Novel Antimicrobial Peptides from Saline Environments Active against E. faecalis and S. aureus: Identification, Characterisation and Potential Usage.

Microorganisms inhabiting saline environments have been known for decades as producers of many valuable bioproducts. These substances include antimicrobial peptides (AMPs), the most recognizable of which are halocins produced by halophilic Archaea. As agents with a different modes of action from that of most conventionally used antibiotics, usually associated with an increase in the permeability of the cell membrane as a result of a formation of channels and pores, AMPs are a currently promising object of research focused on the investigation of antibiotics with non-standard modes of action. The aim of this study was to investigate antimicrobial activity against multidrug-resistant human pathogens of three peptides, which were synthetised based on sequences identified in metagenomes from saline environments. The investigations were performed against Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Subsequently, the cytotoxicity and haemolytic properties of the tested peptides were verified. An in silico analysis of the interaction of the tested peptides with molecular targets for reference antibiotics was also carried out in order to verify whether or not they can act in a similar way. The P1 peptide manifested the growth inhibition of E. faecalis at a MIC50 of 32 µg/mL and the P3 peptide at a MIC50 of 32 µg/mL was shown to inhibit the growth of both E. faecalis and S. aureus. Furthermore, the P1 and P3 peptides were shown to have no cytotoxic or haemolytic activity against human cells.

Skin Microbiome in Prurigo Nodularis.

Prurigo nodularis (PN) is a chronic condition characterized by the presence of nodular lesions accompanied by intense pruritus. The disease has been linked to several infectious factors, but data on the direct presence of microorganisms in the lesions of PN are scarce. The aim of this study was to evaluate the diversity and composition of the bacterial microbiome in PN lesions by targeting the region V3-V4 of 16S rRNA. Skin swabs were obtained from active nodules in 24 patients with PN, inflammatory patches of 14 patients with atopic dermatitis (AD) and corresponding skin areas of 9 healthy volunteers (HV). After DNA extraction, the V3-V4 region of the bacterial 16S rRNA gene was amplified. Sequencing was performed using the Illumina platform on the MiSeq instrument. Operational taxonomic units (OTU) were identified. The identification of taxa was carried out using the Silva v.138 database. There was no statistically significant difference in the alpha-diversity (intra-sample diversity) between the PN, AD and HV groups. The beta-diversity (inter-sample diversity) showed statistically significant differences between the three groups on a global level and in paired analyses. Staphylococcus was significantly more abundant in samples from PN and AD patients than in controls. The difference was maintained across all taxonomic levels. The PN microbiome is highly similar to that of AD. It remains unclear whether the disturbed composition of the microbiome and the domination of Staphylococcus in PN lesions may be the trigger factor of pruritus and lead to the development of cutaneous changes or is a secondary phenomenon. Our preliminary results support the theory that the composition of the skin microbiome in PN is altered and justify further research on the role of the microbiome in this debilitating condition.

Assessment of the Potential Role of Selected Single Nucleotide Polymorphisms (SNPs) of Genes Related to the Functioning of Regulatory T Cells in the Pathogenesis of Psoriasis.

Recent studies have indicated a key role of the impaired suppressive capacity of regulatory T cells (Tregs) in psoriasis (PsO) pathogenesis. However, the genetic background of Treg dysfunctions remains unknown. The aim of this study was to evaluate the association of PsO development with selected single nucleotide polymorphisms (SNPs) of genes in which protein products play a significant role in the regulation of differentiation and function of Tregs. There were three study groups in our research and each consisted of different unrelated patients and controls: 192 PsO patients and 5605 healthy volunteers in the microarray genotyping group, 150 PsO patients and 173 controls in the ARMS-PCR method group, and 6 PsO patients and 6 healthy volunteers in the expression analysis group. The DNA microarrays analysis (283 SNPs of 57 genes) and ARMS-PCR method (8 SNPs in 7 genes) were used to determine the frequency of occurrence of SNPs in selected genes. The mRNA expression of selected genes was determined in skin samples. There were statistically significant differences in the allele frequencies of four SNPs in three genes (TNF, IL12RB2, and IL12B) between early-onset PsO patients and controls. The lowest p-value was observed for rs3093662 (TNF), and the G allele carriers had a 2.73 times higher risk of developing early-onset PsO. Moreover, the study revealed significant differences in the frequency of SNPs and their influence on PsO development between early- and late-onset PsO. Based on the ARMS-PCR method, the association between some polymorphisms of four genes (IL4, IL10, TGFB1, and STAT3) and the risk of developing PsO was noticed. Psoriatic lesions were characterized with a lower mRNA expression of FOXP3, CTLA4, and IL2, and a higher expression of TNF and IL1A in comparison with unaffected skin. In conclusion, the genetic background associated with properly functioning Tregs seems to play a significant role in PsO pathogenesis and could have diagnostic value.

