Low Levels of HIV Stigma Among Transgender Women Receiving HIV Care.

Despite the prevalence of HIV among transgender women (TGW), gaps exist in understanding the impact of HIV-related stigma (HRS) on TGW with HIV. This is a small cross-sectional pilot study examining HRS in TGW (n=18) with HIV in Miami, FL, who completed a survey during an HIV clinical visit. In contrast with previous studies, results demonstrated low levels of HRS and suggest the potential of increasing acceptance of TGW with HIV as a contributing factor. Larger studies are needed to explore factors underlying HRS with the aim of further reducing stigma among TGW with HIV.

There and Back Again: A Perspective on 20 Years of CYP4Z1.

CYP4Z1, a highly expressed CYP gene in breast cancer, was one of the last CYPs to be identified in the human genome, some twenty years ago. CYP4 enzymes typically catalyze w-hydroxylation and metabolize w3 and w6 polyunsaturated fatty acids (PUFAs) to bioactive lipid metabolites that can influence tumor growth and metastasis. These attributes of CYP4Z1 make it an attractive target for new chemotherapeutic drug design, as a potential biomarker for selection of patients that might respond favorably to drugs and for developing enzyme inhibitors as potential therapeutic agents. This review summarizes the current state of knowledge regarding the advancing biochemistry of CYP4Z1, its role in breast cancer and the recent synthesis of selective chemical inhibitors of the enzyme. We identify gaps that need to be filled to further advance this field and present new experimental data on recombinant CYP4Z1 expression and purification of the active catalytic form. Significance Statement In breast cancer, an unmet need is the availability of highly effective therapeutic agents, especially for triple negative breast cancer. The relevance of the work summarized in this mini-review is that it identifies a new potential drug target, CYP4Z1, and discusses ways in which the gene product's catalytic activity might be modulated in order to combat this malignancy and limit its spread.

Cup-cage constructs in revision total hip arthroplasty for pelvic discontinuity.

Aims: Pelvic discontinuity is a challenging acetabular defect without a consensus on surgical management. Cup-cage reconstruction is an increasingly used treatment strategy. The present study evaluated implant survival, clinical and radiological outcomes, and complications associated with the cup-cage construct. Methods: We included 53 cup-cage construct (51 patients) implants used for hip revision procedures for pelvic discontinuity between January 2003 and January 2022 in this retrospective review. Mean age at surgery was 71.8 years (50.0 to 92.0; SD 10.3), 43/53 (81.1%) were female, and mean follow-up was 6.4 years (0.02 to 20.0; SD 4.6). Patients were implanted with a Trabecular Metal Revision Shell with either a ZCA cage (n = 12) or a TMARS cage (n = 40, all Zimmer Biomet). Pelvic discontinuity was diagnosed on preoperative radiographs and/or intraoperatively. Kaplan-Meier survival analysis was performed, with failure defined as revision of the cup-cage reconstruction. Results: The five-year all-cause survival for cup-cage reconstruction was 73.4% (95% confidence interval (CI) 61.4 to 85.4), while the ten- and 15-year survival was 63.7% (95% CI 46.8 to 80.6). Survival due to aseptic loosening was 93.4% (95% CI 86.2 to 100.0) at five, ten, and 15 years. The rate of revision for aseptic loosening, infection, and dislocation was 3/53 (5.7%), 7/53 (13.2%), and 6/53 (11.3%), respectively. The mean leg length discrepancy improved (p < 0.001) preoperatively from a mean of 18.2 mm (0 to 80; SD 15.8) to 7.0 mm (0 to 35; SD 9.8) at latest follow-up. The horizontal and vertical hip centres improved (p < 0.001) preoperatively from a mean of 9.2 cm (5.6 to 17.5; SD 2.3) to 10.1 cm (6.2 to 13.4; SD 2.1) and 9.3 cm (4.7 to 15.8; SD 2.5) to 8.0 cm (3.7 to 12.3; SD 1.7), respectively. Conclusion: Cup-cage reconstruction provides acceptable outcomes in the management of pelvic discontinuity. One in four constructs undergo revision within five years, most commonly for periprosthetic joint infection, dislocation, or aseptic loosening.

