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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
============
Total knee arthroplasty (TKA) is a promising treatment for end-stage osteoarthritis (OA) of the knee for alleviating pain and restoring the function of the knee. Some of the cases with bilateral TKA are symptomatic, necessitating revision arthroplasty in both the knees. A bilateral revision TKA can be done either in two stage or simultaneously as a single stage procedure. However, the decision to perform simultaneous bilateral revision TKA is debatable because of possible higher complexity and complication rate. Very few cases have been reported in the literature on this issue. There are various advantages of doing simultaneous bilateral revision TKA compared with staged bilateral revision TKA. These include single operation and single anesthesia as well as better rehabilitation of both knees, apart from a significant reduction in the hospital stay and hospital costs.
Case presentation
=================
A 67-year-old hypothyroid and hypertensive female presented to us with unstable and painful knees 14 years after primary bilateral TKA for advanced OA. She began developing pain in both the knees for last six months, followed by instability in both knees (right \> left). She was managed symptomatically with painkillers, bracing, and physiotherapy but her pain and instability were not relieved.
On clinical examination, the active and passive knee range of motion was painful. The flexion was 0° to 100°, anterior--posterior laxity of 5--10 mm, and a mild valgus laxity. The plain radiographs showed malalignment and loosening of the implants (Figures [1](#FIG1){ref-type="fig"}-[2](#FIG2){ref-type="fig"}). The leucocyte counts, C-reactive protein, and erythrocyte sedimentation rate (ESR) were within normal limits. A three-phase bone scan was also found to be negative for infection.
{#FIG1}
{#FIG2}
Bilateral revision TKAs were performed using modified Insall's midline approach with lateral retraction of the patella (Figure [3](#FIG3){ref-type="fig"}) \[[@REF1]\]. A joint wound swab was taken and sent for gram stain, culture, and sensitivity. It was found to be negative for any microorganisms. The original cemented TKA implants were removed carefully, preserving as much bone as possible. Revision TKA was done on both sides sequentially, under the same anesthesia, using Scorpio® Total Stabilizer (Stryker®, Mahwah, NJ) constrained implants with long femoral and tibial stems.
{#FIG3}
The knees were protected in hinged braces postoperatively. The drains were removed 48 hours postoperatively; continuous passive motion (CPM) and active knee flexion exercises were started on postoperative day one and gradually increased to 0°--90° of flexion (Figure [4](#FIG4){ref-type="fig"}).
{#FIG4}
The postoperative radiographs showed satisfactory implant positions (Figures [5](#FIG5){ref-type="fig"}-[6](#FIG6){ref-type="fig"}). The patient had no complaints and was able to flex the knee to 80° easily. The range of motion and quadriceps strengthening exercises continued without forced flexion. She gradually resumed full weight-bearing with the help of the walker. Three months after surgery, the brace was removed, and active pain-free range of motion of 0°--115° was achieved with complete stability. At four months, the patient had returned to full activity without the brace or cane. At the final follow-up of four years, the knee was fully stable, and the patient was pain-free with no loosening or wear of the implants.
{#FIG5}
{#FIG6}
Discussion
==========
Symptomatic instability and pain following primary TKA requires revision surgery. In one retrospective study of 49 TKA patients with bilateral simultaneous revision, no postoperative cardiovascular complications, stroke, or death were noted \[[@REF2]\]. The minor reported complications included transient, self-limited confusion (in three cases); pulmonary embolism (in one patient), which was treated successfully with an inferior vena cava filter and extended anticoagulation; posterior compartment syndrome (in one case), which was treated by fasciotomy; and stiff knee in one patient (that was manipulated under anesthesia at three months). In a retrospective cohort study, Carter, et al. \[[@REF3]\] found that 33 of 141 morbidly obese patients (23.4%) who had revision TKA had a complication compared to 10 of 96 patients with a BMI 18.5 - 25 (10.4%) (p = 0.011). The most common complication was wound healing.
Kevin, et al. reviewed 60,355 revision TKA procedures done in the USA and noted that the most common causes of revision TKA were an infection in 25.2%, implant loosening in 16.1%, and implant failure/breakage in 9.7% cases \[[@REF4]\]. They found that revision of all the components was the most common type of procedure done (35.2%). Singh, et al. found a high prevalence (46.5%) of overall moderate to severe activity limitation at two years and 50.5% at five years following revision TKA \[[@REF5]\]. Significantly higher odds of moderate to severe overall activity limitation was noted both at two and five-year follow-ups in patients with a BMI of 40 or higher, age greater than 80 years, higher Deyo-Charlson score, and in females.
Kasmire, et al. studied predictors of functional outcome after revision TKA by using various parameters, such as short-form 36 (SF-36), Western Ontario and McMaster Osteoarthritis Index (WOMAC), and Knee Society Scores (KSS) \[[@REF6]\]. The data was collected preoperatively and at two years follow-up in their 175 revision TKAs done for aseptic failure. All of the above-mentioned parameters improved significantly after revision TKA (p \< 0.001). Lower preoperative pain and higher clinical KSS were found to be predictors of a better outcome.
Sheth, et al. found that the complication rates were different for bilateral TKA done simultaneously and as staged procedures \[[@REF7]\]. These authors reported aseptic revision (1.17% vs. 0.9%), septic revision (0.8% vs. 0.7%), mortality (0.28% vs. 0.1%), and adverse events (2.49% vs. 1.97%). According to Bohm, et al., simultaneous bilateral primary TKA patients required more blood transfusions, a shorter hospital stay, more transfers to a rehabilitation facility, and less frequency of knee infections than staged bilateral TKA patients \[[@REF8]\]. However, these patients had a higher rate of cardiac complications and in-hospital mortality rate. The three-year revision, however, was same in both the groups.
In a meta-analysis of 14 studies, Hu, et al. showed that the prevalence of mortality immediately postoperatively, mortality at 30 days postoperatively, and neurological complications were significantly higher in simultaneous TKA compared to staged TKA patients \[[@REF9]\]. The prevalence of thromboembolic disease, infection, and cardiac complications were not significantly different between simultaneous TKA compared to staged TKA patients. According to Hersekli, et al., the amount of blood loss, intensive care unit days and perioperative complications were same between single- and two-staged operations (p \> 0.05) \[[@REF10]\]. However, hospital stay and overall cost were significantly less in single-staged operations.
We faced the challenge in decision-making regarding the staging of the procedures in this reported case, where revision of the components was necessary for both knees. We could not find proper guidelines regarding bilateral revision TKA as there are only a few documented reports of simultaneous bilateral revision TKA. There is limited evidence to support the one-stage practice of doing bilateral revision TKAs, as its safety remains controversial. We chose to do a single-staged bilateral revision TKA in this case, as a two-staged procedure would have required two anesthesias, longer hospital stay, more hospital bills, and surgery-related complications, which were overcome
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J Med Radiat Sci 65 (2018) 275--281
Introduction {#jmrs290-sec-0005}
============
There is a growing interest globally in making sure that graduates emerge from higher education with the capabilities and competencies that will equip them not only to be 'work ready' on graduation but also prepared for the development of technology, new models of service delivery and advances for practice in the future.[1](#jmrs290-bib-0001){ref-type="ref"}, [2](#jmrs290-bib-0002){ref-type="ref"}, [3](#jmrs290-bib-0003){ref-type="ref"} In a profession, such as medical imaging, the health workforce needs graduates who are ready to understand and apply emerging technology alongside meeting the demands of ever changing healthcare systems.[4](#jmrs290-bib-0004){ref-type="ref"}
This paper reports on the outcomes of a survey undertaken as part of preparation for the review and redesign of clinical placements in a medical imaging programme in New Zealand. The project embraced the goal of defining work ready plus graduates for the medical imaging workforce. Identification of the capabilities required of a medical imaging technologist (MIT) in their graduate years was critical for the development of the clinical experience programme, as it is clinical placement and emersion in work that is most likely to develop capability and work readiness skills in graduates. It was envisaged that by defining, for our regional context, the capabilities and work skills employers seek in our graduates, we would have the data we needed to review and if necessary rewrite the graduate profile and utilise fully and effectively the real‐life clinical experiences that support the development of these capabilities. The results are also impacting positively on lecturers teaching methods as they consider how they can develop these capabilities in students through teaching, learning, and assessment methodologies.
The theoretical underpinning for this study was Scott\'s fellowship work for the Australian Teaching and Learning Council and the professional and graduate capability framework published for the Australian tertiary environment.[1](#jmrs290-bib-0001){ref-type="ref"} The Professional Capability Framework as used by Western Sydney University was used as the foundation for the development of a survey tool as it was current and had been validated in a range of disciplines that included health professions. In addition, it looks beyond graduation and standards for practice (as required by the New Zealand Medical Radiation Technologist Registration Board and the Medical Radiation Practice Board of Australia towards the generic skills graduates need to flourish in a profession in the future.[1](#jmrs290-bib-0001){ref-type="ref"}, [5](#jmrs290-bib-0005){ref-type="ref"} Hence the term work ready plus. Using a validated and comprehensive professional and graduate capability framework ensured that all potentially relevant capability options had been considered. It was deemed generalisable to the New Zealand health care environment due to the similarities between both the health and education systems.
Figure [1](#jmrs290-fig-0001){ref-type="fig"} summarises the key elements of the professional capability framework. The overlapping aspects of professional capability are identified -- personal, interpersonal and cognitive which have been validated in a range of investigations, mainly focused on professional leadership.[1](#jmrs290-bib-0001){ref-type="ref"}, [5](#jmrs290-bib-0005){ref-type="ref"} These domains are underpinned by relevant role‐specific and generic competencies (the skills and knowledge found to be essential to the specific role of an MIT). The key terms "competence" and "capability" are problematic and therefore often confused. We adopted the definition that competence is the possession of the skills and knowledge necessary to perform the duties set down for a specific role. The New Zealand Medical Radiation Technologist Registration Board (MRTB) reviewed and updated their competencies for New Zealand registration in March 2017, so a list of competencies was current and available. We have adopted a definition of capability that goes beyond the skills to practice as a safe and competent practitioner, to embrace the concept of being work ready plus. Being "work ready *plus"* requires capabilities for not just today, for current practice but for the future. Capabilities include the ability to work with others from a range of professions and backgrounds, manage the unexpected, adopt new technology, to be changed implementation savvy, inventive, sustainability responsive, to learn from experience and to operate with a clear understanding of one\'s ethical position.[1](#jmrs290-bib-0001){ref-type="ref"}
{ref-type="ref"} Permission was obtained to reproduce this figure.](JMRS-65-275-g001){#jmrs290-fig-0001}
These capabilities require a mixture of emotional and cognitive intelligence, including the ability to determine when and when not to deploy these competences.[1](#jmrs290-bib-0001){ref-type="ref"} We believed this concept was less developed for the medical imaging profession in New Zealand.
The Professional Capability Framework developed through a scholarship awarded by the Australian Teaching and Learning Council formed the basis for the development of a survey that asked practicing MITs and MIT clinical managers at the three largest placements sites in New Zealand to rate the capabilities deemed critical in a graduate to ensure they are "work ready *plus"*.[1](#jmrs290-bib-0001){ref-type="ref"}
The items used in the survey fall into three domains which align with the capability domains identified in Figure [1](#jmrs290-fig-0001){ref-type="fig"}. These domains are discussed in more detail in Scott, Coates and Anderson[5](#jmrs290-bib-0005){ref-type="ref"} and Fullan and Scott.[6](#jmrs290-bib-0006){ref-type="ref"}
This paper shares the results from the survey and discusses the impact these are having on curriculum review and development.
Method {#jmrs290-sec-0006}
======
This study was carried within all the public (three District Health Board, which includes 2 hospitals on the Northshore, 3 Inner City and 1 in South Auckland), Radiology Services, in the Auckland Region, where Unitec Institute of Technology\'s MIT students are placed for clinical experience during their 3‐year training programme. Data collection period, April and August 2017.
A prospective survey was selected as the method of data collection tool as it allowed us to collect anonymous responses from stakeholders with minimal disruption to the work environment. The survey was distributed electronically.
The SurveyMonkey online tool was used to develop a rating scale questionnaire, using the statements and domains from the Australian Capability Framework.[1](#jmrs290-bib-0001){ref-type="ref"}
The survey was trialled by three clinicians and during this process one question was removed that was perceived repetitive. The final questionnaire had 39 capability statements that were clustered into three domains: personal, interpersonal and cognitive. The first question of the questionnaire requested participants consent before proceeding with the survey.
An open survey link was sent to MIT clinical managers for internal circulation. A participant information sheet was attached to the email invitation email. Participants were assured of the anonymity of their responses and this was achieved by using the anonymity function on SurveyMonkey.
Ethics approval was granted by the Unitec Research Ethics Committee (UREC) -- No 2017--1002.
Analysis design {#jmrs290-sec-0007}
---------------
For the demographic variables of the survey, the data were represented either in the form of tables or graphs.
Owing to the subjective nature of the data related to capabilities that participants were requested to provide in ordinal form (ranking), the average ranking measure was considered most appropriate to statistically determine which answer choice was most preferred overall. The answer choice with the largest average ranking is the most preferred choice.
The calculations were conducted using Microsoft Excel. The questionnaire was organised with a total of 39 statements which were grouped into the three domain categories: personal capabilities included 15 statements, interpersonal capabilities had 11 statements and cognitive capabilities had 13 statements. Thus, the ranking for personal capabilities was from 1 to 15, interpersonal from 1 to 11 and cognitive from 1 to 13.
The average ranking was calculated as follows:$${{Average}\mspace{720mu}{Ranking}} = \frac{x_{1}w_{1} + x_{2}w_{2} + \ldots + x_{n}w_{n}}{Total},$$where *w* represented the weight of ranked position and *x* represented the response count for the answer choice.
Weights are applied in reverse order. The respondent\'s most preferred choice, which is ranked 1, has the largest weight and their least preferred choice has a weight of 1. In our case, the personal capabilities had 15 statements. The highest ranked statement had a weight of 15, second highest had 14, third highest had 13 and so on with the last ranked statement having a weight of 1. Similar weights, depending on the number of statements, were applied to the interpersonal and cognitive capabilities.
Results {#jmrs290-sec-0008}
=======
A total of 52 responses were received from a maximum sample size of 265. This indicates a response rate of 19.6%. However, it is not possible to exactly predict the size of the actual sample pool, as the surveys were distributed via the clinical managers to their staff. From the responses, 90% (47) of the respondents were female and the remaining 10% (5) were males. In terms of the position/title of the respondents, the majority of the respondents (76%) were senior qualified MITs and 15% team leader/clinical specialist. 74% (39
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1. Introduction {#sec1-jcm-08-02039}
===============
Venous thromboembolism (VTE) is a common cause of morbidity and mortality in hospitalized and non-hospitalized patients \[[@B1-jcm-08-02039]\]. The American Society of Hematology and American College of Chest Physicians guidelines recommend low-molecular-weight heparin (LMWH) as a first-line pharmacological option for most patients at risk of VTE \[[@B2-jcm-08-02039],[@B3-jcm-08-02039]\]. Several prophylactic doses and types of LMWH are used worldwide, which is reflected by differences in national summaries of product characteristics (SPCs) and dosing regimens of randomized controlled trials (RCTs). There is no high-quality evidence or guidance on the optimal prophylactic LMWH dose. Preceding systematic reviews on thrombosis prophylaxis have not specifically assessed benefits and harms associated with different LMWH doses \[[@B4-jcm-08-02039],[@B5-jcm-08-02039],[@B6-jcm-08-02039],[@B7-jcm-08-02039],[@B8-jcm-08-02039],[@B9-jcm-08-02039],[@B10-jcm-08-02039]\]. In addition, there have been very few direct comparisons of prophylactic LMWH dose regimens, and therefore indirect evidence could provide a 'second best' estimate of benefits and harms.
There is no generally accepted definition of different prophylactic LMWH dose categories, which is why we previously categorized LMWH thrombosis prophylaxis regimens as either 'low-dose' or 'intermediate-dose', based on different registered doses in SPCs worldwide \[[@B11-jcm-08-02039]\]. Using this approach in a previous meta-analysis, we found that intermediate-dose LMWH, compared with placebo or no treatment, was associated with a significant decrease in symptomatic VTE, at the cost of an increase in major bleeding \[[@B11-jcm-08-02039]\]. The main objective of the current study was to perform a systematic review with meta-analysis and trial sequential analysis (TSA) comparing benefits and harms of low-dose LMWH versus placebo or no treatment for thrombosis prophylaxis in all types of patients at risk of VTE \[[@B12-jcm-08-02039]\].
2. Materials and Methods {#sec2-jcm-08-02039}
========================
We conducted this systematic review according to a pre-published protocol on PROSPERO (<https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42019124722>) following the methodology suggested by Jakobsen et al, the Cochrane Handbook for Systematic Reviews of Interventions, the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement, and the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) recommendations \[[@B12-jcm-08-02039],[@B13-jcm-08-02039],[@B14-jcm-08-02039],[@B15-jcm-08-02039]\].
2.1. Study Selection {#sec2dot1-jcm-08-02039}
--------------------
### 2.1.1. Patients {#sec2dot1dot1-jcm-08-02039}
Studies were considered for inclusion irrespective of language, blinding, publication status, or sample size. We included RCTs with adult patients allocated to receive thrombosis prophylaxis using either low-dose LMWH, placebo, or no treatment, regardless of their underlying disease or whether they were admitted to the hospital or visited the outpatient clinic.
### 2.1.2. Interventions {#sec2dot1dot2-jcm-08-02039}
The experimental intervention was low-dose LMWH, irrespective of LMWH type or duration of treatment. We a priori defined 'low dose' in our protocol according to the SPCs as approved by the US Food and Drug Administration, the European Medicines Agency, and several national authorities ([Table 1](#jcm-08-02039-t001){ref-type="table"}). If different LMWHs or (weight-adjusted) doses were used in one trial, we classified the dose according to what was used most frequently. We included trials evaluating ultra-low-molecular-weight heparins and LMWHs not listed in [Table 1](#jcm-08-02039-t001){ref-type="table"} (e.g., LMWHs we were unable to classify into a specific dose) in a sensitivity analysis. The control intervention was placebo or no treatment. Co-interventions such as mechanical compression devices were allowed if they were applied in both treatment groups.
### 2.1.3. Outcomes {#sec2dot1dot3-jcm-08-02039}
Predefined co-primary outcomes were all-cause mortality, symptomatic VTE, and major bleeding. Secondary outcomes were serious adverse events (SAE), clinically relevant non-major bleeding, and any VTE (including both symptomatic and asymptomatic events). All outcomes were assessed at maximum follow-up. VTE was defined as deep vein thrombosis or pulmonary embolism, and the diagnosis was accepted when objectified by an imaging technique or autopsy. We made no distinction between distal or proximal, or lower versus upper extremity thrombosis. Major bleeding and clinically relevant non-major bleeding were defined according to trial criteria. SAE were defined according to the International Conference on Harmonisation of Good Clinical Practice definitions (ICH-GCP) \[[@B16-jcm-08-02039]\].
2.2. Data Sources and Searches {#sec2dot2-jcm-08-02039}
------------------------------
We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, PubMed/MEDLINE, EMBASE and Web of Science ([Table S1](#app1-jcm-08-02039){ref-type="app"}). References of identified studies were screened to identify further relevant trials. Finally, we searched the World Health Organization's International Clinical Trials Registry and ClinicalTrials.gov for ongoing trials ([Table S2](#app1-jcm-08-02039){ref-type="app"}). The search was last updated on 10 June 2019.
2.3. Data Extraction and Quality Assessment {#sec2dot3-jcm-08-02039}
-------------------------------------------
Two authors (RJE, WB) independently identified trials for inclusion. Trials excluded on the basis of full text were listed with reasons for exclusion. We extracted information on characteristics (year of publication, country, numbers of sites and patients enrolled), participants (age, sex, eligibility criteria), interventions (type, dose, and duration of LMWH treatment), and outcomes. We resolved differences in opinion through discussion. Two authors (R.J.E., W.B.) independently assessed risks of bias of the included trials according to the revised Cochrane risk of bias tool version 2 \[[@B17-jcm-08-02039]\] in the following five domains: "Bias arising from the randomization process'', "Bias due to deviations from intended interventions'', "Bias due to missing outcome data'', "Bias in measurement of the outcome'', "Bias in selection of the reported result''. RCTs were classified as 'overall low risk of bias' when all bias domains were judged as 'low risk'. Conversely, trials were classified as 'overall high risk of bias' when 'some concerns' or 'high risk' was judged in one or more domains \[[@B18-jcm-08-02039]\]. Publication bias was assessed by inspecting funnel plots for signs of asymmetry when 10 or more trials were included in the analyses \[[@B12-jcm-08-02039],[@B14-jcm-08-02039]\].
2.4. Data Synthesis and Analysis {#sec2dot4-jcm-08-02039}
--------------------------------
We calculated relative risk (RR) with both conventional 95% confidence intervals (CIs) and TSA-adjusted CI if there were two or more trials for each outcome.
### 2.4.1. Assessment of Significance {#sec2dot4dot1-jcm-08-02039}
We used adjusted thresholds for statistical significance to correct for multiplicity issues due to repeated testing. An alpha of 0.025 was used for the co-primary and secondary outcomes to keep the family-wise error rate at a maximum of 5% \[[@B14-jcm-08-02039]\]. In case of statistically significant RR, we calculated numbers needed to treat (NNT) or numbers needed to harm (NNH) with 97.5% CI.
### 2.4.2. Meta-Analysis {#sec2dot4dot2-jcm-08-02039}
Data were pooled using both a fixed-effect and a random-effects model. In case of discrepancy between the models, we emphasized the most conservative estimate. Analyses were performed on an intention-to-treat basis whenever possible or otherwise using an 'available-case analysis'.
### 2.4.3. Trial Sequential Analysis {#sec2dot4dot3-jcm-08-02039}
Conventional meta-analyses may result in type-I errors due to risks of random error when few data have been collected or due to repeated significance testing when a meta-analysis is updated with new trials \[[@B19-jcm-08-02039],[@B20-jcm-08-02039],[@B21-jcm-08-02039],[@B22-jcm-08-02039],[@B23-jcm-08-02039]\]. TSA is a sequential meta-analysis method that
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INTRODUCTION {#sec1-1}
============
Bonding of composite resin to dentin has always been a challenge for the clinicians. Although there is an improvement in handling and bonding characteristics of the newer dental materials,\[[@ref1][@ref2]\] the collagen structure and stability of the dentin bond strength are still a challenge. Studies have proved that collagen in the hybrid layer is affected by enzymatic degradation, leading to bond failure over time.\[[@ref3][@ref4]\]
Collagen fibrils in tissue are stabilized by lysyl oxidase-mediated covalent intermolecular cross-linking.\[[@ref5]\] The biomechanical properties of type I collagen can be improved by introducing more cross-links within and/or between the fibrils with the treatment of specific chemical agents.
Grape seed extract, which is a naturally occurring cross-linker, mainly composed of proanthocyanidins (PAs), could be a good candidate to fulfill such role. It is a natural plant metabolite and showed to possess low toxicity and ability to induce exogenous cross-links.\[[@ref6]\] PA-based compounds can improve dentin collagen physical properties.\[[@ref7][@ref8]\] Another such important agent is ascorbic acid. It is an essentially required component in the synthesis of hydroxyproline and hydroxylysine in collagen. Hydroxyproline serves to stabilize the collagen triple helix. Hydroxylysine is necessary for the formation of the intermolecular crosslinks in collagen. It is believed that ascorbate modulates collagen production through its effect on prolyl hydroxylation.\[[@ref9]\] Hence, we can employ it as an effective chairside procedure to overcome the disadvantage of reduced bond strength of composite resin to dentin.
The present study was carried out to evaluate if collagen cross-linking agents such as PA and sodium ascorbate can affect the shear bond strength of composite resin bonded to the dentinal surfaces of the teeth.
The null hypothesis was that "there was no difference in shear bond strength of resin composite with dentin with or without treated with collagen cross-linking agent PA and sodium ascorbate."
SUBJECTS AND METHODS {#sec1-2}
====================
One hundred freshly extracted human permanent molars were used in this study with the following exclusion/inclusion criteria.
Inclusion criteria {#sec2-1}
------------------
Permanent molars with intact crownAll teeth should be free of caries, any visible discoloration, or crack and without any restoration.
Exclusion criteria {#sec2-2}
------------------
Teeth which did not meet our inclusion criteria were excluded from the study.
The roots of the specimens were mounted in self-cure acrylic resin, with occlusal surface parallel to the floor and extending 4 mm above the surface. Dentin surface was prepared with the help of slow-speed sectioning diamond disc under copious water supply and remove complete enamel portion and exposing the dentinal surface, then the surface of dentin was finished with wet 600 grit silicon carbide paper under running water to produce a standardized smear layer. After that, the dentinal surfaces of the specimens were acid etched with 35% phosphoric acid (SwissTec, COLTENE, Switzerland) as per manufacturers\' recommendation.
The specimens were randomly divided into three groups based on the surface treatment of dentin as follows:
Group I (*n* = 20) -- No dentin pretreatment was doneGroup II (*n* = 40) -- 10% sodium ascorbate pretreatment. This group was further divided into two subgroups based on the pretreatment time as follows:Subgroup IIa (*n* = 20) -- The etched dentin surface was treated with 10% sodium ascorbate solution for 5 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bondingSubgroup IIb (*n* = 20) -- The etched dentin surface was treated with 10% sodium ascorbate solution for 10 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bonding.Group III (*n* = 40) -- 6.5% PA pretreatment. This group was further subdivided into subgroups based on pretreatment time as follows:Subgroup IIIa (*n* = 20) -- The etched dentin surface was treated with 6.5% PA solution for 5 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bondingSubgroup IIIb (*n* = 20) -- The etched dentin surface was treated with 6.5% PA solution for 10 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bonding.
Bonding for composite resin build-up {#sec2-3}
------------------------------------
Before application of bonding agent, a transparent plastic tube (3 mm in diameter and 5 mm height) was placed on the prepared occlusal surface, and the outer diameter was delineated with lead pencil as a reference mark for the application of the bonding agent. Then, plastic tube was removed, and bonding agent (One Coat Bond SL, SwissTec, COLTENE, Switzerland) was applied according to the manufacturer\'s instructions. Then, it was cured with light-emitting diode light (Woodpecker, Guilin Woodpecker Medical Instruments Co., Ltd., China) for 30 s (according to manufacturer\'s instruction).
Composite resin buildup {#sec2-4}
-----------------------
Transparent plastic tube was then fixed manually on the prepared surface of each tooth. A microhybrid composite resin (SwisTec, COLTENE, Switzerland) was applied in five 1 mm layers, and each layer was photopolymerized for 40 s, reaching 5 mm in total height, during which the light was moved around the tube to assure curing of the entire composite resin cylinder. The plastic tube was then removed, and excess composite resin was removed using a polishing disc (Sof-Lex™; 3M Espe, St. Paul, MN, USA). The samples were then stored in distilled water at 37°C for 48 h.
Thermocycling and shear bond strength testing {#sec2-5}
---------------------------------------------
After 48 h, samples were transferred from distilled water to normal water at 37°C for 24 h and then thermocycled for 500 cycles between 5°C and 55°C with a dwell time of 30 s each and transfer time between two baths was 5 and 10 s.
After thermocycling, shear bond strength tests were performed on a universal testing machine (Unitek, 9450 PC, FIE, India) at a cross-head speed of 1 mm/min until the composite cylinder was dislodged from the tooth. Shear bond strength was calculated as the ratio of fracture load and bonding area, expressed in megapascals (MPa). The results were tabulated and subjected to statistical analysis.
RESULTS {#sec1-3}
=======
Obtained data analyzed and expressed in mean ± standard deviation (SD). Mean was compared by one-way analysis of variance and Tukey *post hoc* test *P* = 0.05 was taken as statistically significant.
The results obtained through statistical analysis depicted that dentin pretreatment significantly increases the shear bond strength of the composite resin with dentin; duration of application of pretreatment materials on dentin also plays a significant role \[Tables [1](#T1){ref-type="table"}-[3](#T3){ref-type="table"}\].
######
Shear bond strength in different groups irrespective of time of treatment
######
Shear bond strength in different groups taking time of treatment into consideration
######
Shear bond strength comparison between group/subgroup (Tukey\'s HSD test)
Mean difference was found to be maximum between Groups I and IIb and minimum between Groups IIIa and IIIb. All the between-group comparisons except between Groups IIIa and IIIb were significant statistically \[[Table 3](#T3){ref-type="table"}\]. On the basis of above evaluation, the following order of shear bond strength was observed in different groups/subgroups:
IIb \> IIa \> IIIb \~ IIIa \> I" with "IIb \> IIa \> IIIb \> IIIa \> I".
It was observed that shear bond strength values in Group I was of lower order, whereas shear bond strength values in Group II were of higher order. Shear bond strength values in Group III were of middle order.
In Group I, shear bond strength ranged from 13.14 to 17.00 MPa with a mean value of 15.36 and a SD of 1.16 MPa. In Group IIa, shear bond strength ranged from 16.31 to 23.07 MPa with a mean value of 20.09 and a SD of 1.87 MPa. In Group IIb, shear bond strength ranged from 18.74 to 29.99 MPa, with a mean value of 22.55 and a SD of 2.51 MPa. In Group IIIa, shear bond strength ranged from 12.36 to 20.26 MPa, with a mean value of 17.01 and a SD of 2.10 MPa. In Group IIIb, shear bond strength ranged from 17.11 to 20.66 MPa, with a mean value of 18.46 and a SD of 0.97 MPa \[[Table 2](#T2){ref-type="table"}\].
DISCUSSION {#sec1-4}
==========
The results obtained through statistical analysis depicted that sodium ascorbate and PA application
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Introduction
============
Sickle cell disease (SCD) is a genetic disorder in which polymerization of deoxygenated sickle hemoglobin (HbS) leads to decreased deformability of the normally flexible erythrocytes. These rigid sickle-shaped red blood cells (RBC) can occlude the microvasculature leading to the sudden onset of painful vaso-occlusive episodes (VOC).^[@b1-1050083],[@b2-1050083]^ After HbS deoxygenates in the capillaries, it takes some time (seconds) for HbS polymerization and the subsequent flexible-to-rigid transformation. If the transit time of RBC through the microvasculature is longer than the polymerization time, sickled RBC will lodge in the microvasculature.^[@b3-1050083]^ Any trigger that decreases microvascular blood flow will prolong the transit time, promoting the entrapment of sickled RBC, resulting in vaso-occlusion. This physiology of SCD, described decades ago,^[@b4-1050083],[@b5-1050083]^ is fundamental to understanding the triggering of VOC. Patients report that stress, cold, and pain itself can trigger the onset of VOC^[@b6-1050083]^ but the frequency of VOC is highly variable. To date, the mechanism of how such events might trigger regional vaso-occlusion has not been fully elucidated.
Psychological stress is an exacerbating factor in many chronic illnesses, such as SCD,^[@b7-1050083]--[@b10-1050083]^ coronary artery disease and myocardial ischemia.^[@b11-1050083],[@b12-1050083]^ Stress is significantly associated with increased pain intensity, reductions in social and physical activities and greater health care utilization.^[@b8-1050083],[@b13-1050083],[@b14-1050083]^ Day-to-day stressors have been associated with onset of pain and the course of VOC in SCD.^[@b9-1050083],[@b10-1050083]^ Stress is well-known to modulate autonomic nervous system (ANS) activity which in turn plays a major role in the regulation of regional blood flow.^[@b15-1050083]^ Interestingly, SCD children with greater mental-stress-induced autonomic reactivity had more severe clinical disease.^[@b16-1050083],[@b17-1050083]^ SCD subjects also have augmented ANS-mediated vasoconstriction in response to sighing, hypoxia, and pain.^[@b18-1050083]--[@b20-1050083]^ Therefore, autonomic dysregulation in SCD represents a plausible physiological link between mental stress and sickle RBC retention in the microvasculature.^[@b16-1050083],[@b18-1050083]--[@b21-1050083]^ Further understanding of this proposed mechanism of VOC triggering would not only help to predict disease manifestations, but would also open up opportunities for therapeutic intervention in disorders such as SCD in which preservation of microvascular blood flow is important.^[@b22-1050083]^
To address the role of mental stress in the physiology of SCD, we objectively quantified microvascular blood flow, measured by photoplethysmography, in response to standardized mental stress tasks in subjects with SCD and in controls. We also assessed cardiac ANS balance by analysis of heart rate variability in response to mental stress. We correlated photoplethysmogram-derived physiological indices with subjective indices of perceived stress assessed from standardized anxiety questionnaires. The aim of this study was to determine the relationship of peripheral and cardiac ANS responses with mental stress in SCD.
Methods
=======
The study was conducted under an institutional review board-approved protocol at the Children's Hospital Los Angeles with approved consent/assent. Twenty SCD subjects with Hb SS, S-β^0^, S-β^+^ or SC genotype and 16 age- and race-matched controls from the patients' family and friends were recruited.
Experimental setup and study protocol
-------------------------------------
All studies were performed in an ANS laboratory under strictly controlled settings.^[@b18-1050083]^ Neuropsychological stress was assessed at baseline using the State-Trait Anxiety Inventory (STAI) questionnaire.^[@b23-1050083]^ The STAI Y-1 and Y-2 evaluate "anxiety at this moment, aka *state anxiety*" and "how people generally feel, aka *trait anxiety*", respectively.
Following 5 minutes of baseline recording, the stress induction protocol was presented through psychological software (E-prime 2.0, Psychology Software Tools, USA). The protocol consisted of a memory task (N-back)^[@b24-1050083]^ and a conflict test (Stroop),^[@b25-1050083],[@b26-1050083]^ presented in a randomized order, followed by a pain anticipation (PA) test ([Figure 1](#f1-1050083){ref-type="fig"}). During the N-back task, the subjects were asked to respond when the current letter matched the letter from n steps (n=zero, one, two, or three back) earlier in the sequence. During the Stroop task, the participants were asked to identify the font color of a word, not the written name of the word. We measured state anxiety between tasks. During the PA task, subjects read the following sentence on their computer screen: "You will receive a maximum pain stimulus in one minute. When you cannot tolerate the pain any longer, say STOP and the device will cool down to normal level immediately". However, no pain stimulus was actually applied.
{#f1-1050083}
Physiological measurements and analysis parameters
--------------------------------------------------
All the physiological monitoring sensors were attached to the subjects' left arm. Microvascular blood flow was measured using photoplethysmography (Nonin Medical Inc., USA) and laser Doppler flowmetry (Perimed, Sweden). Respiration (thoracic and abdominal bands, zRip DuraBelt, Philips), the electrocardiogram and continuous blood pressure (Nexfin, Amsterdam) were recorded.
Recorded data from all devices were exported for processing and analysis in MATLAB. The photoplethysmogram amplitude was normalized to its own 95^th^ percentile value during the full study. The average microvascular blood flow was calculated over the 5 min baseline period, the N-back, Stroop and PA tasks. The percent decrease in the amplitude of the photoplethysmogram or microvascular perfusion waveforms ([Figure 2](#f2-1050083){ref-type="fig"}; 2^nd^ and 3^rd^ signals, respectively) from the baseline mean was interpreted as a vasoconstriction response.^[@b18-1050083],[@b27-1050083]^
{#f2-1050083}
Cardiac autonomic balance was assessed by analysis of the R-to-R interval and heart rate variability^[@b19-1050083],[@b28-1050083],[@b29-1050083]^ during baseline and mental stress tasks. The following power spectral indices were calculated: low frequency power, reflecting a combination of cardiac sympathetic and parasympathetic activity; high frequency power, reflecting parasympathetic activity;^[@b29-1050083],[@b30-1050083]^ and the ratio of low frequency power to high frequency power, reflecting sympathovagal balance.^[@b30-1050083]^
Percent changes in mean microvascular blood flow and mean spectral indices from baseline to tasks were calculated. The Student *t*-test (or Wilcoxon sign rank) or χ^2^ test was used to test baseline group differences and task differences. Robust regression was used to correlate vasoconstriction response and state anxiety during the PA task. Repeated measures analysis of variance was used to test differences in N-back and Stroop sublevels and accuracy scores. All statistical analyses were performed using STATA/IC 14.1 (StataCorp LP, TX, USA) with nominal significance set at *P*≤0.05.
The methods are described in detail in the *Online Supplementary Methods S1*.
Results
=======
Data from a total of 20 SCD patients and 16 controls were analyzed. Transfused and non-transfused subjects with SCD were grouped together and healthy and sickle cell trait subjects (controls) were combined after it had been demonstrated that these factors were not statistically significant in the analyses. The percentage of HbS (HbS%) was considered to be zero in patients with sickle cell trait as the cellular distribution of HbS differs in sickle cell trait and does not contribute to sickling under the conditions of the experiments in this study, making the HbS% in sickle cell trait not comparable to that in transfused or non-trait sickle phenotypes. The subjects' characteristics are summarized in [Table 1](#t1
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INTRODUCTION {#s1}
============
Upper track urothelial carcinoma (UTUC) is less common than bladder urothelial carcinoma and accounts for 5--10% of all urothelial carcinoma.^[@b1]^ The incidence of ureteral urothelial carcinoma (UUC) is approximately half that of pyelocaliceal urothelial carcinoma.^[@b2]^ UUC has a worse prognosis than pyelocaliceal urothelial carcinoma.^[@b3],[@b4]^ Owing to different anatomical considerations and oncological outcomes in UTUC, these different malignant entities must be evaluated independently.^[@b5]^
The gold standard treatment for UTUC is radical nephroureterectomy with excision of the bladder cuff, regardless of the tumour location.^[@b6]^ Recently, conservative surgery such as endoscopic ablation or segmental ureteral resection, which allows preservation of the upper urinary renal unit, has also been applied.^[@b7]^ However, preoperative histological evaluation through biopsy of the upper urinary tract is difficult, because ureteroscopy is an invasive procedure and usually requires general anaesthesia. Furthermore, the accuracy of ureteroscopic biopsy in predicting tumour stage and grade is limited, and the limitations of endoscopic biopsy must be balanced against the possible advantage of avoiding radical surgery.^[@b8]^ Thus, accurate preoperative prediction of tumour grade could be helpful in selecting more appropriate therapeutic options.
CT urography (CTU) is an imaging modality with high diagnostic accuracy in the detection of UTUC and has replaced intravenous excretory urography and ultrasonography as the first-line imaging test for investigating high-risk patients.^[@b9]^ Even though several studies have investigated diffusion-weighted MRI (DW-MRI) as an imaging assessment for predicting tumour grade of UTUC,^[@b10]--[@b11]^ characteristic CTU findings that can predict tumour grade of UUC have not been identified, to the best of our knowledge.
In this study, we aimed to evaluate the correlation between CTU imaging variables, including tumour size and imaging features, and histological grade of UUC, and to identify CTU imaging features that allow prediction of high-grade UUC, which should be treated by radical surgery.
METHODS AND MATERIALS {#s2}
=====================
Patients
--------
This retrospective single-centre study was approved by the institutional review board at and written informed consent was not required. We searched institutional patient information systems to identify all consecutive patients with UUC who had undergone nephroureterectomy between January 2005 and July 2016. A total of 79 consecutive patients who underwent surgery with removal of a surgical specimen for histological analysis were registered. The inclusion criteria for this study were as follows: (i) tumours only located in the ureter, (ii) patients had undergone CTU scan prior to surgery and (iii) histologicalal confirmation of UUC with clear statement of histological grade according to the WHO 2004 classification system. Four patients were excluded because histological grade was not available in the pathological reports, and two patients did not undergo a CTU scan. Ultimately, 73 patients (52 males and 21 females; mean age, 68.92 ± 9.08 years) with 81 UUCs were included in our study. All pathological data were reviewed by a board-certificated pathologist, and all tumours were classified into low-grade and high-grade groups according to the WHO 2004 classification system and pathologic T stage of the tumours was assessed according to the TNM staging system.
CTU technique
-------------
All CTU examinations were performed using various CT scanners from 16-channel to 128-channel MDCT scanners (Somatom Sensation 16, Siemens Healthcare, Brilliance 64, Philips Medical Systems, Best, Netherlands or Somatom Definition Flash 128, Siemens Healthcare Forchheim, Germany). Scanning parameters of the most frequently used CT scanner (Brilliance 64, Philips Medical Systems, Best, Netherlands) were as follows: tube voltage, 120 kVp; effective tube current, 300 mAs; section thickness, 5 mm; pitch and speed, 0.891:1; rotation time, 0.75 s and collimation, 64 × 0.625 mm for 64-channel MDCT. Before acquisition of contrast-enhanced scans, simple unenhanced scans were obtained, after which 2 ml kg^--1^ non-ionic contrast material containing 300--350 mg ml^−1^ of iodine \[iomeprol (Iomeron 300, Bracco Altana Pharma, Konstanz, Germany), iopamidol (Pamiray 300, Dongkook Pharmaceutical, Seoul, Republic of Korea) or iobitridol (Xenetix 300, Guerbet, Villepinte, France)\] was intravenously administered at a rate of 3.0 ml s^−1^ using a standard power injector. For CTU, in addition to the unenhanced scan, two-phase studies were performed with combinations of corticomedullary and excretory phases at our institution. The corticomedullary phase began 30--40 s after contrast administration, and excretory phases began 300 s after contrast administration, respectively.
Image analysis
--------------
Two radiologists (DJS and STH with 17 and 3 years of experience, respectively, in interpreting genitourinary images) independently reviewed all CTU images on a picture archiving and communication system workstation (INFINITT PACS, INFINITT Healthcare, Seoul, Republic of Korea). The readers knew all patients had been diagnosed with UUC, but were informed of neither the histological grade nor the findings listed in the initial radiological report. They evaluated the following CTU imaging features: tumour location, tumour size, tumour enhancement value, multiplicity, periureteral infiltration, enlarged retroperitoneal lymph nodes with a short axis of more than 1 cm, and hydronephrosis grade. Tumour location was categorized into three groups (proximal, middle, and distal) according to anatomic ureteral segmentation. Tumour size was determined as the maximal length or diameter of the whole tumour presenting as ureteral soft-tissue mass or enhancing wall thickening on the axial, sagittal, or coronal CTU images. In patients with multiple lesions, the largest one was selected for size measurement. Tumour enhancement value was calculated as the difference between attenuation values in the corticomedullary phase and unenhanced phase. On corticomedullary phase images, the readers drew a circular ROI that included the enhancing solid portion of the tumour, avoiding adjacent mesenteric fat. The ROI was as large as possible to minimize noise. A ROI of the same size was placed in the corresponding location on the unenhanced scan image.
The readers also reported hydronephrosis grade according to the modified version of the Society for Foetal Urology Hydronephrosis Grading System ([Table 1](#t1){ref-type="table"}).
######
Modified version of the Society for Fetal Urology Hydronephrosis Grading System
Grade 0 1 2 3 4
--------------------------------- --------------- ------------------------------ -------------------------------------- -------------------------------------------------------------- --------------------------------------------------
Ureter and pelvocalyceal system No dilatation Local dilation of the ureter Ureteral and renal pelvis dilatation Ureteral and renal pelvis dilatation plus calices dilatation Further dilatation of ureter, pelvis and calices
Renal parenchymal thickness Normal Normal Normal Normal Thin
Statistical analysis
--------------------
Descriptive statistics of means, standard deviations and frequencies were used to describe patient characteristics. Univariate logistic regression modelling, Mann--Whitney *U* tests, and *Χ*^2^ tests were used to assess the correlation between CTU imaging variables and histological tumour grade. Multiple logistic regression analysis using a backward selection method was performed to identify significantly independent CTU imaging variables that could predict high-grade tumours. Spearman correlation analysis was used to assess the correlation between tumour size and hydronephrosis grade. *Χ*^2^ test and linear-by-linear association were used to investigate the correlation of hydronephrosis grade and peritumoural intfiltration with pathologic T stage. A receiver operating characteristic (ROC) curve was constructed to identify the cut-off value of effective factors that provided the best diagnostic accuracy. Interobserver agreement was calculated using kappa statistics for nominal values, including hydronephrosis grade, peritumoural infiltration, multiplicity and presence of enlarged retroperitoneal lymph nodes. Intraclass correlation was calculated for continuous values including tumour size and contrast enhancement value. The scores were used to define agreement as follows: 0.41--0.60 denoted moderate agreement; 0.61--0.80, good agreement and greater than 0.81, excellent agreement.
Statistical analysis was done using IBM SPSS Statistics version 22.0 for Windows (IBM Corp., Armonk, NY). A *p* value of less than 0.05 was considered statistically significant.
RESULTS {#s3}
=======
Images of the 73 patients with 81 UUCs were reviewed. The lesions were unilateral in all cases. 15 patients (20.5%) had low-grade UUCs ([Figure 1](#f1){ref-type="fig"}) and 58 patients (79.5%) had high-grade UUCs ([Figure 2](#f2){ref-type="fig"}). 22 (27.1%) lesions were located in the proximal ureter, 14 (17.2%) in the middle ureter, and 45 (55.5%) in the distal ureter. Eight (5.8%) patients had multiple lesions in the ipsilateral ureter. Clinicopathological characteristics of the patients are summarized in [Table 2](#t2){ref-type="table"}.
![A 74-year-old
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Introduction {#S1}
============
Despite significant improvements in outcome,([@R1]--[@R3]) relapse remains the leading cause of treatment failure for children with acute lymphoblastic leukemia (ALL) and occurred in 11 to 36% of those with high-risk B-precursor ALL.([@R4]--[@R10]) Mechanisms by which genomic variation influence relapse risk could involve somatically acquired mutations or inherited genetic variations, which could affect intrinsic resistance to chemotherapy([@R11]--[@R13]) or host pharmacokinetics of anti-leukemic agents.([@R14]--[@R16])
Some studies report that black and Hispanic children with ALL have inferior outcomes to non-Hispanic white children.([@R17]--[@R21]) Reasons for these differences are likely multifactorial, including differences in treatment adherence and access to therapy,([@R22]--[@R24]) in the incidence of favorable and unfavorable presenting features and cytogenetics,([@R25]--[@R27]) and in the frequency of genetic variants affecting pharmacokinetics and pharmacodynamics of antileukemic agents which segregate with ancestry.([@R28]) It remains uncertain whether racial disparities persist with modern intensive ALL regimens.
We performed a genome wide association study (GWAS) in a large cohort of children with high-risk B-ALL to identify inherited genetic variations associated with relapse. We performed an analysis adjusting for both treatment and ancestry to identify single nucleotide polymorphisms (SNPs) which increased risk across ancestries (ancestry-agnostic SNPs). Because racial disparities in relapse persisted in this trial, we also performed analyses within each of the three largest ancestral groups (white, black, Hispanic) to identify ancestry-specific variations associated with relapse. We also interrogated relapse SNPs for associations with risk of central nervous system (CNS) relapse, relapse among patients randomized to receive either escalating-dose methotrexate and asparaginase (i.e., Capizzi regimen) or high-dose methotrexate during the first interim maintenance (IM1), and for associations with the pharmacokinetics of antileukemic agents or the intrinsic sensitivity of leukemia cells to chemotherapy. Finally, to assess robustness of relapse SNPs across different therapies, we tested for replication in an independent cohort.
Methods {#S2}
=======
Patients and treatment {#S3}
----------------------
For the discovery cohort, germline DNA was obtained at remission in children and young adults with newly diagnosed B-precursor ALL enrolled on COG AALL0232 (NCT00075725, <https://clinicaltrials.gov/ct2/show/NCT00075725>).([@R8]) This protocol involved a 2×2 factorial randomization for induction steroid (prednisone ×28 days vs. dexamethasone ×14 days) and interim maintenance 1 regimen (Capizzi escalating-dose methotrexate with pegylated-asparaginase vs. high-dose methotrexate). Exclusion criteria are described in [Figure 1](#F1){ref-type="fig"} and the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. The replication cohort comprised children treated on prior generation protocols who would have met the eligibility criteria of AALL0232 ([Supplementary Methods and Supplementary Table 1](#SD1){ref-type="supplementary-material"}).
All studies were approved by the institutional review boards of participating institutions, and all patients and/or guardians provided age appropriate consent/assent in accordance with the Declaration of Helsinki.
Genotyping and genetic ancestry {#S4}
-------------------------------
Genotyping and genetic imputation was performed as described in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. Genetic ancestry was defined using STRUCTURE v2.2.3.([@R29]) For categorization of patients into discrete ancestral groups, individuals were classified based on inferred genetic ancestry as white \[Northern European (CEU) \>90%\], black \[West African (YRI) \>70%\], Hispanic \[Native American([@R30]) \>10% and Native American greater than West African\], or Other, including Asian \[East Asian (CHB/JPT) \>90%\].
Quality control steps for both patients and SNPs are detailed in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}.
Identification of relapse associated SNPs {#S5}
-----------------------------------------
The approaches to perform GWASs for relapse are detailed in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. GWASs were performed to identify SNPs using an ancestry-agnostic ([Supplementary Table 2 and Supplementary Figure 1](#SD1){ref-type="supplementary-material"}) and an ancestry-specific approach ([Supplementary Figures 2a--c](#SD1){ref-type="supplementary-material"}).
Treatment arm and site specific annotation of relapse SNPs {#S6}
----------------------------------------------------------
SNPs associated with relapse were further characterized in subsets of patients based on their IM1 randomization (the Capizzi arm with escalating-dose methotrexate plus pegylated-asparaginase vs. the high-dose methotrexate arm) while adjusting for induction randomization, rapid early response, and ancestry as categorical variables. Additionally, SNPs were tested for their association with CNS relapse (isolated or combined with other sites), with isolated hematologic or other extramedullary relapse treated as competing risks. Significant association thresholds for all analyses were determined by profile information criteria (Ip),([@R31]) which balances false positives and negatives while addressing the effects of multiple testing.
Association with orthogonal pharmacologic data {#S7}
----------------------------------------------
SNPs associated with relapse (ancestry-specific or ancestry-agnostic) were evaluated for association with drug resistance in HapMap cells lines (prednisone, asparaginase, mercaptopurine, methotrexate polyglutamate accumulation), primary ALL cells from newly diagnosed patients (prednisone, vincristine, mercaptopurine, asparaginase, *in vivo* leukocyte count decrease following methotrexate), or for association with increased drug clearance (asparaginase allergy, methotrexate clearance, dexamethasone clearance), as described in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. SNPs were considered supported by orthogonal data if the risk allele for relapse was associated (at P\<0.05) with *in vitro* drug resistance, decreased methotrexate polyglutamate accumulation, smaller leukocyte decrease after methotrexate, more rapid drug clearance, or greater incidence of asparaginase allergy.
Evaluation of relapse-associated SNPs in replication cohort {#S8}
-----------------------------------------------------------
Relapse-associated SNPs were evaluated in an independent replication cohort (n=719) for their association with relapse using a Cox proportional hazard regression, with patients censored at the time of competing events (i.e. remission death, second malignancy) or last follow-up and adjusting for treatment categorized into 6 groups ([Supplementary Table 2](#SD1){ref-type="supplementary-material"}).([@R5], [@R9], [@R32]) AALL0232 ancestry-agnostic SNPs were evaluated in all patients while adjusting for treatment and ancestry. AALL0232 ancestry-specific SNPs were evaluated in the same ancestry subset of the replication cohort while adjusting for treatment and, in blacks and Hispanics, percent ancestry. The replication cohort SNPs were evaluable if they passed quality control steps as described for the discovery cohort ([Supplementary Methods](#SD1){ref-type="supplementary-material"}). Differences in genotyping platforms between the discovery and replication cohorts, as well as the smaller size of the replication cohort, resulted in 595 of the 1,017 relapse SNPs from the discovery cohort being evaluable in the replication cohort. Validated SNPs were those associated with relapse at P\<0.05 and with identical risk alleles.
Quantitative contribution of SNPs to ancestral differences in relapse {#S9}
---------------------------------------------------------------------
To identify SNPs which most contributed to ancestry-associated differences in relapse risk, a classification and regression tree analysis was performed separately in blacks and Hispanics considering treatment arm and validated ancestry-agnostic and ancestry-specific SNPs as potential branches. Branches were limited to two levels with each new branch needing to contain at least 20% of the initial ancestral patient group (representing \~1% or at least 22 patients from the discovery cohort for the smallest group, those with black ancestry). The impact of these SNPs on the risk of relapse associated with black or Hispanic ancestry was then evaluated in a competing risk regression model of relapse including the SNPs, treatment, and ancestry.
Statistical analysis {#S10}
--------------------
Statistical and bioinformatics analyses were performed using R versions 3.2.2, including the "survival", "cmprsk", "rpart", and "forestplot" packages. Association studies of orthogonal phenotypes were performed either in R or PLINK version 1.07.
Results {#S11}
=======
Patient Characteristics {#S12}
-----------------------
Of 3,084 children and young adults enrolled on AALL0232, germline genotype and relapse data were available for 2,652, and 2,225 were included in the GWAS for relapse ([Figure 1](#F1){ref-type="fig"}). To identify covariates to include in the GWAS, we examined the importance of treatment group and ancestry on relapse risk. Consistent with findings in the entire randomized cohort,([@R8]) patients treated with Capizzi-methotrexate had a higher relapse risk than those treated with high-dose methotrexate ([Supplementary Table 3](#SD1){ref-type="supplementary-material"}). Because patients with slow early response did not differ by their induction steroid assignment but did differ by IM1 randomization, patients with slow early response were combined for multivariable and GW
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Introduction {#S0001}
============
The rotator cuff tear is the most common tendinopathy in humans and over 200,000 cuff repairs are performed annually in the United States \[[1](#CIT0001),[2](#CIT0002)\]. The decreased morbidity associated with arthroscopic repairs has contributed to the popularity and broad indications for this surgical intervention \[[1](#CIT0001),[3](#CIT0003)\]. Tendon reattachment even if biomechanically strong at the time of repair often fails and approximately 50% of patients with full-thickness tears of the rotator cuff report symptoms at 6 months after surgery \[[1](#CIT0001),[4](#CIT0004),[5](#CIT0005)\].10.1080/21623945.2019.1609201-F0005Figure 5.Schematic representation of the changes in the number and cross-section area of fat clumps and of adipocyte number in the proximal, medial and distal SSP muscle after a complete SSP tendon detachment. IMF increased closer to the tendon tear compared to the proximal SSP muscle. Detached muscles had more clumps in the distal and medial sections and of larger size in the distal section. There were more adipocytes in the distal and medial detached SSP muscles compared to proximal and cross-sectional area was smaller in the distal SSP muscle. The fat clumps are represented by ovals and adipocytes by smaller filed black shapes. Results from the statistical analysis are indicated: 0.001 ≤ P \< 0.01 (\*), 0.0005 ≤ P \< 0.001 (\*\*), P \< 0.0005 (\*\*\*).
The unsatisfactory success of rotator cuff repair surgeries has been attributed in many cases to muscle atrophy and fat accumulation both assessed by medical imaging methods \[[6](#CIT0006)--[8](#CIT0008)\]. The benefits of arthroscopy to repair the cuff and of advanced imaging methods to measure rotator cuff muscle fat content are undeniable but enhancing postoperative outcomes remain a challenge and basic knowledge on the mechanisms of intramuscular fat accumulation is needed \[[9](#CIT0009)--[11](#CIT0011)\].
Animal models of rotator cuff tendon injury and repair capture important aspects of the human disease \[[12](#CIT0012)--[16](#CIT0016)\]. Imaging of the rabbit's SSP muscle documented fat accumulations both extra- and intra-muscular, and were evident as early as 4 weeks after SSP tendon detachment and progressed up to 12 weeks \[[17](#CIT0017)\]. The fat signal increased from proximal-to-distal with the highest amount of fat detected in the distal quarter of the SSP muscle, the site closer to tendon detachment \[[17](#CIT0017)\]. Both fat accumulation and muscle atrophy were present at week 1 and 2 after immediate repair but only fat accumulation persisted at 6 weeks \[[18](#CIT0018),[19](#CIT0019)\]. In a different study, delayed tendon reattachment did not reverse SSP fat accumulation \[[20](#CIT0020)\]. The rabbit experimental model of rotator cuff tear and repair reproduced accurately the human pathology and represents a valuable avenue to decipher the pathophysiology of IMF accumulation associated with rotator cuff tear \[[12](#CIT0012),[21](#CIT0021)\].
The mechanisms for adipose tissue expansion have been studied extensively. In the context of obesity resulting from a high-fat diet, large fat accumulations are noticeable in subcutaneous and visceral deposits \[[22](#CIT0022)--[24](#CIT0024)\]. Overnutrition induced adipocyte hypertrophy in upper-body subcutaneous fat while a cycle between hypertrophy and hyperplasia characterized deposits below the waist \[[25](#CIT0025)\]. The IMF deposit, considered a small fat deposit, is made up of white adipocytes and its accumulation characterizes late stages of muscular dystrophies \[[24](#CIT0024),[26](#CIT0026)\]. The pathophysiology of adipocytes leading to IMF accumulation associated with rotator cuff tear remains unknown.
We hypothesized that IMF accumulation observed after rotator cuff tears results from adipocyte hypertrophy rather than hyperplasia leading to the enlargement of resident muscle fat clumps. The purpose of the current study was to characterize, at the microscopic level and over time, the expansion of the adipose tissue in the SSP muscle of rabbits after detachment of the distal SSP tendon.
Materials and methods {#S0002}
=====================
Animals and surgical procedure {#S0002-S2001}
------------------------------
This study was approved by the University of Ottawa Animal Care Committee. Adult female New Zealand rabbits (n = 45) weighing 3.0 kg were purchased from Charles River, Saint-Constant, Quebec, Canada and allowed to acclimate for one week upon arrival. For the experimental group, a supraspinatus tenotomy was performed unilaterally in 30 rabbits by sectioning completely the SSP tendon from the greater tuberosity of the humerus using a surgical blade under general anaesthesia \[[14](#CIT0014)\]. Left and right shoulders were alternated. To prevent postoperative adhesions, the stump of the tendon was wrapped with a polyvinylidene membrane (5 µm, Durapore, Millipore, Bedford MA USA). Animals were housed individually, divided into three equal groups, killed at 4, 8 or 12 weeks after surgery and the operated shoulders were collected for histological analysis. For the control group, 15 unoperated rabbits were equally divided into three groups, killed at 4, 8 and 12 weeks and both shoulders were collected. The harvesting method of shoulders was described in our previous publication. Complete SSP muscles were dissected from the scapula, wrapped and frozen at −20°C until processed for histology analysis \[[17](#CIT0017)\]. Radiology and macroscopic data on this group of animals have already been reported [\[17\];](#CIT0017) the current microscopy analysis at the cellular level builds on those studies.
Histology specimen preparation {#S0002-S2002}
------------------------------
Harvested SSP muscles were fixed in 4% paraformaldehyde and rinsed twice for 1 h in phosphate buffered saline to begin processing for histology. Muscle specimens were frozen to preserve fat structures during sectioning. From each muscle, three cross-section slices of 1-mm thickness were cut at the proximal quarter, middle-half, and distal quarter sites of the supraspinatus muscle. Muscle slices were stained for 2 weeks with 5% potassium dichromate and 2% osmium tetroxide followed by paraffin embedding \[[14](#CIT0014)\]. Using a microtome, 6µm-thick microscopy slides were prepared. Fixation in osmium tetroxide stained adipocytes black.
Histology evaluation and microscopy image analysis {#S0002-S2003}
--------------------------------------------------
A total of 180 slides from detached tendons and from unoperated tendons, at time points 4, 8 or 12 weeks, in the proximal, middle or distal quarters of the SSP muscle were analysed by light microscopy ([Table 1](#T0001)).10.1080/21623945.2019.1609201-T0001Table 1.Summary of the samples studied including numbers of rabbits, shoulders, and tissue sections for both fat clump and adipocyte analyses.SSP Muscle Quarter/WeeksDetached vs ControlRabbits (N)Shoulders\
(N)Muscle Sections (Clumps)\
(N)Muscle Sections (Cells) (N)Proximal Quarter4Detached1010810Control51010108Detached10101010Control510101012Detached1010910Control5101010Middle Quarter4Detached10101010Control5109108Detached1010810Control510101012Detached1010910Control5101010Distal Quarter4Detached10101010Control51010108Detached10101010Control510101012Detached10101010Control5101010 **Fat ClumpsAdipocytes**Total Muscle Sections/Fields AnalyzedDetached8490 (270 fields)Control8990 (270 fields)Total Fat Clumps/Adipocytes AnalyzedDetached18,54210,389Control14,3456706
Fat clumps were measured on entire SSP muscle cross-sections digitized at 6.7x magnification and backgrounds were cropped using Corel Photo-Paint 11. Images were then imported for computer-assisted quantitative image analysis using software ImageJ software (version 1.34s; National Institute of Health, Bethesda, MD, USA). Scales were set by using calliper measurements of two reference points on the slide and converted to pixels. Pictures were converted into binary black and white images (8-bit; grey scale). A fat clump was defined as an area of fat stained black, not in contact with another stained area ([Figure 1](#F0001)). The 'threshold' function was manually adjusted to select only black pixels. The 'watershed' function was used to mark the boundaries of individual fat clumps. The 'analyse particle' command was used to measure clump numbers and areas with 'cellularity' set at 0--1 and 'size' set at 0-infinity. The command 'measure all' was used to automatically generate all measurements.10.1080/21623945.2019.1609201-F0001Figure 1.Representative micrographs of IMF accumulation in the distal quarter of the SSP muscle cross-sections. (a). SSP muscle sections at 4, 8 and 12 weeks after tendon detachment. (b). SSP muscle sections in control animals at the same time points. IMF was stained using osmium tetroxide and is visible at black-stained areas. Note the higher accumulation of fat in the tendon detached group compared to controls at all time points studied. Original magnification at 6.7x.
Adipocyte number per field and average cross-sectional area were measured using computer-assisted image analysis of the same microscopic slices captured at 25x magnification. Three different fields of equal and fixed areas (0.149 mm^2^ each) were chosen using the following criteria: included black staining, not contiguous with the other selected fields, included minimal empty space
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1. Introduction {#sec1-membranes-08-00069}
===============
Polymer electrolytes are regarded as one of the most promising candidates in advanced electrochemical applications, such as "smart" windows, displays, sensors, and more importantly, rechargeable lithium batteries \[[@B1-membranes-08-00069],[@B2-membranes-08-00069],[@B3-membranes-08-00069],[@B4-membranes-08-00069]\]. For this last one, in particular, the research has focused for decades on gel-type membrane \[[@B5-membranes-08-00069]\], generally achieved by immobilizing a liquid solution (for instance, a polar aprotic organic solvent or mixtures with a lithium salt) into a hosting polymeric matrix, such as poly(ethylene oxide) (PEO) and its derivatives (e.g., polyacrylonitrile (PAN), poly(vinylidene fluoride) (PVDF), poly(methyl methacrylate) (PMMA)) \[[@B6-membranes-08-00069],[@B7-membranes-08-00069]\]. Respect to liquid electrolytes, in fact, gel polymer electrolytes (GPEs) are able to conjugate high ion conductivities with good mechanical strength, flexible geometry, reducing of liquid leaking and, thus, higher safety \[[@B8-membranes-08-00069]\].
Owing to its ability to dissolve a large variety of salts, through interaction of its ether oxygen with cations, PEO has been one of the most extensively studied polymer used to prepare solid-state electrolytes, lighter, thinner, and safer for lithium-ion polymer batteries \[[@B9-membranes-08-00069],[@B10-membranes-08-00069]\].
Thought, the low ionic conductivities at room temperature (10^−6^--10^−8^ S cm^−1^), the Li^+^ transference number lower than 0.5 and the poor mechanical strength, still hinder the large scale diffusion of PEO-based device. Conversely, PAN ensures an ionic conductivity of circa 10^−3^ S cm^−1^, satisfactory flame and mechanical resistances, but the dimensional stability of gels is poor \[[@B11-membranes-08-00069],[@B12-membranes-08-00069]\]. After GPE preparation, in fact, a phase separation between the encapsulated electrolyte solution and the PAN matrix typically occurs, leading to a leakage problem and, thus, the passivation phenomena of the lithium electrode when in contact with the gel, as well as failure of the electrode/electrolyte contact both resulting in a dramatic reduction of the ionic conductivity.
One of the strategy undertaken to bypass the drawbacks is the blending method, according to which two or more polymers are mixed to obtain a blend electrolyte. As already probed \[[@B13-membranes-08-00069],[@B14-membranes-08-00069],[@B15-membranes-08-00069],[@B16-membranes-08-00069]\] the method allows to easily control a large number of factors, directly affecting the thermal, mechanical and electrical properties of the final polymer electrolytes. By mixing PMMA and PVdF polymers, Nicotera and coworkers obtained a blend with remarkable improvement of mechanical stability respect to unblended polymers \[[@B17-membranes-08-00069]\]. Helan et al. have been reported outstanding thermal stability up to 230 °C for PAN/PMMA blends, but with quite low ionic conductivity, of the order of 2 × 10^−7^ S cm^−1^ \[[@B18-membranes-08-00069]\]. Very interesting electrical behavior and dimensional stability have been obtained by Choi et al. on PEO-PAN blend gel electrolytes, despite no evidence regarding mechanical resistance being provided \[[@B19-membranes-08-00069]\].
An alternative approach for creating gel electrolyte system with improved mechanical properties and electrochemical performances foresees the incorporation of nanoscale organic/inorganic fillers within the polymer matrix \[[@B20-membranes-08-00069]\]. The addition of SiO~2~ \[[@B21-membranes-08-00069]\], Al~2~O~3~ \[[@B22-membranes-08-00069]\], TiO~2~ \[[@B23-membranes-08-00069]\], and other metal oxides \[[@B24-membranes-08-00069],[@B25-membranes-08-00069]\] generally act as solid plasticizers, softening the polymer backbone and, thus, enhancing the segmental motion of the hosting polymer which, in turn, results in improved ion conductivity.
Among inorganic fillers, layered nanoparticles based on clays have been actively investigated lately since they offer a large number of interesting properties such as high cation exchange capacity, large chemically active surface area, outstanding swelling ability, intercalation, catalytic activity, and high chemical and thermal stability. Finally, the properties of the smectite nanoclays can be tailored using simple chemical methods such as intercalation with organic or inorganic guest molecules. From the above, the dispersion of proper clay minerals within the polymer matrix could enhance the ionic conductivity improving at the same time the strength and heat resistance of the GPE.
Smectite clay with different particle sizes has been effectively tested as filler for the preparation of PEO nanocomposite electrolytes, demonstrating a discrete improvement of ionic conduction \[[@B26-membranes-08-00069]\]. Kurian et al. \[[@B27-membranes-08-00069]\] have shown that the surface modification of clay by ion exchange reactions with cationic organic surfactants such as alkyl amines, enhance the chemical affinity with the polymer matrix, leading to exfoliation of the clay particles and improving the gel's strength. Organic montmorillonite (MMT) prepared by ion exchange with HTAB was dispersed in PAN polymer, obtaining a composite GPEs with improved thermal stability and ionic conductivity \[[@B28-membranes-08-00069]\].
Despite the efforts, however, there is still the need to design a gel electrolyte able to guarantee adequate electrical performance without sacrificing mechanical strength and thermal resistance. In the present study, PAN/PEO blend (80:20 weight ratio) polymers were used in order to prepare nanocomposite GPEs with an organo-modified clay. Specifically, hydrated sodium calcium aluminum magnesium silicate hydroxide (SWy-2, Nanocor) was the natural montmorillonite/smectite clay selected since it is relatively inexpensive, widely available and has small particle size as well as it shows good intercalation capability. The organo-modification of the SWy-2 (org-SWy) was achieved by ion exchange reaction with hexadecyltrimethyl ammonium bromide (CTAB). The filler loading of org-SWy in the GPE was 10 wt % with respect to the polymers PAN/PEO. For the gel preparation, a mixture of ethylene carbonate (EC) and propylene carbonate (PC), with molar ratio EC:PC 1:0.4, was used as plasticizer, while lithium trifluoromethanesulfonate (LiTr) was the salt chosen.
In order to compare the effect of the clay on the gel properties, also not blended and filler-free GPE membranes were also prepared.
All the GPEs were investigated by thermal (DSC), morphological (scanning electronic microscopy-SEM) and mechanical (DMA) analysis, while the ion transport studies were conducted by electrochemical impedance spectroscopy (EIS) and by multinuclear NMR spectroscopy. In particular, the ^1^H, ^7^Li, and ^19^F pulse-field-gradient (PFG) method was employed to obtain a direct measurement of the self-diffusion coefficients both of ions and solvents plasticizers (EC/PC), while the spin-lattice relaxation time (T~1~) was obtained by the inversion recovery sequence.
The combination of the electrochemical and NMR data has provided a wide description of the ions dynamics inside the so complex systems, as well as information on ion associations and interactions between polymers, filler and ions.
2. Materials and Methods {#sec2-membranes-08-00069}
========================
2.1. Materials {#sec2dot1-membranes-08-00069}
--------------
Poly(ethylene oxide) (PEO, M.W. 5,000,000), polyacrylonitrile (PAN), lithium trifluoromethanesulfonate (LiCF~3~SO~3~ or LiTr, 99.95%), ethylene carbonate (EC, 98%), propylene carbonate anhydrous (PC, 99.7%), and hexadecyltrimethyl ammonium bromide (CTAB, 98%) were purchased from Sigma Aldrich, Milan, Italy and used as received.
Natural smectite Wyoming montmorillonite (SWy-2) has obtained from the Source Clay Minerals Repository, University of Missouri Columbia, MO, USA. The cation exchange capacity (CEC), measured by the Co(II) procedure, is equal to 80 mequiv. per 100 g of clay, charge density 0.6 e^−1^/unit cell (the unit cell is the Si~8~O~20~ unit) and particle size around 200 nm. The structural formula is Na~0.62~\[Al~3.01~Fe(III)~0.41~Mg~0.54~Mn~0.01~Ti~0.02~\](Si~7.98~Al~0.02~) O~20~(OH)~4~.
2.2. Synthesys of Organo-Modified Clay (Org-Swy) {#sec2dot2-membranes-08
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1. Introduction {#sec1}
===============
Acute respiratory distress syndrome (ARDS) incorporates a cluster of clinical features including shortness of breath and tachypnea and is defined by the Berlin criteria as having an acute onset with the development of hypoxemia and bilateral pulmonary opacities on radiographic imaging \[[@B1]\]. There are many causes of ARDS; however, most often, it is triggered by infections, blood transfusions, direct lung injury, and toxins \[[@B2]\]. Treatment includes removal of the inciting cause, supportive therapy, and the attainment of sufficient blood oxygenation. Adequate oxygenation in ARDS is often achieved by endotracheal intubation and mechanical ventilation. Noninvasive positive pressure ventilation (NIPPV) has been less frequently indicated as an alternative form of adequate oxygenation. Typically, its use is focused and intended on preventing complications that are associated with invasive ventilation such as barotrauma, vocal cord injury, and ventilator-associated pneumonia \[[@B3]\].
Bupropion is an atypical oral antidepressant medication commonly used to treat depression, tobacco dependence, obesity, and hypoactive sexual disorder. Its mechanism of action involves the inhibition and reuptake of norepinephrine and dopamine which can induce a state of euphoria. Because of these effects, bupropion has been reported to be abused recreationally \[[@B4], [@B5]\]. We describe a case of ARDS induced by bupropion inhalation that was treated with NIPPV.
2. Case Presentation {#sec2}
====================
A 30-year-old male with a past medical history significant for polysubstance abuse presented to the emergency department (ED) two hours after ingesting 90 mg of oxycodone and 30 mg of diazepam along with intranasal bupropion. On arrival, he complained of extreme fatigue, respiratory distress, and confusion.
In the ED, he was noted to be lethargic and short of breath. Initial vital signs revealed heart rate of 104 beats per minute, respiratory rate of 35 per minute, and O~2~ saturation of 75% on room air. Respiratory exam revealed diffuse bilateral coarse breath sounds on inspiration and expiration. His laboratory results were notable for a leukocytosis of 14.1 K, creatinine of 1.25 g/dl, lactate of 4.1, and an elevated procalcitonin level. Venous blood gas analysis showed a pH of 7.18 with a pCO~2~ of 51 mmHg. Chest X-ray (CXR) demonstrated diffuse bilateral alveolar infiltrates ([Figure 1(a)](#fig1){ref-type="fig"}). Computed tomography (CT) of the chest showed diffuse bilateral airspace disease characterized by groundglass and consolidative opacities with relative peripheral lung sparing and perihilar predominance ([Figure 2](#fig2){ref-type="fig"}). He received naloxone with mild improvement in mental status and was initiated on Bilevel Positive Airway Pressure (BiPAP) with an inspiratory positive airway pressure (IPAP) of 15 cm H~2~O, expiratory positive airway pressure (EPAP) of 5 cm H~2~O with 40% fraction of inspired oxygen (FiO~2~). Empiric broad spectrum antibiotics including vancomycin, cefepime, and metronidazole were initiated along with intravenous methylprednisolone 40 mg every 12 hours.
Initially while on BIPAP, he remained tachypneic and tachycardic. His respiratory rate improved over the following 6 hours. He was gradually weaned off BiPAP to nasal cannula oxygen over the course of 36 hours while receiving ongoing corticosteroid and antibiotic therapy.
Repeat CXR on hospital day \#6 showed markedly improved bilateral airspace opacities ([Figure 1(b)](#fig1){ref-type="fig"}). After 6 days, he was discharged in stable condition without requiring supplemental oxygen.
3. Discussion {#sec3}
=============
ARDS is associated with a high mortality that has declined from over 50% to 30% over the last four decades \[[@B6], [@B7]\]. This is primarily due to implementation of lung-protective ventilation protocols and intensified research after formation of the National Heart, Lung, and Blood Institute (NHLBI) ARDS network \[[@B8], [@B9]\]. This case highlights a mild manifestation of ARDS as a result of bupropion inhalation, an exceedingly rare etiology.
The Food and Drug Administration (FDA) classifies bupropion as a psychiatric medication with low abuse potential \[[@B10]\]. However, several case reports and studies have indicated increasing recreational use of bupropion mostly intravenously or intranasally \[[@B11]--[@B13]\]. Lewis et al. conducted a review on bupropion inhalation in a total of 67 patients. Seizures were noted as a common adverse effect occurring in 30% of patients. Acute lung toxicity was not reported as a complication \[[@B14]\]. We were not able to find a second reported case of bupropion-inhalation-induced ARDS.
Endotracheal intubation and mechanical ventilation are mostly required to ensure adequate oxygenation, but this was avoided in this patient as we maintained adequate oxygenation with BiPAP alone. NIPPV is advantageous in such patients as it does not expose them to the potential complications of invasive ventilation and may shorten their hospital length of stay \[[@B3]\]. There is, however, an ongoing debate concerning the most effective mode of NIPPV \[[@B15], [@B16]\]. Recent studies show that noninvasive ventilation can be used in mild cases of ARDS with acute nonhypercapnic hypoxemic respiratory failure \[[@B17]\]. In these cases, BiPAP via facemask is the most commonly used strategy \[[@B18]\]. Another approach with high-flow nasal cannula was shown to have a similar degree of treatment failure and incidence of subsequent intubation as BiPAP. High-flow nasal cannula, however, was associated with decreased 90-day mortality as compared to BiPAP \[[@B19]\]. A randomized, single-center trial by Pohlman et al. showed that delivering noninvasive ventilation via helmet instead of by facemask was associated with a significant reduction in intubations. There was also a decrease in intensive care unit length of stay and mortality at 90 days \[[@B20]\]. However, a subset analysis of the observational "LUNG-SAFE" study for patients with severe ARDS (defined as a PaO~2~/FiO~2~ ratio below 150 mmHg) showed increased mortality with noninvasive ventilation \[[@B21]\].
Per our review, data on the use of noninvasive ventilation in the management of ARDS remains inconclusive and conflicting. NIPPV may decrease the incidence of ventilator-associated complications; however, given that its efficacy is not well established, patients with ARDS should still be treated in a critical care setting for close monitoring with invasive ventilation available on standby.
4. Conclusion {#sec4}
=============
Advances in the treatment of the underlying etiologies and improvement in mechanical ventilation strategies have led to a decrease in the overall mortality associated with ARDS. Despite these developments, it remains a diagnosis with an exceedingly high mortality. New emerging data indicates that NIPPV can be a beneficial and equivalent approach for a subset of patients with ARDS, although the optimal type of noninvasive ventilation and patient group that would benefit most is yet to be determined.
Conflicts of Interest
=====================
All authors declare no conflict of interest.
{#fig1}
{#fig2}
[^1]: Academic Editor: Mabrouk Bahloul
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Historically pulmonary emphysema was described in 1834 by Laennec on the basis of observations made on the cut surface of postmortem human lungs being the lesion attributed to the atrophy of lung tissue from pulmonary hyperinflation.^([@B1])^ Hence, emphysema was redefined as a "abnormal and permanent dilation of distal air spaces of terminal bronchiole".^([@B2])^ In addition, evidences of destruction of alveolar wall and fibrosis must not be ignored in this disease pathogenesis.^([@B3])^
These anatomopathological changes result in loss of respiratory surface and blood irrigation, decrease of elastic recognition and pulmonary hyperexpansion, and it could also affect part of acinus or its structure.^([@B4])^
Pulmonary emphysema is caused by enzymatic imbalance between proteases and anti-proteases that results in destruction of the alveolar wall due to proteolytic enzymes action, which affects the extracellular matrix (ECM)^([@B5])^ and its component integrity especially the elastic fibres.^([@B6])^
Experimental model of pulmonary emphysema is based on nebulization or instillation of proteolytic enzyme, such as panain *(Carica papaya)*,^([@B7])^ porcine pancreatic elastase,^([@B4])^ and human neutrophil elastase.^([@B8])^ This proteolytic process, associated with uniform destruction of ECM of pulmonary acinus, ends up in morphohistological and physiological changes in lungs that resemble those changes find in emphysema in humans.^([@B9],[@B10])^
Dilatation of distal air spaces of terminal bronchiole ([Figure 1](#f01){ref-type="fig"}) and reduction of area occupied by elastic fibres ([Figure 2](#f02){ref-type="fig"}) evidenced histologically the pulmonary emphysema in experimental models that use porcine pancreatic elastase.
Figure 1Photomicrographs of lung parenchyma (hematoxylin-eosin) x 100 increased. (A) Naïve lung and (B) emphysematous lung showing hyperdistension of alveolar ducts associated with the rupture of alveolar septa
Figure 2Photomicrographs of lung parenchyma (Verhoeff), x 400 increased. Lung naïve showing integrity of elastic component of alveolar wall, opposing to areas revealed throughout septa associated with thickening of elastic fibres in alveolar wall and decreasing of proportion of elastic fibres in emphysematous lung (B)
Aprendendo Por Imagens
Características histopatológicas do enfisema pulmonar em modelo experimental
Petta
Antonio Di
1
Universidade de São Paulo, São Paulo, SP, Brasil.
Autor correspondente: Antonio Di Petta − Rua Rodolfo Marcos Teófilo, 49 − Freguesia do Ó -- CEP: 02862-100 − São Paulo, SP, Brasil − Tel.: (11) 3851-0028 − E-mail:
antoniodipetta\@usp.br
Historicamente, Laennec (1834) descreveu o enfisema pulmonar a partir de observações em cortes necroscópicos superficiais de pulmões humanos, atribuindo a lesão à atrofia do tecido pulmonar resultante da hiperinsuflação pulmonar.^([@B1])^ O enfisema foi, então, redefinido como uma "dilatação anormal e permanente dos espaços aéreos distais do bronquíolo terminal".^([@B2])^ Além do mais, a evidência da destruição da parede alveolar e de fibrose não deve ser ignorada na patogenia da doença.^([@B3])^
Essas alterações anatomopatológicas resultam na perda da superfície respiratória e de irrigação sanguínea, na diminuição do recolhimento elástico e na hiperexpansão pulmonar, podendo atingir parte do ácino ou toda sua estrutura.^([@B4])^
O enfisema pulmonar é obviamente uma doença causada pelo desequilíbrio enzimático existente entre proteases e antiproteases, resultando na destruição da parede alveolar ocasionada pela ação de enzimas proteolíticas, que degradam a matriz extracelular (MEC)^([@B5])^ e afetam a integridade de seus componentes, particularmente as fibras elástica.^([@B6])^
Modelos experimentais de enfisema pulmonar baseiam-se na nebulização ou instilação de enzimas proteolíticas, como papaína (*Carica papaya*),^([@B7])^ elastase pancreática de porco,^([@B4])^ e elastase neutrofílica humana.^([@B8])^ Esse processo proteolítico, associado à destruição uniforme da MEC do ácino pulmonar, resulta em alterações morfo-histológicas e fisiológicas dos pulmões equivalentes às alterações encontradas no enfisema em seres humanos.^([@B9],[@B10])^
A dilatação dos espaços aéreos distais do bronquíolo terminal ([Figura 1](#f03){ref-type="fig"}) e a redução da área ocupada pelas fibras elásticas ([Figura 2](#f04){ref-type="fig"}) evidenciam histologicamente o enfisema pulmonar em modelos experimentais instilados por elastase pancreática de porco.
Figura 1Fotomicrografias do parênquima pulmonar (hematoxilina-eosina), aumento x100. (A) Pulmão *naive* e (B) pulmão enfisematoso, demonstrando hiperdistensão dos ductos alveolares com ruptura dos septos alveolares
Figura 2Fotomicrografias do parênquima pulmonar (*Verhoeff*), aumento de 400x. (A) Pulmão *naive* demonstrando integridade dos componentes elásticos da parede alveolar, contrastando com áreas desnudas ao longo dos septos associadas ao adensamento de fibras elásticas na parede alveolar e diminuição da concentração de fibras elásticas no pulmão enfisematoso (B)
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1. Introduction {#sec1-jintelligence-05-00001}
===============
Generally, Black people, White people, and East Asian people have different average IQs, but the specific reasons for this remain controversial. Of various bodies of evidence, one has been nominated as "one of the most powerful" for explaining these racial IQ differences: the IQs of adoptees of different races raised by White adoptive parents \[[@B1-jintelligence-05-00001]\] (p. 25). Several psychologists have reviewed studies of transracial adoptees' IQs and used them as evidence for two claims. The first is that East Asian adoptees raised by Whites have higher average IQs than the general White population; that White adoptees raised by Whites in turn have higher average IQs than Black adoptees raised by Whites; and that multiracial adoptees have an expected IQ that is a weighted average of their ancestral groups' mean IQs (so that adoptees born to a White genetic parent and a Black genetic parent, for example, have an average IQ midway between those of Whites and Blacks). This would suggest that racial IQ differences persist even among children raised in nurturing adoptive homes, intimating that differences in home environments cannot explain racial IQ differences. This leads into the second claim: that most of the racial IQ differences between East Asians, Whites, and Blacks are genetic \[[@B1-jintelligence-05-00001],[@B2-jintelligence-05-00001],[@B3-jintelligence-05-00001]\]. In this paper, I call the conjunction of these two claims the *hereditarian* hypothesis or model, after Rushton and Jensen \[[@B3-jintelligence-05-00001]\] (pp. 239--240, 259).
This paper reanalyzes the adoption studies cited to support these claims, and puts forth an alternative explanation: most of the average racial IQ differences in those studies are spurious, arising from methodological issues and disregard of contrary results. When all of the cited data are considered with these issues in mind, they are not compelling evidence for large and consistent IQ differences between East Asian, White, and Black adoptees raised by White parents. This paper then introduces further adoption data which have yet to be considered in the race and IQ debate. The totality of the data turn out to be at least as consonant with a *nil* hypothesis or model: the IQs of adoptees raised by Whites in comparable environments are hardly affected by the adoptees' race.
2. Methodological Issues {#sec2-jintelligence-05-00001}
========================
The innovation of this paper is that it accounts for four methodological issues which imperil analyses of transracial adoption studies of IQ. Two of the issues are examples of the generic scientific problem of confounded comparisons of groups, and the third is a type of selection bias.
Firstly, if a sample of adoptees of one race or ethnicity scores above another ethnic group's general-population average, one cannot automatically attribute the above-average score to the adoptees' ethnicity. The adoptees are *adoptees*, and adoptees are typically raised in unrepresentative environments which tend to be more nurturing and high in socioeconomic status. Unusually wholesome environments could then explain the adoptees' above-average IQ, rather than the adoptees' race; race and environment would be confounded. (Indeed, the adoptees themselves are likely to be unrepresentative of their own race, and a sceptic could conceivably attribute the adoptees' above-average IQ to their very unrepresentativeness, regardless of environment.). The two most obvious ways to control away this confounding are to compare adoptees against only other adoptees, or to adjust the adoptees' observed IQs downwards to allow for the adoptees' better environments.
The second methodological issue is the Flynn effect. James R. Flynn and his colleagues have documented steady rises in average IQ in several countries \[[@B4-jintelligence-05-00001],[@B5-jintelligence-05-00001],[@B6-jintelligence-05-00001],[@B7-jintelligence-05-00001],[@B8-jintelligence-05-00001]\]. These rises makes IQ test norms progressively more outdated over time, so adoptees who take an IQ test would have exaggerated IQ scores relative to people who took the same test earlier. In particular, all IQ tests must be standardized against a reference population, offen the general population of a given country, and when a group of adoptees takes the IQ test at a later date, the time lag exaggerates the adoptees' performance relative to the reference population. Had the reference population taken the test at the same time as the adoptees, the reference population would have set a higher benchmark for the adoptees. The same mechanism means it is usually illegitimate to directly compare adoptees' IQs across studies, because adoptees in different studies are usually tested in different years, and with different tests. Like environment, the year in which a test is taken and the choice of test can be confounded with race. To avoid such confounds, analysts can either subtract out the Flynn effect from each set of results, or make comparisons only of groups which took the same IQ test at approximately the same time.
