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BackgroundThe current prevalence of chronic obstructive pulmonary disease (COPD) in France is estimated to be 2.6 million and is predicted to increase to 2.8 million by 2025. Presently, there is a lack of data on COPD management within the private healthcare setting. The aim of this study was to investigate the management of COPD exacerbations by pulmonologists within private practices in France.MethodsA prospective, online, qualitative survey was distributed to private practice pulmonologists in France. The survey covered all aspects of COPD management from diagnosis and therapeutic management, to secondary prevention and organization of care. Survey responses were collected between 27 January 2018 and 18 June 2018 and all data were summarized descriptively.ResultsThe survey had a response rate of 20.6%, with 116 out of 563 pulmonologists providing responses. Overall, 87.4% of respondents stated that the management of COPD represented over 15% of their total clinical activity. Most respondents indicated that they work closely with general practitioners and a large multidisciplinary team to manage patients with numerous comorbidities. Following a COPD exacerbation, the majority of respondents (78.4%) were in favor of using respiratory-connected devices (class 2a-connected medical device according to the French HAS classification and available on medical prescription) to assist with patient follow-up at home.ConclusionsCOPD management forms part of the core clinical activity for pulmonologists within the private practice setting in France. Patients with COPD generally have multiple comorbidities and are managed by a multidisciplinary team in line with French guidelines. The use of respiratory-connected devices was highlighted as an important new strategy for improving patient care following a COPD exacerbation.

BackgroundThe symptomatic response to continuous positive airway pressure (CPAP) therapy in COPD-obstructive sleep apnea overlap syndrome (OVS) compared to OSA syndrome (OSA) alone has not been well studied so far. The aim of this study is to explore main differences in the clinical response to CPAP treatment in OVS compared to OSA alone.Study design and methodsUsing prospective data from the French National Sleep Apnea Registry, we conducted an observational study among 6320 patients with moderate-to-severe OSA, available spirometry, and at least one follow-up visit under CPAP therapy.ResultsCPAP efficacy measured on the residual apnea-hypopnea index and median adherence were similar between OVS and OSA patients. In both groups, the overall burden of symptoms related to sleep apnea improved with CPAP treatment. In a multivariable model adjusted for age, gender, body mass index, adherence to treatment and residual apnea-hypopnea index, OVS was associated with higher odds for persistent morning headaches (OR: 1.37 [95% CI; 1.04; 1.79]; P = 0.02), morning tiredness (OR: 1.33 [95% CI: 1.12; 1.59]; PInterpretationCPAP therapy was effective in normalizing the apnea-hypopnea index and significantly improved OSA-related symptoms, regardless of COPD status. CPAP should be offered to patients with OVS on a trial basis as a significant improvement in OSA-related symptoms can be expected, although the range of response may be less dramatic than in OSA alone.

We recently reported a family-based genome wide association study (GWAS) for pediatric stroke pointing our attention to two significantly associated genes of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family ADAMTS2 (rs469568, p = 8x10-6) and ADAMTS12 (rs1364044, p = 2.9x10-6). To further investigate these candidate genes, we applied a targeted resequencing approach on 48 discordant sib-pairs for pediatric stroke followed by genotyping of the detected non-synonymous variants in the full cohort of 270 offspring trios and subsequent fine mapping analysis. We identified eight non-synonymous SNPs in ADAMTS2 and six in ADAMTS12 potentially influencing the respective protein function. These variants were genotyped within a cohort of 270 affected offspring trios, association analysis revealed the ADAMTS12 variant rs77581578 to be significantly under-transmitted (p = 6.26x10-3) to pediatric stroke patients. The finding was validated in a pediatric venous thromboembolism (VTE) cohort of 189 affected trios. Subsequent haplotype analysis of ADAMTS12 detected a significantly associated haplotype comprising the originally identified GWAS variant. Several ADAMTS genes such as ADAMTS13 are involved in thromboembolic disease process. Here, we provide further evidence for ADAMTS12 to likely play a role in pediatric stroke. Further functional studies are warranted to assess the functional role of ADAMTS12 in the pathogenesis of stroke.

