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1. Introduction {#sec1-sensors-20-00472} =============== The relationship between water contents in the plant and yield has been well established \[[@B1-sensors-20-00472],[@B2-sensors-20-00472]\]. The use of standardized parameters based on physiological thresholds that lead to photosynthetic impairment if surpassed have been successfully used \[[@B3-sensors-20-00472],[@B4-sensors-20-00472],[@B5-sensors-20-00472],[@B6-sensors-20-00472]\]. Nevertheless, assessing the water status of a few leaves seldom represents the water status of the field, and thus irrigation decisions would benefit from noninvasive and nondestructive methods covering larger ranges \[[@B7-sensors-20-00472],[@B8-sensors-20-00472]\]. The use of remote sensing for assessing plant water status on different scales, providing near real-time assessments, might become an adequate option. Notwithstanding, the approach has to be tested under field conditions with crops sensitive to water deficiency. Potato (Solanum tuberosum L.)---considered to be the third most prevalent edible crop in production after wheat and rice \[[@B9-sensors-20-00472]\]---was used as an example. Its sensitivity to water scarcity (highly associated with a shallow root system) \[[@B10-sensors-20-00472]\] and its cultivation in drought-prone zones caused by climate change \[[@B11-sensors-20-00472]\] are the drivers spurring research to find ways to maximize potato yields while reducing water resources. The use of physiological thresholds has contributed to this goal; thus, Ramírez et al. \[[@B6-sensors-20-00472]\] recommend keeping leaves at maximum stomatal conductance (associated with leaf pore openness and gas exchange) at saturated light \>0.15 mol H~2~O m^−2^ s^−1^ to guarantee appropriate tuber yield whilst saving water. However, the use of this kind of physiological traits for water status inspection requires expensive, exhaustive, invasive, yet microscale (leaf) assessments. Thus, infrared thermography is seen as a promising technology that can detect variations in the canopy temperature ($T_{canopy}$), and ultimately, estimate the crop water stress index (CWSI), an indicator highly correlated with stomatal conductance \[[@B6-sensors-20-00472],[@B8-sensors-20-00472],[@B12-sensors-20-00472]\] (see [Section 2.4](#sec2dot4-sensors-20-00472){ref-type="sec"} for further details). On the basis of the black body radiation theory, an infrared thermography (IRT) camera can detect the spectral radiance emitted by an object due to its temperature \[[@B13-sensors-20-00472],[@B14-sensors-20-00472],[@B15-sensors-20-00472]\]. Such a radiance, which ranges from 7 to 14 $\mathsf{\mu}$m, is converted into electrical signals and then displayed as a 2D array or IRT image. One of the shortcoming of current IRT image sensors is their typical resolution of 320 × 240 pixels, which precludes appropriate identification of the spatial structure in the captured scene. Unlike its CCD -based visible RGB counterpart, IRT sensors are based on a microbolometer, a sophisticated array of small thermal sensors with a complicated manufacturing process \[[@B16-sensors-20-00472]\]. To overcome this problem, companies recently started to offer IRT camera systems that include a fixed visual (RGB) camera to help identify regions of interest through additional image processing as well as to avoid convoluted image registration methods \[[@B17-sensors-20-00472],[@B18-sensors-20-00472]\]. Numerous agricultural studies have reported the use of infrared thermography along with particular acquisition methodologies to analyze different plant parameters and conditions. For example, Pitarma et al. \[[@B19-sensors-20-00472]\] evaluated tree health through the use of IRT images. In this study, identification of healthy and unhealthy tissues in the tree's surface was performed through an analysis of infrared thermographic data. Similarly, in \[[@B20-sensors-20-00472]\], a review of pest detection applications based on infrared thermography was reported. This comprehensive study includes the detection and identification of the main pest in plant crops such as maize, rice, and soybean. With regard to water-stress estimation in plants, Möller et al. \[[@B21-sensors-20-00472]\] conducted an investigation on the water status of grapevines using thermal and visible images acquired from a platform located 15 m above the canopy (nadir-pointing view). This study aimed to analyze the correlation between the calculated CWSI and the measured leaf stomatal conductance, resulting in a correlation of $R^{2} = 0.91$. Image processing steps in the estimation of $T_{canopy}$ include image registration (i.e., alignment of multisensor images), which requires the placement of aluminum crosses over the field, and the selection of canopy pixels in the thermal image based on the transformation of the RGB image to hue-saturation-intensity color space followed by a manual thresholding procedure. A similar study was conducted in \[[@B22-sensors-20-00472]\] for cotton crops where leaf water potentials showed a linear relationship with CWSI values ($R^{2} = 0.816$). Here, a nadir-pointing view IRT camera was located 5 m above the crops. Since there was no additional sensor to detect where the canopy was located in the scene, canopy pixels detection was based solely on estimated temperature thresholds, thus avoiding the use of an additional sensor. In the context of potato crops, Prashar et al. \[[@B23-sensors-20-00472]\] implemented a high-throughput field phenotyping methodology to estimate CWSI using IRT images. Image acquisition was performed from a fork-lift at a height of about 8 m, with an angular inclination to cover up to 27--36 plots in the scene. Then, manual selection of the central regions in the plots was performed to estimate the CWSI. Similarly to the previous study, the inclusion of canopy pixels in the analysis was based on temperature thresholding. Consequently, several factors can influence the accurate estimation of $T_{canopy}$ and the subsequent calculation of the CWSI. First, the use of an independent, nonfixed sensor imposes an image registration problem where convoluted image processing algorithms could be required. The use of control points in the scene could alleviate the computational load while increasing the overall acquisition time. Second, the optical setup, involving the camera viewing angle ($\theta$) and the distance to the object ($d_{object}$), defines the canopy scene to be analyzed, and an appropriate combination of such parameters is needed to reduce the number of nonleaf pixels. Third, since the pixel resolution of thermal cameras is notably low, a single pixel can detect both soil and leaf thermal radiation such that thresholding based solely on temperature can yield a high level of uncertainty in the estimation of $T_{canopy}$. Finally, the studies mentioned above do not provide a detailed explanation regarding the various image processing procedures involved in the use of thermal images; this is possibly due to the dependence on commercial software. This research has the following specific objectives: (1) To provide a thorough description of infrared thermography for the estimation of canopy temperature, and subsequently the CWSI, in the context of potato production; (2) to generate an open-source software to perform such tasks that can be freely used by the agricultural remote sensing community; (3) to demonstrate that thermographic sensors along with the described acquisition methodology are appropriate for defining irrigation thresholds, which can reduce water consumption. 2. Materials and Methods {#sec2-sensors-20-00472} ======================== 2.1. Plant Material and Study Area {#sec2dot1-sensors-20-00472} ---------------------------------- The potato cultivar used was UNICA (CIP code: 392797.22), an early variety adapted to warm and dry environments and slightly tolerant to salinity \[[@B24-sensors-20-00472]\]. Two field trials were performed at the International Potato Center (CIP) and the National Agrarian University---La Molina (UNALM) experimental stations in Lima, Peru (12.08° S, 76.95° W, 244 m.a.s.l.) during October 2017--January 2018 (first experiment---CIP, E1) and June--September, 2018 (second experiment---UNALM, E2). The study site is characterized by a semi-warm and humid climate \[[@B25-sensors-20-00472]\]. During the growing season, the minimum temperature ($T_{min}$), maximum temperature ($T_{max}$), relative humidity ($RH$), solar radiation ($R_{s}$), and maximum vapor pressure deficit ($VPD_{max}$, estimated using the equation used by Ramírez et al. \[[@B26-sensors-20-00472]\]) were 16.4 ± 0.19 °C, 23.2 ± 0.23 °C, 86.5 ± 0.45%, 16.0 ± 0.43 MJ m^−2^ day^−1^, and 1.06 ± 0.03 kPa, respectively (in E1); and 14.2 ± 0.05 °C, 18.7 ± 0.19 °C, 89.7 ± 0.04%, 7.5 ± 0.46 MJ m^−2^ day^−1^, and 0.64 ± 0.03 kPa, respectively (in E2). Specific values for environmental conditions per experimental period are provided in [Table 1](#sensors-20-00472-t	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== Target condition being diagnosed {#Sec2} -------------------------------- Despite successful vaccination programmes, meningococcal disease (MD) remains a leading infectious cause of septicaemia and death in children worldwide \[[@CR1]--[@CR5]\]. Early diagnosis of MD significantly improves outcomes with reduced morbidity and mortality \[[@CR4], [@CR5]\]. The challenge is, however, that during the prodrome invasive MD is indistinguishable from many self-limiting viral infections \[[@CR4]--[@CR6]\]. This invariably leads to a very cautious approach to the management of these children with many receiving parenteral antibiotics pending culture results \[[@CR7]\]. Despite this cautious approach, children are still being diagnosed late due to the difficulties in identifying those children who are infected with MD from those who have a simple viral illness \[[@CR4], [@CR7]\]. Over the years, a number of studies have explored the value of widely available biomarkers including the use of CRP, Procalcitonin and white cell counts in the initial diagnosis of possible MD \[[@CR8]--[@CR12]\]. Whilst these tests have value, none of them have the necessary diagnostic accuracy to allow them to be used as rule out tests at presentation \[[@CR8]--[@CR12]\]. Loop-mediated-isothermal AMPlification (LAMP) for MD is a rapid form of PCR that targets the ctrA gene sequence. The ctrA gene sequence is genetically conserved across all pathogenic (capsular) stains of the bacterium Neisseria meningitidis that is responsible for MD \[[@CR13]\]. This technique is faster than traditional PCR techniques and requires much simpler equipment \[[@CR13]--[@CR17]\]. It is possible that LAMP technology could be used as a rapid point of care test (POCT) for the early diagnosis of MD in children. This could be achieved through the rapid testing of blood samples or throat swab specimens in emergency departments, primary care facilities or pharmacies. Clinical pathway {#Sec3} ---------------- It is very difficult to diagnose early meningococcal disease with current guidance recommending decision-making based on the clinical presentation and laboratory results \[[@CR7], [@CR12]\]. Unfortunately, no single biomarker, combination of biomarkers or clinical guideline has been found to be ideal \[[@CR8]--[@CR12], [@CR18]\]. This has resulted in a very cautious approach resulting in the overtreatment of many children \[[@CR8], [@CR11], [@CR12], [@CR14]\]. Despite such a cautious approach, children are still being diagnosed late \[[@CR12]\]. LAMP could potentially be used at two points within existing care pathways. Firstly at presentation to identify early invasive meningococcal disease in children who present with a minor illness \[[@CR14]\]. Alternatively, LAMP could be used in place of the current gold standard (quantitative PCR or sterile site culture) to quickly confirm or exclude the diagnosis allowing for a more tailored treatment including early ambulation. The development of LAMP technology for the diagnosis of early MD could therefore represent a significant breakthrough that could alter the care of thousands of children every year worldwide. Why perform this review? {#Sec4} ------------------------ This systematic review is required because there are a growing number of individual studies that have reported on the diagnostic accuracy of LAMP technology in diagnosing MD \[[@CR13], [@CR14], [@CR19]\]. These studies have used a similar approach; LAMP directed at the conserved CtrA region of the bacteria Neisseria meningitidis, but in different populations using different specimens, i.e. blood, CSF and throat swabs \[[@CR13], [@CR14], [@CR19]\]. There are, to our knowledge, no existing systematic reviews. This review may help researchers and policymakers identify the most suitable sample (blood, CSF, throat swab), and it may help to determine the role of LAMP within the existing diagnostic pathway. Objectives {#Sec5} ---------- The objective of this systematic review is to determine the diagnostic accuracy of LAMP technology in the diagnosis of invasive meningococcal disease in children (\< 18 years of age). Methods/design {#Sec6} ============== We will perform a literature search for relevant studies and then screen and select studies for inclusion against eligibility criteria. Data extraction will be performed in duplicate on the selected studies with meta-analysis and report writing. We will adhere to standards of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) in reporting the findings of this review \[[@CR20]\]. The content of this protocol follows the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) recommendations \[[@CR21]\]. (Please see the Additional file [1](#MOESM1){ref-type="media"} for the completed PRISMA-P checklist.) This review is registered with the International Prospective Register of Systematic Reviews (PROSPERO) \[[@CR22]\]. The registration number is \[CRD42017078026\]. Inclusion criteria {#Sec7} ------------------ Table [1](#Tab1){ref-type="table"} below outlines the inclusion criteria for this review. These criteria are discussed in more detail below.Table 1Eligibility criteriaStudy characteristicsInclusion criteriaPopulationChildren \< 18 years of age with suspected meningococcal diseaseIndex testsLoop-mediated-isothermal AMPlification for Neisseria meningitidisReference testQuantitative PCR and/or culture of sterile site (blood and/or CSF) specimensOutcomesTrue and false positives, true and false negativesStudy designsAll prospective, retrospective and randomised control studies that report measures of diagnostic accuracy of Loop-mediated-isothermal AMPlification for Neisseria meningitidis ### Types of studies {#Sec8} All prospective, retrospective and RCT studies that assess the performance of LAMP in assessing children (\< 18 years of age) with potential invasive meningococcal disease will be included. There are no language restrictions. ### Participants {#Sec9} The participants were children (\< 18 years of age) with signs or symptoms of invasive meningococcal disease. ### Index tests {#Sec10} The index test being investigated is the LAMP test for meningococcal DNA. For the purpose of this protocol, this is further defined as LAMP testing specific to the ctrA gene of Neisseria meningitidis. Index testing can be performed using blood, cerebrospinal fluid and throat swabs. Commercially and non-commercially available tests will be considered. ### Target conditions {#Sec11} Meningococcal infection (invasive meningococcal disease) is the target condition. ### Reference standards {#Sec12} The reference standard used to confirm the presence of the target condition in this study is quantitative PCR to detect Neissseria meningitidis DNA in a sterile site sample (normally blood or CSF). A positive blood or cerebrospinal bacterial culture of Neissseria meningitidis will also be used. No other reference standard will be accepted. Exclusion criteria {#Sec13} ------------------ Studies that only assess carriage rates in healthy children will be excluded. Search methods for identification of studies {#Sec14} -------------------------------------------- ### Electronic search strategy {#Sec15} An electronic search strategy has been developed in collaboration with the Queen's University Belfast Medical Librarian (RF). To identify all prospective, retrospective and RCTs, we will search MEDLINE, Embase, Web of Science, Scopus and The Cochrane Library inclusive of Cochrane Controlled Trials Register. An example Medline search strategy is attached as Additional file [2](#MOESM2){ref-type="media"}. There are no language restrictions for this review. ### Searching other resources {#Sec16} In addition, we will hand-search reference lists of relevant articles. A targeted grey literature search will include contacting the manufacturers of commercially available LAMP test for meningococcal disease and a search of conference abstracts. Data collection {#Sec17} --------------- ### Selection of studies {#Sec18} Two reviewers (TW, MDS) will independently screen the study eligibility and extract data. Screening will be a two-step process with initial title/abstract screening followed by full-text screening. Disagreements among reviewers will be resolved through consensus or third-party reviewer (DF). Reports that are duplicates or co-publications of studies will be identified. Following full-text screening, a list of excluded studies with reasons for exclusion will be provided in an appendix of the final report. We will begin with screening published and unpublished records and select those that meet the inclusion/exclusion criteria. Our search of literature will involve both primary studies and systematic reviews. The latter will be used only to identify additional primary studies. ### Data extraction and management {#Sec19} TW and MDS will develop a data extraction form, and this will be piloted initially to achieve a good level of agreement between the data extractors. The following data will be extracted in duplicate by TW and MDS:Study characteristics: author, year of publication, country, design, sample size, clinical setting, number studied, number of drop-outs with reason, and funding source.Population characteristics: inclusion/exclusion criteria and patient demographics such as age and gender.LAMP testing: timing of sampling, method of sampling (e.g. throat swab, blood or CSF), time to result and commercial availability of the test.Gold standard: Quantitative PCR (e.g.TaqMan® PCR) or sterile site bacterial culture (i.e. blood/CSF)Outcomes: From this 2 × 2 table, we will calculate true positives, false positives, true negatives, and false negatives. ###	{ "pile_set_name": "PubMed Central" }
Introduction ============ Haptoglobin (Hp) is a plasma protein that binds extracorpuscular hemoglobin (Hb) and prevents it from inflicting iron‐mediated oxidative tissue damage.^[@b1]^ The Hp gene has 2 major alleles, Hp1 and Hp2. Of these, the latter is a mutated version of the former and unique to humans.^[@b2]^ This common polymorphism (rs72294371) leads to 3 structurally and functionally distinct proteins, Hp 1‐1, 2‐1, and 2‐2, with reported genotype frequencies in subjects of Western European descent of 16%, 48%, and 36%.^[@b2]--[@b3]^ While Hp 1‐1 forms dimers, Hp 2‐1 and Hp 2‐2 form larger linear and cyclic polymers, respectively.^[@b2]^ Hp 2‐2 was reported to be less efficient than Hp 1‐1 in binding Hb and preventing oxidation by stabilizing heme iron within Hb, thereby displaying lower antioxidant activity.^[@b4]--[@b5]^ Based on these biological mechanisms, it has been suggested that the Hp 2‐2 genotype may confer an elevated risk of developing cardiovascular diseases (CVD). Epidemiological studies conducted in general populations, however, have shown contradictory results. On one hand, Hp 2‐2 was linked to the severity of myocardial infarction^[@b6]^ and to more frequent (but less severe) peripheral arterial occlusive disease.^[@b7]^ On the other hand, a large longitudinal population‐based survey has reported a ≈2‐fold risk for coronary artery disease (CAD) death in people with the Hp 1‐1 genotype.^[@b8]^ Two smaller case‐control studies found that the Hp 1 allele was more frequent in patients with CAD^[@b9]^ and lacunar stroke,^[@b10]^ compared with healthy controls. In the past years the focus has shifted towards the role of Hp genotype in CVD of diabetic patients. Because diabetes is featured by a high level of oxidative stress, relevance of the antioxidant Hp may be considerably higher in diabetic patients.^[@b11]^ Actually, in a meta‐analysis of 5 studies involving 1829 patients with diabetes, the pooled odds ratio (95% CI) for CVD was 2.03 (1.46, 2.81) in a comparison of the Hp 2‐2 genotype with other genotypes.^[@b12]^ In line with this finding, Hp 2‐2 was associated with incident CVD in type 2 diabetes mellitus patients (odds ratio \[95% CI\], 4.96 \[1.85, 13.33\]). In conflict with these findings, the Framingham Offspring Study reported that Hp 2‐2 was associated with significantly lower CAD prevalence among diabetic patients than Hp 1‐1 (odds ratio \[95% CI\], 0.43 \[0.21, 0.90\]).^[@b13]^ As a potential explanation for the substantial heterogeneity in the Hp ‐ CVD association between the various studies effect modification by levels of HbA~1c~ has been proposed.^[@b14]^ Indeed, the reported dysfunction of Hp 2‐2 in preventing Hb‐mediated oxidative damage was found to be accentuated with glycosylated Hb (HbA~1c~) in cell culture experiments.^[@b4]^ Testing this hypothesis in humans, Cahill and colleagues^[@b14]^ reported the Hp 2‐2 genotype to be associated with elevated CAD risk among subjects with elevated HbA~1c~ in the Nurses\' Health Study (odds ratio \[95% CI\], 10.12 \[1.08, 94.97\]) and validated their finding in a cohort of type 2 diabetic subjects (hazard ratio \[95% CI\], 7.55 \[2.79, 20.47\]). Based on this and earlier studies, Hp genotyping among diabetic patients and antioxidant treatment in diabetic patients with the Hp 2‐2 genotype has been propagated ^[@b2],[@b12]^ but further confirmatory data are required to change clinical routine. Given its potentially large impact on diabetes management, we investigated the association of Hp genotype with CVD conditional on elevated HbA~1c~ in the prospective, population‐based Bruneck Study. Methods ======= Study Population and Data Collection ------------------------------------ The Bruneck Study is a prospective, population‐based survey on the epidemiology and pathogenesis of atherosclerosis and cardiovascular disease.^[@b15]--[@b18]^ At baseline in 1990 the study population comprised an age‐ and sex‐stratified random sample of all inhabitants of Bruneck (125 men and 125 women from each of the fifth through eighth decades of age, for an age‐ and sex‐stratified random sample of n=1000, all of western European descent). No subjects were enrolled after study initiation. In 1995, 826 subjects participated in the first quinquennial re‐examination and DNA samples for genotyping were available in 816 individuals. During follow‐up from 1995 to 2010, detailed information about fatal and nonfatal new‐onset CVD was carefully collected. Follow‐up was 100% complete for clinical endpoints, which was made possible by the extremely low population mobility of 0.2% in the Bruneck area. The study protocol was approved by the ethics committees of Bolzano and Verona and conformed to the Declaration of Helsinki. All study subjects provided written informed consent. Risk factors were assessed by means of validated standard procedures as described previously.^[@b15],[@b19]^ At the baseline and follow‐up examinations in 1995, 2000, 2005, and 2010, venous blood was sampled in the morning after an overnight fast for laboratory measurements, including fasting plasma glucose and HbA~1c~ (Diabetes Control and Complications Trial‐aligned assay; equipment and reagents from BioRad, Milan, Italy, at both baseline and follow‐up examinations). In accordance with the study of Cahill et al, HbA~1c~ was dichotomized according to a cut‐off of 6.5% (48 mmol/mol, International Federation of Clinical Chemistry units). Diabetes mellitus was coded present for subjects with fasting glucose levels ≥7 mmol/L (≥126 mg/dL) or a medical record confirmed prediagnosis of definite disease status. Based on the literature^[@b14]^ we anticipated few subjects with the Hp 1‐1 genotype and, to maximize statistical power, decided prior to analysis to pool the Hp 1‐1 and 2‐1 genotypes, forming a group of Hp1 allele carriers, which is a common approach.^[@b14]^ We ascertained leisure time‐related physical activity by a standardized questionnaire^[@b20]^ rating the intensity of activities according to the compendium of physical activities^[@b21]^ and calculated average metabolic equivalent hours per week to estimate long‐term physical activity. We assessed food intake by a standardized food‐frequency questionnaire (FFQ) based on the gold standard Harvard FFQ by Willett and colleagues and adapted the FFQ to the dietary peculiarities in the survey area. We validated our FFQ with a dietician‐supervised short‐term assessment of food intake. Validity was high and similar to that previously found for the same FFQ in other populations.^[@b22]^ The Alternative Healthy Eating Index (AHEI) was calculated from these data as a quantitative measure of healthy dietary behavior.^[@b23]^ Body mass index (BMI) was calculated as weight in kilograms over height in meters squared. Haptoglobin Genotyping ---------------------- The Haptoglobin genotype was determined by PCR as described previously^[@b24]^ with slight modifications. In brief, 20 μL reactions contained 20 ng genomic DNA, 2 units Qiagen Taq Polymerase (Qiagen), 1× Qiagen PCR buffer (Qiagen, Hilden, Germany), 1× Q solution (Qiagen), 200 μmol/L of each dNTP (Peqlab) and 0.25 μmol/L of each primer (A/B or C/D). Oligonucleotide primers A and B were used for amplification of a 1757‐bp Hp 1 allele‐specific sequence and a 3481‐bp Hp 2 allele‐specific sequence. Primers C and D were used to amplify a 349‐bp Hp 2 allele‐specific sequence. The primers A (5′‐GAGGGGAGCTTGCCTTTCCATTG‐3′), B (5′‐GAGATTTTTGAGCCCTGGCTGGT‐3′), C (5′‐CCTGCCTCGTATTAACTGCACCAT‐3′) and D (5′‐CCGAGTGCTCCACATAGCCATGT‐3′) were purchased from Microsynth. The amplification reactions were conducted on a DNA Engine Cycler (BioRad) under the following conditions: initial denaturation 3 minutes 94°C; 94°C 30 seconds, 57°C (primers A/B) and 62°C (primers C/D) 30 seconds, 72°C 2 minutes, 35 cycles; final extension 10 minutes 72°C. After amplification 8 μL PCR product A/B and 2 μL PCR product C/D were mixed and separated together on a 1% agarose gel. Endpoints --------- The composite CVD endpoint included incident fatal and non‐fatal myocardial infarction and stroke. Presence of myocardial infarction was assessed by World Health Organization criteria (definite disease status),^[@b25]^ while stroke was classified according to the criteria of the National Survey of Stroke.^[@b26]^ Events were ascertained by careful review of medical records provided by general practitioners, death certificates, and Bruneck Hospital files. A major advantage of the Bruneck Study is that virtually all inhabitants of Bruneck are referred to 1 local hospital that cooperates closely with the general practitioners. This allows retrieval of complete medical information. Subjects who had experienced CVD events before the study baseline in 1995 were included in the main analysis and observed for recurring incident CVD between	{ "pile_set_name": "PubMed Central" }
Cortical oscillations in the beta frequency band (15 to 35 Hz) are thought to play a central role in cortical processing of information related to movement and cognition ([@r1], [@r2]). In Parkinson's disease (PD), cortical beta oscillations become abnormally synchronized through their interaction with the dopamine-depleted basal ganglia network ([@r3], [@r4]). These pathological beta oscillations are suppressed when continuous high-frequency deep brain stimulation (DBS) is applied to either subthalamic nucleus (STN) or the internal segment of the globus pallidus ([@r5][@r6]--[@r7]), suggesting a strong dependence on a distributed subcortical network architecture for their generation and maintenance. A central goal in the field is to define the neuronal interactions through which abnormally strong and sustained beta oscillations emerge following dopamine depletion. Addressing these pathologically exaggerated activities may also give key insights as to how oscillations are transmitted in the healthy brain ([@r1]). The spiking activity of neurons across the basal ganglia locks to specific phases of cortical oscillations in the parkinsonian brain ([@r8][@r9][@r10][@r11]--[@r12]). The high stability of these conditions of synchronization is likely determined by synaptic connectivity between different neuronal populations in the network, be it physiological, pathological, or compensatory. Elucidating the mechanisms of network synchronization is made harder by the large variance in baseline firing rates and presence/absence of autonomous firing in different basal ganglia cell types. Several hypotheses have been raised as to which network connections are necessary and sufficient for cortical oscillations to be abnormally propagated and/or amplified. These theories include 1) the excitatory--inhibitory coupling between the STN and the external segment of the globus pallidus (GPe) ([@r13][@r14]--[@r15]); 2) alteration of striatal output ([@r12]), possibly due to interneuron dysfunction ([@r16][@r17]--[@r18]); and 3) enhanced pallidostriatal positive feedback ([@r19]). These hypotheses have been based around the idea that oscillatory dynamics are relatively stable. However, recent studies have revealed that beta band activity in healthy ([@r20]) and parkinsonian basal ganglia local field potentials (LFPs) and frontal electroencephalograms (EEG) ([@r21], [@r22]) occurs in transient beta bursts (β bursts). It is currently unclear whether cortical and basal ganglia phase locking occurs predominantly during such bursts. If this is the case, the question of how beta oscillations are propagated and amplified can potentially be approached by identifying the neuronal interactions that occur before, during, and after these transient events. Such questions have assumed clinical significance due to the development of closed-loop stimulation approaches to DBS ([@r23]), as they have the potential to reveal the most effective way to disrupt the emergence of pathological oscillations. Here we use multiple neuronal signals and analytical approaches in PD patients and dopamine-depleted rodents to demonstrate that cortical β bursts are associated with highly stable cortical and basal ganglia phase locking. These conditions of synchronization are established significantly earlier than the threshold commonly used to define the onset of β bursts using oscillation amplitude. Moreover, this initial period of synchronization is associated with the cell type-specific phase trajectories of basal ganglia spikes on the cortical beta oscillation. These findings have important implications for identifying the process through which neuronal oscillations propagate through different brain areas and for the design of closed-loop DBS. Results {#s1} ======= The definition of a β burst varies extensively across different studies, in terms of both the threshold used to categorize an epoch of activity as a β burst, as well as the temporal dynamics of the identified burst ([@r20][@r21]--[@r22], [@r24]). In this study, the occurrence of a cortical β burst was inclusively defined as any period lasting \>50 ms when the instantaneous beta amplitude of the Fz--Cz EEG (in the case of PD patients) or frontal electrocorticogram (ECoG, in parkinsonian rats) exceeded the 75th percentile of the amplitude calculated across the entire recording ([@r21], [@r22]). While frontal EEG signals may include contributions from subcortical sources, in this context they have been widely used as a read-out of cortical activity and provide similar information to ECoG ([@r11], [@r25]). PD Patients. {#s2} ------------ ### Corticosubthalamic phase locking follows the time course of EEG β-burst amplitude in PD patients. {#s3} β bursts have been extensively reported in the STN LFPs of PD patients, but it is unclear to what extent they reflect STN unit activity. To address this issue, we utilized the background unit activity (BUA) signal recorded in and around the STN of PD patients undergoing intraoperative functional mapping for subsequent DBS therapy. The BUA signal is a continuous time series (like EEG/ECoG) and provides insight into the synchronous spike discharges of local neuronal ensembles ([@r12]). BUA recorded from the STN of PD patients exhibited enhanced rhythmic activity at beta frequencies ([Fig. 1 A and B](#fig01){ref-type="fig"}). Moreover, subthalamic BUA was strongly coherent with the EEG in the beta frequency band ([Fig. 1C](#fig01){ref-type="fig"}). Beta oscillations and significant coherence with EEG were confined to BUA signals within the physiologically defined boundary of the STN ([Fig. 1 B and C](#fig01){ref-type="fig"}). Having established that synchronous beta oscillations were present, we used these signals to address the question of whether the phase locking of subthalamic neuronal activity to the EEG followed the time course of EEG β bursts. To this end, after filtering EEG and BUA in the beta frequency band (EEG~β~ and STN-BUA~β~) we computed phase locking, as measured by the phase synchrony index (PSI) of the Hilbert transform-derived phase of these signals, around the onset of the EEG β bursts (hemispheres = 13, patients = 7, n = 18 recordings). Significant modulation of the time course of all PSI analyses was assessed using a cluster-based test to compare the burst-aligned data with data that were randomly selected with no relation to burst onset (see [Methods](#s13){ref-type="sec"} for details). PSI in time, which measures the strength of phase locking over short epochs (50 ms) across each individual burst ([SI Appendix, Fig. S1A](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1819975116/-/DCSupplemental)), was elevated around the β-burst onset, and the duration of locking increased with longer burst durations ([Fig. 1E](#fig01){ref-type="fig"}). Importantly, the onset of phase locking (in time) began on average −115, −114, and −35 ms relative to the onset of short (50 to 150 ms), medium (150 to 250 ms), and long (250 to 350 ms) bursts, respectively, suggesting that increased corticosubthalamic synchronization actually precedes the amplitude-based burst threshold. PSI across bursts, which measures the consistency of the phase relationship between bursts at each individual time point ([SI Appendix, Fig. S1B](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1819975116/-/DCSupplemental)), began on average −80, −74, and −79 ms for short, medium, and long bursts relative to the EEG β-burst threshold ([Fig. 1F](#fig01){ref-type="fig"}). The time course of PSI across bursts followed that of the amplitude less closely than that of PSI in time ([Fig. 1F](#fig01){ref-type="fig"}). These findings demonstrate that in PD patients, corticosubthalamic synchronization precedes the onset and follows the time course of EEG β bursts, and the conditions of this transient synchronization are consistent across bursts. ![EEG β bursts are phase locked to spiking activity in the STN of PD patients. (A) BUA signals were recorded from 5 microelectrodes in the STN area together with EEG from the Fz--Cz position. As the microelectrodes traversed the STN, some electrodes were inside and some outside the structure. The layout of the 5 electrodes is shown within the red dotted circle. (B) Average BUA power spectrum from inside (red; n = 256 BUAs) and outside (gray; n = 222 BUAs) the STN, highlighting that enhanced rhythmic activity in the beta band (15 to 35 Hz) is observed inside the STN. (C) The proportion of BUA signals that are coherent with the EEG is selectively increased in the beta frequency range when electrodes were inside but not outside of the STN (number of recordings as in B). (D) An exemplary EEG β burst (black) from subject 1 (right hemisphere), together with simultaneously recorded STN-BUA (red). Onset of a cortical β burst was defined as the time point that the instantaneous beta amplitude exceeded the 75th percentile of the amplitude of the whole recording and remained elevated for at least 50 ms. Dashed line indicates the amplitude threshold for determining the onset of the EEG β burst. The beta amplitude of both the EEG and STN BUA simultaneously crosses the burst threshold and remains above it for around 200 ms (gray box). This increase in amplitude is visible in the raw (black) and filtered (gray) EEG signal. Bursts of activity in the STN BUA (raw, red; beta filtered, pink) are clearly aligned to the trough of the EEG beta oscillation during the burst. (E and	{ "pile_set_name": "PubMed Central" }
1. Introduction {#s0005} =============== Anti-N-methyl-[d]{.smallcaps}-aspartate receptor encephalitis (ANMDARE) is a disorder presenting with subacute onset of seizures, psychosis, memory and language deficits, abnormal movements, and breathing and autonomic disturbances [@bb0005], [@bb0010]. Although initially identified in association with ovarian teratomas, in a majority of children and young adults, ANMDARE occurs without tumors [@bb0010]. Three-fourths of patients with ANMDARE make substantial recovery with early diagnosis, tumor resection and immunotherapy, but relapses may occur in a quarter of them [@bb0005]. In 2012, Schmitt et al. [@bb0015] described an electroencephalographic (EEG) pattern characterized by rhythmic 1--3 Hz delta activity with superimposed bursts of rhythmic 20--30 Hz beta activity overriding on each delta wave, which they named 'extreme delta brush' (EDB). This pattern since then is believed to be diagnostic of ANMDARE, occurs in 30% [@bb0015] to 58% [@bb0020] of patients with ANMDARE, and is associated with abnormal magnetic resonance imaging (MRI), high seizure burden, prolonged hospitalization and poor outcome [@bb0015]. We describe an adolescent girl with non-paraneoplastic ANMDARE, who despite persistence of EDB for nearly 2 years had a speedy and sustained response to immunotherapy. 2. Case report {#s0010} ============== A 16-year-old right-handed girl presented in November 2016 with the history of seizures and episodic confusion of 3 months duration. She had a few seizures at the age of 5 years and was diagnosed with benign rolandic epilepsy, for which she received carbamazepine for over one year. The drug was discontinued and she remained seizure-free from the age of 6 until seizure recurrence at the age of 16. The new onset seizures were characterized by motor restlessness and confusion lasting for nearly 2 min and occurred 2 to 3 times per week, and failed to respond to adequate doses of levetiracetam and clobazam. She was a brilliant student until the recent seizure recurrence, after which there was a dramatic decline in her school performance. On neurological examination, the findings were confined to the higher mental function testing. She had profound difficulties with writing and calculation. She had right--left disorientation and could not identify her fingers. The rest of the neurological and systemic examinations, including the optic fundi, and motor and sensory systems were normal. A diagnosis of Gerstmann\'s syndrome due to a rapidly evolving epileptogenic lesion involving the left angular gyrus region was considered. A 3 Tesla MRI of the brain was normal ([Supplementary Fig. 1](#f0020){ref-type="graphic"}). Her EEG revealed polymorphic left hemispheric slow activity, maximally over the left parietal region with EDB morphology ([Fig. 1](#f0005){ref-type="fig"}). Both the serum and cerebrospinal fluid (CSF) tested strongly positive for NMDA receptor antibodies (by indirect immunofluorescence on transfected cells at our laboratory). There was no evidence of ovarian lesions on ultrasound of the abdomen and pelvis. Brain fluorodeoxyglucose-positron emission tomography (FDG-PET) was normal ([Supplementary Fig. 2](#f0025){ref-type="graphic"}). We treated the patient with injections of methyl prednisolone 1 g/day along with intravenous immune globulin (400 mg/kg/day) for 5 days. She had a seizure recurrence 10 days later characterized by a painful sensation that started in the right upper limb spreading to the right lower limb, along with posturing of the right upper limb with progression to a generalized tonic--clonic seizure. Chronic second-line immunotherapy with injections of rituximab 1 g/m^2^ was started and the same dose was repeated after 2 weeks. There were no further episodes of seizures on oxcarbazepine monotherapy. Her scholastic performance came back to baseline within the next 3 months, and she completed her 12th grade examination with high scores 6 months after the initiation of immunotherapy. Her serial EEGs done at nearly 4--6 month intervals are depicted in [Fig. 2](#f0010){ref-type="fig"}. They revealed persistent focal electrical dysfunction over the left centro-temporo-parietal region with extreme delta brushes over the left fronto-centro-temporal region and infrequent left central spikes. In view of the persisting EDB, we continued biannual injections of rituximab (two doses of 1 g/m^2^, 2 weeks apart). Her serum and CSF anti-NMDA-R antibody remained positive when repeated in November 2018, at 2 years following commencement of immunotherapy. The EEG performed during the same time continued to show EDB ([Fig. 2](#f0010){ref-type="fig"}D). She continued to take oxcarbazepine 300 mg in the morning and 450 mg at night.Fig. 1The first EEG shows 1--2 Hz, polymorphic delta activity over the left hemisphere, with maximum expression over the centro-temporo-parietal region, along with superimposed rhythmic beta activity, which is better expressed over the fronto-centro-parietal region (EDB pattern). (High frequency filter = 70 Hz, low frequency filter = 0.5 Hz, notch filter = off, sensitivity = 10 μV/mm, and display speed = 30 mm/s.)Fig. 1Fig. 2Serial EEGs depict the persistence of the left hemispheric polymorphic slow activity and EDB pattern. The EEG dated May 22, 2018 (C), in addition, shows left central spike discharges. (High frequency filter = 70 Hz, low frequency filter = 0.5 Hz, notch filter = off, sensitivity = 7--10 μV/mm, and display speed = 30 mm/s.)Fig. 2 3. Discussion {#s0015} ============= We have described here a confirmed case of non-paraneoplastic ANMDARE in an adolescent girl, who despite complete resolution of neurological symptoms and signs within 3 months of initiation of immunotherapy, had persistence of EDB and serum antibody positivity for over 2 years. Our patient presented with the four symptoms that are diagnostic of Gerstmann syndrome: finger agnosia, acalculia, left--right disorientation, and agraphia. Although the MRI and FDG-PET turned out to be normal, the EEG disclosed a focal disturbance of electrical function over the dominant hemisphere associated with EDB. Our case illustrates the immense value of scalp EEG, even in this era of advanced neuroimaging, providing not only the localization, but also the first hint to the etiology of the lesion. Although ANMDARE is typically recognized as a multistage disease characterized by prodromal flu-like illness, followed by acute onset of neuropsychiatric manifestations, difficult to control seizures, movement disorders and autonomic instability [@bb0005], [@bb0010], atypical forms of the disease with isolated focal manifestations are being increasingly reported both in children [@bb0025] and adults [@bb0030]. To the best of our knowledge, our patient\'s presentation with Gerstmann syndrome is unique. While brain MRI is unremarkable in 50% of patients with ANMDARE [@bb0005], [@bb0010], EEG is abnormal in most [@bb0020], [@bb0035]. In a recent study of 53 patients (35 adults and 18 children), only 2 (4%) patients had normal EEG [@bb0040]. Focal disturbance of electrical function was encountered in 73% patients. Overall, close to one-third of patients with ANMDARE show a EDB pattern, although its incidence has varied widely in different case series depending upon patient selection, clinical state and timing of EEG [@bb0015], [@bb0020], [@bb0035], [@bb0040]. Generally, with immunotherapy, the EDB pattern gradually becomes less prominent and less frequent, and resolves completely within a few weeks or months [@bb0020], [@bb0045]. In a serial EEG study of 5 children with ANMDARE and the EDB pattern, it resolved within 6 months in 3 children, but persisted in 2 of them [@bb0045]. While an infant of 6 months at onset, in whom the EDB pattern on EEG persisted for 10 months, was left with significant cognitive and motor impairment despite control of seizures with medication, a 14-year-old boy had the EBD pattern persist for 17 months and made a complete recovery [@bb0045]. To the best of our knowledge, our patient had the longest persistence of EDB reported to date. Several studies have linked markedly abnormal initial EEGs, especially the presence of the EDB pattern, to prolonged hospitalization, high seizure frequency, abnormal MRI, poor response to immunotherapy and unfavorable outcome [@bb0015], [@bb0035], [@bb0040]. However, the adverse prognostic implication of this pattern is less apparent in some recent studies [@bb0020], [@bb0050]. Because of its frequent co-occurrence with brief rhythmic discharges, and electrographic and electro-clinical seizures, EBD has been labeled as an ictal rather than an interictal phenomenon [@bb0055]. Our patient did not have any of these clinical or EEG characteristics, despite the early presence of EDB and its persistence for 2 years. EDB may not be unique for ANMDARE and has been reported in febrile infection-related epilepsy syndrome, hypoxic encephalopathy, brain tumor, focal cortical dysplasia, stroke and glycine receptor antibody-associated epilepsy [@bb0020], [@bb0060]. Furthermore, benzodiazep	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== The Istituti Fisioterapici Ospitalieri (IFO) comprise two scientific institutes: the Regina Elena National Cancer Institute (IRE) & the Dermatological Institute S. Gallicano (ISG), both located in Rome, Lazio, Italy. Despite the overwhelming challenges the entire healthcare community faced due to coronavirus disease 2019 (COVID-19) diffusion in our Region, IFO confirmed its mission to provide expert care without compromising continuity of assistance for its patients. Here we describe the strategic decisions made and the plans developed to limit the spread of COVID-19, while taking the opportunities going along with the outbreak. We also report on the effects of our actions, hoping that our experience could be helpful in promoting resilience, emergency plan and continuity of care provision within a public healthcare system. Activation of a crisis unit and adoption of general measures {#Sec2} ------------------------------------------------------------ Since the beginning of the COVID-19 outbreak in Italy, we designed institutional protocols and internal guidelines early on to guarantee cancer treatment for our patients while minimizing COVID-19-related risks ensuring a safe environment for care providers. The first measure was the activation of a Crisis Unit, chaired by the Medical Director and including the main representatives of different units involved in the virus containment[1](#Fn1){ref-type="fn"}, that consisted in meeting once a week to discuss the current clinical scenario, and updating institutional protocols and internal guidelines through a shared decision-making process. The weekly meeting of the Crisis Unit was organized more frequently when needed until 30 May 2020 when institutional activities were fully restored. Afterwards, the meetings, held via a web-based conference platform, were attended every 14 days. Protective and organizational measures have been implemented to provide healthcare professionals with the necessary training needed against the spread of the virus, including prevention, containment measures and hygiene recommendations: use of personal protective equipment (PPE), wearing PPE equipments in healthcare settings, PPE disposal in biohazard containers, use of screening questionnaires for patients admitted to hospital. The training of healthcare providers and PPE supply had a key role. Further measures to preserve safety of our employees have been also implemented, including the use of smart working via a dedicated platform developed upon the healthcare emergency. Until 18 May 2020, about 250 employees were currently on smart working. Residential meetings were replaced by video conferences. Our Institute adopted internal and external communication measures to highlight the health risks and behavior necessary to manage those risks. The Press Office and Communication Office worked synergistically to inform and constantly update both healthcare providers and patients regarding the virus prevention and containment measures by using printed brochures, visual alerts and poster distributed in the hospital. Filters to and within the hospital {#Sec3} ---------------------------------- Restrictions of patients and employees' access to the hospital, employ assistance and cancer center continuity of care support were implemented. ### Patient admission to the hospital {#Sec4} A triage was applied before entering the hospital. Patients were receiving a phone call 24--48 h before hospital admission to monitor their health status and to potentially identify suspected cases. If tested positive, patient was not admitted to the hospital. Furthermore, no visitors were allowed to IFO for the duration of this crisis. Figure [1](#Fig1){ref-type="fig"}a shows a flowchart pertaining to patients admitted to the hospital and encompassing four filters. The prerequisite for triage was the presence of acute symptoms, defined as fever (\> 37.5 °C), assessed by a thermal scanner, suspicion of respiratory infection, and at least one of the following data: respiratory rate \> 30/min, oxygen saturation (SpO~2~) \< 0.95% without oxygen supplementation, and dyspnea. The patient was also interviewed about potential contact with confirmed or suspected COVID-19 patients. The triage allowed us to detect an increasing proportion of suspected patients over time, ranging from 0.3--1.2%, up to June 2020, who were either referred to hospitals dedicated to COVID-19 treatment or sent home, thus avoiding their admission to the wards whenever possible (Fig. [1](#Fig1){ref-type="fig"}b). As of 30 June 2020, we identified 756 suspected patients. Restriction measures dropped visitors and/or caregivers percentage proportion from 42.9% in March 2020 to 9,15% in June 2020. Over time, the parameter most frequently associated with the condition of "symptomatic case" and/or a "case with critical and/or risk contacts" changed from being in contact with a COVID-19-confirmed case and showing dyspnoea during the early stages of the outbreak to the presence of oxygen saturation \< 0.95% (Fig. [1](#Fig1){ref-type="fig"}c). Fig. 1Flowchart of patients admitted to the hospital (a), number of hospital admissions during lockdown (b) and number of cases with symptoms or with critical contacts (c) The nose--throat swab is the current standard procedure to confirm a COVID-19 diagnosis. Relying on a Microbiology and Virus Unit and associated laboratories, our Institute has been part of the Lazio CoroNET network and has analyzed almost 21,576 nose--throat swabs (from 30 March to 30 June 2020). Eighty-seven nose-throat swabs have been analyzed for an external company located in our Region. To optimize the access to swabs while preserving health system sustainability and limiting unjustifiable requests, we have defined prescribing criteria for nose--throat swabs by identifying seven potential case scenarios as illustrated in Fig. [2](#Fig2){ref-type="fig"}. Fig. 2Prescribing criteria to request nose--throat swabs ### Active surveillance and epidemiological survey of healthcare workers {#Sec5} Healthcare personnel taking care of patients with cancer need to be safeguarded thus following PPE guidelines. A surveillance registry has been established to identify those, who may be categorized as contacts at risk and subsequently monitored by the hospital risk manager via a risk assessment questionnaire. As a result, healthcare workers can be categorized as low and high risk. Low-risk healthcare workers undergo active surveillance for 2 weeks monitoring their body temperature and symptoms daily. High-risk healthcare workers are tested for COVID-19 by nose--throat swab and if they test positive, they are quarantined at home; if they test negative, they undergo active surveillance for 2 weeks. Figure [3](#Fig3){ref-type="fig"} shows a flowchart pertaining the actions taken within IFO to manage contagion containment and isolation of suspected cases. Fig. 3Flowchart pertaining the actions taken within IFO to manage contagion containment and isolation of suspected cases A total of 370 subjects underwent surveillance and 2,151 were screened between 18 March and 30 June 2020. Reverse transcription polymerase chain reaction (RT-PCR) was positive in 8 healthcare workers and in 16 patients (seven inpatients and nine outpatients). A total of 1,598 serological tests have been carried out of which 1,563 for employees and 35 for homeless people. Overall, 51 employees were found positive and underwent nose-throat swabs with only one being positive and subsequently undergoing active surveillance. Reorganization of care provision to make our hospital closer to patients {#Sec6} ------------------------------------------------------------------------ Our institution reshaped the provision of care to address the urgency of not deferrable procedures (either surgeries, biopsy or chemotherapy/radiotherapy) while banning the access only for follow-up visits, according a case-by-case evaluation of the risk/benefit ratio \[[@CR1]\]. To reduce potential viral exposure at pharmacy departments and avoid in-person hospital visits to refill prescriptions, at home delivery service has been quickly implemented for those receiving either oral anticancer therapy or biologics. During the national lockdown, we have reached 119 patients with about 450 delivered treatments, thus markedly reducing hospital visits while promoting better patients' quality of life and favoring improved adherence to therapy. Patients who are not currently receiving active therapy may be well-suited for telemedicine consultation and/or routine follow-up. A switch to telemedicine has been recently recommended in breast cancer patients \[[@CR2]\] and virtual visits have been proposed as promising tools to ensure continuity of cancer services and assistance to patients \[[@CR3]\]. We have rapidly developed a dedicated telemedicine platform, named \#IFOConTeOnline, through expedited physician credentialing, training and modification based on changing regulations. With 9,178 web interactions on our platform, we have reached more than 2,000 patients and provided telemedicine services, consultations, symptom control and counselling to both cancer patients and their caregivers as well as to external patients with mild and ordinary dermatological conditions \[[@CR4]\]. To support people living with cancer during COVID-19 isolation and to mitigate the effects of quarantine on mental and physical well-being, we relied on digital technology to implement two phone helplines to promote patient mental health and coping with the pandemic crisis. Finally, we have realized that ensuring psychological support of frontline workers is also necessary to prevent burnout and attrition. Conclusions {#Sec7} =========== The COVID-19 outbreak has presented both challenges and learning opportunities for cancer centers \[[@CR5]\]. As suggested by Trapani et al., the key to success in COVID-19 and cancer is to ensure a continuum of healthcare never disconnecting the cancer cause from the population health needs \[[@CR6]\]. Overall, the proactive management and containment measures, including the triage protocols, have significantly aided the identification of as many as 756 patients with suspected symptoms related to COVID-19, thus limiting their admission to our cancer center. Furthermore, the activation of the active surveillance	{ "pile_set_name": "PubMed Central" }
Introduction {#Sec1} ============ The ocean acts as a buffer against global warming^[@CR1]^. However, understanding all processes and fluxes that lead to carbon sequestration is very complex and acts at multiple scales, from genes to ecosystems via physiological processes^[@CR2],[@CR3]^. In particular, the ecosystem community structure and composition, from large organisms to molecular descriptions, is essential when studying biogeochemical processes and their variability^[@CR2]^. In that community, gelatinous zooplanktons are still poorly represented compared to other groups^[@CR4]^ such as silicifiers, calcifiers, or crustacean. In particular, their role in biogeochemical processes is still debated mainly due to the lack of information on the fate of the gelatinous biomass, even-though several recent studies support their inclusion in ecosystem models^[@CR5]--[@CR7]^. While there is no evidence that their abundances are globally increasing^[@CR8]^, one observes their impact on carbon fluxes^[@CR9]--[@CR11]^ and the structuration of the trophic food webs^[@CR12]--[@CR14]^. Modeling the ecosystem structure to better represent biogeochemical processes in the ocean is, therefore, of primary importance. However, due to scaling issues and lack of holistic information on the plankton community, marine ecosystems are still very simplified compared to their terrestrial counterparts^[@CR15]^. Today, the new generation of Ocean Biogeochemical Models called Dynamic Green Ocean Models allows resolving the biological complexity of marine ecosystems better thanks to the inclusion of multiple plankton functional types^[@CR15]^. Similarly, trait-based modeling uses functional traits such as body size, shape, with a particular emphasis on trade-off to represent the ecosystem functioning^[@CR16],[@CR17]^. Overall, general plankton compartments such as macrozooplankton or microphytoplankton summarize ecosystems in order to facilitate the parameterization of non-linear parameters. Nonetheless, such parameters remain difficult to determine because of their multi-scale implication, from physiology to ecosystems. One of the main challenges in ecological modeling consists of acknowledging the whole biological complexity while remaining computationally tractable. To handle both opposite constraints, one advocates that a formal selection of the modeled species is a reliable solution^[@CR18]^. The sensitivity of a model to one parameter might depend on the value of other parameters. In this setting, analyzing the sensitivity of the model to single parameters in isolation is not satisfying. Moreover, standard sensitivity analyses do not consider the number of simulations that are needed to obtain accurate results^[@CR19]^. Following previous applications in engineering, the use of the Statistical Model Checking Engine (SMCE) overcomes this weakness by bringing formal confidence (trust) in the results while enhancing the range of sensitivity analysis towards considering global dependencies between parameters. First, instead of fixing parameter values to their mean observed values and performing sensitivity analysis of one parameter at a time, SMCE embeds uncertainty on parameter values inside the proposed models by assigning a probabilistic distribution to each parameter value (i.e., uniform distribution per default). SMCE performs model simulations by picking parameter values within their attached distributions (i.e., by considering the variances of parameters) and executing standard simulations. Thus, following several simulations, which implies considering several and distinct parameter choices, the SMCE performs a generalization of standard sensitivity analyses, not by analyzing the sensitivity of a single average simulation but rather by analyzing all feasible simulations and proposing general statistics of the whole; i.e., accurate statistical guarantees to perform predictive simulations while taking into account experimental uncertainties^[@CR20]^. Overall, considering a predictive goal at both physiological and ecosystem levels, the SMCE produces a global set of parameter values that guarantees that the model matches experimental observations despite slight parameter variations. In the Mediterranean Sea, Pelagia noctiluca is the most abundant scyphozoan species^[@CR21]^. This holoplanktonic species is present all year long and has been observed without interruption since 1994^[@CR22]^. Mostly present offshore, P. noctiluca reaches coastal waters thanks to wind events or sea currents variations^[@CR23],[@CR24]^. Moreover, this species can realize nycthemeral migrations between 0 and 300--500 m deep. The development of the jellyfish P. noctiluca from oocyte to adult is strongly related to temperature and environmental food conditions^[@CR25]--[@CR30]^. It is a non-specific predator^[@CR21]^ that responds rapidly to changes in the biotic and abiotic environment^[@CR31],[@CR32]^. Indeed, like most of the scyphozoan species, P. noctiluca has been known to shrink its body mass when prey concentration becomes limiting^[@CR33]--[@CR37]^. Because P. noctiluca is a multi-scale player (i.e., strong interaction from microbial communities to high trophic level), estimating its model parameters is difficult. The goal of this paper is to apply the SMCE to infer the parameters of an ecophysiological model and discuss the putative importance of a jellyfish in marine ecosystems and biogeochemical processes in the Mediterranean Sea. According to our knowledge, the SMCE approach was never used before in this context. To this purpose, we (i) build an ecophysiological model for P. noctiluca to describe the fundamental physiological processes that are involved in carbon fluxes, (ii) infer the model's parameters using the SMCE and (iii) discuss the potential contribution of P. noctiluca egestion to POC fluxes despite missing knowledge. The benefits of the SMCE consist not only in performing an accurate parameter estimation on several scales (from laboratory physiological experiments to in situ biomass distribution) simultaneously but also in emphasizing correlations between parameters of its ecophysiological model through automatic analysis. Here P. noctiluca is used as a scaling-up example of how one can apply state of the art verification methods in computer sciences to better estimate parameters of an ecophysiological model. Results {#Sec2} ======= Conceptual model {#Sec3} ---------------- To reproduce the jellyfish growth and degrowth in captivity and wild conditions, an ecophysiological model was built based on seven physiological processes (predation, ingestion, assimilation, respiration, excretion, reproduction, egestion) and constrained by 17 parameters (see Fig. [1](#Fig1){ref-type="fig"} for illustration and methods for details). Eleven parameters (p~max~, b~p~, t~10p~, R~o~, b~r~, t~10r~, α, β, a~re~, b~re~, W~e~; Table [1](#Tab1){ref-type="table"}) were defined by previous experimental data whereas six unknown parameters (k~p~, a~max~, k~a~, c~re~, spn, c~e~; Table [1](#Tab1){ref-type="table"}) were inferred using SMCE. During the simulations, the jellyfish carbon mass (CM) prediction was constrained by two forcing variables: temperature and zooplanktonic biomass. In controlled conditions, the temperature was fixed at 18 °C following Lilley, et al.^[@CR38]^ whereas prey concentration was null or estimated from a prey concentration range (F~lab~; Table [1](#Tab1){ref-type="table"}) in degrowth and growth experiments, respectively. In the in situ conditions, these variables were obtained by an annual climatology of sea surface temperature and zooplankton concentration from 2011 to 2015 in the bay of Villefranche-sur-Mer, France.Figure 1Conceptual diagram and equations used in the Pelagia noctiluca ecophysiological model. Arrows between compartments represent the biological carbon transfer following the ecophysiological processes. Symbols and units of the different variables are described in Table. [S1](#MOESM1){ref-type="media"}. The red and green colors represent all parameters deduced from the literature dataset and Statistical Model Checking Engine (SMCE) respectively.Table 1Values, symbols, description and units of the different parameters used in the model.Ecophysiological processParametersValuesUnitsDescriptionPredation (P)p~max~0.1399gC.ind^−1^.d^-1^Theoretical predation rate of 1 g CM individual at 0 °C and with unlimited food; log(C) = b.log(B) + a (adapted from Acuna, et al.^[@CR66]^)b~p~0.8856dimensionlessk~p~\[3 × 10^−5^: 2 × 10^−5^: 2 × 10^−4^\]gC.L^−1^Half saturation coefficient for predation (this study)Respiration (R)R~o~2.80μmolO~2~.d^−1^Theoretical respiration rate of 1 g WM individual at 0 °C (adapted from Lilley, et al.^[@CR34]^)b~r~0.934dimensionlessAllometric exponent for the effect of individual mass on respiration rate (adapted from Lilley, et al.^[@CR34]^)Reproduction (Re)a~re~0.07dimensionlessAllometric exponent for the production of eggs (adapted from Lilley, et al.^[@CR34]^)b~re~4.66dimensionlessW~e~1.52 × 10^−6^gC.eggs^−1^Eggs carbon mass^[@CR34]^c~*	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== According to the statistical report released from Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2015, thirty out of every 100,000 people in the general population suffered from a primary intracranial tumor requiring craniotomy for tumor resection. Glioma accounted for 60.8% of all primary intracranial tumors \[[@CR1]\], of which most of these operations require general anesthesia. These patients have vulnerable central nervous systems because of their intracranial tumors (e.g., gliomas are aggressive lesions that invade central neuronal structures anatomically, and at the neuropathologocial level disturb neuronal connections to impair neural function). Compared to intact brains, neuronal network connection disruptions from intracranial lesions and central nervous system inhibition by anesthetics yield a "double insult" to neurosurgical patients, so to speak, and may make them more sensitive to general anesthesia and surgery, leading to both short and long-term neurologic and neurocognitive dysfunction. Anesthesiologists attempt to choose the anesthetic regimen cautiously, taking into account anesthetics' effects on cerebral physiology (e.g., intracranial pressure and cerebral perfusion pressure), as well as to minimize the seeming disturbance to neuronal function by facilitating rapid emergence and orientation during recovery. The less postoperative neuronal function is affected by anesthetics, the more accurately the patient's intracranial disease-related status is reflected, and this is beneficial to neurosurgeons so as to correctly evaluate neural function. The optimal postoperative treatment based on neurological evaluation contributes to reduction of neurologic complications and is critical for long-term quality of life. Therefore, it is important to investigate how different mechanisms of general anesthesia affect postoperative neural function and anesthesia recovery in patients with supratentorial (frontal-parietal-temporal) gliomas in both a qualitative and quantitative (over time) manner, as this may guide anesthetic choice in supratentorial craniotomies for gliomas to ensure a reasonable, safe, and economical perioperative anesthetic regimen. Inhalational anesthesia and intravenous anesthesia are widely used in current neurosurgical anesthesiology \[[@CR2]\], and sevoflurane combined with remifentanil or propofol combined with remifentanil are both accepted as general anesthesia strategies. Comparative studies have been done to investigate the effects of different anesthetics on craniotomies \[[@CR3]\], both primary and secondary outcomes focused on the changes in intraoperative physiological parameters \[[@CR3], [@CR4]\], laboratory results \[[@CR3]\], and anesthesia recovery indices during emergence \[[@CR5], [@CR6]\]; however, neuronal disease-specific study is lacking, and there is no evidence related to how postoperative neurological function and neurocognitive outcomes are affected by different general anesthesia strategies \[[@CR7]\]. Patients with neurological diseases, e.g., brain tumors, are usually excluded in studies examining the correlation between anesthesia and neural function \[[@CR8]\], as those diseases are major confounders leading to nervous system dysfunction, however, anesthetic effects on neural function cannot be ignored in neurosurgical patients, let alone the previously mentioned "vulnerable" brain tumor patient who is even more susceptible to anesthetics \[[@CR9]\]. Recent findings have reported patients with supplementary motor area (SMA) lesions presenting with intraoperative neurological deficits for awake craniotomy which could not be explained anatomically by their intact corticospinal tracts as detected by cortical stimulation or cortical mapping \[[@CR10], [@CR11]\], and these deficits were reversible after operations over time without further intervention. Although this finding may have been related to lesion location, which is called "SMA symptom", this phenomenon cannot exclude the possibility of residual anesthetic-induced neurological deficits in both the intraoperative and early postoperative periods. It has been reported that in some supratentorial brain mass patients, a small plasma concentration of sedative could significantly worsen neurologic deficits before any operative intervention \[[@CR9]\], but there is no evidence related to how residual anesthetics affect neurologic deficits after operations. Repeated neurological evaluations after neurosurgeries can help to assess whether surgical interventions are successful and whether long-term neurologic outcomes are primarily related to diseases and surgeries, while short-term neurologic outcomes are subject to perioperative care beyond the above factors; despite only a few hours of surgical intervention for brain tumor, the overlap of general anesthesia and its effects, after surgery is completed, can linger for hours to days, which may worsen neurologic function and confuse providers. However, in the early postoperative stage, it is unknown whether sevoflurane combined with remifentanil and/or propofol combined with remifentanil could result in unexpected neurologic function deterioration, and whether one strategy is inferior or superior to another. In this study, we will use sevoflurane combined with remifentanil or propofol combined with remifentanil to maintain anesthesia during craniotomy in patients with supratentorial gliomas to determine whether they have comparable effects on neurologic function and neurocognition in the early postoperative period. Since both sevoflurane and propofol are GABAergic anesthetics, we hypothesize that these two general anesthesia methods equivalently affect early postoperative neural function in this group of patients. Methods {#Sec2} ======= Study purpose {#Sec3} ------------- $1$ Primary purpose: To investigate neurologic function in the early postoperative period for patients with supratentorial intracranial gliomas under inhalational general anesthesia compared to intravenous general anesthesia. The hypothesis is that the difference in postoperative National Institute of Health Stroke Scale (NIHSS or modified NIHSS) score changes under the two general anesthesia methods are not different, with the score difference within − 1 to 1. $2$ Secondary purpose: To compare the effects of inhalational general anesthesia versus intravenous general anesthesia on neurocognition, hemodynamics, cerebral physiology, anesthesia recovery quality, pain scores, anesthesia expenses, and stress responses in patients with supratentorial intracranial gliomas undergoing elective craniotomy. Trial design {#Sec4} ------------ The study is a prospective, single-center, open label, randomized parallel arm equivalent clinical trial comparing sevoflurane and propofol, both combined with remifentanil, general anesthesia (Sevoflurane-remifentanil versus Propofol-remifentanil group) in patients with supratentorial gliomas; it will be carried out in the neurosurgical operation room in Beijing Tiantan Hospital (a large city), Capital Medical University, Beijing, China. Populations {#Sec5} ----------- Adult male and female patients scheduled for elective craniotomy under general anesthesia with supratentorial (frontal-parietal-temporal) gliomas diagnosed by magnetic resonance imaging (MRI) are eligible for the study. All patients must sign institutional approval and informed consent before enrollment, the written informed consent will be obtained 1 day or a few days before operation, when patients are seen by anesthesiologists in the ward. The information and reasons why eligible patients are not recruited to the trial will be documented. The inclusion and exclusion criteria are listed in Table [1](#Tab1){ref-type="table"}. Table 1Inclusion and exclusion criteriaInclusion CriteriaPatients aged between 18 to 65 years old with American Society of Anesthesiology (ASA) status I \~ II who are scheduled for elective craniotomy for the treatment of supratentorial gliomas must fulfill the following:1Frontal-Parietal-Temporal glioma diagnosed by preoperative MRI2Glasgow score of 15 without preoperative symptomatic elevated intracranial pressure3New and/or recurrent intracranial gliomas are allowedExclusion Criteria1Unable to comprehend and cooperate with the neurologic examination2Emergency craniotomy or changed to emergency from elective craniotomy3Insular lobe is invaded by glioma4Scheduled intraoperative motor evoked potential monitoring5Patients with traumatic brain injury, intracerebral hemorrhage, or cerebral vascular diseases6Patients with prolonged emergence, postoperative mechanical ventilation, and/or sedation dependence due to a definite reason (e.g., surgery itself or tumor location)7Hypothalamic dysfunction8Radiotherapy and/or chemotherapy before surgery9Uncontrolled hypertension or severe heart disease that impairs cardiac function (New York Heart Association Functional Classification ≥ III)10History of related anesthetic allergy11Severe endocrine system dysfunction that impair metabolic index12Impaired mental status13Drug and/or alcohol abuse14Pregnant and/or lactation period patients15Neuromuscular diseases16Infectious and/or immune diseases with positive biomarker(s)17Body mass index \> = 35 Patients will be recruited in Beijing Tiantan Hospital, Beijing, China. The potentially eligible patient will be screened and contacted by a trial team member who explains the study and ascertains the patient's interest 1 or 2 days before the scheduled operation day. If interested in enrolment, the patient will receive the detailed trial explanation and written consent form. Randomization and blinding {#Sec6} -------------------------- Permuted-block randomization will be used with a block size of 4 and an allocation ratio of 1:1 to either sevoflurane-remifentanil group or propofol-remifentanil group. Random allocation sequence will be based on a computer-generated random digits table. One investigator who will not participate in anesthetic management or follow-up will implement the randomization and enroll patients, and allocation will be concealed in a sealed opaque envelope until patient enters the operating room. Randomization will occur for patients conforming to the above criteria, and written informed consent will then be obtained from themselves or their next-of-kin. Since the two general anesthetic administration routes are distinct (inhalational versus intravenous), patients and anesthesiologists cannot be blinded. An independent team who is not involved in the intraoperative management will be in charge of postoperative follow-up and is blinded to the intervention.	{ "pile_set_name": "PubMed Central" }
The Mediterranean region has unquestionably played a central role in human history, partly deriving from the navigable nature of the sea that connects southern Europe, western Asia, and North Africa. This unique setting has led to its being one of the most important and dynamic areas throughout prehistory. The period of the Mesolithic to the Bronze Age (in western Eurasia) covers two major cultural shifts that are arguably among the most important transitions in human prehistory, heralding the change from hunter-gatherer subsistence to food production and later the emergence of metallurgy, changes that fundamentally transformed human culture. Recent large-scale studies of ancient human genomic variation (e.g., refs. [@r1][@r2][@r3][@r4][@r5][@r6][@r7]--[@r8]) have focused mainly on central and northern Europe and have revealed that changes during the Neolithic and later during the Bronze Age were driven by population movements into Europe from the southeast and east, first by early farmers from Anatolia and the Levant ([@r1][@r2]--[@r3], [@r9], [@r10]) and second by herders from the Pontic-Caspian steppe ([@r4], [@r6]). These migrations profoundly reshaped the genetic and cultural landscape of Europe. However, studying the genetic impacts of these cultural transitions in southernmost Europe, especially the Mediterranean, has usually focused on single time periods ([@r10][@r11][@r12][@r13][@r14][@r15]--[@r16]). The full Neolithic package reached the Iberian Peninsula and northern (modern-day) Morocco ca. 7,500 Cal BP, with the Cardial pottery culture coming from the central Mediterranean ([@r17]). This was rapidly followed by a regional diversification of ceramics and lithics with the Cardial pottery type present in most of the Mediterranean fringe and the interior of the Iberian Peninsula represented by the Boquique type (e.g., El Portalón de Cueva Mayor) ([@r18]) potentially introduced through the north via the Pyrenees ([@r19]). In southern Iberia (Andalusia), however, the early Neolithic is characterized by a type of impressed non-Cardial ceramic decorated a la Almagra ([@r20]). This type of pottery culture reached central Andalusia by 7,300 Cal BP, soon replacing the Cardial pottery, and is found at the Murciélagos de Zuheros cave ([@r21]). It has been proposed that North Africa played a significant part in the origins of the Neolithic in southern Spain ([@r22]), although this has recently been challenged ([@r23]). The prehistory of the Iberian Peninsula as a whole is of particular interest, given its specific geographic location at the westernmost edge of the continent, naturally making it the furthest point from the documented prehistoric migrations originating from eastern Eurasia. This location holds potential for a complex demographic history with migrations from diverse sources, as it is connected with mainland Europe in the north, is surrounded by two potential maritime migration routes along the Mediterranean Sea and the Atlantic Ocean, and furthermore is in close geographic proximity to North Africa. Previous studies on early Iberian farmers have shown that these populations represent the descendants of migrants from Anatolia ([@r6], [@r13]) followed by admixture with local hunter-gatherers ([@r5], [@r6], [@r9][@r10]--[@r11], [@r13], [@r24]). Furthermore, modern-day southwestern Europeans are genetically closer to Early and Middle Neolithic Europeans than are modern-day central Europeans, who are more closely related to Late Neolithic and Bronze Age populations ([@r1], [@r2], [@r4], [@r6], [@r11], [@r25][@r26][@r27]--[@r28]), suggesting diverse and regionally distinctive demographic histories. To investigate the demographic history of the westernmost edge of the prehistoric Eurasian migrations, we have sequenced the genomes of 13 individuals excavated from six prehistoric Iberian sites in the north and south of modern-day Spain ([SI Appendix, Section S1 and Table S2.1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)). These sites cover the Neolithic, Late Neolithic/Copper Age (LNCA), and Bronze Age chronologies between 7,245 and 3,500 Cal BP ([SI Appendix, Fig. S3.2 and Table S3.2](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)), including the oldest sequenced genome in southern Iberia, from the Murciélagos de Zuheros cave. This individual is directly dated to 7,245--7,024 y Cal BP and represents the first genome of an individual from the Neolithic Almagra Pottery Culture, the early agriculturalists of the south of the Iberian Peninsula. For the El Portalón cave, we generated additional DNA sequence data for published individuals ([@r11]) as well as sequencing five additional individuals, enabling the genomic analysis of a population that spans a temporal sequence comprising the Neolithic, Copper Age, and Bronze Age periods (directly dated to between 7,165 and 3,500 y Cal BP). These genomic data, combined with published data ([@r6], [@r9], [@r11], [@r13], [@r24]), allow us to comprehensively study demographic changes through time in the Iberian Peninsula in general and in one single location in particular. We contrast these developments with contemporary populations in other parts of Europe where two major population turnovers took place during this time span ([@r1], [@r4], [@r6], [@r25]). Our genomic analysis was combined with stable isotope analysis to investigate the role of aquatic resources in the diets of the individuals tested here. Previous genomic studies have revealed increasing amounts of genetic hunter-gatherer admixture in the farmer population after the initial arrival of Neolithic people in Europe ([@r1][@r2]--[@r3], [@r11], [@r13]), implying the continued survival of hunter-gatherer populations or at least their lineages. In contrast, paleodietary stable isotope studies of the Iberian late Neolithic--Early Bronze Age have indicated that aquatic resources are not abundant in the diet, despite their likely availability at many sites ([@r29][@r30][@r31][@r32][@r33][@r34]--[@r35]). Detecting aquatic resources can be difficult using only bulk isotope analysis, and it has been posited that freshwater and terrestrial C3 diets can be more easily distinguished using stable carbon isotope values of amino acids ([@r36], [@r37]). Ten of the sequenced individuals were investigated for amino acid stable carbon isotopes of bone collagen to reconstruct paleodietary preferences ([@r38], [@r39]), in particular the presence of aquatic food intake ([SI Appendix, Section S3](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)) ([@r36], [@r40]). Results and Discussion {#s1} ====================== We sequenced the genomes of 10 individuals from northern and southern Spain either contextually or directly radiocarbon dated to the Neolithic, LNCA, and Bronze Age ([Fig. 1A](#fig01){ref-type="fig"} and [SI Appendix, Table S4.1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)), and we increased the sequencing depth of three individuals from a previous study (ATP16, ATP2, and ATP12) ([@r11]) using additional bone material ([SI Appendix, Table S2.1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)). Altogether, our 13 sequenced genomes range from 0.01× to 12.9× ([SI Appendix, Table S4.1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)) with six individuals having \>2.0× genome coverage. Our sequence data show postmortem damage and fragmentation, as expected from endogenous ancient DNA (aDNA) molecules ([@r41]). Eleven individuals were genetically inferred to be males and two to be females. We obtained contamination estimates based on the X chromosome in male samples ([@r42]), which were all ≤5% or lower ([SI Appendix, Table S4.1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)). Mitochondrial contamination estimates based on reads mapping to the mitochondrial genome ([@r43]) suggest \>5% mitochondrial contamination for two samples (POR003 and ATP019) ([SI Appendix, Table S4.1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)), and the sequence data from these individuals were subsequently filtered to retain fragments displaying postmortem damages to conservatively restrict the analysis to these sequences ([@r44]). ![(A) Sampling locations of individuals included in this study. Sites with newly generated sequencing data ([SI Appendix, Table S4.1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717762115/-/DCSupplemental)) are labeled. (B)	{ "pile_set_name": "PubMed Central" }
Introduction ============ Over the course of a chronic illness, patients face challenges on many fronts. On a basic level, they endeavor to understand what is happening to them and deal with their illness. This may include navigating the health care system and understanding their medication regimen. They interact with information that may change their abilities to engage in these behaviors and make health decisions. While existing literature has investigated how people seek health-related information, there is a need for additional research on how information facilitates changes in patients' understanding of their health, which may in turn lead to long-term changes in health management. This study investigated the relationship between information and health management of those with a chronic condition---fibromyalgia. Fibromyalgia is a complex, poorly understood condition characterized by chronic widespread pain, joint stiffness, and systemic symptoms (eg, mood disorders, fatigue, cognitive dysfunction, and insomnia) \[[@ref1]-[@ref3]\]. Due to the diversity of symptoms and problems that patients experience, fibromyalgia has an impact on multiple facets of patients' lives \[[@ref4]-[@ref6]\]. Because fibromyalgia patients often appear healthy and their symptoms are invisible, patients continually struggle with stigma, social isolation, and a search for legitimacy \[[@ref7]-[@ref10]\]. In addition, patients struggle with the medically unexplained nature of the syndrome \[[@ref6],[@ref7]\]. In the case of many illnesses, diagnosis can serve to give meaning to an illness experience, but with fibromyalgia, initial relief is replaced with the realization that the diagnosis does not lead to increased understanding, treatment options, or respect from others \[[@ref11]-[@ref13]\]. This is where information might potentially play a role. Though patients with fibromyalgia have shown little long-term improvement \[[@ref14]\], previous research has shown that becoming knowledgeable about one's condition is an important factor in acceptance or coming to terms with pain \[[@ref15]\], and pain acceptance is associated with less pain, disability, symptoms, mood disturbance, as well as better general health, functioning, and greater well-being (eg, \[[@ref16],[@ref17]\]). Because fibromyalgia is a condition for which there are limited treatment options, self-management is increasingly being recommended \[[@ref18]\]. Fibromyalgia patients consult many sources to try to understand their condition and possible treatments, including health care professionals, the Internet, health organizations, magazines, television, radio, support groups, and other people \[[@ref19],[@ref20]\]. Fibromyalgia patients and other patients with chronic conditions may use online resources such as online discussion forums and blogs to exchange information, understand their illness, and offer social support \[[@ref21]-[@ref23]\]. Online participation may lead to benefits such as reduction of social isolation \[[@ref22]\], patient empowerment \[[@ref24]\], and improved psychological, social, and cognitive health \[[@ref25]\]. Previous research has also reported that fibromyalgia patients' information needs change over the course of the illness \[[@ref26]\]. At first, individuals may be preoccupied with finding a cure. Searches for information on exercise, medications, and research increase over time. However, it is unclear what drives this evolution in information behavior, and moreover, what information behaviors may lead to successful self-management. The motivation for the current study was to provide insight concerning this gap. This paper explores three aspects of fibromyalgia patients' illness journeys: (1) health management, (2) information seeking, and (3) information consumption and use processes. Methods ======= Sample and Recruitment ---------------------- Multiple mechanisms were used to recruit a convenience sample that self-identified as having fibromyalgia (N=23). A recruitment goal was established to recruit a sample that was diverse in terms of three characteristics: age (˂47 years and ≥47 years), illness duration (≤4 years and ˃4 years), and social media participation style (non-user/lurker, infrequent participator, active participator), with substantive representation in each of the subcategories per category. A lurker was defined as someone who read social media content but did not author content themselves, an infrequent participator was someone who authored social media, but infrequently, and a frequent participator was someone who authored social media content quite often. These definitions are based on those in previous studies, with modifications to account for participation on other types of social media \[[@ref27],[@ref28]\]. The decision to focus on these dimensions was based on previous work that showed there was great variation in the age and illness duration of fibromyalgia patients and that social media participation style was significantly associated with other aspects of illness adjustment \[[@ref29]\]. The age threshold was based on the mean age in prior studies \[[@ref19],[@ref26]\], and the illness duration threshold was set in order to emphasize the first several years after onset. The recruitment mechanisms included an email contact list from a previous survey \[[@ref26]\], a university staff and student listserv, face-to-face support groups, health-related discussion forums, and Twitter ([Table 1](#table1){ref-type="table"}). In the case of face-to-face support groups, the leaders of support groups for fibromyalgia, chronic pain, and chronic fatigue syndrome were contacted, and permission was sought to visit the support groups to introduce the study and invite members to participate. The health-related discussion forums included websites such as Reddit, HealingWell, and ProHealth, which feature forums dedicated to fibromyalgia and other conditions that are often co-morbid with fibromyalgia, such as chronic fatigue syndrome. In each case, a description of the study and an invitation to participate was posted in relevant forums. In the case of Twitter, users who self-identified as having fibromyalgia were contacted and invited to participate. ###### Recruitment mechanisms and participants recruited. Recruitment mechanism Participants, n --------------------------------------- ----------------- Participant pool from previous survey 4 Listserv^a^ 7 Social networking sites 5 Face-to-face support groups 6 Twitter 1 ^a^Includes those referred by someone on the listserv. Interviews ---------- The first interview focused on participants' health history and information seeking and use. Participants were also asked to draw a timeline representing their illness journey ([Figure 1](#figure1){ref-type="fig"}). Timelines have been used in previous health-related research (eg, \[[@ref30]-[@ref32]\]). When the exercise was introduced, participants were asked to think about their illness journey and "to draw something that represent\[ed\] it." They were told there were no rules as to what they drew and that the timeline need not be a line. The aim of the prompt was to leave the activity as open as possible, so that participants would feel free to depict the journey as they experienced it. The purpose of this activity was to help participants access their memories of their illness history. The second interview was used to explore participants' social media participation histories, using an interface called the Online Scrapbook that was designed for the study. This interface enabled participants to view their social media participation over time. There were multiple reasons to incorporate the interface, including reminding participants of what they had previously authored, as well as providing them a fresh look at it through an interactive visualization. As this paper focuses on thematic analysis of the interview content, the interface will not be discussed in further depth. The interview guides for the two interviews have been included in the [Multimedia Appendices 1](#app1){ref-type="supplementary-material"} and [2](#app2){ref-type="supplementary-material"}. Participants were interviewed either once or twice, depending on the extent to which they participated in social media and their geographic proximity. If participants participated only minimally in social media or lived far away, they were usually interviewed once, and the questions from the second interview were integrated into the first interview. Three interviews were conducted via Skype or phone due to issues of geographic proximity. All other interviews were conducted in person. To ensure that participants were comfortable during the interview, the location for the interview was left up to the participant, and almost all interviews occurred either in participants' homes or in coffee shops. Altogether, the study involved 37 interviews with 23 participants, and the mean total interview time per participant was 2 hours and 26 minutes. ![P21's illness journey timeline.](jmir_v18i10e269_fig1){#figure1} Data Analysis ------------- The analysis method was derived from two approaches: interpretative phenomenological analysis and constructivist grounded theory. The primary aim of interpretative phenomenological analysis is to explore how participants make sense of their world and focuses on participants' interpretations of the object or event \[[@ref33],[@ref34]\]. Grounded theory focuses on how social and psychological processes occur in a given environment or situation \[[@ref35],[@ref36]\]. These two foci facilitated a study that investigated lived experience as well as social interactions and context. The interview transcripts and a purposively sampled subset of posts authored by participants who engaged in online discussion forums such as Reddit served as the basis for the analysis. Because some participants were extremely prolific in their social media content production and it was not possible to manually analyze all of the posts, it was necessary to select a subset of posts that provided a sense of the diversity of each participant's social media production. The posts that participants authored were analyzed in the context of the threads, or dialogues, in which the posts were embedded. The content was analyzed using Atlas.ti Version 1.0.1. In order to protect the identities of the participants, each participant was assigned an identification number. There were four pilot participants; thus, the 23 participants in the study will be referred to as P05-P27. The analytic procedure involved initial line-by-line coding, followed by conceptualization of	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== Enhancing the grain yield potential of wheat is a key focus of wheat breeders. Grain yield is the product of various yield components. Wheat grain yield per unit area is the product of grain yield per spike (GYS) and the number of spikes per unit area. The latter depends on sowing density and is highly affected by environmental factors. The GYS is determined by grain number per spike (GpS) and thousand grain weight (TGW), which are variably correlated in different wheat collections/populations. A significant negative correlation between these two traits has been reported in bi-parental populations \[[@CR1]--[@CR3]\], but no significant correlation was observed between TGW and GpS in collections of Chinese landraces \[[@CR4]\], French winter wheat cultivars \[[@CR5]\] and CIMMYT-derived spring wheat collections \[[@CR6]\]. On the other hand, a significant positive correlation between TGW and GpS was reported in modern Chinese cultivars \[[@CR4]\]. TGW in wheat has been one of the target traits for selection during domestication and breeding \[[@CR7], [@CR8]\]. For example, in China, an increase in wheat yield potential from \~1 T ha^−1^ in 1992 to \~5.4 T ha^−1^ today is mainly due to the genetic increase in TGW from \~20 g to \~45 g, respectively \[[@CR9]\]. However, genetic gain in TGW has not reached its limit and thus provides an opportunity to increase yield potential \[[@CR9]\]. It is estimated that an increase in yield of 140--160 kg ha^−1^ can be obtained by just a 1-g increase in TGW \[[@CR10]\]. However, genes and their roles in controlling TGW in wheat are still largely unknown. In wheat, TGW is a quantitative trait controlled by several genes/QTL distributed on all chromosomes \[[@CR8], [@CR11]\]. For example, Su et al. \[[@CR12]\] discovered eight TGW-related QTL on chromosomes 2D, 4B, 5A, 7A and 7B, explaining up to 16.2% of the phenotypic variation. Similarly, four QTL for TGW on chromosomes 1D, 2A, 5D, and 6A explained 5.9 to 20.1% of phenotypic variation in different environments \[[@CR13]\]. Nevertheless, from this plethora of QTLs, few loci/genes have been cloned by map-based cloning approaches mainly because of the large and complex hexaploid genome (\~17 Gb) that consists of three homeologous genomes (A, B, D) and an abundance of repeat sequences (80%) \[[@CR14]\]. Studies on comparative genomics have shown high synteny and collinearity among different grass genomes, such as those of wheat, barley, rice, millet, maize and sorghum. This pattern of genome organization in the members of the grass family provides a powerful approach for gene discovery in common wheat \[[@CR15]\]. A large number of genes have been discovered in common wheat by synteny-based cloning, in which the gene sequences of model crops such as rice and barley have been used as references to identify orthologous genes in wheat. For example, the genes TaTGW6 \[[@CR16], [@CR17]\], TaCwi-A1 \[[@CR18]\], TaSus2-2B \[[@CR19]\], TaSus2-2A, TaSus1-7A \[[@CR20]\], TaGW2-6A, 6B \[[@CR9], [@CR12], [@CR21]\], TaCKX6-D1 \[[@CR22]\], TaSAP1-A1 \[[@CR23]\], TaGS1a \[[@CR24]\], TaGS-D1 \[[@CR25]\], and TaGASR-A1 \[[@CR26]\] were discovered using rice-wheat synteny and using molecular markers in marker-assisted wheat breeding. Hence, the isolation and characterization of genes controlling grain size in common wheat will help breeders maximize yield potential by establishing gene-based breeding programs. The FLOURY ENDOSPERM2 (Flo2) gene is a member of a conserved gene family in plants. In rice, this gene has been shown to have a tetratricopeptide repeat (TPR) motif consisting of 3--16 tandem repeats of 34 aa residues that mediate protein--protein interactions in the nucleus \[[@CR27], [@CR28]\]. The OsFlo2 gene was cloned in the indica cultivar 'Kasalath'; this gene was found to have 23 exons and 22 introns and coded for a protein consisting of 1720 amino acid residues that had three TPR motifs in the middle \[[@CR27]\]. The expression of Flo2 was constitutive in both vegetative tissues and developing seeds, and the expression was relatively high level in developing seeds. The flo2 mutants exhibit a significant reduction in amylose content and grain weight and exhibit altered expression of various starch synthesis-related genes, indicating its key role in regulating rice grain weight and starch quality \[[@CR27], [@CR28]\]. In this article, we report the rice-wheat synteny-based isolation of Flo2 orthologs in hexaploid wheat, the association of TaFlo2-A1 sequence polymorphisms with TGW and the comparison of temporal expression profiles of TaFlo2-A1 haplotypes in flag leaves and developing caryopses. Methods {#Sec2} ======= Plant materials {#Sec3} --------------- For cloning the TaFlo2 gene in hexaploid wheat, Chinese Spring (CS) and two sets of cultivars with lower and higher TGW were used; the set of cultivars with higher TGW included Dixiuzao (49.5 g), Enmai4 (49.2 g), Liying 5(49.4 g) and Laizhou 953 (52.2 g), and the set with lower TGW included Jinyang 60 (23.5 g), Baihuamai (24.1 g), Sanyuehuang (25.2 g) and Zipi (25.5 g). The Chinese Spring nulli-tetrasomic lines were used to assign TaFlo2 genes to wheat homeologous chromosomes. The Chinese Micro Core Collection (MCC, 262 accessions) and Pakistani wheat collection (130 accessions) were used to confirm the association between TaFlo2-A1 haplotypes and TGW. To avoid the effect of population structure, normalized MCC subpopulations were used for association analysis \[[@CR19], [@CR29]\]. The Pakistani collection was selected based on previous reports \[[@CR30], [@CR31]\] considering the effect of population structure on association analyses. Cloning and characterization of TaFlo2 sequences {#Sec4} -------------------------------------------------- The genomic sequence of the rice OsFlo2 gene (NC_008397) was used as a query for BLAST searches against the wheat sequences database in the URGI (<https://urgi.versailles.inra.fr>/). All wheat scaffold sequences with high similarity to the rice OsFlo2 sequence were assembled to construct a putative TaFlo2 gene using DNAMAN (<http://www.lynnon.com>). Based on the scaffold sequences, six conserved primer pairs were used to specifically amplify TaFlo2 coding and promoter sequences from the three wheat sub-genomes: A, B and D (Table [1](#Tab1){ref-type="table"}). The TaFlo2 mRNA of 4902 bp was cloned in Chinese Spring using three primer pairs designed from the predicted mRNA sequence (Table [1](#Tab1){ref-type="table"}). Genomic DNA was extracted from young seedlings of each line using the CTAB method \[[@CR32]\]. A 20-μl reaction volume comprising 0.5 μl (5 μM) of each primer, 2× Taq mix (GenStar, Beijing, China) and 100 ng of DNA was used for PCR amplification that consisted of a cycle profile of 5 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 60 °C and 4 min at 72 °C; and a final extension of 10 min at 72 °C. The PCR products were detected by electrophoresis in 1% agarose gels with nucleic acid dye (Tiangen, Beijing, China), and gel images were captured using a UV spectrometer (BioRad, Hercules, CA, USA). The targeted PCR products were obtained from the agarose gels and purified using the TIANgel MIDI Purification Kit (Tiangen, Beijing, China). The purified PCR products were then ligated into the pGEM-T Easy cloning vector (TransGen Biotech, Beijing, China). The ligation product was transformed to 50 μl of Trans1-T1 competent cells by the heat shock method (Tiangen, Beijing, China). Positive clones from each transformation were selected based on positive PCR tests and were sequenced (Beijing Genomics Institute). The sequences were analyzed using DNAMAN software (<http://www.lynnon.com>).Table 1Primer sequences used in this studyPrimer namePrimer sequence (5′-3′)Position on scaffold sequenceAnnealing temperature (°C)PCR product sizeFunctionFlo2-1FTGTGCTGGAATCACCCACTC793--812601061cloning TaFlo2 /polymorphism detectionFlo2-1RGCGCGGCGAAAACTAATCAT1853--	{ "pile_set_name": "PubMed Central" }
