Host-Microbe-Drug-Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy.

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT.

Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.

ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions.

DNA methylation of enhancers is dynamic, cell-type specific, and vital for cell fate progression. However, current models inadequately define its role within the hierarchy of gene regulation. Analysis of independent datasets shows an unanticipated overlap between DNA methylation and chromatin accessibility at enhancers of steady-state stem cells, suggesting that these two opposing features might exist concurrently. To define their temporal relationship, we developed ATAC-Me, which probes accessibility and methylation from single DNA library preparations. We identified waves of accessibility occurring rapidly across thousands of myeloid enhancers in a monocyte-to-macrophage cell fate model. Prolonged methylation states were observed at a majority of these sites, while transcription of nearby genes tracked closely with accessibility. ATAC-Me uncovers a significant disconnect between chromatin accessibility, DNA methylation status, and gene activity. This unexpected observation highlights the value of ATAC-Me in constructing precise molecular timelines for understanding the role of DNA methylation in gene regulation.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

The PLOD2/succinate axis regulates the epithelial-mesenchymal plasticity and cancer cell stemness.

Aberrant accumulation of succinate has been detected in many cancers. However, the cellular function and regulation of succinate in cancer progression is not completely understood. Using stable isotope-resolved metabolomics analysis, we showed that the epithelial mesenchymal transition (EMT) was associated with profound changes in metabolites, including elevation of cytoplasmic succinate levels. The treatment with cell-permeable succinate induced mesenchymal phenotypes in mammary epithelial cells and enhanced cancer cell stemness. Chromatin immunoprecipitation and sequence analysis showed that elevated cytoplasmic succinate levels were sufficient to reduce global 5-hydroxymethylcytosinene (5hmC) accumulation and induce transcriptional repression of EMT-related genes. We showed that expression of procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) was associated with elevation of cytoplasmic succinate during the EMT process. Silencing of PLOD2 expression in breast cancer cells reduced succinate levels and inhibited cancer cell mesenchymal phenotypes and stemness, which was accompanied by elevated 5hmC levels in chromatin. Importantly, exogenous succinate rescued cancer cell stemness and 5hmC levels in PLOD2-silenced cells, suggesting that PLOD2 promotes cancer progression at least partially through succinate. These results reveal the previously unidentified function of succinate in enhancing cancer cell plasticity and stemness.

Vaccine-induced inflammation and inflammatory monocytes promote CD4+ T cell-dependent immunity against murine salmonellosis.

Prior infection can generate protective immunity against subsequent infection, although the efficacy of such immunity can vary considerably. Live-attenuated vaccines (LAVs) are one of the most effective methods for mimicking this natural process, and analysis of their efficacy has proven instrumental in the identification of protective immune mechanisms. Here, we address the question of what makes a LAV efficacious by characterising immune responses to a LAV, termed TAS2010, which is highly protective (80-90%) against lethal murine salmonellosis, in comparison with a moderately protective (40-50%) LAV, BRD509. Mice vaccinated with TAS2010 developed immunity systemically and were protected against gut-associated virulent infection in a CD4+ T cell-dependent manner. TAS2010-vaccinated mice showed increased activation of Th1 responses compared with their BRD509-vaccinated counterparts, leading to increased Th1 memory populations in both lymphoid and non-lymphoid organs. The optimal development of Th1-driven immunity was closely correlated with the activation of CD11b+Ly6GnegLy6Chi inflammatory monocytes (IMs), the activation of which can be modulated proportionally by bacterial load in vivo. Upon vaccination with the LAV, IMs expressed T cell chemoattractant CXCL9 that attracted CD4+ T cells to the foci of infection, where IMs also served as a potent source of antigen presentation and Th1-promoting cytokine IL-12. The expression of MHC-II in IMs was rapidly upregulated following vaccination and then maintained at an elevated level in immune mice, suggesting IMs may have a role in sustained antigen stimulation. Our findings present a longitudinal analysis of CD4+ T cell development post-vaccination with an intracellular bacterial LAV, and highlight the benefit of inflammation in the development of Th1 immunity. Future studies focusing on the induction of IMs may reveal key strategies for improving vaccine-induced T cell immunity.

Digital Twins of Acute Hypoxemic Respiratory Failure Patients Suggest a Mechanistic Basis for Success and Failure of Noninvasive Ventilation.

