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Introduction {#sec1}
============
Cutaneous leishmaniasis (CL) is the disease caused by *Leishmania* spp., an intracellular parasite. More than 70% of all CL cases are seen in 10 countries worldwide, which are Afghanistan, Algeria, Brazil, Colombia, Costa Rica, Ethiopia, the Islamic Republic of Iran, Peru, Sudan, and the Syrian Arab Republic \[[@cit0001]\]. It was reported that the causative agents of the disease are *L. tropica* and *L. major* in Syria \[[@cit0002], [@cit0003]\].
Turkey is one of the countries where CL is also prevalent \[[@cit0001]\]. Studies conducted in Turkey have shown that the predominant causative agent of CL is *L. tropica* \[[@cit0004]\]. Cutaneous leishmaniasis is endemic in Turkey, especially in the Southeast and the Mediterranean region \[[@cit0005]\]. However, in the last 5--6 years, many Syrian people have immigrated to Turkey due to the civil war in Syria. As a result, the incidence of the disease has increased considerably, especially in the areas of intense emigration \[[@cit0006], [@cit0007]\]. Hatay province of Turkey, located in the Mediterranean region, is one of the cities most affected by the migration due to its border with Syria.
Cutaneous leishmaniasis is more common especially in the childhood and young age group (0--20 years of age) \[[@cit0008], [@cit0009]\]. Although the studies reported from Turkey previously described the clinical and demographic data of the disease, a relatively small number of studies have focused on this age group in detail \[[@cit0010]\].
Aim {#sec2}
===
The aim of this retrospective study was to evaluate the clinical and demographic data of Syrian immigrant and Turkish children diagnosed with CL, who were consulted in our Dermatology Outpatient Clinic, and to compare Syrian patients (SP) and Turkish patients (TP) with respect to age, gender, disease duration, and localization, number and type of lesions. So far, this is the first study in which pediatric CL patients from the Hatay region have been examined in detail.
Material and methods {#sec3}
====================
In the present study, we included CL patients aged 0-18 years and recorded their clinical and demographic data. The patients were admitted to our outpatient clinic in the period 2015--2017 and in the first half of 2018. A total of 121 patients (55 (63.2%) male, 32 (36.8%) female SP; 19 (55.88%) male and 15 (44.1%) female TP) were included in the study. The diagnosis of CL was made by showing parasites in the smears taken from the lesions. Demographic and clinical data of patients were recorded from patient files. Statistical analysis was performed by dividing the patients into 0--6, 7--12, and 13--18 age groups.
Statistical analysis {#sec3.1}
--------------------
Data were analyzed using SPSS for Windows (version 21; IBM Corp, Armonk, New York, USA), χ^2^, Kruskal-Wallis, Mann-Whitney *U*, and Yates's continuity correction tests were used for statistical analysis. Values were presented as the mean ± standard deviation and *p* \< 0.05 was considered as significant.
Results {#sec4}
=======
Patients' ages ranged from 1 to 18 years (mean: 9.63 ±5.2). The mean ages of TP and SP were 12.06 ±4.47 (3--18 years) and 8.68 ±5.18 (1--18 years) respectively. Fifty percent of the TP were in the 13-18 age group, and 39% of the SP were in the 7--12 age group ([Table 1](#t0001){ref-type="table"}). There was no relationship between age groups and gender in all patients (*p* = 0.629). The SP and TP also did not show any relationship regarding gender and age group within themselves (SP *p* = 0.644, TP *p* = 0.460).
######
Distribution of TP and SP by age group and gender
Age TP SP Total
-------- ---- ---- ------ ---- ------- ----- -----------
0--6 3 2 14.7 19 14 38 38 (31.4)
7--12 5 7 35.3 22 12 39 46 (38)
13--18 11 6 50 14 6 23 37 (30.6)
Total 19 15 100 55 32 100 121 (100)
The average disease duration was 5.15 ±3.57 months (range: 1 week--12 months) (definition of "disease duration": the time period between the appearance of the first lesion and the arrival of the patient at the hospital for examination). There was no significant difference between the age groups regarding disease duration (*p* = 0.943). The disease durations in SP and TP were 1 week-12 months (mean: 4.73 ±3.39 months) and 1--12 months (mean: 6.25 ±3.86 months) respectively (*p* = 0.049). In the same analysis, we did not include the disease durations of 6 patients because when the outlier analysis was performed, the disease duration was deflecting. The disease durations of these patients were in the range 30--60 months and 4 of them were SP while 2 of them were TP.
The total number of lesions was 247 (*n* = 42 in TP, *n* = 205 in SP), and the mean number of lesions per patient was 2.04 ±2.01. There was one lesion in 61.2% of the patients and 2 lesions in 19.8% of the patients. When TP and SP were analyzed separately, 82.4% (*n* = 28) of the TP had one lesion, 11.8% (*n* = 4) had 2 lesions, and 5.9% (*n* = 2) had 3 lesions, while 52.9% (*n* = 46) of SP had one lesion, 23% (*n* = 20) had 2 lesions, and 23.9% (*n* = 21) had ≥ 3 lesions. Although there was no statistically significant difference between the age groups in terms of number of lesions (*p* = 0.207, Kruskal-Wallis test), the mean number of lesions was the highest in the 7--12 age group (2.54 ±2.43). When TP and SP were evaluated within themselves, mean lesion number was 2.35 ±2.28 (1--11 lesions) in SP, and 1.23 ±0.55 (1--3 lesions) in TP. Between two groups, the number of lesions per patient was significantly higher in SP (*p* = 0.002) compared to TP and two and multiple lesions were significantly higher in SP (*p* = 0.005). Regarding age group, when TP and SP were evaluated separately, the number of lesions in SP was the highest in the 7--12 age group (mean: 3.029, *p* = 0.049). This was followed by the 13--18 age group. There was no significant difference with respect to number of lesions in all three age groups of TP (*p* = 0.653; Bonferroni corrected Mann-Whitney *U* test) ([Table 2](#t0002){ref-type="table"}).
######
Distribution of number of lesions according to age groups
Group Age groups Lesion number Mean ± SD *p*~1~ *p*~2~
--------------- ------------ --------------- ----------- -------- --------
SP 0.049 0.002
0--6 50 1.51 ±0.795
7--12 103 3.02 ±2.657
13--18 52 2.60 ±2.854
Total 205 2.35 ±2.282
TP 0.653
0--6 7 1.40 ±0.894
7--12 14 1.16 ±0.577
13--18 21 1.23 ±0.437
Total 42 1.23 ±0.553
Overall total 247 2.04 ±2.018
p1 -- number of lesions according to age groups in Syrian and Turkish patients, p2 -- evaluation of lesion numbers of Syrian and Turkish patients.
It was found that the lesions were most frequently located in the head/neck (HN) region (*n* = 93, 76.9%) in the patients. 44.1% (*n* = 41) of the patients with HN localization were in the 7--12 age group. This was followed by the age groups 0--6 (32.3%, *n* = 30) and 13--18 (23.7%, *n* = 22). When the age groups of SP and TP were evaluated within themselves, HN localization was most frequently observed in the 7--12 age group in both groups (SP; *n* = 29, 42.6%; TP; *n* = 12, 48%). This was followed by the 0-6 age group in SP, and the 13--18 age group in TP. When the SP and TP were compared in terms of location of the lesions, it was found that the localization of HN was more frequent in TP than in SP (73.5% and 59.8% respectively, | {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
With the increasing affordability of personal genome testing (PGT), the incorporation of patient genotype data into the practice of medicine is becoming more pervasive. Multiple medical centers across the country have begun introducing genetic and genome-wide analysis to make pharmacogenetic testing available to patients [@pone.0068853-Johnson1], [@pone.0068853-Pulley1]. PGT companies offering direct-to-consumer (DTC) tests have empowered individuals to independently obtain their personal genomic profiles, which provide them with a view of their genetic risks for hundreds of diseases and atypical drug responses. Further, applications of genetic testing are expanding to pre-conception genetic screening [@pone.0068853-Srinivasan1], selection of embryos for *in vitro* fertilization [@pone.0068853-Johnson2], non-invasive screening for fetal chromosomal abnormalities [@pone.0068853-Fan1], and the diagnosis of complex medical conditions [@pone.0068853-Worthey1].
Despite this expansion, most medical schools have not kept pace in providing state-of-the-art education in genetics and genomics to medical trainees [@pone.0068853-Salari1]. Healthcare authorities and medical educators now agree that there is a strong need to train medical students and physicians to understand basic principles of genomics and to be able to interpret PGT results [@pone.0068853-Guttmacher1], [@pone.0068853-Wiener1]; however, there has been significant debate over the best educational models to deploy [@pone.0068853-Salari2], [@pone.0068853-Walt1]. Several institutions, including ours, have considered offering students the opportunity to undergo PGT themselves as part of an updated medical school genetics curriculum, with some institutions ultimately deciding against it [@pone.0068853-Walt1]. At Stanford School of Medicine, after a school-wide task force rigorously evaluated potential risks and benefits, PGT was offered to students as part of a first-of-its-kind medical school elective course on genomics and personalized medicine, where students learn principles of genetics and genomics through a combination of interactive lectures and hands-on analysis of genomic data, using either their personal genotype data or publicly available datasets [@pone.0068853-Salari2].
Given the novelty of this educational initiative, there was no data on how PGT impacts student learning and whether its use in the classroom enhances education. Therefore, we used a survey instrument administered before and after the course to examine associations between the use of PGT and student knowledge and attitudes about genomics. Based on previous evidence of the benefit of participatory learning in medical education [@pone.0068853-Genzen1], [@pone.0068853-Knoell1], [@pone.0068853-Mazmanian1], we hypothesized that the use of personal genome data in the classroom would improve knowledge and the learning experience for students.
Materials and Methods {#s2}
=====================
Subjects {#s2a}
--------
Subjects were medical and graduate students enrolled in an elective 8-week course on genomics and personalized medicine (Genetics 210; <http://gene210.stanford.edu/>) offered in the Summer 2010 quarter at Stanford School of Medicine. Forty-six students were enrolled in the course, and participation in this study was voluntary and anonymous.
Genotyping {#s2b}
----------
The course started with two weeks of instruction and class discussion led by a clinical geneticist (L.H.), a genetic counselor (K.E.O.), and a bioethicist/lawyer about the risks, benefits, uses, and limitations of PGT; these sessions provided the students with necessary background to provide informed consent should they proceed with PGT. At the end of the second week of instruction, students decided whether to personally undergo genotyping using the PGT services of either one of two PGT companies (23andMe or Navigenics). Of note, at the time of the course offering, 23andMe provided customers with their genotypes for all ∼600 K SNPs on their microarray while Navigenics provided genotypes for only the ∼300 SNPs used in their clinical reports. The subsequent six weeks of instruction included lectures and hands-on data analysis exercises on various topics related to human genetics, genomics, and personalized medicine (see course website for more details of the curriculum; <http://gene210.stanford.edu/>).
Each week students were led through classroom exercises to analyze various aspects of whole-genome single nucleotide polymorphism (SNP) data. A dataset comprising 12 diverse individuals from the HapMap project genotyped on Illumina HumanHap 650 K SNP microarrays were provided to all students. Students who underwent PGT were able to complete data analysis exercises using their personal genotype data, and students who did not undergo testing used publicly available genotype data from the 12 HapMap patients. A number of safeguards were implemented to ensure student privacy, confidentiality, and safety, including the provisioning of free genetic and medical counseling and mechanisms that students using their own data could ask questions if they had difficulty in resolving the class exercises without disclosing their genotype results [@pone.0068853-Salari2].
Survey Instrument {#s2c}
-----------------
At the start and conclusion of the course, we electronically administered a survey that assessed student attitudes and knowledge about genomics and personalized medicine. The survey (extending the questionnaire developed by Ormond *et al*. [@pone.0068853-Ormond1]) included basic demographic information; assessed attitudes and knowledge about PGT; and solicited students\' feedback on the experience of undergoing testing as it related to the class and their learning experience (**[Methods S1](#pone.0068853.s003){ref-type="supplementary-material"}**). Student attitudes were assessed either via yes/no questions or by asking for extent of agreement with statements on a 5-point Likert scale. Knowledge was assessed by both subjective and objective questionnaire items. For objective knowledge assessment, 6 multiple-choice questions and one free response question were asked; responses were scored blinded to subjects\' genotyping status. Separate from the surveys presented in this study, students were also invited to participate in individual interviews discussing their experience (presented separately [@pone.0068853-Vernez1]). The Stanford University Institutional Review Board approved all study methodology.
Data Analysis {#s2d}
-------------
We analyzed responses from students who completed both pre- and post-course surveys and attended at least 50% of the eight class sessions. Student responses to the pre- and post-course surveys were linked using a randomly assigned numeric code to maintain anonymity. We considered separating the students who underwent PGT before the course from those who underwent it during the course, but since preliminary statistical comparisons were underpowered to show differences, we elected to combine these groups in our study analysis. Student attitudes assessed on a 5-point Likert scale were collapsed and reported as the percentage of students who agree or strongly agree with the stem statement.
Paired pre-course and post-course responses were tested for change using paired non-parametric statistics (McNemar\'s test for binary response questions and the Wilcoxon signed-rank test for Likert items). Comparisons between responses of genotyped and non-genotyped students were made using Fisher\'s exact test for binary response questions and the Mann-Whitney *U*-test for Likert items. The change in student knowledge assessed by pre-course and post-course knowledge scores was evaluated by paired *t*-test. The difference in knowledge improvement between genotyped and non-genotyped students was assessed by Student\'s *t*-test.
Results {#s3}
=======
Forty-three class participants completed the pre-course survey (93% response rate) and 34 class participants completed the post-course survey (74% response rate). We present data from 31 students who completed both pre- and post-course surveys, and attended at least 50% of the class sessions (67% of the course enrollees). Demographics from this study population are presented in [**Table 1**](#pone-0068853-t001){ref-type="table"}; subjects were evenly split between genders, with slightly fewer medical versus non-medical trainees, and most frequently in their first year of training. Thirteen of the 17 students (76%) who indicated on the pre-course survey that they planned to undergo PGT did ultimately undergo testing. Seven students were initially unsure, of which 3 proceeded with testing. Another seven students had already undergone PGT prior to the course (all by 23andMe); these students indicated that they did not plan to undergo testing again and used their previously obtained data in the course. Due in part to a more limited genotype dataset provided by Navigenics, all 16 students who underwent PGT in the course did so via 23andMe. Thus, 23 students formed the genotyped group, and 8 students formed the non-genotyped group. There were no significant differences in demographics between the genotyped and non-genotyped groups ([**Table 1**](#pone-0068853-t001){ref-type="table"}) or between class participants who completed the study and those who were lost to follow-up (**[Table S1](#pone.0068853.s001){ref-type="supplementary-material"}**).
10.1371/journal.pone.0068853.t001
###### Subject characteristics.
{#pone-0068853-t001-1}
Genotyped[a](#nt101){ref-type="table-fn"} Non-genotyped[a](#nt101){ref-type="table-fn"}
| {
"pile_set_name": "PubMed Central"
} |
There are several errors in Table 2. Please refer to the corrected version of Table 2 here:
{#pone-a8dfd4ee-f4b7-443e-bf78-ebb0dab4e55b-g001}
**Competing Interests:**No competing interests declared.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
The role of growth hormone (GH) in growth promotion is well known by clinicians, however, less appreciated is its effect on metabolism in the well state. In 1936, Bernardo Houssay, M.D. in the New England Journal of Medicine, proposed that the anterior pituitary gland after the liver and pancreas plays a key role in glucose metabolism. That key role was later shown to be due in part to GH \[[@B1]\].
The effect of GH on glucose metabolism involves two phases: an initial phase which involves a decrease in glucose (an insulin-like effect) and a second phase which includes its effects on gluconeogenesis and fat mobilization \[[@B2]\]. In both states of growth hormone deficiency and excess, these effects on glucose metabolism will be altered.
Altered glucose and fat metabolism are important components of fatty liver (FL) and type 2 diabetes (T2DM), both states of hepatic insulin resistance (IR). In fact, a seminal case of fatty liver (FL) resolution with GH administration in a 17-year-old patient with panhypopituitarism treated initially with levothyroxine and hydrocortisone alone was reported by*Takano*et al. in 1997 \[[@B3]\]. This suggests that GH treatment of T2DM which may be associated with GHD has the potential to reduce the IR which may be present.
We outline a case of untreated childhood onset growth hormone deficiency (CO-GHD) who presented with type 2 diabetes mellitus (T2DM) and also steatohepatitis. We discuss her management and evidence from the basic sciences and clinical studies which show that her presentation with T2DM and steatohepatitis was likely associated with untreated GHD and that with GH supplementation her condition was ameliorated. Lastly, we discuss the implications of this case.
2. Case Presentation {#sec2}
====================
A 17-year-old nonobese Caucasian female who had a history of a medulloblastoma diagnosed at 7 years of age was treated with radiation therapy. She subsequently developed TSH and GnRH deficiencies. Though GHD was suspected based on height (z-score of -- 3.1; see [Figure 1](#fig1){ref-type="fig"}), treatment had not been initiated based on the initial management focus being to treat her medulloblastoma. At 15 years of age when her bone age showed full skeletal maturity, her parents were informed that GH therapy could not be pursued because her linear growth was complete.
On presentation, the patient\'s height was 141.3 cm (z= -3.1) and weight was 53 kgs (36^th^ percentile for age). Body mass index was 25.8 kg/m^−2^ (86^th^ percentile for age). Surveillance labs done at the oncology clinic showed glucosuria. Further testing showed HbA1c of 9.6% and on another day her fasting glucose was 277 mg/dL. Based on these results, diabetes mellitus was diagnosed.
When glutamic acid decarboxylase (GAD-65; Esoterix), islet-cell (Esoterix), insulin (Esoterix), and zinc transporter 8 (ARUP Laboratories) antibodies as well as DNA panel for maturity onset diabetes of youth (MODY) genes (HNF4*α*, GCK, IPF1, HNF1*α*, and HNF1*β*, \[Athena Diagnostics\]) returned all negative along with an elevated fasting C-peptide level of 3 ng/mL (normal: 0.4 - 2.1), T2DM was diagnosed. With the initiation of traditional basal/bolus insulin therapy using conventional dosing, a rapid escalation to peak total daily insulin dose of 2.9 units/kg/day (\~ 155 units/day) was required to treat her refractory hyperglycemia. Treatment nonadherence was thought to be the unlikely cause of her increased insulin requirements based on the agreement between her insulin dosing and prescription refill data.
A comprehensive evaluation for conditions associated with IR was negative. However, based on Arginine/Clonidine stimulation testing showing peak GH level of 0.8 (normal: ≥ 10 ng/mL), a diagnosis of GHD was made. GH supplementation was initiated at 0.3 mgs daily and titrated based on IGF-1 levels.
After GH was started, her systolic and diastolic blood pressures (BP) which were mildly elevated between 124-136 and 77-89, respectively, became more normal. Despite this, lisinopril 5 mgs once daily was added for microalbuminuria.
With the diagnosis of T2DM and our patient having a significant family history of adverse cardiovascular risk factors, she was started on atorvastatin 10 mgs once daily. Within 2 months of therapy, her LDL cholesterol (LDL-C) decreased to 74 mg/dL. [Table 1](#tab1){ref-type="table"} shows serial lipid profiles.
Though her diabetes was not fully reversed with GH, her HbA1c decreased to 5.9% and 5.8% at 6 and 19 months, respectively. Her insulin therapy requirement decreased to 1.9 units/kg/day (\~ 100 units) at 12 months after the start of GH.
Magnetic Resonance Imaging (MRI) of the brain and abdomen indicated a small anterior pituitary gland and liver masses, respectively. Liver biopsy showed steatohepatitis with bridging fibrosis ([Figure 2](#fig2){ref-type="fig"}). With GH therapy, her liver transaminases trended to normalcy ([Table 1](#tab1){ref-type="table"}). Repeat MRI abdomen at 20 months after the start of GH showed stability of the liver lesions when compared to that done at 14 months. These hyperintense lesions like the initial ones were located in the liver\'s parenchyma and the appearance of the liver was otherwise normal.
With GH therapy, the patient\'s stamina improved. She was now able to work for 20 hours weekly without becoming fully exhausted and her Quality of Life-Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA) and Quality of Life Satisfaction (QLS) scores, both questionnaire-based, improved ([Figure 3](#fig3){ref-type="fig"}).
3. Discussion {#sec3}
=============
This case demonstrates that GH supplementation in an adolescent with CO-GHD led to improvements in transaminases, insulin requirements, and glucose control.
Several mouse models have corroborated the association of GHD with IR. In mice with liver GH receptor (GH-R) knockout, metabolic syndrome (MetS), steatohepatitis, increased inflammation, liver fibrosis, and hepatic tumor develop \[[@B4]\]. Additionally, a similar mouse model resulted in hyperinsulinemia, hyperglycemia, and IR. With the restoration of the liver\'s IGF-1 expression, there was an improvement in both insulin sensitivity and serum lipid profile. This, however, did not protect against hepatic inflammation induced by steatosis. This shows that GH and not IGF-1 directly affects lipid uptake and lipogenesis \[[@B5]\]. Also in a prior study again involving a liver specific GH receptor knockout mice, de novo lipogenesis was increased; however, this increase was not associated with the classic insulin mediated pathway \[[@B6]\]. So, our patient\'s IR and hepatic steatosis can be explained by her GHD based on data from some studies as well as the effect of GH in inhibiting this.
Evidence for the association of hepatic steatosis with GHD is also provided by the abnormalities involving the downstream pathway of GH signal transduction. Mice with hepatocyte-specific deletion of Janus kinase 2 (JAK2L), involved in the postreceptor phase of GH signaling, were lean but had FL. They also showed increased levels of GH, triglycerides, and plasma free fatty acids. Since GH in some instances can cause lipolysis, GH-deficient*little*mice which were crossed to JAK2L mice had both a rescuing of the FL and an increased expression of a fatty acid transporter \[[@B7]\]. Though this provides a mechanism for the FL observed with the liver specific disruption of GH signaling, in the same mice, elevated GH levels occurring as a consequence of disrupted signaling can cause an increase in resting energy expenditure \[[@B8]\]. In this situation, steatohepatitis is prevented based on increased fatty acid utilization. So the putative lipolytic action of GH can be offset by hyperinsulinemia; hence, the action of GH is variable and depends on the physiological context.
Additionally, mice with signal transducer and activator of transcription (STAT) 5 mutations, another downstream signal from GH, also develop steatohepatitis \[[@B9]\]. Since pathologies involving the downstream pathways in GH signal transduction are associated with steatohepatitis, this supports the role of GH in lipid metabolism and the notion that the FL changes in our patient may be due to GHD.
Clinical reports about Laron syndrome, primary GH insensitivity involving a molecular defect in human GH-R, have also documented the development of FL, IR, and T2DM \[[@B10]\]. In addition, men with hypopituitarism have a high prevalence of nonalcoholic FL disease (NAFLD) in the absence of GH therapy. When these men were treated with GH, there was histological improvement in the liver. This demonstrates that NAFLD is predominantly attributable to GH \[[@B11]\].
Though a follow-up liver biopsy was not done, the trend to normal in our patient\'s transaminases ([Table 1](#tab1){ref-type="table"}) supports the effect of GH on the liver and the likelihood that these changes were induced by GHD. Since our patient\'s steatohepatitis improved with GH treatment | {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1-1}
============
Liver cirrhosis has an increasing incidence in the general population. It is estimated that, in Europe, there are 170,000 annual deaths due to this cause with major differences between regions. Thus, in the south-east cirrhosis mortality rate is 10-20 times higher than the rest of the continent \[**[@R1]**\].
Romania is situated on the second place with figures of 64 males and 26,7 females considering the death rate per 100,000 population. The main causes of the disease are represented by chronic alcohol consumption, infections with hepatitis viruses B and C, and obesity related metabolic syndromes \[**[@R2]**\].
In these circumstances, abdominal surgery in patients with liver cirrhosis has an increasing frequency.
Liver cirrhosis is a chronic and slowly progressive disease characterized by a replacement of normal liver tissue with scars. Liver function is progressively altered leading to hepatic failure.
The effects of cirrhosis are not limited to liver function, but also affect other organs and systems. The renal, cardiac and respiratory functions are also adversely affected. These, along with coagulation disorders and immune deficiency create premises of a high-risk surgical ground \[**[@R3]**\].
Surgery for "acute abdomen" in such patients has a high mortality rate and requires special measures of resuscitation \[**[@R4]**\].
Copeland and al. proposed, in 1991, The Physiologic and Operative Score for enUmeration of Mortality and Morbidity (POSSUM) as a scoring system for the surgical audit \[**[@R5]**\]. Considering both pre- and intra-operative commonly measured parameters, this score is easy to use and has a wide application for general surgery in emergency and elective procedures \[**[@R6]**,**[@R7]**\].
The aim of the present study is to evaluate the accuracy of POSSUM score in predicting outcomes after emergency abdominal surgery in patients with liver cirrhosis.
Material and method {#sec1-2}
===================
Our study is based on a prospective analysis between January 2008 and December 2012 on 115 consecutive patients with liver cirrhosis hospitalized and operated for acute surgical abdomen in Surgery Clinic of "Sf. Pantelimon" Hospital in Bucharest. The criteria for the patients' inclusion in the group were the following:
\- Liver cirrhosis as a background disease
\- The presence of an abdominal surgical problem whose solution was imposed in the first 24 from admission as an emergency solution
In order to assess the determinant factors of postoperative morbidity and mortality in these patients we considered the following:
\- Evolutive stage of the liver disease
\- Physiological status of the patient at the moment of operation
\- Type and magnitude of the surgical procedure
For the staging of the liver cirrhosis we choose Child -- Pugh classification. This classification is an useful instrument in emergency conditions because of its small number of parameters which can be rapidly evaluated -- serum albumin level, total bilirubin, prothrombin time INR, the presence and grade of ascites and grade of encephalopathy \[**[@R8]**\].
This score has proven its usefulness in many studies involving patients with liver cirrhosis, whether it was the rupture of esophageal varices, hepatocellular carcinoma, Budd-Chiari syndrome or portal subclinical encephalopathy \[**[@R9]**-**[@R12]**\].
In a study conducted over a period of 12 years Mansour used Child -- Pugh score as a prognosis factor for non hepatic abdominal surgery \[**[@R13]**\].
Despite the criticism that brings into question that the score is based upon components empirically chosen or that its variables are not independent predictors, studies show that the data obtained with this score have statistical significance \[**[@R14]**\].
In light of the above, Child-Pugh score remains an instrument which is easy to use in the evaluation of the prognosis of the patient with liver cirrhosis and its predictive value is at least comparable to some elaborated scores such as MELD \[**[@R15]**\], elements which have imposed their use in our study.
The physiological status of patients and magnitude of surgery procedure were evaluated based on the POSSUM score.
This score quantifies twelve significant and independent physiological variables which evaluate the physiological status of the patient at the time of surgery. The patient\'s respiratory and cardiac function are assessed by using information that can be obtained quickly and easily by clinical and laboratory common methods - the presence and degree of dyspnea and its degree of severity, chest X-ray appearance, presence of arrhythmias on the electrocardiogram, blood pressure, pulse, the presence of angina or an underlying heart disease requiring inotropic therapy or anticoagulant. In addition to these are the laboratory tests such as hemoglobin, WBC count, blood urea nitrogen, sodium and potassium. Moreover, the patient\'s age and Glasgow score are also considered. The values obtained for the physiological score calculated at the time of intervention can have values between 12 and 88 (**[Table 1](#F1){ref-type="fig"}**).
{#F1}
Surgical trauma is evaluated based on six factors of the severity of procedure. This is classified into four categories taking into account the extent of surgery, emergency or elective nature of the operation, amount of blood lost during surgery, duration, peritoneal contamination and the presence / extension of a neoplasm (Table 2).
Once the score is known, it is possible to estimate the risk of morbidity (R2) and mortality (R1) by using two equations. For morbidity the equation is the following:
Log (R2 / (1-R2) = -5.91 + (O.16 X physiological score) + (0.19 X severity score of the surgical procedure)
For mortality the equation is the following:
Log (R1 / 1 R1) = -7.04 + (0.13 X physiological score) + (0.16 X severity score of the surgical procedure).
The physiological score was assessed at the time of surgery and the operative score at the moment of the patient's discharge.
Comparing the obtained results with those estimated by using POSSUM score, we tried an appreciation of the influence upon postoperative outcome of the physiological status and evolution stage of liver cirrhosis in these patients.
Statistical data analysis was based on X²(chi-square) test and the p value of less than 0,05 was considered statistically significant.
{#F2}
Results {#sec1-3}
=======
Our group was composed of 115 patients, 45 females (39%) and 70 males (61%) and their ages ranged from 29 to 85 years, with an average of 60.27.
The etiology of liver cirrhosis in patients represented in our group was especially chronic consumption of ethanol and hepatitis virus infections, groups including 102 cases - 60 and 42 patients, respectively. To these 8 cases of cardiac cirrhosis and other 5 in which the etiology could not be documented, being labeled as cryptogenic, were added.
The classification of patients into Child groups depending on the evolutionary stage of liver disease was the following: 33 of them (28.7%) were classified in group Child A, 54 (47%) in Child B stage and 28 (24.3%) in group Child C(**[Fig. 1](#F3){ref-type="fig"}**).
{#F3}
The preoperative assessment based on the physiological POSSUM score resulted in dividing the patients into the following groups score: below 15,5 cases; 23 cases with a score between 16 and 18, 28 cases with 19 to 21 points, 32 cases with 22 to 24 points, 18 cases with 25 to 27 points, 7 cases with 28 to 30 points, 2 cases over 30 points (**[Fig. 2](#F4){ref-type="fig"}**).
{#F4}
The pathological conditions in our group of 115 patients who required emergent surgery were the following: 22 cases of complicated umbilical hernias, 12 complicated postoperative incision hernias, 8 complicated inguinal hernia, 35 acute cholecystitis, 14 peptic ulcer bleeding, 6 perforated ulcer, 3 penetrating abdominal wounds, 4 acute appendicitis, 2 traumatic ruptures of spleen, 2 choledocholithiasis with cholangitis, 7 bowel obstructions.
Surgery procedures practiced in emergency conditions -- in the first 24 hours from admission -- on our group of patients were the following: splenectomies -- 2 cases, umbilical hernia repair -- 22 cases, inguinal hernia repair -- 8 cases, incision hernia repair -- 12 cases, suture of ulcer perforation -- 3 cases, excision of ulcer -- 2 cases, gastric resection -- 5 cases, gastrotomy with hemostasis \"in situ\" for bleeding ulcer -- 10 cases, resection of small intestine -- 3 cases, appendectomy -- 4 cases, right hemicolectomy -- one case, transverse colon segmental resection -- one case, Hartmann operations -- 2 cases, laparoscopic cholecystectomy -- 21 cases, open cholecystectomy -- 14 cases, laparotomy -- 3 cases, choledochotomy with Kehr drainage -- 2 cases.
Calculating POSSUM operator score for our patients resulted in a classification into the following groups: 31 patients with a POSSUM operator score below 10, 42 patients with a score between 11 and 13; 14 patients had a score of 14 to 16; 26 | {
"pile_set_name": "PubMed Central"
} |
{
"pile_set_name": "PubMed Central"
} | |
1. Introduction {#s0005}
===============
Severe myoclonic epilepsy of infancy (SMEI), also known as Dravet syndrome, is an epileptic encephalopathy that presents during the first year of life and is one of the most severe types of infant epilepsy that is resistant to drugs [@bb0005; @bb0010].
Patients with Dravet syndrome display multiple seizure types including tonic--clonic, myoclonic, absence, and focal seizures. In addition to epilepsy, SMEI is associated with cognitive delays and behavioral disorders [@bb0010].
De novo SCN1A mutations are a major cause of SMEI [@bb0015]. In addition, mutations in *SCN1B*, *SCN2A*, and *GABRG2* also cause SMEI, and recently, a Dravet-like phenotype in which *PCDH19* and *CHD2* genes are involved was described [@bb0020; @bb0025].
Pharmacoresistance is one of the major problems in SMEI, and many antiepileptic drugs do not seem to be able to control all the different kinds of seizures, including generalized tonic--clonic seizures (GTCS) and myoclonic, absence, and focal seizures, which characterize this syndrome.
Eslicarbazepine acetate (ESL) is a relatively new antiepileptic drug which has been approved in 2009 by the European Medicines Agency and in 2013 by the US Food and Drugs Administration as adjunctive therapy in adults with partial onset seizures with and without secondary generalization. Eslicarbazepine acetate shows an important action on the blockade of voltage-gated sodium channel (VGSC). It has been recently reported that there are some differences between AEDs acting on VGSC that are also related to their different affinities for the channel based on the functional state of VGSC. In fact, three distinctive states have been shown for VGSC which include the resting state, the open state, and the inactivated state. The affinity of ESL for the inactivated state of these channels is similar to other AEDs (carbamazepine), while its affinity for the resting state is three times lower than the other drugs, thus preventing sustained repetitive neural firing but disturbing physiological mechanisms to a lesser extent. Another peculiarity of ESL has been reported by an experimental study which showed that this drug could reduce VGSC availability through enhancement of slow inactivation, as reported for lacosamide, while it seems not able to alter fast inactivation of VGSC, unlike carbamazepine (CBZ) and oxcarbazepine (OXC) [@bb0030]. In addition, the eslicarbazepine metabolite effectively inhibited high- and low-affinity CaV3.2 inward currents with greater affinity than CBZ [@bb0035].
We report a short-term open study about two patients affected by SMEI who both showed favorable response with regard to seizure frequency reduction when treated with ESL and good tolerability.
2. Methods {#s0010}
==========
The first patient is a 17-year-old Italian boy with SMEI and a severe clinical outcome, in whom we identified a punctiform mutation (5053del AA fs1685X1691) in the α subunit of the neuronal sodium channel SCN1A gene. He started to have febrile and afebrile seizures at the age of 6 months. No drug treatment, including valproate, lamotrigine, carbamazepine, phenobarbital, vigabatrin, topiramate, clonazepam, ethosuximide, phenytoin, felbamate, nitrazepam, rufinamide, zonisamide, or lacosamide had proven effective ([Table 1](#t0005){ref-type="table"}). The best results in the reduction of seizure frequency was obtained with a combination of phenobarbital, clonazepam, and zonisamide, showing a reduction of about 50% of partial complex seizures during sleep and 70% of daytime GTCS. During this time, the EEGs and clinical and epileptic characteristics of this patient showed typical features of SMEI such as slowing of background EEG activity with multifocal and diffuse discharges prevalent in the frontal regions, myoclonic seizures at the age of 2 years and 6 months, ataxia, and progressive intellectual disability.
At the age of 16 years and 6 months, we added ESL in combination with phenobarbital, clonazepam, and zonisamide at the initial dosage of 400 mg once daily and after two weeks at 800 mg/day. After just a few weeks from the start of therapy, we observed a complete response with disappearance of monthly GTCS and sleep frontal-partial complex seizures (with 3--4 occurrences per night previously observed). We did not observe adverse events during the treatment period of six months with a significant improvement in behavior, particularly oppositional defiant disorder and the maintenance of attention.
The second patient is a 21-year-old Italian girl. At the age of 11, we had identified a punctiform mutation (931G \> T E311X) in the α subunit of the neuronal sodium channel SCN1A gene. The febrile and afebrile complex-partial seizures started at the age of 3 months.
Similar to the first patient, the drug treatment for this patient included acetazolamide and stiripentol that had not been proven effective ([Table 1](#t0005){ref-type="table"}). In particular, at the age of 8, a combination of phenobarbital, clonazepam, and acetazolamide (250 mg/day) caused a decrease in seizure frequency of about 40%. The number of seizures was previously much higher and associated with several eyelid myoclonic seizures at a frequency of hundreds of occurrences daily and several massive myoclonias from the age of 3 years. Magnetic resonance imaging showed moderate brain atrophy, which did not progress during maturation, and brain interictal SPECT at the age of 2 showed hypoperfusion of the left frontal cortex. The EEG showed diffuse and multifocal paroxysmal activity that was more evident in the bioccipital regions. In the first year of life, the child showed unimpaired neuromotor development and hypotonia. She presented progressive intellectual disability, which became profound, as well as severe ataxia, making it impossible for her to walk. In addition, her speech was absent.
At the age of 10, a VNS device was implanted without any results.
At the age of 20, we added eslicarbazepine acetate to phenytoin, clonazepam, and valproate at the initial dosage of 400 mg once daily, which was increased after two weeks to 800 mg/day. We have also observed in this case good response with regard to seizure frequency reduction in just a few weeks after starting therapy. The girl does not present any daily seizures, just one seizure every four days currently.
3. Results {#s0015}
==========
We observed a significant and fast effect using ESL as add-on therapy to other AEDs in both patients similar to what was reported in literature for adult patients with partial-onset seizures [@bb0040]. The peculiarity in our cases was the age when ESL therapy was started, 16 years and 6 months for the boy and 20 years for the girl, respectively, underlying the possibility of using ELS with good efficacy as an add-on therapy in young patients [@bb0045]. Of particular interest is ESL\'s efficacy in both patients with SMEI carrying SCN1A mutation because it is well known that seizure control is very difficult to achieve in this syndrome. The reason why ESL was effective in patients whose seizures are typically drug-resistant is not yet known; we could theorize that it could be due to the peculiar action of ESL on VGSC. Indeed, carbamazepine, which alters fast inactivation of VGSC, can worsen seizures and should not be used in patients with SMEI [@bb0045]. It is interesting to note that in our cases, ESL has been used in combination with different AEDs, although benzodiazepine was the only AED common in both cases. Thus, it does not seem plausible that the effectiveness of ESL in patients with SMEI is attributable to the association between different drugs, but by analyzing the drugs in particular, we can observe their similar mechanisms, especially those on GABAergic system and voltage-gated sodium channel. Another peculiarity of our report is the effective dose of ESL, which was a low recommended dosage. Moreover, we observed a fast response to the initial dose of ESL [@bb0050].
4. Conclusions {#s0020}
==============
Adjunctive eslicarbazepine led to seizure reduction in two patients with severe myoclonic epilepsy of infancy. Although this is a short-term open study, the results are promising but need confirmation.
Conflict of interest {#s0025}
====================
The authors report no conflicts of interest.
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited
######
Response and time of administration of AEDs in both patients.
AEDs Time of administration Response[a](#tf0005){ref-type="table-fn"}
----------------- ------------------------ ------------------------------------------- ---- ---
Valproate 2 years 18 years 1 | {
"pile_set_name": "PubMed Central"
} |
One and a half billion people live in conflict-affected and fragile states. At the last estimate in 2012, 172 million people were directly affected by war, including refugees, internally displaced people and those who were affected but did not flee.[@R1]^,^[@R2] Children are twice as likely to be malnourished and twice as likely to die by the age of five years in low-income countries affected by conflict compared with similar but stable countries. Their families are twice as likely to live without clean water.[@R3] Conflict does more than short-term damage; it decimates a country's infrastructure and impairs the social contract between the state and citizens. Food supplies are disrupted; health services collapse. Pregnant women and people who are ill do not receive the life-saving services they need.[@R4] Less often measured are the long-term consequences of conflict on people's mental health and social functioning.
People made vulnerable by conflict are being bypassed by global progress. The World Bank warned in 2011 that no low-income, conflict-affected country was on course to achieve any of the millennium development goals (MDGs).[@R3] Indeed, four years later, of 55 conflict-affected and fragile states, 37 (67%) had met only two or fewer of the 15 MDG targets.[@R5] The inequity is not simply about the differences between stable and unstable countries. Even within countries, conflict-affected areas fare worse than areas with less or no conflict. In the Democratic Republic of Congo, for example, under-five mortality in the conflict-affected South Kivu province is nearly double that of Kinshasa province.[@R6]
Despite this experience, the sustainable development goals (SDGs) for the year 2030 include barely more guidance on conflict than did the MDGs, which did not specifically mention conflict. SDG 16 explicitly recognizes the need to resolve conflict and mitigate its circumstances, but this intention does not translate into specific action points for other SDGs, such as SDG 3, which focuses on health. Unless we learn how to achieve the targets in conflict settings, the benefits of the SDGs will not reach many of the people who need them most.
The first question is whether the list of 17 SDGs and 169 targets should be adapted by each country. There is experience of simpler, more modest goals being associated with greater success. In Afghanistan, the government and its partners worked together to adapt the MDGs to meet their needs, setting more realistic interim targets and agreeing on objectives and approaches adapted to the country's unique realities.[@R7]
Methods of measurement need to be realistic too. Widely used mechanisms to monitor progress in health at a national scale, such as the demographic and health surveys and the multiple indicator cluster surveys, sometimes leave out whole conflict-affected areas and routinely exclude internally displaced persons and refugees.[@R8] There are alternatives, such as data collected by nongovernmental organizations that work in conflict zones. These data, which are collected to identify needs and monitor the progress of humanitarian interventions, are arguably underused for monitoring development goals.[@R8] Population data are also scarce among displaced and disrupted communities, although humanitarian organizations commonly conduct and update small-scale censuses of difficult-to-access areas. Using these sources, while not a solution to the problem, could be a first step towards better monitoring.
We also need to be more realistic about the level of investment needed to effect even modest changes. Conflict-affected countries often have greater needs both in terms of capital costs to rebuild destroyed infrastructure and of recurring costs to operate in environments with transport and security challenges. Yet conflict-affected countries have received less investment than others. In 2012, for example, the Central African Republic received one-fifth the per-capita direct assistance for health that Malawi did.[@R9] With less than 200 km of paved roads in South Sudan, a country larger than France, air transport is often the only option. Overall, levels of international humanitarian funding routinely face a large shortfall: in 2014, 7.2 out of 18 billion United States dollars of the United Nations (UN) coordinated appeal for humanitarian action worldwide went unfunded.[@R9]
Realistic goals, better measurement of progress and increased investment would help, but these will not be enough to make a difference without a fourth adaptation: the most difficult one. We need to change what we do and how we do it. Investment in scalable, cost-effective interventions, such as family planning, insecticide-impregnated bed nets and integrated community case management, have helped stable countries to make dramatic reductions in maternal and child mortality. Countries in crisis could and should benefit from similar public health interventions, but have often instead been served by short-term actions, such as provision of field hospitals and mobile clinics, which have higher costs, smaller scale and less potential for sustained impact. People already burdened by conflict receive aid that reaches fewer people, is more expensive and has a shorter impact than aid in non-conflict settings.
This does not mean implementing the same large-scale, long-term health programmes in fragile states as in stable low-income countries. Interventions should be effective and cost-effective and have wide coverage but they need to be adapted to the context of conflict. For example, integrated community case management for childhood diseases, traditionally considered a development intervention, has been adapted and scaled up in conflict-affected areas to increase access to care. In South Sudan, for example, the International Rescue Committee (IRC) supports over 2600 community health workers, many of them in the most intense areas of conflict such as Unity state. These community workers have continued to work even when conflict has shut down formal health structures. Tools were developed for illiterate community health workers, in a setting in which literacy rates are very low. Supply-chain adaptations, such as the use of boats and a network of transit villages to move materials, were developed to address problems of flooding and the lack of infrastructure.
Similarly, although family planning has nominally been included in the United Nations Population Fund (UNFPA) minimum initial service package for reproductive health, its family planning component has seldom been implemented in acute or chronic emergencies. In the last 10 years, a coalition of organizations with experience in reproductive health in conflict settings has demonstrated that modern and long-acting contraceptive methods can be provided at low cost and with high quality, and that there is demand for these, even in the most precarious and transient conditions.[@R10]^,^[@R11] As a result, the full range of contraception methods are being provided by Save the Children, CARE, IRC and other agencies as a standard in places like the eastern Democratic Republic of Congo and northern Uganda, even in the midst of security upheavals. In other areas, such as Zaatari camp in Jordan, the United Nations High Commissioner for Refugees and its partner agencies are meeting the demand for modern contraception which existed before the Syrian refugee crisis and which continues.[@R12]
These approaches not only deliver proven, cost-effective interventions at much larger scale than most classic emergency interventions, but also help to mitigate the risks to humanitarian workers and health workers. Hospital workers in the Syrian Arab Republic and vaccine workers in Pakistan appear to have been deliberately targeted during recent conflicts. Attacks of this type are not new, but they are being increasingly documented. Community health workers are less visible than facility workers, and can provide vital services with less danger to themselves in settings where formal health workers are deliberately targeted. They consequently have more options for continuing services in conflict.
Such new approaches to aid in conflicts will not happen without a new understanding of coordination that lasts beyond the acute phase of conflict, and that includes both acute and long-term actors. The cluster system, a UN-led, country-specific coordination system for acute emergencies, has helped to bring better geographical distribution of aid and better communication between agencies. What is needed, however, is coordination not just among acute responders but also among health actors with complementary mandates and spheres of action. Agencies with expertise in specific interventions, such as UNFPA or Planned Parenthood affiliates in family planning, need to support acute responders, such as Médecins Sans Frontières. Agencies just arriving into a conflict area need to collaborate with agencies within a country who can contribute greater knowledge of the local context and assets such as networks of community health workers. Establishing more effective public health responses will also require better coordination with governments ‒ a challenging task in many conflict settings, but an important one. Last but not least, an approach combining large-scale public health interventions with responses in conflict settings will require coordination across different kinds of donors and sometimes different teams within the same agency who may not be used to coordinating among themselves.
As the world embarks on another 15-year enterprise in global aid planning, implementation and tracking, we owe it to populations affected by conflict -- who are some of the most vulnerable people on earth -- to apply public health principles to deliver better aid. Changing the politics that drive conflict is beyond the sphere of the global health community. However, it is well within our power to maintain our high standards of effectiveness and common sense regarding cost‒effectiveness, and to coordinate better between global health actors. With a smarter, more adapted, more ambitious approach to assistance in conflict settings, we have an opportunity to make the SDGs more effective and more equitable than previous development goals.
None declared.
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Turner's syndrome (TS) is depicted as a total or partial absence of one X chromosome, and occurs in approximately 1/2200 of live born females \[[@CR1]\]. Nearly 43-49 % of the patients are cases with classical TS who are monosomic for X chromosome (45,X). The remaining patients are mosaic cases carrying normal and abnormal cell lines together (most of them had 45,X/46,XX karyotype) (15-23 %), those with isochromosome in long arm of X chromosome (i(Xq)) (14 %), those with ring X chromosome (r(X)) (3-11 %) and those with 46,XX karyotype but having partial losses in one X chromosome (9 %). Y chromosome fragments are detected in 10-11 % of the cases \[[@CR2]--[@CR6]\].
Patients with classical TS demonstrate characteristic clinical features such as short stature, web neck, cardiovascular and renal abnormalities and hearing loss; and besides, gonadal dysgenesis in TS results in pubertal delay or failure, infertility and premature ovarian failure (POF) in most patients \[[@CR7], [@CR8]\]. Although correlation between genotype and phenotype is not well understood, mosaic cases present milder phenotypic abnormalities compared to those with 45,X karyotype \[[@CR9]\]. Mosaic TS patients are more likely to experience normal pubertal development, regular menstrual cycles and to conceive spontaneously compared to those with 45,X monosomy \[[@CR2]\]. Since mosaic patients are diagnosed following karyotype analysis due to recurrent pregnancy loss, repeated in vitro fertilization (IVF) failure and history of an abnormal offspring, our knowledge concerning reproductive and obstetric outcomes relies on case reports and case series \[[@CR4], [@CR10]--[@CR12]\] and more comprehensive studies investigating the fertility outcomes of these patients.
The purpose of this study was to evaluate reproductive and obstetric outcomes of natural conception and IVF procedures in mosaic TS cases.
Materials and methods {#Sec2}
=====================
This retrospective study evaluated 706 female patients who underwent karyotyping between 2009--2013 in the laboratory of Medical Genetics Department of our tertiary health institution. The approval from the local ethics committee had been obtained prior to the initiation of the study. Informed consent from all individual participants included was obtained. Chromosomal analysis was performed by G-banding technique at high resolution. A hundred metaphases were counted for each patient and International System for Human Cytogenetic Nomenclature (ISCN, 2009) guidelines were used when performing karyotype analysis \[[@CR13]\].
Mosaicism ratios of the cases were calculated by proportioning total number of abnormal cell lines. In karyotype analysis, mosaic cell line ratio of ≤ %10 was defined as low-grade mosaicism, and \> %10 as high-grade mosaicism \[[@CR14]\].
Medical history regarding prior hormone therapy, prior assisted reproductive techniques attempts in infertility cases, obstetric outcomes in patients who had conceived through spontaneous or IVF cycles and perinatal outcomes were obtained by face-to-face interview or assessment of hospital medical records. All patients were assessed with physical examination and underwent a set of diagnostic tests including thyroid function tests, abdominal ultrasonography and echocardiography.
Statistical analysis {#Sec3}
--------------------
SPSS version 15.0 (Statistical Package for Social Science, Spss Inc. Chicago, IL, USA) was used for statistical analysis. Parametric variables were given as median (range), mean ± standard deviation or ± standard error. Chi-square test was used for the comparison of parametric variables. Non-parametric tests such as Mann--Whitney U test was used to compare non-parametric variables between cases with high-grade mosaicisim and low-grade mosaicism. Possibility of a total of 2-year fecundability was calculated at 6-month intervals by time-table analysis. The effect of menarche age, marriage age and mosaicism ratio on the time until spontaneous or IVF pregnancy was assessed by Cox-regression analysis. p \< 0.05 was considered as statistically significant.
Results {#Sec4}
=======
A total of 22 mosaic TS patients were extracted from 706 patients who underwent genetic karyotyping for varying indications including, recurrent implantation failure (5.1 %), recurrent pregnancy loss (defined as three or more consecutive miscarriage) (2.2 %), POF (2.2 %) and history of an offspring with any chromosomal or structural abnormality (4 %). Clinical characteristics of our study population were presented in Table [1](#Tab1){ref-type="table"}. Menarche was achieved with hormone replacement therapy in three cases at the ages of 16,17 and 18 years and continued regularly. Menstruation was regular in 18 cases at the time of study enrollment whereas it was irregular in the two and the remaining two, who were diagnosed as POF at the ages of 28 and 38, were on hormone replacement therapy. There was one case with a short stature (\<150 cm) phenotype and no cases with cardiovascular or renal abnormality, hearing loss and mental retardation among the included patients. The following systemic disorders were diagnosed in the patients; hypothyroidism in three, type 2 diabetes in three, asthma in one and generalized anxiety disorder in one patients. Uterine hypoplasia was observed in one case. In addition, one patient underwent hysteroscopic septum resection for uterine septum, another case underwent Strasmann metroplasty for bicornuat uterus and one underwent hysteroscopy and cavity expansion with fundal and lateral incisions for T-shaped uterus.Table 1Clinical characteristics of the study groupPatients age at the time of enrollment (years)^a^37 (26--47)Patients age at the time of diagnosis (years)^a^34.5 (18--46)Age of menarche (years)^a^13 (11--18)Age at marriage (years)^a^25 (15--40)Patients age at first pregnancy (years)^a,b^23 (18--32)Time from the marriage to the first conception (months)^a,b^12 (6--49)Height of the patients (cm)^a^163 (132--174)Body mass index at the enrollment (kg/m^2^)^c^28.43 ± 1.21^a^Data presented as median and range, ^b^In the cases who conceived spontaneously, ^c^ data presented as mean ± standart error
A total of 22 female patients, who were diagnosed as mosaic TS after karyotyping and who attended our IVF clinic with the diagnoses of recurrent implantation failure (*n* = 10), recurrent pregnancy loss (*n* = 9), infertility due to POF (*n* = 1), and history of a prior offspring with a chromosomal abnormality (*n* = 2), were included in the study. Out of 22 patients, five despite IVF treatment and one who never sought treatment, could not ever conceive. Out of remaining 16 cases, 11 conceived spontaneously and five conceived following IVF cycles; resulting in a total of 52 pregnancies of which 17 (32.7 %) resulted in live birth and 35 (67.3 %) resulted in abortion.
Of 22 mosaic TS patients' karyotypes, 17 were 45,X/46,XX and five were 45,X/46,XX/47,XXX. There were no cases including 45,X/46,Xr(X); 45,X/46,X(i(Xq)); Y chromosome fragment or 46,XX karyotype with structural abnormalities in X chromosome. One patient was determined to have 45,X/46,XX inv(9)p11q13 karyotype. The comparison regarding the number and the percentages of pregnancies, miscarriages and live births between the different karyotypes of TS were presented in Table [2](#Tab2){ref-type="table"}.Table 2Comparison of the ratios of live birth and miscarriage or terminated fetus between groupsMaternal karyotype45,X/46,XX (*n* = 17)45,X/46,XX/47,XXX (*n* = 5)All cases (*n* = 22)pNo of pregnancies (n)351752.678Live birth (n (%))10 (28.6)7 (41.2)17 (32.7).611Miscarriage (n (%))25 (71.4)10 (58.8)35 (67.3).712*p* \< .05 Statistically significant
Mosaic cell line ratio was below 10 % in 17 and above 10 % in five cases. The comparison regarding the number and the percentages of pregnancies, miscarriages and live births between high-grade and low-grade mosaicism cases were presented in Table [3](#Tab3){ref-type="table"}. Miscarriages included 29 spontaneous abortions, four biochemical abortions and two induced abortions (due to anencephaly and trisomy 21). Mean abortion week was found to be 8.16 ± 2.98 (±SD) in cases who experienced spontaneous abortion (Biochemical abortions were excluded from the calculation).Table 3Comparison of the ratios of live birth and miscarriage or terminated fetus between low and high grade mosaic cell line groupsMosaic cell line ratioCases with low grade mosaic cell line (*n* = 17)Cases with high grade mosaic cell line (*n* = 5)All cases (*n* = 22)pNo of Pregnancies (n)46652.062Miscarriage (n (%))30 (65.2)5 (83.3)35 (67.3 | {
"pile_set_name": "PubMed Central"
} |
Measurement of cerebral blood flow (CBF) is of interest, both in the clinical setting and for research. Absolute quantification of global CBF is of key importance when investigating factors affecting the entire brain, eg, altered physiological states or aging.[1](#jmri25442-bib-0001){ref-type="ref"}, [2](#jmri25442-bib-0002){ref-type="ref"}, [3](#jmri25442-bib-0003){ref-type="ref"}
Different invasive and noninvasive methods have been used to measure global CBF.[4](#jmri25442-bib-0004){ref-type="ref"}, [5](#jmri25442-bib-0005){ref-type="ref"} Phase‐contrast mapping (PCM) magnetic resonance imaging (MRI) allows measurement of total flow in the cerebral arteries, and calculation of global CBF by subsequently normalizing to brain volume.[6](#jmri25442-bib-0006){ref-type="ref"} Measurement of global CBF by PCM MRI has a number of advantages compared to other techniques. It is completely noninvasive, fast, and can be combined with other noninvasive MRI techniques for absolute quantification of global cerebral metabolic rate of oxygen[7](#jmri25442-bib-0007){ref-type="ref"}, [8](#jmri25442-bib-0008){ref-type="ref"} or be used for scaling of regional CBF measured by arterial spin labeling.[9](#jmri25442-bib-0009){ref-type="ref"}, [10](#jmri25442-bib-0010){ref-type="ref"} The technique has been used in a number of studies on variation in CBF in healthy individuals[11](#jmri25442-bib-0011){ref-type="ref"}, [12](#jmri25442-bib-0012){ref-type="ref"} and in larger population‐based studies of aging showing that global CBF decreases with age and that decreased global CBF is associated with accelerated signs of brain aging and increased all‐cause mortality.[2](#jmri25442-bib-0002){ref-type="ref"}, [3](#jmri25442-bib-0003){ref-type="ref"}, [6](#jmri25442-bib-0006){ref-type="ref"}, [13](#jmri25442-bib-0013){ref-type="ref"}, [14](#jmri25442-bib-0014){ref-type="ref"}, [15](#jmri25442-bib-0015){ref-type="ref"}
Values of global CBF obtained by PCM MRI in healthy subjects[6](#jmri25442-bib-0006){ref-type="ref"}, [16](#jmri25442-bib-0016){ref-type="ref"} are generally in good agreement with accepted textbook CBF values of ∼50 mL/100 g/min.[17](#jmri25442-bib-0017){ref-type="ref"}, [18](#jmri25442-bib-0018){ref-type="ref"} Previous studies have also shown excellent reproducibility for CBF measurements in vivo[12](#jmri25442-bib-0012){ref-type="ref"}, [16](#jmri25442-bib-0016){ref-type="ref"}, and shown PCM MRI to be accurate for measuring flow in phantoms[19](#jmri25442-bib-0019){ref-type="ref"} and in large vessels.[20](#jmri25442-bib-0020){ref-type="ref"}
In order to confidently interpret the physiological significance of these measurements, in vivo validation of the accuracy of PCM MRI for CBF measurements is required. However, comparative studies with accepted reference methods are generally lacking. For human studies, ^15^O‐H~2~O positron emission tomography (PET) is generally considered the best available method for absolute CBF measurements.[9](#jmri25442-bib-0009){ref-type="ref"}, [21](#jmri25442-bib-0021){ref-type="ref"}, [22](#jmri25442-bib-0022){ref-type="ref"}, [23](#jmri25442-bib-0023){ref-type="ref"}, [24](#jmri25442-bib-0024){ref-type="ref"} One previous study failed to show a correlation of CBF measured by PCM MRI and ^15^O‐H~2~O PET, but these measurements were obtained months apart and did not account for a number of important physiological covariates.[16](#jmri25442-bib-0016){ref-type="ref"}
The aim of the present study was to validate PCM MRI for measurement of global CBF by same‐day measurements of global CBF by PCM MRI and ^15^O‐H~2~O PET.
Materials and Methods {#jmri25442-sec-0006}
=====================
Twenty‐two healthy males (mean age: 27.4 years, range: 18--40 years) participated in the study. The measurements were obtained as a part of a placebo‐controlled, crossover study investigating the effect of erythropoietin on cerebral metabolism. Measurements from PCM MRI have previously been published.[8](#jmri25442-bib-0008){ref-type="ref"} In the present analysis only data during placebo treatment are included.
The study was approved by the Danish National Committee on Health Research Ethics (H‐4‐2012‐167) and was conducted according to the Declaration of Helsinki. Written informed consent was obtained from all participants.
General Experimental Setup {#jmri25442-sec-0007}
--------------------------
All experimental measurements in each subject were performed on the same day. Height and weight were measured at the time of inclusion in the study. After an overnight fast, a short catheter was inserted in the radial artery of the nondominant hand for blood sampling. After the ^15^O‐H~2~O PET scans, the subjects had a small meal before being transported to the MRI facility. The PET and MRI scans were performed 2--6 hours apart.
Before the PET scans a venous blood sample was obtained and analyzed for hemoglobin. Arterial blood samples were obtained between the two PET scans and again after the MRI CBF measurement, and analyzed for oxygen saturation (SaO~2~), and partial pressures of O~2~ (PaO~2~) and of CO~2~ (PaCO~2~) using a Radiometer ABL800 Flex system (Radiometer, Copenhagen, Denmark). The arterial blood sample obtained at the MRI session was also analyzed for hemoglobin.
PET {#jmri25442-sec-0008}
---
PET scans were performed on a Siemens High Resolution Research Tomograph (HRRT) brain PET scanner (Siemens, Knoxville, TN).[25](#jmri25442-bib-0025){ref-type="ref"}, [26](#jmri25442-bib-0026){ref-type="ref"} The scanner had an axial field of view of 25 cm and a near isotropic resolution of 2 mm. During PET scanning the subject\'s head rested in a foam‐cushioned headrest, and a head strap was used to minimize head movement. Initially, a 6‐minute transmission scan with a rotating ^137^Cs single‐photon point source was performed for attenuation correction.
Approximately 800 MBq of ^15^O radiolabeled water (half‐life: 123 sec) produced online was injected as a bolus using an Automatic Water Injection System (Scansys Laboratorieteknik, Værløse, Denmark).
Arterial blood sampling was initiated 15 seconds before isotope injection. Emission scans were acquired after injection of intravenous bolus of tracer for 7 minutes in dynamic frames of 1 × 30 seconds, 18 × 5 seconds, 9 × 10 seconds, 10 × 15 seconds, and 2 × 30 seconds. Radioactivity concentration in the arterial blood was continuously measured by an automatic blood sampling system and drawn at 8 mL/min (Allogg ABSS, Mariefred, Sweden). The detectors in the system were cross‐calibrated against the PET scanner and the sampling frequency was 2 Hz. The inner diameter of the tube connected to the arterial catheter was 1.2 mm. The clocks of the scanner and sampling system were synchronized.
Two consecutive scans were acquired for each subject with an interval of at least 10 minutes between the two injections to allow for washout and isotope decay.
Scans were reconstructed using 3D‐ordered subset expectation maximum and point spread function (3D OSEM‐PSF).[27](#jmri25442-bib-0027){ref-type="ref"} Each map consisted of 207 image planes in a 256 × 256 matrix with an isotropic voxel size of 1.22 × 1.22 × 1.22 mm^3^. All images were corrected for randoms, scatter, attenuation (TXTV method),[28](#jmri25442-bib-0028){ref-type="ref"} decay and dead time, and filtered with a 3D Gaussian 5 mm filter.
Postprocessing {#jmri25442-sec-0009}
--------------
Quantitative regional CBF‐maps were calculated using a 1‐tissue 2‐compartment model: $$\frac{dC_{t}\left( t \right)}{dt} = K_{1}C_{a}\left( t \right) - k_{2}C_{t}\left( t \right)$$where *C* ~*t*~ is the tissue compartment concentration, *C* ~*a*~ is the arterial concentration, *K | {
"pile_set_name": "PubMed Central"
} |
This article is part of the thematic issue \"Frontiers in pharmaceutical nanotechnology\".
Introduction
============
Nanocrystals for pharmaceutical use were invented in the early 1990s \[[@R1]--[@R4]\]. They are composed of 100% substance, are stabilized with only small amounts of surfactants, and possess particle sizes below 1 µm. According to the Ostwald--Freundlich equation nanocrystals possess a higher curvature leading to an enhanced dissolution pressure and thus to an enhanced kinetic saturation solubility \[[@R5]\]. Due to their small size they possess an increased surface area, resulting in an increased dissolution rate expressed by the Noyes--Whitney equation. In addition, they also possess an increased adhesiveness and thus, represent a universal, powerful and well-known formulation principle to overcome poor aqueous solubility and poor bioavailability of class-II and class-VI active ingredients of the biopharmaceutics classification system (BCS) \[[@R6]--[@R7]\]. Nanocrystals are already used in various pharmaceutical drug products for oral use. Examples are Rapamune^®^ (Sirolimus, Wyeth), Emend^®^ (Aprepitant, Merck), Tricor^®^ (Fenofibrate, Abbott), Megace ES^®^ (Megestrol, Par Pharm) or Triglide^®^ (First Horizon Pharmaceuticals). In 2009 the first parenteral drug product, Invega Sustenna^®^ (Paliperidone palmitate, Johnson & Johnson), was approved by the FDA. However, besides oral or parenteral administration, nanocrystals can also be used to improve the bioactivity of poorly soluble active ingredients via other routes of administration. Examples include pulmonal, ocular or dermal application \[[@R8]--[@R12]\].
Nanocrystals can be produced by different methods. Examples are precipitation, wet milling, high-pressure homogenization or combinations of these methods \[[@R1]--[@R4]\]. Regardless of the process used, all these methods will yield nanosuspensions, i.e., nanocrystals dispersed in a liquid. As liquid formulations are not always a convenient dosage form for the final drug product, in most cases nanosuspensions need to be formulated into other, more convenient, dosage forms. Depending on the route of administration this could be tablets, pellets, powders, gels or creams. However, prior to the formulation into final drug products, the aqueous nanosuspensions need to be stored, which certainly requires a sufficient stability of the nanosuspension. For this, besides chemical and physical stability, also the microbial stability needs to be considered.
One method to avoid microbial contamination of aqueous formulations during storage is the use of preservatives. In previous studies it was already found that preservatives can strongly impair the physical stability of the nanosuspensions. Reasons for this are changes of pH value or of the conductivity of the dispersion medium, or the adsorption of the preservatives onto the surface of the particles, which changes the charge of the particles (zeta potential) and forces agglomeration of the nanocrystals. To avoid instabilities of nanosuspensions only very hydrophilic and non-charged preservatives, which will not interact with the nanocrystals, should be used. Due to the above-mentioned reasons, only a few preservatives are available for the preservation of nanocrystals. Suitable preservatives for the preservation of nanocrystals include different alcohols, i.e., pentylene glycol or mixtures of phenoxyethanol and ethyl hexyl glycerol \[[@R13]--[@R15]\].
The limited number of preservatives and sometimes the incompatibility of these preservatives with other excipients in the final formulation are inconvenient for a successful formulation of nanosuspensions. Therefore, to enable a more convenient formulation of nanocrystals in the future, this work aimed at investigating an alternative method to maintain the microbial stability of nanosuspensions during storage.
Considering that bacterial growth strongly depends on the temperature, it was hypothesized that freezing of non-preserved nanosuspensions might prevent bacterial growth of the nanosuspensions during storage. However, the harsh conditions during freezing and thawing might also impair the physical stability of the nanocrystals and might cause agglomeration of the particles, which would then lead to a loss of the "nano properties". Hence, in this case the method could not be exploited to preserve nanosuspensions without preservative.
To investigate whether the freeze--thaw method is suitable for the production of long-term stable non-preserved nanosuspensions with high microbial quality, previously developed nanosuspensions containing the flavonoid rutin as model substance and either Plantacare 2000 or Poloxamer 188 (PLX 188) as stabilizers were produced by high-pressure homogenization as described previously \[[@R16]--[@R19]\]. Each of the nanosuspensions obtained was allocated into two parts. One part was preserved, and the other part remained non-preserved. All formulations were stored at different temperatures for a period of three months and size, zeta potential, antioxidant capacity and the microbial quality were determined and monitored over this period of time ([Fig. 1](#F1){ref-type="fig"}).
{#F1}
Results and Discussion
======================
Production and characterization of nanosuspensions
--------------------------------------------------
High-pressure homogenization yielded rutin nanosuspensions with a relatively broad size distribution, i.e., polydispersity indices (PdI values) above 0.3 and some larger particles with sizes above 4 µm ([Table 1](#T1){ref-type="table"}). Because of this, the nanosuspensions were expected to be prone to Ostwald ripening, i.e., particle growth during storage was expected. Limited physical stability of suspensions is advantageous if a study aims at investigating different stabilizing and destabilizing effects, because in comparison to highly physically stable formulations, destabilizing effects can be detected earlier during storage, making a discrimination between stabilizing and destabilizing effects clearer.
######
Overview of results obtained from the characterization of the nanocrystals at the day of production.
------------------- ----------------- ------------- ----------------------- ------------------- -------------- ------------- -------------
stabilizer preservative DLS data zeta potential \[mV\] LD data \[µm\]^a^
z-ave \[nm\] PdI in water in medium *d*(*v*)0.50 *d*(*v*)0.95
PLX 188 no preservative 408 ± 45 0.31 ± 0.08 −29.4 ± 2.9 −24.8 ± 2.6 1.3 ± 0.17 4.09 ± 0.15
with preservative 412 ± 29 0.30 ± 0.05 −27.0 ± 7.8 −24.6 ± 3.0 1.3 ± 0.12 4.13 ± 0.15
Plantacare 2000 no preservative 436 ± 30 0.32 ± 0.06 −30.6 ± 4.0 −39.5 ± 2.7 1.2 ± 0.11 4.02 ± 0.11
with preservative 447 ± 15 0.33 ± 0.06 −29.3 ± 2.7 −37.6 ± 3.3 1.2 ± 0.12 3.99 ± 0.11
------------------- ----------------- ------------- ----------------------- ------------------- -------------- ------------- -------------
^a^ *d*(*v*): volumetric median diameter.
The suspension stabilized with PLX 188 yielded sizes of about 410 nm. Slightly larger nanocrystals with a slight agglomeration were obtained when Plantacare was used as stabilizer ([Table 1](#T1){ref-type="table"} and [Table 2](#T2){ref-type="table"}). From this it was expected that non-preserved nanosuspensions stabilized with PLX 188 might possess a slightly better physical stability than the Plantacare-stabilized formulations. Upon the addition of the preservatives only very minor changes in size were observed for both formulations ([Table 1](#T1){ref-type="table"}) and for the Plantacare-stabilized formulation even a slight deagglomeration was determined ([Table 2](#T2){ref-type="table"}). Also the zeta potential values did not change significantly in both water and original dispersion medium ([Table 1](#T1){ref-type="table"}), again indicating no, or only a very limited, impairment of the stabilization mechanisms of the nanocrystals by the hydrophilic preservative \[[@R20]\].
######
Microscopic images of the nanosuspensions stabilized with Poloxamer 188 (left) and Plantacare 2000 (right) at the day of production. Magnification: 400-fold.
--------------- ----------------------------------------------- -----------------------------------------------
stabilized with Poloxamer 188 stabilized with Plantacare 2000
non-preserved  
preserved  | {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-ijms-19-03456}
===============
Potassium (K^+^) is an essential macronutrient and is essential for plant growth and development \[[@B1-ijms-19-03456]\]. K^+^ is associated with or involved in several physiological processes that support plant growth and development, such as photosynthesis, enzyme activation, osmoregulation, electrical neutralization, pH and ion homeostasis, anion-cation balance, membrane electrical potential, protein and starch synthesis, sugar and nutrient transport, and stomatal movement \[[@B2-ijms-19-03456]\]. K^+^ also plays a major role in enhancing the tolerance of plants to various stresses \[[@B3-ijms-19-03456],[@B4-ijms-19-03456]\]. The concentrations of K^+^ in the soil solution range from only 0.1--1 mM, and can be much lower at the root surface due to local depletion \[[@B5-ijms-19-03456]\]. K^+^ deficiency in most arable fields is limiting for optimal plant growth \[[@B6-ijms-19-03456],[@B7-ijms-19-03456]\]. K^+^ deprivation leads to a strong increase in chlorophyll degradation; K^+^ deficiency-related symptoms include brown scorching and curling of leaf tips, as well as interveinal chlorosis \[[@B8-ijms-19-03456]\]. Reduced leaf area under K^+^ deficiency has also been reported \[[@B9-ijms-19-03456],[@B10-ijms-19-03456]\]. In addition, K^+^ deficiency affects root development, as primary root growth is negatively affected \[[@B11-ijms-19-03456],[@B12-ijms-19-03456]\]. Various K^+^ shortage-activated signaling cascades exist; these cascades involve reactive oxygen species (ROS) \[[@B13-ijms-19-03456]\], phytohormones (ethylene, auxin, and jasmonic acid) \[[@B14-ijms-19-03456],[@B15-ijms-19-03456]\], calcium \[[@B16-ijms-19-03456]\], and phosphatidic acid \[[@B17-ijms-19-03456]\]. Among these signaling cascades, calcium signaling is the most important signaling system within plant cells. In this review, the possible roles of calcium signaling in plant responses to low-K^+^ stress are discussed ([Figure 1](#ijms-19-03456-f001){ref-type="fig"}).
2. Molecular Mechanisms of Calcium Signaling Involved in Plant Responses to K^+^ Deficiency {#sec2-ijms-19-03456}
===========================================================================================
2.1. Generation of Calcium in Response to K^+^ Deficiency {#sec2dot1-ijms-19-03456}
---------------------------------------------------------
The concentration and distribution of cytosolic free calcium form the basis of calcium signaling. Under normal conditions, levels of cytosolic free calcium are low, but some organelles, including the vacuole, endoplasmic reticulum, mitochondria and so on, contain high concentrations of calcium, henceforth referred to as the calcium pool. Elevations in intracellular calcium \[Ca^2+^\]~i~ have been recorded in the responses of both lower and higher plants to a wide variety of both biotic and abiotic stimuli \[[@B18-ijms-19-03456],[@B19-ijms-19-03456]\]. In plants, \[Ca^2+^\]~i~ levels that are altered in response to multiple abiotic stresses result in calcium signatures that exhibit temporal and spatial features \[[@B20-ijms-19-03456],[@B21-ijms-19-03456],[@B22-ijms-19-03456]\]. These calcium signatures can take the form of single calcium transients \[[@B23-ijms-19-03456],[@B24-ijms-19-03456]\], oscillations \[[@B25-ijms-19-03456],[@B26-ijms-19-03456],[@B27-ijms-19-03456]\], or repeated spikes \[[@B28-ijms-19-03456],[@B29-ijms-19-03456]\]. Alterations to cytosolic calcium signals can be perceived by calcium sensors, which can result in a series of downstream responses, such as protein modification and transcriptional regulation \[[@B30-ijms-19-03456],[@B31-ijms-19-03456],[@B32-ijms-19-03456],[@B33-ijms-19-03456],[@B34-ijms-19-03456],[@B35-ijms-19-03456],[@B36-ijms-19-03456],[@B37-ijms-19-03456],[@B38-ijms-19-03456]\]. Calcium sensors in *Arabidopsis* root are involved in both K^+^ uptake and responses to K^+^ deficiency. Low K^+^ induces \[Ca^2+^\] to increase in *Arabidopsis* guard cells \[[@B39-ijms-19-03456]\] and in the pollen tubes \[[@B40-ijms-19-03456]\]. The results of a recent study revealed that K^+^ deficiency triggers two successive but distinct calcium signals in roots, and that those two signals exhibit spatial and temporal specificity \[[@B16-ijms-19-03456]\]. Calcium flows into or out of the cytoplasm via calcium channels located within the plasma membrane and endomembrane system \[[@B18-ijms-19-03456],[@B41-ijms-19-03456],[@B42-ijms-19-03456]\]. Most calcium channels are nonselective for ions \[[@B41-ijms-19-03456],[@B43-ijms-19-03456]\]. In plants, these calcium channels mainly include nonspecific cation channels located within the cell membrane \[[@B43-ijms-19-03456],[@B44-ijms-19-03456]\], including members of the cyclic nucleotide-gated channel (CNGC) family \[[@B26-ijms-19-03456],[@B45-ijms-19-03456],[@B46-ijms-19-03456],[@B47-ijms-19-03456]\] and the glutamate receptor channel (GLR) family \[[@B48-ijms-19-03456],[@B49-ijms-19-03456],[@B50-ijms-19-03456]\], hyperosmolality-gated calcium-permeable channels \[[@B51-ijms-19-03456],[@B52-ijms-19-03456]\], and annexins proteins \[[@B53-ijms-19-03456]\], and mechanosensitive channels (MCAs) \[[@B54-ijms-19-03456]\], as well as two-pore calcium channels (TPCs) \[[@B55-ijms-19-03456],[@B56-ijms-19-03456]\] located in the vacuolar membrane. However, the mechanisms of their action remain unclear.
2.2. Initial Sensing of K^+^ Deficiency by Calcium Channels {#sec2dot2-ijms-19-03456}
-----------------------------------------------------------
In plants, although some calcium channel activation occurs via depolarization \[[@B57-ijms-19-03456],[@B58-ijms-19-03456],[@B59-ijms-19-03456]\], most channels operate at highly-negative membrane voltages, and are often described as hyperpolarization-activated calcium channels (HACCs) \[[@B41-ijms-19-03456],[@B53-ijms-19-03456],[@B60-ijms-19-03456],[@B61-ijms-19-03456],[@B62-ijms-19-03456],[@B63-ijms-19-03456]\]. After low-K^+^ stress, calcium channels located within the root epidermis and root hair zone can be activated by hyperpolarization of the PM \[[@B60-ijms-19-03456],[@B64-ijms-19-03456]\]. Calcium increases in the cytosol can activate additional calcium channels located within the inner membrane, thereby causing the calcium pool to release calcium. For instance, two-pore channel 1 (TPC1), which is a voltage-gated channel and is located within the vacuolar membrane, is involved in the influx of calcium to the cytoplasm from the vacuole \[[@B55-ijms-19-03456]\]. In addition, the low-K^+^-inducing \[Ca^2+^\]~i~ increase is also mediated by ROS-activated calcium channels in the PM \[[@B61-ijms-19-03456]\]. ROS are important signaling molecules that mediate many physiological stimuli and lead to the generation of \[Ca^2+^\]~i~ signals under stress \[[@B65-ijms-19-03456],[@B66-ijms-19-03456]\]; increases in ROS are induced by K^+^ deficiency \[[@B67-ijms-19-03456],[@B68-ijms-19-03456]\]. Elevated \[Ca^2+^\]~i~ can induce NADPH oxidase-mediated production of ROS, which in turn activates calcium-permeable ion channels, thereby resulting in further calcium influx \[[@B62-ijms-19-03456],[@B69-ijms-19-03456]\]. Additionally, ROS have been also suggested to participate in long distance signaling with calcium, and are likely involved in generating calcium waves \[[@B70-ijms-19-03456],[@B71-ijms-19-03456],[@B72-ijms-19-03456],[@B73-ijms-19-03456]\].
2.3. Calcium Sensors Involved in the Sensing of K^+^ Deprivation {#sec2dot3-ijms-19-03456}
----------------------------------------------------------------
How plant cells sense transient increases in \[Ca^2+^\]~i~ in response to low-K^+^ stress is ambiguous. Calcium signals are likely perceived | {
"pile_set_name": "PubMed Central"
} |
All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
In arid and semi-arid environments, environmental gradients differ at local scales because shrubs generally improve soil fertility and produce microclimates under their canopies that favor plant-plant interactions \[[@pone.0133559.ref001]\]. According to the stress-gradient hypothesis (SGH) \[[@pone.0133559.ref002]\], interactions among plants vary with environmental conditions, shifting from competition to facilitation as environmental stress increases. Such environmental stress comprises stresses from abiotic and biotic (i.e., consumers) sources. It has been found that plant seedlings growing beneath shrubs have different probabilities of survival than do conspecific seedlings growing in open interspaces between shrubs \[[@pone.0133559.ref003]\].
The occurrence of facilitation interactions under high abiotic stress conditions has been verified by numerous studies in many ecosystems and has provided a crucial basis for empirical studies and theory governing plant-plant interactions in stress-prone Mediterranean environments (see \[[@pone.0133559.ref001]\] and references therein, \[[@pone.0133559.ref003]\]) where tree seedlings benefit from habitat amelioration, especially during summer drought \[[@pone.0133559.ref003]--[@pone.0133559.ref009]\]. However, information on biotic impacts caused by consumers in these environments is still scarce (see \[[@pone.0133559.ref010]\] and references therein). Positive plant-plant interactions should be especially important in plant communities subjected to both climatic stress and biotic stress \[[@pone.0133559.ref002]\], as is the case in Mediterranean-type ecosystems. Surprisingly, the role of herbivory, a common and widespread interaction in arid and semiarid environment, has attracted little attention in this context \[[@pone.0133559.ref011]--[@pone.0133559.ref014]\].
In Mediterranean central Chile, the exotic mammalian herbivores have been altering the structure and functioning of ecosystems \[[@pone.0133559.ref015]\] by, promoting homogenization of species like *Acacia caven* and preventing the recolonization of clearings by woody matorral species on the slopes \[[@pone.0133559.ref008], [@pone.0133559.ref015]\]. It is expected that the impacts of introduced herbivores on native vegetation will be different from the native fauna \[[@pone.0133559.ref016]\], but how plant-plant facilitation relationship may be modulated by animal-plant interactions, specifically in this case, by herbivory, has been frequently neglected.
Valiente-Banuet, Rumebe, \[[@pone.0133559.ref017]\] have indicated that facilitation (or the nurse effect) prevented Tertiary species extinction when climatic conditions became more xeric during the Quaternary, the period during which the current Mediterranean climate emerged. This effect should be of particular importance for the persistence of fleshy-fruited Tertiary species with recalcitrant seeds \[[@pone.0133559.ref017]\] as palms (Arecaceae). Originating in the Cretaceous, palms experienced a spectacular radiation in the Tertiary Early Cenozoic and severe extinction rates due to rainforest decline during the drier Quaternary \[[@pone.0133559.ref018]\]. Thus, relict palms in arid ecosystems, such as the endemic wine palm (*Jubaea chilensis* (Molina) Baill.) in Mediterranean central Chile, constitute a proper candidate for evaluating the relative importance of abiotic and biotic (herbivory) stresses for seedling survival and growth, and for assessing the relationship between animal-plant and plant-plant interactions in arid environments.