Genome-Wide DNA Methylation and Gene Expression in Patients with Indolent Systemic Mastocytosis.

Mastocytosis is a clinically heterogenous, usually acquired disease of the mast cells with a survival time that depends on the time of onset. It ranges from skin-limited to systemic disease, including indolent and more aggressive variants. The presence of the oncogenic KIT p. D816V gene somatic mutation is a crucial element in the pathogenesis. However, further epigenetic regulation may also affect the expression of genes that are relevant to the pathology. Epigenetic alterations are responsible for regulating the expression of genes that do not modify the DNA sequence. In general, it is accepted that DNA methylation inhibits the binding of transcription factors, thereby down-regulating gene expression. However, so far, little is known about the epigenetic factors leading to the clinical onset of mastocytosis. Therefore, it is essential to identify possible epigenetic predictors, indicators of disease progression, and their link to the clinical picture to establish appropriate management and a therapeutic strategy. The aim of this study was to analyze genome-wide methylation profiles to identify differentially methylated regions (DMRs) in patients with mastocytosis compared to healthy individuals, as well as the genes located in those regulatory regions. Genome-wide DNA methylation profiling was performed in peripheral blood collected from 80 adult patients with indolent systemic mastocytosis (ISM), the most prevalent subvariant of mastocytosis, and 40 healthy adult volunteers. A total of 117 DNA samples met the criteria for the bisulfide conversion step and microarray analysis. Genome-wide DNA methylation analysis was performed using a MethylationEPIC BeadChip kit. Further analysis was focused on the genomic regions rather than individual CpG sites. Co-methylated regions (CMRs) were assigned via the CoMeBack method. To identify DMRs between the groups, a linear regression model with age as the covariate on CMRs was performed using Limma. Using the available data for cases only, an association analysis was performed between methylation status and tryptase levels, as well as the context of allergy, and anaphylaxis. KEGG pathway mapping was used to identify genes differentially expressed in anaphylaxis. Based on the DNA methylation results, the expression of 18 genes was then analyzed via real-time PCR in 20 patients with mastocytosis and 20 healthy adults. A comparison of the genome-wide DNA methylation profile between the mastocytosis patients and healthy controls revealed significant differences in the methylation levels of 85 selected CMRs. Among those, the most intriguing CMRs are 31 genes located within the regulatory regions. In addition, among the 10 CMRs located in the promoter regions, 4 and 6 regions were found to be either hypo- or hypermethylated, respectively. Importantly, three oncogenes-FOXQ1, TWIST1, and ERG-were identified as differentially methylated in mastocytosis patients, for the first time. Functional annotation revealed the most important biological processes in which the differentially methylated genes were involved as transcription, multicellular development, and signal transduction. The biological process related to histone H2A monoubiquitination (GO:0035518) was found to be enriched in association with higher tryptase levels, which may be associated with more aberrant mast cells and, therefore, more atypical mast cell disease. The signal in the BAIAP2 gene was detected in the context of anaphylaxis, but no significant differential methylation was found in the context of allergy. Furthermore, increased expression of genes encoding integral membrane components (GRM2 and KRTCAP3) was found in mastocytosis patients. This study confirms that patients with mastocytosis differ significantly in terms of methylation levels in selected CMRs of genes involved in specific molecular processes. The results of gene expression profiling indicate the increased expression of genes belonging to the integral component of the membrane in mastocytosis patients (GRM2 and KRTCAP3). Further work is warranted, especially in relation to the disease subvariants, to identify links between the methylation status and the symptoms and novel therapeutic targets.

