Somatic Mosaicism in Brain Disorders.

Research efforts over the past decade have defined the genetic landscape of somatic variation in the brain. Neurons accumulate somatic mutations from development through aging with potentially profound functional consequences. Recent studies have revealed the contribution of somatic mosaicism to various brain disorders including focal epilepsy, neuropsychiatric disease, and neurodegeneration. One notable finding is that the effect of somatic mosaicism on clinical outcomes can vary depending on contextual factors, such as the developmental origin of a variant or the number and type of cells affected. In this review, we highlight current knowledge regarding the role of somatic mosaicism in brain disorders and how biological context can mediate phenotypes. First, we identify the origins of brain somatic variation throughout the lifespan of an individual. Second, we explore recent discoveries that suggest somatic mosaicism contributes to various brain disorders. Finally, we discuss neuropathological associations of brain mosaicism in different biological contexts and potential clinical utility.

Noninflammatory 97-amino acid High Mobility Group Box 1 derived polypeptide disrupts and prevents diverse biofilms.

BACKGROUND: Bacterial biofilm communities are embedded in a protective extracellular matrix comprised of various components, with its' integrity largely owed to a 3-dimensional lattice of extracellular DNA (eDNA) interconnected by Holliday Junction (HJ)-like structures and stabilised by the ubiquitous eubacterial DNABII family of DNA-binding architectural proteins. We recently showed that the host innate immune effector High Mobility Group Box 1 (HMGB1) protein possesses extracellular anti-biofilm activity by destabilising these HJ-like structures, resulting in release of biofilm-resident bacteria into a vulnerable state. Herein, we showed that HMGB1's anti-biofilm activity was completely contained within a contiguous 97 amino acid region that retained DNA-binding activity, called 'mB Box-97'. METHODS: We engineered a synthetic version of this 97-mer and introduced a single amino acid change which lacked any post-translational modifications, and tested its activity independently and in combination with a humanised monoclonal antibody that disrupts biofilms by the distinct mechanism of DNABII protein sequestration. FINDINGS: mB Box-97 disrupted and prevented biofilms, including those formed by the ESKAPEE pathogens, and importantly reduced measurable proinflammatory activity normally associated with HMGB1 in a murine lung infection model. INTERPRETATION: Herein, we discuss the value of targeting the ubiquitous eDNA-dependent matrix of biofilms via mB Box-97 used singly or in a dual host-augmenting/pathogen-targeted cocktail to resolve bacterial biofilm infections. FUNDING: This work was supported by NIH/NIDCD R01DC011818 to L.O.B. and S.D.G. and NIH/NIAID R01AI155501 to S.D.G.

Stress and depression-associated shifts in gut microbiota: A pilot study of human pregnancy.

Background: Psychosocial stress and mood-related disorders, such as depression, are prevalent and vulnerability to these conditions is heightened during pregnancy. Psychosocial stress induces consequences via several mechanisms including the gut microbiota-brain axis and associated signaling pathways. Previous preclinical work indicates that prenatal stress alters maternal gut microbial composition and impairs offspring development. Importantly, although the fecal and vaginal microenvironments undergo alterations across pregnancy, we lack consensus regarding which shifts are adaptive or maladaptive in the presence of prenatal stress and depression. Clinical studies interrogating these relationships have identified unique taxa but have been limited in study design. Methods: We conducted a prospective cohort study of pregnant individuals consisting of repeated administration of psychometrics (Perceived Stress Scale (PSS) and Center for Epidemiological Studies Depression Scale (CES-D)) and collection of fecal and vaginal microbiome samples. Fecal and vaginal microbial community composition across psychometric responses were interrogated using full-length 16S rRNA sequencing followed by α and ß-diversity metrics and taxonomic abundance. Results: Early pregnancy stress was associated with increased abundance of fecal taxa not previously identified in related studies, and stress from late pregnancy through postpartum was associated with increased abundance of typical vaginal taxa and opportunistic pathogens in the fecal microenvironment. Additionally, in late pregnancy, maternal stress and depression scores were associated with each other and with elevated maternal C-C motif chemokine ligand 2 (CCL2) concentrations. At delivery, concordant with previous literature, umbilical CCL2 concentration was negatively correlated with relative abundance of maternal fecal Lactobacilli. Lastly, participants with more severe depressive symptoms experienced steeper decreases in prenatal vaginal α-diversity. Conclusion: These findings a) underscore previous preclinical and clinical research demonstrating the effects of prenatal stress on maternal microbiome composition, b) suggest distinct biological pathways for the consequences of stress versus depression and c) extend the literature by identifying several taxa which may serve critical roles in mediating this relationship. Thus, further interrogation of the role of specific maternal microbial taxa in relation to psychosocial stress and its sequelae is warranted.