The third issue, attrition, is less common, affecting only longitudinal studies. Even if a longitudinal study compares adoptees against only other adoptees (eliminating the first confound) who took the same IQ test at similar times (eliminating the second confound), attrition can take place between waves. When researchers lose track of some subjects between waves of a longitudinal study, the pattern of subjects lost to follow-up can vary between subgroups of subjects, degrading the statistical comparability of those subgroups. Taking the specific case of adoptees' IQs, if a longitudinal study included e.g., White and e.g., Black adoptees, and the White adoptees lost to follow-up were disproportionately lower scorers, whereas the Black adoptees lost to follow-up were not, the White--Black IQ difference among the remaining adoptees would be inflated. Race can correlate with selection for retention in the study. One can adjust for this type of selection bias by making a counterfactual estimate of how the subgroups would have scored had no one been lost to follow-up.
The fourth issue is that published reviews of transracial-adoption IQ studies have not considered all of the studies which were available and germane, and this selective treatment of the data may introduce bias. Consequently, as Rushton and Jensen put it, "\[t\]o be compelling, \[\...\] researchers must take the totality of available evidence into account" \[[@B9-jintelligence-05-00001]\] (p. 921). The remainder of this paper should give an idea of whether past writers on transracial adoption and IQ have met this standard.
3. A Hereditarian Interpretation {#sec3-jintelligence-05-00001}
================================
Before contesting the hereditarian model of transracial adoption studies, this paper must first describe it. To that end, this section takes stock of the studies already discussed in the literature, and why they might seem to support the hereditarian hypothesis. [Table 1](#jintelligence-05-00001-t001){ref-type="table"} gives a list in publication-year order, where samples of adoptees with two Black genetic parents each are labelled "Black--Black" and samples of adoptees with one White and one Black genetic parent each are labelled "Black--White". The 130 East Asian adoptees in the Winick, Meyer, and Harris \[[@B10-jintelligence-05-00001]\] and Frydman and Lynn \[[@B11-jintelligence-05-00001]\] studies were all Korean, while the 25 East Asian adoptees in the Clark and Hanisee \[[@B12-jintelligence-05-00001]\] sample comprised "12 from Vietnam, 8 from Korea, 3 from Cambodia, and 2 from Thailand" (p. 596). The racial designations may not be rigorous but the current paper uses them for argument's sake.
A hereditarian interpretation of the data in [Table 1](#jintelligence-05-00001-t001){ref-type="table"} begins by "plac\[ing\] greatest weight on the Minnesota Trans-Racial Adoption Study because it is the largest and best-known of these studies and is the only one that included a longitudinal follow-up" \[[@B1-jintelligence-05-00001]\] (p. 25). Scarr and Weinberg \[[@B13-jintelligence-05-00001]\] reported results from the first wave of that study, and Weinberg, Scarr, and Waldman \[[@B14-jintelligence-05-00001]\] the follow-up results. In both waves the wholly White adoptees had a higher mean IQ than the Black--White adoptees, and the Black--White adoptees had a higher mean IQ than the Black--Black adoptees, in line with the hereditarian model. Moreover, those IQ gaps widened between the study's two waves, consistent with genes exerting greater power as the adoptees aged.
The other studies of Black adoptees \[[@B15-jintelligence-05-00001],[@B16-jintelligence-05-00001]\] do not fit the hereditarian model nearly as well, as the race-IQ correlations they found were all either small or associated higher IQ with greater Black ancestry. However, one can downplay these other studies on the ground that they lacked longitudinal follow-up testing, and by arguing that genetic effects could've been masked by their subjects' lower age (since "as people age, their genes exert ever more influence" \[[@B3-jintelligence-05-00001]\] (p. 259)).
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All relevant data can be found at the following URL: <https://doi.org/10.6084/m9.figshare.4640092.v1>.
Introduction {#sec001}
============
Length-weight relationships (LWR) are used for estimating the weight corresponding to a given length \[[@pone.0171811.ref001]\]. As most observations in fisheries surveys are typically obtained as the number of specimens and length of each sampled specimen, LWR are widely used to transform the survey data into estimates of the biomass which is important for modelling aquatic ecosystems. Therefore, estimation of the LWR is common practice and essential in fisheries science \[[@pone.0171811.ref002]\].
There are two parameters in the LWR model (*W* = *a*×*L*^*b*^), the coefficient *a* and the exponent *b*. Parameter *a* is the condition factor, describing body shape which can be classified as four groups: eel-like, elongated, fusiform and short and deep \[[@pone.0171811.ref002]\]. Parameter *b* is the allometric growth parameter, which indicates isometric growth in body proportions if *b*≥3 where fish have more girth as it grows longer; the species tends to be more streamlined if the exponent *b*\<3 \[[@pone.0171811.ref002]\].
Numerous studies found that factors, such as geographic, seasonal, inter-annual, and environmental conditions, can affect the estimates of *a* and *b* in the LWR model \[[@pone.0171811.ref002]--[@pone.0171811.ref005]\]. Instead of constructing several models reflecting various situations (different regions and years), it is more reasonable to make use of generalized linear mixed model to cover all the spatial and temporal effects in a single model. Linear mixed-effects modelling, a mature model in the statistics community, has been used in multiple fields. Cnaan et al. (1997) provided two detailed case studies to sufficiently introduce the use of the general linear mixed effects model for the regression analysis of correlated data \[[@pone.0171811.ref006]\]. Xu et al. (2015, 2014) developed nonlinear mixed-effects model to study the individual-tree diameter growth and linear mixed effects model for individual-tree crown width of China-Fir trees in Southeast China \[[@pone.0171811.ref007], [@pone.0171811.ref008]\]. Baayen et al. (2008) provided an introduction of mixed-effects models and illustrated its advantages, which would allow the researcher to simultaneously consider all factors that potentially contribute to the understanding of the structure of the data \[[@pone.0171811.ref009]\].
The linear mixed-effects model provides a powerful and versatile approach to analyze a wide variety of data structures, in which the linear predictor contains random effects in addition to the usual fixed effects \[[@pone.0171811.ref007]\]. The random effects vary with respect to one or more grouping variables, e.g. regions and years, adding its contribution of variation to the residual error, which can account for the additional resources of random variation \[[@pone.0171811.ref007], [@pone.0171811.ref008], [@pone.0171811.ref010]\].
Yellow Croaker (*Larimichthys polyactis*) is a warm-temperate demersal fish species widely distributed throughout the northwest Pacific Ocean. As a commercially important species, Yellow Croaker experienced heavy fishing pressure in China. From catch data derived from *China Fishery Statistical Yearbook*, we found the variations of Yellow Croaker stock ([S1 Fig](#pone.0171811.s001){ref-type="supplementary-material"}). Yellow Croaker were abundant in 1950s and the beginning of 1960s; but the stock collapsed in the 1970s \[[@pone.0171811.ref011]\]. After a series of restoration efforts (i.e. seasonal fishery closure and protection of the spawning ground), the Yellow Croaker stock has been recovering since 1990 and the catch has increased continually in the subsequent two decades \[[@pone.0171811.ref012]--[@pone.0171811.ref014]\]. However, the characteristics of the stock have changed; as individuals are smaller, younger and reaching maturity earlier \[[@pone.0171811.ref013],[@pone.0171811.ref015],[@pone.0171811.ref016]\].
Quite many scientists have done much work about the growth of Yellow Croaker around the world. Li *et al*. (2013) applied simple linear regression to analyze LWRs of Yellow Croaker in Bohai Sea-Northern Yellow Sea from 1960 to 2004 and in the Southern Yellow Sea from 1960 to 2010 for male and female respectively \[[@pone.0171811.ref003]\]. Zhang *et al*. (2010) investigated the biological characteristics of Yellow Croaker in the central and southern Yellow Sea, including the LWRs in 1960, 1985, 1998 and 2008 \[[@pone.0171811.ref017]\]. Lin *et al*. (2004) studied the population biology of Yellow croaker in the East China Sea, which evaluated the LWR in 1963, 1983 and 2001 \[[@pone.0171811.ref018]\]. All these studies of LWR used simple linear regression to develop region specific or year specific models, rather than linear mixed-effects models, to detect the spatial and temporal variations of Yellow Croaker.
In this study, our objective was to analyze the biological characteristics of Yellow Croaker in recent years. Specifically, we estimated the LWR of Yellow Croaker used linear mixed-effects model, condition factors relative to reference years, and explore the possible relationship between parameters in LWR model and environmental factors, based on the observations along north coast of China among 2008 and 2011 to 2015.
Materials and methods {#sec002}
=====================
Data collection {#sec003}
---------------
Specimens were collected along the Shandong and Jiangsu province through 2008, and 2011--2015. These regions along northern Chinese coast include Yellow River Estuary (YE), Coastal Waters of Northern Shandong (NS), Jiaozhou Bay (JB), Coastal Waters of Qingdao (QD), Haizhou Bay (HB), and South Yellow Sea (SY) ([Table 1](#pone.0171811.t001){ref-type="table"}, [S1 Table](#pone.0171811.s006){ref-type="supplementary-material"}, [Fig 1](#pone.0171811.g001){ref-type="fig"}), which are important spawning and feeding grounds of Yellow Croaker \[[@pone.0171811.ref019]\]. The maps of surveying area were plotted using package maps and mapdata of the R (version: R i386 3.3.1) \[[@pone.0171811.ref020], [@pone.0171811.ref021]\].
10.1371/journal.pone.0171811.t001
###### Sample size and location of Yellow Croaker among regions and years.
{#pone.0171811.t001g}
Regions 2008 2011 2012 2013 2014 2015 Total
------------------------------------- ---- ------ ------ ------ ------ ------ ------ -------
Yellow River Estuary YE 40 40
Coastal Waters of Northern Shandong NS 35 35
Jiaozhou Bay JB 426 26 63 515
Coastal Waters of Qingdao QD 419 533 952
Haizhou Bay HB 921 81 100 579 1681
South Yellow Sea SY 148 11 308
**Total** 426 947 482 121 816 590 3382
The second column shows the abbreviations of regions.
{ref-type="table"}.](pone.0171811.g001){#pone.0171811.g001}
In Coastal waters of Northern Shandong, specimens were randomly collected from fishermen immediately after landing. For all other five regions, stratified random bottom trawling surveys were implemented to collect samples. In total, 3,275 individuals were collected, with sample size variations among years and regions (see [Table 1](#pone.0171811.t001){ref-type="table"} and [S1 Table](#pone.0171811.s006){ref-type="supplementary-material"}). For each individual, standard length was measured to the nearest mm and total weight was measured to the nearest gram.
The surveys, which held in the *Seasonal Fishery Closure* (June, July and August) were approved by the Department of Marine and Fishery of Shandong and Jiangsu Province. No specific permissions were required for all the other surveys, because these surveys are traditional surveys, which did not cover any marine protected area or private area and this study did not involve endangered or protected species.
LWR modelling {#sec004}
-------------
We fitted an exponential function to the weight at length data that took the form \[[@pone.0171811.ref001]\]: $$W = aL^{b}$$ where *W* is the wet weight of an individual fish (g), *L* is the standard length (cm), *a* is the condition factor, and *b* is the allometric growth parameter. Because the variance of W increases when
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INTRODUCTION {#SEC1}
============
Next-generation sequencing (NGS) and microarray technologies uncovered thousands of long non-coding RNAs (lncRNAs) encoded in the human genome ([@B1],[@B2]). The majority of those lncRNAs are transcribed and processed in a similar manner to mRNAs, however, lack protein-coding potential ([@B3],[@B4]). Although it is still unclear how many of those lncRNAs have a significant biological function, some of them have been found to be crucial players in the regulation of cellular processes such as proliferation, differentiation or development, as well as in a progression of a variety of human diseases including cancer ([@B5]). It has been shown that lncRNAs are key determinants of epigenetic regulation, modulation of chromatin structure, scaffolding or decoy function of mRNAs and post-transcriptional mRNA regulation ([@B11]).Gene regulation by lncRNAs can be a result of cis-action on nearby genes, or in trans by modulating mRNA stability, mRNA translation, or microRNA and RNA-binding-protein function ([@B16]).
Cellular senescence was initially defined by Hayflick in 1965 as the limited lifespan of primary human fibroblasts in culture ([@B24]). It is a state of irreversible growth arrest which can be induced by different stimuli such as telomere shortening, DNA damage, oxidative stress or oncogene activation ([@B25]). Serrano *et al.*, were the first to observe that primary human and mouse fibroblasts enter senescence following the induction of oncogenic RAS, a process termed oncogene-induced senescence (OIS) ([@B26]). Cellular senescence has been studied most extensively as a strong tumor-suppressive mechanism against the emergence of oncogenes ([@B27]). Moreover, there is evidence indicating for a role of senescence in age-related conditions and diseases, including cancer, cardiovascular diseases, neurodegeneration, diabetes, sarcopenia and declining immune function in the elderly ([@B28]). In contrast, senescent cells can also contribute to tumorigenesis by secreting interleukins (e.g. IL-6, IL-8 and IL-1a), metalloproteases (e.g. MMP-1 and MMP-3) and other cytokines (e.g. granulocyte-macrophage colony-stimulating factor (GM-CSF)), as part of the senescence-associated secretory phenotype ([@B25],[@B30],[@B33]). Therefore, senescence may either suppress or promote tumor progression depending on the context where it occurs ([@B38],[@B39]). Given the impact of senescence on human physiology and pathology, it is of interest to understand the molecular mechanisms underlying senescence in order to utilize it for diagnosis and therapy.
A number of factors have been implicated in regulating senescence, including transcription factors, RNA binding proteins and microRNAs, such as p53, Ets ([@B40]), HuR ([@B41]), AUF1 ([@B42]) and TTP ([@B43]), and miR-377 ([@B44]), miR-22 ([@B45]). In contrast, despite increasing interest in the expression and function of lncRNAs, their possible implication in senescence remains largely unexplored. Recent works indicated a role of MIR31HG and SALNR in senescence ([@B46],[@B47]), but a focused functional genetic screen was not described before. We therefore sought to identify senescence-associated lncRNAs using our established cellular system that induces senescence in primary human BJ fibroblasts ([@B48]). Using transcriptomic profiling we identified a number of differentially expressed lncRNAs following oncogene induction. Next, using functional screen, we discovered that one of the lncRNAs whose expression was induced upon oncogenic stress---lncRNA-OIS1---is required for OIS. We demonstrate that lncRNA-OIS1 is required for senescence by controlling a nearby DPP4 gene with a tumor suppressive activity. Collectively, our results provide a new lncRNA-mediated regulatory pathway for controlling DPP4 during OIS. Our findings support the role of lncRNAs as transcriptional regulators in critical processes such as cellular senescence and a potential role in cancer.
MATERIALS AND METHODS {#SEC2}
=====================
Cell culture, transfection, retroviral and lentiviral transduction {#SEC2-1}
------------------------------------------------------------------
BJ/ET/Ras^V12^, TIG3/ET/RAS^V12^, Ecopack 2 and HEK293-T cells were cultured in Dulbecco's modified Eagle's medium (Gibco), supplemented with 10% fetal calf serum (FCS) (Hyclone) and 1% penicillin/streptomycin (Gibco). Senescence was induced by treatment with 100 nM 4-OHT (Sigma) for 14 days. Retroviruses were made by calcium phosphate transfection of Ecopack 2 cells and harvest at 40 and 64 h later. Lentiviruses were made by polyethylenimine (PEI) transfection of HEK293T. Medium was refreshed after 16 h and collect the lentivirus by filtering through a 0.45 μm membrane (Milipore Steriflip HV/PVDF) 40 h post-transfection and stored at −80°C. Cells were selected with the proper selection medium 48 h after transduction for at least 4 days until no surviving cells remained in the no-transduction control plate.
RNA-seq and analysis {#SEC2-2}
--------------------
RNA-seq samples were processed with TruSeq RNA library prep kit v2 (Illumina) and sequenced in a HiSeq 2500 (Illumina). Sequenced reads were aligned to the human genome (hg19) using TopHat2 ([@B49]) and gene expression levels were counted using HTseq ([@B50]) and normalized using quantile normalization. To avoid inflation of lowly expressed genes among the genes called as differentially expressed, we applied a dynamic cut-off which takes into account that technical variation varies with expression level. Specifically, in the comparison between two conditions, we divided the genes into 20 bins according to their average expression level, and calculated the standard deviation (SD) of fold-change within each bin. Genes whose expression was changed by at least 1.75-fold and this fold-change was above the bin's 1.75 SD (dashed curve in Figures [1B](#F1){ref-type="fig"} and [3B](#F3){ref-type="fig"}) were called as differentially expressed. To further avoid false positive calls among lowly expressed genes we set a floor level of five counts (i.e. any level below five was set to five). Functional enrichment analysis was done using DAVID ([@B51]). Global characterization of pathways that were deregulated upon knockdown of lncRNA-OIS1 was done using gene set enrichment analysis (GSEA) ([@B52]).
{#F1}
*In situ* hybridization {#SEC2-3}
-----------------------
*In situ* hybridization (ISH) was performed using double-FAM labeled locked nucleic acid (LNA) probes (Exiqon) as described previously ([@B53]). Briefly, cells were fixed, permeabilized and pre-hybridized in hybridization buffer and then hybridized at 55°C for 1 h with LNA probes for lncRNA-OIS1: 5-TTGAAAACCCATCACTCCT-3, or with a scramble probe 5-TGTAACACGTCTATACGCCCA-3 as negative control, all at 25 nM. Cells were subsequently incubated with 3% hydrogen peroxide to block potential endogenous peroxidase, and then probes were detected with peroxidase-conjugated anti-fluorescein-Ab (Roche applied Sciences) diluted 1:400 followed by addition of Cy3-labeled TSA substrate for 10 min (Perkin Elmer). All cells were mounted with ProLong^®^GoldAntifade Mountant containing DAPI nuclear stain (ThermoFisher Scientific). Images were acquired using a Zeiss Axio Imager Z1 epi-fluorescence microscope equipped with an AxioCamMRm CCD camera and a Plan-APOCHROMAT 63×/1.4 objective (Zeiss). Within the same experiment, images were acquired at the same exposure conditions.
BrdU proliferation assay {#SEC2-4}
------------------------
BJ and TIG3 Cells were pulsed for 3 h with 30 μM bromodeoxyuridine (BrdU, Sigma), washed two times with phosphate-buffered saline (PBS) and then fixed with 4% formaldehyde, wash two times with PBS and treated with 5M HCl/0.5% Triton to denature DNA and neutralized with 0.1M Na~2~B~4~O~7~, incubated with anti-BrdU (Dako) for 2 h in RT after 30 min blocking with 3% bovine serum albumin (BSA) in 0.5% Tween PBS, washed in blocking buffer (PBS, Tween 0.5%, 3% BSA) three times, and finally incubated with FITC-conjugated anti-mouse Alexa FLOUR 488 secondary antibody (Dako) for 1 h, washed three times, stained with
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Introduction {#s1}
============
Osteoarthritis is a multifactorial disease characterized by destruction of the articular cartilage due to genetic, mechanical and environmental components [@pone.0003740-Spector1], affecting more than 20 million people in the US [@pone.0003740-Lawrence1]. Despite its high prevalence there are few studies concerning the molecular pathobiology and the involvement of genetic factors in the pathogenesis of osteoarthritis [@pone.0003740-Miyamoto1], [@pone.0003740-Kizawa1]. Several clinical studies have implicated the causative role of obesity in osteoarthritis development [@pone.0003740-Lohmander1], [@pone.0003740-Lementowski1], however there are few molecular studies correlating metabolism with osteoarthritis [@pone.0003740-Aspden1], [@pone.0003740-Ostalowska1]. Achieving a deeper understanding of osteoarthritis molecular mechanisms requires global strategies aimed at modelling the functional interrelationships between genes as complex interdependent networks.
Lately, it has become evident that genetic alterations in non-coding genes can also contribute to the pathogenesis of human disease [@pone.0003740-McManus1]. A new class of small non-coding RNAs, named microRNAs, regulate gene expression by inhibition of translation or mRNA cleavage [@pone.0003740-EsquelaKerscher1]. MicroRNAs have been implicated in important cellular processes such as lipid metabolism [@pone.0003740-Esau1], apoptosis [@pone.0003740-He1], differentiation [@pone.0003740-Chen1] and organ development [@pone.0003740-Callis1]. Furthermore microRNAs expression signatures have been associated with well-defined clinicopathological features and disease outcome [@pone.0003740-Calin1]. It is known that microRNAs exert their biological functions through suppression of their target genes. Several bioinformatic algorithms have been constructed in order to predict microRNA gene targets. Most of these algorithms search for sequence complementarity between the microRNA and the 3′ UTR of the gene target. These algorithms predict hundreds of potential gene targets, which can not all be experimentally validated. Previous studies have tried to identify microRNA gene targets using cDNA microarray data [@pone.0003740-Huang1]. However, it has been shown that a microRNA (miR-10b) regulates gene expression only at the protein level, while mRNA levels were not affected [@pone.0003740-Ma1]. In addition, very recently Selbach et al, showed that a microRNA can repress the production of hundred of proteins [@pone.0003740-Selbach1]. Therefore, it becomes evident that proteomic data are needed in order to accurately detect microRNA gene targets. Up to now there are few studies trying to characterize the cartilage proteome. More specifically, recently Vincourt et al, performed a detailed two dimensional electrophoresis-based proteomic analysis of articular cartilage [@pone.0003740-Vincourt1]. In addition Wu et al. performed a comparative proteomic analysis of cartilage from healthy donors and osteoarthritis patients, however the number of samples used was very small [@pone.0003740-Wu1].
For all above reasons, we undertook to associate specific microRNAs and proteins with the development of osteoarthritis and clinicopathological parameters, in order to identify new signalling pathways involved in its pathogenesis. Here, we report a novel approach of studying multi-aetiological diseases and identifying new genes involved in the pathogenesis of a complex disease. Integration of microRNA microarray and proteomic analysis data together with computational approaches, such as microRNA gene target prediction algorithms and gene network construction, revealed the role of microRNAs in cartilage destruction and linked inflammatory and metabolic gene networks with cartilage homeostasis.
Materials and Methods {#s2}
=====================
Cartilage tissue samples {#s2a}
------------------------
Articular cartilage samples were obtained from femoral heads, femoral condyles and tibial plateaus of patients with primary osteoarthritis undergoing hip or knee replacement surgery at the Orthopaedics Department of University Hospital of Larissa. A total of 33 patients were included in this study (twenty eight females and five males; mean age 68.91±6.97 years, range 57--83; mean Body Mass Index (BMI) 30.51±5.23, range 22.67--43.96) who had undergone total knee replacement surgery. Each sample was categorized according to its gross morphology, as severely damaged and was taken from the main defective area of maximal load. Macroscopic findings were validated by histological studies performed on 5 mm serial sections of cartilage samples and graded using the Mankin score. Specimens with osteoarthritis had Mankin score 10--14. Normal cartilage was obtained from small free cartilaginous fragments from ten individuals (six females and four males; mean age 61.70±18.17, range 27--78; mean BMI 23.80±4.34, range 19.03--35.06) with 0 Mankin score, undergoing fracture repair surgery, with no history of joint disease. Both patients and healthy individual\'s cartilage samples were obtained upon individuals\' verbal informed consent. The method of obtaining verbal approval by all individuals was approved by Institutional Review Board of the University Hospital of Larissa. Also the protocol was approved by the local ethics committee of University Hospital of Larissa.
Detection of microRNA expression {#s2b}
--------------------------------
Expression levels of 365 microRNAs were evaluated with TaqMan microRNA microarray assays as previously described [@pone.0003740-Thum1]. Validation of these results was performed using the mirVana qRT--PCR miRNA Detection Kit and qRT--PCR Primer Sets, according to the manufacturer\'s instructions (Ambion Inc, TX, USA). The U6 small nuclear RNA was used as an internal control.
MicroRNA Northern Blot Analysis {#s2c}
-------------------------------
For microRNA Northern Blot Analysis, 10ug of RNA were separated on 12% denaturating polyacrylamide gels and transferred to GeneScreen Plus membrane (PerkinElmer, Waltham). MiRCURY LNA Probes for miR-483 and miR-22 (Exiqon, Denmark) were end-labeled with T4 polynucleotide kinase. Prehybridization of the filters was carried out in 50% formamide, 0.5% SDS, 5· SSPE, 5·Denhardt\'s solution and 20 mg/ml sheared, denatured salmon sperm DNA. Hybridizations were performed in the same solution at 42°C. The labeled probes were heated for 1 min at 95°C before addition to the filters in the prehybridization solution. After hybridization, the membranes were washed in 0.1 SSC, 0.1% SDS at 42°C twice for 10 min.
Reverse-Phase protein microarray analysis {#s2d}
-----------------------------------------
Chondrocyte cell lysates were boiled for 5 min and were loaded into 384-well plates in serial dilutions (neat, 1∶2, 1∶4, 1∶8, and 1∶16) with negative control wells containing only lysis buffer. These samples were printed in duplicate onto nitrocellulose-coated glass slides (Schleicher & Schuell Bioscience, Keene, NH) using a ring-and-pin robotic arrayer (GMS 417, Affymetrix, Santa Clara, CA). The arrays were stained as previously described [@pone.0003740-Sheehan1] on an autostainer (DAKO, Carpinteria, CA) using a biotinyl-linked catalyzed signal amplification system (DAKO). Specificity of each antibody was tested by western blot analysis.
Statistical analysis {#s2e}
--------------------
All calculations were performed on a Microsoft computer, using the SPSS software (version 12.0). Correlation of between microRNA and protein expression levels with BMI was identified by correlation coefficients, calculated by Pearson rank correlation (*r*) and Spearman rank correlation. Statistical methods regarding the proteomic analysis are described analytically in the suppl. [Methods](#s2){ref-type="sec"} section. Construction and statistical significance of gene networks was performed by Ingenuity pathway analysis. Statistical significant networks were considered those with p value higher than 10^−5^. In addition clustering of the protein data in functional groups was performed using DAVID NIH Bioinformatics Database with a p value cut-off of 10^−5^. Quantification of western blots was performed by standard densitometric analysis. All transfection experiments were performed in triplicate and the results were compared by student\'s t-test analysis.
Additional methods {#s2f}
------------------
Detailed experimental methods are described in the supplemental methods section ([**Methods S1**](#pone.0003740.s001){ref-type="supplementary-material"}).
Results {#s3}
=======
MicroRNA gene signature of osteoarthritis {#s3a}
-----------------------------------------
To identify microRNAs involved in osteoarthritis, we tested the expression of 365 microRNAs in articular cartilage obtained from patients with osteoarthritis undergoing knee replacement surgery and from normal individuals with no history of joint disease. We identified 16 microRNAs differentially expressed in osteoarthritic compared to normal cartilage ([**Figure 1A**](#pone-0003740-g001){ref-type="fig"}). Specifically we detected nine up-regulated and seven down-regulated microRNAs in osteoarthritic cartilage compared to normal ([**Table S1**](#pone.0003740.s002){ref-type="supplementary-material"}). Real-time PCR and Northern blot analysis ([**Figure 1B, C**](#pone-0003740-g001){ref-type="fig"} **;** [**Table S2**](#pone.0003740.s003){ref-type="supplementary
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Introduction {#sec1-1}
============
Obstructive sleep apnea (OSA) is an increasingly prevalent condition that is characterised by repetitive upper airway obstructions resulting in intermittent hypoxia and sleep fragmentation caused by arousals \[[@ref1]\]. Among adults, 30--70 years of age, approximately 13% of men and 6% of women, have moderate to severe forms of OSA \[[@ref2]\]. OSA is often closely associated with other conditions which are recognised causes of morbidity and mortality such as obesity, metabolic syndrome, atherosclerosis, systemic inflammation, insulin resistance and type 2 diabetes mellitus \[[@ref3], [@ref4]\]. Recently, there has been a great interest in the interaction between OSA and metabolic dysfunction. There is no consistent data suggesting that OSA is a risk factor for dyslipidemia. Indeed, conflicting results have been observed in cross-sectional and interventional studies \[[@ref5]\]. Taking into account components of the metabolic syndrome, some reports found increased levels of triglycerides \[[@ref6]-[@ref9]\] and reduced levels of high-density lipoproteins (HDL) in patients with OSA \[[@ref8]-[@ref10]\], while others studies did not find the correlation between OSA and dyslipidemia \[[@ref11],[@ref12]\]. Of note, the majority of the studies were not specifically designed to evaluate the lipid profile. Therefore, more evidence is still needed. Increased understanding of the independent associations between OSA, metabolic syndrome and insulin resistance is important to develop appropriate therapeutic strategies to reduce the high cardiometabolic risks in patients with OSA.
The aim of this cross-sectional study was to evaluate the prevalence of lipid abnormalities in patients suspected for OSA referred to our sleep laboratory for polysomnography.
Material and Methods {#sec1-2}
====================
The study included 200 patients. It was conducted at University Clinic of Pulmonology and Allergy in Skopje. Inclusion criteria for patients were age from 35 to 60 years and persistence of minimum 2 of 3 clinical symptoms of OSA. The symptoms were snoring, witnessed apnea and daytime sleepiness. Exclusion criteria were previous history and treatment of diabetes and lipid abnormalities.
The study was approved by Ethical Committee of the Faculty of Medicine with No. 03-941/2, and before the study procedures, informed consent was obtained from all patients. Body mass index (BMI) was calculated, and patients were divided into two groups according to the BMI. All patients underwent polysomnography (Respironix, model Alice 5). All results from polysomnography were scored manually according to standard criteria \[[@ref13]\]. Apnea, hypopnea and arousals were also identified according to the standard criteria and summarised in the form of a respiratory disturbance index (RDI). All patients with RDI above 15 were diagnosed with OSA.
In the morning after 12 hours fasting, a blood sample was collected from all patients. Blood levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL), were assessed. Biochemical measurements were conducted using an Architect Abbott C8000 auto analyser.
Statistical analyses were performed using Statistical software (Stat Soft). Data were expressed as *mean (X)* and *standard deviation (SD)*. Comparisons between variables were made using the unpaired t test for parametric data. The multiple linear regressions were used to determine the association between OSA and metabolic parameters. Statistical significance was considered at p less than 0.05.
Results {#sec1-3}
=======
From all study patients, 51 were female with an average age of 49 ± 9 years, and 149 were men average age of 47 ± 9 years. There was no significant difference in age, BMI and RDI between male and female. There was the significant difference in the occurrence of OSA in men versus women, 109 (73.2%) of males and 31 (62.8%) of females were OSA positive (p \< 0.03).
According to BMI, patients in the study were divided into two groups. There were 120 non-obese patients with BMI ≤ 30 kg/m\^2, and 80 obese patients with BMI \> 30 kg/m\^2. In a non obese group with BMI ≤ 30, 62 patients were OSA negative and 58 patients were OSA positive. In an obese group with BMI \> 30, 14 patients were OSA negative, and 66 patients were OSA positive ([Figure 1](#F1){ref-type="fig"}).
{#F1}
OSA patients had statistically significant higher BMI, triglycerides, total cholesterol and lower HDL when compared to OSA negative patients ([Table 1](#T1){ref-type="table"}). There was no statistical difference in age and LDL levels between these two groups of patients.
######
Comparison between OSA positive and OSA negative patients
OSA negative RDI \< 15 (76 patients) OSA positive RDI \> 15 (124 patients)
--------------- -------------------------------------- --------------------------------------- ------- ------- -------
RDI 5.04 3.81 43.78 18.71 0.000
Age (years) 47.29 9.73 48.22 8.49 NS
BMI (kg/m\^2) 27.58 3.14 31.11 4.35 0.000
TG (mmol/l) 1.60 0.36 1.76 0.26 0.000
TC (mmol/l) 4.94 0.50 5.22 0.34 0.000
HDL (mmol/l) 1.45 0.23 1.34 0.23 0.001
LDL (mmol/l) 2.85 0.53 2.94 0.49 NS
OSA = Obstructive sleep apnea; RDI = Respiratory disturbance index; BMI = Body mass index; TG = Triglycerides; TC = Total cholesterol; HDL= High-density lipoprotein cholesterol; LDL = Low-density lipoprotein cholesterol.
In the study, both OSA positive and OSA negative patients were divided according to BMI in two groups, first group with BMI ≤ 30 and the second group with BMI \> 30. OSA positive patients with BMI ≤ 30 had statistically significant higher levels of triglycerides and total cholesterol, and statistically significant lower level of HDL compared to OSA negative patients with BMI ≤ 30. There were no statistically significant differences in age and LDL levels between these groups ([Table 2](#T2){ref-type="table"}).
######
Comparison between OSA positive and OSA negative patients with BMI ≤ 30
BMI ≤ 30
--------------- ---------- ------ ------- ------- -------
RDI 4.65 3.41 38.68 16.92 0.000
Age (years) 47.08 9.56 47.62 8.38 NS
BMI (kg/m\^2) 26.55 2.40 27.38 1.80 0.035
TG (mmol/l) 1.53 0.28 1.66 0.18 0.003
TC (mmol/l) 4.90 0.49 5.10 0.28 0.010
HDL (mmol/l) 1.55 0.22 1.35 0.19 0.000
LDL (mmol/l) 2.84 0.52 2.79 0.36 NS
RDI = Respiratory disturbance index; BMI = Body mass index; TG = Triglycerides; TC = Total cholesterol; HDL= High-density lipoprotein cholesterol; LDL = Low-density lipoprotein cholesterol.
OSA positive patients with BMI\>30 had higher triglycerides, total cholesterol and LDL and lower HDL versus OSA negative patients with BMI\>30, but without statistically significant differences ([Table 3](#T3){ref-type="table"}.)
######
Comparison between OSA positive and negative patients with BMI \> 30
BMI \>30
--------------- ---------- ------- ------- ------- -------
RDI 6.81 5.01 48.26 19.17 0.000
Age (years) 48.21 10.76 48.74 8.62 NS
BMI (kg/m\^2) 32.14 1.59 34.38 3.11 0.011
TG (mmol/l) 1.85 0.49 1.90 0.28 NS
TC (mmol/l) 5.12 0.53 5.33 0.35 NS
HDL (mmol/l) 1.37 0.25 1.30 0.23 NS
LDL (mmol/l) 2.94 0.60 3.08 0.55 NS
RDI = Respiratory disturbance index; BMI = Body mass index; TG = Triglycerides; TC = Total cholesterol; HDL= High-density lipoprotein cholesterol; LDL = Low-density lipoprotein cholesterol.
When all parameters were analysed with multiple linear regressions, only BMI, total cholesterol levels and LDL levels were found to be independent predictors of OSA ([Table 4
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Introduction {#Sec1}
============
Recent times have seen higher demand for sustainable and green products reproduced from waste materials^[@CR1]^. In particular, this has called for more research and development endeavors to recycle waste materials into biodegradable products with low environmental impacts^[@CR2]^. The potential of several types of cellulose-based materials from agro-wastes such as palm oil, pineapple, kenaf, sisal, etc. as reinforcing materials in cement composites has been discovered decades ago, but yet to be thoroughly understood and applied in real-world construction^[@CR3],[@CR4]^. Cellulose can be extracted from various plants while oil palm empty fruit bunch is one of its sources. Malaysia is the second-largest producer of palm oil and the country's palm oil industry produces about 90 million tonnes of lignocellulosic biomass, including empty fruit bunches, oil palm trunks, and oil palm fronds, as well as palm oil mill effluent^[@CR5]^. Reinforcement of natural fibers and cementitious matrices from various sources of plant fiber have found to improve the mechanical strength of the composites and currently is being applied in various industrial sectors including construction, automobiles, aerospace's, etc.^[@CR6],[@CR7]^. In particular, renewable waste materials such as agro-waste have enhanced the mechanical properties of cement mortar^[@CR8]^. However, direct incorporation of natural fiber material into mortar leads to low concrete workability, decay problems, low resistance to chemical attack, and other structural problems^[@CR9]^. The most common approach to overcome these problems is through surface modification of plant fibers using various chemical treatments (alkali, silane, ozone, etc.) and physical treatment (plasma, UV radiation, corona, etc.)^[@CR10]--[@CR12]^. Another approach to improve the properties of natural fibers is through a top-down approach by deconstructing the structure of the plant itself into nano or microfibrous materials such as nanocrystalline cellulose (CNCs) and microcrystalline cellulose (MCC)^[@CR13]^.
In recent days, construction industries conduct various researches to discover new eco-friendly reinforcement materials to reduce cement usage. Previous few examples of the reinforcement material that contributes to minimizing the cement usage include steel, glass, carbon, etc.^[@CR14],[@CR15]^. In order to replace this material, various high-performance nanostructures such as carbon nanotubes have been utilized to improve the performance of cement composites^[@CR16],[@CR17]^. However, troubles in dispersion and cost-effectiveness are the major crisis with these materials, which are needed to be addressed before commercialization^[@CR7]^. At present, the performance of nano and micro cellulose materials as reinforcement of cementitious composites is getting research attention worldwide. Positive result discovered by Cao *et al*.^[@CR12]^ through the use of CNCs extracted by using the acid hydrolysis process. An enhancement of cement mortar up to 30% for the flexural strength with the addition of 0.2% CNCs was found^[@CR12]^. Another achievement found with the addition of CNCs in oil well cement studied by Reza *et al*.^[@CR18]^, revealed that CNCs reduced the porosity up to 33%, surface area to 66% and 0.7% of the total design water by mass. Also, CNCs have raised the compressive and tensile strength by 60% in the first 24 hours of composite's age.
However, research on CNCs based cementitious composites extracted from palm oil fruit bunches are very rare in the existing literature. Therefore, this present study intends to report the effect of CNCs suspension on the mechanical properties of mortar via different: i) curing environment, ii) percentage of CNCs added and iii) morphological observation. A series of experiments were conducted to examine the effect of different curing methods (water, lime and wrapping curing) to find the best method of curing, as well as the microstructural and mechanical properties of the cement composites after adding CNCs.
Materials and Methods {#Sec2}
=====================
The cement mortar mix used in this study were prepared by mixing the CNCs aqueous suspension together with fine aggregates and cement at 0.5 water/cement ratio^[@CR19]^. The amount of CNCs liquid suspension added to cement composites was from 0% to 0.8% by volume of cement content. The dispersion behavior of the CNCs in aqueous suspension was found to be more stable compared to the powder form.
Preparation of the CNCs {#Sec3}
-----------------------
The cellulose was supplied by a local company from Waris Nove Sdn. Bhd. at Kuantan, Pahang Malaysia that commercially produces α-cellulose from palm oil wastes for industrial applications. The extraction process began by referring to the extraction methods from Dong *et al*.^[@CR20]^ and Lu and Hsieh^[@CR21]^ with the production of CNCs from α-cellulose. The first step is to extract the microcrystalline cellulose (MCCs) from α-cellulose^[@CR20]^. In order to produce MCC, 2.5 N hydrochloric acid (HCl) was mixed with α-cellulose and incubated for 15 minutes at the controlled temperature of 105 °C. This is followed by the addition of cold water to the hot mixture, stirred and the mixture was left overnight. The mixture was filtered and then washed with water until the pH reached 6\~7. The filtered sample was dried in a hot air oven for 60 minutes at 60 °C. The finally the sample was ground and sieved using 60 µm sieve aperture before it was extracted for CNCs.
Based on Kumar *et al*.^[@CR22]^, during the CNCs extraction, 64% w/v sulphuric acid (H~2~SO~4~) was used for the acid hydrolysis process. The acid solution was initially preheated to 45 °C and MCCs were added at a ratio of 10:1 (diluted H~2~SO~4~: MCCs). Subsequently, the solution was stirred for 60 minutes, mixed with 1/10 fold of chilled distilled water, and centrifuged at 6000 rpm for 15 minutes to remove any excessive acid. Then, the remaining precipitate underwent dialysis for 5-7 days for neutralization. Finally, the solution was centrifuged and subjected to sonification for 15 minutes to form CNCs aggregates. The final product was refrigerated at 4 °C until the further application.
In general, when MCCs was mixed with sulphuric acid (through acid hydrolysis process) it changes the MCCs to CNCs^[@CR23]--[@CR25]^. Sulfuric acid hydrolysis of cellulose is a heterogeneous process where the acid diffuses into the pulp fiber and cleaves the glycosidic bonds in the cellulose polymer. Depending on reaction times, temperature, and how the heating rate is controlled, the hydrolysis could also occur on the crystalline regions and some of the hydroxyl groups on the crystalline surface and convert into sulfate groups (e.g., conversion of cellulose-OH to cellulose-OSO~3~−H^+^). CNCs can be generated which is in a milky white color but not as brownish or blackish color. The brownish and blackish color solution shows that the CNC is burned due to improper selection of acid concentration and temperature. Therefore, in this study concentration of sulphuric acid used was 64% w/v with a temperature of 45 °C for 60 minutes. With this condition of acid hydrolysis, the end product of CNC comes out to be milky white in color. Figure [1(a)](#Fig1){ref-type="fig"} shows the MCCs powder after the α-cellulose pre-treatment process and Fig. [1(b)](#Fig1){ref-type="fig"} displays the CNCs aqueous suspension after the acid hydrolysis process (milky white in color). The schematic illustration of the CNCs extraction process was simplified in Fig. [2](#Fig2){ref-type="fig"}. The chemical composition of the extracted CNCs from palm oil wastes used in this study was evaluated via X-ray Fluorescence (XRF) assessment and tabulated in Table [1](#Tab1){ref-type="table"}. The main constituents of the CNCs were detected as carbon and oxygen. A low amount of sulfur was also detected, most probably from the leftover acid during the acid hydrolysis process with the H~2~SO~4~ solution.Figure 1Two different types of cellulose production (**a**) MCC powder (**b**) CNCs aqueous suspension.Figure 2Schematic illustration of CNCs extraction process.Table 1Chemical composition of cellulose nanocrystals used as an admixture in cement composites.Chemical CompositionMass Percentage (%)Carbon, C43.76Oxygen, O55.12Sulfur, S1.01Others0.11
Cement mortar samples preparation and strength tests {#Sec4}
----------------------------------------------------
### Cement and sand preparation {#Sec5}
Portland Cement Type I was used throughout the study. This was obtained from a Tasek Cement Company located at Ipoh, Malaysia. Based on ASTM C778-113^[@CR26]^, two types of sand with different sizes were blended in the equal portion into the mortar mixes, i.e passing 850-µm sieve and retained at 600-µm sieve and passing 600-µm sieve and retained at 150-µm sieve^[@CR26]^.
### Mortar samples casting {#Sec6}
The specimens consisted of 50 mm cubes and 40 mm by 40 mm by 160 mm prism for compressive and flexural strength tests, respectively. By following the ASTM C109/C109M^[@CR19]^, the specimens were prepared based on the optimum mix design of 1:2.75
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Introduction
============
Caridean shrimps (infraorder Caridea) belonging to the genus *Lysmata*, Risso 1816, are heavily targeted in the marine ornamental trade ([@ref-20]; [@ref-8]; [@ref-36]) and have experienced increases in market demand ([@ref-35]). Many marine ornamentals provide needed ecosystem services in home aquariums, such as algal--grazing and scavenging and are a biological alternative to other mechanical and chemical methods for aquarium maintenance ([@ref-21]; [@ref-35]). In the United States, Florida is the center of the ornamental fishery ([@ref-30]), where *Lysmata boggessi* and *Lysmata wurdemanni* are collected as bycatch between October and May from commercial stone crab traps and are landed year-round in the commercial bait shrimp trawl fishery ([@ref-8]; [@ref-33]). These shrimp are desired among aquarists specifically for their ability to regulate the pest anemone *Aiptasia* spp., Gosse 1858 ([@ref-34]), which is often introduced to aquaria via live rock. Previous studies suggest that their recent increase in popularity is in part driven by this biological control ([@ref-35]).
The overall increase in landings of marine invertebrates that provide ecosystem services has raised concerns regarding the impact of harvest on wild stocks and their surrounding environment ([@ref-1]; [@ref-19]; [@ref-35]; [@ref-8]). For instance, [@ref-19] found that many ornamental fisheries operated on small spatial scales and that harvest could result in a nearly 50% localized reduction in population size. Unfortunately, there are severe data gaps in the life history, reproductive biology, population structure and growth characteristics for many of these organisms ([@ref-21]; [@ref-35]), which is especially true for ornamental crustaceans and *Lysmata* spp. The alarming result is that ornamental fisheries in Florida have not been managed using fisheries data and no management strategies currently inform the catch targets (i.e., maximum sustainable yield, total allowable catch) necessary to ensure stock sustainability ([@ref-19]). If these data gaps were to lead to fisheries mismanagement and the overexploitation of ecosystem service providers such as *Lysmata*, then an unintended ecological consequence would be the loss of those same services from the wild ([@ref-35]; [@ref-8]).
In addition to their fishery and ecosystem value, shrimp from the genus *Lysmata* are notable for their rare sexual system, protandric simultaneous hermaphroditism (PSH). Individuals displaying PSH settle and mature initially as males and over a series of transitional molts develop functional female gonads to become simultaneous hermaphrodites ([@ref-25]; [@ref-39]). The latter sex phase, characterized by the presence of ovotestes and gonopores ([@ref-16]) is capable of functioning as either sex but cannot self-fertilize ([@ref-13]). Phylogenetic studies have determined that PSH is a fixed and conserved trait within the genus ([@ref-4], [@ref-5]; [@ref-11]), which is thought to be advantageous for increasing mating opportunities in low density populations and fitness specifically for large male phase shrimp ([@ref-3], [@ref-6]; [@ref-31]). Interestingly, the size at which *L. wurdemanni* males transition to hermaphrodite varies with season and appears flexible, which is suspected to be a strategy to decrease reproductive output when conditions are unfavorable ([@ref-7]; [@ref-12]). If true across the genus, this adaptive capability may be an important consideration in the management of other harvested populations.
[@ref-9] produced a snapshot of the life history parameters of a Florida *L. boggessi* population, but they did not capture the important detailed seasonality to these parameters. We implemented a similar methodology as used by [@ref-9] and [@ref-12] to assess life history and reproductive characteristics, but focused on a fished population of *L. boggessi* on the west Florida shelf. These shrimp are found in nearshore waters and are landed year round as bycatch in the commercial bait shrimp (*Farfantepenaeus duorarum*) industry. The primary objective was to describe the seasonality of sex phase ratio and size at sex change at the population level and fecundity, embryo volume and reproductive investment at the individual level.
Materials and Methods
=====================
Study site, sampling protocol and shrimp measurements
-----------------------------------------------------
We sampled a segment of the *L. boggessi* population on the Florida west coast using fisheries-dependent techniques for a full year. This genetically homogeneous population ranges from Key West to approximately Cedar Key, Florida ([@ref-8]). Shrimp were collected by a trained observer at night and as bycatch once per month from December 2012 to November 2013 via roller-frame trawlers ([@ref-18]) in a shallow subtidal region off the west coast of Florida. This area, locally referred to as The Reef, is located adjacent to the St. Martin's Aquatic Preserve, 6--8 km offshore of Citrus County, Florida ([Fig. 1](#fig-1){ref-type="fig"}). The depth ranged from 2 to 5 m and the benthic substrate was composed of heterogeneous coarse sand, seagrass and low relief hard bottom areas. Seagrass areas were dominated by *Thalassia testudinum* and *Syringodium filiforme* and the hard bottom was characterized by exposed limestone covered with a thin (\<2 cm) layer of sediment. *L. boggessi* shelter diurnally within crevices found in hard bottom ([@ref-9]), but their cryptic and nocturnal nature poses a logistical challenge for collecting specimens. We were able to circumvent this limitation by using commercial trawlers, which provided sufficient catch efficiency to achieve the sample sizes required for a statistically robust population assessment. Nocturnal segregation of shrimps by size or ontogenetic phase was not suspected in *L. boggessi* based on previous population studies of *L. wurdemanni* ([@ref-12]). Therefore, we are confident that the haphazard trawler coverages collected representative samples of *L. boggessi* on The Reef.
{#fig-1}
During each sampling event the trawl vessel simultaneously deployed two roller-frame trawls (4.27 m height × 0.61 m width), one on the port and one on the starboard side, 8--10 times between sunset and 02:30 h. Trawl durations were 30--45 min. A mesh size of 25.4 mm was used near the mouth of the trawl net and throughout the tapered body, with a finer mesh catch bag (19.1 mm) woven into the tailing end. The vessel tracks, average speeds and tow times were recorded with a hand-held Garmin^™^ GPS Map 60CSX. *L. boggessi* landed during each deployment were counted and approximately 30 individuals on alternating deployments were haphazardly sub-sampled for further measurements back at the laboratory. The sub-sampled shrimp were fixed onboard the fishing vessel in 10% neutral buffered formalin and were transferred after 48 h to 70% ethanol for preservation.
Preserved specimens were viewed under a Leica^®^ Model S8AP0 dissecting stereomicroscope to measure body size, determine sex phase and assess embryonic development in gravid hermaphrodites. Carapace length (CL) was measured using Leica^®^ Application Suite V4 image analysis software to 0.1 mm from the mid-dorsal posterior margin of the carapace to the posterior edge of the eye orbital. Sex phase was determined by observing the second pair of pleopods for either the presence or absence of the appendices masculinae, where presence indicated male phase and absence indicated hermaphroditic phase ([@ref-16]). Hermaphrodites were considered gravid if they retained an embryonic mass on the ventral side of their abdomen. Masses were delicately removed with forceps and the embryos were then counted at 10× power under the stereomicroscope to determine batch fecundity. We were not able to confidently categorize embryo maturation based on eyespot and yolk sac development alone, so we used only early embryo masses void of either feature for this analysis. Therefore, fecundity estimates were not corrected for embryo loss. To calculate embryo volume, 10 embryos were haphazardly sub-sampled from each removed mass and measured along their short and long axes to a precision of 0.001 mm. Lastly, hermaphrodites and their corresponding embryo mass were dried at 60 °C for 24 h and weighed separately to approximate reproductive output.
Population-level life history parameters in *Lysmata boggessi*
--------------------------------------------------------------
Temporal differences in sex phase ratio, in terms of the proportion of male phase shrimp and size at sex change (CL~50~) were assessed from the monthly sub-samples. Sex phase ratio was estimated as the number of male phase individuals divided by the total number of males plus hermaphrodites in the population during each month ([@ref-10]). CL~50~ was calculated via binomial logistic regression with 95% confidence interval bounds. The CL~50~ estimates represented the CL at which *L*.
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"I soon noticed that many cases of congenital hypoplasia of the mandible occurred and that the organovegetative and psychic life of the infants was more disturbed when the hypoplasia was more pronounced. I have never seen babies live for more than sixteen or eighteen months who presented hypoplasia such that the lower maxilla was pushed more than 1 cm behind the upper.(Pierre Robin 1934)"
Introduction {#Sec1}
============
In 1923 Pierre Robin described a constellation of findings that bears his name today \[[@CR1]\]. The triad of findings included micrognathia, glossoptosis and respiratory obstruction; however, considerable confusion in the medical literature delineating Robin sequence has been demonstrated \[[@CR2], [@CR3]\]. Pediatricians often encounter the entity "Robin sequence"; however, there are still many unanswered questions surrounding this disorder. Robin sequence can still be associated with significant morbidity and even mortality \[[@CR4]\]. Glossoptosis associated with airway compromise is most often the culprit instigating respiratory insufficiency (Fig. [1](#Fig1){ref-type="fig"}). However, other causes can cause breathing problems, and these patients should be carefully investigated preferably by a multidisciplinary team \[[@CR5]\]. Traditionally tracheotomy has been considered the definitive treatment in securing a stable airway when the airway was compromised. However, tracheotomy can be associated with significant morbidity and even mortality \[[@CR6], [@CR7]\].Fig. 1Typical cases of glossoptosis. Patient has a cleft of the small palate (not visible on photo). Note retrusion of mandibula with regard to maxilla
Distraction of the mandible has become an accepted method to treat the micrognathia and subsequently the airway compromise \[[@CR8]--[@CR12]\]. Distraction osteogenesis (DO) is a technique in which bone is gradually lengthened after performing an osteotomy. After a short latency period, the bone segments are distracted. The bone segments are separated from each other at a slow, steady rate. Similar to fracture healing new bone will subsequently be formed between these segments. After the acquired bone length is achieved the consolidation period ensues in which the bone segments are held in their advanced positions. This is needed because the newly formed bone has to mature and consolidate. During DO the distraction proceeds at a slow, steady state ensuing not only bone lengthening but also concomitant soft tissue expansion. Subsequently will not only new bone be formed, but the muscles, blood vessels, nerves and mucosa will also be elongated. Ilizarov popularized distraction on the lower extremity in the 1940s \[[@CR13]\], although Codvilla introduced distraction nearly 100 years ago \[[@CR14]\]. Following in the footsteps of Ilizarov, mandibular distraction was first performed experimentally by Snyder \[[@CR15]\]. The first clinical report of mandibular distraction in the English literature was reported by McCarthy et al. in 1992 \[[@CR16]\]. Like Ilizarov did, mandibular distraction was performed with an external device. Since then, numerous reports have been published demonstrating the feasibility in relieving airway obstruction \[[@CR8]--[@CR12]\]. However, an external distraction system is cumbersome to take care of; it leaves external scars and always needs a second operation to remove the distraction device. In an attempt to alleviate these disadvantages, an internal and resorbable distraction device (located under the skin) was developed \[[@CR17]\]. The goal of this manuscript is to review our results of performing mandibular distraction with a resorbable system in patients with Robin sequence and life-threatening airway compromise.
Methods {#Sec2}
=======
For this study we looked at the patients we treated early, i.e. in the first 3 months after births. Patients were considered for distraction only after a diagnosis of Robin sequence was made (glossoptosis, micrognathia and airway compromise). The medical ethical board approved this study. Patients were seen by a multidisciplinary team consisting of a pediatrician, ENT surgeon, geneticist, dietician and plastic surgeon. Non-invasive treatment options such as prone positioning and nasal continuous positive pressure are sufficient measures for most newborn babies with Robin sequence. Only patients that could not be treated conservatively and would traditionally be considered candidates for a tracheotomy were candidates for distraction osteogenesis. Before intervention patients were observed with continuous pulse oximetry and blood gas evaluation (pCO2, HCO 3 etc). Saturation measured over 12 h in all patients was \< 90% for \> 5% of the 12 h \[[@CR10]\]. Polysomnography was only used if the aforementioned results were not comparable to the clinical picture. Patients received an endoscopy by the ENT surgeon prior to DO to exclude any other cause of airway obstruction (e.g. tracheomalacia, stenosis etc) besides the glossoptosis.
The first patient treated (Table [1](#Tab1){ref-type="table"}) had already a tracheotomy, while the others were treated primarily for airway compromise. The aim in the first patient was to relieve him of his tracheostoma.Table 1Patient characteristicsNameDate of birth (day.month.year)Age at surgery (days)Amount of distraction (mm)Associated malformations (syndrome)OutcomeDuration of hospital stay (days)1. JH09.19.20068320COL 11a2 gene mutation (anocular Stickler syndrome)Admission with tracheacanule. Minor local symptoms of infection at pin site. Removal 10 months post op.182. NS10.04.20071518COL 11a2 gene mutation (anocular Stickler syndrome)Successful detubation on day 9 post op. Minor local symptoms of infection at one pin site163. SS10.03.20071916NoneSuccessful detubation on day 8 post op.114. LB11.16.20071720No mutation on Col2A1 and Col11A1 genes. No definite exclusion of Stickler because of severe myopiaSuccessful detubation on day 11 post op. Technical failure of one distraction screw 5 weeks after surgery185. LN01.17.20081318NoneSuccessful detubation on day 8 post op.236. LK03.30.20089418NoneSuccessful detubation on day 5 post op.147. RS06.26.20082720Megaencephaly and retardation, no genetic mutation foundSuccessful detubation on day 8 post op.278. LW02.08.20104522NoneSuccessful detubation on day 5 post op.169. RS06.19.201016202.19 Mb deletion in 3q22.2q22.3. Further research is ongoingSuccessful detubation on day 7 post op.1510. GH11.03.20092218NoneSuccessful detubation on day 6 post op.2011. JH07.31.20081118NoneSuccessful detubation on day 8 post op.1712. AE04.23.20102416Suspicion of Stickler due to familiar myopiaSuccessful detubation on day 8 post op.14*op.* operation
All patients were treated with the Lactosorb internal distractor distributed by W. Lorenz Surgical, a Biomet company. The precise placement has been described previously by Burstein \[[@CR17]\]. Briefly, the surgical approach was a submandibular incision (2--2.5 cm) with dissection to the mandibular body and angle while preserving the mandibular branch of the facial nerve. The two dissolvable plates were placed after the vector of distraction was determined from a mandibular X-ray or a CT scan. An osteotomy was performed after the plates were fixated with soluble screws (Fig. [2](#Fig2){ref-type="fig"}). The distractor wire was subsequently placed subperiostealy and protruded the skin through an incision placed above the ear (Fig. [3](#Fig3){ref-type="fig"}). After the placement of the distractor, we waited for 36--48 h before the distraction was started. A postoperative X-ray was made. Distraction was performed at a rate of 1 mm twice daily (Figs. [4](#Fig4){ref-type="fig"} and [5](#Fig5){ref-type="fig"}). After surgery all patients were treated in the pediatric intensive care unit, until the intubation tube could be removed. On average this was performed 5--7 days after the actual distraction was initiated, i.e. when 10--14 mm of bone lengthening was achieved. Distraction was continued until the mandibular alveolus was in a normal position with regard to the maxillary alveolus or until the maximum technical length of distraction with this device (20--25 mm) was achieved (Fig. [6](#Fig6){ref-type="fig"}). After a consolidation phase of 4 weeks the distraction screw was removed in the outpatient clinic with patients receiving only paracetamol 30 min before removal of the screw. An X-ray was performed before the distraction screw was removed to demonstrate bone consolidation.Fig. 2Location of osteotomyFig. 3Placement of internal device with distractor wire visible above ear. This could easily be concealed with a baby hatFig. 4After osteotomy the mandibular is gradually lengthened with the distraction. **a** Prior to distraction. This brings the tongue forward (**b**) and alleviates the respiratory obstructionFig. 5Comparison of resorbable plate size with 2-euro coinFig. 6Example of patient before (**a**) and after (**b**) surgery. Notice the extra space in the oropharynx after the distraction and that the nasogastric tube has been removed
Results {#Sec3}
=======
Twelve patients with Robin sequence were included (Table [1](#Tab1){ref-type="table"}). All our patients had
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1. Introduction {#sec1-nanomaterials-10-00919}
===============
Biological apatite (non-stoichiometric hydroxyapatite) (BAp) is the main inorganic constituent in human bones and teeth \[[@B1-nanomaterials-10-00919]\]. Pure hydroxyapatite (HAp, Ca~10~(PO~4~)~6~(OH)~2~) has inorganic components of Ca and P ions; however, biological apatite is amorphous and contains several other ions, such as carbonate, Mg^2+^, SO~4~^3−^, Na^+^, CO~3~^2−^, K^+^ and Cl^−^, among others \[[@B2-nanomaterials-10-00919]\]. In addition, the 2D plate-like structure is the predominant phase in biological apatite, due to its intrinsic tendency to grow in a plate-like structure under physiological conditions \[[@B2-nanomaterials-10-00919],[@B3-nanomaterials-10-00919]\]. The fabrication of BAp with a similar chemical composition to natural bone with a controlled 2D plate-like structure is extremely difficult, especially when the mineralization occurs in a complex bioinspired condition. Designing BAp 2D nanoplates is a crucial factor for the improvement of its biological properties \[[@B2-nanomaterials-10-00919],[@B4-nanomaterials-10-00919],[@B5-nanomaterials-10-00919]\]. It has been reported that the composition, size, shape and controlled structure of bioactive bone minerals play a crucial role in determining the physical and chemical properties enabling its biomedical applications \[[@B5-nanomaterials-10-00919]\]. Although there have been many attempts to synthesize BAp minerals with nanoplate morphology, the proposed methods are replete with several problems, including the use of a toxic template and an organic solvent, and creating hazardous by-products \[[@B5-nanomaterials-10-00919],[@B6-nanomaterials-10-00919],[@B7-nanomaterials-10-00919]\], and it is difficult to obtain a 2D plate-like structure simulating the morphology of natural bone \[[@B3-nanomaterials-10-00919],[@B8-nanomaterials-10-00919],[@B9-nanomaterials-10-00919]\]. To drive homogeneous nucleation, a very high degree of supersaturation is required. This is dependent on the crystal-like arrangement and formation of ions meeting the thermodynamic criteria of the critical dimensions. Therefore, the synthesis of BAp nanocrystals using efficient green chemistry routes can be an alternative method of incorporating other biological minerals into precipitated bone minerals. Green synthesis has been intensively employed in recent studies because it is simple, biocompatible, eco-friendly and cost-effective \[[@B5-nanomaterials-10-00919],[@B10-nanomaterials-10-00919]\]. Biomimetic synthesis of its outstanding properties, including the absence of toxic chemicals, high pressure, temperature and/or energy, and its ability to easily scale-up for the large-scale synthesis of nanomaterials \[[@B9-nanomaterials-10-00919]\]. Therefore, the ecofriendly synthesis method is employed in the present study to incorporate bone minerals and control the growth rate of crystal size. Polyphenolic provenance from plants is comprehensively employed in the synthesis of inorganic nanomaterials as stabilizing, reducing and chelating factors \[[@B11-nanomaterials-10-00919],[@B12-nanomaterials-10-00919],[@B13-nanomaterials-10-00919],[@B14-nanomaterials-10-00919],[@B15-nanomaterials-10-00919],[@B16-nanomaterials-10-00919]\], and as eco-friendly alternatives to chemical and physical routes. Fenugreek (FG) is an annual plant belonging to the legume family, which has been used as one of the most promising traditional medicinal herbs. It has been widely used for medicinal purposes such as remedying pain, anti-cancer effects, anti-diabetic medication, anti-microbial effects and as an anti-oxidant reagent \[[@B17-nanomaterials-10-00919],[@B18-nanomaterials-10-00919],[@B19-nanomaterials-10-00919]\]. This is due to the pharmacological activity of the major compounds in FG seeds, such as linoleic acid, palmitic acid, pinene, 4-Pentyl-1-(4-propylcyclohexyl)-1-cyclohexene and linoleic acid methyl ester \[[@B10-nanomaterials-10-00919],[@B17-nanomaterials-10-00919],[@B20-nanomaterials-10-00919]\]. FG is also being studied for its cardiovascular benefits \[[@B18-nanomaterials-10-00919]\].
In particular interest, previous studies including our own ([Table 1](#nanomaterials-10-00919-t001){ref-type="table"}) showed that FG plants can be a good source of essential minerals, such as Mg^+2^, Zn^+2^, Ca^+2^, PO~4~^3−^, K^+^, Na^+^ and Fe^2+^ \[[@B17-nanomaterials-10-00919],[@B18-nanomaterials-10-00919]\]. Therefore, the incorporation of such ions into the synthesized bone minerals can have a favorable impact on fast bone formation. On the other hand, it is speculated that, by increasing the concentration of the hydroxyl groups in the precursor solution, the interaction between the organic molecules existing in the FG extract and the metallic ions of the calcium phosphate is increased, which means higher interference by apatite crystals during the biosynthesis process. This phenomenon might lead to denser structures being formed by FG organic molecules of the BAp minerals. A denser structure leads to smaller interplanar spacings in the obtained bone minerals. Accordingly, the crystallite size can be reduced to its smallest size to obtain apatite with 2D fine plate-like structures. Controlling the structure of apatite crystals at the nano-level is vital for acquiring a favorable commercial product. In this work, BAp minerals with a 2D plate-like morphology mimicking the natural bones' composition and structural features, fabricated by biosynthesis of the plant extraction method, have been prepared successfully. The plant polyphenolics possess high cognation for metal ions because of the existence of the hydroxyl groups of phenolic compounds and their molecular structure. As an effort to prepare BAp 2D plate-like structures mimicking the morphology of biological apatite, a biosynthesis process was developed based on the use of natural macromolecules from FG extracts. The bioactive ceramic formed in FG extraction has been called biosynthesized BAp nanoplate, because its composition and structure are similar to those of natural bone rather than of sintered stoichiometric HAp, and it has important characteristics such as low crystallinity and nanoscale sizes that are important for the reabsorption and remodeling found in bone \[[@B21-nanomaterials-10-00919],[@B22-nanomaterials-10-00919]\]. The BAp with the 2D nanostructures formed in this FG extract is believed to exhibit even higher bioactivity and biocompatibility than stoichiometric HAp \[[@B22-nanomaterials-10-00919],[@B23-nanomaterials-10-00919]\]. Furthermore, BAp ultrafine 2D plate-like structures were prepared by employing FG seed extract using biosynthesis wet-chemical precipitation as a simple, efficient, economic and non-toxic route.
2. Materials and Methods {#sec2-nanomaterials-10-00919}
========================
2.1. Biosynthesis Process {#sec2dot1-nanomaterials-10-00919}
-------------------------
In total, 20 g of commonly used FG seeds were washed several times before being boiled for 15 min \[[@B24-nanomaterials-10-00919]\] in 100 mL Milli-Q water (18.2 MΩ·cm), as typically prepared in traditional medicine. The extracted solution was filtered twice through Whatman no. 1 filter paper. The extract solution was then used for the synthesis of BAp powders by the wet-chemical precipitation route, as described in our previous report \[[@B4-nanomaterials-10-00919]\]. Briefly, calcium cations (1 M Ca(NO~3~)~2~·4H~2~O) (Sigma Aldrich, South Korea) and phosphate anions (0.6 M (NH~4~)~2~HPO~4~) (Sigma Aldrich, South Korea) were separately dissolved in 100% concentration FG extract solution. The phosphate solution was added dropwise at a rate of 0.4 mL min^−1^, under vigorous mixing, into the calcium solution. Ca/P ratio was controlled to be 1.67, the stoichiometric value of HAp. The resultant precipitate slurries were dispersed in a mixing solution of pure Milli-Q water and ethanol (volume ratio = 1:1) and then left to dry in a vacuum for 24 h. The dried powder was further heat-treated at 650 °C for 4 h, with a heating rate of 20 °C/min \[[@B4-nanomaterials-10-00919]\].
The chemical element concentration of the FG seed extract and the biosynthesized powder (0.1 mg dispersed in 5 mL Milli-Q water) were analyzed at 670.783 nm wavelength using a Varian (Inc., Melbourne, Australia) Vista Pro (MPX) radial inductively coupled plasma atomic emission spectroscopy (ICP-OES) instrument. Standards from 0 to 5 mg/L metallic ions were prepared from Fluka, TraceCERT 1000 mg/L stock standard. In addition, due to the limitation of ICP-OES in detecting SO~4~, Cl and CO~3~, the colorimetric analysis method was used to measure the concentration of these elements in both the FG extracts and obtained BAp powders. Electrical conductivity
|
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Introduction {#section1-0963689719854363}
============
Urethral strictures represent a pathologic narrowing of the urethral lumen. This pathology affects mainly men and is manifested by lower urinary tract symptoms: poor stream, hesitancy, terminal dribbling, incomplete voiding, etc^[@bibr1-0963689719854363]^. Female urethral strictures are rare. Urinary tract infections, overactive bladder, and stress incontinence are common conditions affecting women´s urinary tracts^[@bibr2-0963689719854363]^. Management of the female urethral strictures involves urethral dilatation and urethral reconstruction using vaginal flaps, vaginal grafts, and oral mucosal grafts^[@bibr3-0963689719854363]^.
In men, iatrogenic injury is the most common cause of urethral stricture followed by idiopathy, trauma, and inflammation. Systemic diseases (e.g., lichen sclerosus) can also lead to urethral strictures. Scarring of the urethral tissue is the causative process leading to the replacement of the vascular tissue of the corpus spongiosum, which leads to ischemic spongiofibrosis of the urethra. Urethral stricture is often manifested by other complications that can be presented as recurrent or chronic infection, the formation of bladder calculi, fistulas, development of sepsis, or renal failure. Therefore, urethral strictures present a serious health condition that significantly impairs quality of life and may lead to the failure of vital organs if left untreated^[@bibr4-0963689719854363]^.
It is estimated that the incidence of the urethral strictures is approximately 1% in males over the age of 55. However, the real incidence of male urethral stricture disease is unknown and strongly depends on certain populations, geography, and income^[@bibr5-0963689719854363]^.
The choice of surgical technique depends on the location and length of the stricture. Approximately 50% of urethral strictures are located in the bulbar urethra, 30% in the penile urethra, with the rest in a combination of both. There is a significant interest in optimizing surgical techniques to avoid further interventions and obtain a satisfactory high long-term success rate. Urethral dilatation and direct visualization internal urethrotomy are the preferred techniques for urethral stricture management. However, the management of long urethral strictures remains challenging, as outcomes seem to be poor^[@bibr6-0963689719854363],[@bibr7-0963689719854363]^. The success rate of urethroplasty techniques is between 80--90%. The outcomes of anastomotic and graft substitution urethroplasties showed a satisfactory long-term success rate.
In pioneering works, different tissues such as the buccal mucosa and free-skin grafts have been applied to treat these pathological conditions. However, both are accessible in limited quantities and their utilization is accompanied by donor site morbidity and various post-surgery complications, so alternative treatment procedures are needed to improve long-term outcomes^[@bibr8-0963689719854363],[@bibr9-0963689719854363]^. Tissue engineering (TE) offers several promising approaches that may help to overcome these problems. TE, in the context of the urethral repair, uses different cell types alone or in combination with different functional biomaterials and specific growth factors to engineer functional urethral tissue suitable for transplantation purposes. Choosing the right cell type together with a supporting matrix (scaffold) is a crucial step in urethral TE^[@bibr10-0963689719854363][@bibr11-0963689719854363]--[@bibr12-0963689719854363]^.
So far, there have been only a few clinical studies describing the use of cell-seeded templates in urethral reconstruction. Cells isolated from the buccal mucosa and bladder tissue were applied^[@bibr13-0963689719854363]^. According to our search criteria, only one article describing clinical application on in vitro cultivated cells was suitable for review.
The main goal of this article is to focus on the various types of cells involved in urethral reconstruction using approach of TE.
Materials and Methods {#section2-0963689719854363}
=====================
Literature search methodology {#section3-0963689719854363}
-----------------------------
A search was performed (3 January 2019) of the PubMed/Medline databases. Keywords related to TE were combined with synonyms for the urethra, urethral tissue, urothelium, smooth muscle cells (SMCs), stem cells, and urethral TE. The search was restricted to the last 10 years, the English language, and studies performed on humans or animals. A Prisma Flow Diagram represents the outline of the literature search ([Figure 1](#fig1-0963689719854363){ref-type="fig"}).
{#fig1-0963689719854363}
Results {#section4-0963689719854363}
=======
Urine-derived stem sells {#section5-0963689719854363}
------------------------
Urine-derived stem cells (UDSCs) were the point of interest in the following studies. Either human or animal urine samples were the source of these cells. Urethral catheterization, spontaneously voided urine, bladder irrigation, and bladder washing were the methods used for urine harvesting. To collect the cells, urine samples were centrifuged. Cells were cultured in initiation media, which mainly consisted of a mixture of embryonic fibroblast (EFM) and keratinocyte serum free medium ([Figure 2](#fig2-0963689719854363){ref-type="fig"}).
{#fig2-0963689719854363}
Tayhan et al. used six fresh urine samples harvested from healthy patients via urethral catheterization. Human UDSCs and urine-derived urothelial cells were studied. Immunocytochemical analysis was performed to characterize isolated cells. Antibodies against cytokeratin 7 were used as urothelial cell markers. Antibodies against CD45 and CD90 were used to determine the presence of the mesenchymal stem cells (MSCs). Results showed that epithelial cell colonies were observed up to 2 days after initial seeding. Overall, 80--90% confluency of human UDSCs was reached within 12 days. Some of these cells were also positive for cytokeratin 7. Those that were positive for CD90 were negative for CD45. This study demonstrated the presence of both cell types in fresh urine samples^[@bibr14-0963689719854363]^.
Yang et al. also focused on the characterization of UDSCs, but in this study cells were of the animal origin (rabbit). A total of 12 urine and 13 bladder wash samples were used for cell isolation. For the characterization, cell proliferation assay, flow cytometry, Western blot, and immunocytochemistry were used. A differentiation experiment was also performed and stem cells were successfully differentiated into smooth muscle, urothelial, and osteogenic cell lines^[@bibr15-0963689719854363]^.
UDSCs seeded on small intestinal submucosa (SIS) were examined in two studies^[@bibr16-0963689719854363],[@bibr17-0963689719854363]^. The aim was to engineer a cell-seeded construct that could be applicable for urethral repair. In one study, modified three-dimensional (3D) porous SIS was colonized with human UDSCs, which were differentiated into urothelial and smooth muscle cell lines. The cell source was 12 voided urine samples. The culture medium for smooth muscle differentiation consisted of Dulbecco's modified Eagle's medium, EFM, platelet-derived growth factor-BB, and transforming growth factor β1 (TGF-β1). Cells were analyzed after 7 and 14 days. Cell-seeded constructs were cultured under static and dynamic conditions and also applied in vivo. To confirm urothelial and myogenic differentiation, immunohistochemical tests were performed. The results showed the multilayered mucosal structure was formed under dynamic conditions with similar features to the native urothelial tissue^[@bibr16-0963689719854363]^.
In another study, autologous rabbit UDSCs were obtained from bladder irrigation solution samples. The media for urothelial and smooth muscle differentiation were the same as in the study above. Seeded UDSCs were labeled with PKH67 to establish cell differentiation. Labeled cell-seeded constructs were transplanted into rabbits to repair the ventral urethral defect. Histological analyses and retrograde urethrograms were performed at various time points. The results revealed that transplanted UDSCs could differentiate into required cell lineages and, when seeded on SIS, the urethral defect could be regenerated^[@bibr17-0963689719854363]^.
Using human UDSCs to optimize their differentiation into a functional urothelium together with the emphasis on proper urothelial barrier function was the
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1. Introduction {#sec1}
===============
Deep resources such as oil, gas, and solid mineral have drawn more interest. Generally, the deeper drill is characterized by higher pressure and temperature, which make the drilling and borehole stability harder \[[@B1]--[@B7]\]. However, in Mexico Bay, North Sea Basin, Sichuan Basin, and the South Sea of China \[[@B8]\], for example, the gas and oil reservoirs in layers over 200°C have been successfully exploited.
When the fluid circles, the upper surrounding rock will be heated; when the fluid ceases to work, however, the lower one will be heated. Balanced by the fluid column pressure and the rock confining pressure \[[@B9], [@B10]\], the heated rock will fail to expand, generating thermal stress as a result \[[@B11]\]. Maury and Guenot claim that the thermal stress contributes most to the instability of the borehole \[[@B12]\]. The outcome they obtained shows that when the temperature of the midhard rock rises up by 1 centigrade, the stress can increase by 0.4 MPa, up to 1 MPa for the harder rock as a result. The thermal stress in 25 MPa to 50 MPa is practically common in 4000 meters boreholes. Consequently, the initial borehole stress and the common thermal stress can work together leading to collapse and fracture.
Wang et al.\'s research \[[@B13]\] shows that the Westerly granite can generate thermal cracking when heated up to 75°C. And the threshold value of 60\~70°C is suggested by Chen et al. \[[@B14]\]. Impacted by hydrostatic stress and thermal cracking, the granite\'s peak of the permeability, up to 3.5 × 10\~4 mD/°C, to the initial one reaches up to 93 \[[@B15]\]. This indicates that a field characterized by high permeability is developed around the borehole, triggering another stress field. In the borehole, the initial stress, temperature, and the stress field were triggered by overlapping the fluid together, which led to the deformation instability and leakage \[[@B9], [@B16], [@B17]\]. Consequently, the instability may make the drill stick or damage the casing.
Since the 1980s, in order to dispose the permanent nuclear waste, people began researching the coupling of THM (thermo-hydro-mechanical) \[[@B18], [@B19]\]. A global International cooperation project named DECOVLEX was established in 1992. Since then, a series of experiments, including modeling, have been conducted and some invaluable outcomes have been obtained as a result \[[@B20]--[@B24]\]. At the fourth stage of this project, the aim mainly was to study the mechanics of crystalline rock and the process in which the mechanical and hydraulic properties of the EDZ (excavation damage zone) are transformed. This process can harden or soften the rock \[[@B25]\]. In this paper, the thermal physical and mechanical properties of the granite are developed and researched under high temperature and three-dimensional stress. By utilizing*ANASYS-APDL*(ANSYS Parameter Design Language-APDL) \[[@B26], [@B27]\], the dynamic evolution equations of elastic modulus, Poisson ratio, uniaxial compressive strength, and permeability of granite with temperature are built and run. The temperature-fluid-stress coupling model to analyze the granite\'s stability is established and simulated to figure out the temperature\'s influence on collapse pressure, fracture pressure, and stress near the borehole, which can provide theoretical guidance for borehole stability and safety drilling in granite formations.
2. Thermophysical and Mechanical Properties {#sec2}
===========================================
2.1. Overview of Experiment {#sec2.1}
---------------------------
The sample, obtained from a 1000 meter deep borehole in Mount Yan, North China, is about 100 mm with a diameter of 50 mm. The density is about 2.54 g/cm^3^. TAW-1000 deep pore pressure servo experimental system was employed to test the sample. It consists of quartz, feldspar, and hornblende. All the samples were processed on the basis of Chinese national standard of GB50128-94 (shown in [Figure 1](#fig1){ref-type="fig"}). In order to avoid being contaminated by the hydraulic oil, we encapsulated the sample with a 3 mm thickness hot pyrocondensation pipe.
The experiments were conducted in a 1000°C electrothermal furnace whose space is 300 × 200 × 120 mm. The samples were placed at the center of the furnace, to whose front and rear it is about 3 mm far from the sample. All the samples were divided into 5 groups, with each was heated to room temperature, 100°C, 200°C, 300°C, 400°C and insulated for 2 hours, respectively. Compared with the original sample in [Figure 2](#fig2){ref-type="fig"}, these heated to 300°C and 400°C is dark red, owing to the Fe^3+^ transformed from Fe.
2.2. Longitudinal Wave Velocity Characteristics {#sec2.2}
-----------------------------------------------
[Figure 3](#fig3){ref-type="fig"} plots the link between longitudinal wave velocity and the temperature. The curve shows that the speed varies inversely with the temperature. This can be accounted for as follows: (I) as free water inside the rock evaporates, the pore becomes bigger; (II) when the temperature increases, the thermal stress will be triggered between minerals, due to their different coefficients of thermal expansion and anisotropy, generating new fractures or expanding the old.
2.3. Uniaxial Compression Tests {#sec2.3}
-------------------------------
### 2.3.1. Uniaxial Strength and Strain {#sec2.3.1}
[Figure 4](#fig4){ref-type="fig"} plots the link between temperature and uniaxial strength. It shows that the threshold temperature is 200°C, in accordance with the result obtained from [Figure 5](#fig5){ref-type="fig"}. Below 200°C, the sample mainly undergoes brittle fracture, specially divided into compacting and linear elastic phases. On the other hand, over 400°C, the sample mainly undergoes the shear and tensile fractures.
Below 200 centigrade, the peak stress increases slowly but rapidly when it is over 200 centigrade. It shows that the threshold temperature is 200 centigrade, which accords with the outcome obtained from the link between the temperature and the uniaxial strength.
### 2.3.2. Elasticity Parameters of the Sample {#sec2.3.2}
The thermal damage is introduced to reflect the fluctuation of the elastic modulus of the samples before and after the heating the sample. The thermal stress will be produced between different mineral compositions due to the temperature change \[[@B28]\]. The thermal damage is calculated as follows: $$\begin{matrix}
{D\left( T \right) = 1 - \frac{E_{(T)}}{E_{(0)}}.} \\
\end{matrix}$$
The elastic modulus decreases with the increase of the temperature. Additionally, *e* the relationship between the elastic modulus and temperature is fitted by the data, and its fitting formula is *E* = −0.0145*T* + 29.997, with a goodness of 0.955.
[Figure 6](#fig6){ref-type="fig"} displays the increase of thermal damage after the sample was heated. As same as aforementioned, the threshold temperature obtained from the*D*-*T* curve is also 200 centigrade. When the temperature is from 0 centigrade to 100 centigrade and over 200 centigrade, the thermal damage of the sample is increasing while the thermal damage is unchanged from 100 centigrade to 200 centigrade.
The Poisson ratio is characterized by polymeric. As shown in [Figure 7](#fig7){ref-type="fig"}, with the increasing of the temperature, the Poisson ratio of the granite samples is more and more mounting. The proportion between Poisson ratio and temperature is mainly accounted for two reasons: (I) the increase of the temperature leads to the changes of the sample\'s interior structure, the water content, and the porosity; (II) and the temperature and stress are beyond the sample\'s elasticity.
### 2.3.3. Damage States of Samples {#sec2.3.3}
The sample was experimentally damaged under uniaxial pressure in three ways as shown in [Figure 8](#fig8){ref-type="fig"}: (I) under room temperature, the sample undergoes the brittle fractures developing along the axial direction. (II) Under 100--200°C, the sample undergoes the shear fracture. If loaded, the softer part would be damaged without losing its bearing capacity. (III) Under 300--400°C, being sheared and tensioned, the sample undergoes the column fractures.
2.4. Triaxial Compression Tests {#sec2.4}
-------------------------------
### 2.4.1. Mechanical Properties with Different Confining Pressure {#sec2.4.1}
[Figure 9](#fig9){ref-type="fig"} displays the link between triaxial compressive strength and confining pressure. It shows that as the confining pressure rises, the triaxial compressive strength virtually and nonlinearly increases. With *R* = 0.996, the nonlinear link can be expressed as $$\begin{matrix}
{\sigma_{s} = 0.834\sigma_{w}^{2} - 14.05\sigma_{w} + 269.65.} \\
\end{matrix}$$
The link between elastic modulus
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Introduction {#s1}
============
Based on the current trends in fossil energy production and use, deforestation, and population growth, it is expected that the increase of global mean surface temperatures for 2081--2100 relative to 1986--2005 is projected to be in the ranges of 0.3 to 1.7°C (RCP2.6), 1.1 to 2.6°C (RCP4.5), 1.4 to 3.1°C (RCP6.0), and 2.6 to 4.8°C (RCP8.5), which will have dramatic effects on economics, agriculture, and environment (AR5, IPCC, [@B26]). Plant traits are sensitive to climate warming and ecologists use plant trait-climate relationships to simulate plant physiology and growth in current and future climate situations (Farquhar and Sharkey, [@B18]; Wang et al., [@B66]; Jing et al., [@B28]). Therefore, understanding the patterns of plant physiological and morphological responses to global warming is of great importance in simulating and predicting the impact of global change on natural systems and agriculture.
Predictions of response to global warming may be derived from experimental and observational studies (Tilman, [@B58]; Wang et al., [@B65], [@B67]; Knapp et al., [@B33]). While both types of study are common, relatively few authors have investigated whether they produce similar predictions or reflect reality (Dunne et al., [@B16]; Knapp et al., [@B32]). Experimental global change studies are typically limited in scope both spatially and temporally (Rustad et al., [@B49]). Observational studies often have broader spatial and temporal scales but suffer from a lack of control over covariates in biophysical and biochemical parameters of weather and soil. To minimize the weaknesses of each approach, it has been suggested that more research should explicitly unite observational and experimental work, perhaps by nesting experiments at multiple sites within a larger observational context or through summarized meta-analysis (Dunne et al., [@B16]; Jing et al., [@B28]).
Many manipulative experiments controlling physical and environmental factors have been conducted around the world to investigate the potential effects of global change on plants and terrestrial ecosystems (Sage and Kubien, [@B51]; Rustad, [@B50]; Wang et al., [@B67]). However, the methodology used in these experiments was different in their research settings, treatment intensities and durations and targeted species. The impact of short-term vs. long-term warming on plants traits would probably be different due to plants\' acclimation capacity in photosynthesis, respiration and other physiological processes and these impacts would vary among different plant functional types (PFTs) under natural or controlled settings (Smith and Dukes, [@B54]). Plants\' physiological and morphological responses to short-term warming treatment, however, are often used to parameterize the sub-models of photosynthesis, stomatal conductance, and respiration in plant growth and terrestrial ecosystem models, which would likely unrealistically simulate plant energy, carbon, and water fluxes in the long term. Indoor or outdoor settings and pot sizes could also affect the magnitude of ecophysiologial responses to temperature increase by implicating root growth and plant above-ground and below-ground tissue interactions (Arp, [@B3]). To accurately predict the impacts of climatic change and develop proper adaptive agricultural management practices, it is imperative to understand how temperature changes of different intensities and duration and changes manipulated under different experimental settings affect photosynthetic carbon gain, loss and allocation through a comprehensive analysis of relevant studies.
Previous research and meta-analyses have indicated that global warming will promote plant photosynthesis, dark respiration, leaf nitrogen content, specific leaf area, and other metabolisms (Poorter et al., [@B41]). It has been reported that the modulation of leaf traits and trait relationships by site climatic properties was modest (Wright et al., [@B71]). However, the modulation of leaf traits by warming treatment of different intensities and duration has not been extensively analyzed. Understanding how these processes vary among different species and plant functional types is a major goal for plant ecology and crucial for modeling how nutrient fluxes and vegetation boundaries will shift under global warming. The effect of the intensities and the treatment duration of global warming manipulative experiments on the plant physiology and growth among different plant functional groups, however, remain unclear. Therefore, the main objective of this study was to investigate the effects of global warming treatment with different magnitudes and durations on plant response in ecophysiological traits. Specifically, we aim to: (1) assess the impact of global warming of different magnitudes and durations on plant ecophysiological traits at leaf level; (2) detect the variations of ecophysiological traits response of different plant functional types to warming treatment of different durations; (3) explore the effect of different experimental settings on the response of a plant\'s traits to global warming. Accordingly, we propose: (1) due to plant acclimation capacity, short-term vs. long-term warming has different impacts on plant traits, with short-term warming having a more stimulating effect on the physiological functions of plants; (2) different experimental facilities may change the response of plants traits to warming treatment. To test these hypotheses, we conducted a comprehensive meta-analysis of the warming manipulating studies published from 1980 to 2018.
