Untargeted proteomic differences between clinical strains of methicillin-sensitive and methicillin-resistant Staphylococcusaureus.

Staphylococcus aureus is a common disease-causing bacterium that has developed resistances to a wide variety of antibiotics. This increasing antibiotic resistance has made management of these infections difficult. A better understanding of the general differences among clinical S. aureus strains beyond the well characterized resistance mechanisms may help in identifying new anti-microbial targets. This study aimed to identify and compare the general differences in protein profiles among clinical strains of S. aureus sensitive and resistant to methicillin. The proteomic profiles of five methicillin sensitive (MSSA) and five methicillin resistant (MRSA) S. aureus strains were analyzed by ultra-performance liquid chromatography-mass spectrometry. Protein identification was done using Progenesis QI for Proteomics and the UniProt S. aureus database. Proteins that play roles in virulence, metabolism, and protein synthesis were found to be present at different abundances between MSSA and MRSA (Data available via ProteomeXchange with identifier PXD021629). This study shows differences in protein profiles between antibiotic sensitive and antibiotic resistant clinical strains of S. aureus that may affect the resistance mechanism. Further research on these differences may identify new drug targets against methicillin resistant S. aureus strains.

Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro.

Cataracts cause vision loss and blindness by impairing the ability of the ocular lens to focus light onto the retina. Various cataract risk factors have been identified, including drug treatments, age, smoking and diabetes. However, the molecular events responsible for these different forms of cataract are ill-defined, and the advent of modern cataract surgery in the 1960s virtually eliminated access to human lenses for research. Here, we demonstrate large-scale production of light-focusing human micro-lenses from spheroidal masses of human lens epithelial cells purified from differentiating pluripotent stem cells. The purified lens cells and micro-lenses display similar morphology, cellular arrangement, mRNA expression and protein expression to human lens cells and lenses. Exposing the micro-lenses to the emergent cystic fibrosis drug Vx-770 reduces micro-lens transparency and focusing ability. These human micro-lenses provide a powerful and large-scale platform for defining molecular disease mechanisms caused by cataract risk factors, for anti-cataract drug screening and for clinically relevant toxicity assays.

A simplified method for producing human lens epithelial cells and light-focusing micro-lenses from pluripotent stem cells.

Here we describe a modified method for harvesting tens-of-millions of human lens epithelial-like cells from differentiated pluripotent stem cell cultures. To assess the utility of this method, we analysed the lens cell population via: light microscopy; single cell RNA-sequencing and gene ontology analyses; formation of light-focusing micro-lenses; mass spectrometry; and electron microscopy. Both individually and collectively, the data indicate this simplified harvesting method provides a large-scale source of stem cell-derived lens cells and micro-lenses for investigating human lens and cataract formation.

Human Group IIA Phospholipase A2-Three Decades on from Its Discovery.

Phospholipase A2 (PLA2) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA2) enzymes were the first of the five major classes of human PLA2s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA2, has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA2 field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function.

A study of Pt(II)-phenanthroline complex interactions with double-stranded and G-quadruplex DNA by ESI-MS, circular dichroism, and computational docking.

The binding interactions of a series of square-planar platinum(II)-phenanthroline complexes of the type [Pt(PL)(AL)]2+ [where PL = variously methyl-substituted 1,10-phenanthroline (phen) and AL = ethane-1,2-diamine (en)] were assessed with a G-quadruplex DNA (5'-TTG GGG GT-3', G4DNA) and a double-stranded DNA (5'-CGC GAA TTC GCG-3', dsDNA) sequence by ESI-MS. The results indicate a strong correlation between G4DNA affinity and increasing phenanthroline methyl substitution. Circular dichroism (CD) spectroscopy and molecular docking studies also support the finding that increased substitution of the phenanthroline ligand increased selectivity for G4DNA. ESI-MS was used to probe the interaction of a range of square-planar Pt(II)-phenanthroline complexes with double-stranded and G-quadruplex DNA.

