Genome-Wide DNA Methylation in Policemen Working in Cities Differing by Major Sources of Air Pollution.

DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and it can serve as a useful biomarker of prior environmental exposure and future health outcomes. This study focused on DNA methylation profiles in a human cohort, comprising 125 nonsmoking city policemen (sampled twice), living and working in three localities (Prague, Ostrava and Ceske Budejovice) of the Czech Republic, who spent the majority of their working time outdoors. The main characterization of the localities, differing by major sources of air pollution, was defined by the stationary air pollution monitoring of PM2.5, B[a]P and NO2. DNA methylation was analyzed by a genome-wide microarray method. No season-specific DNA methylation pattern was discovered; however, we identified 13,643 differentially methylated CpG loci (DML) for a comparison between the Prague and Ostrava groups. The most significant DML was cg10123377 (log2FC = -1.92, p = 8.30 × 10-4) and loci annotated to RPTOR (total 20 CpG loci). We also found two hypomethylated loci annotated to the DNA repair gene XRCC5. Groups of DML annotated to the same gene were linked to diabetes mellitus (KCNQ1), respiratory diseases (PTPRN2), the dopaminergic system of the brain and neurodegenerative diseases (NR4A2). The most significant possibly affected pathway was Axon guidance, with 86 potentially deregulated genes near DML. The cluster of gene sets that could be affected by DNA methylation in the Ostrava groups mainly includes the neuronal functions and biological processes of cell junctions and adhesion assembly. The study demonstrates that the differences in the type of air pollution between localities can affect a unique change in DNA methylation profiles across the human genome.

Accuracy of molecular drug susceptibility testing amongst tuberculosis patients in Karakalpakstan, Uzbekistan.

OBJECTIVES: In this retrospective study, we evaluated the diagnostic accuracy of molecular tests (MT) for the detection of DR-TB, compared to the gold standard liquid-based drug susceptibility testing (DST) in Karakalpakstan. METHODS: A total of 6670 specimens received in the Republican TB No 1 Hospital Laboratory of Karakalpakstan between January and July 2017 from new and retreatment patients were analysed. Samples were tested using Xpert MTB/RIF and line probe assays (LPA) for the detection of mutations associated with resistance. The sensitivity and specificity of MTs were calculated relative to results based on DST. RESULTS: The accuracy of MT for detection of rifampicin resistance was high, with sensitivity and specificity over 98%. However, we observed reduced sensitivity of LPA for detection of resistance; 86% for isoniazid (95% CI 82-90%), 86% for fluoroquinolones (95% CI 68-96%), 70% for capreomycin (95% CI 46-88%) and 23% for kanamycin (95% CI 13-35%). CONCLUSIONS: We show that MTs are a useful tool for rapid and safe diagnosis of DR-TB; however, clinicians should be aware of their limitations. Although detection of rifampicin resistance was highly accurate, our data suggest that resistance mutations circulating in the Republic of Karakalpakstan for other drugs were not detected by the methods used here. This merits further investigation.

The Biological Effects of Complete Gasoline Engine Emissions Exposure in a 3D Human Airway Model (MucilAirTM) and in Human Bronchial Epithelial Cells (BEAS-2B).

The biological effects induced by complete engine emissions in a 3D model of the human airway (MucilAirTM) and in human bronchial epithelial cells (BEAS-2B) grown at the air-liquid interface were compared. The cells were exposed for one or five days to emissions generated by a Euro 5 direct injection spark ignition engine. The general condition of the cells was assessed by the measurement of transepithelial electrical resistance and mucin production. The cytotoxic effects were evaluated by adenylate kinase (AK) and lactate dehydrogenase (LDH) activity. Phosphorylation of histone H2AX was used to detect double-stranded DNA breaks. The expression of the selected 370 relevant genes was analyzed using next-generation sequencing. The exposure had minimal effects on integrity and AK leakage in both cell models. LDH activity and mucin production in BEAS-2B cells significantly increased after longer exposures; DNA breaks were also detected. The exposure affected CYP1A1 and HSPA5 expression in MucilAirTM. There were no effects of this kind observed in BEAS-2B cells; in this system gene expression was rather affected by the time of treatment. The type of cell model was the most important factor modulating gene expression. In summary, the biological effects of complete emissions exposure were weak. In the specific conditions used in this study, the effects observed in BEAS-2B cells were induced by the exposure protocol rather than by emissions and thus this cell line seems to be less suitable for analyses of longer treatment than the 3D model.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

BACKGROUND: Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. METHODS: Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. RESULTS: Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. CONCLUSION: GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

Pre-Clinical Tools for Predicting Drug Efficacy in Treatment of Tuberculosis.