BackgroundMore advanced knowledge is needed on how COPD alters the clinical presentation of obstructive sleep apnea (OSA) and how the association of both diseases, known as 'overlap syndrome' (OVS), impacts on cardiovascular health.ObjectiveTo investigate differences between patients with OVS and those with moderate-to-severe OSA alone.MethodsA cross-sectional study conducted in the French National Sleep Apnea Registry between January 1997 and January 2017. Univariable and multivariable logistic regression models were used to compare OVS versus OSA alone on symptoms and cardiovascular health.Results46,786 patients had moderate-to-severe OSA. Valid spirometry was available for 16,466 patients: 14,368 (87%) had moderate-to-severe OSA alone and 2098 (13%) had OVS. A lower proportion of OVS patients complained of snoring, morning headaches and excessive daytime sleepiness compared to OSA alone (median Epworth Sleepiness Scale score: 9 [interquartile range (IQR) 6-13] versus 10 (IQR 6-13), respectively; P ConclusionsIn adults with moderate-to-severe OSA, OVS was minimally symptomatic, but exhibited higher odds for prevalent coronary heart disease, heart failure and peripheral arteriopathy.

Antimicrobial peptides (AMPs) can be found in various organisms, and could be considered an alternative for pesticides used to control plant pathogens, including those affecting citrus. Brazil is the largest producer and exporter of frozen concentrated orange juice in the world. However, the citrus industry has been affected by several diseases such as citrus canker and huanglongbing (HLB), caused by the bacteria Xanthomonas citri subsp. citri (X.citri) and Candidatus Liberibacter asiaticus (CaLas), respectively. In order to control these pathogens, putative AMPs were prospected in databases containing citrus sequences. Furthermore, AMPs already reported in the literature were also used for in vitro and in vivo assays against X.citri. Since CaLas cannot be cultivated in vitro, surrogates as Sinorhizobium meliloti and Agrobacterium tumefaciens were used. This study reports the evaluation of six AMPs obtained from different sources, two of them from Citrus spp. (citrus-amp1 and citrus-amp2), three from amphibians (Hylin-a1, K0-W6-Hy-a1 and Ocellatin 4-analogue) and one from porcine (Tritrpticin). Peptides K0-W6-Hy-a1, Ocellatin 4-analogue, and citrus-amp1 showed bactericidal activity against X.citri and S. meliloti and bacteriostatic effect on A. tumefaciens. These results were confirmed for X.citri in planta. In addition cytotoxicity evaluations of these molecules were performed. The AMPs that showed the lowest hemolytic activities were Triptrpticin, citrus-amp1 and citrus-amp2. Citrus-amp1 and citrus-amp2 not presented toxicity in experiments using in vivo model, G. mellonella and U87 MG cells. To verify the interaction of these AMPs with bacteria and erythrocyte cell membranes, vesicles mimicking these cells were built. Citrus-amp1 and Tritrpticin exhibited higher affinity to bacterial membranes, while Ocellatin 4-analogue and Hylin-a1 showed higher affinity to erythrocyte membranes; exclude their use in citrus. This work demonstrates an essential alternative, trough AMPs obtained from Citrus spp., which can be feasibly used to control bacterial pathogens.

Superantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens.We examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis.TSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis.Our studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis.

Secreted factors of Staphylococcus aureus can activate host signaling from the epidermal growth factor receptor (EGFR). The superantigen toxic shock syndrome toxin-1 (TSST-1) contributes to mucosal cytokine production through a disintegrin and metalloproteinase (ADAM)-mediated shedding of EGFR ligands and subsequent EGFR activation. The secreted hemolysin, α-toxin, can also induce EGFR signaling and directly interacts with ADAM10, a sheddase of EGFR ligands. The current work explores the role of EGFR signaling in menstrual toxic shock syndrome (mTSS), a disease mediated by TSST-1. The data presented show that TSST-1 and α-toxin induce ADAM- and EGFR-dependent cytokine production from human vaginal epithelial cells. TSST-1 and α-toxin also induce cytokine production from an ex vivo porcine vaginal mucosa (PVM) model. EGFR signaling is responsible for the majority of IL-8 production from PVM in response to secreted toxins and live S. aureus. Finally, data are presented demonstrating that inhibition of EGFR signaling with the EGFR-specific tyrosine kinase inhibitor AG1478 significantly increases survival in a rabbit model of mTSS. These data indicate that EGFR signaling is critical for progression of an S. aureus exotoxin-mediated disease and may represent an attractive host target for therapeutics.