Multivessel Coronary Artery Disease Presenting as a False-Negative Nuclear Stress Test: A Case of Balanced Ischemia.

Myocardial perfusion imaging (MPI) is fundamental to comparing coronary vessel perfusion levels and guides in identifying ischemic areas. However, false negatives, such as balanced ischemia, are important considerations in interpreting these results. In this case report, we describe a 77-year-old female who presented with cardiac chest pain with normal laboratory results, electrocardiogram, and imaging. However, given her history and risk factors, left heart catheterization was performed, which showed triple vessel coronary artery disease.

Cytochrome P450 Family 4F2 and 4F11 Haplotype Mapping and Association with Hepatic Gene Expression and Vitamin K Hydroxylation Activity.

This study evaluated the underlying mechanistic links between genetic variability in vitamin K metabolic pathway genes (CYP4F2 and CYP4F11) and phylloquinone hydroxylation activity using genotype- and haplotype-based approaches. Specifically, we characterized genetic variability in the CYP4F2/CYP4F11 locus and compared common single allele genotypes and common haplotypes as predictors of hepatic gene expression, enzyme abundance, and phylloquinone (VK1) ω-hydroxylation kinetics. We measured CYP4F2 and CYP4F11 mRNA levels, CYP4F2 and CYP4F11 protein abundances, and the VK1 concentration-dependent ω-hydroxylation rate in matched human liver nucleic acid and microsome samples, utilizing a novel in vitro population modeling approach. Results indicate that accounting for the CYP4F2*3 allele alone is sufficient to capture most of the genetic-derived variability in the observed phenotypes. Additionally, our findings highlight the important contribution that CYP4F11 makes toward vitamin K metabolism in the human liver.

Evaluation of stress distribution on an endodontically treated maxillary central tooth with lesion restored with different crown materials: A finite element analysis.

Objectives: The biomechanical response of teeth with periapical lesions that have been restored using various substructure materials, as well as the stress mapping in the alveolar bone, has not been thoroughly described. In this context, the objective of this study is to investigate the structural stress distributions on root canal-treated maxillary right central incisors with lesions restored using different crown materials under linear static loading conditions through finite element analysis (FEA). Methods: In the study, five FEA models were utilised to represent healthy teeth and teeth restored with different substructure materials: (A) a healthy tooth, (B) a lesioned, root canal-treated, composite-filled tooth, (C) a lesioned, fiber-posted, zirconia-based crown, (D) a tooth with lesions, a fiber post, and Ni-Cr infrastructure crown, (E) a tooth with a lesion, a fiber post, and an IPS E-max infrastructure crown. A force of 100 N was applied at an angle of 45° to the long axis of the tooth from 2 mm cervical to the incisal line on the palatal surface. Deformation behaviour and maximum equivalent stress distributions on the tooth sub-components, including the bony structure for each model, were simulated. Results: Differences were observed in the stress distributions of the models. The maximum stress values of the models representing the restorations with different infrastructures varied, and the highest value was obtained in the model of the E-max crown (Model E: 136.050 MPa). The minimum stress magnitudes were obtained from Model B the composite-filled tooth (80.39 MPa); however, it was observed that the equivalent stresses in all the models showed a similar distribution for all components with varying magnitudes. In periapical lesion areas, low stresses were observed. In all models, the cervicobuccal collar region of the teeth had dense equivalent stresses. Conclusion: Different restorative treatment methods applied to root canal-treated teeth with periapical lesions can impact the stress in the alveolar bone and the biomechanical response of the tooth. Relatively high stress values in the cortical bone at the cervical line of the tooth have been observed to decrease towards the apical region. This observation may suggest a potential healing effect by reducing pressure in the periapical lesion area. Clinical significance: Composite resin restorations can be considered the first-choice treatment option for the restoration of root canal-treated teeth with lesions. In crown restorations, it would be advantageous to prefer zirconia or metal-supported prostheses in terms of biomechanics.

