An exploratory study investigating the impact of the bladder tumor microbiome on Bacillus Calmette Guerin (BCG) response in non-muscle invasive bladder cancer.

PURPOSE: Intravesical Bacillus Calmette-Guerin (BCG) is standard of care for intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC). The effect of the bladder microbiome on response to BCG is unclear. We sought to characterize the microbiome of bladder tumors in BCG-responders and non-responders and identify potential mechanisms that drive treatment response. MATERIALS AND METHODS: Patients with archival pre-treatment biopsy samples (2012-2018) were identified retrospectively. Prospectively, urine and fresh tumor samples were collected from individuals with high-risk NMIBC (2020-2023). BCG response was defined as tumor-free 2 years from induction therapy. Extracted DNA was sequenced for 16S rRNA and shotgun metagenomics. Primary outcomes were species richness (α-diversity) and microbial composition (ß-diversity). Paired t-tests were performed for α-diversity (Observed species/Margalef). Statistical analysis for ß-diversity (weighted and unweighted UniFrac distances, weighted Bray-Curtis dissimilarity) were conducted through Permanova, with 999 permutations. RESULTS: Microbial species richness (P < 0.001) and composition (P = 0.001) differed between BCG responders and non-responders. Lactobacillus spp. were significantly enriched in BCG-responders. Shotgun metagenomics identified possible mechanistic pathways such as assimilatory sulfate reduction. CONCLUSION: A compositional difference exists in the tumor microbiome of BCG responders and non-responders with Lactobacillus having increased abundance in BCG responders.

Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA.

Inherited and germ-line de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA-origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres-suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at a replication fork, followed by integration via homologous recombination, generates inverted interstitial triplications.

Transperineal Prostate Biopsy is Associated With Lower Tissue Core Pathogen Burden Relative to Transrectal Biopsy: Mechanistic Underpinnings for Lower Infection Risk in the Transperineal Approach.

OBJECTIVE: To understand the mechanistic basis for reduced infectious complications in transperineal (TP) prostate biopsy, we sought to determine whether TP prostate biopsy is associated with a lower degree of pathogen introduction into the prostate relative to transrectal (TR) biopsy. MATERIALS AND METHODS: In men scheduled for prostate biopsy for standard clinical indications, rectal and perineal skin swabs, and 2 extra biopsy cores, were obtained. Specimens underwent DNA extraction followed by next-generation sequencing and standard laboratory culture. Microbial quantity and composition were determined and compared between prostate core biopsy tissue from individuals who underwent TP vs TR biopsy. RESULTS: Twenty-three men were accrued to the study. Biopsy core tissue from the TP group had less microbial diversity (15.0 vs 25.8 phylogenetic clades/sample, P = .0004) and had a lower quantity of known pathogens (36.3 vs 104.2 normalized counts of pathogens/sample, P = .018) relative to the TR group. TP group tissue core flora was more attributable to the perineal than rectal source (P = .047). Viable Escherichia coli was isolated from 45% of the TR group cores, but none in the TP group (P = .014). CONCLUSION: Biopsy tissue from individuals who undergo TP biopsy harbors a lower human pathogenic bacterial load than those who undergo TR biopsy, with a minimal risk of viable E. coli. Our results elucidate a possible mechanism for reduced infectious risk associated with TP biopsy relative to TR biopsy and a rational basis for widespread implementation of TP biopsy.

Tranexamic acid in patients with complex stones undergoing percutaneous nephrolithotomy: a randomised, double-blinded, placebo-controlled trial.

