Assessing Human Immunodeficiency Virus (HIV) Prevention Through Knowledge and Condom Use Among Female Sex Workers at the Border of Haiti and Dominican Republic.

Geographic border studies are relatively scare, but have the potential to inform bilateral health policies that affect the well-being of female sex workers (FSWs) who work at these borders as well as those individuals who solicit their services, both groups being at high risk for human immunodeficiency virus (HIV). We applied bivariate and multivariate techniques to examine FSWs' HIV knowledge and condom use across three partner types, at the Haiti Dominican Republic border, using data from the Study on Sex Workers (n = 241, 2014). Condom use was significantly lower among FSWs on the Haitian side of the border compared to the Dominican side, yet levels of HIV knowledge were similar; specifically, 81% of respondents on the Dominican side reported using condoms every time they had sex with a client, compared to 38% of peers in Haiti (p < 0.001). After introducing controls, FSWs in Haiti continued to have lower odds of using condoms with clients (p < 0.001), noncommercial partners (p < 0.001), and regular partners (p < 0.05) compared to peers in the Dominican Republic. This unique border study highlights disparities in FSWs' condom use regardless of HIV knowledge. The lack of consistent condom use by FSWs in Haiti has the potential to exacerbate the HIV epidemic at the border and impact both nations' HIV incidence rates.

Identification of a Paracrine Signaling Mechanism Linking CD34high Progenitors to the Regulation of Visceral Fat Expansion and Remodeling.

Accumulation of visceral (VIS) is a predictor of metabolic disorders and insulin resistance. This is due in part to the limited capacity of VIS fat to buffer lipids allowing them to deposit in insulin-sensitive tissues. Mechanisms underlying selective hypertrophic growth and tissue remodeling properties of VIS fat are not well understood. We identified subsets of adipose progenitors (APs) unique to VIS fat with differential Cd34 expression and adipogenic capacity. VIS low (Cd34 low) APs are adipogenic, whereas VIS high (Cd34 high) APs are not. Furthermore, VIS high APs inhibit adipogenic differentiation of SUB and VIS low APs in vitro through the secretion of soluble inhibitory factor(s). The number of VIS high APs increased with adipose tissue expansion, and their abundance in vivo caused hypertrophic growth, fibrosis, inflammation, and metabolic dysfunction. This study unveils the presence of APs unique to VIS fat involved in the paracrine regulation of adipogenesis and tissue remodeling.

Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly.

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, ß-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar ß-propeller proteins.

GFI1 functions in transcriptional control and cell fate determination require SNAG domain methylation to recruit LSD1.

Proper hematopoietic cell fate decisions require co-ordinated functions of transcription factors, their associated co-regulators, and histone-modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here, we describe a functional axis involving GFI1, SMYD2, and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -(8)KSKK(11)- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and co-ordinate control of hematopoietic cell fate in both normal and malignant settings.

Platelet-Rich Plasma Injection With Arthroscopic Acromioplasty for Chronic Rotator Cuff Tendinopathy: A Randomized Controlled Trial.

BACKGROUND: Platelet-rich plasma (PRP) has been proposed to augment tendon healing through improving tissue structure during the initial repair phase. PURPOSE: To investigate both the clinical and tissue effects of the coapplication of PRP injection with arthroscopic acromioplasty (AA) in patients with chronic rotator cuff tendinopathy. DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: The study comprised 60 randomized patients diagnosed with rotator cuff tendinopathy (55% women) aged between 35 and 75 years. Patients were randomized to AA alone or in combination with an injection of autologous PRP into the subacromial bursa (AA + PRP). Efficacy of treatment was assessed by analysis of patient-reported outcomes up to 2 years after treatment (Oxford Shoulder Score [OSS]) and by analysis of tendon biopsy specimens taken 12 weeks after treatment. RESULTS: There was no significant difference in the OSS between AA alone and AA + PRP at any time point in the study. From 12 weeks onward, there was a significant increase in the OSS for both groups compared with their baseline scores (P < .001). Bonar scoring determined no significant change in tissue structure with the coapplication of PRP compared with surgery alone. The number of blood vessels and tendon cellularity were significantly decreased in tissue biopsy specimens taken from PRP-treated patients. The expression of p53-positive apoptotic cells increased after AA + PRP but decreased after AA alone. CONCLUSION: Arthroscopic acromioplasty significantly improves long-term clinical outcomes up to 2 years. The coapplication of PRP did not affect clinical outcomes. PRP significantly alters the tissue characteristics in tendons after surgery with reduced cellularity and vascularity and increased levels of apoptosis. CLINICAL RELEVANCE: The coapplication of PRP did not improve clinical outcomes and may have potential deleterious effects on healing tendons. REGISTRY NUMBER: ISRCTN 10464365.

