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1.  Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle 
Journal of cellular biochemistry  2010;110(4):875-881.
TPM1κ is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1κ expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1κ protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1κ mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1κ specific antibody, we demonstrated the expression and incorporation of TPM1κ protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1κ in myofibrillogenesis and sarcomeric function.
doi:10.1002/jcb.22599
PMCID: PMC2895019  PMID: 20564186
Ambystoma mexicanum; Heart; Skeletal muscle; Growth & development; Reverse Transcriptase Polymerase Chain Reaction; Tropomyosin
2.  A cluster randomised controlled trial protocol of an adapted intervention for alcohol use disorders in people living with HIV and AIDS: impact on alcohol use, general functional ability, quality of life and adherence to HAART 
BMC Psychiatry  2017;17:44.
Background
Interventions for alcohol use disorders (AUDs) in HIV infected individuals have been primarily targeted at HIV risk reduction and improved antiretroviral treatment adherence. However, reduction in alcohol use is an important goal. Alcohol use affects other key factors that may influence treatment course and outcome. In this study the authors aim to administer an adapted intervention for AUDs to reduce alcohol use in people living with HIV/AIDS (PLWHA).
Methods
This study is a cluster randomised controlled trial at 16 HIV care clinics. A motivational interviewing and cognitive behavioural therapy based intervention for AUDs, developed through adaptation and piloted in Zimbabwe, will be administered to PLWHA with AUDs recruited at HIV clinics. The intervention will be administered over 16 sessions at 8 HIV clinics. This intervention will be compared with an equal attention control in the form of the World Health Organization Mental Health Gap Action Programme (WHO mhGAP) guide, adapted for the Zimbabwean context. General function, quality of life, and adherence to highly active antiretroviral treatment (HAART) will be secondary outcomes. Booster sessions will be administered to both groups at 3 and 6 months respectively.
The primary outcome measure will be the Alcohol Use Disorder Identification Test (AUDIT) score. The World Health Organisation Disability Assessment Schedule 2.0 (WHODAS 2.0), World Health Organisation Quality of Life (WHOQoL) HIV, viral load, and CD4 counts will be secondary outcome measures. Outcome assessments will be administered at baseline, 3, 6, and 12 months. Moderating factors such as perceived social support, how people cope with difficult situations and post-traumatic exposure and experience will be assessed at baseline. Trained research assistants will recruit participants. The outcome assessors who will be trained in administering the outcome and moderating tools will be blinded to the treatment arms allocated to the participants. However, the principal investigator, participants and intervention staff will be unblinded.
Data will be analysed using STATA Version 14. Primary and secondary outcomes will be measured at four time points that is; at baseline, 3, 6, and 12 months respectively. All participants will be included in the analysis of primary and secondary outcome measures. The mean AUDIT scores will be compared between groups using student t-tests. Multilevel logistic regression analysis will be performed for binominal variables and multilevel linear regression for continuous variables. Descriptive statistics will be computed for baseline and follow-up assessments.
Discussion
The study will be the first to address problematic alcohol use in PLWHA in Zimbabwe. It seeks to use local resources in delivering a modified, brief, evidence-based, and culturally contextualised intervention. The study results will determine the effectiveness of adapting psychological interventions for AUDs in HIV infected adults using a task-sharing framework.
Trial registration
Pan African Clinical Trial Registry, PACTR201509001211149. Registered 22 July 2015.
doi:10.1186/s12888-017-1208-3
PMCID: PMC5273845  PMID: 28129756
Alcohol use disorders; Motivational interviewing; Cognitive behavioural therapy; Intervention; Zimbabwe
3.  Expression of various sarcomeric tropomyosin isoforms in equine striated muscles 
Open Veterinary Journal  2017;7(2):180-191.
In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.
doi:10.4314/ovj.v7i2.17
PMCID: PMC5498770
Absolute copy number; Horse; qRT-PCR; Relative expression; TPM
4.  Zimbabwe’s national AIDS levy: A case study 
Background
We conducted a case study of the Zimbabwe National AIDS Trust Fund (‘AIDS Levy’) as an approach to domestic government financing of the response to HIV and AIDS.