OBJECTIVES: To assess the efficacy and safety of single-dose tranexamic acid on the blood transfusion rate and outcomes of patients with complex kidney stones undergoing percutaneous nephrolithotomy (PCNL). PATIENTS AND METHODS: In a randomised, double-blinded, placebo-controlled trial, 192 patients with complex kidney stone (Guy's Stone Scores III-IV) were prospectively enrolled and randomised (1:1 ratio) to receive either one dose of tranexamic acid (1 g) or a placebo at the time of anaesthetic induction for PCNL. The primary outcome measure was the occurrence rate of perioperative blood transfusion. The secondary outcome measures included blood loss, operative time, stone-free rate (SFR), and complications. ClinicalTrials.gov identifier: NCT02966236. RESULTS: The overall risk of receiving a blood transfusion was reduced in the tranexamic acid group (2.2% vs 10.4%; relative risk, 0.21, 95% confidence interval [CI] 0.03-0.76, P = 0.033; number-needed-to-treat: 12). Patients randomised to the tranexamic acid group had a higher immediate and 3-month SFR compared with those in the placebo group (29% vs 14.7%, odds ratio [OR] 2.37, 95% CI 1.15-4.87, P = 0.019, and 46.2% vs 28.1%, OR 2.20, 95% CI 1.20-4.02, P = 0.011, respectively). Faster haemoglobin recovery occurred in patients in the tranexamic acid group (mean, 21.3 days; P = 0.001). No statistical differences were found in operative time and complications between groups. CONCLUSIONS: Tranexamic acid administration is safe and reduces the need for blood transfusion by five-times in patients with complex kidney stones undergoing PCNL. Moreover, tranexamic acid may contribute to better stone clearance rate and faster haemoglobin recovery without increasing complications. A single dose of tranexamic acid at the time of anaesthetic induction could be considered standard clinical practice for patients with complex kidney stones undergoing PCNL.

Multiplexing mutation rate assessment: determining pathogenicity of Msh2 variants in Saccharomyces cerevisiae.

Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time-consuming and tedious. Established methods such as fluctuation tests and mutation accumulation experiments are low-throughput and often require significant optimization to ensure accuracy. We established a new method to determine the mutation rate of many strains simultaneously by tracking mutation events in a chemostat continuous culture device and applying deep sequencing to link mutations to alleles of a DNA-repair gene. We applied this method to assay the mutation rate of hundreds of Saccharomyces cerevisiae strains carrying mutations in the gene encoding Msh2, a DNA repair enzyme in the mismatch repair pathway. Loss-of-function mutations in MSH2 are associated with hereditary nonpolyposis colorectal cancer, an inherited disorder that increases risk for many different cancers. However, the vast majority of MSH2 variants found in human populations have insufficient evidence to be classified as either pathogenic or benign. We first benchmarked our method against Luria-Delbrück fluctuation tests using a collection of published MSH2 missense variants. Our pooled screen successfully identified previously characterized nonfunctional alleles as high mutators. We then created an additional 185 human missense variants in the yeast ortholog, including both characterized and uncharacterized alleles curated from ClinVar and other clinical testing data. In a set of alleles of known pathogenicity, our assay recapitulated ClinVar's classification; we then estimated pathogenicity for 157 variants classified as uncertain or conflicting reports of significance. This method is capable of studying the mutation rate of many microbial species and can be applied to problems ranging from the generation of high-fidelity polymerases to measuring the frequency of antibiotic resistance emergence.

Intestinal Epithelial Cell-Derived LKB1 Suppresses Colitogenic Microbiota.

Dysregulation of the immune barrier function of the intestinal epithelium can often result in dysbiosis. In this study we report a novel role of intestinal epithelial cell (IEC)-derived liver kinase B1 (LKB1) in suppressing colitogenic microbiota. IEC-specific deletion of LKB1 (LKB1ΔIEC) resulted in an increased susceptibility to dextran sodium sulfate (DSS)-induced colitis and a definitive shift in the composition of the microbial population in the mouse intestine. Importantly, transfer of the microbiota from LKB1ΔIEC mice was sufficient to confer increased susceptibility to DSS-induced colitis in wild-type recipient mice. Collectively, the data indicate that LKB1 deficiency in intestinal epithelial cells nurtures the outgrowth of colitogenic bacteria in the commensal community. In addition, LKB1 deficiency in the intestinal epithelium reduced the production of IL-18 and antimicrobial peptides in the colon. Administration of exogenous IL-18 restored the expression of antimicrobial peptides, corrected the outgrowth of several bacterial genera, and rescued the LKB1ΔIEC mice from increased sensitivity to DSS challenge. Taken together, our study reveals an important function of LKB1 in IECs for suppressing colitogenic microbiota by IL-18 expression.

LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection.

Although genetic polymorphisms in the LRRK2 gene are associated with a variety of diseases, the physiological function of LRRK2 remains poorly understood. In this study, we report a crucial role for LRRK2 in the activation of the NLRC4 inflammasome during host defense against Salmonella enteric serovar Typhimurium infection. LRRK2 deficiency reduced caspase-1 activation and IL-1ß secretion in response to NLRC4 inflammasome activators in macrophages. Lrrk2-/- mice exhibited impaired clearance of pathogens after acute S. Typhimurium infection. Mechanistically, LRRK2 formed a complex with NLRC4 in the macrophages, and the formation of the LRRK2-NLRC4 complex led to the phosphorylation of NLRC4 at Ser533. Importantly, the kinase activity of LRRK2 is required for optimal NLRC4 inflammasome activation. Collectively, our study reveals an important role for LRRK2 in the host defense by promoting NLRC4 inflammasome activation.

Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes.

Aspirin overutilization for the primary prevention of cardiovascular disease.

BACKGROUND: Aspirin is commonly used for the primary prevention of cardiovascular disease (CVD) in the US. Previous research has observed significant levels of inappropriate aspirin use for primary CVD prevention in some European populations, but the degree to which aspirin is overutilized in the US remains unknown. This study examined the association between regular aspirin use and demographic/clinical factors in a population-based sample of adults without a clinical indication for aspirin for primary prevention. METHODS: A cross-sectional analysis was performed using 2010-2012 data from individuals aged 30-79 years in the Marshfield Epidemiologic Study Area (WI, USA). Regular aspirin users included those who took aspirin at least every other day. RESULTS: There were 16,922 individuals who were not clinically indicated for aspirin therapy for primary CVD prevention. Of these, 19% were regular aspirin users. In the final adjusted model, participants who were older, male, lived in northern Wisconsin, had more frequent medical visits, and had greater body mass index had significantly higher odds of regular aspirin use (P<0.001 for all). Race/ethnicity, health insurance, smoking, blood pressure, and lipid levels had negligible influence on aspirin use. A sensitivity analysis found a significant interaction between age and number of medical visits, indicating progressively more aspirin use in older age groups who visited their provider frequently. CONCLUSION: There was evidence of aspirin overutilization in this US population without CVD. Older age and more frequent provider visits were the strongest predictors of inappropriate aspirin use. Obesity was the only significant clinical factor, suggesting misalignment between perceived aspirin benefits and cardiovascular risks in this subgroup of patients. Prospective studies that examine cardiac and bleeding events associated with regular aspirin use among obese samples (without CVD) are needed to refine clinical guidelines in this area.

Regional epidemiologic assessment of prevalent periodontitis using an electronic health record system.

An oral health surveillance platform that queries a clinical/administrative data warehouse was applied to estimate regional prevalence of periodontitis. Cross-sectional analysis of electronic health record data collected between January 1, 2006, and December 31, 2010, was undertaken in a population sample residing in Ladysmith, Wisconsin. Eligibility criteria included: 1) residence in defined zip codes, 2) age 25-64 years, and 3) ≥1 Marshfield dental clinic comprehensive examination. Prevalence was established using 2 independent methods: 1) via an algorithm that considered clinical attachment loss and probe depth and 2) via standardized Current Dental Terminology (CDT) codes related to periodontal treatment. Prevalence estimates were age-standardized to 2000 US Census estimates. Inclusion criteria were met by 2,056 persons. On the basis of the American Academy of Periodontology/Centers for Disease Control and Prevention method, the age-standardized prevalence of moderate or severe periodontitis (combined) was 407 per 1,000 males and 308 per 1,000 females (348/1,000 males and 269/1,000 females using the CDT code method). Increased prevalence and severity of periodontitis was noted with increasing age. Local prevalence of periodontitis was consistent with national estimates. The need to address potential sample selection bias in future electronic health record-based periodontitis research was identified by this approach. Methods outlined herein may be applied to refine oral health surveillance systems, inform dental epidemiologic methods, and evaluate interventional outcomes.

