The Role of AdhE on Ethanol Tolerance and Production in Clostridium thermocellum.

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.

How bile acids and the microbiota interact to shape host immunity.

Bile acids are increasingly appearing in the spotlight owing to their novel impacts on various host processes. Similarly, there is growing attention on members of the microbiota that are responsible for bile acid modifications. With recent advances in technology enabling the discovery and continued identification of microbially conjugated bile acids, the chemical complexity of the bile acid landscape in the body is increasing at a rapid pace. In this Review, we summarize our current understanding of how bile acids and the gut microbiota interact to modulate immune responses during homeostasis and disease, with a particular focus on the gut.

Latent profiles of social reactions to sexual assault disclosure among bisexual women.

OBJECTIVE: Bisexual women experience higher rates of sexual assault (SA) and posttrauma distress compared to monosexual women. We examined the patterns of reactions to SA disclosure (positive and two types of negative) and bisexual minority stress (antibisexual stigma from heterosexual and lesbian/gay people, internalized binegativity) experienced by young bisexual women who experienced adult SA. We also examined differences in assault-related experiences, posttraumatic stress disorder and depression symptoms, and hazardous drinking based on the identified patterns. METHOD: The current sample was drawn from two parent samples of women and included survivors who had disclosed an adult SA: (a) n = 149 bisexual women (98% cisgender, ages 18-35, 87.9% White) recruited via Amazon's Mechanical Turk and (b) n = 81 cisgender bisexual women (ages 18-25, 84% White) recruited via online ads. Latent profile analyses and analyses of variance were conducted. RESULTS: Analyses identified four latent profiles characterized by: (a) low stigmatizing responses, (b) high negative SA disclosure reactions, (c) high antibisexual stigma, and (d) highest stigmatizing responses. The fourth profile had higher posttraumatic stress disorder and depression symptoms and hazardous drinking compared to the other profiles. CONCLUSIONS: Results suggest women in the profile characterized by highest negative reactions to SA disclosure and highest bisexual minority stress (antibisexual stigma and internalized binegativity) may experience greatest distress postassault. Results highlight the importance for researchers, policymakers, and clinicians to consider and address both SA-related and identity-related stigma among bisexual survivors. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Development and clinical implementation of a digital workflow utilizing 3D-printed oral stents for patients with head and neck cancer receiving radiotherapy.

OBJECTIVES: We describe the development of 3D-printed stents using our digital workflow and their effects on patients enrolled in the lead-in phase of a multi-center, randomized Phase-II trial. MATERIALS AND METHODS: Digital dental models were created for patients using intraoral scanning. Digital processes were implemented to develop the mouth-opening, tongue-depressing, and tongue-lateralizing stents using stereolithography. Time spent and material 3D-printing costs were measured. Physicians assessed mucositis using the Oral Mucositis Assessment Scale (OMAS) and collected MD Anderson Symptom Inventory (MDASI) reports and adverse events (AEs) from patients at various time points (TPs). OMAS and MDASI results were evaluated using paired t-test analysis. RESULTS: 18 patients enrolled into the lead-in phase across 6 independent clinical sites in the USA. 15 patients received stents (average design and fabrication time, 8 h; average material 3D-printing cost, 11 USD). 10 eligible patients with complete OMAS and MDASI reports across all TPs were assessed. OMAS increased significantly from baseline to week 3 of treatment (mean difference = 0.34; 95 % CI, 0.09-0.60; p = 0.01). MDASI increased significantly from baseline to week 3 of treatment (mean difference = 1.02; 95 % CI, 0.40-1.70; p = 0.005), and week 3 of treatment to end of treatment (mean difference = 1.90; 95 % CI, 0.90-2.92; p = 0.002). AEs (grades 1-3) were reported by patients across TPs. Mucositis and radiation dermatitis were primarily attributed to chemoradiation. CONCLUSIONS: 3D-printed stents were successfully fabricated and well tolerated by patients. As patients enroll in the randomized phase of this trial, data herein will establish a baseline for comparative analysis.

The endocast morphology of LES1, Homo naledi.