Accordingly, we formulated four hypotheses (i) Shrub canopies, by creating a more humid, mesic, and shaded environment relative to open interspaces, differentially affect palm seedling performance. (ii) Shrubs interact positively with wine palm seedlings to facilitate recruitment, buffering the seedlings performance from potentially limiting stresses (SGH) or (iii) seedling performance is facilitated because shrubs protect the seedlings against browsers. In order to study these hypotheses, we monitored growth and herbivore defoliation of 300 wine palm seedlings over a period of 18 months. Seedlings were transplanted outside and under the canopy of the seven most representative shrub species and were grown with and without exclosure protection from browsing. This study can improve our understanding of the relative importance of biotic and abiotic mechanisms for shrub facilitation in the Mediterranean hotspot ecosystem region, which has experienced an increasing frequency, duration, and severity of drought in recent decades \[[@pone.0133559.ref019]\] and has also faced risks associated with the presence of a high proportion of invasive non-native vertebrate herbivores \[[@pone.0133559.ref008], [@pone.0133559.ref015], [@pone.0133559.ref020]\]. Also, this study will help us to examine to fate of wine palm in a context of increasing of aridity and herbivory pressure.
Materials and Methods {#sec002}
=====================
Study species and site {#sec003}
----------------------
This study was conducted in the Oasis La Campana private reserve in central Chile (hereafter La Campana; 71°04\'W; 32°57'S). The CONAF/Parque Nacional la Campana and Reserva Oasis de La Campana issued all of the required permits for the work conducted in those sites. Located in the core zone of the Campana-Peñuelas UNESCO Biosphere Reserve, La Campana covers 17,095 ha and has irregular topography varying from 300 to 1,800 m altitude. The area has a Mediterranean climate, closely equivalent to that of southern California but with a six-month offset. The mean annual rainfall and temperature are 109 mm and 20μC, respectively \[[@pone.0133559.ref021]\]. Rainfall is concentrated in spring and autumn, alternating with hot and dry summers and cold winters. Mean temperatures for December-February are 18°C, and a mean of only 30 mm of precipitation falls from November to April. Winters are mild and moist, and the mean winter temperature, from June to August, is 11°C \[[@pone.0133559.ref022]\].
Mediterranean-type ecosystems have been substantially altered by human activities worldwide, but it is probable that such ecosystems persist to a greater extent in central Chile than in other regions of the world \[[@pone.0133559.ref023]\]. The natural recovery of sclerophyllous vegetation in many open and abandoned areas of central Chile is a slow process \[[@pone.0133559.ref024], [@pone.0133559.ref025]\] due to unsuitable conditions for seedling recruitment and a lack of seed sources. The limiting factors for recruitment include seed deposition in unsafe sites for seed germination and for seedling establishment \[[@pone.0133559.ref024], [@pone.0133559.ref025]\].
The most common evergreen trees and shrubs in La Campana are *Cryptocarya alba* (Lauraceae), *Lithraea caustica* (Anacardiaceae), *Quillaja saponaria* (Quillajaceae), *Maytenus boaria* (Celastraceae), *Kageneckia oblonga* (Rosaceae), and *Peumus boldus* (Monimiaceae) \[[@pone.0133559.ref026]\]. Sclerophyllous forests commonly exhibit a patchy spatial structure, but they may present a continuous canopy in ravines, deep creeks, along permanent water courses and on south-facing slopes \[[@pone.0133559.ref027]\]. On dry north-facing slopes or in frequently disturbed sites \[[@pone.0133559.ref028]\], the sclerophyllous vegetation is often replaced by a xerophytic thorn scrub, with a combination of deciduous shrubs, such as *Retanilla trinervia* (Rhamnaceae), *Colliguaja odorifera* (Euphorbiaceae), *Baccharis linearis* (Compositae), *Acacia caven* (Leguminosae) and *Puya berteroniana* (Bromeliaceae) \[[@pone.0133559.ref026]\]. The palm forest, where this study was conducted, is narrowly distributed within native vegetation and frequently neglected component with numerically abundant populations of the endemic wine palm *Jubaea chilensis* (Molina) Baill ([Fig 1](#pone.0133559.g001){ref-type="fig"}). The wine palm population has been severely fragmented and currently occurs mainly on slopes and in deep canyons of the central Chile Coastal Range \[[@pone.0133559.ref029]\]. It is probable that these sites, with soils of granitic origin, are favorable because of their maritime, moisture-laden air and limited temperature oscillations \[[@pone.0133559.ref023]\].
{#pone.0133559.g001}
La Campana, the heart of the wine palm\'s range, contains the world's largest remnant population of the endemic wine palm, harboring ≈ 60,000 wine palms that represent more than 58% of the total number of native stands \[[@pone.0133559.ref029]\]. The wine palm has a limited distribution but is numerically abundant where it is found (22--113 ind. ha^-1^) \[[@pone.0133559.ref030]\]. It is usually dominant in its favored habitat of slopes and | {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
Stress myocardial perfusion imaging using single-photon emission computed tomography (SPECT) is commonly used for risk stratification and therapeutic decision making in patients with coronary artery disease (CAD).[@B1][@B2][@B3] Since ischemia is a strong predictor of adverse outcomes, such as death or myocardial infarction (MI),[@B4][@B5] detecting ischemia is an important part of the diagnostic strategy for patients with stable CAD. In addition to the diagnostic and prognostic utility of myocardial perfusion SPECT, the extent of ischemia is one of the primary measures that drives decisions regarding revascularization.[@B6][@B7][@B8] Patients with moderately or severely abnormal myocardial perfusion SPECT have significantly higher mortality rates if treated with medical therapy alone.[@B5][@B7] Previous studies on myocardial perfusion SPECT have focused on the prognostic utility of stress imaging as the initial test for patients with CAD.[@B8][@B9] However, there are only a few studies that show a relationship between the presence and severity of ischemia and prognosis during CAD treatment.[@B10][@B11][@B12] Therefore, according to the current guidelines and appropriate use criteria for follow-up of CAD, routine stress imaging is not recommended, except for special high-risk groups after coronary revascularization.[@B13][@B14] Hence, in the current study, we aimed to assess the clinical implications of serial myocardial perfusion SPECT in patients with CAD who were receiving either medication or revascularization therapy.
MATERIALS AND METHODS
=====================
Study population
----------------
We identified consecutive patients who underwent serial myocardial perfusion SPECT, had abnormal results on a first study \[which was defined as summed stress score (SSS) ≥3\],[@B15] and had follow-up adenosine stress SPECT at an interval ≥6 months between the two studies that were performed between January 1, 2000 and June 31, 2014. Patients were also excluded if they had MI \<3 months before initial SPECT, previous revascularization therapy, serious non-coronary heart disease, including cancer with a life expectancy less than one year, incomplete nuclear data, multiple coronary revascularizations between the two SPECT procedures, and no clinical follow-up information. As a result, a total of 1153 patients with serial SPECT studies were included ([Fig. 1](#F1){ref-type="fig"}). Because of the retrospective nature of the study, a waiver for individual informed consent was granted by the Institutional Review Board.
Myocardial perfusion imaging
----------------------------
Thallium-201 (Tl-201) SPECT was the default stress myocardial perfusion imaging used during the study period. Images were acquired with a standardized protocol.[@B16] Adenosine was intravenously administered at a rate of 140 mcg/kg/minute for 6 min. Three minutes after the initiation of the adenosine infusion, a dose of Tl-201 (range=92.5--148 MBq, as determined by the patient\'s body weight) was intravenously injected. Six minutes after adenosine infusion, post-stress myocardial perfusion images were acquired using two-head gamma cameras equipped with low-energy, all-purpose collimators. The specific acquisition parameters were dependent on the camera.
Image interpretation
--------------------
Semi-quantitative visual interpretation was performed by independent expert interpreters, using 17 segments for the severity and extent of abnormalities on stress imaging.[@B17] Each segment was scored using a 5-point scoring system (0=normal; 1=equiv-ocal; 2=moderate; 3=severe reduction in radioisotope uptake; 4=absence of detectable tracer uptake in a segment), as previously described.[@B18] The score that was summed from the stress scan was defined as the SSS: SSS was determined by adding the scores of the 17 segments on the stress images. The SPECT study was considered to be abnormal if the SSS was 3 or greater. According to the result of follow-up myocardial perfusion SPECT, patients were categorized into normal and abnormal groups.
Procedure and follow-up
-----------------------
Coronary angiography was recommended for patients on the basis of their clinical presentation and the results of the noninvasive stress test. Significant stenosis on coronary angiography was defined as \>50% stenosis in an epicardial coronary artery. In patients with significant stenosis, the decision to perform revascularization or medical therapy was at the discretion of the individual cardiologist. Percutaneous coronary intervention (PCI)[@B19] or coronary artery bypass graft (CABG) surgery was performed using standard techniques.[@B20] Medical treatment was performed with a medical regimen that consisted of at least antiplatelet, antianginal, and lipid-lowering therapies.[@B3] After index myocardial perfusion SPECT, patients received either medical treatment or revascularization treatment.
Definitions
-----------
The primary outcome of interest was the occurrence of major adverse cardiac events (MACE), which was the composite of all-cause death, nonfatal MI, or unplanned revascularization after follow-up myocardial perfusion SPECT. When patients received multiple serial myocardial perfusion SPECTs, the first follow-up SPECT with an interval of ≥6 months after the index myocardial perfusion SPECT was selected for analysis. An MI was defined as elevated cardiac enzymes (troponin I or myocardial band fraction of creatine kinase) more than the upper limit of the normal value with ischemic symptoms or electrocardiography findings that were indicative of ischemia. After follow-up myocardial perfusion SPECT, any further PCI or CABG (excluding planned staged PCI) was considered an unplanned revascularization. Death, non-fatal MI, and unplanned revascularizations were verified by reviewing medical records.
Statistical analysis
--------------------
The continuous and categorical covariates are summarized as a mean±standard deviation or count (%). According to the follow-up myocardial perfusion SPECT results, all patients were divided into normal and abnormal groups. The baseline patient characteristics were compared between the two groups using the t test or Fisher exact test for continuous and categorical variables, respectively. The cumulative incidence of MACE for the normal and the abnormal groups was obtained using the Kaplan-Meier method and compared between the two groups using the log-rank test. To examine the effect of abnormal results on MACE and its individual events, the unadjusted and adjusted Cox proportional hazards regression models were fitted. Covariates that were statistically significant in univariate analysis and/or those that were clinically relevant were considered candidate variables for multivariate models. In the Cox model, the proportionality assumptions were assessed using the Scho-enfeld residual test, and no relevant violations were detected.
To reduce treatment selection bias and potential confounding, propensity score (PS)-matching analysis was performed. The PS of obtaining the abnormal myocardial perfusion SPECT results was estimated using the nearest-neighbor matching method with a caliper width of 0.2. The considered variables for the PS were age, sex, body mass index, hypertension, diabetes mellitus, current smoking, hyperlipidemia, prior revascularization treatment, chronic pulmonary disease, chronic renal failure, creatinine, total cholesterol, left ventricular (LV) ejection fraction \<50%, and the use of beta blockers, calcium channel blockers (CCB), angiotensin converting enzyme inhibitor (ACEi), angiotensin receptor blocker (ARB) agents, or statins, which are listed in [Table 1](#T1){ref-type="table"}. In general, covariate balancing was considered to be achieved as long as the absolute standardized difference of the means or proportions was \<0.25. In the PS analyses, no violations of covariate balancing were detected ([Supplementary Fig. 1](#S2){ref-type="supplementary-material"}, only online). For the PS-matched cohorts, continuous variables were compared using the paired t test or the Wilcoxon signed-rank test, as appropriate, and the categorical variables were compared using the McNemar\'s or Bowker\'s test of symmetry, as appropriate. A subgroup analysis was performed according to the treatment groups. Furthermore, univariate and multivariate logistic regression analyses were performed to identify independent clinical predictors of having abnormal results on follow-up myocardial perfusion SPECT. In the multivariable logistic regression, we employed a backward variable selection approach based on the *p* values. The significance level for staying in the model was set to 0.05. All statistical analyses were performed using SPSS (version 19.0 software; IBM Corp., Armonk, NY, USA) and R software (version 2.13; R Foundation for Statistical Computing, Vienna, Austria; <http://www.r-project.org>). Additionally, the R package MatchIt was used to conduct the PS analysis.[@B21] All tests were two-tailed, and *p*\<0.05 was considered statistically significant.
RESULTS
=======
Overall population
------------------
### Baseline characteristics
The median follow-up interval between the index and follow-up myocardial perfusion SPECT procedures was 474 days \[interquartile range (IQR)=243--1107 days\]. Abnormal results on follow-up myocardial perfusion SPECT were noted in 591 patients (51.3%). The baseline clinical characteristics and myocardial perfusion SPECT results, according to the results of the follow-up myocardial perfusion SPECT procedures and treatment strategy, are presented in [Table 1](#T1){ref-type="table"}. The patients in the abnormal group were more likely to be male and have a higher incidence of hypertension, diabetes mellitus, and a low ejection fraction. On myocardial perfusion SPECT, the abnormal group had greater baseline perfusion defects than the normal group. The revascularization therapy group was older and more likely to have diabetes mellitus, greater extent of CAD, and high perfusion defect on the initial myocardial perfusion SPECT ([Supplementary Table 1](#S1){ref-type="supplementary-material"}, only online).
### Clinical outcomes
Patients were followed for a median | {
"pile_set_name": "PubMed Central"
} |
{
"pile_set_name": "PubMed Central"
} | |
Zhang J, Gao X, Han Y, *et al*. Treatment effects of systematic two-stent and provisional stenting techniques in patients with complex coronary bifurcation lesions: rationale and design of a prospective, randomised and multicentre DEFINITION II trial. *BMJ Open* 2018;8:e020019. doi: 10.1136/bmjopen-2017-020019.
The sample size estimation in Statistical Analysis Part should be:
From previous studies, we hypothesized that the rate of a 1-year TLF would be 7% in the systematic two-stent technique group and 14% in the provisional stenting group. Accordingly, a total sample size of 600 is needed to detect a power of 0.8 (Type Ⅱ error = 0.2, = 0.05, 2-tailed). Because of the considerable uncertainty, the enrolment is extended to 660 patients (10% increment).
Instead of:
From previous studies, we hypothesized that the rate of a 1-year TLF would be 15% in the systematic two-stent technique group and 25% in the provisional stenting group. Accordingly, a total sample size of 600 is needed to detect a power of 0.8 (Type Ⅱ error = 0.2, = 0.05, 2-tailed). Because of the considerable uncertainty, the enrolment is extended to 660 patients (10% increment).
| {
"pile_set_name": "PubMed Central"
} |
{#sp1 .92}
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction
===============
Fluid flowmeter calibration methods use a variety of techniques; they include wide ranges of operational parameters; they cover wide ranges of precision and accuracy, \[[@b1-jresv97n5p509_a1b]--[@b3-jresv97n5p509_a1b]\]. Increasingly, the diverse improvements being sought for flowmeters are producing corresponding improvements in the characteristics of flowmeter calibration systems. Of these systems, the piston displacement-type calibrator offers advantages such as compactness, mobility, efficient change of fluid, and prospects for state-of-the-art performance characteristics.
To characterize the performance of a piston displacement calibrator, which produces a pulsed output signal that is proportional to the volumetric flowrate, the objective is, generally, to determine the "pulses per volume displacement" ratio (or its reciprocal) where the pulse output is assumed to come from a source such as a linear encoder. This pulse output is also assumed to be proportional to the piston displacement. High accuracy calibrator performance requires examination of these assumptions.
A number of techniques can be used to determine volumetric displacement. It is assumed in what follows that the displaced fluid is a liquid, but the principles apply to gases or mixtures of gases and liquids as well. It is also assumed that both temperature and pressure effects on all the components of the piston displacement system, the cylinder, encoder, and fluid should be considered in order that high accuracy performance can be achieved.
Depending upon the desired uncertainty level for the performance of the calibrator, one or more of the pressure and temperature effects on the components of the system may be negligible. When this is so, it may be permissible to disregard such effects to simplify data processing or to reduce the size of the controlling software for the system. Alternatively, and more preferably, all effects can be included in computer software. In this way, the terms which are negligible will not influence the results when higher levels of uncertainty, i.e., less precise performance can be tolerated or is desirable from benefits vs. costs perspectives. More importantly, where high accuracy is required, more of the figures available via the software capabilities can be accepted as significant.
The volumetric-type calibrator system, using encoded piston displacement as both the flow source and as the flow determination scheme, is sketched in [Fig. 1](#f1-jresv97n5p509_a1b){ref-type="fig"}. The piston motion produces and measures a fluid volumetric flowrate that is proportional to the encoder frequency. The piston in the cylinder has a seal that is assumed to seal perfectly for all piston velocities. The corresponding fluid flowrate through the meter and the meter output frequency enable a calibration of the flowmeter. Using these elements, three steps are considered.
The first step is determining the calibrator factor which is an "encoder pulses per volume displaced" ratio (or its reciprocal) at the defined set of reference conditions. These results can be obtained experimentally in a number of ways: (1) by physical measurement, (2) by the so-called "draw" technique, or (3) by using a transfer standard such as a single or, preferably, a tandem arrangement of calibrated turbine flowmeters.
The second step is the use of the characterized calibrator to calibrate a pulsed-output flowmeter such as a turbine meter or other device where the meter factor is a "pulses per volume" quantity or its reciprocal. This process is done with one or more selected fluids and at specified conditions. In what follows turbine flowmeters will be considered. Conventionally, turbine meter results are produced in the form of a meter factor which has units of pulses per volume or volume per pulses (referenced to specified conditions). This meter factor is determined over the desired ranges of fluid conditions and flowrate expressed in terms of a ratio of inertial-to-viscous effects such as a Reynolds number, or equivalent parameter.
The third step is the use of the characterized turbine flowmeter to calculate a fluid flowrate under actual conditions of use. The results can be produced with respect to specified reference conditions or to the actual conditions, depending upon the needs of the meter operator.
The purpose of this paper is to describe these three steps and give the pertinent relationships that pertain to each procedure. The resulting equations are intended to be used in the software packages used with these types of calibrators and metering units. In this way, it is expected that the measurement performance of both the calibrators and the metering units can be maximized.
2. Calibrator Characterization
==============================
2.1 Geometrical Determination at Reference Conditions
-----------------------------------------------------
To perform the required measurements at reference conditions and then calculate the calibrator factor in units of pulses per fluid volume displaced, the system analyzed is that sketched in [Fig. 2](#f2-jresv97n5p509_a1b){ref-type="fig"}. The assumption of reference conditions is a conceptual situation that is impractical to achieve precisely but is done solely for convenience, as will be clear in what follows. For the conditions selected to be the reference conditions which are denoted by the "0" subscript, a specific piston stroke produces a displacement volume, *V*~C0~, and the encoder produces the corresponding number of pulses, *N*~E0~. A list of symbols is given in the Glossary. The reference conditions of calibrator temperature and the pressure in the calibrator are *T*~C0~ and P~C0~, respectively. These properties are assumed to be constant and steady in the calibrator volume. These conditions should be monitored, quantified, and assessed with respect to the performance level of the calibrator. Furthermore, the fluid inside and outside the calibrator is assumed to have the same temperature as the cylinder of the calibrator and no heat is being transferred to or from the calibrator. In all that follows, the reference conditions of *T*~0~ and *P*~0~ will be assumed to be the same for all components.
The specified piston stroke, *L*~E0~, produces the pulse total, *N*~E0~, where $$N_{E0} = L_{E0}K_{E0},$$and *K*~E0~ is the encoder constant in pulses per length at the reference temperature condition, *T~0~.* The calibration constant can be written $$K_{C0} = \frac{N_{E0}}{V_{C0}} = \frac{L_{E0}K_{E0}}{{\overline{A}}_{C0}L_{E0}} = \frac{K_{E0}}{{\overline{A}}_{C0}},$$in units of pulses per volume, where, at the reference conditions, *Ā*~C0~ is the averaged cylinder cross-sectional area over the piston stroke, *L*~E0~. This calibrator constant, *K*~C0~ is assumed to be constant over the operating range of the calibrator.
For the calibrator configuration shown in [Fig. 1](#f1-jresv97n5p509_a1b){ref-type="fig"}, the area *Ā*~C0~ is an annular one between the cylinder of the calibrator and the rod or tube attached to the piston. The precision with which *K*~C0~ is determined can be written using root-sum-square combinations of component precisions: $$\begin{array}{l}
{\frac{\Delta K_{C0}}{K_{C0}} < \left\lbrack {\left( \frac{\Delta N_{E0}}{N_{E0}} \right)^{2} + \left( \frac{\Delta V_{C0}}{V_{C0}} \right)^{2}} \right\rbrack^{1/2}} \\
{= \left\lbrack {\left( \frac{\Delta K_{E0}}{K_{E0}} \right)^{2} + \left( \frac{\Delta{\overline{A}}_{C0}}{{\overline{A}}_{C0}} \right)^{2}} \right\rbrack^{1/2},} \\
\end{array}$$where the numerators of the respective terms refer to the maximum errors of each of the component measurements. [Equation (3)](#fd3-jresv97n5p509_A1b){ref-type="disp-formula"} indicates that high levels of precision in *K*~C0~ can be attained when large pulse sums, *N*~E0~, and large displaced volumes, *V*~C0~, are used. Correspondingly, these precision levels can be achieved with accurate and sensitive linear encoders and accurately measured and large cross-sectional areas.
2.2 Geometrical Determination at Non-Reference Conditions
---------------------------------------------------------
To perform the required measurements at non-reference temperature and pressure conditions, it is assumed that these conditions are constant and steady as shown in [Fig. 3](#f3-jresv97n5p509_a1b){ref-type="fig"}. For a specified piston stroke, *L*~E~, the corresponding encoder pulse total, *N*~E~, is $$N_{E} = N_{E}K_{E},$$where the encoder constant, *K*~E~, is assumed to depend only on temperature according to $$K_{E} = K_{E0}\left\lbrack {1 - \alpha_{E}\left( {T_{E} - T_{E0}} \right)} \right.,$$where *α*~E~ is the pertinent linear expansion coefficient for the encoder and *T*~E~ and *T*~E0~ are, respectively, the encoder temperatures at non-reference and reference conditions.
The cross-sectional area | {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
The mutation of p53 protein is one of the frequent causes of human cancer since it has a key role in numerous cell stability and proliferation functions^[@CR1]^. On the other hand, MDM2 is an oncogene protein that negatively regulates p53^[@CR2]^. MDMX (MDM4) is also a homolog for MDM2 and acts as p53 inhibitor^[@CR3]^. In vivo and in vitro studies have shown the overexpression of both MDM2 and MDMX proteins in several cancer types^[@CR4]^ (e. g. MDMX overexpression up to 92% of AML cases^[@CR5]^). However, their mechanisms of action are slightly different as MDM2 mainly degrades p53 protein using its E3 ubiquitin ligase activity, while MDMX suppresses p53 by decreasing its transactivation activity and increasing MDM2 function^[@CR6]^.
Both MDM2 and MDMX (MDM2/X) are significantly overexpressed in cancer cells harboring wild type p53^[@CR7],[@CR8]^. As a result, the inhibition of MDM2/X-p53 interaction leads to the restoration of p53 activity and subsequent tumor suppression^[@CR9]^. From the structural point of view, MDM2/X share a similar p53-binding domain of the N-terminal hydrophobic pocket. This site engages hydrophobic bonds with p53 residues of Phe19, Trp23, and Leu26, which leads to the p53 transcriptional activity suppression^[@CR10],[@CR11]^.
To pharmacologically antagonize the MDM2/X-p53 interaction, several molecules have been developed. In this path, small molecule inhibitors, such as Nutlin-3 were mostly unsuccessful in clinical trials^[@CR12]^. Although its derivative, RG7112, has shown some promising results, its unsuitable inhibition of MDMX was one of the main drawbacks^[@CR13]--[@CR15]^. Alongside small molecules, some lead peptide sequences have also been developed, mostly by the alteration in the p53-MDM2/X binding site (ETFSDLWKLLPE), including 12/1 (MPRFMDYWEGLN)^[@CR16]^, \_ENREF_17PMI (TSFAEYWNLLSP)^[@CR17]^ and pDI (LTFEHYWAQLTS)^[@CR18]^. In what follows, there are several studies to improve the kinetics and dynamics of these peptides such as synthesizing stapled peptides^[@CR19],[@CR20]^. One of the first examples was SAH-8, designed by stapling p53-MDM2/X binding site sequence^[@CR21]^. More recently, ATSP-7041 has been developed as stapled pDI peptide which later led to ALRN-6924 peptide with promising anticancer results^[@CR22]^. Nevertheless, the main limitation in this area is the common low affinity of peptides to MDMX protein which reduces their effectiveness as anticancer agents^[@CR23],[@CR24]^. As a result, the optimization of lead peptides to obtain a "potent" and "dual" inhibitor of MDM2/X is an ongoing challenge. "Potent" is a common term for high affinity and stable therapeutic candidate which targets MDM2/X. On the other hand, any agent introduced to inhibit MDM2 is not robust enough to have an acceptable cytotoxic effect unless it could also inhibit MDMX.^[@CR18]^. Consequently, "dual" refers to the capability of the peptides to target both MDM2 and MDMX proteins. Most of the previous studies focused on the rational design of p53-based peptides are usually lacking the consideration of dual inhibition of MDM2/X^[@CR25],[@CR26]^. In this regard, one of the promising approaches is using in silico and theoretical workflows such as Molecular Dynamic (MD) simulation to optimize and verify drug candidate peptides^[@CR27]--[@CR29]^. Accordingly, in this study, we applied a computational structure-based approach inclusively on experimentally validated p53-based peptides, including p53, pDI, pDIQ, and pMI to; A) study their "potent" and "dual" inhibitor activities against MDM2/X, B) find critical residues playing roles in higher affinity to MDM2/X and helicity conservation, C) follow a computational high-throughput point mutation screening on pDI, (LTFEHYWAQLTS) and pDIQ, (ETFEHWWSQLLS) to analyze and validate structurally and functionally. To these ends, 27 different MD simulations and follow-up analyses were performed to assign the suitable features of native and mutated peptides against MDM2/X. The appropriate correlation between achieved theoretical and confirmed experimental results highlighted the suitability of the suggested methodology in this study, which could lead to the implicit rational peptide design in this area.
Materials and methods {#Sec2}
=====================
Atomic coordinates of peptide-MDM2/X structures {#Sec3}
-----------------------------------------------
The structures of peptides-MDM2/X complexes were extracted from Protein Data Bank (PDB). First, due to the lack of pDI-MDM2 structure with the total number of residues, the two close PDB codes of 3G03 and 3JZR (<https://www.rcsb.org/>) were taken. The final coordinates were achieved by the superimpositions and refinement of the mentioned PDB structures utilizing Chimera software. The coordinates of other structures were directly extracted from PDB codes of 3FDO, 4HFZ, 3DAB, 3EQS, 3EQY, 3JZS, and 3JZQ for pDI-MDMX, p53-MDM2, p53-MDMX, PMI-MDM2, PMI-MDMX, pDIQ-MDM2, and pDIQ-MDMX, respectively. The "repair PDB" command of FoldX v.3 program was employed to optimize the structures and add the missing atoms.
High-throughput mutation screening of pDI and pDIQ using FoldX {#Sec4}
--------------------------------------------------------------
To screen a large number of possible mutations of pDI and pDIQ peptides, the FoldX program was applied. The FoldX program computes the interaction energy robustly based on the empirical data extracted from protein engineering studies which makes it a notable assistance for peptide designing. First,\_ENREF_29 the "PSSM" interface of FoldX was utilized to mutate all the residues of peptides to 20 native amino acids (mutation to the residue itself was also carried out) in pDI-MDM2/X and pDIQ-MDM2/X complexes. Using BuildModel and AnalyseComplex interfaces upon each mutation, the final optimized structure and the interaction energy of the peptide-MDM2/X complexes were obtained. The FoldX force field was used to calculate the free energy of unfolding (ΔG) as described below.$$\documentclass[12pt]{minimal}
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\begin{document}$$\Delta G = \left( { 0.33 \times \Delta G_{{{\text{vdw}}}} } \right) + \Delta G_{{{\text{solvH}}}} + \Delta G_{{{\text{solvP}}}} + \Delta G_{{{\text{wb}}}} + \Delta G_{{{\text{hbond}}}} + \Delta G_{{{\text{el}}}} + \Delta G_{{{\text{kon}}}} + T\Delta S_{{{\text{mc}}}} + T\Delta S_{{{\text{sc}}}}$$\end{document}$$
where the terms are free energy difference of total van der Waals energy of all atoms (Δ*G*~vdw~); solvation energy for hydrophobic and polar groups (Δ*G*~solvH~ and Δ*G*~solvP~ , respectively); the water bridges (Δ*G*~wb~); the hydrogen bonds (Δ*G*~hbond~); the electrostatic bonds (Δ*G*~el~ and the electrostatic bonds of subunit molecules (Δ*G*~kon~). Meanwhile, Δ*S*~mc~ is defined as the entropy change when the backbone is changed to a particular conformation, and Δ*S*~sc~ is the entropy change when the side chain is transformed into an appropriate structure. *T* is the temperature^[@CR30]^.
The input structures of pDI-MDM2/X and pDIQ-MDM2/X for the FoldX program were described in the "[Atomic coordinates of peptide-MDM2/X structure](#Sec3){ref-type="sec"}s" section. After each mutation on the peptides' residues, the variance of interaction energy (ΔE) was calculated. Subsequently, the extracted structures with the lowest ΔE were selected for further studies. The final atomic coordinates of selected mutant peptides_MDM2/X complexes were used as reference structures for MD simulations.
Molecular dynamics simulations {#Sec5}
------------------------------
GROMACS-2018 package^[@CR31]^ was applied to perform 200 ns simulation on 27 different systems (total time of 5.4 μs) using the GROMOS96 54a7 force field. Solvation of each system was conducted via the SPC water model in the a triclinic box that the distances of molecules' centers to the edges were 15 Å. All the simulation systems were neutralized by the suitable number of Na + and Cl- ions with the final ionic concentration of 150 mM. The van der Waals interactions | {
"pile_set_name": "PubMed Central"
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Background {#Sec1}
==========
Early supported discharge (ESD) with continued rehabilitation in the home has been shown to be beneficial among patients with mild to moderate stroke. The ESD- model for rehabilitation was introduced in the late 1990s and includes an interdisciplinary team with appropriate recourses that coordinates the discharge and plan, supervise and continue the rehabilitation in the home environment \[[@CR1]\]. This form of rehabilitation accelerates the discharge from hospital, reduce long term dependency and admission to institutional care \[[@CR1]--[@CR4]\]. However, the criticism has been raised that most of the randomized controlled trials on ESD services were published more than 10 years ago \[[@CR5], [@CR6]\]. Today, the majority of patients are being discharged home early after stroke, due to access of hyper-acute therapies, and implement early interventions for carotid stenosis. There is a need to evaluate efficacy and safety of ESD in current stroke care settings and to adapt ESD service to local conditions for appropriate implementation \[[@CR1], [@CR5], [@CR6]\].
In Sweden, the majority (91% in 2014) of patients with stroke are cared for at a stroke unit (<http://www.riks-stroke.org>), but there has been no major expansion of ESD. Despite recommendations in the National Guidelines for stroke care \[[@CR7]\], the proportion of patients with stroke that receive ESD after stroke unit care has varied, dramatically across Sweden. Västerbotten County (Umeå Stroke Center and Skellefteå hospital) is one of the counties with high a proportion of ESDs (<http://www.riks-stroke.org>, \[[@CR8]\]).
We previously described the method, content, and outcome of ESDs according to the Umeå Stroke Center model in Västerbotten County \[[@CR9]\]. This model showed that it was possible to adopt and implement ESD for patients with stroke in Umeå. The model included important key elements for an effective ESD service \[[@CR6], [@CR8]\], such as a multidisciplinary team with experience in stroke rehabilitation, appropriate resources, periodic team meetings, and continuous evaluations of outcome with standardized measurements. Our previous results showed that ESD services reduced patient dependence in activities in daily living (ADL) and increased patient mobility, without increasing the risk of accidental falls or other injuries \[[@CR9]\]. The patients were very satisfied with the ESD-service. However, that observational implementation study did not include a control group.
The present study aimed to evaluate patient-reported outcomes measures (PROMs) among patients with stroke that received modern stroke unit care, and compare PROMs between those that received or did not receive ESD. The ESD was delivered according to a previously described model \[[@CR9]\]. We hypothesized that patients that received ESD would exhibit improved PROMs regarding satisfaction with rehabilitation (primary outcome), activity in daily living (ADL), tiredness/fatigue, pain, dysthymia/depression, general health status and information about stroke (secondary outcomes) compared to controls.
Methods {#Sec2}
=======
For this case control, observational study, we retrieved data from the Swedish Stroke Registry, Riksstroke \[[@CR10]\], and from the Longitudinal Integration Database for Health Insurance and Labor Market Studies (LISA). Information from the Swedish Stroke Registry was linked to the LISA database through personal identification numbers. This study was approved by the Regional Ethics Review Board at Umeå University (Dnr 2012--179-32 M, 2014--273-32 M).
Register {#Sec3}
--------
Currently, all 72 hospitals that treat patients with acute stroke participate in the Swedish Stroke Registry, Riksstroke \[[@CR10]\], which started in 1994. The primary aim of Riksstroke is to monitor and support improvements in the quality and implementation of new methods in stroke care in Sweden. The registry includes patients with ischemic and hemorrhagic stroke and data on first-ever and recurrent strokes. The acute phase questionnaire of Riksstroke contains basic patient characteristics (age, sex, living conditions, history of previous stroke, and comorbidities), diagnosis, level of consciousness on arrival, pharmaceutical treatments, complications, and the sequence of care (type of stroke care, organization, and department). In Riksstroke the hospitals that care for patients with acute stroke have been divided into three categories: university hospitals (9 pcs), specialized nonuniversity hospitals (23 pcs) and community hospitals (40 pcs) \[[@CR11]\]. Riksstroke also includes 3-month and 12-month follow up questionnaire that describe patient-reported outcomes and rehabilitation after stroke. The 3-month and 12-month questionnaire is administrated by the hospitals and filled in by the patients.
The LISA database at Statistics Sweden includes information on all Swedish citizens, starting at 16 years of age. In particular, it includes socioeconomic factors, like disposable income, education, and country of birth.
Participants and setting {#Sec4}
------------------------
All patients registered in the Riksstroke registry with a first-ever diagnosis of acute stroke between 1 January, 2010 and 31 December 2013 were included in the present study, when they fulfilled the following criteria: diagnosis of ischemic or hemorrhagic stroke; mild to moderate stroke severity at admission (measured as level of consciousness on a scale of 1--3, according to the Reactive Level Scale, RLS85) \[[@CR12]\]; living at home; and independency in ADL at stroke onset. Patients that met the inclusion criteria were divided into an ESD intervention group and a control group (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1Flow chart of the study inclusion procedure. ESD: early supported discharge
ESD intervention group {#Sec5}
----------------------
Västerbotten County have three hospitals with different primary catchment areas that cared patients in acute and sub-acute phases of stroke. Two of these hospitals, the Umeå Stroke Center (university hospital) and Skellefteå hospital (community hospital) had similar organizations regarding stroke care~~.~~ Both hospitals had stroke unit care followed by ESD according to a previously described model \[[@CR9]\]. These hospitals provided ESD to a comparatively large proportion of patients (41.2% at Umeå Stroke Center and 57.5% at Skellefteå hospital) compared to other hospitals in Sweden. The intervention group (ESD group) consisted of consecutive patients with stroke that received modern stroke unit care, followed by the ESD-service. During the study period, ESD was delivered to 41.2% of all patients with stroke admitted to the Umeå Stroke Center and 57.5% admitted to Skellefteå. Patients with severe stroke and those who died during hospitalization were not included in the ESD intervention group.
Control group {#Sec6}
-------------
The control group consisted of patients treated at stroke units in hospitals that cared for patients with acute stroke. At these hospitals, a low (\< 5% of all stroke patients) proportion of patients were given ESD. Therefore, the control group, which fulfilled the same inclusion criteria as the intervention group also included some patients that received ESD (\< 5%). Patients with severe stroke and those who died during hospitalization were not included in the control group. Hospitals in Sweden that only partially implemented ESD (5--20% of stroke patients) and those who registered other models of home rehabilitation were excluded.
Variables {#Sec7}
---------
Patient characteristics for both groups included sex, age, stroke subtype, treatment with thrombolysis, domestic companions, mobility, hypertensive treatment, diabetes, atrial fibrillation, and smoking before stroke onset. The level of consciousness upon admission and the length of hospital stay were also considered baseline characteristics of the two groups. These variables were retrieved from the Riksstroke registry. Information on education and country of birth was retrieved from the LISA database. The level of education was classified as primary school, secondary school, or university level. Country of birth was categorized as Sweden, Nordic countries (Sweden excluded), Europe (Nordic countries excluded), or other countries.
The outcome variables for this study were PROM results from the 3-months follow up recorded in Riksstroke. The primary outcome was satisfaction with the rehabilitation after discharge. Secondary outcome variables were; satisfaction with information provided about stroke, tiredness/fatigue, pain, dysthymia/depression, general health status and ADL dependence (mobility, toileting, and dressing). Data from the PROM values recorded in Riksstroke at the 3-months follow up have been validated against established measurements with the finding of accurate reliability (<http://www.riks-stroke.org>).
Some questions had multiple choice responses, and for our analysis, the responses were dichotomized. For example, patients were asked the questions: "How satisfied or dissatisfied are you with the rehabilitation or training you received after your stay in the hospital?" and "How satisfied or dissatisfied are you with the stroke information provided?"; and they responded with one of the following options: very satisfied, satisfied, dissatisfied, very dissatisfied, no need of rehabilitation, needed, but did not receive rehabilitation, or I do not know. And dependent categories were taken as reference (code = ref) for variables measuring a favourable outcome (satisfaction with rehabilitation, information about stroke, general health and Adl function) When assessing outcome variables that means an unfavourable result (tiredness/fatigue, pain, depression) we used a 'positive' reference (code = ref).
Similarly, the questions: "Do you feel tired/fatigue?", "Do you have any pain?", and "Do you feel depressed?" had response options; never, almost never, sometimes, often, constantly, or do | {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Bladder cancer is one of the most common malignant tumors. Most cases progress to high-grade invasive cancer despite long-standing intravesical therapies. Novel therapeutic treatment options are urgently needed to improve the overall treatment success rates for localized bladder cancer^\[^[@r1]^\]^. Therefore, stable, reliable, simple, and reproducible orthotopic animal models are critically important. Suitable animal models provide an opportunity to study the mechanism of pathogenesis and allow the research and development of novel therapeutic agents.
In this study, we have successfully established a model of orthotopic bladder tumor in mice using a drift angle stylet to injure the mucous membrane of the urinary bladder. The tumor cells grew on the wall of the urinary bladder after tumor cell injection. This method is convenient, rapid, and stable for establishing bladder tumor in mice and demonstrates the growth, infiltration, and metastasis of the tumor.
Materials and Methods
=====================
Preparation of the drift angle stylet
-------------------------------------
The stylet of the 24\# venous retention needles (Becton Dickinson Infusion Therapy Systems, Inc., America) was bent in a 5° to 7° angle at a distance of 15 mm from the needlepoint to form a Φ=2.61 mm to 3.66 mm circle when the stylet was rotated ([**Figure 1**](#f1){ref-type="fig"}).
{#f1}
Cell strain
-----------
Mice urinary bladder transitional cancer cell line BTT-T739, which originated from inbred line T739 mice, is a carcinogen-induced \[N-butyl-N-(4-hydroxybutyl) nitrosamine\] poorly differentiated transitional carcinoma. The cell line was provided by Professor Yang Xiaofeng from Shanxi Medical University. Human urinary bladder T24 cell strain was purchased from Shanghai Institutes for Biological Science, CAS. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO~2~. The cells were harvested by trypsin/EDTA treatment. Viability was determined using the trypan blue exclusion method. The cells were suspended in prepared PBS at a concentration of 1×10^7^/mL.
Animal
------
The present study was approved by the local ethics committee and followed the guidelines of the National Research Council Guide for the Care and Use of Laboratory Animals. Female inbred line T739 mice, 4 weeks to 6 weeks old and weighing 20 g to 22 g, were purchased from Beijing HFK Bio-Technology Co. Ltd. The animal produce license number was SCXK (Jing) 2009-0004. Female Balb/C-nu-nu mice, 4 weeks to 6 weeks old and weighing 20 g to 22 g, were purchased from Vital River Laboratories. The animal produce license number was SCXK (Jing) 2006-0009. The animals were bred in the animal laboratory (license number of SYXK (Jin) 2007-0002). The temperature and humidity of the environment was kept at (26±1.5)°C and between 40% and 60%, respectively. The mice were provided with sterilized drinking water and sterile complete nutrition feed.
The orthotopic transplantation of the tumor cell
------------------------------------------------
The mice in the experimental group were narcotized by sodium pentobarbital at a dosage of 60 mg/kg. The pipe casing was lubricated with liquid paraffin and inserted into the bladder cavity, and then urine was removed. The drift angle stylet was slowly inserted into the pipe casing. The pipe casing was fixed with one hand, the stylet was rotated for five rounds with the other hand, and then pulled out. Cell suspension (100 µL) of approximately 1×10^6^ cells was injected immediately. The mice in the normal control group were injected with 1×10^6^ cells directly without mucosa injury. The comparison results between the mucosa injured and uninjured urinary bladder are shown in [**Figure 2**](#f2){ref-type="fig"}.
{#f2}
Observation method
------------------
The tumor cell-inoculated mice were fed routinely. The activity and body weight of the mice were observed daily, and survival time was recorded. All mice that died of natural causes were dissected and checked for tumor formation on the urinary bladder and abdominal cavity and hematuria. Metastasis and wet weight of the urinary bladder were recorded after the dissection. The urinary bladder and tumor metastasized organs were fixed in 10% formaldehyde for pathological examination. The observation time of the T739 mice was 40 d, whereas that of the Balb/C-nu-nu mice was 60 d.
Results
=======
**Tumor developmen**t
---------------------
A total of 60 T739 mice were inoculated with BTT cells in the experimental group. The results showed that the bladder tumor incidence was 100% and the average wet weight of the tumor was (0.54±0.37) g. Ten T739 mice were inoculated with BTT cells in the mucosa uninjured control group; the bladder tumor incidence was 0% ([**Table 1**](#t1){ref-type="table"}**,** [**Figure 3**](#f3){ref-type="fig"}).
###### Comparison of tumor incidence between the mucosa uninjured bladder and mucosa injured bladder by drift angle stylet.
Groups Number of mice Cell inoculation amount Tumor developed amount Tumor incidence (%)
----------------------------- ---------------- ------------------------- ------------------------ ---------------------
T739 without injury 10 1×10^6^ 0 0
T739 with injury 60 1×10^6^ 60 100
Balb/C-nu-nu without injury 10 1×10^6^ 0 0
Balb/C-nu-nu with injury 60 1×10^6^ 60 100
{#f3}
A total of 60 Balb/C-nu-nu nude mice were inoculated with T24 cells in the experimental group. The results showed that the bladder tumor incidence was 100% and the average wet weight was (0.11±0.13) g. The bladder tumor incidence in the normal control group was 0% ([**Table 1**](#t1){ref-type="table"}**,** [**Figure 3**](#f3){ref-type="fig"}).
Metastasis
----------
Metastases in the liver, kidney, mesentery, and peritoneum with bloody ascites were observed in 23% of the 60 T739 mice inoculated with BTT cells. Abdominal cavity adhesion was observed, and metastasis occurred either on several viscera (liver, kidney, mesentery, and peritoneum) or only one viscera (kidney) ([**Figure 4**](#f4){ref-type="fig"}). Only one of the 60 Balb/C-nu-nu mice inoculated with T24 cells had metastasis in the kidney ([**Figure 4**](#f4){ref-type="fig"}).
{#f4}
Survival time
-------------
T739 mice in the experimental group began to die on the 16th day after BTT cell inoculation. The animals had serious athrepsia with hematuria. The average survival time of the mice was (26.69±9.24) d. In addition, a number of mice died from extreme athrepsia and cachexia ([**Table 2**](#t2){ref-type="table"}). Balb/C-nu-nu mice in the experimental group began to die on the 18th day after T24 cell inoculation. The average survival time of the mice was (34.59±9.8) d. The mice had extreme athrepsia and cachexia ([**Table 2**](#t2){ref-type="table"}**,** [**Figure 5**](#f5){ref-type="fig"}).
###### Average survival time and bladder weight of the mice transplanted with tumor cells.
Groups Number of mice Average survival time (days) Average tumor wet weight
----------------------------- ---------------- ------------------------------ --------------------------
T739 without injury 10 \>40 0.02±0.22
T739 with injury 60 26.69±9.24 0.54±0.37
Balb/C-nu-nu without injury 10 \>60 0.02±0.32
Balb/C-nu-nu with injury 60 34.59±9.80 0.11±0.13
{#f5}
Pathological examination
------------------------
The bladder mucosa of the normal control mice was intact, and no metastasis was observed ([**Figure 6A**](#f6){ref-type="fig"}). Huge solid | {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Osteoporosis is a prevalent bone disease that is defined by a low bone mass and an increased risk of fractures \[[@B1]\]. Adequate regulation of bone remodeling in adulthood is essential to maintain a healthy bone mass \[[@B2]\]. However, an imbalance between bone formation and bone resorption in pathological conditions or during the aging process can result in conditions that lead to osteoporosis \[[@B3]\]. In the past, the process of bone remodeling was considered to be regulated by two major cell types: bone-forming osteoblasts and bone-resorbing osteoclasts \[[@B4]\]. Currently available agents used to treat osteoporosis include estrogen, raloxifene, the bisphosphonates (e.g., alendronate and risedronate, etc.), and calcitonin. Their mechanisms are based on the inhibition of osteoclastic bone resorption to prevent further bone loss \[[@B5]\]. However, many osteoporotic patients already had lost a substantial amount of bone mass before diagnosis \[[@B6]\]. Furthermore, many side effects of antiresorptive agents have been reported, causing many patients to discontinue their use \[[@B7]--[@B9]\].
Drugs with anabolic effects have received much recent attention for osteoporosis therapy. These pharmacologic agents can ultimately stimulate new bone formation, enhance bone density, reduce bone fracture, and promote bone health. Currently, there is only one available anabolic agent on the market approved for the treatment of osteoporosis, recombinant human parathyroid hormone (hPTH). However, hPTH is limited for use in cases of severe osteoporosis \[[@B10]\], and the clinical treatment period is limited to 18 months \[[@B11]\]. Consequently, it is necessary to develop other potential anabolic agents with fewer undesirable side effects to prevent or reverse osteoporosis.
The Chinese yam (*Dioscorea*) has been widely used as a herbal medicine in China for more than 2000 years. Ancient Chinese medicinal books or folk remedies have provided evidence that *Dioscorea* might have effects in regulating bone metabolism. Therefore, we initiated a project to evaluate the effects of an ethanol extract of rhizomes of *Dioscorea alata* L. cv. Phyto, Dispo85E, on bone formation and to delineate the mechanisms involved. In our study, we found that Dispo85E promoted bone formation by inducing mesenchymal stem cells (MSCs) differentiation into osteoblasts rather than adipocytes and that it also possessed antiosteoporotic activity *in vivo*.
2. Materials and Methods {#sec2}
========================
2.1. Animals {#sec2.1}
------------
Specific pathogen-free (SPF) C57BL/6 female mice, 8 to 10 weeks of age, were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Animals were housed at a constant temperature and fed with laboratory chow (PMI, Brentwood, Mo, USA) and water *ad libitum*. The protocol of the experiments was approved by the Animal Research Committee of National Yang-Ming University (Guide for Animal Experiments, National Yang-Ming University).
2.2. Preparation of Dispo85E {#sec2.2}
----------------------------
Dried and peeled tubers of *Dioscorea alata* L. cv. Phyto (2.92 kg) were extracted with 85% ethanol (EtOH) (15 L each, three times) for 24 hours. The extract was filtered through an 11 *μ*m Whatman filter, then concentrated under vacuum evaporator and lyophilized, giving a yield of 3.05%. The extract was stored at −20°C before use.
2.3. HPLC Analysis of Dispo85E {#sec2.3}
------------------------------
Twenty milligrams of dried Dispo85E was dissolved in 2 mL of dichloromethane (DCM). After centrifugation, the supernatant was evaporated to dryness, adjusted to a concentration of 5 mg/mL in DCM and then subjected to normal-phase high performance liquid chromatography (NP-HPLC) with a 100 *μ*L injection volume. The pellet was evaporated to dryness, adjusted to a concentration of 10 mg/mL in water and then subjected to reverse-phase high performance liquid chromatography (RP-HPLC) with a 100 *μ*L injection volume. RP-HPLC profiling was performed using a Mightysil RP-18 column (4.6 × 250 mm, 5 mm) at room temperature. The mobile phase was methanol and water in gradient mode (10 : 90--100 : 0 over 120 minutes). The effluent was monitored at 254 nm, and a constant flow rate was set at 0.8 mL/minute. NP-HPLC profiling was performed with a Cosmosil 5SL-II column (4.6 × 250 mm, 5 mm) at room temperature. The mobile phase was DCM and methanol (MeOH) in gradient mode as follows: 100%--98% DCM in MeOH (0--15 minutes), 98%-95% DCM (15--25 minutes), 95%--90% DCM (25--35 minutes), 90%--80% DCM (35--45 minutes), and 80%--70% DCM (45--55 minutes). The effluent was monitored at 254 nm, and a constant flow rate was set at 0.8 mL/minute.
2.4. Cell Culture {#sec2.4}
-----------------
Primary mouse bone marrow cells and C3H10T1/2 cells were cultured as described previously \[[@B12], [@B13]\]. Briefly, bone marrow cells were obtained from the femoral bone of SPF-grade C57BL/6 female mice. The bone marrow cells were collected by flushing the diaphysis with *α*-minimum essential medium (*α*-MEM, Gibco BRL) through a 23-gauge needle. After flushing, the bone marrow cells were filtered through a no. 53 sterile nylon mesh to obtain a single cell suspension. The cells were cultured in osteogenic medium (*α*-MEM medium supplemented with 15% fetal calf serum (FCS, Gibco), 50 *μ*g/mL ascorbic acid (Sigma), 10 mm sodium *β*-glycerophosphate (Sigma), and 10 nm dexamethasone (Sigma)). The pluripotent murine mesenchymal cell line C3H10T1/2 was cultured in Dulbecco\'s modified Eagle\'s medium (DMEM) supplemented with 10% FCS.
For the alkaline phosphatase (ALP) activity assay, primary bone marrow cells were seeded in a 96-well microplate at 2.5 × 10^5^/well and cultured in osteogenic induction medium. After two days, half of the medium was changed and the cells were incubated with Dispo85E at the indicated concentrations for 4 days. ALP activity for each cell lysate was assayed. C3H10T1/2 cells were grown to confluence in standard medium. One day after the cells reached confluence, the medium was replaced with permissive osteogenic/adipocytic medium \[[@B14]\] (DMEM with 10% FCS, 50 *μ*g/mL ascorbic acid, 10 *μ*M sodium *β*-glycerophosphate, 10 nm dexamethasone, 10 nm all-trans retinoic acid (Sigma), 10 *μ*g/mL insulin (Sigma), and 50 *μ*M isobutylmethylxanthine (IBMX, Sigma)) containing drugs at the indicated concentrations. The medium was changed every 4 days. After 12 days, ALP activity for each cell lysate was assayed.
For the nodule formation assay, primary bone marrow cells were seeded in a 24-well microplate at 1 × 10^6^/well and cultured in osteogenic induction medium. After two days, half of the medium was changed and the cells were incubated with Dispo85E at the indicated concentrations. The culture medium was renewed every 4 days. After 14 days, the mineralization of bone marrow cells was analyzed.
For lipid staining, C3H10T1/2 cells were grown to confluence in standard medium. One day after the cells reached confluence, the medium was replaced with adipogenic medium (DMEM with 10% FCS, 10 *μ*g/mL insulin, 1 *μ*M dexamethasone, and 0.5 mm IBMX) containing drugs at the indicated concentrations for 4 days. The cells were then incubated in a standard medium containing 5 *μ*g/mL insulin and drugs at the indicated concentrations. The medium was changed every 4 days. The oil red O staining was performed at postinduction day (PID) 12. Nile red staining was performed at PID16.
2.5. Alkaline Phosphatase Activity Assay {#sec2.5}
----------------------------------------
ALP activity was assayed at 37°C by a method modified from that of Qu et al. \[[@B15]\]. In brief, cell layers were washed twice with phosphate buffered saline (PBS) and extracted into lysis solution (10 mm Tris, 0.1% Triton X-100 buffer (pH 7.5)). Enzyme activity was determined colorimetrically using p-nitrophenylphosphate (p-NPP, Sigma) as a substrate. The reaction mixture contained 8 mm p-NPP, 2 mm mgCl~2~, and 0.5 M 2-amino-2-methyl-1-propanol, pH 10. After 10 minutes of incubation, the color change of p-NPP to p-nitrophenol was monitored at 405 nm. Cell viability was determined by resaz | {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Dancing is a very rare seizure semiology. To the best of our knowledge, there have been only a few case reports of ictal dancing or dancing-like movement. The previous reports provided either of description only or video only; or the case could hardly be called dancing. The "dancing epilepsy" was shown in a case of refractory complex partial seizures \[[@CR1]\]. However, this article did not show the electroencephalography. Therefore, we cannot confirm whether the dancing was truly ictal or only a manifestation of post-ictal confusion. A report of 'tap dancing in epilepsy' showed a video with simultaneous electroencephalography in which rhythmic leg and foot tapping \[[@CR2]\]. However, there was only slow wave activity, which indicates "post-ictal" dancing rather than "ictal". In a recent report of a patient with left temporal lobe epilepsy \[[@CR3]\], the authors provided a video with simultaneous electroencephalography but it could hardly be called dancing. Here, we present a case of dancing semiology in a patient with refractory temporal lobe epilepsy.
Case presentation {#Sec2}
=================
A 42-year-old right-handed woman suffered from weekly repetitions of unconscious dancing for 5 years, despite of multiple antiepileptic drugs including levetiracetam 3000 mg/d, valproate 900 mg/d, and pregabalin 300 mg/d. She was admitted to the epilepsy monitoring unit of a tertiary referral center for the feasibility of epilepsy surgery. On the initial clinical examination, she had no other symptoms or signs. The routine laboratory tests were negative. Magnetic resonance imaging showed right hippocampal atrophy (Fig. [1](#Fig1){ref-type="fig"}). Her habitual seizure was recorded during video-electroencephalography monitoring. The seizure began with the right hand automatism and ictal speech, which suggest that the ictal onset zone would be on the right side. An evolution of rhythmic delta activity was observed in the right temporal area beginning 16 s after the automatism (see Additional file 1: Video S1). As the ictal discharge spreads to the left temporal area, which means the secondary generalization, the ictal speech disappeared. After 20 s from the secondary generalization, she had rhythmical movement of her legs, similar to stepping through a dance, and the simultaneous video-electroencephalography showed regional slow waves over both of her frontal areas. When the ictal rhythm has switched to the left side, the left upper limb automatism and immobility of the right upper limb represented the rhythmic theta activity, which is still seen in the left temporal area. Taken together, we could identify the kicking and stepping like a dance as well as shaking left arm. According to her husband, the movement would have involved twirling dance, making a right turn when she was standing. However, it was not shown on the video. The dancing lasted even after the rhythmic discharge, which definitized the post-ictal dancing. We surmise that the "dancing" movement might be derived from some combination of automatism consisted of complex, rhythmic, and sequencial movement. She was completely amnestic with respect to the episode. Her ictal speech and the ictal electroencephalography imply that the right hippocampus atrophy should be the epileptogenic focus. There has been only one seizure for 3 months since a stereotactic gamma knife surgery applied to the atrophic right hippocampus.Fig. 1Magnetic Resonance Imaging. T2-weighted oblique coronal magnetic resonance imaging revealed right hippocampal atrophy
Additional file 1:Simultaneous Video-electroencephalography Monitoring. Description of data: The seizure begins with right hand automatism and ictal speech, followed by an evolution of rhythmic delta activity in the right temporal area. After the secondary generalization, she showed rhythmical 2movement of legs, similar to stepping through a dance. Subsequent post-ictal paraphasia as well as confusion were documented. (WMV 12mb)
The authors declare that they adhered to CARE guidelines/methodology.
Conclusions {#Sec3}
===========
This patient showed a semiology which can be classified as complex partial seizure followed by hypermotor seizure consisting of rhythmic movements of the extremities termed dancing \[[@CR4]\]. Although it is difficult to distinguish dancing from hypermotor semiology, the former connotes more complex, rhythmical and sequencial movement of multiple body regions. Here, we report the most plausible dancing in the ictal and post-ictal state, documented by simultaneous video and electroencephalography. The underlying mechanism and symptomatogenic zone remain to be investigated.
Not applicable.
Funding {#FPar1}
=======
Not applicable.
Availability of data and materials {#FPar2}
==================================
All data analyzed during this study are included in this published article and its supplementary information files.
Authors' contributions {#FPar3}
======================
KKT made substantial contributions to acquisition of data. KKT and LSK analyzed and interpreted the video-electroencephalography and magnetic resonance imaging. KKT and CK involved in drafting the manuscript and revising it critically for important intellectual content. LSK has decided to submit this manuscript for publication. All authors read and approved the final manuscript.
Authors' information {#FPar4}
====================
Sang Kun Lee:
Professor at Seoul national university hospital
Department of Neurology, Comprehensive Epilepsy Center, Seoul national university hospital, Seoul, Republic of Korea
Member of American Epilepsy Society
The current Editor-in-Chief of Journal of Epilepsy Research and Journal of Korean Epilepsy Society
Consulting Editor of Epilepsy Research
Kon Chu, MD:
Associate Professor at Seoul national university hospital
Department of Neurology, Comprehensive Epilepsy Center, Seoul national university hospital, Seoul, Republic of Korea
General Councilor of Korean Society for Stem Cell Research
Member of Basic Science Committee of Korean Epilepsy Society
Keun Tae Kim: MD
Fellowship at Seoul national university hospital
Department of Neurology, Comprehensive Epilepsy Center, Seoul national university hospital, Seoul, Republic of Korea
Competing interests {#FPar5}
===================
The authors declare that they have no competing interests.
Consent for publication {#FPar6}
=======================
Written informed consent for publication of the clinical details, clinical image and video was obtained from the patient.
Ethics approval and consent to participate {#FPar7}
==========================================
This study is in accordance with the Declaration of Helsinki. This report has been approved by the Ethics Board of Seoul national university hospital biomedical research institute. All authors agreed the publish statements of BMC Neurology.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-ijerph-17-01813}
===============
The current evidence-based paradigm requires that the most reliable scientific evidence is used to increase patient safety by predicting how a certain intervention might affect health and well-being. This is true both in a strictly clinical context, where doctors and patients need to evaluate the potentiality of interventions for the single case, and in public health, where policy interventions are needed at the population level. How should such evidence best be generated? This is an ongoing debate in medicine and public health, but also in philosophy of science.
Recently, there has been some discussions about the epistemic primacy of statistical approaches, such as randomised controlled trials (RCTs), for establishing causality and predict the health outcomes of medical interventions. In the recent article 'Understanding and Misunderstanding Randomized Controlled Trials', for instance, Deaton and Cartwright summarise a number of arguments against the dominant reliance on RCTs over every other form of causal evidence \[[@B1-ijerph-17-01813]\]. This article has received considerable attention, partially because it is written within a growing movement that calls for evidential pluralism in the medical and health sciences \[[@B2-ijerph-17-01813],[@B3-ijerph-17-01813],[@B4-ijerph-17-01813],[@B5-ijerph-17-01813],[@B6-ijerph-17-01813],[@B7-ijerph-17-01813]\]. The main idea is that evidence from RCTs can provide useful causal information, but only when combined with evidence from other methods for causal inquiry. The purpose of causal evidence would then be to develop the theoretical knowledge and to advance the understanding of 'the hows and whys' underlying the statistical correlations.
Scholars who argue that we need plural methods to establish causality use different types of arguments and have different emphasis. Some traditions, such as critical realism, are primarily critical of the ontological bias in scientific methodology; they argue that standard ways to evaluate causal evidence mistakenly rely on a positivist, Humean conception of the nature of causality \[[@B2-ijerph-17-01813],[@B8-ijerph-17-01813],[@B9-ijerph-17-01813]\]. Others are more concerned with epistemological issues of how we can know about causality using different methods, without considering the ontological question of what causality is \[[@B5-ijerph-17-01813],[@B10-ijerph-17-01813]\]. However, the ontological and epistemological traditions generally agree that what counts as 'the best causal evidence' differs depending on the research question and the research context. This stance is in opposition to a formal, probabilistic tradition which is primarily interested in the mathematical relationship between a certain hypothesis and a certain evidence, without considering the specific causal content or explanation to the evidence itself \[[@B11-ijerph-17-01813]\].
Although the formal approach is still dominant, there is increasing awareness among scientists that the evaluation of causal hypotheses cannot be restricted to purely statistical standards \[[@B12-ijerph-17-01813],[@B13-ijerph-17-01813]\]. In a recent commentary in *BMJ Evidence Based Medicine*, a large group of clinicians, medical researchers and philosophers made a common appeal to evidence-based medicine to expand its notion of 'evidence': "The rapid dominance of evidence based medicine has sparked a philosophical debate concerning the concept of evidence... The undersigned include 42 clinicians and philosophers from interdisciplinary research networks working specifically on questions related to causality in medicine worldwide. Our research has developed out of a conviction that philosophical analysis ought to have a direct impact on the practice of medicine. In particular, if we are to understand what is meant by 'evidence', what is the 'best available evidence' and how to apply it in the context of medicine, we need to tackle the problem of causality head on... Establishing causality often requires the use of multiple methods, since no single method will be universally applicable or perfect for this purpose\[[@B14-ijerph-17-01813]\] (p. 1)"
How should this call for evidential pluralism be understood? How useful are the various methods available for causal inquiry? Further, how should different types of causal evidence be evaluated? Among philosophers of science, various answers to this question have been proposed. For instance, Russo and Williamson (2007) state that a causal claim must be supported both by evidence of difference-making from population studies and from evidence of the underlying mechanism, and that each of these approaches compensate for the weakness of the other \[[@B15-ijerph-17-01813]\]. Others are trying to expand formal approaches to the inclusion of different types of methods and the amalgamation of different types of evidence \[[@B16-ijerph-17-01813],[@B17-ijerph-17-01813]\].
In this paper, we propose our own version of evidential pluralism. This version is based on the philosophical idea that any type of scientific claim, including causal claims within medicine and public health, should seek to say something about a system's intrinsic properties, as well as their mutual influence and causal interaction. Our aim here is to introduce a framework to support scientists in developing appropriate methodological approaches for exploring causality. This is relevant for ensuring that all the evidence necessary for the safe treatment of individuals and populations is considered.
2. Dispositions and Science {#sec2-ijerph-17-01813}
===========================
In her discussion about causal evidence and scientific methods, Cartwright urges that the whole scientific enterprise is about finding properties, or potentialities, which she calls *stable capacities* \[[@B18-ijerph-17-01813]\]. For instance, an RCT might show that, for a certain sample of population, aspirin relieves headache in more instances than a sugar pill does. This observation is interesting insofar as it allows us to make a claim about a capacity of aspirin, which is generated from its properties. Such capacity, we can say, then works as the truth-maker of causal claims: 'aspirin has a capacity to (causally) relieve headache'.
What Cartwright calls 'capacities' are also commonly referred to as *causal powers*, or *dispositions*, with a very similar meaning \[[@B19-ijerph-17-01813],[@B20-ijerph-17-01813]\]. Despite some disagreements over philosophical details, proponents of capacities, powers and dispositions generally agree that these are properties or potentials of things or systems, that can become manifested under certain conditions. The philosophy of dispositions descends from an ancient tradition going back to Aristotle, but which has had a revival in recent decades. Indeed, in philosophy of science, there has been a tendency to shift the attention away from ideal and extrinsic laws of nature onto the intrinsic causal powers of things and systems \[[@B18-ijerph-17-01813],[@B21-ijerph-17-01813]\]. Shifting the attention to the dispositions, or properties, of a system corresponds to looking at the causal mechanism, the interactions and the dynamics at place. It is no longer sufficient to notice a lawlike regularity in nature, for instance, that holds under some ideal, normal or similar conditions. Rather, one now wants to understand the intrinsic dispositions and causal capacities of the system that allow such regular behaviour \[[@B20-ijerph-17-01813]\]. In other words, one is now seeking a deep causal understanding about *why* and *how* something might or might not happen. This contrasts with the investigation into *whether* and *how often* something happens, which is typically tested via statistical significance, relative frequencies, and so on \[[@B22-ijerph-17-01813]\].
There is a flourishing literature about the metaphysics of dispositions and powers (recent edited volumes include \[[@B9-ijerph-17-01813],[@B23-ijerph-17-01813],[@B24-ijerph-17-01813],[@B25-ijerph-17-01813],[@B26-ijerph-17-01813]\]). The question here is whether thinking in terms of dispositions can help the discussion about causal evidencing in medicine and public health, and if so, how? If we acknowledge that the health sciences should be concerned with understanding the underlying causal powers, capacities or dispositions of observable processes and events, then what are the specific advantages and disadvantages of different types of scientific evidence for this purpose? There has been comparative little discussion on these topics, at least to our knowledge.
In what follows, we will address these questions in more detail. As the theoretical framework for our discussion, we adopt a particular idea within the literature on dispositions: the theory of causal dispositionalism, developed by Anjum and Mumford over the last decade (for a detailed overview of the theory, see \[[@B27-ijerph-17-01813]\]). The aim is to make the methodological implications of this specific philosophy of causality more explicit and show the relevance of considering philosophy of causality for medicine and public health.
3. Dispositionalism about Causality {#sec3-ijerph-17-01813}
===================================
In the Anjum--Mumford dispositionalist theory, causes come from dispositions or 'causal powers'. What is a disposition in this specific view, and how exactly does it relate to causality?
A disposition is, in this view, an *intrinsic* property, belonging to some particular thing, individual or process. That dispositions are intrinsic in this way is crucial for causality in medicine. For instance, an intervention cannot be said to cause an effect unless it has an intrinsic disposition toward the recovery. Dispositional properties can exist unmanifested. A woman can be fertile without ever becoming pregnant, but once fertility is manifested in pregnancy, causality has happened. From this philosophical perspective, causality requires more than a single disposition. In order to manifest its effect, the disposition must interact with a number of other dispositions, or | {
"pile_set_name": "PubMed Central"
} |
Background
==========
Schizophrenia is a severe mental disorder with grave personal and social costs \[[@B1]\]. Approximately 1% of the population develops schizophrenia during their lifetime. Over the years, many genes have been reported to be responsible for the susceptibility to schizophrenia \[[@B2]\]. In general, schizophrenia is considered to be a complex disease with multiple genetic and environment etiological factors. Linkage analysis, association and positional cloning studies and candidate gene approaches \[[@B3]\] have been successful in identifying risk genes. The way in which multiple genes, each possibly having a small individual contribution, leads to vulnerability and then the pathophysiology, remains to be elucidated. In order to figure out the relationship among those genes, we should investigate not only in gene-gene interaction level but also a whole picture at the protein level. Recent works to map the protein-protein interaction (PPI) in human to curate human metabolism and regulatory networks offer the relationships among different disease genes \[[@B4],[@B5]\]. The protein clusters in the network may represent the modules with biological functions \[[@B6]\]. It is also reported that if the disease candidate genes are treated as a phenotype, these genes are likely to be function together in the normal cell \[[@B7]\].
In this study, we provided a novel strategy by taking advantages of PPI to discover the regulatory mechanisms among disease candidate genes. We speculated that disease candidate genes may cluster together in a functional network at a protein level. Protein complexes interact with preferred partners to form a biological module serving a specific collective function \[[@B8]\]. When using a network-clustering method by calculating the pairwise distance in the protein interaction network \[[@B6]\], two major protein clusters were found which were involved in synaptic transmission and signal transduction protein cluster. We proposed a model to explain the interaction between NRG1 and CACNG2 which not only fell into the synaptic transmission cluster at protein interaction level but also associated at the gene-gene interaction level.
Recent molecular studies implicate neuregulin1 (NRG1) as the most promising risk factor for schizophrenia \[[@B9],[@B10]\]. Liu and colleagues also found suggestive linkage evidence of schizophrenia to loci near NRG1 on chromosome 8p21 in an ethnically distinct Taiwanese sample \[[@B11]\]. There is also evidence that this genetic risk is elevated when accompanied by genetic changes in the gene for ErbB4, one of neuregulin\'s binding partners. NRG1-mediated ErbB signalling has important roles in neural development \[[@B12]-[@B14]\], as well as in the regulation of neurotransmitter receptors thought to be involved in the pathophysiology of schizophrenia \[[@B15]\]. Hahn and colleagues suggest that enhanced endogenous NRG1-ERBB4 signalling may be responsible for N-methyl-D-aspartate receptors (NMDARs) hypofunction of the disease state \[[@B16]\]. NMDA receptors are a major subtype of glutamate receptors and mediate slow excitatory postsynaptic potentials (EPSPs). Glutamate is the major excitatory neurotransmitter in the brain, and it has been proposed that disruption in glutamate signalling may underlie many of the symptoms of schizophrenia \[[@B17]\]. NRG1 reduces the tyrosine phosphorylation of NMDA receptors, a modification that is triggered by the binding of NMDA or glutamate. NMDAR hypofunction may contribute to the symptomatic features of schizophrenia \[[@B18]\]. ERBB4 associates with NMDAR via DLG4 (also called PSD95), and the binding to DLG4 is probably involved in the enhanced activation of ERBB4. This association provides a physical link between ERBB4 and the NMDAR. These findings add to our basic understanding of glutamatergic transmission, which has been implicated in the pathogenesis of schizophrenia.
CACNG2, also known as stargazin, was found to interact directly with AMPA receptor and allow interaction of the receptor with the scaffold proteins of the postsynaptic density, such as DLG4 \[[@B19],[@B20]\]. In a previous linkage study of schizophrenia that included Taiwanese samples, CACNG2 was also reported as a vulnerability gene for neuropsychologically defined subgroups of schizophrenic patients \[[@B21]-[@B23]\]. Bats and colleagues found that a mutation in PDZ domain of CACNG2 will increase AMPA receptor diffusion. CACNG2 regulates trafficking of AMPA-type glutamate receptors and stabilizes them at the postsynaptic density when neurotransmitters are received \[[@B20]\].
Results and discussion
======================
Products of candidate disease genes form two major clusters in a schizophrenia-related protein interaction sub-network
----------------------------------------------------------------------------------------------------------------------
There are two major types of reactions which are complex formation and covalent modification in the signalling pathway. Both types of reactions have protein-protein interactions (PPI), which can be detected by high throughput methods. It has been shown that proteins which are involved in the same pathway, are likely to cluster together in the PPI network \[[@B8]\]. If the candidate genes may increase the risk of acquiring a disease synergistically, it implies that these genes are likely to work together in the normal cell \[[@B7]\]. Taking these two observations together, it is likely that the products of candidate genes may cluster together in a protein network. Therefore, we have collected 36 reported candidate genes from the literature and used them as a query set to retrieve the nearest neighbours of the candidate proteins. There were in total 831 human proteins retrieved from 6 major PPI databases (see Materials and Methods section). Interestingly, the retrieved interactions linked the gene products of these candidate genes in a big cluster even though these genes were found by a different approach (Figure [1](#F1){ref-type="fig"}). This result implies that the products of these candidate genes may be important candidates and they will work together in the cell.
{#F1}
In order to cluster proteins that are close to one another, the distances between every pair of these 831 proteins were computed by using a standard algorithm based on shortest-path of network topology \[[@B6]\]. On the basis of these pairwise distances, two major clusters (Figure [2](#F2){ref-type="fig"}) were found by using a visualization tool, called Generalized Association Plot (GAP) \[[@B24]\]. By examining the enriched gene ontology terms for the members of these two protein clusters \[[@B25],[@B26]\], the possible function of these clusters was identified. As shown in figure [1](#F1){ref-type="fig"}, the small cluster (cluster 1), which contains PPP3CC, NOTCH4, RASD2 and BMP6 genes, may be involved in signal transduction. The large cluster (cluster 2), which contains the NRG1 and CACNG2 genes, may be mainly involved in synaptic transmission and sometimes in neural development. NRG1 and CACNG2 were both reported as vulnerability genes from an association study of schizophrenia that included Taiwanese samples \[[@B11],[@B22]\]. These two genes were found to have strong interaction on the basis of linkage and association studies (unpublished data). Thus, it would be interesting to see how these functionally unrelated genes act synergistically in the development of schizophrenia.
{#F2}
Discovering the protein interactions between NRG1 with CACNG2 according to biological interpretationIn order to explore the detailed relation between NRG1 and CACNG2, the cluster 2 sub-network was extracted from the original large network for further study. This cluster contains 7 gene products of candidate genes, which content DPYSL2, CRTC1, DISC1, CACNG2, KCNJ12, PTK2B and NRG1, and 204 interacting proteins; part of this cluster is shown in Figure [3](#F3){ref-type="fig"}. The NRG1 protein is connected to CACNG2 protein via the ERBB and DLG protein families, which are known to be involved in glutamatergic signalling process \[[@B12]\]. Since cluster 2 proteins were retrieved by the nearest neighbor approach, this subset of proteins will definitely lose some interacting proteins excluded from the sub-network in the disease forming process. Hence, the second or even third neighbors may be needed to propose a biologically plausible mechanism. This step was done manually and the goal is to recover the proteins that may affect synaptic transmission. Therefore, membrane receptors that may link the function of NRG1 and CACNG2 were added to this sub-network. Two major protein families were added, which were the NMDA receptor subunits and the AMPA receptor subunits, respectively. Both receptors are calcium channels that are triggered by glutamate \[[@B27]\]. Thus, they have the potential to act synergistically. DLG4 protein is an important intermediate between NRG1 and CACNG2, because DLG4 protein is interacting with ERBB4, which may receive a signal from NRG1. On the other hand, DLG4 is interacting with both the NMDA and AMPA receptors. As a result, both receptors may receive the NRG1 signal from neighboring neuron cells.
![**Sub-network linked NRG1 with CACNG2 according to biological interpretation**. This small network linked NRG1 with CACNG2 via the ERBB4 and | {
"pile_set_name": "PubMed Central"
} |
1. Introduction
===============
Many attempts have been made to use breeding or molecular biological methods to modify the ability to produce secondary metabolites in medicinal plants. Among the challenges being addressed, manipulations of the morphine biosynthesis in the opium poppy (*Papaver somniferum* L.), particularly the conversion of narcotic morphine to codeine, which is of high importance as an antitussive and a synthetic source of dihydrocodeine, or to thebaine, which is also an important starting material for the semi-synthesis of the analgesic oxycodone, will contribute to the control of narcotics, and to the supply of useful alkaloids for the production of pharmaceuticals.
The gradual elucidation of enzymology of the alkaloid biosynthesis in *P. somniferum* led to genetical engineering of alkaloid biosynthetic pathway using native genes. The first report was on the introduction of a gene encoding berberine bridge enzyme (BBE) to *P. somniferum* in antisense orientation \[[@B1-pharmaceuticals-05-00133]\]. To date, several reports on metabolic engineering of *P. somniferum* have appeared, such as RNAi-mediated gene silencing of codeinone reductase (COR) \[[@B2-pharmaceuticals-05-00133]\], overexpression of COR \[[@B3-pharmaceuticals-05-00133]\], overexpression and antisense co-suppression of (*S*)-*N*-methylcoclaurine-3\'-hydroxylase (CYP80B3) \[[@B4-pharmaceuticals-05-00133]\], overexpression and RNAi-mediated gene silencing of salutaridinol-7-*O*-acetyltransferase (SalAT) \[[@B5-pharmaceuticals-05-00133]\], and RNAi-mediated gene silencing of SalAT \[[@B6-pharmaceuticals-05-00133]\]. Mutant poppy *top1* \[[@B7-pharmaceuticals-05-00133]\] which accumulates thebaine and oripavine as major alkaloids instead of morphine was also established by the treatment of mutagen (ethyl methanesulphonate) and screening of progeny plants.
The T-DNA insertional mutant clone of *P. somniferum* PsM1-2, which we developed by the infection of the *Agrobacterium rhizogenes* strain MAFF03-01724, regenerated shoots from embryogenic callus that lacked the ability to produce morphine. Codeine was detected as a major alkaloid in this *in vitro* shoot culture \[[@B8-pharmaceuticals-05-00133]\]. By the improvement of the alkaloid analysis and proceeding studies on this mutant, thebaine (*ca.* 55 μg/g dry weight) and codeine (*ca.* 20 μg/g dry weight) were found to be the major opium alkaloids in the *in vitro* regenerated shoots \[[@B9-pharmaceuticals-05-00133]\]. The information provided from this mutant, which shows an altered alkaloid composition, might make an important contribution to the further modification of alkaloid production in *P. somniferum*, and therefore we carried out genetic and phenotypic analyses on this mutant.
Recently, long unidentified enzymes involved in the two demethylation steps in the conversion of thebaine to morphine were successfully identified as non-heme dioxygenases \[[@B10-pharmaceuticals-05-00133]\]. These two enzymes, namely, thebaine 6-*O*-demethylase (T6ODM) and codeine *O*-demethylase (CODM), represent the first known 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze *O*-demethylation. The altered alkaloid composition in the PsM1-2 mutant may be due to the genetic mutation in the conversion steps from thebaine to morphine. In the present study, an expression analysis of these two enzymes together with selected genes involved in the morphine biosynthesis was carried out to reveal the molecular mechanism of the mutation.
2. Results and Discussion
=========================
2.1. Morphological Characteristics of the PsM1-2 Mutants
--------------------------------------------------------
The days to flowering, number of petals, appearance of split on the boundary of the petal, and height of the aerial part at the seed-filling stage of soil-cultivated T~0~ mutant and selfed progenies are summarized in [Table 1](#pharmaceuticals-05-00133-t001){ref-type="table"}. The T~0~ primary mutant showed delay of flowering and dwarfness. In addition, a deep split was observed on the boundary of the petal ([Figure 1](#pharmaceuticals-05-00133-f001){ref-type="fig"}). Delay of flowering was consistantly observed in the progenies. The number of petals, which was not altered at the T~0~, varied in the T~1~, T~2~ and T~3~ progenies. A deep split at the boundary of the petal was observed in 45% of T~1~ plants, 33% to 83% of T~2~ plants, and 8.3% and 10% of T~3~ plants.
pharmaceuticals-05-00133-t001_Table 1
######
Summary of the morphological characteristics of PsM1-2 T~0~ mutant, selfed progenies, and WT plant.