OBJECTIVES: To clarify the mechanistic basis for the success or failure of noninvasive ventilation (NIV) in acute hypoxemic respiratory failure (AHRF). DESIGN: We created digital twins based on mechanistic computational models of individual patients with AHRF. SETTING: Interdisciplinary Collaboration in Systems Medicine Research Network. SUBJECTS: We used individual patient data from 30 moderate-to-severe AHRF patients who had failed high-flow nasal cannula (HFNC) therapy and subsequently underwent a trial of NIV. INTERVENTIONS: Using the digital twins, we evaluated lung mechanics, quantified the separate contributions of external support and patient respiratory effort to lung injury indices, and investigated their relative impact on NIV success or failure. MEASUREMENTS AND MAIN RESULTS: In digital twins of patients who successfully completed/failed NIV, after 2 hours of the trial the mean (sd) of the change in total lung stress was -10.9 (6.2)/-0.35 (3.38) cm H2O, mechanical power -13.4 (12.2)/-1.0 (5.4) J/min, and total lung strain 0.02 (0.24)/0.16 (0.30). In the digital twins, positive end-expiratory pressure (PEEP) produced by HFNC was similar to that set during NIV. In digital twins of patients who failed NIV vs. those who succeeded, intrinsic PEEP was 3.5 (0.6) vs. 2.3 (0.8) cm H2O, inspiratory pressure support was 8.3 (5.9) vs. 22.3 (7.2) cm H2O, and tidal volume was 10.9 (1.2) vs. 9.4 (1.8) mL/kg. In digital twins, successful NIV increased respiratory system compliance +25.0 (16.4) mL/cm H2O, lowered inspiratory muscle pressure -9.7 (9.6) cm H2O, and reduced the contribution of patient spontaneous breathing to total driving pressure by 57.0%. CONCLUSIONS: In digital twins of AHRF patients, successful NIV improved lung mechanics, lowering respiratory effort and indices associated with lung injury. NIV failed in patients for whom only low levels of positive inspiratory pressure support could be applied without risking patient self-inflicted lung injury due to excessive tidal volumes.

Sociodemographic and clinical indicators of children and young people with cerebral palsy and reported unmet social needs.

AIM: To determine the frequency, type, clinical, and sociodemographic associations of unmet social needs in children with cerebral palsy (CP). METHOD: We conducted a cross-sectional study of parents and carers of children with CP attending a specialist hospital clinic between July and September 2022. Unmet social needs were self-identified using a survey, guided by the WE CARE survey instrument and adapted to the local context. Sociodemographic and clinical data were obtained from medical records. We performed descriptive analysis of participants' unmet social needs, sociodemographic factors, and clinical factors, and examined for associations using a χ2 test and logistic regression. RESULTS: A total of 105 parents and carers completed the survey. Of these, 68 (64.8%) reported one or more unmet social need, with 24 (22.9%) reporting three or more unmet needs. A higher number (three or more) of unmet needs was associated with Gross Motor Function Classification System levels IV and V (odds ratio [OR] = 3.77, 95% confidence interval [CI] = 1.44-9.86) and intellectual disability (OR = 4.63, 95% CI = 1.61-13.31), but were not significant when corrected for neighbourhood socioeconomic disadvantage. The greatest socioeconomic disadvantage was associated with housing concerns (p = 0.002), food (p = 0.026), and financial insecurity (p = 0.02). INTERPRETATION: Unmet social needs are experienced by most families of children with CP. This study highlights the importance of systematic pathways to identify and address unmet social needs.

Development of a new social prescribing intervention for families of children with cerebral palsy.

AIM: To co-design a social prescribing intervention (the EPIC-CP programme: Equitable Pathways and Integrated Care in Cerebral Palsy) with children with cerebral palsy (CP), their families, and clinicians to address unmet social needs. METHOD: The study was conducted (August 2021 to March 2023) at the paediatric rehabilitation departments of the three tertiary paediatric hospitals in New South Wales, Australia. Eligible participants attended or worked at one of the departments, including children with CP, parents/caregivers, and clinicians. Mixed-methods co-design was used in intervention co-production and prototyping. The project was overseen by research advisors with lived experience of CP. RESULTS: More than 200 participants contributed to the co-design research. Families experienced a substantial burden of unmet social needs. Co-designed interventions involved systematic identification of unmet social needs with (1) targeted community resources and (2) engagement with a 'community linker' who supported children/young people and their families to access health, education, and social services that matched their identified needs and preferences. Research participants co-developed the programme logic model and prototype. This was piloted in research action cycles and iteratively refined until consensus was achieved. INTERPRETATION: We co-designed a social prescribing programme responsive to the needs of its end-users and purposefully developed to be embedded in the Australian health setting. A pilot randomized controlled trial will further evaluate this intervention.