OBJECTIVES: Homo naledi is near the extreme of small brain size within Homo but is easily recognized as Homo in other aspects of endocast morphology. This study adds new evidence of the endocast morphology of H. naledi by describing the Lesedi Hominin 1 (LES1) endocranium from the Lesedi Chamber and compares it to the previously known H. naledi individual Dinaledi Hominin 3 (DH3) as well as other hominin taxa. MATERIALS AND METHODS: We examined interlandmark distances with both univariate and multivariate methods in multiple hominin taxa and both species of Pan. For each distance, we compared groups using adjusted Z-scores (Azs). Our multivariate analyses included both principal component analyses (PCA) and linear discriminant analyses (LDA). RESULTS: DH3 and LES1 each have absolute third frontal convolution measures that enter the upper half of the variation for Homo sapiens, Homo erectus, and Homo neanderthalensis. Examined relative to the cube root of endocranial volume, H. naledi ranks among the highest values in these samples of Homo. Both absolute and relative values for H. naledi specimens are far above Pan, Australopithecus, and Paranthropus, suggesting an expanded Broca's area. CONCLUSIONS: Both qualitative and quantitative analyses show consistency between LES1 and other H. naledi endocasts and confirm the shared morphology of H. naledi with H. sapiens, H. neanderthalensis, and some specimens of H. erectus.

Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings.

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Enhancing anaerobic digestion of lignocellulosic biomass by mechanical cotreatment.

BACKGROUND: The aim of this study was to increase the accessibility and accelerate the breakdown of lignocellulosic biomass to methane in an anaerobic fermentation system by mechanical cotreatment: milling during fermentation, as an alternative to conventional pretreatment prior to biological deconstruction. Effluent from a mesophilic anaerobic digester running with unpretreated senescent switchgrass as the predominant carbon source was collected and subjected to ball milling for 0.5, 2, 5 and 10 min. Following this, a batch fermentation test was conducted with this material in triplicate for an additional 18 days with unmilled effluent as the 'status quo' control. RESULTS: The results indicate 0.5 - 10 min of cotreatment increased sugar solubilization by 5- 13% when compared to the unmilled control, with greater solubilization correlated with increased milling duration. Biogas concentrations ranged from 44% to 55.5% methane with the balance carbon dioxide. The total biogas production was statistically higher than the unmilled control for all treatments with 2 or more minutes of milling (α = 0.1). Cotreatment also decreased mean particle size. Energy consumption measurements of a lab-scale mill indicate that longer durations of milling offer diminishing benefits with respect to additional methane production. CONCLUSIONS: Cotreatment in anaerobic digestion systems, as demonstrated in this study, provides an alternative approach to conventional pretreatments to increase biogas production from lignocellulosic grassy material.

Solubilization of sugarcane bagasse by mono and cocultures of thermophilic anaerobes with and without cotreatment.

Cotreatment, mechanical disruption of lignocellulosic biomass during microbial fermentation, is a potential alternative to thermochemical pretreatment as a means of increasing the accessibility of lignocellulose to biological attack. Successful implementation of cotreatment requires microbes that can withstand milling, while solubilizing and utilizing carbohydrates from lignocellulose. In this context, cotreatment with thermophilic, lignocellulose-fermenting bacteria has been successfully evaluated for a number of lignocellulosic feedstocks. Here, cotreatment was applied to sugarcane bagasse using monocultures of the cellulose-fermenting Clostridium thermocellum and cocultures with the hemicellulose-fermenting Thermoanaerobacterium thermosaccharolyticum. This resulted in 76 % carbohydrate solubilization (a 1.8-fold increase over non-cotreated controls) on 10 g/L solids loading, having greater effect on the hemicellulose fraction. With cotreatment, fermentation by wild-type cultures at low substrate concentrations increased cumulative product formation by 45 % for the monoculture and 32 % for the coculture. These findings highlight the potential of cotreatment for enhancing deconstruction of sugarcane bagasse using thermophilic bacteria.

Normal-Tension Glaucoma Complicated by a Giant Internal Carotid-Ophthalmic Artery Aneurysm.

