A dedicated C-6 ß-hydroxyacyltransferase required for biosynthesis of the glycolipid anchor for Vi antigen capsule in typhoidal Salmonella.

Vi antigen is an extracellular polysaccharide produced by Salmonella enterica Typhi, Citrobacter freundii, and some soil bacteria belonging to the Burkholderiales. In Salmonella Typhi, Vi-antigen capsule protects the bacterium against host defenses, and the glycan is used in a current glycoconjugate vaccine to protect against typhoid. Vi antigen is a glycolipid assembled in the cytoplasm and translocated to the cell surface by an export complex driven by an ABC transporter. In Salmonella Typhi, efficient export and cell-surface retention of the capsule layer depend on a reducing terminal acylated-HexNAc moiety. Although the precise structure and biosynthesis of the acylated terminus has not been resolved, it distinguishes Vi antigen from other known glycolipid substrates for bacterial ABC transporters. The genetic locus for Vi antigen-biosynthesis encodes a single acyltransferase candidate (VexE), which is implicated in the acylation process. Here, we determined the structure of the VexE in vitro reaction product by mass spectrometry and NMR spectroscopy to reveal that VexE catalyzes ß-hydroxyacyl-ACP dependent acylation of the activated sugar precursor, uridine-5'-diphospho-GlcNAc, at C-6 to form UDP-6-O-[ß-hydroxymyristoyl]-α-d-GlcNAc. VexE belongs to the lysophosphatidyl acyltransferase family, and comparison of an Alphafold VexE model to solved lysophosphatidyl acyltransferase structures, together with modeling enzyme:substrate complexes, led us to predict an enzyme mechanism. This study provides new insight into Vi terminal structure, offers a new model substrate to investigate the mechanism of glycolipid ABC transporters, and adds biochemical understanding for a novel reaction used in the synthesis of an important bacterial virulence factor.

Quantitative trait locus mapping and functional genomics of an organophosphate resistance trait in the western corn rootworm, Diabrotica virgifera virgifera.

The western corn rootworm, Diabrotica virgifera virgifera, is an insect pest of corn and population suppression with chemical insecticides is an important management tool. Traits conferring organophosphate insecticide resistance have increased in frequency amongst D. v. virgifera populations, resulting in the reduced efficacy in many corn-growing regions of the USA. We used comparative functional genomic and quantitative trait locus (QTL) mapping approaches to investigate the genetic basis of D. v. virgifera resistance to the organophosphate methyl-parathion. RNA from adult methyl-parathion resistant and susceptible adults was hybridized to 8331 microarray probes. The results predicted that 11 transcripts were significantly up-regulated in resistant phenotypes, with the most significant (fold increases ≥ 2.43) being an α-esterase-like transcript. Differential expression was validated only for the α-esterase (ST020027A20C03), with 11- to 13-fold greater expression in methyl-parathion resistant adults (P < 0.05). Progeny with a segregating methyl-parathion resistance trait were obtained from a reciprocal backcross design. QTL analyses of high-throughput single nucleotide polymorphism genotype data predicted involvement of a single genome interval. These data suggest that a specific carboyxesterase may function in field-evolved corn rootworm resistance to organophosphates, even though direct linkage between the QTL and this locus could not be established.

Spontaneous motility of passive emulsion droplets in polar active gels.

We study by computer simulations the dynamics of a droplet of passive, isotropic fluid, embedded in a polar active gel. The latter represents a fluid of active force dipoles, which exert either contractile or extensile stresses on their surroundings, modelling for instance a suspension of cytoskeletal filaments and molecular motors. When the polarisation of the active gel is anchored normal to the droplet at its surface, the nematic elasticity of the active gel drives the formation of a hedgehog defect; this defect then drives an active flow which propels the droplet forward. In an extensile gel, motility can occur even with tangential anchoring, which is compatible with a defect-free polarisation pattern. In this case, upon increasing activity the droplet first rotates uniformly, and then undergoes a discontinuous nonequilibrium transition into a translationally motile state, powered by bending deformations in the surrounding active medium.

