A precise swaying map for how promiscuous cellobiose-2-epimerase operate bi-reaction.

Promiscuous enzymes play a crucial role in organism survival and new reaction mining. However, comprehensive mapping of the catalytic and regulatory mechanisms hasn't been well studied due to the characteristic complexity. The cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) with complex epimerization and isomerization was chosen to comprehensively investigate the promiscuous mechanisms. Here, the catalytic frame of ring-opening, cis-enediol mediated catalysis and ring-closing was firstly determined. To map the full view of promiscuous CE, the structure of CsCE complex with the isomerized product glucopyranosyl-ß1,4-fructose was determined. Combined with computational calculation, the promiscuity was proved a precise cooperation of the double subsites, loop rearrangement, and intermediate swaying. The flexible loop was like a gear, whose structural reshaping regulates the sway of the intermediates between the two subsites of H377-H188 and H377-H247, and thus regulates the catalytic directions. The different protonated states of cis-enediol intermediate catalyzed by H188 were the key point for the catalysis. The promiscuous enzyme tends to utilize all elements at hand to carry out the promiscuous functions.

Five-Year results of a multi-specialty induction course for surgical training.

Background: The Essential Surgical Skills Course (ESSC) is a multi-specialty induction "boot camp" style course that has been run successfully for five years. The aim of the current paper is to create an accurate guide for the replication of the course by other teams and assess the course's fitness for purpose, through the survey feedback provided by trainees. Methods: The course's fitness for purpose was assessed through cumulative five-year survey feedback from trainees. This observational study describes the design and process of content adjustment according to feedback. Results: The course its five-year span offered twelve different procedural skills in four different specialties. Feedback for each session was persistently >8/10. Key themes identified as beneficial include teacher-to-trainee ratio (often 1:1), teaching style, course structure and responsiveness. Conclusions: The ESSC was found to be fit for purpose for the induction of trainees into surgical training. The key factors contributing to the success of the course include the structured method of curriculum design, outstanding teaching delivery methods, teacher-to-trainee ratio, the availability of appropriate faculty and infrastructure and the willingness to learn from trainee feedback and adjust the content of the course accordingly. It acts as a paradigm for courses aimed to prepare surgical trainees for a "step-up" in their careers.

Human immune and gut microbial parameters associated with inter-individual variations in COVID-19 mRNA vaccine-induced immunity.

COVID-19 mRNA vaccines induce protective adaptive immunity against SARS-CoV-2 in most individuals, but there is wide variation in levels of vaccine-induced antibody and T-cell responses. However, the mechanisms underlying this inter-individual variation remain unclear. Here, using a systems biology approach based on multi-omics analyses of human blood and stool samples, we identified several factors that are associated with COVID-19 vaccine-induced adaptive immune responses. BNT162b2-induced T cell response is positively associated with late monocyte responses and inversely associated with baseline mRNA expression of activation protein 1 (AP-1) transcription factors. Interestingly, the gut microbial fucose/rhamnose degradation pathway is positively correlated with mRNA expression of AP-1, as well as a gene encoding an enzyme producing prostaglandin E2 (PGE2), which promotes AP-1 expression, and inversely correlated with BNT162b2-induced T-cell responses. These results suggest that baseline AP-1 expression, which is affected by commensal microbial activity, is a negative correlate of BNT162b2-induced T-cell responses.

Dietary compounds activate an insect gustatory receptor on enteroendocrine cells to elicit myosuppressin secretion.

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae.

LEARN: A multi-centre, cross-sectional evaluation of Urology teaching in UK medical schools.

OBJECTIVE: To evaluate the status of UK undergraduate urology teaching against the British Association of Urological Surgeons (BAUS) Undergraduate Syllabus for Urology. Secondary objectives included evaluating the type and quantity of teaching provided, the reported performance rate of General Medical Council (GMC)-mandated urological procedures, and the proportion of undergraduates considering urology as a career. MATERIALS AND METHODS: LEARN was a national multicentre cross-sectional study. Year 2 to Year 5 medical students and FY1 doctors were invited to complete a survey between 3rd October and 20th December 2020, retrospectively assessing the urology teaching received to date. Results are reported according to the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). RESULTS: 7,063/8,346 (84.6%) responses from all 39 UK medical schools were included; 1,127/7,063 (16.0%) were from Foundation Year (FY) 1 doctors, who reported that the most frequently taught topics in undergraduate training were on urinary tract infection (96.5%), acute kidney injury (95.9%) and haematuria (94.4%). The most infrequently taught topics were male urinary incontinence (59.4%), male infertility (52.4%) and erectile dysfunction (43.8%). Male and female catheterisation on patients as undergraduates was performed by 92.1% and 73.0% of FY1 doctors respectively, and 16.9% had considered a career in urology. Theory based teaching was mainly prevalent in the early years of medical school, with clinical skills teaching, and clinical placements in the later years of medical school. 20.1% of FY1 doctors reported no undergraduate clinical attachment in urology. CONCLUSION: LEARN is the largest ever evaluation of undergraduate urology teaching. In the UK, teaching seemed satisfactory as evaluated by the BAUS undergraduate syllabus. However, many students report having no clinical attachments in Urology and some newly qualified doctors report never having inserted a catheter, which is a GMC mandated requirement. We recommend a greater emphasis on undergraduate clinical exposure to urology and stricter adherence to GMC mandated procedures.

