Activity of protein kinase CK2 uncouples Bid cleavage from caspase-8 activation.

In the present study, we quantitatively analysed the interface between apoptosis initiation and execution by determining caspase-8 activation, Bid cleavage and mitochondrial engagement (onset of mitochondrial depolarisation) in individual HeLa cervical cancer cells following exposure to tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL). Employing resonance-energy-transfer probes containing either the caspase-8 recognition site IETD or full-length Bid, we observed a significant delay between the times of caspase-8 activation and Bid cleavage, suggesting the existence of control steps separating these two processes. Subsequent analyses suggested that the divergence of caspase-8 activation and Bid cleavage are critically controlled by kinase signalling: inhibiting protein kinase CK2 by using 5,6-dichloro-l-(beta-D-ribofuranosyl-1)-benzimidazole (DRB) or by overexpression of a dominant-negative CK2alpha catalytic subunit largely eliminated the lag time between caspase-8 activation and Bid cleavage. We conclude that caspase-8 activation and Bid cleavage are temporally uncoupled events, providing transient tolerance to caspase-8 activities.

Apoptosis repressor with caspase recruitment domain, a multifunctional modulator of cell death.

Apoptosis repressor with caspase recruitment domain (ARC) is a highly potent and multifunctional inhibitor of apoptosis that is physiologically expressed predominantly in post-mitotic cells such as cardiomyocytes, skeletal muscle cells and neurons. ARC was also found to be up-regulated in many forms of malignant tumours. ARC impairs the cellular apoptotic responsiveness to a wide range of stresses and insults, including extrinsic apoptosis initiation via death receptor ligands, dysregulation of cellular Ca(2+) homeostasis and endoplasmatic reticulum (ER) stress, genotoxic drugs, ionizing radiation, oxidative stress and hypoxia. ARC is subject to both transcriptional and post-translational regulation and exhibits its function through a multitude of molecular interactions with upstream transducers of apoptosis signals. This review summarizes, structures and comments on the published knowledge regarding ARC and its roles in modulating apoptotic cell death responsiveness in physiological and pathophysiological contexts.

First insight into microbial community composition in a phosphogypsum waste heap soil.

The aim of this study was to investigate the soil microbial communities of a phosphogypsum waste heap. The soil microbial community structures can differ over time, as they are affected by the changing environmental conditions caused by a long-term exposure to different kinds of pollutions, like is the case of soil in the post-production waste area in Wislinka (in the northern part of Poland) currently undergoing restoration. Our analyses indicated that the most abundant phyla were Proteobacteria, Acidobacteria, and Actinobacteria, and generally such an abundance is common for most of the studied soils. The most dominant class were Alphaproteobacteria, with their participation in 33.46% of the total reads. Among this class, the most numbered order was Sphingomonadales, whereas among this order the Sphingomonadaceae family was the most abundant one. The Sphingomonadaceae family is currently in the center of interest of many researchers, due to the ability of some of its members to utilize a wide range of naturally occurring organic compounds and many types of environmental contaminants. This kind of knowledge about microbial populations can support efforts in bioremediation and can improve monitoring changes in the contaminated environments.

The choice of the DNA extraction method may influence the outcome of the soil microbial community structure analysis.

Metagenomics approaches and recent improvements in the next-generation sequencing methods, have become a method of choice in establishing a microbial population structure. Many commercial soil DNA extraction kits are available and due to their efficiency they are replacing traditional extraction protocols. However, differences in the physicochemical properties of soil samples require optimization of DNA extraction techniques for each sample separately. The aim of this study was to compare the efficiency, quality, and diversity of genetic material extracted with the use of commonly used kits. The comparative analysis of microbial community composition, displayed differences in microbial community structure depending on which kit was used. Statistical analysis indicated significant differences in recovery of the genetic material for 24 out of 32 analyzed phyla, and the most pronounced differences were seen for Actinobacteria. Also, diversity indexes and reproducibility of DNA extraction with the use of a given kit, varied among the tested methods. As the extraction protocol may influence the apparent structure of a microbial population, at the beginning of each project many extraction kits should be tested in order to choose one that would yield the most representative results and present the closest view to the actual structure of microbial population.

Vascular transcriptome profiling identifies Sphingosine kinase 1 as a modulator of angiotensin II-induced vascular dysfunction.

Vascular dysfunction is an important phenomenon in hypertension. We hypothesized that angiotensin II (AngII) affects transcriptome in the vasculature in a region-specific manner, which may help to identify genes related to vascular dysfunction in AngII-induced hypertension. Mesenteric artery and aortic transcriptome was profiled using Illumina WG-6v2.0 chip in control and AngII infused (490 ng/kg/min) hypertensive mice. Gene set enrichment and leading edge analyses identified Sphingosine kinase 1 (Sphk1) in the highest number of pathways affected by AngII. Sphk1 mRNA, protein and activity were up-regulated in the hypertensive vasculature. Chronic sphingosine-1-phosphate (S1P) infusion resulted in a development of significantly increased vasoconstriction and endothelial dysfunction. AngII-induced hypertension was blunted in Sphk1-/- mice (systolic BP 167 ± 4.2 vs. 180 ± 3.3 mmHg, p < 0.05), which was associated with decreased aortic and mesenteric vasoconstriction in hypertensive Sphk1-/- mice. Pharmacological inhibition of S1P synthesis reduced vasoconstriction of mesenteric arteries. While Sphk1 is important in mediating vasoconstriction in hypertension, Sphk1-/- mice were characterized by enhanced endothelial dysfunction, suggesting a local protective role of Sphk1 in the endothelium. S1P serum level in humans was correlated with endothelial function (arterial tonometry). Thus, vascular transcriptome analysis shows that S1P pathway is critical in the regulation of vascular function in AngII-induced hypertension, although Sphk1 may have opposing roles in the regulation of vasoconstriction and endothelium-dependent vasorelaxation.

