Viral and cellular determinants of polarized trafficking of viral envelope proteins from insect-specific and insect-vectored viruses in insect midgut and salivary gland cells.

Systemic viral infection of insects typically begins with the primary infection of midgut epithelial cells (enterocytes) and subsequent transit of the progeny virus in an apical-to-basal orientation into the hemocoel. For insect-vectored viruses, an oppositely oriented process (basal-to-apical transit) occurs upon secondary infection of salivary glands and is necessary for virus transmission to non-insect hosts. To examine this inversely oriented virus transit in these polarized tissues, we assessed the intracellular trafficking of two model viral envelope proteins (baculovirus GP64 and vesicular stomatitis virus G) in the midgut and salivary gland cells of the model insect, Drosophila melanogaster. Using fly lines that inducibly express either GP64 or VSV G, we found that each protein, expressed alone, was trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked apically in most but not all cells, whereas GP64 was consistently trafficked basally. We demonstrated that a YxxØ motif present in both proteins was critical for basal trafficking in midgut enterocytes but dispensable for trafficking in salivary gland cells. Using RNAi, we found that clathrin adaptor protein complexes AP-1 and AP-3, as well as seven Rab GTPases, were involved in polarized VSV G trafficking in midgut enterocytes. Our results indicate that these viral envelope proteins encode the requisite information and require no other viral factors for appropriately polarized trafficking. In addition, they exploit tissue-specific differences in protein trafficking pathways to facilitate virus egress in the appropriate orientation for establishing systemic infections and vectoring infection to other hosts. IMPORTANCE: Viruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. Using Drosophila as a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium.

Trends in weight gain recorded in English primary care before and during the Coronavirus-19 pandemic: An observational cohort study using the OpenSAFELY platform.

BACKGROUND: Obesity and rapid weight gain are established risk factors for noncommunicable diseases and have emerged as independent risk factors for severe disease following Coronavirus Disease 2019 (COVID-19) infection. Restrictions imposed to reduce COVID-19 transmission resulted in profound societal changes that impacted many health behaviours, including physical activity and nutrition, associated with rate of weight gain. We investigated which clinical and sociodemographic characteristics were associated with rapid weight gain and the greatest acceleration in rate of weight gain during the pandemic among adults registered with an English National Health Service (NHS) general practitioner (GP) during the COVID-19 pandemic. METHODS AND FINDINGS: With the approval of NHS England, we used the OpenSAFELY platform inside TPP to conduct an observational cohort study of routinely collected electronic healthcare records. We investigated changes in body mass index (BMI) values recorded in English primary care between March 2015 and March 2022. We extracted data on 17,742,365 adults aged 18 to 90 years old (50.1% female, 76.1% white British) registered with an English primary care practice. We estimated individual rates of weight gain before (δ-prepandemic) and during (δ-pandemic) the pandemic and identified individuals with rapid weight gain (>0.5 kg/m2/year) in each period. We also estimated the change in rate of weight gain between the prepandemic and pandemic period (δ-change = δ-pandemic-δ-prepandemic) and defined extreme accelerators as the 10% of individuals with the greatest increase in their rate of weight gain (δ-change ≥1.84 kg/m2/year) between these periods. We estimated associations with these outcomes using multivariable logistic regression adjusted for age, sex, index of multiple deprivation (IMD), and ethnicity. P-values were generated in regression models. The median BMI of our study population was 27.8 kg/m2, interquartile range (IQR) [24.3, 32.1] in 2019 (March 2019 to February 2020) and 28.0 kg/m2, IQR [24.4, 32.6] in 2021. Rapid pandemic weight gain was associated with sex, age, and IMD. Male sex (male versus female: adjusted odds ratio (aOR) 0.76, 95% confidence interval (95% CI) [0.76, 0.76], p < 0.001), older age (e.g., 50 to 59 years versus 18 to 29 years: aOR 0.60, 95% CI [0.60, 0.61], p < 0.001]); and living in less deprived areas (least-deprived-IMD-quintile versus most-deprived: aOR 0.77, 95% CI [0.77, 0.78] p < 0.001) reduced the odds of rapid weight gain. Compared to white British individuals, all other ethnicities had lower odds of rapid pandemic weight gain (e.g., Indian versus white British: aOR 0.69, 95% CI [0.68, 0.70], p < 0.001). Long-term conditions (LTCs) increased the odds, with mental health conditions having the greatest effect (e.g., depression (aOR 1.18, 95% CI [1.17, 1.18], p < 0.001)). Similar characteristics increased odds of extreme acceleration in the rate of weight gain between the prepandemic and pandemic periods. However, changes in healthcare activity during the pandemic may have introduced new bias to the data. CONCLUSIONS: We found female sex, younger age, deprivation, white British ethnicity, and mental health conditions were associated with rapid pandemic weight gain and extreme acceleration in rate of weight gain between the prepandemic and pandemic periods. Our findings highlight the need to incorporate sociodemographic, physical, and mental health characteristics when formulating research, policies, and interventions targeting BMI in the period of post pandemic service restoration and in future pandemic planning.