Methods
Data came from three sources: a literature review, including a search for grey literature, review of government documents from the Zimbabwe National AIDS Council (NAC), and key informant interviews with representatives of the Zimbabwean government, civil society and international organizations.
Findings
The literature search yielded 139 sources, and 20 key informants were interviewed. Established by legislation in 1999, the AIDS Levy entails a 3% income tax for individuals and 3% tax on profits of employers and trusts (which excluded the mining industry until 2015). It is managed by the parastatal NAC through a decentralized structure of AIDS Action Committees. Revenues increased from inception to 2006 through 2008, a period of economic instability and hyperinflation. Following dollarization in 2009, annual revenues continued to increase, reaching US$38.6 million in 2014. By policy, at least 50% of funds are used for purchase of antiretroviral medications. Other spending includes administration and capital costs, HIV prevention, and monitoring and evaluation. Several financial controls and auditing systems are in place. Key informants perceived the AIDS Levy as a ‘homegrown’ solution that provided country ownership and reduced dependence on donor funding, but called for further increased transparency, accountability, and reduced administrative costs, as well as recommended changes to increase revenue.
Conclusions
The Zimbabwe AIDS Levy has generated substantial resources, recently over US$35 million per year, and signals an important commitment by Zimbabweans, which may have helped attract other donor resources. Many key informants considered the Zimbabwe AIDS Levy to be a best practice for other countries to follow.
doi:10.1080/17290376.2015.1123646
PMCID: PMC4762022  PMID: 26781215
health care financing; HIV; AIDS; Zimbabwe
5.  Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans 
Molecular Biology International  2016;2016:3105478.
In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.
doi:10.1155/2016/3105478
PMCID: PMC5040813  PMID: 27703814
6.  Expression of tropomyosin 2 gene isoforms in human breast cancer cell lines 
Oncology Reports  2016;35(6):3143-3150.
In humans, four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) are known to produce a multitude of isoforms via alternate splicing and/or using alternate promoters. Expression of tropomyosin has been shown to be modulated at both the transcription and the translational levels. Tropomyosins are known to make up some of the stress fibers of human epithelial cells and differences in their expression has been demonstrated in malignant breast epithelial cell lines compared to 'normal' breast cell lines. We have recently reported the expression of four novel TPM1 isoforms (TPM1λ, TPM1µ, TPM1ν, and TPM1ξ) from human malignant tumor breast cell lines that are not expressed in adult and fetal cardiac tissue. Also, we evaluated their expression in relation to the stress fiber formation. In this study, nine malignant breast epithelial cell lines and three 'normal' breast cell lines were examined for stress fiber formation and expression of tropomyosin 2 (TPM2) isoform-specific RNAs and proteins. Stress fiber formation was assessed by immunofluorescence using Leica AF6000 Deconvolution microscope. Stress fiber formation was strong (++++) in the 'normal' cell lines and varied among the malignant cell lines (negative to +++). No new TPM2 gene RNA isoforms were identified, and TPM2β was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2β RNA or protein expression at high, statistically significant degrees. Previously, we had shown that TPM1δ and TPM1λ positively and inversely, respectively, correlated with stress fiber formation. The most powerful predictor of stress fiber formation was the combination of TPM2β RNA, TPM1δ RNA, and the inverse of TPM1λ RNA expression. Our results suggest that the increased expression of TPM1λ and the decreased expression of TPM1δ RNA and TPM2β may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells.
