A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling.

The integrated stress response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling downregulates translation and activates expression of stress-responsive factors that promote return to homeostasis and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions and mediating the therapeutic effects of the small-molecule ISR inhibitor ISRIB are unknown. Using hydrogen-deuterium exchange-mass spectrometry and cryo-electron microscopy, we identified a central α-helix whose orientation allosterically coordinates eIF2B conformation and assembly. Biochemical and cellular signaling assays show that this 'switch-helix' controls eIF2B activity and signaling. In sum, the switch-helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a conserved allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.

ESSENCE: An Implementation Research Program to Scale Up Depression Care in Rural Communities.

BACKGROUND: Task sharing may involve training nonspecialist health workers (NSHWs) to deliver brief mental health interventions. This approach is promising for reducing the global mental health treatment gap. However, capacity is limited for training large cadres of frontline workers in low- and middle-income countries, hindering uptake of these interventions at scale. METHODS: The ESSENCE (enabling translation of science to service to enhance depression care) project in Madhya Pradesh, India, aims to address these challenges through two sequential randomized controlled trials. First, a training trial will evaluate the effectiveness and cost-effectiveness of digital training, compared with conventional face-to-face training, in achieving clinical competency of NSHWs in delivering an intervention for depression. This initial trial will be followed by an implementation trial aimed at evaluating the effectiveness of a remote enhanced implementation support, compared with routine implementation support, in addressing barriers to delivery of depression care in primary care facilities. RESULTS: This project involved developing and pilot testing a scalable smartphone-based program for training NSHWs to deliver a brief psychological intervention for depression screening. This initial research guided a randomized trial of a digital training approach with NSHWs to evaluate the effectiveness of this approach. This trial will be followed by a cluster-randomized trial to evaluate the effectiveness of remote implementation support in ensuring efficient delivery of depression care in primary care facilities. NEXT STEPS: Findings from these trials may inform sustainable training and implementation support models to integrate depression care into primary care for scale-up in resource-constrained settings.

Distinct evolution of type I glutamine synthetase in Plasmodium and its species-specific requirement.

Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids. Interestingly, a putative gene for glutamine synthetase (GS) is retained despite glutamine being an abundant amino acid in human and mosquito hosts. Here we show Plasmodium GS has evolved as a unique type I enzyme with distinct structural and regulatory properties to adapt to the asexual niche. Methionine sulfoximine (MSO) and phosphinothricin (PPT) inhibit parasite GS activity. GS is localized to the parasite cytosol and abundantly expressed in all the life cycle stages. Parasite GS displays species-specific requirement in Plasmodium falciparum (Pf) having asparagine-rich proteome. Targeting PfGS affects asparagine levels and inhibits protein synthesis through eIF2α phosphorylation leading to parasite death. Exposure of artemisinin-resistant Pf parasites to MSO and PPT inhibits the emergence of viable parasites upon artemisinin treatment.

An uncommon case of self-strangulation by ligature and the importance of a crime scene visit by a forensic pathologist.

Self-strangulation is an uncommon method of suicide. The body was found lying on the floor in front of the "multi-gym" inside the gym in the basement of the deceased's house. It was initially presented as a case of sudden death, but during autopsy, a ligature mark was noted over the deceased's neck and bilateral temporal regions along with findings supportive of ligature strangulation. A visit was made to the crime scene. A plausible reconstruction of events suggested that the deceased had used the metallic rope of the multi-gym for this purpose. The rope was connected to weights from one end, passed through a pulley and connected to a rod at the other end. Its width and pattern matched with the ligature mark. The deceased wound the rod end of the rope around his neck and entangled the rod to the rope over his head so that the weight attached to the other end tightened the rope around his neck and strangled him. As the rope unravelled, gravity caused the body to fall to the ground while the rope with the rod resumed its normal position due to the pull of the weight attached at the opposite end. This case is reported for its rarity and the unusual means used to commit suicide by self-strangulation.

Significance of Plasmodium berghei Amino Acid Transporter 1 in Food Vacuole Functionality and Its Association with Cerebral Pathogenesis.