Ultrafast manipulation of the NiO antiferromagnetic order via sub-gap optical excitation.

Wide-band-gap insulators such as NiO offer the exciting prospect of coherently manipulating electronic correlations with strong optical fields. Contrary to metals where rapid dephasing of optical excitation via electronic processes occurs, the sub-gap excitation in charge-transfer insulators has been shown to couple to low-energy bosonic excitations. However, it is currently unknown if the bosonic dressing field is composed of phonons or magnons. Here we use the prototypical charge-transfer insulator NiO to demonstrate that 1.5 eV sub-gap optical excitation leads to a renormalised NiO band-gap in combination with a significant reduction of the antiferromagnetic order. We employ element-specific X-ray reflectivity at the FLASH free-electron laser to demonstrate the reduction of the upper band-edge at the O 1s-2p core-valence resonance (K-edge) whereas the antiferromagnetic order is probed via X-ray magnetic linear dichroism (XMLD) at the Ni 2p-3d resonance (L2-edge). Comparing the transient XMLD spectral line shape to ground-state measurements allows us to extract a spin temperature rise of 65 ± 5 K for time delays longer than 400 fs while at earlier times a non-equilibrium spin state is formed. We identify transient mid-gap states being formed during the first 200 fs accompanied by a band-gap reduction lasting at least up to the maximum measured time delay of 2.4 ps. Electronic structure calculations indicate that magnon excitations significantly contribute to the reduction of the NiO band gap.

Allergenicity reduction of the bio-elicited peanut sprout powder (BPSP) and toxicological acceptance of BPSP-supplemented diets assessed with ICR mice.

The allergenic and toxicological acceptances of the bio-elicited peanut sprout powder (BPSP) have not been assessed. BPSP was generated from peanut kernels germinated at 26-28 °C for 72 h (designated as 72 h-NGS). The 72 h-NGS were subsequently sliced, incubated, dried, defatted and pulverized to generate bio-elicited peanut sprout powder (BPSP). Protein solubility of BPSP increased 2.6-fold compared to 72 h-NGS. SDS-PAGE analysis revealed BPSP production triggered extensive degradation of the high-molecular weight peanut allergic proteins, mainly Ara h 1 and Ara h 3. Western blotting detected with peanut allergic patients' IgE indicated decreased in vitro reactivity. Food safety assessment of BPSP was performed with ICR mice fed with basal (control) and three doses of formulated BPSP-supplemented diets containing 0.11 g (normal), 2.5 g (high) and 25 g (super high) BPSP /kg BW. Animals appeared healthy with steady body weight gain in all groups during the entire 35-day dietary intervention. Hematological and serum biochemical analyses revealed no significant difference among groups. Histopathological examination on the tissue sections of primary organs further supported safety with no pathologies. The in vitro allergic reduction and toxicological safety in the BPSP-supplemented dietary intervention in the ICR mice study, support moving forward with BPSP-involved product development. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05537-7.

The Galleria mellonella-Enteropathogenic Escherichia coli Model System: Characterization of Pathogen Virulence and Insect Immune Responses.

The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies using this system. We found that EPEC causes disease in G. mellonella larvae when injected intrahemocoelically but not orally. Disease manifests as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, increased pupal duration, and hemocytopenia. Disease symptoms are dose-dependent and can be used as metrics for measuring EPEC virulence in future studies. The type III secretion system was only partially responsible for EPEC virulence in G. mellonella while the majority of the virulence remains unknown in origin. EPEC elicits insect anti-bacterial immune responses including melanization, hemolymph coagulation, nodulation, and phagocytosis. The immune responses were unable to control EPEC replication in the early stage of infection (≤3 h post-injection). EPEC clearance from the hemocoel does not guarantee insect survival. Overall, this study provided insights into EPEC virulence and pathogenesis in G. mellonella and identified areas of future research using this system.