doi:10.3892/or.2016.4732
PMCID: PMC4869935  PMID: 27108600
TPM2 isoforms; RT-PCR; western blot analysis; stress fiber; immunofluorescence
7.  Expression of myotilin during chicken development 
Several missense mutations in the Z-band protein, myotilin, have been implicated in human muscle diseases such as myofibrillar myopathy, spheroid body myopathy, and distal myopathy. Recently, we have reported the cloning of chicken myotilin cDNA. In this study, we have investigated the expression of myotilin in cross-striated muscles from developing chicken by qRT-PCR and in situ hybridizations. In situ hybridization of embryonic stages shows myotilin gene expression in heart, somites, neural tissue, eyes and otocysts. RT-PCR and qRT-PCR data, together with in situ hybridization results point to a biphasic transcriptional pattern for MYOT gene during early heart development with maximum expression level in the adult. In skeletal muscle, the expression level starts decreasing after embryonic day 20 and declines in the adult skeletal muscles. Western blot assays of myotilin in adult skeletal muscle reveal a decrease in myotilin protein compared with levels in embryonic skeletal muscle. Our results suggest that MYOT gene may undergo transcriptional activation and repression that varies between tissues in developing chicken. We believe this is the first report of the developmental regulation on myotilin expression in non-mammalian species.
doi:10.1002/ar.22964
PMCID: PMC4135462  PMID: 25125173
chick embryos; myotilin; Z-bands; skeletal muscle; heart
8.  Expression of Tropomyosin 1 Gene Isoforms in Human Breast Cancer Cell Lines 
Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1γ and TPM1δ were the dominant transcripts of TPM1, there was no clear evidence for TPM1δ protein expression. Four novel human TPM1 gene RNA isoforms were discovered (λ, μ, ν, and ξ), which were not identified in adult and fetal human cardiac tissues. TPM1λ was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ, λ, and ν protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γ RNA expression but significantly and inversely correlated with TPM1δ and TPM1λ expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study.
doi:10.1155/2015/859427
PMCID: PMC4480939  PMID: 26171250
9.  Expression of TPM1κ, a Novel Sarcomeric Isoform of the TPM1 Gene, in Mouse Heart and Skeletal Muscle 
We have investigated the expression of TPM1α and TPM1κ in mouse striated muscles. TPM1α and TMP1κ were amplified from the cDNA of mouse heart by using conventional RT-PCR. We have cloned the PCR amplified DNA and determined the nucleotide sequences. Deduced amino acid sequences show that there are three amino acid changes in mouse exon 2a when compared with the human TPM1κ. However, the deduced amino acid sequences of human TPM1α and mouse TPM1α are identical. Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1κ is significantly lower compared to TPM1α in both mouse heart and skeletal muscle. It was also found that the expression level of TPM1κ transcripts in mouse heart is higher than it is in skeletal muscle. To the best of our knowledge, this is the first report of the expression of TPM1κ in mammalian skeletal muscle.
doi:10.1155/2014/896068
PMCID: PMC4020292  PMID: 24876965
10.  Expression of tropomyosin in relation to myofibrillogenesis in axolotl hearts 
The anatomy, function and embryonic development of the heart have been of interest to clinicians and researchers alike for centuries. A beating heart is one of the key criteria in defining life or death in humans. An understanding of the multitude of genetic and functional elements that interplay to form such a complex organ is slowly evolving with new genetic, molecular and experimental techniques. Despite the need for ever more complex molecular techniques some of our biggest leaps in knowledge come from nature itself through observations of mutations that create natural defects in function. Such a natural mutation is found in the Mexican axolotl, Ambystoma mexicanum. It is a facultative neotenous salamander well studied for its ability to regenerate severed limbs and tail. Interestingly it also well suited to studying segmental heart development and differential sarcomere protein expression due to a naturally occurring mendelian recessive mutation in cardiac mutant gene “c”. The resultant mutants are identified by their failure to beat and can be studied for extended periods before they finally die due to lack of circulation. Studies have shown a differential expression of tropomyosin between the conus and the ventricle indicating two different cardiac segments. Tropomyosin protein, but not its transcript have been found to be deficient in mutant ventricles and sarcomere formation can be rescued by the addition of TM protein or cDNA. Although once thought to be due to endoderm induction our findings indicate a translational regulatory mechanism that may ultimately control the level of tropomyosin protein in axolotl hearts.
doi:10.1186/2050-490X-1-8
PMCID: PMC4431041  PMID: 25984327
Ambystoma mexicanum; Cardiac lethal mutation; Non-beating ventricle; Conus; Ectopic expression; Translational repression
11.  Nonsyndromic Familial Oligodontia with Multiple Dens Invaginatus: A Case Report of an Unusual Case 
Case Reports in Dentistry  2013;2013:983580.