Combination therapy has, to some extent, been successful in limiting the emergence of drug-resistant tuberculosis. Drug combinations achieve this advantage by simultaneously acting on different targets and metabolic pathways. Additionally, drug combination therapies are shown to shorten the duration of therapy for tuberculosis. As new drugs are being developed, to overcome the challenge of finding new and effective drug combinations, systems biology commonly uses approaches that analyse mycobacterial cellular processes. These approaches identify the regulatory networks, metabolic pathways, and signaling programs associated with M. tuberculosis infection and survival. Different preclinical models that assess anti-tuberculosis drug activity are available, but the combination of models that is most predictive of clinical treatment efficacy remains unclear. In this structured literature review, we appraise the options to accelerate the TB drug development pipeline through the evaluation of preclinical testing assays of drug combinations.

The Transgenerational Transmission of the Paternal Type 2 Diabetes-Induced Subfertility Phenotype.

Diabetes is a chronic metabolic disorder characterized by hyperglycemia and associated with many health complications due to the long-term damage and dysfunction of various organs. A consequential complication of diabetes in men is reproductive dysfunction, reduced fertility, and poor reproductive outcomes. However, the molecular mechanisms responsible for diabetic environment-induced sperm damage and overall decreased reproductive outcomes are not fully established. We evaluated the effects of type 2 diabetes exposure on the reproductive system and the reproductive outcomes of males and their male offspring, using a mouse model. We demonstrate that paternal exposure to type 2 diabetes mediates intergenerational and transgenerational effects on the reproductive health of the offspring, especially on sperm quality, and on metabolic characteristics. Given the transgenerational impairment of reproductive and metabolic parameters through two generations, these changes likely take the form of inherited epigenetic marks through the germline. Our results emphasize the importance of improving metabolic health not only in women of reproductive age, but also in potential fathers, in order to reduce the negative impacts of diabetes on subsequent generations.

Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds.

In vitro spermatogenesis (IVS) has already been successfully achieved in rodents by organotypic and soft matrix culture systems. However, the former does not allow single cell input, and the latter presents as a simple thick layer in which all cells are embedded. We explored a new culture system using a mouse model by employing an alginate-based hydrogel and 3D bioprinting, to control scaffold design and cell deposition. We produced testicular constructs consisting of printed cell-free scaffolds (CFS) with prepubertal testicular cells (TC) in their easy-to-access macropores. Here, the pores represented the only cell compartment (TC/CFS). Double-cell compartment testicular constructs were achieved by culturing magnetic-activated cell sorting-enriched epithelial cells in the pores of interstitial cell-laden scaffolds (CD49f+/CLS). Cell spheres formed in the pores in the weeks following cell seeding on both CFS and CLS. Although restoration of the tubular architecture was not observed, patches of post-meiotic cells including elongated spermatids were found in 66% of TC/CFS. Differentiation up to the level of round spermatids and elongated spermatids was observed in all and 33% of CD49f+/CLS constructs, respectively. Organ culture served as the reference method for IVS, with complete spermatogenesis identified in 80% of cultivated prepubertal tissue fragments. So far, this is the first report applying a 3D bioprinting approach for IVS. Further optimization of the scaffold design and seeding parameters might be permissive for tubular architecture recreation and thereby increase the efficiency of IVS in printed testicular constructs. While it remains to be tested whether the gametes generated on the alginate-based scaffolds can support embryogenesis following IVF, this IVS approach might be useful for (patho)physiological studies and drug-screening applications.

Transgenerational inheritance of susceptibility to diabetes-induced male subfertility.