Galleria mellonella (Lepidoptera: Pyralidae) Hemocytes Release Extracellular Traps That Confer Protection Against Bacterial Infection in the Hemocoel.

Extracellular traps (ETs) released from vertebrate and invertebrate immune cells consist of chromatin and toxic granule contents that are capable of immobilizing and killing microbes. This recently described innate immune response is not well documented in insects. The present study found that ETs were released by hemocytes of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) in vivo and ex vivo after bacterial stimulation. ET release (ETosis), hemolymph coagulation, and melanization likely contributed to the immobilization and killing of the bacteria. The injection of G. mellonella hemocyte deoxyribonucleic acid (DNA) in the presence of bacteria increased bacterial clearance rate and prolonged insect survival. Taken together, these results indicate the presence of insect hemocyte extracellular traps (IHETs) that protect the insect against microbial infection in the hemocoel and represent the first documentation of ETs in insects in vivo.

The Anti-Cancer Effects of a Zotarolimus and 5-Fluorouracil Combination Treatment on A549 Cell-Derived Tumors in BALB/c Nude Mice.

Zotarolimus is a semi-synthetic derivative of rapamycin and a novel immunosuppressive agent used to prevent graft rejection. The pharmacological pathway of zotarolimus restricts the kinase activity of the mammalian target of rapamycin (mTOR), which potentially leads to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical influence on tumorigenesis. This study aims to examine the anti-tumor effect of zotarolimus or zotarolimus combined with 5-fluorouracil (5-FU) on A549 human lung adenocarcinoma cell line implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. We established A549 xenografts in nude mice, following which we randomly divided the mice into four groups: control, 5-FU (100 mg/kg/week), zotarolimus (2 mg/kg/day), and zotarolimus combined with 5-FU. Compared the results with those for control mice, we found that mice treated with zotarolimus or zotarolimus combined with 5-FU retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; decreased inflammation cytokines levels (e.g., IL-1ß, TNF-α, and IL-6); reduced inflammation-related factors such as cyclooxygenase-2 (COX-2) protein and nuclear factor-κB (NF-κB) mRNA; enhanced anti-inflammation-related factors including IL-10 and inhibitor of NF-κB kinase α (IκBα) mRNA; and inhibited metastasis-related factors such as transforming growth factor ß (TGF-ß), CD44, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). Notably, mice treated with zotarolimus combined with 5-FU had significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with the groups of mice treated with 5-FU or zotarolimus alone. The in vivo study confirmed that zotarolimus or zotarolimus combined with 5-FU could retard lung adenocarcinoma growth and inhibit tumorigenesis. Zotarolimus and 5-FU were found to have an obvious synergistic tumor-inhibiting effect on lung adenocarcinoma. Therefore, both zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents for treatment of human lung adenocarcinoma.

Effect of Intensive vs Standard Blood Pressure Treatment Upon Erectile Function in Hypertensive Men: Findings From the Systolic Blood Pressure Intervention Trial.

