Endocrine Disruption by Heavy Metals on Steroidogenesis in Model Systems

In this study human adrenocortical carcinoma cell line NCI-H295R was used as an in vitro biological model to study the effect of heavy metals on steroidogenesis. The cell cultures were exposed to different concentrations of cadmium (1.90; 3.90; 7.80; 15.60; 31.20; 62.50 μM of CdCl2), mercury (1.0; 5.0; 25; 50; 100 μM of HgCl2), nickel (3.90; 7.80; 15.60; 31.20; 62.50; 125; 250; 500 μM of NiCl2) and compared to control. Cell viability was measured by the metabolic activity (MTT) assay for estimation of mitochondria structural integrity. Quantification of sexual steroid production directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Cadmium decreased the release of progesterone and testosterone already at a very low concentration (1.90 μM) of CdCl2, while the cell viability remained relatively high (> 75%) up to 7.80 μM of CdCl2 and significantly (P<0.01) decreased at 15.60 μM and higher concentrations of CdCl2. Concentration-dependent depression in testosterone production was detected particularly for higher concentration of HgCl2. Progesterone production was also decreased, but at the lower concentrations (1.0 and 5.0 μM) of HgCl2 this decline was lower compared to depression of testosterone. The cell viability significantly decreased at 25 μM and higher concentration of HgCl2. Results of the our study indicate dose dependent decreases in both sexual steroid hormones by NCI-H295R cell culture following a 48 h in vitro NiCl2 exposure. The lowest concentration of progesterone was significantly (P<0.01) detected in groups with the higher doses (≥ 500 μM) of NiCl2, which elicited significant cytotoxic effect. The testosterone production was decreased as well, but this decline was more pronounced compared to depression of progesterone. These results suggest that heavy metals have detrimental effects on steroid hormone synthesis even at very low concentrations and consecutively on reproductive physiology

Mechanistic Oral Absorption Modeling and Simulation for Formulation Development and Bioequivalence Evaluation: Report of an FDA Public Workshop

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138394/1/psp412204.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138394/2/psp412204_am.pd

Comparison of lipid membrane-water partitioning with various organic solvent-water partitions of neutral species and ionic species: Uniqueness of cerasome as a model for the stratum corneum in partition processes

YesLipid membrane-water partitions (e.g., immobilized artificial membrane systems where the lipid membrane is a neutral phospholipid monolayer bound to gel beads) were compared to various organic solvent-water partitions using linear free energy relationships. To this end, we also measured the retention factors of 36 compounds (including neutral and ionic species) from water to liposomes made up of 3-sn-phosphatidylcholine and 3-sn-phosphatidyl-l-serine (80:20, mol/mol), employing liposome electrokinetic chromatography in this work. The results show that lipid membranes exhibit a considerably different chemical environment from those of organic solvents. For both neutral species and ionic species, partitions into the more polar hydroxylic solvents are chemically closer to partition into the lipid membrane as compared to partitions into the less polar hydroxylic solvents and into aprotic solvents. This means that solutes partition into the polar parts of lipid membranes, regardless of whether they are charged or not. In addition, cerasome (i.e., liposome composed mainly of stratum corneum lipids) was compared with regular phospholipid liposomes as a possible model for human stratum corneum in partitions. It was found that the cerasome-water partition exhibits a better chemical similarity to skin permeation. This is probably due to the unique structures of ceramides that occur in cerasome and in the stratum corneum lipid domain. We further show that membranes in membrane-water partitions exhibit very different properties

Horizontally acquired glycosyltransferase operons drive salmonellae lipopolysaccharide diversity.

The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions

Transcriptome analysis reveals tumor microenvironment changes in glioblastoma

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells

The EGFRvIII transcriptome in glioblastoma, a meta-omics analysis.

BACKGROUND: EGFR is among the genes most frequently altered in glioblastoma, with exons 2-7 deletions (EGFRvIII) being amongst its most common genomic mutations. There are conflicting reports about its prognostic role and it remains unclear whether and how it differs in signalling compared with wildtype EGFR. METHODS: To better understand the oncogenic role of EGFRvIII, we leveraged four large datasets into one large glioblastoma transcriptome dataset (n=741) alongside 81 whole-genome samples from two datasets. RESULTS: The EGFRvIII/EGFR expression ratios differ strongly between tumours and ranges from 1% to 95%. Interestingly, the slope of relative EGFRvIII expression is near-linear, which argues against a more positive selection pressure than EGFR wildtype. An absence of selection pressure is also suggested by the similar survival between EGFRvIII positive and negative glioblastoma patients. EGFRvIII levels are inversely correlated with pan-EGFR (all wildtype and mutant variants) expression, which indicates that EGFRvIII has a higher potency in downstream pathway activation. EGFRvIII-positive glioblastomas have a lower CDK4 or MDM2 amplification incidence than EGFRvIII-negative (p=0.007), which may point towards crosstalk between these pathways. EGFRvIII-expressing tumours have an upregulation of 'classical' subtype genes compared to those with EGFR-amplification only (p=3.873e-6). Genomic breakpoints of the EGFRvIII deletions have a preference towards the 3' end of the large intron-1. These preferred breakpoints preserve a cryptic exon resulting in a novel EGFRvIII variant and preserve an intronic enhancer. CONCLUSIONS: These data provide deeper insights into the complex EGFRvIII biology and provide new insights for targeting EGFRvIII mutated tumours