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The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor
Get PDFEnterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium, including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB – both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3–4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP, a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells
Identification of pig-specific Cryptosporidium species in mixed infections using Illumina sequencing technology
Get PDFNowadays molecular methods are widely used in epidemiological studies of Cryptosporidium infections in humans and animals. However to gain better understanding of parasite species or genotypes, especially when mixed infections are noticed, highly sensitive tools with adequate resolution power need to be employed. In this article, we report an application of the next generation sequencing method (NGS) for detection and characterisation of Cryptosporidium species concurrently present in pig faeces. A mixture of Cryptosporidium DNA obtained from two faecal samples was amplified at the 18 SSU rRNA gene locus and the resulting amplicons were subsequently used for MiSeq sequencing. Although initial molecular analyses indicated the possible presence of another Cryptosporidium species other than Cryptosporidium scrofarum and Cryptosporidium suis, deep sequencing only confirmed the presence of pig-specific Cryptosporidium
New inhibitors of ALKBH dioxygenases overexpressed in neck and head cancer
Get PDFAlkB protein from E. coli (EcAlkB) is a profoundly studied member of 2-oxoglutarate and Fe(II) – dependent dioxygenase superfamily that removes alkyl lesions from DNA bases via oxidative demethylation restoring native bases in DNA. In human cells, there are nine EcAlkB homologs, ALKBH1-8 and FTO. These dioxygenases, repair alkylation lesions to DNA and RNA but also show other biological functions. Cancerous tissues develop greater efficiency of DNA repair systems in comparison
to healthy ones. Overexpression of DNA repair proteins in tumor leads to the removal of DNA lesions before they become toxic to cancer cell, in this way assuring tumor welfare and creating major mechanism of resistance to anticancer therapy. Our main approach was to determine the levels of expression of particular ALKBHs in cancer tissues by Western-blot in head and neck cancer tissues. We have demonstrated that dioxygenases ALKBH
1, 3, 4, 5, and FTO are highly expressed in these tumors. Statistical analysis allowed to create protein expression tree of ALKBHs tested. We have found that FTO and ALKBH5, and ALKBH1 and ALKBH3 show similar expression patterns in healthy and cancerous tissues. To answer the question whether ALKBHs under study form any oligomers, we performed size exclusion chromatography and observed monomeric forms of ALKBH3, 4 while ALKBH1, 5, and FTO formed protein complexes. Natural substances, rhein and emodin, show anti-cancer, anti-inflammatory,
and anti-microbial activities. Rhein inhibits activity of
EcAlkB, ALKBH2, ALKBH3, and FTO. We synthesized and
investigated new rhein and emodin derivatives as a potential inhibitors of ALKBHs and found that chloridoemodins in the form of mono- and di-chlorides are the most active inhibitors of ALKBHs among compounds tested. Moreover, they were more selective against cancerous than healthy cells and seem to be a
promising anthraquinones in anti-cancer therapy.Funding: Pol/Nor/196258/59/2013
Evaluation of the role of downregulation of SNF5/INI1 core subunit of SWI/SNF complex in clear cell renal cell carcinoma development
Get PDFClear cell renal cell carcinoma (ccRCC) is characterized by stabilization of hypoxia-inducible factor (HIF1), and mutations in von Hippel-Lindau (VHL) gene. Additionally, in about 40% of ccRCC cases the mutation in PBRM1 (POLYBROMO1) gene coding for a non-core subunit of SWI/SNF chromatin remodeling complex was found suggesting potential impairment of this complex function in ccRCC. In this study we assessed the extent to which the core SWI/SNF complex subunit - INI1 (hSNF5/SMARCB1) is affected in ccRCC and whether it has any consequences on the development of this type of cancer. The evaluation of INI1 protein level in samples from 50 patients with diagnosed ccRCC, including three displaying rhabdoid features, showed the INI1 positive staining in rhabdoid cells while the conventional ccRCC cells exhibited reduced INI1 level. This indicated the rhabdoid component of ccRCC as distinct from other known rhabdoid tumors. The reduced INI1 protein level observed in all conventional ccRCC cases used in this study correlated with decreased SMARCB1 gene expression at the transcript level. Consistently, the overexpression of INI1 protein in A498 ccRCC cell line resulted in the elevation of endogenous SMARCB1 transcript level indicating that the INI1-dependent regulatory feedback loop controlling expression of this gene is affected in ccRCC Moreover, the set of INI1 target genes including i.e. CXCL12/CXCR7/CXCR4 chemokine axis was identified to be affected in ccRCC. In summary, we demonstrated that the inactivation of INI1 may be of high importance for ccRCC development and aggressiveness
Models in the research process of psoriasis
Get PDFPsoriasis is an ancient, universal chronic skin disease with a significant geographical
variability, with the lowest incidence rate at the equator, increasing towards the poles. Insights into
the mechanisms responsible for psoriasis have generated an increasing number of druggable targets
and molecular drugs. The development of relevant in vitro and in vivo models of psoriasis is now a
priority and an important step towards its cure. In this review, we summarize the current cellular
and animal systems suited to the study of psoriasis. We discuss the strengths and limitations of the
various models and the lessons learned. We conclude that, so far, there is no one model that can
meet all of the research needs. Therefore, the choice model system will depend on the questions
being addressed
Plant MicroRNAs - Novel Players in Natural Medicine?
