtville, 3119AC, 760 m, 16.xi.1986, Londt & Quickelberge, stream macchia; 1_ Visrivier 50 km E Calvinia on Williston Rd, 31°26'15"S:20°16'48"E, 990 m, 10.xi.1998, Londt, river edge vegetation; 2_ 10 km W Williston, 3120BD, 1060 m, 15.xi.1986, Londt & Quickelberge, sand/Acacias’; 2^Cacadu R. nr Lady Frere, 3127CA, 27.x.1978, Londt & Miller, river bank; 1^23 km N Middelpos, 31°44'S:20°14'E, 29.xi.1990, 1170 m, Whittington & Londt, at Kookfontein R.; 1_ nr Perdefontein, E Middelpos, 31°45'S:20°44'E, 11.xii.1988, Eardley (SANC); 1_ Queenstown [31°54'S:26°53'E], 16.i–10.ii.1923, Turner (BMNH); 1^Dunedin [31°57'S:22°25'E], 1.xi.1979, Whitehead (SAMC); 4_ 1^Murraysburg Dist. [31°57'S:23°46'E], xi.1935, Museum Staff (SAMC); 1_ 1^23 km SE Middelpos, 32°01'S:20°25'E, 1200 m, 28.xi.1990, Whittington & Londt, banks of Visrivier; 1^Cape of Good Hope, Nels Poort [32°07'S:23°00'E], 5.xii.1933, Ogilvie (BMNH); 1_ Ingleside, Cradock [32°11'S:25°37'E], iv.1914, Brincker (BMNH); 1_ Cradock, i.1915, Brincker (BMNH); 1^Goshen nr Cathcart [32°13'S:27°03'E], iii.1954, S.A. Museum (SAMC); 1_ 1^Graaff-Reiner Urquhart Park Caravan Park, 32°15'S:24°33'E, 4–6.xii.1988, Londt, riverine vegetation, sandy ground; 1^Urquhart Park Caravan Park in Graaff-Reinet, 32°15'S:24°33'E, 7.xii.1989, Londt,Acaciasavannah nr dam; 1_ Graaf-Reinet [Graaff-Reinet, 32°17'S:24°28'E], 19.xi.1959, Greathead (BMNH); 8_ 7^Rietvlei Nieuveld Escarpment [32°20'S:21°30'E], i.1949, Zinn & Hesse (SAMC); 1_ Beaufort West Dist. [32°21'S:22°35'E], ii.1958, SAM (SAMC); 1^Renoster R. Tankwa Karoo [32°25'S:20°00'E], xi.1952, Mus. Expd. (SAMC); 5_ 10^1? Aberdeen [32°29'S:24°05'E], xi.1935, Mus. Staff (SAMC); 1_ Katberg [32°31'S:26°38'E], 1219 m, xi.1932, Turner (BMNH); 1^Merweville Lainsburg Dist. [32°40'S:21°31'E], i.1959, Zinn (SAMC); 4_ 2^Merweville, i–ii.1947, Zinn (SAMC); 1^Dikbome Merweville Koup, i.1953, Zinn (SAMC); 3_ 2^Gardiner's Drift Adelaide [32°42'S:26°18'E], iii.1954, S.A.Museum (SAMC); 2_ Somerset East [32°43'S:25°35'E], 1–9.xii.1930, Turner (BMNH); 4_ Somerset East, 25–30.xi.1930, Turner (BMNH); 1_ Cookhouse [32°45'S:25°49'E], 8.ii.1925, Munro HK (NMNH); 2_ 1^Cookhouse, iii.1954, S.A. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Trichardis testacea (Macquart, 1838) Figs 1–3, 49, 50, 60 Type material examined: I have not studied the Macquart types as I am unable to establish their whereabouts. I have studied a number of specimens, in three different collections, that have been labelled as ‘types’ or ‘cotypes’ of testacea Hermann. While it is difficult to establish exactly which of these were actually used by Hermann for his 1906 publication it is certain that at least some of them were not available to him. As Hermann’s description was based on males (number not stated) from ‘Capland, Willowmore (Dr. Brauns)’ I suggest that the following three specimens can be accepted as syntypes: SOUTH AFRICA: 1_ ‘Willowmore [33°17'S:23°29'E] / Capland / Dr. Brauns’, ‘Type von / Trichardis / testacea _ / Herm.’ [orange], ‘Capland / Trichardis / testacea / Type Hr.’ [pink] (ZSMC); 1_ ‘Capland / Willowmor [Willowmore] / 1.2 1906 / Dr. Brauns’, ‘Type von / Trichardis / _ testacea / Herm.’ [orange] (ZSMC); 1_ ‘Cotype’, ‘Capland / Willowmor / 6 12 1906 / Dr. Brauns’, ‘Diptera / Asilidae / Trichardis / testacea / Hermann’, ‘Cotype / Trichardis / testacea / Hermann’ [green] (AMGS). [g ] ( ) The following specimens are females or were either collected at another locality or in the year following the appearance of the description and so should be excluded from any list of syntypes: SOUTH AFRICA: 1^‘Lichtenburg [26°09'S:26°10'E] / Transvaal / Dr. Brauns’, ‘Type von / Trichardis ^/ testacea / Herm.’ [orange] (ZSMC); 1_ [without type label] ‘Capland / Willowmor / 15.2 1907 / Dr. Brauns’, ‘Trichardis / testacea / _ Lw/Herm.’ [white] (ZSMC); 1^‘Capland / Willowmor / 1 1 1907 / Dr. Brauns’, ‘Trichardis / 204 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Londt, Euphorbia & Aloe sp. nr farm “Resolution”; 8_ 5^Grahamstown, Resolution [farm] [33°10'S:26°37'E], 6.ii. (2_), 1.iv. (1^), 1.xii. (1_), 13.xii 1928 (1^), 9.i. (1_), 11.i. (1_), 12.i. (1^), 15.i. (1^), 18.i. (1^), 24.i. (1_), 26.i.1929 (2_), Walton; 3_ 2^same place, 1930, Walton (SAMC); 1^Spitzkop Laingsburg [33°12'S:20°50'E], iii.1937, Mus. Staff (SAMC); 2_ Oukloof Beaufort West [33°15'S:22°06'E], i.1949, Zinn & Hesse (SAMC); 1_ 1^Alicedale New Year's Dam, 3326AC, 5.xi.1978, Miller & Londt, dam edge; 5_ 2^1? Willowmore [33°17'S:23°29'E], 1.xii. (2_ 1^), 7.xii. (1?), 10.xii.1920 (2_ 1^), 3.i.1927 (1_), Brauns; 1_ Willowmore, Brauns (NMNH); 1_ 1^Willowmore, 1.xii.1920, Brauns (NMNH); 1_ 1^Willowmore, 10.xii.1909 (1_), ii.1914, Brauns (SAMC); 2_ 3^Willowmore, ii.1908 (2_), xi.1909 (2^), 25.xii.1915 (1^), Brauns (MRAC); 1_ Willowmore, 12.xii.1906, Brauns (BMNH); 2_ 1^Willowmore, 15.i.1907 (1^), 15.xii. 25.xii. (1_), 1915 (1_), Brauns (MCMI); 1^Willowmore, 1907 Brauns (ZSMC – While labelled a syntype, this specimen was collected a year after Herman's description); 1^Willowmore, Brauns (BMNH); 1? Willowmore Modderfontein, 7.xii.1920, Brauns; 2^Grahamstown [33°18'S:26°32'E], iii.1971, Londt; 1_ Grahamstown, 29.xi.1964, Brothers (AMGS); 2_ Grahamstown, 21.xii.1971, Greathead (BMNH); 1^2 km S Grahamstown, 33°20'S:26°31'E, 800 m, 20.xi.1990, Londt & Whittington, Dassie Krantz forest; 2^Rooinek Lainsburg Dist. [33°20'S:20°55'E], i.1949, Zinn & Hesse (SAMC); 1_ 30 km E of Touws R. to Hondewater [33°20'S:20°02'E], xii.1962, SAM (SAMC); 1^Dunbrody [33°28'S:25°33'E], 1897, O'Neil (SAMC); 1_ nr Highgate Ostrich Farm Oudtshoorn [33°35'S:22°12'E], 10.xii.1986, Gess (AMGS); 1_ Frischgewaagd Oudtshoorn, 33°39'29"S:22°13'18"E, 7–8.xii.1986, Gess (AMGS); 1^Dr Hoek Uitenhage [33°45'S:25°24'E], 11.iii.1919, Munro; 1_ Table farm Grahamstown, 12–25.ii.1971, Gess (AMGS); 1_ Strowan Grahamstown, 11.vii.1968, Gess (AMGS); 2_ 8^Hilton Grahamstown, 5.xi.1969 (1^), 21.xii.1976 (1_), 13.xii.1977 (1_), 14.ii. (1^), 23.ii. (1^), 28.ii. (1^), 17.iii. (2^), 21.iii.1978 (1^), 30.i.1986 (1^), Gess (AMGS); 1_ 1^Pluto's Vale Grahamstown, 6.iii.1960, Jacot-Guillarmod (AMGS); 2_ 2^Boesmans R. [33°42'S:26°40'E], iii.1954, S.A.Museum (SAMC); 1^Algoa Bay [Port Elizabeth, 33°58'S:25°35'E], 25.xii.1896, Brauns (NHMW); 1^Mossel Bay [34°11'S:22°08'E], 15–28.iii.1922, Turner (BMNH); 1_ Mossel Bay, ii.1922, Turner (BMNH); 1_ Cap [Cape] (NHMW); 3^C.B.Sp. [on general label not attached to specimens = Cape of Good Hope] (OXUM). ZIMBABWE: 1^Zambezi, Victoria Falls [17°55'S:25°51'E], vii.1914, Brincker (BMNH); 1_ 1^Sarowa B.B. [?], 17.x.1923, Stevenson (SAMC). Distribution and biology: A fairly commonly encountered, widely distributed and easily recognised southern African species (Fig. 60), being recorded from Namibia, Botswana, Zimbabwe and many localities in South Africa. The species is absent from the winter- rainfall area, the eastern highlands and subtropical coastal areas of southern Africa. Adults fly between July and April (Table 1). AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Museum (SAMC); 1_ Fort Willshire nr Alice [32°47'S:26°50'E], 21.i.1959, Jacot-Guillarmod (AMGS); 5_ 3^Umdala Fort Beaufort [32°48'S:26°39'E], iii.1954, S.A.Museum (SAMC); 2^Mountain Zebra Nat Park, 3225AD, 17–21.xii.1985, Londt, Bushveld vegetation; 2_ 3^16 km E Cradock Farm “Who can tell”, 3225BB, 11.iii.1972, Irwin; 1^Meiringspoort, 3322BC, 11–12.xii.1979, Londt & Stuckenberg, rocky hillside & stream edge; 1_ 22 km NW Grahamstown, Clifton Farm, 3326AB, 3–5.i.1986, Londt & Gess, arid area; 1_ Tierberg Res. Stat. Prince Albert Dist., 33°07'42"S:22°16'24"E, 26.xi–5.xii.1987, Gess (AMGS); 1_ Fort Brown [33°08'S:26°37'E], 23.ii.1928, Walton (AMGS); 1^Fort Brown area, 33°08'38"S:26°38'37"E, 314 m, 21.x.2004, 205 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Personal experience and label data indicate that the species lives predominantly in dry woodland (Acacia and Mopane) and is often associated with sandy stream banks or dry river courses where they are found resting on the ground. Little biological information is available. I am aware of five prey records, four in NMSA collection and one in AMGS. These are: 1_ 3^(from Oudtshoorn, 16 km E Cradock, Soutpan & Middelpos respectively) pinned with sweat bees (Hymeno- ptera: Halictidae), 1^(16 km E Cradock) pinned with a spider-hunting wasp (Hymeno- ptera: Pompilidae). Engel and Cuthbertson (1934) record for testacea ‘Many of both sexes were taken along native paths in mopani forest in December, 1930, at Nympani Vlei near Gatooma. One female has been compared with the type’. Similar species: T. testacea has an entirely pruinose postpronotal lobe and in this respect can be grouped with apicalis, grisescens, ornata, picta, terminalis, turneri and zinidi. Although the species is distinctive in having a number of unique features, such as the clearly marked wings, it is perhaps most similar to ornata. Trichardis turneri: Oldroyd 1974: 120; 1980: 356 (catalogue). Redescription (based on paratype _ from type locality in excellent condition): Head: Dark red-brown except for outer borders of face which are brown-orange, exten- sively silver pruinose (weak on face centrally), white setose. Antenna brown-orange, postpedicel red-brown with brown-orange base, setae orange and black; postpedicel Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 206 not markedly clavate (L:D=3.2:1). Mystax white. Ocellar tubercle with 2 macrosetae. Proboscis dark red-brown, palpi red-brown. Thorax: Brown-orange with dark red-brown areas, extensively silver pruinose, yellow and white setose. Postpronotum entirely pruinose, mesonotum dark red-brown with brown-orange borders, apruinose except for lateral and posterior margins, macrosetae pale yellow, setulae white. Scutellum apruinose except for narrow anterior margin. Anepisternum with pale yellow posterior macroseta, extensively pruinose except for a small anteroventral area. Proepimeron pruinose but weakly posteriorly, katepisternum pruinose but weakly anteriorly, anepisternum entirely pruinose. Legs: Brown-orange, femora orange-brown, pulvilli and empodium of similar length. Hind femur orange- brown, length:height ratio 3.3:1, ventral tubercles well-developed. Hind tibia lacking ventrodistal spur. Wing: 5.2×2.2 mm. Costal vein strongly developed as far as wing tip, then very weakly developed along posterior margin of wing and absent from alula. Membrane extensively microtrichose but microtrichiae absent from parts of proximally situated cells; discal and r5cells entirely microtrichose. LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Abdomen: Dark red-brown, apruinose, macrosetae pale yellow setulae white. T2 dark red-brown, apruinose. p _ genitalia (Figs 51, 52): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view without projections and with about 13 distally arranged macrosetae; mediodistal projection stout, with upturned sclerotised tip. Gonostyli stout, converging distally and ending in upturned slightly curved tips. Aedeagal prongs small, fairly straight. Holotype (examined): SOUTH AFRICA: _ ‘Holo- / type’ [circular, red edge], ‘S.W. Africa. / R. E. Turner. / Brit. Mus. / 1931-12.’, ‘Trichardis / turneri Oldr / det. H. Oldroyd 1972 / Holotype’ [white], ‘Cape Province: / Somerset East. [32°43'S:25°35'E] Nov. 25-30 1930’ (BMNH). Paratypes (examined; all BMNH): SOUTH AFRICA: 1_ ‘Para- / type’, ‘Aliwal North [30°42'S:26°42'E], / Cape Province. / Dec. 1922’, ‘S. Africa. / R. E. Turner. / Brit. Mus. / 1932-45’; 6_ 2^1? ‘Para- / type’ [circular with yellow border], ‘Cape Province / Somerset East. / Nov. 25–30.1930.’, ‘SW Africa / R.E. Turner / Brit. Mus. / 1931–12’; 1^‘Para- / type’, ‘Cape Province / Somerset East. / 10-22.xii.1930.’, ‘S. Africa / R.E. Turner / Brit. Mus. / 1931–37’. Other material examined: SOUTH AFRICA: 1_ 2^Parys [26°54'S:27°27'E], ARC, Fry (SAMC); 1^Bothaville [27°22'S:26°37'E], 30.xii.1964, Brothers (AMGS); 1_ 1^Verdun Kommadagga Dist. [30°18'S:20°59'E], 1.xii.1985, Gess (AMGS); 2_ Dreunberg Burghersdorp [30°59'S:26°19'E], xi.1939, Mus. Staff (SAMC); 1_ 2^3 km E Cradock, 32°10'02"S:25°40'09"E, 956 m, 29.x.2004, Londt, Acacia scrubland with many wild flowers; 1_ Urquhart Park Caravan Park Graaff-Reinet, 32°15'S:24°33'E, 4–6.xii.1988, Londt, riverine vegetation, sandy ground; 1_ Klein Visrivier c. 10 km W Somerset East, 32°44'S:25°30'E, 800 m, 6.xii.1989, Londt, rocky ridge & stream; 2_ 2^Willowmore [33°17'S:23°29'E], 20.i.1908 (1_), ii.1914 (1_), xii.1914 (1^), 25.xii.1914 (1^), Brauns; 1^Willowmore, 10.i.1908, Brauns (SAMC); 1_ Bathurst [33°31'S:26°50'E], 14.i.1959, Jacot-Guillarmod (AMGS). Distribution and biology: A South African endemic restricted to the central parts of the country (Fig. 58). Adults recorded in summer between October and February (Table 1). The species has been collected on sandy ground in Acacia scrubland and in the vicinity of a stream. The biology is otherwise unknown. Similar species: T. turneri has an entirely pruinose postpronotal lobe and in this respect can be grouped with apicalis, grisescens, ornata, picta, terminalis, testacea and zinidi. The species is distinctive, especially with respect to male genital form. Holotype (examined): SOUTH AFRICA: _ ‘Holo- / type’ [circular, red edge], ‘S.W. Africa. / R. E. Turner. / Brit. Mus. / 1931-12.’, ‘Trichardis / turneri Oldr / det. H. Oldroyd 1972 / Holotype’ [white], ‘Cape Province: / Somerset East. [32°43'S:25°35'E] Nov. 25-30 1930’ (BMNH). Paratypes (examined; all BMNH): SOUTH AFRICA: 1_ ‘Para- / type’, ‘Aliwal North [30°42'S:26°42'E], / Cape Province. / Dec. 1922’, ‘S. Africa. / R. E. Turner. / Brit. Mus. / 1932-45’; 6_ 2^1? ‘Para- / type’ [circular with yellow border], ‘Cape Province / Somerset East. / Nov. 25–30.1930.’, ‘SW Africa / R.E. Turner / Brit. Mus. / 1931–12’; 1^‘Para- / type’, ‘Cape Province / Somerset East. / 10-22.xii.1930.’, ‘S. Africa / R.E. Turner / Brit. Mus. / 1931–37’. Trichardis zinidi sp. n. Etymology: Named for the collector Dr I. Abu-Zinid, who donated a number of East African Asilidae to the Natal Museum. Redescription (based on holotype in excellent condition): Head: Orange-brown anteriorly dark red-brown posteriorly, colours masked by silver pruinescence covering entire head, pale yellow and white setose. Antenna brown-orange, postpedicel dark red-brown, yellow setose; postpedicel clavate (L:D=2.6:1). Mystax shiny yellowish. Ocellar tubercle with 4 macrosetae. Proboscis and palpi dark red- brown. Thorax: Orange-brown and dark red-brown patches, gold-silver pruinose, shiny pale yellow setose. Postpronotum entirely pruinose, mesonotum extensively apruinose centrally margins silver pruinose, macrosetae and setulae shiny yellowish. Scutellum apruinose except for narrow anterior margin. Anepisternum with pale yellow posterior macroseta, pruinose except for large anteroventral area. Proepimeron, katepisternum and anepisternum entirely pruinose. Legs: Orange-brown, femora dark red-brown dorsally, pulvilli and empodium of similar length. Hind femur orange-brown, dark red- brown dorsally, length:height ratio 3.6:1, ventral tubercles poorly developed. Hind tibia lacking ventrodistal spur. Wing: 4.8×1.8 mm. Costal vein strongly developed as far as wing tip, then very weakly developed along posterior margin of wing and absent from alula. Membrane extensively microtrichose; discal cell microtrichose but weak proxi- mally, cell r5microtrichose in distal half only. Abdomen: Dark red-brown, extensively silver pruinose, shiny pale yellowish setose. T2 dark red-brown, extensively pruinose (central area apruinose). _ genitalia (Figs 53, 54): Epandrium in lateral view slightly longer than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in ventral view lacking projections and with about 6 medially directed distal macrosetae; mediodistal projections stout, converging distally, with fairly sharply upturned sclerotised distal part. Gonostyli well-developed, bulky, converging distally to upturned flange-like tips. Aedeagal prongs small, more or less straight, with small terminal filamentous tubules. Holotype: KENYA: _ ‘Kenya: Kajiado Dist. / Nguruman area 700 m / 01°50'S:36°56'E / coll: I. Abu-Zinid / Date: 28.iv.1990’ (NMSA). Holotype: KENYA: _ ‘Kenya: Kajiado Dist. / Nguruman area 700 m / 01°50'S:36°56'E / coll: I. Abu-Zinid / Date: 28.iv.1990’ (NMSA). Paratypes: KENYA: 1^same data as holotype (NMSA). TANZANIA: 1^‘Tanzania: 10 km N–NE / di Mto Wa Mbu [?], presso bosco / di euforbia (1100 m), alla luce’ (MZUF). UNKNOWN: 1^‘Afrique / Laga Arba [?] / 25 juillet’, ‘Museum Paris’, ‘Trichardis / H. Oldroyd det. 1965’ (MNHN). Paratypes: KENYA: 1^same data as holotype (NMSA). LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 207 Holotype: KENYA: _ ‘Kenya: Kajiado Dist. / Nguruman area 700 m / 01°50'S:36°56'E / coll: I. Abu-Zinid / Date: 28.iv.1990’ (NMSA). Paratypes: KENYA: 1^same data as holotype (NMSA). TANZANIA: 1^‘Tanzania: 10 km N–NE / di Mto Wa Mbu [?], presso bosco / di euforbia (1100 m), alla luce’ (MZUF). UNKNOWN: 1^‘Afrique / Laga Arba [?] / 25 juillet’, ‘Museum Paris’, ‘Trichardis / H. Oldroyd det. 1965’ (MNHN). Etymology: Named after the country of origin, India. Description (based mainly on holotype, that is in fair condition, but some details taken from paratype; the holotype is missing both mesothoracic legs and parts of the right metathoracic leg, mesonotum and scutellum appear to have been eaten away by dermestid beetles; the paratype is in fair condition although somewhat ‘greasy’): Head: Dark red-brown, extensively dull silver pruinose except for central face, setae pale yellow and white. Antennae red-brown, pale yellow setose except for a few black setae on pedicel; postpedicel elongate spindle-shaped (L:D=3.7:1), with few pale setulae dorsally. Mystax pale yellowish, fairly well-developed. Ocellar tubercle with 2 strong pale yellowish macrosetae. Occipital setae whitish. Proboscis and palpi dark red-brown. Thorax: Dark red-brown, largely apruinose with dull silver pruinose parts, fine setae yellowish, more major setae shiny yellowish. Postpronotum largely apruinose except for narrow medial part, mesonotum largely apruinose except for margins, macrosetae shiny pale yellow, setulae yellowish. Scutellum dark red-brown, apruinose except for anterior margin. Anepisternum with slender pale yellow posterior macroseta, extensively pruinose except anteroventrally. Proepimeron anteriorly pruinose, posteriorly apruinose; katepisternum posteriorly pruinose, anteriorly apruinose; anepisternum largely apruinose. Legs: Orange-brown, pulvilli and empodium of similar length. Hind femur uniformly orange-brown, length:height ratio 3.6:1, ventral tubercles poorly developed, major setae pale yellowish. Hind tibia lacking ventrodistal spur. Wing: 4.0×1.6 mm. Costal vein extends around most of wing margin, weakly along anal cell, absent from alula. Wing membrane extensively microtrichose—discal cell microtrichose, except for tiny proximal part, cell r5microtrichose, but weakly so proximally. Thorax: Dark red-brown, largely apruinose with dull silver pruinose parts, fine setae yellowish, more major setae shiny yellowish. Postpronotum largely apruinose except for narrow medial part, mesonotum largely apruinose except for margins, macrosetae shiny pale yellow, setulae yellowish. Scutellum dark red-brown, apruinose except for anterior margin. Anepisternum with slender pale yellow posterior macroseta, extensively pruinose except anteroventrally. Proepimeron anteriorly pruinose, posteriorly apruinose; katepisternum posteriorly pruinose, anteriorly apruinose; anepisternum largely apruinose. Abdomen (entire abdomen macerated): Terga red-brown with orange-brown central parts, apruinose, setae transparent yellowish. T2 orange-brown, apruinose. p p y g p _ genitalia (Figs 55, 56): Epandrium in lateral view slightly shorter than basal part of gonocoxite (i.e. excluding distal projection of gonocoxite and gonostylus). Proctiger small, moderately dorsoventrally compressed. Hypandrium greatly reduced and simple. Gonocoxite in lateral view somewhat extended proximally and tip somewhat clavate, in ventral view without median projections distally and with long mediodistal setae; mediodistal projection moderately developed with pointed distal end. Trichardis zinidi sp. n. TANZANIA: 1^‘Tanzania: 10 km N–NE / di Mto Wa Mbu [?], presso bosco / di euforbia (1100 m), alla luce’ (MZUF). UNKNOWN: 1^‘Afrique / Laga Arba [?] / 25 juillet’, ‘Museum Paris’, ‘Trichardis / H. Oldroyd det. 1965’ (MNHN). Distribution and biology: Recorded from Kenya and Tanzania. Adults collected in April and July (Table 1). Little label data relating to habitat preference exists, although one specimen is labelled ‘presso bosco di euforbia’ which suggests an arid environment. The life history and prey preferences are unknown. Similar species: T. zinidi has an entirely pruinose postpronotal lobe and in this respect can be grouped with apicalis, grisescens, ornata, picta, terminalis, testacea and turneri. The species is, however, most similar to apicalis. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 208 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 ORIENTAL SPECIES While this paper focuses on the Afrotropical fauna I have identified a single new species of Trichardis from India that represents the first record of the genus from the Oriental Region. ORIENTAL SPECIES While this paper focuses on the Afrotropical fauna I have identified a single new species of Trichardis from India that represents the first record of the genus from the Oriental Region. Trichardis indica sp. n. Etymology: Named after the country of origin, India. Holotype: INDIA: _ ‘Region Himalayenne / Kurséong [26°56'N:80°18'E] (1500 m alt.)’, ‘Museum Paris / Inde / P. Caïus 1924’ (MNHN). Paratype: 1^same data as holotype Holotype: INDIA: _ ‘Region Himalayenne / Kurséong [26°56'N:80°18'E] (1500 m alt.)’, ‘Museu Inde / P. Caïus 1924’ (MNHN). Etymology: Named after the country of origin, India. Gonostylus uniquely shaped—fairly broad basal part, in lateral view, with a long slender subapically positioned dorsal projection that projects out to a similar degree to mediodistal lobe of gonocoxite. Aedeagal prongs more or less straight, with small terminal tubules. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 209 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Note: I have seen and recorded below another specimen from India that was not available at the time this description was drafted. While I list it here, it has no type status and indeed needs to be confirmed as belonging to T. indica: 1_ ‘Coimbatore [11°02'N:76°59'E] / S. India 15-iv-37’ ~ ‘B.M. – C.M. Expdn. / to South India. / April–May 1937’ (BMNH). Distribution and biology: The species is known with certainty from the type locality only. Phenology is uncertain, but a specimen that may be conspecific was collected in April. Nothing is known of its biology. Similar species: This species is of particular interest as it is clearly morphologically most similar to the group of Afrotropical species that has here been called the ‘cribrata species group’. The group is made up of eight species, including indica and the following African taxa—crassipala, cribrata, eburacta, hesperia, malawi, similis and spicata. These are generally small, darkly sclerotised species with entirely microtrichose wings. They are difficult to key without reference to the male genitalia that serve to easily separate the species. T. indica has distinctive male genitalia. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Key to Afrotropical species of Trichardis Although the following key attempts to separate species without reference to characters of the male genitalia it is preferable that well preserved males are used and that identi- fications are confirmed by comparisons of the genitalia with the illustrations provided. 1 Anepisternal macroseta absent; pulvilli reduced in size and clearly shorter than empodium [unknown for rueppelii]; T2 entirely pruinose; _ genitalia as in Figs 27, 28 (Niger, Yemen and various Palaearctic countries) ............................................. .................................. leucocoma (Wulp, 1899) & rueppelii (Wiedemann, 1828) – Anepisternal macroseta present; pulvilli normal and approximately equal in length to empodium; T2 at least partly, if not entirely apruinose .................................... 2 2 Postpronotal lobe entirely pruinose ...................................................................... 3 – Postpronotal lobe partly to extensively apruinose .............................................. 10 3 Scutellum extensively apruinose .......................................................................... 4 – Scutellum extensively pruinose ............................................................................ 8 4 Cell r5extensively microtrichose; usually 2 ocellar macrosetae; hind femur with a few well-developed ventral tubercles (some higher than broad); _ genitalia as in Figs 51, 52 (South Africa) ................................................. turneri Oldroyd, 1974 – Cell r5 microtrichose only in distal half; usually 4 ocellar macrosetae; hind femur with at most poorly developed ventral tubercles (broader than high) .................. 5 5 Anepisternum entirely pruinose (even if only weakly anteroventrally) ............... 6 – Anepisternum apruinose anteroventrally .............................................................. 7 6 T2 yellowish; costal vein continues around wing margin beyond wing tip; wing membrane with distinct dark markings; _ genitalia as in Figs 49, 50 (Botswana, Namibia, South Africa, Zimbabwe) ............................. testacea (Macquart, 1838) – T2 dark red-brown; costal vein continues weakly, if at all, around wing margin beyond wing tip; wing membrane without distinct markings; _ genitalia as in Figs 35, 36 (Chad) ..................................................................................... ornata sp. n. 7 Discal cell extensively microtrichose; hind femur dark red-brown; T2 dark red- brown; _ genitalia as in Figs 53, 54 (Kenya, Tanzania) ...................... zinidi sp. n. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 210 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 – Discal cell entirely lacking microtrichiae; hind femur brown-orange; T2 orange; _ genitalia as in Figs 7, 8 (Namibia, Mozambique, South Africa, Zimbabwe) ......... ...........................................................................................apicalis Oldroyd, 1974 8 Anepisternum extensively apruinose; T2 apruinose ............................................. 9 – Anepisternum entirely pruinose; T2 extensively pruinose (Namibia, South Africa) ..............................................................................................picta Hermann, 1906 9 Scutellar disc entirely pruinose; male terminalia not markedly elongate, as in Figs 19, 20 (Ethiopia, Gambia, Kenya, Senegal) ..................... Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Key to Afrotropical species of Trichardis grisescens Engel, 1924 – Scutellar disc partly apruinose (i.e. weak centrally); male terminalia markedly elongate, as in Figs 47, 48 (Botswana, Namibia, Zimbabwe) ................................ ...................................................................................... terminalis Oldroyd, 1974 10 Hind tibia with well-developed ventrodistal spur............................................... 11 – Hind tibia lacking ventrodistal spur.................................................................... 12 11 Mystax entirely blackish; T2 entirely apruinose; _ genitalia as in Figs 25, 26 (Somalia) ......................................................................................... lavignei sp. n. – Mystax with both shiny yellowish and black macrosetae; T2 apruinose except for silver pruinose patches posterolaterally; _ genitalia as in Figs 23, 24 (DR Congo, Tanzania)...................................................................katangaensis Oldroyd, 1970 12 Discal cell lacking microtrichiae, especially proximally and adjacent to veins..13 – Discal cell extensively microtrichose (members of cribrata species group) ..... 18 13 Mystax entirely white, yellow or orange, lacking black setae; antennal segments predominantly yellowish or light brownish ........................................................ 14 – Mystax with some black setae; antennal segments entirely or almost entirely dark red-brown............................................................................................................ 16 14 Antennal postpedicel clavate (L:D < 3.0); costal vein ends at wing tip; veins R5+M1, M2 and M3+CuA1 not reaching wing margin (Fig. 4); cell r5almost entirely lacking microtrichiae; _ genitalia as in Figs 15, 16 (Namibia, South Africa)..................... .......................................................................................................... effrena sp. n. – Antennal postpedicel spindle shaped (L:D > 3.0); costal vein continues around wing tip and along much of hind margin of wing; veins R5+M1, M2 and M3+CuA1 reaching wing margin (e.g. Fig. 3); cell r5at least weakly microtrichose ........... 15 15 A yellowish or orange species; _ genitalia as in Figs 31, 32 (Eritrea) ................... ..........................................................................................................mellina sp. n. – A dark red-brown to blackish species; _ genitalia as in Figs 17, 18 (Gambia) ...... ........................................................................................................... glabra sp. n. 16 Postpronotal and postalar lobes orange and contrasting with dark red-brown to blackish mesonotum; legs ventrally orange-brown; smaler species (wing length ca 4.0 mm); _ genitalia as in Figs 41, 42 (Socotra I., Yemen) .................................... ..................................................................................... pohli Geller-Grimm, 2002 – Postpronotal and postalar lobes blackish and not contrasting with mesonotum; legs uniform dark red-brown to blackish; bigger species (wing length ca 5.5 mm).. 17 17 Mesonotal macrosetae, anepisternal seta(e) and ocellar setae black; _ genitalia as in Figs 33, 34 (Socotra I., Yemen) .............................. nigrescens (Ricardo, 1903) Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 211 – Mesonotal macrosetae, anepisternal seta(e) and ocellar setae yellowish; _ genitalia as in Figs 5, 6 (Abd el Kuri I., Yemen) .........................................abdelkuri sp. n. Key to Afrotropical species of Trichardis 18 Pleural pruinescence confined to anepisternum (i.e. proepimeron, katepisternum and anepimeron uniformly pruinose); in males the greatly expanded (trumpet-like) distal tips of the three aedeagal openings are clearly visible; _ genitalia as in Figs 29, 30 (Malawi, Tanzania, Zimbabwe) ............................................. malawi sp. n. – Anterior part of katepisternum and posterior part of proepimeron shiny apruinose; male aedeagal openings not markedly trumpet-like ........................................... 19 19 Anepimeron with an extensive apruinose area; _ genitalia as in Figs 21, 22 (Gambia, Senegal) .......................................................................................... hesperia sp. n. Note: The Oriental species, indica sp. n., from India, keys out here and can be separated from hesperia on characters of the _ genitalia, Figs 55, 56) – Anepimeron entirely pruinose ............................................................................ 20 20 Hypopygium dark red-brown and not contrasting with proximally situated abdominal terga .................................................................................................................... 21 – Hypopygium dark somewhat orange and contrasting with proximally situated abdominal terga that are dark red-brown ............................................................ 22 21 _ genitalia as in Figs 11, 12 (Lesotho, Mozambique, South Africa, Zimbabwe) ... ........................................................................................... cribrata (Loew, 1858) – _ genitalia as in Figs 45, 46 (Mozambique) ..................................... spicata sp. n. 22 _ genitalia as in Figs 9, 10 (Burkina Faso, Niger)....................... crassipala sp. n. – _ genitalia as in Figs 13, 14 (Ivory Coast, Nigeria) ..................... eburacta sp. n. – _ genitalia as in Figs 43, 44 (Malawi) .............................................. similis sp. n. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Taxonomy Hoplistomerus Macquart – Antennal postpedicel moderately elongate to clavate; proboscis small and hardly protruding beyond lower epistomal margin; hind leg of male usually lacking a distoventral tibial spur; hind femur usually with tubercles, which, when present, do not coalesce; usually small species (wing length <7 mm) lacking obvious golden abdominal setation. _ genitalia: Gonocoxite rarely with median projection (when present it is short and medially directed) and commonly with mediodistal macrosetae ................................................................................................Trichardis Hermann ...................................................................................... Hoplistomerus Macquart – Antennal postpedicel moderately elongate to clavate; proboscis small and hardly protruding beyond lower epistomal margin; hind leg of male usually lacking a distoventral tibial spur; hind femur usually with tubercles, which, when present, do not coalesce; usually small species (wing length <7 mm) lacking obvious golden abdominal setation. _ genitalia: Gonocoxite rarely with median projection (when present it is short and medially directed) and commonly with mediodistal macrosetae ................................................................................................Trichardis Hermann When attempting to draw up a key to Afrotropical Trichardis it became evident that there was at least one fairly distinctive ‘species group’. The species in this group, comprising seven Afrotropical species (crassipala, cribrata, eburacta, hesperia, malawi, similis, spicata), were difficult to separate using easily observed external characters, but possess highly distinctive male genitalia. As only one of these species (cribrata) had been described, I suggest that the group be called the ‘cribrata species group’. Species in this group are widely distributed, occurring mainly in West Africa, but also in East and Southern Africa. Of interest is the fact that the newly described Oriental species (indica) appears also to belong to this group. There may be other groups that can be detected, but as these can only be very poorly defined I prefer to refrain from making further comment on the matter apart from saying that I believe ‘related’ or ‘sister species’ tend to key out together in the key provided above. Taxonomy Londt (2007a) discussed the situation regarding the characterisation of Afrotropical laphriine genera previously considered to belong to the subfamily Laphystiinae (i.e. Perasis, Hoplistomerus and Trichardis), saying that these rather similar genera had not been adequately separated using published keys. Londt (2007a) gave a provisional key to these genera stating that it would probably have to be updated following revisions of the genera. These studies have now been completed, and, although the provisional key works reasonably well for most of the species, a new key is provided below and is con- sidered superior. Key to genera of Afrotropical Laphriinae previously classified as Laphystiinae 1 C continues fairly strongly along entire wing margin, including anal cell and alula; postpronotal lobe without macrosetae; covered with tiny setulae only; hind femora slender (length/breadth ratio >5) and lacking tubercles; abdominal terga lacking discal setae beyond T1. _ genitalia: Epandrium in dorsal view deeply incised (i.e. lobes evident for about half length); hypandrium moderately well-developed and gonocoxites widely separated ventrally..................................... Perasis Hermann 1 C continues fairly strongly along entire wing margin, including anal cell and alula; postpronotal lobe without macrosetae; covered with tiny setulae only; hind femora slender (length/breadth ratio >5) and lacking tubercles; abdominal terga lacking discal setae beyond T1. _ genitalia: Epandrium in dorsal view deeply incised (i.e. lobes evident for about half length); hypandrium moderately well-developed and gonocoxites widely separated ventrally..................................... Perasis Hermann – C continues fairly strongly along wing margin to Cu+A1 before becoming much weaker along anal cell and completely absent from alula; postpronotal lobe with at 212 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 least a few long and sometimes strongly developed macrosetae; hind femora robust (length/breadth ratio <4), with a swollen appearance and usually equipped with tubercles; abdominal terga with discal setae beyond T1. _ genitalia: Epandrium in dorsal view hardly incised (i.e. not divided into lobes); hypandrium usually absent or at best poorly developed and gonocoxites usually closely associated ventrally ............................................................................................................... 2 y 2 Antennal postpedicel elongate; proboscis large and protruding well beyond lower epistomal margin; hind tibia of male always with a distoventral spur; hind femur always with well-developed tubercles, the largest often coalescing with each other; usually large species (wing length >7 mm), commonly with golden abdominal setation. _ genitalia: Gonocoxite always with median projection which is usually elongate and distally directed, and lacking mediodistal macrosetae ...................... ...................................................................................... ACKNOWLEDGEMENTS Curators of the museums who have kindly hosted me or sent specimens for study are gratefully acknowledged for their participation and assistance. Dr Mike Mostovski (Natal Museum) is thanked for his encouragement and helpful editorial advice. Mrs Heidi Snyman (Ezemvelo KZN Wildlife) is thanked for generating the distribution maps. The University of KwaZulu-Natal allocated funding in support of my research, while the Natal Museum provided laboratory space and library services. I also wish to acknowledge the assistance of all the conservation authorities who have issued collecting permits over the many years I have been working on Afrotropical Asilidae. My wife Ann is thanked for all the assistance she has rendered me especially in relation to my field work. Biology Trichardis appears to be a genus largely restricted to grassland and savannah biomes. Fig. 57 demonstrates its absence from tropical and subtropical areas where forests dominate, and from the winter-rainfall area of the south-western parts of South Africa, which is dominated by succulents and fynbos. Nothing is known about the immature stages of any Trichardis species and biological data relating to the adults are fragmentary. It appears that all species are normally encountered resting on the ground (on sand or peddles) in savannah or woodland biomes (see Londt 1994). Londt (2006) mentions Trichardis in a discussion of asilid predation and this information is added here too. Only six prey records are known to me, four for T. testacea and two for T. picta. Except for a single dipteran (Tachinidae), all prey items are hymenopterans (4 Halictidae, 1 Masaridae, 1 Pompilidae). The possible predilection for Hymenoptera, and Halictidae in particular, is interesting, but probably not significant as these insects are also commonly found resting on the ground and therefore easily accessible as prey. Distribution and phenology Trichardis is widely distributed throughout the Afrotropical region (Fig. 57) where the vast majority of species are to be found. The genus is, however, also represented by a few widely scattered species within the Palaearctic and Oriental regions. Only one species, T. leucocoma, has been recorded from two regions. This species is primarily Palaearctic as there are only a few records of it in the Afrotropical region. The limited data available to me suggest, however, that it may be a more widely distributed than currently appreciated. Of the 25 Afrotropical species, eight (32%) are found only in Southern Africa (apicalis, cribrata, effrena, picta, spicata, terminalis, testacea, turneri). Nine (36%) are known only from Eastern Africa (abdelkuri, lavignei, mellina, nigrescens, ornata, pohli, rueppelii, similis, zinidi), three of these being confined to the small Yemenese islands of Socotra and Abd el Kuri (abdelkuri, nigrescens, pohli). Three (12%) species are Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 213 known only from Western Africa (eburacta, glabra, hesperia). Two species (8%) are widespread between East and West Africa (crassipala, grisescens), one (4 %) is distri- buted between East and Central Africa (katangaensis), and another is found in both East and Southern Africa (malawi). known only from Western Africa (eburacta, glabra, hesperia). Two species (8%) are widespread between East and West Africa (crassipala, grisescens), one (4 %) is distri- buted between East and Central Africa (katangaensis), and another is found in both East and Southern Africa (malawi). Bearing in mind that the genus is found straddling the equator, without exception, adult Trichardis appear to be active during the warmer, summer months of the year (Table 1). Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 GELLER-GRIMM, F. 1999. Raubfliegen-Typenmaterial des Senckenberg-Museums in Frankfurt am Main, das überwiegend von Wiedemann und Jaennicke bearbeitet wurde (Insecta, Diptera, Asilidae). Senckenbergiana biologica 78 (1/2): 205–217. g g ( ) ––––––2002. Robber flies (Diptera: Asilidae) of the Socotra Archipelago, Yemen. Fauna of Arabia 19: 467–489. HERMANN, F. 1906. Beitrag zur Kenntnis der Asiliden (II.) (Dipt.). Zeitschrift für Systematische Hymenoptero- logie und Dipterologie 6: 129–144. g p g ––––––1920. Beitrag zur allgemeinen Systematik der Asiliden. Zoologische Jahrbücher, Abteilung für Systematik 43: 161–194. y HULL, F.M. 1962. Robber flies of the World. The genera of the family Asilidae. Bulletin of the United Stat National Museum 224 (1): 1–430, (2): 431–907. ( ) ( ) KERTÉSZ, C. 1909. Catalogus dipterorum hucusque descriptorum. IV. Oncodidae, Nemestrinidae, Mydaida Apioceridae, Asilidae. Budapest: Museum Nationale Hungaricum, pp. 1–348. p p g pp LEHR, P. A. 1988. Family Asilidae. In: Soos, A. & Papp, L., eds, Catalogue of Palaearctic Diptera. Vol. 5. Amsterdam: Elsevier, pp. 197–326. pp LOEW, H. 1858 [1857]. Bidrag till kannedomen om Afrikas Diptera [part]. Öfversigt af Kongliga Vetenskaps- Akademiens Förhandlingar (Stockholm) 14: 337–383. g ( ) 1860. Die Dipteren-Fauna Südafrika’s. Erste Abtheilung. Abhandlungen des Naturwissenschaftlichen Vereins für Sachsen und Thüringen in Halle 2 (1858–1861): 57–402. f g , J.G.H. 1994. Afrotropical Asilidae (Diptera) 26. Ethological observations, and a possible ecological classification based on habitats. Annals of the Natal Museum 35: 97–122. f –2004. A revision of Laphystotes Oldroyd, with the description of a new species, and a key to the genera of afrotropical Laphriinae (Diptera: Asilidae). African Entomology 12 (1): 19–28. –2006. Predation by Afrotropical Asilidae (Diptera): An analysis of 2000 prey records. African Entomology 14 (2): 317–328. –2007a. A review of Afrotropical Perasis (Hermann, 1905) (Diptera: Asilidae: Laphriinae). Zootaxa 1521: 9–18. 2007b. A review of the genus Hoplistomerus Macquart, 1838 (Diptera: Asilidae: Laphriinae). African Invertebrates 48 (2): 167–198. UART, P.J.M. 1838. Diptères exotiques nouveaux ou peu connus. Mémoires de la Société (Royale) des Sciences, de l'Agriculture et des Arts à Lille 1 (2): 5–207. , g ( ) MCALPINE, J. F. 1981. Morphology and terminology—Adults. In: McAlpine, J.F. et. al., eds, Manual of Nearctic Diptera. Vol. 1. Monograph 27. Ottawa: Agriculture Canada, Research Branch, pp. 9–63. pp OLDROYD, H. 1970. Studies of African Asilidae (Diptera). 1. Asilidae of the Congo basin. Bulletin of the British Museum (Natural History). Entomology, Supplement 24 (7): 207–334. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use REFERENCES AUSTEN, E.E. 1914. On Diptera collected in the western Sahara by Dr. Ernst Hartert, with descriptions of new species. Novitates Zoologicae 21: 265–274. p g ECKER, T. 1907. Die Ergebnisse meiner dipterologischen Frühjahrsreise nach Algier und Tunis. 906. [part Zeitschrift für systematische Hymenopterologie und Dipterologie 7: 33–61. f f y y p g p g EFFLATOUN, H. C. 1937. A monograph of Egyptian Diptera. Part V. Family Asilidae (Section II). Mémoires de la Société Royal Entomologique d'Égypte 4 (3): 199–443 + pls IV–VIII. S y g q gyp (3) 99 3 p ENGEL, E.O. 1924. Studien über afrikanische Dipteren (Asiliden). Wiener Entomologische Zeitung 41: 100– 110. ENGEL, E.O. & CUTHBERTSON, A. 1934. Systematic and biological notes on some Asilidae (Diptera) of Southern Rhodesia with a description of a species new to science. Proceedings of the Rhodesia Scientific Association 34 (1): 35–47. NGEL, E.O. & CUTHBERTSON, A. 1934. Systematic and biological notes on some Asilidae (Diptera) of Souther Rhodesia with a description of a species new to science. Proceedings of the Rhodesia Scientifi –––1939. Systematic and biological notes on some brachycerous Diptera of Southern Rhodesia. Journal of the Entomological Society of Southern Africa 11: 181–195. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 214 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Species Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun abdelkuri – – – – – – – – – – • – apicalis – – – – • • • • • – – – crassipala • • – – – – – – – – • – cribrata • – – • • • • • • – – – eburacta – – – – – – – – – • – • effrena – – – – – – • • • – – – glabra – – – – – – – – – • • – grisescens – – – – • – • • • • • – hesperia – • – – – – – – – – • – katangaensis – – – • • • – – – – – – lavignei – – – – – – – – – – • • leucocoma – • – – – – – • • • • – malawi – – – – • • – – – – – – mellina – – – – • – – – – – – • nigrescens – – – • – • • – – – – – ornata – • – – – – – – – – – – picta – – – • • • • • • – – – pohli – – – – – – – • – • – – rueppelii – – – – – – – – – – – – similis – – – – – • – – – – – – spicata – – – – – • – – – – – – terminalis – – • • • • – • – – – – testacea • • • • • • • • • • – – turneri – – – • • • • • – – – – zinidi • – – – – – – – – • – – No. species 4 5 2 7 10 11 8 10 7 7 7 3 TABLE 1 Phenology of Afrotropical Trichardis species. Index to Trichardis species described or revised in this paper (valid names in bold face) Page abdelkuri sp. n. ..................................... 175, 211, 216 albipila Becker, 1907 = leucocoma ......................189 apicalis Oldroyd, 1974 ..........................176, 210, 216 crassipala sp. n. .................................... AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 ( y) gy pp ( ) ––––––1974. An introduction to the robber flies (Diptera: Asilidae) of southern Africa. Annals of the Natal Museum 22 (1): 1–171. ––––––1980. Family Asilidae. In: Crosskey, R.W., ed., Catalogue of the Diptera of the Afrotropical Region. London: British Museum (Natural History), pp. 334–373, 1218, 1226, 1229. ( y) pp RICARDO, G. 1903. Insecta: Diptera. In: Forbes, H.O., ed., The natural history of Sokotra and Abd-el-Kur etc. Bulletin of the Liverpool Museum (Special): 357–378. p THEODOR, O. 1980. Diptera: Asilidae. Fauna Palaestina, Insecta II. Jerusalem: Israel Academy of Scienc and Humanities. WIEDEMANN, C.R.W. 1828. Aussereuropaische zweiflügelige Insekten als Fortsetzung des Meigenschen Werkes v. 1. Pt 8. Familie: Räuberfliegen (Asilici). Hamburg: Schulzischen Buchhandlung, 1828–1830. WIEDEMANN, C.R.W. 1828. Aussereuropaische zweiflügelige Insekten als Fortsetzung des Meigenschen Werkes v 1 Pt 8 Familie: Räuberfliegen (Asilici) Hamburg: Schulzischen Buchhandlung 1828 1830 WIEDEMANN, C.R.W. 1828. Aussereuropaische zweiflügelige Insekten als Fortsetzung des Meigenschen Werkes v. 1. Pt 8. Familie: Räuberfliegen (Asilici). Hamburg: Schulzischen Buchhandlung, 1828–1830. WULP, F.M. VAN DER. 1899. Asilidae from Aden and its neighbourhood. Transactions of the Entomological Society of London 1899: 81–98. f g ( ) g g, WULP, F.M. VAN DER. 1899. Asilidae from Aden and its neighbourhood. Transactions of the Entomological Society of London 1899: 81–98. LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 215 TABLE 1 Phenology of Afrotropical Trichardis species. Species Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun abdelkuri – – – – – – – – – – • – apicalis – – – – • • • • • – – – crassipala • • – – – – – – – – • – cribrata • – – • • • • • • – – – eburacta – – – – – – – – – • – • effrena – – – – – – • • • – – – glabra – – – – – – – – – • • – grisescens – – – – • – • • • • • – hesperia – • – – – – – – – – • – katangaensis – – – • • • – – – – – – lavignei – – – – – – – – – – • • leucocoma – • – – – – – • • • • – malawi – – – – • • – – – – – – mellina – – – – • – – – – – – • nigrescens – – – • – • • – – – – – ornata – • – – – – – – – – – – picta – – – • • • • • • – – – pohli – – – – – – – • – • – – rueppelii – – – – – – – – – – – – similis – – – – – • – – – – – – spicata – – – – – • – – – – – – terminalis – – • • • • – • – – – – testacea • • • • • • • • • • – – turneri – – – • • • • • – – – – zinidi • – – – – – – – – • – – No. species 4 5 2 7 10 11 8 10 7 7 7 3 TABLE 1 Phenology of Afrotropical Trichardis species. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 178, 211, 216 cribrata (Loew, 1858) .......................... 179, 211, 217 cribratus = cribrata ..............................................179 eburacta sp. n. ...................................... 181, 211, 217 effrena sp. n. .........................................182, 210, 217 glabra sp. n. ..........................................183, 210, 218 grisescens Engel, 1924..........................184, 210, 218 grisescens Hermann, 1920 = grisescens Engel, 1924 ...................................184 hesperia sp. n. ....................................... 185, 211, 218 indica sp. n. ...................................................208, 224 katangaensis Oldroyd, 1970 .................186, 210, 219 lavignei sp. n. ........................................188, 210, 219 leucocoma (Wulp, 1899).......................188, 209, 219 leucocomus ............................................................189 lucifer Oldroyd, 1974 = picta .......................195, 221 Index to Trichardis species described or revised in this paper (valid names in bold face) Page lucifera Oldroyd, 1980 = picta ............................195 malawi sp. n. ........................................ 191, 211, 220 mellina sp. n. ........................................192, 210, 220 nigrescens (Ricardo, 1903) ..................193, 210, 220 ornata sp. n. .........................................194, 209, 221 picta Hermann, 1906 ............................195, 210, 221 pohli Geller-Grimm, 2002....................197, 210, 222 rueppelii (Wiedemann, 1828) ......................198, 209 rueppellii = rueppelii ...........................................198 rufescens Austen, 1914 = leucocoma...................189 ruppelii = rueppelii ..............................................198 similis sp. n........................................... 199, 211, 222 spicata sp. n. ........................................ 200, 211, 222 terminalis Oldroyd, 1974 ....................201, 210, 223 testacea (Macquart, 1838) ...................203, 209, 223 testacea Hermann, 1906 = testacea (Macquart, 1838) .............................203 turneri Oldroyd, 1974 .........................205, 209, 223 zinidi sp. n. ...........................................207, 209, 224 Page abdelkuri sp. n. ..................................... 175, 211, 216 albipila Becker, 1907 = leucocoma ......................189 apicalis Oldroyd, 1974 ..........................176, 210, 216 crassipala sp. n. .................................... 178, 211, 216 cribrata (Loew, 1858) .......................... 179, 211, 217 cribratus = cribrata ..............................................179 eburacta sp. n. ...................................... 181, 211, 217 effrena sp. n. .........................................182, 210, 217 glabra sp. n. ..........................................183, 210, 218 grisescens Engel, 1924..........................184, 210, 218 grisescens Hermann, 1920 = grisescens Engel, 1924 ...................................184 hesperia sp. n. ....................................... 185, 211, 218 indica sp. n. ...................................................208, 224 katangaensis Oldroyd, 1970 .................186, 210, 219 lavignei sp. n. ........................................188, 210, 219 leucocoma (Wulp, 1899).......................188, 209, 219 leucocomus ............................................................189 lucifer Oldroyd, 1974 = picta .......................195, 221 Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 216 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Figs 5–10. Trichardis species, male genitalia: (5, 6) lateral and ventral of T. abdelkuri sp. n., paratype, Jebel Saleh; (7, 8) lateral and ventral of T. apicalis Oldroyd, 1974, paratype, Ndumu Game Reserve; (9, 10) lateral and ventral of T. crassipala sp. n., paratype, Ouagadougou. Scale lines = 1 mm. Figs 5–10. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Trichardis species, male genitalia: (5, 6) lateral and ventral of T. abdelkuri sp. n., paratype, Jebel Saleh; (7, 8) lateral and ventral of T. apicalis Oldroyd, 1974, paratype, Ndumu Game Reserve; (9, 10) lateral and ventral of T. crassipala sp. n., paratype, Ouagadougou. Scale lines = 1 mm. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 217 Figs 11–16. Trichardis species, male genitalia: (11, 12) lateral and ventral of T. cribrata (Loew, 1858), Mhlopeni Nat. Res.; (13, 14) lateral and ventral of T. eburacta sp. n., paratype, Comoé National Park; (15, 16) lateral and ventral of T. effrena sp. n., paratype, Witsand Nat. Res. Scale lines = 1 mm. Figs 11–16. Trichardis species, male genitalia: (11, 12) lateral and ventral of T. cribrata (Loew, 1858), Mhlopeni Nat. Res.; (13, 14) lateral and ventral of T. eburacta sp. n., paratype, Comoé National Park; (15, 16) lateral and ventral of T. effrena sp. n., paratype, Witsand Nat. Res. Scale lines = 1 mm. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 218 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Figs 17–22. Trichardis species, male genitalia: (17, 18) lateral and ventral of T. glabra sp. n., paratype, Bansang; (19, 20) lateral and ventral of T. grisescens Engel, 1924, holotype; (21, 22) lateral and ventral of T. hesperia sp. n., paratype, Kayes. Scale lines = 1 mm. Figs 17–22. Trichardis species, male genitalia: (17, 18) lateral and ventral of T. glabra sp. n., paratype, Bansang; (19, 20) lateral and ventral of T. grisescens Engel, 1924, holotype; (21, 22) lateral and ventral of T. hesperia sp. n., paratype, Kayes. Scale lines = 1 mm. LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 219 Figs 23–28. Trichardis species: (23, 24) T. katangaensis Oldroyd, 1970, male genitalia, paratype, Kapanga: (23) lateral, (24) ventral; (25, 26) T. lavignei sp. n., holotype: (25) male genitalia, dorsolateral, (26) posterior view of distal end of right hind tibia; (27, 28) T. leucocoma (Wulp, 1899), male genitalia, paratype, Shaik Othman: (27) lateral, (28) ventral. Scale lines = 1 mm. Figs 23–28. Trichardis species: (23, 24) T. katangaensis Oldroyd, 1970, male genitalia, paratype, Kapanga: (23) lateral, (24) ventral; (25, 26) T. lavignei sp. n., holotype: (25) male genitalia, dorsolateral, (26) posterior view of distal end of right hind tibia; (27, 28) T. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) leucocoma (Wulp, 1899), male genitalia, paratype, Shaik Othman: (27) lateral, (28) ventral. Scale lines = 1 mm. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 220 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Figs 29–34. Trichardis species, male genitalia: (29, 30) lateral and ventral of T. malawi sp. n., paratype, Monkey Bay; (31, 32) lateral and ventral of T. mellina sp. n., paratype, Ghinda; (33, 34) lateral and ventral of T. nigrescens (Ricardo, 1903), Homhil. Scale lines = 1 mm. Figs 29–34. Trichardis species, male genitalia: (29, 30) lateral and ventral of T. malawi sp. n., paratype, Monkey Bay; (31, 32) lateral and ventral of T. mellina sp. n., paratype, Ghinda; (33, 34) lateral and ventral of T. nigrescens (Ricardo, 1903), Homhil. Scale lines = 1 mm. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 221 Figs 35–40. Trichardis species, male genitalia: (35, 36) lateral and ventral of T. ornata sp. n., holotype, (note: X indicates broken structures); (37–40) T. picta Hermann, 1906: (37, 38) lateral and ventral of holotype, (39, 40) lateral and ventral of holotype of T. lucifer Oldroyd, 1974. Scale lines = 1 mm. Figs 35–40. Trichardis species, male genitalia: (35, 36) lateral and ventral of T. ornata sp. n., holotype, (note: X indicates broken structures); (37–40) T. picta Hermann, 1906: (37, 38) lateral and ventral of holotype, (39, 40) lateral and ventral of holotype of T. lucifer Oldroyd, 1974. Scale lines = 1 mm. 222 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Figs 41–46. Trichardis species, male genitalia: (41, 42) lateral and ventral of T. pohli Geller-Grimm, 2002, Sokotra I.; (43, 44) lateral and ventral of T. similis sp. n., paratype, Kasungu National Park; (45, 46) lateral and ventral of T. spicata sp. n., paratype, 30 km S Caia. Scale lines = 1 mm. Figs 41–46. Trichardis species, male genitalia: (41, 42) lateral and ventral of T. pohli Geller-Grimm, 2002, Sokotra I.; (43, 44) lateral and ventral of T. similis sp. n., paratype, Kasungu National Park; (45, 46) lateral and ventral of T. spicata sp. n., paratype, 30 km S Caia. Scale lines = 1 mm. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 223 Figs 47–52. Trichardis species, male genitalia: (47, 48) lateral and ventral of T. terminalis Oldroyd, 1974, holotype; (49, 50) lateral and ventral of T. testacea (Macquart, 1838), holotype; (51, 52) lateral and ventral of T. turneri Oldroyd, 1974, Graaff-Reinet. Scale lines = 1 mm. Figs 47–52. Trichardis species, male genitalia: (47, 48) lateral and ventral of T. terminalis Oldroyd, 1974, holotype; (49, 50) lateral and ventral of T. testacea (Macquart, 1838), holotype; (51, 52) lateral and ventral of T. turneri Oldroyd, 1974, Graaff-Reinet. Scale lines = 1 mm. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 224 AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 Figs 53–56. Trichardis species, male genitalia: (53, 54) lateral and ventral of T. zinidi sp. n., holotype; (55, 56) lateral and ventral of T. indica sp. n., holotype. Scale lines = 1 mm. Figs 53–56. Trichardis species, male genitalia: (53, 54) lateral and ventral of T. zinidi sp. n., holotype; (55, 56) lateral and ventral of T. indica sp. n., holotype. Scale lines = 1 mm. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) 225 LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) Fig 57. The distribution of Afrotropical Trichardis species. istributions of southern African Trichardis species: ■ – T. apicalis Oldroyd, 1974, 9 – T Oldroyd, 1974, • – T. turneri Oldroyd, 1974. Fig 57. The distribution of Afrotropical Trichardis species. Fig 58. Distributions of southern African Trichardis species: ■ – T. apicalis Oldroyd, 1974, 9 – T. terminalis Oldroyd, 1974, • – T. turneri Oldroyd, 1974. Fig 58. Distributions of southern African Trichardis species: ■ – T. apicalis Oldroyd, 1974, 9 – T. terminal Oldroyd, 1974, • – T. turneri Oldroyd, 1974. Fig 58. Distributions of southern African Trichardis species: ■ – T. apicalis Oldroyd, 1974, 9 – T. terminalis Oldroyd, 1974, • – T. turneri Oldroyd, 1974. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use AFRICAN INVERTEBRATES, VOL. 49 (2), 2008 226 Fig 59. Distributions of southern African Trichardis species: – T. cribrata (Loew, 1858), 9 – T. effrena sp. n., 1974, • – T. picta Hermann, 1906. Fig 59. Distributions of southern African Trichardis species: – T. cribrata (Loew, 1858), 9 – T. LONDT: AFROTROPICAL TRICHARDIS (ASILIDAE: LAPHRIINAE) effrena sp. n., 1974, • – T. picta Hermann, 1906. Fig 60. Distribution of Trichardis testacea (Macquart, 1838) in southern Africa. Fig 60. Distribution of Trichardis testacea (Macquart, 1838) in southern Africa. Downloaded From: https://bioone.org/journals/African-Invertebrates on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use
https://openalex.org/W4288279636	https://zenodo.org/record/3369736/files/Psychiatry%20Diagnoses%20Coercion%20in%20Government%2C%20Towards%20a%20Unified%20Theory%20of%20Psychiatric%20Disorders.pdf	English	null	Psychiatry Diagnoses Coercion in Government, Towards a Unified Theory of Psychiatric Disorders	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	1,734	1.1 Psychiatric disorders, a unified theory Executive function (EF) or executive control refers to the higher order cognitive processes involved in the conscious control of behavior, thought, and emotion, essential for successful navigation through a complex social world inundated with intricate norms and moral values. (Forbes, 2010) Over 2500 scientific articles have been published on this topic in the past 25 years. (Alvarez, 2006) The executive (ego) function recruits from a wide range of functional abilities that are orchestrated in part by the frontal lobes, the term “frontal functions” often used synonymously with “executive functions”. (Alvarez, 2006) Impairment of the ego function, usually defined as executive dysfunction, executive function disorder, or executive disorder in short, affects the higher order cognitive functions in this “orchestrator of the mind”, and psychiatric disorders are classified based on the severity of impairment. Psychiatry Diagnoses Coercion in Government, Towards a Unified Theory of Psychiatric Disorders Johan Nygren, johanngrn@gmail.com ABSTRACT: Executive function (EF) refers to the higher order cognitive processes involved in the conscious control of behavior, thought, and emotion. Impairment of the executive function characterizes all psychiatric diagnoses. This article presents the theory that subordinating the executive function of a person through coercion is a direct cause of executive dysfunction, and that the psychiatric disorders that are diagnosed by psychiatric science reflect coercion used in government. 1.2 Executive disorder, not mental disorder The loss of executive function, i.e., the ability to manipulate memes, makes it difficult for the individual to absorb, process, and integrate new information. (Leigh, 2010) In medical terms, failure of the executive function can be understood as analogous to failure of any organ function. It is the paralysis of this control mechanism of the brain that defines psychiatric disorders, and it is responsible for mental illness, a secondary disease caused by executive disorder, the loss of authority over the contents of the mind. The executive function is above the mind in a hierarchy of control, and per deductive reasoning, the term mental disorder is a causal impossibility, the equivalent of spontaneous generation theory for mental illness, and now an obsolete medical theory. 1.3 Mental illness as “meme illness” Hoyle Leigh, psychiatrist and MD who co-founded the Yale Institute of Behavioral Medicine in the 70s, and went on to write Genes, Memes, Culture and Mental Illness: Towards an Integrative Model in 2010, defines mental illness in that it is caused by pathological memes, and studies diseases of the mind as memetic diseases. (Leigh, 2010) The word mental, from Latin mentalis "of the mind," can be understood as a primitive version of meme theory, with memes being the units of information that make up the mind, the constituents of the mind. (Dawkins, 1976; Blackmore, 1999) 2.1 Meme theory in medical science, and psychiatry “Before Pasteur popularized the notion that bacteria cause disease, healthcare was effectively a disease vector, and we will come to look at centralized legal systems as vectors for what we have called diseases of the mind.“ The concept of disease transmission and contagion was well established before microorganisms were identified. As the conception upon diseases transmission reflects the society’s state of progress, we passed from the miasma theory and spontaneous generation theory, to germ theory. (Karamanou, 2012) Likewise, the concept of meme-transmission reflects society’s progress, from possessed spirits or an infection of the uterus, to the most important concept in the history of psychiatric science, the meme theory. (Dawkins, 1976; Blackmore, 1999) 2.3 Meme illness a symptom of executive disorder Impairment of the executive function from coercion decreases the brain’s ability to filter and select memes, to self-regulate (Leigh, 2010), and this loss of executive control is responsible for proliferation of pathological memes and disease progression of meme illness. 2.2 Executive disorder a symptom of coercion Subordinating the executive function of a person through coercion, under a monopoly on violence, decreases their executive control. Decreased executive control, impairment of the executive function, causes executive dysfunction. Using these propositions only, it can be proven that overruling executive control in a person causes executive dysfunction. 1.4 Executive disorder by other causes than coercion It goes without saying that executive dysfunction can have other causes than government coercion, brain damage like in the case of Phineas P. Gage (Bigelow, 1850) being an often used example. Psychiatry is per definition the study of mental disorders, an obsolete medical theory, in this article redefined as executive disorder from coercive government. 2.4 Hysteria as a case study “Never doubt that a small group of thoughtful, committed citizens can change the world. Indeed, it’s the only thing that ever has.” “Hysteria” was an executive disorder in the 19th century that affected females. The lack of female voting rights meant that females suffered more government coercion than males, with more severe impairment of the executive function. Psychiatry in the 19th century pointed the cause of “hysteria” to the female uterus, a sex organ that distinguishes females from males, a fiction that served to legitimize social inequality between males and females. Hysteria is an ideal case study to prove the theory that psychiatry diagnoses coercion in government, because of what the Women’s Rights Movement achieved. After the war and the passage of women’s suffrage in England and the United States, it was believed that female hysteria declined and even disappeared (Gilman, 1993). 2.6 Human ritual sacrifice and the social control hypothesis The social control hypothesis (Winkelman, 2014) proposes that human sacrifice legitimizes and stabilizes a stratified society. Evidence for human sacrifice is found throughout the archaeological record of early civilization. (Watts, 2016) This begs a simple question – is there any comparable system around today to promote social stratification by human sacrifice? Psychiatric diagnoses as religious myths (Whitley, 2008) legitimize the subordination of humans who have not given consent, appeal to “objective truth” and the “will of science” to strip people of individual agency, and institute an example, a deterrent to disobedience. Psychiatry is human sacrifice to legitimize coercive government. (Moncrieff, 2010) 2.5 Executive disorder from coercion is somatic, not mental Executive disorders that are caused by coercion, are a response to physical and visceral - somatic - pecking orders, somatic trauma that originates from genetic imperatives for dominance hierarchy behaviour in humans. (Price, 1967) This assertion is proven by the observation that physical removal of the coercive entity that causes an executive disorder will regain executive function in the individual. The mental component in psychiatric disorders is a consequence of the loss of executive control. 3.1 Psychiatry diagnoses coercion in government Overruling executive control in a person causes executive dysfunction, and accounts for all executive dysfunction that is diagnosed by psychiatric science. 3.2 Psychiatry is a disease vector The field of psychiatric science, a proto-science and religion (Whitley, 2008) more than a branch of medical science, has given legitimacy to the subordination of non-consenting human beings by a master class (Whitley, 2008; Moncrieff, 2010), and stabilized master-slave type social stratification (Watts, 2016) enforced through instincts for dominance hierarchies with pecking orders (Price, 1967). This role of psychiatry as a political device (Moncrieff, 2010) has increased the prevalence of executive disorder, it has served as a disease vector that has decreased health, the exact opposite of medical science. In other words, psychiatric disorders are nosocomial diseases, diseases whose development are favoured by a hospital environment. 3.3 The cure for executive disorder and meme illness The evidence that executive disorder is caused by coercion, and that meme illness results from a loss of executive control, opens the door to a new form of treatment for psychiatric disorders. It can be shown, beyond any reasonable doubt, that physical removal of the coercive entity that causes an executive disorder, will return executive control to the patient and alleviate all symptoms, curing the executive disorder. That should be the role of the medical doctor, and psychiatric science, going forwards. 4. Conclusion Executive disorder is caused by coercion, decreasing the brain’s ability to filter and select memes, causing "meme illness", a symptom of loss of executive control, and psychiatric disorders are classified based on the severity of impairment. The conclusion of this unified theory of psychiatric disorders must be to go further than just point to where psychiatry failed, and to instead look at how it could be redeemed. To ask the question: could the "medical gaze" (Foucault, 1963) be re-focused, and rehumanize instead of dehumanize? Science as a protocol is not a map but a compass, and like germ theory led to a revolution in healthcare, meme theory could raise medical science out of mind-body dualism, and return executive control to the individual. References Forbes, Chad & Grafman, Jordan. (2010). The Role of the Human Prefrontal Cortex in Social Cognition and Moral Judgment . Annual review of neuroscience. 33. 299-324. 10.1146/annurev-neuro-060909-153230. Forbes, Chad & Grafman, Jordan. (2010). The Role of the Human Prefrontal Cortex in Social Cognition and Moral Judgment . Annual review of neuroscience. 33. 299-324. 10.1146/annurev-neuro-060909-153230. Alvarez, J. A., & Emory, E. (2006). Executive Function and the Frontal Lobes: A Meta-Analytic Review. Neuropsychology Review, 16(1), 17–42. https://doi.org/10.1007/s11065-006-9002-x Leigh, H., & Leigh, H. (2010). Genes, Memes, Culture, and Mental Illness. Springer New York. https://doi.org/10.1007/978-1-4419-5671-2 Dawkins, Richard. (1978, ©1976) The selfish gene /New York : Oxford University Press. Blackmore, S. J. (1999). The meme machine. Oxford [England]: Oxford University Press. Bigelow, Henry Jacob (1850). Dr. Harlow's case of Recovery from the passage of an Iron Bar through the Head. American Journal of the Medical Sciences 20:13–22 (Republished in Macmillan 2000). Karamanou, Marianna & Panayiotakopoulos, George & Tsoucalas, Gregory & Kousoulis, Antonis & Androutsos, George. (2012). From miasmas to germs: A historical approach to theories of infectious disease transmission. Le Infezioni in Medicina, n. 1, 52-56. Gilman, S. (1993). Hysteria before Freud. Berkeley: University of California Press Price, J. (1967). THE DOMINANCE HIERARCHY AND THE EVOLUTION OF MENTAL ILLNESS. The Lancet, 290(7509), 243–246. https://doi.org/10.1016/s0140-6736(67)92306-9 Winkelman, M. (2014). Political and Demographic-Ecological Determinants of Institutionalised Human Sacrifice. Anthropological Forum, 24(1), 47–70. https://doi.org/10.1080/00664677.2014.860888 Watts, J., Sheehan, O., Atkinson, Q. D., Bulbulia, J., & Gray, R. D. (2016). Ritual human sacrifice promoted and sustained the evolution of stratified societies. Nature, 532(7598), 228–231. https://doi.org/10.1038/nature17159 Whitley, R. (2008). Is psychiatry a religion? Journal of the Royal Society of Medicine, 101(12), 579–582. https://doi.org/10.1258/jrsm.2008.080044 Moncrieff, J. (2010). Psychiatric diagnosis as a political device. Social Theory & Health, 8(4), 370–382. https://doi.org/10.1057/sth.2009.11 Foucault, M. (1963). Naissance de la clinique: Une archéologie du regard médical. Paris: Presses universitaires de France.