Progenies Lines Number of Plants Days to Flowering (Mean ± SD) (days) Number of Petals: Percentage (%) Split on Petal Boundary (%) Plant Height (Aerial Part) (Mean ± SD) (cm)
----------------- ------- ------------------------------ --------------------------------------------------- ---------------------------------- ----------------------------- ---------------------------------------------
T~0~ WT 1 47 \*^1^ 4: 100 0 60.0
T~0~ 1 71 \*^1^ 4: 100 100 38.0
T~1~ WT 6 53.5 ± 4.8 4: 100 0 42.4 ± 5.8
T~1~ 60 100.6 ± 14.6 ^\#\#\#\#^ 3: 1.7, 4: 41.7, 5: 35.0, 6: 16.7, 7: 3.3, 8: 1.7 45.0 52.1 ± 8.5 ^\#\#^
T~2~ WT 12 53.3 ± 4.0 3: 25.0, 4: 75.0 8.3 36.0 ± 7.6
\#1-27(HT) 15 90.8 ± 12.6 ^\#\#\#\#^ 5: 60.0, 6: 33.3, 10: 6.7 60.0 44.7 ± 5.4 ^\#\#^
\#2-17(HT) 6 79.8 ± 2.5 ^\#\#\#\#^ 5: 50.0, 6: 33.3, 8: 16.7 83.3 45.1 ± 3.4 ^\#^
\#2-1(LT) 12 83.3 ± 6.8 ^\#\#\#\#^ 5: 66.7, 6: 16.7, 7: 8.3, 8: 8.3 33.3 35.6 ± 7.8
\#2-6(LT) 10 76.4 ± 3.6 ^\#\#\#\#^ 5: 10.0, 6: 40.0,7: 30.0, 8: 10.0,12: 10.0 80.0 39.4 ± 3.1
T~3~ WT 6 109.4 ± 0.9 \*^2^ 4: 100 0 80.3 ± 5.8
\#1-27(HT)L\#2 10 129.2 ± 11.9 \*^3,\ \#\#\#^ 4: 40.0, 5: 50.0, 6: 10.0 10.0 45.8 ± 7.9 ^\#\#\#\#^
\#2-17(HT)\#2-1 12 131.1 ± 7.3 \*^4,\ \#\#\#\#^ 3: 8.3, 4: 75.0, 5: 16.7 8.3 47.0 ± 13.4 ^\#\#\#\#^
\*^1^: Days after transplanting; \*^2^: n = 5; \*^3^: n = 9; \*^4^: n = 11; ^\#^*p* \< 0.05; ^\#\#^*p* \< 0.01; ^\#\#\#^*p* \< 0.005; and ^\#\#\#\#^*p* \< 0.001 *vs.* WT.
2.2. Alkaloid Composition in the PsM1-2 Mutants
-----------------------------------------------
The soil-cultivated PsM1-2 T~0~ primary mutant accumulated 16.3% (% dry weight) of thebaine as a major opium alkaloid in the latex, which was not detected in the WT ([Figure 2](#pharmaceuticals-05-00133-f002){ref-type="fig"}; [Table 2](#pharmaceuticals- | {
"pile_set_name": "PubMed Central"
} |
Learning pointsManagement decisions appropriate for a non-athlete might be inappropriate in an athlete as they may result in disqualification, financial loss, or put the athlete at additional risk of complications.Cardiologists with expertise in sports cardiology should be involved in the management of athletes as it is often complex and requires a holistic approach.Left atrial appendage occlusion devices can play an important role in reducing stroke risk in selected cases.
Introduction
============
Caring for athletes with cardiac disease requires an approach that caters to the specific needs of the individual. Often athletes require their care to fit around training and competition requirements and this can come into conflict with the best care their clinicians feel they can offer. Medications and interventions with proven symptomatic and prognostic benefit may affect athletes' performance and lead to poor adherence. Moreover, they may result in disqualification from competitive sports which are likely to carry both personal and financial consequences. In some individuals, engaging in demanding physical activity and competitive sports against medical advice may carry significant health risks. Therefore, shared decision-making is vital and alternative management strategies are often warranted to ensure appropriate adherence to prescribed treatment.
This case illustrates this conflict in a professional rugby player with a cardiomyopathy and atrial fibrillation (AF) on anticoagulation who wished to continue to play and discusses how an alternative approach was able to optimize his care.
Timeline
========
Date Events
--------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
November 2017 Admission with decompensated heart failure and new diagnosis of atrial fibrillation (AF).Transthoracic echocardiogram (TTE) showed evidence of left ventricular (LV) dilatation and severe LV systolic dysfunction.Discharged on heart failure medications and anticoagulation for stroke prevention.
January 2017 Unobstructed coronary arteries on angiography.Early recurrence of AF following direct current cardioversion.
February 2017 Left ventricular systolic function remained severely impaired on outpatient TTE.
April 2018 Cardiac magnetic resonance showed dilated cardiomyopathy with ejection fraction (EF) 37%. No scaring or fibrosis seen on late gadolinium enhancement.
July 2018 Right arm weakness and paraesthesia in keeping transient ischaemic attack.Screening for connective tissue disorders, HIV, syphilis, and Fabry disease was negative.
October 2018 Left ventricular systolic function returned to 'near normal' (EF 50%). Euvolaemic.Advised not to play rugby due to high bleeding risk on anticoagulation.
November 2018 Playing rugby on anticoagulation.Referred for consideration of left atrial appendage occlusion (LAAO) device.
July 2019 Successful LAAO device implantation.Anticoagulants stopped.
Case presentation
=================
A 27-year-old male professional rugby player was admitted to his local district general hospital with a 2-day history of chest tightness and breathlessness. He had no other significant past medical history and was not taking any regular medications. He admitted to regularly taking cocaine and performance-enhancing steroids. He was haemodynamically stable with normal saturation. The main findings on physical examination were an irregularly irregular pulse and bibasal crackles. His admission electrocardiogram (ECG) showed AF and his chest X-ray findings were in keeping with pulmonary oedema. Initial bloods tests were within normal range. Transthoracic echocardiogram (TTE) revealed bi-atrial dilatation and a moderately dilated left ventricle (left ventricular end diastolic diameter 7 cm) with mild concentric left ventricular (LV) hypertrophy with an ejection fraction (EF) 35--40%.
He was acutely managed with intravenous diuretics and initiated on evidence-based heart failure medications including beta-blockers and angiotensin-converting enzyme inhibitors. In view of his drug history, a working diagnosis of drug-induced dilated cardiomyopathy (DCM) was made. Once stabilized, he was discharged on bisoprolol 2.5 mg and ramipril 2.5 mg. In anticipation of a direct current cardioversion (DCCV), he was started on rivaroxaban 20 mg. He was counselled not to participate in any competitive sports.
To further investigate his cardiomyopathy, he underwent an outpatient coronary angiogram which revealed unobstructed coronary arteries. In addition, a cardiac magnetic resonance (CMR) confirmed a dilated left ventricle with globally impaired systolic function and a calculated EF of 37%. There was no evidence of scarring or fibrosis on delayed enhancement images. He was unable to maintain sinus rhythm following DCCV and relapsed back into persistent AF. His CHA~2~DS~2~-VASc score was 1 (LV dysfunction) which does not strictly mandate anticoagulation; however, he made an informed decision to continue rivaroxaban.
On a follow-up TTE performed 3 months later, LV systolic function remained unchanged. His ramipril was increased and he was initiated on eplerenone. Left ventricular function gradually improved on optimal medical therapy and, at 8 months of follow-up, had returned to near-normal (EF 50--55%). He remained in AF and experienced a brief episode of left arm weakness and paraesthesia suggestive of a transient ischaemic attack (TIA) despite being compliant with anticoagulation. As his CHA~2~DS~2~-VASc score increased to 3 (TIA, LV dysfunction) he now had a clear indication for anticoagulation. Connective tissue disorders, syphilis, and Fabry disease screening were negative.
At 1-year follow-up, he was asymptomatic (New York Heart Association 1) but remained in AF and continued to participate in competitive rugby, whilst on oral anticoagulation despite counselling regarding the high bleeding risk. He was reluctant to terminate his professional rugby career prematurely and sought alternative stroke prevention strategies that obviated the need for continuous anticoagulation. He was referred to a Tertiary Cardiology Centre for further management of his AF and consideration of a left atrial appendage occlusion (LAAO) device. A 27 mm Watchman Flx (Boston Scientific, MA, USA) device was successfully deployed under general anaesthetic in the left atrial appendage with a good seal and no leaks ([*Figures 1*--*4*](#ytz242-F1){ref-type="fig"}, [Supplementary material](#sup1){ref-type="supplementary-material"}). He was discharged home on a 6-week course of aspirin and clopidogrel therapy with a follow-up transoesophageal echocardiogram to assess LAAO device position and guide cessation of antiplatelet strategy.
{#ytz242-F1}
{#ytz242-F2}
{#ytz242-F3}
{#ytz242-F4}
Discussion
==========
Recommendations regarding participation in competitive sports should be given following comprehensive evaluation of the athlete's disease characteristics and a thorough risk assessment. The work-up includes a 12-lead ECG, echocardiography, CMR, 24-h Holter monitor, and cardiopulmonary exercise testing. Disqualification from competitive sports is likely to carry both personal and financial consequences for athletes.[@ytz242-B1] Ensuring that the athlete is involved in the decision-making process is therefore paramount.
This athlete was strongly advised not to engage any competitive sports, in line with the European Association of Preventive Cardiology (EAPC) recommendations. The EAPC position paper states that athletes with DCM should not participate in competitive sports if any of the following are present[@ytz242-B1]: symptomatic, orEjection fraction \<40%, orextensive late gadolinium enhancement (i.e. \>20%) on CMR, and/orfrequent/complex ventricular tachyarrhythmias on ambulatory ECG monitoring and exercise testing, orhistory of unexplained syncope.
Once his LV systolic function had improved there was no further restriction on exercise from a cardiomyopathy perspective.
On admission, his CHA~2~DS~2~-VASc score was 1 (LV dysfunction) which is not a strict indication to initiate oral anticoagulation \[Class IIA, level of evidence (LOE B)\] as the evidence supporting a net clinical benefit of oral anticoagulation in patient with a single stroke risk factor (excluding gender) is limited.[@ytz242-B2] Oral anticoagulation was started in anticipation of a DCCV and, after appropriate counselling, he made an informed decision to continue on rivaroxaban. However, his CHA~2~DS~2~-VASc increased to 3 (TIA, LV dysfunction) and he had definitive indication to continue term-anticoagulation (Class I, LOE A).[@ytz242-B2] As a professional rugby | {
"pile_set_name": "PubMed Central"
} |
Full text
=========
A new term, \'selective estrogen receptor modulator\'; (SERM), has infiltrated the estrogen receptor (ER) literature lately \[[@B1]\]. It is nothing more than the reaffirmation of an old fact, namely that different estrogens have different effects, in different tissues. The major natural estrogens -estradiol, estriol and estrone - bind ERs with differing affinities, hence variations in their tissue distribution and concentrations influence the extent of their estrogenic effects. Studies with synthetic estrogens have focused on antiestrogenic ligands, which bind ERs and interefere with the actions of the natural estrogens. Tamoxifen is the prototypical antiestrogen, and newer second-generation antagonists, such as raloxifene, are in various stages of clinical trials \[[@B2]\]. Both tamoxifen and raloxifene are SERMs, because their antiestrogenic effects are restricted to only certain tissues. Tamoxifen has been used for more than 20 years to treat ER-positive breast cancers \[[@B3]\]. It was first demonstrated to be effective in advanced disease, later in adjuvant settings, and most recently as a breast cancer preventant in women at high risk. Thus, in various settings tamoxifen is an inhibitory ER ligand in the breast, and this property explains both its efficacy and its widespread use.
The picture is not all rosy, however. True to its SERM nature, tamoxifen is not antiestrogenic in all tissues. For example, in the uterus tamoxifen is a potent estrogen, where, like estradiol (when unopposed by progestins), it induces epithelial hyperplasia and endometrial cancers \[[@B4]\]. The excitement surrounding raloxifene stems from the fact that, like tamoxifen, it is an antagonist in the breast, but, unlike tamoxifen, it lacks estrogenic activity in the uterus \[[@B2],[@B5]\]. In summary, tamoxifen can be either an agonist or an antagonist in normal tissues.
Unfortunately, the same duality of function operates in malignant tissues, including breast cancers. Almost without exception, breast cancers that initially respond well to tamoxifen by growth cessation or regression eventually resume growing despite the continued presence of the antagonist. How can this \'acquired resistance\'; be explained? Most tamoxifen-resistant tumors continue to express ER \[[@B6]\], suggesting that resistance is not simply due to outgrowth of a nonresponsive, ER-negative sub-population. Indeed, tamoxifen-resistant tumors remain responsive to growth inhibition by pure antiestrogens (but clinical data are sparse) and other hormonal therapies \[[@B3],[@B5]\]. Paradoxic reports of tumor stasis and even regression after tamoxifen withdrawal in resistant patients \[[@B7]\] suggest that in at least some resistant tumors the antagonist has switched to an agonist. Thus, for several years the notion has been advanced that the term \'resistance\' inappropriately describes such tumors, and that tamoxifen is not simply inactive (as implied by the term \'resistance\'), but, instead, that it has switched to an agonist, and actively stimulates tumor growth \[[@B8],[@B9]\].
That the same ligand can have opposing transcriptional and biologic effects has long been puzzling, but recent advances in our understanding of the molecular biology of steroid receptors has shed light on this paradox. We now know that transcriptional regulation by liganded, DNA-bound receptors is influenced by their association with multiprotein activator or repressor complexes. Detailed analyses of the identity and function of the constituent \'coregulatory\' proteins in these complexes are being carried out in many laboratories. They break down into two classes - coactivators and corepressors - and involve proteins with a variety of functions, including the following: enzymes such as acetylases, deacetylases, methyltransferases, ubiquitin ligases, proteases, ATPases and kinases; proteins with activator or repressor domains that stabilize or destabilize protein-protein interactions; scaffolding proteins involved in the assembly of multiprotein complexes; and even nonpeptide factors such as the steroid receptor RNA activator \[[@B10],[@B11]\].
What does this have to do with tamoxifen? It turns out that the activity of the tamoxifen-ER complex can be exquisitely modulated by the nature of the associated coregulatory proteins. Binding of corepressors, such as the silencing mediator for retinoid and thyroid receptors or nuclear receptor corepressor, suppresses the partial agonist activity of tamoxifen. At least one antagonist-specific coactivator, the L7 switch protein for antagonist, enhances the partial agonist activity of tamoxifen \[[@B9]\]. As a result of these basic molecular studies, there is now intense interest in correlating tamoxifen resistance in breast cancer with the underexpression of corepressors or the overexpression of coactivators. These proteins could clearly represent the next targets for therapeutic interventions. Additionally, although we have learned a great deal about steroid receptor coregulatory proteins in recent years, most investigators believe that only a minor subset have been identified to date. This is because the many subtle structural variations in the conformation of receptors that result from the binding of different ligands yield multiple subtly different targets on the receptor\'s surface for the binding of a variety of coregulators. It is this variability that can, in part, explain the tissue specificity and paradoxic agonist activity of ligands like tamoxifen.
The hunt is therefore also on to identify the large number of endogenous coregulatory proteins that are probably lurking in tissues, and, additionally, to synthesize their pharmacologic equivalents with a view to manipulating the functional direction of ligand-receptor complexes. In a recent paper, Norris *et al* \[[@B12]\] described a novel method to define an array of synthetic peptides that interact specifically with estradiol- or tamoxifen-occupied ER, and regulate their transcriptional activity. Several methods have recently been developed to select members of random peptide libraries based on their binding affinity to known protein targets \[[@B13]\]. In the method of phage-display, a library of phage, each displaying a different cloned peptide sequence on its surface, is exposed to a plastic plate coated with the target protein. Specifically bound phage are eluted, the phage are amplified, and the process is repeated for several rounds, after which the selected clones of interest are isolated from the phage, the DNAs are sequenced, and the peptides they encode are deduced. Norris *et al* \[[@B12]\] used tamoxifen- or estradiol-occupied ER as the target protein bound to the plate, and they ensured that the receptors would be in the appropriate DNA-bound structural conformation by precoating the plastic with DNA containing estrogen response elements. The screen led to the isolation of several, 15 amino acid peptides, representing three major classes: α /β I, which interacts with estradiol-occupied ER; α /β III or V, which interact with tamoxifen-occupied ER; and α II, which interacts with ER in the presence of either ligand, in the presence of a pure antiestrogen, and even in the absence of ligand.
The α /β I peptide SSNHQSSRLIELLSR interacts with ER only in the presence of estradiol, and not in the presence of SERMs like tamoxifen, raloxifene, GW7604, idoxifene, nafoxidene or the pure antiestrogen ICI182,780. In the presence of agonists, it also interacts with the progesterone receptor B-isoform, and glucocorticoid receptors. When overexpressed, α /β I and α II peptides reduce the transcriptional activity of estradiol, whereas α /β III or V have no effect, which is consistent with their inability to bind ER in the presence of the agonist. On the other hand, peptides α /β III or V are quite tamoxifen-specific for ER, but also bind antagonist-occupied progesterone receptors. Six peptides of the α /β V class were isolated, that had the consensus sequence (S/M)X(D/E)(W/F)(W/F)XXXL. α /β III or V, as well as α II, inhibit the partial agonist effect of tamoxifen, but do not alter transcription by estradiol-occupied ER. The inhibitory activity of these synthetic peptides thus resembles that of the natural corepressors SMRT and N-CoR \[[@B9]\]. It would be of interest to determine whether the complementary DNAs encoding these synthetic peptides could be used as probes to isolate additional endogenous corepressors from complementary DNA libraries. At present the list of known corepressors is much smaller than that of known coactivators \[[@B11]\], and it is unclear whether this discrepancy represents a true cellular condition, or whether it is an artifact due to the technical complexity of screening for corepressors.
Norris *et al* \[[@B12]\] speculated that each class of peptides recognizes different protein contact sites on the ER protein; contact sites that are generated specifically by the class of ligand bound to the receptors. They postulated that these contact sites could be targets for drug discovery. Analogous suggestions have previously been made for the use of corepressor or coactivator-occupied receptors to screen for new ligands \[[@B9]\]. The studies of Norris *et al* \[[@B12]\], along with those of others cited herein, indicate that we are at the brink of important insights into the molecular mechanisms by which ER and their ligands regulate hormone dependence and resistance in breast cancers. These insights will bring completely new approaches to treating these tumors, and if their promise is confirmed they will allow us to predict, and pehaps even prevent or reverse, development of resistance. It is an exciting time to be studying the roles of steroid hormones in breast cancer!
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
The assessment of human daily physical activity in population studies requires accurate, cheap, and feasible measurement technology [@pone.0061691-Corder1], [@pone.0061691-Wareham1], [@pone.0061691-Wong1]. Accelerometers are increasingly being used for physical activity assessment and most of the accelerometers that have been used in population studies express their output in proprietary units usually referred to as "counts" [@pone.0061691-Hagstromer1], [@pone.0061691-Colley1].
Accelerometer devices, based on acceleration sensors which allow for raw data storage expressed in g-units or SI units at a relatively high sampling frequency have been used in gait analysis [@pone.0061691-Brandes1], [@pone.0061691-MoeNilssen1] and ambulant activity classification [@pone.0061691-Aminian1], [@pone.0061691-Veltink1] for a number of years. The output of raw accelerometers is not summarized by the monitor allowing for increased control over data processing by the end-user in contrast to the traditional accelerometers. Technological developments in recent years have made raw accelerometry feasible for population research, allowing weeklong data collection.
A measured acceleration signal consists of a gravitational component, a movement component, and noise [@pone.0061691-Veltink1]. During static conditions or conditions of steady state non-rotational movement, the gravitational component is visible as the offset of one or more sensor axes and can then be used for detection of the sensor orientation relative to the vertical plane [@pone.0061691-Veltink1]. The separation of the gravitational component from the acceleration signal is complicated by the fact that in the presence of rotational movements the frequency domains of the movement-related component and the gravitational component can overlap, thus making simple frequency-based filtering inappropriate for perfect separation.
The first two studies that identified the challenge of separating the components of acceleration lacked a comparison against a reference method [@pone.0061691-Redmond1], [@pone.0061691-VanSomeren1]. Studies by Bouten et al. and Bourke et al. used a reference method, but were limited to laboratory experiments that may not generalise to accelerometer data collected under real life conditions [@pone.0061691-Bourke1], [@pone.0061691-Bouten1]. None of the studies as mentioned above systematically evaluated how metric accuracy varies across magnitudes and frequencies of acceleration. Characterisation of the latter may be important to gain insight into metric performance under real-life conditions.
The use of gyroscopes in addition to acceleration sensors could be regarded as the solution for separating the gravitational component from the acceleration signal [@pone.0061691-Roetenberg1], [@pone.0061691-Sabatini1], [@pone.0061691-Yun1]. However, these devices do not yet meet feasibility requirements for use in large scale observational research. Raw accelerometry has been applied in various epidemiological studies since it became sufficiently feasible in the period 2008--2010. Most of these studies are not published yet, but already amount to over ten thousand participants. None of these datasets include gyroscopic data and therefore require an accelerometer-specific solution.
The main objective of the present study was therefore to evaluate the ability of different methods (metrics) of processing acceleration signals to remove the gravitational component of acceleration by comparison against a reference method under a range of standardised kinematic conditions. A second objective was to assess the shared variance between these metrics in human physical activity data collected during daily life and the impact of metric selection on the accuracy with which daily energy expenditure can be estimated.
Methods {#s2}
=======
Ethics Statement {#s2a}
----------------
Ethical approvals were obtained from the Cambridgeshire research ethics committee, Cambridge (UK) and from the Regional Ethical Review Board in Umeå (Sweden).
Study Design {#s2b}
------------
The main experiment in this study was done with a robot and did not involve testing of human participants. Two additional sets of experiments were performed, the first to test the degree to which metrics convey similar information when applied to wrist and hip signals, and the second to assess the implication of such differences for estimation of daily physical activity-related energy expenditure.
Robot Experiment {#s2c}
----------------
An industrial robot (TX90, Stäubli Tec-Systems GmbH, Bayreuth, Germany; see [**Figure 1**](#pone-0061691-g001){ref-type="fig"}) was used to rotate accelerometers (GENEA, Unilever Discover, Sharnbrook Bedfordshire, UK) in the vertical plane following a general minimum-jerk oscillatory motion (single plane). The motion was applied to establish a standardized alternating contribution of gravity to the accelerometer output. The robot consists of an articulated arm with six joints from which the fifth joint counted from the base of the robot was used in this study. The oscillating motion was continuous (non-damping) around a single horizontal axis. The trajectory was programmed using a 7th order polynomial function with kinematic constraints **([Supporting Information S1](#pone.0061691.s001){ref-type="supplementary-material"})**. A high order function was needed to reduce the natural vibrations transmitted between the robot and its own base [@pone.0061691-Piazzi1], [@pone.0061691-Kyriakopoulos1]. An example of the angular position over time for one experimental condition is given in [**Figure 2**](#pone-0061691-g002){ref-type="fig"}.
{#pone-0061691-g001}
{#pone-0061691-g002}
The frequency of oscillation, the radius of rotational movement (shortest distance to centre of rotation), and the angular range of motion were systematically varied. The range of frequency conditions was limited by the maximal amount of mass moment of inertia and torques that could be absorbed by the robot and supporting frame. For all frequencies ranging from 0.05 Hz to 1.2 Hz, eighteen tri-axial accelerometers were positioned along the length of a 70 cm bar mounted to the flange of the robot at 10 cm from the centre of rotation. The application of eighteen accelerometers in parallel allowed for assessment of the relationship between metric output and the radius of movement. To reduce mass moment of inertia at the higher frequencies of oscillation (\>1.1 Hz) a shorter bar (20 cm) was used, see [**Figure 1**](#pone-0061691-g001){ref-type="fig"}. The shorter bar provided space for the attachment of only five accelerometers. The torque can be further reduced by reducing the range of angular rotation; some experimental conditions were defined by this constraint. For reference purposes, all eighteen accelerometers were also tested under static conditions (no robot movement) at angles 0° and 22.5°. Each experimental condition was done for three minutes. An overview of all experimental conditions is shown in [**Table 1**](#pone-0061691-t001){ref-type="table"}. For monitoring potential vibrations, a source of experimental error, one additional accelerometer was attached to the base of joint 5 for all experimental conditions. The base of joint 5, i.e. the robotic with its joint 1 up to joint 4, should in theory not move during these experiments.
10.1371/journal.pone.0061691.t001
###### Experimental conditions of the robot setup.
{#pone-0061691-t001-1}
Frequencies Angle range\* Number of accelerometers (range in position relative to axis of rotation)
-------------------------------------------------------- --------------- ---------------------------------------------------------------------------
0 Hz 0° and 22.5° 18 (0.13--0.78 m)
0.05 to 0.55 Hz (steps of 0.05) 0--90° 18 (0.13--0.78 m)
0.60, 0.70, and 0.80 Hz 0--45° 18 (0.13--0.78 m)
0.90, 1.00, and 1.10 Hz 0--20° 18 (0.13--0.78 m)
1.20 and 1.30 Hz 0--45° 5 (0.13--0.29 m)
1.4 to 2.6 (steps of 0.1), 2.8, 3.0, 3.2, 3.6 and 4 Hz 0--20° 5 (0.13--0.29 m)
\[\*for 0° the bar is in horizontal position and for 90° the bar is pointing upwards relative to the axis of rotation\].
Human Experiments {#s2d}
-----------------
In order to facilitate the interpretation of the robot experiment in the context of human daily (free-living) physical activity, we asked 47 men and 50 women (healthy, aged 22--65 yrs) to wear accelerometers on their wrist and on their hip for seven days during free-living as previously described [@pone.0061691-vanHees1]. We also re-anal | {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Prion diseases, such as bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and Creutzfeldt-Jakob disease (CJD), kuru and Gerstmann-Sträussler-Scheinker (GSS) syndrome in humans, are a group of neurodegenerative disorders caused by prions, self-replicating β-sheet-rich infectious polymeric assemblies of misfolded host-encoded cellular prion protein (PrP^C^)^[@CR1]--[@CR4]^. Whilst rare, prion diseases are an area of intense research interest, as it is increasingly recognised that other degenerative brain diseases, such as Alzheimer's and Parkinson's diseases, also involve the accumulation and spread of aggregates of misfolded host proteins through an analogous process of seeded protein polymerisation^[@CR2],[@CR5]--[@CR8]^. Consequently, study of 'prion-like' mechanisms has been recognised to have much a wider relevance to the understanding of neurodegenerative disorders^[@CR9]--[@CR11]^.
PrP^C^ is a cell surface, predominantly α-helical, glycosylphosphatidylinositol (GPI)-anchored glycoprotein that is sensitive to protease treatment and soluble in detergents^[@CR1]^. In contrast, prions may acquire protease-resistance and are classically designated as PrP^Sc^ (refs. ^[@CR12],[@CR13]^[@CR13]. PrP^Sc^ is found only in prion-infected tissue and is β-sheet-rich aggregated material, partially resistant to protease treatment, and insoluble in detergents^[@CR14]^.
Transmission experiments to transgenic mice provide strong supporting evidence that alternative conformers or assembly states of PrP^Sc^ encode multiple prion strains, which differ in their pathogenic properties^[@CR2],[@CR15]^. Transgenic mice expressing only human PrP with either valine or methionine at residue 129 have shown that this common human polymorphism constrains the propagation of distinct human prion conformers, and the occurrence of associated patterns of neuropathology consistent with the conformational selection model of prion propagation^[@CR16]--[@CR20]^. Heterozygousity at codon 129 is thought to confer resistance to prion disease by inhibiting homologous protein--protein interactions essential for efficient prion replication with the presence of methionine or valine at residue 129 controlling the propagation of distinct human prion strains^[@CR2],[@CR21]^. Biophysical measurements suggest that this powerful effect of residue 129 on prion strain selection is likely to be mediated via its effect on the conformation of the disease-associated PrP^Sc^ form, or its precursors or on the kinetics of their formation, as it has no measurable effect on the structure, folding or stability of PrP^C[@CR22]^.
The acquired prion disease kuru, which was epidemic amongst the Fore linguistic group of the Papua New Guinea highlands when first studied in the 1950′s, and which was transmitted during mortuary feasts, imposed strong genetic selection on the Fore, essentially eliminating residue 129 homozygotes^[@CR23]^. A novel variant of prion protein, V127, unique to the affected population in the epicentre of the kuru epidemic, was also identified^[@CR24]^. In this variant, the glycine at residue 127, which is fully conserved amongst vertebrate PrP primary structures, is substituted by valine. The V127 polymorphism was found on one copy of the *PRNP* gene in unaffected individuals within the population, suggesting that this polymorphism conferred resistance to prion disease, having been selected for in response to the kuru epidemic^[@CR23],[@CR24]^. The protection afforded by this polymorphism was modelled using transgenic mice expressing human PrP^[@CR25]^, and showed that heterozygous mice expressing both alleles containing glycine and valine at residue 127 (G/V127), echoing the human resistance genotype, exhibited profoundly reduced susceptibility to infection with kuru and classical CJD prions. Most importantly, however, and in complete contrast to the protective effect of the residue 129 polymorphism, homozygous mice expressing human PrP with solely valine at residue 127 (V127), showed total resistance to all inoculated human prion strains. A comparison of the incubation periods between hemizygous mice expressing wild-type G127 human PrP only, with heterozygous mice expressing both G127 and V127 PrP, indicated a dose-dependent dominant-negative inhibitory effect of V127 PrP on prion propagation, resulting in prolonged incubation periods and variable attack rates in heterozygotes^[@CR25]^. These data indicated that V127 PrP is intrinsically resistant to prion propagation and can inhibit propagation involving wild-type (WT) G127 PrP. In essence, this single amino acid substitution, at a residue completely conserved in vertebrate evolution, has as potent a protective effect on the host as a null mutation. Consequently, the structural and mechanistic basis of the protective effect of the V127 mutation is of keen interest as it may provide key insights into the mechanism of prion conformational conversion and recruitment.
As a first step in characterising the effect of this protective polymorphism on PrP, we undertook a detailed investigation of the effect of the residue 127 polymorphism on the biophysical properties of the native cellular PrP^C^ conformation using a combination of X-ray crystallography, NMR and equilibrium unfolding. We show that this mutation imposes local changes in backbone conformation which facilitate formation of intermolecular hydrogen bonds between native-state dimers and imposes conformational restrictions on this region of the protein. In addition, it significantly alters millisecond timescale conformational rearrangements in regions of PrP proposed to be important in prion transmission^[@CR26]--[@CR28]^. These effects may modulate the conversion of native PrP^C^ to a disease-associated form or on pathway intermediates relevant to the disease process, and provide a mechanistic explanation for the protective effect of this mutant.
Results {#Sec2}
=======
Choice of PrP variants studied {#Sec3}
------------------------------
Persons who were exposed to kuru and survived the epidemic were predominantly heterozygotes at PrP residue 129^[@CR23]^. The V127 protective polymorphism in human PrP was always present on an M129 allele^[@CR24]^, consequently our main interest was with the V127/M129 PrP variant. However, we took the opportunity, given the known biological effect of the residue 129 polymorphism to also study the V127 variant with valine at residue 129 (V127/V129), and both forms of wild-type PrP (G127/M129 and G127/V129) with the aim of dissecting the effects of both of these protective polymorphisms.
V127 PrP structures closely resemble wild-type G127 PrP {#Sec4}
-------------------------------------------------------
To determine whether the overall structure of PrP^C^ was affected by the protective V127 variant we crystallised recombinant human PrP (residues 119--231), with valine at residue 127, (V127/M129 and V127/V129), complexed with the Fab fragment of the anti-PrP antibody ICSM18, as performed previously with G127/M129 PrP (Supplementary Table [1](#MOESM1){ref-type="media"} and Supplementary Fig. [1](#MOESM1){ref-type="media"})^[@CR29]^. The crystal structures of both V127 variants (V127/M129, 2.3 Å resolution, pdb 6SV2 and V127/V129, 2.5 Å resolution, pdb 6SUZ) closely resembled that of WT G127/M129 (pdb 2W9E, Fig. [1a](#Fig1){ref-type="fig"} and Supplementary Fig. [2](#MOESM1){ref-type="media"})^[@CR29]^. The structured C-terminal domain (residues 125--225) comprises three α-helices (α1--α3) and a short, two-stranded, anti-parallel β-sheet (Fig. [1](#Fig1){ref-type="fig"} and Supplementary Fig. [3](#MOESM1){ref-type="media"}). Residue 127 immediately precedes the first β-strand of the β-sheet whereas residue 129 lies within it. The residues surrounding 127 and 129 are well defined in both crystal structures (Figs. [2](#Fig2){ref-type="fig"} and [3](#Fig3){ref-type="fig"}) and show that the side-chains of both residues are predominantly located on the protein surface. Neither the 127 nor 129 polymorphisms substantially perturb the backbone or sidechain positions, or hydrogen bonding, of residues within the β-sheet (Fig. [1b](#Fig1){ref-type="fig"} and Supplementary Fig. [2a--c](#MOESM1){ref-type="media"}). Both circular dichroism (CD) and heteronuclear NMR spectra (Supplementary Figs. [4](#MOESM1){ref-type="media"}--[6](#MOESM1){ref-type="media"}) are consistent with the crystal structures accurately reflecting the solution structure of the proteins. The global stability and unfolding behaviours of the V127/M129 and V127/V129 variants (Supplementary Fig. [7](#MOESM1){ref-type="media"} and Supplementary Table [2](#MOESM1){ref-type="media"}) are also not significantly affected by the substitution of valine for glycine at position 127, reflecting the lack of major structural perturbation.Fig. 1Effect of the V127 polymorphism on the structure of human PrP^C^.**a** V127/M129 (PDB 6SV2 -- green) and wild-type G127/M129 human PrP (PDB 2W9E -- light blue^[@CR29]^) crystal structures, superimposed in cartoon representation. Residues 125--223 are shown. | {
"pile_set_name": "PubMed Central"
} |
Key Points {#Sec1}
==========
CBD has been reported to exert a number of physiological, biochemical, and psychological effects that have the potential to benefit athletes.The available evidence is preliminary, at times inconsistent, and largely based on preclinical studies involving laboratory animals.Rigorous, controlled investigations clarifying the utility of CBD in the sporting context are warranted.
Introduction {#Sec2}
============
*Cannabis sativa* contains numerous chemical compounds with potential bioactive effects, including at least 144 cannabinoids \[[@CR56], [@CR76]\]. The most studied of the cannabinoids are Δ^9^-tetrahydrocannabinol (Δ^9^-THC), renowned for its distinctive intoxicating effects \[[@CR73], [@CR123]\], and cannabidiol (CBD)---a non-intoxicating cannabinoid that is particularly enriched in industrial hemp cultivars grown for seed and fibre \[[@CR61]\]. CBD was first isolated in 1940 and initially considered to be biologically inactive, with no apparent therapeutic or "subjective" drug effects \[[@CR1]\]. However, in 1973, Carlini et al. \[[@CR27]\] demonstrated anticonvulsant effects of CBD in a preclinical model, which were later mirrored in humans suffering from intractable epilepsy \[[@CR46]\]. A subsequent rise in research into CBD \[[@CR206]\] has uncovered interactions with numerous molecular targets \[[@CR92]\] and a range of potential therapeutic applications \[[@CR138]\]. Following successful phase 3 clinical trials \[[@CR53], [@CR54], [@CR172]\], the oral CBD solution, Epidiolex®, has also recently gained Food and Drug Administration approval as a regulated prescription medication to treat certain forms of paediatric epilepsy.
Recently, interest in CBD has intensified among the general population as evidenced by an exponential rise in internet searches for 'CBD' in the United States (USA) \[[@CR108]\]. Some professional athletes (e.g. golfers, rugby players) also appear to be using CBD (e.g. 'Team cbdMD' <https://www.cbdmd.com/>), despite there being no published studies demonstrating beneficial effects on sport or exercise performance. In many jurisdictions, including the USA and Europe, access to regulated, prescription CBD (i.e. Epidiolex®) is limited to patients with intractable epilepsy. However, a wide range of low dose (e.g. 5--50 mg·d^−1^) CBD-containing "nutraceuticals" (primarily in oil or capsule form) have become readily available online and over-the-counter (e.g. pharmacies, health food stores) \[[@CR20], [@CR125]\]. This includes some varieties that are marketed specifically to recreational and elite athletes (e.g. cbdMD, fourfivecbd). The use of these products is likely to become even more widespread if the World Health Organization's recommendation that CBD no longer be scheduled in the international drug control conventions is adopted by the United Nations member states \[[@CR201]\].
Cannabis has been prohibited in all sports during competition since the World Anti-Doping Agency first assumed the responsibility of establishing and maintaining the list of prohibited substances in sport 15 years ago \[[@CR89]\]. In 2018, however, CBD was removed from the Prohibited List \[[@CR199]\], presumably on the basis of mounting scientific evidence that the cannabinoid is safe and well-tolerated in humans \[[@CR16], [@CR169]\], even at very high doses (e.g. 1500 mg·day^−1^ or as an acute dose of 6000 mg) \[[@CR170]\]. While several recent reviews have described the impact of cannabis on athlete health and performance \[[@CR99], [@CR176], [@CR188]\], the influence of CBD alone has yet to be addressed.
The aim of this narrative review was to explore evidence on the physiological, biochemical, and psychological effects of CBD that may be relevant to sport and/or exercise performance and to identify relevant areas for future research. Given the absence of studies directly investigating CBD and sports performance, this review draws primarily on preclinical studies involving laboratory animals and a limited number of clinical trials involving non-athlete populations.
Cannabidiol (CBD): Molecular Targets, Pharmacokinetics and Dosing {#Sec3}
=================================================================
Molecular Targets {#Sec4}
-----------------
The distinctive intoxicating effects of Δ^9^-THC (as well as some of its therapeutic effects) involve the activation of CB~1~R (the cannabinoid type 1 receptor) \[[@CR12]\]. This ubiquitous receptor is expressed throughout the central nervous system, the peripheral nervous system, and in the cardiovascular system, gastrointestinal (GI) tract, skeletal musculature, liver, and reproductive organs \[[@CR205]\]. Unlike Δ^9^-THC, CBD is not an agonist of CB~1~R, although it may act as a negative allosteric modulator (NAM) at this site (i.e. decreasing the potency and/or efficacy of other ligands without activating the receptor itself) \[[@CR92], [@CR106]\]. Δ^9^-THC also acts as an agonist at CB~2~R (the cannabinoid type 2 receptor) \[[@CR12]\] and there is emerging evidence of CBD functioning as a partial agonist at this site \[[@CR171]\]. CB~2~R is primarily located on immune system cells but is also expressed in the cardiovascular system, GI tract, bone, liver, adipose tissue, and reproductive organs \[[@CR205]\]. CBD may also influence the endocannabinoid system indirectly via the inhibition of fatty acid amide hydrolase (FAAH), a key enzyme involved in the degradation of the principle endocannabinoid signalling molecule, anandamide (AEA) \[[@CR92], [@CR110]\]. The inhibition of FAAH is predicted to lead to an increase in brain and plasma concentrations of AEA, which acts as a partial agonist at CB~1~R and CB~2~R, thereby increasing endocannabinoid tone \[[@CR92], [@CR110]\]. Increases in endocannabinoid tone may also occur as a result of CBD inhibiting AEA transport via effects on fatty acid-binding proteins (and this mechanism may have more relevance than FAAH inhibition in humans) \[[@CR57]\].
CBD also interacts with many other non-endocannabinoid signalling systems \[[@CR92]\]. Briefly, at concentrations ≤ 10 μM, CBD has been reported to interact with the serotonin 1A \[5-HT~1A~\] receptor, the orphan G protein-coupled receptor 55, as well as the glycine, opioid, and peroxisome proliferator-activated receptors, various ion channels (e.g. the transient potential vanilloid receptor type 1 channel \[TRPV1\] and other transient potential vanilloid channels) and various enzymes (e.g. cyclooxygenase (COX)1 and COX2, cytochrome P450 enzymes) \[[@CR11], [@CR92]\] (see Ibeas et al. \[[@CR92]\] for review). CBD also possesses antioxidant properties \[[@CR92]\].
It is important to recognise that the molecular targets of CBD are still being established, with many of those identified in in vitro cellular assays still to be validated as occurring in vivo. As such, the functional relevance of many of these interactions remains to be established.
Pharmacokinetics {#Sec5}
----------------
CBD is often consumed orally as oil; however, it can also be ingested in other forms (e.g. gel capsules, tinctures, beverages, and confectionery products) and applied topically \[[@CR20], [@CR125]\]. High concentration CBD "vape oils" (i.e. for use in e-cigarette devices) are also available in some countries, as are some CBD-dominant forms of cannabis (sometimes known as "light cannabis") that can be smoked or vaporised \[[@CR20], [@CR125]\]. Pure, synthetic, crystalline CBD was also vaporised in a recent laboratory study \[[@CR160]\].
Taylor et al. \[[@CR170]\] recently conducted a comprehensive analysis of oral CBD oil pharmacokinetics in healthy participants. When administered as a single, oral dose (1500--6000 mg), the time to reach peak plasma concentrations (*t*~max~) was \~4--5 h and the terminal half-life was \~14--17 h. Although *t*~max~ did not increase dose-dependently in this investigation \[[@CR170]\], another study \[[@CR19]\], involving a much lower oral dose of CBD (300 mg), did indicate a shorter *t*~max~ (i.e. \~2--3 h). Peak plasma concentrations (*C*~max~) were \~0.9--2.5 μM in Taylor et al. \[[@CR170]\], but increased \~4.9-fold when CBD was administered with a high-fat meal (i.e. \~5.3 μM at 1500 mg dose) \[[@CR170]\]. Both studies observed a large amount of inter-individual variation in pharmacokinetic responses \[[@CR19], [@CR170]\].
The pharmacokinetics of inhaled CBD are yet to be well characterised. However, smoked "light cannabis" (with a lower Δ^9^-THC and higher CBD content than other varieties) has been reported to elicit high serum CBD concentrations at 30 min post-treatment (that decline over time) \[[@CR146]\]. A recent study in which participants vaporised 100 mg of CBD likewise observed high blood CBD concentrations 30 min post-treatment \[[@CR160]\]. As neither study collected blood samples within \< 30 min of CBD administration, *t*~max~ and *C*~max~ are unknown \[[@CR146], [@CR160]\].
CBD is metabolised by several cytochrome P \[CYP\] 450 enzymes ( | {
"pile_set_name": "PubMed Central"
} |
###### Strengths and limitations of this study
- This study is the first to evaluate the risk of bias in the randomised controlled trials referenced in the guidelines for cardiopulmonary resuscitation.
- A detailed protocol for risk of bias assessments was used to ensure reproducibility and transparency in the evaluation.
- Various subgroup analyses were performed after stratification according to topics, impact factor, and the years of introduction and update of the Consolidated Standards of Reporting Trial statement.
- The risk of bias assessed using the Cochrane Collaboration's tool can be subjective.
- We did not contact authors to resolve the unclear information when judging the risk of bias.
Introduction {#s1}
============
Randomised controlled trials (RCTs) provide the most reliable evidence for the impacts of medical interventions.[@R1] However, RCTs can be biased by faults in the design, performance, analyses and reporting. Bias is a systemic error that underestimates or overestimates the true effects of an intervention.[@R2] Bias can invalidate the results of RCTs, potentially leading to patients receiving non-beneficial or harmful treatments.[@R3]
The Cochrane Collaboration's tool for assessing the risk of bias in randomised trials was developed to clarify the trial process and increase accuracy.[@R2] The Cochrane Collaboration's tool has been used to assess the risk of bias in RCTs conducted in various fields, including paediatrics, orthopaedics, urology, neurology and ophthalmology.[@R3] Recently, the risk of bias in 20 920 RCTs included in the Cochrane Review was reported.[@R8] In the emergency medicine area, the Cochrane Collaboration's risk of bias tool was used to evaluate RCTs that assessed simulation-based medical education.[@R9] However, to our knowledge, no study has evaluated the risk of bias in the RCTs referenced in the guidelines for cardiopulmonary resuscitation (CPR). Clinical guidelines are an important tool for knowledge transfer and affect millions of clinicians and patients.[@R10] The main advantage of guidelines is that they reduce unjustified variations in patient care, but biased guidelines are potentially harmful and ineffective for the patient.[@R11] Although the Cochrane Collaboration's tool for assessing the risk of bias was used to conduct an evidence evaluation of RCTs in the 2015 American Heart Association (AHA) guidelines for CPR and emergency cardiovascular care (ECC),[@R12] the assessment of individual items of bias or overall features was not presented.
The purpose of this study was to identify the risk of bias in the RCTs referenced in the 2015 AHA guidelines update for CPR and ECC,[@R12] which is used worldwide and considered as the basis for resuscitation.
Methods {#s2}
=======
We identified the RCTs cited as references in the 2015 AHA guidelines update for CPR and ECC. Articles containing the search term 'random' in the title or abstract were extracted. After reviewing the contents of the abstract and text, the studies that were included in the actual randomisation process were confirmed. We included clinical studies in which participants were human patients and excluded animal studies. Studies that analysed existing RCTs and RCTs published in a letter format were also excluded.
We analysed the RCTs using the Cochrane Collaboration's tool for assessing the risk of bias in randomised trials.[@R1] The following six domains of the Cochrane Collaboration's tool were selected to evaluate the risk of bias: random sequence generation and allocation concealment for selection bias, blinding of participants and personnel for performance bias, blinding of outcome assessment for detection bias, incomplete outcome data for attrition bias and selective reporting for reporting bias. We did not prespecify other sources of bias that are not identified by the above six domains described because we were not able to simply define the other sources of bias in RCTs of various topics included in the guidelines. The risk of bias for each domain was reported as 'low', 'unclear', or 'high'. The criteria for assessing the risk of bias are shown in online [supplementary appendix 1](#SP1){ref-type="supplementary-material"}. As shown in the study by Zhai *et al*, real-time randomisation was added to the domain of allocation concealment.[@R5] It was considered low risk when the treatment was assigned at the time of randomisation and allocation concealment was guaranteed. If participants do not know the study objectives, the domain of blinding of participants and personnel was judged as a low risk of bias. In addition, studies with objective/permanent end-points, such as mortality or laboratory data, were evaluated as having a low risk of bias for the blinding of outcome assessment. Two independent reviewers (YC and CK) scored the RCT articles in each domain, and a third reviewer (BK) resolved the discrepancies. Kappa values for the inter-rater agreement between the two reviewers were calculated for each of the six domains.
10.1136/bmjopen-2018-023725.supp1
We identified the number of RCTs in 5-year intervals. The percentages of RCTs with high, unclear and low risks of bias were determined for each of the six Cochrane domains. We examined the risk of bias in RCTs by grouping them into seven topics (basic life support \[BLS\], adult advanced cardiovascular life support \[ACLS\], postcardiac arrest care, acute coronary syndrome, neonatal and paediatric resuscitation, education, and others). We also investigated the risk of bias in RCTs based on the type of intervention (drug trial or non-drug trial). The journal's impact factor (IF) in 2017 and the Journal Citation Report (JCR) categories were identified. We designated journals without an IF as having an IF of zero. The risk of bias in the six domains was identified in the following journal IF groups: IF\<5 (low IF), 5≤IF\<10 (intermediate IF) and IF≥10 (high IF). The risk of bias for each Cochrane domain was evaluated in RCTs divided into three periods (≤1995, 1996--2009 and ≥2010) based on the year of introduction and the update of the Consolidated Standards of Reporting Trials (CONSORT) statement. We used R V.3.4.0 ([www.R-project.org](www.R-project.org)) to plot the numbers and proportions of RCTs over 5-year intervals. Microsoft Excel was used for data collection and creating the graphs showing the risk of bias.
Patient and public involvement {#s2a}
------------------------------
No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for the design or implementation of the study. No patients were asked to advise on data interpretation or writing the manuscript describing the results. The results of the research are not planned to be disseminated to study participants or the relevant patient community.
Results {#s3}
=======
Three hundred RCTs referenced in the 2015 AHA guidelines were identified. After the exclusion of 27 articles, 273 RCTs were selected for analyses. Studies were excluded because they were animal studies (n=23), studies that analysed existing RCTs (n=3), and RCTs published in a letter format (n=1) (online [supplementary appendices 2, 3](#SP1){ref-type="supplementary-material"}). The RCT articles included in this study were published from 1980 to 2015. The number of RCTs has increased during this period ([figure 1](#F1){ref-type="fig"}), and 90.5% (247/273) of RCTs were published after 1996. A total of 42.1% (115/273) of the RCTs included in this study were published after 2010.
{#F1}
[Table 1](#T1){ref-type="table"} shows the baseline characteristics of the RCTs. The median number (IQR) of participants randomised and the number of participants analysed were 140 (59--372) and 130 (54--335), respectively. Education (33.0%, 90/273) was the most common topic cited in the guidelines, followed by neonatal and paediatric resuscitation (18.3%, 50/273) and acute coronary syndrome (14.7%, 40/273). The most common JCR category was critical care medicine (27.5%, 75/273), followed by medicine, general and internal (22.0%, 60/273).
######
Characteristics of the randomised controlled trials referenced in the 2015 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care (n=273)
Characteristics Values
------------------------------------------------- ---------------
Number of participants randomised, median (IQR) 140 (59--372)
Number of participants analysed, median (IQR) 130 (54--335)
*Topics, n (%)*
BLS 19 (7.0)
ACLS 32 (11.7)
Postcardiac arrest care 21 (7.7)
Acute coronary syndrome 40 (14.7)
Neonatal and paediatric resuscitation 50 (18.3)
Education 90 (33.0)
Others 21 (7.7)
*Type of intervention, n (%)*
Drug trial 37 (13.6)
Non-drug trial 236 (86.4)
*JCR category, n (%)*
Critical care | {
"pile_set_name": "PubMed Central"
} |
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Introduction {#s1}
============
The prevalence of heart failure including systolic and diastolic functional impairments continues to increase representing a major health problem [@pone.0109164-Murray1], [@pone.0109164-Owan1]. Several diagnostic techniques have been proposed to study systolic and diastolic myocardial performances [@pone.0109164-Karamitsos1], [@pone.0109164-Kowallick1], [@pone.0109164-Pennell1].
Left ventricular (LV) systolic torsion and diastolic recoil describe the myocardial twisting and untwisting motion resulting from apical counter-clockwise and basal clockwise rotation during systole (when viewed from the apex and normalized for LV length). Torsion and recoil rate have shown to be sensitive markers for systolic and diastolic dysfunction [@pone.0109164-Delhaas1], [@pone.0109164-Dong1], [@pone.0109164-Opdahl1]. The phenomenon can be captured by the simple twist angle (difference in apical and basal rotation) [@pone.0109164-Swoboda1], [@pone.0109164-Goffinet1], the circumferential-longitudinal shear angle [@pone.0109164-Russel1], [@pone.0109164-Buchalter1] or torsion (twist per cardiac length) [@pone.0109164-Sorger1], [@pone.0109164-Yoneyama1]. Recent studies showed that torsion might represent an important compensatory mechanism to maintain an adequate systolic function in patients with chronic hypertension as examined in a large community-based population based on cardiovascular magnetic resonance (CMR) myocardial tagging [@pone.0109164-Yoneyama1], [@pone.0109164-Donekal1]. Recoil rate seems particularly appealing to study diastolic function. Its applicability has been demonstrated in isolated diastolic dysfunction [@pone.0109164-Park1], in heart failure with preserved ejection fraction [@pone.0109164-Wang1] and heart disease characterized by both systolic and diastolic dysfunction such as hypertrophic cardiomyopathy (HCM) [@pone.0109164-Abozguia1]. CMR myocardial tagging is considered the reference standard for the evaluation of torsion at the current time. However, this technique has not found widespread implementation into clinical routine since practical obstacles, e.g. the need for additional sequence acquisition and time-consuming post-processing limit its clinical applicability. Recent advances in LV deformation quantification based on CMR feature tracking (CMR-FT) allow straightforward quantification of myocardial physiology from routine steady-state free precession (SSFP) images [@pone.0109164-Hor1], [@pone.0109164-Kowallick2]. The technique has been used to analyse ventricular strain in health and disease [@pone.0109164-Morton1], [@pone.0109164-Schuster1]. However, the feasibility of CMR-FT for quantitative assessment of LV torsion has never previously been demonstrated.
We therefore aimed to first develop a model that allows uniform selection of apical and basal slices at standardized LV levels and to evaluate its feasibility and reproducibility to quantify LV systolic torsion and diastolic recoil at rest and during dobutamine stress using CMR-FT.
Materials and Methods {#s2}
=====================
Ten healthy volunteers underwent CMR on a 1.5 Tesla scanner (Intera R 12.6.1.3, Philips Medical Systems, Best, The Netherlands). The study protocol was approved by the Institutional Review Board at the University of Nebraska Medical Center and complies with the Declaration of Helsinki. Written informed consent was obtained from all participants.
CMR Imaging {#s2a}
-----------
All CMR measurements were performed in the supine position using a 5-channel cardiac surface coil. LV dimensions and function were assessed with an ECG- gated SSFP cine sequence during brief periods of breath-holding in 12 to 14 equidistant short-axis planes (slice thickness 8 mm; gap 1--2 mm) completely covering the LV. The field of view was 360×480 mm and matrix size 196×172. Dobutamine stress imaging was performed as previously described [@pone.0109164-Nagel1]. Complete short-axis stacks were acquired at rest and with 10 and 20 µg·kg^−1^·min^−1^ of dobutamine.
Haemodynamic analysis {#s2b}
---------------------
End-diastolic (EDV) and end-systolic volume (ESV), stroke volume (SV), ejection fraction and cardiac index (CI), were calculated using MassK-Mode in QMass Version 7.6 (Medis Medical Systems, Leiden, The Netherland). Ventricular volumes were adjusted to body surface area. All parameters were analysed at rest, 10 and 20 µg·kg^−1^·min^−1^ of dobutamine stress. Furthermore, heart rate and mean blood pressure were measured at rest and with both levels of dobutamine stimulation.
Cardiac magnetic resonance myocardial feature tracking {#s2c}
------------------------------------------------------
Myocardial feature tracking was performed using dedicated software (TomTec Imaging Systems, 2D CPA MR, Cardiac Performance Analysis, Version 1.1.2.36, Unterschleissheim, Germany). All short-axis slices were included in the analysis between the most apical slice showing LV cavity at end-systole and the most basal slice including a complete circumference of myocardium at end-systole. LV endocardial and epicardial borders were manually traced in all slices using a point-and-click approach with the initial contour set at end-diastole. Endocardial and epicardial border surfaces were manually delineated and the automated tracking algorithm was applied. The software automatically tracks 48 subendocardial and subepicardial tissue voxels throughout the cardiac cycle. Tracking performance was visually reviewed to ensure accurate tracking of the ventricular myocardium. In case of insufficient border tracking manual adjustments were made to the initial contour and the algorithm was reapplied. Tracking was repeated for three times in each short-axis view and results were based on the average of the three repeated measures.
Definition of torsion and recoil rate {#s2d}
-------------------------------------
Torsion and peak recoil rate were calculated using MATLAB software (The Math Works, MA, USA). Averaged LV rotation profiles were calculated from the angular displacement of all 48 tissue voxels in all slices. LV rotation was averaged across the three repeated measurements. The rotation of points that would typically be located in between adjacent slices was linearly interpolated resulting in a complete 3D LV model of angular displacement ([**Figure 1**](#pone-0109164-g001){ref-type="fig"}). When viewed from the apex, LV torsion was calculated as the difference in counter-clockwise apical rotation (**φ** ~apex~) and clockwise rotation at the base (**φ** ~base~) divided by the inter-slice distance (D) [@pone.0109164-Sorger1], [@pone.0109164-Yoneyama1]:
![3 D model of LV rotational displacement.\
Rotation between time points (angular difference between red and green contours) is computed in each slice, and it is then linearly interpolated between slices. The 3D ventricular model is automatically fitted to the contours [@pone.0109164-Lamata1] and is used here only for illustration purposes, and not for the definition of the location of the most apical and basal points.](pone.0109164.g001){#pone-0109164-g001}
The distance (D) was calculated as the sum of the slice thickness (8 mm) and gap (1--2 mm) of imaging planes that were located in between the basal and apical slices where rotation was measured [@pone.0109164-Yoneyama1] ([**Figure 2**](#pone-0109164-g002){ref-type="fig"} **and** [**3**](#pone-0109164-g003){ref-type="fig"}). Depending on these measurement positions of rotation obtained at different LV locations and the distance between the most apical (defined as 0% of LV distance) and the most basal slice (defined as 100% of LV distance) the following four methods to calculate torsion were examined: Model 1 (difference in rotation between 25% and 75%), model 2 (difference in rotation between 0% and 100%), model 3 (difference in rotation between 25% and 100%) and model 4 (difference in rotation between 0% to 75%) ([**Figure 3**](#pone-0109164-g003){ref-type="fig"}). The analysis included subepicardial and subendocardial torsion as well as LV global torsion (averaged subendocardial and subepicardial torsion). Furthermore, subendocardial, subepicardial and global peak recoil rates were calculated from the maximum slope (−dθ/dt) of the diastolic limb of the torsion-time curve ([**Figure 2**](#pone-0109164-g002){ref-type="fig"}).
{#pone-0109164-g002}
![Definitions to calculate torsion.\
CMR feature tracking was performed in all slices of a short-axis stack. Four models (1--4 | {
"pile_set_name": "PubMed Central"
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Introduction
============
In 2010, the Carnegie Foundation made recommendations to fundamentally change medical education.[@b1-amep-5-289] It called for curricular reforms that would provide earlier exposure to clinical work, more hands-on experiential learning, and a greater focus on care coordination and working with interprofessional teams. The call for medical education reform parallels the call for health care reform. Medical schools must make certain that efforts toward curricular reform align with the rapidly changing health care delivery system.
As the health care delivery landscape changes, medical schools must develop creative strategies for preparing future physicians to provide quality care in this new environment. One such change is the increasing adoption of the patient-centered medical home (PCMH). The PCMH promises to deliver care that is more accessible, effective, efficient, and safe, while simultaneously bending the cost curve.[@b2-amep-5-289] The core tenets of the PCMH are a personal physician, a physician-directed medical practice, a whole-person orientation, integration and coordination of care, quality and safety assurance, enhanced access, and outcome-based payment.[@b3-amep-5-289] As health centers across the country adopt this model of care, future physicians need to be prepared to incorporate these principles in their practices.
Despite the growing prominence of the PCMH as effective models for health care delivery, few medical schools have integrated formal education on the PCMH into their curricula. Studies show that few medical students are exposed to this model in their formal education or have opportunities to gain hands-on experience using this model of care.[@b4-amep-5-289]--[@b6-amep-5-289] Only 41% of family medicine departments in one survey reported implementing a PCMH curriculum for students, and teaching modalities largely focused on educational conferences rather than longitudinal exposure.[@b5-amep-5-289] Integrating the tenets of the PCMH into medical school curricula is important to ensure that students have a comprehensive understanding of the different models of health care delivery and can operate effectively as physicians.
Student-run free clinics have gained popularity as a unique experiential learning opportunity that provides hands-on experience for students while serving as a safety net for medically underserved and uninsured populations. Approximately 110 student-run free clinics exist in the US.[@b7-amep-5-289] They are operated by health professional students under the supervision of a licensed clinician and provide care to medically uninsured populations.[@b7-amep-5-289],[@b8-amep-5-289] Free clinics place students as early as their first year of medical school on the front lines of problem solving and care coordination for patients who have no or little access to care.[@b7-amep-5-289] The infrastructure of these clinics varies, with students seeing patients after hours in an established care facility or in community-based settings, such as mobile health clinics, shelters, or churches.[@b9-amep-5-289] There is literature to support that patients receive quality care at these student-run clinics and are generally satisfied with the experience.[@b10-amep-5-289] These patients most commonly present to these clinics requesting acute care, chronic disease management, screenings, or social services. However, a notable absence exists in the literature regarding how student-run clinics can be used to expose students to new systems of health care delivery, such as the PCMH.
The PCMH model is particularly applicable to the care of patients who are economically and medically underserved. The lack of health insurance and basic necessities often renders this population vulnerable to discontinuous or fragmented care. A PCMH model can help to provide continuous and patient-centric care that addresses social determinants of health through linkages with key community agencies.[@b11-amep-5-289] Therefore, students who care for the underserved in a free clinic setting should be aware of this model.
Given the limited exposure to the PCMH within current curricula, new physicians may be ill-prepared to practice within a PCMH model of care.[@b5-amep-5-289] Integrating the PCMH model into the medical school curriculum would allow students to learn how to establish long-term relationships with patients, work in interprofessional teams, integrate methods of quality assurance and patient safety into practice, and develop proficiency in the use of electronic health records (EHRs).[@b12-amep-5-289] The objective of this paper is to provide a detailed description of the processes by which the Weill Cornell Community Clinic (WCCC, New York, NY, USA), a student-run free clinic, adopted elements of the PCMH model into an existing experiential learning program.
Methods
=======
Description of the WCCC
-----------------------
The WCCC was established by medical students in 2003 with the mission of providing care to patients who were uninsured or had insufficient health insurance to cover the cost of care. The activities of the clinic are coordinated by a board of medical students working closely with volunteer physicians and a Faculty Medical Director. Each board member is responsible for specific tasks, such as scheduling patient visits, directing patient care, and coordinating psychosocial screenings and services. At the student level, two Clinical Directors and two Executive Directors oversee the board. Clinical Directors are fourth year medical students who serve a 1-year term and supervise the care of all patients ([Table 1](#t1-amep-5-289){ref-type="table"}). Executive Directors are two MD--PhD students during their graduate training who deal with administrative tasks of the WCCC. The board also operates under the close supervision of a Faculty Medical Director, a physician who provides overall guidance for the program, ensures that patients are provided with quality care, and encourages students to participate as active learners. The Faculty Medical Director was selected by the Dean's office as a faculty member who has practiced in primary care with experience supervising and teaching students. In order to ensure support for this position, the Faculty Medical Director receives 0.1 full time equivalents for her work with the WCCC. The WCCC operates on a \$50,000 annual budget funded by a combination of monetary donations and grants in addition to in-kind donations from the Weill Cornell Physicians Organization, Weill Cornell Internal Medicine Associates, Weill Cornell Medical College, and NewYork-Presbyterian Hospital.
Patients are seen every Monday from 5--8 PM in clinical space that is donated by the Weill Cornell Internal Medicine Associates, the adult ambulatory care practice for attendings and Internal Medicine residents. Each patient is seen by a team of students consisting of a Junior Clinician, a first or second year student, and a Senior Clinician, a third or fourth year student, under the supervision of a licensed physician volunteer. The responsibilities of this supervising physician are to discuss and examine each patient with the medical students and develop a plan of action. Supervising physicians volunteer their time at the WCCC and must be a practicing physician and a faculty member at the Medical College. In addition, on a weekly basis, all patients are reviewed with the Faculty Medical Director to discuss patient follow-up and review the laboratory data of patients who were seen.
In 2009, the EHR system EpicCare (Epic Systems Corporation, Verona, WI, USA) was introduced into the WCCC. This provided an opportunity to reflect on the clinic practices and adopt different models of health care delivery that would maximize the use of an EHR. The PCMH model was considered because of its potential to provide students with exposure to comprehensive patient care, give them an opportunity to assume different roles as providers, and allow them to participate in interdisciplinary teams.
Tenets of the PCMH
------------------
### Students assume the role of the personal physician
In a PCMH, each patient has an ongoing relationship with a personal physician who provides continuous and comprehensive care[@b13-amep-5-289] ([Table 2](#t2-amep-5-289){ref-type="table"}). The physician coordinates communication between members of the health care team and establishes relationships with administrative staff and outside consultants. In an effort to provide students at the WCCC with the opportunity to take on the role of a lead physician, third and fourth year students rotate through the WCCC for 6 weeks as part of their primary care clerkship and take on the responsibilities of the personal physician while directing the patient care team. These Senior Clinicians continue to follow the patients after each clinical session and ensure that all necessary tests and referrals are completed. The Senior Clinician also becomes the patient's advocate, helping to fill out necessary forms for social services and making sure the patient sees necessary service providers, such as referred specialists and social workers.
In order to ensure a seamless transition and patient hand-off, each outgoing Senior Clinician provides detailed information about the patient and describes any outstanding tests, referrals, or procedures that are pending to the Clinical Directors. This helps to promote a smooth transition of care when new Senior Clinicians rotate through the WCCC. The Medical Faculty Director also ensures that new students joining the WCCC are aware of pertinent outstanding patient issues.
In an attempt to meet the high level of interest among the preclinical students, Junior Clinicians sign up for individual nights rather than blocks of time, as with the Senior Clinicians. Thus, a limitation of the nature of such a student-run free clinic can be a lack of continuity as volunteers come and go. In order to provide the opportunity for preclinical students to be involved in the long-term care of a patient, the Continuity of Care Program was piloted. This pilot project was designed to provide additional experience in communicating with patients and their families and to provide students with first-hand experience of the after visit care process. Each student in the program is paired with a patient and attends all of their | {
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Introduction {#h0.0}
============
The therapeutic benefit of drugs depends on achieving high potency toward the target (an enzyme, cell, bacterium, parasite, virus, etc.) while avoiding damage to the host organism. One approach to killing tumor cells has been to use highly potent bacterial and plant toxins that act catalytically in the cytosol of the targeted cells. Most commonly, specificity has been sought by linking these toxins chemically or genetically to antibodies that bind to cell surface materials enriched on tumor cells. These proteins, initially termed "immunotoxins" and now more generally referred to as "targeted toxins" (TTs), have been under development for decades, but few have reached clinical use ([@B1]--[@B3]). This appears to be due to inadequate specificity for the tumor versus the host (i.e., a low therapeutic index), low efficiency of delivery to the cytosol, and other factors. Several approaches have been explored for improving the therapeutic indices of TTs (reviewed in reference 2) or to increase the uptake of TTs into the cytosol of tumor cells ([@B4]).
Our research group has used a different approach to achieve tumor cell specificity. This approach exploits the fact that anthrax toxin from *Bacillus anthracis* activity depends on proteolytic activation of the receptor-bound protective antigen (PA) protein by cell surface proteases ([@B5]--[@B7]). Replacing the site normally cleaved by furin and related proteases with sequences recognized by matrix metalloproteases or urokinase plasminogen activator has yielded potent agents having high specificity and efficacy in mouse tumor models. The protease-activated PA assembles into an oligomeric-protein-conducting channel that efficiently delivers the anthrax toxin catalytic effector proteins to endosomes and then translocates them to the cytosol. The native anthrax effector proteins lethal factor (LF) and edema factor can be replaced with a fusion protein containing the N-terminal 254 amino acids of anthrax toxin lethal factor (lethal factor N terminus \[LFn\]) and the *Pseudomonas aeruginosa* exotoxin A (PE) catalytic domain (PEIII). Once in the cytosol, PEIII will transfer ADP-ribose to eukaryotic elongation factor 2 (eEF2), resulting in protein synthesis inhibition and cell death. This system is highly effective in terms of cytosolic delivery and tumor-specific activation. It has been tested successfully on a number of tumor types ([@B8]) and is expected to be active on nearly all types of solid tumors.
One factor that affects the potency of all TTs but that has received limited attention is the issue of the stability of the effector proteins once they have reached the cytosol. It was noted in 1989 that many protein toxins have a strong bias against the presence of lysine residues in their catalytic domains ([@B9]). In retrospect, it is now evident that this feature limits the attachment of ubiquitin and the resulting proteasomal degradation of toxins ([@B10]). The cytosolic stability and resulting potencies of several toxins have been shown to depend on the N-end rule, which specifies that the N-terminal amino acid of a polypeptide determines the efficiency with which side chain lysine residues are ubiquitinated for proteasomal targeting ([@B11]). The N-end rule applies to LF and LFn-based fusion proteins ([@B12], [@B13]), indicating that it will impact the efficacy of anthrax toxin-based TTs.
Ubiquitin is a small eukaryotic protein that plays a major role in signal transduction and many other processes in addition to its role in protein degradation. Ubiquitin contains a diglycine motif at its C terminus that is conjugated to the epsilon amine of a lysine within the target protein. According to the N-end rule noted above, ubiquitination occurs on proteins with specific destabilizing N-terminal residues ([@B11]), and ubiquitination may occur on several sites within one protein. Ubiquitinated proteins are targeted for degradation by the 26S proteasome system ([@B14]). Ubiquitin itself may be ubiquitinated after its conjugation to a target protein, leading to creation of polyubiquitin chains. These chains may be built upon any of the seven lysine residues within ubiquitin by ubiquitin ligases, although Lys48 is most often used ([@B15]). Furthermore, polyubiquitination of Lys63 instead of Lys48 seems to result in less protein degradation ([@B16]). Ubiquitination is a balanced process with deubiquitinating enzymes (DUBs) counteracting ubiquitination. The DUBs recognize the C-terminal diglycine motif of ubiquitin and release ubiquitin from labeled proteins. This specific release of cargo from ubiquitin fusions is described by Varshavsky as the ubiquitin fusion technique ([@B17]), which can be used to conditionally stabilize or destabilize a protein.
In this study, we examined how the insertion of ubiquitin variants within the TT LFn-PEIII fusion protein altered enzymatic activity, cytotoxicity, and stability of the TTs. We used ubiquitin variants that allowed us to study the accessibility of the TTs to DUBs and polyubiquitination. The results obtained indicate that intracellular release of the catalytic PEIII domain is achievable and that ubiquitination of the TTs controls their persistence in the cytosol and thus determines their potency.
RESULTS {#h1}
=======
Design of TTs. {#h1.1}
--------------
Six different TTs were constructed and analyzed in this study ([Fig. 1A](#fig1){ref-type="fig"}). The five ubiquitin-containing TTs are based on the TT anthrax fusion toxin FP59 that contains LFn at the N terminus and PEIII at its C terminus. An alignment of the amino acid sequences shows the differences in the ubiquitin fusion proteins ([Fig. 1B](#fig1){ref-type="fig"}). The TT designated "Ub" ([Fig. 1A](#fig1){ref-type="fig"}) contains the human wild-type ubiquitin with the C-terminal diglycine motif that is specifically recognized by DUBs. The TT Ub~UN~ has the same sequence but with three glycines (including the diglycine motif) replaced by a sequence of three alanines, which renders the sequence uncleavable by DUBs. The Ub~K48~ and Ub~K63~ mutants have all lysine residues of ubiquitin replaced by arginine residues except for one lysine residue, either Lys48 or Lys63, respectively. Ub~Knull~ contains ubiquitin with all seven lysines replaced by arginine. Ub~K48~, Ub~K63~, and Ub~Knull~ all retain the intact C-terminal diglycine motif. All proteins were successfully purified in yields of at least 1.5 mg per liter of culture ([Fig. 2A](#fig2){ref-type="fig"}). The molecular masses of all six proteins were confirmed by electrospray ionization mass spectrometry. Since the accessibility of PEIII within a larger polypeptide or toxin may limit its activity ([@B18]), we confirmed the catalytic activity of the five TTs containing ubiquitin or variants of ubiquitin in comparison to the fusion protein FP59 ([Fig. 2B](#fig2){ref-type="fig"}). All samples show a band for biotin-containing ADP-ribosylated eEF2 at a molecular mass of 100 kDa and an additional band of the same intensity at around 40 kDa. The latter is a degradation product of eEF2, an artifact of the purification of eEF2 from *Saccharomyces cerevisiae* yeast. This fragment contains the site of ADP-ribosylation of eEF2 and is thus likewise ADP-ribosylated and detected ([@B19]).
{#fig1}
{#fig2}
*In vitro* deubiquitination of TTs. {#h1.2}
-----------------------------------
In order to analyze and compare the cleavability of the ubiquitin-containing TTs, the fusion proteins were incubated with HN6 cells (a human head and neck cancer cell line) lysates. The lysates contain active DUBs that are expected to cleave the C-terminal diglycine motif of the ubiquitin ([@B20]). All TTs containing ubiquitin with the natural C-terminal diglycine sequence were cleaved within the 2-h reaction time ([Fig. 3A, B](#fig3){ref-type="fig"}, and C). The cleavage product, LFn-ubiquitin, has an expected molecular mass of 41 kDa and is | {
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{#sp1 .39}
| {
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Dear Editor,
We reviewed the manuscript recently published by Cumhur Cure M, et al. \[[@CR1]\], regarding colchicine and COVID-19. The authors discussed the effects of colchicine in intracellular and extracellular pH conditions and argued that low pH levels secondary to colchicine may increase the viral load of SARS-CoV-2 and, therefore, cytokine storms will be more severe.
In contrast, we consider that colchicine can be a good therapeutic option because of several effects in the immunology system involved in SARS-CoV-2 infection and in the acute respiratory distress syndrome (ARDS). Colchicine has effects on the chemotaxis of inflammatory cells such as neutrophils and monocytes and on the intracellular transportation of vesicles such as endosomes and exosomes. Colchicine also inhibits the expression of E-selectin, an adhesion molecule important for binding leukocytes to endothelial cells, and the recruitment of monocytes and neutrophils to inflamed tissue. Finally, colchicine reduces neutrophil production of free radicals like superoxide \[[@CR2]\]. The recruitment of neutrophils in COVID-19 is a key factor in the severity of cases \[[@CR3]\]. Moreover, colchicine has also been associated with disrupting inflammasome activation, thereby suppressing caspase-1 activation and the subsequent release of IL-1β and IL-18 \[[@CR4]\]. In SARS-CoV-1, the inflammasome activation has been associated with this disruption \[[@CR5]\].
The authors also cited an article published in 1986 by Maurizi M, et al. \[[@CR6]\] regarding two patients who developed ARDS after treatment with toxic doses of colchicine. The first patient received approximately 80 mg of colchicine or 1.6 mg/kg, while the other patient received 15 to 20 mg or 0.25--0.3 mg/kg of colchicine for acute gout. Both patients had ARDS between 24 and 72 h after the colchicine doses \[[@CR1]\]. Both articles \[[@CR1], [@CR6]\] attributed a direct toxic action of colchicine on pneumocyte microtubules, and the inhibition of surfactant production was deemed the probable cause of death of those patients. The dose used in those cases is not recommended due the toxicity. The usual adult oral dose in acute gout is 1.2 mg/day or as a prophylactic, 0.5--1 mg/day; however, dose must be adjusted in patients with renal impairment. The high fatality rate was reported after acute ingestions exceeding 0.5 mg/kg \[[@CR7]\], and therefore, there is no support for that outcome in therapeutic doses.
Classically colchicine is commonly used to treat different inflammatory diseases such as gout, as well as some autoinflammatory diseases, and cardiac conditions including viral pericardial syndromes. Recently, a patient with cardiac tamponade secondary to COVID-19 was treated with colchicine in addition to corticosteroids and antimalarials with positive clinical response \[[@CR9]\]. Other research demonstrates the positive effect of colchicine in respiratory syncytial virus replication and suppression of secondary airway inflammation given the promoted expression of IFN-α and IFN-β1 of colchicine and regulation of anti-oxidative factor production \[[@CR10], [@CR11]\]. In addition, there is a wide range of preclinical and clinical literature on the effect of colchicine inhibiting viral disease like adenoviral and adeno-associated viral \[[@CR12]\], herpes simplex virus type 1 \[[@CR13]\], Epstein-Barr virus \[[@CR14]\], and hepatitis virus \[[@CR15], [@CR16]\], among others.
On the other hand, Cumhur Cure M, et al. \[[@CR1]\] discussed the interactions of colchicine with other drugs. We would agree that colchicine may indeed have interactions with other drugs; therefore, we recommend adjusting the dose in close consideration of the interactions with inhibitors of CYP3A4 as antibiotics and antivirals even though some of them are used in COVID-19 as lopinavir/ritonavir.
In our experience, we reported 5 patients (age 38--61 years) with comorbidities (arterial hypertension, type 2 diabetes, among others) in treatment with colchicine for iatrogenic allogenosis 1 to 3 weeks before COVID-19 test positive. They developed mild symptoms such as headache, cough without dyspnea, and arthralgias. It should be noted that some close contacts presented severe symptoms and three of them died \[[@CR8]\].
According to a potential benefit of colchicine in COVID-19 patients, since March 26, 2020, a total of twelve studies have been registered in [www.clinicaltrials.gov](http://www.clinicaltrials.gov) and the European Union Clinical Trials Register considering the clinical utility of this well-known medication. Most of these studies correspond to randomized, open-label, phase 2 clinical trials. It is aspired to include more than 11,000 patients on three continents. The experimental arm in most studies includes an oral colchicine regimen with loading and maintenance doses plus the standard of care for COVID-19. Primary outcomes to evaluate include change in clinical condition (according to the semiquantitative ordinal scale suggested by WHO), requirement for invasive mechanical ventilation/intensive care unit, delta in the score for the Sequential Organ Failure Assessment, length of hospital stay, all-cause mortality, and change in prognostic biomarkers. The evaluation of these outcomes will be carried out in a time frame up to 30 days, and hopefully, their results will be available soon.
**Publisher's note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
None.
Written informed consent for publication of their clinical details and clinical images was obtained from the patient.
| {
"pile_set_name": "PubMed Central"
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Introduction {#Sec1}
============
In Europe, on average 70% of total expenditure on dental care is private expenditure and 16% of this private expenditure is covered by complementary dental insurance (CDI) \[[@CR1]\]. However, most CDI products currently on the market in Belgium, France, Germany, and the Netherlands are not optimal because (1) they provide little protection against financial risk and do not improve access to otherwise unaffordable dental treatment (e.g., implants and crowns) and (2) moral hazard and adverse selection are not sufficiently counteracted. The suboptimal character of CDI can be explained by supply-side aspects (the limits of insurability) and demand-side aspects (behavioral economics). On the basis of these potential explanations, strategies will be drawn to optimize dental insurance.
We begin by presenting a framework for optimal insurance design, as well as the current situation of CDI in Belgium, France, Germany, and the Netherlands.
Optimal health insurance design {#Sec2}
===============================
Health insurance has two important advantages for the consumer, but also two disadvantages (see Table [1](#Tab1){ref-type="table"} below). On the one hand, health insurance reduces the financial risk for the insured and provides access to health care that would otherwise be unaffordable \[[@CR2]\]. On the other hand, insurance increases costs due to loading costs---the administrative and other expenses of the insurer---and moral hazard. In relation to dental care, perhaps more so than in relation to other types of care, a choice is possible between cheaper basic treatments and more expensive 'luxury' treatments (e.g., placing a metal crown versus a porcelain crown), which may result in substantial moral hazard (both consumer- and supplier-induced moral hazard).Table 1Advantages and disadvantages of health insuranceAdvantagesDisadvantagesReduction of financial risk for the insuredLoading costsAccess to health care that would otherwise be unaffordableMoral hazard
Optimal health insurance should maintain the advantages and reduce the disadvantages as much as possible. First, it should reduce the insured's financial risk as far as possible. Because trivial risks lead to losses that can be borne by the insured without any noticeable burden, optimal insurance should not provide coverage for trivial risks. This avoids relatively high administrative expenses and loss settlement costs (loading costs) that are very high for these risks compared with the pure risk premium.
Secondly, optimal insurance should provide 'access to health care that would otherwise be unaffordable'. This implies that cover limits should be avoided and expensive care should be covered as much as possible.
Third, the optimality of CDI can be increased by restricting the loading costs of dental insurance by increasing insurers' efficiency.
Fourth, to reduce moral hazard, optimal insurance should involve cost-sharing arrangements such as deductibles and co-insurance, and apply managed care. A deductible makes the enrollee responsible for all costs up to a defined threshold. A co-insurance rate makes the enrollee responsible for a percentage of costs. Optimal insurance contracts should also have a stop-loss, a limit on out-of-pocket expenses \[[@CR3]\].
Fifth, optimal insurance should also counteract adverse selection as much as possible. Adverse selection occurs when the insured knows more information about his expected losses than the insurer knows or uses in his premium setting and underwriting process. Adverse selection can be counteracted by selective underwriting (using medical questionnaires), risk rating (setting higher premiums for groups presenting high risk, e.g., age-related) and product differentiation (designing benefits so to attract lower risks).
The features of optimal health insurance are summarized in the first column of Table [4](#Tab4){ref-type="table"}.
