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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.  Zimbabwe's national AIDS levy: A case study 
Abstract
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; financement des soins de santé; le VIH; le sida,; le Zimbabwe
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  Reducing Overselective Stimulus Control with Differential Observing Responses 
Overselective stimulus control refers to discriminative control in which the number of controlling stimuli is too limited for effective behavior. Experiment 1 included 22 special-education students who exhibited overselective stimulus control on a two-sample delayed matching task. An intervention added a compound identity matching opportunity within the sample observation period of the matching trials. The compound matching functioned as a differential observing response (DOR) in that high accuracy verified observation and discrimination of both sample stimuli. Nineteen participants learned to perform the DOR and two-sample delayed matching accuracy increased substantially for 16 of them. When the DOR was completely withdrawn after 10 sessions, accuracy declined. In Experiment 2, a more gradual withdrawal of DOR requirements showed that highly accurate performance could be maintained with the DOR on only a proportion of trials for most participants. The results show that DOR training may lead to a general improvement in observing behavior.
doi:10.1002/jaba.363
PMCID: PMC5235982  PMID: 27861843
differential observing responses; intellectual disabilities; matching to sample; overselective stimulus control
18.  Changes in Plasma Levels of Oxidized Lipoproteins and Lipoprotein Subfractions with Atazanavir-, Raltegravir-, Darunavir-Based Initial Antiviral Therapy and Associations with Common Carotid Artery Intima-Media Thickness: ACTG 5260s 
Antiviral therapy  2016;22(2):113-126.
BACKGROUND
The role of oxidized lipoproteins (high-density [HDLox] and low-density [LDLox]) and total lipoprotein particle (Lp) number and size in HIV-related cardiovascular disease (CVD) is unclear. The goal of this study was to evaluate changes of these biomarkers and their associations with rate of carotid intima media thickness progression over 3 years (ΔCIMT) in chronic HIV infection.
METHODS
Prospective study of 234 HIV-infected antiretroviral treatment naïve participants without CVD who were randomized to receive tenofovir-emtricitabine plus atazanavir/ritonavir, darunavir/ ritonavir, or raltegravir (RAL) and achieved plasma HIV-1 RNA <50 copies/ml by week 24 and thereafter. Biomarker changes over 24, 48 or 96 weeks from baseline and pairwise treatment group comparisons were examined. Associations of these biomarkers with ΔCIMT were analyzed with mixed effects linear regression.
RESULTS
HDLp number increased with both protease inhibitors (PIs) over 48 weeks, while LDLp number declined with RAL; Lp size did not change. Over 96 weeks, normalized HDLox declined with both PIs; LDLox increased in all groups. Few treatment group differences were observed across all biomarkers. Associations between ΔCIMT and oxidized lipoproteins at all timepoints were not apparent (p≥0.10). There was some evidence of slower ΔCIMT for higher HDLp number (p=0.06) and for lower LDLp number (p=0.08) measured at baseline.
CONCLUSIONS
Unexpectedly, LDLox increased modestly in all treatment groups after ART initiation. Associations of plasma HDLox and LDLox with ΔCIMT were not apparent. While plasma levels of abnormal lipoproteins have been shown to be associated with CVD outcomes, clear associations with sub-clinical atherosclerosis progression were not apparent in our study.
doi:10.3851/IMP3093
PMCID: PMC5364070  PMID: 27661466
Oxidized Lipoproteins; Lipoprotein Subfractions; cardiovascular disease; Human Immunodeficiency Virus; Inflammation; Protease inhibitors; Integrase inhibitors; lipoprotein function; HIV-1 infection
19.  Development of rare bacterial monosaccharide analogs for metabolic glycan labeling in pathogenic bacteria 
ACS chemical biology  2016;11(12):3365-3373.
Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.
Graphical Abstract
doi:10.1021/acschembio.6b00790
PMCID: PMC5161589  PMID: 27766829
20.  Point-of-care device to diagnose and monitor neonatal jaundice in low-resource settings 
Significance
Neonatal jaundice, a condition caused by the accumulation of bilirubin in the bloodstream, affects approximately half of all newborns. In high-resource settings, babies with elevated serum bilirubin levels are identified through routine hospital laboratory testing. When identified, jaundice is easily treated using blue-light phototherapy. Low-cost, rugged phototherapy lights have been developed and shown to be effective in low-resource settings. However, jaundice regularly goes undetected in these settings due to a lack of diagnostic tools to measure bilirubin levels. Left untreated, jaundice can lead to permanent neurological damage and mortality, the vast majority of which currently occurs in low-resource settings. In this paper, we present a low-cost method to measure total bilirubin at the point of care in low-resource settings.