![](brforeignmedrev70071-0252){#sp1 .245} ![](brforeignmedrev70071-0253){#sp2 .246}	{ "pile_set_name": "PubMed Central" }
Introduction {#S0001} ============ Dry eye disease (DED) is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms.[@CIT0001] Being the most common reason for seeking medical eye care, DED is a common public health problem, with the prevalence ranging from approximately 5--50% worldwide.[@CIT0002]--[@CIT0004] Although the pathogenesis of dry eye disease is not fully understood, it is recognized that inflammation has a prominent role in the development and propagation of this debilitating condition. DED inflammation disrupts the normal homeostasis of the ocular surface, resulting in a vicious circle of ocular surface injury, and anti-inflammatory and immunosuppressive drugs are considered the pharmacologic agents of choice for controlling the inflammatory cascade of this disease.[@CIT0005]--[@CIT0007] Even so, there is still a great potential for the development in anti-inflammatory and damage-repair treatments of dry eye disease. Vitamin B12 (VB12) is a dietary essential nutrient and is important for metabolic functions of the nervous system, whose deficiency is associated with an impairment of sensory innervation, and may cause optic neuropathy, eye movement disorders, and corneal epitheliopathy with decreased vision and photophobia.[@CIT0008]--[@CIT0010] Recently, several studies have reported the parenteral and topical use of VB12 for ocular pain and symptoms in dry eye treatment.[@CIT0011],[@CIT0012] Oxytocin (OXT), a nonapeptide produced in the hypothalamus, exerts a wide spectrum of central and peripheral effects. Nasal spray of OXT, which has been proved to be safe in children,[@CIT0013] is currently suggested as a potential treatment for various psychopathologies of social cognition and behavior in humans.[@CIT0014] In addition to its reproduction and psychiatry-related functions, OXT has also been proved to display a potent anti-inflammation and wound-healing effect in various inflammatory disease,[@CIT0015],[@CIT0016] as well as an analgesic and nerve healing effect in animal models.[@CIT0017],[@CIT0018] However, the anti-inflammatory and nerve healing effect of OXT has yet to be applied in ocular diseases. Nebulization, applied mainly in pneumology and rhinology diseases, is a widely used means of drug delivery to the upper and lower airways. Nebulizers are devices that convert a drug liquid in solution or suspension into small droplets.[@CIT0019] Its theoretic advantage over classic means of delivery is that, as a non-invasive painless treating system, it delivers drug and humidifies the target region simultaneously, enables dose modification and dose compounding, and directly reaches the target organ, which avoids systemic side-effects and enhancing local efficacy.[@CIT0020] To date, no applications of nebulization therapy havebeen reported in ophthalmic diseases. In the light of the facts mentioned above, we developed a new ocular nebulization therapy delivering vitamin B12 and oxytocin separately for the treatment of DED. The main objective of this study was to estimate the efficacy of ocular nebulization in dry eye patients, as well as to compare the characteristic of VB12 and OXT aerosols using both clinical and in vivo confocal microscopy (IVCM) evaluations. Materials and methods {#S0002} ===================== Study design and participants {#S0002-S2001} ----------------------------- This prospective cohort study included 38 patients with DED recruited from the outpatient department of the Department of Ophthalmology at Peking University Third Hospital between August 2017 and June 2018. The study protocol was approved by the Human Research and Ethics Committee of Peking University Third Hospital, and the research was adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each participant before enrollment. Inclusion criteria included adult patients 1) with a diagnosis of DED in accordance with the criteria proposed by the Tear Film and Ocular Surface Society (TFOS) Dry Eye Workshop (DEWS);[@CIT0001] 2) willing to cooperate during the examination and treatment procedure; and 3) with the ability to participate in follow-up examinations for at least 3 months during nebulization treatment. Exclusion criteria consisted of the following: a) patients with active allergies, infections, or inflammatory diseases of the ocular surface unrelated to dry eye within 6 months; b) patients who currently used treatments for DED (other than artificial tears); c) patients who took oral neurotrophic and anti-inflammatory drugs in the past 3 months; d) a history of ocular trauma or surgery within 1 year; e) patients with alterations in the lacrimal drainage system such as punctal occlusion; f) patients who used contact lenses within the past month; g) patients with uncontrolled systemic diseases; and h) pregnant or nursing mothers. Clinical evaluation {#S0002-S2002} ------------------- Each participant had three clinic visits: before starting the nebulization therapy (baseline), 1 month after treatment, and 3 months after starting treatment. Both the patients and the observers were blinded of the nebulization group throughout the process. During each visit, all participants had a complete masked ophthalmic evaluation conducted in the following order: evaluation of symptoms using the Ocular Surface Disease Index (OSDI) questionnaire, along with a self-assessment of light sensitivity and dryness (each on a scale of 0--4, with 0 for never and 4 for severe), a complete examination of the ocular surface of both eyes, including tear meniscus height (TMH),[@CIT0021] tear break-up time (BUT),[@CIT0001] and corneal fluorescein staining (CFS) evaluated using the Van Bijsterveld scale,[@CIT0022] and IVCM analysis of the central cornea. Additionally, adverse events during the treatment, including pain, pressure, dizziness, blurred vision, and abdominal discomfort, were recorded during the 3-month follow-up. TMH {#S0003} === The central lower TMH were measured using a slit lamp microscope (with a graticule in 0.05 mm units).[@CIT0021] Three consecutive readings were obtained, and the final results are presented as medians. BUT {#S0004} === A total of 5 mL of 2μ sodium fluorescein was instilled onto the bulbar conjunctiva using a micropipette, without inducing reflex tearing. The patient was asked to blink naturally without squeezing three to five times and was then asked to stare straight ahead without blinking under the cobalt blue light until he or she received other instructions. A stopwatch was used to record the time between the last complete blink and the first appearance of a dry spot or disruption in the tear film.[@CIT0001] The procedure was repeated three times, and the final score is presented as an average value. Corneal fluorescein staining {#S0005} ============================ For the corneal staining evaluation, the cornea was divided into four sectors. Each sector was graded from 1 to 3 using the following criteria: 1 representing few separated spots; 2 representing many separated spots; and 3 representing confluent spots.[@CIT0022] In vivo confocal microscopy {#S0006} =========================== All participants underwent laser IVCM of the central cornea in both eyes by a masked investigator (XL) using Heidelberg Retina Tomograph 3 with the Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany). Each image represents a coronal section of 384 × 384 pixels which is equivalent to 400 × 400 μm of the cornea. Before each examination, a drop of 0.4μ oxybuprocaine chlorohydrate (Santen, Osaka, Japan) was instilled into the lower conjunctival fornix. The examination was conducted approximately at the corneal apex. For each eye, 1 image of basal epithelial layer, and 3 most representative images of the sub-epithelial layer were selected by a single masked investigator (XL). Three independent observers (JF, YL and YZ) measured each image, among which the mean value of series of the 3 sub-epithelial layer readings was recorded, and the average of the measurements by the observers was used for further analysis. Evaluated parameters included the following:Basal epithelial cell density (cells/mm^2^): basal epithelial cells were immediately above the Bowman's layer and identified by the lack of a visible nucleus, polygonal cell shape, hyper reflective cell borders and a relatively higher density of cells per frame.[@CIT0023] The counting was carried out within a region of interest of standardized size (region of interest \[ROI\] = 100 × 100 μm) under a manual cell counting procedure conducted using ImageJ software (<http://imagej.nih.gov/ij/)>, in which cells that were partially within the area analyzed were counted only along the left and upper margins.[@CIT0024]Dendritic cell (DC) density (cells/mm^2^): Dendritic cells were morphologically identified as bright individual dendritiform structures with cell bodies,[@CIT0025] which was manually counted in each corneal section at the level of subbasal nerves, selecting the images in which there was a greater density of DCs. The counting was carried out using the manual cell counting procedure same as basal epithelial cell density in the ROI of 400×400 μm.Nerve tortuosity: the grade of nerve tortuosity was classified in four grades according to the tortuosity grading scale reported previously.[@CIT0026]Sub-basal nerve density (mm/mm^2^): defined as the total length of all nerve fibers within a frame (400×400 μm). It was traced and calculated using NeuronJ (<http://www.imagescience.org/meijering/software/neuronj/>), a semi-automated nerve analysis plug-in of ImageJ.[@CIT0027] To note, change	{ "pile_set_name": "PubMed Central" }
![](jove-47-2423-thumb) Protocol ======== To guarantee high quality and reproducibility, we recommend the use of Standard Operating Procedures (SOP). In this video, published SOPs as developed and used in our laboratory are applied. ^1^ 1. Middle Cerebral Artery Occlusion ----------------------------------- 1. Mice are anaesthetized with an appropriate anaesthetic regime in consult with veterinary staff. (E.g. induction with 1.5 - 2 % Isoflurane and maintainance with 1.0 - 1.5 % Isoflurane in 2/3 N2O and 1/3 O2 using a vaporizer). Body temperature of the mice is maintained at 36.5°C ± 0.5°C during surgery with a heating plate. A feedback controlled heating pad, which warms according to the rectal temperature of the mouse, is highly recommended.Disinfect the skin and surrounding fur with an appropriate agent (e.g. 70% ethyl alcohol) and dry it afterwards. 2. A midline neck incision is made and the soft tissues are pulled apart. 3. The left common carotid artery (LCCA) is carefully dissected free from the surrounding nerves (without harming the vagal nerve) and a ligature is made using 6.0/7.0 string. 5.0 string can also be used. 4. The left external carotid artery (LECA) is then separated and a second knot is made. 5. Next, the left internal carotid artery (LICA) is isolated and a knot is prepared with a 6.0 filament. 6. After obtaining good view of the left internal carotid artery (LICA) and the left pterygopalatine artery (LPA), both arteries are clipped, using a microvascular clip. 7. A small hole is cut in the LCCA before it bifurcates to the LECA and the LICA. A monofilament made of 8.0 nylon coated with silicon hardener mixture (see below) is then introduced into the LICA, until it stops at the clip. Attention has to be paid not to enter the occipital artery. (Figure 1) 8. The clipped arteries are opened while the filament is inserted into the LICA to occlude the origin of the LMCA in the circle of Willis. 9. The third knot on the LICA is closed to fix the filament in position. 10. The mice receive saline 0.5 mL subcutaneously as volume replenishment. For pain relief, Lidocaine gel is topically applied in the wound. 11. If reperfusion is intended, mice stay for 30 - 90 min occlusion in a heated cage, the wound could be closed with a small suture clip. Afterwards, a second anaesthesia is performed, the third knot on the ICA is momentary opened and the filament withdrawn. 12. The remaining sutures are shortened and the skin is adapted with a surgical suture. 13. All animals receive a second volume replenishment as described above. 14. Animals are put in a heated cage for two hours to control for body temperature. The animals must be checked daily after surgery for signs of discomfort. The mice could show some post surgical weight loss. They receive mashed food in a petri-dish placed on the floor to encourage eating. The food is replaced daily for seven days. 2. Sham Operation ----------------- 1. For sham operations the filament is inserted to occlude LMCA and withdrawn immediately to allow instant reperfusion (1.8). The subsequent operation is identical to that performed on the animals undergoing cerebral ischaemia (1.9 - 1.14) including a second anaesthesia. 3. Preparation of the Filament ------------------------------ 1. Sterility of the filament should be considered. The use of sterilized equipment as well as an appropriate handling of the filament afterwards is a prerequisite for a sterile surgery. Disinfection of the filament is difficult, since many of the common sterilization methods may worsen the quality of the filament. However methods such as radiation, for example with UV light or γ rays, or chemical sterilization, for example with highly reactive gases such as ethylene oxide, are applicable. 2. 8.0 nylon filament is cut into lengths of 11 mm under the microscope 3. The filament tip must be coated completely and evenly over a length of 8 mm with a hardener mixture of Xantopren M Mucosa and Activator NF Optosil 4. Representative Results ------------------------- Depending on the duration of blood flow restriction, different motor and behavioural deficits result. Both after 30 and 60 min of cerebral ischaemia, animals in most cases show decreased resistance to lateral push and circling due to disturbance in locomotion. Milder lesions manifest as a flexor position in the front limbs. These easily observable signs can be used as a basic score for the success of the operation.^2^ Morphologically the lesion can be assessed using either histology or Magnetic Resonance Imaging (MRI). Sixty minutes occlusion of the middle cerebral artery produces tissue pannecrosis in an area including both the striatum and the neocortex, whereas 30 minutes of ischaemia cause mainly neuronal cell death limited to the striatum. ^3^ ( Figure 2) In terms of infarct volume, we expect a standard deviation lower than 30% in a set of operations. Mortality depends on the occlusion time with around 5 % after 30 min of ischaemia and 10 - 20 % after 60 min. Another minimal invasive possibility is the use of Laser Doppler flowmetry (LDF) during the operation, which allows a direct control of its success. In an individual animal, reduction to 10 - 20 % of preocclusion values clearly indicates successful induction of focal cerebral ischemia. ^4^ However, LDF cannot be used as a method for interindividual comparisons, since LDF can only measure quantitative changes (in percentages) of blood flow within a small and limited tissue sample volume. It does not give information about the spatial extent of blood flow reduction. ^5^ There are several tests to assess behavioural aspects after stroke, including gait analysis ^6,7^, Rotarod ^8^, Pole test ^9,10^, adhesive removal test ^11,12^, staircase test ^13,14^, ladder rung test^15,16^ and Morris water maze^17^. In all these tests, mice subjected to MCAo perform less successfully than control animals. Figure 1. Scheme of the vessel architecture supplying the brain (depicted in background) in the mouse. Different strains may show variations, for example the occipital artery sometimes leaves from the internal carotid artery. Figure 2. Schematic illustration of typical lesion sizes after different time points of reperfusion in the proximal MCAo model. In the middle, the typical course of functional activity and cerebral blood flow after MCAo are depicted. (MCAo: Middle cerebral artery occlusion, LDF: Laser Doppler flow measurement) Discussion ========== The model of transient, proximal MCA occlusion^18,19^ presented here mimics one of the most common types of ischaemic stroke in patients.^20^ Based on variable reperfusion times, the model offers different grades of damage ranging from transient ischaemic attack (TIA) to large infarcts including major parts of the ischemic hemisphere. This allows the researcher to study different pathophysiological mechanisms after stroke. ^20,21^ Surgery can be performed in a short time period and produces highly reproducible lesions. Nevertheless, this requires a thorough control of confounders.^22^ Small differences in operation technique may account for different effects on the infarct.^23,24^ Furthermore, due to variances in cerebral vascular anatomy, different mouse strains show a different outcome.^25,26^ Body temperature affects neurological damage, with hypothermia leading to smaller lesions^27^ and hyperthermia to more severe deficits.^28^ Accordingly, temperature control and maintenance is highly relevant in this model.^29^ In addition, blood pressure and blood gases are important confounders of outcome and need to be monitored.^30,31^ The use of quick and minimal invasive methods (non-invasive blood pressure measurement, suitable and easy accessible blood collection sites) is recommended. The choice of the anaesthetic is also highly important, as some may have neuroprotective effects, and/or be vasodilators, as for example Isoflurane.^32^ Consequently, exposure to anaesthesia should be as short as possible and standardized. We exclude animals which have undergone surgery for longer than 15 min. Shaving the surgical site produces microabrasions and inflammation and releases hair fragments. This may further increase inflammation and foster local infection, which may impact on stroke pathophysiology. Housing conditions, especially the use of enrichment, may affect stroke outcome and should be standardized and described in research reports. ^6,33,34^ The use of environmental enrichment and its effect on reproducibility is a matter of discussion.^35^ Another important confounder is the stroke induced risk for infection especially after longer times of ischaemia^36^, which leads to additional morbidity and increased mortality. ^37,38^ As infections become symptomatic at around day 3 to 5, this has important consequences for long term follow-up in such models. To produce results relevant for the development of novel treatment strategies for stroke, standardization and quality control is highly important in preclinical translational stroke research. ^39^ \"Good Laboratory Practice\" ^40,41^ mandates: 1. an appropriate and detailed description of animals used; 2. sample size calculation and reporting on the expected	{ "pile_set_name": "PubMed Central" }
![](transedinobsoc81673-0180){#sp1 .162} ![](transedinobsoc81673-0181){#sp2 .162-1} ![](transedinobsoc81673-0182){#sp3 .163} ![](transedinobsoc81673-0183){#sp4 .164} ![](transedinobsoc81673-0181-a){#f1 .162-1}	{ "pile_set_name": "PubMed Central" }
Although the CA2 region was first described by Lorente de Nó in 1934^[@R8]^ relatively little is known about its functional properties and behavioral role. To examine the importance of this region, we generated a transgenic mouse line (Amigo2-Cre) that expresses Cre recombinase predominantly in CA2 pyramidal neurons (PNs) in adult mice ([Extended Data Fig. 1](#F5){ref-type="fig"}). Because this line expresses Cre throughout the brain during early development, as well as in certain limited areas outside of CA2 in the adult, we stereotactically injected Cre-dependent adeno-associated virus (AAV) into the hippocampus of adult Amigo2-Cre mice to limit viral expression to CA2 pyramidal cells. To determine the specificity of CA2 expression in the transgenic line, we bilaterally injected into dorsal hippocampus a Cre-dependent AAV to express yellow fluorescent protein (YFP) in Cre^+^ cells ([Fig. 1a](#F1){ref-type="fig"}). We observed selective and robust YFP expression in CA2 PNs throughout dorsal hippocampus^[@R9]-[@R11]^ ([Fig. 1b](#F1){ref-type="fig"}; [Extended Data Fig. 2a](#F6){ref-type="fig"}). We confirmed that the Cre^+^ cells were indeed CA2 PNs by demonstrating co-staining for RGS14^[@R12]^ (97.38 ± 0.31% overlap; n = 4 mice, 2546 cells; [Fig. 1c-e](#F1){ref-type="fig"} and [Extended Data Fig. 3](#F7){ref-type="fig"}) and other known CA2 PN markers ([Extended Data Fig. 2](#F6){ref-type="fig"}). In contrast, there was no co-staining for a CA1 PN marker ([Extended Data Fig. 2](#F6){ref-type="fig"}). Additionally, the electrophysiological properties of the YFP^+^ neurons differed significantly from those of CA1 PNs ([Extended Data Table 1](#T1){ref-type="table"}) and largely matched the values previously reported for CA2 pyramidal neurons^[@R7]^. Only a minute fraction of YFP^+^ neurons were also GABA^+^ (0.16 ± 0.16%; n = 3 mice, 1539 cells), demonstrating the specific targeting of CA2 excitatory PNs ([Fig. 1f, g](#F1){ref-type="fig"} and [Extended Data Fig. 3](#F7){ref-type="fig"}). Finally, our AAV injections resulted in the targeting of the vast majority of CA2 PNs in the dorsal hippocampus, measured by the percentage of RGS14^+^ cells that were also YFP^+^ (82.33 ± 2.37%, n = 4 mice, 2992 cells). Next, we mapped CA2 synaptic input and output using viral tracing strategies that take advantage of the genetic targeting of CA2 PNs in the Amigo2-Cre mice, and largely confirmed results of previous studies using conventional^[@R13]^ and genetic-based^[@R14]^ approaches. Monosynaptic inputs to CA2 PNs were determined using an EnvA pseudotyped ΔG rabies virus strategy^[@R15]^ ([Extended Data Fig. 4](#F8){ref-type="fig"}). Unilateral viral injections revealed bilateral inputs from CA3 and CA2 ([Fig. 2a, b](#F2){ref-type="fig"}) and strong unilateral input from both lateral and medial EC layer II neurons ([Fig. 2c, d](#F2){ref-type="fig"}). In addition, synaptic inputs were detected from medial septum and diagonal band ([Fig. 2e](#F2){ref-type="fig"}), median raphe nucleus ([Fig. 2f](#F2){ref-type="fig"}), and the supramammillary nucleus of the hypothalamus ([Fig. 2g](#F2){ref-type="fig"}). Surprisingly, we observed only sparse labeling of EC layer III neurons with the rabies virus approach. Our laboratory previously concluded that EC LIII axons provide strong excitatory drive to CA2 PNs, based on the finding that large excitatory postsynaptic potentials (EPSPs) are evoked in CA2 PNs with a focal stimulating electrode placed in the stratum lacunosum (SLM) of the CA1 region^[@R7]^, where axons from LIII EC neurons are thought to provide the predominant source of excitatory inputs. Our present results, combined with recent results^[@R13],[@R14]^ suggest that these synaptic responses recorded in CA2 PNs may result from activation of LII fibers that course through or near SLM in CA1. Output projections from CA2 were determined by expressing YFP in CA2 PNs (as in [Fig. 1](#F1){ref-type="fig"}) and examining brains for YFP fluorescent axons. Unilateral viral injections resulted in strong bilateral labeling in hippocampal CA1, CA2, and CA3 regions, with densest projections observed in stratum oriens (SO) and weaker projections detected in stratum radiatum (SR) ([Fig. 2h, i](#F2){ref-type="fig"}). We did not observe extra-hippocampal outputs. These anatomical results generally support previous^[@R13],[@R14]^ findings. However, we failed to observe vasopressinergic input to CA2 from the paraventricular nucleus of the hypothalamus^[@R13]^, which may reflect an inability of the transsynaptic rabies tracing system to label peptidergic inputs^[@R15]^. We also did not observe CA2 output to the supramammillary nucleus^[@R13]^, suggesting this output may represent an inhibitory projection from CA2 as we selectively labeled PNs. Moreover, we did not observe CA2 output to EC layer II^[@R16]^, perhaps because the anterograde tracing failed to detect weak connections. To examine directly the functional and behavioral relevance of CA2, we employed the Amigo2-Cre mouse line to inactivate output from CA2 PNs selectively. We injected into the dorsal hippocampus of the Amigo2-Cre mice a Cre-dependent AAV to express tetanus neurotoxin (TeNT) light chain fused to green fluorescent protein (eGFP-TeNT) in CA2 PNs to block their synaptic output. We first verified the efficacy of this approach and characterized the influence of CA2 on its CA1 PN targets by co-expressing the light-activated cation channel channelrhodopsin-2 (ChR2)^[@R17]^ with either TeNT or YFP using Cre-dependent AAVs. Low intensity illumination (using 2-ms pulses of 470 nm light at 3 mW·mm^−2^) focused on CA2 reliably triggered action potentials in CA2 PNs, as seen by the presence of fast action currents in cell-attached patch clamp recordings ([Fig. 3a-c](#F3){ref-type="fig"}). Similar spiking was seen in neurons that co-expressed either YFP ([Fig. 3b](#F3){ref-type="fig"}) or TeNT ([Fig. 3c](#F3){ref-type="fig"}) with ChR2, indicating that the TeNT did not inhibit excitability. Next, we determined the strength of synaptic transmission from CA2 to CA1 PNs using whole-cell current-clamp recordings to measure light-evoked postsynaptic potentials (PSPs) in CA1 PNs from hippocampal slices in which ChR2 and YFP were expressed in CA2 PNs ([Fig. 3d](#F3){ref-type="fig"}). In agreement with anatomical mapping ([Fig. 2h, i](#F2){ref-type="fig"}) and paired recordings^[@R7]^, focal photostimulation delivered to CA1 SO and SR regions evoked robust monosynaptic PSPs (mean latency 1.22 ± 0.06 ms, n = 119 observations) in nearby CA1 PNs ([Fig. 3e](#F3){ref-type="fig"}). Increasing the light intensity recruited progressively larger PSPs, presumably due to an increase in the number of optically-activated CA2 axons ([Fig. 3e, f](#F3){ref-type="fig"}). In stark contrast, in slices in which TeNT was co-expressed with ChR2 in CA2 PNs, illumination over a wide range of intensities produced little or no synaptic response in CA1 neurons ([Fig. 3e, f](#F3){ref-type="fig"}), demonstrating the efficacy of the TeNT lesion. What are the behavioral consequences of inactivation of CA2? To address this question, we compared the behavior of control mice (CA2-YFP) with mice in which CA2 PNs were inactivated (CA2-TeNT), using viral injections in dorsal hippocampus^[@R11]^. Functional inactivation of dorsal CA2 did not alter locomotor activity or anxiety-like behavior ([Extended Data Fig. 5](#F9){ref-type="fig"}). Surprisingly, CA2-inactivation also did not significantly alter hippocampal-dependent spatial memory assessed by the Morris water maze (although there was a trend for the CA2-inactivated mice to learn the task more slowly; [Extended Data Fig. 6](#F10){ref-type="fig"}). Nor was there any change in hippocampal-dependent contextual fear memory or amygdala-dependent auditory fear memory ([Extended Data Fig. 7](#F11){ref-type="fig"}). The finding that CA2 PNs integrate synaptic input from lateral EC (which conveys non-spatial information^[@R18]^) with subcortical input from both the serotonergic median raphe nucleus^[@R19]^ and the	{ "pile_set_name": "PubMed Central" }
Introduction {#Sec1} ============ Water is a seemingly simple yet practically complex liquid with extraordinary phase behavior, which enables many of life's intricacies. While water is possibly the most studied liquid, there remain many areas where its behavior is still mysterious^[@CR1]^. A prime example for this is the freezing and the supercooling of water occurring in our daily lives and scientific research^[@CR2],[@CR3]^. Ice formation and the preceding supercooled state of microdroplets in atmospheric clouds are crucial elements for precipitation and reflection of solar radiation^[@CR4][@CR5]^. Furthermore, chilling, freezing, freeze avoidance, and supercooling are important strategies to combat cold environment for ectothermic animals^[@CR6],[@CR7]^, treat malignant diseases via cryotherapy^[@CR8]^, and preserve food and various biological samples, such as cells, tissues, and organs^[@CR9],[@CR10]^. Recent advances have shown that supercooling can be a promising alternative approach for the preservation of cells, tissues, and especially organs^[@CR11]^. Nevertheless, an important hurdle for supercooling preservation, as well as other applications of supercooling, is that simultaneous low temperature (\<−10 °C), large volume (\>1 ml), and long period (\>1 week) of supercooling for aqueous solutions cannot be readily achieved^[@CR12]--[@CR14]^. High-pressure-based approaches have provided supercooled states of water down to −92 °C briefly^[@CR1]^, according to the water phase diagram. They are, however, expensive, might further complicate preservation of biological samples, and their long-term fate is unknown. Few experiments have unstably supercooled large volumes, several hundred milliliters, of water to −12 °C^[@CR15]^, albeit also for periods on the order of seconds. Similarly, in Dorsey's classical work on freezing of supercooled water, he was able to achieve a temperature of −19 °C for a few milliliters of water very briefly during his constant cooling experiments^[@CR16]^. A method that overcomes these hurdles and enables long-term supercooling of large aqueous samples at low temperatures could find applications in biopreservation, as well as many other areas which have previously been practically prohibited. Under normal atmospheric conditions, ice melts at 0 °C, the ice-water equilibrium temperature (T~e~). Nevertheless, the observed freezing temperature (T~f~) for pure water could fall below T~e~ since successful ice nucleation at 0 °C can take a long time. Water, in the liquid phase, below the equilibrium temperature is said to be "supercooled" where ΔT = T~e~ − T~f~ measures the degree of supercooling. Supercooled water is intrinsically metastable and can spontaneously transform to lower-energy-level ice crystals through the formation of ice nuclei, which can be readily achieved by ice seeding^[@CR17]^, ultrasonicating^[@CR18]^, or presenting ice-nucleating agents^[@CR19]^. On the contrary, it is very difficult to maintain supercooled water unfrozen, especially for a large volume, under a high degree of supercooling, or for a long period, as each of these increases the possibility of ice nucleation and water freezing (Supplementary Note [1](#MOESM1){ref-type="media"}). For instance, ΔT of a water droplet decreases logarithmically with increasing volume under a constant cooling rate^[@CR20]^. Similarly, supercooling frequency (f~s~, f~s~ = number of unfrozen droplets/number of total droplets) of an ensemble of droplets decreases exponentially with increasing droplet volume, storage time, and nucleation rate (J)^[@CR21],[@CR22]^, while J itself increases exponentially with ΔT^[@CR23]^. Consequently, simultaneous long-term (\>1 week), large volume (\>1 ml), and deep supercooling (DSC) (ΔT \> 10 °C) of water has not yet been achieved. There are two general ice nucleation mechanisms, homogenous and heterogeneous crystallization. Homogeneous crystallization occurs due to random aggregation of interior water molecules to create a critically large nucleus of ice crystal, which could only be achieved and observed below −20 °C^[@CR24]^. Heterogeneous crystallization, on the other hand, stems from ice nucleus formation catalyzed by a substrate and/or with the aid of foreign objects at much higher temperatures^[@CR25]^. Consequently, water freezing is generally initiated by heterogeneous nucleation, and the water/air interface is the primary nucleation site as revealed in theoretical^[@CR26],[@CR27]^, experimental^[@CR14],[@CR28]^, and numerical^[@CR29],[@CR30]^ studies. Here, we describe an unexpected method based on sealing of the water surface by an immiscible hydrocarbon-based liquid, such as oils, pure alkanes, and pure primary alcohols. This method, as we demonstrate through a series of experiments, enables stable supercooling of large volumes of water for long periods at temperatures well below −10 °C by eliminating the primary ice nucleation site on the water/air interface. The supercooled water can withstand vibrational and thermal disturbances with all sealing agents, and even ultrasonic disturbance if it is sealed by alcohols. In addition, we utilize this DSC approach to preserve human red blood cells (hRBCs) for as long as 100 days. Results {#Sec2} ======= Water deep supercooling via surface sealing {#Sec3} ------------------------------------------- When water molecules aggregate on the water surface (water/air interface) to form an ice nucleus, they need to overcome an energy barrier γ^ia^ − γ^wa^ (γ: interfacial tension, symbols i, w, and a refer to ice, water, oil, and air, respectively) per unit area as the ice/air interface replaces original water/air interface. In comparison, the energy barrier for homogeneous ice nucleation within bulk water is proportional to the water/ice interfacial tension, γ^wi^. This interfacial tension can be expressed via the Young's equation as γ^wi^ = γ^ia^ − γ^wa^ cosθ~iwa~ ≥ γ^ia^ − γ^wa^ (θ~iwa~: water contact angle on ice/water/air interface, Supplementary Note [1](#MOESM1){ref-type="media"} and Supplementary Figure [1a](#MOESM1){ref-type="media"}). This inequality indicates that heterogeneous ice nucleation on the surface is thermodynamically more favorable than homogeneous nucleation in bulk as complete wetting (θ~iwa~ = 0°) is not generally observed^[@CR27]^, and a receding contact angle of 12° has been reported^[@CR31]^. Therefore, if the water surface is sealed by an oil phase, the energy barrier of ice nucleation at the water-oil interface would be γ^io^ − γ^wo^ (symbol o refers to oil phase). Similarly, the homogenous nucleation energy barrier can be now expressed in terms of another triple interface, namely the oil/water/ice as γ^wi^ = γ^io^ − γ^wo^ cos θ~iwo~, where θ~iwo~, for many oils can be nearly 0° as they are very repellent to ice (Supplementary Note [1](#MOESM1){ref-type="media"} and Supplementary Figure 1b)^[@CR28],[@CR32]^. In the case of θ~iwo~ ≅ 0, the energy barrier approaches the limiting case γ^io^ − γ^wo^ ≅ γ^wi^. This analysis indicates that the energy barrier of heterogeneous crystallization at the surface is elevated almost to the level of homogeneous one when the water/air interface is replaced by an oil/water interface. Accordingly, we hypothesized that surface sealing of water with an appropriate oil phase could suppress primary heterogeneous ice nucleation at the surface and enable extended storage of deeply supercooled water. Water deep supercooling via surface sealing with oils {#Sec4} ----------------------------------------------------- First, we cooled a large ensemble of polystyrene tubes containing 1 ml of ultra-pure water to −13 °C (Fig. [1a, b](#Fig1){ref-type="fig"}). This resulted in \>90% of samples to be frozen after 24 h and nearly all samples to be frozen after 5 days. In contrast, the ultra-pure water samples could be kept in the liquid phase for a week, at the same temperature, if their surfaces were sealed by various types of immiscible oils, such as light mineral oil (MO), olive oil (OO), heavy paraffin oil (PO), and nutmeg oil (NO). Interestingly, the curdling of OO during DSC does not trigger water freezing, though the cumulative freezing frequency (f~f~, f~f~ = 1 − f~s~) increases significantly compared to water sealed by other oils (Fig. [1a](#Fig1){ref-type="fig"}). In supplementary experiments, we observed that the water degassed by vacuuming for 24 h, has similar f~f~ as normal water, with or without oil sealing (Supplementary Figure [2](#MOESM1){ref-type="media"}). These experiments indicate that air dissolved in the water does not play a major role in ice nucleation in our experiments. Given this result and the consistent efficacy of surface sealing by different oils on freezing reduction, we infer that the air	{ "pile_set_name": "PubMed Central" }
1.. Introduction {#sec1} ================== The technique of single-crystal X-ray diffraction enables the routine and precise structure determination of biological macromolecules at high resolution (Blow, 2002[@bb9]; Rupp, 2010[@bb51]). It has been applied extensively to proteins, DNA and RNA, with the PDB recently celebrating the amazing milestone of 100 000 deposited structures. This is essentially the result of a multitude of innovations and technological developments spanning the last few decades (Abola et al., 2000[@bb1]; Fersht, 2008[@bb22]). Nonetheless, behind this success hides the struggle to produce purified samples, to obtain crystals with suitable diffraction quality and to subsequently reproduce/optimize them (Bergfors, 2009[@bb7]; Khurshid et al., 2014[@bb31]). The poor yield of suitable crystals can be explained by the concept called the 'curse of dimensionality': there are so many dimensions associated with the parameter space to be explored that it is problematic or impossible to perform an analysis that has any statistical significance. The underlying reason is that the combinations of reagents employed in crystallization trials alter the combinations of variables associated with the main parameters of crystallization (McPherson et al., 1995[@bb39]); for example, the variables that are related to the nature of the protein and the experiment, the type of protein--protein interactions and so on. Hence, initial crystallization screening is a stochastic process (Carugo & Argos, 1997[@bb13]; Lomakin et al., 1999[@bb33]) that usually requires various approaches and conditions. A crystallization condition traditionally contains a precipitant, a buffer and an additive. There are now hundreds of well known crystallization reagents and the possible combinations used to formulate conditions in a systematic manner has grown to a very large number that cannot be captured in any practical way because the amount of sample and the screening technology are limiting (Carter & Carter, 1979[@bb12]; Gorrec, 2014[@bb28]). As a consequence, many laboratories have chosen an approach with a minimum number of conditions. A widespread minimal approach is to employ a set of conditions selected empirically to form a 'sparse-matrix' screen (Jancarik & Kim, 1991[@bb30]). In the last two decades, advances in automation and the increase in the number of crystal structures solved and deposited have stimulated the optimization of sparse-matrix screens, mostly in the form of sets of 96 conditions, as this is an automation-friendly format (Kimber et al., 2003[@bb32]; Rupp & Wang, 2004[@bb52]; Newman et al., 2005[@bb55]; Stock et al., 2005[@bb54]; Newstead et al., 2008[@bb42]; Fazio et al., 2014[@bb21]). Nevertheless, we have argued that the minimal approach may mean undersampling of conditions and therefore essential hits could be missed (Gorrec, 2013[@bb27]). Subsequently, novel formulations should still be investigated when possible. Besides, screens should evolve in parallel with the increasing complexity of the samples and the technical difficulties encountered during the process of structure determination. Notably, the demands of cryo-crystallography (Petsko, 1975[@bb46]; §[](#sec3.1.1){ref-type="sec"}3.1.1) as well as current and future solutions to the phase problem (Taylor, 2003[@bb56]; §[](#sec3.1.2){ref-type="sec"}3.1.2) should be taken into account. Previously, we presented an innovative approach in a screen formulation called MORPHEUS (Gorrec, 2009[@bb26]). In order to reduce bias towards a subset of samples, the conditions were formulated de novo by integrating a larger number of reagents than traditionally employed. For this, novel mixes of reagents were investigated. Reagents that aided protein stabilization, crystallization and crystal cooling were included in the final formulation. The multiplexing of reagents has been performed previously for cryoprotectants (Garman & Mitchell, 1996[@bb24]), precipitants (Majeed et al., 2003[@bb36]), buffer systems (Newman, 2004[@bb40]) and additives (McPherson & Cudney, 2006[@bb38]) (§[](#sec3.1.3){ref-type="sec"}3.1.3). The original MORPHEUS combined all of these innovations. It is worth highlighting two other particular design principles of MORPHEUS. Firstly, the inclusion of PDB-derived small molecules (as potential ligands) that were gathered into mixes of additives, sorted according to their chemical nature to avoid incompatibilities. Secondly, mixes of precipitants, additives and buffers were combined within a 96-condition three-dimensional grid screen using fixed ratios to facilitate easier screen preparation and follow-up optimizations. Here, we present MORPHEUS II, a 96-condition protein crystallization screen formulated in continuity with previous work. MORPHEUS II follows the design principles of MORPHEUS; however, we introduced less common additives, for example metals that are amendable to the collection of anomalous data sets directly from screening conditions. We also included nondetergent sulfobetaines (NDSBs), polyamines, amino acids and monosaccharides, which are known to enhance the solubility and stability of many proteins. To complete the formulation of MORPHEUS II, four unusual 'glycerol-like' polyols have been added as cryoprotectants to aid flash-cooling. Finally, innovative buffer systems were included as part of the formulations. The suitability of the resulting conditions is shown by the crystallization of eight different protein samples and their efficiency is compared with commercially available conditions (§[](#sec3.2){ref-type="sec"}3.2). 2.. Materials and methods {#sec2} =========================== 2.1.. Screen formulation {#sec2.1} -------------------------- The mixes of ligands, precipitants and buffers were combined using a fixed ratio of volumes for the stock solutions as employed in the original MORPHEUS screen: 0.5 stock precipitants + 0.1 stock additives + 0.1 stock buffer system + 0.3 water. Methods used to select the PDB-derived ligands, to design the screen and to prepare the stock solutions were also as described previously (Gorrec, 2009[@bb26]). Further details can be found in the Supporting Information concerning the four precipitant mixes (Supplementary Table S1) and the three buffer systems (Supplementary Table S2). Although the additive-to-protein ratio preferably needs to be maximized (Danley, 2006[@bb17]), the concentrations of the mixes Divalent cations II, Akalis, Oxometalates and Lanthanides had to be lowered compared with other, more soluble and less reactive additives, such as monosaccharides and carboxylic acids. Relatively low concentrations (around 1 mM) are suitable for these particular additives according to others (Petsko, 1985[@bb47]; Trakhanov & Quiocho, 1995[@bb59]). By empirical experimentation, stable and suitable combinations of reagents were found. Unfortunately, some traditional heavy atoms had to be excluded, such as those of the platinum group (chloride salts of platinum, osmium, iridium, ruthenium, rhodium and palladium) since they could not be solubilized and/or were unstable in solution. Of course, they can still be tested later on crystals already formed when necessary. The buffer system was removed from conditions B5--B8 to avoid precipitation (probably owing to formation of a chelate between a divalent cation and one of the corresponding buffers). Also following an empirical approach, it was found that four small polyols that are not currently found in any commercially available screens vitrified samples during flash-cooling as efficiently as glycerol (i.e. typically 20--25% required to cryoprotect conditions): 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,5-pentanediol and 1,1,1-tris(hydroxymethyl)propane. They were thus integrated into the precipitant mixes (Table 1[▸](#table1){ref-type="table"} and Supplementary Table S1). X-ray diffraction tests with the mixes of polyols and polyethylene glycols (PEGs) flash-cooled in cryoloops were then used to adjust the concentration of cryoprotectants and ensured that the resulting diffraction patterns were free of background from ice. NDSBs are another group of reagents often used in sample preparation and crystallization additive screens. They have a relatively low frequency of occurrence in crystal structures, which suggests that their role may be less specific and therefore they have been integrated to the precipitant mixes. 2.2.. Crystallization experiments {#sec2.2} ----------------------------------- The protein samples can be briefly described as follows: concanavalin A ('Con', molecular weight 27 kDa, concentration 13 mg ml^−1^, Sigma catalogue No. L7647 dissolved in 0.1 M Tris pH 8.5), polymerase III clamp--exonuclease complex ('Pol', 80 kDa, 10 mg ml^−1^; Rêgo et al., 2013[@bb58]), ESCRT-II complex ('E2H', 115 kDa, 7 mg ml^−1^; Teo et al., 2004[@bb57]), bar domain ('Bar', 6 mg ml^−1^, 29 kDa; Peter et al., 2004[@bb45]), HIV capsid ('HIV	{ "pile_set_name": "PubMed Central" }
The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. Introduction and background =========================== Baastrup\'s disease was first described in 1933 as a cause of postural back pain, which was thought to be related to the adjacent \'kissing\' of the spinous processes \[[@REF1]\]. The diagnosis was based on \'symptoms\' of positional back pain with extension, and plain radiographs showing close approximation of the spinous processes (Figure [1](#FIG1){ref-type="fig"}). ![Sagittal computerized tomography (CT) scan showing \'kissing\' spinous processes\ Sagittal lumbar CT shows the close approximation and common hyperostosis seen with the spinous processes of Baastrup\'s disease (thick white arrows). Also shown is associated disc space narrowing at multiple levels especially L4-5 and L5-S1 (thin white arrows) and \'vacuum\' phenomenon characteristic of advanced disc degeneration (dashed white arrows) with disc necrosis and intradiscal clefts at L4-5 greater than at L5-S1.](cureus-0009-00000001449-i01){#FIG1} Gradually throughout the years, individual case reports in patients with clinical symptoms of Baastrup\'s disease were published describing ligamentous stenosis and large dorsal epidural cysts causing neurogenic claudication, as well as interspinous fluid collections believed to be related to the epidural cyst. Review of these individual case reports clearly demonstrates a spectrum of abnormalities involving weakening of the interspinous ligament that can lead to the formation of interspinous fluid clefts, facet and dorsal epidural cysts, intervertebral disc narrowing, and spinal instability with anterolisthesis and ligamentous posterior spinal stenosis \[[@REF2]-[@REF3]\]. By relating the biomechanical role of the interspinous and supraspinous ligaments and the different types of radiologic findings seen in Baastrup\'s disease, interspinous bursitis and segmental instability this review highlights the underlying interrelationship of what is often different stages of the same lumbar segmental degeneration. Review ====== Biomechanical studies show that the interspinous ligament works in combination with the supraspinous ligament and the ligamentum flavum to provide flexion resistance to the lumbar spine \[[@REF4]\]. The interspinous ligament is one of the mechanisms that maintains sagittal stability of the spinal segment. Inflammatory reaction with fluid and cyst formation within the interspinous ligament can result from chronic repetitive weakening and stretching of the ligament. The ventral area near the posterior epidural space is the weaker section and where cysts develop \[[@REF5]\]. Degeneration and weakness of this ligament leads to spinous process approximation, but more importantly a tendency to develop instability and anterolisthesis, or forward shifting of the superior vertebrae over the inferior vertebrae, at the involved segment \[[@REF6]\]. The different pathologic changes identified on magnetic resonance imaging (MRI) scans with both Baastrup's disease, interstitial bursitis and dorsal epidural cysts are part of the degenerative and biomechanical process that occurs after the interspinous and supraspinous ligaments deteriorate and lose tensile strength \[[@REF7]\]. There are obviously very few pathologic studies of Baastrup\'s disease but bursas are commonly found in the interspinous space. In autopsies, interspinous cysts with inflammatory changes, bone erosion, and bone hypertrophy have been frequently described and are significantly more common with advanced age, being found in up to 40% of specimens \[[@REF8]\]. The actual contact of the spinous processes seen on plain radiographs as the basis for diagnosing Baastrup\'s disease may often be a minor part of all the patient\'s radiologic findings and may be indicative of underlying segmental degeneration. Computerized tomography (CT) scans show bony hypertrophy of the touching spinous processes combined with reactive sclerosis as well as facet joint hypertrophy \[[@REF9]\]. As MRI became the standard imaging technique of the spine, intraspinal cysts and other facet pathology began to be 'associated' with interspinous fluid bursas. There is a wide range of different bursas and cysts seen in these patients commonly identified in the facet joints. MRI scans reveal a spectrum of abnormalities in the interspinous ligament from fluid cystic change, thickening of the adjacent ligamentum flavum, and anterolisthesis of the involved vertebrae. There was often multilevel disease on MRI. Bursas and clefts were most commonly seen at L4-5, but also seen at L3-4 and L5-S1. Connections between these bursas and the epidural space have been found in 50% of the cases \[[@REF9]\]. Incorporating the biomechanics together with the radiologic findings, we hypothesized that repetitive shear due to weakening of the interspinous and supraspinous ligaments leads to the development of interspinal adventitious bursas and cysts, and extension of an inflammatory process within the ligamentum flavum. This progressive degenerative process contributes to the frequently observed soft tissue canal stenosis reported in many cases, although many patients present initially with localized positional lumbar pain and not neurogenic claudication (Figure [2](#FIG2){ref-type="fig"}). ![Interspinous fluid with L4-5 one level stenosis on T2 sagittal magnetic resonance imaging (MRI) studies\ A: L5-S1 large interspinous hyperintense fluid (two solid white arrows) with no ligamentous enlargement. L4-5 minimal ligamentous enlargement posteriorly (dashed white arrow) with early canal stenosis. L4-5 intervertebral disc has early signs of T2 signal desiccation.\ B: Narrowed and dessicated intervertebral disc at L4-5 with annular bulge (dashed black arrow) with minimal dorsal posterior spinal cyst (dashed white arrow) causing posterior central canal stenosis connected to interspinous fluid (solid white arrows).\ C: Grade 1 spondylolisthesis at L4-5 (dashed black arrow), posterior fibrous ligamentum flavum hypertrophy (dashed white arrow) and fluid in interspinous space (solid white arrows).\ D: Degenerated narrowed L4-5 intervertebral disc with annular bulge and grade 1 spondylolisthesis (superior dashed black arrow). Grade 2 spondylolisthesis at L5-S1 with marked endplate degeneration (inferior dashed black arrow). Posterior interspinous cyst (solid red arrow) with marked ligamentous hypertrophy and stenosis at L4-5 (two dashed white arrows). Marked edema and displacement of cauda equina roots.](cureus-0009-00000001449-i02){#FIG2} Originally, due to Baastrup\'s description relating the problem to the spinous processes, treatment was directed to the interspinous abnormality seen on plain radiographs and originally consisted of surgery for removal of the spinous process with poor and inconsistent results. Beks reviewed 64 patients who had spinous process resection with very poor results \[[@REF9]\]. Only 11/64 patients reported pain relief. Radiographic studies in the form of x-rays (i.e., no CT or MRI) were retrospectively reviewed and showed that all 53 of the failed surgical patients had other spinal pathology including lumbar spondylosis in 55%, disc degeneration in 23%, and spinal stenosis in 13%. He correctly attributed the unrecognized pathology to be the reason resection of the spinous process was unsuccessful. The conclusion was Baastrup\'s disease is not a disease entity by itself; rather it is a result of mechanical changes in the interspinous and supraspinous ligaments, degenerative lumbar discs, and facets with interspinous bursas, osteophyte formation, and spondylosis \[[@REF2]\]. Initially, individual case reports were published of finding epidural cysts and masses causing stenosis associated with Baastrup's disease, but it was later recognized that these two problems were much more commonly associated \[[@REF10]-[@REF11]\]. Chen, et al. reviewed 10 cases of posterior dorsal intraspinal cysts and noted concurrent degenerative disc disease and variable degrees of stenosis, spondylolisthesis at or below the level of the cyst with marked facet degeneration, and three patients had facet joint effusions \[[@REF12]\]. They concluded that Baastrup\'s disease is associated with interspinous fluid and if the fluid bursa is large enough it could extend into the posterior epidural space causing canal stenosis. They also noted that not all cysts communicate with the facet joints or dorsal epidural space. MRI also showed severe stenosis due to a posterior non-cystic fibrous mass often with a linear fluid signal from the interspinous space. In surgery, an interspinous cleft was found and easily probed without excising the interspinous ligament. The histology reports showed the composition to be a collagen matrix mass with peripheral inflammation. They suggested that the Baastrup\'s cyst may change over time becoming more fibrous. Kwong, et al. performed another radiologic retrospective review of 1008 CT scans and found that 41% had evidence of Baastrup\'s disease, most commonly at L4-5, and the frequency of this finding increased with	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1-nanomaterials-09-01643} =============== The wide-ranging use of antibiotics, dyes, and heavy metals and their reckless release in water has drawn intensive attention due to their toxicity and non-biodegradability \[[@B1-nanomaterials-09-01643],[@B2-nanomaterials-09-01643],[@B3-nanomaterials-09-01643]\]. In recent years, many strategies, such as electrochemical oxidation \[[@B4-nanomaterials-09-01643]\], coagulation and flocculation \[[@B5-nanomaterials-09-01643]\], adsorption \[[@B6-nanomaterials-09-01643]\], membrane filtration \[[@B7-nanomaterials-09-01643]\], and advanced oxidation \[[@B8-nanomaterials-09-01643]\], have been applied for water purification. Photocatalysis, a typical advanced oxidation technique, has become the route with the most potential to conquer these intensifying environmental problems via the utilization of solar light \[[@B9-nanomaterials-09-01643],[@B10-nanomaterials-09-01643],[@B11-nanomaterials-09-01643]\]. Hence, various semiconductors, such as g-C~3~N~4~, TiO~2~, ZnS, ZnO, CuS, and MoS~2~, have been confirmed as cost-effective photocatalysts for the heterogeneous photocatalytic purification of polluted water \[[@B12-nanomaterials-09-01643],[@B13-nanomaterials-09-01643],[@B14-nanomaterials-09-01643],[@B15-nanomaterials-09-01643],[@B16-nanomaterials-09-01643],[@B17-nanomaterials-09-01643]\]. Due to their wide band gap, nontoxicity, and high stability, cerium dioxide (CeO~2~) and TiO~2~ have been widely applied in photocatalytic reactions \[[@B18-nanomaterials-09-01643],[@B19-nanomaterials-09-01643],[@B20-nanomaterials-09-01643]\]. In addition, CeO~2~ exhibits strong UV-light sorption capacity and a high resistance to photocorrosion \[[@B21-nanomaterials-09-01643]\]. The adsorption and desorption capacities of oxygen ions are critical for the catalytic process of CeO~2~. The oxygen storage capacity of CeO~2~ is greatly affected by the redox activity of the Ce(III)/Ce(IV) coupling, further depending on the type and content of oxygen vacancies in the lattice structure \[[@B22-nanomaterials-09-01643]\]. The defect structure of Ce^3+^--O~v~--Ce^4+^ (O~v~- oxygen vacancy) and the formation of Ce^3+^ ions induce a red shift in the band gap of CeO~2~ \[[@B23-nanomaterials-09-01643]\]. In fact, the crystal defect of CeO~2~ is greatly related to its crystal structure and morphologies. Various microstructures of CeO~2~ have been fabricated for practical applications via different approaches in previous works, including bowknot-like crystallites, nanocubes, X-architecture, nanopolyhedra, square-like nanoparticles, nanosheets, nano-octahedrons, ribbon-like nanofibers, urchin-like hierarchical structures, flower-like microspheres, and well-aligned nanorod arrays \[[@B24-nanomaterials-09-01643],[@B25-nanomaterials-09-01643],[@B26-nanomaterials-09-01643],[@B27-nanomaterials-09-01643],[@B28-nanomaterials-09-01643],[@B29-nanomaterials-09-01643],[@B30-nanomaterials-09-01643],[@B31-nanomaterials-09-01643],[@B32-nanomaterials-09-01643],[@B33-nanomaterials-09-01643]\]. Unfortunately, CeO~2~ with indirect band gap energy (3.2 eV) is strictly limited in the photocatalytic system of the UV-light region. Hence, nonmetal or metal ions such as S, N, P, Er, Fe, Sm, and Y have been doped in CeO~2~ to extend light harvesting to the visible-light region, leading to enhanced photocatalytic activity \[[@B34-nanomaterials-09-01643],[@B35-nanomaterials-09-01643],[@B36-nanomaterials-09-01643],[@B37-nanomaterials-09-01643],[@B38-nanomaterials-09-01643],[@B39-nanomaterials-09-01643],[@B40-nanomaterials-09-01643]\]. Apart from a doping strategy, noble metals such as Au and Ag loaded on CeO~2~ have been confirmed to present much better photocatalytic activity \[[@B41-nanomaterials-09-01643]\]. In addition, the fabrication of heterojunctions coupled with other semiconductors can simultaneously enhance the separation efficiency of charge carriers and restrain the recombination rate of photoexcited electron--hole pairs through the interface structure of different semiconductors, especially core--shell structures \[[@B42-nanomaterials-09-01643],[@B43-nanomaterials-09-01643],[@B44-nanomaterials-09-01643]\]. To further avoid photocorrosion in solar energy-driven reaction systems, carbon has been employed for the synthesis of heterojunction composites due to its unique physicochemical properties and low cost \[[@B45-nanomaterials-09-01643],[@B46-nanomaterials-09-01643],[@B47-nanomaterials-09-01643],[@B48-nanomaterials-09-01643]\]. Carbon with a porosity structure and a high surface area not only exhibits excellent affinity for pollutant molecules, but also efficiently captures and transfers the photoexcited electron, leading to an enhancement in photocatalytic activity \[[@B49-nanomaterials-09-01643],[@B50-nanomaterials-09-01643]\]. However, the combined effect of Ag doping and carbon coating on the enhanced photocatalytic capacity of CeO~2~ nanosheets has been scarcely reported in previous works. This work focused on the effect of Ag quantum dots (QDs) on the photocatalytic activity of carbon-coated CeO~2~ (CeO~2~\@C) nanosheets in the visible-light region. In this strategy, Ag QDs were anchored in situ on CeO~2~\@C nanosheets to form Ag/CeO~2~\@C. The photocatalytic capacity of Ag/CeO~2~\@C was affected by the carbon dosage, the Ag-doping content, the Cr(VI) concentration, the pH value, and inorganic ions. The combined effects of the surface plasma resonance (SPR) of Ag QDs, an electron trapper of carbon shells, and the redox activity of the Ce(III)/Ce(IV) coupling were responsible for enhanced visible-light harvesting and efficient charge transfer and separation, leading to excellent photocatalytic activity in the CeO~2~ nanosheets \[[@B39-nanomaterials-09-01643],[@B41-nanomaterials-09-01643],[@B45-nanomaterials-09-01643],[@B51-nanomaterials-09-01643],[@B52-nanomaterials-09-01643]\]. The possible photocatalytic mechanism of Ag/CeO~2~\@C is discussed in detail. 2. Materials and Methods {#sec2-nanomaterials-09-01643} ======================== 2.1. Preparation of Catalysts {#sec2dot1-nanomaterials-09-01643} ----------------------------- CeO~2~ nanosheets were prepared via a hydrothermal route. Briefly, 1.0 mmol of cerium nitrate hexahydrate (Ce(NO~3~)~3~·6H~2~O) and 2 mmol of hexamethylenetetramine (C~6~H~12~N~4~) were dissolved in 70 mL of deionized water through vigorous stirring. Then, 3 mL of acetic acid (CH~3~COOH) was added to the above solution and stirred at room temperature for 2 h. This mixture was transferred to a 100 mL Teflon-lined autoclave and treated at 433 K for 9 h. After being cooled down to room temperature, the suspension was filtered, washed with ethanol and deionized water, dried at 333 K for 6 h, and calcined at 773 K for 3.0 h to obtain CeO~2~ nanosheets. Carbon-coated CeO~2~ (CeO~2~\@C) nanosheets were also synthesized through a hydrothermal route. Here, 0.1 g obtained CeO~2~ bulks, 0.2 g glucose (C~6~H~12~O~6~), and 1.0 g polyvinyl pyrrolidone (PVP, M = 58,000) were dispersed into 30 mL of deionized water through intensive stirring at room temperature for 2.0 h and then treated at 453 K for 15.0 h in a 50 mL Teflon-lined autoclave. After being cooled down to room temperature, the above suspension was centrifuged, washed, dried at 343 K for 5.0 h, and calcined at 773 K for 2.0 h in an N~2~ flow rate of 40 mL·min^−1^ to obtain CeO~2~\@C nanosheets (called CeO~2~\@C-1). With the above process, CeO~2~\@C composites with varying carbon contents were obtained with different mass ratios of CeO~2~/glucose. CeO~2~\@C-2 and CeO~2~\@C-3 were obtained via the addition of glucose contents of 0.4 g and 0.6	{ "pile_set_name": "PubMed Central" }
BoNTs are a family of bacterial toxins, classified as one of the six most dangerous potential bioterrorism agents (Category A and Tier 1 select agent in the United States)[@b1]. They are also widely used to treat a growing list of medical conditions[@b2][@b3], including muscle spasms, chronic pain, overactive bladders, as well as having cosmetic applications. There are seven well-established serotypes of BoNTs (BoNT/A--G), traditionally defined based on a lack of cross-neutralization by different antisera raised against each toxin type. All BoNTs share the same structure and function[@b4][@b5][@b6]. They are composed of a light chain (LC, ∼50 kDa) and a heavy chain (HC, ∼100 kDa) connected by an inter-chain disulfide bond. The HC contains two sub-domains: the C-terminal H~C~ that mediates binding to receptors, and the N-terminal H~N~ that mediates translocation of the LC across endosomal membranes. The LC acts as a protease in neurons to cleave a set of proteins: BoNT/A, C and E cleave at distinct sites on a peripheral membrane protein known as SNAP-25; BoNT/B, D, F and G cleave at different sites on homologous vesicle proteins VAMP1, 2 and 3 (vesicle-associated membrane proteins); and BoNT/C also cleaves the plasma membrane protein syntaxin 1. These proteins are prototypes of the SNARE (soluble NSF attachment protein receptor) protein family, whose members mediate various membrane fusion events in eukaryotic cells[@b7][@b8]. Cleavage of any one of the three neuronal SNARE proteins blocks fusion of synaptic vesicles to plasma membranes, thus preventing neurotransmitter release from neurons. Recognizing all distinct serotypes of BoNTs is essential for developing effective countermeasures against this family of toxins. BoNT/A and BoNT/B were first identified in 1919 by Georgina Burke[@b9]. The last of the seven serotypes, BoNT/G, was discovered in 1969 (ref. [@b10]), and no new BoNT serotype has been found for the past four decades. Recent progress in genomic sequencing has revealed multiple subtypes (designated with Arabic numbers, for example, BoNT/A1), which can be recognized by the same antiserum but contain substantial sequence variations[@b11][@b12][@b13]. Furthermore, there are also multiple mosaic toxins. For instance, a 'type H' was reported in 2013 but was later designated as a mosaic toxin, as its LC shares ∼80% identity with the LC of a BoNT/F subtype, BoNT/F5, and its H~C~ shares ∼84% identity with the H~C~ of BoNT/A1 (refs [@b14], [@b15], [@b16], [@b17]). Consistently, this toxin can be neutralized by antibodies against BoNT/A (ref. [@b16]). The genes encoding BoNTs can be on a plasmid, a phage, or the chromosome, indicating that these genes are mobile and capable of horizontal gene transfer[@b18][@b19][@b20][@b21][@b22][@b23]. Some strains contain two or even three different BoNT genes[@b15][@b24][@b25]. These strains are usually designated with a capital letter for the toxin type that is expressed at higher levels than the other one, followed with a lower case letter for the second toxin type (for example, BoNT/Af strain). In addition, it has also been reported that some BoNT/A strains contain a complete BoNT/B gene, but only BoNT/A is expressed[@b26][@b27][@b28]. Thus, the BoNT/B gene is considered a silent gene and the strains are known as BoNT/A(B) strains. A recent survey of infant botulism cases reported that ∼8% isolates are BoNT/A(B) strains[@b29]. Here we searched published genomic sequences and identified a novel BoNT gene encoded on the chromosome of Clostridium botulinum strain 111. This strain was originally identified from an infant botulism patient in Japan in 1996 (ref. [@b30]). The initial characterizations indicated that the toxicity of this strain is due to BoNT/B[@b30]. Later studies confirmed that this strain expresses a subtype of BoNT/B (BoNT/B2) encoded on a plasmid[@b31][@b32]. The sequence of this novel BoNT gene was deposited into the GenBank database in February 2015, as a part of the genomic sequence of C. botulinum 111. We characterized the protein encoded by this gene at functional levels and established it as a new BoNT serotype with a unique substrate profile. Results ======= Searching genomic databases revealed a novel BoNT gene ------------------------------------------------------ In an attempt to survey the evolutionary landscape of BoNTs, we performed iterative Hidden Markov model searches of the Uniprot sequence database. Our search identified all known BoNT subtypes and mosaic toxins, as well as the related tetanus neurotoxin ([Fig. 1a](#f1){ref-type="fig"}; [Supplementary Fig. 1](#S1){ref-type="supplementary-material"}). To our surprise, the search revealed a potentially new BoNT, tentatively designated BoNT/X ([Fig. 1a](#f1){ref-type="fig"}, GenBank no.: BAQ12790.1), from the recently reported genomic sequence of C. botulinum strain 111. BoNT/X showed the least protein sequence identity with the other BoNTs in pairwise comparisons ([Fig. 1b](#f1){ref-type="fig"}). Furthermore, the low sequence similarity is evenly distributed along the entire BoNT/X sequence ([Fig. 1c](#f1){ref-type="fig"}), indicating that it is not a mosaic toxin. Despite this low sequence identity, the overall domain arrangement of BoNTs is conserved in BoNT/X ([Fig. 1c](#f1){ref-type="fig"}), including a zinc-dependent protease motif HEXXH (residues 227--231, HELVH) in the LC (ref. [@b33]), and a SXWY motif in the H~C~ (residues 1,274--1,277, SAWY), which recognizes the lipid receptor gangliosides[@b34]. Similar to the other BoNTs, the BoNT/X gene is located in a gene cluster[@b23]. All seven established BoNTs are co-expressed with another 150 kDa protein known as NTNHA (non-toxic non-hemagglutinin protein), which forms a pH-dependent complex with BoNTs and protects them from proteases in the gastrointestinal tract[@b35]. The BoNT/X gene is also preceded by a potential NTNHA gene ([Fig. 1d](#f1){ref-type="fig"}). Besides BoNT and NTNHA, a typical BoNT gene cluster contains genes encoding one of the two types of accessory proteins: (1) the HA cluster encoding three conserved proteins HA17, HA33 and HA70, which form a complex with BoNT/NTNHA and facilitate absorption of toxins across the intestinal epithelial barrier[@b36][@b37][@b38]; or (2) the OrfX cluster encoding conserved OrfX1, OrfX2, OrfX3 and P47 proteins with unknown function[@b23]. The BoNT/X gene is located in an OrfX gene cluster, as are BoNT/E, F and members of BoNT/A. Interestingly, the BoNT/X cluster has two unique features ([Fig. 1d](#f1){ref-type="fig"}): (1) there is an additional OrfX2 gene that does not exist in any other BoNT clusters (we designated it OrfX2b); (2) the reading frame of OrfX genes is usually opposite to BoNT/NTNHA genes, but it has the same direction as the BoNT/X gene in the BoNT/X cluster ([Fig. 1d](#f1){ref-type="fig"}). These findings suggest that BoNT/X is a unique branch of the BoNT family. The LC of BoNT/X cleaves VAMP2 at a novel site ---------------------------------------------- To characterize BoNT/X, we first focused on its LC (X-LC, residues 1--439) and produced it as a His6-tagged protein in Escherichia coli. LCs of BoNT/A (A-LC) and BoNT/B (B-LC) were produced and assayed in parallel as controls. Incubation of X-LC with rat brain detergent extracts (BDE) did not affect syntaxin 1 or SNAP-25, but abolished VAMP2 immunoblot signals ([Fig. 2a](#f2){ref-type="fig"}). LCs of BoNTs are zinc-dependent proteases[@b33]. As expected, EDTA prevented cleavage of SNARE proteins by X-, A- and B-LCs ([Fig. 2a](#f2){ref-type="fig"}). Furthermore, incubation of X-LC with the purified recombinant cytosolic domain of VAMP2 (residues 1--93) converted VAMP2 into two lower-molecular-weight bands ([Fig. 2b](#f2){ref-type="fig"}), confirming that X-LC cleaves VAMP2. To identify the cleavage site, we analysed the VAMP2 (1--93) protein, with or without pre-incubation with X	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1-life-06-00037} =============== Halophilic microorganisms thrive in the briny waters of salt lakes and evaporative solar salterns \[[@B1-life-06-00037]\]. Saturated salinity, from 25%--35%, presents osmotic obstacles for life, but we see representatives from all three domains thriving in these conditions \[[@B2-life-06-00037]\]. Hypersaline habitats select for archaea at a higher abundance than bacteria and eukarya in the community \[[@B3-life-06-00037]\]. The present study focuses on this particular group, which likely share genetic strategies for life at high salinity \[[@B2-life-06-00037]\]. Microbial diversity studies show a wide array of halophilic archaea present in hypersaline ecosystems \[[@B4-life-06-00037]\] that are adapted to thrive in the saltiest places on Earth. Beyond salt, high solar radiation is a feature of these environments; thus, their microbial inhabitants must have evolved to overcome the challenge of ultraviolet (UV) exposure. Halophilic archaea are highly resistant to UV light, first noted by Dundas and Larsen \[[@B5-life-06-00037]\]. For example, one Halorubrum species was previously found to be nearly ten-fold more UV-resistant than Escherichia coli \[[@B6-life-06-00037]\]. This is due in part to their robust UV DNA repair systems, including photoreactivation and nucleotide excision repair \[[@B7-life-06-00037],[@B8-life-06-00037],[@B9-life-06-00037],[@B10-life-06-00037],[@B11-life-06-00037]\], but there are clearly additional processes that afford the high level of resistance to solar radiation exhibited by halophilic archaea. Several studies have proposed other potential photoprotective strategies; for example, carotenoids in the cell membranes of halophilic archaea may provide UV resistance \[[@B6-life-06-00037],[@B12-life-06-00037],[@B13-life-06-00037]\], although the mechanism of which remains unclear. These organisms have a unique composition of carotenoids that set halophilic archaea apart \[[@B14-life-06-00037],[@B15-life-06-00037],[@B16-life-06-00037],[@B17-life-06-00037]\]. In fact, some halophiles may have an array of genes that allow their complex interplay with light \[[@B18-life-06-00037]\], including carotenoid biosynthetic pathways, and also genes encoding retinal-containing proteins like bacteriorhodopsin, or "purple membrane" protein, which pumps protons out of the cell upon exposure to light \[[@B19-life-06-00037]\] and may aid in the generation of ATP. An environmental genomics approach to saltern communities found the presence of a large variation of rhodopsin-like genes \[[@B20-life-06-00037]\], indicating the significance of photobiology for halophiles. One photoprotective mechanism suggested in the literature for decades \[[@B22-life-06-00037],[@B23-life-06-00037],[@B24-life-06-00037],[@B25-life-06-00037],[@B26-life-06-00037]\] relates to the genomes of organisms with high Guanine+Cytosine (G + C) content, such as halophilic archaea. Their genomes typically exceed 60% G + C ([Table 1](#life-06-00037-t001){ref-type="table"}), which effectively reduces the number of thymines (Ts) present. Thymine dimers (T\^T), which form from the UV-induced cyclization of two adjacent thymines on the same DNA strand ([Figure 1](#life-06-00037-f001){ref-type="fig"}), were long thought to be the primary DNA damage arising from sunlight \[[@B23-life-06-00037]\]. Logically, the limitation of T residues is expected to reduce the incidence of adjacent Ts on the same DNA strand and thus, the possibility of T\^Ts. Reduction of DNA damage would reduce mutagenesis during replication of unrepaired lesions. Kennedy et al. \[[@B26-life-06-00037]\] suggested that this strategy of thymine limitation could explain the UV resistance of halophilic archaea, but the idea remains untested. The most important oversight in this premise is that T\^T lesions are but a subclass of cyclobutane pyrimidine dimers (CPDs), which include each possible adjacent bipyrimidine pair: (5' to 3') T\^T, C\^C, C\^T, and T\^C. Haynes \[[@B21-life-06-00037]\] noted that the sensitivity of microorganisms to UV light was not mathematically proportional to the thymine frequency in the genome, which pointed to other lethal lesions. Any set of two adjacent pyrimidines may cyclize upon exposure to a photon of UV light. Though CPDs represent the majority of solar-induced DNA damage events \[[@B28-life-06-00037]\], other potential lesions resulting from the UV-irradiation of bipyrimidine sequences are pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). The proportion of CPDs to 6-4PPs is dependent on wavelength of light \[[@B29-life-06-00037]\] and on flanking sequences \[[@B30-life-06-00037],[@B31-life-06-00037]\]. Though it is logical to assume T\^T lesions may be limited in halophilic archaea, other photolesions may form; therefore, the photoreactivities of each bipyrimidine pair and the relationship between G + C content and their incidences must be considered in order to assess any photoprotective benefit \[[@B32-life-06-00037],[@B33-life-06-00037]\]. Therefore, we sought to address the hypothesis that thymine limitation is photoprotective using a genome photoreactivity score that incorporates all bipyrimidine pairs and their respective photoreactivities, while accounting for all potential types of UV damage (e.g. CPDs and 6-4PPs). We developed an equation to quantify the theoretical photoreactivity of a genome based on the frequencies of each bipyrimidine doublet within it, weighted by their intrinsic photoreactivities (as determined by \[[@B33-life-06-00037]\]) and genome size. Comparison with other groups and a robust statistical analysis sheds light on an interesting story in genome evolution. 2. Materials and Methods {#sec2-life-06-00037} ======================== 2.1. Comparing G + C Content of Halophilic Archaea Versus Other Prokaryotes {#sec2dot1-life-06-00037} --------------------------------------------------------------------------- A list of all prokaryotes with full-genome sequences available (n = 5074) and their corresponding G + C contents was downloaded from the NCBI database \[[@B21-life-06-00037]\]. One representative genome for each species was selected at random, yielding sample groups of n = 29 halophilic archaea and n = 2231 other prokaryotes. 2.2. Genome Sampling {#sec2dot2-life-06-00037} -------------------- Tabulated lists of all species with full-genome sequences available were downloaded from the NCBI database \[[@B21-life-06-00037]\] for the sample groups bacteria (taxid: 2, n = 4829), halophilic archaea (taxid: 183963, n = 33), archaea excluding halophilic archaea (taxid: 2157, n = 209), cyanobacteria (taxid: 1117, n = 90) and enterobacteriaceae (taxid: 91347, n = 736). Steps were taken to minimize sample bias: for the halophilic archaea group, one representative strain from each species was selected at random, yielding a sample group of n = 29 halophilic archaea. For the archaea, cyanobacteria, and enterobacteriaceae groups, one representative from each genus was selected at random, yielding sample groups of n = 68 (non-halophilic) archaea, n = 32 cyanobacteria, and n = 42 enterobacteriaceae. For the bacteria, the first 101 strains of a unique genus to be randomly selected constituted the final sample group of n = 101 bacteria. The full-genome sequences corresponding to each sampled strain were downloaded as .fasta files from the NCBI database \[[@B21-life-06-00037]\]. 2.3. Determining Bipyrimidine Incidences {#sec2dot3-life-06-00037} ---------------------------------------- To determine the incidences (i.e., relative frequencies) of each bipyrimidine in the sampled genomes, a novel word-counting program "DinucleotideCounts" was written in the scripting language R (script available at: \[[@B34-life-06-00037]\]). This program determines the frequency of each dinucleotide, the frequency of each nucleotide, and the size of any .fasta-formatted DNA sequence. The bipyrimidine frequencies within sampled genomes were determined via the DinicleotideCounts program. Bipyrimidine incidences (TC~i~, TT~i	{ "pile_set_name": "PubMed Central" }
Introduction ============ Hematopoietic stem cells (HSC) reside in a special bone marrow (BM) niche, which regulates their localization, self-renewal and differentiation. Studies have identified several major cell types of the niche, including mesenchymal stem cells (MSC), osteolineage cells (OLC), adipocytes and vascular endothelial cells (EC).^[@b1-1031969]--[@b5-1031969]^ Besides the key cellular components, some growth and survival factors are also indispensable components of the niche, including C-X-C motif chemokine 12 ligand (CXCL12), vascular cell adhesion molecule1 (VCAM1),^[@b6-1031969],[@b7-1031969]^ stem cell factor (SCF)^[@b4-1031969]^ and osteopontin.^[@b8-1031969]^ A sophisticated network of interactions between these multiple BM cells, extracellular factors and adhesion molecules is essential to regulate different HSC properties during homeostasis and keep normal hematopoiesis in check. Acute myeloid leukemia (AML) has been considered for decades to be a disorder intrinsic to hematopoietic cells; however, evidence is accumulating that the microenvironment exerts more than a mere bystander effect. Leukemic cells can remodel the niche into a permissive environment favoring leukemic stem cell (LSC) expansion over normal HSC maintenance.^[@b9-1031969]^ Recently, emerging evidence even points to a role for the BM niche as a driver of disease maintenance/progression. Krause et al. showed that osteoblast-specific activation of the parathyroid hormone receptor enhances MLL-AF9 oncogene-induced AML in mouse transplantation models.^[@b10-1031969]^ To date, there are still few studies concerning the role of the bone marrow niche in initiating and maintaining AML and relevant mechanisms remain elusive. TWIST1 is a highly conserved transcription factor belonging to the basic helix-loop-helix family and is implicated in diverse developmental systems.^[@b11-1031969]--[@b13-1031969]^ Studies have revealed that TWIST1 is a key regulator of MSC self-renewal, enhances their life-span, inhibits MSC osteo/chondrogenic differentiation and promotes adipogenic differentiation.^[@b14-1031969]--[@b16-1031969]^ Twist1 haploisufficiency leads to Saethre-Chotzen syndrome, which is characterized by alterations in osteogenic precursor cell proliferation, differentiation and survival.^[@b17-1031969]^ Recent studies have demonstrated that TWIST1 promotes angiogenesis by inducing EC proliferation and migration, and deregulation of this mechanism mediates pathological angiogenesis.^[@b18-1031969],[@b19-1031969]^ Arthur et al. showed that overexpression of Twist1 in MSC enhances the capacity to maintain human CD34^+^ cells in long-term culture-initiating cell assays through increasing Cxcl12 expression.^[@b20-1031969]^ However, the effects of TWIST1 on multiple niche elements and its modulation of normal HSC maintenance and leukemia progression in vivo have not been functionally characterized so far. To explore this issue, we generated a murine model of a Twist1-deficient microenvironment. We showed that the major niche cellular components and factors changed remarkably upon Twist1 deletion, causing severe dysfunction of normal HSC. Nevertheless, these alterations of the BM microenvironment promoted MLL-AF9 oncogene-induced AML progression in mouse transplantation models, not only pointing to TWIST1 as an instructive signal modulating the stem cell niche, but also emphasizing the importance of the niche for AML development. Methods ======= Mice ---- Twist1^flox/flox^ mice were purchased from Mutant Mouse Regional Resource Centers. ER-Cre mice were a gift from Professor Weiping Yuan. C57BL/6 and B6.SJL mice were purchased from the animal facility of State Key Laboratory of Experimental Hematology. Twist1^flox/flox^ mice were crossed with ER-Cre mice to generate ER-Cre;Twist1^fl/fl^ and ER-Cre;Twist1^+/+^ mice. Eight- to 12-week old mice were used. Cre expression was induced by daily intraperitoneal injection of tamoxifen (75 mg/kg of total body weight in corn oil; Sigma-Aldrich, St. Louis, MO, USA) for 5 days. All animal procedures complied with the animal care guidelines approved by the Institutional Animal Care and Use Committees of the State Key Laboratory of Experimental Hematology. Transplantation assays ---------------------- For non-competitive BM transplantation, to create the chimeras described in Online Supplementary Figure S1A, 2x10^[@b6-1031969]^ whole BM cells from B6.SJL (CD45.1) mice were transplanted into ER-Cre;Twist1^+/+^ or ER-Cre;Twist1^fl/fl^ (CD45.2) recipients that were lethally irradiated (9.5 Gy from a Cesium source, 4-24 h before transplantation). Sixteen weeks later, tamoxifen was injected to induce Twist1 deletion. For competitive transplantation, 300 BM long-term HSC (CD45.1) from tamoxifen-treated ER-Cre;Twist1^+/+^ or ER-Cre;Twist1^fl/fl^ chimeric mice were mixed with 2×10^[@b5-1031969]^ congenic BM support cells and injected into lethally irradiated CD45.2 recipients. For the MLL-AF9 AML model, 5x10^[@b5-1031969]^ GFP^+^ leukemic cells were transplanted into Twist1-deleted or control chimeric recipients. Flow cytometry analysis and cell sorting ---------------------------------------- The BM cell suspensions were flushed from femora and tibiae. Spleen cells were pestled by the plug of a 10 mL syringe. The cells were then filtered through a 74 mm nylon mesh. For flow cytometric analysis of stromal cells, BM was flushed using phosphate-buffered saline with 2% bovine serum, the bones were minced with scissors, then the plugs were digested in 1 mg/mL collagenase I (OLC) or IV (MSC and EC) (Sigma-Aldrich) dissolved in Hank's balanced salt solution with 10% fetal bovine serum for 90 min (collagenase I) or 30 min (collagenase IV) at 37°C. The dissociated cells were collected and kept on ice. Cells were incubated with conjugated antibodies. Stained cells were analyzed with FACS LSR II or sorted with a FACS Aria II instrument (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed by FlowJo software. Statistical analysis -------------------- The significance of differences between two groups was determined using unpaired two-tailed Student t tests. Data are presented as means ± standard deviations. Overall survival curves were plotted according to the Kaplan-Meier method with the log-rank test applied for comparisons. ^\^P\<0.05, ^\\^P\<0.01, ^\\\^P\<0.001. Details of other experimental procedures are given in the Online Supplementary Methods. Results ======= Microenvironmental Twist1 deficiency leads to decreased numbers of mesenchymal stem cells and mature osteoblasts, an increased proportion of endothelial cells, and altered expression of cell factor genes ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- To explore the role of TWIST1 in the BM niche and its regulation of HSC, we generated microenvironment Twist1-deleted and control chimeric mice according to the method described by Schreck and Saez.^[@b21-1031969],[@b22-1031969]^ In brief, 2×10^[@b6-1031969]^ BM cells from B6.SJL wild-type (WT) mice (CD45.1) were transplanted into ER-Cre;Twist^[@b1-1031969]^^fl/fl^ and ER-Cre;Twist1^+/+^ recipients (CD45.2) (Online Supplementary Figure S1A). Sixteen weeks later, about 90% of the cells in the peripheral blood of recipients were donor-derived cells (Online Supplementary Figure S1B). Tamoxifen was then injected intraperitoneally for 5 days to induce Twist1 deletion. Two weeks after the last injection, mRNA detection demonstrated that Twist1 had been knocked out in all the MSC, OLC, and EC isolated from Twist1^Δ/Δ^ mice with similar knockout levels (Online Supplementary Figure S1C), while the expression of Twist2 was almost unchanged (data not shown). To define components of the Twist1-deleted BM microenvironment that may be altered, stromal populations and extracellular factors were assessed in Twist1-deleted and control chimeric mice. We observed that conditional deletion of Twist1 led to a significant decrease in the number of MSC (CD140a^+^CD51^+^CD45/Ter119/CD31-)^[@b23-1031969]^ in the BM compared with that in control mice, as determined by flow cytometry ([Figure 1A](#f1-1031969){ref-type="fig"}). The decrease in MSC number was further confirmed by a fibroblastic colony-forming unit assay (Online Supplementary Figure S2A). Furthermore, Twist1-deleted MSC showed a decrease in proliferative cells and an increase in apoptotic cells (Online Supplementary Figure S2B,C), indicating the mechanism underlying the reduced number of MSC. ![Twist1 deficiency in the bone marrow microenvironment leads to decreased frequency of	{ "pile_set_name": "PubMed Central" }