The Analysis of a Genome-Wide Association Study (GWAS) of Overweight and Obesity in Psoriasis.

There is evidence that the concomitance of psoriasis and obesity may originate from the interplay between multiple genetic pathways and involve gene−gene interactions. The aim of this study was to compare the genetic background related to obesity among psoriatic patients versus healthy controls by means of a Genome-Wide Association Study (GWAS). A total of 972 psoriatic patients and a total of 5878 healthy donors were enrolled in this study. DNA samples were genotyped for over 500,000 single nucleotide polymorphisms (SNPs) using Infinium CoreExome BeadChips (Illumina, San Diego, CA, USA). Statistical analysis identified eleven signals (p < 1 × 10−5) associated with BMI across the study groups and revealed a varying effect size in each sub-cohort. Seven of the alternative alleles (rs1558902 in the FTO gene, rs696574 in the CALCRL gene, as well as rs10968110, rs4551082, rs4609724, rs9320269, and rs2338833,) are associated with increased BMI among all psoriatic patients and four (rs1556519 in the ITLN2 gene, rs12972098 in the AC003006.7 gene, rs12676670 in the PAG1 gene, and rs1321529) are associated with lower BMI. The results of our study may lead to further insights into the understanding of the pathogenesis of obesity among psoriatic patients.

The Role of Lysine-Specific Demethylase 1 (LSD1) in Shaping the Endothelial Inflammatory Response.

BACKGROUND/AIMS: Inflammation is the body's natural response to stress in the broadest sense. The regulatory mechanisms that control this process, some of which are still unclear, are needed to balance the immune response, but also when insufficient, can cause immunodeficiency resulting in infection, cancer, neurodegeneration or other serious disorders. In this study, we focused on defining the role of lysine-specific demethylase 1 (LSD1), an enzyme involved in modulating the methylation state of lysine, including histone and non-histone proteins, in shaping the inflammatory profile of endothelial cells. METHODS: To determine the role of LSD1 in the inflammatory response of ECs, cells were stimulated with lipopolysaccharide (100 ng/ml LPS) in the presence and absence of an LSD1 inhibitor (2-PCPA). A transcription model of LSD1 deficient cells (HMEC-1 LSD1 KD) obtained by lentiviral shRNA transduction was also used. The indicated cellular models were analyzed by gene profiling, monitoring of p65 shuttling by Western blotting and immunofluorescence staining. Also chromatin immunoprecipitation (ChIP) was performed to identify the interactions between selected: IL-6/p65 and LSD1. RESULTS: Analysis of both experimental models revealed an altered inflammatory response following both LSD1 inhibition and LSD1 silencing. We observed decreased U-937 monocytes recruitment to LPS-activated endothelial cells and decreased extracellular secretion of many proinflammatory cytokines, also confirmed at the transcript level by RT-qPCR. Monitoring of the LPS-induced p65 translocation revealed inhibition of the NF-kB subunit in LSD1 KD vs nonT as well as due to pretreatment of 2-PCPA cells. Gene profiling performed with RNA microarrays confirmed the obtained biochemical data at the transcript level. CONCLUSION: In conclusion, the conducted studies showed a proinflammatory profile of LSD1 activity in endothelial cells, revealed by the inhibition of the enzyme activity and confirmed at the transcriptional level by the inhibition of its expression. Although we found significant changes in the modification of interactions between monocytes and endothelial cells as well as in cytokine/chemokine release and expression that were consistent with the altered NF-κB-p65 translocation into the nucleus, we did not identify a direct interaction between LSD1 and the transcription factor. Our finding may have important implications for prevention of cardiovascular diseases at their first stage - activation of the endothelium as well as for tumor cell biology, providing evidence for the use of LSD1 inhibitors to reduce the inflammatory response, which enhances tumor tissue remodeling, angiogenesis and metastasis.

Upper-airway dysbiosis related to frequent sweets consumption increases the risk of asthma in children with chronic rhinosinusitis.