Materials and Methods {#s2}
=====================
Data Collection
---------------
Journal articles were searched on the Web of Science database with the keyword "leaf traits & warming," "leaf traits & temperature increase" and etc. The articles were later cross-checked with review articles and book chapters. The articles were imported into EndNote software and formed a database. All articles about warming effects on leaf traits were screened to ensure that all the articles available were included for the analysis. The articles published from 1980 to 2018 and meeting the following two conditions were included in the analysis: (1) the control group in the experiment was treated at ambient temperature situation; (2) physiological and morphological measurements were performed on both ambient and manipulated groups. Articles were rejected if: (1) plant physiological changes under warming treatments led to death of or severe damage to the plant; (2) there were other stressing factors impacting the warming treatments. Finally, 80 papers meeting the requirements were included in the database ([Supplementary Material S1](#SM1){ref-type="supplementary-material"}). Data was obtained directly from the table or was extracted using the GetData Graph Digitizer software from the selected articles. In these studies, the magnitude of warming treatment ranged between 0.3 and 25°C, with only two studies showing a warming treatment above 20°C above AT ([Supplementary Material S1](#SM1){ref-type="supplementary-material"}). Response variables collected from these articles included net photosynthetic rate (A~net~), stomatal conductance (G~s~), leaf nitrogen (LN), dark respiration (R~d~), and specific leaf area (SLA). When A~net~, R~d~, and G~s~ of one species with the same unit were all provided in the study (including measurements conducted on the same leaves/individuals and those across individuals), the R~d~/A~net~, and A~net~/G~s~ in the control and warming treatments were calculated. In addition to the above responsive variables under different treatments, plant species, sample size, growth facilities, and duration of warming treatment were also collected. To ensure the independent nature of the data, we excluded duplicate results collected from the same studies. However, our analyses were not completely independent because individual study often provided data with more than one treatment (e.g., different warming treatment intensities) and/or different response variables. To examine the influence of non-independence of data, we first averaged those data from the same published study by PFTs so that only one comparison was used from a published study for each PFT. Nonetheless, we found that most of the response patterns were unchanged; therefore, all data were used in our study.
Categorization of the Studies
-----------------------------
Temperature treatment was divided into two categories: AT (ambient temperature) and ET (elevated temperature). Plant species were classified into different photosynthetic pathways (C~3~, C~4~, or CAM), growth forms (herb or wood) and economic values (crop or non-crop). Experimental facilities were categorized into indoor (growth chambers or greenhouses) and outdoor (open top chambers or fully-open) settings and \<10 L and \>10 L growing pots. In our dataset, exposure time (i.e., how long plants were exposed to warming) ranged from \<10 days to \>10 years. To analyze the possible different responses under various warming durations, we banded the temperature treatment into two categories: short-term (\<1 year) and long-term (\>1 year). Warming treatments that were applied through air warming were included in the analyses. We listed the species, PFTs information and relevant experimental methodology used in this study ([Supplementary Material S1](#SM1){ref-type="supplementary-material"}).
Meta-Analysis Methods
---------------------
To avoid the adverse effects of different units, we used the response ratio r = X~t~/X~c~ to estimate the magnitude of the effect of warming treatment, where X~t~ is the treatment mean and X~c~ is the control mean. For ease of comparison, we calculated the natural logarithm of the response ratio (lnr). The standard deviation (SD) and the sample size (*n*) for each observation were collected to calculate the variance of the effect size.
The lnr was calculated without and with being standardized by warming magnitude (Equations 1, 2).
log
e
r
=
log
e
(
X
t
X
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Kuntz‐Melcavage KL, Gahagan KDC, Fracasso MR, Marsteller JA. Enhancing knowledge of authorization requests through registry development. Learn Health Sys. 2018;2:e10050 10.1002/lrh2.10050
All authors are employed by Johns Hopkins HealthCare LLC, which is the company that funded this study.
1. INTRODUCTION {#lrh210050-sec-0001}
===============
Health insurers are a crucial part of health care systems and have a responsibility to provide wise medical coverage decisions for their members. Coverage decisions are determined by regulations, benefits, and medical policies, with each insurer developing its own company‐specific policies. It is essential that medical coverage policies be written in a manner that promotes the best health for insured members while aligning with the mission of a company. Sometimes, there is insufficient evidence on a new procedure or drug to know for sure which patients will benefit from it most and which may not benefit at all.
As part of an academic health system, our organization strives to maximize the knowledge obtained from data we collect. Current research in the industry relies heavily on administrative claims[1](#lrh210050-bib-0001){ref-type="ref"}, [2](#lrh210050-bib-0002){ref-type="ref"} that are useful for providing data about use and costs. However, there are other sources of information that can be informative to medical policy decisions that remain largely untapped. Claims data do not contain information that is submitted as part of pre‐authorization requests (such as clinical measurements and laboratory values). Additionally, data from claims do not contain information about members who were denied authorization for a procedure. A source of data beyond claims can be helpful for providing a more complete profile of a member population seeking services. We are currently working to expand knowledge at the juncture between the medical‐policy setting and the subset of members who are affected by coverage decisions. The mechanism we have chosen to increase our knowledge in this area is the development of registries.
The need for a registry to record member‐level information surrounding certain procedures first became apparent, as the medical policy unit at our company planned a revision to the coverage policy for bariatric surgery. Anecdotal stories and opinions about bariatric surgery were prevalent, but no one was able to provide information derived from an aggregate collection of authorization requests.
To address the uncertainty about details contained in requests for bariatric surgery, we developed a registry of bariatric surgery requests containing data to inform future policy decisions. The registry records information from medical records, submitted as part of the pre‐authorization process for bariatric surgery. This paper describes the development of the registry and workflow to incorporate the registry into the process of medical pre‐authorization review. Results from a descriptive analysis of the population seeking surgery are presented, along with an examination of characteristics of members whose pre‐authorization requests for bariatric surgery are initially denied. Two examples of how the registry has broadened our knowledge about the pre‐authorization requests received by our company are presented.
This report is relevant for health systems because it demonstrates an approach for gathering meaningful data from an operational process. Data are the foundation for information, which ultimately leads to knowledge. Analyzing data from authorization requests has allowed our company to be more aware of approval patterns for requests and has provided the basis for us to learn more about factors that influence medical decisions.
2. METHOD {#lrh210050-sec-0002}
=========
This study was reviewed and approved by the Institutional Review Board of our institution (IRB number IRB00084964).
2.1. Registry design {#lrh210050-sec-0003}
--------------------
The initial step in developing a registry was to determine the proper electronic platform to be used. We chose to use Microsoft Access (Microsoft Corp, Redmond, Washington) because of 2 features considered to be of great importance: It is compatible with a variety of data analysis programs, and it provides the ability to develop a user‐friendly electronic data entry form. Both of these considerations were important for ensuring the functionality and usefulness of the registry. After deciding upon the software to be used for registry development, we identified the data fields to be included in the registry. Variables that are important when making authorization decisions were identified through literature reviews and interviews with medical directors. Health conditions that are associated with candidates for bariatric surgery were identified from literature, while conversations with medical directors revealed laboratory measures that were worthy of recording. A data dictionary was developed to provide a framework for the fields to be developed in the database (Table [1](#lrh210050-tbl-0001){ref-type="table"}). Each of the fields is able to be populated from information included in authorization requests. Because of the limited demographic information included in pre‐authorization requests, the registry similarly contains limited demographic information.
######
Kuntz‐Melcavage et al
Name Description
------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------
Database identifier Unique number assigned to database entry
Study identifier Number assigned to member seeking pre‐authorization for bariatric surgery
Member gender Gender of member seeking pre‐authorization
Age Age of member seeking pre‐authorization for bariatric surgery at time of application
Line of business Line of business of which member seeking pre‐authorization is a member
Date of letter seeking authorization Date that appears on the cover letter that was sent to seek pre‐authorization for bariatric surgery
Date of disenrollment If member disenrolls during study period; date on which member disenrolled
Date of baseline measurements Date on which baseline measurements of weight and BMI were made
Baseline weight Baseline weight (rounded to nearest whole number)
Baseline BMI Baseline BMI (body mass index)
Date of 1‐month postbaseline measurements Date on which measurements of weight and BMI 1 month after baseline were made
One‐month postbaseline weight Weight at timepoint approximately 1 month after baseline measurement (rounded to nearest whole number)
One‐month postbaseline BMI BMI at timepoint approximately 1 month after baseline
Date of 2‐month postbaseline measurements Date on which measurements of weight and BMI 2 months after baseline were made
Two‐month postbaseline weight Weight at timepoint approximately 2 months after baseline (rounded to nearest whole number)
Two‐month postbaseline BMI BMI at timepoint approximately 2 months after baseline
Date of 3‐month postbaseline measurements Date on which measurements of weight and BMI 3 months after baseline were made
Three‐month postbaseline weight Weight at timepoint approximately 3 months after baseline (rounded to nearest whole number)
Three‐month postbaseline BMI BMI at timepoint approximately 3 months after baseline
Date of 4‐month postbaseline measurements Date on which measurements of weight and BMI 4 months after baseline were made
Four‐month postbaseline weight Weight at timepoint approximately 4 months after baseline (rounded to nearest whole number)
Four‐month postbaseline BMI BMI at timepoint approximately 4 months after baseline
Date of 5‐month postbaseline measurements Date on which measurements of weight and BMI 5 months after baseline were made
Five‐month postbaseline weight Weight at timepoint approximately 5 months after baseline (rounded to nearest whole number)
Five‐month postbaseline BMI BMI at timepoint approximately 5 months after baseline
Date of 6‐month postbaseline measurements Date on which measurements of weight and BMI 6 months after baseline were made
Six‐month postbaseline weight Weight at timepoint approximately 6 months after baseline (rounded to nearest whole number)
Six‐month postbaseline BMI BMI at timepoint approximately 6 months after baseline
Date of 7‐month postbaseline measurements Date on which measurements of weight and BMI 7 months after baseline were made
Seven‐month postbaseline weight Weight at timepoint approximately 7 months after baseline (rounded to nearest whole number)
Seven‐month postbaseline BMI BMI at timepoint approximately 7 months after baseline
Comorbidities Comorbidity noted in letter requesting authorization for bariatric surgery
Comorbidities 2 Comorbidity noted in letter requesting authorization for bariatric surgery
Comorbidites 3 Comorbidity noted in letter requesting authorization for bariatric surgery
Comorbidities 4 Comorbidity noted in letter requesting authorization for bariatric surgery
Comorbidities 5 Comorbidity noted in letter requesting authorization for bariatric surgery
TSH laboratory results Laboratory results for thyroid‐stimulating hormone
Total cholesterol Laboratory results for total cholesterol level
Triglycerides Laboratory results for triglycerides level
LDL cholesterol Laboratory results for LDL cholesterol level
HbA1c laboratory results Laboratory results for hemoglobin A1c level
Complete? Has all available information been entered to the comorbidities/lab form?
*H*. *pylori* laboratory results Laboratory results for *Helicobacter pylori* test
Type of surgery requested The type of surgery that is requested (open or laparoscopic)
Initial determination Initial authorization determination (yes or no)
If no, action taken after initial denial If initial authorization determination was *no*, what action was taken after denial (peer‐to‐peer discussion, appeal 1, appeal 2, none)
Did reapplication occur? Did reapplication occur
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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi:10.1111/hae.14070
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INTRODUCTION
============
Globally, the incidence rate of malaria has decreased from 72 to 59 cases *per* 1,000 at risk inhabitants between 2010 and 2017, a 18% reduction^[@B1]^ . An estimated 219 million cases of malaria occurred worldwide in 2017 compared to 239 million cases in 2010. However, the estimates for 2015-2017 were almost similar, suggesting no progress in reducing the malaria burden during these last three years. In 2017, malaria resulted in an estimated 435,000 deaths globally compared to 607,000 deaths in 2010, with a 28% decrease in mortality^[@B1]^ . This reduction was attributed to the availability of highly effective antimalarial drugs and long-lasting insecticide nets (LLINs), as well as a mutual investment to provide treatment and preventive measures to the people in need^[@B1]\ -\ [@B3]^ . Most malaria cases were reported from the African World Health Organization (WHO) (92%), followed by the South-East Asia Region WHO (5%), and the Eastern Mediterranean Region WHO (2%). Notably, the highest numbers of malaria cases and deaths (93%) were reported from the African WHO, mostly in children under five years of age^[@B1]^ . To accelerate progress in reducing the burden of malaria, WHO endorsed the Global Technical Strategy for Malaria 2016--2030 (GTS) which set a vision to eliminate malaria in 35 countries by 2030 and in at least ten countries by 2020^[@B1]^ .
Bhutan has achieved a significant reduction of malaria incidence from 1,868 indigenous cases in 2006 to only six indigenous cases in 2018^[@B4]\ -\ [@B8]^ . The geographical distribution of malaria infection has also decreased from 15 of the 20 districts in 2006 to only two districts in 2018^[@B4]\ ,\ [@B6]^ . The dramatic decline in malaria cases is believed to be due to the high coverage of LLINs, intensified surveillance and early diagnosis and treatment^[@B5]\ ,\ [@B6]\ ,\ [@B9]\ ,\ [@B10]^ . Bhutan plans to eliminate malaria by 2025^[@B9]^ . However, imported and reintroduced cases along the international border with some Indian States remain a significant concern as seven of the seven Bhutanese districts (Chukha, Dagana, Pemagatshel, Samtse, Samdrup Jongkhar, Sarpang, and Zhemgang) share porous international borders with Assam, West Bengal, Arunachal Pradesh and Sikkim, in India. Among these States, Assam and West Bengal borders have intense cross-border activity, and most cases are reported in areas bordering Assam^[@B5]\ ,\ [@B11]\ ,\ [@B12]^ . Percentages of imported malaria cases have increased from 79.7% of the total malaria cases detected in Bhutan in 2016 to 82.33% in 2017, and 88.9% in 2018^[@B4]\ ,\ [@B10]\ ,\ [@B13]^ .
In Bhutan, *Plasmodium* species that cause malaria are *P. falciparum* and *P. vivax* . *Anopheles* species recorded and considered as malaria vectors in Bhutan are *An. minimus* , *An. fluviatilis, An. dirus, An. pseudowillmori* and *An. culicifacies* ^[@B13]^ . However, no studies on vectors including their ecology and behaviors have been conducted in Bhutan. The primary malaria control intervention adopted in the country includes mandatory screening for plasmodial infections in any fever case, early detection and treatment, active case finding to detect foci of transmission, community awareness and education. In addition, vector control by the universal coverage of LLINs, indoor residual spraying (IRS), clearing bushes and avoiding stagnation of water in the surrounding have also been implemented. While malaria incidence has dramatically declined, there is not much information on asymptomatic reservoirs in the country. Some evidences suggest that a significant proportion of asymptomatic reservoirs are present in both, high and low transmission settings^[@B14]\ ,\ [@B15]^ . The diagnosis of asymptomatic plasmodial infections in people living in low transmission settings cannot be made by commonly used diagnostic methods, *i.e.,* microscopic examination and rapid diagnostic tests (RDT)^[@B16]^ . To achieve malaria elimination, it is essential to ascertain the burden of asymptomatic reservoirs in the population at risk, as well as in migrant workers from malaria-endemic countries, particularly India^[@B14]\ ,\ [@B17]^ , and proactively detect and treat asymptomatic plasmodial infections with effective antimalarial drugs^[@B18]\ -\ [@B21]^ .
This study aimed to estimate the prevalence of asymptomatic plasmodial infections in the population living in at risk malaria areas in Bhutan, as well as in migrant workers from India. This information is essential to support the implementation of malaria elimination strategies in pursuit of elimination by 2025^[@B9]^ .
MATERIALS AND METHODS
=====================
Study area and sample size
--------------------------
A cross-sectional survey was conducted to determine the prevalence of asymptomatic *P. vivax* and *P. falciparum* infections targeting populations living in risk areas for malaria in seven districts of Bhutan, *i.e.,* Chukha, Dagana, Pemagatshel, Samtse, Sarpang Samdrup Jongkhar, and Zhemgang ( [Figure 1](#f01){ref-type="fig"} ). The study period coincided with the peak of the malaria season in April to May 2016. Based on the records maintained by 16 health centers in the risk areas they consisted of 6,319 households and 28,583 people, accounting for approximately 4% of the country's population. For the estimation of the prevalence of asymptomatic plasmodial infections ( *P. vivax and P. falciparum* ), catchment areas of two health centers in each of these districts located in malaria risk areas (based on the number of cases detected from 2011-2015) were intentionally selected^[@B22]^ . From each selected health center, approximately four villages (primary sampling units) were randomly selected and from each selected village, a maximum of ten households (secondary sampling units) were selected using a systematic random sampling technique (based on data maintained by VDCP and health centers). When the selected village had less than ten households, additional village(s) were randomly sampled, from the eligible list of villages in the catchment area of the selected health centers. From each household, a single member in the eligible list of household members was randomly sampled based on the inclusion criteria: (i) individual residing in a household that received LLINs distributed by the Vector-Borne Disease Control program in 2014; (ii) had not been diagnosed or treated for malaria during the last 21 days (based on the maximum incubation period^[@B23]^ ), (iii) aged ≥18 years on the date of the survey, (iv) resident in the locality for ≥ 1 years and (v) agreed to participate in the study.
Figure 1- Bhutan's chiwog map (small administrative units are in dotted lines) and the district map (bold lines) shows the sampling sites (shaded in black color) to estimate the prevalence of asymptomatic malaria in the community and in migrant workers at the three border entry points (triangles).
Using a 95% confidence interval (95% CI), an error of 2% and an anticipated asymptomatic prevalence of 5% (the expected prevalence of 5% was assumed although previous small-scale studies reported a prevalence of asymptomatic plasmodial infections \< 1%^[@B5]^ ). The required sample size was 457 participants. To account for the loss and missing information (approximately 10%), the sample size was rounded to 500. Taking the maximum design effect of 1.5^[@B18]^ , 750 individuals were included in the analysis using the formula N= (Zα/2)^[@B2]^ \*P (1-P)\*DEFF/ ME^[@B2]^ , where N is the sample size, Zα/2 is the critical α level, P is the anticipated asymptomatic malaria prevalence, DEFF is the design effect, and ME is the marginal error.
Migrant workers were enrolled in three main entry points, *i.e.,* Phuntsholing, Gelephu and Samdrup Jongkhar. All migrant workers underwent compulsory medical examination for entry into Bhutan. To this end, there were five registered private diagnostic centers in Phuntsholing, two in Gelephu and one in Samdrup Jongkhar. The sample size required for the estimation of the prevalence of asymptomatic malaria in this group of migrant workers was estimated using the same formula (without adjusting for the design effect DEFF assuming a within-private-diagnostic-centers variance of zero). Therefore, the estimated sample size for migrant workers entering Bhutan through these entry points was 500 individuals. Since over 50% of the workers entered through Phuntsholing, and around 50% through Gelephu and Samdrup Jongkhar, 250 individuals were randomly sampled in Phuntsholing and 125 individuals in Gelephu and the same number in Samdrup Jongkhar.
Approval of the study protocol was obtained from the Research Ethics Board of Health (REBH), Ministry of Health, Royal Government of Bhutan (approval No. REBH/Approval/2016/016). Written informed consent was obtained from the head of each household (HH) family. The interviewers explained the purpose, the risks and benefits of the study in the participant language and participation in the survey were voluntary. Written information on the survey was translated into Dzongkha for Bhutanese and Hindi for migrant workers and provided to all participants. Participants' demographics
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1. Introduction {#sec1}
===============
*Histoplasma capsulatum* (*H. capsulatum*) is a dimorphic fungus, which is endemic to North America (Central United States; Ohio-Mississippi valley) and Latin America \[[@B1]--[@B3]\]. It is associated with exposure to bat caves and avian droppings \[[@B4], [@B5]\].*H. Capsulatum* peritonitis should be suspected in patients on peritoneal dialysis (PD) from endemic areas who have the potential for exposure. Review of data from United States Renal Data Systems from 1992 to 1997 demonstrated age-adjusted incidence ratio of fungal peritonitis of 9.8 compared with general population and represents 4.5% of total peritonitis episodes in the PD population \[[@B6]\]. The vast majority of these cases are caused by*Candida* species. Mortality secondary to PD associated peritonitis is organism specific: 28% for fungi, 16% for enteric organisms, and 15% for staphylococcal species. The presence of certain additional factors in PD patients increases the risk for fungal peritonitis. Almost all published series have found an association with both recent antibacterial use and episodes of bacterial peritonitis \[[@B7]--[@B12]\]. When these series are combined, 65 percent of patients had been exposed to antibiotics within 30 days of the onset of fungal peritonitis, and 48 percent had experienced an episode of bacterial peritonitis within the same time frame. Other risk factors include emergency PD, HIV infection, abdominal surgeries, extraperitoneal fungal infections, and environmental exposures. Details of previously reported cases of*H. capsulatum* are also discussed.
2. Case Report {#sec2}
==============
A 63-year-old female who was visitor from Veracruz, Mexico, presented to the emergency room with complaints of progressively worsening abdominal pain and distention for three days. She also had fever and altered mentation. Her past medical history was significant for hypertension, diabetes mellitus, hyperlipidemia, and end-stage renal disease. She had been on PD for four years and denied any recent changes in technique. She had two episodes of peritonitis in the past while in Mexico but was unaware of the details of those episodes. Her surgical history was significant for appendectomy, cholecystectomy, and tubal ligation and she denied any recent abdominal procedure. She denied smoking, alcohol intake, or use of recreational drugs.
On examination, her blood pressure was 172/85 mm of Hg, pulse 88/min, oral temperature 39.5°C (103.1°F), respiratory rate 14/min, and oxygen saturation on room air 94%. She was lethargic and confused. She had abdominal distention and diffuse tenderness without any rebound or guarding. Her PD catheter exit site was clean and dry. Laboratory studies showed white blood cell count of 14.5 × 10^3^/*μ*L with 87.1% granulocytes, hemoglobin of 6.3 g/dL, and hematocrit of 18.4%. Serum chemistries showed sodium of 130 mmol/L, potassium of 2.7 mmol/L, chloride of 90 mmol/L, bicarbonate of 27 mmol/L, blood urine nitrogen of 30 mg/dL, and creatinine of 7.7 mg/dL. Her liver function tests were within normal limits. Computed tomography of abdomen and pelvis without intravenous or oral contrast showed peritoneal thickening consistent with peritonitis, and there was no evidence of perforation or obstruction ([Figure 1](#fig1){ref-type="fig"}). PD fluid analysis showed white cell count of 2173 per mm^3^ with 96% neutrophils and red blood cells of \<3000 per mm^3^.
Blood and PD fluid cultures were sent, and she was empirically treated for bacterial peritonitis with intraperitoneal cefazolin and ceftazidime. PD fluid gram stain revealed budding yeast; blood and PD fluid cultures did not reveal bacterial growth. Given the high suspicion of fungal peritonitis, immediate removal of the PD catheter was discussed with the patient. She chose not to have the catheter removed, leave to Mexico, and get treated by her own nephrologist. Hence oral fluconazole was started for presumed*Candida* peritonitis. However, six days later, the fungal culture \[Mycosel Agar and Brain Heart Infusion Agar\] of the PD fluid grew*H. Capsulatum*.
3. Discussion {#sec3}
=============
As previously noted, fungal peritonitis is an uncommon cause of peritonitis in PD patients. There are currently no established guidelines for the diagnosis of fungal peritonitis. The International Society of Peritoneal Dialysis (ISPD) recommends repeating PD fluid cell count at day 3 of culture negative peritonitis and employing special culture techniques for isolating uncommon organisms including fungi. \[[@B13]\]. Isolation of*Histoplasma* from culture may take up to 12 weeks but can happen as early as 1-2 weeks. Nonculture techniques available for diagnosis include molecular techniques like polymerase chain reaction, serological tests like complement fixations, and immunodiffusion tests for precipitins \[[@B14]\].
If yeast is seen on initial gram stain, then prompt antifungal treatment should be started as we did in our patient. Although the mainstay of therapy in the past has been Amphotericin B, its toxicity has frequently precluded its use \[[@B15]\]. Experience with the newer imidazoles/triazoles and flucytosine suggests that these agents are well tolerated and efficacious \[[@B13]\].
Regarding the removal of PD catheter, ISPD guidelines recommend prompt removal once fungal infection is identified. Additionally, an appropriate antifungal agent should be continued to 2 weeks after removing the catheter \[[@B13]\]. There are isolated reports of treating fungal peritonitis without removing the catheter, but with varying degrees of success. However, this should be considered an option only if patient\'s medical condition precludes removal of the catheter \[[@B14]\].
Since peritonitis due to*H. capsulatum* is extremely rare, there are no established guidelines for treatment of this condition. The six reported cases were treated with 6--12-month course of Itraconazole with or without Amphotericin B as noted in [Table 1](#tab1){ref-type="table"} \[[@B16]--[@B20]\].
4. Conclusion {#sec4}
=============
Although*H. capsulatum* peritonitis is extremely rare, morbidity and mortality associated with it are high. Diagnosis requires high degree of suspicion based on geography and occupation. In such patients, if yeast is seen on gram stain it would be prudent to remove the PD catheter and consider Itraconazole as first choice of therapy for extended period of time.
Consent
=======
The patient left to Mexico immediately after discharge. Her son-in-law, who has the power of attorney over her healthcare issues, provided informed consent over the telephone for publishing this case report.
Conflicts of Interest
=====================
The authors attest that they do not have any conflicts of interest pertaining to this case report.
{#fig1}
######
Management of reported cases of PD patients with *Histoplasma* peritonitis.
Cases Treatment regimen Treatment Catheter removed
------------------------------- -------------------------------- --------------------- ------------------
Case 1 \[[@B16]\] Oral Itraconazole 12 months Yes
Case 2 & 3 \[[@B17], [@B18]\] Amphotericin B Unknown Yes
Case 4 \[[@B18], [@B19]\] Fluconazole and Amphotericin B 1 month and 10 days No
Case 5 \[[@B14]\] Oral Itraconazole 6 months Yes
Case 6 \[[@B20]\] Oral Itraconazole 12 months Yes
[^1]: Academic Editor: Rumeyza Kazancioglu
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Introduction
============
One key characteristic of fast-growing solid tumors is the development of intratumoral hypoxia, which promotes malignant tumor progression. Cancer cell migration and invasion play important roles in the metastatic cascade. Cell migration is the transition process from the local, noninvasive confined tumor cells to the migrating, metastatic cancer cells when the cells obtain the ability to dissociate from intracellular adhesions and become motile [@B1],[@B2]. Invasion, on the other hand, is the process by which malignant cells move through the basement membrane and gain access to blood vessels and lymphatic channels. As cancer cell migration and invasion are essential prerequisites for tumor metastasis, identifying agents that effectively block hypoxia-induced cancer migration and invasion would promote development of effective anti-cancer therapeutic approaches.
Hypoxia-inducible factor-1 (HIF-1), a master transcriptional factor in response to hypoxia, activates a group of downstream genes that promote tumor malignant progression. HIF-1 is composed of an inducible subunit, HIF-1α and a constitutively expressed subunit, HIF-1ß [@B3]. Heterodimerization of HIF-1α and HIF-1ß is required to form the transcriptionally active HIF-1 that binds to the hypoxic response elements within the promoter regions of target genes [@B4]. HIF-1α degrades quickly under normal oxygen tension [@B5] but it is inducible and stabilized under low oxygen tension (hypoxia) [@B6]. Increased HIF-1α levels have been shown to correlate with decreased patient survival in many cancers including breast cancer (BCa) [@B7]. The activation of HIF-1α stimulates a group of HIF-1α-regulated genes including vascular endothelial growth factor (VEGF) [@B3],[@B8]. As a result, the cells are converted towards malignant progression. HIF-1 activity is mainly dependent on the level of HIF-1α protein, the inducible and regulatory subunit of the HIF-1 dimer complex [@B9], [@B10].
We have previously shown that p16, a tumor suppressor gene and cyclin D kinase inhibitor and a negative cell cycle regulator [@B11], can neutralize the transactivation ability of HIF-1α on its target gene VEGF [@B12]. In this study, we evaluated whether hypoxia stimulates breast cancer migration and invasion, and whether ectopic expression of p16 is capable of modulating hypoxia-mediated cell migration and invasion. We also evaluated whether cocultured stromal cells (fibroblasts) promotes BCa cell invasion and whether p16 inhibits that effect, as well as whether HIF-1α of fibroblasts specifically has a role in promoting cocultured BCa cell invasiveness.
Materials and Methods
=====================
Cell lines, cell culture conditions, and reagents
-------------------------------------------------
Human breast cancer (BCa) cell line MDA-MB-231 was obtained from American Type Culture Collection (ATCC, Manassas, VA). Human breast cancer cell lines LM2, a MDA-MB-231 derivative line that has high lung metastatic ability [@B15], was a generous gift from Dr. J. Massague of Memorial Sloan-Kettering Cancer Center (NY). Murine mammary carcinoma cell line JygMC(A) was from Dr. H. Azuma of Osaka Medical College, Osaka, Japan [@B13]. Mouse MEF fibroblasts expressing wild-type (WT) and knockout (KO) HIF-1α were from Dr. T Seagroves of University of Tennessee Health Science Center (TN).
MDA-MB-231 cells were grown in RPM1-1640 (Gibco, Life technologies Corporation, Grand Island, NY) with 10% fetal bovine serum (FBS) (Gibco), LM2 cells were grown in Dulbecco\'s Modified Eagle medium (DMEM) (Cellgro, Mediatech, Inc, Manassas, VA) with 10% FBS. JygMC(A) cells were grown in DMEM (Cellgro) with 10% FBS. Both MEF lines were grown in DMEM medium with 25 mM HEPES (Gibco) with 10% FBS. All cell lines were grown in medium containing 100 units/ml penicillin, and 100 μg/ml streptomycin. The cell cultures were incubated at 37°C either under normoxia (5% CO~2~, 21% O~2~) or hypoxia (5% CO~2~, 1% O~2~, balanced with N~2~) conditions.
Generation and screen of stably transfected, Dox inducible (Tet-on) p16-expressing breast cancer cell lines
-----------------------------------------------------------------------------------------------------------
The construction of the Tet-on p16 lentiviral system Lenti-Tet-on p16 (pLenti-Tet-p16-pgkpuro and pcFUW-rtTA3-IRES-puro) was described previously [@B12]. Breast cancer cells (MDA-MB-231, LM2, and JygMC(A), respectively) were co-transduced by pLenti-Tet-p16-pgkpuro and pcFUW-rtTA3-IRES-puro at multiplicity of infection (moi) of 10 each, together with 6 μg/ml polybrene (Millipore, Bedford, MA). The stably transfected cells were enriched in medium containing 1 μg/ml puromycin (Clontech, Mountain View, CA). The resultant stably transfected cells are named as MDA/Tet-on-p16, JygMC(A)/Tet-on-p16, and LM2/Tet-on-p16, respectively. For induction of p16 transgene expression in Tet-on-p16 stably transfected cells, 1 μg/ml doxycyline (Dox) (Clontech) was used in the medium for incubation.
Immunohistochemistry
--------------------
The procedure followed the method as described previously [@B12]. Briefly, for immunohistochemical (IHC) staining, cultured cells were grown on SlideFlasks with bottom detachable slides (Nalge Nunc, Naperville, IL) that could be used for IHC staining directly later. The samples (slides) were first incubated with 1% H~2~O~2~ for 30 min. The samples were incubated with first antibody against human p16 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 16 h at 4^0^C, then by corresponding second antibody and the Universal Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer\'s protocol. The reaction was visualized with DAB solution (75 mg 3,3\'-Diaminobenzidine and 30 ml 50% H~2~O~2~ in 150 ml PBS) for 3-10 min.
Cell migration assay
--------------------
The breast cancer MDA-MB-231 cells stably transfected with inducible Tet-on p16 (MDA/Tet-on p16) were incubated in the absence or presence of 1 μg/ml Dox for 72 h. The cells were then harvested and used for the following migration assay. The cell migration was measured by a modified Boyden\'s chamber method using BD Falcon Cell Culture Inserts incorporating polyethylene terephthalate (PET) track-etched membrane with a pore size of 8.0 μm (BD Bioscience, Belgium). The inserts were precoated on the under-surface (between upper and lower chambers) with 10 µg/ml fibronectin (Thermo Fisher Scientific) at 37^0^C for 3 h. Above mentioned harvested cells in suspension of serum-free medium (SFM) with 1% BSA were incubated at 37°C for 30 min under normoxic (5% CO~2~, 21% O~2~) or hypoxic (5% CO~2~, 1% O~2~, balanced with N~2~) conditions. Subsequently, cell suspensions were seeded into the upper chamber of an insert at a density of 10,000 cells per well, and 300 µl serum-free medium with 1% BSA was placed in the lower chamber to act as a chemoattractant in the 24-well cell culture plate. The cells were further incubated at normoxia and hypoxia (see above) at 37°C for 3 h. The inserts were then removed and nonmigrating cells remaining on the upper side of the filter were scraped off. The cells that had migrated to the lower surface of the insert were stained using Giemsa staining solution (Sigma, St. Louis, MO). After extensive washing with water, the migrated cells were counted in five different fields under a microscope at x200 magnification. Migratory activity was expressed as the number of cells (that is, the sum of total cell numbers in five randomly selected fields of view) that migrated to the lower side of the filter.
*In vitro* invasion assay with commercially precoated invasion inserts
----------------------------------------------------------------------
The BCa cells were examined by the invasion assay under normoxia by using commercially available invasion inserts. For each Dox inducible BCa/Tet-on p16 cell line, the cells were either incubated in medium with or without 1 μg/ml Dox for 72 h. The cells were then harvested and used for the following *in vitro* invasion assay in the 6-well-plate BD Biocoat Matrigel Invasion Chambers (BD Biosciences Bio-Coat Matrigel Invasion Chamber) according to the manufacturer\'s procedure. Briefly, the chamber was first rehydrated with serum-free medium (SFM) for 2 hr at 37^0^C. After rehydration, the chambers were placed in the lower compartment loaded with medium containing 5% FBS. Meanwhile, the above-mentioned cells were suspended and adjusted to 1.25x10^5^ cells/ml in SFM with or without 1 μg/ml Dox. The cell suspension (2 ml or 2
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The first case of coronavirus disease 2019 (COVID-19) in Indonesia was reported on March 2, 2020.[@bib1] Since then, the number of people who are infected with SARS-CoV-2 has grown exponentially, a pattern that can usually be observed in an uninhibited outbreak.[@bib2] ^,^ [@bib3] Some experts have stated that the number will continue to soar in the subsequent few weeks if no significant intervention is performed.[@bib4] The government then announced a public health emergency on March 31, 2020.[@bib5]
Following the public health emergency announcement, the Indonesian government imposed a temporary ban on all foreign travelers and also published several other regulations.[@bib6] One major regulation was a guideline for large-scale social restriction (Permenkes No. 9 Tahun 2020; Pedoman Pembatasan Sosial Bersakala Besar). The local governments with a high number of cases or deaths in their regions were allowed to impose such restriction with approval from the Indonesian Ministry of Health.[@bib7] Such measures included banning mass gatherings, closing schools and offices, curbing nonessential businesses, and banning public transport.[@bib7]
Furthermore, the Indonesian Ministry of Health selected several hospitals as the referral hospitals to handle the emerging infectious disease (KH.01.07/MENKES/169/2020). Dr Sardjito General Hospital was one of the selected hospitals located in the Special Region of Yogyakarta.[@bib8] The hospital, which is also the university hospital for Universitas Gadjah Mada, was chosen because of its full-range intensive care unit and 2 isolation wards for airborne infectious diseases.[@bib9] Finally, to accommodate more patients with COVID-19, the hospital also remodeled a ward as a new isolation ward.
Compared with other countries located outside the equatorial zone, the number of COVID-19 cases has been relatively small in Indonesia.[@bib10] However, the outbreak is adding extra burdens on the health care system worldwide, including Indonesia.[@bib11] Neurosurgical service is one of the systems affected.[@bib12] Several articles have discussed how neurosurgical services should be managed during this difficult time[@bib13] ^,^ [@bib14]; however, there is no specific report on neurosurgical services in developing countries. This article focuses on the neurosurgical service in Dr. Sardjito General Hospital, Yogyakarta, Indonesia, as a representative of low- and middle-income countries. Moreover, although the number of COVID-19 cases has remained rather low in Yogyakarta, we believe that hospital preparedness and contingency plans for a pandemic should be formulated, especially for neurosurgery cases, to manage the potential development of this pandemic and possible future outbreaks.
Here we review what has been done by related stakeholders to curb the impact of the disease. We also describe our hospital\'s policy for performing neurosurgical services during the COVID-19 outbreak and explore what can be corrected in the future, especially in the setting of low- and middle-income countries.
Methods {#sec1}
=======
We collected information on the effect of the outbreak in Indonesia from the COVID-19 online database created by the Indonesian Ministry of Health. The government selected the National Institute of Health Research and Development (Badan Penelitian dan Pengembangan Kesehatan Kementerian Kesehatan \[Balitbangkes\]) as COVID-19 national referral laboratory. In addition, the government also selected several regional laboratories as COVID-19 diagnostic laboratories.[@bib15] All facilities had to meet the Biosafety Level 2 standard and have reverse-transcription polymerase chain reaction (RT-PCR) capabilities.[@bib16] By the middle of April 2020, the Indonesian government had tested more than 30,000 samples (i.e., approximately 130 tests per 1 million population).[@bib17]
Furthermore, we gathered information from online COVID-19 records generated by the Yogyakarta government to assess the effect of the outbreak in the Special Region of Yogyakarta. By April 13, 2020, the Yogyakarta administration had performed 516 tests, for a rate of approximately 144 tests per 1 million people.[@bib18] The data on confirmed COVID-19 cases from all laboratories throughout Indonesia were then sent to Balitbangkes to be validated and verified.[@bib15] All confirmed cases, which included inpatients, recovered patients, and deaths, had been confirmed by RT-PCR.[@bib15] Finally, the data were sent to the Public Health Emergency Operation Center of the Indonesian Ministry of Health.
The data on the number of neurosurgical procedures performed between February 2, 2020, and April 10, 2020, at Dr. Sardjito General Hospital were obtained from the hospital\'s Division of Neurosurgery. Emergency cases were defined as head tumor and cerebrovascular problems with acute deterioration, shunt malfunction, cauda equina syndrome, acute obstructive hydrocephalus, or head and spinal cord trauma. In addition, urgent cases such as non--rapid-onset hydrocephalus, head tumor (with urgency depending on history), or problems with recent onset of upper motor neuron symptoms or signs (e.g., cervical or thoracic myelopathy) were included in the elective surgical planning. Preoperative procedures during the pandemic, including tests to detect COVID-19, are described in the Discussion section.
We included data from the previous months to help the reader differentiate the number of surgical procedures performed before and during the outbreak. Moreover, we compared the types of neurosurgical procedures performed at our center before and after the announcement of the first COVID-19 case in Yogyakarta. Finally, we also analyzed the changes in the number of outpatients visiting Dr. Sardjito General Hospital\'s neurosurgical clinic.
Results {#sec2}
=======
COVID-19 in Indonesia {#sec2.1}
---------------------
The number of confirmed cases in Indonesia had skyrocketed in the last few weeks. By the middle of April, the government had tested more than 30,000 samples (i.e., approximately 130 tests per 1 million population) and found more than 4800 confirmed cases, with 459 fatalities ([Figure 1](#fig1){ref-type="fig"} ).[@bib17] ^,^ [@bib19] The highest number of death was in the capital city, Jakarta, which had 2335 infected cases and 241 deaths at the midpoint of April 2020.[@bib20] Moreover, compared with other nations, Indonesia had a considerably higher case fatality rate (i.e., 9.56%).[@bib19] On the other hand, the mortality rate was rather low at roughly 1 death per 588,000 people.[@bib19] Figure 1The confirmed cases in 34 provinces in Indonesia as of April 14, 2020. DKI Jakarta and Special Region of Yogyakarta are highlighted owing to their status as the capital city of Indonesia and the province in which our hospital is located, respectively.
COVID-19 in the Special Region of Yogyakarta {#sec2.2}
--------------------------------------------
The infection rate in Yogyakarta was not as high as that seen in Jakarta. The first case was detected on March 13, 2020, in a 3-year-old child who had a history of a family trip to Depok, West Java. As of April 13, 2020, there were 55 confirmed cases, including 6 deaths.[@bib18] Sleman, where our hospital is located, was the region with the highest number of positive cases (i.e., 29 cases). Moreover, Yogyakarta has not been declared as an area with local transmission.[@bib21] Nonetheless, it is strongly suggested that each stakeholder remain vigilant.
The 4 Phases of the Pandemic at Dr. Sardjito Hospital {#sec2.3}
-----------------------------------------------------
We divided our experience during this period into 4 phases to ease the understanding of the nature of the pandemic ([Figure 2](#fig2){ref-type="fig"} ). Phase 1 (March 2--13, 2020) was the period of indigency. There were confirmed cases already in Indonesia during this period; however, knowledge of the disease was limited at that time. As a result, there was no clear diagnosis, and inadequate equipment to manage it. Phase 2 is defined as the period between the initial detection of the outbreak in Yogyakarta and when the government relaxes the strict regulations regarding the outbreak. Early in this phase, the number of cases outside Yogyakarta had soared, and the Indonesian Ministry of Health and the Indonesian Medical Association had instituted several measures in an effort to curb the impact.[@bib22] ^,^ [@bib23] In phase 3, the transition phase, the infection rate has subsided but the end of the outbreak has not been declared. Finally, phase 4 is defined as the period after the outbreak resolved. At the time of this writing, we have not yet entered the last 2 phases; we described them here to explain our expectations of the end of the pandemic.Figure 2Summary of our experiences during the outbreak, described in a four-phase model, in Dr. Sardjito General Hospital. Note that Phase 3 and 4 have not yet passed by our institution. Therefore, the purpose of stating those two phases is to explain our expectations.
Number of Neurosurgical Procedures Performed at Dr. Sardjito General Hospital {#sec2.4}
-----------------------------------------------------------------------------
Our center\'s staff comprises 6 consultant neurosurgeons and 23 residents capable of performing both emergency and elective procedures.[@bib8] In the last 9 weeks before the first confirmed case in Indonesia, an average of 4 emergency operations were performed each week. However, early in phase 2, there was a drop in the number of emergency operations, to an average of 2.4 per week ([Figure 3](#
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INTRODUCTION
============
Chlorhexidine is an antiseptic and disinfectant used against a broad of bacteria, viruses and fungi \[[@B1]\]. Since its introduction in 1954, it is used in the hospital settings for medical and surgical products and widely in over-the-counter products \[[@B1][@B2]\]. Many health professionals are unaware of its presence in different products, so it is often a 'hidden' allergen.
The most common allergic reactions described to chlorhexidine are delayed reactions (type IV hypersensitivity), T cell mediated, and occur after exposure to the antiseptic for topical use. Contact dermatitis is the most frequent manifestation \[[@B2][@B3][@B4]\]. Immediate reactions (type I hypersensitivity), have also been reported, but much less frequently, and symptoms can range from urticaria to anaphylaxis with a risk of cardiorespiratory arrest and death \[[@B2][@B3][@B4]\].
It has not been described cross reactivity between chlorhexidine and other antiseptic agents \[[@B1]\].
CASE REPORT
===========
A 75-year-old male with hypertension receiving beta-blocker and bladder cancer underwent transurethral tumor resection in 2014. Surveillance postsurgical cystoscopy under local anesthesia was performed every 6 months. During the 2nd procedure he developed generalized cutaneous pruritus with no other symptoms with spontaneous resolution after one hour. This reaction was interpreted as allergy to cefoxitin and it was recommended to avoid 2nd generation cephalosporins.
Twenty minutes after the 4th cystoscopy, he developed generalized urticaria, oropharyngeal tightening, dyspnea, hypotension (75/40 mmHg) and loss of consciousness with cardiorespiratory arrest. Cardiopulmonary resuscitation was initiated immediately with endovenous administration of adrenaline (1 mg), clemastine (2 mg) and hydrocortisone (200 mg) and orotracheal intubation with invasive ventilation. The patient recovered over the next 2 hours and was extubated on the same day.
The patient was referred to the immunoallergology outpatient clinic and a complete workup was performed. Local disinfection and anesthesia were performed with iodopovidone (Betadine, manufacturer, city, country) and lidocaine + chlorhexidine gel (Optilube, manufacturer, city, country). Prophylactic antibiotic therapy was performed only in 2nd procedure (cefoxitin) and ortho-phthalaldehyde (Cidex, manufacturer, city, country) was not used as cystoscope disinfectant.
The allergologic investigation revealed negative skin prick test (SPT) to iodopovidone and latex, and negative cutaneous tests (standard concentration \[[@B5]\] to PPL, MDM, amoxicillin, penicillin, cefoxitin). Specific IgE (sIgE) available (latex, penicillin, amoxicillin) were negative. Provocation tests to lidocaine and cefoxitin were negative.
SPT to chlorhexidine (2%) was strongly positive (11 mm × 10 mm wheal), with a positive sIgE - 4 kU/L (normal value: \<0.35 kU/L). [Table 1](#T1){ref-type="table"} summarizes the allergologic workup.
###### Allergologic workup carried out in our immunoallergology outpatient clinic
Reagent Skin prick test Intradermal test (standard ENDA concentration) \[[@B5]\] Specific IgE (RV \<0.35 kU/L) Challenge
------------------- ----------------- ---------------------------------------------------------- ------------------------------- --------------- -----------------
Antiseptic agents
Iodopovidone Negative Not advised Not available Tolerated
Chlorhexidine Positive Not advised 4 kU/L Contraindicated
Local anesthetics
Lidocaine Negative Not advised Not available Negative (SC)
Antibiotics
PPL and MDM Negatives Negative \- \-
Amoxicillin Negative Negative Negative Not performed
Penicillin Negative Negative Negative Not performed
Cefaclor Negative Negative Negative Not performed
Cefoxitin Negative Negative Not available Negative (IV)
Cefazolin Negative Negative Not available Not performed
Cefuroxime Negative Negative Not available Not performed
Other
Latex Negative \- Negative Tolerated
ENDA, European Network for Drug Allergy; RV, reference value; SC, subcutaneous; PPL, ; MDM, ; IV, intravenous.
We also performed a basophil activation test (BAT) using chlorhexidine digluconate 20% (1062 mg/mL) at 0.05%, 0.005%, 0.005%, and 0.00005% \[[@B6]\]. The basophil population was identified as HLA-DR-CD123+ CD203c+ cells and activation by CD63 expression. BAT was positive at 0.005%, 0.0005%, and 0.00005% with activation of 5.02%, 8.58%, and 11.9% and stimulation index of 3.22, 5.5, and 7.63 respectively ([Fig. 1](#F1){ref-type="fig"}).
{#F1}
The diagnosis of severe allergic reaction to chlorohexidine was confirmed. The patient was advised to avoid products containing chlorhexidine. Subsequent cystoscopy was uneventful using lidocaine gel as local anesthetic and iodopovidone as disinfectant. Moreover, he was informed to be aware of chlorhexidine as a component of over the counter products and the need to avoid them.
DISCUSSION
==========
The first case of anaphylaxis to chlorhexidine has been reported in 1984 in Japan \[[@B1][@B3]\]. Although rare, the number of clinical case reports of anaphylaxis (type I hypersensitivity) to this antiseptic is increasing. Odedra et al. \[[@B1]\] published that from 1994 to 2013, 65 case reports of chlorhexidine-related anaphylaxis were diagnosed. The majority was among surgical patients (urology and cardiothoracic) \[[@B6]\]. From 1984 to 2014, 36 cases of perioperative anaphylaxis to chlorhexidine were published \[[@B2]\].
Most reactions have been reported after application of chlorhexidine to damaged skin surfaces (wounds, burns, surgical incision); and to mucous membranes (urethra, eyes, nose) or after insertion of medical devices (central venous catheters, CVC) impregnated with chlorhexidine \[[@B4]\].
The prevalence of perioperative anaphylaxis range from 0.05%--2% and is increasing \[[@B2]\]. True incidence attributed to chlorhexidine is unknown, with several authors suggesting that is rare, but some studies referring incidences ranging from 5.5% to 8.8% \[[@B2]\]. Sharp et al. (Australia, 2016) \[[@B2]\] in a review to chlorhexidine-induced anaphylaxis in surgical patient (total of 68 anaphylactic reactions) showed that most frequent cases occur due to the presence of chlorhexidine in urinary catheter lubricant (n = 30 \[44.12%\]), CVC (n=24 \[35.29%\]) and topical solutions (n=11 \[16.18%\]).
It appears to occur more frequently in men with mean age of 58 years, previously reporting a mild cutaneous reaction on chlorhexidine exposure \[[@B1]\].
Patients rarely have history of atopic disease. The clinical presentation is variable. In most cases patients developed erythematous rash/urticarial at the time of reaction and hypotension, with some presenting cardiorespiratory arrest \[[@B1][@B2]\]. Bronchospasm is rarely reported \[[@B1][@B2]\]. Our patient was older than the mean presented, however the reaction occurred during a cystoscopy. This procedure and the severity of the symptoms were similar to the most commonly described.
To our knowledge, in the last five years (2014--2018), a total of 24 cases of chlorhexidine-related anaphylaxis were published ([Table 2](#T2){ref-type="table"}). The male gender is the most affected (83%). Mean age was 51 ± 15 years (range, 3--78 years) in agreement with what has already been described. The majority of the diagnosis was established through SPT. Twenty-one patients performed SPT, 20 were positive. The diagnosis in patient with negative SPT was determined by positive provocation test. Fifteen patients performed sIgE and were all positive (mean, 7.12 kU/L; range, 0.04--30 kU/L). Only 3 performed BAT and were positive.
###### Published cases of chlorhexidine-
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Introduction {#s0005}
============
*Pseudomonas* is a diverse genus known for their ubiquity in the environment and production of secondary metabolites [@bb0005]. Some pseudomonad strains are well-suited to be biocontrol agents [@bb0010], producing a wide range of bioactive metabolites. The general antibiotics produced by *Pseudomonas* include phenazine derivatives, pyoluteorin (Plt), pyrrolnitrin (Prn), hydrogen cyanide (HCN), 2,4-diacetylphloroglucinol (DAPG) and insect toxin [@bb0005]. The ability to release products with antimicrobial activity is the major mechanism by which pseudomonads suppress pathogens. Since the persistent use of chemical pesticides jeopardizes the health of some species like amphibians [@bb0015], biocontrol agents, including plant growth-promoting rhizobacteria (PGPR) such as *Pseudomonas chlororaphis* strains, become the focus of study [@bb0020].
*P. chlororaphis* is an important non-pathogenic biocontrol agent and is studied widely. Many strains show antagonistic activity against a number of disease-causing pathogens, such as *Fusarium oxysporum* f. sp. *radicis-lycopersici* [@bb0025], *Colletotrichum lagenarium*, *Phytophthora capsici*, *Pythium aphanidermatum*, *Pythium ultimum*, *Sclerotinia sclerotiorum*, *F. oxysporum* f. sp. *cucumerinum*, *Corticium sasakii*, *Rhizoctonia solani* [@bb0030] and *Gaeumannomyces graminis* var. *tritici* [@bb0025]. *P. chlororaphis* competitively colonizes the roots, and is distributed worldwide [@bb0030], [@bb0035], [@bb0040]. Additionally, *P. chlororaphis* is well known for its ability to adapt well to the environment using several mechanisms, including the degradation of aromatic compounds [@bb0035], complex regulatory systems [@bb0045], [@bb0050] and metabolism of nitrile compounds [@bb0055]. However, no systematic study of *P. chlororaphis* has been performed. It is important to study the shared and different traits of *P. chlororaphis* at the genomic level to better apply *P. chlororaphis* in agriculture and to promote the production of certain antibiotics. Whole-genome sequencing makes a comparative analysis among strains of *P. chlororaphis* possible. Up until January 2014, four *P. chlororaphis* strains have been whole-genome sequenced.
*P. chlororaphis* HT66 was isolated from the rhizosphere of rice in Shanghai, China by our group. This strain shows broad-spectrum resistance to plant pathogens and produces phenazine-1-carboxamide (PCN). *P. chlororaphis* GP72 was isolated from the green pepper rhizosphere by our group, and its genomic information was reported in 2012 [@bb0060]. This strain shows broad-spectrum antimicrobial activity, and can synthesize two phenazine derivatives, phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). *P. chlororaphis* 30--84 was isolated from the rhizosphere of wheat. Strain 30--84 is regarded as a biocontrol strain because of its ability to control take-all disease. It provides protection mainly by producing phenazines, such as 2-OH-PCA, 2-hydroxyphenazine and PCA [@bb0065]. *P. chlororaphis* O6 was isolated from the rhizosphere of field-grown wheat. It produces phenazines similar to strain 30--84 [@bb0070].
This study first reports the sequencing of the HT66 genome. To identify the shared and divergent genomic characteristics among *P. chlororaphis* strains, we performed a comparative genomic analysis of the four known *P. chlororaphis* strains, HT66, GP72, 30--84 and O6. There are 4833 conserved genes among the four strains and 733 strain-specific genes in the genome of HT66. We focus on their characteristics, such as biocontrol activities, rhizosphere colonization, biosafety, and production of phenazines. Our research provides a theoretical foundation to develop and improve the antagonistic activities of *P. chlororaphis* for agricultural applications, as well as to use *P. chlororaphis* to produce phenazines with high-yield.
Results and discussion {#s0010}
======================
General genome features and comparative genomes {#s0025}
-----------------------------------------------
The general features of the four *P. chlororaphis* genomes are summarized in [Table 1](#t0005){ref-type="table"}. The genome sizes of HT66, GP72, 30--84 and O6 range from 6.66 to 7.30 Mb, with the number of CDSs ranging from 5869 to 6455. Compared with other pseudomonads, such as *Pseudomonas fluorescens* and *Pseudomonas putida*, these four genomes have higher GC contents [@bb0075], [@bb0080], suggesting that this is a general characteristic of *P. chlororaphis*.
The COG database was used to functionally categorize predicted proteins [@bb0085], and we made a comparison of COG categories among the four strains (HT66, GP72, 30--84 and O6). The results are shown in [Fig. 1](#f0005){ref-type="fig"}. The COGs for the four strains showed highly similar distributions, especially COGs F and J, suggesting that the four strains have comparable biological niches. The percentage of genes in COG H was slightly higher in GP72 (approximately 3.95% of the total genes with COG annotations) than in the other three strains (3.77% in HT66, 3.78% in 30--84 and 3.78% in O6), suggesting that genes associated with coenzymes take more proportion in the genome of GP72.
We established a phylogenetic tree for completely sequenced representative strains of pseudomonad based on multilocus sequence analysis (MLSA) ([Fig. 2](#f0010){ref-type="fig"}). The tree showed that these four strains of *P. chlororaphis* fall into the same clade. The most closely related pseudomonad species to this clade was *P. fluorescens*, followed by *Pseudomonas syringae*. *P. chlororaphis* may share a more recent common ancestor with *P. fluorescens* than with *P. syringae*.
Conserved and specific regions in the genome can be identified through global alignments. BLASTatlas gives an overview of the whole genome homology, and the reference genome can be compared at the gene and/or protein level against many genomes [@bb0090]. In this study, the reference genome of HT66 was compared to the other three query genomes ([Fig. 3](#f0015){ref-type="fig"}).
mGenomeSubtractor performs a mpiBLAST based on in silico subtractive hybridization to identify conserved and strain-specific proteins. In this analysis, proteins with homology (H) values of less than 0.42 or more than 0.81 are defined arbitrarily as strain-specific or conserved, respectively [@bb0095]. The degree of protein conservation among HT66 and the other three genomes were determined by blastp based on homology value. The distribution of homology values for the 6455 predicted CDSs from *P. chlororaphis* HT66 compared with the other three genomes is shown in [Fig. 4](#f0020){ref-type="fig"}A. Genes conserved among all of four *P. chlororaphis* isolates comprised 4833 CDSs, representing 74.9%, 79.3%, 82.3% and 77.5% of the coding capacity in HT66, GP72, 30--84 and O6, respectively. Comparisons between HT66 and the other three strains (GP72, 30--84, and O6) revealed that 730 CDSs, 11.3% of the total coding capacity, were HT66 specific (E-value \< 10^− 5^) ([Fig. 4](#f0020){ref-type="fig"}B). Among the 730 genes, we found that 192 genes formed 18 gene clusters. The 192 genes were individually blasted on NCBI (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>). Four clusters showed high homology with *Pseudomonas protegens* Pf-5. One may be related to the metabolism of polysaccharides, one is similar with hemophore and may be responsible for the acquisition of heme, one may represent an *ofa* cluster responsible for the production of orfamide A which was first found in *P. chlororaphis*, and one\'s function remained undetermined. Two additional clusters showed high homology, with one being similar to *P. putida* HB3267, which may be related to phage, and the other having homology with *Pseudomonas* sp. UW4, which has unknown functions.
Genomic islands (GIs) {#s0030}
---------------------
GIs were identified by IslandViewer [@bb0100]. Since the genome of GP72 contains many contigs, the GI predictions were not reliable (data not shown). The genome of HT66 contains 23 putative GIs ([Fig. 5](#f0025){ref-type="fig"}A), which
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Full text
=========
Progesterone is critical in mammary gland development. Breast cancer evolves from normal tissue through increasingly abnormal cellular changes that include increased expression of progesterone receptor (PR), and PR is an established marker of response to endocrine therapy. PR is expressed as two proteins (PRA and PRB) with different functions, and *in vitro* evidence reveals PRA to inhibit PRB function. This suggests that PRA may repress progesterone action and that the ratio of PRA:PRB may be an important determinant in tissue sensitivity to ovarian steroid hormones.
This study examined the expression of PRA and PRB proteins in normal breast tissue (*n* =13) during the menstrual cycle, and in premalignant (*n* =45) and malignant (*n* =39) breast tissues, to determine differences in relative isoform expression.We used dual immunofluorescent histochemistry on formalin-fixed, paraffin-embedded tissue sections using mouse monoclonal antibodies that bind to either PRB or PRA.
In most normal breast cases PR staining was confined to scattered epithelial cells expressing equivalent levels of PRA and PRB. However, 50% of cases in the luteal phase (*n* =6) showed reduced PRA expression. In proliferative premalignant lesions without atypia (PDWA, *n* =15), there was a marked increase in intensity and number of cells expressing PR, but inter-cell homogeneity was maintained. Atypical proliferative benign lesions (ADH, *n* =15; DCIS, *n* =15), showed high levels of both PRA and PRB expression with notable inter-cell heterogeneity in relative isoform content. This was also observed in malignant breast tumours (*n* =39). Furthermore, breast tumours expressing an overall predominance of one isoform were associated with features of higher histological grade.
In conclusion, our results show a change from inter-cell homogeneity of PRA:PRB in normal tissue to extensive heterogeneity in the malignant state, suggesting a progressive loss of control of relative PRA and B expression that may occur early in cancer development and may eventually be associated with features of poorer prognosis.
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By the end of 2018, nearly 8000 complete tailed phage genomes were published online and a further 22,000 partial genomes were stored in databases gathered under the umbrella of the International Nucleotide Sequence Database Collaboration ([@B34]; [@B49]). The classification of this massive group is the formal responsibility of the Bacterial and Archaeal Viruses Subcommittee of the International Committee on the Taxonomy of Viruses (ICTV). In recent years, we (the Subcommittee) have focused on classifying newly described phages into species and genera ([@B41], [@B40]; [@B3]; [@B37]; [@B4]). However, once our attention shifted toward higher order relationships, we found that the ranks currently used in phage taxonomy (species, genus, subfamily, family, and order) are no longer sufficient for the description of phage diversity. The limitation is particularly acute in the case of the order *Caudovirales*---arguably the most abundant and heterogeneous group of viruses ([@B50]; [@B53]; [@B47]). Indeed, the diversity of caudoviruses surpasses that of any other virus taxon. A recent analysis of the gene content of the dsDNA virosphere demonstrated that the global network of dsDNA viruses consists of at least 19 modules, 11 of which correspond to caudoviruses ([@B30]). Each of the eight remaining modules encompasses one or more families of eukaryotic or archaeal viruses. Consequently, each of the 11 caudovirus modules could be considered a separate family. Despite this remarkable diversity, the vast majority of caudoviruses is classified into three families *Myoviridae*, *Podoviridae*, and *Siphoviridae*, which were historically established on morphological features, forming an artificial classification ceiling. These observations prompted us to work on the update of current taxonomic order within the *Caudovirales* order.
As an initial step of this major reclassification of the tailed phages, we, the members of the Subcommittee proposed creation of two novel families corresponding to distinct modules revealed in the abovementioned gene-sharing network analyses ([@B30]; [@B11]). The first of these, named *Ackermannviridae*, encompasses phages related to *Salmonella virus ViI* that were formerly assigned to the genus *Viunalikevirus* ([@B2], [@B5]). In the present work, we focus on the second new family, named *Herelleviridae*. The phages belonging to this new family are large myoviruses related to the Bacillus phage SPO1, Staphylococcus phage Twort, Staphylococcus phage K, Listeria phage P100, and Enterococcus phage $\documentclass[12pt]{minimal}
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}{}$\varphi$\end{document}$EF24C. Most of these viruses were previously grouped in the *Spounavirinae* subfamily or recognized as related to it. When this subfamily was first devised ([@B40]), the unifying characteristics of its members included: the hosts belong to the bacterial phylum *Firmicutes*; strictly virulent lifestyle; myovirion morphology (i.e., icosahedral capsid and long contractile tail); terminally redundant, nonpermuted dsDNA genome of 127--157 kbp in length; and "considerable amino acid similarity" ([@B35]). The strictly virulent lifestyle of these viruses has been somewhat disputed ([@B54]; [@B60]) but still remains a rule of thumb for inclusion into the taxon. Since the initial description of the subfamily, the number of its members has grown significantly, and its taxonomic structure has been contested several times ([@B35]; [@B10]; [@B30]; [@B37]; [@B11]; [@B4]). Thus, we wanted not only to delineate a new family but also resolve its internal structure.
Unfortunately, there is no one-size-fits-all method for the classification of viruses at all taxonomic ranks. Virus taxonomy has always suffered from the lack of universal marker genes that could be used for phylogenetic reconstruction of the evolutionary relationships. Additionally, differing mutation rates between viral lineages, horizontal gene transfer, and genomic mosaicism limit usefulness of many of the available phylogenetic and phylogenomic methods that have become the gold standard in evolutionary biology ([@B18]; [@B44]). Thus, our strategy for reclassification included a plethora of classification tools that employ very different approaches. Our analyses ranged from coarse-grained, high-throughput, holistic clustering methods where similarity is computed from comparison of all viral genes \[vContact, GRAViTy ([@B11]; [@B7]; [@B8])\] to detailed genome and proteome comparisons \[Victor, Dice, GOAT and Phage Proteomic Tree ([@B52]; [@B45]; [@B44])\] and individual gene phylogenies \[IQtree ([@B46])\]. This multifaceted approach allowed us to gradually descend from the definition of the new family to the study of its internal structure. Interestingly, despite the diversity of the applied methods their results turned out to be complementary and predominantly concordant. All methods painted a robust picture of the new family as a distinct and diverse taxon and supported the same general scheme for its structure ([Table 1](#T1){ref-type="table"}).
######
New classification of the 93 spounaviruses and spouna-like viruses in the new family *Herelleviridae*$\documentclass[12pt]{minimal}
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*Herelleviridae* *Bastillevirinae* *Agatevirus* *Bacillus virus Agate*, *Bacillus virus Bobb*, *Bacillus virus Bp8pC* (Bp8p-T)
*Bequatrovirus* (formerly *B4virus*) *Bacillus virus AvesoBmore*, *Bacillus virus B4* (B5S), *Bacillus virus Bigbertha, Bacillus virus Riley, Bacillus virus Spock, Bacillus virus Troll*
*Bastillevirus* *Bacillus virus Bastille, Bacillus virus CAM003, Bacillus virus Evoli, Bacillus virus HoodyT*
*Caeruleovirus* (formerly *Bc431virus*) *Bacillus virus Bc431*, *Bacillus virus Bcp1*, *Bacillus virus BCP82*, *Bacillus virus JBP901*
*Nitunavirus* (formerly *Nit1virus*) *Bacillus virus Grass*, *Bacillus virus NIT1*, *Bacillus virus SPG24*
*Tsarbombavirus* *Bacillus virus BCP78* (BCU4), *Bacillus virus TsarBomba*
*Wphvirus* *Bacillus virus BPS13*, *Bacillus virus Hakuna*, *Bacillus virus Megatron* (Eyuki), *Bacillus virus WPh*, *Bacillus virus BPS10C*
Unassigned *Bacillus virus Mater*, *Bacillus virus Moonbeam*, *Bacillus virus SIOphi*
*Brockvirinae* *Kochikohdavirus* *Enterococcus virus ECP3*, *Enterococcus virus EF24C* (phiEFC24C-P2), *Enterococcus virus EFLK1*
Unassigned *Enterococccus virus EFDG1*
*Jasinskavirinae* *Pecentumvirus* (formerly *P100virus*) *Listeria virus A511*, *Listeria virus P100*, *Listeria virus List36*, *Listeria virus LMSP25* (LMTA-57, LMTA-94), *Listeria virus LMTA148*, *Listeria virus LMTA34*, *Listeria virus LP048*, *Listeria virus LP064* (LP-125), *Listeria virus LP083-*2 (LP-124), *Listeria virus AG20*, *Listeria virus
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1.. Introduction
================
Methionine is an essential amino acid for animals and is involved in numerous metabolic processes \[[@b1-sensors-10-03562]--[@b4-sensors-10-03562]\]. In addition to being a building block in protein synthesis, methionine, after being transformed into S-adenosylmethionine, serves as a methyl donor in transmethylation reactions involved in the biosynthesis of lipids, biotin, and polyamines \[[@b5-sensors-10-03562]\]. Since methionine cannot be synthesized *de novo* in mammal cells, its supplementation in animal diets is required to provide optimal growth and physiological performance of the animals. Plant proteins, however, are poor in methionine and its optimal level in animal diets is provided by supplementation with crystalline methionine \[[@b6-sensors-10-03562]\] or methionine analogs such as 2-keto-4-(methylthio) butyric acid \[[@b7-sensors-10-03562]\] and hydroxymethionine \[[@b8-sensors-10-03562],[@b9-sensors-10-03562]\]. Therefore, timely and accurate pre-quantification of this amino acid in feed ingredients is necessary to improve cost efficiency of feed formulation and prevent its overdosage. According to Klasing and Austic \[[@b10-sensors-10-03562]\] and Baker \[[@b11-sensors-10-03562]\], excess of individual amino acids due to feed mixing errors can be potentially harmful to the animal, with methionine considered to be the amino acid possessing the highest toxicity. Feed compounds such as cysteine, vitamin B~12~, arginine, choline, and sulfate that are related to methionine metabolism can affect the apparent methionine requirement of animals and additionally complicate the estimation of the optimal dosage of this amino acid in animal diets \[[@b12-sensors-10-03562]\].
Chemical assays including high performance liquid chromatography (HPLC) are commonly used to quantify methionine level in feed ingredients. The analysis, however, involves pretreatment of the samples with performic acid followed by acid digestion \[[@b13-sensors-10-03562],[@b14-sensors-10-03562]\]. The procedure results in a complete protein degradation and liberation of methionine which differs from protein digestion under physiological conditions. Feed-derived methionine, which is available to animals to assimilate, can be more accurately estimated by animal or microbial assays which are considered to correspond more directly to the physiological needs of animals \[[@b15-sensors-10-03562],[@b16-sensors-10-03562]\]. Although considered standard, animal assays are laborious, expensive, and time consuming \[[@b17-sensors-10-03562]--[@b19-sensors-10-03562]\]. The types of animal assays that have been used for quantifying methionine availability have been reviewed extensively by Froelich and Ricke \[[@b18-sensors-10-03562]\] and will not be discussed further in the current review. Microbial assays appear to be easier and more affordable for routine analysis. Rapid development and recent improvements in molecular techniques allow for constructing successful and accurate amino acid biosensors via more precise genetic targeting of specific genes in microbial cells. This review discusses methionine biosynthesis and regulation in *Escherichia coli* and the potential of genetically modifying this microorganism into practical whole cell biosensors for methionine bioavailability quantification.
2.. Microbial Biosensors
========================
Recently, numerous microbial biosensors have been created and used in medical diagnostics, food technology, biotechnology, and environmental monitoring. Microbial biosensors couple a biological element (enzymes, viable or non-viable microbial cells) and a transducer or a device which allows for rapid, accurate and sensitive detection of target analytes \[[@b20-sensors-10-03562],[@b21-sensors-10-03562]\]. Their popularity is due to highly specific selectivity to the substrate of interest, relative inexpensiveness, and portability \[[@b22-sensors-10-03562],[@b23-sensors-10-03562]\]. Versatile microorganisms have proven to be useful in development of biosensors. The bacterium *Vibrio harveyi* and *Mycena citricolor*, a fungal microorganism, demonstrated high sensitivity for detecting cyanide and sodium monofluoroacetate respectively \[[@b24-sensors-10-03562]\]. A microbial biosensor for sensitive, selective, rapid, and direct determination of *p*-nitrophenyl (PNP) -substituted organophosphates was developed based on PNP oxidation metabolic pathway of the *Moraxella* sp. \[[@b25-sensors-10-03562]\]. *Flavobacterium* sp. were employed for development of a biosensor for methyl parathion pesticide \[[@b26-sensors-10-03562]\]. The variety and versatility among microbial species useful in the construction of biosensors for environmental application is more extensively discussed elsewhere \[[@b20-sensors-10-03562],[@b27-sensors-10-03562]\] and will not be further discussed here.
In the food industry, microbial biosensors, derived from *Gluconobacter oxydans* and yeast have gained popularity for detecting total sugars, sucrose, and ethanol \[[@b28-sensors-10-03562],[@b29-sensors-10-03562]\]. Respiratory activities of *Gluconobacter oxydans* DSM 2343 cells, immobilized on chitosan, were used in the quantification of glucose. A linear relationship (R^2^ = 0.99) between sensor's response and substrate concentration was achieved in the range of 0.05 to 0.1 mM glucose \[[@b23-sensors-10-03562]\]. By using a microbial biosensor based on immobilized *Saccharomyces ellipsoideus* yeast cells, Rotariu *et al*. \[[@b29-sensors-10-03562]\] were able to determine ethanol concentrations up to 50 mM in alcoholic beverages including two types of beer, vodka, and cognac. The comparison to the chemical assay used for the analyses of the same analyte revealed good correlation (correlation coefficient 0.998) between the biosensor and the spectrometric method. An *Acetobacter pasteurianus*-based biosensor has been proposed as an alternative to chemical methods available for quantifying lactate which is used as an indicator for specific fermentations activities including those of milk, yogurt, and wine \[[@b30-sensors-10-03562],[@b31-sensors-10-03562]\]. *Aeromonas phenologenes*-, *Pseudomonas fluorescens*-, and *Bacillus subtilis*-based biosensors were proposed to serve as alternatives in quantification of amino acids including tyrosine, tryptophan, and glutamate \[[@b32-sensors-10-03562],[@b33-sensors-10-03562]\].
3.. *E. coli* as a Biosensor
============================
Among all microorganisms, *E. coli* is one of the most highly investigated bacteria for the purposes of biosensor fabrication. It is easy to cultivate, with simple nutritive requirements and rapid growth \[[@b34-sensors-10-03562]\]. *E. coli* is a Gram negative microorganism with very well known genetics which enables the construction of a wide variety of biosensors \[[@b20-sensors-10-03562],[@b21-sensors-10-03562]\]. The complete *E. coli* genome has been sequenced and the information deposited to the National Center for Biotechnology Information (NCBI) \[[@b35-sensors-10-03562]\]. Thus, each DNA sequence of interest is routinely available to the public and can be used for a wide range of potential further genetic manipulations. In promoter-based *E. coli* biosensors, a gene promoter, inducible by the analyte of interest, is fused to a reporter that generates a signal in response to the analyte that can be easily monitored and measured. A strong SOS *E. coli* promoter fused to a *lux* gene resulted in the development of a construct which served in a dose-dependent detection of 6 genotoxic chemicals including mitomycin C, *N*-methyl-*N*-nitro-*N*-nitrosoguanidine, nalidixic acid, dimethylsulfate, hydrogen peroxide, and formaldehyde \[[@b36-sensors-10-03562]\]. An *E. coli* BL21 DE3 (RIL) biosensor strain displayed a specific response and high sensitivity to different aromatic aldehydes. The response was measured by monitoring the fluorescence of a reporter (green fluorescent protein) fused to an alcohol dehydrogenase inducible promoter (*Sso2536adh*) belonging to the archaeon *Sulfolobus solfataricus* \[[@b37-sensors-10-03562]\]. A plasmid-borne transcriptional fusion between the *E. coli* nitrate reductase (*narG*) promoter and the *Photorhabdus luminescens lux* operon was used to generate a modified *E. coli* with a highly bioluminescent phenotype in the presence of nitrate that enabled the detection of nitrate to a level of 5 × 10^−5^ mol L^−1^ (0.3 ppm) \[[@b38-sensors-10-03562]\]. Following the same approach, biosensors for toluene, arsenite and arsenate, and lead have also been generated \[[@b39-sensors-10-03562]--[@b41-sensors-10-03562]\].
In addition to environmental testing and analyses, *E. coli*-based biosensors were found to be useful in the food industry as well. *E. coli* derived β-galactosidase, glucose oxidase, and horseradish peroxidase were immobilized on a glassy carbon electrode to generate a biosensor for quantification of lactose in raw milk \[[@b42-sensors-10-03562]\]. Simultaneous determination of various mono- and disaccharides was
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Introduction {#s1}
============
An ecological trap arises when an artificial habitat is introduced into a natural environment, attracts animals to its vicinity and the subsequent association leads to negative ecological consequences for the animal [@pone.0015646-Battin1]. Animals may prefer an artificial habitat over natural habitats if it mimics the set of ecological cues which signify a good quality habitat, despite other ecological processes rendering the habitat of low quality and leading to poorer reproduction or survival. Robertson and Hutto [@pone.0015646-Robertson1] suggest that ecological traps derive from habitat alteration that operates in one of three ways; (1) increasing the attractiveness of an environment by enhancing the set of cues that animals recognise as attractive; (2) decreasing the suitability of a habitat; or (3) doing both (1) and (2) simultaneously. Alternatively, artificial habitats of high quality, where individuals increase in condition, reproduce better or have improved survival, all of which may ultimately lead to positive population growth rates, act as population sources.
Objective testing of whether ecological traps exist is well embedded in the literature concerning terrestrial systems [@pone.0015646-Robertson1], yet few studies have investigated whether they exist in marine environments. Artificial structures that aggregate fish (fish aggregation devices; FADs) have been previously suggested to act as ecological traps by acting as a super-stimulus and misleading fish to make inappropriate habitat selections [@pone.0015646-Hallier1]. Coastal sea-cage fish farms are widespread artificial structures in coastal waters, producing over 2.5 million tons of fish each year [@pone.0015646-FAO1]. They have previously been described as analogous to FADs, attracting and aggregating large assemblages of wild fish in their immediate vicinity [@pone.0015646-Dempster1]. Attraction and aggregation of tons of wild fish to the immediate surrounds of Norway\'s coastal salmon farms [@pone.0015646-Dempster1], [@pone.0015646-Dempster2] meets Robertson and Hutto\'s [@pone.0015646-Robertson1] first condition for the formation of an ecological trap. However, whether the fish farm area is poorer in habitat quality for wild fish than natural adjacent habitats, thus meeting Robertson and Hutto\'s [@pone.0015646-Robertson1] second condition, remains unknown. Relative habitat quality is a key component in determining the extent to which fish farms may act as population sources or ecological traps for wild fish.
Along the Norwegian coastline, 1198 coastal sea-cage salmonid farm concessions used 1.2 million tons of fish food to produce 829 000 t in 2008 [@pone.0015646-Norwegian1]. Farming is concentrated in particular fjords, with farms spaced several kilometres apart. Wild saithe are the most abundant species associated with salmon farms within fjord systems [@pone.0015646-Dempster2], [@pone.0015646-Dempster3]. Saithe use farms as a loose network of preferred habitats, moving repeatedly among farms and remaining resident at specific farms for weeks to months [@pone.0015646-Uglem1]. Atlantic cod are also attracted to fish farms in number [@pone.0015646-Dempster2] and may reside in their vicinity for months at a time [@pone.0015646-Uglem2], [@pone.0015646-Uglem3]. Attraction of wild fish to salmon farms is likely to have a range of fitness consequences due to the modified environment fish farms induce, both in the altered trophic network around farms and the close proximity of hundreds of thousands to millions of farmed salmonids. Diet, body fat content, fatty acid composition and parasite loads may all be altered when wild fish closely associate with farms [@pone.0015646-Diamant1], [@pone.0015646-FernandezJover1], [@pone.0015646-FernandezJover2]. Simultaneous analysis of this suite of factors at an extensive number of locations is required to resolve whether farms function as population sources or ecological traps [@pone.0015646-Chalfoun1].
Here, we tested the hypotheses that the diets, indices of condition and parasite loads of cod and saithe associated with salmon farms differed from those of fish present at locations distant from salmon farms. To ensure broad generality of the results, we sampled fish in three intensive fish farming areas along the latitudinal extent of salmon farming in Norway (59°N to 70°N).
Materials and Methods {#s2}
=====================
Study locations and experimental design {#s2a}
---------------------------------------
Saithe and cod were sampled from the three salmon farming areas (Ryfylke 59°N, Hitra 63°N and Øksfjord 70°N) from the same Atlantic salmon (*Salmo salar*) farms and during the same season (summer) as aggregation sizes were determined [@pone.0015646-Dempster2]. Within each salmon farming area, fish were sampled at three farms and two to six non-farm control locations ([Fig. 1](#pone-0015646-g001){ref-type="fig"}). Farm-associated fish were captured within 5 m of cages containing salmon. The number of non-farm locations varied from two to six depending on the area and species of wild fish sampled (Saithe: Ryfylke 2, Hitra 4, Øksfjord 3; Cod: Ryfylke 3, Hitra 6, Øksfjord 3). Control fish were sampled from locations 4 to 20 km distant from the nearest farm ([Fig. 1](#pone-0015646-g001){ref-type="fig"}) to limit the possibility of sampling fish at non-farm locations that had interacted recently with a farm. The 4 km minimum limit was based on telemetry-derived observations of the predominant movements of wild cod and wild saithe [@pone.0015646-Uglem1], [@pone.0015646-Uglem2], [@pone.0015646-Uglem3] in the vicinity of fish farms.
{#pone-0015646-g001}
All fish were sampled with standardised hook and line fishing gear. Collections by hook and line select for feeding fish, but are more suitable for accurate counts of the number of external parasites than other catch methods such as trawling or gill nets which may remove external parasites through abrasion. Moreover, capture by any other method beside the cages at fish farms is impractical due to possible negative interactions of fishing gear with fish farming structures. Collections were made at each location from June to September 2007 during the period where feed input to salmon farms is high [@pone.0015646-Norwegian1].
Size, diet and condition indices {#s2b}
--------------------------------
Upon capture, fish were immediately examined for the presence of external parasites (see parasite sampling section below) and then placed on ice. Fish were weighed and measured to the nearest 0.5 cm (fork length; FL). Each fish was dissected and liver and gonad weights were obtained. Sex for each fish was determined by macroscopic examination of the gonads. In gadoid species, such as Atlantic cod, lipids are stored primarily in the liver [@pone.0015646-Lambert1] making liver weight a measure of spawner quality [@pone.0015646-Marshall1]. Therefore, we calculated three condition indices: body condition, the hepatosomatic index and the gonadosomatic index. Fulton\'s condition index (FCI) was calculated with the formula: FCI = (W/FL^3^)×100, where W = wet weight--stomach content weight and FL = fork length (cm). The hepatosomatic index (HSI) was calculated using the formula: HSI = (LW/W)×100, where LW = liver weight and W = wet weight--stomach content weight. The gonadosomatic index (GSI) was calculated using the formula: GSI = (GW/W)×100, where GW = gonad weight and W = wet weight--stomach content weight.
Stomach contents from the foregut were examined and prey species were identified to the lowest taxonomic level possible and weighed. Prey categories were later reduced to 11 for saithe (waste salmon feed, Brachyura, Osteichthyes, Polychaeta, Caridea, zooplankton, Phaeophyceae, Bivalvia (principally *Mytilus* sp.), Ophiuridae, Hydroida (principally *Ectopleura larynx*), and other organic matter) and 13 for cod (waste salmon feed, Brachyura, Osteichthyes, Polychaeta, Caridea, Phaeophyceae, Bivalvia (*Mytilus* sp.), Holothuria, Ophiuridae, Echinoidea, Octopoda, Amphipoda and other organic matter).
Parasite sampling {#s2c}
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1. Introduction
===============
Type 1 diabetes (T1D) is an incurable autoimmune disease that destroys the pancreas's ability to naturally produce insulin, a hormone necessary for the absorption and storage of glucose. Failure to adequately monitor and manage blood glucose and insulin levels can lead to major vascular complications and organ damage from hyperglycemia \[ [@JR0047-1] \] on the one hand and seizures, loss of consciousness, and death from hypoglycemia on the other \[ [@JR0047-2] \]. In the United States, over a million adults live with T1D, and recent estimates are that over 200,000 youth (individuals under the age of 20) live with it as well \[ [@JR0047-3] \]. While the United States Food and Drug Administration has recently approved an "artificial pancreas" device that will automatically manage blood sugar and release insulin into the bloodstream of a person with diabetes \[ [@JR0047-4] \], its relative newness, cost, and the requirement that patients be at least 14 years old limit its accessibility to a substantial portion of the T1D population. An alternative that some diabetics have pursued is the creation of their own insulin management system using resources such as OpenAPS (Open Artificial Pancreas System, openaps.org). To date, users of OpenAPS have self-reported improved glycohemoglobin levels and increases in the amount of time that they were in a desired blood-sugar range. These same users observed, however, that the amount of technical know-how and efforts needed to create and maintain their self-developed artificial pancreas system are likely substantial obstacles that will limit broader adoption \[ [@JR0047-5] \].
As such, diabetes management for most individuals with T1D involves frequent manual blood tests to measure the amount of sugar in one's blood coupled with the manual administration of insulin multiple times during the day. In some cases, devices such as insulin pumps or continuous glucose monitors (CGM) may be used. Such devices typically obtain and store several days' worth of specific data about how much insulin was administered and the blood sugar level readings for a patient over certain time intervals. Regardless of whether readings and administrations are done manually with a glucometer or with a pump or CGM, there are many measures being obtained by a person with diabetes even during a single day. What happens to those measures depends on the individual collecting the information and the technological infrastructure that they use, with some data being stored briefly mentally and others on proprietary cloud systems.
This current paper is a case study showing the comparative realizations that were made by a T1D-affected family upon examination of aggregated blood sugar information obtained using two data collection methods. One method was manual testing involving regular finger pricking and use of a separate glucometer accompanied by systematic handwritten logging. The other involved use of a CGM device. The diabetic individual whose data are examined and shared in this report is the third author (Chris), who explicitly asked (and with consent of his parents, as he was 9 years old at the time of this writing) that his identity be disclosed.
2. Objectives
=============
The primary objective of this report is to illustrate what regularities are detected in aggregate visualizations of different blood glucose data collection methods of end users. In this case, the end users are the child with diabetes and his parents. One data collection method involved manual data collection and recording over the course of three and a half years, and the other involved data collected by a CGM. With this family, manual data collection turned out to exhibit blind spots due to daily family routines and transition to formal schooling that were only fully realized when data were aggregated. On the other hand, automated data collection created more data but tended to invite only examinations of data from preceding hours but not aggregate analysis for recurring daily patterns.
3. Methods
==========
The quantitative data for this report were obtained by the second and third author and other family members prior to any plans for a research study. As such, this is a post hoc analysis of existing data. Since those who collected data are co-authors and written records exist as a paper trail, we are able to describe the data collection process with some accuracy.
The diabetic individual, Chris, had been diagnosed at approximately 20 months of age. His parents (including his father, the second author), began to prepare journals of Chris's blood sugar data from the time of his diagnosis (August, 2010). The written log initially included records of how much of which insulin type was administered and when along with a detailed specification of what foods were eaten and the equivalent number of carbohydrates that would lead to an increase in blood glucose. The log (see ► [Figure 1](#FI0047-1){ref-type="fig"} ) served as the sole record of Chris's blood sugar information in the family, and served as a boundary object \[ [@JR0047-6] \] around which Chris, his parents, and ultimately health care practitioners, would discuss recent trends observed over past days and make necessary adjustments. The records that were manually recorded by Chris's family were considered to be quite thorough relative to many other patient families and were important resources for both the parents and health care providers to examine when planning meals, activities, and insulin dosage.