Sepsis and septic shock are the leading causes of death in intensive care units (ICUs) and have significant mortality rates (as high as 60%) and health-care cost burdens (40% of total ICU expenditures)[@b1]. Recent epidemiological studies have shown that approximately 47% of ICU patients with severe sepsis have positive cultures for Gram-positive bacteria, and the contribution of these bacteria to sepsis has been steadily increasing since 1979[@b2][@b3]. The clinical manifestation of sepsis is highly variable and influenced by several factors, including the health and immune status of the patient and the infectious agent involved. In the ICUs, where sepsis is a common occurrence following surgery, attention needs to be directed to the hospital-specific attributes, including (but not limited to) altered metabolism and immune-modulation due to opioid administration, to gain mechanistic insights into poly-microbial sepsis and its potential remedy[@b4][@b5]. Due to their analgesic and sedative properties, opioids are widely used in ICUs to optimize patient comfort and facilitate mechanical ventilation[@b6]. The immunosuppressive effects of opioids are well documented[@b5] and raise safety issues, especially in ICU patients. In humans, higher circulating morphine levels are observed in patients with sepsis, severe sepsis, and septic shock[@b7], and several murine sepsis models show that morphine treatment induces bacterial translocation from the gut lumen into the peritoneal organs and circulatory system[@b8][@b9]. We have previously shown that morphine accelerates the progression of LPS-induced sepsis by modulating Toll-like receptor (TLR) pathways and altering endotoxin tolerance[@b10]. However, the exact mechanisms by which opioids modulate sepsis progression remain largely elusive. In the present study, we used cecal ligation and puncture (CLP), a well-established model for inducing poly-microbial sepsis, in C57BL/6J mice treated with either opioids or placebo. The survival rates of mice were analyzed to investigate the effects of opioids on sepsis progression. We demonstrated that both morphine and methadone treatment resulted in high mortality following CLP when compared with placebo-treated animals. Furthermore, morphine promoted bacterial dissemination and increased production of the pro-inflammatory cytokine interleukin-17A (IL-17A). IL-17A is a member of the interleukin-17 (IL-17) family, which consists of a subset of cytokines that participate in both acute and chronic inflammatory responses. In various diseases, IL-17A is involved in host defense and implicated in excessive inflammation and overt tissue damage[@b11]. Although a few reports implicate IL-17A in poly-microbial sepsis[@b12][@b13], its role in the progression of Gram-positive sepsis is still unknown. Our study showed that overexpression of IL-17A following morphine treatment resulted in increased gut permeability, a higher bacterial load, sustained inflammation, and, subsequently, higher mortality. Concomitantly, neutralization of IL-17A protected morphine-treated animals from sepsis-induced mortality. We (and others) have previously shown that the source of the bacteria that contributes to morphine-induced sepsis is derived from the commensal pool of the gut microbiome[@b8][@b9]. In this study, we further showed that morphine treatment induced enrichment of the Gram-positive bacteria Staphylococcus and Enterococcus in the gut lumen, the same species that were isolated from various systemic organs following CLP. Activation of TLR2 by the disseminated Gram-positive bacteria led to overexpression of IL-17A, resulting in higher mortality in the morphine-treated animals. These results are consistent with the clinical observation that Staphylococcus aureus is one of the most common Gram-positive isolates from patients with sepsis, and infection with Enterococcus species is considered as an independent factor associated with a greater risk of hospital death[@b1]. In summary, the current study provides insight into the influence of opioids on sepsis progression, showing that IL-17A may be a potential therapeutic target for the treatment of sepsis caused by Gram-positive infection, especially in ICU patients who are on a moderate to severe pain management regimen. Results ======= Opioids increase mortality in a poly-microbial sepsis model of CLP ------------------------------------------------------------------ To determine the effects of opioids on the progression of poly-microbial sepsis, wild-type (WT) mice were subjected to a CLP procedure and implanted with either a placebo or a slow release morphine pellet subcutaneously. As shown in [Fig. 1a](#f1){ref-type="fig"}, the survival rates were significantly reduced in morphine-treated mice following CLP. At 24 hours after CLP, all placebo-treated mice were alive compared with only 66.67% in the morphine-treated group. None of the morphine-treated mice survived beyond 96 hours, whereas 78.57% of the placebo-treated mice survived for the entire period of observation (7 days). All of the sham-operated mice treated with the placebo survived, whereas 80% of the sham-operated mice treated with morphine survived until the end of 7 days. To investigate the influence of other prescription opioids on the outcome of CLP-induced sepsis, mice were injected with methadone or saline following CLP. Methadone showed similar effects to morphine on the survival rates following CLP ([Fig. 1b](#f1){ref-type="fig"}). A total of 66.67% of the saline-treated CLP mice lived for 7 days, whereas no methadone-treated mice survived beyond the fifth day after CLP. All of the sham-operated mice injected with saline or methadone survived for 7 days. The morphine- and methadone-induced mortality following CLP was significantly reduced by the opioid receptor antagonist naltrexone ([Fig. 1c,d](#f1){ref-type="fig"}), indicating that opioid treatment exacerbated the outcome of poly-microbial sepsis in an opioid receptor-dependent manner. Morphine promotes bacterial dissemination and inhibits bacterial clearance following CLP-induced sepsis ------------------------------------------------------------------------------------------------------- Next, the bacterial load was determined in the peritoneal lavage fluid, mesenteric lymph node (MLN), liver, spleen, and blood at different time points following CLP in the presence or absence of morphine. As shown in [Fig. 2](#f2){ref-type="fig"}, at 24 hours, morphine alone induced bacterial translocation into the peritoneal cavity, MLN, liver, and spleen, confirming that morphine itself can compromise the gut epithelial barrier, as previously reported[@b8]. In the placebo-treated CLP animals, the bacterial load in the peritoneal lavage fluid, MLN, liver, spleen, and blood reached its highest levels at 24 hours, and subsequently decreased at 72 hours. At 168 hours after the CLP, almost all bacteria that disseminated into the peritoneal organs were cleared in the placebo-treated animals. In the morphine-treated CLP mice, a significant increase in the amount of bacteria was observed in the peritoneal organs and blood compared with the placebo-treated mice at both 24 and 72 hours, indicating that morphine promoted bacterial dissemination and inhibited bacterial clearance. Bacterial translocation in liver homogenates was further evaluated using bacterial 16 s ribosomal DNA (rDNA). Consistent with data obtained using bacterial cultures and colony forming units, the bacterial load as measured by 16 s rDNA was also significantly higher in the morphine treated-CLP animals than all other groups ([Supplementary Fig. 1](#S1){ref-type="supplementary-material"}). Because none of the animals survived beyond 96 hours in the morphine-treated CLP group, data regarding the bacterial load were not available for the last time point. Morphine treatment promotes Gram-positive bacterial dissemination and modulates the gut microbiome -------------------------------------------------------------------------------------------------- We next serotyped the bacterial species that disseminated into the MLN, liver, and spleen following CLP. In placebo-treated CLP animals, the most common bacterial species detected in the MLN, spleen, and liver were non-hemolytic Escherichia coli ([Fig. 3a](#f3){ref-type="fig"}), which are common Gram-negative commensal bacteria resident in the gut lumen; very few Enterococcus species were detected in the MLN and spleen. However, all MLN, spleen, and liver isolates from morphine-treated animals with or without CLP procedures revealed a significant prevalence of the Gram-positive families Staphylococcus and Enterococcus ([Fig. 3a](#f3){ref-type="fig"}). These data were validated using Illumina sequencing of 16 S rDNA from liver samples of morphine-treated CLP mice, which showed a prevalence of both the Enterobacteriaceae and Enterococcus families ([Supplementary Fig. 2a](#S1){ref-type="supplementary-material"}). Analysis of the gut microbiome showed that morphine treatment induced enrichment of mostly the Firmicutes phylum and specifically the Gram-positive bacterial species Staphylococcus sciuri, Staphylococcus cohnii, and Staphylococcus aureus as well as Enterococcus durans, Enterococcus casseliflavus, Enterococcus faecium, and Enterococcus faecalis in the gut microbiome ([Fig. 3b,c](#f3){ref-type="fig"}). Interestingly, these species belonged to the same families that were observed to translocate to the peritoneal organs following morphine treatment. Morphine-induced alterations of the gut microbiome were antagonized by the opioid receptor antagonist naltrexone ([Fig. 3b,c](#f3){ref-type="fig"}), further validating that morphine treatment modulated the gut microbiome and thereby influenced the outcome of poly-microbial sepsis in an opioid receptor-dependent manner. This was also reflected in the bacterial sequences obtained from liver samples. When the beta-diversity of 16 S rDNA sequences from liver samples were compared between Placebo-	{ "pile_set_name": "PubMed Central" }
Introduction {#Sec1} ============ Atherosclerosis is generally considered to be positively associated with hypertension^[@CR1]^. A single nucleotide polymorphism (SNP) (rs3782886) in breast cancer suppressor BRCA1-related associated protein (BRAP) has been associated with the risk of developing myocardial infarction^[@CR2]^. Another study reported that BRAP activates inflammatory cascades, increasing the risk of carotid atherosclerosis^[@CR3]^. Even though the minor allele frequency (MAF) of rs3782886, and aldehyde dehydrogenase 2 (ALH2) polymorphism rs671 are significantly inversely associated with both systolic and diastolic blood pressure^[@CR4]^. Since strong linkage disequilibrium (LD) values between rs3782886 and rs671 have been reported^[@CR5]^, rs671 could have same the characteristics of rs3782886. However, the mechanism underlying the association between hypertension and these genetic factors (rs3782886 and rs671) have not yet been elucidated. Atherosclerosis is involved in aggressive endothelial repair. Therefore, the favorable association of genetics factor (rs3782886 and rs671) with hypertension may be caused by stimulating endothelial repair. Additionally, several studies have demonstrated a close association between bone marrow activity (hematopoietic activity) and endothelial maintenance^[@CR6],\ [@CR7]^. We have showed in a previous study that active endothelial repair, represented by high levels of hematopoietic (CD34-positive) stem cells, is positively associated with atherosclerosis but could have beneficial effect on prevention of hypertension among elderly participants^[@CR8]^. Hematopoietic bone marrow activity declines with age^[@CR9]^ and aging is also a well-known cause of endothelial injury^[@CR10],\ [@CR11]^. Since hematopoietic activity also influences reticulocyte levels, high levels of reticulocytes among older participants may indicate a high capacity of endothelial maintenance. Therefore, the levels of reticulocytes could play a role in the association between genetic factors (rs3782886 and rs671) and hypertension. Furthermore, platelet activation is involved in the initial mechanism of an endothelial repair^[@CR12],\ [@CR13]^ and a platelet count indicates the activity of an endothelial repair^[@CR1],\ [@CR14]^. Therefore, evaluating the number of platelets may be an efficient tool to determine the mechanism underlying the association between genetic factors (rs3782886 and rs671) and hypertension. An extensive prevalence of SNPs should have the same beneficial effect on the participants' daily activities, rather than impose a disadvantage. Therefore, determining the mechanism by which genetic characteristics prevent hypertension inducing progression of atherosclerosis could provide novel knowledge to elaborate strategies for risk estimation and prevention of hypertension. To determine the potential mechanism underlying the association between the genetic factors (rs3782886 and rs671) and hypertension, we conducted a cross-sectional study comprised of 1,313 elderly Japanese individuals (aged 60--98) who had previously participated in a general health check-up, in 2017. Methods {#Sec2} ======= Study population {#Sec3} ---------------- The methods that relates to present risk survey including genetic factor also have been described elsewhere^[@CR1],\ [@CR14]--[@CR16]^. The study population was comprised of 1,401 Japanese residents (504 men and 897 women) aged between 60 and 99 years old from Goto City (western Japan) who had undergone an annual medical check-up in 2017. Participants without platelet data (n = 2) or SNP data (n = 1) were excluded from the study population. Additionally, to control for the influence of chronic disease, participants with hypo-nutrition (BMI ≤ 18.0 kg/m^2^) (n = 85) were also excluded. The remaining participants, comprising 1,313 (494 men and 819 women) with a mean age of 72.9 (standard deviation (SD): 7.3; range: 60--98), were enrolled in the study. All of the procedures involving human participants in this study were performed in accordance with the ethical standards of the institution research committee and the 1964 Helsinki Declaration, and its later amendments for comparable ethical standards. Written consent forms in Japanese were made available to ensure the comprehensive understanding of the study objectives, and informed consent was provided by the all participants. Ethics Committee of Nagasaki University Graduate School of Biomedical Sciences (project registration number: 14051404) approved this study. Data collection and laboratory measurements {#Sec4} ------------------------------------------- Specially trained interviewers were tasked with obtaining the medical histories and habitual statuses of the participants. The body weight and height were measured using an automatic body composition analyzer (BF-220; Tanita, Tokyo, Japan), from which the body mass index (BMI; kg/m^2^) was calculated. Systolic (SBP) and diastolic blood pressure (DBP) were recorded at rest. Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg and/or taking anti-hypertensive medication. Fasting blood samples were collected in a heparin sodium tube, an EDTA-2K tube, and a siliconized tube. The quantities of platelets, red blood cells (RBC), and reticulocytes in the samples from the EDTA-2K tube were measured using an automated procedure at SRL, Inc. The levels of reticulocytes were determined using the following formula: reticulocytes (×10^4^ cells/μL) = (reticulocytes, ‰) × RBC (×10^4^ cells/μL)/1,000 (for men: 5.37 ± 2.01 \[×10^4^ cells/μL\]; for women: 4.78 ± 1.56 \[×10^4^ cells/μL\]). The concentrations of triglyceride (TG), HDL-cholesterol (HDL-C), hemoglobin A1c (HbA1c), and creatinine were measured using the standard laboratory procedures. All measurements were performed at SRL, Inc. (Tokyo, Japan). The glomerular filtration rate (GFR) was estimated using a recently adapted established method introduced by a working group of the Japanese Chronic Kidney Disease Initiative^[@CR17]^, which yielded an estimate of GFR (mL/min/1.73 m^2^) = 194 × (serum creatinine (enzyme method))^−1.094^ × (age)^−0.287^ × (0.739 for women). The genomic DNA was extracted from 2 mL of whole peripheral blood using the Gene Prep Star NA-480 (Kurabo Industries Ltd., Osaka, Japan), which was genotyped for SNP rs3782886 (BRAP on chromosome 12q24.12) and rs671 (ALDH2 on chromosome 12q24.2) using the TaqMan method with a LightCycler 480 (Roche Diagnostics, Basel, Switzerland). Expected numbers and percentages were calculated. Deviations from the Hardy--Weinberg equilibrium were evaluated via χ^2^ test for each allele (rs3782886 and rs671). Statistical analysis {#Sec5} -------------------- The study population was classified by reticulocyte levels (high and low) according to the median values (5.21 × 10^4^ cells/μL for men and 4.65 × 10^4^ cells/μL for women). The characteristics of the study population in relation to the reticulocyte levels were expressed as the mean ± standard deviation (SD) for the contentious variables except for TG, and as prevalence for the medication status and habitual status. Since TG showed a skewed distribution, the characteristics of the study population were expressed as median \[first quartile and third quartile\], followed by a logarithmic transformation. Differences between mean values or proportional values of monitored characteristics were analyzed in relation to reticulocyte levels. Significant differences were evaluated using t-test for continuous variables and χ^2^ test for proportional data. Logistic regression models were used to calculate the odds ratios (OR) and 95% confidence intervals (CI) were used to determine associations between hypertension and platelets, as well as between hypertension and reticulocyte levels. The numbers of platelets and reticulocytes in relation to the genotype (rs3782886 and rs671) by reticulocyte levels were also calculated. In addition, OR and 95% CI were also calculated using a logistic regression model to determine the influence of SNP (rs3782886 and rs671) on hypertension by the levels of reticulocytes. Two different approaches were used to make adjustments for confounding factors. First, one model was adjusted only for sex and age (Model 1). For the second model (Model 2), we included several other potential confounding factors, namely BMI (kg/m^2^), alcohol consumption (never drinker, former drinker, current drinker \[23--45 g/week, 46--68 g/week, ≥ 69 g/week\]), smoking status (never, former, current), TG (mg/dL), HDL-C (mg/dL), HbA1c (%), and GFR (mL/min/1.73 m^2^). As Japanese men are known to have high rates of drinking, while Japanese women have low rates of drinking^[@CR18]^, to evaluate the influence of SNP (rs3782886 and rs671) on the status of individual who had never had a drink (never drinker), sex-specific analyses were performed. Furthermore, to evaluate the influence of drinking status (never drinker) on platelet and reticulocyte count, sex-adjusted value of platelet and reticulocyte count in relation to drinking status stratified by reticulocyte levels were calculated by using covariance analysis. All statistical analyses were performed using the	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== Praziquantel (PZQ) is virtually the sole treatment regimen for Schistosomiasis in sub-Saharan Africa \[[@CR1]\]. This oral schistosomicidal agent is constituted of a racemate mixture, with activity both in vivo and in vitro \[[@CR2], [@CR3]\]. Evidence from studies conducted on Schistosoma (S.) mansoni and S. japonicum although not very clear, indicate the mode of action of PZQ is the targeting of calcium channels and antigen exposure rendering the worm susceptible to elimination by antibodies \[[@CR1], [@CR4]\]. After oral administration, PZQ is rapidly absorbed, metabolized and excreted by the kidney. Metabolism of PZQ is primarily via the cytochrome P450 system leading to the production of toxic metabolic intermediates, which are potentially harmful to hepatocytes \[[@CR5]\]. Plasma levels of PZQ are also reported to be reduced by inducers but elevated by inhibitors of cytochrome P450 activity \[[@CR6], [@CR7]\]. Several studies, predominantly in Asian populations, where S. japonicum infections are endemic, state conflicting findings on hepatotoxicity associated with PZQ treatment against the helminth \[[@CR8], [@CR9]\]. PZQ treatment is reported to be associated with elevated serum concentrations of liver aminotransferase \[[@CR8]\]. However, in a large retrospective study from China, there was insignificant (less than 1%) incidence of hepatotoxicity among populations treated for S. japonicum with PZQ \[[@CR9]\]. Therapy for Schistosomiasis in sub-Saharan Africa has mainly been documented based on intestinal S. mansoni infections \[[@CR1]\]. As a result, there is paucity of data on urinary S. haematobium and its associated drug metabolism effects on organs involved in metabolizing and excretion of PZQ. This leaves a gap in knowledge about the protective or destructive effect of metabolizing the drug in S. haematobium infection. It has further been shown that varied degrees of reduction in incidence and infection rates of S. haematobium are reported with mostly single PZQ dosage of 40 mg/kg/day in both children and adults \[[@CR1]\]. There are also indications of drug resistance to single doses of PZQ for treating schistosomiasis \[[@CR10]\]. This heightens the need to probe the outcome of repeated PZQ treatment on urinary schistosome counts against its implication on liver and renal function. The aim of this study was to assess the effect of PZQ on schistosome egg count, liver and renal function after 3 doses of 60 mg/kg/day (PZQ60) in three months for treating urinary S. haematobium infection. Methods {#Sec2} ======= This was a nested-Case Control study conducted among children and adults of ages 6--30 years from the urban Asokwa District in Kumasi, Ghana (see plate 1). This was part of a larger study to assess plasmodium transmission in persons infected with Schistosomiasis ([NCT02769013](https://www.clinicaltrials.gov/ct2/show/NCT02769013)). Ethical approval was obtained from the Committee for Human Research Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Ghana. All participants were required to sign an informed consent. For minors below 16 years, a signed assent form from the participant and an informed consent from a parent or guardian were obtained. Cases were respondents diagnosed to have S. haematobium by routine microscopic examination of urine samples. Controls from the same communities, without laboratory or clinical detection of urinary schistosomiasis infection were age and sex matched with cases. Study area {#Sec3} ---------- Apromase, Deduako, Emena and Kokoben were the study communities in the urban Asokwa District with a population of 140,161 inhabitants in 36, 183 households (Fig. [1](#Fig1){ref-type="fig"}) \[[@CR11]\]. These communities are located between latitude 6°30′ and 7°00′ North and longitude 1°30′ and 2°00 West of Kumasi, the capital city of the Ashanti Region of Ghana. The four communities have Saman (Kokoben and Apromase), Oda (Deduako) and Subin (Emena) as names of three rivers running through it. Fig. 1Map of study communities and sites (Rivers) in Ashanti region, Ghana Climatic conditions are tropical with temperatures varying from 20.2 °C to 37.1 °C. Rainfall pattern is seasonally bimodal with major rains extending from late April to August with a minor one from September to October \[[@CR12]\]. The average annual rainfall for the area is 6.25 mm with peaks of 214.3 mm and 16.2 mm in June and September respectively. The dry season (harmattan) is from November to March with humidity ranging between 53 and 93%. Screening and enrolment {#Sec4} ----------------------- A census of the selected communities was conducted with the ages and number of inhabitants per building collected along with corresponding GPS coordinates using Personal Digital Assistants (PDAs). Households within the buildings were selected and their members asked for written informed consent to be screened in the study. Twenty millilitres (20 ml) of urine samples were collected once, from consenting participant into well-labelled 30 ml urine containers. The urine samples were collected within the hours of 6:00 am and 12 noon. Subsequently, the samples were kept in cold boxes at temperature of 4--6 °C until transported to the laboratory at Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) about 15--20 min drive from the study sites. Asymptomatic schistosomiasis positive (SP) cases and schistosomiasis-negative (SN) controls based on screening results were invited to participate in the study. Sampling procedures {#Sec5} ------------------- A total of 1258 participants were screened for schistosomiasis out of which 104 were positive. All 104 schistosome positive participants (Fig. [2](#Fig2){ref-type="fig"}) were placed on PZQ60 treatment. Controls were selected from the same communities and matched in a 2: 1 ratio with cases by sex and age. Out of the 104 positive cases, 32 started the treatment phase with 28 successfully completing the course with samples analyzed. On the other hand, 53 controls had all samples analyzed. Fig. 2Sampling of study participants Design of Experiment {#Sec6} -------------------- Biochemical parameters and schistosome counts were analysed before and after treatment for both cases and controls (Fig. [3](#Fig3){ref-type="fig"}). In between pre- and post-treatment, the biochemical and schistosome counts were monitored before the 2nd and 3rd dosages of PZQ60 for cases. Fig. 3Flowchart of experimental design Laboratory processes {#Sec7} -------------------- ### Processing of urine and S. haematobium quantification {#Sec8} Freshly voided urine was collected between 6:00 am and 12:00 pm in a sterile wide-mouthed screw-top plastic container (30 ml) and transported to the laboratory on ice at 4 °C to 8 °C. Urine processing and quantification were done as described by Cheesebrough \[[@CR13]\]. Briefly, blunt-ended forceps were utilized to place a polycarbonate membrane filter of pore size of 12.0 μm (Whatmann Nuclepore) on the filter-support of a filter-holder (Swinnex 25 mm support chamber). The filter holder was re-assembled and attached to a 10 ml syringe filled with well-mixed urine which was filtered with the aid of the plunger. The filter was carefully removed and transferred with the face upwards to a clean glass-slide. A drop of physiological saline was added, covered with a cover-slip, and examined by two independent expert microscopists using the 10X objective (Carl Zeiss Microscope) with the condenser iris closed sufficiently to give good contrast. The entire filter was examined systematically for the presence of S. haematobium eggs. The number of the eggs counted per 10 ml of urine was recorded and the average of the two counts was calculated. A slide was declared negative when no parasites were detected. ### Blood sampling and processing {#Sec9} Blood samples were collected from the antecubital vein with the aid of a tourniquet and the puncture site cleaned with 70% alcohol prep. Blood drawn into separator gel tubes (5 ml) were centrifuged at 1780 x g for 10 min at 4 °C to obtain the sera which were subsequently stored at -80 °C. ### Biochemical analysis {#Sec10} Assays for the liver function; albumin, globulin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) and renal function (urea and creatinine), were conducted using a chemistry analyzer (HumaStar 200, Human, Germany). Elevated levels of AST, ALT and GGT are known indications of liver damage which may be caused by drug metabolism, infections and alcohol consumption. Increase in the levels of globulin is also found to correlate with infection or an inflammatory state. Aliquots of the serum	{ "pile_set_name": "PubMed Central" }
Main {#Sec1} ==== RNA interference (RNAi) is a natural cellular process that regulates gene expression and provides an innate defence mechanism against invading viruses and transposable elements^[@CR1]^. The finding that dsRNA initiates RNAi was among the most significant recent contributions to cell biology^[@CR2]^, and since the discovery that RNAi can be mediated by 21 nucleotide (nt) duplexes^[@CR3]^, researchers have worked to harness their potential for addressing biological questions and treating human disease. Some reagents, such as small interfering RNAs (siRNAs), are applied directly to cells, tissues and organisms; others are engineered to be expressed in cells, such as hairpin structures that provide siRNAs when processed. The basic premise underlying the broad utility of RNAi is that, in theory, we can design siRNAs (or vectors encoding them) to target virtually any gene of interest. RNAi technologies use a cell\'s natural machinery to move exogenously applied siRNAs to the appropriate cellular compartment, where they encounter the correct mRNA target and induce its degradation. Initial work on RNAi in flies and worms moved quickly to larger mammals and fuelled excitement for potential clinical applications. However, in a similar way to other developing fields in human therapy, such as gene- and antibody-therapy, early excitement has been tempered as a realistic understanding emerges of the milestones that must be reached before the eventual approval of human therapy. Over recent years various complex barriers to achieving efficient RNAi have become evident. These hurdles include: specificity for the target gene; delivery to the correct cell or tissues; the durability of RNAi activity and the ability to redose (if needed); and considerations of the stability of the target mRNA and encoded protein. We have also become aware of the problems posed by the various platforms used to elicit RNAi. However, with setbacks come opportunities. For example, early work in which siRNAs were applied to mouse airway epithelial cells to reduce viral burdens in vivo elicited inhibition of target gene expression^[@CR4]^, but it was later found that the induction of an innate immune response probably contributed to the efficacy in these and other experiments^[@CR5],[@CR6]^. Altering the chemical make-up of the synthetic RNA diminished the immune response, as did avoiding known pro-inflammatory sequences^[@CR7],[@CR8]^. This finding also opened investigators\' eyes to the possibility of purposefully using immunostimulatory RNAi as a direct therapeutic or adjuvant^[@CR9]^. Although the clinical utility of RNAi has not yet been realized, ongoing patient trials provide opportunities for success. The numbers of RNAi-based preclinical and clinical trials have grown over the past several years and have included studies in retinal degeneration, dominantly inherited brain and skin diseases, viral infections, respiratory disorders, cancer and metabolic diseases ([Table 1](#Tab1){ref-type="table"}).Table 1Clinical trials for RNAi therapy^\^Clinical settingDrugIndication(s)Target(s)SponsorStatusOcular and retinal disordersTD101Pachyonychia congenitaKeratin 6A N171K mutantPachyonychia Congenita ProjectCompleted, Phase IQPI-1007Non-arteritic anterior ischaemic optic neuropathyCaspase 2Quark Pharm., Inc.Active, Phase IAGN211745Age-related macular degeneration; choroidal neovascularizationVEGFR1Sirna Therapeutics, Inc.Completed, Phase I, IIPF-655Diabetic macular oedema (DME); age-related macular degeneration (AMD)RTP801Quark Pharm., Inc.Active, Phase ISYL040012Glaucomaβ2 adrenergic receptorSylentisActive, Phase I, IIBevasiranibDiabetic macular oedemaVEGFOpko Health, Inc.Completed, Phase IIBevasiranibMacular degenerationVEGFOpko Health, Inc.Completed, Phase IICancerCEQ508Familial adenomatous polyposisβ-cateninMDRNA, Inc.Active, Phase IALN-PLK1Liver tumoursPLK1Alnyam Pharm.Active, Phase IFANGSolid tumoursFurinGradalis, Inc.Active, Phase IICALAA-01Solid tumoursRRM2Calando Pharm.Active, Phase ISPC2996Chronic myeloid leukemiaBCL-2Santaris Pharm.Ongoing, Phase I, IIALN-VSP02Solid tumoursVEGF, kinesin spindle proteinAlnylam Pharm.Active, Phase INCT00672542Metastatic melanomaLMP2, LMP7, and MECL1Duke UniversityActive, Phase IAtu027Advanced, recurrent or metastatic solid malignanciesPKN3Silence TherapeuticsActive, Phase IKidney disordersQPI-1002/I5NPAcute kidney injuryp53Quark Pharm., Inc.Terminated, Phase IQPI-1002/I5NPDelayed graft function kidney transplantp53Quark Pharm., Inc.Active, Phase I, IIQPI-1002/I5NPKidney injury acute renal failurep53Quark Pharm., Inc.Completed, Phase ILDL loweringTKM-ApoBHypercholesterolaemiaAPOBTekmira Pharm. Corp.Terminated, Phase IPRO-040,201HypercholesterolaemiaAPOBTekmira Pharm. Corp.Terminated, Phase IAntiviralSPC3649Hepatitis C virusmiR-122Santaris PharmActive, Phase IIpHIV7-shI-TAR-CCR5RZHIVHIV Tat protein, HIV TAR RNA, human CCR5City of Hope Medical Center/BenitecActive, Phase 0ALN-RSV01RSV in volunteersRSV nucleocapsidAlnylam Pharm.Completed, Phase IIALN-RSV01RSV in lung transplant patientsRSV nucleocapsidAlnylam Pharm.Completed, Phase IALN-RSV01RSV in lung transplant patientsRSV nucleocapsidAlnylam Pharm.Active, Phase IIAPOB, apolipoprotein B; BCL-2, B-cell CLL/lymphoma 2; CCR5, C-C chemokine receptor type 5; LDL, low-density lipoprotein; LMP2, also known as proteasome subunit beta type 9 (PSMB9); LMP7, also known as proteasome subunit beta type 8 (PSMB8); MECL1, also known as proteasome subunit beta type 10 (PSMB10); Pharm., Pharmaceuticals; PKN3, protein kinase N3; PLK1, polo-like kinase 1; RRM2, ribonucleoside-diphosphate reductase subunit M2; RSV, respiratory syncytial virus; RTP801, also known as DNA damage-inducible transcript 4 protein (DDIT4); VEGF, vascular endothelial growth factor.^\^From [ClinicalTrials.gov](http://clinicaltrials.gov/). Here, we provide an overview of RNAi and discuss strategies to use the pathway for directed gene silencing for therapy. We describe delivery systems that might be suitable for different circumstances, and bring to the reader\'s attention issues that must be surmounted for widespread use in vivo. Harnessing small RNA biogenesis The development of RNAi for therapy is based on our understanding of small RNA biogenesis pathways. The two main types of small RNAs involved in gene silencing are microRNAs (miRNAs) and siRNAs, and their processing and targeting is summarized in [Fig. 1](#Fig1){ref-type="fig"} (further details can be found in recent reviews^[@CR10],[@CR11],[@CR12]^).Figure 1The miRNA and siRNA pathways of RNAi in mammals.Primary microRNAs (pri-miRNAs) are transcribed by RNA polymerases^[@CR156],[@CR157],[@CR158]^ and are trimmed by the microprocessor complex (comprising Drosha and microprocessor complex subunit DCGR8) into \~70 nucleotide precursors, called pre-miRNAs^[@CR67],[@CR159],[@CR160]^ (left side of the figure). miRNAs can also be processed from spliced short introns (known as mirtrons)^[@CR161]^. pre-miRNAs contain a loop and usually have interspersed mismatches along the duplex. pre-miRNAs associate with exportin 5 and are exported to the cytoplasm^[@CR162],[@CR163]^, where a complex that contains Dicer, TAR RNA-binding protein (TRBP; also known as TARBP2) and PACT (also known as PRKRA) processes the pre-miRNAs into miRNA--miRNA\* duplexes^[@CR116],[@CR164],[@CR165]^. The duplex associates with an Argonaute (AGO) protein within the precursor RNAi-induced silencing complex (pre-RISC). One strand of the duplex (the passenger strand) is removed. The mature RISC contains the guide strand, which directs the complex to the target mRNA for post-transcriptional gene silencing. The \'seed\' region of an miRNA is indicated; in RNAi trigger design, the off-target potential of this sequence needs to be considered. Long dsRNAs (right side of the figure) are processed by Dicer, TRBP and PACT into small interfering RNAs (siRNAs). siRNAs are 20--24-mer RNAs and harbour 3′OH and 5′ phosphate (PO~4~) groups, with 3′ dinucleotide overhangs^[@CR3],[@CR166],[@CR167]^. Within the pre-RISC complex, an AGO protein cleaves the passenger siRNA strand. Then, the mature RISC, containing an AGO protein and the guide strand, associates with the target mRNA for cleavage. The inset shows the properties of siRNAs. The thermodynamic stability of the terminal sequences will direct strand loading. Like naturally occurring or artificially engineered miRNAs, the potential \'	{ "pile_set_name": "PubMed Central" }
Background ========== Accurate and timely diagnosis of malaria infections is a critical part of case management in malaria control programmes aiming to reduce malaria morbidity and mortality. Sensitive and effective detection of infected individuals also plays a vital role in areas where transmission has been reduced markedly due to intensified control measures and elimination is being considered. Malaria diagnosis is traditionally achieved by microscopic examination of blood smears. Microscopy is able to detect parasite species and determine parasite densities. The limit of detection by thick film microscopy is in the range of 5 to 100 parasites/μL of blood \[[@B1]-[@B3]\]. However, the quality of microscopy can vary significantly \[[@B1],[@B4]\] because its accuracy largely relies on the experience and training of the microscopists to make and stain a blood slide correctly and read it accurately. Malaria rapid diagnostic tests (RDTs) have been developed and tested over the past 2 decades as an alternative to microscopy, particularly for areas where quality microscopy is absent or hard to maintain. RDTs are lateral flow devices that detect parasite proteins using antibodies. The tests are easy to perform and provide rapid results in 15 to 20 minutes without the need for electricity, expensive equipment or extensive training. The limit of detection, by good quality RDTs, is similar to that of thick film microscopy for P. falciparumbut often poorer for P. vivax\[[@B1]\]. Today, over 50 brands of malaria RDTs are manufactured, and over 150 individual products commercially available. Polymerase chain reaction (PCR) is a DNA-based molecular detection method that is more sensitive than microscopy and RDT, and has been widely used for diagnosis, confirmation of diagnosis, epidemiology studies and drug efficacy assessment. In theory, PCR is capable of detecting a single parasite in a blood sample, and its sensitivity often is only limited by the volume of the blood. PCR provides accurate determination of parasite species, better sensitivity in detecting low density of parasites and better detection of mixed species/strain infections \[[@B2],[@B5],[@B6]\]. In the field, PCR has been mostly used in epidemiological studies instead of point of care diagnosis due to its requirement for sophisticated equipment, reagents and several hours of turn over time. In most malaria control settings, where the goal is to significantly reduce malaria morbidity and mortality, quality assured microscopy and RDTs have been shown to effectively detect parasites in the majority of symptomatic patients and thus guide treatment. In contrast, for malaria elimination settings it is critical to detect all infections, including those with low and sub-microscopic parasite densities in asymptomatic carriers as they represent a parasite reservoir in the community capable of effectively transmitting infections to mosquitoes \[[@B7],[@B8]\] and seeding transmission foci \[[@B9]\]. It is well known that malaria epidemiology varies between country and region, and particularly between islands, because the dominant vector species, the characteristics of human populations and factors that influence transmission such as rainfall, temperature, housing conditions and population movement differ. Therefore, the challenges to malaria elimination in different settings will vary. Each area needs to investigate the malaria epidemiology and carefully tailor its diagnosis strategy to the local context. Temotu Province in Solomon Islands is preparing for malaria elimination. An assessment of the epidemiological characteristics of malaria infections in the island population, particularly the prevalence and distribution of asymptomatic infections, and infections with low or sub-microscopic parasite densities will contribute to the understanding of the requirements of diagnostics in malaria elimination. A baseline malaria parasitological survey was conducted in Temotu Province in October-November 2008 as the first step in a provincial malaria elimination programme. This baseline survey provided opportunities to obtain point prevalence and epidemiological characteristics of malaria infections, as well as to assess how well different diagnostic methods performed in this particular setting. During the survey, blood samples collected from the population were examined by microscopy, with a subsample examined by PCR and RDTs. The overall malaria point prevalence was estimated at 2-3% with predominant P. vivaxinfections \[[@B10]\]. Of concern for diagnosis was the high prevalence of asymptomatic infections (86%) reported in this relatively low transmission setting. To better understand malaria epidemiology in the Province, the basis of the observed low disease rate and to better evaluate the performance of different diagnostic methods, a detailed comparison of the microscopy, RDT and PCR was undertaken, including re-examination of the blood smears from discrepant samples and the determination of parasite densities. The results revealed that a large proportion of malaria infections in the province were at low and sub-microscopic parasite densities. The finding has an important practical implication to the malaria elimination strategy in the province. Methods ======= Survey site and sample collection --------------------------------- The survey was conducted by the Pacific Malaria Initiative Survey team in the Temotu Province, a remote island group in the Solomon Islands, approximately 166°E, 11°S with a population of approximately 17,000. The survey site, consent process and sample collection methods are as previously described \[[@B10]\]. Preparation of blood films, microscopic examination and quality assurance ------------------------------------------------------------------------- Thick and thin blood films from finger prick blood of 9,491 survey participants were prepared, air dried, stained and examined as reported earlier \[[@B10]\]. All slides which were considered positive on the initial examination, negative on the initial examination but positive by PCR and a 10% sample of initial microscopy negative samples, were subjected to a second quality assurance (QA) examination (of 300 fields in the thick film). All slides which recorded discrepant results between the initial examination and the QA examination were then subjected to a third \"referee\" examination (of 500 fields in the thick film), which was recorded as the final microscopy result. Identification of parasite species was performed by reference to the thin smears where parasites densities were sufficiently high enough. Otherwise, parasite species identification was made by reference to the thick smear only. Malaria microscopists who participated in this survey were all certified to WHO malaria microscopy standards by prior participation and assessment in WHO accredited malaria microscopy competency assessment courses in Brisbane or Honiara. Microscopists who performed the initial slide examinations were all certified to competency levels 1, 2 or 3. Microscopists who performed the QA examination were all certified to 1 or 2, and microscopists who performed the third \"referee\" examinations were certified to competency level 1. Parasite density counts ----------------------- Parasite densities were determined by counting the number of parasites against 500 white blood cells (WBC) and calculated assuming 8,000 WBC per μL of blood. PCR assays ---------- Genomic DNA was extracted from a subset of 1,784 filter paper samples. These included samples that were microscopy positive, samples from all febrile patients and 10% of the microscopy negative samples from each village. Following microscopy QA, some of the samples initially included as microscopy positive were reclassified as microscopy negative. This increased the percentage of negative samples examined to 15.9%. The DNA was extracted using QIAamp DNA Mini Kits and a QIAcube robot (QIAGEN, Crawley, UK), and eluted to a 100 μl volume. A multiplex PCR was performed to determine parasite species using published primers and PCR conditions \[[@B11]\] with the number of cycles increased from 43 to 45. The positive results were confirmed using a second round P. falciparumand P. vivaxspecific PCR. RDTs ---- ICT Malaria Combo Cassette Test ML02 (ICT diagnostics) was used as per the manufacturer\'s instructions when a participant was febrile and on a subset of 400 children from villages around Lata, the Provincial capital. For QA, a post purchasing sample of these RDTs were tested at the Research Institute for Tropical Medicine, Manila, Philippines, a WHO-FIND lot testing laboratory and were found suitable. All RDTs were used within one month of the commencement of the survey. Results ======= Malaria prevalence determined by microscopy ------------------------------------------- A total of 9,491 blood samples were collected and examined by microscopy. After the initial microscopy and two rounds of microscopy QA, 256 samples were determined positive giving an overall point prevalence of 2.7%. P. falciparumand P. vivaxmade up 17.5% (45/256) and 82.4% (211/256) of positive samples respectively. Two of the 256 positive samples were excluded from density related analyses because parasite densities were not determined due to poor quality of the slides. A low percentage of microscopy positive subjects were symptomatic ----------------------------------------------------------------- The proportion of microscopy positive individuals having an aural temperature ≥38°C at the time of examination was 17.8% (8/45) for P. falciparumand 2.9% (6/209) for P. vivax, respectively, giving an overall fever rate of 5.5% (14/254). The majority of fevers, 50.0% (4/8) and 66.7% (4/6) for P. falciparumand P. vivax, were observed in the 5-14 year age group. Parasite densities in six of the eight P. falciparum-infected fever subjects were \>500/μL. In contrast, 66.7% of P. vivaxinfected fever subjects had parasite densities \<100/ μL. A large proportion of microscopy positive subjects had low parasite densities ----------------------------------------------------------------------------- Parasite densities were determined for 254 of the 256 microscopy positive subjects and divided into three groups: \<100, 100-500 and \>500/μL. Overall, 61.0% (155/254) of these subjects, including 40.0% (18/45) of the P. falciparumand 65.6% (137/209) of	{ "pile_set_name": "PubMed Central" }
This article was republished on November 20, 2013, to replace an incorrectly published version. Please download this article again to view the correct version. Competing Interests:No competing interests declared.	{ "pile_set_name": "PubMed Central" }
Sakamoto S, Inoue H, Kaneko MK, et al. Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy. Cancer Sci. 2019;110:3595--3602. 10.1111/cas.14196 Sakamoto and Inoue contributed equally to this work. ADCC : antibody‐dependent cellular cytotoxicity CAR : coxsackievirus and adenovirus receptor CDC : complement‐dependent cytotoxicity CTA : cancer tissue array FcγRIIIA : Fcγ receptor IIIA NK : natural killer SCLC : small cell lung cancer 1. INTRODUCTION {#cas14196-sec-0001} =============== Coxsackievirus and adenovirus receptor (CAR) protein that encoded by CXADR gene, is a single‐pass transmembrane protein and is involved in the formation and/or maintenance of epithelial tight junctions.[1](#cas14196-bib-0001){ref-type="ref"} CAR has an essential role in the development of the heart and lymphatic system in mice.[2](#cas14196-bib-0002){ref-type="ref"}, [3](#cas14196-bib-0003){ref-type="ref"} Because CAR is the primary cellular receptor for adenoviruses,[1](#cas14196-bib-0001){ref-type="ref"} its expression level is considered an important factor for adenovirus infection and adenoviral vector‐mediated treatment. It has been reported that high CAR expression levels occur in various human cancers.[4](#cas14196-bib-0004){ref-type="ref"}, [5](#cas14196-bib-0005){ref-type="ref"}, [6](#cas14196-bib-0006){ref-type="ref"}, [7](#cas14196-bib-0007){ref-type="ref"}, [8](#cas14196-bib-0008){ref-type="ref"}, [9](#cas14196-bib-0009){ref-type="ref"}, [10](#cas14196-bib-0010){ref-type="ref"} Moreover, CAR promotes tumor growth in some cancer types. CXADR silencing in lung cancer with high CAR expression suppressed tumor formation ability in xenografts,[11](#cas14196-bib-0011){ref-type="ref"} whereas CXADR knockdown in oral squamous cell carcinoma resulted in the inhibition of both anchorage‐independent growth and metastatic tumor formation in vivo.[12](#cas14196-bib-0012){ref-type="ref"} These previous studies suggest that CAR might be an appropriate target molecule for cancer therapy. Previously, we found that LNCaP‐CR cells, a highly tumorigenic subline of the LNCaP human prostate cancer cell line,[13](#cas14196-bib-0013){ref-type="ref"} express higher levels of CAR than their parental cells.[14](#cas14196-bib-0014){ref-type="ref"} We also developed mouse mAbs against human CAR and found that one of these antibodies (clone 6G10A) significantly inhibited tumor growth in xenografts of human prostate, pancreatic, and colorectal cancer.[14](#cas14196-bib-0014){ref-type="ref"} Based on these findings, we proposed that an anti‐CAR antibody might be a feasible candidate for cancer immunotherapy.[14](#cas14196-bib-0014){ref-type="ref"} Because the immunogenicity of antibodies needs to be reduced for their therapeutic use in humans, mouse‐human chimerization of mouse mAbs is an important step in the development of therapeutic antibodies.[15](#cas14196-bib-0015){ref-type="ref"} In the current study, we generated a mouse‐human chimeric anti‐CAR antibody (ch6G10A) from 6G10A mouse anti‐CAR antibody (mu6G10A), and characterized ch6G10A by flow cytometry, western blotting, ADCC/CDC analyses, and in vivo anti‐tumor activity against a prostate cancer cell line. In addition, we carried out a CTA analysis to investigate CAR expression levels in lung, prostate, and brain tumors, and examined the anti‐tumor activities of anti‐CAR antibodies against SCLC using mouse xenograft models. 2. MATERIALS AND METHODS {#cas14196-sec-0002} ======================== 2.1. Cell lines and reagents {#cas14196-sec-0003} ---------------------------- KHYG‐1/FcγRIIIA cells, a human NK cell line stably expressing Fcγ receptor IIIA (FcγRIIIA), were previously established[16](#cas14196-bib-0016){ref-type="ref"} and were maintained in RPMI‐1640 medium (Nissui Pharmaceutical) supplemented with 10% FBS (PAN Biotech GmbH) and 100 units of human interleukin (IL)‐2 (FUJIFILM Wako Pure Chemical Corporation). Human SCLC cell lines NCI‐H69, DMS53, and DMS114 were maintained in RPMI‐1640 medium containing 10% FBS. Human prostate cancer cell line DU‐145, human SCLC cell line DMS273, GFP‐labeled subline DMS273‐GFP, highly metastatic subline G3H,[17](#cas14196-bib-0017){ref-type="ref"} and C5B (S. Sakamoto, H. Inoue & M. Kawada, unpubl. data, manuscript in preparation) were maintained in DMEM (Nissui) containing 10% FBS. 2.2. Generation of mouse‐human chimeric anti‐human CAR {#cas14196-sec-0004} ------------------------------------------------------ A mouse anti‐human CAR mAb, mu6G10A, was developed as described previously.[14](#cas14196-bib-0014){ref-type="ref"} Generation of mouse‐human chimeric anti‐human CAR (ch6G10A) was previously described.[18](#cas14196-bib-0018){ref-type="ref"} Briefly, the appropriate V ~H~ and V ~L~ cDNAs of mu6G10A and C ~H~ and C ~L~ of human IgG~1~ were subcloned into pcDNA3.3/Neo or pcDNA3.1/Zeo vectors (Thermo Fisher Scientific Inc.), respectively. Antibody expression vectors were transfected into CHO‐S cells (Thermo Fisher Scientific Inc.) using Lipofectamine 2000 reagent (Thermo Fisher Scientific Inc.). Stable transfectants of CHO‐S/ch6G10A were selected by culturing the transfectants in medium containing 1 mg/mL G418 (Nacalai Tesque, Inc.) and 0.5 mg/mL zeocin (InvivoGen). Stable transfectants were cultured for 14 days in CHO‐S‐SFM II medium (Thermo Fisher Scientific Inc.), and then ch6G10A immunoglobulin was purified from the culture supernatant using Protein G Sepharose (GE Healthcare United Kingdom, Ltd). Purity of ch6G10A was evaluated by SDS‐PAGE and Coomassie Brilliant Blue staining. 2.3. Flow cytometry {#cas14196-sec-0005} ------------------- Cells were fixed in 4% neutral buffered formalin for 10 minutes and suspended in PBS containing 0.1% NaN~3~. Cells were resuspended in PBS containing 0.5% BSA and incubated with anti‐CAR antibodies for 1 hour at 4°C and then with PE‐labeled secondary antibodies for 1 hour at room temperature. After washing with PBS containing 0.1% NaN~3~, cells were analyzed by FACSCalibur (BD Biosciences). 2.4. Western blot analysis {#cas14196-sec-0006} -------------------------- Western blot analysis was carried out as described previously.[19](#cas14196-bib-0019){ref-type="ref"} Briefly, cultured cell pellets were lysed with lysis buffer (20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1% Triton X‐100, 10% glycerol, 1 mmol/L EDTA, 50 mmol/L NaF, 50 mmol/L β‐glycerophosphate, 1 mmol/L Na~3~VO~4~, 1× cOmplete Protease Inhibitor Cocktail; Roche). Lysates were centrifuged at 13 000 g for 15 minutes at 4°C, and the supernatants were boiled in SDS sample buffer containing 0.5 mol/L β‐mercaptoethanol. These samples were separated by 12.5% SDS‐PAGE and transferred to PVDF membranes (Merck KGaA). Protein levels were detected using the following antibodies: rabbit polyclonal antibodies specific for CAR (H‐300/sc‐15405; Santa Cruz Biochemical) and rabbit polyclonal antibodies specific for HSP90 (H‐114/sc‐7947; Santa Cruz Biochemical). 2.5. Immunohistochemical analysis of cancer tissue arrays {#cas14196-sec-0007} --------------------------------------------------------- Cancer tissue arrays (BC041115c for lung cancer, BC19013 for prostate cancer, and GL803b for brain cancer) were purchased from US Biomax. The arrays were deparaffinized and rehydrated. Then, antigens were unmasked using citrate buffer for 10 minutes at 96°C. Prior to antibody reactions, endogenous peroxidase was	{ "pile_set_name": "PubMed Central" }
Acute ischemic stroke patients with large established infarction treated with reperfusion therapy are more likely to have a poor functional outcome and reperfusion edema or haemorrhage.^[@R1]^ The Alberta Stroke Program Early Computed Tomography Score (ASPECTS) score is a 10-item score assessing brain parenchyma hypodensity in predefined anterior circulation regions on brain noncontrast CT as a marker of early ischemic changes.^[@R2]^ Use of the score was part of the imaging inclusion criteria in several large randomized clinical trials on endovascular clot retrieval in ischemic stroke.^[@R3][@R4][@R5]^ The ASPECTS score is widely used to identify acute ischemic stroke patients with established infarction who may not benefit or may be harmed by reperfusion therapy. Although the ASPECTS score correlates well with functional outcome in ischemic stroke patients evaluated for thrombolysis, correlation with functional outcome after thrombectomy is less robust, especially when a cutoff value of the score is used to exclude patients from treatment.^[@R2],[@R6][@R7][@R8]^ Benefit of thrombectomy is preserved in patients with ASPECTS scores lower than the cutoff value proposed in the ASPECTS validation study (i.e., 8) and in patients with scores lower than the cutoff value commonly used for selection of patients for reperfusion therapy (i.e., 6 or 7).^[@R2],[@R8]^ CT perfusion to evaluate ischemic core and penumbra volume of ischemic stroke has shown to correlate well with acute MRI diffusion-weighted imaging (DWI) lesion volume.^[@R9],[@R10]^ Compared to acute brain MRI, CT perfusion is more readily available and rapidly acquired.^[@R9]^ Software processing of CT perfusion source images can generate ischemic core and penumbra maps that can accurately identify the acute infarct and tissue at risk for infarction.^[@R10]^ The addition of CT perfusion imaging, however, is more time-consuming compared to noncontrast CT and CT angiography imaging alone. We therefore aimed to compare the accuracy of brain noncontrast CT ASPECTS and CT perfusion core volume to identify patients with established infarction. METHODS {#s1} ======= Patient population. {#s1-1} ------------------- We retrospectively analyzed a convenience series of 59 confirmed acute anterior circulation ischemic stroke patients between May 2003 and December 2011 who presented within 6 hours from stroke onset and underwent acute multimodal CT as well as MRI. Each patient underwent a noncontrast CT, CT angiography, and CT perfusion as part of the standard acute stroke assessment. All patients then went on to have MRI brain within 100 minutes from CT perfusion. Thrombolytic treatment was initiated between CT and MRI in eligible patients. As this study was performed before validation of endovascular clot retrieval therapy in acute ischemic stroke, no patient received a thrombectomy. Imaging protocol. {#s1-2} ----------------- CT imaging was performed on a 16-slice Philips (Best, the Netherlands) Mx8000 or a 64-slice Philips Brilliance. A 40 mL bolus of contrast agent (Ultravist 370; Bayer HealthCare, Whippany, NJ) at a rate of 6 mL/s was used to acquire CT perfusion images at 45 time points of 1.33 seconds. Four to eight adjacent 5- to 6-mm slices covered 24--40 mm sections, using a 16- or 64-slice scanner, respectively. We used commercially available MIStar software (Apollo Medical Imaging Technology, Melbourne, Australia) to process CT perfusion images and generate cerebral blood volume, cerebral blood flow, mean transit time, and delay time, as well as ischemic core and penumbra maps.^[@R10][@R11][@R12]^ Core and penumbra maps are graphically represented as red and green areas, respectively, superimposed on the noncontrast CT ([figure 1](#F1){ref-type="fig"}). The software automatically selects global arterial input function from a large intracranial artery and a venous outflow function from a large draining cerebral venous sinus. A model-free singular value decomposition is used to deconvolve the tissue enhancement curve and the arterial input function with automated delay and dispersion correction.^[@R13],[@R14]^ Core infarct is defined as the brain region with a relative cerebral blood flow of less than 30% (compared to the contralateral hemisphere) within a region with a delay time of more than 3 seconds and a minimum cluster size of 5 mm. The perfusion lesion is defined as the region with a delay time of more than 3 seconds and the penumbra as perfusion lesion after exclusion of the ischemic core. Areas of no blood flow, chronic infarction, or CSF regions are automatically masked from the perfusion maps: no blood flow pixels are removed by eliminating areas where cerebral blood flow = 0 and a Hounsfield threshold and geometrical analysis is used to remove skull and CSF or ventricle pixels. ![Noncontrast CT, core (red) and penumbra (green) CT perfusion maps, and acute diffusion-weighted imaging (DWI) MRI in 3 different patients\ (A) Agreement among all imaging modalities (Alberta Stroke Program Early Computed Tomography Score \[ASPECTS\] score 5, CT perfusion core volume 58 mL, MRI DWI volume 106 mL). (B) Established large infarct with high ASPECTS score (ASPECTS score 9, CT perfusion core volume 52 mL, MRI DWI volume 90 mL).](NEUROLOGY2016763458FF1){#F1} MRI brain was performed on a 1.5 T MRI (Siemens \[Munich, Germany\] Avanto) and included axial isotropic DWI, echoplanar spin-echo sequence, time of flight magnetic resonance angiography, and bolus-tracking perfusion-weighted imaging.^[@R15]^ Data collection. {#s1-3} ---------------- Prior to data collection, patients with missing imaging data were excluded (n = 5). ASPECTS was performed by 4 independent vascular neurologists at 2 centers. Assessors were unblinded to the affected hemisphere. CT images were assessed using nonstandard, variable soft copy, narrow window and level settings centered between 35--45 HU width and 35--45 HU level ([aspectsinstroke.com](http://www.aspectsinstroke.com/)). After collection of all imaging data, a consensus meeting was organized to solve disagreement between raters and to obtain a single ASPECTS score per patient. We used commercial MIStar software to calculate the ischemic core volume on CT perfusion, applying the abovementioned thresholds. CT perfusion imaging analysis was performed after ASPECTS rating. We used an area of interest tool for semiautomated selection of lesion outline to delineate acute MRI DWI lesions based on signal intensity (contrast). The tool automatically measured the volume of the DWI lesion by adding the highlighted area in each slice the lesion was visualized. A DWI lesion volume of at least 70 mL was chosen to represent a large infarct volume beyond which the benefit of IV reperfusion therapy may be limited.^[@R10],[@R16],[@R17]^ Assessment of MRI DWI lesions was performed after acute CT perfusion imaging analysis. Before perfusion postprocessing, CT perfusion source images were coregistered to the corresponding MRI DWI images. Next, the MRI DWI areas of interest were transferred to the coregistered acute CT perfusion core--penumbra maps for statistical volume analysis. Volume coverage of the acute MRI DWI lesion by the CT perfusion core lesion was then calculated. Statistical analysis. {#s1-4} --------------------- All statistical analyses were performed with SPSS version 20 (IBM Corp., Armonk, NY). The area under the receiver operating characteristic curve (AUC) was calculated to estimate the accuracy of noncontrast CT ASPECTS and CT perfusion core volume to predict a large acute MRI DWI lesion volume (\>70 mL) and the method of Delong et al. was applied to compare the AUC between ASPECTS and CT perfusion.^[@R18]^ Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and correct classification rate were then calculated for different ASPECTS cutoff scores and CT perfusion core volume cutoffs to predict a DWI lesion of at least 70 mL using standard 2 × 2 contingency table methodology. Next, the Youden index was used to determine the optimum ASPECTS cutoff score and CT perfusion core volume. Intraclass correlation coefficient (ICC) was used to estimate interrater agreement for ASPECTS scoring (2-way mixed effects model with measures of absolute agreement). AUC was used to determine accuracy for ASPECTS, CT perfusion core volume, and MRI DWI lesion volume to predict poor functional outcome at 90 days after the stroke, defined as a modified Rankin Scale score of 3--6. The correlation between CT perfusion core volume and MRI DWI lesion volume was measured by calculating Pearson correlation coefficient (Pearson R) and the coefficient of determination (R^2^). ICC was calculated as a measure for agreement between CT perfusion core volume and DWI lesion volume. A scatterplot and Bland-Altman plot was used to further examine the correlation between CT perfusion and MRI DWI lesion volume. A Wilcoxon rank sum test was used to assess statistical differences between onset and imaging time in patients with high vs low ASPECTS scores. Standard protocol approvals, registrations, and patient consents. {#s1-5} ----------------------------------------------------------------- The study was approved by the Hunter New England Human Research Ethics Committee and all patients gave informed consent. RESULTS {#s2} ======= Patient characteristics. {#s2-1} ------------------------ The median patient age was 76 years (interquartile range \[IQR\] 68--84). Forty-nine percent of the population was male. Median NIH Stroke Scale score at admission was 15 (IQR 12--17). Median onset to admission time was 157 minutes (IQR 113--166). Median	{ "pile_set_name": "PubMed Central" }
INTRODUCTION ============ Protein interactions take place physically between interface residues of two complementary proteins. Studies focusing on protein interfaces have revealed that binding energies are not uniformly distributed along the protein interfaces. Instead, there are certain critical residues called 'hot spots'. These residues comprise only a small fraction of interfaces yet account for the majority of the binding energy ([@B1; @B2; @B3]). These residues are observed to be critical for function and stability of the protein association ([@B1]). There are several sites collecting the experimental hot spots. Thorn and Bogan ([@B4]) deposited hot spots from alanine scanning mutagenesis experiments, in a database called ASEdb. BID is an effort to organize protein interaction data compiled from the literature and presents amino acids at the protein--protein binding interfaces ([@B5]). Yet, these servers provide hot spots for only a limited number of proteins. Computational methods can introduce alternative approaches to experimental techniques to detect and catalog hot spots ([@B6]). Several groups have developed energy-based methods to predict hot spots ([@B7; @B8; @B9]). Molecular dynamics studies can also be used to investigate the energetic contributions of interface residues ([@B10; @B11; @B12]). While both energy and MD-based methods are very efficient, they are at the same time costly and not applicable in large-scale hot spot prediction. Residues in protein interfaces ([@B13]) and functional sites ([@B14]) were observed to be mutating at a slower pace compared to the rest of the protein surface. There are several studies focusing on the detection of hot spots based on conservation. A very recent study based on sequence environment and evolutionary profile of residues predicts computational hot spots ([@B15]). Correlation between hot spot residues and structurally conserved residues were found to be remarkable ([@B16; @B17; @B18; @B19]). These hot spots are also found to be buried and tightly packed with other residues ([@B18]) resulting in densely packed clusters of networked hot spots, called 'hot regions'. Here, we present HotSprint, a database documenting computational hot spots in the protein interfaces combining conservation and solvent accessibility of residues in the protein interfaces. HotSprint contains protein interfaces extracted from the structures in Protein Data Bank (PDB) and is the first database, to our knowledge, which exploits sequence conservation to detect hot spots on a large scale. Total 49 512 interfaces are extracted from 34 817 PDB entries as of February 2006. Conserved residues of 35 776 protein interfaces are found using Rate4Site algorithm ([@B20]). NACCESS is used to obtain the solvent accessibility of residues ([@B21]). In summary, HotSprint marks residues that are highly conserved and tightly packed in protein interfaces as hot spots. METHODOLOGY AND RESULTS ======================= Interface datasets ------------------ The interfaces, used for the identification of the computational hot spots in the HotSprint, are taken from the updated version of interface dataset generated by Keskin et al. ([@B22]). Interfaces were generated by the atomic distance criteria: if the distance between any atoms of two residues, one from each chain, is less than the summation of their van der Waals radii plus a tolerance 0.5 Å, these residues are named as interface residues. If the distance between non-interacting and interacting residues in the same chain is smaller than 6 Å, the non-interacting residue is named a 'nearby' (neighboring) residue. Nearby residues are important for the information about the architecture of the interface and provided in our database. All 15 268 multi-chain PDB structures are used to extract two chain interfaces and then interfaces having less than 10 residues are eliminated. The resulting dataset contains 49 512 two-chained interfaces that are denoted by six-letter nomenclature where the first four letters denote the PDB ID, and the last two letters are the chain identifier. Detection of computational hot spots in protein interfaces ---------------------------------------------------------- HotSprint database can be accessed through a web interface where users can search for computational hot spots in protein interfaces. The evolutionarily conserved residues are found by Rate4Site algorithm ([@B20]). Rate4Site makes use of topology and branch lengths of the phylogenetic trees constructed from multiple sequence alignments (MSA) of proteins and estimates conservation rates of amino acids based on the empirical Bayesian rule. MSAs of proteins constituting interfaces are taken from HSSP (Homology-Derived Secondary Structure of Proteins) ([@B23]) database as of 14 January 2006. All MSAs obtained from HSSP are converted to FASTA format to be used in Rate4Site step. In addition, some residues are more frequently observed to be hot spots, so each of the 20 amino acids has a different propensity to be a hot spot. Hot spot propensities are used to rescale the conservation scores. Further, hot spots prefer to reside in protein cavities ([@B24]), therefore surface area accessibility of interface residues are incorporated into our hot spot scoring formula. The computational hot spot score of ith residue in a chain is defined as pScore~i~ = score~i~ x P~k~, where score~i~ is the conservation score from Rate4Site ([@B25]), P~k~ is the propensity of residue type k (i.e, k = ALA, VAL, etc.) to be conserved in the interface (details are given in the Supplementary Data). For an amino acid in a protein interface to be considered as a computational hot spot, we propose that following formulation should be satisfied: pScore~i~ \> t and ΔASA \> t~ASA~ and ASA~complex~ \< t~ASAx~ where t, t~ASA~ and t~ASAx~ are user-defined thresholds, the default values are set to 6.2, and 49 and 12 Å^2^, respectively. ΔASA is the ASA change of the residue upon complexation, ΔASA = ASA~monomer~ − ASA~complex~, ASA of the residue in the monomer and complex form, respectively. In ASA calculations, NACCESS ([@B21]) is used and buried ASAs of interface are calculated for each interface. Thus, this formulation combines amino acid conservation scores obtained from Rate4Site \[scaled with amino acid conservation propensities (e.g. aromatic residues are observed to be hot spots independent of their sequence position)\] and ASA of the residue. [Figure 1](#F1){ref-type="fig"} summarizes the flowchart to detect computational hot spots in interfaces. Figure 1.The flowchart of the procedure to predict hot spots and deposit them in the HotSPrint. We have evaluated prediction performance of our formulation by comparing the results with the experimental hot spot data extracted from ASEdb ([@B4]). We assessed success of the formulations using the statistical analysis using 'Accuracy' and 'f-measure'. Our formulation yields 76.83%, 60.1%, 86.56%, 63.06% and 65.69% for accuracy (percentage of correctly predicted hot spot and non-hot spot residues over all interface residues), sensitivity (ratio of correctly predicted hot spots to all hot spots residues on the interface), specificity (ratio of correctly predicted non-hot spots to all non-hot spot interface residues), positive predictive value (number of correctly predicted hot spots divided by number of interface residues predicted as hot spot) and f-measure \[2 × sensitivity × ppv/(sensitivity + ppv) where ppv is the positive predictive value\], respectively. Ofran and Rost recently developed a sequence environment and evolutionary profile-based method to predict computational hot spots ([@B15]). They considered residues contributing ≥2.5 kcal/mol as hot spots. When we adopt the same convention, their positive predictive value (referred as positive accuracy in their text) of ∼60%, outperforms ours (∼46%). However, our sensitivity (57%, coverage in their text) is remarkably higher than theirs (15%). Web interface and querying the HotSprint database ------------------------------------------------- HotSprint provides an easy query screen with three distinct query boxes: (i) hot spot search in protein interfaces for a given PDB ID, (ii) advanced search box and (iii) conservation and ASA querying of the complete protein (including non-interface residues). The computational hot spots in the interfaces can be identified based on one of the three options mentioned in Supplementary Data. One may either choose (i) the default hot spot criterion as defined in the Methods section (pScore + ASA, conservation score rescaled with conservation propensity + contribution of ASA), (ii) only conservation criterion (score) or (iii) conservation score rescaled with conservation propensity (pScore) in the query page. The first query box allows the user to fetch associated interfaces of a given protein using its PDB identifier. The default thresholds in these expressions can also be modified by the user. If there exists only a single interface associated with the input PDB identifier (e.g. for PDB ID: 1axd), then information for that interface (1axdAB) is displayed. However, there may be more than one interface extracted from that protein. In this case, interface identifiers of interfaces associated with that PDB are displayed (e.g. for the PDB ID 1yp2, four interfaces are available 1yp2AB, 1yp2AD, 1yp2BC and 1yp2CD). When one selects one of the interface identifiers listed, information for that interface is presented. [Figure 2](#F2){ref-type="fig"} demonstrates the result page yielded after querying the interface 1yp2AB among the associated interfaces of 1yp2. Figure 2.	{ "pile_set_name": "PubMed Central" }
Introduction {#s1} ============ Reputations, information that signals the potential cooperativeness of others (Tennie et al., [@B62]), are known to facilitate cooperation (Milinski et al., [@B41]; Feinberg et al., [@B22]). Due to their impact, however, reputations are subject to distortion (e.g., spreading lies or gossip) by others seeking to flatter allies and tarnish rivals (Mayzlin, [@B39]). Although this fact underscores the importance of disregarding unfounded reputations, this ability appears limited. Bad reputations are difficult to ignore and can continue to affect one's judgment even after they are shown to be false (Suzuki et al., [@B60]). The persistent effects of bad reputations are problematic given their power to cause avoidance (Chevalier and Mayzlin, [@B14]) and ostracism (Feinberg et al., [@B22]) of the target individuals. Elucidating the neural mechanisms underlying this persistence is therefore of theoretical and practical interest. There has been growing interest in the neural basis of reputation processing (Frith and Frith, [@B26]; Izuma, [@B30]), and a few studies have investigated brain mechanisms underlying the difficulty of "unlearning" reputations through reinforcement learning based on social interactions. For example, Delgado et al. ([@B18]) measured brain activity while participants were playing an iterated trust game and examined how it was modulated by the presence or absence of prior reputation about trading partners. When no reputation was available, the striatum showed greater responses to the partner's cooperation than to cheating; when good or bad reputation was provided in advance, however, such differential striatal activity depending on the partner's behavior diminished. These findings have been elaborated by Fouragnan et al. ([@B24]) who conducted a similar experiment and analyzed the data using a computational model of reinforcement learning. They demonstrated that prior reputation about partners attenuated striatal activity in response to the trial-by-trial prediction error---the difference between the expected value of trusting a partner and the actual outcome from having trusted (i.e., cooperation \[reward\] or cheating \[loss\])---during a trust game. In contrast, the neural underpinnings of the failure to intentionally disregard false reputations after being given verbal instructions that undermine their credibility (Suzuki et al., [@B60]) remain poorly studied. The present study approached this issue from the perspective of evaluation transfer in evaluative conditioning (Martin and Levey, [@B38]; Jones et al., [@B31]; Gawronski and Bodenhausen, [@B27]; but see also Hofmann et al., [@B28]). This refers to the transfer of the evaluation from an unconditioned stimulus (US) to a conditioned stimulus (CS).[^1^](#fn0001){ref-type="fn"} For instance, suppose you are told that Ken, a bank employee, embezzled money from client accounts. A subsequent encounter with Ken will remind you of his embezzlement, and you are likely to conclude that Ken is untrustworthy because of his cheating behavior. In addition, reputation learning may also cause the transfer of the evaluation from the reputation (US) to the target individual (CS) such that the person acquires an ability to activate a positive or negative evaluation on their own. That is, the negative evaluation made about embezzlement becomes associated with Ken himself and consequently Ken alone generates a negative evaluation. In neural terms, evaluation transfer would be operationalized as the CS alone activating evaluation-related brain structures. In general, item evaluation is considered to be an essential component of emotional processing (Russell, [@B53]). Thus, many cortical and subcortical structures linked with emotions are assumed to be involved in the evaluation of stimuli, including ventral portions of the prefrontal cortex (Kringelbach and Rolls, [@B35]), anterior parts of the insular (Singer et al., [@B58]) and cingulate cortices (Rushworth and Behrens, [@B52]), the amygdala (Morrison and Salzman, [@B42]), and the striatum (O'Doherty, [@B44]). Of particular relevance to reputation learning, these neural structures have been implicated in the evaluation of others' behavior in previous studies (Sanfey et al., [@B56]; Delgado et al., [@B18]; Buckholtz et al., [@B10]; Rilling et al., [@B50]; Schiller et al., [@B57]; Mende-Siedlecki et al., [@B40]). Thus, after learning one's reputation, the target individual may activate the evaluation-related brain region on his/her own, constituting a neural correlate of evaluation transfer. More specifically, with regard to bad reputations, the involvement of the lateral and ventral portions of the prefrontal cortex and the anterior insula might be expected since these regions have been implicated in anger and disgust (Murphy et al., [@B43]; Vytal and Hamann, [@B65]; but see also Lindquist and Barrett, [@B37]), which are negative emotions closely related to appraisals of harmfulness and immorality (Hutcherson and Gross, [@B29]). A hallmark of evaluation transfer is the difficulty with which one can intentionally negate its effects (Gawronski and Bodenhausen, [@B27]). Suppose that Ken's reputation as an embezzler is later found to be invalid. Then, you will not reason that Ken is untrustworthy from the inaccurate reputation that he embezzled money. However, if evaluation transfer has occurred, Ken will still elicit negative evaluations independently of the validity of his reputation, and therefore, will continue to be distrusted. It has indeed been reported that the effect of evaluation transfer cannot be neutralized voluntarily (Sweldens et al., [@B61]; Balas and Gawronski, [@B4]), and that negative evaluations are especially transferable (Rydell and Jones, [@B54]; Bell et al., [@B8]; Campbell and Warren, [@B11]). It can therefore be hypothesized that the persisting effects of a bad reputation are related to a transfer of the negative evaluation about the bad reputation to the target person. Here, we report a functional magnetic resonance imaging (fMRI) experiment testing this hypothesis. In this fMRI study, participants memorized faces paired with either a good or a bad reputation. Next, they viewed the faces alone and inferred whether each person would be likely to cooperate, first while retrieving the memorized reputations and then while trying to disregard them as false. If reputation learning transfers the negative evaluation of bad reputations to target persons, and if the transfer is related to the persisting distrust, the following two predictions would be made. Prediction 1: Face stimuli that are paired with bad reputations during a learning task will activate an evaluation-related brain region during an inference task, irrespective of whether participants attempt to retrieve or disregard the reputations. Prediction 2: Participants showing higher activity of the region described in Prediction 1 will infer that the persons with bad reputations are less likely to cooperate, irrespective of whether participants attempt to retrieve or disregard the reputations. Materials and Methods {#s2} ===================== Participants {#s2-1} ------------ Thirty-two undergraduate and graduate students (18 males and 14 females; age 20--31 years) gave informed consent to participate in this study, which was approved by the Ethics Committees of the National Center for Geriatrics and Gerontology, Japan, and the Graduate School of Environmental Studies, Nagoya University, Japan. Four participants (2 males and 2 females) were excluded from analyses because one withdrew due to fatigue, two expressed suspicion about a cover story for the experiment, and one showed perfect performance in the good-reputation condition of the baseline session of action inference.[^2^](#fn0002){ref-type="fn"}^,^[^3^](#fn0003){ref-type="fn"} Stimuli {#s2-2} ------- Twenty-four neutral faces of Japanese individuals (12 males and 12 females) from the Facial Information Norm Database (Watanabe et al., [@B66]) were used as stimuli. They were divided into three groups and each was assigned to one of the three conditions: good-, bad-, and no-reputation. The assignment of reputations to faces was counterbalanced across participants. The three groups of faces were matched for number of males and females and mean trustworthiness rating (1, very untrustworthy, to 5, very trustworthy) assessed in a preliminary survey with 102 participants (M = 2.96, 2.96, 2.97; unpublished data). All face stimuli were presented in gray scale and were cropped into square shapes (270 × 270 pixels) so that only the central facial features (eyes, eyebrows, nose, and mouth) were visible. Experimental Procedure {#s2-3} ---------------------- The experiment consisted of four tasks in the following order: baseline session of action inference, reputation learning, retrieval, and disregard sessions of action inference (Figure [1](#F1){ref-type="fig"}). The baseline session of action inference was performed on a laptop computer (HP ProBook 4740s, Hewlett-Packard Japan, Ltd., Tokyo, Japan) outside an MRI scanner. The other tasks were administered inside the scanner, with a short rest outside of the scanner after completion of the reputation-learning task.[^4^](#fn0004){ref-type="fn"} Inside the scanner, stimuli were presented with VisuaStim digital goggles (Resonance Technology, Inc., Northridge, CA, USA), and responses were collected via	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1} =============== Fritillaria cirrhosa, which belongs to the family Lilliaceae, is primarily distributed in the southwestern China. Bulbs ofFritillaria cirrhosa (BFC) have long been used both for food and folk medicine in many Asian countries \[[@B1]\]. The materials from wild collections can be harvested at least for five years \[[@B2]\]. In the past, the original plants of BFC were mainly obtained from their wild species and thus were unable to meet the demands of the industry \[[@B3]\]. In recent years, there is a significant breakthrough in artificial plantation technologies. The bulbs of cultivatedFritillaria cirrhosa (BCFC) have become the mainstream of BFC in market, which provide the adequate resources for further exploitation and utilization of BFC. Studies on phytochemistry of BFC indicated that chemical components of BFC included steroidal alkaloids, saponins, terpenoids, glycosides, and many other compounds \[[@B4]\]. As revealed by modern studies, steroidal alkaloids were the major biological active compositions of BFC \[[@B5]\]. Pharmacological studies found that alkaloids from BFC exhibited remarkable antitussive, expectorant, antiasthmatic properties \[[@B6], [@B7]\], hypotensive effects \[[@B8]\], antibacterial activity, antitumor effects \[[@B9]--[@B11]\], anti-inflammatory effects \[[@B12], [@B13]\], and so on. However, the content of total alkaloids of BFC is quite low and varies wildly, approximately ranging from 0.02% to 0.3%, which is regarded as major quality constraint for its applications. To the best of our knowledge, there are few systematic publication focusing on optimization of the extraction and enrichment for large-scale production of alkaloids from BCFC; thus, there is an urgent need to optimize the extracting and enriching conditions. Orthogonal array design (OAD) has been widely used to optimize the factors which influenced the extraction yield or extract profiles of bioactive components from natural materials \[[@B14]\]. It has been used to optimize experimental conditions with fewer numbers of experiments \[[@B15], [@B16]\]. In this study, OAD was also used to optimize the extraction conditions. There are several conventional methods, such as polyamide chromatography, gel chromatography, and silica gel column, available for the enrichment of active constituents \[[@B14]\]. However, these methods have several disadvantages, including long time consuming poisonous residual solvents and low recoveries \[[@B17]\]. Recently, growing attention has been taken to enrich and purify targeted components from crude biological samples using macroporous resins for their convenience, low operating costs, low solvent consumption, high chemically stability, and easy regeneration \[[@B14], [@B17]\]. In this study, the absorption and desorption on macroporous resins were utilized for the enrichment of alkaloids from BCFC. The present study focused on the optimization of extraction and enrichment of alkaloids from BCFC to develop a cost-effective method for large-scale extraction and enrichment of alkaloids. In extraction process, we tried to optimize four independent variables by using OAD. For enrichment of alkaloids, the optimum macroporous resin was screened and the major process parameters were determined. Meanwhile, the experimental isotherm data were analyzed using pseudo-first- and second-order models, the Langmuir and Freundlich equations. Finally, macroporous resin chromatography was used for the enrichment of alkaloids from BCFC. 2. Materials and Methods {#sec2} ======================== 2.1. Plant Materials and Reagents {#sec2.1} --------------------------------- BCFC were purchased from the dealers who cultivate theFritillaria cirrhosa at Chengdu International Trade City Hehuachi Chinese Medicinal Herbal Market (Chengdu, Sichuan province, China) and identified by Professor Shu Wang (Department of Pharmacognosy, West China College of Pharmacy, Sichuan University, Chengdu, China). The sample (Wang 20120510) has been deposited in the pharmacognosy lab of West China College of Pharmacy, Sichuan University. Anhydrous ethanol, ammonia water, ethylenediamine, and chloroform were analytical grade obtained from Tianjing Kemiou Chemical Reagents Co. (Tianjing, China). Acetonitrile of chromatographic grade was purchased from Sigma Aldrich, Inc. (Steinheim, Germany). Imperialine and peimisine were isolated from BFC in our laboratory \[[@B12], [@B13]\]. The ultrapure water was prepared by a Milli-Q water system (Millipore, USA). 2.2. Selection of Extraction Parameters {#sec2.2} --------------------------------------- The preprepared powder of BCFC (50 g) was soaked in 70 mL ammonia for different times (0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, and 4.0 h) and then extracted under reflux with different concentrations of ethanol (50%, 60%, 70%, 80%, 90%, and 100%) with different ratios of liquid/solid (mL : g) (5 : 1, 10 : 1, 15 : 1, 20 : 1, 25 : 1, and 30 : 1) for different given times (60 min, 90 min, 120 min, 150 min, 180 min and 240 min), while the temperature of the water bath was set as 50°C, 60°C, 70°C, 80°C, 90°C, and 95°C, respectively, and kept steady (within ±2.0°C), for different extraction times (once, twice, thrice, and four times) \[[@B18]\]. 2.3. Orthogonal Experimental Design of Extraction Procedure {#sec2.3} ----------------------------------------------------------- Based on single-factor experimental results, four independent parameters (ethanol concentration (v/v, C), solid-liquid ratio (mL/g, R), extraction time (min, t), and temperature (°C, T) were confirmed as the major influencing factors; then a L~9~(3^4^) orthogonal experimental design was conducted to evaluate the effects of four independent variables on the extraction efficiency of total alkaloids, which was reflected by extraction yield of total alkaloids of BCFC. The range and levels of individual variables are shown in [Table 1](#tab1){ref-type="table"}. The experiment design is shown in [Table 2](#tab2){ref-type="table"}, along with experimental data. All experiments were run in twice. 2.4. Preparation of Sample Solution and Absorbents {#sec2.4} -------------------------------------------------- The extract process was done according to optimal extraction conditions obtained in [Section 2.3](#sec2.3){ref-type="sec"}. The extracting solution was filtered, the filtrates were combined and evaporated using a vacuum rotary evaporator at 45°C and driedin vacuo for 72 h to yield ethanol extracts. The sample solution was prepared by dissolving the dried ethanol extracts in distilled water. The physical properties of the 16 kinds of adsorbents are summarized in [Table 3](#tab3){ref-type="table"}. For prior use, the adsorbents needed to be soaked for 24 h with 100% ethanol and then washed with 1 mol/L HCl and NaOH solution successively and finally washed with distilled water to remove monomers and porogenic agents trapped inside the pores during the synthesis process \[[@B17]\]. 2.5. Adsorption Resins Screening {#sec2.5} -------------------------------- ### 2.5.1. Adsorption Resins Screening {#sec2.5.1} The optimum resin was screened by static adsorption and desorption tests. The adsorption tests on different resins were performed as follows: preweighed amounts of hydrated resins (equal to 0.5 g dry resin) and 100 mL sample solution (0.3271 mg/mL) were added into 250 mL flasks which were continually shaken for 24 h at 30°C with 120 rpm. The solutions after adsorption were separated from the resins and analyzed by Alpha-1900PC UV-Vis spectrophotometer and Shimadzu-10AT HPLC coupled to Sedex-75 evaporative light-scattering detector (HPLC-ELSD) according to the method described previously \[[@B19], [@B20]\]. The static desorption tests were conducted as follows: after reaching adsorption equilibrium, the resins were washed with 100 mL distilled water and then desorbed with 50 mL 80% ethanol solution. The flasks were continually shaken for 12 h at 30°C with 120 rpm. The adsorption and desorption properties of different resins for alkaloids including adsorption capacity, desorption capacity, and desorption ratio of each resin were quantified with the following equations \[[@B14], [@B21], [@B22]\]: $$\begin{matrix} {Q_{e} = \left( {C_{0} - C_{e}} \right) \times \frac{V_{i}}{W},} \\ {Q_{d} = \frac{C_{d} \times V_{d}}{W},} \\ {D = \frac{C_{d} \times V_{d}}{\left\lbrack {\left( {C_{0} - C_{e}} \right) \times V_{i}} \right\rbrack} \times 100,} \\ \end{matrix}$$ where Q ~e~ is the adsorption capacity at adsorption equilibrium (mg/g dry	{ "pile_set_name": "PubMed Central" }
Introduction {#s1} ============ Inbreeding, defined as the mating between two related individuals, increases the occurrence of homozygous deleterious alleles. The loss of heterozygosity leads to a decrease in the fitness of offspring, known as inbreeding depression [@pone.0095963-Charlesworth1] although inbreeding may also be associated with benefits [@pone.0095963-Szulkin1]. Inbreeding depression has been reported for most taxa and has led to a variety of inbreeding-avoidance mechanisms [@pone.0095963-Pusey1] e.g. by sex differences in dispersal [@pone.0095963-Pusey2], [@pone.0095963-Perrin1], [@pone.0095963-Bilde1], in life-history [@pone.0095963-Bukowski1], [@pone.0095963-Clarke1], or by mate choice [@pone.0095963-Tregenza1]. The latter requires a kin-recognition mechanism (but see [@pone.0095963-Yasui1]) and can occur before but also after mating [@pone.0095963-Lihoreau1], [@pone.0095963-Penn1]. Post-mating sexual selection requires multiple mating by females which increases copulation costs that should be offset, at least in part, by benefits [@pone.0095963-Zeh1]. Such benefits are particularly enigmatic if they are only of an indirect nature [@pone.0095963-Jennions1]. Indeed, avoidance or reduction of inbreeding costs through post-copulatory mate choice have been identified as a major benefit of female multiple mating in several taxa, such as house mice [@pone.0095963-Firman1], birds [@pone.0095963-Birkhead1], field crickets [@pone.0095963-Tregenza2], [@pone.0095963-Bretman1], [@pone.0095963-Bretman2], and spiders [@pone.0095963-Welke1]. In mating systems with classical sex roles (unselective males maximise fitness by increasing mating rates while reproductive success of females does not increase linearly with the number of mates [@pone.0095963-Bateman1]), females show a larger investment per offspring [@pone.0095963-Smith1], [@pone.0095963-Parker1] and suffer more from inbreeding through the loss of their individual fitness than males that only invested some sperm. Thus, selection to avoid inbreeding in the context of individual fitness costs should act particularly strong on females. However, in mating systems characterised by low male mating rates, males suffer similar fitness costs from inbreeding as females and avoidance of inbreeding should also be favoured in males. Indeed, when male mating rates are lower than female mating rates, selection should act more strongly on males than on females, particularly when polyandrous females possess means of cryptic female choice. These conditions are met in monogynous or bigynous mating systems, which are especially common in spiders [@pone.0095963-Welke2], [@pone.0095963-Fromhage1], [@pone.0095963-Schneider1], [@pone.0095963-Herberstein1]. Males in such mating systems restrict themselves to mating with a single or maximally two females while females appear to favour multiple mating [@pone.0095963-Schneider1], [@pone.0095963-Miller1]. It has been suggested that females oppose monopolisation by a single male through post-copulatory discrimination against less compatible males and there is some evidence that females cryptically discriminate against the sperm of related males [@pone.0095963-Welke1]. However, to date no study has directly measured natural risks and costs of inbreeding for an individual in such mating systems. Inbreeding is particularly likely if a small number of individuals split off from the original population and establish a new population representing only a fraction of the gene pool of the source population [@pone.0095963-Mayr1]. Furthermore, the co-settlement of siblings may promote the risk of inbreeding in the newly founded population. Analogous to the classical scenarios of founding populations and bottlenecks, although only short-term, species that actively expand their range will likely experience a decrease in genetic diversity at the forefront of range expansion in comparison to populations in the centre of a species\' range [@pone.0095963-Eckert1]. This may result in an increased risk of inbreeding at least in the short term. Individuals that reproduce in a new patch may be faced with a reduced choice of mating partners that are perhaps even siblings. The lack of compatible mating partners can entail fitness costs as even one generation of inbreeding can lead to drastic fitness losses of the offspring, e.g. in terms of reduced competitive fertilisation success as reported for male Telegryllus oceanicus [@pone.0095963-Simmons1] or reduced adult lifespan in the spider Argiope australis (Welke & Schneider unpublished data). However, some species are tolerant to short-term inbreeding as for example Stegodyphus lineatus [@pone.0095963-Bilde1], Oedothorax apicatus [@pone.0095963-Bilde2] and Anelosimus cf. jucundus [@pone.0095963-Aviles1]. The degree of inbreeding depression can vary depending on the size and age of the mating population [@pone.0095963-Jamieson1], as well as the potentially involved purging of deleterious recessive alleles [@pone.0095963-Barrett1], [@pone.0095963-Crnokrak1]. Generally, species that are successful colonisers are expected to show some tolerance towards the negative effects of inbreeding [@pone.0095963-Purcell1] or a dispersal mode that does ensure genetic diversity even in newly founded sites. Here, we use the spider Argiope bruennichi (Araneae) that unites a mono- and bigynous mating system and has recently extended its range from southern Europe and Asia to Northern Europe [@pone.0095963-Kumschick1]. The rapid colonisation implies that A. bruennichi can be considered a successful disperser. In combination with the observation that the species has started its range expansion from a large source population, it is likely that newly established populations even by small numbers of individuals encompass some genetic variation. A. bruennichi disperses aerially by ballooning and bridging to move within habitats. This passive mode of dispersal, particularly ballooning, entails a large component of chance as individuals can only influence direction by selecting certain wind conditions to fly [@pone.0095963-Suter1], [@pone.0095963-Bonte1]. The expansion would likely occur through small numbers of individuals establishing new populations and as new meadows are colonised, individual females can expect high reproductive success. A. bruennichi spiderlings hatch simultaneously from large clutches after winter and likely disperse in groups from the same brood when conditions are favourable. This will lead to a situation in which many siblings from a single female are likely present in a meadow that also contains other families. Spiderlings may disperse short or long distances. This scenario creates both, inbreeding risk as siblings encounter one another and costs of inbreeding (note that costs of inbreeding require a population that is not inbred). Such a scenario match data derived from mating experiments and field observations that demonstrated selection to avoid the costs of inbreeding [@pone.0095963-Welke1]. Hence, we predict that genetic diversity is present in small recently colonised meadows but that sibling matings will occur. As a consequence, we predict the presence of inbreeding depression from within-population matings, which should be absent in among-population matings. Hence, we expect a larger variation in hatching success resulting from the former matches in comparison from the latter ones and we expect this to be matched by the occurrence of sibling matches within populations. We collected A. bruennichi egg-sacs and juveniles from four similar sized populations located near the northern edges of the species range. We assessed genetic diversity by analysing 16 microsatellite loci and a part of the mitochondrial COI gene. Furthermore, we assembled mating pairs that stemmed from the same egg-sac, from different egg-sacs of the same population or from two different populations and correlated the genetic distance of the mating partners with mating behaviour and hatching success. We predicted differences in mating behaviour with increasing relatedness of the mating partners and expected genetic distance to be positively correlated with hatching success. While the sampled populations are all located within the recently colonised range of the species, they likely differ in their short-term settlement history in that they may have been populated early in the invasion process or in recent years. Material and Methods {#s2} ==================== Study species {#s2a} ------------- Argiope bruennichi [@pone.0095963-Scopoli1] did not occur in Northern Europe until the beginning of the 20^th^ century with the exception of an isolated group around Berlin [@pone.0095963-Krehenwinkel1]. It expanded its range since around 1930 [@pone.0095963-Kumschick1], [@pone.0095963-Guttmann1] and colonised Northern Germany including the region around Hamburg since 1975	{ "pile_set_name": "PubMed Central" }