Long-term Recordings of Arcuate Nucleus Kisspeptin Neurons Across the Mouse Estrous Cycle.

The arcuate nucleus kisspeptin (ARNKISS) neurons represent the GnRH pulse generator that likely drives pulsatile gonadotropin secretion in all mammals. Using an improved GCaMP fiber photometry system enabling long-term continuous recordings, we aimed to establish a definitive profile of ARNKISS neuronal activity across the murine estrous cycle. As noted previously, a substantial reduction in the frequency of ARNKISS neuron synchronization events (SEs) occurs on late proestrus and extends into estrus. The SE amplitude remains constant throughout the cycle. During metestrus, we unexpectedly detected many multipeak SEs where many SEs occurred rapidly, within 160 seconds of each other. By applying a machine learning-based, k-means clustering analysis, we were further able to detect substantial within-stage variability in the patterns of pulse generator activity. Estrous cycle-dependent changes in SE activity occurred around the time of lights on and off. We also find that a mild stressor such as vaginal lavage reduces ARNKISS neuron SE frequency for up to 3 hours. These observations provide a comprehensive account of ARNKISS neuron activity across the estrous cycle, highlight a new pattern of multipeak SE activity, and introduce a new k-means clustering approach for analyzing ARNKISS neuron population behavior.

Applied Anatomy of the Lower Eyelid Preseptal Space for Treating Ectropion and Retraction with Soft-Tissue Fillers.

BACKGROUND: Lower eyelid malposition can result from age-related changes, such as ectropion, or postsurgical changes, such as retraction after lower lid blepharoplasty. The current accepted treatment is surgical, but soft-tissue fillers have been used as well, with good outcome. The underlying anatomy, which is incompletely described, would be useful information for practitioners desiring to provide minimally invasive injections of the lower eyelid. The authors describe a minimally invasive injection technique adjusted to the complex anatomy of the lower eyelid for the treatment of ectropion and retraction of the lower eyelid. METHODS: A total of 39 periorbital regions of 31 study participants were retrospectively analyzed using photographs before and after reconstruction of the lower eyelid with soft-tissue fillers. Two independent raters assessed the degree of ectropion and lower eyelid retraction (0 to 4, best to worst) before and after the reconstruction and the overall aesthetic improvement using the Periorbital Aesthetic Improvement Scale. RESULTS: The median degree of ectropion and lower eyelid retraction score improved statistically significantly from 3.00 (SD, 1.5) to 1.00 (SD, 1.0) ( P < 0.001). The mean volume of soft-tissue filler material applied per eyelid was 0.73 cc (SD, 0.5). The median Periorbital Aesthetic Improvement Scale score after the treatment was rated as 4.00 (SD, 0.5), indicating improvement of the periorbital functional and appearance. CONCLUSIONS: Anatomic knowledge of the lower eyelid and of the preseptal space is of clinical relevance when reconstructing the lower eyelid with soft-tissue fillers. The targeted space provides optimal lifting capacities for improved aesthetic and functional outcome. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Enhancing Ocular Drug Delivery: The Effect of Physicochemical Properties of Nanoparticles on the Mechanism of Their Uptake by Human Cornea Epithelial Cells.

This study investigates the effect of nanoparticle size and surface chemistry on interactions of the nanoparticles with human cornea epithelial cells (HCECs). Poly(lactic-co-glycolic) acid (PLGA) nanoparticles were synthesized using the emulsion-solvent evaporation method and surface modified with mucoadhesive (alginate [ALG] and chitosan [CHS]) and mucopenetrative (polyethylene glycol [PEG]) polymers. Particles were found to be monodisperse (polydispersity index (PDI) below 0.2), spherical, and with size and zeta potential ranging from 100 to 250 nm and from -25 to +15 mV, respectively. Evaluation of cytotoxicity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that incubating cells with nanoparticles for 24 h at concentrations up to 100 µg/mL caused only mild toxicity (70-100% cell viability). Cellular uptake studies were conducted using an in vitro model developed with a monolayer of HCECs integrated with simulated mucosal solution. Evaluation of nanoparticle uptake revealed that energy-dependent endocytosis is the primary uptake mechanism. Among the different nanoparticles studied, 100 nm PLGA NPs and PEG-PLGA-150 NPs showed the highest levels of uptake by HCECs. Additionally, uptake studies in the presence of various inhibitors suggested that macropinocytosis and caveolae-mediated endocytosis are the dominant pathways. While clathrin-mediated endocytosis was found to also be partially responsible for nanoparticle uptake, phagocytosis did not play a role within the studied ranges of size and surface chemistries. These important findings could lead to improved nanoparticle-based formulations that could improve therapies for ocular diseases.

