T7 polymerase-driven transcription is downregulated in metacyclic promastigotes and amastigotes of Leishmania mexicana.

In our previous work we established a T7 polymerase-driven Tetracycline-inducible protein expression system in Leishmania mexicana (Biagi, 1953). We used this system to analyse gene expression profiles during development of L. mexicana in procyclic and metacyclic promastigotes and amastigotes. The transcription of the gene of interest and the T7 polymerase genes was significantly reduced upon cell differentiation. This regulation is not locus-specific. It depends on untranslated regions flanking open reading frames of the genes analysed. In this paper, we report that the previously established conventional inducible protein expression system may not be suitable for studies on differentiation of species of Leishmania Ross, 1903 and protein expression systems might have certain limitations.

Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels.

Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.

Lead exposure induces changes in 5-hydroxymethylcytosine clusters in CpG islands in human embryonic stem cells and umbilical cord blood.

Prenatal exposure to neurotoxicants such as lead (Pb) may cause stable changes in the DNA methylation (5mC) profile of the fetal genome. However, few studies have examined its effect on the DNA de-methylation pathway, specifically the dynamic changes of the 5-hydroxymethylcytosine (5hmC) profile. Therefore, in this study, we investigate the relationship between Pb exposure and 5mC and 5hmC modifications during early development. To study the changes in the 5hmC profile, we use a novel modification of the Infinium™ HumanMethylation450 assay (Illumina, Inc.), which we named HMeDIP-450K assay, in an in vitro human embryonic stem cell model of Pb exposure. We model Pb exposure-associated 5hmC changes as clusters of correlated, adjacent CpG sites, which are co-responding to Pb. We further extend our study to look at Pb-dependent changes in high density 5hmC regions in umbilical cord blood DNA from 48 mother-infant pairs from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohort. For our study, we randomly selected umbilical cord blood from 24 male and 24 female children from the 1st and 4th quartiles of Pb levels. Our data show that Pb-associated changes in the 5hmC and 5mC profiles can be divided into sex-dependent and sex-independent categories. Interestingly, differential 5mC sites are better markers of Pb-associated sex-dependent changes compared to differential 5hmC sites. In this study we identified several 5hmC and 5mC genomic loci, which we believe might have some potential as early biomarkers of prenatal Pb exposure.

Distribution of CYP17α polymorphism and selected physiochemical factors of uterine leiomyoma in Barbados.

Uterine leiomyoma is a major reproductive health disease among women and in particular Black women. The present study sought to determine whether a single nucleotide polymorphism (SNP) of CYP17 (rs743572) was associated with the risk of developing uterine leiomyoma (UL) in affected women in Barbados; a majority Black population. It also sought to determine if BMI, waist circumference and oestradiol levels were associated with UL in this group. A total of 96 random persons were assessed in a case-control study using a PCR-RFLP assay, and measurements of body mass index, waist circumference, and oestradiol levels were also assessed. Our results showed no genetic association with the risk of UL and this gene. The genetic distribution of CYP 17α- alleles resembled a normal Hardy-Weinberg distribution, and a relatively low risk of 0.25 at a confidence interval at 95%, of UL disease development. However, a significant association was found between oestradiol levels and fibroids, as well as oestradiol levels and BMI, at P < 0.05 among cases. Therefore our study indicates that significant associations between physiochemical factors comprising BMI, waist circumference, and oestrogen levels are disease indicators in this population. In conclusion, our findings suggest that obesity and its associated risk factors are important in a majority Black Caribbean population, although the sample size needs to be increased.