Oligodontia is a rare dental anomaly with a prevalence of 0.3% in permanent teeth and much less frequency in the primary dentition. Familial oligodontia represents an absence of varying numbers of primary and/or secondary teeth as an isolated trait. It is a complex and multifactorial condition. Many explanations—evolutionary, genetic, and environmental—have been proposed as the etiology. Simultaneous with oligodontia are often the different positional changes of the existing teeth, their morphology, size, and growth disturbances of the maxillofacial skeleton. Early recognition is vital to provide adequate treatment and prevent squeal. Multidisciplinary referral or consultation is thus important in treatment planning to improve function and esthetics. The present paper reports a rare case of familial oligodontia associated with multiple dense invaginatus and microdontia.
doi:10.1155/2013/983580
PMCID: PMC3835485  PMID: 24319603
12.  Delayed seroconversion to STLV-1 infection is associated with mutations in the pol and rex genes 
Virology Journal  2013;10:282.
Background
Simian T-cell lymphoma/leukemia virus-1 (STLV-1) infection of non-human primates can serve as a model for human T-cell lymphoma/leukemia virus infection.
Methods
Two tantalus and 2 patas monkeys were transfused with intraspecies whole blood infected with STLV-1. Infection was determined by ELISA, western blot and DNA PCR analyses. The entire genome of the STLV-1 Tan 90 strain and some of the STVL-1 Pat74 strain were amplified using over-lapping primer-pairs and subsequently sequenced.
Results
Followup studies conducted over 2 years indicated that all 4 monkeys remained healthy despite being infected with STLV-1, as determined by PCR, cloning and sequencing analyses. ELISA and Western blot analyses indicated that both patas monkeys seroconverted within 2 months of transfusion, while one tantalus monkey required one year to seroconvert and the other never fully seroconverted. The tantalus monkey which never fully seroconverted, failed to react to HTLV-1 p24 Gag antigen. Sequence analyses indicated that, while unique, the deduced p24 Gag amino acid sequence of the STLV-1 Tan 90 strain used for infection was still highly homologous to the HTLV-1 p24 Gag amino acids present in the ELISA and WB assays. However, a mutation in the pol sequence of STLV-1 Tan 90 encoded a putative stop codon, while a common deletion in the pol/rex regulatory gene causes significant changes in the Pol, and p27 Rex proteins. These same mutations were also observed in the viral DNA of both recipient infected tantalus monkeys and were not present in the STLV-1 Pat 74 strain.
Conclusion
Our data suggest that seroconversion to STLV-1 infection may be prolonged due to the above mutations, and that compensatory molecular events must have occurred to allow for virus transmission.
doi:10.1186/1743-422X-10-282
PMCID: PMC3851238  PMID: 24025129
13.  The complete genomic sequence of an in vivo low replicating BLV strain 
Virology Journal  2009;6:120.
DNA was extracted from lamb lymphocytes that were infected in vivo with a BLV strain after inoculation with the peripheral blood mononuclear cells from a persistently sero-indeterminate, low viral load, BLV-infected Holstein cow (No. 41) from Argentina. The DNA was PCR amplified with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 41 DNA was cloned, sequenced, and compared phylogenetically to other BLV sequences including an in vivo high replicating strain (BLV ARG 38) from the same herd in Argentina. Characterization of BLV ARG 41's deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.
doi:10.1186/1743-422X-6-120
PMCID: PMC3224937  PMID: 19650931
14.  Role of Myofibril-Inducing RNA in cardiac TnT expression in developing Mexican axolotl 
The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene “c”, for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts.. Myofibril-Inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac Troponin-T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart
doi:10.1016/j.bbrc.2007.03.064
PMCID: PMC2034438  PMID: 17408593
cardiac Troponin T; axolotl; myofibril; alternative splicing
15.  Genetic heterogeneity in human T-cell leukemia/lymphoma virus type II. 
Journal of Virology  1993;67(3):1175-1184.