The classification of obstructive sleep apnea is on the basis of sleep study criteria that may not adequately capture disease heterogeneity. Improved phenotyping may improve prognosis prediction and help select therapeutic strategies.This study used cluster analysis to investigate the clinical clusters of obstructive sleep apnea.An ascending hierarchical cluster analysis was performed on baseline symptoms, physical examination, risk factor exposure and co-morbidities from 18,263 participants in the OSFP (French national registry of sleep apnea). The probability for criteria to be associated with a given cluster was assessed using odds ratios, determined by univariate logistic regression.Six clusters were identified, in which patients varied considerably in age, sex, symptoms, obesity, co-morbidities and environmental risk factors. The main significant differences between clusters were minimally symptomatic versus sleepy obstructive sleep apnea patients, lean versus obese, and among obese patients different combinations of co-morbidities and environmental risk factors.Our cluster analysis identified six distinct clusters of obstructive sleep apnea. Our findings underscore the high degree of heterogeneity that exists within obstructive sleep apnea patients regarding clinical presentation, risk factors and consequences. This may help in both research and clinical practice for validating new prevention programs, in diagnosis and in decisions regarding therapeutic strategies.

Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 μg of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naïve mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to assess the possible use of viola in therapeutic approaches in human autoimmune diseases.

The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.

Superantigens (SAgs) are important virulence factors in S. aureus. Recent studies identified their presence in animal coagulase-negative staphylococci (CNS). The emergence of human-associated SAg+ CNS would mark a prodigious shift in virulence capabilities. We examined CNS isolates from healthy human nares and diseased individuals, and determined that no known SAgs were present.

Broad-spectrum antimicrobials kill indiscriminately, a property that can lead to negative clinical consequences and an increase in the incidence of resistance. Species-specific antimicrobials that could selectively kill pathogenic bacteria without targeting other species in the microbiome could limit these problems. The pathogen genome presents an excellent target for the development of such antimicrobials. In this study we report the design and evaluation of species-selective peptide nucleic acid (PNA) antibacterials. Selective growth inhibition of B. subtilis, E. coli, K. pnuemoniae and S. enterica serovar Typhimurium in axenic or mixed culture could be achieved with PNAs that exploit species differences in the translation initiation region of essential genes. An S. Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials. Analysis of the genomes of E. coli and S. Typhimurium gave a conservative estimate of >150 PNA targets that could potentially discriminate between these two closely related species. This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.

RationaleIn obstructive sleep apnea patients (OSA), continuous positive airway pressure (CPAP) adherence is crucial to improve symptoms and cardiometabolic outcomes. The choice of mask may influence CPAP adherence but this issue has never been addressed properly.ObjectiveTo evaluate the impact of nasal pillows, nasal and oronasal masks on CPAP adherence in a cohort of OSA.MethodsNewly CPAP treated OSA participating in "Observatoire Sommeil de la Fédération de Pneumologie", a French national prospective cohort, were included between March 2009 and December 2011. Anthropometric data, medical history, OSA severity, sleepiness, depressive status, treatment modalities (auto-CPAP versus fixed pressure, pressure level, interface type, use of humidifiers) and CPAP-related side effects were included in multivariate analysis to determine independent variables associated with CPAP adherence.Results2311 OSA (age = 57(12) years, apnea+hypopnea index = 41(21)/h, 29% female) were included. Nasal masks, oronasal masks and nasal pillows were used by 62.4, 26.2 and 11.4% of the patients, respectively. In univariate analysis, oronasal masks and nasal pillows were associated with higher risk of CPAP non-adherence. CPAP non-adherence was also associated with younger age, female gender, mild OSA, gastroesophageal reflux, depression status, low effective pressure and CPAP-related side effects. In multivariate analysis, CPAP non-adherence was associated with the use of oronasal masks (OR = 2.0; 95%CI = 1.6; 2.5), depression, low effective pressure, and side effects.ConclusionAs oronasal masks negatively impact on CPAP adherence, a nasal mask should be preferred as the first option. Patients on oronasal masks should be carefully followed.