Effect of platelet-rich plasma on healing tissues in acute ruptured Achilles tendon: a human immunohistochemistry study.

BACKGROUND: Platelet-rich plasma (PRP), an autologous derivative of whole blood that contains a supraphysiological concentration of platelets, is thought to invoke an earlier and improved tissue healing response. This notion has been supported by in-vitro and animal studies in bone, cartilage, tendon, and muscle. To our knowledge no published study exists of the effects of PRP in human tissues in vivo. The aim of our study was to investigate the response of ruptured Achilles tendon treated with PRP. METHODS: Tendon tissue biopsy samples were obtained from 20 patients with ruptured Achilles tendon by means of ultrasound-guided needle biopsies from the healing area of the Achilles tendon 6 weeks after treatment with PRP or placebo controls (10 patients each). All samples were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin and alcian blue. Immunohistochemistry markers were used to identify collagen I and III, lymphocytes (CD45), proliferation (KI67), and blood vessels (CD34). All images were masked and analysed with Image J software. FINDINGS: Cellularity and glycosaminoglycans content were significantly higher in PRP-treated tendons than in controls (p=0·01 and p<0·001, respectively). Fibre structure of the tissue was significantly better in the PRP group than in the control tissue (p<0·001). Although both groups showed high collagen I staining, content of collagen I was significantly higher in PRP-treated tendons than in control tendons (p=0·0079), whereas collagen III content was not different (p=1·0). The ratio of collagen III to collagen I was significantly lower in PRP samples (p=0·007). There was no significant difference in CD45 expression (p=0·33). However, PRP samples had fewer blood vessels than did control samples (p=0·023). The overall modified Bonar score was significantly lower in PRP samples, which indicates improved early tendon healing. INTERPRETATION: This is the first study, to our knowledge, to report the immunohistochemical response of ruptured human Achilles tendon to PRP. The findings reveal that locally applied PRP enhanced the maturity of the healing tendon tissues by promoting better collagen I deposition, decreased cellularity, less vascularity, and higher glycosaminoglycan content when compared with control samples. Further work is required to determine the longer term effects of the use of PRP in musculoskeletal diseases. FUNDING: National Institute for Health Research.

Structures of the Gß-CCT and PhLP1-Gß-CCT complexes reveal a mechanism for G-protein ß-subunit folding and Gßγ dimer assembly.

G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein ßγ (Gßγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gß and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gßγ assembly process, the Gß-CCT and the PhLP1-Gß-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gß interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gß with the CCTγ subunit. PhLP1 binding stabilizes the Gß fold, disrupting interactions with CCT and releasing a PhLP1-Gß dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.

NHS patients, staff, and visitor viewpoints of smoking within a hospitals' ground: a qualitative analysis.

BACKGROUND: Smoking is a public health concern and an avoidable cause of morbidity and mortality. Widening tobacco control policies might help shift social norms, the acceptability of exposing others to second-hand smoke, and cultural attitudes towards smoking. This study explored patient, staff, and visitor viewpoints of smoking within the grounds of a National Health Service hospital. METHODS: Analysis of free text responses given as part of a larger repeat cross sectional questionnaire study. Free text qualitative responses analysed using thematic analysis. Pinderfields Hospital, a UK National Health Service hospital in the county of Yorkshire, provides a health service to around half a million people living in the Wakefield and North Kirklees area. Surveys were distributed 10th-18th September and 17th-21st December 2012. Of the n=952 participants who completed an anonymous survey n=306 participants provided a response to the optional free text question. RESULTS: Thematic analysis revealed 5 distinct themes: (1) smoking is a dirty problem; (2) smokers are free to do as they wish; (3) the poor smoker; (4) smoke in our space: the battleground; and (5) no smoking please. Of the n=272 represented by the five themes, generally people accepted that smoking is socially unacceptable but their understanding of smoking behaviours and attitudes towards management and control of smoking differed. There was a strong sense that action is needed to separate the space smokers and non-smokers share. We identified a distinct group of participants that supported a hard line approach and suggested enforcing the no smoking policy through fines and monitoring. CONCLUSIONS: Smoking on hospital grounds remains a contentious issue. Participants acknowledge that smoking is an increasingly unacceptable social behaviour but their understanding and acceptance of smokers vary. There is a strong sense of dislike about the impact of smoke and smokers on the shared hospital environment, with a focus on the hospital entrance. Participants suggest separating smokers and non-smokers and moving smokers away from the hospital entrance with the introduction of smoking shelters. These results suggest a complex narrative that should be investigated further to inform the implementation of the no-smoking policy across hospital settings.