DNA from the peripheral blood mononuclear cells of 17 different individuals infected with human T-cell lymphoma/leukemia virus type II (HTLV-II) was successfully amplified by the polymerase chain reaction (PCR) with the primer pair SK110/SK111. This primer pair is conserved among the pol genes of all primate T-cell lymphoma viruses (PTLV) and flanks a 140-bp fragment of DNA which, when used in comparative analyses, reflects the relative degree of diversity among PTLV genomes. Cloning, sequencing, and phylogenetic comparisons of these amplified 140-bp pol fragments indicated that there are at least two distinct genetic substrains of HTLV-II in the Western Hemisphere. These data were confirmed for selected isolates by performing PCR, cloning, and sequencing with to 10 additional primer pair-probe sets specific for different regions throughout the PTLV genome. HTLV-II isolates from Seminole, Guaymi, and Tobas Indians belong in the new substrain of HTLV-II, while the prototype MoT isolate defines the original substrain. There was greater diversity among HTLV-II New World strains than among HTLV-I New World strains. In fact, the heterogeneity among HTLV-II strains from the Western Hemisphere was similar to that observed in HTLV-I and simian T-cell lymphoma/leukemia virus type I isolates from around the world, including Japan, Africa, and Papua New Guinea. Given these geographic and anthropological considerations and assuming similar mutation rates and selective forces among the PTLV, these data suggest either that HTLV-II has existed for a long time in the indigenous Amerindian population or that HTLV-II isolates introduced into the New World were more heterogeneous than the HTLV-I strains introduced into the New World.
PMCID: PMC237482  PMID: 8437209
16.  Maternal Near Miss: A Valuable Contribution in Maternal Care 
Background
MMR has always been recognized as an important indicator of quality of health services. The MMR in India has so far not reached up to the required MDG 2015. If we look into this matter with the eagle’s eye view, then there are certain gray areas which need attention. For this, it is not the maternal mortality but the maternal near miss which has to be focused.
Objectives
To audit the maternal near miss in our institution and to review the pathways that lead to severe maternal morbidity and death.
Methods
Prospective observational study from September 2013 to August 2015 in Dr. Bhim Rao Ambedkar Memorial Hospital, Raipur. Maternal near miss cases were identified based on WHO criteria 2009, recorded, and studied.
Results
There were 13,895 live births, 211 maternal near miss, and 102 maternal deaths. Maternal near miss to mortality ratio was 2:1. Maternal near miss incidence ratio was 15.18/1000 live births. Mortality index was 32.58 %. Hemorrhage and hypertensive disorders of pregnancy toped the list of the leading causes of near miss morbidity. The near miss events were more common in the primipara (39 %), with age group 21–30 years and in the third trimester at the time of admission.
Conclusion
Auditing maternal near miss can help in reducing their morbidity and mortality in our institution. Similar audit between other institute, state, and countries may help to hasten the slow progress of reducing maternal mortality.
doi:10.1007/s13224-015-0838-y
PMCID: PMC5016444  PMID: 27651607
Maternal near miss; Maternal death; Maternal near miss audit
17.  Skills and compensation strategies in adult ADHD – A qualitative study 
PLoS ONE  2017;12(9):e0184964.
Objective
The primary objectives of this study were to investigate how adult patients with ADHD coped with their symptoms prior to diagnosis and treatment, what skills and compensation strategies they had developed and what their self-perceptions of these strategies were.
Methods
We used a qualitative approach to analyze interviews with 32 outpatients of a specialty care unit at a university hospital.
Results
Patients reported frequent use of diverse compensatory strategies with varying degrees of effectiveness. These were classified into five categories (organizational, motoric, attentional, social, psychopharmacological). In certain circumstances, ADHD symptoms were even perceived as useful.
Conclusion
Before diagnosis and treatment, patients with ADHD may develop a variety of skills to cope with their symptoms. Several of these skills are perceived as helpful. Knowledge of self-generated coping strategies may help better understand patients and their histories and thus facilitate patient cooperation. Moreover, knowing ways in which such patients cope with their symptoms may help elucidate reasons for late or under-diagnosing of the disorder.
doi:10.1371/journal.pone.0184964
PMCID: PMC5617155  PMID: 28953946
18.  Hepatic 11β-hydroxysteroid dehydrogenase type 1 activity in obesity and type 2 diabetes using a novel triple tracer cortisol technique 
Diabetologia  2014;57(7):1446-1455.