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1') with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.

BACKGROUND: Elevated cholesterol levels in children can be a risk factor for cardiovascular diseases in later life. In adults, it has been shown that blood lipid levels are strongly influenced by polymorphisms in the fatty acid desaturase (FADS) gene cluster in addition to nutritional and other exogenous and endogenous determinants. Our aim was to investigate whether lipid levels are determined by the FADS genotype already in children and whether this association interacts with dietary intake of n-3 fatty acids. METHODS: The analysis was based on data of 2006 children from two German prospective birth cohort studies. Total cholesterol, HDL, LDL and triglycerides were measured at 10 years of age. Six single nucleotide polymorphisms (SNPs) of the FADS gene cluster were genotyped. Dietary n-3 fatty acid intake was assessed by food frequency questionnaire. Linear regression modeling was used to assess the association between lipid levels, n-3 fatty acid intake and FADS genotype. RESULTS: Individuals carrying the homozygous minor allele had lower levels of total cholesterol [means ratio (MR) ranging from 0.96 (p = 0.0093) to 0.98 (p = 0.2949), depending on SNPs] and LDL [MR between 0.94 (p = 0.0179) and 0.97 (p = 0.2963)] compared to homozygous major allele carriers. Carriers of the heterozygous allele showed lower HDL levels [β between -0.04 (p = 0.0074) to -0.01 (p = 0.3318)] and higher triglyceride levels [MR ranging from 1.06 (p = 0.0065) to 1.07 (p = 0.0028)] compared to homozygous major allele carriers. A higher n-3 PUFA intake was associated with higher concentrations of total cholesterol, LDL, HDL and lower triglyceride levels, but these associations did not interact with the FADS1 FADS2 genotype. CONCLUSION: Total cholesterol, HDL, LDL and triglyceride concentrations may be influenced by the FADS1 FADS2 genotype already in 10 year old children. Genetically determined blood lipid levels during childhood might differentially predispose individuals to the development of cardiovascular diseases later in life.

The sea urchin (Echinodermata: Echinoidea) masticatory apparatus, or Aristotle's lantern, is a complex structure composed of numerous hard and soft components. The lantern is powered by various paired and unpaired muscle groups. We describe how one set of these muscles, the lantern protractor muscles, has evolved a specialized morphology. This morphology is characterized by the formation of adaxially-facing lobes perpendicular to the main orientation of the muscle, giving the protractor a frilled aspect in horizontal section. Histological and ultrastructural analyses show that the microstructure of frilled muscles is largely identical to that of conventional, flat muscles. Measurements of muscle dimensions in equally-sized specimens demonstrate that the frilled muscle design, in comparison to that of the flat muscle type, considerably increases muscle volume as well as the muscle's surface directed towards the interradial cavity, a compartment of the peripharyngeal coelom. Scanning electron microscopical observations reveal that the insertions of frilled and flat protractor muscles result in characteristic muscle scars on the stereom, reflecting the shapes of individual muscles. Our comparative study of 49 derived "regular" echinoid species using magnetic resonance imaging (MRI) shows that frilled protractor muscles are found only in taxa belonging to the families Toxopneustidae, Echinometridae, and Strongylocentrotidae. The onset of lobe formation during ontogenesis varies between species of these three families. Because frilled protractor muscles are best observed in situ, the application of a non-invasive imaging technique was crucial for the unequivocal identification of this morphological character on a large scale. Although it is currently possible only to speculate on the functional advantages which the frilled muscle morphology might confer, our study forms the anatomical and evolutionary framework for future analyses of this unusual muscle design among sea urchins.