Randomized controlled trial of the "WISER" intervention to reduce healthcare worker burnout.

OBJECTIVE: Test web-based implementation for the science of enhancing resilience (WISER) intervention efficacy in reducing healthcare worker (HCW) burnout. DESIGN: RCT using two cohorts of HCWs of four NICUs each, to improve HCW well-being (primary outcome: burnout). Cohort 1 received WISER while Cohort 2 acted as a waitlist control. RESULTS: Cohorts were similar, mostly female (83%) and nurses (62%). In Cohorts 1 and 2 respectively, 182 and 299 initiated WISER, 100 and 176 completed 1-month follow-up, and 78 and 146 completed 6-month follow-up. Relative to control, WISER decreased burnout (-5.27 (95% CI: -10.44, -0.10), p = 0.046). Combined adjusted cohort results at 1-month showed that the percentage of HCWs reporting concerning outcomes was significantly decreased for burnout (-6.3% (95%CI: -11.6%, -1.0%); p = 0.008), and secondary outcomes depression (-5.2% (95%CI: -10.8, -0.4); p = 0.022) and work-life integration (-11.8% (95%CI: -17.9, -6.1); p < 0.001). Improvements endured at 6 months. CONCLUSION: WISER appears to durably improve HCW well-being. CLINICAL TRIALS NUMBER: NCT02603133; https://clinicaltrials.gov/ct2/show/NCT02603133.

COVID-19 serological survey using micro blood sampling.

During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for more than 9 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.

Takotsubo syndrome: a predominantly female CV disorder, from the perspective of primary care.

We describe two cases of Takotsubo syndrome and discuss the issues relating to diagnosis and patient communication that they raise.

Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis.

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Šresolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148-181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.

Structure of polymerized type V pilin reveals assembly mechanism involving protease-mediated strand exchange.

Bacterial adhesion is a general strategy for host-microbe and microbe-microbe interactions. Adhesive pili are essential for colonization, biofilm formation, virulence and pathogenesis of many environmental and pathogenic bacteria1,2. Members of the class Bacteroidia have unique type V pili, assembled by protease-mediated polymerization3. Porphyromonas gingivalis is the main contributor to periodontal disease and its type V pili are a key factor for its virulence4. However, the structure of the polymerized pilus and its assembly mechanism are unknown. Here we show structures of polymerized and monomeric states of FimA stalk pilin from P. gingivalis, determined by cryo-electron microscopy and crystallography. The atomic model of assembled FimA shows that the C-terminal strand of a donor subunit is inserted into a groove in the ß-sheet of an acceptor subunit after N-terminal cleavage by the protease RgpB. The C terminus of the donor strand is essential for polymerization. We propose that type V pili assemble via a sequential polar assembly mechanism at the cell surface, involving protease-mediated strand exchange, employed by various Gram-negative species belonging to the class Bacteroidia. Our results reveal functional surfaces related to pathogenic properties of polymerized FimA. These insights may facilitate development of antibacterial drugs.

Catalytic Cycle of Neisseria meningitidis CMP-Sialic Acid Synthetase Illustrated by High-Resolution Protein Crystallography.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of α-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the α-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

A Bump-Hole Approach for Directed RNA Editing.

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity.

Neurodevelopmental Outcomes in Early Childhood.

Technological advances in neonatal-perinatal medicine have led to a steady increase in the survival of preterm infants. Although the increase in survival is a remarkable success, children born preterm remain at high risk for brain injury and long-term neurodevelopmental deficits. Children born preterm may have abnormal muscle tone or movements, cognitive deficits, language impairments, and behavioral problems. This article reviews neurodevelopmental outcomes and factors that influence outcomes in preterm children during early childhood.

POSTOPERATIVE INSULIN REQUIREMENTS IN BARIATRIC SURGERY.