Trends in Opioid Misuse Among Individuals Aged 12 to 21 Years in the US.

Importance: Although opioid misuse has been decreasing among US youths and adolescents in recent years, it is unclear what has contributed to this trend and how this trend differs by age group and sex over time. Objective: To identify trends in opioid misuse among youths and young adults across and between ages, birth cohorts, and sexes. Design, Setting, and Participants: Cross-sectional National Survey on Drug Use and Health (NSDUH) public-use files were used to produce nationally representative pseudocohorts. The survey population includes the civilian US population in the 50 states and Washington, DC. Individuals without a fixed address and institutionalized individuals were excluded. Respondents to the NSDUH are a population-based sample selected using a stratified cluster design. For the years (January 1, 2002, to December 31, 2019) and ages (12-21 years) analyzed, the sample sizes ranged from 1607 to 3239 respondents. Data were analyzed from January 1, 2022, to April 12, 2023, for the main outcome by age, sex, and pseudocohort. Main Outcomes and Measures: Respondents were asked whether they misused prescription opioids or used heroin in the past year. The analysis hypotheses were formulated and tested after data collection. Results: In a total of 5 pseudocohorts, data from 114 412 respondents aged 12 to 21 years were analyzed; the unweighted distribution of male sex (complement was female) ranged from 47.7% to 52.6% (mean [SD], 50.6% [1.1%]). Response rates ranged from 45.8% to 71.3%. High school-aged youths and young adults had distinctly lower rates of opioid misuse in later pseudocohorts compared with earlier ones. Rates of misuse among individuals aged 16 years were 2.80% (95% CI, 1.06%-4.54%) higher in 2002 vs 2008; among those aged 18 years, rates were 4.36% (95% CI, 1.85%-6.87%) higher in 2002. Similarly, rates of misuse among individuals aged 16 years were 3.93% (95% CI, 2.15%-5.71%) higher in 2008 vs 2014; among those aged 17 years, rates were 3.41% (95% CI, 1.94%-4.88%) higher in 2008. Similar patterns were observed by sex. In earlier cohorts, younger female participants had higher rates of opioid misuse than their male counterparts and older male participants had higher rates than their female counterparts. Sex differences decreased in later cohorts. Conclusions and Results: The findings of this cross-sectional study of US youths and young adults suggest that high school-aged individuals consistently misused fewer opioids in later pseudocohorts overall and by sex. Sex differences in opioid rates also diminished in later pseudocohorts. A decrease in drug availability and general exposure to the harms of opioid use could be contributing to these findings. Future planned research using this pseudocohort approach will examine polysubstance use and evaluate how substance use differs by other sociodemographic characteristics.

Maternal anxiety, depression and stress affects offspring gut microbiome diversity and bifidobacterial abundances.