Up-regulation of Glutamate in Painful Human Supraspinatus Tendon Tears.

BACKGROUND: Pain related to rotator cuff tendinopathy is a common problem, but little is known regarding the origin and cause of pain from the tendon substance. No study to date has looked at the association between tissue changes and patient outcomes. PURPOSE: To describe the peripheral neuronal phenotype in painful rotator cuff tears and to determine correlations between tissue changes and clinical outcome measures. STUDY DESIGN: Controlled laboratory study. METHODS: Tissue samples of the supraspinatus were taken from patients undergoing surgery to repair a rotator cuff tendon tear. Patients were classified as having small/medium or large/massive tears. Control tissue was obtained from patients undergoing surgery for posttraumatic shoulder instability. Immunohistochemical techniques were performed using antibodies to known nociceptive and neuronal markers as well as general tissue structural markers. RESULTS: There was no correlation between tissue changes and patient-reported outcomes. A significant increase in the expression of glutamate was seen in tendon tears. There were differences in the expression of metabotropic and ionotropic glutamate receptors. Expression changes were also observed for markers of the sensory and autonomic systems; however, no differences were found in neurotrophins. CONCLUSION: Glutamate and the glutaminergic system play a key role in painful human tendon tears; however, the exact role is still uncertain, as glutamate is highly involved in both pain and metabolic pathways. CLINICAL RELEVANCE: This study has identified a number of markers that could be potential therapeutic targets.

Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex.

Cellular senescence is a crucial tumor suppressor mechanism. We discovered a CAPERα/TBX3 repressor complex required to prevent senescence in primary cells and mouse embryos. Critical, previously unknown roles for CAPERα in controlling cell proliferation are manifest in an obligatory interaction with TBX3 to regulate chromatin structure and repress transcription of CDKN2A-p16INK and the RB pathway. The IncRNA UCA1 is a direct target of CAPERα/TBX3 repression whose overexpression is sufficient to induce senescence. In proliferating cells, we found that hnRNPA1 binds and destabilizes CDKN2A-p16INK mRNA whereas during senescence, UCA1 sequesters hnRNPA1 and thus stabilizes CDKN2A-p16INK. Thus CAPERα/TBX3 and UCA1 constitute a coordinated, reinforcing mechanism to regulate both CDKN2A-p16INK transcription and mRNA stability. Dissociation of the CAPERα/TBX3 co-repressor during oncogenic stress activates UCA1, revealing a novel mechanism for oncogene-induced senescence. Our elucidation of CAPERα and UCA1 functions in vivo provides new insights into senescence induction, and the oncogenic and developmental properties of TBX3.

TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

Glucocorticoids induce specific ion-channel-mediated toxicity in human rotator cuff tendon: a mechanism underpinning the ultimately deleterious effect of steroid injection in tendinopathy?

BACKGROUND: Glucocorticoid injection (GCI) and surgical rotator cuff repair are two widely used treatments for rotator cuff tendinopathy. Little is known about the way in which medical and surgical treatments affect the human rotator cuff tendon in vivo. We assessed the histological and immunohistochemical effects of these common treatments on the rotator cuff tendon. STUDY DESIGN: Controlled laboratory study. METHODS: Supraspinatus tendon biopsies were taken before and after treatment from 12 patients undergoing GCI and 8 patients undergoing surgical rotator cuff repair. All patients were symptomatic and none of the patients undergoing local GCI had full thickness tears of the rotator cuff. The tendon tissue was then analysed using histological techniques and immunohistochemistry. RESULTS: There was a significant increase in nuclei count and vascularity after rotator cuff repair and not after GCI (both p=0.008). Hypoxia inducible factor 1α (HIF-1α) and cell proliferation were only increased after rotator cuff repair (both p=0.03) and not GCI. The ionotropic N-methyl-d-aspartate receptor 1 (NMDAR1) glutamate receptor was only increased after GCI and not rotator cuff repair (p=0.016). An increase in glutamate was seen in both groups following treatment (both p=0.04), while an increase in the receptor metabotropic glutamate receptor 7 (mGluR7) was only seen after rotator cuff repair (p=0.016). CONCLUSIONS: The increases in cell proliferation, vascularity and HIF-1α after surgical rotator cuff repair appear consistent with a proliferative healing response, and these features are not seen after GCI. The increase in the glutamate receptor NMDAR1 after GCI raises concerns about the potential excitotoxic tendon damage that may result from this common treatment.

