Development and validation of the Cannabis Exposure in Pregnancy Tool (CEPT): a mixed methods study.

BACKGROUND: Evidence of associations between prenatal cannabis use (PCU) and maternal and infant health outcomes remains conflicting amid broad legalization of cannabis across Canada and 40 American states. A critical limitation of existing evidence lies in the non-standardized and crude measurement of prenatal cannabis use (PCU), resulting in high risk of misclassification bias. We developed a standardized tool to comprehensively measure prenatal cannabis use in pregnant populations for research purposes. METHODS: We conducted a mixed-methods, patient-oriented tool development and validation study, using a bias-minimizing process. Following an environmental scan and critical appraisal of existing prenatal substance use tools, we recruited pregnant participants via targeted social media advertising and obstetric clinics in Alberta, Canada. We conducted individual in-depth interviews and cognitive interviewing in separate sub-samples, to develop and refine our tool. We assessed convergent and discriminant validity internal consistency and 3-month test-retest reliability, and validated the tool externally against urine-THC bioassays. RESULTS: Two hundred fifty four pregnant women participated. The 9-item Cannabis Exposure in Pregnancy Tool (CEPT) had excellent discriminant (Cohen's kappa = -0.27-0.15) and convergent (Cohen's kappa = 0.72-1.0) validity; as well as high internal consistency (Chronbach's alpha = 0.92), and very good test-retest reliability (weighted Kappa = 0.92, 95% C.I. [0.86-0.97]). The CEPT is valid against urine THC bioassay (sensitivity = 100%, specificity = 82%). CONCLUSION: The CEPT is a novel, valid and reliable measure of frequency, timing, dose, and mode of PCU, in a contemporary sample of pregnant women. Using CEPT (compared to non-standardized tools) can improve measurement accuracy, and thus the quality of research examining PCU and maternal and child health outcomes.

Longitudinal assessment of bacterial vaginosis prior to and during incident pregnancy: an observational study in Kenyan adolescent girls and young women.

OBJECTIVE: To determine bacterial vaginosis (BV) status at multiple time points among adolescent girls and young women (AGYW) and assess the impact of pregnancy on their BV status. DESIGN: Longitudinal cohort study. SETTING: Thika, Kenya. PARTICIPANTS: AGYW aged 16-20 years enrolled prior to first sex or reporting only a single lifetime partner. MAIN OUTCOME MEASURES: The primary outcome was relative risk (RR) of BV during pregnancy compared with before pregnancy by analysing longitudinal trends in BV over time. BV risk was estimated using Poisson regression models. RESULTS: A total of 121 AGYW became pregnant in the parent cohort and had BV results before, during or after pregnancy. Point prevalence of BV was 11.0% at visits >12 months pre-pregnancy, 13.0% at 3-12 months pre-pregnancy, 22.1% at <3 months pre-pregnancy and 13.4% during pregnancy. Compared with visits during pregnancy, RR of BV was 1.65 (95% CI: 1.00 to 2.71; p=0.05) at visits <3 months pre-pregnancy, 0.97 (95% CI: 0.62 to 1.52; p=0.90) at visits 3-12 months pre-pregnancy and 0.82 (95% CI: 0.44 to 1.53; p=0.53) at visits 12 months pre-pregnancy. An adjusted analysis including age, income, residence, date of first sex, recent sexual activity and positive sexually transmitted infection test resulted in small changes in risk estimates, with adjusted RR of BV of 1.66 (95% CI: 1.04 to 2.67; p=0.04) at visits <3 months pre-pregnancy compared with visits during pregnancy. CONCLUSIONS: BV risk during pregnancy was lower than during the immediate pre-pregnancy period. Hormonal changes in pregnancy may reduce BV.

Low prevalence of bacterial vaginosis in Kenyan adolescent girls and rapid incidence after first sex.