Uncovering mechanisms underlying fetal programming during pregnancy is of critical importance. Atypical neurodevelopment during the pre- and immediate postnatal period has been associated with long-term adverse health outcomes, including mood disorders and aberrant cognitive ability in offspring. Maternal factors that have been implicated in anomalous offspring development include maternal inflammation and tress, anxiety, and depression. One potential mechanism through which these factors perturb normal offspring postnatal development is through microbiome disruption. The mother is a primary source of early postnatal microbiome seeding for the offspring, and the transference of a healthy microbiome is key in normal neurodevelopment. Since psychological stress, mood disorders, and inflammation have all been implicated in altering maternal microbiome community structure, passing on aberrant microbial communities to the offspring that may then affect developmental outcomes. Therefore, we examined how maternal stress, anxiety and depression assessed with standardized instruments, and maternal inflammatory cytokine levels in the pre- and postnatal period are associated with the offspring microbiome within the first 13 months of life, utilizing full length 16S sequencing on infant stool samples, that allowed for species-level resolution. Results revealed that infants of mothers who reported higher anxiety and perceived stress had reduced alpha diversity. Additionally, the relative taxonomic quantitative abundances of Bifidobacterium dentium and other species that have been associated with either modulation of the gut-brain axis, or other beneficial health outcomes, were reduced in the offspring of mothers with higher anxiety, perceived stress, and depression. We also found associations between bifidobacteria and prenatal maternal pro-inflammatory cytokines IL-6, IL-8, and IL-10. In summary, specific microbial taxa involved in maintaining proper brain and immune function are lower in offspring born to mothers with anxiety, depression, or stress, providing strong evidence for a mechanism by which maternal factors may affect offspring health through microbiota dysregulation.

National polydrug use patterns among people who misuse prescription opioids and people who use heroin. Results from the National Household Survey on Drug Use and Health.

BACKGROUND: Polysubstance use among people who misuse opioids (PWMO) is highly prevalent, but understudied. We defined, estimated, and analyzed national polysubstance use patterns among PWMO using National Household Survey on Drug Use and Health data (2017-2019). METHODS: We obtained estimates of past-month patterns of polydrug use using cluster analysis and latent class/profile analysis. We considered misuse of prescription opioids and use of heroin, cocaine (including crack), marijuana, alcohol, and "other" substances. RESULTS: We identified a five-cluster solution for binary indicators of past-month use and a six-cluster solution for frequency of use. The largest binary cluster (37%) included misuse of prescription opioids and use of alcohol. The second-largest cluster (15%) included misuse of prescription opioids, alcohol, marijuana, and "other" substances. Among those who used heroin, 36% used methamphetamine. In terms of frequency of use, the largest cluster among people who misuse opioid who used multiple substances (almost 40%) misused prescription pain relievers, alcohol, and marijuana infrequently. The second-largest cluster (23%) used marijuana almost daily and misused prescription pain relievers an average of 6.6 days. PWMO in a cluster of almost daily heroin use indicated use of methamphetamine, marijuana, and prescription opioids. Those who used methamphetamine, were using it more than 15 days a month. CONCLUSIONS: We have developed reference measures of polydrug patterns among US household population and estimated their demographic characteristics. We identified clusters of high-risk polydrug use. These findings have implications for the development of prevention and treatment solutions in the United States.

CRITICAL WINDOW VARIABLE SELECTION FOR MIXTURES: ESTIMATING THE IMPACT OF MULTIPLE AIR POLLUTANTS ON STILLBIRTH.

Understanding the role of time-varying pollution mixtures on human health is critical as people are simultaneously exposed to multiple pollutants during their lives. For vulnerable subpopulations who have well-defined exposure periods (e.g., pregnant women), questions regarding critical windows of exposure to these mixtures are important for mitigating harm. We extend critical window variable selection (CWVS) to the multipollutant setting by introducing CWVS for mixtures (CWVSmix), a hierarchical Bayesian method that combines smoothed variable selection and temporally correlated weight parameters to: (i) identify critical windows of exposure to mixtures of time-varying pollutants, (ii) estimate the time-varying relative importance of each individual pollutant and their first order interactions within the mixture, and (iii) quantify the impact of the mixtures on health. Through simulation we show that CWVSmix offers the best balance of performance in each of these categories in comparison to competing methods. Using these approaches, we investigate the impact of exposure to multiple ambient air pollutants on the risk of stillbirth in New Jersey, 2005-2014. We find consistent elevated risk in gestational weeks 2, 16-17, and 20 for non-Hispanic Black mothers, with pollution mixtures dominated by ammonium (weeks 2, 17, 20), nitrate (weeks 2, 17), nitrogen oxides (weeks 2, 16), PM2.5 (week 2), and sulfate (week 20). The method is available in the R package CWVSmix.

Antibacterial and anti-inflammatory effects of Lactobacillus reuteri in its biofilm state contribute to its beneficial effects in a rat model of experimental necrotizing enterocolitis.