The food vacuole plays a central role in the blood stage of parasite development by digesting host hemoglobin acquired from red blood cells and detoxifying the host heme released during hemoglobin digestion into hemozoin. Blood-stage parasites undergo periodic schizont bursts, releasing food vacuoles containing hemozoin. Clinical studies in malaria-infected patients and in vivo animal studies have shown the association of hemozoin with disease pathogenesis and abnormal host immune responses in malaria. Here, we perform a detailed in vivo characterization of putative Plasmodium berghei amino acid transporter 1 localized in the food vacuole to understand its significance in the malaria parasite. We show that the targeted deletion of amino acid transporter 1 in Plasmodium berghei leads to a swollen food vacuole phenotype with the accumulation of host hemoglobin-derived peptides. Plasmodium berghei amino acid transporter 1-knockout parasites produce less hemozoin, and the hemozoin crystals display a thin morphology compared with wild-type parasites. The knockout parasites show reduced sensitivity to chloroquine and amodiaquine by showing recrudescence. More importantly, mice infected with the knockout parasites are protected from cerebral malaria and display reduced neuronal inflammation and cerebral complications. Genetic complementation of the knockout parasites restores the food vacuole morphology with hemozoin levels similar to that of wild-type parasites, causing cerebral malaria in the infected mice. The knockout parasites also show a significant delay in male gametocyte exflagellation. Our findings highlight the significance of amino acid transporter 1 in food vacuole functionality and its association with malaria pathogenesis and gametocyte development. IMPORTANCE Food vacuoles of the malaria parasite are involved in the degradation of red blood cell hemoglobin. The amino acids derived from hemoglobin degradation support parasite growth, and the heme released is detoxified into hemozoin. Antimalarials such as quinolines target hemozoin formation in the food vacuole. Food vacuole transporters transport hemoglobin-derived amino acids and peptides from the food vacuole to the parasite cytosol. Such transporters are also associated with drug resistance. Here, we show that the deletion of amino acid transporter 1 in Plasmodium berghei leads to swollen food vacuoles with the accumulation of hemoglobin-derived peptides. The transporter-deleted parasites generate less hemozoin with thin crystal morphology and show reduced sensitivity to quinolines. Mice infected with transporter-deleted parasites are protected from cerebral malaria. There is also a delay in male gametocyte exflagellation, affecting transmission. Our findings uncover the functional significance of amino acid transporter 1 in the life cycle of the malaria parasite.

Impact of COVID-19 vaccination: a global perspective.

Introduction: The COVID-19 pandemic has caused widespread morbidity, mortality, and socio-economic disruptions worldwide. Vaccination has proven to be a crucial strategy in controlling the spread of the virus and mitigating its impact. Objective: The study focuses on assessing the effectiveness of COVID-19 vaccination in reducing the incidence of positive cases, hospitalizations, and ICU admissions. The presented study is focused on the COVID-19 fully vaccinated population by considering the data from the first positive case reported until 20 September 2021. Methods: Using data from multiple countries, time series analysis is deployed to investigate the variations in the COVID-19 positivity rates, hospitalization rates, and ICU requirements after successful vaccination campaigns at the country scale. Results: Analysis of the COVID-19 positivity rates revealed a substantial decline in countries with high pre-vaccination rates. Within 1-3 months of vaccination campaigns, these rates decreased by 20-44%. However, certain countries experienced an increase in positivity rates with the emergence of the new Delta variant, emphasizing the importance of ongoing monitoring and adaptable vaccination strategies. Similarly, the analysis of hospitalization rates demonstrated a steady decline as vaccination drive rates rose in various countries. Within 90 days of vaccination, several countries achieved hospitalization rates below 200 per million. However, a slight increase in hospitalizations was observed in some countries after 180 days of vaccination, underscoring the need for continued vigilance. Furthermore, the ICU patient rates decreased as vaccination rates increased across most countries. Within 120 days, several countries achieved an ICU patient rate of 20 per million, highlighting the effectiveness of vaccination in preventing severe cases requiring intensive care. Conclusion: COVID-19 vaccination has proven to be very much effective in reducing the incidence of cases, hospitalizations, and ICU admissions. However, ongoing surveillance, variant monitoring, and adaptive vaccination strategies are crucial for maximizing the benefits of vaccination and effectively controlling the spread of the virus.