Get PDFMicroRNAs (miRNAs) represent a class of small non-coding RNAs that act as efficient gene expression regulators and thus play many important roles in living organisms. Due to their involvement in several known human pathological and pathogenic states, miRNA molecules have become an important issue in medicine and gained the attention of scientists from the pharmaceutical industry. In recent few years, a growing number of studies have provided evidence that miRNAs may be transferred from one species to another and regulate gene expression in the recipients’ cells. The most intriguing results revealed that stable miRNAs derived from food plants may enter the mammals’ circulatory system and, after reaching the target, inhibit the production of specific mammalian protein. Part of the scientific community has perceived this as an attractive hypothesis that may provide a foundation for novel therapeutic approaches. In turn, others are convinced about the “false positive” effect of performed experiments from which the mentioned results were achieved. In this article, we review the recent literature that provides evidence (from both fronts) of dietary, plant miRNA uptake and functionality in various consumers. Additionally, we discuss possible miRNA transport mechanisms from plant food sources to human cells
Two novel C-terminal frameshift mutations in the β-globin gene lead to rapid mRNA decay
Get PDFBACKGROUND:
The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level.
METHODS:
Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation.
RESULTS:
We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level.
CONCLUSION:
Our results show that a lack of natural stop codon due to the frameshift in exon 3 of β-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway
An optimized method for high quality DNA extraction from microalga Prototheca wickerhamii for genome sequencing
Get PDFBackground: The complex cell wall structure of algae often precludes efficient extraction of their genetic material. The purpose of this study was to design a next-generation sequencing-suitable DNA isolation method for unicellular, achlorophyllous, yeast-like microalgae of the genus Prototheca, the only known plant pathogens of both humans and animals. The effectiveness of the newly proposed scheme was compared with five other, previously described methods, commonly used for DNA isolation from plants and/or yeasts, available either as laboratory-developed, in-house assays, based on liquid nitrogen grinding or different enzymatic digestion, or as commercially manufactured kits.
Results: All five, previously described, isolation assays yielded DNA concentrations lower than those obtained with the new method, averaging 16.15 ± 25.39 vs 74.2 ± 0.56 ng/μL, respectively. The new method was also superior in terms of DNA purity, as measured by A260/A280 (−0.41 ± 4.26 vs 2.02 ± 0.03), and A260/A230 (1.20 ± 1.12 vs 1.97 ± 0.07) ratios. Only the liquid nitrogen-based method yielded DNA of comparable quantity (60.96 ± 0.16 ng/μL) and quality (A260/A280 = 2.08 ± 0.02; A260/A230 = 2.23 ± 0.26). Still, the new method showed higher integrity, which was best illustrated upon electrophoretic analysis. Genomic DNA of Prototheca wickerhamii POL-1 strain isolated with the protocol herein proposed was successfully sequenced on the Illumina MiSeq platform.
Conclusions: A new method for DNA isolation from Prototheca algae is described. The method, whose protocol involves glass beads pulverization and cesium chloride (CsCl) density gradient centrifugation, was demonstrated superior over the other common assays in terms of DNA quantity and quality. The method is also the first to offer the possibility of preparation of DNA template suitable for whole genome sequencing of Prototheca spp
Performance of machine-learning scoring functions in structure-based virtual screening
Get PDFClassical scoring functions have reached a plateau in their performance in virtual screening and binding affinity prediction. Recently, machine-learning scoring functions trained on protein-ligand complexes have shown great promise in small tailored studies. They have also raised controversy, specifically concerning model overfitting and applicability to novel targets. Here we provide a new ready-to-use scoring function (RF-Score-VS) trained on 15 426 active and 893 897 inactive molecules docked to a set of 102 targets. We use the full DUD-E data sets along with three docking tools, five classical and three machine-learning scoring functions for model building and performance assessment. Our results show RF-Score-VS can substantially improve virtual screening performance: RF-Score-VS top 1% provides 55.6% hit rate, whereas that of Vina only 16.2% (for smaller percent the difference is even more encouraging: RF-Score-VS top 0.1% achieves 88.6% hit rate for 27.5% using Vina). In addition, RF-Score-VS provides much better prediction of measured binding affinity than Vina (Pearson correlation of 0.56 and -0.18, respectively). Lastly, we test RF-Score-VS on an independent test set from the DEKOIS benchmark and observed comparable results. We provide full data sets to facilitate further research in this area (http://github.com/oddt/rfscorevs) as well as ready-to-use RF-Score-VS (http://github.com/oddt/rfscorevs_binary)