Transient elastography for screening of liver fibrosis: Cost-effectiveness analysis from six prospective cohorts in Europe and Asia.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease and alcohol-related liver disease pose an important challenge to current clinical healthcare pathways because of the large number of at-risk patients. Therefore, we aimed to explore the cost-effectiveness of transient elastography (TE) as a screening method to detect liver fibrosis in a primary care pathway. METHODS: Cost-effectiveness analysis was performed using real-life individual patient data from 6 independent prospective cohorts (5 from Europe and 1 from Asia). A diagnostic algorithm with conditional inference trees was developed to explore the relationships between liver stiffness, socio-demographics, comorbidities, and hepatic fibrosis, the latter assessed by fibrosis scores (FIB-4, NFS) and liver biopsies in a subset of 352 patients. We compared the incremental cost-effectiveness of a screening strategy against standard of care alongside the numbers needed to screen to diagnose a patient with fibrosis stage ≥F2. RESULTS: The data set encompassed 6,295 participants (mean age 55 ±â€¯12 years, BMI 27 ±â€¯5 kg/m2, liver stiffness 5.6 ±â€¯5.0 kPa). A 9.1 kPa TE cut-off provided the best accuracy for the diagnosis of significant fibrosis (≥F2) in general population settings, whereas a threshold of 9.5 kPa was optimal for populations at-risk of alcohol-related liver disease. TE with the proposed cut-offs outperformed fibrosis scores in terms of accuracy. Screening with TE was cost-effective with mean incremental cost-effectiveness ratios ranging from 2,570 €/QALY (95% CI 2,456-2,683) for a population at-risk of alcohol-related liver disease (age ≥45 years) to 6,217 €/QALY (95% CI 5,832-6,601) in the general population. Overall, there was a 12% chance of TE screening being cost saving across countries and populations. CONCLUSIONS: Screening for liver fibrosis with TE in primary care is a cost-effective intervention for European and Asian populations and may even be cost saving. LAY SUMMARY: The lack of optimized public health screening strategies for the detection of liver fibrosis in adults without known liver disease presents a major healthcare challenge. Analyses from 6 independent international cohorts, with transient elastography measurements, show that a community-based risk-stratification strategy for alcohol-related and non-alcoholic fatty liver diseases is cost-effective and potentially cost saving for our healthcare systems, as it leads to earlier identification of patients.

Validation of a Model for Identification of Patients With Compensated Cirrhosis at High Risk of Decompensation.

BACKGROUND & AIMS: It is important to rapidly identify patients with advanced liver disease. Routine tests to assess liver function and fibrosis provide data that can be used to determine patients' prognoses. We tested the validated the ability of combined data from the ALBI and FIB-4 scoring systems to identify patients with compensated cirrhosis at highest risk for decompensation. METHODS: We collected data from 145 patients with compensated cirrhosis (91% Child A cirrhosis and median MELD scores below 8) from a cohort in Nottingham, United Kingdom, followed for a median 4.59 years (development cohort). We collected baseline clinical features and recorded decompensation events. We used these data to develop a model based on liver function (assessed by the ALBI score) and extent of fibrosis (assessed by the FIB-4 index) to determine risk of decompensation. We validated the model in 2 independent external cohorts (1 in Dublin, Ireland and 1 in Menoufia, Egypt) comprising 234 patients. RESULTS: In the development cohort, 19.3% of the patients developed decompensated cirrhosis. Using a combination of ALBI and FIB-4 scores, we developed a model that identified patients at low vs high risk of decompensation (hazard ratio [HR] for decompensation in patients with high risk score was 7.10). When we tested the scoring system in the validation cohorts, the HR for decompensation in patients with a high-risk score was 12.54 in the Ireland cohort and 5.10 in the Egypt cohort. CONCLUSION: We developed scoring system, based on a combination of ALBI and FIB-4 scores, that identifies patients at risk for liver decompensation. We validated the scoring system in 2 independent international cohorts (Europe and the Middle East), so it appears to apply to diverse populations.

Determination of glyoxal and methylglyoxal in serum by UHPLC coupled with fluorescence detection.

Glyoxal (GO) and methylglyoxal (MGO) are two important biomarkers in diabetes. Analytical methods for determination of GO and MGO in serum samples are either HPLC with UV-Vis (low sensitivity) or MS/MS (expensive) detection. These disadvantages have hampered the introduction of these biomarkers as a routine analyte for diabetes diagnostics into the clinical laboratory. In this study, we introduce a UHPLC method with fluorescence detection for the measurement of GO and MGO in serum samples by pre-column derivatization at neutral pH with 5, 6-diamino-2,4-dihydroxypyrimidine sulfate (DDP) to form lumazines. The method was validated as per FDA guidelines. Using this method, we have determined GO and MGO in a variety of animal serum samples, and for example, determined the GO and MGO concentration in adult bovine serum to be 852 ±â€¯27 and 192 ±â€¯10 nmol/L, respectively. In human serum, GO and MGO levels in non-diabetic subjects (n = 14) were determined to be 154 ±â€¯88 and 98 ±â€¯27 nmol/L, and in serum samples from subjects with diabetes (n = 14) 244 ±â€¯137 and 190 ±â€¯68 nmol/L, respectively. In addition, interference studies showed that physiological serum components did not lead to an artificial increase in the levels of GO and MGO.