https://openalex.org/W4389731055	http://j.uniyar.ac.ru/index.php/vyrgu/article/download/1460/1237	Russian	null	Spiritual and moral aspects of personality and reflexivity in the context of research on spiritual capacities	Vestnik Âroslavskogo gosudarstvennogo universiteta imeni P.G. Demidova. Seriâ, Gumanitarnye nauki/Vestnik Âroslavskogo gosudarstvennogo universiteta im. P. G. Demidova. Seriâ, Gumanitarnye nauki	2,023	cc-by	2,570	Spiritual and moral aspects of personality and reflexivity in the context of research on spiritual capacities G. V. Ozhiganova1 1Institute of Psychology of the Russian Academy of Sciences, 13 Yaroslavskaya str., Moscow, 129366 1Institute of Psychology of the Russian Academy of Sciences, 13 Yaroslavskaya str., Moscow, 129366 DOI: 10.18255/1996-5648-2023-4-604-611 Vestnik YarGU. Seriya Gumanitarnye nauki. 2023. Vol. 17, No 4 journal homepage: http://j.uniyar.ac.ru/index.php/vyrgu Vestnik YarGU. Seriya Gumanitarnye nauki. 2023. Vol. 17, No 4 journal homepage: http://j.uniyar.ac.ru/index.php/vyrgu PSYCHOLOGY © Yaroslavl State University, 2023 This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) Г. В. Ожиганова1 Г. В. Ожиганова1 1Институт психологии РАН, ул. Ярославская, 13, Москва, 129366, Российская Федерация 1Институт психологии РАН, ул. Ярославская, 13, Москва, 129366, Российск DOI: 10.18255/1996-5648-2023-4-604-611 УДК 159.9.072 В статье отражены результаты изучения связи аспектов духовно-нрав ственной сферы: духовно-нравственные качества, альтруизм, стремление к смыслу (выступающих как субкомпоненты морального компонента ду ховных способностей) с рефлексивностью (рассматриваемой в качестве суб компонента ментального компонента духовных способностей). Отмечено, что существуют разные типы рефлексии: продуктивный и непродуктив ный. Цель исследования – выяснить, существует ли связь аспектов духов но-нравственной сферы с рефлексивностью (ее разными типами). На основе выборки 274 человек установлена значимая положительная связь духов но-нравственной сферы личности с продуктивным типом рефлексии (си стемная рефлексия) и значимая отрицательная связь или отсутствие связи с непродуктивными типами рефлексии (интроспекция и квазирефлексия), что позволяет идентифицировать субкомпонент ментального компонен та духовных способностей «рефлексивность» с продуктивной (системной) рефлексией. Ключевые слова: духовно-нравственная сфера; духовно-нравственные качества; альтруизм; стремление к смыслу; духовные способности; рефлексия; рефлексивность; высшие рефлексивные способности DOI: 10.18255/1996-5648-2023-4-604-611 Research article Full text in Russian The article reflects the results of studying the relationship of the spiritual and moral aspects: spiritual and moral qualities, altruism, striving for meaning (as sub-components of the moral component of spiritual capacities) with reflexivity (considered as a sub-component of the mental component of spiritual capacities). It is noted that there are different types of reflection: productive and unproductive. The purpose of the study is to find out whether there is a connection between aspects of the spiritual and moral sphere with reflexivity (its different types). On the basis of a sample of 274 people, a significant positive relationship of the aspects of spiritual and moral sphere of personality with a productive type of reflection (systemic reflection) was established and a significant negative connection or lack of connection with unproductive types of reflection (introspection and quasi-reflection) was found, which makes it possible to identify the sub-component of the mental component of spiritual abilities "reflexivity" with productive (systemic) reflection. Keywords: spiritual and moral sphere; spiritual and moral qualities; altruism; striving for meaning: spiritual capacities; reflection; reflexivity; higher reflexive capacities INFORMATION ABOUT AUTHORS Ozhiganova, Galina V. E-mail: symposium2016@rambler.ru Cand. Sc. (Psychology) INFORMATION ABOUT AUTHORS Ozhiganova, Galina V. E-mail: symposium2016@rambler.ru Cand. Sc. (Psychology) 604 Вестник ЯрГУ. Серия Гуманитарные науки. 2023. Том 17, № 4 веб-сайт: http://j.uniyar.ac.ru/index.php/vyrgu ПСИХОЛОГИЯ ИНФОРМАЦИЯ ОБ АВТОРАХ Ожиганова, Галина Валентиновна E-mail: symposium2016@rambler.ru Кандидат психологических наук, ведущий научный сотрудник, Лаборатория психологии способностей и ментальных ресурсов им. В. Н. Дружинина © ЯрГУ, 2023 Актуальность исследования проблемы связи рефлексивности с ду ховно-нравственной сферой личности обусловлена усилением вредонос ных манипулятивных технологий воздействия на сознание современного человека и необходимостью противостоять искажению реальности в умах людей, противодействовать насаждению идей, противоположных высшим общечеловеческим ценностям; важно предотвратить искоренение духов татья открытого доступа под лицензией CC BY (https://creativecommons.org/licenses/by/4.0/ 605 Г. В. Ожиганова ных идеалов, ассоциируемых с гуманизмом и справедливостью, необходи мо открыть дорогу рефлексии, обусловленной духовно-нравственной ори ентацией личности. ных идеалов, ассоциируемых с гуманизмом и справедливостью, необходи мо открыть дорогу рефлексии, обусловленной духовно-нравственной ори ентацией личности. Духовно-нравственная сфера личности определяется как «совокуп ность духовно-нравственных идеалов и ценностей; личностных смыслов, отражающих субъективное отношение к ним; духовных потребностей и нравственных мотивов поведения; нравственных чувств; стремления личности к поведению в соответствии с принятыми ценностями; опыта со ответствующего действия; способностей к духовно-нравственному самоо пределению, самореализации, самосовершенствованию» [1, c. 175]. Выделяются следующие критерии развитости духовно-нравственной сферы личности: - смысложизненные устремления на основе моральных принципов и идеалов; - смысложизненные устремления на основе моральных принципов и идеалов; - принятие высоких нравственных стандартов и их претворение в жизнь; - принятие высоких нравственных стандартов и их претворение в жизнь; - осмысленность бытия: рефлексия своих поступков и корректировка поведения; - осмысленность бытия: рефлексия своих поступков и корректировка поведения; - саморегуляция исходя из принятых высоких нравственных норм морегуляция исходя из принятых высоких нравственных норм. Определение духовно-нравственной сферы и критерии ее развитости приводят к мысли о связи аспектов духовно-нравственной сферы с прояв лением рефлексивности. В данном исследовании, исходя из предложенной нами психологиче ской модели духовных способностей (включающей три компонента: мо ральный, ментальный, трансцендентный) [2–4], рассматриваются такие аспекты духовно-нравственной сферы, как духовно-нравственные каче ства, альтруизм и стремление к смыслу. Согласно нашему представлению, эти аспекты выступают в качестве субкомпонентов морального компонен та духовных способностей. Рефлексивность же относится к ментальному компоненту духовных способностей. Духовные способности понимаются как свойства личности, отражающие единство её интеллектуальной и нрав ственной сфер. Духовные способности характеризуются согласованностью функционирования всех составляющих [3–4]. В этом исследовании рассма тривается связь вышеуказанных субкомпонентов морального и ментально го компонентов духовных способностей. Обратимся к понятиям рефлексивности и рефлексии, которые в неко торых случаях могут использоваться как взаимозаменяемые. Несмотря на переплетение понятий рефлексии и рефлексивности, обо значается и их различие. ИНФОРМАЦИЯ ОБ АВТОРАХ Рефлексия раскрывается как процесс познания себя, включающий понимание себя, других людей, анализ и осмысление ситуаций и жизни в целом, а также как состояние, в котором пребывает че ловек, предаваясь процессу самопознания. Рефлексивность же понимается как свойство личности, как устойчивая черта – склонность к рефлексии [5]. 606 606 Г. В. Ожиганова лу), будут связаны с субкомпонентом ментального компонента духовных способностей «рефлексивность», идентифицируемым с продуктивным ти пом рефлексии. лу), будут связаны с субкомпонентом ментального компонента духовных способностей «рефлексивность», идентифицируемым с продуктивным ти пом рефлексии. р ф Эмпирические гипотезы: р ф Эмпирические гипотезы: 1. Существует значимая положительная связь аспектов духовно-нрав ственной сферы (духовно-нравственные качества личности, альтруизм, стремление к смыслу) с рефлексивностью – продуктивным типом рефлек сии (системная рефлексия). 2. Связь аспектов духовно-нравственной сферы (духовно-нравствен ные качества личности, альтруизм, стремление к смыслу) с непродук тивными типами рефлексии (интроспекция и квазирефлексия) будет отсутствовать. Методы исследования Духовно-нравственная сфера личности 1. Для изучения духовно-нравственных качеств личности исполь зовалась методика «Духовная личность» А. Хусейна, М. Анаса (адапта ция Г. В. Ожигановой) [13]. 2. Для исследования альтруизма – методика «Измерение альтруисти ческих установок» [14]. 2. Для исследования альтруизма – методика «Измерение альтруисти ческих установок» [14]. 3. Для исследования стремления к смыслу – тест смысложизненных ориентаций (СЖО) [15]. Рефлексивность 3. Для исследования стремления к смыслу – тест смысложизненных ориентаций (СЖО) [15]. Рефлексивность Рефлексивность Методика «Дифференциальный тип рефлексии» [12] диагностиру ет три типа рефлексии: продуктивную (системную) рефлексию и два типа непродуктивной рефлексии: интроспекцию (самокопание) и квазирефлек сию (фантазирование). Выборка. В исследовании участвовали 274 респондента – студенты и служащие из разных городов России (Москва, Уфа, Санкт-Петербург); 141 –женского и 133 – мужского пола); возраст 18–55 лет. Духовно-нравственная сфера личности и рефлексивность… Исследования рефлексивности позволили прийти к выводу о много гранности этого феномена, существования как его позитивных, так не гативных проявлений, то есть, его неоднозначности. Так, М.А. Холодная описывает эффект расщепления рефлексии [6]. В экспериментальном исследовании Н. Дишона с коллегами исследовалось воздействие рефле кивности на социальную идентификацию: проверялось, в какой степени саморефлексия может оказывать влияние на самооценку в контексте при нятия решений, таким образом, выявлялись причинно-следственные свя зи. В результате было установлено, что саморефлексия усиливала социаль ную идентификацию [7]. В экспериментальном исследовании, проведенном Л. А. Да Силвой выявлено, что успешное освоение интеллектуальных операций обусловлено их рефлексией обучаемым, а успешность реф лексии определяется рефлексивностью как личностным качеством [8]. В то же время в исследованиях А.В. Карпова и его сотрудников, показано, что рефлексивность не всегда связана с положительными результатами психологических проявлений личности. Так, возрастание рефлексивности сопряжено с повышением качества принятия управленческих решений, но только до определенного уровня, а затем качество принятия решений начинает снижаться [9]. Согласно исследованиям А. А. Карпова и А. В. Кар пова, выраженность многих метакогнитивных показателей связана с высо ким уровнем психологических защит, нейротизма и пр. [10]. В исследованиях Д. А. Леонтьева с коллегами были выявлены раз ные типы рефлексии, как продуктивные, так и непродуктивные [11–12]. На основании предложенной Д. А. Леонтьевым дифференциальной моде ли рефлексии были установлены четыре типа рефлексии: 1) арефлексия, 2) интроспекция, 3) квазирефлексия, 4) системная рефлексия. Эти типы реф лексии отражают сфокусированность на разных объектах: 1) арефлексия – только на внешнем объекте; 2) интроспекция – на самом субъекте (самоко пание); 3) квазирефлексия – на посторонних объектах (фантазирование); 4) системная рефлексия – на себе и объекте одновременно (взгляд на себя со стороны). Она представляет собой продуктивный вид рефлексии по срав нению с первыми тремя, относимыми к непродуктивному виду. Наличие разных типов рефлексии приводит к вопросу о том, как они бу дут соотноситься с духовно-нравственной сферой личности, что имеет от ношение к духовным способностям. Цель исследования – сопоставление аспектов духовной сферы (духов но-нравственные качества личности, альтруизм, стремление к смыслу, ко торые выступают как субкомпоненты морального компонента духовных способностей), с различными типами рефлексии; выяснение, какой тип реф лексии соотносится с духовными способностями. Теоретическая гипотеза: субкомпоненты морального компонента ду ховных способностей, имеющие отношение к духовно-нравственной сфере (духовно-нравственные качества личности, альтруизм, стремление к смыс 607 Г. В. Ожиганова Результаты исследования и их обсуждение Для изучения связи аспектов духовно-нравственной сферы таких, как духовно-нравственные качества, альтруизм, стремление к смыслу с рефлексивностью, включающей разные типы рефлексии: системную, интроспекцию и квазирефлексию, был проведен корреляционный анализ (табл. 1). Были установлены значимые положительные связи продуктивной (си стемной) рефлексии со всеми рассматриваемыми аспектами духовно-нрав ственной сферы: духовно-нравственные качества, альтруизм, стремление к смыслу. Выявлено отсутствие достоверной связи непродуктивной реф лексии (интроспекция) с альтруизмом и значимые отрицательные связи со стремлением к смыслу и духовно-нравственными качествами. Уста новлено отсутствие достоверной связи непродуктивной рефлексии (квази 608 608 Духовно-нравственная сфера личности и рефлексивность… рефлексия) с духовно-нравственными качествами; значимая отрицатель ная связь со стремлением к смыслу и крайне слабая на уровне тенденции значимая положительная связь с альтруизмом. Комментируя полученную крайне слабую на уровне тенденции, но значимую корреляцию квазиреф лексии (фантазирование) с альтруизмом, следует сказать следующее: фантазирование может отражать стремление человека к заботе о других, но оставаться лишь на уровне фантазий, не связанных с реальностью. Таблица 1 Результаты исследования связи продуктивной и непродуктивной рефлексии с аспектами духовной сферы (субкомпонентами морального компонента духовных способностей) Таблица 1 Результаты исследования связи продуктивной и непродуктивной рефлексии с аспектами духовной сферы (субкомпонентами морального компонента духовных способностей) Рефлексивность Духовно-нравственная сфера Показатели Духовно- нравственные качества Альтруизм Стремление к смыслу Продуктивная рефлексия (системная рефлексия) 0,323* 0,306* 0,328* р значения 0,000 0,000 0,000 Непродуктивная рефлек сия (интроспекция) -0,170 0,057 -0,391*** р значения 0,005 0,348 0,000 Непродуктивная рефлек сия (квазирефлексия) -0,064 0,124* -0,202*** р значения 0,292 0,039 0,001 Примечание. * - р ≤ 0,05; - р ≤ 0,01; * - р ≤ 0,001 ц езультаты исследования связи продуктивной и непродуктивной рефлексии с аспектами духовной сферы (субкомпонентами морального компонента духовных способностей) Установленные значимые положительные корреляции продуктивной (системной) рефлексии со всеми рассматриваемыми аспектами духов но-нравственной сферы (которые представляют собой субкомпоненты мо рального компонента духовных способностей) позволяют говорить о связи вышеуказанных субкомпонентов морального компонента и такого субком понента ментального компонента духовных способностей, как рефлексив ность, причем речь идет именно о продуктивной рефлексии. Продуктивная (системная) рефлексия предполагает возможность удерживать в фокусе внимания одновременно и себя, и объект, включает способность смотреть на себя со стороны. Она создает условия для приближения к объективной оценке своих действий, мыслей и чувств, возможность работать над собой для коррекции своего поведения. Будучи сопряженной с духовно-нрав ственной сферой, системная рефлексия позволяет развивать положитель ные качества, ориентируя на продуктивную жизнедеятельность, служе ние людям, совершение духовно-нравственных поступков. Таким образом, 609 Г. В. Результаты исследования и их обсуждение Ожиганова продуктивная (системная) рефлексия, демонстрируя связь с аспектами духовно-нравственной сферы, такими как духовно-нравственные каче ства, альтруизм, стремление к смыслу (выступающими в качестве субком понентов морального компонента духовных способностей) позволяет гово рить о возможности существования не просто рефлексивных способностей, но о рефлексивных способностях, имеющих духовно-нравственную окра ску, то есть, о высших рефлексивных способностях. у р ф В целом отсутствие достоверной связи или значимая отрицательная связь непродуктивной рефлексии с показателями духовно-нравственной сферы (духовно-нравственные качества, альтруизм, стремление к смыс лу), свидетельствует о том, что непродуктивная рефлексия не соотносима с духовными проявлениями, отражающими ориентацию на высшие цен ности истины, добра, красоты, справедливости. Интроспекция и квази рефлексия характеризуются излишней сконцентрированностью на соб ственных фантазиях, эгоцентрических потребностях, что препятствует заботе о благополучии других людей, служению обществу, то есть, прояв лению духовно-нравственных аспектов личности, связанных с духовными способностями. Выводы Полученные в исследовании результаты свидетельствуют о значимой положительной связи духовно-нравственной сферы личности с рефлек сивностью, что: - позволяет говорить уже не о рефлексивных способностях, а о выс ших рефлексивных способностях, относимых к категории духовных, отра жающих духовно-нравственную ориентацию личности. - позволяет говорить уже не о рефлексивных способностях, а о выс ших рефлексивных способностях, относимых к категории духовных, отра жающих духовно-нравственную ориентацию личности. - показывает согласованность функционирования субкомпонентов мо рального компонента духовных способностей (духовно-нравственные ка чества, альтруизм, стремление к смыслу) с субкомпонентом ментального компонента духовных способностей «рефлексивность». - отражает идентификацию субкомпонента ментального компонен та духовных способностей «рефлексивность» с продуктивной (системной) рефлексией. - отражает идентификацию субкомпонента ментального компонен та духовных способностей «рефлексивность» с продуктивной (системной) рефлексией. Отсутствие достоверной связи или значимая отрицательная связь аспек тов духовно-нравственной сферы, выступающих в качестве субкомпонен тов морального компонента духовных способностей (духовно-нравственные качества, альтруизм, стремление к смыслу) с непродуктивными типа ми рефлексии (интроспекция и квазирефлексия), показывает несовмести мость непродуктивной рефлексии с проявлением духовных способностей. 610 Духовно-нравственная сфера личности и рефлексивность… Духовно-нравственная сфера личности и рефлексивность… 15. Леонтьев Д. А. Тест смысложизненных ориентаций (СЖО). М.: Смысл, 2000, 18 с. Ссылки 1. Шитякова Н. П., Верховых И. В. Духовно-нравственное воспитание школь ников: проблемы, теории, технологии. Челябинск: Изд-во Челяб. гос. пед. ун-та, 2016. 197 с. 2. Ожиганова Г.В. Психологические аспекты духовности: духовные способно сти // Психологический журнал. 2010. № 5. С. 39–53. 3. Ожиганова Г. В. Духовная личность. М.: Институт психологии РАН, 2020. 288 с. 4. Ожиганова Г. В. 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Психология рефлексивных процессов управ ления. М. ; Ярославль: ДиаПресс, 2000. 283 с. 10. Карпов А. А., Карпов А. В. Взаимосвязь психометрического интеллек та с организацией метакогнитивных процессов и качеств личности // Психологиче ский журнал. 2016. Т. 37, № 2. С. 69–78. 11. Леонтьев Д. А., Аверина А. Ж. Феномен рефлексии в контексте пробле мы саморегуляции // Психологические исследования: электрон. науч. журн. 2011. Т. 4, № 16. С. 1-17. DOI 10.54359/ps.v4i16.860. 12. Леонтьев Д. А., Осин Е. Н. Рефлексия «хорошая» и «дурная»: от объясни тельной модели к дифференциальной диагностике // Психология: Журнал Высшей школы экономики. 2014. Т. 11, № 4. С. 110–135. 13. Ожиганова Г. В. Адаптация опросника «Духовная личность» на русскоя зычной выборке // Экспериментальная психология. 2019. Т. 12, № 4. С. 160–176. DOI 10.17759/exppsy.2019120413 14. Ясин М. И. Методика измерения альтруистических установок // Психоло гический журнал. 2020. Т. 41, № 1. С. 77–85. DOI 10.31857/S020595920007898-3. онтьев Д. А. Тест смысложизненных ориентаций (СЖО). М.: Смысл, 2000, 15. Леонтьев Д. А. Тест смысложизненных ориентаций (СЖО). М.: Смысл, 2000, 18 с. 611
https://openalex.org/W4385201692	https://www.qeios.com/read/880NA2/pdf	English	null	Review of: "Why Engineering Education is Losing Charm among Students in India? A Discussion"	null	2,023	cc-by	488	Qeios, CC-BY 4.0 · Review, July 24, 2023 Review of: "Why Engineering Education is Losing Charm among Students in India? A Discussion" Thanikachalam Vedhathiri1 1 National Institute of Technical Teachers Training and Research (NITTTR) Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. The author has taken an important area of research. To provide needed suggestions for improvement, the following issues are to investigated: 1. State-wide unemployment of engineers. Not all the states have well-established product design and manufacturing units. There are states where they don't have even one industry. 1. State-wide unemployment of engineers. Not all the states have well-established product design and manufacturing units. There are states where they don't have even one industry. 2. Branch wide unemployment. Many branches of engineering are very much affected due to advances in disruptive technologies, lack of establishing advanced manufacturing units, shortage of industry -specific curriculum, and not including courses in advances in product analysis, design, prototype development, testing, improving, selection of appropriate manufacturing methods, and maintenance. 3. Absence of needed industrial linkages for offering co-operative education 4. Absence of outcome-oriented instructional design. 5. Absence of continuous training of the faculty members and students on the fast-changing industrial practices. 6. Absence of adjunct faculty who are from industries. 5. Absence of continuous training of the faculty members and students on the fast-changing indust 5 bse ce o co t uous t a g o t e acu ty e be s a d stude ts o t e ast c a g g dust a p act ces 6. Absence of adjunct faculty who are from industries. 7. Absence of assessment of human resources needs in every industry. 7. Absence of assessment of human resources needs in every industry. 8. Not introducing industrial training to the students. 8. Not introducing industrial training to the students. 9. Improper goals, objectives, mission and vision in many engineering institutions. 9. Improper goals, objectives, mission and vision in many engineering institutions. 10. Absence of quality improvement in program planning and implementation. 10. Absence of quality improvement in program planning and implementation. 11. Failure of Institute of Applied Manpower Research, Delhi in conducting research on the human resource requirements in every sector and publishing the outcome. 11. Failure of Institute of Applied Manpower Research, Delhi in conducting research on the human resource requirements in every sector and publishing the outcome. 15. Doubling and trebling the capacity of the intake without assessing the industrial needs. are investigated and remedial measures are introduced, more institutes will be closed. There will be a very low return on investments (ROI) in the colleges established in every town. Qeios ID: 880NA2 · https://doi.org/10.32388/880NA2 Qeios, CC-BY 4.0 · Review, July 24, 2023 Potential competing interests: No potential competing interests to declare. Qeios ID: 880NA2 · https://doi.org/10.32388/880NA2 1/2 Qeios, CC-BY 4.0 · Review, July 24, 2023 Qeios ID: 880NA2 · https://doi.org/10.32388/880NA2 2/2
https://openalex.org/W4317987503	https://zenodo.org/records/7567268/files/IJISRT23JAN658.pdf	English	null	The Advantage of Animated Advertisements in Today's Era	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	5,225	International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Junaid Hushain Research scholar, Department of Management Studies Jai Narain Vyas University, Jodhpur, Rajasthan, India Junaid Hushain Research scholar, Department of Management Studies Jai Narain Vyas University, Jodhpur, Rajasthan, India Dimple Sharma Research scholar, Department of Management Studies Jai Narain Vyas University, Jodhpur, Rajasthan, India Dr. Kamal Kant Post-Doctoral Fellow-ICSSR, Department of Management Studies Jai Narain Vyas University, Jodhpur, Rajasthan, India (animate2explain, 2017). For instance, in the realm of digital, Videos and ads be practical marketing tools. Abstract:- There is no doubt that animation is quickly emerging as one of the most significant trends and approaches in advertising in the modern era. Animated commercials offer a video that is entertaining in addition to being promotional, so there is no need to wonder why that is the case. The Consumer will be unable to tear their eyes away from the sights and motion. Considering the significance of the purpose of this research is to provide a comparison and analysis of the most recent animated commercials. The study examines the differences and similarities between animated and traditional forms of advertising. Today, the CEO and President of Darvideo Animation Studio, Yuriy Polyashko, Because the audience's tolerance for waiting is short; animation has become a popular kind of entertainment. Everyday encounters and a crucial marketing strategy that keeps the audience from dullness (Polyashko, 2019). He continues by saying that scientific advancements support the since "90% of the information is communicated to the brain," the concept of animated or visual material is entirely visual, establishing the legitimacy of and defending the use of animation in marketing devices. According to statistics supplied by Unbounce, animated infographics videos with motion graphics also boost conversion by 20%. Animoto estimates that 4 out of 5 consumers prefer animated infographics and motion graphic films. The Advantage of Animated Advertisements in Today's Era Dr. Meeta Nihalani Associate Professor, Department of Management Studies, Jai Narain Vyas University, Jodhpur, Rajasthan, India Volume 8, Issue 1, January – 2023 Volume 8, Issue 1, January – 2023 II. When digital computers first became a part of everyday life in the 1940s, the discipline of computer animation was born (Jim, 2020). In 1960, computer graphics were developed, which paved the way for the invention of two-dimensional (2D) animation. The 1970s. 3-dimensional animation (3D) first appeared in the late 1980s, and The use of 3D animation in film production peaked in the mid-1990s. These advances led to computer animation and visuals (CGA) development. Many academics have talked about how vital animation is to the advertising sector. German professor Patrick Vonderau teaches media and communication studies at Martin Luther University Halle-Wittenberg, stressed the importance of animation and video games. Computer-generated imagery in marketing and how goods, services, and other things are sold (M. C. Thompson, 2019). He believes that "advertising has influenced modern media; nonetheless, advertising was shaped using animation. According to Ruchi and Deepti Goel Upadhyay, professors of communication studies at the University of Delhi, marketing companies heavily rely on animation in their advertising strategy since it Additionally, motion in commercials makes them more alluring and enticing viewers of all ages, including youngsters, teens, and even adults (Upadhyay, 2017). According to New Jersey native and StudioBinder copywriter Alyssa Maio, the animation is the process of photographing subsequent models or drawings to try to provide the impression of movement in a series (Maio, 2020). She continues by stating that animation represents movement in a succession of images. According to Yasha Vora, a graphic designer at the Souled Store, the animation is a sort of creativity that brings anything to life. She defined animation as Maio, "techniques of recording consecutive drawings or models to generate and appearance of movement and motion" (Vora, 2018). According to her study, advertising is a type of communication intended to persuade potential consumers to buy the commodities, products, and services being marketed. She continues by saying that from the late 19th and early 20th centuries, mass manufacturing and technological breakthroughs have led to an evolution in advertising. Indian professor of fine arts, Deepak Kochhar, describes advertising as a strategy a business or brand uses to market its goods or services to consumers (Kochhar, 2019). Only when viewers recall the commercial can it be said that the ad was successful. II. The advent of animation into the realm of advertising in the late 19th century had a good and enticing impact on customers' buying intentions as well as their memory of the advertisement's content, according to Sameh Al-Natourm, Robert Krider, and Andrew Gemino, marketing academics (Sameh Al Natourm, 2013). Accordingly, marketing academics Jarmo et al. confirmed in their research that animation boosts attention, which improves the viewer's memory (Jarmo Kuisma, 2010). The artist and advertising specialist Huntley Baldwin from the International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 animated commercials, focusing on realistic animation rendering—and its usefulness in today's advertising world. animated commercials, focusing on realistic animation rendering—and its usefulness in today's advertising world. United States highlighted the strength of animation in its ability to get away with things that reality cannot achieve, such as creating a world of imagination and enchantment for a product that makes "puffery digestible" (Thompson M. C., 2019). Baldwin explained the four factors that made animation a good choice for ads. It first draws spectators' attention. Second, animation gives goods, services, and products a distinctive brand. Third, animation simplifies complicated concepts into straightforward expressions that everybody can understand. Finally, it can bring an improbable, abstract concept to life. I. There is no denying the fact that videos are a much more engaging and successful form of advertising than images. That is clear, given how readily films can capture viewers' attention and turn them into potential customers for the promoted offering, service, or product. As a result, video advertising has been increasingly popular recently. Statistics demonstrate that most individuals (86%) prefer to watch videos for businesses and items (McCormick, 2021). Additionally, research from Wordstream has revealed that advertisers that use videos - incredibly animated and visual material, increase income by 49% more quickly and significantly. Those who do not rely on videos. For instance, an animated commercial may create Virtual worlds with fictitious people that are not necessarily required to be realistic; as a result, Storytelling appeals to the imagination of the audience (animate2explain, 2017). Additionally, while most individuals read pages and paragraphs, not everyone will see and comprehend an animated film. Forbes reports that 95% of users, when watching animated videos, just 10% of viewers can comprehend the material. The subject of the writings. Animated commercials have become a popular type. Consequently, in 2021, animation use in advertising decreased and expanded by 40% (Buzzflick, 2021). On social media, 82% prefer watching a video to reading or checking a post. 64% of customers who see the video go on to purchase the products, services, or product advertisements. People process and enjoy visual material 60,000 times faster on average. Videos, particularly animated multimedia, are more engaging than text-based information. It takes less than a tenth of a second to capture viewers' attention (Hoque, 2020). It has long been recognized that animation has significantly impacted society. While advertising. Celebrities, advertising firms, production companies, animation studios, Businesses, and sectors have adopted animation to capture and represent companies, goods, and services while hooking the Consumer psychologically and logically (Thompson M. C., 2019). However, animated Real-world aspects in advertising are being replaced with cartoons, objects, and settings. The extraordinary dependence on animation in advertising has increased as technology has progressed. It has been increasing Animation is undoubtedly growing in importance as a form of advertising: trends and approach. Told, animated advertisements provide us with promotional and amusing videos, so there is no need to ponder why. Customer addicted. This thesis seeks to demonstrate the significance of animated advertising in the modern world. III. To further underline the significance and applicability of animated ads in today's environment, the next section will continue to show the benefits and drawbacks of animated advertising over live-action commercials. It is essential to remember that there is no such thing as the optimal or optimally optimal strategy for advertising. The decision is highly debatable because the advertisement's purpose and the targeted audience's demographics must be taken into account to establish which kind of advertising is more successful (522 Blog, 2013). However, to determine which alternative is more practical, it is vital to understand the primary benefits and drawbacks of both choices. One thing is sure, regardless of whether you decide to go with real-life or animated advertisements: firms and enterprises need to incorporate videos into their marketing plan, as this is a fruitful approach to boost user engagement and awareness (Hoque, 2020). I. To give a general review of www.ijisrt.com 431 IJISRT23JAN658 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165  The Animation Procedures in Advertising A live-action commercial has three stages (Johnston, 2019). Pre-production, production, and post-production are in order. Pre-production is creative. Scripting, graphic design, and hiring comprise this stage. actors/interviewers, International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Animators produce animated camera motions by moving the camera onscreen and producing keyframes. Animators use tilts, pans, close-ups, and other real-world camera methods to create animations. A corporation considers many factors before making a product video ad. Due to the positives and downsides, animation or live-action video is a top choice for commercial companies. The Animation Procedures in Advertising using live-action photography. According to Kristin C. Au, senior product designer at Slack, substantial developments in CGI have enabled animation to achieve a new high degree of realism through the use of 3D methods, hence making the animation and plot more relevant and engaging to the viewer (Au, 2014). into thinking a product is real. The 3D model's shadows, lighting, texturing, and camera movement creates a realistic look. Photorealistic computer animation may present unique ideas in strange settings. Animators may construct worlds and environments inaccessible to live-action camera crews. Many firms may use animation for advertising. Animation is more formal and flexible than real-life filming. Animation allows for a clean, soft aesthetic. Abstracts and intricate concepts are harder to demonstrate in real life than digitally. In CGI, where reality does not matter, colors may be manipulated to extremes. into thinking a product is real. The 3D model's shadows, lighting, texturing, and camera movement creates a realistic look. Photorealistic computer animation may present unique ideas in strange settings. Animators may construct worlds and environments inaccessible to live-action camera crews. Many firms may use animation for advertising. Animation is more formal and flexible than real-life filming. Animation allows for a clean, soft aesthetic. Abstracts and intricate concepts are harder to demonstrate in real life than digitally. In CGI, where reality does not matter, colors may be manipulated to extremes. This will provide the corporation with an improved version of its product, which will help with advertising by attracting consumers' visual and sensory attention. This is not tricking or manipulating the audience but rather offering the Consumer an idealized depiction of the product, in contrast to real-life filming, which may have defects that post-production cannot eradicate. Live shooting does not ensure a clean, polished look. Advertisements aim to promote a product, object, or service by emphasizing the beauty and eliminating faults. Humans want perfection and want to invest in near perfection. Animated ads may regulate and attain this degree of excellence to increase sales. Lighting is another factor. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Natural illumination cannot always make product details apparent, crisp, and exact. The team will be at the mercy of light and time if the shot is outside. Production houses typically employ expensive artificial studio lighting. Due to CGI's independence and improvements, animators may generate excellent lighting without relying on external elements (weather, studio light...). Post-production is a big part of filmmaking and advertising. The live-action video filming has X hours/days, so what is shot cannot be modified. If the corporation wanted to make changes after the final phases, it would not be easy to refilm since it would cost twice. This causes live-action film time and expense unpredictability. Live-action filming has several benefits in any case. A real-life camera may capture actual emotions and human feel. Demonstrating the ad's goal. A genuine camera's narrow depth of focus, light, and reflections assist create the ad's ambient impression. To summarise, find realistic components. Detailed product design, light, natural color palette, and smooth movement. Creating hyperrealistic 3d models using ty flow simulation for 3ds max. Ad animation is fluent, detailed, and animated. The liquid appears and flows realistically. The animated camera, discussed above, enhances the realism of live shooting. Animators produce animated camera motions by moving the camera onscreen and producing keyframes. Animators use tilts, pans, close-ups, and other real-world camera methods to create animations. A corporation considers many factors before making a product video ad. Due to the positives and downsides, animation or live-action video is a top choice for commercial companies. Similarly, lecturers from the University of Johannesburg, Fortunate Tatenda Mauyakufa, and Anup Pradhan have emphasized the increased reliance on animation in advertising and movies (Pradhan, 2018). In their essay, they explain that this change is now conceivable since animated (realistic and non-realistic) cartoons may replace performers. Additionally, it has become challenging to identify manufactured landscapes from natural ones. They further emphasize the animation's ability to produce and improve realistic people, sounds, objects, and surroundings. The public is more interested in animated material due to its adaptability and lack of limitations. From the above, it is clear that realistic animation has been enthusiastically received and embraced by the global advertising sector. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 into thinking a product is real. The 3D model's shadows, lighting, texturing, and camera movement creates a realistic look. Photorealistic computer animation may present unique ideas in strange settings. Animators may construct worlds and environments inaccessible to live-action camera crews. Many firms may use animation for advertising. Animation is more formal and flexible than real-life filming. Animation allows for a clean, soft aesthetic. Abstracts and intricate concepts are harder to demonstrate in real life than digitally. In CGI, where reality does not matter, colors may be manipulated to extremes. This will provide the corporation with an improved version of its product, which will help with advertising by attracting consumers' visual and sensory attention. This is not tricking or manipulating the audience but rather offering the Consumer an idealized depiction of the product, in contrast to real-life filming, which may have defects that post-production cannot eradicate. Live shooting does not ensure a clean, polished look. Advertisements aim to promote a product, object, or service by emphasizing the beauty and eliminating faults. Humans want perfection and want to invest in near perfection. Animated ads may regulate and attain this degree of excellence to increase sales. Lighting is another factor. Natural illumination cannot always make product details apparent, crisp, and exact. The team will be at the mercy of light and time if the shot is outside. Production houses typically employ expensive artificial studio lighting. Due to CGI's independence and improvements, animators may generate excellent lighting without relying on external elements (weather, studio light...). Post-production is a big part of filmmaking and advertising. The live-action video filming has X hours/days, so what is shot cannot be modified. If the corporation wanted to make changes after the final phases, it would not be easy to refilm since it would cost twice. This causes live-action film time and expense unpredictability. Live-action filming has several benefits in any case. A real-life camera may capture actual emotions and human feel. Demonstrating the ad's goal. A genuine camera's narrow depth of focus, light, and reflections assist create the ad's ambient impression. To summarise, find realistic components. Detailed product design, light, natural color palette, and smooth movement. Creating hyperrealistic 3d models using ty flow simulation for 3ds max. Ad animation is fluent, detailed, and animated. The liquid appears and flows realistically. The animated camera, discussed above, enhances the realism of live shooting.  Live Action vs. Animated Advertisements There are two broad categories of media content: live- action and animation. Live-action advertisements feature real- life videos of people, animals, objects, and environments. In contrast, animated advertisements entail "using and modifying drawn characters to make them appear moving" (YumYum, 2020). According to Fable Studio, Bristol-based advertising and marketing business, the selection of live-action or animated material is heavily influenced by the intended demographic; yet, animation has frequently been a technique for grabbing the viewer's interest and attention. Chang-Hyun Jin categorizes animation as CGI animation, silhouette animation, cartoon animation, film animation, clay animation, puppet animation, drawn animation, and realistic animation (Jin, 2011). He notes that new technology, such as 3D objects, has made it possible to reproduce animation, remarkably realistic animation. They are increasingly utilized in the television commercial and advertising sector, not only because of their realistic looks but also because of the decreased cost and production time. In addition, while depending on animation to finish their commercial concepts, many businesses want to achieve and produce realistic components. In other words, they are creating the most realistic-looking animation possible. Today, with the progress of computer-generated imagery (CGI), animators are increasingly able to achieve realistic rendering, prompting businesses to reassess the conventional marketing strategy of www.ijisrt.com 432 IJISRT23JAN658 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165  The Effect of Animated ads on the Consumer Brainstorming follows The concepts, characters/objects, and The tone and script are set. Afterward, Animators produce storyboards. The storyboard depicts a Draft/scratch ad to be evaluated as in the animatic. An animatic is a moving storyboard. Storyboard all recordings (voice-overs, audio, sound effects), pace, and timing of the ad. The asset characters, items, objects, and settings are created by modeling. The commercial is made. Animation follows. All non-movable inventions come to life. The animator will use Camera movement, lighting, shadows, and polish. After when animation is complete, animators render and combine it. Thus, the storyboard went from scratch to animation. Advertisement. The final output is sound. Finishing touches Fix the video's audio and colors once the ad is ready for distribution, in advertising, and after attaining Many adjustments results. A corporation may alter information or color every year. It can if it breaks or the firm wants to change the ultimate product. Live- action reshoots are expensive and difficult to change. Because it has already been done, making a video would be time- consuming. Changing ads' assets is easy. Cartoons, although it is complicated and time-consuming, you can edit your video. Real action (without spending tonnes of money, time, and energy) Therefore, animated ad imagery and digital models are built from scratch, unlike live action, where the product already exists. Thus, animated advertising has no such thing as Live- action ads are limited by what is physically achievable. Possible. The popularity of animated adverts in today's advertising environment is the best indication that they have a substantial effect and influence on customers. The audience in New York praised the rising immersion of enormous 3D animated billboard commercials for their engaging and realistic images (Weaver, 2022). Figure 1 shows a 3D commercial for an orange Nike sneaker box that bursts open to expose a rotating display of Air Max designs while hinting at the motivation behind each shoe. According to Ethan Jakab, co-founder of "Blunt Action," "these eye-catching 3D billboards are fast becoming highly popular since people are loving the realistic illusion; therefore making them stop and look twice" (Holtz, 2022). The unique campaign elicited a strong response on social media and in person among those traveling through Shinjuku Station, one of the busiest railway stations in the world. Most responses commended the Nike sneaker box's realistic movement toward the audience. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 locations/logistics, scheduling/time-management shooting list and crew selection. During manufacture, Live-shooting days, All pre-production decisions will be reenacted for the camera. They are capturing the ad's visuals. After filming, Proceed to post-production. We are finalizing footage processing, editing, editorial work, Motion graphics, sound checks, and voice-overs. Three things Live-action ads have stages. Animated ads have different methods. (2013). First animators must: Project understanding, meaning knowing the Budget, delivery time, brand image, audience, and purpose of the commercial. The animation enabled a zoom into a tiny component detail. For example, the manufactured water drop was depicted gliding down a leaf and falling via a hole. The use of animation enabled the client to create the desired atmosphere. The atmosphere they like to associate with their goods. In this instance, the bright, jungle, nature-like setting. Lastly, animation enabled the customer to make revisions and demands during the project's duration. It was engaging only with the animation agency. Consequently, spending time and reducing the ad crew and group.  The Benefits of Animation in Advertisements Animated ads are cheaper. A production house's cost and Budget would be higher if it filmed a product or service commercial. Because production companies use crews. Teamwork unifies varied ideas, yet it may be costly. The firm must pay the production house for every contracted crew member (light operator, cameraman, DP, retoucher, editor, set, make-up, etc.). Thus, the Company pays for every individual engaged in fulfilling his specialized role and rent equipment for X days or weeks of filming. Animated advertising costs less because it may be created by a smaller team or even a single individual (a "one-man show"). "One man show" refers to an animator who can do everything himself. He may be the DOP, cameraman, videographer, lighting guy, editor, and producer. The animator will use 2D and 3D animation tools to create a commercial. 2 Animation involves a long, well-thought-out process that includes generating a storyboard and much time. Realistic ads employ 3D software. Modeling space and products take patience. Real-life renders require software animation expertise. Using live-action would entail a lengthy process and production staff for the commercial enterprise. Animated advertising may be a "one-man show," reducing expenditures for the Company. Animated ads do not involve locating, recreating, or hiring a set. Nothing is shot in person for animated advertising. Depending on the render's realism, all parts of a live shot are duplicated. You must find or rent a great setting or replicate one in real life. CGI allows the animator to construct the required location, so there is no need to find a place to shoot the commercial. Animation allows the animator and firm to be creative and unrestricted in setting construction. Animation's flexibility and creativity extend beyond places to goods, characters, settings, emotions, and more. From a 3D polygon, the animation becomes lifelike. CGI has improved to the point that it can generate animations that fool the viewer g A live-action commercial has three stages (Johnston, 2019). Pre-production, production, and post-production are in order. Pre-production is creative. Scripting, graphic design, and hiring comprise this stage. actors/interviewers, A live-action commercial has three stages (Johnston, 2019). Pre-production, production, and post-production are in order. Pre-production is creative. Scripting, graphic design, and hiring comprise this stage. actors/interviewers, www.ijisrt.com IJISRT23JAN658 433 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165  The Effect of Animated ads on the Consumer They also liked the intriguing image, which made them want to watch the commercial again and again. Some others claimed that the commercial was animated yet appeared genuine, making it even more appealing and enticing. The popularity of animated adverts in today's advertising environment is the best indication that they have a substantial effect and influence on customers. The audience in New York praised the rising immersion of enormous 3D animated billboard commercials for their engaging and realistic images (Weaver, 2022). Figure 1 shows a 3D commercial for an orange Nike sneaker box that bursts open to expose a rotating display of Air Max designs while hinting at the motivation behind each shoe. According to Ethan Jakab, co-founder of "Blunt Action," "these eye-catching 3D billboards are fast becoming highly popular since people are loving the realistic illusion; therefore making them stop and look twice" (Holtz, 2022). The unique campaign elicited a strong response on social media and in person among those traveling through Shinjuku Station, one of the busiest railway stations in the world. Most responses commended the Nike sneaker box's realistic movement toward the audience. They also liked the intriguing image, which made them want to watch the commercial again and again. Some others claimed that the commercial was animated yet appeared genuine, making it even more appealing and enticing. Fig 1 (3D Commercial For An Orange Nike Sneaker)  Critical Thinking (benefit of animated ad)  Critical Thinking (benefit of animated ad) It would have been possible to film a live-action advertisement for this product. However, because the customer requested an animated advertisement, there was greater freedom in accomplishing the client's ad concept. The first advantage is boosting the product's visual appeal and vitality while enhancing its functionality and perceived worth. Additionally, it made the product appear more avant-garde and artistic. Moreover, since it is a simple object with a simple mechanism that is readily transportable, it is convenient. Shooting it in real life may have been somewhat tedious. Animation afforded a conceivable dull system a visual boost using captivating visuals and animation bringing life and motion to a static object, which can never be achieved using live shots. Fig 1 (3D Commercial For An Orange Nike Sneaker) IV. CONCLUSION CONCLUSION Another realistic 3D advertisement that drew viewers' attention was the cat commercial in Japan. In Shinjuku, a three- story-high high-tech billboard unveiled a new realistic 3D ad depicting a gigantic 3D calico cat, as shown in figure 2. The cat gets up around 7 a.m., sleeps in the afternoon, and finally turns off the screen at 1 a.m. (Imada, 2021). MicroAd Digital Signage and Yunika Vision created this advertisement to demonstrate the future possibilities of 3D advertising (Hidrly, 2021). This charming 3D cat, like the other 3D commercial, went viral all over the internet, with viewers providing excellent feedback and comments. According to Takayuki Ohkawa, a representative of one of the businesses that designed this billboard, the primary motivation for developing this ad was the very dark environment that engulfed the city as a result of the pandemic, and this cat was an attempt to reinvigorate and lighten up the city. Their endeavor was well received, and their goal of 30 was met. A pedestrian, Ryoko Kikuchi, reported that when she was walking home from the movies, the enormous cat sitting down licking its paws and meowing was too sweet and made her heart melt. Many individuals felt the same way and communicated on numerous social media sites, including Twitter, Tiktok, YouTube, and Instagram. The majority of responses expressed people's excitement at this rare realistic sight on the streets of Japan. Others stated that they were attracted by the interactive quality of this ad, in which the cat appears to be engaging with the humans. According to Mr. Shimoda, a marketing professional, the impact was more than expected, as many people gathered in front of the billboard to film the cat's various moves and antics (NYT, 2021). IV. CONCLUSION An excellent marketing method for enhancing the presentation of a product or service is 3D animation. Modern corporations include animated advertisements in their digital marketing tactics to ensure strong sales for their products and services. Animation is unquestionably more formalistic and adaptable in comparison to the actual shooting. Creating a tidy and gentle appearance requires a great deal of versatility. In animated form, This is one of many examples illustrating abstract and intricate nature. Creating real-world concepts is more complex than digital ones. Animating will permit enterprises to enhance the appearance of the product or service they want to offer. IV. CONCLUSION Despite the visuals, digital models must be created from scratch in animated advertisements. Unlike live-action videos where the product already exists, the client can create the product from scratch. Due to the adaptable nature of animation work, he may easily communicate his needs. So departing Unlike live-action advertisements, which are limited to what is achievable, there is no place for the phrase "impossible." physically attainable. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 REFERENCES [1]. Au, K. C. (2014). Animation: 2D versus 3D and their combined effect (Doctoral dissertation, Massachusetts Institute of Technology). [2]. Eid, M. G. (2022). Benefits of animated advertisements in today's world (Doctoral dissertation, Notre Dame University-Louaize) Fig 2 (3D Ad Depicting a Gigantic 3D Calico Cat) [3]. Hidrėlėy. (2021). Giant Hyper-Realistic 3D Cat Billboard Appears In Tokyo, Mesmerizes The Passersby. Bored Panda. [4]. Holtz, R. M. (2022). A Times Square Billboard Displays 3D Money. Artdeko. [5]. Hoque, A. (2020). 5 reasons why business should use animation. Digital Glue. [6]. Imada, K. (2021). A super realistic giant 3D cat has appeared on a billboard in Shinjuku. Time Out. [7]. Jarmo Kuisma, J. S. (2010). “Effects of Animation and Format on the Perception and Memory of Online Advertising. The Journal of Interactive Marketing. [8]. Jim. (2020). A Brief History of Computer Animation. Miglia. [9]. McCormick, K. (2021). 75 Staggering Video Marketing Statistics for 2021. WordStream. [10]. Thapa, S. (2022). 20 Most Expensive NFTs Sold (So Far). Screen Rant . Fig 2 (3D Ad Depicting a Gigantic 3D Calico Cat) [11]. Thompson, J. (2022). A Short Introduction to NFTs. Futurice. The significant increase in demand, as well as investments in the digital world and animation, is evidence that the dynamics of the world are shifting. There is no denying the fact that digitization and animation have become deeply ingrained in our lives and that this trend will continue to shape the globe in the years to come. [12]. Thompson, M. C. (2019). Animation & Advertisement”. Palgrave Macmillan. 435 www.ijisrt.com IJISRT23JAN658 IJISRT23JAN658 434 www.ijisrt.com Volume 8, Issue 1, January – 2023 Volume 8, Issue 1, January – 2023 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 [13]. Upadhyay, D. G. (2017). Effectiveness of use of Animation in Advertising: A Literature Review. The International Journal of Scientific Research in Network Security and Communication. [14]. Vora, Y. (2018). Impact of Animation in Advertising. [13]. Upadhyay, D. G. (2017). Effectiveness of use of Animation in Advertising: A Literature Review. The International Journal of Scientific Research in Network Security and Communication. [14]. Vora, Y. (2018). Impact of Animation in Advertising. IJISRT23JAN658 IJISRT23JAN658 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 [13]. Upadhyay, D. G. (2017). Effectiveness of use of Animation in Advertising: A Literature Review. The International Journal of Scientific Research in Network Security and Communication. [13]. Upadhyay, D. G. (2017). Effectiveness of use of Animation in Advertising: A Literature Review. The International Journal of Scientific Research in Network Security and Communication. [14]. Vora, Y. (2018). Impact of Animation in Advertising. [14]. Vora, Y. (2018). Impact of Animation in Advertising. IJISRT23JAN658 IJISRT23JAN658 436 www.ijisrt.com
W3212253138.txt	https://www.nature.com/articles/s41419-021-04353-9.pdf	en		Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies	Cell death and disease	2,021	cc-by	8,223	www.nature.com/cddis ARTICLE OPEN Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies Isha Kapoor1, Juraj Bodo 2 , Brian T. Hill3 and Alexandru Almasan 1,4 ✉ 1234567890();,: © The Author(s) 2021 Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a ﬁrst-in class, oral, covalent Bruton’s tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3′-UTR, and subsequently activated AKTSer473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis. Cell Death and Disease (2021)12:1061 ; https://doi.org/10.1038/s41419-021-04353-9 INTRODUCTION B-cell lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL), the most prevalent subtypes of non-Hodgkin lymphoma (NHL), are characterized by chronic activation of the B-cell receptor (BCR) signaling [1, 2]. Bruton’s tyrosine kinase (BTK) is a central kinase in the BCR axis that drives a signaling cascade leading to activation of NF-κB and phosphatidylinositol-3-kinase (PI3K) prosurvival pathways in CLL and the activated B-cell (ABC) subset of DLBCL [1, 3]. Ibrutinib, an FDA-approved, ﬁrst-in-class orally administered BTK inhibitor that binds covalently to the C481 residue of BTK, has demonstrated impressive clinical activity in newly diagnosed and treatment-relapsed/refractory patients with CLL and many subtypes of NHL [4, 5]. However, ibrutinib also binds to other homologous cysteine-containing kinases, such as ITK, EGFR, TEC, and BMX, which result in toxic off-target side-effects, eventually leading to discontinuation of ibrutinib [6–8]. The clinical activity of ibrutinib as a single agent in DLBCL has a preferential beneﬁt for patients with ABC-DLBCL but its utility is limited [9–12]. Despite the efﬁcacy of ibrutinib, clinical responses are variable/partial, often leading to drug resistance and aggressive relapse of the disease. Up to 5% of ibrutinib-treated patients progress with more aggressive ABC-DLBCL [9, 10]. Toxicities, such as atrial ﬁbrillation, bleeding, or arterial hypertension, albeit limited, caused by inhibition of other non-BTK targets, such as ITK and EGFR underscores the need for more selective BTK inhibitors with fewer off-target effects [6, 11]. Unlike ibrutinib, acalabrutinib, is an FDA-approved secondgeneration, highly selective, potent, covalent BTK inhibitor with minimal off-target effects [6]. Interestingly, these BTK inhibitors showed a similar preclinical activity proﬁle, molecular, and biologic effects in primary CLL cells [6, 13–15]. Acalabrutinib monotherapy has shown good tolerability and efﬁcacy in treatment-naïve, relapsed/refractory CLL patients. As a single agent, acalabrutinib demonstrated a high response rate (~81%) in ibrutinib-intolerant CLL patients [8]. Recently presented head-to-head comparison shows similar clinical activity but improved safety proﬁle of 1 Department of Cancer Biology, Lerner Research Institute, Cleveland, OH, USA. 2Department of Laboratory Medicine, Institute of Pathology and Laboratory Medicine, Cleveland, OH, USA. 3Department of Hematology and Medical Oncology, Taussig Cancer Institute, Cleveland, OH, USA. 4Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. ✉email: almasaa@ccf.org Edited by Dr. Marco (J) Herold Received: 27 July 2021 Revised: 13 October 2021 Accepted: 18 October 2021 Official journal of CDDpress I. Kapoor et al. 2 acalabrutinib vs ibrutinib [16]. Recent studies showed constitutive activation of the PI3K/AKT pathway in 25–52% of DLBCL patients, and correlated overexpression of phosphorylated Akt (pAKT) with signiﬁcantly poorer progression-free survival in approximately onefourth of DLBCL patients [17]. We and others have previously shown that downregulation of PTEN, a major negative regulator of the PI3K/AKT signaling is signiﬁcantly associated with chemotherapy resistance and poor survival in patients with DLBCL with AKT hyperactivation [17–19]. Yet, how PTEN mediates resistance to BTK inhibition in B-cell malignancies is not clear. Following prolonged treatment, CLL and DLBCL patients can acquire resistance to BTK inhibitors, ibrutinib or acalabrutinib, through mutations in BTK and its substrate phospholipase C gamma 2 (PLCG2), MYD88, and CARD11 [18, 20]. In addition to the acquisition of these mutations, other mechanisms can confer resistance to BTK inhibition, such as upregulation of druggable survival pathways, clonal evolution due to other genetic alterations [18], or aberrant expression of miRNAs [21, 22]. Such mechanisms may be overcome by rational therapeutic combinations of targeted agents that block adaptive pathways promoting drug resistance. Several studies have reported the involvement of aberrant expression of micro-RNAs (miRNAs) in the development of chemo-sensitivity or -resistance in various cancers, including CLL and DLBCL [21, 23]. miRNAs are small (~20–22 nucleotides) noncoding regulatory RNAs that bind to a speciﬁc target mRNA through a sequence that is complementary to the 3′-UTR of the target mRNA [23]. Several miRNAs regulate oncogenic or tumorsuppressive pathways, such as the NF-κB or BCR signaling cascade in B-cell malignancies [21, 22, 24, 25]. Our previous studies have shown aberrant regulation of miR-377 in germinal center-type DLBCL that targets BCL-xL, and thus drives acquired resistance to BCL-xL inhibition by venetoclax [23]. Therefore, we investigated the underlying molecular signatures of BTKi resistance in sensitive vs acquired acalabrutinib-resistant (Aca-R) and ibrutinib-resistant (IB-R) cells following chronic exposure to these therapeutics. By comparing sensitive vs acquired BTKi-R cells, we have deﬁned BTKi-R as a 14q32 miRNA cluster-dependent regulation of PTEN/AKT/mTOR in CLL and DLBCL in the absence of BTK or PLCG2 mutations. Our data reveal novel mechanistic insights into the role of cooperative PTENtargeting by 14q32 cluster miRNAs: miRNA-494 and miR-495, as well as miR-453, miR-899, miR-737, and miR-433 in BTKi-R cells. These ﬁndings provide a rationale for cooperative inhibition of overexpressed oncogenic miRNAs to overcome resistance to BTK inhibition in lymphoid malignancies by upregulation of PTEN leading to AKT/mTOR activation. RESULTS Acquired resistance to chronic BTK inhibition leads to upregulation of 14q32 cluster miRNAs Aca-R ABC-DLBCL (TMD8), IB-R ABC-DLBCL (RIVA, TMD8), and CLL (MEC-1) cell lines were generated by culturing the parental cell lines in vitro with progressively increasing concentrations of acalabrutinib or ibrutinib, as previously described [18]. Cell viability analysis showed a ~40% increase in cell death in TMD8 (Fig. 1a), but not in Aca-R-derivative cells after 24 h of acalabrutinib treatment. Similarly, MTS assays showed a high sensitivity to increasing concentrations of acalabrutinib administered for 72 h with an IC50 of 78 nM for TMD8 cells. These Aca-Rderivative cells were resistant to much higher concentrations than the IC50 of the parental cells (Supplementary Fig. S1a). To investigate the mechanism of Aca-R, we examined the expression pattern of PTEN in parental and resistant cells. Immunoblot analyses show that the levels of PTEN were low in resistance (TMD8-Aca-R) compared to parental cells (Fig. 1b). Additionally, levels of pAKT (AKTSer473) were upregulated in TMD8Aca-R compared to parental cells. Importantly, pBTK (BTKY223) levels were diminished, indicating that chronic acalabrutinib treatment blocks BTK activation in Aca-R cells. Notably, the qRTPCR analysis indicated that pten mRNA levels were decreased by ~4-fold, even after acute treatment with acalabrutinib in TMD8Aca-R vs parental cells, indicating that the reduced PTEN levels could be attributed to decreased pten mRNA levels (Fig. 1c). Taken together, these ﬁndings indicate the importance of the PTEN/AKT axis in mediating Aca-R. We have previously shown that acquired resistance to ibrutinib is associated with PTEN downregulation and activation of the PI3K/AKT pathway [18]. To investigate the mechanism of resistance to BTK inhibition, we examined the expression patterns of miRNAs located in the 14q32 cluster that we previously found to be involved in the resistance to BCL-xL inhibition in CLL and DLBCL [23]. Examination of the expression patterns of nine miRNAs located in the 14q32 cluster by qRT-PCR analyses indicated their increased expression in both Aca-R (Fig. 1d) and IB-R (Fig. 1e–g) DLBCL and CLL cell lines. Of these miRNAs, expression of miR-494 and miR-543 was increased by ~3-fold and 2-fold, respectively in TMD8-Aca-R cells (Fig. 1d). Similarly, expression of miR-494, miR-495, and miR-543 were increased by ~4.8-fold, ~3-fold, and ~2-fold, respectively in TMD-IB-R (Fig. 1e), ~6-fold, 4-fold, and 5-fold, respectively in MEC-1-IB-R (Fig. 1f) and ~7.5-fold, 4-fold, and 5-fold, respectively in RIVA-IB-R cells (Fig. 1g). Taken together, these ﬁndings indicate an association of aberrant expression of 14q32 cluster miRNAs with resistance to BTK inhibition. BTK inhibition downregulates 14q32 cluster miRNAs and upregulates PTEN expression Since BTKi resistance following chronic exposure to acalabrutinib or ibrutinib resulted in increased expression of 14q32 cluster miRNAs and lower levels of PTEN, we examined the effects of acute acalabrutinib and ibrutinib treatment on 14q32 cluster miRNAs in BTKi-sensitive ABC-DLBCL and CLL cells. Parental TMD8 cells treated with acalabrutinib demonstrated downregulation of 14q32 cluster miRNAs, with decreased levels of miR-494 (~50%), miR-495 (~40%), and miR-543 (~30%) (Fig. 2a). BTK inhibition by ibrutinib in TMD8 cells resulted in ~90% reduced expression of miR-494 and miR-543, and ~80% decreased expression of miR-495 (Fig. 2b). Similarly, ~90% reduced expression of miR-494, miR-495, and miR-543 were observed in both MEC-1 (Fig. 2c) and RIVA cells (Fig. 2d) treated with ibrutinib. Taken together, these ﬁndings indicate the potential role of aberrant expression of 14q32 cluster miRNAs in mediating BTKi resistance. BTK inhibition decreases the expression of 14q32 cluster miRNAs and increases that of PTEN in patient-derived primary CLL cells Given the differences in expression of 14q32 cluster miRNAs and PTEN in Aca-R and IB-R CLL and DLBCL cells in vitro, we tested whether miRNA expression might also be altered in patientderived primary CLL cells, in response to BTK inhibition or standard-of-care clinical therapy. qRT-PCR analysis in three paired CLL patient samples pre- and post-ibrutinib-treated in the clinic revealed a decrease in the levels of miR-494, miR-495, and miR543 in #CLL3 (ibrutinib-sensitive) after clinical ibrutinib treatment in contrast to #CLL1 (ibrutinib-resistant) and #CLL2 (partial remission) (Fig. 3a). Additionally, in vitro ibrutinib treatment of CLL patient samples revealed an increase in the expression of miR494, miR-495, and miR-543 in treatment-relapsed vs naïve patients (Fig. 3b). Similarly, in vitro acalabrutinib treatment of naïve (#CLL4, #CLL5) vs treatment-relapsed (#CLL6, #CLL7) CLL patients showed signiﬁcant downregulation of miR-494 (58%), miR-495 (64%), and miR-543 (68%) in #CLL4 and #CLL5 (treatment naïve) (43% reduction in miR-494; 79% miR-495, and 68% miR-543) in contrast to #CLL6 and #CLL7 (treatment-relapsed) (Fig. 3c). Additionally, in vitro acalabrutinib treatment of naïve (#CLL4, #CLL5) vs Cell Death and Disease (2021)12:1061 I. Kapoor et al. 3 Fig. 1 BTK inhibition downregulates miRNAs in the 14q32 cluster region in CLL and DLBCL cells. a Cell death analysis in parental TMD8 and acalabrutinib-resistant derivatives (TMD8-Aca-R) in response to 24 h acalabrutinib treatment determined by Annexin V/PI staining. All data were expressed as mean ± SD of the percentage of cell death. b Expression levels of pAKTSer473, AKT, and PTEN in whole-cell extracts of untreated parental and Aca-R TMD8 cells. GAPDH was used as a loading control. c mRNA fold change of pten in parental vs Aca-R TMD8 cells with or without acalabrutinib (5 µM) for 48 h. d Relative expression changes of miRNAs in the 14q32 domain cluster, as determined by qRTPCR in untreated parental and Aca-R TMD8 cells. miRNAs fold change in Aca-R cells is normalized to untreated parental cells. SD is indicated as error bars (N = 3). e–g miRNAs fold change in parental vs IB-R TMD8, MEC-1, and RIVA cells. miRNAs fold change in ibrutinib-treated cells is normalized to untreated parental cells. (p < 0.05, p < 0.01, *p < 0.001). Standard deviation (SD) is indicated as error bars (N = 3). treatment-relapsed (#CLL6, #CLL7) CLL patients revealed signiﬁcant increase in pten mRNA levels in #CLL4 (2.9-fold) and #CLL5 (3.2-fold) in contrast to #CLL6 and #CLL7 (Fig. 3d). Taken together, these results indicate the role of aberrant expression of 14q32 cluster miRNAs in mediating therapeutic resistance. PTEN is a direct target of miR-494 Previously, we have shown that ibrutinib treatment regulates transcriptional activation of PTEN in CLL and DLBCL cells [18]. Using target prediction software to identify miRNAs that have a putative PTEN target, we found six miRNAs located in the 14q32 cluster, of which miR-494 had the highest score (Fig. 4a). Interestingly, qRT-PCR and immunoblot analysis of TMD8-Aca-R cells transfected with a miR-494 inhibitor revealed a signiﬁcant increase in the expression of pten mRNA (upper panel) and protein (lower panel) levels (Fig. 4b). Similar results were obtained in Cell Death and Disease (2021)12:1061 TMD8-IB-R (Fig. 4c), MEC-1-IB-R, and RIVA-IB-R cells (Supplementary Fig. S2a, b). To conﬁrm that miR-494 is directly involved in PTEN regulation, adding miR-494 mimics led to a substantial decrease in both endogenous PTEN mRNA (upper panel) and protein (lower panel) expression levels in parental TMD8 (Fig. 4d), MEC-1 and RIVA cells (Supplementary Fig. S2c, d). Additionally, the qRT-PCR analysis indicated that overexpression of miR-494 inhibitor in TMD-Aca-R, TMD8-IB-R, and MEC-1-IB-R (Supplementary Fig. S3a–c) results in increased expression of proapoptotic bim mRNA levels while overexpression of miR-494 mimic in MEC-1 and TMD8 cells (Supplementary Fig. S3d, e) results in decreased expression of bim mRNA levels. Taken together, these results indicate that both PTEN and BIM is regulated at the posttranscriptional level by miR-494 and could be a direct target. To determine potential targets of miR-494, we performed prediction analysis of the 14q32 miRNA cluster region and I. Kapoor et al. 4 Fig. 2 Expression of 14q32 cluster miRNAs is decreased after BTK inhibition in CLL and DLBCL. a Expression of nine miRNAs in the 14q32 cluster, as determined by qRT-PCR in TMD8 cells with or without acalabrutinib (5 µM) for 48 h. miRNA fold change in acalabrutinib treated cells is normalized to untreated cells. b–d Relative expression changes of nine miRNAs in the 14q32 cluster, as determined by qRT-PCR in TMD8, MEC-1, and RIVA cells treated with ibrutinib (10 µM) for 48 h. miRNAs fold change in ibrutinib-treated cells is normalized to untreated cells. (p < 0.05, p < 0.01, p < 0.001). SD is indicated as error bars (N = 3). PTEN 3′-UTR alignment using mirDB [26, 27]. This analysis identiﬁed two conserved complementary sequences at positions 2313 and 2798 in the 3′-UTR of PTEN mRNA with which miR-494 is likely to base-pair (Fig. 4d). To examine whether PTEN is a direct target of miR-494, luciferase reporter containing wild-type (WT) PTEN 3′-UTR or miR-494-binding site mutant PTEN 3′-UTR were transfected together with miR-494 mimic (Fig. 4e) or inhibitor (Fig. 4f) and negative control in TMD8 and TMD8-IB-R cells, respectively and luciferase activity was measured after 48 h. Ectopic miR-494 mimic expression in TMD8 cells downregulated WT-3′-UTR-associated luciferase activity by ~50% as compared with the negative control mimic (Fig. 4e). In contrast, transfection with mutant 3′-UTR luciferase reporter, miR-494 mimic expression was unable to suppress luciferase activity at all (Fig. 4d). Transfection with a miR-494 inhibitor in TMD8-IB-R cells completely reversed the luciferase activity, resulting in a 4-fold increase in wild-type WT-3′-UTR-associated luciferase activity as compared to negative control inhibitor (Fig. 4f). Taken together, these results indicate a direct binding of miR-494 to the predicted and previously reported [25] target sites in the PTEN 3′-UTR. AKT inhibition potentiates miRNA inhibition-induced apoptosis in BTK-R CLL and DLBCL Given that PTEN expression was downregulated by miR-494, we next examined whether these cells could be further sensitized to apoptosis by AKT inhibition, in combination with miR-494 or miR-495. Previously, we have shown that AKT activation was elevated in our IB-R cells, and PTEN was signiﬁcantly downregulated, thus making IB-R cells more sensitive to induction of apoptosis by AKT inhibition [18]. Pharmacological inhibition of AKT showed ~20 and ~19% increase in apoptosis in TMD8 and Aca-R cells, respectively by inhibition of AKT together with miR494 (Fig. 5a) or miR-495 (Fig. 5b). Similarly, inhibition of miR-494 (Fig. 5c) and miR-495 (Fig. 5d) in TMD8-IB-R cells showed ~24 and ~21% increase in apoptosis with AKT inhibition, respectively. Similar results were obtained in MEC-1-IB-R cells with inhibition of AKT in combination with miR-494 (~33%) and miR-495 (~25%) (Supplementary Fig. S4a, b) in comparison to miR-494 or miR-495 inhibition alone. Together, these results indicate the dependency of BTKi-R cells on miR-494 or miR-495-dependent regulation of PTEN/AKT and that inhibition of AKT phosphorylation/activation increases miR-494 and miR-495 inhibition-induced apoptosis in BTK-R cells. Cooperative miRNA inhibition potentiates apoptosis by targeting PTEN miR-494 inhibition suppressed AKT and mTOR activity, as indicated by a decrease in pAKTSer473 and inhibition of phosphorylation of downstream targets of mTORC1, p70S6Thr389 kinase, and p4EBP1. Notably, the expression of PTEN protein levels was increased in TMD8-IB-R cells by miR-494 inhibition. AKT and p70S6 kinase levels, however, did not change signiﬁcantly (Fig. 6a). These ﬁndings indicate that an increase in PTEN protein expression in response to miR-494 inhibition in TMD8-IB-R cells was associated Cell Death and Disease (2021)12:1061 I. Kapoor et al. 5 Fig. 3 Expression of 14q32 cluster miRNAs is decreased and that of PTEN increased after BTK inhibition in patient-derived primary CLL cells. a Expression of miR-494, miR-495, and miR-543 was analyzed in primary cells obtained from three paired CLL patients’ samples pre- and post-ibrutinib-treated in the clinic. b Relative expression changes in miR-494, miR-495, and miR-543 was analyzed in primary cells obtained from nine treatment naïve vs ﬁve treatment-relapsed CLL patients’ samples after in vitro treatment with ibrutinib. Mann–Whitney nonparametric analysis was performed to compare them. Two-sided p value is 0.0004. c, d Relative expression of miR-494, miR-495, miR-543, and pten was analyzed in primary cells obtained from treatment naïve (#CLL4, #CLL5) and relapsed (#CLL6, #CLL7) CLL patients’ samples after in vitro treatment with acalabrutinib (5 µM). (p < 0.05, p < 0.01, p < 0.001). SD is indicated as error bars (N = 1). with decreased activity of AKT and mTOR. In addition, miR-494 inhibition resulted in increased expression of caspase 3 cleavage (Fig. 6a). Similar results were obtained in MEC-1-IB-R cells (Supplementary Fig. S5a) and miR-494 inhibition conferred a ~20% increase in ibrutinib-induced apoptosis in cells (Fig. 6a; upper panel). Similar results were obtained with miR-495 inhibition in TMD-IB-R (Fig. 6b) and MEC-l-IB-R cells (Supplementary Fig. S5b; lower panel). Next, we examined whether these miRNAs may coordinately impact cell viability by regulating PTEN expression. Cell viability analysis in TMD8-IB-R (Fig. 6c) and MEC-1-IB-R (Fig. 6d) cells demonstrated an ~20% increase in apoptosis in cells transfected with a combination of miR-494 and miR-495 inhibitors compared to transfection with either miR-494 or miR-495 inhibitors alone, indicating a cooperative action of these miRNA inhibitors on cell survival. DISCUSSION Despite the extensive heterogeneity of B-cell lymphoid malignancies, accumulating evidence has supported the association between deregulated expression of miRNAs and therapeutic resistance in various cancers, including CLL and DLBCL [21–23, 25]. Our recent ﬁndings uniquely characterize the development of acquired IB-R by PTEN downregulation that impedes ibrutinib-induced apoptosis, as demonstrated by AKT activation [18]. In our present study, we show for the ﬁrst time that similar to IB-R cells, AKT activation was elevated in our Aca-R cells and PTEN was signiﬁcantly downregulated following chronic exposure to acalabrutinib. Nevertheless, how PTEN mediates resistance to BTK inhibition was unknown. In search of the molecular mechanism responsible for decreased PTEN expression, we have uniquely characterized a novel role for Cell Death and Disease (2021)12:1061 14q32 cluster miRNAs-dependent regulation of the PTEN/AKT/ mTOR axis in mediating resistance to BTK inhibition in CLL and DLBCL cells in the absence of BTK or PLCG2 mutations. Interestingly, there is a growing interest in the maternally imprinted DLK1-DIO3 region on chromosome 14q32 because ~53 miRNAs are embedded in two adjacent clusters, many of which have been reported to be deregulated in various cancers, such as, APL [28], melanoma [29], and lung adenocarcinomas [30]. Previously, we have reported miRNA-377-dependent regulation of BCL-xL in venetoclax resistance in B-cell lymphoid malignancies and germinal center-type DLBCL [23]. Here we show that chronic BTK inhibition leads to upregulation of the 14q32 miRNA cluster in CLL and DLBCL cells. Similar to what was observed for ibrutinib, acquired resistance to acalabrutinib also resulted in overexpression of these miRNAs in CLL and DLBCL cells and downregulation of PTEN, revealing aberrant expression of 14q32 cluster miRNAs as a common mechanism of resistance to BTK inhibition. Overexpression of the 14q32 cluster miRNAs has been associated with the CCCTC-binding factor (CTCF)mediated regulation of the maternally expressed gene 3 differentially methylated region (MEG3-DMR) [31] or global genomic hypomethylation of 14q32 locus as reported in various cancers [32], including CLL [33]. While methylation has been well documented in GC-DLBCL [34] there are limited reports on the role of methylation in the ABC-subtype of DLBCL that we have examined as this subtype is responsive to BTK inhibitor therapy. However, chronic exposure to BTKi that induces global hypomethylation of the 14q32 locus that may result in the upregulation of 14q32 miRNAs cluster in BTKi-resistant in comparison to parental CLL and ABC-DLBCL cells warrants further investigation. Importantly, we show increased expression of miR-494, miR-495, and miR-543 and PTEN downregulation in response to BTK inhibition in therapy-relapsed patient-derived I. Kapoor et al. 6 Fig. 4 PTEN is a direct target of miR-494 in CLL and DLBCL. a Expression levels of PTEN mRNA (top) and protein (bottom) in TMD8-Aca-R and b Expression levels of PTEN mRNA (top) and protein (bottom) in TMD8 cells after transfection with miR-494 inhibitor, mimic, or negative control, as indicated. GAPDH was used as a loading control. c Expression levels of PTEN mRNA (top) and protein (bottom) in TMD8-IB-R cells after transfection with miR-494 inhibitor or negative control, as indicated. GAPDH was used as a loading control. d, e Luciferase reporter activity in TMD8-IB-R and TMD8 and cells co-transfected with PTEN WT-3′-UTR or mutant 3′-UTR constructs and miR-494 inhibitor or miR-494 mimic, respectively together with negative control as indicated. (p < 0.01, *p < 0.001). SD is indicated as error bars (N = 3). f schematic representation of the PTEN 3′-UTR with positions of targeting miRNAs. Conserved sites for miR-494, miR-495, and miR-543 are indicated in bold. Other non-conserved sites for other miRNAs, as mentioned in Table 1, are indicated as dotted lines on PTEN 3′-UTR. primary CLL cells vs those treatment-naïve. Therefore, these clinically relevant data along with our BTKi-R models support a broader mechanism of therapeutic resistance that may be critical for conferring resistance to BTK inhibition. miRDB target prediction software identiﬁed six miRNAs that target PTEN, among which miR-494 had the highest prediction score (Table 1). A target with a prediction score >80 is associated with a high conﬁdence level of the validity of the ﬁndings (miRDB. org) [26, 27]. We focused on miR-494 for two reasons: (i) miRDB analysis identiﬁed two conserved complementary 8-mer sequences in the 3′-UTR of PTEN mRNA that miR-494 is likely to base-pair with, (ii) its location at 14q32, the aberrantly regulated chromosome 14 region that has been previously described in B-cell lymphomas [23]. In support of our preclinical ﬁndings, other studies also reported the involvement of 14q32 cluster cancerrelated miRNAs in promoting chemotherapy resistance and malignant transformation in various carcinomas [30, 35–44]. We provide evidence that BTK inhibition downregulates 14q32 cluster miRNAs and upregulates PTEN in CLL and DLBCL cell lines, an observation that is strengthened by our ﬁndings in patient-derived primary CLL cells treated with BTK inhibitors in vitro (Fig. 3c, d) and in samples from patients undergoing ibrutinib therapy in the clinic. Importantly, we deﬁne PTEN as a direct target of miR-494 in CLL and DLBCL cells by two independent approaches: (i) miR-494 modulation both by an inhibitor and a mimic, and (ii) a luciferase reporter assay, consistent with independent investigations showing PTEN as a direct target in various cancers, including hepatocellular carcinoma [42], colorectal cancer [45], and nonsmall cell lung cancer [46]. We identiﬁed 14q32 cluster region miRNAs, such as miR-494, miR-495, and miR-543, to be associated with BTKi resistance and demonstrated that miR-494 mediated PTEN downregulation and AKT activation was responsible for decreased apoptosis. Cell Death and Disease (2021)12:1061 I. Kapoor et al. 7 Fig. 5 miRNA inhibition potentiates AKT-induced apoptosis in BTK inhibitor-resistant CLL and DLBCL. a TMD8-Aca-R cells were transfected with miR-494 (100 nM) and b miR-495 (50 nM) inhibitors and treated with ± MK2206 (5 µM) for 24 h. Cell death analysis was determined by Annexin V-PI staining. Control cells were treated with DMSO. TMD8-IB-R cells were transfected with miR-494 (c) and miR-495 (d) inhibitors together with miRControl and treated with MK2206 (5 µM) for 24 h. Cell viability was determined by Annexin V-PI staining. Control cells were treated with DMSO. (p < 0.05, p < 0.01, p < 0.001). All data were expressed as mean ± SD of the percentage of cell death. SD is indicated as error bars (N = 3). Consistently, our previous studies demonstrated that inhibition of PI3K/AKT signaling sensitizes IB-R cells to apoptosis in a PTEN- and BIM-dependent manner [18]. Now we show that miR494-mediated PTEN regulation is involved more broadly in BTKresistance through AKT activation. Pharmacologic AKT inhibition potentiates miR-494 or miR-495 inhibition-induced apoptosis in BTKi-R CLL and DLBCL cells. Several studies have reported that tumor-promoting miRNAs targeting PTEN, such as miR-494 are involved in drug resistance [42, 46], and that their inhibition by anti-miRNA-based therapeutic strategies induce sensitization to apoptosis [42]. Consistently, we show that inhibition of miR-494 or miR-495 either alone or in combination potentiates induction of apoptosis in BTKi-R cells. Moreover, previously, we and others have shown coordinated therapeutic regulation of miRNAs from the 14q32 cluster region, spanning from the DLK1 to DIO3 genes, also known as the DLK1DIO3 region [23, 28, 32, 47, 48]. Several of these miRNAs, miR-494 [40, 45], miR-495 [49], miR-543 [38, 44], but also miR-889 [38], miR337 [41], and miR-433 [41] are all targeting the PTEN 3′-UTR (Fig. 7). Importantly, simultaneous inhibition of two miRNAs located in the 14q32 cluster region potentiates the inhibitory Cell Death and Disease (2021)12:1061 action of the anti-miRNA-based strategy and coordinately sensitize BTKi-resistant cells to apoptosis. AKT/mTOR signaling activation has been characterized as an important resistance mechanism in IB-R mantle cell lymphoma [50], Waldenstrom macroglobulinemia [51], CLL, and DLBCL [18], as well as venetoclax-resistant [52], or ﬂudarabine-resistant [53] B-cell lymphoid malignancies, where inhibition represented a powerful approach to overcome drug resistance and induce apoptosis. Consistently, activation of AKT and downstream targets of mTOR signaling was elevated in our BTKi-R cells, thus making BTKi-R cells more sensitive to AKT inhibition and induction of apoptosis. Indeed, miR-494 or miR-495-dependent inhibition diminished AKT/mTOR activation and sensitized cells to apoptosis. These ﬁndings suggest the therapeutic ability of an anti-miRNA-based strategy to block adaptive signaling responses in resistant subclones to overcome drug resistance and induce apoptosis. Previous studies of AKT inhibition in lymphoma patients with MK2206 have shown modest clinical activity [54]. However, recently, large phase III trials in advanced prostate and breast cancer have shown signiﬁcant improvement in progression-free survival by the addition of the AKT inhibitors ipatasertib [55] and capivasertib [56] to standard I. Kapoor et al. 8 Fig. 6 Cooperative miR-494 and miR-495 inhibition enhances cell survival through AKT/mTOR signaling. a TMD8-IB-R cells were transfected with miR-494 (200 nM) and b miR-495 (100 nM) inhibitors and treated with ± ibrutinib (10 µM) for 24 h. (Upper panel) Cell death analysis was determined by Annexin V-PI staining. Control cells were treated with DMSO. (p < 0.01). SD is indicated as error bars (N = 3). (Lower panel) Expression levels of PTEN, pAKT Ser473, AKT, pP70S6-T389, pP70S6, p4EBP1, and cleaved caspase 3 were determined in wholecell extracts of TMD-IB-R cells transfected with miR-494 (200 nM) and miR-495 inhibitors, respectively (100 nM) by immunoblotting. GAPDH was used as a loading control. c TMD8-IB-R and d MEC-1-IB-R cells were transfected with miR-494 (200 nM) and miR-495 (100 nM) either alone or in combination for 24 h. Cell death analysis was determined by Annexin V-PI staining. Control cells were treated with DMSO. (p < 0.05, *p < 0.01). SD is indicated as error bars (N = 3). Cell Death and Disease (2021)12:1061 I. Kapoor et al. 9 Table 1. Putative binding sites of 14q32 cluster miRNAs to PTEN 3′ UTR. miRNA Target score Target rank Conserved sites (Nr/ position) Non-conserved sites** (Nr/ position) miR-494 97 18 2 (2313, 2798) 3 (5217, 5861, 5905) Site type 8-mer miR-495 90 60 2 (3221, 3232) 2 (2153, 6272) 8-mer miR-543 69 160 1 (2311) 2 (3929, 6216) 8-mer miR-337 92 48 - 1 (109) 8-mer miR-889 80 100 - 4 (1290, 1616, 2176, 6260) 8-mer miR-433 67 308 - 3 (3743, 5009, 5128) 7–8-mer Conserved sites as shown in Fig. 4f. *Non-conserved/poorly conserved sites are shown as dotted lines in Fig. 4f. treatments. Due to the potential for resistance to continuous treatment with BTK inhibitors, the addition of an AKT inhibitor to such treatment regimens is a rationale strategy. In summary, our ﬁndings provide novel molecular insights into BTK inhibitor resistance mechanisms beyond point mutations in BTK or PLC-γ and support a link between aberrant expression of the 14q32 cluster miRNAs in Aca-R and IB-R cells and the ability of anti-miR-494 or miR-495 to upregulate PTEN to overcome drug resistance and induce apoptosis by diminishing AKT activation (Fig. 7). Importantly, cooperative inhibition of miRNAs leading to induction of apoptosis exploits the resistant cells’ dependency on PTEN/AKT via coordinate regulation of multiple PTEN-targeting miRNAs residing in the 14q32 cluster. Thus, the 14q32 miRNAs cluster/PTEN/AKT/mTOR axis emerges as a determinant of acquired BTKi-R in CLL and DLBCL. Overexpression of miR-494 or miR-495 downregulates PTEN and promotes pro-survival AKT activation in acquired BTKi resistance. Therefore, we propose a combination of miRNA and AKT inhibition as a rational combination strategy to sensitize BTKi-R cells to apoptosis. MATERIALS AND METHODS Cell lines and patient samples Human cell lines MEC-1 (CLL) and ABC-DLBCL (RIVA and TMD8) were cultured in RPMI-1640 medium supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA), and antibiotic-antimycotic (Gibco, Life Technologies, Gaithersburg, MD). Ibrutinib-R (IB-R) and Acalabrutinib-R (Aca-R) cells were cultured with 5% FBS. Cell lines were routinely screened for Mycoplasma, variations in growth rates, changes in morphological characteristics, and their response to stress with Annexin V FITC-PI staining; their passage number did not exceed 20. Ibrutinib was obtained from Santa Cruz (Chicago, IL); Acalabrutinib (ACP-196) from Selleck Chemicals (Houston, TX). Peripheral blood samples were obtained from CLL patients after informed consent according to protocols approved by the institutional review board (IRB) according to the Declaration of Helsinki. Patient characteristics were as described earlier [18]. Lymphocytes from these blood samples were puriﬁed by Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ) gradient centrifugation. Generation of acalabrutinib-resistant (Aca-R) cell lines Acalabrutinib-resistant TMD8 cells were generated by in vitro culture of the parental cell lines for prolonged periods of time with progressively increasing concentrations of acalabrutinib. Brieﬂy, cells were intermittently incubated with a low concentration (six-fold lower than IC50) of acalabrutinib for short intervals over time and allowed to recover after washing off the drug. The acalabrutinib concentration and treatment time were gradually increased until cells remained viable after continuous exposure to the drug that was double the concentration of their IC50 value. The Aca-R cells were routinely tested for resistance to acalabrutinib and cultured without the drug for 72 h before they were used in experiments, as described previously for IB-R cells [18]. Cell viability and apoptosis assays The number of viable cells in culture was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium inner Cell Death and Disease (2021)12:1061 salt (MTS) assay (Promega, Madison, WI), and the percentage reduction in metabolic activity was calculated as previously described [18]. The percentage of cells undergoing apoptosis was measured by phosphatidylserine externalization using ﬂuorescein-conjugated Annexin V/PI double staining (BD Biosciences, San Jose, CA). The analysis was done on a BD FACS MACSQuant ﬂow cytometer (BD Biosciences), and the raw data were processed using the FlowJo software. The results were normalized to the survival of control cells that have been treated with DMSO. Immunoblotting To prepare lysates, cells were collected and washed twice with ice-cold PBS. Cell lysates were prepared in RIPA buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1% phosphatase inhibitor cocktail (Sigma, St. Louis, MO), and 1 mM PMSF (Sigma) for 30–45 min at 4 °C. The protein concentration in each sample was determined using the Bradford reagent (Bio-Rad, Hercules, CA); 50 µg protein was resolved on 10% SDS-PAGE followed by transferring to nitrocellulose membrane (Millipore, Danvers, MA). The immunoblotting was performed with primary antibodies for PTEN Cat No. #9188 S, cleaved caspase 3 Cat No. #9661 S, AKT Cat No. #9272, pAKTSer473 Cat No. #9271 S, pP70S6T389 Cat No. #9205 S, pP70S6 Cat No. #2708 S, p4EBP1Ser65 Cat No. #9451 S, pBTKY223 Cat No. #5082 S BTK Cat No. #8547 S (Cell Signaling Technologies, Danvers, MA), GAPDH Cat No. sc-365062 (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary HRP-conjugated anti-mouse Cat No. 31450 and -rabbit Cat No. 31460 antibodies were purchased from Thermo Fisher Scientiﬁc (Pittsburgh, PA). The immunoreactive bands were visualized by chemiluminescence according to the manufacturer’s recommendations (Thermo Fisher, Waltham, MA). Luciferase reporter assay and miRNA modiﬁcation Wild-type and mutant PTEN 3′-UTR luciferase reporters were kind gifts from Dr. Chuanshu Huang (NYU School of Medicine, Tuxedo, NY) [25]. Luciferase assays were performed as described previously [23]. Brieﬂy, TMD8 and TMD8-IB-R cells (30,000 cells/well) were placed in a 24-well plate and 24 h later were co-transfected using Lipofectamine 3000 (InvitrogenThermo Fisher, Waltham, MA), with 100 ng WT-3′-UTR or Mut-3′-UTR luciferase reporter constructs, 0.5 ng renilla luciferase reporter plasmid (Promega, Madison, WI) and either miR-494/495-inhibitor (50 nM), -mimic (50 nM), or negative control. Cell lysates were assayed for ﬁreﬂy and renilla luciferase activities 48 h after transfection using the dual-luciferase reporter assay system (Promega, Madison, WI) and a Victor [3] multilabel plate reader (Perkin Elmer, Waltham, MA). Renilla luciferase activity served as a control for transfection efﬁciency. Data were shown as the ratio of ﬁreﬂy luciferase activity to renilla luciferase activity. RNA isolation and real-time quantitative-PCR Total RNA was extracted using the TRIZOL reagent (Life Technologies) from parental and BTKi- R cell lines or CLL primary cells after ibrutinib or acalabrutinib treatment according to the manufacturer’s instructions. One microgram of the RNA samples was reverse-transcribed using the TaqMan reverse transcription kit and ampliﬁed using the SYBR Green Master Mix (Applied Biosystems) and examined on a 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA). Levels of mRNA were analyzed using a quantitative real-time reverse transcriptase PCR (qRT-PCR) kit with primers synthesized by IDTR for pten (forward: 5′-CCAATGTTCAGTGGCGGAACT-3′; reverse: 5′-GAACTTGTCTTCCCGTCGTGTG-3′), as described previously [18]. I. Kapoor et al. 10 Fig. 7 Schematic representation of 14q32 cluster miRNAs-dependent resistance to BTK inhibition in B-cell lymphoid malignancies. BTK inhibitor (ibrutinib or acalabrutinib) downregulates miRNAs in the 14q32 cluster region, such as miR-494, miR-495, miR-543, resulting in increased PTEN expression and induction of apoptosis via antagonizing AKT in BTKi-sensitive cells (upper panel). In BTKi-R cells, overexpressed miRNAs in the 14q32 cluster region downregulate PTEN and promote pro-survival AKT activation resulting in reduced apoptosis. AKT inhibition or cooperative miRNA inhibition rescues apoptosis in BTKi-R cells by restoring PTEN and inhibition of AKT. The intensities of each band were normalized to the corresponding β-actin bands. For miRNA analyses, Megaplex RT primers (Applied Biosystems, Waltham, MA), which are 380 stem-looped reverse transcripts that allow cDNA synthesis for mature miRNAs were used except miRNA cDNA synthesis was performed using primer 5′-CAGTGCGTGTCGTGGAGT-3′. The TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) was used to make cDNAs for mature miRNAs. The SYBR Green Master Mix (Applied Biosystems) was used to amplify miR-494 using speciﬁc primers (forward: 5′-GGGT GAAACATACACGGGA-3′; reverse: 5′- GTCGTATCCAGTGCGTGTCGTGG AGTCGGCAATTGCACTGGATACGACGAGGTT-3′), miR-495 (forward: 5′-GCC AAACAAACATGGTGCACTT-3′; reverse:5′-GTTGGCTCTGGTGCAGGGTCCGA GGTATTCGCACCAGAGCCAACAAGAAG-3′); miR-377 (forward: 5′-GAGCA GAGGTTGCCCTTG-3′; reverse: 5′-ACAAAAGTTGCCTTTGTGTGA-3′); miR-154 Cell Death and Disease (2021)12:1061 I. Kapoor et al. 11 (forward:5′-TAGGTTATCCGTGTTGCCTT-3′; reverse: 5′-AATAGGTCAACCGTGTATGATTC-3′); miR-136 (forward: 5′-GGACTCCATTTGTTTTGATGATG-3′; reverse: 5′-AGACTCATTTGAGACGATGATGG-3′); miR379 (forward: 5′-AGAGA TGGTAGACTATGGAACGT-3′; reverse: 5′-GTGGACCATGTTACATAGGTCAG-3′), miR-127 (forward: 5′-AGCCTGCTGAAGCTCAGAGG-3′; reverse: 5′-GCCAAGCT CAGACGGATCC-3′), miR-337 (forward: 5′-ACACTCCAGCTGGGTCAAGAG CAAT-3′; reverse: 5′-CTCAACTGGTGTCGTGGA-3′), miR-543 (forward: 5′ CCAGCTACACTGGGCAGCA GCAATTCATGTTT-3′; reverse: 5′-CTCAACTGGTG TCGTGGA-3′). The U6 small nuclear RNA primers (forward: 5′- CTCGCTT CGGCAGCACA-3′; reverse: 5′-AACGCTTCACGAATTTGCGT-3′) was used as an internal normalization control since the levels did not change in primary CLL patients’ samples and CLL or DLBCL cell lines. miRNA overexpression or knockdown was achieved using a speciﬁc miRNA mimic or inhibitor or miControl (Ambion, Life Technologies, Austin, TX) by the AMAXA Nucleofector Kit V (Lonza, Walkersville, MD) according to the manufacturer’s protocol. Statistical analysis Each experiment was repeated at least three times. For all the quantitative analyses represented in the graphs, the values are expressed as the mean values ± SD. The signiﬁcance of the differences between mean values were assessed using a two-tailed Student’s t-test and a one-way ANOVA with Bonferroni’s multiple comparison test was performed. All comparisons were calculated using Microsoft Excel version 2106 and GraphPad Prism version 5.00. 