INTRODUCTION: Necrotizing enterocolitis (NEC) remains a significant surgical emergency in neonates. We have demonstrated the efficacy of Lactobacillus reuteri (Lr) in protecting against experimental NEC when administered as a biofilm by incubation with maltose loaded dextranomer microspheres. Lr possesses antimicrobial and anti-inflammatory properties. We developed mutant strains of Lr to examine the importance of its antimicrobial and anti-inflammatory properties in protecting the intestines from NEC. METHODS: Premature rat pups were exposed to hypoxia/hypothermia/hypertonic feeds to induce NEC. To examine the importance of antimicrobial reuterin and anti-inflammatory histamine, pups received either native or mutant forms of Lr, in either its planktonic or biofilm states, prior to induction of NEC. Intestinal histology was examined upon sacrifice. RESULTS: Compared to no treatment, administration of a single dose of Lr in its biofilm state significantly decreased the incidence of NEC (67% vs. 18%, p < 0.0001), whereas Lr in its planktonic state had no significant effect. Administration of reuterin-deficient or histamine-deficient forms of Lr, in either planktonic or biofilm states, resulted in significant loss of efficacy. CONCLUSION: Antimicrobial and anti-inflammatory effects of Lr contribute to its beneficial effects against NEC. This suggests that both infectious and inflammatory components contribute to the etiology of NEC.

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

The extracellular innate-immune effector HMGB1 limits pathogenic bacterial biofilm proliferation.

Herein, we describe an extracellular function of the vertebrate high-mobility group box 1 protein (HMGB1) in the proliferation of bacterial biofilms. Within host cells, HMGB1 functions as a DNA architectural protein, similar to the ubiquitous DNABII family of bacterial proteins; despite that, these proteins share no amino acid sequence identity. Extracellularly, HMGB1 induces a proinflammatory immune response, whereas the DNABII proteins stabilize the extracellular DNA-dependent matrix that maintains bacterial biofilms. We showed that when both proteins converged on extracellular DNA within bacterial biofilms, HMGB1, unlike the DNABII proteins, disrupted biofilms both in vitro (including the high-priority ESKAPEE pathogens) and in vivo in 2 distinct animal models, albeit with induction of a strong inflammatory response that we attenuated by a single engineered amino acid change. We propose a model where extracellular HMGB1 balances the degree of induced inflammation and biofilm containment without excessive release of biofilm-resident bacteria.

Polyethylene Glycol 3350 Changes Stool Consistency and the Microbiome but not Behavior of CD1 Mice.

OBJECTIVES: Polyethylene Glycol 3350 (PEG3350) is a laxative commonly used to treat constipation in children. The Food and Drug Administration has received reports of increased anxiety, aggression, and obsessive--compulsive behaviors in children administered PEG3350. Thus, we assessed whether daily administration of PEG3350 leads to anxiety-like behavior in mice. METHODS: Outbred CD-1 IGS mice were administered either a high or a low dose of PEG3350 via daily oral gavage for 2 weeks. As a laxative comparison and control, additional mice were given a high or low dose of magnesium citrate or vehicle (water). Weight and stool consistency were assessed after each gavage to determine laxative effectiveness. Anxiety-like behaviors were assessed using light/dark, open field, and elevated plus maze (EPM) tests at baseline, after 2 weeks of daily gavage, and after a 2 week washout in experiment 1, and after 2 weeks of daily gavage in experiment 2. Stool samples were collected for microbiome analysis in experiment 2 at baseline, after 2 weeks of daily gavage, and after 2 weeks washout. RESULTS: PEG3350 and magnesium citrate significantly changed stool consistency, as well as microbiome alpha and beta diversity. Anxiety-like behaviors were not, however, different in mice administered low or high doses of PEG3350 or magnesium citrate. CONCLUSIONS: Although changes in stool consistency and the gut microbiome occurred, administration of PEG3350 did not alter anxiety-like behaviors.

Lactobacillus reuteri in its biofilm state promotes neurodevelopment after experimental necrotizing enterocolitis in rats.