BACKGROUND: Innate immunity response to local dysbiosis seems to be one of the most important immunologic backgrounds of chronic rhinosinusitis (CRS) and concomitant asthma. We aimed to assess clinical determinants of upper-airway dysbiosis and its effect on nasal inflammatory profile and asthma risk in young children with CRS. METHODS: We recruited one hundred and thirty-three children, aged 4-8 years with doctor-diagnosed CRS with or without asthma. The following procedures were performed in all participants: face-to-face standardized Sinus and Nasal Quality of Life questionnaire, skin prick test, taste perception testing, nasopharynx swab, and sampling of the nasal mucosa. Upper-airway dysbiosis was defined separately by asthma-specific microbiome composition and reduced biodiversity. Multivariate methods were used to define the risk factors for asthma and upper-airway dysbiosis and their specific inflammatory profile of nasal mucosa. RESULTS: The asthma-specific upper-airway microbiome composition reflected by the decreased ratio of Patescibacteria/Actinobacteria independently of atopy increased the risk of asthma (OR:8.32; 95%CI: 2.93-23.6). This asthma-specific microbiome composition was associated with ≥ 7/week sweet consumption (OR:2.64; 95%C:1.11-6.28), reduced biodiversity (OR:3.83; 95%CI:1.65-8.87), the presence of Staphylococcus strains in the nasopharynx (OR:4.25; 95%CI:1.12-16.1), and lower expression of beta-defensin 2, IL-5, and IL-13 in the nasal mucosa. The reduced biodiversity was associated with frequent antibiotic use and with a higher nasal expression of IL-17 and T1R3 (sweet taste receptor). In asthmatic children, reduced sweet taste perception was observed. CONCLUSIONS: Specific upper-airway dysbiosis related to frequent sweet consumption, frequent antibiotic courses, and altered nasal immune function increases the risk of asthma in young children with CRS.

Cortisol concentration affects fat and muscle mass among Polish children aged 6-13 years.

BACKGROUND: Cortisol is a steroid hormone acting as a stress hormone, which is crucial in regulating homeostasis. Previous studies have linked cortisol concentration to body mass and body composition. METHODS: The investigations were carried out in 2016-2017. A total of 176 children aged 6-13 years in primary schools in central Poland were investigated. Three types of measurements were performed: anthropometric (body weight and height, waist and hip circumferences), body composition (fat mass FM (%), muscle mass - MM (%), body cellular mass - BCM (%), total body water - TBW (%)), and cortisol concentration using saliva of the investigated individuals. Information about standard of living, type of feeding after birth, parental education and maternal trauma during pregnancy was obtained with questionnaires. RESULTS: The results of regression models after removing the environmental factors (parental education, standard of living, type of feeding after birth, and maternal trauma during pregnancy) indicate a statistically significant association between the cortisol concentration and fat mass and muscle mass. The cortisol concentration was negatively associated with FM (%) (Beta=-0.171; p = 0.026), explaining 2.32 % of the fat mass variability and positively associated with MM (%) (Beta = 0.192; p = 0.012) explaining 3.09 % of the muscle mass variability. CONCLUSIONS: Cortisol concentration affects fat and muscle mass among Polish children. TRIAL REGISTRATION: The Ethical Commission at the University of Lodz (nr 19/KBBN-UL/II/2016).

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe.

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies.

Association of saliva 25(OH)D concentration with body composition and proportion among pre-pubertal and pubertal Polish children.

OBJECTIVES: Due to increasing problems with obesity and vitamin D deficiency among children, studies that tackle both problems together are needed. METHODS: Data were collected from 182 randomly selected children aged 6-13 years in primary schools in central Poland. Measures included anthropometric dimensions, body composition, questionnaires completed by participants' parents, and saliva samples. The level of 25(OH)D was assessed from the saliva samples using an enzyme-linked immunosorbent assay kit. The children were divided into two groups: pre-pubertal (girls below 10 years and boys below 11 years) and pubertal individuals (girls above 10 years and boys above 11 years). RESULTS: The 25(OH)D concentrations were higher in late spring (June) among pre-pubertal children than in the autumn (November-December) among pubertal children. The level of 25(OH)D was positively correlated with body cell mass (BCM,%) among all children (pubertal: R = 0.20, P = .044; pre-pubertal: R = 0.23, P = .041) and inversely associated with waist-to-hip ratio (WHR) among pubertal children of both sexes (R = -0.25; P = .031). The stepwise regression analysis revealed that investigation in spring (June) and breastfeeding was associated with increased muscle mass (MM, %) (beta = 0.253, P = .003 and beta = 0.225, P = .005, respectively) and total body water (TBW, %) (beta = 0.276, P = .004 and beta = 0.246, P = .011, respectively) and was associated with decreased body mass index (BMI; beta = -0.222, P = .024 and beta = -0.269, P = .009, respectively) and fat mass (%) (beta = -0.288, P = .003 and beta = -0.266, P = .005, respectively). CONCLUSIONS: Season of salivary sampling and breastfeeding status were more strongly associated with body components, BMI and WHR, than 25(OH)D concentrations.