Purpose. We describe a patient with normal tension glaucoma (NTG) of several years whose management was complicated by the presence of a giant internal carotid-ophthalmic artery aneurysm. Observations. A 72-year-old woman presented to our glaucoma clinic with accelerated deterioration of her vision in her left eye (OS) over a 1-month period. Her ophthalmic history was most notable for bilateral NTG diagnosed 3 years prior which had been treated with several laser trabeculoplasty OS and topical bimatoprost 0.01% eye drops in both eyes (OU). Upon evaluation, her visual acuity OS had worsened, and visual field (VF) testing showed extensive progressive losses temporally and pericentrally OS over a year with stable IOP measurements and no neurological complaints. Given her atypical NTG progression, she was referred for an urgent neurological evaluation which revealed an unruptured giant left internal carotid-ophthalmic aneurysm. Following the successful treatment of the aneurysm with platinum coils, she continued to demonstrate additional bilateral ophthalmic changes including further progression of VF loss and RNFL thinning OS > OD on follow-up. Conclusion and Importance. Overall, this report describes a unique complication in the management of a patient with chronic bilateral NTG in the form of a giant internal carotid-ophthalmic aneurysm. Moreover, it highlights the need for clinicians to maintain a degree of suspicion for compressive lesions of the optic nerve when presented with atypical progression of VFs and/or visual acuity loss in glaucomatous patients.

Adolescent Idiopathic Scoliosis: Increased Body Mass Associated with Decreased Bracing Outcomes.

Bracing reduces the need for surgical intervention in patients with adolescent idiopathic scoliosis (AIS). However, bracing outcomes with variable body mass index (BMI) are understudied. The authors sought to determine the association of BMI with bracing outcomes. The authors performed a retrospective cohort study of 104 patients presenting with AIS. Initial Risser score, hours of bracing per day, BMI percentile, and curve magnitude pre- and postbracing were collected. There was no detectable difference between years of brace wear or primary curve magnitude at time of presentation between both groups. Overall, 29% (25/87) of underweight/normal weight patients and 59% (10/17) of overweight/obese patients had curves ≥ 45 degrees at the end of bracing (p = 0.016). Odds of having a curve ≥ 45 degrees after bracing were 3.5 (95% confidence interval: 1.2 to 10.3, p = 0.021) times higher for overweight/obese patients compared with underweight/normal weight patients. Increased overlying adipose tissue may reduce the corrective forces required to straighten the spine. (Journal of Surgical Orthopaedic Advances 33(1):029-032, 2024).

Aerobic anoxygenic phototrophs play important roles in nutrient cycling within cyanobacterial Microcystis bloom microbiomes.

BACKGROUND: During the bloom season, the colonial cyanobacterium Microcystis forms complex aggregates which include a diverse microbiome within an exopolymer matrix. Early research postulated a simple mutualism existing with bacteria benefitting from the rich source of fixed carbon and Microcystis receiving recycled nutrients. Researchers have since hypothesized that Microcystis aggregates represent a community of synergistic and interacting species, an interactome, each with unique metabolic capabilities that are critical to the growth, maintenance, and demise of Microcystis blooms. Research has also shown that aggregate-associated bacteria are taxonomically different from free-living bacteria in the surrounding water. Moreover, research has identified little overlap in functional potential between Microcystis and members of its microbiome, further supporting the interactome concept. However, we still lack verification of general interaction and know little about the taxa and metabolic pathways supporting nutrient and metabolite cycling within Microcystis aggregates. RESULTS: During a 7-month study of bacterial communities comparing free-living and aggregate-associated bacteria in Lake Taihu, China, we found that aerobic anoxygenic phototrophic (AAP) bacteria were significantly more abundant within Microcystis aggregates than in free-living samples, suggesting a possible functional role for AAP bacteria in overall aggregate community function. We then analyzed gene composition in 102 high-quality metagenome-assembled genomes (MAGs) of bloom-microbiome bacteria from 10 lakes spanning four continents, compared with 12 complete Microcystis genomes which revealed that microbiome bacteria and Microcystis possessed complementary biochemical pathways that could serve in C, N, S, and P cycling. Mapping published transcripts from Microcystis blooms onto a comprehensive AAP and non-AAP bacteria MAG database (226 MAGs) indicated that observed high levels of expression of genes involved in nutrient cycling pathways were in AAP bacteria. CONCLUSIONS: Our results provide strong corroboration of the hypothesized Microcystis interactome and the first evidence that AAP bacteria may play an important role in nutrient cycling within Microcystis aggregate microbiomes. Video Abstract.