Introduction {#sec1} ============ Silicosis is considered as one of the serious types of pneumoconiosis and a potentially fatal occupational fibrotic lung disease \[[@B1]\], which is highly prevalent in developing countries, especially in China, South Africa, and Brazil \[[@B2]\]. Accumulating evidence shows that silicosis is caused by long-term occupational or environmental exposure to free crystalline silica (CS) particles \[[@B3]\]. There are estimated to be tens of millions of workers exposed to CS worldwide and industrialization processes have made this situation even worse \[[@B4]\]. To date, the increasing mortality of silicosis has made silica exposure a high-priority public health concern in countries worldwide \[[@B5]\]. It has been accepted that the hallmarks of lung silicosis consists of massive inflammation and significant lung fibrosis \[[@B6],[@B7]\]. When respirable CS particles are inhaled, their entry into alveoli induces oxidative stress through the formation of reactive oxygen (ROS) and nitrogen species due to the generation of siloxil radicals after crystalline-silica fracturing \[[@B8]\]. Therefore, it leads to extensive fibroblast proliferation and deposition of extracellular matrix (ECM) with the lungs and further triggers cytotoxicity, oxidative stress, pulmonary inflammation, and eventually silicosis \[[@B9]\]. In the meantime, the progression of lung fibrosis is accompanied with infiltration of inflammatory cells including eosinophils, neutrophils, and macrophages. Macrophages, for instance, can phagocyte silica particles and become activated to release a multitude of mediators, such as histamine and serotonin, which are retained in lung throughout inflammation and mediate cascade reaction leading to fibrotic response \[[@B10]\]. In addition, alveolar macrophages, as well as their produced TNF-α, interleukin (IL)-1β and IL-6 has been suggested to play a crucial role in inflammation, as a hallmark of exposure to silica \[[@B11]\]. Besides the aberrant induction of pulmonary inflammation, the intratracheal silica instillation leads to the enlargement of thoracic lymph nodes. The mitogenic responses to T cell receptor stimulation are apparently reduced in lymphocytes from silica-exposed lymph nodes with markedly increased activation-induced cell death, suggesting that silicotic lung apoptosis has been also implicated in the development of the initial inflammation \[[@B12]\]. During the process, Fas-L expression was inversely associated with mast cells, collagen/elastic deposition. As the progression of silicosis involves a fibrotic phase in which the ECM is deposited and lung parenchyma is remodeled, researches have been focussing on the intervening the inflammation processes and CS-induced fibrosis of silicosis \[[@B13]\]. Therefore, there is an urgent need to identify antioxidative and anti-inflammatory agents that might be promising for the prevention and treatment of silicosis. N-acetylcysteine (NAC), a semi-essential amino acid with a thiol side chain, is the acetylated precursor of cysteine. It is clinically used to treat a wide variety of medical issues and conditions \[[@B14]\], such as acute respiratory distress syndrome, heavy metal-induced toxicity, and ameliorate certain psychiatric disorders \[[@B15]\] for its multiple beneficial properties, including enhancing bone regeneration, reducing post-surgical complications \[[@B16]\], and preventing mutagenic irradiation \[[@B17]\]. Furthermore, NAC has been reported to possess antioxidant properties by scavenging reactive oxygen species (ROS) and also inhibit the activity of cyclooxygenase-2 and membrane lipid peroxidation induced by inflammation \[[@B18],[@B19]\]. Coombes et al. suggest that NAC has reno-protective activities in chronic kidney disease \[[@B20]\] and neuro-protective effect on spinal cord-injury \[[@B21]\]. Preliminary data in rat model revealed that oral treatment with high-dose NAC during early silica exposure can ameliorate the activity of proinflammatory cytokines, down-regulating ROS, and mitochondrial apoptosis signaling, thus attenuating subsequent lung fibrosis, suggesting the potential of NAC in the treatment for silica-induced lung fibrosis \[[@B22],[@B23]\]. In the present study, we employed a mouse model of silicosis and different doses of NAC, in order to determine the potential pulmonary protective effects of NAC and the underlying mechanism. Methods {#sec2} ======= Animal groups and treatments {#sec2-1} ---------------------------- Total 138 female wild-type C57BL/6J mice aged 6--8 weeks were purchased from the Experimental Animal Center of Hainan Medical College and randomly divided into the following groups: (1) blank control group (n=18)-animals without any treatment; (2) CS-induced model group (n=30); and (3) NAC-treated groups-animals were underwent CS exposed, and then administrated with NAC via gavage every day at a dose of 1.73 mg/20g (low-dose, n=30, NAC-L group), 3.46 mg/20g (moderate-dose, n=30, NAC-M group), or 5.19 mg/20g (high-dose, n=30, NAC-H group), respectively. For CS-induced model group, suspensions of CS (CAS: 7631-86-9, 2 μm, U.S. Silica Co., WV, Sigma--Aldrich) were prepared in normal saline (0.9% \[w/v\] NaCl). Each animal was received CS administration (2.5 mg suspended in 60 μl of saline) through intratracheal instillation and then administrated with saline via gavage every day, as previously described \[[@B24]\]. On months 0 (24 h), 1, 2, 3, 4, and 5 after treatment, animals were killed and harvested. For blank control group, three mice were used at every time point. Total five mice were used for analysis for the other experimental groups at every time point. The present study was performed in strict accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse experiments were approved by the Ethics Committee on Animal Research of the Hainan Medical College. Preparation of bronchoalveolar lavage fluid {#sec2-2} ------------------------------------------- Bronchoalveolar lavage fluid (BALF) was processed and collected as previously described \[[@B25]\]. Briefly, mice were intraperitoneally injected with 10% chloral hydrate for euthanasia. Immediately after euthanasia, the trachea of each mouse was exposed through a midline incision and cannulated with a sterile 22-gauge Abbocath-T catheter. Approximately 1.0 ml of BALF was retrieved per mouse, and BALF samples were kept on ice to avoid cell lysis. Serum and tissue sample collection {#sec2-3} ---------------------------------- Mice in the above groups were killed and blood samples were obtained from abdominal aorta. After centrifuged at 1500 rpm for 10 min, serum was collected and stored at −80°C. The lung tissues were removed and immediately frozen in liquid nitrogen. Some of the lung tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline for 24 h. The remaining lung tissues were frozen in liquid nitrogen for RNA detection or ELISA assay. ELISA assay {#sec2-4} ----------- The mouse TNF-α ELISA Kit (CSB-E04741m, CUSABIO), mouse IL-1β (CSB-E08054m, CUSABIO), mouse IL-4 (CSB-E04634m, CUSABIO), and mouse IL-6 (CSB-E04639m) were used to determine the concentrations of TNF-α, IL-1β, IL-6, and IL-4 in plasma or BALF, respectively following the manufacturer's instructions. Analysis of mRNA expression by RT-PCR {#sec2-5} ------------------------------------- Total RNA was extracted from mice lung tissues with TRIzol reagent (TaKaRa Biotech), and cDNA was synthesized from 1 μg of total RNA using the Bestar qPCR RT Kit (DBI). RT-PCR was performed using SYBR^®^ Premix Ex Taq™ (TaKaRa) with Roche 480 using the following primers: inducible nitric oxide synthase (iNOS) (F: 5′-GTTCTCAGCCCAACAATACAAGA-3′ and R: 5′-GTGGACGGGTCGATGTCAC-3′); NADPH oxidase 1(NOX1) (F: 5′-CCTGATTCCTGTGTGTCGAAA-3′ and R: 5′-TTGGCTTCTTCTGTAGCGTTC-3′); NOX2 (F: 5′-GTATTGTGGGAGACTGGACG-3′ and R: 5′-ACAGACTTGAGAATGGAGGC-3′); NOX4 (F: 5′-GGTGTCTGCATGGTGGTGGTATT-3′ and R: 5′-CAGCCAGGAGGGTGAGTGTCTA-3′); SOD2 (F: 5′-CAGACCTGCCTTACGACTATGG-3′ and R: 5′-CTCGGTGGCGTTGAGATTGTT-3′); xanthine oxidase (XO) (F: 5′-ATGACGAGGACAACGGTAGAT-3′ and R: 5′-TCATACTTGGAGATCATCACGGT-3′); internal standard β-actin (F: 5′-CATTGCTGACAGGATGCAGA-3′ and R: 5′-CTGCTGGAAGGTGGACAGTGA-3′). The expression data were normalized to actin and quantitated using the Stratagene Mx3000P Real time PCR (Agilent, U.S.A.). Relative expression was calculated using 2^−ΔΔC^~T~ method. H&E staining, IHC, and Masson's trichrome staining {#sec2-6} -------------------------------------------------- Morphological	{ "pile_set_name": "PubMed Central" }
Introduction ============ More than two-thirds of pregnant women exceed the Institute of Medicine (IOM) 2009 gestational weight gain (GWG) recommendations \[[@ref1]\]. According to weight management guidelines for obesity treatment \[[@ref2]\], prenatal care should provide an ideal clinical framework for treatment delivery with frequent visits, weight recording, an established definition of acceptable weight gain, and opportunities for in-person counseling. However, despite several efforts to prevent excessive GWG in clinical trials, it remains unclear if lifestyle interventions can be efficacious, particularly in women with overweight or obesity \[[@ref3],[@ref4]\]. Poor effectiveness in these trials is explained by intervention designs that fail to take advantage of the entire prenatal care continuum because program initiation is often delayed until mid or late gestation and weight management counseling and intervention are limited to one or two in-person sessions \[[@ref3]\]. As more patients have access to mobile phones and 67% of pregnant women subscribe to electronic health information delivery during pregnancy \[[@ref5]\], eHealth interventions designed to target healthy weight gain provide an opportunity for high-intensity and cost-effective interventions to be delivered to all patients throughout prenatal care. The aim of this study was to test whether a personalized gestational weight management program (SmartMoms) delivered in-person or via an intensity-matched mobile phone app could decrease the proportion of women with overweight and obesity that exceed the IOM 2009 guidelines for GWG by 25%. Methods ======= This study targeted overweight and obese (body mass index \[BMI\] 25.0-39.9 kg/m^2^) women aged 18 to 40 years expecting a singleton pregnancy in their first trimester. Women with a known fetal anomaly, hypertension (systolic \>160 mm Hg or diastolic \>90 mm Hg), history of or current psychotic or eating disorder, human immunodeficiency virus, preexisting diabetes (self-report or determined by glycated hemoglobin A~1c~ and/or 75 g oral glucose tolerance test in the first trimester), or with contraindications to exercise (by PARmed-X and American College of Obstetricians and Gynecologists committee opinion \#67 \[[@ref6]\]) were excluded. With support of local obstetricians, participants were recruited from brochures placed in various clinics and by study staff during the patients' first prenatal appointment \[[@ref7]\]. Participants were randomized by unblinded intervention staff equally to one of three groups between 10.4 to 13.6 weeks of gestation: (1) no intervention (usual care group), (2) receipt of the SmartMoms intervention in-person (in-person group), or (3) receipt of the SmartMoms intervention via mobile phone (remote group), with randomization stratified by pregravid BMI. The block randomization schedule and sealed numbered randomization envelopes were prepared by the biostatistician. Usual care (control) participants were under the usual care of their obstetrician and did not receive weight management services from the intervention team. The SmartMoms intervention was designed to assist an expectant mother in gaining weight within the recommended 2009 IOM guidelines for her respective BMI class. SmartMoms is grounded in the ability to objectively quantify dietary adherence to an energy intake prescription based on measured body weight and to provide patients with data-driven feedback about their energy intake \[[@ref8]-[@ref12]\]. SmartMoms participants received dietary intake advice, exercise advice, and a weight graph created from the dynamic GWG models to determine the trimester-specific increase in energy intake required by each participant to adhere to the IOM GWG recommendations \[[@ref13]\]. To promote these lifestyle changes, participants received a structured intervention that consisted of 18 lessons and behavior modification strategies. SmartMoms participants received behavior modification counseling weekly between weeks 13 and 24 of gestation and biweekly from week 25 until delivery. Importantly, the content of the lesson materials were identical and only the mode of delivery differed between the two intervention groups. Participants in both the intervention groups were provided with a wireless Internet-connected bathroom scale and a pedometer (in-person group: Omron Healthcare, Lake Forest, IL, USA; remote group: Fitbit Zip, FitBit, San Francisco, CA, USA) to self-monitor body weight and step counts daily. The mobile phone app is similar to a virtual weight management system described for weight loss in which body weight and daily steps are automatically transmitted in real time to personalized charts \[[@ref14]\]. The SmartMoms intervention includes an IOM 2009 GWG weight graph personalized for each patient and behavioral modification tools including daily self-monitoring of weight, dietary intake, and physical activity \[[@ref14]\]. SmartMoms participants were provided with an individualized calorie intake above their estimated prepregnancy energy requirement \[[@ref13]\] or energy gap represented by an ideal weight gain zone \[[@ref15]\], and were coached how to adjust energy intake and/or physical activity to adhere to the IOM 2009 GWG guidelines. The in-person group tracked step counts with pen and paper, and the IOM weight graph was reviewed in hard copy during counseling sessions with interventionists. Clinic assessments were performed by certified staff who were blinded to group assignment. Maternal weight was measured fasting and in a hospital gown. Total GWG and GWG per week were calculated between the initial (10-13 weeks) and final (35-36 weeks) study visits. GWG per week was used to calculate the proportion of women based on prepregnancy BMI with recommended or excessive GWG per the 2009 IOM GWG guidelines \[[@ref16]\]. Adherence to the SmartMoms intervention was defined as the percentage of days participants weighed and recorded step counts in comparison to the expected number of days. Study economics, including costs incurred for travel to and from treatment sessions and time spent with the counselor while accounting for session attendance and intervention adherence, were calculated for each participant. The clinic economics included cost of interventionist time (training, session preparation, participant contacts, routine staff meetings) and equipment (scale, pedometer) cost. Statistical analyses were completed using SAS/STAT version 9.4 software of the SAS System for Windows (Cary, NC, USA). Sample size was based on the hypothesis that the proportion of overweight and obese pregnant women in the usual care group exceeding IOM guidelines for GWG would be 58% and that lowering this proportion by at least 25% would be clinically significant. Intention-to-treat comparison of continuous variables (eg, GWG, birth weight) between the three treatment groups used one-way analysis of variance with post hoc pairwise intervention group comparisons. Comparisons of categorical variables (eg, prevalence of excess GWG) between the three treatment groups used Fisher exact test. Equality of adherence to IOM GWG guidelines was tested through one-sided z tests for proportions. Finally, differences in study costs and intervention adherence were assessed through two-sample t tests. All tests were performed with significance level alpha=.05, and findings were considered significant when P\<alpha. Data are reported as least square (LS) mean and standard error (SE) unless otherwise noted. Results ======= Recruitment of participants from community clinics from February 1, 2013 to April 14, 2014, yielded three groups of pregnant women who were similar ([Table 1](#table1){ref-type="table"}). The majority of participants were white and nulliparous or primiparous. No study-related serious adverse events were reported. Gestational Weight Gain and Guideline Adherence ----------------------------------------------- The SmartMoms intervention (in-person and remote groups combined) was effective at reducing GWG in overweight and obese pregnant women (usual care: LS mean 12.8, SE 1.5 kg; SmartMoms: LS mean 9.2, SE 0.9 kg; P=.04). The in-person group gained significantly less total weight ([Figure 1](#figure1){ref-type="fig"}) during pregnancy than the usual care group (LS mean 8.0, SE 1.3 kg vs LS mean 12.8, SE 1.5 kg; P=.04) and weight gain in the remote group was equivalent to the in-person group (LS mean 10.0, SE 1.3 kg; P=.04 equivalence) and modestly lower than weight gain with usual care (LS mean 10.0, SE 1.2 kg vs LS mean 12.8, SE 1.5 kg; P=.07). Compared to usual care, the rate of GWG was significantly lower in the in-person group (LS mean 0.49, SE 0.06 kg/week vs LS mean 0.31, SE 0.05 kg/week; P=.01) and the rate of GWG in the in-person group was equivalent to the remote group (LS mean 0.39, SE 0.05 kg/week; P=.04) within 200 grams of weight gained per week. The proportion of women with excess GWG ([Figure 2](#figure2){ref-type="fig"}) was significantly lower in the in-person (56%, 10/18; P=.03) and remote groups (58%, 11/19; P=.04) compared to usual care (84.6%, 11/13). ###### Baseline characteristics by treatment group (N=54). ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Characteristic Usual care (n=17) In-person (n=18) Remote (n=19) P^a^ ------------------------------------------ ----------------------------------------------------------------------------- ------------------ --------------- --------- --- Age (years), mean (SD) 29.5 (5.1) 29.2 (4.8) 29.0 (4.2) .96 Race, n (%) \ \ \ .24 \ Black 6 (	{ "pile_set_name": "PubMed Central" }
Ghazvini K, Karbalaei M, Kianifar H, Keikha M. The first report of Streptococcus pluranimalium infection from Iran: A case report and literature review. Clin Case Rep. 2019;7:1858--1862. 10.1002/ccr3.2374 1. INTRODUCTION {#ccr32374-sec-0001} =============== Streptococcus pluranimalium is unusual streptococcal species, which is rarely isolated from human infection. Limited information is available about the pathogenicity of this species; the present study is the first report from Iran indicating infection with S pluranimalium; this study can be a novel insight in pathogenicity of this species. The members of genus streptococcus are the gram‐positive bacteria, which naturally live in the skin, mucosa membrane, respiratory tract, gastrointestinal tract, and urinary tract. Streptococcal infections were primarily described by Billroth in 1874.[1](#ccr32374-bib-0001){ref-type="ref"} So far, 163 species of this genus have identified, about half of which are reported from human infections.[1](#ccr32374-bib-0001){ref-type="ref"}, [2](#ccr32374-bib-0002){ref-type="ref"} Streptococcus pluranimalium was first isolated and reported by Devriese et al (1999) from domestic animal infections.[3](#ccr32374-bib-0003){ref-type="ref"} S pluranimalium can cause a wide range of infections, including mastitis, tonsillitis, genital tract infection, and brain abscesses in cattle, respiratory tract infection in canary, septicemia, and endocarditis in chicken, as well as tonsillitis in cat and goat.[4](#ccr32374-bib-0004){ref-type="ref"}, [5](#ccr32374-bib-0005){ref-type="ref"}, [6](#ccr32374-bib-0006){ref-type="ref"} Moreover, there are several reports nowadays about S pluranimalium from human infections, containing subdural empyema, endocarditis, brain abscesses, and septicemia.[7](#ccr32374-bib-0007){ref-type="ref"} Since the source of this bacterium is blood, milk, and other infectious secretions of animals, it seems that the microorganism has some animal reservoirs, and transmitted to human in the form of zoonosis.[8](#ccr32374-bib-0008){ref-type="ref"}, [9](#ccr32374-bib-0009){ref-type="ref"} According to review of the literatures, vancomycin, aminoglycosides, and cephalosporins are known as the first choice of therapeutic agents for S pluranimalium infection.[5](#ccr32374-bib-0005){ref-type="ref"} Despite many studies regarding the pathogenic or opportunistic nature of this bacterium, the fact is still not clearly understood.[7](#ccr32374-bib-0007){ref-type="ref"}, [10](#ccr32374-bib-0010){ref-type="ref"} The aim of present study was to report the first case of human septicemia due to S pluranimalium in Iran. 2. CASE PRESENTATION {#ccr32374-sec-0002} ==================== An Iraqi 2.5‐month‐old infant with clinical manifestations such as lethargy, vomiting, and anorexia was brought to the emergency department of the pediatric hospital in Mashhad, Iran, in November 2018. The initial examinations were done, which included pupil dilation, temperature: 37.2°C, and blood pressure: 75/55. The laboratory results are listed in Tables [1](#ccr32374-tbl-0001){ref-type="table"}, [2](#ccr32374-tbl-0002){ref-type="table"}, [3](#ccr32374-tbl-0003){ref-type="table"}, and [4](#ccr32374-tbl-0004){ref-type="table"}. ###### Arterial blood values Index pH pCO~2~ (mm Hg) pO~2~ (mm Hg) HCO~3~ ^−^ (mmol/L) -------------- ----------- ---------------- --------------- --------------------- Patient case 7.55 25.6 116.4 26.4 Normal range 7.35‐7.45 35‐45 80‐100 22‐28 John Wiley & Sons, Ltd ###### Analysis of complete blood count (CBC) Index WBC RBC Hb HCT Platelets -------------- ------------------------------------------------ ----------- --------- ------- ------------- Patient case 22.760 (PMN: 87%, Lymph: 13%) 2.86 7.7 24.2 1284 Normal 5000‐19500 (PMN: 1000‐9000, Lymph: 2500‐16500) 2.70‐4.50 11‐17.1 33‐55 10000‐45000 John Wiley & Sons, Ltd ###### Analysis of urine Index WBC RBC Epithelial cells pH SG (specific gravity) -------------- ----- ------ ------------------ ------- ----------------------- Patient case 20 Many 1‐5 5 1.010 Normal 0 0 1‐3 4.6‐6 1.010‐1.020 John Wiley & Sons, Ltd ###### Analysis of biochemical factors in blood serum Index Urea Creatinine Na K AST ALT ALP CRP -------------- --------- ------------ --------- --------- ------ ------- --------- ------ Patient case 23 0.6 137 4.72 33 51 465 78.5 Normal 2.0‐7.0 0.2‐0.4 130‐140 3.5‐6.0 9‐80 13‐45 113‐360 10 John Wiley & Sons, Ltd However, the sonography results of lungs, kidneys, and urinary tract were normal, and also the result of patient\'s UC (urine culture) was negative. Nonetheless, patient immediately transported to the PICU ware, and pulse oximetry was done for patient. A chronic intraventricular hemorrhage (IVH) grade 1 was observed in the sonography of the right caudothalamic groove of brain (Figure [1](#ccr32374-fig-0001){ref-type="fig"}A). Also, an obvious hypoechoic mass containing internal cystic region lacking vascularity was observed with 31x51 dimensions in the left temporal lobe. LP (lumbar puncture) was taken twice from the patient, and the results included albumin: 3.0‐3.4, CSF culture (twice): negative. In another step, the blood culture was done three times for the patient by BACTEC method; the blood culture results were positive. After three days, the greenish‐pinpoint (Colony size less than 1 mm) colonies were appeared on chocolate agar medium (containing 5% CO~2~) (Figure [1](#ccr32374-fig-0001){ref-type="fig"}B). The characterized properties of bacteria included gram‐positive cocci, α‐hemolytic, and negative catalase. However, the phenotypic tests were nonconclusive; for example, the laboratory results for this bacterium were negative for hippurate hydrolysis, inulin, and VP, as well as optochin‐sensitive (Figure [1](#ccr32374-fig-0001){ref-type="fig"}C). Finally, the desired bacterium was identified as S pluranimalium by using the VITEK 2 system, automated instrument for rapid and accurate microbial identification (ID) and antibiotic susceptibility testing (AST) (bioMérieux). Furthermore, the species identification was confirmed using 16S rRNA sequencing method (99% similarity with S pluranimalium strain DSM 15636). The phylogenetic relationship of our isolate and the closely related S pluranimalium were investigated using 16S rRNA gene sequence by MEGA 5 software, the Neighbor‐Joining (NJ) method and Kimura\'s two parameter (K2P) distance correction model with 1000 bootstrap replications; the phylogenic analysis confirmed the high homogeneity of S pluranimalium strains (Figure [2](#ccr32374-fig-0002){ref-type="fig"}). ![The results of clinical and laboratorial tests related to the S pluranimalium. A, Sonography of brain; B, The white small (needle‐like) colonies on blood agar medium (the Fig. was from Siddaiah et al); and (C) The sensitivity to optochin disk on blood agar](CCR3-7-1858-g001){#ccr32374-fig-0001} ![The phylogenic tree of our isolate and closely related S pluranimalium by 16S RRNA gene sequence](CCR3-7-1858-g002){#ccr32374-fig-0002} Based on the CLSI instruction, the antibiogram test was done by disk diffusion method in Mueller‐Hinton agar medium supplemented with 5% sheep blood. In this method, the sensitivity of *	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== Sepsis remains a major cause of mortality, and there is recent data to suggest that the incidence of sepsis is increasing \[[@CR1]\]. Treatment options remain limited to early antibiotic therapy, resuscitation and aggressive support of failing organ systems. Multiorgan dysfunction syndrome (MODS), the most severe end of the spectrum of sepsis syndrome, may involve the myocardium \[[@CR2],[@CR3]\], and this has clear prognostic implications, with some reports pointing to a direct correlation between the onset of myocardial dysfunction and mortality in septic shock \[[@CR4]\]. The pathophysiology of septic cardiomyopathy has not been fully elucidated. Although apoptosis is postulated to play an important role in the pathophysiology of septic cardiomyopathy \[[@CR5],[@CR6]\], post-mortem studies have demonstrated relatively little cell death, even in cases of undoubted severe septic cardiomyopathy \[[@CR7],[@CR8]\]. This has led to the suggestion that activation of pro-survival pathways may occur in association with pro-apoptotic pathway activation and that septic cardiomyopathy, although ultimately dysfunctional for the organism, is a reactive process of metabolic downregulation, serving a cellular preservation function broadly similar to preconditioning \[[@CR6]\]. Preconditioning is the phenomenon whereby prior exposure to a stimulus, often one normally held to be injurious, triggers phenotypic changes that confer resistance to subsequent insults. It was first described by Murry et al. in 1986 \[[@CR9]\], following the discovery that brief episodes of non-lethal myocardial ischaemia, followed by brief periods of reperfusion, provided protection against a subsequent more prolonged period of ischaemia/reperfusion. The concept of endotoxin-induced increased tissue tolerance first emerged in the 1960s \[[@CR10]\]. These early descriptions of 'endotoxin tolerance' in animal models were followed by discoveries that a similar phenomenon occurs in humans \[[@CR11]\]. Sepsis preconditioning of cardiac tissue may also occur in response to brief, non-fatal episodes of sepsis and bears undoubted observational similarities to ischaemic preconditioning, being inhibited, for example, by cycloheximide, a well-known blocker of 'late ischaemic preconditioning' \[[@CR12]\]. The mechanisms whereby sepsis confers protection to the heart against subsequent ischaemia/reperfusion injury, however, are unknown, and whether this phenomenon most closely approximates myocardial stunning, preconditioning or other unrelated effects remains to be firmly established. In the current study, we hypothesised that sepsis would protect the myocardium from subsequent ischaemia/reperfusion (IR) injury. We further hypothesised that the mitogen-activated protein kinases (MAPK) and nuclear factor-kappaB (NF-κB) pathways mediated this protective effect. Methods {#Sec2} ======= Ex vivo isolated heart model {#Sec3} ------------------------------ All animal studies were carried out with the approval of the National University of Ireland Galway Animal Care Research Ethics Committee and under licence issued by the Public Health Division, Department of Health, Ireland. Specific pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), 250 to 300 g in body weight, were used for caecal ligation and perforation (CLP)-isolated heart experiments. Animals were randomised to control (n = 7) or CLP (n = 7) groups. The CLP procedure was carried out under general anaesthesia. Briefly, following midline laparotomy, the caecum was identified and ligated, following which needle perforation was carried out, and wound layers were closed. Animals were returned to housing in individually ventilated cages. At 48 h following operation, animals were harvested for isolated heart protocol using the Langendorff suspension. Animals were sacrificed by means of decapitation. The heart was then removed and suspended from a canula inserted into the aortic root facilitating perfusion with prewarmed Krebs Ringer at 37°C, oxygenated by means of a gas mixture containing 5% CO~2~ and 95% O~2~, maintaining pH of solution at 7.4. Episodes of ventricular fibrillation were promptly treated with lignocaine bolus 200 mg, administered via a t-piece connected to the aortic canula such that it passes directly through the coronary arteries. Measurements of systolic and diastolic left ventricular (LV) functional characteristics were made with a saline-filled balloon placed in the left ventricle. This balloon was connected to a pressure transducer facilitating provision of a digital readout as a continuous waveform. Balloon volume was adjusted to achieve LVEDP 4 to 8 mmHg, following which the volume of the balloon was maintained constant, thus providing a pre-load-independent measure of contractility. Parameters measured were systolic and diastolic left ventricular pressure (LVP), developed (sys-dias) LVP, maximal and minimal derivatives of LVP (dp/dt LVP~max~, dp/dt LVP~min~) and heart rate. Global myocardial ischaemia was induced by means of clamping the inflow line for a period of 30 min, followed by 90 min reperfusion. Upon completion of the protocol, the hearts were stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC), a dye taken up only by viable cells. Myocardial infarct size was determined by direct visualisation, dissection and separation of viable and infarcted heart tissue, which were then weighed and expressed as percentage of total heart weight. In vitro hypoxic/ischaemic studies {#Sec4} ------------------------------------ Preparation of primary cultures of neonatal cardiomyocytes was achieved by trypsin/collagenase digestion of ventricles harvested from 3- to 5-day-old neonatal Sprague Dawley rats following decapitation. Cells were cultured in a 1:1 mixture of Dulbecco\'s modified Eagle\'s medium (DMEM) and Ham\'s F12 (D8437 Sigma-Aldrich Ireland Limited, Wicklow, Ireland) containing 17 mM glucose, supplemented with 1 mM sodium pyruvate (Gibco, Life Technologies, Grand Island, NY, USA), 10% newborn foetal calf serum, 5 μg/ml bovine insulin, 5 ng/ml human transferrin, 5 μg/ml sodium selenite, 100 U/ml penicillin and 100 μg/ml streptomycin. All plates and flasks used to culture cardiomyocytes were previously coated with 2% gelatin solution for 24 h and aspirated prior to plating of cells, to ensure adherence of cardiomyocytes. To prevent fibroblast proliferation, 100 mM 5-bromo-2-deoyuridine (BrdU) was added to the cardiomyocyte media for 48 h following plating, at which point media were replaced. Each well was then re-fed with prewarmed media prior to commencing experimentation. This is required to provide sufficient metabolic substrate for viability and normal function throughout the experimental period. Human alveolar epithelial cells (A549) were purchased from ATCC, Middlesex, UK. HK2 human adult kidney cells were a kind gift from Prof. Michael Ryan (University College Dublin, Ireland). Cells were cultured in DMEM medium supplemented with heat-inactivated 10% foetal bovine serum, 2 mmol/l glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. To investigate putative signaling pathways involved in the protective effect of cellular exposure to endotoxin prior to incubation under hypoxic conditions, inhibitors of MAP kinases extracellular-regulated protein kinase (ERK) 1/2, p38 MAPK (p38), and c-Jun NH2-terminal protein kinase (JNK) in addition to pyrrolidinedithiocarbamate (PDTC) (an NF-κB inhibitor) and anti-HMGB1 antibody were added to cell culture at standard concentrations. For cell viability experiments, A549 and HK2 cells were grown to confluence in 96-well flat-bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) and cultured in pre-equilibrated and prewarmed RPMI, DMEM and a 1:1 mixture of DMEM:Ham\'s F12 media, respectively. The cells were allowed 24 h to polarise prior to experimentation. The medium was then re-fed, supplemented with LPS, LPS with MAPK inhibitors, PDTC, anti-HMGB1 antibody or vehicle under conditions of normoxia (humidified 5% CO~2~, 95% air) or hypoxia (humidified O~2~/N~2~/CO~2~ in the following ratio: 2:93:5), following randomisation. Viability was measured by cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, under conditions of normoxia (humidified 95% air, 5% CO~2~) and hypoxia (humidified O~2~/N~2~/CO~2~ in the following ratio: 2:93:5), for an experimental duration of 48 h, on all four cell types. The MTT assay provides an estimate of mitochondrial activity from which cell viability is deduced. Data analysis {#Sec5} ------------- Results are expressed as mean (SD) for normally distributed data. Data were analysed using t test comparison. A p value of 0.05 was considered statistically significant. Results {#Sec6} ======= Sepsis preconditioning protects the heart {#Sec7} ----------------------------------------- Seven animals underwent the CLP procedure to provide a model of polymicrobial sepsis. These septic animals were then sacrificed and hearts suspended in Langendorff preparation. The hearts were also isolated from seven non-septic animals which provided the control group in this series of experiments. ### Indices of myocardial function {#Sec8} Hearts from septic animals had reduced baseline developed LVP ((d)LVP) compared to those from control animals, mean (SE) 101	{ "pile_set_name": "PubMed Central" }
Introduction ============ Chronic hypoxia is an important factor contributing to the near-irreversibility of hypoxia-induced pulmonary hypertension (HPH), a condition characterized by structural remodeling of small pulmonary arteries (PAs). These important morphological changes are characterized by an abnormal increase in numbers of smooth muscle (SM)-like cells \[cells expressing α-SM actin (α-SMA)\] in the PA wall ([@b1-ijmm-42-01-0270],[@b2-ijmm-42-01-0270]), which results in thickening of muscular and elastic vessels, muscularization of distal vessels, and eventual development of pulmonary hypertension. The small pulmonary arteries and veins may be distinguished based on the relative abundance of SM cells or SM-like cells in vessel walls. However, the origin of SM-like cells has remained elusive. Epithelial-mesenchymal transition (EMT) is a process wherein epithelial cells undergo phenotypic changes and develop into mesenchymal/SM-like cells ([@b3-ijmm-42-01-0270]). Similarly, endothelial cells may acquire a mesenchymal or SM-like phenotype, a process referred to as endothelial-mesenchymal transition (EndMT). Evidence of EndMT has been reported in the context of cardiac and vascular development, wound healing and various diseases, including fibrosis, diabetic nephropathy, heterotopic ossification and cancer ([@b4-ijmm-42-01-0270]--[@b10-ijmm-42-01-0270]). Transdifferentiated cells co-express the endothelial marker CD31 and the SM-like cell type marker α-SMA. Thus, EndMT is regarded as another important mechanism for the generation of SM-like cells. The endothelial cell appears to be one of the targets in hypoxia, and endothelial cell dysfunction has a direct and indirect role in the process of pulmonary vascular remodelling ([@b11-ijmm-42-01-0270]). Ranchoux et al ([@b12-ijmm-42-01-0270]) reported that EndMT may be a source of α-SMA-expressing cells. The EndMT participates in vascular remodeling as a characteristic of pulmonary hypertension; however, the underlying mechanism has remained to be fully elucidated. Hypoxia-inducible transcription factor-1α (HIF-1α) is a critical regulator of the cellular response to hypoxia. HIF-1α activity in the endothelium of PAs has been observed to significantly increase in pulmonary hypertension ([@b13-ijmm-42-01-0270]). The HIF-1α-regulated gene Twist ([@b14-ijmm-42-01-0270],[@b15-ijmm-42-01-0270]) has an important role in EMT, cell movement and proliferation ([@b16-ijmm-42-01-0270]--[@b18-ijmm-42-01-0270]). However, the role of HIF-1α/Twist in EndMT has remained to be fully characterized. In the present study, a significant upregulation of HIF-1α in hypoxic pulmonary microvascular endothelial cells (PMVECs) was demonstrated, and knockdown of HIF-1α and Twist1 effectively blocked hypoxia-induced EndMT. It appeared that HIF-1α regulated the EndMT through binding to the promoter of the Twist1 gene and subsequently activating Twist1 transcription and expression. Materials and methods ===================== Animal model ------------ A total of 36 male Sprague Dawley rats (age, 6--8 weeks; weight, 200--250 g) were obtained from the Fourth Military Medical University. All experimental procedures were approved by the Animal Use and Care Committee for Research and Education of The Fourth Military Medical University (Xi\'an, China). Control rats were housed under a 12-h light/dark cycle at room temperature (humidity: 50--60%) and were provided water and a standard laboratory diet ad libitum. Rats were housed for 28 days in a chamber containing 10% oxygen for exposure to continuous hypobaric hypoxia. A gradual decrease in oxygen concentration was employed in order to acclimatize the rats to the hypoxic conditions ([@b19-ijmm-42-01-0270]). Over 30 min, the pressure was slowly increased at the beginning of hypoxia by 0.5 atm, and the oxygen concentration was reduced to 10%. The gradual decrease in the oxygen concentration had no observable negative effect on the rats. After 4 weeks, the HPH models were successfully replicated. Rats in the normoxic group (control group) were housed continuously in room air (n=6 per group). At the end of hypoxia exposure, the right ventricular systolic pressure (RVSP) and the ratio of ventricular weight \[right ventricle/(left ventricle+septum), denoted as RV/(LV+S)\] were measured. Increases in RVSP and RV/\[LV+S\] were considered as being indicative of HPH. Cell culture and chemical reagents ---------------------------------- The lung was isolated from healthy adult rats under sterile conditions. After washing repeatedly with sterile D-Hank\'s buffer, tissues at the edge and surface of the lung (thickness, \~1 mm) were removed, dissected into small pieces and cultured in M200 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with a growth factor cocktail \[fibroblast growth factor, heparin, hydrocortisone and epidermal growth factor (Gibco; Thermo Fisher Scientific, Inc.)\] and 20% fetal bovine serum FBS (HyClone; GE Healthcare, Little Chalfont, UK). Primary cultures used were confirmed to be uncontaminated with SM cells by immunostaining for α-SMA and CD31 or VIII factor. In the normoxic group, PMVECs were cultured at 37°C in a humidified atmosphere of 5% CO~2~ in air. In the hypoxic groups, PMVECs were incubated in a hypoxic chamber containing 1% O~2~, 5% CO~2~ and 94% N~2~ for 7 days, and SM-like cells were then obtained. In another experiment, PMVECs were also stimulated with transforming growth factor (TGF)-β (10 ng/ml) in normoxic conditions for 7 days. Antibodies against α-SMA (MA1-744) were obtained from Thermo Fischer Scientific, Inc. (Waltham, MA, USA). Monoclonal antibodies against CD31 (ab64543) and Twist1 (ab50581) were purchased from Abcam (Cambridge, MA, USA). collagen (Col) 1A1 (sc-293182) and Col3A1 (sc-271249) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-HIF-1α monoclonal antibody (MAB5382) was purchased from EMD Millipore (Billerica, MA, USA). Secondary antibodies (anti-rabbit or anti-mouse IgG antibody conjugated to horseradish peroxidase; 7074 and 7076; 1:2,000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Recombinant human TGF-β was purchased from R&D Systems (Minneapolis, MN, USA). Immunofluorescence ------------------ Frozen tissue sections or cultured cells grown on coverslips were washed with PBS and fixed in 1% paraformaldehyde. Cells were subsequently permeabilized with 0.2% Triton X-100 in PBS at room temperature for 5 min. The cells and tissue sections were blocked by 2% goat serum (OriGene Technologies, Beijing, China) at 37°C for 1 h. Then they were incubated with primary antibodies (CD31, 1:500; α-SMA, 1:1,000) overnight at 4°C. Subsequently, samples were incubated with secondary antibody (Alexa Fluor^®^ 594 donkey anti-mouse IgG; R37115; 1:500; Molecular Probes; Thermo Fischer Scientific, Inc.) for 2 h at room temperature. Fluorescence was examined by confocal laser scanning microscopy. Immunohistochemistry -------------------- Sagittal sections of the right lung were placed in 4% paraformaldehyde and processed for paraffin embedding. Sections (5 μm) were prepared and mounted on glass slides prior to overnight incubation at 4°C with anti-Twist1 antibody (1:500). Slides were washed and incubated with corresponding secondary antibodies conjugated with alkaline phosphatase (Expose Rabbit specific HRP/DAB detection IHC kit; ab80437; Abcam). Sections were evaluated using an Olympus BX50 optical microscope (Olympus Corporation, Tokyo, Japan) equipped with an image analysis program (Image Pro Plus version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). Electron microscopy ------------------- Transmission electron microscopy (TEM) analysis was performed using glutaraldehyde-fixed PMVECs embedded in resin (Epon 812 epoxy resin). The clean slide was placed in 0.25% Formvar solution and then placed vertically on filter paper to air dry. Then the slide was exposed vertically to the surface of the water. The supporting membrane on the surface of the glass slide was removed and the 75-mesh copper grids placed on the membrane, removed from water and allowed to dry. Thin sections (50--80 nm) were collected on the formvar-coated copper grids and stained with 3% uranyl acetate and lead citrate solutions for 20--30 min respectively at room temperature. Following air drying at room temperature, the sections were examined under an electron microscope (JEM1200EX II; JEOL, Tokyo, Japan); images were captured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ---------------------------------------------------------------------- Total RNA was extracted from lung tissues and PMVECs with TRIzol (Invitrogen; Thermo Fisher Scientific	{ "pile_set_name": "PubMed Central" }
Passive smoking is the inhalation of second-hand smoke (SHS) by persons other than the "active" tobacco smoker. This exposure to SHS may cause disease, disability, and death \[[@b1-ophrp-10-049]\]. Passive smoking has been shown to be associated with the same diseases as active smoking (such as cardiovascular diseases, lung cancer, and respiratory diseases) \[[@b2-ophrp-10-049]\]. Analysis of the effects of SHS has come from studies of nonsmokers who are married to a smoker, with similar findings also reported in studies of workplace exposure to tobacco smoke \[[@b3-ophrp-10-049]\]. SHS consists of mainstream smoke (the smoke exhaled by a smoker) and sidestream smoke (the smoke released from a cigarette into the surrounding air) \[[@b4-ophrp-10-049]\]. The main component of SHS is sidestream smoke, which is about 4 times more toxic than mainstream smoke \[[@b5-ophrp-10-049]\], and has the most impact on the health of passive smokers \[[@b6-ophrp-10-049]\]. Looking forward, an objective of the Health Plan 2020 (HP2020) in Korea is to lower the exposure to SHS for those individuals who do not smoke tobacco by assigning designated non-smoking areas in public places in Korea such as squares and city parks \[[@b7-ophrp-10-049]\]. This supports the National Health Promotion Act (2017) which prohibits smoking in all middle and high school facilities, and smoking bans in college classrooms \[[@b8-ophrp-10-049]\]. Previous studies on the exposure to SHS have generally focused on the subjective reports of study participants regarding their exposure to SHS \[[@b9-ophrp-10-049]\]. Most studies lack the quantitative measurement of biomarkers of exposure to SHS such as cotinine, and nicotine-derived nitrosamine ketone in urine. The measurement of cotinine in hair and urine is accurate, and non-invasive, and is an effective indicator of exposure to SHS \[[@b1-ophrp-10-049]\]. In the current issue of Osong Public Health and Research Perspectives, a study by Park et al. measured the exposure to SHS amongst non-smoking, nursing college students, by measuring urinary cotinine, and analyzed the demographics, health, and smoking-related factors that may influence exposure to SHS \[[@b10-ophrp-10-049]\]. This cross-sectional study examined nursing college students (N = 196) who had not smoked tobacco in the previous year. To measure the exposure to SHS in students, the authors examined urinary cotinine levels, and social factors that may be influential, such as asking a smoker to put out their cigarette. The study observed that 32 students (16.3%) had urinary cotinine levels suggestive of exposure to SHS. There were 80 students (40.8%) affected by risk factors that increased exposure to SHS. In addition, students who were exposed to SHS were 4.45 times more likely to have increased urinary cotinine levels. It was reported that students who asked smokers to extinguish their cigarette, were 0.34 times less likely to test positive for urinary cotinine. The authors concluded that non-smoking, nursing college students, that displayed self-assertive behavior by requesting smokers to extinguish their cigarette, could avoid exposure to SHS. It was also suggested that systematic support for the enforcement, and expansion of no-smoking zones, should be a requirement in all college facilities. Conflicts of Interest The author declares no conflicts of interest.	{ "pile_set_name": "PubMed Central" }
Introduction {#s1} ============ Among trace elements, zinc (Zn) is a micronutrient influencing growth and affecting the development and integrity of the immune system \[[@R1]\]. Also, it has important roles in physiological functions of the human body. It has a critical role for the function of over 300 enzymes \[[@R2]\]. Furthermore, it plays an important role in function of the liver. There are hepatic and extrahepatic actions for Zn in the prevention of alcoholic liver injury \[[@R3]\]. Zinc deficiency has been involved in the pathogenesis of a number of clinical findings in chronic liver disease. These include the possible role of Zn deficiency in the pathogenesis of hepatic encephalopathy, by inducing alterations in urea metabolism \[[@R4]\]. In a study, Gur et al. reported decreased level of plasma zinc in cirrhotic patients due to hepatitis B \[[@R5]\]. Hepatitis B and hepatitis C are the most common causes of liver cirrhosis in many countries like Iran \[[@R6]\]. As nutritional habits are different among various populations, it is logical to determine Zn level in Iranian cirrhotic patients as an important trace element. There are some differences in the etiology, disease progress, prognosis and treatment plan of hepatitis B and C, and it is possible that plasma zinc concentration differs in two groups. The aims of the present study were 1) determination of plasma zinc level in a sample of Iranian cirrhotic patients due to hepatitis B or C, 2) to determine if there is any difference between plasma zinc levels between the 2 groups. Materials and Methods {#s2} ===================== In a cross sectional study, adult cirrhotic inpatients due to hepatitis B or hepatitis C referred to gastrointestinal and liver disease center of Taleghani Hospital, Tehran, Iran were enrolled for the study. This place is one of the important referral centers of liver disease in Iran. Diagnosis of cirrhosis and hepatitis were determined by clinical, laboratory and liver biopsy from the patients. Exclusion criteria were patients who had been co-infected with hepatitis B and C and those who had taken a zinc tablet or any complementary medicine with zinc in its content. Because of the difference in the zinc amount of foods, we preferred to select patients nourished with a similar regimen prepared by the kitchen of the hospital during the study. After informed consent, 5 cc blood samples were kept from the forearm of each patient in fasting state in the morning. Blood samples were centrifuged in 5000 rpm for 5 minutes and plasma was separated. Then plasma samples were kept in the Frazer at the -25 degree centigra . Because the usual method for assessment of zinc in plasma or serum is the atomic absorption \[[@R7]\], we selected the same method for zinc assessment. Concentrations of 0.1, 0.3, 0.5 and 0.7 ppm of zinc sulfate were prepared as standard samples. Atomic absorptions of them were determined for drawing standard curve. After completion of 60 samples of patients, plasma zinc levels were evaluated by using atomic absorption (Perkin Elmer 1100B). Results {#s3} ======= 60 cirrhotic patients due to hepatitis B or Hepatitis C were included in the study during 7 months (April 2008 to November 2008). Of them 36 were hepatitis B patients and 24 were hepatitis C patients. Demographic data of patients including age distribution, sex and smoking habits were shown in [Table 1](#s3tbl1){ref-type="table"} Analysis of chi-square showed that there is not any difference between 2 groups regarding to sex, age distribution and smoking habits (P = 0.82, 0.53 and 0.8 respectively). Mean ± standard deviation (SD) plasma zinc levels determined 0.34±0.22 mg/L and 0.37±0.22 mg/L in hepatitis B and hepatitis C patients respectively. Analysis of t-test showed that there is not any significant difference between 2 groups regarding to plasma zinc level (P = 0.745). ###### Demographic of patients. Parameter Total Parameter N= 60 HCV Patients (N= 24) HBV Patients (N=36) P-value ---------------- ----------------------- ---------------------- --------------------- --------- ------ Age \< 40 12 6 6 0.53 41-60 40 14 26 61\> 8 4 4 Sex Female 21 8 13 0.82 Male 39 16 23 Smoking Habits No 22 10 12 0.80 Yes 38 14 24 Discussion {#s4} ========== Plasma trace elements concentrations are frequently reported to be a good indicator for diagnosis and prognosis of some diseases \[[@R8]\]. Previous studies showed a decrease in zinc level in cirrhotic patients. Pramoolsinsap et al. have stated that serum zinc levels were significantly decreased in patients with chronic active hepatitis, cirrhosis, and hepatocellular carcinoma \[[@R9]\]. Lin et al. announced that the zinc concentration in the serum of Chinese patients with hepatic cirrhosis was significantly less than a control group \[[@R10]\]. It should be noted that zinc level is usually related to the nutritional pattern of each population. It has been shown that zinc deficiency is widespread in people living in developing countries like Iranian populations who consume rice-based diets \[[@R11]\]. The phytate and fiber present in cereal diets can form insoluble complexes with zinc leading to its decreased bioavailability \[[@R12]\]. Since nutritional impairment is common in cirrhotic patients \[[@R13]\], it seems that determination of zinc level in Iranian cirrhotic patients due to hepatitis B and C and comparison with a normal amount of healthy people is necessary as an indicator of nutritional status. Some demographic data may alter zinc plasma concentration in human. Lopez et al. reported that Serum Zn concentrations were slightly higher in men than in women and also there is some elevated serum Zn levels in smoking men rather than non smokers \[[@R14]\]. In the present study, since there is no significant difference between the 2 groups regarding gender, age distribution and smoking habits, these parameters could not lead to biases in interpretation of zinc level in both of the groups. The results showed that plasma zinc levels of both groups were below the normal range as mentioned by the similar investigations \[[@R9]\]\[[@R10]\]. There are some differences in the normal range of zinc in various populations, but a recent study reported the normal range of 0.89 ± 0.16 mg/L for plasma zinc in healthy volunteers in Tehran \[[@R15]\]. With a comparison result of the present study (0.34±0.22 mg/L and 0.37±0.22 mg/L) with a range of the latter study, it seems that plasma zinc level of the cirrhotic patients are less than half of normal values. As nutritional parameters, this study significantly indicates a zinc level deficiency in Iranian cirrhotic patients due to hepatitis B or C in comparison with healthy volunteers. The results of the study are more considerable because of the effective role of the zinc supplement in pharmacotherapy of viral hepatitis. Yuasa et al. have shown that zinc may play an important role as a negative regulator of hepatitis C virus (HCV) replication in genome-length HCV RNA-replicating cells. They mentioned that zinc appears to offer a novel approach to the development of future plans for the treatment of intractable chronic hepatitis C \[[@R16]\]. Himoto et al. examined the effects of polaprezinc, a complex of zinc and L-carnosine, on inflammatory activity and fibrosis in the HCV infected patients. They reported that polaprezinc exerts an anti-inflammatory effect on the liver in patients with HCV-related Chronic liver disease by reducing iron overload \[[@R17]\]. Based on the result of the study, administration of zinc may be recommended for Iranian cirrhotic patients due to hepatitis B or C. In future more studies recommend for the role of zinc administration on clinical, pathological status and pharmacotherapy response of Iranian cirrhotic patients due to hepatitis B or C.	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== School safety is fundamental to fostering positive short and long-term outcomes for children, including positive mental health, school connectedness, student retention and academic success (Battistich, Schaps, & Wilson, [@CR6]; Horner et al., [@CR37]). We define a safe school as one that minimizes violence, promotes student mental health, and fosters a social climate that promotes positive development. Unfortunately, concerning rates of school violence persist in the US: in 2017, 19.0% of students were bullied, 15.7% carried a weapon at least once in a month (3.8% on school property), and 6% had been threatened or injured with a weapon (Kann et al., [@CR44]). Rates of violent, aggressive, and bullying behaviors are similarly concerning among younger students with 8.1% of elementary and 21.8% of middle school students reporting daily or weekly bullying in 2015--16 (Diliberti, Jackson, & Kemp, [@CR22]). Violence victimization is associated with distress, adjustment difficulties, and mental health problems. Exposure to violence, including direct victimization and well as exposure in the school environment, is a potent risk factor for poor mental health outcomes including depression and anxiety (Kennedy, Bybee, Sullivan, & Greeson, [@CR47]). The CDC (Perou et al., [@CR76]) identifies mental health problems, including depression and anxiety, as a critical public health issue among youth with significant impact on the individual, family, and community. Mental health issues that go untreated early in life are associated with further problems, including increased likelihood of academic failure, dropout, substance use, relationship conflicts, violence, and suicide (World Health Organization, [@CR94]). In the short term, mental health problems evident in middle school predicts school absences a year later (Suldo, Thalji, & Ferron, [@CR91]). In addition, many children, particularly those living in low resource communities, experience disproportionate risk of violence and subsequent mental health consequences without sufficient treatment and prevention services needed to reduce risk of poor outcomes (O'Connell, Boat, & Warner, [@CR73]). Childhood vulnerability is exacerbated in high stress environments when children and youth receive limited support from adults (O'Connell et al., [@CR73]). Yearly, an estimated 13--20% of children aged 3--17 years experience a mental disorder, and more than half of lifetime psychiatric diagnoses have an initial age of onset before age 14 (Kessler et al., [@CR48]). School climate plays a major role in shaping the lives of students, affecting violence (Brookmeyer, Fanti, & Henrich, [@CR14]), mental health and wellness (Jacobson & Rowe, [@CR39]), truancy and achievement (Astor, Guerra, & Van Acker, [@CR2]). The National School Climate Council recommends an encompassing definition of school climate that includes experiences of school life, that reflect the norms, goals, values, teaching, organization structure, and relationships. Relationships include connections among students, teachers, and staff; feelings of commitment to the institution; and connection to a community (Pittman & Richmond, [@CR78]). The relationship between student outcomes and school climate are evident longitudinally (Anderman, [@CR1]; Goodman, [@CR30]). Researchers have found that poor school-based relationships were associated with initiation of deviant behavior (Dornbusch, Erickson, Laird, & Wong, [@CR23]; McNeely & Falci, [@CR65]). Consequently, promoting a positive school climate is an important mechanism by which interventions can reduce risk of poor health outcomes including violence and mental distress. Researchers suggest that schools, particularly those with concentrated poverty, may benefit from multicomponent prevention approaches that improve positive discipline management and support positive psychosocial climates, effectively identify youth experiencing mental distress and improves the physical as well as social environment (Gottfredson, Gottfredson, Payne, & Gottfredson, [@CR31]). In order to effectively address challenging issues such as school safety, communities need to deliver multicomponent interventions targeting prevention efforts across levels of social ecology (Komro, Flay, Biglan, & Wagenaar, [@CR55]; PriCowan, Vaillancourt, Rossen, & Pollitt, [@CR80]). Even the best individual interventions have relatively limited scope in terms of outcomes when offered alone, and, consequently, small effects when taken to scale; therefore, multicomponent interventions have greater potential to achieve positive outcomes at the school or community level than a single intervention alone (Komro et al., [@CR55]). Evidence-based interventions (EBIs) that address the multifaceted nature of school safety such as Restorative Justice (RJ), Mental Health First Aid (MHFA) and Crime Prevention Through Environmental Design (CPTED) when deloyed as a single, coordinated, multicomponent intervention approach, are promising approaches to improving the school environment. Yet, more complex, multicomponent interventions are also more challenging to implement. Such interventions require effective implementation strategies to adopt the constellation of EBIs and adapt them to suit the needs of the context, providers and target population. Interventions will fail to achieve their desired effects if not implemented well (Durlak, [@CR24]). Researchers have acknowledged that evidence-based interventions must be complemented by implementation strategies to achieve desired public health outcomes (Kirchner, Waltz, Powell, Smith, & Proctor, [@CR51]). Implementation strategies are highly specified, theory-based methods to enhance EBI delivery in community settings and are key to bridging the research-to-practice gap (Kilbourne et al., [@CR50]). Study designs that assess implementation strategy utility and evaluate EBI effectiveness, such as with hybrid designs, provide vital information for stakeholders about using implementation strategies with new innovations to maximize public health impact. To inform the optimal implementation strategies for these effective interventions, we develop and test an intervention focused on promoting a positive school climate guided by a school-based 3-person leadership team (3-PLT) using a hybrid Type II design. A hybrid Type II design tests the effectiveness of the intervention and determines the feasibility and potential utility of an implementation strategy (Curran, Bauer, Mittman, Pyne, & Stetler, [@CR21]). The 3-PLT includes representatives from the police (School Resource Officer, SRO), school (e.g., administration), and mental health services (i.e., social work), the latter of whom leads the team as a newly appointed climate specialist (CS). The team, led by the CS, work together to support the integration of the key intervention components: (1) RJ practices, (2) MHFA training, and (3) CPTED (see Fig. [1](#Fig1){ref-type="fig"}). The CS coordinates these efforts as a staff member within the school through a process of interactive problem solving and supports, consistent with the implementation facilitation strategy (Ritchie, Dollar, Kearney, & Kirchner, [@CR83]). We will focus on change among students in an early developmental period---elementary school students aged 8--12 years---in a county with significant social and economic challenges. The purpose of this research is to study the effectiveness and implementation of three complementary interventions delivered concurrently to enhance school safety through improved school climate. School climate represents a critical mechanism by which interventions, including multicomponent school safety and mental health interventions, can reduce risk of violence and mental distress among youth. Fig. 1Proposed Conceptual Relationship Between Evidence-Based Interventions, Implementation Strategies and Study Outcomes. Adapted from Proctor et al. ([@CR77]) and Lyon (Lyon, [@CR59]) Methods/design {#Sec2} ============== Aims and objectives {#Sec3} ------------------- The overarching objective of this study is to provide a safe school environment to positively impact health, wellbeing, social, educational, violence and delinquency outcomes among youth. This is accomplished through the concurrent delivery of a multicomponent approach consisting of 3 integrated interventions: Restorative Justice (RJ), Mental Health First Aid (MHFA) and Crime Prevention Through Environmental Design (CPTED). The implementation strategy used is a facilitation approach based on the Implementation Facilitation, Enhanced REP, and the iPARiHS framework (Kilbourne et al., [@CR49]; Ritchie et al., [@CR83]) as part of the 3-PLT (see Fig. [1](#Fig1){ref-type="fig"}). Elementary school safety is understudied yet represents a critical period in which to develop positive mental health, build constructive and trusting relationships with adults, and prevent early experiences of violence. Primary study aim {#Sec4} ----------------- The primary study aim is to examine the overall effectiveness of the intervention, including change in violence (e.g., fights, bullying, victimization), over time compared with a control group of students who receive school practice as usual. Secondary study aim 1 {#Sec5} --------------------- Employ implementation facilitation from an appointed school climate specialist (CS) to support delivery of a multicomponent, integrated intervention and evaluate feasibility and potential utility to support sustainment. Secondary study aim 2 {#Sec6} --------------------- Examine specific mechanisms associated with change in mental health (e.g., anxiety, depression, well-being), including perceptions of school climate as a moderator. Secondary study aim 3 {#Sec7} --------------------- Estimate the costs of the intervention and its implementation and conduct a cost-benefit analysis for positive outcomes, such as improve school climate perceptions	{ "pile_set_name": "PubMed Central" }
1.. Introduction ================ In Sri Lanka, alcohol use is a growing public health issue \[[@b1-ijerph-06-02408]--[@b3-ijerph-06-02408]\], and, because it is a predominantly male habit there, alcohol use disproportionately harms Sri Lankan males. Adolescents and young adults in the country are highly vulnerable to the onset and continuation of the habit \[[@b4-ijerph-06-02408]\]. Beer is probably the most popular alcoholic beverage among Sri Lankan youth, and a significant surge in beer sales has been observed over the last few years \[[@b5-ijerph-06-02408]--[@b7-ijerph-06-02408]\]. In 2003, 19.3 million liters of beer were produced in the country and the corresponding figure for the year 2007 was 29.5 million liters, a 53 percent increase. Socio-cultural factors, such as urbanization and westernization and environmental factors, such as availability and affordability, may have contributed to this upward trend in the sale of alcohol. The consumption patterns of alcohol by different age groups and the social-cognitive determinants of alcohol use, such as drinking motives, have not been clearly understood in the context of Sri Lanka. This is partly due to the widespread production of illegal liquor in a country where no records of the quantity and sale of illegal liquor are kept \[[@b8-ijerph-06-02408]\]. Another partial reason is the shortage of epidemiological and psycho-social data pertaining to the problem. Preventive programs targeted at young people should be based on a scientific knowledge of the distribution and determinants of alcohol use if they are to be effective in curbing the problem. Thus, there is a significant need to understand how and why young people in Sri Lanka use alcohol. The reasons and motives for drinking alcohol are closely associated to the drinking patterns and consequences of alcohol use \[[@b9-ijerph-06-02408],[@b10-ijerph-06-02408]\]. Different age groups may develop different motivations towards alcohol use that are shaped by many factors, including culture and environment \[[@b11-ijerph-06-02408],[@b12-ijerph-06-02408]\]. Thus, motivation is considered a key concept in behavioral and psycho-social models of alcohol use. Since different drinking motives are associated with different types of drinking behavior \[[@b9-ijerph-06-02408],[@b10-ijerph-06-02408]\] and culture plays a significant role in motivating or de-motivating people toward various behaviors, proper understanding of motives that direct young people to drink would help public health and education authorities formulate effective public health policies and develop cost-effective measures to curb the alcohol problem. Researchers have identified a number of domains of drinking motives \[[@b9-ijerph-06-02408],[@b10-ijerph-06-02408]\], and among them, personal enjoyment, social pressure, and tension reduction have been identified as prominent. Further, each domain is shown to be related to different aspects of alcohol use. For example, the personal enjoyment (enhancement) motive was found to be associated with heavy drinking, whereas social motives were identified to be associated with lighter drinking patterns and were more prevalent among young alcohol users \[[@b13-ijerph-06-02408],[@b14-ijerph-06-02408]\]. Tension-reduction (anxiety reduction) motives were found to be related to solitary drinking and to problem drinking \[[@b13-ijerph-06-02408],[@b15-ijerph-06-02408]\]. However, most of these studies have been conducted in the West, limiting the understanding of how these relationships work in non-western cultures. In the Sri Lankan context, there are certain culture-specific motivational factors of drinking behavior. Young males in Sri Lanka drink to become more prominent among peers and to attract females in social gatherings. Since the prevalence of alcohol use in the country is very low among women compared to men (5% verses 53%), alcohol use often symbolizes manhood, and thus, drinking behaviors are occasionally used by males to dominate family members and neighbors. A new scale was developed based on the existing drinking motives questionnaires \[[@b9-ijerph-06-02408],[@b10-ijerph-06-02408],[@b13-ijerph-06-02408]\] that incorporates some of these culture-specific motivational factors. To date and to our knowledge, no studies have been conducted on the motivation toward alcohol use among people in Sri Lanka. This study's purpose was to test the 3-factor structure of a scale developed to measure drinking motives and to examine the relationships between drinking motives and drinking patterns in a sample of males aged 16--30 years in Sri Lanka. It was hypothesized that the drinking motives scale consist of three factors: personal enjoyment, social pressure, and tension reduction. It was also hypothesized that drinking motives were related to drinking frequency. 2.. Methods =========== A cross-sectional survey design was used. An anonymous, self-administered questionnaire was employed to collect data. Boards for the protection of human subjects from universities in both the United States and Sri Lanka approved the study protocol. 2.1.. Sampling -------------- Purposive sampling method was used to select sample subjects. Efforts were made to obtain an equal number of adolescents (16--19 years of age) and young adults (20--30 years of age) for the sample, so that the sample would represent the young males in the study population. Men aged 16--30 years, who were in the streets, universities and other educational institutes, in work places, or at homes during the survey period and who identified themselves as either occasional or regular drinkers, were invited to participate in the survey after explaining to them briefly the purpose of the survey and the anonymous nature of the data collection method. Those who agreed to participate in the study were surveyed using the questionnaire. The survey was conducted in two settings: urban and semi-urban. Three medical undergraduate students were recruited and trained as data collectors. The people in the area respect and have a high confidence in those working or studying in the medical field, thus recruitment of medical students as data collectors increased the response rate. Only about 5% of the subjects who were asked to participate refused to do so. Data were collected for a period of two weeks in January 2007 and a total of 448 subjects participated in the study. 2.2.. The Instrument -------------------- The survey instrument consisted of several variables: age, drinking frequency, and 20 questions on drinking motives. Drinking frequency of the participants was obtained using the item I use alcohol followed by four response categories: daily, 2--3 times a week, 2--3 times a month, and 2--3 times a year. A literature survey was done and expert opinions were sought to identify motives towards alcohol use in young people in Sri Lanka \[[@b10-ijerph-06-02408],[@b16-ijerph-06-02408]--[@b19-ijerph-06-02408]\]. The 3-dimensional drinking motives scale developed by Cooper, Russell, Skinner, and Windle (1992) was used as the base in the development of our new scale. In addition, ideas and opinions expressed on drinking motives by more than 100 young males, by whom the first author came into contact during his health education and prevention activities in the community, were also considered when selecting culturally-appropriate items for the scale. The scale developed by Cooper and colleagues (1992) has a total of 15 items and the new scale has 20 items. Some items from the scale developed by Cooper and colleagues, such as I use alcohol because it helps me to forget my worries, were included in the new scale. New culturally-appropriate items, such as I drink alcohol because it will enhance my creative ability and I drink alcohol because it helps me to control others, were added to the new scale. The new scale varies from the contemporary drinking motive scales that have been developed and used in the West. A jury of experts consisting of a psychiatrist, two social scientists, and a psychologist examined the items for content validity. Based on their evaluation, the final scale of motive towards alcohol use was constructed using 20 rating items (see [Table 1](#t1-ijerph-06-02408){ref-type="table"} for scale items). Four Personal Enjoyment (PE) items, 10 Tension-Reduction (TR) items, and six Social Pressure (SP) items were included in the scale. For example, I drink alcohol because I like the taste was a PE item, and I drink alcohol because it is customary for men on special occasions was a SP item. There were four response categories for each of these 20 items which were scored as follows: to a greater extent equals 3; to some extent equals 2; to little extent equals 1; and not relevant equals 0. The internal consistencies of the three factors using Cronbach's alpha methods were as follows: alpha~PE~ = 0.48; alpha~TR~ = 0.74; and alpha~SP~ = 0.62. To assess the impact of each factor on drinking habits, each item in each of the three subscales were added up to obtain 3 subscale scores. 2.3.. Data Analysis ------------------- The collected data were checked for consistency. Descriptive and bivariate analyses of the data set were done using SPSS 15.0 \[[@b20-ijerph-06-02408]\]. Confirmatory Factor Analysis (CFA) of the 20 item motives scale toward alcohol use was conducted using Lisrel 8.80 \[[@b21-ijerph-06-02408]\]. 3.. Results =========== After cleaning and consistency checking, analysis was done using 412 sample subjects. The age of the participants ranged from 16--30 years (*M = 20.27	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1} =============== Lung cancer remains a major health problem worldwide. In 2012 lung cancer was the most commonly diagnosed cancer worldwide making up 13.0% of the total incidence of cancer. It was also the most common cause of death from cancer worldwide, accounting for nearly one in five cancer deaths (19.4% of the total) \[[@B1]\]. Lung cancer is clinically divided into two main groups based upon the size and appearance of malignant cells: small cell lung cancer (SCLC) (16.8%) and non-small cell lung cancer (NSCLC) (80.4%) \[[@B2]\]. The most effective option for treatment of lung cancer is surgical resection, when feasible \[[@B3]\]. However, majority of patients are diagnosed at an advanced or metastatic stage of disease in which case chemotherapy and/or concurrent administration of chemotherapy and radiation is the most beneficial form of treatment \[[@B4]\]. Nevertheless, even with treatment, the 5-year survival rate in patients is only 16.6% \[[@B5]\], with poor survival rates mainly being attributed to late stage diagnosis and high frequency of drug resistance. Obtaining a better understanding regarding the molecular mechanisms involved in lung carcinogenesis is of utmost importance in the aim to identify the diagnostic and prognostic markers for early detection and targeted treatment of lung cancer. Apoptosis plays an important role during development and in the maintenance of multicellular organisms through the removal of damaged, aged, or autoimmune cells \[[@B6]\]. The apoptotic process can be divided into the extrinsic and intrinsic pathway. Each pathway will ultimately result in the activation of cell death proteases, which in turn initiates a cascade of proteolysis involving effector caspases that carries out the completion of the apoptotic process \[[@B7]\]. In contrast to normal cells, cancer cells have the ability to evade apoptosis to promote cell survival under the conditions of environmental stress. There are a number of mechanisms by which cancer cells are able to suppress apoptosis. For example, the tumor suppressor genep53 is a widely mutated gene in human tumorigenesis \[[@B8]\].p53 mutation will inhibit the activation of DNA repair proteins leading to a decrease in the initiation of apoptosis \[[@B7]\], allowing for cells to divide and grow uncontrollably, forming malignant tumors. Furthermore, cancer cells are able to disrupt the balance between pro- (BCL-2,BCL-XL) and antiapoptotic factors (BAX,BIM, andPUMA) \[[@B9]\]. Increased expression of proapoptotic Bcl-2 protein contributes not only to the development of cancer but also to resistance against a wide variety of anticancer agents, such as cisplatin (DDP) and paclitaxel \[[@B10]--[@B12]\]. MicroRNAs (miRNAs) are a subset of noncoding RNAs of about 20 to 25 nucleotides long which posttranscriptionally regulate gene expression via inhibition of mRNA translation, by binding to specific target sites in their 3′-untranslated region (3′UTR), or inducing degradation of target mRNA through cleavage \[[@B13]\]. An individual miRNA is able to modulate the expression of multiple genes; correspondingly, a single target can be modulated by many miRNAs \[[@B14]\]. MiRNAs were reported to be involved in a vast range of biological processes, including apoptosis (see [Figure 1](#fig1){ref-type="fig"}) \[[@B15]--[@B22]\]. As miRNAs play a key role in an assortment of biological processes, an altered miRNA expression is likely to contribute to human diseases including cancer \[[@B23]\]. Previous studies have shown that compared to normal tissues, malignant tumors and tumor cell lines were found to have widespread deregulated miRNA expression \[[@B24]--[@B28]\]. MiRNAs are critical apoptosis regulators in tumorigenesis and cancer cells are able to manipulate miRNAs to regulate cell survival in oncogenesis. Many studies carried out in the past several years are aimed at elucidating the specific miRNAs associated with apoptosis in cancer and their related target genes. In this review we will examine the recent progress of research on miRNA-mediated regulation of apoptosis in lung cancer and its future therapeutic applications. 2. Antiapoptotic miRNAs {#sec2} ======================= Evasion of apoptosis is a significant hallmark of tumor progression, and one mechanism by which miRNAs influence development of cancer is through the regulation of the apoptotic process as shown in various studies \[[@B29]--[@B32]\]. miRNA expression can be either upregulated or downregulated and evidence has shown that dysregulated miRNAs can behave as oncogenes or tumor suppressor genes in lung cancers \[[@B18], [@B28], [@B33]\]. Amplification of miRNAs can lead to the downregulation of tumor suppressors or other genes that are involved in apoptosis \[[@B34]\]. miR-197. For example, the expression of miR-197 is increased in cancer tissues in comparison to normal specimens. Fiori et al. (2014) demonstrated that knockdown of miR-197 in NIH-H460 and A549 cells promoted induction of apoptosis, evident by the observation of caspases 3--7 activation and increased apoptotic population by Annexin staining. Furthermore, the direct interaction of miR-197 with the 3′UTR ofBMF andNOXAwas demonstrated by the luciferase reporter assay \[[@B35]\]. When activated by intra- or extracellular stimuli, proapoptotic Bmf binds to and neutralizes antiapoptotic Bcl-2 family members on the mitochondrial membrane, thus allowing proapoptotic proteins Bak and Bax to dimerize and promote the release of cytochrome c, ultimately leading to cell death \[[@B36]\]. Noxa is a BH-3 only proapoptotic protein transcriptionally activated byp53. Collectively, miR-197 is able to act upon different levels of thep53 pathway to counteract the induction of apoptosis, thus allowing cells to proliferate uncontrollably \[[@B35]\]. miR-21. miR-21 is found to be frequently upregulated in a number of cancers; however its potential role in tumorigenesisin vivo is not fully explored. Using transgenic mice with loss-of-function and gain-of-function miR-21 alleles, Hatley and colleagues elucidated the role of miR-21 in NSCLC pathogenesisin vivo\[[@B37]\]. It was determined that miR-21 regulates tumor proliferation and survival, which are two integral components of NSCLC pathogenesis, by targeting negative regulators of the RAS pathway as well as by targeting proapoptotic genes \[[@B37]\]. In regards to the apoptotic pathway, overexpression of miR-21in vivo leads to decreased protein levels of Apaf-1, an important component of the intrinsic mitochondrial apoptotic pathway, as well as decreased expression of FasL, a key initiator of the extrinsic apoptotic pathway. Furthermore,RHOB, with a tumor suppressor role, is a target of miR-21 and its dysregulation leads to an increase in cell growth and inhibition of apoptosis \[[@B38]\]. Together these results suggest that relieving miR-21 downregulation of these proapoptotic and tumor suppressor genes could provide a means to enhance the effect of current chemotherapy. miR-212. Acetylcholinesterase (AChE), a component of the cholinergic system, has the ability to influence apoptotic sensitivity bothin vitro andin vivo\[[@B39]--[@B41]\]. In NSCLC tissues AChE levels are low and are associated with tumor aggressiveness, increase risk of postoperative recurrence, and low survival rate \[[@B42]\]. Lu et al. (2013) determined thatAChE expression in NSCLC was posttranscriptionally modulated by miR-212 binding to its 3′UTR. Interestingly, alterations in neither AChE nor miR-212 expression significantly affected cell survival; however it was observed that during DDP-induced apoptosis miR-212 levels were reduced with a concurrent increase in AChE protein levels. This suggests that miR-212 plays a role in DDP resistance by directly inhibiting AChE and preventing apoptosis. Therefore, interference against miR-212 may potentially be a means to improve the pharmacotoxicological profile of DDP in NSCLC \[[@B43]\]. miR-17-5p and miR-20a. The miR-17-92 cluster, which is composed of seven miRNAs and resides in intron 3 of theC13orf25 gene at 13q31.3, is frequently overexpressed in lung cancers \[[@B44]\]. Matsubara et al. (2007) demonstrated that inhibition of two components of the miR-17-92 cluster, miR-17-5p, and miR-20a, with antisense oligonucleotides can induce apoptosis selectively in lung cancer cells that overexpress miR-17-92 \[[@B45]\]. Previously, miR-17-5p and miR-20a have been shown to directly targetE2F1 \[[@B46]\]; thus inhibition of these miRNAs may cause the induction of apoptosis in part through the induction ofE2F1 and subsequent cell cycle progression intoS phase \[[@B47]\]. However additional studies would have to be carried out to determine the actual targets for the miR-17-92 cluster to gain a better understanding of the development of this cancer. 3. Proapoptotic miRNAs {#sec3} ====================== MiRNAs that are downregulated are considered tumor suppressor genes. Tumor suppressor miRNAs usually prevent tumor development by negatively regulating oncogenes and/or genes that control cell differentiation or apoptosis \[[@B48]\]. MiRNAs that act as tumor suppressors can be downregulated as a result of deletions, epigenetic silencing, or loss of expression of transcription factors (see [Table 2](#tab2){ref-type="table"}) \[[@B49]\]. 3.1. B-Cell Lymphocyte 2 (BCL-2) Family Related miRNAs {#sec3.1} -------------------------------------------------------- Members of the evolutionarily conservedBCL-2 family are thought to be the central regulators of apoptosis. The expression level ofBCL-2 differs for different cell types; however high levels and aberrant patterns ofBCL-2 expression were reported in a wide variety of	{ "pile_set_name": "PubMed Central" }
Related literature {#sec1} ==================== For the isostructural compound 5-chloro-2-hydroxybenzaldehyde 4-ethylthiosemicarbazone, see: Lo et al. (2011[@bb1]) Experimental {#sec2} ============== {#sec2.1} ### Crystal data {#sec2.1.1} C~10~H~12~BrN~3~OSM ~r~ = 302.20Monoclinic,a = 22.040 (4) Åb = 11.844 (2) Åc = 9.5102 (19) Åβ = 101.69 (3)°V = 2431.1 (8) Å^3^Z = 8Mo Kα radiationμ = 3.54 mm^−1^T = 123 K0.20 × 0.10 × 0.05 mm ### Data collection {#sec2.1.2} Rigaku Saturn70 diffractometerAbsorption correction: multi-scan (CrystalClear; Rigaku, 2008[@bb2]) T ~min~ = 0.661, T ~max~ = 0.8384201 measured reflections2331 independent reflections1760 reflections with I \> 2σ(I)R ~int~ = 0.032 ### Refinement {#sec2.1.3} R\[F ^2^ \> 2σ(F ^2^)\] = 0.042wR(F ^2^) = 0.114S = 0.952331 reflections155 parameters3 restraintsH atoms treated by a mixture of independent and constrained refinementΔρ~max~ = 0.70 e Å^−3^Δρ~min~ = −1.01 e Å^−3^ {#d5e409} Data collection: CrystalClear (Rigaku, 2008[@bb2]); cell refinement: CrystalClear; data reduction: CrystalClear; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008[@bb3]); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008[@bb3]); molecular graphics: SHELXTL (Sheldrick, 2008[@bb3]); software used to prepare material for publication: publCIF (Westrip, 2010[@bb4]). Supplementary Material ====================== ###### Click here for additional data file. Crystal structure: contains datablock(s) I, global. DOI: [10.1107/S1600536813008787/ng5322sup1.cif](http://dx.doi.org/10.1107/S1600536813008787/ng5322sup1.cif) ###### Click here for additional data file. Structure factors: contains datablock(s) I. DOI: [10.1107/S1600536813008787/ng5322Isup2.hkl](http://dx.doi.org/10.1107/S1600536813008787/ng5322Isup2.hkl) ###### Click here for additional data file. Supplementary material file. DOI: [10.1107/S1600536813008787/ng5322Isup3.cml](http://dx.doi.org/10.1107/S1600536813008787/ng5322Isup3.cml) Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?ng5322&file=ng5322sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?ng5322sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?ng5322&checkcif=yes) Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [NG5322](http://scripts.iucr.org/cgi-bin/sendsup?ng5322)). The authors would like to thank the China Scholarship Council (CSC). Comment ======= A Schiff ligand was synthesized through one-pot reaction with high yield using 5-bromo-2-hydroxybenzaldehyde and 4-ethyl-3-thiosemicarbazide (Fig. 1). The title compound can be used as tridentate chelating ligand to construct spin-crossover complexes. Isostructural 5-chloro-2-hydroxybenzaldehyde-4-ethylthiosemicarbazone was reported previously (Lo et al., 2011). In the title compound, a strong intramolecular hydrogen bond O---H···N is observed. An intermolecular N---H···S hydrogen bond connects two molecules into a supramolecular dimer as shown in Figure 2. Experimental {#experimental} ============ 5-Bromo-2-hydroxybenzaldehyde (4.02 g, 20 mmol) in 50 ml ethanol and 4-ethyl-3-thiosemicarbazide (2.38 g, 20 mmol) were reacted for 6 h at 350 K. Slow evaporation of the yellow solution gave large colorless crystals. Refinement {#refinement} ========== Carbon-bound H-atoms were placed in calculated positions (C---H 0.95, 0.98 and 0.99 Å) and were included in the refinement in the riding model approximation, with U~iso~(H) =1.5U~eq~(C) for methyl H atoms and 1.2U~eq~(C) for the others. The hydroxy and amino H atoms were located in a difference Fourier map, and were refined with distance restraints of O---H 0.85±0.01 and N---H 0.88±0.01 Å; with U~iso~(H) =1.2U~eq~(N and O). Figures ======= ![Displacement ellipsoid plot (50% probability level) of the title compound, with atom numbering of structurally unique non-H atoms and the H atoms.](e-69-0o762-fig1){#Fap1} ![The packing diagram of the title compound, with H atoms omitted for clarity. Hydrogen bonds are shown as dashed lines.](e-69-0o762-fig2){#Fap2} Crystal data {#tablewrapcrystaldatalong} ============ ----------------------- --------------------------------------- C~10~H~12~BrN~3~OS F(000) = 1216 M~r~ = 302.20 D~x~ = 1.651 Mg m^−3^ Monoclinic, C2/c Mo Kα radiation, λ = 0.710747 Å Hall symbol: -C 2yc Cell parameters from 3650 reflections a = 22.040 (4) Å θ = 3.1--27.5° b = 11.844 (2) Å µ = 3.54 mm^−1^ c = 9.5102 (19) Å T = 123 K β = 101.69 (3)° Block, colourless V = 2431.1 (8) Å^3^ 0.20 × 0.10 × 0.05 mm Z = 8 ----------------------- --------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== ------------------------------------------------------------------ -------------------------------------- Rigaku Saturn70 diffractometer 2331 independent reflections Radiation source: Rotating Anode 1760 reflections with I \> 2σ(I) Confocal monochromator R~int~ = 0.032 Detector resolution: 28.5714 pixels mm^-1^ θ~max~ = 26.0°, θ~min~ = 3.1° dtprofit.ref scans h = −27→20 Absorption correction: multi-scan (CrystalClear; Rigaku, 2008) k = −9→14 T~min~ = 0.661, T~max~ = 0.838 l = −11→10 4201 measured reflections ------------------------------------------------------------------ -------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ------------------------------------- ------------------------------------------------------------------------------------- Refinement on F^2^ Primary atom site location: structure-invariant direct methods Least-squares matrix: full Secondary atom site location: difference Fourier map R\[F^2^ \> 2σ(F^2^)\] = 0.042 Hydrogen site location: inferred from neighbouring sites wR(F^2^) = 0.114 H atoms treated by a mixture of independent and constrained refinement S = 0	{ "pile_set_name": "PubMed Central" }
	{ "pile_set_name": "PubMed Central" }
