Effects of prior Zika infection on male fertility outcomes

Zika virus (ZIKV), a mosquito-borne, fever-causing pathogen, swept through Latin America in 2015 and 2016. Previously thought to be a mild febrile illness of little clinical importance, unusual characteristics of ZIKV were newly identified during the novel American pandemic – prolonged shedding in semen, sexual transmissibility, and congenital anomalies. One study examined ZIKV’s effect on semen measures, demonstrating a decline in sperm count and testosterone after acute ZIKV infection that recovered after several weeks. Fever itself can impair sperm production, confounding interpretation of these results. To investigate the long-term effect of ZIKV infection on human male fertility, we conducted a cohort study of young healthy men in two cities that experienced intense ZIKV transmission 1-2 years prior to the study; namely, León, Nicaragua, and Iquitos, Peru. Healthy men aged 18-40 years were enrolled between July 2018 and March 2019, after the ZIKV epidemic had subsided at each site. A baseline questionnaire recorded demographic, clinical, and epidemiological data; these data and symptoms of infection were updated at quarterly visits. The participating men provided semen and blood samples quarterly for 12 months in León, and 9 months in Iquitos, although loss to follow up was high (>40%). Fresh semen and blood analysis were performed on-site. Semen and serum markers of fertility were compared between ZIKV-seropositive and seronegative men, averaging each man’s results across their visits. Data were analyzed using Student’s t-test for continuous data comparisons of means between groups of interest. We enrolled 110 men (50 in Peru, 60 in Nicaragua). Median age was 23 years (IQR 19-27) and 55% were students. More than 90% had evidence of prior dengue infection by IgG, and 20% reported prior chikungunya infection. 39% were seropositive for ZIKV infection at enrollment. There was no association between age and odds of ZIKV positivity. None were febrile at enrollment, though 6 reported a fever within the previous week. 26% of men reported having impregnated a partner, with no difference by ZIKV status. There were no differences at enrollment or averaged across the study duration by ZIKV exposure in semen pH, total sperm count, ejaculate volume, percent progressive motile sperm, vitality, or round cell count. We observed lower average FSH levels in participants seropositive for Zika (p=0.04). These preliminary data suggest that any effect of Zika virus infection on male fertility is likely short-lived.Bachelor of Science in Public Healt

Disruption of genomic neighbourhood at the imprinted IGF2-H19 locus in Beckwith-Wiedemann syndrome and Silver-Russell syndrome

Hyper- and hypomethylation at the IGF2-H19 imprinting control region (ICR) result in reciprocal changes in IGF2-H19 expression and the two contrasting growth disorders, Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). DNA methylation of the ICR controls the reciprocal imprinting of IGF2 and H19 by preventing the binding of the insulator protein, CTCF. We here show that local changes in histone modifications and CTCF-cohesin binding at the ICR in BWS and SRS together with DNA methylation correlate with the higher order chromatin structure at the locus. In lymphoblastoid cells from control individuals, we found the repressive histone H3K9me3 and H4K20me3 marks associated with the methylated paternal ICR allele and the bivalent H3K4me2/H3K27me3 mark together with H3K9ac and CTCF-cohesin associated with the non-methylated maternal allele. In patient-derived cell lines, the mat/pat asymmetric distribution of these epigenetic marks was lost with H3K9me3 and H4K20me3 becoming biallelic in the BWS and H3K4me2, H3K27me3 and H3K9ac together with CTCF-cohesin becoming biallelic in the SRS. We further show that in BWS and SRS cells, there is opposing chromatin looping conformation mediated by CTCF-cohesin binding sites surrounding the locus. In normal cells, lack of CTCF-cohesin binding at the paternal ICR is associated with monoallelic interaction between two CTCF sites flanking the locus. CTCF-cohesin binding at the maternal ICR blocks this interaction by associating with the CTCF site downstream of the enhancers. The two alternative chromatin conformations are differently favoured in BWS and SRS likely predisposing the locus to the activation of IGF2 or H19, respectively. © The Author 2011. Published by Oxford University Press