A family with the Arg103Pro mutation in the NEUROD1 gene detected by next-generation sequencing - Clinical characteristics of mutation carriers.

UNLABELLED: Until now only a few families with early onset autosomal diabetes due to the NEUROD1 gene mutations have been identified. Moreover, only some of them meet strict MODY (maturity-onset diabetes of the young) criteria. Next-generation sequencing (NGS) provides an opportunity to detect more pathogenic mutations in this gene. Here, we evaluated the segregation of the Arg103Pro mutation in the NEUROD1 gene in a pedigree in which it was detected, and described the clinical characteristics of the mutation carriers. METHODS: We included 156 diabetic probands of MODY families, among them 52 patients earlier tested for GCK-MODY and/or HNF1A-MODY by Sanger sequencing with negative results. Genetic testing was performed by targeted NGS sequencing using a panel of 28 monogenic diabetes genes. RESULTS: As detected by NGS, one patient had the missense Arg103Pro (CGC/CCC) mutation in the gene NEUROD1 changing the amino-acid structure of the DNA binding domain of this transcription factor. We confirmed this sequence difference by Sanger sequencing. This family had previously been tested with negative results for HNF1A gene mutations. 17 additional members of this family were invited for further testing. We confirmed the presence of the mutation in 11 subjects. Seven adult mutation carriers (all but one) from three generations had been already diagnosed with diabetes. There were 3 individuals with the Arg103Pro mutation diagnosed before the age of 30 years in the family. The range of age of the four unaffected mutation carriers (3 minors and 1 adult) was 3-48 years. Interestingly, one mutation carrier had a history of transient neonatal hypoglycemia, of which the clinical course resembled episodes typical for HNF4A-MODY. CONCLUSIONS: We report a family with autosomal dominant diabetes related to a new NEUROD1 mutation, one of very few meeting MODY criteria. The use of the NGS method will facilitate identification of more families with rare forms of MODY.

Qualitative Parameters of the Colonic Flora in Patients with HNF1A-MODY Are Different from Those Observed in Type 2 Diabetes Mellitus.

Background. Type 2 diabetes mellitus (T2DM) is determined by genetic and environmental factors. There have been many studies on the relationship between the composition of the gastrointestinal bacterial flora, T2DM, and obesity. There are no data, however, on the gut microbiome structure in monogenic forms of the disease including Maturity Onset Diabetes of the Young (MODY). Methods. The aim of the investigation was to compare the qualitative parameters of the colonic flora in patients with HNF1A-MODY and T2DM and healthy individuals. 16S sequencing of bacterial DNA isolated from the collected fecal samples using the MiSeq platform was performed. Results. There were significant between-group differences in the bacterial profile. At the phylum level, the amount of Proteobacteria was higher (p = 0.0006) and the amount of Bacteroidetes was lower (p = 0.0005) in T2DM group in comparison to the control group. In HNF1A-MODY group, the frequency of Bacteroidetes was lower than in the control group (p = 0.0143). At the order level, Turicibacterales was more abundant in HNF1A-MODY group than in T2DM group. Conclusions. It appears that there are differences in the gut microbiome composition between patients with HNF1A-MODY and type 2 diabetes. Further investigation on this matter should be conducted.

Genetic testing for monogenic diabetes using targeted next-generation sequencing in patients with maturity-onset diabetes of the young.

INTRODUCTION: Molecular diagnosis of monogenic diabetes mellitus is important for individualized patient care. Next-generation sequencing (NGS) enables a simultaneous analysis of multiple genes in a single test. OBJECTIVES: We aimed to assess the feasibility of using NGS for detecting mutations in a set of known monogenic diabetes gene mutations in a cohort of Polish patients with maturity-onset diabetes of the young (MODY) with earlier negative Sanger sequencing results for HNF1A-MODY or GCK-MODY. PATIENTS AND METHODS: We selected a panel of 28 chromosomal genes in which mutations have been reported to cause monogenic diabetes. The MiSeq platform was used for NGS. An exon-capture assay was designed to include coding regions and splice sites. A total of 54 patients with existing negative Sanger sequencing screening results for HNF1A or GCK gene mutations were selected for the study. RESULTS: NGS results were generated for all 54 patients and 9 positive controls with previously identified HNF1A or GCK gene mutation. All selected positive controls were confirmed by NGS. Among 28 genes, mutations were detected in 16. The type of the analyzed genetic changes was described in the NGS study as high (n = 3) or moderate (n = 76). Among the detected mutations, there were 4 known GCK gene mutations that had been previously missed in Sanger sequencing. So far, Sanger sequencing allowed us to confirm 21 gene mutations detected by NGS, and segregation with diabetes in 14 pedigrees. CONCLUSIONS: Our pilot study using NGS for monogenic diabetes screening in the MODY cohort confirmed that it improves the detection of diabetes-related sequence differences. The screening with NGS should also include diabetic patients for whom Sanger-based screening for particular subtypes of MODY provided negative results.