Pharmacokinetics of ganciclovir and valganciclovir in the adult horse.

Equine herpes myeloencephalopathy, resulting from equine herpes virus type 1 (EHV-1) infection, is associated with substantial morbidity and mortality in the horse. As compared to other antiviral drugs, such as acyclovir, ganciclovir has enhanced potency against EHV-1. This study investigated the pharmacokinetics of ganciclovir and its oral prodrug, valganciclovir, in six adult horses in a randomized cross-over design. Ganciclovir sodium was administered intravenously as a slow bolus at a dose of 2.5 mg/kg, and valganciclovir was administered orally at a dose of 1800 mg per horse. Intravenously administered ganciclovir disposition was best described by a three-compartment model with a prolonged terminal half-life of 72 ± 9 h. Following the oral administration of valganciclovir, the mean observed maximum serum ganciclovir concentration was 0.58 ± 0.37 µg/mL, and bioavailability of ganciclovir from oral valganciclovir was 41 ± 20%. Superposition predicted that oral dosing of 1800-mg valganciclovir two times daily would fail to produce and maintain effective plasma concentrations of ganciclovir. However, superposition suggested that i.v. administration of ganciclovir at 2.5 mg/kg every 8 h for 24 h followed by maintenance dosing of 2.5 mg/kg every 12 h would maintain effective ganciclovir serum concentrations in most horses throughout the dosing interval.

Modelling Subarctic watershed dissolved organic carbon response to hydroclimatic regime.

Shifts in hydroclimatic regimes associated with global climate change may impact freshwater availability and quality. In high latitudes of the northern hemisphere, where vast quantities of carbon are stored terrestrially, explaining landscape-scale carbon (C) budgets and associated pollutant transfer is necessary for understanding the impact of changing hydroclimatic regimes. We used a dynamic modelling approach to simulate streamflow, DOC concentration, and DOC export in a northern Canadian catchment that has undergone notable climate warming, and will continue to for the remainder of this century. The Integrated Catchment model for Carbon (INCA-C) was successfully calibrated to a multi-year period (2012-2016) that represents a range in hydrologic conditions. The model was subsequently run over 30-year periods representing baseline and two future climate scenarios. Average discharge is predicted to decrease under an elevated temperature scenario (22-27 % of baseline) but increase (116-175 % of baseline) under an elevated temperature and precipitation scenario. In the latter scenario the nival hydroclimatic regime is expected to shift to a combined nival and pluvial regime. Average DOC flux over 30 years is predicted to decrease (24-27 % of baseline) under the elevated temperature scenario, as higher DOC concentrations are offset by lower runoff. Under the elevated temperature and precipitation scenario, results suggest an increase in carbon export of 64-81 % above baseline. These increases are attributed to greater connectivity of the catchment. The largest increase in DOC export is expected to occur in early winter. These predicted changes in DOC export, particularly under a climate that is warmer and wetter could be part of larger ecosystem change and warrant additional monitoring efforts in the region.

Surgical management of primary appendiceal malignancy.

AIM: Primary appendiceal neoplasms are rare, with carcinoid being more common than carcinoma. Preoperative diagnosis is infrequent. We report a series of 24 primary appendiceal neoplasms treated over a 5-year period. METHOD: All primary appendiceal neoplasms diagnosed in a university teaching hospital between April 2003 and June 2008 were identified from the prospective histopathology database. Patient records were reviewed for clinical, operative and pathological data. RESULTS: Fourteen carcinomas and 10 carcinoids were identified in the 5-year period. The former presented at median age 55.8 years with median symptom duration of 35 days, compared with 48 years and 3 days, respectively, for carcinoids. An abdominal mass was commoner in carcinomas (8/14 vs 1/10). Six patients with carcinoma underwent appendicectomy followed by completion right-hemicolectomy, two of whom had residual disease; seven underwent primary right-hemicolectomy and one had abscess drainage. Five patients with appendiceal carcinoma died of the disease during a median follow-up of 633 (256-1158) days. Six patients underwent appendicectomy for acute appendicitis, one had a primary right-hemicolectomy for a caecal mass. Three had a subsequent right-hemicolectomy for a high-risk carcinoid. An appendiceal carcinoid was an incidental finding in three right-hemicolectomy specimens removed for other indications. No metastases or deaths were recorded at median follow-up 451 (51-975) days. CONCLUSION: Appendiceal carcinomas follow a more protracted clinical course than carcinoids, which usually present as acute appendicitis. Caution is needed when diagnosing simple appendicitis in older patients with longer symptom duration, particularly if a mass is present. Patients with appendiceal carcinoma should be offered completion right-hemicolectomy. Carcinoma has a poor prognosis.