Results from a Genome-Wide Association Study (GWAS) in Mastocytosis Reveal New Gene Polymorphisms Associated with WHO Subgroups.

Mastocytosis is rare disease in which genetic predisposition is not fully understood. The aim of this study was to analyze associations between mastocytosis and single nucleotide polymorphisms (SNPs) by a genome-wide association study (GWAS) approach. A total of 234 patients were enrolled in our study, including 141 with cutaneous mastocytosis (CM; 78 children and 63 adults) and 93 with systemic mastocytosis (SM, all adults). The control group consisted of 5606 healthy individuals. DNA samples from saliva or blood were genotyped for 551 945 variants using DNA microarrays. The prevalence of certain SNPs was found to vary substantially when comparing patients and healthy controls: rs10838094 of 5OR51Q1 was less frequently detected in CM and SM patients (OR = 0.2071, p = 2.21 × 10-29), rs80138802 in ABCA2 (OR = 5.739, p = 1.98 × 10-28), and rs11845537 in OTX2-AS1 (rs11845537, OR = 6.587, p = 6.16 × 10-17) were more frequently detected in CM in children and adults. Additionally, we found that rs2279343 in CYP2B6 and rs7601511 in RPTN are less prevalent in CM compared to controls. We identified a number of hitherto unknown associations between certain SNPs and CM and/or SM. Whether these associations are clinically relevant concerning diagnosis, prognosis, or prevention remains to be determined in future studies.

Genetic and Epigenetic Aspects of Atopic Dermatitis.

Atopic dermatitis is a heterogeneous disease, in which the pathogenesis is associated with mutations in genes encoding epidermal structural proteins, barrier enzymes, and their inhibitors; the role of genes regulating innate and adaptive immune responses and environmental factors inducing the disease is also noted. Recent studies point to the key role of epigenetic changes in the development of the disease. Epigenetic modifications are mainly mediated by DNA methylation, histone acetylation, and the action of specific non-coding RNAs. It has been documented that the profile of epigenetic changes in patients with atopic dermatitis (AD) differs from that observed in healthy people. This applies to the genes affecting the regulation of immune response and inflammatory processes, e.g., both affecting Th1 bias and promoting Th2 responses and the genes of innate immunity, as well as those encoding the structural proteins of the epidermis. Understanding of the epigenetic alterations is therefore pivotal to both create new molecular classifications of atopic dermatitis and to enable the development of personalized treatment strategies.

Pathogenesis of psoriasis in the "omic" era. Part III. Metabolic disorders, metabolomics, nutrigenomics in psoriasis.

Psoriasis is a systemic disease that is strictly connected with metabolic disorders (insulin resistance, atherogenic dyslipidemia, arterial hypertension, and cardiovascular diseases). It occurs more often in patients with a more severe course of the disease. Obesity is specially an independent risk factor and it is associated with a worse treatment outcome because of the high inflammatory activity of visceral fatty tissue and the production of inflammatory mediators involved in the development of both psoriasis and metabolic disorders. However, in psoriasis the activation of the Th17/IL-17 and the abnormalities in the Th17/Treg balance axis are observed, but this pathomechanism does not fully explain the frequent occurrence of metabolic disorders. Therefore, there is a need to look for better biomarkers in the diagnosis, prognosis and monitoring of concomitant disorders and therapeutic effects in psoriasis. In addition, the education on the use of a proper diet as a prophylaxis for the development of the above disorders is an important element of holistic care for a patient with psoriasis. Diet may affect gene expression due to epigenetic modification which encompasses interactions of environment, nutrition and diseases. Patients with psoriasis should be advised to adopt proper diet and dietician support.