Aims/hypothesis
Dysregulation of 11β-hydroxysteroid dehydrogenase (11β-HSD) enzyme activities are implicated in the pathogenesis of obesity and insulin resistance. The aim of the study was to determine whether hepatic 11β-HSD type 1 (11β-HSD-1) enzyme activity differs in people with and without obesity and type 2 diabetes.
Methods
We measured hepatic 11β-HSD-1 activity in the overnight fasted state in 20 lean non-diabetic participants (LND), 21 overweight/obese non-diabetic participants (OND) and 20 overweight/obese participants with type 2 diabetes (ODM) using a non-invasive approach. One mg doses of [9,12,12-2H3]cortisol (D cortisol) and [4-13C]cortisone ([13C]cortisone) were ingested, while [1,2,6,7-3H]cortisol ([3H] cortisol) was infused intravenously to enable concurrent measurements of first-pass hepatic extraction of ingested D cortisol and hepatic conversion of ingested [13C]cortisone to C13 cortisol derived from the ingested cortisone (a measure of 11β-HSD-1 activity in the liver) using an isotope dilution technique. One-way ANOVA models and Kruskal–Wallis tests were used to test the hypothesis.
Results
Plasma D cortisol and C13 cortisol concentrations were lower in OND than in LND (p<0.05) over 6 h of the study. There was no difference (p=0.15) in C13 and D cortisol concentrations between OND and ODM and between LND and ODM for the same study period. Hepatic conversion of [13C]cortisone to C13 cortisol was similar between groups.
Conclusions/interpretation
Hepatic conversion of [13C]cortisone to C13 cortisol did not differ between the groups studied. We conclude that hepatic 11β-HSD-1 activity is similar in individuals who are overweight/obese or who have type 2 diabetes.
doi:10.1007/s00125-014-3240-x
PMCID: PMC5611844  PMID: 24771091
Diabetes; 11β-Hydroxysteroid dehydrogenase; Obesity
19.  Antibody responses to P. falciparum blood stage antigens and incidence of clinical malaria in children living in endemic area in Burkina Faso 
BMC Research Notes  2017;10:472.
Background
High parasite-specific antibody levels are generally associated with low susceptibility to Plasmodium falciparum malaria. This has been supported by several studies in which clinical malaria cases of P. falciparum malaria were reported to be associated with low antibody avidities. This study was conducted to evaluate the role of age, malaria transmission intensity and incidence of clinical malaria in the induction of protective humoral immune response against P. falciparum malaria in children living in Burkina Faso.
Methods
We combined levels of IgG and IgG subclasses responses to P. falciparum antigens: Merozoite Surface Protein 3 (MSP3), Merozoite Surface Protein 2a (MSP2a), Merozoite Surface Protein 2b (MSP2b), Glutamate Rich Protein R0 (GLURP R0) and Glutamate Rich Protein R2 (GLURP R2) in plasma samples from 325 children under five (05) years with age, malaria transmission season and malaria incidence.
Results
We notice higher prevalence of P. falciparum infection in low transmission season compared to high malaria transmission season. While, parasite density was lower in low transmission than high transmission season. IgG against all antigens investigated increased with age. High levels of IgG and IgG subclasses to all tested antigens except for GLURP R2 were associated with the intensity of malaria transmission. IgG to MSP3, MSP2b, GLURP R2 and GLURP R0 were associated with low incidence of malaria. All IgG subclasses were associated with low incidence of P. falciparum malaria, but these associations were stronger for cytophilic IgGs.
