Examining the use, confidence, and barriers to follow Advanced Life Support in Obstetrics (ALSO) course guides in managing obstetric emergencies in Sudan.

BACKGROUND: The Advanced Life Support in Obstetrics (ALSO) course is a globally recognized interprofessional training program designed to assist healthcare professionals in acquiring and sustaining the necessary knowledge and skills to handle obstetric emergencies effectively. This survey aimed to assess the use, barriers, and confidence in using the ALSO course guidelines in managing obstetric emergencies in Sudan. METHODS: This descriptive cross-sectional study involved 103 physicians from the Sudan ALSO group in Sudan. A structured, close-ended questionnaire was distributed electronically to the participants. Data analysis was conducted using Statistical Package of Social Sciences Software version 26. RESULTS: More than half of the participants were specialists (54.4%). Although all respondents claimed to adhere to the ALSO guidelines for managing shoulder dystocia, a lower percentage followed them for neonatal resuscitation (75.0%) and maternal venous thrombosis management (68.9%). Only 62.1% of participants felt confident performing neonatal resuscitation. The main barriers to implementing the ALSO course guidelines were the respondents' preference for other guidelines and their belief that the guidelines were not applicable in their specific settings. CONCLUSION: The majority of participants displayed a high level of confidence, indicating a positive perception of the guide's effectiveness. However, there is room for improvement, particularly in areas such as neonatal resuscitation and forceps-assisted births, where confidence levels were lower. Addressing barriers, including the preference for other guidelines and the applicability of the guide in specific settings, is crucial to ensure widespread adoption. Refresher training programs, contextual adaptations, and the integration of guidelines may help overcome these barriers and enhance the overall implementation of the ALSO guide in managing obstetric emergencies in Sudan.

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein.

RATIONALE: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. METHODS: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. RESULTS: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. CONCLUSION: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

Violence in Sudan: A Looming Public Health Disaster.

Sudan's ongoing conflict, rooted in colonial-era policies and resource competition, has led to widespread displacement, poverty, and social service breakdowns. The escalating power struggle between the Sudanese Army Forces (SAF) and the paramilitary Rapid Support Forces (RSF) exacerbates the humanitarian crisis by severely undermining the nation's health services and infrastructure. This leads to long-lasting social and economic consequences, creating a need for a coordinated response from national and international organizations to provide emergency healthcare, rebuild infrastructure, and train and retain healthcare workers. Moreover, the recent takeover of a National Public Health Laboratory in Khartoum, which contains dangerous biological material, is considered extremely dangerous. The expulsion of technicians and power cuts prevent the proper management of biological materials (e.g., polio, measles, and cholera isolates). This editorial sheds light on the deep-seated repercussions of the conflict in Sudan, with a specific focus on the toll it takes on health services and infrastructure. It calls for an all-encompassing, synergistic approach that places the health and welfare of impacted communities at the forefront. Through concerted collaboration between national entities and the global community, there lies the potential to pave the way for recuperation, fortitude, and enduring stability in regions ravaged by conflict.

Analysis of the N-glycosylation profiles of the spike proteins from the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a "glycan shield"). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics.

N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry.

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.

Membrane-partitioned cell wall synthesis in mycobacteria.

Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in Mycobacterium smegmatis, we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. smegmatis, indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis.

Role of LmeA, a Mycobacterial Periplasmic Protein, in Maintaining the Mannosyltransferase MptA and Its Product Lipomannan under Stress.

The mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, a periplasm, and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan chain. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis Here, we examined the role of LmeA under stress conditions. We found that lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in an lmeA deletion (ΔlmeA) mutant under both stress conditions. The transcript levels of mptA in the ΔlmeA mutant were similar to or even higher than those in the wild type, indicating that the decrease of MptA protein was a posttranscriptional event. The ΔlmeA mutant was unable to maintain the cellular level of LM under stress, consistent with the decrease in MptA. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase, particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity.IMPORTANCE Mycobacteria differentially regulate the cellular amounts of lipoglycans in response to environmental changes, but the molecular mechanisms of this regulation remain unknown. Here, we demonstrate that cellular lipoarabinomannan (LAM) levels rapidly decline under two stress conditions, stationary growth phase and nutrient starvation, while the levels of another related lipoglycan, lipomannan (LM), stay relatively constant. The persistence of LM under stress correlated with the maintenance of two key mannosyltransferases, MptA and MptC, in the LM biosynthetic pathway. We further showed that the stress exposures lead to the upregulation of lmeA gene expression and that the periplasmic protein LmeA plays a key role in maintaining the enzyme MptA and its product LM under stress conditions. These findings reveal new aspects of how lipoglycan biosynthesis is regulated under stress conditions in mycobacteria.

Deletion of PimE mannosyltransferase results in increased copper sensitivity in Mycobacterium smegmatis.

The unique cell envelope structure of Mycobacterium tuberculosis is fundamental to its pathogenesis. Phosphatidylinositol (PI)-anchored glycolipids, such as phosphatidylinositol mannosides (PIMs), lipomannan and lipoarabinomannan, are essential components of the cell envelope widely conserved among mycobacteria, but their roles in the cell envelope integrity are not fully understood. We previously identified PimE in Mycobacterium smegmatis, a nonpathogenic model organism, as a mannosyltransferase that catalyzes the fifth mannose transfer for the biosynthesis of hexamannosyl PIMs. Our analyses, reported here, further demonstrate that the growth of the pimE deletion mutant (ΔpimE) is defective in the presence of copper. We first found that the small colony phenotype of ΔpimE on a solid Middlebrook 7H10 agar surface was alleviated when grown on M63 agar. Comparative analysis of the two media led us to identify copper in Middlebrook 7H10 as the cause of growth retardation seen in ΔpimE. We further demonstrated that ΔpimE is sensitized to several antibiotics, but the increased sensitivities were independent of the presence of copper. We conclude that the deletion of the pimE gene does not cause growth defects under optimal growth conditions, but makes the cell envelope vulnerable to toxic compounds such as copper and antibiotics.