Male infertility is a worldwide problem associated with genetic background, environmental factors, and diseases. One of the suspected contributing factors to male infertility is diabetes mellitus. We investigated the molecular and morphological changes in sperms and testicular tissue of diabetic males. The study was performed in streptozotocin-induced type 1 diabetes mouse model. Diabetes decreased sperm concentration and viability and increased sperm apoptosis. Changes in protamine 1/protamine 2 ratio indicated reduced sperm quality. The testicular tissue of diabetic males showed significant tissue damage, disruption of meiotic progression, and changes in the expression of genes encoding proteins important for spermiogenesis. Paternal diabetes altered sperm quality and expression pattern in the testes in offspring of two subsequent generations. Our study revealed that paternal diabetes increased susceptibility to infertility in offspring through gametic alternations. Our data also provide a mechanistic basis for transgenerational inheritance of diabetes-associated pathologies since protamines may be involved in epigenetic regulations.

Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide testing by MTBDRsl in Armenia.

OBJECTIVE/BACKGROUND: The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, second-line aminoglycosides (amikacin [AMK] and kanamycin [KAN]), and cyclic peptides (capreomycin [CAP]) in Mycobacterium tuberculosis. According to data from Global Drug Resistance Surveillance Report (2007), Armenia is counted as a high-burden country for multidrug-resistant tuberculosis (MDR-TB). The estimated burden of MDR-TB in 2012 was 9.4 (7-12) and 43 (38-49) among retreatment TB cases. A total of 92 laboratory confirmed cases were reported to the World Health Organization (57 new and 35 previously treated) out of 511 cases tested for MDR-TB. METHODS: A set of 77 drug-resistant TB isolates during 2011 and 2012 period, being either acid-fast bacterium positive or negative but culture-positive resistant to isoniazid, rifampin, or both according to the GenoType MTBDR plus assay, were consecutively tested using GenoType MTBDRsl. rrs gene analysis and the results from GenoType MTBDRsl were compared with phenotypic drug resistance testing. The DNA preparation method was performed as recommended by the manufacturer (Genotype MTBDR plus version 1.0 and Genotype MTBDRsl version 2.0 Hain Lifescience Nehren, Germany). RESULTS: Aminoglycosides are key drugs for the treatment of MDR-TB. A total of 77 drug-resistant TB and four extensively drug-resistant M. tuberculosis isolates from Armenian TB patients were analyzed to characterize mutations within rrs and to compare with phenotypic drug resistance testing. Simultaneously, the following were identified: 65 (84.41%) rrs wild type (WT), 1 (1.3%) rrs WT MUT1 and MUT2 (WT; A1401G and G1484T), 1 (1.3%) rrs WT1, MUT1 (A1401G), 9 (11.7%) rrs WT1, MUT1 (A1401G), and 1 (1.3%) rrs WT1, MUT1. Mutation at position 1401 in rrs leads to resistance to KAN (7/77=9%), AMK (9/77=11.68%), and CAP (5/77=6.49%). Eleven (14.28%) streptomycin-resistant strains had a rrs mutation. CONCLUSION: Isolates with rrs structural gene mutations were cross-resistant to streptomycin, KAN, CAP, and AMK. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to KAN and AMK. Being the first assessment, these data establish the presence of phenotypic drug-resistant and extensively drug-resistant strains using molecular profiling and are helpful in understanding aminoglycoside resistance on a molecular level.

Target antigens for Hs-14 monoclonal antibody and their various expression in normozoospermic and asthenozoospermic men.

BACKGROUND: Poor semen quality is one of the main causes of infertility. We have generated a set of monoclonal antibodies to human sperm and used them to investigate sperm quality. Some of these antibodies found differences in the expression of proteins between normal sperm and pathological sperm displaying severe defects. One of them was the Hs-14 antibody. The aim of this paper was to determine the target protein of the Hs-14 monoclonal antibody and to investigate the expression of the Hs-14-reacting protein on the sperm of asthenozoospermic men with sperm motility defect and of healthy normozoospermic men. METHODS: Indirect immunofluorescence, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry. RESULTS: The Hs-14 antibody binds fibronectin, ß-tubulin and valosin-containing protein - new name for this protein is transitional endoplasmic reticulum ATPase (TERA). Since the Hs-14 reaction with TERA remained the strongest at the highest antibody dilution, and Hs-14 consistently labelled the same spot or band as the monospecific anti-TERA antibody on immunoblots, we assume that TERA is an Hs-14-specific protein. Binding of fibronectin and ß-tubulin might represent nonspecific cross-reactivity or Hs-14 reaction with similar epitopes of these proteins. A significant difference (P < 0.001) in immunofluorescence staining with Hs-14 was found between the normozoospermic and asthenozoospermic men. CONCLUSION: The Hs-14 antibody enables discrimination between sterile or subfertile asthenozoospermic and fertile normozoospermic men. Decreased levels of TERA in men can be used as a biomarker of reduced fertility.