An Examination of Students' Perspectives of Medical English Course Quality in Guangdong Medical Universities.

Phenomenon: In China, medical English courses are critical to medical education, equipping Chinese students with the linguistic tools necessary for international medical practice and collaboration. However, a disconnect persists between the pedagogical approaches of medical practitioners and language educators, leading to a curriculum that emphasizes grammatical accuracy over practical communication skills. This misalignment results in student disengagement and falls short of addressing the real-world demands of the medical profession. With the growing importance of English proficiency in the global health sector, the need for significant improvements in medical English education is evident. This study delves into the underlying causes of student demotivation and aims to reconcile educational delivery with the evolving expectations of the medical field. Insights gained from this research will inform targeted interventions, promising to enhance medical English courses and support improved educational experiences for Chinese medical undergraduates. Approach: This cross-sectional quantitative study surveyed 3,046 second-year medical students from four medical universities in Guangdong Province, China, leveraging means-analysis and Expectancy-Disconfirmation Theory (EDT) as its foundation. The research was conducted at the end of the 2022-2023 academic year, utilizing a questionnaire to assess students' perceptions of their medical English courses. Importance-Performance Analysis (IPA) was the primary analytical tool to discern discrepancies between students' expectations and experiences. Findings: The IPA revealed that course content, classroom environment, and instructor effectiveness were pivotal factors influencing the perceived quality of the medical English courses. Students expressed a need for practical and relevant course material, with current content and textbooks falling short of preparing them for future medical communication demands. Additionally, while learning technologies were acknowledged, there was a discernible preference against their excessive application, suggesting a misalignment between student satisfaction and learning outcomes. Insights: This study highlights the need for innovative staffing models, refined qualifications for part-time instructors, development of collaborative and practical teaching materials, and focused training for medical English instructors. It also emphasizes the judicious integration of e-learning to enhance the learning experience. These insights aim to improve instruction quality by informing potential pedagogical adjustments and resource allocations in medical English education.

Cross-tissue patterns of DNA hypomethylation reveal genetically distinct histories of cell development.

BACKGROUND: Establishment of DNA methylation (DNAme) patterns is essential for balanced multi-lineage cellular differentiation, but exactly how these patterns drive cellular phenotypes is unclear. While > 80% of CpG sites are stably methylated, tens of thousands of discrete CpG loci form hypomethylated regions (HMRs). Because they lack DNAme, HMRs are considered transcriptionally permissive, but not all HMRs actively regulate genes. Unlike promoter HMRs, a subset of non-coding HMRs is cell type-specific and enriched for tissue-specific gene regulatory functions. Our data further argues not only that HMR establishment is an important step in enforcing cell identity, but also that cross-cell type and spatial HMR patterns are functionally informative of gene regulation. RESULTS: To understand the significance of non-coding HMRs, we systematically dissected HMR patterns across diverse human cell types and developmental timepoints, including embryonic, fetal, and adult tissues. Unsupervised clustering of 126,104 distinct HMRs revealed that levels of HMR specificity reflects a developmental hierarchy supported by enrichment of stage-specific transcription factors and gene ontologies. Using a pseudo-time course of development from embryonic stem cells to adult stem and mature hematopoietic cells, we find that most HMRs observed in differentiated cells (~ 60%) are established at early developmental stages and accumulate as development progresses. HMRs that arise during differentiation frequently (~ 35%) establish near existing HMRs (≤ 6 kb away), leading to the formation of HMR clusters associated with stronger enhancer activity. Using SNP-based partitioned heritability from GWAS summary statistics across diverse traits and clinical lab values, we discovered that genetic contribution to trait heritability is enriched within HMRs. Moreover, the contribution of heritability to cell-relevant traits increases with both increasing HMR specificity and HMR clustering, supporting the role of distinct HMR subsets in regulating normal cell function. CONCLUSIONS: Our results demonstrate that the entire HMR repertoire within a cell-type, rather than just the cell type-specific HMRs, stores information that is key to understanding and predicting cellular phenotypes. Ultimately, these data provide novel insights into how DNA hypo-methylation provides genetically distinct historical records of a cell's journey through development, highlighting HMRs as functionally distinct from other epigenomic annotations.