INTRODUCTION: The effect of intensive blood pressure control upon erectile function in men with hypertension, but without diabetes, is largely unknown. AIM: To examine the effects of intensive systolic blood pressure (SBP) lowering on erectile function in a multiethnic clinical trial of men with hypertension. METHODS: We performed subgroup analyses from the Systolic Blood Pressure Intervention Trial ([SPRINT]; ClinicalTrials.gov: NCT120602, in a sample of 1255 men aged 50 years or older with hypertension and increased cardiovascular disease risk. Participants were randomly assigned to an intensive treatment group (SBP goal of <120 mmHg) or a standard treatment group (SBP goal of <140 mmHg). MAIN OUTCOME MEASURE: The main outcome measure was change in erectile function from baseline, using the 5-item International Index of Erectile Function (IIEF-5) total score, and erectile dysfunction ([ED]; defined as IIEF-5 score ≤21) after a median follow-up of 3 years. RESULTS: At baseline, roughly two-thirds (66.1%) of the sample had self-reported ED. At 48 months after randomization, we determined that the effects of more intensive blood pressure lowering were significantly moderated by race-ethnicity (p for interaction = 0.0016), prompting separate analyses stratified by race-ethnicity. In non-Hispanic whites, participants in the intensive treatment group reported slightly, but significantly better change in the IIEF-5 score than those in the standard treatment group (mean difference = 0.67; 95% CI = 0.03, 1.32; P = 0.041). In non-Hispanic blacks, participants in the intensive group reported slightly worse change in the IIEF-5 score than those in the standard group (mean difference = -1.17; 95% CI = -1.92, -0.41; P = 0.0025). However, in non-Hispanic whites and non-Hispanic blacks, further adjustment for the baseline IIEF-5 score resulted in nonsignificant differences (P > 0.05) according to the treatment group. In Hispanic/other participants, there were no significant differences in change in the IIEF-5 score between the two treatment groups (P = 0.40). In a subgroup of 280 participants who did not report ED at baseline, the incidence of ED did not differ in the two treatment groups (P = 0.53) and was without interaction by race-ethnicity. CLINICAL IMPLICATIONS: The effect of intensive treatment of blood pressure on erectile function was very small overall and likely not of great clinical magnitude. STRENGTH & LIMITATIONS: Although this study included a validated measure of erectile function, testosterone, other androgen, and estrogen levels were not assessed. CONCLUSION: In a sample of male patients at high risk for cardiovascular events but without diabetes, targeting a SBP of less than 120 mm Hg, as compared with less than 140 mm Hg, resulted in statistically significant effects on erectile function that differed in accordance with race-ethnicity, although the clinical importance of the differences may be of small magnitude. Foy CG, Newman JC, Russell GB, et al. Effect of Intensive vs Standard Blood Pressure Treatment Upon Erectile Function in Hypertensive Men: Findings From the Systolic Blood Pressure Intervention Trial. J Sex Med 2020;17:238-248.

PIP2 mediates functional coupling and pharmacology of neuronal KCNQ channels.

Retigabine (RTG) is a first-in-class antiepileptic drug that suppresses neuronal excitability through the activation of voltage-gated KCNQ2-5 potassium channels. Retigabine binds to the pore-forming domain, causing a hyperpolarizing shift in the voltage dependence of channel activation. To elucidate how the retigabine binding site is coupled to changes in voltage sensing, we used voltage-clamp fluorometry to track conformational changes of the KCNQ3 voltage-sensing domains (VSDs) in response to voltage, retigabine, and PIP2. Steady-state ionic conductance and voltage sensor fluorescence closely overlap under basal PIP2 conditions. Retigabine stabilizes the conducting conformation of the pore and the activated voltage sensor conformation, leading to dramatic deceleration of current and fluorescence deactivation, but these effects are attenuated upon disruption of channel:PIP2 interactions. These findings reveal an important role for PIP2 in coupling retigabine binding to altered VSD function. We identify a polybasic motif in the proximal C terminus of retigabine-sensitive KCNQ channels that contributes to VSD-pore coupling via PIP2, and thereby influences the unique gating effects of retigabine.

A randomized trial of the effectiveness of the neutropenic diet versus food safety guidelines on infection rate in pediatric oncology patients.

BACKGROUND: The neutropenic diet (ND) is prescribed to avoid introduction of bacteria into a host's gastrointestinal tract and reduce infection. Due to a lack of evidence to support the ND, there continues to be debate among pediatric oncologists regarding its usefulness. This prospective randomized controlled trial evaluated the difference in neutropenic infection rates in pediatric oncology patients randomized to Food and Drug Administration approved food safety guidelines (FSGs) versus the ND plus FSGs during one cycle of chemotherapy. PROCEDURE: Pediatric patients receiving cancer treatment with myelosuppressive chemotherapy were eligible. Neutropenic infection was the primary outcome and defined as (i) fever with neutropenia or (ii) hospital admission and treatment for clinical infection and neutropenia. The rate of neutropenic infection was compared with Student's t-test for independent samples. Documented infections were identified by comprehensive chart review and compared between groups using a χ2 test. RESULTS: One hundred fifty patients were randomly assigned to FSGs (n = 73) or ND + FSGs (n = 77). The most common diagnoses were acute lymphoblastic leukemia (32%) and sarcoma (32%). There was no significant difference between the groups in the percentage of patients who developed neutropenic infection: FSGs 33% versus ND + FSGs 35% (P = 0.78). Patients randomized to ND + FSGs reported that following the diet required more effort than those on FSGs alone. CONCLUSION: The ND offers no benefit over FSGs in the prevention of infection in pediatric oncology patients undergoing myelosuppressive chemotherapy and adherence requires more effort for patients and families. Institutions caring for children with cancer should consider replacing ND guidelines with FSGs.

