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Viability and genetic stability of potato spindle tuber viroid mutants with indels in specific loops of the rod-like secondary structure
Get PDFABSTRACT
Maintenance of the rod-like structure of potato spindle tuber viroid (PSTVd), which contains over 20 loops and bulges between double-stranded helices, is important for viroid biology. To study tolerance to modifications of the stem-loop structures and PSTVd capacity for mutation repair, we have created 6 mutants carrying 3-4 nucleotides deletions or insertions at three unique restriction sites, EagI, StyI and AvaII. Differences in the infectivity of these in vitro generated PSTVd mutants can result from where the mutations map, as well as from the extent to which the secondary structure of the molecule is affected. Deletion or insertion of 4 nucleotides at the EagI and StyI sites led to loss of infectivity. However, mutants with deletion (PSTVd-Ava-del) or insertion (PSTVd-Ava-in) of 3 nucleotides (221GAC223), at the AvaII site (loop 20) were viable but not genetically stable. In all analyzed plants, reversion to the wild type PSTVd-S23 sequence was observed for the PSTVd-Ava-in mutant a few weeks after agroinfiltration. Analysis of PSTVd-Ava-del progeny allowed the identification of 10 new sequence variants carrying various modifications, some of them having retained the original three nucleotide deletion at the AvaII site. Interestingly, other variants gained three nucleotides in the deletion site but did not revert to the original wild type sequence. The genetic stability of the progeny PSTVd-Ava-del sequence variants was evaluated in tomato leaves (early infection) and in both leaves and roots (late infection), respectively
Taxonomic and chemical assessment of exceptionally abundant rock mine biofilm
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An exceptionally thick biofilm covers walls of ancient gold and arsenic Złoty Stok mine (Poland) in the apparent absence of organic sources of energy.
Methods and Results
We have characterized this microbial community using culture-dependent and independent methods. We sequenced amplicons of the 16S rRNA gene obtained using generic primers and additional primers targeted at Archaea and Actinobacteria separately. Also, we have cultured numerous isolates from the biofilm on different media under aerobic and anaerobic conditions. We discovered very high biodiversity, and no single taxonomic group was dominant. The majority of almost 4,000 OTUs were classified above genus level indicating presence of novel species. Elemental analysis, performed using SEM-EDS and X-ray, of biofilm samples showed that carbon, sulphur and oxygen were not evenly distributed in the biofilm and that their presence is highly correlated. However, the distribution of arsenic and iron was more flat, and numerous intrusions of elemental silver and platinum were noted, indicating that microorganisms play a key role in releasing these elements from the rock.
Conclusions
Altogether, the picture obtained throughout this study shows a very rich, complex and interdependent system of rock biofilm. The chemical heterogeneity of biofilm is a likely explanation as to why this oligotrophic environment is capable of supporting such high microbial diversity
Mono-epoxy-tocotrienol-α enhances wound healing in diabetic mice and stimulates in vitro angiogenesis and cell migration
Get PDFDiabetes mellitus is characterized by hyperglycemia and capillary hypoxia that causes excessive production of free radicals and impaired antioxidant defense, resulting in oxidative stress and diabetes complications such as impaired wound healing. We have previously shown that modified forms of tocotrienols possess beneficial effects on the biosynthesis of the mevalonate pathway lipids including increase in mitochondrial CoQ. The aim of this study is to investigate the effects of mono-epoxy-tocotrienol-α on in vitro and in vivo wound healing models as well as its effects on mitochondrial function. Gene profiling analysis and gene expression studies on HepG2 cells and human dermal fibroblasts were performed by microarray and qPCR, respectively. In vitro wound healing using human fibroblasts was studied by scratch assay and in vitro angiogenesis using human dermal microvascular endothelial cells was studied by the tube formation assay. In vivo wound healing was performed in the diabetic db/db mouse model. For the study of mitochondrial functions and oxygen consumption rate Seahorse XF-24 was employed. In vitro, significant increase in wound closure and cell migration (p < 0.05) both in normal and high glucose and in endothelial tube formation (angiogenesis) (p < 0.005) were observed. Microarray profiling analysis showed a 20-fold increase of KIF26A gene
expression and 11-fold decrease of lanosterol synthase expression. Expression analysis by qPCR showed significant increase of the growth factors VEGFA and PDGFB. The epoxidated compound induced a significantly higher basal and reserve mitochondrial capacity in both HDF and HepG2 cells. Additionally, in vivo wound healing in db/db mice, demonstrated a small but significant enhancement on wound healing upon local application of the compound compared to treatment with vehicle alone. Mono-epoxy-tocotrienol-α seems to possess beneficial effects on wound healing by increasing the expression of genes involved in cell growth, motility and angiogenes as well as on mitochondrial function
Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae.
Get PDFRad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria
Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways
Get PDFIn 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
(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
Evidence for interactions between homocysteine and genistein: insights into stroke risk and potential treatment
Get PDFElevated plasma homocysteine (2-amino-4-
sulfanylbutanoic acid) level is a risk factor for stroke.