Pathogenesis of psoriasis in the "omic" era. Part II. Genetic, genomic and epigenetic changes in psoriasis.

Psoriasis is a multifactorial disease in which genetic, environmental and epigenetic factors regulating gene expression play a key role. In the "genomic era", genome-wide association studies together with target genotyping platforms performed in different ethnic populations have found more than 50 genetic susceptible markers associated with the risk of psoriasis which have been identified so far. Up till now, the strongest association with the risk of the disease has been proved for HLA-C*06 gene. The majority of other psoriasis risk SNPs are situated near the genes encoding molecules involved in adaptive and innate immunity, and skin barrier function. Many contemporary studies indicate that the epigenetic changes: histone modification, promoter methylations, long non-coding and micro-RNA hyperexpression are considered as factors contributing to psoriasis pathogenesis as they regulate abnormal keratinocyte differentiation and proliferation, aberrant keratinocytes - inflammatory cells communication, neoangiogenesis and chronic inflammation. The circulating miRNAs detected in the blood may become specific markers in the diagnosis, prognosis and response to the treatment of the disease. The inhibition of expression in selected miRNAs may be a new promising therapy option for patients with psoriasis.

Pathogenesis of psoriasis in the "omic" era. Part IV. Epidemiology, genetics, immunopathogenesis, clinical manifestation and treatment of psoriatic arthritis.

Psoriatic arthritis (PsA) is a chronic, progressive, inflammatory arthropathy associated with psoriasis as well as a complex pathogenesis. Genetic and environmental factors trigger the development of the immune-mediated auto-inflammatory response in different sites: skin, bone marrow, entheses and synovial tissues. Studies of the last two decades have changed the view of PsA from a mild, non-progressive arthritis to an inflammatory systemic disease with serious health consequences, not only associated with joint dysfunction, but also with an increased risk of cardiovascular disease and socioeconomic consequences with significantly reduced quality of life. The joint damage starts early in the course of the disease, thus early recognition and treatment with modern biological treatments, which may modify the natural history and slow down progression of this debilitating disease, is essential for the patient long-term outcome.

Pathogenesis of psoriasis in the "omic" era. Part I. Epidemiology, clinical manifestation, immunological and neuroendocrine disturbances.

Psoriasis is a common, chronic, inflammatory, immune-mediated skin disease affecting about 2% of the world's population. According to current knowledge, psoriasis is a complex disease that involves various genes and environmental factors, such as stress, injuries, infections and certain medications. The chronic inflammation of psoriasis lesions develops upon epidermal infiltration, activation, and expansion of type 1 and type 17 Th cells. Despite the enormous progress in understanding the mechanisms that cause psoriasis, the target cells and antigens that drive pathogenic T cell responses in psoriatic lesions are still unproven and the autoimmune basis of psoriasis still remains hypothetical. However, since the identification of the Th17 cell subset, the IL-23/Th17 immune axis has been considered a key driver of psoriatic inflammation, which has led to the development of biologic agents that target crucial elements of this pathway. Here we present the current understanding of various aspects in psoriasis pathogenesis.

1H-Benzo[d]Imidazole Derivatives Affect MmpL3 in Mycobacterium tuberculosis.

1H-benzo[d]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequencing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1H-benzo[d]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.

Association between body height and month of birth among women of European origin in northern and southern hemispheres.

OBJECTIVES: The purpose of this study was to examine the potential association between month of birth and body height among women in northern and southern hemispheres. METHODS: Body heights of adult women of European origin born between 1935 and 1981 who lived in Poland (N = 3,933) and in Australia (N = 1,118) were examined in relation to month of birth by analysis of variance. RESULTS: No association between month of birth and body height was observed in either Polish or Australian women. For Polish women, a clear, statistically significant secular trend in body height was confirmed for the analyzed period (P < .0001). No such trend occurred among the Australian women. CONCLUSIONS: Results do not confirm a significant association between month of birth and adult body height in women. It is, however, important to see a difference in secular trends, which was large in Polish women and nonexistent in Australian females.

High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.