Insights into Migration and Development of Coral Black Band Disease Based on Fine Structure Analysis

In many diverse ecosystems, ranging from natural surfaces in aquatic ecosystems to the mammalian gut and medical implants, bacterial populations and communities exist as biofilms. While the process of biofilm development has been well-studied for those produced by unicellular bacteria such Pseudomonas aeruginosa, little is known about biofilm development associated with filamentous microorganisms. Black band disease (BBD) of corals is characterized as a polymicrobial biofilm (mat) community, visually-dominated by filamentous cyanobacteria. The mat migrates across a living coral host, completely lysing coral tissue and leaving behind exposed coral skeleton. It is the only known cyanobacterial biofilm that migrates across a substratum, thus eliciting questions about the mechanisms and unique characteristics of this system. Fragments of the coral Montastraea annularis, five artificially infected with BBD and two collected from a naturally BBD-infected colony, were used to address these questions by detailed examination using scanning and transmission electron microscopy (SEM and TEM). In areas close to the interface of coral tissue and the mature disease band two types of clusters of cyanobacteria were observed, one with random orientation and one with parallel orientation of filaments. The latter exhibited active secretion of extracellular polysaccharide (EPS) while the randomly oriented clusters did not. Within the well developed band cyanobacterial filaments were observed to be embedded in EPS and were present as layers of filaments in parallel orientation. These observations suggest that BBD cyanobacteria orient themselves and produce EPS in a sequential process during migration to form the complex BBD matrix.

En muchos ecosistemas diversos, que van desde ecosistemas acuáticos hasta los intestinos de mamíferos e implantes médicos, las poblaciones y comunidades de bacterias existen como biopelículas (biofilms). El proceso de desarrollo de las biopelículas ha sido bien estudiado para aquellos producidos por bacterias unicelulares como Pseudomonas aeruginosa, pero se conoce muy poco acerca del desarrollo de biopelículas asociadas con microorganismos filamentosos. La Enfermedad de Banda Negra (EBN) de coral es caracterizada como una comunidad polimicrobiana que forma una biopelícula (lecho), visualmente-dominada por una cianobacteria filamentosa. El lecho migra a través de un huésped de coral vivo, rompiendo completamente el tejido del coral y dejando atrás el esqueleto de coral expuesto. Es la única biopelícula cianobacteriana que migra a través de un sustrato, por lo tanto esto genera preguntas acerca de los mecanismos y las características únicas de este sistema. Fragmentos del coral Montastraea annularis, cinco artificialmente infectados con EBN y dos colectados de una colonia EBN-infectada, fueron usados para abordar estas preguntas mediante exámenes detallados con microscopía electrónica de barrido y de transmisión (MEB y MET). En zonas cercanas a la interfaz de tejido del coral y la banda de la enfermedad madura, se han observado dos tipos de grupos de cianobacterias, uno con orientación aleatoria y otro con una orientación paralela de los filamentos. Este último exhibe la secreción activa de polisacáridos extra-celulares (PEC), mientras que los grupos orientados al azar no lo hicieron. Dentro de la banda de filamentos cianobacterianas bien desarrollados se observó que estaban integradas en PEC y que se presentaban como capas de cianobacteria con orientación paralela. Estas observaciones sugieren que la cianobacteria de EBN se orienta a sí misma y produce PEC en un proceso secuencial durante la migración para formar la matriz complejo de EBN.