Renal safety of critical care sedation with sevoflurane: a systematic review and meta-analysis.

Volatile anesthetic agents are increasingly widely used for critical care sedation. There are concerns that sevoflurane presents a risk of renal injury when used in this role. RCTs comparing the use of critical care sevoflurane sedation with any control in humans were systematically identified using MEDLINE, Cochrane CENTRAL, web of Science, and CINAHL (until May 2022), if they presented comparative data on renal function or serum inorganic fluoride levels. Pooled SMDs (95% CI) were calculated where possible after assessment of quality with GRADE and risk of bias with ROB-2. Eight studies analyzing 793 patients were included. The median duration of use of critical care sevoflurane sedation was 4.8 [IQR 3.5-9.2] hours; however, most trials also included a period of prior intraoperative use. No significant difference was found in serum creatinine at 1 day (SMD 0.05, 95% CI - 0.12 to 0.21), 48 h (SMD = - 0.04; 95% Cl - 0.25 to 0.17), 72 h (SMD = - 0.15; 95% CI - 0.45 to 0.15), and at discharge (SMD = - 0.1; 95% CI - 0.3 to 0.13) between the sevoflurane group and the control groups. Creatinine clearance was measured in two studies at 48 h with no significant difference (SMD = - 0.13; 95% Cl - 0.38 to 0.11). Levels of serum inorganic fluoride were significantly elevated in patients where sevoflurane was used. Sevoflurane was not associated with renal failure when used for critical care sedation of fewer than 72-h duration, despite the elevation of serum fluoride. Longer-term studies are currently inadequate, including in patients with compromised renal function, to further evaluate the role of sevoflurane in this setting.Trial registration PROSPERO (CRD42022333099).

Validation of at-the-bedside formulae for estimating ventilator driving pressure during airway pressure release ventilation using computer simulation.

BACKGROUND: Airway pressure release ventilation (APRV) is widely available on mechanical ventilators and has been proposed as an early intervention to prevent lung injury or as a rescue therapy in the management of refractory hypoxemia. Driving pressure ([Formula: see text]) has been identified in numerous studies as a key indicator of ventilator-induced-lung-injury that needs to be carefully controlled. [Formula: see text] delivered by the ventilator in APRV is not directly measurable in dynamic conditions, and there is no "gold standard" method for its estimation. METHODS: We used a computational simulator matched to data from 90 patients with acute respiratory distress syndrome (ARDS) to evaluate the accuracy of three "at-the-bedside" methods for estimating ventilator [Formula: see text] during APRV. RESULTS: Levels of [Formula: see text] delivered by the ventilator in APRV were generally within safe limits, but in some cases exceeded levels specified by protective ventilation strategies. A formula based on estimating the intrinsic positive end expiratory pressure present at the end of the APRV release provided the most accurate estimates of [Formula: see text]. A second formula based on assuming that expiratory flow, volume and pressure decay mono-exponentially, and a third method that requires temporarily switching to volume-controlled ventilation, also provided accurate estimates of true [Formula: see text]. CONCLUSIONS: Levels of [Formula: see text] delivered by the ventilator during APRV can potentially exceed levels specified by standard protective ventilation strategies, highlighting the need for careful monitoring. Our results show that [Formula: see text] delivered by the ventilator during APRV can be accurately estimated at the bedside using simple formulae that are based on readily available measurements.

Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines.

BACKGROUND: Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse. RESULTS: In this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences. CONCLUSIONS: Amongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4 × and 14 × more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community.

Pre-hospital continuous positive airway pressure after blast lung injury and hypovolaemic shock: a modelling study.