Behavior of bicarbonate ion between capillary blood and saliva during acute exercise

This study includes the monitoring of the behavior of the bicarbonate ions concentration in the capillary blood and in saliva, with the main objective of establishing reliable parameters of the relationship of this variable between these two fluid compartments. The tests were conducted under a modified Balk protocol in the cycloergometer with progressively increasing load of 50 to 50 watts and measurement of bicarbonate ion was held in a gas analyzer ABL 800 FLEX Radiometer, assigned by UNIMED of São Carlos. It was necessary to develop 70 glass capillary 110μL and one adapter to collect saliva, manufactured in PVC. The volunteers (n=10) were selected athletes of the city of São Carlos and local students, all healthy and not using any type of drug, with an average age of 24 ± 4 years. Statistical analysis was done through the InStat software, using the comparison of the average concentration of bicarbonate ion between blood capillary and saliva at each stage (1-7º), getting a p-value \u3c0.0001 and significant confidence interval of 0 .95. Average concentration of bicarbonate were: Blood/Saliva: (1) 23.5 /3.2 - (2) 23.1/2.9 - (3) 21.4/4.0 - (4) 19.7/3.2 - (5) 15.9/2.9 - (6) 19.5/4.0 - (7) 16.6/4.1 mmol/L. The results allowed the establishment of a correlation between the fluids and demonstrates the effectiveness of the proposed method

Disruption of genomic neighbourhood at the imprinted IGF2-H19 locus in Beckwith–Wiedemann syndrome and Silver–Russell syndrome

Hyper- and hypomethylation at the IGF2-H19 imprinting control region (ICR) result in reciprocal changes in IGF2-H19 expression and the two contrasting growth disorders, Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS). DNA methylation of the ICR controls the reciprocal imprinting of IGF2 and H19 by preventing the binding of the insulator protein, CTCF. We here show that local changes in histone modifications and CTCF–cohesin binding at the ICR in BWS and SRS together with DNA methylation correlate with the higher order chromatin structure at the locus. In lymphoblastoid cells from control individuals, we found the repressive histone H3K9me3 and H4K20me3 marks associated with the methylated paternal ICR allele and the bivalent H3K4me2/H3K27me3 mark together with H3K9ac and CTCF–cohesin associated with the non-methylated maternal allele. In patient-derived cell lines, the mat/pat asymmetric distribution of these epigenetic marks was lost with H3K9me3 and H4K20me3 becoming biallelic in the BWS and H3K4me2, H3K27me3 and H3K9ac together with CTCF–cohesin becoming biallelic in the SRS. We further show that in BWS and SRS cells, there is opposing chromatin looping conformation mediated by CTCF–cohesin binding sites surrounding the locus. In normal cells, lack of CTCF–cohesin binding at the paternal ICR is associated with monoallelic interaction between two CTCF sites flanking the locus. CTCF–cohesin binding at the maternal ICR blocks this interaction by associating with the CTCF site downstream of the enhancers. The two alternative chromatin conformations are differently favoured in BWS and SRS likely predisposing the locus to the activation of IGF2 or H19, respectively

Long-range chromatin interactions at the mouse Igf2/H19 locus reveal a novel paternally expressed long non-coding RNA

Parental genomic imprinting at the Igf2/H19 locus is controlled by a methylation-sensitive CTCF insulator that prevents the access of downstream enhancers to the Igf2 gene on the maternal chromosome. However, on the paternal chromosome, it remains unclear whether long-range interactions with the enhancers are restricted to the Igf2 promoters or whether they encompass the entire gene body. Here, using the quantitative chromosome conformation capture assay, we show that, in the mouse liver, the endodermal enhancers have low contact frequencies with the Igf2 promoters but display, on the paternal chromosome, strong interactions with the intragenic differentially methylated regions 1 and 2. Interestingly, we found that enhancers also interact with a so-far poorly characterized intergenic region of the locus that produces a novel imprinted long non-coding transcript that we named the paternally expressed Igf2/H19 intergenic transcript (PIHit) RNA. PIHit is expressed exclusively from the paternal chromosome, contains a novel discrete differentially methylated region in a highly conserved sequence and, surprisingly, does not require an intact ICR/H19 gene region for its imprinting. Altogether, our data reveal a novel imprinted domain in the Igf2/H19 locus and lead us to propose a model for chromatin folding of this locus on the paternal chromosome