Complementary dental insurance in Belgium, France, Germany, and the Netherlands {#Sec3}
===============================================================================
Complementary dental insurance (CDI) provides coverage for care that is either not covered or not fully covered by the mandatory basic insurance (MBI). In the four countries studied, private expenditure on dental care (i.e., not covered by MBI) expressed as a percentage of total expenditure on dental care ranges between 30% in Germany and 74% in the Netherlands (Table [2](#Tab2){ref-type="table"}). CDI represents 3% (Belgium) to 55% (Netherlands) of total expenditure on dental care. Out-of-pocket expenditure on dental care is the highest in Belgium (42%) and the lowest in the Netherlands (19%).Table 2Dental care: expenditure and insuranceBelgiumFranceGermanyNetherlandsAverage expenditure per person on dental care€150€160€314€179Private expenditure (i.e., not covered by MBI)^a^45%65%30%74%Complementary dental insurance (CDI)^a^3%^b^43%5.4%55%Percentage of population with CDI5%95%18%62%Source: \[[@CR1]\]^a^As share of total expenditure on dental care^b^Authors' estimate
The coverage provided by CDI is complementary to that provided by MBI, which covers 26% (the Netherlands) to 70% (Germany) of total expenditure on dental care. Table [3](#Tab3){ref-type="table"} provides an overview of the dental care covered by MBI in the four countries.Table 3Dental care covered by mandatory basic health insuranceBelgiumFranceGermanyNetherlandsConservative care (e.g., fillings)+++^a^+++^a^++++^b^Orthodontics (e.g., braces)++++−Prosthetics (e.g., implants, bridges, crowns)−++−Periodontics (gum disease treatment)++++−+++: good coverage++: medium coverage+: low coverage−: no coverage^a^Extra billing is possible^b^Conservative dental care is covered for children only (under age 18)
No optimal dental insurance {#Sec4}
---------------------------
Currently, CDI offered in Belgium, France, Germany, and the Netherlands cannot be described as 'optimal' (Table [4](#Tab4){ref-type="table"}). German CDI responds best to the criteria of optimal health insurance.Table 4Complementary dental insurance: presence of features of optimal health insurance designBelgiumFranceGermanyNetherlandsNo upper limit on coverage−−+−No coverage of trivial risks−−−−Deductible−−−−Co-insurance+−++Cap on out-of-pocket expenses−−−−Selective underwriting−^a^−^a^+−^a^Risk rating++++Product differentiation++++Managed care−+++−: The feature is not present in CDI products offered+: The feature is present in CDI products offered^a^Selective underwriting is applied only for a limited number of contracts, i.e., those with the highest upper limits on coverage
Only in Germany are there no upper limits on coverage for dental care (after an initial period). CDI in the other three countries does not provide access to otherwise unaffordable health services and protection against unpredictable high financial risks. For example, replacing four teeth by implants and crowns can cost about €10,000. Upper limits of only €250 (the Netherlands) or €1000 (Belgium) do not provide protection against high financial risks nor do they make this kind of dental care more accessible. In France, even the most extensive complementary covers provide a maximum amount of only €750 per year for implants. With total costs for an implant easily amounting to about €2500, €1750 still needs to be paid for out-of-pocket after complementary insurance has kicked in. In all four countries, CDI provides coverage of trivial risks.
In all four countries, cost sharing is applied, but only in the form of co-insurance. Co-insurance rates vary between 0 and 50% in Belgium, 0 and 55% in Germany, and 0 and 25% in the Netherlands. In France, co-insurance is generally not used. In Germany and the Netherlands, many products are offered with 0% co-insurance. Deductibles are not used. Caps on out-of-pocket expenses, which protect the consumer against high financial risk, are not applied in any of the four countries studied.
Selective underwriting is primarily used in Germany, and to a lesser extent in the other three countries. In Belgium, selective underwriting is used by only one insurer offering CDI. In France, where a 7% tax has to be paid for contracts that apply selective underwriting, most CDI contracts abstain from selective underwriting. In Germany, a medical questionnaire needs to be filled out for most CDI products. The insurer can decline to cover the candidate or charge an additional premium or exclude missing teeth or the use of certain techniques from the scope of coverage. Insurance products without selective underwriting usually have contractual clauses excluding reimbursement for problems that existed well before the start of the contract and for treatments running at the moment of the conclusion of the contract ('pre-existing conditions'). In the Netherlands, selective underwriting is rarely applied (only for the high-end dental coverage).
In all four countries, risk rating and product differentiation are used to a certain extent (e.g., age-related premiums). Products are designed and marketed to attract certain market segments.
In all countries except Belgium, preferred provider networks are used as an element of managed care for CDI. In all countries, waiting times are used as a means to contain costs and to counteract adverse selection.
Why is dental insurance suboptimal? {#Sec5}
===================================
Both | {
"pile_set_name": "PubMed Central"
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Background
==========
The post thrombotic syndrome
----------------------------
The post thrombotic syndrome (PTS) (sometimes called \'post-phlebitic syndrome\') is a chronic condition that develops in 20--40% of patients within 1--2 years after symptomatic deep venous thrombosis (DVT). A severe form, which can include venous ulcers, affects 1/4 to 1/3 of patients with PTS \[[@B1]\]. In contrast to research gains in the areas of diagnosis, prevention and treatment of acute venous thromboembolism (VTE), PTS has been understudied and as a result, treatment options are limited.
Patients with PTS experience pain, heaviness, swelling, or other symptoms in the affected limb, which are typically aggravated by standing or walking and improve with rest and recumbency. Edema, venous ectasia, hyperpigmentation, eczema, and varicose collateral veins may be apparent. In severe cases, ulceration can occur \[[@B2]\].
Since PTS is a direct consequence of DVT, its prevalence is influenced by the incidence of DVT. Despite advances in VTE prevention and treatment, the annual incidence of VTE has not decreased over time, and remains at 1.0--1.6 per 1000 persons per year, with a per-person lifetime incidence of 2--5% \[[@B3],[@B4]\]. Because of its prevalence and chronicity, PTS is expensive, both in terms of direct medical costs and indirect costs such as loss of productivity and thus it is burdensome to patients and society \[[@B5]\].
Risk factors for the development of PTS
---------------------------------------
While hereditary and acquired risk factors that predispose to the development of VTE are widely known \[[@B6]\], factors that influence the development of PTS after DVT have not been well elucidated. The only clearly identified clinical risk factor for PTS is recurrent, ipsilateral DVT \[[@B7],[@B8]\]. The site (proximal vs. distal), size and occlusiveness of the initial DVT and the intensity (i.e. target INR) of long term anticoagulation for DVT do not appear to reliably predict PTS \[[@B1],[@B7]-[@B10]\]. It is not clear why some patients develop PTS while others recover from their DVT. As a result, physicians are unable to provide their DVT patients with reliable, individualized prognostic information.
Potential role of inflammation, d-Dimer and thrombophilia in the post-thrombotic syndrome
-----------------------------------------------------------------------------------------
While the pathophysiology of PTS is incompletely understood, it is likely that the acute thrombus itself, associated mediators of inflammation, and the process of vein recanalization in the weeks following DVT induce damage to venous valves, leading to valvular incompetence (reflux). Valvular incompetence and/or persistent venous obstruction by thrombus cause venous hypertension, which promotes capillary leakage of plasma proteins, erythrocytes and leukocytes and the development of venous ectasia and varicosities. The result is edema, tissue hypoxia, and ultimately, in some cases, skin ulceration \[[@B11]-[@B14]\].
Inflammation and thrombosis are closely interrelated \[[@B15]-[@B17]\]. It has been appreciated over the last few years that arterial atherothrombosis is, at least in part, an inflammatory disease, and that elevations in markers of inflammation, such as C-reactive protein (CRP), increase the risk of future clinical cardiovascular events \[[@B18]\]. Clinically, patients with DVT exhibit cardinal signs of inflammation such as redness, warmth, swelling, pain and fever. Several clinical studies have examined the association between levels of inflammatory markers and venous thrombosis, and found that a two- to six-fold increase in the risk of DVT associated with elevations in plasma levels of CRP, interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1 or tumor necrosis factor (TNF)-alpha \[[@B19]\]. A recent paper showed that CRP is elevated in patients with acute DVT (median 37.5 mg/L) compared with controls (5.0 pg/L), and that levels decline during the first 5 days of DVT treatment. Similar trends were noted for IL-6 \[[@B20]\], leading the authors to conclude that the thrombotic process produces a systemic inflammatory response and to speculate whether the observed decrease in levels was partly caused by treatment with heparin, which is known to have anti-inflammatory properties distinct from its anticoagulant properties \[[@B21]\]. Whether levels of markers of inflammation are predictive of PTS has not previously been examined.
D-dimer is a degradation product of cross-linked fibrin that reflects fibrinolysis and is an indirect marker of coagulation activation. Recent work performed by our group and by others suggests that D-dimer levels appear to be a significant predictor of first VTE \[[@B22]\] and of recurrent VTE \[[@B23]-[@B28]\], whether measured during or after anticoagulation. Independent of VTE recurrence, persistent coagulation activation, as reflected by elevated D-dimer levels, may also predict a higher risk of developing PTS after DVT \[[@B29]\].
Thrombophilia refers to an inherited or acquired predisposition to VTE. Over 10% of the general population is affected by one or more identifiable inherited thrombophilias which have been shown to underlie at least 1/3 of cases of VTE \[[@B30]\]. The most common inherited thrombophilias are the single nucleotide polymorphisms Factor V Leiden, which renders coagulation factor V resistant to the anticoagulant effects of activated protein C \[[@B31]\], and Prothrombin G20210A, which leads to elevated plasma prothrombin levels \[[@B32],[@B33]\]. Recent studies have also established that elevation of Factor VIII level is an inherited risk factor both for first and for recurrent VTE \[[@B34]-[@B36]\], independent of age, sex, Factor V Leiden, Prothrombin G20210A or markers of acute phase activation such as CRP \[[@B35],[@B37],[@B38]\].
While results of a few studies to date suggest that thrombophilia does not increase the risk of developing PTS \[[@B7],[@B8],[@B10]\], not all thrombophilias have been examined and interactions among disorders have not been studied. Further investigation of the potential link between thrombophilia and PTS is warranted.
Management of PTS
-----------------
PTS could be averted by primary prevention of the initial DVT with the judicious use of thromboprophylaxis \[[@B39]\], and by preventing recurrent ipsilateral DVT by prescribing adequate anticoagulation for the initial DVT \[[@B40]\]. However, at least 1/2 of all cases of DVT occur unpredictably, hence are not preventable \[[@B41],[@B42]\]. There is no definitive evidence that using thrombolysis to treat DVT reduces the incidence of PTS \[[@B43]\]. The treatment of established PTS is limited and frustrating for patients. Severe PTS can be managed with long-term use of an intermittent compression extremity pump \[[@B44],[@B45]\]. Management of venous ulcers is labor intensive and protracted, and involves compression therapy, leg elevation, topical dressings, and sometimes surgery \[[@B2],[@B46]\]. Ulcers are often recalcitrant to treatment, and tend to recur \[[@B47]\].
A significant reduction in the overall burden of PTS is unlikely to be achieved by attempts to prevent the initial DVT or by treatment of established PTS. Rather, strategies that focus on preventing the development of PTS in patients with DVT are more likely to be effective and cost-effective in reducing the patient and societal impact of PTS.
Elastic compression stockings for the prevention of PTS
-------------------------------------------------------
Graduated elastic compression stockings (ECS) work by providing graded compression to the leg that is highest at the ankle, which assists the calf muscle pump, reduces venous hypertension and valvular reflux, and consequently reduces edema, improves tissue microcirculation, and prevents skin breakdown \[[@B48],[@B49]\]. Both knee-length and thigh-length stockings appear to have equal physiological effects, but knee-length ECS are easier to apply and are more comfortable \[[@B50]\]. The effectiveness of daily use of ECS to prevent PTS is supported by two studies \[[@B8],[@B51]\] but challenged by another \[[@B52]\]. All three studies have limitations that could affect their validity and generalizability. The first trial, Brandjes\' study of 194 patients with symptomatic proximal DVT, provides evidence supporting the effectiveness of ECS. Patients were randomized to daily use of custom-made, knee-length ECS applied within 2--3 weeks of diagnosis for at least 2 years (class II compression, i.e. 30--40 mm Hg pressure at the ankle), or no stocking. Use of ECS resulted in a 50% reduction in the incidence of PTS, diagnosed using a modification of Villalta\'s clinical PTS scale \[[@B53]\], or an absolute decrease from 47% to 20% of mild/moderate PTS and from 23% to 11% of severe PTS \[[@B51]\]. In contrast, a randomized trial conducted by Ginsberg suggested that ECS were not of benefit in preventing PTS \[[@B52]\]. A strength of Ginsberg\'s study was the use of a control comparison group that wore sham stockings (i.e. stockings that were 1--2 sizes too big to be effective). However, because of the small number of patients with PTS, benefit or harm of up to 30% could not be definitively excluded. Recently, Prandoni published the results of a trial performed at a single center in Italy to evaluate the effectiveness of ECS to prevent PTS \[[@B8]\]. Among 180 patients with proximal DVT, those randomized to wear daily E | {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Overweight and obesity correspond to an abnormal or excessive fat accumulation, defined as a Body Mass Index (BMI) ≥ 25.0 kg/m^2^ and ≥30 kg/m^2^, respectively \[[@B1]\]. These chronic situations described as epidemic should be considered as pandemic with a prevalence that has more than doubled since 1980 and which continues to increase dramatically to reach about 1.5 billion overweight or obese people worldwide \[[@B1], [@B2]\]. Overweight and obesity represent a major risk factor of ischemic heart diseases, ischemic stroke, type 2 diabetes, osteoarthritis, some cancers (endometrial, breast, and colon) \[[@B1], [@B2]\], and some of the leading diseases in terms of frequency, quality of life impairment, morbidity, mortality, and heath expenses \[[@B3]\].
Public health campaigns and industry-supported changes in our food supply have obviously failed to control the pandemic to date. Many individuals appear to conclude that the benefits of weight management strategies are not worth the cost (i.e., time, money, and continued unrewarding efforts) \[[@B4], [@B5]\]. This underlines the critical need to implement new, practical, and affordable strategies to complete the armamentarium to fight excess weight.
Balneotherapy (BT), or SPA therapy, is defined as the treatment of disease by bathing, usually at a SPA resort, using hot or cold water rich in minerals, and including also drinking, inhalation, massage through moving water, mud-baths, relaxation, or stimulation \[[@B6]\]. The medical community, mainly in Europe and Asia, has long used BT to improve the symptoms of several chronic diseases, such as osteoarthritis for which recent studies show evidence-based benefit \[[@B7]--[@B9]\]. Obesity is another common indication of BT, with preliminary positive results \[[@B10]--[@B13]\]. In France, BT has been subsidized by national health insurance for more than 50 years, but scientific evidence to support long-term BT benefit on weight loss has been requested by national health authorities.
Long-term weight control may be facilitated by an appropriate weight loss maintenance strategy such as motivational interviewing, with a weak but significant difference reduction in body mass compared to the control group \[[@B14]\]. Numerous reports have concluded that this modest weight loss contributes to important health benefits \[[@B15]--[@B17]\].
The objective of the present study was to assess the one-year effectiveness of a 3-week program of BT for overweight or obese patients and whether a monthly phone dietary motivational interview (DMI) during the follow-up could improve weight loss.
2. Methods {#sec2}
==========
The study was a Zelen double consent randomised controlled trial \[[@B18], [@B19]\] to assess the effectiveness of BT compared to usual care (UC) with or without a monthly phone DMI during follow-up, using a 2 × 2 factorial design. After inclusion by their GP, subjects were equally allocated by a centralized randomisation to one of the following groups: BT alone, BT and DMI, UC alone, and UC and DMI. Zelen double consent was applied to BT randomisation but not to DMI intervention. BT should be done in the 2 months following inclusion. All patients were followed up by the same GP at baseline, 7 months and 14 months after inclusion (i.e., about one year after BT).
2.1. Participants {#sec2.1}
-----------------
Inclusion criteria were generally healthy adults between the ages of 20 and 70 years consulting their GP for excess weight, with a BMI \> 27 kg/m^2^ and ≤35 kg/m^2^. Exclusion criteria were BT contraindication (severe general weakness, inflammatory bowel disease, cirrhosis, severe disability, psychosis and dementia, or immunodeficiency), major eating disorders (bulimia nervosa, compulsive overeating), pregnancy, previous BT for weight problems, poor proficiency, and involvement in another clinical trial. All participants signed an informed consent to participate.
2.2. Interventions {#sec2.2}
------------------
BT was a 3-week program in Brides les Bains, Capvern les Bains, Vals les Bains, Vichy, or Vittel BT resorts. The core of the program included 18 daily sessions of (i) individual mineral water bubble bathing at 37°C during 10 minutes; (ii) mineral water manual massages during 10 minutes; (iii) mud body wrap applied at 42°C during 10 minutes; (iv) mineral water pool supervised callisthenic exercises (34°C, 15 minutes); (v) daily dinking of resort mineral water. Mineral waters were mainly sulfate (Brides les bains, Capvern les bains, Vittel) and bicarbonate waters (Vals les bains, Vichy). The specific chemical composition of the five resorts\' water is reported in [Table 1](#tab1){ref-type="table"}. Dieticians and personal trainers provided nutrition and physical activity counselling. However, no particular caloric restriction or physical training was mandatory during the patient\'s stay. Meals were taken freely either at the resort restaurants or anywhere in the town, offering low-fat/low-calorie menu. This program was the usual one for overweight or obese patients, and the staff was not informed which patients were taking part in the clinical trial.
Patients in the UC group received usual weight management advice from the GP consisting of verbal and/or written advice based on the French national guideline, and a brochure "Health comes when eating" \[[@B20]\] was given to the patients at inclusion. Patients were advised to reduce calories, fat, and alcohol, to increase fruit, vegetable, and whole cereal intake, and to incorporate low-intensity, long-duration physical activity into their lifestyle.
DMI was a monthly half-an-hour phone interview with a dietician, consisting in an overview of the current situation, to examine the last 24 hours intake and physical activity, followed by meal composition and food behaviour advice as well as to define 3 or 4 objectives for the next month including at least one about physical activity. DMI started one month after the end of BT for patients attending BT and 3 months after inclusion for UC patients.
2.3. Data Collection {#sec2.3}
--------------------
Data collected at inclusion were gender, height, birth date, marital status, professional occupation, overweight family history, medical history, weight loss objective, previous drug and nondrug overweight treatment, and drug, examination, dietary, and physical activity prescribed for overweight. Other data collected at inclusion and at the two follow-up visits were the date of visit, weight, body fat mass, waistline measurements, blood pressure and heart rate, physical activity, and tobacco and alcohol consumption. Patient quality of life was assessed using SF12 at inclusion and at the two follow-up visits. Weight was measured by GPs, as well as by a dietician at the end of BT, using the same scale provided for the study.
2.4. Outcome {#sec2.4}
------------
The primary outcome was body mass index (BMI) loss at 14 months of follow-up. Secondary outcomes were weight loss, a weight loss ≥ 5% at 14 months of follow-up and intervention tolerance.
2.5. Sample Size {#sec2.5}
----------------
A sample size of at least 139 subjects per group was required to demonstrate a 25% difference between BT and UC groups, with a two-tailed alpha risk of 5%, a power of 90%, and a BMI loss of 1.4 kg/m^2^ (±0.9 kg/m^2^) one year after BT therapy, according to the results of an open pilot study.
2.6. Statistics {#sec2.6}
---------------
According to the Zelen double consent design, a patient could decline the BT or UC randomised allocation to switch to the alternative treatment \[[@B18], [@B19]\]. This occurred for about half of the patients ([Figure 1](#fig1){ref-type="fig"}). With such a complete dilution of the randomisation, it was not relevant to perform intent-to-treat (ITT) analysis. Therefore, in order to reduce a treatment-selection and potential confounding factors as in an observational study, a propensity score to attend BT or to be managed by UC was used. The main analysis compared patients attending BT to optimally matched patients managed by UC. Patients were matched by sex, overweight or obese status, DMI randomisation, and propensity score nearest neighbour with a threshold of 0.1 \[[@B21]\]. A secondary analysis compared all BT and UC patients with DMI stratification and propensity score adjustment.
The baseline comparison between 2 groups used the Student\'s *t*-test and Chi-square test according to the variables. Propensity scores were estimated using a logistic model with all patient inclusion characteristics. The effect of BT versus UC, DMI versus no DMI, and the interaction between both interventions were assessed using generalized linear model for BMI and weight loss and using logistic model for weight loss ≥ 5% frequency. Adjusted mean and 95% confidence intervals \[95%CI\] were estimated using Least Squares Mean. A two-tailed *P* value \< 0.05 was considered statistically significant. Statistical analysis was performed using SAS software.
The study protocol was ethically approved by the Persons Protection Committee (Paris---Eudract number 2006-A00309-42) and registered to Clinicaltrials.gov number NCT01258114.
3. Results {#sec3}
==========
From March 2007 to July 2008, 71 GPs assessed 298 patients, 74 randomised to BT | {
"pile_set_name": "PubMed Central"
} |
1. Introduction
===============
Fish, marine oils, and their concentrates all serve as sources of the two marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as do some products from algae. To demonstrate an effect of EPA + DHA on heart health, a number of randomized, controlled intervention studies with clinical endpoints like overall mortality or a combination of adverse cardiac events were conducted in populations with elevated cardiovascular risk. One early intervention study with oily fish, rich in EPA + DHA, and some early studies with fish oil or fish oil concentrate or even purified EPA at doses ranging between 0.9 and 1.8 g/day indeed demonstrated effects in terms of fewer sudden cardiac deaths, fatal or non-fatal myocardial infarctions, or a combination of adverse cardiac events \[[@B1-nutrients-06-00799],[@B2-nutrients-06-00799],[@B3-nutrients-06-00799],[@B4-nutrients-06-00799],[@B5-nutrients-06-00799],[@B6-nutrients-06-00799]\]. More recent trials did not demonstrate such effects \[[@B7-nutrients-06-00799],[@B8-nutrients-06-00799],[@B9-nutrients-06-00799],[@B10-nutrients-06-00799],[@B11-nutrients-06-00799],[@B12-nutrients-06-00799]\]. Recent meta-analyses found no significant benefits on total mortality, cardiovascular mortality, and other adverse cardiac or cardiovascular events \[[@B13-nutrients-06-00799],[@B14-nutrients-06-00799],[@B15-nutrients-06-00799],[@B16-nutrients-06-00799],[@B17-nutrients-06-00799],[@B18-nutrients-06-00799]\]. This is in contrast to findings in epidemiologic studies, where intake of EPA + DHA had been found to correlate generally with an up to 50% lower incidence of adverse cardiac events \[[@B18-nutrients-06-00799],[@B19-nutrients-06-00799]\], and in even sharper contrast to epidemiologic studies based on levels of EPA + DHA, demonstrating e.g., a 10-fold lower incidence of sudden cardiac death associated with high levels of the fatty acids, as compared to low levels \[[@B20-nutrients-06-00799],[@B21-nutrients-06-00799]\]. This seemingly contradictory evidence has led the American Heart Association to recommend "omega-3 fatty acids from fish or fish oil capsules (1 g/day) for cardiovascular disease risk reduction" for secondary prevention, whereas the European Society for Cardiology recommends "Fish at least twice a week, one of which to be oily fish", but no supplements for cardiovascular prevention \[[@B22-nutrients-06-00799],[@B23-nutrients-06-00799]\]. The more recent guidelines on treating patients with stable ischemic heart disease or patients after a myocardial infarction, targeting similar patient populations, do not recommend EPA + DHA \[[@B24-nutrients-06-00799],[@B25-nutrients-06-00799]\]. At least in Europe, cardiologists do not routinely use EPA + DHA to reduce cardiovascular risk.
A similar picture emerges for atrial fibrillation: In epidemiologic studies, consumption of EPA + DHA or higher levels of EPA + DHA were associated with lower risk for developing atrial fibrillation, while intervention studies found no effect \[[@B26-nutrients-06-00799],[@B27-nutrients-06-00799],[@B28-nutrients-06-00799]\]. Pertinent guidelines do not mention EPA + DHA \[[@B29-nutrients-06-00799]\]. A similar picture also emerges for severe ventricular rhythm disturbances \[[@B20-nutrients-06-00799],[@B21-nutrients-06-00799],[@B30-nutrients-06-00799],[@B31-nutrients-06-00799]\].
Why is it that trial results are at odds with results from epidemiology? What needs to be done to better translate the epidemiologic findings into trial results? The current review will try to shed some light on this issue, with a special consideration of the Omega-3 Index.
2. The Omega-3 Index as a Cardiovascular Risk Factor
====================================================
At least some nutritional surveys do not provide valid data \[[@B32-nutrients-06-00799]\]. This may explain, why the relation of EPA + DHA in the diet to clinical events has been found to be looser than the relation of levels of EPA + DHA measured in blood to clinical events (e.g., \[[@B20-nutrients-06-00799],[@B33-nutrients-06-00799]\]). A detailed discussion of the pros and cons of the various fatty acid compartments in which levels of omega-3 fatty acids (whole blood, whole plasma, plasma phospholipids, and others) should be measured is outside the scope of this review and can be found elsewhere \[[@B34-nutrients-06-00799]\]. The following points argue for the use of erythrocytes: erythrocyte fatty acid composition has a low biological variability, erythrocyte fat consists almost exclusively of phospholipids, erythrocyte fatty acid composition reflects tissue fatty acid composition, pre-analytical stability, and other points \[[@B34-nutrients-06-00799],[@B35-nutrients-06-00799],[@B36-nutrients-06-00799],[@B37-nutrients-06-00799],[@B38-nutrients-06-00799]\]. In 2004, EPA + DHA in erythrocyte fatty acids were defined as the Omega-3 Index and suggested as a risk factor for sudden cardiac death \[[@B39-nutrients-06-00799]\]. Integral to the definition was a specific and standardized analytical procedure, conforming the quality management routinely implemented in the field of clinical chemistry \[[@B39-nutrients-06-00799]\] In fatty acid analysis, methods have a large impact on results: when one sample was sent to five different laboratories offering determination of an Omega-3 Index, results differed by a factor of 3.5 \[[@B34-nutrients-06-00799]\]. While results may be internally valid in one laboratory, a difference by a factor of 3.5 makes it impossible to compare results among laboratories. Therefore, the Omega-3 Index was renamed HS-Omega-3 Index^®^. In contrast, the laboratories adhering to the HS-Omega-3 Index methodology perform regular proficiency testing, as mandated in routine Clinical Chemistry labs \[[@B34-nutrients-06-00799]\]. So far, the HS-Omega-3 Index is the only analytical procedure used in several laboratories. A standardized analytical procedure is a prerequisite to generate the data base necessary to transport a laboratory parameter from research into clinical routine. Moreover, standardization of the analytical procedure is the first important criterion for establishing a new biomarker for cardiovascular risk set forth by the American Heart Association and the US Preventive Services Task Force \[[@B40-nutrients-06-00799],[@B41-nutrients-06-00799]\].
As exemplified by [Table 1](#nutrients-06-00799-t001){ref-type="table"}, the HS-Omega-3 Index has been measured in many populations. Of note, a lower HS-Omega-3 Index was always associated with a poorer clinical condition ([Table 1](#nutrients-06-00799-t001){ref-type="table"}).
nutrients-06-00799-t001_Table 1
######
Mean HS-Omega-3 Index values in various populations, Mean (±standard deviation (SD)). Please note that in every population studied, a lower value was found to be associated with a worse condition than a higher value. References are given, if not, unpublished, *n =* number of individuals measured.
Population HS-Omega-3 Index
--------------------------------------------------------------------------------------------------------------------------------- ------------------
**Western countries (high incidence of coronary heart disease)**
*Germany*
Unselected Individuals (*n* = 5000) 7.15 (±2.19)%
Patients with atherosclerosis \[[@B42-nutrients-06-00799]\], (*n* = 190) 5.94 (±1.41)%
Patients with hyperlipidemia \[[@B43-nutrients-06-00799]\], (*n* = 47) 7.00 (±1.90)%
Pregnant women, week 24 (*n* = 103) 7.66 (±1.83)%
Patients with congestive heart failure (*n* = 895) 3.47 (±1.20)%
Patients with major depression \[[@B44-nutrients-06-00799]\], (*n* = 90) 3.93 (±1.50)%
*Spain*
Individuals with high risk for, but without cardiovascular disease \[[@B45-nutrients-06-00799]\], (*n* = 198) (SD not reported) 7.10%
*Norway*
Patients with myocardial infarction \[[@B46-nutrients-06-00799]\] (SD not reported)
With ventricular fibrillation (*n* = 10) 4.88%
Without ventricular fibrillation (*n* = 185) 6.08%
*Europe*
Unselected data from routine determinations, *n* = 10,000 6.96 (+2.15)%
*USA*
Healthy in Kansas City \[[@B47-nutrients-06- | {
"pile_set_name": "PubMed Central"
} |
DOI: [10.1002/bjs5.50177](https://doi.org/10.1002/bjs5.50177)
*The Editor‐in‐Chief welcomes topical correspondence from readers relating to articles published in BJS Open. Letters should be submitted via ScholarOne Manuscripts and, if accepted, will be published online*.
We read with interest the work of Charehbili and colleagues[1](#bjs550285-bib-0001){ref-type="ref"}, which 'aimed to investigate whether there is a superiority of chlorhexidine--alcohol over iodine--alcohol for preventing SSI'.
This cluster‐randomized crossover trial was conducted in five hospitals and 3665 patients were included. The authors found that the incidence of surgical‐site infection (SSI) was not different between the groups: 3·8 per cent among patients in the chlorhexidine--alcohol group *versus* 4·0 per cent in those in the iodine--alcohol group (odds ratio 0·96, 95 per cent c.i. 0·69 to 1·35).
We commend the authors for performing this interesting study, as these results are useful for the choice of the most appropriate preoperative antiseptic. However, we have several statistical suggestions and queries that we would like to communicate to the authors.
The authors concluded that 'Preoperative skin disinfection with chlorhexidine--alcohol is similar to that for iodine--alcohol with respect to reducing the risk of developing an SSI'. This may be due to an underpowered study.
In fact, sample size was estimated by simulation. Although this approach is efficient, the authors do not provide enough details on the parameters they used. Thus it is not easy to replicate calculations. As mentioned by the CONSORT statement[2](#bjs550285-bib-0002){ref-type="ref"}: 'the reports of cluster randomized trials should state the assumptions used when calculating the number of clusters and the cluster sample size'.
The authors mention R software for the simulations that led to the final estimation of sample size. But several R packages are available and one may suppose that a package such as clusterPower was used[3](#bjs550285-bib-0003){ref-type="ref"}. Not knowing which package was used does not permit the analysis to be replicated. Moreover, algorithms used may vary between packages and lead to different estimations of sample size.
In a cluster‐randomized crossover trial, a sequence of interventions is assigned to a cluster (group) of individuals. Each cluster receives each intervention in a separate period of time and this leads to 'cluster periods\'[4](#bjs550285-bib-0004){ref-type="ref"}. There is usually a correlation between patients in the same cluster. In addition, within a cluster, patients within the same period may be more similar to one another than to patients in other periods[5](#bjs550285-bib-0005){ref-type="ref"}.
In a cluster‐randomized crossover trial, the sample size estimated by not taking into account the above‐mentioned features must be multiplied by a defined inflation factor. The latter can be approximated by (1 + (*n* − 1)ρ) − η[6](#bjs550285-bib-0006){ref-type="ref"}, [7](#bjs550285-bib-0007){ref-type="ref"}, where *n* is the average number of patients in a cluster during one of the periods, ρ is the intraclass correlation (ICC), and η the interperiod correlation. See, for example, Turner *et al*.[8](#bjs550285-bib-0008){ref-type="ref"} or Moerbeek and Teerenstra[9](#bjs550285-bib-0009){ref-type="ref"} (p. 94) for other approaches to estimate sample size in this context.
Parameters ρ and η can be retrieved from literature or estimated using assumptions or approximations[10](#bjs550285-bib-0010){ref-type="ref"} (p. 203), for instance: the logarithm of the ICC can be approximated by the logarithm of the prevalence of disease (here, the SSI rate)[11](#bjs550285-bib-0011){ref-type="ref"}; the interclass correlation is intrinsically lower than the ICC[12](#bjs550285-bib-0012){ref-type="ref"}.
Data were analysed using a multilevel model, which is appropriate. Treatment period was considered as a fixed effect and hospitals as random effect. Treatment period could also be considered as a random effect. In their simulations, Morgan *et al*.[5](#bjs550285-bib-0005){ref-type="ref"} actually demonstrated that 'hierarchical models without random effects for period‐within‐cluster, which do not account for any extra within‐period correlation, performed poorly with greatly inflated Type I errors in many scenarios'.
The authors did not report variance components of outcomes: within‐ and between‐participant variance, the ICC, as recommended by some authors[13](#bjs550285-bib-0013){ref-type="ref"}.
In a cluster‐randomized crossover trial, three components of variation are available: variation in cluster mean response; variation in the cluster period mean response; and variation between individual responses within a cluster period[4](#bjs550285-bib-0004){ref-type="ref"}. Small changes in the specification of the within‐cluster--within‐period correlation, or the within‐cluster--between‐period correlation, can increase the required number of clusters[4](#bjs550285-bib-0004){ref-type="ref"}. Thus, as the above‐mentioned correlation parameters were not reported by Charehbili *et al*.[1](#bjs550285-bib-0001){ref-type="ref"}, the number of clusters required may be larger than that used in the study.
A simulation study showed an association between an increase in cluster size variability and a decrease in statistical power[14](#bjs550285-bib-0014){ref-type="ref"}. The authors did not address this point.
In summary, the results of this study are interesting, but readers should interpret them with caution, according to the statistical methods used for design and analysis of cluster‐randomized crossover trials.
Disclosure {#bjs550285-sec-0002}
==========
The authors declare no conflict of interest.
| {
"pile_set_name": "PubMed Central"
} |
Debates over fundamental concepts in quantum theory such as reality, locality and quantum entanglement not only deepen our understanding of quantum mechanics but also promote the development of quantum information technology. One of such debates, which has existed since the beginning of quantum mechanics, is whether the quantum state corresponds to a real physical state (i.e., it is ontic) or whether it merely represents an observer's knowledge about the underlying reality (i.e., it is epistemic)[@b1][@b2][@b3][@b4]. A major reason for doubting the reality of the quantum state is that an epistemic interpretation of the quantum state could provide an intuitive explanation for many counterintuitive but fundamental quantum phenomena, such as the measurement postulate and wave function collapse[@b3][@b4].
Recently Harrigan and Spekkens formulated a theory regarding the ontic and epistemic concepts of the quantum states[@b2]. Their model is based on a reasonable assumption that every quantum system possesses a real physical state denoted *λ*, which is called the ontic state. Every ontic state corresponds to a different ontic *λ*, on which the possibility of measurement outcomes depends. Under this assumption, quantum states are supposed to be distinguishable via two models. A state is said to be of the *ψ*-ontic model if distinct pure quantum states always match distinct real states. On the other hand, a state is of the *ψ*-epistemic model if distinct states may result in the same ontic *λ*. Pusey, Barrett, and Rudolph (PBR) further develop the argument for the *ψ*-ontic and *ψ*-epistemic models through a no-go theorem. The theorem states that *ψ*-epistemic model cannot reproduce the predictions of quantum theory if the preparation independence assumption is taken[@b3]. Patra *et al.*[@b4] conclude on a similar result but with a different assumption of continuity for a single quantum system (Hardy also obtains a similar result independently[@b5]). Further theoretical works focusing on the no-go theorems for *ψ*-epistemic models have been presented in refs [@b6], [@b7], [@b8], [@b9], [@b10], [@b11], [@b12], [@b13], [@b14]. Given these growing theoretical works, experimental tests of the *ψ*-ontic and *ψ*-epistemic models are needed.
The first experiment testing of the existence of *ψ*-epistemic models was implemented by Nigg *et al.*[@b15] with two atoms in an ion trap following the PBR theorem[@b3]. Very recently, Ringbauer *et al.* reported an experiment to rule out the maximal *ψ*-epistemic model with single photons in 3 and 4 dimensions[@b16]. The no-go theory of continuous *ψ*-epistemic models[@b4] was also experimentally tested using attenuated coherent states of light to simulate the high-dimensional single photon quantum states by Patra *et al.*[@b17]. However, due to the nonideal state preparation (the phase fluctuations of the coherent states) in the experiment[@b17], one additional assumption that the ontic state depends on control measurements should be included in the explanation of the experiment. Furthermore, the no-go theorem is proposed for a state of single particles whereas a coherent state with pretty large possibility of multi-photons was used in the experiment[@b17]. Therefore, further experiments to test the epistemic models with a state of single particles and with fewer assumptions are needed.
In this paper, we report an experimental test of the existence of the continuous *ψ*-epistemic models with high-dimensional single photon quantum states. We produce the heralded narrowband single photon quantum states in dimensions 3, 7, 16, 25 and 32 to test the no-go theorem and our results support the no-go theorem for continuous *ψ*-epistemic models. In comparison with Patra *et al.*'s experiment[@b17], our experiment benefits from the ideal single photon source and avoids the nonideal state preparation loophole. Thus the no-go theory of continuous *ψ*-epistemic models is tested without additional assumptions except for the fair-sampling assumption for the single photon detection loophole made in all of the above experiments[@b15][@b16][@b17] and often made in nonlocality experiments[@b18].
Results
=======
The no-go theorem and the experimental setup
--------------------------------------------
The difference between the *ψ*-ontic models and the continuous *ψ*-epistemic models (also named *δ*-continuous *ψ*-epistemic models) is shown in [Fig. 1](#f1){ref-type="fig"}. The *δ*-continuous *ψ*-epistemic models assume that there are ontic states *λ* in the initial ensemble (e.g. *ψ*~1~) that will remain part of the slightly perturbed ensemble (e.g. *ψ*~2~) (see Method for a detail definition of the *δ*-continuity). There are two parameters to characterize the *δ*-continuous *ψ*-epistemic models: the parameter *δ* characterizes how continuous the model is, and characterizes how epistemic it is. As proven in ref. [@b4], there is a fundamental constraint (the no-go theorem) on *δ*-continuous models for single systems: there are no *δ*-continuous *ψ*-epistemic models with reproducing the measurement statistics of quantum states in a Hilbert space of dimension *d*. Mathematically, the *δ*-continuous *ψ*-epistemic models predict the following (with preparations *Q*~*k*~ corresponding to distinct quantum states \|*ψ*~*k*~〉 all contained in a ball of radius *δ*):
Here *P*(*k*\|*M*, *Q*~*k*~) stands for the probability of outcome *k* with a measurement *M* carried out on a system in ontic state *λ* which is prepared with procedure *Q*~*k*~ and associated with a probability distribution *P*(*λ*\|*Q*~*k*~). The parameter can be viewed as a measure of the extent to which distributions over real states overlap in the neighborhood of a given quantum state[@b4], or it can be seen as related to the variational distance between the distributions *P*(*λ*\|*Q*~*k*~)[@b3].
According to quantum theory, however, there invariably exist some states for which the left-hand side of [Eq. (1)](#eq19){ref-type="disp-formula"} is equal to 0. As a typical example, we consider *d* distinct states (*j*, *k* = 1, ...*d*), which are all at mutual distance from the state . They have *δ*-continuity property since \|*ψ*~*k*~〉 contain in a ball of radius *δ* centered on \|*ψ*〉. If the basis of the measurement *M* is chosen to be \| *j*〉, then *P*(*k*\|*M*, *Q*~*k*~) = 0 for all *k* = 1, ...*d* and thus the left hand side of [Eq. (1)](#eq19){ref-type="disp-formula"} is equal to 0. Hence, the *δ*-continuous *ψ*-epistemic models' existence can be experimentally tested.
We use the time bins of the narrowband single photon (labeled by the time *jτ* when it is detected) as the basis states \| *j*〉 to realize the quantum state \|*ψ*~*k*~〉 in the experiment. As shown in [Fig. 2](#f2){ref-type="fig"}, narrowband paired photons are generated through spontaneous four-wave mixing (SFWM) in a two-dimensional ^85^Rb magneto-optical trap (MOT) (see Methods for detail)[@b19][@b20]. A detection (the single photon counter: PerkinElmer SPCM-AQRH, *C*~*dk*~ = 500 ± 100 *c*/*s*, *D*~0~, *D*~1~/*D*~2~) of the Stokes photon heralds the presence of its paired anti-Stokes photon, which also determines the start point of the experiment. After collection of the photons with single mode fibre (SMF), the anti-Stokes photon passes through an electro-optical amplitude modulator (EOAM, fiber-based, 10 GHz, Eospace), which is used to shape the waveform of the photons[@b21][@b22]. With the EOAM and an arbitrary waveform generator (AWG, Agilent 81150A), the anti-Stokes photon can be prepared with the waveform consisting of a train of *d* pulses with one missing. To perform under a stable time base, the AWG's master clock is phase-locked to an external 10 MHz reference (SRS FS725) with exceptionally low phase noise (−130 dBc/Hz at 10 Hz offset) and one second Allan variance (\<2 × 10^−11^). The modulated anti-Stokes photons then pass through a 180 *m* fiber spool to end the preparation process, which is long enough to store the entire photon wave package. The coincidence counts are implemented by a time-to-digital converter (Fast Comtec P7888) with a 2 ns bin width. The verification of the single-photon nature of the heralded anti-Stokes photons is carried out with standard Hanbury-Brown-Twiss interferometer (a fiber-based 50/50 beam splitter and | {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Our understanding of the genetic basis of Parkinson's disease (PD) has increased tremendously over the past 20 years. Mutations in the gene encoding alpha-synuclein (α-syn) were the first to be associated with genetic PD. Another monogenic causative factor in PD patients is *leucine-rich repeat kinase 2* (*LRRK2*), of which more than 100 variants have been identified \[[@CR1]\]. Asymptomatic carriers of *LRRK2* mutations constitute an ideal population for identifying predictive biomarkers of PD for several reasons: 1) a high risk of conversion to PD, 2) dopaminergic neuronal loss demonstrated by positron emission tomography (PET) scanning, and 3) similarity of the clinical phenotype of LRRK2-associated PD to that of patients with sporadic PD (sPD). While the exact involvement of LRRK2 in PD pathogenesis remains only partially understood, converging evidence suggests a role for LRRK2 in modulating inflammation \[[@CR2], [@CR3]\]. As PD has been proposed to start as an inflammatory disease \[[@CR4], [@CR5]\], it is plausible to suggest that there may be a link between *LRRK2* mutations and inflammation.
Several research groups, including ours, have explored the potential of CSF alpha-synuclein (α-syn) forms as diagnostic or progression biomarkers for PD. Total α-syn (t-α-syn) levels were reported to be lower in PD, whereas oligomeric (o-α-syn) and phosphorylated Ser129-α-syn (pS129-α-syn) appear to be elevated \[[@CR6]--[@CR9]\]. CSF core biomarkers of Alzheimer's disease (AD) pathology have also been widely explored in PD cases. While a drop in CSF Amyloid-beta (Aβ-42) levels have been reported in PD \[[@CR10]\], the biomarker profile of total tau (tTau), and phosphorylated threonine 181 tau (pTau) were variable \[[@CR11], [@CR12]\]. More importantly, the potential of the aforementioned proteins as markers for PD at preclinical stage remains largely unexplored. Carriers of *LRRK2* mutations have an elevated risk of developing PD and they therefore represent a useful population in which to identify biomarkers of prodromal PD \[[@CR13]\]. However, there is a paucity of data on different forms of α-syn, AD-related proteins and inflammatory biomarkers in *LRRK2* mutation carriers \[[@CR14]--[@CR16]\]. In the present study, our primary objective was to identify a panel of CSF biomarkers for the early detection of PD, preferably at the presymptomatic stage. A secondary objective was to study whether CSF levels of particular biomarkers were associated with severity of clinical symptoms of PD. Towards that end, we measured the levels of different α-syn species, AD-related proteins and 40 different inflammatory markers in CSF samples from a well-characterized Norwegian cohort of 74 subjects with *LRRK2* mutations: 23 symptomatic individuals and 51 asymptomatic mutation carriers. In parallel, we included 60 patients with sporadic (i.e. idiopathic) PD (sPD) and 43 healthy control subjects (first-degree relatives of *LRRK2* mutation carriers (Ctrl)).
Methods {#Sec2}
=======
Patient selection and CSF sampling {#Sec3}
----------------------------------
Patient selection criteria and the method of CSF collection were as described in previous publications \[[@CR16], [@CR17]\]. In total, 74 Norwegian individuals from 12 different families with *LRRK2* mutations were assessed in the current study. Twenty-three patients were clinically diagnosed with PD, whereas 51 patients were healthy, asymptomatic *LRRK2* mutation carriers when enrolled in the study. These families have been extensively described in previous reports \[[@CR17]--[@CR19]\]. In addition, 60 patients with sPD and 43 age-matched controls were recruited for this study from St. Olav's Hospital at the University Hospital of Trondheim in Norway. The control group was composed of first-degree relatives of *LRRK2* mutation carriers who were not carrying *LRRK2* mutations. PD clinical diagnoses were made by experienced senior clinicians based on guidelines described by Gelb and colleagues \[[@CR20]\] and disease stage was assessed according to the Hoehn and Yahr (H&Y) scale. All patients with sPD were screened and confirmed to be negative for known *LRRK2* mutations. Patients with an age at onset of ≤50 years were confirmed to be negative for known pathogenic mutations in *Parkin* and *PINK1*. All family members of LRRK2 patients were examined for clinical features of PD by movement disorder specialists and found to be asymptomatic, although a few had mild premotor signs and an increased Unified Parkinson's Disease Rating Scale (UPDRS) score (three cases scored \> 10 on UPDRS-III) \[[@CR21]\]. The *LRRK2*-mutant PD patients were taking levodopa, and some were taking dopamine agonists and/or monoamine oxidase-B (MAO-B) inhibitors.
To collect CSF, lumbar punctures were performed in overnight fasted patients between 8 and 10 am. CSF samples were aliquoted in 1.2--1.5 ml low-binding tubes, and one vial was sent for routine laboratory analysis (i.e., white and red blood cell count, total protein and glucose levels, according to the Parkinson's Progression Markers Initiative \[PPMI\] protocol), whereas the majority of the vials were frozen fewer than 15 min after collection following centrifugation at 2000 g at 4 °C then sub-aliquoted and stored at − 80 °C until further analysis. All patients provided signed informed consent, and the study was approved by the Regional Committee for Medical and Health Research Ethics.
Measurement of alpha-synuclein species {#Sec4}
--------------------------------------
All immunoassays used to measure the different species of α-syn were developed in-house and described in previous reports \[[@CR9], [@CR22], [@CR23]\] . Briefly, to capture t- or pS129-α-syn, a 384-well ELISA microplate was coated with 0.1 or 0.5 μg/ml Syn-140 (a sheep anti-α-syn polyclonal antibody) in 200 mM NaHCO~3~, pH 9.6 (50 μl/well) by overnight incubation at 4 °C, while 0.2 μg/ml of our mouse conformation-specific antibody, Syn-O2, was used to capture o-α-syn. After incubation with 100 μl/well of blocking buffer (PBS-T containing 2.25% gelatin) for 2 h at 37 °C, 50 μl/well of the CSF samples (diluted 1:2 in artificial CSF) along with serial dilutions of recombinant human t-, pS129-or o-α-syn (50 μl) were dispensed in each well, and the plate was incubated at 37 °C for 2.5 h. After washing with PBS-T, 50 μl/well of 11D12 (a mouse anti-α-syn monoclonal antibody) \[[@CR9]\], PS129 (a mouse anti-pS129-α-syn monoclonal antibody) \[[@CR9]\], or FL-140 (rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), for measuring t-, pS129-α-syn, or o-, respectively, were added to the corresponding wells and incubated at 37 °C for 2 h. Next, the plates were washed and incubated for 2 h at 37 °C with 50 μl/well of species-appropriate secondary antibody (goat anti-rabbit IgG HRP or donkey anti-mouse IgG HRP, Jackson ImmunoResearch Laboratories Inc., USA, 1:20,000 dilution). After washing, the plates were incubated with 50 μl/well of enhanced chemiluminescent substrate (Super Signal ELISA Femto, Pierce Biotechnology, USA). The chemiluminescence, expressed in relative light units, was immediately measured using a PerkinElmer Envision multi-label plate reader (PerkinElmer, Finland). CSF samples were measured in a blinded fashion and randomized for analysis, with all LRRK2 symptomatic/asymptomatic, PD and HC samples being tested together on the same ELISA microplates. A series of internal controls was also run to check for run-to-run variations. The concentrations of α-syn species in the samples were calculated using the corresponding standard curves.
Measurement of AD biomarkers {#Sec5}
----------------------------
CSF Aβ42, Aβ40, total tau (tTau), and phosphorylated threonine 181 tau (pTau) were measured using MILLIPLEX® MAP Human Amyloid Beta Tau Magnetic Bead Panel (Luminex xMAP) run on the Bio-Plex® 3D instrument (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's instructions. The kit allows simultaneous quantification of Aβ40, Aβ42, tTau, and pTau.
Measurement of inflammatory markers {#Sec6}
-----------------------------------
A magnetic human chemokine bioplex assay (Bio-Rad Laboratories, Hercules, CA) was used to measure 40 chemokines from human CSF samples (6Ckine/CCL21, BCA-1/CXCL13, CTACK/CCL27, ENA-78/CXCL5, Eotaxin/CCL11, Eotaxin-2/CCL24, Eotaxin-3/CCL26, Fractalkine /CX3CL1, GCP-2/CXCL6,GM-CSF, Gro-α/CXCL1, | {
"pile_set_name": "PubMed Central"
} |
1.. Introduction {#s1}
================
Metabolic syndrome (MetS) refers to a cluster of metabolic disorders such as hyperglycemia, insuline resistance, type II diabetes mellitus, and hyperlipidemia which cause obesity and cardiovascular diseases [@b1]. Diet quality is a strong predictor of this syndrome [@b1],[@b2]. A diet, which is high-carbohydrate and low fibre, increases directly postprandial levels of blood glucose and insuline, which leads to insuline resistance [@b3]. Insuline resistance is the cause of type II diabetes mellitus and cardiovascular diseases [@b4]. Therefore, eating well-quality diets would be well-advised for controlling metabolic syndrome [@b5]. White rice is the most commonly starchy food staple, especially in developing countries of Asia and Africa [@b6]. However, its high glycemic index leads to white rice could not be frequent consumption food for diabetes patients [@b7]. Pre-germinated brown rice (PGBR) may be more benefits than white rice. PGBR, a newly developed type of rice, was made by soaking brown rice kernels in water to slightly germinated them [@b8]. There are few studies proved the effects of PGBR on the prevention and treatment of metabolic syndrome [@b9]--[@b11]. The aim of this study was to investigate the effects of PGBR used to treat metabolic syndrome patients.
2.. Materials and methods {#s2}
=========================
2.1.. Settings and subjects study {#s2a}
---------------------------------
The protocol of this study was approved by the ethics committee of the National Institute of Nutrition (NIN), Hanoi, Vietnam (number 1656/QĐ-VDD) and also approval from all participants, before recruiting subjects. 80 subjects with the metabolic syndrome (MetS) were recruited in the intervention. All of them live in Bac Ninh City. Bac Ninh City is in the Red River Delta of northern Vietnam, approximately 40 km from the capital, Hanoi. All subjects were fully informed concerning the process of the study, and the study was designed in with the Helsinki Declaration on Human Studies. Eligibility criteria were: age from 55 to 70 years old, live in Bac Ninh City, and were diagnosed having MetS. A subject was defined as having MetS if he/she was any three of the following six factors: waist circumference \> 90 cm for men and \> 80 cm for women; blood triglyceride level ≥ 1.69 mmol/L; serum HDL-cholesterol concentration \< 1.04 mmol/L for men and \< 1.3 mmol/L for women; fasting plasma glucose \> 6.1 mmol/L; systolic blood pressure ≥ 130 mmHg and/or diastolic blood pressure ≥ 85 mmHg. Exclusion criteria included the following: subjects who were taking medicines that controlled blood glucose or lipid metabolic; subjects who were suffering from serious heart diseases, brain diseases, kidney diseases, liver diseases, or gastrointestinal diseases; subjects who were consuming PGBR daily; subjects who had body mass index (BMI) under 18.5; subjects who were not feeling well after starting the intervention. After screening, 86 subjects met the requirements. During 3 months of the intervention, 3 subjects in each group were excluded because of the individual health status.
The subjects were divided randomly into two groups: a PGBR group (n = 43) and a placebo group (n = 43). The subjects in PGBR group received 1 PGBR capsule (2.0 kg) containing 200 g PGBR/day for 10 days/time, and subjects in the placebo group took 1 capsule (2.0 kg) containing 200 g white rice/day for 10 days/time. The amount of energy, lipid, and protein is almost equivalent to the placebo and PGBR capsules. All subjects were advised to avoid change in their lifestyle, and no subjects were taking any medicines that affected cholesterol levels or body fat reduction during the time of the study.
We measured blood samples at the laboratory of NIN, Hanoi. Blood samples were taken an over-night fast. The PGBR and placebo groups were measured on the same day.
2.2.. Anthropometric measurements {#s2b}
---------------------------------
Weight, height, waist and hip circumferences were measured two times. Bodyweight and height were measured in light clothing and without shoes. BMI was calculated as body weight per height squared (kg/m^2^). Waist circumference was measured mid-way between the lower rib margin and the iliac crest, while hip circumference was measured at the broadest circumference around the buttocks.
2.3.. Nutrition survey {#s2c}
----------------------
We used the Semi-Quantitative Food Frequency Questionnaire for a nutrition survey at baseline and completion. Energy and nutrient intake were measured based on the Vietnamese Food Composition Table 2007 [@b12].
2.4.. Physical activity survey {#s2d}
------------------------------
We used the structured questionnaire for a physical activity survey at baseline and completion. The levels of physical activity were built based on the Global Recommendations on Physical Activity for Health [@b13].
2.5.. Blood sample collection {#s2e}
-----------------------------
Blood samples were taken in the morning and after an overnight fast and were measured two times. Fasting glucose was measured by Accu-Chek glucometer. Venous blood samples were kept frozen at 8 °C for analysis: serum total cholesterol, HDL cholesterol, LDL cholesterol, concentrations of insuline, fasting triglyceride.
2.6.. Blood pressured {#s2f}
---------------------
Blood pressure was measured by using the mercury sphygmomanometer. All subjects were measured in a quiet, air-conditioned room (the room temperature at 25 °C). The participants had to rest before and during measurements for about 20 min.
2.7.. Homeostatic Model Assessment for Insuline Resistance (HOMA-IR) {#s2g}
--------------------------------------------------------------------
HOMA-IR was calculated using the following formula: fasting insuline (mU/l) × fasting glucose (mmol/l)/22.5. Fasting glucose and insuline were measured using an enzymatic method and chemiluminescence immunoassay, respectively.
2.8.. Statistical analysis {#s2h}
--------------------------
Data were analyzed by using SPSS for windows 16.0. We calculated the change in percentage for each variable at completion and baseline. All variables of the PGBR and placebo groups were compared using an unpaired *t*-test, and data at baseline and completion were compared using a paired *t*-test. Varibales with confirmed homoscedasticity were compared by Student\'s *t*-test. *p* values of less than 0.05 were considered statistically significant for all analyses. Mann-Whitney test and Wilconxon test were used in non-normal distribution. Mc Nemar test -- non parametrict test -- was used on paired nominal data.
3.. Results {#s3}
===========
3.1.. Subjects {#s3a}
--------------
Eighty subjects completed the intervention; 3 subjects from the placebo group and 3 subjects from the PGBR group withdrew from the study, due to personal reasons. All subjects completed over 90% of the study process. No subjects in either group had any symptoms related to the intake of test substances.
3.2.. Nutrition facts for PGBR and placebo capsules {#s3b}
---------------------------------------------------
[Table 1](#publichealth-07-01-005-t01){ref-type="table"} shows the amount of energy, protein, lipid, carbohydrate, ash, sodium, and γ-aminobutyric acid (GABA) per 100 g GPBR and per 100 g white rice.
###### Nutrition facts per 100 g of PGBR and white rice.
Nutrition facts per 100 g PGBR White rice
--------------------------- -------- ------------
Energy (kcal) 357 345
Protein (g) ≥ 7 ≥ 5
Lipid (g) ≥ 2 ≥ 0.1
Carbohydrate (g) ≥ 60 ≥ 75
Fibre (g) 3--4 0.5--0.9
Inositol (mg) ≥ 10 \-
GABA (mg) 12--20 1--3
3.3.. Baseline characteristic {#s3c}
-----------------------------
[Table 2](#publichealth-07-01-005-t02){ref-type="table"} shows the baseline parameters of both groups. There were no significant differences in parameters for age, sex, education levels, height, weight, BMI, abdominal circumference, systolic blood pressure, diastolic blood pressure, fasting glucose, insuline, HOMA-IR, total cholesterol, triglyceride, LDL cholesterol, HDL cholesterol, percentage of constipation, physical activity levels, diet incides between the PGBR and the placebo groups.
###### Baseline characteristics of PGBR and placebo groups.
Total sample PGBR group Placebo group
--------------------------------- ------------------------------------- -------------- --------------- ------------
Age(y) 65.1 ± 3.81 65.2 ± 3.78 65.0 ± 3.85
Sex
Male 16 (20%) 8 (20%) 8 (20%)
Female 72 (80%) 36 (80%) 36 (80%)
Education
Primary and secondary 38 (47.5%) 18 (45.0%) 20 (50 | {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
There is a clinical need for efficient and cost-effective grafts in urethral reconstructive surgery. Current surgical techniques require harvesting from autologous sites eg. buccal mucosa and penile skin tissue^[@CR1]^. This increases the risk of complications, the possible lack of tissue availability and patient discomfort. Certain oral pathologies are contraindications for buccal mucosal graft harvesting^[@CR2]^. Both cell-free and cell-seeded urethral grafts have been investigated. To date, cell-based grafts have shown better functional results in preclinical large animal models and inconclusive results in subsequent clinical trials^[@CR3]^. In 2015 and 2017, the clinical outcome of a commercial cell-seeded tissue engineered buccal mucosa product, was published^[@CR4],[@CR5]^. This product requires a buccal biopsy to be taken, followed by *ex-vivo* cell culturing in a GMP facility and a 3-week manufacturing time. The cost-effectiveness and efficacy of these grafts have been debated^[@CR6],[@CR7]^. Therefore, it is unlikely that this approach will become standard clinical practice for the patient with limited financial resources or insurance coverage. To overcome these problems we have targeted the development of a cell-free urethral graft that is cost-effective and adequate also to be used in third world countries.
*In vitro* the mechanical niche is an important parameter to be considered for controlling cell fate. For example, mesenchymal stem cells are known to differentiate into different cell lineages, depending on the substrate stiffness^[@CR8]^. Previous studies have shown that fluid content or collagen fiber density relates to the stiffness and influenced material properties of the collagen gels^[@CR9],[@CR10]^. The more extracted fluid from the collagen gels, the stiffer the collagen material gets. In this manuscript we address the question if collagen fiber density of a collagen graft influences the functional regeneration of the rabbit urethra? Two different cell-free bovine tubular collagen grafts were manufactured, one with polarized and low collagen fiber density distribution (LD-graft) and one control with uniform and high collagen fiber density distribution (HD-graft). Polarization was achieved by specific compression and the collagen density increased towards the luminal side of the tube. These grafts were implanted in rabbits and were examined post-implantation by histology and immunohistochemistry, and the functional outcome was assessed by voiding cystourethrography and mating.
Materials and Methods {#Sec2}
=====================
Preparation of collagen grafts {#Sec3}
------------------------------
Tubular collagen grafts were fabricated under sterile conditions, utilizing liquid type I bovine collagen (5 mg/mL, Symatese, F). 8.5 mL of collagen solution were added to 0.8 mL of 10 × MEM and neutralized with 1 M NaOH (approx. 1.85 mL). The neutralized solution was set in a mold previously used for manufacturing tubular constructs of 3 mm inner diameter^[@CR10]^. To fabricate the LD-graft, the gelated collagen was manually compressed by rolling it on filter paper, followed by an air-drying step (Fig. [1A](#Fig1){ref-type="fig"}). To fabricate the HD-graft, only air-drying was applied to achieve a desired liquid content of 1% w/w (Fig. [1A](#Fig1){ref-type="fig"}). Monitoring of the water content of collagen gels/grafts were done with a balance (Mettler Toledo, CH). The fabricated grafts were kept in PBS supplemented with 1% Penicillin/Streptomycin (Gibco, CH) and 2.5 mg/mL Fungizone (Gibco) until used.Figure 1Manufacturing technique of the low-density graft (LD-graft) and high-density graft (HD-graft) (**A**) Two methods were utilized to manufacture the density controlled collagen grafts by monitoring the water content removed either by rolling compression or air-drying. The polarized collagen fiber distribution characteristics of the LD-graft were achieved by utilizing both a rolling compression followed by air-drying. The uniform collagen fiber distribution characteristics of the HD-graft were achieved by only air-drying the collagen. (**B**) By weighing and monitoring the collagen gel during production, we could achieve our polarized (compressed and air-dried: LD-graft) and uniform (only air-dried: HD-graft) collagen fiber distribution characteristics. Note: Images in Fig. 1A are Sirius red stained paraffin sections with scale bars of 200 µm.
Scanning electron microscopy {#Sec4}
----------------------------
The samples were fixed with 1% tannic acid and 1.25% glutaraldehyde, then washed with 0.1 M cacodylate, and dehydrated in increasing ethanol concentrations prior to critical point drying. Thereafter coated with gold/palladium and imaged at a voltage of 10 kV using a scanning electron microscope (SEM, XLF30, Philips).
Mechanical evaluation {#Sec5}
---------------------
Burst pressure, ultimate tensile strength (UTS) and Young's modulus of grafts (N = 4 in each group) were measured with an electronic manometer (Extech instruments HD750) or an Instron tensile machine (Norwood, MA, USA) as described previously^[@CR10]^.
Surgical implantation of the graft and evaluation in rabbits {#Sec6}
------------------------------------------------------------
Following approval by the Animal Ethics Committees of the Canton of Vaud (Authorization number: VD-2740), Switzerland and of the Faculty of Medicine of the University of Malaya (Authorization number: 2013-07-19/SUR/R/TCR), Kuala Lumpur the experiments were performed on New Zealand white male rabbits (2.5--3.5 kg; Charles River Laboratories France, and Harlan and Bred, Singapore) in Lausanne and Kuala Lumpur. All experiments were done in compliance with national directives for the care and use of laboratory animals. The surgical steps and post-operative monitoring were done as previously described^[@CR10]^. Rabbits from the long-term study group were involved in the in-house breeding program of the Animal Experimental Unit of the Faculty of Medicine, University Malaya.
Cysto-urethrography {#Sec7}
-------------------
Animals were submitted under general anesthesia for macroscopic evaluation and voiding cysto-urethrography (Visipaque 270 mg/mL). All images were collected with a Philips BV Pulsera. The diameter of the urethra was measured utilizing a scale. Knowing that the graft was sutured at 0.5 cm proximal to the base of the glans and it measured 2 cm in length, the position of the graft could be determined on the radioscopic images. It was then possible to estimate the potential presence of stenosis at the anastomotic sites. Stenosis was defined as a persistent 50% reduction of the diameter of the urethra at the same location.
Histology and immunohistochemistry {#Sec8}
----------------------------------
Entire penises were harvested and fixed in 4% PFA. Specimens were embedded in paraffin, and 8 µm thick sections were prepared. Antibodies used: Mouse anti-α-smooth muscle actin (1:150, Abcam, CH), Goat anti-uroplakin-2 (1:150, Labforce, CH), Mouse anti-caldesmon (1:400, Sigma, D), Rabbit anti-CD31 (1:100, Abcam, CH), Donkey anti-mouse Alexa 647 (1:800, Abcam, CH), and Donkey anti-goat Alexa 546 (1:800, Abcam, CH). Images were taken with a Leica DM5500 microscope (Leica, D) and with a LSM 700 microscope (Zeiss, D). Alpha smooth muscle actin (α-SMA) expression was quantified with Fiji imaging program (ImageJ) as previously described^[@CR10]^.
Statistical analysis {#Sec9}
--------------------
Statistical analyses were performed using Prism v5.0a (GraphPad), using the test mentioned in the figure legends. A p-value of less than 0.05 was considered significant. All error bars in diagrams represent the standard deviation (SD).
Data Availability {#Sec10}
-----------------
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Results {#Sec11}
=======
Manufacture of HD- and LD-grafts {#Sec12}
--------------------------------
By applying a fast compression step, resulting in a less than 50% liquid loss followed by a slow air-drying step, we could control the liquid content of our produced grafts and therefore also the collagen grafts final density (Fig. [1B](#Fig1){ref-type="fig"}). This procedure yields a controlled polarized collagen fiber distribution, with a more dense structure at the compressed outer surfaces while the internal luminal part of the collagen tubular wall was left with a less dense collagen structure as shown by SEM (Fig. [2A](#Fig2){ref-type="fig"}). This graft hereafter is referred to as the LD-graft. In comparison, in the only air-dried collagen grafts, the latter become highly dense and with a uniform non-polarized collagen structure (Fig. [2B](#Fig2){ref-type="fig"}). This graft is hereafter referred to as the HD-graft. The denser HD-grafts showed significantly better mechanical properties, in terms of measured burst pressure, UTS and Young's Modules compared to the LD-graft (Supplementary | {
"pile_set_name": "PubMed Central"
} |
Background
==========
The methylenetetrahydrofolate reductase enzyme (MTHFR) catalyzes a reaction that produces 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for homocysteine in the synthesis of methionine. The 677C\>T mutation of the *MTHFR*gene has been associated with a thermolabile enzyme with decreased activity that may cause an increase in plasma homocysteine concentrations \[[@B1]\] when folate status is poor. This polymorphism is one of the most widely studied clinically relevant polymorphisms in humans, as it is related to cardiovascular disease \[[@B2]\] and neural tube defects (NTD; 601634) \[[@B3]\].
A large number of studies have provided a broad overview of the prevalence of the 677C\>T polymorphism in different human populations, showing that the distribution of frequencies is diverse \[[@B4]\]. These differences have been also observed between groups of different ages in the same Spanish population (older and younger than 24 years) \[[@B5]\] and in a Swiss population (older and younger than 60 years) \[[@B6]\], as well as in a Japanese population \[[@B7]\].
In some populations, such the Toscanians in Italy \[[@B8]\] and Mexicans \[[@B9]\], the homozygous mutated genotype (TT) has reached frequencies greater than 30%. On the other hand, in Africans the frequency of the TT genotype is very low (less than 1%) \[[@B10],[@B11]\], but, in African-Americans, it has already reached 2% \[[@B12]\]. Studies based on the distribution of genotypic and allelic frequencies of the 677C\>T polymorphism and the 1298A\>C polymorphism in the *MTHFR*gene in Israeli, Japanese and Ghanaian Africans populations \[[@B13]\] concluded that the 677T mutation in the *MTHFR*gene emerged as a founder haplotype with some selective advantage. Recently, preliminary evidence of genetic selection of this polymorphism related to folate intake has been reported \[[@B14]\].
The aim of the present study is to analyze the changes in frequencies of the 677C\>T polymorphism during the 20^th^century and particularly the evolution of the frequencies during the decade of 1980--1989, by comparing the genotype frequencies between living subjects born in this period versus samples of spontaneous abortions (SA) that occurred during in the same time period.
Methods
=======
Subjects
--------
This study was approved by the Ethics Committee at the University Hospital \"Virgen de la Victoria\" (Málaga). One of the study groups consisted of 344 fetal tissue samples from SA, obtained from the Department of Pathology of the University Hospital Carlos Haya (Málaga). These samples were selected after checking the clinical history and by the inclusion criteria of containing histologically confirmed fetal tissue collected in the 1980s from SA at less than 3 months (11 ± 1.70 week) and of unknown etiology. These fetal samples were compared with a control population of 276 subjects born in the 1980s with an average age of 22 ± 4.58.
Another population of subjects born in the south of Spain in the 20th century were genotyped (1305 subjects, 697 women and 608 men) and divided into four groups according to birth date: 1900 to 1925 (n = 206); 1926 to 1950 (n = 320), 1951 to 1975 (n = 408), 1976 to 2000 (n = 371). Individuals were selected randomly from different areas of the province of Malaga, in southern Spain, and from different social statuses to avoid a selection bias. All the selected individuals were Caucasian and residents of the study area. The parents of all subjects included in the study were also Caucasian and born in Spain. The possibility of a founder effect or genetic drift was investigated and rejected. All the selected individuals were also genotyped for an insertion/deletion polymorphism in the angiotensin converting enzyme (ACE) gene and/or the 2756A\>G polymorphism in the methionine synthase gene (MTR), in order to determine whether or not our adult and young populations were genetically homogeneous. No significant differences were observed in allelic or genotypic frequencies for these genes between the different groups.
The population studied was randomly selected according to age. Subjects 0--12 years old were selected from dried blood spots from neonatal screening papers; subjects 10--24 years old were recruited from students in primary and secondary schools and in university; subjects 25--50 years old and \>51 years old were recruited using their Andalusia Health Service identity cards. After approval by the University Hospital Ethical Committee, all the subjects were contacted, and, from those whose written consent was obtained, 10 ml of blood was taken. The investigation in this study conforms to the principles outlined in the Declaration of Helsinki.
Genetic analysis
----------------
The fetal samples were extracted from the archived formalin-fixed, paraffin-embedded tissue sections. Genomic DNA was extracted from fetal tissue using the method described by Coombs et al. (1999) \[[@B15]\]. Genomic DNA of the second and third groups was extracted from peripheral leukocytes using the AquaPure Genomic DNA Blood Kit (Bio-Rad).
Genotyping was performed using Real Time PCR with allele specific Taqman^®^probes and primers described by Ulvik et al. (2001) \[[@B16]\] and the following optimized protocol for 45 cycles: 10 s -- 94°C, 40 s -- 54°C, 15 s -- 72°C. The PCR mix (25 μl total volume) consisted of 5 μl of genomic DNA, 0.5 μl of sense primer, 0.62 μl of anti-sense primer, 0.85 μl Taqman^®^probe FAM, 0.43 μl Taqman^®^probe TET, 20 μl PCR-buffer iQ-SupermixTM (Bio-Rad) (containing 100 mM KCl, 40 mM Tris-HCl, (pH 8.4) 1.6 mM dNTP (dATP, dCTP, dGTP and dTTP), iTaq^®^polymerase (50 units/mL) and 6 mM MgCl2) and 17.75 μl H2O.
Statistical and mathematical analysis
-------------------------------------
All samples were genotyped, and the allelic and genotypic frequencies were compared. Differences were analyzed statistically using the chi-square test or Fisher\'s exact test. Correlations are expressed using Pearson\'s coefficient (r).
Compliance of genotype distributions with Hardy-Weinberg (HW) equilibrium was evaluated by chi-square analysis. For all tests, a p-value \< 0.05 was considered to be statistically significant. Values are expressed as the mean ± SD.
The genetic selection model was calculated for the evolution of the 677C\>T genotypes. The genetic selection could be classified as codominant or incompletely dominant and directional with the heterozygous genotype having an intermediate fitness. For this kind of selection, the most appropriate mathematical model is dq = \[spq(2hp+q-h)\]/\[p^2^+ 2pq(1-hs) + q^2^× (1-s)\], where dq is the change of frequency of the allele with lower fitness, s is the fraction of that genotype lost to selection, h is the degree of dominance (between 0, for no dominance and 1, for complete dominance), and p is the frequency of the allele with higher fitness.
Results
=======
We analyzed the genotype frequencies of the 677C\>T polymorphism in a population born during the 20th century. A total of 1305 subjects were divided into four groups of 25 years according to birth date. The genotype frequencies were compared between the four quarters of the century and showed very significant changes (p \< 0.001) in the group born in the last quarter of the 20th century (1976--2000), when compared to any of the other groups. The changes show a decrease of the CC genotype and an increase of the TT genotype in the last 25 years of the 20th century. (Table [1](#T1){ref-type="table"})
######
Distribution of C677T genotype frequencies in the 20th century.
**Frequencies by date of birth**
-------------- ---------------------------------- ----- ------- ----- ------- ----- ---------- -----
**CC** 0.393 81 0.394 126 0.37 151 0.299\* 111
**CT** 0.495 102 0.488 156 0.49 200 0.461 171
**TT** 0.112 23 0.119 38 0.14 57 0.24\*\* 89
**C allele** 0.641 264 0.638 408 0.615 502 0.53\*\* 393
**T allele** 0.359 148 0.363 232 0.385 314 0.47\*\* 349
Note: \*p \< 0.05; \*\*p \< 0.01 (significance values in the last quarter of the century are relative to any other quarter of the century)
Considering that each 25 year period corresponds to a generation, allelic frequencies did not change during the first 75 years of the century (HW equilibrium). However, we found that allelic and genotypic frequencies for the 677C\>T polymorphism in the last quarter of the century are significantly different compared to the previous generation (1951--1975). The genotype frequencies in the last quarter of the century are not the expected by a HW calculation using the allelic frequencies of the previous generation. This could be described as a consequence of genetic selection found in this population, in the absence of other causes. Applying the mathematical model described above to our population, the calculated fitness | {
"pile_set_name": "PubMed Central"
} |
Background
==========
Papaya mosaic virus (PapMV) is a member of the large family of *Flexiviridae* in the genus *Potexvirus*. The virus has a flexuous rod shape of 500 nm in length and 13 nm in diameter \[[@B1]\]. The CP, made mostly of alpha helices \[[@B2]\], is composed of 215 amino acids and has an estimated molecular weight of 23 kDa \[[@B3]\]. We showed previously that non-infectious nanoparticles made of recombinant PapMV CP are similar in shape and appearance to wild-type virus purified from plants \[[@B3]\]. PapMV nanoparticles were used previously as a vaccine platform technology to improve the immunogenicity of a peptide antigen fused to the nanoparticle structure \[[@B4]-[@B8]\]. The PapMV vaccine platform can induce a long-lasting memory response to an antigen fused on its surface \[[@B4]\]. Previous studies showed the capacity of PapMV nanoparticles to trigger a CTL response, in both *in vitro* and *in vivo* models, when the CTL epitope was fused to the C-terminus of the CP \[[@B6],[@B7],[@B9]\]. Although PapMV tolerates insertion of several peptides to its C-terminus \[[@B4]-[@B7],[@B10]\], a recent study revealed that N-terminal fusion of some peptides is also tolerated \[[@B8]\]. Depending on the nature of the amino acid sequence, some peptides can interfere with the CP assembly or with nanoparticle stability, which can affect their ability to stimulate an humoral response. A modification of the fusion site on the CP can help to resolve this issue. In this study, we compared the efficacy of nanoparticles harbouring fusion of a CTL epitope at either the N- or the C-terminus to trigger a cellular immune response.
The crystalline and highly ordered structure of the nanoparticles is critical to triggering an efficient humoral response, as also reported by many other groups \[[@B11]-[@B13]\]. However, it is still unknown if assembly into the highly ordered nanoparticle structure, made of several hundreds of PapMV CP, is more efficient than assembly of a smaller disc-like structure (aggregate of 20 subunits) in triggering the CTL response. Since the mechanisms of induction of humoral and CTL immune responses rely on different immune cells and mechanisms, we also evaluated the importance of highly ordered assembly of recombinant PapMV CP into nanoparticles in triggering a CTL response by comparing the immunogenicity of nanoparticles and discs.
Results and discussion
======================
Engineering of PapMV nanoparticles fused to the influenza CTL epitope
---------------------------------------------------------------------
In this study, we used the CTL epitope NP~147-155~, a 9-mer H-2Kd epitope specific for Balb/C mice derived from influenza virus to search for the optimal position for fusion of this epitope to the vaccine platform. The CTL epitope was flanked by 5 native residues of influenza NP protein at both the N- and C-termini to favour natural processing of the peptide, and was fused genetically to either the N- (before F13) or C-terminus of PapMV CP \[Figure [1](#F1){ref-type="fig"}A\]. Following expression in *E. coli*, recombinant PapMV particles harbouring the peptide on their surface (named PapMV NP-12 and PapMV NP-C) were affinity purified on a Ni^2+^ column via the 6xHis tag located at the C-terminus \[Figure [1](#F1){ref-type="fig"}B\]. Purified proteins were subjected to ultracentrifugation to pellet the nanoparticles. The lower molecular weight forms, composed of discs (20 subunits of CP) remained in the supernatant. As shown by dynamic light scattering (DLS), two structures were obtained with this purification protocol: (1) typical long rod-shaped structures of approximately 90 nm in length for PapMV NP-12 and PapMV NP-C \[Figure [1](#F1){ref-type="fig"}C--D\], and (2) smaller discs of approximately 20 nm in length for PapMV NP-12 and PapMV NP-C \[Figure [1](#F1){ref-type="fig"}D\]. The DLS method provides a global view of the size of the particles in solution that fits our qualitative observations by electron microscopy, and revealed the difference in size between discs (black line) and nanoparticles (dotted line) for both constructs (Figure [1](#F1){ref-type="fig"}D). It also shows that structures formed by NP-12 and NP-C constructs are similar in size, confirming observations by transmission electron microscopy showing that PapMV NP-12 and NP-C nanoparticles are similar in length, structure and appearance \[Figure [1](#F1){ref-type="fig"}C\].
{#F1}
We previously reported that residue F13 of PapMV CP is critical for the interaction between the PapMV CP subunits when assembling into nanoparticles \[[@B14]\]. We showed that this hydrophobic residue fits snugly inside the hydrophobic pocket of the neighbouring CP \[[@B2]\]. Interestingly, insertion of the NP~147-155~ epitope just before F13 in the N-terminal fusions clearly does not interfere with the interaction between PapMV CP monomers that is crucial for self-assembly of nanoparticles.
PapMV NP-12 nanoparticles are better inducers of the CTL response
-----------------------------------------------------------------
To evaluate the potential of PapMV nanoparticles to induce a CD8+ mediated cellular response, we immunised 6- to 8-week-old Balb/C mice three times at 2-week intervals by the intraperitoneal route with 100 μg of PapMV (without fusion), NP-12 or NP-C nanoparticles. Two weeks after the second boost, spleens were harvested and ELISPOT assays using the NP~147-155~ peptide were performed to quantify the level of IFN-γ secreted by CD8+ cells \[Figure [2](#F2){ref-type="fig"}\]. Secretion of IFN-γ is proportional to the level of precursors of CD8+ cytotoxic lymphocytes specific to the fused CTL epitope in vaccinated mice. The result showed that, compared to all the other treatments, mice immunized with PapMV NP-12 nanoparticles secrete significantly more IFN-γ \[Figure [2](#F2){ref-type="fig"}\].
{#F2}
Fusion of a peptide to the PapMV vaccine platform could affect its stability, and potentially the ability to mount an immune response to the fused epitope \[[@B8]\]. As temperature can affect protein stability, we thus measured the influence of temperature on the aggregation of recombinant nanoparticles using DLS. Upon heating, proteins initiate partial denaturation through exposure of their hydrophobic residues to the solvent. This conformational change triggers formation of aggregates that can be measured easily by DLS. We found that PapMV NP-C nanoparticles initiated aggregation at 25°C while PapMV (without fusion) and NP-12 nanoparticles were more stable and initiated aggregation at 37°C or higher \[Figure [3](#F3){ref-type="fig"}A\]. Therefore, the higher stability of NP-12 nanoparticles at 37°C or higher appears to correlate with an optimal CTL response in mice.
{#F3}
Based on this observation, it was anticipated that stabilization of NP-C nanoparticles by chemical cross-linking should improve their immunogenicity. Therefore, we compared the capacity of NP-C and NP-12 nanoparticles either cross-linked with glutaraldehyde or not to induce a CTL response. The cross-linked nanoparticles were very stable even at temperatures exceeding 37°C \[Figure [3](#F3){ref-type | {
"pile_set_name": "PubMed Central"
} |
**PURPOSE:** The face and craniofacial skeleton (CFS) make up a complex 3D structure that is critical to human function and cosmesis. Traumatic injury to the CFS requires fracture treatment to both allow the recovery of mechanical function and forms a foundation for the restoration of soft tissue anatomy. CFS reconstruction aims to restore pre-injury appearance, however in severe injuries shape information for the skull and facial bones may be missing. This presents a particular challenge in bi-frontal injuries and pan-facial fractures where the mirror imaging of the intact side of the head cannot be used to guide reconstruction.