Establishment and partial characterisation of a new cell line derived from adult tissues of the tsetse fly Glossina morsitans morsitans.

BACKGROUND: Insect cell lines play a vital role in many aspects of research on disease vectors and agricultural pests. The tsetse fly Glossina morsitans morsitans is an important vector of salivarian trypanosomes in sub-Saharan Africa and, as such, is a major constraint on human health and agricultural development in the region. METHODS: Here, we report establishment and partial characterisation of a cell line, GMA/LULS61, derived from tissues of adult female G. m. morsitans. GMA/LULS61 cells, grown at 28 °C in L-15 (Leibovitz) medium supplemented with foetal bovine serum and tryptose phosphate broth, have been taken through 23 passages to date and can be split 1:1 at 2-week intervals. Karyotyping at passage 17 revealed a predominantly haploid chromosome complement. Species origin and absence of contaminating bacteria were confirmed by PCR amplification and sequencing of fragments of the COI gene and pan-bacterial 16S rRNA gene respectively. However, PCR screening of RNA extracted from GMA/LULS61 cells confirmed presence of the recently described Glossina morsitans morsitans iflavirus and Glossina morsitans morsitans negevirus, but absence of Glossina pallipides salivary gland hypertrophy virus. GMA/LULS61 cells supported infection and growth of 6/7 different insect-derived strains of the intracellular bacterial symbiont Wolbachia. CONCLUSIONS: The GMA/LULS61 cell line has potential for application in a variety of studies investigating the biology of G. m. morsitans and its associated pathogenic and symbiotic microorganisms.

Gut microbial ß-glucuronidases influence endobiotic homeostasis and are modulated by diverse therapeutics.

Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial ß-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation. We demonstrate that a range of FDA-approved drugs prevent this reactivation by intercepting the catalytic cycle of the enzymes in a conserved fashion. Finally, we find that inhibiting GUS in conventional mice reduces free serotonin and increases its inactive glucuronide in the serum and intestines. Our results illuminate the indispensability of gut microbial enzymes in sustaining endobiotic homeostasis and indicate that therapeutic disruptions of this metabolism promote interindividual response variabilities.

Heart Transplantation for Uhl Anomaly in an Adult.

Uhl anomaly is characterized by the morphologic absence of right ventricular myocardium and is an exceedingly rare cause of nonischemic cardiomyopathy. We report the first case of a successful heart transplantation in a 41-year-old patient who presented in cardiogenic shock from Uhl anomaly causing decompensated right ventricular failure.

Prognostic Significance of Immune Cell Infiltration in Muscle-invasive Bladder Cancer Treated with Definitive Chemoradiation: A Secondary Analysis of RTOG 0524 and RTOG 0712.