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature newborns with no known cure. Up to half of survivors subsequently exhibit cognitive impairment and neurodevelopmental defects. We created a novel probiotics delivery system in which the probiotic Lactobacillus reuteri (Lr) was induced to form a biofilm [Lr (biofilm)] by incubation with dextranomer microspheres loaded with maltose (Lr-DM-maltose). We have previously demonstrated that a single dose of the probiotic Lr administered in its biofilm state significantly reduces the incidence of NEC and decreases inflammatory cytokine production in an animal model of the disease. The aim of our current study was to determine whether a single dose of the probiotic Lr administered in its biofilm state protects the brain after experimental NEC. We found that rat pups exposed to NEC reached developmental milestones significantly slower than breast fed pups, with mild improvement with Lr (biofilm) treatment. Exposure to NEC had a negative effect on cognitive behavior, which was prevented by Lr (biofilm) treatment. Lr administration also reduced anxiety-like behavior in NEC-exposed rats. The behavioral effects of NEC were associated with increased numbers of activated microglia, decreased myelin basic protein (MBP), and decreased neurotrophic gene expression, which were prevented by administration of Lr (biofilm). Our data indicate early enteral treatment with Lr in its biofilm state prevented the deleterious effects of NEC on developmental impairments.

The dental plaque biofilm matrix.

The extracellular matrix is a critical component of microbial biofilms, such as dental plaque, maintaining the spatial arrangement of cells and coordinating cellular functions throughout the structure. The extracellular polymeric substances that comprise the matrix include carbohydrates, nucleic acids, proteins, and lipids, which are frequently organized into macromolecular complexes and/or are associated with the surfaces of microbial cells within the biofilm. Cariogenic dental plaque is rich in glucan and fructan polysaccharides derived from extracellular microbial metabolism of dietary sucrose. By contrast, the matrix of subgingival dental plaque is a complex mixture of macromolecules that is still not well understood. Components of the matrix escape from microbial cells during lysis by active secretion or through the shedding of vesicles and serve to anchor microbial cells to the tooth surface. By maintaining the biofilm in close association with host tissues, the matrix facilitates interactions between microorganisms and the host. The outcome of these interactions may be the maintenance of health or the development of dental disease, such as caries or periodontitis. The matrix affords microbial cells protection against chemical and physical insults and hinders the eradication of pathogenic dental plaque. Therefore, strategies to control the matrix are critical to maintain oral health. This review discusses recent advances in our understanding of the composition, origins, and function of the dental plaque matrix, with a focus on subgingival dental plaque. New strategies to control subgingival dental plaque based on targeting the biofilm matrix are also considered.

Predictive model of multiple emergency department visits among adults: analysis of the data from the National Survey of Drug Use and Health (NSDUH).

BACKGROUND: In this methodological paper, we use a novel, predictive approach to examine how demographics, substance use, mental and other health indicators predict multiple visits (≥3) to emergency departments (ED) within a year. METHODS: State-of-the-art predictive methods were used to evaluate predictive ability and factors predicting multiple visits to ED within a year and to identify factors that influenced the strength of the prediction. The analysis used public-use datasets from the 2015-2018 National Surveys on Drug Use and Health (NSDUH), which used the same questionnaire on the variables of interest. Analysis focused on adults aged ≥18 years. Several predictive models (regressions, trees, and random forests) were validated and compared on independent datasets. RESULTS: Predictive ability on a test set for multiple ED visits (≥3 times within a year) measured as the area under the receiver operating characteristic (ROC) reached 0.8, which is good for a national survey. Models revealed consistency in predictive factors across the 4 survey years. The most influential variables for predicting ≥3 ED visits per year were fair/poor self-rated health, being nervous or restless/fidgety, having a lower income, asthma, heart condition/disease, having chronic obstructive pulmonary disease (COPD), nicotine dependence, African-American race, female sex, having diabetes, and being of younger age (18-20). CONCLUSIONS: The findings reveal the need to address behavioral and mental health contributors to ED visits and reinforce the importance of developing integrated care models in primary care settings to improve mental health for medically vulnerable patients. The presented modeling approach can be broadly applied to national and other large surveys.