INTRODUCTION: La pauvre qualité de la semence est l'une des causes d'infertilité. Nous avons généré une série d'anticorps monoclonaux contre le sperme humain et nous l'avons utilisée pour examiner la qualité du sperme. Certains de ces anticorps ont montré des différences d' expression des protéines entre le sperme normal et le sperme pathologique qui a des défauts sévères. L'un d'eux a été l'anticorps Hs-14. Le but de cet article était de déterminer la protéine cible de l'anticorps monoclonal Hs-14 et d'établir l'expression de la protéine réagissant avec Hs-14 sur le sperme des hommes asthénozoospermiques qui ont des défauts de la mobilité du sperme et sur celui des hommes normozoospermiques. MÉTHODES: Immunofluorescence indirecte, electrophorèse sur gel polyacrylamide à une ou deux dimensions, immunoblotting et spectrométrie de masse. RÉSULTATS: L'anticorps Hs-14 s'attache à la fibronectine, à la ß-tubuline et à la protéine TERA (ATPase transitoire de réticulum endoplasmique). Etant donné que la réaction du Hs-14 avec TERA a été la plus forte à la dilution la plus grande de l'anticorps, et que Hs-14 marquait systématiquement la même tache ou bande que l'anticorps mono-spécifique anti-TERA sur les immunoblots, nous supposons que TERA est une protéine spécifique pour Hs-14. L'attachement à la fibronectine et à la ß-tubuline pourrait représenter une réaction croisée non spécifique ou la réaction du Hs-14 avec des épitopes similaires de ces protéines. Une différence significative (P < 0.001) en immunofluorescence avec Hs-14 a été révélée entre hommes normozoospermiques et asthénozoospermiques. CONCLUSIONS: L'anticorps Hs-14 permet de différencier les hommes stériles ou subfertiles asthénozoospermiques des hommes fertiles normozoospermiques. Les niveaux de la TERA chez les hommes pourraient être utilisés comme un marqueur biologique d'une fertilité réduite.

Adverse effect of tetracycline and doxycycline on testicular tissue and sperm parameters in CD1 outbred mice.

Tetracycline and doxycycline are commonly used antibiotics in acne treatment during puberty in humans. The long-term effect of these antibiotics on male reproductive tract development has not been fully elucidated. For this reason we tested the effect of antibiotics on the reproductive parameters of mice males during puberty with the therapeutic dose used in humans, and with lower and higher doses. The outbred mouse strain CD1 with higher heterozygosity was exposed for 14 days at puberty. Adult males at the age of 70 days were used for the measurements. We observed a significant decrease in anogenital distance and thickness of the seminiferous epithelium in the treated animals. Pathological changes in the testes had an impact on sperm quality; a higher number of sperm positively stained with Annexin V and TUNEL and a lower number of acrosome-intact sperm was detected. In conclusion, the treatment of male mice with antibiotics in puberty led to long-lasting effects on reproductive organs and spermatozoa in adult males.

Effect of tetrabrombisphenol A on induction of apoptosis in the testes and changes in expression of selected testicular genes in CD1 mice.

Tetrabromobisphenol A (TBBPA) is a substance widely used in industry as a flame retardant. TBBPA was found in the environment and was detected even in the human body. The effect of this chemical was observed in different cell lines in vitro and it is supposed that TBBPA may affect various hormonal systems in vivo. In this study we examined the effect of TBBPA on the reproductive parameters of two generations of outbred mice in vivo. Experimental and control animals of F1 generation were bred in various conditions to enable evaluation of the possible trans-generational effect. An increased incidence of apoptosis in the testes and changes in the morphometry of seminiferous tubules was detected in the experimental animals. In addition, changes in the expression pattern of selected genes encoding proteins that play an important role during spermatogenesis were observed. In contrast, sperm quality and reproduction were not affected by TBBPA.