Design strategies for composite matrix and multifunctional polymeric scaffolds with enhanced bioactivity for bone tissue engineering.

Over the past few decades, various bioactive material-based scaffolds were investigated and researchers across the globe are actively involved in establishing a potential state-of-the-art for bone tissue engineering applications, wherein several disciplines like clinical medicine, materials science, and biotechnology are involved. The present review article's main aim is to focus on repairing and restoring bone tissue defects by enhancing the bioactivity of fabricated bone tissue scaffolds and providing a suitable microenvironment for the bone cells to fasten the healing process. It deals with the various surface modification strategies and smart composite materials development that are involved in the treatment of bone tissue defects. Orthopaedic researchers and clinicians constantly focus on developing strategies that can naturally imitate not only the bone tissue architecture but also its functional properties to modulate cellular behaviour to facilitate bridging, callus formation and osteogenesis at critical bone defects. This review summarizes the currently available polymeric composite matrices and the methods to improve their bioactivity for bone tissue regeneration effectively.

COVID-19-Associated Mucormycosis in a Tertiary Care Hospital in India: A Case Series.

Mucormycosis is a disease that usually occurs in immunocompromised patients or those with uncontrolled diabetes mellitus. The second wave of the coronavirus disease 2019 (COVID-19) pandemic in India was accompanied by an unexpected rise in mucormycosis cases, ranging from the most commonly occurring Rhino-orbital-cerebral mucormycosis (ROCM) to rare cases of pulmonary and gastrointestinal mucormycosis. The majority of cases that presented to our hospital were individuals with underlying diabetes mellitus who received steroids for COVID-19 before being diagnosed with mucormycosis. In this case series, we present five rhino-orbital-cerebral mucormycosis cases that were histopathologically positive and treated at a tertiary-care hospital in India. Magnetic resonance imaging (MRI) of all of the patients demonstrated orbital apex syndrome and diffuse or focal infiltration of the cavernous sinus. Cases were treated with anti-fungal drugs, transcutaneous retrobulbar injection of amphotericin B (TRAM B), along with appropriate surgical excision and debridement of the involved tissue. The essential elements for successfully managing this fatal infection are control of the predisposing factors, early detection, anti-fungal drugs, and surgical debridement of the involved tissues.

Malaria parasite heme biosynthesis promotes and griseofulvin protects against cerebral malaria in mice.

Heme-biosynthetic pathway of malaria parasite is dispensable for asexual stages, but essential for mosquito and liver stages. Despite having backup mechanisms to acquire hemoglobin-heme, pathway intermediates and/or enzymes from the host, asexual parasites express heme pathway enzymes and synthesize heme. Here we show heme synthesized in asexual stages promotes cerebral pathogenesis by enhancing hemozoin formation. Hemozoin is a parasite molecule associated with inflammation, aberrant host-immune responses, disease severity and cerebral pathogenesis. The heme pathway knockout parasites synthesize less hemozoin, and mice infected with knockout parasites are protected from cerebral malaria and death due to anemia is delayed. Biosynthetic heme regulates food vacuole integrity and the food vacuoles from knockout parasites are compromised in pH, lipid unsaturation and proteins, essential for hemozoin formation. Targeting parasite heme synthesis by griseofulvin-a FDA-approved antifungal drug, prevents cerebral malaria in mice and provides an adjunct therapeutic option for cerebral and severe malaria.

Assessing costs of developing a digital program for training community health workers to deliver treatment for depression: A case study in rural India.