Newborns are at increased risk of jaundice, a condition in which excess bilirubin accumulates in blood. Left untreated, jaundice can lead to neurological impairment and death. Jaundice resulting from unconjugated hyperbilirubinemia is easily treated with exposure to blue light, and phototherapy systems have been developed for low-resource settings; however, there are no appropriate solutions to diagnose and monitor jaundice in these settings. To address this need we present BiliSpec, a low-cost reader and disposable lateral flow card designed to measure the concentration of total bilirubin from several drops of blood at the point of care. We evaluated the performance of BiliSpec, using blood from normal volunteers spiked with varying amounts of bilirubin; results measured using BiliSpec correlated well with a reference laboratory bilirubinometer (r = 0.996). We then performed a pilot clinical study using BiliSpec to measure total bilirubin in neonates at risk for jaundice at Queen Elizabeth Central Hospital in Blantyre, Malawi. Concentrations measured using BiliSpec correlated well with those measured using a laboratory reference standard in 94 patient samples ranging from 1.1 mg/dL to 23.0 mg/dL in concentration (r = 0.973). The mean difference between bilirubin levels measured with BiliSpec and the reference standard was 0.3 mg/dL (95% CI: −1.7–2.2 mg/dL).
doi:10.1073/pnas.1714020114
PMCID: PMC5754796  PMID: 29203650
neonatal jaundice; point-of-care; lateral flow; low-resource setting
21.  Simple Technique Overcoming a Persistent Problem in Cleft Palate Repair 
Purpose
Cleft palate repair mandates use of a mouth gag and Dingmans moth gag is the most commonly used for the same; but the use of Dingmans mouth gag may have the demerit of the suture getting tethered at various places of the instrument during cleft palate closure particularly in the hands of the beginner surgeon.
Method
This article discusses about a simple technique of using a rubber dam sheet to cover the frame of the mouth gag.
Conclusion
The technique discussed in this article is simple, cost effective method to overcome the potential disadvantage of suture adherence during repair of palatal tissue. The technique also has the potential to reduce the total operative time which needs a further study to validate the same.
doi:10.1007/s12663-016-0906-6
PMCID: PMC5083704  PMID: 27833354
Rubber dam; Dingman’s mouth gag; Cleft palate
22.  Random Allocation of Blastomere Descendants to the Trophectoderm and ICM of the Bovine Blastocyst1 
Biology of Reproduction  2016;95(6):123.
The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (∼60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage.
doi:10.1095/biolreprod.116.141200
PMCID: PMC5333943  PMID: 27760750
blastocyst; embryo biopsy; H3 arginine methylation; patterning; preimplantation development; time-lapse microscopy
23.  A Methodological Pipeline for Serial-Section Imaging and Tissue Realignment for Whole-brain Functional and Connectivity Assessment 
Background
Understanding the neurobiological basis of cognition and behavior, and disruptions to these processes following injury and disease, requires a large-scale assessment of neural populations, and knowledge of their patterns of connectivity.
New Method
We present an analysis platform for large-scale investigation of functional and neuroanatomical connectivity in the rodents. Retrograde tracers were injected and in a subset of animals behavioral tests to drive immediate-early gene expression were administered. This approach allows users to perform whole-brain assessment of function and connection in a semi-automated quantitative manner. Brains were cut in the coronal plane, and an image of the block face was acquired. Wide-field fluorescent scans of whole sections were acquired and analyzed using Matlab software.
Results
The toolkit utilized open-source and custom platforms to accommodate a largely automated analysis pipeline in which neuronal boundaries are automatically segmented, the position of segmented neurons are co-registered with a corresponding image acquired during vibratome sectioning, and a 3-D representation of neural tracer (and other products) throughout the entire brain is generated.