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Biomed Pharmacother. 2017;96:974–81. 37. Bräuer-Hartmann D, Hartmann JU, Wurm AA, Gerloff D, Katzerke C, Falzacappa MVV, et al. PML/RARα-regulated miR-181a/b cluster targets the tumor suppressor RASSF1A in acute promyelocytic leukemia. Cancer Res. 2015;75:3411–24. 38. Liu G, Zhou JP, Dong M. Down-regulation of miR-543 expression increases the sensitivity of colorectal cancer cells to 5-Fluorouracil through the PTEN/PI3K/AKT pathway. Biosci Rep. 2019;39:BSR20190249. 39. Geraldo MV, Nakaya HI, Kimura ET. Down-regulation of 14q32-encoded miRNAs and tumor suppressor role for miR-654-3p in papillary thyroid cancer. Oncotarget. 2017;8:9597–607. 40. Zhu L, Wang X, Wang T, Zhu W, Zhou X. MiR-494-3p promotes the progression of endometrial cancer by regulating the PTEN/PI3K/AKT pathway. Mol Med Rep. 2019;19:581–8. 41. Cai Y, He T, Liang L, Zhang X, Yuan H. Upregulation of microRNA-337 promotes the proliferation of endometrial carcinoma cells via targeting PTEN. Mol Med Rep. 2016;13:4827–34. 42. 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MiR-494 is regulated by ERK1/2 and modulates TRAIL-induced apoptosis in non-small-cell lung cancer through BIM down-regulation. Proc Natl Acad Sci USA. 2012;109:16570–5. 47. Jishnu PV, Jayaram P, Shukla V, Varghese VK, Pandey D, Sharan K, et al. Prognostic role of 14q32.31 miRNA cluster in various carcinomas: a systematic review and meta-analysis. Clin Exp Metastasis. 2020;37:31–46. 48. Honda S, A Chatterjee A, Leichter AL, Myagi H, Minato M, Fujiyoshi S, et al. A MicroRNA Cluster in the DLK1-DIO3 Imprinted Region on Chromosome 14q32.2 Is Dysregulated in Metastatic Hepatoblastomas. Front Oncol. 2020;10:513601. 49. Tan M, Mu X, Liu Z, Tao L, Wang J, Ge J, et al. microRNA-495 promotes bladder cancer cell growth and invasion by targeting phosphatase and tensin homolog. Biochem Biophys Res Commun. 2017;483:867–73. 50. Zhao X, Lwin T, Silva A, Shah B, Tao J, Fang B, et al. Uniﬁcation of de novo and acquired ibrutinib resistance in mantle cell lymphoma. Nat Commun. 2017;8:14920. 51. 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Sweeney C, Bracarda S, Sternberg CN, Chi KN, Olmos D, Sandhu S, et al. Ipatasertib plus abiraterone and prednisolone in metastatic castration-resistant prostate cancer (IPATential150): a multicentre, randomised, double-blind, phase 3 trial. Lancet. 2021;398:131–42. 56. Schmid P, Abaham J, S Chan S, Wheatley D, Brunt AM, Nemsadze G, et al. Capivasertib plus paclitaxel versus placebo plus paclitaxel as ﬁrst-line therapy for metastatic triple-negative breast cancer: the PAKT trial. J Clin Oncol. 2020;38:423–33. development of methodology and writing, review, and revision of the paper; B.T.H. and J.B. provided technical and material support, review and revision of the paper. All authors read and approved the ﬁnal paper. FUNDING This work was supported by the National Institutes of Health (NIH) CA184137 and the Cleveland Clinic Velosano Bike Ride. COMPETING INTERESTS B.T.H. has received research funding and consulting fees from Pharmacyclics/Abbvie and AstraZeneca. The remaining authors declare no competing interests. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41419-021-04353-9. Correspondence and requests for materials should be addressed to Alexandru Almasan. Reprints and permission information is available at http://www.nature.com/ reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. AUTHOR CONTRIBUTIONS I.K. and A.A. performed study concept and design; I.K. performed experiments, analysis and interpretation of data, and statistical analysis; I.K. and A.A. performed © The Author(s) 2021 Cell Death and Disease (2021)12:1061
https://openalex.org/W4382290846	https://ejournal.uinsatu.ac.id/index.php/legacy/article/download/5430/1802	Indonesian	null	IMPLIKASI UNDANG-UNDANG NOMOR 6 TAHUN 2014 TERHADAP PERAN SENTRAL PENGEMBAN ADAT DI DUSUN SADE LOMBOK TENGAH	Legacy	2,022	cc-by-sa	4,654	Abstract Law No. 6 of 2014 gives legitimacy to the existence of customary villages. In the context of statehood, one of the forming elements of the State of Indonesia is the customary law society. Moreover, customary development in the hamlet of Sade Central Lombok also has an important role in improving the economic level of the community, because it is directly related to improving the quality of life and the fulfillment of daily life for the community. One of the economics developments, come from utilizing the tourism, must also be adapted to natural and community conditions, so that its authenticity is maintained, does not damage local wisdom, which is in the main characteristic. Improving the economic standard of a community, cannot only depend on the capabilities and initiatives of indigenous peoples, but must be supported by customary village, who are the main figures to be listened to and obeyed. Therefore, a strategy initiated by customary stakeholders is needed, in order to optimize the quality of the economic level. Keywords: sade village, Economic level Improvement, Tourism, customarry village, Customarry Mentalling. Naskah dikirim : 27/01/2022, direvisi: 6/02/2022, diterima: 26/02/2022 Naskah dikirim : 27/01/2022, direvisi: 6/02/2022, diterima: 26/02/2022 Naskah dikirim : 27/01/2022, direvisi: 6/02/2022, diterima: 26/02/2022 Keywords: sade village, Economic level Improvement, Tourism, customarry village, Customarry Mentalling. Nurlaili Rahmawati, Fildzah Izzah Ishmah Fakultas Syariah dan Hukum UIN Syarif Hidayatullah Jakarta Jl. Ir. H. Juanda No. 95 Ciputat – Tangerang Selatan Email : rnurlaili086@uinjkt.ac.id, fildzah.izzati18@mhs.uinkjkt.ac.id. Fakultas Syariah dan Hukum UIN Syarif Hidayatullah Jakarta Jl. Ir. H. Juanda No. 95 Ciputat – Tangerang Selatan Email : rnurlaili086@uinjkt.ac.id, fildzah.izzati18@mhs.uinkjkt.ac.id. Abstrak Undang-Undang Nomor 6 Tahun 2014 memberikan legitimasi akan keberadaan desa adat. Dalam konteks kenegaraan, Salah satu unsur pembentuk Negara Indonesia adalah masyarakat hukum adat. Lebih dari itu pengemban adat di dusun Sade Lombok Tengah juga mempunyai peran penting dalam meningkatan taraf ekonomi masyarakat, karena berkaitan langsung dengan peningkatan kualitas hidup dan pemenuhan kehidupan sehari-hari bagi masyarakat. Pengembangan ekonomi salah satunya berasal dari pemanfaatan potensi pariwisata desa adat. Pengembangan pariwisata desa adat, juga harus disesuaikan dengan kondisi alam dan masyarakat, agar keasliannya tetap terjaga dan tidak merusak kearifan ekonomi lokal, yang menjadi ciri khas utama. Peningkatan taraf ekonomi suatu masyarakat, tidak bisa hanya bergantung kepada kemampuan dan inisiatif masyarakat adat saja, tetapi harus didukung oleh pemangku adat dan kepala desa adat yang menjadi figur utama untuk didengarkan serta ditaati. Oleh karena itu, Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 diperlukan strategi yang diinisiasi oleh pemangku adat agar dapat mengoptimalisasi kualitas taraf ekonomi. Serta harus adanya analisa mendalam terhadap faktor yang mempengaruhi peningkatan, maupun penurunan kualitas taraf ekonomi masyarakat melalui desa wisata. Kata Kunci: Dusun Sade, Peningkatan Taraf Ekonomi, Pariwisata, Desa Adat, Pengemban Adat. ( gg ) ( p y ) 2 Pasal 1 angka (1) Undang-undang Republik Indonesia Nomor 6 Tahun 2014 Tentan 1 Muhammad Ali Akbar Felani, “Kedudukan Desa Adat Dalam Sistem Pemerintah Desa Di Indonesia (Studi di Desa Lingga Kab Karo)” (Repository Umsu: 2021): 4. 1 Muhammad Ali Akbar Felani, “Kedudukan Desa Adat Dalam Sistem Pemerintah Desa Di I d i (St di di D Li K b K )” (R it U 2021) 4 1 Muhammad Ali Akbar Felani, “Kedudukan Desa Adat Dalam Sistem Pemerintah D Indonesia (Studi di Desa Lingga Kab Karo)” (Repository Umsu: 2021): 4. , Indonesia (Studi di Desa Lingga Kab Karo)” (Repository Umsu: 2021): 4. A. PENDAHULUAN Desa merupakan hirarki pemerintahan terendah dalam suatu pemerintahan berada dibawah kabupaten dengan entitas masyarakat hukum tertua yang bersifat asli. Hal ini terletak pada kewenangan otonomi dan tata kelola pemerintahannya, yang diatur berdasarkan atas hak asal usul dan adat istiadat setempat yang diakui sah oleh konstitusi.1 sehingga desa merupakan entitas pemerintahan yang langsung bersinggungan dengan rakyat. Jadi apa yang dikatakan Bung Hatta “Indonesia tidak akan bercahaya karena obor besar di Jakarta, tetapi Indonesia baru akan bercahaya dengan lilin-lilin di desa” itu benar, karena desa merupakan ujung tombak dalam pemerintahan baik dalam pembangunan ekonomi, pendidikan, sosial, budaya, dll. Sedangkan pengertian desa adat dan desa disamakan oleh Undang- Undang Nomor 6 Tahun 2014, penyebutan antara desa dan desa adat sendiri, disesuaikan dengan penyebutan yang berlaku di daerah setempat. Meskipun ada beberapa pendapat yang menjelaskan bahwa desa dan desa adat memiliki prinsip yang berbeda dan terpisah.2 Apabila ditinjau secara prinsip, desa adat sendiri tidak lepas dari sejarah warisan adat istiadat yang menjadi susunan pemerintahan lokal, dengan berbagai ketentuan adat dengan berbagai ketentuan adat dan norma setempat yang diwariskan turun temurun. Desa Adat justru memiliki hak 21 21 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 asal-usul yang mendominasi daripada desa biasa. Karena nilai dan norma yang bersifat asli dan mengakar dari daerah itu sendiri.3 Desa dan desa adat diatur dalam Undang-Undang Nomor 6 Tahun 2014. Dalam undang-undang ini termaktub bahwa pemerintah desa memiliki kewenangan dalam mengatur kebutuhan, kepentingan, dan aspirasi masyarakat setempat berdasarkan prakarsa masyarakat, hak asal-usul, dan atau hak tradisional.4 Kewenangan ini salah satunya merujuk kepada kewenangan dalam mengembangkan desa dalam aspek perekonomian. Pengembangan perekonomian desa sangat didorong melalui bagaimana inovasi serta gerakan perangkat pejabat desa ataupun pemangku adat di suatu desa. Karena pemangku adat menjadi orang yang sangat didengarkan petuahnya. Sehingga adanya keselarasan dan komunikasi yang baik, antara pemangku adat ataupun perangkat pemerintahan desa, bisa menjadi faktor penting dalam mendukung perkembangan taraf kehidupan masyarakat, termasuk dalam peningkatan kualitas ekonomi. Salah satu desa adat yang ada di Indonesia yaitu Dusun Sade. Terletak di Desa Rembitan, Kecamatan Pujut, Kabupaten Lombok Tengah, Nusa Tenggara Barat. Dusun Sade merupakan salah satu dari sekian banyaknya desa adat yang mengembangkan perekenomiannya melalui pariwisata, yang mana hal ini dimanfaatkan oleh masyarakat setempat. Walaupun wisata bukan satu-satunya tempat mereka bergantung, karena saat ini ada beberapa profesi lain yang mereka geluti. 2015): 24. 4 Penjelasan atas Undang-Undang Republik Indonesia Nomor 6 Tahun 2014 Tentang 3 Dra. Suparmini, M.Si Agustina Tri Wijayanti, M.Pd, “Masyarakat Desa dan Kota (Tinjauan Geografis, Sosiologis Dan Historis)”, (Fakultas Ilmu Sosial Universitas Negeri Yogyakarta: 2015): 24. Dra. Suparmini, M.Si Agustina Tri Wijayanti, M.Pd, Masyarakat Desa dan Kota (Ti Geografis, Sosiologis Dan Historis)”, (Fakultas Ilmu Sosial Universitas Negeri Yogy 2015): 24. 23 ) 7 I Nyoman Nurjaya, “Pengelolaan Sumber Daya Alam dalam perspektif Antropologi Hukum” (Jakarta: Prestasi Pustaka Publisher, 2008). , ( j , ) 6 Mestika Zed, “Metode Penelitian Kepustakaan”, (Jakarta: Yayasan Pustaka Obor Indonesia, 2004): 2-3. 5 Anselm Strauss dan Juliet Corbin, “Dasar-Dasar Penelitian Kualitatif: Tata Langkah dan Teknik-Teknik Teoritisasi Data”, (Jakarta: Pustaka Pelajar, 2010): 11-13. A. PENDAHULUAN Sebagai desa adat, Dusun Sade tentu memiliki pemangku adat yang menjadi salah satu titik sentral dalam menjadi panutan atau yang didengar petuahnya dalam berbagai aspek. Berangkat dari hal inilah, yang menjadi titik menarik bagi penulis untuk diteliti dari Dusun Sade, yaitu peran serta pengaruh figur pemangku adat dalam meningkatkan taraf ekonomi masyarakat melalui pengembangan wisata. Selain itu apa saja faktor 22 22 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 penghambat dan pendukung dalam pengembangan sektor ekonomi di Dusun Sade melalui desa wisata. Penelitian yang digunakan oleh penulis adalah berupa penelitian kualitatif. Jenis penelitian kualitatif berorientasi pada temuan-temuan baru yang tidak dapat dilakukan dengan mekanisme dan tata cara statistik atau dengan cara kuantifikasi (penghitugan). Penelitian ini terfokus pada keilmuan berupa sosiologis, perilaku, aktivitas sosial, dll.5 Berdasarkan jenis penelitian berdasarkan teknik pengumpulan data, penulis menggunakan penelitian kepustakaan (library research), dengan melakukan proses membaca, menulis, mengumpulkan dan mengolah data dari karya tulis ilmiah sebagai sumber utama penulisan. Sumber pustaka yang digunakan oleh penulis sendiri, berasal dari sumber buku, artikel, jurnal dan berita.6 5 Anselm Strauss dan Juliet Corbin, “Dasar-Dasar Penelitian Kualitatif: Tata Langk Teknik Teknik Teoritisasi Data” (Jakarta: Pustaka Pelajar 2010): 11 13 Hukum, Volume 13 Nomor 26 Agustus 2017): 163 9 Pasal 6 angka (2) Undang-Undang Nomor 6 Tahun 2014 Tentang Desa. g ( ) g g g 10 Pasal 97 angka (1) Undang-Undang Nomor 6 Tahun 2014 Tentang Desa. 8 Abd Hadi, “Desa Adat Dalam Sistem Ketatanegaraan Republik Indonesia Sebagai Implikasi Hukum Setelah Berlakunya UU Nomor 6 Tahun 2014 Tentang Desa” (DIH Jurnal Ilmu Hukum, Volume 13 Nomor 26 Agustus 2017): 163 g ( ) g g g 11 Pasal 97 angka (2), (3), dan (4) Undang-Undang Nomor 6 Tahun 2014 Tentang Des Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Kewenangan desa adat diberikan dalam rangka menunjang kemandirian desa untuk mengurus kepentingan masyarakatnya. Kewenangan yang diberikan kepada desa adat berdasarkan hak asal usul yang meliputi: pertama mengatur dan melaksanakan pemerintahan berdasarkan susunan asli; kedua mengatur dan mengurus ulayat atau wilayah adat; ketiga melestarikan nilai sosial budaya desa adat; keempat menyelesaikan sengketa adat berdasarkan hukum adat yang berlaku di desa adat dalam wilayah yang selaras dengan prinsip hak asasi manusia dengan mengutamakan penyelesaian secara musyawarah; kelima menyelenggarakan sidang perdamaian desa adat sesuai dengan ketentuan undang-undang; keenam memelihara ketenteraman dan ketertiban masyarakat desa adat berdasarkan hukum adat yang berlaku di desa adat; dan yang ketujuh mengembangkan kehidupan hukum adat sesuai dengan kondisi sosial budaya masyarakat desa adat.12 13 I Nyoman Suarsana dan Purwadi, “Etnografi Dusun Sade Desa Rembitan Lombok Tengah Nusa Tenggara Barat”, (Program Studi Antropologi, Fakultas Sastra dan Budaya Universitas Udayana, 2016): 8-9. y , ) 14 I Nyoman Suarsana dan Purwadi: 8-9. 12 Pasal 103 Undang-Undang Nomor 6 Tahun 2014 Tentang Desa. y , ) 14 I Nyoman Suarsana dan Purwadi: 8-9. B.1 Desa Adat dalam Undang-Undang Nomor 4 Tahun 2016 Indonesia adalah Negara dengan berbagai macam suku, agama, budaya, adat istiadat, bahasa daerah, dll. Sehingga semboyan bhinneka tunggal ika secara de facto mencerminkan kemajemukan budaya bangsa dalam bingkai Negara Kesatuan Republik Indonesia. Wilayah Negara yang terbentang luas dari Sabang di ujung Aceh sampai Merauke di tanah Papua. Memiliki sumber daya alam (natural resources) yang melimpah bak untaian zamrud di bentang garis katulistiwa, serta sumber daya budaya (cultural resources) yang beragam coraknya.7 Salah satu kekayaan budaya Indonesia yaitu desa adat sebagai sistem pengelompokan sosial (sosial alignment) dengan hukum adatnya (tertulis/tidak tertulis) sebagai instrument pengawasan sosial (sosial control) dalam kehidupan masyarakat hukum adat. Desa adat mempunyai beraneka macam sebutan/istilah, misalnya desa/dusun di Jawa, desa/banjar pakraman di Bali, nagari di Sumatera Barat, marga di Sumatera Selatan, 23 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 huta/nagori di Sumatera Utara, gampong di Aceh, kadagmangan di Kalimantan tengah, dll. Sedangkan sebutan hukum adat tertulis dalam kehidupan komunal masyarakat di daerah dikenal berbagai macam nama, misalnya awig-awig di Bali dan Lombok, pepakem di Cirebon, kitab simbur cahaya di Lampung, dll., dan sebgian besar hukum adat tidak tertulis.8 Dengan adanya Undang-Undang Nomor 6 Tahun 2014 tentang Desa memberikan legitimasi terhadap desa adat; yang memberikan pengakuan bahwa desa itu terdiri atas desa dan desa adat.9 Ada 3 syarat dalam penetapan desa adat, yaitu:10 a. Kesatuan masyarakat hukum adat beserta hak tradisional hukum adat masih hidup, baik yang bersifat territorial, genealogis, maupun yang bersifat fungsional; b. Kesatuan masyarakat hukum adat beserta hak tradisional nya dipandang sesuai dengan perkembangan masyarakat; serta c. Kesatuan masyarakat hukum adat beserta hak tradisionalnya sesuai dengan prinsip Negara Kesatuan Republik Indonesia. Ketentuan tentang syarat penetapan desa adat, dalam hal kesatuan masyarakat hukum adat beserta hak tradisional nya ini menjadikan desa adat melaksanakan fungsi pemerintahan (local self government), maka ada syarat mutlak, yaitu: tidak mengancam kedaulatan dan integritas Negara Kesatuan Republik Indonesia, substansi norma hukum adat tidak bertentangan ketentuan peraturan perundang-undangan, keberadaanya telah diakui oleh undang-undang, serta ditambah salah satu pranata lain dalam kehidupan masyarakat hukum adat seperti memiliki persamaan bersama dalam kelompok, pranata pemerintahan adat, harta kekayaan, dan perangkat norma hukum adat.11 24 12 Pasal 103 Undang Undang Nomor 6 Tahun 2014 Tentang Desa y 16 I Nyoman Suarsana dan Purwadadi, 9-11. y 17 I Nyoman Suarsana dan Purwadadi, 34. Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Untuk berkomunikasi sehari-hari, masyarakat Dusun Sade menggunakan bahasa daerah setempat, yaitu Bahasa Sasak. Namun tidak menutup kemungkinan, bahwa masyarakat Sade juga bisa berkomunikasi denggan menggunakan bahasa nasional (Bahasa Indonesia), bahkan salah satu syarat sebagai ketua Dusun Sade harus bisa berkomunikasi menggunakan Bahasa Indonesia. Saat ini juga sudah ada beberapa masyarakat yang bisa berkomunikasi dengan Bahasa Inggris karena dipengaruhi oleh faktor pendidikan, profesi, serta kemajuan pengembangan pariwisata.15 Sebagian besar masyarakat dusun Sade berprofesi sebagai petani, buruh tani, peternak, pelayan restoran, pedagang makanan atau pernak pernik, pemandu wisata, serta mayoritas perempuan sebagai penenun. Keadaan tanah di Dusun Sade sendiri sebenarnya kurang mendukung secara maksimal dalam hal bercocok tanam, karena hanya bergantung pada system tadah hujan, dan tidak adanya saluran irigasi. Hal ini yang menjadi salah satu kekurangan bagi masyarakat yang banyak bergantung terhadap kegiatan bercocok tanam.16 y 18 I Nyoman Suarsana dan Purwadadi, 34. 15 I Nyoman Suarsana dan Purwadi: 8-9. B.2 Demografi Dusun Sade Dusun Sade merupakan dusun yang terletak di Desa Rembitan, Kecamatan Pujut, Kabupatan Lombok Tengah, Provinsi Nusa Tenggara Barat. Secara kependudukan terdapat kurang lebih 150 kepala keluarga, dengan total kurang lebih 700 warga yang bermuki di Desa Sade dua tahun terakhir. Hal ini mengalami peningkatan setelah sensus penduduk tahun 2013 yang bejumlah kurang lebih 529 penduduk.13 Masyarakat Dusun Sade merupakan salah satu komunitas masyarakat di Pulau Lombok yang masih cukup erat dalam memegang dan mempertahankan budaya dan adat istiadatnya. Seiring dengan berjalannya waktu, banyak adat istiadat yang mulai terkikis dan terganti. Contohnya ajaran islam Watu Telu yang menjalankan sholat hanya tiga waktu, yang mana ajaran ini semakin terkikis seiring dengan pengetahuan mereka mengenai ajaran agama yang semakin kaffah.14 25 y , 20 I Nyoman Suarsana dan Purwadadi, 36-40. Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Pemilihan pengemban adat cukup selektif, karena ada beberapa faktor yang harus ada dalam diri pengemban adat. Pengemban adat haruslah berbudi pekerti yang baik, jujur, tegas, bijaksana, cukup umur, memiliki pengetahuan yang mumpuni mengenai agama, budaya dan adat istiadat setempat. Serta dekat dan memiliki hubungan kekerabatan dengan pengemban adat sebelumnya. Seorang pengemban adat juga harus seorang laki-laki yang sudah dewasa, yang mana seorang perempuan ataupun anak muda tidak diperbolehkan menjadi pengemban adat.19 Pengemban adat di dusun Sade memiliki posisi sentral dan sangat strategis secara wewenang, tanggung jawab, dan kewajiban. Hal ini karena pengemban adat memiliki peran ganda dalam berbagai aspek, yaitu pengemban adat harus menjadi kepala dusun dalam system pemerintahan formal, kepala adat, pemimpin agama, hakim dalam memutuskan dan menyelesaikan konflik, bahkan menjadi khatib saat shalat jum’at. Selain itu pengemban adat bertugas mengkoordinir seluruh tugas kedinasan maupun adat.20 19 I Nyoman Suarsana dan Purwadadi, 34-36. I Nyoman Suarsana dan Purwadadi, 34-36. y , I Nyoman Suarsana dan Purwadadi, 36-40. B.3 Pemerintahan Adat Dusun Sade Pemerintahan Dusun Sade, diatur oleh pemangku adat yang bernama pengemban adat.17 Pengemban adat memiliki fungsi dan peran dan sentral dalam pengambilan keputusan penting serta dalam penyelesaian konflik di Dusun Sade. Apabila Peran Pengemban Adat tidak mampu menyelesaikan konflik atau permasalahan yang terjadi di Dusun Sade, maka perangkat pemerintahan di tingkat desa yang akan menyelesaikannya. Dalam hal pengambilan keputusan, pengemban adat yang ada di dusun Sade lebih diprioritaskan dan lebih utama karena berkaitan langsung dan mengetahui nilai adat istiadat serta budaya setempat. Pengemban ada juga merupakan sosok yang sekaligus menjabat sebagai ketua dusun apabila dilihat secara hierarkis pemerintahan di bawah tingkat desa.18 26 19 I N S d P d di 34 36 B.4 Peran dan Pengaruh Figur Pengemban Adat Dalam Pengembangan Taraf Ekonomi Masyarakat Melalui Desa Adat Apabila kita mencermati bagaimana wewenang serta kewajiban pemangku adat Dusun Sade yang cukup sentral dan dominan dalam berbagai aspek, bahkan dalam ranah kedinasan sebagai kepala dusun maupun kepala adat, maka dapat kita simpulkan bahwa pemangku adat juga seharusnya memiliki peran dan pengaruh sentral, dalam pengembangan taraf ekonomi msyarakat. Ada beberapa peran dan pengaruh penting figur seorang pengemban adat, dalam mendukung serta meningkatkan pengembangan ekonomi masyarakat, yaitu: a. Menjadi akomodasi dalam menyampaikan komunikasi dan kebijakan yang diberikan oleh pemerintah Desa ataupun tingkat pemerintahan a. Menjadi akomodasi dalam menyampaikan komunikasi dan kebijakan yang diberikan oleh pemerintah Desa ataupun tingkat pemerintahan 27 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 diatasnya. Terkait hal-hal yang dapat meningkatkan sumber daya manusia dan optimalisasi pengembangan pariwisata. diatasnya. Terkait hal-hal yang dapat meningkatkan sumber daya manusia dan optimalisasi pengembangan pariwisata. diatasnya. Terkait hal-hal yang dapat meningkatkan sumber daya manusia dan optimalisasi pengembangan pariwisata. b. Sebagai penyeimbang antara kearifan lokal desa adat dan dusun Sade dan juga pengaruh hal eksternal yang bersifat modern, sehingga autensititas desa adat tetap terjaga, namun tidak menutup diri dari kemajuan zaman yang berperan dalam meningkatkan taraf ekonomi masyarakat dan sumber daya manusia. b. Sebagai penyeimbang antara kearifan lokal desa adat dan dusun Sade dan juga pengaruh hal eksternal yang bersifat modern, sehingga autensititas desa adat tetap terjaga, namun tidak menutup diri dari kemajuan zaman yang berperan dalam meningkatkan taraf ekonomi masyarakat dan sumber daya manusia. c. Pengemban adat sebagai tokoh sentral dan sangat dekat dengan masyarakat bisa menjadi pengamat, penampung aspirasi, dan melakukan evaluasi bersama masyarakat terkait adanya peningkatan maupun penurunan kualitas ekonomi yang di dapat dari sektor pariwisata, kemudian dikomunikasikan dengan pemerintahan desa. c. Pengemban adat sebagai tokoh sentral dan sangat dekat dengan masyarakat bisa menjadi pengamat, penampung aspirasi, dan melakukan evaluasi bersama masyarakat terkait adanya peningkatan maupun penurunan kualitas ekonomi yang di dapat dari sektor pariwisata, kemudian dikomunikasikan dengan pemerintahan desa. d. Ikut serta atau menjadi pelopor dalam membuat kebijakan berkaitan dengan pengembangan ekonomi yang melibatkan antara masyarakat, wisatawan, dan pemerintah. d. Ikut serta atau menjadi pelopor dalam membuat kebijakan berkaitan dengan pengembangan ekonomi yang melibatkan antara masyarakat, wisatawan, dan pemerintah. e. Penafsir atas norma-norma dan ketentuan adat yang terdapat di masyarakat, sebagai suatu pendukung agar masyarakat tetap bisa mengembangkan diri dan mengembangkan kualitas ekonomi. B.4 Peran dan Pengaruh Figur Pengemban Adat Dalam Pengembangan Taraf Ekonomi Masyarakat Melalui Desa Adat Salah satu nya terdapat ketentuan yang menyatakan bahwa masyarakat bisa tinggal di Dusun Sade untuk mengembangkan kualitas ekonomi, sumber daya alam maupun sumber daya manusia, tanpa mengabaikan ketentuan adat dan norma agama serta sosial. 21 Nur Rohmi Aida, “6 Hal Unik yang Hanya Ada di Desa Sade Lombok” , Kompas 9/5/2019, 05:05 WIB, https://travel.kompas.com/read/2019/05/09/050500027/6-hal-unik-dan- hanya-ada-di-desa-sade-lombok?page=al y p g 22 Novita Prihandini, “Identifikasi modal (sosial, alam, finansial, fisik, dan manusia) Pada Dusun Wisata Sade di Kabupaten Lombok Tengah” (Fakultas Ekonomi dan Bisnis Universitas brawijaya, 2017): 6-7. B.5 Faktor Pendukung dan Penghambat Peningkatan Taraf Ekonomi Masyarakat Melalui Pengembangan Desa wisata Dusun Sade memiliki beberapa aset atau modal dalam yang menjadi komposisi utama sebagai pengembangan pariwisata. Pertama, keadaan sosial budaya yang unik dan autentik. Keadaan sosial budaya yang unik dan autentik menjadi daya tarik sendiri bagi para wisatawan domestik maupun internasional, baik untuk diteliti mupun sebagai objek wisata semata. Selain itu, terdapat rumah adat yang menjadi corak khusus dari Dusun Sade. Adanya kain tenun khas Lombok yang diproduksi langsung oleh para 28 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 perempuan Dusun Sade yang dekat dengan Pantai Tanjung An, Pantai Kuta Lombok, dan Pantai Selong Belanak.21 perempuan Dusun Sade yang dekat dengan Pantai Tanjung An, Pantai Kuta Lombok, dan Pantai Selong Belanak.21 Sebagai desa yang memiliki potensi pariwisata yang baik, masyarakat menjadikan peluang ini sebagai tempat mengais rezeki, contoh nya dengan berjualan pernak-pernik, kain tenun, jajanan khas daerah setempat, serta menjadi pemandu wisata. Kualitas pariwisata di dusun Sade secara berangsur-angsur memang mengalami peningkatan, walaupun tidak bisa dipertimbangkan sebagai sebuah peningkatan yang signifikan dari waktu ke waktu. Hal ini dinilai dari bagaimana masyarakat mampu menyelaraskan kebutuhan para wisatawan, dan tingkat pendapatan per kapita masyarakat yang di dapat dari pengembangan wisata. Apabila dianalisa terkait keadaan sosial budaya, lingkungan, sumber daya alam, dan sumber daya manusia. Dapat disimpulkan dan dipetakan apa saja faktor yang mendukung serta menghambat Peningkatan Taraf Ekonomi masyarakat melalui pengembangan desa wisata di dusun Sade. 1. Faktor Pendukung p , 25 Hendri Adrian, Wayan Resmini, “Pengaruh Globalisasi Terhadap Nilai-Nilai Budaya Pada Rumah Tradisional Masyarakat Sade Lombok Tengah”, (CIVICUS: Pendidikan Penelitian Pengabdian Pendidikan Pancasila dan kewarganegaraan, Vol. 6, No. 2 , September 2018): 19. 26 Lalu Dwarno Dimahandi, “Peran Pemerintah Desa Dalam Pengembangan Desa Wisata Sade (Studi Pada Pemerintahan Desa Rembitan Kabupaten Lombok tengah”, (Universitas Brawijaya Fakultas Ilmu Administrasi): 94-95. j y ) 27 Novita Prihandini, “Identifikasi modal (sosial, alam, finansial, fisik, dan manusia) Pada Dusun Wisata Sade di Kabupaten Lombok Tengah” (Fakultas Ekonomi dan Bisnis Universitas brawijaya, 2017): 9. 24 Novita prihandini, 8-9. 1. Faktor Pendukung a. Terdapat pengemban adat sebagai sosok sentral yang sangat didengarkan oleh Masyarakat Dusun Sade. Hal ini merupakan faktor pendukung apabila pengemban adat memaksimalkan perannya sebagai kepala dusun maupun ketua adat untuk menstimulus, mendukung, dan ikut mengevaluasi pengembangan pariwisata di Dusun Sade.22 b. Prinsip gotong royong dan kekeluargaan yang terjalin diantara masyarakat menjadi faktor pendukung untuk menyatukan persepsi dalam bekerja sama membangun Dusun Sade yang tentunya tetap 29 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 berpegang terhadap kearifan lokal, sehingga keautentikan desa wisata tidak luntur.23 berpegang terhadap kearifan lokal, sehingga keautentikan desa wisata tidak luntur.23 berpegang terhadap kearifan lokal, sehingga keautentikan desa wisata tidak luntur.23 c. Letak wilayah dusun sade yang strategis, karena didukung oleh pemandangan beberapa pantai yang terbentang indah. c. Letak wilayah dusun sade yang strategis, karena didukung oleh pemandangan beberapa pantai yang terbentang indah. d. Kemampuan masyarakat dalam menenun kain khas Lombok, mengelola makanan khas daerah, membuat pernak-pernik dan bermain music. Hal ini menjadi daya jual yang tinggi untuk menarik perhatian wisatawan.24 e. Hal-hal iconic yang masih terjaga sampai saat ini, seperti rumah adat khas Dusun Sade, yang berlantai kotoran sapi dan tanah liat, yang dianggap memiliki daya rekat yang baik serta menyebabkan rumah tidak berdebu.25 f. Masyarakat yang sudah mulai terbuka untuk mengembangkan diri sebagai cara untuk meningkatkan kualitas ekonomi, seperti mengikuti pelatihan Bahasa Inggris, menjadi pemandu wisata, dan belajar pengetahuan dasar.26 2. Faktor Penghambat vita Prihandini, 8. vita prihandini, 8-9. 23 Novita Prihandini, 8. rihandini, 8. rihandini, 8-9. vita prihandini, 8 9. endri Adrian, Wayan Resmini, “Pengaruh Globalisasi Terhadap Nilai-Nilai Budaya Pad ah Tradisional Masyarakat Sade Lombok Tengah”, (CIVICUS: Pendidikan Penelitia 30 23 Novita Prihandini, 8. 24 Novita prihandini, 8-9. 25 Hendri Adrian, Wayan Resmini, “Pengaruh Globalisasi Terhadap Nilai-Nilai Budaya Pada Rumah Tradisional Masyarakat Sade Lombok Tengah”, (CIVICUS: Pendidikan Penelitian Pengabdian Pendidikan Pancasila dan kewarganegaraan, Vol. 6, No. 2 , September 2018): 19. 26 Lalu Dwarno Dimahandi, “Peran Pemerintah Desa Dalam Pengembangan Desa Wisata Sade (Studi Pada Pemerintahan Desa Rembitan Kabupaten Lombok tengah”, (Universitas Brawijaya Fakultas Ilmu Administrasi): 94-95. 27 Novita Prihandini, “Identifikasi modal (sosial, alam, finansial, fisik, dan manusia) Pada Dusun Wisata Sade di Kabupaten Lombok Tengah” (Fakultas Ekonomi dan Bisnis Universitas brawijaya, 2017): 9. Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 kurang dari harga sewajarnya atau bahkan tidak diberi upah sama sekali.28 kurang dari harga sewajarnya atau bahkan tidak diberi upah sama sekali.28 kurang dari harga sewajarnya atau bahkan tidak diberi upah sama sekali.28 c. Masyarakat dusun sade juga banyak yang menjadikan potensi pariwisata bukan sebagai pekerjaan tetap, tapi menjadi salah satu pekerjaan sampingan selain bertani, menjadi buruh atau beternak. Penyebabnya juga karena masyarakat belum mampu memaksimalkan potensi wisata sebagai hal utama untuk mengembangkan taraf ekonomi mereka.29 d. Masyarakat Dusun Sade lebih sering berutang dalam menutupi kebutuhan sehari-hari. Hal ini dikarenakan masyarakat tidak pandai menabung, dan jumlah kebutuhan tidak selaras dengan jumlah pemasukan.30 e. Fasilitas seperti toilet/WC dan tempat beristirahat yang kurang terawat dan tidak dimaksimalkan dengan baik.31 f. Sosialisasi dan dukungan materi dari pemerintah tidak disertai dengan langkah konkrit dalam memberikan edukasi dan pelatihan kepada masyarakat dalam mengelola pariwisata, memaksimalkan lahan kering dan pembuatan saluran irigasi, serta mengoptimalkan fasilitas yang ada, demi kenyamanan masyarakat dan para wisatawan yang datang berkunjung.32 31 32 Moh Ardhi Akbar, “Pengembangan Desa Wisata Budaya Berbasis Masyarakat Di Dusun Sade Desa Rembitan Kabupaten Lombok Tengah”, (Jurusan Ilmu Pemerintahan, FISIP Universitas Muhammadiyah Malang: 2018): 24-25. 28 Novita Prihandini: 7. 31 28 Novita Prihandini: 7. 29 Novita Prihandini: 7. 30 Novita Prihandini: 7. 31 Novita Prihandini: 7. 32 Moh Ardhi Akbar, “Pengembangan Desa Wisata Budaya Berbasis Masyarakat Di Dusun Sade Desa Rembitan Kabupaten Lombok Tengah”, (Jurusan Ilmu Pemerintahan, FISIP Universitas Muhammadiyah Malang: 2018): 24-25. 29 Novita Prihandini: 7. 31 Novita Prihandini: 7. 30 Novita Prihandini: 7. 2. Faktor Penghambat a. Sumber air yang kurang memadai karena tidak adanya cadangan air dan saluran irigasi, sehingga di beberapa lokasi, masyarakat ataupun wisatawan harus mengantri dalam menggunakan air. Hal ini juga berpengaruh terhadap kurang maksimalnya fasilititas untuk MCK (Mandi, Cuci, Kakus).27 b. Masyarakat masih belum memaksimalkan potensi perkembangan wisata di daerah Sade. Hal ini terlihat dari banyaknya masyarakat yang tidak bisa menentukan harga, ketika memberikan jasa sebagai pemandu wisata, sehigga wisatawan kadang hanya memberikan upah 30 C. PENUTUP Dusun Sade merupakan salah satu desa Adat yang ada di Pulau Lombok yang masih memegang erat budaya dan adat istiadat nya. Sebagai desa adat yang juga mengembangkan perekonomiannya melalui potensi pariwisata, sosok pengemban adat yang sekaligus menjabat sebagai kepala dusun memiliki posisi dan figur sentral nya, hal ini yang membuat peran serta 30 Novita Prihandini: 7. 31 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 pengaruh nya cukup penting dalam mengoptimalkan taraf ekonomi masyarakat melalui desa wisata. Pengemban adat berperan sebagai pengawas, penyeimbang, penafsir ketentuan adat yang lebih terbuka dan komunikator antara keadaan yang terjadi di masyarakat dengan perangkat pemerintahan di atasnya. Hal ini menjadi sangat penting, apabila kita juga menilik keadaan ekonomi masyarakat yang terbilang masih jauh dalam kata cukup. Masyarakat juga belum mampu memaksimalkan, pengelolaan fasilitas, keadaan alam dan lingkungan, serta potensi pariwisata di dusun mereka sendiri. DAFTAR PUSTAKA Abd Hadi, “Desa Adat Dalam Sistem Ketatanegaraan Republik Indonesia Sebagai Implikasi Hukum Setelah Berlakunya UU Nomor 6 Tahun 2014 Tentang Desa” (DIH Jurnal Ilmu Hukum, Volume 13 Nomor 26 Agustus 2017 Adrian, Hendri dan Resmini, Wayan, ‘Pengaruh Globalisasi Terhadap Nilai- Nilai Budaya Pada Rumah Tradisional Masyarakat Sade Lombok Tengah’, CIVICUS : Pendidikan-Penelitian-Pengabdian Pendidikan Pancasila dan Kewarganegaraan, Vol. 6, No. 2, 2018. Anselm Strauss dan Juliet Corbin, “Dasar-Dasar Penelitian Kualitatif: Tata Langkah dan Teknik-Teknik Teoritisasi Data”, Jakarta: Pustaka Pelajar, 2010. Akbar, Moh Ardhi, ,“Pengembangan Desa Wisata Budaya Berbasis Masyarakat di Dusun Rembitan Kabupaten Lombok Tengah, Jurusan Ilmu Pemerintahan, FISIP, Universitas Muhammadiyah Malang, 2018. Dimahandi, Lalu Dwarno, ‘Peran Pemerintah Desa Dalam Pengembangan Desa Wisata Sade (Studi Pada Pemerintahan Desa Rembitan Kabupaten Lombok Tengah)’, Universitas Brawijaya Fakultas Ilmu Administrasi, 2017. Felani, Muhammad Ali Akbar, ‘Keduduk an Desa Adat Dalam Sistem Pemerintahan Desa di IndonesiaDESA (Studi di Desa Lingga Kab. Karo)’, Repository UMSU. 2021. I Nyoman Nurjaya, “Pengelolaan Sumber Daya Alam dalam perspektif Antropologi Hukum” Jakarta: Prestasi Pustaka Publisher, 2008. 32 Legacy : Jurnal Hukum dan Perundang-undangan Vol 2 No 1 - Maret 2022 Kompas.com, (2019, 9 Maret). 6 Hal Unik dan Hanya Ada di Desa Sade Lombok, Kompas, diakses pada 29 Desember 2021. https://travel.kompas.com/read/2019/05/09/050500027/6-hal- unik-dan-hanya-ada-di- desa-sade-lombok?page=all Undang-undang Republik Indonesia Nomor 6 Tahun 2014 Tentang Desa, DPR RI, https://www.dpr.go.id/dokjdih/document/uu/UU_2014_6.pdf Suarsana, I Nyoman, Purwadi, ‘Etnografi Dusun Sade Desa Rembitan Lombok Tengah Nusa Tenggara Barat’, Program Studi Antropologi, Fakultas Sastra dan Budaya Universitas Udayana, 2016. Suparmini, Wijayanti Agustina Tri, 2015, ‘Masyarakat Desa dan Kota( Tinjauan Geografi, Sosiologi, dan Historis)’, Fakultas Ilmu Sosial Universitas Negeri Yogyakarta. Prihandini, Novita , ‘Identifikasi Modal (Sosial, Alam, Finansial, Fisik, dan Manusia) Pada Dusun Wisata Sade di Kabupaten Lombok Tengah’, Fakultas Ekonomi dan Bisnis Universitas Brawijaya, 2017. Zed, Mestika, Metode Penelitian Kepustakaan. Jakarta: Yayasan Pustaka Obor Indonesia, 2009 33
https://openalex.org/W4283653993	https://www.frontiersin.org/articles/10.3389/fmicb.2022.894849/pdf	English	null	Escherichia coli Can Eat DNA as an Excellent Nitrogen Source to Grow Quickly	Frontiers in microbiology	2,022	cc-by	11,928	ORIGINAL RESEARCH published: 28 June 2022 doi: 10.3389/fmicb.2022.894849 ORIGINAL RESEARCH published: 28 June 2022 doi: 10.3389/fmicb.2022.894849 Escherichia coli Can Eat DNA as an Excellent Nitrogen Source to Grow Quickly Lili Huang 1, Yehui Zhang 1, Xinmei Du 1, Ran An 1* and Xingguo Liang 1,2* 1 College of Food Science and Engineering, Ocean University of China, Qingdao, China, 2 Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China Is DNA or RNA a good nutrient? Although scientists have raised this question for dozens of years, few textbooks mention the nutritional role of nucleic acids. Paradoxically, mononucleotides are widely added to infant formula milk and animal feed. Interestingly, competent bacteria can bind and ingest extracellular DNA and even integrate it into their genome. These results prompt us to clarify whether bacteria can “eat” DNA as food. We found that Escherichia coli can grow well in the medium with DNA as carbon and nitrogen sources. More interestingly, in the presence of glucose and DNA, bacteria grew more rapidly, showing that bacteria can use DNA as an excellent nitrogen source. Surprisingly, the amount of DNA in the culture media decreased but its length remained unchanged, demonstrating that E. coli ingested long DNA directly. The gene expression study shows that E. coli mainly ingests DNA before digestion and digests it in the periplasm. Biﬁdobacterium biﬁdum can also use DNA as the nitrogen source for growth, but not efﬁciently as E. coli. This study is of great signiﬁcance to study DNA metabolism and utilization in organisms. It also lays a foundation to understand the nutritional function of DNA in intestinal ﬂora and human health. Edited by: Hiroyuki Yamada, Japan Anti-Tuberculosis Association, Japan Reviewed by: Mary Catherine O’Connell Motherway, University College Cork, Ireland Yin Lin Ge, Qingdao University, China Rosemary Redﬁeld, University of British Columbia, Canada Correspondence: Ran An ar@ouc.edu.cn Xingguo Liang liangxg@ouc.edu.cn Edited by: Hiroyuki Yamada, Japan Anti-Tuberculosis Association, Japan Reviewed by: Mary Catherine O’Connell Motherway, University College Cork, Ireland Yin Lin Ge, Qingdao University, China Rosemary Redﬁeld, University of British Columbia, Canada Correspondence: Ran An ar@ouc.edu.cn Xingguo Liang liangxg@ouc.edu.cn *Correspondence: Ran An ar@ouc.edu.cn Xingguo Liang liangxg@ouc.edu.cn INTRODUCTION In the natural environment, extracellular or free DNA can be found everywhere, but its utilization by organisms has not been clariﬁed. In the soil, for example, DNA concentration is between 0.08 and 2 µg/g (Nielsen et al., 2007). In the ocean, 0.044 mg/L of DNA has been found (Lorenz and Wackernagel, 1994). Extracellular DNA is circulating throughout animal bodies in the blood and other ﬂuids, including urine, milk, amniotic ﬂuid, and bronchial lavage (Hoskins, 1978; Vlassov et al., 2007). It is also present in the wound contaminated by bacteria (Brandt et al., 1995; Ulmer et al., 1996). In contrast, bacteria are also everywhere and can utilize almost any organic molecules from organisms as “food.” However, few textbooks on nutrition mention the nutritional role of nucleic acids, although scientists have raised the question of “Is DNA or RNA a good nutrient?” for dozens of years. Paradoxically, mononucleotides are added to infant formula milk as the essential component, and RNA-rich formula is widely used in the feed of animal oﬀspring. DNA and RNA products have also been used as functional foods. One reason for nucleic acids being ignored as nutrition is that researchers believe its content (especially for DNA) is less than proteins or polysaccharides. Actually, it is a misunderstanding, especially in the world of prokaryotes. In an Escherichia coli cell, for example, nucleic acids account for about 7% of the wet weight, and it is only less than protein, which is 15% (Lewin and Dover, 2002). The content of DNA in E. coli is Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 12 March 2022 Accepted: 31 May 2022 Published: 28 June 2022 Citation: Huang L, Zhang Y, Du X, An R and Liang X (2022) Escherichia coli Can Eat DNA as an Excellent Nitrogen Source to Grow Quickly. Front. Microbiol. 13:894849. doi: 10.3389/fmicb.2022.894849 Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 12 March 2022 Accepted: 31 May 2022 Published: 28 June 2022 Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 12 March 2022 Accepted: 31 May 2022 Published: 28 June 2022 Keywords: DNA, Escherichia coli, nitrogen source, nutrient, nutrition Citation: Huang L, Zhang Y, Du X, An R and Liang X (2022) Escherichia coli Can Eat DNA as an Excellent Nitrogen Source to Grow Quickly. Front. Microbiol. 13:894849. doi: 10.3389/fmicb.2022.894849 June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 1 DNA as Excellent Nitrogen Source Huang et al. about 1.0% (0.5–1.5% wet weight). From the perspective of chemical structure, it is easy to see that nucleic acid and its derivatives are higher in carbon (C), nitrogen (N), and phosphate (P). Sulfur (S) has also been found in some bacteria. For example, Deng et al. have found that the genome of many bacteria involves sulfur atoms attaching to phosphates on DNA (Zhou et al., 2005). Accordingly, it is worth studying nucleic acids deeply from the perspective of nutrition, not just from that of molecular biology and biotechnology. Many exciting ﬁndings can be expected in the areas of nucleic acid metabolism and nutrition. was proposed that the two DNA strands are separated in the periplasm, and one strand breaks down there and the other ssDNA strand transfers to the cytoplasm. The phosphatase can remove phosphate groups from 5′- or 3′-nucleotides in the periplasm (Roy et al., 1982; Rittmann et al., 2005; Pinchuk et al., 2008; McDonough et al., 2016). Released phosphate can traverse the inner membrane via the phosphate-speciﬁc transport system (Pst/PhoU) (McDonough et al., 2014), and nucleosides can pass through nucleoside transporters (e.g., NupC). There are controversies about the biological signiﬁcance (for evolution, repair, or nutrition) of this eﬃcient and active intake of dsDNA (Seitz and Blokesch, 2013). Bacteria have been shown to utilize DNA as element sources of P, C, and N for growth under special conditions (Paul et al., 1988; Jørgensen et al., 1993; Redﬁeld, 1993; Kroer et al., 1994; Lennon, 2007; Pinchuk et al., 2008; Mulcahy et al., 2010; Seper et al., 2011; McDonough et al., 2016). For example, V. cholera has been shown to assimilate phosphate from extracellular DNA and nucleotides, and PhoX, UshA, and CpdB have been identiﬁed as the major periplasmic phosphatases (McDonough et al., 2016). The extracellular deoxyribonucleases of Dns and Xds digest DNA ﬁrst before assimilation (Seper et al., 2011). The genus Shewanella, one kind of metal-reducing bacteria, could use extracellular DNA as carbon and energy sources (Pinchuk et al., 2008). Citation: The possibility of digesting DNA into nucleotides for direct DNA and RNA synthesis was also discussed (Paul et al., 1988; Jørgensen et al., 1993; Redﬁeld, 1993; Kroer et al., 1994). Mulcahy et al. (2010) showed that P. aeruginosa may secrete an extracellular deoxyribonuclease (DNase) to degrade extracellular DNA and utilize it as the C, N, and P source. Some marine bacteria were also reported to utilize DNA as a nutrient source of C, P, and N, but the culture media contained HEPES or other compounds of organic carbon and nitrogen (Lennon, 2007). Escherichia coli, one model bacterium for molecular biology, has also been shown to be capable of consuming DNA as the sole source of carbon and energy (Palchevskiy and Finkel, 2006). However, these studies on DNA utilization are scattered and not so persuasive from the viewpoint of nutrition. We did not ﬁnd any research report using DNA as the nitrogen source in the presence of other carbon sources, although the content of nitrogen in DNA is even higher than some amino acids. It is well known that glucose is the best carbon source for most organisms, and DNA cannot be a better carbon source than glucose. Actually, there are a large number of plants in the natural environment that provide potential carbon sources, but the nitrogen source is relatively poor. In theory, as the most signiﬁcant biomolecule, DNA should be an excellent nutrient. However, there is no strong evidence, especially considering that the amount of DNA is usually trivial in a cell. To solve this conﬂict, a breakthrough in understanding DNA nutrition is highly expected. In this study, we found DNA can be utilized as an excellent nitrogen source by E. coli, one of the most important model bacteria. When cultured in the coexistence of glucose and DNA (as the sole nitrogen source), E. coli grew unexpectedly quickly. As the nitrogen source, DNA was even comparable to glutamic acid, an amino acid. We also found that DNA was “eaten” directly and eﬃciently without degradation. We believe that the ability of E. coli to assimilate DNA as a nutrient indicates that bacteria utilize DNA very actively as a “delicious” food ingredient of high quality. A model for how E. coli assimilates and utilizes DNA is proposed. Frontiers in Microbiology \| www.frontiersin.org Materials Salmon sperm DNA (GC content is 41.20%, 100–250 bp) was purchased from Tokyo Pharmaceutical Factory Limited (Tokyo, Japan) and was solved in water to a concentration of 10.0 g/L. The purity of nucleic acid was evaluated using UV-Vis spectra. At 280 nm, no absorption peak was observed, and the Abs260/Abs280 was higher than 1.8 (Supplementary Figure 1). Oligo DNA nutrients for culturing DNA or as primers for RT- PCR used in this study (see sequences in Supplementary Table 1) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Staphylococcus aureus (ATCC6538) was purchased from the China Center of Industrial Culture Collection (CICC). Escherichia coli (No. 1.3344) and Biﬁdobacterium biﬁdum (B. biﬁdum, No. 1.5091) were purchased from the China General Microbiological Culture Collection Center (CGMCC). Media (nutrient broth and TPY broth) were purchased from Hope Bio-technology (Qingdao, China). Nutrient broth (pH 7.0 ± 0.2) contains the following components (per liter): peptone 10.0 g, beef extract powder 3.0 g, NaCl 5.0 g; TPY broth (pH 6.5 ± 0.1) contains (per liter) casein hydrolysate 10.0 g, peptone from soybean meal 5.0 g, yeast extract 2.0 g, glutose 5.0 g, L- cysteine monohydrochloride 0.5 g, K2HPO4 2.0 g, MgCl2 0.5 g, ZnSO4 0.25 g, CaCl2 0.15 g, FeCl3 0.10 mg, and Tween-80 1.0 ml. Glutamic acid (98.5%) was purchased from Sinopharm Co., Ltd. (Beijing, China). For all plates, agar was added to a concentration of 15.0 g/L. DNase was purchased from Tiandz Gene Technology Co., Ltd. (Beijing, China). Proteinase K (20.0 g/L) was purchased y p From the viewpoint of genomics, bacteria were believed to “eat” extracellular dsDNA for recombination (or for “sex”) (Goodgal, 1982; Kahn and Smith, 1984; Dubnau, 1991, 1999; Redﬁeld et al., 1997). Later, Finkel et al. showed that the mechanisms of DNA uptake may have been widely conserved during evolution (Finkel and Kolter, 2001; Palchevskiy and Finkel, 2006). Very interestingly, competent bacteria were found to bind and transport long extracellular DNA through their cell envelope and integrate the “eaten” DNA into their chromosome (Smith et al., 1981; Solomon and Grossman, 1996; Palchevskiy and Finkel, 2006). The molecular machines set on the cell surface work for this transportation using the energy from ATP (Finkel and Kolter, 2001). More interestingly, a model June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 2 DNA as Excellent Nitrogen Source Huang et al. from Sangon Biotech Co., Ltd. (Shanghai, China). Evaluation of Extracellular DNA Degradation Activity Using an Agar Plate Assay y Extracellular DNA degradation activity of S. aureus and E. coli was examined using a previously described method (Jeﬀries et al., 1957; Pressler et al., 2019). Brieﬂy, a 10.0 µl aliquot of an overnight culture (washed with PBS buﬀer (pH 7.2), then resuspended in PBS buﬀer) was spotted onto nutrient broth agar plates containing 2.0 g/L of DNA and incubated overnight at 37◦C. For visualization, plates were ﬂooded with 3.0 ml of perchloric acid solution (50% v/v perchloric acid in distilled water) and kept for 5 min. DNA-digested areas appeared as a clear zone around the colony against an opaque background. The extracellular DNA degradation activity was determined accordingly because there is a direct relation between the diameter of the cleared zone and nuclease activity (Jeﬀries et al., 1957; Pressler et al., 2019). Culture of Bacterial Strains in Various Media g g Cultures were grown in nutrient broth, TPY broth, and corresponding basal medium (containing nucleic acid) at 37◦C for the same cultivation time intervals. The cell-free supernatants (CFS) were obtained by centrifugation of the strains at 13,400 g for 10 min (4◦C), followed by ﬁlter-sterilization using a sterile ﬁlter with a pore size of 0.22 µm. Absorbance (Abs) at 260 nm was measured using an ultraviolet spectrophotometer (Nanodrop ND 2000, Thermo Scientiﬁc). The percentage for Abs decrease was calculated as [(Ac-At)/Ac×100%)], where Ac and At are absorbance values measured at 0 h and after incubation of a certain time. The media containing the nucleic acid of the same concentration were used as a control (Ac). First, E. coli were cultured in nutrient broth at 37◦C under atmospheric conditions, whereas B. biﬁdum was cultured in TPY broth at 37◦C under anaerobic conditions in an anaerobic incubator (Mitsubishi Gas Chemical, Tokyo, Japan). To ensure that no other residual sources of carbon and nitrogen remained in the cultures, bacterial strains were then propagated in an M9 minimal medium with glucose as the source of carbon and NH4Cl as the source of nitrogen. This deﬁned growth medium (pH 7.0 ± 0.2) contains 22.0 mM glucose (4.0 g/L), 18.6 mM NH4Cl, 22.0 mM KH2PO4, 0.10 mM CaCl2, 47.0 mM Na2HPO4, 8.5 mM NaCl, and 2.0 mM MgSO4. Growth Curves of E. coli With DNA, Deoxyribonucleotides, and Deoxyribonucleosides Initially, the overnight cultures were taken out of the incubator and put on ice to block the growth of bacteria. After cooling on ice for 15 min, the colonized bacteria strains were centrifuged (4,500 g, 4◦C, 10 min) and washed three times with 10.0 ml of the corresponding basal medium (without DNA, dNMPs, or deoxyribonucleosides). Then the cells were harvested, and resuspended in the basal medium, followed by the addition of the nucleic acid. All bacterial strains were adjusted by the medium to an optical density (OD) of 0.05 (∼1.0 × 108 CFU/ml) at 600 nm, and then incubated aerobically (for E. coli) or anaerobically (for B. biﬁdum) at 37◦C. The number of E. coli (CFU/ml) values were calculated using a standard curve obtained by counting the colonies on an agar plate. For analyzing samples cultured for various time intervals, each of the bacteria suspensions (200 µl) was taken and added to a 96-well plate and measured the OD at 600 nm and 37◦C immediately (in some cases, if the OD value exceeds 0.8, the samples are further diluted to the OD value between 0.1 and 0.8). All growth experiments were Nucleic Acid Degradation Assays by Agarose Gel Electrophoresis For nucleic acid-dependent growth using various nucleic acid derivatives (including DNA, deoxyribonucleotides (dNMPs), and deoxyribonucleosides) as the sources of carbon and nitrogen, nucleic acid was added to the carbon or/and nitrogen-free medium to a ﬁnal concentration ranging from 0.10 to 10.0 g/L. The nucleic acid was added to the medium prior to ﬁltering through a 0.22-µm-pore-size membrane (Jinteng, Tianjin, China). The proteinase K was used to remove any potentially contaminated protein present in the commercial DNA preparation under the following conditions (10.0 ml): 10.0 g/L DNA, 0.5 g/L of proteinase K, 37◦C, 30 min, 10.0 mM NaH2PO4 (pH 7.5), 5.0 mM CaCl2. After the reaction, the solution was extracted by phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol, followed by ethanol precipitation. The CFS of each strain previously ﬁlter-sterilized was used to check its activity for nucleic acid digestion. After incubation for a certain time, the DNA left (without digestion) was analyzed on a 1.2% agarose gel. The reaction yield was obtained by quantitative analysis of the decrease in the intact DNA band (Molecular Imager Gel Doc XR+ imaging system, Image Lab 3.0). The digestion yield was calculated by comparing the intensity of the band with that before digestion. Materials Other chemical reagents such as HClO4, phenol/chloroform/isoamyl alcohol, were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The RNA prep pure kit and FastKing RT kit for RNA extraction and reverse transcription were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). independently repeated at least three times. The error for OD values is within 4–18%. Cultures were grown in nutrient broth, TPY broth, or M9 minimal medium lacking DNA, dNMPs, or deoxyribonucleosides served as the control. Frontiers in Microbiology \| www.frontiersin.org DNA Is Comparable to Glutamic Acid as the Nitrogen Source for E. coli Growth To avoid incorporation or entrainment of other carbon (C) or nitrogen (N) sources, we chose the M9 medium (containing 22.0 mM glucose, 18.6 mM NH4Cl, 22.0 mM KH2PO4, 0.10 mM CaCl2, 47 mM Na2HPO4, 8.5 mM NaCl, and 2.0 mM MgSO4) as the basal medium, using glucose as the sole C and NH4Cl as the sole N source. Glucose is the only organic nutrient in the M9 medium. As shown in Figure 1A (red blank triangles), E. coli grew well in M9 within the ﬁrst 12 h (as the logarithmic phase). Very interestingly, when salmon sperm DNA (100–250 bp long) was used to replace NH4Cl, E. coli grew much more quickly and the logarithmic phase was as short as about 3 h (blue solid triangles, Figure 1A). In the stationary phase, the OD value was even higher than that in the complete medium of M9. Certainly, when neither DNA nor NH4Cl was used, no growth of E. coli was observed because no N source was present in the medium (blue blank triangles). This eﬀect depends to some extent on the DNA concentration, and 0.2–1.0 g/L DNA shows a better eﬀect (Supplementary Figure 2A). It is well known that amino acids are considered the best nitrogen source for bacteria among simple organic compounds. Here, we chose glutamic acid for comparison because it is one of the key compounds for the metabolism of protein, nucleic acids, and glucose. For a more fair comparison, we use 0.8 g/L of DNA (containing about 17% nitrogen on average) or 1.43 g/L of glutamic acid (containing 9.5% nitrogen) with the same concentration of nitrogen (∼0.136 g/L). As shown in Figure 2A, when glutamic acid was used to replace DNA, the growth speed (0.30 × 108 CFU/ml/h) for the ﬁrst 3 h was not as fast as the case for DNA (1.27 × 108 CFU/ml/h) (comparing triangles and circles). Interestingly, when both glutamic acid and DNA were present, the growth speed became much faster (2.0 × 108 CFU/ml/h, see squares in Figure 2A). Similar results were obtained when 0.625–5.4 g/L of glutamic acid was used (Supplementary Figure 2B). Notably, this is diﬀerent from the cases in that DNA was present together with other nitrogen sources such as NH4Cl or components in NB, in which the speed became slower (Figures 1B,C,E). These results show again that DNA can be used as an excellent nitrogen source for E. RNA Isolation and Reverse Transcription PCR Total RNA was extracted from bacteria using RNA preparation pure kit according to the manufacturer’s instructions. The RNA samples were diluted with RNase-Free distilled water, and the optical density (OD) values at 260 nm and 280 nm were used to evaluate the RNA concentration and purity. Next, to determine June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 3 DNA as Excellent Nitrogen Source Huang et al. the mRNA expression of each gene, a total of 10.0 µl reverse transcription system was used as per the instructions of the FastKing RT Kit. The generated cDNA was stored at −20◦C. RT-qPCR was performed on a PikoReal 96 Real-time PCR System (Thermo Scientiﬁc, USA). 16S rRNA was used for the normalization of mRNA expression, and the relative level of each mRNA was determined using the 2−11Ct method (Landry and Levin, 2014). The PCR conditions were comprised of pre- denaturation at 95◦C for 10 min, 40 cycles of denaturation at 95◦C for 15 s, annealing at 60◦C for 30 s, and extension at 72◦C for 30 s. All RT-qPCR experiments were conducted with three duplicate wells, and each gene experiment was repeated three times. present, the growth speed was also not so quick in the ﬁrst 3 h (black triangles in Figure 1E). These results show that the extremely quick growth of E. coli only occurs in the case that DNA was used as the sole N source (or nutrient-poor medium) in the presence of glucose. Notably, even in the case that DNA was the only organic compound, E. coli grew well, although both the growth speed and the ﬁnal concentration were not as good as that in the M9 medium (comparing G−N−(DNA) with G+N+ (no DNA) in Figure 1C). This result demonstrates that DNA can be used as the sole carbon and sole nitrogen source for E. coli growth. Amazingly, E. coli can grow in the presence of only DNA and inorganic salts. Another exciting result was that even in NB, the addition of DNA could improve both the growth speed and the bacterium density of E. coli (comparing black solid triangles and blank triangles in Figure 1E), indicating that DNA can be used as a nutrient (probably being mainly used as the nitrogen source even in the case of rich nutrition). RNA Isolation and Reverse Transcription PCR In addition, similar results were obtained when the inoculum was replaced by the following culture, in which E. coli was cultured in a medium with DNA as the sole nitrogen source but not complete M9 (Supplementary Figure 3). Statistical Analysis Data are presented as means ± SEM. The GraphPad PRISM 7.0 software (GraphPad Software Corporation, USA) was used for the analysis of the experimental data. The analysis of variance (ANOVA) was used to estimate the statistical parameters. Frontiers in Microbiology \| www.frontiersin.org DNA Is Comparable to Glutamic Acid as the Nitrogen Source for E. coli Growth coli at least in some special cases. pp y g In the case that glucose was replaced by DNA (G−N+, Figure 1B) or both glucose and NH4Cl were replaced by DNA (G−N−, Figure 1C), E. coli did not grow so quickly compared with that DNA replacing NH4Cl (G+N−). In addition, the OD values were lower than the positive control (G+N+, M9 containing glucose and NH4Cl) in the stationary phase. Interestingly, even when both NH4Cl and DNA were present as the N source, the quick growth as shown in Figure 1A was not observed, and the OD value became a little bit higher than that for M9 (Figure 1D). More interestingly, even for nutrient broth (NB), which is very rich in various nutrients, the growth speed was slower for the ﬁrst 3 h as compared with the combination of DNA and glucose (see black blank triangles in Figure 1E). Obviously, as compared with NB, the M9 medium contains very poor nutrition. Surprisingly, when both DNA and NB were As shown in Figure 2B, E. coli grew slowly in the ﬁrst 2 h, indicating that the gene expression of proteins using DNA and/or glutamic acid as a nitrogen source was not ready. Obviously, the diﬀerence in growth speed was biggest during the 30 min from 2.5 to 3.0 h of culture. The corresponding average growth speeds for glutamic acid, DNA, and both glutamic acid and DNA were determined to be 0.46 × 108, 4.96 × 108, and 8.80 × 108 CFU/ml/h, respectively (data not shown). This result shows that there is a synergistic eﬀect between DNA and glutamic acid. In addition, it also indicates that E. coli can use DNA as an N June 2022 \| Volume 13 \| Article 894849 4 DNA as Excellent Nitrogen Source Huang et al. FIGURE 1 \| Growth curves for Escherichia coli in the presence (DNA) or absence (no DNA) of DNA as the sole carbon and/or nitrogen source in M9 minimal medium. N is the number of bacteria in 1.0 ml. (A) Lacking NH4Cl (G+N−). (B) Lacking glucose (G−N+). (C) Lacking glucose and NH4Cl (G−N−). (D) Complete M9 minimal medium (G+N+). For (A–D), M9 medium (G+N+) was used as the positive control (red solid triangles). (E) Growth of E. coli in nutrient broth (NB) with DNA added as an organic nutrient. DNA Is Comparable to Glutamic Acid as the Nitrogen Source for E. coli Growth For (E), the result is shown for comparison of M9 medium lacking NH4Cl (G+N−) with DNA added as an organic nutrient. The concentrations of DNA and glucose are 0.8 g/L and 4.0 g/L, respectively. Growth of E. coli was carried out at 37◦C for 30 h. OD600 values were measured after certain time intervals. Assays were carried out in triplicate, and average values were used (mean and standard deviation are shown). FIGURE 1 \| Growth curves for Escherichia coli in the presence (DNA) or absence (no DNA) of DNA as the sole carbon and/or nitrogen source in M9 minimal medium. N is the number of bacteria in 1.0 ml. (A) Lacking NH4Cl (G+N−). (B) Lacking glucose (G−N+). (C) Lacking glucose and NH4Cl (G−N−). (D) Complete M9 minimal medium (G+N+). For (A–D), M9 medium (G+N+) was used as the positive control (red solid triangles). (E) Growth of E. coli in nutrient broth (NB) with DNA added as an organic nutrient. For (E), the result is shown for comparison of M9 medium lacking NH4Cl (G+N−) with DNA added as an organic nutrient. The concentrations of DNA and glucose are 0.8 g/L and 4.0 g/L, respectively. Growth of E. coli was carried out at 37◦C for 30 h. OD600 values were measured after certain time intervals. Assays were carried out in triplicate, and average values were used (mean and standard deviation are shown). FIGURE 2 \| Comparison of DNA with glutamic acid as the nitrogen source for Escherichia coli growth in M9 media (A). The enlarged part of the ﬁrst 3 h is shown in (B). N is the number of bacteria in 1.0 ml. Solid triangles: 0.8 g/L DNA; Solid circles: 1.43 g/L glutamic acid (Glu); Solid squares: both DNA and glutamic acid are present; Blank triangles: no nitrogen source is present. Glucose of 4.0 g/L is used as the carbon source. FIGURE 2 \| Comparison of DNA with glutamic acid as the nitrogen source for Escherichia coli growth in M9 media (A). The enlarged part of the ﬁrst 3 h is shown in (B). N is the number of bacteria in 1.0 ml. Solid triangles: 0.8 g/L DNA; Solid circles: 1.43 g/L glutamic acid (Glu); Solid squares: both DNA and glutamic acid are present; Blank triangles: no nitrogen source is present. Glucose of 4.0 g/L is used as the carbon source. Escherichia coli Can “Eat” DNA Directly and Grow Quickly Using DNA as the Nitrogen Source analysis by electrophoresis is large, we further checked the absorbance (Abs) change in the supernatant at 260 nm with the culturing time after removing the E. coli by centrifugation (Figure 3B). Interestingly, the Abs decreases with a similar trend during culture as that of the E. coli growth curve (Figure 1A), demonstrating that E. coli utilized DNA to grow. Again, Abs did not change much after 6 h of culture, indicating that the utilization stopped or reached the equilibrium between intake and release of molecules with Abs at 260 nm. A decrease in DNA was also observed for other culture media (G−N−, G−N+, G+N+, and NB), and the DNA was not digested completely in all cases (Supplementary Figure 4). How does E. coli use DNA in the presence of glucose and grow so quickly? Two possible paths can be proposed. One is that E. coli secretes nuclease to digest DNA to oligonucleotides, nucleotides, or nucleosides around the cells, and then these relatively small molecules are ingested. The other one is that E. coli ingests long DNA molecules directly without fragmenting them, followed by digestion and utilization in the cells. The latter hypothesis can explain better the quick growth of E. coli. To ﬁgure out which speculation is correct, we analyzed the change in DNA amount in the medium at various time intervals. pp y g We further quantitatively analyzed the expressed mRNA of two proteins (ComE and HofQ) related to DNA uptake by reverse transcription-quantitative real-time PCR (RT-qPCR). Protein ComE (coding by the comE gene) is in charge of DNA binding, and protein HofQ (coding by the hofQ gene) ingests long dsDNA directly into E. coli (Goodgal, 1982; Palchevskiy and Finkel, 2006). As shown in Figures 3C,D, the transcription level for both proteins became much higher in the presence of DNA (cultured for 3 h). In the absence of DNA, even for the NB medium, the transcription level was also much lower. Interestingly, these two Figure 3A shows the result of electrophoresis analysis of DNA in the culture media (G+N−, glucose as C source, and DNA as N source) after removing E. coli cultured for a certain time. It can be seen that the amount of DNA (100–250 bp) decreased to some extent during culture, but the DNA length did not change. DNA Is Comparable to Glutamic Acid as the Nitrogen Source for E. coli Growth coli after being cultured for 3 h was used. (E) Expression of comE and hofQ genes after 24 h of the culture in various media. 16S rRNA was used for the normalization of gene expression; the relative level was determined using the 2−11Ct method (Landry and Levin, 2014). DNA Is Comparable to Glutamic Acid as the Nitrogen Source for E. coli Growth coli cell is 0.5 µm3 and its dry weight is about 1.5 × 10−13 g, the dry weight of 6.0 × 108 CFU/ml E. coli is equivalent to 0.09 g/L. Accordingly, considering the eﬃciency of anabolism, it can be estimated that 5–20% of organic nutrition (0.8 g/L DNA and 4.0 g/L glucose) was consumed. source very eﬃciently once E. coli prepares well corresponding proteins to digest and utilize DNA. The consumption of DNA and glutamic acid was also estimated. At the stationary phase for DNA as the sole nitrogen source (Figure 2A), about 6.0 × 108 CFU/ml E. coli was obtained. Suppose that the volume of an E. June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 5 Huang et al. DNA as Excellent Nitrogen Source FIGURE 3 \| Analysis of DNA uptake to Escherichia coli. (A) Analysis of DNA size change by electrophoresis analysis (agarose) at various time intervals of culture in the media using DNA as the sole nitrogen source (G+N−). L: 100–1,000 bp ladder; M: growth media only, Con: control (medium containing DNA without E. coli). (B) Decrease in absorbance at 260 nm at various time intervals of culture (G+N−). For (A) and (B), the supernatant after centrifugation was used. (C) Expression of comE (binding protein for extracellular DNA uptake) in E. coli. (D) Expression of hofQ (a protein for extracellular DNA uptake) in E. coli. For (C) and (D), the E. coli after being cultured for 3 h was used. (E) Expression of comE and hofQ genes after 24 h of the culture in various media. 16S rRNA was used for the normalization of gene expression; the relative level was determined using the 2−11Ct method (Landry and Levin, 2014). FIGURE 3 \| Analysis of DNA uptake to Escherichia coli. (A) Analysis of DNA size change by electrophoresis analysis (agarose) at various time intervals of culture in the media using DNA as the sole nitrogen source (G+N−). L: 100–1,000 bp ladder; M: growth media only, Con: control (medium containing DNA without E. coli). (B) Decrease in absorbance at 260 nm at various time intervals of culture (G+N−). For (A) and (B), the supernatant after centrifugation was used. (C) Expression of comE (binding protein for extracellular DNA uptake) in E. coli. (D) Expression of hofQ (a protein for extracellular DNA uptake) in E. coli. For (C) and (D), the E. Frontiers in Microbiology \| www.frontiersin.org Escherichia coli Can “Eat” DNA Directly and Grow Quickly Using DNA as the Nitrogen Source As shown in Figure 4A, when DNA was present in the medium, endA was expressed at a much higher level, especially after 3 h of culture. After 24 h, however, the expression level decreased greatly. Certainly, in the absence of DNA in the medium, endA was only expressed at a background level, which was lower than that of 16S rRNA (as the base for comparison). These results show that the presence of exogenous DNA did improve the expression level of endA to digest DNA (see also Supplementary Figure 5 for the results in other media). However, a new question has arisen about whether the DNA is digested completely outside of the E. coli, or in its periplasm. We further tried to clarify this as follows. proteins were expressed in a similar order in the presence of DNA: G+N−> G−N−> G−N+ > G+N+ ≈NB. Obviously, the expression of comE and hofQ was at a higher level when DNA was used as the sole nitrogen source. Surprisingly, their expression was also upregulated even when another nitrogen source (NB or NH4Cl) was present, indicating that DNA can be ingested once a relatively high concentration of DNA (e.g., 0.8 g/L) is present. It should be noted that DNA accounts for about 0.5% weight of E. coli cells (5.0 g/L). After 24 h of culture (Figure 3E), even when the growth stopped for G+N−(DNA), a high level of expression of comE and hofQ was kept, although it is a little bit lower than that at 3 h (comparing the data for G+N−(DNA) in Figure 3E with those in Figures 3C,D). For all the other media containing DNA, the gene expression of comE and hofQ was also kept at a relatively high level (Figure 3E). These results indicate that the presence of a high concentration of DNA can stimulate the expression of genes in E. coli for DNA uptake. It should be noted that these two proteins only “eat” relatively long dsDNA. Accordingly, it can be concluded that E. coli can detect the exogenous DNA and be able to “eat” it as food for growth. It is well known that a tiny amount of DNase can destroy long DNA eﬃciently because even if 1% digestion occurs, most of the 100 bp DNA should be destroyed to shorter ones. Escherichia coli Can “Eat” DNA Directly and Grow Quickly Using DNA as the Nitrogen Source For the ﬁrst 6 h, the intensity of the DNA band decreased by approximately 20%, and almost no obvious decrease could be observed after that. Considering that the error for quantitative June 2022 \| Volume 13 \| Article 894849 6 Huang et al. DNA as Excellent Nitrogen Source FIGURE 4 \| Gene expression and digestion activity of DNase (EndA) from Escherichia coli. (A) Gene expression of deoxyribonuclease EndA of E. coli in the presence or absence of DNA in the medium (G+N−). 16S rRNA was used for normalization, and the relative level was determined using the 2−11Ct method (Landry and Levin, 2014). (B) The digestion of DNA (0.8 g/L) by DNase (0.01 ng/L) at 37◦C for various times. (C) Investigation of the extrogenous DNase activity of E. coli on agar nutrient broth containing 0.2% (w/v) ﬁlter-sterilized DNA. The pictures were taken after incubation at 37◦C for 3 h or 24 h. The clear region (black color region here) appearing around the colony reﬂects the areas where DNA in the agar was digested. S. aureus (ATCC6538) for 24 h was used as the positive control. FIGURE 4 \| Gene expression and digestion activity of DNase (EndA) from Escherichia coli. (A) Gene expression of deoxyribonuclease EndA of E. coli in the presence or absence of DNA in the medium (G+N−). 