Epidemiology of alcohol use and alcohol use disorder among female sex workers in Mbeya City, Tanzania.

Alcohol misuse is a global concern, contributing to 5.3% of total deaths and 132.6 million disability-adjusted life years worldwide. In Sub-Saharan African countries, the prevalence of Alcohol Use Disorder (AUD) has risen, especially among female sex workers, due to increased availability and advertising. However, there are limited studies on alcohol use and AUD among female sex workers in Tanzania. This study aimed to determine the prevalence, patterns, and factors associated with alcohol use and AUD among sex workers in Mbeya city, Tanzania. In this cross-sectional study, 212 female sex workers in Mbeya city, Tanzania, seeking enrolment in the National Institute for Medical Research Mbeya Medical Research Centre's registration cohort from July to November 2022. Structured interviews covered socio-demographics, alcohol screening (AUDIT-C and Timeline Follow Back Calendar), and sexual behaviours data. Data were analysed using Stata version 17. Descriptive analysis assessed alcohol consumption and AUD prevalence. Factors associated with alcohol use and AUD at bivariate analysis were identified using Chi-square/Fisher's exact tests. All variables with p-value ≤ 0.20 were entered into a multivariable logistic regression model to identify factors associated with alcohol use and AUD. Among 212 participants, 86.6% reported alcohol use in the past 12 months, 85% in the past 30 days, and 98.5% met AUD criteria. Factors linked to recent alcohol consumption included primary education or higher, income above the median, and more than 10 sexual partners. Education level, marital status, income, and having dependents were significantly associated with heavy drinking episodes. The prevalence of AUD, alcohol use, and heavy episodic drinking were high among female sex workers in Mbeya city. Socio-demographic factors and risky sexual behaviours were associated with alcohol use and heavy episodic drinking highlighting the need for targeted interventions to combat alcohol abuse among female sex workers within the HIV program.

Prehospital portable ultrasound for safe and accurate prehospital needle thoracostomy: a pilot educational study.

BACKGROUND: Simulated needle thoracostomy (NT) using ultrasound may reduce potential injury, increase accuracy, and be as rapid to perform as the traditional landmark technique following a brief educational session. Our objective was to determine if the use of an educational session demonstrating the use of handheld ultrasound to Emergency Medical Services (EMS) staff to facilitate NT was both feasible, and an effective way of increasing the safety and efficacy of this procedure for rural EMS providers. METHODS: A pre/post-educational intervention on a convenience sample of rural North American EMS paramedics and nurses. Measurement of location and estimated depth of placement of needle thoracostomy with traditional landmark technique was completed and then repeated using handheld ultrasound following a training session on thoracic ultrasound and correct placement of NT. RESULTS: A total of 30 EMS practitioners participated. Seven were female (23.3%). There was a higher frequency of dangerous structures underlying the chosen location with the landmark technique 9/60 (15%) compared to the ultrasound technique 1/60 (1.7%) (p = 0.08). Mean time-to-site-selection for the landmark technique was shorter than the ultrasound technique at 10.7 s (range 3.35-45 s) vs. 19.9 s (range 7.8-50 s), respectively (p < 0.001). There was a lower proportion of correct location selection for the landmark technique 40/60 (66.7%) when compared to the ultrasound technique 51/60 (85%) (p = 0.019). With ultrasound, there was less variance between the estimated and measured depth of the pleural space with a mean difference of 0.033 cm (range 0-0.5 cm) when ultrasound was used as compared to a mean difference of 1.0375 cm (range 0-6 cm) for the landmark technique (95% CI for the difference 0.73-1.27 cm; p < 0.001). CONCLUSIONS: Teaching ultrasound NT was feasible in our cohort. While time-to-site-selection for ultrasound-guided NT took longer than the landmark technique, it increased safe and accurate simulated NT placement with fewer identified potential iatrogenic injuries.

Distinct classes of misfolded proteins differentially affect the growth of yeast compromised for proteasome function.