OBJECTIVE: Though insulin dose reduction months after surgery is a well-studied outcome, there are limited data on immediate postoperative changes. The goals of the present study were to ( 1) To determine peri-operative glycemic control in patients with type 2 diabetes mellitus (DM) on insulin who have undergone Roux-en-Y gastric bypass (RYGB) and ( 2) to compare pre- and postoperative insulin regimens and dosages in these patients. METHODS: A retrospective chart review was conducted on patients with type 2 DM on insulin who underwent RYGB surgery. Blood glucose (BG) levels and insulin doses were compared prior to surgery, on the day of surgery (DOS), and postoperative days (POD) 1 and 2. Subgroup analysis was performed to see if insulin dose was related to glucose control. RESULTS: There were 114 subjects with a mean (SD) age of 52.8 ± 9.8 years, body mass index (BMI) 46.2 ± 8.0 kg/m2, glycated hemoglobin A1c (HbA1c) 8.3% (67 mmol/mol) ± 1.7%, and 66% on insulin plus noninsulin medications and 34% on insulin only. Mean blood glucose (BG) significantly decreased from the DOS (185 ± 43 mg/dL) through POD2 (160 ± 36, P<.0001). The median daily insulin dose significantly decreased from before surgery on usual diet (75 units [36, 116 interquartile range (IQR)]) through POD2 (6 [2, 15 IQR]), P<.0001). The median insulin dose per body weight decreased significantly from before surgery on usual diet (0.58 units/kg [0.35, 0.84 IQR]) through POD2 (0.04 [0.02, 0.11 IQR]), P<.0001). The subgroup with relatively good control experienced a larger percentage reduction in insulin requirements versus subjects with poor control. CONCLUSION: An 87.5% reduction in total daily insulin dose was seen by POD2. This will assist in developing algorithms for insulin titration postbariatric surgery. ABBREVIATIONS: BG = blood glucose DM = diabetes mellitus DOS = day of surgery HbA1c = glycated hemoglobin IQR = interquartile range IV = intravenous NPH = neutral protamine Hagedorn POD = postoperative day RYGB = Roux-en-Y gastric bypass SSRI = sliding scale regular insulin.

Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity.

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

Vending machine assessment methodology. A systematic review.

The nutritional quality of food and beverage products sold in vending machines has been implicated as a contributing factor to the development of an obesogenic food environment. How comprehensive, reliable, and valid are the current assessment tools for vending machines to support or refute these claims? A systematic review was conducted to summarize, compare, and evaluate the current methodologies and available tools for vending machine assessment. A total of 24 relevant research studies published between 1981 and 2013 met inclusion criteria for this review. The methodological variables reviewed in this study include assessment tool type, study location, machine accessibility, product availability, healthfulness criteria, portion size, price, product promotion, and quality of scientific practice. There were wide variations in the depth of the assessment methodologies and product healthfulness criteria utilized among the reviewed studies. Of the reviewed studies, 39% evaluated machine accessibility, 91% evaluated product availability, 96% established healthfulness criteria, 70% evaluated portion size, 48% evaluated price, 52% evaluated product promotion, and 22% evaluated the quality of scientific practice. Of all reviewed articles, 87% reached conclusions that provided insight into the healthfulness of vended products and/or vending environment. Product healthfulness criteria and complexity for snack and beverage products was also found to be variable between the reviewed studies. These findings make it difficult to compare results between studies. A universal, valid, and reliable vending machine assessment tool that is comprehensive yet user-friendly is recommended.

G protein activation without a GEF in the plant kingdom.

Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the "self-activating" trait, indicate the existence of divergent Gα regulatory mechanisms in the plant kingdom. In the grasses, purifying selection on the regulatory gene was lost after the physical decoupling of the RGS protein and its cognate Gα partner. More broadly these findings show extreme divergence in Gα activation and regulation that played a critical role in the evolution of G protein signaling pathways.

The effects of a supplemental, theory-based physical activity counseling intervention for adults with type 2 diabetes.

BACKGROUND: Physical activity (PA) is a cornerstone in the management of type 2 diabetes (T2DM). This pilot investigation explores the effects of a standard diabetes education program compared with a supplemental PA intervention on diabetes-related health outcomes. METHODS: Using a prospective 2-armed design, 96 adults with T2DM were randomly assigned to either standard care (diabetes education program; n = 49) or standard care supplemented with an 8-week, individualized-counseling and community-based PA component (n = 47). Measurements were taken at baseline, 3, 6, and 12 months. Primary outcomes were changes in PA (self-report) and HbA1c. Between group changes were compared using analysis of covariance (ANCOVA) and changes over time using repeated-measures ANOVA. RESULTS: In comparison with standard care, the supplemental group demonstrated an increase in PA (Ps < 0.01) and cardiorespiratory fitness (Ps < 0.05) from baseline to all follow-up time-points. HbA1c levels declined (P < .05) from baseline to all time points in the standard care group. Reduction in cholesterol-ratio (P < .01), increase in HDL (P < .05), and reductions in blood pressure, resting heart rate and BMI (approaching statistical significance Ps < 0.10) were also reported for both groups. CONCLUSIONS: PA counseling in addition to standard care is effective for promoting PA behavior change and positive health-related outcomes among individuals with T2DM.