16S rRNA was used for normalization, and the relative level was determined using the 2−11Ct method (Landry and Levin, 2014). (B) The digestion of DNA (0.8 g/L) by DNase (0.01 ng/L) at 37◦C for various times. (C) Investigation of the extrogenous DNase activity of E. coli on agar nutrient broth containing 0.2% (w/v) ﬁlter-sterilized DNA. The pictures were taken after incubation at 37◦C for 3 h or 24 h. The clear region (black color region here) appearing around the colony reﬂects the areas where DNA in the agar was digested. S. aureus (ATCC6538) for 24 h was used as the positive control. EndA has been proposed to be related to utilizing DNA as nutrition (Heun et al., 2012). By gene analysis, we found that the endA gene had a high degree of sequence identity with a known DNase of P. ﬂuorescens extracellular deoxyribonuclease (VVP09331.1). The deduced amino sequence reveals that this DNase has a potential signal peptide (SignalP 5.0 software, score 0.998), demonstrating that it can be secreted outside the E. coli cell (Cherny and Sauer, 2019). Frontiers in Microbiology \| www.frontiersin.org Escherichia coli Can “Eat” DNA Directly and Grow Quickly Using DNA as the Nitrogen Source We checked this by adding only 0.01 ng/L of DNase I to the medium containing DNA but without E. coli (Figure 4B). As expected, To check whether E. coli secretes deoxyribonuclease (DNase) to digest DNA, the expression of the endA gene (to produce EndA protein, an endodeoxyribonulclease) was investigated. June 2022 \| Volume 13 \| Article 894849 7 Huang et al. DNA as Excellent Nitrogen Source FIGURE 5 \| Escherichia coli can use deoxyribonucleotides (A) and deoxyribonucleosides (B) as the sole nitrogen source and 4.0 g/L of glucose as the carbon source for growth. N is the number of bacteria in 1.0 ml. The concentration for each deoxyribonucleotide or deoxyribonucleoside was 1.0 g/L. (C) Effect of adding DNase I (0.01 ng/L) to the G+N−medium containing DNA on E. coli growth. FIGURE 5 \| Escherichia coli can use deoxyribonucleotides (A) and deoxyribonucleosides (B) as the sole nitrogen source and 4.0 g/L of glucose as the carbon source for growth. N is the number of bacteria in 1.0 ml. The concentration for each deoxyribonucleotide or deoxyribonucleoside was 1.0 g/L. (C) Effect of adding DNase I (0.01 ng/L) to the G+N−medium containing DNA on E. coli growth. Deoxyribonucleotides and Deoxyribonucleosides Are Used as the Excellent Nitrogen Source for E. coli Growth no DNA could be observed after 3 h, showing that all DNA was digested to shorter ones. Obviously, the digestion here was much faster as compared with those shown in Figure 3A, indicating that EndA may be mainly present in the periplasm but not completely outside the cell. This was further proved by checking the ability of E. coli to secrete DNase (EndA) by another approach, in which E. coli were spotted onto nutrient broth agar containing 0.2% (2.0 g/L) ﬁlter-sterilized DNA and kept for 3 or 24 h (Figure 4C). For the E. coli cultured for only 3 h, almost no DNase activity was observed; even after 24 h of culture, the DNase activity was much less compared with the positive control of S. aureus. Interestingly, when the cells of E. coli (G+N− (DNA), 3 h) were disrupted, and the liquid of cell disruption was used for DNA digestion, DNA was digested completely even within 1 h (data not shown). More interestingly, even for the control without DNA in the medium, the liquid of cell disruption also showed some DNase activity, indicating that DNase may be present to a certain level in the periplasm whether DNA is present in the medium or not. Certainly, when the cell-free supernatant (DNA is present in the medium or not during culture) was used, almost no digestion was observed even after 30 h of incubation (data not shown). no DNA could be observed after 3 h, showing that all DNA was digested to shorter ones. Obviously, the digestion here was much faster as compared with those shown in Figure 3A, indicating that EndA may be mainly present in the periplasm but not completely outside the cell. This was further proved by checking the ability of E. coli to secrete DNase (EndA) by another approach, in which E. coli were spotted onto nutrient broth agar containing 0.2% (2.0 g/L) ﬁlter-sterilized DNA and kept for 3 or 24 h (Figure 4C). For the E. coli cultured for only 3 h, almost no DNase activity was observed; even after 24 h of culture, the DNase activity was much less compared with the positive control of S. aureus. Interestingly, when the cells of E. coli (G+N− (DNA), 3 h) were disrupted, and the liquid of cell disruption was used for DNA digestion, DNA was digested completely even within 1 h (data not shown). Frontiers in Microbiology \| www.frontiersin.org Deoxyribonucleotides and Deoxyribonucleosides Are Used as the Excellent Nitrogen Source for E. coli Growth More interestingly, even for the control without DNA in the medium, the liquid of cell disruption also showed some DNase activity, indicating that DNase may be present to a certain level in the periplasm whether DNA is present in the medium or not. Certainly, when the cell-free supernatant (DNA is present in the medium or not during culture) was used, almost no digestion was observed even after 30 h of incubation (data not shown). It is easy to imagine that DNA has to be hydrolyzed into small molecules (monomers and even smaller ones) for synthesizing the macromolecules required for E. coli growth. To check whether E. coli can ingest directly deoxyribonucleotides (dNMPs) and deoxyribonucleosides, we used dNMPs and deoxyribonucleosides to replace DNA in the medium of G+N−. As shown in Figure 5A, E. coli could grow well using dNMPs in an M9 medium without other nitrogen sources. Similar to the case using DNA as the sole nitrogen source, E. coli grew quickly in the ﬁrst 3 h. No obvious diﬀerences were observed for dAMP, dCMP, dGMP, and dTMP. In the case that deoxyribonucleosides (without phosphate) were used, E. coli also grew quickly in the ﬁrst 3 h. However, the ﬁnal concentrations of E. coli were about 40% lower. For E. coli growth, E. coli can use the phosphate in dNMPs as a P source, but for deoxyribonucleosides media, E. coli can only use K2HPO4 as the P source. Again, no obvious diﬀerences were observed for four deoxyribonucleosides (dA, June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 8 Huang et al. DNA as Excellent Nitrogen Source FIGURE 6 \| Growth of Biﬁdobacterium biﬁdum using DNA as the sole carbon and/or nitrogen source. N is the number of bacteria in 1.0 ml. (A–C) M9 minimal medium lack of NH4Cl (G+N−), glucose (G−N+), both glucose and NH4Cl (G−N−), respectively. (D) DNA was added into the full M9 minimal medium (G+N+). (E) DNA was added into TPY broth. The concentration of DNA was 0.5 g/L. Media without DNA were used as controls. B. biﬁdum was cultured at 37◦C for 30 h under anaerobic conditions. Assays were carried out in triplicate and OD600 values were measured. FIGURE 6 \| Growth of Biﬁdobacterium biﬁdum using DNA as the sole carbon and/or nitrogen source. N is the number of bacteria in 1.0 ml. Deoxyribonucleotides and Deoxyribonucleosides Are Used as the Excellent Nitrogen Source for E. coli Growth (A–C) M9 minimal medium lack of NH4Cl (G+N−), glucose (G−N+), both glucose and NH4Cl (G−N−), respectively. (D) DNA was added into the full M9 minimal medium (G+N+). (E) DNA was added into TPY broth. The concentration of DNA was 0.5 g/L. Media without DNA were used as controls. B. biﬁdum was cultured at 37◦C for 30 h under anaerobic conditions. Assays were carried out in triplicate and OD600 values were measured. For all cases, B. biﬁdum grew to a stationary stage after 15– 18 h (Figures 6A–C). Interestingly, the ﬁnal concentration of B. biﬁdum in the case where DNA was used as the sole nitrogen source (G+N−) was higher than other two cases (G−N+, G−N−). In G+N−medium, although B. biﬁdum grew faster in the ﬁrst several hours to some extent compared with G−N+ and G−N−media, the very quick growth (in the case of E. coli) was not observed. This result indicates that the direct ingestion of dsDNA may only occur for E. coli, which is a kind of gram- negative bacteria with the periplasmic space. When DNA was additionally added to M9 (G+N+) or TYP media (medium), no obvious synergistic eﬀect of glucose as carbon and DNA as a nitrogen source was observed, indicating again that E. coli and B. biﬁdum use DNA diﬀerently. In spite of the above results, we can conclude that B. biﬁdum can use DNA for growth, especially in the absence of other nitrogen sources. dG, dC, and dT). Similar results were also obtained for other media (G+N−, G−N−, G+N+, NB, Supplementary Figures 6 and 7). When 0.01 ng/L of DNase I was added to the culture medium of G+N−(DNA), both the growth speed and the ﬁnal concentration of cultured E. coli increased to some extent (Figure 5C). This result indicates again that E. coli can ingest both big dsDNA molecules (>100 bp) and smaller ones such as dNMPs and deoxyribonucleosides. Similar results were obtained for other media (G+N−, G−N−, G+N+, NB, Supplementary Figure 8). As described previously, in summary, E. coli prefers to ingest dsDNA directly and digest it in its periplasm. In contrast, E. coli can also ingest and utilize directly the fragments and monomers of DNA, which are present due to other factors. These results indicate again that DNA and its digested products of small molecules are all delicious food for E. coli. Biﬁdobacterium biﬁdum (Gram-Positive Bacteria) Can Also Use DNA as the Sole Carbon and/or Nitrogen Source The utilization of exogenous nucleic acids to synthesize the DNA and RNA in the bacteria should be energetically favorable. In addition, nitrogen-rich nutrients are usually insuﬃcient compared with carbon sources. Accordingly, we believe that the utilization of DNA, as well as RNA by intestinal ﬂora, is closely related to human health. The nucleic acids in the food may be also a good nutrient for us. Not like proteins or RNA, dsDNA has a very simple secondary structure (duplex) so bacteria can easily evolve proteins to ingest dsDNA directly. Although E. coli can also ingest and utilize directly dNMPs or deoxyribonucleosides as the nitrogen source (Figure 5), it does not mean E. coli use this approach (digest dsDNA to small molecules and ingest) as the main one for DNA utilization. However, it demonstrates that DNA is a good nutrient and E. coli may use any kind of DNA and its derivatives. It can be speculated that DNA and its derivatives are preferred to be utilized even when their concentration is much lower than that in the medium we used. Although many reports claim that bacteria use their own extracellular nucleases to degrade DNA (Croft et al., 1968; Akrigg and Mandelstam, 1978; Focareta and Manning, 1987; Pinchuk et al., 2008; Mulcahy et al., 2010; Gödeke et al., 2011; Seper et al., 2011; Heun et al., 2012; Korczynska et al., 2012; Liechti and Goldberg, 2013; Jaskólska et al., 2018; Cherny and Sauer, 2019), it is not clear where these nucleases work outside the cell or in the periplasm. For example, V. cholerae has been shown to secrete two nucleases (exonuclease Xds and endonuclease Dns) to break down extracellular DNA into nucleotides as a nutrient (Focareta and Manning, 1987; Seper et al., 2011). Bacillus subtilis was reported to release DNase to degrade and utilize foreign DNA when it lacks essential nutrients (Akrigg and Mandelstam, 1978). In this study, we pointed out that nuclease EndA is used as the major nucleases to digest extracellular DNA in the periplasm. EndA (a 235-aa protein) contains an N-terminal signal sequence that is predicted to be located in the periplasm, which is also conﬁrmed experimentally (Cordonnier and Bernardi, 1965; Heun et al., 2012). We believe that for most gram-negative bacteria, their periplasm is the main location for DNA digestion. Biﬁdobacterium biﬁdum (Gram-Positive Bacteria) Can Also Use DNA as the Sole Carbon and/or Nitrogen Source Our results clearly show that E. coli can ingest dsDNA directly and utilize it as a nutrient, especially as an excellent nitrogen source, which is even comparable to amino acids such as glutamic acid (Figures 1–3). Considering that DNA usually exists in the form of dsDNA either inside or outside a cell (released from organisms), it is more eﬃcient for E. coli to “eat” dsDNA to the periplasm and digest DNA there for further utilization. Finally, we checked whether B. biﬁdum (a gram-positive bacterium) can use DNA as a nutrient (Figure 6). M9 (G+N+) was also used as the base medium, and NH4Cl and/or glucose were removed in some cases (G+N−, G−N+, and G−N−). June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 9 DNA as Excellent Nitrogen Source Huang et al. It is less eﬃcient than E. coli secreting DNase to the culture medium (outside the cell to the environment) to digest DNA to monomers before ingestion. In addition, secretion of DNase to the environment is not proﬁtable because E. coli usually live together with many other bacteria. The digested DNA monomers and fragments are easily snatched by E. coli’s competitors. In contrast, the DNase concentration will become much lower if secreted to the culture medium. the aspect of bacteria, they should not miss these nutrient-rich materials (especially rich in nitrogen) as food. On average, the content of nitrogen in nucleic acids is higher than that in amino acids or proteins. This study has conﬁrmed that salmon sperm DNA can be used as nitrogen and carbon sources by E. coli, one of the intestinal bacteria. Especially in the case that no other nitrogen source was present, E. coli can utilize dsDNA very quickly in the presence of glucose (Figure 1). Here, very simple media are used because we want to make the results clear without the distribution of other organic nutrients. The possibility of E. coli utilizing contaminated molecules as a nitrogen source is extremely low because similar results were obtained when artiﬁcially synthesized DNA was used (Supplementary Figure 9). The size of DNA seems a very important factor in its utilization. We believe that, even when E. coli lives in a nutrient-rich medium, DNA, as well as RNA, can be utilized eﬃciently. Notably, for most bacteria, DNA and RNA account for 10–20% of their dry weight. Biﬁdobacterium biﬁdum (Gram-Positive Bacteria) Can Also Use DNA as the Sole Carbon and/or Nitrogen Source The weak DNase activity in the media may be caused by the release of DNase from dead bacteria or leak from their outer membrane. Based on the above analysis, as shown in Figure 7, we proposed a model for the utilization of extracellular DNA by E. coli. DNA passes across the outer membrane into the periplasm through porins (HofQ, responsible for the transport of DNA across the outer membrane) or nucleoside- speciﬁc channel-forming protein (van Alphen et al., 1978; Bremer et al., 1990). The DNase (EndA) in the periplasm can quickly digest dsDNA to short DNA fragments (<10 bp) or deoxyribonucleotides and deoxyribonucleosides, followed by intake to the cytoplasm of E. coli. Usually, a DNA duplex shorter than 10 bp is easily dissociated to ssDNA form (Lehman et al., 1962). Accordingly, we believe that the short DNA fragments are mainly transformed into the cytoplasm but not further digested in the periplasm. The deoxyribonucleotides and deoxyribonucleosides are mainly formed in the cytoplasm. Then, these small nitrogen-rich molecules are utilized for the synthesis of various molecules for E. coli growth by entering the metabolism cycles. Ribose can be used as the carbon source and nucleobases can be used as the nitrogen source. This speculation is supported by the fact that PDE (phosphodiesterase) has no signal peptide. PDE can only hydrolyze ssDNA in the cytoplasm to yield mono- and dideoxyribonucleotides (Lehman, 1960). The relatively high level of expression of some corresponding genes to further hydrolyze dNMPs, deoxyribonucleosides, and nucleobases (xdhA, preA, pgm gene) supports the model we proposed (Supplementary Figure 10). As expected, expression is also improved for the genes for salvage nucleotide synthesis (deoD, apt, pyrG gene, Supplementary Figure 11) and de novo nucleotide synthesis (purH, pyrF gene, Supplementary Figure 12). Earlier studies reported the abundance of extracellular DNA in both terrestrial and aquatic environments (Dell’Anno et al., 1998; Finkel and Kolter, 2001; Trulzsch et al., 2001; Rittmann et al., 2005; Vlassov et al., 2007), where bacteria may use the DNA as a nutrient. Actually, besides those from food, there are two major sources of DNA in the mammalian gut lumen. One is eukaryotic DNA shed from the mucosal epithelium, and the other pool is the DNA from the indigenous microﬂora. Frontiers in Microbiology \| www.frontiersin.org Biﬁdobacterium biﬁdum (Gram-Positive Bacteria) Can Also Use DNA as the Sole Carbon and/or Nitrogen Source coli phosphodiesterase which hydrolyzes deoxyribonucleic acid only in the single-stranded form to yield mono- and dinucleotides; NupC: nucleoside transporter C; CFP: channel-forming protein; NS: nucleoside; NB: nucleobase; R5P: ribose 5-phosphate; F6P: fructose 6-phosphate; AC: acetyl-CoA; FA: fatty acid; OM: outer membrane; PS: periplasm; IM: inner membrane. Proteins of PII series are in charge of uptake of dsDNA. FIGURE 7 \| Model for utilization of extracellular DNA as a nutrient by Escherichia coli. DNA is caught by proteins on the outer membrane and ingested to the periplasm, where DNase digests DNA into small molecules. The expressed DNase is secreted from the cytoplasm to the periplasm. Small DNA molecules (∼7 nt) and monomers are ingested into the cytoplasm by some speciﬁc proteins on the inner membrane. After digested to deoxyribonucleosides and even bases and deoxyribose, these small molecules enter the cycles of TCA (tricarboxylic acid cycle), PPP (pentose phosphate pathway), and GNG (gluconeogenesis). The small molecules can also be ingested from the environment directly to the cytoplasm. PDE: E. coli phosphodiesterase which hydrolyzes deoxyribonucleic acid only in the single-stranded form to yield mono- and dinucleotides; NupC: nucleoside transporter C; CFP: channel-forming protein; NS: nucleoside; NB: nucleobase; R5P: ribose 5-phosphate; F6P: fructose 6-phosphate; AC: acetyl-CoA; FA: fatty acid; OM: outer membrane; PS: periplasm; IM: inner membrane. Proteins of PII series are in charge of uptake of dsDNA. CONCLUSION recombined with the bacteria genome (Smith et al., 1981; Goodgal, 1982; Kahn and Smith, 1984; Stewart and Carlson, 1986; Dubnau, 1991, 1999; Solomon and Grossman, 1996; Syvanen and Kado, 1998). In our opinion, this kind of system is mainly used for DNA utilization as a nutrient. Only in the case that the intake dsDNA has almost the same sequences as the bacterium genome, recombination can occur. It seems that over a long evolutionary time, the beneﬁts of maintaining a system for horizontal genetic transfer outweigh the costs. Actually, several groups also claim that this dsDNA ingestion system might serve a nutritional purpose by utilizing extracellular DNA (Redﬁeld, 1993; Solomon and Grossman, 1996; Redﬁeld et al., 1997; Burton and Dubnau, 2010; Allemand et al., 2012). Therefore, it is quite reasonable that E. coli and other organisms take advantage of this system to eﬃciently “eat” dsDNA. recombined with the bacteria genome (Smith et al., 1981; Goodgal, 1982; Kahn and Smith, 1984; Stewart and Carlson, 1986; Dubnau, 1991, 1999; Solomon and Grossman, 1996; Syvanen and Kado, 1998). In our opinion, this kind of system is mainly used for DNA utilization as a nutrient. Only in the case that the intake dsDNA has almost the same sequences as the bacterium genome, recombination can occur. It seems that over a long evolutionary time, the beneﬁts of maintaining a system for horizontal genetic transfer outweigh the costs. Actually, several groups also claim that this dsDNA ingestion system might serve a nutritional purpose by utilizing extracellular DNA (Redﬁeld, 1993; Solomon and Grossman, 1996; Redﬁeld et al., 1997; Burton and Dubnau, 2010; Allemand et al., 2012). Therefore, it is quite reasonable that E. coli and other organisms take advantage of this system to eﬃciently “eat” dsDNA. In conclusion, E. coli can utilize DNA as a good nutrient by directly “eating” it. In the periplasm, the eaten DNA is digested and the obtained fragments or monomers are transferred into the cytoplasm as nutrients. The ingested DNA can be used to synthesize almost all the biomolecules for E. coli’s reproduction. Biﬁdobacterium biﬁdum can also use DNA as the nitrogen source and carbon source, although the utilization is carried out diﬀerently. DNA and its derivatives should be utilized by most organisms as good nutrients. Many scientiﬁc new understandings are expected in the area of nucleic acid metabolism and nutrition. Biﬁdobacterium biﬁdum (Gram-Positive Bacteria) Can Also Use DNA as the Sole Carbon and/or Nitrogen Source The amount of eukaryotic DNA has been estimated as ∼5 mg/day in the stomach, 200–500 mg/day in the small intestine, 20–50 mg/day in the colon, and ∼6–15 mg/day in the lumen (Croft et al., 1968; Croft and Cotton, 1973). In some special cases, e.g., acute episodes of diarrhea, eukaryotic DNA can reach 1–10 g/day in the small intestines of humans (Banwell et al., 1970, 1971). From The direct ingestion of dsDNA to bacteria has also been reported in a proposed model where the intake DNA is June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 10 Huang et al. DNA as Excellent Nitrogen Source FIGURE 7 \| Model for utilization of extracellular DNA as a nutrient by Escherichia coli. DNA is caught by proteins on the outer membrane and ingested to the periplasm, where DNase digests DNA into small molecules. The expressed DNase is secreted from the cytoplasm to the periplasm. Small DNA molecules (∼7 nt) and monomers are ingested into the cytoplasm by some speciﬁc proteins on the inner membrane. After digested to deoxyribonucleosides and even bases and deoxyribose, these small molecules enter the cycles of TCA (tricarboxylic acid cycle), PPP (pentose phosphate pathway), and GNG (gluconeogenesis). The small molecules can also be ingested from the environment directly to the cytoplasm. PDE: E. coli phosphodiesterase which hydrolyzes deoxyribonucleic acid only in the single-stranded form to yield mono- and dinucleotides; NupC: nucleoside transporter C; CFP: channel-forming protein; NS: nucleoside; NB: nucleobase; R5P: ribose 5-phosphate; F6P: fructose 6-phosphate; AC: acetyl-CoA; FA: fatty acid; OM: outer membrane; PS: periplasm; IM: inner membrane. Proteins of PII series are in charge of uptake of dsDNA. FIGURE 7 \| Model for utilization of extracellular DNA as a nutrient by Escherichia coli. DNA is caught by proteins on the outer membrane and ingested to the periplasm, where DNase digests DNA into small molecules. The expressed DNase is secreted from the cytoplasm to the periplasm. Small DNA molecules (∼7 nt) and monomers are ingested into the cytoplasm by some speciﬁc proteins on the inner membrane. After digested to deoxyribonucleosides and even bases and deoxyribose, these small molecules enter the cycles of TCA (tricarboxylic acid cycle), PPP (pentose phosphate pathway), and GNG (gluconeogenesis). The small molecules can also be ingested from the environment directly to the cytoplasm. PDE: E. REFERENCES Focareta, T., and Manning, P. A. (1987). Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization of the deoxyribonuclease (DNase) together with its periplasmic localization in Escherichia coli K-12. Gene 53, 31–40. doi: 10.1016/0378-1119(87) 90090-4 Akrigg, A., and Mandelstam, J. (1978). Extracellular manganese-stimulated deoxyribonuclease as a marker event in sporulation of Bacillus subtilis. Biochem. J. 172, 63–67. doi: 10.1042/bj1720063 Gödeke, J., Heun, M., Bubendorfer, S., Paul, K., and Thormann, K. M. (2011). Roles of two Shewanella oneidensis MR-1 extracellular endonucleases. Appl. Environ. 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J. Clin. Invest. 49, 183–195. doi: 10.1172/JCI106217 Hoskins, L. C. (1978). Host and microbial DNA in the gut lumen. J. Infect. Dis. 137, 694–698. doi: 10.1093/infdis/137.5.694 Brandt, T., Breitenstein, S., von der Hardt, H., and Tummler, B. (1995). DNA concentration and length in sputum of patients with cystic ﬁbrosis during inhalation with recombinant human DNase. Thorax 50, 880–882. doi: 10.1136/thx.50.8.880 Jaskólska, M., Stutzmann, S., Stoudmann, C., and Blokesch, M. (2018). QstR- dependent regulation of natural competence and type VI secretion in Vibrio cholerae. Nucleic Acids Res. 46, 10619–10634. doi: 10.1093/nar/gky717 Bremer, E., Middendorf, A., Martinussen, J., and Valentin-Hansen, P. (1990). Analysis of the tsx gene, which encodes a nucleoside-speciﬁc channel-forming protein (Tsx) in the outer membrane of Escherichia coli. Gene 96, 59–65. doi: 10.1016/0378-1119(90)90341-N Jeﬀries, C. D., Holtman, D. F., and Guse, D. G. (1957). 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Many scientiﬁc new understandings are expected in the area of nucleic acid metabolism and nutrition. A study on the utilization of RNA as a nutrient by bacteria is underway in our lab. June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 11 DNA as Excellent Nitrogen Source Huang et al. SUPPLEMENTARY MATERIAL XL and LH developed and designed the experiments for the study. LH and YZ performed the experiments. LH, XD, and XL analyzed and interpreted the data. XL, RA, and LH wrote the paper. All authors contributed to the article and approved the submitted version. XL and LH developed and designed the experiments for the study. LH and YZ performed the experiments. LH, XD, and XL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2022.894849/full#supplementary-material analyzed and interpreted the data. XL, RA, and LH wrote the paper. All authors contributed to the article and approved the submitted version. analyzed and interpreted the data. XL, RA, and LH wrote the paper. All authors contributed to the article and approved the submitted version. FUNDING This study was supported by the Fundamental Research Funds for Co-construction of Universities in Qingdao to XL; Natural Science Foundation of Shandong Province, China ZR2019BC096 to RA; and National Natural Science Foundation of China 31571937 to XL. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. REFERENCES J., Baker, D. L., Dodge, A. H., Sinicropi, D., et al. (1996). Engineering actin-resistant human DNase I for treatment of cystic ﬁbrosis. Proc. Natl. Acad. Sci. U. S. A. 93, 8225–8229. doi: 10.1073/pnas.93.16.8225 Palchevskiy, V., and Finkel, S. E. (2006). 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Utilization of DNA as a sole source of phosphorus, carbon, and energy by Shewanella spp.: ecological and physiological implications for dissimilatory metal reduction. Appl. Environ. Microbiol. 74, 1198–1208. doi: 10.1128/AEM.02026-07 Zhou, X., He, X., Liang, J., Li, A., Xu, T., Kieser, T., et al. (2005). A novel DNA modiﬁcation by sulphur. Mol. Microbiol. 57, 1428–1438. doi: 10.1111/j.1365-2958.2005.04764.x Pressler, K., Mitterer, F., Vorkapic, D., Reidl, J., Oberer, M., and Schild, S. (2019). Characterization of Vibrio cholerae’s Extracellular Nuclease Xds. Front. Microbiol. 10:2057. doi: 10.3389/fmicb.2019.02057 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Redﬁeld, R. J. (1993). Genes for breakfast: the have-your-cake-and- eat-it-too of bacterial transformation. J. Hered. 84, 400–404. doi: 10.1093/oxfordjournals.jhered.a111361 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Redﬁeld, R. J., Schrag, M. R., and Dean, A. M. (1997). The evolution of bacterial transformation: sex with poor relations. Genetics 146, 27–38. doi: 10.1093/genetics/146.1.27 Rittmann, D., Sorger-Herrmann, U., and Wendisch, V. F. REFERENCES doi: 10.1128/AEM.02674-06 Finkel, S. E., and Kolter, R. (2001). DNA as a nutrient: novel role for bacterial competence gene homologs. J. Bacteriol. 183, 6288–6293. doi: 10.1128/JB.183.21.6288-6293.2001 Lewin, B., and Dover, G. (2002). Genes. Oxford: Oxford University Press. June 2022 \| Volume 13 \| Article 894849 Frontiers in Microbiology \| www.frontiersin.org 12 Huang et al. DNA as Excellent Nitrogen Source Liechti, G. W., and Goldberg, J. B. (2013). Helicobacter pylori salvages purines from extracellular host cell DNA utilizing the outer membrane- associated nuclease NucT. J. Bacteriol. 195, 4387–4398. doi: 10.1128/JB. 00388-13 environmental Gram-negative bacteria. FEMS Microbiol. Rev. 37, 336–363. doi: 10.1111/j.1574-6976.2012.00353.x Seper, A., Fengler, V. H., Roier, S., Wolinski, H., Kohlwein, S. D., Bishop, A. L., et al. (2011). Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae bioﬁlm formation. Mol. Microbiol. 82, 1015–1037. doi: 10.1111/j.1365-2958.2011.07867.x Lorenz, M. G., and Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58, 563–602. doi: 10.1128/mr.58.3.563-602.1994 Smith, H. O., Danner, D. B., and Deich, R. A. (1981). Genetic transformation. Annu. Rev. Biochem. 50, 41–68. doi: 10.1146/annurev.bi.50.070181.000353 McDonough, E., Kamp, H., and Camilli, A. (2016). Vibrio cholerae phosphatases required for the utilization of nucleotides and extracellular DNA as phosphate sources. Mol. Microbiol. 99, 453–469. doi: 10.1111/mmi.13128 Solomon, J. M., and Grossman, A. D. (1996). Who’s competent and when: regulation of natural genetic competence in bacteria. Trends Genet. 12, 150–155. doi: 10.1016/0168-9525(96)10014-7 McDonough, E., Lazinski, D. W., and Camilli, A. (2014). Identiﬁcation of in vivo regulators of the Vibrio cholerae xds gene using a high-throughput genetic selection. Mol. Microbiol. 92, 302–315. doi: 10.1111/mmi.12557 Stewart, G. J., and Carlson, C. A. (1986). The biology of natural transformation. Annu. Rev. Microbiol. 40, 211–235. doi: 10.1146/annurev.mi.40.100186.001235 Syvanen, M., and Kado, C. I. (1998). Horizontal Gene Transfer. United Kingdom: Academic Press. Mulcahy, H., Charron-Mazenod, L., and Lewenza, S. (2010). Pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of DNA as a nutrient source. Environ. Microbiol. 12, 1621–1629. doi: 10.1111/j.1462-2920.2010.02208.x Trulzsch, K., Roggenkamp, A., Pelludat, C., Rakin, A., Jacobi, C., and Heesemann, J. (2001). Cloning and characterization of the gene encoding periplasmic 2’,3’-cyclic phosphodiesterase of Yersinia enterocolitica O:8. Microbiology 147, 203–213. doi: 10.1099/00221287-147-1-203 Nielsen, K. M., Johnsen, P. J., Bensasson, D., and Daﬀonchio, D. (2007). Release and persistence of extracellular DNA in the environment. Environ. Biosaf. Res. 6, 37–53. doi: 10.1051/ebr:2007031 Ulmer, J. S., Herzka, A., Toy, K. Frontiers in Microbiology \| www.frontiersin.org June 2022 \| Volume 13 \| Article 894849 REFERENCES (2005), Phosphate starvation-inducible gene ushA encodes a 5′ nucleotidase required for growth of Corynebacterium glutamicum on media with nucleotides as the phosphorus source. Appl. Environ. Microbiol. 71, 4339–4344. doi: 10.1128/AEM.71.8.4339-4344.2005 Copyright © 2022 Huang, Zhang, Du, An and Liang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Roy, N. K., Ghosh, R. K., and Das, J. (1982). Monomeric alkaline phosphatase of Vibrio cholerae. J. 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https://openalex.org/W4362672524	https://www.frontiersin.org/articles/10.3389/fopht.2023.1126338/pdf	English	null	Super-resolution STED imaging in the inner and outer whole-mount mouse retina	Frontiers in ophthalmology	2,023	cc-by	16,122	OPEN ACCESS OPEN ACCESS EDITED BY David W. Marshak, University of Texas Health Science Center, United States REVIEWED BY Christophe P. Ribelayga, University of Houston, United States Jacqueline Reinhard, Ruhr-University, Germany CORRESPONDENCE Timm Schubert timm.schubert@cin.uni-tuebingen.de †These authors have contributed equally to this work and share ﬁrst authorship SPECIALTY SECTION This article was submitted to Retina, a section of the journal Frontiers in Ophthalmology RECEIVED 17 December 2022 ACCEPTED 07 March 2023 PUBLISHED 06 April 2023 CITATION Kremers L, Sarieva K, Hoffmann F, Zhao Z, Uefﬁng M, Euler T, Nikic´ -Spiegel I and Schubert T (2023) Super-resolution STED imaging in the inner and outer whole-mount mouse retina. Front. Ophthalmol. 3:1126338. doi: 10.3389/fopht.2023.1126338 Leon Kremers 1,2,3,4†, Kseniia Sarieva 1,2,5†, Felix Hoffmann 1, Zhijian Zhao 1, Marius Uefﬁng 1, Thomas Euler 1,2, Ivana Nikic´- Spiegel 2 and Timm Schubert 1,2 1Institute for Ophthalmic Research, University of Tübingen, Tübingen, Germany, 2Werner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, Germany, 3Institute for Experimental Epileptology and Cognition Research, University of Bonn, Bonn, Germany, 4International Max Planck Research School for Brain and Behavior, Bonn, Germany, 5Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible ﬂuorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest ﬁlamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. TYPE Methods PUBLISHED 06 April 2023 DOI 10.3389/fopht.2023.1126338 retina, horizontal cells, ganglion cells, diffraction limit, super-resolution, synapses, mouse, STED OPEN ACCESS When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole- mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease. Kremers L, Sarieva K, Hoffmann F, Zhao Z, Uefﬁng M, Euler T, Nikic´ -Spiegel I and Schubert T (2023) Super-resolution STED imaging in the inner and outer whole-mount mouse retina. Front. Ophthalmol. 3:1126338. doi: 10.3389/fopht.2023.1126338 Introduction A recent study has identiﬁed bulbs along HC dendrites, which likely represent the synaptic contacts between HCs and BCs (33). Intriguingly, these bulbs are located below the very distal HC dendritic tips, suggesting that they do not contact cones, but co- localize with synaptic landmarks, such as mitochondria and GABAC receptors. The presence of such a HC-to-BC feedforward signalling spatially separated from the HC-photoreceptor contacts is appealing, because HC feedback to photoreceptors is thought to operate locally and to be minimally inﬂuenced by global HC computations (34). Still, HC feedforward signalling is still understudied and functional experiments testing the involvement of HC-to-BC signalling in the generation of BC responses are missing. STED microscopy differs from confocal microscopy by including an additional “donut-shaped” depletion laser, which is aligned to the central Gaussian-shaped excitation laser (Figures 1A–C). Here, the ﬂuorophore is ﬁrst excited by the excitation laser, and then it is either subdued by the depletion laser or spontaneously emits ﬂuorescence (Figure 1D). The donut shape of the depletion laser enables ﬂuorescence emission from the centre while restricting it in the spatial surround. This scales the effective point spread function (PSF) down by reducing the volume from which ﬂuorescence is generated and detected (Figures 1C, D) (4, 20). The main challenge when using STED imaging lies in the additional artefacts created by biological tissue: absorption, spherical aberration and light scattering intensify with increasing tissue depth (19), resulting in the generation of out- of-focus ﬂuorescence and minimising the STED effect. To some extent, one can compensate for these effects by increasing the intensities of both excitation and depletion lasers. However, given that STED requires high laser intensities by itself, further increasing the laser power comes with a trade-off in photo-damaging effects and thermal drift as well as reducing the signal-to-noise ratio (SNR), which is already quite low in STED microscopy per se. Even though in-depth super-resolution imaging is challenging, there are questions in neuroscience that can be only addressed within thick specimens. For instance, STED imaging was applied in acute hippocampal slices at depths of 90–120 μm, where it helped to resolve the actin dynamics of dendritic spines (11). Taken together, the complex synaptic connectivity pattern of mouse HC cells with distinct synaptic sites (33) and synaptic mechanisms (28), makes this interneuron one of the most fascinating cells in the mouse retina. Introduction central nervous system quite well (22–24). Given these features, valuable insights can be gained by applying super-resolution microscopy to the retina (25, 26). Super-resolution microscopy combines the advantages of ﬂuorescent imaging with resolutions below the diffraction limit of light and has been abundantly used to image biological specimens (1–4). It is especially relevant for neuroscience, where important subcellular structures (e.g. synaptic structures) have sizes below the diffraction limit (5, 6). Thus, it is not surprising that different super- resolution techniques were applied, e.g., to resolve clustering of neurotransmitter receptors (7, 8) or protein complex composition in presynaptic terminals (9, 10). So far, the majority of these experiments were carried out either in cell cultures or thin tissue sections. In contrast, there are only a handful of examples of super- resolution imaging in thick specimens (11, 12), as imaging deep tissue is challenging for most super-resolution techniques (13). Speciﬁcally, the coordinate-stochastic approaches (e.g. PALM, STORM) (14–17) have more fundamental restrictions for imaging of non-superﬁcial structures due to the total internal reﬂectance ﬂuorescence (TIRF) microscopy conﬁguration that limits ﬂuorescence illumination to the thin layer immediately adjacent to the glass coverslip (18). In contrast, coordinate-targeted approaches, such as stimulated emission depletion (STED) microscopy, can potentially image in deeper layers (30 - 50 μm) of biological samples (e.g. the whole-mount mouse retina) by taking the advantage of optical sectioning originating from the confocal basis of the setup (19) (Figures 1A, B). At the surface of the retinal tissue, retinal ganglion cells (RGCs) sample the visual input via the vertical photoreceptor-bipolar cell pathway and project the information via the optic nerve to higher areas of the brain (Figure 1B) (27). Using immunolabeling against some neuroﬁlament structures, speciﬁc types of RGCs can be easily visualised. This approach offers two advantages: ﬁrst, it labels only a subfraction of all RGC types, and thus, enables the identiﬁcation of individual cells. Second, labelling the neuroﬁlament structures visualises all cellular compartments of an individual RGC – soma, dendrites, and axon. Therefore, neuroﬁlament staining provides a suitable starting point for establishing a high-resolution imaging approach. Horizontal cells (HCs) are interneurons in the outer retina. The complex feedback synapses between photoreceptors and HCs in the mouse retina are anatomically and functionally well described (28). In addition, they have been shown to form synaptic contacts with bipolar cells (BCs) using electron microscopy (29–32). COPYRIGHT © 2023 Kremers, Sarieva, Hoffmann, Zhao, Uefﬁng, Euler, Nikic´ -Spiegel and Schubert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 01 01 Frontiers in Ophthalmology frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 10.3389/fopht.2023.1126338 Introduction In particular, at the level of synaptic and subsynaptic organisation, HCs may always be good for a surprise (34). In this study, we developed a reliable protocol for STED imaging in the whole-mount mouse retina. First, we optimised the sample preparation and imaging settings for imaging RGC neuroﬁlament structures close to the surface of the retinal tissue. Second, we adapted our approach for imaging synaptic structures of HCs deeper in the outer retina. Frontiers in Ophthalmology frontiersin.org Animals & whole-mount retina tissue preparation In addition to the excitation laser (green) the microscope includes a red shifted depletion laser (red) which is converted into a donut shape by passing through a gradient phase plate. Depletion and excitation lasers are aligned with dichroic mirrors and focused on the sample. Emission photons (orange) are passed back through a pinhole. Distinct emission wavelengths are separated in the prism scan head and transmitted to multiple hybrid detectors (HyDs). NA, numerical aperture. (B) Illustration of the whole-mount mouse retina for STED imaging of retinal ganglion cells (RGCs) in the inner retina and horizontal cells (HCs) in the outer retina depicted in cyan. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (C) Lateral resolution is improved with STED microscopy: The donut-shaped depletion laser depletes emission from the periphery of the excited volume and reduces the effective point spread function. (D) Jablonski diagram depicting the energetic principles behind the ﬂuorescent depletion effect. Excitation light lifts the ﬂuorophore from its ground state (S0) into a higher energetical level (excitation). The ﬂuorophore drops into the S1 state via vibrational relaxation. When the ﬂuorophore drops from its S1 to its S0 state energy is released in the form of ﬂuorescent emission (emission). However, when the ﬂuorophore in its S1 state is depleted the energy level decreases without emitting ﬂuorescence (depletion). FIGURE 1 Principle of STED microscopy and application in the mouse retina (A) Schematic organisation of an inverted STED microscope used in this work. In addition to the excitation laser (green) the microscope includes a red shifted depletion laser (red) which is converted into a donut shape by passing through a gradient phase plate. Depletion and excitation lasers are aligned with dichroic mirrors and focused on the sample. Emission photons (orange) are passed back through a pinhole. Distinct emission wavelengths are separated in the prism scan head and transmitted to multiple hybrid detectors (HyDs). NA, numerical aperture. (B) Illustration of the whole-mount mouse retina for STED imaging of retinal ganglion cells (RGCs) in the inner retina and horizontal cells (HCs) in the outer retina depicted in cyan. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Animals & whole-mount retina tissue preparation Retinae from adult (4-13 weeks old) male and female mice of the C57BL/6J wildtype line were used for this study. The animals were deeply anaesthetized with isoﬂurane (CP-Pharma, Germany) and were sacriﬁced by cervical dislocation. All animals were As a part of the brain, the mammalian retina has a deﬁned layered structure (21) that enables easy access to neuronal compartments. Moreover, the chemical and electrical synapse types in the retina represent those found across the rest of the 02 frontiersin.org 10.3389/fopht.2023.1126338 Kremers et al. A B D C FIGURE 1 Principle of STED microscopy and application in the mouse retina (A) Schematic organisation of an inverted STED microscope used in this work. In addition to the excitation laser (green) the microscope includes a red shifted depletion laser (red) which is converted into a donut shape by passing through a gradient phase plate. Depletion and excitation lasers are aligned with dichroic mirrors and focused on the sample. Emission photons (orange) are passed back through a pinhole. Distinct emission wavelengths are separated in the prism scan head and transmitted to multiple hybrid detectors (HyDs). NA, numerical aperture. (B) Illustration of the whole-mount mouse retina for STED imaging of retinal ganglion cells (RGCs) in the inner retina and horizontal cells (HCs) in the outer retina depicted in cyan. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (C) Lateral resolution is improved with STED microscopy: The donut-shaped depletion laser depletes emission from the periphery of the excited volume and reduces the effective point spread function. (D) Jablonski diagram depicting the energetic principles behind the ﬂuorescent depletion effect. Excitation light lifts the ﬂuorophore from its ground state (S0) into a higher energetical level (excitation). The ﬂuorophore drops into the S1 state via vibrational relaxation. When the ﬂuorophore drops from its S1 to its S0 state energy is released in the form of ﬂuorescent emission (emission). However, when the ﬂuorophore in its S1 state is depleted the energy level decreases without emitting ﬂuorescence (depletion). A B A D C D C C D FIGURE 1 Principle of STED microscopy and application in the mouse retina (A) Schematic organisation of an inverted STED microscope used in this work. Cryosectioning and immunolabeling of horizontal sections of the outer retina The whole-mount retina preparations (for imaging of the inner retina) were ﬁxed using 4% paraformaldehyde (PFA) in 0.1 M PBS for 20 minutes at 4°C, washed with 0.1 M PBS (6 x 20 minutes at 4° C) and blocked with blocking solution (10% normal goat serum (NGS) and 0.3% Triton X-100 in 0.1 M PBS) overnight at 4°C. Afterwards, the samples were incubated with primary antibodies (see Table 1 below) solution with 0.3% Triton X-100 and 5% NGS in 0.1 M PBS for 4-9 days at 4°C. The samples were then washed with 0.1 M PBS (6 x 20 minutes at 4°C) and incubated with secondary antibody (see Table 2 below) solution in 0.1 M PBS overnight at 4° C. After another washing step (6 x 20 minutes at 4°C), the retinae To prepare retinal pieces for horizontal sectioning (and imaging of the outer retina), retinal pieces, isolated as previously described, were ﬁxed in 4% PFA solution for 20 minutes as described above. After ﬁxation, the retinal pieces were washed in 0.1 M PBS (3 x 10 minutes) at 4°C, separated from the nitrocellulose membrane and passed through a series of incubations in sucrose-PBS solutions with increasing concentration. All sucrose incubations were performed at 4°C. First, retinae were kept in 10% sucrose solution TABLE 1 The following primary antibodies were used. Target protein Host species Clonality Antibody isotype Dilution factor Catalogue no. Manufacturer Neuroﬁlament H (SMI32) Mouse Monoclonal IgG1 1:100 801701 BioLegend, USA Calbindin Guinea pig Polyclonal IgG 1:500 214 005 Synaptic Systems, Germany GABA r2 receptor Rabbit Polyclonal IgG 1:500 AGA-007 Alomone Labs, Israel TABLE 2 The following species-speciﬁc secondary antibodies were used. TABLE 2 The following species-speciﬁc secondary antibodies were used. g p p y Host species Target species Isotype Conjugated dye Dilution factor Catalogue no. Animals & whole-mount retina tissue preparation (C) Lateral resolution is improved with STED microscopy: The donut-shaped depletion laser depletes emission from the periphery of the excited volume and reduces the effective point spread function. (D) Jablonski diagram depicting the energetic principles behind the ﬂuorescent depletion effect. Excitation light lifts the ﬂuorophore from its ground state (S0) into a higher energetical level (excitation). The ﬂuorophore drops into the S1 state via vibrational relaxation. When the ﬂuorophore drops from its S1 to its S0 state energy is released in the form of ﬂuorescent emission (emission). However, when the ﬂuorophore in its S1 state is depleted the energy level decreases without emitting ﬂuorescence (depletion). 03 Frontiers in Ophthalmology frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 10.3389/fopht.2023.1126338 were embedded in mounting media on a glass slide. Different types of mounting media were used. ProLong Gold (ThermoFisher Scientiﬁc, USA) and Vectashield (Vector Laboratories, USA) were used according to the Manufacturers’ protocol. Abberior TDE Mounting Medium O (Abberior, Germany), was used according to manufacturer prescriptions with elongation of each incubation step to 1 hour (see description below). The samples were covered with high-precision coverslips (No. 1.5H, Carl-Roth GmbH, Germany), sealed with transparent nail polish and left overnight at 4°C. handled in accordance with the European and national government regulations following the European animal welfare law. The eyes were quickly enucleated, and all further dissection steps were performed in 0.1 M phosphate saline buffer (PBS) (pH 7.4). Cornea, lens and vitreous body were carefully removed. The retina was dissociated from the eyecup and mounted RGC side- up as a whole retina or cut in three or four pieces on black nitrocellulose membrane (0.8 mm pore size, Millipore, Ireland). Frontiers in Ophthalmology Confocal and STED imaging for 1 to 2 h, until all pieces sank to the bottom. Retinae were then transferred into 20% sucrose solution and incubated for an additional 1 to 2 hours, again until all pieces subsided, before being transferred to a 30% sucrose solution, in which they were kept overnight. Retinal pieces were transferred into tissue freezing medium for preincubation before being mounted to the sample holders of an Epredia CryoStar NX50 Cryotome (Thermo Fisher Scientiﬁc, USA). In order to section the retinae along the xy axis as horizontally as possible, tissue freezing medium was applied to the sample holders of the cryotome and frozen solid. The cryotome was then used to cut an even plane into the frozen medium big enough to ﬁt one retinal piece. Retinal pieces were aligned on a glass slide wrapped in paraﬁlm with the RGC layer facing downwards. Single pieces were then picked up by carefully descending the plane of frozen medium with the sample holder on it. Additional freezing medium was used to fully cover the retina, before the sample was quickly frozen with liquid nitrogen. The sample holder was then placed back into the cryotome and 50 μm thick horizontal sections of the retina were cut. Cut sections were picked up using superfrost slides (Thermo Fisher Scientiﬁc, USA) and dried for 1 hour at 37°C on a heating plate. Confocal and STED imaging were both performed at the same microscope setup using a DMi8 inverse microscope (Leica, Germany) with three oil immersion objectives with 20x (NA 0.75), 63x (NA 1.4) and 100x (NA 1.4) magniﬁcation (Leica, Germany) in combination with the TCS SP8 STED setup (Leica, Germany). The microscopy setup included three pulsed excitation lasers, one with 488 nm (Leica, Germany) and two additional lasers with 532 nm and 635 nm wavelengths (OneFive, Switzerland). For depletion, a continuous-wave 592 nm and a gated/pulsed 775 nm STED laser were used. The intensities of the depletion lasers were experimentally chosen and were 0.65 W (43% of maximum value) for the 592 nm laser and 0.45 W (30% of maximum value) for the 775 nm laser. The microscope function was controlled was controlled with the LAS X software (Leica, Germany). Fluorescent emission was split and quantiﬁed using the integrated prism scan head and photomultiplier tubes (PMTs) and/or hybrid detectors (HyDs). For STED imaging only HyDs were used. Confocal and STED imaging The scan head allowed for the free selection of the emission wavelength spectra to be captured and measured. For STED imaging, xy-pixel and z-step size were chosen in accordance with the Nyquist–Shannon sampling theorem. This meant that STED imaging was only performed with the 100x objective and using an image size of 2048 x 2048 pixels. An additional 3-4 x zoom was applied, resulting in an effective pixel size of 14 - 20 x 14 - 20 nm. Minimal z-step size was manually calculated and set to 130 nm. Bidirectional scanning was enabled, and each line was scanned three times with pixel intensities being accumulated. For each z-section of an image stack three lines or frames were imaged and intensity values averaged. Excitation and depletion laser intensities as well as PMT/HyD gain were determined via testing of signal strength and photobleaching. Laser intensities and gain thus differed on a case-to-case basis but were kept consistent within experiments. Confocal imaging was performed by disabling the depletion lasers while keeping the excitation lasers on. If the same region was imaged with both STED and confocal microscopy, confocal imaging was typically performed before STED to prevent photobleaching from the high intensity depletion laser. When the same region was imaged in multiple ﬂuorescent channels, ﬂuorophores with higher excitation/ emission wavelengths were imaged ﬁrst. Imaging data was saved as lif-ﬁles, with z-stacks being represented as different series within one ﬁle. Retinal slices mounted on microscopy slides were surrounded by PAP pen (Science Services, Germany) and solutions were directly applied on the slides. After cryosectioning, the retinal sections were washed in 0.1 M PBS (6 x 20 minutes) at 4°C. 0.1 M PBS was removed and unspeciﬁc antigens were blocked by incubating the retinae in 10% NGS and 0.3% Triton-X-100 in 0.1 M PBS overnight. The blocking solution was then removed, and primary antibodies were added (see Table 1). The antibodies were diluted in 5% NGS and 0.3% Triton-X-100 in 0.1 M PBS to the concentration speciﬁed by the respective manufacturer. To accommodate the distinct diffusion time of the antibody within the cut samples of minor thickness, retinal slices were incubated for two days. After incubation, unbound antibodies were removed by washing the retinae again (6 x 20 minutes) with 0.1 M PBS at 4°C before adding the secondary antibody. Confocal and STED imaging Secondary antibodies, which are conjugated to ﬂuorophores of choice (see Table 2), were diluted in 0.1 M PBS to the concentration speciﬁed by the manufacturer and retinae were incubated with the solution overnight. To remove excess antibodies, the retinae were again washed in 0.1 M PBS (6 x 20 minutes) at 4°C. All retinae were mounted with a 2,2′- thiodiethanol-based embedding medium (TDE, Abberior, Germany. Some retinae were mounted with the 120 μm thick spacers (Secure-seal spacer, Invitrogen, USA) between slice and coverslip. One drop of 10% TDE solution was added to cover the retinal pieces and left to incubate for 1 hour at 4°C. Afterwards, the 10% solution was exchanged with 25% solution and incubated again for 1 hour, before being exchanged again for a 39% TDE solution. The retinae were incubated in the 39% solution for 45 min before the medium was substituted for the ﬁnal 97% TDE solution. The samples were left to incubate for an additional 45 minutes before and high-precision coverslips were carefully put on top of the retinal pieces and spacers. The sample was sealed by applying nail polish to the edges of the coverslip and the polish was left to dry overnight at 4°C before imaging experiments were performed. Cryosectioning and immunolabeling of horizontal sections of the outer retina Manufacturer Goat Mouse IgG Alexa Fluor 488 1:100 A-11001 Invitrogen Antibodies, USA Goat Mouse IgG STAR488 1:100-1000 ST488-1001-500UG Abberior, Germany Goat Mouse IgG Chromeo 488 1:1000 15031 Active Motif, USA Goat Mouse IgG ATTO532 1:100 610-153-121 Rockland, USA Goat Mouse IgM (heavy chain) Alexa Fluor 633 1:100-400 A-21046 Invitrogen Antibodies, USA Goat Mouse IgG STAR635P 1:100 ST635P-100–1- 500UG Abberior, Germany Goat Mouse IgG ATTO647N 1:100 50185 Sigma Aldrich, USA Goat Rabbit IgG Alexa Fluor 488 1:750 A-11008 Invitrogen Antibodies, USA Goat Rabbit IgG ATTO488 1:100 18772-1ML-F Sigma-Aldrich, USA Goat Rabbit IgG ATTO633 1:100 41176-1ML-F Sigma-Aldrich, USA Goat Guinea pig IgG STAR488 1:100 ST488-1006-500UG Abberior, Germany Goat Guinea pig IgG Alexa Fluor 647 1:500 106-605-003 Jackson Immuno Research, UK 04 Frontiers in Ophthalmology frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 Frontiers in Ophthalmology Statistics f (x) = k e−(x−u)2 2s2 + m Data representation and statistical analysis was performed using Prism 9 software (GraphPad, US) or in the R programming language. For Prism 9, data sets were copied into grouped tables as required and statistics were calculated using the analysis function. For complex data featuring various groups, each with multiple subcategories, 2-way ANOVA combined with a Tukey’s multiple comparisons test was performed. Paired data with only two categories was subjected to Shapiro-Wilk normality testing and two-tailed paired t-test was used for analysis of normally distributed data, Wilcoxon signed-rank test was used otherwise. Unpaired data with only two categories was analysed using an unpaired t-test or Wilcoxon rank sum test. Signiﬁcance was deﬁned as: p > 0.05 = ns, p< 0.05 = , p< 0.01 = , p< 0.001 = , p< 0.0001 = , p< 0.00001 = *, p< 0.000001 = **. Mean values in text and ﬁgures are given as mean ± standard deviation (SD). The x values with f (x) = m + 0:5 (fmax − m) were deﬁned and the distance between both x values was calculated as the FWHM: For structures in the inner retina (RGC structures), we used an unconstrained NLS ﬁt. In the outer retina (HC structures and GABA receptors), we used multiple constraints in our model: First, we deﬁned s as s ≥25 determining the width of the Gaussian curve with a minimal FWHM of 58.8 nm. With our imaging system, sample probes and previous results from the RGC layer we did not expect FWHM values below this limit. Second, under the assumption that the brightest pixels along the line represent the structure we are interested in, we deﬁned fmax ≥maximum intensity, and thus, prevented the model from being biassed by unspeciﬁc background noise. Third, we deﬁned u as 50 ≤u ≤length of line -50 nm to ensure that the model doesn’t ﬁt the curve to unspeciﬁc noise at the borders of the extracted vector. Overall, constraining our model produced ﬁts that improved in describing the observed signal. The process was repeated with the previously deﬁned coordinates for all corresponding images. It has to be emphasized that the measured FWHMs depend strongly on the structures measured and only give an approximation of the spatial resolution that can be achieved in this sample. Image processing and analysis Acquired images were processed and analysed by LAS X (Leica Microsystems) and ImageJ (National Institutes of Health, USA) software. Stabilisation and deconvolution of STED images were performed by Huygens Software (Version 17.10.0p6 64b, SVI, Netherlands). The deconvolution was performed with the Classical Maximum Likelihood Estimation (CMLE) algorithm under experimentally deﬁned settings. Images were deconvoluted using theoretical PSFs calculated for the DMi8 STED microscope and the Frontiers in Ophthalmology Frontiers in Ophthalmology 05 frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 10.3389/fopht.2023.1126338 100x (NA 1.4) objective used in this work. No lateral drift and bleaching corrections were performed, and most settings were kept at default values. The background level was estimated by software, the quality threshold was 0.001, the number of iterations was 50, the SNR was set to 7 for STED images. In ImageJ, the original and deconvolved z-stacks were typically transformed into a single maximum intensity projection and saved as raw ﬁles for further analysis. For presentational purposes image contrast and brightness were automatically optimised, channels depicted in deﬁned colours and scale bars inserted. All quantiﬁcations and processing were performed in unprocessed (= not deconvolved or brightness- adjusted data) unless otherwise speciﬁed. 100x (NA 1.4) objective used in this work. No lateral drift and bleaching corrections were performed, and most settings were kept at default values. The background level was estimated by software, the quality threshold was 0.001, the number of iterations was 50, the SNR was set to 7 for STED images. In ImageJ, the original and deconvolved z-stacks were typically transformed into a single maximum intensity projection and saved as raw ﬁles for further analysis. For presentational purposes image contrast and brightness were automatically optimised, channels depicted in deﬁned colours and scale bars inserted. All quantiﬁcations and processing were performed in unprocessed (= not deconvolved or brightness- adjusted data) unless otherwise speciﬁed. For the resolution enhancement factor (REF), the ratios between corresponding confocal and STED FWHMs as well as STED and deconvolved STED FWHMs were calculated and saved along the absolute FWHMs. Furthermore, the formulae of the ﬁtted curves, the extracted intensity values and the determined coordinates were also saved in the same Excel (.xlsx) ﬁle. Image processing and analysis For calculating the theoretical REF (REFth) the following formula was applied: REFth = dth conf dth STED = 0:61 l nsina = l 2nsina ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 + a Imax Is q The signal-to-background ratio (SBR) was calculated as Isignal Ibackground, where Isignal was deﬁned as the maximum pixel intensity along the previously deﬁned line selection and Ibackground as the term m from the ﬁtted Gaussain curve. We omitted values where Ibackground ≤ 1 as we believe that these backgrounds were unreasonably dark. Image analysis was performed using the open-source software ImageJ in its Fiji distribution and custom scripts written in the R programming language. For direct extraction of intensity values, a line selection was performed in ImageJ and values along the selection were extracted for all channels manually or using a custom written ImageJ macro. For determination of the full width at half maximum (FWHM), line proﬁles were ﬁtted (Gaussian curve, non-linear least square (NLS) approximation) using R free programming software. In short, raw image ﬁles were imported into R and transformed into intensity value matrices. Coordinates were selected using the shiny plug-in for R and intensity values along a vector between both coordinates were saved. A Gaussian curve was ﬁtted to these values using the NLS approach and the nls-multstart package. The general formula for the ﬁtted curve was deﬁned as: For dendritic bulb identiﬁcation, pixel intensities for the Calbindin and SMI32 stainings were normalised in ImageJ. Here the brightest pixel of a z-slice was set to 255 and the darkest to 0. All other pixel intensities were scaled accordingly. The normalised pixel intensities of Calbindin and SMI32 stainings were then subtracted for a line deﬁned across the bulb/non-bulb. Statistics The smallest measured FWHM that we could achieve in xy with this STED microscope (as measured with 100 nm ﬂuorescent beads) was 106.8 ± 4.1 nm (mean ± SD, n = 2) nm with the 532 nm excitation/775 nm depletion laser pair. However, we expect that smaller ﬂuorescent beads/quantum dots would allow us to measure smaller FWHMs and get a better estimate of the best resolution. Frontiers in Ophthalmology frontiersin.org Results To establish STED microscopy (Figures 1A, B) in the mouse retina, we used whole-mount preparations labelled with a primary antibody against neuroﬁlament H (from here on: SMI32 labelling) and secondary antibodies conjugated with synthetic dyes. The required labelling density was achieved by increasing the concentrations of both primary (2x, Table 1) and secondary (≤10x, Table 2) antibodies compared to commonly used concentrations of respective antibodies. We improved the sample preparation by choosing the best-performing spacer type between Frontiers in Ophthalmology 06 frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 10.3389/fopht.2023.1126338 slide and coverslip, mounting medium, and synthetic dyes (see Methods). We tried both conventional dyes (Alexa Fluor) and new- generation dyes (STAR, ATTO, Chromeo) speciﬁcally developed for STED microscopy. We tested photostability and selected dyes with the lowest bleaching effects at 488 and 635 nm excitation. For both green and far-red dyes, new-generation dyes were more photostable than Alexa Fluor dyes. For far-red dyes (ATTO647N and STAR635P), we never observed bleaching with our experimental conditions. spectrum and is incompatible with large-Stokes shift dyes. However, in our experiments, only a minor difference in resolution with Vectashield and Abberior TDE could be observed (resolution of STED images determined as the full width at half maximum (FWHM, see Methods); with Abberior TDE: 118.2 ± 23.2 nm (n = 24 structures, n = 3 images); with Vectashield: 144.9 ± 35.1 nm (n = 20 structures, n = 3 images; mean ± SD, p = 0.01, Wilcoxon rank sum test). Finally, we chose Abberior TDE as the most stably performing mounting medium with its only constraint being the short lifetime of the specimens at around 1.5 - 2 weeks in our hands. To avoid physical squeezing of the retinal tissue upon mounting, we placed spacers in between the slide and coverslip. For this purpose, we used commercially available silicon spacers. Optimization of acquisition settings for STED microscopy The choice of mounting medium was determined by its refractive index (n). We tested four different mounting media: Abberior Liquid AntiFade (n = 1.38), Vectashield (n = 1.47), Abberior TDE (n = 1.51) and ProLong Gold (n = 1.47). Abberior Liquid AntiFade had a lower refractive index than the immersion oil (n = 1.52) and was thus excluded from further experiments. We hypothesised that ProLong Gold should be the most stable medium as it was the only polymerizing mounting medium in our study. However, we observed strong axial drift when switching from confocal to STED mode, which we could not correct for. Vectashield mounting medium is not recommended by STED manufacturers because it absorbs light in the red range of the The main feature of a scanning STED microscope is the depletion laser (Figures 1C, D). Our STED microscope was equipped with two depletion laser lines with 592 and 775 nm wavelengths. While the 592 nm depletion laser was used together with the 488 nm excitation laser, the 775 nm depletion laser depleted the emission of ﬂuorophores excited with either the 532 or 633 nm excitation laser. The orange beam (592 nm) is a continuous-wave laser, whereas the far-red one (775 nm) is a pulsed/gated laser (Figure 2B). The resolution of STED imaging A B D C FIGURE 2 Representative SMI32-labelled structures in the inner retina acquired with different STED lasers (A) Formula for the lateral resolution of STED microscope with the saturation factor Imax/Is with Imax as the maximally applied laser intensity and Is as the STED laser intensity at which half of the ﬂuorescence is lost. (B) Temporal conditions of STED imaging. Ideally, all depleting photons act when ﬂuorophores are in the singlet-excited state S1 and ﬂuorescence must be registered after the stimulating photon’s action. Experimental time sequences are shown for the excitation (green), the depletion (red), and the emission signal detection (black) for continuous-wave STED (592 nm, top) and gated/pulsed STED (775 nm, bottom) microscopy. (C, D) Representative confocal (left) and STED (right) images acquired with different excitation and STED depletion lasers (592 nm in C, and 775 nm in D). For C, the excitation wavelength was 488 nm, and the dye was STAR488. For D, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bars: 1 µm. Optimization of acquisition settings for STED microscopy A C B B B D D D FIGURE 2 Representative SMI32-labelled structures in the inner retina acquired with different STED lasers (A) Formula for the lateral resolution of STED microscope with the saturation factor Imax/Is with Imax as the maximally applied laser intensity and Is as the STED laser intensity at which half of the ﬂuorescence is lost. (B) Temporal conditions of STED imaging. Ideally, all depleting photons act when ﬂuorophores are in the singlet-excited state S1 and ﬂuorescence must be registered after the stimulating photon’s action. Experimental time sequences are shown for the excitation (green), the depletion (red), and the emission signal detection (black) for continuous-wave STED (592 nm, top) and gated/pulsed STED (775 nm, bottom) microscopy. (C, D) Representative confocal (left) and STED (right) images acquired with different excitation and STED depletion lasers (592 nm in C, and 775 nm in D). For C, the excitation wavelength was 488 nm, and the dye was STAR488. For D, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bars: 1 µm. FIGURE 2 Representative SMI32-labelled structures in the inner retina acquired with different STED lasers (A) Formula for the lateral resolution of STED microscope with the saturation factor Imax/Is with Imax as the maximally applied laser intensity and Is as the STED laser intensity at which half of the ﬂuorescence is lost. (B) Temporal conditions of STED imaging. Ideally, all depleting photons act when ﬂuorophores are in the singlet-excited state S1 and ﬂuorescence must be registered after the stimulating photon’s action. Experimental time sequences are shown for the excitation (green), the depletion (red), and the emission signal detection (black) for continuous-wave STED (592 nm, top) and gated/pulsed STED (775 nm, bottom) microscopy. (C, D) Representative confocal (left) and STED (right) images acquired with different excitation and STED depletion lasers (592 nm in C, and 775 nm in D). For C, the excitation wavelength was 488 nm, and the dye was STAR488. For D, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bars: 1 µm. 07 07 Frontiers in Ophthalmology frontiersin.org 10.3389/fopht.2023.1126338 Kremers et al. depends – among other factors – on the saturation factor (Imax/Is, Figure 2A) (35). The saturation factors for our acquisition settings were calculated by the LAS X software. Optimization of acquisition settings for STED microscopy With the 592 nm continuous-wave depletion laser, we could achieve a maximum saturation factor of 7.5 without severe bleaching of the 488 nm dye at laser intensity 0.65 W (Figure 2C). In contrast, with the 775 nm laser the saturation factors were as high as 30 for the 633 nm excitation laser (Figure 2D) and 28.6 for the 532 nm excitation laser at a relatively low laser intensity of 0.45 W, likely due to the fact, that a high density of 775 nm photons is ‘pumped’ into the pulsed events whereas the photon number in the between-pulse intervals is minimal (Figure 2B). Therefore, we suggest that using the pulsed depletion laser results in both better resolution and minor photo- damaging compared with the continuous wave depletion laser. the FWHM is calculated from differently sized biological structures, it strongly depends on the structures selected and can vary between conditions. Thus, it does not strictly represent the theoretical maximal resolution of the microscope (see Methods). To correct for this effect, a resolution enhancement factor (REF) was calculated (35), which was deﬁned as the ratio of STED FWHM to confocal FWHM of the same structure. Here, the REF peaked at 2.21 ± 0.36 (mean ± SD) and ranged from 1.50 to 3.09 in the dendrites of RGCs (Figure 4C). The maximal theoretical REF for a saturation factor 30 for this experiment is 6.79 (see Methods). In some retinal samples, lateral and axial drift occurred at the stage of sample imaging. We tackled this problem using the Huygens Software (SVI) where appropriate. Although the image stabilisation tool performed well in lateral (xy) direction, allowing us to obtain good 3D stacks and time series, it provided no satisfying solution for axial drift (along the z-axis). The same software was used for image deconvolution (Figure 5A). We used a Classical Maximum Likelihood Estimation algorithm and adapted the program settings to obtain reliable deconvolution results. We compared both the FWHM and the SBR of STED and deconvolved STED images. Super-resolution microscopy of horizontal cell axon terminals in the outer retina Super-resolution STED microscopy at larger depths in samples remains challenging due to scattering within biological tissue, which leads to decreased depletion efﬁciency in deeper layers. This restricts efﬁcient STED imaging to the superﬁcial 50-70 μm of the sample. In the retina this, corresponds to the ganglion cell layer with the somata and axons of RGCs, and the inner plexiform layer, which roughly comprises of dendrites and synaptic connections of RGCs, BCs and amacrine cells (ACs) (Figures 1B, 3A). SMI32 labelling reveals intermediate ﬁlaments in axonal bundles of RGCs (Figure 3B) as well as dense cytoskeletal network structures in RGC somata (Figure 3C) at a depth of 25-30 μm. Imaging in the inner plexiform layer at a depth of 40-50 μm visualised the dendritic arborisation of RGCs (Figure 3D). However, as discussed above, the signal-to-background ratio (SBR) drops when increasing the imaging depth. Compared with the inner retinal structures, we did not achieve an increase in resolution when we imaged SMI32-labelled HC axon terminals in the STED mode at larger depth in the outer retina (Figures 6A, B, F, G). One possible reason for this is scattering of both the excitation and depletion lasers during their passage through the tissue and, thus, misalignment of the spatially optimal laser conﬁguration and a decrease of the STED effect. Theoretically, adaptive optics may allow the application of STED in deeper tissue while retaining super-resolution (36, 37). However, this approach would likely increase imaging duration and photobleaching. A more straightforward way, avoiding adaptive optics, is physical horizontal sectioning of the retinal whole-mount so that the structures of interest lay just below/at the section surface (Figure 6C, see Methods). We chose horizontal cryotome sectioning due to its widespread availability. Here, we cut off the inner layers of the whole-mounted retina to expose HC structures to the surface and image them (Figure 6C). As an approximation of spatial resolution, the FWHM was calculated by selecting thin ﬁlamentous structures and ﬁtting a Gaussian curve to the intensity values of respective confocal and STED images using a custom-written R script (see Methods). The described approach allowed paired comparison between the resolution of confocal and STED images (Figure 4A). The mean FWHM of ﬁlamentous structures in confocal images was 254.1 ± 23.0 nm, coming close to the diffraction limit of approx. 200 nm. Optimization of acquisition settings for STED microscopy While the deconvolution only marginally (though signiﬁcantly) increased the resolution of STED images (STED, FWHM = 125.7 ± 36.0 nm; STED deconvolved, 107.2 ± 17.6 nm, n = 26 structures, mean ± SD, p< 0.001, Wilcoxon signed-rank test) (Figures 5B, C), it did increase the SBR (STED, 3.3 ± 0.7; STED deconvolved, 16.2 ± 7.2, n = 26 structures, mean ± SD, p< 0.0001, paired t-test) and smoothened intensity proﬁles of given structures (Figures 5B–D). In general, we obtained more comparable ﬂuorescence intensities by adjusting the excitation laser intensity for every specimen separately and increasing it with larger imaging depths. We deﬁned the xy-pixel size as 14 - 20 nm according to the Nyquist- Shannon sampling theorem. Other imaging settings were adjusted experimentally. The bit depth was 12 bit, and we used 3x line accumulation with or without 3x frame averaging. Frame averaging was commonly used to decrease unspeciﬁc noise, however, in our case, the laser exposure was sometimes too high and led to thermal effects and physical deterioration of the sample upon imaging from the same focal plane. Frontiers in Ophthalmology frontiersin.org Super-resolution microscopy of horizontal cell axon terminals in the outer retina In comparison, the FWHM of STED images was 118.2 ± 23.2 nm and therefore surpassed the theoretical diffraction limit (n = 24 structures measured, n = 3 images, mean ± SD) (Figure 4B). As Intact and horizontally cryosectioned whole-mounted retinae were stained against SMI32 using ATTO532 as the ﬂuorophore. One set of horizontal cryosections was surrounded by a 120 mm thick silicon spacer to minimise the physical pressure of the coverslip on the retina, to avoid squeezing and an eventual change of the FWHM of ﬁne structures. In the other set, the 08 frontiersin.org 10.3389/fopht.2023.1126338 Kremers et al. A B D C FIGURE 3 Confocal and STED imaging of SMI32-positive retinal ganglion cell structures in the inner retina (A) Scheme of experimental design for imaging RGC structures in the inner retina. Retinae were dissected, mounted on ﬁlter paper, immunolabelled, mounted on glass slides and imaged. (B-D). Representative example images of RGCs’ axon bundles (B), soma (C) and dendrites (D) imaged in the confocal (conf.) and STED mode at a depth of ~30 µm (axons, soma) and ~50 µm (dendrites). For B, the excitation wavelength was 635 nm, and the dye was STAR635P. For C and D, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bars: 5 µm; for insets: 1 µm. A A C B D C B D FIGURE 3 Confocal and STED imaging of SMI32-positive retinal ganglion cell structures in the inner retina (A) Scheme of experimental design for imaging RGC structures in the inner retina. Retinae were dissected, mounted on ﬁlter paper, immunolabelled, mounted on glass slides and imaged. (B-D). Representative example images of RGCs’ axon bundles (B), soma (C) and dendrites (D) imaged in the confocal (conf.) and STED mode at a depth of ~30 µm (axons, soma) and ~50 µm (dendrites). For B, the excitation wavelength was 635 nm, and the dye was STAR635P. For C and D, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bars: 5 µm; for insets: 1 µm. retinal sections were directly touching the coverslip without any spacer (Figures 6D, E). Images were taken in each condition ﬁrst using confocal mode and then switching to STED, and thus, imaging the exact same region. Again, we calculated the FWHMs of ﬁlamentous structures as a measure for the resolution in both confocal and STED images (Figures 6B, E). Frontiers in Ophthalmology Super-resolution microscopy of horizontal cell axon terminals in the outer retina We did not ﬁnd any signiﬁcant change of the FWHM for STED compared to confocal images of HC structures in the intact retina (confocal, 319.5 ± 131.6 nm; STED, 358.3 ± 138.9 nm; mean ± SD; p = 0.11, paired t-test) (Figures 6A, B, F). Interestingly, in the intact whole-mount, the variability of FWHMs for STED images was strongly increased at the HC level (Figure 6F), likely illustrating the random effect of scattering in heterogeneous and deep tissue. In contrast, in both cryosection conditions (with and without spacers), we did not see such a pronounced effect on the variability. However, here we found signiﬁcantly improved STED FWHMs with spacer (confocal 367.4 ± 86.4 nm; STED, 271.6 ± 85.6 nm; mean ± SD; p< 0.0001, paired t- test) and without spacer (confocal, 302.7 ± 94.0 nm; STED, 230.1 ± 85.4 nm; mean ± SD; p< 0.0001, paired t-test), thus indicating that horizontal sectioning indeed reduces aberrations in STED microscopy (Figures 6D–F). While not signiﬁcantly different (confocal p = 0.09 and STED p = 0.44, 2-way ANOVA test), both confocal and STED FWHMs tended to be slightly smaller in cryosections mounted without silicon spacers, possibly reﬂecting a Frontiers in Ophthalmology Frontiers in Ophthalmology 09 frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 B C A B C FIGURE 4 Super-resolution STED imaging of SMI32-positive retinal ganglion cell dendrites in the inner retina reveals spatial resolution enhancement (A) Example structures (top, dendrite of a SMI32-positive RGC) for resolution estimation and with FWHMs for dendritic structure for confocal (blue) and STED (orange) (bottom). FWHM intensity proﬁles taken at the indicated position in example confocal and STED images. (B) Comparison of FWHMs in confocal (blue) and STED (orange) images (p< 0.0001 = , Wilcoxon signed-rank test, n = 1 animal; n = 24 ﬁlamentous structures from 3 images, horizontal bars indicate means, grey lines connect corresponding values). (C) Histogram of resolution enhancement factor quantiﬁed as ratio between the FWMHs of confocal and STED images (n = 24). For A, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bar: 1 µm in A. B A C B A FIGURE 4 Super-resolution STED imaging of SMI32-positive retinal ganglion cell dendrites in the inner retina reveals spatial resolution enhancement (A) Example structures (top, dendrite of a SMI32-positive RGC) for resolution estimation and with FWHMs for dendritic structure for confocal (blue) and STED (orange) (bottom). Super-resolution microscopy of horizontal cell axon terminals in the outer retina FWHM intensity proﬁles taken at the indicated position in example confocal and STED images. (B) Comparison of FWHMs in confocal (blue) and STED (orange) images (p< 0.0001 = , Wilcoxon signed-rank test, n = 1 animal; n = 24 ﬁlamentous structures from 3 images, horizontal bars indicate means, grey lines connect corresponding values). (C) Histogram of resolution enhancement factor quantiﬁed as ratio between the FWMHs of confocal and STED images (n = 24). For A, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bar: 1 µm in A. D C A B D C FIGURE 5 Deconvolution increases resolution and SBR for retinal ganglion cell dendrites (A) Representative example STED image without (top) and with deconvolution (bottom). (B) Top: Zoomed-in region as indicated in (A) Bottom: FWHM intensity proﬁles taken at indicated position without (STED) and with deconvolution (Deconv.) (C) Quantiﬁcation of the effect of deconvolution on FWHMs (p< 0.001 = , Wilcoxon signed-rank test, n = 1 animal; n = 26 structures from 1 image, horizontal bars indicate means, grey lines connect corresponding values). (D) Quantiﬁcation of the effect of deconvolution on SBR (p< 0.0001 = *, paired t-test, n = 1 animal; n = 26 structures from 1 image, horizontal bars indicate means, grey lines connect corresponding values). For A and B, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bars: 5 µm in A, 1 µm in B. B A A B FIGURE 5 FIGURE 5 Deconvolution increases resolution and SBR for retinal ganglion cell dendrites (A) Representative example STED image without (top) and with deconvolution (bottom). (B) Top: Zoomed-in region as indicated in (A) Bottom: FWHM intensity proﬁles taken at indicated position without (STED) and with deconvolution (Deconv.) (C) Quantiﬁcation of the effect of deconvolution on FWHMs (p< 0.001 = , Wilcoxon signed-rank test, n = 1 animal; n = 26 structures from 1 image, horizontal bars indicate means, grey lines connect corresponding values). (D) Quantiﬁcation of the effect of deconvolution on SBR (p< 0.0001 = *, paired t-test, n = 1 animal; n = 26 structures from 1 image, horizontal bars indicate means, grey lines connect corresponding values). For A and B, the excitation wavelength was 635 nm, and the dye was ATTO647N. Scale bars: 5 µm in A, 1 µm in B. 10 Frontiers in Ophthalmology frontiersin.org A B D E F G C GURE 6 TED imaging of horizontal cell structures in the outer retina (A) Comparison of confocal (left) and STED (right) images of SMI32-stained HC axon rminals at a depth of ~130 µm in the retinal whole-mount imaged through the inner retina. Each image was ﬁrst taken in confocal mode and then the STED mode to allow a direct comparison. (B) Confocal (blue) and STED (orange) FWHMs of the same neuritic structure as indicated in ), (C) Scheme of alternative experimental design for imaging structures in the deeper outer retina. Retinae were horizontally cryosectioned, mounted n glass slides, immunolabelled, and imaged. (D) Example confocal and STED images of SMI32-stained HC axonal structures taken with (top) and thout (bottom) silicon spacers between slide and coverslip. (E) Confocal and STED FWHMs of the same neuritic structures in (D), (F) Quantiﬁcation FWHMs of corresponding HC structures in confocal (blue) and STED (orange) imaging mode for whole-mount condition (whole-mount HC) and orizontally cryosectioned retina (cryo HC) with and without spacers (whole-mount HC n = 1 animal/24 structures, Cryo HC + spacer n = 1 animal/26 uctures, Cryo HC n = 1 animal/30 structures, p< 0.0001 = , ns = not signiﬁcant, paired t-tests, horizontal bars indicate means, grey lines connect orresponding values). FIGURE 5 (G) Violin plot showing the resolution enhancement factor calculated as the ratio of the corresponding FWHMs in confocal and TED images for the three experimental conditions, horizontal bars indicate means (p< 0.0001 = , p< 0.00001 = *, ns = not signiﬁcant, Wilcoxon nk sum test). For A and D, the excitation wavelength was 532 nm, and the dye was ATTO532. Scale bars: 5 µm in A,D. ers et al. 10.3389/fopht.2023.1126338 10.3389/fopht.2023.1126338 Kremers et al. A B D E C B A A B A C C E D E D E G F G F FIGURE 6 STED imaging of horizontal cell structures in the outer retina (A) Comparison of confocal (left) and STED (right) images of SMI32-stained HC axon terminals at a depth of ~130 µm in the retinal whole-mount imaged through the inner retina. Each image was ﬁrst taken in confocal mode and then in the STED mode to allow a direct comparison. (B) Confocal (blue) and STED (orange) FWHMs of the same neuritic structure as indicated in (A), (C) Scheme of alternative experimental design for imaging structures in the deeper outer retina. Retinae were horizontally cryosectioned, mounted on glass slides, immunolabelled, and imaged. (D) Example confocal and STED images of SMI32-stained HC axonal structures taken with (top) and without (bottom) silicon spacers between slide and coverslip. (E) Confocal and STED FWHMs of the same neuritic structures in (D), (F) Quantiﬁcation of FWHMs of corresponding HC structures in confocal (blue) and STED (orange) imaging mode for whole-mount condition (whole-mount HC) and horizontally cryosectioned retina (cryo HC) with and without spacers (whole-mount HC n = 1 animal/24 structures, Cryo HC + spacer n = 1 animal/26 structures, Cryo HC n = 1 animal/30 structures, p< 0.0001 = , ns = not signiﬁcant, paired t-tests, horizontal bars indicate means, grey lines connect corresponding values). (G) Violin plot showing the resolution enhancement factor calculated as the ratio of the corresponding FWHMs in confocal and STED images for the three experimental conditions, horizontal bars indicate means (p< 0.0001 = , p< 0.00001 = *, ns = not signiﬁcant, Wilcoxon rank sum test). For A and D, the excitation wavelength was 532 nm, and the dye was ATTO532. Scale bars: 5 µm in A,D. 11 Frontiers in Ophthalmology frontiersin.org 10.3389/fopht.2023.1126338 Kremers et al. 10.3389/fopht.2023.1126338 better ﬁt with the working distance of the microscope objective. Identiﬁcation of dendritic bulbs of horizontal cells After conﬁrming that the antibody staining and STED imaging protocols work as anticipated for the outer mouse retina, we applied our approach to the outer synaptic circuits. Bulb structures on HC dendrites have been recently identiﬁed as putative synaptic sites between HCs and BCs (33). Therefore, we aimed at investigating this synapse type using super-resolution microscopy. We ﬁrst identiﬁed HC bulb structures in the cryosectioned retina using confocal microscopy. The calcium-binding protein Calbindin is expressed throughout HCs, with anti-Calbindin staining being used to visualise the whole cell including soma, dendrites, and axon terminals. In HCs, neuroﬁlaments are only expressed in the axon terminal system and can thus be used to distinguish between dendritic and axonal structures (38). GABA r2 is a subunit of GABAC receptors, which in the outer retina is exclusively expressed on BCs, thus indicating postsynaptic BC sites. Before performing a triple staining, primary antibody functionality was tested in single and double stainings, observing structures known to express the targets of the antibodies with confocal microscopy (Calbindin for HC somata, dendrites and axons, SMI32 for HC axons, GABA r2 for GABA r2-expressing receptor clusters in the HC layer). FIGURE 5 To correct for differently sized biological structures, we calculated the REF in all conditions (Figure 6G), which validated the previous results: STED in whole-mounted retinae did not alter the resolution in HC axon terminals (0.95 ± 0.35, mean ± SD). In contrast, the relative resolution in the cryosectioned groups was improved by the factors 1.44 ± 0.08 (mean ± SD; with spacer) and 1.38 ± 0.07 (mean ± SD; with spacer). With the saturation factor of 28.6 in this experiment, the maximal theoretical REF is 6.6. The REF in HC structures in cryosectioned retinae did not signiﬁcantly differ from the whole-mounted RGC axons imaged in the same set of experiments (REF: 1.41 ± 0.50, n = 1 animal, n = 23 RGC structures; mean ± SD; p = 0.46 compared to HC with spacer and p = 0.77 compared to HC without spacer, Wilcoxon rank sum test). Interestingly, the mean SBRs of SMI32-positive