Differences in ebullitive methane release from small, shallow ponds present challenges for scaling.

Small, shallow waterbodies are potentially important sites of greenhouse gas release to the atmosphere. The role of ebullition may be enhanced here relative to larger and deeper systems, due to their shallow water, but these features remain relatively infrequently studied in comparison to larger systems. Herein, we quantify ebullitive release of methane (CH4) in small shallow ponds in three regions of North America and investigate the role of potential drivers. Shallow ponds exhibited open-water season ebullitive CH4 release rates as high as 40 mmol m-2 d-1, higher than previously reported for similar systems. Ebullitive release of CH4 varied by four orders of magnitude across our 15 study sites, with differences in flux rates both within and between regions. What is less clear are the drivers responsible for these differences. There were few relationships between open water-season ebullitive flux and physicochemical characteristics, including organic matter, temperature, and sulphate. Temperature was only weakly related to ebullitive CH4 release across the study when considering all observation intervals. Only four individual sites exhibited significant relationships between temperature and ebullitive CH4 release. Other sites were unresponsive to temperature, and region-specific factors may play a role. There is some evidence that where surface water sulphate concentrations are high, CH4 production and release may be suppressed. Missouri sites (n = 5) had characteristically low ebullitive CH4 release; here bioturbation could be important. While this work greatly expands the number of open-water season ebullition rates for small and shallow ponds, more research is needed to disentangle the role of different drivers. Further investigation of the potential thresholding behaviour of sulphate as a control on ebullitive CH4 release in lentic systems is one such opportunity. What is clear, however, is that efforts to scale emissions (e.g., as a function of temperature) must be undertaken with caution.

The effect of coincidental SARS-CoV-2 infection on pre-operative cardiopulmonary exercise testing.

This case report demonstrates the significant impact active infection with SARS-CoV-2 can have on functional capacity evaluated by cardiopulmonary exercise testing, even in minimally symptomatic individuals. A 75-year-old man underwent cardiopulmonary exercise testing before a right hemicolectomy; SARS-CoV-2 was incidentally diagnosed following his test. The patient underwent a period of isolation and recovery before a second pre-operative cardiopulmonary exercise test 6 weeks later. His resting pulmonary function tests did not vary between tests but his peak work, anaerobic threshold, oxygen pulse, pulse oximetry nadir, ventilation perfusion matching and heart rate response to exercise all improved significantly after this recovery period. These are unique results that add to the existing knowledge of the pathophysiology and management of SARS-CoV-2 in the peri-operative setting. While our patient demonstrated dramatic improvement in his functional capacity following 6 weeks of recovery, he remained in a high-risk group for surgery according to our local guidelines. Cardiopulmonary exercise testing has a valuable role in individualised risk assessment and shared decision-making in complex, urgent surgical cases where the benefits of delaying surgery to recover from SARS-CoV-2 infection should be balanced against the potential risks.

The effect of rapidly discharging psychiatric inpatients from Mental Health Act section during COVID-19: a cohort study.