Optimized Method to Quantify Dopamine Turnover in the Mammalian Retina.

Measurement of dopamine (DA) release in the retina allows the interrogation of the complex neural circuits within this tissue. A number of previous methods have been used to quantify this neuromodulator, the most common of which is HPLC with electrochemical detection (HPLC-ECD). However, this technique can produce significant concentration uncertainties. In this present study, we report a sensitive and accurate UHPLC-MS/MS method for the quantification of DA and its primary metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in mouse retina. Internal standards DA-d4 and DOPAC-d5 result in standard curve linearity for DA from 0.05-100 ng/mL (LOD = 6 pg/mL) and DOPAC from 0.5-100 ng/mL (LOD = 162 pg/mL). A systematic study of tissue extraction conditions reveals that the use of formic acid (1%), in place of the more commonly used perchloric acid, combined with 0.5 mM ascorbic acid prevents significant oxidation of the analytes. When the method is applied to mouse retinae a significant increase in the DOPAC/DA ratio is observed following in vivo light stimulation. We additionally examined the effect of anesthesia on DA and DOPAC levels in the retina in vivo and find that basal dark-adapted concentrations are not affected. Light caused a similar increase in DOPAC/DA ratio but interindividual variation was significantly reduced. Together, we systematically describe the ideal conditions to accurately and reliably measure DA turnover in the mammalian retina.

Prevalence and natural history of histologically proven chronic liver disease in a longitudinal cohort of patients with type 1 diabetes.

UNLABELLED: Although a higher prevalence of raised liver enzymes and altered echotexture on ultrasound have been reported in patients with type 1 diabetes mellitus (T1DM), the histological spectrum and natural history of chronic liver disease (CLD) in T1DM is unknown. We investigated the prevalence and outcome of histologically proven CLD in a longitudinal cohort of patients with T1DM. We identified patients who have had liver biopsy from a computerized database (DIAMOND; Hicom Technology, Brookwood, UK) containing longitudinal data for over 95% of type 1 diabetes patients from an overall catchment population of 700,000 people. Gender-matched patients with oral hypoglycemic-treated (T2OH) and insulin-treated type 2 diabetes (T2IN) who had liver biopsy formed two comparative cohorts. We collated clinical and histological data, as well as long-term outcomes of all three groups, and compared T1DM cirrhosis incidence to UK general population data. Of 4,644 patients with T1DM, 57 (1.2%) underwent liver biopsy. Of these, 53.1% of patients had steatosis, 20.4% had nonalcoholic steatohepatitis, and 73.5% had fibrosis on index liver biopsy. Cirrhosis was diagnosed in 14 patients (24.6%) during follow-up. T1DM with age under 55 years had an odds ratio of 1.875 (95% confidence interval: 0.936-3.757) for cirrhosis incidence, compared to the general population. Longitudinal liver-related outcomes were similar comparing the T1DM cohort and respective type 2 diabetes cohorts--when adjusted for important confounders, diabetic cohort type did not predict altered risk of incident cirrhosis or portal hypertension. CONCLUSION: Type 1 diabetes is associated with a previously unrecognized burden of CLD and its complications.

An efficient continuous flow approach to furnish furan-based biaryls.

Suzuki cross-couplings of 5-formyl-2-furanylboronic acid with activated or neutral aryl bromides were performed under continuous flow conditions in the presence of (Bu)4N(+)F(-) and the immobilised t-butyl based palladium catalyst CatCart™ FC1032™. Deactivated aryl bromides and activated aryl chlorides were cross-coupled with 5-formyl-2-furanylboronic in the presence of (Bu)4N(+)OAc(-) using the bis-triphenylphosphine CatCart™ PdCl2(PPh3)2-DVB. Initial evidence indicates the latter method may serve as a universal approach to conduct Suzuki cross-couplings with the protocol successfully employed in the synthesis of the current gold standard Hedgehog pathway inhibitor LDE225.

Insignoic acids A - E, unusual α, ß-unsaturated keto fatty acids isolated from the exocarp of Australian rainforest tree Endiandra insignis (Lauraceae).