Maintenance of the proteome (proteostasis) is essential for cellular homeostasis and prevents cytotoxic stress responses that arise from protein misfolding. However, little is known about how different types of misfolded proteins impact homeostasis, especially when protein degradation pathways are compromised. We examined the effects of misfolded protein expression on yeast growth by characterizing a suite of substrates possessing the same aggregation-prone domain but engaging different quality control pathways. We discovered that treatment with a proteasome inhibitor was more toxic in yeast expressing misfolded membrane proteins, and this growth defect was mirrored in yeast lacking a proteasome-specific transcription factor, Rpn4p. These results highlight weaknesses in the proteostasis network's ability to handle the stress arising from an accumulation of misfolded membrane proteins.

Harmonizing Experimental Data with Modeling to Predict Membrane Protein Insertion in Yeast.

Membrane proteins must adopt their proper topologies within biological membranes, but achieving the correct topology is compromised by the presence of marginally hydrophobic transmembrane helices (TMHs). In this study, we report on a new model membrane protein in yeast that harbors two TMHs fused to an unstable nucleotide-binding domain. Because the second helix (TMH2) in this reporter has an unfavorable predicted free energy of insertion, we employed established methods to generate variants that alter TMH2 insertion free energy. We first found that altering TMH2 did not significantly affect the extent of protein degradation by the cellular quality control machinery. Next, we correlated predicted insertion free energies from a knowledge-based energy scale with the measured apparent free energies of TMH2 insertion. Although the predicted and apparent insertion energies showed a similar trend, the predicted free-energy changes spanned an unanticipated narrow range. By instead using a physics-based model, we obtained a broader range of free energies that agreed considerably better with the magnitude of the experimentally derived values. Nevertheless, some variants still inserted better in yeast than predicted from energy-based scales. Therefore, molecular dynamics simulations were performed and indicated that the corresponding mutations induced conformational changes within TMH2, which altered the number of stabilizing hydrogen bonds. Together, our results offer insight into the ability of the cellular quality control machinery to recognize conformationally distinct misfolded topomers, provide a model to assess TMH insertion in vivo, and indicate that TMH insertion energy scales may be limited depending on the specific protein and the mutation present.

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inward rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.

Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates.

Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum-associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood. In this study, we asked whether the hydrophobic character of a TMH acts as an energetic barrier to retrotranslocation. To this end, we designed a dual-pass model ERAD substrate, Chimera A*, which contains the cytoplasmic misfolded domain from a characterized ERAD substrate, Sterile 6* (Ste6p*). We found that the degradation requirements for Chimera A* and Ste6p* are similar, but Chimera A* was retrotranslocated more efficiently than Ste6p* in an in vitro assay in which retrotranslocation can be quantified. We then constructed a series of Chimera A* variants containing synthetic TMHs with a range of ΔG values for membrane insertion. TMH hydrophobicity correlated inversely with retrotranslocation efficiency, and in all cases, retrotranslocation remained Cdc48p dependent. These findings provide insight into the energetic restrictions on the retrotranslocation reaction, as well as a new computational approach to predict retrotranslocation efficiency.

Specific α-arrestins negatively regulate Saccharomyces cerevisiae pheromone response by down-modulating the G-protein-coupled receptor Ste2.

G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gßγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself. Ste2 is also downregulated by endocytosis, both constitutive and ligand induced. Ste2 internalization requires its phosphorylation and subsequent ubiquitinylation by membrane-localized protein kinases (Yck1 and Yck2) and a ubiquitin ligase (Rsp5). Here, we demonstrate that three different members of the α-arrestin family (Ldb19/Art1, Rod1/Art4, and Rog3/Art7) contribute to Ste2 desensitization and internalization, and they do so by discrete mechanisms. We provide genetic and biochemical evidence that Ldb19 and Rod1 recruit Rsp5 to Ste2 via PPXY motifs in their C-terminal regions; in contrast, the arrestin fold domain at the N terminus of Rog3 is sufficient to promote adaptation. Finally, we show that Rod1 function requires calcineurin-dependent dephosphorylation.