INTRODUCTION {#s1} ============ Epithelial ovarian cancer (EOC) represents the leading cause of death in gynaecological malignancies \[[@R1]\]. EOC progresses mainly through extensive intraperitoneal and/or retroperitoneal (lymphatic) tumour spread, while distant metastases are rarely seen \[[@R2]\]. So far, the biologic background of these two different modes of progression is poorly understood. Recent investigations have shown that the tumour cell differentiation status (epithelial-mesenchymal-transition) may influence the route of metastases in ovarian cancer \[[@R3]\]. Accordingly, we could previously show that strong expression of an 85 kDa fragment of the epithelial marker E-Cadherin in ovarian cancer cells appears to be associated with intraperitoneal metastases \[[@R4]\]. Contrarily, other analyses revealed that mesenchymal tumour cells exhibited locally restricted tumour growth \[[@R3]\]. Highly metabolic tumour cells in locally growing tumour masses are dependent on vascular endothelial growth factor (VEGF) mediated angiogenesis \[[@R5]\]. The VEGF family consists of VEGF-A,-C,-D and induces different cascades via their receptor-tyrosine-kinases VEGFR-1, VEGFR-2, VEGFR-3 to exhibit their various biological effects. In this context, VEGF-C and -D bind VEGFR-3 and promote additional lymphangiogenesis \[[@R6]\]. The aim of the present study was to identify the diagnostic potential of the VEGF family for identification of ovarian cancer patients being at high risk for retroperitoneal metastases. Identifying these patients may enable tailored therapeutic strategies to improve their prognosis and reduce morbidity. RESULTS {#s2} ======= Tumours with lymph node metastases exhibit high levels of VEGF-C expression {#s2_1} --------------------------------------------------------------------------- Tumour samples of all included 100 patients were analysed for VEGF-A, VEGF-C and VEGF-D expression by Western Blot, including 80 (80%) with 'extensive intraperitoneal' tumour growth and 20 patients (20%) with 'predominant retroperitoneal' tumour involvement. Patient characteristics were balanced between both groups except for an expected higher rate of large bowel resections (69% vs 40% p=0.005) and lower number of resected lymph nodes (median 40 vs 49 lymph nodes p=0.043) in patients with 'extensive intraperitoneal' tumour growth (Table [1](#T1){ref-type="table"}). ###### Patient characteristics according to the pattern of tumour dissemination Overall cohort Extensive intraperitoneal group Predominant retroperitoneal group p-value ------------------------------------------------------- ---------------- --------------------------------- ----------------------------------- --------------- No. of patients 100 (100%) 80 (80.0%) 20 (20.0%) Age at diagnosis (years) 0.84b Median 62 62 62 Range 33-84 33-84 47-75 Tumour stage \< 0.001a pT1c 3 (3%) 0 (0%) 3 (15%) pT2b 1 (1%) 0 (0%) 1 (5%) pT2c 2 (2%) 0 (0%) 2 (10%) pT3a 2 (2%) 0 (0%) 2 (10%) pT3b 13 (13%) 1 (1.25%) 12 (60%) pT3c 79 (79%) 79 (98.75%) 0 (0%) Lymph node status \< 0.001a N0 40 (40%) 40 (50%) 0 (0%) N1 60 (60%) 40 (50%) 20 (100%) Number of resected lymph nodes 0.043b Median 41 40 49 Range 1-97 1-94 1-97 Mode of tumour dissemination \< 0.001a Solely intraperitoneal group (pT2b-pT3c, pN0) 40 (40%) 40 (50%) 0 (0%) Predominant retroperitoneal group (pT1a-3b, pN1) 20 (20%) 0 (0%) 20(100%) Two-sided group (pT3c, pN1) 40 (40%) 40 (50%) 0 (0%) Grading 0.18c G1 4 (4%) 3 (3.75%) 1 (5%) G2 24 (24%) 22 (27.5%) 2 (10%) G3 72 (72%) 55 (68.75%) 17 (85%) Histologic subtype 0.09a Serous 85 (85%) 71 (88.75%) 14 (70%) Endometrioid 4 (4%) 2 (2.5%) 2(10%) Clear cell 3 (3%) 1 (1.25%) 2(10%) Mixed differentiation 6 (6%) 5 (6.25%) 1 (5%) Not determined/unknown 2 (2%) 1 (1.25%) 1 (5%) Surgical procedures Large bowel resection 63 (63%) 55 (68.75%) 8 (40%) 0.002a Small bowel resection 8 (8%) 7 (9.6%) 1 (5%) 0.35a Upper abdomen interventions Liver 29 (29%) 25 (31,25%) 4 (20%) 0.23a Spleen 19 (19%) 17 (21.25%) 2 (10%) 0.20a Pancreas 3 (3%) 2 (2.5%) 1 (5%) 0.50a Postoperative residual tumour 0.58a Microscopic 78 (78%) 62 (77.1%) 16 (80%) Macroscopic 21 (21%) 17 (21.6%) 4 (20%) Preoperative CA 125 level (kU/l) 0.32b Median 386 401 307 Range 14-13089 14-13089 34-2802 Postoperative CA 125 level (kU/l) 0.79b Median 103 101 121 Range 11-1267 11-1267 28-896 Overview of the clinical characteristics of all included patients with EOC (n = 100). A total of 20 patients with predominant retroperitoneal tumour dissemination are opposed to 80 patients with extensive intraperitoneal tumour involvement. Both groups were tested with Pearson-Chi-Quadrat test (a), ANOVA analysis (b), as well as with the Kruskal Wallis (c) test upon differences. Statistically significant P values are printed in bold. FIGO: International Federation of Gynecology and Obstetrics; kU/l: kilo Units per liter. For VEGF-A no significant difference in expression levels between the different groups was found ('predominant retroperitoneal' vs. 'extensive intraperitoneal' metastases: median 1.18 vs 1.09, p=0.50, Figures [1](#F1){ref-type="fig"}, [2A](#F2){ref-type="fig"}). ![Expression of VEGF-A, VEGF-C, VEGF-D according to the mode of tumour progression\ Representative expression of VEGF-A, VEGF-C, VEGF-D in the different types of tumour dissemination. Protein lysates from the breast cancer cell line MCF7 were used as positive controls for VEGF-A, --C and --D. Equal amounts of protein lysate (20 μg) were loaded per well.](oncotarget-08-43218-g001){#F1} ![Quantitative expression of VEGF-A, VEGF-C, VEGF-D	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1} =============== Radical cystectomy (RC) remains the standard of care for local treatment of non metastatic muscle invasive bladder cancer (MIBC). However, cancer specific survival is approximately 50% depending on the presence of extravesical extension and/or lymph nodes metastases \[[@bib1]\]. In daily practice, more than 50% of patients die of distant metastases within two years after cystectomy, suggesting the presence of micro-metastases at time of surgery \[[@bib2]\]. Therefore, peri-operative chemotherapy (adjuvant or neoadjuvant) has been developed to increase overall survival, with an absolute benefit of 5% reported for neoadjuvant chemotherapy (NAC) and international guidelines recommend NAC based on the available level I evidence \[[@bib3],[@bib4]\]. The chemotherapy administration time and the optimal chemotherapy regimen to be delivered remain open to discussion. As dose-dense methotrexate, vinblastine, doxorubicin and cisplatin (dd-MVAC) has been shown to be associated with higher response rates in bladder metastatic disease \[[@bib5]\], a better efficacy can also be suspected in the peri-operative setting. Recently, Choueiri et al. and Plimack et al. reported interesting results from two phase II trials using dd-MVAC as neoadjuvant chemotherapy in MIBC. After three to four cycles, the pathologic downstaging (pT1 N0M0) was quite similar (49% and 53%), the pathologic complete response rates (pT0) were 26% and 38%, respectively \[[@bib6],[@bib7]\]. Our objective was to design a randomized phase III controlled study comparing the efficacy of GC and dd-MVAC in term of progression-free survival in patients for whom chemotherapy has been decided, before or after radical cystectomy. 2. Material and methods {#sec2} ======================= 2.1. - Study design {#sec2.1} ------------------- This randomized phase III study assesses the efficacy of dd-MVAC and GC peri-operative chemotherapy (adjuvant or neoadjuvant) in patients with bladder cancer disease defined by a T2, T3 or T4a N0 (≤10 mm on CT scan) M0 staging for patients receiving neoadjuvant chemotherapy or pT3 or pT4 or pN+ and M0 for patients receiving adjuvant chemotherapy. Secondary endpoints include overall survival, safety, response rate in the neoadjuvant setting. From February 2013 to March 2018, a total of 500 patients have been randomized in the French GETUG/AFU V05, controlled phase III trial, including 28 participating centers with referent urologist and oncologist investigators ([Fig. 1](#fig1){ref-type="fig"}).Fig. 1Participating centers of the GETUG/AFU V05 multicentre, randomised phase III trial.Fig. 1 As previously mentioned, the peri-operative chemotherapy schedule proposed was: [Standard Arm A: GC]{.ul}.-GEMCITABINE 1250 mg/m2: Day 1 and Day 8-CISPLATIN 70 mg/m2: Day 1Every 3 weeks, for a total of 4 cycles [Experimental Arm B: dd-MVAC]{.ul}.-METHOTREXATE 30 mg/m2: Day 1-VINBLASTINE 3 mg/m2: Day 2-DOXORUBICIN 30 mg/m2: Day 2 - CISPLATIN 70 mg/m2: Day 2 - G-CSF: Day 3 to Day 9Every 2 weeks, for a total of 6 cycles The chemotherapy response was evaluated according to RECIST 1.1 criteria. The treatment toxicity was evaluated according to NCI CTCAE (v 4.0). The progression-free survival estimation rate of this trial was determined at 3 years. In our prospective randomized study all the patients were followed for 5 years. 2.2. - Study Procedures {#sec2.2} ----------------------- At baseline before screening, a CT with contrast of the chest, abdomen and pelvis was performed for all patients, in association with a systematic bone scan and a complete biological evaluation. Follow up visits and their schedules and measurements are clearly reported in [Fig. 2](#fig2){ref-type="fig"}.Fig. 2Study procedures, schedule and parameters of the patients follow-up.Fig. 2 In our clinical trial, a dose reduction of chemotherapy in case of toxicity was allowed. Considering the GC group (standard arm), the cisplatin dose was adapted to renal function (creatinine clearance \> 60 ml/mn: 70 mg/m2; between 50 and 60 ml: 50 mg/m2; between 40 and: 35 ml mg/m2; creatinine clearance \< 40 ml/mn: end of the chemotherapy). As regards haematologic toxicity (neutropenic fever), a dose reduction of 15% was recommended for the two molecules. Considering the dd-MVAC group (experimental arm), the cisplatin dose was adapted to renal function as previously described. A dose reduction of 15% was also recommended for the four molecules in case of grade 4 toxicity and the chemotherapy stopped in the absence of recovery within 14 days. The study protocol was approved by the Ethics Committee CPP ROUEN NO on 19 April, 2012 and the competent authority on 27 February, 2012. All patients signed the informed consent form to be enrolled in this randomized phase III study *(Clinical trial registry: clinicaltrials.gov - NCT 018 12369).* After the exclusion of any analysis of 7 patients who did not meet the inclusion/exclusion criteria, it remained 493 patients for the primary analysis (intent-to-treat population). Baseline characteristics by chemotherapy arm are reported in [Table 1](#tbl1){ref-type="table"}, whereas tumour staging at randomization by type of peri-operative chemotherapy are detailed in [Table 2](#tbl2){ref-type="table"}.Table 1Baseline characteristics by chemotherapy arm.Mean (standard deviation) for quantitative data. Frequency (percentage) for qualitative data. Comparisons between GC and dd-MVAC groups are performed with a Student T-test or Chi-2 test. P-value \< 0.05 would assume a statistical difference between GC and dd-MVAC groups.Table 1GCdd-MVACp-valuen = 245n = 248[Demography]{.ul}Age63 (7.6)62.6 (7.9)0.62SexMale206 (84%)202 (81%)0.51Female39 (16%)46 (19%)[Physical examination]{.ul}Body Mass Index26.6 (4.7)26 (4.4)0.16Body Surface Area1.9 (0.2)1.9 (0.2)0.52WHO status0171 (70%)165 (67%)0.59172 (29%)82 (33%)Not done2 (1%)1 (0%)[Medical History]{.ul}No10 (4%)6 (2%)0.43Yes235 (96%)242 (98%)whoseneuropathy3 (1%)1 (0%)0.60hearing disorder35 (15%)46 (19%)0.28high blood pressure100 (43%)89 (37%)0.23infarc9 (4%)11 (5%)0.88coronary insuff.5 (2%)9 (4%)0.45diabetes14 (6%)4 (2%)0.03tobacco198 (84%)197 (81%)0.48aromatic amines14 (6%)7 (3%)0.16[Biology and renal function]{.ul}Hemoglobin (g/100 mL)14.3 (6.8)13.9 (1.5)0.33Neutrophil polynuclear cells (1000/mm^3^)7.5 (34.7)6.9 (22.3)0.83Platelets (1000/mm^3^)272.6 (78.2)274.3 (85.4)0.81Total bilirubin (mg/L)5.0 (2.6)5.2 (2.5)0.34ALT (UI/L)23.6 (14.6)22.8 (10.5)0.49AST (UI/L)20.4 (7.2)20.6 (7.2)0.75Alkaline phosphatase (UI/L)77.9 (26.2)77.9 (31.5)0.97Creatinine (mg/L)9.6 (3.3)9.1 (2.1)0.05Clearance of creatinine (mL/min)89.3 (27.6)90.2 (25)0.69Table 2Staging at randomization by type of peri-operative chemotherapy and armFrequency (percentage). For adjuvant chemotherapy, pTNM staging is performed on cystectomy. For neoadjuvant chemotherapy, TNM staging is performed on TURBT. Staging according to 2009 TNMclassification.Table 2Adjuvant chemotherapyNeoadjuvant chemotherapyGCdd-MVACGCdd-MVACn = 26n = 30n = 219n = 218TumourT11 (4%)1 (3%)00T2a01 (3%)141 (64%)138 (63%)T2b3 (12%)2 (7%)66 (30%)59 (27%)T3a8 (31%)12 (40%)4 (2%)7 (3%)	{ "pile_set_name": "PubMed Central" }
CASE REPORT =========== A 36-year-old man visited the emergency department. He had suffered from a sudden onset of chest pain, headache, palpitation, and dizziness. His pulse was regular, bounding, and at a rate of 119 per minute, and his blood pressure was at 190/120 mmHg. Electrocardiography and echocardiography were normal. Chest X-ray showed an oval-shaped mass lesion on the left paravertebral area ([Fig. 1A](#F1){ref-type="fig"}). Chest computed tomography (CT) and magnetic resonance imaging (MRI) showed a left paravertebral mass in the region of T4-T6, measuring at 3×6 cm, which was not invasive to the adjacent organs ([Fig. 1B](#F1){ref-type="fig"}). There was no specific finding on abdomen CT. The biochemical studies showed elevated levels of serum norepinephrine (12.905 ng/mL; normal limit \[nl\], 0 to 0.8 ng/mL), urine norepinephrine (4,946 µg/day; nl, 15 to 80 µg/day), urine metanephrine (4.2 mg/day; nl\<0.8 mg/day), and urine vanillylmandelic acid (26.6 mg/day; nl, 0 to 8 mg/day). The serum and urine epinephrine levels were normal. The presence of the tumor was confirmed using an I-123 metaiodobenzylguanidine (MIBG) scan, along with its isolated nature ([Fig. 2](#F2){ref-type="fig"}). In order to control the blood pressure and prepare for surgery, we administered α and β blocking agents to the patient. During the first three weeks, Concor (5 mg), Norvasc (5 mg), Atacand (8 mg), and Xyrem (0.25 mg) were administered. When visited the outpatient department, his blood pressure was near normotensive, but tachycardia and chest discomfort were still present. Therefore, we changed the medication to Cardura-XL (4 mg) for five weeks. The patient no longer felt chest discomfort, but tachycardia was still present. The heart rate was controlled (100 beat/min) after we administered Concor (2.5 mg). Two months after his first visit, we performed the operation. The tumor was found lying on the left paravertebral sulcus, which was hypervascular and well demarcated ([Fig. 3A](#F3){ref-type="fig"}). We performed thoracoscopic resection, but there were transient changes in the blood pressure, which ranged from 130/90 to 200/120 mmHg; therefore, we converted to thoracotomy. In the intraoperative period, we controlled the blood pressure with Brevibloc and Perdipine. After the tumor was resected, the blood pressure dropped sharply. Therefore, we stopped administering antihypertensive drugs, and then the blood pressure remained stable. The resected tumor measured 6.5×4.0×3.0 cm and weighed 47.5 g. Histopathological examination confirmed the tumor to be pheochromocytoma ([Fig. 3B](#F3){ref-type="fig"}). During the postoperative period, the patient remained normotensive without the need of antihypertensive drugs. He was discharged on the postoperative day 8. Two months later, the follow-up biochemical studies showed decreased levels of serum norepinephrine (1.108 ng/mL), urine norepinephrine (149.3 µg/day), urine metanephrine (1 mg/day), and urine vanillylmandelic acid (7.1 mg/day). The patient was asymptomatic with normal blood pressure and without taking antihypertensive drugs for more than 1 year. DISCUSSION ========== Pheochromocytoma develops from the chromaffin cells of the sympathetic nervous system \[[@B1]-[@B3]\]. In most cases, pheochromocytoma originates in the adrenal gland, but it can arise anywhere on the paraganglion tissues that contain chromaffin \[[@B1]\]. Extra-adrenal pheochromocytoma, often known as paraganglioma, is rare in adults (approximately 10%) and it occurs in thorax in 1% of the cases \[[@B1],[@B4],[@B5]\]. Pheochromocytomas and paragangliomas are associated with several well-known inherited syndromes such as multiple endocrine neoplasia type IIA, IIB, or III, von Hippel-Lindau disease, von Recklinghausen\'s disease, and Sturge-Weber syndrome. Patients with any of these syndromes have familial histories \[[@B4],[@B6],[@B7]\]. Most pheochromocytomas are catecholamine-secreting, hyperfunctioning tumors, which cause symptoms such as hypertension, headache, palpitations, sweating, and weight loss resulting from the excess production of catecholamines. Such excess production leads to elevated levels of catecholamines in the blood, or increases the level of metabolites in the urine \[[@B6]\]. Thus, blood and urine examinations are useful for diagnosing functional pheochromocytoma. Paragangliomas are easily located using a CT scan, a MRI, or an I-131 MIBG \[[@B1],[@B6],[@B8]\]. Surgical resection is the preferred treatment for paraganglioma. Before the operation, a combination of α and β adrenergic blockades are required to control blood pressure and to prevent intra-operative hypertensive crises \[[@B2],[@B3]\]. Preoperative adrenergic blockades are used until the patient\'s symptoms subside. The end point is normotension or near normotension, with slight postural hypotension and elimination or reduction of paroxysmal spells \[[@B3],[@B6]\]. We also administered α and β blocking agents to the patient for two months until the operation date. Intraoperative monitoring by an experienced anesthesia team is very important because the patient\'s blood pressure and heart rate fluctuate very severely \[[@B2],[@B6]\]. When the operators touch the tumor directly, the blood pressure and heart rate increase rapidly; therefore, blunt dissection through thoracotomy should be excluded and video-assisted thoracic surgery is performed rarely on thoracic lesions \[[@B1]\]. In our case, we attempted to perform thoracoscopic resection, but we experienced severe fluctuation of the vital signs; therefore, we converted to thoracotomy for resection of the tumor. After the tumor was resected, the blood pressure dropped sharply. Thus, the central venous line should be prepared in order to facilitate rapid volume resuscitation and an appropriate α agonist injection \[[@B6]\]. In our case, the blood pressure also dropped sharply after resection of the tumor. Because paragangliomas have a higher recurrence rate than pheochromocytomas, long-term postoperative follow-up is necessary \[[@B1],[@B6]\]. When previous symptoms reoccur, it indicates recurrence of a tumor. Annual measurement of blood pressure and the urinary catecholamine metabolites is necessary. Performing CT or MRI may be helpful, and I-123 MIBG scanning is often used for detecting metastasis and recurrence of the disease \[[@B6]\]. No potential conflict of interest relevant to this article was reported. ![Radiologic studies of the patient. (A) Chest posteroanterior shows well defined mass density at left paravertevral area (black arrow). (B) Chest magnetic resonance imaging shows a 7 cm sized well dermacated mass lesion at left posterior mediastinum (white arrow). Adrenal gland and adjacent structures are normal findings.](kjtcs-46-88-g001){#F1} ![I-123 metaiodobenzylguanidine (MIBG) tumor scan shows hot uptake in left paraspinal area (arrow) and another metastatic lesion is not observed.](kjtcs-46-88-g002){#F2} ![(A) Operative findings. About 3×6 cm sized well dermacated, hypervascular mass at left paravertebral area extending from T4-T6 level. (B) Histopathology of the resected specimen. It shows characteristic zellballen pattern with thin fibrovascular septa (H&E, ×200).](kjtcs-46-88-g003){#F3}	{ "pile_set_name": "PubMed Central" }
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Introduction {#s1} ============ The nucleus accumbens (NAc) is a forebrain structure that regulates the vigor of reward-seeking. Its excitatory inputs likely encode motivational states and the presence of reward-associated cues (Mannella et al., [@B33]). For example, paraventricular thalamic (PVT) input regulates food-seeking behavior under conditions of hunger and threat (Labouèbe et al., [@B27]; Choi and McNally, [@B12]; Do-Monte et al., [@B15]; Cheng et al., [@B11]; Choi et al., [@B13]), while basolateral amygdala (BLA) input encodes the motivational value of reward-associated cues (Ambroggi et al., [@B2]; Stuber et al., [@B47]; Esber and Holland, [@B18]). Few studies, however, have directly compared the behavioral consequences of pathway-specific manipulations, so it remains unclear how each input distinctly contributes to effective reward-seeking. Maladaptive alterations in the strength of NAc inputs are thought to underlie discrete aspects of psychopathologies, including the aversive symptoms of drug withdrawal (Neumann et al., [@B36]; Zhu et al., [@B52]) and stress susceptibility in animal models of depression (Bagot et al., [@B4]). Thus, pathway-specific inactivation of these inputs is critical to gaining insight into how this circuitry contributes to healthy and unhealthy behavior alike. Archaerhodopsin (ArchT)-mediated photoinhibition of axon terminals is commonly used to test the involvement of specific long-range neural projections in behavior. Sustained activation of this outward proton pump in axon terminals effectively decreases evoked transmitter release but alkalizes affected axon terminals, which has the unintended consequence of increasing spontaneous vesicle release (El-Gaby et al., [@B17]; Mahn et al., [@B30]). It is unclear whether ArchT's off-target effects undermine the interpretation of its behavioral effects or still permit assessment of pathway-specific function. If the aberrant spontaneous vesicle release recruits local circuit feedforward inhibition, as previously suggested (Mahn et al., [@B30]), the intended pathway-specific nature of the manipulation may be compromised. The shortcomings of ArchT terminal inhibition have been well characterized in acute slice preparations (El-Gaby et al., [@B17]; Mahn et al., [@B30]), but in vivo applications of this technique are still widely used to study the circuit-level basis of specific behaviors (Herrera et al., [@B22]; Yamamoto and Tonegawa, [@B51]; Mangieri et al., [@B32]), particularly concerning NAc inputs (Stefanik et al., [@B45]; Zhu et al., [@B52]; Reed et al., [@B41]; Trouche et al., [@B49]). Thus, there remains a need to validate the pathway-specific nature of this manipulation in behaving animals. Here, we compare photoinhibition targeted to the axon terminals or cell bodies of NAc inputs. We test the efficacy of these two approaches for uncovering pathway-specific contributions of the PVT-NAc and BLA-NAc pathways to behavior. We first demonstrate in brain slice recordings that ArchT photoinhibition of glutamatergic fibers effectively reduced evoked excitatory synaptic currents. We also report that it increased asynchronous transmitter release and consequently interneuron spiking, which broadly suppressed glutamate release via presynaptic GABA~B~ receptors. In vivo, excitatory axon terminal photoinhibition increased feeding and effortful reward-seeking irrespective of the pathway targeted. These effects are comparable to those obtained with broad inhibition (O'Connor et al., [@B37]) or lesions (Bowman and Brown, [@B9]) of NAc projection neurons. In contrast, cell body inhibition of NAc afferents from the PVT and BLA revealed pathway-specific contributions to distinct aspects of reward-seeking when food was available and during extinction, respectively. These data underscore the off-target behavioral consequences of ArchT-mediated terminal inhibition while highlighting cell body inhibition as a valuable alternative for pathway-specific optogenetic silencing. Materials and Methods {#s2} ===================== Experimental Model and Subject Details {#s2-1} -------------------------------------- Adult wild-type and transgenic C57BL/6J mice were used, including tdTomato Cre-reporter mice (JAX\#007914) and parvalbumin-Cre mice (JAX\#008069, Jackson Laboratory, Sacramento, CA, USA). All animals were bred in-house and kept on a reverse light cycle with a 12 h photoperiod. Animals underwent surgery at approximately 3 months of age (25--30 g). Six weeks later they were placed on a restricted feeding schedule and maintained at 85--90% of their pre-surgery body weight. The number of male and female mice were balanced within groups. All experiments were conducted following the Canadian Council of Animal Care and the McGill Animal Care Committee. Method Details {#s2-2} -------------- ### Viral Constructs and Surgery {#s2-2-1} Before surgery, animals were anesthetized using a ketamine (Ventoquinol, 100 mg/kg) and xylazine (Bayer, 10 mg/kg) cocktail. The skull of the animal was then secured to a stereotaxic frame (Kopf Instruments) and prepared for intracranial virus injections according to the standard stereotaxic procedure. Seven-hundred nanoliter of virus (5.0 × 10^12^ GC/ml) was injected bilaterally over 10 min using a Nanoject II Injector with an oil-filled glass micropipette pulled to a tip diameter of 10 μm (Drummond Scientific, 3-000-203-G/X). For axonal photoinhibition experiments, rAAV5-CaMKIIα-eArchT3.0-eYFP (UNC Vector Core) was delivered into the BLA (AP −1.8 mm, ML ±2.8 mm, DV −5.15 mm) and PVT (AP −1.1 mm, ML ±0.35 mm, DV −3.3 mm) of different cohorts of wildtype mice. Optical fibers with a 200 μm core were implanted in the NAc 10 min later (10° angle, AP 1.5 mm, ML ±1.35 mm, DV −4.6 mm). Animals used for brain slice electrophysiology experiments included PV reporter mice and were prepared in the same manner, but optical fibers were not implanted. For afferent-specific cell body photoinhibition experiments, retroAAV2-CAG-ArchT-GFP (Neurophotonics Centre at Université Laval) was delivered to the NAc (AP 1.5 mm, ML ±0.62 mm, DV −4.7 to −4.2) and an optical fiber was placed above the BLA (AP −2.06 mm, ML ±3 mm, DV −4.02 mm) or PVT (10° angle, AP −1.20 or −0.95 mm, ML ±0.56 mm, DV −2.82 mm) of different cohorts of wildtype mice. ### Behavioral Testing {#s2-2-2} Mice were trained in sound-attenuating chambers (Med Associates), in which levers were extended on either side of a centrally located food receptacle. A house light and speaker were located on the opposite side of the chamber. All behavioral data were collected using the Med Associates software. Food-restricted mice were tethered to optical fiber and placed in these operant chambers daily for 40-min sessions. One lever was randomly designated the active lever. Initially, each press on this lever was reinforced with the delivery of 30 μl of a 15% sucrose solution (m/v) to the food receptacle and a tone presentation (4.8 kHz, 80 dB, 5 s duration). After mice earned 40 rewards in a single session, we switched them to a variable ratio (VR3) reinforcement schedule. The number of active lever presses required for reward delivery and tone presentation then varied randomly between 1 and 5. Inactive lever presses were always inconsequential. Both levers remained extended throughout each session. Photoinhibition experiments were carried out after animals consistently attained 20 rewards per daily session. Photoinhibition involved bilateral intracranial light delivery (532 nm, 10 mW) for two 8-min periods within the 40-min session. The timing of the photoinhibition periods was counterbalanced across 2 days of testing. Animals subsequently experienced two sessions under extinction conditions, in which presses on the active lever were no longer reinforced. Both extinction sessions were preceded by three daily sessions on the VR3 reinforcement schedule. ### Brain Slice Electrophysiology {#s2-2-3} Mice were anesthetized and perfused with a modified artificial cerebrospinal fluid that contained, in mM, 92 NMDG, 20 HEPES, 25 glucose, 30 NaHCO~3~, 1.25 NaH~2~PO~4~, 2.5 KCL, 5 sodium ascorbate, 3 sodium pyruvate, 2 thiourea, 10 MgSO~4~, 0.5 CaCl~2~ (pH 7.35). Two-hundred micrometer thick coronal slices containing the NAc were prepared using a VT-1200 vibratome (Leica) and held at 34°C for 10 min in this same solution. Slices were then transferred to a "holding ACSF" at room temperature, which was identical except that NaCl (92 mM) was included instead of NMDG and the MgSO~4~ and CaCl~2~	{ "pile_set_name": "PubMed Central" }
Correction to: BMC Biol https://doi.org/10.1186/s12915-018-0606-4 Upon publication of the original article, \[[@CR1]\], the authors noticed that the first authors' affiliation contained an error. In the first affiliation it reads "Queensland Brian Institute", whereas it should in fact be "Queensland Brain Institute".	{ "pile_set_name": "PubMed Central" }
Several recent reports point to enterovirus (EV) infections as key environmental triggers of type 1 diabetes (T1D)[@b1][@b2][@b3][@b4][@b5][@b6][@b7]. Conclusions are based on multiple proofs that include the histopathologic detection of EV antigens/genome in the islets of Langerhans of diabetics at different stages of the disease[@b8][@b9][@b10][@b11][@b12]. Recent findings suggest that EVs are causing chronic low-level infection in the islet cells of newly diagnosed T1D patients[@b12]. EVs are small, non-enveloped, single-strand positive-sense RNA viruses belonging to the family Picornaviridae. The EV capsid is composed of four structural proteins named VP1, VP2, VP3, and VP4. The main structural differences among VP1, VP2, and VP3 lie in the loops that connect the beta-strands with the N- and C-terminal sequences extending from the beta-barrel domain[@b13]. These amino acid (AA) sequences give each EV its distinct morphology and antigenicity. The VP4 component lies on the inner surface of the shell and is essential for virion stability. Evolution has resulted in a large number of antigenically distinguishable members that have been categorized as EV "serotypes"[@b13]. Not considering human rhinoviruses, the EV genus contains human agents of the A, B, C, D, and unnamed species that, together, comprise 109 different types. Each of the serotypes correlates with a specific immune response of the host and protection from disease. The serotype-specific protective immune response is directed to the capsid proteins VP1, VP2, and VP3, as the VP4 has no role in the interaction with neutralizing antibodies (i.e., those directed to variable regions of surface capsid proteins). Non-neutralizing antibodies recognizing VP regions that are conserved among different EV types are also produced. The significance of the non-neutralizing antibody response is currently under investigation[@b14]. Immunization with different EV types allowed to produce a variety of monoclonal antibodies (MAbs) that are either type-specific (i.e., responsible of virus neutralization) or directed to conserved regions of capsid proteins[@b15][@b16][@b17][@b18]. Reactivity of the latter antibodies may be limited to sets of different EV types or may be directed to a wider range of EV types. Though molecular methods are held to be more informative than classical serologic methods for virus identification[@b19], "pan-EV antibodies" capable of reacting with all or with the majority of EV types remain desirable reagents for detecting these agents both in the diagnosis of infectious diseases[@b20][@b21][@b22][@b23] and in immunohistochemistry[@b24]. The greater part of immunohistochemical studies in which a conserved region of enteroviral VP1 has been detected in the islets of Langerhans of T1D cases employed the pan-EV MAb 5D-8.1[@b10][@b25]. This MAb has been produced in mice immunized with inactivated coxsackievirus B5 (CV-B5) and has been shown, by immunostaining, to react with multiple EV types[@b18][@b20][@b23]. The 5D-8.1 epitope in VP1 has been partially characterized using competition assays with synthetic peptides and demonstrated to be EV-specific[@b26]. However, subsequent studies suggested that MAb 5D-8.1 may also recognize human proteins, including an isoform of creatine kinase and a mitochondrial ATP synthase[@b27]. Comprehensibly, these results casted doubts on the conclusions of previous immunohistochemical studies of pancreatic tissue in T1D cases[@b25][@b28]. Due to the relevance of MAb 5D-8.1 in diabetes research, we re-investigated this antibody in parallel with the pan-EV MAb 9D5 that is used for diagnosing EV infections in virology laboratories[@b20][@b21]. MAb 9D5 has been obtained from mice immunized with inactivated CV-B3 and shown to react with multiple EV types[@b20]. Reactivity of the two MAbs was defined with the help of innovative microarray technology, substitution scan of peptide epitopes, immunostaining of acutely and persistently infected cell lines, neutralization assays. The MAbs' epitopes were then examined versus the human proteome and versus proteins of diverse viral agents in order to delineate their specificity and define possible cross-reactivities. Results ======= Epitopes of Mab 5D-8.1 and MAb 9D5 ---------------------------------- Secondary goat anti-mouse IgG Ab did not show background interactions with antigen-derived peptides. As shown in [Fig. 1a,c](#f1){ref-type="fig"}, MAb 5D-8.1 gave defined spots for the VP1 sequence of CV-B1, CV-B4, E-30, and reduced reactivity with CV-A1. The peptide scan indicated IPALTAVETGHT as the consensus sequence containing the epitope of MAb 5D-8.1. Comparable signals were produced by MAb 9D5 ([Fig. 1b,d](#f1){ref-type="fig"}) and attributed to the consensus motif SIGNAYSMFYDG. Thus, relative to the VP1 sequence of CV-B4 (GI: 61031)[@b29], the sequence containing the epitope of 5D-8.1 was close to the N-terminus at AA residues 28--39, whereas that of 5D9 was located towards the C-terminus at residues 187--198. Substitution scan of peptide IPALTAVETGHT against MAb 5D-8.1 allowed to delimit the antibody binding site to a conserved core motif 4-IPALTAAET-12. AA positions 4I, 7L and 8T showed the highest degree of sequence conservation with a nearly complete loss in binding of MAb 5D-8.1 upon exchange by other amino acids. AA positions 5P and 11E were well-conserved, exchange by Q and by H, respectively, caused a 60--80% loss in antibody binding. Compared to this, AA positions 6A and 10A exhibited a slightly higher susceptibility for substitution by selected amino acids with a maximal decrease of 50% of spot intensities. 9A and 12T showed the highest tolerance for exchange by other amino acids. Replacement by F (9A) and A (12T) was accepted without loss in antibody binding. Three dimensional analysis of the VP1 protein and the two MAb epitopes ---------------------------------------------------------------------- For each viral capsid organization level, the Solvent-Accessible Surface Area (SASA) was calculated in order to estimate the degree-of-burial of antibody epitopes within the protein. The resolved structures of six reference enterovirus strains were obtained from the RCSB database and a 1.4 Å sphere probe was used to represent a water molecule. The exposed surface area was first calculated, and then normalized with the maximum allowed solvent-accessible area[@b30]. Normalized SASA values take the form of Relative Solvent Accessibility (RSA), a quantity which varies between 0 (for completely buried residues) and 100 (for maximally exposed residues). Results are summarized in [Fig. 2](#f2){ref-type="fig"}. The alignment of the VP1 regions recognized by the two MAbs is shown in [Fig. 3a](#f3){ref-type="fig"}. The target residues are mainly accessible in the monomeric form for the two epitopes (N-terminal 5D-8.1, yellow; C- terminal 9D5, green). The exposed residues are highly conserved among different EV types, evidencing their importance in the capsid assembly process. Localization of the VP1 protein within the capsomere ([Fig. 3b](#f3){ref-type="fig"}) shows that the 5D-8.1 epitope is located in a domain where exposed residues are stabilized by a beta sheet structure. [Figure 3c](#f3){ref-type="fig"} shows the two epitopes in the VP1 protein assembled into the capsid. Detection of MAb epitope sequences among human and viral proteins ----------------------------------------------------------------- The predicted reactivity of antibodies 5D-8.1 and 9D5 with human proteins and viral agents was explored using BLASTp queries vs. the human proteome and viruses. Results are summarized in [Table 1](#t1){ref-type="table"}. It is deduced that the two MAbs may recognize linear targets producing significant alignments with sufficiently low E values. Interestingly, MAb 5D-8.1 produced significant alignments for creatine kinase U-type and a mitochondrial ATP synthase, among other proteins. These targets may represent the antigens indicated as cross-reactive by Korsgren and collaborators in 2013[@b27]. Similarly, MAb 9D5 may be expected to bind to a variety of human proteins, including the leucine-rich repeat-containing protein 66. However, much better alignments (i.e., much lower E values) have been obtained for enteroviruses, rhinoviruses, and agents of the Rabovirus and Sapelovirus genera that represent the most recent members of the Picornaviridae family[@b31][@b32]. The expected reactivity of MAbs 5D-8.1 and 9D5 with different EV types of the A, B, C, D species is shown in [Table 2](#t2){ref-type="table"}. Whereas reactivity of the two MAbs is largely equivalent, 5D-8.1 has a wider coverage of the A species as compared to MAb	{ "pile_set_name": "PubMed Central" }
Introduction {#Sec1} ============ The detection and classification of voltage deflections in extracellular recordings caused by action potentials of neurons - called spikes - is known as spike sorting. It is necessary if single neuronal activities must be resolved from multi-neuronal firing activity. The assignment of spikes to neurons is only possible because the spike shapes of neurons differ due to their morphology, their spatial position with respect to the recording electrode(s), the intrinsic membrane properties of the neuron and the surrounding medium (Camuñas-Mesa and Quiroga [@CR6]; Gold et al. [@CR18]). Furthermore, at least to a first approximation, spikes from the same neuron have similar waveforms. It is, therefore, possible to group extracellular potentials based on their waveform assuming that spikes within one group were actually emitted by the same neuron. In principle, extracellular recordings can be seen as having two different linearly added components, background activity (noise and small action potentials from far away neurons) and spikes of close-by cells (Buzsáki [@CR4]; Camuñas-Mesa and Quiroga [@CR6]). Therefore, for any given piece of data, spike sorting typically solves three problems (Einevoll et al. [@CR9]; Lewicki [@CR27]): First, spikes are detected in the noisy recording. Second, spikes are extracted from the data, aligned and their dimensionality is reduced using feature extraction. Third, spikes, which are now represented by a small number of features, are grouped into clusters of similar spike shapes that putatively originate from the same neuron. However, there is another option: Spikes can be assigned to neurons by using knowledge from a preceeding clustering step, which we refer to as spike classification. At first the classification problem seems to be redundant because spikes are assigned to putative neurons already during the clustering step. But there are several reasons why it is important to treat the classification problem separately from the clustering step. Among these, classification is usually much faster than clustering, an important advantage for online applications, where it might be desirable to use a fast classifier that was derived from an initial offline clustering for real-time spike sorting. Additionally, many clustering procedures scale poorly with the number of spikes and their application becomes infeasible for very long recordings. Then, only a subset of spikes can be clustered and the rest simply classified. One way to build such a classifier is to calculate the average of all elements for each cluster. This cluster center is called the template. Each unclassified spike is then compared to each template and is subsequently assigned to the template that was most similar to it according to some appropriate similarity measure. This procedure is often referred to as template matching. Here, we focus exclusively on the detection and the classification problem: If the number of neurons and their templates are known, e.g., as the result of an offline spike sorting procedure, what is the best way to perform template matching? Different approaches to solve this problem were proposed (see e.g., (Abeles and Goldstein [@CR1]; Friedman [@CR15]; Gerstein and Clark [@CR16]; Keehn [@CR24]; Salganicoff et al. [@CR41]) but also recent approaches (Vargas-Irwin and Donoghue [@CR47]; Zhang et al. [@CR50])) including filter based methods (Roberts and Hartline [@CR39]) (see Fig. [1](#Fig1){ref-type="fig"} for an illustration). Although template matching is of great importance for the analysis of extracellular recordings, it was not thoroughly investigated yet what the best strategy is. Even current commercial products rely on very simple strategies like Euclidean distance (Cambridge Electronic Design Limited [@CR5]; Plexon Inc [@CR34]) with manual threshold selection.Fig. 1Illustration of different template matching techniques for a toy example (artificial data; y-axis arbitrary units). A short piece of a 4 electrode recording was simulated by copying two templates and a simulated ripple into Gaussian white noise. The first column of panels shows the data, the second column the templates (and derived filters), the third column the respective single electrode template matching outputs, and the last column the final multichannel template matching output. For all three methods spikes would have to be detected in the template matching outputs by thresholding (in a with a threshold on minima, for b and c on maxima of the output). The second to last column shows the contribution of individual electrodes to the final template matching output. a Euclidean distance of the data to each template. Known templates are subtracted at each possible position from the data (for each electrode individually) and the norm of the residual (over all electrodes) is computed. If this residual is close to zero, the template is assumed to be present in the data at the respective temporal position. b Convolution of the data with the multi-electrode templates. For each multi-electrode template the data from every electrode is convolved with the respective single-electrode template individually and the results are summed. c Convolution of the data with matched filters. Matched filters are computed from the template and the noise covariance matrix (in this example including the statistics of the "ripple")Fig. 2Flowchart of the BOTM method (left) and illustration of the most important steps (right) on an example overlapping spike from Benchmark 1 (Q) (top, right). BOTM outputs are computed for each template (box labeled "BOTM") and thresholded to detect and classify spikes. To resolve overlapping spikes two alternative approaches are possible. Box labeled "Option 1": from the individual template BOTM outputs more BOTM outputs are constructed that reflect the presence of overlapping spikes (light grey traces on the right, here shown only for overlaps of maximal 2 spikes and 3 samples temporal difference); the one with the highest peak is marked in dark grey). Box labeled "Option 2 (SIC)": Once a spike is detected by threshold crossing on the BOTM outputs, it is subtracted from each BOTM output and the threshold is reapplied iterativelyFig. 3Example of different template matching outputs on a public benchmark data set with a single electrode simulation. a Raw simulated data of two different data files from the benchmark. The true spike times (shown as ticks below the data) are known since the data was simulated. At the position of the true spike times the respective templates are superimposed over the data. b Output of Euclidean distance template matching. Colored traces correspond to the template matching output of the respective template in a. Even on the high signal-to-noise-ratio data file (left) not all spikes can be reliably detected by thresholding the output: the first spike of the orange neuron is obscured by the partial overlap. In the low SNR case (right) the output cannot be thresholded in a useful way to detect spikes. c Convolution-based template matching gives in both cases clear peaks at the positions of the spikes. But not always is the template matching output with the highest peak also associated with the correct template. d The matched filter is in this case very close to the convolution-based template matching. But for the difficult data set (right) all spikes would be classified as the blue neuron by a simple threshold. e Thresholding the BOTM output recovers spike times and their correct identities in both examples (with the exception of the overlapping spike in the difficult data set). This overlap would have to be resolved by a post-processing step. Since the color of the template matching outputs with the highest peak is not always clearly visible, a line with the same color is placed at the respective peaks (maxima in c-e, minima in b) and can be compared to the true spike identities in aFig. 4Illustration of the BOTM responses on a 160 ms piece of benchmark 2 (H). a Intracellular recording of a single neuron. b Extracellular recording using a tetrode located nearby the neuron recorded in a. At the time points at which a spike was detected in the intracellular recording the spike-triggered average (template) of the target neuron superimposes the recordings (cyan traces) for each electrode. c BOTM output and sorted spikes. In this recording, 10 putative neurons were found by the initial spike sorting, one of which was assigned to the target neuron (magenta). Each putative neuron has an associated BOTM output. For sake of clarity, neurons for which no ground truth was available are depicted in grey. Small ticks over the traces show the sorting output of the BOTM + SIC procedure. The three spikes of the target neuron were correctly detected and classified. The spike in the middle was classified as part of an overlapping spike. The dotted line represents the detection threshold In the toy example in Fig. [1](#Fig1){ref-type="fig"} three template matching procedures are illustrated and it can be seen that they respond differently to noise, artifacts (in this case a narrow band oscillation or "ripple") and overlapping spikes. Convolution (Fig. [1](#Fig1){ref-type="fig"}b) has the advantage that noise on electrodes which are too far away to measure the activity of a neuron, and, thus, do not carry information about the presence of the neuron's template, are suppressed (topmost electrode). Additionally, overlapping spikes produce detectable peaks in the output. However, an artifact or "ripple" may also lead to strong responses. If the noise and ripple characteristics are known, matched filters can be used instead, which have the potential to suppress unwanted signal components. The implementation in this case can be done by using the ripple on the seemingly useless electrode 2 - on which the templates	{ "pile_set_name": "PubMed Central" }
Introduction {#S1} ============ A feature of almost all cancers is their ability to escape from the immune system. This process is called immuno-editing and is composed of three different steps: elimination, equilibrium, and escape. During the elimination phase, the immune system recognizes the antigens expressed by tumor cells and eliminates them. If any cells are able to escape from the process of eradication they pass into the second phase in which they modify their antigens to render them unrecognizable by the immune system. In this stage, tumor cells start growing until the mass reaches a considerable volume. This is the third phase of escape, in which the immune system loses control of the tumor which can then spread and become detectable and thus clinically relevant ([@B1]). In this landscape, conventional cancer therapies show some limitations. The inability of chemotherapy and radiotherapy to selectively target cancer cells leads to a very high toxicity. Also, the development of chemo-resistance leaves then surgery as the last chance, if available. Another important aspect not to be underestimated, is the lack of conventional therapies able to create long-lasting immunity preventing metastasis and the relapse of cancer. Cancer immuno-therapy is a promising new strategy to fight cancer and it consists of the activation and arming of the immune system against tumors. There are many different approaches among which, oncolytic virus therapy (OVT) is one of the most encouraging. As stated in the name, OVT takes advantage of the oncolytic nature of some viruses \[oncolytic viruses (OVs)\] in order to kill tumor cells. The advantage of these viruses is their ability to infect and replicate in tumor cells without harming normal tissues. Tumor cells are indeed a good target for OVs. They show a reduction in many of the specific mechanisms used by host cells to respond to viral infection (such as the type I IFN pathway) allowing viruses to replicate successfully in these cells ([@B2]). Moreover, advances in genetic engineering have led to the production of viruses lacking the thymidine kinase gene forcing the virus to replicate only in those cells that have an up-regulation of the RAS pathway like cancer cells ([@B3]). Oncolytic therapy is not just a dream. Several viruses have already reached the clinical stages. The best example is given by Talimogene Laherparepvec, known as T-Vec. This is a modified herpex simplex virus (HSV) that has two viral gene deletions and is armed with the human GM-CSF gene. T-Vec has been shown---in a phase II study---to increase the number of tumor-specific CD8^+^ T cells and to reduce the number of regulatory and suppressor T cells ([@B4]). Moreover, T-Vec has been tested in a phase III trial in patients with melanoma ([@B5], [@B6]) resulting, in 2015, in the FDA approval for the treatment of melanoma patients with injectable but non-resectable lesions in the skin and lymph nodes ([@B6]). Pushed from the good results obtained with T-Vec, over the last few years a variety of OVs have been tested in clinical trials. Safety profiles have reached an excellent standard through modification of OVs to increase specificity and reduce side effects. Despite these promising results, anti-tumor efficacy is still limited (especially when the viruses are used alone) and as a result, new strategies are needed for further improvement of OVT. In this review, we would like to highlight the promising therapeutic effect of OVT mostly focusing on the ability of OVT to activate the immune system, and how to further improve the anti-tumor efficacy of current OVT by modulating the host immune responses to the viruses and tumor cells. Anti-Tumor Effects by OVT {#S2} ========================= The inhibition of the IFN pathway, the major anti-viral response of the cells, is frequently disfunctioned in cancer cells. As a result, OVs can easily infect the transformed cells and fulfill their function. However, the purpose of oncolytic viral therapy is not only to kill cancer cells but also to activate the immune system, silenced by the tumor microenvironment. In order to achieve this, OVT can act in different ways. OVs are able to create long-lasting memory. As discussed above, transformed cells have the ability to escape from the immune system by mutating their antigens and becoming invisible to leukocytes in a process called immuno-editing. When OVs infect tumor cells, an inflammatory reaction is triggered. This is due to the fact that viruses are able to induce immunogenic cell death (ICD). This process is a particular form of apoptosis in which the death of cancer cells is able to induce an effective anti-tumor response via the recruitment and activation of dendritic cells (DCs) and the consequent stimulation of specific T lymphocytes. In ICD, the process of apoptosis is not "sterile" but it triggers the endoplasmic reticulum with the consequent release of some dangerous metabolites called damage-associated molecular patterns (DAMPs). ICD is characterized by the release of three particular molecules that can be classed as DAMPs: calreticulin, ATP, and HMGB1 ([@B7]). APCs in the tumor microenvironment recognize these key metabolites and they are able to generate an immune response. Moreover, when OVs infect and destroy cancer cells, tumor associated or/and specific antigens are released into the microenvironment allowing the immune system to recognize them and to generate a response, breaking down the immuno-editing process. As reported by Breitbach et al., this local stimulation of the immune system is able to create a systemic and long-lasting anti-cancer response from immune cells, which also occurs in advanced stage patients ([@B8]). In many tumors, the tumor microenvironment is "a cold place" in which the process of immuno-editing has created an immuno-suppressive environment. As reported by Bell's group, tumor cell infection with OV creates an inflammatory site with the consequent release of cytokines that activate the immune system, making the "cold" tumor "warm" ([@B8]). In this way, the primary immune response which can be seen as a negative response triggered against OVs (the activation of the immune system against the virus itself) can create anti-tumor immunological memory with very long-term benefits to protect the host against relapse (Figure [1](#F1){ref-type="fig"}). ![Anti-tumor immunity by oncolytic virus (OV) therapy: OVs can be modified in order to let them to replicate only in transformed cells. This process stimulates the immune system which is recruited into the tumor, skewing the neoplastic mass from an immuno-suppressive environment to an inflammatory site. Macrophages and T lymphocytes are key players in this process, producing cytokines that can recruit other immune cells and actively destroy cancer cells. This action generates an immunological memory that avoids cancer recurrence and synergizes with the oncolytic action of the viruses, potentially leading to tumor remission.](fimmu-09-00866-g001){#F1} The anti-tumor responses that OVTs can generate are very promising and give hope for new anti-cancer therapies. However, the true potential of OVs cannot be realized until some natural barriers (intrinsic of many tumors) are overcome. Among these, the hardest problems to solve is the large size of the tumor that can deny OVs access to the tumor core along with other physical barriers such as the elevated interstitial pressure and, in case of intravenous delivery, the poor tumor vasculature. Anti-Viral Effects by OVT {#S3} ========================= The battle between anti-tumor and anti-viral immunity exists because triggering the immune system will clear virus and along with it the lytic effect of viral infection, however, anti-tumor immunity will then also be diminished. We need to discover whether or not to focus on engineering a virus that very efficiently kills tumor cells or a virus that stimulates the most robust anti-tumor response. However, OV infection is the reason why anti-tumor immunity is generated. Upon targeted infection of tumor cells, the immune system detects viral infection and as well as innate immunity, adaptive responses are also triggered. These involve T cells which are primed to lyse cells containing foreign antigens (which usually derive from virus but can also be derived from the cancerous cell itself). This ultimately leads to targeted lysis of not only infected cells but also, the tumor cells themselves. It is therefore important to design OVs that can replicate and spread within tumors quickly to induce the maximal anti-tumor effect before viral clearance ([@B9]). In light of the fact that anti-viral responses may dampen the effect of OVs by clearing virus prematurely, manipulating the anti-viral immune response by blocking antibody responses to the virus means that the virus has extra time to take effect and kill tumor cells through anti-tumoral T-cell responses generated by the presentation of tumor antigens by infected cells ([@B10]). Recently, we demonstrated an innovative way to bypass anti-viral immunity, showing that repeated local administration of adenovirus can improve the efficacy of anti-cancer therapy in a Syrian hamster model ([@B9]). Li et al. demonstrate that if the virus is injected intratumorally, humoral immunity has no effect on the clearance of the virus which suggests innate immunity and cytotoxic T cells plays a central role. Moreover, they demonstrate that pre-existing immunity against the virus does not affect therapeutic efficacy. As a consequence of this, repeated administration of the virus to the tumor site can trigger a robust immune response against the virus. In this way, a great number of tumor cells are destroyed due to viral infection and the release of several antigens into the tumor microenvironment triggers a huge immune response against the tumor ([@B9]). Anti-viral immunity occurs as the immune system responds to the presence of virus in tumor cells within the body. This attracts various types of immune cell to the site of infection including innate cells (e.g.,	{ "pile_set_name": "PubMed Central" }
Introduction ============ Viruses are obligatory parasites that critically rely on their ability to transmit their genome from infected to uninfected host organisms. Being an inert particle, viruses have successfully evolved to exploit the behavior and physiology of their host. Viral infection induces the activation of various endogenous responses that enable it to permeate through cell membranes and other barriers to reach the cytoplasm or nucleus. Herpesviruses have a large double stranded DNA genome enclosed in the viral protein shell (capsid) surrounded by a tegument layer which is enclosed in a lipid envelope with at least eight distinct viral envelope glycoproteins. A characteristic property of these viruses is that after primary infection they establish lifelong latent infection in the infected host with periodic reactivation and re-infection. KSHV or human herpesvirus-8 (HHV-8) is a member of the γ2-lymphotropic-oncogenic herpesviruses. KSHV is etiologically associated with Kaposi's sarcoma (KS) and with at least two lymphoproliferative malignancies, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). It is the newest member of the human herpesvirus family and is closely related to γ-1 Epstein--Barr virus (EBV), γ-2 herpesvirus saimiri (HVS) and Rhesus monkey rhadinovirus (RRV; Ganem, [@B17],[@B18]). KSHV has a double stranded DNA genome of about ∼160-kb encoding more than 90 ORFs designated 4--75 by their homology to HVS ORFs. The genome contains gene blocks conserved with other herpesviruses as well as divergent regions encoding more than 20 KSHV unique genes (K genes). KSHV encodes several proteins that are homologs of host proteins with immunomodulatory, anti-apoptotic, signal induction, transcriptional regulation, and other functions (Cesarman et al., [@B11]; Neipel et al., [@B48]). The first step of any viral infection is governed by binding and entry into target cells. Therefore, to control KSHV infection, a detailed understanding of how KSHV infects its target cells utilizing the varied set of cellular receptors, envelope glycoproteins, signaling, and modes of entry is essential. Recent advances indicate that KSHV interacts with multiple host cell surface receptors of adherent target cells and these interactions induce a network of rapid intracellular signaling pathways, which facilitate the various steps of successful infection. Here, we review the important steps involved in KSHV entry into target cells utilizing viral envelope--cellular receptor interactions, and signal cascades inducing dynamic cell membrane changes leading to a productive latent infection. KSHV Tropism ============ To better understand the underlying multistep complex entry mechanism(s) initiated by KSHV, one must appreciate the broad variety of cell types infected both in vivo and in vitro. In vivo KSHV has a broad tropism as suggested by the detection of its genome and transcripts in a variety of in vivo cell types such as CD19+ peripheral blood B cells, endothelial cells, monocytes, keratinocytes, and epithelial cells (Ganem, [@B18]). Latent KSHV DNA is present in vascular endothelial and spindle cells of KS lesions, associated with expression of latency-associated ORF73 (LANA-1), ORF 72 (v-cyclin D), K13 (v-FLIP), and K12 (Kaposin) genes and microRNAs (Boshoff et al., [@B9]; Dupin et al., [@B14]; Ganem, [@B18]). Lytic infection is also detected in \<1% of infiltrating inflammatory monocytic cells of KS lesions (Dourmishev et al., [@B13]; Ganem, [@B18]). Available evidences suggest that B cells and monocytes are the major reservoir of in vivo latent infection. Cell lines with B cell characteristics, such as BC-1, BC-3, BCBL-1, HBL-6, and JSC have been established from PEL tumors (Dourmishev et al., [@B13]; Ganem, [@B18]). In PEL cells, in addition to the above set of latent genes, K10.5 (LANA-2) gene is also expressed (Parravicini et al., [@B50]; Ganem, [@B18]). About 1--3% of PEL cells spontaneously enter lytic cycle and virus induced from these cells by chemicals serve as the source of virus. Multiple genome copies of both KSHV and EBV exist in latent form in BC-1, HBL-6, and JSC cells while BCBL-1 and BC-3 cells carry only the KSHV genome (Ganem, [@B18]). An endothelial cell line carrying KSHV has not been established from KS lesions since KS cells grow poorly in cell culture and viral DNA is lost within a few passages (Ganem, [@B18]). Kaposi's sarcoma associated herpesvirus has been shown in vitro to infect several types of human cells such as B, endothelial, epithelial, fibroblast cells, CD34+ stem cell precursors of dendritic cells (DCs), and monocytes (Ganem, [@B18]). KSHV also infects owl monkey kidney cells, baby hamster kidney (BHK-21) cells, Chinese hamster ovary (CHO) cells, and mouse fibroblasts cells (Parravicini et al., [@B50]; Akula et al., [@B2],[@B3], [@B4]; Birkmann et al., [@B8]; Bechtel et al., [@B6]; Inoue et al., [@B27]; Garrigues et al., [@B19]; Jarousse et al., [@B29]). Infection of primary B cells by KSHV does not result in immortalization and a lytic KSHV replication is seen in activated B cells. Another characteristic feature of in vitro infection of human microvascular dermal endothelial cells (HMVEC-d), human umbilical vein endothelial cells (HUVEC), human foreskin fibroblasts (HFF), human endothelial cells immortalized by telomerase (TIME), and human endothelial cells (HEK-293), monkey kidney cells (VERO, CV-1), and mouse fibroblasts (Bechtel et al., [@B6]) by KSHV is the expression of latency-associated genes and the absence of productive lytic replication and thus providing a reasonable model for studying in vitro latency. However, latent infection of KSHV in vitro is not persistent and leads to the loss of viral genome over time (Grundhoff and Ganem, [@B24]). Analysis of in vitro KSHV interaction with adherent target cells and quantitation of infection has been hampered by the absence of a lytic replication cycle and hence a plaque assay. Since in vitro KSHV infection results in the expression of latency-associated genes, various methods have been devised to assess the different phase(s) of KSHV infection (Parravicini et al., [@B50]; Table [1](#T1){ref-type="table"}). ###### *Methods employed to study the various stages of in vitro* KSHV infection**. Stage of infection Detection methods --------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1\. Binding \[H3\] Thymidine labeled virus and FITC labeled (FACS) binding assay; viral DNA (ORF73 gene) DNA quantitation by real-time DNA PCR; electron and confocal microscopy. 2\. Signal induction Quantitation of signal molecules induction by Western blots and ELISA; use of chemical inhibitors or dominant-negative signal molecules. 3\. Viral DNA internalization (entry) Real-time DNA PCR for KSHV ORF73 gene after removal of unbound/partially bound virus by trypsin--EDTA; electron and confocal microscopy. 4\. Cytoplasmic trafficking of KSHV Confocal microscopy by colocalizing virus with microtubules and endosomal vesicles; physiological ligand uptake assays. 5\. Nuclear delivery of KSHV DNA Real-time DNA PCR for ORF73 gene in the isolated nuclei of infected cells. 6\. Viral gene expression, host gene manipulation Real-time RNA PCR for KSHV and host gene expression; confocal microscopy and FACS for KSHV ORF73 gene and GFP expression. KSHV Binding and Entry into Target Cells ======================================== Kaposi's sarcoma associated herpesvirus uses multiple envelope glycoproteins to complete the binding and entry processes. KSHV binding to the target cells and identity of the receptors involved in binding and entry were elucidated by using labeled virus binding to the target cells at 4°C as well as other methods (Table [1](#T1){ref-type="table"}). These studies have demonstrated that KSHV binds and enters a variety of target cells which include human (293, HFF, HeLa, HMVEC-d, HUVEC, TIME, BCBL-1, BJAB, Raji), monkey (Vero, CV-1), hamster (BHK-21, CHO), and mouse (Du17) cells. This is demonstrated by the detection of viral DNA, limited viral gene expression, and GFP expression (Table [1](#T1){ref-type="table"}). Real-time DNA PCR of internalized KSHV DNA demonstrates a rapid internalization of viral DNA in the infected endothelial and HFF cells (Krishnan et al., [@B34]). KSHV Envelope Glycoproteins-Mediators of Binding and Entry ========================================================== Kaposi's sarcoma associated herpesvirus envelope glyc	{ "pile_set_name": "PubMed Central" }
###### Summary box What is already known about this subject? ========================================= 1. Current treatment of acute gastroenteritis as recommended by the WHO includes the use of oral rehydration solution and zinc. 2. Studies carried out on racecadotril in children have proved its efficacy in reducing duration and severity of diarrhoea. 3. The European Society of Paediatric Gastroenterology, Hepatology and Nutrition has stated that racecadotril may be used in the management of acute gastroenteritis in children. What are the new findings? ========================== 1. Racecadotril did not significantly impact the duration or severity of diarrhoeal disease in children with severe gastroenteritis. 2. One of the first studies using racecadotril to treat children in East Africa. How might it impact on clinical practice in the foreseeable future? =================================================================== 1. Defining which populations of children with acute gastroenteritis would have a maximum benefit from racecadotril. This could allow recommendations on its use to be refined. Introduction {#s1} ============ Diarrhoea is one of the most common causes of morbidity and mortality in children under 5 years in the developing world. Kenya is ranked in the top 10 countries bearing the burden of high mortality rates in children suffering from diarrhoeal disease with a prevalence of 16.7%.[@R1] [@R2] In the 1970s--1980s, trends in mortality showed a significant decrease with the advent of oral rehydration solution (ORS). Since then this decline has slowed with current data at 530 000 deaths per year.[@R3] Racecadotril is a diesterified derivative of thiorphan.[@R4] Once absorbed after oral administration, it is converted to its parent compound (thiorphan) which acts to increase the half-life of enterocyte methionine-enkephalin (a potent antisecretory agent).[@R5] Racecadotril has been in use for the past 20 years in Europe and has been consistently useful in the management of diarrhoeal disease with an acceptable adverse effect profile.[@R6] Although zinc has been shown to significantly affect the duration and severity of diarrhoea and forms part of the core treatment recommendations by the WHO, none of the trials studying efficacy of racecadotril have included it in their protocols.[@R7] [@R11] Despite a number of studies performed in different parts of the world, there has been no published data involving racecadotril from the African continent. Of the few studies carried out in the developing world, majority have been inpatient studies with methodological flaws noted in many.[@R7] This study was carried out to bridge gaps in knowledge about the use of racecadotril and its usefulness when used with zinc in African children with severe gastroenteritis. Methodology {#s2} =========== Study design {#s2a} ------------ This was a prospective randomised, double-blind, placebo-controlled, parallel-group study conducted at the Kenyatta National Hospital. This is a tertiary level, national referral hospital and has four dedicated paediatric wards. Participants were assigned to the control arm or treatment arm with a 1:1 ratio. Eligibility criteria {#s2b} -------------------- The inclusion criterion was: children aged 3--60 months requiring admission for severe acute gastroenteritis, as evidenced by a Vesikari score of \>11 ([figures 1](#BMJGAST2016000124F1){ref-type="fig"} and [2](#BMJGAST2016000124F2){ref-type="fig"}) with written parental consent.[@R12] [@R13] ![The Vesikari clinical severity scoring system parameters and scores. Table adapted from Vesikari Clinical Severity Scoring System Manual.[@R13]](bmjgast2016000124f01){#BMJGAST2016000124F1} ![Grading of severity of diarrhoea according to the Vesikari score. Table adapted from Vesikari Clinical Severity Scoring System Manual.[@R13]](bmjgast2016000124f02){#BMJGAST2016000124F2} Exclusions were: children who had severe vomiting (scoring 3 on the Vesikari score for maximum number of vomiting episodes per day); those with a clinical diagnosis of dysentery or a known diagnosis of liver or renal failure; children who had prescriptions of probiotics or any other antidiarrhoeal medication (loperamide, attapulgite, activated charcoal, diphenoxylate and kaolin). Interventions {#s2c} ------------- All children were admitted and started on WHO recommended treatment by the attending physician. They received either intravenous fluids as per WHO plan C (30 mL/kg followed by 70 mL/kg over 1 and 5 hours, respectively, in infants and over 30 min and 2.5 hours for those over 12 months of age) or low osmolality ORS as per WHO plan B (75 mL/kg over 4 hours). Zinc was prescribed at 10--20 mg/day. Participants were recruited within 24 hours of admission after initial hydration had been completed. Eligibility was determined by calculating the Vesikari score based on the information recorded by the primary physician in the patients\' chart. This included the patients\' history up to and the examination at the time of admission. A computer program was used to generate random numbers in blocks with varying sizes. These were used to assign patients randomly to either the test or control arm of the study. Both the drug and placebo were supplied by the manufacturer, Racedot---Macleods (India) and distributed by Sai Pharmaceuticals (Kenya). These were packed in tamper proof brown bags, sealed and labelled by a study pharmacist based off site. The test arm received racecadotril at a dose recommended by the manufacturer: 10 mg per dose for children below 12 months of age and 30 mg for those over 12 months of age. This was administered as granules dissolved in 10 mL of water and taken three times a day for a maximum duration of 3 days. The control arm had a placebo preparation administered in the same way. An initial dose of the drug or placebo was given at the time of enrolment. The parents were taught how to administer the drug at this time and this was confirmed by return demonstration. The participants were followed up using daily interviews asking about: the number of stools, the consistency using a Bristol stool chart,[@R14] the presence of blood in stool, any new symptoms and the introduction of an antidiarrhoeal or other medication. Treatment was given either until the stools were formed or for a total of 3 days, whichever came first. Children who were discharged before complete resolution of symptoms were followed up by phone interviews until recovery. Outcome measures {#s2d} ---------------- The primary outcome measure was the number of stools in the first 48 hours after introduction of the drug. The secondary outcomes included the duration of inpatient stay, the duration of illness and the number of adverse events associated with racecadotril. Sample size calculation {#s2e} ----------------------- Estimation of sample size was carried out using the formula for comparison of two sample means. The study was powered at 90% with a significance level of 95%. Assumptions for the formula were based on the study by Cojocaru et al[@R8] using his outcome of total number of stools at 48 hours. A provision of 10% was made for attrition and a final sample size of 60 participants in each arm was derived. The attached online [supplementary file](http://bmjopengastro.bmj.com/doi/suppl/10.1136/bmjgast-2016-000124) contains the complete sample size calculation and references. Statistical methods {#s2f} ------------------- Baseline data for the two groups were collected and analysed by comparing proportions to determine the presence of any significant differences. Once the data were collected and analysed, it was noted to be positively skewed. Medians with IQRs were calculated for the outcome measures and the two groups were compared using the Mann-Whitney U test according to intention-to-treat analysis. STATA V.11.0 software was used for the above. Ethical consideration {#s2g} --------------------- The study was conducted after the approval of the Ethics and Research Committees of the Aga Khan University Hospital and the Kenyatta National Hospital/University of Nairobi. The development of known side effects, suspected adverse effects or new symptoms were communicated to a Drug Safety Monitoring Board (DSMB). Adverse events were graded based on the Division of Allergy and Infectious Diseases (DAIDS) table for grading the severity of adult and paediatric adverse events summary sheet.[@R15] Results {#s3} ======= A total of 154 children with severe gastroenteritis were screened for the study during the data collection period from 3 February to 26 March 2014. The details of recruitment and allocation are shown in [figure 3](#BMJGAST2016000124F3){ref-type="fig"}. Analysis included all 60 participants in each group. Three participants from the drug group and two from the placebo group that did not complete the protocol were included in the analysis. All results for the outcome measures (except adverse events) are given as medians with IQRs.	{ "pile_set_name": "PubMed Central" }
![](edinbmedj74792-0084){#sp1 .T21} ![](edinbmedj74792-0085){#sp2 .T22} ![](edinbmedj74792-0086){#sp3 .T23} ![](edinbmedj74792-0087){#sp4 .T24} ![](edinbmedj74792-0088){#sp5 .T25} ![](edinbmedj74792-0089){#sp6 .T26} ![](edinbmedj74792-0090){#sp7 .T27} ![](edinbmedj74792-0091){#sp8 .T28} ![](edinbmedj74792-0092){#sp9 .T29} ![](edinbmedj74792-0093){#sp10 .T30} ![](edinbmedj74792-0094){#sp11 .T31} ![](edinbmedj74792-0095){#sp12 .T32} ![](edinbmedj74792-0096){#sp13 .T33} ![](edinbmedj74792-0097){#sp14 .T34} ![](edinbmedj74792-0098){#sp15 .T35} ![](edinbmedj74792-0099){#sp16 .T36} ![](edinbmedj74792-0100){#sp17 .T37} ![](edinbmedj74792-0101){#sp18 .T38} ![](edinbmedj74792-0102){#sp19 .T39} ![](edinbmedj74792-0103){#sp20 .T40} ![](edinbmedj74792-0104){#sp21 .T41} ![](edinbmedj74792-0105){#sp22 .T42} [^1]: Read 3rd November 1926.	{ "pile_set_name": "PubMed Central" }
Introduction {#sec1} ============ Mesoporous semiconductor oxide electrodes are used in different applications including electrochemical sensing, electrochromic devices and photoelectrochemical generation of fuels or electrical energy.^[@ref1]−[@ref5]^ All these applications rely on the external manipulation or tracking of the charge transfer between an optically and/or chemically active layer and an external contact. The macroscopic rate of charge transfer between the mesoporous film and a conductive substrate is the result of a sequence of intermingled microscopic processes. These processes, which are associated with carrier generation, recombination, transport and transfer, take place on different time scales and compete kinetically with each other. Due to a high concentration of trap states electron transport in mesoporous semiconductor oxide films is orders of magnitude slower than in single crystals. In photoelectrocatalytic and photovoltaic applications electron collection at the external contact competes with charge recombination in the bulk of the semiconductor and at the interfaces within the porous film, limiting solar conversion efficiency. The use of electrochemical doping (charge transfer reductive doping), where electron/proton or electron/Li^+^ pairs incorporated within the oxide "passivate" the electron traps,^[@ref6]−[@ref8]^ is a very efficient way to temporarily improve photoelectrochemical activity, and as such, constitutes an elegant way to improve charge separation within these materials. Although many efforts are being pursued in this direction, a comprehensive study of these electronic defects is still missing. To reach such a level of understanding, one needs to carefully identify the nature of the recombination centers and transport-limiting traps in mesoporous semiconductor electrodes, which is a significant challenge for both experimentalists and theoreticians. It is well established that the fundamental processes associated with the transport,^[@ref9]−[@ref12]^ transfer,^[@ref13],[@ref14]^ and recombination^[@ref15]^ of photogenerated charge carriers in mesoporous semiconductor electrodes are determined by both the distribution of band gap states and their population, i.e., the position of the Fermi level within the film. Electrochemical methods such as cyclic voltammetry and electrochemical impedance spectroscopy have proven useful in characterizing electronic states in nanostructured semiconductor oxide electrodes, though the chemical nature of the traps remains controversial.^[@ref5],[@ref16]^ Specifically, charge/discharge measurements provide information on the distribution of electrochemically active states in mesoporous electrodes, where electron accumulation is compensated by the adsorption of ions at the oxide surface. For TiO~2~ electrodes in an aqueous acidic electrolyte the generation of Ti^3+^ centers is compensated by proton uptake ([eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"})Importantly, in the case of small cations (such as H^+^ or Li^+^), charge injection and compensation not only take place at the semiconductor/electrolyte interface, but can become a three-dimensional process via insertion of ions into subsurface regions of the nanocrystals. This process is often referred to as electrochemical or charge transfer reductive doping.^[@ref6],[@ref17]^ The reversibility of this charge accumulation opens up the possibility of driving fast reduction reactions at the semiconductor/electrolyte interface.^[@ref18]^ On the other hand, electrochemical doping was found to modify, at least temporarily, the electrode performance in different applications ranging from dye-sensitized solar cells^[@ref8],[@ref19],[@ref20]^ to photocatalysis^[@ref6]−[@ref8],[@ref21]−[@ref24]^ and supercapacitors.^[@ref25]^ The doping of mesoporous TiO~2~ electrodes with Li (which is isovalent with H) has recently been demonstrated to enhance electron transport and improve efficiency in perovskite solar cells.^[@ref26]^ Whereas different studies discuss an increase of the electrode performance upon electrochemical doping phenomenologically by accelerated charge transport and reduced recombination,^[@ref6]−[@ref8],[@ref19],[@ref22]^ the underlying microscopic details remain to be elucidated. For TiO~2~, theoretical studies have recently addressed at the single particle level the geometry and energetics of electron trap states in the bulk^[@ref27]^ and at the semiconductor/electrolyte interface.^[@ref28]−[@ref31]^ Furthermore, intrinsic trapping properties of grain boundary interfaces have been studied. Deep electron traps located at the grain boundary are found to slow down charge transport unless high current densities ensure a high average occupation of transport-limiting traps.^[@ref9]^ Such trap filling effects have recently been highlighted for deep traps in oriented TiO~2~ nanotube arrays by dynamic photocurrent measurements.^[@ref10]^ In addition, these states may act as recombination sites exerting a further deleterious impact on the photocurrent.^[@ref32]^ The high structural and electronic complexity of mesoporous semiconductor oxide electrodes makes an investigation of the nature, concentration, and location of electronic trap states and the elucidation of their impact on charge recombination and transport very challenging. Designing appropriate model systems to understand the action of these states is difficult. Indeed, their complexity must be high enough to realistically mimic processes in technologically relevant materials, but low enough to result in clear structure--activity relationships that can be supported by both experiments and theoretical modeling. In this work, we combine first-principles theoretical calculations with the electrochemical characterization of nanostructured rutile TiO~2~ films to demonstrate that particle/particle interfaces introduce deep traps. These interfaces represent favorable locations for proton segregation, which can be induced by the electrochemical doping of the porous electrodes. A long lasting (hours to days), but reversible accumulation of electrons and protons (i.e., e^--^/H^+^ or H^0^ doping) at the interface is tracked by cyclic voltammetry via the shift toward more positive potentials of a pair of capacitive peaks, which is associated with trap states at the particle/particle interface. The passivation of recombination centers by e^--^/H^+^ doping leads to a transient photocurrent enhancement due to improved electron/hole separation for those electrodes, which are characterized by a high concentration of particle/particle interfaces (i.e., random particle networks). For electrodes lacking a high density of particle/particle interfaces (i.e., arrays of oriented nanocolumns), only a minor improvement of the photocurrent is observed upon doping. This study highlights the importance of particle/particle interfaces in mesoporous films and provides strategies to actively manipulate the density of electronic states and their population by electrochemical methods. The resulting long-lasting (\>15 h) improvement of photoelectrode performance after electrochemical doping is explained by using theoretical calculations that are in qualitative agreement with experiments. Experimental Section {#sec2} ==================== Thin Film Preparation {#sec2.1.1} --------------------- Slurries of rutile TiO~2~ nanoparticles (Sachtleben, Nano Rutile) were prepared by grinding 1 g of TiO~2~ powder with 3.2 mL of H~2~O, 60 μL of acetylacetone (99+%, Aldrich), and 60 μL of Triton X (Aldrich) and were spread with a glass rod onto fluorine-doped tin oxide (FTO) conducting glass (Pilkington, TEC 15, resistance 15 Ω/□) using Scotch tape as a spacer. Alternatively, Ti foils (Goodfellow, 99.6+%, 250 μm) were used to investigate a possible impact of the substrate type on the electrochemical and photoelectrochemical properties. However, no such effect was observed for the experiments reported here. The nanoparticle (NP) films were annealed and sintered for 1 h at 450 °C in air. After sintering a film thickness of 3.5 ± 2.0 μm was determined by scanning electron microscopy (SEM). The films are formed by a random network of elongated particles with a length of ∼50 nm and a width of ∼20 nm ([Figure S1a,b](#notes-1){ref-type="notes"} and ref ([@ref33])) and are of pure rutile phase ([Figure S2](#notes-1){ref-type="notes"}). As shown in a previous study, nanoparticles are elongated in the \[001\] direction. Furthermore, it was estimated that a high fraction of the exposed surface is formed by (110) facets.^[@ref33],[@ref34]^ Electrodes formed by a rutile TiO~2~ nanocolumn (NC) array were prepared using a hydrothermal synthesis.^[@ref35]^ Concretely, 21.6 mL of a 6 M HCl solution were mixed with 360 μL of Tetra-n-butylorthotitanat (98%, Merck Millipore). The solution was placed in a Teflon-lined steel autoclave (45 mL, Parr Instruments) containing FTO substrates and was heated to 150 °C for 15 h. After synthesis, the electrodes were thoroughly rinsed with water. These films consist of rutile TiO~2~ nanocolumns with a rectangular cross section and with a width of 80--180 nm and a length of ∼1.5 μm ([Figures S1c,d and S2](#notes-1){ref-type="notes"}). As highlighted previously, individual nanocolumns are porous and consist of a bundle of oriented and single crystalline nanowires with a diameter of 10--20 nm.^[@ref36]^ The nanowires are elongated along the \[001\] direction and are expected to expose (110) facets at the surface.^[@ref35]^ The endings of the nanowires can be observed at higher magnifications at the top parts of the nanocolumns (inset in [Figure S1c](#notes-1){ref-type="notes"}). For both types of electrode a copper wire was attached to the conducting substrates with silver epoxy. The contact area and the uncovered parts of the substrate were finally sealed by epoxy resin. Theoretical Calculations {#sec2.2} ------------------------ Spin polarized density	{ "pile_set_name": "PubMed Central" }
Introduction ============ Most coaches and professionals linked to sports teams agree on the importance of both an adequate climate and good social relations, and the high perception of skill among team members in order to optimize group efficacy. Thus, many expert investigators in group dynamics have identified cohesion and efficacy as two of the most important properties within a sports team ([@b6-jhk-34-129]; [@b8-jhk-34-129]; [@b13-jhk-34-129]; [@b20-jhk-34-129]; [@b22-jhk-34-129]; [@b29-jhk-34-129]). Notwithstanding, there are few studies that have attempted to analyze the evolution of these psychological variables over the season, as well as what kind of variables could influence this process ([@b14-jhk-34-129]; [@b12-jhk-34-129]). Considering each of these concepts specifically, the term team cohesion is defined by [@b7-jhk-34-129] as "a dynamic process that is reflected in part by the tendency of a group to stick together and remain united in the pursuit of its instrumental objectives and/or for the satisfaction of member affective needs". As expressed in the definition, this concept is changing and variable over time and is affected by a series of environmental, personal, leadership, and team factors that promote the level of cohesion. If it is taken into account that one of team factors refers to the group's desire for success, this indicates that the greater these desires and higher expectations of success, the higher will be the degree of cohesion shown by the players to achieve their goals. In addition, within the conceptual model of Carron et al., cohesion consists of four dimensions based on two levels of distinction. The first level distinguishes individual attractions to the group and group integration; the second level distinguishes the task and social aspects of group involvement. Thus, four constructs are identified: Group integration-task (GI-T), Group integration-social (GI-S), Individual attractions to the group-task (ATG-T) and Individual attractions to the group-social (ATG-S). In our study we only used one of the levels, because the aim was to examine the evolution of task cohesion aspects, which reflects the degree to which group members work together to achieve common goals, and social cohesion, which reflects the degree to which team members empathize with each other and enjoy the companionship of the group ([@b5-jhk-34-129]; [@b10-jhk-34-129]; [@b9-jhk-34-129]) in different moments of the season. These dimensions change as a function of the diverse above-mentioned factors. Therefore, as seen in previous studies, success expectations can have a close relation with levels of player-perceived cohesion over the season ([@b19-jhk-34-129]). With regard to perceived efficacy, diverse studies have attempted to appraise this construct through players' and coaches' opinions ([@b1-jhk-34-129]; [@b2-jhk-34-129]; [@b11-jhk-34-129]; [@b17-jhk-34-129]; [@b20-jhk-34-129]). Thus, there are various types of efficacy related to the sport context ([@b4-jhk-34-129]), among which are noteworthy coach-perceived efficacy and teammate-perceived efficacy, self-efficacy, and collective efficacy. [@b2-jhk-34-129] first defined self-efficacy as an individual's belief in his or her capacity to successfully organize and perform a specific task. Coach-perceived efficacy is the coach's assessment of each player's capacities and skills with regard to the good development of the play actions ([@b11-jhk-34-129]), whereas teammate-perceived efficacy is the players' belief in the capacities of their teammates to adequately meet the demands of the sport ([@b17-jhk-34-129]). Lastly, collective efficacy is defined as the group's shared beliefs in their capacities to organize and perform the actions required to achieve certain goals ([@b2-jhk-34-129]). As with team cohesion, the perception of efficacy will evolve as a function of a series of antecedents. These sources of information may be past performance, success expectations, the group's physiological state, leadership, the motivational climate, team cohesion, etc. ([@b2-jhk-34-129]). Thus, as previously noted by [@b19-jhk-34-129], among the diverse antecedents of efficacy, success expectations, understood as the belief in the attainment of a series of goals or sports success, can play an important role in the perception of efficacy ([@b26-jhk-34-129]). The relevance to examine these aspects is due to the narrow relationship between cohesion and different types of efficacy ([@b12-jhk-34-129]; [@b16-jhk-34-129]; [@b20-jhk-34-129]; [@b24-jhk-34-129]; [@b25-jhk-34-129]; [@b28-jhk-34-129]) and these variables with respect to performance ([@b8-jhk-34-129]; [@b13-jhk-34-129]; [@b19-jhk-34-129]; [@b22-jhk-34-129]; [@b29-jhk-34-129]). In accordance to this issue, several works have shown that players who perceived greater cohesion level and perceived efficacy achieve higher performance at the end of the league ([@b8-jhk-34-129]; [@b13-jhk-34-129]; [@b19-jhk-34-129]; [@b22-jhk-34-129]; [@b29-jhk-34-129]). Therefore, it might be interesting to know how these variables progress during a season, and how success expectations, which have been shown as cohesion and efficacy antecedent might influence those variables ([@b4-jhk-34-129]; [@b19-jhk-34-129]). Moreover, it is important to note that this line of research has not been pursued much in the sport sphere, although some investigations have indirectly examined cohesion and efficacy in different moments over a season ([@b14-jhk-34-129]; [@b12-jhk-34-129]; [@b21-jhk-34-129]). The most relevant studies about the evolution of cohesion and efficacy were carried out in diverse sports such as handball ([@b14-jhk-34-129]; [@b12-jhk-34-129]), and basketball ([@b14-jhk-34-129]). These works present a similar line of results: [@b14-jhk-34-129] and [@b12-jhk-34-129] found that all the factors of cohesion decreased their levels as the season advanced and approached the end. Likewise, the levels of players' perceived collective efficacy were higher at the start than at the end of the season ([@b14-jhk-34-129]; [@b12-jhk-34-129]; [@b21-jhk-34-129]). Our research at the same time that shows new information about evolution of those variables in football players, aims to examine whether success expectations perceived at the beginning of the season might influence the perception of cohesion and efficacy at the end of the league. Unlike most studies, this checking was developed through the composition of two groups regarding whether expectations was achieved or was not achieved and how dependent variables changes in both groups during the league. Thus, the main goal of the study is to examine the evolution of players' perception of cohesion and efficacy over the season and their relation with success expectations. Hence, as first hypothesis, we propose that the diverse factors of cohesion, perceived efficacy, and success expectations will undergo significant changes over the season. The second hypothesis states that success expectations will emerge as an antecedent in the evolution of cohesion and efficacy. Material and Methods ==================== Participants ------------ The research sample comprised 265 male soccer players, aged between 15 and 19 years (M = 16.96, SD = 0.76). All the players who made up the sample belonged to the 15 federated teams that played in the XI group of the Sub18 National League, and each participant held a federative card with his personal and sports data. The final sample was formed by 146 players who completed the questionnaires at the start and at the end of the season, and the players who did not complete the two measurements, or who had completed them on different teams (due to a possible change during the season) were eliminated. The team coaches (N = 15), aged between 29 and 45 years, with at least 7 years experience, also participated in the study. The study received ethical approval from the University of Extremadura. All participants were treated according to American Psychological Association ethics guidelines regarding consent, confidentiality, and anonymity of responses. Measures -------- Cohesion. To assess cohesion we used the Spanish version of the Group Environment Questionnaire (GEQ: [@b10-jhk-34-129]), carried out by [@b15-jhk-34-129]. This instrument has 18 items grouped into four factors. Despite this issue, due that our aim was to analyze the evolution of social and task dimensions, we used the two global factors grouped items in task cohesion (9 items, i.e., "The team members unite their efforts to achieve the goals during the training sessions and the games")	{ "pile_set_name": "PubMed Central" }
Introduction ============ There is an established literature on gender differences in humor perception and humor styles. Men have been noted to prefer humor that has sexual or aggressive themes whereas women appear to prefer neutral or absurd humor ([@B1]). Whereas earlier studies showed that sexist humor (i.e., humor that upholds gender role stereotypes) is preferred over non-sexist humor ([@B4]), other studies report that both men and women prefer humor that has the opposite gender as the butt ([@B33]; [@B23]). Furthermore, men typically rate themselves higher than women in humor initiation whereas women tend to rate themselves higher in humor appreciation, but when humor is studied in actual conversational contexts a more nuanced picture emerges (see [@B15]; [@B13], [@B14]; [@B26]). Similarly, whereas some studies have found that humor produced by men is judged to be more humorous than that produced by women ([@B3]), other studies have not found this effect ([@B12]), and still other work suggests a bias operating, whereby men are perceived to be the "funnier sex" regardless of how their humorous creations are actually judged ([@B20]; [@B10]). Taken as a whole, the literature on gender and humor eludes easy generalization (see [@B18], for a review). Methodologically, early studies have been criticized for their use of decontextualized or "canned" humor samples instead of spontaneously generated humor that arises naturally in conversation ([@B8]; [@B16]). The literature on gender differences in humor preferences has also been critiqued for its reliance on classifications of humor type (as "hostile" or "sexual" or "sexist") based on experimenter intuitions rather than eliciting participants' own perceptions of humor type, which may not coincide with those of the experimenter (e.g., [@B23]). Moreover, studies that have sought to measure the construct of a sense of humor have led to many promising instruments, such as the three Witz-Dimensionen (3WD) instrument by [@B25], and to new adaptations of established instruments for use with non-English speakers (see [@B22]). At the same time, it is recognized that for a construct as slippery and contextual as humor, it is important to consider multiple, converging measures across different groups and settings. This recognition of the complexity of studying humor, together with a growing shift in regarding gender as performative, has led to a shift in humor scholarship in the direction of studying humor as it is enacted by men and women in a range of social contexts (e.g., [@B9]; [@B6]), and in a range of laboratory contexts. Our own previous work has explored the relationship between cognitive, neurocognitive, and psycholinguistic aspects of humor detection and comprehension (e.g., [@B28]; [@B34]; [@B32], [@B35]; [@B11]; [@B17]). Our work on humor production has sought to develop controlled ways of eliciting humor to study its cognitive and social underpinnings. For example, we developed a concept comparison task in which participants were asked to produce "catchy" ways in which the concepts were related, which invariably elicited humorous responses, e.g., MONEY and CHOCOLATE: one swells the wallet, the other, the hips ([@B12]). Another task involved generating rejoinders to proverbs, e.g., Absence makes the heart grow fonder, but also makes the eyes wander ([@B30]). Other prior work in our laboratory has examined the role of culture in judgments about when humor (vs. silence) is an appropriate response to embarrassing situations encountered in daily life ([@B31]). Finally, we have examined how individuals' perceptions of their own humor styles compare with their perceptions of humor styles of members of their gender category and/or same or different cultural group ([@B24]; [@B27], [@B29]). As an extension of our interest in gender and cultural dimensions of humor, the aim of the present research was to characterize how gender and country of origin (as a proxy for culture) may shape how individuals conceptualize an ideal sense of humor. The motivation for this study was a previous study which examined the role of gender in lay conceptions of an ideal sense of humor ([@B7]) in a large sample of United States-based participants of different ages and backgrounds. Participants in this study were asked to provide a brief narrative describing the humor characteristics of a person they knew who embodied an outstanding sense of humor. [@B7] reported that a majority of the participants identified a male figure as the person who embodied an outstanding sense of humor. Indeed, of the 141 respondents (49 men, 92 women), nearly 84% of men and 67% of women selected a male figure. The researchers also classified the humor characteristics mentioned into five categories: creativity (witty, clever, quick comeback), caring (humor used to put others at ease), real life (grounding the humor in real life experiences), jokes (having a repertoire of jokes), and hostility/sarcasm (satirical, biting humor) and noted that creativity, caring and real life were mentioned most often, and that there were no discernible differences in the weighting of these characteristics as a function of either participant gender or target gender. Over 25 years have passed since the [@B7] study. While gender continues to be a salient element structuring society, women have also become more visible in a number of domains of public life, including in the realm of comedy. It is possible that gender stereotypes may have become less entrenched in the present day. We therefore wondered if the preference for a male figure as the embodiment of an outstanding sense of humor noted previously still holds among young adults in the present age. We also wondered whether individuals from other countries would show a similar preference, given that they might be less likely to be influenced by Western gender stereotypes (including stereotypes regarding men as being the canonical humor initiator), but might have their own cultural stereotypes about humor, gender, and the relation between the two. Although there have been a few prior studies of humor stereotypes in different nationalities, the focus of our study was on how individuals from the United States compared to those from two other countries in articulating characteristics of an ideal sense of humor, as embodied in someone they knew. Our interest was to uncover patterns of commonalities as well as differences across groups and across genders. In searching the literature, we could find only one other empirical study conducted since the study by [@B7] that used their open-ended prompt. This study, by [@B21], was conducted on men and women in Singapore. It, too, found that the embodiment of an outstanding sense of humor was male. Of the 18 men and 46 women in the study, 76% of respondents selected a male target ([@B21]). The researchers further noted that the preference for a male target was more pronounced in men, but no additional analyses were reported in terms of specific humor characteristics mentioned by men and women. Thus, we felt another study was warranted. The Present Research -------------------- Our study had two goals. The first was to investigate if the male preference first reported by [@B7] still holds. To examine this, we pooled data from United States-based college students tested from 2004 to the present. The second goal was to investigate if the pattern of a male preference as the embodiment of an ideal sense of humor is restricted to United States participants or is generalizable to other samples. In particular, we considered samples drawn from Iran and from Turkey, as these particular groups have been understudied in the humor literature; where country-based differences have been studied, they have either tended to be within north American/European samples or have compared Western with east Asian samples (e.g., China). Turkey is considered geographically and culturally as a bridge between Asia and Europe. Thus, we aimed to compare participants raised in a Western (American), a Middle Eastern (Iranian), and a blended (Turkish) culture. We did not have a priori expectations of how participants across the three groups would respond on the task; our study is exploratory with regard to the cultural dimension, as our sample sizes were limited and varied in other respects (e.g., age) and we recognize that much more follow up investigation would be needed to fully understand the nature of any differential patterns uncovered. Materials and Methods {#s1} ===================== Participants ------------ Participants included male and female United States born (American) and international students (born in Iran or in Turkey) recruited from a university town in the southwestern region of the United States, from a university in Istanbul, and from online responses. The American sample consisted of 279 undergraduate students (including 201 women) who ranged in age from 18 to 23 years, with a mean of 21 years. The majority self-identified as white, and the numbers of Latinx, African American, or Asian Americans were too few to permit separate subgroup analyses. The Iranian sample comprised 71 participants (47 women) who ranged in age from 17 to 54 years, with a mean age of 31.32 years, and the Turkish sample consisted of 79 undergraduate students (48 women) ranging in age from 18 to 25 years with a mean of 21.7. The American and Turkish participants completed the task as part of a class activity; the Iranian sample was recruited by placing an announcement in social media and participants completed an online version of the task. All participants received and answered the prompt in their primary language. The Iranian and Turkish data were translated into English by native Farsi- and Turkish speakers who had advanced English proficiency. Most of the data were coded by the same researcher (with gender of participants masked) to provide consistency in coding. A subset of the data were also intercoded to ensure some level of consensus (at least 80%). Materials and Procedure -----------------------	{ "pile_set_name": "PubMed Central" }