Anti-inflammatory bioassay-guided compound isolation from the exocarp of the Australian rainforest tree Endiandra insignis (family Lauraceae) has led to the discovery and structural elucidation of unusual α, ß-unsaturated twenty-four carbon fatty acids and their positional isomers, insignoic acids A - E (1a - 5c). The stereochemistry and position of the double bond within the aliphatic chain were independently determined via NMR spectroscopy and Ozone-Induced Dissociation (OzID) Mass Spectrometry, respectively. Compounds (1a - 5c) displayed good to moderate anti-inflammatory activity in the range of 8-84 µM. The low therapeutic index observed when assessing the cell viability in the RAW macrophage cell lines, prompted us to investigate the anticancer potential of these unusual fatty acids. The anti-cancer activity was assessed in A-431 carinoma cell lines and MM649 melanoma cell lines. Insignoic acid C (3a-f) exhibited the highest level of potency with an IC50 value of 5-7 µM against both the cell lines. The insignoic acids are the first of their kind known for incorporating an alpha-beta unsaturated system flanked next to a keto group with an additional level of oxygenation at C-6 in a 24­carbon fatty acid backbone.

Translating the potential of the urine steroid metabolome to stage NAFLD (TrUSt-NAFLD): study protocol for a multicentre, prospective validation study.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) affects approximately one in four individuals and its prevalence continues to rise. The advanced stages of NAFLD with significant liver fibrosis are associated with adverse morbidity and mortality outcomes. Currently, liver biopsy remains the 'gold-standard' approach to stage NAFLD severity. Although generally well tolerated, liver biopsies are associated with significant complications, are resource intensive, costly, and sample only a very small area of the liver as well as requiring day case admission to a secondary care setting. As a result, there is a significant unmet need to develop non-invasive biomarkers that can accurately stage NAFLD and limit the need for liver biopsy. The aim of this study is to validate the use of the urine steroid metabolome as a strategy to stage NAFLD severity and to compare its performance against other non-invasive NAFLD biomarkers. METHODS AND ANALYSIS: The TrUSt-NAFLD study is a multicentre prospective test validation study aiming to recruit 310 patients with biopsy-proven and staged NAFLD across eight centres within the UK. 150 appropriately matched control patients without liver disease will be recruited through the Oxford Biobank. Blood and urine samples, alongside clinical data, will be collected from all participants. Urine samples will be analysed by liquid chromatography-tandem mass spectroscopy to quantify a panel of predefined steroid metabolites. A machine learning-based classifier, for example, Generalized Matrix Relevance Learning Vector Quantization that was trained on retrospective samples, will be applied to the prospective steroid metabolite data to determine its ability to identify those patients with advanced, as opposed to mild-moderate, liver fibrosis as a consequence of NAFLD. ETHICS AND DISSEMINATION: Research ethical approval was granted by West Midlands, Black Country Research Ethics Committee (REC reference: 21/WM/0177). A substantial amendment (TrUSt-NAFLD-SA1) was approved on 26 November 2021. TRIAL REGISTRATION NUMBER: ISRCTN19370855.

Isolation and characterization of charge-tagged phenylperoxyl radicals in the gas phase: direct evidence for products and pathways in low temperature benzene oxidation.

The phenylperoxyl radical has long been accepted as a critical intermediate in the oxidation of benzene and an archetype for arylperoxyl radicals in combustion and atmospheric chemistry. Despite being central to many contemporary mechanisms underpinning these chemistries, reports of the direct detection or isolation of phenylperoxyl radicals are rare and there is little experimental evidence connecting this intermediate with expected product channels. We have prepared and isolated two charge-tagged phenyl radical models in the gas phase [i.e., 4-(N,N,N-trimethylammonium)phenyl radical cation and 4-carboxylatophenyl radical anion] and observed their reactions with dioxygen by ion-trap mass spectrometry. Measured reaction rates show good agreement with prior reports for the neutral system (k(2)[(Me(3)N(+))C(6)H(4)˙ + O(2)] = 2.8 × 10(-11) cm(3) molecule(-1) s(-1), Φ = 4.9%; k(2)[((-)O(2)C)C(6)H(4)˙ + O(2)] = 5.4 × 10(-11) cm(3) molecule(-1) s(-1), Φ = 9.2%) and the resulting mass spectra provide unequivocal evidence for the formation of phenylperoxyl radicals. Collisional activation of isolated phenylperoxyl radicals reveals unimolecular decomposition by three pathways: (i) loss of dioxygen to reform the initial phenyl radical; (ii) loss of atomic oxygen yielding a phenoxyl radical; and (iii) ejection of the formyl radical to give cyclopentadienone. Stable isotope labeling confirms these assignments. Quantum chemical calculations for both charge-tagged and neutral phenylperoxyl radicals confirm that loss of formyl radical is accessible both thermodynamically and entropically and competitive with direct loss of both hydrogen atom and carbon dioxide.