BACKGROUND: Bacterial vaginosis is a risk factor for sexually transmitted infections, including HIV. Adult African women have a high prevalence of bacterial vaginosis, but it is not known when first bacterial vaginosis occurs. OBJECTIVE: This study aimed to describe bacterial vaginosis in younger African women, before and after first sex, and to determine the incidence of bacterial vaginosis and significant correlates of bacterial vaginosis incidence and recurrence. STUDY DESIGN: In a prospective observational cohort study enrolling adolescents with limited sexual experience, young women aged 16 to 21 years were recruited in Thika, Kenya. Eligible participants were HIV and herpes simplex virus 2 seronegative and reported 0 or 1 lifetime sexual partner. The Nugent score was determined at quarterly visits from vaginal Gram stains. The trends in bacterial vaginosis were described over time; hazard ratios were calculated using Cox regression, and relative risk of bacterial vaginosis was estimated using generalized estimating equations and Poisson regression. RESULTS: A total of 400 participants with a median age of 18.6 years (interquartile range, 16-21) were enrolled. Of note, 322 participants (80.5%) reported no history of sex, whereas 78 participants (19.5%) reported sex with 1 partner. At enrollment, bacterial vaginosis (Nugent score of ≥7) was uncommon (21/375 [5.6%]). Overall, 144 participants had bacterial vaginosis at least once, for an incidence rate of 16.5 cases per 100 person-years. Before first sex, bacterial vaginosis was present at 2.8% of visits, compared with 13.7% of visits after first sex. An adjusted model of bacterial vaginosis incidence observed that first sex was associated with more than a 2-fold increased bacterial vaginosis risk (adjusted hazard ratio, 2.44; 95% confidence interval, 1.25-4.76; P=.009). Chlamydia diagnosis (adjusted hazard ratio, 1.73; 95% confidence interval, 1.1-2.8; P=.02), and herpes simplex virus 2 seropositivity (adjusted hazard ratio, 2.88; 95% confidence interval, 1.17-7.09; P=.021) were both associated with incident bacterial vaginosis. A multivariate generalized estimating equation model, including all episodes of bacterial vaginosis, demonstrated risk factors, including first sex, sexually transmitted infections, urban residence, recent sex, and no income; the most important risk factor was first sex (adjusted relative risk, 1.92; 95% confidence interval, 1.12-3.31; P=.018). The probability of bacterial vaginosis increased with each subsequent episode; mean Nugent scores increased after each bacterial vaginosis episode. CONCLUSION: Using detailed longitudinal observation, this study found that Kenyan adolescents have almost no bacterial vaginosis before first sex and that initiation of sexual activity was the strongest risk factor for both prevalent bacterial vaginosis and incident bacterial vaginosis.

Protease activities of vaginal Porphyromonas species disrupt coagulation and extracellular matrix in the cervicovaginal niche.

Porphyromonas asaccharolytica and Porphyromonas uenonis are common inhabitants of the vaginal microbiome, but their presence has been linked to adverse health outcomes for women, including bacterial vaginosis and preterm birth. However, little is known about the pathogenesis mechanisms of these bacteria. The related oral opportunistic pathogen, Porphyromonas gingivalis, is comparatively well-studied and known to secrete numerous extracellular matrix-targeting proteases. Among these are the gingipain family of cysteine proteases that drive periodontal disease progression and hematogenic transmission to the placenta. In this study, we demonstrate that vaginal Porphyromonas species secrete broad-acting proteases capable of freely diffusing within the cervicovaginal niche. These proteases degrade collagens that are enriched within the cervix (type I) and chorioamniotic membranes (type IV), as well as fibrinogen, which inhibits clot formation. Bioinformatic queries confirmed the absence of gingipain orthologs and identified five serine, cysteine, and metalloprotease candidates in each species. Inhibition assays revealed that each species' proteolytic activity can be partially attributed to a secreted metalloprotease with broad substrate specificity that is distantly related to the P. gingivalis endopeptidase PepO. This characterization of virulence activities in vaginal Porphyromonas species highlights their potential to alter the homeostasis of reproductive tissues and harm human pregnancy through clotting disruption, fetal membrane weakening, and premature cervical remodeling.

Reconstruction of Microbial Haplotypes by Integration of Statistical and Physical Linkage in Scaffolding.

DNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or "haplotypes." However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics, and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here, we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis.

Maternal consumption of artificially sweetened beverages during pregnancy is associated with infant gut microbiota and metabolic modifications and increased infant body mass index.