Division of labor in social insects has made the evolution of collective traits possible that cannot be achieved by individuals alone. Differences in behavioral responses produce variation in engagement in behavioral tasks, which as a consequence, generates a division of labor. We still have little understanding of the genetic components influencing these behaviors, although several candidate genomic regions and genes influencing individual behavior have been identified. Here, we report that mixing of worker honeybees with different genotypes influences the expression of individual worker behaviors and the transcription of genes in the neuronal substrate. These indirect genetic effects arise in a colony because numerous interactions between workers produce interacting phenotypes and genotypes across organisms. We studied hygienic behavior of honeybee workers, which involves the cleaning of diseased brood cells in the colony. We mixed ∼500 newly emerged honeybee workers with genotypes of preferred Low (L) and High (H) hygienic behaviors. The L/H genotypic mixing affected the behavioral engagement of L worker bees in a hygienic task, the cooperation among workers in uncapping single brood cells, and switching between hygienic tasks. We found no evidence that recruiting and task-related stimuli are the primary source of the indirect genetic effects on behavior. We suggested that behavioral responsiveness of L bees was affected by genotypic mixing and found evidence for changes in the brain in terms of 943 differently expressed genes. The functional categories of cell adhesion, cellular component organization, anatomical structure development, protein localization, developmental growth and cell morphogenesis were overrepresented in this set of 943 genes, suggesting that indirect genetic effects can play a role in modulating and modifying the neuronal substrate. Our results suggest that genotypes of social partners affect the behavioral responsiveness and the neuronal substrate of individual workers, indicating a complex genetic architecture underlying the expression of behavior.

BACKGROUND: Berberine is a plant alkaloid that is widely used as an anti-infective in traditional medicine. Escherichia coli exposed to berberine form filaments, suggesting an antibacterial mechanism that involves inhibition of cell division. Berberine is a DNA ligand and may induce filamentation through induction of the SOS response. Also, there is biochemical evidence for berberine inhibition of the cell division protein FtsZ. Here we aimed to assess possible berberine mechanism(s) of action in growing bacteria using genetics tools. METHODOLOGY/PRINCIPAL FINDINGS: First, we tested whether berberine inhibits bacterial growth through DNA damage and induction of the SOS response. The SOS response induced by berberine was much lower compared to that induced by mitomycin C in an SOS response reporter strain. Also, cell filamentation was observed in an SOS-negative E. coli strain. To test whether berberine inhibits FtsZ, we assessed its effects on formation of the cell division Z-rings, and observed a dramatic reduction in Z-rings in the presence of berberine. We next used two different strategies for RNA silencing of ftsZ and both resulted in sensitisation of bacteria to berberine, visible as a drop in the Minimum Inhibitory Concentration (MIC). Furthermore, Fractional Inhibitory Concentration Indices (FICIs) showed a high level of synergy between ftsZ silencing and berberine treatment (FICI values of 0.23 and 0.25 for peptide nucleic acid- and expressed antisense RNA-based silencing of ftsZ, respectively). Finally, over-expression of ftsZ led to a mild rescue effect in berberine-treated cells. CONCLUSIONS: The results argue against DNA binding as the primary mechanism of action of berberine and support the hypothesis that its antibacterial properties are due to inhibition of the cell division protein FtsZ. In addition, the genetic approach used here provides a means to rapidly test the activity of other putative FtsZ inhibitors.

BACKGROUND:Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development. METHODOLOGY/PRINCIPAL FINDINGS:Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)). When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required. CONCLUSIONS:RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

Background: The classification of obstructive sleep apnea is on the basis of sleep study criteria that may not adequately capture disease heterogeneity. Improved phenotyping may improve prognosis prediction and help select therapeutic strategies. Objectives: This study used cluster analysis to investigate the clinical clusters of obstructive sleep apnea. Methods: An ascending hierarchical cluster analysis was performed on baseline symptoms, physical examination, risk factor exposure and co-morbidities from 18,263 participants in the OSFP (French national registry of sleep apnea). The probability for criteria to be associated with a given cluster was assessed using odds ratios, determined by univariate logistic regression. Results: Six clusters were identified, in which patients varied considerably in age, sex, symptoms, obesity, co-morbidities and environmental risk factors. The main significant differences between clusters were minimally symptomatic versus sleepy obstructive sleep apnea patients, lean versus obese, and among obese patients different combinations of co-morbidities and environmental risk factors. Conclusions: Our cluster analysis identified six distinct clusters of obstructive sleep apnea. Our findings underscore the high degree of heterogeneity that exists within obstructive sleep apnea patients regarding clinical presentation, risk factors and consequences. This may help in both research and clinical practice for validating new prevention programs, in diagnosis and in decisions regarding therapeutic strategies. [ABSTRACT FROM AUTHOR]

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces. [ABSTRACT FROM AUTHOR]