Untargeted lipidomic differences between clinical strains of methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common cause of infectious diseases in humans. It has become resistant to many antibacterial agents making management of infections difficult. A better understanding of differences among S. aureus strains that are sensitive and resistant to antibiotics may offer insights into the resistant phenotype and identify new antimicrobial targets. This study aimed at comparing general differences in lipid profiles among clinical strains of S. aureus sensitive and resistant to antibiotics. The cell wall thickness and cell surface charge were also compared. METHODS: Five methicillin sensitive (MSSA) and five methicillin resistant (MRSA) S. aureus strains were compared both individually and as MSSA and MRSA groups in the absence of antibiotics. Lipids were compared by ultra-performance liquid chromatography-mass spectrometry, cell wall thickness was compared by scanning transmission electron microscopy and whole-cell surface charge was compared using a cytochrome c binding assay. RESULTS: Twenty-two lipid species were identified in all ten strains of S. aureus. The abundance of three lipid species (two lysyl-phosphatidylglycerol and one diglycosyldiacylglycerol) were found to be different between MSSA and MRSA. Differences in cell wall thickness were identified between strains but not between MSSA and MRSA. No difference in whole-cell surface charge was observed between MSSA and MRSA. CONCLUSION: This study shows differences in membrane lipids between antibiotic sensitive and antibiotic resistant clinical strains of S aureus that may affect resistance mechanisms related to cell membrane structure and fluidity. Further research on these differences may identify new drug targets against resistant strains.

Choice of antibody is critical for specific and sensitive detection of androgen receptor splice variant-7 in circulating tumor cells.

Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes.

Transient Elastography in Community Alcohol Services: Can It Detect Significant Liver Disease and Impact Drinking Behaviour?

Introduction: Alcohol is the leading cause of cirrhosis in Western populations. The early identification of high-risk drinkers followed by intervention is an effective way to reduce harm. We aim to assess the feasibility of integrating transient elastography (TE) into community alcohol services, and to determine its impact on modifying drinking behaviours. Method: A prospective cohort study was conducted at a community alcohol clinic in Nottingham, UK (April 2012 to March 2014). Patients (>18 years) with a primary alcohol problem were recruited. Those known to liver services or those known to have chronic liver disease were excluded. Significant liver fibrosis was defined by a liver stiffness of >8 kilopascal (kPa). Follow-up was for a minimum of six months. Data were descriptively analysed for significant differences between patients with a normal liver stiffness versus raised liver stiffness. Results: 156 patients were invited; n = 87 attended and n = 86 underwent successful TE. The majority were male (n = 53, 70.0%), and the mean age was 46.3 years (SD ± 9.8). Median liver stiffness was 6.9 kPa (range 3.1-75.0kPa). Clinically significant liver fibrosis was identified in n = 33 (38.4%), of which n = 6 were in the cirrhotic range (≥15 kPa). The baseline median self-reported alcohol intake for normal stiffness was 126 units per week (range 24-378) and in raised stiffness was 149.0 units per week (range 39.0-420.0); this difference was nonsignificant (p = 0.338). The median reduction in self-reported alcohol intake in the whole cohort was 65.0 units per week (range 27.0-88.0, p < 0.001); in the normal liver stiffness group it was 25.0 units per week (range 18.0-75.0, p = 0.154), and in the raised liver stiffness group it was 78.5 units per week (range 36.0-126.0, p < 0.001). Conclusion: The study demonstrated that transient elastography is a feasible tool to stratify clinically significant liver disease in community alcohol services. It can stimulate a change in high-risk drinking behaviour and a normal liver stiffness result does not provide false reassurance to participants.

Direct observation of the gas phase reaction of the cyclohexyl radical with dioxygen using a distonic radical ion approach.