Artificial sweetener consumption by pregnant women has been associated with an increased risk of infant obesity, but the underlying mechanisms are unknown. We aimed to determine if maternal consumption of artificially sweetened beverages (ASB) during pregnancy is associated with modifications of infant gut bacterial community composition and function during the first year of life, and whether these alterations are linked with infant body mass index (BMI) at one year of age. We studied 100 infants from the prospective Canadian CHILD Cohort Study, selected based on maternal ASB consumption during pregnancy (50 non-consumers and 50 daily consumers). BMI was higher among ASB-exposed infants. Infant stool (16S rRNA gene sequencing) and urine (untargeted metabolomics) were acquired in early (3-4 months) and late (12 months) infancy. We identified four microbiome clusters, of which two recapitulated the maturation trajectory of the infant gut bacterial communities from immature (Cluster 1) to mature (Cluster 4) and two deviated from this trajectory (Clusters 2 and 3). Maternal ASB consumption did not differ between clusters, but was associated with community-level shifts in infant gut bacterial taxonomy structure and depletion of several Bacteroides sp. in Cluster 2. In the complete dataset, urine succinate and spermidine levels at 3 months were higher in ASB-exposed infants, and urine succinate was positively associated with BMI at one-year-old. Overall, gestational exposure to ASB was associated with gut microbiota structure in infants from Cluster 2, and gut microbiota structure was associated with infant BMI. Gestational exposure to ASB was positively associated with infant urine succinate and spermidine. Succinate was found to mediate 29% of the effect of ASB exposure on BMI at one-year-old, revealing a potential role of this metabolite in increased infant weight linked to gestational ASB consumption. As we face an unprecedented rise in childhood obesity, future studies should evaluate the causal relationships between maternal ASB consumption (a modifiable exposure), gut microbiota and metabolites, infant metabolism, and body composition.

Genetic Variation in Toll-Like Receptor 5 and Colonization with Flagellated Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency-defined by a dominant negative stop codon polymorphism, rs5744168-is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, Mobiluncus curtisii, and Mobiluncus mulieris). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed M. mulieris or M. curtisii and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed M. mulieris and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed M. curtisii did not. In contrast, partially purified flagellin from both Mobiluncus species stimulated a TLR5-mediated response in vitro We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by Mobiluncus species versus those encoded by BVAB1.

A Gut Microbial Mimic that Hijacks Diabetogenic Autoreactivity to Suppress Colitis.

The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic ß cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ T cells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin ß7-, perforin-, and major histocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood T cells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic T cells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.

New Systems for Studying Intercellular Interactions in Bacterial Vaginosis.

Bacterial vaginosis (BV) affects almost a quarter of US women, making it a condition of major public health relevance. Key questions remain regarding the etiology of BV, mechanisms for its association with poor reproductive health outcomes, and reasons for high rates of treatment failure. New model systems are required to answer these remaining questions, elucidate the complex host-microbe and microbe-microbe interactions, and develop new, effective interventions. In this review, we cover the strengths and limitations of in vitro and in vivo model systems to study these complex intercellular interactions. Furthermore, we discuss advancements needed to maximize the translational utility of the model systems. As no single model can recapitulate all of the complex physiological and environmental conditions of the human vaginal microenvironment, we conclude that combinatorial approaches using in vitro and in vivo model systems will be required to address the remaining fundamental questions surrounding the enigma that is BV.

Relationship of Specific Bacteria in the Cervical and Vaginal Microbiotas With Cervicitis.

BACKGROUND: Cervicitis is an inflammatory condition of the cervix associated with upper genital tract infection and reproductive complications. Although cervicitis can be caused by several known pathogens, the etiology frequently remains obscure. Here we investigate vaginal bacteria associated with bacterial vaginosis as potential causes of cervicitis. METHODS: Associations between vaginal bacteria and cervicitis were assessed in a retrospective case-control study of women attending a Seattle sexually transmitted disease clinic. Individual bacterial species were detected using 2 molecular methods: quantitative polymerase chain reaction (qPCR) and broad-range 16S rRNA gene PCR with pyrosequencing. The primary finding from this initial study was evaluated using qPCR in a second cohort of Kenyan women. RESULTS: The presence of Mageeibacillus indolicus, formerly BVAB3, in the cervix was associated with cervicitis, whereas the presence of Lactobacillus jensenii was inversely associated. Quantities of these bacteria did not differ between cervicitis cases and controls, although in a model inclusive of presence and abundance, M. indolicus remained significantly associated with cervicitis after adjustment for other cervicitis-causing pathogens. M. indolicus was not associated with cervicitis in our study of Kenyan women, possibly due to differences in the clinical definition of cervicitis. CONCLUSIONS: Colonization of the endocervix with M. indolicus may contribute to the clinical manifestations of cervicitis, but further study is needed to determine whether this finding is repeatable and applicable to diverse groups of women. Colonization of the cervix with L. jensenii could be a marker of health, perhaps reducing inflammation or inhibiting pathogenic infection.

Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology.

The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.

Multiple peptidoglycan modification networks modulate Helicobacter pylori's cell shape, motility, and colonization potential.

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape.

Peptidoglycan crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach colonization.

The mechanisms by which bacterial cells generate helical cell shape and its functional role are poorly understood. Helical shape of the human pathogen Helicobacter pylori may facilitate penetration of the thick gastric mucus where it replicates. We identified four genes required for helical shape: three LytM peptidoglycan endopeptidase homologs (csd1-3) and a ccmA homolog. Surrounding the cytoplasmic membrane of most bacteria, the peptidoglycan (murein) sacculus is a meshwork of glycan strands joined by peptide crosslinks. Intact cells and isolated sacculi from mutants lacking any single csd gene or ccmA formed curved rods and showed increased peptidoglycan crosslinking. Quantitative morphological analyses of multiple-gene deletion mutants revealed each protein uniquely contributes to a shape-generating pathway. This pathway is required for robust colonization of the stomach in spite of normal directional motility. Our findings suggest that the coordinated action of multiple proteins relaxes peptidoglycan crosslinking, enabling helical cell curvature and twist.

Functional analysis of the Helicobacter pylori flagellar switch proteins.

Helicobacter pylori uses flagellum-mediated chemotaxis to promote infection. Bacterial flagella change rotational direction by changing the state of the flagellar motor via a subcomplex referred to as the switch. Intriguingly, the H. pylori genome encodes four switch complex proteins, FliM, FliN, FliY, and FliG, instead of the more typical three of Escherichia coli or Bacillus subtilis. Our goal was to examine whether and how all four switch proteins participate in flagellation. Previous work determined that FliG was required for flagellation, and we extend those findings to show that all four switch proteins are necessary for normal numbers of flagellated cells. Furthermore, while fliY and fliN are partially redundant with each other, both are needed for wild-type levels of flagellation. We also report the isolation of an H. pylori strain containing an R54C substitution in fliM, resulting in bacteria that swim constantly and do not change direction. Along with data demonstrating that CheY-phosphate interacts with FliM, these findings suggest that FliM functions in H. pylori much as it does in other organisms.

Persistence of genital human papillomavirus infection in a long-term follow-up study of female university students.

BACKGROUND: Little is known about the epidemiology of human papillomavirus (HPV) infections that persist for more than a few years. METHODS: Four to 12 years after participation in a longitudinal study of incident HPV infection, a cohort of former university students returned for a follow-up visit that included HPV genotyping of cervical and vulvovaginal swab specimens and collection of colposcopy-directed biopsy specimens. RESULTS: Of 147 women with HPV infection detected during their undergraduate years, 24 (16.3%) were positive for 1 or more of the same HPV types at follow-up. Overall, 27 (4.8%) of 567 type-specific HPV infections persisted, and DNA sequence analyses of a subset revealed that all were variant specific. Long-term HPV persistence was positively associated with frequent but sporadic detection of the same HPV type early during the course of the infection and with abnormal Pap tests and genital warts; it was negatively associated with marriage and was not associated with the number of intercurrent sex partners. CONCLUSIONS: HPV variant and behavioral risk factor analyses indicated that long-term detection of the same HPV type was more consistent with viral persistence than with reinfection. Although long-term persistence was not common, it was associated with detection of HPV-related pathologies.

Identification of Helicobacter pylori genes that contribute to stomach colonization.

Chronic infection of the human stomach by Helicobacter pylori leads to a variety of pathological sequelae, including peptic ulcer and gastric cancer, resulting in significant human morbidity and mortality. Several genes have been implicated in disease related to H. pylori infection, including the vacuolating cytotoxin and the cag pathogenicity island. Other factors important for the establishment and maintenance of infection include urease enzyme production, motility, iron uptake, and stress response. We utilized a C57BL/6 mouse infection model to query a collection of 2,400 transposon mutants in two different bacterial strain backgrounds for H. pylori genetic loci contributing to colonization of the stomach. Microarray-based tracking of transposon mutants allowed us to monitor the behavior of transposon insertions in 758 different gene loci. Of the loci measured, 223 (29%) had a predicted colonization defect. These included previously described H. pylori virulence genes, genes implicated in virulence in other pathogenic bacteria, and 81 hypothetical proteins. We have retested 10 previously uncharacterized candidate colonization gene loci by making independent null alleles and have confirmed their colonization phenotypes by using competition experiments and by determining the dose required for 50% infection. Of the genetic loci retested, 60% have strain-specific colonization defects, while 40% have phenotypes in both strain backgrounds for infection, highlighting the profound effect of H. pylori strain variation on the pathogenic potential of this organism.