Comparison with Existing Methods
Current whole brain connectivity measures primarily target mice and use anterograde tracers. Our focus on segmented units of interest (e.g., NeuN labeled neurons) and restricting measures to these units produces a flexible platform for a variety of whole brain analyses (measuring activation, connectivity, markers of disease, etc.).
Conclusions
This open-source toolkit allows an investigator to visualize and quantify whole brain data in 3-D, and additionally provides a framework that can be rapidly integrated with user-specific analyses and methodologies.
doi:10.1016/j.jneumeth.2016.03.021
PMCID: PMC5695690  PMID: 27039972
connectome; immediate-early genes; Neuroimaging; Functional networks; Neural tracing; Tissue Realignment
24.  Parkinson disease polygenic risk score is associated with Parkinson disease status and age at onset but not with alpha-synuclein cerebrospinal fluid levels 
BMC Neurology  2017;17:198.
Background
The genetic architecture of Parkinson’s Disease (PD) is complex and not completely understood. Multiple genetic studies to date have identified multiple causal genes and risk loci. Nevertheless, most of the expected genetic heritability remains unexplained. Polygenic risk scores (PRS) may provide greater statistical power and inform about the genetic architecture of multiple phenotypes. The aim of this study was to test the association between PRS and PD risk, age at onset and cerebrospinal fluid (CSF) biomarkers (α-synuclein, Aβ1–42, t-tau and p-tau).
Methods
The weighted PRS was created using the genome-wide loci from Nalls et al., 2014 PD GWAs meta-analysis. The PRS was tested for association with PD status, age at onset and CSF biomarker levels in 829 cases and 432 controls of European ancestry.
Results
The PRS was associated with PD status (p = 5.83×10−08) and age at onset (p = 5.70×10−07). The CSF t-tau levels showed a nominal association with the PRS (p = 0.02). However, CSF α-synuclein, amyloid beta and phosphorylated tau were not found to be associated with the PRS.
Conclusion
Our study suggests that there is an overlap in the genetic architecture of PD risk and onset, although the different loci present different weights for those phenotypes. In our dataset we found a marginal association of the PRS with CSF t-tau but not with α-synuclein CSF levels, suggesting that the genetic architecture for the CSF biomarker levels is different from that of PD risk.
Electronic supplementary material
The online version of this article (10.1186/s12883-017-0978-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12883-017-0978-z
PMCID: PMC5688622  PMID: 29141588
Parkinson disease; Genetics; Age at onset; Biomarkers; Polygenic risk score
25.  Oxidized lipoproteins are associated with markers of inflammation and immune activation in HIV-1 infection 
AIDS (London, England)  2016;30(17):2625-2633.
Objective
The pathogenesis of immune dysfunction in chronic HIV-1 infection is unclear, and a potential role for oxidized lipids has been suggested. We hypothesize that both oxidized low- and high-density lipoproteins (HDLox, LDLox) contribute to HIV-1 related immune dysfunction.
Study
In the AIDS Clinical Trials Group (ACTG) A5260, 234 HIV-infected antiretroviral therapy (ART)-naïve participants were randomized to receive tenofovir-emtricitabine plus protease inhibitors or raltegravir and had HIV-1 RNA <50 copies/ml by week 24 and thereafter.
Methods
Associations between biomarkers of inflammation (IL-6, hs-CRP, D-Dimer), immune activation (sCD163, sCD14, sIL-2r, CD38, HLA-DR), inflammatory monocytes (CD14+CD16+), T cell senescence (CD28, CD57) and exhaustion (PD1) and HDLox, LDLox were assessed at entry and after ART (week 96) with Spearman (partial) correlations.
Results
HDLox declined and LDLox increased over 96 weeks of ART. Positive associations were observed at baseline and over time between HDLox, (but not consistently for LDLox) and most markers of inflammation and immune activation (but not senescence/exhaustion), even after adjustment for multiple comparisons, demographics, entry CD4 count and HIV-1 RNA. HDLox was positively associated with IL-6 (r=0.19–0.29, p<0.01), and sCD163 (r=0.14–0.41 p≤0.04) at all timepoints.
Conclusions
These prospective longitudinal data suggest that oxidized lipoproteins may contribute to persistent immune activation on ART.
doi:10.1097/QAD.0000000000001238
PMCID: PMC5083154  PMID: 27603288
Oxidized lipoproteins; HIV; inflammation; immune activation

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