Digital technology has emerged as a promising approach for training and building capacity of community health workers in low-income and middle-income countries (LMICs). Little is known about the cost of developing digital training programs in LMICs, which hinders the adoption, implementation, and scaling up of the programs in routine primary care settings. This study assessed the costs of developing a digital program for training community health workers to deliver a psychological treatment for depression in a rural district of Madhya Pradesh, India. We developed survey instruments to document required resources in development, including involved personnel (their roles, responsibilities, time spent, and salaries or payments), information technologies (e.g., smartphones, software programs), and infrastructure-related costs (e.g., vehicle, office space, utilities). Costs were estimated from an accounting perspective. Over a 10-month developmental period, the total costs were 208,814 USD, with the largest portion on human resources (61%, with 14% on management and supervision), followed by information technologies (33%), and infrastructure-related costs (6%). These findings could inform policymakers in LMICs on costs of developing online-training programs, which will be especially useful during the COVID-19 pandemic.

Sudden death due to non-traumatic rupture of splenic artery aneurysm.

INTRODUCTION: Splenic artery aneurysm is a rare form of vascular pathology that carries a high risk of mortality once it gets ruptured. It has a prevalence of 1% and occurs due to thinning and dilatation of the arterial wall. CASE: We describe a case of a 35-year-old policeman who died suddenly. At medico-legal autopsy, intraperitoneal clotted blood about 1000 g and liquid blood about 3000 ml were seen. On further exploration, ruptured splenic artery aneurysm about 2.0 cm in diameter became visible near the hilum. CONCLUSION: Rare cases typically present as sudden and unexpected death with intraperitoneal bleed and may be confused with blunt trauma abdomen. Therefore, splenic artery aneurysm is an appropriate differential diagnosis for sudden deaths and intraperitoneal bleeding, respectively.

Socio-demographic profile of suicidal hanging deaths in Delhi and the National Capital Region - a retrospective study.

The aim of this retrospective socio-demographic analysis is to identify those at higher risk of suicidal hanging in the region of Delhi and the National Capital Region. All deaths due to suicidal hanging from January 2016 to December 2019 reported in the Vardhman Mahavir Medical College and Safdarjung Hospital were included. Suicidal hanging accounted for 2.67% of total autopsied cases; 21-30 years old represented 42.62% of the victims. Male:female ratio was 1.7:1 and 38.37% of cases were from the adjoining areas of Vasant Kunj and Vasant Vihar. Therefore, a preventive strategy should focus concern on young adults, the male sex, and areas of Vasant Kunj and Vasant Vihar.

Digital training for non-specialist health workers to deliver a brief psychological treatment for depression in India: Protocol for a three-arm randomized controlled trial.

BACKGROUND: Training non-specialist health workers (NSHWs) at scale is a major barrier to increasing the coverage of depression care in India. This trial will test the effectiveness of two forms of digital training compared to conventional face-to-face training in changing the competence of NSHWs to deliver a brief evidence-based psychological treatment for depression. METHODS: This protocol is for a three-arm, parallel group randomized controlled trial comparing three ways of training NSHWs to deliver the Healthy Activity Program (HAP), a brief manualized psychotherapy for depression, in primary care. The arms are: digital training (DGT); digital training combined with individualized coaching support (DGT+); and conventional face-to-face training (F2F). The target sample comprises N = 336 government contracted NSHWs in Madhya Pradesh, India. The primary outcome is change of competence to deliver HAP; secondary outcomes include cost-effectiveness of the training programs, change in participants' mental health knowledge, attitudes and behavior, and satisfaction with the training. Assessors blind to participant allocation status will collect outcomes pre- (baseline) and post- (endpoint) training to ascertain differences in outcomes between arms. Training program costs will be collected to calculate incremental costs of achieving one additional unit on the competency measure in the digital compared to face-to-face training programs. Health worker motivation, job satisfaction, and burnout will be collected as exploratory outcome variables. DISCUSSION: This trial will determine whether digital training is an effective, cost-effective, and scalable approach for building workforce capacity to deliver a brief evidence-based psychological treatment for depression in primary care in a low-resource setting. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04157816.

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

Digital Training for Non-Specialist Health Workers to Deliver a Brief Psychological Treatment for Depression in Primary Care in India: Findings from a Randomized Pilot Study.