Introduction {#s1} ============ Rabies virus (RABV) is a prototypical member of the Lyssavirus genus of the Rhabdoviridae family, and the causative agent of rabies. Rabies is associated with severe neurological symptoms and high mortality rate, causing more than 50,000 human deaths annually, mainly in Asia and Africa (Sudarshan et al., [@B49]). Although rabies has been studied for over 100 years, it remains incurable and a serious threat to human health. To develop effective antiviral drugs, a deeper understanding of the pathogenic mechanisms of RABV infection is required. The life cycle of RABV occurs exclusively in the cytoplasm with the transcription of five viral genes that encode the viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the viral RNA polymerase (L), respectively. The N, P, and L proteins, together with the viral RNA genome, form a helical ribonucleoprotein (RNP) complex that is responsible for viral RNA transcription and replication (Albertini et al., [@B2]). The M protein recruits RNPs to the cell membranes and interacts with the G protein to facilitate virion budding and release (Mebatsion et al., [@B33]). Furthermore, the M protein has been reported to regulate the balance of viral RNA synthesis and be related to the pathogenicity of RABV (Finke and Conzelmann, [@B11]; Pulmanausahakul et al., [@B42]; Wirblich et al., [@B51]). The degeneration of neuronal processes is considered to play an essential role in the fatal neurological symptoms of rabies (Baloul and Lafon, [@B5]; Scott et al., [@B45]; Kojima et al., [@B24]). An increasing number of studies indicate that microtubules are vital to the development and maintenance of axons and dendrites throughout the life of the neuron, and microtubule abnormalities are closely associated with neurodegenerative diseases (Matamoros and Baas, [@B30]; Stevenson et al., [@B48]). Thus, we hypothesized that the neuronal degeneration that occurs during RABV infection might be linked to microtubule abnormalities. Microtubules are hollow, cylindrical structures composed of associated protofilaments of α and β-tubulin dimers and play important roles in multiple cellular processes, such as intracellular transport, signaling pathways, and cellular division (Downing, [@B9]; Hammond et al., [@B18]; Akhmanova and Steinmetz, [@B1]). The functional versatility of microtubules is dependent on microtubule dynamics, which are regulated by microtubule-associated proteins (MAPs; Akhmanova and Steinmetz, [@B1]; Alfaro-Aco and Petry, [@B3]). Among these, MAP4 binds to the microtubule lattice to stabilize microtubules, and thereby prevent depolymerization (Kadavath et al., [@B23]). Alternatively, stathmin depolymerizes microtubules by sequestering tubulin subunits and promoting microtubules shrinkage, so as that the growth of microtubules was blocked. This procedure was termed as "microtubule catastrophe" (Howell et al., [@B20]). In addition, microtubule dynamics have been found to be regulated by post-translational modifications, such as acetylation, phosphorylation, or palmitoylation of α-tubulin (Hammond et al., [@B18]; Song and Brady, [@B47]). Among these modifications, the acetylation which occurs on lysine 40 of α-tubulin is fairly common and considered to be a well-known marker of stable microtubules (L\'Hernault and Rosenbaum, [@B27]; Perdiz et al., [@B41]). Furthermore, α-tubulin can be deacetylated by histone deacetylase 6 (HDAC6), a primary α-tubulin deacetylase (Matsuyama et al., [@B31]; Yang et al., [@B54]). Numerous viruses have been reported to utilize microtubules to facilitate the intracellular transport of virions or subviral particles, including human immunodeficiency virus type 1 (HIV-1), vaccinia virus, herpesvirus, and circovirus (Sanderson et al., [@B44]; Nishi et al., [@B37]; Pasdeloup et al., [@B40]; Cao et al., [@B8]; Fernandez et al., [@B10]). In addition, several viruses have been reported to take advantage of microtubules to regulate the formation of viral inclusion bodies, such as reovirus and orthopoxvirus (Parker et al., [@B39]; Howard and Moss, [@B19]). Conversely, some viruses induce microtubule depolymerization to facilitate viral infection, \[e.g., rotavirus, Epstein-Barr virus, and Sendai virus (SeV)\] (Ogino et al., [@B38]; Martin et al., [@B29]; Liu et al., [@B28]). Although a recent study demonstrates that α-tubulin is incorporated into RABV particles (Tu et al., [@B50]), the role of the microtubule cytoskeleton in the process of RABV infection, especially regarding viral transcription and replication remains poorly understood. Therefore, the aim of this study was to investigate: (1) the effects of RABV infection on the microtubule cytoskeleton; and (2) the role of microtubule dynamics in viral transcription and replication. Materials and methods {#s2} ===================== Cells, virus, and reagents -------------------------- Mouse neuroblastoma N2a cells, baby hamster kidney (BHK-21) cells, and human embryonic kidney epithelial (HEK) 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco/Invitrogen, USA) at 37°C with 5% CO~2~. The challenge virus standard-11 strain of fixed rabies virus (CVS) was propagated in N2a cells. All the cells and virus used in this study were stored in our laboratory. Cells were infected with CVS at a multiplicity of infection (MOI) of 1 and the supernatant was harvested after 72 h post-infection (hpi). Virus preparations were titrated on N2a cells, and then stored at −80°C. Drug treatment -------------- The chemicals used to treat the RABV-infected N2a cells included the microtubule-depolymerizing drug nocodazole (Noco) (S1765; Beyotime, China), the microtubule-polymerizing drug paclitaxel (Taxol) (S1150; Selleckchem, USA), the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (T1952; Sigma) and the HDAC inhibitor sodium butyrate (NaBut) (S1539; Beyotime). The N2a cells were infected with RABV for 4 h and then treated with the various drugs. The solvent dimethyl sulfoxide (DMSO) was used as a control. Cell viability assay -------------------- N2a cells cultured in 96-well-plates were incubated with each drug or DMSO for 20 h. Cell viability was measured using a CCK-8 Cell Counting Kit (A311-02; Vazyme, China) and expressed as the percent of the control culture as described previously (Zan et al., [@B55]). Transient transfection ---------------------- N2a cells were seeded into 6-well-plates or 35 mm glass bottom dishes (Shengyou Biotechnology Co. Ltd., Hangzhou, China). On the following day, the N2a cells were transfected with p-CMV-Myc-M (Zan et al., [@B56]) or the pCMV-myc empty vector using Exfect Transfection Reagent (Vazyme Biotech Co. Ltd., Nanjing, China), and the transfection procedure was carried out according to the manufacturer\'s instructions. Confocal microscopy ------------------- N2a, BHK-21, or 293T cells grown overnight into 35-mm glass bottom dishes were infected with RABV (MOI = 1) or not. At the indicated hours post-infection (hpi), the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and incubated at 4°C with primary antibodies overnight. Cells were then incubated with FITC-conjugated-secondary antibodies (KPL) and/or Alexa Fluor 546-conjugated secondary antibodies (Invitrogen) at 37°C for 1 h. Cellular nuclei were stained with 10 μg/ml DAPI (Roche) for 5 min, and viewed by an LSM780 laser scanning confocal microscopy (Carl Zeiss). All images were taken using multitrack scanning for each fluorophore to prevent bleed-through. FITC fluorescence was detected after excitation at 488 nm with an emission long-band filter at 505--530 nm (green). Alexa Fluor 546 fluorescence was detected after excitation at 561 nm with an emission long-pass filter at 550--600 nm (red). DAPI was detected after excitation at 405 nm with an emission long-pass filter at 445--450 nm (blue). The pinholes were set to an Airy unit of 1 (equal in size to an Airy disk). Images were acquired with the identical capture settings and analyzed using Zen 2012 software (Zeiss). Rabbit anti-α-tubulin antibody (11224-1-AP; Proteintech, China), rabbit anti-acetylated α-tubulin antibody (5335S, Cell Signaling Technology), mouse anti-RABV-N antibody, and mouse anti-RABV-M antibody (Zan et al., [@B55]) were used as primary antibodies. RNA preparation and quantitative real-	{ "pile_set_name": "PubMed Central" }
1. Introduction =============== Postpartum depression (PD) is one of the commonest psychological problems to affect postpartum women\'s health. Its prevalence rate worldwide was reported to be 11.9% and a higher rate of developing countries.^\[[@R1],[@R2]\]^ PD could develop symptoms as depressed emotion, fatigue, and even suicidal behavior.^\[[@R1],[@R3]\]^ Mothers with PD were associated with autistic disorder and developmental retardation in their offspring.^\[[@R3],[@R4]\]^ Thus, it is urgent to prevent pregnant women from PD. Nutrition during pregnancy is a vital factor of PD. Accumulating evidence has proved that deficiencies in unsaturated fatty acids and vitamin D were more commonly in patients with PD.^\[[@R5],[@R6]\]^ The possible mechanism might be involved in regulation of proinflammatory cytokines and serotonergic neurotransmitters.^\[[@R6]\]^ Physiological increases in nutrition related biochemical indicators, such as blood glucose, lipid profile, and uric acid (UA), are reported during pregnancy. Some previous studies have reported an association between serum lipid concentrations and the risk of depression,^\[[@R5],[@R7]\]^ while its mechanisms were not well understood. Some other studies demonstrated an increased risk of anxiety and depression in postpartum women who had a rapid reduction in the concentrations of these biochemical indicators.^\[[@R8]\]^ The physiological increases may vary in pregnant women from different regions. Chinese economy has enjoyed a rapid development over the past 3 decades and the lifestyle in Chinese people is changing dramatically, especially reflected on food consumption. An increased intake of animal-based foods, but a decreased intake of plant-based foods is characterized as modern Chinese.^\[[@R9]--[@R11]\]^ In this study, we conducted a case-control study to explore the association between nutrition assessments during pregnancy and the risk of PD in Chinese women. 2. Method ========= 2.1. Study design ----------------- This study was hospital-based case-control designed with a study period from January 2016 to June 2019. Participants with PD were recruited in the case group according to the criteria as follows: 1. first time diagnosis for PD; 2. singleton and term delivery; 3. without other postpartum disease; 4. without severe co-morbidities including neoplasm, cardiovascular disease, mental disorder, or severe infection during pregnancy; 5. no smoking or drinking during pregnancy; 6. age from 20 to 40 years. The controls were selected from puerpera who had childbirth in our hospital as the same period of the cases and were free of PD. Protocol of this study was in accordance with the Declaration of Helsinki and the research ethics principles of the Committee of our hospital. Eligible participants attended this study with signed informed consent. Baseline information was extracted from medical records of our hospital information system, including maternal age, maternal weight, neonatal weight, gestational weeks, neonatal gender, type of delivery. 2.2. PD ------- The Chinese version of Edinburgh postnatal depression scale (EPDS) was employed to identify PD,^\[[@R12]\]^ which has a fine reliability and validity and has been widely applied in China.^\[[@R13]--[@R15]\]^ EPDS contained 10 items including mood, pleasure, remorse, anxiety, fear, coping ability, insomnia, sadness, crying, and NSSI.^\[[@R12]\]^ Score in each item range of 0 to 3 points.^\[[@R12]\]^ Puerpera obtained an EPDS score ≥10 points to suggest the presence of PD, while an EPDS score \< 10 points to suggest free of PD.^\[[@R12]\]^ 2.3. Food consumption during pregnancy -------------------------------------- A food frequency questionnaire (FFQ) was designed to collect food consumption before the childbirth preceding month. Stable food (rice, wheat, other), vegetable (light, dark), fruit, fish, meat (pork, beef, and mutton, other red meat), poultry, and water were list on the FFQ (8, 9, 31). Participants were interviewed by well-trained investigators. Frequency and amount to food consumption before the childbirth preceding month were recorded. Several options were applied for food frequency, including never, times per day, times per week, and times per month. 2.4. Biochemical indicators detection during pregnancy ------------------------------------------------------ A routine fasting blood sample (5--10 mL) was collected from each participant in the third trimester of pregnancy. The samples were stored at −80°C until used. Biochemical indicators including fasting blood-glucose (GLU), total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), HDL, and UA were detected by valid methods as described previously.^\[[@R5],[@R7]\]^ Dyslipidemia in pregnant women was diagnosed when TC, TG, LDL concentrations were above the 95th percentile, and HDL concentration was below the 5th percentile for gestational age.^\[[@R16]\]^ Hyperglycemia in pregnant women was defined as GLU ≥7.0 mmol/L.^\[[@R17]\]^ Hyperuricemia was defined as UA ≥ 390 μmol/L.^\[[@R18],[@R19]\]^ 2.5. Statistical analysis ------------------------- R version 3.5.1 (The R Foundation, Vienna, Austria) was applied for statistical analysis. Categorical variables were given as frequency and percentile, while continuous variables were given as mean and standard deviation. Based on variable types, Student t test or Chi-squared test was used to evaluate variable difference in the 2 groups. A logistic regression model was applied to evaluate the association between nutrition related biochemical indicators (GLU, TC, TG, LDL, HDL, and UA) and PD. Crude odds ratio (OR) and its corresponding 95% confidence interval (CI) were computed. After then, adjusted ORs were calculated by adjusting for several possible risk factors (maternal age, maternal weight, neonatal weight, gestational weeks, neonatal gender, delivery type, systolic blood pressure, and diastolic blood pressure). A P-value \< .05 was set to describe statistical difference. 3. Results ========== 3.1. Characteristics of eligible participants --------------------------------------------- A total of 565 participants were eligible in this study, which comprised 182 puerpera with PD and 383 puerpera without. Flowchart exhibiting participant selection is showed in Figure [1](#F1){ref-type="fig"}. Average age in case group was 31.9 ± 5.3 and 31.5 ± 5.7 years in control group. Vaginal delivery and caesarean section delivery were 68.1% and 31.9% in case group, and 63.4% and 36.6% in control group. Other baseline information about eligible participants is summarized in Table [1](#T1){ref-type="table"}. ![Flowchart exhibiting the selection of eligible individuals.](medi-99-e21647-g001){#F1} ###### Characteristics of eligible participants with postpartum depression and without. ![](medi-99-e21647-g002) 3.2. Association between pregnant biochemical indicators and PD --------------------------------------------------------------- GLU in case group (5.0 ± 1.0 mmol/L) was significantly higher than the control group (5.3 ± 2.1 mmol/L). Logistics analysis demonstrated that patients with PD have a 2.67-fold (95%CI = 1.67--4.11, P-value = .012) higher odds exposed to elevated GLU level. After adjusting for several possible risk factors, the result remained. Serum lipid indicators including TC, TG as well as LDL in case group were also statistically higher than the control group, but HDL was found to be lower in case group. Compare with the controls, ORs for increased TC, TG, and LDL during pregnancy were 1.73 (95%CI = 1.22--2.46, P-value = .023), 2.43 (95%CI = 1.55--3.81, P-value \<.001), and 3.41 (95%CI = 2.09--5.57, P-value \< .001), respectively in patients with PD. Additionally, an inverse association between decreased HDL and PD (OR = 3.41, 95%CI = 2.09--5.57, P-value \< .001) was identified. The positive associations mentioned above remained after matching with some risk factors. However, with respect to UA, no statistically association was identified (OR = 2.23, 95%CI = 0.82--6.26, P-value = .137). Detail information is shown in Figure [2](#F2){ref-type="fig"} and Table [2](#T2){ref-type="table"}. ![Comparison of nutrition biomarkers between participants with postpartum depression and without postpartum depression. ^∗^=P-value \< .05, ^∗∗^=P-value \< .01, GLU = GLU, HDL = high density lipoprotein, LDL = low density lipoprotein, TC = total cholesterol, TG = triglyceride, UA = uric acid.](medi-99-e21647-g003){#F2} ###### Association between pregnant related biochemical indicators and postpartum depression. ![](medi-99-e21647-g004) 3.3. Food consumption before the childbirth preceding month ----------------------------------------------------------- According to FFQ, patients with PD were found to have	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== After the completion of the Human Genome Project \[[@CR1]\] and a successful case-control experiment in identifying age-related markers using single-nucleotide polymorphism (SNP) \[[@CR2]\], the number of genome-wide association study (GWAS) has been rising exponentially \[[@CR3]\]. GWAS provides an efficient way to scan the whole genome to locate SNPs associated with the trait of interest which may potentially lead to identification of the susceptibility gene through linkage disequilibrium. Unlike linkage analysis that requires data collection from genetically related subjects, GWAS is applicable to a more general setting involving independent subjects. This makes GWAS highly desirable because for many diseases, it may not be feasible to recruit enough related subjects for linkage analysis. For example, the parents of human subjects with late onset diseases are usually not available. Furthermore, many statistical programs such as PLINK \[[@CR4]\] have been developed to manage and analyze high dimensional GWAS data from independent subjects. Due to reduced costs for SNP arrays, in recent years, many family studies have collected GWAS data \[[@CR5]--[@CR7]\]. If existing methods designed for independent subjects are adopted to analyze these data, the power of association tests will be greatly reduced because only a subset of data can be used. On the other hand, employing all the data in the analysis (i.e. ignoring the correlation between genetically related subjects) may result in false positive findings \[[@CR8]\]. Yu et al. (2006) \[[@CR9]\] proposed a compromise between these two approaches that used all related subjects while adjusting for the relatedness by random effects in a linear mixed model. This approach has been widely studied and the original algorithm has been improved to be applicable to larger scale studies \[[@CR10]\]. However, this approach can only handle a univariate phenotype such as a positive or negative diagnosis. Many complex diseases such as mental health disorders have multiple phenotypic traits that are correlated \[[@CR11]\]. These multivariate phenotypes may point to a shared genetic pathway and underscore the relevance of pleiotropy (i.e., a gene or genetic variant that affects more than one phenotypic trait, Solovieff et al. (2013) \[[@CR12]\]). Furthermore, a statistical model searching for loci that are simultaneously associated with multiple phenotypes has higher power than a model that only considers each phenotype individually \[[@CR13]\]. Our research team recently developed a multivariate association test based on the Fisher combination function that can be applied to analyze GWAS data with multivariate phenotypes \[[@CR14]\]. This method, however, can only handle independent subjects. Taken together, advanced methods that can handle multivariate phenotypes and related subjects simultaneously are highly desirable. The crucial problem in GWAS is to deal with confounders such as population structure, family structure, and cryptic relatedness. Astle and Balding \[[@CR15]\] reviewed approaches to correcting association analysis for confounding factors. When family-based samples are collected, analysis based on the transmission disequilibrium test is robust to population structure. Several methods have been developed for multivariate phenotype data collected from family-based samples \[[@CR16]--[@CR20]\]. However, the major challenge of this type of studies is to recruit enough families in order to conduct the analysis with sufficient power. This type of studies also have limited applications to late-onset diseases. Recently, Zhou and Stephens \[[@CR21]\] proposed a multivariate linear mixed model (mvLMMs) for identifying pleiotropic genes. This approach can handle a mixture of unrelated and related individuals and thus has broader applications. However, it was recommended for a modest number of phenotypes (less than 10) due to computational and statistical barriers of the EM algorithm \[[@CR21]\]. For related subjects with multivariate phenotypes, there are two sources of correlations between multivariate phenotypes: one is the correlation arising from genetically related subjects whose phenotypes are more highly correlated because of shared genotypes; and the other is the correlation between multiple phenotypes which exists even when independent subjects are employed. This study proposes a new statistical method that can model both sources of correlations. We also compare the performance of the proposed method with that of the mvLMMs method. The rest of this paper is organized as follows. In the "[Methods](#Sec2){ref-type="sec"}" section, we review our previous work on multivariate phenotypes in independent subjects and also extend the method to handle related subjects. The "[Results](#Sec5){ref-type="sec"}" section summaries the results of simulation studies and statistical analysis on the Study of Addiction: Genetics and Environment (SAGE) data. Future directions and major findings are presented in "[Discussion](#Sec12){ref-type="sec"}" and "[Conclusions](#Sec13){ref-type="sec"}" sections, respectively. Methods {#Sec2} ======= Suppose that for each subject, we measure R different phenotypes and run an assay with S SNPs. The resulting measurements can be organized with two data matrices. The genotype data are stored in a S×N matrix where N is the total number of subjects and each element of the matrix is coded as 0, 1, or 2 copies of the reference allele. The phenotype data are stored in an N×R matrix where each row records the individual's multivariate phenotypes. Studying the association between genotypes and phenotypes, thus, involves measuring and testing the association between each row of the genotype matrix and the entire phenotype matrix. Since one SNP is consider at a time, the association test is repeated S times for all SNPs. Specifically, Let β ~1~,...,β ~R~ be the effect sizes of a candidate SNP on R different phenotypes. The null hypothesis of testing the pleiotropic gene is $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$H_{0}: \beta_{1}= \ldots = \beta_{R} = 0. $$ \end{document}$$ If this H ~0~ is rejected, we claim that the corresponding SNP is associated with the pre-determined multivariate phenotypes. When the phenotype is univariate, the association test for GWAS data can be carried out using commonly adopted software such as R \[[@CR22]\] or PLINK \[[@CR4]\]. For multivariate phenotypes in independent subjects, Yang et al. (2016) \[[@CR14]\] has conducted a comprehensive review of various multivariate methods and proposed a method using the Fisher combination function. They further showed that their proposed method is better than other existing methods. The following sections briefly review their method and extend it to handle related subjects by employing a linear mixed model to adjust for relatedness. Review of previous work on independent subjects with multivariate phenotypes {#Sec3} ---------------------------------------------------------------------------- To illustrate the method proposed by Yang et al. (2016) \[[@CR14]\], we define the notations for genotypes and phenotypes. Let i(=1,...,N) be the index of individuals. Define $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$y^{r}_{i}$\end{document}$ as the rth phenotype of individual i (r=1,...,R) and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$g^{s}_{i}$\end{document}$ as the sth genotype of individual i (s=1,...,S). Therefore, the vector $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\boldsymbol {y}^{r} = (y^{r}_{1},\ldots,y^{r}_{N})$\end{document}$ represents the rth marginal phenotype collected from N individuals and the vector $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\boldsymbol {g}^{s} = (g^{s}_{1},\ldots,g^{s}_{N})$\end{document}$ represents the genotypes of the sth SNP from N individuals. When R=1 (i.e., the phenotype is univariate), a regression model is commonly adopted to model *y* ^r^ as a function of *g* ^s^ with covariates in the model to adjust for confounding factors or to increase the precision of estimates. When R\>1 (i.e., multivariate phenotypes), Yang et al. (2016) \[[@CR14]\] proposed a two-step approach. In the first step, for each phenotype r, a marginal p-value, p	{ "pile_set_name": "PubMed Central" }
INTRODUCTION ============ Pregnancy in a rudimentary noncommunicating uterine horn has already been described in the literature.^[@B1]--[@B8]^ Only 9 cases have been reported of a placenta accreta in a pregnant rudimentary uterine horn, but this situation led to hemorrhagic rupture of the uterus in 8 women.^[@B1],[@B3]^ In the literature, pregnant rudimentary uterus ruptures, with or without placenta accreta, tend to occur during the second or third trimester of pregnancy, and in most cases the consequent hemorrhage threatens women\'s lives.^[@B1],[@B3]--[@B5],[@B7]^ However, early diagnosis may be possible and would allow the resection of the rudimentary uterine horn with scarce maternal morbidity.^[@B6]^ We report the case of a rudimentary uterine horn pregnancy with a placenta percreta revealed and managed laparoscopically at 7-weeks gestation, with favorable outcomes, and we argue for the laparoscopic removal of rudimentary uterine horns when they are revealed in young women who desire further pregnancies. CASE REPORT =========== A 28-year-old, healthy, nulliparous woman underwent a routine pelvic ultrasound examination at 7-weeks gestation. As regards her antecedents, she had a right ectopic fallopian pregnancy 4 months previously that had been treated laparoscopically at another establishment. She was not aware of any particular findings during this surgical procedure that led to the removal of the ectopic pregnancy by a right salpingotomy. The ultrasound examination revealed an image, suggesting a left ectopic pregnancy measuring 3cm with Doppler embryo heart activity. However, a pseudo-pattern of asymmetrical bicornuate uterus was noted as well as a gestational sac surrounded by myometrial tissue and completely separated from the uterine cervix. No communication with the contralateral uterine cavity could be found. These arguments suggested a rudimentary noncommunicating horn pregnancy. The β-human chorionic gonadotropin titer was 10,091 µUI/mL. Even though the woman was free of pain, a surgical procedure was proposed because of the expected risk of rudimentary uterus rupture. The laparoscopy was carried out using one umbilical ancillary 12-mm trocar and 3 ancillary 5-mm trocars placed downward. The peritoneal cavity was free of any blood or liquid collection. A right unicornuate uterus with a seemingly normal right fallopian tube and a left pregnant rudimentary uterus with a normal fallopian tube were present ([Figure 1](#F1){ref-type="fig"}). The 2 uteri were linked by a fibrous, less congestive flap. The pelvic peritoneum had several disseminated, red, congestive, nodular lesions that appeared to be endometriotic implants. No apparent signs of uterine rupture were revealed. A left hysterectomy with left salpingectomy using a classical procedure (bipolar coagulation and section by scissors of the mesosalpinx, fibrous flap and board ligament) was carried out. The procedure took 45 minutes, and no incidents were encountered. Biopsies were performed on 2 endometriotic lesions, the other being coagulated. Postoperative outcomes were favorable, and the woman was discharged 3 days later. Abdominal ultrasound examination showed that both kidneys were present. ![Left pregnant rudimentary uterine horn with a normal fallopian tube, linked by a fibrous flap to the right unicornuate uterus. The pelvic peritoneum has several disseminated, red, congestive, nodular endometriotic lesions.](jsls-12-1-101-g01){#F1} Histopathologic examination revealed a rudimentary left horn uterus with a 2-cm pregnancy of 7-week\'s gestation. There was no communication with the right unicornuate uterine cavity. The chorioamniotic sac contained the embryo and the placenta infiltrating through the primitive müllerian myometrium. The placental infiltration had completely pierced the myometrium thickness and was found exteriorized on the anterior uterine side. This finding corresponded to a placenta percreta ([Figure 2](#F2){ref-type="fig"}). ![Placenta percreta in the rudimentary uterine horn (the arrow shows the complete piercing of the uterine myometrium thickness).](jsls-12-1-101-g02){#F2} DISCUSSION ========== This case report is peculiar not only because of the rarity of the pathology, but also due to the onset of circumstances and early management of the pregnancy, avoiding any hemorrhagic incident. To our knowledge, in the literature, few cases have been reported of pregnant rudimentary uterine horn where pregnancy had not led to a symptomatic uterine rupture.^[@B1],[@B2],[@B6]^ In 2 similar cases, the pregnant rudimentary uterine horn diagnosis was carried out early by MRI examination.^[@B6]^ Despite the high accuracy of MRI, we considered that it was not necessary in this case because of both the strong ultrasound presumption of pregnant rudimentary uterus and the decision to carry out a laparoscopic procedure anyway. Otherwise, we agree that MRI is useful in preoperative assessment when the ultrasonography is not able to affirm a rudimentary uterine horn pregnancy and to rule out an ectopic pregnancy. Early diagnosis allows safe laparoscopic removal of the pregnant rudimentary uterus with rapid favorable outcomes. In the literature, the laparoscopic procedure often had not been done in circumstances of severe hemoperitoneum due to uterine rupture with major threats to the woman\'s life.^[@B2]--[@B5],[@B7]^ Even though in the literature uterine rupture is reported to mainly occur during the second trimester of pregnancy, we consider that no safe bet can be made on the pregnant rudimentary uterine horn capacity to relax during the first trimester. Subsequently, we consider that a prompt surgical procedure ought to be resorted to without delay. Peritoneal endometriosis is often associated with obstructive malformations of the genital tract,^[@B9]^ and this finding is an argument in favor of the pathogenic menstrual blood reflux theory of endometriosis. It is worth noting that despite the extended peritoneal area involved in active endometriosis and the antecedent of right salpingotomy, pregnancy could occur in the left rudimentary uterine horn. It appears that 2 mechanisms may be involved in the occurrence of this pregnancy.^[@B4],[@B8]^ The first supposes that spermatozoons go up to the peritoneum by the right permeable fallopian tube, transmigrate intraperitoneally and fecundate the ovule that had been released either by the left or right ovary. Nahum et al^[@B8]^ showed that intraperitoneal sperm and ovum transmigration appears to occur respectively in 50% and 40% of all cases of human pregnancy. However, several authors^[@B10]^ suggest that peritoneal endometriosis would be harmful to spermatozoic mobility and survival. An alternative hypothesis is that fertilization might have occurred within the peritoneal cavity with subsequent intraperitoneal transmigration of the resulting fertilized ovum and contralateral tubal pick up.^[@B8]^ Whatever the mechanism, the onset of pregnancy despite these unfavorable circumstances may be considered worthy of speculation. The placenta accreta is seemingly frequently found in rudimentary uterine horn pregnancies. On the strength of cases previously reported in the literature, Oral et al^[@B3]^ estimated that the prevalence of placenta accreta in rudimentary uterine horn pregnancies may be greater than 10%. The hemorrhagic risk due to placenta accreta and that of spontaneous uterine rupture due to the thinness of the myometrium represent in our opinion 2 sufficient arguments to recommend the immediate surgical removal of a pregnant rudimentary uterine horn as soon as its diagnosis is carried out. This attitude allows a safe laparoscopic procedure with rapid favorable outcomes and avoids hemorrhagic injuries requiring emergency surgical procedures by laparotomy. On the basis of the high rate of spontaneous sperm transmigration across the peritoneal cavity, Nahum et al^[@B8]^ had recently suggested that salpingectomy should be preferable to the salpingotomy in women with ectopic pregnancy and unilaterally damaged fallopian tube to avoid recurrences. Similarly, on the basis of the major risk related to the pregnancies located in a rudimentary uterine horn, we ought to go forward and suggest laparoscopic removal of rudimentary uterine horns when they are revealed in young women with a desire for further pregnancies. In our opinion, the option of a planned laparoscopic procedure is always less morbid than whatever surgical procedure is carried out in pregnancy circumstances.	{ "pile_set_name": "PubMed Central" }
Introduction {#S1} ============ Disulfide bonds between pairs of cysteines contribute importantly to the folding and stability of many proteins. In bacteria, this advantageous modification is generally limited to proteins that are exported to the cell envelope or beyond. Many proteins involved in bacterial virulence (such as toxins, adhesins, flagella, fimbriae, pili, and type II and III secretion systems) require disulfide bonds for their stability and activity^[@R1]^. Thus, inactivation of enzymes that make protein disulfide bonds interferes with the activity of multiple bacterial virulence factors. Inhibitors of these enzymes could have profound effects on pathogen virulence. In Gram-negative bacteria, disulfide bonds are introduced into substrate proteins as they cross through the cytoplasmic membrane into the cell envelope^[@R2],[@R3]^. The periplasmic enzyme DsbA, a member of the thioredoxin family, oxidizes pairs of cysteines in substrate proteins through its Cys-X-X-Cys active site^[@R4]^. The resulting reduced DsbA is re-oxidized by the membrane protein DsbB, regenerating DsbA's activity. DsbB itself is reoxidized by membrane-imbedded quinones, from which electrons are transferred to the electron transport chain ([Figure 1](#F1){ref-type="fig"}). While the DsbB/DsbA system is widespread in bacteria, some bacteria, e.g. the Actinobacteria and Cyanobacteria, use a somewhat different pathway^[@R5]^. This alternate pathway retains a DsbA-like protein, but uses the membrane protein VKOR instead of DsbB to oxidize DsbA^[@R6]-[@R8]^. Bacterial VKOR is a homologue of the vertebrate protein, vitamin K epoxide reductase, an endoplasmic reticular enzyme involved in blood coagulation and the target of the anticoagulant warfarin (Coumadin©). While bacterial VKORs show no homology to DsbB, like DsbB they encode two extra-cytoplasmic soluble domains containing essential pairs of cysteines, one of which is a Cys-X-X-Cys motif. The interactions between the redox-active cysteines of DsbA and VKOR proceed by the same steps seen between DsbA and DsbB^[@R7]^. Therefore, a vkor gene from Mycobacterium tuberculosis (MtbVKOR) expressed in E. coli complements a dsbB null mutant; while E. coli DsbB (EcDsbB) complements a vkor null mutant of Mycobacterium smegmatis^[@R9]^. Although some bacterial VKORs, like their eukaryotic homologues, are sensitive to coumarin-based anticoagulants, EcDsbB is not^[@R6],[@R9]^. Furthermore, VKOR is essential for Mycobacterial growth^[@R9],[@R10]^, while neither DsbB nor DsbA is essential for aerobic E. coli growth. The fact that EcDsbB and MtbVKOR are functionally homologous but do not share amino acid homology suggested a methodology for identifying specific inhibitors of either enzyme. This methodology is greatly facilitated by the ability to detect inhibitory effects on disulfide bond formation in growing E. coli cells. The methodology is further enhanced by a sensitive assay for disulfide bond formation in E. coli, provided by a version of the enzyme β-galactosidase that is exported to the E. coli periplasm where it is inactivated by the introduction of non-native disulfide bonds^[@R11]^. This disulfide-sensitive β-galactosidase (β-Gal^dbs^ ) is the product of a hybrid gene encoding a β-galactosidase fused to a periplasmic domain of the membrane protein MalF^[@R4],[@R12]^. In E. coli cells with an intact disulfide bond pathway, the activity of β-Gal^dbs^ is two to three orders of magnitude lower than when disulfide bond-forming enzymes are absent. Thus, wild-type cells expressing the β-Gal^dbs^ form white colonies on agar media that contain the chromogenic β-galactosidase indicator, X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) while dsb mutant colonies expressing the same β-Gal^dbs^ are blue. Importantly for limiting the array of targets in this screen, our previous genetic studies revealed that only null mutations in dsbA or dsbB restore high levels of β-galactosidase activity. Much weaker restoration of β-galactosidase activity results from certain non-null mutations of the dsbA or dsbB genes or in genes encoding proteins required for cytoplasmic membrane protein assembly^[@R12],[@R13]^. These latter mutations restore only \~1% of the β-galactosidase activity, presumably because strong mutations in these genes are lethal. Employing this highly sensitive assay of disulfide bond activity, we carried out a High Throughput Screen (HTS) to identify compounds potentially useful in the development of antibiotics. The rationale follows: 1)Disulfide-bonded proteins are important for bacterial virulence; 2)Detecting inhibition of MtbVKOR or EcDsbB can be achieved in growing E. coli cells; 3)Detecting high levels of β-Gal^dbs^ activity requires strong inhibition of either MtbVKOR or EcDsbB; 4) Strong inhibitors of other pathways that restore β-galactosidase activity are not likely to be detected; 5)Screening compounds in parallel for inhibition of the non-homologous MtbVKOR and EcDsbB enzymes provides reciprocal controls, allowing us to tentatively eliminate inhibitors that are influencing β-galactosidase activity by affecting membrane protein assembly or acting directly on DsbA. Such inhibitors would show up as hits in the screen for both strains while specific inhibitors would only register as hits against one strain or the other. Here, we report the results of a screen of 51,487 compounds that yielded six specific inhibitors of EcDsbB, but none of MtbVKOR. Chemical modifications led to more potent inhibitors that directly inhibit purified EcDsbB activity and inhibit anaerobic growth of E. coli. Furthermore, these small molecules inhibit DsbBs of other gram-negative pathogenic bacteria. Results {#S2} ======= Target- and cell-based screen using agar-multiwell plates {#S3} --------------------------------------------------------- We screened inhibitors of disulfide bond formation in two E. coli strains in parallel. One expresses chromosomally encoded EcDsbB and the other is deleted for the dsbB gene but complemented by the Mtbvkor gene expressed from an IPTG-inducible promoter. Both strains express the β-Gal^dbs^ from the chromosome. In these strains, strong inhibition of disulfide bond formation should lead to a substantial increase in β-galactosidase activity^[@R4],[@R12]^. Since the enzyme directly responsible for disulfide bond formation in both strains is DsbA, inhibitors of DsbA or of other processes enhancing the activity of β-galactosidase would raise the activity in both MtbVKOR- and EcDsbB-based strains. In contrast, compounds that specifically inhibit EcDsbB would increase β-galactosidase activity in the EcDsbB-dependent strain but not in the MtbVKOR-dependent strain and vice versa. Assaying the effects of compounds on the two strains in parallel allowed us to pick candidate inhibitors that were specific to either EcDsbB or MtbVKOR. Therefore, we eliminate from further consideration compounds giving positive signals with both strains, even though these might include compounds that inhibit both enzymes directly. Initially, we compared screens with 384-well plates in which wells contained liquid or agar-based growth media. It appeared that aeration interfered with growth in the liquid media leading us to choose the agar media where growth occurred on the media surface. When these mini-agar wells included X-Gal, high levels of β-galactosidase activity in the presence of an inhibitor were readily seen in the blue color resulting from X-Gal hydrolysis ([Supplementary Results, Supplementary Figure 1](#SD1){ref-type="supplementary-material"}). Using such agar-filled wells required special procedures that quickly became routine (Online Methods). We observed that while the EcDsbB strain exhibited no visible trace of blue in the wells, the MtbVKOR-complemented strain did show a pale blue color, indicating that the MtbVKOR did not completely restore levels of disulfide bond formation^[@R9]^. This difference did not interfere with the principles of the screen, but did result in a greater number of false positives in the MtbVKOR screen. The HTS was carried out with 50,374 compounds from the collection of Harvard University's Institute for Chemistry and Chemical Biology (ICCB) and 1,113 compounds from the National Institute of Allergy and Infectious Diseases (NIAID) collection of inhibitors of M. tuberculosis H37Rv growth^[@R14]^. Each compound was assayed in duplicate for each strain. Only a small number of EcDsbB hits were observed, seen easily by the clear blue/white difference. Because of the weaker expression of MtbVKOR, real potential hits	{ "pile_set_name": "PubMed Central" }
1. INTRODUCTION {#sec1-1} =============== Worldwide, cervical cancer is the third most common cancer among women and the second most common cause of death, with 300,000 deaths per year ([@ref1]). PAP test is cheap, simple and most commonly used technique for cervical cancer and preinvasive cervical lesions screening. Atypical squamous cells (ASC) is a term that refers to inflammatory, reactive and reparative processes which are atypical and of higher level and whose quality and quantity is insufficient to be classified as cervical intraepithelial lesions (CIN). ASC was first described in Betheda classification in 1988, and revised in 2001 and 2014 when it was divided into ASCUS (atypical squamous cells of undetermined significance) and ASC-H (atypical squamous cells-high level of lesion cannot be excluded). ASC continues to be defined as general category with subcategorisation as ASCUS and ASC-H ([@ref2], [@ref3]). Every year, over 2 million women in the USA are found with atypical squamous cells in PAP test ([@ref4]). Frequency of ASCUS lesions is between 1,6% and 9% ([@ref5]), and their frequency shouldn't exceed two to three times frequency of low grade lesions (LSIL) ([@ref6]). Metaanalysis showed that progression of ASCUS can progress into invasive cervical cancer may occur in 0,25% within 2 years, HSIL in 7,13%, while regression occurs in 68,19% ([@ref7]). Out of 1000 women with atypical squamous lesions one already has invasive cervical cancer ([@ref8]). Aims of this study were to examine: a) Frequency and age of the examinees with ASCUS lesions; b) Frequency of HPV infection in ASCUS lesions; c) Regression, stagnation and progression of ASCUS lesion during six-month period. 2. MATERIALS AND METHODS {#sec1-2} ======================== Prospective study was conducted over a period of 3 years, from January 2012 to December 2014. In private gynecological ambulance „Dr Mahira Jahić" 1784 PAP tests were examined as routine screening in first year. Patients with ASCUS were reevaluated after local treatment with PAP test and have been monitored in period of one year. ASCUS treatment includes repeated cytology, HPV typization and colposcopy. Protocol of monitoring depended on the result of repeated PAP test. PAP test was normal in 1530 patients and they were advised to make control test once a year. ASCUS was found in 133 patients and 50 of them agreed to be monitored. Analysis of vaginal secretion and HPV typization have been done in these 50 patients and PAP test has been repeated after six months. All cytology samples were screened in the same laboratory. PAP tests were taken by conventional method, colored by method of Papanicolaou and examined with Kruss microscope. Analysis of vaginal discharge was performed from a PAP test and HPV typization-In situ by hybridization of HPV. X² test was used for statistical analysis of data. 3. RESULTS {#sec1-3} ========== Analysis of 1784 PAP smears showed normal results and benign cellular changes in 86,6% (N-1530), and abnormal in 13% (N-254) of women. ASCUS was found in 7,4% (N-133) and ASC-H in 0,5% (N-9), LSIL in 4,4% (N-80), HSIL in 1,3% (N-24), CIN II in 1,2% (N-20), CIN III in 0,2% (N-4) ([Table 1](#T1){ref-type="table"}). ###### Results of 1784 PAP tests ![](MA-70-296-g001) Patients with ASC-H lesion 0,5% (N-9) were transferred to biopsy of cervix and PH results showed 22% (N-2) Carcinoma in situ (CIS), 33% (N-3) CIN II, 22% (N-2) CIN I and 22% (N-2) chronic cervicitis. 25% (N-10) of patients with ASCUS lesion didn't give birth and 70% (N-35) didn't have abortion. 94% (N-47) were married and 6% (N-3) weren't. 14% (N-7) of women used contraception ([Table 2](#T2){ref-type="table"}). Progression occurred in 18% ([@ref9]), persistence in 74% (37) and regression in 8% out of 50 women with ASCUS lesion after treatment and repeated PAP test ([Table 3](#T3){ref-type="table"}). There wasn't progression into high grade lesions (HSIL) in this period of time. Bacterial vaginosis has been found in 10% (N-4) of patients with ASCUS and CIN I in 33% (N-3) while patients with normal PAP results didn't have it. Patients with CIN I in 88% (N-7) were positive on HPV of high risk. Patients with persistent ASCUS result were positive in 51% (N-19). Grade of lesion was higher with the higher incidence of HPV infection ([Table 4](#T4){ref-type="table"}). The number of CIN I lesions found in women with ASCUS is bigger and statistically significant (p\<0,05) in relation to number of CIN I findings found in regular examinations of 1784 women ([Table 5](#T5){ref-type="table"}). ###### Vital characteristcs of patients with ASCUS lesion ![](MA-70-296-g002) ###### Results of PAP tests of 50 women with ASCUS lesions after 6 months ![](MA-70-296-g003) ###### Distribution of patients (N-50) after repeated PAP test in relation to infection ![](MA-70-296-g004) ###### Difference in frequency of CIN I findings in regular screening (N-1784) in relation to repeated ASCUS findings (N-50) ![](MA-70-296-g005) 4. DISCUSSION {#sec1-4} ============= The average age of patients with ASCUS lesion was 37,6 ± 12,6 years, and frequency of 7.4% is in accordance with data cited in literature ([@ref9]), but Jahic in her research found ASCUS in 12.9% and CIN I in 5.4% ([@ref10]). Cytologic diagnosis of ASCUS is 1.7 times more often than frequency of LSIL which was 4.4% of total number of analyzed PAP tests. The category of ASCUS is by far the most commonly reported abnormal cervical cytology interpretation. Lack of specific criteria for the diagnosis of ASCUS results in differences in the incidence of these lesions in works ([@ref11]). One of the obstacles in the interpretation of cytologic finding is presence of inflammation. Miguel and ass. analyzed 50 cervical and vaginal smears in which 15 of them, at first, were interpreted as ASCUS and 34 as normal ([@ref12]). After report using Bethesda criteria for ASCUS, he found changes that associate with the presence of Candida sp., and reclassified 10 out of 16 cases as normal findings ([@ref13]). Candida was present in 24% (N-12) of patients provided that in ASCUS lesion was more often 21% (N-8) and 3 women were reevaluated as normal findings. HPV of high risk was positive in 52% (N-26) and negative in 48% (N-24) of patients. Boardman found presence of high risk HPV in 68% of 527 women with ASCUS ([@ref14]). Other authors state frequency of HPV in ASCUS lesion from 49 to 51% ([@ref15]) and 46% in CIN I ([@ref16]). Our study showed similar representation of HPV infection in 51% (N-19) of women with ASCUS. During monitoring, regression was found in 8% ([@ref4]), persistence in 74% (37) and progression to CIN I in 18% (N-9). Progressions to high grade lesions such as HSIL weren't found in this study, although some authors state it is possible in 7.13%. Frequency of HPV of high risk was bigger with higher grade of lesion. Similar incidence of 20% of CIN I was found in Barcelos' study of analysis of ASCUS and cytologic criteria. Barcelos states that it is necessary to seriously monitor patients with ASCUS lesions. In her monitoring of patients with normal/inflammatory cytology, 85.7% had normal findings after six-months monitoring and one was CIN I, so she recommended that repeated PAP test should be performed in six months time if cytologic diagnosis after ASCUS lesion is normal. Basically, changes that are ASCUS lesion are of low grade but they enable selection of women who should be examined more often because of the possible progression of lesion or possible higher grade of lesion hidden behind this diagnose. Reports say that 10-30% of LSIL and HSIL are diagnosed in women with ASCUS lesion who were monitored ([@ref17]). Rinku finds cervicitis as a result of biopsy after ASCUS in 11 out of 17 cases, but repeated PAP test after 4-8 months shows ASCUS in all patients. After monitoring in next six months 6 was ASCUS, 2 LSIL and 1 HSIL ([@ref18]). Persson warns that there will be approximately 30% of women with ASCUS and HPV positive finding for high risk	{ "pile_set_name": "PubMed Central" }
Introduction ============ Humans possess robust metacognitive capacities to evaluate their performance on various tasks and make predictions about how such performance might alter in the future ([@niw018-B54]; [@niw018-B50]; [@niw018-B42]). Metacognitive evaluations are often studied by eliciting confidence judgments. For example, a student may predict their success on an upcoming exam by reflecting on their current level of knowledge and preparation (a prospective metacognitive judgment; P-metacognition). After taking the exam, the same student may then estimate his or her grade before receiving feedback (a retrospective metacognitive judgment; R-metacognition). Metacognitive capacity -- the extent to which judgments track performance -- is dissociable from first-order task performance and associated with distinct neural substrates (see [@niw018-B25]; [@niw018-B59], for reviews). However, it is unknown whether prospective and retrospective judgments draw on distinct or common resources. Behaviorally, few studies have directly compared the accuracy of P- and R-judgments for the same task and stimulus material. [@niw018-B16] compared probability judgments made about future and past events such as "What is the probability that next (last) week IBM stock will finish the week higher than it began the week?" He found that, when the same subjects make both past and future judgments, the Brier score (a measure of probability judgment accuracy) was better for past than future judgments. However, in this case the events to be judged are external to the subject and not evaluations of self-performance. Siedlecka et al. (2016) compared prospective and retrospective judgments of performance while participants solved anagrams. Participants rated their confidence that a particular word was the solution, either before or after their binary response of "yes" or "no," and before or after seeing the suggested solution. Confidence ratings given after the decision were more accurate than when ratings were prospective. [@niw018-B52] showed that rhesus macaques were able to make accurate confidence judgments -- bets on performance -- both before and after responding to a delayed-match-to-sample memory task, suggesting temporal flexibility in the use of confidence responses in nonhuman primates. However in this study, first-order performance also differed between the prospective and retrospective judgment tasks, making direct comparison of metacognitive accuracies difficult. There is evidence for neural dissociations between P- and R-metacognition ([@niw018-B17]; [@niw018-B25]). For example, [@niw018-B66] found that damage to the right ventromedial prefrontal cortex was associated with a decrease in metacognitive accuracy for judgments about future recall (feeling of knowing), but did not affect accuracy for retrospective confidence judgments. In contrast, [@niw018-B60] found that patients with lateral frontal lesions were impaired on retrospective confidence judgments, but not judgments about future task performance. Studies using functional MRI have shown that prospective metacognition activates medial PFC ([@niw018-B66], [@niw018-B67]; [@niw018-B51]), while retrospective metacognitive accuracy in a short-term memory task is correlated with rostrolateral PFC activity ([@niw018-B79]). A related line of research has demonstrated that post-decision confidence judgments also recruit rostrolateral PFC ([@niw018-B24]; [@niw018-B29], [@niw018-B26]). Together these studies suggest that humans and nonhuman primates have the capacity to make P- and R-metacognitive judgments about the same stimulus material, and that R-metacognition is typically more accurate than P-metacognition. However, it is clear that there are conceptual and methodological differences between different types of prospective metacognitive judgment. For some prospective judgments, such as a feeling-of-knowing, a specific representation of stimulus strength is available (albeit perhaps weakly) to the system on each trial. For other types of judgment, such as predicting one's success at a sporting event, judgments must instead be made based on an aggregate likelihood of success, with little or no information pertaining to individual trials. Finally, compared to P-judgments, R-judgments are able to draw on additional trial-specific cues of response fluency, response identity and stimulus type or difficulty, potentially explaining their increased accuracy (Siedlecka et al. 2016). Thus, while previous studies have quantified differences in R- and P-metacognitive accuracy, the influence of different cues and their temporal dynamics (e.g. the recent history of performance and confidence on the task) on each judgment type have received less attention ([@niw018-B63]). The dissociations between P- and R-metacognition noted above referred to metacognitive accuracy (or discrimination), the extent to which moment-to-moment variations in confidence track task performance. In contrast, bias (or calibration) reflects the tendency to be over- or underconfident ([@niw018-B27]). While metacognitive accuracy has been shown to differ between tasks ([@niw018-B64]; [@niw018-B7]; [@niw018-B49]; [@niw018-B28]; [@niw018-B1]), perhaps reflecting differences in the cues that subjects use to construct their confidence in each domain, bias may be more stable, transcending temporal focus: people have been found to have high or low confidence in their performance, irrespective of task ([@niw018-B64]; [@niw018-B58]; [@niw018-B70]; [@niw018-B1]; [@niw018-B35]). Several studies have found that subjects are overconfident in their judgments ([@niw018-B45]; [@niw018-B8]; [@niw018-B15]; [@niw018-B34]; [@niw018-B5]), but in some experiments underconfidence is found ([@niw018-B22]; [@niw018-B12]; [@niw018-B77]). In particular, while overconfidence may be the default in more difficult tasks, underconfidence may appear for easier tasks ([@niw018-B8], [@niw018-B9]), a phenomenon known as the hard--easy effect ([@niw018-B31]). In the present study, we set out to quantify influences on prospective and retrospective judgments of self-performance. We employed the same visual discrimination task for both judgment types, thereby matching performance and task characteristics across temporal focus. We elicited numerical probabilities of success, allowing assessment of both overconfidence (bias) and accuracy of confidence ratings. Retrospective ratings were provided on every trial, whereas prospective judgments of upcoming task performance were made every five trials, before seeing the stimulus. By using repeated, similar stimuli of constant difficulty, we allowed subjects to build up knowledge of their own performance over time ([@niw018-B38]). The elicitation of subjective judgments was incentivized to ensure subjects treated both prospective and retrospective judgments with similar importance. To assess metacognitive accuracy, we calculate both the area under the type 2 ROC and measures of probability judgment calibration and discrimination ([@niw018-B27]). We hypothesised that P- and R-metacognitive judgments would draw on separate cues, such as fluency for retrospective judgments, and recent outcome history for prospective judgments, and that metacognitive accuracy and calibration would be greater for retrospective compared to prospective judgments. In contrast, based on evidence that overconfidence is pervasive and domain-general, we hypothesized that overconfidence would be similar across the two judgment types. Methods and Materials ===================== Participants ------------ The experiment was conducted in December 2012 at the Laboratory of Experimental Economics in Paris (LEEP) of the University of Paris 1. Subjects were recruited by standard procedure from the LEEP database and gave written informed consent to take part in the experiment. A total of 47 subjects (26 men; age 18--29 years, mean age, 22.1 years) participated in this experiment for pay. The session lasted around 90 min and subjects were paid on average €19.7. We excluded subjects from analysis due to insufficient variation (SD \< 0.02) of R-confidence (4 subjects) or P-confidence (4 subjects) for estimation of metacognitive accuracy (see below). The final sample included 39 subjects for analysis. Stimuli ------- The experiment was conducted using Psychophysics Toolbox version 3 ([@niw018-B13]) running in Matlab. The stimuli consisted of two circles with a variable number of dots in each circle. All dots were of the same size and the average distance between dots was kept constant. One of the two circles always contained 50 dots while the other contained 50 + c dots. The position of the target circle (on the left or right) was randomly chosen on each trial. Before the experiment, we estimated the value of c needed to obtain a success rate of 71% using a psychophysical staircase ([@niw018-B44]; see below). This dot difference (c) was kept constant throughout the main experiment, such that all trials were of equal objective difficulty. The position of the circle containing the greater number of dots was randomly assigned to be on the left or right on each trial. Task and procedure ------------------ ### Practice and thresholding Subjects initially performed practice trials of the dots task without confidence ratings, in which full feedback was given. We used these trials to calibrate task difficulty. The calibration phase used a one-up two-down staircase ([@niw018-B44]): after two consecutive correct answers one dot is removed, and	{ "pile_set_name": "PubMed Central" }
Background ========== It is particularly challenging to provide policy-relevant information on women in the area of substance use and misuse, as the topic covers such broad territory. Problem substance use has been found to arise from a complex interplay of biological, genetic, psychological, social, cultural, relational, environmental and spiritual factors. Because of the interplay of these factors, it remains a challenge for policy makers and program planners to develop and implement the very broad, collaborative, systemic responses required and to do so in a manner that links prevention, enforcement, harm reduction and treatment strategies. In spite of the overall challenges, advances in gender-specific policy and programming are especially worthy of consideration and could be of tremendous benefit to the health of women and their families. This chapter presents findings from both surveillance sources and select research reports. As there has not been recent or adequate surveillance specific to substance use by Canadians, cross-sectional data from the Canadian Community Health Survey (CCHS) Cycle 1.1 (2000) are highlighted. While trends in women\'s substance use and substance use problems could not be provided, an overview of key research findings is offered that supports a comprehensive understanding of the topic from a gender perspective and places the findings of the CCHS in the appropriate context. Readers are encouraged to consult existing research reports mentioned throughout the chapter to further their understanding of this complex topic. Physical Health Impact of Problem Substance Use ----------------------------------------------- Health Canada\'s Straight Facts about Drugs and Drug Abuse \[[@B1]\] outlines the health consequences of high doses of all classes of psychoactive substances, including stimulants, hallucinogens, cannabis and central nervous system depressants (opioid analgesics, alcohol, inhalants/solvents, benzodiazepines, barbiturates). For some drug classifications, evidence of sex differences in the physical health impact of substance use is available. \[[@B2]\] To take alcohol as an example, women metabolize alcohol and other psychoactive substances more slowly than men, allowing harmful metabolites to remain longer in the body. Women are more likely than men to develop cirrhosis of the liver after consuming lower levels of alcohol over a shorter period of time. \[[@B3],[@B4]\] These findings also apply to brain shrinkage and impairment, \[[@B5]-[@B7]\] breast cancer, \[[@B8]-[@B12]\] gastric ulcers \[[@B13]\] and alcoholic hepatitis. \[[@B14]\] Although there is not adequate research on sex differences in the impact of illicit drugs, gender differences are beginning to be documented. \[[@B15]\] Injecting drug use (IDU) is a key risk factor among women for the transmission of blood-borne diseases such as HIV/AIDS and hepatitis C. \[[@B16]\] Also linked to illicit drug use are high-risk sexual behaviours (e.g. sex trade work), which, in turn, are associated with a range of negative health impacts. \[[@B17]\] Mental Health, Trauma and Substance Use --------------------------------------- Research has shown that as many as two thirds of women with substance misuse problems may have a concurrent mental health problem, such as depression, post-traumatic stress disorder, panic disorder and/or an eating disorder. \[[@B18]\] Research also shows that a large proportion of women with substance use problems are victims of domestic violence, incest, rape, sexual assault and child physical abuse. \[[@B19]-[@B22]\] Victimization has been linked to a variety of negative outcomes, including post-traumatic stress disorder, depression, anxiety, suicidal behaviour and low self-esteem among women in the general population. \[[@B23],[@B24]\] Compared with non-abused clients, women in treatment for problem substance use who have been victimized are more likely to suffer from depression and suicidal ideation, have lower self-esteem, negative psychological adjustment and more post-traumatic stress symptoms. \[[@B25]-[@B30]\] Pregnancy, Mothering and Substance Use -------------------------------------- Substance use by pregnant women and mothers has received a great deal of attention, and there is a voluminous literature documenting the adverse effects of alcohol, tobacco and other drugs on the fetus. Alcohol use during pregnancy, particularly in combination with poor nutrition, poor general health, experience of trauma and mental health problems, and lack of prenatal care, has been found to have the most harmful effects. \[[@B31],[@B32]\] Maternal use of licit and illicit drugs can also result in problems that have short- or long-term consequences for those prenatally exposed. However, study of the impact of these drug categories is hampered by methodological flaws that fail to take into account the use of more than one drug. \[[@B33]\] Rather than the provision of effective outreach, engagement and treatment, the responses in some countries and to some extent in Canada towards women who use illicit substances during pregnancy have been blame, attempts to force women into treatment, and even criminal sanctions. \[[@B34]-[@B37]\] The Health Canada document entitled Best Practices: Fetal Alcohol Syndrome/Fetal Alcohol Effects and the Effects of Other Substance Use during Pregnancy \[[@B32]\] provides a comprehensive view of the issues and of promising fetal alcohol syndrome (FAS) prevention practices grounded in both the literature and the expertise of Canadians working in the field. Programs such as the Breaking the Cycle Program in Toronto and the Sheway Program in Vancouver are examples of effective programs being developed in Canada to serve women at high risk of having a child affected by FAS. Stigma and Barriers to Treatment for Women ------------------------------------------ In the forefront of psychosocial influences on women\'s misuse of alcohol and other drugs is the stigma arising from societal attitudes towards women\'s substance use. \[[@B38]\] This stigma causes women to feel considerable guilt and shame as their substance use/misuse continues and creates barriers to their accessing help. \[[@B39]-[@B43]\] The stigma associated with women\'s substance use affects service providers as well. Women often encounter mis-information, denial, inaction and even punitive attitudes towards their substance use by professionals in a position to intervene, and thus they may not be identified as needing help. \[[@B37],[@B44]-[@B46]\] Stigma intersects with structural and other barriers that arise from experience of victimization and mental health problems, fear of having one\'s children apprehended, minority status, income and rural location, to name but a few. Health Canada\'s report Best Practices: Treatment and Rehabilitation of Women \[[@B40]\] has been developed as a key resource for health care providers in helping to reduce barriers to treatment access for diverse groups of substance-using women. The barriers to accessing supportive treatment services are even greater for pregnant and parenting women. Programs that accept both mothers and children are very scarce. Finding affordable and safe care for their children as well as residential care is often an overwhelming obstacle for mothers needing treatment. \[[@B47]\] Although outpatient counselling and other supports for pregnant and parenting women have been made available, appalling barriers remain for mothers in need of residential treatment for problem substance use in Canada. Gender and Harm Reduction Approaches ------------------------------------ Harm reduction is a newer policy approach to substance misuse, which arose initially as a response to the spread of AIDS among injection drug users. \[[@B48]\] The primary goal of harm reduction policy and practice is a reduction in the negative consequences associated with use rather than complete cessation of substance use. Key harm reduction programs and policies in Canada relating to illicit drugs include methadone maintenance; needle/syringe exchange; supervised injection sites; increased attention to the decriminalization of use of small quantities of cannabis; and education, information and diversion programs. Such initiatives can be beneficial to women who are drug users and/or who have partners who are drug users in helping prevent HIV transmission, improve their access to drug treatment and health care services, prevent birth defects and disabilities in their children, make incarceration experiences safer, and stabilize their health overall. Particularly promising is harm reduction programming offered to high-risk, marginalized pregnant women and their support networks, an approach that has been shown to prevent FAS and other alcohol- and drug-related disabilities, support retention of custody, and increase the health and social stability of both mothers and children. \[[@B47]\] The Current Study ----------------- We explored the use of alcohol, licit and illicit substances by Canadian women and subgroups of women (including girls, elderly women, Aboriginal women and pregnant women). Additionally, the health consequences of substance use, and the coexistence of mental health, trauma and substance use issues, were explored. Methods ======= Data ---- Cross-sectional data from the Canadian Community Health Survey (CCHS) Cycle 1.1 (2000--2001) \[[@B49]\] are presented below. The data are weighted to represent the Canadian population. Age-adjusted data are presented where appropriate. The sample comprised 125,574 individuals, aged 12 years and older, from across all provinces and territories. Data from other sources (including select research reports, such as the Canadian Profile on Alcohol, Tobacco and Other Drugs \[[@B50]\]) are similarly provided to highlight what is known about the use of alcohol, licit and illicit drugs by women, and the effects of this use. Where pertinent, comparisons between females and males are made. It is important to note that comparability issues arise when different surveys, data sources and research reports are compared. Key issues of concern are related to design (e.g. the jurisdictions included in the survey) and methodology (e.g. the form of the survey -- telephone or face-to-face interviews). For a full understanding of the comparability of the research studies reported on here, the reader is encouraged to consult the original source. Likewise, it is important to point out some of the key limitations of general population surveys. These include the difficulty in obtaining information from and about hard-to-reach populations (heavy	{ "pile_set_name": "PubMed Central" }
Background ========== Atherosclerosis is a highly prevalent chronic inflammatory disorder that causes extensive mortality \[[@b1-medscimonit-25-8820]\], but the details of the underlying mechanism are unknown. In recent decades, accumulating evidence has revealed the essential regulatory role played by inflammation throughout atherosclerotic progression \[[@b2-medscimonit-25-8820]\]. Moreover, proinflammatory tumor necrosis factor-α (TNF-α) promotes atherosclerosis progression and subsequent cardiovascular events via induction of inflammatory responses \[[@b3-medscimonit-25-8820]\]. TNF-α is a typical member of the cytokine family, which promotes inflammation, differentiation, and apoptosis of endothelial cells (ECs). Atherosclerosis is increasingly thought to be an age-associated chronic disorder, and endothelial dysfunction always precedes the development of atherosclerosis. Endothelial senescence is an important mediator of vascular inflammation and dysfunction \[[@b4-medscimonit-25-8820],[@b5-medscimonit-25-8820]\]. Sirtuins, a group of NAD+-dependent class III histone/protein deacetylases (HDACs), are involved in essential processes such as proliferation, apoptosis, senescence, and differentiation \[[@b6-medscimonit-25-8820],[@b7-medscimonit-25-8820]\]. Sirtuin1 (SIRT1) is the most extensively characterized and best-studied member of the sirtuin family. SIRT1 acts as a regulatory switch in vascular endothelial cells homoeostasis \[[@b8-medscimonit-25-8820],[@b9-medscimonit-25-8820]\] and is critically involved in regulating inflammation-related disorders via inhibition of proinflammatory cytokine secretion \[[@b10-medscimonit-25-8820]--[@b12-medscimonit-25-8820]\]. Previous studies have demonstrated that hypoxia, lipopolysaccharide (LPS), TNF-α, and high glucose decreased SIRT1 expression in ECs \[[@b9-medscimonit-25-8820],[@b10-medscimonit-25-8820],[@b13-medscimonit-25-8820]\]. SIRT1 expression is modulated at diverse levels, and previous studies have shown that post-transcriptional regulation is important in modifying SIRT1 expression. Previous reports have shown that the SIRT1 3′-untranslated region (3′ UTR) is critical for its mRNA stability. Additionally, a series of microRNAs (miRNAs), such as miR-34a, miR-23b-3p, and miR-132-3p, modulate SIRT1 expression \[[@b9-medscimonit-25-8820],[@b14-medscimonit-25-8820],[@b15-medscimonit-25-8820]\]. miRNAs are small noncoding RNAs (containing approximately 22 nucleotides) responsible for post-transcriptional modulation of gene expression and suppression of mRNA translation and stability, typically through binding to the 3′ UTR of target mRNAs \[[@b16-medscimonit-25-8820]\]. Several miRNAs have been found to exert a vital role in cardiac and endothelial function, which is associated with cardiovascular disease progression \[[@b17-medscimonit-25-8820],[@b18-medscimonit-25-8820]\]. miR-155, a typical miRNA with multiple functions, was recently identified as an element of the inflammatory signaling pathway in atherosclerosis \[[@b19-medscimonit-25-8820],[@b20-medscimonit-25-8820]\]. miR-155 regulates gene expression associated with inflammation in various cell types in vitro and affects atherogenesis in vivo \[[@b21-medscimonit-25-8820]--[@b26-medscimonit-25-8820]\]. miR-155 can enhance or suppress lesion formation at a range of atherosclerotic stages. Therefore, miR-155 may be linked to the pathogenesis of atherosclerosis \[[@b27-medscimonit-25-8820]\]. Recent evidence has demonstrated that some clinical drugs can modulate miRNA expression, suggesting that miRNAs may be therapeutic targets of these agents \[[@b9-medscimonit-25-8820]\]. Statins and sartan are widely applied to treat dyslipidemia and related vascular disorders. However, whether valsartan and simvastatin modulate the effects of proinflammatory cytokines on endothelial function is largely unknown. Despite the acknowledged importance of SIRT1 and miR-155 in the modulation of endothelial function, the relationship between them and the regulatory effects of inflammation on endothelial function during atherosclerosis remain unclear. Therefore, this study was designed to assess the possible association between SIRT1 and miR-155 and to determine how miR-155 regulates cardiovascular homeostasis. We found that simvastatin or valsartan can ameliorate TNF-α-induced HUVEC senescence through inhibition of miR-155. Material and Methods ==================== Endothelial cell culture ------------------------ Human umbilical vein endothelial cells (HUVECs) (ScienCell, CA, USA) were cultured in endothelial cell medium (ECM) (ScienCell) containing 5% FBS, 1% endothelial cell growth supplement (ECGS) (ScienCell), and penicillin/streptomycin and were cultured at 37˚C with 5% CO~2~ and 21% O~2~. ECs were seeded at a proper density in accordance with each experimental procedure. HUVECs were treated with ±TNF-α (20 ng/ml) (PeproTech, USA). miR-155 inhibition and overexpression ------------------------------------- To inhibit miR-155 expression, HUVECs at 50%--70% confluency were transfected using miR-155 inhibitor (GenePharma, Shanghai, China) or miRNA inhibitor negative control (NC) (50 nmol/l) for 36 h using Lipofectamine 3000 (Invitrogen, Life Technologies, NY, USA) in Opti-MEM, following the manufacturer's instructions. After transfection, Opti-MEM was exchanged for medium containing TNF-α, and cells were cultured for another 12 h. HUVECs were transfected using miR-155 mimic (GenePharma) to overexpress miR-155, or with NC mimic (50 nmol/l) for 48 h prior to additional analysis. Western blotting ---------------- After washing, cold RIPA with protease inhibitors (Roche) was used to lyse cells for 15 min. Then, after centrifugation, the cell supernatant was kept frozen at −80°C until use. Bradford assays (Bio-Rad Laboratories; CA, USA) were employed to assess protein concentrations. Samples (20 μg) were separated via SDS-PAGE, after which dry transfer to nitrocellulose membranes was conducted. After being blocked in 5% BSA for 1 h, primary antibodies were used to probe the membranes overnight at 4°C, including antibodies targeting human SIRT1, acetylated-p53 at K382 (Ac-p53), p21, forkhead box O1 (FoxO-1) (Cell Signaling Technology; USA), Ac-FoxO-1, total p53 (1: 2000 dilution), and senescence marker protein-30 (SMP-30) (all from Santa Cruz Biotechnology). β-actin served as an internal control and was purchased from Sigma Aldrich. After incubation with the appropriate secondary antibodies (1: 10 000 dilution; GE Healthcare, Buckinghamshire, UK), the immunostained protein bands were visualized with an ECL system (ProteinSimple; Santa Clara, USA), followed by quantification using ProteinSimple image software. All samples had 3 biological replicates, and each biological replicate was assayed in duplicate; therefore, the data for each time point are an average of 6 individual replicate runs. Representative images of the immunostained bands are presented in the figures. qPCR ---- TRIzol (Invitrogen) was used for EC total RNA extraction in accordance with standard protocols. A Nanodrop 2000 spectrophotometer (Invitrogen) was employed to determine RNA concentration and purity. cDNA was produced from 500 ng of total RNA utilizing a qScript microRNA Quantification System kit (microRNAs; Quanta Biosciences) or PrimeScript RT Master Mix (mRNAs; TaKaRa) in line with standard protocols. qPCR was conducted utilizing PerfeCTa SYBR Green SuperMix (microRNAs; Quanta Biosciences) and Power SYBR^®^ Green PCR Master Mix (mRNAs; Applied Biosystems; USA) using an ABI 7500 machine (Applied Biosystems). The primer sequences were commercially obtained from Invitrogen and were as follows (forward and reverse, respectively): 1. SIRT1, 5′-GCT GTG AAA TTA CTG CAA GAG TG-3′ and 2. 5′-AAT ACC ATC CCT TGA CCT GAA G -3′; 3. CYPA, 5′-TTG CTG TTC CTT AGA ATT TTG CC-3′ and 4. 5′-ACC CTG ACA CAT AAA CCC TG -3′; 5. miR-155, 5′-TTA ATG CTA ATC GTG ATA GGG GT-3′; 6. U6, 5′-GAT GAC ACG CAA ATT CGT G-3′. mRNA and miRNA expression were normalized to CYPA and U6, respectively. The ΔΔCt method was utilized to calculate fold changes in expression. All mRNA and miRNA samples had 3 biological replicates, and each biological replicate was also assayed in triplicate; therefore, the data for each time point are an average of 9 individual replicate runs. Luciferase reporter assay ------------------------- Firefly luciferase cDNA fused with the amplified 3′ UTR of human SIRT1 from a genomic DNA sample was cloned into psiCHECK-2 (Promega, USA). Luciferase in this vector was replaced by the produced wild-type (WT) and mutant 3′ UTR of SIRT1. HUVECs were added to 24-well plates, grown to 70--80% confluency, and then transfected using psiCHECK-2 that had been cloned so as to contain the WT or mutant S	{ "pile_set_name": "PubMed Central" }
All relevant data are within the manuscript and its Supporting Information files. Introduction {#sec001} ============ Swans are among the largest existing flying birds of the waterfowl family Anatidae. They are represented around the world (except in Africa and Antarctica) by six species from the genus Cygnus: four in the temperate and artic zones of North America and Eurasia (Cygnus olor, Cygnus buccinator, Cygnus cygnus and Cygnus columbianus), one in the temperate zones of South America (Cygnus melancoryphus) and another one in the south of Australia and New Zealand (Cygnus atratus) \[[@pone.0226331.ref001],[@pone.0226331.ref002]\]. Environmental aspects related to swans have triggered numerous conservation strategies around the world, for example the successful re-introduction of the trumpeter swan C. buccinator by wildlife American agencies after it was close to extinction during the 1930's \[[@pone.0226331.ref003],[@pone.0226331.ref004]\]. Currently, the presence, population abundances and reproductive success of swans have been used as proxies for environmental changes in threatened wetlands \[[@pone.0226331.ref005],[@pone.0226331.ref006],[@pone.0226331.ref007]\]. The black-necked swan C. melancoryphus is the only representative of the genus in South America, and nearly 100,000 swans \[[@pone.0226331.ref008]\] inhabit freshwater and coastal wetlands located between ca. 28°-52°S \[[@pone.0226331.ref009]\]. These water birds prefer habitats with abundant subaquatic banks of macrophytes serving as their primary food source. Thus, C. melancoryphus has been described as an herbivorous species \[[@pone.0226331.ref010],[@pone.0226331.ref011],[@pone.0226331.ref012]\], similar to all the other swans which are primarily vegetarians \[[@pone.0226331.ref001],[@pone.0226331.ref002]\]. Because of their low digestive efficiency, these birds dedicate nearly 50% of their daily activity to foraging \[[@pone.0226331.ref011],[@pone.0226331.ref012]\]. Therefore, the population abundances of herbivorous swans exert a significant foraging pressure over the aquatic macrophyte standing stocks as it has been shown for C. melancoryphus in Chile and Argentina \[[@pone.0226331.ref010],[@pone.0226331.ref013]\], C. atratus in eastern Australia \[[@pone.0226331.ref014]\], C. olor in eastern USA \[[@pone.0226331.ref015]\] and C. columbianus in Canada \[[@pone.0226331.ref016]\]. The Río Cruces wetland is one of the most well-known estuarine wetlands along the Chilean coast (ca. 39°S; [Fig 1](#pone.0226331.g001){ref-type="fig"}), characterized for its high diversity of aquatic macrophytes and water birds \[[@pone.0226331.ref017]\] and its tectonic origin \[[@pone.0226331.ref018]\]. In 1981, the central area of the wetland was declared the first Ramsar site in Chile (Ramsar site Santuario de la Naturaleza Carlos Anwandter, [https://www.ramsar.org](https://www.ramsar.org/)). Until 2004, the Río Cruces wetland was the main reproductive and nesting area of C. melancoryphus in South America \[[@pone.0226331.ref019]\], a fact probably related to the abundance of the aquatic macrophyte Egeria densa \[[@pone.0226331.ref020]\], which represents the primary food source for C. melancoryphus and other herbivorous water birds in this region such as coots (Fulica spp.) \[[@pone.0226331.ref021]\]. ![Geographic location of the Río Cruces wetland and its tributary rivers in southern Chile.\ The area of swan captures is highlighted in green. NL and SL: northern and southern limit of the wetland, respectively.](pone.0226331.g001){#pone.0226331.g001} Although C. melancoryphus has been described as a migratory species \[[@pone.0226331.ref022],[@pone.0226331.ref023],[@pone.0226331.ref024]\], the observed patterns of movement within the Río Cruces wetland characterize this water bird as rather resident \[[@pone.0226331.ref010],[@pone.0226331.ref019]\], similar to the behavior reported for swans from the northern hemisphere \[[@pone.0226331.ref025]\]. Between 1987 and 2003, the swan population of the Río Cruces wetland showed particularly high emigration rates with inter-annual variations between 2,000 and 12,000 birds \[[@pone.0226331.ref024]\], which were apparently related to large-scale climatic forcing by El Niño Southern Oscillation events (ENSO). However, after reaching an approximate number of 5,400 birds in early 2004, the swan population dropped to less than 600 birds during 2005 \[[@pone.0226331.ref026]\] due to massive migration. It was argued that the putative cause of that migration was an episodic change in water quality leading to the disappearance of E. densa from vast areas of the wetland, which was concomitant with the onset of production of a new pulp mill plant located nearly 25 km upstream from the wetland \[[@pone.0226331.ref027]\]. Since 2012, a gradual recovery of E. densa was observed across the wetland, followed by a gradual recovery of the population of swans \[[@pone.0226331.ref026],[@pone.0226331.ref027],[@pone.0226331.ref028]\] reaching numbers as high as 16,000 birds in early 2019 ([https://www.conaf.cl](https://www.conaf.cl/)). Thus, the variability in population abundance and permanence of black-necked swans within the Río Cruces wetland appears to be strongly coupled to the cover of the aquatic macrophyte E. densa. Consequently, the dietary composition of these water birds is expected to be a reliable proxy for temporal changes in the cover of the most common aquatic macrophytes in coastal wetlands. To test this hypothesis, we studied the diet of C. melancoryphus by analyzing their feces and the interannual variability of the most common macrophyte species in the Río Cruces wetland as derived from remote sensing data. Material and methods {#sec002} ==================== CV thanks the support from the Graduate School of Faculty of Sciences, Vicerrectoría de Investigación y Creación Artística (project I-2015-10) and Graduate Office of Universidad Austral de Chile (Assistance Scholarship, 2015--2016). FAL received funds from CONICYT PIA ANILLOS ACT172037. All the authors acknowledge the support of CONAF- Valdivia; especially the assistance in the field of Luis Miranda and Roberto Rosas and the logistical support of Mario Maturana (the administrator of the Ramsar site). The captures of the swans were authorized by the National Forest Corporation (Corporación Nacional Forestal) (resolutions N°01/2015 PCM/RAA; N°1/2016; N°327840/2016; N°417735/2016) and by the Agricultural and Livestock Service (Servicio Agrícola y Ganadero) (resolutions N°1786/2016; Nº3670/2016; N°255/2017) at the Agricultural Ministry of Chile (Ministerio de Agricultura). Study area {#sec003} ---------- The Río Cruces wetland is an extensive inundated area formed by co-seismic continental subsidence during the 1960 Valdivia earthquake with the largest magnitude Mw 9.5 ever recorded by seismic instruments \[[@pone.0226331.ref018]\]. During this event, the Río Cruces river banks were flooded to form an extensive area with shallow water levels (less than 2 m depth), which was colonized by subaquatic macrophyte banks dominated by E. densa \[[@pone.0226331.ref020]\]. Eight tributary rivers join the Río Cruces central axis forming together a wetland area of approximately 6000 ha ([Fig 1](#pone.0226331.g001){ref-type="fig"}). The wetland is an estuarial system characterized by a tidal variability between 40 and 50 cm \[[@pone.0226331.ref029]\]. The climate in this region is temperate and rainy, with precipitations between 1300 and 3500 mm per year and an annual cycle with minimum and maximum rainfall during January-March and May-August, respectively \[[@pone.0226331.ref017]\]. The diet of swans {#sec004} ----------------- The direct analysis of swan feces is the most common approach to study their dietary habits, probably because is not expensive and does not harm the birds. Other techniques include analyses of gut content \[[@pone.0226331	{ "pile_set_name": "PubMed Central" }
Introduction {#s1} ============ South Africa, a land of stark contrasts, contains a diverse natural beauty that can easily be compared with some of the world's most majestic outdoor scenes. One of the new seven wonders of the natural world, Table Mountain, parades its splendour to the capital of South Africa, Cape Town. Unfortunately, South Africa is also considered by many to be one of the tuberculosis (TB) capitals of the world. The incidence of TB in South Africa is estimated to have increased by over 400% in the past 15 years. This is confounded by a staggering co-infection rate of approximately 73% with the human immunodeficiency virus (HIV).[@R01] One of the most dreaded complications of TB pericarditis is pericardial scar formation. Due to scarring, the pericardium becomes calcified and contracts over the cardiac chambers, thereby encasing the heart in a fibrocalcific skin that impedes diastolic filling.[@R02] Constrictive pericarditis (CP) is the natural consequence of about 17 to 40% of cases of TB pericardial infection.[@R03] The definitive treatment of CP is surgical removal of the pericardium, a procedure with a significant peri-operative mortality rate of approximately 15%.[@R04] South Africa is on the forefront of research on TB heart disease and has recently published the large, multi-centre IMPI trial.[@R05] One of the goals of the IMPI trial was to assess the impact of corticosteroids in the management of TB pericarditis. The major findings of the study included (1) corticosteroids had no impact on mortality rates in patients with TB pericarditis, (2) corticosteroids decreased the incidence of pericardial constriction by 46%, and (3) HIV-positive patients who received corticosteroids had a significantly increased risk of developing HIV-associated malignancies. In established TB, early and effective treatment with shortcourse anti-TB therapy is the mainstay of management. Various strategies have been investigated as adjuncts to anti-TB drugs in the prevention of pericardial constriction. The ongoing discussions and numerous investigations into a wide array of agents as possible 'magic bullets' in the prevention of pericardial constriction (post-TB infection) illustrates both the interest in the field, and also the lack of a satisfying solution to this problem. The following strategies have previously been evaluated: Mycobacterium indicus pranii immunotherapy,[@R05] corticosteroids,[@R05] pericardiocentesis,6 open surgical drainage (pericardial window),[@R07] thalidomide,[@R08] instilling intrapericardial fibrinolytic therapies,[@R09]-[@R11] and a wide array of non-steroidal anti-inflammatory medication. Not one of these therapies has, to date, been internationally recognised as an acceptable standard of therapy, and the choice of adjuvant treatment varies significantly among experts in the field. Colchicine is an inhibitor of microtubule polymerisation. It acts by binding to tubulin and is registered for the acute treatment of gout crystal arthropathies. The plant source of colchicine, the autumn crocus (Colchicum autumnale), was described as treatment for arthritis in the Ebers Papyrus in 1500 BC.[@R12] In modern medicine, colchicine has however played a wider role in the treatment of pericarditis of various aetiologies, both acute and chronic. This has been investigated in a prospective, randomised trial named COPE (Colchicine for Acute Pericarditis),13 and the major findings concluded that colchicine significantly reduced the recurrence rates and symptom persistence due to pericarditis. To date however, the use of colchicine has, to the best of our knowledge, never been systematically assessed in the context of pericardial TB. The purpose of this research was to assess the merit for the use of colchicine in the context of TB pericarditis. Methods {#s2a} ======= This research was conducted in the Northern Cape province of South Africa at a secondary-level hospital in Kimberley between August 2013 and April 2015. The research was approved by the ethics committee of the University of the Free State and the study was registered with the National Health Research Committee. The research was conducted in accordance with the Declaration of Helsinki. This pilot study was designed as a prospective, doubleblind, randomised, control cohort. All patients presenting to the Kimberley Hospital complex (KHC) with pericardial effusions were assessed for inclusion and exclusion criteria. In the absence of contra-indications, patients underwent therapeutic pericardiocentesis if the procedure was deemed safe and possible. Standard therapy was initiated in accordance with the South African National Tuberculosis Management Guidelines:14 weight-adjusted anti-TB drugs (Rifafourf^®^) and oral corticosteroids. (prednisone: 1.5 mg/kg per day for four weeks; 1.0 mg/kg per day for two weeks; 0.5 mg/kg per day for one week; 0.25 mg/kg per day for one week). HIV co-infected patients not previously on treatment were initiated on fixeddose combination (FDC) antiretroviral treatment six weeks after initiation of TB treatment (FDC: Tenofovir Disoproxil Fumarate 300 mg, Emtricitabine 200 mg and Efavirenz 600 mg). Patients were randomly assigned to the intervention group with the use of a web-based randomisation system that ensured assignment concealment. The intervention group received colchicine (dose 1.0 mg per day) for a total of six weeks, whereas the control group received a placebo for the same period ([Fig](#F1){ref-type="fig"}. 1). ![Flow diagram illustrating study methodology.](cvja-27-351-g001){#F1} Patients subsequently underwent serial echocardiographic examinations on an out-patient basis and adherence checks, including pill counts, were done at follow-up visits. The primary outcome assessed was the development of pericardial constriction and this diagnosis was made echocardiographically at four months post initial presentation. Upon completion of the follow-up period of all patients, the blinding was unveiled and data were presented for statistical analysis. Two groups of patients were included: (1) definite TB pericarditis: the presence of TB bacilli was observed on microscopic examination of pericardial fluid; cultures of pericardial fluid were positive for Rifampicin-sensitive Mycobacterium tuberculosis (MTB); pericardial fluid was positive for MTB on direct polymerase chain reaction (PCR) (Gene Xpert); and (2) probable TB pericarditis: proof of TB was found elsewhere (positive cultures for MTB on sputum or cerebrospinal fluid); pericardial fluid with adenine deaminase (ADA) level \> 40 U/l; a total diagnostic index score \> 6 on using the Tygerberg clinical prediction score ([Table 1](#T1){ref-type="table"}).[@R15] ###### The Tygerberg clinical prediction score for the diagnosis of TB pericarditis. A total diagnostic score \> 6 yields a sensitivity of 82% and a specificity of 76% for the diagnosis of TB pericarditis Admission variable Diagnostic index ----------------------------- -------------------- Weight loss 1 Night sweats 1 Fever 2 Serum globulin \> 40 g/l 3 Leukocyte count \< 10 × 109 3 The exclusion criteria were: patients with renal or hepatic impairment (creatinine clearance rate \> 85 ml/min or transaminases \> 1.5 upper limit of normal); and pregnant patients or patients intending to become pregnant within four months. The gold-standard diagnostic test for the diagnosis of CP is the demonstration of increased interventricular interdependence during cardiac catheterisation. Doppler echocardiography and other novel echocardiographic techniques have provided us with reliable non-invasive alternatives to the diagnosis of CP. In this study, the diagnosis of CP was made by means of echocardiography by adhering to the principles in the article by Dal-Bianco et al. on the echocardiographic diagnosis of CP.[@R16] Initial echocardiographic assessment ensured that no features of constriction were present at the time of enrolment in the study. Follow-up echocardiograms were performed four months after the initiation of therapy. The echocardiograms were performed and co-reviewed by two experienced echocardiographers (who had both attended a dedicated workshop at a tertiary-level academic hospital aimed at the echocardiographic diagnosis of CP). A GE Vivid E6® ultrasound machine was used to perform a systematic examination according to the basic minimum standards as stipulated by the British Society of Echocardiography.[@R17] Numerous other echocardiographic parameters were assessed, including the presence of a septal shudder, respiratophasic septal shift, left atrial enlargement and echocardiographic features of pericardial thickening ([Figs 2](#F2){ref-type="fig"}--[4](#F4){ref-type="fig"}). ![A. Tissue Doppler of the medial aspect of the mitral valve annulus demonstrating early diastolic tissue velocity of 0.14 m/s. B. Tissue Doppler of the lateral aspect of the mitral valve annulus showing early diastolic tissue velocity of 0.12 m/s. The lower tissue velocity on the lateral aspect is the opposite of the normal phenomenon (annulus reversus).](cvja-27-352-g002){#F2} ![Pulse-wave Doppler at the level of the mitral valve leaflet tips demonstrating a respiratophasic variation in the early diastolic transmitral inflow velocities in excess of 25%.](cvja-27-352-g003){#F3} ![Dilated and distended inferior vena cava. No respiratory variation	{ "pile_set_name": "PubMed Central" }