BACKGROUND: In non-traumatic respiratory failure, pre-hospital application of CPAP reduces the need for intubation. Primary blast lung injury (PBLI) accompanied by haemorrhagic shock is common after mass casualty incidents. We hypothesised that pre-hospital CPAP is also beneficial after PBLI accompanied by haemorrhagic shock. METHODS: We performed a computer-based simulation of the cardiopulmonary response to PBLI followed by haemorrhage, calibrated from published controlled porcine experiments exploring blast injury and haemorrhagic shock. The effect of different CPAP levels was simulated in three in silico patients who had sustained mild, moderate, or severe PBLI (10%, 25%, 50% contusion of the total lung) plus haemorrhagic shock. The primary outcome was arterial partial pressure of oxygen (Pao2) at the end of each simulation. RESULTS: In mild blast lung injury, 5 cm H2O ambient-air CPAP increased Pao2 from 10.6 to 12.6 kPa. Higher CPAP did not further improve Pao2. In moderate blast lung injury, 10 cm H2O CPAP produced a larger increase in Pao2 (from 8.5 to 11.1 kPa), but 15 cm H2O CPAP produced no further benefit. In severe blast lung injury, 5 cm H2O CPAP inceased Pao2 from 4.06 to 8.39 kPa. Further increasing CPAP to 10-15 cm H2O reduced Pao2 (7.99 and 7.90 kPa, respectively) as a result of haemodynamic impairment resulting from increased intrathoracic pressures. CONCLUSIONS: Our modelling study suggests that ambient air 5 cm H2O CPAP may benefit casualties suffering from blast lung injury, even with severe haemorrhagic shock. However, higher CPAP levels beyond 10 cm H2O after severe lung injury reduced oxygen delivery as a result of haemodynamic impairment.

Optimising respiratory support for early COVID-19 pneumonia: a computational modelling study.

BACKGROUND: Optimal respiratory support in early COVID-19 pneumonia is controversial and remains unclear. Using computational modelling, we examined whether lung injury might be exacerbated in early COVID-19 by assessing the impact of conventional oxygen therapy (COT), high-flow nasal oxygen therapy (HFNOT), continuous positive airway pressure (CPAP), and noninvasive ventilation (NIV). METHODS: Using an established multi-compartmental cardiopulmonary simulator, we first modelled COT at a fixed FiO2 (0.6) with elevated respiratory effort for 30 min in 120 spontaneously breathing patients, before initiating HFNOT, CPAP, or NIV. Respiratory effort was then reduced progressively over 30-min intervals. Oxygenation, respiratory effort, and lung stress/strain were quantified. Lung-protective mechanical ventilation was also simulated in the same cohort. RESULTS: HFNOT, CPAP, and NIV improved oxygenation compared with conventional therapy, but also initially increased total lung stress and strain. Improved oxygenation with CPAP reduced respiratory effort but lung stress/strain remained elevated for CPAP >5 cm H2O. With reduced respiratory effort, HFNOT maintained better oxygenation and reduced total lung stress, with no increase in total lung strain. Compared with 10 cm H2O PEEP, 4 cm H2O PEEP in NIV reduced total lung stress, but high total lung strain persisted even with less respiratory effort. Lung-protective mechanical ventilation improved oxygenation while minimising lung injury. CONCLUSIONS: The failure of noninvasive ventilatory support to reduce respiratory effort may exacerbate pulmonary injury in patients with early COVID-19 pneumonia. HFNOT reduces lung strain and achieves similar oxygenation to CPAP/NIV. Invasive mechanical ventilation may be less injurious than noninvasive support in patients with high respiratory effort.

Tissue-Specific Downregulation of Fatty Acid Synthase Suppresses Intestinal Adenoma Formation via Coordinated Reprograming of Transcriptome and Metabolism in the Mouse Model of Apc-Driven Colorectal Cancer.

Altered lipid metabolism is a potential target for therapeutic intervention in cancer. Overexpression of Fatty Acid Synthase (FASN) correlates with poor prognosis in colorectal cancer (CRC). While multiple studies show that upregulation of lipogenesis is critically important for CRC progression, the contribution of FASN to CRC initiation is poorly understood. We utilize a C57BL/6-Apc/Villin-Cre mouse model with knockout of FASN in intestinal epithelial cells to show that the heterozygous deletion of FASN increases mouse survival and decreases the number of intestinal adenomas. Using RNA-Seq and gene set enrichment analysis, we demonstrate that a decrease in FASN expression is associated with inhibition of pathways involved in cellular proliferation, energy production, and CRC progression. Metabolic and reverse phase protein array analyses demonstrate consistent changes in alteration of metabolic pathways involved in both anabolism and energy production. Downregulation of FASN expression reduces the levels of metabolites within glycolysis and tricarboxylic acid cycle with the most significant reduction in the level of citrate, a master metabolite, which enhances ATP production and fuels anabolic pathways. In summary, we demonstrate the critical importance of FASN during CRC initiation. These findings suggest that targeting FASN is a potential therapeutic approach for early stages of CRC or as a preventive strategy for this disease.

Using reactive dissipative particle dynamics to understand local shape manipulation of polymer vesicles.