{#FI0047-1}
The journals changed in structure and content over time as carbohydrate and serving size information was internalized by family members, and they stopped logging that information. Eventually, only blood sugar levels and times of day were recorded. As part of another study related to educational and learning activities that take place by way of self-quantification in and out of the classroom \[ [@BR0047-7] -- [@JR0047-9] \], these journals were introduced to the first author. With the consent of Chris and his parents, the glucose measurements were digitized by the first author and research assistants. One month of data (March, 2013) was missing. In total, there were 7,437 entries from all the journals from August 2010 to February 2014. Details about the numbers of readings are provided in ► [Table 1](#TB0047-1){ref-type="table"} .
######
Number of digitized handwritten glucose data measurements per year.
**Year** **2010** **2011** **2012** **2013** **2014**
------------------------------------------ ---------- ----------- ----------- ----------- --------------------
**Total Glucose Entries** 617 2595 2184 1899 142
**Time Period** 4 months 12 months 12 months 11 months 1 month and 5 days
**Estimated number of readings per day** 5.14 7.21 6.07 5.75 4.06
Family circumstances changed in February, 2014 that made continued handwritten journaling difficult (February, 2014). Blood sugar readings were still obtained but not retained in a permanent record. In summer of 2014, Chris's parents were able to purchase a CGM device and switched to it as the primary source and repository of blood glucose information. None of the CGM data were collected with the intention of performing a pre-planned self-experiment or to be used for third party analysis.
The CGM data were obtained through a Dexcom G4 Platinum (► [Figure 2](#FI0047-2){ref-type="fig"} ), which was configured to obtain a blood glucose reading every five minutes. The Dexcom unit was linked to Chris's parents' smart-phones so that they could be alerted to extraordinary deviations from desired blood sugar ranges (typically between 80 and 180 mg/dL). Because several dozen data points were accessible to Chris and his parents on an ongoing basis, these blood sugar data were not manually recorded by any individual. The information available when they were checking immediate blood sugar levels and the previous few hours was deemed adequate for routine in-the-moment decision making on their part with respect to deciding on Chris's insulin dosage.
{#FI0047-2}
Due to some misunderstandings about how and for how long data were stored by the device, the readings from the Dexcom were not downloaded to be viewed or retained separately by the family until October of 2016, when the family switched endocrinologists (due to retirement of their longstanding provider) and were explicitly requested by the new attending endocrinologist to export the Dexcom data and bring specific reports to their first consultation. For the purposes of this paper, 14 days of CGM data are considered. That is the equivalent of 6,720 blood sugar data points. This number of days is considered for two reasons. One is that after preparing and reviewing an aggregate visualization of these fourteen days, Chris's family noticed clear evidence of a regularity that they wanted to change for Chris's benefit. Thus, those 14 days were prior to any intervention that was meant to mitigate the detected regularity. Secondly, this number of data points is comparable to the number of data points obtained in the manual record, albeit over a different time scale and density. By comparing roughly equal numbers of manually and CGM collected data points, we feel that the density of sampling and the inferences made from large numbers of visualized data points becomes foregrounded in our analysis rather than the absolute number of measurements.
In addition to the aforementioned numerical data, the first author met with Chris and his family on three occasions for 1--
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1. Introduction {#sec1}
===============
According to the commentary on the 2008 WHO classification of lymphoid tumors, follicular lymphoma *in situ* (FLIS) refers to lymph nodes with a background of hyperplastic germinal centres harbouring distinct areas with Bcl-2 overexpression in centroblasts and centrocytes \[[@B1]\]. In FLIS, there is a B-cell population with immunophenotypic and genotypic features of follicular lymphoma; however, these B-cells are exclusively localised to germinal centres in morphologically reactive lymph nodes \[[@B2]\].
FLIS was first recognised in 2002 \[[@B3]\]; however, it is not presently clear cut whether this condition is a precursor to full blown follicular lymphoma (FL) \[[@B4]\]. In a series of 34 cases of FLIS identified by Jegalian et al., six had prior or concurrent FL and five had FLIS composite with another lymphoma. Of patients with negative staging at diagnosis and available followup (twenty-one patients), only one developed FL \[[@B5]\].
We present a case recently diagnosed in a middle-aged African lady which to our knowledge is the first reported occurrence in the West African subregion of the world.
2. Case Report {#sec2}
==============
A 48-year-old civil servant presented with axillary lymphadenopathy of insidious onset, discovered on routine mammography. The lymph node was excised and sent for histological analysis. She had neither clinically obvious enlarged lymph nodes elsewhere nor a previous history of lymphadenopathy.
Histological examination was done with the aid of immunohistochemistry. The predominant abnormality on H&E examination was a reactive change characterised by follicular hyperplasia, sinus histiocytosis, and expansion of the interfollicular T-cell zones with increased numbers of inter-follicular dendritic cells associated with patchy aggregates of melanophages. These features were considered indicative of a dermatopathic lymphadenopathy by some of the consultant pathologists. However, some others disagreed and felt there was some subtle evidence of lymphoma. This resulted in the use of immunohistochemistry. Immunohistochemistry done in our laboratory showed CD20 positivity in B-cell areas of the lymph node and follicular germinal centres which were CD10 and Bcl-2 positive. Due to our limited immunohistochemistry experience and the few immunohistochemistry panels at our disposal, the blocks were sent to the Department of Cellular Pathology in Queen\'s Hospital, Rom Valley Way, Romford, Essex, UK.
Their analysis revealed secondary follicles with germinal centres that were variably colonised by small CD10 and Bcl-6 positive cells that also overexpressed Bcl-2. The involved follicles had a low proliferation fraction as determined by Ki67 immunohistochemistry. S100 stained the increased numbers of interfollicular dendritic cells (see [Figure 1](#fig1){ref-type="fig"}). These features indicated an intrafollicular neoplasia/*in situ* follicular lymphoma.
3. Discussion {#sec3}
=============
Follicular lymphoma (FL) is the second most common non Hodgkin lymphoma (NHL) in the Western world \[[@B6]\]. It has an average incidence of 2.6 per 100,000 and median age in the 6th decade \[[@B6]\] and is slightly more common amongst females \[[@B7]\].
In FLIS, the enlarged lymph nodes are usually incidental findings, and the patient has no generalised lymphadenopathy \[[@B4]\]. However, there may be a coexisting FL in the same lymph node or in other nodes as has been reported in a few cases \[[@B5], [@B8]\]. In the index patient, the lymph nodes were discovered on routine mammography.
In the series of lymph nodes affected by *in situ* follicular lymphoma analysed by Jegalian et al. \[[@B5]\], they found a majority of females (56%) and a peak age of occurrence between the fifth and sixth decades. This report corresponds with the clinical profile of our index patient who is female and aged 48 years.
Follicular lymphoma is a mature B-cell neoplasm thought to be derived from follicular centre B lymphocytes. The lymphoid cells express the immunophenotypic markers associated with germinal centre B-cells, including CD10 and Bcl-6 \[[@B9]\]. In FL, the *Bcl-2* gene on chromosome 18 is merged with the immunoglobulin heavy gene locus on chromosome 14 (t 14:18) (q32;q21) \[[@B2]\]. This results in constitutive activation of the *Bcl-2* gene which is antiapoptotic and leads to accumulation of follicular centre B-cells which may otherwise have died through apoptosis.
Bcl-2 is not expressed in normal follicle centre cells. Inappropriate expression of the *Bcl-2* oncogene has long been believed to be the initial event in malignant transformation to FL \[[@B9]\]. Immunohistochemical staining for Bcl-2 protein also provides a very useful tool for distinguishing between reactive and neoplastic lymphoid follicles as normal germinal centres almost never express Bcl-2 protein \[[@B10], [@B11]\].
To diagnose FLIS, the following criteria are used \[[@B4], [@B5], [@B8], [@B12]\]: preserved nodal architecture with open sinuses and preserved paracortical regions. On H&E sections, the follicles appear to be reactive. Immunohistochemistry shows follicles positive for CD10, Bcl-6, and CD20. Bcl-2 positive germinal centre cells are confined to the germinal centres of the follicles, do not replace the entire follicle centre, and are not seen in the interfollicular regions or elsewhere in the lymph node. The involved follicles also have a lower proliferation rate with ki67 than the adjacent reactive follicles.
Molecular studies, for example, fluorescence *in situ* hybridization analysis for t(14:18), are only necessary for cases in which immunohistochemistry findings are ambiguous \[[@B12]\]. In the index case, the immunohistochemistry results were not ambiguous and diagnosis of FLIS was made without FISH.
FLIS has been reported in association with other conditions such as nonlymphoid malignancies and Crohn\'s disease \[[@B12]\]. In these reports, the FLIS was an incidental finding.
FLIS may transform to follicular lymphoma. In the case series reviewed by Jegalian et al. \[[@B5]\], one out of thirty-four patients reviewed eventually developed follicular lymphoma at the same site. Bonzheim et al. \[[@B2]\] have, however, proposed a clonal evolution from FLIS to manifest follicular lymphoma. This finding has not been supported by other studies with long-term follow-up of patients already diagnosed with FLIS \[[@B3], [@B5]\].
The index patient, a year after this diagnosis, is presently on followup and has not presented with symptoms or signs of a follicular lymphoma.
*In situ* FL needs to be differentiated from cases of partial involvement of a lymph node by a follicular lymphoma (PFL). In PFL, the architecture of the lymph node is altered when compared to the preserved architecture for FLIS. Also, in FLIS, the germinal centres stain strongly for Bcl-2 and CD10, while in PFL these markers show variable intensity \[[@B5]\]. Other criteria adopted by a panel of experts during the workshop on "early lesions in lymphoid neoplasms" organised by the European Association of Hematopathology in Uppsala, Sweden (2010) include the following: the follicular size is normal in FLIS while being expanded in PFL, the follicles are widely scattered in FLIS while they are grouped together in PFL, the follicular cuff is intact in FLIS while it is attenuated in PFL, and FLIS is composed of almost pure centrocytes while PFL is composed of centrocytes with few centroblasts \[[@B13]\].
4. Conclusion {#sec4}
=============
This report highlights a hitherto unreported entity in the West African subregion of the world and highlights the need for immunohistochemistry in the diagnosis of lymph node pathology---a resource which is very limited in this environment.
Learning Points {#sec5}
===============
FLIS is a rare entity.This is the first case reported in this subregion.Immunohistochemistry is very relevant in lymph node pathology---a resource which is scarcely available in this environment.
The authors declare that there is no conflict of interests.
Orah Nnamdi Obumneme carried out the literature research and prepared the draft paper. Akinde Ralph interpreted the H&E sections. Igbokwe Uche interpreted the immunohistochemistry. Irurhe Nicholas did the mammography. Banjo Adekunbiola edited the final paper.
{#fig1}
[^1]: Academic Editors: A. Rajput and A. N. Walker
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Introduction {#s1}
============
Chinese herbal medicines (CHMs) were the main treatment method used in ancient times by the Chinese to combat disease. As early as the Qin and Han Dynasty (around 221 BCE to 220 CE), *Sheng Nong\'s Herbal Classic* recorded 365 medicines. By the time of the Ming Dynasty (1368--1644), the number of CHMs listed in the book of *Compendium of Materia Medica* had increased to 1892. Most herbal medicines in such publications have been used constantly throughout medical history and are still applied in practice today. For example, according to *Sheng Nong\'s Herbal Classic, Coptis chinensis* Franch. was found to relieve abdominal pain and diarrhea and this herb is still widely used in China for the treatment of diarrhea or dysentery. Further, *Panax notoginseng* (Burk.) F. H. Chen, a traditional herb was initially used to stop bleeding, promote blood circulation and ease pain, was recorded in the *Compendium of Materia Medica*. It is now commonly used in cases of trauma and cardiovascular, and cerebrovascular diseases. A recent meta-analysis further demonstrated that several *P. notoginseng* preparations are beneficial for patients with unstable angina pectoris (Song et al., [@B121]).
Despite the positive effects of CHMs, little is known about their effective constituents, bioactive ingredients, and mechanisms of action. Therefore, in addition to the clinical applications mentioned in classic texts, understanding the specific active ingredients and clarifying the mechanisms of action of these compounds would facilitate the improved application of CHMs. The discovery of the drug artemisinin best illustrates the importance of CHM to the world (Tu, [@B133]), inspiring the notion that, through study of their bioactive ingredients, CHMs can help people around the world to conquer life-threatening diseases.
Non-coding RNA molecules (ncRNAs), which mainly comprise miRNA, lncRNA, and circRNA, do not encode proteins; however, as the most abundant class of RNA (at least 90%) (Sana et al., [@B113]), ncRNAs have important functions in gene regulation and are involved in pathological processes contributing to many diseases (Batista and Chang, [@B4]; Memczak et al., [@B97]; Zhang et al., [@B177]), particularly cancer, and cardiovascular and nervous system diseases. Moreover, circRNA and lncRNA act as competitive endogenous RNAs (ceRNA), which are natural miRNA sponges that influence miRNA-induced gene silencing via miRNA response elements (Tay et al., [@B127]). Thus, complex regulatory networks exist, comprising circRNA, lncRNA, miRNA, and target genes. Unraveling of this complexity has laid the foundation for a comprehensive understanding of the pathology and treatment of diseases influenced by gene regulatory networks, rather than only core disease-related genes (Boyle et al., [@B9]). Excitingly, recent studies (Feng et al., [@B30]; Tian F. et al., [@B128]; Zhou Y. et al., [@B196]) have revealed that some miRNA, lncRNA, circRNA, and ceRNA crosstalk can be regulated by bioactive ingredients from CHMs, which often have multiple targets ([Table 1](#T1){ref-type="table"}). By influencing regulatory mechanisms, including pro-apoptosis (Feng et al., [@B30]), anti-proliferation and anti-migration (Liu T. et al., [@B83]), anti-inflammation (Fan et al., [@B28]), anti-atherosclerosis (Han et al., [@B42]), anti-infection (Liu et al., [@B79]), anti-senescence (Zhang J. et al., [@B178]), and suppression of structural remodeling (Liu L. et al., [@B81]), these ingredients exert protective functions in cancer, cardiovascular disease, nervous system disease, inflammatory bowel disease, asthma, infectious diseases, and senescence-related diseases.
######
Detailed information on bioactive ingredients targeting ncRNAs.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**No**. **Name of ingredient** **Original plant** **ncRNA target** **Gene/protein/pathway** **Disease**
--------- ------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------
1 Berberine *Coptis chinensis* Franch., *Berberis soulieana* Schneid, *Berberis poiretii* Schneid., *Berberis vernae* Schneid, *Berberis wilsoniae* Hemsl., and *Platycladus orientalis* (Linn.) Franco. miR-99a\~125b (Feng et al., [@B30])\ RAC1, NFκB1, MYC, JUN, and CCND1 (Feng et al., [@B30])\ Multiple myeloma (Luo et al., [@B89]; Feng et al., [@B30]; Yin et al., [@B166])
miR-21 (Luo et al., [@B89])\ IL6/STAT3, PDCD4 (Luo et al., [@B89])\
miR-19a/92a (Yin et al., [@B166]) P53 signaling pathway (Luo et al., [@B89])\
P53, ERB, and MAPK signaling pathways (Feng et al., [@B30])
miR-23a (Wang N. et al., [@B141]) NEK6, P53, P21, GADD45α (Wang N. et al., [@B141]) Hepatocellular carcinoma (Wang N. et al., [@B141])
miR-152\ DNMT1, DNMT3A, DNMT3B, CDK4\ Colorectal cancer (Su et al., [@B123]; Huang et al., [@B48])
miR-429\ (Su et al., [@B123]; Huang et al., [@B48])\
miR-29a (Huang et al., [@B48])\ Pin1-β-catenin-cyclin D1 signaling pathway (Su et al., [@B123])
miR-296-5p (Su et al., [@B123])
miR-34a\ CDK4, CyclinD1, CyclinE, CDK2 (Yang L. H. et al., [@B159]) Melanoma (Yang L. H. et al., [@B159])
miR-154\
miR-26a\
miR-124 (Yang L. H. et al., [@B159])
miR-93 (Chen et al., [@B17]) miR-93/PTEN/AKT signaling pathway (Chen et al., [@B17]) Ovarian cancer (Chen et al., [@B17])
miR-203 (You et al., [@B167]) BCL-w (You et al., [@B167]) Gastric cancer (You et al., [@B167])
miR-27a miR-27b (Wu et al., [@B154]) PPAR-γ (Wu et al., [@B154]) Obesity (Wu et al., [@B154])
lncRNA MRAK052686 (Yuan et al., [@B172]) NRF2 (Yuan et al., [@B172]) Steatotic liver (Yuan et al., [@B172]; Li C. H. et al., [@B69])
miR-373 (Li C. H. et al., [@B69]) EGR1, AKT1\
AKT-mTOR-S6K signaling pathway (Li C. H. et al., [@B69])
miR-29a-3p (Mao et al., [@B94]) IRS1 (Mao et al., [@B94]) Insulin resistance (Mao et al., [@B94])
2 Artesunate *
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Takeuchi H, Savitzky AH, Ding L, et al. Evolution of nuchal glands, unusual defensive organs of Asian natricine snakes (Serpentes: Colubridae), inferred from a molecular phylogeny. Ecol Evol. 2018;8:10219--10232. 10.1002/ece3.4497
1. INTRODUCTION {#ece34497-sec-0001}
===============
In the 20th Century, many biologists were focused on commonalities among taxa, as represented by studies using model organisms (Alberts et al., [2008](#ece34497-bib-0001){ref-type="ref"}). On the other hand, appreciating the diversity of life and its evolutionary origins has been another essential pursuit in biology (Rosenzweig, [1995](#ece34497-bib-0037){ref-type="ref"}; Whittaker, [1972](#ece34497-bib-0051){ref-type="ref"}). Because evolution of novel phenotypic characters, such as wings of birds and mammary glands of mammals, can facilitate the diversification of a lineage (Wagner & Lynch, [2010](#ece34497-bib-0049){ref-type="ref"}), investigation of the evolutionary history of such novel characters can provide basic information that clarifies the processes underlying species diversification.
Snakes (Serpentes) comprise a distinct monophyletic taxon within the Squamata (Pyron, Burbrink, & Wiens, [2013](#ece34497-bib-0033){ref-type="ref"}), including over 3,500 species that are distributed on all continents except Antarctica (Wallach, Williams, & Boundy, [2014](#ece34497-bib-0050){ref-type="ref"}). In spite of their seemingly uniform appearance, snakes exhibit prominent morphological and ecological diversity (Greene, [1997](#ece34497-bib-0011){ref-type="ref"}; Lillywhite, [2014](#ece34497-bib-0021){ref-type="ref"}) and have often evolved novel organs that serve particular ecological functions. A well‐known example of a novel defensive structure is the rattle of rattlesnakes, which is used to warn potential predators of the snakes' venomous bite (Greene, [1997](#ece34497-bib-0011){ref-type="ref"}). The rattle evolved once in the ancestor of extant rattlesnakes (Castoe & Parkinson, [2006](#ece34497-bib-0005){ref-type="ref"}; Greene, [1997](#ece34497-bib-0011){ref-type="ref"}), and it has been lost secondarily in some island populations, where selection for defense is reduced in the absence of mammalian predators (Martins, Arnaud, & Murillo‐Quero, [2008](#ece34497-bib-0024){ref-type="ref"}; Rowe, Farrell, & May, [2002](#ece34497-bib-0038){ref-type="ref"}).
The nuchal gland system is another example of a novel defensive structure that has evolved in snakes (Mori et al., [2012](#ece34497-bib-0027){ref-type="ref"}). Nuchal glands were originally described in a Japanese natricine snake, *Rhabdophis tigrinus* (Figure [1](#ece34497-fig-0001){ref-type="fig"}; Nakamura, [1935](#ece34497-bib-0031){ref-type="ref"}). The organs, which superficially resemble secretory structures, are embedded in the dermal layer of the dorsal skin of the neck. The nuchal glands of *R. tigrinus* contain cardiotonic steroid toxins known as bufadienolides (Hutchinson et al., [2007](#ece34497-bib-0019){ref-type="ref"}), which are sequestered from toads consumed as prey and can be redeployed as a defensive mechanism (Hutchinson et al., [2007](#ece34497-bib-0019){ref-type="ref"}). The glands of some other species also contain bufadienolides (Mori et al., unpublished). Ontogenetically, the nuchal glands are of mesodermal origin (Fukada, [1958](#ece34497-bib-0010){ref-type="ref"}; Mori et al., [2012](#ece34497-bib-0027){ref-type="ref"}), which is different from any other skin glands of terrestrial vertebrates, all of which arise from ectoderm (Savitzky et al., [2012](#ece34497-bib-0039){ref-type="ref"}). The glands lack a secretory epithelium and consist of a homogeneous population of fluid‐filled cells surrounding a dense aggregation of capillaries. There is no central lumen or duct, and the glands simply rupture through the skin to expel their fluid contents when the snake is under predatory attack (Mori et al., [2012](#ece34497-bib-0027){ref-type="ref"}).
{#ece34497-fig-0001}
Nuchal glands and the structurally similar nucho‐dorsal glands (which extend the full length of the body; Smith, [1938](#ece34497-bib-0041){ref-type="ref"}) are currently known in 17 species of Asian Natricinae (Mori et al., [2012](#ece34497-bib-0027){ref-type="ref"}; Mori, Jono, Ding, et al., [2016](#ece34497-bib-0028){ref-type="ref"}). Hereafter, we refer to all such structures as nuchal glands, for simplicity. No other animals have been reported to possess organs similar in their structural details to the nuchal glands. The 17 species that possess such glands belong to three nominal genera, *Balanophis*,*Macropisthodon*, and *Rhabdophis*. Interestingly, *Macropisthodon* and *Rhabdophis* also include species that do not have nuchal glands (Table [1](#ece34497-tbl-0001){ref-type="table"}). This distribution might indicate the occurrence of (a) multiple independent origins of these unusual organs, (b) their secondary loss, and/or (c) improper generic assignment of some species.
######
A species list for the three nominal genera, *Balanophis*,*Macropisthodon*, and *Rhabdophis*
Species Glands Source
-------------------------------- -------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
***Balanophis ceylonensis*** P Smith ([1938](#ece34497-bib-0041){ref-type="ref"})
***Macropisthodon flaviceps*** A/P Smith ([1938](#ece34497-bib-0041){ref-type="ref"})
***M. plumbicolor*** P Mori, Jono, Takeuchi, Ding et al. ([2016](#ece34497-bib-0030){ref-type="ref"}) and Smith ([1938](#ece34497-bib-0041){ref-type="ref"})
*M. rhodomelas* P Smith ([1938](#ece34497-bib-0041){ref-type="ref"})
***M. rudis*** A Smith ([1938](#ece34497-bib-0041){ref-type="ref"}) and Takeuchi and Mori ([2012](#ece34497-bib-0045){ref-type="ref"})
***Rhabdophis adleri*** P Mori, Jono, Ding et al. ([2016](#ece34497-bib-0028){ref-type="ref"})
*R. akraios* U Doria, Petri, Bellati, Tiso and Pistarino ([2013](#ece34497-bib-0006){ref-type="ref"})
*R. angelii* U Mori et al. ([2012](#ece34497-bib-0027){ref-type="ref"})
*R. auriculatus* U Mori et al. ([2012](#ece34497-bib-0027){ref-type="ref"})
*R. barbouri* U Mori et al. ([2012](#ece34497-bib-0027){ref-type="ref"})
***R. callichromus*** P Mori et al. ([2012](#ece34497-bib-0027){ref-type="ref"}) and Smith ([1938](#ece34497-bib-0041){ref-type="ref"})
*R. chrysargoides* U Mori et al. ([2012](#ece34497-bib-0027){ref-type="ref"})
***R. chrysargos*** A Smith ([1938](#ece34497-bib-0041){ref-type="ref"})
***R. conspicillatus*** A Mori, Jono, Takeuchi and Das ([2016](#ece34497-bib-0029){ref-type="ref"})
***R. formosanus
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1. Introduction {#sec1}
===============
In cardiac anesthesia BIS monitoring is increasingly used to monitor anesthesia depth as well monitoring cerebral ischemia, which may be particularly important during cardiopulmonary bypass (CPB) \[[@B1], [@B2]\] and cardiopulmonary resuscitation \[[@B3], [@B4]\]. We describe a patient who, while being monitored with BIS, suffered a transient ischemic attack (TIA) of the brainstem.
2. Case Report {#sec2}
==============
A 76-year-old man was scheduled for a three-vessel coronary artery grafting (CAG) using CPB.
His medical history included hypertension, a minor inferior myocardial infarction, amaurosis fugax, surgical resection of a vocal cord carcinoma, and an eyelid correction.
Physical examination showed a 70 kg, 1.69 m tall patient with a blood pressure of 170/70 mmHg and a pulse of 52. A bruit was heard over the heart, both carotid arteries and femoral arteries consistent with an aortic valve sclerosis. Cardiac ultrasound showed a hypokinetic inferior left ventricular wall, a good left and right ventricular function and an aortic valve sclerosis with minor aortic valve insufficiency. Coronary angiogram showed occlusion of the RCA, 90% occlusion of the LAD and 70% occlusion of the RCX. Patient took acetylsalicylic acid, chlortalidone, amlodipine, atorvastatin, metoprolol, isosorbide mononitrate, and isosorbide dinitrate. The patient was scheduled for a coronary bypass and premedicated with paracetamol 1000 mg and midazolam 7.5 mg, orally. Once in the operating theatre the patient was prepared for the operation with an 14 G IV infusion and a 20 G arterial cannula in the left radial artery as well as standard monitoring with a 5 lead ECG, pulsoximetry, and a noninvasive blood pressure band on the right arm. A BIS Quatro Sensor (XP) was placed and the monitor (Aspect Medical Systems, Inc. model A-2000 BIS Monitor) started. During this period the patient was alert and communicative. Without provocation, the patient suddenly complained of dizziness, stopped breathing, became unresponsive, and his eyes deviated upwards en laterally. The BIS monitor then gave its first reading of 60.
We initially ventilated the patient by mask and gave naloxone to rule out an accidental sufentanil bolus. Subsequently the patient was intubated without medication. We requested an emergency neurological consultation. During this period the blood pressure and pulse remained stable at around 170/85 with a pulse of 55.
Neurological examination showed a Glasgow Coma Score (GCS) of E-1, M-1, and V-T: eyes closed, no motor response to painful stimuli, and no sounds. He had pinpoint pupils and pupillary reactions showed minimal constriction to light. Corneal reflexes were present while oculocephalic reflexes were absent. Both eyes were deviated upwards and to the left. Tendon reflexes were increased on the left side, with bilateral extensor plantar responses.
We performed a CT-scan, which showed central and cortical atrophy with subthalamic and periventricular hypodensities consistent with older vascular damage. The CT-angiogram showed generalized atherosclerotic changes in all brachiocephalic vessels, especially in the common and internal carotid arteries and a slight stenosis of the origin of the left vertebral artery.
After the CT-scan, we restarted the BIS monitoring which still showed the BIS value at about 50 to 60. Eleven minutes after resuming BIS monitoring the patient opened his eyes, started breathing and responding to voice commands, and BIS values increased to around 80--85. The patient was extubated and wanted to know the result of the operation. On neurological examination, the patient was alert and responded adequately. There was a bilateral downbeat nystagmus in downgaze. Tendon reflexes remained increased on the left side, while both plantar responses were flexor. Based on the neurological examination, the transient nature of the neurological deficit, and the CT-scan, we concluded that the patient had suffered a transient ischemic attack of the brainstem.
During the whole event which lasted 102 minutes the patient had remained hemodynamically stable. He was transferred to the neurological intensive care where the rest of his stay remained unremarkable, and he recovered without any neurological deficit.
3. Discussion {#sec3}
=============
The primary reason to use an EEG monitor in anesthesia is to prevent awareness. Indeed, individuals suffering from this experience can have serious mental disorders. Awareness in a general population was found to be just below 0,2% and in a high-risk population just below 1% \[[@B5], [@B6]\]. Two large scale prospective trials (SAFE trial and B-Aware trial \[[@B5], [@B6]\]) demonstrated a reduction of 80% in the incidence of awareness using the BIS monitor. Furthermore, BIS and other EEG devices can be used to titrate anesthetics towards a desired level of hypnosis, with the aim to prevent exaggerated plasma concentration, which could lead to hemodynamic instability and prolonged awakening.
There are some clinical conditions where BIS is unusually low. These are conditions were the cerebral functions are impaired by hypoperfusion, ischemia, hypoglycaemia, and hypothermia \[[@B7]\]. Patients with a neurological disease can present with unusual BIS values and anticonvulsant drugs may reduce BIS values. BIS may also detect microembolic injury \[[@B8]\].
In our case, BIS monitoring coincided with the clinical findings of reduced cerebral activity and later the return of consciousness. The BIS value is derived from two frontal leads. As such it will primarily monitor frontal lobe cortical electrical activity and indirectly frontal cerebral perfusion. As there was no change in blood pressure and pulse rate we think that the overall cerebral perfusion pressure was not reduced during the ischemic attack. Therefore, together with the diagnosis of brainstem TIA, we think that the BIS value is decreased by another mechanism then a perfusion disturbance. The cerebral cortex receives extensive afferent projections from brainstem nuclei. There is a long list of pathological brain conditions known to affect the EEG. Ischaemia is one of these conditions. Ischaemia of the lower brainstem (with a clinical picture characterized by coma, respiratory abnormalities, and pinpoint pupils) results in diffuse low-voltage activity and bilateral slowing of the EEG although a posterior alpha rhythm may be preserved. Hence a logical explanation for the significant decrease in the BIS signal observed in our patient may be the decrease in frontal cortical activity during ischemia of the brainstem. However, people using the BIS should be aware that the simplifying algorithm built in to the monitor limits its diagnostic possibilities and there are many factors, unknown to the user, which can alter the BIS value. The BIS monitoring did not lead to a change in treatment policy.
Brainstem TIAs are supposedly a rare occurrence. The timing of our patients attack was quite spectacular: 20 minutes earlier and he would have been found asphyxiated in his bed and his death would most likely have been attributed to a coronary event. Thirty seconds later he would have been under anesthesia and subjected to hypothermia, hypotension, and full heparinization from the CPB. We can only speculate what the neurological outcome would have been. If the neurological outcome had been bad, it would have been labeled as a complication of the CPB. It begs the question how often the brainstem TIAs really happen in our cardio vascular-compromised population.
4. Summary {#sec4}
==========
This 76-year-old patient suffered a brainstem TIA before cardiac surgery. The TIA was registered on BIS and resulted in a drop in BIS to a value of 60. When consciousness returned spontaneously, the BIS increased to 85. We believe that the lack of input from the brainstem to the frontal cortex resulted in the reduced cortical electrical activity as registered with the BIS.
[^1]: Academic Editor: Michael G. Irwin
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Introduction {#Sec1}
============
In manufacturing industries, industry 4.0 and digital transformation are interrelated fields that both motivate the development of digital twins. *Industry 4.0* is a concept attracting much research and development over the last decade, including reference models \[[@CR1]\], applications \[[@CR2]\], standards \[[@CR3]\] and supporting methods \[[@CR4]\]. A core idea of Industry 4.0 is to connect physical devices (e.g., manufacturing systems and the objects they produce), digital components (e.g. ERP or MES systems) and human actors along production processes for the sake of seamless integration and continuous monitoring and control \[[@CR5]\]. *Digital twins* (DT) support this core idea and can be defined as "a dynamic virtual representation of a physical object or system across its lifecycle, using real-time data to enable understanding, learning and reasoning" \[[@CR6]\]. *Digital transformation*, in general, denotes adopting digital technologies, such as industry 4.0 related technologies or DTs, in the digitalization of an organization's business model and its operations (cf. Sect. [2.3](#Sec5){ref-type="sec"}). Several researchers emphasize the importance of industry 4.0 for digital transformation \[[@CR21]\] or, vice versa, that digital transformation motivates the implementation of industry 4.0 \[[@CR20]\]. However, DTs as an element of digital transformation or digital transformation as driver for DT development are not included in the aforementioned work. A literature analysis (see Sect. [5](#Sec11){ref-type="sec"}) confirmed that digital twin research predominantly focuses on technological questions of DT design and operations. So far, organizational and business model related aspects of DTs are only sparsely covered in research which motivated this paper. In response to this, the paper's objective is *to investigate how DT solutions are integrated into organizational structures and business models of manufacturing enterprises, and what motivates the development of DT from a digital transformation perspective.*
Enterprise Modeling (EM) is a versatile approach and is able to tackle various organizational design problems by means of multi-perspective conceptual modeling. EM captures organizational knowledge about the motivation and business requirements for designing IS \[[@CR7]\]. Hence it has the potential of capturing and representing the organizational motivation for DT design. A key aspect of operating and managing DTs is to configure and adjust them according to the situational changes in operations. Capability Management, and in particular Capability Driven Development (CDD), has been proven applicable for managing information systems (IS) in changing context \[[@CR10]\]. E.g., CDD supports generation of monitoring dashboards from models that include context elements, measurable properties, KPIs as well as rule-based based adjustments based on context data. In concrete terms, the goal of this paper is *to analyze the suitability of EM and capability management for the purpose of supporting the development and management of DTs from an organizational perspective.* We have chosen the 4EM and CDD methods for the purpose of this study because they have already established integration mechanisms between themselves and with other modeling languages.
The rest of the paper is structured as follows. Section [2](#Sec2){ref-type="sec"} gives background to EM, CDD, and digital transformation. Section [3](#Sec6){ref-type="sec"} describes our research approach. Section [4](#Sec7){ref-type="sec"} presents two case studies. Section [5](#Sec11){ref-type="sec"} summarizes the main requirements for developing Industry 4.0 solutions found in literature. Section [6](#Sec12){ref-type="sec"} discusses the requirements from Sects. [4](#Sec7){ref-type="sec"} and [5](#Sec11){ref-type="sec"} with respect to CDD. Section [7](#Sec15){ref-type="sec"} discusses an example of a capability model for the purpose of DT development. Section [8](#Sec16){ref-type="sec"} provides concluding remarks.
Background {#Sec2}
==========
Enterprise Modeling and 4EM {#Sec3}
---------------------------
EM is the process of creating an enterprise model that captures all the enterprise's aspects or perspectives that are required for a given modeling purpose. An enterprise model consists of a set of interlinked sub-models, each of them focusing on a specific perspective, like, processes, goals, concepts, actors, rules, IS components.
4EM \[[@CR7]\] is a representative of the Scandinavian strand of EM methods. At its core is participatory stakeholder involvement and the modeling process is usually organized in the form of facilitated workshops. 4EM shares many underlying principles of the, so called, multi-perspective, approaches that recommend analyzing organizational problems from various perspectives, e.g. AKM \[[@CR12]\] and MEMO \[[@CR11]\]. 4EM consists of six interconnected sub-model types for modeling a specific aspect or perspective of the enterprise -- Goals Model, Business Rules Model, Concepts Model, Business Process Model, Actors and Resources Model, as well as Technical Components and Requirements Model. 4EM also supports integration with other modeling languages and methods by allowing to define new inter-model relationships between the 4EM components and components of the modeling language to be integrated.
Capability Driven Development {#Sec4}
-----------------------------
In \[[@CR10]\] the concept of *capability thinking* and a method to capability management are introduced. It is an organizational mindset that puts capabilities in focus of the business model and IS development. Capability thinking emphasizes that capabilities are not self-emergent, instead they should be planned, implemented, controlled, and adjusted. In doing so they need to be addressed from the perspectives of (1) vision (e.g. goals and KPIs), (2) enterprise designs such as processes and IS architectures, (3) situation context incl. measurable properties, as well (4) best practices such as process variants and patterns for dealing with context changes. Capability as a concept allows reasoning about these four aspects of the business in an integrated way because enterprises need to know how to realize the business vision and designs as well as what needs to be changed depending on real-life situations. The definition of *capability is the ability and capacity that enables an enterprise to achieve a business goal in a certain context* \[[@CR10]\]. Successful implementation of capability thinking will lead to *capability management* as a systematic way to plan, design, develop, deploy, operate, and adjust capabilities.
CDD is a method supporting the four perspectives of capability thinking. CDD consists of a number of method components each focusing on a specific task of the capability cycle, such as Capability Design, Context Modeling, Patterns and Variability Modeling, Capability Adjustment Algorithm Specification, as well as method extensions for dealing with certain business challenges such as supporting business process outsourcing and managing service configuration with the support of open data \[[@CR17]\].
Digital Transformation {#Sec5}
----------------------
In scientific literature, digital transformation often is discussed in the general context of digitalization and considered the most complex digitalization phase \[[@CR13]\]. Its focus is on the disruptive social and economic consequences which, due to the potential of digital technologies to substantially change markets, lead to new technological application potentials and the resulting changes in economic structures, qualification requirements for employees and working life in general. \[[@CR14]\] proposes to distinguish between transformation of the value proposition and the value creation when analysing and planning digital transformation. These two "dimensions" can be divided into different steps of digitalization which form the prerequisite for the next step. In \[[@CR15]\] we have proposed the steps for the dimensions of operations and product digitization.
In the operations dimension, the steps are (1) replacing paper documents with digital representations, (2) end-to-end automated processing of this digital representation within a process and (3) integration of relevant processes within the enterprise and with partners. On the product dimension, the departure point for digitization are physical products without built-in information or communication technology. Digitization steps are (1) to enhance the product/service by providing complementary services (maintenance information, service catalogs) without actually changing it, (2) to extend functionality and value proposition of products by integration of sensors and actuators, and (3) redefinition of the product or service which leads to a completely new value proposition. A completed digital transformation requires all three steps in both dimensions.
Research Approach {#Sec6}
=================
This study is part of a research program aiming to provide methodological and tool support for organizations in dynamic contexts, e.g., supporting the process of digital transformation and capability management. It follows the five stages of Design Science research \[[@CR16]\], namely, problem explication, requirements definition, design and development of the design artifact, demonstration, as well as evaluation. This study concerns the first two steps for the design artifact supporting DT design and management from an organizational perspective. This part of our research started from the following research question which is based on the motivation presented in Sect. [1](#Sec1){ref-type="sec"}: *RQ: In the context of digital transformation, how are digital twin initiatives emerging and what are the driving forces for starting implementation projects?*
The research method used for working on this research question is a combination of literature study and descriptive case study. Based on the research question, we identified industrial cases of digital transformation suitable for studying the origin of DT developments, i.e. we performed qualitative case studies in order to obtain relevant and original data (see Sect. [4](#Sec7){ref-type="sec"}). Qualitative case study is an approach to research that facilitates exploration of a phenomenon within its context using a variety of data sources. This
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Background {#Sec1}
==========
The concept of the autonomy of a child in the context of healthcare is both complex and challenging globally. In South Africa the controversy surrounding children and autonomy has come into sharp focus since the promulgation of the Children's Act 38 of 2005 (hereinafter referred to as "the Children's Act" or "the Act"). Much of the debate revolves around the concept of maturity and the child's developing capacity to consent.
The process of development generally concerns progressive advances from one state, usually primitive or simple, toward another, usually more complex or advanced. Where this process typically terminates is what is colloquially (and formally) understood as maturity. There are various dimensions of maturity including emotional, biological, cognitive and social. However, for the sake of firmer pertinence to our research question we concern ourselves herein with cognitive and social maturity as they bear directly on Western liberalism and African communitarianism, the former often emphasising rationality and individualism and the latter; sociality of persons albeit not refuting the significance of other dimensions of maturity such as the emotionality of the deciding subject in decision-making. We can thus conceive of a child as a developing *person* with evolving capacities like autonomy, mental (decisional) capacity and capacity to assume responsibility. Notwithstanding possession of capacities, we must first plainly conceive of a child as a *person*; a human being. Although this plain conception of a child is indeed attractive as it admits no prejudice toward children as rights-holders, a fuller and more adequate definition is required to define when a subject becomes a person[1](#Fn1){ref-type="fn"} and at what age we should consider a person no longer to be regarded as a child but rather an adult \[[@CR1]\].
The Constitution of the Republic of South Africa (hereinafter referred to as "the Constitution") aligns itself with the African Charter on the Rights and Welfare of the Child (ACRWC) and the United Nations Convention on the Rights of the Child (UNCRC) \[[@CR2], [@CR3]\] with regard to exalting children as independent legal actors -- as stipulated in the Act[2](#Fn2){ref-type="fn"}, and in defining which persons are entitled to provisions entailed therein. It provides that a child is a *person* under the age of 18 years[3](#Fn3){ref-type="fn"} \[[@CR2], [@CR4]\]. It also follows that a person is considered to have attained majority at this age. Furthermore, under the Children's Act, a child is considered a rights-holder, not merely a property or extension of her parents or an object of adult concern \[[@CR2], [@CR5]\]. Children are indeed persons with an evolving capacity for individual autonomy \[[@CR6]\] hence deserve the right to express their views freely in matters affecting them[4](#Fn4){ref-type="fn"}. The relevant sections in the Children's Act attend to the various jurisdictions of a child but of particular interest to us is section 129 of the Children's Act which pertains to the consent of children to medical treatment. Section 129 expressly dictates the prerequisites for the medical treatment of a child and stipulates as follows:
'(2) A child may consent to his or her own medical treatment or to the medical treatment of his or her child if-the child is over the age of 12 years; andthe child is of sufficient maturity and has the mental capacity to understand the benefits, risks, social and other implications of the treatment.' \[[@CR4]\]
In the past, when the Child Care Act 75 of 1983 was still in effect, only children above the age of 14 years could consent to medical treatment \[[@CR7]\]. What necessitated law reform was a realisation of a number of shortcomings experienced with the Child Care Act[5](#Fn5){ref-type="fn"} and a need to fully acknowledge children as rights-holders. A lower threshold for age of consent was thus seen as a means to promote access to health services, promote participation of children in health decisions affecting them in accordance with international trends \[[@CR7], [@CR8]\]. Over the years there has been mounting empirical evidence suggesting lowering age thresholds for decisional capacity in children. For example it has been demonstrated that children below 12 years can make well considered decisions \[[@CR9]\] and that children as young as nine years old can understand issues pertinent to decision making in clinical trials \[[@CR10]\] however the statutory age of consent to medical treatment as stipulated in various countries appears arbitrary as it varies from 12 to 19 years \[[@CR11]\].
A child contemplated under the Children's Act must satisfy two requirements before accessing medical treatment on his or her own, that is, without parental, guardian, or care-giver's consent being required. The first requirement is that the child must have reached 12 years of age to consent. The second requirement is that the child must have 'sufficient maturity' and decisional capacity to understand the 'benefits, risks, social and other implications of the treatment.'[6](#Fn6){ref-type="fn"} \[[@CR12]\] However, there are a few deficiencies in this section of the Act with regard to definitions, regulations and sufficient descriptions \[[@CR8]\]. Firstly, the Act does not provide a definition regarding what ought to be considered medical treatment. Hence, for the purposes of this article, we define medical treatment as a non-invasive intervention usually in the form of a drug[7](#Fn7){ref-type="fn"}. Secondly, the Act also does not provide a definition of sufficient maturity. Hence, we will comprehend that the Act infers by 'sufficient maturity' a degree of cognitive development that affords a child the kind of engagement necessary in decision-making comparable to that of fully developed persons, namely*,* adults[8](#Fn8){ref-type="fn"}. We will provide an alternative rendition of 'sufficient maturity' in the course of this article. Thirdly, there is no provision in the Act specifying how the health practitioner ought to assess a child's decisional capacity. This is compounded by the fact that there currently is no standard objective tool for assessing the decisional capacity of children \[[@CR9]\].
Moreover, considering that South Africa is a culturally diverse country \[[@CR5]\] another concern with regard to the implementation of the Act involves the potential consequence of conferring (autonomy) rights on children without commensurate responsibilities to their community[9](#Fn9){ref-type="fn"}. For '\[i\]n the African context, for example, individual autonomy is of smaller status than the pursuit of the communal good'[10](#Fn10){ref-type="fn"} \[[@CR5]\]. In view of this, it appears the conferring of autonomy rights on children without cementing their reciprocal duties erodes interdependent relations between the child and his or her community \[[@CR13], [@CR14]\].
Our research question may be posited as follows: given the newest developments in child law as regards the conception of the child and his or her participation in society, how may appeals to different moral theories (African communitarianism and Western liberalism) aid in finding better and alternative means of determining *how* and *by whom* decisions about medical treatment of the child should be made? Perhaps there has not been a time better suited to address questions of this nature than today given the near-universal advocacy for children's rights and the resurgence of activism and scholarly criticism against old hegemonic conceptions such as the status of children in civil society, person and personhood and so forth.
In advancing forth our argument we first assert children as rights-holders, give an overview of the doctrine of informed consent and the principle of respect for individual autonomy and the legal conception of a person in the setting of the Constitution; discuss African communitarianism with regard to its notion of a person and personhood and the child and the implications thereof in the consent of children to medical treatment. And in pursuit of a case-specific definition for sufficient maturity we appeal to the notion of capacity for responsibility. Lastly, in view of both legal liberal and African communitarian moral vantages we conclude by giving due attention to the enquiry whether a 12 year old is of sufficient maturity to consent to medical treatment with the conviction that no moral theory should be assigned an absolute (moral) value *a priori*, that is, antecedent to the context within which it is to be observed and/or contemplated.
Discussion {#Sec2}
==========
Asserting children as rights-holders {#Sec3}
------------------------------------
As a point of departure, a child is a developing person. When he or she obtains decisional capacity of such degree that affords him or her the kind of engagement necessary in decision-making comparable to that of fully developed persons, *viz.* adults, we will comprehend that as what is inferred by the Act as 'sufficient maturity'. It appears to follow from this that a child with sufficient maturity ought to be equally afforded autonomy rights in decision-making, including medical treatment as is the case in adults. For '\[c\]onferring rights on children is viewed as '*recognising their moral equality with adults, thereby underscoring the moral worth of all human beings, irrespective of their situation*.' (emphasis added) \[[@CR12]\], and by autonomy rights we understand broadly those entitlements persons have which allow them the freedom of involvement in matters affecting them as members of civil society, be they public or private (also referred to herein as participatory rights); different perhaps to rights in general which are often conceived as entitlements persons have plainly by virtue of being persons . Having said that, do these rights also extend to those children who do not possess sufficient maturity and/or decisional capacity? The Act is unambiguous on this issue. Where a child is judged to lack sufficient maturity and decisional capacity to understand the benefits, risks,
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For addressing the question of cardiovascular importance of hypoglycemia, it is important to clarify its context. First, hypoglycemia is a result of treatment of hyperglycemia by oral insulin secretagogues or insulin. Chronic hyperglycemia usually expressed by HbA~1c~ level is considered a risk factor for cardiovascular disease, although this epidemiological association does not necessarily mean the existence of causal association, so the possibility cannot be excluded that HbA~1c~ may be only a marker of atherosclerotic vascular disease. Thus, in the present review the evidence related to hyperglycemia and hypoglycemia as factors contributing to the development of cardiovascular events will be discussed and the main following issues will be addressed: The relationship of hyperglycemia to cardiovascular disease will be documented based on analysis of epidemiological and clinical interventional studies. Furthermore, the evidence will be summarized that hypoglycemic episodes contribute to the development of cardiovascular events in patients with type 2 diabetes treated by hypoglycemia-inducing drugs. Finally, it will be demonstrated how the conclusions from the described studies translated in practical recommendations for personalized treatment of type 2 diabetes.
Is hyperglycemia related to cardiovascular disease? {#s2}
===================================================
The evidence about a relationship between hyperglycemia and cardiovascular disease comes from epidemiological studies and epidemiological post hoc analyses of clinical trials. For consideration of a biological variable, e.g., HbA~1c~, as a cardiovascular risk factor, it is important to analyze its relationship with cardiovascular disease also outside the diabetic range. The epidemiological study European Prospective Investigation into Cancer in Norfolk (EPIC-Norfolk) included 4,662 men and 5,570 women. Relative risks for cardiovascular disease (nonfatal or fatal coronary heart disease and strokes) adjusted for age and risk factors were calculated after 6-year follow-up period. An increase in HbA~1c~ of 1% (11 mmol/mol) was associated with relative risk for cardiovascular disease of 1.21 (95% CI 1.13--1.29 for males and 1.11--1.31 for females; *P* \< 0.001). Moreover, the increased risk associated with diabetes seemed to be mediated entirely through HbA~1c~ level, since diabetes was no longer a significant predictor when HbA~1c~ was included into multivariate model ([@B1]). Very similar results were found by another large prospective epidemiological study---Atherosclerosis Risk in Communities (ARIC)---which included 11,092 adults without history of diabetes or cardiovascular disease. After 15-year follow-up, an increase in HbA~1c~ of 1% (11 mmol/mol) was associated with hazard ratio (HR) of 1.19 (1.11--1.27) for coronary heart disease and 1.34 (1.22--1.48) for stroke ([@B2]).
Epidemiological analysis from the UK Prospective Diabetes Study (UKPDS) showed a similar association. A reduction in HbA~1c~ by 1% (11 mmol/mol) was associated with a 14% decrease in fatal and nonfatal myocardial infarction (MI) (*P* \< 0.0001), as well as 12% decrease in fatal and nonfatal stroke (*P* = 0.035). The relationship between HbA~1c~ and incidence of cardiovascular end points was linear to the level of HbA~1c~ of 5.5% (37 mmol/mol) ([@B3]). On the other hand, an epidemiological analysis from the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study showed that within the range of HbA~1c~ studied (5.5--10.5%; 37--91 mmol/mol), there was evidence for a threshold effect: While for microvascular events this value was 6.5% (48 mmol/mol), for macrovascular events and death the threshold was 7% (53 mmol/mol). Above this threshold, the risks increased significantly so that every 1% (11 mmol/mol) higher HbA~1c~ was associated with a 40% higher risk of microvascular events (*P* \< 0.0001), a 38% higher risk of macrovascular outcomes (*P* \< 0.0001), and a 38% higher risk of all-cause mortality (*P* \< 0.0001) ([@B4]). Epidemiological analysis of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study showed that 1% (11 mmol/mol) increase in average HbA~1c~ during 3.4 years' duration of the study was associated with 22% increase in mortality (*P* = 0.0001). Interestingly, the relationship between mortality and HbA~1c~ was linear in the range of 6--9% (42--75 mmol/mol) only in the intensively treated group (*P* \< 0.0001), while no significant relationship (*P* = 0.17) was observed in the standard treatment group ([@B5]).
Does the reduction of high blood glucose lead to a cardiovascular benefit? {#s3}
==========================================================================
Studies in newly diagnosed patients with type 2 diabetes. {#s4}
---------------------------------------------------------
### University Group Diabetes Program (UGDP). {#s5}
The first study to approach this question in patients with type 2 diabetes was the UGDP. This study included 1,027 patients and was statistically underpowered (with \~200 patients in each treatment category group: placebo, tolbutamide, phenphormin, insulin standard, or insulin variable regimens) to detect beneficial effect of any treatment modality. The first analysis, published in 1970, showed that despite better glycemic control, a significantly higher cardiovascular mortality was observed in a group treated by tolbutamide in comparison with placebo and both insulin regimens ([@B6]). Further analysis from UGDP showed that patients treated with tolbutamide had significantly higher incidence of fatal MI in comparison with patients on placebo (*P* = 0.01), while patients on variable insulin regimen had borderline significantly higher incidence of fatal MI (*P* = 0.06) compared with patients treated with placebo. There was no difference in incidence of nonfatal MI events among the four groups ([@B7]). With respect to the incidence of hypoglycemia in UGDP, the number of patients who had glucose levels \<50 mg/dL was zero for placebo, four for tolbutamide, three for standard insulin regimen, and five for variable insulin regimen.
### The UK Prospective Diabetes Study (UKPDS). {#s6}
The UKPDS study included 4,203 patients with newly diagnosed type 2 diabetes. The main results of the UKPDS study were published in 1998 in two articles. UKPDS 33 reports results of 3,867 patients with newly diagnosed type 2 diabetes who were randomized to intensive glycemic control policy with sulfonylureas or insulin or to conventional treatment policy---primarily with diet. More drugs were added in both groups of patients if fasting plasma glucose was ≥15 mmol/L. The patients in the intensive group had median HbA~1c~ of 7.0% (53 mmol/mol) during 10-year follow-up, while patients in the conventional group achieved median HbA~1c~ of 7.9% (63 mmol/mol) ([@B8]).
While significant risk reduction by 12% (*P* = 0.029) in the incidence of any diabetes-related end point in the intensive treatment group was observed, nonfatal and fatal MI incidence was reduced by 16% with a borderline significance (*P* = 0.052) ([@B8]). Major hypoglycemic episodes defined as the mean proportion of patients per year with one or more episode occurred with chlorpropamide (1.0%), glibenclamide (1.4%), insulin (1.8%), and diet (0.7%). Interestingly, after 10-year poststudy follow-up as more events occurred, risk reductions for MI (15%, *P* = 0.01) and all-cause mortality (13%, *P* = 0.007) became significant ([@B9]).
The results of subgroup analysis of 1,704 overweight patients with type 2 diabetes randomized to intensive treatment by metformin or sulfonylurea/insulin or to conventional treatment were published separately ([@B10]). Patients treated primarily by intensive metformin treatment had a median HbA~1c~ level of 7.4% (57 mmol/mol) during the follow-up, while patients in the conventional treatment group had median HbA~1c~ level of 8.0% (64 mmol/mol). Patients allocated to metformin compared with the conventional group had significantly reduced risk for diabetes related death by 42% (*P* = 0.017), as well as for fatal/nonfatal MI by 39% (*P* = 0.01). Patients allocated to metformin had lower risk for all-cause mortality (*P* = 0.021) and for stroke (*P* = 0.032) compared with patients allocated to insulin or sulfonylurea. Major hypoglycemic episodes occurred in 0.6% patients/year treated with metformin ([@B10]). One of the explanations of lower cardiovascular preventive effect of sulfonylurea or insulin treatment in comparison with metformin in UKPDS might be that metformin-treated patients had lower incidence of severe hypoglycemic episodes.
### Outcome Reduction with an Initial Glargine Intervention (ORIGIN). {#s7}
The ORIGIN study included a total of 12,537 participants, among whom 88% had diabetes and 12% had prediabetic dysglycemias. Patients were assigned either to insulin glargine or to standard care treatment. After the median follow-up of 6.2 years, there was no significant difference in rates of cardiovascular outcomes between the study groups. Rates of severe hypoglycemia were higher in the glargine-treated group (1.00 vs. 0.31/100
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Introduction {#s1}
============
As the longest and most architecturally complex cells in the body, neurons face the unique challenge of regulating membrane and protein levels in distal compartments. Neurons have highly elaborate dendritic arbors. These dendrites possess synapses, points of contact where electrochemical transmission of information occurs. Most of the excitatory synapses are situated on dendritic spines, tiny protrusions with a head and neck comprising a geometry that is essential for shaping electrical signals ([@bib55]; [@bib30]; [@bib53]; [@bib26]; [@bib29]; [@bib54]) and providing biochemical compartmentalization ([@bib28]; [@bib6]; [@bib14]). For synapses to function appropriately, the levels of receptor proteins at the postsynaptic density must also be finely tuned. Synapses are often located hundreds of micrometers away from the neuronal cell body. Adding to this spatial problem is the challenge of regulating protein abundance on the membrane in a temporally precise manner, as demanded by fast-acting processes such as synaptic potentiation.
Integral membrane proteins destined for the cell surface are canonically thought to be synthesized in the somatic rough endoplasmic reticulum, transported to the Golgi apparatus, and then secreted into the plasma membrane via exocytosis. It is now known that many proteins are translated locally in dendrites, a highly regulated process essential for normal development and plasticity ([@bib49]; [@bib10]; [@bib25]). Endoplasmic reticulum (ER) extends into dendrites, forming a continuous tubular network with regions of varying structural complexity and occasional entry into spines ([@bib48]; [@bib15]; [@bib16]). Together with endosomes, the ER is perfectly positioned to provide a local source of membrane and integral membrane proteins, such as glutamate receptors. However, the Golgi apparatus is absent in most distal dendrites. This puzzling observation has been resolved by recent work demonstrating that dendritic and somatic protein trafficking are highly segregated, and that glutamate receptors are trafficked through a specialized Golgi apparatus-independent pathway from the dendritic ER to the plasma membrane via recycling endosomes ([@bib9]). Structural changes in ER contribute to normal synaptogenesis during development and maturation ([@bib16]). The involvement of this system in activity-induced synaptogenesis is unknown.
Long-term potentiation (LTP), the long-lasting enhancement of synaptic strength due to repetitive activity, is thought to underlie learning and memory. This process has been studied extensively in the hippocampus, a key brain region responsible for new memory formation. Insertion of glutamate receptors from an extrasynaptic reserve pool into the postsynaptic compartment is required for LTP in hippocampal area CA1 ([@bib23]). LTP is also accompanied by structural changes in dendritic spines ([@bib8]; [@bib2]). In the young rat hippocampus, LTP produces new dendritic spines ([@bib52]), contrasting with adult rat hippocampus where new spine outgrowth is stalled in favor of synapse enlargement ([@bib7]; [@bib3]). While Golgi apparatus-independent trafficking has not been studied directly in the context of lasting LTP, recycling endosomes (RE) are known to supply AMPA receptors ([@bib40]), and recycling endosome exocytosis is required for spine formation and growth shortly after the induction of LTP ([@bib41]). Expanded knowledge about the involvement of Golgi apparatus-independent pathways in developmental synaptic plasticity could provide new targets for rescuing dysregulated synaptogenesis in cases of profound developmental disorders ([@bib18]).
Here, three-dimensional reconstruction from serial section electron microscopy (3DEM) revealed morphological changes in SER and endosomal compartments 2 hr following the induction of LTP. The findings are consistent with the involvement of the Golgi-bypass secretory system in supporting synaptic plasticity in the developing hippocampus.
Results {#s2}
=======
An acute within-slice experimental protocol ([@bib52]) was used to compare the effects of TBS and control stimulation on subcellular membranous compartments in dendrites. In brief, two stimulating electrodes were positioned \~800 µm apart with a recording electrode halfway in between them in CA1 stratum radiatum of P15 rat hippocampus in one slice from each of two animals ([Figure 1A](#fig1){ref-type="fig"}). Baseline responses were collected from both electrodes. TBS was delivered at one stimulating electrode and control stimulation was delivered at the other stimulating electrode, counterbalanced in position relative to CA3 for each experiment. There was a significant increase in the field excitatory postsynaptic potential (fEPSP) slope immediately after TBS ([Figure 1B,C](#fig1){ref-type="fig"}). Slices were fixed 120 min later. EM image volumes were collected from tissue on a diagonal \~120 µm below and to the side of each stimulating electrode. Segments of spiny dendrites, synapses, and all subcellular membrane compartments were reconstructed in three dimensions (see Materials and methods for details).
![Within-slice experimental design and electrophysiological outcome.\
(**A**) Illustration of an acute slice from a P15 rat hippocampus with a recording electrode (rec.) in the middle of CA1 stratum radiatum between two bipolar stimulating electrodes (S1 and S2). S1 and S2 are separated by 600-800 µm. The two experiments were counterbalanced for which of the two electrodes delivered TBS or control stimulation. Tissue samples collected for 3DEM were located \~120 µm beneath and to the side of the stimulating electrodes. D.G., dentate gyrus; Sub., subiculum. (**B**) Representative waveforms from control (CON, blue) and TBS (LTP, red) sites. Each waveform is the average of the final 10 responses to each stimulating electrode obtained for the last 20 min before delivery of TBS at *time 0* (light color) and for 20 minutes before the end of the experiment at 120 min after TBS (dark color). The stimulus intensity was set at population spike threshold to activate a large fraction of the axons in the field of each stimulating electrode. The positive deflection in the post-TBS waveform at \~3-4 ms reflects synchronous firing of pyramidal cells with LTP. (**C**) Changes in the slope of the field excitatory postsynaptic potential (fEPSP), expressed as a percentage of the average baseline response to test-pulses, were recorded for 20 min before delivery of TBS at *time 0* (red) or control stimulation (blue). Responses were recorded for n=2 slices for 120 min after the first TBS train, then fixed and processed for 3DEM as described in Methods. Error bars are SEM. Adapted from [@bib52] where it was originally published under a CC BY-NC-ND 4.0 license <https://creativecommons.org/licenses/by-nc-nd/4.0/>).\
10.7554/eLife.46356.003Figure 1---source data 1.Excel spreadsheet containing the raw numbers that generated the graphs and waveforms for these experiments.](elife-46356-fig1){#fig1}
Limited entry of SER into dendritic spines {#s2-1}
------------------------------------------
Consistent with previous reports on hippocampal dendrites ([@bib48]; [@bib15]), the SER formed an anastomosing network throughout the dendritic shaft with occasional entry into a subset of dendritic spines ([Figure 2A](#fig2){ref-type="fig"}; see [Figure 2---figure supplement 1](#fig2s1){ref-type="fig"} for all analyzed dendrites reconstructed with SER). While the dendritic spine density more than doubled 2 hr following TBS, a similar increase in the occurrence of SER in spines did not occur ([Figure 2B,C](#fig2){ref-type="fig"}).
![The limited occupancy of spines by SER does not increase during spinogenesis in the LTP condition.\
(**A**) Sample serial section EMs (left) and representative 3D reconstructions of dendrites (right) from control (top) and LTP (bottom) conditions, illustrating dendrites (yellow), SER (green), and synapses (red). Synaptic area was measured as the total surface area of the PSD. Arrows point to SER-containing spines. (**B**) Spine density (\#/µm) binned for PSD area. Significant increase in spines following TBS was carried by spines in the category with the smallest PSD areas (\<0.05 µm^2^; ANOVA F(~1,12~)=50.707, p=0.00001, η^2^ = 0.81). No statistically significant changes occurred in the frequency of spines with larger synapses (PSD area 0.05 to 0.1 µm^2^, ANOVA F(~1,12~)=1.079, p=0.31941; PSD area 0.1 to 0.15 µm^2^, ANOVA F(~1,12~)=0.09638, p=0.76154; PSD area 0.15 to 0.2 µm^2^, ANOVA F(~1,12~)=3.5065, p=0.08569; PSD area \>0.2 µm^2^, ANOVA F(~1,11~)=3.0778, p=0.10484). Control n = 8, LTP n = 8 dendrites. (**C**) Decrease in percentage of spines containing SER following TBS (ANOVA F(~1,12~)=10.599, p=0.00688, η^2^ = 0.87). Control n = 8, LTP n = 8 dendrites. (**D--F**) SER content for spines with PSD areas less than 0.05 µm^2^. (**D**) No statistically significant difference between control and LTP conditions in density of spines with SER (ANOVA F(~1,12~)=2.59, p=0.13322). Control n = 8, L
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(To access the full article, please see PDF)
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All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
Low temperature is a major constraint on the growth, geographical distribution, and yield of some plants. Cold resistance of many plants\[[@pone.0188514.ref001]--[@pone.0188514.ref004]\], e.g. *Eucalyptus nitens*, *Miscanthus*, *Medicago sativa* and *North American Rhododendron* can be improved by prior exposure to a period of low, nonfreezing temperatures, which known as cold acclimation (CA) \[[@pone.0188514.ref005]--[@pone.0188514.ref007]\]. For instance, CA improves the tolerance of North American Rhododendron from -7°C to -53°C \[[@pone.0188514.ref004]\]. Up to date, CA is a key strategy to increase the physiological adaptation of tea plants to low temperatures \[[@pone.0188514.ref008]\]. During CA, many physiological and biochemical processes are altered in plants. Those processes include the cytoskeleton rearrangement as an integrating system perceiving the signals \[[@pone.0188514.ref009]\], accumulated membrane phospholipids and modifications in lipid composition of different organelles. For example, the proportion of MGDG (monogalactosyldiacylglycerols) was decreased and the proportion of DGDG (digalactosyldiacylglycerols) was increased in the chloroplast in Rye \[[@pone.0188514.ref010]--[@pone.0188514.ref014]\]. Moreover, plants introduce the accumulation of antifreeze proteins and cryoprotectants like soluble sugars and proline \[[@pone.0188514.ref015]--[@pone.0188514.ref016]\]. The increased synthesis of soluble sugars, including sucrose, glucose, raffinose, and fructose, contributes directly to membrane stabilization in *Alcantarea imperiali* \[[@pone.0188514.ref017]\], and *Camellia sinensis* \[[@pone.0188514.ref008]\]. The raised content of proline in *Triticum aestivum* \[[@pone.0188514.ref018]\], *Arabidopsis* \[[@pone.0188514.ref019]\] and *Camellia sinensis* \[[@pone.0188514.ref008]\] was also observed during CA. Antioxidant metabolism is known to improve the scavenging activity of reactive oxygen species (ROS) and maintain redox balance during CA \[[@pone.0188514.ref020]\]. During CA, a high ratio of abscisic acid (ABA) to gibberellin content has been shown to increase freezing tolerance in some woody taxa \[[@pone.0188514.ref021]\].
Upon cold stress, the expression of various cold-regulated (COR) genes are induced to protect plants \[[@pone.0188514.ref022]\]. The expression of COR genes is regulated by both the CBF (C-repeat-binding factor)-mediated ABA-independent pathway and the bZIP (basic region/leucine zipper)-mediated ABA-dependent pathway \[[@pone.0188514.ref023]\]. CBF transcription factors regulate \~12% of the cold-responsive transcriptome \[[@pone.0188514.ref024]\]. ICE1 (inducer of CBF expression 1)-CBF-COR cold-response pathway in plants is critical for configuring cold-induced transcriptomic changes \[[@pone.0188514.ref025]--[@pone.0188514.ref026]\]. Genes of the ICE1-CBF cold-response pathway have been reported in woody and herbaceous plants \[[@pone.0188514.ref027]--[@pone.0188514.ref029]\]. Studies have shown that the cascade regulation of *ICE1*, *CBF*, and *COR* is the main pathway for cold acclimation \[[@pone.0188514.ref030]--[@pone.0188514.ref031]\]. In *Arabidopsis*, *ICE1* express constitutively and is not responsive to cold stress, whereas ICE1 undergoes sumoylation to become functionally active \[[@pone.0188514.ref032]\]. Three CBFs (*CBF1-3*) were found in *Arabidopsis*. *CBF1* and *-CBF3* positively regulates the downstream CBF-target genes, while *CBF2* negatively regulates them \[[@pone.0188514.ref033]\]. Wang at al. \[[@pone.0188514.ref034]\] found that the ICE1-CBF-COR pathway was conserved in tea plants. To date, several COR genes have been discovered in tea plant including one *CsICE1* (FE861156), two *CsCBFs*, designated as *CsCBF1* (EU563238), *CsCBF2* (KC702795), and three dehydrin homologs designated as *CsDHN1* (GQ228834.1), *CsDHN2* (FJ436978) and *CsDHN3* (KY270880) \[[@pone.0188514.ref034]--[@pone.0188514.ref036]\].
Several studies revealed the key enzymes' activities during sugar synthesis, and associated genes expression during CA in plants. Sucrose is synthesized in the cytosol by the sucrose-phosphate synthase (SPS) and degraded by either sucrose synthase or invertase (INV) into a monosaccharide or derivative \[[@pone.0188514.ref037]\]. Raffinose synthase (RS) for raffinose synthesis was also explored in recent researches upon cold stress \[[@pone.0188514.ref038]\]. Yue et al. \[[@pone.0188514.ref008]\] analyzed the expression patterns of 32 genes during the natural CA in tea plant (var. *sinensis* cv. *Longjing43*) and found that expression of *CsSPS*, *CsINV5 and CsRS2* was significantly induced. To date, it is known that the proline biosynthesis is catalyzed by P5C synthase (P5CS) and P5C reductase (P5CR) in plants \[[@pone.0188514.ref039]--[@pone.0188514.ref040]\]. Another key enzyme in the proline synthesis pathway is Ornithine-D-aminotransferase (OAT) \[[@pone.0188514.ref041]\]. Degradation of proline is catalyzed by Pro-dehydrogenase (ProDH) and P5C-dehydrogenase (P5CD) \[[@pone.0188514.ref042]\]. In tea plants, the sequences of *CsP5CS* (KJ143742.1), *CsOAT* (KJ641844.1) and *CsP5CR* (KY368574), *CsP5CDH*(KY368572) and *CsProDH* (KY368573) are available at NCBI (<https://www.ncbi.nlm.nih.gov/>).
Tea plants (*Camellia sinensis* (L.) O. Kuntze), one of the important economic wooden plants in the world, are mainly grown in subtropical and tropical regions. Two basic classes of varieties can be classified as var. *assamica*, a quick-growing tree well suited to tropical climates, and var. *sinensis*, a slower-growing bush that can withstand colder climates than *assamica* \[[@pone.0188514.ref043]--[@pone.0188514.ref044]\]. Tea plants are vulnerable to cold injury during winter such as in East Asia (China, Japan), especially in northern China. Recent studies have explored the response of tea plants to cold stress and natural CA \[[@pone.0188514.ref045]--[@pone.0188514.ref048]\]. However, a comparative study on cold resistance between cold-resistant and cold-susceptible cultivars has not been reported yet. The present study was conducted to explore the molecular mechanism of cold resistance by treating the cold-resistant *camellia var*. *sinensis* CV. *Shuchazao* (SCZ) and cold-susceptible *camellia var*. *assamica* CV *Yinghong9* (YH9) under CA and de-acclimation (DA). We found difference in biochemical changes, including EL50 (temperature leading to 50% tissue damages due to leakage of electrolyte), Fv/Fm (maximum quantum yield of PSII photosystems), sugars and proline. Then we examined the expression of 14 genes related to these biochemical changes. Comparison of gene expression and study of biochemical changes in the responses to cold in two tea cultivars led to our finding of the difference in cold tolerance. Our results indicated that the increased expression of *CsCBF1* and *CsDHNs* coupling with the accumulation of sucrose has played a role in conferring higher cold resistance in tea cultivar SCZ. The results provide understanding in biochemical and gene regulatory mechanisms of cold resistance in tea plants.
Materials and methods {#sec002}
=====================
Plant material {#sec003}
--------------
The clone cuttings of *Camellia sinensis* cv. *Shuchazao* and *Camellia sinensis* var. *assamica* cv.*Yinghong9* were obtained from the Dechang Tea Plantation in Anhui (116° 56\' 24\'\' E, 31° 27\' N) and the Tea Research Institute of Guangdong Academy of Agricultural Sciences (113° 22\' 48\'\' E, 24° 10\' 12\'\' N), China, respectively. One-year-old cutting-propagated plants were transferred to a growth chamber with temperature cycles of
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Pes cavus is an increase of normal plantar concavity, where the anterior and posterior weight-bearing areas of the foot are brought closer together. A wide spectrum of foot deformities includes a plantarflexed first ray, forefoot pronation and adduction, and hindfoot varus or high calcaneal pitch.^[@bibr1-2058-5241.2.160077],[@bibr2-2058-5241.2.160077]^
Cavovarus deformity can be classified according to the severity of malalignment ranging from a subtle and flexible cavovarus foot to a severe and fixed cavovarus deformity.
There are many aetiologies of unequal frequency that account for cavovarus foot deformities. Traumatic causes are rare (improperly treated fracture or subluxation of the tarsal bones or scarring from a burn of the sole of the foot). Cavovarus deformity has been long associated with neurological disease such as cerebral palsy, Charcot-Marie-Tooth (CMT) disease or other hereditary sensory and motor neuropathies (myelodysplasia, Friedreich ataxia, etc).
CMT disease results from defects in the genetic code for the protein of the peripheral myelin sheath and is classified into subtypes varying in progression. CMT IA is the most common form including peripheral nerve myelin degeneration and decreased motor nerve conduction. In most cases, the disease process is progressive rather than static; therefore, the deformities worsen and surgical treatment must be considered to prevent the progression to a fixed and symptomatic deformity.^[@bibr3-2058-5241.2.160077]^
However, in recent years, a mild variation of the cavovarus deformity has been increasingly observed to exist without an identifiable underlying deficit.^[@bibr4-2058-5241.2.160077]^ In our experience, this primary pes cavus (idiopathic) is diagnosed by elimination in more than half the cases and most authors believe that it is the consequence of a latent neurological disorder. Thus, neurological disorders must be looked for in the family history and clinical and electrophysiologica evaluation of the patient is necessary to eliminate any very subtle neurological lesion.
Patho-anatomy {#section1-2058-5241.2.160077}
=============
There are several types of pes cavus, depending on the site of the deformity. Some authors have divided the deformity into posterior, anterior or mixed cavus which includes both deformities.^[@bibr5-2058-5241.2.160077]^
The most frequent anterior pes cavus is characterised by lowering of the forefoot in plantarflexion ([Fig. 1](#fig1-2058-5241.2.160077){ref-type="fig"}). In total pes cavus, the increase of the slope of the forefoot involves the whole of the metatarsal range, whereas in medial pes cavus, it decreases from the medial to the outer side which causes pronation of the forefoot.
{#fig1-2058-5241.2.160077}
The posterior cavus or calcaneocavus is characterised by an isolated high calcaneal pitch of greater than 30° related to a weakness of the gastrocnemius muscle leading to a calcaneus deformity of the hindfoot.
The exact cause in the cavus foot is a longstanding issue, and both intrinsic and extrinsic muscle imbalance may play a role in the final deformity.^[@bibr6-2058-5241.2.160077]^ An imbalance between the antagonistic muscles, in particular the peroneus longus and tibialis anterior, is often listed as a cause.^[@bibr7-2058-5241.2.160077]^ Manoli et al^[@bibr8-2058-5241.2.160077]^ consider the primary deforming force to be the plantarflexed first metatarsal, which is thought to be a result of peroneus longus overaction. Relative weakness of the peroneus brevis and tibialis anterior muscles with strong tibialis posterior and peroneus longus muscles cause plantar flexion of the first metatarsal bone and varus of the hindfoot.^[@bibr9-2058-5241.2.160077]^ Recruiting extensor hallucis longus and extensor digitorum longus as secondary ankle dorsiflexors will lead to 'cock-up' deformity of the hallux and clawtoe deformity of the lesser toes. To allow the toe pulp to touch the ground, the flexor muscles of the toes contract, producing clawing of the toes, which is also aggravated by a deficiency of the interosseous muscles. Clawing of the toes accentuates the slope of the metatarsals due to the exaggerated pressure on the metatarsal heads, which in turn increases the tension in the plantar aponeurosis. Additional contracture of the plantar fascia will accentuate the windlass mechanism and further depress the metatarsal heads.
Hindfoot varus is described as being forefoot or hindfoot driven. In forefoot-driven varus, excessive plantarflexion of the first metatarsal and supination of the midfoot leads to the hindfoot moving into varus, whereas hindfoot-varus-driven is related to simple varus malalignment of the heel ([Fig. 2](#fig2-2058-5241.2.160077){ref-type="fig"}).
{#fig2-2058-5241.2.160077}
Hindfoot varus also increases the risk of damage to the lateral structures of the foot and ankle. Thus, peroneal tendons can suffer as a consequence of the hindfoot varus^[@bibr10-2058-5241.2.160077]^ but may also be responsible for the hindfoot varus in cases with relative weakness or paralysis of one or both. The relationship between the varus heel and chronic instability has been well documented; moreover, the heel varus overloads the lateral structures of the foot and ankle and may lead to varus ankle arthritis.
Because of hindfoot inversion, the Achilles tendon will shift medially and act as a secondary invertor. Furthermore, patients with cavus feet often have tight calves and a short and tight gastrocnemius leading to increase of the plantar pressures in the forefoot and the plantar fascia and act as a deforming hindfoot inverting force.
Radiographic evaluation {#section2-2058-5241.2.160077}
=======================
Plain film radiographs are essential in surgical planning, not only to identify the site of the deformity but also to quantify the degree of correction that is required and to decide whether to perform an osteotomy or an arthrodesis. The apex of the deformity can vary. Usually the deformity is located in the mid-foot at the transverse tarsal articulation or at the naviculocuneiform joint.^[@bibr1-2058-5241.2.160077]^
Weight-bearing radiographs of the foot include at least three views:
1. A lateral view of the weight-bearing ankle and foot allows the cavus to be demonstrated and measured.
2. A frontal view of the ankle (Meary view or Salzman view) demonstrates the frontal deformity of the hindfoot.^[@bibr11-2058-5241.2.160077]^
3. A dorsoplantar view of the forefoot shows adduction of the forefoot and opening of the metatarsal plate.
Numerous geometric measurements have been proposed on lateral weight-bearing radiographs to quantify cavus deformity ([Fig. 3](#fig3-2058-5241.2.160077){ref-type="fig"}). In France, the angle of the medial arch is widely used (Djian-Annonier angle) and in pes cavus foot it is less than 120°. A Hibb's angle (angle between the long axis of the calcaneum and first metatarsal) of more than 45° indicates cavus.^[@bibr12-2058-5241.2.160077]^
{#fig3-2058-5241.2.160077}
The intersection point between the first metatarsal axis and the sagittal axis of the talus corresponds to the apex of the deformity which is important when considering osteotomies. The cavus foot is defined as a Meary's angle (the angle between the long axes of the talus and first metatarsal) greater than 5°. In posterior cavus foot, the calcaneal pitch angle is greater than 30°. An associated equinus deformity of the ankle is characterised by a tibio-talar angle greater than 105°.
On the lateral view, a stacking effect may be observed because the first
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Related literature {#sec1}
==================
For the triclinic polymorph of the title compound, see: Chekhlov (2007[@bb3]).
Experimental {#sec2}
============
{#sec2.1}
### Crystal data {#sec2.1.1}
H~3~O^+^·ClO~4~ ^−^·C~20~H~24~O~6~*M* *~r~* = 478.9Monoclinic,*a* = 8.6586 (1) Å*b* = 26.7718 (3) Å*c* = 19.1518 (2) Åβ = 100.0011 (10)°*V* = 4372.05 (8) Å^3^*Z* = 8Cu *K*α radiationμ = 2.09 mm^−1^*T* = 124 K0.26 × 0.18 × 0.13 mm
### Data collection {#sec2.1.2}
Oxford Diffraction Xcalibur Atlas Gemini ultra diffractometerAbsorption correction: multi-scan (*CrysAlis RED*; Oxford Diffraction, 2008[@bb4]) *T* ~min~ = 0.098, *T* ~max~ = 1.00036184 measured reflections6865 independent reflections5283 reflections with *I* \> 3σ(*I*)*R* ~int~ = 0.048
### Refinement {#sec2.1.3}
*R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.051*wR*(*F* ^2^) = 0.124*S* = 2.076865 reflections595 parametersH atoms treated by a mixture of independent and constrained refinementΔρ~max~ = 0.49 e Å^−3^Δρ~min~ = −0.32 e Å^−3^
{#d5e700}
Data collection: *CrysAlis CCD* (Oxford Diffraction, 2008[@bb4]); cell refinement: *CrysAlis RED* (Oxford Diffraction, 2008[@bb4]); data reduction: *CrysAlis RED*; program(s) used to solve structure: *SIR2002* (Burla *et al.*, 2003[@bb2]); program(s) used to refine structure: *JANA2006* (Petříček *et al.*, 2006[@bb5]); molecular graphics: *DIAMOND* (Brandenburg & Putz, 2005[@bb1]); software used to prepare material for publication: *JANA2006* and *publCIF* (Westrip, 2010[@bb6]).
Supplementary Material
======================
Crystal structure: contains datablocks global, I. DOI: [10.1107/S1600536810048622/hb5736sup1.cif](http://dx.doi.org/10.1107/S1600536810048622/hb5736sup1.cif)
Structure factors: contains datablocks I. DOI: [10.1107/S1600536810048622/hb5736Isup2.hkl](http://dx.doi.org/10.1107/S1600536810048622/hb5736Isup2.hkl)
Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?hb5736&file=hb5736sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?hb5736sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?hb5736&checkcif=yes)
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [HB5736](http://scripts.iucr.org/cgi-bin/sendsup?hb5736)).
This work was supperted by the institutional research plan No. AVOZ10100521 of the Institute of Physics, the project Praemium Academiae of the Academy of Sciences of the Czech Republic and the Czech Ministry of Education, Youth and Sports, Project MSM 4977751303.
Comment
=======
The crystal structure of dibenzo-18-crown-6 hydronium perchlorate was previously published by A.N.Chekhlov (2007). The published structure determined at room temperature is triclinic(space group P-1, a = 8.582 Å, b = 10.486 Å, c = 26.293 Å, α = 79.45°,β = 82.00° and γ = 79.36°, V =2272.5 Å) with asymmetric unit consisting of two independent molecules of macrocycle with complexed hydronium ions. The neutrality of the compound is ensured by two perchlorate anions. The data of crystal structure, presented in this paper, were collected at room temperature (testing stage) and at 120 K (final data collection). We found the complex monoclinic, *P*2~1~/*c* space group, with unit-cell parameters a = 8.6535 Å, b = 26.7823 Å, c = 19.1707 Å, β = 99.9987° and doubled unit cell volume V = 4372.05 Å^3^. The difference between both structures is in their system of hydrogen bonds. In Chekhlov\'s structure, the hydronium ion is held by three hydrogen bonds inside the crown cavity. In presented structure, hydronium ion and crown-ether form only two hydrogen bonds. The third hydrogen atom of hydronium ion is shared with perchlorate anion which makes it to point out of the cavity. This hydrogen bond causes that the perchlorate anions are not disordered as it was observed in Chekhlov\'s structure. Consequently, sharp maxima in difference Fourier map could be used for localizing hydrogen positions in both oxonia cations (Fig 3) and the found hydrogen positions could be refined without restraints. The distance between hydronium and oxygen atoms in macrocycles are 1.637 Å (O21---H1···O3) and 1.864 Å (O21---H9···O5) for one crownether molecule and 1.895 Å (O22---H10···O11) and 1.661 Å (O22---H8···O9) for the other one. The length of hydrogen bond between hydronium ion and perchlorate is 1.732 Å (O21---H7···O17) and 1.687 Å (O22---H6···O14). The distances between hydrogen atoms and oxygen atoms in hydronium correspond to the extent of their participation in hydrogen bonding system: O---H distance close (but still longer) to the standard value 0.983 Å has been only found for the weakest hydrogen bond O22---H10···O11. For stronger hydrogen bonds O---H distance becomes significantly longer, taking the maximum value 1.29 (4) for O22---H6···O14. The O---H and corresponding H···O distances for oxonia are summarized in Table 2. The hydronium ions are enclosed in the crown-ether cavities by phenyl ring of neighbouring molecules. This arrangement is stabilized due to CH-π interactions between phenyl rings and CH~2~ groups of crownether (the distance between centroid of phenyl ring C11→C16 and H37*b* in ethylen group is 2.989 Å and between centriod of phenyl ring C31→C36 and H17*a* in ethylen group is 2.870 Å) and due to the face-to-edge orientation of phenyl rings (distance between the centriod of phenyl ring C21→C26 and H13 of phenyl ring C11→C16 is 3.207 Å and between the centriod of phenyl ring C1→C6 and H33 in phenyl ring C31→C36 is 3.004 Å).
Experimental {#experimental}
============
Dibenzo-18-crown-6, perchloric acid and acetonitrile were purchased by Fluka. Crystals were prepared by slow evaporation of equimolar mixture of dibenzo-18-crown-6 (0.05*M*) and perchloric acid (0.05*M*) in acetonitrile to yield colourless prisms of the title compound.
Figures
=======
{#Fap1}
{#Fap2}
{#Fap3}
Crystal data {#tablewrapcrystaldatalong}
============
----------------------------------- ----------------------------------------
H~3~O^+^·ClO~4~^−^·C~20~H~24~O~6~ *F*(000) = 2016
*M~r~* = 478.9 *D*~x~ = 1.455 Mg m^−3^
Monoclinic, *
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1. Introduction {#S1}
===============
Congenital coronary artery anomalies are defined as a coronary pattern that is found in less than 1% of the general population, with a prevalence ranging from 0.3%−5.6% \[[@R1]\]. In few types of the anomalies, there is an association with sudden death and premature coronary disease \[[@R2]\]. Congenital absence of left main coronary artery (LMCA) and anomalous origins of left anterior descending artery (LAD) and left circumflex artery (LCX) arising from right sinus of Valsalva is rarely reported. Here we are presenting a 62-year-old male who presented with non-ST NSTEMI who found to have anomalous absence of left main coronary artery and anomalous origins of left anterior descending artery and left circumflex artery from right sinus of Valsalva.
2. Report of the Case {#S2}
=====================
62 years old man with past medical history of hypertension, dyslipidemia and type II diabetes mellitus presented with acute chest pain. The pain started suddenly, pressure like, at the left side, 9/10 in intensity, radiates to left arm and was associated abdominal discomfort, nausea and diaphoresis. The pain was relieved by sublingual nitroglycerin. Electrocardiography showed tall positive T waves at inferior leads ([Figure 1](#F1){ref-type="fig"}). His troponin was initially 0.5 ng/L then increased after 4 hours to 1.2 ng/L. He was started on aspirin, clopidogrel and heparin. Transthoracic echocardiography showed ejection fraction estimated to be 60% without wall motion abnormality. He was taken for cardiac catheterization, which showed 95% occlusion of proximal left circumflex artery (LCX) and 60% occlusion of distal Left anterior descending artery (LAD). LCX and LAD were originated from right coronary cusp ([Figure 2](#F2){ref-type="fig"}). He was treated with drug-eluting stent for proximal LCX ([Figure 3](#F3){ref-type="fig"}). He was discharged on aspirin and ticagrelor in a stable medical condition.