AIMS: In March 2020, the UK government ordered mental health services to free up bed space to help manage the COVID-19 pandemic. This meant service users detained under the Mental Health Act were discharged at a higher rate than normal. We analysed whether this decision compromised the safety of this vulnerable group of service users. METHODS: We utilised a cohort study design and allocated service users to either the pre-rapid discharge, rapid discharge or post-rapid discharge group. We conducted a recurrent event analysis to assess group differences in the risk of experiencing negative outcomes during the 61 days post-discharge. We defined negative outcomes as crisis service use, re-admission to a psychiatric ward, community incidents of violence or self-harm and death by suicide. RESULTS: The pre-rapid discharge cohort included 258 service users, the rapid discharge cohort 127 and the post-rapid discharge cohort 76. We found no statistical association between being in the rapid discharge cohort and the risk of experiencing negative outcomes (HR: 1.14, 95% CI: 0.72-1.8, p = 0.58) but a trend towards statistical significance for service users in the post-rapid discharge cohort (HR: 1.61, 95% CI: 0.91-2.83, p = 0.1). CONCLUSIONS: We did not find evidence that service users rapidly discharged from section experienced poorer outcomes. This raises the possibility that the Mental Health Act is applied in an overly restrictive manner, meaning that sections for some formally detained service users could be ended earlier without compromising safety.

Correlated expression patterns of microRNA genes with age-dependent behavioural changes in honeybee.

The hive-living honeybees (Apis mellifera) show age-dependent behavioural changes; young bees usually nurse the broods in the colony and the older bees engage in foraging activities. These developmentally regulated behavioural changes were previously shown to be correlated with genome-wide transcriptional changes in the honeybee brain. The indigenous small regulatory RNA molecules, known as microRNAs (miRNAs), are potent regulators of gene expression and also are developmentally regulated. Thus, we wanted to study if there might be correlation of differential expression of miRNA genes in the brain with age-dependent behavioural changes of the bees. We determined expression patterns of a set (n= 20) of predicted miRNA genes, by quantitative real-time PCR assays, in the brains of young and old bees that were engaged in nursing or foraging activities in the colony, respectively. Our data show correlated up-regulation of miRNA-124, miRNA-14, miRNA-276, miRNA-13b, let-7 and miRNA-13a in the young nurse bees. miRNA-12, miRNA-9, miRNA-219, miRNA-210, miRNA-263, miRNA-92 and miRNA-283 showed correlated expression patterns in the old forager bees. The modular changes of miRNA genes in the young nurse and old forager bees suggest possible roles of miRNAs in age-dependent behavioural changes in bees. The correlated expression of intronic miRNA genes and their host genes as well as of miRNA genes physically clustered in the genome are also observed.

The adsorption and release of sulfur in mineral and organic soils of the Athabasca Oil Sands Region, Alberta, Canada.

Mineral soil and fibric peat from acid-sensitive western boreal catchments in the Athabasca Oil Sands Region of Alberta, Canada were evaluated for their ability to adsorb and release SO(4)(2-). Laboratory batch studies indicated that SO(4)(2-) adsorption in mineral soil from both the A and B horizons exhibits a limited response to elevated SO(4)(2-) concentrations, with the slope of initial mass isotherms <0.2 for all soils, likely due to low iron and aluminum oxide content. Although S retention is the dominant process in peat soils in the region, drought simulations in the lab using fibric peat collected from a poor fen exhibited as much as a five-fold increase in SO(4)(2-) concentration after drying and rewetting. Given the limited SO(4)(2-) adsorption capacity of mineral soils and the potential drought-induced S release from peatlands in this region where increased S deposition is expected, further investigation of acidification impacts is warranted.

Infective abdominal aortic aneurysm due to Haemophilus influenza identified via the polymerase chain reaction.

Infective abdominal aortic aneurysms due to Haemophilus influenza are rarely reported. We report a case in a 65 year old female presenting with abdominal pain, weight loss, pyrexia and elevated inflammatory markers. The patient was found to have an abdominal aortic aneurysm clinically and on CT scanning. At surgery, an inflammatory aneurysm was successfully repaired using an autogenous vein panel-graft. Tissue samples were analysed using the polymerase chain reaction, identifying H. influenza as the causative organism. H. influenza is a scarcely reported cause of infective aortic aneurysms. The mechanism of infection is unknown. Reference is made to existing reports of such infection.

Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

Basolateral localization of the Caenorhabditis elegans epidermal growth factor receptor in epithelial cells by the PDZ protein LIN-10.

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.

Gelatin colloids in the resuscitation of trauma.