Brain Point of Care Ultrasound in Young Children Receiving Computed Tomography in the Emergency Department: A Proof of Concept Study.

Background: Point of Care Ultrasound (POCUS) is an important tool in pediatric emergency medicine. In neonatal intensive care medicine ultrasound is often used to evaluate the brains of sick neonates. In theory, POCUS could be used in the ED in young children to evaluate the brain for abnormal pathology. Objectives: To examine the ability of PEM faculty to use brain POCUS to identify clinically significant brain injuries in children with head injuries and/or abnormal neurological exams, and generate sensitivity and specificity of brain POCUS in assessing such findings. Methods: This study used a convenience sample of patients seen in a tertiary care pediatric centre who required a CT head. A team of physicians who were trained at a workshop for brain POCUS were on call to perform the POCUS while being blinded to the results of the CT. Results: 21 children were enrolled in the study. Five (24%) of the patients had a CT that was positive for intracranial bleeds. Of the 5 patients with a positive CT, 3 had a brain POCUS scan that was also positive. The two false negative brain POCUS scans were on patients with small bleeds (no surgical intervention required) on CT, as reported by radiology. The sensitivity of brain POCUS was 60% (CI 15% - 95%) with a specificity of 94% (CI 70%-100%). The diagnostic accuracy of brain POCUS was 86% (CI 64% - 97%). Conclusion: This small proof of concept study shows that brain POCUS is an imaging modality with reasonable sensitivity and specificity in identifying intracranial pathologies that are present on CT. Its use may be most beneficial to expedite definitive imaging and subspeciality involvement.

PrEP initiation and discontinuation among transgender women in the United States: a longitudinal, mixed methods cohort study.

INTRODUCTION: Transgender women in the United States experience high HIV incidence and suboptimal Pre-exposure prophylaxis (PrEP) engagement. We sought to estimate PrEP initiation and discontinuation rates and characterize PrEP discontinuation experiences among a prospective cohort of transgender women. METHODS: Using a sequential, explanatory, mixed-methods design, 1312 transgender women at risk for HIV acquisition were enrolled from March 2018 to August 2020 and followed through July 2022 (median follow-up 24 months; interquartile range 15-36). Cox regression models assessed predictors of initiation and discontinuation. In-depth interviews were conducted among 18 participants, including life history calendars to explore key events and experiences surrounding discontinuations. Qualitative and quantitative data were integrated to generate typologies of discontinuation, inform meta-inferences and facilitate the interpretation of findings. RESULTS: 21.8% (n = 286) of participants reported taking PrEP at one or more study visits while under observation. We observed 139 PrEP initiations over 2127 person-years (6.5 initiations/100 person-years, 95% CI: 5.5-7.7). Predictors of initiation included identifying as Black and PrEP indication. The rate of initiation among those who were PrEP-indicated was 9.6 initiations/100 person-years (132/1372 person-years; 95% CI: 8.1-11.4). We observed 138 PrEP discontinuations over 368 person-years (37.5 discontinuations/100 person-years, 95% CI: 31.7-44.3). Predictors of discontinuation included high school education or less and initiating PrEP for the first time while under observation. Four discontinuation typologies emerged: (1) seroconversion following discontinuation; (2) ongoing HIV acquisition risk following discontinuation; (3) reassessment of HIV/STI prevention strategy following discontinuation; and (4) dynamic PrEP use coinciding with changes in HIV acquisition risk. CONCLUSIONS: PrEP initiation rates were low and discontinuation rates were high. Complex motivations to stop using PrEP did not consistently correspond with HIV acquisition risk reduction. Evidence-based interventions to increase PrEP persistence among transgender women with ongoing acquisition risk and provide HIV prevention support for those who discontinue PrEP are necessary to reduce HIV incidence in this population.