A novel probiotic therapeutic in a murine model of Clostridioides difficile colitis.

For prophylactic therapy, mice received an oral antibiotic cocktail followed by clindamycin injection, followed by probiotic administration (planktonic vs. biofilm state), followed by C. difficile oral gavage. For treatment therapy, mice received antibiotics and C. difficile first, followed by probiotic administration. Clinical sickness scores (CSS) and intestinal histologic injury scores (HIS) were assigned.In the Prophylactic Therapy model, CSS: 67% of untreated mice exposed to C. difficile demonstrated CSS ≥ 6, which is consistent with C. difficile infection (p< .001 compared to unexposed mice). In mice treated with planktonic Lr, 55% had a CSS ≥ 6, but only 19% of mice treated with Lr in its biofilm state had CSS ≥ 6 (p< .001). Mice receiving Lr + DM-Maltose lost the least amount of weight compared to mice receiving saline (p = .004676) or to mice receiving Lr (p= .003185). HIS: 77% of untreated mice exposed to C. difficile had HIS scores ≥4, which is consistent with C. difficile infection. In mice treated with planktonic Lr, 62% had HIS ≥4, but only 19% of mice treated with Lr in its biofilm state had HIS ≥4. (p< .001). Additionally, mice treated with Lr in its biofilm state had better survival compared to untreated mice and to mice treated with planktonic Lr (p ≤ 0.05). Similar findings for weight loss, CSS, HIS and survival were obtained for Treatment Therapy.A single dose of Lactobacillus reuteri in its biofilm state reduces the severity and incidence of experimental C. difficile infection when administered as both prophylactic and treatment therapy.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

The conserved mosaic prophage protein paratox inhibits the natural competence regulator ComR in Streptococcus.

Horizontal gene transfer is an important means of bacterial evolution. This includes natural genetic transformation, where bacterial cells become "competent" and DNA is acquired from the extracellular environment. Natural competence in many species of Streptococcus, is regulated by quorum sensing via the ComRS receptor-signal pair. The ComR-XIP (mature ComS peptide) complex induces expression of the alternative sigma factor SigX, which targets RNA polymerase to CIN-box promoters to activate genes involved in DNA uptake and recombination. In addition, the widely distributed Streptococcus prophage gene paratox (prx) also contains a CIN-box, and here we demonstrate it to be transcriptionally activated by XIP. In vitro experiments demonstrate that Prx binds ComR directly and prevents the ComR-XIP complex from interacting with DNA. Mutations of prx in vivo caused increased expression of the late competence gene ssb when induced with XIP as compared to wild-type, and Prx orthologues are able to inhibit ComR activation by XIP in a reporter strain which lacks an endogenous prx. Additionally, an X-ray crystal structure of Prx reveals a unique fold that implies a novel molecular mechanism to inhibit ComR. Overall, our results suggest Prx functions to inhibit the acquisition of new DNA by Streptococcus.

An enhanced Lactobacillus reuteri biofilm formulation that increases protection against experimental necrotizing enterocolitis.

One significant drawback of current probiotic therapy for the prevention of necrotizing enterocolitis (NEC) is the need for at least daily administration because of poor probiotic persistence after enteral administration, increasing the risk of the probiotic bacteria causing bacteremia or sepsis if the intestines are already compromised. We previously showed that the effectiveness of Lactobacillus reuteri ( Lr) in preventing NEC is enhanced when Lr is grown as a biofilm on the surface of dextranomer microspheres (DM). Here we sought to test the efficacy of Lr administration by manipulating the Lr biofilm state with the addition of biofilm-promoting substances (sucrose and maltose) to DM or by mutating the Lr gtfW gene (encoding an enzyme central to biofilm production). Using an animal model of NEC, we determined that Lr adhered to sucrose- or maltose-loaded DM significantly reduced histologic injury, improved host survival, decreased intestinal permeability, reduced intestinal inflammation, and altered the gut microbiome compared with Lr adhered to unloaded DM. These effects were abolished when DM or GtfW were absent from the Lr inoculum. This demonstrates that a single dose of Lr in its biofilm state decreases NEC incidence. Importantly, preloading DM with sucrose or maltose further enhances Lr protection against NEC in a GtfW-dependent fashion, demonstrating the tunability of the approach and the potential to use other cargos to enhance future probiotic formulations. NEW & NOTEWORTHY Previous clinical trials of probiotics to prevent necrotizing enterocolitis have had variable results. In these studies, probiotics were delivered in their planktonic, free-living form. We have developed a novel probiotic delivery system in which Lactobacillus reuteri (Lr) is delivered in its biofilm state. In a model of experimental necrotizing enterocolitis, this formulation significantly reduces intestinal inflammation and permeability, improves survival, and preserves the natural gut microflora compared with the administration of Lr in its free-living form.