Moreover, it has been suggested that high levels of homocysteine in the acute phase of an ischemic stroke can predict mortality, especially in stroke patients with the large-vessel atherosclerosis subtype. In clinical studies, supplementation with genistein(5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) decreased plasma homocysteine levels considerably. Therefore, genistein could be considered as a potential drug for prevention and/or treatment of stroke. However, the mechanism of the effect of genistein on homocysteine level remains to be elucidated. In this report, direct functional interactions between homocysteine and genistein are demonstrated in in vitro experimental systems for determination of methylenetetrahydrofolate reductase (MetF) and glutathione peroxidase(GPx) activities, reconstructed with purified compounds, and in a simple in vivo system, based on measurement of growth rate of Vibrio harveyi and Bacillus subtilis cultures. Results of molecular modelling indicated that homocysteine can directly interact with genistein. Therefore, genisteinmediated decrease in plasma levels of homocysteine, and alleviation of biochemical and physiological effects of one of these compounds by another, might be ascribed to formation of homocysteine-genistein complexes in which biological activities of these molecules are abolished or alleviated
Bad Phages in Good Bacteria: Role of the Mysterious orf63 of λ and Shiga Toxin-Converting 824B Bacteriophages
Get PDFLambdoid bacteriophages form a group of viruses that shares a common schema of
genome organization and lifecycle. Some of them can play crucial roles in creating the
pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli
(STEC) acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages).
The results obtained so far present the evidence for the relation between the exo-xis
region of the phage genome and lambdoid phage development, however molecular
mechanisms of activities of the exo-xis genes’ products are still unknown. In view of this,
we decided to determine the influence of the uncharacterized open reading frame orf63
of the exo-xis region on lambdoid phages development using recombinant prophages, λ
and Stx phage 824B. We have demonstrated that orf63 codes for a folded protein, thus,
it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend
this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution,
likely a trimer and consistent with its small size (63 aa.), is comprised of two helices, likely
intertwined to form the oligomer. We observed that the deletion of phage orf63 does not
impair the intracellular lambdoid phage lytic development, however delays the time and
decreases the efficiency of prophage induction and in consequence results in increased
survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63
negatively influences expression of themajor phage genes and open reading frames from
the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that
lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages
development, especially at the stage of the lysis vs. lysogenization decision. Besides,
orf63 probably participates in the regulation of the level of expression of essential phage
genes and open reading frames from the exo-xis region during prophage induction
Mating of 2 Laboratory Saccharomyces cerevisiae Strains Resulted in Enhanced Production of 2-Phenylethanol by Biotransformation of L-Phenylalanine
Get PDF2-Phenylethanol (2-PE) is an aromatic alcohol with a rosy scent which is widely used in the food, fragrance, and cosmetic industries. Promising sources of natural 2-PE are microorganisms, especially yeasts, which can produce 2-PE by biosynthesis and biotransformation. Thus, the first challenging goal in the development of biotechnological production of 2-PE is searching for highly productive yeast strains. In the present work, 5 laboratory Saccharomyces cerevisiae strains were tested for the production of 2-PE. Thereafter, 2 of them were hybridized by a mating procedure and, as a result, a new diploid, S. cerevisiae AM1-d, was selected. Within the 72-h batch culture in a medium containing 5 g/L of L-phenylalanine, AM1-d produced 3.83 g/L of 2-PE in a shaking flask. In this way, we managed to select the diploid S. cerevisiae AM1-d strain, showing a 3- and 5-fold increase in 2-PE production in comparison to parental strains. Remarkably, the enhanced production of 2-PE by the hybrid of 2 yeast laboratory strains is demonstrated here for the first time
Fungal lifestyle reflected in serine protease repertoire
Get PDFFungi are able to switch between different lifestyles in order to adapt to environmental changes. Their ecological strategy is connected to their secretome as fungi obtain nutrients by secreting hydrolytic enzymes to their surrounding and acquiring the digested molecules. We focus on fungal serine proteases (SPs), the phylogenetic distribution of which is barely described so far. In order to collect a complete set of fungal proteases, we searched over 600 fungal proteomes. Obtained results suggest that serine proteases are more ubiquitous than expected. From 54 SP families described in MEROPS Peptidase Database, 21 are present in fungi. Interestingly, 14 of them are also present in Metazoa and Viridiplantae – this suggests that, except one (S64), all fungal SP families evolved before plants and fungi diverged. Most representatives of sequenced eukaryotic lineages encode a set of 13–16 SP families. The number of SPs from each family varies among the analysed taxa. The most abundant are S8 proteases. In order to verify hypotheses linking lifestyle and expansions of particular SP, we performed statistical analyses and revealed previously undescribed associations. Here, we present a comprehensive evolutionary history of fungal SP families in the context of fungal ecology and fungal tree of life
Immunogenicity of DNA Vaccine against H5N1 Containing Extended Kappa B Site: In Vivo Study in Mice and Chickens
Get PDFInfluenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis