Search

Discusses selected bioinorganic aspects of carbonic anhydrase and describes experiments that will reinforce the students' understanding of the presence and essential role that metal ions have in some biological systems. (SL)

Discusses the kinetic behavior associated with the interaction of metal ions with apocarbonic anhydrase, focusing on the formation of two metallocarbonic anhydrase--the biochemically active Co(II) and the inactive Ni(II)derivatives. (GA)

Overt hepatic encephalopathy is a generally reversible neurologic complication of cirrhosis. Overt hepatic encephalopathy has been associated with poor hospitalization- and mortality-related outcomes, which is important given increasing hepatic encephalopathy-related hospitalizations over time. The aim of this narrative review is to provide an overview of hospital- and mortality-related outcomes in patients with overt hepatic encephalopathy and the pharmacologic therapies that may improve these outcomes. Guideline-recommended prophylaxis with lactulose (first-line therapy) or secondary prophylaxis with rifaximin plus lactulose decreases hospital admissions and mortality rates. Rifaximin or lactulose treatment was beneficial for reducing the hospitalization rate in patients with hepatic encephalopathy compared with no treatment. Further, retrospective studies have shown that rifaximin with or without lactulose was effective for decreasing the number of hepatic encephalopathy episodes, hepatic encephalopathy-related hospitalizations, and duration of hospitalization. Ornithine phenylacetate, an ammonia-reducing agent currently in development, is also being investigated in hospitalized patients with hepatic encephalopathy. Overall, data support that prophylaxis for the prevention of hepatic encephalopathy recurrence improves outcomes in patients with cirrhosis and a history of hepatic encephalopathy.

Background: Experimental and epidemiological studies indicate an association between exposure to particulate matter (PM) air pollution and increased risk of type 2 diabetes. In view of the high and increasing prevalence of diabetes, we aimed to quantify the burden of type 2 diabetes attributable to PM2·5 originating from ambient and household air pollution.Methods: We systematically compiled all relevant cohort and case-control studies assessing the effect of exposure to household and ambient fine particulate matter (PM2·5) air pollution on type 2 diabetes incidence and mortality. We derived an exposure–response curve from the extracted relative risk estimates using the MR-BRT (meta-regression—Bayesian, regularised, trimmed) tool. The estimated curve was linked to ambient and household PM2·5 exposures from the Global Burden of Diseases, Injuries, and Risk Factors Study 2019, and estimates of the attributable burden (population attributable fractions and rates per 100 000 population of deaths and disability-adjusted life-years) for 204 countries from 1990 to 2019 were calculated. We also assessed the role of changes in exposure, population size, age, and type 2 diabetes incidence in the observed trend in PM2·5-attributable type 2 diabetes burden. All estimates are presented with 95% uncertainty intervals.Findings: In 2019, approximately a fifth of the global burden of type 2 diabetes was attributable to PM2·5 exposure, with an estimated 3·78 (95% uncertainty interval 2·68–4·83) deaths per 100 000 population and 167 (117–223) disability-adjusted life-years (DALYs) per 100 000 population. Approximately 13·4% (9·49–17·5) of deaths and 13·6% (9·73–17·9) of DALYs due to type 2 diabetes were contributed by ambient PM2·5, and 6·50% (4·22–9·53) of deaths and 5·92% (3·81–8·64) of DALYs by household air pollution. High burdens, in terms of numbers as well as rates, were estimated in Asia, sub-Saharan Africa, and South America. Since 1990, the attributable burden has increased by 50%, driven largely by population growth and ageing. Globally, the impact of reductions in household air pollution was largely offset by increased ambient PM2·5.Interpretation: Air pollution is a major risk factor for diabetes. We estimated that about a fifth of the global burden of type 2 diabetes is attributable PM2·5 pollution. Air pollution mitigation therefore might have an essential role in reducing the global disease burden resulting from type 2 diabetes.Funding: Bill & Melinda Gates Foundation.

Alcoholic hepatitis (AH) is a severe condition with poor short-term prognosis. Specific treatment with corticosteroids slightly improves short-term survival but is associated with infection and is not used in many centers. A reliable method to identify patients who will recover spontaneously will minimise the numbers of patients who experience side effects of available treatments.We analysed the trajectory of serum bilirubin concentration over the course of hospital admissions in patients with AH to predict spontaneous survival and the need for treatment.data from 426 patients were analysed. Based on bilirubin trajectory, patients were categorized into three groups: 'fast fallers' (bilirubin0.8 x admission value at day 7), 'static' (bilirubin of0.9 -1.2 x admission value) and 'rapid risers' (bilirubin of ≥1.2 x admission bilirubin). Fast fallers had significantly better 90-day survival compared to other groups (log rank p.001), and showed no benefit of corticosteroid therapy (OR for survival at 28 days of treatment, 0.94, 95% CI 0.06 - 8.41). These findings remained even amongst patients with severe disease based on initial DF, GAHS or MELD scores.We present an intuitive method of classifying patients with AH based on the trajectory of bilirubin over the first week of admission. It is complimentary to existing scores that identify candidates for corticosteroid treatment or assess response to treatment. This method identifies a group of patients with AH who recover spontaneously and can avoid corticosteroid therapy.

Background & Aims Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Expression of serine-threonine kinase receptor-associated protein (STRAP) is elevated in CRCs and is associated with poor outcomes. We investigated the role of STRAP in Apc mutation–induced intestinal tumor initiation and progression. Methods We generated Strap intestinal epithelial knockout mice (StrapΔIEC) by crossing mice containing floxed alleles of Strap (Strapfl/fl) with Villin-Cre mice. Then we generated ApcMin/+;Strapfl/fl;Vill-Cre (ApcMin/+;StrapΔIEC) mice for RNA-sequencing analyses to determine the mechanism of function of STRAP. We used human colon cancer cell lines (DLD1, SW480, and HT29) and human and mouse colon tumor–derived organoids for STRAP knockdown and knockout and overexpression experiments. Results Strap deficiency extended the average survival of ApcMin/+ mice by 80 days and decreased the formation of intestinal adenomas. Expression profiling revealed that the intestinal stem cell signature, the Wnt/β-catenin signaling, and the MEK/ERK pathway are down-regulated in Strap-deficient adenomas and intestinal organoids. Correlation studies suggest that these STRAP-associated oncogenic signatures are conserved across murine and human colon cancer. STRAP associates with MEK1/2, promotes binding between MEK1/2 and ERK1/2, and subsequently induces the phosphorylation of ERK1/2. STRAP activated Wnt/β-catenin signaling through MEK/ERK-induced phosphorylation of LRP6. STRAP was identified as a target of mutated Apc and Wnt/β-catenin signaling as chromatin immunoprecipitation and luciferase assays revealed putative binding sites of the β-catenin/TCF4 complex on the Strap promoter. Conclusions STRAP is a target of, and is required in, Apc mutation/deletion-induced intestinal tumorigenesis through a novel feed-forward STRAP/MEK-ERK/Wnt-β-catenin/STRAP regulatory axis.

Several lines of research suggest that BCL-XL-mediated anti-apoptotic effects may contribute to pathogenesis of myeloproliferative neoplasms driven by JAK2V617F and serve as therapeutic target. Here, we used a knock-in JAK2V617F mouse model and confirmed that Bcl-xl was overexpressed in erythroid progenitors. MPN-induced phenotype in the peripheral blood by conditional knockin of JAK2V617F was abrogated by conditional knockout of Bcl2l1, which presented anemia and thrombocytopenia independently of JAK2 mutation status. The Mx1-Cre Jak2V617W/VF /Bcl2l1f/f mice presented persistent splenomegaly as a result of extramedullary hematopoiesis and pro-apoptotic stimuli in terminally differentiated erythroid progenitors. The pan-BH3 mimetic inhibitor obatoclax showed superior cytotoxicity in JAK2V617F cell models, and reduced clonogenic capacity in ex vivo assay using Vav-Cre Jak2V617F bone marrow cells. Both ruxolitinib and obatoclax significantly reduced spleen weights in a murine Jak2V617F MPN model but did not show additive effect. The tumor burden reduction was observed with either ruxolitinib or obatoclax in terminal differentiation stage neoplastic cells but not in myeloid-erythroid precursors. Therefore, disrupting BCL2 balance is not sufficient to treat MPN at the stem cell level, but it is certainly an additional option for controlling the critical myeloid expansion of the disease.

This study tests biodegradation resistance of a custom synthesized novel ethylene glycol ethyl methacrylate (EGEMA) with ester bond linkages that are external to the central polymer backbone when polymerized. Ethylene glycol dimethacrylate (EGDMA) with internal ester bond linkages and EGEMA discs were prepared in a polytetrafluoroethylene (PTFE) mold using 40 μl macromer and photo/co-initiator mixture cured for 40 s at 1000 mW/cm(2). The discs were stored in the constant presence of Streptococcus mutans (S. mutans) in Todd Hewitt Yeast + Glucose (THYE+G) media up to 9 weeks ([Formula: see text] for each macromer type) and physical/mechanical properties were assessed. Initial measurements EGEMA versus EGDMA polymer discs showed equivalent degree of conversion (45.69% ± 2.38 vs. 46.79% ± 4.64), diametral tensile stress (DTS; 8.12± 2.92 MPa vs. 6.02 ± 1.48 MPa), and low subsurface optical defects (0.41% ± 0.254% vs. 0.11% ± 0.074%). The initial surface wettability (contact angle) was slightly higher [Formula: see text] for EGEMA (62.02° ± 3.56) than EGDMA (53.86° ± 5.61°). EGDMA showed higher initial Vicker’s hardness than EGEMA (8.03 ± 0.88 HV vs. 5.93 ± 0.69 HV; [Formula: see text]). After 9 weeks of S. mutans exposure, EGEMA ([Formula: see text]) showed higher resistance to biodegradation effects with a superior DTS than EGDMA ([Formula: see text]) [Formula: see text]. Visible and scanning electron microscopy images of EGEMA show less surface cracking and defects than EGDMA. EGDMA had higher loss of material (18.9% vs. 8.5%, [Formula: see text]), relative changes to fracture toughness (92.5% vs. 49.2%, [Formula: see text]) and increased water sorption (6.1% vs. 1.9%, [Formula: see text]) compared to EGEMA discs. The flipped external ester group linkage design is attributed to EGEMA showing higher resistance to bacterial degradation effects than an internal ester group linkage design methacrylate.

A Rayleigh distribution of particles exposing (1 1 1) surface populations is consistent with the particle size dependence of the turnover rates of the electrochemical reduction of dioxygen catalyzed by a platinum cathode.

Emerging evidence suggests that elevated concentrations of triglyceride-rich lipoprotein remnants (TRLs) derived from hepatic and intestinal sources contribute to the risk of atherosclerotic cardiovascular events. Natural selection studies support a causal role for elevated concentrations of remnant cholesterol and the pathways contributing to perturbations in metabolic pathways regulating TRLs with an increased risk of atherosclerotic cardiovascular disease events. New therapies targeting select catalytic pathways in TRL metabolism reduce atherosclerosis in experimental models, and concentrations of TRLs in patients with a vast range of triglyceride levels. Clinical trials with inhibitors of angiopoietin-like 3 protein and apolipoprotein C-III will be required to provide further guidance on the potential contribution of these emerging therapies in the paradigm of cardiovascular risk management in patients with elevated remnant cholesterol.

Purpose: Soft-tissue sarcomas (STS) are a rare, heterogeneous group of mesenchymal tumors. For decades the mainstay of treatment for advanced, unresectable STS has been palliative chemotherapy. High levels of activated MET receptor have been reported in various sarcoma cell lines, together with elevated vascular endothelial growth factor (VEGF) levels in patients with STS, suggesting that dual targeting of the VEGF and MET pathways with the multi-receptor tyrosine kinase inhibitor cabozantinib would result in clinical benefit in this population. Patients and Methods: We performed an open-label, multi-institution, single-arm phase II trial of single-agent cabozantinib in adult patients with advanced STS and progressive disease after at least 1 standard line of systemic therapy. Patients received 60 mg oral cabozantinib once daily in 28-day cycles, and dual primary endpoints of overall response rate and 6-month progression-free survival (PFS) were assessed. Changes in several circulating biomarkers were assessed as secondary endpoints. Results: Six (11.1%; 95% CI, 4.2%–22.6%) of the 54 evaluable patients enrolled experienced objective responses (all partial responses). Six-month PFS was 49.3% (95% CI, 36.2%–67.3%), with a median time on study of 4 cycles (range, 1–99). The most common grade 3/4 adverse events were hypertension (7.4%) and neutropenia (16.7%). Patients' levels of circulating hepatocyte growth factor (HGF), soluble MET, and VEGF-A generally increased after a cycle of therapy, while soluble VEGFR2 levels decreased, regardless of clinical outcome. Conclusions: Cabozantinib single-agent antitumor activity was observed in patients with selected STS histologic subtypes (alveolar soft-part sarcoma, undifferentiated pleomorphic sarcoma, extraskeletal myxoid chondrosarcoma, and leiomyosarcoma) highlighting the biomolecular diversity of STS.

Acute kidney injury (AKI) and frailty are major drivers of outcomes among patients with cirrhosis. What is unknown is the impact of physical frailty on the development of AKI. We included adults with cirrhosis without hepatocellular carcinoma listed for liver transplantation at nine US centers (n=1,033). Frailty was assessed using the Liver Frailty Index (LFI); "frail" was defined by LFI≥4.2. Chronic kidney disease as a baseline estimated glomerular filtration rate

Chronic disruption of rhythms (CDR) impacts sleep and can result in circadian misalignment of physiological systems which, in turn, is associated with increased disease risk. Exposure to repeated or severe stressors also disturbs sleep and diurnal rhythms. Prebiotic nutrients produce favorable changes in gut microbial ecology, the gut metabolome, and reduce several negative impacts of acute severe stressor exposure, including disturbed sleep, core body temperature rhythmicity, and gut microbial dysbiosis. In light of previous compelling evidence that prebiotic diet broadly reduces negative impacts of acute, severe stressors, we hypothesize that prebiotic diet will also effectively mitigate the negative impacts of chronic disruption of circadian rhythms on physiology and sleep/wake behavior. Male, Sprague Dawley rats were fed diets enriched in prebiotic substrates or calorically matched control chow. After 5 weeks on diet, rats were exposed to CDR (12 h light/dark reversal, weekly for 8 weeks) or remained on undisturbed normal light/dark cycles (NLD). Sleep EEG, core body temperature, and locomotor activity were recorded via biotelemetry in freely moving rats. Fecal samples were collected on experimental days –33, 0 (day of onset of CDR), and 42. Taxonomic identification and relative abundances of gut microbes were measured in fecal samples using 16S rRNA gene sequencing and shotgun metagenomics. Fecal primary, bacterially modified secondary, and conjugated bile acids were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Prebiotic diet produced rapid and stable increases in the relative abundances of Parabacteroides distasonis and Ruminiclostridium 5. Shotgun metagenomics analyses confirmed reliable increases in relative abundances of Parabacteroides distasonis and Clostridium leptum, a member of the Ruminiclostridium genus. Prebiotic diet also modified fecal bile acid profiles; and based on correlational and step-wise regression analyses, Parabacteroides distasonis and Ruminiclostridium 5 were positively associated with each other and negatively associated with secondary and conjugated bile acids. Prebiotic diet, but not CDR, impacted beta diversity. Measures of alpha diversity evenness were decreased by CDR and prebiotic diet prevented that effect. Rats exposed to CDR while eating prebiotic, compared to control diet, more quickly realigned NREM sleep and core body temperature (ClockLab) diurnal rhythms to the altered light/dark cycle. Finally, both cholic acid and Ruminiclostridium 5 prior to CDR were associated with time to realign CBT rhythms to the new light/dark cycle after CDR; whereas both Ruminiclostridium 5 and taurocholic acid prior to CDR were associated with NREM sleep recovery after CDR. These results support our hypothesis and suggest that ingestion of prebiotic substrates is an effective strategy to increase the relative abundance of health promoting microbes, alter the fecal bile acid profile, and facilitate the recovery and realignment of sleep and diurnal rhythms after circadian disruption.

Context Vascular aging, including endothelial dysfunction secondary to oxidative stress and inflammation, increases the risk for age-associated cardiovascular disease (CVD). Low testosterone in middle-aged/older men is associated with increased CVD risk. Objective We hypothesized that low testosterone contributes to age-associated endothelial dysfunction, related in part to greater oxidative stress and inflammation. Methods This cross-sectional study included 58 healthy, nonsmoking men categorized as young (N = 20; age 29 ± 4 years; testosterone 500 ± 58 ng/dL), middle-aged/older with higher testosterone (N = 20; age 60 ± 6 years; testosterone 512 ± 115 ng/dL), and middle-aged/older lower testosterone (N = 18; age 59 ± 8 years; testosterone 269 ± 48 ng/dL). Brachial artery flow-mediated dilation (FMDBA) was measured during acute infusion of saline (control) and vitamin C (antioxidant). Markers of oxidative stress (total antioxidant status and oxidized low-density lipoprotein cholesterol), inflammation (interleukin [IL]-6 and C-reactive protein [CRP]), and androgen deficiency symptoms were also examined. Results During saline, FMDBA was reduced in middle-aged/older compared with young, regardless of testosterone status (P < 0.001). FMDBA was reduced in middle-aged/older lower testosterone (3.7% ± 2.0%) compared with middle-aged/older higher testosterone (5.7% ± 2.2%; P = 0.021), independent of symptoms. Vitamin C increased FMDBA (to 5.3% ± 1.6%; P = 0.022) in middle-aged/older lower testosterone but had no effect in young (P = 0.992) or middle-aged/older higher testosterone (P = 0.250). FMDBA correlated with serum testosterone (r = 0.45; P < 0.001), IL-6 (r = −0.41; P = 0.002), and CRP (r = −0.28; P = 0.041). Conclusion Healthy middle-aged/older men with low testosterone appear to have greater age-associated endothelial dysfunction, related in part to greater oxidative stress and inflammation. These data suggest that low testosterone concentrations may contribute to accelerated vascular aging in men.

Rate coefficients for the reaction of NO 3 radicals with a series of aromatic aldehydes were measured in a 7300 L simulation chamber at ambient temperature and pressure by relative and absolute methods. The rate coefficients for benzaldehyde (BA), ortho-tolualdehyde (O-TA), meta-tolualdehyde (M-TA), para-tolualdehyde (P-TA), 2,4-dimethyl benzaldehyde (2,4-DMBA), 2,5-dimethyl benzaldehyde (2,5-DMBA) and 3,5-dimethyl benzaldehyde (3,5-DMBA) were k1= 2.6 ± 0.3, k2= 8.7 ± 0.8, k3= 4.9 ± 0.5, k4= 4.9 ± 0.4, k5= 15.1 ± 1.3, k6= 12.8 ± 1.2 and k7= 6.2 ± 0.6, respectively, in the units of 10 −15 cm 3 molec. −1 s −1 at 298 ± 2 K. The rate coefficient k13 for the reaction of the NO 3 radical with deuterated benzaldehyde (benzaldehyde-d 1) was found to be half that of k1 . The end product of the reaction in an excess of NO 2 was measured to be C 6 H 5 C(O)O 2 NO 2 . Theoretical calculations of aldehydic bond energies and reaction pathways indicate that the NO 3 radical reacts primarily with aromatic aldehydes through the abstraction of an aldehydic hydrogen atom. The atmospheric implications of the measured rate coefficients are briefly discussed.

Background Adults with atherosclerotic cardiovascular disease (ASCVD) at very high-risk for recurrent events who have low-density lipoprotein cholesterol ≥ 70 mg/dL despite maximally-tolerated statin therapy are recommended to initiate ezetimibe or a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. Objective Compare the initiation of ezetimibe and a PCSK9 inhibitor after a myocardial infarction (MI) among very high-risk ASCVD patients by race/ethnicity and sex. Methods We analyzed data from 374,786 adults ≥ 66 years of age with Medicare fee-for-service coverage who had an MI between July 1, 2015 and December 31, 2018, were not taking ezetimibe or a PCSK9 inhibitor, and had very high-risk ASCVD defined by the 2018 American Heart Association/American College of Cardiology multi-society cholesterol guideline. Pharmacy claims through December 31, 2018 were used to determine ezetimibe and PCSK9 inhibitor initiation. Results Overall, 6980 (1.9%) beneficiaries initiated ezetimibe, and 1433 (0.4%) initiated a PCSK9 inhibitor. Adjusted hazard ratios (aHR) for ezetimibe initiation among non-Hispanic Black, Hispanic, and Asian versus non-Hispanic White beneficiaries were 0.77 (95% confidence interval [95%CI]: 0.70–0.86), 0.92 (95%CI: 0.76–1.11) and 0.73 (95%CI: 0.59–0.89), respectively. Compared to non-Hispanic White beneficiaries, the aHRs for PCSK9 inhibitor initiation were 0.63 (95%CI: 0.48–0.81) among non-Hispanic Black, 0.70 (95%CI: 0.43–1.13) among Hispanic, and 0.93 (95%CI: 0.62–1.39) among Asian beneficiaries. The aHRs for ezetimibe and PCSK9 inhibitor initiation comparing women to men were 1.11 (95%CI: 1.06–1.17) and 1.13 (95%CI: 1.01–1.25), respectively. Conclusion There are race/ethnic and sex disparities in the initiation of ezetimibe and a PCSK9 inhibitor following MI among very high-risk ASCVD patients.

Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell's intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN.

The prognosis of most patients with AML is poor due to frequent disease relapse. The cause of relapse is thought to be from the persistence of leukemia initiating cells (LIC's) following treatment. Here we assessed RNA based changes in LICs from matched patient diagnosis and relapse samples using single-cell RNA sequencing. Previous studies on AML progression have focused on genetic changes at the DNA mutation level mostly in bulk AML cells and demonstrated the existence of DNA clonal evolution. Here we identified in LICs that the phenomenon of RNA clonal evolution occurs during AML progression. Despite the presence of vast transcriptional heterogeneity at the single cell level, pathway analysis identified common signaling networks involving metabolism, apoptosis and chemokine signaling that evolved during AML progression and become a signature of relapse samples. A subset of this gene signature was validated at the protein level in LICs by flow cytometry from an independent AML cohort and functional studies were performed to demonstrate co-targeting BCL2 and CXCR4 signaling may help overcome therapeutic challenges with AML heterogeneity. It is hoped this work will facilitate a greater understanding of AML relapse leading to improved prognostic biomarkers and therapeutic strategies to target LIC's.

Clinical Practice Points • Aggressive NK-cell leukemia/lymphoma (ANKL) is a rare natural killer (NK)-cell malignancy, and although it typically presents as rapidly progressive disease, cases may present more indolently, progressing over months rather than weeks. • Unlike the more common extranodal NK-cell leukemia/lymphoma, ANKL may not involve the nasopharynx and can be Epstein-Barr virus (EBV) negative. • Diagnosing ANKL can be challenging. Although not yet standard of care, next-generation sequencing and digital image analysis can be used to clarify this rare diagnosis and confirm EBV negativity. • Treatment of ANKL utilizes asparaginase-based regimens, unlike the anthracycline-based regimens used in peripheral T-cell lymphomas or myeloid disorders. Ensuring the correct diagnosis is crucial in determining the appropriate treatment regimen for this uncommon disease.

O-glycosylation is a post-translational protein modification essential to life. One of the enzymes involved in this process is protein O-fucosyltransferase 1 (POFUT1), which fucosylates threonine or serine residues within a specific sequence context of epidermal growth factor-like domains (EGF-LD). Unlike most inverting glycosyltransferases, POFUT1 lacks a basic residue in the active site that could act as a catalytic base to deprotonate the Thr/Ser residue of the EGF-LD acceptor during the chemical reaction. Using quantum mechanics/molecular mechanics (QM/MM) methods on recent crystal structures, as well as mutagenesis experiments, we uncover the enzyme catalytic mechanism, revealing that it involves proton shuttling through an active site asparagine, conserved among species, which undergoes tautomerization. This mechanism is consistent with experimental kinetic analysis of Caenorhabditis elegans POFUT1 Asn43 mutants, which ablate enzyme activity even if mutated to Asp, the canonical catalytic base in inverting glycosyltransferases. These results will aid inhibitor development for Notch-associated O-glycosylation disorders.

Introduction: Metabolic resuscitation of organ donors and the attenuation of oxidative stress incurred by organs following brain death and transplantation have the potential to improve organ yield and allograft function. Thiamine (vitamin B1) is a vital coenzyme in both energy metabolism and the production of antioxidants that has not been studied in the donor population. Research Aim: To determine the frequency of subclinical thiamine deficiency in brain-dead organ donors and its correlation with demographics, length of hospitalization, donor management, lactic acidosis, and the requirement for vasoactive support. Design: Prospective cohort study of brain-dead donors managed at a single organ procurement organization’s organ recovery facility. Results: A total 64 donors were enrolled; 24 donors had thiamine levels drawn upon arrival and 40 donors had levels drawn at the time of organ procurement. Whole blood thiamine levels were inversely correlated with the time from death (P = .007) and 20% (8/40) of donors had levels below the normal range at the time of organ procurement. Demographic features of the donor were not associated with thiamine levels although longer hospital stays prior to death were associated with lower levels ( P < .05). The presence and resolution of lactic acidosis was not associated with whole blood thiamine level. Higher thiamine levels were associated with earlier discontinuation of vasoactive support ( P = .04). Discussion: Whole blood thiamine deficiency was not uncommon at the time of organ procurement. Thiamine may be associated with the requirement for hemodynamic support.

Bioorthogonal phosphines were introduced in the context of the Staudinger ligation over twenty years ago. Since that time, phosphine probes have been used in myriad applications to tag azide-functionalized biomolecules. The Staudinger ligation also paved the way for the development of other phosphorus-based chemistries, many of which are widely employed in biological experiments. Several reviews have highlighted early achievements in the design and application of bioorthogonal phosphines. This review summarizes more recent advances in the field. We discuss innovations in classic Staudinger-like transformations that have enabled new biological pursuits. We also highlight relative newcomers to the bioorthogonal stage, including the cyclopropenone-phosphine ligation and the phospha-Michael reaction. The review concludes with chemoselective reactions involving phosphite and phosphonite ligations. For each transformation, we describe the overall mechanism and scope. We also showcase efforts to fine-tune the reagents for specific functions. We further describe recent applications of the chemistries in biological settings. Collectively, these examples underscore the versatility and breadth of bioorthogonal phosphine reagents.

The homologous recombination deficiency (HRD) score integrates three DNA-based measures of genomic instability, and has been understudied in prostate cancer. Given the recent FDA approval of two PARP inhibitors for prostate cancer, HRD score analysis could help to refine treatment selection. We assessed HRD score (defined as the sum of loss-of-heterozygosity, telomeric allelic imbalance, and large-scale state transitions) in three cohorts of primary prostate cancer, including a Johns Hopkins University (JHU) cohort with germline mutations in BRCA2, ATM, or CHEK2 (n = 64), the TCGA cohort (n = 391), and the PROGENE cohort (n = 102). In the JHU cohort, tumors with germline BRCA2 mutations had higher HRD scores (median = 27) than those with germline ATM or CHEK2 mutations (median = 16.5 [p = 0.029] and 9 [p

Objective To identify the prevalence of renal insufficiency (RI) in children with a history of prematurity and acute kidney injury (AKI). Study design This prospective cohort study evaluated renal function in children born preterm at 5-9 years of age. Univariable analyses compared perinatal and follow-up data from subjects with and without AKI history, and with and without current RI. Regression analyses were attempted to model RI as a function of AKI and other clinical risk factors. Results Fifteen of 43 (35%) participants had previously undiagnosed RI. Only children with no AKI history or neonatal stage 1 AKI presented for follow-up. Children born preterm with a history of stage 1 AKI had higher serum creatinine (sCr) at follow-up, but were not more likely to have RI compared to children without stage 1 AKI history (RI prevalence 30% and 36% in AKI and non-AKI group, respectively). Conclusion The high prevalence of RI in this preterm cohort at middle childhood follow-up highlights the need for routine kidney health assessments in this population. Large multicenter studies are needed to further characterize the impact of premature birth and mild AKI on renal function throughout childhood.

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor involved in over 400 cellular reactions. During embryogenesis, mammals synthesize NAD de novo from dietary L-tryptophan via the kynurenine pathway. Biallelic, inactivating variants in three genes encoding enzymes of this biosynthesis pathway (KYNU, HAAO, and NADSYN1) disrupt NAD synthesis and have been identified in patients with multiple malformations of the heart, kidney, vertebrae, and limbs; these patients have Congenital NAD Deficiency Disorder. Here we have identified a further three families with biallelic variants in HAAO and four families with biallelic variants in KYNU. These patients present similarly with multiple malformations of the heart, kidney, vertebrae, and limbs, of variable severity. We show that each variant identified in these patients results in loss-of-function, revealed by significant reduction in NAD levels via yeast genetic complementation assays. For the first time missense mutations are identified as a cause of malformation and shown to disrupt enzyme function. These missense and frameshift variants cause moderate to severe NAD deficiency in yeast, analogous to insufficient synthesized NAD in patients. We hereby expand the genotypic and corresponding phenotypic spectrum of Congenital NAD Deficiency Disorder.

D-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of D-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D(2)O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.