Introduction: Task sharing holds promise for scaling up depression care in countries such as India, yet requires training large numbers of non-specialist health workers. This pilot trial evaluated the feasibility and acceptability of a digital program for training non-specialist health workers to deliver a brief psychological treatment for depression. Methods: Participants were non-specialist health workers recruited from primary care facilities in Sehore, a rural district in Madhya Pradesh, India. A three-arm randomized controlled trial design was used, comparing digital training alone (DGT) to digital training with remote support (DGT+), and conventional face-to-face training. The primary outcome was the feasibility and acceptability of digital training programs. Preliminary effectiveness was explored as changes in competency outcomes, assessed using a self-reported measure covering the specific knowledge and skills required to deliver the brief psychological treatment for depression. Outcomes were collected at pre-training and post-training. Results: Of 42 non-specialist health workers randomized to the training programs, 36 including 10 (72%) in face-to-face, 12 (86%) in DGT, and 14 (100%) in DGT+ arms started the training. Among these participants, 27 (64%) completed the training, with 8 (57%) in face-to-face, 8 (57%) in DGT, and 11 (79%) in DGT+. The addition of remote telephone support appeared to improve completion rates for DGT+ participants. The competency outcome improved across all groups, with no significant between-group differences. However, face-to-face and DGT+ participants showed greater improvement compared to DGT alone. There were numerous technical challenges with the digital training program such as poor connectivity, smartphone app not loading, and difficulty navigating the course content-issues that were further emphasized in follow-up focus group discussions with participants. Feedback and recommendations collected from participants informed further modifications and refinements to the training programs in preparation for a forthcoming large-scale effectiveness trial. Conclusions: This study adds to mounting efforts aimed at leveraging digital technology to increase the availability of evidence-based mental health services in primary care settings in low-resource settings.

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation.

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

Inhibition of the integrated stress response by viral proteins that block p-eIF2-eIF2B association.

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.

Assessing health worker competence to deliver a brief psychological treatment for depression: development and validation of a scalable measure.

Increased interest in disseminating and implementing psychological treatments has focused on the need for evidence-based training programs for providers, especially those without specialized training. To evaluate provider-training programs, validated outcome measures are necessary; however, the scalable measurement of training outcomes has been largely overlooked. Current methods of assessing providers' ability to deliver psychological treatments are generally time-consuming and costly, representing a major bottleneck in scaling up mental health care for commonly occurring disorders such as depression. The present study describes the development and initial validation of a scalable measure for assessing provider competence in delivering a brief behavioral activation treatment for depression, called the Healthy Activity Program, adapted for primary care settings. The measure focuses on testing knowledge about the treatment and applied knowledge regarding how to skillfully deliver the treatment, both essential features of competence. The measure was tested on a sample of 531 respondents with a variety of educational levels and professional backgrounds and found to meet the requirements of the Rasch model. Three versions of the measure each of equal difficulty were derived to allow repeat testing of training outcomes over time. A scalable measure of provider competence is an essential first step towards supporting the wider dissemination and implementation of brief psychological interventions for depression, especially in low-resource settings.

Structural insights into ISRIB, a memory-enhancing inhibitor of the integrated stress response.

The integrated stress response (ISR) regulates protein synthesis under conditions of stress. Phosphorylation of translation initiation factor eIF2 by stress-sensing kinases converts eIF2 from substrate to competitive inhibitor of its dedicated nucleotide exchange factor, eIF2B, arresting translation. A drug-like molecule called integrated stress response inhibitor (ISRIB) reverses the effects of eIF2 phosphorylation and restores translation by targeting eIF2B. When administered to mice, ISRIB enhances cognition and limits cognitive decline due to brain injury. To determine ISRIB's mechanism of action, we solved an atomic structure of ISRIB bound to the human eIF2B decamer. We found that ISRIB acts as a molecular staple, pinning together tetrameric subcomplexes of eIF2B along the assembly path to a fully active, decameric enzyme. In this Structural Snapshot, we discuss ISRIB's mechanism, its ability to rescue disease mutations in eIF2B and conservation of the enzyme and ISRIB-binding pocket.