Biological cells have long been of interest to researchers due to their capacity to actively control their shape. Accordingly, there is significant interest in generating simplified synthetic protocells that can alter their shape based on an externally or internally generated stimulus. To date, most progress has been made towards controlling the global shape of a protocell, whereas less is known about generating a local shape change. Here, we seek to better understand the possible mechanisms for producing local morphological changes in a popular protocell system, the block copolymer vesicle. Accordingly, we have combined Dissipative Particle Dynamics (DPD) and the Split Reactive Brownian Dynamics algorithm (SRBD) to produce a simulation tool that is capable of modeling the dynamics of self-assembled polymer structures as they undergo chemical reactions. Using this Reactive DPD or RDPD method, we investigate local morphological change driven by either the microinjection of a stimulus or an enzymatically-produced stimulus. We find that sub-vesicle-scale morphological change can be induced by either a solvent stimulus that swells the vesicle membrane, or by a reactant stimulus that alters the chemistry of the block polymer in the membrane corona. Notably, the latter method results in a more persistent local deformation than the former, which we attribute to the slower diffusion of polymer chains relative to the solvent. We quantify this deformation and show that it can be modulated by altering the interaction parameter of the parts of the polymer chain that are affected by the stimulus.

Emergence of Cumyl-PEGACLONE-related fatalities in the Northern Territory of Australia.

Suspected unnatural or unexpected deaths in the Northern Territory of Australia are reportable to the coroner, and investigation of such cases typically includes a post-mortem examination with comprehensive toxicological screening. An autopsy case series of five Cumyl-PEGACLONE-related fatalities over a recent eighteen-month period is presented. Databases of the Northern Territory coroner's office and the Royal Darwin Hospital Forensic Pathology Unit were searched to identify deaths related to synthetic cannabis use between July 1, 2018 and December 31, 2020. Toxicological analysis was performed at Forensic Science South Australia using a combination of liquid chromatography, gas chromatography and mass spectrometry. Cumyl-PEGACLONE, a synthetic cannabinoid receptor agonist (SCRA) with a gamma-carbolinone core, was detected in five cases (range in post-mortem blood 0.73-3.0 µg/L). Concurrent alcohol use and underlying cardiovascular disease were considered relevant factors in most cases. Toxicological Significance Scoring was carefully considered in all five cases, and in four cases, the presence of Cumyl-PEGACLONE was considered to be highly significant (TSS = 3). Synthetic cannabis use has not previously been identified in Northern Territory drug trends, and only one fatality related to the use of gamma-carbolines was identified in a recent Australia-wide study on synthetic cannabinoid-related fatalities. Deaths related to Cumyl-PEGACLONE use are emerging in the Northern Territory of Australia; this has public health implications. Although the exact mechanism(s) of death related to Cumyl-PEGACLONE are not fully established, this additional descriptive case series reaffirm an association with underlying cardiovascular disease, and suggest that concurrent use with alcohol may be relevant.

Methionine biosynthesis and transport are functionally redundant for the growth and virulence of Salmonella Typhimurium.

Methionine (Met) is an amino acid essential for many important cellular and biosynthetic functions, including the initiation of protein synthesis and S-adenosylmethionine-mediated methylation of proteins, RNA, and DNA. The de novo biosynthetic pathway of Met is well conserved across prokaryotes but absent from vertebrates, making it a plausible antimicrobial target. Using a systematic approach, we examined the essentiality of de novo methionine biosynthesis in Salmonella enterica serovar Typhimurium, a bacterial pathogen causing significant gastrointestinal and systemic diseases in humans and agricultural animals. Our data demonstrate that Met biosynthesis is essential for S. Typhimurium to grow in synthetic medium and within cultured epithelial cells where Met is depleted in the environment. During systemic infection of mice, the virulence of S. Typhimurium was not affected when either de novo Met biosynthesis or high-affinity Met transport was disrupted alone, but combined disruption in both led to severe in vivo growth attenuation, demonstrating a functional redundancy between de novo biosynthesis and acquisition as a mechanism of sourcing Met to support growth and virulence for S. Typhimurium during infection. In addition, our LC-MS analysis revealed global changes in the metabolome of S. Typhimurium mutants lacking Met biosynthesis and also uncovered unexpected interactions between Met and peptidoglycan biosynthesis. Together, this study highlights the complexity of the interactions between a single amino acid, Met, and other bacterial processes leading to virulence in the host and indicates that disrupting the de novo biosynthetic pathway alone is likely to be ineffective as an antimicrobial therapy against S. Typhimurium.