Chemoradiation therapy (CRT) is a treatment for muscle-invasive bladder cancer (MIBC). Using a novel transcriptomic profiling panel, we validated prognostic immune biomarkers to CRT using 70 pretreatment tumor samples from prospective trials of MIBC (NRG/RTOG 0524 and 0712). Disease-free survival (DFS) and overall survival (OS) were estimated via the Kaplan-Meier method and stratified by genes correlated with immune cell activation. Cox proportional-hazards models were used to assess group differences. Clustering of gene expression profiles revealed that the cluster with high immune cell content was associated with longer DFS (hazard ratio [HR] 0.53, 95% confidence interval [CI] 0.26-1.10; p = 0.071) and OS (HR 0.48, 95% CI 0.24-0.97; p = 0.040) than the cluster with low immune cell content. Higher expression of T-cell infiltration genes (CD8A and ICOS) was associated with longer DFS (HR 0.40, 95% CI 0.21-0.75; p = 0.005) and OS (HR 0.49, 95% CI 0.25-0.94; p = 0.033). Higher IDO1 expression (IFNγ signature) was also associated with longer DFS (HR 0.44, 95% CI 0.24-0.88; p = 0.021) and OS (HR 0.49, 95% CI 0.24-0.99; p = 0.048). These findings should be validated in prospective CRT trials that include biomarkers, particularly for trials incorporating immunotherapy for MIBC. PATIENT SUMMARY: We analyzed patient samples from two clinical trials (NRG/RTOG 0524 and 0712) of chemoradiation for muscle-invasive bladder cancer using a novel method to assess immune cells in the tumor microenvironment. Higher expression of genes associated with immune activation and high overall immune-cell content were associated with better disease-free survival and overall survival for patients treated with chemoradiation.

Prevalence of opioid dependence in Scotland 2015-2020: A multi-parameter estimation of prevalence (MPEP) study.

BACKGROUND AND AIMS: Drug-related deaths in Scotland more than doubled between 2011 and 2020. To inform policymakers and understand drivers of this increase, we estimated the number of people with opioid dependence aged 15-64 from 2014/15 to 2019/20. DESIGN: We fitted a Bayesian multi-parameter estimation of prevalence (MPEP) model, using adverse event rates to estimate prevalence of opioid dependence jointly from Opioid Agonist Therapy (OAT), opioid-related mortality and hospital admissions data. Estimates are stratified by age group, sex and year. SETTING: Scotland, 2014/15 to 2019/20. PARTICIPANTS: People with opioid dependence and potential to benefit from OAT, whether ever treated or not. Using data from the Scottish Public Health Drug Linkage Programme, we identified a baseline cohort of individuals who had received OAT within the last 5 years, and all opioid-related deaths and hospital admissions (whether among or outside of this cohort). MEASUREMENTS: Rates of each adverse event type and (unobserved) prevalence were jointly modelled. FINDINGS: The estimated number and prevalence of people with opioid dependence in Scotland in 2019/20 was 47 100 (95% Credible Interval [CrI] 45 700 to 48 600) and 1.32% (95% CrI 1.28% to 1.37%). Of these, 61% received OAT during 2019/20. Prevalence in Greater Glasgow and Clyde was estimated as 1.77% (95% CrI 1.69% to 1.85%). There was weak evidence that overall prevalence fell slightly from 2014/15 (change -0.07%, 95% CrI -0.14% to 0.00%). The population of people with opioid dependence is ageing, with the estimated number of people aged 15-34 reducing by 5100 (95% CrI 3800 to 6400) and number aged 50-64 increasing by 2800 (95% CrI 2100 to 3500) between 2014/15 and 2019/20. CONCLUSIONS: The prevalence of opioid dependence in Scotland remained high but was relatively stable, with only weak evidence of a small reduction, between 2014/15 and 2019/20. Increased numbers of opioid-related deaths can be attributed to increased risk among people with opioid dependence, rather than increasing prevalence.

The changing metabolic landscape of bile acids - keys to metabolism and immune regulation.

Bile acids regulate nutrient absorption and mitochondrial function, they establish and maintain gut microbial community composition and mediate inflammation, and they serve as signalling molecules that regulate appetite and energy homeostasis. The observation that there are hundreds of bile acids, especially many amidated bile acids, necessitates a revision of many of the classical descriptions of bile acids and bile acid enzyme functions. For example, bile salt hydrolases also have transferase activity. There are now hundreds of known modifications to bile acids and thousands of bile acid-associated genes, especially when including the microbiome, distributed throughout the human body (for example, there are >2,400 bile salt hydrolases alone). The fact that so much of our genetic and small-molecule repertoire, in both amount and diversity, is dedicated to bile acid function highlights the centrality of bile acids as key regulators of metabolism and immune homeostasis, which is, in large part, communicated via the gut microbiome.