Use-dependent activation of neuronal Kv1.2 channel complexes.

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.

A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

Dehydrated Basella alba Fruit Juice as a Novel Natural Colorant: Pigment Stability, In Vivo Food Safety Evaluation and Anti-Inflammatory Mechanism Characterization.

Flesh of Basella alba L. mature fruits bearing deep-violet juice provides a novel and potential source of natural colorant. To minimize the pigment purification process and warrant safety acceptability, B. alba colorant powder (BACP) was prepared using mature fruits through a practical batch preparation and subjected to fundamental pigment characterization, food safety assessment and bio-function evaluation. Yield of the dehydrated B. alba colorant powder (BACP) was 37 g/kg fresh fruits. Reconstituted aqueous solution of the BACP exhibited an identical visible spectrum (400-700 nm) as that of fresh juice. Color of the solution (absorbance at 540 nm) was stable in a broad pH ranged from 3 to 8 and enhanced by co-presence of calcium and magnesium ions, while was rapidly bleached by ferrous and ferric ions. For in vivo food safety evaluation, ICR mice were daily gavage administered with BACP up to 1000 mg/kg body weight for 28 days. Organ weight determination, serum biochemical analysis and histopathological examination of hearts, livers, lungs and kidneys revealed no obvious health hazard. In vitro anti-inflammatory activity of BACP was characterized in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. BACP exerted potent anti-inflammatory activity by down-regulation of inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2), TNF-α, IL-1ß, IL-6 and IL-12 and the blockage of IκB kinase (IKK)/IκB/nuclear factor-κ B (NFκB) activation cascade. These results supported that BACP may serve as a beneficial alternative of natural food colorant.

Atom-by-atom engineering of voltage-gated ion channels: magnified insights into function and pharmacology.

Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino acids into proteins. We also review insights into the function and pharmacology of voltage-gated ion channels that have emerged from unnatural amino acid mutagenesis approaches.

Identification of peanut seed prolamins with an antifungal role by 2D-GE and drought treatment.

This work revealed peanut seed prolamins likely displaying a defensive role besides the known nitrogen storage. Drought stress and proteomic approaches were used in varieties of peanuts to explore the prolamin member in association with a test against Aspergillus flavus spore germination. The stress effect was showed by aerial biomass, leaf content of malondialdehyde, and seed contamination by A. flavus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles were not informative for the antifungal polypeptides. From two-dimensional gel electrophoresis, the suspected polypeptides were those with pI 5.45-5.75 and sizes of 22.0-30.5 kDa specifically in Spanish-type peanuts. Regarding to the drought effect in most of these peanuts, the spot peak volume analysis deduced three novel prolamin-related antifungal polypeptides at pI 5.75-5.8 with 30.5, 27.5-28.5, and 22.0-22.5 kDa, which was confirmed after isoelectric purification at pH 5.60. The data could not yet conclude their correlation with resistance to drought and to seed infection by A. flavus.

Decomposition of slide helix contributions to ATP-dependent inhibition of Kir6.2 channels.

Regulation of inwardly rectifying potassium channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. We investigated the molecular mechanisms that link ligand binding to the channel gate in ATP-sensitive Kir6.2 channels. In these channels, the "slide helix" forms an interface between the cytoplasmic (ligand-binding) domain and the transmembrane pore, and many slide helix mutations cause loss of function. Using a novel approach to rescue electrically silent channels, we decomposed the contribution of each interface residue to ATP-dependent gating. We demonstrate that effective inhibition by ATP relies on an essential aspartate at residue 58. Characterization of the functional importance of this conserved aspartate, relative to other residues in the slide helix, has been impossible because of loss-of-function of Asp-58 mutant channels. The Asp-58 position exhibits an extremely stringent requirement for aspartate because even a highly conservative mutation to glutamate is insufficient to restore normal channel function. These findings reveal unrecognized slide helix elements that are required for functional channel expression and control of Kir6.2 gating by intracellular ATP.

Presence of Flazin in Miso and Identification of Four Tryptophan-Derived ß-Carbolines Formed during Fermentation.

In miso, due to the substantial presence of genistein, flazin is often overlapped and masked by genistein in HPLC analysis. Flazin in the miso extracts could be resolved with genistein through medium-pressure liquid chromatography run under a nonacidified methanol-water system and subsequently fractionated by semipreparative HPLC and identified by NMR spectroscopic analysis. As referenced, flazin was detected in all 11 locally marketed miso products, with contents ranging from 3.5 to 124.8 µg/g. In lab-made miso fermented at 28 and 37 °C for 8 weeks, flazin formed faster at 37 °C than at 28 °C. Based on the time-dependent HPLC chromatographic changes of the miso extracts during fermentation, the presence of tryptophan-derived ß-carboline intermediates was deduced. Tryptophan was then supplemented for miso fermentation, and four peak substances were targeted for isolation by sophisticated approaches. Four ß-carbolines were purified and instrumentally identified, i.e., P1: 1-(1,3,4,5-tetrahydroxypentyl)-9H- pyrido[3,4-b]indole, P2 (diastereomer of P1): 1-(1*,3,4,5-tetrahydroxypentyl)- 9H-pyrido[3,4-b]indole, and Miso 101: 1-(1,3,4,5-tetrahydroxypentyl)-9H- pyrido[3,4-b]indole 3-carboxylic acid, and Miso 111 (diastereomer of Miso 101): 1-(1*,3,4,5-tetrahydroxypentyl)-9H-pyrido[3,4-b]indole 3-carboxylic acid. Each of the purified ß-carbolines along with tryptophan and flazin exhibited varied ABTS·+ scavenging and xanthine oxidase inhibitory activities.

Investigating the prognostic impact of NY-ESO-1 expression and HLA subtypes in metastatic synovial sarcoma.

BACKGROUND: To better understand the importance of the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) and human leukocyte antigen (HLA) subtypes in treatment decision-making, further investigation of their prevalence and prognostic impact among patients with metastatic synovial sarcoma (mSS) is needed. PATIENTS AND METHODS: This was a retrospective clinico-biological cohort study of adults with mSS. Patient data were collected from the French Sarcoma Group NetSARC database and supplemented by electronic medical records. Primary tumor samples were collected and analyzed for NY-ESO-1 expression by immunohistochemistry (IHC) and HLA-A∗02 status by RNA sequencing (RNA-seq). The primary cohort included patients with available primary tumor samples; the impact of a larger sample size was explored by including patients who had either a primary or metastatic sample (termed the exploratory cohort). P values are provided for descriptive purposes. RESULTS: In 92 patients with primary tumor samples, ∼25% (n = 23) were positive for NY-ESO-1 and HLA-A∗02 expression (dual positive). Among 106 patients with IHC data, 61% (n = 65) were NY-ESO-1 positive, and among 94 patients with RNA-seq data, 45% (n = 42) were HLA-A∗02 positive. The median overall survival (OS) for positive versus negative NY-ESO-1 status was 35.3 and 21.7 months, respectively (unadjusted P = 0.0428). We observed no difference in median OS for HLA-A∗02-positive versus -negative and dual-positive patients versus others (both unadjusted P > 0.05). Multivariate analyses of OS showed no prognostic impact for NY-ESO-1 among primary tumor samples and in the exploratory cohort. However, in the latter we observed an association between NY-ESO-1 expression and OS in the first-line (P = 0.0041) but not in the second-line setting. CONCLUSIONS: The primary tumor cohort showed no association between NY-ESO-1 expression and OS (including stratification by HLA-A∗02 subtype and treatment line) when adjusting for important prognostic factors, possibly due to small sample sizes.

Changes in sick notes associated with COVID-19 from 2020 to 2022: a cohort study in 24 million primary care patients in OpenSAFELY-TPP.