HC structures were similar in the intact whole-mounted and both cryosectioned conditions (whole-mounted, 8.8 ± 15.3, n = 24 structures; cryosectioned with spacer, 12.6 ± 20.4, n = 25 structures; cryosectioned without spacer, 7.5 ± 8.6, n = 28 structures, all imaged in the STED mode, mean ± SD). However, as expected from the diffuse nature of SMI32-staining, there were large differences between the SBRs of single structures (Figure 6D), as indicated by the high SD values. Structures with low SBR were frequently encountered in all three conditions. structures (see examples in Figure 7A). Surprisingly, only few SMI32-negative structures could be observed, suggesting that HC dendrites are not isolated from axons but co-fasciculate, and thus, cannot be easily distinguished under the microscope. However, several round and ‘blobby’ SMI32-negative thickenings could still be detected emerging from double positive structures, which we assumed to be HC dendritic bulbs (Figure 7A). Such identiﬁed bulbs were further examined by extracting normalised SMI32 and Calbindin intensity values along a line selection which was put through each bulb (Figures 7B, C). Bulbs were more positive for Calbindin than for SMI32 and underlying SMI32 signals did not follow the shape of Calbindin signals (Figure 7B). Intensity values were also extracted from line selections of control structures (dendritic thickenings, non-bulbs), which we assumed to be double-positive (Figure 7A top panel, C). In some rare cases Calbindin or SMI32 staining intensities reached a plateau which was likely caused by signal saturation (Figure 7B top panel, C). FIGURE 5 However, within-bulb differences between the weaker SMI2 and stronger Calbindin signals could still be observed (theoretically, the signal difference would have been even more prominent without saturation) (Figure 7D). For further comparison and statistical analysis, mean staining intensities along the lines were calculated for each structure. SMI32 and Calbindin signals did strongly correlate in many non-bulb controls but never in the bulbs (Figure 7D). Indeed, the average intensity difference was signiﬁcantly higher for the HC bulbs than for the non-bulb structures (bulbs, 102.55 ± 27.3; non-bulbs, -12.5 ± 47.8; mean ± SD; p< 0.000001, Wilcoxon rank sum test) (Figure 7E). Thus, although SMI32-positive/Calbindin-positive axons and SMI32- negative/Calbindin-positive dendrites of HCs did strongly co- fasciculate, the reliable identiﬁcation of dendritic bulbs was possible. Frontiers in Ophthalmology frontiersin.org Identiﬁcation of putative GABAergic synapse at bulb sites To image HC bulbs and GABA r2 receptors with higher resolution, STED images of identiﬁed bulb structures were acquired. Bulbs were identiﬁed as described above using large confocal z-stacks, which were screened for bulbs directly at the microscope (Figure 8A). For this triple labelling approach, each primary antibody was paired with multiple secondary antibodies, coupled to different ﬂuorophores, and imaged using both confocal and STED microscopy to identify ﬂuorophores working optimally with three-colour STED (Figure 8B). Fluorophores of the Alexa Fluor family were tested but found to easily bleach. In the end, ﬂuorophores of the STAR and ATTO families were chosen for their superior photostability, although in confocal imaging, they can be dimmer compared to Alexa Fluor dyes. To avoid spectral overlap in the triple staining and make use of the available excitation and depletion lasers, STAR488 was chosen for the Calbindin, ATTO532 for SMI32, and ATTO633 for GABA r2 staining. Bulbs were then zoomed-in using confocal live-view before Calbindin, SMI32 and GABA r2 stainings were subsequently imaged with the STED mode. Deconvolution was applied afterwards to increase SBR and better resolve the ﬁne GABA receptor clusters. Interestingly, bulbs Dendritic HC bulbs were characterised as Calbindin-positive and SMI32-negative structures that possibly co-localize with GABA r2 (33). Bulb identiﬁcation was performed using lower magniﬁcation confocal microscopy (100x objective, 2.1× digital zoom) and imaging Calbindin and SMI32 in large z-stacks, spanning the whole HC layer. Calbindin and SMI32 images were superimposed, and single z-slices were manually scanned for bulb 12 frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 A B D E C FIGURE 7 Identiﬁcation of dendritic horizontal cell bulbs (A) Three example images showing three HC bulbs and one non-bulb identiﬁed in confocal single plane images z-stack slices. Retinal sections were imaged for Calbindin and SMI32. Bulb structures (bulb) positive for only Calbindin and non-bulb structure (non-bulb) positive for both SMI32 and Calbindin were identiﬁed (small white squares). (B) Intensity values for Calbindin and SMI32 stainings were extracted along a line through the three bulbs taken from (A) Plots show Calbindin and SMI32 intensity distribution across the three bulbs (dashed blue lines). (C) Intensity values for Calbindin and SMI32 stainings of a dendritic non-bulb taken from (A) (top panel). Plot shows Calbindin and SMI32 intensity distribution across the non-bulb (blue dashed line). (D) Mean intensities of Calbindin (red) and SMI32 (cyan) stainings along the lines through individual non-bulbs (left) and bulbs (right). Frontiers in Ophthalmology Identiﬁcation of putative GABAergic synapse at bulb sites Error bars indicate 95% conﬁdence interval. The vertical grey lines connect corresponding Calbindin and SMI32 intensities within the same dendritic structures. (E) Violin plot showing the average intensity difference. Difference per pixel was calculated for multiple bulbs and non-bulb control structures, horizontal bars indicate means (n = 1 animal, n = 22 structures for bulbs, n = 22 structures for non-bulbs, p< 0.000001 = **, Wilcoxon rank sum test). For A and B, the excitation wavelength was 532 nm, and the dye was ATTO532 for SMI32. For Calbindin, the excitation wavelength was 488 nm, and the dye was STAR488. Scale bars: 10 µm in A, 2 µm in B,C. C B A C B A D D E E FIGURE 7 Identiﬁcation of dendritic horizontal cell bulbs (A) Three example images showing three HC bulbs and one non-bulb identiﬁed in confocal single plane images z-stack slices. Retinal sections were imaged for Calbindin and SMI32. Bulb structures (bulb) positive for only Calbindin and non-bulb structure (non-bulb) positive for both SMI32 and Calbindin were identiﬁed (small white squares). (B) Intensity values for Calbindin and SMI32 stainings were extracted along a line through the three bulbs taken from (A) Plots show Calbindin and SMI32 intensity distribution across the three bulbs (dashed blue lines). (C) Intensity values for Calbindin and SMI32 stainings of a dendritic non-bulb taken from (A) (top panel). Plot shows Calbindin and SMI32 intensity distribution across the non-bulb (blue dashed line). (D) Mean intensities of Calbindin (red) and SMI32 (cyan) stainings along the lines through individual non-bulbs (left) and bulbs (right). Error bars indicate 95% conﬁdence interval. The vertical grey lines connect corresponding Calbindin and SMI32 intensities within the same dendritic structures. (E) Violin plot showing the average intensity difference. Difference per pixel was calculated for multiple bulbs and non-bulb control structures, horizontal bars indicate means (n = 1 animal, n = 22 structures for bulbs, n = 22 structures for non-bulbs, p< 0.000001 = ****, Wilcoxon rank sum test). For A and B, the excitation wavelength was 532 nm, and the dye was ATTO532 for SMI32. For Calbindin, the excitation wavelength was 488 nm, and the dye was STAR488. Scale bars: 10 µm in A, 2 µm in B,C. r2-positive clusters had an FWHM under 200 nm, surpassing the xy resolution limit of confocal light microscopy. Identiﬁcation of putative GABAergic synapse at bulb sites In fact, many FWHMs peaked at 110 to 120 nm (140.1 ± 443.8 nm, mean ± SD, n = 100 receptor clusters), which is within the ‘working range’ of the resolution limit of super-resolution STED microscopy (Figure 8E). In conclusion, we were able to reliably identify GABA receptor clusters on HC dendritic bulbs using super-resolution STED identiﬁed in confocal mode were often only weakly visible with higher-magniﬁcation (4.0× digital zoom) STED microscopy. However, bulbs that could be observed with STED correlated with multiple GABA receptor blobs/clusters (Figure 8C). Finally, we determined the FWHMs of GABA receptor clusters in deconvolved STED images (Figure 8D). Our quantiﬁcation showed that the vast majority (93 out of 100) of analysed GABA r2-positive clusters had an FWHM under 200 nm, surpassing the xy resolution limit of confocal light microscopy. In fact, many FWHMs peaked at 110 to 120 nm (140.1 ± 443.8 nm, mean ± SD, n = 100 receptor clusters), which is within the ‘working range’ of the resolution limit of super-resolution STED microscopy (Figure 8E). In conclusion, we were able to reliably identify GABA receptor clusters on HC dendritic bulbs using super-resolution STED Frontiers in Ophthalmology 13 frontiersin.org 10.3389/fopht.2023.1126338 Kremers et al. A B D E C FIGURE 8 GABAC receptor clusters can be localised on horizontal cell dendritic bulbs with high-resolution STED (A) Schematic showing experimental design to image dendritic HC bulbs with high-resolution STED microscopy. Individual bulbs are identiﬁed in sections of large confocal z-stacks (Bulb ID, see also Figure 3). Bulbs were zoomed into, imaged as STED z-stacks and visualised using maximum z-projections and deconvolution. (B) Triple staining experiment against Calbindin (red), SMI32 (yellow) and GABA r2 (cyan) showing a dendritic bulb (white square) imaged in the STED mode. The centre of the bulb is negative for SMI32 but positive for calbindin labelling. White square indicates the position of the bulb depicted as close-ups in C. (C) Zoomed-in bulb with triple staining against Calbindin, SMI32 and GABA r2 showing a HC dendritic bulb (from white square in B) imaged in STED mode and deconvolved. Note that GABA r2 receptor clusters (arrows) are located at the edges of bulb. (D) Example r2-positive GABA receptor cluster (top, small white square from C) with Gaussian ﬁt and FWHM (bottom). FWHM intensity proﬁle is taken at the indicated position (orange line). Identiﬁcation of putative GABAergic synapse at bulb sites (E) Histogram showing distribution of FWHMs of GABA r2 clusters (in 10 nm bins) (n = 1 animal, n = 100 clusters). Dashed blue bar indicates xy resolution limit for confocal light microscopy (~ 200 nm), dashed grey bar shows FWHM mean for GABA receptor clusters (~ 140 nm, see Results section). For SMI32, the excitation wavelength was 532 nm, and the dye was ATTO532. For Calbindin, the excitation wavelength was 488 nm, and the dye was STAR488. For GABA r2, the excitation wavelength was 635 nm, and the dye was ATTO633. Scale bars: 5 µm in B, 1 µm in C, 0.25 µm in D. A B A B B D E C C D D E E E FIGURE 8 GABAC receptor clusters can be localised on horizontal cell dendritic bulbs with high-resolution STED (A) Schematic showing experimental design to image dendritic HC bulbs with high-resolution STED microscopy. Individual bulbs are identiﬁed in sections of large confocal z-stacks (Bulb ID, see also Figure 3). Bulbs were zoomed into, imaged as STED z-stacks and visualised using maximum z-projections and deconvolution. (B) Triple staining experiment against Calbindin (red), SMI32 (yellow) and GABA r2 (cyan) showing a dendritic bulb (white square) imaged in the STED mode. The centre of the bulb is negative for SMI32 but positive for calbindin labelling. White square indicates the position of the bulb depicted as close-ups in C. (C) Zoomed-in bulb with triple staining against Calbindin, SMI32 and GABA r2 showing a HC dendritic bulb (from white square in B) imaged in STED mode and deconvolved. Note that GABA r2 receptor clusters (arrows) are located at the edges of bulb. (D) Example r2-positive GABA receptor cluster (top, small white square from C) with Gaussian ﬁt and FWHM (bottom). FWHM intensity proﬁle is taken at the indicated position (orange line). (E) Histogram showing distribution of FWHMs of GABA r2 clusters (in 10 nm bins) (n = 1 animal, n = 100 clusters). Dashed blue bar indicates xy resolution limit for confocal light microscopy (~ 200 nm), dashed grey bar shows FWHM mean for GABA receptor clusters (~ 140 nm, see Results section). For SMI32, the excitation wavelength was 532 nm, and the dye was ATTO532. For Calbindin, the excitation wavelength was 488 nm, and the dye was STAR488. For GABA r2, the excitation wavelength was 635 nm, and the dye was ATTO633. Discussion A very crucial factor of every super-resolution microscopy approach is resolution estimation. For this purpose, we ﬁtted a Gaussian to the line proﬁles of ﬁlamentous structures using the NLS algorithm. From the Gaussian ﬁts, we determined the FWHMs and compared the values between imaging conditions. This approach, although widely used (41–43), is prone to errors (19) and relatively laborious. It requires isolated ﬁlamentous or punctuated nanometre-sized structures, which are not necessarily easy to ﬁnd even in the samples labelled against cytoskeletal or calcium-binding proteins. In contrast, another approach, Fourier ring correlation analysis, could be performed without any prior information about the sample (44). Given the abundance of the antigen and the antibodies concentration, we had sufﬁcient labelling density. However, given that the intermediate ﬁlament has a width of ~10 nm and is labelled by indirect immunodetection with antibodies having size of ~20 nm, the smallest measurable biological structure is theoretically restricted to approximately 50 nm in our case (45). Furthermore, the labelling with IgG antibodies may introduce artefacts into the imaging (40) making some densely packed antigens inaccessible for labelling. This challenge can be overcome by targeted labelling with small molecules (e.g. nanobodies, protein/peptide-directed labelling, aptamers or click chemistry-based labelling of single amino acids (46–49)). Super-resolution imaging of deeper layers of specimens has been established for different brain structures (11, 39). Here, we developed a reliable protocol for STED imaging in the whole-mount mouse retina. So far, the number of STED protocols for mouse retinal tissue is very limited (26). To optimise the imaging procedure, we adapted sample preparation and microscope settings. As the ﬁrst retinal model system, we labelled neuroﬁlaments in RGCs and found that – at a depth of up to ~30 μm – the spatial xy resolution could be increased by a factor of > 2. Next, we aimed at imaging synaptic structures of HCs in the outer retina at a depth of ~130 μm. Due to scattering of the laser light and misalignment of the excitation and depletion laser beams, super- resolution imaging in deeper layers of the outer retina did not yield resolution improvements. However, we established a method to circumvent this problem and to increase the resolution of STED microscopy when imaging in those deeper retinal layers: horizontal cryosectioning and removal of the inner retinal tissue allowed us to access HC neurites with STED imaging. Identiﬁcation of putative GABAergic synapse at bulb sites Scale bars: 5 µm in B, 1 µm in C, 0.25 µm in D. 14 Frontiers in Ophthalmology frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 microscopy and could show that the FWHM of the GABA receptor clusters is clearly under the resolution limit of confocal microscopy. Discussion Here, we visualised dendritic bulbs, which likely represent a novel type of synapse capable of GABAergic feedforward signalling from HCs to BCs. Thus, imaging in the whole-mount retina can help to describe the protein composition and scaffold at retinal synapses. Taken together, we are convinced that our protocol further expands the application portfolio of STED microscopy. Resolution increment is determined by the saturation factor of the depletion laser In combination with the cryosectioning, a protocol for an immunocytochemistry triple staining for STED imaging was developed and tested in both whole-mount retinae and horizontal retinal cryosections. Antibodies against Calbindin, SMI32, and GABA r2 were chosen for assessing HC synapses. However, we expect that the protocol also works with other antibodies. In general, all tested antibody stainings functioned as expected, although problems with the GABA r2 antibody such as insufﬁcient penetration of the tissue occurred from time to time. Possible explanations might be a lower afﬁnity of the antibody, or more generally, in the antigen properties of GABAC receptors, whose epitopes may be hidden within the double lipid membrane or beneath other synaptic proteins. Additionally, GABA receptor clusters are smaller and sparser than SMI32 and Calbindin complexes, resulting in a lower overall number of target epitopes. As Calbindin is present throughout the HC cytosol, its staining often appears to be diffuse and HC structures appear blurry, especially with higher magniﬁcation. Alternative approaches to stain whole HCs, such as immunostaining against GFP in transgenic animals or direct injection of ﬂuorophores into single HCs, may result in signals with a better SBR (50). For single-colour STED microscopy and for imaging the ﬁne structures as GABA receptor clusters, we chose the 775 nm depletion beam because with it we obtained a higher saturation factor than with the 592 nm laser. The two lasers differed in both laser architecture and temporal properties of excitation, depletion and detection. The 775 nm pulsed/gated laser allowed us to achieve a saturation factor of 30, which was theoretically sufﬁcient for resolution scaling up to 6.79 times if compared with confocal microscopy (35). The main constraint with increasing depletion laser intensity was specimen overheating. Another problem that we faced was the loss of SBR with increasing imaging depth and light scattering in the tissue. To our knowledge, it is a common problem with several possible solutions (19, 20). We tried deconvolution and obtained a pronounced SBR improvement. To further increase contrast and improve the axial resolution, one can use a 3D STED approach with additional donut-shaped illumination perpendicularly to the optical axis (40) or adaptive optics (36, 37), or alternatively horizontal cryosectioning (see below). Frontiers in Ophthalmology Frontiers in Ophthalmology 15 frontiersin.org 10.3389/fopht.2023.1126338 Kremers et al. 10.3389/fopht.2023.1126338 subsequently calculated, partially correct for different-sized structures and conﬁrm the effects observed with the absolute FWHMs. Resolution increment is determined by the saturation factor of the depletion laser The robustness of curve ﬁtting and thus FWHM calculation also strongly depended on the SBR of the structures selected. For example, in some STED images, the SBR was frequently inadequate, and the accuracy of the ﬁtted curves had to be manually reviewed for each structure. Due to the low number of emitted and detected photons, a low SBR is a general issue with STED imaging. Thus, for each experiment appropriate settings must be determined and the right balance between signal and resolution has to be deﬁned. Theoretically, STED microscopy can reach xy resolutions in the low nm range and, using ﬂuorescent beads, PSFs with as little as 5.8 nm width have been recorded (54). However, these resolutions cannot be currently achieved in biological samples and, while proof-of-concept studies could produce resolutions of around 20 nm (55), the measured maximal resolution is in practice often limited by target size, optical distortions, photobleaching, and labelling strength. In this study, FWHMs of below 100 nm, and thus far beyond the diffraction limit of confocal microscopy, were observed. Still, even under best possible STED imaging conditions, the FWHMs of the HC structures were on average above 200 nm. One reason for this could simply be the relatively large size of the neuroﬁlament structures stained in HCs. This possibility is supported by the fact that although STED resolutions in this experiment are beyond the diffraction limit, they still represent a signiﬁcant improvement compared to the confocal FWHMs of the same structures. Another argument in favour of this view comes from imaging individual and sparse GABA receptor clusters at HC bulbs: here, the mean FWHM is around 140 nm, and therefore, is close to the mean FWHM of RGC neuroﬁlaments. Comparing neuroﬁlament structures of retinal ganglion cells and horizontal cells In this study, we used the FWHM in biological samples as an indicator for the effective resolution. We demonstrated that both the absolute FWHMs as well as the REF of HC structures were signiﬁcantly enhanced in cryosections compared with the intact retinal whole-mount, and thus, similar to the resolution of superﬁcial RGC axons and dendrites in the whole-mount retinae. It should be emphasised that in our experiments, the FWHM measured for individual RGC dendrites was lower than the FWHM in co-fasciculating HC neurites. Thus, the measured FWHM does not necessarily reﬂect the absolute theoretical resolution of the microscope but, for instance, depends on the width of the measured structure, on the thickness of the biological specimen, the size/type of the antibodies and the SBR. Still, the FWHM has been used as a reliable indicator for resolution in past studies (52, 53). Furthermore, the relative resolutions, which were Retinal cryosectioning is compatible with standard immunocytochemistry and STED super-resolution microscopy One major challenge of our approach was to show whether cryosectioning of the retina is still compatible with standard triple staining protocols using STED-compatible ﬂuorophores. Here, we demonstrate the feasibility of performing complex super-resolution STED experiments in retinal cryosections. The newly developed protocol was applied to study HC dendritic bulbs, which likely represent a recently identiﬁed synapse for GABAergic feedforward signalling from HCs to BCs (33). As STED microscopy is based on confocal microscopy technology, sharing the pinhole, it is intrinsically capable of optical sectioning, and thus, should be theoretically able to image in deeper planes of thick specimens. However, the spatial resolution of STED microscopy strongly depends on the exact alignment of the depletion laser donut around the excitation laser beam, which can be disturbed by scattering in biological samples. Multiple adaptive optics approaches, which measure and compensate for the distortions for each point, have been developed but involve multiple drawbacks, mainly high costs and increased imaging time and photobleaching. The method described here averts optical distortions by removing biological tissue above the plane of interest. Additionally, it enables the application of conventional STED microscopy without requiring special settings or computations and thus decreases costs, effort, and imaging time compared to adaptive optics. Cryosectioning is a well-established and widely used technique. Still, some problems, including freezing artefacts as well as wrinkled and displaced sections, are commonly reported and could inﬂuence tissue integrity and STED resolution (51). Even small artefacts, hardly visible under confocal microscopes, could inﬂuence images taken with STED. Thus, precise and meticulous work is important throughout the whole freezing and cryosectioning process. Huygens deconvolution was applied to STED images to further increase the resolution and improve the SBR. Deconvolution algorithms are computational methods that try to recalculate the original optical scene in the sample by subtracting effects of known optical distortions and diffractions from the image (56) and that were used with STED microscopy before with excellent results (57, 58). In our case however, only minor improvements of FWHM could be observed after deconvolution, although SBR mostly appeared to be increased. A problem with the employed deconvolution algorithm might be that it is “blind”, meaning that it uses a theoretical PSF that was calculated for the microscope setup present. A better approach would include measuring real PSFs using the exact imaging conditions, which might enable the deconvolution to predict optical aberrations more accurately. Frontiers in Ophthalmology Horizontal cell bulbs likely represent GABAergic presynapses This allowed the distinction between dendrites and axons and by calculating the intensity differences between Calbindin and SMI32 signals quantitative characteristics of bulbs could be deﬁned. We originally expected to ﬁnd half of the HC structures double-positive, representing axons, and the other half only positive for Calbindin, representing dendrites. However, very few Calbindin-only-positive structures could be observed. Space limitations in the outer plexiform layer and a high density of HC structures likely result in strong co-fasciculation of HC dendrites and axons and an overlay of single- and double-positive structures. Still, by imaging large z- stacks with low magniﬁcation in the confocal mode, single bulb structures were frequently identiﬁed. These structures typically emerged from double-positive ﬁlamentous structures and occasionally overlapped with additional SMI32 signals, which might be the result of HC axons stratifying along the bulbs. Nevertheless, these structures showed no correlation between Calbindin and SMI32 signals and were thus further considered dendritic bulbs. within a single interneuron) and the presence of similarly complex synapses between photoreceptors, BCs and HCs in close proximity. Nonetheless, structural super-resolution studies such as the present work might provide novel access points for further experiments. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Funding This work was funded by the Deutsche Forschungsgemeinschaft (DFG; INST 2388/62-1 to MU) and the Tistou and Charlotte Kerstan Foundation to MU. Acknowledgments We thank Merle Harrer and Gordon Eske for excellent technical support, and Sylvia Bolz and Christine Henes for assistance with the cryotome. We thank Karin Dedek, Christian Puller and Bettina Kewitz for helpful discussions. We thank Dominic Gonschorek and Jonathan Oesterle for critical reading of the manuscript and discussion. We acknowledge support by the Open Access Publishing Fund of the University of Tübingen. Author contributions LK, KS: Conceptualisation, investigation, methodology, data analysis, writing (original draft), visualisation. TS: Conceptualisation, investigation, writing (original draft), visualisation, supervision. IN-S: Conceptualisation, visualisation, supervision, writing (review and editing). FH: writing (review and editing), visualization. TE: writing (review and editing), visualisation, supervision. MU: writing (review and editing), visualisation, funding acquisition. ZZ: investigation, data analysis, visualisation, writing (review and editing). All authors contributed to the article and approved the submitted version. By detecting the r2 subunit of GABAC receptors, which in the retina are exclusively expressed on BCs (65), at Calbindin-positive/ SMI32-negative bulbs, we could speciﬁcally investigate the role of bulbs in HC to BC GABAergic signalling. Some GABA r2 signals were detected on or in direct proximity to bulbs, thus indicating co- localization of bulbs with BC postsynaptic sites, and supporting the notion that bulbs are HC-to-BC synapses. Still, the presence of additional synaptic markers (e.g. presynaptic proteins) would provide further evidence for the type and mechanism of bulb synapses. For instance, synaptic proteins of the release machinery in HC dendrites as well as GABA and GAD65 have been found in mammalian HCs (33, 64, 66). It might be compelling to monitor these targets with super-resolution microscopy and compare their localization within bulbs to the GABA receptors. Further intriguing imaging targets are voltage-gated calcium channels, although antibodies against them are either extremely subtype-speciﬁc and have a low efﬁciency or are very unspeciﬁc, resulting in cross- reactions with other targets (67). In general, staining as many targets simultaneously as possible would enable the extraction of much more information about the synapse ultrastructure, however, immunocytochemistry gets increasingly difﬁcult the more simultaneous stainings are applied and chances for unspeciﬁc bindings or ﬂuorescent crosstalk grow. Functional experiments unravelling the mechanisms and function of bulb synapses would be highly desirable, but selectively monitoring or manipulating HC- to-BC synapses is challenging due to the complexity of outer retinal circuits (e.g. simultaneous feedback and feedforward signalling Ethics statement The animal study was reviewed and approved by Tierschutzbeauftragte der Universität Tübingen. Frontiers in Ophthalmology Horizontal cell bulbs likely represent GABAergic presynapses Horizontal cells are essential for the generation of centre- surround receptive ﬁelds in BCs (59). While most studies focused on the complex HC feedback mechanisms to photoreceptors, evidence for direct HC-to-BC synaptic contacts has been found in both non-mammalian and mammalian retinae (31, 32, 60–62). HC feedforward signalling is likely GABAergic and dependent on 16 frontiersin.org frontiersin.org frontiersin.org Kremers et al. 10.3389/fopht.2023.1126338 vesicular release (63, 64). Recently, bulbs on HC dendrites have been observed in 3D electron microscopy reconstructions and identiﬁed as possible synaptic contacts, with most bulbs contacting either other HC bulbs or BC dendrites (33). Furthermore, Behrens and colleagues demonstrated the presence of mitochondria in dendritic bulbs and used immunolabeling to reveal that bulbs of individually stained horizontal cells co-localize GABA r2 receptors. In this study, bulb identiﬁcation was performed by using Calbindin as a marker for the entire HC and additionally labelling SMI32 to counterstain HC axon terminals. This allowed the distinction between dendrites and axons and by calculating the intensity differences between Calbindin and SMI32 signals quantitative characteristics of bulbs could be deﬁned. We originally expected to ﬁnd half of the HC structures double-positive, representing axons, and the other half only positive for Calbindin, representing dendrites. However, very few Calbindin-only-positive structures could be observed. Space limitations in the outer plexiform layer and a high density of HC structures likely result in strong co-fasciculation of HC dendrites and axons and an overlay of single- and double-positive structures. Still, by imaging large z- stacks with low magniﬁcation in the confocal mode, single bulb structures were frequently identiﬁed. These structures typically emerged from double-positive ﬁlamentous structures and occasionally overlapped with additional SMI32 signals, which might be the result of HC axons stratifying along the bulbs. Nevertheless, these structures showed no correlation between Calbindin and SMI32 signals and were thus further considered dendritic bulbs. vesicular release (63, 64). Recently, bulbs on HC dendrites have been observed in 3D electron microscopy reconstructions and identiﬁed as possible synaptic contacts, with most bulbs contacting either other HC bulbs or BC dendrites (33). 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W3133980288.txt	https://www.nature.com/articles/s41467-021-21864-3.pdf	en		A randomised controlled trial to reduce highest priority critically important antimicrobial prescription in companion animals	Nature communications	2,021	cc-by	10,926	ARTICLE https://doi.org/10.1038/s41467-021-21864-3 OPEN A randomised controlled trial to reduce highest priority critically important antimicrobial prescription in companion animals 1234567890():,; David A. Singleton 1 ✉, Angela Rayner2, Bethaney Brant1, Steven Smyth1, Peter-John M. Noble1, Alan D. Radford1 & Gina L. Pinchbeck1 Robust evidence supporting strategies for companion animal antimicrobial stewardship is limited, despite frequent prescription of highest priority critically important antimicrobials (HPCIA). Here we describe a randomised controlled trial where electronic prescription data were utilised (August 2018–January 2019) to identify above average HPCIA-prescribing practices (n = 60), which were randomly assigned into a control group (CG) and two intervention groups. In March 2019, the light intervention group (LIG) and heavy intervention group (HIG) were notiﬁed of their above average status, and were provided with educational material (LIG, HIG), in-depth benchmarking (HIG), and follow-up meetings (HIG). Following notiﬁcation, follow-up monitoring lasted for eight months (April–November 2019; postintervention period) for all intervention groups, though HIG practices were able to access further support (i.e., follow-up meetings) for the ﬁrst six of these months if requested. Postintervention, in the HIG a 23.5% and 39.0% reduction in canine (0.5% of total consultations, 95% conﬁdence interval, 0.4-0.6, P = 0.04) and feline (4.4%, 3.4-5.3, P < 0.001) HPCIAprescribing consultations was observed, compared to the CG (dogs: 0.6%, 0.5-0.8; cats: 7.4%, 6.0-8.7). The LIG was associated with a 16.7% reduction in feline HPCIA prescription (6.1% of total consultations, 5.3-7.0, P = 0.03). Therefore, in this trial we have demonstrated effective strategies for reducing veterinary HPCIA prescription. 1 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Leahurst Campus, Chester High Road, Neston, UK. 2 CVS (UK) Limited, 1 Owen Road, Diss, UK. ✉email: D.A.Singleton@liverpool.ac.uk NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications 1 ARTICLE C NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 ompanion animals are increasingly being recognised as an important contributor to the development1,2, carriage3 and transmission of antimicrobial resistant (AMR) bacteria both between animals and to/from humans, primarily due to frequent antimicrobial prescription driving selection for resistance in both species groups1,4–8, and the close proximity in which companion animals reside with humans8,9. Recent population studies facilitated by the expanding availability of companion animal electronic health records (EHRs)10,11 have identiﬁed encouraging trends in antimicrobial prescription, including reducing antimicrobial frequency, both in general10 and for some clinical presentations12–14. However, antimicrobials still remain amongst the most commonly prescribed pharmaceutical agents in companion animals15. Of particular concern, cefovecin, a 3rd generation cephalosporin considered a “highest priority critically important antimicrobial” (HPCIA) by the World Health Organisation16, remains the most commonly prescribed antimicrobial in cats10,11; such prescriptions frequently lacking clearly recorded clinical reasoning to justify their prescription17. HPCIAs, also including ﬂuoroquinolones which are also relatively frequently prescribed to companion animals10, are recommended to ideally be reserved for human use alone16, with current companion animal prescribing guidance suggesting HPCIAs should only be prescribed when there is clear evidence of resistance to ﬁrst-line antimicrobials18. As cefovecin is frequently prescribed in the absence of such evidence17, this raises signiﬁcant questions as to how appropriate such frequent prescription is in cats. Along with more qualitative studies19–23, EHRs17,24,25 have also identiﬁed key motivators for antimicrobial prescription, revealing a complex interplay between animal, owner, clinical presentation, individual veterinary surgeon, and the overarching culture of the veterinary practice in which they are employed. On a population-level, considerable inter-practice variation in antimicrobial prescription frequency, including the HPCIAs, has been identiﬁed10, with those practices with higher levels of professional accreditation being relatively less frequent prescribers of systemically-administered antimicrobials to dogs, compared to their non-accredited peers25. Much of the work to mitigate the companion animal contribution to AMR has focused on improving antimicrobial prescription, under the banner of antimicrobial stewardship, largely through the production of evidence-based antimicrobial prescribing guidance18,26–29 and practice benchmarking30,31. Although these are to be welcomed, robust evidence supporting their impact on companion animal antimicrobial prescribing practices is absent. In this regard, there is much to learn from medical practice, where robustly evidenced antimicrobial stewardship schemes have been established for some time32–38. In the absence of evidence, individuals tend to over-estimate perceived negative traits in their peers, thus serving as justiﬁcation for their own behaviour (e.g., alcohol consumption frequency)39. A similar tendency regarding antimicrobial prescription amongst veterinary surgeons has been previously observed19, leading us to hypothesise that lack of knowledge of relative frequencies of antimicrobial prescription might in itself serve as a driver for more frequent prescription. As such, use of benchmarking, thus drawing attention to an individual’s departure from a “social norm”, might have some utility in counteracting such tendencies. Indeed, use of prescribing benchmarks within a social norms framework has already shown some potential in prompting medical general practitioner-reﬂection and behavioural change34. Hence, in this work we present a randomised controlled trial demonstrating the efﬁcacy of social norm messaging, via integrated EHR-driven antimicrobial prescription benchmarking and in-practice educational support, to signiﬁcantly reduce HPCIA 2 prescription frequency, and antimicrobial prescription frequency in general. Participants were recruited from a cohort of above average HPCIA-prescribing practices within a single veterinary practice group (CVS Group Ltd.), with the hypothesis that such interventions would effectively reduce HPCIA prescription frequency post-intervention. Results Heavy intervention group trial engagement. Following above average total HPCIA prescription frequency notiﬁcation (28-29 March 2019), HIG practices were given the option to voluntarily participate in a further reﬂection and education programme; two HIG practices opted out (Fig. 1). As all practices had previously consented to routinely provide data, data continued to be collected from all practices both before and following intervention, irrespective of their active participation in this trial. All 18 consenting HIG practices participated in an initial review with a hub clinical lead (15 held in April, two in May and one in June 2019), and 16 held a separate practice meeting (held April–June 2019). Of these, 15 requested a hub clinical lead follow-up review (held April–July 2019), seven requested a further hub clinical lead follow-up review (held May–July 2019), and 16 held a ﬁnal hub clinical lead review (held August–October 2019). No signiﬁcant variations in baseline characteristics between groups were observed (Table 1). Antimicrobial prescription frequency. Pre-intervention, 0.71% (95% conﬁdence interval, CI, 0.57–0.86, n consultations = 88,298), 0.72% (CI 0.60–0.83, n consultations = 107,223), and 0.68% (CI 0.47–0.90, n consultations = 63,366) of canine consultations were associated with total HPCIA prescription in the CG, LIG and HIG, respectively (Table 2). No signiﬁcant variations between intervention groups were observed (P = 0.95). In cats, 7.44% CG (CI 6.27-8.61, n consultations = 33,613), 7.03% LIG (CI 6.32–7.74, n consultations = 39,803), and 8.03% HIG (CI 7.16–8.90, n consultations = 25,661) consultations were associated with total HPCIA prescription (Table 3). No signiﬁcant variations between intervention groups were observed (P = 0.48). The “respiratory”, “kidney disease” and “pruritus” MPCs in dogs, and “respiratory” and “trauma” MPCs in cats were associated with increased total HPCIA prescription frequency, relative to other MPCs. Post-intervention, HIG canine total HPCIA prescription frequency signiﬁcantly reduced by 23.5% (0.49% of canine HIG consultations, CI 0.37–0.61, P = 0.04, n consultations = 63,301), but no signiﬁcant change was observed in the LIG (0.79% of canine LIG consultations, CI 0.61-0.97, P = 0.78, n consultations = 105,518), compared to the CG (0.64% of canine CG consultations, CI 0.47–0.82, n consultations = 81,582) (Table 2; full model results Supplementary Table 2). For cats, a signiﬁcant 39.0% and 16.7% decrease in HPCIA prescription frequency was observed in both the HIG (4.35% of feline HIG consultations, CI 3.41–5.29, P < 0.01, n consultations = 25,808), and LIG (6.14% of feline LIG consultations, CI 5.29-7.00, P = 0.03, n consultations = 37,339), respectively, compared to the CG (7.37% of feline CG consultations, CI 6.02–8.72, n consultations = 30,362) (Table 3; full model results Supplementary Table 3). Both models provided a good ﬁt to underlying data, with the ﬁxed terms explaining 10% of canine and 40% of feline variance (Supplementary Table 2 and 3). Fourteen and twenty HIG practices recorded post-intervention total HPCIA prescription frequency decreases in dogs (Fig. 2A) and cats (Fig. 3A), respectively. In the LIG, nine and sixteen practices were associated with post-intervention HPCIA prescription frequency reductions in dogs and cats respectively, whereas in the CG, ten NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 ARTICLE Fig. 1 Trial practice group allocation. Schematic diagram of the process used to select above average highest priority critically important antimicrobial (HPCIA) prescribing veterinary practices owned by CVS Group Limited, followed by drop-off rate following the initial benchmarking intervention. practices were associated with post-intervention HPCIA prescription frequency reductions in both species. Regarding HIG month-by-month variation, signiﬁcant reductions in total HPCIA prescription frequency were observed for three and eight post-intervention months in dogs (Table 4; Fig. 2B; full model results Supplementary Table 4) and cats (Table 4; Fig. 3B; full model results Supplementary Table 5), respectively, with a particularly steep decline being observed between March and April 2019 in cats. The LIG was associated with a signiﬁcant HPCIA prescription frequency reduction in cats in June and November 2019, compared to the CG. Addition of temporal ﬁxed terms was found to improve model ﬁt (Supplementary Table 4 and 5). Overall, no signiﬁcant temporal variation in HPCIA prescription frequency was observed in the CG in either species, whereas a signiﬁcant linear reduction was observed in both dogs (P = 0.005) and cats (P < 0.001) in the HIG. Whereas in the LIG a signiﬁcant HPCIA prescription frequency linear reduction was observed for cats (P = 0.01), no signiﬁcant trend was observed for dogs (full model results Supplementary Table 6). In HIG dogs, HPCIA prescription frequency was found to signiﬁcantly reduce in one MPC post-intervention (Table 2; full model results Supplementary Table 7), whereas three MPCS were associated with reductions in cats (Table 3; full model results Supplementary Table 8). In the LIG for dogs, no MPCS showed signiﬁcant reductions in HPCIA prescription frequency, although the “vaccination” MPC was associated with a signiﬁcant increase. In cats in the LIG no MPCs showed a signiﬁcant reduction in HPCIA prescription frequency. Only the “other healthy” and “gastroenteric” MPCs in dogs were associated with improved ﬁt compared to a null model in dogs, whereas six MPCs were associated with improved ﬁts in cats. Considering antimicrobial prescription more broadly, the HIG was also associated with a 18.9% and 17.3% signiﬁcant decrease in the percentage of dog (Table 2; full model results Supplementary Table 2) and cat (Table 3; full model results Supplementary Table 3) consultations prescribed a systemic antimicrobial, compared to the CG, respectively. Signiﬁcant reductions in total antimicrobial prescription frequency were also observed in both species in the HIG. In terms of other pharmaceutical agents, we found no signiﬁcant post-intervention group differences in anti-inﬂammatory prescription frequency, nor frequency of euthanasia in either species (dogs, Table 2, Supplementary Table 9; cats, Table 3, Supplementary Table 9). Antimicrobial prescription choice. A summary of antimicrobial prescription choice variation as a percentage of total antimicrobial prescriptions by class, and for beta-lactams by sub-class, is available in Table 5 (dogs; full model results Supplementary Table 10) and Table 6 (cats; full model results Supplementary Table 11), and as a percentage of total consultations in Supplementary Table 12 (dogs) and 13 (cats). Though no comparisons were signiﬁcant, there was some evidence of post-intervention increases in clavulanic acid potentiated amoxicillin prescription in cats in the HIG, overtaking 3rd generation cephalosporins as the most prescribed antimicrobial. Bacterial diagnostic test frequency. Pre-intervention there were 3,932 cytological test orders, representing 1.09% of CG (CI 0.67–1.51), 1.03% LIG (CI 0.47–1.58) and 1.18% HIG (CI 0.54–1.83) consultations. Of 4,783 post-intervention cytological test orders, 1.29% of CG (CI 0.77–1.81), 1.31% LIG (CI 0.51–2.11) and 1.60% HIG (CI 1.03–2.17) post-intervention consultations were associated with an order. However, no signiﬁcant increase was observed, compared to the CG (LIG P = 0.53; HIG P = 0.89). Pre-intervention, there were 4517 bacterial culture and susceptibility test orders, representing 1.16% of CG (CI 0.77–1.55), 1.01% LIG (CI 0.64–1.39) and 1.76% HIG (CI 1.05–2.47) consultations. Of 4623 post-intervention bacterial culture and susceptibility test orders, 1.19% of CG (CI 0.83–1.55), 1.12% LIG (CI 0.68–1.55) and 1.81% HIG (CI 1.20–2.42) post-intervention consultations were associated with an order. However, no signiﬁcant increase was observed, compared to the CG (LIG P = 0.94; HIG P = 0.20). Full NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 Table 1 Practice, canine and feline pre-intervention (August 2018–March 2019 inclusive) baseline characteristics. Variable Control group Light intervention group Practice characteristics Median vet FTEa/practice [range] 3.8 [1.1–17.4] 4.1 [1.0–13.4] % of total FTE locum cover 6.5 5.0 CANINE Median n consultations/practice 2,657.5 [460.0–23,889.0] 3,814.5 [1,004.0–13,476.0] [range] Median n unique animals/practice 1,442.5 [335.0–14,436.0] 2,104.0 [626.0–6,613.0] [range] Main presenting complaint—% of total consultations (95% conﬁdence interval, CI) Vaccination 32.5 (29.5–35.4) 29.4 (26.9–31.9) Other healthy 21.8 (17.7–26.0) 27.7 (23.6–31.7) Post-operative check 9.8 (6.8–12.8) 8.4 (7.0–9.8) Gastroenteric 3.0 (2.4–3.7) 3.2 (2.7–3.7) Respiratory 1.0 (0.7–1.2) 0.9 (0.8–1.0) Pruritus 4.8 (3.8–5.9) 4.7 (3.7–5.6) Trauma 4.2 (3.3–5.0) 4.4 (3.7–5.0) Tumour 1.4 (1.1–1.7) 1.5 (1.2–1.7) Kidney disease 0.3 (0.2–0.3) 0.3 (0.2–0.4) Other unwell 21.3 (18.1–24.5) 19.6 (16.6–22.7) Animal characteristics—% of total consultations (95% CI) Sex: Male 51.2 (50.4–51.9) 51.0 (49.9–52.1) Neutered 67.4 (65.4–69.4) 66.4 (62.2–70.6) Insured 34.9 (21.9–48.0) 31.6 (22.2–41.0) Vaccinated 79.8 (78.1–81.6) 79.7 (77.6–81.8) Median age [range] 5.8 [0.0–20.7] 5.9 [0.0–24.7] FELINE 868.0 [231.0–8,608.0] 1,609.0 [424.0–6,365.0] Median n consultations/practice [range] Median n unique animals/practice 577.0 [161.0–5,996.0] 1,031.0 [340.0–3,922.0] [range] Main presenting complaint—% of total consultations (95% CI) Vaccination 36.7 (33.0–40.5) 33.9 (31.7–36.2) Other healthy 20.4 (16.6–24.2) 27.8 (23.7–31.9) Post-operative check 7.9 (5.1–10.7) 6.6 (5.4–7.9) Gastroenteric 2.3 (1.6–3.0) 2.2 (1.8–2.5) Respiratory 1.4 (1.0–1.9) 1.2 (0.8–1.6) Pruritus 2.5 (1.8–3.2) 2.0 (1.6–2.5) Trauma 4.6 (3.5–5.7) 4.6 (3.7–5.5) Tumour 0.7 (0.5–0.9) 0.9 (0.6–1.2) Kidney disease 0.8 (0.5–1.0) 0.7 (0.6–0.9) Other unwell 22.6 (19.3–25.9) 20.1 (17.6–22.6) Animal characteristics—% of total consultations (95% CI) Sex: Male 48.5 (47.5–49.5) 49.0 (47.8–50.1) Neutered 81.6 (79.0-84.3) 83.0 (80.9–85.0) Insured 24.9 (11.1–38.6) 21.7 (14.2–29.2) Vaccinated 68.9 (65.6–72.2) 71.5 (68.1–74.9) Median age [range] 6.7 [0.0–26.7] 7.3 [0.0–27.0] Heavy intervention group P 5.0 [1.1–11.5] 6.8 0.59 0.24 3,127.0 [443.0–9,145.0] 0.17 1,745.5 [310.0–4,785.0] 0.21 33.5 (29.9–37.0) 21.7 (18.7–24.7) 7.1 (6.1–8.2) 3.1 (2.5–3.7) 1.0 (0.7–1.2) 4.7 (3.9–5.6) 4.4 (3.5–5.3) 1.7 (1.3–2.0) 0.3 (0.2–0.4) 22.4 (20.0–24.7) 0.61 0.66 0.46 0.31 0.29 0.33 0.31 0.12 0.15 0.94 52.5 (51.3–53.8) 67.9 (65.1–70.8) 25.2 (15.7–34.8) 81.9 (79.4–84.3) 6.2 [0.0–24.0] 0.17 0.81 0.88 0.29 0.62 1,062.0 [457.0–2,626.0] 0.34 740.0 [373.0–1,895.0] 0.32 37.7 (33.9–41.4) 21.7 (18.3–25.1) 5.8 (5.0–6.6) 2.2 (1.7–2.7) 1.4 (1.1–1.8) 2.3 (1.9–2.7) 4.2 (3.6–4.8) 0.9 (0.7–1.2) 1.1 (0.7–1.4) 22.7 (20.4–25.1) 0.76 0.62 0.50 0.44 0.36 0.43 0.33 0.43 0.10 0.94 49.6 (48.5–50.7) 80.0 (77.1–82.9) 17.3 (12.1–22.5) 72.0 (69.2–74.7) 7.3 [0.0–26.8] 0.38 0.35 0.76 0.39 0.57 Practice characteristics summarised as of March 2019; statistical comparisons between groups performed via Kruskal-Wallis tests. Data is derived from 88,298 canine and 33,613 feline pre-intervention CG consultations, 107,223 canine and 39,803 feline pre-intervention LIG consultations, and 63,366 canine and 25,661 pre-intervention HIG consultations. aFull-time equivalent (40 h), inclusive of locum veterinary surgeon FTE. regression model results for both comparisons are available in Supplementary Table 9; neither model provided improved ﬁt compared to a null model. Use of antimicrobial prescription benchmarking portal. Prior to the intervention (1st February–27th March 2019 inclusive), there was no signiﬁcant variation between practices logging into their antimicrobial benchmarking portal: CG (n = 0), LIG (n = 3) and HIG (n = 3) (Fisher’s Exact Test, P = 0.23). Postintervention (1st April – 30th November 2019 inclusive) however, signiﬁcant variation was observed between practices logging into the portal within the CG (n = 3), LIG (n = 8) and HIG (n = 16), respectively (Fisher’s Exact Test, P < 0.001), with signiﬁcant variation being observed between both the CG and HIG (P < 0.001), 4 and LIG and HIG (P = 0.03), but not between the CG and LIG (P = 0.16). The two HIG practices that declined further participation did not interact with their portal pre- or post-intervention. A pronounced increase in LIG and HIG practice portal engagement was observed in April 2019, declining towards the end of the trial (Fig. 4). Discussion Here we describe use of EHR data from veterinary practices to initiate voluntary antimicrobial prescribing behavioural change in veterinary prescribing. In so doing, we have outlined a data-led and educational support framework by which HPCIA prescription frequency can be signiﬁcantly reduced, while preserving clinical autonomy. These insights are now being used to inform a national NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications Pre-intervention Pre-intervention Post-intervention Light intervention group Control group 0.777 0.143 0.110 0.912 0.575 0.012 0.285 0.305 0.304 0.491 0.645 0.232 0.130 0.997 0.981 0.977 0.541 (16.33–18.46) (9.05–10.95) (7.61–8.36) (0.31–0.42) (0.11–0.31) (0.49–0.94) (0.42–0.89) (0.14–0.66) (0.23–1.22) (1.56–2.90) (0.32–0.93) (0.00–0.60) (0.56–3.65) (1.02–2.17) 17.40 10.00 7.98 0.36 0.21 0.71 0.66 0.40 0.73 2.23 0.62 0.30 2.11 1.60 19.89 (18.16–21.02) 1.16 (0.94–1.38) P 0.79 (0.61–0.97) Post-intervention (0.04–0.20) (0.27–0.59) (0.26–0.81) (0.37–2.67) (0.87–3.80) (0.57–2.04) (0.36–0.87) (0.25–1.48) (1.46–7.83) (0.88–2.00) (16.20–18.36) (9.33–11.16) (7.11–8.11) (0.35–0.67) 21.41 (19.90–22.92) 1.07 (0.95–1.19) 0.12 0.43 0.53 1.52 2.34 1.30 0.61 0.87 4.65 1.44 17.28 10.25 7.61 0.51 0.68 (0.47–0.90) Pre-intervention (0.02–0.06) (0.19–0.43) (0.17–0.49) (0.00–0.77) (0.00–0.97) (0.87–1.53) (0.30–0.90) (0.00–0.33) (0.37–2.83) (0.89–1.50) 21.05 (19.53–22.57) 1.15 (1.01–1.29) 0.04 0.31 0.33 0.39 0.34 1.20 0.60 0.15 1.60 1.20 0.668 0.553 0.088 0.008 0.869 0.616 0.295 0.764 0.260 0.109 0.989 0.354 0.0002 0.0001 0.173 0.099 15.43 8.42 7.44 0.22 (13.91–16.95) (7.41–9.43) (6.61–8.27) (0.15–0.30) 0.044 P 0.49 (0.37–0.61)b Post-intervention Heavy intervention group Antimicrobial prescription considered in total, by authorised administration route, and by priority classiﬁcation; the latter also being summarised by main presenting complaint. Also included are P value outputs of a series of mixed effects panel regression models, modelling prescription category against intervention group, pre- and post-intervention. Full regression model outputs are available in Supplementary Tables 2 (Antimicrobial prescription), 7 (HPCIA prescription by main presenting complaint) and 9 (other prescriptions). Multiple authorised administration route antimicrobials can be prescribed in a single consultation. Data is derived from 88,298 pre-intervention and 81,582 post-intervention CG consultations; 107,223 pre-intervention and 105,518 post-intervention LIG consultations, and 63,366 pre-intervention and 63,301 post-intervention HIG consultations. aHighest priority critically important antimicrobial. bEmboldened text refers to ﬁndings which were found to be signiﬁcantly different on comparison to the control group. Primary outcome HPCIAa 0.71 (0.57–0.86) 0.64 (0.47–0.82) 0.72 (0.60–0.83) Antimicrobial prescription—% of total consultations (95% conﬁdence interval, CI) Total 17.44 (16.23–18.65) 17.48 (16.02–18.95) 17.44 (16.40–18.48) Systemic 10.49 (9.56–11.42) 10.51 (9.45–11.58) 10.32 (9.43–11.22) Topical 7.58 (6.95–8.22) 7.57 (6.83–8.31) 7.79 (7.39–8.18) Systemic HPCIA 0.40 (0.29–0.52) 0.32 (0.21–0.43) 0.39 (0.34–0.44) HPCIA prescription by main presenting complaint—% of relevant consultations (95% CI) Vaccination 0.10 (0.06–0.14) 0.13 (0.06–0.19) 0.15 (0.10–0.19) Other healthy 0.70 (0.45–0.95) 0.72 (0.37–1.07) 0.47 (0.30–0.63) Post-operative check 0.75 (0.15–1.36) 0.65 (0.21–1.10) 0.62 (0.38–0.87) Gastroenteric 0.60 (0.25–0.95) 0.09 (0.00–0.21) 0.59 (0.30–0.88) Respiratory 2.05 (0.67–3.43) 1.56 (0.17–2.96) 1.88 (0.99–2.78) Pruritus 1.69 (0.87–2.51) 1.23 (0.60–1.86) 2.39 (1.57–3.22) Trauma 0.55 (0.25–0.86) 0.25 (0.06–0.45) 0.49 (0.24–0.74) Tumour 0.71 (0.38–1.04) 0.68 (0.19–1.16) 0.62 (0.11–1.14) Kidney disease 2.06 (0.16–3.97) 1.03 (0.00–2.52) 1.22 (0.00–2.59) Other unwell 1.39 (1.11–1.68) 1.32 (1.05–1.58) 1.58 (1.08–2.08) Other prescriptions—% of total consultations (95% CI) Anti-inﬂammatory 20.26 (18.99–21.54) 20.62 (19.46–21.79) 19.91 (18.86–20.95) Euthanasia 0.99 (0.80–1.18) 1.04 (0.81–1.26) 1.09 (0.90–1.29) CANINE Variable Table 2 Canine antimicrobial and anti-inﬂammatory prescription, and euthanasia as a percentage of total canine consultations by intervention group, split into pre-intervention (August 2018–March 2019) and post-intervention (April–November 2019) phases. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 ARTICLE NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications 5 6 Pre-intervention Pre-intervention Post-intervention Light intervention group Control group 0.030 0.103 0.098 0.297 0.026 0.933 0.179 0.742 0.672 0.691 0.311 0.202 0.059 0.420 0.382 0.321 0.684 (13.35–16.20) (10.64–13.36) (2.90–3.45) (5.19–6.91) (0.54–1.74) (3.89–6.23) (2.45–4.38) (1.29–5.08) (12.92–22.95) (10.77–16.69) (17.89–28.27) (5.86–11.38) (7.05–13.12) (9.94–13.21) 14.78 12.00 3.18 6.05 1.14 5.06 3.42 3.18 17.93 13.73 23.08 8.62 10.08 11.58 17.81 (16.68–18.93) 2.33 (1.97–2.68) P 6.14 (5.29–6.99)b Post-intervention (0.35–2.36) (4.29–7.55) (3.05–6.03) (4.00–8.88) (24.15–40.23) (14.05–25.35) (24.71–33.66) (8.80–17.68) (11.67–19.71) (13.01–16.97) (14.34–16.18) (11.91–13.58) (2.62–3.28) (7.08–8.82) 18.67 (17.06–20.28) 2.13 (1.89–2.38) 1.35 5.92 4.54 6.44 32.19 19.70 29.19 13.24 15.69 14.99 15.26 12.75 2.95 7.95 8.03 (7.16–8.90) Pre-intervention (0.39–0.78) (2.16–5.04) (1.83–4.39) (0.94–3.61) (7.54–19.38) (5.73–13.41) (9.63–18.28) (4.10–10.39) (4.87–13.79) (6.14–10.12) (12.59–15.52) (9.93–12.68) (2.84–3.40) (3.31–5.20) 18.71 (17.16–20.25) 2.29 (1.89–2.70) 0.59 3.60 3.11 2.27 13.46 9.57 13.96 7.25 9.83 8.13 14.06 11.31 3.12 4.25 4.35 (3.41–5.29) Post-intervention Heavy intervention group 0.578 0.645 0.090 0.001 0.833 0.306 0.062 0.066 0.001 0.095 0.412 0.001 0.002 0.002 0.073 <0.0001d <0.0001c P Antimicrobial prescription considered in total, by authorised administration route, and by priority classiﬁcation; the latter also being summarised by main presenting complaint. Also included are P value outputs of a series of mixed effects panel regression models, modelling prescription category against intervention group, pre- and post-intervention. Full regression model outputs are available in Supplementary Tables 3 (Antimicrobial prescription), 8 (HPCIA prescription by main presenting complaint) and 9 (other prescriptions). Multiple authorised administration route antimicrobials can be prescribed in a single consultation. Data is derived from 33,613 pre-intervention and 30,632 post-intervention CG consultations; 39,803 pre-intervention and 37,339 post-intervention LIG consultations, and 25,661 preintervention and 25,808 post-intervention HIG consultations. aHighest priority critically important antimicrobial. bEmboldened text refers to ﬁndings which were found to be signiﬁcantly different on comparison to the control group. c0.0000006. d0.0000005. Primary outcome HPCIAa 7.44 (6.27–8.61) 7.37 (6.02–8.72) 7.03 (6.32–7.74) Antimicrobial prescription—% of total consultations (95% conﬁdence interval, CI) Total 16.68 (15.19–18.17) 16.62 (15.35–17.88) 14.97 (13.46–16.49) Systemic 14.08 (12.60–15.55) 13.84 (12.61–15.08) 12.43 (11.13–13.74) Topical 3.03 (2.70–3.35) 3.27 (2.94–3.60) 2.98 (2.64–3.33) Systemic HPCIA 7.35 (6.12–8.58) 7.29 (5.98–8.59) 6.93 (6.23–7.63) HPCIA prescription by main presenting complaint—% of relevant consultations (95% CI) Vaccination 0.76 (0.34–1.19) 1.04 (0.54–1.55) 1.02 (0.51–1.52) Other healthy 5.17 (3.42–6.93) 6.29 (4.34–8.25) 5.65 (4.49–6.82) Post-operative check 5.81 (1.88–9.75) 5.65 (0.96–10.35) 3.50 (2.06–4.94) Gastroenteric 4.86 (3.19–6.53) 4.26 (1.78–6.74) 4.45 (2.34–6.56) Respiratory 27.35 (20.07–34.63) 25.05 (14.19–35.90) 24.99 (19.21–30.77) Pruritus 15.71 (11.25–20.17) 14.93 (9.64–20.22) 17.76 (13.63–21.90) Trauma 28.86 (22.65–35.08) 26.96 (21.66–32.26) 27.39 (24.18–30.60) Tumour 13.20 (10.09–16.32) 13.77 (9.30–18.23) 12.03 (9.25–14.82) Kidney disease 15.28 (7.27–23.28) 15.10 (6.75–23.45) 13.02 (8.34–17.69) Other unwell 14.13 (11.79–16.47) 12.98 (10.98–14.91) 13.25 (11.49–15.01) Other prescriptions—% of total consultations (95% CI) Anti-inﬂammatory 19.03 (17.12–20.93) 19.46 (18.17–20.76) 17.50 (16.23–18.76) Euthanasia 2.14 (1.64–2.65) 2.15 (1.64–2.65) 2.29 (1.99–2.58) FELINE Variable Table 3 Feline antimicrobial and anti-inﬂammatory prescription, and euthanasia as a percentage of total feline consultations by intervention group, split into pre-intervention (August 2018–March 2019) and post-intervention (April–November 2019) phases. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 Fig. 2 Canine highest priority critically important antimicrobial prescription frequency. Canine highest priority critically important antimicrobial (HPCIA) prescription frequency as a percentage of total consultations, measured by (a) practice pre-intervention (August 2018–March 2019 inclusive) and post-intervention (April–November 2019 inclusive), and (b) month. Lines in plot a refer to linear regression ﬁts, modelling intervention status by practice, with lines referring to regression ﬁt estimates around a 95% conﬁdence interval (shaded region). An asterisk refers to the two practices in the HIG which declined to participate in the post-benchmarking intervention reﬂection and education programme. Lines in plot b refer to HPCIA prescription frequency as a percentage of total consultations; the red solid line in both plots a and b refers to ﬁndings from the control group (CG); the blue hashed line refers to the light intervention group (LIG), and the green dotted line refers to the heavy intervention group (HIG). The orange dashed line in plot b shows the month at which the initial notiﬁcation took place, and the orange solid box outlines the months in which the HIG could access further support if requested. Shaded regions refer to 95% conﬁdence intervals. Data is derived from 88,298 preintervention and 81,582 post-intervention CG consultations; 107,223 preintervention and 105,518 post-intervention LIG consultations, and 63,366 pre-intervention and 63,301 post-intervention HIG consultations. ARTICLE Fig. 3 Feline highest priority critically important antimicrobial prescription frequency. Feline highest priority critically important antimicrobial (HPCIA) prescription frequency as a percentage of total consultations, measured by (a) practice pre-intervention (August 2018–March 2019 inclusive) and post-intervention (April–November 2019 inclusive), and (b) month. Lines in plot a refer to linear regression ﬁts, modelling intervention status by practice, with lines referring to regression ﬁt estimates around a 95% conﬁdence interval (shaded region). An asterisk refers to practices in the HIG which declined to participate in the postbenchmarking intervention reﬂection and education programme. Lines in plot b refer to HPCIA prescription frequency as a percentage of total consultations; the red solid line in both plots a and b refers to ﬁndings from the control group; the blue hashed line refers to the light intervention group, and the green dotted line refers to the heavy intervention group (HIG). The orange dashed line in panel b shows the month at which the initial notiﬁcation took place, and the orange solid box outlines the months in which the HIG could access further support if requested. Shaded regions refer to 95% conﬁdence intervals. Data is derived from 33,613 preintervention and 30,632 post-intervention CG consultations; 39,803 preintervention and 37,339 post-intervention LIG consultations, and 25,661 pre-intervention and 25,808 post-intervention HIG consultations. NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 Table 4 Canine and feline HPCIA prescription as a percentage of total consultations, summarised by month between August 2018 and November 2019. Month CANINE August, 2018 (pre-intervention) September, 2018 October, 2018 November, 2018 December, 2018 January, 2019 February, 2019 March, 2019 April, 2019 (post-intervention) May, 2019 June, 2019 July, 2019 August, 2019 September, 2019 October, 2019 November, 2019 FELINE August, 2018 (pre-intervention) September, 2018 October, 2018 November, 2018 December, 2018 January, 2019 February, 2019 March, 2019 April, 2019 (post-intervention) May, 2019 June, 2019 July, 2019 August, 2019 September, 2019 October, 2019 November, 2019 Control group Light intervention group Heavy intervention group HPCIAa % (CI)b HPCIA % (CI) P HPCIA % (CI) 0.73 (0.48–0.98) 0.71 (0.48–0.94) 0.78 (0.53–1.02) 0.67 (0.54–0.81) 1.00 (0.70–1.30) 0.62 (0.41–0.84) 0.75 (0.52–0.99) 0.49 (0.29–0.70) 0.60 (0.36–0.85) 0.88 (0.62–1.14) 0.63 (0.37–0.89) 0.43 (0.26–0.60) 0.79 (0.54–1.04) 0.66 (0.42–0.90) 0.61 (0.43–0.78) 0.53 (0.25–0.81) 0.79 (0.62–0.95) 0.77 (0.62–0.92) 0.64 (0.51–0.76) 0.66 (0.49–0.83) 0.93 (0.69–1.16) 0.84 (0.64–1.05) 0.55 (0.38–0.72) 0.56 (0.42–0.70) 0.76 (0.52–1.00) 0.78 (0.54–1.03) 0.71 (0.52–0.90) 0.69 (0.48–0.89) 0.95 (0.74–1.16) 0.77 (0.52–1.01) 0.90 (0.68–1.11) 0.78 (0.52–1.04) 0.369 0.452 0.174 1.000 0.639 0.501 0.164 0.986 0.909 0.338 0.437 0.536 0.568 0.382 0.335 0.912 0.62 (0.37–0.88) 0.65 (0.41–0.90) 0.56 (0.34–0.78) 0.79 (0.53–1.05) 0.83 (0.51–1.15) 0.68 (0.32–1.03) 0.82 (0.45–1.18)c 0.59 (0.39–0.80) 0.49 (0.33–0.64) 0.46 (0.26–0.66) 0.49 (0.27–0.72) 0.56 (0.36–0.77) 0.37 (0.18–0.56) 0.38 (0.23–0.53) 0.65 (0.41–0.89) 0.50 (0.24–0.76) 0.623 0.741 0.122 0.780 0.513 0.129 0.043 0.418 0.222 0.022 0.220 0.399 0.005 0.042 0.910 0.322 8.49 (7.42–9.57) 7.60 (5.95–9.25) 6.81 (5.55–8.07) 7.01 (5.93–8.08) 7.77 (5.65–9.89) 6.83 (5.22–8.44) 7.86 (6.10–9.62) 7.42 (6.10–8.75) 8.10 (6.50–9.69) 7.68 (6.36–9.00) 8.35 (6.00–10.70) 6.86 (5.29–8.43) 7.49 (6.03–8.95) 6.95 (5.61–8.30) 6.44 (5.48–7.40) 7.07 (5.30–8.83) 7.90 (7.16–8.63) 7.40 (6.45–8.35) 7.54 (6.24–8.83) 6.78 (5.66–7.90) 7.63 (6.21–9.06) 5.44 (4.51–6.37) 7.03 (6.09–7.98) 6.37 (5.58–7.15) 6.71 (5.53–7.89) 5.98 (4.76–7.20) 6.69 (5.43–7.94) 6.34 (5.43–7.25) 6.47 (5.52–7.42) 6.19 (4.79–7.60) 5.51 (4.70–6.33) 5.16 (4.26–6.07) 0.337 0.799 0.857 0.574 0.626 0.312 0.060 0.557 0.325 0.195 0.033 0.788 0.110 0.740 0.265 0.019 8.47 (7.41–9.53) 8.70 (7.31–10.09) 8.03 (6.73–9.33) 7.14 (5.95–8.32) 8.31 (6.88–9.73) 7.74 (6.73–8.76) 7.65 (6.47–8.82) 8.29 (6.93–9.64) 4.88 (3.84–5.92) 4.77 (3.32–6.22) 3.97 (2.82–5.13) 4.05 (2.99–5.11) 4.28 (2.72–5.83) 4.84 (3.15–6.52) 4.27 (3.41–5.12) 3.86 (2.80–4.92) 0.878 0.308 0.770 0.394 0.397 0.874 0.166 0.572 0.001 0.001 <0.0001d 0.005 <0.0001e 0.006 0.020 0.0006 P P value outputs of mixed effects panel regression, modelling intervention group against month are also included. Full regression model outputs are available in Supplementary Tables 4 (dogs) and 5 (cats). Data is derived from 169,880 canine and 64,245 feline CG consultations; 212,741 canine and 67,142 feline LIG consultations, and 126,667 canine and 51,469 feline HIG consultations. aHighest priority critically important antimicrobial. b95% Conﬁdence interval. cEmboldened text refers to ﬁndings which were found to be signiﬁcantly different on comparison to the control group. d0.000002. e0.00004. antimicrobial stewardship scheme, led by RCVS Knowledge (https:// knowledge.rcvs.org.uk/home/), ﬁrmly demonstrating a professionwide commitment to responsible usage of antimicrobials. In the LIG and HIG, use of the SAVSNET antimicrobial benchmarking portal signiﬁcantly increased post-notiﬁcation, with increases being most apparent in the 2 months following notiﬁcation, suggesting that notiﬁcations informing practitioners of their relatively “unusual” status in relation to HPCIA prescription frequency prompted enhanced portal engagement. However, while post-intervention 80% of HIG practices interacted with the portal, only 40% of LIG practices did so. Furthermore, while engagement waned to at or below preintervention levels within two months of notiﬁcation in the LIG, interest exceeded pre-intervention levels for 6 months postnotiﬁcation in the HIG. It is probable that either the additional in-depth benchmarking report provided to practices in the HIG, or post-notiﬁcation offer of assistance from the hub clinical leads might have enhanced initial interest compared to the LIG. Though the relative contributions of each was not able to be elucidated here, individuals are more likely to re-evaluate existing 8 behaviours if modifying behaviour might bring reward, or not doing so might bring punishment40. Although the supportive, optional nature of the trial was emphasised throughout, requested hub clinical lead intercession might have nevertheless introduced a perception of potential reward or punishment linked with engagement with the trial. It is also possible that the letter and email sent to LIG practices was not disseminated beyond clinical directors in some cases, whereas the opportunity for practicewide meetings held in HIG practices encouraged the engagement of more staff. It was not possible to distinguish between individual practice staff members interacting with the benchmarking portal; hence, engagement might have been limited to a single or few member(s) of each practice. There was concern that notiﬁcation of relatively high HPCIA prescription alone could prompt practice policy changes not reﬂective of latest clinical evidence, such changes being potentially detrimental to animal welfare or employee wellbeing. Structured reﬂection and education programmes have been shown to be effective at achieving sustained improvements in anti-infective prescription habits in the medical ﬁeld33, and in this NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications 0.38 2.75 18.03 3.96 0.03 6.83 0.20 7.12 0.05 1.25 0.99 0.09 2.92 13.39 3.89 0.03 7.46 0.19 12.56 5.50 8.28 44.84 4.00 73.93 20.73 0.07 1.10 (4.16–6.84) (6.22–10.33) (41.34–48.34) (0.11–7.89) (69.43–78.43) (16.50–24.95) (0.01–0.13) (0.65–1.54) – (0.00–0.24) (2.18–3.66) (11.78–15.00) (2.69–5.09) (0.00–0.05) (5.83–9.09) (0.07–0.32) (10.68–14.44) – (0.41–1.56) Post-intervention 0.30 2.97 19.40 2.73 0.04 5.52 0.29 8.86 0.02 0.71 10.60 5.38 43.48 4.58 73.31 20.43 0.06 1.31 (8.08–13.13) (3.44–7.31) (40.85–46.11) (2.21–6.94) (68.49–78.14) (15.65–25.22) (0.00–0.13) (0.83–1.80) – (0.00–0.62) (2.32–3.62) (18.24–20.56) (2.06–3.40) (0.01–0.08) (3.73–7.31) (0.06–0.51) (6.71–11.00) (0.00–0.05) (0.46–0.95) (4.34–7.99) (5.23–9.51) (41.97–48.32) (1.84–6.33) (71.08–82.60) (12.70–22.40) (0.00–0.12) (0.85–1.84) (0.00–0.03) (0.00–0.36) (2.57–4.49) (13.06–17.01) (1.61–2.79) (0.01–0.06) (4.53–7.72) (0.08–0.60) (11.28–15.61) – 0.61 (0.38–0.84) 6.17 7.37 45.15 4.09 76.84 17.55 0.05 1.35 0.01 0.15 3.53 15.03 2.20 0.03 6.13 0.34 13.45 Post-intervention Pre-intervention (9.00–12.88) (4.88–9.39) (37.35–46.07) (0.58–9.97) (69.84–77.76) (14.87–22.83) (0.00–0.19) (1.13–2.30) – (0.00–0.92) (2.17–3.33) (16.35–19.71) (2.76–5.17) (0.01–0.06) (4.96–8.70) (0.05–0.34) (5.29–8.95) (0.00–0.11) (0.81–1.70) Pre-intervention 10.94 7.14 41.71 5.27 73.80 18.85 0.10 1.71 Light intervention group % (95% CI) Control group % (95% CI)a P 0.444 0.618 0.259 0.684 0.178 0.711 1.000 0.165 0.296 0.644 0.086 0.209 0.397 0.423 0.824 0.280 0.137 1.000 0.612 9.81 6.60 41.87 6.83 74.46 16.13 0.29 2.24 0.02 0.09 2.50 19.97 3.55 0.03 6.21 0.44 8.25 0.06 0.69 (7.71–11.90) (4.21–8.99) (39.20–44.53) (1.49–12.18) (69.46–79.46) (13.14–19.11) (0.03–0.54) (1.61–2.86) (0.00–0.05) (0.00–0.26) (1.51–3.49) (18.74–21.19) (2.32–4.78) (0.00–0.06) (4.27–8.15) (0.00–1.10) (7.12–9.38) (0.00–0.13) (0.38–1.00) Pre-intervention 2.54 18.02 3.20 0.02 5.65 0.21 12.76 0.05 0.81 6.04 8.03 41.83 5.36 78.44 15.17 0.02 0.97 (3.11–8.97) (4.85–11.21) (38.81–44.86) (1.54–9.18) (72.21–84.68) (11.23–19.11) (0.00–0.06) (0.50–1.45) – – (1.88–3.21) (15.82–20.21) (2.35–4.04) (0.00–0.04) (4.28–8.83) (0.00–0.46) (10.88–14.65) (0.00–0.12) (0.45–1.17) Post-intervention Heavy intervention group % (95% CI) P 0.699 0.281 0.204 0.849 0.242 0.125 0.482 0.355 0.117 0.729 0.253 0.862 0.182 0.593 0.209 0.813 0.253 0.232 0.317 Also included are P value outputs of a series of mixed effects panel regression models, modelling antimicrobial class or sub-class against intervention group, pre- and post-intervention. Full regression model outputs are available in Supplementary Table 10. Data is derived from 18,599 pre-intervention and 15,966 post-intervention CG antimicrobial prescriptions; 22,678 pre-intervention and 20,827 post-intervention LIG antimicrobial prescriptions, and 13,199 pre-intervention and 11,024 post-intervention HIG antimicrobial prescriptions. a95% Conﬁdence interval. bFindings presented in italics are expressed as a percentage of total beta-lactam prescription within intervention groups i.e., in the control group pre-intervention, amoxicillin comprised 5.3% of beta-lactam prescriptions. cHighest Priority Critically Important Antimicrobial. Aminoglycoside Amphenicol Beta-lactamb Amoxicillin Clavulanic acid potentiated amoxicillin 1st generation cephalosporin 2nd generation cephalosporin 3rd generation cephalosporin c Penicillin Other beta-lactams Fluoroquinolonec Fusidic acid Lincosamide Macrolidec Nitroimidazole Nitroimidazole-macrolide Other antimicrobials Sulphonamide Tetracycline CANINE Antimicrobial class Table 5 Canine antimicrobial prescription choice as a percentage of total antimicrobial prescriptions, presented by class and for beta-lactams, by sub-class, and by intervention group split into pre- and post-intervention time periods. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications ARTICLE 9 10 1.35 1.11 0.32 2.37 0.96 10.50 3.06 1.10 1.16 9.19 2.91 0.02 1.08 0.30 4.11 1.89 3.42 74.82 6.76 40.12 0.84 0.05 52.09 0.10 (0.97–2.81) (1.76–5.08) (71.43–78.20) (1.20–12.32) (31.32–48.93) (0.47–1.21) (0.00–0.14) (42.00–62.17) (0.00–0.32) – (0.88–1.44) (7.65–10.73) (1.67–4.16) (0.00–0.06) (0.60–1.56) (0.10–0.50) (3.09–5.13) – (0.38–1.83) Post-intervention 0.59 0.12 2.99 0.01 1.22 1.55 11.53 3.04 3.95 1.91 73.08 6.95 37.07 1.31 0.04 54.57 0.02 (2.77–5.13) (1.48–2.35) (71.04–75.12) (2.49–11.41) (30.73–43.40) (0.33–2.29) (0.00–0.13) (45.64–63.51) (0.00–0.05) – (1.10–2.01) (10.36–12.70) (1.14–4.93) – (0.31–0.87) (0.02–0.21) (2.45–3.53) (0.00–0.04) (0.81–1.63) 1.02 0.75 0.11 4.62 1.29 11.58 2.93 2.31 1.91 73.49 6.73 43.17 1.47 0.02 48.72 (1.38–3.23) (1.44–2.40) (70.69–76.29) (2.31–11.16) (36.18–50.15) (0.54–2.40) (0.00–0.07) (41.97–55.48) – – (0.92–1.66) (9.92–13.24) (1.02–4.83) – (0.50–1.00) (0.02–0.20) (3.82–5.42) – (0.61–1.42) Post-intervention Pre-intervention (2.54–4.18) (0.80–4.20) (69.92–79.14) (1.96–16.25) (28.44–47.16) (0.43–1.00) (0.00–0.06) (42.80–62.09) (0.00–0.15) – (0.56–1.36) (9.00–12.01) (1.61–4.51) – (0.57–1.66) (0.01–0.63) (1.60–3.13) – (0.41–2.28) Pre-intervention 3.36 2.50 74.53 9.11 37.80 0.72 0.02 52.45 0.06 Light intervention group % (95% CI) Control group % (95% CI)a P 0.515 0.318 0.742 0.931 0.636 0.257 0.612 0.908 0.182 – 0.708 0.217 0.808 0.317 0.509 0.347 0.300 – 0.961 1.31 3.14 11.79 2.81 0.02 1.44 0.31 2.99 63.11 0.03 3.62 1.92 70.63 6.84 29.14 0.96 (2.74–4.49) (1.07–2.77) (67.22–74.05) (3.70–9.99) (23.72–34.57) (0.19–1.72) – (57.45–68.77) (0.00–0.09) – (1.00–5.29) (10.68–12.91) (1.88–3.75) (0.00–0.07) (0.91–1.97) (0.00–0.66) (2.39–3.59) – (0.29–2.33) Pre-intervention 1.52 1.47 11.82 3.37 0.03 1.21 0.18 3.80 2.25 3.05 71.29 9.33 52.59 1.59 0.07 36.43 (1.55–2.95) (1.87–4.22) (68.72–73.86) (4.86–13.80) (43.32–61.87) (0.42–2.75) (0.00–0.16) (29.10–43.75) – – (0.87–2.07) (10.03–13.61) (2.23–4.52) (0.00–0.08) (0.75–1.68) (0.00–0.40) (2.98–4.62) – (0.64–2.40) Post-intervention Heavy intervention group % (95% CI) P 0.837 0.363 0.277 0.957 0.776 0.715 1.000 0.057 0.182 – 0.894 0.880 0.808 1.000 0.637 0.347 0.198 – 0.942 Also included are P value outputs of a series of mixed effects panel regression models, modelling antimicrobial class or sub-class against intervention group, pre- and post-intervention. Full regression model outputs are available in Supplementary Table 11. Data is derived from 6296 pre-intervention and 5575 post-intervention CG antimicrobial prescriptions; 6762 pre-intervention and 6183 post-intervention LIG antimicrobial prescriptions, and 4351 pre-intervention and 4107 post-intervention HIG antimicrobial prescriptions. Also included are P value outputs of a series of mixed effects panel regression models, modelling antimicrobial class or sub-class against intervention group, pre- and post-intervention. Full regression model outputs are available in Supplementary Table 11. Data is derived from 6296 pre-intervention and 5575 post-intervention CG antimicrobial prescriptions; 6762 pre-intervention and 6183 post-intervention LIG antimicrobial prescriptions, and 4351 pre-intervention and 4107 post-intervention HIG antimicrobial prescriptions. a95% Conﬁdence interval. bHighest Priority Critically Important Antimicrobial. cFindings presented in italics are expressed as a percentage of total beta-lactam prescription within intervention groups i.e., in the control group pre-intervention, amoxicillin comprised 9.1% of beta-lactam prescriptions. Aminoglycoside Amphenicol Beta-lactamb Amoxicillin Clavulanic acid potentiated amoxicillin 1st generation cephalosporin 2nd generation cephalosporin 3rd generation cephalosporinc Penicillin Other beta-lactams Fluoroquinolonec Fusidic acid Lincosamide Macrolidec Nitroimidazole Nitroimidazole-macrolide Other antimicrobials Sulphonamide Tetracycline FELINE Antimicrobial class Table 6 Feline antimicrobial prescription choice as a percentage of total antimicrobial prescriptions, presented by class and for beta-lactams, by sub-class, and by intervention group split into pre- and post-intervention time periods. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 Fig. 4 SAVSNET antimicrobial prescription benchmarking portal engagement. Number of practices logging into the SAVSNET antimicrobial prescription benchmarking portal by month (February–November 2019 inclusive) and intervention group. The purple boxes refer to ﬁndings from the control group (n = 20 practices); the blue boxes refer to the light intervention group (n = 20 practices), and the green orange boxes refer to the heavy intervention group (HIG, n = 20 practices). The pink shaded region shows the month at which the initial notiﬁcation took place. trial we compared both a light (LIG) and heavy (HIG) reﬂection and educational intervention. While signiﬁcant reductions in HPCIA prescription frequency were seen in both species in the HIG, signiﬁcant decreases were only observed in the LIG for cats. Across the veterinary sector impressive reductions have been achieved over the past ﬁve years, especially in pigs and poultry41,42, utilising a variety of statutory43 and voluntary44 improvement measures. Though over this time reductions in antimicrobial prescription frequency in companion animals have been noted10,12,14, HPCIA use has remained an issue, particularly in cats10,11,17. Unlike other veterinary sectors, no statutory policies have been introduced to prompt improvements in antimicrobial prescription in companion animals. It is unknown what impact such enforced measures might have on animal welfare, and thus we consider ﬁndings presented here to be an encouraging sign that practitioners might be willing to voluntarily engage with improvement efforts, potentially negating need for ﬁrmer regulatory approaches. This study further demonstrated the relative ease by which EHRs can be utilised to both identify participants and monitor key outcomes in near real-time. Such efﬁciency advantages have been previously outlined in medical research, enabling rapid scaling of interventions to instigate national quality improvement34. Only comparatively recently have EHRs become available for research and surveillance in the veterinary sector45, and we believe this work serves as a promising demonstration of what could be achieved using EHR data-led approaches, expanding beyond practitioner-focused interventions to those encompassing owners, or pragmatic efﬁcacy assessment of surgical and pharmaceutical interventions in routine practice, for example. Though the intervention package provided in this trial represented a comprehensive approach to encouraging evidence-based behaviour change, it did limit our ability to determine which individual components might have been of greatest impact. Interestingly, though both LIG and HIG practices received a high feline HPCIA prescription frequency notiﬁcation at the beginning ARTICLE of the trial, reductions in the LIG were more modest compared to the HIG, where all HIG practices, including both HIG practices that refused engagement with the reﬂection and education programme, reported decreases post-notiﬁcation. Though variation in scale of reduction was evident, circumstantially, these ﬁndings might suggest that hub clinical lead involvement was a motivational factor in prompting behavioural change. In either case, the aforementioned refusals do indicate a limiting factor in intervention scalability to a wider audience. Furthermore, this trial beneﬁted from utilising existing quality improvement management structures within a single large practice group for the HIG. Thus, there remains a question as to whether this intervention would be feasible amongst other practices, including those that are relatively infrequent HPCIA prescribers, over a longer period of time than the eight months observed here. Though the scale of feline HPCIA prescription frequency reduction in the LIG was approximately half that of the HIG, LIG interventions would arguably be more amenable to national deployment. Ensuring widespread efﬁcient dissemination, even if such efforts produce a smaller overall effect, might therefore prove preferable compared to high intensity, smaller scale action. However, there are many unique practice management systems utilised in the UK, and securing SAVSNET software compliance with all systems presents somewhat of a barrier to wider participation in either intervention approach at the current time. While it was encouraging to see no immediate reversion to preintervention prescribing rates in the two un-supported months where hub clinical leads did not interact with HIG practices, we regard this as insufﬁcient evidence to demonstrate “sustained” improvements at this time, and would recommend longer followup periods in the future. Some MPCs in cats and dogs in the HIG were associated with signiﬁcant HPCIA prescription frequency reductions, some of which (e.g., trauma) were previously associated with frequent HPCIA prescription10,24, despite often lacking clear clinical justiﬁcation for their prescription24. These ﬁndings suggest a generalised culture change not necessarily being restricted to reﬂection on individual disease presentations. This result is supported by signiﬁcant reductions in systemic and overall antimicrobial prescription frequency in both species. In HIG dogs, these wider reductions were greater than that contributed by HPCIA reductions alone, suggesting that the trial had a wider impact on discouraging antimicrobial prescription more generally. However, in HIG cats the opposite was seen; HPCIA reductions were greater than overall decreases, suggesting a tendency of some practitioners to move from prescribing a HPCIA to prescribing another non-HPCIA antimicrobial, instead of avoiding prescription altogether. Whilst no prescription choice comparisons were signiﬁcantly different, 3rd generation cephalosporin feline prescription does appear to have decreased to a degree in the HIG, while clavulanic acid potentiated amoxicillin prescription increased, suggesting a preferred alternative to 3rd generation cephalosporins. Clavulanic acid potentiated amoxicillin is an authorised, widely used antimicrobial in veterinary practice10. However, like 3rd generation cephalosporins, use of clavulanic acid potentiated amoxicillin has also been associated with resistance development4, is considered of ‘high’ importance by the WHO16, and accordingly is only infrequently prescribed to humans in the UK46. It is therefore possible that in seeking to reduce risk for development of resistance in response to HPCIA prescription that this trial has inadvertently enhanced or at least sustained resistance development risk for broader beta-lactam resistance. Thus, future stewardship efforts will need to expand scope beyond HPCIAs to also consider how to promote responsible use of all antimicrobials, and indeed other medicines too. NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 For instance, we have previously reported a tendency for antimicrobials and anti-inﬂammatories to be prescribed at the same time, despite perhaps limited clinical evidence to suggest necessity for both pharmaceutical agents15. However, we have also noted a recent reversing trend for respiratory disease whereby antimicrobial prescription frequency has decreased whilst anti-inﬂammatory prescription frequency has increased12. These ﬁndings perhaps reﬂect increasing recognition of nonbacterial mediators for respiratory disease47, or increased attention to prescribing guidance18. It was interesting to note that no signiﬁcant variation in anti-inﬂammatory prescription was observed here, perhaps demonstrating more generalised “decoupling” of anti-inﬂammatory and antimicrobial prescription. Though measuring frequency of use represents a relatively simple method for demonstrating change, reduced use is not necessarily representative of more responsible use. Hence, it is probable that more nuanced methods for ascertaining whether a pharmaceutical agent has been prescribed appropriately will be needed, such as consideration of speciﬁc clinical signs and/or diagnostic test ﬁndings at point of prescription. As part of these developments, we would advocate increased attention on use of other pharmaceutical agents that might form effective alternatives to antimicrobial prescription, whilst also satisfying the recognised need of a practitioner to provide a clear demonstration of action via provision of a therapeutic product to the client19. Though prescribers retained full autonomy to prescribe what they considered best for the animal under their care, we incorporated euthanasia frequency as a relatively crude measure of any increase in adverse health effects associated with change in prescription decision-making prompted by this trial. While no signiﬁcant increases were observed in either intervention group, compared to the CG, we recognise that this method lacks sensitivity, not taking into account a range of potential sub-optimal outcomes that might compromise animal welfare. Though effectively and efﬁciently quantifying such adverse effects from EHRs at the scale required for this trial presents a signiﬁcant challenge48, we recommend further development of text-mining and statistical methodologies to explore such nuances for subsequent trials. Use of bacterial infection-associated diagnostic tests was not signiﬁcantly altered in this trial. Low frequency of use of such tests has been identiﬁed as a barrier to effective stewardship49, likely reﬂecting low conﬁdence in their ability to provide timely, useful clinical insights50. Indeed, across all groups test orders were low, indicating a preference for empirical antimicrobial prescription throughout the trial. Used correctly, these tests do play an important role in correctly managing a patient51; however, there is clearly more work needed to convince practitioners —and owners—of the beneﬁts of regularly pursuing these diagnostic routes. That said, during this trial a lack of equipment and training for cytological examinations within practices was identiﬁed, which resulted in wide-scale equipment and training provision (unpublished observations). Thus, there is hope that signiﬁcant impact will be generated beyond the conﬁnes of this trial. To conclude, in this trial we outlined a data-led benchmarking, reﬂection and education antimicrobial stewardship framework that successfully reduced HPCIA, systemic and overall antimicrobial prescription frequency in dogs and cats in practices belonging to a single large practice group. However, whilst initially encouraging, further work is required to understand the relative impact of different antimicrobials on conferring clinically meaningful resistance, and how to incentivise increased use of diagnostic testing in preference to empirical antimicrobial prescription. This work provides a robust evidence base for future antimicrobial stewardship interventions in companion animal 12 practice, and ﬁndings are now being used to inform development of a national stewardship scheme, in collaboration with RCVS Knowledge and CVS Group (UK) Limited. Methods Data collection. This trial used data collected by the Small Animal Veterinary Surveillance Network (SAVSNET) project, which harnesses voluntarily provided EHR from booked consultations in a sentinel network of UK veterinary practices, held in a SQL database and queried via Microsoft SQL Server Management Studio 18. Each EHR includes anonymised information pertaining to the animal and owner, the clinical narrative, and products dispensed during such consultations. Antimicrobial prescription was identiﬁed via reference to products dispensed, and classiﬁed into systemic (oral or injectable) or topical (topical, aural, ocular) authorised administration routes using a semi-automated rule-based text-mining method, using the Veterinary Medicine Directorate’s Product Information Database and the electronic Medicines Compendium (Datapharm Communications) as a guide for veterinary and human-authorised products, respectively10. Fluoroquinolones, macrolides and 3rd generation cephalosporins were considered HPCIAs16. Every consultation was further classiﬁed by the attending veterinary professional into one of ten main presenting complaints (MPCs), indicating the main reason the animal was presented to the veterinary practice10. In addition, CVS Group Ltd. provided data relating to staff numbers per practice (in full-time equivalents, FTE), and the number of cytological or bacterial culture and susceptibility tests ordered by practices included in this trial. SAVSNET holds ethical approval to collect EHR data from the University of Liverpool (ethical approval reference: RETH000964); additional approval was granted to encompass interventions and data collection speciﬁc to this trial from the Universities’ Veterinary Research Ethics Committee (ethical approval reference: VREC745). Study practice selection. This three-armed randomised controlled trial (RCT) initially utilised EHRs voluntarily supplied by 157 UK veterinary practices (385 sites/branches) belonging to CVS Group Ltd., that had been participating in SAVSNET between 1 August 2018 and 15 January 2019. Pre-intervention, practicelevel median HPCIA prescription as a percentage of total canine (n = 409,279) and feline (n = 164,827) consultations were 0.5% [range 0.0–2.3] and 5.3% [range 0.0–13.9], respectively. Practices were eligible for trial inclusion if they were within the 40% most frequent total HPCIA (including systemic and topical formulations) prescribing bracket as a percentage of total consultations in both dogs (in excess of 0.5% of canine consultations) and cats (in excess of 5.4% of feline consultations) (n practices = 43). Practices where either cats (n practices = 17) or dogs (n practices = 8) were placed in the 40% most frequent total HPCIA prescribing bracket, with the opposing species being placed in the 40-60% most frequent total HPCIA prescribing bracket [dogs, range 0.4-0.5%; cats, 4.5-5.3%], were also considered eligible. The primary outcome was HPCIA prescription frequency post-intervention, compared to the CG. A sample size estimation indicated that to detect a 10% relative decrease in the primary outcome (standard deviation = 10%, power = 80% and α = 0.05), 17 practices would be required in each group. Hence, of the 68 initially eligible practices, 60 practices were randomly and evenly allocated into three intervention groups: the control group (CG, n practices=20, sites=40), low group (LIG, n practices=20, sites=57) or high group (HIG, n practices=20, sites=51) by block random allocation, utilising the ‘complete random allocation’ function available through the ‘randomizr’ R package version 0.20.0 (Fig. 1)52. Practice allocation was completed by DS. Intervention. For the LIG and HIG, the trial consisted of two phases: (1) an initial notiﬁcation of above median HPCIA prescription frequency status, followed by (2) a voluntary reﬂection and education programme, with intensity varying by intervention group. On 28 March 2019, LIG practices received a posted letter and email (both addressed to the clinical director of that practice) stating their above average HPCIA-prescribing status (Supplementary Note 1). They also received a copy of the practice group’s antimicrobial prescribing policy (based on current prescribing guidance)18, a reminder and interpretive guidance for an online anonymised antimicrobial prescription benchmarking portal already freely available to all SAVSNET-participating practices (Supplementary Note 2)30, and access to AMR educational videos (can be viewed here: https://savsnetvet.liverpool.ac.uk/ savsnetamr/iv?id=61). Practices could opt-out of or access these as many times as they wished, and clinical directors were able to distribute these materials amongst staff members as they saw ﬁt; they were not prompted to do so. On 28 and 29 March 2019, HIG practices also received a posted letter and email (both addressed to the clinical director of that practice) stating their above average HPCIA-prescribing frequency status. This letter included all LIG materials, and further included an in-depth benchmarking report (Supplementary Note 3) and explanatory video, in addition to the videos available to LIG practices (can be viewed here: https://savsnetvet.liverpool.ac.uk/savsnetamr/iv?id=62). Practices were further invited, via their clinical director, to participate in a reﬂection and education programme delivered by a “hub clinical lead” (a member of the senior clinical team recruited internally by CVS Group Ltd. based on clinical experience). NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 This programme consisted of an initial in-person review between a hub clinical lead and the clinical director and/or head veterinary surgeon of the practice; presence of additional members of staff were left to the discretion of each practice. Reviews were structured around a series of questions aimed at identifying factors (e.g., stafﬁng, equipment, attitudes) that might have contributed to their relatively high HPCIA prescription frequency status (full question list available: Supplementary Note 4). Staff were able to suggest their own action points, and were encouraged to hold a separate practice-wide meeting to discuss ﬁndings. Hub clinical leads checked in by email with HIG practices on a monthly basis following initial intervention, reminding practices to arrange the aforementioned practice-wide meeting if not already done so. Practices could also hold additional follow-up reviews with the hub clinical lead if they wished. A ﬁnal hub clinical lead review near conclusion of the study was also requested, each utilising a development of the initial contributory factors question sheet (Supplementary Note 4). Though individual LIG and HIG practices were aware of their involvement in a trial, they were not informed of which other practices were involved, nor interventions being performed in opposing intervention groups. The CG received no intervention beyond sustained access to the antimicrobial prescription benchmarking portal through SAVSNET, and remained unaware of their involvement in this trial. Presence and frequency of SAVSNET prescription benchmarking portal access was monitored throughout the trial for all practices included in this trial. It was not practical to blind study team members to group allocation. Outcomes. Post-intervention monitoring was carried out between 1 April 2019 and 30 November 2019 (inclusive). The primary outcome measure was postintervention canine or feline total HPCIA (including systemic and topical formulations) prescription frequency as a percentage of total consultations. Secondary outcome measures included post-intervention total, systemic and topical antimicrobials; systemic HPCIA prescription frequency; total HPCIA prescription frequency by month; total HPCIA prescription frequency by MPC; relative antimicrobial class prescription frequency; anti-inﬂammatory prescription, and euthanasia frequency. Post-intervention cytological or bacterial culture and susceptibility test order numbers, and interactions with the online antimicrobial prescription benchmarking portal were also summarised. Statistical analyses. The statistical programme “R” was used for all analyses (version 4.0.3). Descriptive proportions and 95% conﬁdence intervals were adjusted for clustering within practices (bootstrap method, n = 5,000 samples)53. Baseline characteristic variation between practices within each intervention group were explored via Kruskal-Wallis tests. Mixed effects panel regression models, modelling practice as the random effect, were used for making intervention group comparisons, utilising the R package ‘plm’ (version 3.2-5)54. For each of the outcome measures described, practice-level pre-intervention (August 2018 – March 2019) and post-intervention (April – November 2019) values were compared with intervention group (modelled as interacting variables). For total HPCIA prescription frequency, intervention group was also compared across all pre- and post-intervention months. The CG was used as the reference category for all analyses. Intervention groups were also individually modelled via an orthogonal polynomial method to analyse temporal trend, considering up to quartic ﬁts (Supplementary Table 1)55. Model assumptions were met for all models; goodness of ﬁt was assessed via comparison against a null model. The R package “ggplot2” (version 3.3.2)56 was used for all visualisations, with the ‘lm’ function being used to produce linear regression lines in Figs. 2 and 3. Analyses were performed on an intention-to-treat basis, as EHR data were available for all practices regardless of participation opt-out status. Number of practices within each group interacting with SAVSNET’s antimicrobial prescription benchmarking portal were compared pre- and post-intervention via a two-sided Fisher’s Exact Test; post-hoc pairwise Fisher’s test comparisons were also completed utilising the ‘pairwiseNominalIndependence’ function available via the R package “rcompanion” (version 2.3.26)57. The trial was completed according to CONSORT guidelines58, and statistical signiﬁcance was deﬁned as P < 0.05 throughout. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 23. Data availability 24. Source data are provided with this paper. Other data that support the ﬁndings of this study are available from the corresponding author (D.A.S.) on reasonable request. Some of the data are not publicly available due to them containing information that could compromise research participant privacy. Source data are provided with this paper. Received: 24 July 2020; Accepted: 12 February 2021; 25. 26. Rantala, M. et al. Antimicrobial resistance in Staphylococcus spp., Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs. Acta Vet. Scand. 45, 37–45 (2004). Trott, D. J. et al. Canine model for investigating the impact of oral enroﬂoxacin on commensal coliforms and colonization with multidrugresistant Escherichia coli. J. Med Microbiol 53, 439–443 (2004). Guardabassi, L., Schwarz, S. & Lloyd, D. H. Pet animals as reservoirs of antimicrobial-resistant bacteria. J. Antimicrob. Chemother. 54, 321–332 (2004). Schmidt, V. M. et al. Routine antibiotic therapy in dogs increases the detection of antimicrobial-resistant faecal Escherichia coli. J. Antimicrob. Chemother. (2018), https://doi.org/10.1093/jac/dky352. O’Neill, J. Tackling drug-resistant infections globally: ﬁnal report and recommendations. http://amr-review.org/home (2016). Cuny, C., Wieler, L. H. & Witte, W. Livestock-associated MRSA: the Impact on Humans. Antibiot 4, 521–543 (2015). Cantón, R. & Bryan, J. Global antimicrobial resistance: from surveillance to stewardship. Part 1: surveillance and risk factors for resistance. Expert Rev. Anti. Infect. Ther. 10, 1269–1271 3p (2012). Lei, L. et al. mcr-1 in enterobacteriaceae from companion animals, Beijing, China, 2012–2016. Emerg. Infect. Dis. 23, 710–711 (2017). Guardabassi, L., Loeber, M. E. & Jacobson, A. Transmission of multiple antimicrobial-resistant Staphylococcus intermedius between dogs affected by deep pyoderma and their owners. Vet. Microbiol 98, 23–27 (2004). Singleton, D. A. et al. Patterns of antimicrobial agent prescription in a sentinel population of canine and feline veterinary practices in the United Kingdom. Vet. J. 224, 18–24 (2017). Buckland, E. L. et al. Characterisation of antimicrobial usage in cats and dogs attending UK primary care companion animal veterinary practices. Vet. Rec. 179, 489 (2016). Singleton, D. A. et al. Small animal disease surveillance 2019: respiratory disease, antibiotic prescription and canine infectious respiratory disease complex. Vet. Rec. 184, 640–645 (2019). Singleton, D. A. et al. Small animal disease surveillance 2019: pruritus, pharmacosurveillance, skin tumours and ﬂea infestations. Vet. Rec. 185, 470–475 (2019). Singleton, D. A. et al. Small animal disease surveillance: gastrointestinal disease, antibacterial prescription and Tritrichomonas foetus. Vet. Rec. 184, 211–216 (2019). Singleton, D. et al. New approaches to pharmacosurveillance for monitoring prescription frequency, diversity, and co-prescription in a large sentinel network of companion animal veterinary practices in the United Kingdom, 2014–2016. Prev. Vet. Med. 159, 153–161 (2018). WHO. Critically important antimicrobials for human medicine. https://www. who.int/foodsafety/areas_work/antimicrobial-resistance/cia/en/ (2019). Burke, S. et al. Use of cefovecin in a UK population of cats attending ﬁrstopinion practices as recorded in electronic health records. J. Feline Med Surg. 19, 687–692 (2017). BSAVA. BSAVA/SAMSoc guide to responsible use of antibacterials: PROTECT ME. https://www.bsavalibrary.com/content/book/10.22233/ 9781910443644 (2018). Mateus, A. L., Brodbelt, D. C., Barber, N. & Stark, K. D. Qualitative study of factors associated with antimicrobial usage in seven small animal veterinary practices in the UK. Prev. Vet. Med 117, 68–78 (2014). Dickson, A. et al. Understanding the relationship between pet owners and their companion animals as a key context for antimicrobial resistance-related behaviours: an interpretative phenomenological analysis. Heal. Psychol. Behav. Med. 7, 45–61 (2019). King, C. et al. Exploring the behavioural drivers of veterinary surgeon antibiotic prescribing: a qualitative study of companion animal veterinary surgeons in the UK. BMC Vet. Res. 14, 332 (2018). Smith, M. et al. Pet owner and vet interactions: exploring the drivers of AMR. Antimicrob. Resist. Infect. Control 7, 46 (2018). Tompson, A. C. et al. What drives antimicrobial prescribing for companion animals? A mixed-methods study of UK veterinary clinics. Prev. Vet. Med. 183, 105117 (2020). Singleton, D. A. et al. Pharmaceutical prescription in canine acute diarrhoea: a longitudinal electronic health record analysis of ﬁrst opinion veterinary practices. Front. Vet. Sci. 6, 218–232 (2019). Singleton, D. A. et al. A large multi-centre study utilising electronic health records to identify antimicrobial prescription risk factors for dogs and cats. Emerg. Infect. Dis. 26, 1778–1791 (2020). Beco, L. et al. Suggested guidelines for using systemic antimicrobials in bacterial skin infections: part 2— antimicrobial choice, treatment regimens and compliance. Vet. Rec. 172, 156–160 (2013). NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21864-3 27. Lappin, M. R. et al. Antimicrobial use Guidelines for Treatment of Respiratory Tract Disease in Dogs and Cats: Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases. J. Vet. Intern. Med. 31, 279–294 (2017). 28. Hubbuch, A. et al. Antimicrobial prescriptions in cats in Switzerland before and after the introduction of an online antimicrobial stewardship tool. BMC Vet. Res. 16, 229 (2020). 29. Lutz, B. et al. Antimicrobial prescriptions and adherence to prudent use guidelines for selected canine diseases in Switzerland in 2016. Vet. Rec. Open 7, e000370 (2020). 30. Radford, A. et al. Prescribing antibiotics in small animals practices [2]. Vet. Rec. 181, 71 (2017). 31. Hopman, N. E. M. et al. Implementation and evaluation of an antimicrobial stewardship programme in companion animal clinics: A stepped-wedge design intervention study. PLoS One 14, e0225124 (2019). 32. Martinez-Gonzalez, N. A. et al. The impact of interventions to improve the quality of prescribing and use of antibiotics in primary care patients with respiratory tract infections: a systematic review protocol. BMJ Open 7, e016253 (2017). 33. Thakkar, K. et al. A quality improvement programme to increase compliance with an anti-infective prescribing policy. J. Antimicrob. Chemother. 66, 1916–1920 (2011). 34. Hallsworth, M. et al. Provision of social norm feedback to high prescribers of antibiotics in general practice: a pragmatic national randomised controlled trial. Lancet 387, 1743–1752 (2016). 35. Gjelstad, S. et al. Improving antibiotic prescribing in acute respiratory tract infections: cluster randomised trial from Norwegian general practice (prescription peer academic detailing (Rx-PAD) study. BMJ 347, f4403 (2013). 36. Le Corvoisier, P. et al. Long-term effects of an educational seminar on antibiotic prescribing by GPs: a randomised controlled trial. Br. J. Gen. Pract. 63, e455–e464 (2013). 37. Gerber, J. S. et al. Effect of an outpatient antimicrobial stewardship intervention on broad-spectrum antibiotic prescribing by primary care pediatricians: a randomized trial. JAMA 309, 2345–2352 (2013). 38. Rognstad, S. et al. Characteristics of GPs responding to an educational intervention to minimise inappropriate prescriptions: subgroup analyses of the Rx-PAD study. BJGP Open 2, bjgpopen18X101373 (2018). 39. Hagman, B. T., Noel, N. E. & Clifford, P. R. Social Norms Theory-Based Interventions: Testing the Feasibility of a Purported Mechanism of Action. J. Am. Coll. Heal. 56, 293–298 (2007). 40. Aunger, R. & Curtis, V. Behaviour centred design: towards an applied science of behaviour change. Health Psychol. Rev. 10, 425–446 (2016). 41. VMD. Joint report on antibiotic use and antibiotic resistance, 2013–2017. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/ attachment_data/ﬁle/775075/One_Health_Report_2019_v45.pdf (2019). 42. VMD. 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Fluoroquinolones to treat uncomplicated acute cough in primary care: predictors for unjustiﬁed prescribing of antibiotics. J. Antimicrob. Chemother. 65, 1521–1525 (2010). 48. Anholt, R. M., Berezowski, J., Jamal, I., Ribble, C. & Stephen, C. Mining freetext medical records for companion animal enteric syndrome surveillance. Prev. Vet. Med 113, 417–422 (2014). 49. Currie, K., King, C., Nuttall, T., Smith, M. & Flowers, P. Expert consensus regarding drivers of antimicrobial stewardship in companion animal veterinary practice: a Delphi study. Vet. Rec. 182, 691 (2018). 50. De Briyne, N., Atkinson, J., Pokludova, L., Borriello, S. P. & Price, S. Factors inﬂuencing antibiotic prescribing habits and use of sensitivity testing amongst veterinarians in Europe. Vet. Rec. 173, 475 (2013). 51. Nuttall, T. Bacterial isolation and antimicrobial susceptibility trends: why these are important and how they can be used. Vet. Rec. 183, 19–20 (2018). 52. Coppock, A. Randomizr. (2019). 53. AOD. AOD R Packages. (2016). 14 54. Croissant, Y. & Millo, G. Panel data econometrics in R: The plm package. (2020). 55. UCLA. R library contrast coding systems for categorical variables. https://stats. idre.ucla.edu/r/library/r-library-contrast-coding-systems-for-categoricalvariables/ (2020). 56. H., W. ggplot2: elegant graphics for data analysis. https://ggplot2.tidyverse.org (2020). 57. Mangiaﬁco, S. S. Summary and analysis of extension program evaluation in R, version 1.18.1. https://rcompanion.org/handbook/ (2016). 58. Moher, D. et al. CONSORT 2010 explanation and elaboration: updated guidelines for reporting parallel group randomised trials. BMJ 340, c869 (2010). Acknowledgements SAVSNET is grateful for the support and major funding of BBSRC, the Dogs Trust, and BSAVA. CVS Group (UK) Limited provided support in-kind for this research via creation and postage of physical materials, and through secondment of existing staff onto this project. We particularly thank Marcus Evans, Richard Killen and Joe Williams. We also acknowledge the signiﬁcant time investment of the hub clinical leads (Kate Allgood, Lisa Baker, Sinead Bennett, Rachel Clay, Ryan Davis, Elizabeth McLennan-Green, Mark Moreton and Nicola Rolph) involved in implementing many of the HIG interventions. More broadly, we thank data providers in veterinary practice (VetSolutions, Teleos, CVS and independent practitioners) and participating veterinary diagnostic laboratories (Axiom Veterinary Laboratories, Batt Laboratories, BioBest, Idexx, NationWide Laboratories Microbiology Diagnostics Laboratory at the University of Liverpool, the Department of Pathology and Infectious Diseases at the University of Surrey, and the Veterinary Pathology Group). Finally, we are grateful for the help and support provided by SAVSNET team administrator Susan Bolan. Author contributions D.A.S. and G.L.P. conceived of and devised the trial design and prepared trial materials, including intervention letters, educational videos and benchmarking reports. D.A.S. collated and prepared data, conducted analysis and wrote the manuscript, with supervision from G.L.P.. P.J.M.N. and A.D.R. supported trial design, and provided oversight of manuscript drafts. B.B. initially recruited all trial practices onto the SAVSNET project, and provided communications support between SAVSNET and trial practices throughout. S.S. provided database support, created online platforms for educational videos to be viewed, and devised the practice antimicrobial benchmarking portal monitoring capability. A.R. assisted trial design, acted as a liaison between SAVSNET and CVS Group Ltd., and trained HCLs in how to correctly implement devised interventions. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-21864-3. Correspondence and requests for materials should be addressed to D.A.S. Peer review information Nature Communications thanks David Brodbelt and the other, anonymous, reviewer for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2021 NATURE COMMUNICATIONS \| (2021)12:1593 \| https://doi.org/10.1038/s41467-021-21864-3 \| www.nature.com/naturecommunications
https://openalex.org/W2915338759	http://scielo.iics.una.py/pdf/anales/v51n3/1816-8949-anales-51-03-13.pdf	Spanish; Castilian	null	Donation and transplant of organs in Paraguay	Anales de la Facultad de Ciencias Médicas	2,018	cc-by	2,607	Donación y trasplante de órganos en Paraguay Arellano, Nelson1 1 Jefe Unidad Trasplante Hepático. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. 1 Jefe Unidad Trasplante Hepático. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. La palabra donación adquiere su verdadera importancia cuando se la asocia a la palabra trasplante y representa la solidaridad dentro de una sociedad. de su humanismo al tratar a los estigmatizados pacientes con SIDA, habiendo acuñado la frase “Hay que atacar la enfermedad y no a los enfermos”, misión que continúan sus allegados gracias a la materialización de uno de sus sueños, la Fundación de lucha contra el SIDA que lleva su nombre (2). Una sociedad donde interactuamos todos sus integrantes y dependemos unos de otros. Para ilustrar la dependencia de unos y otros, permítanme hablarles de tres protagonistas, que representan a otros tantos, que no se conocen y sin saberlo forman parte de una misma historia, unidos como eslabones de una cadena. Séneca a través de una de sus citaciones, con una vigencia que sorprende, nos permite recordar y honrar la memoria del Dr. Marco Aguayo «Cuando el sol se eclipsa para desaparecer, se ve mejor su grandeza» (3). En el momento en que se envió ese primer mensaje de solidaridad, ese primer regalo de vida por el Dr. Aguayo y su familia, tal vez nuestra sociedad no estaba preparada para escuchar. Esta es la historia de la Donación y Trasplante en el Paraguay, donde tres protagonistas: el donante, el paciente en lista de espera y el paciente trasplantado, que representan tres situaciones diferentes, en épocas distintas, están unidos sin embargo con un solo fin, LA VIDA. - El paciente en lista de espera, es el segundo protagonista de esta historia. Ana Almirón Riquelme, una niña, es el segundo eslabón de esta cadena, fallecía en abril del año 2013, aquejada por una enfermedad cardiaca terminal. Con tan solo 6 años de edad, había repetido el mismo mensaje de solidaridad, durante casi dos años, estando en lista de espera, mensaje enviado una primera vez dos décadas atrás por la familia Aguayo, a la misma sociedad, autoridades, médicos, conciudadanos, que se rehusaban a escuchar. - El donante, primer protagonista, primer eslabón: en el mes de septiembre de 1992, sucedía lo irreversible, tras una hemorragia cerebral, un colega, el Dr. An. Fac. Cienc. Méd. (Asunción) / Vol. 51 - Nº 3, 2018 http://dx.doi.org/10.18004/anales/2018.051(03)13-016 Autor correspondiente: Prof. Dr. Nelson Arellano, Jefe Unidad de Trasplante Hepático. Hospital de Clínicas. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. Email: narellanodr@hotmail.fr Fecha de recepción el 20 de Diciembre del 2018; aceptado el 26 de Diciembre del 2018. Donación y trasplante de órganos en Paraguay Germán Martínez Vierci, periodista, compatriota el, no encontrando respuestas en su país, se ve obligado a viajar al extranjero para beneficiar de un trasplante hepático, sumándose de esta manera el desarraigo a lo grave de su estado de salud, una enfermedad que no conoce ni respeta edades, sexos, religiones ni clases sociales. La citada ley, reglamenta no solamente la donación, sino también el trasplante, demostrando como si fuera necesario, que aislada la donación no tiene significado, así como los eslabones de una única cadena, de una misma historia que debe contar con todos sus actores para que sea completa. El Sr. Martínez Vierci, agradecido por esta nueva oportunidad de vida que le ofrecía el trasplante permitiéndole seguir disfrutando de su familia, inició, impulsó y orquestó con una precisión quirúrgica la “Ley Anita”, promulgada en el 2018, intentando con ello evitar que compatriotas sigan padeciendo de las insuficiencias de una estructura de salud que no ofrece soluciones adecuadas a problemas tan complejos, teniendo como copartícipes de este proyecto Docentes dedicados al trasplante de la Facultad de Medicina de la UNA, colegas de otras instituciones, pacientes en lista de espera así como pacientes trasplantados, encontrando en el Parlamento de nuestra nación la caja de resonancia ideal. En cuanto a los profesionales de la salud dedicados a la Donación y al Trasplante, soy un convencido de que somos simplemente actores secundarios, intermediarios privilegiados de esta historia de máxima expresión de amor y solidaridad entre dos seres humanos, dos familias, dentro de una sociedad, donde una persona le ofrece a otra, sin conocerla y sin pedir nada a cambio la posibilidad de seguir viviendo. Finalmente, deberíamos decidir como ciudadanos de una sociedad que se quiere solidaria, si deseamos ser protagonistas de esta que es nuestra historia, o ser simplemente observadores y críticos al costado del camino. Donación y trasplante de órganos en Paraguay Marco Aurelio Aguayo Rodríguez, de 33 años, egresado de nuestra bicentenaria alma mater (1), especializado en el exterior en infectología, se convertía en el primer donante en nuestro país. Su familia en medio del profundo dolor que les embargaba, nos ofrecía el primer regalo de vida y al mismo tiempo enviaba un mensaje silencioso de solidaridad a la sociedad paraguaya. “Anita” con su hermosa e inocente sonrisa esperó largos e interminables meses, su espera fue demasiado larga por un regalo de vida que nunca llegó. El Dr. Aguayo, que nos había dado muestras El Dr. Aguayo, que nos había dado muestras Autor correspondiente: Prof. Dr. Nelson Arellano, Jefe Unidad de Trasplante Hepático. Hospital de Clínicas. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. Email: narellanodr@hotmail.fr Fecha de recepción el 20 de Diciembre del 2018; aceptado el 26 de Diciembre del 2018. 13 Arellano N et al • Donación y trasplante de órganos en Paraguay An. Fac. Cienc. Méd. (Asunción) / Vol. 51 - Nº 3, 2018 Arellano N et al • Donación y trasplante de órganos en Paraguay An. Fac. Cienc. Méd. (Asunción) / Vol. 51 - Nº 3, 2018 Arellano N et al • Donación y trasplante de órganos en Paraguay An. Fac. Cienc. Méd. (Asunción) / Vol. 51 - Nº 3, 2018 cada sonrisa de una madre que ve a su hijo recuperado lo será también. Sus padres, inconsolables por la pérdida, nos seguían mostrando el camino a seguir, decidiendo donar las corneas, transformándose Anita de esta manera, de paciente en lista de espera a donante, ofreciendo lo que nuestra sociedad le había negado. Sus padres, inconsolables por la pérdida, nos seguían mostrando el camino a seguir, decidiendo donar las corneas, transformándose Anita de esta manera, de paciente en lista de espera a donante, ofreciendo lo que nuestra sociedad le había negado. La “Ley Anita”, que lejos de ser una ley coercitiva, simplemente nos recuerda que como partes integrantes de una sociedad, tenemos derechos pero al mismo tiempo obligaciones, y debemos de tomar un minuto de nuestro tiempo, discutir en familia y tomar una decisión: ser donantes o no. - El paciente trasplantado, es el tercer protagonista de esta historia, el Sr. Donación y trasplante de órganos en Paraguay Tal vez sea muy pronto para dimensionar los cambios que se generaran a partir de esta nueva ley en la vida de muchos de nuestros conciudadanos aquejados por una enfermedad terminal, tal vez no se vea la calma que nos embarga al despertar una pequeña luz de esperanza a los profesionales dedicados al trasplante, desesperados por una realidad que era difícil de entender, donde el día a día nos quebraba un poco más y nos hacía interrogarnos si nuestros sueños no eran simplemente una quimera, en un país que no merece y sin embargo se encuentra asfixiado por los vicios propios del ser humano. Prof. Dr. Nelson Arellano - Editor Invitado REFERENCIAS BIBLIOGRAFICAS Y si todos fuéramos “Anita” Prof. Dr. Nelson Arellano - Editor Invitado Prof. Dr. Nelson Arellano - Editor Invitado Donation and transplant of organs in Paraguay Arellano, Nelson Jefe Unidad Trasplante Hepático. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. Jefe Unidad Trasplante Hepático. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. and not the sick”, a mission that his close ones continue through one of his dreams, Anti- AIDS Foundation that bears his name (2). The word donation acquires its true importance when it is associated with the word transplant and represents solidarity within a society. A society where all its members interact and we depend on each other. Seneca through one of his quotes, with a surprising long-lasting force, allows us to remember and honor the memory of Dr. Marco Aguayo «When the sun is eclipsed to disappear, you can see its greatness» (3). To illustrate the dependence, allow me to talk about three protagonists, who represent many others, who do not know each other and unknowingly form part of the same story, united as links of a chain. At the time that first message of solidarity was sent, that first gift of life by Dr. Aguayo and his family, perhaps our society was not prepared to listen. This is the story of Donation and Transplant in Paraguay, where three protagonists: the donor, the patient on the waiting list and the transplanted patient, who represent three different situations, at different times, are nevertheless united with a single purpose, LIFE. - The patient on the waiting list is the second protagonist of this story. Ana Almirón Riquelme, a girl, is the second link of this chain, she died in April 2013, suffering from a terminal heart disease. With only 6 years old, she repeated the same message of solidarity for almost two years being on the waiting list, message sent a first time two decades ago by the Aguayo family, to the same society, authorities, doctors and fellow citizens, who refused to listen. - The donor, first protagonist, first link: in September 1992, the irreversible happened, after a brain hemorrhage, a colleague, Dr. Marco Aurelio Aguayo Rodríguez, 33 years old, a graduated of our bicentennial alma mater (1), specialized abroad in infectology, became the first donor in our country. “Anita” with her beautiful and innocent smile waited for long and endless months, she waited for too long for a gift of life that never came. REFERENCIAS BIBLIOGRAFICAS 1. http://dle.rae.es/?w=alma+mater 1. http://dle.rae.es/?w=alma+mater 2. https://www.facebook.com/fundacion.marcoaguayo 3. El libro de los Valores / The Book of Values. Ilustrado por Sandra Ardila. Ediciones Robinbook, 2005. ISBN 9788493423056. p. 157. Todo lo que se pudo hacer, será algún día olvidado y no tiene verdaderamente importancia, lo realmente importante es que para mucha gente habrá un antes y un después de la “Ley Anita”, cada vida que vuelva a nacer luego de una donación será una nueva victoria, 14 An. Fac. Cienc. Méd. (Asunción) / Vol. 51 - Nº 3, 2018 Autor correspondiente: Prof. Dr. Nelson Arellano, Jefe Unidad de Trasplante Hepático. Hospital de Clínicas. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. Email: narellanodr@hotmail.fr Fecha de recepción el 20 de Diciembre del 2018; aceptado el 26 de Diciembre del 2018. Donation and transplant of organs in Paraguay His family in the midst of sorrow, offered us the first gift of life and at the same time sent a silent message of solidarity to the Paraguayan society. Her parents, devastated by their loss, kept showing us the path to follow, deciding to donate the girl’s corneas, transforming Anita, from patient on waiting list to donor, offering what our society had denied her. Dr. Aguayo, gave us profs of his humanism by treating the stigmatized patients with AIDS and by phrases like “We must attack the disease Autor correspondiente: Prof. Dr. Nelson Arellano, Jefe Unidad de Trasplante Hepático. Hospital de Clínicas. Facultad de Ciencias Médicas, Universidad Nacional de Asunción. San Lorenzo, Paraguay. Email: narellanodr@hotmail.fr Fecha de recepción el 20 de Diciembre del 2018; aceptado el 26 de Diciembre del 2018. 15 Santa Cruz F et al • Estudio preliminar de Infecciones Urinarias Intrahospitalarias en Salas ... An. Fac. Cienc. Méd. (Asunción) / Vol. 51 - Nº 3, 2018 Santa Cruz F et al • Estudio preliminar de Infecciones Urinarias Intrahospitalarias en Salas ... An. Fac. Cienc. Méd. (Asunción) / Vol. 51 - Nº 3, 2018 - The transplanted patient, is the third protagonist of this story, Mr. German Martínez Vierci, journalist and fellow citizen, not finding answers in his country, is forced to travel abroad for a liver transplant, adding the distance and displacement to his serious preexisting condition, a disease that does not discriminate ages, sexes, religions or social classes. The aforementioned law covers not only the donation, but also the transplant, demonstrating as if it were necessary, that the donation has no meaning without a transplant, as a chain of a single link, as a story without all of its characters. Regarding the health professionals dedicated to Donation and Transplant, I am convinced that we are simply supporting actors, privileged intermediaries of this maximum expression of love and solidarity between two human beings, two families, within one society, where one person, without knowing the person and without asking anything in return, offers another the opportunity to live. Mr. And if we were all “Anita” Prof. Dr. Nelson Arellano - Editor Invitado Prof. Dr. Nelson Arellano - Editor Invitado It may be too early to understand the changes that this law will generate in the lives of many of our fellow citizens suffering from a terminal illness, and maybe it isn´t evident the calm brought by a small light of hope that overwhelms us as professionals dedicated to organ transplant, within a desperate and difficult to understand reality, where the day by day broke us a little more each day and even made us wonder if our dreams were not just a pipe dream, in a non-deserving country suffocated by the vices of the human kind. Donation and transplant of organs in Paraguay Martinez Vierci, grateful for the new life opportunity offered to him in the form of a transplant that allowed him to continue enjoying his family, initiated, promoted and orchestrated with surgical precision the “Anita Law”, promulgated in 2018; all of this in the attempt to avoid the suffering of our people whom already suffer from the inadequacies of a health structure that did not offer adequate solutions for such complex problems; having as partners in this project Faculty of the School of Medicine of the University of Asuncion dedicated to transplants, colleagues from other institutions, patients on the waiting list as well as transplanted patients, finding in the Parliament of our nation the ideal auditorium. Finally, we should decide as citizens, if we want to be protagonists of our own history, member of a society that wants to be solidary or simply be observers and critics at the side of the road. BIBLIOGRAPHIC REFERENCES 1. http://dle.rae.es/?w=alma+mater 2. https://www.facebook.com/fundacion.marcoaguayo 3. The Book of Values / The Book of Values. Illustrated by Sandra Ardila. Editions Robinbook, 2005. ISBN 9788493423056. p. 157. 1. http://dle.rae.es/?w=alma+mater 2. https://www.facebook.com/fundacion.marcoaguayo 3. The Book of Values / The Book of Values. Illustrated by Sandra Ardila. Editions Robinbook, 2005. ISBN 9788493423056. p. 157. 1. http://dle.rae.es/?w=alma+mater 2. https://www.facebook.com/fundacion.marcoaguayo 3. The Book of Values / The Book of Values. Illustrated by Sandra Ardila. Editions Robinbook, 2005. ISBN 9788493423056. p. 157. Everything that could and could not be done, will one day be forgotten and it doesn´t really matter, the important thing is that for many people there will be a before and after the “Anita Law”. Every reborn person after a donation will be a new victory, every smile of a mother who sees her son recovered will be too. The “Anita Law”, far from being a coercive law, simply reminds us we are an integral parts of a society, we have rights but at the same time obligations, and we must take a minute of our time, and discuss as a family and make a decision: to be donors or not. 16
https://openalex.org/W2128866712	https://diposit.ub.edu/dspace/bitstream/2445/177124/1/114264.pdf	English	null	Insulin-induced Recruitment of Glucose Transporter 4 (GLUT4) and GLUT1 in Isolated Rat Cardiac Myocytes	Journal of biological chemistry/The Journal of biological chemistry	1,997	cc-by	9,423	Yvan Fischer‡§, Julia Thomas‡, Lidia Sevilla¶i, Purificacio´n Mun˜ oz¶, Christoph Becker‡, Geoffrey Holman‡‡, Izabela J. Kozka‡‡, Manuel Palacı´n¶, Xavier Testar¶, Helmut Kammermeier‡, and Antonio Zorzano¶ From the ‡Institute of Physiology, Medical Faculty, RWTH Aachen, Pauwelsstrasse 30, Aachen D-52057, Federal Republic of Germany, ¶Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain, and ‡‡Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom Yvan Fischer‡§, Julia Thomas‡, Lidia Sevilla¶i, Purificacio´n Mun˜ oz¶, Christoph Becker‡, Geoffrey Holman‡‡, Izabela J. Kozka‡‡, Manuel Palacı´n¶, Xavier Testar¶, Helmut Kammermeier‡, and Antonio Zorzano¶ From the ‡Institute of Physiology, Medical Faculty, RWTH Aachen, Pauwelsstrasse 30, Aachen D-52057, Federal Republic of Germany, ¶Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain, and ‡‡Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom Using isolated rat cardiomyocytes we have examined: 1) the effect of insulin on the cellular distribution of glucose transporter 4 (GLUT4) and GLUT1, 2) the total amount of these transporters, and 3) the co-localization of GLUT4, GLUT1, and secretory carrier membrane pro- teins (SCAMPs) in intracellular membranes. Insulin in- duced 5.7- and 2.7-fold increases in GLUT4 and GLUT1 at the cell surface, respectively, as determined by the non- permeant photoaffinity label [3H]2-N-[4(1-azi-2,2,2-trif- luoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)propyl-2- amine. The total amount of GLUT1, as determined by quantitative Western blot analysis of cell homogenates, was found to represent a substantial fraction (;30%) of the total glucose transporter content. Intracellular GLUT4-containing vesicles were immunoisolated from low density microsomes by using monoclonal anti- GLUT4 (1F8) or anti-SCAMP antibodies (3F8) coupled to either agarose or acrylamide. With these different im- munoisolation conditions two GLUT4 membrane pools were found in nonstimulated cells: one pool with a high proportion of GLUT4 and a low content in GLUT1 and SCAMP 39 (pool 1) and a second GLUT4 pool with a high content of GLUT1 and SCAMP 39 (pool 2). The existence of pool 1 was confirmed by immunotitration of intracel- lular GLUT4 membranes with 1F8-acrylamide. Acute in- sulin treatment caused the depletion of GLUT4 in both pools and of GLUT1 and SCAMP 39 in pool 2. In conclu- sion: 1) GLUT4 is the major glucose transporter to be recruited to the surface of cardiomyocytes in response to insulin; 2) these cells express a high level of GLUT1; and 3) intracellular GLUT4-containing vesicles consist of at least two populations, which is compatible with recently proposed models of GLUT4 trafficking in adipocytes. In mammalian cells, the facilitative uptake of glucose is mediated by a group of specialized glucose transporters (for reviews, see Refs. 1–4). 1 The abbreviations used are: GLUT, glucose transporter; SCAMP, secretory carrier membrane protein; LDM, low density microsome; PM, plasma membrane; ATB-BMPA, 2-N-[4(1-azi-2,2,2-trifluoroethyl)ben- zoyl]-1,3-bis-(D-mannos-4-yloxy)propyl-2-amine; PBS, phosphate-buff- ered saline. Vol. 272, No. 11, Issue of March 14, pp. 7085–7092, 1997 Printed in U.S.A. Vol. 272, No. 11, Issue of March 14, pp. 7085–7092, 1997 Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Yvan Fischer‡§, Julia Thomas‡, Lidia Sevilla¶i, Purificacio´n Mun˜ oz¶, Christoph Becker‡, Geoffrey Holman‡‡, Izabela J. Kozka‡‡, Manuel Palacı´n¶, Xavier Testar¶, Helmut Kammermeier‡, and Antonio Zorzano¶ From the ‡Institute of Physiology, Medical Faculty, RWTH Aachen, Pauwelsstrasse 30, Aachen D-52057, Federal Republic of Germany, ¶Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain, and ‡‡Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom Peripheral insulin-sensitive tissues, such as fat, skeletal muscles, and heart, express a unique transporter isoform (GLUT4),1 which is largely confined to an intracellular storage site in the basal, nonstimulated state and becomes recruited to the cell surface under the influence of insulin (5, 6) but also other stimuli, such as contraction (7–9) and hypoxia or anoxia (10, 11). This recruitment process is likely to account for a large part of the increase in the rate of glucose uptake observed on stimulation with these agents (12–16). It has been recently reported that mice expressing a defec- tive GLUT4 gene show cardiac hypertrophy (17), which sup- ports the view that GLUT4 is important for normal function and properties of cardiac myocytes. This fact emphasizes the necessity of a thorough understanding of the mechanisms in- volved in the control and function of this protein in heart tissue. An important issue is the delineation of the GLUT4 trafficking pathway(s) in cardiomyocytes. In this respect, im- munoelectron microscopy studies performed in the rat heart have observed that GLUT4 is localized in cardiac myocytes, under nonstimulated conditions, in small tubulovesicular ele- ments adjacent to the sarcolemma and the transverse tubular system and in the trans-Golgi region (18), and that insulin stimulates the recruitment of intracellular GLUT4 carriers to the sarcolemma and to the T-tubular system. However, there is no information on biochemical grounds regarding the charac- teristics of the intracellular GLUT4 pool(s) in cardiomyocytes and the proteins essential for recruitment to the cell surface in response to insulin. On the other hand, many cells types, such as endothelial cells and erythrocytes and also fat and muscle cells, contain another transporter isoform, GLUT1, which is thought to be at least in part responsible for the basal uptake of glucose (19, 20). In heart and skeletal muscle, the content of GLUT1 mRNA and protein was shown to largely decrease during postnatal devel- opment (21, 22), whereas the reverse is true for GLUT4 (21– 23). However, the adult heart still appears to contain consid- erable amounts of GLUT1, in contrast to skeletal muscles (24, 25). This paper is available on line at http://www-jbc.stanford.edu/jbc/ 7085 This paper is available on line at http://www-jbc.stanford.edu/jbc/ § To whom correspondence should be addressed. Tel.: 49-241- 8088811; Fax: 49-241-8888434; E-mail: yvan@physiology.rwth-aachen. de. Yvan Fischer‡§, Julia Thomas‡, Lidia Sevilla¶i, Purificacio´n Mun˜ oz¶, Christoph Becker‡, Geoffrey Holman‡‡, Izabela J. Kozka‡‡, Manuel Palacı´n¶, Xavier Testar¶, Helmut Kammermeier‡, and Antonio Zorzano¶ From the ‡Institute of Physiology, Medical Faculty, RWTH Aachen, Pauwelsstrasse 30, Aachen D-52057, Federal Republic of Germany, ¶Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain, and ‡‡Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom Moreover, recent studies have shown that in heart muscle cells GLUT1 is recruited to the plasma membrane by several * This work was supported in part by Grant Fi 551/1-2 from the Deutsche Forschungsgemeinschaft, Grant PB92/0805 from the Direc- cio´n General de Investigacio´n Cientı´fica y Te´cnica, Grant GRQ94-1040 from Generalitat de Catalunya, Spain, and Grants HA94-125 and HA95-125 from the Deutscher Akademischer Austauschdienst in con- junction with Acciones Integradas Hispano-Aleman˜as. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. i Recipient of a predoctoral fellowship from the Ministerio de Educa- cio´n y Ciencia, Spain. Supported in part by a grant from Fondo de Investigaciones Sanitarias. § To whom correspondence should be addressed. Tel.: 49-241- 8088811; Fax: 49-241-8888434; E-mail: yvan@physiology.rwth-aachen. de. y i Recipient of a predoctoral fellowship from the Ministerio de Educa- cio´n y Ciencia, Spain. Supported in part by a grant from Fondo de Investigaciones Sanitarias. MATERIALS AND METHODS Antibodies—Antisera directed against the C-terminal peptides of either GLUT1 or GLUT4 (and used to purify the photoaffinity-labeled transporters) were raised in rabbits in the laboratories of G. H. (GT1 and GT4), and A. Z. (OSCRX against GLUT4) or were a kind gift from Dr. Samuel W. Cushman and Dina R. Yver (Bethesda, MD; 9301 pA and 8105p aG4). A polyclonal antibody generated against the C terminus of GLUT1 obtained from Biogenesis Inc. was used for immunoblotting assays. Monoclonal antibodies 1F8 (against GLUT4) and 3F8 (against SCAMPs) were kindly provided by Dr. Paul F. Pilch (Boston Universi- ty). A rabbit polyclonal antibody against rat b1-integrin was kindly given by Dr. Carles Enrich (University of Barcelona) (30). Monoclonal antibody NCL-DYS 1 against the midrod of dystrophin was obtained from NovoCastra. The polyclonal antibody against the rat a1-subunit of the Na1-K1-ATPase was from Upstate Biotechnology Inc. (Lake Placid, NY). Polyclonal antibody 18B11, against TGN 38, was kindly given by Dr. Ignacio Sandoval (Centro de Biologia Molecular, Madrid, Spain). In some assays, antibodies 1F8 (5–7 mg) and 3F8 (3 mg) were incu- bated overnight at 4 °C with goat anti-mouse IgG or goat anti-mouse IgM coupled to agarose (75 ml of bead volume). Beads were collected by a 6-s spin in a Microfuge and washed in PBS. LDM preparations (15–25 mg of proteins) were incubated with 1F8- or 3F8-agarose overnight at 4 °C in the absence of detergents (0.1% bovine serum albumin and 1 mM EDTA in PBS; final volume, 200 ml). The agarose beads and vesicles bound to them were collected by a 6-s spin in a Microfuge. The vesicles that were bound to the immobilized antibody were washed in PBS. The adsorbed material was eluted with electrophoresis sample buffer. Isolation of Cardiomyocytes and Glucose Transport Assays—Car- diomyocytes from adult female Sprague-Dawley rats (180–220 g, fed ad libitum) were obtained as described previously (31). Treatment of car- diomyocytes for all experiments was performed in medium A containing 6 mM KCl, 1 mM Na2HPO4, 0.2 mM NaH2PO4, 1.4 mM MgSO4, 128 mM NaCl, 10 mM HEPES, 1 mM CaCl2, and 2% bovine serum albumin (fatty acid free, pH 7.4) at 37 °C, equilibrated with oxygen. The rate of 2-de- oxy-D-glucose uptake was determined as described elsewhere (31). MATERIALS AND METHODS Chemicals—125I-Protein A and 125I-sheep anti-mouse antibody were purchased from ICN (Meckenheim, Germany). 125I-Goat anti-mouse antibody, ECL, and 2-deoxy-D-[3H]glucose were from Amersham Corp. The photoaffinity label (3H-ATB-BMPA) used to quantify the glucose transporters was prepared as described elsewhere (29). All chemicals for media used for cell isolation, glucose transport assays, and labeling experiments were from Merck; antipain, a-hemolysin, protein A-Sepha- rose, g-globulin, goat-anti mouse IgG, and goat anti-mouse IgM coupled to agarose were obtained from Sigma; aprotinin, pepstatin, and leupep- tin were from ICN; ThesitTM and bovine serum albumin (fraction V, fatty acid free) were purchased from Boehringer Mannheim); purified bovine insulin was a kind gift from Prof. Axel Wollmer (Aachen, Ger- many). All chemicals were the highest purity grade available. Concen- trated stock solutions of insulin (in medium A, see below) were stored at 220 °C in appropriate aliquots and diluted just prior to addition to the isolated cardiomyocytes. Immobilon polyvinylidene difluoride was ob- tained from Millipore. All electrophoresis reagents and molecular weight markers were obtained from Bio-Rad. Preparation of Purified Membrane Fractions from Cardiomyocytes— Cardiomyocytes were incubated for 30 min at 37 °C in the presence or absence of insulin (10 nM) and were washed once with TES buffer and then immediately frozen in liquid nitrogen in a ratio of 107 cells/2.7 ml of TES. Membrane fractionation was performed as described previously (26). Protocols of Vesicle Immunoisolation—Protein A-purified monoclonal anti-GLUT4 antibody (1F8) or a corresponding amount of nonspecific antibodies (g-globulins) was coupled to acrylamide beads (Reacti-gel GF 2000, Pierce) at a concentration of 1 mg of antibody/ml of resin accord- ing to the manufacturer’s instructions. Before use, the beads were saturated with 1% bovine serum albumin in PBS (134 mM NaCl, 2.6 mM KCl, 6.4 mM Na2HPO4, and 1.46 KH2PO4, pH 7.4) for at least 30 min (at room temperature) and washed with PBS. Intracellular membranes (low density microsomes (LDMs)) were incubated with beads overnight at 4 °C (50 mg of LDMs, 20-ml beads). The beads were spun down; the supernatant was taken for later analysis; the beads were washed five times with PBS; and the adsorbed material was eluted with electro- phoresis sample buffer (0.1 M Tris-HCl, 20% glycerol, and 2% sodium dodecyl sulfate, pH 6.8), incubated for 5 min at 95 °C, cooled, and microcentrifuged. The supernatant fraction from the vesicle immuno- adsorption assay and the immunoadsorbed extract were subjected to immunoblot analysis. This is an Open Access article under the CC BY license. This is an Open Access article under the CC BY license. This is an Open Access article under the CC BY license. This is an Open Access article under the CC BY license. This is an Open Access article under the CC BY license. This is an Open Access article under the CC BY license. Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes 7086 glucose transporter recovery. First, larger amounts of anti-GLUT1 an- tisera (up to 300 ml of GT1 or 50 ml of 9301pA) or GLUT4 antisera (up to 300 ml of GT4 or 50 ml of p8105 aG4) or longer incubation times (up to 18 h) did not result in increased GLUT1 or GLUT4 signals. Second, no detectable amount of glucose transporters could be recovered from a second immunoprecipitation with the same antiserum. Third, the total signals obtained were independent of the antiserum used (GT4 or GT1, raised in the laboratory of G. H., versus p8105 aG4 or 9301pA). Finally, as shown in a previous study, the order of addition of antibodies has no influence on the results (13). types of glucose transport stimuli, including insulin (26, 27), metformin (26), serotonin (27), and catecholamines (28). Thus, it is conceivable that GLUT1 plays an important role in the regulation of glucose uptake in the heart under certain physi- ological conditions. In view of the importance of glucose carriers in cardiac func- tion, we have used freshly isolated cardiac myocytes from adult rats: 1) to quantify the effect of insulin on the recruitment of GLUT4 and GLUT1 carriers to the cell surface; 2) to compare the amounts of these proteins expressed in intact cardiac myo- cytes; and 3) to explore the nature of the intracellular GLUT4 compartment. Determination of Total Cellular Amounts of GLUT4 and GLUT1 by Quantitative Western Blot Analysis—Nonstimulated cardiomyocytes (;30 mg protein/sample) were washed once with TES buffer (20 mM Tris, 1 mM EDTA, 250 mM sucrose, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4) and then homogenized with a Potter (clearance, 0.2 mm) in a total volume of 4.7 ml. The samples were then spun down for 30 min at 50,000 g at 4 °C, and the pellet of this centrifugation was resuspended in 350 ml of TES buffer. This crude membrane fraction was used to perform immunoblot analysis, as described in a separate section below. MATERIALS AND METHODS Photoaffinity Labeling of Glucose Transporters with [3H]ATB- BMPA—The labeling of glucose transporters was performed according to a method developed previously (12, 29), which was adapted and extensively validated in cardiomyocytes (27, 28). In brief, the labeling of cell surface transporters was carried out as follows. Cardiomyocytes (;5 mg protein/sample in a total volume of 6 ml) were incubated for 30 min at 37 °C in the absence (control) or in the presence of insulin (10 nM). Parallel samples were used for the determination of glucose trans- port. The cells allotted to the photoaffinity labeling were then washed and resuspended in 500 ml of medium A (with or without insulin); 60 ml of the nonpermeant, photoreactive bismannose compound 3H-ATB- BMPA (300 mCi, 60 mM final concentration) were added immediately before the samples were irradiated for 3 min with UV light under continuous gentle shaking. Following irradiation the cells were washed with medium A and then solubilized at 4 °C with 2% ThesitTM (in a phosphate buffer containing the proteinase inhibitors antipain, aproti- nin, pepstatin, and leupeptin, 1 mg/ml each). The immunotitration experiments illustrated in Figs. 4 and 5 were performed with antibodies bound to acrylamide beads prepared as described above. LDM membranes (50 mg) were incubated overnight at 4 °C with different mixtures of two batches of beads (one batch with 1F8 and one with g-globulin as nonspecific antibodies), corresponding to varying amounts of 1F8 (0–7 mg) in a constant total bead volume of 20 ml. The adsorbed and nonadsorbed membranes were then processed as described above. Immunoblot Analysis—SDS-polyacrylamide gel electrophoresis was performed on a membrane protein following the method of Laemmli (33). Proteins were transferred to Immobilon as previously reported (34) in buffer consisting of 20% methanol, 200 mM glycine, and 25 mM Tris, pH 8.3. Following transfer, the filters were blocked with 5% nonfat dry milk and 0.02% sodium azide in PBS for 1 h at 37 °C and were incu- bated with antibodies in 1% nonfat dry milk and 0.02% sodium azide in PBS. Transfer was confirmed by Coomassie Blue staining of the gel after the electroblot. Detection of the immune complex with the rabbit polyclonal antibodies was accomplished using 125I-protein A for 4 h at room temperature. This is an Open Access article under the CC BY license. This is an Open Access article under the CC BY license. The absolute amounts of GLUT4 and GLUT1 were quantified by comparing the signals obtained from several dilutions of crude mem- branes with those of known standards. These standards were purified intracellular membranes from isolated adipocytes (GLUT4) or from human erythrocytes (GLUT1) in which the amount of D-glucose-dis- placeable cytochalasin B binding sites had been determined as de- scribed elsewhere (32). RESULTS Effects of Insulin on Cell Surface Content of GLUT4 and GLUT1 and Quantification of Total Glucose Transporter Con- tent of Cardiomyocytes—To quantify the effect of insulin on cell surface GLUT4 and GLUT1 we used the selective, nonper- meant, photoreactive bismannose compound 3H-ATB-BMPA, which has proven to give a more accurate picture of cell surface changes than classical membrane fractionation methods com- bined with Western blot analysis (12, 14, 16). ATB-BMPA labeling was performed according to a protocol that has previ- ously been successfully used in adipocytes (12, 13, 35) or skel- etal muscles (14–16) and that we have recently adapted to isolated cardiac muscle cells (27, 28). Fig. 1 summarizes the quantitative data on the effects of insulin on the content of glucose transporters at the surface of cardiomyocytes. The hormone induced 5.7- and 2.7-fold in- creases in the amounts of GLUT4 and GLUT1, respectively, in this compartment (Fig. 1, left and middle panels). By compar- ison, insulin caused a 12.0 6 0.74-fold stimulation of glucose transport in the same experiments (Fig. 1, right panel). It is worth mentioning that in the basal, i.e. nonstimulated, state and in terms of absolute signals, the level of GLUT4 labeling at the surface of cardiomyocytes already exceeded that of GLUT1 labeling by a factor of ;2.5 (not shown), so that with insulin treatment, there was about four times more GLUT4 than GLUT1 at the cell surface. Thus, the insulin-dependent in- crease in glucose transport is largely explained by a recruit- ment of glucose transporters (mainly GLUT4). The difference in the extent of GLUT translocation and glucose transport stimulation might be due either to a change in intrinsic activity of recruited transporters or to a slightly higher accuracy of the transport assay in comparison with the labeling method. Although the results presented above point to GLUT4 as the major transporter responsible for insulin-dependent glucose uptake, reports indicating a relatively high expression of GLUT1 in cardiac tissue (24, 25) prompted us to directly com- pare the total amounts of GLUT4 and GLUT1 in isolated car- diomyocytes by Western blot analysis. For this purpose, non- stimulated cardiomyocytes were homogenized as described under “Materials and Methods,” and their content in GLUT4 and GLUT1 was estimated by comparing the cell samples with known standards obtained from intracellular adipocyte mem- branes (GLUT4 standard) or erythrocyte membranes (GLUT1 standard). MATERIALS AND METHODS Detection of the immune complex with monoclonal Preliminary experiments were performed to ensure that amounts of antibodies used to immunoprecipitate GLUT1 and GLUT4 from homog- enized or permeabilized cardiomyocytes were saturating with respect to FIG. 1. Quantitative analysis of the effects of insulin on cell surface labeling of GLUT4 and GLUT1 and on glucose transport in intact cardiomyocytes. Cardiomyocytes were exposed to insulin (10 nM) for 30 min at 37 °C. The samples were then subjected to labeling, solubilization, immunoprecipitation, and gel electrophoresis, and the amount of labeled transporters was determined as described under “Materials and Methods.” The rate of 2-deoxy-D-glucose uptake was measured in parallel samples (right panel). Results shown are means of 16–18 independent experiments 6 S.E. (bars). The labeling data are expressed as values normalized to control. The statistical significance of the differences from control was assessed by paired Student’s t test; ppp, p , 0.001 versus control. Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes 7087 7087 nsulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes FIG. 1. Quantitative analysis of the effects of insulin on cell surface labeling of GLUT4 and GLUT1 and on glucose transport in intact cardiomyocytes. Cardiomyocytes were exposed to insulin (10 nM) for 30 min at 37 °C. The samples were then subjected to labeling, solubilization, immunoprecipitation, and gel electrophoresis, and the amount of labeled transporters was determined as described under “Materials and Methods.” The rate of 2-deoxy-D-glucose uptake was measured in parallel samples (right panel). Results shown are means of 16–18 independent experiments 6 S.E. (bars). The labeling data are expressed as values normalized to control. The statistical significance of the differences from control was assessed by paired Student’s t test; ppp, p , 0.001 versus control. antibodies was performed using sheep anti-mouse 125I-labeled anti- body. Antibody 3F8 was detected using horseradish peroxidase linked to a goat anti-IgM mouse secondary antibody and visualized using an ECL system. The autoradiograms were quantified using scanning den- sitometry. Immunoblots were performed under conditions in which autoradiographic detection was in the linear response range. (36). Importantly, the Ki value found for the inhibition of glu- cose transport by ATB-BMPA in cardiomyocytes (;180 mM; not shown) was similar to that determined in erythrocytes (29), rat adipocytes (13), and 3T3-L1-adipocytes (36). The Ki was un- changed with insulin treatment (13, 36). MATERIALS AND METHODS Moreover, incorpora- tion efficiency of ATB-BMPA into GLUT1 and GLUT4 was consistently found to be the same in different cell types, includ- ing rat adipocytes (13, 37), 3T3-L1-adipocytes (36), and Xeno- pus oocytes (37), and to be very high under the conditions used (which were the same as in this study; Refs. 13, 29, and 36). Finally, insulin had no effect on the total level of ATB-BMPA incorporation as determined in cardiomyocytes permeabilized with the pore-forming agent a-hemolysin or by sonication (data not shown). This finding is in line with previous investigations, which have shown that the total amount of labeled GLUT1 and GLUT4 is the same in basal or insulin-stimulated 3T3-L1 adi- pocytes (38) or muscle (14). Overall, these observations indicate that the labeling of ATB-BMPA to cell surface glucose trans- porters is independent of the cell type and is not affected by insulin. RESULTS In addition, the distribution of SCAMPs has also been examined, because these proteins have been reported to co-localize with GLUT4 in isolated rat adipocytes (39, 40). Western blot analysis of plasma and LDM membranes with anti-SCAMP antibodies revealed two distinct bands showing apparent molecular masses of 37 and 39 kDa (Fig. 2), as pre- viously shown in adipocytes and skeletal muscle (39, 40). Acute insulin treatment resulted in a redistribution of SCAMP 39 from LDMs to the plasma membranes (Fig. 2). Thus, insulin caused a significant increase in the abundance of SCAMP 39 in plasma membranes (94 6 5% increase) and a concomitant significant decrease in LDMs (levels in the insulin-treated group accounted for 55 6 8% of the unstimulated control group) (Fig. 2). A similar tendency was found with regard to SCAMP 37, which did not reach significance in PM (37 6 34% increase in PM and 26 6 7% decrease in LDMs after insulin treatment) (Fig. 2). p y To this end, subcellular fractionation of isolated rat cardiom- yocytes was performed as previously reported (26). This proce- dure results in the isolation of several membrane fractions. One of these fractions (PM, see “Materials and Methods”) was enriched in the plasma membrane marker enzyme ouabain- sensitive p-nitrophenylphosphatase (41) by a factor of 13.5 (2.40 versus 0.17 nmol/h/mg of protein), whereas the specific activity of the sarcoplasmic reticulum marker, the EGTA-sen- sitive Ca11-ATPase (42), was decreased by a factor of 3.6 (0.41 versus 1.88 nmol/h/mg of protein) when compared with crude cell homogenates. PM was also highly enriched in cell surface markers such as b1-integrin (Fig. 2), the a1-subunit of the Na1-K1-ATPase, and dystrophin, as determined by immuno- blot analysis (not shown). Another fraction (LDMs) contained substantial amounts of GLUT4, GLUT1, and TGN 38 (a trans- Golgi marker) and was nominally free from plasma membrane markers or EGTA-sensitive Ca11-ATPase activity (data not shown). We next characterized the degree of co-localization of GLUT4, GLUT1, and SCAMPs in LDMs. To this end, we per- formed two different types of vesicle immunoadsorption assays with LDMs obtained from unstimulated cardiomyocytes: 1) immunoadsorption of GLUT4-containing vesicles with 1F8 an- tibody (monoclonal antibody against GLUT4) noncovalently bound to agarose beads, and 2) immunoisolation of GLUT4 vesicles with 1F8 covalently linked to acrylamide beads. Im- munoadsorption of GLUT4 vesicles with 1F8-agarose resulted in the specific recovery of near 60% of total GLUT4 present in LDMs (Fig. RESULTS Using this method, we found contents of 1.2 pmol of GLUT4 and 0.47 pmol of GLUT1/mg of protein (n 5 2). In other words, GLUT1 makes up nearly 30% of the total glucose trans- porter content in these cells. This substantial level of GLUT1 expression was essentially confirmed by experiments in which we have attempted to determine the total amounts of GLUT1 and GLUT4 by using ATB-BMPA in cardiomyocytes permeabi- lized with either a-toxin or by sonication. These experiments even yielded a higher proportion of GLUT1 (up to 50%; data not shown), although we cannot rule out that the labeling efficiency of intracellular GLUT4 might be lower than that of GLUT1. It should be noted that the quantitative evaluation of the effects of insulin and the comparison of GLUT1 and GLUT4 relies on the assumption that the hormone does not modify the reactivity of the cell surface transporters, and that both iso- forms display the same labeling efficiency. In this context, it was previously shown that the Kd of ATB-BMPA binding is similar for both carrier isoforms and is not altered by insulin Characterization of GLUT4-containing Vesicles and Co-lo- calization of GLUT4, GLUT1, and SCAMPs in Intracellular FIG. 2. Effect of insulin on GLUT4, GLUT1, b1-integrin, and SCAMP distribution in intracellular and plasma membranes from cardiomyocytes. Isolated cardiomyocytes were incubated for 30 min with or without insulin (10 nM), as indicated, before PM and LDM fractions were obtained as described under “Materials and Methods.” GLUT4, GLUT1, b1-integrin, and SCAMPs were detected by immuno- blot analysis by using specific antibodies. Equal amounts of membrane proteins (5 mg for GLUT4, 15 mg for GLUT1, 30 mg for b-integrin, and 10 mg for SCAMPs) from PMs or LDMs were laid on gels. Representa- tive autoradiograms, obtained after various times of exposure, of six to eight separate experiments are shown. Insulin-induced Recruitment of GLU 7088 FIG. 3. Immunoadsorption of GLUT4 and GLUT1 in intracel- lular membranes from unstimulated cardiomyocytes. LDM mem- branes obtained from nonstimulated cardiac myocytes were immuno- adsorbed with 1F8-agarose beads (A) or with 1F8-acrylamide beads (B) (1) or with beads covered with nonspecific antibodies (2). After the incubation, the adsorbed fraction (pellet, P) and nonadsorbed fraction (supernatant, S) were electrophoresed and immunoblotted to determine the abundance of GLUT4 and GLUT1. Representative autoradiograms, obtained after various times of exposure, of six to eight separate exper- iments are shown. RESULTS T4 and GLUT1 in Cardiac Myocytes Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes 7088 Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes GLUT1 in Cardiac Myocytes FIG. 2. Effect of insulin on GLUT4, GLUT1, b1-integrin, and SCAMP distribution in intracellular and plasma membranes from cardiomyocytes. Isolated cardiomyocytes were incubated for 30 min with or without insulin (10 nM), as indicated, before PM and LDM fractions were obtained as described under “Materials and Methods.” GLUT4, GLUT1, b1-integrin, and SCAMPs were detected by immuno- blot analysis by using specific antibodies. Equal amounts of membrane proteins (5 mg for GLUT4, 15 mg for GLUT1, 30 mg for b-integrin, and 10 mg for SCAMPs) from PMs or LDMs were laid on gels. Representa- tive autoradiograms, obtained after various times of exposure, of six to eight separate experiments are shown. FIG. 2. Effect of insulin on GLUT4, GLUT1, b1-integrin, and SCAMP distribution in intracellular and plasma membranes from cardiomyocytes. Isolated cardiomyocytes were incubated for 30 min with or without insulin (10 nM), as indicated, before PM and LDM fractions were obtained as described under “Materials and Methods.” GLUT4, GLUT1, b1-integrin, and SCAMPs were detected by immuno- blot analysis by using specific antibodies. Equal amounts of membrane proteins (5 mg for GLUT4, 15 mg for GLUT1, 30 mg for b-integrin, and 10 mg for SCAMPs) from PMs or LDMs were laid on gels. Representa- tive autoradiograms, obtained after various times of exposure, of six to eight separate experiments are shown. FIG. 3. Immunoadsorption of GLUT4 and GLUT1 in intracel- lular membranes from unstimulated cardiomyocytes. LDM mem- branes obtained from nonstimulated cardiac myocytes were immuno- adsorbed with 1F8-agarose beads (A) or with 1F8-acrylamide beads (B) (1) or with beads covered with nonspecific antibodies (2). After the incubation, the adsorbed fraction (pellet, P) and nonadsorbed fraction (supernatant, S) were electrophoresed and immunoblotted to determine the abundance of GLUT4 and GLUT1. Representative autoradiograms, obtained after various times of exposure, of six to eight separate exper- iments are shown. Membranes—In view of the fact that GLUT4 and GLUT1 showed differential recruitment to the cardiomyocyte surface in response to insulin, and regarding the high level of GLUT1 expression in these cells, we decided to characterize the intra- cellular insulin-sensitive GLUT4 pool and the degree of co- localization of GLUT4 and GLUT1 in this compartment. Recovery of GLUT4, GLUT1, and SCAMPs on immunoisolation of intracellular membranes from unstimulated cardiac myocytes LDM membranes obtained from nonstimulated cardiac myocytes were immunoadsorbed with 1F8-agarose beads, 1F8-acrylic beads, 3F8- agarose beads, or beads linked to nonspecific antibodies. After the incubation, the adsorbed and nonadsorbed fractions were electrophore- sed and immunoblotted to determine the abundance of GLUT4, GLUT1, and SCAMPs. Autoradiograms were subjected to scanning densitome- try. Data are means 6 S.E. of three to six experiments (with the exception of data on GLUT1 after immunoadsorption with 3F8-agarose beads, which are the mean of two observations) and expressed as percentages of specific immunoadsorption. FIG. 4. Immunotitration of GLUT4 and GLUT1 in intracellular membranes from unstimulated cardiomyocytes. LDM mem- branes obtained from nonstimulated cardiac myocytes were immuno- adsorbed with increasing amounts of 1F8 acrylic beads (corresponding to the 1F8 quantities indicated). GLUT4 and GLUT1 were detected in the adsorbed fractions by Western blotting. Upper panel, representative autoradiograms. Lower panel, quantitative analysis. The amounts of immunoadsorbed GLUT4 and GLUT1 were determined by densitome- try of autoradiograms such as those shown above and normalized to the maximal amount precipitated with a saturating dose of 1F8 in the same experiment. Data are means from two to five independent experiments. (Note that the percentage values plotted here are not expressed in the same way as those shown in Table I, which were normalized to the total amount in LDM; e.g. 100% GLUT1 as plotted in this figure would be equivalent to ;64% in Table I.) Immunoadsorption agent GLUT4 SCAMP 39 GLUT1 % of specific immunoadsorption 1F8-agarose 60.1 6 2.2 24.2 6 7.1 15.2 6 2.7 1F8-acrylamide 79.2 6 0.9 55.4 6 7.1 63.7 6 6.6 3F8-agarose 36.6 6 5.7 63.0 6 15.6 24.7 1F8-agarose (Table I). When using 1F8 coupled to acrylic beads, four times as much GLUT1 and more than twice as much SCAMP 39 were specifically immunoprecipitated when compared with 1F8-agarose (i.e. 64% of all GLUT1 contained in LDMs and 55% SCAMP 39; Fig. 3 and Table I). Thus, this contrasts with the relatively small increase of 20% in GLUT4 recovery (i.e. 80% of total GLUT4 with 1F8-acrylamide versus 60% with 1F8-agarose; Fig. 3 and Table I). Taken together, these results might be interpreted in terms of the existence of two distinct intracellular GLUT4 membrane populations in LDMs from unstimulated cardiomyocytes: GLUT4 vesicles showing a low GLUT1 and SCAMP 39 content and GLUT4 vesicles showing a high GLUT1 and SCAMP 39 content. FIG. 4. Immunotitration of GLUT4 and GLUT1 in intracellular FIG. 4. Immunotitration of GLUT4 and GLUT1 in intracellular membranes from unstimulated cardiomyocytes. LDM mem- branes obtained from nonstimulated cardiac myocytes were immuno- adsorbed with increasing amounts of 1F8 acrylic beads (corresponding to the 1F8 quantities indicated). GLUT4 and GLUT1 were detected in the adsorbed fractions by Western blotting. Upper panel, representative autoradiograms. Lower panel, quantitative analysis. The amounts of immunoadsorbed GLUT4 and GLUT1 were determined by densitome- try of autoradiograms such as those shown above and normalized to the maximal amount precipitated with a saturating dose of 1F8 in the same experiment. Data are means from two to five independent experiments. (Note that the percentage values plotted here are not expressed in the same way as those shown in Table I, which were normalized to the total amount in LDM; e.g. 100% GLUT1 as plotted in this figure would be equivalent to ;64% in Table I.) Immunoadsorption of LDMs was also carried out with anti- bodies directed against SCAMPs (antibody 3F8 linked to aga- rose beads). With this approach, nearly 63% of SCAMP 39 but only 37% of GLUT4 and 25% of GLUT1 were recovered (Table I). This finding suggests the existence of a subpopulation of LDMs that is enriched in SCAMP 39 compared with GLUT4. This type of analysis could not be extended to GLUT1 vesicles, since the anti-GLUT1 antibodies used showed little efficiency in immunoadsorbing GLUT1 vesicles (data not shown). The conclusion that LDMs may contain at least two popula- tions of GLUT4-containing vesicles, on the grounds of the im- munoprecipitation experiments described above, is limited by the fact that it is based on the comparison of data obtained from two different protocols (adsorption with 1F8-agarose versus adsorption with 1F8-acrylamide; Table I). To directly examine the hypothesis of different vesicle populations, we performed immunotitration experiments using varying amounts of 1F8 (bound to acrylamide) to adsorb GLUT4 vesicles in LDMs from nonstimulated cells and determined the amount of recovered GLUT4, GLUT1, and SCAMPs by Western blot analysis. We found that about 10 times less 1F8 antibody (;0.7 mg) was required to reach a saturating degree of GLUT4 adsorption than was the case for either GLUT1 (7 mg of 1F8; Fig. 4) or SCAMPs (Fig. 5). These observations thus confirm the exist- ence of an intracellular vesicle pool that is enriched in GLUT4 but poor in GLUT1 and SCAMPs. amount of GLUT4 (246%), GLUT1 (245%), and SCAMP 39 (259%) (Fig. 6A). Similarly, insulin reduced the GLUT4 con- tent in GLUT4 vesicles obtained with 1F8-agarose (55% de- crease; Fig. 6B). amount of GLUT4 (246%), GLUT1 (245%), and SCAMP 39 (259%) (Fig. 6A). Similarly, insulin reduced the GLUT4 con- tent in GLUT4 vesicles obtained with 1F8-agarose (55% de- crease; Fig. 6B). RESULTS 3 and Table I); however, on adsorption with 1F8- acrylamide, approximately 80% of all GLUT4 was specifically recovered in the immunoprecipitates (Fig. 3 and Table I). Based on this, we reasoned that this different efficiency of the two protocols to immunoisolate GLUT4 vesicles may be used to characterize the extent of co-localization of GLUT4 with other proteins such as GLUT1 and SCAMPs in LDMs. As shown in Fig. 3 and Table I, after immunoadsorption with 1F8-agarose, only 15% of GLUT1 originally contained in LDMs was immu- noadsorbed. Similarly, only 24% of SCAMP 39 was adsorbed to In keeping with prior observations (26) and with the data shown in Fig. 1, the incubation of cardiomyocytes with insulin caused a significant increase in GLUT4 and GLUT1 in plasma membranes by 138 6 35% and 61 6 18%, respectively (Fig. 2). The results indicate, in agreement with data illustrated in Fig. 1, that insulin causes a greater recruitment to the cell surface of GLUT4 than GLUT1. Under the same conditions, no alter- ations in the abundance of b1-integrin (Fig. 2), the a1-subunit of the Na1-K1-ATPase, or dystrophin were detected (data not shown). Concomitantly, there was a significant drop in the abundance of GLUT4 and GLUT1 in LDMs after insulin treat- ment (levels after insulin accounted for 40 6 8% and 48 6 6% of control values, respectively; n 5 6–8 observations). 7089 Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes DISCUSSION Similarly, fasting over 24–48 h was shown to dramatically diminish the basal rate as well as the insulin sensitivity of myocardial glu- cose uptake in vivo, with a concomitant reduction in the amount of cardiac GLUT1 (25). It is also worth mentioning that streptozotocin-induced diabetes also causes a decrease in GLUT1 protein in the rat heart (46). FIG. 5. Immunotitration of GLUT4 and SCAMPs in intracellu- lar membranes from unstimulated cardiomyocytes. LDM mem- branes were immunoadsorbed with increasing amounts of 1F8-acryl- amide, and the content of GLUT4 and SCAMPs in the adsorbed material was determined as described in the legend of Fig. 4. Upper panel, representative autoradiograms. Lower panel, quantitative anal- ysis. Data are means from four independent experiments and expressed as in Fig. 4. FIG. 5. Immunotitration of GLUT4 and SCAMPs in intracellu- lar membranes from unstimulated cardiomyocytes. LDM mem- branes were immunoadsorbed with increasing amounts of 1F8-acryl- amide, and the content of GLUT4 and SCAMPs in the adsorbed material was determined as described in the legend of Fig. 4. Upper panel, representative autoradiograms. Lower panel, quantitative anal- ysis. Data are means from four independent experiments and expressed as in Fig. 4. The fact that insulin induces a more modest translocation of GLUT1 than GLUT4 suggests that the specific function of GLUT1 in these cells lies in the mediation of other effects than that of insulin. One possibility worth being explored is that the intracellular GLUT1 pool of heart cells is more responsive to stimuli such as contraction or anoxia than to insulin. In this context, one may speculate that GLUT1 becomes redistributed to this pool after cardiomyocyte isolation (e.g. as a consequence of the arrest of contractile activity). factor of ;2.5 (not shown), and the preferential recruitment of GLUT4 by insulin will accentuate the importance of GLUT4 as the predominant transporter in the plasma membrane. Thus, these results clearly indicate that GLUT4 is responsible for a large part of insulin-stimulated glucose uptake in these cells. This is in line with prior observations in adipocytes and skel- etal muscles (12–14, 16). Characterization of Intracellular GLUT4-containing Mem- branes—We have further explored the intracellular insulin- sensitive GLUT4 pool by performing vesicle immunoisolation analysis in LDMs from rat cardiomyocytes. DISCUSSION insulin-tre with antib (upper pan “Materials quently su three to ei Insulin-induced Recruitment of GLUT4 and G 7090 roidism) (44), an intervention known to selectively reduce the myocardial level of GLUT1 but not GLUT4 (45). Similarly, fasting over 24–48 h was shown to dramatically diminish the basal rate as well as the insulin sensitivity of myocardial glu- cose uptake in vivo, with a concomitant reduction in the amount of cardiac GLUT1 (25). It is also worth mentioning that streptozotocin-induced diabetes also causes a decrease in GLUT1 protein in the rat heart (46). The fact that insulin induces a more modest translocation of GLUT1 th GLUT4 gg t th t th ifi f ti f FIG. 5. Immunotitration of GLUT4 and SCAMPs in intracellu- lar membranes from unstimulated cardiomyocytes. LDM mem- branes were immunoadsorbed with increasing amounts of 1F8-acryl- amide, and the content of GLUT4 and SCAMPs in the adsorbed material was determined as described in the legend of Fig. 4. Upper panel, representative autoradiograms. Lower panel, quantitative anal- ysis. Data are means from four independent experiments and expressed as in Fig. 4. FIG. 6. Effect of insulin on the content of GLUT4, GLUT1, and SCAMPs in immunoadsorbed intracellular GLUT4-containing vesicles. LDM membranes obtained from basal (i.e. unstimulated) or insulin-treated cardiac myocytes were immunoadsorbed with 1F8 (1) or with antibodies from nonimmune serum (2) coupled to agarose beads (upper panel) or to acrylamide beads (lower panel) as described under “Materials and Methods.” The immunoadsorbed material was subse- quently subjected to immunoblot. Representative autoradiograms of three to eight separate experiments are shown. Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes 7090 FIG. 6. Effect of insulin on the content of GLUT4, GLUT1, and SCAMPs in immunoadsorbed intracellular GLUT4-containing vesicles. LDM membranes obtained from basal (i.e. unstimulated) or insulin-treated cardiac myocytes were immunoadsorbed with 1F8 (1) or with antibodies from nonimmune serum (2) coupled to agarose beads (upper panel) or to acrylamide beads (lower panel) as described under “Materials and Methods.” The immunoadsorbed material was subse- quently subjected to immunoblot. Representative autoradiograms of three to eight separate experiments are shown. roidism) (44), an intervention known to selectively reduce the myocardial level of GLUT1 but not GLUT4 (45). DISCUSSION Quantification of GLUT1 and GLUT4 and Insulin-dependent Recruitment to the Plasma Membrane—A first issue addressed in this study is the direct comparison of the contents of GLUT4 and GLUT1 in isolated rat cardiomyocytes and of the relative contribution of these isoforms to the effect of insulin on glucose uptake. As shown in Fig. 1, GLUT4 is recruited to the plasma membrane to a larger extent than GLUT1 in response to insu- lin, as quantified by ATB-BMPA labeling. This is confirmed by experiments such as that illustrated in Fig. 2, in which the relative effects of insulin on the level of GLUT4 and GLUT1 were evaluated in purified plasma membranes by Western blot analysis. It is also worth mentioning that the absolute level of ATB-BMPA labeling of GLUT4 at the surface of nonstimulated cardiomyocytes already exceeded that of GLUT1 labeling by a Finally, it was verified that the GLUT4-containing vesicles immunoadsorbed with 1F8-acrylamide, as described above, represents an insulin-sensitive pool. Therefore, the action of insulin on the abundance of GLUT4, GLUT1, and SCAMP 39 was studied in GLUT4 vesicles immunoadsorbed with a satu- rating amount of 1F8-acrylamide (to obtain GLUT4 vesicles with a high content in GLUT1 and SCAMP 39; see above). In this fraction, there was an insulin-dependent reduction in the Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes FIG. 6. Effect of insulin on the content of GLUT4, GLUT1, and SCAMPs in immunoadsorbed intracellular GLUT4-containing vesicles. LDM membranes obtained from basal (i.e. unstimulated) or insulin-treated cardiac myocytes were immunoadsorbed with 1F8 (1) or with antibodies from nonimmune serum (2) coupled to agarose beads (upper panel) or to acrylamide beads (lower panel) as described under “Materials and Methods.” The immunoadsorbed material was subse- quently subjected to immunoblot. Representative autoradiograms of three to eight separate experiments are shown. T4 and GLUT1 in Cardiac Myocytes 7090 roidism) myocardi fasting ov basal rat cose upt amount o streptozo GLUT1 p The fac GLUT1 t FIG. 5. Immunotitration of GLUT4 and SCAMPs in intracellu- lar membranes from unstimulated cardiomyocytes. LDM mem- branes were immunoadsorbed with increasing amounts of 1F8-acryl- amide, and the content of GLUT4 and SCAMPs in the adsorbed material was determined as described in the legend of Fig. 4. Upper panel, representative autoradiograms. Lower panel, quantitative anal- ysis. Data are means from four independent experiments and expressed as in Fig. 4. FIG. 6. E SCAMPs vesicles. GLUT4 populations (see Fig. 7). Immunocytochemical studies have localized GLUT4 in sev- eral distinct intracellular sites in adipocytes (47) and cardiom- yocytes (18). Furthermore, kinetic studies are indicative of the existence of two intracellular GLUT4 pools in adipocytes. First, mathematical analysis of kinetic data of GLUT4 endocytosis and exocytosis obtained with the photoaffinity reagent ATB- BMPA (6, 35) predicts that GLUT4 localizes to at least two distinct intracellular compartments in fat cells (48). Second, data of subcellular trafficking of both GLUT4 and GLUT1 and chimeric transporters in adipocytes (49) are best explained by a model postulating two intracellular pools (50). According to the predictions of this model, GLUT4 is internalized into an endo- somal compartment and then sorted into an insulin-recruitable compartment; in contrast, GLUT1 is endocytosed into the en- dosomal compartment and recycles from this compartment to the cell surface (50). Further support to the idea that GLUT4 is present in separate intracellular compartments comes from very recent compartment ablation analysis; thus, ablation of the endosomal compartment in 3T3-L1 adipocytes reduces by 40% the amount of cellular GLUT4 (51). 