The reconstruction of 3D facial surface geometry from pre-injury 2D photographs has recently been established through large scale morphable face modeling.^1^ As well, in forensic sciences, models with variable soft-tissue depths^2^ are used to determine face shape from skull geometry. This study aims to 'reverse-engineer' a forensics' tissue depth model to determine pre-injury CFS shape from reconstructed 3D facial geometry. It is hypothesized that 3D forensics data can be used to fill in missing gaps in CFS geometry with sufficient accuracy to guide pre-operative planning for CFS reconstruction.
**METHODS:** The forensics' tissue depth model was applied to 3D facial geometries acquired through segmentation of head CT data. Age, sex and BMI were used as input parameters to guide the application of the forensics' tissue depth model data to each face. The tissue depths between the face and CFS were determined by finding the Euclidian distance transform (nearest neighbor) employed by the original forensics study and via calculation using normal vectors generated from the face surface. Calculated tissue depth was evaluated against measured thickness on the head CT between the segmented CFS (bone) and the skin.
**RESULTS:** Tissue depth determined by nearest neighbor and normal vector measurements yielded accurate reconstructions of the frontal and zygoma bones (within 1mm, or +/- 2 voxels). However, only the normal vector technique succeeded in estimating tissue depth in bone regions where the face and skull have differing concavity (i.e. eye sockets, maxilla). Agreement was more limited in the lower facial skeleton where greater variation of soft tissue structures occur.
**CONCLUSION:** The reversed forensics tissue depth model was found to appropriately infer bony anatomy for the upper CFS from 3D face geometry. The 3D skull shaping provided by this work yields sufficient accuracy to warrant its inclusion into a translational pipeline of tools for pre-operative planning for CFS reconstruction.
**References:**
1\. Booth J. et al. Int J Comput Vis. 2017, 1--22.
2\. Shrimpton S. et al. *Forensic Sci. Int.* 2014, *234*, 103--110.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
In wild sika deer, *Cervus nippon yesoensis*, in Hokkaido, Japan, sarcocysts of protozoan parasites of the genus *Sarcocystis* are highly prevalent, and four *Sarcocystis* species have been identified in the region to date: *S. ovalis*, *S. pilosa*, *S. tarandi*-like, and *S. truncata*-like ([@bib28]; [@bib19]; [@bib12]). Among these species, *S. ovalis* has been reported to have a corvid as its definitive host in the region ([@bib13]). Although the definitive hosts of the remaining species remain unknown, phylogenetic and epidemiological evidence seems to indicate that members of the Felidae (or unknown animals) are the likely definitive hosts of *S. tarandi*-like and *S. truncata*-like ([@bib4]; [@bib10]). The remaining species, *S. pilosa*, was originally isolated from *C. nippon* in Lithuania ([@bib21]), where seven *Sarcocystis* species were characterized in farmed sika deer by means of morphological and molecular methods ([@bib22]). Seven partially different *Sarcocystis* species, including *S. pilosa*, were also characterized in wild sika deer (*C. nippon centralis*) in mainland Japan ([@bib2]). The definitive host of *S. pilosa* is suspected to be a member of the Canidae, because *S. pilosa* falls phylogenetically within a clade that includ *Sarcocystis* spp. using Canidae as their definitive host. Further, sarcocysts that are morphologically similar to *S. pilosa* have been described in *C. nippon centralis* and *C. nippon yesoensis* in Japan, and this type of sarcocyst is experimentally able to infest and reproduce in dogs ([@bib25], [@bib26]; [@bib3]). The red fox, *Vulpes vulpes schrencki*, is a very common canid in Hokkaido, and has been observed feeding on deer carrion ([@bib29]). It therefore seems likely that red foxes serve to maintain *S. pilosa* in sika deer in Hokkaido. To clarify the *Sarcocystis* life cycle and the cause of the high prevalence of these parasites in deer, a survey of fecal sporocysts in red fox fecal samples was conducted.
2. Materials and methods {#sec2}
========================
2.1. Red fox fecal sample collection {#sec2.1}
------------------------------------
As described by [@bib18], red fox fecal samples were collected along road verges, agricultural fields, and paths that were likely to be utilized by red foxes in eastern Hokkaido. Fecal collection was conducted in May 2018 (n = 44) and in December 2018 (n = 21). To aim to collect feces from different foxes, the sites where feces were picked up were separated by at least 2 km. To confirm that fecal samples belonged to red foxes, fecal DNA was extracted as described by [@bib20], and the 12S ribosomal RNA (rRNA) gene was analyzed as described by [@bib31]. To inactivate *Echinococcus multilocularis* eggs, which are highly prevalent in red foxes in the study area, fecal samples were incubated at 70 °C for 12 h before being stored at −30 °C until use.
2.2. Fecal sporocyst examination {#sec2.2}
--------------------------------
Fecal sporocysts were examined in 1 g of feces using a modified sucrose flotation method (specific gravity, 1.27) ([@bib14]). For species identification, sporocysts were collected from the supernatant of the centrifuge tube by simple sedimentation in saline.
2.3. DNA extraction and PCR sequencing of collected sarcocysts {#sec2.3}
--------------------------------------------------------------
Genomic DNA from collected sporocysts was extracted using a PowerSoil DNA Isolation Kit (Mobio Laboratories, Solana Beach, CA) according to the manufacturer\'s instructions. The 18S rRNA and cytochrome *c* oxidase subunit I (COI) genes were amplified and sequencing was performed using previously described primers ([@bib12]). Direct sequencing was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA), and the obtained sequences were compared with those deposited in GenBank/EMBL/DDBJ.
3. Results and discussion {#sec3}
=========================
Of the 65 fecal samples analyzed, one (collected in December 2018) contained *Sarcocystis* sporocysts at approximately 500 sporocysts per gram of feces. The fecal sample was confirmed to belong to *V. vulpes* based on the 12S rRNA gene sequence. To confirm the presence of sporocysts in the feces, fecal examination was repeated again, and revealed reproducibility of the examination. The fecal sample was also positive for eggs of *E. multilocularis* and Capillariidae, and negative for *Cystoisospora* oocysts. The Capillariidae species might be *Calodium hepaticum* (syn. *Capillaria hepatica*), which reproduces in the liver of vole, and the eggs pass thorough the intestines of foxes that prey on vole. In other feces, eggs of hookworm, *Toxocara canis*, *Dibothriocephalus nihonkaiensis*, and oocysts of unidentified *Cystoisospora* were detected by the flotation.
The detected *Sarcocystis* sporocysts contained four sporozoites and were 15.0 μm (SD = 0.2) long and 9.3 μm (SD = 0.4) wide ([Fig. 1](#fig1){ref-type="fig"}) (mean values for 100 sporocysts). 18S rRNA (1669bp) and COI (1029bp) gene sequences of the sporocysts were deposited in GenBank/EMBL/DDBJ with accession numbers LC496069 and LC496070, respectively. Both sequences were 99.95%--100% identical to those of *S. pilosa* obtained from sarcocysts from sika deer in Hokkaido and Lithuania, and 99.23%--99.87% identical to such samples from mainland Japan ([Supplemental Table S1](#appsec1){ref-type="sec"}).Fig. 1Morphology of detected sporocysts under light microscopy. Scale bar: 10 μm.Fig. 1
Although *Sarcocystis* is considered highly endemic in sika deer from Hokkaido ([@bib26]; [@bib12]), very little is known about the life cycles of these *Sarcocystis* spp. Of the four prevalent species in the region, we focused on *S. pilosa*, the definitive hosts of which are suspected to belong to the Canidae. We therefore collected and analyzed red fox fecal samples and we detected *S. pilosa* sporocysts in one sample. Although it was previously experimentally demonstrated that red foxes can act as a definitive host for *S. hjorti*, which is closely related to *S. pilosa* ([@bib5]), this is the first record of *S. pilosa* sporocysts in feces excreted by red foxes in the wild. The finding indicates that the red fox serves as a definitive host of *S. pilosa*, and also that red foxes could be an infection source for deer in the region.
In this study, only one fecal sample from red foxes contained sporocysts, representing 1.5% of the analyzed fecal samples and 4.8% of fecal samples collected in the winter. Prevalence of *Sarcocystis* sporocysts in feces of red foxes has been evaluated in many studies: 1.9% in Bulgaria ([@bib16]), 3.8% in Ireland ([@bib30]), 10.1% and 17.9% in the USA ([@bib7]; [@bib6]), and 84.4% in Newfoundland ([@bib15]). Species were unfortunately not identified in those studies, and thus intermediate host animals were also not determined. Although simple comparison might not be appropriate because of biological and geographical differences, the rate of sporocyst-positive feces in the present study might relatively be low. However, the number of fecal samples examined in our study was statistically insufficient, and further investigation is necessary to evaluate the true prevalence. In addition, it is indispensable in future work to confirm that *S. pilosa* can reproduce in fox intestine by histological and/or intestinal scraping examinations, to eliminate the possibility that we have observed pseudoparasitism caused by ingestion by red foxes of carcasses of other animals that do serve as a definitive host of *S. pilosa*.
Red fox feeding habits in Hokkaido were inferred by analyzing residues in collected feces, which revealed that the annual percentage occurrence of material derived from deer was 16.7%, with the highest utilization of deer occurring in May ([@bib29]). Considering the feeding habits of red foxes, the prevalence of *Sarcocystis* infection in red foxes may be higher than that observed in our study. Moreover, given that *S. pilosa* endemicity among deer in the region is \>90% ([@bib12]), difference in the prevalence of these parasites between the definitive and intermediate hosts is considered unlikely. | {
"pile_set_name": "PubMed Central"
} |
1. Introduction
===============
The occurrence and undesirable complications from healthcare-associated infections have been well recognized in the literature for the last several decades \[[@B1-ijerph-09-03330]\]. The most common sources of infectious agents causing healthcare associated infections, described in a scientific review of 1,022 outbreak investigations \[[@B2-ijerph-09-03330]\] are (listed in decreasing frequency): the individual patient, medical equipment or devices, the hospital environment, the healthcare personnel, contaminated drugs, contaminated food, and contaminated patient care equipment. Although the person-to-person transmission route is the most likely, the role of the environment should not be ignored and hospital linen may contribute to the spread of nosocomial infections \[[@B3-ijerph-09-03330],[@B4-ijerph-09-03330]\].
Healthcare textiles include bed sheets, blankets, towels, personal clothing, patient apparel, uniforms, gowns, drapes for surgical procedures \[[@B5-ijerph-09-03330]\]. Contaminated textiles and fabrics often contain high numbers of microorganisms from body substances, including blood, skin, stool, urine, vomitus, and other body tissues and fluids. Although contaminated textiles in healthcare facilities can be a source of substantial numbers of pathogenic microorganisms, reports of healthcare associated diseases linked to contaminated fabrics are few, therefore the overall risk of disease transmission is very low \[[@B5-ijerph-09-03330]\].
Cleaning in general has two main functions: first: non-microbiological, to improve or restore appearance, and prevent deterioration. Second, microbiological, to reduce the numbers of microbes present, together with any substances that support their growth or interfere with disinfection \[[@B4-ijerph-09-03330]\]. The purpose of laundering hospital textiles is therefore to ensure clean and safe textiles for patients and staff and thus enable uninterrupted implementation of healthcare \[[@B5-ijerph-09-03330],[@B6-ijerph-09-03330]\]. The most common found microorganisms on hospital textiles are: Gram negative bacteria, coagulase negative staphylococci, *Bacillus* sp. and typical skin flora \[[@B7-ijerph-09-03330]\].
Most people working in hospitals assume that laundry returned to them is in fact clean and therefore safe. Laundry may certainly have had the dirt removed, but it is far from sterile and experience encourages infection control teams to take laundering very seriously in outbreaks that seem to have no obvious cause \[[@B8-ijerph-09-03330]\].
2. Reports on the Survival of Microorganisms on Hospital Textiles after Laundering
==================================================================================
Literature in the field of survival of microorganisms on hospital textiles after laundering is very diverse and perhaps even confusing and contradictory. Each publication states a different laundering temperature as appropriate. It is therefore important to note that a successful laundering procedure is dependent on several factors and each much be optimized. These factors with a possible synergistic effect include: duration of laundering procedure, mechanical action of laundering procedure, dosage and type of added detergents and disinfection agents, bath ratio, type of linen, filling ratio, *etc*. According to Sinner the four basic interconnected factors of the laundering procedure are: duration, mechanical action, chemicals and temperature \[[@B9-ijerph-09-03330]\]. If one of these factors is decreased such as for example temperature, then other factors such as chemicals, mechanical action or time must be increased to achieve the same laundering and disinfecting effect. This also explains the differences in the published efficient laundering conditions. The exact correct optimized combination of all the mentioned factors is therefore important in order to achieve a hygienic laundering procedure for hospital textiles. Wilcox and Jones \[[@B10-ijerph-09-03330]\] stated that many isolates of *Enterococcus faecium* survived exposure to laundering temperatures specified in the U.K. Department of Health guidelines for disinfecting foul and used and infected linen (60 °C for 10 min). Another published report by Orr and co-workers \[[@B11-ijerph-09-03330]\] even confirms survival of certain strains of enterococci at laundering temperatures as high as 71 °C. They therefore concluded that hospital linen is a possible source of enterococcal cross-infection.
The survival of enterococci on textiles at laundering temperatures as high as 60 °C was also confirmed by a study \[[@B12-ijerph-09-03330]\] where biomonitors (*Enterococcus faecium* inoculated onto textile swatches with pre-inoculated defibrinated sheep blood) were washed in a simulated common hospital laundry procedure. It was found that *Enterococcus faecium*, as well as *Staphylococcus aureus*, *Enterobacter aerogenes* and *Pseudomonas aeruginosa* all survived the chosen laundering conditions at 60 °C, but none of the challenge organisms survived laundering at 75 °C. On the other hand other studies \[[@B13-ijerph-09-03330],[@B14-ijerph-09-03330]\] confirm that optimizing the laundering procedure including using high-tech environmental detergents and innovative disinfection agents renders an appropriate disinfection effect, even at laundering temperatures as low as 30 °C as noted in the study \[[@B13-ijerph-09-03330]\] with challenge organisms *Enterococcus faecium* and *Enterobacter aerogenes.* In another study \[[@B14-ijerph-09-03330]\] the optimum laundering temperature was found to be at 40 °C.
Walter and co-workers \[[@B15-ijerph-09-03330]\] reported that *Staphylococcus aureus* survived a 10 min laundering at 54 °C followed by drying. *Klebsiella pneumoniae* also survived the same laundering procedure, but did not survive the drying procedure. The same research also indicated that the challenge bacteria *Staphylococcus aureus* was not found after a 60 °C laundering procedure; thus recommending a 60 °C laundering procedure for linen in healthcare facilities.
In the report by Smith and co-workers \[[@B16-ijerph-09-03330]\] it was found that soiled hospital terry towels initially contaminated with Gram-positive rods (predominantly *Klebsiella*, *Enterobacter* and *Serratia* spp.) and Gram-positive bacteria (predominantly *Staphylococci*) in the range between 10^7^ to 10^9^/100 cm^2^ were washed in different laundering procedures. It was found that the washing cycle with a temperature of 60 °C followed by the drying cycle at 93.3 °C was sufficient to maintain linen hygiene.
Christian and co-workers \[[@B17-ijerph-09-03330]\] also conducted experimental research on low temperature laundering of hospital textiles using economically reasonable chemicals and wash conditions. They examined the disinfection effect of laundering procedures against aerobic chemoorganotrophs, staphylococci and total coliforms. They found that low temperature washing procedures (47.8 °C) using increased concentrations of bleach eliminated all bacterial groups as effectively as the high temperature procedures (77 °C).
In the research by Blaser and co-workers \[[@B7-ijerph-09-03330]\] a comparison of laundering procedures at 71.1 °C and 22 °C was conducted. The argument of such low-temperature washing was the vast amount of energy used for laundering at 71.1 °C. The 22 °C laundering procedure included the use of low-temperature chemicals. The initial counts on the use soiled terry towels and sheet were between 10^6^ to 10^8^/100 cm^2^ with predominantly Gram-negative rods (especially *Enterobacteriaceae* and *Pseudomonadaceae*) and *Staphylococcus* species as the most common Gram-positive organisms. It was found that the bacterial counts from low-temperature and high-temperature washed fabrics were comparable. The authors therefore concluded that low-temperature washing for eliminating pathogenic bacteria from hospital laundry is as effective as high-temperature laundering.
It has been reported that *Clostridium difficile* \[[@B18-ijerph-09-03330]\] spores can survive temperatures and chemical treatment of typical hospital laundering cycles and that cross-contamination of *Clostridium difficile* spores can occur on bed linen during a wash cycle. Therefore the persistent nature of this organism must be considered by infection control personnel when implementing programs for laundering soiled and contaminated hospital linen. Articles reporting on the survival of microorganisms on hospital textiles after laundering, together with their main conclusions are summarized in [Table 1](#ijerph-09-03330-t001){ref-type="table"}.
ijerph-09-03330-t001_Table 1
######
Reports on the survival of microorganisms on hospital textiles after laundering.
Described laundering conditions Added disinfection agent or bleach Surviving microorganism Reference
---------------------------------------- -------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------- -----------------------------------------------------
10 min at 60 °C No *Enterococcus faecium* Wilcox & Jones, 1995 \[[@B10-ijerph-09-03330]\]
10 min at 60 °C or 3 min at 71 °C No Certain strains of *Enterococcus faecalis* and *Enterococcus faecium* Orr *et al*. 2002 \[[@B11-ijerph-09-03330]\]
less than 10 min at 60 °C 3 mL Peroxyacetic acid/ kg textiles *Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa* and *Enterobacter aerogenes* Fijan *et al*. 2007 \[[@B12-ijerph-09-03330]\]
20 min at 30 °C 10 mL Sodium hypochlorate/kg textiles | {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1-1}
============
Knowledge of internal dental morphology is an extremely important step in planning and administering endodontic therapy.\[[@ref1]\] The numerous anatomical variations existing in the root canal system may contribute to the failure of root canal therapy. Failure to explore and instrument even one of the canals results in improper cleaning of root canal system and can lead to endodontic treatment failure.\[[@ref2]\] The mandibular first molar, the earliest permanent posterior tooth to erupt, seems to be the tooth that most often requires root canal treatment. The usual canal distribution is two canals in the mesial root and one or two canals in the distal root. Baugh D and Wallace J. also described the presence of a middle mesial canal.\[[@ref3]\] Additionally, Stoner *et al*. and Beatty and Iterian\[[@ref4][@ref5]\] have reported on more obscure cases in which a third canal was located in the distal root. Martinez and Bandaneli\[[@ref6]\] showed two cases with six canals. Astonishingly, Reeh\[[@ref7]\] has even reported a case with seven canals, consisting of four canals in the mesial root and three in the distal root. The role of advanced diagnostic tools cannot be overlooked in the diagnosis and management of such a complex canal system.
It has been postulated that secondary dentin apposition during tooth maturation would form dentinal vertical partitions inside the root canal cavity, thus creating root canals. A third root canal may also be created inside the root canal cavity of mandibular molars by this process. Such third canals are usually situated centrally between the two main root canals, the buccal and lingual of the mesial and distal roots. The diameter of these third middle canals is usually smaller than that of the other two.\[[@ref6]\] This case report presents the management of a mandibular first molar with six root canals, three in mesial and three in distal root. Additionally, this case report highlights the use of Cone Beam Computed Tomography (CBCT) as a diagnostic tool in Endodontics.
Case Report {#sec1-2}
===========
A 38 year-old female patient presented with a chief complaint of pain in the mandibular right posterior region for the past two weeks. Clinical examination revealed a carious left mandibular first molar (36). The clinical and radiographic findings led to a diagnosis of chronic irreversible pulpitis, necessitating endodontic therapy. Preoperative radiographic evaluation of the involved tooth pointed towards the presence of more than one canals in the distal root \[[Figure 1](#F1){ref-type="fig"}\]. Anaesthesia of mandibular left first molar was achieved with inferior alveolar nerve block using 2% Lignocaine. The tooth was isolated using a rubber dam and an endodontic access cavity preparation was done. Clinical examination revealed four distinct orifices, two located mesially (mesiobuccal, and mesiolingual) and two distally (distobuccal and distolingual). However, on careful examination of the access cavity with endo-explorer, an additional orifice was detected between the two main canals, both mesially and distally. \[Figures [2a](#F2){ref-type="fig"} and [b](#F3){ref-type="fig"}\]
{#F1}
{#F2}
{#F3}
Glide path and patency was achieved using no. 6, 8, and 10 k- files. Working-length radiographs were taken at different angulations with a file placed in each of the three mesial and three distal orifices \[[Figure 3](#F4){ref-type="fig"}\], and confirmed with electronic apex locator (Raypex 5). Cleaning and shaping was performed using a crown down preparation with rotary Protaper instruments (Maillefer, Dentsply, Ballaigues, Switzerland) under profuse irrigation with 3% sodium hypochlorite solution. After drying the root canals with sterile paper points, obturation was carried out with Protaper gutta percha cones (Maillefer, Dentsply, Ballaigues, Switzerland) using zinc oxide eugenol sealer. The access cavity was temporarily restored with Cavit \[Figure [4a](#F5){ref-type="fig"} and [b](#F6){ref-type="fig"}\].
{#F4}
{#F5}
{#F6}
CBCT imaging confirmed the presence of six canals in the concerned tooth. However, the middle mesial and middle distal canals were found confluent with their respective mesial/distal buccal canals at the junction of middle and apical one third, indicating towards the presence of three orifice and two apical foramina in each root \[Figure [5a](#F7){ref-type="fig"} and [b](#F7){ref-type="fig"}\].
{#F7}
Discussion {#sec1-3}
==========
A thorough knowledge of root canal morphology and canal configuration of the teeth plays an important role in the success of endodontic therapy.\[[@ref8]\] Studies have described the presence of aberrant canals in the mandibular first molar with three canals in the mesial as well as distal roots.\[[@ref3][@ref9][@ref10][@ref11]\] The third mesial and distal canal is defined as being independent when a distinct coronal orifice and apical foramen are observed, or confluent when converging into one of the other two main canals and terminating at a common apical foramen.\[[@ref7]\] Many authors have agreed on the presence of three foramina in the mesial root; however, only a few reported the presence of three independent canals, which presents itself as a rare anatomical variant. In a study of 760 mandibular molars, Fabra *et al*.\[[@ref12]\] found that 20 molars (2.6%) had three canals in the mesial root. Endodontic success in teeth with the aforementioned number of canals requires a careful clinical and radiographic inspection. Diagnostic aids such as CBCT, Dentascan, multiple preoperative radiographs, examination of the pulp chamber floor with a sharp explorer, troughing of the grooves with ultrasonic tips, staining the chamber floor with 1% methylene blue dye, performing the sodium hypochlorite "champagne bubble test," and visualizing canal bleeding points are all important aids in locating the root canal orifices.\[[@ref13]\] The search for an extra orifice is also aided by the use of microscopes, magnifying loupes and fiber-optic trans-illumination to locate the developmental line between the mesiobuccal and mesiolingual orifices.\[[@ref14]\]
A significant constraint in conventional radiography is that it produces a 2D image of a 3D object, resulting in the superimposition of the overlying structures. Therefore, such radiographs are of limited value in cases with complex root canal anatomy. Interpretation and appraisal based on a 2D radiograph may alert the clinician to the presence of aberrant anatomy; however, may not be able to present the variable and complex morphological structure of the root canals and their interrelations. Hence, CBCT has been specifically designed to produce undistorted three dimensional non invasive information of the root canal anatomy. Gopikrishna *et al*.\[[@ref15]\] used spiral computerized tomography for the confirmatory diagnosis of a morphological aberration in the maxillary first molar. Matherne *et al*. (2008)\[[@ref16]\] conducted an ex vivo investigation to compare a Charge Coupled Device Photostimuable Phosphor Plates (CCDPSP) digital radiography system with CBCT to detect the number of root canals in 72 extracted teeth. They found that with digital radiography, endodontists fail to identify at least one root canal in 40% of teeth.
Treating extra canals may be challenging; however, the inability to find and properly treat the root canals may cause failures. With advance diagnostic aids like CBCT and Dentascan, these challenges can be overcome. Although the incidence of root and canal variations is rare, every effort should be made to find and treat all the root canals for successful clinical results.
**Source of Support:** Nil.
**Conflict of Interest:** None declared.
| {
"pile_set_name": "PubMed Central"
} |
Sir,
A forty-nine year-old male underwent live related renal transplantation thirteen years ago for end stage renal disease secondary to bilateral vesicoureteric reflux. He was on prednisolone 10 mg per day and azathioprine 125 mg per day for maintenance immunosuppression. He presented with pain in his abdomen and vomiting that had been prevalent since the past day. Evaluation was suggestive of hollow viscous perforation which was confirmed by computed tomography of the abdomen. As laparotomy revealed a perforation with growth in the jejunum, a jejunal segment resection was done followed by jejunojejunal anastamosis. Histopathological investigation revealed that the growth was a diffuse, large B cell lymphoma \[[Figure 1](#F0001){ref-type="fig"}\]. Immunohistochemistry confirmed it to be a B cell variant of diffuse large cell NonHodgkin\'s lymphoma of the small bowel. Viral serology showed significant Epstein barr virus (viral capsid antigen) IgM and Epstein barr virus (nuclear antigen) IgG levels being normal. The patient was maintained on CHOP (Cyclophosphamide, Adriamycin, Vincristine, and Prednisolone) regimen by a medical oncologist.
{#F0001}
Renal transplant recipients carry a three to five fold higher risk of malignancy as compared to healthy people. Posttransplant lymphoproliferative disease (PTLD) is second only to skin cancers in contributing to 12% of posttransplant malignancies and its incidence increases with time.\[[@CIT1]\] The risk of developing PTLD is highest within the first year after the transplant, and the probability decreases thereafter.\[[@CIT2]\] The incidence increases with the age of the patient and the duration and cumulative dose of immunosuppressive therapy. It is associated with high morbidity and mortality. Cumulative doses and longer duration of systemic glucocorticoids and azathioprine increase the incidence of nonHodgkin\'s lymphoma (NHL).\[[@CIT3]\] The single most important risk factor for developing PTLD is EBV infection. This association is well established and 80--90% of PTLD cases are associated with primary EBV infection or reactivation of previously acquired EBV.\[[@CIT4]\]
Most cases of PTLD occur as nodal diseases, but they can present with localized symptoms involving organs such as the gastrointestinal tract, lungs, skin, liver, central nervous system, and infiltrative lesions in the allograft. Extranodal involvement occurs in more than 50% of the cases. The gastrointestinal tract is predominantly involved with an increased propensity for ulceration and perforation.\[[@CIT5]\] In our patient, the points of interest were: a) presentation as perforation, b) the neoplastic growth resected turning out as a diffuse B cell variant of nonHodgkin\'s lymphoma with positive Epstein barr viral serology.
| {
"pile_set_name": "PubMed Central"
} |
Lead is one of the most useful elements in industry, but serves no useful function in the human body. Environmental and industrial lead exposures continue to pose major public health problems in the exposed population.\[[@CIT1]\] Over the years, it has become increasingly evident that low-level lead exposure resulting in blood lead levels between 10 and 15 μg/dL can lead to deleterious effects like cognitive impairment and behavioral deficits, high blood pressure (BP) and impaired renal function.\[[@CIT2][@CIT3]\] Lancereaux\[[@CIT4]\] provided the first description of kidney disease and interstitial nephritis by postmortem examination of a lead-poisoned artist. It was not until the late 1920s when an epidemic of chronic nephritis in Queensland, Australia, was linked to childhood lead poisoning that the full spectrum of lead-induced nephropathy became apparent.\[[@CIT5][@CIT6]\] This was followed by cases of renal diseases from the US in individuals consuming lead-contaminated illegally distilled moonshine whisky.\[[@CIT7]\]
ENVIRONMENTAL LEAD EXPOSURE {#sec1-1}
===========================
Environmental lead exposure continues to be a public health problem. In the past, lead-based paint was a major source of lead poisoning among children. The painted surfaces of old houses contained significant amounts of lead. Direct ingestion of lead paint, lead-contaminated house dust and water by children has been identified as a major contributor to lead poisoning among the children. Many studies have confirmed that lead-contaminated dust is a major determinant of lead concentrations in blood.\[[@CIT8]\] Similarly, a highly significant correlation between lead concentration in drinking water and blood lead concentrations has been reported.\[[@CIT9]\] Children are more susceptible to the effects of environmental lead than adults because of the increased gastrointestinal absorption of lead in children. Children are more vulnerable because they absorb lead 5--10 times more effectively than adults and have a greater exposure because of their exploratory behavior and frequent hand to mouth activity.\[[@CIT10]\] Adults at the highest risk are those exposed to lead fumes or dust in the industry.\[[@CIT11][@CIT12]\]
OCCUPATIONAL LEAD NEPHROPATHY {#sec1-2}
=============================
An association between lead poisoning and renal diseases in humans has been recognized and documented by several studies.\[[@CIT8][@CIT12][@CIT13]\] Elemental lead and inorganic lead compounds are absorbed by ingestion or inhalation, but organic lead compounds, e.g. tetraethyl lead, may also be absorbed by skin contact. Organic lead compounds are the most toxic. Absorption of lead from the lungs is very efficient, especially if the particles are less than 1 μm in diameter. The gastrointestinal absorption of lead varies with the age of the individual; children absorb around 50% of what they ingest but adults only absorb 10--20% of what they ingest. Lead is very similar to calcium chemically. Thus, once in the body, it is handled as if it were calcium. Lead serves no useful purpose in the human body and its presence in the body can lead to toxic effects, regardless of the exposure pathway.
The kidney is the critical organ after long-term occupational or environmental exposure to lead. Excessive exposure to lead may cause acute or chronic nephrotoxic effects. Two types of nephropathy, acute and chronic nephropathy, have been observed in humans. Acute Pb nephropathy is characterized functionally by a generalized deficit of tubular transport mechanisms (Fanconi syndrome) and morphologically by the appearance of degenerative changes in the tubular epithelium and the nuclear inclusion bodies containing Pb protein complexes.\[[@CIT15][@CIT16]\] These effects, which are usually reversible with chelation therapy, have been reported mainly in children manifested by glycosuria and aminoaciduria. Chronic occupational exposure to lead has also been linked to a high incidence of renal dysfunction, which is characterized by glomerular and tubulointerstitial changes, resulting in chronic renal failure, hypertension and hyperuricemia. Chronic lead nephropathy is an irreversible renal disease that develops over months or years of excessive exposure.\[[@CIT17][@CIT18]\] This has been reported in adults who had ingested leaded paint during childhood and those who consumed illicitly distilled alcohol (moonshine whisky).\[[@CIT14][@CIT15]\] In the chronically exposed adults, Pb nephropathy occurs as a progressive tubulointerstitial nephritis that is difficult to diagnose at the early stage. Incipient Pb nephropathy is not associated with urine abnormalities easily detected by dipsticks. The tests evaluating the glomerular filtration rate (GFR) (creatinine clearance, blood urea nitrogen, serum creatinine) are the only ones that can be used to detect the renal effect caused by the occupational exposure to Pb.\[[@CIT14][@CIT19][@CIT20]\] But, when these tests are abnormal, the nephropathy has already reached the irreversible phase that may lead to renal sufficiency.\[[@CIT19]\] Chronic low-level exposure to lead is also associated with an increased urinary excretion of low molecular weight proteins and lysosomal enzymes.\[[@CIT21]\] Epidemiologic studies have shown an association between blood lead levels and BP, and hypertension is a cardinal feature of lead nephropathy.\[[@CIT13][@CIT20][@CIT22][@CIT24]\]
Most lead-associated renal effects or toxicity are a result of the ongoing chronic or current high acute exposure. They can also be attributable to a previous chronic lead exposure. The lowest level at which Pb has an adverse effect on the kidney remains unknown. Both glomerular and tubular effects have been reported.\[[@CIT20]\] The glomerular effects range from an increased prevalence of high molecular weight proteinuria to a nephrotic syndrome.\[[@CIT18][@CIT23]\] The reported tubular changes consist of an enhanced urinary excretion of enzymes.
BIOMARKERS OF NEPHROTOXICITY {#sec1-3}
============================
The prevention of renal diseases induced by exposure to industrial or environmental Pb largely relies on the capability to detect nephrotoxic effects at a stage when they are still reversible or at least not yet compromising the renal function. During the past decade, various tests have been proposed for the early detection of the toxic effects at different sites on the nephron. Some of these tests have been validated and some need epidemiological validation.
Currently, there are some early and sensitive indicators available that are considered predictive or indicative of renal toxicity from lead exposure. Recent studies have shown more than 20 potential markers of renal effects that can be arbitrarily classified into three broad categories.\[[@CIT25]--[@CIT28]\] [Table 1](#T0001){ref-type="table"} shows the different biomarkers used in Pb-induced nephrotoxicity.
######
Showing the different biomarkers used in Pb-induced nephrotoxicity.
No. Reference Exposure Exposure duration (years) B Pb conc. (μg/dL) Biomarkers used
----- -------------------------------- --------------- --------------------------- -------------------- ------------------------
1 Lilis *et al*.\[[@CIT25]\] Occupational \> 10 79 GFR, SCr
2 Cramer *et al*.\[[@CIT26]\] Occupational 9 103 GFR, HP
3 Wedeen *et al*.\[[@CIT27]\] Occupational 5 48 GFR, HP, TMPAH
4 Wedeen *et al*.\[[@CIT28]\] Occupational 14 51 GFR, HP
5 Hong *et al*.\[[@CIT29]\] Occupational 7 68 GFR, TMG
6 Lilis *et al*.\[[@CIT30]\] Occupational 12 80 SCr, BUN
7 Dekort *et al*.\[[@CIT31]\] Occupational 12 47 SCr, BUN
8 Verschoor *et al*.\[[@CIT33]\] Occupational \< 2--10 47 UNAG, URBP
9 Staeseen *et al*.\[[@CIT34]\] Environmental NA 10 SCr
10 Omae *et al*.\[[@CIT35]\] Occupational 0.1--26 37 CCr, CUA, Uβ2μG, Cβ2μG
11 Hu\[[@CIT36]\] Environmental NA 6 Ccr
12 Kim *et al*.\[[@CIT37]\] NA NA 10 Scr
13 Fels *et al*.\[[@CIT38]\] Environmental NA 13 SCr, UE, UP, ULMWP
14 Hsiao *et al*.\[[@CIT39]\] Occupational 13 40 SCr
15 Sonmez *et al*.\[[@CIT40]\] Occupational 0.14 25 UNAG
16 Muntner *et al*.\[[@CIT41]\] NA NA 5 SCr
B Pb conc., blood lead concentration; NA, not available; GFR, glomerular filtration rate; SCr, serum creatinine; HP, histopathology; TMPAH, transport max. for PAH; TMG, transport max for glucose; BUN, blood urea nitrogen; UNAG, urine N-acetyl-β-D-glucosaminidase; URPB, urine retinal binding protein; CCr, creatinine clearance; CUA, uric acid clearance; Uβ2μG, urine β2μG; UP, urine proteins; ULMWP, urinary low molecular weight proteins.
Functional markers {#sec2-1}
------------------
Creatinine in serum (crt-S).Creatinine in urine (crt-U).Urinary proteins of low or high molecular weight.3.1.High molecular weight | {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-jcm-08-01780}
===============
While numerous guidelines for the management of mild traumatic brain injury (TBI) exist, there is still controversy regarding the treatment of head trauma patients with antithrombotic therapy (ATT). The management of mild TBI on ATT is complicated by the heterogeneity of patients with different medications and varying patient characteristics. Most authors define mild TBI based on a Glasgow Coma Scale (GCS) of 13 to 15, others include any impact to the brain, not necessarily causing symptoms \[[@B1-jcm-08-01780]\]. For patients using ATT, several studies show an increased risk for abnormal computed tomography (CT) findings, even with a normal neurological exam and a history lacking neurological symptoms \[[@B2-jcm-08-01780],[@B3-jcm-08-01780],[@B4-jcm-08-01780]\]. Therefore, in most centers patients on ATT receive a routine CT at presentation, even if common definitions of mild TBI are not fulfilled and head trauma is merely reported, or visible signs of head trauma are present.
Vitamin K antagonists (VKA) were shown to increase the risk for clinically significant TBI and mortality \[[@B5-jcm-08-01780],[@B6-jcm-08-01780],[@B7-jcm-08-01780]\] and numerous studies indicate an increased risk and mortality for patients on all other kinds of ATT \[[@B8-jcm-08-01780],[@B9-jcm-08-01780],[@B10-jcm-08-01780],[@B11-jcm-08-01780],[@B12-jcm-08-01780]\].
Delayed traumatic intracranial hemorrhage (DIH) can occur up to several weeks after trauma to the head \[[@B13-jcm-08-01780]\] and was reported to occur more frequently in patients with ATT, ranging from 0.2% to 6% \[[@B14-jcm-08-01780],[@B15-jcm-08-01780],[@B16-jcm-08-01780],[@B17-jcm-08-01780]\].
Due to studies showing a high number of DIH, international guidelines and recommendations in literature suggest admitting patients with ATT for observation after negative CT \[[@B18-jcm-08-01780],[@B19-jcm-08-01780],[@B20-jcm-08-01780],[@B21-jcm-08-01780]\]. Many trauma centers adopted management protocols highly cautious of DIH and performed repeat CT after head trauma for asymptomatic patients. These extensive management protocols with in-hospital observation and repeat CT were evaluated in numerous studies, and most authors concluded that a routine repeat CT is not necessary \[[@B22-jcm-08-01780],[@B23-jcm-08-01780]\]. Some authors even question the necessity for clinical observation after negative CT \[[@B18-jcm-08-01780],[@B24-jcm-08-01780],[@B25-jcm-08-01780]\].
2. Materials and Methods {#sec2-jcm-08-01780}
========================
Our level I trauma center follows a high level of precaution for head injury patients. CT are performed based on the Canadian CT Head Rule, which, however, excludes patients with ATT \[[@B26-jcm-08-01780]\]. At the time of data collection, all cases of head trauma with ongoing ATT regardless of clinical signs for TBI received a CT and were admitted for a minimum of 24 h of in-hospital observation. Patients receiving VKA, Clopidogrel or direct oral anticoagulants (DOAC) received a routine repeat CT before discharge to detect delayed hemorrhages. Patients using acetylsalicylic acid (ASA) or low molecular weight heparin (LMWH) underwent clinical observation but did not receive a routine repeat CT.
After two years of said management, we analyzed our clinical protocol to determine the frequency of delayed intracranial hemorrhage in patients with head trauma and antithrombotic therapy, adjusting our practice and thereby contributing to the ongoing international debate on the management of head trauma patients on ATT.
The study was performed in a level I trauma center with authorization by the local Institutional Review Board (1632/2014). Between January 2012 and April 2014 patients aged 18 years or older were retrospectively included if they were admitted for observation after blunt head trauma with ongoing ATT and no pathologies in their initial CT. Management of these patients followed the described standard clinical protocol. This included an initial CT, clinical and GCS assessment including history of unconsciousness and laboratory tests including S100 and coagulation studies at time of admission. We did not routinely perform laboratory tests for evaluation of therapeutic levels during the observation period (viscoelastic tests, platelet function or anti-Xa assays) for ATT other than VKA. In-hospital observation for a minimum of 24 h followed, and patients received their applicable protocols:Patients using ATT with an expected higher risk for DIH based on the literature, such as vitamin K antagonists (VKA), direct oral anticoagulants (DOAC) and Clopidogrel received a routine repeat CT prior to discharge from hospital.Patients using ATT with an expected low risk for DIH including acetylsalicylic acid (ASA) and prophylactic doses of low molecular weight heparin (LMWH) did not receive a routine repeat CT and were discharged after observation only. Due to the greater number of patients receiving ASA compared to other ATT we included only patients from January 2013 until December 2013 in this study.
The primary endpoint of this study was the occurrence of delayed intracranial hemorrhages. Data was collected and analyzed using SPSS version 24 and descriptive statistics were performed. Due to the low number of delayed intracranial hemorrhages the variables age, GCS and prothrombin time were tested using the Mann--Whitney--U test while the remaining nominal variables were tested using Fisher's exact test. The significance level alpha for all implemented tests was set to α \< 0.05.
3. Results {#sec3-jcm-08-01780}
==========
During the study period 793 patients fulfilled the inclusion criteria, with a majority of 453 (57.1%) women and 340 (42.9%) men. A routine repeat CT was performed in 395 cases and in-hospital observation without routine repeat CT in 398 patients. The average patient age at presentation was 81 years (range 32--102). The most prevalent ATT was acetylsalicylic acid in 368 patients (46.4%), followed by vitamin K antagonists in 255 (32.2%) and Clopidogrel in 86 patients (10.8%), see [Table 1](#jcm-08-01780-t001){ref-type="table"}. Since patients using ASA were included from only one year whereas all other patients were collected from a two-year observation period, the distribution of different types of ATT is not representative of the total population at our institution.
Only blunt trauma was included in the study, with low energy trauma due to falls accounting for 95.2% of all cases. Most patients presented with a normal neurological status, only 16.5% of patients showed any neurological symptoms at presentation. The average GCS at presentation at the hospital was 15 and in 75.5% there was no history of unconsciousness or amnesia reported by either the patients or others. Lesions to the head such as abrasions and lacerations were present in 57.4%. The mean prothrombin time of the 255 patients using VKA was 32%, and 91.8% of the VKA patients were in their therapeutic range at time of admission.
The timing of the routine repeat CT as well as discharge from hospital followed the clinical protocol and occurred after a minimum observation time of 24 h, which resulted in an average of two nights of in-hospital observation, depending on the time of admission, with discharge after morning rounds.
In total, there were 11 cases (1.2%) of delayed intracranial hemorrhages. The routine repeat CT group showed nine DIH, resulting in 2.3% of cases detected with routine repeat CT. In the observation-only group, 16 patients showed a worsening of GCS or other symptoms indicating TBI. Two of these repeat CTs based on clinical judgement found minor DIH (0.5% of the observation group). One of the 11 patients with DIH needed an urgent decompressive craniectomy due to subdural hematoma with midline shift on the second day of observation. This was an 84-year-old female with vitamin K antagonist therapy, who showed no clinical signs of traumatic brain injury, no exterior injury to the head and an international normalized ratio of 2.9 at admission. The repeat CT was performed due to neurological deterioration with reduced Glasgow Coma Scale 27 h after admission. The patient underwent immediate decompressive craniectomy and was consecutively discharged to a neurological rehabilitation facility with a mild left-sided hemiparesis. The other patients with small delayed intracranial hemorrhage did not undergo surgical intervention and were discharged from hospital after an average observation period of 12 days (range 5--23). A review of the cases of delayed intracranial hemorrhages in the repeat CT group by a radiologist revealed, that small epi- and subdural hematomas, minimal intracerebral and subarachnoid hematomas were visible but not described in the initial CT report in four of the eleven cases. Excluding the four cases of initially undiagnosed pathologies in the CT report, an adjusted number of seven DIH (1.8%) were found in the repeat CT group, and in 0.9% overall. There were no significant differences between patients with or without delayed intracranial hemorrhage regarding age, sex, mechanism of injury, extent of external head injury, S100 level and coagulation studies or neurological status at admission. Characteristics of patients with DIH in our study population can be seen in [Table 2](#jcm-08 | {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-biomolecules-10-00575}
===============
Aflatoxin is a type of secondary metabolite produced mainly by microscopic fungal species *Aspergillus flavus* and *Aspergillus parasiticus* in the environment of high temperature and humidity (temperature 25--30 °C, moisture \> 15%) \[[@B1-biomolecules-10-00575]\]. According to the International Agency for Research on Cancer (IARC) \[[@B2-biomolecules-10-00575]\], aflatoxins have been classified as a grade I carcinogenic substance. Among them, aflatoxin B~1~ (AFB~1~) is the most toxic, with strongest carcinogenicity; it contaminates more than 100 kinds of foods such as grain, oils, milk, condiments, nuts, tea and dairy products \[[@B3-biomolecules-10-00575],[@B4-biomolecules-10-00575]\]. Since AFB~1~-caused food contamination comprises about 75% out of total mycotoxin contaminations \[[@B5-biomolecules-10-00575]\], maximum residue limits (MRLs) for AFB~1~ in grains have been set (from 2 to 20 μg kg^−1^) in many countries, including the European Union (EU), the United States of America and China \[[@B6-biomolecules-10-00575],[@B7-biomolecules-10-00575],[@B8-biomolecules-10-00575]\].
To better monitor the threat of AFB~1~ contamination, various methods have been developed in the past few decades \[[@B9-biomolecules-10-00575],[@B10-biomolecules-10-00575],[@B11-biomolecules-10-00575],[@B12-biomolecules-10-00575]\]. Although the results are reliable and accurate, instrumental techniques \[[@B13-biomolecules-10-00575]\] need expensive equipment and complicated sample pretreatment. Biosensors based on the antibody immunoprobes such as enzyme-linked immunosorbent assay (ELISA) \[[@B14-biomolecules-10-00575]\] and fluorescence-linked immunosorbent assay (FLISA) \[[@B15-biomolecules-10-00575],[@B16-biomolecules-10-00575]\] can achieve quantitative detection with good performance of specificity, sensitivity and simplicity, but the heterogeneous immunoassays require multiwashing procedures and long analysis times. To address the above issues, lateral flow immunochromatography assays have been considered as a promising method for onsite screening of mycotoxins \[[@B17-biomolecules-10-00575],[@B18-biomolecules-10-00575],[@B19-biomolecules-10-00575]\]. Moreover, immunochromatography assays based on fluorescent markers (time-resolved fluorescent nanobeads (TRFN), quantum dot nanobeads (QB) and quantum dots (QD), etc.) have gradually become a popular research field in recent years for their advantages of sensitivity, accuracy, automated detection, shorter detection time, and so on \[[@B20-biomolecules-10-00575],[@B21-biomolecules-10-00575],[@B22-biomolecules-10-00575]\]. Several fluorescence immunochromatography assays for highly sensitive detection of AFB~1~ have been reported \[[@B20-biomolecules-10-00575],[@B21-biomolecules-10-00575],[@B23-biomolecules-10-00575],[@B24-biomolecules-10-00575],[@B25-biomolecules-10-00575]\].
Although many methods based on immune interactions have been developed for the detection of toxic and harmful substances, it is impossible to compare the performance of those methods for identifying the most appropriate approach due to the utilization of distinct antibodies/antigens, markers and the detection conditions. In recent years, only a few reports have used comparative methods under the same conditions \[[@B26-biomolecules-10-00575],[@B27-biomolecules-10-00575],[@B28-biomolecules-10-00575],[@B29-biomolecules-10-00575],[@B30-biomolecules-10-00575],[@B31-biomolecules-10-00575]\]. For instance, Xie et al. \[[@B27-biomolecules-10-00575]\] established flow immunochromatography to detect *Escherichia coli O157:H7* in milk, in which fluorescent microspheres and colloidal gold were compared in terms of antibody labeling efficiency, sensitivity, antibody consumption and coefficient of variation. Wu et al. \[[@B28-biomolecules-10-00575]\] systematically and comprehensively compared the performance of fluorescent microsphere and quantum dot immunochromatographic strips for quantitative detection of aflatoxin M~1~ (AFM~1~) in milk. However, to the best of our knowledge, among the widely used fluorescent labeling materials of TRFN, QB and QD, there are no clear statements on which labeling material is better for AFB~1~ detection in foods by immunochromatography. In this paper, in order to find a more suitable fluorescent detection method for quantitative detection of AFB~1~ in grains, TRFN, QB and QD were used as labels to establish fluorescent immunochromatography (TRFN-FICA, QB-FICA and QD-FICA) for the first time by comparing antibody labeling efficiency, detection sensitivity, antibody and antigen consumption, and accuracy under the same conditions ([Figure 1](#biomolecules-10-00575-f001){ref-type="fig"}).
2. Materials and Methods {#sec2-biomolecules-10-00575}
========================
2.1. Materials and Apparatus {#sec2dot1-biomolecules-10-00575}
----------------------------
### 2.1.1. Materials {#sec2dot1dot1-biomolecules-10-00575}
Time-resolved fluorescent nanobeads (TRFN, 1%, solid content, *w*/*v*; carboxylate-modified Eu (III)-chelate-doped polystyrene nanobeads; excitation = 365 nm, emission = 610 nm) were purchased from Bangs Laboratories, Inc. (Fishers, Hamilton, IN, USA). Carboxylated quantum dot nanobeads (QB, 1 uM, *w*/*v*, excitation = 365 nm, emission = 610 nm) and quantum dots (QD, 1.0 mg/mL, *w*/*v*; carboxylate-modified CdSe/ZnS core/shell nanocrystals with amphiphilic polymer coating; excitation = 365 nm, emission = 610 nm) were purchased from NanoGen (Beijing, China). Anti-AFB~1~ monoclonal antibody (mAb) and coating antigen (AFB~1~-CMO-BSA) were donated by Beijing WDWK Biotech Co., Ltd., (Beijing, China). N-hydroxysuccinimide (NHS) and 1-ethyl-3-\[3-(dimethylamino) propyl\] carbodiimide (EDC) were obtained from Aladdin (Shanghai, China). AFB~1~, aflatoxin B~2~ (AFB~2~), AFM~1~, aflatoxin M~2~ (AFM~2~), aflatoxin G~1~ (AFG~1~), aflatoxin G~2~ (AFG~2~), zearalenone (ZEN), ochratoxin A (OTA), deoxynivalenol (DON) and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Chicken IgY and rabbit antichicken IgY-IgG were obtained from Biodragon Immunotechnologies Co., Ltd. (Beijing, China). Other chemical substances were purchased from Beijing Chemical Reagent Company (Beijing, China). All solvents and other chemicals were of analytical reagent grade and did not require further purification. A working standard of AFB~1~ was prepared from the 2 mg mL^−1^ stock solution by serial dilution with a sample buffer solution (0.3 M Tris-HCl containing 0.5% polyvinyl pyrrolidone and 0.4% Tetronic 1307, pH 8.0).
The nitrocellulose (NC) membrane (Unistart CN95) was acquired from Sartorius Stedim Biotech GmbH (Goettingen, Germany). The sample pad (glass fiber) and the absorbent pad were supplied by Shanghai Liangxin Co., Ltd. (Shanghai, China). The microtiter plates were supplied by Guangzhou JET BIOFIL Co., Ltd. (Guangzhou, China).
### 2.1.2. Apparatus {#sec2dot1dot2-biomolecules-10-00575}
An XYZ3060 dispensing platform was purchased from Bio Dot Inc. (Irvine, CA, USA). The CM4000 guillotine-cutting module was purchased from Kinbio Tech Co., Ltd. (Shanghai, China). The fluorescence immunochromatography quantitative analyzer was purchased from WDWK Bio Co., Ltd. (Beijing, China). Ultrapure water was purified with the Milli-Q system from Millipore Corp. (Bedford, MA, USA). The size distributions and surface morphologies of the three labels were determined by transmission electron microscope (JEM 1200EX, Tokyo, Japan). The mAb labels were characterized with a particle size analyzer (Malvern Instruments Ltd., Worcestershire, UK).
2.2. Preparation of Three Labeled Antibody Probes {#sec2dot2-biomolecules-10-00575}
-------------------------------------------------
The TRFN-mAb was prepared based on the procedures described in the previous literature with slight modification \[[@B26-biomolecules-10-00575],[@B30-biomolecules-10-00575]\]. Briefly, 5 μL of TRFN was | {
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In the most recent version of the projections of the Office of the Actuary in the Centers for Medicare & Medicaid Services, national health spending growth is expected to average 5.5% per year for 2017-2026 in the United States (US): approximately 1.0% higher than projected gross domestic product (GDP) growth.^[@bibr1-2192568219878133]^ This results in an increase in the health-related costs as a percentage of GDP from 17.9% in 2016, to nearly 20% by 2026, reaching a total of \$5.7 trillion by 2026.^[@bibr1-2192568219878133]^ When looking granularly at specific medical fields, a significant portion of the rising costs of health care in the US relates to the diagnosis and treatment of spinal pathology. It is estimated that 12% to 30% of US adults have an active back problem with approximately 6% having made a visit to a physician for these conditions at one point in their lives, costing upward of \$100 billion to the system each year.^[@bibr2-2192568219878133],[@bibr3-2192568219878133]^ Specifically with regard to spine surgery, fusions and laminectomies were the third and fifth most commonly performed surgical procedures in the United States in 2015, respectively.^[@bibr4-2192568219878133]^ Given the rising costs associated with spine surgery and an aging population, it becomes increasingly clear that the current trajectory is not sustainable, and further scrutiny will be placed on the field in assessing the effectiveness, efficiency, and safety of care delivered. As more healthcare systems invest in healthcare analytics and "big data" (large, complex datasets such as those found in electronic medical records), the opportunity arises to employ predictive analytics via machine learning (ML)/artificial intelligence (AI) approaches to improve quality, reduce waste and error, and minimize cost.^[@bibr5-2192568219878133],[@bibr6-2192568219878133]^
Recent developments in the technologies related to healthcare data collection and analytics have led to a rapid rise in the application of AI within health-related fields. One such application is ML, a branch of AI that involves the construction and application of statistical algorithms that continuously learn and make observations from existing data, and then create a predictive model based on that data.^[@bibr7-2192568219878133]^ With advances in computer processing capability, data storage, and networking, these computer-based algorithms can perform the intricate and extremely complex mathematical operations of classification or regression (specifically nonlinear regression) on immense amounts of data to detect intricate and potentially previously unknown patterns in that data.^[@bibr8-2192568219878133]^ ML algorithms have been able to analyze complex and large volumes of electronic medical record data to produce predictions for a wide range of clinical problems.^[@bibr9-2192568219878133]^ For example, Rajkomar et al^[@bibr9-2192568219878133]^ demonstrated that ML models outperformed traditional, clinically used models in predicting mortality, unexpected readmission, and increased length of stay (LOS) in a study cohort of all admissions in 2 major hospitals from 2009 to 2016. Various investigators have been developing image analysis methods using ML algorithms that have shown promising results in fields such as dermatology, radiology, and ophthalmology. For example, Esteva et al^[@bibr10-2192568219878133]^ have trained ML algorithms to classify skin cancer with a level of competence comparable to dermatologists. These early examples provide insight into early contemporary use of AI in medicine and provide a view of technology that may transform the medical field over the decades to come.
While not the first medical field to adopt an "AI approach" to problem solving, the spine surgery field has recently seen an outpouring of publications related to research in this area. An initial topic of focus by researchers was related to the cost of spine care, as there has been heightened emphasis on moving to a value-based (quality/cost) health care market. For example, as Medicare payments are standardized by procedures performed regardless of hospital LOS, ML systems have been designed with the ability to accurately predict spine surgery-related LOS, discharge to nonhome facility, and early unplanned readmissions using only presurgical or predischarge variables.^[@bibr11-2192568219878133][@bibr12-2192568219878133][@bibr13-2192568219878133]-[@bibr14-2192568219878133]^ These models can help identify/target certain high-risk patients and the variables that contribute to that risk status, allowing hospitals to allocate specific clinical and social resources to reduce costly LOS and readmissions. This can help to maximize efficiency of care delivered, while also keeping constant or even increasing the quality of care delivered.
As spinal surgery has evolved with an explosion of new techniques and technologies in recent decades, there still remains a lack of quality, high-level evidence to support much of the spine care rendered in the US, especially with the cost associated with many of the treatments and devices. As there are numerous surgical treatments in spine surgery that do not easily lend themselves to traditional randomized controlled trials (due to either cost or ethical considerations, among other reasons), an opportunity arises that is ripe for solutions derived from ML approaches. Multiple clinical registries are being collected that contain large quantities of high-quality, spine health care data, such as the 1000-patient Spinal Laminectomy versus Instrumented Pedicle Screw (SLIP) II study.^[@bibr15-2192568219878133][@bibr16-2192568219878133]-[@bibr17-2192568219878133]^ These registries contain demographics, surgery-related variables, patient-reported and complication outcome measures, and notably, they even contain digital imaging with metadata. Leveraging of these vast data repositories can help develop predictive algorithms that are able to incorporate the full range of variables (including complex imaging) in order to guide treatment recommendations.
Because of this lack of high-level evidence, there remains much heterogeneity in the current surgical treatment of spinal disorders, with significant clinical and economic implications.^[@bibr18-2192568219878133][@bibr19-2192568219878133]-[@bibr20-2192568219878133]^ For instance, national surveys of US spine surgeons conducted by Mroz et al^[@bibr21-2192568219878133]^ found 69% disagreement for recurrent lumbar disk herniation, while another study demonstrated 75% disagreement among surgeons on the approach to treat patients with lower back pain,^[@bibr22-2192568219878133]^ implying that 2 similar patients with the same pathology could receive entirely different care. Furthermore, a cost analysis based on the results of the national survey mentioned above revealed that there is also a variation in costs based on spine surgeon specialty, practice type, surgical volume and geographical location.^[@bibr23-2192568219878133]^ Recent ML/AI approaches to this problem have been published that attempt to assist surgeons' decisions with predictions of patient outcomes. Utilizing data from repositories created from AOSpine prospective, multicenter studies, Merali et al^[@bibr17-2192568219878133]^ developed a supervised ML model that accurately predicts a positive outcome on an individual patient after surgery for degenerative cervical myelopathy, with an average area under the curve of 0.70, classification accuracy of 77%, and sensitivity of 78% on an independent testing cohort. Shah et al^[@bibr24-2192568219878133]^ were able to build an ML model that predicts probability of failure of nonoperative management in spinal epidural abscess, while Karhade et al^[@bibr25-2192568219878133]^ successfully developed an ML algorithm that predicts in-hospital and 90-post discharge mortality in this patient group. The same group was able to predict short-term postoperative mortality in individual patients with spinal metastatic disease with an ML model, aiding in decision-making and informed discussions with the patient regarding surgical intervention this challenging patient population.^[@bibr26-2192568219878133]^ All of these previously mentioned studies have now published their prognostic tools in an open-access, digital interface to be integrated into practice, supporting clinicians in developing treatment plans that are more standardized across the world.
Along with prediction of positive patient outcomes, clinician researchers have also used AI/ML to forecast negative outcomes as well, as recent publications have explored the likelihood of complications from spine surgery. In multiple articles, the same group led by Cho et al utilized an artificial neural network-based ML algorithm to predict surgical complications in patients undergoing elective anterior cervical discectomy and fusion, posterior lumbar fusion, and adult spinal deformity surgeries. Their models were able to specifically predict the risk of cardiac-related, wound-related, venous thromboembolism--related, and mortality in these patients, outperforming the American Society of Anesthesiologists Physical Status Classification scoring in predicting individual risk prognosis.^[@bibr27-2192568219878133],[@bibr28-2192568219878133]^ Another publication by Sheer et al^[@bibr29-2192568219878133]^ describes their method to create a ML model that successfully predicts major intraoperative/perioperative complications following adult spinal deformity surgery with an accuracy of 87%. Utilizing large databases of patient information, Han et al^[@bibr30-2192568219878133]^ were able to analyze over 1 000 000 patients that had previously undergone spine surgery and developed multiple ML predictive models that identify risk factors for postoperative complications. Karhade et al^[@bibr24-2192568219878133]^ were even able to predict prolonged opioid prescription after surgery for lumbar disc herniation in an ML algorithm. These surgery- and patient-specific models can help to aid in surgical planning, as well as patient counseling and shared decision making. If these models identify modifiable risk factors in the preoperative setting of a nonurgent surgery, time and effort could be dedicated to improved medical management of that comorbidity prior | {
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{
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See related research by Switzer et al., <http://breast-cancer-research.com/content/14/5/R125>
Unlike the constitutive nitric oxide (NO) synthase isoforms, the inducible isoform of NO synthase - nitric oxide synthase 2 (NOS2) - is capable of producing sustained intracellular levels of NO, and it is increasingly appreciated that protein S-nitrosylation, the covalent modification of cysteine thiol by NO, is important for NOS2-dependent signal transduction.
In the previous issue of *Breast Cancer Research*, Switzer and coworkers delineate a novel pathway for S-nitrosylation in regulation of estrogen receptor (ER)-negative breast cancer invasion \[[@B1]\]. This work adds to the growing appreciation that S-nitrosylation can regulate myriad pathways important for tumorigenesis, including gene transcription, apoptosis and DNA repair \[[@B2]-[@B4]\], and it complements another recent study by these coworkers showing that S-nitrosylation and activation of epidermal growth factor receptor is associated with the induction of epithelial-to-mesenchymal transition as well as chemoresistance in ER-negative breast cancer \[[@B5]\]. More generally, these data begin to explain why expression of NOS2 correlates with aggressive tumor phenotypes and poor clinical outcomes in a variety of malignancies, including breast cancer, lung cancer, colon cancer and prostate cancer \[[@B6]-[@B9]\].
The small GTPase Ras was one of the earliest described regulatory targets of S-nitrosylation. Modification of a single cysteine residue that is located in the nucleotide-binding region of wild-type Ras and is conserved among all Ras isoforms (Cys118 in human H-Ras) stimulates guanine nucleotide exchange and downstream pathways, including activation of mitogen-activated protein kinase signaling \[[@B10]\]. S-nitrosylation of wild-type Ras by endothelial NO synthase has been shown to promote pancreatic tumor growth \[[@B11]\]. Switzer and colleagues identify a role for S-nitrosylation of wild-type Ras in ER-negative breast cancer \[[@B1]\]. They find that the only elements common to genes upregulated in high NOS2-expressing breast cancer are binding sites for the Ets-1 transcription factor. Using a model ER-negative breast cancer cell line, they further show that NO induces S-nitrosylation of wild-type Ras, leading to phosphorylation and activation of Ets-1 through the Ras/MEK/ERK pathway. Knockdown of Ets-1 inhibits the NO-dependent expression of basal-like breast cancer markers and attenuates NO-dependent cancer cell invasion. These findings delineate a mechanism by which NOS2 promotes an aggressive tumor phenotype in ER-negative breast cancer.
While NOS2 expression is high in ER-negative tumors \[[@B9]\], it is not constitutively expressed in the triple-negative breast cancer cell line MDA-MB-468 (lacking ER, progesterone receptor and epidermal growth factor receptor 2) that was utilized in this and related studies \[[@B1],[@B5]\]. Indeed, the loss of NOS2 expression in cultured tumor cells *ex vivo*is not uncommon and could reflect a deficiency in cytokine signaling and/or an inability to replicate the hypoxic tumor environment. Regardless of mechanism, this deficit in NOS2 expression can make challenging the study of the (patho)physiological functions of the enzyme in cell culture systems. To model NOS2-based signaling as it occurs in the solid tumor, Switzer and colleagues employ a long-lived NO donor as well as adenoviral expression of NOS2 \[[@B1]\]; in earlier work, they also co-cultured these cells with macrophages stimulated to produce high levels of NOS2-derived NO \[[@B5]\]. While these conditions may adequately recapitulate pathophysiological NO levels within the tumor, further work is needed to determine the extent to which these manipulations fully reconstitute NOS2-dependent signal transduction. These questions may in part be addressed by defining the precise mechanisms underlying enhanced expression of NOS2 in breast cancer and other malignancies. In addition to the environmental factors of cytokine secretion and hypoxia, aberrant regulation of NOS2 transcription, mRNA stability, and proteasomal degradation in the tumor cells present potential mechanisms.
There is increasing recognition that denitrosylases - in particular, S-nitrosoglutathione reductase (GSNOR) and thioredoxin (Trx) - are critical modulators of S-nitrosothiol (SNO) homeostasis \[[@B12]\]. As the name implies, GSNOR metabolizes S-nitrosoglutathione, a small molecule intermediate in S-nitrosylation and denitrosylation. This enzyme may have general housekeeping functions, one of which is to protect against indiscriminate protein S-nitrosylation (nitrosative stress) that may occur as a result of excessive NOS2 activity. In this regard, GSNOR deficiency has been tied to inhibition of DNA repair and to hepatocellular carcinoma \[[@B3]\], and the enzyme is also deficient in some lung cancers \[[@B13]\]. Trx reduces protein SNOs directly, and inhibition of thioredoxin reductase (TrxR) can increase protein SNOs by blocking Trx turnover. Trx has another major role as a modulator of oxidative stress, and it is in this capacity that chemotherapeutics targeting TrxR are believed to affect cancer cell killing. Given the potential for protein S-nitrosylation to stimulate tumor growth and invasion, TrxR inhibitors may have unexpected deleterious effects. More generally, the balance of SNO formation/degradation is clearly an important factor in malignant cell transformation and thus warrants further investigation.
Given the potential role of NOS2 in tumor chemoresistance and aggressiveness, it is important to note that NOS2 inhibitors have had some success in inhibiting tumor growth \[[@B14],[@B15]\]. At the same time, interventions that increase SNO levels, either through application of NO donors and low-mass SNOs or through inhibition of SNO-metabolizing enzymes, are being proposed as therapeutics in numerous diseases \[[@B16]\]. Given recent findings in the cancer field, it may be necessary to weigh the risk-benefit of therapies designed to increase protein S-nitrosylation for those individuals at high risk of developing cancer.
Abbreviations
=============
ER: estrogen receptor; GSNO: S-nitrosoglutathione reductase; NO: nitric oxide; NOS2: nitric oxide synthase 2; SNO: S-nitrosothiol; Trx: thioredoxin; TrxR: thioredoxin reductase.
Competing interests
===================
The authors declare that they have no competing interests.
Acknowledgements
================
This work was supported in part by National Institutes of Health grants HL106121 (to MWF and HEM) and HL092994 (to HEM).
| {
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Background {#Sec1}
==========
Over the past decade, there has been an increasing emphasis among university educators on globalization and internationalization, and global health programs and international experience have become key areas of focus for university professionals. According to China's Ministry of Education, in 2017, the number of Chinese students studying abroad exceeded 600,000 for the first time, reaching 608,400, which was an increase of 11.74% compared with 2016 \[[@CR1]\]. Researchers may benefit from experiencing the cultural differences inherent in exchange programs, and they may bring an awareness of these differences into their curricula, thereby broadening international training opportunities through work, education, and research activities \[[@CR2]--[@CR7]\]. After returning home, many health professionals work at medical universities or affiliated hospitals. As an implicit responsibility of these roles, health professionals must mentor medical students through engagement in clinical work and scientific research. The research ability of medical students during their postgraduate studies depends on many aspects, including the background and extent of their advisors' education and scientific research ability. Our previous research \[[@CR8]\] has shown that overseas study experience improved health professionals' scientific research ability, but no previous research has examined the impact of overseas experience on those professionals' students' scientific research ability. Is there a gap in the research ability of students of health professionals who studied or trained overseas ("returning" professionals) and that of students of health professionals who did not train overseas ("resident" professionals)? Do returning professionals, as postgraduate advisors, have a greater positive impact on their students' scientific research ability, compared with resident professionals? Does overseas experience play a positive role in the development of the research ability of medical students in China? In this study, we set out to answer these questions and to encourage the construction of a health professionals' overseas-experience database to help postgraduate students choose highly effective advisors. We also hope that our research results will be helpful in forming better training programs for medical students and in improving medical education policy.
Methods {#Sec2}
=======
Harbin Medical University (HMU) is the only Western medical institution in Harbin, in China's Heilongjiang Province. It is a government institution that offers a five-year medical bachelor's degree course, a three-year master's degree course, and a three-year PhD degree course. HMU has recently begun to restructure and reform its medical education program to offer comprehensive solutions to national medical education problems.
This study was conducted from September 2016 to April 2018. During this period, only 1--2% of master's students (*n* = 1561) published a Science Citation Index (SCI) article prior to graduation. In contrast, around 70% of PhD students (*n* = 1083) published SCI articles before graduating. Thus, we included only PhD students in our analyses. PhD students in China are differentiated by their year of enrollment. PhD students in the class of 2015 graduated in June 2018 and their articles may not have been published by the end of the research period, so we selected PhD students enrollment in 2012--2014 for our study. Health professionals with a minimum of 6 months study-abroad experience were categorized as "returning," because we designated 6 months as the minimum experience required to be an independent researcher. Therefore, health professionals with less than 6 months study-abroad experience were excluded from this study. In the study, both "returning" and "resident" professionals had PhD degrees. These professionals were 56 scientific researchers from HMU and 76 clinical doctors from HMU's affiliated hospitals.
We analyzed 257 students of returning health professionals (Group A)-106 from HMU (Group A~1~), who were trained to be scientific researchers, and 151 from HMU's affiliated hospitals (Group A~2~), who were trained to be clinical doctors. To collect information effectively, we constructed the questionnaire presented in Additional file [1](#MOESM1){ref-type="media"} Table S1, which was distributed to each of the 257 students. To ensure confidentiality and a high response rate, three investigators meet the students face to face and distributed the questionnaires to them. The students were required to complete the questionnaires while the investigator was present, and the investigators were not allowed to disclose any information or data collected.
The SCI is internationally recognized as an authoritative scientific literature search tool. We used the SCI impact factor (IF) as an indicator of scientific and research capability. Because authorship contribution is determined differently across institutions and countries and there is no international system to weigh these differences against, we ranked the authors to reflect contribution differences according to HMU's 2013 official promotion system. This system uses the following formulae: first author or corresponding author = IF \* 100%; second author = IF \* 50%; and third or later author = IF \* 25% \[[@CR9]\].
We compared the publishing histories of the students of returning professionals with those of the students of resident professionals to estimate the impact of advisors' overseas experience on their students' research capacity. Other relevant advisor information was included, such as total IF, number of articles published while abroad, duration of overseas study, and age at travel abroad. We tried to find out the correlations between these overt factors and students' scientific research ability and identify which factor should be considered most for students when choosing advisors.
The following data were collected from 257 students of returning professionals: (1) student's and advisor's names; (2) student's and advisor's ages; (3) student's and advisor's sexes; (4) student's enrollment year (Grade); (5) student's school and department; (6) student's total IF for articles published during their PhD study; (7) student's number of articles published during their PhD study; (8) advisor's duration of study or training abroad (months); (9) advisor's age when they went abroad; (10) advisor's total IF for articles published while abroad; and (11) advisor's number of articles published while abroad. We used SPSS, Version 19 (IBM Corp., Armonk, NY) to estimate multiple linear regression models to identify the factors associated with student's IF for articles and with the number of articles students published during their PhD study. To explore the relationship between health professionals' international experience and the academic output of their students, we selected 257 age-, enrollment year-, and specialty-matched students of resident professionals (Group B)---106 scientific research students from HMU (Group B~1~) and 151 clinical medicine students from affiliated hospitals (Group B~2~). We compared the total IF and the number of articles published between the returning and resident groups. We selected the control group (Group B) based on exact matches for age, year, and specialty with Group A participants. And there were 78 students of Group A who did not have a match. In total, there were 694 students enrolled in 2012--2014, 335 of whom were students of returning professionals. To avoid arbitrarily selecting matching controls, when there was more than one match, we randomly selected one control, without considering student's research backgrounds and advisor's name. We replaced study cases for whom we could not find an exact match with another student who had at least one match. We distributed the same questionnaires to the 257 control students of resident professionals, and questions regarding the advisor's information of studying abroad were not filled.
Results {#Sec3}
=======
In this study, the questionnaire response rate is 100%. Of the 257 students of returning professionals (Table [1](#Tab1){ref-type="table"}, Group A), there were 82 students in the 2012 graduating class, 87 in the 2013 class, and 88 in the 2014 class. Students' ages ranged from 26 to 34 years (mean = 31.29 years). In Group A, there were 106 scientific research students from HMU (Group A~1~: 37 students in the 2012 class, 32 students in the 2013 class, and 37 students in the 2014 class), with an age range of 26--34 years (mean = 31.32 years), and 151 clinical medicine students from affiliated hospitals (Group A~2~: 45 students in the 2012 class, 55 students in the 2013 class, and 51 students in the 2014 class), with an age range of 26--34 years (mean, 31.26 years). Similarly, there were 257 age-, enrollment year-, and specialty-matched students of resident professionals with the same grade and age proportion as those in Group A (Table [2](#Tab2){ref-type="table"}, Group B), consisting of 106 scientific research students (Group B~1~: 37 students in the 2012 class, 32 students in the 2013 class, and 37 students in the 2014 class), with an age range of 26--34 years (mean = 31.32 years), and 151 clinical medicine students (Group B~2~: 45 students in the 2012 class, 55 students in the 2013 class, and 51 students in the 2014 class), with an age range of 26--34 years (mean = 31.26 years). Table 1Data of 257 students of returning professionals (Group A), including 106 scientific research students (Group A~1~) and 151 clinical medicine students (Group A~2~)MaximumMinimumMeanTotal IF of papers published during PhDGroup A 58.23Group A 0Group A 4.01Group A~1~ 58.23Group A~1~ 0Group A~1~ 4.17Group A~2~ 26.82Group A~2~ 0Group A~2~ 3.91Number of papers published during PhDGroup A 10Group A 0Group A 1.16Group A~1~ 10Group A~1~ 0Group A~1~ 1.15Group A~2~ 4Group A~2~ 0Group A~2~ 1.16Tutor's duration of studying abroad (months)Group A | {
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All relevant data are within the manuscript and its Supporting Information files.
1 Introduction {#sec001}
==============
The formation of stable aggregates is very common in nature. For example, long-range attraction through chemotaxis can lead to aggregation of Dictyostelium cells \[[@pone.0222371.ref001]\] or eukaryotic cells during development to form organs and blood vessels \[[@pone.0222371.ref002]\]). But the formation of aggregates can also arise from Brownian motion and contact adhesion. Numerous examples can be cited, from inert particles such as colloids \[[@pone.0222371.ref003]\] to living cells, but also in ecology where animals like mussels produce stable patterns by clustering \[[@pone.0222371.ref004]\]. It has been shown that the living entities can, through this process, optimize at the same time protection against predation and access to food. In cancer, tumor cells circulating in the blood stream form aggregates that will become a metastatic tumor when settling in an organ \[[@pone.0222371.ref005], [@pone.0222371.ref006]\]. The merging of metastatic lumps, forming a larger aggregate, can also occur \[[@pone.0222371.ref007], [@pone.0222371.ref008]\].
It is now recognized that cells cultured in 2D at the bottom of a plastic Petri dish do not behave as they would do in their natural environment. For example, in vitro, an organization in 3D-clusters makes the aggregates more resistant to treatments compared to the same cells plated in 2D, in a Petri dish \[[@pone.0222371.ref009]\]. Several factors can explain these different behaviors \[[@pone.0222371.ref010]\]: first, the fact that the dimensionality is not the same (2D versus 3D) is important; second, cell-plastic interactions are often very strong and prevail over cell-cell interactions; finally, the plastic dish has a very high stiffness, often non realistic (for brain cells for example).
Therefore, new approaches that allow cells to grow in 3D aggregates *in vitro* are being pursued. Aggregates and spheroids *in vitro*, formed on non-adhesive substrates, are considered as pseudo-tumors that can be used to study tumor development in more realistic conditions. Recently, in the context of brain tumors (gliomas), we developed a new PEG-based hydrogel that allows the formation of a tumor-like structure, which can be used to study the effect of drugs in conditions more realistic than those of a 2D Petri dish. In some of those gels, we grafted poly(L-lysine) (PLL), because of its ability to promote unspecific cell adhesion via electrostatic interactions between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. Addition of PLL in the gels allows us to modulated cell-substrate adhesion. The stiffness of the gel can also be tuned, in order to mimic the stiffness of the natural matrix (in our case, the brain) corresponding to a specific cell line. In \[[@pone.0222371.ref009]\], we chose to study the behavior of two glioblastoma (which is the most aggressive type of gliomas) cell lines. Glioblastomas are currently non-curable and the development of an *in vitro* system that could mimic the development of these tumors could be used in order to test new drugs or radiotherapeutic strategies. Compared to other tissues, the stiffness of the brain is low (lower than 1 kPa \[[@pone.0222371.ref011]\]), so we chose to design soft gels. We observed a significant difference in cell growth between PLL-containing (adhesive substrate) and PLL-free soft PEG hydrogels (non-adhesive substrate), showing the role of non-specific adhesion factors such as PLL in the migration, proliferation and aggregation in two glioblastoma cell line cultures. More precisely, we showed that on a non-adhesive substrate, the aggregates are larger and less numerous than on an adhesive substrate.
The formation of aggregates has been studied theoretically with a perikinetic equation, first in the context of colloid aggregation \[[@pone.0222371.ref012]--[@pone.0222371.ref015]\]. In \[[@pone.0222371.ref016]\], the same concepts are used to study and fit the evolution of the number of cell aggregates on a non adherent substrate. The evolution of the mean projected aggregate area is more difficult to model, especially when aggregates are composed of cells that can deform, modulate their cell-cell adhesion and reorganize in 3D. For example, it has been observed that after their formation, cell aggregates often go through a compaction phase \[[@pone.0222371.ref010], [@pone.0222371.ref017], [@pone.0222371.ref018]\] that reduces their projected area.
Other theoretical approaches consider the formation of aggregates under the point of view of phase separation: like two immiscible liquids, when mixed in a liquid medium cells move and seek a lower energy state through adhesion with other cells. The evolution of a system from a state where the concentration of particles is uniform to a final state where patterns appear is a spontaneous phase transition driven by motion of particules, the latter being either passive by diffusion (for example, in colloids), or active (as for mussels or cells) and adhesion. This phase separation corresponding to the formation of aggregates has been described by mean-field models, based on the Cahn-Hilliard \[[@pone.0222371.ref019], [@pone.0222371.ref020]\]. With chemotaxis, Keller-Segel equations can also be used to describe pattern formation \[[@pone.0222371.ref021]\]. In development biology, discrete approaches, in particular with cellular Potts models \[[@pone.0222371.ref022]\] have been used to model the formation of patterns or the segregation of two cell types in aggregates.
However, to our knowledge, a model that describes the formation of aggregates from the early stages of a population of individual migrating cells to their aggregation and the late aggregate compaction, does not exist. For instance, in \[[@pone.0222371.ref016]\], the decrease of the mean aggregate area is modeled with an exponential function, but there is no direct connection with processes at the cellular level. In order to be able to describe the individual cells, an agent-based model should be chosen. One advantage of agent-based models is that one can easily implement the local rules of cell-cell interaction (\[[@pone.0222371.ref023]--[@pone.0222371.ref026]\], for a good review, see \[[@pone.0222371.ref027]\]).
In \[[@pone.0222371.ref009]\], we presented the snapshots of already formed aggregates. Here, we add new experimental results by following the whole process, from the early stage where the cell population is composed only of individual cells, to their aggregation, and later to the compaction of the aggregates. We confirm the differential migration and aggregation of cells on the substrates with different adhesivity and for two different cell lines.
We combine these experimental results with a theoretical study based on two models: first, we show that a spaceless model of perikinetic aggregation can reproduce the experimental evolution of the number of aggregates. Second, we developed a minimal off-lattice agent-based model, whose rules are defined in order to reproduce the important phenomena that drive the behavior of cell assemblies: cell and aggregate motion, cell-cell adhesion, cell proliferation and aggregate compaction. We show that this model reproduces very well the experimental temporal evolution of both the number of aggregates and their area, on adhesive and non-adhesive soft gels, for the two cell lines and that it gives access to quantitative values of three parameters.
2 Materials and methods {#sec002}
=======================
2.1 Preparation of the hydrogels and glioma cell lines {#sec003}
------------------------------------------------------
In \[[@pone.0222371.ref009]\], we showed that the PEG concentration of our artificial substrate optimal for the survival and growth of the two glioma cell lines is around 3% PEG. This concentration corresponds to an elastic modulus around 300 Pa, close to the value measured for brain tissue \[[@pone.0222371.ref028]\]. We use this concentration in all the following experiments. All the experimental methods can be found in \[[@pone.0222371.ref009]\]. Briefly: poly(L-lysine) hydrobromide (PLL-HBr 30,000 Da, Sigma-Aldrich, Saint-Quentin Fallavier, France) was first functionalized with an acrylate residue. Hydrogels were prepared from 3% (w/v) PEG-DA 6 kDa precursor (Sigma-Aldrich), dissolved in DPBS with 0.01% (w/v) of DMPA solubilized in VP. Precursor solutions were photopolymerized under UV (UV-LED LC-L1; Hamamatsu, 2 W/cm^2^, λ = 365 nm) for 40 s in homemade cylindrical dishes. Photopolymerized hydrogels were then incubated during 1 day in a high volume of DMEM for the hydrogel structure to be hydrated and thermodynamically stable before cell seeding. After this day of hydration and two rinsings with fresh medium, cells were seeded upon hydrogels at 2 10^5^ cells/well (or 10^6^ for the high-density experiments). To avoid cell medium acidification, the cell culture medium was replaced by fresh medium every day.
