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Ultrasensitive electrochemical genosensor for direct detection of specific RNA sequences derived from avian influenza viruses present in biological samples
Get PDFAn electrochemical genosensor based on an epoxyphenanthroline–Fe(III)–NH2-ssDNA layer for the detection of RNA derived from Avian Influenza is presented. The biosensor preparation consists of: (I) modification of gold electrodes with aminoethanethiol, (II) modification of the self-assembled monolayer of aminoethanethiol with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline using “click” chemistry, (III) a first step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (IV) a second step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (V) immobilization of the single stranded amino-DNA probe via “click” chemistry between epoxy and amino groups. The interactions between the ssDNA probe and RNA targets were explored with Osteryoung Square Wave Voltammetry. The genosensor showed a remarkable detection limit of 3 copies/μL (5 aM) for RNA extracted from A/swan/Poland/305/06 (H5N1) containing a fully complementary sequence. A linear dynamic range for this sequence was observed from 3.0×103 to 3.0×105 [copies/μl]. RNA extracted from A/mallard/Poland/446/09 (H7N7), containing a non-complementary sequence, generated a much
weaker response. Moreover, the developed genosensor
allows to distinguish RNA present in biological samples
having 2, 3 and 4 mismatches. This biosensing approach
can become a potential alternative tool for detecting
RNA samples in biomedical research and early clinical diagnosis of avian influenza viruses
Crystal structure and directed evolution of specificity of NlaIV restriction endonuclease
Get PDFSpecificity engineering is challenging, and particularly difficult for enzymes that have the catalytic machinery and specificity determinants in close proximity. Restriction endonucleases have been used as a paradigm for protein engineering, but successful cases are rare. Here, we present the results of a directed evolution approach to the engineering of a dimeric, blunt end cutting restriction enzyme NlaIV (GGN/NCC). Based on the remote similarity to EcoRV endonuclease, regions for random mutagenesis and in vitro evolution were chosen. The obtained variants cleaved target sites with an up to 100-fold kcat/KM preference for AT or TA (GGW/WCC) over GC or CG (GGS/SCC) in the central dinucleotide step, compared to the only ~17-fold preference of the wild-type enzyme. To understand the basis of the increased specificity, we determined the crystal structure of NlaIV. Despite the presence of DNA in the crystallization mix, the enzyme crystallized in the free form. We therefore constructed a computational model of the NlaIV-DNA complex. According to the model, the mutagenesis of the regions that were in the proximity of DNA did not lead to the desired specificity change, which was instead conveyed in an indirect manner by substitutions in the more distant regions
Structural bases of peptidoglycan recognition by lysostaphin SH3b domain.
Get PDFStaphylococcus simulans lysostaphin cleaves pentaglycine cross-bridges between stem peptides in the peptidoglycan of susceptible staphylococci, including S. aureus. This enzyme consists of an N-terminal catalytic domain and a cell wall binding domain (SH3b), which anchors the protein to peptidoglycan. Although structures of SH3bs from lysostaphin are available, the binding modes of peptidoglycan to these domains are still unclear. We have solved the crystal structure of the lysostaphin SH3b domain in complex with a pentaglycine peptide representing the peptidoglycan cross-bridge. The structure identifies a groove between β1 and β2 strands as the pentaglycine binding site. The structure suggests that pentaglycine specificity of the SH3b arises partially directly by steric exclusion of Cβ atoms in the ligand and partially indirectly due to the selection of main chain conformations that are easily accessible for glycine, but not other amino acid residues. We have revealed further interactions of SH3b with the stem peptides with the support of bioinformatics tools. Based on the structural data we have attempted engineering of the domain specificity and have investigated the relevance of the introduced substitutions on the domain binding and specificity, also in the contexts of the mature lysostaphin and of its bacteriolytic activity
URINARY PROTEOMIC MARKERS OF IGA NEPHROPATHY, LUPUS NEPHRITIS AND MEMBRANOUS NEPHROPATHY
Get PDFINTRODUCTION: Chronic kidney disease (CKD) is a worldwide public health problem, related to increased morbidity and mortality. Glomerulopathies represent major causes of CKD and require complicated diagnostics. Standard of care includes kidney biopsy in order to confirm the type of nephropathy. However, biopsy brings specific risks. Therefore, non-invasive diagnostic and prognostic methods are sought. Urinary proteomics emerged as safe and promising tool, but still requires development and improvements. Our previous studies which are part of European Patent Application from 10th June 2016 (WO/2017/212463), identified urinary markers of IgA nephropathy. They included among others: alpha-1B-glycoprotein (A1BG), alpha-l-acid glycoprotein 1 (ORM-1), ferritin light chain (FTL) and serotransferrin (TF). The aim of this study was to evaluate them in comparison to patients with glomerulopathies of different etiologies, such as lupus nephritis (LN) and membranous nephropathy (MN).
METHODS: This proteomic study included patients with CKD (41 IgAN, 33 LN, 26 MN, 6 with erytrocyturia of unknown etiology) and 19 healthy controls. Urine samples were obtained from a midstream of the: first-morning (FM) and second- or third-morning (SPOT) sample. The SPOT samples were processed up to 2 h and FM samples up to 4 h after collection, by agitating and gently inverting 4-6 times, portioned into 2-ml aliquots and stored at -80°C for further measurements. Western Blotting was used for analysis of the SPOT and FM samples, ELISA and mass spectrometry for SPOT urine only. The results were related to demographic data, standard laboratory tests and GFR estimated with use of Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.