14. Lund, S., Holman, G. D., Schmitz, O., and Pedersen, O. (1993) FEBS Lett. 330, 312–318 15. Lund, S., Holman, G. D., Schmitz, O., and Pedersen, O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5817–5821 16. Wilson, C. M., and Cushman, S. W. (1994) Biochem. J. 299, 755–759 17. Katz, E. B., Stenbit, A. E., Hatton, K., Depinho, R., and Charron, M. J. (1995) Nature 377, 151–155 , 18. Slot, J. W., Geuze, H. J., Gigengack, S., James, D. E., and Lienhard, G. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7815–7819 18. Slot, J. W., Geuze, H. J., Gigengack, S., James, D. E., a (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7815–7819 (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7815–7819 19. Zorzano, A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinksi, B. E A. E., and Pilch, P. F. (1989) J. Biol. Chem. 264, 12358–12363 20. Marette, A., Richardson, J. M., Ramlal, T., Balon, T. W., Vranic, M., Pessin, J. E., and Klip, A. (1992) Am. J. Physiol. 263, C443–C452 21. Wang, C., and Hu, S. M. (1991) Biochem. Biophys. Res. Commun. 177, 1095–1100 22. Santalucia, S., Camps, M., Castello, A., Mun˜oz, P., Nuel, A., Testar, X., Palacı´n, M., and Zorzano, A. (1992) Endocrinology 130, 837–846 23. Studelska, D. R., Campbell, C., Pang, S., Rodnick, K. REFERENCES 1. Gould, G. W., and Holman, G. D. (1993) Biochem. J. 295, 329– 2. Bell, G. I., Burant, C. F., Takeda, J., and Gould, G. W. (1993) J. Biol. Chem. 268, 19161–19164 3. Mueckler, M. (1994) Eur. J. Biochem. 219, 713–725 3. Mueckler, M. (1994) Eur. J. Biochem. 219, 713–725 4. Gould, G. W., Jess, T. J., Andrews, G. C., Herbst, J. J., Plevin, R. J., and Gibbs, E. M. (1994) J. Biol. Chem. 269, 26622–26625 4. Gould, G. W., Jess, T. J., Andrews, G. C., Herbst, J. J E. M. (1994) J. Biol. Chem. 269, 26622–26625 5. Smith, R. M., Charron, M. J., Shah, N., Lodish, H. F., and Jarett, L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6893–6897 6. Satoh, S., Nishimura, H., Clark, A. E., Kozka, I. J., Vannucci, S. J., Simpson, I. A., Quon, M. J., Cushman, S. W., and Holman, G. D. (1993) J. Biol. Chem. 268, 17820–17829 7. Douen, A. G., Ramlal, T., Rastogi, S., Bilan, P. J., Cartee, G. D., Vranic, M., Holloszy, J. O., and Klip, A. (1990) J. Biol. Chem. 265, 13427–13430 Holloszy, J. O., and Klip, A. (1990) J. Biol. Chem. 265, 13427–1 FIG. 7. Hypothetical model of GLUT4 and GLUT1 distribution in rat cardiomyocytes. According to this model, intracellular vesicles containing GLUT4 (filled rectangles) consist of at least two pools in nonstimulated cardiomyocytes: one (pool 1) with a low content of GLUT1 (open diamonds) and SCAMPs (dotted ovals) and the other (pool 2) with a relatively high content of GLUT1 and SCAMPs. Both pools are acutely recruitable by insulin. For details see “Discussion.” FIG. 7. Hypothetical model of GLUT4 and GLUT1 distribution in rat cardiomyocytes. According to this model, intracellular vesicles containing GLUT4 (filled rectangles) consist of at least two pools in nonstimulated cardiomyocytes: one (pool 1) with a low content of GLUT1 (open diamonds) and SCAMPs (dotted ovals) and the other (pool 2) with a relatively high content of GLUT1 and SCAMPs. Both pools are acutely recruitable by insulin. For details see “Discussion.” 8. Brozinick, J. T., Jr., Etgen, G. J., Jr., Yaspelkis, B. B., III, and Ivy, J. L. (1994) Biochem. J. 297, 539–545 9. Kolter, T., Uphues, I., Wichelhaus, A., Reinauer, H., and Eckel, J. (1992) Biochem. Biophys. Res. Commun. 189, 1207–1214 10. Cartee, G. D., Douen, A. G., Ramlal, T., Klip, A., and Holloszy, J. O. (1991) J. Appl. Physiol. 70, 1593–1600 11. Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes Insulin-induced Recruitment of GLUT4 and GLUT1 in Cardiac Myocytes FIG. 7. Hypothetical model of GLUT4 and GLUT1 distribution in rat cardiomyocytes. According to this model, intracellular vesicles containing GLUT4 (filled rectangles) consist of at least two pools in nonstimulated cardiomyocytes: one (pool 1) with a low content of GLUT1 (open diamonds) and SCAMPs (dotted ovals) and the other (pool 2) with a relatively high content of GLUT1 and SCAMPs. Both pools are acutely recruitable by insulin. For details see “Discussion.” This suggests the existence of an additional GLUT1 pool that is not part of LDMs (and is therefore distinct from pools 1 and 2). Acknowledgments—The expert and devoted technical assistance of Christiane Lo¨ken and Ilinca Ionescu is gratefully acknowledged. We also thank Dr. Samuel W. Cushman and Dina R. Yver for providing some of the antibodies used in the labeling experiments. REFERENCES Wheeler, T. J., Fell, R. D., and Hauck, M. A. (1994) Biochim. Biophys. Acta 1196, 191–200 12. Calderhead, D. M., Kitagawa, K., Tanner, L. I., Holman, G. D., and Lienhard, G. E. (1990) J. Biol. Chem. 265, 13800–13808 ( ) , 13. Holman, G. D., Kozka, I. J., Clark, A. E., Flower, C. J., Saltis, J., Habberfield, 13. Holman, G. D., Kozka, I. J., Clark, A. E., Flower, C. J., Saltis, J., Habberfield, A D Simpson I A and Cushman S W (1990) J Biol Chem 265 13. Holman, G. D., Kozka, I. J., Clark, A. E., Flower, C. J., Saltis, J., Habberfield, A. D., Simpson, I. A., and Cushman, S. W. (1990) J. Biol. Chem. 265, 18172–18179 DISCUSSION Immunoadsorption assays with 1F8-agarose resulted in a high recovery of GLUT4 but in a low recovery of GLUT1 and SCAMP 39; in contrast, immunoadsorption with 1F8-acrylamide led to a large increase in the amount of GLUT1 and SCAMP 39 recovered, with a relatively modest increase in the degree of GLUT4 recovery (Table I). In other words, immunoadsorption with 1F8-agarose yields GLUT4 vesicles with a low GLUT1 and low SCAMP 39 content (GLUT4 pool 1), whereas immunoadsorption with 1F8- acrylamide yields, in addition, GLUT4 vesicles with a high GLUT1 and high SCAMP 39 content (GLUT4 pool 2). Further- more, immunotitration experiments with LDM membranes show that a much smaller amount of 1F8 is required to reach a saturating degree of GLUT4 recovery than was the case for GLUT1 (Fig. 4) or SCAMPs (Fig. 5). 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https://openalex.org/W3105000478	http://e-journals.unmul.ac.id/index.php/psikoneo/article/download/4671/pdf	Indonesian	null	Hubungan Antara Motivasi Kerja dan Kepuasan Kerja Dengan Pelayanan Prima	Psikoborneo	2,018	cc-by-sa	3,343	ISSN: 2477-2666/E-ISSN: 2477-2674 ISSN: 2477-2666/E-ISSN: 2477-2674 Psikoborneo, Vol 6, No 4, 2018:499- 504 1 Email: inacucusanita@gmail.com Hubungan Antara Motivasi Kerja dan Kepuasan Kerja Dengan Pelayanan Prima Ina Cucu Sanita1 Program Studi Psikologi Fakultas Ilmu Sosial dan Ilmu Politik Universitas Mulawarman Samarinda ABSTRACT. This study aims to determine the relationship between work motivation and job satisfaction with excellent service (service excellence) for nurses of BLUD Hospital Dr. H. Soemarno Sosroatmodjo Tanjung Selor. This study uses quantitative research methods. The subjects of this study were nurses of BLUD Regional General Hospital Dr. H. Soermarno Sosroatmodjo Tanjung Selor, Bulungan Regency, with a sample of 70 nurses selected by using random sampling technique. The collected data were analyzed with the help of the Statistical Packages for Social Sciences (SPSS) 24.0 for Windows program. The results showed that: (1) there was no relationship between work motivation and service excellence in RSUD nurses with beta coefficient (β) = 0.000, and t count> t table (-0.003 <1.995) and p = 0.997 (p <0.05); (2) there is a positive and significant relationship of job satisfaction with excellent service (service excellence) in RSUD nurses with a coefficient of beta (β) = 0.927, and the value of t count> t table (19,326> 1,995) and p value = 0,000 (p <0.05 ); (3) there is a relationship between work motivation and job satisfaction with service excellence in RSUD nurses with f count> f table (203.668> 3.19) and p = 0.000 (p <0.05). Contribution of influence (R2) on work motivation and job satisfaction with service excellence on nurses is 0.859. PENDAHULUAN Memberikan pelayanan yang bermutu dan terakreditasi, hal tersebut dapat tercapai apabila rumah sakit dapat mengmbangkan kinerja karyawannya secara maksimal terutama pada tenaga medis yaitu perawat, memberikan motivasi pada karyawan agar lebih baik lagi menjalankan tugas, menjalin komunikasi yang baik, memiliki kepedulian terhadap orang lain, memiliki kondisi yang layak dan nyaman dalam bertugas dan memiliki rasa tanggung jawab agar tercapailah semua misi dari rumah sakit tersebut. Pelayanan prima adalah suatu tindakan atau sikap yang dapat diberikan kepada pelanggan berupa produk/jasa serta memiliki kepedulian kepada pelanggan dengan memberikan layanan terbaik. Pelayanan prima merupakan pelayanan dengan memiliki standar kualitas tinggi dan selalu mengikuti perkembangan kebutuhan pelanggan setiap saat secara akurat (handal) dan konsisten, berorientasi kepada kepuasan pelanggan, menerapkan manajemen mutu total dan selalu mengikuti perkembangan, Rahmayanty (2010). Untuk memberikan pelayanan kepada pasien perawat memiliki bagian penting dari rumah sakit, mereka dituntut untuk memberikan perlakuan mambantu pasien sehingga pasien sampai pada proses penyembuhan, menurut Schaufeli dan Jauczur (1994), dalam menjalankan peran dan fungsinya, seorang perawat dituntut untuk memiliki keahlian, pengetahuan, dan konsentrasi yang tinggi. Peran seorang perawat tidak kalah penting dengan profesi seorang dokter, dalam tugas sehari-hari perawat disibukkan dengan hal-hal rutin dan monoton, berbagai usaha menolong menyelamatan nyawa seseorang serta harus bertindak cepat dan tenang dalam melakukan pertolongan. Kepuasan kerja juga merupakan suatu sikap positif pegawai terhadap berbagai situasi di tempat pekerjaan (Hermawan, 2016). Pelayanan prima dalam konteks pelayanan rumah sakit berarti pelayanan yang diberikan kepada pasien yang berdasarkan standar kualitas untuk memenuhi kebutuhan dan keinginan pasien sehingga pasien dapat memperoleh kepuasan yang akhirnya dapat meningkatkan kepercayaannya kepada rumah sakit tersebut (Endarini, 2001). Dan pelayanan prima di Rumah Sakit melibatkan seluruh karyawan dari manajer puncak sampai ke pekarya. Para profesi yang meliputi berbagai bidang kedokteran atau kesehatan merupakan ujung tombak pelayanan di Rumah Sakit, yang tidak hanya dituntut profesional akan tetapi juga diharapkan peran serta aktifnya dalam manajemen Rumah Sakit termasuk manajemen mutu (Sunartini, 2000). j ( ) Rumah sakit dituntut memiliki standar pelayanan dan fasilitas yang memadai namun, permasalahan yang terjadi adalah, tidak seluruh rumah sakit mampu memberikan pelayanan terhadap masyarakat secara prima, dalam hal pelayanan rawat inap maupun gawat darurat. Pelayanan prima yang diberikan kepada pasien harus berdasarkan pada standar prosedur operasional untuk dapat memenuhi harapan atau bahkan melebihi harapan pasien dengan memberikan nilai tambah dalam unsur pelayanan, sehingga tercapai kepuasan dan dapat mempengaruhi pasien terhadap instansi pelayanan kesehatan. Fenomena yang terjadi di RSUD BLUD dr.H. ISSN: 2477-2666/E-ISSN: 2477-2674 ISSN: 2477-2666/E-ISSN: 2477-2674 Keywords: service excellence, job satisfaction, work motivation Keywords: service excellence, job satisfaction, work motivation ABSTRAK. Penelitian ini bertujuan untuk mengetahui hubungan motivasi kerja dan kepuasan kerja dengan pelayanan prima (service excellence) pada perawat BLUD RSUD Dr. H. Soemarno Sosroatmodjo Tanjung Selor. Penelitian ini menggunakan metode penelitian kuantitatif. Subjek penelitian ini adalah perawat BLUD RSUD Dr. H. Soermarno Sosroatmodjo Tanjung Selor Kabupaten Bulungan, dengan sampel sebanyak 70 perawat yang dipilih dengan menggunakan teknik random sampling. Data yang terkumpul dianalisis dengan bantuan Program Paket Statistik untuk Ilmu Sosial (SPSS) 24.0 untuk Windows. Hasil penelitian menunjukkan bahwa: (1) tidak ada hubungan antara motivasi kerja dengan service excellence pada perawat RSUD dengan koefisien beta (β) = 0,000, dan t hitung> t tabel (-0,003 <1,995) dan p = 0,997 (p <0,05). ); (2) terdapat hubungan yang positif dan signifikan antara kepuasan kerja dengan pelayanan prima (service excellence) pada perawat RSUD dengan koefisien beta (β) = 0,927, dan nilai t hitung> t tabel (19,326> 1,995) dan p nilai = 0,000 (p <0,05); (3) terdapat hubungan motivasi kerja dan kepuasan kerja dengan service excellence pada perawat RSUD dengan nilai f hitung> f tabel (203.668> 3.19) dan p = 0.000 (p <0.05). Kontribusi pengaruh (R2) terhadap motivasi kerja dan kepuasan kerja terhadap service excellence pada perawat sebesar 0,859. Kata Kunci: keunggulan layanan, kepuasan kerja, motivasi kerja Kata Kunci: keunggulan layanan, kepuasan kerja, motivasi kerja 499 Psikoborneo, Vol 6, No 4, 2018:499- 504 PENDAHULUAN Perhatian (Attention), adalah kepedulian penuh dengan keinginan pelanggan maupun pemahaman atas saran dan kritiknya. 4. Perhatian (Attention), adalah kepedulian penuh dengan keinginan pelanggan maupun pemahaman atas saran dan kritiknya. Berdasarkan rangkaian permasalahan di atas, penulis tertarik untuk melakukan penelitian dengan judul “Hubungan Antara Motivasi Kerja dan Kepuasan Kerja dengan Pelayanan Prima pada Perawat RSUD BLUD dr.H. Soemarno Sosrotmodjo”, tepatnya di kota Tnjung Selor, kab Bulungan. 5. Tindakan (Action), adalah berbagai kegiatan nyata yang harus dilakukan dalam memberikan layanan kepada pelanggan. 6. Tanggung Jawab (Accountability), adalah suatu sikap keberpihakan kepada pelanggan sebagai wujud kepedulian untuk menghindarkan atau memininimalkan kerugian. 6. Tanggung Jawab (Accountability), adalah suatu sikap keberpihakan kepada pelanggan sebagai wujud kepedulian untuk menghindarkan atau memininimalkan kerugian. Motivasi Kerja Menurut Sunyoto (2014) motivasi adalah suatu proses dimana kebutuhan-kebutuhan mendorong seseorang untuk melakukan serangkaian kegiatan yang mengarah ke tercapainya sebuah tujuan tertentu. Selain itu, perusahaan juga harus memperhatikan mengenai bagaimana menjaga dan mengelola motivasi karyawan dalam bekerja agar selalu tinggi dan fokus pada tujuan perusahaan. Menjaga motivasi karyawan itu sangatlah penting karena motivasi itu adalah motor penggerak bagi setiap individu yang mendasari mereka untuk bertindak dan melakukan sesuatu (Prasetyo, 2003). Menurut Robbins (2013), motivasi adalah kesediaan untuk mengeluarkan tingkat upaya yang tinggi ke arah tujuan organisasi, yang dikondisikan oleh kemampuan upaya itu untuk memenuhi suatu kebutuhan individual PENDAHULUAN Soemarno Sosrotmodjo berdasarkan hasil wawancara observasi dan screening yang dilakukan peneliti, berdasarkan keterangan pasien merasa bahwa pelayanan prima yang diberikan oleh perawat di RSUD BLUD dr.H. Soemarno Sosrotmodjo kurang, dan seharusnya perawat di RS bisa lebih baik lagi karena ini merupakan RS satu- satunya di Bulungan, begitu pendapat pasien berinisial LH tersebut. Berikut ini hasil screening yang dilakukan oleh peneliti pada pasien di RSUD BLUD dr.H. Soemarno Sosrotmodjo Tanjung Selor terhadap 25 orang pasien tentang pelayanan prima yang dilakukan oleh para perawat. Tabel 1. Data Hasil Screening Pasien Pelayanan Prima Jumlah Persentase Rendah 25 100% Sedang - - Tinggi - - Total 25 100% Tabel 1. Data Hasil Screening Pasien Pelayanan Prima Jumlah Persentase Rendah 25 100% Sedang - - Tinggi - - Total 25 100% Tabel 1. Data Hasil Screening Pasien Tabel 1. Data Hasil Screening Pasien Pelayanan Prima Jumlah Persentase Rendah 25 100% Sedang - - Tinggi - - Total 25 100% Berdasarkan tabel 1 diatas menunjukkan bahwa dari 25 pasien ini mengacu pada interval tingkat pelayanan prima yang dilakukan oleh perawat pada pasien, maka diperoleh bahwa 25 atau 100% berada pada tingkat rendah. dikatakan rendah dikarenakan tingkat pelayanan prima yang telah 500 Psikoborneo, Vol 6, No 4, 2018:499- 504 ISSN: 2477-2666/E-ISSN: 2477-2674 dilakukan oleh perawat kepada pasien berada direntang interval rendah, dikatakan rendah berarti perawat tidak memenuhi syarat pelayanan prima dengan baik. menunjung program layanan prima, meliputi kemampuan dalam bidang kerja yang akan ditekuni, melaksanakan komunikasi yang efektif, mengembangkan motivasi dan menggunakan public relation sebagai instrument dalam membina hubungan ke dalam dan keluar organisasi. Rumah sakit dituntut memiliki standar pelayanan dan fasilitas yang memadai namun, permasalahan yang terjadi adalah, tidak seluruh rumah sakit mampu memberikan pelayanan terhadap masyarakat secara prima, dalam hal pelayanan rawat inap maupun gawat darurat. Pelayanan prima yang diberikan kepada pasien harus berdasarkan pada standar prosedur operasional untuk dapat memenuhi harapan atau bahkan melebihi harapan pasien dengan memberikan nilai tambah dalam unsur pelayanan, sehingga tercapai kepuasan dan dapat mempengaruhi pasien terhadap instansi pelayanan kesehatan. 2. Sikap (Attitude), adalah perilaku atau perangi yang harus ditonjolkan ketika menghadapi pelanggan, sperti berpikir positif dan menghargai orang lain. 3. Penampilan (Appereance), adalah penampilan seseorang baik yang bersifat fisik maupun non fisik, yang mampu merefleksikan kepercayaan diri dan kreadibilitas dari pihak lain. 3. Penampilan (Appereance), adalah penampilan seseorang baik yang bersifat fisik maupun non fisik, yang mampu merefleksikan kepercayaan diri dan kreadibilitas dari pihak lain. 4. Pelayanan Prima Pelayanan prima adalah palayanan yang unggul, yang merupakan sikap atau cara karyawan dalam melayani pelanggan secara memuaskan (Elhaitammy dalam Tjiptono, 2004). Menurut Barata (2004) bahwa Pelayanan prima adalah kepedulian kepada pelanggan dengan memberikan layanan terbaik. Untuk mencapai suatu pelayanan yang prima pihak perusahaan haruslah memiliki keterampilan tertentu, diantaranya memperhatikan penampilan, bersikap yang ramah, memperlihatkan gairah kerja dan sikap selalu siap untuk melayani, tenang dalam bekerja, menguasai pekerjaan dengan baik, mampu berkomunikasi dengan baik, dan mampu menangani keluhan pelanggan secara professional Barata mengembangkan budaya pelayanan prima berdasarkan pada A6, yaitu mengembangkan pelayanan prima dengan menyelaraskan faktor- faktor yaitu: Kemampuan, Sikap, Penampilan, Perhatian, Tindakan dan Tanggung Jawab dasar yang harus diperhatikan agar pelayanan prima dapat berhasil dilaksanakan: Menurut Herzberg (dalam Sunyoto (2014)), faktor motivasi kerja terbagi menjadi dua, yaitu intrinsik yang berarti bersumber dari dalam diri seseorang, sedangkan ekstrinsik yang bearti bersumber dari luar diri seseorang. 1) Faktor-faktor Intrinsik 1. Kemampuan (Ability), adalah pengertahuan dan keterampilan yang multlak diperlakukan untuk 1. Kemampuan (Ability), adalah pengertahuan dan keterampilan yang multlak diperlakukan untuk 501 ISSN: 2477-2666/E-ISSN: 2477-2674 Psikoborneo, Vol 6, No 4, 2018:499- 504 a) Tanggung jawab (responsibility), besar kecilnya tanggung jawab yang dirasakan diberikan kepada seorang tenanga kerja. a) Tanggung jawab (responsibility), besar kecilnya tanggung jawab yang dirasakan diberikan kepada seorang tenanga kerja. positif pada suatu pekerjaan, yang merupakan dampak/hasil evaluasi dari berbagai aspek pekerjaan tersebut. Kepuasan kerja merupakan penilaian dan sikap seseorang atau karyawan terhadap pekerjaannya dan berhubungan dengan lingkungan kerja, jenis pekerjaan, hubungan antar teman kerja, dan hubungan sosial di tempat kerja. b) Kemajuan (advancement), besar kecilnya kemungkinana tenaga kerja dapat maju dalam pekerjaannya. c) Pekerjaan itu sendiri, besar kecilnya kemungkinana tenaga kerja mencapai prestasi kerja yang tinggi. Menurut Robbins dan Judge (2013), kepuasan kerja memiliki lima dimensi yaitu: Menurut Robbins dan Judge (2013), kepuasan kerja memiliki lima dimensi yaitu: d) Capaian (achievement), besar kecilnya kemungkinan tenaga kerja mencapai prestasi kerja yang tinggi. 1. Pekerjaan itu sendiri dengan indikator: tugas, kesempatan belajar, dan tanggung jawab. 2. Gaji saat ini, dengan indikator: sistem penggajian dan keadilan penggajian. e) Pengakuan (recognition), besar kecilnya pengakuan yang diberikan kepada tenaga kerja atas unuk-kerjanya 3. Kesempatan promosi, dengan indikator: peluang promosi. 4. Pimpinan, dengan indikator: gaya memimpin. 2) Faktor-faktor Ekstrinsik a) Administrasi dan kebijakan perusahaan, derajat kesesuaian yang dirasakan tenaga kerja dari semua kebijakan dan peraturan yang berlaku dalam perusahaan. 5. Rekan kerja, dengan indikator: dukungan antar rekan kerja. Kepuasan Kerja Menurut Jex dan Britt (2008) menyatakan bahwa kepuasan kerja pada dasarnya merupakan sikap pegawai terhadap pekerjaannya. Sikap tersebut adalah pernyataan evaluative baik yang menyenangkan atau yang tidak menyenangkan, mengenai objek atau peristiwa. Sikap tersebut mencerminkan bagaimana seseorang merasakan sesuatu. Kepuasan kerja menunjukkan kesesuaian antara harapan seseorang akan sesuatu dengan apa yang benar-benar diterima, sehingga tingkat kepuasaan kerja pegawai secara individu berbeda- beda. Perbedaan disebabkan karena masing-masing individu memiliki perbedaan baik dalam nilai yang dianut, sikap, perilaku maupun motivasi untuk bekerja. METODE PENELITIAN b) Penyeliaan, derajat kewajaran penyeliaan yang dirasakan diterima oleh tenaga kerja. Jenis penelitian yang digunakan adalah dengan menggunakan penelitian kuantitatif, yang menekankan analisisnya pada data-data numerical (angka) yang diolah dengan metode statistika. Teknik sampling yang digunakan dalam penelitian ini adalah teknik probability sampling, yaitu teknik pengambilan sampel yang memberikan peluang yang sama bagi setiap unsur atau anggota populasi untuk dipilih menjadi anggota sampel. Teknik probability sampling yang digunakan dalam penelitian ini adalah simple random sampling. Menurut Sugiyono (2014) simple random sampling adalah teknik sampling sederhana yang dilakukan secara acak tanpa memperhatikan strata yang ada dalam populasi. Subjek dalam penelitian ini adalah perawat yang masih aktif bekerja di RSUD BLUD Soemarno Sosroatmodjo Tanjung Selor yang berjumlah 70. Metode penelitian ini menggunakan skala likert. c) Gaji, derajat kewajaraan dari gaji yang diterima sebagai imbalan unjuk-kerjanya. d) Hubungan antarpribadi, derajat kesesuaian yang dirasakan dalam berinteraksi dengan tenaga kerja lainnya. e) Kondisi kerja, derajat kesesuaian kerja dengan proses pelaksanaan tugas pekerjaannya. e) Kondisi kerja, derajat kesesuaian kerja dengan proses pelaksanaan tugas pekerjaannya. Kesimpulan Berdasarkan hasil penelitian dan pembahasan, maka dapat ditarik kesimpulan sebagai berikut: p p g 1. Terdapat hubungan antara motivasi kerja dan kepuasan kerja dengan pelayanan prima pada perawat RSUD BLUD dr. H. Soermano Sosrotmodjo Tanjung Selor. Hal ini menyatakan semakin tinggi pelayanan prima seorang perawat maka akan tinggi pula tingkat motivasi kerja dan kepuasan kerja pada perawat. Hal tersebut dapat dilihat dari berbagai aspek variable dependent yang beberapa berhubungan secara signifikan terhadap aspek-aspek variable independent dan dari hasil uji hipotesis yang menyatakan ada hubungan yang signifikan dengan nilai F = 203.668, R2 = 0.859, dan p = 0.000 pada perawat RSUD BLUD Tanjung Selor antara motivasi kerja dan kepuasan kerja dengan pelayanan prima. Kontribusi hubungan (R2) motivasi kerja dan hubungan kerja dengan pelayanan prima adalah sebesar 0.859, hal ini menunjukkan bahwa 85.9 persen dari pelayanan prima dapat dijelaskan oleh motivasi kerja dan kepuasan kerja. Sedangkan sisanya 14.1 persen dijelaskan oleh variable lain atau sebab-sebab lain yang tidak diteliti dalam penelitian ini. Sejalan dengan penelitian yang dilakukan oleh Natshir (2008) beberapa factor yang mempengaruhi pegawai dalam memberikan pelayanan prima pada pasien di rumah sakit yaitu, motivasi kerja, kepuasn kerja, tingkat stress, kondisi fisik pekerjaan, system kompensasi, desain pekerjaan, aspek ekonomi dan karakteristik pegawai seperti umur dan masa kerja. 2. 2. Tidak ada hubungan positif dan signifikan antara motivasi kerja dengan pelayanan prima pada perawat perawat RSUD BLUD dr. H. Soemarno Sosroatmodjo Tanjung Selor. Berarti semakin rendah motivasi kerja yang dirasakan maka akan semakin renda pelayanan prima yang dilakukan. 2. Tidak ada hubungan positif dan signifikan antara motivasi kerja dengan pelayanan prima pada perawat perawat RSUD BLUD dr. H. Soemarno Sosroatmodjo Tanjung Selor. Berarti semakin rendah motivasi kerja yang dirasakan maka akan semakin renda pelayanan prima yang dilakukan. Seperti yang disampaikan oleh Anoraga (1992) kepuasan kerja berhubungan dengan sikap dari karyawan terhadap pekerjaan itu sendiri, situasi kerja, kerja sama antara pimpinan dan sesama karyawan, dimana sikap merupakan salah satu aspek dari pelayanan prima yang berarti sikap atau perilaku karyawan harus menonjol ketika menghadapi pelanggan. 3. Ada hubungan positif dan signifikan antara kepuasan kerja dengan pelayanan prima pada perawat RSUD BLUD dr. H. Soemarno Sosroatmodjo Tanjung Selor. Berarti semakin tinggi kepuasan kerja pada perawat maka akan semakin tinggi pelayanan prima yang dilakukan oleh perawat. Penelitian sebelumnya dalam jurnal pengaruh pelayanan prima terhadap kepuasan pelanggan salon agata singaraja. Kemampuan memiliki pengaruh terhadap kepuasan pelanggan. KESIMPULAN DAN SARAN Berdasarkan tabel 2, hasil yang diperoleh dari uji hipotesis menunjukkan bahwa F hitung > F tabel yang artinya motivasi kerja dan kepuasan kerja terhadap pelayanan prima memiliki hubungan dan dengan nilai F = 203.668, R2 = 0.859, dan p = 0.000 pada perawat RSUD BLUD Tanjung Selor, hal ini menunjukkan bahwa H1 diterima dan H0 ditolak. HASIL PENELITIAN DAN PEMBAHASAN Pengujian hipotesis yang disajikan dalam bentuk analisis regresi model penuh bertujuan untuk menguji hipotesis penelitian yang berbunyi “Ada hubungan motivasi kerja dan kepuasan kerja dengan pelayanan prima”. Berikut rangkuman hasil analisis model penuh disajikan dalam tabel di bawah ini: Menurut Robbins and Judge, (2011) mendefinisikan kepuasan kerja sebagai perasaan 502 ISSN: 2477-2666/E-ISSN: 2477-2674 Psikoborneo, Vol 6, No 4, 2018:499- 504 Tabel 2. Hasil Uji Analisis Regresi Model Penuh Variabel F Hitung F Tabel R2 P Pelayanan Prima (Y) 203.668 3.19 0.859 0.000 Motivasi Kerja (X1) Kepuasan Kerja (X2) Kesimpulan Hal ini sesuai dengan teori Barata (2003) yang mengemukakan bahwa apabila perusahaan memberikan karyawan kemampuan (ability) yang meliputi pengetahuan dan keterampilan tertentu yang mutlak diperlukan untuk menunjang pelayanan prima yang meliputi kemampuan dalam bidang kerja yang ditekuni, melaksanakan komunikasi yang efektif, mengembangkan motivasi, dan mengembangkan public relation sebagai instrument dalam membina hubungan ke dalam dan keluar organisasi atau perusahaan. Saran Berdasarkan hasil penelitian yang diperoleh, maka dapat dikemukakansaran-saran sebagai berikut: 1. Bagi seluruh perawat diharapkan bekerja secara loyalitas dan lebih berkomitmen dalam memberikan pelayanan kepada pasien sehingga pekerjaan yang dilakukan tidak semata-mata hanya karena insentif dan kepada seluruh perawat/tenaga medis dapat meningkatkan pelatihan agar dapat meningkatkan kualitas pelayanan yang akan diberikan kepada pasien. 503 ISSN: 2477-2666/E-ISSN: 2477-2674 Psikoborneo, Vol 6, No 4, 2018:499- 504 2. Bagi seluruh perawat RSUD BLUD dr.H. Soemarno Sosroatmodjo Tanjung Selor dapat meningkatkan kerja sama tim seperti membuat program-program kerja sama tim, melakukan outbound, piknik dan mengadakan gathering, sehingga hal tersebut dapat meningkatkan semangat kerja, meningkatkan kualitas pekerjaan, loyalitas dan sesame rekan kerja perawat menjadi lebih interaktif. 2. Bagi seluruh perawat RSUD BLUD dr.H. Soemarno Sosroatmodjo Tanjung Selor dapat meningkatkan kerja sama tim seperti membuat program-program kerja sama tim, melakukan outbound, piknik dan mengadakan gathering, sehingga hal tersebut dapat meningkatkan semangat kerja, meningkatkan kualitas pekerjaan, loyalitas dan sesame rekan kerja perawat menjadi lebih interaktif. Elhaitammy. (2011). Pengaruh Service Quality Terhadap Kepuasan Pelanggan Pada Lembaga Pendidikan Solocom Di Surakarta. BENEFIT Jurnal Manajemen dan Bisnis, 15(2), 101- 108. Endarini, S. (2001). Pelayanan prima. Yogyakarta: Kanwil Departemen Kesehatan Propinsi DIY. George., & Jones. (2005). Analisis Pengaruh Motivasi Kerja Terhadap Kepuasan Kerja (Studi Kasus pada Karyawan Restoran di Pakuwon Food Festival Surabaya). Jurnal Manajemen dan Kewirausahaan, 12(1), 100- 112. 3. Bagi pemimpin RSUD BLUD dr.H. Soemarno Sosroatmodjo Tanjung Selor diharapkan dapat meningkatkan gaya kepemimpinan yang lebih baik lagi kepada seluruh karyawan seperti memberikan motivasi kerja, meningkatkan sikap kerja yang positif, seperti memberikan feed back yang positif kepada hasil pekrjaan perawat/tenaga medis. Hermawan, G. (2016). Hubungan Iklim Organisasi dengan Kepuasan Kerja Karyawan PT. Jembayan Muarabara Desa Separi Tenggarong Seberang. Jurnal Psikologi, 4(2), 373-384. 4. Bagi pihak RSUD Tanjung Selor ada beberapa saran yang akan diberikan yaitu: 4. Bagi pihak RSUD Tanjung Selor ada beberapa saran yang akan diberikan yaitu: 1) Dalam menempatkan perawat sebaiknya melakukan assessment terlebih dahulu agar pekerjaan yang diberikan pada perawat sesuai dengan keilmuan atau pendidikannya, sehingga hal tersebut dapat mengembangkan prestasi masing-masing perawat. 4. Bagi pihak RSUD Tanjung Selor ada beberapa saran yang akan diberikan yaitu: Jex, M. S., & Britt, W. T. (2008). Organizational Psychology: a scientistpractitioner approach- 2nd ed. New Jersey: John Wiley & Sons, inc 1) Dalam menempatkan perawat sebaiknya melakukan assessment terlebih dahulu agar pekerjaan yang diberikan pada perawat sesuai dengan keilmuan atau pendidikannya, sehingga hal tersebut dapat mengembangkan prestasi masing-masing perawat. Saran Rahmayanty, N. (2010). Manajemen Pelayanan Prima (Mencegah Pembelotan dan Membangun Customer Loyality), Graha Ilmu, Yogyakarta. Reksoatmodjo, T. N. (2009). Statistika untuk Psikologi dan Pendidikan. Jakarta: Aditama 2) Dapat memberikan penilaian kerja dengan insentif sesuai dengan beban kerja yang dilakukan oleh perawat Robbins, S. P & Judge, Timothy A. 2013. Organizational Behavior Edition 15. New Jersey: Pearson Education 3) Kemudian pihak RSUD diharapkan memberikan jabatan yang sesuai dengan kompetensi tiap-tiap perawat, agar perawat memiliki semangat kerja yang tinggi sehingga hal tersebut tidak mempengaruhi pelayanan yang diberikan perawat terhadap pasien. Schaufeli., & Jauczur. (1994). Hubungan Antara Kematangan Emosi Dan Burnout Pada Perawat Rumah Sakit Pku Muhammadiyah Bantul Yogyakarta. Journal Psychology, 10 (1). Sugiyono. (2015). Statistik untuk Penelitian. Bandung: Penerbit Alfabeta. DAFTAR PUSTAKA Sunartini. (2000). Pelayanan Prima di Rumah Sakit, Peran Profesi dan Unit Penunjang. Jurnal: Dalam Menuju Paradigma Sehat dengan Pelayanan Prima. Yogyakarta: RSUP Dr. Sardjito dan Fakultas Kedokteran UGM. Anoraga, P. (2006). Psikologi Kerja. Jakarta: PT. Rineka Cipta. Ashar, S. M. (2014). Psikologi Industri dan Organisasi. Jakarta: Penerbit Universitas Indonesia. Barata, A. A. (2004). Dasar – Dasar Pelayanan Prima. Jakarta : PT Elex Media Komputindo. 504
https://openalex.org/W2106339428	https://ccforum.biomedcentral.com/counter/pdf/10.1186/cc8896	English	null	Non-invasive ventilation in acute respiratory failure related to 2009 pandemic Influenza A/H1N1 virus infection	Critical care	2,010	cc-by	1,516	Non-invasive ventilation in acute respiratory failure related to 2009 pandemic Infl uenza A/H1N1 virus infection João Carlos Winck1,2 and Anabela Marinho1 See related research by Rello et al., http://ccforum.com/content/13/5/R148 João Carlos Winck1,2 and Anabela Marinho1 See related research by Rello et al., http://ccforum.com/content/13/5/R148 See related research by Rello et al., http://ccforum.com/content/13/5/R148 Non-invasive ventilation (NIV) is considered ﬁ rst-line intervention for diﬀ erent causes of acute respiratory failure [1]. However, Rello and colleagues [2] show high rates of NIV failure in pandemic Inﬂ uenza A/H1N1 virus infection (PH1N1). association with antibiotics and diuretics. On day 2, a nasopharyngeal swab was positive for PH1N1. Th e patient was subsequently transferred to a negative- pressure ward. He was still tachypneic, with basal crackles and a PaO2/fraction of inspired oxygen (FiO2) ratio of 246. NIV (BiPAP Vision; Philips Respironics, Murrysville, PA, USA) through an oro-nasal mask in bi- level positive airway pressure mode (inspiratory positive airway pressure [IPAP] = 16 cm H2O, expiratory positive airway pressure [EPAP] = 8 cm H2O) was started. Due to patient preference, the mode was changed to continuous positive airway pressure (CPAP) at 10 cm H2O and an FiO2 of 25%. After 1 hour, PaO2/FiO2 increased to 364, and CPAP was stopped after 12 hours. We describe a patient with PH1N1 in whom NIV was eﬀ ective. A 53-year-old male was admitted in November 2009 with cough, dyspnea, and hemoptysis. His tempera- ture was 38.9°C, he was tachypneic, with diﬀ use rhonchi and bilateral crackles, and oxygen saturation was 96% (4 L/min oxygen). Arterial partial pressure of oxygen (PaO2) and arterial partial pressure of carbon dioxide (PaCO2) were 76 and 23 mm Hg, respectively. Creatine kinase (2,278 U/L) and brain natriuretic peptide (3,544 pg/ mL) were increased. Acute myocardial infarction was excluded. Chest x-ray showed bilateral interstitial inﬁ l- trates and cardiomegaly. Echocardiogram showed severe left ventricular systolic dysfunction. PH1N1 pneumonia was suspected, and oseltamivir was administered in Recently, Djibré and colleagues [3] demonstrated the eﬀ ectiveness of NIV in acute respiratory distress syn- drome related to PH1N1 pneumonia. Our case further supports its role in a hypoxemic patient with cardiogenic pulmonary edema and PH1N1 pneumonitis. Winck and Marinho Critical Care 2010, 14:408 http://ccforum.com/content/14/2/408 Acknowledgments 5. Ramsey CD, Funk D, Miller III DF, Kumar A: Ventilator management for hypoxemic respiratory failure attributable to H1N1 novel swine origin infl uenza virus. Crit Care Med 2010, 3 (suppl). 5. Ramsey CD, Funk D, Miller III DF, Kumar A: Ventilator management for hypoxemic respiratory failure attributable to H1N1 novel swine origin infl uenza virus. Crit Care Med 2010, 3 (suppl). Winck and Marinho Critical Care 2010, 14:408 http://ccforum.com/content/14/2/408 Winck and Marinho Critical Care 2010, 14:408 http://ccforum.com/content/14/2/408 Winck and Marinho Critical Care 2010, 14:408 http://ccforum.com/content/14/2/408 or presenting with exacer bation of chronic obstructive pulmonary disease might beneﬁ t from this alternative therapy as it has been reported. 2. Rello J, Rodríguez A, Ibañez P, Socias L, Cebrian J, Marques A, Guerrero J, Ruiz-Santana S, Marquez E, Del Nogal-Saez F, Alvarez-Lerma F, Martínez S, Ferrer M, Avellanas M, Granada R, Maraví-Poma E, Albert P, Sierra R, Vidaur L, Ortiz P, Prieto del Portillo I, Galván B, León-Gil C; H1N1 SEMICYUC Working Group: Intensive care adult patients with severe respiratory failure caused by Infl uenza A (H1N1)v in Spain. Crit Care 2009, 13:R148. 3. Djibré M, Berkane N, Salengro A, Ferrand E, Denis M, Chalumeau-Lemoine L, Parrot A, Mayaud C, Fartoukh M: Non-invasive management of acute respiratory distress syndrome related to Infl uenza A (H1N1) virus pneumonia in a pregnant woman. Intensive Care Med 2010, 36:373-374. 4. Agarwal R, Reddy C, Aggarrwal AN, Gupta D: Is there a role for noninvasive ventilation in acute respiratory distress syndrome? A meta-analysis. Respir Med 2006, 100:2235-2238. 5. Ramsey CD, Funk D, Miller III DF, Kumar A: Ventilator management for hypoxemic respiratory failure attributable to H1N1 novel swine origin infl uenza virus. Crit Care Med 2010, 3 (suppl). 6. Rodriguez A, Lisboa T, Rello J: Pandemic infl uenza A (H1N1)v in the intensive care unit: what have we learned? Arch Bronchoneumol 2010, 46(suppl 2):24-31. 7. Conti G, Larrsson A, Nava S, Navalesi P: On the role of non-invasive (NIV) to treat patients during the H1N1 infl uenza pandemic [http://dev.ersnet.org/ uploads/Document/63/WEB_CHEMIN_5410_1258624143.pdf]. doi:10.1186/cc8896 Cite this article as: Winck JC, Marinho A: Non-invasive ventilation in acute respiratory failure related to 2009 pandemic Infl uenza A/H1N1 virus infection. Critical Care 2010, 14:408. 2. Rello J, Rodríguez A, Ibañez P, Socias L, Cebrian J, Marques A, Guerrero J, Ruiz-Santana S, Marquez E, Del Nogal-Saez F, Alvarez-Lerma F, Martínez S, Ferrer M, Avellanas M, Granada R, Maraví-Poma E, Albert P, Sierra R, Vidaur L, Ortiz P, Prieto del Portillo I, Galván B, León-Gil C; H1N1 SEMICYUC Working Group: Intensive care adult patients with severe respiratory failure caused by Infl uenza A (H1N1)v in Spain. Crit Care 2009, 13:R148. Published: 19 March 2010 Published: 19 March 2010 doi:10.1186/cc8896 Cite this article as: Winck JC, Marinho A: Non-invasive ventilation in acute respiratory failure related to 2009 pandemic Infl uenza A/H1N1 virus infection. Critical Care 2010, 14:408. Author details 6. Rodriguez A, Lisboa T, Rello J: Pandemic infl uenza A (H1N1)v in the intensive care unit: what have we learned? Arch Bronchoneumol 2010, 46(suppl 2):24-31. 7. Conti G, Larrsson A, Nava S, Navalesi P: On the role of non-invasive (NIV) to treat patients during the H1N1 infl uenza pandemic [http://dev.ersnet.org/ uploads/Document/63/WEB_CHEMIN_5410_1258624143.pdf]. 1Pneumology Department, Faculdade de Medicina da Universidade do Porto, São João Hospital, Alameda Professor Hernâni Monteiro; 4303-451 Porto, Portugal. 2Faculdade de Medicina da Universidade do Porto, São João Hospital, Alameda Professor Hernâni Monteiro; 4303-451 Porto, Portugal Abbreviations CPAP, continuous positive airway pressure; FiO2, fraction of inspired oxygen; NIV, non-invasive ventilation; PaO2, arterial partial pressure of oxygen; PH1N1, pandemic Infl uenza A/H1N1 virus infection. yl 3. Djibré M, Berkane N, Salengro A, Ferrand E, Denis M, Chalumeau-Lemoine L, Parrot A, Mayaud C, Fartoukh M: Non-invasive management of acute respiratory distress syndrome related to Infl uenza A (H1N1) virus pneumonia in a pregnant woman. Intensive Care Med 2010, 36:373-374. 3. Djibré M, Berkane N, Salengro A, Ferrand E, Denis M, Chalumeau-Lemoine L, Parrot A, Mayaud C, Fartoukh M: Non-invasive management of acute respiratory distress syndrome related to Infl uenza A (H1N1) virus pneumonia in a pregnant woman. Intensive Care Med 2010, 36:373-374. Competing interests The authors declare that they have no competing interests. 2. Rello J, Rodríguez A, Ibañez P, Socias L, Cebrian J, Marques A, Guerrero J, Ruiz-Santana S, Marquez E, Del Nogal-Saez F, Alvarez-Lerma F, Martínez S, Ferrer M, Avellanas M, Granada R, Maraví-Poma E, Albert P, Sierra R, Vidaur L, Ortiz P, Prieto del Portillo I, Galván B, León-Gil C; H1N1 SEMICYUC Working Group: Intensive care adult patients with severe respiratory failure caused by Infl uenza A (H1N1)v in Spain. Crit Care 2009, 13:R148. Competing interests p p g 4. Agarwal R, Reddy C, Aggarrwal AN, Gupta D: Is there a role for noninvasive ventilation in acute respiratory distress syndrome? A meta-analysis. Respir Med 2006, 100:2235-2238. 4. Agarwal R, Reddy C, Aggarrwal AN, Gupta D: Is there a role for noninvasive ventilation in acute respiratory distress syndrome? A meta-analysis. Respir Med 2006, 100:2235-2238. Acknowledgments Written consent for publication was obtained from the patient. References 1. Brochard L: Noninvasive ventilation for acute respiratory failure. JAMA 2002, 288:932-935. Authors response Alejandro Rodríguez, Thiago Lisboa, Jordi Rello and H1N1 SEMICYUC Working Group p Alejandro Rodríguez, Thiago Lisboa, Jordi Rello and H1N1 SEMICYUC Working Group We appreciate the interest from Winck and Marinho in our article and their insightful observations regarding non-invasive ventilation (NIV) in severe inﬂ uenza A (H1N1)v. Th e use of NIV in hypoxemic respiratory failure is controversial, and the etiology of hypoxemia appears to be an important determinant of its success. A meta- analysis [4] suggests that non-invasive positive-pressure ventila tion does not decrease the need for intubation, so there is not enough evidence to support its use in acute respira tory distress syndrome. Our experience [2] is consistent with other reports [5,6]; 25% to 30% of patients were non-invasively ventilated on admission, but 70% to 85% of these patients required subsequent intubation and invasive ventilation. Th ere are only a few patients with H1N1-related respiratory failure who seem to beneﬁ t from NIV alone, so it should be reserved for patients with milder disease. Guidelines endorsed by the European Respiratory Society and European Society of Intensive Care Medicine [7] conclude that, as a general rule, NIV not be recommended as an alternative to invasive ventilation in patients aﬀ ected by H1N1. In spite of this, selected patients with hypoxemia and additional cardiac compromise (severe left ventri cular systolic dysfunction) Correspondence: jwinck@hsjoao.min-saude.pt 1Pneumology Department, Faculdade de Medicina da Universidade do Porto, São João Hospital, Alameda Professor Hernâni Monteiro; 4303-451 Porto, Portugal Full list of author information is available at the end of the article Correspondence: jwinck@hsjoao.min-saude.pt 1Pneumology Department, Faculdade de Medicina da Universidade do Porto, São João Hospital, Alameda Professor Hernâni Monteiro; 4303-451 Porto, Portugal Full list of author information is available at the end of the article © 2010 BioMed Central Ltd © 2010 BioMed Central Ltd Page 2 of 2
W3197281483.txt	https://downloads.hindawi.com/journals/ije/2021/9965728.pdf	en		Association between RBC Indices, Anemia, and Obesity-Related Diseases Affected by Body Mass Index in Iranian Kurdish Population: Results from a Cohort Study in Western Iran	International journal of endocrinology	2,021	cc-by	8,735	Hindawi International Journal of Endocrinology Volume 2021, Article ID 9965728, 13 pages https://doi.org/10.1155/2021/9965728 Research Article Association between RBC Indices, Anemia, and Obesity-Related Diseases Affected by Body Mass Index in Iranian Kurdish Population: Results from a Cohort Study in Western Iran Maryam Kohsari , Mehdi Moradinazar , Zohreh Rahimi, Farid Najaﬁ, Yahya Pasdar , Atefeh Moradi, and Ebrahim Shakiba Behavioral Disease Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran Correspondence should be addressed to Mehdi Moradinazar; m.moradinazar@gmail.com Received 28 March 2021; Accepted 27 August 2021; Published 6 September 2021 Academic Editor: Christian S. Goebl Copyright © 2021 Maryam Kohsari et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. The relationship between RBC indices and metabolic diseases remains unclear. The association between anemia and obesity is also controversial. The present study aimed to investigate the relationship between RBC indices and metabolic diseases caused by obesity and evaluate the eﬀect of body mass index (BMI) on RBC indices on the Ravansar cohort data. Method. For the purpose of this study, 9826 participants aged 35–65 years (5158 females and 4668 males) were recruited in the analyses. A quadratic prediction ﬁt plot investigated the association between RBC indices with BMI and lipid proﬁle. The odds ratio of obesity-related diseases in each quartile category of RBC indices and anemia was estimated using multivariable logistic regression models. Results. Subjects in the fourth quartiles of RBC count, hematocrit (HCT), hemoglobin (HGB), and red cell distribution width (RDW) had a higher risk for obesity-related diseases compared to the ﬁrst quartiles. However, individuals with the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in fourth quartiles had lower ORs of obesity-related diseases. While BMI reduced the eﬀect of RBC count, HCT, HGB, and RDW on the incidence risk of obesity-related disease, it increased the impact of MCV, MCH, and MCHC. There was a negative association between BMI and RBC indices except for RDW. The BMI eﬀect on RBC indices was diﬀerent in normal and obese individuals. BMI in mild anemia lowered the risk of metabolic diseases, but it increased the risk of metabolic diseases for moderate anemia. Conclusion. A higher risk of obesity-related diseases was observed in the fourth quartiles of RBC count, HCT, HGB, and RDW compared to the ﬁrst quartiles. However, the incidence risk was lower for MCV, MCH, and MCHC. BMI plays an anemiatype dependent role in the relationship. Consideration should be given to the type of anemia in the relationship between BMI and anemia. 1. Introduction Obesity is deﬁned as the body mass index (BMI) ≥30 kg/m2 [1]. The rate of obesity has grown so dramatically in the last three decades that in 2014, almost 30% of the world’s population was considered overweight and obese, and the number is estimated to reach 50% mark by 2030 [2]. The role of obesity in metabolic diseases including diabetes mellitus (DM), cardiovascular disease (CVD), metabolic syndrome (MetS) [3], and hypertension (HTN) [4] is clearly understood. Obesity has a potent correlation with dyslipidemia that contributes to CVD risk developments [5], and an increase in BMI leads to the progression of heart damage [4] and nonalcoholic fatty liver disease (NAFLD) [6]. Recently, the role of red blood cell (RBC) indices was identiﬁed in metabolic diseases. The complete blood count (CBC) test which is routinely administered in medical examinations can be utilized in the early detection of metabolic disorders [7]. However, there are limited studies that indicate the role of RBC indices in the incidence of metabolic diseases. A number of reports have suggested that red cell distribution width (RDW) reduced the risk of MetS [8] and 2 increased the risk of CVD [9] and NAFLD [10]. RDW is an indicator that shows the variation in the size of RBC [9, 10]. NAFLD and CVD are diseases in which inﬂammation plays an inﬂuential role. According to these studies, RDW is associated with inﬂammation that may increase in response to proinﬂammatory cytokines. Cytokines can also interact with erythropoietin in the bone marrow, resulting in the lower production of RBC. Besides, cytokines act as RBC suppressors and raise the number of immature RBC, and RDW increased consequently [9, 10]. On the other hand, increased hematocrit (HCT), hemoglobin (HGB), and red blood cell (RBC) count are associated with an increased chance of MetS [11]. Also, it has been suggested that RBC count has a positive relationship with the severity of HTN [12]. This eﬀect may occur as a result of an additional load on the cardiovascular system by increasing RBC count [13]. Various studies indicated a controversial and contradictory relationship between RBC indices and anemia with lipid proﬁle and BMI. Anemia is considered a risk factor for dyslipidemia [14] and CVD [15]. Some studies indicate a lack of association between increased BMI and obesity with anemia [16–18]. A study conducted in China found that the rate of anemia in overweight women was lower compared to normal subjects [18]. Contradictory results were also found on the Iranian population. In a study of young females in north Iran, Rad et al. demonstrated the absence of a signiﬁcant diﬀerence in anemia prevalence between normal weight and obese females [16]. However, a study of young university students in central Iran (males and females) demonstrated a high incidence of anemia among the population with abnormal BMI [19]. Besides, reports indicated the absence of signiﬁcant correlation [20] and the presence of a negative inverse correlation [21] between mean corpuscular volume (MCV) and BMI. Antwi-Baﬀour et al. illustrated that the lipid proﬁle parameter is positively associated with RBC count and negatively correlated with HGB and HCT [22]. However, studies have discussed the relationship between inﬂammation and anemia [23]. We know that obesity is associated with low-grade chronic systemic inﬂammation. Also, obese people are prone to chronic inﬂammatory diseases such as DM, MetS, liver, and kidney failure, especially with age [24]. Inﬂammation in these people eventually leads to the activation of oxidative stress signaling pathways. Free radicals could cause the peroxidation of erythrocyte membrane lipids and activate Ca2+ permeable nonselective cation channels in the cell membrane. Consequently, phosphatidylserine (PS) translocation enhances from the interior to the cell membrane surface and leads to the erythrocyte suicidal death or eryptosis [25, 26]. In addition, the eﬀect of lipid proﬁle on RBC indices still is ill-deﬁned, although in vitro studies demonstrated that erythrocytes act as a storage of cholesterol for serum lipoproteins, and dyslipidemia may play a role in impairing erythrocyte maturation and deformability [27]. Given that obesity, dyslipidemia, and abnormalities in RBC indices such as anemia all are the risks of CVD. It is crucial to expand our knowledge of the underlying relationships between these factors. As the matter is not also investigated on International Journal of Endocrinology the Iranian population, the present study is primarily conducted to evaluate the association between RBC indices and obesity-related diseases on a Kurdish population in western Iran. We also aimed to examine the eﬀect of lipid proﬁle parameters and BMI on this relationship. 2. Methods 2.1. Study Design and Population. The present study used the data obtained from the Ravansar noncommunicable cohort disease (RaNCD) initial phase, which began in 2014 and ended in 2017. The RaNCD cohort study is part of the Iranian adult (PERSIAN) cohort that studies participants in the age range of 35–65 years and aims to conduct a series of follow-ups for a period of 15 years. Study details have been published [28, 29], and all questionnaires, study instructions, and additional information are available at http:// persiancohort.com. The study was approved by the Ethics Committees of Kermanshah University of Medical Sciences (KUMS.REC.1394.315), Kermanshah, Iran. Subjects aged 35–65 years who were residents of Ravansar for the past nine months were included in the study after they were fully informed of the process and signed written consent. Individuals with underlying kidney disease (101) and pregnant women (125) were excluded from the study to eliminate confounder variables. 2.2. Measurements and Deﬁnition. Fasting blood samples were collected by Venoject tubes. After centrifugation for 10 minutes at 300g, the samples were transferred to cryotubes and were kept at −20°C until the testing time. Serum triglyceride (TG), high-density lipoprotein cholesterol (HDLC), total cholesterol (TC), low-density lipoprotein cholesterol (LDL), and fasting blood glucose (FBG) were analyzed with the enzymatic colorimetric assay by the Mindray-BS380 autoanalyzer (Mindray, USA). RBC indices including RBC count, HCT, HGB, mean corpuscular hemoglobin (MCH), MCHC, and RDW were measured via the CBC test by the Sysmex cell counter. Dyslipidemia was deﬁned based on the presence of one or more abnormalities in the lipid proﬁle, including serum levels of TC ≥ 240 mg/dl, lowdensity lipoprotein (LDL) 160 mg/dl, triglyceride (TG) ≥150 mg/dl, and high-density lipoprotein (HDL) <40 mg/dl [30]. Blood pressure was measured according to the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) classiﬁcation of hypertension to diagnose hypertension [31]. After 10 minutes rest, blood pressure was measured twice for each participant using the cuﬀ on both arms at the heart level with one-minute interval between each measurement. The mean obtained for each arm was used as the ﬁnal blood pressure. Nonalcoholic fatty liver (NAFLD) and cardiovascular diseases (CVD) are based on self-report of participants and use of related medication. NAFLD is reported in nonalcoholic participants with the fatty liver. HTN was deﬁned as SBP ≥140 mm Hg and/or DBP ≥90 m Hg and/or current use of antihypertensive drugs. The presence of three or more of the following criteria identiﬁed the existence of MetS: International Journal of Endocrinology FBS ≥ 100 mg/dl, TG ≥ 150 mg/dl, and reduced HDL-C: <40 mg/dl in males and <50 mg/dl in females, waist circumference (WC) ≥85 cm in males and ≥80 cm in females, and SBP ≥ 130 and DBP ≥ 86 mmHg [32]. Diabetes mellitus was deﬁned as FBS ≥ 126 mg/dl and/or a history of taking medications to treat diabetes [33]. Mild anemia was deﬁned as HGB � 11–11.9 g/dL for females and HGB � 11–12.9 g/dL for males, with moderate anemia as HGB � 8–10.9 g/dL for males and females [34]. The bioimpedance analyzer (BIA) (In Body 770 BIOSPACE, Korea) was used to measure weight. Height was measured with 0.1 cm accuracy using a stadiometer. BMI was calculated by dividing weight (kg) by square of height (m2). BMI was categorized into 18.5–24.9 for normal weight, 25–29.9 for overweight, and greater than 30 for obese. An elastic tape was used to measure upper hip bones for waist circumference. The smoking status was speciﬁed the National Health Interview Survey (NHIS) [35]. The 24-hour physical activity was determined based on average weekday sport, work, and leisure-related activities, classifying the subjects into three categories of low, moderate, and high physical activities [36]. 2.3. Statistical Analysis. Quantitative and qualitative variables were analyzed by the t-test and chi-square test, respectively. Quadratic prediction ﬁt plot with conﬁdence interval was used to assess the correlation between RBC indices with lipid proﬁle and BMI. The relationship between anemia and the risk of obesity-related disorders was presented within the forest plot with an odds ratio (OR) and 95% conﬁdence interval. The association between RBC indices quartiles with dyslipidemia, HTN, NAFLD, MetS, DM, and CVD was investigated by multivariable logistic regression models. For all tests, the statistical signiﬁcance was considered at p level <0.05. Statistical analyses were carried out using Microsoft Excel 2016 and Stata software (version14.2) (Stata Corp, College Station, TX, USA). 3. Results As given in Table 1, the sample included 5158 females (52.5%) with a mean age of 47.5 ± 8.4 years and 4668 males (47.5%) with a mean age 47.0 ± 8.0 years. Overall, 38% of the subjects were considered healthy, 23.8% had one disorder, and 19.6% and 18.6% suﬀered from two disorders and more than two obesity-related disorders, respectively. The prevalence of obesity-related diseases increased with age. Nearly 50% of the individuals over 55 had at least one type of dyslipidemia disorder, and CVD and HTN prevalence doubled compared to the age group of 45–55 years. Subjects with metabolic disorders had higher levels of anthropometric indices and SBP and DBP than the control group. No diﬀerence in hypertension parameters was found for NAFLD patients. Participants with obesity-related diseases had higher mean levels of FBG, TC, TG, and LDL-C, but a lower level of HDL-C. Concerning RBC indices, those who suﬀered from metabolic diseases had signiﬁcantly higher RDW and lower 3 MCV and MCH levels. RBC count, HCT, and HGB levels were signiﬁcantly higher for dyslipidemia subjects. MetS, and DM, with NAFLD and CVD subjects, showed lower levels. There was no diﬀerence in RBC count, HCT, HGB, and MCHC between HTN and controls. All participants with metabolic disorders had signiﬁcantly higher white blood cell (WBC) count. Lymphocyte (lymph) was higher for dyslipidemia and NAFLD patients and lower for HTN and CVD subjects. While only NAFLD showed no diﬀerence in monocyte (mono), this was proved to be higher for other obesity-related subjects. Granulocyte percent (GR %) was higher for dyslipidemia, HTN, and CVD subjects, but lower for NAFLD. Platelet (PLT) count was higher in NAFLD, MetS, and DM patients. The correlation between the level of BMI and hematological parameters was examined by quadratic prediction ﬁt plot along with a conﬁdence interval, Figures 1(a)–1(l). The relationship between BMI and RBC indices (including RBC count, HCT, HGB, MCV, MCH, and MCHC) was positive for normal weights and negative for overweight/obese. RDW in normal weights was negatively related to BMI. In the overweight/obese group, the relationship was positive for RDW. Concerning the association between BMI and PLT count, WBC, and GR%, results were similar to the negative correlation in the normal weights versus the positive correlation in the overweight/obese group. BMI and lymphocyte count correlation was positive for normal weight and was negative for overweight/obese. No distinct diﬀerence was observed between normal and overweight/ obese groups in terms of monocyte count. Figure 2 shows the relationship between lipid proﬁle parameters and RBC indices. TC and RBC indices (A1–A7) were positively related in the normal range but reversed outside. Regarding the RDW, the correlation was inverse. In the overweight/obese group, this relationship was diﬀerent. The positive correlation between TC and RBC indices maintained out of the normal range. Besides, in the overweight/obese group outside the normal range, the TC level increases resulted in an increase in RBC count. TG and RBC indices (B1–B7) in the normal and overweight obese groups were almost similar. Except for the MCHC, which was negatively correlated with TG for normal weights and positively correlated with TG for overweight/obese, the correlation pattern between LDL-C and RBC indices (C1–C7) in both normal and overweight/obese groups was the same as TC results. However, the correlation between LDL-C and RBC count was negative for overweight/obese. HDL-C was inversely related to RBC indices (D1–D7). In overweight/obese individuals, the HDL-C had a positive correlation with MCV and MCHC. Table 2 provides the results of OR with 95% CI according to RBC indices quartiles. After adjusting model 1 for age, gender, smoking status, and physical activity, RBC count, HCT, HGB, and RDW in fourth quartiles had a higher risk for HTN, dyslipidemia, NAFLD, MetS, DM, and CVD compared to the ﬁrst quartiles. HGB and RBC count in the normal values had the highest risk for NAFLD. Also, there was a similar result of HGB for HTN. On the other hand, the risk of obesity-related disorders decreased within increased 3.6 ± 1.3 56.0 ± 9.4 254.2 ± 64.8 189.6 ± 39.4 151.7 ± 91.0 45.9 ± 11.0 104.5 ± 26.2 105.5 ± 39.0 3.4 ± 1.2c 55.6 ± 9.3c 253.0 ± 62.5 181.3 ± 28.4c 102.0 ± 38.0c 51.6 ± 9.3c 99.0 ± 20.4c 93.8 ± 25.6c 3.5 ± 1.2 54.8 ± 9.4 254.5 ± 60.9 190.7 ± 46.7 182.0 ± 99.8 39.8 ± 10.1 105.9 ± 30.1 101.0 ± 34.5 100.1 ± 10.5 96.8 ± 10.4c 102.1 ± 9.9 96.7 ± 10.4c 100.8 ± 9.1 95.4 ± 10.7c 100.9 ± 9.8 96.9 ± 10.5c 100.5 ± 10.5 96.6 ± 10.4c 129.0 ± 21.9 104.3 ± 12.5c 108.6 ± 16.2 108.1 ± 17.1 114.5 ± 19.3 104.8 ± 14.5c 115.3 ± 18.0 107.5 ± 16.7c 119.5 ± 20.3 105.8 ± 15.2c 80.5 ± 12.2 67.8 ± 7.9c 69.8 ± 9.6 69.8 ± 9.9 73.0 ± 11.1 68.0 ± 8.7c 72.8 ± 10.6 69.5 ± 9.8c 74.9 ± 11.2 68.7 ± 9.3c 4.9 ± 0.5 4.9 ± 0.5 4.8 ± 0.01 4.9 ± 0.006b 4.9 ± 0.5 4.8 ± 0.5c 4.99 ± 0.5 4.91 ± 0.5b 4.8 ± 0.5 4.9 ± 0.5c c c a 39.4 ± 4.2 39.4 ± 4.1 38.8 ± 3.9 39.5 ± 4.1 39.7 ± 4.1 39.3 ± 4.1 39.7 ± 3.9 39.4 ± 4.1 38.8 ± 4.1 39.6 ± 4.1c 80.2 ± 7.2 80.7 ± 6.9a 80.2 ± 6.8 80.6 ± 7.0 80.3 ± 6.9 80.7 ± 7.0b 80.0 ± 6.3 80.7 ± 7.0b 80.3 ± 7.0 80.7 ± 7.0a 14.1 ± 1.6 14.1 ± 1.5 13.8 ± 1.5 14.2 ± 1.5c 14.2 ± 1.5 14.1 ± 1.5c 14.2 ± 1.5 14.1 ± 1.5 13.8 ± 1.5 14.2 ± 1.5c b b b b 28.7 ± 3.0 28.9 ± 3.0 28.6 ± 3.0 28.9 ± 3.0 28.8 ± 3.0 29.0 ± 3.0 28.6 ± 2.8 28.9 ± 3.0 28.7 ± 3.0 28.9 ± 3.0b 35.8 ± 1.4 35.8 ± 1.5 35.6 ± 1.6 35.8 ± 1.4c 35.8 ± 1.5 35.8 ± 1.4 35.7 ± 1.7 35.8 ± 1.4 35.7 ± 1.4 35.8 ± 1.5a b c c a 11.09 ± 0.9 11.01 ± 0.9 11.2 ± 1.1 11.0 ± 0.9 11.1 ± 0.9 10.9 ± 1.0 11.0 ± 0.8 11.0 ± 0.9 11.1 ± 1.0 11.0 ± 0.9b 6.5 ± 1.6 6.4 ± 1.5b 6.6 ± 1.5 6.4 ± 1.6c 6.7 ± 1.5 6.2 ± 1.5c 7.0 ± 1.7 6.3 ± 1.5c 6.5 ± 1.6 6.4 ± 1.5c 98.7 ± 9.6 96.2 ± 11.0c 110.4 ± 17.3 106.3 ± 16.5c 71.0 ± 10.1 68.8 ± 9.6c 5.01 ± 0.5 4.8 ± 0.5c 40.1 ± 4.1 38.9 ± 4.0c 80.4 ± 6.9 80.8 ± 7.0b 14.4 ± 1.5 13.9 ± 1.5c 28.9 ± 3.0 28.9 ± 3.0 35.9 ± 1.4 35.8 ± 1.5b 11.1 ± 0.9 10.9 ± 1.0c 6.6 ± 1.6 6.2 ± 1.5c 40.3 ± 8.9 320 (20.9) 641 (41.9) 566 (37.2) 926 (21.3) 1910 (34.8)c 2108 (48.5) 2126 (38.7) 1310 (30.2) 1446 (26.5) 40.8 ± 8.8b 271 (13) 1190 (57.4) 884 (42.6) 41.5 ± 8.8 731 (15.2) 2803 (58.2) 2014 (41.8) 55.1 ± 9.3b 253.5 ± 61.2 184.7 ± 37.6c 134.9 ± 80.5c 46.5 ± 11.3 101.6 ± 25.2c 95.4 ± 27.9c 3.4 ± 1.2C 41.3 ± 8.7c 2516 (30.3)c 3592 (43.2) 2190 (26.5) 1802 (87) 4086 (84.8) 2410 (82.1)c 54.7 ± 9.2 264.4 ± 64.3 186.9 ± 37.9 150.3 ± 88.4 45.42 ± 11.0 102.5 ± 25.8 102.9 ± 33.7 3.5 ± 1.3 41.7 ± 8.7 120 (11.9) 448 (44.4) 440 (43.7) 124 (6) 519 (10.8) 365 (12.4) 557 (26.9) 1516 (73.1) 3126 (64.9) 55.3 ± 9.4a 252.4 ± 61.4c 185.3 ± 38.0 136.1 ± 81.6c 46.5 ± 11.3b 102.0 ± 25.4 96.3 ± 29.6c 3.5 ± 1.2 41.1 ± 8.8a 55.0 ± 9.3 259.0 ± 61.9 191.3 ± 39.1 197.7 ± 98.8 40.0 ± 8.7 105.7 ± 26.0 109.7 ± 39.6 3.5 ± 1.2 41.3 ± 8.7 55.4 ± 9.4 250.8 ± 61.5c 182.4 ± 37.0c 105.6 ± 47.9c 49.7 ± 11.1c 100.1 ± 24.9c 90.3 ± 20.7c 3.4 ± 1.2a 41.0 ± 8.8 2716 (30.8)c 487 (14.1) 2349 (36.7)c 3786 (89.4) 1676 (48.8) 2557 (39.9) 2316 (84) 1269 (37.1) 1487 (23.4) 1950 (94) 4298 (89.2) 1691 (35.1) 2570 (87.6)c 1184 (40.3) 1751 (59.7)c 55.1 ± 9.6 259.8 ± 62.7 190.2 ± 43.7 179.5 ± 119.0 44.0 ± 11.0 104.0 ± 28.1 167.8 ± 62.9 3.6 ± 1.3 41.2 ± 9.0 148 (17.5) 360 (42.5) 338 (40) 129 (6.2) 417 (8.7) 300 (10.3) 525 (17.9) 4234 (91)c 1266 (27.2) 3386 (72.8)c 2756 (87.9) 1247 (39.8) 1888 (60.2) 1828 (89.7) 919 (45.1) 1119 (54.9) 1033 (89.8)c 399 (34.7) 751 (65.3)c 1446 (49.3) 1489 (50.7)c 418 (9) 380 (12.1) 210 (10.3) 117 (10.2) 55.2 ± 9.3 253.1 ± 61.7b 185.0 ± 37.3b 133.6 ± 76.9c 46.6 ± 11.3c 101.9 ± 25.1a 90.3 ± 9.8c 3.5 ± 1.2a 41.1 ± 8.8 2680 (30.0)c 3846 (43.0) 2406 (26.9) 1935 (93.8) 4379 (91.3) 2618 (89.7)c 4425 (95.7)c 2776 (89) 1731 (85) 1044 (91.2)c 55.9 ± 9.5 256.2 ± 65.2 187.4 ± 40.1 150.4 ± 87.6 46.1 ± 11.2 102.9 ± 26.6 107.7 ± 40.2 3.5 ± 1.3 40.5 ± 9.0 320 (19.5) 688 (41.9) 631 (38.6) 237 (11.4) 808 (16.8) 594 (20.2) 310 (6.7) 561 (17.9) 768 (37.7) 190 (16.5) 55.1 ± 9.3b 253.2 ± 61.1 185.1 ± 37.5a 134.9 ± 81.1c 46.4 ± 11.3 101.9 ± 25.2 94.8 ± 27.1c 3.5 ± 1.2a 41.3 ± 8.7b 2516 (30.7)c 3546 (43.3) 2125 (26.0) 1837 (88.6) 4009 (83.2) 2341 (79.8)c 4342 (93.3)c 2575 (82.1) 1270 (62.3) 960 (83.5)c No 8187 (83.3) 4101 (87.8) 198 (4.3) 343 (11) 305 (15) 101 (8.8) 4360 (93.7)c 2581 (82.3) 1357 (66.6) 964 (83.8)c Yes 1639 (16.7) 567 (12.2) 292 (6.3) 554 (17.7) 681 (33.4) 186 (16.2) No 8932 (91.4) 4262 (91.8) 1901 (40.9) 2751 (59.1)c 1444 (46) 1692 (54) 999 (49) 1039 (51) 519 (45.1) 631 (54.9)b CVD Yes 846 (8.6) 379 (8.2) DM Dyslipidemia HTN NAFLD MetS Yes No Yes No Yes No Yes No 4344 (44.2) 5482 (55.8) 1527 (15.5) 8298 (84.5) 1008 (10.3) 8818 (89.7) 3432 (33.3) 6393 (66.7) 2475 (53) 2193 (47)c 673 (14.4) 3995 (85.6) 306 (6.7) 4362 (93.4)c 1605 (34.4) 3063 (65.6) P value <0.05. bP value <0.01. cP value <0.001. a Number % Males Age group 35–45 46–55 56–65 Smoke Physical activity daily METs Low (24–36.5) Moderate (36.6–44.9) High (≥45) BMI Normal Overweight Obesity Mean ± SD WC (cm) SBP (mmHg) DBP (mmHg) RBC (106 μ/L) Hct (%) MCV (fL) HGB (g/L) MCH (pg) MCHC (g/dl) RDW-CV (%) WBC (103 μ/L) Lymphocyte (103 μ/L) Monocyte (103 μ/L) GR % PLT (103 μ/L) TC (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) FBS (mg/dl) Variables Table 1: General and biochemical characteristics of participants. 4 International Journal of Endocrinology International Journal of Endocrinology 14.5 40 4.8 39 HGB (g/dl) 4.9 HCT (%) 38 37 4.7 4.6 36 20 30 40 BMI (kg/m2) 20 (a) 50 35.5 30 40 BMI (kg/m2) 50 10 95% CI normal overweight/obese 20 30 40 BMI (kg/m2) (e) 7 6.5 6 20 30 40 BMI (kg/m2) 95% CI normal overweight/obese (i) 50 34 20 30 40 BMI (kg/m2) 50 10 20 50 95% CI normal overweight/obese (j) 30 40 BMI (kg/m2) 50 95% CI normal overweight/obese (h) 64 62 4 3.5 60 58 56 54 3 30 40 BMI (kg/m2) 260 (g) 36 20 280 240 10 4.5 10 300 95% CI normal overweight/obese 32 10 (d) 11 50 38 50 320 MONO ( 103 U/L) 40 LYMPH (103 U/L) 8 7.5 30 40 BMI (kg/m2) 95% CI normal overweight/obese 11.5 (f ) 42 20 340 95% CI normal overweight/obese 8.5 10 10.5 35 20 50 (c) 36 27 30 40 BMI (kg/m2) 12 RDW- CV (%) MCHC (g/dl) 28 10 20 95% CI normal overweight/obese 36.5 29 78 76 10 (b) 30 MCH (pg) 30 40 BMI (kg/m2) 80 13 95% CI normal overweight/obese 95% CI normal overweight/obese WBC (103 U/L) 13.5 12.5 10 50 14 PLT (103 U/L) 10 82 GR (%) RBC (106 U/L) 5 MCV (fL) 5.1 5 10 20 30 40 BMI (kg/m2) 95% CI normal overweight/obese (k) 50 10 20 30 40 BMI (kg/m2) 50 95% CI normal overweight/obese (l) Figure 1: Quadratic ﬁt plots with conﬁdence intervals between BMI and hematological parameters among normal and overweight/obese groups. MCV, MCH, and MCHC levels. Adjusted model 2 for model 1 plus BMI indicated the dual eﬀects of BMI on the relationship between RBC indices and obesity-related diseases. BMI had a reducing eﬀect on the increased ORs of obesityrelated disorders by RBC count, HCT, HGB, and RDW and decreased the risk of these diseases by RBC indices. However, BMI increased the inﬂuence of MCV, MCH, and MCHC on obesity-related diseases. The association between anemia and the OR of obesityrelated diseases is illustrated in Figure 3. In adjusted model 1 for age, gender, smoking status, and physical activity, subjects with mild anemia had 0.83, 0.91, 0.90, 0.85, 0.94, and 1.06-fold risk of dyslipidemia, HTN, NAFLD, MetS, DM, and CVD, respectively, compared with the subject in ﬁrst quartiles. While the BMI eﬀect in model 2 (model 1 plus BMI) decreased the OR for mild anemia, it increased the risk of obesity-related diseases for the moderate anemia participants. 4. Discussion The current study indicated that the prevalence of dyslipidemia, MetS, CVD, HTN, NAFLD, and DM was 44.2, 33.3, 16.7, 15.5, 10.3, and 8.6% among participants from the Ravansar cohort. According to the literature, no studies have targeted a large homogeneous population in terms of examining the association between complete RBC indices and the risk of metabolic diseases, the eﬀect of BMI alteration on RBC indices, and the inﬂuence of lipid proﬁle on hematological indices. Yet we found an increase of WBC, monocytes, PLT counts, and RDW for metabolic diseases. Overweight/obese individuals with increased BMI had also higher WBC, PLT, GR%, and RDW. Increased WBC count in overweight/obese people can be explained by production of the IL-6, a proinﬂammatory cytokine in adipose tissue that plays a role in bone marrow granulopoiesis, WBC proliferation, and diﬀerentiation [37]. 6 International Journal of Endocrinology A1 B1 4.8 5 4.5 4 400 500 95% CI overweight/obese normal B2 40 38 HCT (%) 40 38 36 34 0 100 200 300 TC (mg/dl) 400 95% CI overweight/obese normal A3 500 TG (mg/dl) 100 200 300 TC (mg/dl) 400 15 14 14 13 500 TG (mg/dl) 1000 15 400 0 MCV (fL) 80 80 95% CI overweight/obese normal 500 TG (mg/dl) D4 85 80 75 1000 75 70 65 0 95% CI overweight/obese normal A5 100 200 300 LDL-C (mg/dl) 400 0 95% CI overweight/obese normal B5 C5 30 30 28 26 24 100 200 300 TC (mg/dl) 95% CI overweight/obese normal 400 500 28 22 24 0 500 TG (mg/dl) 1000 26 24 26 24 0 MCH (pg) 30 MCH (pg) 30 MCH (pg) 32 28 150 D5 32 26 50 100 HDL-C (mg/dl) 95% CI overweight/obese normal 32 28 150 95% CI overweight/obese normal 70 75 0 50 100 HDL-C (mg/dl) C4 85 MCV (fL) 100 200 300 LDL-C (mg/dl) 95% CI overweight/obese normal 80 500 13 11 B4 400 14 12 0 85 150 D3 12 90 200 300 TC (mg/dl) 50 100 HDL-C (mg/dl) 16 95% CI overweight/obese normal A4 100 0 95% CI overweight/obese normal 13 85 0 400 11 0 70 100 200 300 LDL-C (mg/dl) C3 15 95% CI overweight/obese normal 75 35 95% CI overweight/obese normal 16 500 40 30 0 HGB (g/dl) HGB (g/dl) 0 D2 34 1000 12 12 150 95% CI overweight/obese normal 36 B3 13 50 100 HDL-C (mg/dl) 45 95% CI overweight/obese normal 14 0 30 0 15 400 32 32 34 100 200 300 LDL-C (mg/dl) C2 40 38 5 95% CI overweight/obese normal 42 HCT (%) HCT (%) 0 42 36 HGB (g/dl) 1000 95% CI overweight/obese normal A2 MCV (fL) 500 TG (mg/dl) 5.5 4.5 4 0 HCT (%) 200 300 TC (mg/dl) 4.5 HGB (g/dl) 100 5 MCV (fL) 0 D1 6 RBC (106 u/L) 5 RBC (106 u/L) RBC (106 u/L) RBC (106/uL) 5.2 4.6 MCH (pg) C1 5.5 5.5 5.4 0 95% CI overweight/obese normal 100 200 300 LDL-C (mg/dl) 95% CI overweight/obese normal (a) Figure 2: Continued. 400 0 50 100 HDL-C 95% CI overweight/obese normal 150 International Journal of Endocrinology 36 35 37 36 35 100 200 300 TC (mg/dl) 400 500 0 95% CI overweight/obese normal 500 TG (mg/dl) 1000 11 10.5 10 95% CI overweight/obese normal 400 500 100 200 300 LDL-C (mg/dl) 400 0 C7 12 11.5 11 10.5 1000 12 11.5 11 10.5 13 12 11 10 0 95% CI overweight/obese normal 150 D7 14 10 500 TG (mg/dl) 50 100 HDL-C (mg/dl) 95% CI overweight/obese normal 12.5 0 35 95% CI overweight/obese normal 10 200 300 TC (mg/dl) 36 34 0 RDW-CV (%) RDW-CV (%) RDW-CV (%) 12 100 36 B7 12.5 11.5 0 37 95% CI overweight/obese normal A7 12.5 38 RDW-CV (%) 0 D6 37 35 34 34 C6 39 MCHC (g/dl) 37 MCHC (g/dl) MCHC (g/dl) B6 38 MCHC (g/dl) A6 38 7 100 200 300 LDL-C (mg/dl) 95% CI overweight/obese normal 400 0 50 100 HDL-C (mg/dl) 150 95% CI overweight/obese normal (b) Figure 2: Quadratic ﬁt plots with conﬁdence intervals of the relationship between hematological parameters and lipid proﬁles parameters among normal and overweight/obese groups. Besides, obesity is associated with impaired glucose tolerance, leading to inﬂammation in the body tissues [37]. Also, higher levels of WBC, monocyte, and PLT counts in subjects with metabolic diseases, positive association between BMI with WBC, PLT, and monocyte counts, and also GR% in overweight/obese individuals indicate the presence of inﬂammation in these subjects [38]. Increased thrombocytosis in both individuals with metabolic diseases and those who were overweight/obese could result from an inﬂammatory process and the activation of platelet which has a key role in atherothrombosis acceleration [39]. In overweight/obese individuals with increasing LDL-C, the levels of RBC count, HGB, HCT, MCV, MCH, MCHC, and RDW decreased. However, decreasing HDL-C was associated with elevation of the RBC count, HGB, HCT, MCV, MCH, and MCHC levels. Increased BMI in overweight/obese subjects was associated with decreased RBC count, HGB, HCT, MCV, MCH, MCHC levels, and lymphocyte counts. A recent study demonstrated no correlation between BMI and RBC indices except for MCH and MCHC, but it did not specify the relationship and point to a discrepancy by MCH and MCHC between diﬀerent BMI categories [20]. Alrubaie et al. reported a negative correlation between BMI with MCH and MCV [21]. We noticed a diﬀerent association between BMI and RBC indices for normal weights against the overweight/obese. This diﬀerence can result from proinﬂammatory cytokines driven from adipocytes and free radicals from oxidative stress. Increased free radicals by aﬀecting RBCs membrane proteins change their natural structure, increase fragility, decrease survival, and cause anisocytosis by the raised proportion of circulating premature erythrocytes [39]. To compensate for the reduction in red blood cell life, the body increases the production of new red blood cells, which has led to an increase in RBC count [27]. In metabolic diseases, we detected increased levels of RBC, HCT, HGB, RDW, and TC. Increased levels of RBC count, HGB, HCT, and RDW were associated with the risk of metabolic diseases. However, enhanced levels of MCV, MCH, and MCHC were associated with reduced risk of metabolic diseases. In two studies, similar results were obtained, and a positive correlation was detected between HGB, HCT, and RBC count with MetS components, without examination of other RBC indices [7, 11]. Also, Hu et al. demonstrated RDW was a potential prognostic index for liver disease [40]. Furthermore, Jiang et al. determined that HGB can help predict NAFLD [41]. The mechanisms underlying the increased RDW in liver disease are well not understood; however, nutrition deﬁciency is prevalent in liver disease patients, and reports indicated lower folic acid levels in these patients than healthy controls. Decreased folic acid might aﬀect hematopoiesis and amplify the heterogeneity of RBC [42]. Also, an increase in blood concentration and viscosity causes reduced blood ﬂow rate and blood glucose supply to the muscle, leading to insulin resistance. Insulin resistance is one of the known factors involved in NAFLD pathogenesis that leads to mitochondrion oxidation overload and aggravating fat deposition in liver cells [41, 43]. However, Nebeck et al. reported no diﬀerences in RBC indices between MetS individuals and the healthy group [7]. Unlike our MCV HGB Hct RBC count Q4 > 84.8 Q3 81.7_84.7 Q2 78.1_81.6 Q1 < 78 Q4 > 15.3 Q3 14.3_15.2 Q2 13.3_14.2 Q1 < 13.2 Q4 > 42.3 Q3 39.5_42.2 Q2 36.8_39.4 Q1 < 36.7 Q4 > 5.25 Q3 4.89_5.24 Q2 4.54_4.87 RBC indices Q1 < 4.53 Reference Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Reference Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Reference Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Reference Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 HTN OR (95% CI) 1.00 1.03 (0.82_1.30) 0.99 (0.79_1.25) 1.28 (1.01_1.62) 1.20 (0.95_1.53) 1.56 (1.22_2.00) 1.38 (1.07_1.78) 1.00 0.92 (0.73_1.16) 0.94 (0.74_1.18) 1.23 (0.97_1.56) 1.20 (0.94_1.54) 1.36 (1.03_1.78) 1.27 (0.96_1.68) 1.00 0.97 (0.78_1.22) 1.00 (0.80_1.26) 1.22 (0.96_1.54) 1.25 (0.98_1.58) 1.20 (0.92_1.57) 1.16 (0.88_1.52) 1.00 0.90 (0.72_1.12) 0.92 (0.74_1.16) 0.80 (0.64_0.99) 0.83 (0.66_1.04) 0.75 (0.60_0.94) 0.82 (0.65_1.03) Dyslipidemia OR (95% CI) 1.00 1.06 (0.90_1.25) 1.01 (0.86_1.20) 1.39 (1.17_1.65) 1.27 (1.07_1.52) 1.72 (1.43_2.05) 1.52 (1.26_1.82) 1.00 1.07 (0.91_1.26) 1.07 (0.91_1.27) 1.25 (1.05_1.49) 1.21 (1.01_1.45) 1.40 (1.15_1.70) 1.29 (1.05_1.58) 1.00 1.03 (0.88_1.21) 1.06 (0.90_1.24) 1.16 (0.97_1.37) 1.16 (0.98_1.39) 1.47 (1.21_1.78) 1.41 (1.16_1.71) 1.00 1.23 (1.05_1.45) 1.28 (1.09_1.51) 0.97 (0.83_1.14) 1.02 (0.87_1.20) 0.82 (0.69_0.96) 0.90 (0.76_1.06) NAFLD OR (95% CI) 1.00 1.02 (0.79_1.31) 0.95 (0.74_1.23) 1.34 (1.03_1.74) 1.21 (0.93_1.58) 1.26 (0.94_1.68) 1.08 (0.81_1.46) 1.00 1.00 (0.79_1.27) 1.01 (0.79_1.28) 1.14 (0.88_1.49) 1.05 (0.80_1.38) 1.21 (0.87_1.68) 1.10 (0.79_1.54) 1.00 0.88 (0.69_1.12) 0.88 (0.69_1.12) 1.02 (0.78_1.32) 1.00 (0.76_1.30) 0.94 (0.67_1.30) 0.88 (0.63_1.23) 1.00 1.00 (0.77_1.29) 1.03 (0.80_1.33) 0.88 (0.67_1.14) 0.94 (0.72_1.23) 1.01 (0.78_1.30) 1.13 (0.87_1.47) MetS OR (95% CI) 1.00 1.24 (1.04_1.47) 1.16 (0.97_1.39) 1.64 (1.37_1.96) 1.44 (1.20_1.74) 2.05 (1.70_2.47) 1.73 (1.42_2.11) 1.00 1.20 (1.02_1.43) 1.22 (1.02_1.45) 1.40 (1.17_1.69) 1.35 (1.12_1.64) 1.62 (1.32_2.00) 1.47 (1.19_1.83) 1.00 1.02 (0.86_1.20) 1.05 (0.88_1.25) 1.22 (1.02_1.46) 1.25 (1.04_1.50) 1.47 (1.20_1.79) 1.39 (1.13_1.71) 1.00 0.93 (0.79_1.09) 0.98 (0.83_1.16) 0.81 (0.69_0.96) 0.88 (0.74_1.05) 0.74 (0.63_0.88) 0.72 (0.72_1.02) DM OR (95% CI) 1.00 1.26 (0.93_1.70) 1.20 (0.88_1.63) 1.84 (1.36_2.50) 1.68 (1.23_2.28) 2.10 (1.53_2.88) 1.84 (1.33_2.53) 1.00 1.21 (0.91_1.60) 1.18 (0.89_1.57) 1.03 (0.75_1.40) 0.97 (0.71_1.33) 1.26 (0.89_1.77) 1.12 (0.79_1.59) 1.00 1.00 (0.75_1.32) 0.98 (0.74_1.30) 1.06 (0.79_1.43) 1.04 (0.77_1.41) 1.18 (0.85_1.66) 1.09 (0.78_1.54) 1.00 1.09 (0.84_1.42) 1.11 (0.85_1.46) 0.79 (0.60_1.05) 0.83 (0.62_1.02) 0.57 (0.42_0.77) 0.63 (0.46_0.85) Table 2: Odds ratio (ORs) and 95% CI of obesity-related diseases (according to quartiles of RBC indices). CVD OR (95% CI) 1.00 0.86 (0.69_1.07) 0.82 (0.66_1.02) 1.11 (0.88_1.39) 1.03 (0.82_1.30) 1.16 (0.91_1.48) 1.03 (0.81_1.33) 1.00 0.89 (0.72_1.10) 0.88 (0.71_1.10) 1.05 (0.83_1.32) 1.03 (0.81_1.30) 1.07 (0.81_1.40) 1.01 (0.77_1.34) 1.00 1.13 (0.91_1.40) 1.16 (0.93_1.44) 1.11 (0.88_1.41) 1.13 (0.89_1.44) 1.01 (0.76_1.34) 1.00 (0.75_1.32) 1.00 1.04 (0.84_1.30) 1.10 (0.88_1.37) 0.91 (0.73_1.14) 0.98 (0.78_1.23) 0.89 (0.71_1.11) 0.97 (0.77_1.21) 8 International Journal of Endocrinology ∗ Q4 > 11.4 Q3 11_11.4 Q2 10.5_10.9 Q1 < 10.4 Q4 > 37 Q3 36.2_36.9 Q2 35.3_36.1 Q1 < 35.2 Q4 > 30.9 Q3 29.6_30.8 Reference Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Reference Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Reference Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 HTN OR (95% CI) 1.00 0.98 (0.78_1.21) 0.97 (0.78_1.21) 0.81 (0.65_1.02) 0.85 (0.68_1.07) 0.77 (0.61_0.96) 0.84 (0.66_1.05) 1.00 1.13 (0.91_1.41) 1.13 (0.90_1.41) 1.07 (0.85_1.33) 1.12 (0.89_1.40) 0.94 (0.74_1.96) 1.03 (0.81_1.31) 1.00 1.04 (0.84_1.30) 0.97 (0.78_1.22) 1.23 (0.98_1.54) 1.06 (0.84_1.34) 1.21 (0.96_1.52) 1.04 (0.82_1.31) Model 1 adjusted for age, gender, and smoking and physical activity. RDW MCHC MCH Q2 27.9_29.5 RBC indices Q1 < 27.8 ∗∗ NAFLD OR (95% CI) 1.00 1.09 (0.85_1.39) 1.10 (0.85_1.41) 0.95 (0.74_1.23) 1.00 (0.78_1.30) 0.81 (0.62_1.06) 0.93 (0.71_1.22) 1.00 1.01 (0.79_1.28) 1.01 (0.79_1.29) 0.71 (0.55_0.92) 0.74 (0.57_0.95) 0.68 (0.52_0.90) 0.75 (0.57_0.99) 1.00 1.30 (0.99_1.69) 1.16 (0.89_1.53) 1.49 (1.14_1.96) 1.28 (0.97_1.68) 1.80 (1.38_2.35) 1.48 (1.12_1.94) Model 2 adjusted for model 1 plus BMI. Dyslipidemia OR (95% CI) 1.00 1.19 (1.02_1.40) 1.22 (1.04_1.43) 0.91 (0.77_1.06) 0.94 (0.80_1.11) 0.88 (0.75_1.03) 0.98 (0.83_1.15) 1.00 1.27 (1.09_1.49) 1.30 (1.11_1.53) 1.07 (0.91_1.25) 1.13 (0.96_1.32) 1.15 (0.98_1.36) 1.26 (1.06_1.48) 1.00 1.56 (1.31_1.81) 1.45 (1.24_1.69) 2.15 (1.83_2.52) 1.93 (1.63_2.27) 1.91 (1.62_2.26) 1.66 (1.40_1.97) Table 2: Continued. MetS OR (95% CI) 1.00 0.97 (0.83_1.15) 0.99 (0.83_1.17) 0.81 (0.69_0.96) 0.87 (0.74_1.04) 0.75 (0.64_0.89) 0.88 (0.74_1.04) 1.00 1.08 (0.92_1.27) 1.08 (0.91_1.28) 0.91 (0.77_1.07) 0.98 (0.82_1.16) 0.81 (0.81_1.14) 1.08 (0.91_1.29) 1.00 1.46 (1.23_1.72) 1.31 (1.10_1.59) 2.06 (1.74_2.44) 1.73 (1.45_2.06) 2.06 (1.73_2.45) 1.68 (1.40_2.01) DM OR (95% CI) 1.00 1.02 (0.78_1.32) 1.01 (0.77_1.32) 0.73 (0.55_0.96) 0.75 (0.56_0.99) 0.60 (0.45_0.81) 0.68 (0.50_0.91) 1.00 1.02 (0.78_1.34) 1.01 (0.77_1.33) 0.93 (0.70_1.22) 0.96 (0.72_1.27) 0.91 (0.68_1.22) 0.98 (0.73_1.31) 1.00 1.09 (0.82_1.45) 0.98 (0.74_1.31) 1.26 (0.95_1.68) 1.09 (0.81_1.45) 1.46 (1.10_1.94) 1.21 (0.90_1.62) CVD OR (95% CI) 1.00 1.03 (0.82_1.28) 1.07 (0.85_1.33) 0.93 (0.75_1.62) 1.00 (0.80_1.25) 0.81 (0.65_1.01) 0.91 (0.72_1.15) 1.00 1.00 (0.81_1.24) 1.01 (0.82_1.26) 0..91 (0.73_1.13) 0.97 (0.78_1.21) 0.82 (0.65_1.04) 0.92 (0.72_1.16) 1.00 1.17 (0.94_1.46) 1.10 (0.87_1.37) 1.31 (1.05_1.65) 1.13 (0.90_1.42) 1.22 (0.97_1.53) 1.04 (0.82_1.31) International Journal of Endocrinology 9 10 International Journal of Endocrinology 0.83 (0.67_1.02) 0.78 (0.63_0.96) 0.70 (0.39_1.26) 0.81 (0.45_1.47) 0.91 (0.68_1.22) 0.85 (0.63_1.15) 0.62 (0.25_1.50) 0.71 (0.29_1.73) 0.90 (0.65_1.24) 0.87 (0.63_1.21) 0.93 (0.39_2.22) 1.10 (0.46_2.66) 0.85 (0.68_1.05) 0.79 (0.63_0.99) 0.50 (0.26_0.97) 0.61 (0.31_1.20) 0.94 (0.65_1.35) 0.94 (0.65_1.35) 0.81 (0.28_2.29) 0.94 (0.65_1.35) 1.06 (0.81_1.40) 1.00 (0.76_1.33) 1.04 (0.50_2.18) 1.20 (0.56_2.53) Dyslipidemia HTN NAFLD MetS DM CVD 0 0.5 Mild Anemia Model 1 Model 2 1 1.5 2 2.5 3 Moderate Anemia Model 1 Model 2 Figure 3: Forest plot of ORs (95% CIs) of obesity-related diseases according to anemia severity. ﬁndings, Yan et al. reported that an increase in the RDW level was associated with reduced MetS incidence among Chinese population [8]. As the Chinese study was performed on people over 60 years, this contrast might be due to age diﬀerences between the two studies. Some evidence suggests that RDW is associated with pulmonary hypertension mortality [40], and RBC count is related to the severity of hypertension [12]. We found no signiﬁcant diﬀerence in RBC count, HTC, HGB, and MCHC levels between HTN subjects and control groups. However, individuals with HTN had higher RDW levels than control subjects. Furthermore, upper levels of RDW, RBC count, HCT, and HGB was correlated with greater risk of HTN. An association between RBC count and HTN may occur due to an additional load on the cardiovascular system by increasing the RBC count [13]. In the present study, moderate anemia was associated with increased BMI and metabolic diseases. Anemia is an independent risk factor for CVD progression and predicts heart complications [44]. It was observed that anemia was the highest risk factor for CVD. RDW is used in the prognosis of CVD and heart failure, and increased RDW could be an important predictor of the mortality and morbidity in atherosclerosis and heart failure, regardless of the level of hemoglobin [43]. We found that BMI decreased the metabolic disease incidence risk in mild anemia and increased it in moderate anemia, indicating that the type and the severity of anemia should be considered when examining the relationship between anemia and obesity. The eﬀect of BMI on the association between obesity-related disorders and RBC indices (including RBC count, HCT, HGB, and RDW) decreased in the ORs. In contrast, BMI increased the incidence risk of metabolic diseases by aﬀecting MCV, MCH, and MCHC, which shows an inverse relationship between BMI and RBC indices based on our ﬁndings. There is presently no consensus in the literature regarding the relationship between anemia and obesity. A study on Chinese women reported a lower prevalence of anemia in overweight females compared to normal weight participants [18]. Findings of two studies on Iranian population suggested no signiﬁcant diﬀerence in the prevalence of anemia and the levels of hemoglobin, MCV, serum iron, ferritin, and transferrin according to BMI [16, 45]. In contrast, evidence exists that indicate an association between obesity and anemia [19, 46]. The role of iron deﬁciency in obesity is unclear. However, obesity, as a low-grade inﬂammation status, may cause a negative regulation of iron absorption through increased secretion of hepcidin by adipocytes and result in a decrease in iron uptake in small intestine [45, 46]. Current results demonstrated that HGB and HDL-C levels were negatively correlated with obesity. The association between HDL-C and MCV was also a positive linear correlation in overweight/obese. Studies have shown that HDL-C levels are inversely related to the incidence of anemia. HDL-C is positively associated with MCV, which is likely to play a role in megaloblastic anemia [27]. Although dyslipidemia, MetS, and DM group had higher International Journal of Endocrinology mean of RBC count, their level of MCV and MCH was lower compared to the controls. Being the ﬁrst study of an Iranian population on a large scale, our ﬁndings imply that obesity aﬀected the lipid proﬁle and inﬂuenced the RBC indices. Decreased HGB and increased RDW levels with consequent anemia and the elevation of PLT, WBC count, and reduction of lymphocytes resulted in inﬂammation with BMI playing an important role in the process. Abnormal lipid proﬁle in overweight/obese had an inverse relation with RBC indices. We observed an inﬂammatory state with increased WBC, monocytes, and PLT counts and also changes in all RBC indices for metabolic diseases. Changes in RBC indices in overweight/obese had a signiﬁcant impact on the interpretation of laboratory results. Finally, it should be noted that the current study is an explorative study aiming to create new hypotheses which should be further investigated in future studies. 5. Conclusion We found an increase of WBC, monocytes, PLT counts, and RDW for metabolic diseases. There was also a correlation between increased levels of RBC count, HGB, HCT, and RDW and the risk of metabolic diseases. Increased BMI enhanced the WBC, PLT, GR counts, and RDW for overweight/obese. An inverse correlation between LDL-C and the levels of RBC count, HGB, HCT, MCV, MCH, MCHC, and RDW was also observed for them. Furthermore, their increased BMI was associated with reduced RBC count, HGB, HCT, MCV, MCH, MCHC levels, and lymphocyte counts. While the risk of obesity-related diseases in the fourth quartiles of RBC count, HCT, HGB, and RDW was higher than the ﬁrst quartiles; MCV, MCH, and MCHC had lower OR. Moderate anemia was associated with increased BMI and metabolic diseases. Further studies on inﬂammation signaling pathways in adipose tissue are needed to achieve accurate results. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Additional Points Limitations of the Study. Since the study was cross-sectional, a prospective study is needed to conﬁrm the results. The inﬂammatory and lipid peroxidation indices were not examined in our population. Ethical Approval The study was approved by the Ethics Committees of Kermanshah University of Medical Sciences (KUMS.REC.1394.315), Kermanshah, Iran. All participants entered the study after they were fully informed of the process and signed a written consent. 11 Disclosure The funder had no role in the design of the study, collection, analysis, and interpretation of the data or writing and approval of manuscript. Conflicts of Interest The authors declare that they have no conﬂicts of interest. Acknowledgments The authors are deeply grateful to the investigators of PERSIAN for their valuable support for designing the methods and developing the questionnaire. The authors also appreciate their interviewers, RaNCD staﬀ, and Ravansar population for their signiﬁcant cooperation in data collection. This study was supported by the Ministry of Health and Medical Education of Iran and Kermanshah University of Medical Science (grant no: 92472). References [1] W. T. Garvey, “New tools for weight-loss therapy enable a more robust medical model for obesity treatment: rationale for a complications-centric approach,” Endocrine Practice, vol. 19, no. 5, pp. 864–874, 2013. [2] M. Tremmel, U.-G. Gerdtham, P. Nilsson, and S. Saha, “Economic burden of obesity: a systematic literature review,” International Journal of Environmental Research and Public Health, vol. 14, no. 4, p. 435, 2017. [3] D. Petrakis, L. Vassilopoulou, C. 