Characterization of adherent bacteroidales from intestinal biopsies of children and young adults with inflammatory bowel disease.

There is extensive evidence implicating the intestinal microbiota in inflammatory bowel disease [IBD], but no microbial agent has been identified as a sole causative agent. Bacteroidales are numerically dominant intestinal organisms that associate with the mucosal surface and have properties that both positively and negatively affect the host. To determine precise numbers and species of Bacteroidales adherent to the mucosal surface in IBD patients, we performed a comprehensive culture based analysis of intestinal biopsies from pediatric Crohn's disease [CD], ulcerative colitis [UC], and control subjects. We obtained biopsies from 94 patients and used multiplex PCR or 16S rDNA sequencing of Bacteroidales isolates for species identification. Eighteen different Bacteroidales species were identified in the study group, with up to ten different species per biopsy, a number higher than demonstrated using 16S rRNA gene sequencing methods. Species diversity was decreased in IBD compared to controls and with increasingly inflamed tissue. There were significant differences in predominant Bacteroidales species between biopsies from the three groups and from inflamed and uninflamed sites. Parabacteroides distasonis significantly decreased in inflamed tissue. All 373 Bacteroidales isolates collected in this study grew with mucin as the only utilizable carbon source suggesting this is a non-pathogenic feature of this bacterial order. Bacteroides fragilis isolates with the enterotoxin gene [bft], previously associated with flares of colitis, were not found more often at inflamed colonic sites or within IBD subjects. B. fragilis isolates with the ability to synthesize the immunomodulatory polysaccharide A [PSA], previously shown to be protective in murine models of colitis, were not detected more often from healthy versus inflamed tissue.

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy.

As host to the genome, the nucleus plays a critical role as modulator of cellular phenotype. To understand the totality of proteins that regulate this organelle, we used proteomics to characterize the components of the cardiac nucleus. Following purification, cardiac nuclei were fractionated into biologically relevant fractions including acid-soluble proteins, chromatin-bound molecules and nucleoplasmic proteins. These distinct subproteomes were characterized by liquid chromatography-tandem MS. We report a cardiac nuclear proteome of 1048 proteins--only 146 of which are shared between the distinct subcompartments of this organelle. Analysis of genomic loci encoding these molecules gives insights into local hotspots for nuclear protein regulation. High mass accuracy and complementary analytical techniques allowed the discrimination of distinct protein isoforms, including 54 total histone variants, 17 of which were distinguished by unique peptide sequences and four of which have never been detected at the protein level. These studies are the first unbiased analysis of cardiac nuclear subcompartments and provide a foundation for exploration of this organelle's proteomes during disease.

Macrophage stimulating protein is a neurotrophic factor for a sub-population of adult nociceptive sensory neurons.

Macrophage stimulating protein (MSP) is a pleiotropic growth factor that signals via the RON receptor tyrosine kinase. Here we demonstrate that MSP increases the proportion of cultured adult mouse DRG neurons displaying discernable neuritic processes and promotes the elongation and branching of these processes in a dose dependent manner. RON expression in adult DRG is largely restricted to nerve growth factor (NGF)-responsive nociceptive neurons, and MSP mimics the effects of NGF by increasing the expression of several mRNAs that encode functionally important proteins that are characteristically expressed by this neuronal sub-population. MSP mRNA is expressed at high levels in the peripheral target fields of DRG somatic afferents, but is undetectable in DRG, spinal cord or freshly dissected sciatic nerve. These results suggest that MSP is a peripheral target-derived neurotrophic factor for NGF-responsive adult DRG neurons.