Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria

Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals up to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker

Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

In this report, selected non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and nimesulide, and analgesics acetaminophen, alone, as well as in combination with isoflavone genistein as potential glycosaminoglycan (GAG) metabolism modulators were considered for the treatment of mucopolysaccharidoses (MPSs) with neurological symptoms due to the effective blood-brain barrier\ud (BBB) penetration properties of these compounds. We found that indomethacin and nimesulide, but not acetaminophen, inhibited GAG synthesis in fibroblasts significantly, while the most pronounced impairment of glycosaminoglycan production was observed after exposure to the mixture of nimesulide and genistein. Phosphorylation of the EGF receptor (EGFR) was inhibited even more effective in the presence of indomethacin and nimesulide than in the presence of genistein. When examined the activity of phosphatidylinositol-3-kinase (PI3K) production, we observed its most significant decrease in the case of fibroblast exposition to nimesulide, and afterwards to indomethacin and genistein mix, rather than indomethacin used alone. Some effects on expression of individual GAG metabolism-related and lysosomal function genes, and significant activity modulation of a number of genes involved in intracellular signal transduction pathways and metabolism of DNA and proteins were detected. This study documents that NSAIDs, and their mixtures with genistein modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

Synthetic genistein derivatives as modulators of glycosaminoglycan synthesis

Background: Mucopolysaccharidoses (MPS) are severe metabolic disorders caused by 26 accumulation of undegraded glycosaminoglycans (GAGs) in lysosomes due to defects in certain 27 lysosomal hydrolases. Substrate reduction therapy (SRT) has been proposed as one of potential 28 treatment procedures of MPS. Importantly, small molecules used in such a therapy might 29 potentially cross the blood-brain barrier (BBB) and improve neurological status of patients, as 30 reported for a natural isoflavone, 5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one, 31 also known as genistein. Although genistein is able to cross BBB to some extent, its delivery to 32 the central nervous system is still relatively poor (below 10% efficiency). Thus, we aimed to 33 develop a set of synthetically modified genistein molecules and characterize physicochemical as 34 well as biological properties of these compounds. Methods: Following parameters were 35 determined for the tested synthetic derivatives of genistein: cytotoxicity, effects on cell 36 proliferation, kinetics of GAG synthesis, effects on epidermal growth factor (EGF) receptor’s 37 tyrosine kinase activity, effects on lysosomal storage, potential ability to cross BBB. Results: We 38 observed that some synthetic derivatives inhibited GAG synthesis similarly to, or more 39 efficiently than, genistein and were able to reduce lysosomal storage in MPS III fibroblasts. The 40 tested compounds were generally of low cytotoxicity and had minor effects on cell proliferation. 41 Moreover, synthetic derivatives of genistein revealed higher lipophilicity (assessed in silico) than 42 the natural isoflavone. Conclusion: Some compounds tested in this study might be promising 43 candidates for further studies on therapeutic agents in MPS types with neurological symptoms

Metal and antibiotic resistance of bacteria isolated from the Baltic Sea

The resistance of 49 strains of bacteria isolated from surface Baltic Sea waters to 11 antibiotics was analyzedand the resistance of selected strains to three metal ions (Ni2+, Mn2+, Zn2+) was tested. Most isolates belonged to Gammaproteobacteria (78 %), while Alphaproteobacteria (8 %), Actinobacteria (10 %), and Bacteroidetes (4 %) were lessabundant. Even though previous reports suggested relationships between resistance and the presence of plasmids or the abilityto produce pigments, no compelling evidence for such relationships was obtained for the strains isolated in this work. In particular, strains resistant to multiple antibiotics did not carry plasmids more frequently than sensitive strains. A relationbetween resistance and the four aminoglycosides tested (gentamycin, kanamycin, neomycin, and streptomycin), but not tospectinomycin, was demonstrated. This observation is of interest given that spectinomycin is not always classified as anaminoglycoside because it lacks a traditional sugar moiety. Statistical analysis indicated relationships between resistance tosome antibiotics (ampicillin and erythromycin, chloramphenicol and erythromycin, chloramphenicol and tetracycline, erythromycinand tetracycline), suggesting the linkage of resistance genes for antibiotics belonging to different classes. The effectsof NiSO4, ZnCl2 and MnCl2 on various media suggested that the composition of Marine Broth might result in low concentrationsof Mn2+ due to chemical interactions that potentially lead to precipitation. [Int Microbiol 2012; 15(3):131-139