3. Discussion {#S3}
=============
Coronary heart disease one of the leading causes of death in developed countries \[[@R3]\]. Congenital coronary artery anomalies are relatively rare entities with a prevalence that is reported to be approximately 0.3%-5.6%. The variation in prevalence is likely explainable by the diversity of the study populations \[[@R1]\]. Studies suggest that congenital coronary artery anomalies are the second most common cause of sudden death in young athletes, likely due to premature coronary disease \[[@R2]\]. Based on predisposition to CAD, congenital coronary artery anomalies are classified into 3 major groups: first: anomalies that predispose to ischemia such as coronary vessels originating from right atrium; second: anomalies that do not predispose to ischemia, as in anomalous origin of right coronary artery from posterior sinus of Valsalva; third: anomalies that might predispose to ischemia as in congenital absence of LAD \[[@R4]\]. It is usually diagnosed incidentally by CT coronary angiography, interventional coronary angiography, and in some severe and unusual cases, by transthoracic or transesophageal echocardiography, particularly in pediatric populations \[[@R4]\].
In our case, the patient has multiple anomalies: congenital absence of the left main coronary artery (LMCA) which was reported in 0.41%−0.67% of the cases \[[@R5]\]. It is, in most of the cases, clinically benign. However, an association with myocardial infarction and syncope were reported \[[@R6]\]. The second one is anomalous origins of left anterior descending artery (LAD) and left circumflex artery (LCX) from right sinus of Valsalva, were report separately as 0.03% and 0.032% respectively. The combination of both is extremely rare. The clinical significance of this anomaly is according to the course of the LAD. The possibilities of LAD course are: pre-pulmonic anterior to the right ventricular outflow tract which rarely causes ischemia; retro-aortic posterior to the aortic root, as in our case, usually benign; inter-arterial between the aorta and pulmonary artery, often associated with unfavorable outcomes; trans-septal subpulmonic course, which rarely causes ischemia; and retro-cardiac in the posterior atrioventricular groove, which predisposes to coronary disease \[[@R6]\]. The other aspect of the anomaly management is technical difficulties of coronary angiography and percutaneous coronary intervention due to unexpected positions and difficult angles.
4.. Conclusion {#S4}
==============
We reported a case of combined anomalies of the coronary arteries including absence of Left Main Coronary Artery with Anomalous Origin of Left Anterior Descending and Left Circumflex Arteries that presented with NSTEMI. This combination of anomalies is exceedingly rare.
While congenital coronary artery anomalies are quite rare, these entities could result in acute coronary syndrome with technical difficulties during percutaneous coronary interventions. Our case report highlights the need to keep that possible diagnosis of congenital coronary anomalies in mind while managing patients with acute myocardial infarction.
This work is supported, in part, by the efforts of Dr. Moro O. Salifu M.D., M.P.H., M.B.A., M.A.C.P., Professor and Chairman of Medicine through NIH Grant number S21MD012474.
{#F1}
{#F2}
{#F3}
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Introduction
============
The epidemic of diabetes is worsening. The Centers for Disease Control estimates that over 23 million Americans suffer from diabetes, with an incidence of over 1.4 million new cases of diabetes every year.[@b1-vhrm-5-225] As the prevalence of diabetes increases, there is greater concern for the vascular complications that accompany diabetes. Insulin remains the most potent medication available to treat diabetes, and arguably to prevent diabetic complications. Some advocate using insulin much sooner in the course of diabetes.[@b2-vhrm-5-225] On the other hand, others believe that insulin may actually be harmful in obese type 2 diabetic subjects because it increases body fat, which may exacerbate insulin resistance.[@b3-vhrm-5-225],[@b4-vhrm-5-225] This review explores the interplay between insulin and the vascular system with special emphasis on the rapid-acting insulin analogs. Because of the heterogeneity of the literature, this paper will refer to vascular events defined very loosely, including peripheral events, cardiovascular events, cerebrovascular events, and death due to vascular causes.
Is insulin harmful?
===================
The relationship between insulin and vascular events is debatable. One study has associated higher serum insulin levels as an independent predictor of cardiovascular events. The Helsinki Policeman study was a retrospective study of 22-year mortality data of 970 non-diabetic men without coronary artery disease. The authors found an increase in cardiovascular mortality among subjects with higher serum insulin levels.[@b5-vhrm-5-225] A larger study, the Paris Protection Study, followed 7246 non-diabetic men without coronary artery disease for an average of 63 months and concluded that higher fasting plasma insulin levels were an independent risk factor for the development of coronary artery disease.[@b6-vhrm-5-225]
Although these studies may suggest that elevated circulating insulin is a direct cause of vascular disease, this does not seem to be true. Subjects with insulin producing neoplasms do not have an increase in clinically overt atherosclerotic disease.[@b7-vhrm-5-225] This suggests that factors other than hyperinsulinemia are responsible for an increased risk of vascular disease. Insulin resistance is often associated with hypertension, lipid abnormalities, and obesity, all of which are thought to contribute much more than hyperinsulinemia to the development of vascular disease.[@b8-vhrm-5-225] In fact, some have proposed that insulin resistance is another symptom associated with the metabolic syndrome and cardiovascular disease, rather than the root cause of cardiovascular disease. Cardiologists performing a population study on 322 healthy adults have described an "insulin gradient."[@b9-vhrm-5-225] These researchers noted a direct correlation between body weight and blood pressure and insulin levels. The heavier their subjects were, the higher the blood pressure and insulin level. Thus, the higher cardiovascular rates seen with higher insulin levels may be caused by an increase in other risk factors such as hypertension, rather than insulin itself.
Such a relationship is often ignored in articles that claim an association between insulin resistance and cardiovascular events. For example, in the Veteran Affairs-HDL Intervention Trial (VA-HIT), the authors concluded that "the occurrence of a new cardiovascular event was dependent on ... the presence or absence of insulin resistance."[@b10-vhrm-5-225] However, it is interesting to note that the group with insulin resistance had an average body mass index of over 31 while those without insulin resistance had an average body mass index of only 27.[@b10-vhrm-5-225] Furthermore, there was no reporting of blood pressure values. Although it is possible that higher levels of insulin increased the rate of cardiovascular events in this population, it is equally plausible than other factors such as hypertension were responsible.
This then begs the question of whether hypertension is a byproduct of hyperinsulinemia. Although no unequivocal data to answer this question exist, multiple experiments in dogs have shown this not to be the case. There were no pressor effects noted in normal dogs infused with insulin, or in dogs with a 70% reduction in kidney mass on a high salt diet.[@b11-vhrm-5-225] Interestingly, chronic hyperinsulinemia actually caused a reduction in total peripheral vascular resistance as well as arterial pressure.[@b11-vhrm-5-225] This decrease in vascular resistance disappeared when the dogs were made obese via a high-fat diet.[@b11-vhrm-5-225] This series of experiments suggests that in dogs, hyperinsulinemia does not cause hypertension. Whether or not this translates into humans remains to be seen. However, a cross-sectional relational study found obesity to be an independent risk factor for left ventricular hypertrophy, but not insulin resistance or fasting insulin levels.[@b12-vhrm-5-225] These data seem to suggest that obesity and its complications are responsible for cardiovascular risk, not merely high insulin levels.
Although there is a theoretical concern of a mitogenic effect of insulin analogs, this concern appears limited to the long-acting insulin glargine.[@b13-vhrm-5-225] Glargine appears to be a more potent stimulus of DNA synthesis in human osteosarcoma cell lines than the native insulin molecule; the rapid-acting analogs appear to be equivalent to regular insulin.[@b14-vhrm-5-225] Some have postulated that mitogenic potency is related to the half-life of the receptor--ligand binding complex, which would explain why the rapid-acting analogs do not appear to have as much theoretical mitogenic effect as the long-acting analog glargine.[@b15-vhrm-5-225]
Does intensive diabetes therapy with insulin improve vascular events?
=====================================================================
Recently, there have been several large prospective studies examining the relationship between the treatment of hyperglycemia and vascular complications. The first major study to associate a decrease in vascular events with glycemic control was the United Kingdom Prospective Diabetes Study (UKPDS). This prospective observational study included 4585 type 2 diabetic subjects. It concluded that each 1% reduction in mean HbA~1c~ was associated with a risk reduction of 21% for deaths related to diabetes, 14% for myocardial infarction, and 37% of microvascular complications.[@b16-vhrm-5-225]
These patients were followed for another 10 years without diabetic treatment manipulation by the researchers. Despite the difference in HbA~1c~ disappearing after one year, the group intensively treated initially with insulin or sulfonylurea still had a 24% risk reduction for microvascular disease, a 15% in risk reduction for myocardial infarction, and a 13% reduction for death from any cause.[@b17-vhrm-5-225] Because patients initially treated with metformin also had risk reductions, the authors of this study concluded that this legacy effect was not the result of insulin, but rather a possible reduction in advanced glycation end products from the initial intensive treatment of hyperglycemia.[@b18-vhrm-5-225]
The Diabetes Control and Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) illustrated that intensive insulin therapy in type 1 diabetic patients reduced the major diabetic complications of neuropathy, nephropathy, and retinopathy.[@b19-vhrm-5-225] Intensive insulin therapy was associated with less cardiovascular disease as evidenced by decreased intima-media thickness and lower coronary artery calcium accumulation.[@b20-vhrm-5-225] Intensive insulin treatment also decreased the risk of any cardiovascular disease by 42% and the risk of non-fatal stroke, myocardial infarction, or cardiovascular death by 57% in type 1 diabetic subjects.[@b21-vhrm-5-225]
Intensive insulin treatment was also shown to be beneficial to those with the worst vascular disease. Diabetic patients who suffered an acute myocardial infarction had an absolute reduction in mortality of 11% when treated with intensive insulin therapy.[@b22-vhrm-5-225]
Other studies have been less convincing. The recent Action in Diabetes and Vascular Disease (ADVANCE) trial was aimed to address the effect of intensive glucose control on vascular outcomes in subjects with type 2 diabetes. The researchers randomly assigned 11,140 type 2 diabetic patients to standard glucose control or intensive glucose control using to a HbA~1c~ of 6.5% or less.[@b23-vhrm-5-225] Although the first line drug was a sulfonylurea, 40.5% of subjects in the intensive arm and 24.1% of subjects in the standard arm ended up on insulin.[@b23-vhrm-5-225] After a median of 5 years of follow-up, the researchers found that intensive glucose control led to a reduction in nephropathy (4.1 versus 5.2%), but had no effect on retinopathy, major macrovascular events, or death from cardiovascular causes.[@b23-vhrm-5-225]
The Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial also attempted to address the relationship between vascular events and glucose control in patients with type 2 diabetes. One distinguishing feature of this trial is that all subjects had to have either established cardiovascular disease or additional cardiovascular risk factors. Therefore, it was aimed for secondary instead of primary prevention of vascular disease. 10,251 subjects were randomly assigned to intensive therapy (HbA~1c~ \< 6%) or standard therapy (HbA~1c~ 7.0%--7.9%).[@b24-vhrm-5-225] This portion of the study (a blood pressure arm is still ongoing) was discontinued after a mean of 3.5 years of follow up due to an increase in total mortality (257) in the intensive group as opposed to the standard group (203).[@b24-vhrm-5-225] Interestingly, the number of events in the primary outcome (non-fatal myocardial infarction, non-fatal stroke,
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Introduction {#Sec1}
============
A hexanucleotide repeat expansion mutation in *chromosome 9 open reading frame 72* (*C9orf72*) is the most common known genetic cause of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of corticospinal, brainstem and spinal motor neurons. A *C9orf72* mutation underlies approximately 40% of familial and 5% of sporadic ALS cases^[@CR1]--[@CR3]^. This hexanucleotide expansion is also found in approximately 10% of cases of a second neurodegenerative disease, frontotemporal dementia (FTD), a common cause of dementia in middle-aged patients^[@CR3]--[@CR5]^. Mutations in *C9orf72* are a rare risk factor for several additional neurologic and psychiatric disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease phenocopy patients, multiple system atrophy, depressive pseudodementia, bipolar disorder, and schizophrenia^[@CR6]--[@CR16]^. However, it is not known how this genetic mutation leads to these cell type-specific neurodegenerative disorders.
Both loss-of-function and gain-of-function mechanisms have been proposed to mediate *C9orf72*-linked ALS^[@CR17],\ [@CR18]^. Several studies have demonstrated decreased *C9orf72* transcript and protein levels in patients with ALS and FTD^[@CR19]--[@CR23]^. Deletion of the mouse ortholog of the *C9orf72* gene (3110043O21Rik, referred to here as *C9orf72*) has been reported to shorten lifespan and induce modest motor deficits in some, but not all mouse models, and cause profound dysregulation of the immune system^[@CR24]--[@CR29]^. Gain-of-function toxicity of the repeat expansion induced by sense and anti-sense RNA transcripts, as well as dipeptide proteins generated through repeat-associated non-ATG (RAN) translation, are also thought to contribute to neurodegeneration^[@CR17],\ [@CR18]^. Several transgenic mouse lines recently developed using patient-derived gene constructs demonstrate that *C9orf72* repeat expansions induce age-dependent accumulation of RNA foci and dipeptide repeat proteins, along with neurodegeneration and behavioral abnormalities that at least partially recapitulate human disease^[@CR26],\ [@CR28],\ [@CR30],\ [@CR31]^. Although the complex mechanisms underlying *C9orf72*-related disease have not been resolved, understanding the expression pattern of *C9orf72* in the central nervous system (CNS) is not only important for understanding the pathogenesis of ALS, but is also relevant to the wide spectrum of *C9orf72*-associated diseases.
Although the signature of ALS is the loss of corticospinal, brainstem, and spinal motor neurons, multiple cell types have been shown to contribute to the pathogenesis of the disease. Non-neuronal cells including oligodendrocytes, astrocytes, and microglia are also critical players in the pathogenesis of ALS^[@CR17]^. Although some progress has been made in understanding the cell-type specific expression of *C9orf72* ^[@CR26],\ [@CR28],\ [@CR32]^, a comparison of the distribution of *C9orf72* expression across different neuronal and glial cell types in relevant regions of the brain and spinal cord is still lacking. Whether *C9orf72* promoter activity is specifically enriched in affected corticospinal neurons, spinal motor neurons, or oligodendrocytes in regions implicated in ALS is not yet fully understood.
Here, we systematically mapped the promoter activity of the mouse ortholog of *C9orf72* in a genetically engineered strain of mice containing a targeted *LacZ* insertion under the control of the *C9orf72* native promoter. Through quantitative comparisons among different types of neurons and glial cells labelled with retrograde neuronal tracers and cell type-specific markers, we demonstrate that mouse *C9orf72* promoter activity, although widespread throughout the brain and spinal cord, is specifically enriched in corticospinal and spinal motor neurons and in oligodendrocytes, subsets of cells known to undergo degeneration in ALS, in regions affected by ALS. In contrast, *C9orf72* promoter activity was detected in only a small percentage of microglial cells and even fewer astrocytes. Thus, these data suggest that, despite widespread expression, *C9orf72* promoter activity reflects the patterns of degeneration typically seen in this disease, consistent with direct cell autonomous toxicity.
Results {#Sec2}
=======
The distribution of *C9orf72* promoter activity and cellular density are highly correlated in the CNS {#Sec3}
-----------------------------------------------------------------------------------------------------
Mice have a single gene, 3110043O21Rik (here referred to as *C9orf72*), which is orthologous to human *C9orf72* ^[@CR32]^. To determine the distribution of *C9orf72* promoter activity in the central nervous system, we generated chimeric mice using several mouse embryonic stem cell lines heterozygous for an allele with a *LacZ* insertion in the *C9orf72* locus generated by the Knock Out Mouse Project (KOMP)^[@CR29],\ [@CR32]--[@CR35]^. The *LacZ* insertion results in deletion of exons 2--6 of the mouse *C9orf72* gene, producing a knockout allele (Supplementary Fig. [1a](#MOESM1){ref-type="media"}). Analysis of RNA-sequencing (RNA-seq) data from mice generated using the same targeting cassette and embryonic stem (ES) cell background indicates that the transcript structure is largely maintained between wild type (WT) and *C9orf72* ^*LacZ*/+^ mice^[@CR28]^ (Supplementary Fig. [1b](#MOESM1){ref-type="media"}). Although WT transcripts have two alternative starts, exon 1a or 1b, while the *LacZ* allele appears to use only exon 1a, there is no reported evidence for differential usage of exon 1a and 1b among different cell types indicating that the *LacZ* reporter likely reflects the pattern of the wild type gene expression. The heterozygous mice have a normal phenotype until six months of age, after which a fraction of *C9orf72* ^*LacZ*/+^ mice exhibit an age-dependent decrease in survival, with approximately 20% of the heterozygotes dead by 600 days^[@CR29]^. We therefore used young, six to eight-week-old heterozygotes to assess the distribution of *LacZ* as a reporter for *C9orf72* promoter activity.
We confirmed that *C9orf72* promoter activity was not limited to areas known to degenerate in ALS and FTD. X-gal staining to assess regions of β-galactosidase (β-gal) expression (encoded by *LacZ*) revealed widespread promoter activity throughout the brain and spinal cord. The regions with the most intense X-gal signals in the brain corresponded to regions with high cell density, such as the dentate gyrus of the hippocampus and the granular layer of the cerebellum (Fig. [1a](#Fig1){ref-type="fig"}). Regions with the weakest signals corresponded to areas with low cell densities such as the molecular layer of the cerebellum and the corpus callosum (Fig. [1a](#Fig1){ref-type="fig"}). We also observed broad X-gal staining in primary motor cortex (Fig. [1a](#Fig1){ref-type="fig"}) and in the spinal cord (Fig. [1b](#Fig1){ref-type="fig"}). These results suggest that the distribution of *C9orf72* promoter activity largely follows cellular density across brain regions. To more directly test this hypothesis, we compared the distribution of cells stained with the nuclear marker, DAPI, and an antibody specific to β-galactosidase (β-gal; Supplementary Fig. [1c](#MOESM1){ref-type="media"}) and found that the distribution of β-gal signal correlated with the overall cellular density across layers in primary motor cortex, primary somatosensory cortex, and spinal cord grey matter (Fig. [1c-d](#Fig1){ref-type="fig"}). Together, these results indicate that *C9orf72* promoter activity is widely distributed in the CNS, consistent with that reported in previous studies^[@CR26],\ [@CR27],\ [@CR32]^, and largely correlates with overall cellular density.Figure 1The distribution of *C9orf72* promoter activity follows overall cellular density in the central nervous system. (**a**) The overall distribution of *C9orf72* promoter activity in a parasagittal brain section from a *C9orf72* ^*LacZ/*+^ mouse revealed using X-gal staining. *C9orf72* promoter activity was high in brain regions with high cell density (dentate gyrus of the hippocampus, cerebellar granule cell layer, two left panels), low in regions with low cell density (cerebellar molecular cell layer and corpus callosum, middle panels), and medium in motor cortex (rightmost panel). (**b**) The overall distribution of *C9orf72* promoter activity in a coronal section of the lumbar spinal cord. X-gal staining was seen in both the dorsal and ventral horns (left and right panels) (**c**) Representative images showing the distribution of immunofluorescence staining for β-gal in primary motor (top) and somatosensory (bottom) cortex of *C9orf72* ^*LacZ/*+^ mice concurrently labelled with the nuclear stain, DAPI. The intensities of the β-gal (red) and DAPI (blue) signals were summed along the horizontal axis in the region indicated by the white boxes (n = 3 mice) and the intensity values plotted (mean ± SEM). (**d**) A similar analysis was performed for lumber spinal cord (*n* =
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{#sp1 .457}
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Introduction {#Sec1}
============
Superparamagnetic iron oxide nanoparticles (SPION) are widely used for biomedical applications, including magnetic resonance imaging (MRI), magnetic particle imaging (MPI), magnetic fluid hyperthermia (MFH), separation of biomolecules, and targeted drug and gene delivery \[[@CR1]--[@CR3]\]. This widespread list of applications not only results from the magnetic properties of SPION, but also from the capability of synthesizing them in different sizes and shapes. For all of the above applications, SPION should ideally have a high magnetization value, a size below 100 nm and a narrow size distribution \[[@CR4], [@CR5]\].
SPION are typically based on Fe~3~O~4~ and/or Fe~2~O~3~. They can be synthesized using various methods, such as co-precipitation \[[@CR5], [@CR6]\], thermal decomposition \[[@CR7]\], sol--gel \[[@CR8]\], microemulsion \[[@CR9]\], hydrothermal \[[@CR10]\], and electrochemical synthesis \[[@CR11]\]. The co-precipitation technique is among the most successful, most commonly employed and most cost-effective methods for high-yield synthesis. However, strategies are needed to overcome the most important limitation of this method, i.e. the very broad particle size distribution of the resulting SPION mixture \[[@CR5], [@CR6]\].
In this study, we describe a straightforward, easily implementable and broadly applicable centrifugation protocol to obtain relatively monodisperse SPION from a polydisperse starting mixture prepared using the co-precipitation technique. As a result of their refined size distribution, the obtained optimized SPION dispersions showed substantially improved performance in MRI, MPI and MFH compared to the crude starting formulation, as well as to commercial SPION products, such as Resovist® and Sinerem®.
In this context, it is important to keep in mind that not the centrifugation protocol per se, but the eventual development of a SPION formulation with a very well-defined size and with a very narrow size distribution (and its consequent more optimal use for diagnostic and therapeutic purposes) is the objective of our work. Thus far, no systematic study has been published on SPION size-isolation via sequential centrifugation, and no systematic analysis is available in which the performance of five size-isolated SPION sub-fractions (and clinically/commercially relevant controls) is head-to-head compared in MRI, MPI and MFH setups.
Results and discussion {#Sec2}
======================
SPION preparation and size-isolation {#Sec3}
------------------------------------
Prototypic citrate-coated SPION were prepared via the standard co-precipitation technique, under nitrogen atmosphere \[[@CR5], [@CR6]\] (see "[Experimental](#Sec12){ref-type="sec"}" section for details). Based on this highly polydisperse starting batch, which we refer to as the "crude sample", five sequential rounds of centrifugation were performed to obtain much more monodispersed SPION subfractions. To this end, as depicted schematically in Fig. [1](#Fig1){ref-type="fig"}, the crude sample was transferred into 1.5 ml Eppendorf tubes and centrifuged at 14,000 rpm for 20 min. The resulting 1 ml of supernatant was collected and referred to as the "C1 sample". Subsequently, 0.1 ml of the bottom compartment in the Eppendorf tube that contained the largest nanoparticle fraction was resuspended in water. The obtained dispersion was then again centrifuged, the top 1 ml was collected as the "C2 sample", and the bottom 0.1 ml was again resuspended and re-centrifuged. These steps were sequentially repeated to obtain five fractions of relatively monodisperse SPION samples. These fractions are referred to as C1--C5. The crude starting mixture, Resovist® and Sinerem® are referred to as C, R and S, respectively. Multiple systematic experiments were performed to identify the optimal centrifugation speeds and times for obtaining monodispersed SPION with well-defined sizes. The optimum conditions for size-isolation are presented in Fig. [1](#Fig1){ref-type="fig"}. The production efficiencies of the size-isolated fractions C1, C2, C3, C4 and C5 were approximately 7, 29, 23, 18 and 11%, respectively.Fig. 1SPION size-isolation via sequential centrifugation. Schematic overview of the centrifugation protocol to obtain monodispersed SPION with different hydrodynamic diameters from a crude mixture of polydisperse SPION. The polydisperse SPION sample (C) was transferred into 1.5 ml Eppendorf tubes and centrifuged at 14,000 rpm for 20 min. The resulting 1 ml of supernatant was collected (C1). 0.1 ml of the bottom compartment in the Eppendorf tube was resuspended in water and again centrifuged, and the top 1 ml was collected (C2). These steps were repeated multiple times, with optimized centrifugation times and speeds, to obtain three additional fractions of monodisperse SPION samples (C3--C5). The different fractions were subsequently analyzed for magnetic resonance imaging (MRI), magnetic particle imaging (MPI) and magnetic fluid hyperthermia (MFH) performance, and compared to the crude sample (C), to Resovist® and to Sinerem®
Despite the large number of previous publications describing the synthesis of iron oxide nanoparticles, the tools and technologies for their size separation are relatively limited. Techniques employed to control average particle size and polydispersity can be based on the use of magnetic/electric fields, porous media, and mass- and density-based purification \[[@CR12]--[@CR14]\]. Fortin and colleagues for instance synthesized citrate-coated nanocrystals of maghemite and cobalt ferrite by alkaline co-precipitation, and size-sorted the nanoparticles by successive electrostatic phase separation \[[@CR15]\]. Magnetic field-flow fractionation (MFFF) uses a homogeneous external magnetic field applied orthogonal to the direction of flow, to achieve efficient separation of particles \[[@CR12]\]. Nonmagnetic size-exclusion chromatography (SEC) is another frequently used method for size separation of iron oxide nanoparticles. The fractions separated by SEC and MFFF have similar size distributions. However, MFFF is faster and has a higher capacity \[[@CR12], [@CR16]\]. In addition to the above techniques, differential magnetic catch-and-release (DMCR) has recently been established to size-sort magnetic nanoparticles. DMCR, like MFFF, relies on an external magnetic field to separate magnetic species \[[@CR17]\]. High-gradient magnetic separation (HGMS) is a column flow method used to isolate iron oxide nanoparticles from a nonmagnetic medium \[[@CR18]\]. Capillary electrophoresis (CE) is used for the separation of colloidal nanoparticles in an electric field. CE requires specialized equipment, because of the high electric field. Electrical field-flow fractionation (ElFFF) separates iron oxide nanoparticles based on their size and electrophoretic mobility but without the drawbacks of CE \[[@CR12], [@CR16]\]. As compared to the above techniques, the here presented centrifugation method is somewhat more time- and labor-intensive, but it is also easier to perform and more broadly applicable, because it does not require specialized equipment.
Particle size, size distribution and surface charge {#Sec4}
---------------------------------------------------
Figure [2](#Fig2){ref-type="fig"} shows the results obtained using TEM, DLS and NTA on the size and size distribution of the SPION formulations prepared and evaluated in this study. The reported TEM values which correspond to the average size were calculated on the basis of manually measuring at least 100 randomly chosen particles, using Image SP Viewer software. The average core sizes of the samples C1, C2, C3, C4 and C5 were 7.7 ± 1.6, 10.6 ± 1.8, 13.1 ± 2.2, 15.6 ± 2.8 and 17.2 ± 2.1 nm, respectively (Fig. [2](#Fig2){ref-type="fig"}a, b). This indicates that all five fractions are superparamagnetic, as SPION typically present superparamagnetic behavior when their core size is below 20 nm \[[@CR5]\]. The corresponding average hydrodynamic diameters obtained by DLS-based on intensity---for the five samples were 26.3 ± 1.2, 49.4 ± 1.1, 64.8 ± 2.1, 82.1 ± 2.3 and 114.6 ± 4.4 nm (Fig. [2](#Fig2){ref-type="fig"}c). The average sizes obtained using NTA were comparable to the values observed in DLS (Fig. [2](#Fig2){ref-type="fig"}d). The numerical values corresponding to the results presented in Fig. [2](#Fig2){ref-type="fig"}b--d are provided in Additional file [1](#MOESM1){ref-type="media"}: Table S1. The fact that the TEM sizes are smaller than those obtained via DLS and NTA can be explained by keeping in mind that DLS and NTA measure the hydrodynamic diameter of the citrate-coated SPION in aqueous solution incorporating surface-bound water layers in their measurement, while TEM determines the actual core size of dried nanoparticle formulations.Fig. 2Effect of sequential size-isolation on SPION size and size distribution. **a** TEM images and size distributions obtained by TEM. **b**--**d** Analysis of nanoparticle size obtained using TEM, DLS and
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(Current Biology *24*, 2018--2024; September 8, 2014)
A reader has brought to our attention a labeling error in Figure 2 of this manuscript as originally published online and in print: the two arrows pointing upward under panels D4 and D5 should have been labeled "touch." This error has now been corrected in Figure 2 of the article online. The authors apologize for any confusion this error may have caused.
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1. Introduction {#sec1-nanomaterials-08-00083}
===============
Cancer is one of the serious concerns around the world and it is one of the main causes of death worldwide \[[@B1-nanomaterials-08-00083]\]. In a press release by the World Health Organization in 2014, it was reported that cancer accounted for 8.2 million deaths worldwide in 2012 with lung, breast and colorectal cancers identified as most common occurrences worldwide in the year 2012 \[[@B2-nanomaterials-08-00083]\]. This was further supported by a press release by the World Health Organization in 2015. According to the Cancer statistics 2017, in the United States alone, about 1,688,780 new cancer cases and 600,920 cancer deaths are projected to occur \[[@B3-nanomaterials-08-00083]\]. Current cancer treatments rely on radiation and chemotherapeutic agents that work by killing rapidly dividing cells in the body. The main drawback of conventional chemotherapy is the adverse effects on the body as it cannot deliver selective action specifically to the cancer cells, thus damaging the surrounding normal healthy cells or rapidly dividing healthy cells such as the cells of gastrointestinal tract, bone marrow, hair follicles, causing issues like cardiac, hepatic, pulmonary, renal and gastrointestinal toxicities \[[@B4-nanomaterials-08-00083],[@B5-nanomaterials-08-00083],[@B6-nanomaterials-08-00083]\]. Drug delivery systems offer numerous advantages over tradition chemotherapy such as targeted delivery at disease site, sustained release leading to prolonged bioavailability, lower dosage requirement and improved drug solubility among others \[[@B7-nanomaterials-08-00083],[@B8-nanomaterials-08-00083],[@B9-nanomaterials-08-00083],[@B10-nanomaterials-08-00083],[@B11-nanomaterials-08-00083],[@B12-nanomaterials-08-00083]\]. Significant impact has been made by the application of nanotechnology in medicine for theranostic agents development which can diagnose and cure the diseases simultaneously \[[@B13-nanomaterials-08-00083],[@B14-nanomaterials-08-00083]\]. Variety of nanocarriers have been designed and successfully applied for the delivery of the therapeutic agents such as graphene oxide, polymers-based delivery systems, layered double hydroxides, gold nanoparticles, multifunctional nanoparticles and iron oxide magnetite nanoparticles \[[@B8-nanomaterials-08-00083],[@B15-nanomaterials-08-00083],[@B16-nanomaterials-08-00083],[@B17-nanomaterials-08-00083],[@B18-nanomaterials-08-00083],[@B19-nanomaterials-08-00083],[@B20-nanomaterials-08-00083],[@B21-nanomaterials-08-00083]\]. Polymer coated iron oxide magnetite nanoparticles (Fe~3~O~4~) have received much of the attention as the novel cancer therapeutics vectors because of their unique properties such as ease of preparation, easily scalable production, sustained release properties, high encapsulation capacity, biocompatibility with normal cells and tissues, easier surface modification and stable magnetic nature \[[@B9-nanomaterials-08-00083],[@B22-nanomaterials-08-00083],[@B23-nanomaterials-08-00083],[@B24-nanomaterials-08-00083],[@B25-nanomaterials-08-00083],[@B26-nanomaterials-08-00083]\]. All of these characteristics make iron oxide magnetite nanoparticles (Fe~3~O~4~) an ideal candidate for cancer delivery vectors. However, the large surface area to volume ratio and the dipole-dipole attraction causes the agglomeration of nanoparticles, hence the need for surface polymer modification. The magnetic nanocarriers need to be stable in normal saline and water at neutral pH for biological, medical diagnostic and therapeutic applications \[[@B23-nanomaterials-08-00083],[@B27-nanomaterials-08-00083]\]. To avoid agglomeration, the surface of iron oxide magnetite nanoparticles is coated with polymer which also helps in sustained release and better stability in physiological conditions. The polymer poly (ethylene-glycol) (PEG) has been widely applied in drug delivery and is being utilized as protective layer for the nanoparticles. The monomer unit of PEG contains both polar oxygen and two methylene group which are non-polar. This dual polarity makes PEG to be soluble in variety of polar and non-polar solvents and has been widely used to improve aqueous solubility of hydrophobic drugs \[[@B28-nanomaterials-08-00083],[@B29-nanomaterials-08-00083]\]. Gallic acid (3,4,5-trihydroxybenzoic acid) is a bioactive compound found in plants and foods such as white tea, witch hazel and it has been reported to possess antioxidant, anti-inflammatory, anticancer properties and is also known for its protective activity on normal cells which makes them pivotal for cancer therapy \[[@B30-nanomaterials-08-00083]\]. In this study we have redesigned an anticancer nanocomposite formulation of Gallic acid loaded on iron oxide magnetite nanoparticles coated with polyethylene glycol (Fe~3~O~4~-PEG-GA) with improved drug loading and better sustained release properties and was characterized by X-ray diffraction (XRD), dynamic light scattering (DLS), in vitro cytotoxicity assay and drug loading quantification. In previous study we tested Gallic acid nanocomposite formulation \[(P-Fe~3~O~4~-PEG-GA) (in formula P stands for previous)\] against MCF-7, a breast cancer cell line with the IC~50~ value of 11.61 ± 0.12 µg/mL and human normal lung fibroblast cells MRC-5 was used as a model for normal cell in which more than 80% cell viability was observed after 72 h incubation \[[@B31-nanomaterials-08-00083]\]. In this study we tested the free drug GA, empty nanocarrier and the anticancer nanocomposite formulation Fe~3~O~4~-PEG-GA against A549 human lung carcinoma cells, *HT29* human colon adenocarcinoma cell line, repeated on MCF-7 breast cancer cells and normal 3T3 cells for incubation period of 24, 48 and 72 h.
2. Results {#sec2-nanomaterials-08-00083}
==========
2.1. Physicochemical Characterization {#sec2dot1-nanomaterials-08-00083}
-------------------------------------
### 2.1.1. X-ray Diffraction (XRD) Analysis {#sec2dot1dot1-nanomaterials-08-00083}
[Figure 1](#nanomaterials-08-00083-f001){ref-type="fig"}a shows the XRD patterns of iron oxide magnetite nanoparticles (Fe~3~O~4~) alone, poly ethylene glycol (PEG) and anticancer nanocomposite Fe~3~O~4~-PEG-GA. Iron oxide magnetite nanoparticles (Fe~3~O~4~) showed the six characteristics peaks ascribed to Brag reflections due to (220), (311), (400), (422), (511), and (440) and these peaks can be observed at 2*θ* = 30.16°, 35.95°, 43.34°, 54.17°, 57.27° and 62.98° respectively. The pure polymer PEG showed two main characteristics high intensity peaks at about 2*θ* = 19.3° and 23.5°. The pure gallic acid (GA) has been reported to show many peaks between the 2*θ* of 10--50° as reported previously \[[@B31-nanomaterials-08-00083],[@B32-nanomaterials-08-00083]\]. In the XRD patterns of the nanocomposite Fe~3~O~4~-PEG-GA characteristic peaks of iron oxide magnetite nanoparticles (Fe~3~O~4~), PEG and of the pure drug peaks are present with slight lesser intensity. The presence of characteristic peaks of Fe~3~O~4~, PEG and GA in the final anticancer nanocomposite confirms the successful formation of the nanocomposite Fe~3~O~4~-PEG-GA.
### 2.1.2. Dynamic Light Scattering (DLS) {#sec2dot1dot2-nanomaterials-08-00083}
The size of the anticancer nanocomposite Fe~3~O~4~-PEG-GA was determined using Zetasizer by dynamic light scattering (DLS). The sample was dispersed in water and sonicated for 15 min and then analyzed with Zetasizer. The sample was found to have narrow size distribution between 5 and 12 nm with average particle size of 10 nm as shown in [Figure 1](#nanomaterials-08-00083-f001){ref-type="fig"}b. This size distribution is much smaller compared to previously reported (P-Fe~3~O~4~-PEG-GA) which had wide distribution 20--50 nm with average size of 31.44 nm \[[@B31-nanomaterials-08-00083]\].
### 2.1.3. In vitro Release Studies {#sec2dot1dot3-nanomaterials-08-00083}
Release behavior of GA from the nanocomposite Fe~3~O~4~-PEG-GA was conducted in human body simulated buffer saline (PBS) solution of pH 7.4 (human blood pH) and in pH 4.8 (intracellular lysosomal pH) as shown in [Figure 1](#nanomaterials-08-00083-f001){ref-type="fig"}c. For the release studies 10 mg of the nanocomposite was put in 10 mL PBS solution of pH 7.4 and pH 4.8 in thermostat at 37 °C with constant shaking. At different time points 3 mL aliquot was taken out and replaced with new buffer of either solution of pH 7.4 and pH 4.8 respectively and analyzed for the percentage release using UV-Vis spectrophotometer (Waltham, MA, USA).
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Background
==========
Fusarium wilt of *Arabidopsis thaliana* is an ideal pathosystem for mapping, identifying and characterizing genes responsible for host resistance to vascular wilt fungi. *A. thaliana*, which is the preeminent subject of plant molecular genetic and genomic studies, is susceptible to infection by three phylogenetically-distinct pathogenic forms, or formae speciales, of the soil-borne fungus *Fusarium oxysporum*\[[@B1],[@B2]\]. In the field, *F. oxysporum* forma specialis *conglutinans* (FOC), *F. oxysporum* forma specialis *raphani* and *F. oxysporum* forma specialis *matthioli* (FOM) are isolated from diseased *Brassica* species, radish (*Raphanus sativus*) and garden stock (*Matthioli incana*), respectively \[[@B3]\]. Fusarium wilt of *A. thaliana* recapitulates the development of disease symptoms in field hosts \[[@B1]\].
The response of different accessions of *A. thaliana* to different formae speciales varies from complete resistance to ready susceptibility \[[@B1]\]. For example, the standard laboratory accession Columbia-0 (Col-0) is completely resistant to FOM but expresses only partial resistance to FOC1. Taynuilt-0 (Ty-0), on the other hand, is susceptible to FOM but also expresses partial resistance to FOC1.
Two strategies are used to map genes responsible for phenotypic variation in populations \[[@B4]-[@B6]\]. When the population of interest is wild and results from an indeterminate number of undefined crosses, a genome-wide association (GWA) study uses evidence of linkage disequilibrium to associate sequence polymorphisms within or near the genes responsible for the trait. Enabling GWA studies in the plant *A. thaliana* is the primary motivation for the 1001 Genomes Project, which has generated whole genome sequence for hundreds of wild accessions of *A. thaliana*\[[@B7],[@B8]\]. Indeed, the detection of functional sequence diversity in *A. thaliana* using GWA is reported \[[@B9],[@B10]\]. However, GWA studies rarely detect more than a modest fraction of the sequence diversity responsible for variation in existing populations of plant and animal species \[[@B5],[@B9],[@B11]\].
Genetic linkage may be used to map the genes associated with a trait to chromosomal intervals. However, this approach requires that the studied population is derived from controlled crosses between defined parents; and, only the genetic diversity distinguishing the parents of crosses is detected. Nevertheless, linkage analysis has been a powerful and successful approach for detecting and defining the genes responsible for complex traits in *A. thaliana*\[[@B12]\].
With plant species that readily inbreed, such as *A. thaliana*, recombinant inbred (RI) populations are almost exclusively used to map and define genetic loci underlying natural traits \[[@B12],[@B13]\]. RI populations in their simplest form originate from an outcross between parents with dissimilar genotypes. Unique recombinant genotypes of the parents are captured in dozens to hundreds of RI lines that result from propagating individual F~2~ offspring by self-fertilization and single-seed descent. After several filial generations of inbreeding, RI progeny become largely homozygous and thus true-breeding RI lines. However, the effort to propagate and curate an RI population without introducing selection represents a substantial investment in time and effort before QTL analysis begins. The effort to generate an RI population is offset by the fact that RI lines are immortal and can be retested innumerable times and reused in separate studies but need to be genotyped just once. There are now dozens of published RI populations from crosses between wild accessions of *A. thaliana*\[[@B12],[@B14]-[@B16]\]. Recently, a technique for generating haploid *A. thaliana* has made the generation of doubled haploid lines possible \[[@B17]\]. Like RI lines, doubled haploids are homozygous and thus immortal but require fewer generations to create.
Other mating strategies generate recombinant mapping populations in less time and with less effort than it takes to generate RI lines. In particular, BC~1~ populations are generated from crosses in two successive generations. An initial outcross between parental genotypes produces the F~1~ hybrid, which is then backcrossed to its recurrent parent. Each resulting BC~1~ hybrid inherits a set of non-recombinant chromosomes from the recurrent parent and a set of recombinant chromosomes from the F~1~ hybrid. Because crossovers resulting from single meioses can be unambiguously assigned to recombinant chromosomes, the BC~1~ mating scheme is often used to generate a model population for the evaluation of novel approaches to QTL analysis \[[@B18]-[@B20]\]. In addition, backcrossing is a common feature in traditional breeding schemes that seek to introgress new traits into elite crop varieties \[[@B21]\].
The appeal of BC~1~ populations is undermined by the need for extensive genotyping, and very few studies of natural traits in *A. thaliana* have used BC~1~ populations for genome-wide mapping \[[@B1],[@B12],[@B22]\]. Because each BC~1~ hybrid possesses a unique recombinant genotype, it is necessary to genotype each tested BC~1~ hybrid genome-wide. Without whole genome sequence information for the parents of a BC~1~ population, the discovery of sequence polymorphism and their development into an appropriate set of DNA markers for genome-wide mapping is a time-consuming and laborious process.
Nevertheless, prior genetic analysis of a BC~1~ population shows that the qualitative resistance of Col-0 to FOM is a polygenic trait \[[@B1]\]. Six *RFO* QTLs, accounting for the resistance of Col-0 and susceptibility of Ty-0, segregate in a population generated by crossing Col-0 and Ty-0 and then backcrossing the resistant F~1~ hybrid to its susceptible parent Ty-0. Among *RFO* loci, *RFO1* has the strongest association with resistance to FOM. *RFO1* also appears to interact with three other *RFO* loci, namely *RFO2*, *RFO4* and *RFO6*, because the three interacting loci have significant association only when recombinant BC~1~ hybrids also inherit the Col-0 allele of *RFO1* (*RFO1-C*). *RFO2* is a receptor-like protein (RLP) gene that is homologous to the PSY1 peptide receptor gene, *PSY1R*\[[@B23]\]. The *RFO1*-linked gene At1g79670 is named *RFO1* because the Col-0 sequence of At1g79670, as a transgene, enhances the resistance of Ty-0, and the loss-of-function allele of At1g79670 (*rfo1*) compromises the resistance of Col-0 \[[@B1]\]. At1g79670 is a member of the wall-associated kinase-like kinase subfamily of receptor-like kinase (RLK) genes.
Here, I map Fusarium wilt resistance in two new BC~1~ populations (i) to address whether At1g79670 alone is responsible for resistance attributed to the *RFO1* QTL, including interactions with *RFO2*, *RFO4* and *RFO6*, and (ii) to examine whether the same or different *RFO* QTLs mediate resistance to different formae speciales of *F. oxysporum*. In doing so, I present a methodology for genome-wide genotyping that makes the mapping of complex quantitative traits a routine procedure. Importantly, because whole genome sequence is now available for most studied accessions, the same approach could be applied to crosses between any pair of *Arabidopsis* accessions.
Results
=======
Resistance to FOM in *rfo1*
---------------------------
In prior mapping of resistance to FOM, *RFO1* was the most significant of six *RFO* loci in *A. thaliana*, and *RFO1* was epistatic to, or enhanced the resistance of, three other *RFO* loci \[[@B1]\]. In theory, the *RFO1* QTL could represent one gene or multiple genes. To appreciate whether At1g79670 is responsible for all or part of the resistance attributed to the *RFO1* QTL, resistance to FOM was mapped in a new BC~1~ population that included *rfo1*, which is a loss-of-function allele of At1g79670 resulting from a T-DNA insertion in and deletion of coding sequence in the Col-0 genetic background \[[@B1],[@B24]\]. The same crossing scheme that generated the original Col-0 and Ty-0 (C-T) BC~1~ population, was used to generate the new *rfo1* and Ty-0 (*r*-T) BC~1~ population with the exception that *rfo1* replaced wild type as the Col-0 parent: Crossing *rfo1* and Ty-0 produced the F~1~ hybrid that was then backcrossed to Ty-0. Differences in quantitative resistance in the new *r*-T and original C-T populations would include the contribution of At1g79670.
As in the C-T population, resistance to FOM segregated in the *r*-T population as a polygenic trait, and most BC~1~ hybrids exhibited resistance that was intermediate to that of either parent \[[@B1]\]. Wilt disease in the F~1~ hybrid, Ty-0 parent and 190 BC~1~ hybrids was evaluated using a
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Description
===========
****The *daf-2* gene encodes an insulin-like growth factor/IGF-1 receptor that regulates *C. elegans* embryonic and larval development. It has previously been shown that DAF-2 inhibits neurite regeneration of the GABAergic motor neurons and PVD sensory neurons in an age-dependent fashion (Bryne et al., 2014; Kravtsov et al., 2017). Following injury, the posterior lateral microtubule (PLM) neurons are capable of regenerating through axonal fusion, a highly efficient regrowth mechanism in which separated fragments fuse back together (Ghosh-Roy et al., 2010; Neumann et al., 2011; Neumann et al. 2015; Abay et al., 2017). We previously established that a critical event for axonal fusion to occur is the exposure of injury-induced phosphatidylserine (PS) 'save-me' signals (Neumann et al., 2015). The level of PS exposed increases with advancing age (Abay et al., 2017). To determine if *daf-2* is involved in this age-dependent modulation of PS exposure, we visualised and quantified the level of PS exposed after PLM axotomy using a secreted, tagged version of Annexin V (Neumann et al., 2011; Mapes et al., 2012; Neumann et al. 2015). Mutation of *daf-2* had no effect on PS exposure 1 h post-axotomy, with no significant differences observed on either the distal or proximal axon segments (Table 1).
**Table 1.** Quantification of the relative level of PS exposed 1 h post-axotomy.
---------------- ----------------------------------------------------- ---- ------------------------------------------------------- ----
Genotype PS exposed on distal axon (relative to pre-axotomy) n PS exposed on proximal axon (relative to pre-axotomy) n
wild-type 1.53 ± 0.105 28 1.44 ± 0.0855 28
*daf-2(e1370)* 1.51 ± 0.167 26 1.57 ± 0.166 26
---------------- ----------------------------------------------------- ---- ------------------------------------------------------- ----
Reagents
========
One-day-old adult hermaphrodites were used for all experiments, and were grown under standard conditions at 20°C. The BXN301 \[*daf-2(e1370); smIs95(Phsp-16.2::sAnxV::mRFP); zdIs5(Pmec-4::GFP)*\] strain was used along with the CU4204 \[*smIs95(Phsp-16.2::sAnxV::mRFP); zdIs5(Pmec-4::GFP)*\] control strain. The *daf-2(e1370)* allele has been considered temperature sensitive for the dauer phenotype, but not for the long-lived phenotype. At 20°C, *daf-2(e1370)* animals display a greater than 2-fold increase in lifespan compared to the wild-type (Kenyon et al., 1993). Laser axotomy, microscopy and quantification of data was performed as previously described (Abay et al 2017).
Acknowledgments
===============
We thank Ding Xue for sharing strains.
Funding
=======
This work was supported by National Health and Medical Research Council (NHMRC) Project Grant 1101974.
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All relevant data are within the paper, and a statement to this effect has been included in the final paragraph of the methods section.
Introduction {#sec005}
============
Sexually transmitted infections (STIs) have been a longstanding problem in the United States military. Prior to the discovery of antibiotics, these infections were a significant cause of morbidity and mortality, with a significant toll on operational readiness; in fact, before the invention of modern weaponry, venereal disease was more commonly lethal than combat duty \[[@pone.0167892.ref001]\]. During World War I, STIs were only second to influenza in disabling troops from performing their duties, with 7 million person days lost to STIs in the US army \[[@pone.0167892.ref002]\]. In recent decades, the influence of human immunodeficiency virus (HIV), increased numbers of deployed personnel and enhanced tools for STI screening and diagnosis have necessitated ongoing attention to the burden of STIs.
There are many reasons why STI diagnoses are prevalent among military personnel, including the population of healthy, sexually active, risk taking young adults. Demographic characteristics commonly associated with increased risk for STIs, including African-American race, younger age, education at the high school (as opposed to college) level and residence in endemic areas \[[@pone.0167892.ref003],[@pone.0167892.ref004]\], are over-represented in the military compared to the general population. Thus, even at accession, military recruits show a high rate of STIs prior to starting their service \[[@pone.0167892.ref005]\]. In addition, military service may appeal to individuals prone to risk-seeking behavior, and high prevalence of sexual risk behavior is well-described \[[@pone.0167892.ref006],[@pone.0167892.ref007]\]. Last, military service provides more opportunities for health screening, resulting in higher ascertainment of diagnoses; the disparity is particularly noteworthy among women in the military, who receive annual gonorrhea/chlamydia screening, and therefore have higher rates of STI diagnosis than men, who are only screened for HIV infection.
Despite frequent screening and surveillance, it is often difficult to determine the longitudinal impact of STIs on the military. Several studies have found high rates of reinfection among individuals with STIs \[[@pone.0167892.ref003],[@pone.0167892.ref008]\], but the cumulative risk over an individual's military career is less well-described. This paper takes a novel approach to this problem by selecting cohorts of service-members based on the year they joined the armed forces and evaluating the occurrence of STI diagnoses over the course of their service based on administrative records in the centralized military healthcare system. Our goal was to gain a better understanding of the prevalence of sexually transmitted infections in the DOD, to identify where more data are needed, and to potentially identify areas for intervention.
Materials and Methods {#sec006}
=====================
We retrospectively studied data which were collected from the Defense Medical Surveillance System (DMSS) between 1997--2012. The DMSS is a centralized relational database which contains medical encounter data for the US armed forces, including clinical, laboratory and epidemiologic data \[[@pone.0167892.ref009]\]. We sampled 100,005 subjects in cohorts of 6,667 based on year of entry into military service; all cohorts had at least one year of follow-up. We oversampled females by a 2:1 ratio to balance the likely outcome counts: while females represent 14.5% of military personnel, they experience approximately twice the rate of STIs of males. We obtained demographic information, including age at accession, sex, race, home of record (zip code), marital status and military-specific data including date of accession/discharge, service affiliation, job code/rank and deployment status. Medical information included all STI diagnoses (at any anatomic site), hepatitis B virus (HBV) and human papilloma virus (HPV) immunization dates and exemptions, and the date of the first DoD seropositive test among HIV-infected individuals. Both etiologic and syndromic STI diagnoses were considered in our search (see definitions, [Table 1](#pone.0167892.t001){ref-type="table"}).
10.1371/journal.pone.0167892.t001
###### ICD-9 Diagnosis Codes examined in this study.
{#pone.0167892.t001g}
Condition Diagnosis Code
--------------------------------------------- ----------------------------------------------------------------------
Etiologic
*Chlamydia trachomatis* 099.41, 099.5
*Neisseria gonorrhoeae* 098, V02.7
Herpes simplex virus (HSV-genital) 054.1,
Human papilloma virus (HPV-genital) 078.1, 079.4, 795.05, 795.09, 795.15, 795.19, 796.75, 796.79, V73.81
Syphilis 090, 091, 092, 093, 094, 095, 096, 097
*Lymphogranuloma venereum* (LGV) 991
Trichomoniasis 131
Hepatitis B virus (HBV) 0703, 0702
Human immunodeficiency virus (HIV) V08, 042
Syndromic
Cervicitis (unspecified agent, \<45yo) 616.0
Vaginitis (\<45yo) 616.1
Pelvic Inflammatory Disease (PID) 614
Orchitis/Epididymitis, Prostatitis (\<35yo) 604, 601
Urethritis (\<35yo) 099.4
Data analysis {#sec007}
-------------
Descriptive statistics were used to characterize the accession year cohorts. Incident STI diagnoses were counted by each quarter of a calendar year. A bacterial STI was considered incident and only counted once in each quarter. Viral STIs were considered incident only at the first recorded diagnosis. If a syndromic diagnosis was found in the same quarter as an etiologic diagnosis explaining the syndrome (e.g. gonorrhea and urethritis), only the etiologic diagnosis was counted. Incidence rates and unadjusted risk ratios were calculated for each STI diagnosis, and a multivariate Poisson regression was performed to determine the relationship between number of STIs and years in the military. In addition, multivariate logistic regression was used to characterize factors independently associated with a history of diagnosed syndromic or pathogen-specific STIs.
All patient demographics and medical records were anonymous and de-identified prior to analysis. All relevant data are included within the manuscript. This study was approved by the Institutional Review Board of the Uniformed Services University of the Health Sciences.
Results {#sec008}
=======
The total sample size included 70,995 males (71%) and 21,005 females (29%), with baseline demographics shown in [Table 2](#pone.0167892.t002){ref-type="table"}. Owing to the large number of subjects in the sample, all variables of interest demonstrated statistically significant differences in univariate analyses. STI rates were highest among women, African-Americans, enlisted personnel and Air Force/Army compared with other service branches ([Table 3](#pone.0167892.t003){ref-type="table"}). STI rates were higher among individuals without a history of deployment, except for pathogen-specific diagnoses ([Table 3](#pone.0167892.t003){ref-type="table"}), though the end-study prevalence of STIs was higher among individuals who had been deployed than individuals with a history of non-deployment ([Table 2](#pone.0167892.t002){ref-type="table"}).
10.1371/journal.pone.0167892.t002
###### Baseline characteristics of participants and lifetime prevalence of at least one sexually transmitted infection.
{#pone.0167892.t002g}
Overall--women N (%) Syndromic---women N (%) Etiologic---women N (%) Overall---men N (%) Syndromic---men N (%) Etiologic---men N (%)
--------------------------------------------------------------------- ---------------------- ------------------------- ------------------------- --------------------- ----------------------- -----------------------
**Median Age at entry (IQR)**[^1^](#t002fn001){ref-type="table-fn"} 19 (18--22) 19 (18--21) 19 (18--21) 19(18--21) 19 (18--21) 19 (18--21)
**Race-White** 17,876 4,823 (26.9) 2,791 (15.6) 51,994 1,266 (2.4) 1,248 (2.4)
**Race-Black** 6,813 3,254 (47.8) 1,844 (27.0) 9,874 247 (2.5) 789 (8.0)
**Race-Other** 2,789 780 (28.0) 526 (18.9) 5,567 108 (1.9) 144 (2.6)
**Race-Unknown** 1,532 597 (39.0) 324 (21.1) 3,560 130 (
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1. Introduction {#sec1}
===============
High cumulative doses of anthracyclines (300--500 mg/m^2^) are frequently administered to children with cancer. Cardiac toxicity is a serious adverse effect that limits the therapeutic potential of anthracyclines and threatens the cardiac function of pediatric cancer patients leading to debilitating long-term effects resulting in poor quality of life in cancer survivors \[[@B1]--[@B5]\]. This is particularly devastating in children who are cured of their cancer because they have to endure the debilitating cardiac dysfunction for the rest of their lives with limited exercise capacity which may also lead to other chronic illnesses.
B-type-natriuretic peptide (BNP) is a polypeptide hormone predominantly released from the cardiac ventricles in response to volume expansion and pressure overload. BNP is found in the circulation as BNP-32 and the NH~2~-terminal portion of ProBNP (Nt-proBNP). BNP levels are elevated in patients with left ventricular systolic dysfunction and correlate with the severity of symptoms and prognosis \[[@B6]--[@B14]\]. Measuring serum Pro-BNP levels is a reliable way to monitor the cardiac function of patients receiving cardiotoxic drugs such as anthracyclines.
Selenium (Se) is a trace element distributed in a small amount in the soil and certain foods. It is an important antioxidant, and its absence has been associated with cardiomyopathy in people living in areas with poor levels of soil Se. The concentration of Se in grain varies based on the soil content. Dietary Se is found in meat and seafood. It is a cofactor for glutathione peroxidase which catalyzes the reduction of hydrogen peroxide using glutathione. It is an essential element to remove free radicals from the body and to prevent oxidative tissue damage \[[@B15]--[@B19]\]. Se supplementation could potentially prevent cardiac toxicity of anthracyclines \[[@B16]--[@B20]\].
In this study, we assessed anthracycline-induced cardiotoxicity by measuring Pro-BNP levels and echocardiographic (ECHO) findings, and we investigated the potential protective effect of Se supplementation in a group of children with high Pro-BNP levels and/or cardiac dysfunction.
2. Patients and Methods {#sec2}
=======================
Plasma level of Pro-BNP was measured, and echocardiography (ECHO) was performed in 67 pediatric cancer patients (45 boys and 22 girls, ages between 2 and 18 years, median age 12 years) with a variety of tumors (leukemias, lymphomas, solid tumors) after completing anthracycline-containing treatment. Serum Se levels were measured in 37 patients. Sera were stored at −20 degrees centigrade until selenium levels were measured with atomic absorption method. Patients with low level of Se were supplemented with Se (100 mcg/day).
3. Statistical Analysis {#sec3}
=======================
Statistical analysis was performed using SPSS (Version 15.0) software package. Comparisons between the groups were done using Mann-Whitney *U* test, Wilcoxon sign test, and Fisher\'s exact test. Levels of statistical significance were set at a *P* value \< 0.05. The results were expressed as range (minimum and maximum) and median.
4. Results {#sec4}
==========
In eleven patients who had high Pro-BNP levels and/or cardiac failure Pro-BNP levels ranged between 10 and 8022 pg/mL with a median of 226.3 pg/mL (normal \< 120 pg/mL). Fifty-six patients had normal Pro-BNP levels (8.2--119.6 pg/mL, median 32.4 pg/mL). As seen in [Table 1](#tab1){ref-type="table"}, the difference in levels of Pro-BNP between these two groups was significant (*P* \< 0.001). Serum Se levels were low in 10 of these 11 patients with high Pro-BNP levels and/or cardiac failure (20--129 mcg/L, median 62 mcg/L). Twenty-six of 56 patients with normal Pro-BNP levels were also investigated for Se levels (51.3--150 mcg/L, median 99.4 mcg/L). There was a significant difference between Se levels of patients in high Pro-BNP and normal Pro-BNP groups (*P* \< 0.001) ([Table 1](#tab1){ref-type="table"}).
Abnormal ECHO findings were observed in 7 of 11 (63.6%) patients with high Pro-BNP levels and/or cardiac failure group. Only 1 (3.8%) of 26 patients with normal Pro-BNP levels had abnormal ECHO finding. A patient with normal pro-BNP and low Se level died in 1 month because of progressive disease with respiratory failure and cardiac failure. The probability of having abnormal ECHO findings was significantly higher in patients with high Pro-BNP compared to those with normal Pro-BNP (*P* \< 0.001) ([Table 2](#tab2){ref-type="table"}). Eight of 11 patients with low Se and high Pro-BNP levels were supplemented with Se 100 mcg per day for 4--33 months (median 6 months). Three of 8 patients had cardiac failure according to ECHO and were supplemented with Se in addition to digoxin and ACE inhibitors. All 3 patients were doing well with normal ECHO findings and normal Pro-BNP levels after a follow-up periods of 33, 14, and 5 months. Five patients, 3 with normal ECHO and 2 with diastolic dysfunction (one with low Pro-BNP level, other with high Pro-BNP level) also, were supplemented with selenium (100 mcg per day). One patient who had diastolic dysfunction with normal Pro-BNP did well with Se supplementation with normalization of ECHO findings, but she later died due to progression of her cancer. Another patient with diastolic dysfunction as well as 3 patients with normal ECHO had normal Se and Pro-BNP levels after 4--6 months of Se supplementation. Only 3 patients were not supplemented with Se in the high Pro-BNP and/or cardiac failure group, because one of them had normal Se level, the second one died with progressive disease in a very short period of time, and the third one had Pro-BNP level within normal limits after the removal of intracardiac tumor thrombus with open heart surgery ([Table 3](#tab3){ref-type="table"}). In Se-supplemented group, supplementation period was between 4 and 33 months (median 6 months). Before supplementation, Pro-BNP levels were between 10 and 843 pg/mL (median 175 pg/mL). After supplementation, Pro-BNP levels were 2--536 pg/mL (median 73.5 pg/mL) which were significantly lower than pretreatment levels (*P* = 0.018). Pretreatment Se levels were between 20 and 83 mcg/L (median 57 mcg/L). After supplementation Se levels were 65--109 mcg/L (median 103 mcg/L) which were significantly higher than presupplementation level (*P* = 0.028) ([Table 4](#tab4){ref-type="table"}). After achieving normal Se and Pro-BNP levels, Se supplementation was discontinued. During follow-up period with no Se supplementation, 2--6 months after supplementation repeat measurements of Se levels were 75--106 mcg/L (median 83 mcg/L), and Pro-BNP levels were 10--123.5 pg/mL (median 106.5 pg/mL), which were lower for Se (*P* = 0.068) and higher for Pro-BNP (*P* = 0.109) compared to Se-supplemented period ([Table 4](#tab4){ref-type="table"}).
5. Discussion {#sec5}
=============
The main long-term toxicity of anthracyclines is cardiac dysfunction associated with their chronic and/or high-dose administration. Severe cardiomyopathy and congestive heart failure may develop any time after the completion of the treatment. The precise pathogenesis of anthracycline-induced cardiotoxicity is still uncertain, and it is likely to be multifactorial in origin. Nevertheless, pivotal role is attributed to the iron-catalyzed intramyocardial production of reactive oxygen species (ROS), which cause damage of various targets in the myocardial cells \[[@B1]--[@B5]\]. Probrain natriuretic peptide (Pro-BNP) is released by cardiac cells, and serum levels are elevated even before the development of overt cardiac distress symptoms related to impairment of left ventricular systolic or diastolic function leading to increased left ventricular wall stretch. Recent studies have also suggested that ischemia itself may promote release of BNP \[[@B7], [@B20]--[@B25]\]. In the present study, we evaluated cardiotoxicity in 67 pediatric patients with cancer (leukemia, lymphoma, and solid tumor) after they completed treatment with anthracycline-containing regimens. We also evaluated Se levels and the effects of Se supplementation with regard to cardiotoxicity because previous studies with Keshan disease (KD) suggested potential protective role of Se for cardiac dysfunction observed in Se deficiency.
KD, a potentially fatal form of cardiomyopathy, first found in Keshan county, northeast China, is one of the most harmful endemic diseases. The disease is characterized by multifocal myocardial necrosis and fibrosis and leads to congestive heart failure and cardiogenic shock. Although the exact etiology of KD is unclear, Se deficiency is a major contributing factor \[[@B19]\]. Investigations into the epidemiology of KD revealed that individuals living in areas with Se-poor soil were under a high risk of development of the disease. Individuals living
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Pathogenic response to viral infection varies dramatically between individuals infected with the same viral strain and dose, and much of this variation is heritable. The impact of host genetics is evident both on the primary exposure to a virus during early life ([@bib82]) and upon infection with newly emerging viral strains; the latter, where prior immune exposure to a variant viral strain is not cross-protective, being especially common for quickly evolving RNA viruses such as the influenza A virus (IAV) ([@bib55]). Pathogenesis induced by IAV, whether contracted during early childhood or later in life, is thus likely to have a significant heritable component. A greater understanding of this heritability should improve our ability to not only identify populations at risk of enhanced morbidity and mortality during an emerging pandemic, but also to identify successful options for treatment.
The past several years have seen significant progress in identifying and characterizing host genes that modulate susceptibility to IAV infection via knockout mouse studies, *in vitro* screens, and studies of primary immunodeficiencies and allelic variants in humans ([@bib85]). In humans, screening for inborn errors identified a major role for interferon regulatory factor 7 (*Irf7*) in modulating the severity of primary IAV infection ([@bib11]), and allelic variation in *Ifitm3*, which was identified in a high-throughput siRNA screen, was associated with differential severity of IAV-infection outcomes during the 2009 H1N1 pandemic ([@bib18]).
Most of our insights into genes modulating host IAV resistance, however, have come from studies on mice. These include studies using knockout mice---which have identified host genetic factors critical to antiviral responses, including *Tlr3* ([@bib31]) and *Isg15* ([@bib45])---and studies that examine differences between laboratory inbred strains. Inbred strain studies were the first to identify the *Myxovirus resistance* (*Mx*) family of proteins as important for host antiviral response ([@bib81]), and inbred studies have continued to demonstrate the relevance of genetic background to multiple aspects of IAV pathogenesis ([@bib80]; [@bib1]; [@bib43]; [@bib74]).
Yet, despite the identification of clear phenotypic differences between inbred strains, there have been relatively few attempts to dissect the genetic basis of those differences using traditional quantitative trait locus (QTL) mapping approaches such as the use of F2s or backcrosses (although see [@bib4]). This may be, in part, because traditional QTL mapping approaches tend to rely on outbred animals---and when it comes to studying viral pathogenesis, outbreds are in many respects problematic. One important limitation is phenotyping: studying the response to an infection is equivalent to studying the causal effect of an applied treatment in that its strict definition relies on a comparison between otherwise identical individuals subject to infection *vs.* control. But such like-for-like comparisons are biologically and technically challenging to make in an outbred population, where every individual is genetically distinct, and this has undesirable consequences for downstream interpretation: namely, that when genetic determinants of severe IAV pathogenesis are confounded with those influencing baseline phenotypes, the roles of any detected QTL are ambiguous. A related disadvantage of outbreds from the perspective of genetics is the inability to obtain biological replicates, which makes it harder to distinguish which aspects of pathology are stable consequences of genetics *vs.* products of stochastic variability. This is particularly important, since it also makes it almost impossible to follow-up on genuinely extreme responders for additional mechanistic and genetic analysis. Translating strain differences in IAV pathogenesis to meaningful QTL studies ideally requires an experimental paradigm that combines population-level genetic diversity with individual-level replicability.
An exciting opportunity is therefore presented by replicable genetic reference populations, in particular, those based on panels of recombinant inbred (RI) strains. Across a panel of RIs, genetic background varies, providing a basis for QTL mapping; within a RI strain, individuals are genetically identical, providing a basis for replication. The combination allows infection response to be rigorously defined and genomic regions affecting that response to be mapped. It also permits the creation of sophisticated experiments that target a wider range of heritable mechanisms: crossing RIs with each other to form RI intercrosses (RIXs), or crossing them with outside strains, produces replicable systems capable of distinguishing, for example, additive, dominance, and parent-of-origin effects, among others (*e.g.*, [@bib95]; [@bib34], [@bib33]; [@bib42]; [@bib84]; [@bib54]; [@bib26]; [@bib83]; [@bib77]; [@bib47]; [@bib101]; [@bib29]; [@bib93]).
RI genetic reference panels range from inbred lines derived from crosses between two mouse strains to more complex multiparental crosses. The BXD RI panel, derived from two founder strains, C57BL/6J and DBA/2J, has been used to study the impact of genetic variation on susceptibility to IAV infection and map QTL associated with these effects. [@bib5] studied H5N1 infection in females from 66 BXD strains, and [@bib58] studied H1N1 infection in 53 BXD strains, with both studies identifying QTL associated with susceptibility to infection. The Collaborative Cross (CC) RI panel is a multiparental population (MPP) descended from eight inbred founder strains ([@bib84]; [@bib9]), with these founders including representatives from the three major domesticated house mouse subspecies ([@bib99]). As such, the CC captures considerably more genetic diversity and, thanks to its breeding structure, this diversity is also more uniformly distributed across the genome, with as many as eight distinct haplotypes segregating at any given locus within the population ([@bib12]; [@bib79]). The eight CC founder strains have distinct pathogenesis profiles in response to influenza virus ([@bib43]), suggesting that the CC RI panel is capable of a broader phenotypic range than would be observed in less complex populations. Indeed, studies using an incompletely inbred, ancestor population of the CC (pre-CC), demonstrated high levels of phenotypic variation across the population and successfully mapped several QTL associated with variation in susceptibility to IAV infection ([@bib19]; [@bib6]). The CC therefore represents a promising resource for studying how genetically diverse populations respond to IAV infection.
Determining an optimal strategy for how the CC should be used to study the genetic architecture of IAV pathogenesis is nonetheless complicated because (1) the space of possible experimental designs is vast, and (2) information about what types of heritable effects are likely to be present is extremely limited. Regarding (1), with ∼75 CC strains currently available, including all reciprocal F1 hybrids (so called CC-RIXs), there are \>5600 potential replicable configurations. Since only a subset of these configurations can be explored within any realistic experiment, any chosen experimental design necessarily targets some types of heritable effects to the exclusion of others. Regarding (2), to date, most *in vivo* studies of IAV pathogenesis have been confined to candidate genes or additive interactions at single loci; studies investigating the broader question of what types of heritability are at play during IAV infection are largely absent.
To rationally design studies of heritable effects in complex populations such as the CC it is therefore helpful to have advance knowledge of which types of heritable effects might be present. One source of such information is phenotype data collected on the multiparental founders and their F1 hybrid offspring, a combination that can be more formally described as an (inbred) diallel. Diallels have a long history in quantitative genetics, having been used originally in plant breeding studies to judge the relative merits of different strain combinations and subsequently for gaining insight into the heritable architecture of a broad range of phenotypes \[*e.g.*, references in [@bib8], [@bib44], and [@bib62]\], including host--pathogen interactions in, *e.g.*, crickets ([@bib66]) and flies ([@bib92]) (see *Statistical Models and Methods* and *Discussion* for connections to other diallel literature).
Here we use a diallel of the CC founders and their reciprocal F1 hybrids (hereafter, a CC founder diallel) to give an overall predictive picture of the range and relative influence of the different types of heritable effects on IAV pathogenesis that are likely to be present in CC founder-derived MPPs, a group that includes not only replicable MPPs such as the CC and the CC-RIX but also irreplicable ones such as the Diversity Outbred (DO) population ([@bib105]). We take advantage of the diallel design's replicability to measure IAV-induced pathogenesis in a precise way, as the response to an applied treatment defined in terms of postinfection (p.i.) weight-loss differences (deltas) between matched sets of mock and infected individuals ([Figure 1](#fig1){ref-type="fig"}). Adapting a recently developed statistical framework for analyzing treatment-response diallels ([@bib14]), we use those deltas to model how pathogenic response to IAV is modulated by parentage, sex, and their interaction, framed in terms of additive genetics, dominance, epistasis, parent-of-origin, and sex-specific versions thereof.
After observing that, following IAV infection, diallel individuals show a broad, continuous distribution of day 4 (D4) p.i. weight loss, we find, through statistical modeling, that the IAV-induced weight loss includes substantial contributions of host additive, epistatic, and sex-specific effects, with much of the heritable variation closely tracking the genotype state implied by the three distinct functional alleles of the previously identified resistance locus *Mx1*. Confirming previous findings, the functional CAST/EiJ (CAST) *Mx1* allele, in contrast with functional NZO/HlLt
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Bacterial Resistance to Aminoglycoside Antibiotics
==================================================
Aminoglycoside (AG) antibiotics (Box [1](#BX1){ref-type="boxed-text"}) have not been an exception to the fact that after their introduction in clinical practice, resistance has been recorded ([@B85]). In fact, the three classes of aminoglycoside-modifying enzymes, aminoglycoside acetyltransferases (AACs), aminoglycoside phosphotransferases (APHs), and aminoglycoside nucleotidyltransferases (ANTs), have been widely detected in most pathogenic bacteria as a major determinant of resistance; in these bacteria the presence of aminoglycoside-modifying enzymes correlated with patterns of AG susceptibility ([@B76]). The modified AG (either by acetylation, phosphorylation or nucleotidylation) fails to inhibit their bacterial target, the 30S ribosomal subunit ([@B76]). Most genes encoding aminoglycoside-modifying enzymes are plasmid-located (indicative of a potential acquisition by horizontal gene transfer processes) and confer the bacterial hosts with high levels of AG resistance ([@B19]).
**Box 1.** Aminoglycosides: origins, structure, mode of action, resistance and clinical use.
Most aminoglycoside antibiotics are produced by bacterial species of the genus *Streptomyces*, such as the antitubercular streptomycin that is produced by *S. griseus* being the first antibiotic identified from bacteria. Other genera producing aminoglycosides are *Micromonospora* and *Bacillus*. Many semi-synthetic aminoglycosides, such as amikacin, have also been produced ([@B55]; [@B51]; [@B74]).
Structurally, aminoglycosides are formed by an aminocyclitol (commonly, 2-deoxystreptamine) with additional amino sugars bound by glycosidic bonds. There are two large families of 2-deoxystreptamine aminoglycosides, those carrying substitutions at positions 4 and 5 of the 2-deoxystreptamine ring (including neomycin, paromomycin, lividomycin, ribostamycin and butirosin) and those being substituted at positions 4 and 6 of the 2-deoxystreptamine (including kanamycin, amikacin, tobramycin, dibekacin, arbekacin, gentamicin, isepamicin, sisomicin, and netilmicin). The carbon atoms in the sugar bound to position 4 of the 2-deoxystreptamine ring are named with primed numbers ('), and those in the sugar bound to positions 5 or 6 of the 2-deoxystreptamine ring are named with double-primed numbers ("). Other aminoglycosides contain aminocyclitols distinct to 2-deoxystreptamine (this is the case of streptomycin or apramycin) or are formed by fused amino sugar rings (i.e., spectinomycin) ([@B51]; [@B74]). The following figure shows the structure of kanamycin A, an example of 4,6 di-substituted 2-deoxystreptamine aminoglycoside antibiotic.
{#d35e349}
The bacterial target of aminoglycosides is the 30S small ribosomal subunit, and their global effect is the interference with protein synthesis. In bacterial cells, translation is initiated when the 30S ribosomal subunit binds the Shine-Dalgarno sequence (because of sequence complementarity between the Shine-Dalgarno sequence and the 16S rRNA molecule of the 30S ribosomal subunit), which is normally present in the 5′ untranslated region of mRNAs. Next, initiation factors and fMet-tRNA will join the complex that finally will recruit the 50S ribosomal subunit in order to start translation. Aminoglycoside binding to the 30S subunit does not affect the binding of mRNA and the large 50S subunit, so translation can proceed. However, aminoglycosides differ in their binding site at the 30S subunit, hence affecting the protein production at different levels. Whereas spectinomycin blocks translocation (hence being bacteriostatic), streptomycin and most 2-deoxystreptamine aminoglycosides lock the ribosome in a conformation that is prone to introducing erroneous aminoacyl-tRNAs. The accumulation of aberrant proteins in the bacteria results in cell death ([@B19]; [@B51]; [@B73]).
Resistance to aminoglycosides may be due to several mechanisms ([@B19]; [@B51]). Reduced uptake (which can be a consequence of alterations in the composition of bacterial membrane or to metabolic conditions like anaerobiosis) or the action of efflux pumps can lead to limited intracellular concentration of aminoglycosides, hence causing resistance. Mutations in 16S ribosomal RNA or certain ribosomal proteins such as S12 (encoded by *rpsL* gene) lead to aminoglycoside resistance through target modification; this is also achieved after the action of methyltransferases, which introduce methyl groups in guanine or adenine nucleotides of 16S ribosomal RNA. The presence of aminoglycoside-modifying enzymes is, however, the most prevalent mechanism of aminoglycoside resistance; there are three types of aminoglycoside-modifying enzymes: aminoglycoside *N*-acetyltransferases (AAC), *O*-phosphotransferases (APH), and *O*-nucleotidyltransferases (ANT). Aminoglycoside-modifying enzymes are named by using the aforementioned abbreviations, followed by a number in brackets indicating the site of modification in the aminoglycoside molecule (as explained above), a Roman numeral related with substrate profile, and a lower-case letter for differentiating isoenzymes, i.e., AAC(2′)-Ib.
Clinically, due to their oto- and nephrotoxicity and the rising prevalence of resistance, aminoglycosides are commonly reserved as a second line of treatment of serious infections. Due to their low absorption when given orally, aminoglycosides need to be administered through injections. Streptomycin was a first-line drug in the treatment of tuberculosis, and in our days, kanamycin and amikacin are listed as second line drugs against this disease. Spectinomycin is used against *Neisseria gonorrhoeae* infections ([@B78]). *Pseudomonas aeruginosa* infections in cystic fibrosis patients, septicemia, endocarditis and several other infections caused by non-tuberculous mycobacteria, Gram-positive or Gram-negative bacteria can be treated efficiently by using aminoglycosides, either alone or in combinations with other antibacterials such as the beta-lactam antibiotics ([@B55]; [@B51]; [@B9]).
In mycobacteria, however, resistance to AGs resulted mainly from mutations of the ribosome components that prevent the drugs from inhibiting its function ([@B43]; [@B93]; [@B72]). This is due to the fact that most mycobacterial species have either one (like *Mycobacterium. tuberculosis*) or two (like *Mycobacterium fortuitum*) ribosomal operons, hence making dominant those mutations acquired in their components ([@B51]; [@B74]). The presence of aminoglycoside-modifying enzymes, mostly AAC, in mycobacterial species has been reported over the years (as detailed in the following sections), and the role of such AACs has been explored, originally for their contribution to AG resistance, and more recently for their role in other bacterial processes, which has resulted in the interest of developing inhibitors of these enzymes ([@B40]; [@B46]). In this review, we will summarize major findings on two mycobacterial AACs, the AAC(2′)-I and Eis enzymes, that have resulted in a Copernican turn for AACs in mycobacteria, from being putative drug resistance mechanisms, to reach the status of novel drug targets.
Aacs in Non-Tuberculous Mycobacteria
====================================
The first detection of AACs in mycobacteria ([@B39]) was reported in a group of *M. fortuitum* isolates, an opportunistic fast-growing mycobacteria. Biochemical assays of crude extracts from *M. fortuitum* strains revealed the presence of AAC activity, strongly acetylating gentamicin and kanamycin A, along with other AGs. This substrate profile was consistent with that of AAC(3) enzymes that had been previously described in *Pseudomonas* and *Enterobacteriaceae* ([@B7]), although confirmation at the genetic or molecular levels were not done at that time. Surprisingly, the AG susceptibility profile of *M. fortuitum* could not be correlated with the activity of AACs, indicating that in this species AACs were not the major responsible for AG resistance; it was neither correlated with the presence of plasmids, hence suggesting a chromosomal location ([@B39]). In fact, the frequency of resistant mutants to kanamycin and amikacin in *M. fortuitum* and the related species *Mycobacterium chelonae* ranged between 10^-4^ and 10^-7^ ([@B86]). This relatively high frequency of mutations, along with the fact that AAC activity was detected at similar levels between susceptible and resistant strains, led the authors to suggest that ribosome alterations were the main factor responsible of AG resistance in these species ([@B86]). In another study ([@B82]), altered transport or permeability of AGs was identified as a contributor to AG resistance in *M. fortuitum*, since ribosomes from a clinical isolate were inhibited by one tenth of the MIC of AGs: for example, the MIC of kanamycin for *M. fortuitum* was 50 μg/ml, and in cell-free systems,
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Introduction {#s1}
============
It is commonly accepted that in order for a neuronal population to encode the value of a quantity x, it must contain cells tuned to a range of values of x. Thus for example the retina can encode information about the wavelength of light because it contains three different types of cones with different tuning to wavelength, and the primary visual cortex can encode feature orientation because it contains neurons tuned to a range of orientations. This is unproblematic because natural images contain a wide range of light wavelengths and object orientations. However, the same argument applied to stereo vision produces some more challenging conclusions.
The expected vertical disparity in natural viewing depends on position in the retina, with opposite signs in opposite quadrants of the visual field. The range in vertical disparities encountered at a given position depends on a number of assumptions about eye movement and scene statistics, but all attempts to estimate it agree that it is extremely narrowly distributed compared to horizontal disparity [@pcbi.1000754-Liu1], [@pcbi.1000754-Hibbard1], [@pcbi.1000754-Read1]. Thus, if disparity sensors in the brain were to reflect disparity in the natural world, we would expect the distribution of two-dimensional disparity tuning at a given retinotopic location to be highly elongated, virtually one-dimensional, with a wide range of horizontal disparity and a narrow range of vertical disparity, centered on the value expected for that retinotopic location. Yet, vertical disparities which hardly ever occur in normal visual experience can still have demonstrable effects on perception in the lab [@pcbi.1000754-Helmholtz1], [@pcbi.1000754-Ogle1], and there is evidence that stereo matching occurs in all 2D directions, vertical as well as horizontal [@pcbi.1000754-Farell1]. Thus, the brain clearly can extract unusual vertical disparities, on relatively local scales [@pcbi.1000754-SerranoPedraza1], [@pcbi.1000754-Rogers1], [@pcbi.1000754-Kaneko1]. This has led to the conclusion that the brain must contain neurons tuned to a range of vertical disparities, including highly unusual ones, on the assumption that otherwise, these disparities could not be perceived [@pcbi.1000754-Durand1], [@pcbi.1000754-Durand2], [@pcbi.1000754-Gonzalez1].
Motivated by this, a number of physiological studies have examined two-dimensional disparity tuning in cortical neurons in monkey primary visual cortex (V1). Near the fovea, most disparity-tuned neurons are tuned to vertical disparities which are not significantly different from zero, given the confidence interval on the measurement [@pcbi.1000754-Cumming1]. In the visual periphery, neurons tuned to non-zero vertical disparities have been reported [@pcbi.1000754-Durand1], [@pcbi.1000754-Durand2], [@pcbi.1000754-Gonzalez1]. Unfortunately, these studies only reported disparity in head-centric coordinates, which can differ substantially from retino-centric disparity [@pcbi.1000754-Read2]. For example, it is perfectly possible for a neuron tuned to a substantial head-centric vertical disparity, say 0.3°, to be tuned to a vertical disparity of 0° on the retina [@pcbi.1000754-Read1]. Thus, the published data do not enable us to draw any conclusions about 2D disparity tuning on the retina. Furthermore, these studies did not report the retinal location of individual neurons, making it impossible to assess whether a range of vertical disparity tuning is found at a single retinotopic location.
Given this lack of data from physiology, theoretical considerations become important. A clear understanding of how, in principle, neurons could represent two-dimensional disparity is essential for guiding future physiology experiments. We recently argued [@pcbi.1000754-Read3] that a population of model binocular neurons like that shown in [Figure 1](#pcbi-1000754-g001){ref-type="fig"}, tuned to a range of horizontal disparities and orientations but all tuned to zero vertical disparity on the retina, nevertheless encodes information about the vertical disparity of the stimulus. This original model only extracted the magnitude, not the sign, of the local vertical disparity, and we later demonstrated that this was inconsistent with human psychophysics [@pcbi.1000754-SerranoPedraza2]. However, this model did not make optimal use of the information available in the population. In the present paper, I show that this population of disparity sensors does contain information about both the magnitude and the sign of the vertical disparity at that point in the retina, even if all neurons in the population are tuned to the same vertical disparity. With an appropriate decoding technique, information about the two-dimensional disparity can be deduced from activity in this one-dimensional population. This result is of interest in its own right as a theoretical demonstration that it is possible to extract the value of a quantity from a neuronal population, all of whose members respond optimally to the same value of that quantity. From the point of view of understanding stereo vision, it means that two-dimensional disparity may be represented far more efficiently than previously appreciated.
![A neuronal population which explicitly encodes horizontal, but not vertical, disparity.\
The shaded region represents the space of two-dimensional disparity on the retina [@pcbi.1000754-Read2]. The purple disks represent the preferred 2D disparity of an idealized population of disparity sensors. Although these sensors form a one-dimensional population, all tuned to zero vertical disparity, they can nevertheless encode two-dimensional stimulus disparity, e.g. the stimulus disparity represented by the green dot, which has both a horizontal and a vertical component. (Cf [figure 1](#pcbi-1000754-g001){ref-type="fig"} of Serrano-Pedraza & Read [@pcbi.1000754-SerranoPedraza2].)](pcbi.1000754.g001){#pcbi-1000754-g001}
Methods {#s2}
=======
Overview {#s2a}
--------
The essential insight guiding this paper is relatively trivial. According to the stereo energy model of disparity-selective neurons [@pcbi.1000754-Ohzawa1], [@pcbi.1000754-Ohzawa2], cells with obliquely-oriented receptive fields will also have obliquely-oriented disparity tuning surfaces, like the one illustrated in [Figure 2A](#pcbi-1000754-g002){ref-type="fig"}. This cell\'s optimal disparity is marked with a red circle. It has zero vertical component, i.e. the cell responds best to zero vertical disparity. [Figure 2B](#pcbi-1000754-g002){ref-type="fig"} shows two cross-sections through this surface, corresponding to vertical disparity tuning curves for two different horizontal disparities, as indicated by the vertical lines in [Figure 2A](#pcbi-1000754-g002){ref-type="fig"}. At the optimal horizontal disparity (red curve), the cell responds best to zero vertical disparity. But at horizontal disparities away from the optimum (e.g. purple curve), the cell\'s response is reduced, but is now tuned to a non-zero vertical disparity. Thus, while the cell in [Figure 2](#pcbi-1000754-g002){ref-type="fig"} is "tuned to zero vertical disparity" in that its optimum 2D disparity has zero vertical component, when it is probed at horizontal disparities on either side of the optimum, it responds best to vertical disparities on either side of zero. This suggests that, given cells tuned to a range of orientations and horizontal disparities, one could potentially extract the stimulus orientation, horizontal disparity *and* vertical disparity. Of course, it may not be quite that simple. In order to use the cells\' tuning to vertical disparity away from the optimal horizontal disparity, one has to know what the horizontal disparity is. Extracting this may be hard in the presence of vertical disparity, since then none of the cells in the population is tuned to the correct stimulus disparity. Also, because the tuning to vertical disparity occurs only at sub-optimal horizontal disparities, the neuron\'s activity is weaker, so more subject to noise. Thus, this intuitive idea has to be rigorously tested by simulation. This is what is achieved in this paper.