Confocal microscopy of the light organ crypts in juvenile Euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis.

In the hours to days following hatching, the Hawaiian bobtail squid, Euprymna scolopes, obtains its light-emitting symbiont, Vibrio fischeri, from the surrounding environment and propagates the bacteria in the epithelial crypts of a specialized light organ. Three-dimensional analyses using confocal microscopy revealed that each of the three crypts on either side of the juvenile light organ is composed of four morphological regions. Progressing from the lateral pore to the medial blind end of each crypt, the regions consist of 1) a duct, 2) an antechamber, 3) a bottleneck, and 4) a deep region. Only the deep region houses a persistent bacterial population, whereas the duct, antechamber, and bottleneck serve as conduits through which the bacteria enter during initial colonization and exit during diel venting, a behavior in which approximately 90% of the symbionts are expelled each dawn. Our data suggest that, like the duct, the antechamber and bottleneck may function to promote and maintain the specificity of the symbiosis. Pronounced structural and functional differences among the deep regions of the three crypts, along with previously reported characterizations of embryogenesis, suggest a continued developmental progression in the first few days after hatching. Taken together, the results of this study reveal a high degree of complexity in the morphology of the crypts, as well as in the extent to which the three crypts and their constituent regions differ in function during the early stages of the symbiosis.

Myocyte enhancer factor-2 and serum response factor binding elements regulate fast Myosin heavy chain transcription in vivo.

Adult fast muscle fibers express distinct myosin heavy chains (MyHC) in differing proportions, but the mechanisms underlying their differential expression remain undefined. We used a variety of in vitro and in vivo approaches to explore the contribution of transcriptional regulation to adult fast MyHC expression. Here we show that 800-1000 bp of a sequence upstream of the three mouse adult fast MyHC genes (Ia, IIb, and IId/x) are sufficient to drive muscle-specific and fiber-specific expression in vivo. We show that the upstream promoter region of the gene most abundantly expressed in mouse skeletal muscles, IIb MyHC, retains binding activity and transcriptional activation for three positive transcription factors, the serum response factor, Oct-1, and myocyte enhancer factor-2, whereas the other two genes (IIa and IId/x) have nucleotide substitutions in these sites that reduce binding and transcriptional activation. Finally, we demonstrate that regions upstream of 300 bp modulate the effects of these elements. Together, these data demonstrate that the quantitative differences in MyHC expression in mouse skeletal muscle have evolved at least in part through the elimination of positive-acting transcription factor binding sites.

NO means 'yes' in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association.

During colonization of the Euprymna scolopes light organ, symbiotic Vibrio fischeri cells aggregate in mucus secreted by a superficial ciliated host epithelium near the sites of eventual inoculation. Once aggregated, symbiont cells migrate through ducts into epithelium-lined crypts, where they form a persistent association with the host. In this study, we provide evidence that nitric oxide synthase (NOS) and its product nitric oxide (NO) are active during the colonization of host tissues by V. fischeri. NADPH-diaphorase staining and immunocytochemistry detected NOS, and the fluorochrome diaminofluorescein (DAF) detected its product NO in high concentrations in the epithelia of the superficial ciliated fields, ducts, and crypt antechambers. In addition, both NOS and NO were detected in vesicles within the secreted mucus where the symbionts aggregate. In the presence of NO scavengers, cells of a non-symbiotic Vibrio species formed unusually large aggregates outside of the light organ, but these bacteria did not colonize host tissues. In contrast, V. fischeri effectively colonized the crypts and irreversibly attenuated the NOS and NO signals in the ducts and crypt antechambers. These data provide evidence that NO production, a defense response of animal cells to bacterial pathogens, plays a role in the interactions between a host and its beneficial bacterial partner during the initiation of symbiotic colonization.