To date, the specific role of gelatins in trauma resuscitation remains under-investigated. Their adverse affects are well described and relate principally to the provocation of allergic responses whilst their influence upon haemostasis is relatively benign in comparison to the other colloids. However, their benefits are only sparsely documented and the evidence to choose one gelatin over another virtually non-existent. As knowledge of the microcirculatory dysfunction inherent in the shocked state increases, the role of the gelatins in trauma resuscitation is being increasing sidelined by other colloids--notably the starches. Their role beyond a basic resuscitation tool is now uncertain.

Biosynthesis of lipopolysaccharide O antigens.

Lipopolysaccharide O antigens are important virulence determinants for many bacteria. O-antigen synthesis is an interesting problem in cell-surface assembly. There are two known assembly pathways, which differ in the cellular location of their polymerization steps and in the direction of chain polymerization. Some reactions are shared with those for other surface polymers, such as capsular polysaccharides, and may be potential targets for therapeutic intervention.

Bacterial polysaccharide synthesis and gene nomenclature.

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.

Behavior and the limits of genomic plasticity: power and replicability in microarray analysis of honeybee brains.

Transcription is slow relative to many post-transcriptional processes in the brain. Using the rich system of division of labor in the honeybee (Apis mellifera), we found extreme differences in the extent to which behavioral occupations of different durations were associated with gene-expression differences in the brain. Nursing and foraging, occupations lasting > 1 week, were associated with significant expression differences for nearly one-quarter of the genes tested (1208 of 5563 cDNAs tested; P < 0.01, anova), consistent with previous results. In contrast, transitional occupations, performed for 1-2 days after nursing and before the onset of foraging, were associated with either no differences (guards vs. undertakers; 19 cDNAs, fewer than the expectation of 56 false-positives) or few differences (comb builders vs. guards and undertakers; 248 cDNAs), but extensive differences relative to both nursing and foraging (> 500 cDNAs, all contrasts). Statistical power analysis indicated that expression differences of two-, 1.5- and 1.25-fold should have been detected in 100, 92 and 37% of cases, respectively. Replication of previous results at these magnitudes was 95, 71 and 51%, with no genes showing differences in the opposite direction. These results indicate that behavioral plasticity over different time-scales may be associated with substantial differences in the extent of genomic plasticity in the brain.

The crystal structure of dTDP-D-Glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar Typhimurium, the second enzyme in the dTDP-l-rhamnose pathway.

l-Rhamnose is a 6-deoxyhexose that is found in a variety of different glycoconjugates in the cell walls of pathogenic bacteria. The precursor of l-rhamnose is dTDP-l-rhamnose, which is synthesised from glucose- 1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway requiring four enzymes. Significantly this pathway does not exist in humans and all four enzymes therefore represent potential therapeutic targets. dTDP-D-glucose 4,6-dehydratase (RmlB; EC 4.2.1.46) is the second enzyme in the dTDP-L-rhamnose biosynthetic pathway. The structure of Salmonella enterica serovar Typhimurium RmlB had been determined to 2.47 A resolution with its cofactor NAD(+) bound. The structure has been refined to a crystallographic R-factor of 20.4 % and an R-free value of 24.9 % with good stereochemistry.RmlB functions as a homodimer with monomer association occurring principally through hydrophobic interactions via a four-helix bundle. Each monomer exhibits an alpha/beta structure that can be divided into two domains. The larger N-terminal domain binds the nucleotide cofactor NAD(+) and consists of a seven-stranded beta-sheet surrounded by alpha-helices. The smaller C-terminal domain is responsible for binding the sugar substrate dTDP-d-glucose and contains four beta-strands and six alpha-helices. The two domains meet to form a cavity in the enzyme. The highly conserved active site Tyr(167)XXXLys(171) catalytic couple and the GlyXGlyXXGly motif at the N terminus characterise RmlB as a member of the short-chain dehydrogenase/reductase extended family. The quaternary structure of RmlB and its similarity to a number of other closely related short-chain dehydrogenase/reductase enzymes have enabled us to propose a mechanism of catalysis for this important enzyme.