The two glioma cell lines, F98 from rat model and U87-MG from human glioma, were provided by ATCC (CRL 2397 and HTB-14, respectively). Cells were maintained in Dulbecco's Modified Eagle's Medium (DM | {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#s1}
============
Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world with more than 788,000 death per year based on data presented in the World Health Organization (WHO). HCC is the most common liver malignancy with risk factors to the liver such as chronic viral hepatitis of B or C, exposure of alcohol abuse and aflatoxin toxins, and aberrant metabolic stress such as nonalcoholic steatohepatitis (NASH) and obesity \[[@R1]\]. Less than 50% of HCC patients were diagnosed at early stage and majority of them were not qualified for curable surgical resection owing to late stage of tumor \[[@R2]\]. The prognosis for untreated HCC patients has a poor average survival rate 6∼20 months and more than 50% of treated patients developed recurrence and metastasis within 5 years of therapy \[[@R3]\]. A multiple kinase inhibitor Sorafenib was firstly approved for systemic therapy of advanced HCC in 2007 with an average response rate of 2∼3% and extending patient survival for a few months \[[@R4]\]. Another multiple kinase inhibitor regorafenib showed 10∼20% response rate as second-line therapy for advanced HCC was approved by FDA in 2017 \[[@R5]\]. Both multiple kinase inhibitors showed anti-angiogenesis activity by targeting angiogenic and oncogenic receptor tyrosine kinases with modest improvement of HCC patient survival \[[@R6]\]. Moreover, with limited success on HCC cancer genome sequencing for common mutations as therapeutic targets for drug development, it is critical to develop other innovative strategies and novel targets and drugs to prolong survival of HCC patients \[[@R7]\].
Several lines of evidence already supported that aberrant translational machinery such as modulation of ribosome biogenesis, Akt1/mTOR signaling and translational initiation play critical roles in tumor progression \[[@R8], [@R9]\]. Translational initiation is the rate-limiting step of protein synthesis that could alter the overall gene expression or selectively enhance translation of oncogeneic mRNAs to impact cancer development and progression \[[@R10], [@R11]\]. EIF4E, the best-studied eukaryotic initiation factor (eIF), is frequently overexpressed in cancers and activates Akt1/mTOR signaling pathway to phosphorylate 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1) for releasing EIF4E to bind to 5′ cap of mRNA to enable translation of oncogenic genes \[[@R12], [@R13]\]. EIF3 translation initiation complex is the largest eIF protein complex composed of 13 subunits from eIF3a to eIF3m and orchestrates formation and stability of 43S preinitiation complexes (PICs) for translational initiation \[[@R14]\]. Aberrant expression of eIF3 subunits were reported in many malignant tumors and play important roles during tumor progression \[[@R15], [@R16]\]. EIF3C also known as EIF3S8 or eIF3-p110 were found to be upregulated in neurofibromatosis type 2 (NF2), colon cancer, glioma, HCC and breast cancer. Silencing of EIF3C via knockdown or interacting with schwannomin could induce cell apoptosis and suppress cell proliferation and tumor growth \[[@R17]--[@R23]\]. Nevertheless, the underlining molecular mechanisms for upregulated EIF3C to mediate tumor progression and serve as therapeutic target to improve patient survival remain unclear.
Extracellular vesicles were released as heterogeneous plasma membrane vesicles from majority of cell types into body fluids under normal or disease conditions for intercellular communication \[[@R24]\]. Based on their size and biogenesis, extracellular vesicles in diameter could be mainly divided into exosomes (30∼150 nm), microvesicles (or microparticles) (100 nm∼1 µm) and apoptotic bodies (\>1 µm) as cargos for transmitting proteins, lipids, coding RNAs, noncoding RNAs such as microRNAs or even DNA to target cells for inducing various signaling cascades \[[@R25]\]. Numerous studies have shown that cancer cells utilized extracellular vesicles to communicate with stroma cells in the tumor microenvironment similar to cytokines and growth factors VEGF, FGF, TGFβ and Wnt to sustain favorite microenvironment for tumor progression, angiogenesis and metastasis \[[@R26], [@R27]\]. As part of routing clinical liquid biopsy, extracellular vesicles especially nano-sized exosomes could carry various cargos such as proteins, lipids, mRNAs, and miRNAs, and be easily uptook by recipient cells are emerging for developing tools for diagnostic and therapeutic interventions \[[@R28], [@R29]\].
In this study, we explored the roles of EIF3C upregulation in the tumor progression of HCC. Rather than direct stimulation of tumorigenic features such as cell proliferation and cell migration, we found an interesting mechanism that expression of EIF3C in HCC cells increase secretion of exosomes to promote angiogenesis and tumorigenesis of HCC with cross-validations of markers in human HCC tumor samples.
RESULTS {#s2}
=======
EIF3C is overexpressed and associated with poor patient survival in multiple HCC cohorts {#s2_1}
----------------------------------------------------------------------------------------
We found that EIF3C is upregulated in HCC tumor samples in comparison with normal tissues in TCGA HCC dataset (Figure [1A](#F1){ref-type="fig"}). Higher expression of EIF3C in tumor samples is associated with poor patient survival in compared to that of lower expression HCC patients (Figure [1B](#F1){ref-type="fig"}). We validated EIF3C RNA expression in another HCC transcriptome dataset and found that EIF3C gradually increased expression to advanced stages and associated with poor patient survival of HCC patients \[[@R30]\] (Figure [1C](#F1){ref-type="fig"} and [1D](#F1){ref-type="fig"}). Consistently, we also detected higher expression of EIF3C protein level at advanced stages of HCC tissues and in association with poor patient survival by using immunohistochemistry (IHC) assays (Figure [1E--1G](#F1){ref-type="fig"}).
{#F1}
The increasing expression of EIF3C in HCC tissues prompted us to examine its oncogenic properties in HCC cells. We were disappointed that there is no alterations in cell proliferation and expression of tumor progression-related genes including HIF1A, TGFβ1 and VEGF at RNA levels but decrease of trans-well cell migration assays in HCC cells PLC5, SNU449 and Huh7 ([Supplementary Figure 1A--1C](#SD1){ref-type="supplementary-material"}).
Overexpressed-EIF3C in HCC cells increased release of exosomes and enhanced angiogenesis *in vitro* and *in vivo* {#s2_2}
-----------------------------------------------------------------------------------------------------------------
To investigate the roles of overexpressed EIF3C in HCC progression, we performed proteomic study of EIF3C protein complex with immunoprecipitation by using mass spectrometry. Interestingly, we found that EIF3C pulled down 1,738 proteins that participated in functions such as intracellular trafficking and secretion in the clusters of orthologous groups (COGs) and extracellular exosome in cellular component of gene ontology by DAVID Bioinformatics analysis ([Supplementary Figure 2A](#SD1){ref-type="supplementary-material"} and [Supplementary Table 1](#SD2){ref-type="supplementary-material"}). We hypothesized that EIF3C might enhance secretion of exosomes to promote tumor angiogenesis. We found that incubation of conditioned mediums collected from EIF3C-overexpressed HCC cells PLC5, SNU449 and Huh7 (Figure [2A](#F2){ref-type="fig"}) with HUVEC cells could enhance the tube formation of HUVEC in compared with that of mock control for *in vitro* angiogenesis assays (Figure [2B](#F2){ref-type="fig"}). We speculated that the conditioned mediums especially collected from EIF3C-expressed HCC cells might increase the release of vesicles to promote tube formation of HUVEC cells. Indeed, more vesicles ranged in exosome size were observed in EIF3C overexpressed PLC5 than that of mock control detected by electron microscopy (EM) (Figure [2C](#F2){ref-type="fig"}) \[[@R31]\] and nanoparticle tracking analysis (NTA) (Figure [2D](#F2){ref-type="fig"}) \[[@R32], [@R33]\]. Consistently, when incubated HUVEC cells with PKH26-labelled vesicles, more PKH26- | {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Plate tectonics has reshaped the distribution of the Earth's continental and oceanic lithosphere through time. However, when modern-style plate tectonics started is still debated, with proposals ranging from the Neoproterozoic Era to the Archean Eon^[@CR1]--[@CR7]^. The absence from the geological record of high-pressure and low-temperature metamorphism, including blueschists and ophiolites before the Neoproterozoic Era, presents a challenge to understanding early subduction. Archean mantle is about 300 K hotter than the present mantle and high mantle temperature would have significant effects on the viability and style of the subduction process^[@CR8]^. As a result, early ocean plates may have been more buoyant and richer in MgO but weaker than modern ocean lithosphere. The Mg-rich composition of the Archean oceanic crusts may preclude blueschist formation^[@CR9]^. Popular models^[@CR10],[@CR11]^ for early plate tectonics involve shallow flat subduction and delamination of thick crust, precluding low thermal gradient deep subduction. This interpretation is supported by thermodynamic modeling of metamorphic mineral assemblages that the Archean tonalitic--trondhjemitic--granodioritic (TTG) compositions may be formed by shallow subduction of thick oceanic crust^[@CR12]^. The petrological record also shows that the trace element signatures in TTG magmas can only be produced when melts separate from a garnet amphibolite residue, not an eclogite residue^[@CR13]^. However, low Paleoproterozoic thermal gradients are recorded in blueschist facies metamorphism (\~350 °C GPa^−1^) in West Africa^[@CR3]^ and retrograded amphibolitized eclogite boudins (\~300 °C GPa^−1^) in North America^[@CR6]^. These recent findings challenge the timing of onset of modern-style subduction.
A global record of low geothermal gradients in the Paleoproterozoic is a prerequisite to demonstrate a change of plate tectonic processes that dates back to that time. During the Paleoproterozoic Era, there were global-scale continent and mountain building events from West Africa to North America and to North China, leading to Columbia, the first coherent supercontinent in the Earth history^[@CR14],[@CR15]^. The North China craton (NCC, Fig. [1a](#Fig1){ref-type="fig"}) is one of the fundamental Precambrian nuclei of Asia and one of the oldest cratonic blocks in the world (\~3.8 Ga)^[@CR16]^. It preserves a record of a long and complex crustal evolution, but evidence of a thick mantle keel beneath Archean-aged crust is absent^[@CR17]^. The Eastern and Western blocks evolved independently during the Archean and collided along the Trans-North China Orogen (TNCO, Fig. [1b](#Fig1){ref-type="fig"}) during the Paleoproterozoic (\~1.85 Ga) to form the NCC^[@CR16]^. The appearance of Paleoproterozoic carbonatites in the NCC offers an opportunity to evaluate subduction--accretion collision tectonics during the Paleoproterozoic Era. Here, we report pressure--temperature (*P*--*T*) conditions retrieved from an eclogite xenolith in a Paleoproterozoic carbonatite that provides direct petrological evidence of cold oceanic subduction during the Paleoproterozoic Era.Fig. 1Schematic geological map of the North China craton (NCC) with the location of the eclogite xenoliths. The maps are after Zhao et al.^[@CR16]^. **a** The NCC is separated from Yangtze craton by Qinling orogen^[@CR16]^. **b** The Eastern and Western blocks in the NCC were amalgamated by the Paleoproterozoic Trans-North China Orogen (TNCO)^[@CR16]^. The Paleoproterozoic carbonatite and its associated eclogite xenoliths and high-pressure granulites^[@CR17]^ occur in the TNCO. The scale bar is 800 and 400 km in **a** and **b**, respectively
Results {#Sec2}
=======
Geological background {#Sec3}
---------------------
The carbonatites are associated with pyroxenites, pyroxene syenites, and occasionally with granulites (Supplementary Fig. [1a, b](#MOESM1){ref-type="media"}). They occur as dykes and were emplaced in the Fengzhen and Huai'an areas at the crossing between the Khondalite belt and TNCO (Fig. [1b](#Fig1){ref-type="fig"}). These orogens are Paleoproterozoic collisional belts formed by the amalgamation of the Western and Eastern blocks and before that the Ordos and Yinshan blocks, respectively^[@CR16]^. The Khondalite belt is composed of Paleoproterozoic sillimanite--garnet and garnet--biotite gneisses. In the TNCO, late Archean crust is dominated by TTG gneisses. Paleoproterozoic mafic granulites are widely distributed (Fig. [1b](#Fig1){ref-type="fig"}). Their *P*--*T* paths are mostly clockwise, related to collision/exhumation processes, and some of them evolved through eclogite facies^[@CR17]^. The granulites, occurring as lenses and tabular bodies associated with the carbonatites, consist of plagioclase, clinopyroxene, orthopyroxene, amphibole, garnet, and quartz. They are characterized by augen structures consisting of plagioclase and orthopyroxene enveloped by porphyroblastic garnet, indicating rapid exhumation (Supplementary Fig. [1c](#MOESM1){ref-type="media"}). Guo et al.^[@CR18]^ obtained an age of \~1.82 Ga from the Huai'an granulite, with peak *P*--*T* conditions of 750--850 °C and 1.1--1.5 GPa.
Mineralogy and geochronology {#Sec4}
----------------------------
The carbonatite is mainly composed of calcite (70--80%), with minor amounts of diopside, apatite, phlogopite, olivine, and spinel (Supplementary Fig. [2](#MOESM1){ref-type="media"} and Supplementary Table [1](#MOESM1){ref-type="media"}). Apatite occurs as well-developed elongate prismatic crystals (typically \~5 mm in length). In-situ LA-ICPMS U--Pb dating of apatite yields weighted U--Pb ages of 1681(±61) Ma (Fengzhen area) and 1765(±35) Ma (Huai'an area) (Supplementary Fig. [2](#MOESM1){ref-type="media"} and Supplementary Table [2](#MOESM1){ref-type="media"}).
Within the Paleoproterozoic carbonatites, there are rare but well-preserved cm-sized eclogite xenoliths (Supplementary Fig. [1d](#MOESM1){ref-type="media"}). The eclogite is composed of euhedral garnet porphyroblast up to 1 cm in diameter (\~37 vol%), omphacite (\~36 vol%), kyanite (\~9 vol%), quartz (\~9 vol%), zoisite (\~7 vol%), and phengite (\~2 vol%), with accessory amphibole, biotite, and rutile (Supplementary Fig. [3](#MOESM1){ref-type="media"} and Supplementary Table [3](#MOESM1){ref-type="media"}). The mineral proportions were obtained from a SEM--EDS area-scan of one eclogite mount (Fig. [2a](#Fig2){ref-type="fig"}). Garnet has elevated Si (3.019 ± 0.005 pfu on the base of 12 oxygen atoms) and compositional traverses reveal prograde concentric zoning with a homogeneous core overgrown by a rim of increasing pyrope but decreasing almandine, showing a Mg/(Mg + Fe) ratio increasing from the core to the rim (Supplementary Fig. [4](#MOESM1){ref-type="media"}). The garnet core traps abundant mineral inclusions, including kyanite, quartz, zoisite, omphacite, Ca--Na amphibole, Na amphibole, paragonite, and rutile, whereas the rim is relatively free of inclusions (Fig. [2a](#Fig2){ref-type="fig"}). The jadeite content of omphacite in the matrix and inclusions is 23(±1) and 27(±3) mol%, respectively. Zoisite is poor in Fe^3+^. Subparallel zoisite grains in the matrix define a foliation that is characteristic of orogenic eclogite (Fig. [2a](#Fig2){ref-type="fig"}). Phengite has high Si contents (3.40--3.44 pfu). Large matrix phengite is texturally equilibrated with garnet and omphacite, and contains omphacite and kyanite inclusions, interpreted as peak metamorphic minerals. Minor magnesiohornblende occurs | {
"pile_set_name": "PubMed Central"
} |
Introduction {#S0001}
============
Colon cancer is one of the most common malignant cancers and among the leading causes of cancer-related deaths worldwide.[@CIT0001] The combination therapy of oxaliplatin and fluoropyrimidine has been the standard adjuvant therapy in patients with stage III/IV colon cancer. However, these chemotherapies are toxic and sometimes ineffective. Due to its heterogenicity, multiple genetic mutations were regarded as the underlying causes of this disease, for example, mutations in phosphatidylinositol-3 kinase (PI3K) and the mitogen activated protein kinase (MAPK) pathways. Deregulation of MAPK pathway genes have been found, including the amplification and mutation of KRAS, BRAF, and MEK1.[@CIT0002] Therefore, targeting the downstream MEK in the mutated tumors might be a new strategy for colon cancers, especially the patients with KRAS or BRAF mutations. Several MEK inhibitors have entered clinical trial evaluation. However, clinical activity was poor following the treatment with any single MEK inhibitor, and acquired drug resistance appears inevitable.[@CIT0003]--[@CIT0008] Trametinib is a novel oral MEK inhibitor which has been approved by the FDA (Food and Drug Administration) for BRAF-mutated patients alone or in combination with dabrafenib.[@CIT0009]--[@CIT0011] Combination remedies of MEK inhibitors, rather than single medicine therapy, was considered to be more effective in various tumors.[@CIT0012]--[@CIT0014] Therefore, there is an urgent clinical demand for new synergic agents to cooperate with trametinib to enhance the survival of patients with colon cancer.
Interferon-Stimulated Gene 15 (ISG15), a ubiquitin-like protein (UBL),[@CIT0015] is an important oncoprotein and has become a potential diagnostic[@CIT0016] and therapeutic[@CIT0017] target for cancer treatment. The function of ISG15 was largely underestimated mainly due to its low expression in most human malignancies.[@CIT0018] Some studies have found that ISG15 expression was elevated and ISG15-conjugates in many malignant tumors, including melanoma[@CIT0019] and oral squamous cell carcinoma[@CIT0020],[@CIT0021] as well as malignancies of the breast,[@CIT0016] endometrium,[@CIT0018] and bladder.[@CIT0022] However, the roles of ISG15 in tumorigenesis and its responses to anticancer treatments in colon cancer remain largely unknown. In a recent study, Roulois et al[@CIT0023] showed that DNA methylation inhibitor 5-aza-2-deoxycytidine (5-AZACdR) could enhance the expression of ISG15 in LIM1215 colon cancer cells, which implied the nature of ISG15 as a tumor suppressor in colorectal cancer. Controversially, Desai et al[@CIT0018] discovered that ISG15 expression and ISG15-conjugated proteins in two colon cancer cases were up-regulated in comparison to normal colon tissues. Whether ISG15 acts as a tumor suppressor or promotor remains controversial.
In this study, we explored the function of ISG15 in colon cancer cell lines. Our results showed that high expression of ISG15 was an intrinsic feature for colon cancer, and ISG15 promoted the cell proliferation and metastasis. Trametinib could increase the expression of ISG15 shown by gene expression analysis. After treatment of a synergistic combination of trametinib with ISG15 siRNA, proliferation and colony formation was inhibited in vitro. Thus, combined targeting of ISG15 and MEK might be a promising therapeutic strategy for colon cancer treatment.
Materials and Methods {#S0002}
=====================
Tissue Samples and Study Cohort {#S0002-S2001}
-------------------------------
Sixty-six pairs of tumor samples and matched adjacent non-tumor tissues were obtained from the Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). All the patients signed informed consent forms. This study was approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province. ISG15 expression was detected in all specimens. Two pathologists were appointed to evaluate the specimens separately without prior knowledge of the clinical statuses of the specimens.
Immunohistochemistry {#S0002-S2002}
--------------------
Immunohistochemistry (IHC) was performed using the biotin-streptavidin HRP detection system according to the manufacturer's instructions. The tissue chips were incubated with ISG15 antibody (1:100; Abcam, Cambridge, UK) in phosphate-buffered saline (PBS) overnight at 4°C in a humidified container. Biotinylated secondary antibodies (Zhongshan Golden Bridge Biotechnology Co. Ltd., China) were applied. The sections were incubated with HRP-streptavidin conjugates appropriate for detecting ISG15. Proper positive and negative controls were included in each IHC assay.
Western Blotting Analysis {#S0002-S2003}
-------------------------
Total proteins (30 µg) were subjected to fractionation by SDS polyacrylamide gel electrophoresis and immunoblotting assay. Antibodies used in the study included the following: anti-ISG15, anti-PARP, anti-c-Myc, anti-pERK, anti-ERK (all Abcam, Cambridge, UK), and anti-GAPDH (Zhongshan Golden Bridge Biotechnology Co. Ltd., China).
Cell Culture and RNA Interference {#S0002-S2004}
---------------------------------
Human colon cancer cell lines were obtained from the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, including RKO, HCT116 and SW480. And all the cells were tested and authenticated by an AmpFlSTR Identifiler PCR assays in the year of 2019 in TSINGKE Biological Technology Company. The results showed that RKO and SW480 were 100% exact matched, and HCT116 was 98% matched. All the cell lines were confirmed to be mycoplasma negative via a PCR method and directly thawed from the liquid nitrogen jar. Cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (Gibco, USA), 100mU/mL penicillin, and 100 µg/mL streptomycin in a 5% CO2 atmosphere at 37°C. Cells (1x10 5 cells/well) were seeded into six-well plates and transfected with the indicated constructs using Lipofectamine 2000 (Invitrogen/Life Sciences) according to the manufacturer's instructions. After 72 h, the transfected cells were harvested for further analysis. The sequence of the siRNA sense strand was as follows: si-ISG15\#1: 5-'TCCTGGTGAGGAATAACAA-3ʹ, si-ISG15\#2: 5-'CCAUGUCGGUGUCAGAGCUTT-3ʹ.
CCK-8 and Colony Formation Assays {#S0002-S2005}
---------------------------------
CCK-8 assay kit was used for colon cancer cell proliferation analysis. Colon cancer cells were seeded in 96-well plate at a density of 5x10^\^3^ cells per well. After incubation, 10 µL CCK8 was added to the wells at different times. The absorbance was measured at 450 nm by a microplate reader (Bio-Rad, Hercules, CA, USA). Colony formation assay was performed to detect the capacity of cell proliferation. RKO, HCT116 and SW480 cells were seeded into 35 mm dishes with 3x10^\^3^ cells per well and cultured for 10 days. Colonies were then fixed with 10% formaldehyde for 30 min and stained for 5 min with 0.5% crystal violet.
Wound Healing Assay {#S0002-S2006}
-------------------
Colon cancer cells were cultured in 6-well plates and grown until 80--90% confluence. After that, the cell monolayer was scratched with a sterile pipette tip. An Olympus microscope was applied for taking pictures of cell morphology at different time intervals (0,48h). The results were analyzed by ImageJ software.
Matrigel Invasion Assays {#S0002-S2007}
------------------------
The capacity invasiveness was determined in a 24-well transwell plate (8 up pore size; Costar). Briefly, 6x10^\^\ 4^ cells were suspended and added to the upper chamber of each insert coated with 200 mg/mL of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). After 48 h, the cells that traversed the membrane and spread to the lower surface of the filters were stained with haematoxylin and counted.Each experiment was repeated three times independently.
RNA Extraction and qRT-PCR {#S0002-S2008}
--------------------------
Total RNA was isolated through the guanidinium thiocyanate-phenol-chloroform extraction method using TRIzolsolution (Invitrogen, USA), and cDNA synthesis was done using the Advantage RT-forPCRKit (Takara, Japan). The forward and reverse primers were designed to span two different exons for each gene.The primers for human ISG15 were forward:5ʹ-TGGACAAATGCGACGAACC-3ʹ; reverse: 5ʹ-TTCGTCGTTCACTCGCC-3ʹ.CFX96 Real-Time PCR detectionsystem (Bio-Rad, Hercules, CA, USA) was used to perform qRT-PCR. The threshold cycle number was determinedusing iCycler software version 3.0. Reactions were performed in duplicate and the threshold cycle numbers wereaveraged. The data were analysed using the 2\^^−ΔΔCT^ method.
Edu Assay {#S0002-S2009}
---------
Quantification of cellular proliferative capacity was determined by 5-Ethynyl-2ʹ-deoxyuridine assay using a Cell-Light TM EdU Apollo^®^ | {
"pile_set_name": "PubMed Central"
} |
Optimal patient care relies on a cohesive team of health care professionals who work synergistically, focusing on individual strengths to ensure the highest quality of patient care ([@CIT0001], [@CIT0002]). It has been shown that interprofessional collaboration enhances patient safety, decreases medical errors, and improves satisfaction among health professionals ([@CIT0002], [@CIT0003]). Skills necessary to function as part of an interprofessional team require cultivation and development during health professions training ([@CIT0004]). This has made training in interprofessional collaboration an increasingly appreciated component of medical education.
Interprofessional education (IPE) occurs when two or more professions learn with, from, and about each other to improve collaboration and the quality of care ([@CIT0005]). The development and implementation of interprofessional education curriculum, however, can be a difficult task, requiring significant faculty involvement and curriculum development ([@CIT0006]).
Student-led peer teaching conveys a number of benefits to the peer learner, the peer teacher, and the institution as a whole ([@CIT0007], [@CIT0008]). Peer teaching occurs when instructors and learners are at a similar stage in their education ([@CIT0009]). Students engaged in peer learning have been shown to be highly receptive to instruction from peer teachers due to the decreased power differential existing between peer teacher and peer learners ([@CIT0010]). In addition, student-led initiatives are cost-effective and can promote collegiality and socialization, leading to long-term sustainability of student-led programs ([@CIT0008]).
Small-group problem-based learning (PBL) is a highly effective means of education ([@CIT0011]). During PBL sessions students are presented with a problem or clinical scenario and are then required to work together to come up with a solution with minimal input from the facilitator ([@CIT0012]). The problem-based model is designed around individual inquiry and is very effective in developing problem solving skills, independent learning, and teamwork ([@CIT0013]). This educational method presents a unique way of delivering IPE. We believe student-led small-group PBL seminars can be used to effectively deliver IPE and improve students' perceived need for interprofessional education.
To measure the efficacy of IPE sessions, the Interprofessional Education Perception Scale (IEPS) tool was used ([@CIT0014]). The IEPS tool, originally developed by Leucht et al., is an 18-item questionnaire using a six-point Likert scale, designed to assess perceptions of interprofessional education. The questionnaire is then subdivided into four subscales of IPE: competence and autonomy (Subscale 1), perceived need for cooperation (Subscale 2), perception of actual cooperation (Subscale 3), and understanding of others values (Subscale 4). The IEPS was used to evaluate participants' opinions regarding IPE over 16 weeks ([Table 1](#T0001){ref-type="table"}).
######
IEPS subscale classifications
------------ ---------------------------------------------
Subscale 1 Professional competence and autonomy
Subscale 2 Perceived need for professional cooperation
Subscale 3 Perception of interdependence
Subscale 4 Willingness to share ideas
------------ ---------------------------------------------
Methods {#S0002}
=======
First- and second-year medical and pharmacy students at Creighton University were invited to participate in small-group PBL interprofessional seminars led by fellow second-year medical students who served as peer-teachers. The study was approved by the Institutional Review Board at Creighton University.
Seminars were held weekly during the lunch hour for 16 weeks. Each seminar was led by one to two second-year medical students, serving as peer-teachers and consisted of 10--14 student learners, split evenly between medical and pharmacy students. The cases were adaptations of cases seen at the Creighton University Medical Center or cases published in peer-reviewed journals. At the beginning of each seminar the group was given a basic patient presentation. Student learners were then required to ask appropriate questions and find appropriate answers to create a differential diagnosis. Student learners were next asked to identify appropriate diagnostic testing to order. The designated test results and questions regarding history and physical were provided by the peer-teacher when appropriate. Through the course of the seminar the peer-teacher provided little to no guidance in keeping with the PBL design. During each seminar students were allowed to use any reference material they deemed necessary.
Evaluation {#S0003}
==========
A case--control study design was used to evaluate the efficacy of IPE seminars. Following 16 weeks of IPE seminars, student\'s attitudes toward IPE were assessed using the IEPS. Questionnaires were distributed to the entire first- and second-year medical and pharmacy student classes, including those who did not attend. Those students not attending the IPE seminars served as the control group for our study. All first- and second-year medical and pharmacy students were also given a questionnaire regarding barriers to IPE; this survey was designed by the authors of this study ([Table 2](#T0002){ref-type="table"}).
######
IEPS scaled results
*n* Subscale 2 mean±SD Subscale 3 mean±SD
----------------------------------- ----- ---------------------------------------------- ----------------------------------------------
All medical students 62 94.76[a](#TF0001){ref-type="table-fn"}±6.44 75.91[b](#TF0002){ref-type="table-fn"}±10.32
All pharmacy students 35 90.24[a](#TF0001){ref-type="table-fn"}±10.00 84.29[b](#TF0002){ref-type="table-fn"}±10.05
Medical student participants 19 97.81[c](#TF0003){ref-type="table-fn"}±4.68 76.67±11.65
Medical student non-participants 43 93.45[c](#TF0003){ref-type="table-fn"}±7.02 74.76±10.04
Pharmacy student participants 10 95.37^d^±5.71 86.33±10.82
Pharmacy student non-participants 25 88.33[d](#TF0004){ref-type="table-fn"}±10.76 82.67±10.93
All medical students compared to all pharmacy students 'need for professional cooperation' *p*=0.02
all medical students compared to all pharmacy students 'perception of interdependence' *p*=0.002
medical student participants compared to medical student non-participants 'need for professional cooperation' *p*=0.006
pharmacy student participants compared to pharmacy student non-participants 'need for professional cooperation' *p*=0.02.
Data from the IEPS were analyzed along four subscales as described previously ([@CIT0014]). The primary outcome was the difference in responses to the IEPS of those students who attended seminars versus those students who did not attend. The data were scaled to a score of 100 for easier interpretation. It is important to note that data can only be compared within each subscale and not between subscales. Statistical significance was determined using two-tailed *t-*test and a *p* value of 0.05. Student responses with regard to perceived barriers to interprofessional education were also recorded and served as secondary outcome data.
Results {#S0004}
=======
In total, 43 students (24 medical and 19 pharmacy) attended IPE seminars, of those 29 completed the IEPS (67.4% response rates). Sixty eight (43 medical and 29 pharmacy) out of 313 students who did not participate in IPE seminars completed the IEPS survey (21.7% response rate) ([Table 2](#T0002){ref-type="table"}). IEPS responses show that medical and pharmacy students who participated in seminars perceived a significantly higher need for cooperation when compared to those who did not participate (*p=*0.006 and *p=*0.02, respectively). Significance was not observed within subscale 1 or subscale 4 (data not shown). In addition, combined responses from participants and non-participants showed that pharmacy students perceived a significantly higher need for professional cooperation (*p=*0.02) and interdependence (*p=*0.002) when compared to medical students.
In total, 109 (30.62%) out of 356 eligible students responded to the survey regarding current barriers to interprofessional education ([Table 3](#T0003){ref-type="table"}). The most common barrier to participation was 'I am not aware of interprofessional education programs' (61.5% of respondents). The second most common response was 'I do not have time' (52.3% of respondents). Zero respondents indicated, 'Interprofessional education is not an important part of my career'.
######
Student perceived barriers to IPE
Student response \% *n*
--------------------------------------------------------------------- ------ -----
I am interested in IPE but I do not have enough time to participate 52.3 57
I am not aware of currently available IPE activities 61.5 67
I have tried and all IPE sessions are full 5.5 6
I do not think IPE is important in my future 0.0 0
Discussion {#S0005}
==========
The role that student leaders play in curricular development, especially pertaining to interprofessional education, can be of great value to educational institution as a whole. Current student-led initiatives in IPE have used designs such as social gatherings, conferences, lectures, and formal communications between health professions students ([@CIT0008]). Our data demonstrate that a student-led | {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#s1}
============
Cytomegalovirus (CMV) is a re-emerging human herpes virus, which causes substantial morbidity and occasional mortality in immunocompromised patients \[[@CIT0001]\]. Among these, solid organ transplant recipients who maintain an allograft by receiving immunosuppressive drugs are well-known to be at risk of infection. CMV infection after kidney transplantation (KT) reportedly ranges from asymptomatic infection to symptomatic CMV syndrome and tissue-invasive disease \[[@CIT0002]\]. Data from 2 retrospective studies in Thailand revealed the prevalence of CMV seropositivity in both donors and recipients was 99 percent. Despite preexisting immunity in most patientsindicated by CMV seropositivity, they remain at moderate risk of CMV infection. The prevalence of asymptomatic CMV infection and CMV disease in KT recipients were 5%--21% and 7%, respectively. Risk factors include advanced age of the recipient and use of antithymocyte globulin (ATG) for induction or steroid-resistant rejection therapy \[[@CIT0003], [@CIT0004]\]. Patients developing CMV diseases carry a greater risk of allograft failure and death compared with those free from CMV disease \[[@CIT0003]\]. Furthermore, KT recipients who developed drug-resistant CMV infection could suffer increased morbidity from prolonged CMV DNAemia and anti-CMV therapy \[[@CIT0005]\]. Prevention of this infection remains a suitable intervention to limit unfavorable consequences. Two international guidelines from the Transplantation Society and the American Society of Transplantation Infectious Disease Community of Practice encouraged implementation of prevention strategies for CMV infection among KT recipients \[[@CIT0006], [@CIT0007]\]. Although the guideline for prevention of CMV infection in Thai KT care was developed by the Thai Transplant Society, the strategies implemented among transplant centers in Thailand remained variable \[[@CIT0008]\]. Despite the fact that a preemptive approach is recommended in CMV-seropositive KT recipients by the aforementioned guideline, impracticability and financial restrictions could limit its utilization in real-world practice. Additionally, an optimal cut-off value of plasma CMV DNA load has not been standardized among physicians. Furthermore, an investigation of real-life strategies to prevent this specific infection has never been performed, especially in resource-limited settings. Therefore, we aimed to investigate CMV prevention strategies utilized among transplant centers in Thailand. We also investigated differing perspectives in terms of CMV prevention in KT recipients between infectious disease physicians (ID) and nephrologists (NP).
METHODS {#s2}
=======
Questionnaire {#s3}
-------------
A survey was delivered to all 31 transplant centers in Thailand during October and November 2018. One ID and 1 NP, who were directly caring for KT recipients at each transplant center, were included. The names of the transplant centers and physicians were obtained from the Thai Transplant Society in October 2018. An email with a link to the electronic survey was sent to all physicians with a reminder email if a response was not received. The study protocol was approved by the Institutional Review Board of the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
A questionnaire on CMV prevention strategies for KT recipients was developed using a web-based electronic survey website ([www.surveymonkey.com](http://www.surveymonkey.com)). The survey included the respondents' demographic data, such as sex, age, transplant center setting or years in KT practice, and CMV prevention strategies. CMV prevention strategies were defined according to the recently published guidelines \[[@CIT0006], [@CIT0007]\]. Prophylaxis was defined as administration of anti-CMV drugs, either intravenous (IV) ganciclovir or oral valganciclovir, for a defined period of time after KT. A preemptive approach was defined as surveillance of plasma CMV DNA load, quantified by real-time polymerase chain reaction (PCR) assay and initiation of anti-CMV drugs when the cut-off value was reached to prevent progression from asymptomatic CMV infection to disease. Plasma CMV DNA load was reported in both copies/mL and international units (IU)/mL, based on the use of a quantitative real-time PCR technique by COBAS AmpliPrep/COBAS TaqMan (TaqMan CMV Test, Roche Molecular Diagnostics, Branchburg, NJ). One copy/mL was calibrated to 0.91 IU/mL by the calibration of tests with the World Health Organization international standard \[[@CIT0009]\]. Targeted prophylaxis or preemptive approaches were defined as above, but they were focused on a defined high-risk group of patients rather than universal implementation. A hybrid approach was defined as surveillance by plasma CMV DNA quantification after cessation of a defined period of prophylaxis. A CMV-specific immunity-guided approach was defined as an intervention (prophylaxis, preemptive approach, or closed observation) guided by measurement of cell-mediated immunity, such as CMV-specific T-cell immunity assay.
Statistical Analyses {#s4}
--------------------
Demographic data of all physicians involved in the study were analyzed by descriptive analysis. Categorical data were described as frequencies and percentages and compared by Fisher exact test. A *P* value \< .05 was considered statistically significant. Statistical analyses were performed with the statistical software Stata version 15 (StataCorp, LLC, College Station, TX).
RESULTS {#s5}
=======
Demographic Data {#s6}
----------------
There were 43 respondents from 26 of the 31 (84%) transplant centers, including 26 (60%) IDs and 17 (40%) NPs. The demographic data of all physicians included in the survey are summarized in [Table 1](#T1){ref-type="table"}. Fifty-eight percent of respondents were aged 35--44 years and approximately half were males (49%). Two-thirds of the physicians had been working in a public hospital setting (63%) and encountering KT recipients for at least 2 years (74%). The demographic data between the ID and NP groups were comparable; however, there were slightly more male physicians among the IDs compared with the NP group (62% vs 29%; *P* = .06). There were also significantly more physicians from a public hospital setting in the ID group compared with the NP group (81% vs 36%; *P* \< .05).
######
Demographic Data of Infectious Disease Physicians and Nephrologists Who Participated in the Survey
Characteristics Infectious Disease Physicians (n = 26) Nephrologists (n = 17) *P* value
--------------------------------------------------- ---------------------------------------- ------------------------ -----------
Male, n (%) 16 (62) 5 (29) .06
Age, years, n (%)
25--34 5 (19) 9 (53) .04
35--44 18 (69) 7 (41) .11
45--54 3 (12) 1 (6) 1.00
Transplant center setting, n (%)
Public general hospital 10 (39) 2 (12) .09
Public university hospital 11 (42) 4 (24) .33
Nonprofit hospital 1 (4) 8 (47) .001
Others 4 (15) 3 (17) 1.00
Years in kidney transplant recipients care, n (%)
\<1 3 (12) 2 (12) 1.00
1--2 1 (4) 5 (29) .03
2--5 15 (57) 5 (29) .12
5--10 4 (15) 3 (18) 1.00
\>10 3 (12) 2 (12) 1.00
CMV Prevention Strategies {#s7}
-------------------------
Overall responses in terms of CMV prevention strategies and the different perspectives between the ID and NP groups are shown in [Figure 1](#F1){ref-type="fig"}. Forty-one (95%) physicians agreed with a need for CMV prevention in KT recipients, although 33 physicians (77%) stated that they already utilized prevention strategies for their patients. Cytomegalovirus prevention strategies currently implemented include preemptive approaches (48%), universal prophylaxis (45%) (either by IV ganciclovir or oral valganciclovir), hybrid approaches; surveillance after prophylaxis (3%), and CMV-specific immunity-guided approach (3%). When specifically asked about the potential role of CMV prevention in Thai KT recipients, when the majority are CMV seropositive and likely to receive an allograft from a CMV seropositive donor, only 5% of physicians stated no need for prevention in this scenario. The remaining 95% reported that they preferred the preemptive approach (84%) over prophylaxis (12%). However, 81% of the former preferred targeted prophylaxis for those receiving ATG.
{#F1}
Among physicians choosing to implement prophylaxis, the duration of prophylaxis ranged from less than 1 to more than 6 months, with half (51%) choosing to implement prophylaxis over a 3-month course. For the preemptive approach, 65% and 93% of all physicians initiated preemptive therapy when the plasma CMV DNA load reached 2000 and 3000 copies/mL (1820 and 2730 IU/mL) or 3.3 and 3.4 log10 copies/mL (3.2and 3.4 log10 IU/mL), respectively [Figure 2](#F2){ref-type="fig"}. At a plasma CMV DNA load cut-off value of 5000 copies | {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
*Toxoplasma gondii* is an obligate intracellular apicomplexan protozoan parasite that can infect all warm-blooded animals, mainly through oral and congenital infections (Tenter et al., [@B34]; Elmore et al., [@B11]; Zhou et al., [@B39]). This parasite has a significant medical and socioeconomic importance because it infects over two billion people worldwide. It is the causative agent of toxoplasmosis, an important zoonotic disease that can cause serious, even fatal health consequences in immunocompromised individuals, such as AIDS patients and organ transplant recipients (Liu et al., [@B22]; Xiao et al., [@B38]; Chemoh et al., [@B9]). Infection in immunocompetent individuals are generally asymptomatic, however the parasite persists as bradyzoites-containing cysts in brain and muscle tissues for many years. Primary infection in pregnant women can lead to fetus death, deformity, abortion, and long-term damage of the eye and central nervous system (Hill and Dubey, [@B19]).
The remarkable ability of *T. gondii* to invade and colonize virtually all nucleated cells (Morisaki et al., [@B25]), and to adopt a successful intracellular lifestyle depends partly on the sequential secretion of effectors from the specialized secretory organelles micronemes, rhoptries and dense granules (Håkansson et al., [@B17]; Boothroyd and Dubremetz, [@B4]; Nadipuram et al., [@B26]). *T. gondii* tachyzoites enter the host cells through an active invasion mechanism and replicate within a membrane-bound parasitophorous vacuole (PV) inside the surrogate host cell cytoplasm (Coppens et al., [@B10]; Nam, [@B27]). After release from the host cell, newly formed parasites invade new mammalian host cells, and the replication cycle starts again. Most of the dense granule proteins (GRAs) are destined to the PV and parasitophorous vacuole membrane (PVM), and contribute to the biogenesis and maturation of the PV, and nutrient acquisition (Mercier and Cesbron-Delauw, [@B24]).
Some GRA proteins have the ability to traffic to the host cytoplasm or nucleus and interfere with host cell signaling pathways. For example, *GRA6* is a polymorphic dense granule protein and activates the host transcription nuclear factor of activated T cells 4 (NFAT4) in order to manipulate host immune responses to maximize the parasite virulence in a strain-specific manner (Ma et al., [@B23]). *GRA15* is another strain-specific effector that activates NF-κB pathway and induces IL-12 secretion in type II, but not type I or III genotypes. *GRA15*-deficient type II strain cannot activate NF-κB pathway or induces IL-12 secretion, hence *GRA15*-deficient type II strains grow faster compared with wild-type strains (Rosowski et al., [@B29]). *GRA16* and *GRA24* also manipulate host gene expression and signaling pathways (Bougdour et al., [@B5]; Braun et al., [@B6]). *GRA17* mediates the transfer of small molecules between the host cell and PV, and maintains the stability of the PV (Gold et al., [@B16]). *GRA7, GRA25*, and *GRA39* are also virulence factor and can interfere with host cell signaling pathways (Alaganan et al., [@B1]; Shastri et al., [@B30]; Nadipuram et al., [@B26]). *GRA22* and *GRA41* are involved in the parasite egress (Okada et al., [@B28]; LaFavers et al., [@B21]).
Despite the wealth of information regarding *T. gondii*\'s 40+ GRAs including sequence variation and expression of GRA coding genes, and their roles in the infection process, the contribution of many GRAs to the parasite growth and virulence are still unclear. The CRISPR-Cas9 system provides a novel and promising tool for editing *T. gondii* genes (Shen et al., [@B32]) and the genome (Sidik et al., [@B33]; Wang et al., [@B35]; Shen et al., [@B31]). Using CRISPR-Cas9 to target *T. gondii*\'s GRA genes may ultimately offer a new approach to achieving functional cure to toxoplasmosis. In this study, we used the CRISPR-Cas9 technique to edit 17 GRA genes, namely *GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38*, and *GRA40* in the virulent *T. gondii* RH strain and examined the effects of gene loss on the parasite\'s ability to grow and exit from host cells *in vitro* and to cause death in mice.
Materials and methods {#s2}
=====================
Parasite and cell cultures
--------------------------
Tachyzoites of *T. gondii* RH strain (type I) were maintained *in vitro* by passages in human foreskin fibroblast (HFF, ATCC, Manassas, VA, USA). HFF cells were grown in 75-cm^2^ tissue culture flasks containing Dulbecco\'s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 10 μg/ml gentamycin, at 37°C under a 5% CO~2~ atmosphere. To purify tachyzoites, infected HFF cells were lysed through a 27-gauge needle and the tachyzoites were filtered using a 5 μm pore size Millipore filter. The number of purified parasites was counted using a hemocytometer under a phase-contrast microscopy.
Construction of GRA knockout *T. gondii* strains using CRISPR-Cas9
------------------------------------------------------------------
*GRA* knockout strains were constructed by using CRISPR-Cas9 as previously described (Wang et al., [@B36]). All plasmids, primers, and gRNAs used in this study are listed in Table [S1](#SM1){ref-type="supplementary-material"}. Briefly, sgRNA of each *GRA* was engineered into pSAG1::CAS9-U6::sgUPRT by PCR using the Q5 Mutagenesis Kit (NEB). Positive plasmid was extracted with Endo-Free Plasmid DNA Mini Kit Protocols (OMEGA). The resistance cassettes (DHFR^\*^-Ts) were amplified from the plasmid pUPRT-DHFR-D and purified by agarose gel electrophoresis. About 40 μg positive plasmids and 15 μg purified DHFR^\*^-Ts amplicons were co-transfected into freshly harvested *T. gondii* RH tachyzoites by electroporation. *GRA*-deficient tachyzoites were selected with pyrimethamine and examined by PCR analysis. Stable clones were confirmed by reverse transcription PCR (RT-PCR) by comparison to WT strains. Total RNA was extracted from tachyzoites of wild type (WT) or Δ*GRA* mutant *T. gondii* strains using TRIzol (Invitrogen). Reverse transcription was performed using a PrimeScriptTM 1st Strand cDNA Synthesis Kit (TaKaRa). The central region of each target gene cDNA was amplified by RT-PCR using specific primers (see Table [S1](#SM1){ref-type="supplementary-material"} in the Supplemental Material).
Assessment of parasite growth using plaque assay
------------------------------------------------
The growth rates of individual *GRA*-deficient and WT RH strains were determined in HFF cells. Cells were grown to confluence overnight in 6-well cell culture plates. The cells were then infected with tachyzoites of Δ*GRA* mutant and WT RH strains (\~200 tachyzoites/well). Cells were incubated for 3 h to allow the parasites to enter the host cells and were then washed twice with sterile 1 × phosphate-buffered saline (PBS) to remove unbound parasites, and fresh medium was added. The plates were incubated for 7 days at 37°C in 5% CO~2~ environment. Then, the culture medium was removed and infected HFF cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min at ambient temperature. Then, fixed cells were incubated with crystal violet staining solution (2% \[wt/vol\] crystal violet, pH 7.4) for 10 min at ambient temperature. The size of plaques (i.e., areas in the cell culture devoid of cells caused by the proliferating parasites) was determined using inverted microscope as previously described (Wang et al., [@B37]). This experiment was performed in triplicate and the experiments were repeated three independent times.
Egress assay
------------
Asexual reproduction of *T. gondii* culminates with the egress of the newly formed tachyzoites from the surrogate host cell and subsequent parasite invasion into new host cells. Therefore, parasite egress represents a very important event which is indispensable for the parasite dissemination in the host body. In order to determine the role of the GRAs in this important process, we examined the effect of ionophore A23187 modulating Ca2^+^ homeostasis on the egress of the parasite from the host cells. Briefly, HFF cells were incubated with 10^5^ freshly harvested *T. gondii* tachyzoites in 2 ml culture medium for 3 h, followed by washing twice with DMEM medium to remove the unbound parasites. After 30--36 h of incubation, the wells were washed twice with sterile PBS and 3 μM calcium ionophore A23187 | {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Sensory experience plays an essential role in establishing and refining neural organization during development. In fact, abnormal input or experiential modifications due to injury can produce severe changes in neuronal morphology including alterations in dendritic length, cell size, and can even influence the fate of a cell [@pone.0111243-LeviMontalcini1]--[@pone.0111243-Smith1]. Second-order auditory neurons in the chick brain stem have proven useful in investigating the importance of sensory experience during development [@pone.0111243-Rubel1]. Neurons in the cochlear nucleus, nucleus magnocellularis (NM) receive their sole excitatory input from the cochlea via the ipsilateral eighth nerve [@pone.0111243-Boord1], [@pone.0111243-Parks1]. Thus, unilateral cochlear ablation results in a complete loss of excitatory afferent input to the ipsilateral NM but leaves the contralateral side unaffected, allowing for within-subject comparisons of NM neurons on the intact versus the deafened side of the brain. Elimination of auditory nerve activity during a sensitive period of development leads to the death of approximately 20--30% of NM neurons on the deafened side of the brain [@pone.0111243-Born1]. The remaining neurons survive, albeit at a lower metabolic rate and with a reduction in soma size [@pone.0111243-Born1]--[@pone.0111243-Steward1].
The factors that determine which cells die and which cells survive still remain unclear; however, the chain of events that occur within NM as a consequence of deafening are well documented. Within 1 to 3 hrs following cochlear ablation, NM neurons show a threefold increase in intracellular calcium (\[Ca^2+^\]~i~) [@pone.0111243-Zirpel1], and a decrease in RNA and protein synthesis [@pone.0111243-Steward1], [@pone.0111243-Garden1], [@pone.0111243-Garden2]. At 6--12 hrs, deafened NM neurons appear to segregate into two populations: one population suffers a complete cessation of protein synthesis and eventually goes on to die, while the other population continues to synthesize proteins, albeit at a reduced level, and goes on to survive [@pone.0111243-Steward1]. The cessation of protein synthesis appears to be due to the dissociation of polyribosomes in NM neurons following cochlear ablation [@pone.0111243-Rubel2]. One way to visualize the rapid activity-dependent changes that occur in ribosomes following deafening is by using Y10B, a monoclonal antibody that recognizes a ribosomal epitope [@pone.0111243-Garden1], [@pone.0111243-Garden2], [@pone.0111243-Garden3], [@pone.0111243-Hyson1]. Changes in Y10B immunoreactivity match the changes observed in overall protein synthesis [@pone.0111243-Garden3], and these changes correspond with changes observed in ribosomes at the EM level [@pone.0111243-Garden2], [@pone.0111243-Rubel2]. Consequently, Y10B immunoreactivity provides an efficient and meaningful assay for examining changes in NM ribosomes, one of the earliest cellular changes to occur following deafferentation.
Studies directed at identifying the trans-synaptic signals necessary for preventing the early changes that occur in NM neurons following cochlea removal have made use of an *in vitro* slice preparation of the chick auditory brain stem. In this condition, NM activity is eliminated bilaterally since both cochleae are destroyed. In order to mimic the case of unilateral deafening, auditory nerve fibers are electrically stimulated on one side of the slice while fibers on the opposite side of the same slice remain unstimulated. Within 1 hr, NM neurons on the stimulated side of the slice show greater protein synthesis [@pone.0111243-Hyson2] and Y10B labeling [@pone.0111243-Hyson1], [@pone.0111243-Hyson3], [@pone.0111243-Hyson4] than the neurons on the unstimulated side. Through the use of the *in vitro* slice preparation, it has been shown that glutamate\'s action on ionotropic glutamate receptors (iGluRs) is responsible for the electrical activity in postsynaptic NM neurons [@pone.0111243-Hyson3], [@pone.0111243-Nemeth1], and glutamate\'s action on metabotropic glutamate receptors (mGluRs) is responsible for providing trophic support to NM neurons [@pone.0111243-Hyson4], [@pone.0111243-Nicholas1], [@pone.0111243-Zirpel2]. Previous *in vivo* studies have also shown that mGluR activation is necessary for maintaining ribosomal antigenicity of Y10B and for the ultimate survival of NM neurons [@pone.0111243-Carzoli1]. For example, application of selective group I or group II mGluR antagonists results in NM neuronal cell death even with an intact cochlea.
Although it is clear that mGluR activation is [necessary]{.ul} to preserve NM neurons in a healthy state, it is not known if mGluR activation is [sufficient]{.ul} to maintain these neurons or if other activity-dependent factors are also required. The present experiments evaluate whether or not activating mGluRs on deafferented NM neurons is sufficient for maintaining ribosomes in a healthy state during the period immediately following deafferentation. If adding mGluR activation to the deprived NM is sufficient to regulate NM neuronal ribosomes, then application of mGluR agonists should preserve ribosomal Y10B antigenicity, which is normally lost in the absence of auditory experience.
Materials and Methods {#s2}
=====================
Subjects {#s2a}
--------
All subjects were 1 to 4 day old chicks of either sex, hatched from eggs obtained from a local supplier and reared at Florida State University. The procedures used in these experiments were approved by the Animal Care and Use Committee at Florida State University and conform to the guidelines set forth by the National Institutes of Health. All efforts were made to minimize the number of animals used and their suffering.
Slice preparation {#s2b}
-----------------
Subjects were anesthetized with isoflurane and decapitated. A 4 mm segment of the caudal skull containing the brain stem was removed with a razor blade, quickly submerged in room temperature artificial cerebral spinal fluid (ACSF), and was oxygenated using a 95% O~2~, and 5% CO~2~ gas mixture. ACSF consisted of (in mM) 130 NaCl, 3 KCl, 2 CaCl~2~, 2 MgCl~2~, 26 NaHCO~3~1.25 NaH~2~PO~4~ and 10 dextrose. The brain stem was rapidly dissected from the skull segment and mounted onto a custom-built stage for slicing using cyanoacrylate glue with additional support provided by a 30% gelatin compound. Using a vibrating blade microtome, a 300 µm, bilaterally symmetrical, coronal slice containing NM was obtained, transferred to a submersion-type recording chamber, and perfused (2--3 ml/min by gravity) with oxygenated ACSF. The slice was anchored in the recording chamber using nylon-strung metal "harp".
Drug administration {#s2c}
-------------------
Once the brain slice was placed in the chamber, a glass micropipette was lowered into the bath and positioned over the slice toward the lateral side of the IVth ventricle. Drugs were ejected onto one side of the brain slice using periodic pressure pulses (10 psi, 10--50 msec duration) through a picospritzer (General Valve Corporation, Fairfield, N.J.). The slice was oriented such that the laminar flow of ACSF through the chamber carried the drug only to NM on the outflow side of the chamber. Different groups of slices (n = 4 per group) were unilaterally treated with either vehicle, glutamate (1 mM), the general mGluR agonist, ± trans 1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) (400 µM), a selective group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG) (200 µM), or a selective group II mGluR agonist, LY354740 (200 nM) for 1 hr. Drug concentrations in the pipette were selected based on their EC~50~ and predicting an approximately10-fold reduction in concentration by the time the drug flowed to NM. A dose-response curve for glutamate was constructed using concentrations of L-glutamic acid ranging from 0.01 to 10 mM in the ejection pipette (n = 3--4 slices per group). The vehicle was dextrose-free ACSF.
Confirmation of unilateral drug application {#s2d}
-------------------------------------------
Preliminary tests using fast green were performed to monitor the distribution of solution released by pressure application in order to ensure drug was being administered to only one side of the slice and that minimal backflow was affecting the opposite side of the same slice. Additional confirmation was obtained by ejecting a 1% solution of a fluorescent dextran, tetramethylrhodamine (fluoro-ruby) 10,000 MW (Invitrogen), from the micropipette. Distribution of dye clearly showed that ejected substances primarily, if not exclusively, reached only one NM (see [Figure 1](#pone-0111243-g001){ref-type="fig"}). Dye was used in slices to document the application procedure and was not included for drug-treated slices, so as not to interfere with immunoreactivity.
{#sp1 .153}
{#sp2 .154}
{#sp3 .155}
{#sp4 .156}
| {
"pile_set_name": "PubMed Central"
} |
According to sexual conflict theory females are the choosy sex ([@zow090-B63]; [@zow090-B41]; [@zow090-B7]) and should choose males of higher quality as potential mates ([@zow090-B75], [@zow090-B76]; [@zow090-B5]; [@zow090-B31]; [@zow090-B38]; [@zow090-B32]; [@zow090-B30]). By choosing a high quality partner the female provides offspring with resources ([@zow090-B26]) or with a better genetic background ([@zow090-B23]; [@zow090-B4]). Androgen-dependent male sexual traits (STs), including behavioral characteristics, as well as immunocompetence, are theoretically assumed to be key indicators for female mate choice. The expression of androgen-dependent traits and the maintenance of a male's physical health are costly processes causing a tradeoff between competing functional systems. According to the "Immunocompetence handicap" hypothesis, when the female chooses a male, she focuses on signs of his innate health, although high levels of testosterone may negatively affect immunity ([@zow090-B19]). Theoretically, a female can base a choice on any optional trait indicating a male's quality, but the interdependence between such traits and expression of secondary sexual characters, immunocompetence, androgens, or androgen-dependent behavior is assumed.
The Campbell dwarf hamster is a small, polyoestral, seasonally breeding rodent with highly pronounced sexual dimorphism, inhabiting the dry steppes and semi-deserts of Central Asia ([@zow090-B18]; [@zow090-B58]; [@zow090-B17]). Ready to mate females (of different ages) weigh from 22 g to 45 g, and males weigh from 32 g to 60 g. Males have a large specific skin gland in the middle ventral part of the body. They use a secretion of the gland to mark extensive overlapping home ranges. Information about their mating system in nature is quite scarce, so it is safe to speak only about polygamy ([@zow090-B64]; [@zow090-B72]; [@zow090-B61]; [@zow090-B60]). Receptive females attract males from a distance of up to 1 km, and may mate with several males ([@zow090-B72]; [@zow090-B60]). In captivity, adult males act aggressively toward each other from the age of 2 months, and their cohabitation in the same cage often leads to the death of one of them. Success in mating with the female can be related positively to aggressive dominance in male--male conflicts. However, this does not exclude the possibility of selective responses in the female. When a male and female share one cage, they demonstrate pronounced features of social monogamy including male participation in care of pups ([@zow090-B70], [@zow090-B71]; [@zow090-B65]). Fragmental observations in nature also signify to the close contact of a male with the female and juveniles ([@zow090-B72]; [@zow090-B59]; [@zow090-B71]).
We studied experimentally the choice by receptive and sexually motivated (SM) female *Phodopus campbelli* between 2 male full-sibs that differed in the degree of expression of external STs. In addition to morphological characteristics in these males, we estimated levels of testosterone and cortisol in their blood, intensity of specific immune response to antigens, and their aggressive and sexual behavior when males competed for the female. We estimated cortisol concentrations to characterize stress level. Glucocorticoid stress hormones can play a role in sexual selection. In terms of mate choice, individuals can exhibit preferences for mates with either low baseline or peak glucocorticoid levels ([@zow090-B28]). Siblings were used in order to minimize the genetic component of variance for the female choice since it is known that the female may choose a partner according to the difference in MHC genes ([@zow090-B73]; [@zow090-B42]; [@zow090-B36]).
We tested 3 hypotheses in this study: 1) The potential predictors of female mate choice (male STs, intermale aggressiveness and sexual dominance, endocrine and immune characteristics of males) will be correlated with each other. 2) A SM female will choose a partner between 2 male full-sibs divergent in the expression of ST. 3) Mate choice can be explained by a greater expression of ST, an increased level of testosterone, higher behavioral competitiveness, higher (indicator of health), or lower (reciprocal relationship with testosterone) specific immunocompetence, or by a combination of these characteristics.
Materials and Methods
=====================
Males
-----
We obtained 18 litter with 3 or more juvenile males from 57 pairs of hamsters (aged from 6 months to a year) in 2014 and 30 litter from 120 such pairs in 2015. At 25 days we removed juvenile males from parental cages and kept them together by litter (males without females) in Ferplast plastic cages (70 × 40 × 40 cm) for up to 2 months. At 2 months the animals were photographed with a digital camera (Nikon 7000) in fixed positions with their ventrum up against a ruler for measurements of STs on the computer screen ([@zow090-B56]). We fixed animal by hand ventrum up, so that the testes became visible in scrotum. ST measurements included body mass and characteristics of male specific external morphology (both secondary and primary sexual characters): area (length × width) of mid-ventral skin gland, distance between anal and genital openings, and the testes size (average length of testes) in their external outlines from a live animal.
In each litter we chose 2 males with maximal differences in body mass, area of mid-ventral gland, ano-genital distance, and testes size. From 8 to 9 AM we took a blood sample from the sublingual vein (0.3 mL) of each male. The whole procedure of sampling blood lasted no longer than 2 min, which is 2 times less than the time of glucocorticoid signal in the blood in response to handling. Samples were centrifuged at 3,000 rpm for 15 min, and the serum was separated from the hematocrite and frozen at −18°C. Then selected males were placed in individual cages 30 × 22 × 20 cm, where they lived during the experiment ([Figure 1](#zow090-F1){ref-type="fig"}).
{#zow090-F1}
Over 2 years 45 pairs of male sibs were selected for testing. One male died. Thus, for the statistical analysis we used the data set for 88 individuals (44 male pairs).
Females
-------
Young females after removal from the parental cage at the age of 25 days were kept in groups of 7--8 individuals in plastic cages "Ferplast" (70 × 40 × 40 cm). At 3--4 months the majority of sexually mature females remained in diestrus. A week before the experiment we stimulated females by placing an adult male, confined in a small box of metal mesh, in the female's cage for 1 h, which was enough time to stimulate the female's estrous cycle. We tracked the females' cycle daily (1--2 h before testing) by viewing their vaginal smears under a microscope (×20). In the tests we used virgin females 3--4 months old in the phase of transition from proestrus to estrus. Our preliminary observations showed that the female was SM only at this stage of the estrous cycle and actively looked for contact with a male. Although the female remains attractive to male during most of estrus, in the middle and at the end of estrus she opposes his attempts to mate and escapes into an area of the experimental enclosure inaccessible to the male. In Campbell's hamster a vaginal estrus, registered in smears, may not coincide with behavioral receptivity ([@zow090-B16]).
Thirty minutes before the test began the female was paired in a neutral arena with an adult male for 1--2 min to ensure that she was positively motivated. All females we used were not sisters of tested males. During the test, males were able to copulate with a female. In each test (with each pair of males) we used a new female.
Animals housing
---------------
The animals belonged to the laboratory population of hamsters at the A.N. Severtsov Institute of Ecology and Evolution, RAS. This population is descended from hamsters taken in the 1980s from Mongolia (MPR). All hamsters were kept in a room with a constant long-day regime (14 h). Food (formula feed for rats and mice, oat, vegetables, as additives, sunflower seeds, low fat cottage cheese, boiled chicken) and water were *ad libitum*. Thin wood shavings and sawdust were used as bedding, and pieces of cardboard served for shelters. Animal care mandated that we change the bedding once every 2 weeks and check on the reproductive and health state every week.
Test 1: mate choice by the female
---------------------------------
For each pair of male siblings at the age of 3 months ([Figure 1](#zow090-F1){ref-type="fig"}) we selected a SM female (in transition from proestrus to estrus). Before testing we exposed this female to the odor of the litter | {
"pile_set_name": "PubMed Central"
} |
Introduction
============
The prevalence of diabetes mellitus is increasing worldwide with approximately half of all persons with diabetes living in Asia \[[@B1]\]. The herb fenugreek (*Trigonella foenum-graecum* L., Fabaceae family) is used both in cooking and for the treatment of diabetes in many parts of the world, especially in China, Egypt, India and Middle Eastern countries \[[@B2]-[@B4]\]. In low-income countries, individuals with diabetes often do not have access to appropriate medications due to a lack of financial resources \[[@B5]\]. Active compounds of fenugreek included soluble fiber \[[@B6]-[@B8]\], saponins \[[@B9],[@B10]\], trigonelle \[[@B11]\], diosgenin \[[@B12]\], and 4-hydroxyisoleucine \[[@B13],[@B14]\]. Hypoglycemic activities have mainly been attributed to dietary fiber \[[@B6],[@B7]\] and saponin \[[@B9]\]. Fenugreek is a widely used herbal medicine for diabetes, but its efficacy for glycemic control remains unclear.
Animal studies have shown that fenugreek seed extracts have the potential to slow enzymatic digestion of carbohydrates, reduce gastrointestinal absorption of glucose, and thus reduce post-prandial glucose levels \[[@B8]\]. In addition, fenugreek stimulated glucose uptake in peripheral tissues \[[@B15]\] and had insulinotropic properties in isolated rat pancreatic cells \[[@B16]\]. In humans, fenugreek seeds acutely reduced postprandial glucose and insulin levels \[[@B17]-[@B20]\]. In addition, several longer-term clinical trials showed reductions in fasting and post-prandial glucose levels and glycated haemoglobin (HbA1c) \[[@B9],[@B21]-[@B23]\], but some trials did not show benefit \[[@B24],[@B25]\]. Systematic reviews that have evaluated the effect of various alternative therapies for diabetes included only a few clinical trials of fenugreek \[[@B26]-[@B29]\]. We therefore conducted a systematic review and meta-analysis of the effects of fenugreek on glucose homeostasis based on a comprehensive literature search leading to the identification of a reasonably large number of trials with an evaluation of potential explanations for differences in study results.
Methods
=======
Data sources and searches
-------------------------
To identify articles on the effect of fenugreek on glucose homeostasis we searched MEDLINE (PubMed), SCOPUS, Web of Science, BIOSIS, and Cochrane Trials Registry from inception through Nov 29, 2013 using key search terms related to fenugreek ("fenugreek", "trigonella"), an experimental study design ("trial", "clinical trial", "intervention", "therapy"), to identify potentially relevant articles. The search strategy utilized both index terms and free text to search for synonyms of trigonella, fenugreek and diabetes/healthy subjects, and was limited to human studies. Grey literature such as conference proceedings, abstracts, dissertations and technical reports was identified using the same key terms through the electronic search engines Google Scholar, SCIRUS, CINAHL, and ProQuest. No language restriction was applied.
The results (titles, abstracts and citations) of electronic searches were downloaded into EndNote software (EndNote X5, 2011, Thomson Reuters, Philadelphia) and initial screening for eligibility was performed by two independent reviewers (Nithya Neelakantan, Madanagopal Narayanan). When assessment of eligibility based on the title and abstract was insufficient, the full text of the articles was obtained. The second screening of those full text articles was then independently performed by at least two reviewers (Nithya Neelakantan, Madanagopal Narayanan, Rob M van Dam). Disagreements were resolved by consensus. The kappa for the inter-reviewer reliability was 0.78. Study authors were contacted to verify results and methodological quality of retrieved articles where necessary. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement to report our findings \[[@B30]\].
Study selection
---------------
We included clinical trials that compared single herb preparations of fenugreek in any dose or form with a control intervention that was either placebo or no treatment and evaluated effects on markers of glycemia \[fasting blood glucose, 2 hr postload glucose, glycosylated hemoglobin (% HbA1c) and/or fasting serum insulin levels\]. We excluded trials that used combination preparations of fenugreek with other herbs, non-human studies, observational studies, literature reviews/editorials/letters/case reports, and articles not reporting the outcomes of interest. We also excluded trials with interventions that lasted less than 7 days. The number of articles that did not meet the eligibility criteria and the reasons for their exclusion are shown in Figure [1](#F1){ref-type="fig"}.
{#F1}
Data extraction and quality assessment
--------------------------------------
Details of trial design, study setting, population, randomization, blinding, sample size, duration of follow-up, participant characteristics, interventions, total daily dose and outcome characteristics were independently extracted by two reviewers (Nithya Neelakantan, Madanagopal Narayanan), using a standardized data extraction form. Differences in data extraction were resolved by a third reviewer (Rob M van Dam). The quality assessment was conducted using the CONSORT statement for herbal trials \[[@B31]\] by two reviewers (Nithya Neelakantan, Madanagopal Narayanan), with disagreements resolved by consensus.
From each trial, data on mean and SD for all outcomes of interest were extracted. If trials reported fasting blood glucose and 2 hr postload glucose (glucose concentrations 2 hours after the start of the oral glucose tolerance test) in units of mg/dL, this was converted to the standardized international unit \[[@B32]\] of mmol/L by multiplying the glucose values in mg/dL by 0.0555; for fasting serum insulin, we divided the serum insulin values reported in pmol/L by 6.945 and reported the results in mU/L.
Parallel trials generally reported the baseline mean and standard deviation and follow-up mean and standard deviation, but not the standard deviation (SD) of change for the intervention and control groups. For parallel trials, the net changes in each outcome measure were calculated as the change in the intervention group minus the change in the control group. For crossover trials, net changes in the outcome measures were calculated as the value of the outcome measure at the end of the intervention period minus the value of the outcome measure at the end of control period. We estimated the SD of the change on the basis of reported p values for differences in means, if available \[[@B33]\]. We used the p-values cutoff if it was only reported that a p-value was below a threshold (e.g., 0.05 if p \< 0.05 was reported) leading to conservative estimates \[[@B34],[@B35]\]. If p-values were not reported, we imputed SD of the change by using a pooled correlation coefficient between baseline and final measurements from a meta-analysis of correlation coefficients from those trials reporting sufficient data. We derived correlation coefficients for individual trials according a standard formula \[[@B33]\] and we then imputed these correlations into the meta-analysis as transformed z scores (±SEs) to estimate the pooled correlation coefficient \[[@B36]\]. For HbA1c and fasting serum insulin measures, due to small number of trials, we estimated the SDs of the change assuming a conservative 0.5 correlation and performed a sensitivity analysis assuming alternative values of 0.25 or 0.75. To investigate the effect of imputed within-person correlation coefficients, we performed sensitivity analyses with a range of correlation coefficients (0.25, 0.50 and 0.75) \[[@B37]\], the pooled estimates did not change substantially.
Meta-analysis
-------------
The meta-analysis was performed according to the methods described by Curtin et al. \[[@B38]\]. In the combined design meta-analysis, the pooled estimate of treatment effect combining parallel and crossover trial results was the weighted sum of the separate treatment effects estimated, respectively, from parallel and crossover trials divided by the sum of the associated weights. We anticipated large differences in the fenugreek drug preparation format, active components/chemical composition, administration of supplements, and dosages as well as variation in the study population and study design. Therefore, we *a priori* decided to use a random effects model for this meta-analysis. Hence, for each outcome measure, weighted mean differences and corresponding 95% confidence intervals (CI) were calculated by using DerSimonian and Laird random-effects models. We also conducted separate meta-analyses for parallel and crossover trials for the primary outcome measures, fasting blood glucose, and 2 hr post prandial glucose.
Heterogeneity in study results was tested by using the Cochran *Q* statistic (and associated p value), and was quantified by the *I*^2^ statistic. The *I*^*2*^ provides an estimate of the percentage of variation in study results that is explained by between-study heterogeneity rather than sampling error \[[@B39]\]. Potential sources of heterogeneity were investigated using *a priori* defined stratified analyses by study design (parallel or crossover), daily dose of fenugreek extract (\<5 g, 5--10 g or \>10 g), study duration (\<30 days or \> =30 days), randomization (yes or no), blinding (yes or no), baseline BMI (\<25 or \> =25 kg/m^2^), study precision (SE of the effect estimate above or below the median), geographical region (India vs. other countries) and age (above or below the median mean age of all studies | {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec001}
============
Oropharyngeal candidiasis (OPC) occurs in a diverse group of patients. Risk factors for OPC include the use of dentures, corticosteroid inhalers, cigarettes, broad-spectrum antibiotics, and immunosuppressive and chemotherapeutic agents. Patients with HIV, diabetes, and iatrogenic or autoimmune-induced dry mouth are also at substantial risk for OPC. This infection is caused primarily by *Candida albicans*, a ubiquitous polymorphic fungus that is part of the normal microbiota of the gastrointestinal and reproductive tracts of healthy individuals. In order to persistently colonize the oropharynx, *C*. *albicans* must adhere to the epithelial cell lining of the oral mucosa while avoiding being killed by host antimicrobial factors. OPC develops when local host defenses are weakened, permitting the fungus to invade and damage oral epithelial cells. The epithelial cells respond to fungal infection by secreting antimicrobial peptides that directly kill the fungus and by releasing pro-inflammatory cytokines that recruit neutrophils to the focus of infection, where they can kill *C*. *albicans* and limit the extent of epithelial cell damage \[[@ppat.1006056.ref001],[@ppat.1006056.ref002]\]. In this Pearl, we summarize recent advances in our knowledge of the pathogenesis of OPC, focusing on fungal-epithelial interactions.
*C*. *albicans* Invades, Damages, and Stimulates Oral Epithelial Cells {#sec002}
======================================================================
Although OPC is a superficial fungal infection, it is characterized by invasion of the epithelial cell lining of the oropharynx \[[@ppat.1006056.ref003]\]. *C*. *albicans* can invade into oral epithelial cells by two distinct mechanisms, induced endocytosis and active penetration ([Fig 1](#ppat.1006056.g001){ref-type="fig"}). During induced endocytosis, *C*. *albicans* hyphae express the Als3 \[[@ppat.1006056.ref004]\] and Ssa1 \[[@ppat.1006056.ref005]\] invasins, which bind to E-cadherin and a heterodimer composed of the epidermal growth factor receptor (EGFR) and HER2 on the epithelial cell surface \[[@ppat.1006056.ref006]\] ([Fig 1](#ppat.1006056.g001){ref-type="fig"}). These binding events trigger the clathrin-dependent endocytosis machinery and induce epithelial cells to produce pseudopods that engulf the fungus and pull it into the cell. Treatment of mice with a small molecule inhibitor of EGFR/HER2 reduces the severity of OPC, suggesting that induced endocytosis contributes to the pathogenesis of this infection \[[@ppat.1006056.ref006]\]. Additional epithelial cell signaling pathways that are required for maximal endocytosis of *C*. *albicans* include the platelet-derived growth factor BB (PDGF BB) and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9) pathways. Activation of these signaling pathways requires *C*. *albicans* hyphal formation and expression of the Als3 invasin \[[@ppat.1006056.ref007]\]. The relationship among these pathways and those activated by E-cadherin and EGFR/HER2 has not yet been determined.
{#ppat.1006056.g001}
Active penetration occurs when elongating *C*. *albicans* hyphae physically push their way into the epithelial cell ([Fig 1](#ppat.1006056.g001){ref-type="fig"}) \[[@ppat.1006056.ref008]\]. While it is likely that both induced endocytosis and active penetration occur during OPC, it has been difficult to determine the role of active penetration in the pathogenesis of OPC because most invasins are hyphal specific. Thus, *C*. *albicans* mutants that do not form hyphae also fail to express invasins and, consequently, are defective in both active penetration and induced endocytosis.
*C*. *albicans* can also penetrate epithelial cell barriers via a paracellular route by secreting lytic enzymes such as members of the secreted aspartyl proteinase (SAP) family. These proteases degrade E-cadherin and other inter-epithelial cell junctional proteins, enabling the organism to penetrate between epithelial cells \[[@ppat.1006056.ref009]\] ([Fig 1](#ppat.1006056.g001){ref-type="fig"}). Recently, it has been found that *C*. *albicans* infection also stimulates the activity of epithelial cell calpain, a cysteine protease that degrades E-cadherin. Moreover, calpain activity is dramatically enhanced in epithelial cells that are co-infected with *C*. *albicans* and *Streptococcus oralis* \[[@ppat.1006056.ref010]\].
When *C*. *albicans* invades epithelial cells, it damages them. Epithelial cells in turn respond to *C*. *albicans* by secreting proinflammatory cytokines and antimicrobial peptides. Recently, it was discovered that *C*. *albicans*-induced epithelial cell damage is mainly caused by candidalysin, a toxin released by the fungus ([Fig 1](#ppat.1006056.g001){ref-type="fig"}). Candidalysin is encoded by the hyphal-specific gene *ECE1*. The protein is cleaved by the Kex2 protease into eight peptides, of which only one permeabilizes epithelial cell membranes, induces epithelial cell damage, and stimulates epithelial cell cytokine secretion. A *C*. *albicans ece1*Δ/Δ deletion mutant is defective in damaging and stimulating oral epithelial cells in vitro. Furthermore, it has a greatly attenuated virulence in the mouse model of OPC \[[@ppat.1006056.ref011]\]. Thus, candidalysin and epithelial cell damage play a central role in the pathogenesis of OPC.
Although the presence of candidalysin is required to induce a maximal epithelial cell response to *C*. *albicans*, binding of yeast, yeast lysates, and Als3 to epithelial cells is sufficient to induce some epithelial cell responses even in the absence of this toxin. For example, contact with *C*. *albicans* yeast cells, which do not secrete candidalysin, stimulates phosphorylation of the Akt serine/threonine kinase within 5 minutes \[[@ppat.1006056.ref012]\]. In addition, lysates of yeast cells induce secretion of IL-8, possibly by interacting with intercellular adhesion molecule-1 (ICAM-1) \[[@ppat.1006056.ref013]\]. Finally, *Saccharomyces cerevisiae* cells expressing the Als3 invasin stimulate the phosphorylation of EGFR and HER2 within 20 minutes \[[@ppat.1006056.ref006]\]. These data suggest that oral epithelial cells sense and respond to contact with *C*. *albicans*, and that this initial response is subsequently amplified by candidalysin. While EGFR/HER2 and E-cadherin interact with *C*. *albicans* hyphae, the epithelial cell receptor(s) that is activated by yeast-phase *C*. *albicans* is currently unknown.
Interplay between Epithelial Cell-Derived Antimicrobial Peptides and *C*. *albicans* {#sec003}
====================================================================================
Host defense peptides (HDPs) represent one of the first lines of defense against invading microbes. Epithelial cells release multiple types of HDPs, including β-defensins and cathelicidins, that either kill or inhibit the growth of *C*. *albicans*. Different HDPs have different mechanisms of action, and their targets include the fungal cell membrane and mitochondria \[[@ppat.1006056.ref014]\]. HDPs that are highly expressed during the early stage of OPC in mice include murine β-defensin 1 (mBD1), β-defensin 3 (the homolog of human β-defensin 2), and the alarmins | {
"pile_set_name": "PubMed Central"
} |
{#sp1 .26}
{#sp2 .27}
{#sp3 .28}
| {
"pile_set_name": "PubMed Central"
} |
Key question {#Sec1}
============
Does hypo-attenuated leaflet thrombosis occur in bioprosthetic mitral valve?
Key finding {#Sec2}
===========
Hypo-attenuated leaflet thrombosis was observed in patients who underwent bioprosthetic mitral valve replacement.
Take-home message {#Sec3}
=================
Although rare, hypo-attenuated leaflet thrombosis of mitral valve occurs and must be further characterized.
Introduction {#Sec4}
============
A series of reduced aortic-valve leaflet motion and a radiographic finding of hypoattenuating opacities in bioprosthetic aortic valve was first described in 2015 \[[@CR1]\], bringing attention to this previously unrecognized entity. Using 4D volume-rendered computed tomography (CT) scans, this was identified in 40% of the evaluated cohort, occurring in both transcatheter and surgically implanted aortic valve prostheses. Recent analysis of two large registries reported 13% prevalence of this entity, and reported a higher prevalence in valves implanted via transcatheter aortic valve replacement (TAVR) compared to those implanted via surgical aortic valve replacement (SAVR) \[[@CR2]\]. Importantly, its previous perception as a benign finding was questioned by the demonstration of increased incidences of cerebrovascular events associated with subclinical leaflet thrombosis in this series \[[@CR2]\].
Hypo-attenuated leaflet thickening (HALT) of bioprosthetic valve is a hallmark finding of subclinical leaflet thrombosis \[[@CR3]\]. Its characteristics have been described fairly extensively in aortic valves, but its occurrence and characteristics are unknown in bioprosthetic mitral valves (bMV). Mitral valve prosthesis may be at higher risk of leaflet thrombosis for its exposure to lower flow compared to that of prostheses in the aortic position. In addition, possible impact of anticoagulation strategy on HALT would be of interest in the context of current variability in anticoagulation following mitral valve replacement \[[@CR4]\] despite the guideline recommendation \[[@CR5]\]. This study describes the characteristics and prevalence of HALT in surgically-implanted bMV in a single-center series.