Perioperative Behavioral Therapy and Pelvic Muscle Strengthening Do Not Enhance Quality of Life After Pelvic Surgery: Secondary Report of a Randomized Controlled Trial.

BACKGROUND: There is significant need for trials evaluating the long-term effectiveness of a rigorous program of perioperative behavioral therapy with pelvic floor muscle training (BPMT) in women undergoing transvaginal reconstructive surgery for prolapse. OBJECTIVE: The purpose of this study was to evaluate the effect of perioperative BPMT on health-related quality of life (HRQOL) and sexual function following vaginal surgery for pelvic organ prolapse (POP) and stress urinary incontinence (SUI). DESIGN: This study is a secondary report of a 2 × 2 factorial randomized controlled trial. SETTING: This study was a multicenter trial. PARTICIPANTS: Participants were adult women with stage 2-4 POP and SUI. INTERVENTION: Perioperative BPMT versus usual care and sacrospinous ligament fixation (SSLF) versus uterosacral ligament suspension (ULS) were provided. MEASUREMENTS: Participants undergoing transvaginal surgery (SSLF or ULS for POP and a midurethral sling for SUI) received usual care or five perioperative BPMT visits. The primary outcome was change in body image and in Pelvic Floor Impact Questionnaire (PFIQ) short-form subscale, 36-item Short-Form Health Survey (SF-36), Pelvic Organ Prolapse-Urinary Incontinence Sexual Questionnaire short form (PISQ-12), Patient Global Impression of Improvement (PGII), and Brink scores. RESULTS: The 374 participants were randomized to BPMT (n = 186) and usual care (n = 188). Outcomes were available for 137 (74%) of BPMT participants and 146 (78%) of the usual care participants at 24 months. There were no statistically significant differences between groups in PFIQ, SF-36, PGII, PISQ-12, or body image scale measures. LIMITATIONS: The clinicians providing BPMT had variable expertise. Findings might not apply to vaginal prolapse procedures without slings or abdominal apical prolapse procedures. CONCLUSIONS: Perioperative BPMT performed as an adjunct to vaginal surgery for POP and SUI provided no additional improvement in QOL or sexual function compared with usual care.

Enhanced Probiotic Potential of Lactobacillus reuteri When Delivered as a Biofilm on Dextranomer Microspheres That Contain Beneficial Cargo.

As with all orally consumed probiotics, the Gram-positive bacterium Lactobacillus reuteri encounters numerous challenges as it transits through the gastrointestinal tract of the host, including low pH, effectors of the host immune system, as well as competition with commensal and pathogenic bacteria, all of which can greatly reduce the availability of live bacteria for therapeutic purposes. Recently we showed that L. reuteri, when adhered in the form of a biofilm to a semi-permeable biocompatible dextranomer microsphere, reduces the incidence of necrotizing enterocolitis by 50% in a well-defined animal model following delivery of a single prophylactic dose. Herein, using the same semi-permeable microspheres, we showed that providing compounds beneficial to L. reuteri as diffusible cargo within the microsphere lumen resulted in further advantageous effects including glucosyltransferase-dependent bacterial adherence to the microsphere surface, resistance of bound bacteria against acidic conditions, enhanced adherence of L. reuteri to human intestinal epithelial cells in vitro, and facilitated production of the antimicrobial compound reuterin and the anti-inflammatory molecule histamine. These data support continued development of this novel probiotic formulation as an adaptable and effective means for targeted delivery of cargo beneficial to the probiotic bacterium.