A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

CRISPR-Cas9 knockdown of ESR1 in preoptic GABA-kisspeptin neurons suppresses the preovulatory surge and estrous cycles in female mice.

Evidence suggests that estradiol-sensing preoptic area GABA neurons are involved in the preovulatory surge mechanism necessary for ovulation. In vivo CRISPR-Cas9 editing was used to achieve a 60-70% knockdown in estrogen receptor alpha (ESR1) expression by GABA neurons located within the regions of the rostral periventricular area of the third ventricle (RP3V) and medial preoptic nuclei (MPN) in adult female mice. Mice exhibited variable reproductive phenotypes with the only significant finding being mice with bilateral ESR1 deletion in RP3V GABA neurons having reduced cFos expression in gonadotropin-releasing hormone (GnRH) neurons at the time of the surge. One sub-population of RP3V GABA neurons expresses kisspeptin. Re-grouping ESR1-edited mice on the basis of their RP3V kisspeptin expression revealed a highly consistent phenotype; mice with a near-complete loss of kisspeptin immunoreactivity displayed constant estrus and failed to exhibit surge activation but retained pulsatile luteinizing hormone (LH) secretion. These observations demonstrate that ESR1-expressing GABA-kisspeptin neurons in the RP3V are essential for the murine preovulatory LH surge mechanism.

Opioids exacerbate inflammation in people with well-controlled HIV.

Introduction: People with HIV (PWH) are known to have underlying inflammation and immune activation despite virologic control. Substance use including opioid dependence is common in this population and is associated with increased morbidity and reduced lifespan. The primary objective of the present study termed opioid immunity study (OPIS), was to investigate the impact of chronic opioids in PWH. Methods: The study recruited people with and without HIV who had opioid use disorder (OUD). Study participants (n=221) were categorized into four groups: HIV+OP+, n=34; HIV-OP+, n=66; HIV+OP-, n=55 and HIV-OP-, n=62 as controls. PWH were virally suppressed on ART and those with OUD were followed in a syringe exchange program with confirmation of OP use by urine drug screening. A composite cytokine score was developed for 20 plasma cytokines that are linked to inflammation. Cellular markers of immune activation (IA), exhaustion, and senescence were determined in CD4 and CD8 T cells. Regression models were constructed to examine the relationships of HIV status and opioid use, controlling for other confounding factors. Results: HIV+OP+ participants exhibited highest inflammatory cytokines and cellular IA, followed by HIV-OP+ for inflammation and HIV+OP- for IA. Inflammation was found to be driven more by opioid use than HIV positivity while IA was driven more by HIV than opioid use. In people with OUD, expression of CD38 on CD28-CD57+ senescent-like T cells was elevated and correlated positively with inflammation. Discussion: Given the association of inflammation with a multitude of adverse health outcomes, our findings merit further investigations to understand the mechanistic pathways involved.

Improved methods for the detection of heme and protoporphyrin IX adducts and quantification of heme B from cytochrome P450 containing systems.

Heme B is a critical prosthetic group for the function of numerous proteins including the cytochrome P450 (CYP) family of enzymes. CYP enzymes are involved in the metabolism of endogenous and xenobiotic molecules that are of central interest in drug development. Formation of reactive metabolites by CYPs can lead to heme modification and destruction of the enzyme. The structure of the adducted heme can provide key information on the mechanism of inactivation, which is of great interest during preclinical drug discovery. Historically, techniques to extract the modified heme or protoporphyrin IX species involved harsh extraction conditions and esterification of propionate groups to aid chromatography. We have developed a simplified extraction method and LC/MS chromatography system that does not require derivatization to quantify heme B and identify modified heme B species from multiple CYP-containing matrices. The method uses mass defect filter triggered data dependent MS2 scans to rapidly identify heme and protoporphyrin IX adducts. These methods may also be useful for the analysis of other heme variants and hemoproteins.