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https://openalex.org/W3200779258	https://europepmc.org/articles/pmc8467567?pdf=render	English	null	Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies	International journal of molecular sciences	2,021	cc-by	16,931	4 Departamento de Microbiología, Pediatría, Radiología y Salud Pública, Fac University of Zaragoza, 50009 Zaragoza, Spain 5 Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B9820 Merelbeke, Belgium; Helena.Berlamont@UGent.be (H.B.); freddy.haesebrouck@ugent.be (F.H.) Citation: Salillas, S.; Galano-Frutos, 6 ARAID Foundation, Government of Aragon, 50018 Zaragoza, Spain 7 CIBER de Enfermedades Hepáticas y Digestivas CIBERehd, Instituto de Salud Carlos III, 28029 Madrid, Spain 8 Unit of Helicobacter Pathogenesis, CNRS UMR2001, Department of Mi 25-28 Rue du Dr. Roux, 75724 Paris, France; eliette.touati@pasteur.fr 9 Cellular Microbiology, Program Area Infections, Research Center Borstel, Leibniz Lung Center, 23845 Borstel, Germany; umamat@fz-borstel.de (U.M.); uschaible@fz-borstel.de (U.E.S.) y 10 Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), CSIC—Departamento de Química Orgánica, Faculty of Science, University of Zaragoza, 50009 Zaragoza, Spain; jagl@unizar.es (J.A.G.); loladiaz@unizar.es (M.D.D.-d.-V.) 11 CIBER de Enfermedades Respiratorias—CIBERES, Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: jsancho@unizar.es * Correspondence: jsancho@unizar.es Abstract: Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of ﬂavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly speciﬁc for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the ﬂavodoxin structure, several H. pylori-ﬂavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and speciﬁc H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens. International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efﬁcacy, Synergy, Resistance and Mechanistic Studies dra Salillas 1,2,3, Juan José Galano-Frutos 1,2,3 , Alejandro Mahía 1,2,3 , Ritwik Maity 1,2,3 , J J , j , María Conde-Giménez 1,2,3, Ernesto Anoz-Carbonell 1,2,4, Helena Berlamont 5 , Adrian Velazquez-Campoy 1,2,3,6,7 , Eliette Touati 8, Uwe Mamat 9, Ulrich E. Schaible 9 , í í d ill 10 dd b k 5 é í 1 3 4 11 d i Campoy 1,2,3,6,7 , Eliette Touati 8, Uwe Mamat 9, Ulrich E. Schaible 9 , José A. Gálvez 10 , Villegas 10, Freddy Haesebrouck 5 , José A. Aínsa 1,3,4,11 and Javier Sancho 1,2,3,* 1 Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and GBsC-CSIC, University of Zaragoza, 50018 Zaragoza, Spain; sandrasalillasberges@gmail.com (S.S.); juanjogf@gmail.com (J.J.G.-F.); amahia@unizar.es (A.M.); mr.ritwikmaity@outlook.com (R.M.); mcondeg@unizar.es (M.C.-G.); eanoz@unizar.es (E.A.-C.); adrianvc@unizar.es (A.V.-C.); ainsa@unizar.es (J.A.A.) 1 Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and GBsC-CSIC, University of Zaragoza, 50018 Zaragoza, Spain; sandrasalillasberges@gmail.com (S.S.); juanjogf@gmail.com (J.J.G.-F.); amahia@unizar.es (A.M.); mr.ritwikmaity@outlook.com (R.M.); mcondeg@unizar.es (M.C.-G.); eanoz@unizar.es (E.A.-C.); adrianvc@unizar.es (A.V.-C.); ainsa@unizar.es (J.A.A.) 2 Departamento de Bioquímica y Biología Molecular y Celular, Faculty of Science, University of Zaragoza, 50009 Zaragoza, Spain g p 3 Aragon Health Research Institute (IIS Aragón), 50009 Zaragoza, Spain Aragon Health Research Institute (IIS Aragón), 50009 Zara Health Research Institute (IIS Aragón), 50009 Zaragoza, Spai Citation: Salillas, S.; Galano-Frutos, J.J.; Mahía, A.; Maity, R.; Conde-Giménez, M.; Anoz-Carbonell, E.; Berlamont, H.; Velazquez-Campoy, A.; Touati, E.; Mamat, U.; et al. Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efﬁcacy, Synergy, Resistance and Mechanistic Studies. Int. J. Mol. Sci. 2021, 22, 10137. https://doi.org/ 10.3390/ijms221810137 Academic Editor: Francesca Micoli Received: 28 July 2021 Accepted: 16 September 2021 Published: 20 September 2021 Citation: Salillas, S.; Galano-Frutos, J.J.; Mahía, A.; Maity, R.; Conde-Giménez, M.; Anoz-Carbonell, E.; Berlamont, H.; Velazquez-Campoy, A.; Touati, E.; Mamat, U.; et al. Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efﬁcacy, Synergy, Resistance and Mechanistic Studies. Int. J. Mol. Sci. 2021, 22, 10137. https://doi.org/ 10.3390/ijms221810137 Academic Editor: Francesca Micoli Received: 28 July 2021 Accepted: 16 September 2021 Published: 20 September 2021 4 Departamento de Microbiología, Pediatría, Radiología y Salud Pública, Faculty of Medicine, 1. Introduction Helicobacter pylori (H. pylori) is a Gram-negative proteobacterium estimated to in- fect about 50% of the human population worldwide [1]. Although H. pylori infection remains often asymptomatic [2], it is an important cause of peptic ulcer disease, MALT lymphoma, and gastric cancer [3,4], and H. pylori is the only bacterial pathogen considered as a Class I carcinogen [5]. H. pylori eradication is carried out using either triple or quadru- ple chemotherapy, in which several antibiotics and antimicrobial compounds including bismuth are combined with a proton pump inhibitor (PPI) [6]. The increasing develop- ment of resistance to antimicrobials used in H. pylori eradication therapies has led to the inclusion of clarithromycin (Cla)-resistant H. pylori as a Priority 2 pathogen in the WHO global priority pathogens list, urging the development of novel antimicrobials to treat the infection [7]. On the other hand, no speciﬁc or highly selective H. pylori antimicrobial exists, which prevents the design of precision eradication therapies that could minimise both the impact on the patients’ microbiota and the multiplication of resistances due to the massive use of broad-spectrum antimicrobials [8]. To meet the challenges represented by H. pylori eradication, a variety of new targets for pharmacological intervention are being studied [9] including H. pylori ﬂavodoxin (Hp- Fld) [10]. Flavodoxins are bacterial electron carriers, not present in humans, that participate in different redox reactions and that, depending on the bacteria, may be essential or non-essential proteins [10,11]. Hp-Fld [12,13] is an essential protein that mediates the oxidative decarboxylation of pyruvate by pyruvate-oxidoreductase [14]. It belongs to the long-chain ﬂavodoxin class, differing from short-chain ﬂavodoxins by the presence of an extra loop that may play a role in the binding to partner proteins [15]. While all bacterial ﬂavodoxins, either long or short-chain, are quite similar at the structural level, Hp-Fld contains a distinct pocket near the binding site of the FMN (ﬂavin mononucleotide) redox cofactor [12]. As the binding of small molecules at such a pocket might serve to inhibit electron transfer by the FMN cofactor or to impair the binding of partner proteins to Hp- Fld [11], a high throughput screening was run to identify ﬂavodoxin binders from a diverse chemical library of 10,000 compounds [16]. Furthermore, by using an in vitro coupled reaction, several binders proved to inhibit Hp-Fld activity and, interestingly, they were subsequently shown to be either bactericidal (three of them) or bacteriostatic for H. pylori. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 10137. https://doi.org/10.3390/ijms221810137 2 of 25 Int. J. Mol. Sci. 2021, 22, 10137 Keywords: Helicobacter; narrow-spectrum antimicrobial; AMR; ﬂavodoxin; drug discovery 2. Materials and Methods 2.1. Reagents and Chemicals Compounds IV, IV-a, IV-b, IV-c, IV-d (Figure 1) were previously synthesised as de- scribed and their MCC50. for eukaryotic cells were determined [18]. The synthesis of compounds rac-IV-j, rac-IV-k and rac-IV-l is described below. Their MCC50. for eukary- otic cells follow the trends of the respective parent compounds (not shown). The antibiotics metronidazole (Mnz) and clarithromycin (Cla) and the proton pump inhibitors (PPIs) omeprazole and rabeprazole were purchased from Acros Organics, Sigma-Aldrich, Fluo- rochem and Tokyo Chemical Industry, respectively, whereas the bacterial efﬂux inhibitors (EIs) carbonyl cyanide 3-chlorophenylhydrazone (CCCP), reserpine and valinomycin were obtained from Sigma-Aldrich. All chemicals were dissolved in 100% dimethylsulfoxide (DMSO) and stored frozen at −20 ◦C. Resazurin sodium salt solution was prepared at 0.01% (w/v) in distilled water, sterilised by ﬁltering and stored at 4 ◦C for up to two weeks. Flavin mononucleotide (FMN) was acquired from Santa Cruz Biotechnology. 1. Introduction After several rounds of chemical variation and efﬁcacy testing [17,18], a family of novel nitrobenzoxadiazol-based antimicrobials has emerged, led by compound IV (Figure 1) [18]. These antimicrobials have been provisionally described as potentially speciﬁc for H. pylori, but a detailed analysis of their spectrum of antibacterial activity is lacking. On the other hand, these compounds have been tested in a mouse model of H. pylori infection as a monotherapy only [18]. Therefore, further knowledge of the potential synergy of these compounds with compounds currently used in triple/quadruple anti-H. pylori therapies (such as Cla, metronidazole (Mnz), or PPIs) could serve to investigate the efﬁcacy of such novel combination therapies in mice, with the aim of proposing new combination therapies to treat H. pylori infections in humans. In this work, we describe the antimicrobial spectrum of lead compound IV and variants thereof for bacteria belonging to 15 bacterial genera (7 Gram-negatives and 8 Gram- positives), including a more detailed characterization of their activity against 9 species of the Helicobacter genus (H. pylori plus 8 non-H. pylori Helicobacter species: NHPH) capable of infecting humans and/or domestic animals and having been described as causative agents of gastric or hepatic pathologies [19,20]. Besides, we obtain valuable mechanistic information concerning the binding of the Hp-Fld inhibitors to the target protein and their potential capacity to generate new resistances. As it appears, aniline-bearing IV derivatives are narrow-spectrum antimicrobials speciﬁc for H. pylori (and possibly for Int. J. Mol. Sci. 2021, 22, 10137 3 of 25 3 of 25 other gastric Helicobacter species), while those bearing a nitro group are extended-spectrum antimicrobials, which are also effective against most Gram-positive bacteria tested. other gastric Helicobacter species), while those bearing a nitro group are extended-spectrum antimicrobials, which are also effective against most Gram-positive bacteria tested. Figure 1. Compounds tested for antimicrobial activity against bacteria. Figure 1. Compounds tested for antimicrobial activity against bacteria. 2.2.1. rac-(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl p-Toluenenesulfonate (rac-S1) A suspension of p-toluenesulfonyl chloride (1.51 g, 7.92 mmol) in anhydrous dichloromethane (3.0 mL) was added dropwise to a solution of (2,2-dimethyl-1,3-dioxolan- 4-yl)methanol (872 mg, 6.60 mmol) and pyridine (1.6 mL, 19.8 mmol) in anhydrous dichloromethane (4.0 mL) (Figure 2). The reaction mixture was stirred at room temperature for 16 h. Then, the reaction mixture was washed with water (10 mL) and the aqueous phase was extracted with dichloromethane (10 mL). The combined organic layers were dried with anhydrous MgSO4, ﬁltered and evaporated under reduced pressure. The resulting residue was puriﬁed by column chromatography (eluent: Et2O/hexane 1:1) to afford compound rac-S1 (1.60 g, 85% yield) as a white solid. mp: 48–49 ◦C. IR (KBr, νmax/cm−1): 1169, 1347, 1457, 1495, 1595. 1H-NMR (300 MHz, CDCl3, δ): 7.82–7.74 (m, 2H), 7.38–7.31 (m, 2H), 4.31–4.22 (m, 1H), 4.06–3.92 (m, 3H), 3.75 (dd, 1H, J = 9.0, J = 5.1), 2.44 (s, 3H), 1.33 (s, 3H), 1.30 (s, 3H). 13C-NMR (100 MHz, CDCl3, δ): 145.0, 132.6, 129.9, 127.9, 110.0, 72.9, 69.4, 66.1, 26.6, 25.1, 21.6. HRMS (ESI+): m/z [M + Na]+ calculated for C13H18NaO5S 309.0768, found 309.0754. 2.2.2. rac-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)methanol (rac-S2) 2.2. Synthesis of Compounds Rac-IV-j, Rac-IV-k and Rac-IV-l Unless otherwise speciﬁed, all reagents for synthesis were obtained from commercial suppliers and were used without puriﬁcation. TLC was performed on precoated silica gel polyester plates, and products were visualised using UV light (254 nm) and ninhy- drin, anisaldehyde, or potassium permanganate solutions, followed by heating. Column chromatography was performed on silica gel 60 (70–200 µm) with air pressure. Melting points were determined in open glass capillaries with a Gallenkamp apparatus. Infrared spectra were recorded with a Fourier transform infrared spectrometer (Thermo Nicolet Avatar 360 FT-IR (Thermo-Fischer Scientiﬁc, Waltham, MA, USA)). NMR spectra were recorded with a Bruker AV-400 spectrometer (Bruker-Biospin, Rheinstetten, Germany) (400 MHz for 1H-NMR experiments and 100 MHz for 13C-NMR) or a Bruker AV-300 spec- trometer (Bruker-Biospin, Rheinstetten, Germany) (300 MHz for 1H-NMR experiments) in the stated deuterated solvents. 1H and 13C chemical shifts were referenced to internal solvent resonances and reported in ppm relative to tetramethylsilane. J values are given in Hz. High-resolution positive (or negative) electrospray ionisation mass spectra were recorded with a Bruker Daltonics MicroToF-Q spectrometer (BrukerDaltonics, Billerica, MA, USA) with use of ultradilute solutions of the chemical compounds in methanol. A scheme of the synthesis of compounds rac-IV-j, rac-IV-k and rac-IV-l is shown in Figure 2. Int. J. Mol. Sci. 2021, 22, 10137 4 of 25 Figure 2. Scheme of the synthesis of compounds rac-IV-j, rac-IV-k and rac-IV-l. Figure 2. Scheme of the synthesis of compounds rac-IV-j, rac-IV-k and rac-IV-l. 2.2.3. rac-S-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)benzyl) Thioacetate (rac-S3) 2.2.3. rac-S-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)benzyl) Thioacetate (rac-S3) 2.2.3. rac-S-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)benzyl) Thioacetate (rac-S3 ( (( , y , y ) y) y ) ( ) To a cooled (0 ◦C) solution of PPh3 (671 mg, 2.56 mmol) in anhydrous THF (8.0 mL) was added diisopropyl azodicarboxylate (0.50 mL, 2.56 mmol) and the resulting suspension was stirred for 20 min at 0 ◦C under an argon atmosphere. Then thioacetic acid (0.18 mL, 2.56 mmol) and a solution of compound rac-S2 (305 mg, 1.28 mmol) in anhydrous THF (16 mL) were consecutively added. The reaction mixture was stirred under an argon atmosphere for 1 h at 0 ◦C and for 1 additional hour at room temperature. The solvent was removed under reduced pressure and the resulting residue was puriﬁed by column chromatography (eluent 1: hexane/Et2O 9:1, eluent 2: hexane/Et2O 8:2) to afford com- pound rac-S3 (368 mg, 97% yield) as a white solid. mp: 57–58 ◦C. IR (KBr, νmax/cm−1): 1455, 1514, 1612, 1687. 1H-NMR (300 MHz, CDCl3, δ): 7.23–7.16 (m, 2H), 6.87–6.80 (m, 2H), 4.51–4.40 (m, 1H), 4.15 (dd, 1H, J = 8.4, J = 6.6), 4.07 (s, 2H), 4.03 (dd, 1H, J = 9.6, J = 5.4), 3.96–3.83 (m, 2H), 2.33 (s, 3H), 1.45 (s, 3H), 1.40 (s, 3H). 13C-NMR (100 MHz, CDCl3, δ): 195.2, 157.7, 130.1, 129.9, 114.6, 109.7, 73.9, 68.8, 66.8, 32.8, 30.3, 26.7, 25.3. HRMS (ESI+): m/z [M + Na]+ calculated for C15H20NaO4S 319.0975, found 319.0983. 2.2.4. rac-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)methanethiol (rac-S4) To a solution of compound rac-S3 (332 mg, 1.12 mmol) in anhydrous methanol (15 mL) was added dry K2CO3 (185 mg, 1.34 mmol) and the resulting suspension was stirred for 15 min at room temperature under an argon atmosphere. The reaction mixture was then neutralised by dropwise addition of 2M HCl aqueous solution and the solvent was removed under reduced pressure. The resulting residue was dissolved in distilled water (15 mL) and extracted with dichloromethane (2 × 10 mL). The combined organic layers were dried with anhydrous MgSO4, ﬁltered and evaporated under reduced pressure. The resulting residue was puriﬁed by column chromatography (eluent: hexane/Et2O 4:1) to afford compound rac-S4 (241 mg, 85% yield) as a white oily solid. mp: 33–34 ◦C. IR (KBr, νmax/cm−1): 1456, 1513, 1611. 1H-NMR (400 MHz, CDCl3, δ): 7.26–7.20 (m, 2H), 6.89–6.82 (m, 2H), 4.51–4.43 (m, 1H), 4.16 (dd, 1H, J = 8.4, J = 6.4), 4.04 (dd, 1H, J = 9.6, J = 5.6), 3.92 (dd, 1H, J = 9.6, J = 6.0), 3.89 (dd, 1H, J = 8.4, J = 5.8), 3.70 (d, 2H, J = 7.6), 1.73 (t, 1H, J = 7.6), 1.46 (s, 3H), 1.40 (s, 3H). 13C-NMR (100 MHz, CDCl3, δ): 157.6, 133.7, 129.1, 114.7, 109.7, 73.9, 68.9, 66.8, 28.3, 26.8, 25.3. HRMS (ESI+): m/z [M + Na]+ calculated for C13H18NaO3S 277.0870, found 277.0870. 2.2.2. rac-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)methanol (rac-S2) 2.2.2. rac-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)methanol (rac-S2) To a solution of compound rac-S1 (802 mg, 2.80 mmol) and 4-hydroxybenzyl alcohol (417 mg, 3.36 mmol) in anhydrous dimethylformamide (6.0 mL) was added dry K2CO3 To a solution of compound rac-S1 (802 mg, 2.80 mmol) and 4-hydroxybenzyl alcohol (417 mg, 3.36 mmol) in anhydrous dimethylformamide (6.0 mL) was added dry K2CO3 Int. J. Mol. Sci. 2021, 22, 10137 5 of 25 (464 mg, 3.36 mmol) and the resulting suspension was stirred for 16 h at 90 ◦C. The sol- vent was then removed under reduced pressure and the resulting residue was dissolved in distilled water (15 mL) and extracted with dichloromethane (2 × 10 mL). The com- bined organic layers were dried with anhydrous MgSO4, ﬁltered and evaporated under reduced pressure. The resulting residue was puriﬁed by column chromatography (eluent: Et2O/hexane 1:1) to afford compound rac-S2 (339 mg, 51% yield) as a white solid. mp: 47–48 ◦C. IR (KBr, νmax/cm−1): 1453, 1514, 1614, 3402. 1H-NMR (300 MHz, CDCl3, δ): 7.32–7.25 (m, 2H), 6.93–6.87 (m, 2H), 4.61 (s, 2H), 4.53–4.43 (m, 1H), 4.17 (dd, 1H, J = 8.4, J = 6.3), 4.06 (dd, 1H, J = 9.6, J = 5.4), 3.94 (dd, 1H, J = 9.6, J = 6.0), 3.90 (dd, 1H, J = 8.4, J = 5.7), 1.83 (sa, 1H), 1.47 (s, 3H), 1.41 (s, 3H). 13C-NMR (100 MHz, CDCl3, δ): 158.1, 133.6, 128.5, 114.5, 109.7, 73.9, 68.8, 66.8, 64.8, 26.7, 25.3. HRMS (ESI+): m/z [M + Na]+ calculated for C13H18NaO4 261.1098, found 261.1093. 2.2.7. rac-3-(4-(((7-Aminobenzo[c][1,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane- 1,2-diol (rac-IV-k) To a suspension of compound rac-S5 (71 mg, 0.17 mmol) in EtOH/AcOH/H2O 2:2:1 (3.5 mL) was added iron powder (49 mg, 0.88 mmol) and the reaction mixture was sonicated (80 W, 45 kHz) at 30 ◦C for 30 min. The reaction mixture was then ﬁltered through a pad of celite® and the ﬁltrate was diluted with dichloromethane (10 mL) and washed with saturated K2CO3 aqueous solution (2 × 10 mL). The combined aqueous layers were extracted with dichloromethane (10 mL). The combined organic layers were dried with anhydrous MgSO4, ﬁltered and evaporated under reduced pressure. The resulting crude was used in the next reaction step without further puriﬁcation. Bismuth trichloride (11 mg, 0.034 mmol) and 3 drops of distilled water were sequentially added to a solution of the former crude compound in acetonitrile (3.5 mL). The reaction mixture was stirred for 16 h at room temperature. The solvent was then removed under reduced pressure. The resulting residue was dissolved in MeOH/AcOEt 1:1 and adsorbed in silica-gel to be puriﬁed by column chromatography (eluent 1: Et2O, eluent 2: Et2O/MeOH 95:5), affording compound rac-IV-k (44 mg, 75% yield) as an orange solid. mp: 161–162 ◦C. IR (KBr, νmax/cm−1): 3248, 3358, 3453. 1H-NMR (400 MHz, CD3OD, δ): 7.08 (d, 1H, J = 7.6), 7.05–6.98 (m, 2H), 6.81–6.73 (m, 2H), 6.19 (d, 1H, J = 7.6), 4.07 (s, 2H), 4.01–3.96 (m, 1H), 3.96–3.86 (m, 2H), 3.66 (dd, 1H, J = 11.2, J = 4.8), 3.61 (dd, 1H, J = 11.2, J = 5.2). 13C-NMR (100 MHz, CD3OD, δ): 159.4, 152.4, 146.5, 141.3, 138.6, 131.6, 131.1, 115.3, 107.0, 106.3, 71.8, 70.3, 64.1, 39.2. HRMS (ESI+): m/z [M + Na]+ calculated for C16H17NaN3O4S 370.0832, found 370.0853. 2.2.6. rac-3-(4-(((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1,2- diol (rac-IV-j) Bismuth trichloride (26 mg, 0.082 mmol) and 7 drops of distilled water were sequen- tially added to a solution of compound rac-S5 (171 mg, 0.41 mmol) in acetonitrile (8.0 mL). The reaction mixture was stirred for 5 h at room temperature. The solvent was then re- moved under reduced pressure. The resulting residue was dissolved in MeOH/AcOEt 1:1 and adsorbed in silica-gel to be puriﬁed by column chromatography (eluent 1: Et2O, eluent 2: AcOEt), affording compound rac-IV-j (136 mg, 88% yield) as a yellow solid. mp: 151–152 ◦C. IR (KBr, νmax/cm−1): 1304, 1338, 1510, 3289, 3389. 1H-NMR (400 MHz, acetone-d6, δ): 8.56 (d, 1H, J = 8.0), 7.63 (d, 1H, J = 8.0), 7.52–7.46 (m, 2H), 7.00–6.93 (m, 2H), 4.66 (s, 2H), 4.14–4.05 (m, 2H), 4.02–3.93 (m, 2H), 3.83–3.77 (m, 1H), 3.71–3.60 (m, 2H). 13C-NMR (100 MHz, acetone-d6, δ): 159.9, 150.2, 143.8, 141.1, 132.5, 131.3, 127.3, 123.1, 115.8, 71.3, 70.4, 64.1, 36.1. HRMS (ESI+): m/z [M + Na]+ calculated for C16H15NaN3O6S 400.0574, found 400.0577. 2.2.5. rac-4-((4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)benzyl)thio)-7- nitrobenzo[c][1,2,5]oxadiazole (rac-S5) 2.2.5. rac-4-((4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)benzyl)thio)-7- nitrobenzo[c][1,2,5]oxadiazole (rac-S5) To a solution of 4-chloro-7nitrobenzofurazan (285 mg, 1.43 mmol) and compound rac-S4 (242 mg, 0.95 mmol) in anhydrous DMF (22 mL) was added anhydrous pyridine (0.15 mL, 1.90 mmol) and the reaction mixture was stirred for 2 h at 80 ◦C under an argon atmosphere. An extra amount of anhydrous pyridine (77 µL, 0.95 mmol) was then added and the mixture was stirred for 2 additional hours under the same reaction conditions. The solvent was removed under reduced pressure and the resulting residue was puriﬁed by Int. J. Mol. Sci. 2021, 22, 10137 6 of 25 column chromatography (eluent 1: hexane/Et2O 7:3, eluent 2: dichloromethane/hexane 9:1; eluent 3: dichloromethane/Et2O 9:1) to afford compound rac-S5 (178 mg, 45% yield) as a yellow solid. mp: 106–107 ◦C. IR (KBr, νmax/cm−1): 1304, 1330, 1512. 1H-NMR (400 MHz, CDCl3, δ): 8.34 (d, 1H, J = 8.0), 7.38–7.31 (m, 2H), 7.18 (d, 1H, J = 8.0), 6.93–6.86 (m, 2H), 4.51–4.43 (m, 1H), 4.47 (s, 2H), 4.16 (dd, 1H, J = 8.4, J = 6.4), 4.04 (dd, 1H, J = 9.6, J = 5.6), 3.93 (dd, 1H, J = 9.6, J = 5.6), 3.89 (dd, 1H, J = 8.4, J = 5.6), 1.45 (s, 3H), 1.39 (s, 3H). 13C-NMR (100 MHz, CDCl3, δ): 158.6, 149.1, 142.4, 141.0, 132.8, 130.6, 130.0, 125.9, 121.3, 115.1, 109.8, 73.9, 68.8, 66.7, 36.2, 26.8, 25.3. HRMS (ESI+): m/z [M + Na]+ calculated for C19H19NaN3O6S 440.0887, found 440.0879. column chromatography (eluent 1: hexane/Et2O 7:3, eluent 2: dichloromethane/hexane 9:1; eluent 3: dichloromethane/Et2O 9:1) to afford compound rac-S5 (178 mg, 45% yield) as a yellow solid. mp: 106–107 ◦C. IR (KBr, νmax/cm−1): 1304, 1330, 1512. 1H-NMR (400 MHz, CDCl3, δ): 8.34 (d, 1H, J = 8.0), 7.38–7.31 (m, 2H), 7.18 (d, 1H, J = 8.0), 6.93–6.86 (m, 2H), 4.51–4.43 (m, 1H), 4.47 (s, 2H), 4.16 (dd, 1H, J = 8.4, J = 6.4), 4.04 (dd, 1H, J = 9.6, J = 5.6), 3.93 (dd, 1H, J = 9.6, J = 5.6), 3.89 (dd, 1H, J = 8.4, J = 5.6), 1.45 (s, 3H), 1.39 (s, 3H). 13C-NMR (100 MHz, CDCl3, δ): 158.6, 149.1, 142.4, 141.0, 132.8, 130.6, 130.0, 125.9, 121.3, 115.1, 109.8, 73.9, 68.8, 66.7, 36.2, 26.8, 25.3. HRMS (ESI+): m/z [M + Na]+ calculated for C19H19NaN3O6S 440.0887, found 440.0879. 2.2.6. rac-3-(4-(((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1,2- diol (rac-IV-j) 2.3. Bacterial Strains, Culture Media and Growth Conditions H. pylori (ATCC 700392) and Helicobacter hepaticus (ATCC 51449) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), whereas Campylobacter jejuni (ATCC 33560) was donated by Dr. Pilar Mañas from the University of Zaragoza (Spain). Strains of Helicobacter felis (JKM5), Helicobacter suis (HS1 and HS5), Helicobacter heilmannii (ASB1.4 and ASB2), Helicobacter ailurogastricus (ASB7 and ASB9) and Helicobacter bizzozeronii (ASB22 kol15) have been described in Smet et al. [21]. Strains of H. bizzozeronii (10 and Heydar) were provided by Prof. Dr. Mirko Rossi. Helicobacter muridarum and Helicobacter bilis were available from Dr. Eliette Touati from the Institut Pasteur (Paris, France). Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium; SV 5015), Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 15442), Bacillus sp. (CECT 40), Streptococcus pneumoniae (ATCC 49619), Listeria monocytogenes (ATCC BAA-679), Enterococcus faecalis (JH2-2), Corynebacterium diphtheriae (ATCC 39255), Corynebacterium ammoniagenes (ATCC 6872), Mycolicibacterium smegmatis (ATCC 700084) and Staphylococcus aureus (ATCC 29213) were available from the culture collections of the Departments of Microbiology and Biochemistry of the University of Zaragoza (Spain). gy y y g ( p ) The strains P. aeruginosa MPAO1 and P. aeruginosa PW9682, a ∆rmlC mutant of MPAO1 that is defective in lipopolysaccharide (LPS) biosynthesis [22], were obtained from the Transposon Mutant Collection of the University of Washington [23,24]. Klebsiella pneumoniae subspecies pneumoniae strains 3025 (serotype O1:K−, StrR; produces smooth-type LPS with D-galactan I and D-galactan II O-antigens) and CWK43 (rpsL, cps, rfb, StrR, serotype O−:K−; produces truncated rough-type LPS) [25–27] were kindly provided by Prof. Dr. Ian R. Poxton (Department of Medical Microbiology, University of Edinburgh, Edinburgh, U.K.) and Prof. Dr. Chris Whitﬁeld (Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada), respectively. The Stenotrophomonas maltophilia K279a [28] wild-type strain was originally obtained from the laboratory of Prof. Dr. Matthew B. Avison (School of Medical Sciences, University of Bristol, Bristol, U.K.), and the construction of the S. maltophilia K279a ∆rmlBACD [29] mutant lacking the O-antigen of LPS has been described elsewhere. Cultures of H. pylori, H. hepaticus, H. muridarum and H. bilis were grown in blood agar base No. 2 (Oxoid, Basingstoke, Hampshire, UK) supplemented with 8% deﬁbrinated horse blood (Oxoid) under microaerophilic conditions (85% N2, 10% CO2, 5% O2) at 37 ◦C for 48–72 h. 2.2.8. rac-3-(4-(((7-Aminobenzo[c][1,2,5]oxadiazol-4- yl)sulﬁnyl)methyl)phenoxy)propane-1,2-diol (rac-IV-l) To a solution of compound rac-IV-k (24 mg, 0.07 mmol) in glacial acetic acid (2.0 mL) was added 35% H2O2 aqueous solution (57 µL, 0.59 mmol) and the reaction mixture was stirred for 1.5 h at room temperature. The reaction mixture was quenched with 1M Na2SO3 aqueous solution (0.59 mL, 0.59 mmol) and the resulting suspension was diluted with distilled water and extracted with ethyl acetate (4 × 10 mL). The combined organic layers were dried with anhydrous MgSO4, ﬁltered and evaporated under reduced pressure. The Int. J. Mol. Sci. 2021, 22, 10137 7 of 25 resulting residue was dissolved in methanol and adsorbed in silica-gel to be puriﬁed by column chromatography (eluent 1: AcOEt, eluent 2: Et2O/MeOH 9:1), affording compound rac-IV-l (21 mg, 82% yield) as a yellow solid. mp: 73–74 ◦C. IR (KBr, νmax/cm−1): 1032, 3405. 1H-NMR (400 MHz, CD3OD, δ): 7.39 (d, 1H, J = 7.6), 6.99–6.92 (m, 2H), 6.85–6.78 (m, 2H), 6.31 (d, 1H, J = 7.6), 4.54 (d, 1H, J = 13.0), 4.39 (d, 1H, J = 13.0), 4.03–3.96 (m, 1H), 3.96–3.87 (m, 2H), 3.70–3.58 (m, 2H). 13C-NMR (100 MHz, CD3OD, δ): 160.6, 147.3, 146.1, 142.9, 138.1, 132.6, 122.9, 115.6, 111.8, 104.1, 71.7, 70.3, 64.1, 59.4. HRMS (ESI+): m/z [M + Na]+ calculated for C16H17NaN3O5S 386.0781, found 386.0779. 2.3. Bacterial Strains, Culture Media and Growth Conditions 2.4. Evaluation of the Antibacterial Activity against a Diverse Microbial Panel 2.4. Evaluation of the Antibacterial Activity against a Diverse Microbial Panel Serial broth microdilutions were made to determine the Minimal Inhibitory Con- centration (MIC) of IV, IV-a, IV-b, IV-c, IV-d, rac-IV-j, rac-IV-k, rac-IV-l, Mnz and Cla against H. pylori, H. hepaticus, H. muridarum, H. bilis, C. jejuni, S. Typhimurium, E. coli, P. aeruginosa, Bacillus sp., S. pneumoniae, L. monocytogenes, E. faecalis, S. aureus, C. diphtheriae, C. ammoniagenes and M. smegmatis as previously described [18]. Brieﬂy, each well of a 96-well plate was inoculated with 100 µL of fresh liquid bacterial culture at 106 CFU/mL (for Helicobacter strains) or 5 × 105 CFU/mL (for the other bacteria), except for the ﬁrst well of each raw, which received 200 µL of bacterial culture plus 2 µL of compound (from stock solutions at 6.4 mg/mL in 100% DMSO). After performing two-fold serial dilutions, plates were incubated under the culture conditions described above, to evaluate the antimicrobial activity of compounds in a concentration range of 0.031–64 µg/mL. MIC values, deﬁned as the lowest concentration of compound able to inhibit bacterial growth, were determined after addition of 30 µL of resazurin 0.1 mg/mL (Sigma-Aldrich, San Luis, MO, USA). Positive and negative controls were included in all the experiments, which were performed, at least, twice in duplicate. MIC values of omeprazole, rabeprazole, CCCP, reserpine and valinomycin against H. pylori (ATCC 700392) were also determined by the microdilution MIC testing as explained above. g p Susceptibility of H. felis and H. bizzozeronii to compounds IV and rac-IV-j was eval- uated by the agar dilution method, as previously described [30]. Succinctly, compounds were added to agar plates according to two-fold serial dilutions, with ﬁnal concentrations ranging from 0.03 to 128 µg/mL. Agar plates free of compounds were included as pos- itive controls. H. felis and H. bizzozeronii bacteria were grown for 72 h, then harvested and suspended in sterile saline to a density of 3 on the McFarland turbidity scale. Then, plates were seeded by a Steers inoculum replicator (MAST, London, United Kingdom) and incubated in a microaerophilic atmosphere. After 7 days, plates were read, the MIC being determined as the lowest compound concentration that inhibits visible growth. MICs of IV and rac-IV-j against H. suis, H. heilmannii and H. ailurogastricus were determined by using a combined agar and broth dilution method in 24-well plates (Greiner Bio-On, Frickenhausen, Germany), as previously described [31]. 2.3. Bacterial Strains, Culture Media and Growth Conditions For drug susceptibility testing, bacteria were grown under the same conditions in brain heart infusion (BHI) broth (Oxoid) supplemented with 4% foetal bovine serum (FBS) (Pan-Biotech, Aidenbach, Germany) for H. pylori, 10% FBS and 2.5 g/L yeast extract (Scharlab, Barcelona, Spain) for H. hepaticus, and 10% FBS for H. muridarum and H. bilis. In these last two cases, the cultures were also stirred at 150 rpm. Cultures of C. jejuni were grown under the same conditions except for the FBS supplement and shaking. Susceptibil- ity of H. felis and H. bizzozeronii to the compounds was evaluated by incubating bacteria on Mueller-Hinton II agar (Becton Dickinson, Cockeysville, MD, USA), supplemented with 10% deﬁbrinated horse blood and 0.6% Vitox (Oxoid) for 7 days at 37 ◦C under microaerophilic conditions. H. suis, H. heilmannii and H. ailurogastricus were grown for 48 h at pH 5 and 37 ◦C under microaerophilic conditions, using a biphasic medium consisting of Brucella agar (BD, Franklin Lakes, NJ, USA) supplemented with 20% inactivated fetal calf serum (Hyclone, ThermoFisher Scientiﬁc, Waltham, MA, USA), Vitox supplement Int. J. Mol. Sci. 2021, 22, 10137 8 of 25 (Oxoid), and Campylobacter selective supplement (Skirrow, Oxoid), with Brucella broth (Oxoid) added on top. On the other hand, S. Typhimurium, E. coli, P. aeruginosa, Bacillus sp. and E. faecalis were grown under aerobic conditions at 37 ◦C in lysogeny broth (LB) overnight with stirring at 150 rpm. Same conditions were used for M. smegmatis cultures, but it was incubated for 72 h without shaking. Cultures of L. monocytogenes, C. diphtheriae and C. ammoniagenes were grown in BHI broth for 24 h with stirring at 150 rpm, with the exception of the last one whose culture was incubated overnight. S. pneumoniae was grown in BHI broth supplemented with 4% FBS for 10 h, while S. aureus was cultured in Mueller-Hinton broth (Panreac, Castellar del Vallès, Spain) pH 7 for 16 h. Finally, the strains 3025 and CWK43 of K. pneumoniae, K279a and K279a ∆rmlBACD of S. maltophilia, and MPAO1 of P. aeruginosa were routinely grown with shaking (220 rpm) at 37 ◦C in LB medium. For cultivation of P. aeruginosa PW9682, the LB medium was supplemented 17 µg/mL of tetracycline. 2.5. Cloning, Expression, Puriﬁcation and Quantiﬁcation of Recombinant Flavodoxin 2.5. Cloning, Expression, Puriﬁcation and Quantiﬁcation of Recombinant Flavodoxin Recombinant wild-type Hp-Fld, along with N14A, A55W, V113W, Q115W, T116W, K133A and D142Y mutants, were overexpressed in E. coli strain BL21 (DE3, Sigma-Aldrich) and puriﬁed as previously described [11] with slight modiﬁcations. A bacterial culture containing the pET28a-ﬂdA plasmid was grown at 37 ◦C in LB medium supplemented with 20 µg/mL kanamycin (Sigma-Aldrich) to an OD600 of 0.8. Then, ﬂavodoxin expression was induced by addition of 1 mM isopropyl β-D-thiogalactoside (Thermo Fisher Scientiﬁc) and further incubation at 37 ◦C overnight. After centrifugation, bacteria were resuspended in cell disruption buffer (50 mM Tris-HCl buffer, 100 µM EDTA, 100 µM β-mercaptoethanol and 1 µM phenylmethanesulphonyl ﬂuoride, pH 8), mixed with 50 mg of FMN (Santa Cruz Biotechnology, Dallas, TX, USA) and lysed by sonication (Hielscher UP200S, 24 kHz). After centrifugation at 18.000 rpm (in an Avanti J-26XP High Performance Centrifuge (Beckman Coulter, Brea, CA, USA) with a JA-25.50 rotor) for 30 min at 4 ◦C, the supernatant was precipitated with 65% (NH4)2SO4 and centrifuged at 18.000 rpm for 30 min at 4 ◦C. The supernatant was then loaded onto a diethylaminoethyl (DEAE) cellulose column equilibrated with 65% (NH4)2SO4 in 50 mM Tris–HCl buffer, pH 8, and the ﬂavodoxin, which is only weakly bound to the column, was eluted with 65% (NH4)2SO4 in 50 mM Tris–HCl buffer, pH 8 and then dialysed. The eluted fractions were poured onto a DEAE column equilibrated with 50 mM Tris–HCl buffer, pH 8 and the protein was eluted with a linear gradient from 0 to 1 M NaCl in 50 mM Tris–HCl buffer, pH 8. Then, it was dialysed against the corresponding buffer, concentrated, transformed into holoprotein if needed, and quantiﬁed by using the theoretical extinction coefﬁcients (εAPO, 278 nm = 15.96 mM−1cm−1; εHOLO, 278 nm = 37.37 mM−1cm−1; εHOLO, 452 nm = 10.65 mM−1cm−1). Flavodoxin purity was determined by 15% SDS-PAGE, followed by Coomassie staining (Figure S1). The native conformation of wild type and of the ﬂavodoxin variants can be inferred from the corresponding visible spectra (Figure S2) as, in all variants, the distinctive ﬂavin absorption shoulder at 480 nm, characteristic of FMN bound to native ﬂavodoxin, is clearly observed [33]. 2.4. Evaluation of the Antibacterial Activity against a Diverse Microbial Panel In brief, agar plates and broth were prepared to contain two-fold serial dilutions of the tested compounds. Then, they were inoculated with 150 µL of the bacterial culture at 5 × 107 bacteria/mL, so that each well of the 24-well plates contained 200 µL of broth and 400 µL agar, with ﬁnal concentrations of the compounds ranging from 0.03 to 128 µg/mL. Wells containing only bacterial culture were included as positive controls. MICs were determined as the lowest compound con- centration with at least 50% bacterial growth inhibition compared to controls. To determine the impact of culture and pH conditions on the activity of the compounds IV and rac-IV-j, 2 different MIC assays were performed for S. aureus: (i) the combined agar and broth dilution method at pH 5, similar to the described above and (ii) the broth microdilution method (according to the Clinical and Laboratory Standards Institute ([32]) standards) Int. J. Mol. Sci. 2021, 22, 10137 9 of 25 in unsupplemented Mueller Hinton broth at pH 7, as previously reported [31]. In both cases, bacterial growth was analysed after 16–24 h of incubation. The MIC was recorded as the lowest compound concentration for which there was no turbidity. Susceptibility of P. aeruginosa strains MPAO1 and PW9682, S. maltophilia strains K279a and K279a ∆rmlBACD, as well as K. pneumoniae 3025 and CWK43 to IV, IV-a, IV-b, IV-c and IV-d was studied by MIC determination using the broth microdilution method in LB medium essentially as described above, except that each culture with a starting OD600 of 0.01 was mixed with a two-fold serial dilution of each compound in a concentration range between 0.25 and 512 µg/mL. The microtiter plates were incubated at 37 ◦C in a humidiﬁed chamber for 20 h, followed by determination of the OD600 values and incubation of the cultures in the presence of resazurin at 37 ◦C for 30 min. 2.9. Generation of Spontaneous Resistant Mutants H. pylori reference strain ATCC 700392, at 5 × 106 and 5 × 107 CFU/mL in BHI broth (Oxoid) supplemented with 4% FBS, was incubated at 37 ◦C for 5 days under microaerophilic conditions in the presence of compounds IV, IV-d and rac-IV-l at ﬁnal concentrations of 32, 16, 8, 4, 2, 1 and 0.5 times their MICs. Because H. pylori is able to develop resistance to Mnz, this antibiotic was used as a control. The generation of resistant mutants was evaluated by the MIC values of compounds IV, IV-a, IV-c, IV-d, rac-IV-l and Mnz against the treated cultures, compared to the corresponding MIC of the parental wild-type strain. The experiment was performed twice in duplicate. 2.7. Molecular Docking of Compounds IV, IV-a, IV-b, IV-c and IV-d to Hp-Fld Initial structures of compounds IV, IV-a, IV-b, IV-c and IV-d were obtained from their SMILES codes through the online NCI/CADD SMILES translator tool (https://cactus. nci.nih.gov/translate/ (accessed on 10 September 2020), NIH National Cancer Institute, Frederick, MD, USA). They were subsequently optimised at the ab initio level (#HF/6-31G) with Gaussian 09 [34], and the resulting structures were docked onto the three-dimensional structure of wild-type Hp-Fld (PDB ID: 1FUE) with AutoDock4.2.6 (La Jolla, CA, USA) [35]. A ﬁrst round of docking was performed, allowing the compounds to sample the whole protein surface (blind docking), keeping the protein rigid and allowing the compounds’ rotatable bonds to freely rotate. The ﬂavodoxin regions, which showed the highest scored poses of docked compounds, were then subjected to targeted docking (local sampling), now with both entities in ﬂexible mode (protein sidechains’ and compounds’ rotatable bonds allowed to rotate). The amino acid residues that appeared crucial for the binding were identiﬁed. They were replaced by site directed mutagenesis for the sake of testing their potential contribution to complex formation with the inhibitory compounds by ITC. To that end, the afﬁnity of the complexes formed by the chosen compounds and wild-type Hp-Fld was compared to that of the complexes established with the mutant ﬂavodoxins. 2.8. Combined Antimicrobial Effect Once the MICs of EIs CCCP, reserpine and valinomycin against H. pylori were deter- mined, as described above, the antimicrobial effect of several IV-related compounds was evaluated in combination with these EIs. To that end, the MIC of each compound against a H. pylori culture was evaluated in the presence of each EI at a concentration of 1 4 of its MIC. The assay was carried out in the same way as a conventional microdilution MIC testing, and the culture conditions were as described above. We considered that any EI had a signiﬁcant effect on a compound MIC when this was reduced at least four-fold in the presence of that EI. Each experiment was performed twice in triplicate. 2.9. Generation of Spontaneous Resistant Mutants 2.6. Determination of the Hp-Fld-Binding Afﬁnity of Inhibitors Isothermal titration calorimetry (ITC) experiments were carried out in an Auto-iTC200 calorimeter (MicroCal, Malvern-Panalytical, Malvern, UK) in order to characterise the afﬁnity of the wild-type Hp-Fld protein and the different mutants N14A, A55W, V113W, Q115W, T116W, K133A and D142Y for the different compounds. Recombinant Hp-Fld at 10–20 µM concentration in 50 mM EPPS buffer, pH 9 (for IV, IV-a, IV-b, IV-c, rac- IV-j, rac-IV-k and rac-IV-l), or in 50 mM Tris-HCl buffer, pH 9 (for IV-d) was titrated with 120–300 µM ligand solutions in the same buffer, prepared from stock compound solutions in 100% DMSO. At the residual DMSO concentration of 2.5% employed in the working solutions, wild-type ﬂavodoxin has been described to be conformationally stable and biochemically active [16]. Thermodynamic parameters of the binding equilibrium were calculated by non-linear least-squares regression analysis considering a single ligand Int. J. Mol. Sci. 2021, 22, 10137 10 of 25 10 of 25 binding site and using the user-deﬁned ﬁtting routines in the MicroCal LLC ITC module from the Origin 7.0 software (Northampton, MA, USA). binding site and using the user-deﬁned ﬁtting routines in the MicroCal LLC ITC module from the Origin 7.0 software (Northampton, MA, USA). 3.1. Selectivity of the Flavodoxin Inhibitors for Bacteria of the Helicobacter Genus 3.1. Selectivity of the Flavodoxin Inhibitors for Bacteria of the Helicobacter Genus The in vitro efﬁcacy of IV-related compounds, Mnz and Cla, was evaluated (Table 1) against several bacteria from different phyla (Table S2). Their therapeutic activity was reported as the lowest concentration, leading to 50% bacterial growth inhibition in com- parison with drug-free controls (for the Helicobacter species requiring a biphasic growth medium) or as the MIC (for the other bacteria tested in broth). As seen in Table 1, while H. pylori is susceptible to all IV-related compounds (except rac-IV-l) at low concentrations, the growth of many other bacteria tested is not inhibited by most of these derivatives even at concentrations up to 64 or 512 µg/mL, respectively. However, according to MIC breakpoints of antibiotics traditionally used to treat these bacterial infections [39], two compounds, IV and rac-IV-j, show moderate to good antimicrobial activity (≤32 µg/mL) against C. jejuni, and the Gram-positive Bacillus sp., S. pneumoniae, E. faecalis, S. aureus, C. diphtheriae and C. ammoniagenes. Moreover, these compounds show low MICs for the gastric non-H. pylori Helicobacter species tested (H. felis, H. suis, H. heilmannii, H. ailurogastricus and H. bizzozeronii) and against the enterohepatic Helicobacter species H. muridarum and H. bilis. g p p Globally, the effect of the compounds can be described considering three groups of bacteria: Gram-positive, the Gram-negative bacteria belonging to the Helicobacter and Campylobacter genera, and all other Gram-negative ones. The growth of most Gram-positive bacteria tested—except M. smegmatis and L. monocytogenes—is inhibited by compounds IV and rac-IV-j but not by compounds IV-a, IV-b, IV-c, IV-d, rac-IV-k or rac-IV-l. Table 1. In vitro activity of IV-related compounds against several bacteria from different phyla. ro activity of IV-related compounds against several bacteria from different phyla. Table 1. In vitro activity of IV-related compounds against several bacteria from different phyla. Gram Bacterial Species (Strain) Compound MIC (µg/mL) IV IV-a IV-b IV-c IV-d rac- IV-j rac- IV-k rac-IV-l Mnz Cla - H. pylori (ATCC 700392) 2 8 1 2 8 1 16 256 2 ≤0.032 - H. felis (JKM5) 8 32 ≤0.031 - H. suis (HS1) 4 4 0.25 (HS5) 4 2 ≤0.031 - H. heilmannii (ASB1.4) 2 2 0.125 (ASB2) 2 1 ≤0.031 - H. ailurogastricus (ASB7) 4 1 0.125 (ASB9) 4 4 0.5 - H. bizzozeronii (ASB22 kol15) 16 16 ≤0.031 (10) 8 32 ≤0.031 (Heydar) 8 32 ≤0.031 - H. 2.10. Chequerboard Synergy Testing Pairwise interactions between several IV-related compounds and Mnz, Cla, omepra- zole or rabeprazole, as well as between parent compound IV and its derivatives IV-a, IV-b, IV-c, IV-d, rac-IV-j, rac-IV-k and rac-IV-l were studied in H. pylori strain ATCC 700392. In a 96-well plate containing a fresh culture of bacteria at 106 CFU/mL, one compound (at 32 times its MIC) was added to the second column and the other one (at 4 times its MIC) was poured in raw G. Both compounds were serially 2-fold diluted to generate a gradient matrix of both compounds. The ﬁrst and last columns of the plate allowed one to test each compound alone. After incubation as described above for H. pylori growth, plates were treated with resazurin, as in a conventional MIC assay, to reveal the result of the interaction effect. For each pair of compounds, the fractional inhibitory concentra- tion index (FICI) was determined as FICI = FIC1 + FIC2, where FIC1 = (MICcompound 1 in presence of compound 2)/(MICcompound 1 alone) and FIC2 = (MICcompound 2 in presence Int. J. Mol. Sci. 2021, 22, 10137 11 of 25 11 of 25 of compound 1)/(MICcompound 2 alone). The value of the FICI indicates whether two compounds show a synergistic effect (FICI ≤0.5), no interaction (0.5 < FICI ≤4) or an antagonistic effect (FICI > 4) effect [36]. Pairs of FIC values can be represented graphically, and a concave curve indicating synergy. The point of the curve closest to the intersection of the axes corresponds to the most effective combination of compounds that inhibits H. pylori growth [37,38]. 3.1. Selectivity of the Flavodoxin Inhibitors for Bacteria of the Helicobacter Genus hepaticus (ATCC 51449/3B1) 64 >64 >64 >64 >64 64 >64 >64 >64 0.063 - H. muridarum 1 >64 >64 >64 >64 4 >64 >64 >64 >64 - H. bilis 16 >64 >64 >64 >64 16 >64 >64 >64 4 - C. jejuni (ATCC 33560) * 2 >64 >64 >64 >64 4 >64 >64 1 4 - S. Typhimurium (SV 5015) * >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 Int. J. Mol. Sci. 2021, 22, 10137 12 of 25 Table 1. Cont. Gram Bacterial Species (Strain) Compound MIC (µg/mL) IV IV-a IV-b IV-c IV-d rac- IV-j rac- IV-k rac-IV-l Mnz Cla - E. coli (ATCC 10536) * >64 >64 >64 >64 >64 >64 >64 >64 >64 16 - P. aeruginosa (ATCC 15442) >64 >64 >64 >64 >64 >64 >64 >64 >64 8 (MPAO1) >512 >512 >512 >512 >512 (PW9682) >512 >512 >512 >512 >512 - S. maltophilia K279a >512 >512 >512 >512 >512 (K279a ∆rmlBACD) >512 >512 >512 >512 >512 - K. pneumoniae 3025 >512 >512 >512 >512 >512 (CWK43) >512 >512 >512 >512 >512 + Bacillus sp. (CECT 40) 4 >64 >64 >64 >64 4 >64 >64 >64 0.063 + S. pneumoniae (ATCC 49619) 8 >64 >64 64 >64 16 >64 >64 >64 ≤0.032 + L. monocytogenes (ATCCBAA-679) >64 >64 >64 >64 >64 >64 >64 >64 >64 0.25 + E. faecalis (JH2-2) 2 >64 >64 >64 >64 4 >64 >64 >64 0.25 + S. aureus (ATCC 29213) 16 >64 >64 >64 >64 8 >64 >64 >64 ≤0.032 BB pH 5 16 16–64 8–16 MH broth pH 7 16 16 0.5–1 + C. diphtheriae (ATCC 39255) 16 64 >64 8 >64 32 >64 >64 >64 0.063 + C. ammoniagenes (ATCC 7862) 16 >64 >64 >64 >64 16 >64 >64 >64 ≤0.032 + M. smegmatis (ATCC 700084) >64 >64 >64 >64 >64 >64 >64 >64 >64 4 * Flavodoxins that appear described as essential in the DEG database [40]. Not being in the DEG database does not necessarily mean they are essential. Table 1. Cont. Table 1. Cont. The other Gram-negative bacteria tested, which belong to the class gamma-proteobacteria, are generally non-susceptible to any of the compounds, including isogenic LPS mutants of P. aeruginosa, S. maltophilia and K. pneumoniae tested to address potential permeabilization issues of the compounds across the outer membrane of the parental wild-type strains. 3.1. Selectivity of the Flavodoxin Inhibitors for Bacteria of the Helicobacter Genus However, most Gram-negative bacteria of the Helicobacter genus (9 species tested) and the closely related C. jejuni species (they both belong to the class epsilon-proteobacteria and the order Campylobacterales) are inhibited, at least by compounds IV and rac-IV-j. Within this group, H. pylori is inhibited by all the compounds tested (lead compound IV was initially discovered as an Hp-Fld inhibitor); the ﬁve remaining gastric species (H. felis, H. suis, H. heilmannii, H. ailurogastricus and H. bizzozeronii) are inhibited at least by compounds IV and rac-IV-j at concentrations ≤32 µg/mL (the other compounds have not been tested in these species); and two out of three enterohepatic species tested (H. hepaticus, H. muridarum and H. bilis) are inhibited by compounds IV and rac-IV-j, but not by the rest of the compounds. 3.2. Probable Interaction of the Inhibitors at a Pocket near the FMN Binding Site Interaction between wild-type Hp-Fld and compounds IV, IV-a, IV-b, IV-c, IV-d, rac- IV-j, rac-IV-k and rac-IV-l was evaluated by ITC (Figure S3 and Table S1). These studies reveal only small differences in the binding afﬁnities of the compounds to the protein target, as they all exhibit dissociation constants in the micromolar range and a 1:1 protein:ligand stoichiometry. As shown, the interaction between wild-type Hp-Fld and IV-derivatives is Int. J. Mol. Sci. 2021, 22, 10137 13 of 25 13 of 25 entropically driven, and characterised by a moderate afﬁnity. The small contribution of the enthalpy balance is, in some cases, positive and in some others negative, which gives rise to the different tendencies of the binding curves. entropically driven, and characterised by a moderate afﬁnity. The small contribution of the enthalpy balance is, in some cases, positive and in some others negative, which gives rise to the different tendencies of the binding curves. Despite considerable efforts, the crystallization of a complex between Hp-Fld and any of the inhibitors has not been possible yet. Considering the lack of structural information on ﬂavodoxin-inhibitor complexes, molecular docking assays were carried out to try to understand the structural basis of the interaction between the protein and the inhibitors. These studies proposed one main binding hotspot for the inhibitors, which was located at the long-loop characteristic of long-chain ﬂavodoxins. In particular, the best binding poses for compounds IV, IV-a, IV-b, IV-c and IV-d showed interaction with residues V113, Q115, T116 and K133 of the protein (Figure 3A). On the other hand, Hp-Fld shows a characteristic pocket near the FMN binding site, where the binding of small compounds has been proposed, that could interfere either with electron transfer or with the interaction of ﬂavodoxin with other protein partners [11]. The pocket arises from the unusual presence in Hp-Fld of an alanine residue (A55) at the si face of the cofactor, where other ﬂavodoxins commonly bear a bulky residue. Three residues, namely: N14 and D142 (close to each other and interacting with the FMN phosphate and its ribityl moiety) and especially A55 (right at the pocket edge) have been noticed that could affect the binding of inhibitors at the pocket (Figure 3B). Figure 3. Molecular surface and ribbon models showing putative interacting residues of wild-type Hp-Fld with compounds IV, IV-a, IV-b, IV-c and IV-d. 3.2. Probable Interaction of the Inhibitors at a Pocket near the FMN Binding Site (A): Val 113, Gln 115, Thr 116 and Lys 133, predicted by molecular docking, and (B): Asn 14, Ala 55 and Asp 142, close to the structural pocket near the cofactor binding site. The FMN cofactor is represented as grey sticks and the nitrogen and oxygen atoms from the amino acid residues are depicted as red and blue sticks, respectively. Figure 3. Molecular surface and ribbon models showing putative interacting residues of wild-type Hp-Fld with compounds IV, IV-a, IV-b, IV-c and IV-d. (A): Val 113, Gln 115, Thr 116 and Lys 133, predicted by molecular docking, and (B): Asn 14, Ala 55 and Asp 142, close to the structural pocket near the cofactor binding site. The FMN cofactor is represented as grey sticks and the nitrogen and oxygen atoms from the amino acid residues are depicted as red and blue sticks, respectively. Figure 3. Molecular surface and ribbon models showing putative interacting residues of wild-type Hp-Fld with compounds IV, IV-a, IV-b, IV-c and IV-d. (A): Val 113, Gln 115, Thr 116 and Lys 133, predicted by molecular docking, and (B): Asn 14, Ala 55 and Asp 142, close to the structural pocket near the cofactor binding site. The FMN cofactor is represented as grey sticks and the nitrogen and oxygen atoms from the amino acid residues are depicted as red and blue sticks, respectively. In order to assess whether the protein region predicted by the docking analysis is the likely binding site for the inhibitors, residues V113, Q115 and T116 were mutated to tryptophan, which may introduce steric hindrance, and K133 was mutated to alanine, which removes the positive charge of the lysine side chain. At the alternative potential binding site located near the cofactor, N14 was replaced by an alanine in order to remove Int. J. Mol. Sci. 2021, 22, 10137 14 of 25 14 of 25 its hydrogen bonding groups, and A55 and D142 were replaced by bulkier tryptophan and tyrosine residues, respectively. The seven wild type residues replaced are located at the protein surface and the seven mutants Hp-Fld generated were expressed and puriﬁed with normal yields, and showed no indications of reduced conformational stability. The afﬁnity of the complexes formed between wild-type or mutant Hp-Fld with compounds IV and IV-c was determined by ITC (Figures 4 and S4). Figure 4. 3.2. Probable Interaction of the Inhibitors at a Pocket near the FMN Binding Site The destabilization of the IV-c complex in the A55W mutant is not as large as in the IV complex, but its afﬁnity is still below the mean of the IV-c complexes. Besides, if the afﬁnity of each variant for IV and IV-c is averaged, the A55W mutant exhibits the weakest mean (−7.89 kcal/mol), being the only one below 1 SD of the mean of all inhibitors’ means (−8.24 ± 0.20 kcal/mol). Altogether, the ITC binding study does not conﬁrm the putative binding site predicted by the docking studies, but supports, although non-conclusively, the hypothesis that the inhibitors bind at the pocket near the Hp-Fld cofactor. Table 2. Thermodynamic parameters of the complexes formed between wild-type and several mutant ﬂavodoxins with compounds IV and IV-c a. Flavodoxin Variant IV IV-c Kd b (µM) ∆G c (kcal/mol) ∆H d (kcal/mol) −T∆S e (kcal/mol) Kd b (µM) ∆G c (kcal/mol) ∆H d (kcal/mol) −T∆S e (kcal/mol) WT 0.48 −8.58 3.77 −12.35 0.67 −8.39 3.10 −11.49 V113W 0.50 −8.56 3.69 −12.25 1.45 −7.93 1.40 −9.33 Q115W 0.52 −8.54 3.29 −11.83 0.69 −8.37 2.36 −10.73 T116W 1.10 −8.10 2.58 −10.68 1.43 −7.94 1.68 −9.62 K133A 0.85 −8.25 2.38 −10.63 0.77 −8.31 2.10 −10.41 N14A 0.45 −8.62 2.10 −10.72 1.76 −7.82 2.61 −10.43 A55W 2.33 −7.65 4.50 −12.15 1.05 −8.12 1.70 −9.82 D142Y 1.18 −8.05 1.13 −9.18 0.44 −8.64 0.92 −9.56 −8.29 ± 0.35 Mean ± SD −8.19 ± 0.28 Mean ± SD a Obtained from calorimetric titrations in 50 mM EPPS, pH 9. b Relative error in Kd is 10%. c Calculation of Gibbs energy change was based on ∆G = RTlnKd. Absolute error in ∆G is 0.1 kcal/mol. d Absolute error in ∆H is 0.3 kcal/mol. e Entropic contribution was calculated according to: −T∆S = ∆G −∆H. Absolute error in −T∆S is 0.3 kcal/mol [41]. Table 2. Thermodynamic parameters of the complexes formed between wild-type and several mutant ﬂ compounds IV and IV-c a. namic parameters of the complexes formed between wild-type and several mutant ﬂavodoxins with IV a a Obtained from calorimetric titrations in 50 mM EPPS, pH 9. b Relative error in Kd is 10%. c Calculation of Gibbs energy change was based on ∆G = RTlnKd. Absolute error in ∆G is 0.1 kcal/mol. d Absolute error in ∆H is 0.3 kcal/mol. e Entropic contribution was calculated according to: −T∆S = ∆G −∆H. Absolute error in −T∆S is 0.3 kcal/mol [41]. 3.2. Probable Interaction of the Inhibitors at a Pocket near the FMN Binding Site These results are consistent with the fact that among the Helicobacter species (H. pylori, H. hepaticus, H. muridarum and H. bilis) plus C. jejuni, for which the whole battery of inhibitors has been tested (inhibitors IV to rac-IV-l), H. pylori is the only one bearing the alanine residue that generates the pocket near the cofactor (Figure 5), and the only one being inhibited by all the compounds tested (Table 1). 3.2. Probable Interaction of the Inhibitors at a Pocket near the FMN Binding Site Thermodynamic analysis of the interaction between compound IV and wild-type Hp-Fld and mutants N14A, V113W, Q115W, T116W and K133A by ITC. (A) The upper plots show the thermograms (thermal power as a function of time), whereas the lower panels exhibit the binding isotherms (titrant normalised heat effects as a function of the ligand:protein molar ratio in the cell) In these last ones, the solid line corresponds to the best ﬁt. (B) Thermodynamic parameters of each ﬂavodoxin interaction with compound IV. Gibbs energy (∆G), enthalpy (∆H) and entropic contribution (−T∆S) are represented in black, grey and white bars, respectively. Figure 4. Thermodynamic analysis of the interaction between compound IV and wild-type Hp-Fld and mutants N14A, V113W, Q115W, T116W and K133A by ITC. (A) The upper plots show the thermograms (thermal power as a function of time), whereas the lower panels exhibit the binding isotherms (titrant normalised heat effects as a function of the ligand:protein molar ratio in the cell). In these last ones, the solid line corresponds to the best ﬁt. (B) Thermodynamic parameters of each ﬂavodoxin interaction with compound IV. Gibbs energy (∆G), enthalpy (∆H) and entropic contribution (−T∆S) are represented in black, grey and white bars, respectively. The thermodynamic proﬁles of the interactions between wild-type and mutant Hp-Fld with either compound are similar. They indicate that all complexes are strongly stabilised by the binding entropy change and signiﬁcantly destabilised by the enthalpy one (Table 2). The similarity of the thermodynamic proﬁles suggests that inhibitors IV and IV-c bind to all the mutant proteins at the same site as with the wild-type protein. Although no large changes in binding afﬁnity occur in the mutants (Table 2), the data point to A55 as the amino acid residue whose replacement by a potentially interfering one cause the greatest changes in afﬁnity. This is clearly seen in the complex between the protein and compound Int. J. Mol. Sci. 2021, 22, 10137 15 of 25 15 of 25 IV. In this complex, the replacement of A55 is the only one which decreases the afﬁnity (as judged from the value of ∆G of binding) by more than 1 SD from the mean value exhibited by the eight Hp-Fld variants tested. Besides, the only substitution that makes the binding enthalpy of the complex even more unfavourable than in the wild-type protein is that of A55. 3.3. Prospects of a Low H. pylori Resistance Rate to IV-Related Compounds [42] In all sequences, the amino acid residue at position 55 (according to Hp-Fld numeration) is highlighted in red. As seen, the ﬂavodoxins from H. pylori, H. suis, H. bizzozeronii, H. felis, H. heilmannii and H. ailurogastricus exhibit an alanine at this position, whereas a tyrosine is shown in that from H. hepaticus and a tryptophan is exhibited by those from C. jejuni, H. muridarum and H. bilis. Asterisks () highlight positions with a fully conserved residue; colons (:) indicate positions of residues with high similarity; dots (.) indicate positions with residues possessing weak similarity. Figure 5. Multiple sequence alignment of ﬂavodoxins from C. jejuni, H. muridarum, H. bilis, H. hepaticus, H. pylori, H. suis, H. bizzozeronii, H. felis, H. heilmannii and H. ailurogastricus. The alignment has been performed with Clustal Omega. [42] In all sequences, the amino acid residue at position 55 (according to Hp-Fld numeration) is highlighted in red. As seen, the ﬂavodoxins from H. pylori, H. suis, H. bizzozeronii, H. felis, H. heilmannii and H. ailurogastricus exhibit an alanine at this position, whereas a tyrosine is shown in that from H. hepaticus and a tryptophan is exhibited by those from C. jejuni, H. muridarum and H. bilis. Asterisks () highlight positions with a fully conserved residue; colons (:) indicate positions of residues with high similarity; dots (.) indicate positions with residues possessing weak similarity. Figure 5. Multiple sequence alignment of ﬂavodoxins from C. jejuni, H. muridarum, H. bilis, H. hepaticus, H. pylori, H. suis, H. bizzozeronii, H. felis, H. heilmannii and H. ailurogastricus. The alignment has been performed with Clustal Omega. [42] In all sequences, the amino acid residue at position 55 (according to Hp-Fld numeration) is highlighted in red. As seen, the ﬂavodoxins from H. pylori, H. suis, H. bizzozeronii, H. felis, H. heilmannii and H. ailurogastricus exhibit an alanine at this position, whereas a tyrosine is shown in that from H. hepaticus and a tryptophan is exhibited by those from C. jejuni, H. muridarum and H. bilis. Asterisks (*) highlight positions with a fully conserved residue; colons (:) indicate positions of residues with high similarity; dots (.) indicate positions with residues possessing weak similarity. Table 3. In vitro activity of IV-related compounds alone and in combination with CCCP, reserpine and valinomycin against H. pylori strain ATCC 700392. 3.3. Prospects of a Low H. pylori Resistance Rate to IV-Related Compounds Bacterial efﬂux pumps are membrane proteins able to actively transport a number of substances, including drugs, from the cytoplasm to the extracellular environment. Efﬂux reduces the antibacterial activity of some drugs by throwing them out from the cell, and is considered as a low-level antibiotic resistance mechanism that can favour and promote the development of higher levels of resistance. In order to determine whether IV-related compounds are affected by efﬂux systems, the MICs of these molecules were evaluated in combination with sub-inhibitory concentrations of three different efﬂux inhibitors (EIs): CCCP, reserpine and valinomycin. To do that, we ﬁrst determined the MICs of these EIs against H. pylori reference strain ATCC 700392. Those MICs turned out to be of 0.32, 12.5 and 10 µg/mL for CCCP, reserpine and valinomycin, respectively. Each EI was added at a concentration of 1 4 MIC, and it was considered that a compound could be signiﬁcantly transported out by efﬂux pumps when its MIC was decreased by at least 4 times. As shown in Table 3, CCCP, reserpine and valinomycin were able to reduce the MIC of IV by a factor of 4, indicating that compound IV could be affected by efﬂux, although this effect would be just in the limit of what we consider as signiﬁcant. In addition, reserpine and valinomycin lowered the MIC of IV-a by half, an effect which is also achieved by rac-IV-k when it was combined with valinomycin, suggesting that efﬂux could play a weak role (but not Int. J. Mol. Sci. 2021, 22, 10137 16 of 25 16 of 25 signiﬁcant enough) in the activity of these compounds. On the other hand, the same MICs, or even moderately higher (two- to four-fold) MICs, were obtained for compounds IV-b, IV-c, IV-d and rac-IV-j, which indicates that the EIs tested would interfere moderately with the antimicrobial activity of Hp-Fld inhibitors. signiﬁcant enough) in the activity of these compounds. On the other hand, the same MICs, or even moderately higher (two- to four-fold) MICs, were obtained for compounds IV-b, IV-c, IV-d and rac-IV-j, which indicates that the EIs tested would interfere moderately with the antimicrobial activity of Hp-Fld inhibitors. Figure 5. Multiple sequence alignment of ﬂavodoxins from C. jejuni, H. muridarum, H. bilis, H. hepaticus, H. pylori, H. suis, H. bizzozeronii, H. felis, H. heilmannii and H. ailurogastricus. The alignment has been performed with Clustal Omega. 3.3. Prospects of a Low H. pylori Resistance Rate to IV-Related Compounds Compound MIC a (µg/mL) Without any EI CCCP Reserpine Valinomycin IV 2 0.5 0.5 0.5 IV-a 8 8 4 4 IV-b 1 2 2 2 IV-c 2 4 4 4 IV-d 8 32 32 32 rac-IV-j 1 4 2 2 rac-IV-k 16 16 16 8 rac-IV-l >64 >64 >64 >64 Mnz 2 4 4 2 Cla ≤0.032 ≤0.032 ≤0.032 ≤0.032 a The MIC values were assayed in the presence of non-lethal concentrations of the following EPIs: CCCP (0.08 µg/mL), reserpine (3.12 µg/mL) and valinomycin (2.50 µg/mL). Table 3. In vitro activity of IV-related compounds alone and in combination with CCCP, reserpine and valinomycin against H. pylori strain ATCC 700392. a The MIC values were assayed in the presence of non-lethal concentrations of the following EPIs: CCCP (0.08 µg/mL), reserpine (3.12 µg/mL) and valinomycin (2.50 µg/mL). Int. J. Mol. Sci. 2021, 22, 10137 17 of 25 17 of 25 Altogether, these data showed that the antibacterial activity of IV-related compounds was not altered signiﬁcantly by EIs, suggesting that the efﬂux pumps inhibited by those EIs were not able to actively transport any of the compounds of this family to a signiﬁcant extent. Therefore, the intrinsic resistance of H. pylori to therapies based on these ﬂavodoxin inhibitors can be regarded as moderate–low, which would make the development of higher resistance levels less likely. y In support of the latter observation, we were not able to select spontaneous mutants showing resistance to ﬂavodoxin inhibitors. After incubating H. pylori strain ATCC 700,392 (at 5 × 106 and 5 × 107 CFU/mL) in the presence of different concentrations (0.5, 1, 2, 4, 8, 16 and 32 times their MICs) of compounds IV, IV-d, rac-IV-l and Mnz (as a control drug), the generation of spontaneous mutants was checked by MIC testing against those cultures. Whereas no signiﬁcant changes in the MICs of compounds IV, IV-d and rac-IV-l were observed against any of those cultures previously treated with the same compounds compared to the MIC for the wild-type strain, a 16-fold increase in Mnz MIC was recorded against a H. pylori culture previously exposed to a 4-fold MIC concentration of this drug, which indicates that Mnz-resistant mutants have been generated. Accordingly, the ability of H. pylori to develop resistance to those IV-related compounds seems to be much less pronounced than that to develop resistance against Mnz. 3.4. Synergistic Interaction between Some IV-Related Compounds and Rabeprazole The bactericidal effect of compound IV and several derivatives in combination with anti-H. pylori drugs such as Mnz, Cla, omeprazole and rabeprazole was analysed in vitro, as well as that of compound IV combined with some of its derivatives. The synergistic effect was estimated by calculating the fractional inhibitory concentration index (FICI) for each pair of molecules. As the checkerboard assay requires previous knowledge of the MIC of the individual compounds, the MICs of omeprazole and rabeprazole against H. pylori strain ATCC 700,392 were also determined. Notably, the in vitro activity of rabeprazole (MIC = 0.5 µg/mL) was 64 times higher than that of omeprazole (MIC = 32 µg/mL). As seen in Table 4, whereas most compounds did not interact with IV, Mnz, Cla, omeprazole or rabeprazole (FICI > 0.5), compounds IV-b and rac-IV-l showed a synergistic relation (FICI = 0.5) with rabeprazole, indicating that the combined effect of these substances is signiﬁcantly higher than the sum of their individual antimicrobial activities. Synergism was conﬁrmed by plotting the FIC values of IV-b and rac-IV-l against the corresponding FIC values of rabeprazole as in both cases a concave curve was obtained (Figure 6). For those compounds, the most effective combinations for inhibiting H. pylori growth were 0.25 µg/mL of IV-b or 64 µg/mL of rac-IV-l plus 0.125 µg/mL of rabeprazole. Although signiﬁcant synergism, as per our quantitative deﬁnition, was only found between the indicated pairs of substances, there were several compounds that clearly reduced the MICs of others. In particular, IV-a lowered the MIC of IV 64-fold (FIC = 0. 016; Table 4), whereas IV lowered that of Mnz 16-fold (FIC = 0.063). Notably, the combination of Cla with compounds IV-d, rac-IV-j or rac-IV-l reduced their MICs 8-fold (FIC = 0.125), an effect which was also observed for the omeprazole MIC when it was combined with IV- b (FIC = 0.125). On the other hand, the combination of rabeprazole with IV-a or IV-d lowered their MICs 16- and 8-fold (FIC = 0.0625 or 0.125, respectively). Thus, in conclusion, these compounds improved the in vitro anti-H. pylori activity of those other molecules and, remarkably, IV and IV-b increased, respectively, the antimicrobial effect of Mnz and omeprazole, two conventional drugs used to treat H. pylori infections. Int. J. Mol. Sci. 2021, 22, 10137 18 of 25 Table 4. 4. Discussion The emergence of resistant bacteria worldwide is a global health concern, of which the management requires coordinated efforts [43]. In particular, H. pylori resistance to currently used broad-spectrum antimicrobials has led to a decrease in the eradication rates, which brought about the inclusion of H. pylori in the ﬁrst-ever list of antibiotic-resistant priority pathogens published by the WHO in 2017 [7]. In order to address this problem, new selective treatments have been proposed, one of them being the development of new compounds acting on speciﬁc bacterial targets such as ﬂavodoxin [10]. This is an essential protein for H. pylori and it is absent in humans, which makes it a promising pharmacological target [11]. In earlier work, three ﬂavodoxin inhibitors (I, II and IV) were discovered [16] and derivatised [17] to improve their therapeutic indexes against H. pylori. In the second round of optimization, the modiﬁcation of nitro and sulphide groups present in one of the original leads (compound IV) increased its therapeutic activity against some reference strains and several drug-resistant clinical isolates. Moreover, this family of compounds was also able to decrease H. pylori gastric colonization rates in a mice model of infection and to eradicate it in up to 60% of mice treated [18]. The goal of the present study was to develop a better understanding of the antimicrobial spectrum and the mechanism of action of this family of antimicrobials. The compounds have been tested against 8 Gram-positive and 15 Gram-negative bacteria. The Gram-negative bacteria tested comprise 7 genera of the phylum Proteobacteria, of which the Helicobacter genus is represented by 3 enterohepatic species (H. hepaticus, H. muridarum and H. bilis) and 6 gastric species (H. pylori, H. felis, H. suis, H. heilmannii, H. ailurogastricus and H. bizzozeronii). Most IV-related compounds showed potent antimicrobial activity against H. pylori. However, the antimicrobial spectrum (Table 1) was not the same for all of them. Compounds IV and rac-IV-j are extended-spectrum antimicrobials that are effective against the Gram-positive bacteria and Gram-negative bacteria of the Helicobacter genus (plus C. jejuni), but they are ineffective against any of the other Gram-negative bacteria tested, belonging to 5 different genera. As they are effective against potential pathogenic bacteria for humans [10], such as H. pylori, H. suis, H. felis, H. heilmannii, H. bizzozeronii, C. jejuni, some Bacillus species, S. pneumoniae, E. faecalis, S. aureus and C. 3.4. Synergistic Interaction between Some IV-Related Compounds and Rabeprazole FICs and FICIs of IV-related compounds in combination with either IV, Mnz, Cla, omepra- zole or rabeprazole a. Combination Compound IV-Related Compound FICcombination compound FICIV-related compound FICI b IV-a 0.016 0.5 0.516 IV-b 1 1 2 IV-c 1 1 2 IV IV-d 1 1 2 rac-IV-j 0.25 0.5 0.75 rac-IV-k 1 1 2 rac-IV-l 0.25 0.5 0.75 IV 0.063 0.5 0.56 IV-a 0.5 0.25 0.75 IV-b 0.5 0.5 1 Mnz IV-c 0.5 0.5 1 IV-d 0.5 0.5 1 rac-IV-j 0.5 0.5 1 rac-IV-k 0.5 0.5 1 rac-IV-l 0.5 0.5 1 IV 0.5 0.5 1 IV-a 0.5 0.25 0.75 IV-b 0.5 0.5 1 Cla IV-c 0.5 0.25 0.75 IV-d 0.5 0.125 0.62 rac-IV-j 0.5 0.125 0.62 rac-IV-k 0.25 0.5 0.75 rac-IV-l 0.5 0.125 0.62 IV 0.5 0.5 1 IV-a 0.25 0.5 0.75 IV-b 0.125 0.5 0.62 Omeprazole IV-d 0.25 0.5 0.75 rac-IV-j 0.5 0.5 1 rac-IV-k 0.25 0.5 0.75 rac-IV-l 0.5 0.5 1 IV 1 1 2 IV-a 0.5 0.063 0.56 IV-b 0.25 0.25 0.5 Rabeprazole IV-d 0.5 0.125 0.62 rac-IV-j 0.5 0.5 1 rac-IV-k 0.5 0.5 1 rac-IV-l 0.25 0.25 0.5 a Values were determined against H. pylori ATCC 700392. b The fractional inhibitory index (FICI) was cal- culated as follows: FICI = (MICCombination compound in the presence of IV-related compound/MICCombination compound alone) + (MICIV-related compound in the presence of Combination compound/MICIV-related compound alone). A FICI value of ≤0.5 indi- cates synergy, a value from 0.5 to 4 denotes no interaction and a value of >4 expresses antagonism. Table 4. FICs and FICIs of IV-related compounds in combination with either IV, Mnz, Cla, omepra- zole or rabeprazole a. 19 of 25 Int. J. Mol. Sci. 2021, 22, 10137 Figure 6. Synergism between rabeprazole and compounds IV-b (A) and rac-IV-l (B) against H. pylori strain ATCC 700392. The concave curve obtained in both graphs by representing FIC values indicates a synergistic relation between rabeprazole and those IV-related compounds. The closest point to the axes intersection relates to the most effective combination of compounds to inhibit H. pylori growth, which is recorded as FICI. Figure 6. Synergism between rabeprazole and compounds IV-b (A) and rac-IV-l (B) against H. pylori strain ATCC 700392. The concave curve obtained in both graphs by representing FIC values indicates a synergistic relation between rabeprazole and those IV-related compounds. The closest point to the axes intersection relates to the most effective combination of compounds to inhibit H. 3.4. Synergistic Interaction between Some IV-Related Compounds and Rabeprazole pylori growth, which is recorded as FICI. 4. Discussion diphtheriae, they could be used as co-adjuvants, or as an alternative treatment for the corresponding infections. This could be particularly useful for C. jejuni, S. pneumoniae and S. aureus, which were also included in the WHO priority list of antibiotic-resistant bacteria that represent a great threat to human health [7]. The additional compounds (IV-a, IV-b, IV-c, IV-d and rac-IV-k) are ineffective against Gram-positive and Gram-negative alike, except H. pylori, for which they Int. J. Mol. Sci. 2021, 22, 10137 20 of 25 20 of 25 all show a potent inhibitory activity (rac-IV-l, perhaps related to its much high hydrophilic- ity, was ineffective against all bacterial species tested). Within the Helicobacter genus, we have determined that none of them inhibits the growth of enterohepatic species (Table 1), but their activity against other gastric species remains to be tested. These compounds (IV-a, IV-b, IV-c, IV-d, rac-IV-k) are thus narrow-spectrum antibiotics effective against H. pylori and perhaps against other gastric species of the Helicobacter genus, but not even against the enterohepatic Helicobacter species tested or C. jejuni. Importantly, these compounds are not effective against bacteria that may be part of the human microbiota, such as E. faecalis, S. aureus, S. pneumoniae and E. coli [10] at concentrations as high as 64 µg/mL. The narrow antimicrobial spectrum of these compounds may limit the side effects on microbiota of anti-H. pylori therapies based on or including them. py p g The mechanism of action of these antimicrobials is not fully understood. They were initially discovered as compounds that bind to and inhibit the in vitro activity of puriﬁed Hp-ﬂavodoxin. All related inhibitors so far tested (this work and [16–18]) have been shown to bind to the puriﬁed protein. In this respect, most ﬂavodoxins embed the FMN redox cofactor by sandwiching its isoalloxazine redox-active ring between two bulky residues (typically a tryptophan at the 50′ loop and a tyrosine at the 90′ loop). In contrast, at the equivalent position of the 50′ loop, Hp-Fld contains an alanine residue (A55), whose small size contributes to creating a distinct pocket at the si face of the cofactor. The ﬂavodoxin inhibitors have been speculated to bind at that pocket [16]. As shown in Figure 5, this peculiar sequence feature is shared with the ﬂavodoxins of gastric Helicobacter species, but is absent in those of enterohepatic species of this genus and in C. jejuni. 4. Discussion Given the present lack of precise structural information about the inhibitors binding site, we have resourced to protein engineering to try to identify surface residues that may be at the binding site or nearby. Thus, we have mutated seven residues of the ﬂavodoxin surface which are located either at a putative binding site identiﬁed through docking analysis (V113, Q115, T116 and K133A) or at the FMN binding site (A55 which is the residue that creates the pocket, and N14A and D142Y which interact with the phospho-ribityl moiety of the FMN cofactor). ITC analysis (Figures 4 and S4, and Table 2) of the afﬁnity of wild-type and mutant ﬂavodoxins for two representative inhibitors (IV and IV-c) suggests that the residues located at the putative binding site do not interact with the inhibitors or lay close to the interacting site, as their replacement does not perturb the binding. The same appears to be the case of residues 14 and 142, in contact with the phosphate and ribityl moieties of the FMN cofactor. In contrast, the replacement of A55 by tryptophan stands out as the only replacement that clearly lowers the inhibitors’ afﬁnity. The ITC analysis thus lends no support to the putative binding site suggested by the docking analysis, but is consistent with the A55 pocket being the likely binding site for the inhibitors, as previously proposed [16]. This observation will require further conﬁrmation, as the A55W mutant weakens but does not abrogate the binding of the inhibitors. Binding to the Hp-Fld speciﬁc pocket near the cofactor remains a likely structural explanation for the antimicrobial activity of the ﬂavodoxin inhibitors and explains why compounds IV-a, IV-b, IV-c, IV-d, rac-IV-k are antimicrobials for H. pylori, but not for the three enterohepatic Helicobacter species tested, which lack the pocket. However, it does not explain why two inhibitors, IV and rac-IV-j, are effective not only for H. pylori but also for two enterohepatic Helicobacter species (H. muridarum and H. bilis), for C. jejuni and, even more remarkably, for most Gram-positive bacteria tested. These two compounds, IV and rac-IV-j, unlike the other analogues, are the only nitro-derivatives of the tested series and it is possible that the nitro functionality allows them to act by an additional mechanism, which could explain their extended antimicrobial spectrum. 4. Discussion As compounds IV and rac-IV-j are more cytotoxic towards HeLa cells [18] than the other compounds of this family, they might be targeting additional proteins or, once in the bacterial cells, they could be transformed into cytotoxic compounds, as has been described for Mnz, which also contains a nitro group. The greater permeability of monoderm Gram-positive bacteria compared to diderm Gram-negative ones might contribute to compounds IV and rac-IV-j Int. J. Mol. Sci. 2021, 22, 10137 21 of 25 21 of 25 reaching higher concentrations in the cytoplasm of these Gram-positive bacteria, where they could exert their alternative antimicrobial activity, unlike in Gram-negative bacteria. Whatever the case, it seems that replacing the nitro group in IV and rac-IV-j by its reduced amine version greatly increases the selectivity of this family of compounds, and turns them from extended-spectrum to narrow-spectrum antimicrobials highly speciﬁc to H. pylori and perhaps for other gastric Helicobacter species. reaching higher concentrations in the cytoplasm of these Gram-positive bacteria, where they could exert their alternative antimicrobial activity, unlike in Gram-negative bacteria. Whatever the case, it seems that replacing the nitro group in IV and rac-IV-j by its reduced amine version greatly increases the selectivity of this family of compounds, and turns them from extended-spectrum to narrow-spectrum antimicrobials highly speciﬁc to H. pylori and perhaps for other gastric Helicobacter species. No spontaneous mutants were obtained when H. pylori cultures were incubated in the presence of high concentrations of some IV-related compounds under the same conditions, which allowed us to generate Mnz resistant mutants. As determined by Wang et al., Mnz showed an in vitro mutation rate of 6.9 × 10−10 per cell per division, which was less than those of Cla, ciproﬂoxacin, and rifampicin [44]. Given that all these antimicrobials are included in H. pylori eradication therapies [45–50], replacing some of them with IV-related compounds could help to reduce the development of drug resistance in this bacterium. On the other hand, the more severe antibiotic resistance associated with Gram-negative bacteria compared to Gram-positive ones appears to be related to several drug resistance mechanisms, including the overexpression of efﬂux pumps in the former [51–53]. Indeed, H. pylori multidrug-resistance is associated with the activation of efﬂux pumps [54,55]. Thus, compounds inhibiting efﬂux pumps are expected to inﬂuence internal concentrations of substances such as antibiotics, and combined use of EIs with antimicrobials might increase bacterial susceptibility [51,55–57]. 4. Discussion CCCP, reserpine or valinomycin inhibit ABC, DMT, MATE, MFS and/or RND [51,55,58–65], which are the main conserved families of bacterial efﬂux pumps [51,65–67]. As the antimicrobial activity of compound IV derivatives is not greatly affected by those EIs, these IV-related antimicrobials do not seem to be substrates of the most common types of bacterial efﬂux pumps, which adds to their potential to bypass intrinsic resistance mechanisms of H. pylori. py The use of antimicrobial monotherapies might boost the selection of drug-resistant strains, so combinations of drugs could be useful to reduce the selection of antibiotic resis- tance [37,38,68]. In this regard, the interaction between IV-related compounds and other pharmacological entities in use against H. pylori has been evaluated by the checkerboard assay [69]. Most of the derivatives tested did not exhibit synergy when combined with lead compound IV, Mnz, Cla, omeprazole or rabeprazole. However, compounds IV-b and rac-IV-l showed synergy with rabeprazole. While both omeprazole and rabeprazol inhibit urease activity in vitro, rabeprazol does it at a lower concentration [70,71], which agrees with our determined MICs for either PPI. Rabeprazole effect on gastric acid secre- tion seems to be more potent and fast than that of omeprazole [71] and it appears to be less affected by CYP2C19 polymorphisms [45]. These facts, together with the synergy ob- served, makes rabeprazole the PPI of choice in a treatment based on IV-related compounds. Since two compounds that show a synergistic relationship are not expected to act on the same target or, at least, on the same region of the target, the human gastric proton pump (H+/K+-ATPase) and H. pylori urease might be discarded as targets of compounds IV-b and rac-IV-l [71]. Importantly, even if synergism was not reached, compounds IV and IV-b decreased 8- and 16-fold, respectively, the MICs of omeprazole and Mnz against H. pylori, which could enhance their activities in a combinatory therapy, and supports the potential of compounds IV and IV-b as adjuvants in current combinatory therapies containing Mnz or omeprazole. 5. Conclusions Acknowledgments: We acknowledge Pilar Mañas from the University of Zaragoza (Spain) and Mirko Rossi from the University of Helsinki (Finland) for donating some C. jejuni and H. bizzozeronii strains, respectively. We would like to thank Manuel Hein and Dörte Grella (Research Center Borstel) for their excellent technical assistance. Acknowledgments: We acknowledge Pilar Mañas from the University of Zaragoza (Spain) and Mirko Rossi from the University of Helsinki (Finland) for donating some C. jejuni and H. bizzozeronii strains, respectively. We would like to thank Manuel Hein and Dörte Grella (Research Center Borstel) for their excellent technical assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. nﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. De Brito, B.B.; Da Silva, F.A.F.; Soares, A.S.; Pereira, V.A.; Santos, M.L.C.; Sampaio, M.M.; Neves, P.H.M.; De Melo, F.F. Pathogenesis and clinical management of Helicobacter pylori gastric infection. World J. Gastroenterol. 2019, 25, 5578–5589. [CrossRef] [PubMed] 2. Percival, S.; Williams, D.W. Chapter 7–Helicobacter pylori. In Microbiology of Waterborne Diseases; Academic Press: New York, NY, USA, 2014; pp. 119–154. 1. De Brito, B.B.; Da Silva, F.A.F.; Soares, A.S.; Pereira, V.A.; Santos, M.L.C.; Sampaio, M.M.; Neves, P.H.M.; De Melo, F.F. Pathogenesis and clinical management of Helicobacter pylori gastric infection. World J. 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The extended spectrum antimicrobials may ﬁnd use in novel therapies against Gram-positive bacteria, while the narrow-spectrum ones may Int. J. Mol. Sci. 2021, 22, 10137 22 of 25 be useful against H. pylori. Interestingly, these narrow spectrum antimicrobials are not substrates of common efﬂux pumps, appear to have a lower resistance rate than Mnz, show synergy with rabeprazole and may contribute to lowering the development of drug resistance in H. pylori. Besides, they might help to reduce the damage to the microbiota if included in eradication therapies, replacing some of the currently used antimicrobials. be useful against H. pylori. Interestingly, these narrow spectrum antimicrobials are not substrates of common efﬂux pumps, appear to have a lower resistance rate than Mnz, show synergy with rabeprazole and may contribute to lowering the development of drug resistance in H. pylori. Besides, they might help to reduce the damage to the microbiota if included in eradication therapies, replacing some of the currently used antimicrobials. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms221810137/s1. pplementary Materials: The following are available online at https://www.mdpi.com/article/10 / / Author Contributions: Conceptualization, J.S.; methodology, S.S., J.J.G.-F., A.M., R.M., M.C.-G., E.A.- C., H.B., A.V.-C., E.T., U.M., U.E.S., J.A.G., M.D.D.-d.-V., F.H., J.A.A., J.S.; formal analysis, S.S., A.M., R.M., H.B., A.V.-C., U.M., J.A.A., J.S.; resources, E.T., U.M., U.E.S., J.A.G., M.D.D.-d.-V., F.H., J.A.A., J.S.; writing—original draft preparation, S.S., J.S.; writing—review and editing, S.S., J.J.G.-F., A.M., R.M., M.C.-G., H.B., A.V.-C., E.T., U.M., U.E.S., J.A.G., M.D.D.-d.-V., F.H., J.A.A., J.S.; supervision, J.A.A., J.S.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript. Funding: We acknowledge ﬁnancial support from JPIAMR (FLAV4AMR grant); INTERREG POCTEFA aCCeSS, EU (grant Infecmol); MINECO, Spain, (PID2019-107293GB-I00 grant); Gobierno de Aragón, Spain (E45_20R and LMP30_18 grants); and Maria Sklodowska-Curie grant agreement No 801586. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data to this study can be shared upon reasonable request from the corresponding author. References 2020, 21, 1881. [CrossRef] [PubMed] 11. Cremades, N.; Bueno, M.; Toja, M.; Sancho, J. Towards a new therapeutic target: Helicobacter pylori ﬂavodoxin. Biophys. Chem. 2005, 115, 267–276. [CrossRef] [PubMed] 12. Freigang, J.; Diederichs, K.; Schäfer, K.P.; Welte, W.; Paul, R. 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