text
stringlengths 1
18.8k
| meta
dict |
|---|---|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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)
|
{
"pile_set_name": "PubMed Central"
}
|
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)).
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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*.
|
{
"pile_set_name": "PubMed Central"
}
|
"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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
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
|
{
"pile_set_name": "PubMed Central"
}
|
End of preview. Expand
in Data Studio
README.md exists but content is empty.
- Downloads last month
- 1