{#pcbi-1000754-g002}
The simulations consist of two neuronal populations: one encoding population, which takes left and right retinal images and performs the initial encoding of binocular disparity, and one decoding population, which estimates the disparity of the stimulus. The encoding population is like that in [Figure 1](#pcbi-1000754-g001){ref-type="fig"}: it consists of a set of neurons tuned to a range of horizontal disparities, orientations and spatial frequencies, but all tuned to the same vertical disparity. For simplicity, I shall set this vertical disparity to be zero, which is appropriate for the parafoveal region.
The encoding neurons are based on the stereo energy model [@pcbi.1000754-Ohzawa1], normalized so as to report the effective local binocular correlation [@pcbi.1000754-Read3], [@pcbi.1000754-Banks1], [@pcbi.1000754-Filippini1]. The activity of this population is then decoded by a separate, higher-level population, using
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1. Introduction
===============
Tumor angiogenesis, that is, the development of new blood vessels from pre-existing ones, plays an essential role in tumor growth, invasiveness, and metastasis \[[@B1-molecules-17-06854],[@B2-molecules-17-06854]\]. It is estimated that angiogenesis in tumors contributes to more than 90% of all cancer deaths. Tumor angiogenesis is a promising therapeutic target for treatment of cancer. Angiogenesis is initiated by cell proliferation and migration in response to chemotactic agents, such as VEGF, which is widely expressed in the majority of cancers and is a critical component of tumor angiogenesis \[[@B3-molecules-17-06854]\]. VEGF mediates its signals through interactions with the receptor tyrosine kinases expressed on endothelial cells \[[@B4-molecules-17-06854]\]. VEGFRs activation leads to the activation of diverse intracellular signaling molecules, including extracellular signal-regulated kinases (ERKs), phosphoinositide 3-kinase/AKT kinase, and mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) \[[@B5-molecules-17-06854],[@B6-molecules-17-06854]\] that promote the proliferation, migration, differentiation, and survival of endothelial cells in the pre-existing vasculature. Thus, VEGFR2 and those intracellular signaling molecules appear to be critical targets for the suppression of tumor angiogenesis. Multiple angiogenesis inhibitors have been therapeutically validated in treatments for many cancers \[[@B7-molecules-17-06854]\].
Pristimerin ([Figure 1](#molecules-17-06854-f001){ref-type="fig"}) is a natural triterpenoid isolated from Celastrus and Maytenus spp. \[[@B8-molecules-17-06854]\] that has been shown to possess a variety of biological activities. Antitumor properties have also been reported for pristimerin. Previous studies have shown that pristimerin can inhibit tumor cell proliferation by inhibiting the NF-κB pathway and cell cycling \[[@B8-molecules-17-06854],[@B9-molecules-17-06854],[@B10-molecules-17-06854],[@B11-molecules-17-06854],[@B12-molecules-17-06854]\]. In addition, pristimerin has been reported to induce caspase-dependent apoptosis of breast cancer cells \[[@B13-molecules-17-06854]\] and prostate cancer cells *in vitro* \[[@B11-molecules-17-06854]\]. Recently, pristimerin is reported to inhibit xenografted plasmacytoma tumors in mice through the suppression of 20s proteasome chymotrypsin-like activity \[[@B9-molecules-17-06854]\]. Despite reports of the antitumor properties of pristimerin, whether this compound directly affects tumor angiogenesis or has potential value for breast cancer prevention *in vivo* remains unknown.
{#molecules-17-06854-f001}
In this study, we investigated how pristimerin inhibits tumor angiogenesis by targeting key signaling pathways. Our results provide the first evidence that pristimerin significantly inhibits VEGF-stimulated endothelial cell proliferation, migration, tube formation, and tumor angiogenesis by targeting VEGFR2 activation, leading to the suppression of tumor growth.
2. Results and Discussion
=========================
2.1. Pristimerin Inhibits Tumor Growth and Tumor Angiogenesis in a Xenograft Mouse Model
----------------------------------------------------------------------------------------
To investigate the effects of pristimerin on tumor growth and tumor angiogenesis *in vivo*, we used a xenograft human breast cancer model and found that 3 mg/kg of pristimerin applied every other day significantly reduced both tumor volume ([Figure 2](#molecules-17-06854-f002){ref-type="fig"}A) and tumor weight ([Figure 2](#molecules-17-06854-f002){ref-type="fig"}B). As shown in [Figure 2](#molecules-17-06854-f002){ref-type="fig"}A, the tumors in the control group increased in size from 114.95 ± 27.50 to 501.06 ± 135.10 mm^3^, whereas the tumors in the pristimerin-treated group shrank from 115.76 ± 29.80 to 109.32 ± 54.40 mm^3^. Additionally, the average final tumor weight in the pristimerin-treated group was dramatically reduced compaired with that in the control group ([Figure 2](#molecules-17-06854-f002){ref-type="fig"}B), suggesting that pristimerin strongly inhibited tumor growth in this xenograft mouse breast tumor model.
To investigate further whether pristimerin inhibited tumor angiogenesis in the xenograft mouse model, we stained solid tumor sections with a blood vessel staining kit. The mean number of blood vessels in the pristimerin-treated group was 37.06 ± 19.09/HPF compared with 100 ± 24.87/HPF in the control group ([Figure 2](#molecules-17-06854-f002){ref-type="fig"}D), indicating that pristimerin significantly inhibited tumor angiogenesis.
{#molecules-17-06854-f002}
2.2. Pristimerin Inhibits Angiogenesis in Vivo and VEGF-induced Vessel Sprouting *ex Vivo*
------------------------------------------------------------------------------------------
We performed a chick embryo chorioallantoic membrane (CAM) assay to determine the antiangiogenic effects of pristimerin *in vivo*. As illustrated in [Figure 3](#molecules-17-06854-f003){ref-type="fig"}A, new blood vessels formed well on CAMs in the control group after a 2-day incubation, whereas incubation with pristimerin at 40 nmol/egg resulted in a notable inhibition, and pristimerin at 80 nmol/egg drastically impaired CAM neovascularization accompanied by a lack of prominent vessel networks. These results demonstrate that pristimerin suppresses angiogenesis in a CAM model.
The aortic ring assay mimics several stages in angiogenesis, including endothelial cell proliferation, migration, and tube formation and is widely used to evaluate the antiangiogenic effects of putative therapeutic compounds \[[@B14-molecules-17-06854]\]. To further investigate whether pristimerin inhibited VEGF-induced angiogenesis *ex vivo*, we examined the sprouting of vessels from aortic rings in the absence or presence of pristimerin. VEGF (20 ng/mL) significantly stimulated microvessel sprouting around the aortic rings ([Figure 3](#molecules-17-06854-f003){ref-type="fig"}B). Treatment with pristimerin antagonized the VEGF-induced sprouting in a dose-dependent manner. Treatment with pristimerin at 1 μM yielded a notable suppression of microvessel formation *versus* the control ([Figure 3](#molecules-17-06854-f003){ref-type="fig"}A), and 2 μM of pristimerin completely blocked the VEGF-induced microvessel sprouting of rat aortic rings ([Figure 3](#molecules-17-06854-f003){ref-type="fig"}B). The presence of pristimerin resulted in decreasing capillary sprouting from the rat aorta rings.
{#molecules-17-06854-f003}
2.3. Pristimerin Inhibits the VEGF-Induced Chemotactic Motility, Capillary Structure Formation and Proliferation of HUVECs *in Vitro*
-------------------------------------------------------------------------------------------------------------------------------------
The migration of endothelial cells is essential for blood vessel formation in angiogenesis and thus for tumor growth. The effects of pristimerin on the chemotactic motility of HUVECs were assessed in a wound-healing migration assay and in a Transwell cell migration assay. As shown in [Figure 4](#molecules-17-06854-f004){ref-type="fig"}A
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1. Introduction {#sec1}
===============
Cocaine abuse is relatively common in our society. Recently, the Drug Enforcement Agency (DEA) has reported that seventy percent (70%) of cocaine seized at United States (US) borders has been adulterated with levamisole \[[@B1]\]. This agent, which is an antihelminthic and chemotherapeutic drug, indirectly increases the number of D1 dopamine receptors in the brain and has a cholinergic effect that seems to potentiate the effect of cocaine. It also has immune stimulating effect producing antineutrophils cytoplasmic antibodies (ANCAs). Several side effects have been associated with its use, including severe neutropenia and necrotizing vasculitis. Levamisole is a challenging drug to test; it has a half-life about 5.6 hours and specific testing is not routinely available \[[@B2]\]. Recently, several cases with this manifestation have presented in our institution. Although cases have been previously reported in the USA, none had been reported in Puerto Rico or the Caribbean.
2. Case Report {#sec2}
==============
A 45-year-old male cocaine user without any previous systemic illness complains of progressive painful erythematous lesions in both auricles and extremities, constant burning, 10/10 pain in upper and lower extremities, although more prominent in upper extremities. Patient also refers suffering of unquantified fever, congested nose, and fatigue; denies nausea, vomiting, and shortness of breath or changes in mental status. Four days prior to admission, patient was evaluated in a health clinic for the above-mentioned lesions and was referred for a follow-up visit to surgery for a skin biopsy. One day prior to admission, patient was taken to the same clinic for the worsening of his skin lesions; he was transferred to our institution for further evaluation and management.
History was remarkable for smoking (use of) cocaine; last reported use was 10 days prior to evaluation. Physical examination demonstrated several purpuric retiform patches with erythematous borders in the helix of both ears and both upper and lower extremities ([Figure 1](#fig1){ref-type="fig"}). A working diagnosis of vasculitis was made with suspicion of cryoglobulinemia; high-dose steroids were initiated. Toxicology test in urine was negative for cocaine; hepatitis profile and HIV test were nonreactive.
C-ANCA (1.7 IU/mL), P-ANCA (1.7 IU/mL), ANA, and cryoglobulins were positive. Complements levels were decreased C3 = 92.50 mg/dL and C4 = 12.30 mg/dL. WBC = 4.7 × 10^3^/uL, Hb = 10.2 g/dL, Hct = 29.8%, and platelets = 390 × 10^3^ uL. Skin biopsy (punch) demonstrated intravascular thrombosis and mild perivascular lymphocytic infiltrates ([Figure 2](#fig2){ref-type="fig"}). High-dose steroids were continued and plasmapheresis was initiated. Complete clinical resolution of skin lesions was noted after treatment and thirty (30) days of no cocaine use ([Figure 3](#fig3){ref-type="fig"}).
The second patient is a 52-year-old male with medical history of constant crack cocaine and marihuana abuse, complaining of bilateral upper and lower extremities necrotic skin lesions, which were also found on ears, nose, and genital area associated with a burning, 10/10 pain of two (2) weeks of evolution. Chills, nauseas, and vomits were referred as associated symptoms; patient denied fever, shortness of breath, or changes in mental status. History remarkable for smoking cocaine, last reported use was the night before initial evaluation at the emergency room. Physical examination showed necrotic lesions with an erythematous base, tender to palpation, on both ears, nose, upper and lower extremities, and genital areas ([Figure 4](#fig4){ref-type="fig"}). Laboratory evaluation was remarkable for neutropenia: WBC = 3.5 × 10^3^/uL, microcytic anemia, Hb = 9.92 g/dL, Hct = 28%, and platelet count of 290 × 10^3^ uL. Due to the appearance of necrotic tissue, a diagnosis of vasculitis with infected necrotic ulcers was made. High-dose steroids and broad-spectrum antibiotics were initiated. Toxicology test was positive for cocaine and cannabinoids, while the hepatitis profile and HIV test were nonreactive. C-ANCA (2.55 IU/ml), P-ANCA (1.69 IU/ML), and cryoglobulins were positive. ANA was negative in this occasion. Complements levels were decreased, C3 = 55.30 mg/dL, and C4 = 8.86 mg/dL. As with the previous patient, skin biopsy revealed homogenous eosinophilic material with blood vessels and lymphocytic infiltrate in dermis; however, no evidence of intravascular thrombosis was found. As recommended by hematology service, plasmapheresis was started, and antibiotics and high-dose steroids were continued. Clinical resolution was observed, but patient left against medical advice before the treatment was completed.
The third patient is a 60-year-old homeless female who complained of suffering 8/10, burning pain in both ears for about six months prior to the initial evaluation, and that exacerbated since the day before arrival to our institution. Patient denied any associated symptoms. History of smoking (use of) cocaine: last reported use was the day of admission. Physical examination remarkable for purpuric retiform patches with erythematous borders and necrotic tissue on both ears ([Figure 5](#fig5){ref-type="fig"}). Purpuric retiform patches also observed on the tip of the patient\'s nose and inferior aspect of second to fifth right toe. Patient also presented with foul smelling necrotic ulcer in the distal right, lower extremity. Patient was admitted with broad-spectrum antibiotics for her ulcer but high-dose steroids were not initiated. Laboratory data showed neutropenia, WBC = 3.6 × 10^3^/uL, and microcytic anemia, Hb = 11.3 g/dL, Hct = 34.2%, and Plts = 368 × 10^3^ uL. Toxicology was positive for cocaine: yet, hepatitis profile and HIV test were nonreactive. Complements levels C3 (131 mg/dL) and C4 (24 mg/dL) were within normal values. A skin biopsy revealed vascular thrombosis in the small-sized vascular channels in the dermis. P-ANCA and C-ANCA levels could not be obtained. Although clinical resolution was observed during hospitalization, patient decided to left against medical advice.
3. Discussion {#sec3}
=============
Cocaine abuse affects more than five (5) million people in the USA with side effects that are not limited to the parent drug. To enhance profitability and acceptability of the product, it is not uncommon for illicit drugs to undergo several processes. Among these, adulteration (intentional addition of a substance with similar pharmacologic properties which attenuates the effects of the parent drug) \[[@B6]\] presents as a new challenge to the medical community.
Levamisole is an anthelmintic agent used in veterinary medicine that was formerly used as an adjuvant with fluorouracil in the treatment of colorectal cancer; as immunomodulator in rheumatoid arthritis; as treatment in nephrotic syndrome. It is known for augmenting dopamine and endogenous opiates levels in the brain, increasing D1 dopamine receptors, and producing cholinergic effects \[[@B3]\]. Levamisole may inhibit MAO and catechol-O-methyltransferase activity, prolonging the presence of catecholamine neurotransmitters in the synapse, adding to the reuptake-inhibition effect of cocaine, and augmenting its effects (some cases of mood elevation have been reported in humans as a side effect of adjuvant therapy with levamisole for colon carcinoma) \[[@B4]\].
Levamisole\'s immune stimulating effect leads to the production of autoantibodies (antinuclear and antineutrophils antibodies), which leads to ANCA positivity (presented in the first two patients), severe neutropenia, and focal necrotizing features of vascular damage \[[@B5]\]. Levamisole-induced, occlusive, necrotizing vasculitis is an uncommon side effect of this agent \[[@B6]\]. It presents as purpuric retiform lesions with necrotic patches typically manifesting on the ears and extremities, as previously reported in patients who received treatment with levamisole for nephrotic syndrome. Also, it differs from lesions directly related to pure cocaine described as palpable purpura or Wegener\'s granulomatosis like \[[@B7]\]. Microscopically, levamisole can produce a mixed pattern of leukocytoclastic vasculitis, thrombotic vasculitis, and/or vascular occlusion as seen in skin biopsies from children using levamisole for the treatment of nephrotic syndrome \[[@B8]\].
Although levamisole levels were not measured because of its short half-life of 5.6 hours and because routine tests for its detection are not commonly available, we believe that the adulteration of cocaine with levamisole was the cause of the clinical presentation of the three evaluated patients.
Retiform purpuric lesions that later become necrotic, an histopathologic report of intravascular thrombosis with a mild perivascular lymphocytic infiltrates, and ANCA positivity in a patient with recent history of smoking (use of) cocaine should raise the suspicion of a levamisole-induced vasculitis. Treatment is primarily supportive, however steroids have been used in some cases with success \[[@B6
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ANNOUNCEMENT {#s1}
============
The ascomycete fungus Fusarium graminearum is the main pathogen causing *Fusarium* head blight (FHB), an important cereal disease worldwide ([@B1]). The F. graminearum genome from a South American strain was not previously available. Here, we report an annotated assembly for strain CML3066 (DON/15-ADON), isolated in 2009 in Rio Grande do Sul state, Brazil (latitude, −28.327, longitude, −51.271). This strain was isolated from a symptomatic wheat spike with 21.5% FHB incidence ([@B2]).
Genomic DNA of F. graminearum CML3066 was extracted from mycelia grown for 3 days in potato dextrose broth (PDB) medium using a cetyltrimethylammonium bromide (CTAB) protocol ([@B3]) and quantified using a Qubit 2.0 fluorometer (Life Technologies). Libraries were prepared with TruSeq DNA high-throughput (HT) (Illumina) and SMRTbell (PacBio) kits. Genome sequencing was done both on an Illumina HiSeq 2000 platform, producing 100-bp paired-end reads (91× coverage) with no quality control required, and on a PacBio RS II platform with a postquality filter of minimum polymerase read quality of 0.80 and minimum subread length of 500 bp, resulting in 160× coverage with a subread total of 6,392,721,477 bp, 1,358,615 reads, and an *N*~50~ value of 5,621 bp. Default parameters were used for all software unless otherwise noted. The *de novo* assembly was carried out using SOAPdenovo2 v2.0.4 using the Illumina data with a range of k-mer values (61 to 99) and the SMRT analysis portal using the PacBio data. The PacBio assembly was manually gap filled and further scaffolded using Lastz v1.04.03 alignments with the complementary Illumina assembly, resulting in four complete chromosomes from telomere to telomere with no gaps or N bases. Reference sequence statistics were extracted from Geneious v8.1. The genome annotation of CML3066 was done using the MAKER v2.30 ([@B4]) annotation pipeline with RepeatMasker v4.50 ([@B5]). Gene calls were generated using both AUGUSTUS v2.7 ([@B6]) using the F. graminearum species model and GeneMark ([@B7]), which was trained using strain PH-1 ([@B8], [@B9]).
The CML3066 assembly is 36,908,675 bp long with a GC content of 47.9%. The CML3066 genome is predicted to contain 14,188 genes, 286 of which are not present in the PH-1 genome. Using the PH-1 reference, a minimum of 80% of the length of the gene was required to have mapped reads to be considered present. Single nucleotide polymorphism (SNP) calling was performed with SAMtools using default settings. SNP effects were predicted using SnpEff 4.2. Comparison of SNP frequencies along all four chromosomes of both CML3066 and PH-1 ([@B8], [@B10]) revealed that all telomere proximal regions displayed the highest SNP density windows. In addition, chromosomes 1, 2, and 4 were found to have one or two large interstitial regions with a high SNP density. To predict secreted proteins, Blast2GO v3.2 was used to identify signal peptide and transmembrane domains. Prediction of glycosylphosphatidylinositol (GPI)-anchored membrane proteins, cellular protein localization, and effectors was performed using Big-Pi, WoLF PSort and ProtComp v9.0 (Softberry), and EffectorP v1.0 ([@B11][@B12][@B14]), respectively. The secretome was predicted to contain 874 genes. A genome comparison between CML3066 and the reference strain PH-1 is summarized in [Table 1](#tab1){ref-type="table"}.
######
Genome sequence assemblies for F. graminearum strains PH-1 and CML3066
Characteristic Value for strain:
--------------------------------------------------------- ------------------- ------------
Genome size (bp)[^*b*^](#ngtab1.2){ref-type="table-fn"} 36,663,736 36,908,675
No. of chromosomes 4 4
GC content (%)[^*c*^](#ngtab1.3){ref-type="table-fn"} 48.2 47.9
No. of spanned gaps 0 0
No. of predicted genes 14,145 14,188
Reannotated genome ([@B10]).
Including all scaffolds and the mitochondrial genome but excluding the large repetitive sequence at the carboxyl end of chromosome 4.
Excluding the mitochondrial genome.
Data availability. {#s1.1}
------------------
The raw data and assembled/annotated sequences have been deposited in the European Nucleotide Archive (ENA). The study accession number is [PRJEB12819](https://www.ebi.ac.uk/ena/data/view/PRJEB12819). The accession numbers for the assembled chromosomes and mitochondrial genome are [LT222053](https://www.ebi.ac.uk/ena/data/view/LT222053) to [LT222057](https://www.ebi.ac.uk/ena/data/view/LT222057). The secretome and effector predictions can be found at <https://github.com/ana321wood/Secretome_CML3066_Feb2020/blob/master/Secretome_CML3066_Feb2020_AMW.txt>.
This research was supported by the Biotechnology and Biological Sciences Research Council of the United Kingdom (BBSRC) through the Institute Strategic Programmes 20:20 Wheat (BB/J/00426X/1) and Designing Future Wheat (BB/P016855/1), the BBSRC Embrapa joint wheat projects (BB/N004493/1 and BB/N018095/1), the CAPES Foundation of Brazil Ph.D. scholarship (BEX 1266-13-6), and the CNPq research fellow grant 303216/2012-3.
We thank Ludwig Pfenning for depositing the strain in the Coleção Micológica de Lavras (CML) in Brazil. We also thank the staff at the European Nucleotide Archive (ENA), Cambridge, United Kingdom.
[^1]: **Citation** Machado Wood AK, King R, Urban M, Nicolli CP, Del Ponte EM, Hammond-Kosack KE. 2020. Genome sequence of *Fusarium graminearum* strain CML3066, isolated from a wheat spike in southern Brazil. Microbiol Resour Announc 9:e00157-20. <https://doi.org/10.1128/MRA.00157-20>.
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Background {#Sec1}
==========
Diabetes is a chronic disease that is responsible for high rates of morbidity and mortality which can be attributed to atherosclerosis and cardiovascular disease \[[@CR1]\]. It is estimated that type II diabetes doubles the risk of cardiovascular disease even after adjustment of other cardiovascular risk factors \[[@CR2]\]. Despite the increase in the rate of treatment of diabetic patients with statins and glucose lowering drugs achieving target glycated hemoglobin (HBA1C) levels and low-density lipoprotein (LDL) levels \[[@CR3]\], another strategy of effective management of diabetes lies in management of the disease process at earlier stage \[[@CR1]\]. Prediabetes is a collective term that encloses individuals with glucose levels lower than cutoff levels for diabetes but too high to be considered normal. It is the term used for individuals with impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) and/or HbA1C levels ranging from 5.7 to 6.4% \[[@CR3]\]. Prediabetes is not an uncommon condition with an estimated worldwide prevalence of 343 million individuals expected to rise to 471 million by 2035 \[[@CR4]\]. Prediabetes is a serious clinical condition that not only increases the risk of developing diabetes but also increases the burden of cardiovascular disease risk. Compared to normoglycemic individuals, patients with prediabetes show a 20% higher risk of developing cardiovascular disease (CVD) \[[@CR5]\]. Prediabetes is a toxic state in which both micro- and macrovascular complications of diabetes can manifest \[[@CR6]\]. The prompt diagnosis and proper management of prediabetes are necessary to prevent progression to diabetes mellitus and to prevent microvascular and macrovascular complications that manifest early in the prediabetic state \[[@CR7]\]***.***
Aim of the work {#Sec2}
===============
Observes the effect of prediabetes on the severity of coronary artery disease in patients undergoing elective coronary angiography.
Methods {#Sec3}
=======
The current study was carried out at the cardiology department at a university hospital.
Inclusion criteria {#Sec4}
------------------
Patients who were admitted for elective coronary angiography and/or PCI starting from September 2017 to August 2018.
Exclusion criteria {#Sec5}
------------------
No exclusion criteria were applied.
After an informed written consent, all patients involved in the study were subjected to:
**A- History taking and examination** with special emphasis on age, sex, risk factors for coronary artery disease (smoking, HTN, DM, dyslipidemia, positive family history for premature CVDs), history of CKD detected either by reduction in GFR or high serum creatinine, history of prior percutaneous coronary intervention (PCI) or coronary arteries bypass grafting (CABG), or acute coronary syndrome (ACS).
**B- Laboratory tests**: Level of HBA1C and serum creatinine on admission.
**C- Estimation of renal function**: eGFR was estimated using MDRD formula:
eGFR = 186 × (serum creatinine)--1.154 × age--0.0203 (× 1.210 if black) (× 0.742 if female) \[[@CR8]\]
**D- Interventional data**: Number of vessels affected and atherosclerotic burden of CAD assessed by Gensini score \[[@CR9]\]***.*** For patients undergoing PCI, additional data was collected regarding number of stents used, type of stents used, and total length of stents used.
The studied patients were divided according to HbA1C level to 3 groups:
1- Group A: Normoglycemic patients (HBA1C \< 5.7%)
2- Group B: Prediabetic patients (HBA1C 5.7--6.4%)
3- Group C: Diabetic patients (HBA1C \> 6.4%) \[[@CR10]\]
Statistical analysis {#Sec6}
--------------------
Data were collected and revised on PC. Data were tabulated and statistically analyzed using SPSS 17 software, mean and standard deviation (± SD), and range for parametric numerical data, while the median was used for nonparametric numerical data. Student t-test was used to assess the statistical significance of the difference between two study group means. Mann--Whitney test (U test) was used to assess the statistical significance of the difference of a nonparametric variable between two study groups. Chi-squared test was used to examine the relationship between two qualitative variables. Fisher's exact test was used to examine the relationship between two qualitative variables when the expected count is less than 5 in more than 20% of cells.
Results {#Sec7}
=======
Patients were divided to group A (normoglycemic group, *N* = 228), group B (prediabetes group, *N* = 177), and group C (diabetic group, *N* = 326). Prediabetics represented 24% of the study population (Table [1](#Tab1){ref-type="table"}). Table 1Group classificationHBA1CGroup A(*n* = 228)\
(31.2%)Group B(*n* = 177)\
(24.2%)Group C(*n* = 326)\
(44.6%)Mean ± SD5.25 ± 0.246.00 ± 0.228.92 ± 1.60Range4.5--5.65.7--6.46.5--13
Among patients with HBA1C in the prediabetic range, there were only 8 patients who were known prediabetic and on medical treatment. Among the diabetic group, 7% of patients were newly diagnosed, denoting that newly diagnosed prediabetics and diabetics represent 26% of the study population.
Demographic and clinical characteristics {#Sec8}
----------------------------------------
There was no significant difference regarding age among the three groups, yet group C showed higher prevalence of male gender and a lower prevalence of smoking. Both DM and prediabetes group showed significantly higher prevalence of HTN. The normoglycemic group showed a stronger family history of CAD (Table [2](#Tab2){ref-type="table"}). Table 2Demographic and clinical characteristics of the groupsGroup AGroup BGroup CTest\
value*P* valueSig.Post hoc\
analysisNo. = 228No. = 177No. = 326P1P2P3Age (years)56.68 ± 9.2157.10 ± 9.8458.26 ± 8.872.1660.115NS------Sex(male)171 (75.0%)132 (74.6%)213 (65.3%)7.8230.020S0.9240.0150.033Smoking114 (50.0%)99 (55.9%)111 (34.0%)26.5880.000S0.2360.0000.000HTN84 (36.8%)90 (50.8%)198 (60.7%)30.6490.000S0.0050.0000.032Dyslipidemia120 (52.6%)86 (49.0%)165 (50.6%)0.660.7NS------Known CKD12 (5.3%)9 (5.1%)10 (3.1%)2.0020.367NS------Family history of CAD97 (42.5%)36 (20.3%)82 (25.2%)28.8040.000S0.0000.0000.224*P* value \> 0.05, nonsignificant; *P* value \< 0.05, significant; *P* value \< 0.01, highly significant\*: Chi-squared test; •: one-way ANOVA testP1: *P* value group A vs group BP2: *P* value group A vs group CP3: *P* value group B vs group C
Assessment of renal function {#Sec9}
----------------------------
On comparing the three groups, there was no significant difference regarding the mean eGFR or prevalence of CKD (Table [3](#Tab3){ref-type="table"}). Table 3Assessment of renal functionGroup AGroup BGroup CTest\
value*P* valueSig.No. = 228No. = 177No. = 326CreatinineMean ± SD1.03 ± 0.261.05 ± 0.251.02 ± 0.290.980•0.376NSRange0.6--1.60.6--1.90.5--2.9eGFRMean ± SD78.86 ± 23.3176.92 ± 23.2878.41 ± 24.120.361•0.697NSRange35--14237--14225--149CKD54 (23.7%)33 (18.6%)75 (23.0%)1.711\*0.425NS*P* value \> 0.05, nonsignificant; *P* value \< 0.05, significant; *P* value \< 0.01, highly significant\*, Chi-squared test; •, one-way ANOVA testP1: Group A vs group BP2: Group A vs group CP3: Group B vs group C
Prior history of ischemia {#Sec10}
-------------------------
There was no significant difference in history of PCI or CABG prior to the current procedure between the different groups with significantly higher prevalence of prior ACS in patients with prediabetes (Table [4](#Tab4){ref-type="table"}). Table 4History of CAD among the different groupsGroup AGroup BGroup CTest\
value\**P* valueSig.Post hoc analysisNo. (%)No. (%)No. (%)P1P2P3Prior PCI42 (18.4%)39 (22.0%)75 (23.0%)1.7470.417NS------Prior CABG9
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INTRODUCTION
============
Sudden infant death syndrome (SIDS) is death of an infant that is neither attributable to medical history nor explained after autopsy or by death scene investigation. SIDS is the leading cause of death in the first year of life after the neonatal period and is currently responsible for 0.53 deaths per 1000 infants. High incidence, catastrophic impact on affected families and absence of mechanistic insight means that SIDS represents a major medical challenge. Since the 'back to sleep' campaign in 1994, there have been no further reductions in SIDS incidence. A number of causative mechanisms have been proposed to lead to SIDS, but without any unifying theory or correlation with pathological findings ([@b7-0060503]).
A study of 33,034 infants found that 50% of infants who died of SIDS had a prolonged QTc interval in the first week of life ([@b13-0060503]). Approximately 10% of SIDS cases carry functionally significant genetic variants in sodium and potassium channels causing long QT ([@b2-0060503]), or variants in the gap junction protein Connexin43 (Cx43) ([@b16-0060503]). This circumstantial evidence suggests a role for abnormal electrical conduction in SIDS, but the underlying cause(s) in the vast majority of cases remains unexplained.
Most risk factors for SIDS, including prone sleeping position, respiratory disorders and high altitude, are associated with a reduced oxygen environment. Furthermore, hypoxia is associated with a prolonged QT interval in the adult ([@b11-0060503]; [@b15-0060503]). We therefore hypothesised that neonatal hypoxia leading to abnormal electrical conduction is a potential cause of sudden death.
We used non-invasive electrocardiography to characterize the postnatal maturation of the cardiac electrical conduction system in neonatal mice ([@b6-0060503]). We investigated whether reduced ambient oxygen environment or genetically manipulated hypoxic signalling affected maturation of the cardiac electrical conduction system and the subsequent risk of sudden death.
RESULTS
=======
Maturation of ECG morphology in wild-type mice
----------------------------------------------
To assess electrocardiographic changes immediately following birth, unborn pups were removed from pregnant females at embryonic day (E)18.5 and placed with a foster mother. We performed electrocardiography in the same pups sequentially at 0, 1, 3, 6, 12 and 24 hours after birth. ECG morphology changes were detectable at 1 hour after birth, becoming significant at 3 hours. Heart rate increased whereas QRS, QTc and QTc dispersion (where 'c' denotes correction for heart rate) declined rapidly and then plateaued over the 24 hours ([Fig. 1](#f1-0060503){ref-type="fig"}). We recorded resting ECGs from postnatal day (P) 0.5 to P10 ([Fig. 1](#f1-0060503){ref-type="fig"}). The trends of increased heart rate and declining QRS, QTc and QTc dispersion continued over this timescale ([Fig. 1](#f1-0060503){ref-type="fig"}).
{#f1-0060503}
Hypoxia prevents maturation of the ECG
--------------------------------------
Neonates reared in 10% oxygen for 24 hours showed reduced heart rate and increased QTc and QTc dispersion compared with normoxic controls ([Fig. 2](#f2-0060503){ref-type="fig"}). These parameters were similar to those in newborn neonates.
{#f2-0060503}
*αMHC-Cre::VHL^fl/fl^* mice exhibit immature ECG morphology and sudden death
----------------------------------------------------------------------------
αMHC*-Cre::VHL^fl/fl^* mice have cardiac-specific deletion of von Hippel-Lindau protein (VHL), causing constitutive upregulation of cardiac hypoxia inducible factor (HIF) signalling. Their neonates showed decreased heart rate with increased QRS, QTc and QTc dispersion compared with control αMHC-*Cre::VHL^+/+^*or *αMHC-Cre::VHL^fl/+^*littermates at 10 days after birth ([Fig. 2](#f2-0060503){ref-type="fig"}). αMHC-*Cre::VHL^fl/fl^* mice died between P16 and P18. Before death, there were no observable differences between mutant and control littermates in behaviour or weight. *αMHC-Cre::VHL^fl/fl^* mice exhibited frequent cardiac arrhythmia, consistent with sudden cardiac death, as did hypoxic wild-type mice ([Fig. 3](#f3-0060503){ref-type="fig"}).
{#f3-0060503}
###### TRANSLATIONAL IMPACT
**Clinical issue**
Sudden infant death syndrome (SIDS) remains one of the major enigmas in modern medicine. The 'back to sleep' campaign in 1994, which encouraged parents to place infants on their backs to sleep, promoted a reduction in SIDS incidence from 2 to 0.53 infants per 1000 births. Since then, there has been no reduction in this figure. Thus, advice for parents remains limited, and tragedies that might be preventable continue to occur. It has been previously documented that ∼50% of infants that die from SIDS display a prolonged QTc interval in the first few weeks of life, but the mechanisms underlying this observation have been elusive (except in cases where rare channelopathies and other genetic abnormalities are present).
**Results**
In this study, the authors addressed this issue using a recent innovation in electrocardiography that allows non-invasive recording of the electrocardiogram (ECG) in mice. This enabled the first reported catalogue of ECG changes from birth to 10 days postnatally, measuring changes in heart rate, QTc interval and QRS duration. By altering ambient oxygen concentration or genetically manipulating cellular hypoxic signalling in neonatal mice, the authors show that an increase in ambient oxygen concentration after birth is important for driving maturation of cardiac electrical conduction. Reduced oxygen predisposed mice to arrhythmia and sudden death, which was associated with ECG abnormalities. At the cellular level, reduced oxygen caused aberrant gap junction phosphorylation and distribution, and misexpression of ion channels, in the heart. These findings are consistent with known risk factors of SIDS -- such as head covering, high altitude, respiratory infections, central nervous system abnormalities and the prone sleeping position -- all of which are directly or indirectly associated with a hypoxic environment.
**Implications and future directions**
This study provides a link between neonatal hypoxia, ECG abnormalities and sudden death, which might provide an explanation for many SIDS cases. The results support the use of regular ECG screening of infants, and subsequent close monitoring of infants displaying long QTc interval, as well as ensuring a well-ventilated environment in cots and the use of other hypoxia-prevention strategies. The mouse models used in this study will facilitate further investigation into SIDS, and the non-invasive ECG approach used here can be applied by other researchers investigating cardiac conduction defects in mice.
Risk of sudden death on exposure to hypoxia decreases with age in neonates
--------------------------------------------------------------------------
We hypothesised that postnatal electrocardiac maturation is oxygen dependent and that exposure of neonatal mice to hypoxia at later points after birth would result in lower rates of sudden death. When neonates were raised from birth in a hypoxic environment for 24 hours, mortality was 58%. When neonates were raised from birth in normoxia for 1, 6 and 12 hours, then placed into 10% hypoxia for 24 hours, mortality was reduced to 33, 25 and 17%, respectively ([Fig. 3](#f3-0060503){ref-type="fig"}).
Connexin43 distribution and quantification
------------------------------------------
Cx43 is essential for normal electrical conduction in the heart;
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Introduction {#Sec1}
============
One of the remarkable features of the Integer Quantum Hall Effect (QHE) is the impressive precision of the quantization of the plateaus observed in the experiments. While the experimental samples have a very complex microscopic structure, depending on a huge number of non-universal details related to molecular forces and the atomic structure, the conductance appears to be quantized at a very high precision, and the result only depends on fundamental constants. The understanding of this phenomenon, via a connection between the Hall conductivity and a topological invariant \[[@CR4], [@CR40]\] was a major success of theoretical condensed matter in the 80s. The argument was later generalized to non-interacting disordered systems \[[@CR1], [@CR5], [@CR9], [@CR10]\] and to clean multi-particle systems \[[@CR3], [@CR37]\]: however, the definition of conductivity in the interacting case required the presence of an unphysical averaging over fluxes, expected to be unimportant in the thermodynamic limit, but a proof remained elusive for many years. Arguments based on Ward Identities for Quantum ElectroDynamics in $\documentclass[12pt]{minimal}
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\begin{document}$$(2+1)$$\end{document}$-dimensions \[[@CR12], [@CR24], [@CR30]\], or on the properties of anomalies \[[@CR15]\], offered an alternative view on the QHE: they indicated that quantization should persists in the presence of many body interaction, but such conclusions were based on manipulations of divergent series, or of effective actions arising in a formal scaling limit.
The problem of a mathematical proof of the quantization of the Hall conductivity in the presence of many-body interactions remained open for several years. After the works \[[@CR1], [@CR3], [@CR5], [@CR9], [@CR10], [@CR37]\], it was dormant for more than a decade, and then, in recent years, it was actively reconsidered again, starting from \[[@CR27]\], which proved the quantization of the Hall conductance of an interacting electron system using quasi-adiabatic evolution of the groundstate around a flux-torus, under the *assumption* of a volume-independent spectral gap. In \[[@CR22]\] we followed a different approach, and proved the quantization of the interacting Hall conductivity by writing it as a convergent series, and by showing that all the interaction corrections cancel exactly, thanks to Ward Identities. Our result holds for interacting fermionic Hamiltonians of the form $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {H}}_0+U {\mathcal {V}}$$\end{document}$, where $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {H}}_0$$\end{document}$ is quadratic and gapped, with the chemical potential in the middle of a spectral gap of width $\documentclass[12pt]{minimal}
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\begin{document}$$\Delta _0$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {V}}$$\end{document}$ is a many body interaction, and $\documentclass[12pt]{minimal}
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\begin{document}$$|U|\ll \Delta _0$$\end{document}$; this smallness condition is assumed to ensure the convergence of the power series expansion in *U* of the Euclidean correlations. The same result also follows from \[[@CR27]\], in combination with a proof of the stability of the spectral gap for such fermionic Hamiltonians \[[@CR13], [@CR26]\]. See also \[[@CR6], [@CR7]\] for alternative proofs of the main theorem in \[[@CR27]\]. Recently, the bulk-edge correspondence for a class of weakly interacting fermionic systems displaying single-mode chiral edge currents was also proved \[[@CR2]\].
Given these results on the quantization of the Hall conductivity in weakly interacting systems (i.e., with interaction strength smaller than the gap), one naturally wonders what happens for stronger interactions. We focus on the interacting extension of the spinful Haldane model \[[@CR23]\], which has been recently realized in cold atoms experiments \[[@CR31]\] and can be used as the building block of more general topological insulators \[[@CR25]\]. Extensions to related systems is straightforward, in particular to the interacting, spin-conserving, Kane--Mele model, for which the quantization of the edge conductivity has been recently established \[[@CR34]\]. We recall that, in the absence of interactions, the phase diagram of the spinful Haldane model consists of two 'trivial' insulating phases, with vanishing transverse conductivity, and two quantum Hall phases, with transverse conductivity $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma _{12} =\pm \,2 e^2/h$$\end{document}$, separated by two critical curves. By \[[@CR22]\], we know that, away from the critical lines, for interactions *U* smaller than the spectral gap, the Hall conductivity is quantized and independent of *U*. However, what happens close to the critical lines, in cases where the interaction is much larger than the gap? This question, and in particular the possible emergence of new quantum phases, has been extensively investigated in the literature, mainly via mean-field, variational, and numerical studies, see \[[@CR28], [@CR29], [@CR38], [@CR41], [@CR42]\] and references therein. These works show evidence for the appearance of a new interaction-induced phase with $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma _{12} =\pm \,e^2/h$$\end{document}$, but the numerics is inconclusive on whether this phase, in the thermodynamic limit, emerges at arbitrarily small, positive, interactions or, rather, above a finite threshold. The main result of this work excludes the first possibility: no new phases appear close to the transition lines, as long as the interaction strength is sufficiently small, compared with the bandwidth $\documentclass[12pt]{minimal}
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\begin{document}$$t_0$$\end{document}$. More precisely, we compute the Hall conductivity for $\documentclass[12pt]{minimal}
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\begin{document}$$|U|\ll t_0$$\end{document}$ and all the values of the parameters outside two critical curves, across which the model undergoes a 'topological' phase transition: the Hall coefficient remains integer and constant as long as we continuously deform the parameters without crossing the curves; when this happens, the Hall coefficient jumps abruptly to a different integer. The main difficulties in proving such results are related to the fact that the critical lines are non-universal (i.e., interaction-dependent), thus making a naive perturbative approach ineffective. The 'dressing' of the critical lines is analogous to what happens in the theory of second order phase transitions, where the critical temperature is modified by the interaction, and one needs to appropriately tune the temperature as the interaction is switched on, in order to stay at criticality. Technically, we proceed in a similar way: we do not expand around the non-interacting Hamiltonian
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Introduction {#S1}
============
Understanding the endogenous pathways that regulate inflammatory responses is of critical importance for the development of novel therapies for chronic inflammatory disease. Recent approaches for the treatment of inflammatory diseases have focussed around blocking specific inflammatory mediators such as the cytokines TNF-α, IL-1β, and IL-6 ([@B1]--[@B3]). Whilst such approaches have been very effective for certain diseases \[e.g., anti-TNF-α monoclonal antibodies for the treatment of rheumatoid arthritis (RA)\] a large percentage of patients either do not respond to treatment or become refractory to therapeutic antibody treatment ([@B4], [@B5]). It is known that cytokines play a central role in shaping the immune response to invading pathogens and in the context of chronic inflammation. It is now appreciated that there exists a plethora of lipid mediators as well as other immune modulating proteins and peptides that play important roles during both the onset as well as the resolution of inflammation ([@B6]--[@B8]). Annexin-A1 and its N-terminal peptide ac2-26 have been demonstrated to exert potent pro-resolution properties in multiple inflammatory disease models ([@B8], [@B9]). Similarly, a number of lipid mediators including the resolvins, lipoxins, maresins, and protectins have been more recently come to the fore ([@B7], [@B8]). These novel immune modulatory mediators and others may represent new avenues to explore for the treatment of chronic inflammatory disease.
Murine chemerin is a 16 kDa protein produced and secreted as an inactive precursor, pro-chemerin, predominantly by hepatocytes and adipocytes ([@B10]). Pro-chemerin is present in the liver, spleen, skin, and plasma ([@B11], [@B12]). During an inflammatory response, inflammatory proteases produced locally by granulocytes and the coagulation cascade, cleave the carboxyl terminus of pro-chemerin to generate active chemerin isoforms at sites of inflammation ([@B13], [@B14]). Chemerin has been implicated in the pathology of a range of inflammatory diseases including RA, inflammatory bowel disease, psoriasis, diabetes, and cardiovascular disease ([@B15]--[@B19]). However, the exact role played by chemerin and its receptors in inflammatory disease remains unclear.
Our group and others have demonstrated that active chemerin, once generated, is a potent chemoattractant for macrophages, immature dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), and NK cells ([@B20]--[@B23]). Chemerin binds to three G protein-coupled receptors (GPCRs) with high affinity; CMKLR1, GPR1, and CCRL2 ([@B24]). Murine GPR1 is expressed in white adipose tissue, skin, muscle, and in the central nervous system ([@B25]). Although chemerin binding to GPR1 has been reported to induce downstream signalling, chemerin is thought to act as a partial agonist at GPR1 whilst chemerin acts as a full agonist at CMKLR1 ([@B26], [@B27]). CMKLR1 is expressed on various immune cells including macrophages, DCs, NK cells, and pDCs ([@B12]). It is also expressed on endothelial cells and adipocytes ([@B28], [@B29]). *Cmklr1* mediates the chemotactic effects of chemerin, and its activation has been reported to lead to rapid downstream signalling cascades, which are G~i/0~ coupled ([@B23], [@B26]). The Chemerin/*Cmklr1* axis has been implicated in driving the recruitment of immature DCs, pDCs, and NK cells to local sites of inflammation in a number of inflammatory diseases ([@B22], [@B30]--[@B32]). Interestingly, *Cmklr1* has also been reported to play an anti-inflammatory role in a number of inflammatory disease models, although these have predominantly been allergic inflammatory models ([@B12], [@B33]). In addition, our group and others have reported anti-inflammatory effects of synthetic chemerin-derived peptides in a number of inflammation models and these effects seem to be dependent on CMKLR1 ([@B34]--[@B36]).
CCRL2 is a seven transmembrane receptor that lacks the DRYLAIV intracellular motif required for classical downstream signalling by GPCRs ([@B37]). It binds chemerin but does not induce classical downstream signalling nor does it internalise chemerin ([@B20], [@B37], [@B38]). CCRL2 is expressed on a range of cell types including macrophages, DCs, endothelial cells, and epithelial cells amongst others ([@B38], [@B39]). Expression of CCRL2 is upregulated in response to inflammatory stimuli but the function of CCRL2 during inflammation remains incompletely understood ([@B39], [@B40]). Zabel et al. have proposed a model in which CCRL2 binds to the non-signalling N-terminus of chemerin and then presents it to other cells expressing CMKLR1. In this way, CCRL2 could function to concentrate chemerin at local sites to augment chemerin signalling during inflammation ([@B38]). The aim of this study was to further explore the role of the non-signalling chemerin receptor CCRL2 during a self-resolving model of acute inflammation.
We report, for the first time, that animals lacking the chemerin receptor CCRL2 displayed exaggerated neutrophil and inflammatory monocyte recruitment in models of acute inflammation. These effects were due in part to increased levels of chemerin, which augmented production of the neutrophil chemoattractant CXCL1, resulting in increased neutrophil recruitment.
Materials and Methods {#S2}
=====================
Animals {#S2-1}
-------
B6.129-*Ccrl2^tm1Dgen^*/J mice were obtained from Jackson Laboratories (Bar Harbour, ME, USA). These mice, originally produced by Deltagen (San Mateo, CA, USA), were backcrossed for 12 generations onto the C57BL/6J background. All animal studies were conducted with ethical approval from the Dunn School of Pathology Local Ethical Review Committee and in accordance with the UK Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986).
Reagents {#S2-2}
--------
Recombinant murine chemerin (aa17--156) was reconstituted in PBS supplemented with 0.1% BSA. Neutralising anti-chemerin antibody and goat IgG control were reconstituted in PBS. Both chemerin and antibodies were purchased from R&D Systems (Abingdon, UK). Zymosan was purchased from Sigma-Aldrich (Dorset, UK). Recombinant CCL5 was purchased from Peprotech (London, UK). Bio-gel P100 (45--90 µm) fine polyacrylamide beads were obtained from BIO-RAD Laboratories, Hemel Hempstead, Hertfordshire, UK.
Murine Bone Marrow-Derived Macrophages (BMDMs) {#S2-3}
----------------------------------------------
Bone marrow-derived macrophages were generated as previously described ([@B41]). Briefly, fresh bone marrow cells from tibiae and femurs of C57BL/6J mice (8--12 weeks) were isolated and cultured in DMEM media supplemented with 10% heat inactivated fetal bovine serum, 10% L929 cell-conditioned media as a source of macrophage colony-stimulating factor, 100 U/ml penicillin, and 100 µg/ml streptomycin for 7 days. A total of 4 × 10^6^ bone marrow cells were seeded into 10 ml of medium in 100 mm Petri dishes (Sterilin, Abergoed, UK.) and on day 3 an additional 5 ml of medium was added. Cells were harvested by gentle agitation to lift cells off surface.
Human Umbilical Vein Endothelial Cells (HUVEC) {#S2-4}
----------------------------------------------
Human umbilical vein endothelial cells were isolated as previously described ([@B42]) and frozen for storage in liquid N~2~. HUVEC were thawed and resuspended in endothelial cell growth medium with supplement mix C (PromoCell, Germany), 100 U/ml penicillin, and 100 µg/ml streptomycin and cultured in pre-coated 0.5% gelatin (Sigma) flask/dishes in a 37°C 5% CO~2~ incubator.
Cell Activation Assays {#S2-5}
----------------------
Cells were seeded into 6-well plates at a concentration of 1 × 10^6^/ml. They were then exposed to TLR ligands and cytokines for 16 h in a 37°C 5% CO~2~ incubator as described previously. The TLR ligands and cytokines were added to cells at a final concentration of: LPS 100 µg/ml, IFNγ 20 ng/ml, Poly I:C 10 µg/ml, zymosan 10 µg/ml, flagellin 500 ng/ml, IL-4 20 ng/ml, and IL-13 20 ng/ml.
RNA Isolation and Reverse Transcription and RT-PCR {#S2-6}
--------------------------------------------------
Total RNA was extracted using the QIAGEN RNeasy Mini kit as instructed by the manufacturer as described previously ([@B43]). RNA concentration and purity was determined using ND-1000 spectrophotometer (Nanodrop, Thermo Scientific) at 260/280 and 260/230 nm. cDNA was synthesised from 500 to 800 ng of total RNA using QuantiTect Reverse Transcription kit following the manufacturer's instructions. cDNA was amplified for 15 min at 42°C and then 3 min at 95°C. Real-time quantitative PCR was performed using
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INTRODUCTION {#sec1-1}
============
*Gynura* genus belongs to the family Asteraceae, consisting of 12 species in China.\[[@CIT1]\] Many species are edible medicinal plants and the leaves are also used as a vegetable by the locals in Southwestern China.\[[@CIT2]\] *G. divaricata* is a traditional Chinese medicinal plant, which is called "Bai Bei San Qi" in Chinese. It has a long history of use for treatment of diabetes in the folk medicine. The ethanol extract of aerial parts of *G. divaricata* was reported to demonstrate hypoglycemic activity in vivo, the flavonoid compounds were the active constituents.\[[@CIT3][@CIT4]\] It also has been reported that many constituents with antiproliferation activity exist in *G. divaricata*.\[[@CIT5][@CIT6]\] The chemical constituents of *G. divaricata* include flavonols, phenolic acids, cerebrosides, polysaccharides, alkaloids, terpenoids, and sterols.\[[@CIT5]--[@CIT10]\] Flavonols were the principal constituents of the plant, 4 flavonol compounds, including quercetin, isoquercitrin, rutin, and kaempferol-3-O-rutinoside, have been isolated and identified from the aerial parts of the plant.\[[@CIT9]\] This article herein describes the isolation and structure elucidation of the flavonol and phenolic acid compounds from the ethanol extract of *G. divaricata* DC. leaves by NMR and high-performance liquid chromatography-diode array detector-electrospray ionization-mass spectrometry (HPLC-DAD-ESI-MS) methods.
MATERIALS AND METHODS {#sec1-2}
=====================
General {#sec2-1}
-------
The^1^H-NMR and^13^C-NMR spectra were measured with a Bruker Avance-600 FT-NMR spectrometer (Bruker, Coventry, Germany), with TMS internal standard. HPLC-DAD-ESI-MS were recorded on Waters 2995 Series LC and ZQ-4000 Mass spectrometer (Waters Corporation, Milford, MA, USA). Column chromatography was carried out with Silica gel (Qingdao Marine Chemistry Co. Ltd., 200-300 mesh, Qingdao, China), Sephadex LH-20, and Reverse phase octadecylsilyl (RP-ODS) (Pharmacia Co. Ltd., Minnesota, USA). Thin layer chromatography (TLC) was carried out with Silica gel GF~254~ (Qingdao Marine Chemistry Co. Ltd., Qingdao, China), and the compounds were prepared either by spraying with 10% sulfuric acid ethanol or under UV lamp at 254 nm. HPLC-grade acetonitrile was purchased from Merck Company (Merck, Darmstadt, Germany), other solvents were analytical grade from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).
Plant material {#sec2-2}
--------------
The *Gynura divaricata* plant was obtained in 2009 from Guangdong province, China. A voucher specimen (201001) was deposited at the Department of Chemistry, Nanchang University. The leaves of *G. divaricata* were dried at 40°C in an air oven and finely powdered.
Extraction and isolation {#sec2-3}
------------------------
The weighed portion of the crude drug 5 kg was extracted twice with 60% ethanol (v/v) under reflux at 90°C. The extract was evaporated to dryness *in vacuo*. Extract yield with respect to the dried herb was 25%. The dry extract was suspended in water and subjected to sequential liquid-liquid extraction with chloroform, ethyl acetate (EA), and *n*-butanol, the yield of those 3 extracts were 31.2, 56.5, and 89.5 g, respectively. The EA fraction was chromatographed using flash column on a Silica gel eluted with chloroform-methanol step-gradient (starting with 100:0 to 4:1), eluted fractions were combined on their TLC pattern to yield 8 fractions. The chloroform-methanol fraction (10:1) was chromatographed on a Sephadex LH-20 column eluted with chloroform-methanol (1:1) to yield compounds 1 and 2. The chloroform-methanol fraction (6:1) chromatographed on a Sephadex LH-20 column eluted with methanol and further chromatographed on an RP-ODS column gradient eluted with methanol-water (40%-60%, v/v) gave compounds 3 and 7. The chloroform-methanol fraction (4:1) chromatographed on a Sephadex LH-20 column eluted with methanol yields compound 4 \[[Figure 1](#F0001){ref-type="fig"}\].
{#F0001}
The *n*-butanol fraction was chromatographed using flash RP-ODS column gradient eluted with methanol-water (10%-50%, v/v), and the eluted fractions were combined on their HPLC pattern to yield 4 fractions. The methanol-water fraction (25%, v/v) was further chromatographed using flash RP-ODS column and isocratic eluted with methanol-water (18%, v/v) gave compounds 5 and 6. The other minor constituents of *n*-butanol extracts were separated and identified by HPLC-DAD-ESI-MS method.
HPLC-MS instrument and conditions {#sec2-4}
---------------------------------
The HPLC-DAD-ESI-MS system consists of a Waters 2995 Series LC and ZQ-4000 Mass spectrometer (Waters, USA), equipped with a vacuum degasser, a quaternary pump, an autosampler, a thermostatted column compartment, a diode array detector (DAD), and an ion-trap mass spectrometer with electrospray ionization interface, controlled by Waters 2995 Series LC/ZQ-4000 Trap Software. Shimadzu shimpack VP-ODS (150 mm × 4.6 mm i.d., 5 μm particle size) was used for separation. Solvents for the mobile phase were water-0.1% acetic acid (A) and acetonitrile (B). The gradient elution was 0-30 min, linear gradient 10%-30% B; 30-40 min, linear gradient 30%-100% B. The flow rate was 0.8 mL/min and the column was operated at 30°C. Peaks were detected with the DAD at 254 nm. The ESI negative and positive ionization (NI and PI) total ion current (TIC) modes were used for MS detection. The *m/z* values of the monitored ions were from 100 to 800. The other parameters were as follows: capillary voltage, 3.5 kV; cone voltage, 30 V; extraction voltage, 5 V; RF voltage, 0.5 V; source temperature, 90°C; nitrogen gas flow for desolvation, 300 L/h; and temperature of the nitrogen gas for desolvation, 350°C. Samples for assay were dissolved in 45% MeOH as 3 mg/mL solutions and centrifuged at 12,000 rpm (Beckman, USA) for 15 min to remove particles before injection.
RESULTS AND DISCUSSION {#sec1-3}
======================
The compounds were identified using UV, ESI-MS, and NMR spectral data, and determined as quercetin,\[[@CIT11][@CIT12]\] kaempferol,\[[@CIT13][@CIT14]\] kaempferol-3-O-*β*-D-glucopyranoside,\[[@CIT15]\] quercetin-3-O-rutinoside,\[[@CIT15]\] kaempferol-3-O-rutinoside-7-O-*β*-D-glucopyranoside,\[[@CIT16][@CIT17]\] kaempferol-3,7-di-O-*β*-D-glucopyranoside,\[[@CIT16]\] and 3,5-Dicaffeoylquinic acid.\[[@CIT18]\]
Compound 1 was obtained as a yellow powder, the ESI-MS yielded a quasi-molecular ion peak \[M-H\]^-^ at *m/z* 301 and \[M+H\]^+^ at *m/z* 303. The UV spectrum showed λ~max~ at 256 and 370 nm. The^1^H-NMR spectrum showed 2 peaks at δ 6.18 (1H, d, *J* = 2.0 Hz) and 6.40 ppm (1H, d, *J* = 2.0 Hz) consistent with the meta protons H-6 and H-8 on A-ring and an ABX system at 7.68 (1H, d, *J*
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1. Introduction {#sec1}
===============
*Toxoplasma gondii* can infect all warm-blooded vertebrates, including mammals and birds \[[@B1]--[@B3]\]. Genetic diversity of*T. gondii* is widespread due to the biological and epidemiological diversity of this parasite.*T. gondii* isolates can be clustered into six major clades \[[@B4]\], and genetic diversity of*T. gondii* is especially common in South America \[[@B4]\]. Utilizing 11 genetic markers,*T. gondii* isolates in North America and Europe are grouped into four major clonal lineage types (I, II, III, and 12) \[[@B5], [@B6]\] using PCR-RFLP.
Rhoptry kinases are involved in mediating pathogenesis of*T. gondii*\[[@B7]\], and they are also master regulators that manipulate the host inflammatory responses \[[@B8], [@B9]\].*T. gondii*rhoptry protein 17 (ROP17), a member of the ROP2 subfamily \[[@B10]\], was predicted to have a cellular localization on the parasitophorous vacuole membrane (PVM), which may participate in the manipulation of the host signalling pathways \[[@B9]\]. Previous studies have shown the existence of sequence variation in some ROP genes, such as*rop7*,*rop9*,*rop13*, and*rop38* \[[@B11]--[@B14]\]. However, it is yet to be known whether sequence diversity exists in*rop17*gene of*T. gondii*. The objective of the present study was to examine sequence variation in*rop17*gene among*T. gondii*strains representing different genotypes and host and origins.
2. Materials and Methods {#sec2}
========================
2.1. *T. gondii* Isolates {#sec2.1}
-------------------------
Ten*T. gondii* strains collected from different hosts and locations were used for analysis in this study ([Table 1](#tab1){ref-type="table"}). These strains have been genotyped and their genomic DNA has been prepared as described previously \[[@B15]--[@B17]\].
2.2. Amplification of*rop17* Genes and Sequencing {#sec2.2}
-------------------------------------------------
The*rop17* gene was amplified by PCR. Two primers were designed based on the*rop17* sequence of*T. gondii* RH strain available in GenBank (accession number: KC997178): ROP17F, 5′-AGGACAACACTAGGTAGCGAGAACC-3′, and ROP17R, 5′-TGGCGAAGTCAAGAGACGACGCAG-3′. Each reaction was performed in a total volume of 25 *μ*L containing 12.5 *μ*L*Premix Taq*(TaKaRa, Dalian, China), ROP17F (20 pmol) 0.25 *μ*L, ROP17R (20 pmol) 0.25 *μ*L, template DNA (200 ng) 2 *μ*L, and ddH~2~O 8 *μ*L, and the reaction conditions were 94°C for 5 min, then 35 cycles of 30 sec at 94°C, 30 sec at 55°C, and 1 min 20 s at 72°C, and a final extension at 72°C for 10 min. All the PCR products were then cloned into pMD18-T vector (TaKaRa, China) after purification using the DNA purification kit (TIANGEN, China) and then sequenced by Songon Biotech Co., Ltd. (Shanghai, China).
2.3. Sequence Analysis and Reconstruction of Phylogenetic Relationships {#sec2.3}
-----------------------------------------------------------------------
The*rop17* gene sequences of different*T. gondii* strains were aligned using Multiple Sequence Alignment Program, Clustal X 1.83 \[[@B18]\], and the sequence differences were determined according to Chilton et al. \[[@B19]\] and Zhao et al. \[[@B20]\]. Phylogenetic reconstruction was based on the*rop17* gene sequences determined in the present study plus the corresponding sequences of strains TgC7, PRU, and RH available in GenBank (accession numbers: KC997176, KC997177, and KC997178) using three inference methods, namely, neighbor-joining (NJ), maximum likelihood (ML), and maximum parsimony (MP), using the sequence of*Neospora caninum* (NCLIV_027930) as the outgroup. All the analyses were conducted following previous studies \[[@B20], [@B21]\]. Phylograms were drawn using the Tree View program version 1.65 \[[@B22]\].
3. Results and Discussion {#sec3}
=========================
The length of the*rop17* genes from all examined*T. gondii* isolates was 1375 bp and A+T contents varied from 49.45% to 50.11%. The alignment of 10*rop17* sequences plus the corresponding sequences of the RH, PRU, and TgC7 strains available in GenBank revealed nucleotide polymorphisms at 33 positions, with an intraspecific variation of 0--2.1%. The genetic diversity in*rop17* gene was higher than our previous studies for PLP1 \[[@B23]\], ROP7 \[[@B11]\], eIF4A \[[@B24]\], and MIC13 \[[@B25]\] genes and the whole genome, secretome, and kinome of*T. gondii*\[[@B8]\]. 16 variable positions were identified as transitions and the rest variable nucleotides were classified as transversions, and no deletions were detected in the 13*rop17* gene sequences.
Phylogeny reconstruction using MP, NJ, and ML analyses revealed two major clusters ([Figure 1(a)](#fig1){ref-type="fig"}). Topologies of all trees based on nucleotide sequences inferred by three different methods were similar, with only the small difference of bootstrap values. The classical genotypes II and III and atypical Type 12 strain were clustered in one clade. The subtree of NJ analysis further showed that genotype III (strain CTG) was separated from other strains which were supported by bootstrap analysis, and the atypical Type 12 (TgWtSc40 strain) was closely related to classical genotype II (strain PRU) ([Figure 1(b)](#fig1){ref-type="fig"}).*T. gondii* genotype II is one of the parental lineage of Type 12 based on the analysis of the inheritance of multilocus genotypes \[[@B6], [@B26]\]. The somewhat close relationship between Type II and Type 12 strains coincided with analyses of*UPRT* and*SAG1* loci \[[@B6]\]. All the strains belonging to genotype I in this study were clustered together, including strain TgPLh and typical strains GT1 and RH. Atypical strains TgCat1, TgToucan, TgCatBr64, and TgCatBr5 were phylogenetically clustered more closely with Type I strains. Of these, TgCatBr64 and TgCatBr5 strains which originated from cats in Brazil were grouped together. Based on the limited*T. gondii* strains examined in the present study, the*rop17* gene sequences could distinguish the major clonal lineages, but not all, showing the weak differentiation of*T. gondii* genotypes compared to analyses using GRA5, GRA6, and AK gene sequences as genetic markers \[[@B27]--[@B29]\]. Further validation of the*rop17* gene sequences as genetic marker is warranted by sampling more*T. gondii*strains from wider geographical locations and more hosts.
The analyses of sequence variations in nucleotides and amino acids among different strains showed high ratio of nonsynonymous to synonymous polymorphisms (\>1), suggesting that*T. gondii rop17* shows signs of positive selection, although more isolates will be required to determine whether*rop17* gene is under selection at the population level. Under the immunized stresses of host cells, the positive selection occurring in*rop17* gene may increase stress resistance. Ongoing positive selection is also found in several polymorphic dense granule (GRA) antigens \[[@B30], [@B31]\] and some other ROPs \[[@B8]\].
4. Conclusion {#sec4}
=============
In summary, the present study demonstrated the existence of slightly high sequence variability in the*rop17* gene sequences among*T. gondii*strains from different hosts and regions, which may be explored as a new genetic marker for population genetic studies of*T. gondii* isolates, and contributed to discovery of the new strategies for vaccination, treatment, or diagnosis.
Project support was provided by National Natural Science Foundation of China (Grant nos. 31228022, 31172316, and 31230073) and the Science Fund for Creative Research Groups of Gansu Province (Grant no. 1210RJIA006). Associate Professor Chunlei Su at the Department of Microbiology, the University of Tennessee, Knoxville, USA, is gratefully thanked for providing reference*T. gondii*strains.
Conflict of Interests
=====================
The authors declare that there is no conflict of interests in this paper.
{ref-type="ref"}). In principle, DNA barcodes contain variation that can be posed as a character to differentiate species. Although the utility of DNA barcoding for species identification has raised debates over its feasibility (Collins & Cruickshank, [2013](#ece34875-bib-0016){ref-type="ref"}; Krisnamurthy & Francis, [2012](#ece34875-bib-0056){ref-type="ref"}), the method has been increasingly applied during the last decade, especially to facilitate biodiversity studies of very diverse but taxonomically poorly known regions (Blaxter, [2004](#ece34875-bib-0007){ref-type="ref"}; Hajibabaei et al., [2005](#ece34875-bib-0038){ref-type="ref"}), such as Sumatran tropical rainforests.
Sumatran tropical rainforests are very rich in flora and fauna (Davis, Heywood, & Hamilton, [1995](#ece34875-bib-0021){ref-type="ref"}; Laumonier, [1997](#ece34875-bib-0059){ref-type="ref"}; Whitten, Damanik, Anwar, & Hisyam, [2000](#ece34875-bib-0089){ref-type="ref"}); nonetheless, they are only sparsely studied compared to other islands in the Malayan Archipelago (Laumonier, [1997](#ece34875-bib-0059){ref-type="ref"}). In terms of plant diversity, the Sumatran forests are comparable to the forests of Borneo and are richer than those found in Java and Sulawesi (Meijer, [1981](#ece34875-bib-0064){ref-type="ref"}). Sumatra is reported as one of the global centers of vascular plant diversity with a species density of 3,000 to 5,000 species per 10,000 km^2^ (Barthlott, Mutke, Rafiqpoor, Kier, & Kreft, [2005](#ece34875-bib-0006){ref-type="ref"}). Roos, Keßler, Gradstein, and Baas ([2004](#ece34875-bib-0074){ref-type="ref"}) estimated a total number of 10,600 plant species in Sumatra with more than 300 endemic species. Laumonier ([1997](#ece34875-bib-0059){ref-type="ref"}) argued that many scientists mistakenly consider that the flora of Sumatra is sufficiently well known since it is similar to that of the Malaysian peninsula, but many parts, especially the center of the island, are floristically unexplored territories.
Despite the importance of conserving the ecosystem, the total forest area in Sumatra has decreased from over 23 million hectares to probably less than 16 million hectares between 1985 and 1997 (World Bank, [2001](#ece34875-bib-0090){ref-type="ref"}). The southern provinces of Sumatra have lost most of their lowland forests, including those in protected areas (Lambert & Collar, [2002](#ece34875-bib-0058){ref-type="ref"}). Approximately 7.5 million hectares of primary forest loss were recorded in Sumatra during 1990--2010 and an additional 2.3 million hectares of primary forest were degraded (Margono et al., [2012](#ece34875-bib-0062){ref-type="ref"}). Between 2000 and 2010, the deforestation rate was estimated to be above 5% per year in the eastern lowlands of Sumatra (Miettinen, Shi, & Liew, [2011](#ece34875-bib-0066){ref-type="ref"}). The total deforested areas in Sumatra within 2011 alone were recorded to be approximately 2,200 hectares or as much as 3,520 soccer fields (BP‐REDD+, [2015](#ece34875-bib-0067){ref-type="ref"}). The causes of these massive deforestation and forest degradation are a large‐scale conversion into timber or estate crop plantations, illegal logging, and forest fires. By 2010, 3.9 million hectares of Sumatran lowland forests had been converted into oil palm (*Elaeis guineensis*) plantations (Koh, Miettinen, Liew, & Ghazoula, [2011](#ece34875-bib-0050){ref-type="ref"}).
The extensive loss of natural habitat puts a great number of species at risk and may lead to the loss of tropical fauna including forest‐dwelling birds (Koh et al., [2011](#ece34875-bib-0050){ref-type="ref"}), mammals (Maddox, Priatna, Gemita, & Salampessy, [2007](#ece34875-bib-0061){ref-type="ref"}), and orangutan (Gaveau et al., [2009](#ece34875-bib-0032){ref-type="ref"}). Undoubtedly, the destruction also affects the plant diversity (Brook, Sodhi, & Ng, [2003](#ece34875-bib-0008){ref-type="ref"}; Corlett, [1992](#ece34875-bib-0017){ref-type="ref"}; Rembold, Mangopo, Tjitrosoedirdjo, & Kreft, [2017](#ece34875-bib-0073){ref-type="ref"}; Turner et al., [1994](#ece34875-bib-0084){ref-type="ref"}). The rate of species loss in tropical forests seems to be higher than the species exploration due to lack of resources and sound species conservation management such as limited number of taxonomists working in this region, inadequate herbarium collections, and inaccessible taxonomic literature (Kiew, [2002](#ece34875-bib-0047){ref-type="ref"}; Meyer & Paulay, [2005](#ece34875-bib-0065){ref-type="ref"}; Tautz, Arctander, Minelli, Thomas, & Vogler, [2003](#ece34875-bib-0082){ref-type="ref"}). Species explorations become more challenging when the species cannot be identified morphologically. Identification keys based upon morphological characteristics can be difficult to use if features are not present (e.g., in sterile or juvenile specimens) or not well developed.
The use of DNA barcoding might help to overcome the limitations of morphological characters and might help to speed up species identification. This has been made possible because DNA barcoding can identify organisms at any stage of development (e.g., Barber & Boyce, [2006](#ece34875-bib-0005){ref-type="ref"}; Hausmann et al., [2011](#ece34875-bib-0039){ref-type="ref"}; Heimeier, Lavery, & Sewell, [2010](#ece34875-bib-0043){ref-type="ref"}; Ko et al., [2013](#ece34875-bib-0049){ref-type="ref"}), or at particular gender (e.g., Elsasser, Floyd, Herbert, & Schulte‐Hostedde, [2009](#ece34875-bib-0027){ref-type="ref"}), or specimens isolated from small and incomplete tissue, whether it is fresh, broken, or old (e.g., Hajibabaei et al., [2006](#ece34875-bib-0037){ref-type="ref"}; Valentini, Pompanon, & Taberlet, [2008](#ece34875-bib-0086){ref-type="ref"}). DNA barcoding may also help to discover new species and to identify cryptic species (e.g., Hebert, Penton, Burns, Janzen, & Hallwachs, [2004](#ece34875-bib-0041){ref-type="ref"}; Pauls, Blahnik, Zhou, Wardwell, & Holzenthal, [2010](#ece34875-bib-0072){ref-type="ref"}; Ward, Costa, Holmes, & Steinke, [2008](#ece34875-bib-0087){ref-type="ref"}).
DNA barcoding is now well established for animals (Crawford et al., [2013](#ece34875-bib-0019){ref-type="ref"}; Hebert, Cywinska, Ball, & deWaard, [2003a](#ece34875-bib-0040){ref-type="ref"}; Hebert, Ratnasingham, & de Waard, [2003b](#ece34875-bib-0042){ref-type="ref"}; Hebert et al., [2004](#ece34875-bib-0041){ref-type="ref"}; Lim, [2012](#ece34875-bib-0060){ref-type="ref"}; Nagy, Sonet, Glaw, & Vences, [2012](#ece34875-bib-0068){ref-type="ref"}; Ward, Zemlak, Innes, Lasr, & Hebert, [2005](#ece34875-bib
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In many physiological processes, cells migrate by moving through narrow channels defined by the surrounding environment. One example is cancer metastasis, where a cancer cell squeezes through the endothelium to reach the blood stream and eventually forms a secondary tumor elsewhere in the body[@b1][@b2][@b3][@b4]. Over recent years, the study of cancer from a physical sciences point of view has drawn much attention[@b3][@b5][@b6][@b7][@b8][@b9][@b10]: Physical principles are believed to offer an alternative perspective of the disease and may help to optimize treatments[@b11] and detection[@b12]. The model we present in this paper emphasizes the role of the elastic properties of cancer cells and surrounding normal cells on the metastatic potential of the former. Our simulations show that elasticity mismatch *alone* can reproduce features of cancer cell migration observed in experiments.
More precisely, we propose a multiple scale model to study the motility of individual cells in a larger cells-on-substrate assembly that comprises normal and cancer cells. We will focus on the nearly confluent scenario which describes monolayers. Understanding the behavior of cell monolayers is an important biological question that goes beyond the physics of cancer since epithelial tissues, which support the structure of embryos and organs, often have a monolayer structure[@b13]. Examples of cells-on-substrate experiments that are not directly related to cancer include studies of collective behavior[@b14][@b15], wound healing[@b9][@b16][@b17] and colony growth[@b18].
Our work is motivated by recent experiments performed by Lee *et al*.[@b9] which showed that the Young modulus of metastatic human breast carcinoma cells (MDA-MB-231) is about *three times smaller* than the one of human breast epithelial cells (MCF10A). In the same study, the authors showed that the motility of a cancer cell embedded in a confluent monolayer of mostly normal cells was much larger than in the case where the layer is made of cancer cells only. This observation was partly attributed to the fact that short speed "bursts" decorate the trajectory of the cancer cell. These bursts typically occur when a cancer cell, highly deformed due to temporary crowding by the neighboring normal cells, rapidly relaxes to a less deformed shape as the cell escapes the crowded configuration. Hence, it was proposed that the elasticity mismatch between cancer cells and normal cells significantly contributes to the observed "bursty" migration behavior and the concomitantly larger motilities of the cancer cells.
In the experiments, the increased motility of the metastatic cancer cells is probably due to many factors where one is the cell mechanical properties. Additional differences between cancer and normal cells include inter cellular adhesions[@b9] and protrusion activity[@b19]. Here, the model parameters will be chosen so that all cells in the monolayer have identical properties except for their elasticity: Cancer cell are softer, normal cells are stiffer. The main results of our simulation studies demonstrate that elasticity mismatch alone is sufficient to trigger bursty migration behavior and significantly increase the motility of the soft cell. Moreover, the simulated migratory behavior of cancer cells in a layer of mostly normal cells is in qualitative agreement with the experiments[@b9].
The model that we use permits the description of very large cell shape deformations. We will show that this point is crucial to accurately describe bursty migration. The effect of deformability of cells and vesicles has recently been studied in other contexts. Many of these studies were based on a beads-and-springs model for the cell shape and focused on red blood cells in capillaries[@b20][@b21], bacteria in biofilms[@b22][@b23] and tissue growth[@b24]. Such models complement recent Potts model studies of cell sorting[@b25] and vertex model dynamical studies[@b26][@b27] of soft tissues.
The phase-field model that we propose is more general than these other approaches. First, it can be easily extended to include more complexity (i.e., cell internal degree of freedom). Second, the inactive part of the dynamics is self-consistently derived from non-equilibrium thermodynamics principles. In that sense, our approach more closely resembles that used in Refs [@b6],[@b7], which focused on tumor growth, and that used in Refs [@b28], [@b29], [@b30], [@b31], [@b32], [@b33], which focused on single cell behavior. Our phase-field model approach is applied to an *assembly* of cells and it retains shape and motion details at the single cell level. Modelling the system behavior down to the single cell level is important to describe the cooperation between normal and cancer cells that leads to the bursty migration behavior and the increased motility of the latter.
The paper is organized as follows. The next section contains the Results. The first subsection gives a brief summary of our cell monolayer model. Simulations results are given in the next two subsections. The second one reports the cell arrangement predicted by our model for monolayers of inactive (non-migrating) cells. These determine the initial conditions for the simulations of motile cells presented in the third subsection. There, the migratory behavior of a tagged cell in monolayers of varying cell mechanical properties is analyzed. Following is the Discussion. It contains a summary of our findings and discusses avenues to be explored. Finally, the Methods section gives extra details on the statics and dynamics aspects of the model and it gives a brief overview of the numerical procedure.
Results
=======
We model monolayers that comprise normal and cancer cells using a phase-field approach similar to the one recently employed by Najem *et al*.[@b30], who studied chemotropism in neural cells, by Löber *et al*.[@b31], who studied cell crawling on soft substrate, and by Larazo *et al*.[@b32][@b33], who studied the shape of red blood cells under flow in small channels. Here, we treat the monolayer as a 2D system. This is a good level of description since the cells dynamics are constrained in a region close to the plane defined by the substrate. In our model, it is assumed that cells do not grow nor divide. The idea is that each cell is described by a "field" which rapidly varies in the region of the cell boundary. Hence, we denote by *ϕ*~*n*~(*x*,*y*; *t*) the field associated with cell *n* where *x*,*y* are the two spatial coordinates and *t* is the time. An example of a cell field is shown in [Fig. 1A](#f1){ref-type="fig"}. The interior of each cell is defined by regions where *ϕ*~*n*~ = 1 while the exterior is defined by regions where *ϕ*~*n*~ = 0. At the cell boundary, *ϕ*~*n*~ interpolates rapidly between 0 and 1. From now on, all monolayer images that we will present only show the boundary of each cell except for a tagged cell that will be highlighted with a color as in [Fig. 1B](#f1){ref-type="fig"}.
Details of the model are presented in the Methods section and in the [Supplementary Information](#S1){ref-type="supplementary-material"}. However, a brief overview is given in the next subsection.
Model outline
-------------
The most important advantages of our phase-field monolayer model are: 1) The cell boundary does not have to be tracked explicitly. 2) Extremely large deformations can be described. 3) The mechanical properties and the velocities of each cell can be modeled individually. The latter point is particularly important since one of our goals is to make connections with the experiments of Lee *et al*.[@b9]. Our approach differs from vertex models[@b26][@b27] by allowing all types of cell deformations and any degree of cell coverage. The difference between our phase field model and an equivalent Cells Potts model[@b25] is at the level of the dynamics since the former is the continuum limit of the latter.
The time dependent behavior of the monolayer is described by dynamical equations for the cell fields,
where is the monolayer free energy, **v**~*n*~ is the translational velocity of cell number *n* and *δ* denotes a functional derivative. Note that this equation is written in terms of dimensionless units, which will be used throughout the paper. The relationships between dimensionless and real units are detailed in the [Supplementary Information](#S1){ref-type="supplementary-material"}, briefly reviewed at the end of the Methods section. The right-hand side of [Eq. (1)](#eq1){ref-type="disp-formula"} describes cell shape dynamics, which are determined by free energy changes. Details of the model free energy are given in the Methods section and in particular, by [Eqs (7](#eq14){ref-type="disp-formula"}) and ([10](#eq19){ref-type="disp-formula"}). The free energy contains several parameters and its minimum determines the prefered state of the system. Briefly, one parameter controls the elastic response of each cell to shape deformation (*γ*~*n*~ in [Eq. (7)](#eq14){ref-type="disp-formula"}). Another controls the preferred radius of the cells (*R* in see [Eq. (7)](#eq14){ref-type="disp-formula"}), which tend to be circular when they are not perturbed by other cells. Also, there is a parameter that controls the energy penalty for overlapping cells (*κ* in [Eq. (10)](#eq19){ref-type="disp-formula"}, which is chosen to be large). Note that the interactions between cells are strictly repulsive.
The velocity of each cell is the sum of two distinct contributions as described in [Eq. (
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1. Introduction {#sec0005}
===============
The prevalence of endometriosis in women during reproductive life is about 10%--15% \[[@bib0005]\]. It can affect not only peritoneum and ovary but also bowel, urinary tract, pericardium and lungs. Gastrointestinal localizations most commonly occur in the rectosigmoid. Colonic endometriosis can lead to a complete bowel obstruction \[[@bib0010], [@bib0015]\]. In emergency settings it is most frequently treated with stoma placement. This approach brings about all the risks related to emergency surgery and might have important psychological and biological side effects.
We herein present a case of sigmoid endometriosis with complete bowel obstruction treated with endoscopic stenting and delayed one step laparoscopic procedure. We only found another similar case reported in literature \[[@bib0020]\]. This work has been reported in line with the SCARE criteria \[[@bib0025]\].
2. Case report {#sec0010}
==============
A 38 years old woman presented at emergency care with a history of abdominal pain started two days earlier and constipation started nine days earlier, she reported nausea but no vomit.
The patient had personal history of endometriosis and laparoscopic right ovarectomy was carried out a few years before; no similar episodes of abdominal pain were reported. She had no family history of intestinal diseases.
The abdomen was meteoric and tender; vital signs were normal with the exception of tachycardia (105bpm). On laboratory exams the WBC was 14090/mm3 and CRP was 3,1 mg/L.
A plain abdominal X-ray was performed with evidence of small and large bowel distension and an Abdomen CT detected an irregular mass (diameter 2 cm) at the proximal sigmoid colon determining stenosis. In consideration of the occlusive state, of the radiologic findings and of the likelihood of endometriosis, emergency recto-sigmoidoscopy was performed. The procedure revealed only lumen narrowing without mucosal alterations. A metallic auto-expansible stent was placed to treat bowel obstruction and to delay surgery.
Fasting, parenteral rehydration, a double intravenous antibiotic therapy and analgesic drugs were started. Over the next 48 h the bowel obstruction was resolved. The patient underwent a transvaginal utltrasonography (TVUS) with evidence of peritoneal endometriosis in the Douglas pouch and suspected sigmoid deep endometrioid localization. CA-125 levels were increased (114,8 U/L). After 5 days from endoscopy a laparoscopic sigmoidectomy was performed without stoma placement.
Histological investigation revealed the presence of endometrioid foci with inflammation and fibrosis affecting the entire sigmoid wall \[[Fig 1](#fig0005){ref-type="fig"}\].Fig. 1Section of the sick sigma with the endoscopic metallic stent inside.Fig. 1
The patient was discharged at fifth postoperative day in good conditions and was referred to Gynecologists.
At one month surgical follow-up she had no more abdominal pain and constipation.
3. Discussion {#sec0015}
=============
Endometriosis is the growth of ectopic endometrium, most commonly on ovary and pelvic peritoneum \[[@bib0030]\].
It usually leads to pelvic pain, deep dyspareunia, dysmenorrhea and infertility \[[@bib0035], [@bib0040]\]. It can also affects other organs determining different clinical pictures. Even though intestinal localizations occur in about 5--15% of patients, only in about 1% bowel resection is required \[[@bib0010], [@bib0015]\].
Laparoscopy should be considered the diagnostic gold standard for Endometriosis.
At present clinical evaluation, imaging and serologic markers can lead to a correct diagnosis leaving surgery to selected patients with a "see and treat" rationale \[[@bib0045]\]. This is also true for deep infiltrating endometriosis; in fact TVUS has a reported sensitivity of 91% and specificity of 98% in detecting bowel localizations \[[@bib0050]\]. Furthermore elevated serum levels of CA-125 can be considered for diagnosis \[[@bib0055]\].
The low incidence of bowel obstruction due to Endometriosis makes the diagnosis unlikely. Contrast abdominal CT has a low specificity and clinical presentation (constipation, nausea, vomit, abdominal pain, rectal bleeding) is unspecific. Other much more common conditions such as Cancer, Inflammatory Bowel Disease and obstruction due to bowel adhesions have a similar onset \[[@bib0010]\]. This is why the diagnosis is usually made by gross histology once the therapeutic decision has already been taken. In the case described patient's age, personal history and the endoscopic findings guided the diagnostic and therapeutic flow-chart.
A very important aspect of the disease consists of the psycho-physical implication related to therapies that can drastically alter patient\'s quality of life \[[@bib0060]\]. For this reason the best management of endometriosis is by integrate approach of both medical and surgical treatment \[[@bib0045], [@bib0065], [@bib0070]\].
In the literature some cases of acute colonic obstruction due to endometriosis are described. Hartmann's procedure or direct anastomosis with defunctioning stoma were performed, either open or laparoscopic \[[@bib0075], [@bib0080], [@bib0085], [@bib0090]\].
Our patient was treated with endoscopic stenting as a bridge to elective laparoscopic surgery.
We consider that this approach should be taken into account when colonic obstruction due to endometriosis is suspected, especially in young women with positive personal history.
Endoscopic stenting is a relatively safe procedure, potentially avoids the costs of two steps surgical intervention and the psychological drawbacks related to stoma placement. Laparoscopic procedure also allows a higher pregnancy rate after surgery \[[@bib0005]\]. In the literature we only found another similar case reported to have good outcomes \[[@bib0020]\].
Conflict of interest {#sec0020}
====================
All the Authors declare that there is no potential personal conflict of interest or financial disclosures or acknowledgements.
Funding {#sec0025}
=======
This research do not receive any specific grant from funding agencies in the public, commercial or not-for-profit sector.
Ethical approval {#sec0030}
================
Ethical approval has been exempted by our Institution, because our paper is not a research but a case report.
Consent {#sec0035}
=======
Written informed consent has been obtained. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.
Author contribution {#sec0040}
===================
Pietro Calcagno: corresponding author who wrote the paper.
Matteo Viti: contribute by giving the paper concept.
Alessandro Cornelli: the consultant surgeon who managed the patient and run the operation.
Davide Galli: the assistance surgeon in patient's operation.
Corrado D'Urbano: head physician who receive the article and gave final approval.
Guarantor {#sec0045}
=========
Corrado D'Urbano.
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Introduction {#Sec1}
============
The relationships between different anatomical measurements are a fundamental aspect of human physiology, as has been elegantly depicted by Leonardo da Vinci in his seminal work 'The Vitruvian Man' (da Vinci [@CR12]; Vitruvius [@CR46]). This work shows these relationships are highly conserved, even for men of variable length. This ancient idea of describing *relationships* between different properties has reached many other fields of research, for example quantitative genetics (Steppan et al. [@CR36]) and individual differences psychology (Goldberg [@CR15]).
Also the levels of many metabolites in a biological system may be highly interrelated through the biochemical pathways. Perturbations of these biological systems (e.g. diet or disease) may alter enzyme activity and therefore the link between different metabolites. However, the extent of such alterations may also differ between individuals. Thereby also in- or decreases of the inter-individual metabolite level differences may indicate system change. Then not only absolute metabolite level differences between experimental groups, but also the relationships between the metabolites may indicate change. Such Between Metabolite Relationships (BMRs) therefore describe an aspect of metabolism that is complementary to the changes that are common to all individuals (Weckwerth et al. [@CR43]).