Conclusions
On the basis of the data presented in this study, we conclude that the induction of humoral immune response to tested malaria antigens is related to age, transmission season level and incidence of clinical malaria.
doi:10.1186/s13104-017-2772-9
PMCID: PMC5591548  PMID: 28886727
P. falciparum infection; Malaria transmission sessions; Age; Antigens
20.  CD8+ T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity 
CD8+ T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8+ T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8+ T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8+ T cells are generated and provide limited protection against challenge with virulent OVA+ Y. pseudotuberculosis. Perforin expression by OVA-specific CD8+ T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8+ T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8+ T cell status. These studies indicate that CD8+ T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.
doi:10.1016/j.meegid.2016.06.008
PMCID: PMC4957522  PMID: 27268148
Yersinia pseudotuberculosis; CD8+ T cells; YopE; perforin; bottle-neck; ovalbumin
21.  Changes in Markers of T-Cell Senescence and Exhaustion With Atazanavir-, Raltegravir-, and Darunavir-Based Initial Antiviral Therapy: ACTG 5260s 
The Journal of Infectious Diseases  2016;214(5):748-752.
It is unclear whether differential roles of CD4+ versus CD8+ T-cell senescence/exhaustion and effects of antiretroviral therapy (ART) on these processes may contribute to morbidity in treated human immunodeficiency virus type 1 (HIV) infection. In a prospective 96-week trial, 328 HIV–infected ART-naive participants were randomly assigned to receive tenofovir-emtricitabine plus either atazanavir/ritonavir, darunavir/ritonavir, or raltegravir. Markers of CD4+ T-cell senescence (ie, the percentage of CD28−CD57+ cells among CD4+ T cells ) and CD4+/CD8+ T-cell exhaustion (ie, the percentage of PD-1+ cells among CD4+/CD8+ T cells) decreased after ART. There were no changes in markers of CD8+ T-cell senescence after ART and no differential changes in all markers in ART groups. Senescent CD4+ and CD8+ T cells may have differential roles in HIV pathogenesis.
doi:10.1093/infdis/jiw253
PMCID: PMC4978379  PMID: 27354367
antiretroviral therapy; human immunodeficiency virus; inflammation; immune activation; biomarkers
22.  Infiltrating mast cells enhance benign prostatic hyperplasia through IL-6/STAT3/Cyclin D1 signals 
Oncotarget  2017;8(35):59156-59164.
Early evidences have showed that mast cells could infiltrate into benign prostatic hyperplasia (BPH) tissues, but the exact role of mast cells in BPH development remains unclear. In this study, we identified more mast cells existing in human BPH tissues compared with that in the normal prostate. In the in vitro co-culture system, BPH-1 prostate cells promoted activation and migration of mast cells, and mast cells conversely stimulated BPH-1 cells proliferation significantly. Molecular analysis demonstrated that mast cell-derived interleukin 6 (IL-6) could activate STAT3/Cyclin D1 signals in BPH-1 cells. Blocking IL-6 or STAT3 partially reverse the capacity of mast cells to enhance BPH-1 cell proliferation. Our findings suggest that infiltrating mast cells in BPH tissues could promote BPH development via IL-6/STAT3/Cyclin D1 signals. Therefore, targeting infiltrating mast cells may improve the therapeutic effect of BPH.
doi:10.18632/oncotarget.19465
PMCID: PMC5601722  PMID: 28938626
benign prostatic hyperplasia; mast cell; proliferation; chemokine
23.  Midwives’ and patients’ perspectives on disrespect and abuse during labor and delivery care in Ethiopia: a qualitative study 
Background
It is increasingly recognized that disrespect and abuse of women during labor and delivery is a violation of a woman’s rights and a deterrent to the use of life-saving, facility-based labor and delivery services. In Ethiopia, rates of skilled birth attendance are still only 28% despite a recent dramatic national scale up in the numbers of trained providers and facilities. Concerns have been raised that womens’ perceptions of poor quality of care and fear of mistreatment might contribute to this low utilization. This study examines the experiences of disrespect and abuse in maternal care from the perspectives of both providers and patients.
Methods
We conducted 45 in-depth interviews at four health facilities in Debre Markos, Ethiopia with midwives, midwifery students, and women who had given birth within the past year. Students and providers also took a brief quantitative survey on patients’ rights during labor and delivery and responded to clinical scenarios regarding the provision of stigmatized reproductive health services.