[^1]: Session: 167. Preclinical Study with New Antibiotics and Antifungals Friday, October 6, 2017: 12:30 PM	{ "pile_set_name": "PubMed Central" }
The management of trauma and other orthopedic diseases has progressed by leaps and bounds during the last 40 years. Similarly, the cost of treatment has increased proportionately with the advent of joint replacement and of course costly trauma implants. India is a land of contrasts where there are 5-star hospital treatment facilities as well as quacks treating everything in villages and smaller towns. We do not have enough resources to treat all patients to the Western standards even if it may be desirable. In India, we need more of the options which will restore the function, are less costly and more dependent on biology rather than implants. This book does exactly the same. Prof. Tuli, in his inimitable way, has described various biological options for these conditions. Book is written in lucid and clear language. He has given several useful tips and practical solutions for complex problems. The basics and fundamental principles have been brought out well. Many useful but forgotten operations such as Huntington\'s procedure, Papineau\'s bone grafting, and pantalar arthrodesis, which give functional results, have been well described. The book is divided into two sections. Each chapter is divided into headings and subheadings. Each chapter is giving key points of the condition. The chapter on bone and joints infection has been written with an aim to emphasize the importance of early diagnosis and treatment. Some common congenital dysplasias and deformities and common tumors have also been discussed. Chapter 10 discusses poliomyelitis whose new cases are not seen, but the old cases may still be there. Section 2 incorporates regional orthopedic conditions where several common painful but common conditions are discussed. Glenoplasty for recurrent dislocation of shoulder, which was popularized by the author also gets a place in this book. This book is recommended for young and practicing orthopedic surgeons. It will also be a useful revision book for postgraduate students.	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1-metabolites-07-00038} =============== Duchene muscular dystrophy (DMD) is a neuromuscular disorder characterized by progressive loss of muscle mass that renders the mobility of its victims to wheelchair before the age of 12 \[[@B1-metabolites-07-00038]\]. Genetically, X-linked Duchenne muscular dystrophy is caused by the absence of dystrophin protein and is a lethal inherited disorder that mainly affects male. The disease progresses with a gradual development of respiratory insufficiency, cardiomyopathy, and skeletal muscle weakness, frequently causing death by the late teens or early twenties \[[@B2-metabolites-07-00038],[@B3-metabolites-07-00038]\]. Of the three naturally-occurring mammalian (murine, feline, and canine) DMD models \[[@B4-metabolites-07-00038]\], in addition to the two non-mammalian (zebrafish and Caenorhabditis elegans) models \[[@B5-metabolites-07-00038]\], the canine models are thought to be the most suitable ones for studies because of their pathological resemblance and clinical relevance to human patients of DMD \[[@B5-metabolites-07-00038],[@B6-metabolites-07-00038]\]. Like DMD, the genetically homologous Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, differential weakness of flexor and extensor muscles muscles, and mixed atrophy and hypertrophy of specific skeletal muscles \[[@B7-metabolites-07-00038],[@B8-metabolites-07-00038]\]. The GRMD phenotype includes marked atrophy of the long digital extensor (LDE) as compared to sibling unaffected normal dogs, while the biceps femoris (BF), a member of the hamstring muscle group, undergoes less pronounced atrophy \[[@B7-metabolites-07-00038],[@B8-metabolites-07-00038]\]. Recent studies have begun to implicate metabolic defects in muscular dystrophies, including DMD. Qualitative and quantitative lipid analysis of DMD, Becker's Muscular Dystrophy, facioscapulohumeral muscular dystrophy (FSHD), and limb girdle muscular dystrophy-2B (LGMD-2B) patient biopsies found significant increases in glycogen \[[@B9-metabolites-07-00038]\]. Fatty acids were also altered in DMD, BMD, FSHD, and LGMD-2B, although DMD biopsies exhibited unique increases in cholesterol in DMD patients \[[@B9-metabolites-07-00038]\]. A recent microarray analysis of GRMD muscle identified the first evidence of potential alterations in metabolism, specifically alterations in genes associated with lipid metabolism and energy production \[[@B10-metabolites-07-00038]\]. Impairment of bioenergetics has recently been reported in congenital muscle dystrophy Type 1A (MDC1A) and Leigh Syndrome, linking metabolism to disturbances in skeletal muscle cell apoptosis \[[@B11-metabolites-07-00038]\]. In the present study, we undertook a non-targeted metabolomics analysis to determine how GRMD skeletal muscle compared to age-matched control muscle metabolically using gas chromatography-mass spectrometry (GC-MS) to identify underlying metabolic defects specific for GRMD skeletal muscle. 2. Results {#sec2-metabolites-07-00038} ========== 2.1. Determination of Metabolomics Changes in the GRMD Biceps Femoris vs. Control (t-Test) {#sec2dot1-metabolites-07-00038} ------------------------------------------------------------------------------------------ The first statistical analysis we ran was designed to determine if there were significant alterations in the GRMD BF muscle compared to controls. To do this, we analyzed biopsy samples from GRMD-affected (and non-affected sibling) dogs at 6 months of age, at which time phenotypic changes have occurred \[[@B7-metabolites-07-00038],[@B8-metabolites-07-00038]\]. Previous studies identified marked atrophy of the LDE compared to sibling unaffected normal dogs, while the BF, a member of the hamstring muscle group, undergoes less pronounced atrophy \[[@B7-metabolites-07-00038],[@B8-metabolites-07-00038]\]. The limited number of samples run (6 GRMD and 4 controls) are due to the cost of housing and maintaining ill dogs for 6 months and the limited number of animals in the colony. A t-test was then performed on the metabolites to determine BF metabolites affected. Analysis of the less atrophied, early stage disease BF yielded 75 metabolites muscle ([Figure S1A](#app1-metabolites-07-00038){ref-type="app"}), including 19 imputed ([Figure S1B](#app1-metabolites-07-00038){ref-type="app"}), which upon partial least squares discriminant analysis (PLS-DA) produced separation of the control and GRMD groups from the first two components ([Figure 1](#metabolites-07-00038-f001){ref-type="fig"}A). PLS-DA VIP analysis identified pyrophosphate (VIP Score 2.65) and stearamide (VIP Score 2.24) with the highest contributions to this signature ([Figure 1](#metabolites-07-00038-f001){ref-type="fig"}B). t-Test analysis of the two groups identified eight significant metabolites (carnosine, fumaric acid, steramide, myoinositol-2-phosphate, lactamide, proline, oleic acid, glutamic acid, [Figure 1](#metabolites-07-00038-f001){ref-type="fig"}C). We next investigated what these eight significant metabolites had in common in terms of metabolic pathways. To do this, we determined their enrichment, or likelihood of being significantly altered by random chance, in metabolic pathways. Pathway analysis of these eight significant metabolites in GRMD BF identified four affected pathways, including: (1) arginine and proline metabolism; (2) alanine, aspartic acid, and glutamic acid metabolism; (3) butanoate metabolism, and (4) histidine metabolism, with false discovery rates (FDR) of 5--9% ([Figure 2](#metabolites-07-00038-f002){ref-type="fig"}A). The FDR estimates the likelihood that a conclusion that a relationship exists, whereas in reality it does not for an individual comparison \[[@B12-metabolites-07-00038]\]. Its use in the the current context represents the likelihood that the pathways identified as significantly enriched may be false and is generally set at 5%. In the current studies, we have presented data with just higher FDR, as we are necessarily analyzing a smaller number of biological biopsies. Enrichment analysis of the BF t-test significant metabolites against a disease-associated metabolite set (blood) identified \> 20-fold enrichment in histidine metabolism and alanine metabolism ([Figure 2](#metabolites-07-00038-f002){ref-type="fig"}B). Enrichment of arginine and proline metabolism had the lowest p value (p = 5.88 × 10^−4^, FDR = 4.7 × 10^−2^) enriched \> 15-fold ([Figure 2](#metabolites-07-00038-f002){ref-type="fig"}B). To further identify metabolic pathways to which our significant metabolites may be related, we compared them to changes in other disease sets, including urine, CSF, and location-based sets for further clues to the metabolic pathways affected. Metabolic enrichment BF t-test significant metabolites were analyzed against disease-associated metabolite sets (urine, CSF) and location-based metabolite sets and identified nearly 80-fold enrichment for carnosinuria/carnosinemia ([Figure S2A](#app1-metabolites-07-00038){ref-type="app"}), \> 10-fold for spinocerebellar degeneration ([Figure S2B](#app1-metabolites-07-00038){ref-type="app"}), and 5-fold for skeletal muscle ([Figure S2C](#app1-metabolites-07-00038){ref-type="app"}). Of the eight (8) t-test significant metabolites, five (5) were significantly decreased (stearamide, carnosine, fumaric acid, lactamide, and myoinositol-2-phosphate), while three (3) were significantly increased (oleic acid, glutamic acid, and proline) ([Figure 2](#metabolites-07-00038-f002){ref-type="fig"}C). 2.2. GRMD vs. Control Long Digital Extensor Muscle vs. Control (t-Test) {#sec2dot2-metabolites-07-00038} ----------------------------------------------------------------------- The second statistical analysis we ran was designed to determine if there were significant alterations in the more atrophied, later stage disease GRMD LDE compared to controls. The LDE functions to flex the tibiotarsal joint and also serves as a digital extensor \[[@B13-metabolites-07-00038]\] and is significantly atrophied in GRMD to a greater extent than the BF muscle \[[@B7-metabolites-07-00038],[@B8-metabolites-07-00038]\]. In this analysis, we compared biopsy samples from GRMD-affected and non-affected sibling LDE samples at 6 months of age and performed a t-test	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== According to the provisions of the current European Medical Device Directive 93/42/EEC \[[@CR1], [@CR2]\] and the new European Medical Device Regulation (applicable as from May 2020) \[[@CR3]\], each manufacturer of medical devices has to set up a comprehensive system in order to identify, evaluate and integrate clinical data derived from the field application of a medical device after market access during post-market surveillance (PMS). Small and medium-sized enterprises in the field of medical devices are in need for operable systems for post-market data retrieval in order to enhance their PMS strategies and to be prepared for the growing requirements of the new European Medical Device Regulation. A wide range of both internal (own quality management and compliant system) and external (scientific databases, medical congresses, internet-based knowledge & experiences, PMS by competent authorities) sources have to be monitored in order to integrate all accessible data about a medical device's safety and performance. Currently, these detailed, continuous searches are still performed manually with a high input of time and personnel resources, making PMS a daunting task. Literally, an employee has to type all search queries (e.g., "safety AND coronary stent") into the input fields of a large number of different databases, congress sites and public search engines in order to gain a broad, unspecific hit list, interspersed with single, relevant content. This strategy of data retrieval reaches its limits assuming that several search strings have to be applied in order to monitor a whole range of medical devices, each featured by a variety of decisive search questions. Additionally, each notable medical database uses its own, inherent syntax to specify search queries. This incompatibility between databases and the amount of different search tasks combined with the manual application to a multitude of databases results in low efficiency and a high potential for human error. Setting up more complex queries in a simple manner by domain experts enables the definition of the topic of interest in a more specific way and circumvents the problem of retrieving irrelevant content. In the OntoVigilance project (predecessor project of OntoPMS), we developed the Search Ontology (SON) v1.0 \[[@CR4]\], a promising approach for an ontology-based specification of complex search queries. The modular architecture of the SON enables the re-use of ontology parts in different use cases as well as a quick and easy adaptation and extension of the ontology according to the specific requirements. The developed Search Ontology and its application was evaluated in the OntoVigilance project by domain experts. This previous study has proven that the SON is a suitable method for modelling complex queries, but it needed to be optimized to face further requirements of domain experts. On the one hand, the structure of the Search Ontology can be simplified to improve the usability by domain experts. On the other hand, certain extensions are necessary to model all relevant query types. Based on these findings, we further developed and optimized the Search Ontology in the OntoPMS project. The Search Ontology v2.0 is presented in this paper. The SON is generic and can be used in any domain. For the application of the SON in a particular domain, it has to be extended by a domain ontology, such that the classes of the domain ontology are subclasses of the SON classes. We call such domain ontologies Domain-specific Search Ontologies (dSON). Whereas SON stands for exactly one ontology (namely the Search Ontology presented in this paper), dSON represents a class or a type of ontologies. Various Domain-specific Search Ontologies can be developed, such as dSON-PMS for the whole PMS domain or dSON-Niti for modelling queries regarding the material Nitinol. In addition, we developed an Excel template to specify the information required to create a dSON, which significantly simplifies the ontology development by domain experts. For the automatic generation of a dSON from the Excel template, we implemented the Search Ontology Generator (SONG). In contrast to the OntoQueryBuilder (OQB) \[[@CR4]\], the SONG generates the complete dSON including all specified queries in the correct query syntax from the Excel template and provides it for external tools (e.g., the search engine). In this way, the search engine can get the complete dSON by accessing the SONG service without any requests or generating queries at search time. Methods {#Sec2} ======= In Europe, market access of a medical device based on a CE-mark is granted after a successful so-called "conformity assessment process", which includes passing an extensive series of tests, risks analyses and evaluations of clinical data on the medical device's safety and efficacy. Nevertheless, the behaviour of a medical device over time in broad application can be investigated a priori only in a limited manner. Thus, PMS strategies are set up in order to retrieve and summarize application data of medical devices and to identify residual risks. Expressive search queries are needed to precisely define the topic of interest. The problem is that the manual creation of complex queries requires knowledge of the correct query syntax and is time-consuming and error-prone. This paper focusses on developing the Search Ontology Generator (SONG), a framework for ontology-based specification and generation of powerful search queries by domain experts with less effort and without knowing the query syntax. Example PMS question {#Sec3} -------------------- Reports on unfavourable interactions between implant material and patient's tissue have to be identified and evaluated in order to a) control residual and/or unexpected risk, b) determine vulnerable patient subpopulations and c) improve the respective medical implant or material, respectively. An example PMS question could be to find out the unexpected side effects of the metal alloy Nitinol used for construction of endoscopic clipping systems. A search query has to be constructed covering the different aspects of the PMS question such as "unexpected complication", "type of medical device" (endoscopic clipping system) and "used material" (Nitinol) by suitable search terms (e.g., "Nitinol", "Nickel Titanium" and "NiTi" as synonyms of Nitinol or "unexpected complication", "unforeseeable risk", "adverse event", etc. to describe the complication). Furthermore, it can be necessary to specify terms that should not appear in the text (negation), for instance, to exclude descriptions of preclinical tests or studies (e.g., terms like "animal", "study" and "preclinical"). Finally, the desired terms have to be assembled to a valid search query using supported operators and brackets. This example is used in the paper for further explanations. Approach {#Sec4} -------- Figure [1](#Fig1){ref-type="fig"} illustrates the overall architecture of the SONG environment.Fig. 1SONG and its application. The Domain Expert specifies the dSON in Excel. The SONG Manager uploads/downloads files and tests the service. The SONG Service generates the dSON (including complete search queries) and offers different methods for external applications. The Searcher uses the SONG Search App and can select desired concepts or queries for the execution by a search engine The Domain Expert specifies information required to generate a dSON (including search queries) using an easily applicable Excel template. The SONG Manager uploads/downloads files and tests the service using the SONG Config App. The SONG service generates the dSON (in our example the dSON-Niti) in OWL and JSON format out of the Excel template, allows adding new entities to the ontology and provides the generated dSON for external tools, especially for search apps. After each file upload (Excel or OWL) or after an adding of a new entity, the new ontology (OWL and JSON) is generated. The SONG manages the generation of OWL and JSON from Excel as well as JSON from OWL. The OWL format is used for a possible optimization of the generated ontology with an ontology editor or for an integration of external ontologies. Any ontology that contains concepts and their labels can be considered as a dSON and can be easily integrated with our approach. JSON is utilized for communication with external tools. The Searcher uses the SONG Search App, which visualizes the dSON, and can select desired concepts or queries for the execution by a search engine. The next sections introduce the two additional components, search engine and Corpus Builder that were used in the OntoPMS project in combination with SONG. ### Search engine {#Sec5} The SONG can be used with any Lucene-based search engine for the generation of queries in the Lucene query syntax out of the Excel template. In addition, new expressive query operators were implemented in the OntoPMS project, which significantly extend the Lucene query syntax. To identify risks or complications (for PMS) in unstructured documents, complex patterns have to be detected. Such patterns go beyond the standard capabilities of state-of-the-art search engines such as Elasticsearch \[[@CR5]\]. Therefore, we extended these capabilities by creating our own search plugin, providing the required functionalities and improving search quality. The extension was realized as an Elasticsearch plugin and contains, among others, the following additional features: improved tokenization, lemmatization and word decomposition; build-in support for several normal forms / term types; improved quality for ambiguous searches; named entity, date and measurement recognition; additional search modes; NEAR operators. In particular, the search modes and new search operators are extensively used in our dSONs to produce more precise queries. The search modes correspond to	{ "pile_set_name": "PubMed Central" }
	{ "pile_set_name": "PubMed Central" }
Introduction {#S1} ============ Fruits and vegetables are known to have high nutrient content, making them the basis of a healthy diet. Many of these foods can be eaten raw, and although this represents a practical advantage, it also makes them notoriously relevant to foodborne illnesses. Salmonella enterica is one of the most common human pathogens found in fresh produce ([@B3]; [@B24]). Previously, plants were thought to be passive vectors for human pathogens, but recent studies showed that S. enterica can induce plant defense responses ([@B26]; [@B10]; [@B25]; [@B27]). Intriguingly, although the mechanism is not fully understood, this bacterium can overcome plant defense ([@B31]; [@B32]) and survive for weeks inside diverse plants species, including lettuce (Lactuca sativa L.) ([@B13]; [@B21], [@B20]; [@B16]; [@B30]). These findings have prompted further research on the interaction between plants and human pathogens. Artificial inoculation of plants is a common technique used to study plant interaction with phytopathogens ([@B17]; [@B15]). Nevertheless, this approach has some technical limitations when studying plant interaction with enterobacteria, in particular S. enterica and enterohemorrhagic Escherichia coli, due to the relative low number of these bacteria inside the plant. In fact, recent studies have shown that S. enterica population can decrease with time in many plant species in an inoculum concentration-dependent manner ([@B8]; [@B16]). Beyond that, the plant species and the inoculation procedure itself can affect bacterial population dynamics inside plants. For instance, tomato (Solanum lycopersicum) seedlings dip-inoculated with S. enterica at a concentration of 8 Log CFU ml^--1^ maintains the population size 1 day after inoculation (DAI) followed by a decrease after 14 DAI ([@B2]). Similarly, when adult lettuce leaves were dip-inoculated with 8 Log CFU ml^--1^ of S. enterica, the Log CFU cm^--2^ of leaf showed no alteration in bacterial population until three DAI, but a reduction in the population size after 7 DAI ([@B30]). Nonetheless, when lower inoculum concentration of 4.7 Log CFU ml^--1^ of S. enterica was used to infiltrate small areas of fully expanded Nicotiana benthamiana leaves, a 100-fold increase in bacterial population was observed at three DAI ([@B26]). These findings indicate that the inoculation method and/or the initial concentration of the inoculum can influence the bacterial population dynamic in leaves. In the field, plants can be exposed to variable amounts of pathogen load depending on the source of the inoculum. In a survey to quantify Salmonella in irrigation water, [@B1] found an average of 0.03 MPN (most probable number) of cells per 100 ml of water. Additionally, animals are reservoirs of bacterial pathogens of humans and might shed high level of inoculum in their feces. For instance, cattle feces can shed E. coli O157 at concentrations \>4 Log CFU g^--1^ ([@B28]), whereas some animals such as mice are considered super-shedders of S. Typhimurium ([@B11]). Once crops are exposed to these environmental inocula, bacterial cells can internalize into edible leaves through natural openings and wounds ([@B4]; [@B21]; [@B31]). Understanding human pathogen survival inside the leaf apoplast is very important as this niche protects the bacterium from common sanitation procedures of leafy vegetables ([@B29]), posing a risk to reach the human host. Thus, we performed vacuum infiltration procedures using a range of low to high concentrations of bacterial inoculum (3--7 Log CFU ml^--1^) to assess the effect of inoculation dose on bacterial survival and the detection limit of our procedure using contrasting lettuce cultivars over a period of 20 days. The findings of this study will assist with designing of plant phenotypic screening useful for breeding programs. Materials and Methods {#S2} ===================== Plant Material and Growth Conditions {#S2.SS1} ------------------------------------ Approximately 15 lettuce seeds of each cultivar (Red Tide, Lollo Rossa, and Salinas) were germinated in water-soaked filter paper for 2 days at room temperature. Each germinated seed was transplanted to a peat pot pre-soaked with distilled water for 10--20 min. Ideally, sprouted seeds with approximately the same root size should be selected for transplanting. Pots were placed in trays covered with plastic dome, leaving a small space (around 4 cm) to avoid water condensation, and kept at 18 ± 2°C, 240 ± 10 μmol m^--2^ s^--1^ with a 12-h photoperiod, and 80 ± 10% of air relative humidity. One week after transplanting, seedlings were fertilized with 0.05 g of fertilizer per plant mixed with 30 ml of distilled water. Three- to 4-week-old plants were used for inoculation ([Figure 1](#F1){ref-type="fig"}). ![Flowchart of the procedure. Please see the section "Materials and Methods" for a detailed description.](fmicb-11-01516-g001){#F1} Bacterial Inoculum Preparation {#S2.SS2} ------------------------------ S. enterica subsp. enterica serovar Typhimurium strain 14028s was streaked from frozen glycerol culture stock on low-salt Luria Bertani (LSLB) agar plate, supplemented with 60 μg ml^--1^ kanamycin, and incubated overnight at 28°C. Late in the afternoon of the day before the inoculation assay (around 5 pm), one single colony was placed in 100 ml of LSLB medium with 60 μg ml^--1^ kanamycin in a 125-ml Erlenmeyer flask. As a blank control, 5 ml of the LSLB plus antibiotic solution was placed into a clean culture tube. Bacterial and blank solutions were incubated in a rotary shaker at 28°C, 150 rpm, overnight ([Figure 1](#F1){ref-type="fig"}). In the morning of the next day, bacterial and blank solutions were removed from the incubator and the optical density at 600-nm wavelength (OD~600~) was measured using a spectrophotometer. It is important to shake the culture flask before transferring 1 ml to a sterile cuvette to avoid errors during OD readings due to bacterial settling on the bottom of the flask. The OD~600~ should be between 0.8 and 1.0 to ensure that the bacterial growth is still in the log phase. A two-step bacterial dilution was used to prepare the final inoculum at the desired concentration. Step 1: the volume of the bacterial solution needed to obtain a bacterial OD~600~ of 0.2 was calculated using the formula C1 × V1 = C2 × V2, where C = concentration and V = volume. After transferring the desired bacterial solution volume (V2) to a 50-ml centrifuge tube, bacterial cells were harvested by centrifugation at 1,200 × g for 20 min at 22 ± 2°C. The supernatant was discarded, and the pellet was resuspended in sterile distilled water by vortexing. The centrifugation step was essential to remove the excess of LBLS media plus kanamycin, to avoid bacterial growth inhibition within the leaf due to the presence of the antibiotic, as well as to reduce the volume of Salmonella solution handled in the lab. Step 2: 0.0001, 0.01, 1, or 100 ml of the final solution from step 1 (OD~600~ = 0.2) was added to a new flask containing 1,000 ml of sterile distilled water to obtain the final inoculum concentration of OD~600~ 0.00000002 (1 Log CFU ml^--1^), 0.000002 (3 Log CFU ml^--1^), 0.0002 (5 Log CFU ml^--1^), or 0.02 (7 Log CFU ml^--1^). Finally, 0.1 ml of Silwet was added to the inoculum to obtain a final concentration of 0.01%. Inoculum was stirred with a magnetic bar. Vacuum Inoculation of Lettuce Leaves {#S2.SS3} ------------------------------------ Three- to four-week-old lettuce plants (four plants per cultivar) were vacuum-infiltrated with the final bacterial solution of 1.8, 3.5, 5.4, or 7.7 Log CFU ml^--1^. These concentration values were estimated by serial dilution plating of the inoculum. Each potted plant was placed upside-down into a 400-ml beaker containing enough inoculum to immerse the plant shoot completely. Aluminum foil was placed at the base of the plant to avoid the contact of soil with the inoculum. Submerged plants were placed in a vacuum chamber and vacuum was applied for 1 min. To enable a uniform filling of the leaf apoplast with inoculum, the vacuum was released quickly by disconnecting the suction tube to the vacuum chamber, allowing the chamber to depressurize. The leaves should become dark green due to inoculum infiltration ([Figure 1](#F1){ref-type="fig"}). Fresh inoculum was added to the beaker to ensure total immersion of the inoculated leaves and after three plants were inoculated. Inoculated plants were placed back in the trays and partially covered with the plastic dome for the duration of the experiment. Enumeration of Apoplastic Bacterium Population {#S2.SS4} ---------------------------------------------- Bacterial population size was estimated in the second true leaf of the inoculated plants at 2 h post inoculation (	{ "pile_set_name": "PubMed Central" }
Erratum {#Sec1} ======= During production of the original article \[[@CR1]\], a document supplied by the author containing a set of further corrections to the manuscript was overlooked. The article therefore published containing a number of errors which are detailed here. The publisher would like to apologise for these errors, which have all been corrected in the original article. In the 'Results' section of the abstract, the number of responses received to the questionnaire was incorrectly given as 1698. This should have been 1693, as was correctly reported throughout the article itself. The first names of all authors except Martin Ho Yin Wong were mistakenly abbreviated to a first initial. The original article has been updated with the correct, full first names, as they appear in this erratum. The affiliation of Hannah Fosker was incorrect, and has now been updated to: Leicestershire Partnership NHS Trust, Bradgate Mental Health Unit, Glenfield Hospital, Groby Road, Leicester, LE3 9EJ, UK. In the original article, this has been inserted as affiliation 3, and subsequent affiliations have been renumbered to reflect this. The affiliation of Alec Knight has also been amended to the correct address: King's Improvement Science, Health Service and Population Research Department, Institute of Psychiatry, Psychology & Neuroscience, King's College London, IoPPN Main Building, London SE5 8AF, UK. The affiliation of Monica Lakhanpaul has been changed from affiliation 7 (Department of Primary Care and Public Health Sciences, King's College London) to affiliation 1 (Population, Policy and Practice, UCL Institute of Child Health). In the Acknowledgements section, it was stated that: "AK was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care South London at King's College Hospital NHS Foundation Trust." This sentence should have included Peter Littlejohns, to read: "PL and AK were supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care South London at King's College Hospital NHS Foundation Trust." Finally, there were minor formatting errors throughout the article, mainly involving the inconsistent use of hyphens, which have now been corrected. The online version of the original article can be found under doi:10.1186/s12909-015-0510-3.	{ "pile_set_name": "PubMed Central" }
INTRODUCTION ============ The way science is done or everyday practice of science is a major theme of Fred Grinnell\'s most recent work. The author presents a critique of the linear model of science followed by many scientists and its failure to represent how scientists really work. It is not a surprising observation to most of us. What is surprising about this book is the way it provides various avenues to engage in productive discussions about doing research. It is not a perfect book, but it manages to be provocative and an easy read with only six chapters in fewer than 200 pages. The book is divided into two sections: "Science" and "Science and Society." Grinnell presents a candid account of the scientific process to make it relevant to those outside academic science. He also provides a fairly accurate view of scientists and how they work. THE PRACTICE OF SCIENCE ======================= In chapter 1, "Practicing Science," Grinnell highlights what he calls the two conversations of science by illustrating the interaction of two processes: the circle of discovery and the circle of credibility (p. 5, Figure 1.1). He is quick to point out that researchers interact with a small part of the world, limiting those interactions to making observations and doing experiments. The interactions within the research community are primarily collaborative and competitive in nature. As Grinnell progresses in his discussion of these interactions, he reveals some of the uncertainties and conflicts that emerge during the practice of science. Chapter 2, "Discovery: Learning New Things about the World," deals with the nonsystematic, nonlinear nature of the scientific process. For instance, Grinnell points out that scientific papers rarely describe scientific failure and rarely communicate to students the notion that "10 research notebooks' worth of experiments might be required to publish a 10-page research paper" (p. 21). This is an interesting assertion that indicates to the nonscientist reader the level of compression that goes on when myriad observations, deductions, and analyses are meshed into a coherent scientific paper. Another interesting idea presented in this chapter deals with the type of question(s) a research group decides to answer. Very often, Grinnell adds, "resource limitations...prevent new initiatives from starting even if they would be worthwhile to carry out. Investing in one project almost always means that something else will not be accomplished" (p. 35). This statement is important, as it calls to the attention of future researchers the various factors that control the nature of everyday practice of science. Chapter 3, "Credibility: Validating Discovery Claims," makes a case for the uncertainty that surrounds achieving credibility. Grinnell shows in Figure 3.1 (p. 64) his version of the credibility process, which involves a rather complex web of researchers interacting with one another, editors and reviewers, and even the public. It seems as if credibility does not necessarily come right away, and a scientist must use her or his intuition and a large dose of optimism in attempting to achieve credibility. Research grants are shown as a major determinant of the credibility process (p. 79, Figure 3.2), provided the research proposal aligns with the priorities of the funding agency (pp. 80--81). SCIENCE, SOCIETY, ETHICS, AND RELIGION ====================================== The second part of the book presents some interesting notions about research ethics and misconduct with which every graduate student should become familiar. Grinnell provides some interesting notions about the influence interest groups (p. 103) and grant reviewers may have on the integrity of the everyday practice of science. A particular example is the issue of intellectual property (pp. 122--126), which gives an aura of business to the research university. Grinnell takes a neutral posture on these issues, and the reader is allowed to make her/his own inferences about the long-term effects on the integrity of the scientific process. Chapter 5, "Informed Consent and Risk: The Intersection of Human Research and Genetics," deals with various issues, such as principles of human research ethics, ethical challenges, and genetics research and vulnerability. This particular chapter should be most useful to students and teachers discussing recent examples of clinical trials. Chapter 6, "Faith: More Than One Way to Practice the World," makes an intersection between science and religion. Grinnell claims that "science and religion represent distinct human attitudes toward experience based on different types of faith" (p. 161). He compares both categories, and tries to dissect the complementarity between those two. I found this chapter to be the least effective. For instance, when he argues that religion "requires a different kind of faith than science but in no way gives up the demand for reason" (p. 169), he does not provide a convincing argument about the reasoning involved in faith. His suggestion that religion is the source of our values, working in a complementary way with science (p. 181, Figure 6.1), is an extremely controversial assumption with which many of the scientists mentioned in the book may disagree. He concludes the book by saying: "Perhaps solving global problems will require the scientific and religious attitudes---both types of faith---rather than one or the other" (p. 185). Perhaps not. CONCLUDING REMARKS ================== This book is an interesting addition to other books detailing the realities of science practice. The book appears to be aimed at a broad audience, which may include teachers, students, and those interested in science. However, I am not sure whether the book would be appealing to those outside the scientific community. In addition, many of the research examples are in biology, which may preclude some nonbiologists becoming as engaged as I did. I especially enjoyed those sections dealing with the process of inquiry, which may benefit those of us who are in the classroom presenting scientific ideas and literature. However, I would have liked to read about Grinnell\'s take on other forms of inquiry, such as those in the humanities and social sciences. Moreover, although Grinnell deftly presents the role of the scientist in the complex world of laboratory research, the additional role of the scientist as a citizen is not well developed. Instead, he chose to veer toward the notion of science and religion. The word passion is mentioned in the subtitle, but there is very little of it in the book. Perhaps in the near future Grinnell will treat us with an account of the scientist as a socially responsible individual, which leads me to my last remark. This book is a clear testament that we need more books that address issues of how to educate future scientists. While practicing science is most fascinating, understanding different types of inquiry, as well as the process of engaging in productive discussions with our students and colleagues, could make the everyday practice of science more passionate than it already is.	{ "pile_set_name": "PubMed Central" }
Introduction {#Sec1} ============ For people leaving homeless or women's shelters, the transition to community living can be challenging. Because much has to be arranged during this stressful time, people are often in need of practical and emotional support. They can no longer utilize shelter services, which are generally terminated after shelter exit, and most of them have few supports that they can rely on in their new living environment (Herman et al. [@CR19]). Relationships with family members and other potential social supports may need to be repaired first and ties to professional supports in the community may be weak or not yet established due to waiting lists. As a result, people leaving shelters experience a discontinuity of support. Post-shelter services are, therefore, vital in preventing negative outcomes such as recurrent homelessness and re-abuse (Caton et al. [@CR6]; McQuistion et al. [@CR31]; Tan et al. [@CR43]; Tutty [@CR46]). Critical time intervention (CTI) is a time-limited, strengths-based case management model designed to prevent adverse outcomes in vulnerable people at the time of a critical transition in their lives, such as following discharge from institutional settings (Herman et al. [@CR19]). CTI facilitates community integration and continuity of care by ensuring that a person has enduring ties to their community and support systems during these critical periods. It has been recognized by the Substance Abuse and Mental Health Services Administration (SAMHSA [@CR41]), the Public Health Agency of Canada ([@CR34]), and the Coalition for Evidence-Based Policy ([@CR9]) as an evidence-based practice (EBP). In the United States, this intervention has been found to be effective in preventing recurrent homelessness and re-hospitalization as well as reducing psychiatric symptoms and substance use after the transition from shelters, hospitals, and other institutions to community living in people with severe mental illness (Herman et al. [@CR20]; Kasprow and Rosenheck [@CR26]; Susser et al. [@CR42]). Furthermore, CTI is a cost-effective alternative to usual care for mentally ill men moving from a shelter to the community (Jones et al. [@CR24]). Few evidence-based interventions for vulnerable people leaving institutional settings have been tested rigorously outside the United States (de Vet et al. [@CR13]; Jonker et al. [@CR25]). Before EBPs are widely implemented internationally, it is necessary to test whether they are effective, because most of these practices have been developed to address place- and time-specific social issues (de Vet et al. [@CR13]). In addition, different nations usually have distinct systems of care, which may influence the effectiveness of interventions (Toro [@CR44]). Differences between systems of care might require adaptations of an intervention during implementation. These adaptations should be consistent with the model, so that its active ingredients are preserved. By evaluating whether CTI is effective outside the United States, we could possibly add to the evidence base supporting that this intervention's mechanisms of effect are not dependent on a particular social context or health care system. We initiated two multi-center randomized controlled trials (RCTs) to test the effectiveness and model fidelity of CTI for homeless people and abused women in the Netherlands. In modern effectiveness research, the development and use of fidelity criteria is considered obligatory to asses model adherence, that is, the degree to which a given intervention has been implemented in accordance with essential theoretical and procedural aspects of the model (Bond et al. [@CR2]; Hogue et al. [@CR22]). Earlier research shows that faithfully implemented EBPs produce better outcomes. For example, high fidelity to assertive community treatment (ACT) and strengths-based case management has been found to have a positive effect on client-level outcomes (Cuddeback et al. [@CR12]; Fukui et al. [@CR16]; McHugo et al. [@CR30]). So far, only one study has published CTI fidelity scores (Olivet [@CR33]). This study was conducted by the Center for Social Innovation (C4) to assess differences in implementation and client outcomes between face-to-face and online CTI training. Fidelity was measured with the CTI fidelity scale, a quantitative tool developed by Conover and Herman ([@CR11]). The CTI fidelity scale consists of 20 items, which are rated on a five-point scale ranging from not implemented to ideally implemented. Item-level ratings can be combined to compute an overall fidelity score (Conover [@CR10]). In the C4 study, overall fidelity scores were calculated nine months after training and were based on compliance fidelity, which is the degree to which providers implemented the key elements of the CTI model (eight items), and chart quality, which measures how well the intervention was documented (four items). The 15 North American homeless service agencies that participated in the C4 study obtained an average overall score of three on the five-point scale, which corresponds to fairly implemented according to the CTI Fidelity Scale Manual (Conover [@CR10]). The present study was designed to provide insight in the implementation of CTI practice in three different ways. Firstly, we also conducted a fidelity assessment, which would allow us to examine whether a similar fidelity score would be achieved in the Netherlands as was obtained by the C4 study in North America. Secondly, we set out to compare the level of fidelity between two distinct service delivery systems---services for homeless people and services for abused women---which was possible because the two RCTs on CTI employed the same ongoing training and monitoring efforts during the same period in each service delivery system (Lako et al. [@CR28]). Earlier studies of the effectiveness of CTI have already demonstrated that the CTI model can be successfully adapted for several types of populations (Herman and Mandiberg [@CR21]). However, the hypothesis that CTI is suitable for a range of populations would be supported further if similar levels of model fidelity could be obtained in different service delivery contexts with the same implementation approach. Lastly, we aimed to provide insight into facilitators and barriers to CTI practice by conducting focus groups with the case managers trained in CTI (referred to as "CTI workers"). This will provide important information on which key aspects should be paid attention to when implementing CTI. The present study will answer the following three research questions: What is the fidelity of CTI for homeless people and abused women making the transition from shelters to community living in the Netherlands? Is it possible to obtain similar fidelity ratings in two distinct service delivery systems (i.e., services for homeless people and services for abused women) with the same implementation approach? And which factors may have facilitated or impeded CTI workers to adhere to the CTI model in these service delivery systems? Method {#Sec2} ====== Procedure and Participants {#Sec3} -------------------------- This study is part of two RCTs examining the effectiveness of CTI for adult homeless people and abused women who are about to move to housing in the community and are willing to accept case management services during and after shelter exit. The two RCTs were initiated by the Academic Collaborative Center for Shelter and Recovery. The 18 shelter organizations that participated in these trials were members of this platform. In the Netherlands, services for homeless people are operated in a service delivery system that is separate from services provided to abused women; these two distinct service delivery systems will be referred to as services for homeless people and services for abused women in the remainder of this article. Participant recruitment began in December 2010 and was completed in December 2012. In total, we recruited 183 clients from 18 homeless shelters, who had been rehoused within 14 months of entering the shelter, and 136 clients from 19 women's shelters, who had been victim to any violence committed by an intimate partner (intimate partner violence) or committed to protect or restore the family honor (honor related violence) and stayed in the shelter for at least 6 weeks before being rehoused. The trials comply with the criteria for approval by an accredited Medical Research Ethics Committee (aMREC). Upon consultation, the aMREC region Arnhem-Nijmegen concluded that these studies were exempt from formal review (registration numbers 2010/038 and 2010/247). The methods of the two RCTs have been reported elsewhere in more detail (Lako et al. [@CR28]). Written informed consent to share client charts with the research team was obtained before participants were randomly allocated to CTI or care-as-usual. To assess the intervention's fidelity to the CTI model, we randomly selected a sample of 70 charts, stratified by service delivery system, from participants allocated to the experimental condition. \[Socio-demographic characteristics of these 70 participants are presented in the online supplement to this article.\] In the two trials, 164 participants were allocated to CTI. In July 2013, we assessed which client charts were available for the fidelity assessment. Fifteen participants allocated to CTI had never been assigned a CTI worker (n = 15) and, as a result, did not have a CTI client chart that could be included in the assessment. Reasons for not assigning a CTI worker were that participants refused to receive services after randomization (n = 8), organizations were unable to provide CTI due to full case-loads or participants' place of residence (n = 5), or participants were mistakenly assigned to another case manager (n = 2). For 17 participants, who had been allocated to CTI in the last 6 months of recruitment, the intervention had not yet ended and their CTI workers were therefore unable to supply these clients' charts. Earlier research has shown that implementation of an EBP with a sufficient level of fidelity takes time (Fukui et	{ "pile_set_name": "PubMed Central" }
![](indmedgaz72459-0061){#sp1 .381}	{ "pile_set_name": "PubMed Central" }
The measurement of cognitive functioning via neuropsychological (NP) testing is an important component of assessment after mild traumatic brain injury (mTBI), also known as concussion. A consensus statement on concussion in sport \[[@B1]\] concluded that such testing provides valuable information when evaluating a person with mTBI. The US military also mandates that service members are administered NP assessment to detect cognitive impairment associated with mTBI \[[@B2]\]. Traditional NP assessments are typically comprised of well-established measures with large normative databases and demonstrate evidence of adequate psychometric properties (i.e., reliability and validity). However, these tests are usually administered in a one-on-one format by a trained professional with paper, pencil and stopwatch, and require interpretation by a neuropsychologist. This can make them expensive and time intensive, not feasible for assessing large groups (e.g., athletic teams, service members) or using on the sideline or in combat settings. Over the past few decades, alternatives to traditional NP assessment batteries have emerged in the form of computerized neurocognitive assessment tools (NCATs). NCATs offer several potential logistical advantages over traditional NP tests. They can be much less time consuming and do not require administration by a testing specialist. Scoring is automated, and test performance can be easily generated into a summary report for interpretation or an electronic spreadsheet for statistical analysis. Furthermore, NCATs allow for cognitive assessment to be obtained in geographic areas where traditional NP services are limited. They are easier to use for obtaining baseline tests (e.g., preseason, predeployment) that can be used for comparison to assessment after concussion, which can be especially advantageous where examinees may have conditions that prevent comparison to normative reference groups (i.e., abnormal cognitive development, ADHD, among others) \[[@B3],[@B4]\]. Also, the computerized nature of NCATs makes it possible to administer alternative forms of a test with numerous combinations of test stimuli, which mitigate practice effects, and allow for multiple administrations in a short amount of time to track recovery after injury \[[@B3]\]. Moreover, being computerized allows for more accurate measurement of reaction time (RT), possibly making NCATs more sensitive to subtle cognitive effects \[[@B5]\]. Despite potential advantages, NCATs are not without limitations, as discussed by Echemendia et al. \[[@B4]\]. Specifically, alternate forms might not be equivalent, computers settings can cause erroneous RT measurement, there are differences between administering to groups (as is often done for baseline administrations) and to individuals (as is often done postinjury), and the tests are marketed to professionals (e.g., athletic trainers) who may have little or no training in cognitive testing. Additionally, one of the most important limitations of NCATs is that the psychometric properties are not fully established. Although NCATs have gained momentum as a tool in the management of mTBI, particularly in military and athletic settings, commonly used NCATs have not undergone the same level of validation as many traditional tests. According to a Joint Position Paper of the American Academy of Clinical Neuropsychology and the National Academy of Neuropsychology \[[@B3]\], though NCATs may seem to be analogous to traditional NP tests, there are important differences between them that need to be explored. Specifically, modifications of existing measures warrant investigations of the new tests' psychometric properties, such as validity \[[@B6]\]. This manuscript will summarize and evaluate the existing literature regarding the validity of four NCATs commonly used for both clinical and research purposes: Automated Neuropsychological Assessment Metric (ANAM), CNS-Vital Signs (CNS-VS), Axon/CogState/CogSport (CogState) and Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT; please see [Table 1](#T1){ref-type="table"} for a description of each NCAT). The tests that will be covered are commonly used in research and clinical settings. Specifically, CNS-VS has been used in several clinical trials and can be billed to medical insurance, ANAM is required for US military Service Members, CogState is commonly used in Australian athletic settings and ImPACT has been approved by the US FDA to detect cognitive deficits following mTBI. Interestingly, to date, these measures have not generated adequate evidence of validity, yet they are commonly used for TBI-related assessment in sports and military settings. This summary and review will focus on the comparisons of these tests to traditional NP batteries as well as evaluations of the ability of these tests to provide clinically meaningful information regarding cognitive functioning after concussion. The existing state of the literature will be evaluated based on criteria put forth by Randolph et al. in a 2005 \[[@B7]\] review of the literature regarding NP testing after sport-related concussion (SRC). That review and those criteria are discussed below (see [Box 1](#BX1){ref-type="boxed-text"}). This is not a systematic literature review, but is rather meant to serve as a concise summary and reference, with recommendations for future studies and considerations identified. ###### Descriptions of computerized neurocognitive assessment tools reviewed. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Test Subtests and cognitive construct intended to measure Classification and summary scores ---------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ANAM4 Simple Reaction Time (SRT): visuomotor processing speed, simple motor speed and attentionProcedural Reaction Time (PRO): processing speed, visuomotor reaction time and attentionCode Substitution Learning (CDS): visual scanning, visual perception, attention, associative learning and processing speedCode Substitution Delayed (CDD): learning and delayed visual recognition memoryMathematical Processing (MTH): basic computational skills, concentration and working memoryMatching to Sample (M2S): visual-spatial processing, working memory and visual recognition memorySRT, Second Administration (SRT2): index of attention (i.e., reaction time \[RT\] and vigilance) Summary scores:Throughput (TP; number of correct responses per minute of available response time)Standardized subtest TP -- standardized composite TPComposite score (standardized average TP \[z-score\])Classification of impairment: ANAM composite z ≤ -1.28 CNS-VS Verbal Memory Test, Immediate (VBM): word recognition and memory, immediate and delayed recallVisual Memory Test, Immediate (VIM): visual recognition and memory, immediate and delayed recallFinger Tapping Test (FTT): motor speed, fine motor controlSymbol Digit Coding (SDC): information processing speed, complex attention, visual perceptual speedStroop Test (ST): SRT, complex reaction time, inhibition, executive skills, processing speedShifting Attention Test (SAT): executive functioning, RTContinuous Performance Test (CPT): sustained attention, choice reaction time (CRT), impulsivityVerbal Memory Test, Delayed (VBM): word recognition, memory and delayed recallVisual Memory Test, Delayed (VIM): visual recognition, memory and delayed recall Summary scores:Neurocognitive Index (NCI)Composite memoryVerbal memoryVisual memoryPsychomotor speedRTComplex attentionCognitive flexibilityProcessing speedExecutive functionSimple attentionMotor speed\ Composite score: IQ scale and percentile\ Classification of impairment: N/A CogState Detection Task: SRTIdentification Task: processing speedOne Back Task: attention, working memoryOne Card Learning Task: learning and recognition memory Summary scores: score for each subtestComposite score: z-scoresClassification of impairment:-1.64 SD on at least two subtests\ CogState composite \< -1.64 ImPACT Word Memory, Immediate: verbal recognition memoryDesign Memory, Immediate: visual recognition memoryX\'s and O\'s: visual working memory and visual processing/visual motor speedSymbol Match: visual processing speed, learning and memoryColor Match: CRT and impulse control/response inhibitionFour Letters: working memory and visual-motor response speedWord Memory, Delayed: verbal recognition memoryDesign Memory, Delayed: visual recognition memory Summary scores:Verbal memoryVisual memoryVisual motor speedRTImpulse control\ Composite score:Test-specificStandardized scores and percentiles\	{ "pile_set_name": "PubMed Central" }
INTRODUCTION ============ Despite advances in care, morbidity following traumatic brain injury (TBI) remains high ( [@nlw045-B1] , [@nlw045-B2] ). Following the primary direct mechanical injury to brain parenchyma, important secondary injuries develop over hours to days from multiple addressable mechanisms including hypoxemia, ischemia, and excitotoxicity ( [@nlw045-B3] ). Clinical observational studies have found a strong association between early hypoxemia (pre-hospital or emergency room setting) following TBI and poor clinical outcomes ( [@nlw045-B4] ). Preclinical investigations of hypoxemia immediately after experimental TBI have demonstrated exacerbations in brain edema and ischemia, hippocampal neuronal cell death, neuroinflammation, axonal injury, and behavioral deficits ( [@nlw045-B8] ). Patients with TBI in the intensive care setting are also at high risk for delayed secondary hypoxemia due to pulmonary contusions, aspiration, pneumonia, atelectasis, acute respiratory distress syndrome, and as a result of procedural interventions such as endotracheal tube displacement, emergent diagnostic imaging, and surgical interventions. However, little is known about the role that delayed hypoxemia (beyond the pre-hospital and emergency room setting) plays in secondary brain injury following TBI. A small prospective study found that any hypoxemia, defined as O ~2~ saturation \< 90%, within the first 24 hours after TBI was associated with an increase in mortality ( [@nlw045-B13] ). The majority of previous animal studies investigating post-TBI hypoxemia have utilized a 15- to 30-minute exposure of low inspired oxygen (8%--12%) occurring within 1 hour of experimental TBI ( [@nlw045-B8] ). No studies to our knowledge have investigated delayed hypoxemia after TBI in an animal model. Although traumatic axonal injury (TAI) is considered to be the major contributor to long-term cognitive deficits in TBI patients ( [@nlw045-B14] ), only 1 preclinical study has investigated the contribution of immediate postinjury hypoxemia to axonal injury ( [@nlw045-B11] ). Axonal injury following TBI is most commonly characterized histologically by immunohistochemical staining of amyloid precursor protein (APP) and neurofilament. APP under normal conditions traverses the length of the axon, but in response to injury will accumulate at axonal varicosities and retraction bulbs ( [@nlw045-B19] ). Changes in structural integrity of the axonal cytoskeleton can result in increased neurofilament immunoreactivity ( [@nlw045-B16] , [@nlw045-B23] , [@nlw045-B24] ). These 2 approaches, when combined, can provide a more complete histological characterization of axonal injury following TBI ( [@nlw045-B19] , [@nlw045-B25] , [@nlw045-B26] ). To our knowledge, the impact of delayed hypoxemia on axonal injury has not been investigated in preclinical models of TBI. Based on retrospective pediatric intensive care data, we developed a clinically relevant mouse model of delayed systemic hypoxemia following TBI. We used this model to test the hypothesis that delayed posttraumatic hypoxemia following controlled cortical impact (CCI) in young mice would exacerbate white matter injury. MATERIALS AND METHODS ===================== Patient Data ------------ The Institutional Review Board at Washington University approved the retrospective clinical study and waived the requirement for obtaining written informed consent. Data were abstracted from registry of pediatric patients with severe TBI (postresuscitation Glasgow Coma Score (GCS) of 8 or less who were admitted to the pediatric intensive care unit (PICU) at Saint Louis Children's Hospital between July 1, 1999 and December 31, 2011. Patients were excluded if they had a GCS of 3 with fixed and dilated pupils at time of admission, cardiac arrest prior to arrival in the PICU, abusive head trauma, and gunshot wounds to the head. Age at time of injury, sex, postresuscitation GCS, pediatric risk of mortality score (PRISM III), injury severity score, injury mechanism, PICU length of stay, hospital length of stay, PICU-free days at 28 days of admission, Glasgow outcome scale score at hospital discharge, and destination at hospital discharge were recorded. Initial head computed tomography scan was categorized by using the Marshall classification of initial head computed tomography ( [@nlw045-B27] ). PICU-free days at 28 days of admission was defined as the total numbers of days alive outside the PICU at 28 days after admission. Complete arterial blood gas, hourly pulse oximetry, and end tidal CO ~2~ data were abstracted from the medical record. Hypoxemia was defined as a pulse oximetry reading \< 90% or an arterial PO ~2~ \<60 mm Hg. A discreet episode of hypoxemia was defined as complete with the first subsequent normal blood gas or pulse oximetry reading. Animal Studies -------------- ### Injury All procedures were approved by the Washington University Animal Studies Committee, and are consistent with the National Institutes of Health guidelines for the care and use of animals. Five week-old C57BL/6J male mice (Jackson Laboratory, Bar Harbor, ME) weighing 16--20 g were used in these experiments. For studies of axonal injury, mice were killed at 2 different time points: 48 hours and 1 week after injury (n = 20 total for each time point). Two additional cohorts of 20 animals each were utilized for the same 2 time points (48 hours and 1 week after injury) to evaluate the response of microglia (1 week time point only) and astrocytes (48 hours and 1 week). For each time point, 8 mice underwent sham injury and 12 mice underwent CCI ( [@nlw045-B28] ). The mice were anesthetized with 5% isoflurane at induction, followed by maintenance at 2% isoflurane for the duration of the procedure. The head was shaved and head holders were used to stabilize the head within the stereotaxic frame (MyNeurolab, St. Louis, MO). Then, a single 5-mm craniotomy was performed by an electric drill on the left lateral side of the skull centered 2.7 mm lateral from the midline and 3 mm anterior to lambda. Animals were randomized to sham or injury after craniotomy utilizing a computer generated numbers randomization. For injured animals, the 3-mm electromagnetic impactor tip was then aligned with the craniotomy site at 1.2 mm left of midline, 1.5 mm anterior to the lambda suture. The impact was then delivered at 2 mm depth (velocity 5 m/s, dwell time 100 ms). The head holders were released immediately after the injury. All animals then received a loose fitting plastic cap secured over the craniotomy with Vetbond (3M, St. Paul, Minnesota). The skin was closed with interrupted sutures and treated with antibiotic ointment before removing the mouse from anesthesia and allowing recovery on a warming pad. Delayed Hypoxemia ----------------- One day after injury or sham surgery, animals were randomized to normoxemia (room air) or hypoxemia (8% O ~2~ , 4% CO ~2~ ) for 30 minutes. During normoxemia or hypoxemia animals were placed in fresh cages with littermates randomized to the same treatment arm and all mice were subjected to identical transport and handling. Animals randomized to hypoxemia were placed in a Coy Labs Hypoxia Chamber (6′ × 3′ × 4′) (Coy Laboratory, Grass Lake, MI), fitted with airlock, oxygen sensor, carbon dioxide sensor, and gas controllers. Arterial Blood Gas Sampling --------------------------- A separate cohort of 18 mice was utilized for the arterial blood gas experiments. Animals were randomized to injury or sham surgery (n = 9 for each group). One day after injury or sham surgery, animals were anesthetized with 5% isoflurane at induction, followed by maintenance at 2% isoflurane for placement of right carotid artery tunneled catheters. Once completely recovered and awake, animals underwent 30 minutes of low inspired oxygen (8% O ~2~ ,) and arterial blood samples were drawn at the end of the 30 minutes. Arterial blood gas analysis was performed on a Bayer Rapidlab 845 blood gas analyzer (Siemens Medical Diagnostics, Bayer, Tarrytown, NY). Initial screening was performed on 1 sham and 1 injured mouse at each of the following CO2 concentrations: 0.5%, 2%, 4%, and 6%. The conditions that produced normocarbia were then repeated with the 10 remaining mice (n = 5 for each group). Immunohistochemistry -------------------- Mice were killed under isoflurane anesthesia by transcardial perfusion with 0.3% heparin in phosphate-buffered saline. Whole brains were removed and fixed in 4% paraformaldehyde for 48 hours, followed by equilibration in 30% sucrose for at least 48 hours prior to sectioning. Serial 50-μm-thick coronal slices were cut on a freezing microtome starting with the appearance of a complete corpus callosum and caudally to bregma −3.08 mm. Sets of 12 sections spaced every 300 μm were mounted on glass slides and used for immunohistochemical studies. Staining was performed on free-floating sections washed in Tris-buffered saline (TBS) between applications of primary and secondary antibodies. Endogenous peroxidase was blocked by incubating the tissue in TBS + 3% hydrogen peroxide for 10 minutes. Normal goat serum (3%) in TBS with 0.25% Triton X (Tris-buffered saline (TBS)-X) was used to block nonspecific staining for all antibodies. Slices were then incubated	{ "pile_set_name": "PubMed Central" }
Pediatric intestinal failure (IF), as a result of short bowel syndrome (SBS) or gastrointestinal motility disorders, is a condition characterized by insufficient bowel function to maintain hydration and nutrient absorption for growth and development[@b1][@b2]. IF-associated liver disease (IFALD) is a serious complication and the leading cause of morbidity and mortality in children IF patients[@b3][@b4]. However, the mechanisms underlying the development of IFALD are poorly understood. To unravel the mechanisms of IFALD, we performed population based cross-sectional study on serum fibroblast growth factor 19 (FGF19) and bile acid (BA) homeostasis in relation to histological liver damage in pediatric IF patients. The human FGF family includes about 23 members with diverse biological functions[@b5]. FGF19 is secreted by ileum in response to activation of farnesoid X receptor (FXR) with bile acids[@b6][@b7]. FGF19 has been shown to regulate BA homeostasis through a negative feedback control of bile salt synthesis in human[@b8]. FGF19 has also been implicated in the regulation of carbohydrate, lipid and energy metabolism in the liver[@b9][@b10]. The studies recently have been demonstrated that the decreased serum concentration of FGF19 associated with increased BA synthesis in patients with Crohn's disease and intestinal failure[@b11][@b12][@b13]. Annika et al. reported that total or partial loss of ileum decreased serum FGF19 concentration corresponding to hepatic inflammation and fibrosis[@b12]. However, the roles of FXR/FGF19 signaling in BA metabolism among pediatric IF patients remained unknown. We hypothesized that pediatric IF patients decreased serum concentration of FGF19 in association with ileal inflammation, and corresponding to bile acid dysmetabolism, leading to histological liver injury. To this end, we determined the serum concentrations of FGF19, pro-inflammatory cytokines, histological liver injury, biochemical liver function tests and serum BA levels, and analysis the relationship between them. Results ======= IF patients characteristics --------------------------- A total of twenty-three patients at median age 8.0 months (IQR 3.3--58.7) participated in the study ([Table 1](#t1){ref-type="table"}). Causes of IF included short bowel syndrome (necrotizing enterocolitis (NEC): n = 4, small bowel atresia: n = 5, and mid-gut volvulus: n = 3) and intestinal dysmotility disorders (chronic intestinal pseudo-obstruction (CIPO): n = 7 and extensive aganglionosis of hirschsprung's disease: n = 4). No significant differences in age were observed in controls and IF patients. In total, twenty patients preserved Ileocaecal valve and ileum. Five patients were on parenteral nutrition (PN) and eighteen had weaned off PN 0.6 years (0.5--0.9) earlier, after 3 months (1.7--4) on PN. The PN energy comprised 50.79% (47.14--55.36) of glucose and 31.96% (28.03--36.25) of fat. PN fat was given as soy oil-based emulsion \[1.5 g/kg/day (1.10--1.78)\] and combined with fish oil-based emulsion (0.8 g/kg/day) in two patients. The IF patients exhibit histological liver injury ------------------------------------------------- As seen in [Fig. 1A](#f1){ref-type="fig"}, the control liver tissues exhibited normal liver histology. In contrast, the liver sections from pediatric IF patients exhibited liver damages characterized by bile duct proliferation, lymphocytes infiltration, and hepatocyte ballooning ([Fig. 1A](#f1){ref-type="fig"}). Apoptotic hepatocytes were detected by TUNEL staining of liver sections. As expected, few TUNEL-positive cells were observed in the control liver specimens. Conversely, TUNEL-positive hepatocytes increased significantly in liver sections from patients ([Fig. 1A and B](#f1){ref-type="fig"}). When compared with the controls, extensive portal fibrosis was indicated in liver slides of pediatric IF patients ([Fig. 1A and C](#f1){ref-type="fig"}). The FGF19 is associated with liver injury and inflammation ---------------------------------------------------------- As shown in [Fig. 1D](#f1){ref-type="fig"}, in IF patients, serum FGF19 concentration was significantly lower (n = 23, 51.42 ± 47.03 pg/mL, p \< 0.01) compared to controls (n = 21, 102.31 ± 71.23 pg/mL) ([Fig. 1D](#f1){ref-type="fig"}). As seen in [Table 2](#t2){ref-type="table"}, abnormal values in liver enzymes, and parameters of cholestasis were indicated. The values of liver enzymes including alkaline phosphatase (ALP) (51.76 ± 69.24 U/L vs. 90.34 ± 66.99 U/L), alanine aminotransferase (ALT) (23.23 ± 16.01 U/L vs. 55.77 ± 19.91 U/L, p \< 0.01) and aspartate aminotransferase (AST) (36.35 ± 16.66 U/L vs. 61.67 ± 27.51 U/L, p \< 0.01) were increased in patients' serum compared to the controls ([Table 2](#t2){ref-type="table"}). Plasma total bilirubin (5.33 ± 2.55 μmol/L vs. 6.24 ± 1.35 μmol/L) and conjugated bilirubin (3.16 ± 1.43 μmol/L vs. 3.19 ± 2.26 μmol/L) were unregulated in patients that compared to controls ([Table 2](#t2){ref-type="table"}). Correlated analysis showed that serum FGF19 concentrations were inversely correlated with both the levels of ALP (r = −0.34, p = 0.03) and AST(r = −0.3, p = 0.06) ([Table 2](#t2){ref-type="table"}). As indicated in [Fig. 1D](#f1){ref-type="fig"}, we also found that the pro-inflammatory factors serum interleukin-6 (IL-6) (13.28 ± 21.78 pg/ml vs. 50.63 ± 45.99 pg/ml, p \< 0.001) and tumor necrosis factor-alpha (TNF-α) (0.41 ± 0.20 pg/ml vs. 0.89 ± 0.43 pg/ml, p \< 0.01) concentrations were higher in patients compared to controls ([Fig. 1D](#f1){ref-type="fig"} and [Table 2](#t2){ref-type="table"}). In addition, the serum IL-6 (r = −0.32, p \< 0.05) was inversely associated with serum FGF19 ([Table 2](#t2){ref-type="table"}). FXR/FGF19 signaling contributes to BA homeostasis ------------------------------------------------- As shown in [Fig. 2A](#f2){ref-type="fig"}, the total plasma BA significantly elevated in IF patients ([Fig. 2A](#f2){ref-type="fig"}). The BA composition of plasma was markedly altered in pediatric patients with a significant in the proportion of the primary bile acid, including the cholic acid (CA) and α, β, ω-muricholic acid (MCA) ([Fig. 2B--D](#f2){ref-type="fig"}). In contrast, the secondary or tertiary bile acids, including the deoxycholic acid (DCA), taurochenodeoxycholic acid (TCDCA), lithocholic acid (LCA) and hyodeoxycholic acid (HDCA), slightly decreased in IF patients compared to controls ([Fig. 2B--D](#f2){ref-type="fig"}). As a FXR agonist, TCDCA positively related to concentration of serum FGF19 ([Fig. 2E](#f2){ref-type="fig"}). In intestinal, we found that the IF patients without intestinal surgery, suffering from ileal inflammation, had numbers of CD3-positive lymphocytes infiltrating ileal mucosa ([Fig. 3A and B](#f3){ref-type="fig"}). As result of ileal mucosa injury, the FXR expression was reduced significantly in patients when compared to controls ([Fig. 3A and C](#f3){ref-type="fig"}). As shown in [Fig. 3D](#f3){ref-type="fig"}, the CD3-positive lymphocytes in ileal mucosa negatively correlated to the FXR expression ([Fig. 3D](#f3){ref-type="fig"}). In addition, ileal mucosa inflammatory degrees was inversely associated with serum FGF19 levels (r = −0.5, p = 0.04) ([Fig. 3E](#f3){ref-type="fig"}). As seen in [Fig. 4A](#f4){ref-type="fig"}, the total BA contents in IF patients' liver were higher (442.49 ± 312.93 nmol/mg) compared to controls (199.15 ± 134.59 nmol	{ "pile_set_name": "PubMed Central" }
Background {#Sec1} ========== Fasciola gigantica and Fasciola hepatica are omnipresent agents of a zoonotic parasitic disease, fascioliasis, which continues to be a major health burden on animals and humans. Fascioliasis can adversely affect the sustainability of the farm animal industry \[[@CR1]\]. The annual global economic loss due to fascioliasis has been estimated to be in excess of three billion dollars \[[@CR2]\]. Worldwide, at least 2.4 million people have been infected with fascioliasis, with a further 180 million people at risk of being infected \[[@CR3]\]. Despite this high impact and investigations for decades using clinical studies as well as animal models, knowledge about host defense mechanisms against F. gigantica is limited. This challenge is partly due to the fact that Fasciola spp. are very efficient modulators of the host immune response \[[@CR4]\]. The immunomodulatory capacity of F. gigantica, mediated by parasite-derived effector molecules, is believed to play important roles in the establishment of long-lasting infection in the host. Several studies have investigated various excretory/secretory products (ESPs) and virulence effector molecules employed by F. gigantica flukes to ensure their survival and establishment of persistent infection \[[@CR5], [@CR6]\]. Rab proteins are a family of small GTP-binding proteins, part of the Ras superfamily, which regulate intracellular membrane trafficking of several pathogens; including parasites (e.g. Plasmodium, Theileria, Cryptosporidium and Babesia \[[@CR7]\] and Toxoplasma gondii \[[@CR8]\]), bacteria (e.g. Mycobacterium spp. \[[@CR9]\] and Listeria monocytogenes \[[@CR10]\]) and fungi \[[@CR11]\]. Despite their crucial role as regulators of vesicular membrane traffic, the roles of Rab proteins in the pathogenesis of F. gigantica infection remain largely unknown. Understanding the influence of parasite-secreted proteins on the function of immune cells, such as goat peripheral blood mononuclear cells (PBMCs), is essential due to their important role in the immunopathogenesis of fascioliasis \[[@CR12]\]. In a recent study, we cloned and expressed a recombinant F. gigantica 14-3-3 epsilon protein (rFg14-3-3e), and characterized its effects on specific functions of goat PBMCs \[[@CR6]\]. In the present study, we expand our investigation of the effects of F. gigantica ESPs on the functions of these immune cells. Specifically, the gene encoding F. gigantica Rab10 (FgRab10) was cloned and expressed in Escherichia coli. Then, the modulatory effects of the purified recombinant FgRab10 (rFgRab10) protein on the functions of goat PBMCs; including cytokine secretion, proliferation, migration, nitric oxide (NO) production, phagocytosis and apoptosis were investigated. Our results indicate that rFgRab10 protein can significantly influence key functions of goat PBMCs, all are critical facets of the immunopathogenesis of F. gigantica infection. Methods {#Sec2} ======= Animals {#Sec3} ------- Three crossbred goats (3--6 months-old) were obtained from the teaching and research flock at Nanjing Agricultural University. Goats were treated with triclabendazole (50 mg/kg body weight) in order to exclude the possibility of any prior infection with liver flukes. Two weeks post-treatment, a faecal specimen from each goat was examined microscopically to exclude the presence of helminth eggs. Female Sprague Dawley (SD) rats (150--200 g) were purchased from the Experimental Animal Center of Jiangsu Province, China (Certificate: SCXK 2008-0004), and used for the production of antibodies. Rats were raised under specific pathogen-free conditions, and fed with sterilized food and water ad libitum. All efforts were made to minimize the suffering of animals, and daily health checks were performed throughout the experiment. Purification and culture of goat PBMCs {#Sec4} -------------------------------------- Venous blood samples were collected from the jugular vein of goats, with no history of F. gigantica infection or other parasitic infections, into Vacutainer tubes coated with ethylenediaminetetraacetic acid (EDTA). PBMCs were isolated from freshly collected blood using a PBMC isolation kit (TBD, Tianjin, China). Culturing was done by incubating the isolated PBMCs in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, New York, USA). Cultures were maintained in a humidified atmosphere of 5% CO~2~ at 37 °C. The number of PBMCs was adjusted to 10^6^ cells/ml in RPMI 1640 medium and cell viability was assessed using trypan blue dye exclusion method. The number of viable cells was counted in a haemocytometer and only cells with \> 95% viability were used in the experiments. All assays were performed using freshly isolated PBMCs. For the monocyte phagocytosis experiment, adherent monocytes were obtained after incubation of PBMCs for 48 h at 37 °C in 5% CO~2~, followed by removal of the non-adherent cells. In all assays performed to investigate the effects of rFgRab10 protein on certain functions of goat PBMCs and monocytes, cultured cells were exposed to rFgRab10 protein at concentrations of 10 μg/ml, 20 μg/ml, 40 μg/ml or 80 μg/ml. PBMCs treated with SUMO protein expressed in pET-SUMO expression vector and sham-treated with phosphate buffered saline (PBS, pH 7.4) were used as controls in all assays. All experiments were performed in triplicate. Parasite strain {#Sec5} --------------- Adult F. gigantica flukes were harvested from the gall-bladder of naturally infected buffaloes at local abattoirs in Guangxi Zhuang Autonomous Region, PR China. The collected flukes were washed twice in PBS and used for RNA isolation or stored frozen at -80 °C with RNA stabilizer for later analysis. The identity of the flukes collected was ascertained to be F. gigantica based on PCR and sequencing analysis of the second internal transcribed spacer (ITS2) of ribosomal DNA \[[@CR13]\]. This analysis revealed 100% ITS2 sequence homology between the flukes collected and F. gigantica samples examined previously from the same region (GenBank: AJ557569). Cloning and characterization of FgRab10 gene {#Sec6} ---------------------------------------------- Because F. gigantica genome sequences are not available, we searched the NCBI/BLASTx (<https://blast.ncbi.nlm.nih.gov/Blast.cgi>), F. hepatica cDNA library and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based F. hepatica ESPs dataset from previously unpublished proteomics studies to identify homologous Ras family protein gene sequences. This analysis identified F. hepatica Ras family protein gene (GenBank: PIS87503.1), which was utilized to design primers to amplify F. gigantica Ras-related protein Rab10 (FgRab10) gene. Total RNA was isolated from adult F. gigantica flukes using Trizol reagent (Invitrogen, San Diego, USA), and was used to synthesize first-strand cDNA with reverse transcription polymerase chain reaction (RT-PCR) using the RevertAid First Strand cDNA Synthesis Kit \[Thermo Scientific (EU), Vilnius, Lithuania\]. The cDNA was used for the amplification of FgRab10 gene using a forward primer (5\'-CCG [GAA TTC]{.ul} ATG GCT AAG AAG TCG TAC GAT G -3\') and a reverse primer (5\'-ATT T[GC GGC CGC]{.ul} TGT AGG ACA CCA GGA GCA-3\'). EcoR I and Not I restriction sites, which were underlined, were incorporated into the primers. The amplified FgRab10 gene was digested with EcoR I and Not I, and ligated into the corresponding cloning sites in the T-vector pMD19 (Simple) (Takara, Dalian, Liaoning, China). The recombinant plasmid was transformed into Trans5α chemically competent cells (TransGen Biotech, Beijing, China). Positive clones were sequenced by GenScript (Nanjing, Jiangsu, China) in order to confirm the correct insertion/orientation of FgRab10 gene in the vector. The FgRab10 cDNA sequence was translated into amino acid sequences using EditSeq (DNASTAR Lasergene). The signal peptide and conserved domains of FgRab10 were predicted by SignalP 4.1 Server (<http://www.cbs.dtu.dk/services/SignalP/>), and (<https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi>), respectively. Expression and purification of rFgRab10 protein {#Sec7} ----------------------------------------------- Positive clones containing the FgRab10 gene were selected. The *FgRab	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1} =============== With the increasing popularity in seeking complementary and alternative medicine (CAM) as a healthcare service, the prevalent use of herbal medicine as part of treatment is inevitable. Along with the rapid growth in consumption comes the pressing question on the safety of herbal medicine. While much research and investigation on the potential uses of herbal medicine has been done widely, evaluation on the safety of herbal medicines is relatively scarce. The lack of knowledge of the nature and mechanism of interaction of herbal medicines in the human body has brought about exaggerated reports and extreme measures to counter the adverse effects reported. This paper aims to review the safety of Rhizoma Coptidis (RC) and berberine, using the prohibition of use and subsequent lifting of ban on RC and berberine in Singapore as an illustration to highlight the importance of evidence-based studies in Traditional Chinese Medicine (TCM). 2. Rhizoma Coptidis and Berberine {#sec2} ================================= RC is a commonly used TCM herb for "clearing damp-heat, quenching fire and counteracting poison" and is found in prescriptions for various ailments including febrile illness, hepatobiliary diseases, and gastroenteritis \[[@B1]\]. The earliest record of RC dates back toShen Nong Ben Cao Jing (Shen Nong\'s Herbal Classic) compiled in the Han Dynasty of China, in which it was classified as a top-grade drug \[[@B2]\]. The prevalent use of RC as part of a TCM compound formula (fufang in Chinese) was cited in several renowned TCM medical compilations, namely,Shang Han Za Bing Lun (Treatise on Febrile and Miscellaneous Diseases),Wai Tai Mi Yao (Medical Secrets from Royal Library), andBen Cao Gang Mu (Compendium of Materia Medica) \[[@B3]\], some of which are still in use today clinically and in evidence-based studies \[[@B4]--[@B7]\]. TCM formulae containing RC have also been used during pregnancy and in neonates for various conditions including neonatal jaundice (NNJ) \[[@B8]\]. Berberine, an alkaloid isolated from RC, was first isolated in the early nineteenth century \[[@B9]\]. Numerous studies on these two subjects have been conducted. Berberine and RC were also reported to have antimicrobial effects \[[@B10], [@B11]\] and are used to treat bacteria-induced diarrhea \[[@B12]\]. Recent area of interest is the antineoplastic effects of RC and berberine \[[@B13], [@B14]\], specifically in areas of breast cancer \[[@B15], [@B16]\], leukemia \[[@B17], [@B18]\], gastric cancer \[[@B19], [@B20]\], pancreatic cancer \[[@B21]\], and nasopharyngeal cancer \[[@B22]\]. It was also reported that RC and berberine have hepatoprotective \[[@B23]\], nephroprotective \[[@B24]\], neuroprotective \[[@B25]\], and cardioprotective \[[@B26]\] effects. Studies have also explored the effect of berberine and RC in controlling metabolic syndrome \[[@B27]\], hyperlipidemia \[[@B28], [@B29]\], and type II diabetes \[[@B30]\], highlighting berberine and RC as multifaceted drugs with immense therapeutic potential. 3. The Prohibition of Use of RC and Berberine in Singapore {#sec3} ========================================================== TCM was first introduced in Singapore by the influx of Chinese immigrants in the early days as part of culture heritage and healthcare. It provided an affordable and familiar healthcare service for the new Chinese immigrants before the 1960s who found Western medical care foreign and unaffordable. TCM has since developed to become an integrative part of complementary and alternative medicine (CAM). Currently, Western Medicine serves as the mainstream of the healthcare system in Singapore \[[@B31]\]. Nonetheless, TCM remains the most widely used CAM in Singapore, accounting for 88% of CAM use locally \[[@B32]\]. Prior to its ban in 1978, RC was widely consumed as part of oral administered compound formula in Singapore. Its properties of "clearing damp-heat, quenching fire and counteracting poison" as described earlier were suitable to treat diseases caused by tropical weather in Singapore. Mass screening for G6PD deficiency in newborns has been introduced in Singapore since 1965. This is a vital move as preventive measures and effective counseling needed to be instituted early to prevent exposures to triggers \[[@B33]\]. Under this surveillance program, more than two decades of prevalent kernicterus was observed in Singapore. It was found that half of the babies suffering from kernicterus were suffering from deficiency of glucose-phosphate-6 dehydrogenase (G6PD) deficiency \[[@B34], [@B35]\]. G6PD deficiency is an X-linked disorder of the antioxidant homeostasis that is caused by mutations in the G6PD gene \[[@B36]\]. This condition currently affects about four hundred million people worldwide \[[@B37]\], making it the most common enzymopathy in the world. G6PD is an important enzyme that helps to protect the erythrocytes from oxidative damage. Within the restricted metabolism of erythrocytes, G6PD catalyses the first step in the hexose monophosphate pathway, converting glucose-6-phosphate to 6-phosphogluconolactone and reducing the cofactor nicotinamide-adenine dinucleotide phosphate (NADP) to NADPH. The second enzymatic step in the pathway is also associated with the reduction of NADP to NADPH ([Figure 1](#fig1){ref-type="fig"}). As G6PD is the only source of NADPH, which is essential in the protection of erythrocytes from oxidation, premature lysis of erythrocytes may occur in the absence of the enzyme \[[@B38]\]. In severe cases, this results in NNJ and kernicterus, causing permanent damage to the brain, resulting in mental retardation, convulsion, cerebral palsy, hearing deficit, or even death \[[@B37], [@B39], [@B40]\]. Triggers identified include common drugs like aspirin, methylene blue, primaquine and nitrofuran, and common environmental factors like mothballs (naphthalene), henna, and fava beans \[[@B41]--[@B46]\]. Infections have also been identified as trigger of hemolysis in G6PD deficient individuals \[[@B38], [@B47], [@B48]\]. Chinese herbal medicine containing berberine was also identified as triggers of acute hemolysis in G6PD deficient babies \[[@B49]\]. In the 1980s, a study in Singapore found that a high level of "indirect plasma bilirubin" was observed in local babies in general and nearly 100% were visibly jaundiced in the first week of life, compared to about 30% in Caucasian babies born in Singapore \[[@B50]\]. It also found that a retrospective comparison of a cohort of G6PD deficient neonates yielded a result of 22 out of 102 suffering from severe NNJ after exposure to TCM herbal medicinesin utero, as compared to 2 out of 34 for those without. The same study also observed that exposure to mothball also triggered NNJ with a prevalence of 29 out of 100 as compared to the 20 out of 113 who were not exposed to mothballs. The authors then concluded that TCM herbal medicines, particularly RC, were the cause of severe NNJ in G6PD deficient neonates in Singapore \[[@B50]\]. Based on this study, the Department of Health (known as Ministry of Health now) announced the prohibition of use RC and items containing berberine in Singapore. These items have since been regulated under the Poisons Act until 2013 \[[@B31]\]. The prohibition had forced local TCM practitioners to need to source for substitute Chinese herbal medicine (CHM) during treatment, which affected the efficacy of the compound formula prescribed for patients. 4. Safety of RC and G6PD Deficiency Studies {#sec4} =========================================== The implementation of this policy sparked active research and discussion in Hong Kong, Taiwan, and China, where prevalence of G6PD deficiency is pronounced and use of TCM is ubiquitous. More studies observing the safety of RC, as well as its relation to G6PD deficient individuals, were also conducted, bringing greater understanding to the safety of the herb and genetic condition. 4.1. In Vivo andIn Vitro Studies of RC and Berberine {#sec4.1} -------------------------------------------------------- Several studies have supported the retrospective epidemiology findings described by Wong \[[@B50]\] that identified CHM, particularly RC, as the cause of NNJ in G6PD deficient neonates. A study by Ko et al. on the prooxidative effects of Chinese herbal medicine on G6PD deficient erythrocytesin vitro found that RC significantly reduced GSH level and increased the level of methaemoglobin in G6PD deficient blood samples, pointing to the possibility of RC as the cause of neonatal jaundice in G6PD deficient neonates \[[@B51]\]. A study found that chronic intraperitoneal administration of 10 and 20 mg/g of berberine daily for 1 week to adult rats resulted in a significant decrease in mean bilirubin serum protein binding, due to anin vivo displacement effect and a persistent elevation in steady-state serum concentrations of unbound and total bilirubin, possibly caused by inhibition of metabolism \[[@B52]\]. Another study by Yeung et al. yielded similar results, discovering that RC had a significant effect in displacing bilirubin from its serum protein binding as assessed by peroxidase oxidation method \[[@B53]\], which results in elevation of free bilirubin that can readily cross the blood brain barrier, resulting in kernicterus in neonates. Besides the potential damage that RC may cause in G6PD deficient neonates, it was also reported that RC was known to have caused several adverse reactions including respiratory failure, extrapyramidal system reactions, severe arrhythmia, liver function injury,	{ "pile_set_name": "PubMed Central" }