RESULTS: The urinary concentrations of A1BG, ORM-1, FTL and TF were found to be higher in CKD patients than in healthy controls. Moreover, these proteins varied depending on the disease. According to ELISA measurements, patients with IgAN, erytrocyturia and LN had significantly more A1BG and ORM-1 (p < 0.05), whereas TF was more elevated in LN and MN individuals comparing to healthy controls. The western blot analysis revealed significantly elevated level of A1BG, ORM-1 and FTL in IgAN, LN and MN, comparing to healthy control. Additionally, it revealed fragmentation of A1BG in several patients and the bottom range bands tended to be most prominently elevated in IgAN patients. Mass spectrometry confirmed differences between the diseases according to the specific amino acids fragments of each tested protein. Figure 1. Western blot scans for urinary A1BG, ORM-1 and FTL in CKD patients (2-4) and healthy controls (1).
CONCLUSIONS: The urinary concentrations of A1BG, ORM-1, FTL and TF are elevated in CKD patients and vary depending on the type of nephropathy. This observation suggests their differential roles in the pathophysiology of the given diseases, and we believe their evaluation may help distinguishing between nephropathies. Further studies are desired to establish the role of these urinary proteins in non-invasive disease differentiation
Selected issues of anaerobic digestion based on the studies on hydrogen- and methane yielding bioreactors
Get PDFAnaerobic digestion of organic matter results from the metabolic activity of many groups of microorganisms. Interactions between microorganisms during acidogenesis, acetogenesis and methanogenesis, source of inoculum, type of feedstock and operational conditions determine
metabolic pathways in bioreactors and consequently the efficiency of fermentation processes.
In innovative installations it is desirable to separate acidogenesis from acetogenesis and methanogenesis to favour respectively the production of biohydrogen or biomethane under controlled conditions
Quantitative proteomic analysis of differentially expressed proteins in tubers of potato plants differing in resistance to Dickeya solani
Get PDFGraphene oxide down-regulates genes of the oxidative phosphorylation complexes in a glioblastoma.
Get PDFBACKGROUND:
Recently different forms of nanographene were proposed as the material with high anticancer potential. However, the mechanism of the suppressive activity of the graphene on cancer development remains unclear. We examined the effect of oxygenated, reduced and pristine graphene on the gene expression in glioblastoma U87 cell line.
RESULTS:
Conducting microarrays and RT-qPCR analysis we explored that graphene oxide (rather than reduced graphene oxide and pristine graphene) down-regulates the mRNA expression of mitochondrial oxidative phosphorylation (OXPHOS) nuclear genes of complexes I, III, IV and V. The presented results provide first evidence for the hypothesis that the suppressed growth of GBM can be the consequence of down-regulation of OXPHOS protein expression and decreased ATP level.
CONCLUSIONS:
We suggest that changes in the expression of OXPHOS genes identified in our study may mediate the anti-proliferative and anti-migratory effects of graphene oxide in glioblastoma cells. However, further investigations with different cell lines, regarding expression, regulation and activity of OXPHOS genes identified in our study is necessary to elucidate the mechanism mediating the anti-proliferative and anti-migratory effects of graphene oxide in glioblastoma cells
Carbon assimilation profiles of mucoralean fungi show their metabolic versatility
Get PDFMost mucoralean fungi are common soil saprotrophs and were probably among the first land colonisers. Although Mucoromycotina representatives grow well on simple sugar media and are thought to be unable to assimilate more complex organic compounds, they are often isolated from plant substrates. The main goal of the study was to explore the effects of isolation origin and phylogenetic placement on the carbon assimilation capacities of a large group of saprotrophic Mucoromycotina representatives (i.e. Umbelopsidales and Mucorales). Fifty two strains representing different Mucoromycotina families and isolated from different substrates were tested for their capacity to grow on 99 different carbon sources using the Biolog phenotypic microarray system and agar plates containing selected biopolymers (i.e. cellulose, xylan, pectin, and starch) as a sole carbon source. Although our results did not reveal a correlation between phylogenetic distance and carbon assimilation capacities, we observed 20 significant differences in growth capacity on specific carbon sources between representatives of different families. Our results also suggest that isolation origin cannot be considered as a main predictor of the carbon assimilation capacities of a particular strain. We conclude that saprotrophic Mucoromycotina representatives are, contrary to common belief, metabolically versatile and able to use a wide variety of carbon sources
ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy
Get PDFThe nine identified human homologues of E. coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy
Deproteinated Potato Wastewater as a Sustainable Nitrogen Source in Trichosporon domesticum Yeast Lipids Biosynthesis—a Concept of Valorization of Wastewater from Starch Industry
Get PDFThis study determines the ability of an isolated Trichosporon domesticum yeast strain to accumulate intracellular lipids in media with deproteinated potato wastewater (DPW) containing various carbon sources. The yeast strain was isolated from kefir and identified by internal transcribed spacer (ITS) region sequence analysis. The sequence was deposited in GenBank under accession number MH094668, and the strain was deposited in Polish Collection of Microorganisms as T. domesticum PCM 2960. DPW is an inexpensive and valuable source of nitrogen, potassium, phosphorus, and other elements in yeast cultures. DPW supplemented with glucose medium was most effective at stimulating lipid biosynthesis by T. domesticum PCM 2960 and bioreactor incubation resulted in a final lipid yield of 4.8 g L−1. The lipids of the T. domesticum PCM 2960 biomass were characterized by high contents of linoleic acid (Δ9,12C18:2), oleic acid (Δ9C18:1), palmitic acid (C16:0), and α-linolenic acid (Δ9,12,15C18:3). Theoretical calculations for biodiesel properties showed that the methylated esters of lipids from T. domesticum PCM 2960 biomass cannot be used as a biodiesel in diesel engines. Additionally, the ability to produce biofilm as one criterion for pathogenicity was tested. The ability for biofilm formation by the isolated strain was low. This study provides a promising solution for the more economical production of microbial lipids with DPW