Association of CDKN2BAS gene polymorphism with periodontitis and Coronary Artery Disease from South Indian population.

BACKGROUND: Periodontal disease (PD), a chronic inflammatory disorder mediated by progressive destruction of the oral cavity is one of the key factors for many systemic disorders including Coronary Artery Disease (CAD). The upregulation of CDKN2BAS, a long noncoding RNA gene expression in gingival epithelial cells and gingival fibroblasts of periodontitis shows a strong correlation between the severity of atherosclerosis and PD. Considering the crucial role of CDKN2BAS gene polymorphisms (rs496892 G > A and rs7865618 A > G) and its expression the present study sought to identify the possible association with the disease predisposition in South Indian population. METHODS: For the present case-control study a total of 200 subjects that include 100 PD-CAD patients and 100 controls were recruited with prior consent. Genomic DNA and RNA were extracted and utilized for genotyping via ARMS-PCR and PCR-RFLP, and expression using RT-PCR respectively. RESULTS: The results showed a significant association of both the polymorphisms with that of the disease predisposition. The wild type genotypes (GG: OR-0.37; p-0.001; & AA: OR-0.29; p-0.005) conferred protection against the disease, whereas, the heterozygotes (GA: OR-2.45; p-0.004 & AG: OR-3.41; p-0.0001) conferred risk towards the disease, suggesting the involvement of the variant allele in disease causation. These results were further confirmed by haplotype analysis among A-G block (two variant alleles at both loci) with 2.5 fold risk (OR = 2.49, 95% CI = 1.16-5.36, p = 0.02) and G-G block (single risk allele at rs7865618 locus) with 3-fold risk (OR-3.0; p-0.01) towards the disease, suggesting the dominant involvement of rs7865618 in the disease causation. Though the expression of the CDKN2BAS gene is more in patients than controls, the variant genotypes among patients were evaluated to be down-regulated than the other genotypes. CONCLUSION: The present study concludes that the two selected polymorphisms have significant involvement individually and in interaction with each other in the disease predisposition. The expression studies also suggest that the selected polymorphisms in the 9p21.3 locus affect the CDKN2BAS gene expression. However, the results obtained in the present study should be confirmed with large samples in other ethnic cohorts.

Regulation of Gene Expression during the Onset of Ligninolytic Oxidation by Phanerochaete chrysosporium on Spruce Wood.

Since uncertainty remains about how white rot fungi oxidize and degrade lignin in wood, it would be useful to monitor changes in fungal gene expression during the onset of ligninolysis on a natural substrate. We grew Phanerochaete chrysosporium on solid spruce wood and included oxidant-sensing beads bearing the fluorometric dye BODIPY 581/591 in the cultures. Confocal fluorescence microscopy of the beads showed that extracellular oxidation commenced 2 to 3 days after inoculation, coincident with cessation of fungal growth. Whole transcriptome shotgun sequencing (RNA-seq) analyses based on the v.2.2 P. chrysosporium genome identified 356 genes whose transcripts accumulated to relatively high levels at 96 h and were at least four times the levels found at 40 h. Transcripts encoding some lignin peroxidases, manganese peroxidases, and auxiliary enzymes thought to support their activity showed marked apparent upregulation. The data were also consistent with the production of ligninolytic extracellular reactive oxygen species by the action of manganese peroxidase-catalyzed lipid peroxidation, cellobiose dehydrogenase-catalyzed Fe(3+) reduction, and oxidase-catalyzed H2O2 production, but the data do not support a role for iron-chelating glycopeptides. In addition, transcripts encoding a variety of proteins with possible roles in lignin fragment uptake and processing, including 27 likely transporters and 18 cytochrome P450s, became more abundant after the onset of extracellular oxidation. Genes encoding cellulases showed little apparent upregulation and thus may be expressed constitutively. Transcripts corresponding to 165 genes of unknown function accumulated more than 4-fold after oxidation commenced, and some of them may merit investigation as possible contributors to ligninolysis.

Evidence from Serpula lacrymans that 2,5-dimethoxyhydroquinone Is a lignocellulolytic agent of divergent brown rot basidiomycetes.