Introduction ============ The extract of Colchicum autumnale, which is more commonly known as autumn crocus, wild saffron, naked lady, or any of several other names, has been used in the therapy of gout for more than 15 centuries.[@b1-ijn-6-2697] At present, it is still in clinical use for the treatment of gout, as well as several other inflammatory diseases including familial Mediterranean fever and Behçet's disease.[@b2-ijn-6-2697],[@b3-ijn-6-2697] Colchicine and its colchicinoid derivatives possess the ability to bind irreversibly to tubulin, forming tubulin-colchicine complexes, which hinder microtubule formation and inhibit cell mitosis.[@b2-ijn-6-2697]--[@b4-ijn-6-2697] It has been described that colchicine possesses anti-inflammatory properties, mainly mediated by inhibition of leukocyte adhesion and activity.[@b2-ijn-6-2697],[@b5-ijn-6-2697] At higher doses, tubulin-colchicine complexes induce depolymerization of microtubules, resulting in destabilization of the tubulin cytoskeleton.[@b4-ijn-6-2697],[@b6-ijn-6-2697],[@b7-ijn-6-2697] Whereas most cells rely on actin for their cell morphology, endothelial cells of angiogenic tumor vasculature are more dependent on tubulin to maintain their typically enlongated shape.[@b6-ijn-6-2697],[@b8-ijn-6-2697] Therefore, upon colchicinoid-induced microtubule depolymerization, the tumor endothelial cells lose their shape, thereby exposing the vascular basement membrane, which subsequently leads to coagulation, decreased perfusion, and hemostasis.[@b9-ijn-6-2697],[@b10-ijn-6-2697] This process, known as vascular disruption, deprives the surrounding (tumor) cells of oxygen and nutrients, leading to massive tissue necrosis. Currently, however, there is no use for colchicine and colchicinoids in cancer therapy due to their high systemic toxicity.[@b11-ijn-6-2697] Although in preclinical cancer models doses of colchicine higher than 5 mg/kg induce a significant reduction in the perfusion of tumors, the maximum tolerated dose (MTD) of colchicine is limited to around 1 mg/kg.[@b12-ijn-6-2697],[@b13-ijn-6-2697] Even doses below 0.5 mg/kg, as used in the clinical management of gout and familial Mediterranean fever, are frequently accompanied by gastrointestinal comorbidity (eg, nausea, vomiting, and diarrhea) and hematologic disorders, such as thrombocytopenia. [@b14-ijn-6-2697] Colchicine doses higher than 0.5 mg/kg are generally considered toxic, although lower doses may still cause significant side effects, illustrating its narrow therapeutic index. Overdosing of colchicine may eventually lead to multiple organ failure, including bone marrow suppression, hemolysis, liver failure, renal failure, convulsions, and cardiac arrest, and is often lethal.[@b14-ijn-6-2697],[@b15-ijn-6-2697] One strategy to limit the side effects caused by colchicinoid therapy is to design colchicinoid prodrugs, which possess pharmacological activity only upon conversion.[@b16-ijn-6-2697] Colchicinoids have a partition coefficient (log P) of around 1 and a relatively high volume of distribution (±2 L/kg), which implies that upon intravenous injection they immediately redistribute into the tissues, explaining the high risk for side effects.[@b17-ijn-6-2697]--[@b19-ijn-6-2697] Therefore, by creating a colchicinoid prodrug with improved aqueous solubility, its volume of distribution is expected to be reduced, confining its distribution to the circulation and extracellular compartment and lowering its off-target toxicity. Additionally, to keep the prodrug in the proximity of its target cells, that is, the angiogenic endothelial cells, the tissue penetration of the prodrug may be reduced by increasing its molecular weight. Previously, colchicinoid prodrugs based on glycopeptide dendrimers and cobalamin (vitamin B12) have been synthesized and characterized in vitro.[@b20-ijn-6-2697],[@b21-ijn-6-2697] However, to be converted to the active colchicinoid, both conjugates required cellular uptake in the tumor tissue. For exploiting the direct cytotoxic activity of colchicinoids -- the inhibition of tumor cell mitosis -- this is a rational approach. For colchicinoid-induced vascular disruption, however, a colchicinoid prodrug that is converted extracellularly, preferably in the proximity of the tumor vascular endothelium, is needed. This may be achieved by utilizing polymer-based colchicinoid prodrugs that are more readily transformed into the active colchicinoid, such as by hydrolysis of an ester bond which allows conversion in aqueous conditions. Previous work reported the synthesis of a hydrophilic colchicinoid prodrug, where colchicine was derivatized and conjugated to poly(ethylene glycol) (PEG) using a linker liable to hydrolysis.[@b22-ijn-6-2697] The synthesis of nanomedicines by conjugating PEG-chains (PEGylation) to low-molecular-weight drugs increases the hydrophilicity and size of the construct, and shields them from interactions with plasma proteins.[@b23-ijn-6-2697]--[@b25-ijn-6-2697] Upon intravenous injection, instantaneous and random diffusion of the colchicinoid prodrug into cells is impeded by the relatively large PEG moiety, thereby preventing the binding to tubulin and limiting its toxicity. However, due to the enhanced permeability of the imperfect angiogenic vasculature, the nanosized colchicinoid prodrug may be passively targeted to the tumor tissue, where, promoted by the reductive microenvironment in the tumor tissue, it hydrolyzes to the active colchicinoid.[@b26-ijn-6-2697] In the present study, a polymeric colchicinoid prodrug containing a hydrolysable linker was studied in vitro and in vivo for its therapeutic potential and toxicity as vascular disrupting agent. Materials and methods ===================== Synthesis of polymeric colchicinoid prodrug ------------------------------------------- Colchicine was derived and conjugated to PEG~5000~ using methodology reported elsewhere ([Figure 1](#f1-ijn-6-2697){ref-type="fig"}).[@b22-ijn-6-2697] In brief, colchicine was hydroxyl-functionalized by substituting the N-acetyl moiety with an N-2-hydroxyacetyl moiety. Subsequently, the hydroxyl group was reacted with methoxy PEG-acetic acid to obtain the hydrolysable polymeric colchicinoid prodrug. The amount of colchicine derivative per milligram of material (ie, colchicine equivalents) was determined by means of ultra performance liquid chromatography (UPLC) using an Acquity UPLC^®^ BEH C18 1.7 μm column (Waters, Milford, MA) and ultraviolet detection at 350 nm (Acquity UPLC^®^ PDA; Waters). The mobile phase consisted of a gradient from 5%--95% methanol in water (v/v) and trifluoroacetic acid as modifier. In vitro hydrolysis study ------------------------- The hydrolysis kinetics of the colchicinoid prodrug were determined at 4°C and 37°C in phosphate buffer (20 mM, pH 7.4). During 72 hours, samples were taken at regular time intervals, and stored at −20°C before analysis. For each time point, the concentration of colchicinoid prodrug and hydrolyzed prodrug were determined by UPLC, using the methodology described in the previous section. In vitro cytotoxicity --------------------- Human umbilical vein endothelial cells (HUVECs) were grown at 37°C and 5% carbon dioxide in angiogenic growth factor rich EGM^®^-2 medium (Lonza Ltd, Basel, Switzerland). Cells were seeded in 96-well plates (1 × 10^4^ cells/well) for 24 hours before further treatment. Subsequently, the cells were incubated with colchicine and colchicinoid prodrug at concentrations ranging from 0.025--2.5 μM colchicine equivalents. The cytotoxicity of each drug after 6 hours, 24 hours, and 48 hours incubation was determined by colorimetric XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) cell viability assay.[@b27-ijn-6-2697] In vivo vascular disrupting efficacy of colchicinoid prodrug ------------------------------------------------------------ All animal experiments were conducted in agreement with the local applicable Dutch law, "Wet op de dierproeven" (1977),[@b28-ijn-6-2697] and the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (1986).[@b29-ijn-6-2697] The mice were housed in steel cages, and water and food were provided ad libitum. Female pathogen-free C57BL/6 inbred mice of 21--24 g (Charles River Laboratories International, Inc, Wilmington, MA) were subcutaneously inoculated with 1 × 10^6^ B16F10 cells. Ten days after tumor cell inoculation, when tumor size reached \>100 mm^3^, phosphate buffered saline, colchicine (1 mg/kg), and the colchicinoid prodrug	{ "pile_set_name": "PubMed Central" }
I[ntroduction]{.smallcaps} {#sec1-1} ========================== The neonatal intensive care unit (NICU) is a specialized care unit that treats premature and medically fragile newborns and infants. Advancements in medical technology, focused-training of skilled medical personnel, and the establishment of critical care protocols have resulted in greater neonatal survival in the NICU. In the United States, although the number of babies admitted to the NICU has increased over the years to 77.9 per 1000 births,\[[@ref1]\] the infant mortality rate is at an all-time low with only 5.98 infant deaths per 1000 live births.\[[@ref2]\] Because of the growth in infant survival rates and NICU admission rates, a great deal of research has focused on identifying and creating the ideal NICU environment for both the neonate and the staff. Specifically, attention has been dedicated to monitoring and regulating the acoustic environment in the NICU. The current noise standards for the NICU environment were established decades ago and have not been modified. In 1974, the U.S. Environmental Protection Agency (EPA) recommended that indoor hospital areas maintain an average sound level of less than or equal to 45 decibels, A-weighted (dBA) during the day, and 35 dBA at night to maximize opportunity for patient recovery.\[[@ref3]\] In a 1997 Position Statement summarizing the effects of noise upon the developing fetus and the infant, the American Academy of Pediatrics (AAP) cited EPA standards and applied this recommendation to the NICU environment, stating that average sound levels in the NICU should not exceed 45 dBA.\[[@ref4]\] Over the following decades, several research groups have made additional or updated recommendations in an effort to re-evaluate the EPA and AAP standards. While the recommendations for the average sound levels in the NICU environment did not drastically change, new recommendations regarding the presence of transient sounds in the environment (i.e., doors slamming) were issued, with the maximum level for transient sounds (L ~max~ averaged over 1 s) being either 65 or 70 dBA.\[[@ref5][@ref6]\] Despite the presence of these recommendations, existing analyses of the acoustic environment in the NICU have indicated that these noise standards are being exceeded regularly. Studies from separate NICU environments have demonstrated that average noise levels range from 48 to 55 dBA\[[@ref7]\] and 53.9 to 60.6 dBA.\[[@ref8]\] An additional study found that sound levels exceeded the recommended standard more than 70% of the time,\[[@ref9]\] while another found that noise levels only met recommendations 5.51% of the time.\[[@ref10]\] Furthermore, it appears that NICU incubators also serve as a potential source of noise. Sound levels measured inside enclosed and activated incubators were never measured below recommended noise standards.\[[@ref7]\] In an effort to decrease noise levels and comply with recommendations, numerous noise abatement programs have been developed and implemented; however, none have been successful in creating an environment that effectively maintains current standards. Most programs have focused on a combination of staff training programs and minor structural modifications, but results have demonstrated that these adjustments did not result in any significant reduction in noise levels.\[[@ref11][@ref12][@ref13]\] Because acoustic noise cannot be avoided in the NICU, it is important to know the effect of noise on the neonate. A large contrast exists between the intrauterine and NICU acoustic environments. While the fetal peripheral auditory system is fully developed by approximately the 24^th^ week of gestation,\[[@ref14]\] responses to sound emerge earlier. Fetal responses to low frequency sounds emerge approximately six to eight weeks earlier than responses to high frequency sounds.\[[@ref15]\] This evolution of responses may reflect the natural maturation of the fetal central auditory system and have major implications for auditory brain development in infants born prematurely. Studies have demonstrated that exposure to sounds containing frequencies above 500 Hz is atypical for the developing fetus.\[[@ref16]\] Environmental sound is attenuated 40--50 dB at frequencies above 500 Hz by the body barrier of the womb and the impedance mismatch between air and embryonic fluid. In contrast, in the NICU environment, infants are exposed to acoustic frequencies higher than 500 Hz 57% of the time.\[[@ref17]\] Alarms and sounds emitted from necessary life support equipment such as extracorporeal membrane oxygenation machines may contain frequencies as high as 16,000 Hz.\[[@ref18]\] Therefore, infants who are born prematurely and cared for in the NICU are routinely exposed to sound levels and frequencies they are not developmentally prepared to handle.\[[@ref19]\] Numerous studies have demonstrated the adverse effects of the acoustic environment in the NICU by examining the relationship between acoustic events and alterations in infant physiologic state. Studies have demonstrated that high intensity, transient noises are associated with behavioral disturbances and increases in infant muscle tension.\[[@ref20]\] Other studies have documented a relationship between acoustic noise and changes in infant vital signs including heart rate, respiratory rate, O~2~ saturation, blood pressure, and intracranial pressure.\[[@ref21][@ref22][@ref23][@ref24][@ref25]\] Though the presence of a relationship between noise and infant state is well documented, it is important to consider how the effects of noise may be unique to the infant who is born prematurely. The preterm infant is physiologically immature in all of its major systems, including the central nervous system. As a result of global systemic immaturity and the lack of developed homeostatic mechanisms, preterm infants demonstrate distinctive responses to environmental stress in the NICU.\[[@ref26]\] Multiple studies have demonstrated that the effects of noise upon heart rate, respiratory rate, and oxygen saturation were more pronounced in preterm infants than in full-term infants.\[[@ref21][@ref27]\] Moreover, in a study that compared term and preterm infant reactions to acoustic stimuli, researchers found that preterm infants, unlike term infants, did not demonstrate the ability to habituate to the stimuli after repeated exposures.\[[@ref28]\] In addition to instantaneous changes in infant state, the stressful stimuli present in the NICU environment have also been associated with long-term effects on infant development. Physiologic stress responses to acoustic events such as changes in heart rate, intracranial pressure, and oxygen saturation may have a significant impact on the preterm infant's future neurologic development due to altered perfusion and oxygenation of the brain tissue.\[[@ref26]\] This effect is likely intensified by the preterm infant's inability to regulate these systems.\[[@ref29]\] Studies have also suggested that the stressful stimuli present in the NICU environment may be associated with an increased risk for future attention, language, and hearing disorders, which may be partially attributed to the immature auditory system's exposure to certain types of noise in the NICU and limited exposure to speech and language.\[[@ref30][@ref31]\] Because of the negative outcomes associated with NICU stay, researchers agree that the primary purpose for establishing and maintaining noise standards in the NICU should be to create the most ideal environment for the fragile neonate to grow, heal, and thrive. The NICU environment is full of stressful events, and premature infants are forced to expend a significant amount of energy mediating these stressful stimuli. The goal should be to remove as many of these stressors as possible, so that neonates can reserve their energy for healing, which may result in a reduction in their NICU stay and earlier release to their families.\[[@ref32]\] To accomplish this, attention must be given to the infant's sensory systems, and recommendations for all exposures in the NICU must yield a supportive, nurturing setting that optimizes the neurodevelopmental outcomes of preterm infants.\[[@ref10][@ref33][@ref34][@ref35]\] S[tudy]{.smallcaps} A[im]{.smallcaps} {#sec1-2} ===================================== Given the evidence demonstrating an inability for NICUs to comply with current noise level recommendations, the known effects of noise on the physiologic state of the infant, and the long-term impact of certain types of acoustic stimuli on the developmentally immature preterm infant, the aim of the present study is twofold: first, to identify the type, rate, and levels of acoustic events that occur in the NICU, as well as the signal-to-noise ratios (SNRs) corresponding to those events; and second, to identify how these events may affect infant physiologic state as measured by changes in infant heart rate and respiratory rate. This information will lead to a more precise understanding of the characteristics of acoustic events that may impact infant physiologic state and could lead to the development of more practical NICU noise standards that allow for both protection of the infant and reasonable implementation by hospital staff. M[aterials and]{.smallcaps} M[ethods]{.smallcaps} {#sec1-3} ================================================= Sound level recordings {#sec2-1} ---------------------- Larson Davis Spark 706RC, 705+, and 703+ Type II Noise Dosimeters (Larson Davis Laboratories, Provo, Utah) were used to record the acoustic environment in both the open and private room settings in the Level IV NICU at St. Louis Children's Hospital. Recordings took place during the morning and afternoon hours of February 23, 2017. In the private room, dosimeters were placed on a narrow counter behind the infant isolette, and microphones were taped near the level of the infant's head. In the open room, dosimeters were placed on a counter with microphones taped to a post located between two isolettes. [Figure 1](#F1){ref-type="fig"} shows the sound recording diagram for both the open (a) and private (b) room. All dosimeters were set to A-weighted, fast detector setting (0.125-s interval), 1-s sample interval, 30 dB gain, and 3 dB exchange rate. Each dosimeter was calibrated before and after the recording session and was found to be following appropriate American National Standards	{ "pile_set_name": "PubMed Central" }
Introduction {#Sec1} ============ The use of combined modality treatment, including surgery, chemotherapy, and radiation, has resulted in increased disease control in locally-advanced head and neck cancer (HNC) \[[@CR1]\], but improved disease control occurs at the expense of increased acute and late effects from therapy. In the HNC population, acute tumor and treatment effects have garnered tremendous interest and have been extensively investigated. Until recently, late effects and their sequelae have been largely underrecognized and underappreciated. Improved treatment methodologies and the changing epidemiology, most notably the rise in HPV-associated oropharyngeal cancers, have resulted in a rapid increase in the number of HNC survivors. Accordingly, this expanding survivor population has generated a surge of interest in the late effects of HNC therapy. Evolving data demonstrate that acute toxicities may persist long-term and develop into late effects. In addition, late effects may manifest months or years after completion of therapy, persisting for years or even lifelong, far longer than previously believed \[[@CR2], [@CR3]\]. When severe, late effects may profoundly affect function and quality of life \[[@CR4]\]. The most frequently studied late effects of therapy are those that are due to local tissue damage from cancer or its therapy. However, late systemic symptoms, which may have a more ubiquitous and profound impact on long-term function, have remained elusive from the standpoint of both research and management. Systemic symptoms, also known as sickness behaviors, include fatigue, central pain, neurocognitive dysfunction, mood disorders, thermal discomfort, sweating, gastrointestinal symptoms, and sleep disturbances. Systemic symptoms tend to occur in clusters, whichis felt to be due in part a common underlying pathobiology. While the mechanisms and pathways that contribute to systemic symptoms have yet to be fully elucidated, neuroinflammation is believed to be one of the important connective threads. During the acute phase of illness or injury, the body must coordinate complex biologic pathways and behaviors in order to optimize the body's response to disease and promote healing \[[@CR5]--[@CR7]\]. The illness response is mediated in part through peripheral pro-inflammatory and immune-activating cytokines which act as peripheral-to-central nervous system signaling molecules. The peripheral cytokines can induce a neuroinflammatory state and its associated systemic symptoms. Acutely, the illness response characterized by systemic symptoms such as fever, lethargy, and anorexia may be adaptive \[[@CR8], [@CR9]\]. However, if the inflammatory signal is overly exuberant or protracted, functional and anatomical central nervous system changes may develop. This may result in protracted or chronic systemic symptoms \[[@CR9]--[@CR11]\]. HNC and its treatment are both associated with elevations in peripheral pro-inflammatory cytokines \[[@CR12], [@CR13]\]. While the level of pro-inflammatory cytokines has been correlated with the grade of acute toxicity \[[@CR14]\], the relationship between pro-inflammatory cytokines and late effects has not been reported. Furthermore, while available data indicate that head and neck cancer patients experience chronic systemic symptoms such as fatigue, anxiety, and depression \[[@CR10], [@CR15], [@CR16]\], data describing the breadth, severity, and impact of late systemic effects are not available. To that end, we conducted a cross-sectional, observational, mixed-methods study in HNC survivors to determine the prevalence and impact of late systemic symptoms. Herein we report the results of the quantitative analysis. Materials and methods {#Sec2} ===================== Patients {#Sec3} -------- All patients were recruited from the Henry Joyce Cancer Clinic in the Vanderbilt-Ingram cancer center between November 6, 2014 and November 21, 2016. Patients included in this analysis were consented to participate in two clinical trials, the first of which included 92 patients and was entitled "Characterization of Chronic and Unremitting Symptoms in Long Term Survivors of Head and Neck Cancer." The second study, entitled "Characterization of Chronic Systemic Symptoms among Participants in the Fibrosis-Lymphedema Continuum Study in Head and Neck Cancer," included 13 patients who participated in an earlier, R01-funded study and had agreed to be contacted for participation in subsequent clinic trials. A convenience sample of 105 patients completed study measures and were included in the analysis. Study eligibility criteria for both trials included the following: age 21 years or older, the ability to speak English, a history of histologically-proven HNC, completion of treatment a minimum of 12 months prior without evidence of recurrence. All eligible patients were approached and provided with information about the study. Interested patients were contacted by study staff and signed informed consent prior to completing study-related questionnaires. Methods {#Sec4} ------- After signing informed consent, the participants completed the study questionnaires on an electronic web-based electronic data capture application (REDCap™). Questionnaires {#Sec5} -------------- ### Socio-demographic data form (self-report) {#FPar1} Captured birthdate, gender, race, ethnic category, highest educational level, marital status, employment status, area of residence, insurance coverage, and annual household income. ### Disease and treatment data form (medical record review by study staff) {#FPar2} Captured data related to the patient's cancer and treatment including diagnosis date, location, stage of disease, surgical treatment, medical oncology treatment, and radiation oncology treatment. ### Patient-reported outcome measures {#FPar3} Patient-reported outcome (PRO) measures were included to address common local symptoms in the HNC population (Vanderbilt Head and Neck Symptom Survey version 2.0) as well as systemic symptoms (General Symptom Survey, Profile of Mood States-Short Form, Neurotoxicity Rating Scale). In addition, questionnaires were included to address body image and quality of life due to our interest in assessing the relationship between systemic symptoms and these outcomes. ### Vanderbilt Head and Neck Symptom Survey version 2.0 plus General Symptom Survey (VHNSS v2.0 plus GSS) {#FPar4} The VHNSS v2.0 \[[@CR17]\] assesses the prevalence and severity of treatment-related symptoms and their functional impact in patients with head and neck cancer. The VHNSS v2.0 consists of 50-items within 13 domains including nutrition, swallowing, xerostomia, mucositis, excess mucus, speech, hearing, taste change, smell, dental health, mucosal sensitivity, range of motion, and pain. Items are scored on a numeric scale rating the severity of the symptom from 0 (none) to 10 (severe). The VHNSS v2.0 takes approximately 10 min to complete. Cronbach's alpha are \> 0.9 in six symptom clusters and \> 0.7 in the four remaining clusters \[[@CR17], [@CR18]\]. The GSS includes 11 additional items directed toward the systemic effects of cancer and therapy. Items are scored on a scale of 0 (none) to 10 (severe). The General Symptom Survey was specifically developed to assess the systemic symptoms associated with head and neck cancer and its therapy through review of the systemic symptom literature, patient interviews, and expert panel review. Content validity is being tested in an accompanying qualitative analysis to be published separately. Systemic symptoms investigated in this report include the items in the GSS plus two items from the VHNSS ("weight loss" and "loss of appetite"). ### Profile of Mood States-Short Form {#FPar5} The Profile of Mood States-Short Form (POMS-SF) is a psychological evaluation tool used to assess mood states. This tool contains a 37-item scale consisting of adjectives rated on a 5-point Likert-like scale. It is composed of six subscales: depression (maximum possible score 28), vigor (maximum possible score 20), confusion (maximum possible score 20), esteem-related affect (24), tension (maximum possible score 24), anger (maximum possible score 24), and fatigue (maximum possible score 20). Cronbach's alphas range from 0.78 to 0.91 \[[@CR19]\]. ### Neurotoxicity Rating Scale {#FPar6} The Neurotoxicity Rating Scale (NRS) is a self-report measure examining neurocognitive symptoms associated with neurotoxicity of medical treatment. Its 37 items are symptoms rated in severity using a 5-point Likert-like scale bounded by "not present" and "extremely severe" \[[@CR20]\]. Seven items from the NRS (restlessness, no interest in people, distractibility, irritability, decreased motivation, tension, and slowed movements) were chosen for inclusion in this analysis. Although the NRS has not been validated in the oncologic population, the selected items address unique symptoms that may be related to neuroinflammation and are absent in the other tools. ### Body image quality of life inventory {#FPar7} The body image quality of life inventory (BIQLI) is a 19-item instrument which was developed to quantify the effects of body image on various experiences and life contexts \[[@CR21]\]. Participants rate the impact of their own body image using a 7-point bipolar scale from − 3 to + 3, thereby permitting reports of negative, positive, or no impact \[[@CR22]\]. Overall impact of body image can be determined by averaging the scores of all items. The Cronbach's alpha of the scores in this study was 0.90 \[[@CR21]\]. ### Quality of life {#FPar8} QOL was measured using two scales: a 5-item domain-specific QOL and a single-item self-anchoring scale \[[@CR23], [@CR24]\]. Statistical analysis {#Sec6} -------------------- SPSS version 24.0 was used for statistical	{ "pile_set_name": "PubMed Central" }
Introduction {#sec1-1} ============ ^131^I-NaI has been used very effectively over several years for both the diagnosis and therapy of differentiated thyroid carcinoma (DTC). ^131^I whole body scintigraphy has been used for detection of remnant, residual as well as metastatic disease in patients of DTC. Though highly specific, ^131^I whole body scans also show a number of false positive accumulations, while not many. It is essential to correctly identify these false positive lesions at the earliest in order to avoid subsequent unnecessary radioiodine treatment in these cases. Here, we present a case series of three cases of unusual false positive radioiodine uptake on whole body iodine-131 (^131^I scan) in three different organs and three different pathologies that demonstrated this uptake. Case Reports {#sec1-2} ============ Case 1 {#sec2-1} ------ A 36-year-old female, with diagnosis of multifocal papillary carcinoma thyroid (initial presentation with focal hypoechoic space-occupying lesion (SOL) in the right lobe (1.7 cm × 1.2 cm) with vascularity within, had undergone total thyroidectomy and treatment with 54 μCi of ^131^I subsequently. Posttherapy scan showed multifocal iodine-avid foci in the neck. Large dose scan with ^131^I \[[Figure 1a](#F1){ref-type="fig"}\] after 6 months of therapy showed an iodine focus in the right side of the chest \[thyroglobulin (Tg) and thyroid stimulating hormone (TSH) values mentioned in [Table 1](#T1){ref-type="table"}\]. Chest x-ray was normal and as Tg was low; this iodine-avid focus in the right lung was investigated further to rule out a false positive etiology. Fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) was done at that time, which showed a very low grade FDG uptake in a nonactive fibrotic lesion in the right lung on computed tomography (CT), corresponding to the lesion seen in the iodine scan. The repeat diagnostic radioiodine scan was repeated after 1 year, which again showed persistence of the same lesion in the right lung (Tg and TSH values mentioned in [Table 1](#T1){ref-type="table"}). Correlative SPECT-CT images \[[Figure 1b](#F1){ref-type="fig"} and [c](#F1){ref-type="fig"}\] in the lung and mediastinal window and FDG-PET/CT \[[Figure 1d](#F1){ref-type="fig"}\] showed a spiculated nonehnacing lesion with adjacent area of fibrosis in the posterior segment of the right upper lobe and 1 cm-sized round node in the subcarinal region. CT features of the lung lesion on the right were in favor of benign pathology (infective sequela). ![Planar 131I whole body scan (a) showing iodine-avid focus in the right upper lung. Correlative SPECT-CT (b and c) in the lung and mediastinal window and FDG-PET/CT images (d) showing a spiculated nonenhancing lesion with adjacent area of fibrosis in the posterior segment of the right upper lobe and 1 cm-sized round node in the subcarinal region. CT features of the lung lesion on the right were in favor of benign pathology (nonactive infective sequela)](WJNM-15-137-g001){#F1} ###### Serum Tg and TSH values of Patient 1 at various time course ![](WJNM-15-137-g002) Case 2 {#sec2-2} ------ A 38-year-old female, with diagnosis of multicentric follicular variant of papillary carcinoma thyroid who had prior treatment with total thyroidectomy and 49 μCi of ^131^I, showed iodine-avid foci in the neck and mediastinum in the posttherapy scan. Large dose scan with ^131^I \[[Figure 2a](#F2){ref-type="fig"}\] after 6 months therapy showed an iodine foci in the neck with neck uptake being 0.27% and iodine focus in the right lower chest region (Tg and TSH values mentioned in [Table 2](#T2){ref-type="table"}). SPECT-CT was done, which showed the focus in segment VIII of the liver that was hypodense on CT \[[Figure 2b](#F2){ref-type="fig"}\]. PET-CT with FDG \[[Figure 2c](#F2){ref-type="fig"}\] was done at that time, which showed non-FDG avid hypodense segment VIII liver lesion. The ultrasonography (USG) showed a well-defined lesion with posterior acoustic enhancement and no intralesional vascular flow on Doppler imaging. The T2-weighted (T2W) images revealed a well-defined hyperintense lesion, which did not show any enhancement on the postcontrast T1-weighted (T1W) magnetic resonance imaging (MRI) \[[Figure 2d](#F2){ref-type="fig"}-[f](#F2){ref-type="fig"}\]. All these imaging features were suggestive of simple cyst of the liver. Thus, with low Tg and other modalities ascertaining benign etiology, the cystic liver lesion was considered as a false positive. ![The diagnostic 131I scan (a) showing iodine-avid focus in segment VIII of liver; Correlative SPECT CT \[a\] and PET CT images (c) in abdomen window shows a well-defined round hypodense lesion in segment VIII of the liver, which showed iodine avidity on SPECT scan. T2W image (d) showing well-defined hyperintense lesion (star) in segment VIII with no significant enhancement in postcontrast fast spoiled gradient recalled (FSPGR)-echo images (e). Diffusion MRI (f) showed facilitated diffusion with high apparent diffusion coefficient (ADC) values. The correlated USG (g) shows anechoic lesion with posterior acoustic enhancement (arrowhead). All these imaging features were suggestive of simple cyst of the liver](WJNM-15-137-g003){#F2} ###### Serum Tg and TSH values of Patient 2 at various time course ![](WJNM-15-137-g004) Case 3 {#sec2-3} ------ A 36-year-old female with history of follicular variant of papillary carcinoma thyroid for which she was treated with total thyroidectomy and 49 μCi of ^131^I postoperatively, demonstrated an iodine-avid foci in the left side of the chest on large dose scan with ^131^I after 6 months of therapy (Tg and TSH values mentioned in [Table 3](#T3){ref-type="table"}). The chest x-ray was normal and as Tg was low, this iodine-avid focus in the left chest region was investigated further. SPECT-CT showed iodine-avid foci in the left breast \[[Figure 3a](#F3){ref-type="fig"} and [b](#F3){ref-type="fig"}\]. Whole body FDG-PET/CT was normal. Mammogram as well as USG of both the breasts showed no focal abnormality \[[Figure 3c](#F3){ref-type="fig"}\]. The patient retrospectively gave a past history of fibroadenoma excision from the left breast undertaken previously. The findings of low Tg as well as that of other imaging modalities showed normal glandular architecture of the left breast; the left breast lesion was a false positive lesion. ###### Serum Tg and TSH values of Patient 3 at various time course ![](WJNM-15-137-g005) ![The large dose ^131^I scan (a) showing iodine-avid focus in the left breast. Correlative SPECT-CT (b) showing normal glandular breast parenchyma in the area of the iodine-avid uptake. Left side of the craniocaudal (a) and mediolateral (b) view mammography (c) revealed normal scattered glandular parenchyma (star) with no obvious focal pathology/lesion; FDG-PET/CT was normal in this patient. The patient had previous history of fibroadenoma excision](WJNM-15-137-g006){#F3} Discussion {#sec1-3} ========== ^131^I-NaI is considered as a very specific radiopharmaceutical with respect to detecting thyroid pathology, particularly in patients of DTC following thyroidectomy. The major mechanisms considered important for radioiodine uptake are: 1\) Functional sodium/iodide symporter (NIS) expression (in normal tissues, including thymus, breast, salivary glands, and gastrointestinal tract, or various benign and malignant tumors of these organs), 2) Metabolism of radioiodinated thyroid hormone, 3) Retention of radioiodinated body fluids (saliva, tears, blood, urine, exudate, transudate, gastric, and mucosal secretions, etc.) associated with or without structural change, 4) Retention and uptake of radioiodine in inflamed tissue, 5) Contamination by physiologic secretions, and 6) Other unknown factors.\[[@ref1]\] Various authors have over the years reported the importance of identification of false positives in iodine scans in the form of case reports,\[[@ref2]\] where false positive findings have been reported in various organs of the body. False positive radioiodine uptake in the chest has been reported due to acute respiratory infection,\[[@ref3][@ref4	{ "pile_set_name": "PubMed Central" }
Introduction {#Sec1} ============ NK cells represent a subset of lymphoid cells that are components of innate immunity acting as first line of defense against viral infection and other pathogens, and in the early cellular transformation and tumor surveillance^[@CR1]^. The functions of NK cells are partly regulated by the family of KIR receptors (killer cell immunoglobulin-like receptor) coded by 11 genes (2DL1, 2DL2/2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS4, 2DS3/2DS5, 3DL1/3DS1, 3DL2 and 3DL3) and two pseudogenes (2DP1 and 3DP1) located on the chromosome 19q13.4^[@CR2]--[@CR4]^. Some of these genes may present sequence variations; thus, it has been reported a 22 bp deletion in the second extracellular domain of 2DS4 that affect substantially the sequence of amino acids, whereas the exon 2 can be absent in 3DP1^[@CR5]^. Also, it has been found that 2DL5 gene is encoded by different loci (A and B)^[@CR6]^. The KIR family is primarily expressed on NK cells, but they can also be expressed on CD4, CD8 and γδ T cells^[@CR7]--[@CR9]^. There are four promoter types based on intermediate promoters (ProI), which are associated with distinct expression in KIR genes, thus 2DL2, 2DS2 and 2DL3 are the first to be expressed by NK cells after Hemopoietic Stem Cell Transplantation^[@CR10]^. The 3DL3 is not expressed by circulating CD56 dim NK cells, and 2DL4 is expressed by CD56-bright and dim NK cells in a non-variegated manner; and finally, the remaining KIR genes are expressed by CD56-dim NK cells^[@CR10],[@CR11]^. T cells express 3DL2 more than other KIR genes, probably as a result of ProI activation earlier in the development of T cell^[@CR10]^. In addition, the KIR gene family has bi-directional promoters, which control variegated expression, whereas ProI correlates with protein expression^[@CR10]^. Composition of KIRs may be complex, thus, two haplotypes (A and B) and genotypes (AA and Bx, where x can be A or B) have been reported for KIR based on gene content^[@CR12]^ (Fig. [1](#Fig1){ref-type="fig"}). Actually, there are over 500 different Bx genotypes (<http://www.allelefrequencies.net>). KIR genotype AA is homozygous for the A haplotype, which is an inhibitory haplotype formed by the loci 3DL3, 2DL3, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 2DS4 and 3DL2; whereas Bx genotype has either one (AB heterozygous) or two (BB homozygous) B haplotypes, and is an activator haplotype (formed by 3DL3, 2DS2, 2DL2, 2DL5B, 2DS3/2DS5, 2DP1, 2DL1, 3DP1, 2DL4, 3DS1, 2DL5A, 2DS3/2DS5, 2DS1, 2DS4 and 3DL2 genes)^[@CR13]^. The A haplotype usually has a fixed number of genes, while B has a variable gene content with additional activating KIR genes. KIR haplotypes consists of two regions, the centromeric region from 3DL3 to 3DP1, and the telomeric region from 2DL4 to 3DL2; and both regions can be cenA or cenB, and telA or telB depending on the haplotype^[@CR13],[@CR14]^. 2DL5, 2DS3 and 2DS5 genes have been identified in centromeric and/or telomeric region^[@CR14]^. Based on the gene content 9 centromeric regions (cA01, cA02, cA03, cB01, cB02, cB03, cB04, cB05 and cB06) and 8 telomeric regions (tA01, tB01, tB02, tB03, tB04, tB05, tB06 and tB07) have been described^[@CR14]--[@CR17]^. KIR B haplotype can also be classified according to B content genes, and B content score is calculated by adding the number of cenB and/or telB motifs in each genotype^[@CR18]^.Figure 1Composition of KIR haplotypes (A and B) based on gene content. KIR genes are tightly organised head-to-tail over approximately 150 kb within the Leukocyte Receptor Complex (LCR). Inhibitory KIR genes are shown in white, activating genes in black and pseudogenes in gray. Helicobacter pylori (H. pylori) infects the gastric mucosa of over 50% of the world population and represents the main risk factor to develop gastric cancer (GC) and duodenal ulcer^[@CR19],[@CR20]^. Different immune cells are involved in the development of gastric pathologies by causing a chronic, unregulated mucosal inflammation^[@CR21]^. Thus, cells of the innate and adaptive system such as T lymphocytes and natural killer (NK) cells have a critical role in the regulation of the immune response^[@CR21]^. H. pylori causes an increase of NK cells in the gastric mucosa, where they produce TNF-α and INF-γ^[@CR21]--[@CR23]^, and have an important role in the inflammatory process that drive tissue damage. In this work we aimed to study polymorphisms in KIR receptors genes and identify any possible association with GC. Results {#Sec2} ======= The characteristics of the population studied are described in Table [1](#Tab1){ref-type="table"}. We studied two groups of patients, one with a diagnosis of NAG and the other with GC, formed by 130 and 112 patients respectively, and both were compared with an asymptomatic group (n = 146). It can be observed that the NAG group showed significantly higher seroprevalence to H. pylori and to CagA, with an OR of 3.23 and 2.74, respectively, as compared to the asymptomatic patients.Table 1Characteristics of the groups of patients studied.Diagnosis groupn^a^Age, years, median (IQR)^b^Female/MaleH. pylori +/− (%)OR (95% CI)^c^CagA + /− (%)OR (95% CI)^c^Asymptomatic14641 (36--50)54/92109/37 (74.7)74/72 (50.7)Non-atrophic gastritis13047 (39--57)93/37111/19 (85.4)^d^3.23 (1.39--7.54)89/41 (68.5)^d^2.74 (1.39--5.42)Gastric cancer11259 (49--72)49/6387/25 (77.7)2.56 (0.93--7.02)65/47 (58)2.27 (0.99--5.21)^a^n = number of subjects. ^b^IQR = interquartile range. ^c^OR estimated using the asymptomatic group as the reference and adjusted by age and gender. ^d^p \< 0.05. KIR genes {#Sec3} --------- In order to characterize the KIR genotype frequencies in the study groups, genomic DNA was isolated from peripheral blood leukocytes, and the KIR genes responsible for the activating signals (2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DS1), the inhibitory signals (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 3DL1, 3DL2, 3DL3), and the two pseudogenes (2DP1 and 3DP1) were genotyped using single specific primer-polymerase chain reaction (SSP-PCR). KIR genotypes were assembled according to the presence or absence of each gene locus. The frequency data obtained was analyzed between groups to determine differences in KIR genes between asymptomatic and disease groups. The framework genes of centromeric (3DL3 and 3DP1) and telomeric (2DL4 and 3DL2) regions were present in 100% of the three groups studied. The frequencies of 2DL1 (99.3%, 99.2% and 100%), 2DL3 (97.9%, 96.9% and 92.9%), 2DS4 (93.8%, 100% and 88.4%) and 2DP1 (99.3%, 99.2% and 100%) genes were not statistically different among the groups (Asymptomatic, NAG and GC, respectively). The KIR genes with a significant association with disease are shown in Table [2](#Tab2){ref-type="table"}. When compared with healthy controls, most of the activating and inhibitory genes studied were found significantly associated with either NAG	{ "pile_set_name": "PubMed Central" }
![](edinbmedj74360-0090){#sp1 .890}	{ "pile_set_name": "PubMed Central" }
Introduction ============ Arising from notochord remnants such as the clivus, retropharyngeal space, and the ventral part of the cranio-vertebral junction (CVJ), chordoma has been considered a clinically malignant tumor because of its active biological nature. Maximal resection of the tumor is required because these tumors have high recurrence rates, and controversially, the surgical treatment should be minimally invasive to maintain the patient\'s activities of daily living (ADL).^[@B1]--[@B5]^ Gross total resection is difficult because of tumor extension and surgeons should select the most suitable approach.Historically, various midline anterior approaches to the CVJ have been employed. The transoral approach (TOA) is one of the midline anterior approaches, and provides a direct extradural route without brain retraction around the CVJ. Surgical exposure via the TOA is limited by the extent of the patient\'s mouth opening, and TOA has been modified in various ways to extend the surgical field for caudal and rostral tumor extension. These modifications include the transpalatal approach (TPA), the transmaxillary approach, and the mandibular swing-transcervical approach.^[@B6]^ TPA can offer a TOA surgical field for rostral tumor extension by removal of the hard palate. However, these approaches are invasive and reconstruction takes many hours. The recently developed endoscopic endonasal approach (EEA) is useful for treating lesions not only in the sella turcica but also around the clivus. This approach is considered minimally invasive to neurovascular structures, changing the history of treatment of skullbase tumors.^[@B7]--[@B16]^ However, removal of lesions around the CVJ is difficult even when using the EEA, because the hard palate is located at the same level as the CVJ, and the surgical view of lesions around the CVJ when using the EEA is obstructed by the hard palate. The present study is a retrospective analysis of chordomas operated in a single institution and resected by EEA or transoral-transpalatal approach (TO-TPA) during the period 2002--2012. The study aimed to compare the surgical results and find a suitable approach for clival and upper cervical chordomas. Patients and Methods ==================== Between 2002 and 2012, 18 patients---11 females and 7 males---aged 10--78 years (mean 43.5 years), underwent EEA or TO-TPA. Each approach was performed on nine patients. We started using the EEA in conjunction with the Department of Otolaryngology in 2008. Subsequently, the transnasal approach with a microscope was replaced with endoscopy. TO-TPA was performed in conjunction with the Department of Plastic surgery. The patients\' symptoms, upper and lower limits of surgical location, extent of resection, incidence of cerebrospinal fluid (CSF) leakage and complications were analyzed. The upper and lower margins of the tumor were confirmed by pre-operative magnetic resonance imaging (MRI). The degree of resection was classified as gross total removal (GTR, confirmed by surgical observation and MRI after surgery), subtotal removal (STR, more than 90%), or partial removal (PR, less than 90%). Operative time, pre- and post-operative Karnofsky performance status (KPS) scores, and hospitalization were analyzed in both groups. Post-operative KPS was evaluated at discharge. Hospitalization does not include the stay for post-operative radiation therapy. Operative Procedures ==================== I.. Transoral-transpalatal approach ----------------------------------- General anesthesia was performed using standard endotracheal intubation. The patient was positioned supine with the head down using a shoulder pad, without head fixation. A 2-cm incision was made in the mucosa of the midline gingivo-buccal sulcus and the anterior nasal spine was exposed. The submucosal nasal floor was dissected along the palate, and the nasal septum cut and freed from the palatal bone. After this procedure, a Crockard retractor was attached to open the mouth. A U-shaped mucosal incision was made on the palate, along the maxillary alveolar process to the second/third molars ([Fig. 1](#F1){ref-type="fig"}). A midline palatal incision was added along the palatine raphe, sparing the uvula, to form the muco-periosteal flaps, which were supplied by the palatal artery, and were retracted laterally using strings ([Fig. 2](#F2){ref-type="fig"}). The exposed palatal bone was drilled and cut along the margin of the mucosal incision using a chisel, and the horseshoe-shaped palatal bone was removed. By retraction of the muco-periosteal flaps using the Crockard palatal arms, a long surgical field from the posterior nasal cavity to the pharynx was obtained ([Fig. 3](#F3){ref-type="fig"}). In the middle surgical field, the pharyngeal tonsil was observed. The epidural tumor was exposed using a straight or S-shaped pharyngeal incision. The surgical field covered the total length of the clivus, cranio-cervical junction, and C2 body. Bone resection along the tumor margin is required to achieve total resection of the chordoma. In a tumor with subdural invasion, the dural defect was covered with abdominal fat and coated with fibrin glue, and the mucosa was tightly sutured. Cases in which a mucosal defect was present, especially on the pharyngeal tonsil, a septal mucosal flap was used for closure. After tumor removal, the palatal bone was replaced and fixed by suturing the muco-periosteal flap in two layers ([Fig. 4](#F4){ref-type="fig"}). The tracheal tube was removed the next day, but tube feeding was necessary for 1 week after surgery. II.. Endoscopic endonasal approach ---------------------------------- The surgical approach and tumor resection were performed using a straight 4-mm endoscope; observations were made using angled scopes. An image guidance technology navigation system was also used. Resection of the right middle turbinate was performed and a nasoseptal flap was created. When the tumor extended into the subdural space, the binostril four-hand technique was applied to obtain a wider surgical space, and the posterior segment of the nasal septum was resected. Drilling of the floor of the sphenoid sinus and clivus were performed towards the CVJ, if necessary ([Fig. 5](#F5){ref-type="fig"}). The vidian canal was a good landmark for the carotid arteries. The subdural component was removed carefully because the tumor was noted to involve the basilar arteries and brainstem. Reconstruction was performed using a multilayer approach: the fascia as a subdural inlay, a pedicled nasoseptal flap to cover the fascia, and oxidized cellulose with fibrin glue at the dural and osseous edges. Pressure was applied using a 14-F Foley catheter to maintain the multilayer reconstruction. Three or four days after the operation, the catheter was removed. Results ======= The upper limit of the surgical approach was the frontal skull base in both TO-TPA and EEA. For EEA, the upper limit was the posterior wall of the frontal sinus, and for TO-TPA, the upper limit was the optic chiasma. The lower limit of the surgical approach was the C3 vertebral body in TO-TPA, and the C1 level in EEA. [Table 1](#T1){ref-type="table"} shows a clinical summary and the surgical results for the TO-TPA group, and [Table 2](#T2){ref-type="table"} shows those for the EEA group. In the TO-TPA group, GTR was limited in a case of upper cervical chordoma (Case 9). However other large tumors extending from the clivus to the upper cervical spine were not completely removed (STR 4, PR 4) because of lateral tumor extension into the parapharyngeal space. However, in the EEA group, all tumors except one were localized only in the clivus. GTR was achieved in 3 cases and STR in 2 cases, and the incidence of subdural invasion was the same as that in the TO-TPA group (5 cases each). Surgical complications were lower in TO-TPA than EEA. One patient, who had been treated with heavy-ion radiation pre-operatively, complained of pharyngeal fistula after surgery. No post-operative CSF leakage was observed in the TO-TPA group; the subdural part was removed only in the case with a dural defect smaller than 10 mm, with the aim of preventing CSF leakage ([Fig. 6](#F6){ref-type="fig"}). In the EEA group, surgical complications occurred in 4 cases: postoperative meningitis with or without CSF leakage, acute hydrocephalus with brain stem infarction, and diabetes insipidus. They occurred only in tumors with subdural extension, and these EEA complications were caused by active surgical removal in the subdural space ([Fig. 7](#F7){ref-type="fig"}). In the TO-TPA group, mean operative time was 7.52 hours. On the other hand, mean operative time was 5.25 hours in the EEA group. Oral intake was started an average of 15.5 days after surgery in the TO-TPA group, while all patients in the EEA group started oral intake the day after the operation. The mean hospitalization in the EEA group was almost same as that of TO-TPA group: 41 days in the EEA group and 38.9 days in the TO-TPA group. In patients without surgical complication,	{ "pile_set_name": "PubMed Central" }
Introduction {#s1} ============ Pulmonary fibrosis can result from a variety of causes, including lung injury, environmental particle and toxin inhalation, chemotherapy, systemic autoimmune diseases, or as an idiopathic entity in form of idiopathic interstitial pneumonias (IIP) [@pone.0002142-Kim1]--[@pone.0002142-Martinez1]. Idiopathic pulmonary fibrosis (IPF), the most common form of IIP, represents a progressive and lethal disorder with unresolved pathogenesis and unresponsiveness to currently available therapies [@pone.0002142-Walter1]. Distortion of the normal lung architecture in IPF is evident by temporo-spatially heterogeneous histology, including areas of normal parenchyma, mild interstitial inflammation due to mononuclear infiltrates, septal fibrosis with subepithelial fibroblast foci, and honeycombing [@pone.0002142-Visscher1], [@pone.0002142-AL1]. Fibroblast foci represent the hallmark lesions of IPF, as they constitute aggregates of activated myofibroblasts, which promote excessive ECM deposition [@pone.0002142-AL1]. The occurrence of fibroblast foci represents an important prognostic factor, since their numbers have been correlated with survival in IPF [@pone.0002142-King1]. Fibroblast foci occur in subepithelial layers, close to areas of alveolar epithelial cell injury and repair, suggesting that impaired epithelial-mesenchymal crosstalk contributes to the pathobiology of IPF [@pone.0002142-King1], [@pone.0002142-Noble1]. Indeed, it is well accepted that repetitive injury and subsequent repair of alveolar epithelial type II (ATII) cells, in the presence or absence of local inflammation, represent a key pathogenic mechanism in IPF, which leads to aberrant growth factor activation and perpetuation of fibrotic transformation [@pone.0002142-Selman1]. Although several soluble mediators, such as transforming growth factor (TGF)-β1 or interleukin (IL)-1β, have been assigned a clear pathogenic role in IPF and experimental models thereof (9, 10), therapeutic options neutralizing their activity have not been successful in clinical use as of yet. The Wnt family constitutes a large family of highly conserved secreted growth factors essential to organ development, a process often recapitulated in organ failure. The best characterized Wnt signaling pathway is the β-catenin-dependent, or canonical, Wnt signaling pathway [@pone.0002142-Moon1]--[@pone.0002142-Johnson1]. Here, in the absence of active Wnt ligands, β-catenin is constitutively phosphorylated by its interaction with axin, adenomatosis polyposis coli (APC), and glycogen synthase kinase (Gsk)-3β, and subsequently degraded. In the presence of Wnt ligands, two distinct membrane receptors, the frizzled (Fzd) or the low density lipoprotein receptor-related proteins (Lrp) 5 and 6, are activated upon ligand binding. In detail, Wnt stimulation leads to phosphorylation of Lrp6 by Gsk-3β and casein kinase γ in its cytoplasmic region, which leads to the recruitment of axin. Subsequently, β-catenin phosphorylation is attenuated, its degradation inhibited, and accumulated β-catenin undergoes nuclear translocation, where it regulates target gene expression through interaction with members of the T-cell-specific transcription factor/lymphoid enhancer-binding factor (Tcf/Lef) family [@pone.0002142-Moon1], [@pone.0002142-Logan1]. Importantly, increased nuclear β-catenin staining was recently reported in IPF tissue sections [@pone.0002142-Chilosi1], indicative of increased Wnt signaling. In addition, unbiased microarray screens have also revealed an increased expression of Wnt target genes, such as matrix metalloproteinase (Mmp) 7, or secreted frizzled-related protein (Sfrp) 2 in IPF [@pone.0002142-Yang1]--[@pone.0002142-Lewis1]. We therefore hypothesized that canonical Wnt signaling is aberrantly activated in IPF, recapitulating developmentally active programs in this chronic disease. To this end, we achieved our aim to elucidate the expression, localization, and activity of the Wnt/β-catenin pathway in IPF. Results {#s2} ======= Initially, we sought to quantify the mRNA expression of canonical Wnt/β-catenin signaling components in lung tissue samples of transplant donors and IPF patients using quantitative real-time (q)RT-PCR. As depicted in [Figure 1a](#pone-0002142-g001){ref-type="fig"}, canonical Wnt ligands were variably expressed in the human lung. Wnt1, 2, 3a, and 7b were expressed at similar levels in normal lung tissue, while Wnt10b was only little expressed. In IPF lung specimens, Wnt1, 7b, and 10b mRNA levels were markedly upregulated (log-fold change of 1.19±0.43, 1.05±0.43, and 1.58±0.59, respectively), whereas Wnt3a was significantly downregulated (log-fold change −1.93±0.65) ([Figure 1a](#pone-0002142-g001){ref-type="fig"}). ![The mRNA expression profile of canonical Wnt signaling components in IPF.\ The mRNA levels of the Wnt ligands Wnt1, 2, 3a, 7b, and 10b (a), the receptors frizzled (Fzd) 1--4, low density lipoprotein-related protein (Lrp) 5 and 6 (b), and the intracellular signal transducers glycogen synthase kinase (Gsk)-3β, β-catenin, T-cell-specific transcription facor (Tcf) 3, Tcf 4, lymphoid enhancer-binding factor (Lef) 1 (c) were assessed in donor and IPF lung specimen by quantitative real-time PCR (qRT-PCR). Results are derived from 12 donors and 12 IPF patients and presented as mean±s.e.m., \* p\<0.05.](pone.0002142.g001){#pone-0002142-g001} Next, we analyzed the expression of common Wnt receptors and co-receptors. As shown in [Figure 1b](#pone-0002142-g001){ref-type="fig"}, the most abundant receptors in the human lung were Fzd1 and 4, and the co-receptors Lrp5 and 6, but their expression was similar in control and IPF lungs. Interestingly, Fzd2 and 3 were expressed at low levels in control as well as IPF lungs, but significantly increased in IPF (log-fold change 1.04±0.24 and 1.41±0.31, respectively) ([Figure 1b](#pone-0002142-g001){ref-type="fig"}). The main canonical Wnt signal transducers Gsk-3β and β-catenin were both expressed in normal and fibrotic lung tissue, with a significantly increased expression of β-catenin in IPF (log-fold change 0.98±0.28) ([Figure 1c](#pone-0002142-g001){ref-type="fig"}). With the exception of Tcf1, all members of the Tcf/Lef family of transcription factors were expressed in normal and fibrotic lung tissue. Lef1 was significantly upregulated in IPF (log-fold change 0.85±0.34) ([Figure 1c](#pone-0002142-g001){ref-type="fig"}). We went on to localize cell types capable of Wnt ligand secretion, as assessed by immunohistochemistry of Wnt1 and 3a ligands, and cell types capable of Wnt signaling, as assessed by immunohistochemistry of β-catenin and Gsk-3β, in donor and IPF lung tissue ([Figure 2](#pone-0002142-g002){ref-type="fig"}, [3](#pone-0002142-g003){ref-type="fig"}, [4](#pone-0002142-g004){ref-type="fig"}, [5](#pone-0002142-g005){ref-type="fig"}). Wnt1 was mainly expressed in bronchial and alveolar epithelium, with strong staining of alveolar epithelial type II (ATII) cells ([Figure 2](#pone-0002142-g002){ref-type="fig"}). Additionally, Wnt1 was expressed in vascular smooth muscle cells ([Figure 2a, upper panel](#pone-0002142-g002){ref-type="fig"}). In IPF ([Figure 2b](#pone-0002142-g002){ref-type="fig"}), Wnt1 staining was observed in hyperplastic ATII cells and bronchial epithelial cells. An apical staining pattern of Wnt1 in bronchial epithelial cells was observed in IPF, suggesting increased secretion of Wnt1. Interestingly, Wnt1 was also expressed by endothelial cells in IPF tissues ([Figure 2b, lower panel, arrowhead](#pone-0002142-g002){ref-type="fig"}). Wnt3a protein expression was mainly detected in ATII cells ([Figure 3a, b, lower panels](#pone-0002142-g003){ref-type="fig"}) and selected ciliated bronchial epithelial cells ([Figure 3a, b, upper panels](#pone-0002142-g003){ref-type="fig"}) in donor as well as IPF lung tissue. ![Expression and localization of Wnt1 in lung tissues of donor and IPF patients.\ Immunohistochemical staining was performed on tissue sections of donor (a) or IPF lungs (b). Representative pictures with focus on the bronchial (upper panel) or alveolar epithelium (lower panel) are given. Stainings are representative of two independent experiments using at least three different donor or IPF lung tissues (magnification as indicated). Arrowhead indicates positive endothelial cells.](pone.0002142.g002){#pone-0002142-g002	{ "pile_set_name": "PubMed Central" }
Pancreaticoduodenectomy (PD) remains the standard surgical treatment for resectable peri-ampullary tumors. The first PD operation was reported by Codavilli in 1898 in a patient with an epithelioma of the pancreas, but the patient died from cachexia on the 21^st^ post-operative day.\[[@CIT1]\] In 1946, Whipple described a one-stage PD in which the pylorus was resected.\[[@CIT2]\] The first report of pylorus preserving PD (PPPD) was by Watson in 1944 for ampullary carcinoma\[[@CIT3]\] but it did not gain popularity at that time. In both the classic PD and PPPD, the head of pancreas, duodenum, and distal bile duct are resected. The main difference is that in classic PD, the gastric antrum and pylorus are resected with the creation of a gastro-jejunostomy, while in PPPD, the gastric antrum and pylorus are preserved and the line of resection is through the first part of duodenum and a duodeno-jejunostomy is performed \[Figure [1 a](#F0001){ref-type="fig"} and [b](#F0001){ref-type="fig"}\]. ![Pancreaticoduodenectomy, (a) classic, (b) pylorus preserving](SJG-16-75-g001){#F0001} Traverso and Longmire reintroduced the concept of PPPD for benign peri-ampullary lesions in 1978 as they thought it would decrease the incidence of post-gastrectomy complications.\[[@CIT4]\] In 1980, they published their experience in PPPD for malignant lesions which included 18 patients with peri-ampullary, duodenal, and pancreatic carcinomas with encouraging results of normal gastric emptying and acidity.\[[@CIT5]\] Since, PPPD has been applied widely to patients with peri-ampullary lesions, benign, or malignant. In spite of the reported good outcomes of PPPD, many surgeons still question the benefit of this procedure especially the reported high incidence of delayed gastric emptying and, more importantly, the negative impact that pylorus preservation has on tumor clearance, recurrence, and long-term survival. We will try in this review article to answer the question of the safety of PPPD as compared to classic PD (CPD) in terms of operative factors, peri-operative complications, tumor recurrence, survival, and long-term quality of life. OPERATIVE FACTORS {#sec1-1} ================= Operating time {#sec2-1} -------------- Sugiyama in 2000 compared 10 patients with PPPD to 14 patients with CPD.\[[@CIT6]\] Although there was a trend toward a shorter operative time in the PPPD group, it did not reach statistical significance and that was due to a low volume study. A large, multicentre, randomized, controlled trial of 170 patients comparing PPPD with PD also had found no significant difference in the operating time.\[[@CIT7]\] In a meta-analysis, Traverso had confirmed the previous observation where there had been a trend toward a shorter operating time in PPPD but also not statistically significant.\[[@CIT8]\] Two large volume retrospective studies have looked at the operating time difference between PPPD and CPD and it had been clear that the PPPD operating time was significantly shorter than that of CPD.\[[@CIT9][@CIT10]\] That observation has been further supported by a meta-analysis by Karanicolas in 2006 and has found that PPPD was 72 min shorter than PD.\[[@CIT11]\] A more recent meta-analysis\[[@CIT12]\] has also shown that PPPD was 41.3 min shorter. Blood loss and a need for blood transfusion {#sec2-2} ------------------------------------------- Several reports have indicated no significant difference in intra-operative blood loss and blood transfusion between PPPD and PD.\[[@CIT6]--[@CIT8]\] In a meta-analysis, however, although there has been no significant difference in blood loss, more patients in the PD group have required blood transfusions.\[[@CIT12]\] Other studies with a larger patient volume, on the other hand, have shown significantly less blood loss and blood transfusions in the PPPD group\[[@CIT9]--[@CIT11]\] that could be partly due to the fact that there is less dissection in PPPD. This observation is very important, as blood transfusions in pancreatic cancer have been associated with a decreased survival rate.\[[@CIT13]\] So if an operative procedure results in less blood loss it should translate into a longer survival. Operative mortality {#sec2-3} ------------------- In retrospective analyses, peri-operative mortality has been similar in PPPD and PD groups.\[[@CIT9][@CIT10][@CIT14]\] Two meta-analysis studies have shown a trend toward lower peri-operative mortality in the PPPD group.\[[@CIT11][@CIT12]\] A randomized controlled trial comparing 13 patients with CPD to 14 patients with PPPD has shown no significant difference in mortality (15.4% and 28.6%, respectively, P-value 0.65) but these are very high mortality rates for any pancreaticoduodenectomy in comparison to the widely reported 3% in most studies.\[[@CIT15]\] In a multicentre, randomized, controlled trial involving 170 patients, mortality has been 7% in the CPD group vs. 3% in the PPPD group (P-value 0.27)\[[@CIT7]\] POST-OPERATIVE COMPLICATIONS {#sec1-2} ============================ Delayed gastric emptying {#sec2-4} ------------------------ DGE is probably one of the most studied complications following any type of pancreaticoduodenectomy. There has always been the thought that pylorus preservation would increase the chance of DGE. In a large series from Japan including 1066 patients who underwent PPPD, the incidence of DGE was 46%,\[[@CIT16]\] which supported the idea of higher DGE with PPPD. A small volume, randomized controlled trial has shown DGE to be 15% in PD vs. 64% in the PPPD group (P-value 0.2).\[[@CIT15]\] On the other hand, several other studies have not shown the same observation. A retrospective analysis of 113 patients has shown no significant difference in DGE but half of PPPD patients with DGE had co-existing intra-abdominal complications which could have attributed to DGE.\[[@CIT14]\] Two retrospective studies have shown no significant difference in DGE between the two groups.\[[@CIT6][@CIT9]\] This was also confirmed in a multicentre, randomized, controlled trial.\[[@CIT13]\] A retrospective analysis of 239 patients showed that DGE in the CPD group was double that of the PPPD group (6 vs. 13%), but there was a higher percentage of T4 and more extensive resections in the CPD group.\[[@CIT10]\] Several meta-analysis studies have also shown that DGE is not higher in the PPPD group.\[[@CIT8][@CIT11][@CIT12][@CIT17]\] It seems that DGE is not increased by preservation of the pylorus rather, by other factors including postoperative complications especially intra-abdominal collections. The presence of portal venous hypertension and preoperative cholangitis also increases the chance of post-operative DGE.\[[@CIT18][@CIT19]\] Shan\[[@CIT22]\] has made a distinction between subjective DGE and objective DGE as measured by cholescintography and has concluded that although subjective DGE was higher in the PPPD group, objective DGE was similar between the CPD and PPPD groups. He has proposed that loss of the distal stomach mechanoreceptors in the CPD group reduces the patient\'s sensation of subjective DGE. Additionally, Kim\[[@CIT23]\] proposed that pylorospasm could be a cause of DGE in PPPD and has shown a decrease incidence of DGE with the addition of pyloromyotomy. On the other hand, other studies have shown that abnormal gastric motility post surgery is the main cause of DGE regardless of the type of reconstruction.\[[@CIT24][@CIT25]\] Several methods have been tried to further decrease the incidence of DGE in PPPD. The drug erythromycin has been shown to increase contractility of the stomach and decrease the incidence of DGE.\[[@CIT26][@CIT27]\] On the other hand, somatostatin which is sometimes used to decrease the severity of pancreatic anastomosis leak increases the chance of DGE by more than 3-fold.\[[@CIT28]\] An interesting observation was that the use of ante-colic doudeno-jejunostomy as opposed to a retro-colic reconstruction in PPPD decreased the incidence of DGE.\[[@CIT19]--[@CIT21]\] Anastomotic leak {#sec2-5} ---------------- Anastomotic leak, especially from pancreatico-jejunostomy (PJ), is the main factor for morbidity post-PD. A review of 1066 PPPDs in Japan has revealed a leak rate of 16%.\[[@CIT16]\] In a randomized, controlled trial and two meta-analyses, there has been no difference between CPD and PPPD in terms of PJ leak rate.\[[@CIT11][@CIT12][@CIT15]\] Tani\[[@CIT29]\] has shown that the Traveso-type construction (Duodeno-jejunostomy (DJ) distal to PJ) has a lower leak rate than the Billroth I type reconstruction (DJ proximal to PJ). Intestinal acidity and anastomotic ulceration {#sec2-6} --------------------------------------------- Not performing an antrectomy could	{ "pile_set_name": "PubMed Central" }
Introduction {#sec1} ============ Stable integration of retroviral vectors encompassing a therapeutic transgene enables gene correction of severe blood and immune disorders. Over the past 25 years, murine leukemia virus (MLV)-based vectors have shown therapeutic benefit in gene therapy studies for primary immunodeficiencies (PIDs), such as X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency-severe combined immunodeficiency (ADA-SCID), and Wiskott-Aldrich syndrome (WAS).[@bib1], [@bib2], [@bib3], [@bib4] MLV-based vectors were successfully used in the first clinical trials for ADA-SCID.[@bib5], [@bib6], [@bib7] This led to the recent European approval of a retrovirus-based gene therapy product (Strimvelis; GSK GlaxoSmithKline Pharmaceuticals) to treat patients that lack a suitable human leukocyte antigen (HLA)-matched related stem cell donor.[@bib8], [@bib9] However, in clinical trials for other PIDs, several patients developed leukemia or myelodysplasia, raising concerns about the safety of gene therapy.[@bib10], [@bib11], [@bib12], [@bib13], [@bib14] These side effects have been directly attributed to the integration pattern and vector design. Insertional mutagenesis occurred as a consequence of vector integration preference in proximity of proto-oncogenes and activation by strong viral promoter and enhancer elements in the long terminal repeat (LTR) of retroviral vectors. To prevent insertional mutagenesis, self-inactivating (SIN) vectors with deleted enhancer sequences were designed. The lack of promoter/enhancer activity is compensated by weak heterologous promoters to drive transgene expression, such as the elongation factor 1 α short (EFS) and phosphoglycerate kinase (PGK) promoters.[@bib15], [@bib16] Additionally, introduction of genetic insulator sequences has improved the safety of viral vectors.[@bib17], [@bib18], [@bib19], [@bib20], [@bib21], [@bib22] The efficacy of these modified vectors was confirmed in pre-clinical studies and they are now in phase I/II clinical trials for several PIDs.[@bib19], [@bib23], [@bib24], [@bib25], [@bib26], [@bib27] A complementary approach to improve the safety of gene therapy is to alter the integration pattern, directing integration away from potentially unsafe regions. Gammaretroviral integration is not random, but rather is dictated by host cellular cofactors, such as the bromodomain and extraterminal domain (BET)-containing family of proteins (BRD2, BRD3, and BRD4) that serve as anchors on the host chromatin.[@bib28], [@bib29] A motif in the unstructured C-terminal tail of MLV integrase (IN) interacts with the extraterminal (ET) domain of BRDs, where the latter tethers the retroviral pre-integration complex (PIC) to chromatin regions enriched in BET proteins and thereby defines the integration profile.[@bib28], [@bib29], [@bib30] Deletion of the C-terminal domain (Δ23 amino acids \[aa\], IN~1--380~) or a single substitution (IN~W390A~) uncouples the BET interaction, resulting in BET-independent (Bin) MLV vectors that transduce target cells at wild-type (WT) efficiency but with diminished integration in the vicinity of retroviral integration markers.[@bib31] Here, we developed next-generation BinMLV vectors with a potentially safer integration profile and lower propensity to activate nearby genes in an effort to alleviate the risk of insertional mutagenesis by interfering with the chromatin-tethering process. We linked chromatin binding peptide sequences to the C-terminal end of BinMLV IN and demonstrated that fusion of these peptides to BinMLV IN generates vectors that produce at high titers and transduce cells at wild-type efficiency. The addition of the chromodomain of CBX1 to MLV~IN_W390A~ efficiently retargeted integration away from gene regulatory elements. More importantly, the retargeted vector transduced human CD34^+^ hematopoietic stem cells (HSCs) at wild-type efficiency, while genotoxicity assays revealed reduced transformation potential. Results {#sec2} ======= Efficient Transduction and Integration of Next-Generation BinMLV Vectors {#sec2.1} ------------------------------------------------------------------------ To direct BinMLV integration away from potentially unsafe chromosomal regions, we tailored the chromatin-tethering process by fusing tethering peptides (between 16 and 61 aa long) to the C-terminal end of IN~W390A~ in the MLV packaging plasmid. We opted for peptides that bind histone markers that are widely spread across the chromatin ([Figure 1](#fig1){ref-type="fig"}A; [Table 1](#tbl1){ref-type="table"}). On one hand, we used peptides derived from cellular proteins that bind specific epigenetic histone modifications, such as the chromodomain of heterochromatin-binding protein 1β (CBX1, aa 20--73) and the chromodomain of Y-like protein (CDYL; aa 1--60),[@bib32], [@bib33] giving rise to IN~W390A-CBX~ and IN~W390A-CDYL~, respectively. Alternatively, we fused virus-derived peptides, such as the tethering domain of the human papilloma virus (HPV8) E2 protein (aa 240--255)[@bib34] and the N-terminal end of Kaposi sarcoma's latency associated nuclear antigen (LANA; aa 1--31), which bind to core histone 2A and 2B,[@bib35] resulting in IN~W390A-E2~ and IN~W390A-LANA~, respectively.Figure 1Transduction Efficiencies of Next-Generation BinMLV Vectors(A) Schematic representation of MLV-based vector production. Structure of IN~WT~, IN~W390A~, and the peptides fused to IN~W390A~ are highlighted. The N-terminal HHCC zinc binding domain, the catalytic core domain (CCD), and the C-terminal domain (CTD) are indicated. Red arrowheads indicate the position of the W390A point mutation. The size of the fused peptides is proportionally represented. (B) FACS analysis of SupT1 cells transduced with equal RTUs of the indicated vectors at different MOIs. Three days post-transduction, the percentage of EGFP-positive cells was determined. Average values and standard deviations of triplicate measurements are shown. Data represent measurements from a representative experiment. (C) Mean fluorescence intensity of SupT1 cells transduced with the indicated next-generation BinMLV vectors 3 days post-transduction. Average values and standard deviations of triplicate measurements are shown. Data represent measurements from a representative experiment. ψ, packaging signal; CMV, cytomegalovirus promoter; GAG, group-specific antigen; IN, integrase; LTR, long terminal repeat; PolyA, polyadenylation signal; Pol, polymerase; PR, protease; RT, reverse transcriptase; VSV-G, vesicular stomatitis virus glycoprotein G.Table 1Overview of the Tethering Domains Fused to BinMLV VectorsProteinAccession NumberSelected PeptideBinding SiteSequenceCBX1[P83916](ncbi-wgs:P83916){#intref0025}CD~1~ CBXH3K9me2EYVVEKVLDRRVVKGKVEYLLKWKGFSDEDNTWEPEENLDCPDLIAEFLQSQKTH3K9me3CDYL[Q9Y232](ncbi-wgs:Q9Y232){#intref0030}CD~1~ CDYLH3K9me2LMTFQASHRSAWGKSRKKNWQYEGPTQKLFLKRNNVSAPDGPSDPSISVSSEQSGAQQPPAH3K27me2H3K27me3HPV8 E2[P06422](ncbi-wgs:P06422){#intref0035}E2H2A/H2BQTETKGRRYGRRPSSRKSHV LANAE5LC01LANAH2A/H2BMAPPGMRLRSGRSTGAPLTRGSCRKRNRSPE[^3] The respective packaging plasmids were subsequently used to produce vesicular stomatitis virus glycoprotein G (VSV-G) pseudotyped MLV-based vectors encoding an LTR-driven EGFP reporter (MLV~IN_W390A-CBX~, MLV~IN_W390A-CDYL~, MLV~IN_W390A-E2~, and MLV~IN_W390A-LANA~; [Figure 1](#fig1){ref-type="fig"}A). In line with previous results,[@bib31] transduction efficiencies in SupT1 cells were at similar levels for MLV~IN~\_~W390A~ and MLV~IN~\_~WT~ ([Figure 1](#fig1){ref-type="fig"}B). The addition of peptide sequences to the C-terminal end of MLV~IN_W390A~ resulted in BinMLV vectors that transduced as efficiently as MLV~IN~\_~WT~ at different MOIs ([Figure 1](#fig1){ref-type="fig"}B) and resulted in comparable expression levels (measured as mean fluorescence intensities \[MFIs\] at day 3; [Figure 1](#fig1){ref-type="fig"}	{ "pile_set_name": "PubMed Central" }
1. Introduction {#sec1} =============== RIS, structurally, a benzisoxazole derivative, is one of the most frequently prescribed atypical antipsychotics in the management of schizophrenia, mood disorders, and autism \[[@B1]\]. Its mechanism of action is not entirely clear. Although the use of atypical antipsychotic drugs has been successful in the treatment of schizophrenia, they can provoke some complications, including weight gain, sedation, movement problems, sleepiness, vision difficulties, constipation, hyperprolactinemia, and extrapyramidal side effects \[[@B2]\]. Patients using these drugs tend to disrupt treatment primarily due to their adverse effects. In addition, RIS is a highly potent drug, extensively metabolized in the liver and excreted by the kidneys \[[@B3]\]. RIS and its primary active metabolite, 9-hydroxyrisperidone, are eliminated by the kidneys. In patients with moderate to severe renal disease, clearance of the sum of the parent drug and metabolite has been demonstrated to decrease by 60% compared to healthy subjects \[[@B4]\]. There are a few cases reporting atypical antipsychotic drug-associated renal damage in patients (including those using RIS), but the mechanism that leads to RIS-related injury is not well understood \[[@B5]\]. Adverse outcomes potentially attributable to these drugs, such as hypotension, acute urinary retention, and rhabdomyolysis, are known to cause this injury \[[@B6]\]. Moreover, pneumonia, acute myocardial infarction, and ventricular arrhythmia have been associated with these drugs in former population-based studies and renal damage may also co-occur with these events \[[@B7]\]. Nowadays, medicinal plants are a considerable source of drug synthesis. RSV, trans-3,5,4′-trihydroxy stilbene, has potent antioxidative and anti-inflammatory properties \[[@B8]\]. It is a polyphenolic phytoalexin present in many edible plants, including mulberries, peanuts, and grapes \[[@B9]\]. RSV has been demonstrated to exhibit a wide range of health-promoting benefits for the coronary, neurological, hepatic, and cardiovascular systems. It has been shown to inhibit inflammation, viral infection, oxidative stress, platelet aggregation, and the growth of a variety of cancer cells \[[@B10]\]. In addition, RSV has been studied in vivo and in vitro \[[@B11]\] and has been shown to possess a series of pharmacological benefits, including nephroprotective effects \[[@B12]\], as a result of its antioxidant and cytoprotective properties \[[@B13]\]. The antioxidant properties of RSV are mainly dependent upon the upregulation of endogenous cellular antioxidant systems, but the compound also displays direct reactive oxygen species (ROS) scavenging properties \[[@B14]\]. The high potency and low systemic toxicity of RSV make it a promising alternative to conventional therapeutic drugs. The fact that RIS exposure induces an excessive increase in metabolic alterations suggests that RSV could be used as an alternative therapy. To our knowledge, there is no report regarding the protective and therapeutic effects of RSV on RIS-induced renal damage because it has not been studied until now. Therefore, the present study was designed to investigate the possible beneficial impact of oral supplementation with RSV against RIS-induced renal damage in rats for the first time. To achieve our goal, we performed several biochemical and histological analyses in female rats. 2. Materials and Methods {#sec2} ======================== 2.1. Chemicals {#sec2.1} -------------- RIS was bought from Janssen Turkey, Istanbul, and RSV (CAS number 501-36-0) was purchased from Sigma-Aldrich (St. Louis, MO, USA). They were dissolved in distilled water. All other chemicals used were of the best analytical grade. 2.2. Animals {#sec2.2} ------------ In this study, thirty-five female Sprague Dawley rats were used. Healthy adult (twelve weeks old) rats weighing 250--300 g were obtained from the Fırat University Laboratory Animal Production and Research Center. All animal care and follow-up procedures were performed at this center. The experiments were performed in accordance with the protocol approved by Fırat University Faculty of Medicine, Laboratory Animals Ethics Committee (protocol number 2016/25). The rats were kept at 21 ± 1°C for 12 h in a light-dark cycle, were fed standard rat chow, and drank tap water ad libitum. RIS and RSV were administered to the rats for two weeks. 2.3. Experimental Design {#sec2.3} ------------------------ In our study, 35 rats were randomly assigned to five groups with an equal number in each group. We used the simple randomization technique in this experimental study. Thirty-five female Sprague Dawley rats were divided into five groups as follows: group 1, control; group 2, RIS; group 3, RIS+RSV-1; group 4, RIS+RSV-2; and group 5, RIS+RSV-3. The control group was given physiological saline solution by gavage once a day. 2 mg/kg/day RIS was administered by gavage for two weeks to all groups, except the control group. 20 mg/kg/day RSV was given for two weeks to the RIS+RSV-1 group. 40 mg/kg/day RSV was given for two weeks to the RIS+RSV-2 group. 80 mg/kg/day RSV was given for two weeks to the RIS+RSV-3 group. All compounds were suspended in physiological saline solution and administered by gavage once a day. Body weight was recorded at the beginning and end of the study. The treatment course lasted two weeks for all groups. At the end of the second week of the treatment period, the animals were euthanized by exsanguination through cardiac puncture under diethyl ether anesthesia. Before killing, rats were individually weighed, venous blood samples were collected, and serum samples for biochemical analysis were separated by centrifuge at 2800g for 15 min and then stored at −80°C. Kidneys were surgically removed, weighed, and stored at −80°C for subsequent biochemical and pathohistological analysis. The daily dose of RIS varies between 0.25 and 16 mg, and a frequently prescribed dose in previous studies was 2 mg \[[@B15]\]. The maximum recommended human dose (MRHD) of RIS is 0.4--12 mg/day. The experimental dose was calibrated as 2.0 mg/kg (MHRD × 10) on the basis of per kg body weight per day and its suitability to the rat animal model \[[@B16]\]. Therefore, a 2 mg dose was selected for the present study. The assigned dosage of powdered RIS (2 mg/kg once a day for two weeks) was administered by a gastric tube daily between 8:00 and 9:00 a.m. in line with a previous report \[[@B17]\]. RSV (20, 40, and 80 mg/kg body weight/day) was also administered by a gastric tube daily between 8:00 and 9:00 a.m. The dose and duration of RSV were selected according to results from a previous study \[[@B18]\]. 2.4. Biochemical Analysis {#sec2.4} ------------------------- BUN was measured using a commercially available enzymatic colorimetric method and analyzer system (Hitachi 917 modular device; Roche Diagnostics, Basel, Switzerland). Serum Cr was measured by Jaffe\'s method using the same analyzer system \[[@B19]\]. 2.5. TAS and TOS Determination {#sec2.5} ------------------------------ The automated calorimetric measurement methods developed by Erel were used to define the TAS (mmol/L) and TOS (μmol/L) (a serum oxidant parameter). The measurement of TAS and TOS in serum samples was determined by a TAS and TOS kit (REL Assay Diagnostics) \[[@B20], [@B21]\]. 2.6. OSI Determination {#sec2.6} ---------------------- OSI was determined as TOS to TAS ratio and was calculated as follows: OSI (arbitrary unit) = ((TOS, μmol H2O2 eq/L)/(TAS, μmol Trolox eq/L)) \[[@B22]\]. 2.7. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay {#sec2.7} ------------------------------------------------------------------------------- Apoptotic cells were defined using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, cat. number: S7101, USA) according to the manufacturer\'s instructions. Sections (5 μm) taken from the paraffin blocks were entrenched onto polylysine-coated slides, deparaffinized using xylene, dehydrated with a series of alcohol rinses, and then washed with phosphate-buffered saline (PBS). Then, tissues were incubated with a proteinase K solution (0.05%) and with 3% hydrogen peroxide for 5 min to forestall endogenous peroxidase activity. After washing with PBS, the tissues were incubated with Equilibration Buffer for 6 min and in working solution (70% μL reaction buffer + 30% TdT enzyme) at 37°C under moist conditions for 60 min. Tissues were then incubated in stop/wash buffer for 10 min and incubated in anti-digoxigenin-peroxidase for 30 min. Apoptotic cells were examined using the diaminobenzidine substrate. Cross sections contrast stained with methyl green were sealed using a proper covering solution. Mamma tissue was used as	{ "pile_set_name": "PubMed Central" }
Foodborne illness resulting from consumption of food contaminated with pathogenic bacteria has been of vital concern to public health. Consumers today are increasingly concerned about chemical preservatives in food and tend to choose food products that are natural, safe and with multi-health benefits\[[@ref1][@ref2][@ref3]\]. Foodborne illness is a major problem associated with enormous costs. Foodborne pathogens occur widely in nature and it is difficult to prevent them from entering raw foods. Salmonella sp., Listeria monocytogenes, Bacillus subtilis and Escherichia coli account for the largest number of outbreaks, cases and deaths, and are capable of attaching to inert surfaces and subsequently forming bio films on food processing equipment and environment\[[@ref4][@ref5]\]. Staphylococcus aureus causes a range of illnesses and was found to be the most resistant organism\[[@ref6]\]. Salmonella mutants survive and are able to persist in the food chain\[[@ref7]\]. Many Pseudomonas spp. can cause food spoilage. Novel antipseudomonal activity is of particular interest as it is the leading cause of nosocomial infections and has developed mechanisms of resistance to common classes of antibiotics\[[@ref8][@ref9]\]. The resistance of bacteria and other microorganisms to antimicrobial agents has become a wide-spread medical problem especially as nosocomial pathogens. To reduce health hazards and economic losses due to foodborne microorganisms, the use of natural products as antibacterial compounds is gaining importance. However, it is necessary to establish the scientific basis for the therapeutic actions of traditional plant medicines. Several plants have been reported to be used in treating and managing the complicated diseases. The food antimicrobials are classified into natural and synthetic substances depending on their origin. Although, many synthetic antimicrobials are found naturally (benzoic acid in cranberries, sorbic acid in rowanberries, citric acid in lemons, malic acid in apples and tartaric acid in grapes), the perception of natural has become important for many consumers\[[@ref10]\]. The problems mentioned introduced new research directions in the field of bioactive principles from natural sources and their application as food additives or dietary supplements. Momordica charantia L. (Cucurbitaceae) commonly known as 'bitter gourd' and 'bitter melon', 'karela' is a multipurpose herb widely cultivated in many tropical and subtropical regions of the world. The fruits are used as medicinal vegetable in different parts of the world. Apart from their role in food consumption, a wide array of pharmacological activities such as antidiabetic\[[@ref11]\], antioxidant\[[@ref12]\], anticancer activities\[[@ref13]\] and antiulcer\[[@ref14]\] are reported for this plant. MATERIALS AND METHODS {#sec1-1} ===================== Collection of the plant material: {#sec2-1} --------------------------------- Different parts (aerial, peel, pulp and seed) of Momordica charantia L. were collected in September 2011 from Chotila, Surendranagar, Gujarat, India and identified by comparison with specimens (PSN333) available at the Herbarium of the Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India. The parts were separated, washed thoroughly with tap water, shade dried, homogenised to fine powder and stored in airtight bottle. Hydroalcoholic extraction method: {#sec2-2} --------------------------------- The dried powders of all the four parts were extracted individually by cold percolation method\[[@ref15][@ref16][@ref17]\]. The hydroalchoholic extraction was done using methanol and water\[[@ref18]\]. The dried powder was first defatted by hexane and then extracted in 100% methanol (MeOH), 75% MeOH, 50% MeOH, 25% MeOH and 100% water (aqueous). Ten grams of dried powder was taken in 100 ml of hexane in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 120 rpm for 24 h. After 24 h, the extract was filtered with eight layers of muslin cloth; centrifuged at 5000 rpm for 10 min. Supernatant was collected and the solvent was evaporated. The residue was then added to 100 ml of each solvent, that is 100% MeOH, 75% MeOH, 50% MeOH, 25% MeOH and water in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 120 rpm for 24 h. After 24 h, the extract was filtered with eight layers of muslin cloth; centrifuged at 5000 rpm for 10 min, the supernatant was collected and the solvents were partially evaporated using rotary vacuum evaporator (Equitron, India) then kept in petri plates to dry. The extract was stored at 4° in air tight bottles. The residues were weighed to obtain the extractive yield. Antimicrobial activity: {#sec2-3} ----------------------- The microorganisms used in this investigation were obtained from National Chemical Laboratory, Pune, India. The microorganisms were maintained at 4°. The Gram-positive bacteria studied were Staphylococcus aureus ATCC29737 (SA), Staphylococcus albus NCIM 2178 (SAL), Corynebacterium rubrum ATCC14898 (CR), Listeria monocytogenes ATCC19112 (LM), Micrococcus flavus ATCC10240 (MF); Gram-negative bacteria used were Pseudomonas aeruginosa ATCC27853, (PA) Pseudomonas stutzeri NCIM5136 (PSt), Pseudomonas pictorum NCIB9152 (PPi), Pseudomonas putida NCIM2872 (PP), Pseudomonas testosteroni NCIM5098 (PT), Pseudomonas syrigae NCIM5102 (PS); and fungi were Candida albicans ATCC2091 (CA), Candida neoformans NCIM3542 (CN), Candida glabrata NCIM3448 (CG), Candida epicola NCIM3367 (CE). The organisms were maintained on nutrient agar and MGYP medium (Hi-Media, India) for bacteria and fungi respectively, at 4° and subcultured before use. The microorganisms studied are clinically important ones causing several infections and food spoilage. Ampicillin (AMP 10 μg/disc), chloramphenicol (CH 30 μg/disc), tetracycline (T 30 μg/disc), amphotericin B (AP 100 units/disc) and nystatin (NS 100 units/disc) were used as standard to determine antimicrobial susceptibility. Chloramphenicol and ceftazidime (CF) were used during minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination. All antibiotics were purchased from Hi-Media Laboratory Pvt. Ltd., (Mumbai, India). Agar well diffusion method: {#sec2-4} --------------------------- In vitro antimicrobial activity of the different solvent extracts was studied against pathogenic microbial strains by the agar well diffusion method\[[@ref19][@ref20][@ref21][@ref22]\]. Mueller-Hinton No. 2/Sabouraud dextrose agar (Hi-Media) was used for the antibacterial and antifungal susceptibility test, respectively. The different solvent extracts were diluted in 100% dimethyl sulfoxide (DMSO) to give a concentration of 20 mg/ml. The Mueller-Hinton agar/Sabouraud dextrose agar was melted and cooled to 48--50° and a standardised inoculum (1.5×10^8^ CFU/ml, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes; wells (8.5 mm) were prepared in the seeded agar plates. The test compound (100 μl) was introduced into the well. The plates were incubated overnight at 37° and 28° for 24 and 48 h, respectively, for bacteria and fungi. DMSO was used as negative control. The microbial growth was determined by measuring the diameter of the zone of inhibition and the mean values are presented with ±SEM (standard error of mean). Preparation of bacterial inocula and extracts or antibiotics for MIC and MBC study: {#sec2-5} ----------------------------------------------------------------------------------- The inoculum of the test organisms were prepared using the colony suspension method\[[@ref23]\]. Colonies picked from 24 h old cultures, grown on nutrient agar, were used to make suspension of the test organisms in saline solution to give an optical density of approximately 0.1 at 600 nm. The suspension was then diluted 1:100 by transfer of 0.1 ml of the bacterial suspension to 9.9 ml of sterile nutrient broth before use to yield 6×10^5^ CFU/ml. Twofold serial dilutions using 100% DMSO were carried out from the 1250 μg/ml stock plant extract to make six test concentrations ranging from 39 to 1250 μg/ml for each solvent extracts. Twofold dilutions of chloramphenicol and ceftazidime (1--32 μg/ml) were used as a positive control. Determination of minimum inhibitory concentration: {#sec2-6} -------------------------------------------------- The MICs were determined only for the test organisms that had shown \>15 mm zone of inhibition of the crude extracts. Micro broth dilution method performed in sterile flat bottom 96 well micro test plates (Tarsons Products Pvt. Ltd.) was performed to evaluate MIC of the plant extracts\[[@ref24]\]. One hundred and fifty microlitres of Mueller-Hinton broth was introduced into all the 96 wells and 20 μl of varying concentrations of the extract was added in decreasing order along with 30 μl of the test organism suspension. A final volume of 200 μl was achieved	{ "pile_set_name": "PubMed Central" }

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