Materials and methods {#Sec5}
=====================
Patient selection {#Sec6}
-----------------
A cross-sectional study of 175 consecutive patients who underwent mitral valve replacements with bMV at Toyohashi Heart Center, Japan, between 2007 and 2017 was conducted. Fifty-six patients died during follow-up. Thirty-four patients were excluded for history of chronic kidney disease, due to an elevated risk of further renal injury from contrasted CT scan. Eighty-five surviving patients without chronic kidney disease were contacted between June and October 2017 to undergo contrasted multi-dimensional CT scan, of whom 53 agreed to undergo the scan between June and October 2017 to evaluate the present of HALT in bMV (Additional file [1](#MOESM1){ref-type="media"}: Figure S1). Toyohashi Heart Center Institutional Review Board approved this study, and individual patient consent was obtained at the time of CT scanning. Anticoagulant and antiplatelet therapy use were ascertained at the time of hospital discharge from index hospitalization and at the time of CT scanning. At our center, patients undergoing MVR with bMV are discharged on warfarin and 81 mg aspirin unless contraindicated, and anticoagulation is continued for up to 3 months, after which anticoagulation is discontinued unless there are other indications to continue.
CT scanning and HALT definition {#Sec7}
-------------------------------
All patients underwent cardiac-gated contrasted CT scan using a 256-slice CT scanner (Brilliance iCT; Philips Medical Systems, Eindhoven, the Netherlands) at the Toyohashi Heart Center. Cardiac gating was performed with prospective electrocardiogram triggering (75% of R-R interval) to obtain a slice thickness of 2.5 mm. The scan was performed between the tracheal bifurcation and the diaphragm with the following parameters: collimation width, 32 × 0.625 mm; rotation time, 330 ms/revolution; tube voltage, 120 kV; and maximum effective tube current, 412 mA. Image reconstruction was gated prospectively to 30--45% of R-R interval. CT images were reconstructed across the entire cardiac cycle using a cardiac standard filter with a slice thickness of 2.5 mm. CT datasets were transferred to an offline workstation (Intelli Space Portal; Philips Medical Systems) for image analysis. The images were evaluated by an attending imaging cardiologist (T.S) and an attending cardiac surgeon and consensus was obtained in all findings of HALT.
HALT was defined as the presence of a low-density area on bMV during systole on image with the most closure of the valve, which parallels the definition of HALT in aortic valve bioprosthesis \[[@CR3]\]. Transthoracic echocardiogram was obtained at the time of CT scan. Data were collected on patient demographics, comorbidity, presenting symptoms, operative details, postoperative survival and stroke, echocardiographic and CT scan findings, and use of anticoagulant or antiplatelet therapy at the time of index hospital discharge and at the time of CT scans. CT characteristics and echocardiographic findings including ejection fraction (EF), mitral regurgitation (MR), mean mitral pressure gradient (PG), and mitral valve area (MVA) are reported. The prevalence of HALT was calculated by the number of patients with HALT divided by the 53 patients who underwent CT scan as a denominator. All analysis was conducted using SAS version 9.4 (SAS Institute, Cary NC).
Results {#Sec8}
=======
Of the 53 patients who underwent CT evaluation, 3 patients (5.7%) were found to have a HALT on bMV, each affecting different leaflets. The mean time from index MVR to CT scan was 3.4 ± 0.8 years in HALT cohort and 3.4 ± 2.7 years in non-HALT cohort. Table [1](#Tab1){ref-type="table"} summarizes patient characteristics of the entire cohort. Oral anticoagulant and antiplatelet therapy use at the time of hospital discharge and at the time of CT scan are summarized in Table [2](#Tab2){ref-type="table"}. Among the entire cohort of 53 patients, 74% were discharged on the dual therapy of single antiplatelet agent (SAPT) and warfarin, and additional 21% were on warfarin only. At the time of CT scan, 43% were on warfarin only, and 26% were on the dual therapy of SAPT and warfarin. One patient who was found to have HALT was on warfarin with the INR in the therapeutic range at the time of CT scan. Table 1Baseline characteristics of patients with CT scanVariables***N*** = 53Age (years)71.6 ± 8.9Male26 (49.0)Diabetes12 (22.6)COPD0 (0.0)Creatinine (mg/dL)1.02 ± 0.95Chronic kidney disease0 (0.0)NYHA functional class I or II42 (79.2)NYHA functional class III or IV11 (20.8)Previous PMI0 (0.0)Previous CABG1 (1.9)Previous valve surgery4 (7.5)Operative variables Emergency3 (5.7) Urgent0 (0.0) Elective50 (94.3)Concomitant operations MAZE34 (64.2) CABG9 (17.0) AVR14 (26.4) TAP29 (54.7)Cross-clamp time (minutes)105.7 ± 31.2Perfusion time (minutes)154.4 ± 37.5Operation time (minutes)271.5 ± 54.0Valve size (mm) 2515 (28.3) 2727 (50.9) 2910 (18.9) 311 (1.9)Valve type Carpentier-Edwards Pericardial Bioprosthesis12 (22.6) Magna Ease16 (30.2) Mosaic15 (28.3) St. Jude Medical Epic10 (18.9) Stroke within 30 days of operation2 (3.8)Data are displayed as mean ± SD or n (%). *AVR* Aortic valve replacement, *CABG* Coronary artery bypass grafting, *COPD* Chronic obstructive pulmonary disease, *NYHA* New York Heart Association, *PMI* Pacemaker implantation, *TAP* Tricuspid annuloplastyTable 2Oral anticoagulant and antiplatelet therapy use at the time of hospital discharge and at the time of CT scanMedicationsAt discharge (N = 53)At CT (N = 53)SAPT3 (5.7)13 (24.5)DAPT0 (0.0)2 (3.8)SAPT + warfarin39 (73.6)14 (26.4)SAPT + DOAC0 (0.0)0 (0.0)Warfarin11 (20.8)23 (43.4)DOAC0 (0.0)1 (1.9)Unknown0 (0.0)0 (0.0)Data are displayed as n (%). *CT* Computed tomography, *SAPT* Single antiplatelet therapy, *DAPT* Dual antiplatelet therapy, *DOAC* Direct oral anticoagulant
All three | {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Electrophysiology in audiology is an objective tool to check the integrity of the auditory system. Auditory evoked potentials are electrophysiological tests, which give information about a number of events happening in the peripheral and central nervous systems that are generally related to the sensory pathway ([@bib53], [@bib9]). These sound-related evoked potentials are categorized as endogenous and exogenous potentials ([@bib21]). The exogenous potentials are mainly recorded by external event related dimensions of the stimulus. The endogenous potentials are responses which are due to internal events such as perception and cognition ([@bib47], [@bib40]). Studies have considered the possibility of studying auditory discrimination using a technique referred to as event-related potentials ([@bib6]; [@bib7]). Mismatch negativity (MMN) is an event-related potential that has been extensively studied by researchers to assess the pre-attentive auditory discrimination capability and storage of regularities in features of stimulus ([@bib41]).
Pre-attentive processing is the unintentional gathering of information from the environment. First, all gathered information is pre-attentively processed. Then, our brain sieves and processes the prime information. The important information is selected for further analysis by attentive processing ([@bib1]). Our auditory system has an imperative role in gathering sound information for pre-attentive processing. At the point where auditory stimulus or sound waves hits the tympanic membrane, it transmits the message to the auditory cortex by means of auditory nerve for pre-attentive processing. The proficiency to appropriately filter information from pre-attentive auditory processing to attentive auditory processing is crucial for normal development of speech perception ([@bib52]). According to [@bib19], pre-attentive process uses the Gesalt laws of organization which says temporal proximity, physical similarity and good continuity is required to group the sound, which improves speech perception in quiet as well as in noise. For acoustic pre-attentive auditory processing, the temporal cortex is the primary site of activation, but research additionally demonstrated the association of frontal cortex as well ([@bib11], [@bib17]). Studies also suggest that perception of minute variation in complex musical patterns triggers the right ventromedial prefrontal cortex ([@bib11]).
[@bib36] showed MMN as an endogenous potential with a negative component elicited by any discriminable change in regular auditory stimuli. MMN is usually obtained by presenting a train of repetitive homogenous tones at a rate of approximately one tone per second. It is occasionally interspersed with a tone that differs physically ([@bib10]). [@bib35] described MMN as "an electric brain response, a negative component of the event-related potential (ERP), elicited by any discriminable change (deviant) in some repetitive aspect of auditory stimulation (standard), usually peaking at around 100--200 ms from onset". MMN seems to depict a neuronal representation of the difference perceived between the auditory stimuli. Thus, MMN is well advised as an objective tool to check auditory discrimination skills at pre-attentive level. In which case, it could also be of clinical importance as speech perception, by its nature, depends on neuronal responses to changes in stimulus ([@bib20]). Music demands cognition, which requires specific and appropriate timing of many actions, such as perceiving the exact interval and control of pitch which are otherwise not involved in language. Enhanced auditory perception in musicians is likely to result from auditory perceptual learning during years of training, practice and experience. The musician\'s brain is presumed to be a good and appropriate model to investigate neuroplastic changes ([@bib33]). Professional musicians have fine-tuned auditory skills which are achieved by aural training that they receive during their musical training. It is considered as an important component of their vocational formation ([@bib13]). A study done by [@bib54] assessed pre-attentive auditory discrimination skills in amateur musicians and non-musicians. They reported significantly larger MMN in amateur musicians compared to non-musicians. Another study by [@bib3] reporting a strong advantage for musicians in accompanying behavioral task of detecting the deviants while attending to the stimuli for all pattern lengths showed that long-term musical training differentially affects the memory capacity. [@bib28] investigated MMN in non-musicians native speaker of a quality language, Finnish, in which duration is a phonemically contrastive cue, with French musicians compared to French non-musicians. They reported that pre-attentive and attentive duration processing of duration deviants was enhanced in Finn non-musicians and French musicians compared to French non-musicians. They also observed that MMN in French musicians was significantly larger compared to Finns and French non-musicians. Along a similar line, [@bib22] investigated neuronal representation of vowels and temporally manipulated CV syllables among string players and non-musicians with MMN odd ball paradigm. They showed that musicians are not only advantaged in the pre-attentive encoding of temporal cues but also in processing vowels. Previous literature from western countries have investigated MMN in western classical musicians and reported an enhanced pre-attentive auditory discrimination skill in musicians ([@bib54], [@bib3], [@bib28], [@bib46], [@bib22]). There is some basic mechanistic difference between Western and Indian classical music in terms of pitch structure and temporal patterning. Some basic elements of Indian music i.e. taala (rhythmic pattern), shruti (relative musical pitch), raaga (melody) and swara (the musical sound of a single note) are rarely found in western classical music. These features are difficult to perceive for western listeners without special training. In the case of vocal singers, control of pitch is important and is done by biomechanical and aerodynamic systems. Investigators agree that the ability to produce a precise pitch is very important for the professional vocal musician. Literature shows that accurate pitch control mainly depends on auditory perceptual monitoring, proprioceptive feedback of the laryngeal system and phonatory reflex systems ([@bib16], [@bib34]). The Recent literature reported enhanced auditory skills through different behavioral tests in Indian classical musicians ([@bib48], [@bib31], [@bib32], [@bib24], [@bib49], [@bib50]). It is interesting to know the effect of Indian classical vocal music training and practice on pre-attentive auditory discrimination skills in musicians through an electrophysiological test like MMN. There is a lack of literature regarding pre-attentive auditory discrimination skills in Indian classical vocal musicians. Hence, there is a need to compare pre-attentive auditory discrimination skills in Indian classical vocal musicians with non-musicians.
2. Materials and methods {#sec2}
========================
2.1. Participants {#sec2.1}
-----------------
Two groups of participants (the experimental and control group) were involved in the study. The experimental group consisted of 25 female right handed Indian classical vocal musicians with a mean age of 24.52 ± 2.6 years (age range 18--30 years). According to the inclusion criteria, only those with a minimum of 10 years of experience were taken. The participants of the experimental group in this study had an average experience of 12.3 years in Indian classical vocal music. All of them had started musical training after the age of 8 years. They practiced music for 19.2 ± 9.3 h per week regularly. As the control group, 25 female age-matched right-handed participants (age range = 18--30 years, mean = 24.8 ± 2.2 years) were included. None of them had any kind of formal training in music. The reason of taking only female participants was availability of more female participants in experimental as well as control group.
2.2. Participant selection criteria {#sec2.2}
-----------------------------------
The participants selected for the study had hearing threshold within the normal limit as defined by AC and BC thresholds less that 15dBHL from 250 Hz to 8000 Hz and from 250 Hz to 4000 Hz respectively. They also had normal middle ear function as revealed by tympanometry and reflexometry. Otological problems were ruled out by otological evaluation with the help of a qualified otolaryngologist. Auditory Brainstem Responses for site of lesion were recorded to rule out any neurological problem in the subjects. An informed written consent was taken from all participants before involving them in the study.
2.3. Testing environment {#sec2.3}
------------------------
All behavioral as well as electrophysiological tests were carried out in a sound-treated room. The permissible noise levels were as per the guidelines in ANSI S3.1 (1999). Laboratory room was well lit and air-conditioned for the tranquility of the investigators as well as the subjects.
2.4. Instrumentation {#sec2.4}
--------------------
For pure tone audiometry, a calibrated dual channel clinical diagnostic audiometer (Orbitor-922) was used for all participants. For tympanometry and reflexometry, a calibrated GSI-Tympstar Immittance meter was used for all participants. Mismatch Negativity was recorded on all participants using Intelligent Hearing System with smart EP.
2.5. Procedure {#sec2.5}
--------------
The Modified version of Hughson and Westlake\'s procedure given by [@bib5] was used for pure-tone audiometry across octave frequencies from 250 Hz to 8000 Hz for air conduction. Octave frequencies from 500 to 4000 Hz were tested for bone conduction. To carry out tympanometry, a 226 Hz probe tone was used, whereas 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz stimuli were used for ipsilateral and contralateral reflex.
Previous literature reported difficulty in identifying MMN at an individual level ([@bib | {
"pile_set_name": "PubMed Central"
} |
In the wild, feral cats typically eat small mammals, reptiles, birds and insects. It is often not possible to mimic natural feeding behaviours of feral cats. Extruded diets have been the traditional alternative fed to domestic cats. Commercial chicken-based extruded diets (EXT) have complex diet formulations, including protein, fat, carbohydrate, fibre, vitamin and mineral ingredients. Association of American Feed Control Officials (AAFCO)^(^[@ref1]^)^ recommend minimum concentrations of 26 % crude protein (DM basis) and 9 % fat DM basis. If these minimum concentrations are targeted by formulators, commercial feline EXT may contain up to 50--55 % carbohydrates. Plantinga *et al.*^(^[@ref2]^)^ estimated the diet of feral cats expressed on a DM basis would contain 63 % crude protein (CP), 23 % fat and 2·8 % nitrogen-free extract (i.e. digestible carbohydrate).
Given their carnivorous nature, it has been hypothesised that the protein:carbohydrate ratio of feline diets is important for feline health (i.e. obesity, feline diabetes and gut microbiota)^(^[@ref3]^--^[@ref6]^)^. As lower bacterial diversity and great shifts in commensal bacteria are often present in inflammatory bowel diseases, it suggests that a balanced gut microbiota is important for maintaining host health^(^[@ref7]^--^[@ref9]^)^. Several studies have examined the impact of dietary alterations on faecal microbial populations in cats^(^[@ref3]^--^[@ref5]^,^[@ref10]^--^[@ref13]^)^; however, very few have examined the microbial population of cats fed a 'wild-type' diet^(^[@ref12]^,^[@ref13]^)^. Commercially available whole prey may be more similar to the feral cat diet. For example, commercially available 1--3-d-old chicks (CHI) are approximately 72--76 % CP, 16--20 % fat and \<5 % nitrogen-free extract^(^[@ref14]^,^[@ref15]^)^. Previous studies have shown that extruded and whole-prey diets differ in digestibility as well as macronutrient composition^(^[@ref14]^--^[@ref17]^)^, and this may alter the fermentable substrates that are available to the gastrointestinal microbiota for fermentation^(^[@ref18]^,^[@ref19]^)^. The objective of the present study was to compare the faecal microbiota of cats fed an EXT chicken-based diet to those fed commercially available whole CHI.
Experimental methods {#sec1}
====================
Study design {#sec1-1}
------------
The animal protocol was approved by the University of Illinois Animal Care and Use Committee. Faecal samples were collected from neutered male domestic cats (mean age = 5·7 years; body condition score 4·5--5·5 of 9). A completely randomised design was utilised to test the impacts of two dietary treatments ([Table 1](#tab01){ref-type="table"}): (1) EXT (*n* 3 cats; P & G Petcare); and (2) raw CHI (*n* 5 cats; Rodent Pro). The raw chicks were frozen (−20°C) upon arrival, and thawed in the refrigerator for 24 h prior to feeding. Fresh water was available *ad libitum*. A computer was used to randomly allot cats to treatment. Cats were adapted to diets for 10 d, prior to fresh faecal collection (\<15 min from defection). Faecal samples were stored at −80°C until DNA extraction. Table 1.Chemical composition of CHI and an EXT fed to domestic cats[\*](#tfn1_1){ref-type="table-fn"}ItemCHIEXT†DM (%)24·295·4Organic matter (% DM)91·192·8Crude protein (% DM)71·438·9Acid-hydrolysed fat (% DM)20·014·4Gross energy (kcal/g DM)5·95·2[^1][^2]
Sample analysis {#sec1-2}
---------------
Faecal bacterial DNA was isolated according to procedures described previously^(^[@ref20]^)^ using the MO BIO PowerSoil™ Kit (MO BIO Laboratories). Amplification of a 600 bp sequence of the V4--V6 variable regions of the 16S rRNA gene was done using barcoded primers as previously described^(^[@ref21]^)^. PCR amplicons were further purified utilising AMPure XP beads (Beckman-Coulter Inc.). Amplicons were combined in equimolar ratios to create a DNA pool that was used for pyrosequencing. DNA quality of amplicon pools was assessed before pyrosequencing using a 2100 Bioanalyzer (Agilent Technologies). Pyrosequencing was performed at the W. M. Keck Center for Biotechnology at the University of Illinois utilising a 454 Genome Sequencer and FLX titanium reagents (Roche Applied Science).
Data analysis {#sec1-3}
-------------
High-quality (quality value \>25) sequence data derived from the sequencing process was processed using a proprietary analysis pipeline ([www.mrdnalab.com](www.mrdnalab.com)) and as described previously^(^[@ref22]^)^.
Statistical analysis {#sec1-4}
--------------------
Sequence percentages at each taxonomic level were analysed using the Mixed models procedure of SAS (version 9.3; SAS Institute). The fixed effect of diet was tested. Means were separated for treatments using a Fisher-protected least significant difference with Tukey\'s adjustment. Results are reported as least-squares means with *P* ≤ 0·05 defined as significant and *P* ≤ 0·10 as trends for treatment effects.
Results {#sec2}
=======
Regardless of dietary treatment, Firmicutes (62--88 % of all sequences) was the predominant bacterial phylum in cat faeces (data not shown). Fusobacteria (0·2--17 % of all sequences), Proteobacteria (2--16 % of all sequences), Actinobaceria (1·4--18 % of all sequences), Tenericutes (1·4--9 % of all sequences) and Bacteroidetes (0--3 % of all sequences) also were predominant phyla present (data not shown). The proportion of Bacteroidetes was greater (*P* = 0·03) in faeces of cats fed EXT (1·6 % of all sequences) than those fed CHI (0·2 % of all sequences; data not shown).
Proportions of genera, however, depended on dietary treatment ([Table 2](#tab02){ref-type="table"}). The predominant genera in faeces of cats fed CHI were *Clostridium* (11--25 % of sequences), *Blautia* (4--19 % of sequences), unidentified Lachnospiraceae (14--16 % of sequences), *Peptococcus* (7--13 % of sequences), *Fusobacterium* (4--13 % of sequences), *Ruminococcus* (2--9 % of sequences) and *Collinsella* (2--8 % of sequences). The predominant genera in faeces of cats fed EXT were *Megamonas* (2--28 % of sequences), *Megasphaera* (0·01--26 % of sequences), *Blautia* (10--16 % of sequences), *Collinsella* (1--16 % of sequences), *Lactobacillus* (0·2--14 % of sequences), *Clostridium* (8--12 % of sequences) and unidentified Lachnospiraceae (4--7 % of sequences). Table 2.Predominant bacterial genera (expressed as percentage of sequences) in faeces of domestic cats fed CHI (*n* 5) or EXT (*n* 3)[\*](#tfn2_1){ref-type="table-fn"}PhylumFamilyGenusCHIEXT[sem]{.smallcaps}*P* valueActinobacteriaBifidobacteriaceae*Bifidobacterium*ND†0·3----Bifidobacteriaceae*Collinsella*4·96·917·00·94Coriobacteriaceae*Slackia*0·1\<0·1\<0·10·06Unidentified genera0·10·50·10·08FirmicutesAcidaminococcaeae*Phascolarctobacterium*0·61·30·20·09Clostridiaceae*Clostridium*16·310·52·30·12Enterococcaeae*Enterococcus*1·00·30·30·13Erysipelotrichaceae*Allobaculum*0·80·10·20·06Unidentified genera0·7\<0·10·30·22Eubacteriaceae*Eubacterium*5·82·51·10·09Lachnospiraceae*Blautia*9·712·32·70·51*Coprococcus*2·30·61·00·30*Psuedobutyrivibrio*4·01·00·80·04*Roseburia*1·20·30·50·25Unidentified genera15·25·30·5\<0·01Lactobacilliaceae*Lactobacillus*ND7·6----Oscillospiraceae*Oscillib | {
"pile_set_name": "PubMed Central"
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{
"pile_set_name": "PubMed Central"
} | |
INTRODUCTION {#s1}
============
Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all cases \[[@R1]\]. Most NSCLC patients are diagnosed at an advanced stage and have a 5-year survival rate of less than 20% \[[@R1], [@R2]\] because of their advanced stage diagnoses \[[@R3]\]. Lack of early diagnosis markers and high potential for the invasion ability of NSCLC are challenging for NSCLC therapy. Hence, the molecular mechanisms involved in the development and progression of NSCLC must be investigated.
Long noncoding RNAs (lncRNAs), defined as a class of noncoding RNA with a length of more than 200 nucleotides, have critical roles in the gene expression regulation \[[@R4]\], epigenetic control \[[@R5]\], chromatin structure \[[@R6], [@R7]\], development process, genomic imprinting, and pluripotency of embryonic stem cells \[[@R6], [@R8], [@R9]\]. In addition, dysregulation of lncRNAs has been reported to play a vital role in the carcinogenesis, disease progression, and metastasis of human cancers \[[@R6], [@R7], [@R10]--[@R12]\]. Some lncRNAs such as H19, HOTAIR, ANRIL, MALAT1, and SCAL1 \[[@R13]--[@R15]\] have been reported to be associated with the development and progression of lung cancers. However, the roles of lncRNAs in NSCLC development and metastasis remain largely unknown. Hence, the identification of lung cancer-associated lncRNAs and the investigation of their molecular and biological functions in lung cancers are vital.
Myocardial infarction-associated transcript (MIAT) is one of the noncoding RNAs first identified as an lncRNA in 2006 \[[@R16]\]. MIAT is involved in various cellular processes, including myocardial infarction \[[@R16], [@R17]\], microvascular dysfunction \[[@R18]\], paranoid schizophrenia \[[@R19]\], nuclear body formation \[[@R20]\], and neurogenic commitment \[[@R21]\]. Because MIAT physically interacts with SF1 splicing factor, it is supposed to be involved in RNA splicing and regulating gene expression \[[@R22]\]. Recent studies have demonstrated that MIAT constitutes a loop with Oct4 in malignant mature B cells and is essential for cell survival \[[@R23]\]. MIAT is also upregulated and interacts with the polycomb in neuroendocrine prostate cancer to participate in tumorigenesis \[[@R24]\]. However, the expression pattern, biological function, and underlying mechanism of MIAT in NSCLC are still unclear.
In the present study, we investigated the potential mechanisms of MIAT in NSCLC progression. We observed that MIAT was upregulated and played a role in the advanced pathological stage. Moreover, our data revealed that MIAT could interact with MLL and epigenetically activate MMP9 to facilitate cell proliferation, migration, and invasion in NSCLC.
RESULTS {#s2}
=======
MIAT expression was upregulated and correlated with advanced tumor stage {#s2_1}
------------------------------------------------------------------------
To explore whether MIAT played a role in carcinogenesis, we first profiled the expression of MIAT in 60 pairs of NSCLC tissues (30 paired of adenocarcinoma and 30 paired of squamous) and paired adjacent non-tumor tissues. The qPCR data indicated that the expression level of MIAT in tumor tissues was significantly higher than that in the corresponding non-tumor tissues (mean dCT of tumor vs. normal tissue: 2.95 vs. 3.71, *p* = 0.0014; Figure [1A](#F1){ref-type="fig"}). Furthermore, we analysed the association between *MIAT* gene expression and the clinical stage of NSCLC and the state of metastasis. *MIAT* upregulation in the tumor tissues was associated with an advanced stage (stages III, IV, *n* = 24, *p* = 0.001) but not early stage cancer (stages I and II, *n* = 36, *p* = 0.09; Figure [1B](#F1){ref-type="fig"}). Next we tested the MIAT expression in NSCLC cell lines, including A549, H1299, H460, and H520. Among these cell lines, MIAT was relative higher expressed in A549 and H1299 (Figure [1C](#F1){ref-type="fig"}); thus, we chose A549 and H1299 cells to perform the following experiments. Moreover, to investigate the clinical significances of MIAT, we evaluated the correlation between MIAT level and clinicopathological factors. Results revealed that MIAT levels were correlated with tumor size (*p* = 0.0035), TMN stage (*p* = 0.001), and lymph node metastasis (*p* = 0.0185) in NSCLC. Nevertheless, MIAT levels were not associated with age (*p* = 1.000) or gender (*p* = 0.0581) (Table [1](#T1){ref-type="table"}). These results indicated that upregulated expression of MIAT might play a role in NSCLC tumorigenesis.
{#F1}
###### MIAT expression and clinicopathological factors in NSCLC patients (*n* = 60)
Parameter *N* Relative MIAT expression *p*-value
-------------------------------- ----- -------------------------- ----------- --------
Age (year) 1.000
≤ 65 34 20 14
\> 65 26 16 10
Gender
Male 37 26 11 0.0581
Female 23 10 13
Tumor size (maximum diameters)
≤ 3 cm 32 25 7 0.0035
\> 3 cm 28 11 17
Lymph node metastasis
N1 26 11 15 0.0185
N0 34 25 9
TMN stage
I--II 36 28 8 0.0010
III--IV 24 8 16
*P* values when expression levels were compared using Fisher\'s exact test.
Knockdown of MIAT impaired lung cancer cells proliferation and cell cycles arrest *in vitro* {#s2_2}
--------------------------------------------------------------------------------------------
Because the overexpression of MIAT was significantly associated with progression in NSCLC patients, we further modulated MIAT expression to examine whether MIAT regulated the proliferation of A549 and H1299 cells. A cell counting assay revealed that cell growth rate of A549 and H1299 were dose-dependently inhibited with siMIAT compared with the control (Figure [2A](#F2){ref-type="fig"}). Colony formation assay data also revealed that clonogenic survival were inhibited in si-MIAT-treated A549 and H1299 cells (Figure [2B](#F2){ref-type="fig"}). To further examine whether the effect of MIAT on proliferation reflected cell cycle arrest, cell cycle progression was analysed using flow cytometry analysis. The results indicate that MIAT knockdown retarded the G1/S transition in si-MIAT A549 and H1299 cells (Figure [2C](#F2){ref-type="fig"}). We then performed Western blot and found that knockdown of MIAT would decrease the expressions of cyclin D3 and cdk2 in A549 and H1299 cells (Figure [2D](#F2){ref-type="fig"}). These data indicated that MIAT could promote the proliferation phenotype of NSCLC cells.
{#F2}
MIAT silencing impaired cell migration and invasion *in vitro* {#s2_3}
--------------------------------------------------------------
Next, we explored the efficiency of MIAT on migration and invasion in A549 and H1299 cells. The wound healing scratch assay revealed that the ratio of the recovered region were decreased in MIAT knockdown A549 and H1299 cells compared with the control (Figure [3A](#F3){ref-type="fig"}). Furthermore, a matrigel transwell assay demonstrated that decreasing MIAT expression could | {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Brucellosis is the collective name of a group of zoonotic diseases afflicting a wide range of domestic and wild mammals ([@B75]; [@B77]). In domestic livestock brucellosis is manifested mostly as abortions and infertility, and contact with infected animals and consumption of unpasteurized dairy products are the sources of human brucellosis, an incapacitating condition that requires prolonged antibiotic treatment ([@B78]). Eradicated in a handful of countries, brucellosis is endemic or even increasing in many areas of the world ([@B33]; [@B18]; [@B39]).
This disease is caused by facultative intracellular parasites of the genus *Brucella*. Taxonomically placed in the α-2 *Proteobacteria* ([@B51]), the brucellae are close to plant pathogens and endosymbionts such as *Agrobacterium, Sinorhizobium*, and *Rhizobium* and to soil bacteria such as *Ochrobactrum*, the latter including some opportunistic pathogens, and comparative analyses suggest that soil bacteria of this group are endowed with properties that represent a first scaffold on which an intracellular life style develops ([@B70]; [@B50]; [@B5]). The brucellae owe their pathogenicity mainly to their ability to multiply within dendritic cells, macrophages, and a variety of other cells. Due to their ability to control intracellular trafficking and be barely detected by innate immunity, these bacteria are able to reach a safe intracellular niche before an effective immune response is mounted, and to multiply extensively ([@B25]; [@B4]). A mechanism used by *Brucella* to scape from the host immune response is the interference with the toll-like receptor (TLR) signaling pathway by the injection of active effectors such as BtpA and BtpB through the Type IV secretion system T4SS. Both effector proteins contain a TIR domain that interferes with TLR signaling by directly interacting with MyD88 ([@B12]; [@B61], [@B60]; [@B11]) and contribute to the control of dendritic cell (DC) activation during infection. Moreover, *Brucella* has modified outer membrane (OM) components in order to reduce the pathogen-associated molecular patterns (PAMP) of the cell envelope. In Gram-negative bacteria, these PAMP are created by the conserved composition of the OM lipopolysaccharide (LPS) and the free lipids on which the topology of the OM also depends. However, in addition to free-lipid species present in most Gram-negative bacteria (i.e., cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine), *Brucella* also possesses phosphatidylcholine and amino lipids. Phosphatidylcholine is a eukaryotic-type phospholipid required for *Brucella* full virulence ([@B13]; [@B15]). Among the amino lipids, only the ornithine lipids (OL) have been investigated which unlike their counterparts in *Bordetella*, do not trigger the release of IL-6 or TNF-α by macrophages, possibly on account of their longer acyl chains that reduce the OL PAMP ([@B55]). Concerning the LPS, most bacteria carry C1 and C4′ glucosamine disaccharides with C12 and C14 acyl and acyl-oxyacyl chains. This highly amphipathic structure, named lipid A, is adjacent to additional negatively charged groups of the core oligosaccharide, namely the heptose phosphates and 2-keto-3-deoxyoctulosonate carboxyl groups ([@B36]; [@B52]). This lipid A-core PAMP is so efficiently detected by the innate immunity system that some pathogens partially conceal it by removing phosphate groups or substituting them with arabinosamine and/or ethanolamine, or by hydroxylating the acyl chains ([@B67]; [@B43]; [@B48]; [@B54]; [@B44]; [@B69]). In contrast, *Brucella* lipid A is a diaminoglucose disaccharide amide-linked to long (C16, C18) and very long (C28--C30) acyl chains ([@B70]; [@B31]; [@B21]). Furthermore, negative charges in lipid A phosphates and 2-keto-3-deoxyoctulosonate are counterbalanced by four glucosamine units present in the core ([@B38]; [@B21]). As illustrated by the unusually reduced endotoxicity of the *Brucella* LPS this structure is defectively detected by the innate immune response ([@B40]; [@B47]; [@B14]). It remains unknown, however, whether *Brucella* LPS undergoes post-synthetic modifications that have been described for other bacteria that could alter its PAMP potential and contribution to virulence. In this work, we investigated in *Brucella* the role of gene homologs to phosphatases, phospho-ethanolamine (pEtN) transferases, and acyl hydroxylases (**Figure [1](#F1){ref-type="fig"}**) that have been shown in other Gram-negative pathogens to act on LPS and to contribute to overcoming innate immunity defenses.
{#F1}
Materials and Methods {#s1}
=====================
Bacterial Strains and Growth Conditions
---------------------------------------
The bacterial strains and plasmids used in this study are listed in Supplementary Table [S1](#SM1){ref-type="supplementary-material"}. Bacteria were routinely grown in standard tryptic soy broth or agar either plain or supplemented with kanamycin at 50 μg/ml, or/and nalidixic at 5 or 25 μg/ml or/and 5% sucrose. All strains were stored in skim milk at -80°C.
DNA Manipulations
-----------------
Genomic sequences were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database^[1](#fn01){ref-type="fn"}^. Searches for DNA and protein homologies were carried out using the National Center for Biotechnology Information (NCBI^[2](#fn02){ref-type="fn"}^) and the European Molecular Biology Laboratory (EMBL) -- European Bioinformatics Institute server^[3](#fn03){ref-type="fn"}^. Primers were synthesized by Sigma-Genosys (Haverhill, United Kingdom). DNA sequencing was performed by the "Servicio de Secuenciación del Centro de Investigación Médica Aplicada" (Pamplona, Spain). Restriction--modification enzymes were used under the conditions recommended by the manufacturer. Plasmid and chromosomal DNA were extracted with Qiaprep Spin Miniprep (Qiagen) and Ultraclean Microbial DNA Isolation Kits (Mo Bio Laboratories), respectively. When needed, DNA was purified from agarose gels using the Qiack Gel Extraction Kit (Qiagen).
Mutagenesis
-----------
To obtain *BmeΔlptA, BaΔlpxE*, and *BmiΔolsC* in-frame deletion mutants, directed mutagenesis by overlapping PCR were performed using genomic DNA as template and pJQK ([@B63]) as the suicide vector. The corresponding gene was deleted using allelic exchange by double recombination as previously described ([@B15]).
For the construction of the *BmeΔlptA* mutant, we first generated two PCR fragments: oligonucleotides *lptA-*F1 (5′-GAACGCGAGACTATGGAAAC-3′) and *lptA-*R2 (5′-TGGTGAACGCCAGAAGATAGA-3′) were used to amplify a 400-bp fragment including codons 1--26 of *BmelptA* ORF, as well as 324 bp upstream of the *BmelptA* start codon, and oligonucleotides *lptA-*F3 (5′-TCTATCTTCTGGCGTTCACCGCACGACAATCTCTTC-3′) and *lptA*-R4 (5′-AATATTCCATGGCGCATTTC-3′) were used to amplify a 472-bp fragment including codons 506--544 of the *lptA* ORF and 353-bp downstream of the *lptA* stop codon. Both fragments were ligated by overlapping PCR using oligonucleotides *lptA*-F1 and *lptA*-R4 for amplification, and the complementary regions between *lptA*-R2 and *lptA*-F3 for overlapping. The resulting fragment, containing the *lptA* deleted allele, was cloned into pCR2.1 (Invitrogen, Barcelona, Spain), sequenced to ensure maintenance of the reading frame, and subcloned into the *Bam*HI and the *Xba*I sites of the suicide plasmid pJQK. The resulting mutator plasmid (pRCI-32) was introduced in *B. melitensis 16M* by conjugation using the *Escherchia coli* S.17 strain ([@B64]).
| {
"pile_set_name": "PubMed Central"
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Ischemia-reperfusion injury represents a pathological condition characterized by an initial undersupply of blood to an area or organ followed by a restoration of perfusion and concomitant reoxygenation (= reperfusion). Ischemia typically occurs in the presence of embolism or thrombosis but can also be triggered by surgery and transplantation. Anyway, the disturbance in perfusion results in a severe imbalance between metabolic supply and demand, subsequently causing tissue hypoxia \[[@B1]\]. Notably, these initial changes cause time-dependent molecular and structural alterations. In this context, it is also important to consider that all tissues and organs are susceptible to ischemia, but susceptibility to an ischemic insult differs between organ systems. Whereas the brain can endure ischemia only a few minutes, other tissues (e.g., muscle) are able to withstand ischemia for a long time without signs of irreversible damage.
Interestingly, restoration of blood flow and reoxygenation is commonly associated with an exacerbation of tissue injury and a profound inflammatory response ("reperfusion injury") \[[@B1], [@B2]\]. Ischemia-reperfusion injury contributes to pathology in a wide range of conditions.
For example, myocardial ischemia followed by reperfusion typically manifests in microvascular dysfunction, death of myocytes, and myocardial stunning or dysfunction.
Ischemia-reperfusion injury (IRI) of the lung, for example, following transplantation, is characterized by nonspecific alveolar damage, edema formation, and hypoxemia. The clinical spectrum of pulmonary IRI may range from mild hypoxemia to acute respiratory distress syndrome.
In contrast to other organs, the brain is particularly susceptible to ischemia and irreversible neuronal damage already occurs after only 5 minutes of complete ischemia \[[@B3]\]. For brain ischemia, as occurring in the setting of stroke, reestablishing reperfusion seems to be only beneficial, if carried out within a short time period after the onset of ischemia. Reperfusion of ischemic stroke seems to be very critical, as patients may suffer from cerebral reperfusion injury manifesting in fatal cerebral edema formation and intracranial hemorrhage.
IRI of the kidney may occur in the setting of transplantation and cardiac arrest and during cardiac surgery. Here it is important to note that renal injury is usually associated with a high morbidity and mortality. The cortical-medullary region is the most susceptible region to tubular injury, inflammation, and vascular alterations.
Generally, IRI of a single organ causes the release of different proinflammatory mediators, which may subsequently induce inflammation in other organs, thereby potentially contributing to multiple organ dysfunction or even failure \[[@B4]\].
Different pathological processes contribute to tissue injury secondary to ischemia-reperfusion. During ischemia, limited oxygen availability leads to an impaired endothelial cell barrier function with a concomitant increase in vascular permeability and leakage due to decreases of intracellular cAMP levels caused by a reduced adenylate cyclase activity \[[@B1]\]. Furthermore, ischemia-reperfusion induces cell death due to apoptosis, necrosis, and autophagy \[[@B5]\]. During the ischemic period, alterations in the transcriptional control of gene expression likewise occur. Another mechanism implicated in the pathophysiology of injury during ischemia is the inhibition of oxygen-sensing prolyl hydroxylase (PHD) enzymes, because they require oxygen as a cofactor. Hypoxia-triggered inhibition of PHD enzymes induces the posttranslational activation of hypoxia and inflammatory signaling cascades, which in turn regulate the stability of the transcription factors, hypoxia-inducible factor (HIF) and nuclear factor-*κ*B (NF-*κ*B) \[[@B2]\].
Reperfusion of ischemic tissue activates a complex inflammatory response without the involvement of pathogenic triggers, a phenomenon also referred to as sterile inflammation. During the initiation of this inflammatory response, endogenous molecules act as alarmins or danger-associated molecular patterns (DAMPs) \[[@B6]\]. The inflammatory process is stimulated through self-antigens, which are functional components of intact cells but become stimulators of innate immunity when released from injured or dying cells \[[@B6]\]. In 1996, Weiser et al. discovered and described a novel mechanism for reperfusion injury that involves antibody deposition and activation of the complement system leading to an acute inflammatory response \[[@B7]\]. One decade later, the concept of innate autoimmunity was introduced, which is based on the discovery that circulating natural antibodies recognize self-antigens and elicit an acute inflammatory response involving the complement system \[[@B8]\]. Although ischemia-reperfusion is typically established in a sterile environment, activation of innate and adaptive immune responses occurs and contributes to injury, including activation of pattern-recognition receptors such as TLRs and inflammatory cell trafficking into the injured organ \[[@B9]\]. During this inflammatory process, the coagulation system is also activated, because the innate immune system and coagulation system are highly interconnected \[[@B10]\]. As ischemia-reperfusion injury is a common clinical problem and is associated with relevant complications, it is important to identify therapeutic approaches which prevent or at least mitigate ischemia-reperfusion-induced organ injury and organ dysfunction.
This special issue is devoted to the modulation of ischemia-reperfusion injury by different measures and contains eight original papers addressing this clinically relevant topic. These papers are accompanied by two review articles dealing with the effects of anesthetics on ischemia-reperfusion injury. Papers from B. U. Togrul et al., D. Dohman et al., and Y. Demirci et al. are focusing on ischemia-reperfusion injury of the liver. In two of these three papers, different therapeutic interventions on hepatic ischemia-reperfusion injury are evaluated, whereas the third paper is a retrospective study in which the authors investigated the efficacy and safety of intermittent portal triad clamping with low central venous pressure during liver resection. In this context, it has been reported that remote ischemic preconditioning and therapeutic interventions can reduce liver damage after inducing ischemia-reperfusion injury. The studies by S. C. Karahan et al., B. Michèle et al., S. C. Karahan et al., D. Dohman et al., and G. Altun et al. elucidate the effects of different anesthetic techniques and drugs on ischemia-reperfusion injury. These eight papers are entitled as follows:*"The effects of remote ischemic preconditioning and N-acetylcysteine with remote ischemic preconditioning in rat hepatic ischemia-reperfusion injury model"* by B. U. Togrul et al.,*"The effects of spinal, inhalation, and total intravenous anesthetic techniques on ischemia-reperfusion injury in arthroscopic knee surgery"* by S. C. Karahan et al.,*"Efficacy and safety of hepatectomy performed with intermittent portal triad clamping with low central venous pressure\"* by D. Dohman et al.,*"Adalimumab ameliorates abdominal aorta cross clamping induced liver injury in rats"* by Y. Demirci et al.,*"Evidence for the use of isoflurane as a replacement for chloral hydrate anesthesia in experimental stroke: an ethical issue"* by B. Michèle et al.,*"The effect of dexmedetomidine on oxidative stress during pneumoperitoneum"* by S. C. Karahan et al.,*"The comparison of the effects of sevoflurane inhalation anesthesia and intravenous propofol anesthesia on oxidative stress in one lung ventilation"* by D. Dohman et al., and*"Role of ethyl pyruvate on systemic inflammatory response and lung injury in an experimental model of ruptured abdominal aortic aneurysm"* by G. Altun et al.
*Alexander Zarbock* *Ahmet Eroglu* *Engin Erturk* *Can Ince* *Martin Westphal*
| {
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About 1 in every 10 adults worldwide is overweight or obese[@b1] and obesity is a risk factor for morbidity and mortality from cardiovascular diseases, diabetes, certain cancers, and musculoskeletal disorders. Despite strong interest in research addressing obesity, safe and effective pharmacological options for the prevention and treatment of this condition remain elusive[@b2][@b3]. Currently, most drug-discovery efforts are based on *in vitro* assays with candidate targets, but *in vitro* assays do not reconstitute the complexity of whole organisms. This is particularly relevant for drugs modulating feeding behavior and metabolic homeostasis, since both arise from complex interactions within and between the central nervous system, the digestive tract, and fat-storage organs[@b4][@b5][@b6], which cannot be modeled *in vitro*.
An alternative to *in vitro-*based screens is phenotype-based whole organism screens[@b7][@b8][@b9][@b10]. Whole organism drug screens provide several advantages over *in vitro* assays. Active drugs are by definition bio-available and potential toxicity can be evaluated at early project stages. Further, an effective drug need not act through a well-validated target, but can have novel or complex mechanisms of action. However, whole organism drugs screens can be costly and time-consuming. These disadvantages can be partially overcome by using model organisms that can be raised cost-effectively in large quantities, like the vinegar fly *Drosophila melanogaster*. Flies and vertebrates share many metabolic functions, molecular machinery, and analogous organ systems that control nutrient uptake, storage, and metabolism[@b11][@b12][@b13][@b14][@b15]. Like humans, flies regulate circulating sugar levels according to food availability, and store excess energy in the form of glycogen and lipids. These reserves are mobilized during periods of energy consumption[@b12][@b16][@b17]. As seen in many animals, fasted flies increase food foraging and intake[@b18]. Two master metabolic regulators in vertebrates, insulin and leptin, have functional homologues in the fly[@b13][@b14]. In addition, an unbalanced diet can trigger a type-2 diabetes-like insulin resistance and obesity phenotypes in the fly[@b19].
Here we report the development of a high-throughput drug-screen for *Drosophila* larval feeding. We identify the serotonin (5-hydroxytryptamine or 5-HT) receptor antagonist metitepine as a potent anorectic drug and show that all five fly 5-HT receptors are inhibited by this drug. Despite its broad spectrum antagonism of *Drosophila* 5-HT receptors, metitepine requires only receptor 5-HT2A for its *in vivo* anti-feeding activity. Our results highlight the potential of *Drosophila* as a tool for pharmacological study of feeding behavior and provide a powerful method for drug discovery and target identification.
Results
=======
High-throughput feeding assay for *Drosophila* larvae
-----------------------------------------------------
To screen for drugs that modify food intake in whole animals, we developed a high-throughput assay that allowed us to monitor ingestion of fluorescent liquid food by *Drosophila* first instar larvae in 96-well plates read by a plate reader ([Fig. 1a](#f1){ref-type="fig"}). Larvae were dispensed into plates and fed liquid food consisting of sugar and yeast extract and supplemented with fluorescein for visualization. After washing uningested fluorescent food from the wells, we quantified the fluorescein ingested by the larvae that was visible in the digestive tract ([Fig. 1b](#f1){ref-type="fig"}).
To evaluate the dynamic range of our assay, we carried out control experiments to either decrease or increase food intake in the larvae. When the animals were cold-paralyzed while feeding on fluorescent food, ingestion was reduced ([Fig. 1c](#f1){ref-type="fig"}). When larvae were selectively fasted for protein by removing yeast extract from the liquid food before being exposed to fluorescent food, they showed a post-fasting rebound in which they ingested more standard liquid food than control animals continuously fed standard liquid food ([Fig. 1d](#f1){ref-type="fig"}). The dynamic range of feeding suppression was more than two times greater than feeding enhancement in these experiments, perhaps because larvae are continual feeders and may be ingesting at a near maximal rate during basal conditions[@b20].
We next tested a known feeding mutant in our assay by comparing the fluorescent signal accumulated in three different wild-type strains (*w*^1118^, Canton-S, and Oregon-R) and a *klumpfuss*^09036^ mutant (*klu*, ref. [@b20]). *klu* encodes a transcription factor necessary for proper expression of the neuropeptide hugin, whose activity is required for normal feeding behavior in *Drosophila*[@b20]. All three wild-type strains ingested significantly more than the feeding mutant ([Fig. 1e](#f1){ref-type="fig"}). In addition, we confirmed previous reports[@b21] that high concentrations of dietary amino acids suppressed food intake ([Fig. 1f](#f1){ref-type="fig"}).
Drug screen for modulators of food intake
-----------------------------------------
After validating our feeding assay, we screened for small molecules that modulated food intake. In a pilot screen of 415 compounds tested individually at 20 μg/ml, we identified one compound, cycloheximide, which inhibited feeding (data not shown). This established a hit rate of 0.24%. To improve the throughput of the subsequent primary screen, we used pools of 3 to 4 compounds per well (see Methods for details).
3630 small molecules were tested in the primary screen ([Fig. 2a](#f2){ref-type="fig"}). The compounds were obtained primarily from annotated chemical libraries, such that each compound had at least one known cellular target (see Methods for details). The average signal of all the drug-treated wells was less than 3% different from the average of solvent-treated wells, indicating that the drugs did not cause generalized toxicity ([Fig. 2b](#f2){ref-type="fig"}). 279 and 114 compounds were identified as candidate anorectic (feeding suppressant) and orexigenic (feeding stimulant) drugs, respectively, by the criterion that they differed from solvent controls by more than one standard deviation ([Fig. 2a](#f2){ref-type="fig"}). The anorectic compounds caused an average decrease in fluorescent signal of 27%, while the orexigenic compounds caused an average increase of 18% ([Fig. 2b](#f2){ref-type="fig"}).
The 393 candidate small molecules were re-tested individually in the secondary screen ([Fig. 2a](#f2){ref-type="fig"}). Of the anorectic and orexigenic candidates, 32 and 10 compounds, respectively, were reconfirmed as hits, defined as differing from solvent controls by more than one standard deviation ([Fig. 2a, c, d](#f2){ref-type="fig"}, [Supplementary Table 1](#s1){ref-type="supplementary-material"}). We searched for reported molecular targets for each of the 42 hits of the secondary screen using a drug-discovery database (<https://www.collaborativedrug.com/>). Two known insect anti-feedants, gedunin and plumbagin[@b22][@b23], were among these compounds ([Fig. 2d](#f2){ref-type="fig"}), confirming the efficacy of our screen. We chose 14 compounds for verification of a dose-response curve ([Supplementary Table 1](#s1){ref-type="supplementary-material"}), based on their annotation as drugs that target neuromodulators, cell signaling, and/or neuronal activity. From these, only metitepine, a non-selective antagonist of 5-HT receptors, and reserpine, an inhibitor of the vesicular mono-amine transporter (VMAT), showed reliable dose-dependent responses and were selected for further characterization. Reserpine was subsequently discarded because it dramatically reduced larval locomotion at concentrations as low as 10 μM (data not shown). In light of these results, we concluded that the effects of reserpine on feeding were secondary to a general effect on locomotion and muscular tone, as confirmed by the sluggish phenotype of the dVMAT mutant larvae[@b24].
Metitepine decreased food accumulation by more than one standard deviation when tested in combination with other drugs, during the primary screen ([Fig. 2e, f](#f2){ref-type="fig"}), or alone, in the secondary screen ([Fig. 2g](#f2){ref-type="fig"}). Dose-response experiments indicated that the threshold concentration for metitepine efficacy was 10 μM, with increasing effects at 50 and 100 μM ([Fig. 2h](#f2){ref-type="fig"}).
Metitepine decreases feeding persistently, reversibly, and specifically
-----------------------------------------------------------------------
To ask if metitepine decreases larval feeding on conventional fly food, we tested the effect of the drug on larvae fed a standard laboratory cornmeal-agar-molasses diet supplemented with the dye bromophenol blue. We quantified the amount of food ingested by measuring the optical density (O.D.) of the gut in individual larvae. Larvae treated with the drug accumulated less solid food in their digestive tract as reflected by a lower O.D. ([Fig. 3a](#f3){ref-type="fig"}). The effect of metitepine on solid food accumulation was dose-dependent ([Fig. 3b](#f3){ref-type="fig"}) with a threshold efficacy dose of 50 μM.
To rule out the possibility that metitepine was causing a general locomotor defect in larvae, we measured their | {
"pile_set_name": "PubMed Central"
} |
{
"pile_set_name": "PubMed Central"
} | |
Introduction
============
A giant retinal tear (GRT) is defined as a full-thickness break in the retina equal to or larger than 3 clock hours associated with vitreous detachment.[@b1-opth-12-2053] Risk factors for developing a GRT include trauma, high myopia, Marfan syndrome, Stickler's syndrome or other hereditary vitreoretinopathies, and extensive lattice degeneration.[@b2-opth-12-2053],[@b3-opth-12-2053] Additional etiologies for GRT include iatrogenic and idiopathic causes.
The frequent rolled posterior edge of the retinal flap renders repair of retinal detachment (RD) due to GRTs technically challenging. Attempts to improve treatment through the 1970s and 1980s involved prone positioning for fluid-gas exchange, rotating head movements ("steamrolling") to unfold the tear, and manipulation of the retinal flap using intraocular balloons, retinal microincarceration, tissue adhesives, sodium hyaluronate, and retinal tacks, screws, and sutures.[@b4-opth-12-2053],[@b5-opth-12-2053] The advent of perfluorocarbon liquids changed the way that these cases were approached and allowed for higher surgical success rates as it made the surgical technique easier.[@b6-opth-12-2053] Currently, there is still debate about whether adjunctive procedures such as scleral buckling, lensectomy, or silicone oil injection are recommended.
Thus, the optimal surgical approach for these challenging cases remains controversial. The current study represents an update to a previously reported noncomparative case series of patients with GRT-associated RDs undergoing primary management at a university referral center.[@b7-opth-12-2053]
Materials and methods
=====================
In accordance with the guidelines from the Declaration of Helsinki, this study was approved by the Institutional Review Board at the University of Miami, Miller School of Medicine, as a retrospective noncomparative case series of patients undergoing primary RD repair for GRT at the Bascom Palmer Eye Institute between January 2011 and August 2017. The Institutional Review Board at the University of Miami did not require patient consent for retrospective review of medical records, and all patient data were deidentified upon review. Patients with a history of retinopathy of prematurity or retinal tear of less than 3 clock hours were excluded from this study.
Patients were identified using the ICD-10 codes for GRTs (H33.031, H33.032, H33.033, H33.039), and their charts were reviewed to verify coding information. A total of 427 patients were initially reviewed, yielding 80 eyes of 79 patients that fulfilled the abovementioned inclusion and exclusion criteria.
Data collected included demographic information, recorded etiologies, and surgical techniques. Outcome variables that were studied included best-corrected visual acuity (BCVA), occurrence of single operation retinal reattachment, and occurrence of reoperation. Data were collected postoperatively at 1 week, 1 month, 3 months, 6 months, 1 year, and last follow-up date. The surgical technique was at the discretion of the individual surgeon since there was no defined surgical protocol for this study. The data on proliferative vitreoretinopathy (PVR) were not included in this study as there were inconsistencies in the medical records and lack of standardization in its classification among the surgeons. Current data were compared with data from a previously published article from the same institution from 2005 to 2010.[@b7-opth-12-2053] Surgical variables were analyzed using the chi-squared test and Student's *t*-test. All statistical analyses were performed using SPSS version 24.0 (IBM Corporation, Armonk, NY, USA).
Results
=======
This study comprises 80 eyes of 79 patients who had an initial presentation of GRT associated with RD. The study included 62 (78%) male subjects. The mean age at initial presentation was 48.1±16 years, ranging from 4 to 71 years. The mean follow-up interval was 10.3±9 months. Right eyes were involved in 42/80 eyes (53%). Principal associations for the development of GRTs included prior history of blunt trauma (23 eyes, 18%), high myopia ≥6 diopters (33 eyes, 26%), lattice degeneration (14 eyes, 11%), and prior RD in the fellow eye (12 eyes, 10%). Other less common associations of GRTs included Marfan syndrome (two eyes, 2%) and Stickler's syndrome (one eye, 1%).
The size of the GRT was \<180° in 60/80 eyes (75%) ([Table 1](#t1-opth-12-2053){ref-type="table"}). The majority (68% of eyes) of the GRTs were superior and temporal. The associated RD was macular involving in 65%. The majority of eyes (76%) were initially phakic, while 21% were pseudophakic, and 2.5% were aphakic.
Primary scleral buckle (SB) was performed in 3/80 eyes (4%), with two patients receiving the \#41 band and one patient receiving the \#240 band ([Table 2](#t2-opth-12-2053){ref-type="table"}). These eyes were buckled because they were inferior GRTs without rolled edges, and 2 of the 3 eyes had shallow detachments. The majority of eyes (61/80, 76%) were treated with a combined pars plana vitrectomy (PPV) and SB procedure, utilizing a combination of the \#41, \#42, and \#240 bands. All PPVs were performed with either 23 or 25 gauge instrumentation. PPV alone was performed in 16 eyes (20%). In cases of PPV, endolaser was applied in a confluent fashion for one or two rows posterior to the GRT. In the three cases with SB only, cryopexy was used. Lensectomy was performed in 9/61 phakic eyes (15%). Perfluorocarbon liquid was used in 60/77 eyes (78%) undergoing PPV. Among eyes treated with PPV, silicone oil was used in 54/77 eyes (70%), perfluoropropane (C~3~F~8~) was used in 22/77 eyes (29%), and sulfur hexafluoride (SF~6~) was used in 1/77 eyes (1%).
SB was more likely to be placed in phakic eyes compared with pseudophakic/aphakic eyes (52/61 eyes, 85% vs 12/19 eyes, 63%; *P*\<0.036). Recurrent RD occurred in 1/3 eyes (33%) that received only a primary SB. However, eyes that received an initial SB (with or without PPV) had the same recurrent RD rate compared with cases managed without SB (10/64 eyes, 16% vs 1/16 eyes, 6%; *P*=0.33). Eyes associated with trauma had similar rates of recurrent RD compared with those not associated with trauma (*P*=0.55). For internal tamponade, there was no difference in rates of primary success between silicone oil (87%) and C~3~F~8~ (91%, *P*=0.6), but the one eye treated initially with SF~6~ developed a recurrent RD.
Perfluorocarbon liquid was used equally (and frequently) in GRTs involving both \<180° and ≥180°: 47 eyes (78%) vs 16 eyes (80%), respectively ([Table 3](#t3-opth-12-2053){ref-type="table"}). Silicone oil was more frequently used as tamponade in eyes with GRTs ≥180° compared with C~3~F~8~ (14/20, 70% vs 6/20, 30%; *P*=0.01) and also was more commonly used for GRTs \<180° (40/60 eyes, 67% vs 16/60, 27%; *P*\<0.001). GRTs ≥180° were not less likely to be attached at final follow-up and were not more likely to require further surgery for a recurrent RD compared with GRTs \<180° ([Table 3](#t3-opth-12-2053){ref-type="table"}). The percentage with final BCVA ≥20/400 was not different in those presenting with GRTs ≥180° compared with GRTs \<180°. Rate of lensectomy was not different between phakic eyes with GRTs ≥180° compared with GRTs \<180° ([Table 3](#t3-opth-12-2053){ref-type="table"}).
The retina was reattached at the last recorded follow-up examination in 76/80 eyes (95%). Recurrent RD after the primary repair occurred in 11/80 eyes (14%). The silicone oil had been removed in 24/54 eyes (44%) with silicone oil tamponade at last follow-up ([Figure 1](#f1-opth-12-2053){ref-type="fig"}). Of these eyes, 5/24 (21%) had recurrent detachment, and 1/54 eyes (2%) required a silicone oil exchange. Retinal redetachment occurred in 4/11 eyes (36%) after silicone oil was removed, with all redetachments occurring an average of 3 months after initial repair. When comparing with GRT size, 5/14 (36%) of the GRTs ≥180° had oil removed compared with 18/40 (45%) of the GRTs \<180° (*P*=0.55).
Preoperative visual acuity was ≥20/400 in 43 eyes ( | {
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1. Introduction {#S1}
===============
Water is essential for sustaining all life forms and access to clean and safe drinking water is a basic human need. Latest estimates of the Joint Monitoring Programme for Water Supply and Sanitation state that the world is on track to meet the Millennium Development Goals target for drinking water of "halving by 2015 the proportion of people without sustainable access to safe water and basic sanitation" \[[@R1]\] \[[@R2]\]. But this progress has been uneven. An estimated 768 million people did not use an improved source for drinking-water in 2011 and 185 million still depended on surface water (from lakes, rivers, dams, or unprotected dug wells or springs) for their daily drinking-water needs \[[@R3]\] \[[@R4]\].
India has only 3% of the world's fresh water with 20% of its population. Although, 92% - 96% of the urban population could access improved sources of water by 2010, 4% - 8% continues to use unimproved sources \[[@R4]\]-\[[@R6]\]. The World Bank estimates that, in spite of the improvement in provision for drinking water, 21% of all communicable diseases in India are related to unsafe water \[[@R7]\], with diarrhoea alone causing more than one hundred thousand deaths annually \[[@R8]\].
Groundwater constitutes 85% of the source of drinking water in India \[[@R9]\] \[[@R10]\] and none of the major Indian cities have a continuous water supply \[[@R11]\]. The Ministry of Urban Development, Government of India commissioned a study in 1999 to assess the status of water supply, sanitation and solid waste management in 305 selected Metropolitan, Class I and Class II cities and towns (of population more than 100,000). This study found that the coverage by the formal water supply was 94%. Paradoxically, coverage did not ensure quantity, quality, duration of supply or the mode of provision. In spite of 100 percent coverage in twenty-two sampled urban centres, regular daily supply was not ensured due to acute water shortage. Six sampled urban centres did not have individual house service connections. In most of the sampled cities, the duration of water supply ranged between 1 and 6 hours daily. The study also found that 43% of the sampled urban centres depended entirely on surface water, 34% depended entirely on groundwater while 22% used both surface and groundwater sources \[[@R12]\] \[[@R13]\].
The city of Ahmedabad located on the banks of a seasonal river---Sabarmati, has begun to receive water from the Narmada canal of the Sardar Sarovar project since the year 2000. This now supplements the underground water from French wells and Bore wells. This development has slowed the groundwater table depletion, but groundwater extraction is still widespread, especially in areas not provided by municipal water supply \[[@R14]\].
We investigated the drinking water surveillance parameters in one vulnerable ward of Ahmedabad. The WHO defines drinking water surveillance as the "the continuous and vigilant public health assessment and review of the safety and acceptability of drinking-water supplies". It must include an assessment of quality, quantity, accessibility, affordability and continuity of the drinking-water supply \[[@R15]\].
2. Methods {#S2}
==========
Our selection of this study ward was based on the municipal health officials' categorisation of this particular ward as the most vulnerable in the city. It is situated within the old city limits with more than 85% of its population residing in slums.
The data collection for the study took place in the summer of 2012. A questionnaire was developed based on the interviews we conducted with the municipal water supply officials. This was pilot tested in the neighbouring areas before being administered at the study site. The researchers made several visits to the study ward to understand the distribution of the household clusters and water supply across the ward.
We used a mixed methodology of:
2.1. Interviews with Key Informants {#S3}
-----------------------------------
We interviewed field level sanitary and health workers and supervisors posted at the ward office and health centre. We also interviewed water supply personnel (engineers and operators) at the main water storage and distribution facility located in our study ward and personnel at the Water Testing Laboratory.
2.2. Secondary Analysis and Mapping of Field Test Reports {#S4}
---------------------------------------------------------
Field testing for water samples by the sanitary inspector is recorded on a standard format in a register including the date, source of water supply, site of sample collection and the result. We obtained this list of locations and results of water sample field tests in the study ward for a six month period from October 2011 to March 2012. These were analysed and mapped using ArcGIS software.
2.3. A Short Survey {#S5}
-------------------
We obtained lists of population settlements from field health supervisor and used this to survey households in different types of tenements across the ward. The list divided the ward population of 65,403 into two groups; 86% of them living in 11,072 households (HHs) grouped into 47 slum clusters and 14% living in 4344 households (HHs) grouped into 42 housing society clusters. We sorted these cluster types separately in ascending order of population sizes. We sampled every alternate slum cluster from 47 slum clusters and every fifth housing society from 42 housing society clusters. (Slum clusters were oversampled because we were more interested in the poorer population of the ward.) We surveyed two HHs from every sampled cluster, one close to and one away from the municipal water line on the main road which supplies each cluster. This approach enabled us to assess the reach of the municipal water to the entire slum or society cluster and check for any disparities in supply between the proximal and distal households. The survey questionnaire was administered to a member of the household, the female head being the first preference. We finally surveyed 42 HHs out of 11,072 HHs in 21 slum clusters and 14 HHs from 4344 HHs in 8 housing society clusters. In two societies we sampled only one HH each because all the apartments were grouped around the water connection. Therefore the total tally was 56 households from 29 locations ([Figure 1](#F1){ref-type="fig"}). The data was analysed in MS-Excel and EpiInfo7.
The data was stored securely on a hard disk and no personally identifiable information was collected. All participants were assured of confidentiality of their responses and a formal verbal consent was taken before starting the interview. Ethical clearance for the study was sought from the Institutional Ethics Committee of the Public Health Foundation of India. Support letters were also arranged from the Ahmedabad Municipal Corporation.
3. Results and Discussion {#S6}
=========================
Our interviews revealed that Ahmedabad city receives approximately 1 billion litres of water per day, which averages out to nearly 180 litres per head. Supply is maintained for 2 hours every morning throughout the year (6 to 8 am) and during summers, for an additional one hour in the evenings. Water supply is not metered anywhere in Ahmedabad and therefore the actual quantity of water consumed by residents is not recorded.
The Central Water Testing laboratory tests at least 200 to 250 routine water samples (3 to 4 from each of 64 wards) each day. Additional samples are processed based on public complaints and during focal outbreaks of waterborne diseases. These samples are subjected to the following tests---turbidity, alkalinity, hardness, ${Ca}^{2 +},{Mg}^{2 +},{Cl}^{-},{SO}_{4}^{2 -}$, total dissolved solids, before being tested for faecal indicators (total coliform and fecal *E. coli*).
The Sanitary Inspector of every ward sends two water samples to the Central Water Testing laboratory and carries out 10 Orthotoludene field tests for residual chlorine from across the ward each day. The water samples for the field tests as well as the laboratory tests are collected during morning supply hours. There was no map of the existing water supply network and water sampling was not related to the physical organisation of the distribution system. Sampling is based on an arbitrary pattern of collecting samples from select slum clusters, municipal schools, public offices, etc. which has been set in practice over the years and on complaints from residents.
Analysis and mapping of the records of 217 field tested water samples in the study ward over a six month period from October 2011 to March 2012 showed that 1) all samples had been reported fit; 2) most slum clusters were sampled repeatedly; 3) but none of the housing society clusters were covered by the sampling process ([Figure 2](#F2){ref-type="fig"}).
The water supply department personnel of the ward examined the list of population settlements we had obtained from field health supervisor and marked out the most probable source of water (municipal supply, bore well or both) and the probable purification method used in each of the settlements. They estimated that of the 42 housing society clusters, only two supplement their municipal supply with bore-well water and one completely depends on bore well water for all of their water needs, and 12 of the 42 housing society clusters could afford small home-based Reverse Osmosis systems. Our survey showed this to be an underestimate.
3.1. Accessibility and Continuity {#S7}
---------------------------------
Our site visit revealed that, except for a tiny cluster of 250 HHs, 3.2% of the ward population which drew water from common stand posts, municipal water supply reached into the smallest and poorest of homes. Our survey of half of all the slum clusters found that all slum households depended on municipal water supply. Of the 8 housing society clusters, two tapped into groundwater while the rest depended on municipal supply.
3.2. Quantity {#S8}
-------------
A visual estimation of the volume of stored water showed all HHs stored an average of 35 to 50 litres of drinking water and 600 to 1300 litres | {
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Abedi E, Pourmohammadi K, Abbasi S. Dual‐frequency ultrasound for ultrasonic‐assisted esterification. Food Sci Nutr. 2019;7:2613--2624. 10.1002/fsn3.1115
1. INTRODUCTION {#fsn31115-sec-0001}
===============
Starch is an accessible, thickening, and texturizing stabilizer, and of paramount importance when it comes to augmenting the overall quality of the products, reducing costs, and facilitating the processing procedure (Berski et al., [2011](#fsn31115-bib-0009){ref-type="ref"}; Choi & Kerr, [2003](#fsn31115-bib-0013){ref-type="ref"}); however, due to the high affinity toward retrogradation, its application is restricted in certain food products (Kaur, Ariffin, Bhat, & Karim, [2012](#fsn31115-bib-0027){ref-type="ref"}; Kaur, Sandhu, & Lim, [2010](#fsn31115-bib-0029){ref-type="ref"}). In frozen foods and desserts containing starch, resistance to syneresis of water, resistance to freeze and thaw, and reduction in retrogradation are important issues. In this regard, native starch was modified to overcome this limitation. By introducing the functional groups into the starch molecules, chemically modified starches, such as oxidized, hydroxypropylated, and acetylated starches, were employed. When starch is modified, its gelatinization, pasting, and retrogradation characteristics undergo different changes (Kaur et al., [2012](#fsn31115-bib-0027){ref-type="ref"}, [2010](#fsn31115-bib-0029){ref-type="ref"}; Singh, Kaur, & McCarthy, [2007](#fsn31115-bib-0044){ref-type="ref"}). Acetylated starch (AC) was introduced with CH~3~CO group which caused a cleavage in the hydrogen bonds inside the starch chain and resulted in amphiphilic properties (Hong, Chen, Zeng, & Han, [2016](#fsn31115-bib-0025){ref-type="ref"}). AC with a low degree of substitution (DS) is used as an emulsifier, coating, and thickening agent, which is stable and resistant to retrogradation (Chi et al., [2008](#fsn31115-bib-0012){ref-type="ref"}; Singh, Chawla, & Singh, [2004](#fsn31115-bib-0045){ref-type="ref"}). Moreover, in such kinds of modified starch, solubility, swelling power, viscosity, hardness, adhesiveness, cohesiveness, and translucency of the gels are increased, while the initial gelatinization temperature is reduced (González & Perez, [2002](#fsn31115-bib-0018){ref-type="ref"}). The replacement of the modifier groups may boost the free movement of the starch chains within the amorphous regions in the granule, owing to the disruptions taking place among the inter‐ and intramolecular hydrogen bonds. The weakened internal bond structure in the starch granules, due to the derivatized modifier groups, enhances the freeze--thaw stability, reduces the gelatinization temperature, generates high levels of peak viscosity, and leads to starch paste clarity (Han, [2010](#fsn31115-bib-0021){ref-type="ref"}; Han, Lee, Lim, & Lim, [2005](#fsn31115-bib-0022){ref-type="ref"}).
Ultrasonic treatments can accelerate chemical reaction and increase yields. Under sonication, the surface area of starch granules increases due to the formation of channels and/or holes on the surface and inside the granules (Huang, Li, & Fu, [2007](#fsn31115-bib-0026){ref-type="ref"}). Several modifications have been performed upon ultrasonication, including acetylation of dioscorea starch following ultrasound treatment (Zhang, Zuo, Wu, Wang, & Gao, [2012](#fsn31115-bib-0055){ref-type="ref"}), octenyl succinate starch (Chen, Huang, Fu, & Luo, [2014](#fsn31115-bib-0011){ref-type="ref"}), carboxymethyl starch (Gao et al., [2011](#fsn31115-bib-0017){ref-type="ref"}; Shi & Hu, [2013](#fsn31115-bib-0042){ref-type="ref"}), and octenylsuccinates of carboxymethyl starch (Čížová, Sroková, Sasinková, Malovíková, & Ebringerová, [2008](#fsn31115-bib-0014){ref-type="ref"}). However, there is no information about the acetylation of starch under various frequencies of ultrasonication. The aim of this study was to optimize the AC wheat starch in various parameters such as acetic anhydride concentration, pH, and temperature under frequencies (25, 40, and 25 + 40 kHz) of bath ultrasound; the optimization was further investigated on the amount of modification (molar substitution) in the modified starch. Based on the minimal reagent consumption, the optimized samples were selected and their physicochemical properties (solubility, water absorption), X‐ray diffraction, scanning electron microscopy (SEM), Rapid Visco Analyzer (RVA), differential scanning calorimetry (DSC), and Texture Profile Analyzer (TPA) were investigated at three frequencies (25, 40, and 25 + 40 kHz). The freeze and thaw stability of AC wheat starch was further compared with that made with native wheat starch.
2. MATERIALS AND METHODS {#fsn31115-sec-0002}
========================
2.1. Materials {#fsn31115-sec-0003}
--------------
Native wheat starch was obtained from Fars Glucosin Company, Shiraz, Iran. The moisture, fat, ash, and protein of native (N) and AC wheat starch prepared under ultrasonication were specified along with the standard methods of AACC, [2000](#fsn31115-bib-0001){ref-type="ref"}. Amylose content (%) was calculated by iodine method (Pourmohammadi, Abedi, Hashemi, & Torri, [2018](#fsn31115-bib-0038){ref-type="ref"}).
2.2. Acetylation of wheat starch {#fsn31115-sec-0004}
--------------------------------
We added 100 g of starch to 224 ml of distilled water and stirred for 1 hr at 24°C. The pH was set to 6.5, 8, and 9.5 with NaOH solution (1 M), and acetic anhydride (4, 6 and 8%) was slowly added to the suspension at various pHs. Whole acetylation reaction was performed at 10 min. The suspensions were sonicated at an amplitude of 24% (with an input power of up to 400 W and working frequencies of 25, 40, and 25 + 40 kHz) at 45, 60, and 75°C. The time duration of sonication in each frequency was 5 min (25 kHz: 5 min, 40 kHz: 5 min, and 25 + 40 kHz: 5 min). pH was then maintained at 4.5 with HCl solution (1 M) for 5 min. All the experiments were performed in bakers placed at known positions in ultrasonic bath (Pacisa SA) equipped with a temperature controlling system. The ultrasonic bath with internal dimensions of 300 × 150 × 150 mm^3^consisted of a rectangular tank containing four transducers at the bottom. The suspensions were completely washed, lyophilized, and milled using a laboratory mill (AlexanderWerk, Model WEL82), and manually sieved to obtain particle sizes \< 40 μm (Wojeicchowski, Siqueira, Lacerda, Schnitzler, & Demiate, [2018](#fsn31115-bib-0054){ref-type="ref"}).
2.3. Acetyl percentage {#fsn31115-sec-0005}
----------------------
The titrimetric method of Mbougueng, Tenin, Scher, and Tchiégang ([2012](#fsn31115-bib-0035){ref-type="ref"}) was employed in order to specify the acetyl group percentage (AC%) and (DS). AC (5.0 g), 50 ml of distilled water, and 24 ml of 0.45 M NaOH were added in a 240‐mL flask and mixed for 30 min at room temperature. The surplus alkali was back‐titrated using 0.2 M HCl and phenolphthalein as an indicator. The reaction mixture was put to stand for 2 hr, where the alkali was removed from the titrated sample. The native starch was also used as a blank sample. Initially, the acetyl (%) and DS were calculated according to Equations ([1](#fsn31115-disp-0001){ref-type="disp-formula"}) and ([2](#fsn31115-disp-0002){ref-type="disp-formula"}), respectively:$$\%{\mspace{6mu}\text{Acetylation}} = \lbrack\left( {V_{\text{B}} - V_{\text{S}}} \right) \times N_{\text{HCl}} \times 0.043 \times 100\rbrack/W$$
*V* | {
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Introduction {#s1}
============
Lysosomes and the ubiquitin proteasome system (UPS) are major degradation machineries for damaged and unwanted cellular contents in eukaryotic cells. The UPS degrades damaged and unwanted proteins that are tagged with ubiquitin, whereas large protein aggregates and damaged or dispensable organelles are degraded in the lysosome. Besides housekeeping functions, regulatory roles of these two machineries in development and differentiation are emerging in a number of organisms. Malaria parasites critically rely on these two systems, as inhibitors of the UPS and of proteases of the food vacuole, the lysosome-equivalent of *Plasmodium*, have been shown to block parasite development. Hence, these two proteolytic machineries are attractive targets for antimalarial drug development \[[@B1]--[@B6]\].
Erythrocytic malaria parasites critically rely on hemoglobin for amino acids, which is hydrolyzed in the food vacuole by concerted action of several different types of proteases. The major food vacuole-resident hemoglobin degrading proteases are papain-like cysteine proteases falcipains, aspartic proteases plasmepsins, the metalloprotease falcilysin, dipeptidyl aminopeptidase I, and a M1-family alanyl aminopeptidase \[[@B7]--[@B13]\]. A large body of literature indicates that the papain-like cysteine proteases falcipains, particularly falcipain-2 (FP2) and falcipain-3 (FP3), are the major hemoglobin degrading enzymes in *P. falciparum* \[[@B5]\]. FP2, FP3 and their homologs in other malaria parasites prefer leucine at the P2 position in substrates and inhibitors \[[@B14]--[@B24]\]. Several peptidyl inhibitors of papain-like cysteine proteases, including leupeptin (acetyl-Leu--Leu--Arg-aldehyde, with P2 leucine and aldehyde warhead), block development of malaria parasites by inhibiting FP2 and FP3 \[[@B25]\]. This selectivity for a P2 leucine residue has been exploited for optimizing inhibitors of falcipain-like proteases of malaria parasites \[[@B26]--[@B28]\].
Malaria parasites appear to have a typical 26S proteasome \[[@B29]\]. A 26S proteasome is composed of two multi-subunit assemblies: a core protease complex, called the core particle (CP) or 20S proteasome; and a regulatory element, known as the 19S regulatory particle (RP) \[[@B30]\]. The CP is a barrel-shaped complex of four stacked rings, each with 7 α or 7 β subunits, which are arranged as "αββα". Three catalytically distinct β subunits (β1, β2, and β5) in each inner ring, whose active sites line the central lumen of the CP, form a proteolytic chamber where substrates are degraded. Substrates gain access to this chamber through the pores formed by α rings on either side of the CP \[[@B31],[@B32]\], which requires the RP for opening the pores and unfolding of the substrate. Active site mutagenesis of the three protease subunits together with the use of defined peptide substrates revealed that a typical 26S proteasome possesses three types of activities \[[@B33]--[@B35]\]: 1) caspase-like or peptidyl-glutamyl peptide hydrolyzing (PGPH) activity, which cleaves after acidic residues; 2) trypsin-like activity that cleaves after basic residues; and 3) chymotrypsin-like activity, which cleaves after large hydrophobic residues. The majority of available proteasome inhibitors, including MG132, epoxomicin, and lactacystin, block the chymotrypsin activity \[[@B6],[@B36]--[@B39]\].
Dual inhibition of falcipains and the UPS with optimal selectivity could offer higher potency and less risk of development of drug resistance by the parasite than individual inhibitors of these two proteolytic systems. One such compound might be MG132 (Z-Leu-Leu-Leu-CHO), as it contains P2 leucine, a falcipain preferred residue, and an aldehyde group that reacts with catalytic cysteine residue of cysteine proteases and threonine residues of protease units of the proteasome. MG132 is a first choice inhibitor for studying UPS in a variety of organisms, including malaria parasites. It is commonly used at micromolar concentrations to study the UPS in a variety of human cell lines, with reported 50% cytotoxic concentrations in the range of 2.5-21 µM depending on the cell type and treatment duration \[[@B40]--[@B43]\]. MG132 has also been shown to inhibit the papain-like cysteine proteases cathepsin L and B and the calcium dependent cysteine proteases calpains \[[@B44],[@B45]\].
However, the cysteine protease inhibitory property of MG132 remains underappreciated compared to its extensive use as an UPS inhibitor. Hence, we hypothesized that this compound is a dual-target inhibitor of malaria parasites, blocking hemoglobin degradation by inhibiting falcipains and also inhibiting UPS. To test this hypothesis, we assessed the effects of specific inhibitors of cysteine proteases (E64) and UPS (epoxomicin, lactacystin, and MG132) on parasite development, hemoglobin degradation, proteasome and cysteine protease activities in *P. falciparum* extracts, and activity of recombinant falcipains. We demonstrated that MG132 blocks asexual erythrocytic development of *P. falciparum* by inhibiting both hemoglobin degradation and the UPS.
Materials and Methods {#s2}
=====================
Materials {#s2.1}
---------
The *P. falciparum* 3D7 strain was obtained from the Malaria Research and Reference Reagent Resource Centre (MR4). MG132, epoxomicin, and lactacystin were from Santa Cruz Biotechnology; all other biochemical reagents were from Sigma or Serva. Plasmid isolation kits were from Qiagen or MACHEREY-NAGEL; cell culture reagents were from Lonza and Invitrogen; restriction and DNA modifying enzymes were from New England Biolabs; and SYBR Green 1 was from Invitrogen. Human blood was collected from healthy volunteers after written consent under medical supervision at the medical dispensary of the institute, and the protocol (IEC-2/2010) for blood collection for this study has been approved by the Institutional Ethical Committee of CCMB.
Parasite culture {#s2.2}
----------------
In vitro parasite culture was done according to the protocols approved by the Institutional Biosafety Committees of CCMB and UCSF. *P. falciparum* was cultured in human erythrocytes at 2% haematocrit in the presence of a gas mixture (5% CO~2~, 5% O~2~, and 90% N~2~) in RPMI 1640 medium supplemented with 41.1 mg/litre hypoxanthine, 300 mg/litre glutamine, 2.5% human serum, and 0.5% Albumax II \[[@B46]\]. Synchrony was maintained by serial treatment with 5% D-sorbitol \[[@B47]\]. For parasite isolation, the culture was centrifuged at 894g for 5 min, the supernatant was aspirated off, the pellet was treated with ice-cold 0.05% saponin (in PBS) for 5 min to lyse erythrocytes, and the sample was centrifuged at 12,096g for 5 min at 4°C. The supernatant was discarded, the pellet was washed twice with ice-cold PBS to remove residual hemoglobin, and parasites were recovered by centrifugation at 12,096g for 5 min at 4°C. Genomic DNA (gDNA) was isolated from late trophozoite/schizont stage parasites using the Puregene Blood Core Kit B (Qiagen).