OBJECTIVES: Long-term sickness absence from employment has negative consequences for the economy and can lead to widened health inequalities. Sick notes (also called 'fit notes') are issued by general practitioners when a person cannot work for health reasons for more than 7 days. We quantified the sick note rate in people with evidence of COVID-19 in 2020, 2021 and 2022, as an indication of the burden for people recovering from COVID-19. DESIGN: Cohort study. SETTING: With National Health Service (NHS) England approval, we used routine clinical data (primary care, hospital and COVID-19 testing records) within the OpenSAFELY-TPP database. PARTICIPANTS: People 18-64 years with a recorded positive test or diagnosis of COVID-19 in 2020 (n=365 421), 2021 (n=1 206 555) or 2022 (n=1 321 313); general population matched in age, sex and region in 2019 (n=3 140 326), 2020 (n=3 439 534), 2021 (n=4 571 469) and 2022 (n=4 818 870); people hospitalised with pneumonia in 2019 (n=29 673). PRIMARY OUTCOME MEASURE: Receipt of a sick note in primary care. RESULTS: Among people with a positive SARS-CoV-2 test or COVID-19 diagnosis, the sick note rate was 4.88 per 100 person-months (95% CI 4.83 to 4.93) in 2020, 2.66 (95% CI 2.64 to 2.67) in 2021 and 1.73 (95% CI 1.72 to 1.73) in 2022. Compared with the age, sex and region-matched general population, the adjusted HR for receipt of a sick note over the entire follow-up period (up to 10 months) was 4.07 (95% CI 4.02 to 4.12) in 2020 decreasing to 1.57 (95% CI 1.56 to 1.58) in 2022. The HR was highest in the first 30 days postdiagnosis in all years. Among people hospitalised with COVID-19, after adjustment, the sick note rate was lower than in people hospitalised with pneumonia. CONCLUSIONS: Given the under-recording of postacute COVID-19-related symptoms, these findings contribute a valuable perspective on the long-term effects of COVID-19. Despite likely underestimation of the sick note rate, sick notes were issued more frequently to people with COVID-19 compared with those without, even in an era when most people are vaccinated. Most sick notes occurred in the first 30 days postdiagnosis, but the increased risk several months postdiagnosis may provide further evidence of the long-term impact.

Weight trends among adults with diabetes or hypertension during the COVID-19 pandemic: an observational study using OpenSAFELY.

BACKGROUND: COVID-19 pandemic restrictions may have influenced behaviours related to weight. AIM: To describe patterns of weight change among adults living in England with type 2 diabetes (T2D) and/or hypertension during the pandemic. DESIGN AND SETTING: An observational cohort study using the routinely collected health data of approximately 40% of adults living in England, accessed through the OpenSAFELY service inside TPP. METHOD: Clinical and sociodemographic characteristics associated with rapid weight gain (>0.5 kg/m2/year) were investigated using multivariable logistic regression. RESULTS: Data were extracted on adults with T2D (n = 1 231 455, 43.9% female, and 76.0% White British) or hypertension (n = 3 558 405, 49.7% female, and 84.3% White British). Adults with T2D lost weight overall (median δ = -0.1 kg/m2/year [interquartile range {IQR} -0.7-0.4]). However, rapid weight gain was common (20.7%) and associated with the following: sex (male versus female: adjusted odds ratio [aOR] 0.78 [95% confidence interval {CI} = 0.77 to 0.79]); age (older age reduced odds, for example, aged 60-69 years versus 18-29 years: aOR 0.66 [95% CI = 0.61 to 0.71]); deprivation (least deprived Index of Multiple Deprivation [IMD] quintile versus most deprived IMD quintile: aOR 0.87 [95% CI = 0.85 to 0.89]); White ethnicity (Black versus White: aOR 0.95 [95% CI = 0.92 to 0.98]); mental health conditions (for example, depression: aOR 1.13 [95% CI = 1.12 to 1.15]); and diabetes treatment (non-insulin treatment versus no pharmacological treatment: aOR 0.68 [95% CI = 0.67 to 0.69]). Adults with hypertension maintained stable weight overall (median δ = 0.0 kg/m2/year [IQR -0.6-0.5]); however, rapid weight gain was common (24.7%) and associated with similar characteristics as in T2D. CONCLUSION: Among adults living in England with T2D and/or hypertension, rapid pandemic weight gain was more common among females, younger adults, those living in more deprived areas, and those with mental health conditions.