Female Fabry disease patients and X-chromosome inactivation

Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α- galactosidase A (GLA). Once it was thought to affect only hemizygous males. Over the last fifteen years, research has shown that most females carrying mutated allele also develop symptoms, demonstrating a wide range of disease severity, from a virtually asymptomatic to more classical profile, with cardiac, renal, and cerebrovascular manifestations. This variable expression in females is thought to be influenced by the process of X-chromosome inactivation (XCI). The aim of this study was to assess severity of the clinical phenotype, to analyze XCI patterns, and to estimate their effect on disease manifestation in twelve female Fabry disease patients from five unrelated Polish families. Our analyses revealed that patients presented with the broad range of disease expression - from mild to severe, and their clinical involvement did not correlate with XCI profiles. Female carriers of the mutation in the GLA gene with the random XCI may present with the wide range of disease signs and symptoms. Thus, XCI is not a main factor in the phenotype variability of Fabry disease manifestation in heterozygous females

Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids

Flavonoids were found previously to modulate efficiency of synthesis of glycosaminoglycans (GAGs), compounds which are accumulated in cells of patients suffering from mucopolysaccharidoses (MPSs). The aim of this work was to determine effects of different flavonoids (genistein, kaempferol, daidzein) used alone or in combinations, on expression of genes coding for proteins involved in GAG metabolism. Analyses with DNA microarray, followed by real-time qRT-PCR revealed that genistein, kaempferol and combination of these two compounds induced dose- and time-dependent remarkable alterations in transcript profiles of GAG metabolism genes in cultures of wild-type human dermal fibroblasts (HDFa). Interestingly, effects of the mixture of genistein and kaempferol were stronger than those revealed by any of these compounds used alone. Similarly, the most effective reduction in levels of GAG production, in both HDFa and MPS II cells, was observed in the presence of genistein, keampferol and combination of these compounds. Forty five genes were chosen for further verification not only in HDFa, but also inMPS II fibroblasts by using real-time qRT-PCR. Despite effects on GAG metabolism-related genes, we found that genistein, kaempferol and mixture of these compounds significantly stimulated expression of TFEB. Additionally, a decrease inMTOR transcript level was observed at these conditions

Developing nucleic acid-based electrical detection systems

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed

The Role of Dimethyl Sulfoxide (DMSO) in Gene Expression Modulation and Glycosaminoglycan Metabolism in Lysosomal Storage Disorders on an Example of Mucopolysaccharidosis

Obstacles to effective therapies for mucopolysaccharidoses (MPSs) determine the need for continuous studies in order to enhance therapeutic strategies. Dimethyl sulfoxide (DMSO) is frequently utilised as a solvent in biological studies, and as a vehicle for drug therapy and the in vivo administration of water-insoluble substances. In the light of the uncertainty on the mechanisms of DMSO impact on metabolism of glycosaminoglycans (GAGs) pathologically accumulated in MPSs, in this work, we made an attempt to investigate and resolve the question of the nature of GAG level modulation by DMSO, the isoflavone genistein solvent employed previously by our group in MPS treatment. In this work, we first found the cytotoxic effect of DMSO on human fibroblasts at concentrations above 3%. Also, our results displayed the potential role of DMSO in the regulation of biological processes at the transcriptional level, then demonstrated a moderate impact of the solvent on GAG synthesis. Interestingly, alterations of lysosomal ultrastructure upon DMSO treatment were visible. As there is growing evidence in the literature that DMSO can affect cellular pathways leading to numerous changes, it is important to expand our knowledge concerning this issue