Parasite growth inhibition assays {#s2.3}
---------------------------------
Inhibitors of the proteasome (epoxomicin, lactacystin, and MG132), cysteine proteases (L-trans-epoxysuccinyl-leucyl-amido(4-guanidino)butane; E64), and aspartic proteases (pepstatin) were assessed alone and in combinations for inhibition of *P. falciparum* erythrocytic stage development. 200 x stocks of inhibitors were made in DMSO and serially diluted 2-fold in 100 µl culture medium across rows of a 96 well tissue culture plate. Control wells contained DMSO (0.05%) or 500 nM chloroquine. 100 µl of parasite suspension (1% ring-infected erythrocytes at 4% haematocrit) was added to each well, and plates were incubated in a modular incubator chamber (Billups-Rothenberg, Inc.) with the gas mixture at 37°C for 50 hours. At the end of incubation, 100 µl culture medium was carefully removed from each well, and 100 µl lysis buffer (20 mM Tris-Cl, 5 mM EDTA, 0.008% saponin, 0.08% Triton X-100, pH 7.5) with SYBR Green 1 (at the manufacturer's recommended dilution) was added to each well. The plate was incubated at 37°C for 30 min, and fluorescence was measured (Ex: 485 nm, Em: 530 nm, gain setting: 50) using an Infinite M200 multimode microplate reader (TECAN) as described previously \[[@B48]\]. The fluorescence of chloroquine-treated culture was subtracted from inhibitor-treated and DMSO-containing cultures to account for background fluorescence. Fluorescence values of inhibitor-treated cultures were normalized as percentage of DMSO-treated cultures, plotted against inhibitor concentrations, and analyzed using nonlinear regression analysis to determine IC~50~ concentrations for the inhibitors as described earlier \[[@B12]\].
For assessing the effect of inhibitor treatment on parasite morphology, ring stage parasites were cultured with DMSO (0.1%) or inhibitors (21.7 µM E64, 0.024 µM epoxomicin, 0.820 µM lactacyst | {
"pile_set_name": "PubMed Central"
} |
Background
==========
Endothelial cell adhesion molecules (\'ECAMs\') play essential roles in the development of chronic inflammation by recruiting leukocytes, especially lymphocytes to tissues. ECAMs support several forms of leukocyte adhesion including rolling, firm adhesion and extravasation \[[@B1]\]. Infiltration of tissues by leukocytes is a common hallmark of many chronic inflammatory states that include the inflammatory bowel diseases (IBD), ulcerative colitis (UC), and Crohn\'s disease (CD). In the setting of IBD, the expression of ECAMs like ICAM-1, VCAM-1, and MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is observed in experimental models of colitis, \[[@B2]-[@B5]\] and also within the inflamed human colon in Crohn\'s disease and ulcerative colitis \[[@B6],[@B7]\].
Among the adhesion molecules up-regulated in IBD, MAdCAM-1, the mucosal cell adhesion molecule, is thought to be preeminent in the development of chronic gut inflammation. MAdCAM-1 is normally expressed in the gut, and its expression is dramatically amplified during inflammation \[[@B2],[@B3]\]. The functional significance of increased appearance of MAdCAM-1 in IBD is supported by several reports which show that immunoneutralization of either MAdCAM-1 or its ligand, the α4β7 integrin, attenuate inflammation and mucosal damage in animal models of colitis \[[@B8]-[@B10]\]. However, since monoclonal antibodies directed against other ECAMs, particularly VCAM-1, can as well reduce disease activity in colitis models \[[@B11]-[@B14]\], the literature suggests that MAdCAM-1 is probably necessary, but insufficient for the maximal penetrance of experimental and probably also clinical IBD.
Based on these findings, it is apparent that a better understanding of the mechanisms regulating ECAM expression, especially that of MAdCAM-1, might help to devise improved therapies for colitis.
It has been suggested that pathologic activation of the mucosal immune system in response to antigens is a key factor in the pathogenesis of IBD. Furthermore, changes in leukocyte migration and cytokine production appear to contribute the perpetuation of IBD \[[@B15]\]. Based on modern advances, recombinant anti-inflammatory cytokines (i.e. IL-10) treatment is now being developed for experimental colitis and human IBD. IL-10 produced by macrophages and monocytes appears to limit chronic inflammation \[[@B16]-[@B18]\], through a decreased release of inflammatory factors (IL-1, IL-6, IL-12, TNF-α, GM-CSF, GCSF), suppression of adhesive determinants (MHC class II molecule, β7), and by blocking ICAM-1 induction \[[@B19]-[@B24]\].
Conversely, IL-10 gene-knockout mice develop a chronic colitis that is extremely similar to IBD \[[@B25]\]. IL-10 treatment can reduce inflammation in several models of colitis \[[@B26]-[@B30]\] and human IBD \[[@B31]-[@B35]\]. However, the clinical efficacy of systemically administered IL-10 for patients mild to moderately active Crohn\'s disease has not been as effective as hoped \[[@B33]-[@B35]\]. Furthermore the efficacy of IL-10 administration in mouse colitis models is contentious \[[@B36]\].
We have described in vitro that exogenous IL-10 can block the expression of MAdCAM-1 in response to TNF-α, and attenuates lymphocyte adhesion to lymphatic node derived endothelium under cytokine stimulating conditions via NF-kB inhibition \[[@B5]\]. The purpose of the current study was to show that induction of endothelial expression of IL-10 through an IL-10 expression vector attenuates MAdCAM-1 expression in response to TNF-α and optimistically suggests the possibility of targeted Th2-cytokine gene therapy in IBD.
Methods
=======
Reagents
--------
Recombinant mouse TNF-α was purchased from ENDOGEN (Stoughton, MA) amd plasmid containing human IL-10 (phIL-10) was generous gift from Dr. Meng X (Thomas Jefferson University, PA).
Cell culture
------------
The SVEC4-10 line is an endothelial cell line derived by SV40 (strain 4A) transformation of murine small vessel endothelial cells, originally isolated from the axillary lymph node vessels of an adult male C3H/Hej mouse \[[@B37],[@B38]\]. These cell types were all maintained in Dulbecco\'s modified Eagle\'s medium (DMEM) with 10% fetal calf serum with 1% antibiotic/ antimycotic. Cells were seeded into 24-well tissue culture plates at approximately 20,000 cells/cm^2^, and cultures were used immediately upon reaching confluence (usually 3--4 days after seeding).
Lymphocytes
-----------
The mouse CD8^+^T cell lymphoma TK-1 cells (that constitutively expresses the α4β7 integrin \[[@B39]\]) were obtained as a generous gift from Dr. Eugene Butcher (Stanford University, CA). These cells were cultured in RPMI-1640 medium supplemented with 10% FCS and 0.05 mM 2-mercaptoethanol (minus antibiotic/ antimycotic).
IL-10 gene transfer
-------------------
The pCYIL-10 vector is an expression vector which was established by Dr. Xianmin Meng based on the pCY4B vector \[[@B40]\]. Cultured SVEC endothelial cells seeded in 24 wells were transfected at 70% confluency with SuperFect (Qiagen). Briefly, 1 μg plasmid DNA was diluted with 60 μl DMEM and then 5 μl Superfect (3 mg/ml) was added. After incubation at room temperature for 10 min, the complex was mixed with 350 μl medium and added to the each wells. After 2 h at 37°C, 5% CO2, the complex was aspirated and washed 2 times with medium following replace by 1 ml of medium. After incubation at 37°C for 24 h cells were stimulated with TNF-α.
Western analysis of cell lysates
--------------------------------
24 h of IL-10 gene transfer, monolayers were treated with TNF-α (1 ng/ml, 24 h). All cell samples were harvested at 24 hours. Equal quantities of protein (75 μg) from each sample were electrophoretically separated on 7.5% SDS-PAGE gels. Gels were transferred to nitrocellulose membranes (Sigma) and blocked with 5% milk powder in PBS at 4°C (overnight). These membranes were washed twice for 10 min with wash buffer (0.1% milk powder in PBS). Primary rat anti-mouse MAdCAM-1 mAb was added at a concentration of 10 μg/ml and incubated at room temperature for 2 h. These membranes were washed twice with wash buffer. Secondary rabbit anti-rat horseradish peroxidase conjugated secondary antibody (Sigma) was added at a 1:2000 dilution for 2 h. Lastly, membranes were washed 3 times and developed using the enhanced chemiluminescence (ECL) detection system (Amersham, La Jolla, CA). The density of MAdCAM-1 staining was measured by scanning the 60 kD band, using a HP ScanJet™ flatbed scanner. Images were analyzed for density using Image Pro Plus™ image analysis software (Media Cybernetics). The data are expressed as a percentage of TNF-α-induced level of density. In each protocol, treatments were performed at least in triplicate.
TK-1 lymphocyte adhesion assay
------------------------------
Briefly, TK-1 cells were suspended in culture medium and fluorescence labeled by incubating TK-1 cells at 2 × 10 ^6^cells/ml with 0.02 mg fluorescein diacetate (FDA) (Sigma) at 37°C for 30 min. The cells were then washed twice with ice-cold HBSS, spun at 250 g for 5 min to remove unincorporated fluorescence and suspended in HBSS. The TK-1 lymphocyte cell line used in this assay expresses high levels of the α4β7 integrin, \[[@B39],[@B41]\] which can interact with multiple ligands including mucosal addressin-1 (MAdCAM-1), as well as VCAM-1, L-selectin and fibronectin \[[@B42]\]. In this system, TNF-α stimulated TK-1 adhesion to SVEC4-10 endothelial cells is at least 50% MAdCAM-1 dependent \[[@B41]\]. SVEC monolayers were grown in 48-well plates as described, and to activate endothelium, the monolayers were incubated with TNF-α (1 ng/ml) for 24 h. Cytokine treated endothelial cells were washed three times with media. Labeled TK-1 cells were then added to the endothelium at a 5:1 lymphocyte to endothelial cell ratio \[[@B43]\] and allowed to bind for 30 min under static conditions. At the end of the incubation period, the supernatant was removed and the monolayers were washed twice with HBSS. Plates were read on a Fluoroskan Ascent (Labsystems, Helsinki, Finland) set for excitation at 485 nm, and emission at 515 nm. Blank wells (0% TK-1 adhesion) were run as controls that did not contain labeled TK-1 cells. The data are expressed as a percentage of TNF-α-induced level of fluorescence. In each protocol, treatments were performed at least in triplicate.
Statistical analysis
--------------------
All values are expressed as mean ± SD. Data were analyzed using multiple comparisons. Probability (*P*) values of \< 0.05 were considered significant.
Results
=======
Secretion of human IL-10 concentration by transfected endothelial cells
-----------------------------------------------------------------------
To screen for the efficacy of IL-10 transfected SVEC, we initially measured the IL-10 concentration in the medium prior to gene transfection. There was no detectable human IL-10 | {
"pile_set_name": "PubMed Central"
} |
{
"pile_set_name": "PubMed Central"
} | |
Introduction {#S1}
============
Skin with underlying subcutis is armed with neuroendocrine capabilities and represents the largest and one of the most complex organs in the human body ([@R58]). Strategically located at the interface with external environment, skin detects, integrates, and responds to stressors including ultraviolet radiation (UVR) ([@R8]; [@R26]; [@R47]; [@R58]; [@R59]). UVB (290--320 nm) radiation has powerful biological actions not only on cutaneous biology but it also impacts many regulatory pathways involved in immune homeostasis that are both vitamin D-dependent and independent ([@R1]; [@R2]; [@R8]; [@R12]; [@R19]; [@R23]; [@R27]; [@R31]). The mechanisms of UVB-induced immune suppression ([@R63]) are not completely understood. However, evidence is accumulating that DNA damage and other mechanisms such as the photoisomerization of urocanic acid, free-radical formation, and signal transduction-mediated activation of transcription factors and induction of neuroendocrine signaling may also contribute to the resulting pathological conditions as well ([@R8]; [@R23]; [@R39]; [@R58]; [@R63]).
The main regulatory algorithm to maintain body homeostasis is the hypothalamic-pituitary-adrenal (HPA) axis, which requires activation of a complex range of responses involving the endocrine, nervous, and immune systems, collectively known as the stress responses ([@R4]; [@R34]; [@R60]; [@R62]). Neural signals encoded by the limbic system as stressors trigger neurons of the paraventricular nucleus (PVN) of hypothalamus to produce and release corticotropin releasing hormone (CRH) into the hypophyseal portal circulation ([@R60]; [@R62]). Next, CRH binds to CRH receptor type 1 (CRH-R1) on pituitary corticotropes, and induces the release of POMC-derived adrenocorticotropic hormone (ACTH) into the systemic circulation. The melanocortin receptor type 2 (MC2R), expressed in the adrenal cortex, stimulates glucocorticoid (GC) synthesis and secretion after binding of ACTH ([@R4]; [@R21]; [@R60]). GC (i.e., cortisol (COR) in humans and corticosterone (CORT) in rodents) maintain metabolic and stress-responses, suppress immune activity and are self-regulated, with negative feedback to the hypothalamus and pituitary to mute the HPA axis ([@R4]; [@R21]).
The skin has neuroendocrine capabilities that also encompasses all elements of the "cutaneous HPA axis" that follow the organization of the central HPA axis \[reviewed in ([@R50])\]. This concept was based on the evidence that vertebrate skin expresses CRH with functional CRH-R1 \[reviewed in ([@R59])\] and related POMC macromolecule, which is further processed to ACTH ([@R14]; [@R29]; [@R39]; [@R42]; [@R48]), which, after interaction with MC2R, induces steroidogenesis with the final production of highly adaptable COR or CORT ([@R5]; [@R14]; [@R35]; [@R41]; [@R51]; [@R52]; [@R53]; [@R54]; [@R64]). Furthermore, Hiramoto et al ([@R11]) have demonstrated that exposure of the eye to the UVB increases plasma α-MSH levels with systemic stimulation of epidermal melanocytes.
Based on the above we have decided to test the hypothesis that UVB acting on the skin can regulate body homeostasis through activation of central HPA. Using mouse model we evidence that UVB activates the HPA axis both on the local (skin) and systemic (brain, adrenal and plasma) levels, with a latter requiring an intact pituitary. The UVB induced increases in corticosterone production explain immunosuppressive effects of the UVB, while that of β-endorphin could explain a phenomenon of "UV addiction".
Results {#S2}
=======
General design of UVB exposure {#S3}
------------------------------
To prevent retinal or non-retinal eye signal transmission, the heads including eyes, were covered with aluminum foil, and the skin on the back was irradiated with UVB ([Figure 1](#F1){ref-type="fig"}).
Dose and Time Dependent Effects of UVB in the Skin {#S4}
--------------------------------------------------
Previously, we have documented that UVB (290--320 nm), but not less energetic UVA (320--400 nm), is effective in stimulating HPA axis elements in human and mouse skin organ culture *in vitro* ([@R35]; [@R36]; [@R37]). In current experiments we first measured the CORT in the skin *in vivo* using different doses and time after UVB exposure and found that the dose of 400 mJ/cm^2^ (2.1 minimal erythema doses (MED), [Table S1](#SD1){ref-type="supplementary-material"}) and 12 and 24 h after exposure were most optimal for enhancement of the CORT levels ([Figure 2a,b](#F2){ref-type="fig"}). Lower dose (100 mJ/cm^2^) and shorter times of observation (3 and 6 h) showed significantly lower stimulatory effect. Similarly, markedly stronger stimulation of plasma CORT levels was observed at 400 in comparison to 100 mJ/cm^2^ and at 12 in comparison to 3 h after UVB exposure ([Figure S1](#SD1){ref-type="supplementary-material"}). The histopathological analysis demonstrated that UVB at 400 mJ/cm^2^ did not produce noticeable epidermal necrosis nor trigger marked/moderate inflammatory infiltrate ([Figure 2c](#F2){ref-type="fig"}). However, small increase in infiltrating neutrophils and eosinophils was observed at 1--6 h after UVB exposure, which after 12 or 24 h returned to the control ([Figure 2d](#F2){ref-type="fig"}). Therefore, based on the previous ([@R36]) and current *in vivo* experiments we have chosen the dose of 400 mJ/cm^2^ (2.1 MED) and a time of 12 and 24 h after UVB exposure for further experiments.
UVB effects on cutaneous expression of CRH and Ucn {#S5}
--------------------------------------------------
Since the CRH gene is not expressed in C57BL/6 skin ([@R49]; [@R59]), and can be replaced by Ucn in activating HPA axis elements ([@R46]; [@R59]), we examined Ucn cutaneous expression as a potential triggering regulator. We also measured the concentration of CRH since this peptide can be released locally from cutaneous nerve fibers ([@R28]; [@R40]). UVB radiation stimulated the Ucn expression at the gene ([Figure 3a](#F3){ref-type="fig"}) and peptide levels ([Figure 3l](#F3){ref-type="fig"}) with the similar patterns at 12 and 24 h post-radiation. *In situ* localization studies showed increased expression of Ucn in the main skin compartments including the epidermis, adnexal structures and stratum paniculosum ([Figure 3g](#F3){ref-type="fig"}). There was also an increase in CRH peptide concentration in the skin after UVB exposure, as evaluated by ELISA ([Figure 3k](#F3){ref-type="fig"}).
UVB effects on cutaneous expression of POMC, ACTH and β-END {#S6}
-----------------------------------------------------------
Next, we checked cutaneous POMC expression, a "pituitary" element of the systemic HPA axis. UVB light stimulated a 2.5-fold increase in expression of POMC mRNA after 12 h, and which was still present after 24 h, although at lower levels ([Figure 3b](#F3){ref-type="fig"}). Immunoblotting with antibodies directed against ACTH, which recognize the 33 kDA POMC precursor, confirmed increased expression of this molecule ([Figure 3e](#F3){ref-type="fig"}). UVB-induced increased ACTH production was also confirmed with quantitative IHC ([Figure 3h](#F3){ref-type="fig"}) as was expression of POMC-derived β-END stimulated by UVB ([Figure 3i](#F3){ref-type="fig"}).
UVB effects on cutaneous expression of MC2R, CYP11A1, StAR, 3β-HSD and CORT {#S7}
---------------------------------------------------------------------------
The expression of the next crucial element of the HPA axis, the MC2R (responsible for initiation of steroidogenesis upon ACTH activation), was upregulated 1.5 times after 12 h and almost two times after 24 h ([Figure 3c](#F3){ref-type="fig"}). We also investigated StAR gene expression, which is required to transfer cholesterol from the outer to the inner mitochondrial membrane, and showed that its up-regulation occurred after 12 and 24 hrs post-UVB exposure ([Figure 3d](#F3){ref-type="fig"}). Moreover, we evaluated the expression of the rate-limiting enzyme of steroidogenesis, the cytochrome P450scc (CYP11A1), which cleaves the cholesterol side chain to produce pregnenolone, a precursor of all steroids. Western blot analysis revealed high expression of P450scc at 12 h and 24 h post-UVB exposure, compared to appropriate controls ([Figure 3f](#F3){ref-type="fig"}). Furthermore, immunohistochemistry for 3β-HSD, (the enzyme that transforms pregnenolone to progesterone), showed that this antigen is highly expressed in cutaneous adnexal structures and in the stratum paniculosum, but weakly expressed in the epidermis of untreated skin. UVB radiation | {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#s1}
============
One of the most frequently deregulated pathways in human cancers is the phosphoinositide 3-kinase (PI 3-K) and Akt signaling cascade \[[@R1]\]. This is particularly evident in breast cancer where mutations exist in virtually all of the proteins that lead to activation of PI 3-K and its downstream effectors. Activation of cell surface receptors, particularly receptor tyrosine kinases (RTKs), leads to activation of class I PI 3-K, which catalyzes the synthesis of PtdIns-3,4,5-P3 (PIP3) from PtdIns-4,5-P2 (PIP2). PIP3 functions as a true second messenger by recruiting multiple effector molecules, one of which is the Akt/PKB protein kinase that directly binds to PIP3 through its pleckstrin homology (PH) domain. This binding facilitates the phosphorylation of Akt at Thr308 and Ser473 mediated by phosphoinositide-dependent kinase-1 (PDK-1) and mammalian target of rapamycin complex 2 (mTORC2), respectively \[[@R2]\]. Finally, activated Akt translocates to multiple cellular compartments where it phosphorylates a large number of substrates that transduce the signal to a variety of cellular responses that are intimately associated with malignancy, including cell proliferation, growth, motility, survival and metabolic reprogramming \[[@R3]\]. The Akt family comprises three isoforms - AKT1 (PKBα), AKT2 (PKBβ), and AKT3 (PKBγ) - which have non-redundant functions in various physiological as well as pathophysiological conditions \[[@R4]\].
The most frequent genetic lesions in this pathway comprise oncogenic mutations in *PIK3CA*, the gene that encodes the p110α isoform of class I PI 3-K \[[@R5]\]. Other breast cancer mutations prevalent in this pathway include mutational or epigenetic inactivation of Phosphatase and Tensin Homolog (*PTEN*), a lipid phosphatase that terminates PI 3-K signaling by dephosphorylating PIP3. Both oncogenic *PIK3CA* mutations and inactivation of *PTEN* lead to excessive and constitutive activation of Akt and downstream signaling, resulting in uncontrolled proliferation and increased cellular survival \[[@R6]\]. Mutations and amplifications in the Akt genes themselves have also been identified in various human solid tumors. In the context of breast cancer, the *AKT1(E17K)* somatic mutation was first identified in breast cancer but is also found in lung, bladder, endometrial, urothelial and prostate cancers \[[@R7]-[@R13]\]. The frequency of the *AKT1(E17K)* mutation in breast cancers ranges from 4-8%. This oncogenic mutation renders Akt constitutively active by broadening the lipid specificity of the Akt PH domain \[[@R14]\], thus enabling its transforming capacity in fibroblasts *in vitro* and leukemias *in vivo \[[@R7]\]*. Interestingly, in breast cancer *AKT1(E17K)* is mutually exclusive with *PIK3CA* and *PTEN* mutations \[[@R15]\], although in other cancers such as endometrial carcinoma, these mutations frequently co-exist in the same tumor \[[@R12]\]. Furthermore, *AKT1(E17K)* has been found predominantly in estrogen receptor (ER)-positive breast tumors \[[@R16]\]. However, *in vitro* studies have provided inconclusive information regarding the functional advantages this oncogenic mutation confers \[[@R17]\]. Expression of AKT1(E17K) has been shown to enhance cell migration and resistance to chemotherapeutic agents in luminal breast cancer cells \[[@R17], [@R18]\]. Similarly, knock-in of the *AKT1(E17K)* mutation into MCF-7 ER-positive cells in which oncogenic *PIK3CA(E545K)* has been restored to the wild-type allele restores proliferation and tumor growth *in vivo*, arguing that at least in ER-positive luminal breast cancer cells, *AKT1(E17K)* can function as a *bona fide* oncogene \[[@R19]\]. It is also worth noting that an analogous E17K mutation has been identified in *AKT2* in one breast cancer patient \[[@R20]\] and in *AKT3* in melanomas \[[@R21]\]. Moreover, a recurrent MAGI3-Akt3 fusion protein that results in a truncated form of the *MAGI3* gene fused in frame to *AKT3* at the E17 residue of Akt3 has been identified in breast cancers \[[@R22]\]. The mechanisms by which any of these somatic mutations contribute to malignancy have yet to be reported.
To date, no studies have examined the capacity of *AKT1(E17K)* to drive mammary cancer in a genetically engineered mouse model. Previous studies have addressed the contribution of AKT1 activity to mammary tumorigenesis using constitutively active AKT1 transgenes driven by the mouse mammary tumor virus (MMTV) promoter. MMTV-MyrAKT1 mice treated with DMBA to induce chemical carcinogenesis develop ER-positive mammary tumors \[[@R23]\]. In addition, transgenic mice harboring a phospho-mimetic *AKT1(T308D/S473D)* mutant in combination with *HER2* display a decrease in tumor latency and accelerated tumor growth, but decreased incidence of metastases, consistent with AKT1 functioning as a metastasis suppressor \[[@R24], [@R25]\]. Studies using AKT1 and AKT2 knockout mice have arrived at similar conclusions \[[@R26]\].
Since any association between AKT1 and ER has not been explored *in vivo,* and there are no models to evaluate the contribution of *AKT1(E17K)* to mammary tumorigenesis, we generated a mammary-specific inducible *AKT1(E17K)* transgenic mouse. We present evidence indicating that *AKT1(E17K)* is not sufficient for transformation *in vivo*, but induces mammary gland hyperplasia in both virgin and multiparous females. In addition, combination of MMTV-driven *AKT1(E17K)* with MMTV-*HER2* overexpression prevents *HER2-*driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling mediated by AKT1(E17K).
RESULTS {#s2}
=======
AKT1(E17K) escapes negative feedback inhibition but does not enhance proliferation of mammary epithelial cells *in vitro* {#s2_1}
-------------------------------------------------------------------------------------------------------------------------
To study the functional significance of the *AKT1(E17K)* mutation in breast cancer, we developed a system to stably express either wild-type *AKT1* or *AKT1(E17K)* in a doxycycline-inducible manner in the non-tumorigenic immortalized MCF10A breast epithelial cell line. Cells were serum-starved overnight and stimulated with 5% serum. Consistent with previous studies \[[@R17]\], basal phosphorylation of AKT1(E17K) at Ser473 and Thr308 is moderately elevated compared to wild-type AKT1 (Figure [1A](#F1){ref-type="fig"}). However, this does not translate into significantly enhanced phosphorylation of downstream Akt substrates as measured with a substrate-directed Akt motif antibody, as well as antibodies against known Akt substrates (Figure [1A](#F1){ref-type="fig"}). This is despite the fact that in a cell-free system, isolated AKT1(E17K) has significantly elevated protein kinase activity toward the model substrate GSK-3β, again when compared to wild-type AKT1. Apparently, this enhanced intrinsic kinase activity is not sufficient to propagate signals to constitutive downstream substrate phosphorylation in the absence of stimuli. Consistently, AKT1(E17K) cannot promote the proliferation of cells in the absence of serum and growth factors (Figure [1C](#F1){ref-type="fig"}), nor does it provide a proliferative advantage in full growth media (data not shown).
{#F1}
Since multiple feedback loops exist in the PI 3-K and Akt pathway to maintain homeostatic control, and oncogenic mutations in genes that modulate this pathway can often escape this feedback regulation, we next evaluated the kinetics of AKT1(E17K) activation in MCF10A cells. Cells expressing wild-type AKT1 show a robust induction of Akt phosphorylation in response to IGF-1 as early as 2 min, translating into Akt substrate phosphorylation (pPRAS40). This activation attenuates by 1 h post-stimulation and returns to basal levels by 24 h (Figure [2A](#F2){ref-type="fig"}), as would be expected by feedback inhibition. By contrast, cells expressing AKT1(E17K) show sustained Akt activation (pSer473, pThr308, | {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-ijms-17-01969}
===============
Glycosylation is a posttranslational modification that is found on about 50% of all proteins, in particular on secreted and transmembrane proteins of eukaryotes, archaea and to a lesser extent in prokaryotes \[[@B1-ijms-17-01969],[@B2-ijms-17-01969],[@B3-ijms-17-01969]\]. Eukaryotic proteins require glycosylation for proper folding, oligomerization and solubility, while glycans significantly prolong the stability and half-life time in many cases by protection against proteolysis \[[@B4-ijms-17-01969],[@B5-ijms-17-01969]\]. Although *N*-glycosylation is more frequent, *O*-glycosylation can similarly protect against general and specific proteolysis \[[@B6-ijms-17-01969],[@B7-ijms-17-01969],[@B8-ijms-17-01969]\]. Protein trafficking, i.e., the sending of proteins to cellular compartments or to the extracellular matrix, depends on specific, covalently linked glycans \[[@B9-ijms-17-01969]\]. In addition, glycans play an important role in the interaction and recognition of proteins, such as in the context of immunity and cell adhesion \[[@B10-ijms-17-01969],[@B11-ijms-17-01969],[@B12-ijms-17-01969]\]. Glycosylation may even protect against molecular damage by free radicals \[[@B13-ijms-17-01969]\]. In recent years, increasing evidence was found that glycans have distinct effects on the activity of many enzymes, in particular as regulatory modules for substrate binding and turnover. This study gives an overview on the most relevant types of glycosylation of proteases, regarding the structural knowledge and the functions of glycans. The importance of this little investigated field lies in the enormous diversity of possible glycosylation variants and the altered functionality of proteins under healthy or disease conditions.
*N*-glycosylation at sequons of the Asn-Xaa-Ser/Thr type is widespread in proteins of archaea and eukaryotes, whereby proline is largely excluded as Xaa and disfavored as residue following Ser/Thr \[[@B14-ijms-17-01969],[@B15-ijms-17-01969]\]. Some rare sequons are Asn-Xaa-Cys (1%), Asn-Gly (0.5%) and Asn-Xaa-Val (\<0.5%) \[[@B16-ijms-17-01969]\]. The process of *N*-glycosylation is extensively described in the literature on glycobiology \[[@B17-ijms-17-01969]\]. Essentially, a newly synthesized polypeptide emerging from a ribosome binds with a signal peptide to a signal recognition particle, which docks to a receptor in the endoplasmic reticulum (ER) membrane and forms a complex with the Sec machinery, which transfers the polypeptide through a transmembrane channel into the lumen of the ER \[[@B18-ijms-17-01969],[@B19-ijms-17-01969]\]. A signal peptidase cleaves the N-terminal signal peptide and the oligosaccharyltransferase complex attaches a GlcNAc~2~Man~9~Glc~3~ precursor at a suitable sequon of the Asn-Xaa-Ser/Thr type \[[@B20-ijms-17-01969]\]. Subsequently, the *N*-glycosylated polypeptide folds in the oxidizing environment of the ER, supported by protein disulfide isomerase for disulfide formation and by various chaperones \[[@B21-ijms-17-01969],[@B22-ijms-17-01969]\]. Afterwards, glucosidases and mannosidases trim the *N*-glycan precursor to Man~5~GlcNAc~2~ or GlcNAcMan~3~GlcNAc~2~ core glycans, which are extended by glucosyltransferases, accompanied by protein quality control and followed by sorting and further processing on their way through the ER Golgi intermediate compartment into the Golgi \[[@B23-ijms-17-01969],[@B24-ijms-17-01969]\]. Final modifications of the *N*-glycans in the Golgi comprise extensions by transferases that attach *N*-acetyl-glucosamine (GlcNAc), fucose, galactose, mannose, and sialic acid sugars, before sorting to secretory vesicles \[[@B25-ijms-17-01969]\]. Variations of branching generate a large diversity of *N*-glycans with distinct composition under physiological and pathological conditions ([Figure 1](#ijms-17-01969-f001){ref-type="fig"}A) \[[@B26-ijms-17-01969]\].
Earlier structural database analyses reported a relatively low percentage of only 27% *N*-glycosylation of all sequons in human proteins, of which 96% belonged to secreted and membrane proteins and 4% to cytoplasmic and nuclear proteins \[[@B28-ijms-17-01969],[@B29-ijms-17-01969]\]. More recent data suggested around 85% occupancy of sequons, with 50% glycosylation of all proteins in the SWISS-PROT sequence data bank \[[@B2-ijms-17-01969]\]. Thorough mass spectrometric analyses demonstrated that in the mouse *N*-glycoproteome the majority of identified sequons is occupied, e.g., 99% of predicted membrane protein *N*-glycosylation sites \[[@B16-ijms-17-01969]\]. Glycosylated Asn residues in human proteins are preferentially located in turns (78%) compared to β-sheets (12%) and α-helices (10%), which resembles the situation in murine proteins \[[@B16-ijms-17-01969],[@B28-ijms-17-01969]\]. The strict structural constraints for *N*-glycosylation are reflected by a similar localization in proteins of fish, insects, plants and lower eukaryotes \[[@B28-ijms-17-01969],[@B30-ijms-17-01969]\].
About 12% of all glycosylated proteins are exclusively *O*-glycosylated, while about 10% of them are both *N*- and *O*-glycosylated \[[@B2-ijms-17-01969]\]. Seven types of *O*-glycosylation have been found in humans ([Figure 1](#ijms-17-01969-f001){ref-type="fig"}B). The mucin-type with *N*-acetylgalactosamine (GalNAc) linked to Ser or Thr in membrane or secreted proteins is more common than *O*-glycosylation with xylose, galactose, glucose, fucose, and mannose, whereas mostly proteins with *O*-linked GlcNAc localize to the cytoplasm and the nucleus \[[@B27-ijms-17-01969]\]. Although no distinct consensus sequence is known, Pro-rich sequences are favored, such as Pro-Ser/Thr-Pro-Xaa-Pro \[[@B31-ijms-17-01969],[@B32-ijms-17-01969]\]. Usually, the first step of *O*-glycosylation of the mucin-type is performed in the Golgi by a GalNAc transferase, followed by extensions and branching, which results in eight core glycan subtypes \[[@B27-ijms-17-01969],[@B33-ijms-17-01969]\]. *O*-glycosylation of the mucin-type is important for tissue development and immune reactions \[[@B34-ijms-17-01969]\].
Among the rarer types of glycosylation is *O*-mannosylation for quality control and protection against proteolysis \[[@B35-ijms-17-01969]\]. Similarly, *O*-glycosylation of hydroxylysine can occur in mammals, in contrast to *O*-glycosylation of hydroxyproline, which is found in other eukaryotes \[[@B36-ijms-17-01969],[@B37-ijms-17-01969]\]. The uncommon *C*-mannosylation of Trp residues plays a role in protein folding, secretion and signaling \[[@B38-ijms-17-01969],[@B39-ijms-17-01969]\]. Glycosylphosphatidyl-inositol-anchored proteins (GPI-APs) are linked to glycolipids as membrane anchors, which is sometimes termed glypation \[[@B9-ijms-17-01969],[@B40-ijms-17-01969]\]. By contrast, glycation is the uncatalyzed covalent linkage of glucose or fructose with amino groups, which is involved in diabetes and ageing related diseases \[[@B41-ijms-17-01969],[@B42-ijms-17-01969]\]. Apart from this unregulated process, most glycans play very well defined roles in physiology, such as in the regulation of enzymatic activity.
2. Glycosylated Proteases {#sec2-ijms-17-01969}
=========================
Currently, the UniProtKB database lists about 250 glycosylated human proteases out of more than 700, with changing numbers due to putative or ambiguous classification \[[@B43-ijms-17-01969]\]. They exhibit either experimentally identified or predicted *N*-, *O*-, and *C*-glycosylation sites, using the NetNGlyc and NetOGlyc tools, which are currently applicable to mammalian entries and the mucin-type of *O*-glycans \[[@B29-ijms-17-01969],[@B44-ijms-17-01969]\]. The MEROPS database of proteolytic enzymes includes about 1200 known and putative human proteases, including inactive protease homologs \[[@B45-ijms-17-01969]\].
2. | {
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Visual working memory (VWM) is a cornerstone of human visual cognition. It temporarily retains visual information and makes it accessible for cognitive operation, report, and action control (Eriksson et al. [@CR10]; Oberauer [@CR32]; Poth and Schneider [@CR38]; Schneider [@CR48]). VWM has only limited capacity (Luck and Vogel [@CR23]; Shibuya and Bundesen [@CR50]; Sperling [@CR53]). Efficient use of this capacity dictates selectivity: currently relevant information must enter VWM with priority over less relevant information. This prioritization is performed by mechanisms of visual attention (Bundesen [@CR2]; Bundesen et al. [@CR3]; Duncan and Humphreys [@CR9]; Schneider [@CR47]). The bulk of attention research focused on prioritization up to the time of encoding into VWM (Bundesen et al. [@CR4]; Duncan [@CR8]; Poth and Schneider [@CR37]). However, flexible visual cognition requires that changes of priority can be accommodated also when they happen after information has entered VWM.
Indeed, more recent research demonstrated that prioritization continues after encoding into VWM. This research made use of the retro-cuing paradigm (Griffin and Nobre [@CR13]; Landman et al. [@CR18]). Participants memorized a set of visual stimuli, the *memory display*, over a retention interval which was followed by a probe stimulus. The task was to report whether the probe matched an item from the memory display. A so-called *retro*-*cue* (i.e., a "retrodictive" cue) was shown after the memory display but before the probe appeared. In the experiments of current interest, retro-cues could be valid or neutral (Astle et al. [@CR1]; Kuo et al. [@CR17]). A valid retro-cue predicted which of the items from the memory display was going to be relevant for the upcoming comparison with the probe. A neutral retro-cue did not contain any predictive information regarding this comparison. The central result is that valid retro-cues improved comparison performance relative to neutral retro-cues. Over a decade of research accumulated evidence for beneficial effects of valid retro-cues in different versions of the basic paradigm (Astle et al. [@CR1]; Griffin and Nobre [@CR13]; Landman et al. [@CR18]; Makovski and Jiang [@CR25]; Makovski et al. [@CR26]; Souza et al. [@CR52]). Thus, it seems safe to conclude that valid retro-cues prioritize an item from a preceding memory display, while the memory display is retained in VWM.
Still controversial, however, is the question which mechanisms underlie the prioritization within VWM (Souza and Oberauer [@CR51]). Current accounts assume that valid retro-cues improve comparison performance by manipulating the representations of the memory display in VWM. Specifically, some authors propose that they strengthen the VWM representation of the cued item, increasing the utility of this item for the comparison (Kuo et al. [@CR16]; Lepsien et al. [@CR21]; Nobre et al. [@CR31]). Others propose that they free VWM capacity and reduce interference within VWM by having uncued items removed from VWM (Souza et al. [@CR52]; Williams et al. [@CR60]). Again others suggest that valid retro-cues protect the cued item against decay (Matsukura et al. [@CR27]; Murray et al. [@CR30]; Pertzov et al. [@CR34]) or new interfering information (such as from the probe; Makovski et al. [@CR26]; Makovski and Jiang [@CR25]). Finally, some suggest that valid retro-cues grant the cued items priority in the process of being compared to the probe (Astle et al. [@CR1]; Makovski et al. [@CR26]; Nobre et al. [@CR31]). Fundamental to all these accounts is that retro-cues are, as the term implies, retroactive. That is, all accounts assume that valid retro-cues engage mechanisms that, in one way or the other, prioritize information from the past which is now retained in VWM.
Here, we ask whether retro-cues facilitate memory retention in VWM, or whether they enhance the future perceptual processing in service of the comparison task, or both. To this end, we introduce a novel paradigm which allows to disentangle such retrospective and prospective effects of retro-cues (Fig. [1](#Fig1){ref-type="fig"}). Participants briefly viewed a memory display of two colored squares and memorized them over a retention interval. This interval outlasted iconic memory traces and thus called for retention in VWM (for a review, see Irwin and Thomas [@CR14]). Afterward, a probe stimulus was presented that either matched or did not match an item from the memory display, with each alternative occurring in half the trials. The probe was presented at a location different from the stimuli of the memory display. This ensured that comparisons of probe and memorized stimuli needed to rely on VWM, as opposed to more fragile location-specific forms of short-term memory (see Pinto et al. [@CR36]). Participants' task was to indicate whether or not the probe matched an item from the memory display. A retro-cue appeared after the retention interval but before the probe. The retro-cue was either valid or neutral. A valid retro-cue pointed at the location of one of the items from the preceding memory display, the one that was going to be relevant for the upcoming comparison with the probe. Specifically, if the probe matched an item from the memory display, it was always the one indicated by the retro-cue. A neutral retro-cue did not contain any specific location information. We varied the presentation duration of the probe and terminated it with a pattern mask. This enabled us to assess performance as a function of the presentation duration. To disentangle the retrospective and the prospective effects of retro-cues, we fit these data with an exponential model (cf. Bundesen [@CR2]; Wickelgren [@CR59]) and compared the estimated parameters between valid and neutral retro-cues. The exponential model comprised three parameters. First, the level of asymptotic performance which is reached when the probe is shown long enough (see the upper asymptotes of the smooth curves in Fig. [2](#Fig2){ref-type="fig"}). The perceptual processing of the probe should improve with increasing presentation duration (e.g., Petersen and Andersen [@CR35]; Shibuya and Bundesen [@CR50]). At the asymptote, however, performance stops to increase with increasing presentation duration of the probe. Therefore, when the asymptote was reached, perceptual processing (encoding) of the probe should be over and variations of the asymptote should be due to post-perceptual factors. In the present case, variations in the asymptote should reflect the performance level for retaining the items of the memory display in VWM. Second, the rate at which performance increases with increasing presentation duration of the probe toward asymptotic performance (see how steeply the smooth curves increase with increasing presentation duration in Fig. [2](#Fig2){ref-type="fig"}). This is a well-established measure of processing speed (Bundesen [@CR2]; Bundesen et al. [@CR4]; Shibuya and Bundesen [@CR50]; Wickelgren [@CR59]). In contrast to reaction times, this measure has the important advantage that it reflects the specific perceptual (or cognitive) process without contributions of processes such as response selection or motor execution (that are always included in reaction times, see Finke et al. [@CR12], for a related discussion). Applied to the present case, the measure of processing speed should refer to the speed with which the probe is perceptually processed in order to accomplish the comparison with the items of the memory display. Third, a temporal threshold, reflecting the presentation duration of the probe that must be exceeded to increase performance above chance level (cf. Bundesen [@CR2]; Wickelgren [@CR59]).Fig. 1Paradigm of Experiment A. The paradigm of Experiment B was similar but differed in the display durations (and other aspects, see the Methods). Participants fixated a fixation square, after which a memory display with two colored squares appeared (the squares appeared at two out of four possible positions, whereby each pairing occurred equally often). After an interstimulus interval (ISI), a retro-cue was shown. If the retro-cue was valid, it indicated the location of the previous item from the memory display that was going to be relevant for the current trial. If it was neutral, it did not contain any predictive information. After another ISI, a probe was presented whose presentation duration was parametrically varied across trials. The probe was terminated by a pattern mask. In the end of a trial, a question mark prompted participants to indicate whether or not the probe matched an item from the memory displayFig. 2Performance of an individual participant of Experiment A (left) and average performance for the participants of Experiment A (middle) and B (right). Data points represent (average) performance (*d*′) in indicating whether or not probes matched an item from the memory display at each of the presentation durations of the probes. Error bars indicate ± one standard error for within-subjects designs (Loftus and Masson [@CR22]). The two retro-cue conditions are depicted separately. Smooth curves represent least-squares fits of the exponential model to the data of the two retro-cue conditions. As model fitting was performed for each individual in each condition | {
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Introduction
============
CTLs are crucial for the defense against many invading organisms and certain tumors. Presentation of antigenic peptides bound to MHC class I molecules is a prerequisite for stimulation of a CTL response, and therefore plays a pivotal role in providing CTLs with the capacity to respond to foreign antigens [1](#R1){ref-type="bib"}.
Peptides that meet the restrictive binding characteristics of MHC class I molecules for presentation to CTLs are generated after intracellular protein degradation by cytosolic proteases. The central enzyme responsible for protein degradation is the proteasome [2](#R2){ref-type="bib"}. Because of their intimate involvement in antigen processing and presentation [3](#R3){ref-type="bib"}, detailed knowledge on the cleavage preferences of proteasomes will be crucial for understanding CTL epitope generation and thus, for the regulation of specific immune responses.
The 20S proteasome represents the proteolytic core of the larger 26S proteasome complex that encompasses either one or two regulatory particles of at least 18 subunits [4](#R4){ref-type="bib"}. The eukaryotic 20S particle is composed of 14 different but related subunits organized in a barrel-shaped complex with the stoichiometry α~7~β~7~β~7~α~7~. Three subunits of the two inner β-rings (β1, β2, and β5) participate directly in peptide bond cleavage. They represent three distinct proteolytic activities, designated as the chymotrypsin (ChT)-like, trypsin-like, and peptidyl-glutamylpeptide--hydrolyzing (PGPH) activities [5](#R5){ref-type="bib"} [6](#R6){ref-type="bib"}. As the NH~2~-terminal threonine residues responsible for peptide bond cleavage do not prefer certain peptide bonds over others, the basis for the three distinct proteolytic activities most likely resides in the characteristics of the amino acids in the vicinity (pockets) of each active NH~2~-terminal threonine [7](#R7){ref-type="bib"}.
Upon IFN-γ exposure of cells, the three active β-subunits that are constitutively expressed in 20S proteasomes can be replaced by three IFN-γ--inducible homologues, low molecular weight protein (LMP)-2 (=β1i) (for Y \[β1\]), multicatalytic endopeptidase complex--like (MECL)-1 (β2i) (for Z \[β2\]), and LMP-7 (β5i) (for X \[β5\]). Although there is extensive sequence homology, these replacements alter the nature of peptides that are generated by proteasomes [8](#R8){ref-type="bib"} [9](#R9){ref-type="bib"} [10](#R10){ref-type="bib"} [11](#R11){ref-type="bib"} [12](#R12){ref-type="bib"}.
Proteasomes harboring these IFN-γ--inducible subunits are also called immunoproteasomes, as opposed to the constitutively expressed "constitutive" proteasomes, because immunoproteasomes were found to process a number of viral epitopes with greater efficacy in vitro [13](#R13){ref-type="bib"} [14](#R14){ref-type="bib"} [15](#R15){ref-type="bib"} [16](#R16){ref-type="bib"}. Using several artificial fluorogenic substrates in vitro, it was found that immunoproteasomes display a better capacity to cleave after hydrophobic and basic residues but are less well equipped for cleavage after acidic amino acids [17](#R17){ref-type="bib"}. The finding that proteasomes are responsible for the generation of the correct COOH terminus of several CTL epitopes [18](#R18){ref-type="bib"} [19](#R19){ref-type="bib"} and the notion that hydrophobic or positively charged amino acids serve in most cases as COOH-terminal anchor residues of MHC class I ligands led to the concept that immunoproteasomes contribute to more efficient MHC class I antigen processing. While this holds true for most viral antigens, recent studies have shown that some antigenic peptides are efficiently produced by constitutive proteasomes but cannot be generated by immunoproteasomes [20](#R20){ref-type="bib"}. This demonstrates that immunoproteasomes are not necessarily better suited for the processing of all MHC class I ligands.
To better understand the reasons why certain MHC class I ligands are generated with greater efficiency and others with lower efficiency in cells expressing different sets of proteasomes, we have performed an in-depth analysis of peptide fragments generated after proteasomal cleavage.
We have employed a strictly quantitative method to analyze a large collection of peptide fragments produced by either set of proteasome. Our observations allowed the identification of certain amino acids (or their characteristics) in positions distant, or directly flanking the cleavage sites selected by either set of proteasomes. The (quantified) mapping of cleavage sites using a large protein substrate provides the basis for a better understanding of proteasomal cleavage specificity, allowing a refined proteasomal cleavage prediction, which will be helpful for the identification of new CTL epitopes, the design of new (recombinant) vaccines, and for better insight into immunity against infection.
Materials and Methods
=====================
Purification of 20S Proteasomes.
--------------------------------
20S proteasomes were isolated as described previously [9](#R9){ref-type="bib"}. Frozen pellets of LCL-721 cells or LCL-721.174 cells were lysed in a buffer containing 0.1% Triton X-100 on ice and homogenized in a Dounce homogenizer. The 40,000× *g* supernatant of the lysate was bound to DEAE Sephacel. After elution, the protein fraction was concentrated and loaded onto a 10--40% sucrose gradient. After centrifugation, gradient fractions were tested for protease activity using the fluorogenic substrates succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and succinyl-tyrosyl-valyl-alanyl-aspartyl-7-amino-4-methylcoumarin (Suc-YVAD-AMC). Active fractions were pooled and further purified by anion exchange chromatography on a MonoQ HR5/5 FPLC column (Amersham Pharmacia Biotech). The purity of the proteasome preparates, checked by SDS-PAGE, was \>95%. Quantification of native proteasome protein was determined by a variation of the Lowry Method (protein assay; Bio-Rad Laboratories) and BSA as a standard.
Immunoblotting.
---------------
5 μg of purified proteasome polypeptides were separated by 12% SDS-PAGE and transferred to polyvinyldifluoride (DuPont) with a semidry transfer system. Human LMP-7 was detected using a rabbit polyclonal antiserum in conjunction with chemiluminescence (PW8200; Affiniti Research Products, Ltd.).
In Vitro Degradation of Enolase-1.
----------------------------------
150 μg of yeast enolase-1 were incubated in digestion buffer (20 mM Hepes/KOH, pH 7.6, 2 mM MgAc2, and 0.01% SDS) with proteasomes at a molar ratio of 150:1. Digestions were stopped by freezing the samples at −80°C when ∼50% of the substrate was digested (usually after ∼48 h).
Separation and Analysis of Cleavage Products.
---------------------------------------------
For the separation of degradation products, unfractionated enolase digests were subjected to μRP SC 2.1/10 columns (Amersham Pharmacia Biotech) on a Microbore HPLC system (SMART; Amersham Pharmacia Biotech). Buffer A contained 0.1% trifluoroacetic acid; buffer B contained 0.081% trifluoroacetic acid and 80% acetonitrile. Gradients were 0% for 5 min, in 40 min to 40% in buffer B, in 8 min to 75% in buffer B, and up to 85% in another 7 min at a flow rate of 150 μl/min. Fractions were collected by peak fractionation with a maximal volume of 500 μl/peak.
Peak fractions were dried and dissolved in 25 μl of 40% methanol, 1% formic acid, and subsequently analyzed by matrix-associated laser desorption ionization (MALDI) time of flight mass spectrometry (MS) (G2025A; Hewlett Packard) and NH~2~-terminal sequencing (Edman degradation) (pulsed liquid protein sequencer procise 494A; Applied Biosystems). Alternatively, peptides were analyzed on a hybrid quadruple orthogonal acceleration tandem mass spectrometer (Micromass). All these techniques were applied as described previously [21](#R21){ref-type="bib"}. Pmol amounts for each peptide detected in the HPLC fraction were determined by Edman sequencing and used for the quantitative analysis of the data.
Statistical Analysis - Frequencies of Amino Acids.
--------------------------------------------------
To detect statistically significant features in the amino acid distribution flanking the cleavage sites, we compared percent values using a classic chi-squared test for four tables (variance assumed due to counting). This method was used to compare constitutive and immunoproteasomes fragments with each other and with enolase. For a more thorough comparison of the absolute pmol amounts of constitutive and immunoproteasomes, we accounted for the experimental variability and, according to the quasilikelihood approach of Wedderburn et al. [22](#R22){ref-type="bib | {
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Background
==========
Soft tissue defects around the foot and ankle region often present an awkward problem to the orthopaedic surgeons. Tendons and bones are frequently exposed after trauma because of the thinness of the subcutaneous tissue. Traditionally, local flaps were used for resurfacing but their use is limited by their size and arc of rotation. Reverse flow flaps such as anterior tibial artery flap and posterior tibial artery flap require the sacrifice of major arteries. Free flap is currently the treatment of choice for large soft tissue defects of the distal extremity and it solves the problem of donor site morbidity in the immediate vicinity of the flap. It is however a technically demanding procedure for surgeons with less microsurgical experience. Furthermore in a few cases of trauma with damaged or occluded major vessels, where a free flap may be potentially hazardous, the reverse sural artery flap can prove to be one of the few safe options for soft tissue coverage \[[@B1]\]. The accompanying arteries of the lesser saphenous vein and sural nerve have been utilized with success for harvest of reverse flow sural flap \[[@B2]\]. The sural nerve remains the anatomic landmark for the inclusion of vessels in pedicle of the flap. The sural artery reverse flow flap is nourished by the lowermost perforating branch of the peroneal artery. Since the introduction of this flap by Masquelet \[[@B3]\] in 1992, it has become one of the favourite among reconstructive surgeons. It is the flap of first choice in our centre. In the present report, the results of reverse flow sural neurocutaneous flaps done at our centre is presented along with some thought provoking ideas for the future.
Methods
=======
This descriptive case series was conducted between 1997 and 2003, at Queen Mary Hospital, The University of Hong Kong, Division of Hand Surgery, Department of Orthopaedics and Traumatology. It is a tertiary care centre for cases requiring microsurgery of the extremities. From 1997 to 2003, we performed ten reverse sural neurocutaneous flaps on ten patients (See Table [1](#T1){ref-type="table"}). The age ranged from 19 to 85 years, with a mean age of 59.8 years. There were six females (60%) and four male patients (40%). The soft tissue defects were located on non weight bearing heel in four patients (40%), tendo Achilles in two patients (20%), distal tibia in two patients (20%), lateral malleolus in one patient (10%) and medial aspect of the midfoot in one patient (10%). No patient underwent resurfacing of the weight bearing heel. The minimum and maximum flap size harvested (length × breadth in cm.) was 5 × 4 and 14 × 6 respectively. A midline cuff of gastrocnemius was included in one of the flap harvested from the proximal calf area. All benefits and disadvantages of the operation were discussed with the patient before the operation, including the sensory loss in the distribution of the sural nerve. A detailed questionnaire was developed using variables of interest.
######
Patients\' data. Demographic features, etiology, defect site and size, comorbids, flap type and outcome
**Sex** **Age** **Etiology** **Site of defect** **Flap size (cm)** **Comorbids** **Flap type** **Complications**
--------- --------- --------------- ------------------------------- -------------------- --------------- --------------- ------------------------
F 72 Ulcer Medial aspect of mid foot 9 × 7 DM Sural Mild venous congestion
M 74 Pressure sore Lateral malleolus 5 × 4 Paraplegia Sural
F 68 Ulcer NWB heel 8 × 6 DM Sural
F 62 Trauma NWB heel and medial malleolus 11 × 7 Sural
M 48 Trauma Tendoachilles 8 × 6 Sural
F 53 Trauma Shin 8 × 6 Sural
F 19 Trauma NWB heel 14 × 6 Sural
M 52 Trauma Distal tibia 9 × 7 Sural
F 85 Ulcer Achilles tendon 7 × 4 PVD, DM Sural
M 65 Ulcer Posterior heel 8 × 5 DM Sural
Preoperative evaluation included identification of the site of peroneal perforators above the lateral malleolus using Doppler flow meter. Two or three perforators were identified above the lateral malleolus. The pivot point of the pedicle was chosen according to the need of distal coverage, but it was limited by the lower most perforator, about 05 cm. from the tip of the lateral malleolus.
The skin was incised close to the midline, so as to be sure to include the short saphenous vein in the pedicle in all patients. The skin flap was then elevated with the deep fascia. The pedicle was kept wide, around 03 to 04 cm. in diameter. The flap was rotated 180 degrees through an open tunnel in all but one patient. Split thickness skin graft was used to cover the exposed pedicle in all but one patient. All patients were kept in the \'modified plaster of paris boot\' in the post operative period (Figure [1](#F1){ref-type="fig"}).
{#F1}
Results (Also see Table [1](#T1){ref-type="table"})
===================================================
Survival
--------
All flaps survived.
Venous congestion
-----------------
Only one flap developed mild venous congestion in the early post operative period, which resolved with foot elevation.
Weight bearing problems
-----------------------
None of our patients had resurfacing to the weight bearing part of the heel. There was no interference with walking.
Donor and recipient site appearance
-----------------------------------
The donor site had no complications except for the relatively unsightly scar. It was acceptable to all patients. All patients were satisfied with the cosmesis of the recipient site.
Numbness/Neuroma
----------------
Transient numbness along the sural nerve distribution was experienced by the patients. It resolved in all patients by three months. No painful neuroma developed as we routinely buried the nerve stump in the deep muscular plane.
Need for debulking of the flap
------------------------------
No patients required debulking for cosmetic reasons or shoe wear problems.
Case reports
============
Case report 1 (Figures [2A](#F2){ref-type="fig"} and [2B](#F2){ref-type="fig"})
-------------------------------------------------------------------------------
{#F2}
72 years diabetic lady suffering from ulcer on the dorsomedial aspect of the right midfoot was successfully covered using the reverse sural flap from proximal calf area with the modification of inclusion of the midline cuff of gastrocnemius, as described by Al Qattan MM \[[@B4]\].
Case report 2 (Figures [3A](#F3){ref-type="fig"}, [3B](#F3){ref-type="fig"}, [4A](#F4){ref-type="fig"}, [4B](#F4){ref-type="fig"} and [4C](#F4){ref-type="fig"})
----------------------------------------------------------------------------------------------------------------------------------------------------------------
{#F3}
{#F4}
19 years lady sustained road traffic accident resulting in skin necrosis of the posterior aspect of the left heel. Reverse sural flap size 14 × 6 cm. was used to successfully cover the defect.
Discussion
==========
The results of this study show that the reverse sural neurocutaneous flap is an effective method | {
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Background
==========
Increased population density and movement of people around the globe have generated a rise in the number of outbreaks of infectious diseases and led to the emergence of new infectious diseases \[[@B1]\]. Worldwide, 3.575 million people die each year from water-related diseases \[[@B2]\]. The water and sanitation crises claim more lives through disease than any warfare \[[@B2]\]. A key step in the prevention of outbreaks of communicable diseases is the early detection of virulent particles \[[@B3]\]. Rapid detection of active viral pathogens is of central importance for public health risk assessment and environmental protection. Waterborne viruses are particularly important for public safety monitoring due to their environmental stability and low infectious dose; a single virion is sufficient to initiate illness in previously unexposed, healthy adults \[[@B4]\].
Enteroviruses (family Picornaviridae) are a genus of waterborne viruses that infect humans and other mammals. They are a health problem worldwide, leading to 10 to 15 million cases of symptomatic infection in humans annually in the United States alone \[[@B5]\]. Enteroviruses are single, positive-strand RNA viruses that include polioviruses, Coxsackieviruses and echoviruses, among others. Some enteric virus groups have emerged as waterborne pathogens because of their high levels of resistance to current water treatment processes, which include ultraviolet light inactivation and heat inactivation \[[@B6],[@B7]\]. Poliovirus was used here as a model virus because a large body of research data exists on the physical, chemical and biological properties of the virus, vaccination is available, and its ease of cell culturing \[[@B8]-[@B10]\]. In addition, poliovirus remains endemic in four countries. During 2002 the rejection of polio immunization led to a worrying resurgence of polio in some areas of Nigeria, followed by re-infection in 21 other countries; resurgence of the disease was also observed in India. Auxiliary vaccination actions were restarted and by 2007 most re-infected countries had become polio-free again. The goal of global polio eradication was re-set to 2010, but concerns continue to be expressed about the progress of this eradication program \[[@B11]\].
Current methods for enterovirus detection use mammalian cell culture and require complex analyses (visible monolayer cytopathic effects) that require several days of laboratory time \[[@B12]\]. Polymerase chain reaction (PCR) methods for the detection of viruses have been developed, offering specificity, speed and cost advantages over cell culture methods \[[@B13],[@B14]\]. PCR methods alone do not, however, differentiate between the presence of physical (inactive) virus particles and viable (active) virus particles \[[@B6],[@B15]\]. The major disadvantage of most current methods of virus detection is the inability to provide information about whether a viral particle can start an infection or not. Faster methods with increased sensitivity and specificity are needed to quantify active viral pathogens from medical and environmental samples.
Virus-infected individuals can excrete over 1 billion (10^9^) viruses/g of feces. Some enteric viruses can also be excreted in urine from infected individuals. The presence of these viruses in a human population is variable and reflects current epidemic and endemic conditions \[[@B16]\]. In general, the level of infectious enteric virions in sewage ranges from 100 to 10,000 infectious units/L \[[@B17]-[@B23]\]. In contaminated surface water, levels of 1-100 infectious enteric virions/L are common. In less polluted surface water, their numbers are closer to 1-10/100 L. Groundwater sources have been shown to have between 0 and 200 infectious enteric virions/100 L, depending on the level of contamination; however, most contaminated groundwater systems are thought to have very low levels (\< 2/100 L) \[[@B24]\]. These concentrations were generally obtained through targeted studies, since water and wastewater sources are not routinely monitored for enteric viruses. These measurements are typically performed after an extensive concentrating step which reduces sample volume by 100 to 1000-fold before virus detection is performed.
FTIR spectroscopy is a noninvasive measurement method that has previously been applied for identifying various biological components of cells by detecting vibrations of molecules leading to spectral patterns \[[@B12],[@B25]\]. It can be used as part of a sensitive method for the detection of specific cellular molecular changes \[[@B12],[@B26]-[@B30]\]. Quantitative infrared absorption methods such as FTIR spectroscopy differ from ultraviolet/visible molecular spectroscopic methods because of the greater information content of the spectra. FTIR spectroscopy has been applied in medicine, particularly to study the process of herpes virus infection \[[@B31]\] and in the diagnosis of cancers and other disorders \[[@B32]-[@B35]\]. In addition, FTIR spectroscopy has been used for the quantification of blood serum components such as glucose, protein, cholesterol and urea \[[@B36]\].
Cell based sensors detect changes in the physiological state of cells following exposure to an environmental stimulus. Changes in cell state can provide information about the stimulus; for example, cells have been used to sense toxins in water samples \[[@B37],[@B38]\]. Cell based sensors have recently been combine with spectroscopy and applied to viral detection \[[@B39],[@B40]\]. Cantera et al. \[[@B41]\] used an optical system that involved the use of molecular beacons as a way to detect infective virus particles, resulting in a detection limit of 1 PFU. Using live cells to assist in identifying and quantifying viruses in samples helps to bring the detection closer to an *in vivo*setting, allowing the natural and complex interactions between cell and virus to be part of the experimental setup.
Here we present the development of a novel strategy for virus detection using a combination of FTIR spectroscopy and live BGMK cells (Figure [1](#F1){ref-type="fig"}). Specific absorbance patterns are monitored following changes in cell components (such as lipids, proteins, nucleic acids and sugars) subsequent to the virus infection, effectively using the cells as biosensors \[[@B42],[@B43]\].
{#F1}
Results
=======
Microscopy of cell structure on crystal
---------------------------------------
The way in which cells attach in monolayer culture depends on the cell type and the characteristics of the surface. BGMK cells were confirmed to be biocompatible with the ZnSe crystals for transmission measurements. The organization of the actin cytoskeleton exhibited a good distribution throughout the cell volume with many focal adhesion points that resulted in a spread phenotype on the stiff surface, indicating actin assembly by healthy cells. The cell architecture of the BGMK cells attached to the ZnSe crystal can be seen in Figure [2a](#F2){ref-type="fig"}. Actin, vinculin and cell nuclei were stained to study cell adhesion of BGMK cells on the ZnSe crystal. Figure [2b](#F2){ref-type="fig"} shows the cells under bright field microscopy, where they can be seen elongated and spread out over the crystal surface.
{#F2}
Optimal time for virus detection
--------------------------------
BGMK cells were infected with poliovirus PV1 at different multiplicities of infection (m.o.i.) of 10 PFU (0 - 10^6^PFU/ml) and studied at 1, 1.5, 2, 4, 5, 6, 8 and 12 h.p.i. Virus infection regression models were developed to correlate changes in spectral features with time of infection. Changes in the spectra varied depending on the progress of the viral infection, with biochemical alterations appearing in poliovirus infected cells within 2 h.p.i. Example regression models for 1.5, 4, 6 and 8 h.p.i. are shown in Figure [3](#F3){ref-type="fig"} and a summary of the regression model parameters are given in Table [1](#T1){ref-type="table"}. This table shows the comparison between the error of calibration (RMSEC) and the root mean square error of crossvalidation, RMSECV, which is a mesure of a model\'s ability to predict samples that were not used to build the model (leave-one-out-crossvalidation). RMSECV was analyzed to determine the optimum number of latent variables (LVs) to include in the PLS model. The number of LVs of a PLS model is usually optimized by performing a cross-validation and minimizing the corresponding RMSECV. The RMSECV decreases with the inclusion of each additional initial factor, reaching a minimum value with a certain number of latent variables. The best choice of number of latent variables is also supported by the variance capture in Y. The goal is to identify a subset of the measured variables that gives the lowest RMSECV, which is the most useful and accurate regression model. General calibration procedure consists in the selection of the pretreatment (preprocessing of the spectra), wavelength intervals, and number of latent variables to be driven by minimizing the RMSECV \[[@B44]\]. An infection time of 8 h was selected for subsequent experiments based on these results and on the reported viral replication time \[[@B44]\]. For more information on the spectra of 8 h and 12 h uninfected control cells see Additional File [1](#S1){ref-type="supplementary-material"} and Additional File [2](#S | {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Body size, a key trait for all organisms, affects various aspects of survival and reproduction of individuals in their interactions with their physical and biological environments. In animals, divergence of body size among related populations is often related to adaptation to differing environmental conditions^[@CR1]^ and/or mitigation of interactions with other species through resource competition and/or reproductive interference^[@CR2]--[@CR4]^. Revealing the genetic basis for body size differences between closely related populations is important in understanding the adaptive divergence of populations, which may ultimately result in speciation.
Genes involved in the control of animal body size have been examined in various mammals and insects^[@CR5],\ [@CR6]^. Several pathways have been investigated in *Drosophila melanogaster*, including the insulin signalling pathway, which contributes to the regulation of growth rates and body size in response to environmental cues, such as the presence of nutrients^[@CR6]^. For example, overexpression of an insulin-like peptide during development can increase body size^[@CR7]^. Mutations in genes involved in insulin signalling and other developmental pathways regulating larval growth^[@CR8]^ may also be involved in the evolution of overall body size in insects. However, the genetic basis for body size differences among populations or closely related species remains largely unknown. Although several genetic loci or markers in *Drosophila* show strong associations with geographic variation in body size^[@CR9]--[@CR14]^, the relationships between genes or alleles and the regulation of body size that affect body size evolution remain unclear^[@CR6]^.
Here, we explored the genetic basis for inter-population body size differences in the carabid ground beetle *Carabus* (*Ohomopterus*) *japonicus* (Fig. [1a](#Fig1){ref-type="fig"}). The subgenus *Ohomopterus*, which is endemic to the Japanese islands, shows marked variation in body size (body length) within and among species^[@CR15],\ [@CR16]^. Notably, two or three species with different body sizes co-occur in a large region of the subgenus' range; the size differences are the main contributors to the avoidance of hybridisation^[@CR4]^. *C*. *japonicus* is sympatric with one or two larger species existing within the majority of its range^[@CR16]^; however, it exhibits larger bodies in several solitary islands, consistent with the pattern of character release^[@CR17]^. Such inter-population divergence might be an initial step in speciation via recurrent secondary contacts between populations of differently sized individuals.Figure 1The body lengths of adult P, F~1~, and F~2~ *Carabus japonicus* beetles. (**a**) Males and females from Mt. Aburayama and Kabeshima Island were used in the present study. Measurements of body dimensions are shown. BL, body length; HW, head width; HL, head length; TW, thoracic width; TL, thoracic length; EW, elytral width; EL, elytral length. (**b**) BLs of parents and F~1~ and F~2~ individuals in the crossing experiment.
In this study, we used a traditional approach of accessing the loci responsible for quantitative traits (i.e., QTL mapping) combined with whole genome sequencing and gene prediction to determine the genomic region and gene loci responsible for inter-population body size differences. First, we assembled the genomic sequence of *C*. *japonicus* and predicted protein-coding genes on the scaffolds. Second, we obtained F~2~ individuals by crossing females and males from populations with large and small bodies and measured their body size dimensions. Then, we obtained a large number of restriction site-associated DNA (RAD) sequences for F~2~ individuals to construct linkage maps and to perform QTL mapping. The locations of QTLs for body size on the linkage maps were estimated from the correlations between the phenotypic values of a body dimension and genotypes among F~2~ individuals. Finally, we searched for protein-coding genes potentially related to the body size differences by exploring the scaffolds of the *C*. *japonicus* genomic sequence containing QTLs for body size with reference to the known functions of annotated genes.
Results {#Sec2}
=======
By experimental crossings, we obtained 40 male and 40 female F~2~ adults (Fig. [1b](#Fig1){ref-type="fig"}). Although the initial first instar weight (BW1) and total development time from larval hatching to adult eclosion (TDT) showed no difference between the sexes (BW1, *F* ~1,75~ = 0.073, *P* = 0.7879; TDT, *F* ~1,77~ = 2.327, *P* = 0.1312), the growth rate in terms of increasing body weight during the immature stages was larger in females than in males (*F* ~1,74~ = 7.875, *P* = 0.0064), leading to larger body sizes in females. Excluding the effect of sex, the geometric mean of all adult body dimensions (GM) was positively related to the initial larval body weight (proxy of egg size), developmental time from oviposition to adult emergence, and growth rate (Fig. [2](#Fig2){ref-type="fig"}; Table [1](#Tab1){ref-type="table"}). Of these three variables, growth rate had the largest effect, while the remaining two variables had comparable effects. Overall body length (BL; Fig. [1a](#Fig1){ref-type="fig"}) was closely correlated with GM (*r* = 0.962, *P* \< 0.0001) and showed almost identical responses to the four variables (results not shown). Because of the sexual dimorphism in body dimensions, we used sex-adjusted dimensions in the QTL analyses. Hereafter, symbols for individual body dimensions (HL, HW, TL, TW, EL and EW; Fig. [1a](#Fig1){ref-type="fig"}) and overall body size dimensions (GM and BL) are used for log~10~ transformed, sex-adjusted values. All of these variables were significantly correlated with each other except for two cases (Fig. [3](#Fig3){ref-type="fig"}).Figure 2Correlations between developmental trait values and body size dimensions. Open and closed circles indicate male and female individuals, respectively. BW1, BWA and BL are log~10~ transformed values, and GM (geometric mean of all body dimensions) was calculated as the arithmetic mean of all log~10~ transformed body dimensions except BL. Table 1Effects of sex, developmental time (DT), larval weight at hatching, and growth rate on the geometric mean of all adult body dimensions (GM; log10 transformed).VariableEffectSEdf*tP*Sex \[female-male\]0.4190.0631,70.116.67\<0.0001Initial larval weight (BW1)0.6130.0721,70.258.49\<0.0001Development time (TDT)0.1870.0691,70.112.730.0080Growth rate0.8240.0881,70.179.37\<0.0001All variables (other than sex) were standardised. Family was incorporated as a random variable. Figure 3Correlations between body size dimensions. The values in millimetres were log~10~ transformed and adjusted for sex differences. Open and closed circles indicate male and female individuals, respectively. Pearson correlation coefficients (*r*) and *P* values are given in the panels. The body dimensions are described in the legend of Fig. [1](#Fig1){ref-type="fig"}.
Sequence assembly resulted in the construction of 76,540 scaffolds with an N50 of 734,069 bp (max. 6,537,065) and total length of 191,032,029 bp (N, 8%; GC, 35.0%). A total of 14,929 protein-coding genes were predicted; of these, 10,624 (71%) were assigned to known genes by a blast search.
We constructed 23 autosomal linkage groups with a total length of 1,029 cM from 319 RAD loci and an X-chromosome linkage group of 91 cM from 56 RAD loci (a total of 1,120 cM with 375 markers; Fig. [4](#Fig4){ref-type="fig"}). Based on shared scaffolds, the number of autosomal linkage groups could be reduced to 17, which was still larger than the actual haploid autosome number of 13.Figure 4Linkage map of *Carabus japonicus* showing the locations (peak and range) of the body dimension quantitative trait loci (QTLs). Two-way arrows connect linkage groups probably located on the same chromosomes.
Among the autosomal linkage groups, we found two QTLs of large effect for body length (BL) and geometric mean of all body dimensions (GM) on LG5 and LG6 (*R* ^2^ = 0.26--0.35; Figs [4](#Fig4){ref-type="fig"} and [5](#Fig5){ref-type="fig"}; Table [2](#Tab2){ref-type="table"}). Of the components of BL, elytral length (EL), which comprises 63.1% of BL on average, had two QTLs corresponding to those of BL on LG5 and LG6 and an additional QTL on LG6; these QTLs had large effects (*R* ^2^ = 0.36−0.44). In addition, a QTL for | {
"pile_set_name": "PubMed Central"
} |
(J Am Heart Assoc. 2020;9:e015118 DOI: 10.1161/JAHA.119.015118.)31992159
Clinical PerspectiveWhat Is New?The present study is the first population‐based, untargeted metabolomic analysis of left ventricular diastolic function in a biracial (35% black, 65% white) community‐based sample of middle‐aged adults**.**We found that lipid‐ and amino acid--derived metabolites independently associate with patterns of mitral inflow and left ventricular relaxation in people without heart failure.What Are the Clinical Implications?This hypothesis‐generating study serves as an original groundwork to use metabolite biomarkers for the detection of subclinical heart failure in the general population.
Introduction {#jah34809-sec-0008}
============
The prevalence of heart failure in the United States is projected to increase by over 40% to affect \>8 million individuals by 2030.[1](#jah34809-bib-0001){ref-type="ref"}, [2](#jah34809-bib-0002){ref-type="ref"} Together with global epidemiologic trends, this estimated rise in prevalence classifies heart failure as the most rapidly increasing form of cardiovascular disease (CVD) in the United States and globally.[3](#jah34809-bib-0003){ref-type="ref"} Among all heart failure cases, heart failure with preserved ejection fraction (HFpEF) is becoming the major disease subtype, presently responsible for at least 50% of all heart failure cases.[4](#jah34809-bib-0004){ref-type="ref"}, [5](#jah34809-bib-0005){ref-type="ref"} Such data underline a crucial need to characterize and study subclinical HFpEF phenotypes, which may not only allow for early HFpEF risk prediction but also lead to the development of low‐risk interventions to delay or prevent heart failure onset, improving quality of life in an aging population.
Left ventricular diastolic dysfunction (LVDD), defined by impaired relaxation, decreased compliance, and/or increased left ventricular filling pressures, is one key subclinical precursor for HFpEF.[6](#jah34809-bib-0006){ref-type="ref"} This upstream phenotype represents a potential inflection point on the HFpEF causal disease pathway, as evidence suggests that LVDD is reversible.[7](#jah34809-bib-0007){ref-type="ref"}, [8](#jah34809-bib-0008){ref-type="ref"} Molecular phenotyping may represent a cost‐effective approach for identifying individuals with asymptomatic LVDD, while also implicating novel biological pathways in heart failure pathogenesis. Because metabolites reflect the collective output of both genetic and environmental factors[9](#jah34809-bib-0009){ref-type="ref"} and share a proximal relationship with clinical phenotypes, metabolomics profiling represents a particularly powerful molecular tool for such precision medicine initiatives. Unfortunately, few studies have applied this methodology to the study of LVDD.[10](#jah34809-bib-0010){ref-type="ref"}, [11](#jah34809-bib-0011){ref-type="ref"} Given the fact that both impaired systemic and myocardial metabolism have been implicated in HFpEF pathogenesis,[12](#jah34809-bib-0012){ref-type="ref"} there is a need to determine the role of systemic metabolites in left ventricular diastolic function (LVDF) to improve heart failure prevention.
The current study used metabolomics profiling and echocardiography to conduct a metabolome‐wide association study of LVDF in a cross‐sectional analysis of BHS (Bogalusa Heart Study) participants. We hypothesized that serum metabolites, particularly those involved in biological pathways involving fatty acid metabolism, would be associated with diastolic function.
Methods {#jah34809-sec-0009}
=======
The authors declare that all supporting data are available within the article and its online supplementary files.
Study Population {#jah34809-sec-0010}
----------------
The BHS, seated in Bogalusa, Louisiana, is a population‐based study examining the natural course of CVD across the life span. Between 1973 and 2016, 7 surveys of children aged 4 to 17 as well as 10 surveys of adults, who had been previously observed as children, were completed. The current BHS cohort includes 1298 participants, born between January 1959 and June 1979, who were examined twice in both childhood and adulthood for CVD risk factors and outcomes. Among these 1298 BHS participants, we selected the 1050 individuals who had echocardiographic, metabolomic, and respective covariable data measured contemporaneously at the most recent BHS study examination taking place from 2013 to 2016 (Figure [S1](#jah34809-sup-0001){ref-type="supplementary-material"}). The current study sample (n=1050) was highly similar to the overall BHS sample (n=1298) with respect to sociodemographic characteristics and traditional CVD risk factors (Table [S1](#jah34809-sup-0001){ref-type="supplementary-material"}). All study participants provided written informed consent at each examination, and study protocols were approved the Institutional Review Board of the Tulane University Health Sciences Center.
Echocardiography Protocol {#jah34809-sec-0011}
-------------------------
Two‐dimensional and tissue Doppler echocardiography were performed by trained cardiac sonographers at the BHS field office. Participants were placed in a partial left lateral decubitus position for echocardiographic assessment. Ten cycles of each 2‐dimensional and Doppler signal were recorded. Echocardiographic recordings were accomplished using an Aplio 300 ultrasound instrument (Toshiba America Medical Systems, Tustin, CA) with a linear array transducer of 7.5 mHz using a standard protocol.[13](#jah34809-bib-0013){ref-type="ref"}, [14](#jah34809-bib-0014){ref-type="ref"} Diastolic function parameters assessed included early left ventricular filling peak velocity (E), late left ventricular filling peak velocity (A), medial mitral annular velocity (e'), deceleration time of the E wave (DT), isovolumic relaxation time (IVRT), and left atrial maximum volume index.[15](#jah34809-bib-0015){ref-type="ref"} The apical 4‐chamber view was used to assess left atrial volume as well as patterns of mitral inflow including, DT, E wave, A wave, and medial e' wave velocities. The E and A wave velocities were assessed using pulsed‐wave Doppler echocardiography of transmitral flow at the mitral valve leaflet tips, while medial e' velocity was measured using pulsed‐wave tissue Doppler echocardiography of the mitral annulus. The apical 5‐chamber view was used to measure IVRT, specifically by placing the sample volume in the left ventricular outflow tract to concurrently evaluate aortic ejection and the onset of mitral inflow. Doppler sample volumes were placed between the mitral leaflet tips to measure DT. Left atrial volume was quantified by observing windows in which the left atrial length and transverse diameter were maximized. Left atrial volume was then divided by body surface area to estimate left atrial maximum volume index. Heart rate was assessed during the echocardiography protocol.
Metabolomic Profiling {#jah34809-sec-0012}
---------------------
Fasting serum samples were obtained between 2013 and 2016 from BHS participants and stored at −80°C prior to metabolite quantification. Metabolomic analysis of serum samples was consequently performed at Metabolon Inc (Durham, NC). Metabolites were quantified using ultra‐high‐performance liquid chromatography--tandem mass spectroscopy,[16](#jah34809-bib-0016){ref-type="ref"} providing a comprehensive analysis of serum metabolites. Metabolites were identified by automated comparison of ion characteristics to a reference library of chemical standard records that include retention time, molecular weight (m/z), preferred adducts, and in‐source fragments, as well as associated mass spectroscopy spectra. The majority of missing data in mass spectroscopy--based metabolomics experiments is due to detection limitations.[17](#jah34809-bib-0017){ref-type="ref"} Thus, if a metabolite value was missing, we assumed that it was below the detection limit and assigned it the lowest detectable value in the data set for the corresponding metabolite. Recent metabolomics studies have employed a similar technique.[18](#jah34809-bib-0018){ref-type="ref"}, [19](#jah34809-bib-0019){ref-type="ref"}
Untargeted metabolomic analysis yielded 1466 metabolites, including 956 known biochemical compounds. These metabolites belonged to pathways related to amino acids (n=184), lipids (n=408), nucleotides (n=41), peptides (n=35), carbohydrates (n=25), cofactors and vitamins (n=34), xenobiotics (n=220), and energy (n=9). Furthermore, an additional 510 unnamed compounds presently lacking chemical standards were also identified. We excluded metabolites (n=213) detected in \<20% of participants or with limited intra‐assay reliability, defined by a reliability coefficient \<0.30 (n=51) in duplicate samples. After completion of quality control procedures, 1202 metabolites remained for analysis. Among these 1202 metabolites, 167 were missing or under the detection limit in 50% to 80% of the samples. These latter metabolites were categorized and analyzed according to 3 mutually exclusive groups: (1) missing or below the detection limit; (2) below the median of measured values; and (3) greater than or equal to the median.[18](#jah34809-bib-0018){ref-type="ref"}, [19](#jah34809-bib-0019){ref-type="ref"} For the remaining 1035 metabolites, the minimum observed value of each analyte was imputed for below‐the‐detection‐limit or missing | {
"pile_set_name": "PubMed Central"
} |
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