Alkylperoxyl radicals are intermediates in the oxidation of hydrocarbons. The reactive nature of these intermediates, however, has made them elusive to direct observation and isolation. We have employed ion trap mass spectrometry to synthesize and characterize 4-carboxylatocyclohexyl radical anions (*C(6)H(10)-CO(2)(-)) and observe their reactivity in the presence of dioxygen. The resulting reaction is facile (k = 1.8 x 10(-10) cm(3) molecule(-1) s(-1) or 30% of calculated collision rate) and results in (i) the addition of O(2) to form stabilized 4-carboxylatocyclohexylperoxyl radical anions (*OO-C(6)H(10)-CO(2)(-)), providing the first direct observation of a cyclohexylperoxyl radical, and (ii) elimination of HO(2)* and HO* radicals consistent with recent laser-induced fluorescence studies of the reaction of neutral cyclohexyl radicals with O(2). Electronic structure calculations at the B3LYP/6-31+G(d) level of theory reveal viable pathways for the observed reactions showing that formation of the peroxyl radical is exothermic by 37 kcal mol(-1) with subsequent transition states as low as -6.6 kcal mol(-1) (formation of HO(2)*) and -9.1 kcal mol(-1) (formation of HO*) with respect to the entrance channel. The combined computational and experimental data suggest that the structures of the reaction products correspond to cyclohexenes and epoxides from HO(2)* and HO* loss, respectively, while alternative pathways leading to cyclohexanone or ring-opened isomers are not observed. Activation of the charged peroxyl radical *OO-C(6)H(10)-CO(2)(-) by collision induced dissociation also results in the loss of HO(2)* and HO* radicals confirming that these products are directly connected to the peroxyl radical intermediate.

Distinct Microbiota Dysbiosis in Patients with Non-Erosive Reflux Disease and Esophageal Adenocarcinoma.

Non-erosive reflux disease (NERD) and esophageal adenocarcinoma (EAC) are often regarded as bookends in the gastroesophageal reflux disease spectrum. However, there is limited clinical evidence to support this disease paradigm while the underlying mechanisms of disease progression remain unclear. In this study, we used 16S rRNA sequencing and mass-spectrometer-based proteomics to characterize the esophageal microbiota and host mucosa proteome, respectively. A total of 70 participants from four patient groups (NERD, reflux esophagitis, Barrett's esophagus, and EAC) and a control group were analyzed. Our results showed a unique NERD microbiota composition, distinct to control and other groups. We speculate that an increase in sulfate-reducing Proteobacteria and Bacteroidetes along with hydrogen producer Dorea are associated with a mechanistic role in visceral hypersensitivity. We also observed a distinct EAC microbiota consisting of a high abundance of lactic acid-producing bacteria (Staphylococcus, Lactobacillus, Bifidobacterium, and Streptococcus), which may contribute towards carcinogenesis through dysregulated lactate metabolism. This study suggests the close relationship between esophageal mucosal microbiota and the appearance of pathologies of this organ.

Acceptability of chronic liver disease screening in a UK primary care setting: a qualitative evaluation.

OBJECTIVES: The increasing incidence of chronic liver disease (CLD) in the UK may be attributed to a rise in preventable risk factors, including hazardous alcohol use and type 2 diabetes. Transient elastography (TE) can rapidly stratify risk of CLD in primary care populations and provide an opportunity to raise patient awareness of risk factors.This study explores patients' experiences of TE screening in a primary care setting. In addition, patient awareness of CLD risk is explored. STUDY DESIGN AND SETTING: This study used a qualitative process evaluation of a community screening pathway for CLD (Nottingham, UK). Participants completed semistructured interviews, which were audio-recorded, transcribed verbatim and analysed thematically. PARTICIPANTS: Twenty adults were purposively recruited 6 months to 2 years after TE screening. Inclusion criteria included (1) hazardous alcohol use, (2) type 2 diabetes and/or (3) persistently elevated liver enzymes without known cause. RESULTS: Undergoing TE in primary care was seen as acceptable to most participants. Hazardous alcohol use was identified as the primary cause of CLD; no participants were aware of metabolic risk factors. TE improved understanding of personal risk factors and prompted contemplation of lifestyle changes across all TE stratifications. However, participants' perceptions of risk were altered by the healthcare providers' communication of TE scores. CONCLUSIONS: High acceptability of TE, regardless of the risk factor, provides strong support for inclusion of TE stratification in primary care. Findings highlight the positive impact of receiving TE on risk awareness. Future clinical iterations should improve the structure and communication of TE results to patients.