Recent advances in 'omics'-research brought the study of BMRs closer, because metabolomics emerges more and more as a system-wide approach to observe metabolism (Bino et al. [@CR3]; Fiehn [@CR13]; Hall [@CR16]). The data of a metabolomics study usually consists of a list of numerous metabolites, of which the levels are given for every measured sample (e.g. individual and/or time-point). Of prime interest to metabolomics studies may be to find the in- or decrease of specific metabolite levels between different groups of individuals (e.g. before and after an experimental perturbation) (Fig. [1](#Fig1){ref-type="fig"}a). However, this paradigm holds a major shortcoming for the system-wide view provided by metabolomics analyses, because it may disregard metabolite combinations that show interesting variation where the individual metabolites do not.Fig. 1Three paradigms to observe metabolic differences between two groups: **a** Level difference of an individual metabolite (e.g. ANOVA), **b** Level difference in a combination of, i.e. a component of more metabolites (e.g. PLS), **c** Changes in the combined relationship between metabolites (INDSCAL)
Univariate methods that quantify level changes of individual metabolites (e.g., ANalysis Of VAriance, ANOVA (Sokal and Rohlf [@CR35])) disregard the interrelations between levels of different metabolites and thereby the system-wide aspect of metabolism. Therefore in general multivariate methods are used to analyse data generated in metabolomics studies, mostly those from the 'Component Analysis' family such as PCA and PLS-DA (Barker and Rayens [@CR2], Jolliffe [@CR24]). These summarize data into a small number of 'components'---latent variables that gather information about the importance of all measured metabolites. These profiles are constructed based on the levels of all metabolites and express the relative importance of every metabolite in combination with all other metabolites. PCA or PLS-DA models in e.g. case--control studies enable to describe differences in metabolite combinations between groups, even if the levels of single metabolites are not significantly different (Fig. [1](#Fig1){ref-type="fig"}b).
However, neither ANOVA nor PLS-DA explicitly reveals the changes in the relationships between metabolites in different experimental groups. Also unsupervised methods like PCA (Jolliffe [@CR24]) may be insufficient to describe BMRs, because these methods cover all metabolic variation simultaneously. The BMRs---schematically depicted in Fig. [1](#Fig1){ref-type="fig"}c---usually remain entangled with other sources of metabolic change and remain beyond reach of any method in these two metabolomic paradigms.
Several studies focus on relations between metabolites (Steuer [@CR37]), enzymes and genes (van Erk et al. [@CR41]; Zhai et al. [@CR47]). These studies visualise such relations by Correlation Networks that show the relationships between all metabolite/enzymes/genes pairs (Steuer et al. [@CR38]). However, as already mentioned 'Due to the sheer number of pairwise metabolic correlations, large overview network graphs easily get incomprehensible' (Weckwerth et al. [@CR43]) which is specifically relevant in metabolomics. Therefore a method that both specifically focuses on BMRs and is based on interpretable components that describe the behaviour of the entire system (i.e. all pairwise metabolite relations together) is required. It will provide a novel and complementary view on metabolism.
In the field of individual differences psychology, a component method appropriate for the analysis of BMRs called Individual Differences Scaling (INDSCAL) is already available (Carroll [@CR7]). This method translates the changes in covariance or correlations between metabolites upon experimental manipulation into a series of scores and loadings, analogous to those from PCA or PLS-DA. A voluminous yet well-readable publication reveals that INDSCAL is a special version of Parallel Factor Analysis (PARAFAC) (Harshman and Lundy [@CR18]).[1](#Fn1){ref-type="fn"} The PARAFAC model has been used earlier to solve a range of questions in metabolomics studies that focused on changes in metabolite profiles, see e.g. (Montoliu et al. [@CR30]; Jansen et al. [@CR21]; Forshed et al. [@CR14]; Sinha et al. [@CR32]; Verouden et al. [@CR42]) and will therefore provide a view on BMRs intuitive to metabolomics researchers.
First BMRs and the INDSCAL model are presented. Then two metabolomics data sets are analysed with INDSCAL, one with a very prominent response of plant chemistry to herbivory and another with a much more subtle response of obese humans to catechin-enriched green tea extract (GTE). The results of standard data analysis methods used in metabolomics, such as ANOVA, PCA, and PLS-DA are compared to that of INDSCAL.
Theory {#Sec2}
======
In metabolomics experiments, one or more experimental factors can be manipulated (e.g. doses of a toxicant, different populations) to observe their effect on the metabolites present in an organism, often on different time-points after the manipulation. Metabolomic data consists of comprehensive biochemical descriptions of each sample as a list of metabolites with their corresponding levels. An 'experimental group' of multiple individuals---called biological replicates---undergo a combination of experimental factors. Technical and financial limitations usually lead to considerably more measured metabolites than the number of biological replicates.
The 'conceptual model' underlying most metabolomics experiments states that an experimental manipulation may change the levels of several metabolites. When this manipulation is performed on several biological replicates, their response should be similar to the other replicates, up to a certain deviation caused by natural and technical variation. When quantified in a linear model for one factor with groups 1...*k*...*K*, this leads to Eq. [1](#Equ1){ref-type=""}.$$\documentclass[12pt]{minimal}
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\begin{document}$$ {\mathbf{X}}_{k} = 1_{{I_{k} }} {\varvec{\upmu}}^{\text{T}} + 1_{{I_{k} }} {\varvec{\upmu}}_{k}^{\text{T}} + {\mathbf{S}}_{k} \quad {\text{for}}\,k = 1 \ldots K $$\end{document}$$where **X**~*k*~ is the (*I*~*k*~ × *J*) matrix containing the levels of each metabolite, indicated by 1...*j*...*J* in the biological replicates 1~*k*~...*i*~*k*~...*I*~*k*~ of experimental group *k*, **μ** is the length *J* 'centroid' vector of all samples, vector **μ**~*k*~ the centroid vector for group *k* expressed as a deviation from **μ**; matrix **S**~*k*~ contains the deviation of each individual biological replicate from vector **μ**~*k*~; see Supplementary Table 1 for a list of symbols used throughout the paper.
Equation [1](#Equ1){ref-type=""} is generally used to quantify the significance of this experimental manipulation on levels of a small subset of single metabolites. This can be done by ANOVA (Sokal and Rohlf [@CR35]) that estimates the treatment effects expressed in a series of vectors $\documentclass[12pt]{minimal}
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\begin{document}$$ {\varvec{\upmu}}_{k} \left( {k = 1 \ldots K} \right) $$\end{document}$: interesting putative biomarkers are then identified as variables *j* for which variation across the *j*-th elements of $\documentclass[12pt]{minimal}
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Thirty-five percent of adults in the United States and United Kingdom have chronic lower limb superficial venous disease. [@JR1900086oa-1] Varicose veins are more common in females, with a predilection toward the older age group and may run in families. A body mass index \>30 kg/m ^2^ is a risk factor for chronic venous insufficiency. [@JR1900086oa-2] Symptoms include limb heaviness, ache, and edema. Skin changes such as spider veins, varicose veins, hemosiderin deposition, inflammation, lipodermatosclerosis, and ulceration often follow in untreated cases. [@JR1900086oa-2] [@JR1900086oa-3] [@JR1900086oa-4]
The 2013 National Institute for Health and Care Excellence (NICE) guideline on diagnosis and management of varicose veins (updated March 2018) recommends radiofrequency ablation (RFA) or endovenous laser ablation (EVLA) as first line treatment for truncal reflux. Second-line is ultrasound-guided foam sclerotherapy. Open surgery is indicated only if the other methods are unsuitable. Any incompetent tributaries are preferentially treated in the same session. Compression hosiery should not be used longer than 7 days after intervention, and is first choice only in pregnancy or if the previously mentioned interventions are unsuitable. [@BR1900086oa-5] NICE also issued a specific guideline in 2015 on the use of *n* -butyl-2-cyanoacrylate (NBCA) for varicose veins but did not promote its routine use. [@BR1900086oa-6] Almeida et al reported the first human application of NBCA for incompetent great saphenous veins (GSVs) in 2013. All 38 veins under study were obliterated at 48 hours and 92% at 1 year with minor short-lasting adverse effects. [@JR1900086oa-7]
The aim of this systematic review is to assess the efficacy of NBCA in ablating primary truncal varicose veins and eliminating reflux compared with existing endovascular techniques in the immediate, medium, and long-term settings. Secondary outcomes include complications, patient acceptability, and quality of life.
Methods
=======
Protocol and Search Strategy
----------------------------
This review is registered in PROSPERO database (registration code: CRD42018106323) and followed the PRISMA checklist. [@BR1900086oa-8] [@JR1900086oa-9] One author performed a literature search and data extraction up to October 2018 with no set date range and using established MeSH vocabulary in PubMed, EMBASE, Scopus, Cochrane Library, and ScienceDirect. Search terms were: "varicose vein," "saphenous vein," "glue," " *n* -butyl cyanoacrylate," and " *n* -butyl 2 cyanoacrylate." References and article suggestions by search engines were assessed to identify more relevant studies. Duplicates were removed and further exclusions performed after reviewing abstracts. The chosen manuscripts were then scrutinized while applying inclusion and exclusion criteria.
Inclusion and Exclusion Criteria
--------------------------------
Human randomized controlled trials (RCTs), cohort studies, and case reports in English language involving the use of NBCA to treat primary truncal varicose veins (i.e., GSV, small saphenous vein \[SSV\], and anterior accessory saphenous vein \[AASV\]) were included. If more than one modality was used, the said manuscript was only included if the data for NBCA could be fully extracted. Studies excluding NBCA glue or comparing NBCA with treatments other than RFA, EVLA, or foam sclerotherapy were excluded. [@JR1900086oa-1] [@JR1900086oa-2] [@JR1900086oa-10] [@JR1900086oa-11] [Supplementary Table 1](#SM1900086oa-1){ref-type="supplementary-material"} (online only) summarizes patient characteristics for inclusion/exclusion.
Primary and Secondary Outcomes
------------------------------
Primary outcome was successful obliteration of lumen of target vein, defined as occlusion of the entire treated vein segment with no discrete segments of patency exceeding 5 cm, confirmed on color Duplex ultrasound (DUS) after the procedure. [@JR1900086oa-1] Follow-up DUS assessments at 3 days, 7 days, 1 month, 3 months, 6 months, 1 year, and 2 years were examined.
Influence of vein length, diameter, NBCA device, and postoperative compression stockings on early (3 months) and intermediate term (6 months, 1 year) occlusion rate was taken as secondary outcomes. Vein length was taken as a mean value incorporating GSVs, SSVs, and AASVs with no distinction between the three. Where a particular vein diameter was taken at different levels, the mean of these values was calculated.
Clinical, Etiological, Anatomical, and Pathophysiological classification and Varicose Clinical Severity Score (VCSS) were used to measure severity of varicose veins at baseline and postintervention. Quality of life was primarily investigated using the Aberdeen Varicose Vein Questionnaire (AVVQ). [@JR1900086oa-2] "Thrombophlebitis" and "abnormal skin reactions" in treatment zones were included with the general term "phlebitis." [@JR1900086oa-12] [@JR1900086oa-13] All thrombus extensions into the deep venous systems were classified as deep vein thromboses (DVTs). Complications common to the three ablation modalities were evaluated.
Data Extraction
---------------
Any uncertainties in the literature were discussed with the second author and the authors of the original manuscripts where applicable. Risk of methodological bias was explored using the Cochrane Risk of Bias tool for RCTs. [@BR1900086oa-14] [@JR1900086oa-15] Quality assessment was performed using the Downs and Black quality assessment tool (for RCTs) and the National Heart, Lung and Blood Institute: Quality Assessment Tool for Before-After (Pre--Post) Studies With No Control Group (NHLBI-QAT). [@JR1900086oa-16] [@BR1900086oa-17]
Statistical Analysis
--------------------
Continuous variables were represented by means, standard deviations, and ranges. Categorical variables were shown in actual numbers and percentages. Scatter plots were created using Python version 3.7 (Python Software Foundation, Beaverton, DE). Statistical analysis was done using IBM SPSS Statistics software (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY). Spearman\'s correlation and Mann-Whitney U-test were performed on groups of subjects at 3, 6, and 12-month intervals following NBCA treatment. These tests were chosen because continuous variables were not normally distributed. Level of statistical significance was taken as *p* \< 0.05.
Results
=======
Description of Studies
----------------------
The PRISMA flowchart ( [Fig. 1](#FI1900086oa-1){ref-type="fig"} ) depicts the choice of manuscripts at different phases. One case report was identified but not reviewed as it contained heterogeneous data. [@JR1900086oa-18] All were published in peer-reviewed indexed scientific journals. There were 3038 participants (3,220 veins). A subgroup of 128 patients were excluded because of the missing data. [@JR1900086oa-19] [@JR1900086oa-20] [@JR1900086oa-21] Of the 2910 patients who were included, 1981 received NBCA, 445 RFA, and 484 EVLA. Comparison of NBCA with RFA and/or EVLA was performed in three RCTs and two retrospective studies. [@JR1900086oa-10] [@JR1900086oa-12] [@JR1900086oa-19] [@JR1900086oa-21] [@JR1900086oa-22] No studies compared NBCA with sclerotherapy, but this was frequently used an adjunctive treatment. Levels of evidence for therapeutic studies were judged using criteria from the Centre for Evidence-Based Medicine. [@JR1900086oa-23]
{#FI1900086oa-1}
Quality and Risk of Bias Assessment
-----------------------------------
### Randomized Controlled Trials
Risk of bias for RCTs is illustrated in [Table 1](#TB1900086oa-1){ref-type="table"} . Bozkurt and Yilmaz pseudorandomized their patients to alternate EVLA and NBCA. This led to a high risk of selection bias. [@JR1900086oa-10] Randomization was better in the VeClose trial and the study by Eroglu and Yasim. [@JR1900086oa-1] [@JR1900086oa-12] [@JR1900086oa-19] The former also included "roll-in cases" so that investigators could achieve familiarity with the NBCA procedure. DUS assessments were not always performed by blinded personnel. Attrition bias was unclear in two RCTs as drop-outs were not formally analyzed. [@JR1900086oa-13] [@JR1900086oa-20] Effect of adjunctive therapies and postoperative compression stockings was not evaluated. Only one performed power analysis. [@JR1900086
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Introduction
============
The petri dish and test tube methods are the two sub types of microbiological inhibition methods. Compared to petri dish methods, the test tube methods are more suitable for high-throughput screening of antimicrobial drugs residues in animal food because it is neither time consuming nor laborious ([@B15]). *Geobacillus stearothermophilus* is the most widely used indicator bacterium in microbiological inhibition methods in terms of test tubes, as it is not easily contaminated, demands high incubation temperature (55°C) and grows faster in a short time (less than 4 h) than other bacteria. Moreover, it is more sensitive to antimicrobial agents, particularly, β-lactam ([@B10]). Additionally, spores of *G. stearothermophilus* are more resistant to adverse factors than vegetative cells and show stable activity for a long time. Therefore, spores of *G. stearothermophilus* can be added into kit's medium during the kits preparation process, which simplifies the detection procedure and prolongs the shelf life of kits. However, *G. stearothermophilus* is not sensitive enough to many commonly used antibiotics except β-lactam ([@B16]). In past years, a number of studies by microbiological inhibition methods in terms of test tubes were developed to improve the sensitivity of *G. stearothermophilus* to different kinds of antibiotics residues in milk. There are brilliant black reduction test (BRT AIM) ([@B13]), Copan milk test ([@B11]), Doveltest SP-NT ([@B1]), Eclipse100^®^ ([@B2]), and Charm^®^ Blue-Yellow II ([@B12]). Among these kits, Charm^®^ Blue-Yellow II can detect more antibacterial drugs including β-lactam, aminoglycosides, tetracyclines, sulfonamides, and macrolides. However, this method is not sensitive enough to aminoglycosides and macrolides, and extremely insensitive to quinolones. The chicken egg and honey are also consumed daily and important for human health. However, little research by microbiological inhibition methods in terms of test tubes is known about chicken egg and honey. Even Premi^®^Test, the test tube method is widely applied for the detection of antibiotics residues in milk, muscle, kidney, egg, honey and feed etc. However, Premi^®^ Test is not considered ideal to detect residual antibiotics in chicken egg and honey, as it does not show enough sensitivity to aminoglycosides, macrolides and quinolones ([@B19]). Therefore the aim of the present study was to develop a new test tube method with *G. stearothermophilus var* C953, which was more sensitive to a different kind of antimicrobial agents especially aminoglycosides, macrolides and quinolones in milk, chicken egg, and honey.
Materials and Methods {#s1}
=====================
Antimicrobial Standards
-----------------------
β--lactam: penicillin G (PEN), cefquinome (CEF); aminoglycosides: neomycin (NEO), streptomycin (STR); tetracyclines: doxycycline (DOX), tetracycline (TET); macrolides: erythromycin (ERY), spiramycin (SPI); sulfonamides: sulfadiazine (SDZ), sulfadimidine (SDM); lincosamides: lincomycin (LIN); quinolones: danofloxain (DAN), enrofloxacin (ENR); trimethoprim (TMP); and chloramphenicol (CAP) were all purchased from Sigma-Aldrich (St. Louis, MO, United States). Drugs for the preparation of antimicrobial solutions were stored and handled according to the manufacturers' instructions before use. In addition, the methods for the preparation of stock solutions and working standard solutions of antibiotics were shown in [Table 1](#T1){ref-type="table"}.
######
Methods for the preparation of stock solutions and working standard solutions of antibiotics.
Antimicrobial agents Solvents Diluents
---------------------- -------------------------------------- --------------------------------------
β --lactams Phosphate buffer, pH 6.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Aminoglycosides Tris, pH 8.0, 0.01 mol/L Tris, pH 8.0, 0.01 mol/L
Tetracyclines HCl, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Macrolides Phosphate buffer, pH 8.0, 0.01 mol/L Phosphate buffer, pH 8.0, 0.01 mol/L
Sulfonamides NaOH, 0.1 mol/L Sterilized distilled water
Lincosamides Phosphate buffer, pH 8.0, 0.01 mol/L Phosphate buffer, pH 8.0, 0.01 mol/L
Quinolones NaOH, 0.1 mol/L Phosphate buffer, pH 8.0, 0.1 mol/L
TMP Glacial acetic acid Sterilized distilled water
CAP Methanol Sterilized distilled water
Test Organism
-------------
*Geobacillus stearothermophilus var* C953 was obtained from American Type Culture Centre (ATCC), Rockville, MD, United States.
Recovery, Preparation and Conservation of Test Organism
-------------------------------------------------------
A freeze-dried strain of *G. stearothermophilus var* C953 was dissolved in sterile physiological saline (0.85% NaCl). A 100 μL of *G. stearothermophilus var* C953 suspension was inoculated into nutrient agar with 0.035 g/L MnSO~4~ ⋅ H~2~O and incubated in incubator for 24 h at 55°C. After three generations recovery, a single culture from nutrient agar with 0.035 g/L MnSO~4~⋅ H~2~O was inoculated into a new same medium and incubated in incubator for 72 h at 55°C. At the end of incubation, the cells were washed from medium by 10% (v/v) dried skimmed milk. After collection, the cells suspension was dispended into amber vials. Aliquots of cell suspensions stored at 4, -20, and -80°C for 6 h respectively step by step. After that, the frozen cells suspension was freeze-dried by freeze vacuum dryer and stored at -80°C until usage.
Preparation of Kit's Medium Components
--------------------------------------
Plate Count Agar (Becton Dickinson) fortified with glucose (6 g/L; Sigma^®^) was used. The medium was sterilized at 121°C for 15 min. After the medium was cool down to 50 ± 1°C, its pH was adjusted to 7.8 ± 0.1. After that, *G. stearothermophilus var* C953 spore suspension (5 × 10^9^ CFU/L), along with bromocresol purple (0.1 mg/L, Mallinckrodt^®^) and sensitizers such as 50 μg/L trimethoprim (TMP), 40 μg/L chloramphenicol (CAP), 45 μg/L streptomycin (STR) and 60 μg/L enrofloxacin (ENR) were added. A 150 μL of medium was added into each well of microtiter plates by using an electronic pipette (Eppendorf Research^®^Pro) after kit's medium components mixed well. Finally, these microtiter plates were sealed with aluminized film and conserved at 4°C until use.
Control Samples
---------------
Milk samples were collected from the dairy farm of Huazhong Agricultural University (HZAU), Wuhan, Hubei, China. At the time of samples collection, the cows did not receive any antimicrobial substances in the last 9 weeks and were at postpartum stage (between 60 and 90 days). Because bovine milk presented normal values of chemical composition, total bacterial counts (CFU \< 100,000 mL^-1^) and somatic cell counts (SCC \< 400,000 mL^-1^) ([@B5]) during these days. Milk samples were kept at 4°C for approximately 2 days throughout the experiment. The chicken eggs were collected from laying hens (30 weeks old) with a history of no antimicrobial drugs used either in the form of treatment or growth promoter in last 6 weeks at the chicken farm of HZAU. And chicken eggs were kept at 4°C within 1 week before use. Honey samples were purchased from the local bee farmer and the absence of any antimicrobial substances was confirmed by high performance liquid phase tandem mass spectrometry ([@B5]). Moreover, honey samples were stored at 4°C for less than 1 week before use.
Spiked Samples
--------------
Spiked samples were prepared from the respective antibiotics working standard solutions in a single step using antimicrobial drugs-free respective antibiotics diluents, milk, homogeneous eggs and diluted honey (spiked levels see [Tables 2](#T2){ref-type="table"}--[5](#T5){ref-type="table"}). In addition, eight concentrations at different levels were prepared for each drug, and 24 replicates were prepared for each concentration.
######
Limit of detection (LODs) of microbiological system in antimicrobial agents' diluents (3.75 h).
Antimicrobial agents Spiked levels /(μg/L)
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Erratum to: Mol Genet Genomics DOI 10.1007/s00438-009-0432-z {#Sec1}
============================================================
The Author would like to correct their names in the Author group. Instead of abbreviated form the names should change as:
Geetha Govind, Vokkaliga ThammeGowda Harshavardhan, Jayaker Kalaiarasi Patricia, Ramachandra Dhanalakshmi, Muthappa Senthil Kumar, Nese Sreenivasulu, Makarla Udayakumar.
The online version of the original article can be found under doi:10.1007/s00438-009-0432-z.
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Background
==========
Secretory leukocyte protease inhibitor (SLPI) is a member of the chelonianin class of serine protease inhibitors, and is predominantly expressed in secretory epithelial cells of mucosal surfaces, immune cells and has been identified in various tissues \[[@B1]-[@B3]\]. Among serine proteinase inhibitors, SLPI is considered as \"alarm proteinase inhibitor\" that is upregulated during infection or inflammation to compensate for high human neutrophil elastase \[[@B1],[@B4]\]. The C-terminus of SLPI primarily inhibits human elastase, but is capable of inhibiting other serine proteinases such as tryptase and cathepsin G \[[@B5]\]. In addition to its function as an antiprotease, SLPI possesses antimicrobial activity against several bacteria and fungi \[[@B1],[@B4],[@B6]\]. Furthermore, it was shown that SLPI controls cell proliferation by regulation of growth-associated genes such as cyclin D and transforming growth factor (TGF)-β1 \[[@B7]\], modifies the activation of macrophages \[[@B4]\] and regulates the LPS-induced activation of the transcription factor \"nuclear factor kappa B\" (NF-κB) \[[@B8],[@B9]\]. SLPI-deficient mice provided evidence for functional involvement of SLPI in wound healing \[[@B10]\] and lipopolysaccharide (LPS)-mediated inflammation \[[@B11]\]. In context to its role as \"alarm proteinase inhibitor\", SLPI was found to be differentially regulated in inflammatory diseases and cancer. Increased expression or elevated serum levels of SLPI were reported in human sepsis and experimental endotoxemia \[[@B12]\], febrile patients \[[@B13]\], Wegners\'s granulomatosis \[[@B14]\], gastric cancer \[[@B15]\] and pulmonary infection \[[@B16]\]. In contrast, other bacterial or viral infections in lung \[[@B17]\], stomach \[[@B18]\] and cervical epithelial cells \[[@B19]\] were found to be associated with decreased SLPI levels. The underlying mechanisms responsible for the different regulation of SLPI have not been identified, but most likely both microbial and host factors contribute to the up- or downregulation of SLPI in the various diseases. Notably, the reduction of SLPI levels correlated inversely with the severity of inflammation (infiltration of granulocytes) and neutrophil elastase activity in the gastric mucosa of *H. pylori-*infected individuals \[[@B20],[@B21]\] and the bronchoalveolar lavage fluid (BALF) of *Pseudomonas*-infected subjects \[[@B17]\].
Progranulin, also known as acrogranin, proepithelin and PC cell derived-growth factor, is a 68 kDa glycoprotein secreted by many epithelial and immune cells \[[@B22]\]. The full-length protein is subsequently modified by limited proteolysis leading to the generation of 6-25 kDa fragments called granulins \[[@B23]\]. Pathophysiologically, Progranulin has drawn a lot of attention in the last years since it has been identified that mutations of the corresponding *granulin*gene are causally linked to the development of frontotemporal dementia \[[@B24]\]. Individuals with these mutations exhibit tau-negative, but ubiquitin-positive, inclusions in their brain that eventually cause frontotemporal dementia. Both the precursor (Progranulin) and the degraded forms (Granulins) mediate different cellular effects in a variety of pathophysiological conditions such as inflammation, proliferation, carcinogenesis and wound healing \[[@B25]\]. While Progranulin acts as growth factor for epithelial cells, fibroblasts and neurons and has anti-inflammatory properties \[[@B26],[@B27]\], granulins drive inflammation leading to the infiltration of immune cells and induced cytokine expression \[[@B28],[@B29]\]. The conversion of Progranulin to granulins, which is the critical step in the regulation of the balance between both molecular forms, is controlled by SLPI that binds Progranulin and prevents degradation by elastase \[[@B23]\]. The importance of this interaction for the wound healing was demonstrated at the SLPI-deficient mice \[[@B10]\]. The lack of SLPI resulted in higher serine protease-derived activities that were associated with impaired wound healing in these animals \[[@B10]\]. The delayed wound healing was normalized after the addition of Progranulin providing evidence for the importance of the interaction between Progranulin and SLPI.
We recently identified a marked down-regulation of mucosal SLPI levels in *H. pylori*-infected subjects \[[@B18]\]. The role of SLPI for the balance between Progranulin and granulins and the high prevalence of mucosal injuries (ulcer, erosions) in *H. pylori*-infected subjects, prompted us to study the expression levels of Progranulin in context to that of SLPI in relation to *H. pylori*status. Considering the role of SLPI for regulating the activity of elastase, we hypothesized that the *H. pylori*-induced reduction of SLPI would lead to a reduction of mucosal Progranulin levels, since the higher elastase activities in the mucosa of *H. pylori*-infected subjects would degrade the molecule into the granulin fragments. In addition, gastric epithelial cells (either infected with *H. pylori*± transfection of SLPI-siRNA) were used as *in vitro*model to prove the proposed hypothesis.
Methods
=======
Study design and H. pylori status
---------------------------------
The study protocol was conducted according to the declaration of Helsinki and approved by the ethics\' committee of the Otto-von-Guericke University (No. of ethical vote: 143/99) as well as government authorities; all participants signed informed consent before entering the study. Details of the protocol (inclusion, exclusion criteria, and demographic data) were described previously \[[@B20]\]. The initial protocol was aimed at studying the interaction of *H. pylori*infection and low-dose aspirin. Briefly, human healthy volunteers were stratified according to the *H. pylori*status leading to the *H. pylori*-positive (*H. pylori*^*+*^, n = 10) and -negative (*H. pylori*^-^, n = 10) group. After successful eradication therapy, 9 out of 10 *H. pylori*-infected individuals agreed to participate in this study after 3 months again composing the *H. pylori*-eradicated (*H. pylori*^*erad*^) group. In order to investigate the potential interaction between Progranulin and SLPI, mucosal and serum levels as well as gene expression levels of Progranulin were analyzed retrospectively in existing samples and tissue specimens in relation to *H. pylori*status and SLPI expression levels published previously \[[@B20]\]. The analysis of Progranulin expression was performed in the \"pre-treatment\" samples, which correspond to day 0 \[[@B20]\] before \"low-dose\" aspirin was taken by the individuals.
The study includes a correlation analysis of mucosal Progranulin levels with those of SLPI studied in the same cohorts previously; details concerning the analysis of SLPI were reported recently \[[@B20]\].
Determination of Progranulin expression quantitative RT-PCR and ELISA
---------------------------------------------------------------------
Progranulin levels were quantified in the total protein extract from mucosal biopsies at sera stored at -80°C in previous study \[[@B22]\]. Progranulin levels were quantified using the Progranulin ELISA kit (Axxora GmbH, Lörrach, Germany; No: AG-45A-0018PP-KI01) as described by the manufacturer. Protein levels were normalized to ng/μg total protein content of extracted mucosal samples or ng/ml for sera.
Corresponding Progranulin m-RNA levels were determined by quantitative RT-PCR using existing cDNA samples stored at -80°C. Quantitative RT-PCR was performed using an iCycler (BioRad, Munich, Germany) and HotStarTaq Master Mix™ (Qiagen, Hilden, Germany) as described \[[@B23]\]. Initial activation of Taq-polymerase at 95°C for 15 min was followed by 40 cycles with denaturation at 94°C for 30 s, annealing at 60°C for 30 s and elongation at 72°C for 30 s. The fluorescence intensity of the double-strand specific SYBR-Green I, reflecting the amount of actually formed PCR-product, was read real-time at the end of each elongation step. Then specific initial template mRNA amounts were calculated by determining the time point at which the linear increase of sample PCR product started, relative to the corresponding points of a standard curve; these are given as arbitrary units (a.u.). Both PCR products were cloned into the pDIRECT™ (Qiagen, Hilden, Germany), and subsequent dilutions of the corresponding plasmid DNA were used to create a standard curve for the RT-PCR. The correlation coefficients of both Progranulin and β-actin standards were \> 0.95. β-actin mRNA amounts were used to normalize the cDNA contents of the different samples. Final data reflect the ratio in a.u. between Progranulin transcript and β-actin transcript levels. The following primers were used for the RT-PCR analysis: ß-actin (fw:5\'-GCC-ATC-CTG-CGT-CTG-GAC-C-3\' rev: 5\'-ACA-TGG-TGG-TGC-CGC-CAG-ACA-G-3\'; 400 bp), and Progranulin (fw:5\`-ATG-GCC-CAC-AAC-ACT-GAG-CAG-G-3\`, rev: 5\`-TCT-GGG-CAG-GGA-GCT-TCT-TTG-C-3\`, 440 bp). Both cDNA fragments included intron-spanning regions resulting in the generation of a larger PCR product from genomic DNA or its exclusion. Therefore, all identified PCR products can exclusively be attributed to the mRNA pool of the sample.
Immunohistochemical analysis of Progranulin expression in the gastric mucosa
----------------------------------------------------------------------------
To study the cellular origin of Progranulin
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In the 1950s, Barbara McClintock first discovered transposable elements (TEs) by analyzing genetic stocks of corn that were phenotypically unstable. Her discovery implied that a genetic control exerted by genomes was generally used to regulate TE mobilization. Any loss or decrease of this control would consequently result in severe genetic instabilities due to mobilization of TEs. Just such a genetic instability affecting the genome of *Drosophila melanogaster* under the control of a locus called *flamenco* (*flam*) was first reported in 1983. Focused on *flam*, this review retraces the numerous studies that have been performed from its discovery to the understanding of its ability to survey TEs.
A SINGLE GENOMIC MUTATION IS RESPONSIBLE FOR *Gypsy* ACTIVITY, A RETROELEMENT FROM *Drosophila melanogaster*
============================================================================================================
In the 1980s, [@B8] were studying the dominant *ovoD* mutation in *D. melanogaster* . The *Drosophila ovo* gene, which encodes a putative transcription factor (Ovo) with TFIIIA-like zinc fingers, is required for female germline survival and proper oogenesis. The gain of function *ovoD* allele results from an extension of the N-terminal region which gives rise to a neomorphic protein that causes female sterility ([@B25]). [@B8] performed crosses between *OvoD* males and females from a stock of flies from the lab of Madeleine Gans (MG) carrying a *y v f mal* X-chromosome . In the progeny, reversions of the *ovoD* mutation generating recessive *ovo* alleles were frequently observed which allowed fertile daughters to be recovered. Surprisingly, these reversions were also associated with the appearance of mutations in other loci, which could potentially be explained if such crosses were accompanied by the *de novo* mobilization of TEs. [@B26] found that, indeed, a high frequency of *gypsy* insertions was observed in the progeny of this cross and that a hot spot for *gypsy* exists into the *ovo* locus . Insertions of *gypsy* into the *ovo* locus interfere with the coding sequence of the neomorphic allele resulting in a null allele of the gene. Novel gypsy insertions can thus be assayed by the presence of fertile daughters. The gypsy mobilization could then explain both the genetic instability observed in these crosses and the *ovoD* reversion.
Also, [@B16] reported a mutator strain (MS) of *D. melanogaster* characterized by an elevated frequency of spontaneous mutations in the germ line up to 10^-3^ - 10^-4^. Mutations were recovered in both sexes and displayed the characteristics of being unstable with frequent reversion to wild type or to new mutant states. When analyzing the localization of a battery of TE families, they found that the genomic distribution of *P*, *mdg1*, *412* (*mdg2*), *mdg3*, and *copia* did not vary among the individuals of this strain. However, this was not the case for *gypsy* (*mdg4*) whose frequency of transposition was high and copy number greatly increased to 30--40 copies.
These initial studies identified different mutator lines in which the frequency of *gypsy* insertions is high while several other TE families remain stable ([@B26]; [@B17]; [@B23]). Further work ultimately showed that these *gypsy* instabilities within MS strains resulted from the combination of two factors: the presence of transpositionally active *gypsy* copies, and mutation(s) of loci regulating their transposition ([@B18]). These early studies provided an incredible powerful tool to evaluate *gypsy* activity by assessing the occurrence of fertile females resulting from *ovoD* reversion to a null allele. With the *ovoD* fertility test, one could isolate rare events without having to deal with enormous amount of progeny to score. Interestingly, these tools were created even before the understanding of the mechanism of repression.
A β-HETEROCHROMATIC LOCUS CONTROLS SEVERAL RETROELEMENTS: *gypsy*, *ZAM*, AND *Idefix*:
=======================================================================================
A mutation responsible for *gypsy* mobilization was identified within the y ***v*** *f mal* chromosome of MG stocks ([@B32]). Genetic mapping localized this mutation at the basis of the X-chromosome at position 65.9 (20A1-3) close to β-heterochromatin where numerous TEs were known to accumulate ([@B44]). The locus was called *flamenco* (*flam*) because it had the ability to make *gypsy* "dance." Non-permissive or permissive alleles of *flam* were defined according to their ability to restrict or allow *gypsy* mobilization, respectively. A fine-scale analysis of *flam* genetic characteristics uncovered that: (i) Its control on *gypsy* activity occurs under a strict maternal effect since transposition is only allowed in the progeny (male and females) of homozygous permissive females even if fathers are non-permissive. (ii) The mutant allele present in the MS strains is essentially recessive. (iii) Transposition is largely a premeiotic event. (iv) Although *ovoD* reversion is primarily controlled by *flam*, it is influenced by other factors such as age and temperature, reversion being higher in young flies grown at 25°C. (v) The effects of *flam* on *gypsy* expression are restricted to the somatic follicle cells that surround the maternal germline ([@B31]). Thus, *flam* function could be viewed as the maternal transmission of some factors preventing *gypsy* transposition.
In 1997, an unstable line called Rev was recovered after a PM mutagenesis performed on the line bearing the *w^IR6^* allele ([@B20]; Figure [1A](#F1){ref-type="fig"}). The *w^IR6^* allele is due to the insertion of the non-LTR retrotransposon *I-factor* into the first intron of the *white* gene. It gives an orange eye phenotype to flies ([@B19]). From the PM mutagenesis ([@B37]), a fly with a wild-type red-eye phenotype was recovered and established as a line subsequently called Rev because of the eye phenotype reversion from orange to red. It was found that the *white* locus had suffered a 8.4 kb insertion 3 kb upstream from the *white* start site of transcription (TSS; Figure [1C](#F1){ref-type="fig"}). This insertion corresponded to a novel TE from the *gypsy*-family that was previously uncharacterized and that has been named *ZAM* ([@B20]). *ZAM* did not only insert upstream of *white*. *In situ* hybridization and Southern analyses performed on the Rev genome revealed the presence of some 20 copies of *ZAM*, whereas *ZAM* was not found on the chromosomal arms of the original parental line *w^IR6^* (Figure [1B](#F1){ref-type="fig"}; [@B11]). From Rev, a series of mutations affecting eye coloration has been recovered, most of them affecting the *white* locus (Figure [1A](#F1){ref-type="fig"}). This second event of mutation resulted from the insertion of a novel *gypsy-like* transposable element designated *Idefix* that inserted 1.7 kb upstream of the TSS of the *white* gene. This second mutational event was recovered as a recurrent specific mutation in 11 independent individuals (Figures [1A,C](#F1){ref-type="fig"}; [@B11]). Genome analysis of Rev revealed that this line also suffered a recent and massive invasion of *Idefix* (Figure [1B](#F1){ref-type="fig"}).
{#F1}
The Rev line brought to light a new genetic model in which the activity of two TEs, *ZAM,* and *Idefix*, could be tested. Thereafter, transgenic flies were established with sensor-transgenes containing the full-length long terminal repeat (LTR) of *ZAM* or *Idefix* linked to the *LacZ* reporter gene. These transgenes provided a convenient read-out for analyzing the control exerted on these elements. Crosses designed to test the influence of the genetic background on these reporter constructs indicated that *ZAM* and *Idefix* responded to two types of controls: one restricting their expression to specific somatic cells of the ovaries and the other silencing their expression in the majority of *Drosophila* lines with only one exception reported in 2003 as being the Rev line ([@B12]).
Using these tools, a mutation responsible for the high activity of *ZAM* and *Idefix* was identified in Rev. This mutation was localized at the basis of the X-chromosome close to *flam* (Figure [2](#F2){ref-type="fig"}; [@B12]). Although
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1. Introduction {#sec1}
===============
The heat-shock proteins (Hsps) are a group of highly conserved proteins with major physiological roles in protein homeostasis \[[@B1], [@B2]\]. In most cell types even prior to stress Hsps constitute 1%-2% of total protein, suggesting an important role for these proteins in the biology and physiology of the unstressed cell. These particularly concern regulating the folding and unfolding of other proteins. The proteins are named, however, because they were first identified on the basis of their increased synthesis following exposure to elevated temperatures \[[@B3]\]. Subsequently it has been clearly shown that they can be induced following a variety of stressful stimuli. Some Hsps, such as Hsp90 (each Hsp is named according to its mass in kilodaltons) are detectable at significant levels in unstressed cells, increasing in abundance following a suitable stimulus, whilst others such as Hsp70 exist in both constitutively expressed and inducible forms that is activated by stressful stimuli \[[@B4], [@B5]\].
The dual role of Hsps in both normal and stressed cells, evidently requires the existence of complex regulatory processes which ensure that the correct expression pattern is produced. Indeed, such processes must be operative at the very earliest stages of embryonic development since the genes encoding Hsp70 and Hsp90 have been shown to be amongst the first embryonic genes which are transcribed \[[@B6], [@B7]\].
The induction of Hsps in response to various stresses is dependent on the activation of specific members of a family of transcription factors, the heat-shock factors (HSFs) which bind to the heat-shock element (HSE) in the promoters of the genes encoding Hsps \[[@B8]\]. Four HSFs (HSF1 to −4) have been cloned from a number of organisms and their roles have now been characterised. Only HSF1 and HSF3 have been shown to be involved in regulating Hsps in response to thermal stress whereas HSF2 and HSF4 are involved in Hsp regulation in unstressed cells and their levels are regulated in response to a wide variety of biological processes such as immune activation and cellular differentiation \[[@B8]\]. In general, however, the stimuli which induce such alterations in Hsp gene expression under nonstress conditions are poorly characterized and the mechanisms by which they act are unclear. In this paper, we discuss recent studies indicating that Hsps are not only regulated by HSFs alone, but also by transcription factors which are able to interact or cooperate with HSF1 and modulate the transcriptional regulation of Hsps in response to nonstressful stimuli. More recently, as will be explained later, it has also been reported that HSF2, like HSF1 can also play a role as a stress-inducible factor in promoting the induction of Hsps under certain conditions.
2. Transcriptional Regulation of Hsps by the HSF Family {#sec2}
=======================================================
2.1. HSF1 {#sec2.1}
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As mentioned previously, HSF1 has been identified as the HSF that mediates stress-induced Hsp gene expression in response to environmental stressors. Such stresses cause HSF1 oligomerization and nuclear translocalization, followed by enhanced DNA binding on the Hsp gene promoters. Recent studies have shown that HSF1 is negatively regulated by Hsp70 and Hsp90, therefore suggesting a negative-feedback loop for the regulation of Hsp70 and Hsp90 genes following a heat-shock response \[[@B8]--[@B10]\]. HSF1 is known to undergo posttranslational modification by various processes including phosphorylation, acetylation, and sumoylation \[[@B8]\]. Both phosphorylation and sumoylation are involved in regulating the transactivation capacity of HSF1 \[[@B8]\]. More recent, whereas p300 has been shown to acetylate HSF1, deacetylation by the NAD+-dependent sirtuin (SIRT1) is involved in the attenuation phase of the heat-shock response by preventing HSF1 acetylation and DNA binding \[[@B8]\].
The kinases responsible for phosphorylating HSF1 on several serine sites include glycogen synthase kinase 3*β* *(*GSK*β*) and c-jun N-terminal kinase (JNK) \[[@B12], [@B13]\]. The cytokine interleukin 6 (IL-6) has been shown to derepress HSF1 by reducing the activity of GSK*β* \[[@B14]\]. However, a positive role of HSF1 phosphorylation in the stress-induced activation of Hsp gene expression is also known to occur. The exact mechanism of this effect has not been fully elucidated, although the protein kinase CK2 seems to be involved in enhancing transcriptional activity and the DNA binding of HSF1 by phosphorylating the threonine 142 residue \[[@B15]\]. It is suggested that activation may also involve dephosphorylation of HSF1 \[[@B12]\].
The key role of HSF1 has been supported by the findings that cells lacking this crucial factor exhibited defects in Hsp induction following exposure to heat shock \[[@B16]\]. Moreover, cells lacking HSF1 were susceptible to apoptotic cell death following exposure to heat stress \[[@B16]\]. In addition, mice lacking HSF1 also had elevated levels of tumour necrosis factor *α* (TNF-*α*), which resulted in increased mortality after endotoxin and inflammatory challenge \[[@B16]\]. Interestingly, HSF1 has been shown to also modulate other genes such as interleukin-1*β* and c-fos \[[@B17], [@B18]\], suggesting a role for HSF1 in regulating stress responsive genes other than those encoding Hsps.
More recently, it has been reported that HSF1 also functions in the circadian clock as a circadian transcription factor. The circadian clock enables an organism to adapt to conditions by presetting the area in the brain that controls behavioural changes. Circadian transcription factors are known to be regulated in a timely and rhythmic fashion Thus, using a novel technique of differential display of DNA-binding proteins (DDDPs), HSF1 was shown to be highly rhythmic in its transcriptional activity. Moreover, HSF1 enhanced the expression of Hsps at the onset of the dark phase, when the animals start to be behaviourally active. Furthermore, Hsf1-deficient mice have a longer free-running period and therefore more active than wild-type littermates, suggesting a combined role for HSF1 in the mammalian timekeeping and cytoprotection systems \[[@B19]\].
2.2. HSF2 {#sec2.2}
---------
As mentioned earlier, Hsp gene expression is crucial not only for the survival of cells exposed to extracellular stress stimuli, but also during normal cellular processes such as embryonic development and cellular differentiation. HSF2 has now been described as the factor involved in regulating Hsps under nonstressful conditions. For example, it was previously reported that Hsp70 expression is activated when K562 cells are induced by hemin and this process requires activation of HSF2 \[[@B20]\]. HSF2 exists as two isoforms, HSF2*α* and HSF2*β*, due to alternative splicing, where the HSF2*α* isoform is predominantly expressed in adult tissue, while the HSF2*β* isoform is predominantly expressed in embryonic tissue \[[@B21]\]. HSF2 DNA binding activity is high during early embryogenesis in tissues such as the heart, central nervous system, and testis \[[@B21]\]. The importance of HSF2 in development was recently reported and Hsf2-null mice display gametogenesis defects and brain abnormalities characterized by enlarged ventricles \[[@B22]\].
During mitosis, the genome is well known to be compacted in order for chromosomes to be segregated during cytokinesis. However, some gene promoters such as the inducible Hsp70i (heat stress-induced upregulation) remain uncompacted. The factors that control and prevent this process of compaction or bookmarking have been recently characterized. For example, Hsp70i bookmarking is now known to be mediated by HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site \[[@B23]\]. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G1 phase, which demonstrates the biological importance of gene bookmarking. HSF2 has also been shown to be bound to the HSE promoter elements of other heat-shock genes, including Hsp90 and Hsp27, as well as the proto-oncogene c-fos \[[@B24]\]. These data suggest that HSF2 is important for constitutive as well as stress-inducible expression of HSE-containing genes.
It is also known that HSF2 can form heterotrimers with HSF1. Following certain stress, HSF1 is activated and HSF1-HSF2 heterotrimers are formed. Heat-shock stress diminishes the levels of HSF2 and restricts heterotrimerization by limiting the availability of HSF2. It has been suggested that HSF1-HSF2 heterotrimerization provides a switch that integrates the transcriptional activation in response to specific stimuli during developmental processes; for review see \[[@B8]\].
2.3. HSF3 {#sec2.3}
---------
HSF3 was originally identified in avian cells and no reports have yet described HSF3 in other organisms. Like HSF1, HSF3 is also heat-stress responsive \[[@B25]\]. However, the threshold temperature required to activate HSF3 and HSF1 are different in that HSF1 is activated by less severe heat shock than HSF3 \[[@B25]\]. Previously, H
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Introduction {#Sec1}
============
Distal radius fractures are the most common fractures occurring in childhood and a substantial proportion of these patients will develop malunions initially. Fortunately, malunions in children often show a tremendous remodeling potential and initial treatment can usually be restricted to the reassurance of the parents of the involved child. However, although this is a well-known practice for most doctors treating children with fractures, surprisingly few studies (*n* = 7) are available with quantitative data on the dynamics of remodeling. The time needed for the remodeling process is unknown, which impedes the prediction of outcome and, thus, proper patient information. Reported remodeling times (RT) to full correction vary between a mean of 4 months \[[@CR1]--[@CR3]\] and 5 years \[[@CR4]\] in the literature. In addition, the speed of remodeling has been shown to vary between 0.9° to 2.5°/month \[[@CR5]--[@CR7]\]. Greater angulated fractures tend to remodel at a faster rate \[[@CR5], [@CR7]\]. Hence, the use of a general remodeling speed to predict RT to full correction is not feasible. Friberg, therefore, developed a (exponential) model using the primary malunion angulation (*A*~0~) to describe the residual angulation (*A*~T~) in distal radius malunions \[[@CR5]\]. The model, however, lacks accuracy and is, therefore, only rarely used in orthopedic practice.
The aim of the present study is to develop a model which accurately predicts the dynamics of the remodeling process. We use the remodeling data of two previously published studies to modify Friberg's model in order to enhance its accuracy. In addition, we develop a model to calculate the time needed for complete remodeling. These models should allow to provide a more evidence-based patient education and select those malunions that will not sufficiently remodel and require intervention.
Patients and methods {#Sec2}
====================
We used data from two published cohorts of children with distal radius fractures with dorsovolar angulation. Cohort A is from a study on the remodeling of malunions of forearm fractures which presents a table with patient data on 36 children \[[@CR4]\]. From this table, were selected the malunions in the distal third of the forearm in dorsovolar dislocation (*n* = 31). Cohort B was derived from a study on the remodeling speed of distal radius fractures with dorsovolar angulation more than 15° (*n* = 32) \[[@CR7]\]. Angle measurements in both cohorts were identical: the central longitudinal intramedullary axis was determined in both the proximal and (angulated) distal fragment. The angle between these two axes was used as the angulation angle. This method was described by Hansen et al. \[[@CR8]\].
From all the included patients, we assessed age at time of fracture, gender, malunion angulation (*A*~0~) in the dorsovolar direction, angulation at follow-up, and time of follow-up (= RT). Because both studies were retrospective, the follow-up times (= RT) differ. The difference between initial malunion angulation (*A*~0~) and angulation at follow-up (*A*~T~) was defined as remodeling, measured in degrees.
Using the data from the combined cohort, two models were evaluated: Firstly, a prediction model was formulated based on the findings by Friberg \[[@CR5]\]: $\documentclass[12pt]{minimal}
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\begin{document}$$A_{T} = A_{0} \times e^{ - C \times RT}$$\end{document}$ and, secondly, we modified this model with a second coefficient to study the influence of *A*~0~: $\documentclass[12pt]{minimal}
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\begin{document}$$A_{T} = B \times A_{0} \times e^{ - C \times RT}$$\end{document}$ (the coefficients were calculated using the nonlinear regression function of SPSS, see below).
Statistical analysis {#Sec3}
--------------------
All data were analyzed using SPSS (version 15.0, SPSS Inc., Chicago, IL, USA). The results are presented as means (standard deviation, SD). Nonlinear regression was used to estimate the coefficients of the models. For the Friberg-based model, we started with the coefficient found in that study. For the modified model, the starting value for the second coefficient was the value found in the study of Jeroense et al. \[[@CR7]\]. The significance of the difference of the parameters and differences between subgroups was tested using the *t* test. To test the precision of the prediction of the models, we compared predicted and observed RT using parametric techniques (*t* test). The best of the two models was subsequently used to estimate time needed to complete remodeling. All tests are two-tailed and considered significant if *p* \< 0.05.
Results {#Sec4}
=======
Data are based on the analysis of 63 dorsovolar malunions of the distal radius: 31 from the study by Gandhi (A) (cases 1--31 in the patient data table) and 31 patients (32 malunions) from the study by Jeroense (B) (see [Appendix](#Sec11){ref-type="sec"}). There were 38 boys, with a mean age of 8.5 years (range 2--14.5 years). The mean malunion angulation was 25° (SD 7.8), mean remodeling time 22 (SD 18) months, and mean angulation at follow-up 6.7° (SD 5.8). The cohorts showed differences in follow-up time (35 vs. 9 months) and final angulation (see Table [1](#Tab1){ref-type="table"}).Table 1Summary of the data from 62 patientsGandhi cohort (A), *N* = 31Jeroense cohort (B), *N* = 31DifferenceSignificanceAge (years)7.79.11.3 years0.043Remodeling time (months)35925 months0.000Malunion angulation (*A* ~0~)26°24°2.5°0.1Angulation at FU5°8°3.5°0.02Comparison of the two subgroups
Prediction of remodeling {#Sec5}
------------------------
### Friberg's exponential model {#Sec6}
Using Friberg's model for the combined cohort, the prediction coefficient was 0.13 \[confidence interval (CI): 0.1--0.16), with a low precision (*R*^2^ = 0.11). Using the model for subgroup analysis (cohorts A and B), we found significant differences in the coefficient of remodeling with B coefficients of 0.06 (95 % CI: 0.068--0.045) and 0.17 (95 % CI: 0.21--0.13), respectively (*p* \< 0.05).
### Modified exponential model {#Sec7}
We developed a modified model by adding a second coefficient to modify A~0~. The best fit is the model $\documentclass[12pt]{minimal}
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\begin{document}$$A_{T} = 0.51 \times A_{0} \times e^{ - 0.034 \times RT}$$\end{document}$ (51 % of the starting angulation and a coefficient of 0.034 for RT). This improves prediction for the combined cohort: *R*^2^ = 0.47. With this model, the subgroups did not differ (Table [2](#Tab2){ref-type="table"}). Adding age or gender did not improve the model. Analysis excluding the four patients older than 12 years of age only marginally influenced the results of this nonlinear regression.Table 2Models of observed remodeling (*A* ~T~) and initial malunion angle (*A* ~0~) and RT (*n* = 63 malunions)ModelDependent variableIndependent variablesModel95 % CI of coefficient of RT*R* ^2^Model of FribergA~T~RT, *A* ~0~$\documentclass[12pt]{minimal}
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\begin{document}$$A_{T} = A_{0} \times e^{ - 0.13 \times RT}$$\end{document}$0.1--0.160.1Modified modelA~T~RT, *A* ~0~$\documentclass[12pt]{minimal}
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Cryptic polyadenylation within coding sequences (CDS) or incompletely removed introns produce aberrant transcripts that lack in-frame stop codons[@b1]. Translation of such mRNAs may result in proteins prone to malfunction and deleterious effects on cells[@b2][@b3][@b4]. To mitigate these errors, cells have developed quality-control processes to monitor translating mRNAs and detect aberrant mRNAs, such as those with premature polyA tails within their CDS. Defects in components of the surveillance machineries have been implicated in several types of diseases including neurodegeneration and cancer[@b5][@b6].
The ribosome-associated quality control (RQC) is a mechanism that senses the state of mRNA translation and detects ribosome stalling at the site of defective mRNAs, which results in targeting of both the translating mRNA and nascent peptide for degradation[@b7]. RQC can be divided into several steps, surveillance of the translating mRNA and detection of stalled ribosome, ribosomal subunit dissociation, and degradation of the defective mRNA and nascent peptide. Although the processes of ribosomal subunit dissociation and nascent peptide degradation are well studied[@b8][@b9][@b10][@b11][@b12], the mechanism of surveillance of the translating mRNA and detection of stalled ribosome, in particular the molecular sensors of aberrant mRNAs and their mechanism of action, remain largely unknown. Earlier studies suggested that presence of the polyA sequences within the CDS causes ribosome stalling through interactions between the positively charged peptide (poly-lysine) and the negatively charged exit channel of the ribosome[@b8][@b13][@b14]. However, others showed that at least in mammalian cells RQC at poly-lysine sites is codon-sequence dependent as runs of poly-lysine residues coded by AAA codons induced ribosome stalling much more efficiently than equivalent runs of poly-lysine encoded by AAG codons[@b15]. These results indicate that sensing A-rich mRNA sequence in mammalian cells dominates over general polybasic amino-acid-triggered translational regulation. Nevertheless, the mechanisms by which premature polyA sequences are detected in aberrant mRNAs and the following molecular events leading to ribosome stalling are not known.
In yeast, the E3 ubiquitin ligase Hel2 has been implicated in facilitating the earlier steps of RQC at polybasic sequences[@b8]. Notably, Hel2-dependent K63 polyubiquitination is necessary for the initial processes involved in stalled translation surveillance[@b16]. However, the precise functions of Hel2 in detection of stalled ribosomes or its ubiquitination substrates have not been identified. The Zinc Finger Protein 598 (ZNF598) is the human ortholog of Hel2 and contains a RING domain characteristic of E3 ubiquitin ligases and several C2H2-type zinc finger motifs, commonly found in nucleic acid-binding proteins[@b17][@b18]. We previously described ZNF598 protein in a complex with the translation repressor proteins EIF4E2/4EHP and GIGYF2 (ref. [@b19]). Two recent reports showed that ZNF598 is also required for stalling at polyA sequences and linked its E3 ubiquitin ligase activity to translation arrest through ubiquitinating the 40S subunit ribosomal proteins RPS10 and RPS20 (refs [@b20], [@b21]).
Here, we reveal that ZNF598 directly binds to the translating mRNA and tRNAs on ribosomes and triggers ribosome stalling and RQC at premature polyA sequences. We further identified RPS3A as an additional substrate of ZNF598 E3 ubiquitin ligase activity, and UBE2D3 as the ZNF598-interacting E2 ubiquitin ligase. Our findings establish a link between the RNA-binding properties and ubiquitin ligase activity of a uniquely conserved protein in monitoring mRNA translation.
Results
=======
ZNF598 associates with translating ribosomes
--------------------------------------------
Human ZNF598 encodes a ubiquitously expressed 904 amino acid (aa) protein containing one *N*-terminal RING domain characteristic of E3 ubiquitin ligases and four *N*-terminal and one *C*-terminal C2H2-type zinc finger motifs ([Fig. 1a](#f1){ref-type="fig"} & [Supplementary Fig. 1](#S1){ref-type="supplementary-material"}). To evaluate its potential role in translational control, we performed polysome profiling using ZNF598 overexpression (ZNF598-OE) or ZNF598 knockout HEK293 cells (ZNF598-KO; [Supplementary Fig. 2](#S1){ref-type="supplementary-material"}). ZNF598-OE induced a shift from heavy polysomes to monosomes and ZNF598-KO induced a shift to heavier polysomes, indicating translational repression ([Fig. 1b,c](#f1){ref-type="fig"}). This effect was independent of EIF4E2 ([Supplementary Fig. 3](#S1){ref-type="supplementary-material"}) and was neither due to general translational repression mediated by phosphorylation of eukaryotic initiation factor 2α EIF2S1/eIF2α ([Fig. 1d](#f1){ref-type="fig"}) under stress condition. Western blot analysis of the polysome fractions showed that a proportion of ZNF598 protein was associated with heavy polysomes in ZNF598-OE cells ([Fig. 1e](#f1){ref-type="fig"}). Size exclusion chromatography also revealed that ZNF598 protein co-fractionated with ribosomes in a ≥2 MDa complex ([Supplementary Fig. 4](#S1){ref-type="supplementary-material"}), suggesting that ZNF598 either interacted transiently with assembled ribosomes or associated with a subset of actively translating ribosomes. Immunofluorescence analysis of ZNF598 upon exposure to arsenite-induced stress did not reveal any change in its cytosolic distribution, unlike many known cytosolic RNA-binding proteins or 18S ribosomal RNA which accumulate in stress granules[@b22][@b23] ([Supplementary Fig. 5](#S1){ref-type="supplementary-material"}). Together, these observations support a role of ZNF598 in translation.
ZNF598 binds to RNAs associated with translating ribosomes
----------------------------------------------------------
To investigate ZNF598 function and identify its target RNAs, we performed 4-thiouridine (4SU) photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP)[@b24] in ZNF598-OE HEK293 cells ([Supplementary Figs. 2a and 6a](#S1){ref-type="supplementary-material"}). We observed that ZNF598 cross-linked to tRNAs, mRNAs and rRNAs ([Fig. 2a](#f2){ref-type="fig"}, [Supplementary Fig. 6b--g](#S1){ref-type="supplementary-material"} & [Supplementary Data 1](#S1){ref-type="supplementary-material"}). The average ratio of cross-linked reads annotated as tRNAs, mRNAs and rRNAs was ∼4:2:1. Cross-linked reads derived from mRNAs showed evenly distributed enrichment for CDS over untranslated regions (UTRs) ([Fig. 2a,b](#f2){ref-type="fig"}). The cross-linked mRNA read abundance resembled the overall mRNA abundance in HEK293 cells as determined by polyA mRNA-Seq ([Supplementary Fig. 7](#S1){ref-type="supplementary-material"}). The reads mapped to nuclear encoded cytoplasmic tRNAs originated predominantly from their 5′ halves, which were cross-linked to ZNF598 via their D-loops ([Fig. 2c,d](#f2){ref-type="fig"}). Although ZNF598 protein cross-linked to every cytoplasmic tRNA, cross-linked reads unique to tRNA^Lys^(UUU) were ∼10-fold enriched relative to their total cellular abundance, whereas cross-linked reads to tRNA^Lys^(CUU) only displayed an average twofold enrichment ([Fig. 2e](#f2){ref-type="fig"} & [Supplementary Data 2](#S1){ref-type="supplementary-material"}). Cross-linked reads to rRNA ([Fig. 2f](#f2){ref-type="fig"}) originated predominantly from 5S (pos. 96--121) and 18S rRNAs (pos. 686--707 and 745--778) and to a lesser extent from 5.8S and 28S ([Supplementary Figs. 8 and 9](#S1){ref-type="supplementary-material"}). Taken together, the cross-linked RNA targets and positional cross-linking spectra indicate that a fraction of ZNF598 protein was bound to translating ribosomes. In light of the enrichment of cross-linking to (AAA)-decoding tRNA^Lys^(UUU), we hypothesized that ZNF598 may have a role in detecting premature polyA tails or protein-folding problems of poly-lysine rich proteins that would induce ribosome stalling and RQC pathway[@b7][@b13]. The transcript abundance of ZNF598 is ∼10-fold below the average of transcripts encoding ribosomal proteins but similar in abundance to other proteins implicated in RQC ([Supplementary Data 3](#S1){ref-type="supplementary-material"}). Considering that direct molecular contacts are required for photo-cross-linking of ZNF598 to the CDS of mRNAs, tRNAs and rRNAs, we conclude that ZNF598 protein is associated with translating ribosomes and intimately monitors the CDS of translated mRNAs and/or identity of tRNAs occupying the ribosome and triggering RQC upon encounter with premature polyA tails.
ZNF598 initiates RQC at premature polyA sequences
-------------------------------------------------
In yeast, Hel2 facilitates ribosome stalling at both poly-lysine and poly-arginine polybasic amino-acid coding sites[@b8][@b16][@b25][@b26]. Polybasic peptides
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INTRODUCTION {#sec1-1}
============
Surgical site infections (SSIs) are either superficial or deep and may involve the organs, or spaces accessed during an operation. The reported incidence of SSIs in coronary artery bypass grafting (CABG) surgery ranges between 0.3% and 8%.\[[@ref1]\]
There is a strong suggestion that an impairment of vascular supply of the sternum may be one of the most important factors influencing the incidence of deep sternal wound infection (DSWI). Several studies have studied the risk factors for SSIs including DSWI in cardiac surgery. These risk factors included obesity, diabetes mellitus, chronic obstructive pulmonary disease (COPD), connective tissue disease, steroid use, smoking, peripheral vascular disease and renal insufficiency. In addition, intraoperative factors (e.g., use of bilateral internal mammary arteries \[BIMA\] grafting, prolonged cardiopulmonary bypass \[CPB\] duration) and postoperative variables (e.g., prolonged mechanical ventilation, reoperation for bleeding, postoperative transfusions and gastrointestinal, nephrological and respiratory complications) have been shown to be associated with DSWI.\[[@ref2][@ref3][@ref4]\]
The risk for sternal wound infection (SWI) is increased if cardiac surgery involves internal thoracic arteries grafting and a valve procedure, or use of a ventricular assist device.\[[@ref5][@ref6]\] Leg wound infections at donor sites account for \>70% of cases with severe infection following cardiac surgery.\[[@ref7]\]
Cardiac SSIs increase the length of hospital stay (LOS) and increase treatment costs in proportion to the severity of the infection. These costs increase by 3.8%, 14.7% and 29.4% in mild, moderate and severe infections respectively.\[[@ref7]\]
Treatment is often confounded by the emergence of antibiotic-resistant pathogens and in addition, substantial proportions of these infected patients are elderly and have co-existing medical problems. In the past, such elderly patients with significant comorbidities would not have been considered for surgery.\[[@ref8]\] As the population ages, it is reasonable to assume that older and sicker patients will be admitted for surgery, and this will inevitably increase the risk and incidence of SSIs.\[[@ref9]\]
Within our institution, the infection rate of postoperative wounds has been under surveillance by the infection control team since 2005. According to our preliminary data of rates of SSIs among isolated CABG patients, this was found to be significantly disproportionate to other regional institutions. As a result, we decided to audit, study and identify likely perioperative risk factors among our patients who have undergone isolated CABG between 2012 and 2013.\[[@ref10]\]
The most important step in the management of wound infection is prevention, and this is best done by identifying risk factors. The present study was carried out in our centre to identify the incidence of wound infections following isolated CABG and identify the risk factors that may be associated with SSIs in our center.
None of these perioperative risk factors have been studied previously in our population undergoing isolated CABG. Our pool of patients originates from a general Middle Eastern population, known to have a high prevalence of diabetes and obesity, a sedentary lifestyle with a lack of exercise, which represents a change of lifestyle following the discovery of oil in the region.\[[@ref10][@ref11][@ref12][@ref13][@ref14]\]
MATERIALS AND METHODS {#sec1-2}
=====================
Definitions of Infection {#sec2-1}
------------------------
Sternal SSI was defined according to the SSI criteria of the US Centers for Disease Control and Prevention. Sternal infections occurring within 30 days after surgery can include the following types: (1) Superficial incisional (infection above the sternum with no bony involvement); (2) deep incisional (infection involving the sternum); and (3) organ/space (site-specific infection such as mediastinitis).\[[@ref8][@ref15]\]
Leg SSI was defined as redness, swelling, increased pain, excessive bleeding or discharge at the incision site among the patients who had undergone CABG. All SSI cases were diagnosed by attending physicians and confirmed by the nosocomial infection control committee. Patients who did not have any SSI formed the control group.
Study Design {#sec2-2}
------------
From January 2012 to December 2013, 357 isolated CABG procedures were performed at our cardiac center. Totally, 40 postoperative patients diagnosed with an SSI (including sternal SSI, leg SSI and double SSI \[both sternum and leg\]), in accordance with the CDC criteria for SSI surveillance, were selected randomly. These 40 patients formed our study group (SSI) (*n* = 40, group I). This group was matched according to age, sex, nature of procedure and timing within the above study period with a control group (non-SSI) of 40 postoperative patients who did not suffer from an SSI (non-SSI group: *n* = 40, group II).
The eighty selected CABG patients\' data were collected and analyzed retrospectively. Eight potential risk variables were compared between groups I and II.
Potential Risk Factors {#sec2-3}
----------------------
Eight possible perioperative risk factors were analyzed and included the following: A prolonged LOS (LOS by days) which was arbitrarily taken as beyond a 30 days stay, a previous diagnosis of diabetes mellitus, impaired estimated glomerular filtration rate (eGFR \< 60 ml/min) taken as a sign of renal insufficiency, urgency of surgery (i.e. surgery done within 24 h of diagnosis of surgical coronary artery disease), the use of BIMA for grafting, impaired ejection fraction (EF) including moderate and severe (EF% \<45%), prolonged CPB duration taken as \>2 h and an elevated body mass index (BMI) that is, \>25.
Data Analysis {#sec2-4}
-------------
The risk factors for infection were assessed by univariate analysis. Discrete variables were assessed using Chi-squared analysis or Fisher\'s exact test. Variables were assessed using two tailed Student\'s *t*-test. All variables suggested by the univariate analysis were entered into a stepwise binary logistic regression analysis model. The chosen level of significance was 5%. All analysis was performed using the Statistical Package for the Social Sciences (SPSS) 19.0. IBM Corporation.
RESULTS {#sec1-3}
=======
All of the 80 patients who enrolled during the study period underwent isolated CABG only.
Among the eight potential risk factors studied, the factors that had significant differences between the SSI study group and non-SSI control group were an impaired eGFR (*P* = 0.011, odds ratio \[OR\]: 3.8) and an impaired EF% (*P* = 0.015, OR: 5.1) \[Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}\].
######
Association of exposure with SSI
######
Potential preoperative risk factor for cardiac surgical infection for underwent CABG, 2012-2013\*
Patient\'s LOS (days), urgency of surgery, BIMA grafting, prolonged CPB duration and an increased BMI had no significant influence on the incidence of wound infection \[[Table 1](#T1){ref-type="table"}\].
Of the 40 patients in the SSI group, 22 were male, and 18 were female that was equivalent to the non-SSI group. The mean age for the SSI group was 59 versus 61 for the non-SSI group. There was no significant statistical difference in the age and sex of both groups.
Only 2 patients in the SSI group stayed in the hospital beyond 30 days. None of the non-SSI group had a prolonged LOS. The difference was statistically insignificant.
Thirty-five patients (87%) in the SSI group were previously diagnosed with diabetes mellitus (type 1 or type 2) while 30 patients (75%) in the non-SSI group were diabetic. Hence, diabetes was not found to be a preoperative predictor of SSIs (*P* - 0.156).
Urgent isolated CABG was performed on 6 patients (15%) in the SSI group compared to only 4 patients in the non-SSI group. There was no statistically significant difference with a *P* = 0.499 and an OR: 1.5.
Seventeen patients (42%) in the SSI group had a prolonged CPB time of more than 2 h duration while 13 (32%) of patients in the non-SSI group had a prolonged CPB time. Again, prolonged CPB duration was not found to be an intraoperative predictor of SSI (*P* - 0.356).
Bilateral internal mammary harvesting was performed on 6 patients in the SSI group while 11 patients in the non-SSI group had bilateral mammary harvesting performed. Bilateral mammary harvesting was not found to be a risk factor for SSI with a *P* - 0.1/OR: 2.1.
DISCUSSION {#sec1-4}
==========
Surgical site infections are a manifestation of an imbalance between microbial growth and host\'s defenses. The Surgical stress response imposes an impairment of these defenses.\[[@ref16]\]
Loop *et al*., described several risk factors for sternal wound complications in cardiac surgery. Bilateral internal mammary harvesting, diabetes, obesity, blood transfusion and operative time were considered significant risk factors for sternal wound complications.\[[@ref17]\] Other authors had described other risk factors for SSIs in cardiac surgery, with conflicting findings.
Preoperative hospital admission duration, antibiotic prophylaxis use, surgical urgency, reoperation, surgical
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Introduction {#Sec1}
============
In contrast to mammals, lampreys show spontaneous and successful functional recovery after a complete spinal cord injury (SCI) and this is in part due to their impressive ability for axonal regeneration^[@CR1]--[@CR8]^. But, even in lampreys, not all descending neurons of the brain are able to regenerate their axons through the site of injury after a complete spinal cord transection^[@CR4],[@CR9]--[@CR12]^. The lamprey brainstem contains approximately 30 large individually identifiable descending reticulospinal neurons that vary greatly in their ability for axonal regeneration after SCI, even when their axons run in similar paths in a spinal cord that is permissive for axonal regrowth^[@CR4],[@CR12],[@CR13]^. Some identifiable descending neurons of lampreys are considered "good regenerators" (i.e. they regenerate their axon more than 55% of the times; the I3, I4, I5, B2, B5 and B6 neurons) and others are considered "bad regenerators" (i.e. they regenerate their axon less than 50% of the times; the M1, M2, M3, I1, I2, B1, B3, B4 and Mth neurons)^[@CR4],[@CR6],[@CR12]^. This indicates that interactions with the extrinsic spinal cord environment and intrinsic differences between descending neurons affect their regenerative abilities after SCI. Recent work has also shown that identifiable descending neurons of lampreys that are known to be "bad regenerators" slowly die after a complete SCI and are also "poor survivors"^[@CR12],[@CR14],[@CR15]^. The death of these neurons after SCI appears to be apoptotic as indicated by the appearance of TUNEL labelling and activated caspases in their soma^[@CR14]--[@CR18]^. This offers a convenient vertebrate model to study the inhibition or promotion of neuronal survival and axonal regeneration in the same in vivo preparation and at the level of single neurons.
In mammals, SCI leads to a massive release of aminoacidergic neurotransmitters (glycine and GABA:^[@CR19],[@CR20]^; glutamate:^[@CR21]--[@CR23]^). Excessive glutamate release after SCI is responsible for excitotoxicity and neuronal death^[@CR21],[@CR22]^. High extracellular glutamate levels result in excessive activation of glutamate receptors, triggering massive Ca^2+^ influx into cells, which leads to neuronal death^[@CR24]^. Extracellular glycine could also contribute to glutamate excitotoxicity^[@CR20]^, since it is a co-agonist of the *N*-methyl-D-aspartate glutamate receptor^[@CR25]^. The phenomenon of excitotoxicity has been mainly studied in intrinsic spinal cord cells; however, retrograde damage to neurons is also likely due to the fact that Ca^2+^ ions gain access to the axoplasm of damaged axons^[@CR26]^. In contrast to glutamate, it has been reported that GABA could have neuroprotective effects after different types of central nervous system (CNS) damage^[@CR27]--[@CR31]^. The activation of pre-synaptic GABAB receptors causes inactivation of voltage-dependent Ca^2+^ channels (see^[@CR32]^), which could prevent the influx of Ca^2+^ ions due to glutamate release. In addition, it has been shown that GABA can modulate and promote neurite outgrowth in vitro or during development (for reviews see^[@CR33],[@CR34]^). However, a role for GABA and GABAB receptors in neuroprotection and especially in axonal regeneration after SCI has not been reported yet.
In lampreys, glutamate induces an inhibition of neurite outgrowth in reticulospinal neurons in vitro due to Ca^2+^ influx^[@CR35]^. Electrophysiological studies have also suggested that low intracellular Ca^2+^ levels due to downregulation of Ca^2+^ channels could facilitate axonal regeneration in axotomized descending neurons of lampreys^[@CR36]^. More recently, we have reported that, as in mammals, there is a massive release of glutamate, GABA and glycine from most spinal cord neurons close to the lesion site following a complete SCI^[@CR37]--[@CR39]^. Between 1 and 3 days after the injury, we observed the extracellular accumulation of GABA in the form of "*halos*" around some axotomized axons of descending neurons close to the site of injury. Statistical analyses revealed a significant correlation between GABA accumulation and a higher survival ability of the corresponding identifiable descending neurons^[@CR37]^. An electrophysiological study in the spinal cord of lampreys has also found a correlation between higher GABAergic inhibition and a better recovery of function in spinal lesioned animals^[@CR40]^. These data prompted us to hypothesize that, in lampreys, increased GABA signalling after SCI could be favouring the recovery process by promoting survival and axonal regeneration of descending neurons. Here, we address this question for the first time in vivo in any vertebrate and provide gain and loss of function evidence showing that endogenous GABA, acting through GABAB receptors, promotes survival and axonal regeneration of identifiable descending neurons after SCI in lampreys.
Materials and methods {#Sec2}
=====================
Animals {#Sec3}
-------
All experiments involving animals were approved by the Bioethics Committee at the University of Santiago de Compostela and the *Consellería do Medio Rural e do Mar* of the *Xunta de Galicia* (License reference JLPV/IId; Galicia, Spain) or the Institutional Animal Care and Use Committee at the Marine Biological Laboratory (Woods Hole, MA) and were performed in accordance to European Union and Spanish guidelines on animal care and experimentation or the National Institutes of Health, respectively. During experimental procedures, special effort was taken to minimize animal suffering and to reduce the use of animals. Animals were deeply anaesthetized with 0.1% MS-222 (Sigma, St. Louis, MO) in lamprey Ringer solution before all experimental procedures and euthanized by decapitation at the end of the experiments.
Mature and developmentally stable larval sea lampreys, *Petromyzon marinus* L. (*n* = 115; between 95 and 120 mm in body length, 5 to 7 years of age), were used in the study. Larval lampreys were collected from the river Ulla (Galicia, Spain), with permission from the *Xunta de Galicia*, or provided by Lamprey Services, Inc. (Ludington, MI, USA) and maintained in aerated fresh water aquaria at 15--23 °C with a bed of river sediment until their use in experimental procedures. Lampreys were randomly distributed between the different experimental groups.
SCI surgical procedures {#Sec4}
-----------------------
Animals were assigned to the following experimental groups: control unlesioned animals or animals with a complete spinal cord transection that were analyzed 1 week post-lesion (wpl), 2 wpl, 4 wpl, 10 wpl or 12 wpl. Within the 2, 10 and 12 wpl groups, the injured animals were assigned to either control or treatment groups. Table [1](#Tab1){ref-type="table"} summarizes the number of animals assigned to each experimental group and condition. Each experiment was carried out in at least two different batches of animals. Complete spinal cord transections were performed as previously described^[@CR41]^. Briefly, the rostral spinal cord was exposed from the dorsal midline at the level of the 5th gill by making a longitudinal incision with a scalpel (\#11). A complete spinal cord transection was performed with Castroviejo scissors and the spinal cord cut ends were visualized under the stereomicroscope. After spinal transections, the animals were returned to fresh water tanks and each transected animal was examined 24 h after surgery to confirm that there was no movement caudal to the lesion site. Then, the animals were allowed to recover in individual fresh water tanks at 19.5 °C and in the dark.Table 1Table showing the number of animals included in each experimental group and also the total number of identifiable descending neurons that were included in the analysesAnimalsTotal number of neurons included in the analysesM1M2M3I1I2I3I4I5B1B2B3B4B5B6MthChanges in gabab1 expressionControl71111128--5859--109--791 wpl710101412--12969--139--11144 wpl61012129--6665--65--57GABA treatment (2 wpl)Control6\*1111101291112712111212111212Treated568896995101010107910Baclofen treatment (caspase activation, 2 wpl)Control7\*1312111491314814131414121414Treated71213131471413614131414131313GABOB treatment (12 wpl)Control15303029292430302930303030303030Treated14262524282428272825242628232824Baclofen treatment (axonal regeneration, 12 wpl)Control7121213141214141114121414141213Treated11212121221922222122222222222222Gabab1 morpholino (ISH, 2 wpl)Control36----6----------------------Treated47----8----------------------Gabab1 morpholino (axonal regeneration, 10 wpl)Control9181818181818181818181818181818Treated13262626252626262626262626262626Total115Please note that in the in situ hybridization experiments, only the neurons that were unequivocally identified in at least two brain sections were included in the quantifications. In the FLICA experiments,
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Introduction {#Sec1}
============
Almost one century ago D'Arcy Thomson proposed that the spatiotemporal alterations in tissue mechanics inevitably alter its mechanoenvironemtal properties. These local biphasic mechanical properties, such as stiffness and fluidity, determine the system response to generated forces. This theory has been accepted widely^[@CR1]--[@CR7]^.
Rheological techniques are rarely used for medical diagnosis of living tissue due to the invasive nature of rheological tests such as indentation experiments, torsional resonators and oscillatory shear testing devices^[@CR8]--[@CR10]^. These methods do allow for exploration of the viscoelastic properties of the medium in a wide range of frequencies, including the ultra-low frequency range (less than 1 Hz). Although a rheometer can assess the viscoelastic behavior of a medium in ultra-low frequency ranges, it is a time consuming and cumbersome process to use it in such a low frequency range^[@CR11]--[@CR13]^.
Over a decade ago, non-invasive elastography methods were developed with the capability of remotely inducing shear waves in tissue and measuring its elasticity by recording the deflection response by MRI^[@CR14]^ and ultrasound^[@CR15]^. Later the *in vivo* application of elastography in measuring the stiffness of different tissues and organs like liver^[@CR16]--[@CR18]^, breast^[@CR19],[@CR20]^, brain^[@CR21],[@CR22]^, heart^[@CR23],[@CR24]^ and muscle^[@CR25],[@CR26]^ were reported. Although elasticity measurements were the focus of these efforts, a few studies considered the estimation of both elasticity and viscosity of *in vivo* tissues^[@CR20],[@CR22],[@CR27],[@CR28]^.
While it is possible to use shear wave elastography methods for *in vivo* cases, the frequency range to explore the viscoelasticity of tissue is much narrower than with rheology methods^[@CR20],[@CR22],[@CR27]^. With shear wave elastography methods, reaching the ultra-low frequency range is almost impossible because there is always a tradeoff between the resolution of the resulting map and the frequency of vibration and shear waves^[@CR29]^.
In this paper, we introduce the noninvasive, Loss Angle Mapping (LAM) method, which is based on measuring the local displacement and strain behaviors under constant stress as a function of frequency. Technical details were explained in our previous work^[@CR30]^. The LAM method can monitor the viscoelastic properties of the tissue with high resolution due to its high accuracy in displacement measurement at the micrometer level. High frame rate ultrasound strain imaging, which is the essential part of the LAM method, allows capturing the local viscoelastic parameters *in vivo* like breast tissue. The main components of the LAM test are a compression mechanism that is used to exert an approximately step-force on the tissue and a high-frame rate ultrasound system for monitoring the internal local strain responses. In other words, a step-force is used as a stimulus for a certain amount of time, and the transient strain response, which is governed by viscoelastic properties of the medium, is monitored by analyzing a sequence of radiofrequency (RF) data during the excitation^[@CR31],[@CR32]^. In the LAM method the local tissue behavior in the sub-Hertz frequency range is used because at this frequency range the local biphasic behavior of tissue is more evident compared to other frequency ranges^[@CR33]--[@CR37]^.
Results {#Sec2}
=======
Viscoelastic gel phantom {#Sec3}
------------------------
Gel phantom can be used as a simplified mechanical model for breast tissue. Gel consists of collagen type I matrix that is saturated in water^[@CR38]^, similar to breast tissue. The matrix peptide chain in this type of collagen is responsible for the dense electric charge associated with the hydrophilic properties of collagen fibers and culminates in the viscoelastic properties of the medium^[@CR39]^. The same mechanism can happen in breast tissue in which the glycoproteins are responsible for viscoelastic properties due to their hydrophilic nature^[@CR39]^. The viscosity of the fluid for these two media, however, is different. The solid matrix in both gel and tissue makes a porous structure which helps move the fluid when the medium is compressed or under load^[@CR40]^. Fluid viscosity is responsible for the viscoelastic response of the media or tissue. In addition the hydrogen crosslinks between the fibers in a solid matrix can trigger the viscoelastic response^[@CR40]^.
To verify the performance of the proposed model-free method, a viscoelastic inclusion phantom was made. The inclusion part of the phantom was made with 25.14 grams of gelatin (Sigma-Aldrich, St. Louis, MO), 60 ml propylene glycol (Sigma-Aldrich, St. Louis, MO), and 4 grams cellulose (ultrasound scattering; Sigma-Aldrich) in distilled water for a total volume of 300 ml. The background part of the phantom was made with 32.3 grams gelatin, 30 ml Vanicream Lite (Pharmaceutical Specialties, Inc., Rochester, MN), 6 grams cellulose (ultrasound scatterer; Sigma-Aldrich) and potassium sorbate (preservative; Sigma-Aldrich) in distilled water with a total volume of 600 ml^[@CR30]^. The inclusion phantom dimensions were 7.5 cm × 5.5 cm × 5.5 cm (L × W × H), with the cylindrical inclusion having a diameter of 1.5 cm.
An ultrasound B-mode image of the gel phantom can be seen in Fig. [1](#Fig1){ref-type="fig"}. For demonstration purposes, two points were selected: point 1 in the background and point 2 in the inclusion. The results of the temporal strain profile for these points can be seen in Fig. [2a](#Fig2){ref-type="fig"}. Loss angle profiles for these 2 points were obtained in the frequency range less than 10 Hz (Fig. [2b](#Fig2){ref-type="fig"}) and less than 0.35 Hz (c). Similar to the above mentioned 2 points the viscoelastic map created by processing all the spatial points at a frequency of 0.033 Hz using the LAM method as shown in Fig. [2(d)](#Fig2){ref-type="fig"}.Figure 1Ultrasound Verasonics B-mode image of the gel phantom with the inclusion part indicated with a dashed line. 1 = a point in the background; 2 = a point in the inclusion.Figure 2Profiles obtained on the inclusion gel phantom. (**a**) Temporal strain profile of point 1 (background, red) and point 2 (inclusion, blue) in the phantom imaged in Fig. [1.](#Fig1){ref-type="fig"} (**b**) Loss angle profile of point 1 (background, red) and point 2 (inclusion, blue) in a frequency range less than 10 Hz. (**c**) Loss angle profile of point 1 (background, red) and point 2 (inclusion, blue) in a frequency range less than 0.35 Hz. (**d**) Viscoelastic map produced at 0.033 Hz based on the LAM method.
***In vivo*** patient study {#Sec4}
---------------------------
### **Patient study** {#Sec5}
The phantom study results encouraged us to apply the LAM method on breast patients. The patient study was approved by the Institutional Review Board of the Mayo Clinic, Rochester MN, and informed consent was signed by each enrolled patient. This study was also compliant with the Health Insurance Portability and Accountability Act (HIPPA) in the Mayo Clinic.
A total of 156 female patients with visible breast lesions in US images were recruited at Mayo Clinic from November 2014 to September 2016. The data from the first five patients were used to test the device and algorithm and were eliminated from the final study. Thus, a total of 151 breast patients were included in the study. Amongst them after applying the motion compensated cross-correlation metric (MCCC), 45 patients were selected for analysis. The rest of the patients were rejected. The mean patient age was 56 ± 15 years within the age range of 25--85 years.
Breast lesions were categorized using the Breast Imaging Reporting and Data System (BI-RADS). Figure [3](#Fig3){ref-type="fig"} illustrates the distribution of lesion type in this patient population and Table [1](#Tab1){ref-type="table"} shows the BI-RADS distribution among them.Figure 3Distribution of lesion type in patient population.Table 1Distribution of patients.BI-RADS23456Number of patients08102424
**BI-RADS determination of lesions.** The BI-RADS value was determined for each lesion according to the sonographic features found in clinical US images obtained during a clinical procedure at Mayo Clinic. The patient's eligibility for biopsy was decided based on this value. For BI-RADS 5 and 6, a biopsy is always prescribed; as such, cases are highly suggestive of malignancy. A BI-RADS value of 3 or 4 is challenging as this covers a wide range of suspicion, including low, intermediate, and moderate. In our study, all the BI-RADS 3 and BI-RADS 4 were biopsied.
The LAM technique was performed after determination of the lesion BI-RADS and location, and prior to the biopsy procedure.