Characterization of Gla proteoforms and non-Gla peptides of gamma carboxylated proteins: Application to quantification of prothrombin proteoforms in human plasma.

Gamma (γ) carboxylation is an essential post-translational modification in vitamin K-dependent proteins (VKDPs), involved in maintaining critical biological homeostasis. Alterations in the abundance or activity of these proteins have pharmacological and pathological consequences. Importantly, low levels of fully γ-carboxylated clotting factors increase plasma des-γ-carboxy precursors resulting in little or no biological activity. Therefore, it is important to characterize the levels of γ-carboxylation that reflect the active state of these proteins. The conventional enzyme-linked immunosorbent assay for protein induced by vitamin K absence or antagonist II (PIVKA-II) quantification uses an antibody that is not applicable to distinguish different γ-carboxylation states. Liquid chromatography-mass spectrometry (LC-MS) approaches have been utilized to distinguish different γ-carboxylated proteoforms, however, these attempts were impeded by poor sensitivity due to spontaneous neutral loss of CO2 and simultaneous cleavage of the backbone bond in the collision cell. In this study, we utilized an alkaline mobile phase in combination with polarity switching (positive and negative ionization modes) to simultaneously identify and quantify γ-carboxylated VKDPs. The method was applied to compare Gla proteomics of prothrombin (FII) in 10 µL plasma samples of healthy control and warfarin-treated adults. We also identified surrogate non-Gla peptides for seven other VKDPs to quantify total (active plus inactive) protein levels. The total protein approach (TPA) was used to quantify absolute levels of the VKDPs in human plasma.

Mechanism of Arcuate Kisspeptin Neuron Synchronization in Acute Brain Slices From Female Mice.

The mechanism by which arcuate kisspeptin (ARNKISS) neurons co-expressing glutamate, neurokinin B, and dynorphin intermittently synchronize their activity to drive pulsatile hormone secretion remains unclear in females. In order to study spontaneous synchronization within the ARNKISS neuron network, acute brain slices were prepared from adult female Kiss1-GCaMP6 mice. Analysis of both spontaneous synchronizations and those driven by high frequency stimulation of individual ARNKISS neurons revealed that the network exhibits semi-random emergent excitation dependent upon glutamate signaling through AMPA receptors. No role for NMDA receptors was identified. In contrast to male mice, ongoing tachykinin receptor tone within the slice operated to promote spontaneous synchronizations in females. As previously observed in males, we found that ongoing dynorphin transmission in the slice did not contribute to synchronization events. These observations indicate that a very similar AMPA receptor-dependent mechanism underlies ARNKISS neuron synchronizations in the female mouse supporting the "glutamate two-transition" model for kisspeptin neuron synchronization. However, a potentially important sex difference appears to exist with a more prominent facilitatory role for tachykinin transmission in the female.

The Perils of Overly Sensitive Viral Load Testing for Persons With Human Immunodeficiency Virus.

The concept of "undetectable = untransmittable (U = U)" has been revolutionary in both the prevention and treatment of persons with human immunodeficiency virus (HIV). Most studies proving the concept of U = U used an HIV RNA (viral load [VL]) cutoff of 200 copies/mL to define being undetectable. Since then, increasingly sensitive commercial VL assays, sometimes down to a lower limit of detection (LLD) of 20 copies/mL, lead to confusion about the definition of "undetectable" and when someone is truly considered untransmittable. VLs between the LLD and 200 copies/mL have been associated with future virologic failure; however, no data exist to suggest that intervening in those patients leads to any meaningful benefits. In the absence of a demonstrable benefit of reporting such low VLs, we view this practice as harmful. We suggest recommendations for adjusting VL reporting and improving provider counseling, and call for research designs to mitigate the harms of overly sensitive VL testing.