Basidiomycetes that cause brown rot of wood are essential biomass recyclers in coniferous forest ecosystems and a major cause of failure in wooden structures. Recent work indicates that distinct lineages of brown rot fungi have arisen independently from ligninolytic white rot ancestors via loss of lignocellulolytic enzymes. Brown rot thus proceeds without significant lignin removal, apparently beginning instead with oxidative attack on wood polymers by Fenton reagent produced when fungal hydroquinones or catechols reduce Fe(3+) in colonized wood. Since there is little evidence that white rot fungi produce these metabolites, one question is the extent to which independent lineages of brown rot fungi may have evolved different Fe(3+) reductants. Recently, the catechol variegatic acid was proposed to drive Fenton chemistry in Serpula lacrymans, a brown rot member of the Boletales (D. C. Eastwood et al., Science 333:762-765, 2011). We found no variegatic acid in wood undergoing decay by S. lacrymans. We found also that variegatic acid failed to reduce in vitro the Fe(3+) oxalate chelates that predominate in brown-rotting wood and that it did not drive Fenton chemistry in vitro under physiological conditions. Instead, the decaying wood contained physiologically significant levels of 2,5-dimethoxyhydroquinone, a reductant with a demonstrated biodegradative role when wood is attacked by certain brown rot fungi in two other divergent lineages, the Gloeophyllales and Polyporales. Our results suggest that the pathway for 2,5-dimethoxyhydroquinone biosynthesis may have been present in ancestral white rot basidiomycetes but do not rule out the possibility that it appeared multiple times via convergent evolution.

Spatial mapping of extracellular oxidant production by a white rot basidiomycete on wood reveals details of ligninolytic mechanism.

Oxidative cleavage of the recalcitrant plant polymer lignin is a crucial step in global carbon cycling, and is accomplished most efficiently by fungi that cause white rot of wood. These basidiomycetes secrete many enzymes and metabolites with proposed ligninolytic roles, and it is not clear whether all of these agents are physiologically important during attack on natural lignocellulosic substrates. One new approach to this problem is to infer properties of ligninolytic oxidants from their spatial distribution relative to the fungus on the lignocellulose. We grew Phanerochaete chrysosporium on wood sections in the presence of oxidant-sensing beads based on the ratiometric fluorescent dye BODIPY 581/591. The beads, having fixed locations relative to the fungal hyphae, enabled spatial mapping of cumulative extracellular oxidant distributions by confocal fluorescence microscopy. The results showed that oxidation gradients occurred around the hyphae, and data analysis using a mathematical reaction-diffusion model indicated that the dominant oxidant during incipient white rot had a half-life under 0.1 s. The best available hypothesis is that this oxidant is the cation radical of the secreted P. chrysosporium metabolite veratryl alcohol.

Proteomic and functional analysis of the cellulase system expressed by Postia placenta during brown rot of solid wood.

Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these--two in glycoside hydrolase family 5, one in family 10, and one in family 12--have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.

Expression and functional characterisation of TNC, a high-affinity nickel transporter from Neurospora crassa.

Our previous in silico studies identified a high-affinity nickel transporter, TNC, from the metal transportome of Neurospora crassa. A knockout mutant of the tnc gene in N. crassa failed to transport nickel, showed phenotypic growth defects and diminished urease activity under physiological levels of nickel. Transport assays conducted in wild type and knockout mutant strains showed that TNC transports nickel with high affinity but exhibits selectivity for other transition metal ions like cobalt. Heterologous complementation of Schizosaccharomyces pombe nickel uptake mutant by TNC further substantiates its nickel transport function. Transcriptional analysis of the nickel transporter encoding gene, tnc in N. crassa by qRT-PCR showed its constitutive expression in various phases of its life cycle. However, levels of the corresponding protein TNC were down-regulated only by increasing the nickel, but not cobalt concentration in the media. Immunolocalisation data suggested that TNC is distributed in the plasma membrane of N. crassa. Thus, the present study establishes TNC as a functional plasma membrane nickel transporter necessary for physiological acquisition of nickel in the multicellular fungi N. crassa.

Transgenic Nicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper.

The diets of two-thirds of the world's population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T(0) and T(1)) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd(2+) and Zn(2+) did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper.

Characterization of Ctr family genes and the elucidation of their role in the life cycle of Neurospora crassa.

Transcriptional analysis using qRT-PCR of 62 metal ion transporters during conidial germination of Neurospora crassa showed a significant up regulation of a hypothetical copper transporter gene, tcu-1, that belongs to the Ctr family. Herein we characterised the Ctr family genes (tcu-1, tcu-2 and tcu-3) and deciphered their role in various developmental phases of the N. crassa life cycle. Cross complementation assays in copper uptake mutant of Saccharomyces cerevisiae revealed that tcu-1, tcu-2 and tcu-3 are functional homologs of S. cerevisiae copper transporters. Expression studies of Ctr family members in various developmental phases of N. crassa showed differential expression pattern for high-affinity copper transporter, TCU1. Functional analysis of their gene knockout mutants showed that tcu-1 is essential for saprophytic conidial germination, vegetative growth and perithecia development under copper limited conditions while conidiation remained unaffected.