**Histology.** Surgical excision biopsy or US-guided core needle biopsy was performed as a part of clinical care and the histology results for all patients in this study were available. In the latter cases, five core biopsy samples of each lesion were acquired by one of
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Introduction {#s1}
============
Articular cartilage is a highly specialised connective tissue in joints. Its main function is to provide a smooth, lubricated surface for articulation and to take up and distribute high loads. Its remarkable dimensional stability and mechanical properties are due to the composition of its extracellular matrix. The load-bearing function is based on the high osmotic pressure created by negatively charged glycosaminoglycans, which are predominantly aggrecan molecules. In addition, the fibrillar collagen network, mainly composed of type II collagen, provides the tissue with its tensile resistance. As the only cell type in articular cartilage, chondrocytes are entirely responsible for maintaining the metabolic balance of matrix proteins. Accordingly, it has been shown that mechanical forces affect chondrocyte metabolic activity (for a review, see [@pone.0036964-Grodzinsky1]). More precisely, *ex vivo* and *in vitro* models of chondrocyte mechanobiology have generally shown that static compression inhibits the expression of cartilage matrix proteins whereas dynamic compression regimens enhance them [@pone.0036964-Buschmann1]--[@pone.0036964-Jones1].
In this context, mechanotransduction is the molecular process by which cells convert mechanical force into biochemical signalling. Little is currently known regarding the sequence of biochemical events that are involved in mechanotransduction and that eventually result in the modulation of the chondrocyte phenotype. It is therefore necessary to assess the signalling and regulatory pathways activated during mechanical signal transduction in chondrocytes. In this study, we employed microarray analysis to investigate the overall changes in chondrocyte gene expression in response to dynamic compression. We used a cell model system consisting of isolated mouse chondrocytes embedded within an agarose hydrogel. We have previously used these constructs to develop experimental procedures to analyse the effects of compression at the mRNA level (using reverse transcription-polymerase chain reaction experiments) and to determine the phosphorylation state of signalling molecules (using Western blotting) [@pone.0036964-Bougault1], [@pone.0036964-Bougault2]. Here, our study was designed to identify candidate genes involved in the early response of chondrocytes to compression.
![Chondrocytes embedded in agarose gel maintain a well-differentiated phenotype.\
Expression of extracellular matrix proteins, integrins and the Sox9 transcription factor were analysed on Western blots of chondrocytes cultured in 3D for 3 days (d3) or 6 days (d6). The presence or absence patterns of proteins at day 6 are representative of 3 independent experiments. A: Type II (Col II) and type IX (Col IX) collagens accumulate and become cross-linked by day 6. Procollagen II forms (pro) and mature collagen II chains \[α1(II)\] are present. Beta (β) indicates cross-linked α1(II) dimers. Collagen IX chains \[α1(IX)\] are present. Asterisks (\*) indicate cross-linked collagens. B: Type I collagen (Col I) and α11 integrin (Itgα11) are not or only faintly detected, whereas the chondrocyte-specific α10 integrin (Itgα10) is present at day 6. Passaged chondrocytes cultured in monolayer were used as positive controls (Ctrl) for Col I and α11 integrin immunorevelations. Procollagen I (pro) and mature collagen I chains α1(I) and α2(I) are indicated. C: Sox9 chondrogenic transcription factor increases with the duration of culture.](pone.0036964.g001){#pone-0036964-g001}
Taken together, the results presented here indicate that the mitogen-activated protein kinase (MAPK) and the transforming growth factor (TGF)- β pathways are involved in the early response of chondrocytes to dynamic compression. The microarray analysis revealed that only 20 transcripts were modulated more than 2-fold. At a fold modulation threshold of 1.4, an extended list of candidate genes included 325 candidate mechanosensitive genes, of which 85% were down-regulated. This global down-regulation may indicate a general control mechanism for a rapid response to dynamic compression. Many of the observed modulated genes are known to be mechanosensitive in other biological contexts. In addition, modulation of genes or transcripts involved in various aspects of cellular physiology was observed. Our integrated analysis provides new molecular insight into how chondrocytes respond to mechanical forces.
{#pone-0036964-g002}
Results {#s2}
=======
Maintenance of the chondrocyte phenotype and cartilage-characteristic matrix deposition in an agarose hydrogel {#s2a}
--------------------------------------------------------------------------------------------------------------
To investigate the early effects of dynamic compression on gene expression of fully differentiated chondrocytes, we used a previously described cell model system [@pone.0036964-Bougault1], [@pone.0036964-Bougault2]. Briefly, mouse chondrocytes were embedded in agarose just after their isolation from cartilage and these chondrocyte-agarose constructs were cultured for 6 days to allow extracellular matrix deposition. Under these conditions, chondrocytes are viable and their proliferation was confirmed by an increase in DNA content (about 1.5 fold, data not shown). Furthermore, they maintain their round morphology and type II collagen and aggrecan accumulate at the cell periphery [@pone.0036964-Bougault1], [@pone.0036964-Bougault2]. Western blotting was used to obtain more detailed information on the matrix proteins and integrin receptors present in the chondrocyte-agarose constructs just before application of dynamic compression ([Figure 1](#pone-0036964-g001){ref-type="fig"}).
{#pone-0036964-g003}
As expected, after 3 days of culture, chondrocytes synthesised type II collagen, but mainly in the procollagen form ([Figure 1](#pone-0036964-g001){ref-type="fig"} Panel A). Fibrillar collagens such as type II collagen are synthesised as precursor forms that must be cleaved to produce the mature triple helical collagens capable of packing into fibrils (for a review, see [@pone.0036964-Canty1]). After 6 days of culture, mature-form type II collagen was the predominant form and showed interchain covalent cross-links ([Figure 1](#pone-0036964-g001){ref-type="fig"} Panel A). All the enzymes necessary for the post-translational maturation of collagen were therefore active in the 3D scaffolds. In addition, we investigated type IX collagen, which is a minor non-fibrillar collagen present in hyaline cartilage. Western blot analysis confirmed the presence of covalent cross-links between collagen molecules in the chondrocyte-agarose constructs after 6 days of culture ([Figure 1](#pone-0036964-g001){ref-type="fig"} Panel A). To demonstrate the absence of critical proteins that could cause the chondrocytes to transduce mechanical signals in a non-characteristic way, we looked for type I collagen, the classical marker of fibroblasts and dedifferentiated chondrocytes. No type I collagen was detected in Western blots on the chondrocyte-agarose constructs, but it was detected in the positive controls, i.e. extracts of mouse chondrocytes cultured in monolayer ([Figure 1](#pone-0036964-g001){ref-type="fig"} Panel B). Therefore, before the compression experiments, chondrocytes synthesise mature and cross-linked extracellular matrix components that are part of the typical collagen network in cartilage.
{ref-type="table"}. (B) Real-time PCR analysis on three independent experiments confirmed DNA
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A number of individuals are struggling with the category of low-quality-status medically unexplained symptoms (MUSs)[@b1] and the morbidity rate is 1.6--70%, 2.4--87% and 4.6--18% in young, middle aged and elderly populations dividually from 1966 according to the investigation of MUSs[@b2]. Meanwhile, MUSs has been defined as "suboptimal health" (*Yajiankang* in China) in traditional Chinese medicine explained as a borderline state between health and disease. To our disappointment, suboptimal health is more difficult to be diagnosed with a biological disease because of only vague changes in function but no clear signs of organic disease[@b3], which present as low energy level, loss of vitality, altered sleeping patterns and so on[@b4]. It could be parallel with symptoms of chronic fatigue syndrome (CFS)[@b5][@b6] or "THE THIRD STATE" or "GRAY STATE" raised by the former Soviet scholar prospectively. Also state of suboptimal health included several different subtypes, and as a subtype, psychological suboptimal health has attracted more attentions.
Psychological suboptimal health is a prevalent state with a pathophysiological mechanism that is extremely complicated and poorly understood. Although it exhibits objective symptoms without a specific disease and it cannot reach the standards of psychiatric diagnosis such as depression and anxiety neurosis estimated by scores on diagnostic scales, the 10th edition of international Classification of diseases (ICD-10), Classification and Diagnostic Criteria of Mental Disorders in China-Third-Edition (CCMD-3), the 4th edition Diagnostic and Statistical Manual of Mental Disorder (DSM-IV)[@b7], for instance, but we must not ignore potential hazards. As the intermediate state between mental health and psychological disease, the emblematical symptoms indicating someone immersed in the state contain out of humor, panic, negative emotion, easy to get angry, losing interest, insomnia, impaired concentration and so on. What's more, psychological suboptimal health, can result in crippling quality of life and raising costs in medical due to frequent, unnecessary visits to healthcare facilities for checkups and diagnoses.
In virtue of potential damage and ambiguity in pathomechanism, psychological suboptimal health has garnered increasing attention and has been described in experimental reports and defined as "subthreshold depression"[@b8][@b9] or "subthreshold obsessive-compulsive disorder"[@b10], the concepts of which are very similar to psychological suboptimal health. In addition, Blackwood has divided chronic fatigue syndrome (CFS) into two states of psychological and physical in the survey[@b11] and the psychological state of CFS is parallel to psychological suboptimal health. These researchers laid particular emphasis on epidemiologic characteristics, and unfortunately, studies on psychological suboptimal health and the pathogenetic mechanisms involved are rare relatively. What's more, a diagnostic criterion that effective and widely accepted has not been established at home and abroad. In China, scholars and doctors prefer to use a variety of scales and questionnaires to diagnose the intermediate state, including the Symptom Checklist 90 (SCL-90), Cornell Medical Index (CMI), mental functions decline index health assessment (MDI), or other self-made evaluations, combining with subjective judgment. To a certain extent, these approaches are authentic for diagnosis but at the same time, rate of missed diagnosis and misdiagnosis is not satisfying, owing to inconformity indigestibility of scales, concealment of patients, doctors relying too much on experiences and subjective judgment. So diagnostic methods that objective, high reliability and easy to operate need to be developed urgently. Through the approach of evaluating the significant differences at the molecular level, novel biomarkers or a biomarker panel then further could be discovered in the plasma samples from patients with psychological suboptimal health and healthy controls. And they would be used in clinical diagnosis after further validation and evaluation. Moreover, metabolomics technologies are the principal approaches for diseases biomarkers discovering.
Systems biology[@b12] including genomics, proteomics, and metabolomics can be utilized in research of diseases[@b13][@b14][@b15]. As an important component of systems biology, metabolomics technologies have become a powerful tool and platform for detecting endogenous small compounds[@b16][@b17] as candidate biomarkers closely related to pathological and physiological processes of diseases and carrying rich information concerning metabolism as key pathways[@b18]. It may help to unravel the mechanisms of disease occurrence and progression on the metabolic level[@b19]. Also major metabolomics technologies were based on H-nuclear magnetic resonance (^1^H-NMR)[@b20][@b21][@b22], liquid chromatography−mass spectrometry (LC−MS)[@b23], and gas chromatography−mass spectrometry (GC−MS)[@b24][@b25]. Furthermore, it was noteworthy that ^1^H-NMR is the earliest method used in metabolomics analysis with the advantages of possessing a rapid, non-destructive, high-throughput system[@b26], and still is widely used to detect biomarkers of diseases for clinical diagnosis[@b27].
In common sense, medicine should be applied to improve clinical symptoms, but few chemical drugs was suitable. As a well-known traditional Chinese prescription, Baihe Dihuang Tang (BDT) is described initially in "Synopsis of Golden Chamber" (Jinkui *Yaolue*) consisting of two herbal medications: lily bulb (Bulbus Lilii) and rehmannia root (Radix, Rehmanniae). It is used to treat mental instability, absentmindedness, insomnia, and dysphoria in clinical. These major symptoms are closely associated with early depression disorder[@b28] and also perform in psychological suboptimal health state. Furthermore, BDT has been widely used and significantly improved the symptoms of psychological suboptimal health due to a deficiency of *yin (Yin Xu*), according to the theory of TCM and also BDT was applied as the intervention measure in our experimentation.
As far as we know, just several published papers were involved in the research of suboptimal health state with meatabolomics and achieved some results[@b29][@b30][@b31] but the study of the psychological suboptimal health state taking advantage of metabolomics technology is almost a blank. In the present study, plasma metabolomics based on ^1^H-NMR coupled with multivariate statistical analysis are used for investigating metabolites with significant differences at a molecular level and screening potential biomarkers. What the goal is to develop a biomarker panel from the biomarkers through correlation analysis, drug intervention of BDT and evaluation of diagnostic ability that can be used for clinical diagnosis ultimately. A biomarker panel would provide support for objective diagnostic laboratory tests for psychological suboptimal health.
Results
=======
Clinical information of participators
-------------------------------------
According to the scale and clinical diagnosis, 22 patients being in state of psychological suboptimal health and 23 volunteers acting as the healthy control group were screened. From the SCL-90 scores of 143.9 ± 22.6 and 90 as the mean ± SD form and the filter factors mentioned above, a significant difference between two groups was confirmed in clinical. The basic clinical data for the participators are shown in [Table 1](#t1){ref-type="table"}.
^1^H-NMR spectra of plasma
--------------------------
To identify the small endogenous molecules in plasma and survey the level varieties in different states, all samples were processed, and typical Carr-Purcell-Meiboom-Gill(CPMG) ^1^H-NMR spectra of plasma from groups of psychological suboptimal health was depicted ([Fig. 1](#f1){ref-type="fig"}). 32 metabolites were identified according to the Human Metabolome Database (HMDB: <http://www.hmdb.ca/>), the Chenomx NMR suite (Chenomx Inc, Edmonton, AB, Canada) and previously published references[@b32][@b33][@b34]. For a better visualization, the vertical scales for the 2D spectra, including 1H-1H correlation spectroscopy (1H--1H COSY) and 1H--13C heteronuclear multiple quantum correlation (1H--13C HMQC) spectra ([Supplementary Figures S1 and S2](#S1){ref-type="supplementary-material"}) were adjusted based on metabolite peaks. Plasma spectra from healthy controls and the BDT group are shown in [Supplementary Figures S3 and S4](#S1){ref-type="supplementary-material"}. The metabolites identified in the spectra were listed in [Table 2](#t2){ref-type="table"}. Several amino acids, glucose, organic acids, lipids, choline were demonstrated in the spectra.
Validation and assessment of the differences between groups
-----------------------------------------------------------
With the purpose of demonstrating significant differences not only in the clinical scale scores, we analyzed the NMR spectra information using multivariable statistics. Metabolome difference by comparing the numerical integration was observed and partial least squares discrimination analysis (PLS-DA)-based profiling was employed to explore the intrinsic differences between the groups of psychological suboptimal health and mental health. The samples from different groups were separated and classified into two distinct clusters presented in the PLS-DA score plot ([Fig. 2A](#f2){ref-type="fig"}); each point represents an individual sample (to show the group clusters). The model parameters (R^2^X = 0.541, R^2^Y = 0.949, Q^2^ = 0.755) and the validated model (permutation number: 200) indicated no over fitting ([Fig. 2B](#f2){ref-type="fig"}), supporting the result. All of the results indicated the existence of differences between the two groups and the reliability of diagnosis according to the method with scales mentioned previously.
Discovery and screening of potential biomarkers
-----------------------------------------------
To identify
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INTRODUCTION
============
The treatment choice for patients with gastric cancer is radical gastrectomy with esophagojejunal anastomosis and lymphadenectomy when involves to upper body or cardia of the stomach. Anastomotic leaks are life-threatening complications that can increase postoperative mortality. In total gastrectomy, anastomotic leaks are more common than subtotal gastrectomy, with reported incidence rates ranging from 4% to 17%. The mortality rate is still high (12% to 50%) despite traditional standard management such as conservative treatment with nil per os (NPO), abscess drainage, and re-exploration with surgical repair \[[@B1][@B2][@B3]\]. Subsequent surgical repair is difficult in patients with anastomotic leaks because of the associated high operative mortality.
Endoscopic self-expandable metal stent (SEMS) replacement has recently begun to be accepted as a primary treatment of patients with esophagojejunal anastomotic leaks after total gastrectomy. Endoscopic SEMS replacement has 2 significant problems: embedded stents due to tissue hyperplasia and stent migration in the patient with anastomotic leaks. Endoscopists should carefully choose the type of SEMS to avoid these issues. Some endoscopists may prefer to use a partially covered SEMS to prevent migration and reduce leakage between the stent and esophageal wall. However, embedded stents are emerging as a common obstacle that can be difficult to extract when using partially covered SEMS and even a conventional fully covered SEMS. Embedded stents may occur with other issues including secondary stricture, perforation, and bleeding, despite the successful removal of stents using various methods \[[@B4][@B5]\]. Choosing the right type of stent to avoiding stent migration and embedding is crucial to improve successful endoscopic treatment in patients with anastomotic leaks. Conventional fully and partially covered SEMS may not be suitable considering migration and embedding for endoscopic treatment.
In this study, we used a benign fully covered SEMS with an anchoring thread and thick silicone covering membrane to prevent stents from becoming embedded and migrating in patients with gastric cancer and esophagojejunal anastomotic leaks after total gastrectomy. The clinical outcomes, including effectiveness and complications, were evaluated.
MATERIALS AND METHODS
=====================
Patient selection
-----------------
We performed a retrospective review of the data of patients who underwent benign fully covered SEMS placement with the Shim\'s technique for anastomotic leaks after total gastrectomy to treat gastric cancer between January 2009 and December 2016 at the Chonnam National University Hwasun Hospital. Overall, 1,018 patients with gastric cancer underwent total gastrectomy; anastomotic leaks occurred in 24 (2.4%) cases. Open surgery was performed in 679 cases, including 10 (1.5%) anastomotic leaks, and laparoscopic surgery was performed in 339 cases, including 14 (4.1%) anastomotic leaks. Anastomotic leak treatment included endoscopic stent replacement (n=14, 58.3%), endoscopic hemoclips (n=2, 8.3%), surgical repair (n=3, 12.5%), and conservative treatment (n=5, 20.8%). Of these patients, 14 were enrolled in this study ([Fig. 1](#F1){ref-type="fig"}). We obtained consent from all patients and the study was approved by the Institutional Review Board of the Chonnam National University Medical School, Gwangju, Korea (CNUHH-2015-113).
{#F1}
Embedded stent and migration
----------------------------
Conventional fully covered SEMS with anchoring thread has been commonly used endoscopic stent for treating esophagojejunal anastomotic leaks after total gastrectomy in Korea. However, this stent can cause embedding into esophageal mucosa by granulation tissue before extraction of stent. We had experience case of anastomotic leaks with a severely embedded stent after use of a conventional fully covered SEMS (Hanarostent^®^; M.I. Tech, Seoul, Korea). The embedded stent was removed with great difficultly. The rat tooth grasped at the distal end of stent, and the stent was peeled backward through the inner side of the stent under fluoroscopic control. This patient\'s leaks resolved after stent retrieval, but severe stricture occurred mid-esophagus. The patient ultimately underwent a second surgery for severe stricture after 1 year because of a persistent stricture despite repeated endoscopic balloon dilation ([Fig. 2](#F2){ref-type="fig"}).
{#F2}
Fully covered SEMS with an anchoring thread and thick covering membrane
-----------------------------------------------------------------------
We used a benign fully covered SEMS with a thick membrane and a silk thread (benign Hanarostent^®^, fully covered esophageal SEMS with Shim\' technique; M.I. Tech) to treat anastomotic leaks in the next patient. This stent has a specially designed silicone membrane that was modified in thickness (0.2--0.24 mm) and a long end of the silicone membrane (5 mm) to prevent overgrowth and ingrowth of granulation tissue. This stent was also applied with an anchoring thread, using the so called Shim\'s technique to prevent migration \[[@B6]\]. The Shim\'s technique involves attaching the silk thread at the proximal end of the stent, which can then be moved through the nose and attached to the ear lobe with tape, similar to the method for endoscopic nasobiliary drainage.
Endoscopic stent replacement
----------------------------
Anastomotic leaks were diagnosed via esophagram using gastrografin or endoscopy. Abdominal and chest computed tomographies (CTs) were performed to evaluate fluid collection or abscess formation in the abdomen or chest cavity. Endoscopic benign stent placements were performed for the following indications: 1) endoscopic confirmed leaks at esophagojejunal anastomosis site, 2) hemodynamically stable patients with systolic blood pressure greater than 90 mmHg, and 3) no residual cancer after total gastrectomy. The methods used for endoscopic stent replacement are described in [Fig. 3](#F3){ref-type="fig"}. Patients were consciously sedated with intravenous midazolam and fentanyl during the procedure. The stent length was 4 cm longer than the size of the leaks to ensure sufficient coverage of the esophageal and jejunal margins of leakage sites. The stent was carefully deployed to locate the middle portion of the stent at the leakage site through a guide wire under endoscopic and fluoroscopic control. After stent deployment, the silk thread was removed through the nose and fixed to the patient\'s ear lobe using tape. Simple chest radiography was checked to confirm the stent position the day after the procedure and then at 1-week intervals. Per-oral intake began with a small amount of liquid, which then progressed to a regular diet. If an abscess or fluid collection was found on abdominal or chest CT, abscess drainage was performed with intravenous broad-spectrum antibiotics. Follow-up endoscopy was performed to check the healing status of the leak site and stent position within 3 weeks based on the results of simple chest radiography and clinical findings. The endoscopist can directly observe the healing status of anastomotic leaks through the space between stent and esophageal lumen without embedding stent. In malnourished patients, an additional feeding tube was inserted within stent under endoscopic guidance to improve nutritional status and decrease the incidence of leakage in the space between the esophageal wall and the stent. All stents were extracted with/without a fluoroscopic guide after checking complete closure or other complications under endoscopic visualization. Follow-up endoscopy was scheduled 3--6 months after leak closures, and then at yearly intervals.
{#F3}
Statistical analysis
--------------------
The Student\'s t-test and χ^2^ test were conducted for continuous and categorical variables. Continuous variables were expressed as mean (standard deviation) and categorical variables, as a percentage (%). Data were analyzed using SPSS version 21.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
=======
Baseline characteristics of patients
------------------------------------
A total of 14 patients underwent endoscopic benign fully covered SEMS placement for esophagojejunal anastomotic leaks after total gastrectomy to treat gastric cancer ([Table 1](#T1){ref-type="table"}). Patients consistent of 12 (85.7%) men and 2 (14.3%) women with a mean
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1. Introduction {#sec1-jof-05-00074}
===============
Coccidioidomycosis, commonly known as Valley Fever, is caused by the soil-dwelling fungi *Coccidioides immitis* and *C. posadasii*. As is common for fungi that infect humans and animals, species of *Coccidioides* are dimorphic in terms of their life cycle, growing saprobically as multicellular filaments on non-living organic matter and, upon entry into a host lung, growing pathogenically in a yeast-like spherule stage \[[@B1-jof-05-00074]\]. The endospores formed by spherules can disseminate within a host but are not transmissible to new hosts. Outside a living host, species of *Coccidioides* form arthroconidia (asexual spores) that can become airborne via soil disturbance and can be inhaled by potential hosts \[[@B2-jof-05-00074]\]. Although coccidioidomycosis presents in about 40% of patients as a pulmonary infection, a chronic and disseminated form of coccidioidomycosis, often resulting in lifelong treatment, occurs in roughly 5% of patients \[[@B3-jof-05-00074],[@B4-jof-05-00074]\].
Although arthroconidia can be detected in soil and in dust, the specific niches of *Coccidioides* species are unknown \[[@B5-jof-05-00074]\]. The disease has been characterized as endemic to arid environments that include the Southwestern United States, Central and South America and Mexico \[[@B6-jof-05-00074]\]. In the United States, highly endemic hotspots have been documented in California and Arizona \[[@B7-jof-05-00074],[@B8-jof-05-00074],[@B9-jof-05-00074]\]. Although less prevalent, Valley Fever is present in Nevada, Colorado, Texas, New Mexico, Utah and Washington state \[[@B10-jof-05-00074],[@B11-jof-05-00074],[@B12-jof-05-00074],[@B13-jof-05-00074]\].
Molecular and phenotypic analyses have resulted in *Coccidioides* being divided into two species: *C. immitis*, known primarily from California, and *C. posadasii*, recognized originally as the non--California group \[[@B6-jof-05-00074]\]. While clinical diagnosis and treatment of coccidioidomycosis have not depended on identifying isolates to species, medical and scientific communities are becoming more cognizant of the potential need to recognize previously undetected phenotypic, morphological, ecological, and genetic differences between species. Multi-locus genetic analyses and whole-genome studies have attempted to map the distribution of these species. To date, however, the isolates studied have been mainly limited to California, Arizona, Central America, Mexico, and Texas \[[@B14-jof-05-00074],[@B15-jof-05-00074]\], and the genetics of isolates from New Mexico have not been examined. The study reported here employed genetic analyses of 18 isolates collected from patients diagnosed with coccidioidomycosis from diverse locations across New Mexico and the Four Corners region. Although Southern New Mexico has been recognized as part of the endemic region for *Coccidioides* \[[@B12-jof-05-00074],[@B16-jof-05-00074]\], several of the isolates examined here were from Northern and Central New Mexico, suggesting a broad range for *Coccidioides* that includes the Four Corners region. Also noteworthy is the fact that isolates of both *C. immitis* and *C. posadasii* were obtained from patients in New Mexico with *C. immitis* being obtained from a patient who resides in San Juan County, NM.
The risk for coccidioidomycosis has been reported as much higher among some ethnic groups, particularly African Americans and Filipinos \[[@B17-jof-05-00074]\]. In these ethnic groups, the risk for disseminated coccidioidomycosis is ten-fold that of the general population \[[@B18-jof-05-00074]\]. Health records from patients in our study suggest the possibility that Native American Indians represent an additional risk group for disseminated disease.
2. Materials and Methods {#sec2-jof-05-00074}
========================
2.1. Sample Collection and Patient Information {#sec2dot1-jof-05-00074}
----------------------------------------------
Eighteen human clinical specimens from seventeen patients diagnosed with coccidioidomycosis obtained between 2013 and 2017 were submitted to the New Mexico Department of Health (NMDOH) Scientific Laboratory Division (SLD). Coccidioidomycosis is a reportable condition in New Mexico. The NMDOH collects information on all confirmed and probable cases (case status definitions follow CSTE standards) who reside in New Mexico at the time of diagnosis to look for potential risk factors via chart review and data extraction. When possible, patient information was noted with specific interest in type of infection, residency, travel history, occupation, race/ethnicity, gender, age, and medical history ([Table 1](#jof-05-00074-t001){ref-type="table"}).
2.2. Molecular Methods {#sec2dot2-jof-05-00074}
----------------------
DNA was extracted at the NMDOH SLD from *Coccidioides* isolates using the PrepMan^®^ Ultra Reagent method (Applied Biosystems, Foster City, CA) and stored at −80 °C until further processing. The DNA extractions were transferred to the Natvig Laboratory at the University of New Mexico where a 1/10 DNA dilution of each preparation was used for PCR amplification of three AFToL (Assembling the Fungal Tree of Life)-designated nuclear gene regions in addition to a serine proteinase gene region diagnostic for species ([Table 2](#jof-05-00074-t002){ref-type="table"}). For the serine proteinase, MCM7, and RPB1 genes, primers were designed to amplify and sequence regions that we determined from comparisons of sequences in GenBank to be diagnostic in distinguishing between the two *Coccidioides* species. Serine proteinase, specifically, was chosen as a target based on results presented by Koufopanou et al. \[[@B19-jof-05-00074]\]. ITS amplification and sequencing involved the entire ITS1-5.8S rRNA-ITS2 region.
We designed a primer pair, with one primer anchored in a sequence unique to *C. posadasii*, to amplify a mitochondrial intron sequence from *C. posadasii* but not *C. immitis*. A second primer set was designed to amplify a portion of the first cox1 exon along with an upstream intergenic region in both *Coccidioides* species but with the potential to differentiate *Coccidioides* from other Onygenales ([Table 2](#jof-05-00074-t002){ref-type="table"}).
All PCR reactions began with an initial step at 95 °C for 5 min. This was followed by 35 cycles of 94 °C for 30 s, a gene-specific annealing temperature ([Table 2](#jof-05-00074-t002){ref-type="table"}) for 30 s, then 72 °C for 45 s with a final extension at 72 °C for 7 min. PCR products were cleaned with ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) before DNA sequencing using BigDye v3.1 (Applied Biosystems, Foster City, CA, USA) chain termination with the Big Dye STeP protocol \[[@B20-jof-05-00074]\]. Forward and reverse sequences were assembled and edited with Sequencher 5.1 (Gene Codes, Ann Arbor, MI, USA). Sequences were deposited in GenBank under accessions MH748760--MH748777 for serine proteinase; MH748742--MH748759 for MCM7; MH748724--MH748741 for RPB1; and MH725244--MH725261 for ITS ([Table 3](#jof-05-00074-t003){ref-type="table"}).
2.3. Phylogenetic Analysis {#sec2dot3-jof-05-00074}
--------------------------
Sequences for the four gene regions were aligned individually using Clustal Omega version 1.2.45 \[[@B21-jof-05-00074]\]. For outgroup sequences, each alignment included the appropriate homologous gene region from *Aspergillus steynii* from the GenBank accessions ([Table 3](#jof-05-00074-t003){ref-type="table"}). This species was chosen as an outgroup because it had a clear homolog for each of the four gene regions examined. The serine proteinase in particular, appears to undergo rapid evolution and perhaps gene loss, and as a result, it was difficult to identify clear orthologs in many other species of Eurotiomycetes.
Tree-building analyses employed maximum likelihood analysis with PHYLIP (version 3.695) DNAMLK and parsimony analysis with PHYLIP DNAPARS \[[@B22-jof-05-00074]\]. In each case, tree building employed 1000 bootstrap datasets. Analyses were done on each of the four gene alignments separately, as well as on a concaten
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Neurocysticercosis is a leading cause of acquired epilepsy in the developing world ([@R1]*,*[@R2]). The disease occurs when larvae of the pork tapeworm, *Taenia solium*, encyst in the human brain; this process causes a broad range of neurologic signs and symptoms, including seizures, headache, obstructive hydrocephalus, encephalitis, stroke, and cognitive and other mental health disorders ([@R3]*,*[@R4]). Neurocysticercosis is endemic in poor rural communities in Latin America, sub-Saharan Africa, and Asia, where pigs can access and ingest human feces ([Figure 1](#F1){ref-type="fig"}). However, the disease is also of increasing public health concern in the United States, especially in the immigrant population and among persons who have traveled to regions where cysticercosis is endemic ([@R5]).
{#F1}
The World Health Organization designates cysticercosis as a neglected tropical disease (NTD) and has called for international efforts to strengthen surveillance ([@R6]*--*[@R8]). The disease remains neglected partly because the scale of the problem has not been well defined ([@R2]). In most disease-endemic regions, population-level data are sparse because surveillance for neurocysticercosis is nonexistent and diagnostic neuroimaging is typically unavailable. In the United States, there is an opportunity to collect population-based data on neurocysticercosis because of the large immigrant population at risk for infection, the widespread availability of neuroimaging, and the well-established disease surveillance infrastructure. However, only Alaska, Arizona, California, New Mexico, Oregon, and Texas require reporting of neurocysticercosis.
Death rates due to neurocysticercosis in the United States have been reported previously ([@R9]), but national-level assessments of neurocysticercosis that use population--based data are lacking. The objective of our study was to evaluate the frequency and total associated charges for hospitalizations due to neurocysticercosis in the United States and to compare these against other tropical diseases of potential importance in the United States.
Methods
=======
Data Source
-----------
We analyzed hospital discharge data contained in the Nationwide Inpatient Sample (NIS) for 2003--2012 ([@R10]*,*[@R11]). The NIS, a stratified weighted sample of short-term and nonfederal hospitals, is designed to approximate a 20% sample of all community hospitals in the United States. As of 2012, 47 states participated in reporting discharge data to the NIS (only Alabama, Delaware, Idaho, and the District of Columbia had not participated), creating a sample representing 95% of the national population. The NIS is the largest collection of longitudinal inpatient care data in the United States and holds information on ≈8 million hospitalizations from \>1,000 hospitals each year ([@R10]). NIS data are de-identified and include information on demographics, diagnostic and procedural codes, length of stay, discharge status, total charges, and expected payees associated with each hospitalization.
Case Definitions
----------------
We based our case definitions for hospitalization on diagnostic and procedural codes from the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM). The ICD-9-CM code listed in the first diagnostic field is intended to capture the primary reason for hospitalization. However, there is no specific ICD-9-CM code for neurocysticercosis, so coding patterns may vary. For example, a hospitalization for neurocysticercosis might be coded with a first diagnostic field of 123.1 (cysticercosis) or with a neurologic code, such as 345.9 (epilepsy unspecified), in combination with 123.1 in a different diagnostic field.
We used 2 case definitions in this analysis. The first was a conservative case definition for reporting hospitalizations associated with neurocysticercosis. This definition required the ICD-9-CM code for cysticercosis (123.1) in any of the 15 available diagnostic fields and a supporting diagnostic or procedural code associated with a clinical manifestation of neurologic disease in any of the first 5 diagnostic or procedural fields ([Table 1](#T1){ref-type="table"}). We used individual ICD-9-CM codes and coding groups defined by Clinical Classification Software to define these additional diagnostic or procedural codes ([@R13]). This conservative case definition was designed to reduce the likelihood of including hospitalizations for persons carrying an existing diagnosis of neurocysticercosis who were hospitalized for an unrelated condition.
###### Supporting diagnostic or procedural codes from ICD-9-CM used for conservative case definition for reporting hospitalizations associated with neurocysticercosis\*
CCS code Diagnosis or procedure
------------------ ----------------------------------------------------------------------
Diagnostic code
76 Meningitis
77 Encephalitis
78 Other CNS infection and poliomyelitis
83 Epilepsy; convulsions
84 Headache; including migraine
85 Coma; stupor; and brain damage
90 Inflammation; infection of eye
95 Other nervous system disorders
109 Acute cerebrovascular disease
111 Other and ill-defined cerebrovascular disease
112 Transient cerebral ischemia
245 Syncope
650 Adjustment disorders
651 Anxiety disorders
652 Attention-deficit, conduct, and disruptive behavior disorders
653 Delirium, dementia, and amnestic and other cognitive disorders
656 Impulse control disorders, NEC
657 Mood disorders
658 Personality disorders
659 Schizophrenia and other psychotic disorders
662 Suicide and intentional self-inflicted injury
670 Miscellaneous mental disorders
Procedural codes
1 Incision and excision of CNS
2 Insertion; replacement; or removal of extracranial ventricular shunt
177 Computerized axial tomography (CT) scan head
198 Magnetic resonance imaging
199 Electroencephalogram (EEG)
\*A complete list of ICD-9-CM codes used in this study is provided in the online Technical Appendix (<http://wwwnc.cdc.gov/EID/article/21/6/14-1324-Techapp1.pdf>). ICD-9-CM, International Classification of Diseases, 9th Revision, Clinical Modification; CCS, Clinical Classification Software groupings of ICD-9-CM codes ([@R12]); CNS, central nervous system; NEC, not elsewhere classified.
The second case definition was designed to facilitate comparison of hospitalizations for cysticercosis with those for the 16 other NTDs and malaria. The case definition for cysticercosis included all hospitalizations with an ICD-9-CM diagnostic code for cysticercosis (123.1) in any of the first 15 diagnosis fields, but it did not require an additional supportive diagnostic or procedural code. Similarly, the case definitions for the other tropical diseases in the comparative analysis relied on ICD-9-CM codes specific to the disease without a requirement for an additional supportive diagnostic or procedural code. This approach ensured consistency of case definitions across the various diseases at the expense of greater specificity. We assumed that the likelihood of capturing unrelated hospitalizations was similar for the diseases we compared. We excluded Buruli ulcer from our comparison because there is no ICD-9-CM code specific for this disease. However, to our knowledge, Buruli ulcer has not been reported in the United States ([@R14]). We did not report hospitalizations for rabies, African trypanomiasis, or dracunculiasis because the numbers of hospitalizations were too low (\<10/year) to provide accurate estimates. A list of ICD-9-CM codes used in all case definitions is provided in the [Technical Appendix](#SD1){ref-type="local-data"}.
Statistical Methods
-------------------
To account for the sampling design of the NIS, we analyzed all data by using the survey family of commands in Stata 13 (StataCorp LP, College Station, TX, USA). We applied hospital discharge weights provided in the NIS to estimate total national hospitalizations on the basis of the stratified sample. All sampled hospitals, regardless of whether they had a patient who was hospitalized with neurocysticercosis, were included for calculation of SEs and CIs. We examined neurocysticercosis hospitalizations by patient age, sex, race, place of service, discharge status, and length of stay; US region; associated diagnostic and procedural codes; and hospitalization charges. State-level assessment was not possible because of the sampling and stratification strategy used in the NIS. Mean annual hospitalization rates were calculated as the weighted number of hospitalizations per 100,000 population on the basis of the US Census Bureau data for each year during the study period ([@R15]). Age- and sex-adjusted rates were calculated by using the direct standardization method and the 2005 US Census population as the reference population.
We used Gaussian family generalized linear models with logarithmic function link within the Stata survey framework to estimate the crude and adjusted mean length of stay and mean hospitalization charges. We first constructed univariate generalized linear models to evaluate demographic variables of interest, retaining those that were significant at the p\<0.2 level (Wald test) in the final multivariate models. The independent categorical variables we evaluated were sex, age, race, hospital region, and year of hospitalization. Once we built the final models, we estimated the mean length of stay and mean hospitalization charges for diagnoses and procedures commonly seen with neurocysticercosis (i.e., seizures, obstructive hydrocephal
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All relevant data are within the manuscript and its Supporting Information files.
Introduction {#sec001}
============
Oral squamous cell carcinoma (OSCC) is the most lethal type in head and neck squamous cell carcinoma (HNSCC) in the world. Over the past decades, the incidence rate of OSCC has increased among younger generations \[[@pone.0213463.ref001]--[@pone.0213463.ref003]\]. In spite of considerable advances in surgery, radiotherapy and chemotherapy, the 5-year survival rate for OSCC has not improved markedly because patients still frequently arise loco-regional recurrence and lymph node metastasis \[[@pone.0213463.ref004]--[@pone.0213463.ref006]\]. Hence, finding new biomarker(s) and therapeutic molecule(s) is urgent. Recently, a growing evidence indicates that microRNAs (miRNAs) contribute to the initiation and development of oral cancer \[[@pone.0213463.ref007]--[@pone.0213463.ref009]\]. Therefore, exploring unique miRNAs and related molecular pathways underlying OSCC aggressive will provide advantages to improve therapeutic efficacy, as well as to design more effective treatment strategies.
Previously, we established a dysregulated signature of eighty-four miRNAs from OSCC clinical samples using a miRNA microarray \[[@pone.0213463.ref010]\]. From this analysis, we found that miR-450a was significantly overexpressed in tumor tissues than that in corresponding adjacent normal tissues. MiR-450a is an intragenenic miRNA clustered with miR-542-5p, miR-542-3p, miR-503, miR-450b-5p, miR-450b-3p, and miR-424 on chromosomal location Xq26.3. High expression level of miR-450a performs as a potential oncogene in laryngeal squamous cell carcinoma and breast cancer \[[@pone.0213463.ref011], [@pone.0213463.ref012]\]. Up-regulated miR-450a is found in mesenchymal part of epithelial-to-mesenchymal transition (EMT)-activation in human endometrial carcinosarcoma \[[@pone.0213463.ref013]\]. Contrarily, downregulation of miR-450a is required in hepatocellular carcinoma carcinogenesis \[[@pone.0213463.ref014]\]. Although miR-450a was reported to be dysregulated in different cancer types, its functions and underlying mechanisms have not been elucidated, especially in oral cancer.
Transmembrane proteins (TMEMs) are a group of novel proteins, which have key roles in cell differentiation and tumorigenesis in many cancers, such as pancreatic cancer, prostate cancer, ovarian cancer, and renal cell carcinoma \[[@pone.0213463.ref015]--[@pone.0213463.ref019]\]. However, the role of TMEM182 in oral cancer is unknown. In this study, we demonstrated that miR-450a may function as an oncogene by directly targeting 3'-untranslated region (UTR) of TMEM182 and reduce its expression in OSCC. Downregulation of TMEM182 or overexpression of miR-450a has the same effect on reducing cellular adhesion of OSCC cells. In addition, we also found that miR-450a expression was increased by TNF-α through ERK1/2-dependent pathway, more than via NF-κB. Taken together, these findings may provide understanding into oral carcinogenesis and suggest new therapeutic opportunities in this cancer.
Materials and methods {#sec002}
=====================
Human samples {#sec003}
-------------
The study protocol was approved by the Research Ethics Committee of National Health Research Institutes (EC1040409-E) and Institutional Human Experiment and Ethic Committee of National Cheng Kung University Hospital (HR-97-100) for the use of clinical materials for research purpose. Thirty-five paired primary OSCC and their adjacent non-tumorous epithelial samples were obtained from patients with curative surgery at the National Cheng Kung University Hospital (Tainan, Taiwan) from 1999 to 2010. All human tissues were snap-frozen in liquid nitrogen. Total RNA was extracted by miRNeasy Mini Kit (Qiagen, \#217004) followed by instruction manual. The patient's backgrounds and clinical parameters were summarized in [S1 Table](#pone.0213463.s004){ref-type="supplementary-material"}. The prognostic value of miR-450a among The Cancer Genome Atlas (TCGA) HNSCC cohort was analyzed through SurvMicro database (<http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp>) and was uploaded to GEO (Access number GSE36682). Forty paired of OSCC patients cDNA microarrays analysis were performed according to our previous study deposited in GEO (Accession number GSE37991 and GSE45238) \[[@pone.0213463.ref010]\].
Cell culture {#sec004}
------------
Cultured conditions for all human OSCC cell lines were summarized as previously described \[[@pone.0213463.ref015]\]. Human oral keratinocytes (HOK) were purchased from ScienCell (Carlsbad, CA, USA) and cultured according to the manufacturer's instructions. All cells were maintained at 37°Clin a 5% CO~2~ atmosphere properly.
Cytokine and chemical inhibitor treatment {#sec005}
-----------------------------------------
Cells were incubated at low-serum conditional medium for 24 h, before TNF-α (10ng/ml) addition as indicated times. For intrinsic pathway analysis, cells were incubated with each of the following inhibitors: 1 μM for human dysplasia oral keratinocyte (DOK) and 10 μM for human tongue cancer cells (SAS) of NFκB inhibitor (Calbiochem, 481406), 30 μM U0126 (ERK inhibitor)(Cell Signaling, 9903), and 30 μM SB203580 (p38 inhibitor)(Cell Signaling, 5633).
RNA extraction and reverse-transcription PCR (RT-PCR) {#sec006}
-----------------------------------------------------
Total RNA was extracted from OSCC cell lines using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. RNA concentration and purification were checked by NanoDrop ND-1000 spectrophotometer. First-strand cDNAs were synthesized by NxGen^™^M-MuLV reverse transcriptase with oligo dT~12-18~ primer (Invitrogen, Carlsbad, CA). Gene expression analyses were assayed on a Biometra T3000 thermocycler (Biometra GmbH, Germany) as following conditions: 95°C for 5 min, followed by 35--40 cycles of amplification (95°C for 30s, 60°C for 30s, and 72°C for 30s), and 72°C for 10 min. GAPDH was used as a loading control. PCR products were subjected to electrophoresis on 2% agarose gel and visualized on UVP GDS-8000 Bioimaging System (UVP, CA, USA) with 0.01% of SYBRSafe (Invitrogen, Carlsbad, CA, USA) inner staining. Primer sequences are listed in [S2 Table](#pone.0213463.s005){ref-type="supplementary-material"}.
Quantitative real-time PCR (qPCR) {#sec007}
---------------------------------
Mature miR-450a and RNU44 internal control levels were analyzed by miRNA-specific stem-loop primers and TaqMan Universal PCR Master Mix (Applied Biosystems) on an Applied Biosystems StepOne Plus real-time PCR system. Fold changes were calculated by using2^-ΔΔCt^ method using control and reference normalized. Primer sequences are listed in [S2 Table](#pone.0213463.s005){ref-type="supplementary-material"}.
Plasmids {#sec008}
--------
A wild type 3′-UTR of TMEM182 containing the miR-450a binding sites (3\'UTR-WT) and truncated 3′-UTR fragment with deleted miR-450a binding sites (3\'UTR-DEL) were constructed into the XhoI/XbaI sites of pmiRGLO firefly luciferase-expressing vector (Promega, WI, USA). For gene knockdown experiments, the shRNA clones of TMEM182 (sh182 \#1 & \#2) and empty vector pLKO_TRC (shCTRL) were obtained from the National RNAi Core Facility (Academia Sinica, Taiwan). Human TMEM182 cDNA was sub-cloned into empty vector pCDH-CMV-GFP puro+ (vehicle) (System Biosciences) at EcoRI/BamHI sites and termed as TMEM182-flag. Human TMEM182 cDNA was sub-cloned into empty vector pEGFPN1 (BD Biosciences Clontech's) (vehicle) at XhoI/BamHI sites, termed as TMEM182-GFP. The sequence data were compared against the National Center for Biotechnology Information (NCBI) database using BLAST and miRBase (<http://www.mirbase.org/>). Kyte-Doolittle hydrophobicity analysis was accessed to predict TMEM182 transmembrane portions using ExPASY (<https://web.expasy.org/protscale/>). List primer sequences in the following [S2 Table](#pone.0213463.s005){ref-type="supplementary-material"}.
Protein extraction and western blot {#sec009}
-----------------------------------
Cell lysates were prepared as previously reported.\[[@pone.0213463.ref020]\] Equal amounts of protein lysates were separated by 10\~12% SDS polyacrylamide gels and transferred to poly-vinylidene fluoride (PVDF) membrane (Pall Life Sciences, Glen Cove, NY). Immunoblotting was performed with specific antibodies against TMEM182 (ab177360; Abcam). α-tubulin (sc-23950; Santa Cruz) and GAPDH (GeneTex, G
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Background {#Sec1}
==========
The African pygmy hedgehog (*Atelerix albiventris*) has become a very popular pet in Poland in the last few years. It is smaller than the European hedgehog and is a member of the insectivore family *Erinaceidae*, subfamily *Erinaceinae*. African pygmy hedgehogs are domesticated animals and live for about 5--7 years in captivity. They possess 36 brachyodontic teeth: 2(3/2,1/1,3/2,3/3) with the first incisors being notably longer than the rest, and are spaced apart \[[@CR1]\].
In studies of hedgehogs at histopathology of surgically resected tumor or necropsy approximately 40 % of hedgehogs aged from 1 month to 3 years were diagnosed with neoplastic disease \[[@CR2], [@CR3]\]. The most common histologic types of tumors are mammary gland adenocarcinoma, lymphoma, and oral squamous cell carcinoma \[[@CR2], [@CR4], [@CR5]\]. The digestive tract, including the oral cavity, is the third most common site of neoplastic disease in hedgehogs \[[@CR4]\]. Chemotherapy and radiotherapy protocols have not yet been established for the African pygmy hedgehog, and therefore, surgical resection is currently the best treatment in cases where the tumor is benign, well separated from the healthy tissue and without metastases.
Medical knowledge, veterinary care, and the awareness of African pygmy hedgehog owners are ever increasing. The average life span of domesticated animals is prolonged compared with wild animals. This situation predisposes domesticated hedgehogs to more frequent development of tumors, including oral cavity. In addition, it is well known that periodontal disease, tooth root abscesses, and various neoplasms (e.g. squamous cell carcinoma, lymphosarcoma) occur frequently in African pygmy hedgehogs \>3 years old \[[@CR4]\].
Peripheral odontogenic fibroma (previously named as fibromatous epulis of periodontal ligament origin) is a peripheral odontogenic neoplasm, indistinguishable clinically from fibrous hyperplasia, most common in dogs, and rarely occurring in cats. The prognosis following surgical removal is good \[[@CR6], [@CR7]\].
To our knowledge, this is the first case report of surgical resection of a peripheral odontogenic fibroma in the African pygmy hedgehog. The significance of this case report is that it will enable veterinary clinicians to familiarize themselves with the surgical resection of benign oral tumors (peripheral odontogenic fibroma) in the African pygmy hedgehog and consider the peripheral odontogenic fibroma as other primary neoplasm of oral cavity in this species.
Case presentation {#Sec2}
=================
A 5-year-old male African pygmy hedgehog showed two erythematous, round small tumors protruding from the oral cavity. One tumor was visible on the closed mouth on the right side of the head (Figs. [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"}), and the other was located on the left side, inside the oral cavity, above the molar teeth (Fig. [3](#Fig3){ref-type="fig"}). Both tumors were well separated from the gum, pedunculated, soft texture, uneven surfaces, painless. The neoplastic lesion on the left side had a diameter of approximately 5 mm, was pale pink, while the lesion on the right side had a diameter of approximately 11 mm and on its surface small foci of hyperemia were observed. There was no deformation of hard tissue of splanchnocranium during clinical examination. The pet owner complained of problems with food and water intake, because of the neoplastic tumors located on the surface of maxilla. The animal's appetite remained good. Physical examination revealed the animal was in very good condition with a rectal temperature of 36.8 °C.Fig. 1Preoperative view of oral tumors located above the molar teeth, on both sides of the maxilla in an African pygmy hedgehogFig. 2Preoperative view of oral tumors located above the molar teeth, on both sides of the maxilla in an African pygmy hedgehogFig. 3Preoperative view of oral tumors located above the molar teeth, on both sides of the maxilla in an African pygmy hedgehog
Anesthesia was induced with 30 mg/kg of ketamine hydrochloride (Bioketan; Vetoquinol Biowet, Gorzow Wielkopolski, Poland) and 0.15 mg/kg of medetomidine hydrochloride (Domitor; Zoetis, Florham Park, NJ, USA) intramuscularly (IM). To prevent hypersalivation, atropine sulfate (Atropinum Sulfuricum; Polfa, Warszawa, Poland) at 0.03 mg/kg IM was administered. The preferred method for sedating small animals is gas anesthesia with isoflurane or sevoflurane \[[@CR8]\], but in this case using a facial mask was impossible because of the location of the tumors. The animal was placed on a heating mat to prevent hypothermia. A portable veterinary monitor (MEC-1200-Vet; Mindray, Shenzhen, China) was used to constantly monitor the patient's breathing rate and heart rate. Surgical resection of tumors and bleeding were controlled simultaneously using a surgical knife electrocoagulation system (Martin System 2000; Gebrüder Martin GmbH & Co, Tuttlingen, Germany). The results of surgical resection of the oral tumors are shown in Figures (Figs. [4](#Fig4){ref-type="fig"} and [5](#Fig5){ref-type="fig"}).Fig. 4Postoperative view of the excised oral tumors using surgical knife electrocoagulationFig. 5Postoperative view of the excised oral tumors using surgical knife electrocoagulation
After surgical procedures, atipamezole hydrochloride (Antisedan; Zoetis) 0.3 mg/kg IM and meloxicam (Metacam; Boehringer Ingelheim, Ingelheim, Germany) 0.2 mg/kg subcutaneously were administered \[[@CR9]\]. The patient's condition was monitored until it reached full consciousness.
The excised tumors underwent fixation in 7 % buffered formalin, were embedded in paraffin blocks, and 6 μm slides were cut. The preparations were stained using the standard hematoxylin-eosin method \[[@CR10]\], and subsequently evaluated using light microscopy using WHO guidelines for the evaluation of oral cavity tumors. Photomicrographs of the preparations were obtained using computer-amplified image analysis and an optical microscope (Olympus BX53; Olympus, Tokio, Japan). Histopathological analysis was conducted using the cell^\^^A software (Olympus Soft Imaging Solution GmbH, Münster, Germany).
Results and discussion {#Sec3}
======================
Histopathological examination of lesions showed epithelial covered, well-vascularized, cellular fibroblastic tissue comprised of small spindle to stellate fibroblasts with small dark basophilic nuclei dispersed in a dense collagen matrix. Few mononuclear inflammatory cells, areas of hard tissue corresponding with areas of mineralization, and branching cords or islands of epithelium with peripheral basal stratum were also observed (Fig. [6a](#Fig6){ref-type="fig"}, [b](#Fig6){ref-type="fig"}, and [c](#Fig6){ref-type="fig"}). All tumors were removed with an appropriate (approximately 2--5 mm) clinical surgical margin and evaluated later by histopathology. The observed pattern is characteristic for peripheral odontogenic fibroma \[[@CR6], [@CR7]\]. Peripheral odontogenic fibromas have been extensively reported in a variety of domestic mammals and humans \[[@CR11]--[@CR16]\]. However, to our knowledge, there is no information concerning peripheral odontogenic fibroma in the African pygmy hedgehog.Fig. 6Histopathological pattern of peripheral odontogenic fibroma in the pygmy hedgehog. **a** Small spindle to stellate fibroblasts immersed in eosinophilic dense collagen matrix with branching cords and islands of odontogenic epithelium and peripheral palisading of same epithelium. **b** A small amount of mononuclear inflammatory cells accompanying the peripheral odontogenic fibroma. **c** Areas of hard tissue corresponding with areas of mineralization within the tumor
According to the literature, the most common tumors of the gastrointestinal tract in hedgehogs are oral squamous cell carcinoma, intestinal adenocarcinoma, acinic cell carcinoma, metastatic hepatocellular carcinoma, fibrosarcoma, plasmocytoma, and lymphoma \[[@CR4], [@CR17]--[@CR19]\]. Raymond and Garner \[[@CR4]\] diagnosed 35 (53 %) neoplasms in a group of 66 hedgehogs. In three of the 35 animals, more than one type of tumor was present. The authors revealed that 85 % of the tumors were malignant \[[@CR4]\]. Raymond et al. \[[@CR20]\] indicated the presence of malignant mast cell tumor in adult African hedgehog. This tumor was located subcutaneously, along to ventral part of the neck with metastasis to local lymph node \[[@CR20]\]. In other studies, the authors revealed the evidence of a probable retrovirus associated with multicentric sarcomas in two 3 years old hedgehogs, male and female \[[@CR21]\]. In our case, the investigated tumor was benign. Nonetheless, its location in the oral vestibule, which caused the animal's discomfort, pain, and risk of hemorrhage, was an indication for surgery.
Conclusions {#Sec4}
===========
An early and accurate diagnosis is essential for positive prognosis, curative treatment, and fast recovery in hedgehogs. The resection of oral cavity tumors in the African pygmy hedgehog carried out in this
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**Core tip:** Analytical performance of the serum creatinine assays is the critical determinant of estimated glomerular filtration rate (eGFR) accuracy. The most widely used compensated Jaffé creatinine assay suffers from a non-specific bias from pseudo-creatinine chromogens (glucose, ketones), which is not the case with the costly enzymatic assays. We evaluated the influence of creatinine methodology on the performance of chronic kidney disease (CKD)-Epidemiology-calculated eGFR for CKD diagnosis/staging in diabetic patients. Our results indicate that compensated Jaffé creatinine procedure, in spite of the glucose-dependent bias, is not inferior to enzymatic creatinine in CKD diagnosis/staging and therefore may provide a reliable and cost-effective tool for the renal function assessment in diabetic patients.
INTRODUCTION
============
Global prevalence of diabetes mellitus is rising progressively\[[@B1]\]. Chronic morbidity, associated with various debilitating complications, increased risk for adverse health-outcomes and significant impact regarding both the working ability and quality of life identify diabetes as one of the greatest health-care and socio-economic challenges worldwide. Appropriate strategies to tackle diabetes epidemic include education and lifestyle interventions, evidence-based clinical management as well as the screening for and monitoring of diabetes and/or diabetes-related complications using state-of-the art diagnostic tools.
Diabetic kidney disease (DKD) is one of the most prevalent chronic complications of diabetes and the most common single cause of end-stage renal failure\[[@B1],[@B2]\]. It has been amply evidenced that appropriate interventions at an early stage of DKD can be efficient in preventing and/or delaying the progression of kidney disease and improving patient outcomes. Thus, the regular screening for DKD has became one of the cornerstones of diabetes care. Current clinical guidelines recommend at least an annual screening of DKD in patients with type 1 diabetes with a duration above 5 years, in all patients with type 2 diabetes and in all hypertensive diabetic patients\[[@B3]\]. Once detected, DKD is treated according to clinical guidelines and further monitored at regular intervals\[[@B2],[@B3]\]. Two simple laboratory tests are used for both the screening and staging of CKD in diabetes: Urinary albumin excretion (UAE) and serum creatinine-based estimated glomerular filtration rate (SCr-eGFR).
Abnormal UAE has long been identified as a sensitive marker of the glomerular basal membrane damage, which is one of the early pathophysiological events in the development of DKD\[[@B4]\]. However, a significant decline in eGFR is a common finding in a notable proportion of diabetic patients with normal UAE, probably reflecting a diversity in the natural history of DKD\[[@B5]\]. Thus, the pathophysiology of DKD has shifted from the "albuminuric paradigm"\[[@B6]\], and the accumulated evidence implicating the progressive renal function decline as an equally relevant pathway identified reliable and accurate laboratory testing for serum creatinine and SCr-eGFR as a very important issue for the diagnosis, staging and monitoring of CKD in diabetic patients.
SCr has been used as a cost-effective and practical marker of kidney function for decades, despite severe limitations due to both biological and analytical variability\[[@B7]\]. A handful of biological factors such as age, gender, ethnicity and nutritional habits substantially influence serum creatinine levels, while partial tubular reabsorption and secretion of creatinine further compromise its use as the glomerular filtration marker\[[@B8],[@B9]\]. Nevertheless, SCr-based estimation of GFR by the use of appropriate predictive equation remains the recommended surrogate marker for the assessment of kidney function, since the actual measurement of GFR, due to its complexity and high costs, is not available outside the specialized clinical settings. Current guidelines from the Kidney Disease Improving Global Guidelines (KDIGO) CKD Working Group recommend the use of the chronic kidney disease-Epidemiology Collaboration Group (CKD-EPI) equation\[[@B2]\]. CKD-EPI equation offers an improved reproducibility and accuracy at higher GFR levels (\> 60 mL/min per 1.73 m^2^), which is the most prominent disadvantage of the previously recommended Modification of Diet in Renal Disease equation\[[@B10]\].
Analytical performance and specificity of SCr assay are critical determinants of the eGFR accuracy\[[@B11]\]. The relationship between SCr and GFR is exponential, therefore, errors in SCr measurements resulting from imprecision and bias could strongly impact eGFR results and result in misclassification of the patients regarding their kidney function\[[@B2]\]. Despite standardization and harmonization by the calibration traceable to isotope-dilution-mass spectrometry (IDMS), the non-specific bias from pseudo-creatinine chromogens (glucose, proteins, ketone bodies) is still affecting the most widely used compensated Jaffé alkaline picrate colorimetric creatinine assay\[[@B11],[@B12]\]. Enzymatic creatinine methods are free from these interferences, but far more expensive and therefore not widely used. High-volume routine enzymatic creatinine testing may introduce a substantial financial challenge for the laboratories, even in the otherwise fairly resourced health-care systems\[[@B13]\]. Several analytical and clinical studies advocated the replacement of the compensated Jaffé with enzymatic creatinine assays in order to improve reliability of the eGFR, especially in the diabetic population, which is expected to have an increased amount of interfering substances in serum. However, recently published risk-analysis study, using both analytical and biological variability criteria, revealed a low risk for misclassification of CKD based on Jaffé-SCr-eGFR results in the general population\[[@B13]\], while the clinical impact in diabetic population remains unclear.
The aim of this study was to evaluate the influence of creatinine methodology on the performance of CKD-EPI-calculated eGFR for CKD evaluation and staging in a large cohort of diabetic patients.
MATERIALS AND METHODS
=====================
Fasting blood samples were taken from diabetic patients attending our clinic for their regular annual examination, including laboratory measurement of SCr and eGFR. Samples from the patients with concomitant infection, limb-amputation and malignancies, as well as the pregnant patients and the patients with severe kidney disease (stage 5, according to KDIGO-2012 classification) were not included in the study. A subset of samples of the patients with severe hyperglycaemia were included in order to evaluate the interference of glucose on the CKD classification across various eGFR categories. Serum creatinine was measured by both IDMS-traceable compensated Jaffé (cJ-SCr) and enzymatic (e-SCr) (Beckman Coulter, Inc., Pasadena, California, United States) procedures with intra-assay imprecision (CV) of 1.58% and 1.39%, respectively. Hexokinase (Beckman Coulter, Inc., Pasadena, California, United States) and NGSP-traceable immunoturbidimetric assays (Tina Quant, Roche, F.Hoffmann-La Roche, Basle, Switzerland) were used for plasma glucose and HbA~1c~ measurement.
Assay-specific SCr-eGFR was estimated by the 4-variable CKD-EPI equation using respective creatinine values\[[@B10]\]. UAE was measured by an automated immunoturbidimetric procedure (Beckman Coulter, Inc., Pasadena, California, United States) in fresh spot urine samples. Urinary creatinine was measured in the same samples and UAE results expressed as the urinary albumin/creatinine ratio.
Staging of albuminuria and CKD, as well as risk assessment for CKD progression was carried out according to KDIGO-2012 criteria (Table [1](#T1){ref-type="table"}).
######
Kidney Disease Improving Global Guidelines-2012 Prognostic Categories of Chronic Kidney Disease according to estimated glomerular filtration rate and albuminuria (adapted from the Reference 2)
**Albuminuria categories \[albumin/creatinine (mg/mmol)\]**
---------------------------------------- ------------------------------------------------------------- --------------------------- --------------------------- -----------
eGFR categories (mL/min per 1.73 m^2^) G1, ≥ 90 Low risk Moderately increased risk High risk
Normal/high
G2, 60-90 Low risk Moderately increased risk High risk
Mildly decreased
G3a, 45-59 Moderately increased risk High risk Very high risk
Mildly to moderately decreased
G3b, 30-44 High risk Very high risk Very high risk
Moderately to severely decreased
G4, 15-29 Very high risk Very high risk Very high risk
Severely decreased
G5, \< 15 Very high risk Very high risk Very high risk
Kidney failure
eGFR: Estimated glomerular filtration rate.
The results were analyzed in the entire population and in sub-groups according to albuminuria (Table [2](#T2){ref-type="table"}). Normality of distribution was tested by the Kolmogorov-Smirnov test and the significance of differences between the groups was assessed by the Kruskal-Wallis and Mann-Whitney test, as appropriate. Comparison between the creatinine methods in the study population was tested by Passing-Bablok regression analysis. Specific SCr-eGFR data were compared by Bland Altman analysis, and their agreement regarding clinical CKD staging was evaluated by inter-rater agreement (kappa-analysis). Statistical analyses were performed using MedCalc for Windows, version 9.4.2.0 (MedCalc Software,
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The organ size is controlled through changes in rates of cell growth and division[@b1][@b2]. The underlying mechanisms of organ size control, such as Insulin/IGF signaling (IIS) and Target of Rapamycin complex 1 (TORC1) signaling pathways, have been intensively investigated by using proliferating and morphologically simple cells, such as those in epithelial monolayers of the Drosophila imaginal disc[@b3][@b4]. However, it is still largely unknown how the size of postmitotic and morphologically complex cells such as neurons scale with body size and which molecules control such scaling. We therefore explored these scaling mechanisms in Drosophila neurons.
For our investigation, we used the dendritic arbor of one class of sensory neurons in Drosophila adults as a readout ([Figure 1A and 1B](#f1){ref-type="fig"})[@b5][@b6][@b7][@b8][@b9]. Dendrites are the antennae of neurons that receive and integrate sensory and/or synaptic inputs[@b10][@b11]. Our model neuron in this study, previously designated as v\'ada, is one of the dendritic arborization (da) neurons, whose arbor for adult life is regenerated during pupal stages and entirely covers the lateral plate (pleura) in the abdomen ([Figure 1A--1C](#f1){ref-type="fig"})[@b7][@b8].
Results
=======
Scaling of dendritic arbors of the wild-type da neuron with body size
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To examine how body-size changes affect the size and the branching pattern of the dendritic arbor of the wild-type neuron, we starved larvae beyond 91--96 hr after egg laying (AEL). This is because it has been shown that larvae exposed to such starvation after the "critical weight" stop body growth, but develop to become fertile adults that are smaller than normal, without any developmental delay[@b12][@b13][@b14][@b15]. Under this mild starvation condition, the size of the dendritic arbor was significantly decreased in proportion to the decreased body size ([Figure 1D--1G](#f1){ref-type="fig"}). Importantly, the number of branch endings was not changed and the branch density (total length/arbor size and ending number/arbor size) was significantly increased ([Figure 1H--1K](#f1){ref-type="fig"}). We also quantified the branching complexity, by assigning Strahler orders to individual segments (intervals between branching points; see [Figure 1L](#f1){ref-type="fig"})[@b16][@b17][@b18]. The numbers of individual order segments were similar under the fed and starved conditions ([Figure 1M](#f1){ref-type="fig"}), while 2^nd^ to 4^th^ order segments became shorter under the starved condition ([Figure 1N](#f1){ref-type="fig"}). These results are consistent with the hypothesis that the wild-type neuron can respond commensurately to the decrease in body size and/or the starvation and can form a "miniature" dendritic arbor by tuning its dendritic segment length.
Neurons with defective IIS/TORC1 signaling pathways show dendritic "undergrowth"
--------------------------------------------------------------------------------
The above result suggested that the wild-type neuron is able to scale with the body size and/or the nutrition condition, while keeping the branching complexity intact. We wondered if the IIS/TORC1 pathways ([Supplementary Figure S1A](#s1){ref-type="supplementary-material"}) participate in this scaling, and whether defects in the pathways would affect the size and the branching pattern of the dendritic arbor in adults, when larvae were raised under the normal food condition. Neurons with disruptions of *Drosophila insulin receptor* (*dinr*) or *Akt* downsized and simplified the dendritic arbors, as evidenced by decreases in the arbor size, the ending number, and the total length ([Supplementary Figure S1B--S1D and S1H--S1J](#s1){ref-type="supplementary-material"}). In contrast, the branch density (total length/arbor size and terminal number/arbor size) was not significantly altered from that of the wild-type neuron ([Supplementary Figure S1K and S1L](#s1){ref-type="supplementary-material"}). A loss of function mutation of *tor*, overexpression of a dominant negative form of Phosphoinositide 3-kinase (PI3K), or a knockdown of *raptor* encoding an essential component of TORC1 not only decreased the arbor size, but also the branching complexity ([Supplementary Figure S1E--S1G](#s1){ref-type="supplementary-material"}). Thus this "undergrowth" phenotype is distinct from the "miniature" dendrite of the wild-type neuron under the starved condition ([Figure 2L](#f2){ref-type="fig"}), suggesting that a regulatory mechanism other than the IIS/TORC1 pathways may contribute to the dendritic scaling in the normal da neuron.
*CHORD* mutant neurons form miniature dendrites
-----------------------------------------------
To explore the hypothetical mechanism of the dendritic scaling, we conducted a forward genetic screen by employing the mosaic analysis with a repressible cell marker (MARCM) system[@b19]. To facilitate the generation of mosaic clones, we made "SOP-FLP" lines that express FLP recombinase in sensory organ precursors (SOPs; see details in Methods). In our screening under the fed condition, we found a mutant chromosome that generated miniature dendritic patterns in homozygous neurons ([Figure 2B and 2C](#f2){ref-type="fig"}). Indeed a number of quantitative features indicated that the arbor of this mutant neuron was a proportionally scaled down miniature or a microcopy of the wild-type neuron ([Figure 2E--2I](#f2){ref-type="fig"}): decreases in the arbor size and the total dendritic length, an unaltered ending number, an increase in branch density, profiles of the Strahler-order analysis similar to those of the wild-type neuron under the starvation condition (compare [Figure 1M--1N](#f1){ref-type="fig"} with [Figure 2J--2K](#f2){ref-type="fig"}), and an unaltered distribution of angles at individual branching points (data not shown). In a word, the mutant neuron reduced the dendritic segment length ([Figure 2K](#f2){ref-type="fig"}), without simplifying the branching pattern, which is reminiscent of how the wild-type neuron constructed its arbor under the starved condition ([Figure 1F](#f1){ref-type="fig"}).
Whole-genomic sequencing and complementation mapping using Drosophila deficiency stocks identified a 1 bp deletion that leads to a frameshift in the *CHORD*/*morgana* gene (denoted *CHORD^2^* hereafter; [Figure 2A](#f2){ref-type="fig"}). CHORD is an evolutionarily conserved co-chaperone of HSP90, and it negatively regulates Rho-kinase (Rok) activity to suppress overduplication of centrosomes[@b20][@b21]. Introduction of the 4757-base-pair (bp) genomic fragment including the *CHORD* gene almost fully rescued the above phenotypes to normal, demonstrating that *CHORD* is the causative gene for the miniature dendrite phenotype ([Figure 2D and 2E--2K](#f2){ref-type="fig"}).
The miniature arbor of the *CHORD* mutant neuron may mimic a default state of the wild-type neuron
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One possible explanation of the *CHORD* phenotype is that the mutant neuron no longer interprets favorable or unfavorable extracellular conditions (either the nutrition and/or body size) and generates the miniature arbor as an invariable default. Alternatively, the *CHORD* mutant neuron misreads the size of the abdominal lateral plate; consequently, the arbor occupies only a portion (e.g., 50% instead of 100%) of the body surface. To distinguish these possibilities, we examined dendritic arbors of *CHORD* mutant neurons under the starved condition ([Figure 3A and 3B](#f3){ref-type="fig"}). Neither the size nor the branching complexity significantly changed between the two nutrition conditions ([Figure 3C--3I](#f3){ref-type="fig"}), showing that the *CHORD* mutant neuron formed the miniature arbor irrespective of the extracellular condition. This result suggested that the latter possibility was less likely. It appears that the wild-type neuron possesses a CHORD-dependent mechanism that extends the branch segment length beyond a preset value in response to a favorable environment.
Elongation/retraction rate of terminal branches is critical for reproducing the miniature phenotype of the *CHORD* mutant neuron
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We next investigated the dynamics of dendrite formation whereby the *CHORD* mutant neuron produced the miniature arbor during pupal stages. First we found that the mutant arbor underwent a persistent increase in complexity and size, but the overall appearance had already become miniature-like before eclosion ([Supplementary Figure S2A--S2H](#s1){ref-type="supplementary-material"}). Then we performed time-lapse recordings of the wild-type and mutant neurons at 70--75 hr after puparium formation (APF), when dendritic arbors were under active construction, and dissected the dendrite dynamics of elongation/retraction and branching.
We quantified frequencies of elongations and retractions of terminal branches (branches of Strahler order 1, see [Figure 1L](#f1){ref-type="fig"}) and found that these parameters were
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Letter:
Since the initial report of infection with the coronavirus disease 2019 (COVID-19) in December 2019, the number of confirmed COVI-19 cases has exceeded 1.6 million globally.[@bib1] Without a vaccine in sight and given the success of social distancing, we expect the outbreak to last longer than originally projected. Social distancing while at work remains a challenge of us all. Mid-March, we implemented separate call teams, so that each of us only comes into contact with 4 to 5 coworkers at maximum over the course of the pandemic. These changes are necessary to maintain the workforce required to continue to be able to provide mechanical thrombectomy (MT) for our patients.
Every stroke care system must prepare for the worst-case scenario involving a surge that completely overwhelms the capacity of that system to function. Over the last several weeks, we have been learning from our colleagues around the nation---first, news out of Seattle and then New York City, and now most recently from Detroit, with greater than 800 providers testing positive for COVID-19 at Henry Ford. With approximately 25% of health care providers being infected with the virus, we are faced with the challenge of providing the care our patients desperately require while ensuring the safety of our health care providers.[@bib2] We must be mindful that our selfless tendencies as dedicated frontline providers do not become our biggest vulnerability. This challenge requires that we adjust our mindset and also place at the forefront the safety of ourselves and our teams. We make up one of the smallest and most subspecialized units in our hospitals. As such, we are easily incapacitated with quarantine or illness; who will take care of our community then?
This safeguarding requires major changes to our pre-COVID-19 workflow, to which the current pandemic has added multiple layers of complexity. Engagement of multiple specialties, including emergency department (ED) physicians, stroke neurologists, anesthesiologists and neurointensivists, is required. Fears and anxieties are normal human responses to a pandemic, and each must be acknowledged and addressed, never dismissed. In the acute stroke setting, the stakes are greatest because every minute counts. Although it may seem that there is not enough time to get it right, we must remember that this is also no time to get it wrong. That could mean being placed on diversion and not having the capacity to continue to treat our patients with stroke.
It begins with not overly burdening an already-tenuous hospital system. In the current COVID-19 crisis, there is critical bed shortage at greater level of care hospitals. We must therefore reduce the number of "futile transfers" from community hospitals and ensure that only patients who are likely to receive MT are transferred to the thrombectomy-capable center. Turning down a transfer could be the difference between being able to accept the next. This will entail obtaining advanced imaging at spoke hospitals to confirm the presence of proximal large vessel occlusion (LVO) before transport. Patients ruled out for LVO can remain at the spoke for routine care, even those who receive intravenous thrombolysis, particularly those spokes that are primary stroke center certified.
Even for patients in whom an LVO is confirmed, the criteria for transfer to the thrombectomy capable center should be decided on by a multidisciplinary team. During a pandemic and in the context of bed shortages, plus the prospect of bringing in vulnerable patients into an environment in which they might have a greater likelihood of acquiring the virus, hard decisions must be made. Accept the hard truth that your center should be more stringent in your criteria for MT, particularly for patients who don\'t fall within the guideline recommendations. As a team, discuss how you will handle those falling outside the "trial criteria"---very elderly patients, patients with mild, yet disabling stroke symptoms, and patients with distal occlusions. Only patients with a high likelihood of receiving thrombectomy should be transferred.
Once the patient with LVO who is an MT candidate arrives to your ED, all teams must be in alignment. Pre-thrombectomy screening for COVID-19 is a major challenge; often these patients are unable to provide a history and collateral information is typically lacking. The Society of NeuroInterventional Surgery currently recommends that patients with unknown COVID-19 status should be screened for fever and respiratory symptoms.[@bib3] Given that approximately 35% of patients with COVID-19 are asymptomatic and the growing awareness that asymptomatic patients are able to transmit the virus, this screening might not be sufficient.[@bib4] It may therefore be prudent to consider is to obtain a computed tomography (CT) scan of the chest to evaluate for underlying infiltrates at the time of obtaining a CT angiogram and perfusion. Although extending the CT to capture the entire chest entails additional radiation, identifying a lung infiltrate suggestive of likely COVID-19 infection in an asymptomatic patient affords the health care team the ability to don the proper precautions before the procedure.
Another question that has sparked major debate is whether to intubate patients before MT. Some proponents of an "intubate-all" approach argue that it protects the small and highly specialized MT team from exposure. Although all valid points, this approach fails to consider the other facets and team members along the way. Thrombectomy in and of itself is a low-risk procedure for contracting the virus, whereas the intubation and extubation are by far the greatest-risk components and would incur additional risk to your ED and neuro-intensive care unit (ICU) colleagues, respectively. In addition, intubating an elderly patient adds morbidity, and the airway management would require negative-pressure rooms in both the ED and the neuro-ICU. These are resources expected to be either in very short supply or not available at all during peak surge.
The lowest-risk pathway to the system as a whole is to get the LVO patient through the thrombectomy awake and cooperative without requiring pre-procedure intubation. However, the greatest potential risk to both the anesthesia and MT teams is in the event the thrombectomy unexpectedly transforms into an aerosol-generating procedure. A patient coughing or vomiting mid-procedure, would entail the worse possible scenario not just by the greatest-risk exposure to the providers but also in the delay it would incur. Neuroangiography suites are positive-pressure rooms (exceptions would be hybrid angio/operating room suites), necessitating a clearing out of the room by personnel following an aerosol-generating procedure like an intubation. Thus, the "lowest-risk" pathway is not necessarily the safest.
Finding the balance of risk to patient versus provider and resource use (negative-pressure rooms and personal protective equipment \[PPE\]) is delicate, and the decision of which patient should undergo preprocedural intubation is not straightforward. The Society of NeuroInterventional Surgery recommends a lower threshold for intubation for patients with suspected COVID-19, however, without specific criteria for intubation.[@bib3] We suggest that those patients at risk for converting an otherwise non--aerosol-generating procedure (thrombectomy) to a greater-risk procedure due to airway compromise should be intubated before MT. These would include those with severe stroke symptoms, patients with receptive aphasia, any signs of respiratory distress, or vertebrobasilar occlusion should be intubated before MT. Intubation ideally takes place in a negative-pressure room in the ED. Patients should then be transferred to the ICU with the same ventilator so that a closed circuit can be maintained.
The emergence of a faster COVID-19 test that provide results within minutes will provide us with a more efficient and reliable way to rapidly triage patients with LVO and avoid unnecessary intubations. However, until that test becomes widely available, following vigilant screening and maximizing precautions will be of paramount importance to protect our health care providers.
Another consideration is modification to your angio-suites for team protection. At our center, we have designated one of the biplanar rooms a "COVID-19" room, in which patients with suspected or confirmed COVID-19 receive MT. The door to this room has been sealed off, making it impermeable to aerosolized particles to completely isolate it from the control room. We have rehearsed an elaborate protocol to deliver additional supplies and devices into that room should they be needed via the hallway, as well as donning/doffing of PPE for room entry/exit protocol. Due to staff and PPE shortages, only essential personnel should be allowed into the COVID room during the procedure. This means that only one nurse, one technologist, the interventionists, and anesthesiologist are the only staff member to be in the room. Once the procedure is finished, patients are transferred to the ICU, where they are extubated in a negative-pressure room.
Remarkable challenges lie ahead of us, but we remain optimistic knowing that our field is graced with tremendously devoted, talented, and innovative people. Our solutions may not always be perfect, but these are also imperfect times, and we may have to do the best with what we can. However, one certainty remains: on reflecting back on these unprecedented times, it will be known that we answered the call for our patients.
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Introduction {#Sec1}
============
Heart failure is an ever-growing disease with a growing number of patients in end-stage disease, requiring heart transplantation. However, there is a shortage of organs available to replace failing hearts in these patients. Organs from other species, such as swine, have been proposed to meet the demand in these situations as they are anatomically similar to human hearts, genetically manipulatable, have short breeding cycles and are readily available. However, the arduous immunologic barriers between cross-species transplantation has limited its immediate use.
Heterotopic cardiac transplantation in the intra-abdominal position in a large animal model has been essential in the progression of the field of cardiac transplantation. Our group has over 10 years of experience in cardiac xenotransplantation with pig to baboon models, the longest xenograft of which survived over 900 days, with rejection only after reducing immunosuppression^[@CR1]^. This abdominal model facilitates immunologic monitoring through the period from implantation until graft rejection at reduced cost and complexity compared to orthotopic (life-supporting) models, with the additional advantage that, since the native heart remains in place, rejection of the heterotopic xenograft does not result in primary hemodynamic compromise and/or death.
Here, we demonstrate the approach to implantation of a cardiac graft into the intra-abdominal position in a baboon recipient for the study of transplantation and briefly highlight our model's ability to provide insight into not only xenotransplantation but across disciplines. We include details that have provided us with consistent success in this model; performance of the anastomoses, de-airing of the graft, implantation of a long-term telemetry device for invasive graft monitoring, and ideal geometric positioning of the heart and telemetry device in the limited space of the recipient abdomen. We additionally detail surveillance techniques to assess long-term graft function.
This heterotopic model, namely that it provides a readily reproducible method for long-term and whole-organ cardiac perfusion without compromising the recipient, should be seen as a standard model for testing iterative improvements in immunosuppression regimens and xenograft genetic manipulations for the further enhancements of cardiac xenotransplantation and allotransplantation at large. While we describe this in the context of transplantation from our extensive experience using this model, considerations are otherwise similar in any other large animal model. As this model uniquely provides *in vivo* assessment of whole organ function without compromising host physiology, it can be used for assessing cardiac physiology across disciplines, where other models have failed or are limited.
Materials {#Sec2}
=========
Specific pathogen-free (SPF) baboons of either sex weighing 15--30 kg (2--3 years of age) from Oklahoma University of Health Sciences (Norman, OK) were housed in a clean pathogen-free facility and were used as recipients. 6 to 8 week-old genetically modified swine of either sex, with an established genetic backbone known to produce prolonged xenograft survival, alpha 1--3 galactosyltransferase gene knockout (GTKO) and overexpression of human CD46 (hCD46) and thrombomodulin (hTBM), GTKO.hCD46.hTBM, were used as donors (Revivicor Inc., Blacksburg, VA) as our standard donor^[@CR1]^. However, we have also demonstrated success in pigs that additionally express human transgenes for thromboregulation (endothelial protein C receptor, tissue factor pathway inhibitor), complement inhibition (decay accelerating factor), and cellular immune suppression (hCD39, hCD47). SPF baboons were selected for low non-gal antibody titers as previously published^[@CR2]^. Critical materials are listed in Table [1](#Tab1){ref-type="table"} and the immunosuppression regimen has been previously described^[@CR1],[@CR3]--[@CR5]^.Table 1Additional information on critical materials for heterotopic cardiac transplantation: while these are suggestions based on materials we have used, there are likely other suitable alternatives.Heterotopic Cardiac Xenotransplantation-Important MaterialsNameManufactererReference \#9 Fr Cardioplegia Aortic Root CannulaMedtronic (USA)20012Bladder Irrigation Tubing for CardioplegiaBaxter (USA)2C4041Extension IV Tubing Seticumedical (USA)12656-28PlegisolHospira Inc. (USA)409796905Data Sciences International (DSI) Telemetry Device L21DSI (USA)DSI L2110 Fr Hickmann Tunneled Triple Lumen CatheterBARD (USA)606560
Methods {#Sec3}
=======
All procedures described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland School of Medicine. All methods were carried out in accordance with relevant guidelines and regulations.
Heterotopic cardiac transplantation technique {#Sec4}
---------------------------------------------
The intra-abdominal heterotopic model (HHTx) is a two-anastomosis system utilizing arterial supply from the infrarenal aorta of the recipient baboon to perfuse the coronaries of the donor heart with drainage of the cardiac graft through the donor pulmonary artery remnant anastomosed to the intra-abdominal inferior vena cava of the baboon recipient (Fig. [1](#Fig1){ref-type="fig"}). A list of critical materials is provided in Table [1](#Tab1){ref-type="table"}. A brief description is provided followed by step-by-step instructions for performing HHTx. A supplemental video is also provided (supplementary video [1](#MOESM1){ref-type="media"}).Figure 1Intraabdominal placement of a cardiac xenograft with pressure telemetry monitor in the apex. IVC-inferior vena cava, Ao-Aorta, PA-pulmonary artery, EKG-electrocardiogram leads from telemetry device. Of note, whereas the telemetry device depicted here only has one pressure sensor and it is placed in the left ventricle at the apex, the pressure can be placed in the right atrium, pulmonary artery or aorta as well, depending on which hemodynamic parameters of interest are to be studied. Image Copyright: Tim Phelps JHU/AAMM, 2020.
Briefly, the cardiac donor is prepped and draped sterilely, and a midline sternotomy is performed. Pericardium is opened and major vessels are isolated. Silk ties are placed around both superior (SVC) and inferior (IVC) vena cavae. Cold blood cardioplegia is administered through a 9 Fr aortic root canula after ligating the SVC and applying a vascular cross clamp on the aorta. The heart is decompressed by venting the IVC and left atrium or pulmonary vein and cardiectomy is performed. The heart is placed on ice during backtable preparation. The IVC is ligated and pulmonary vein common channel is created and over sewn (Fig. [2](#Fig2){ref-type="fig"}). The cardiac graft is now readily for transplantation into the abdomen.Figure 2Backtable preparation of the heart for transplantation. Ao-ascending aorta, PA-pulmonary artery, Pvv-Pulmonary vein common channel, Cava-superior and inferior vena caval junction. Image Copyright: Tim Phelps JHU/AAMM, 2020.
Retroperitoneal exposure of the infrarenal abdominal aorta and IVC of the recipient is performed and proximal and distal control of these vessels are obtained. Aortotomy and cavotomies are placed approximately 2 centimeters (cm) distal to the renal vessel takeoffs. The aorta-aorta anastomosis is carried out before the pulmonic-caval anastomosis (Fig. [3](#Fig3){ref-type="fig"}).Figure 3Aortic and Pulmonic Anastomosis to the Recipient. Abdomen. Image Copyright: Tim Phelps JHU/AAMM, 2020. Aortic and Pulmonic Anastomosis to the Recipient. Abdomen. Image Copyright: Tim Phelps JHU/AAMM, 2020.
Step-by-step performance of HHTx transplantation is as follows, with clinical pearls based on observation and experience denoted at relevant points of the procedure. All procedures described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of our residing institution. Here we describe these steps in the context of pig-to-baboon xenotransplantation, however, this can be conducted similarly in baboon heterotopic allotransplantation.
### Procurement of the Cardiac graft {#Sec5}
#### Preparation of the cardiac donor {#Sec6}
The donor is sedated with 10 milligrams per kilogram (mg/kg) of Ketamine intramuscular (I.M.) and 2 mg/kg xylazine for transfer to the operating room.
Clinical pearl*In swine, xylazine is used as an adjunct to ketamine, as many are somewhat refractory to the sedative effects of ketamine. While xylazine is known to cause second degree heart block and hypotension requiring atropine in some cases, this has not been our experience, although the true incidence is not known. Alternatively Ketamine and 0.12 mg/kg IV of Dexmedetomidine or Telazole 1 mg/kg can be used as an adjunct to ketamine*^[@CR1]^.
A peripheral intravenous catheter is placed for medication administration. A 24 gauge angiocatheter in an ear or forelimb vein is preferred. Alternatively, a percutaneous femoral venous cannula can be placed if peripheral IV access is difficult to obtain. Additionally, a femoral arterial line is placed in either groin after appropriate sterility is obtained.
In
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