Results
We find that both health care providers and patients report frequent physical and verbal abuse as well as non-consented care during labor and delivery. Providers report that most abuse is unintended and results from weaknesses in the health system or from medical necessity. We uncovered no evidence of more systematic types of abuse involving detention of patients, bribery, abandonment or ongoing discrimination against particular ethnic groups. Although health care providers showed good basic knowledge of confidentiality, privacy, and consent, training on the principles of responsive and respectful care, and on counseling, is largely absent. Providers indicated that they would welcome related practical instruction. Patient responses suggest that women are aware that their rights are being violated and avoid facilities with reputations for poor care.
Conclusions
Our results suggest that training on respectful care, offered in the professional ethics modules of the national midwifery curriculum, should be strengthened to include greater focus on counseling skills and rapport-building. Our findings also indicate that addressing structural issues around provider workload should complement all interventions to improve midwives’ interpersonal interactions with women if Ethiopia is to increase provision of respectful, patient-centered maternity care.
Electronic supplementary material
The online version of this article (doi:10.1186/s12884-017-1442-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12884-017-1442-1
PMCID: PMC5567643  PMID: 28830383
Midwives; Respectful maternity care; Disrespect and abuse; Maternity care; Quality; Patients’ rights; Woman-centred care
24.  Cryo EM of Mitotic Checkpoint Complex-bound APC/C reveals reciprocal and conformational regulation of ubiquitin ligation 
Molecular cell  2016;63(4):593-607.
SUMMARY
The Mitotic Checkpoint Complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated Anaphase-promoting complex/Cyclosome (APC/CCDC20). APC/CCDC20 and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/CCDC20–UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here, electron microscopy reveals conformational variation of APC/CCDC20-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly, and conformational variability of APC/C, modulate UBE2C-catalyzed ubiquitylation of MCC’s CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/CCDC20–MCC modulating E2 activation underlies distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.
eTOC blurb
The Mitotic Checkpoint Complex (MCC) prevents APC/CCDC20-catalyzed ubiquitination of anaphase inhibitors until all chromosomes are properly bioriented. Cryo-EM and biochemistry reveal diverse conformations of APC/CCDC20-MCC modulating E2 activation and targeting, and suggest responsive mechanisms ensuring accurate chromosome segregation.
doi:10.1016/j.molcel.2016.07.003
PMCID: PMC5148128  PMID: 27522463
25.  T2DiACoD: A Gene Atlas of Type 2 Diabetes Mellitus Associated Complex Disorders 
Scientific Reports  2017;7:6892.
We performed integrative analysis of genes associated with type 2 Diabetes Mellitus (T2DM) associated complications by automated text mining with manual curation and also gene expression analysis from Gene Expression Omnibus. They were analysed for pathogenic or protective role, trends, interaction with risk factors, Gene Ontology enrichment and tissue wise differential expression. The database T2DiACoD houses 650 genes, and 34 microRNAs associated with T2DM complications. Seven genes AGER, TNFRSF11B, CRK, PON1, ADIPOQ, CRP and NOS3 are associated with all 5 complications. Several genes are studied in multiple years in all complications with high proportion in cardiovascular (75.8%) and atherosclerosis (51.3%). T2DM Patients’ skeletal muscle tissues showed high fold change in differentially expressed genes. Among the differentially expressed genes, VEGFA is associated with several complications of T2DM. A few genes ACE2, ADCYAP1, HDAC4, NCF1, NFE2L2, OSM, SMAD1, TGFB1, BDNF, SYVN1, TXNIP, CD36, CYP2J2, NLRP3 with details of protective role are catalogued. Obesity is clearly a dominant risk factor interacting with the genes of T2DM complications followed by inflammation, diet and stress to variable extents. This information emerging from the integrative approach used in this work could benefit further therapeutic approaches. The T2DiACoD is available at www.http://t2diacod.igib.res.in/.
doi:10.1038/s41598-017-07238-0
PMCID: PMC5537262  PMID: 28761062

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