Changes in the Transcriptome of Human Astrocytes Accompanying Oxidative Stress-Induced Senescence

Aging is a major risk factor for many neurodegenerative disorders. A key feature of aging biology that may underlie these diseases is cellular senescence. Senescent cells accumulate in tissues with age, undergo widespread changes in gene expression, and typically demonstrate altered, pro-inflammatory profiles. Astrocyte senescence has been implicated in neurodegenerative disease, and to better understand senescence-associated changes in astrocytes, we investigated changes in their transcriptome using RNA sequencing. Senescence was induced in human fetal astrocytes by transient oxidative stress. Brain-expressed genes, including those involved in neuronal development and differentiation, were downregulated in senescent astrocytes. Remarkably, several genes indicative of astrocytic responses to injury were also downregulated, including glial fibrillary acidic protein and genes involved in the processing and presentation of antigens by major histocompatiblity complex class II proteins, while pro-inflammatory genes were upregulated. Overall, our findings suggest that senescence-related changes in the function of astrocytes may impact the pathogenesis of age-related brain disorders

Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination

Studies have suggested that antigen receptor loci adopt contracted conformations to promote long-distance interactions between gene segments during V(D)J recombination. The Tcra/Tcrd locus is unique because it undergoes highly divergent Tcrd and Tcra recombination programs in CD4−CD8− double negative (DN) and CD4+CD8+ double positive (DP) thymocytes, respectively. Using three-dimensional fluorescence in situ hybridization, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus. We found that the 3′ portion of the locus is contracted in DN and DP thymocytes but not in B cells. Remarkably, the 5′ portion of the locus is contracted in DN thymocytes but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving Vδ gene segments distributed over 1 Mb, whereas the unique 3′-contracted, 5′-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3′ of the available Vα gene segments. This would maintain a large pool of distal 5′ Vα gene segments for subsequent rounds of recombination. Thus, distinct contracted conformations of the Tcra/Tcrd locus may facilitate a transition from a Tcrd to a Tcra mode of recombination during thymocyte development

Somatically acquired hypomethylation of IGF2 in breast and colorectal cancer

The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2–5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI

Cohesin, condensin, and the intramolecular centromere loop together generate the mitotic chromatin spring

During mitosis, spindle microtubule force is balanced by the combined activities of the cohesin and condensin SMC complexes and intramolecular pericentric chromatin loops

Genome-wide studies of the multi-zinc finger Drosophila Suppressor of Hairy-wing protein in the ovary

The Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is a globally expressed, multi-zinc finger (ZnF) DNA-binding protein. Su(Hw) forms a classic insulator when bound to the gypsy retrotransposon and is essential for female germline development. These functions are genetically separable, as exemplified by Su(Hw)f that carries a defective ZnF10, causing a loss of insulator but not germline function. Here, we completed the first genome-wide analysis of Su(Hw)-binding sites (SBSs) in the ovary, showing that tissue-specific binding is not responsible for the restricted developmental requirements for Su(Hw). Mapping of ovary Su(Hw)f SBSs revealed that female fertility requires binding to only one third of the wild-type sites. We demonstrate that Su(Hw)f retention correlates with binding site affinity and partnership with Modifier of (mdg4) 67.2 protein. Finally, we identify clusters of co-regulated ovary genes flanked by Su(Hw)f bound sites and show that loss of Su(Hw) has limited effects on transcription of these genes. These data imply that the fertility function of Su(Hw) may not depend upon the demarcation of transcriptional domains. Our studies establish a framework for understanding the germline Su(Hw) function and provide insights into how chromatin occupancy is achieved by multi-ZnF proteins, the most common transcription factor class in metazoans