Congenital cataract causing mutants of αA-crystallin/sHSP form aggregates and aggresomes degraded through ubiquitin-proteasome pathway

Background

Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy.

Methodology/Principal Findings

YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control.

Conclusions/Significance

Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells.     

Niacin deficiency modulates genes involved in cancer: Are smokers at higher risk?

The role of niacin’s metabolite, nicotinamide adenine dinucleotide (NAD), in DNA repair via base-excision repair pathway is well documented. We evaluated if niacin deficiency results in genetic instability in normal human fetal lung fibroblasts (MRC-5), and further, does it leads to enhanced accumulation of cigarette smoke–induced genetic damage? MRC-5 cells were grown discretely in niacin-proficient/deficient media, and exposed to nicotine-derived nitrosamine ketone (NNK, a cigarette smoke carcinogen). Niacin deficiency abated the NAD polymerization, augmented the spontaneous induction of micronuclei (MN) and chromosomal aberrations (CA) and raised the expression of 10 genes and suppressed 12 genes involved in different biological functions. NNK exposure resulted in genetic damage as measured by the induction of MN and CA in cells grown in niacin-proficient medium, but the damage became practically marked when niacin-deficient cells were exposed to NNK. NNK exposure raised the expression of 16 genes and suppressed the expression of 56 genes in cells grown in niacin-proficient medium. NNK exposure to niacin-deficient cells raised the expression of eight genes including genes crucial in promoting cancer such as FGFR3 and DUSP1 and suppressed the expression of 33 genes, including genes crucial in preventing the onset and progression of cancer like RASSF2, JUP, and IL24, in comparison with the cells grown in niacin-proficient medium. Overall, niacin deficiency interferes with the DNA damage repair process induced by chemical carcinogens like NNK, and niacin-deficient population are at the higher risk of genetic instability caused by cigarette smoke carcinogen NNK.     

Length–weight relationships of six indigenous fish species from the River Cauvery and its estuary,India

The present study estimated length–weight relationships (LWRs) for six indigenous fish species (Barilius gatensis, Salmostoma acinaces, S. boopis, Puntius amphibius, Hemibagrus punctatus and Ambassis miops) based on specimens collected from River Cauvery (including estuary) during July 2017–January 2020. The sampling surveys were carried out in three distinct sampling seasons, viz., the pre-monsoon (March–May), the monsoon (July–October) and the post-monsoon (November–February). Majority of the fish specimens dealt in the study were collected from multi-meshed monofilament gill nets (mesh sizes 18, 30, 45, 60, 90, 110, 120 and 150 mm) operated by local fishers. For those sites situated in the protected areas, sampling was carried out by cast nets with prior permission from the local administration and the collected fishes were released back into river after length–weight measurements. The length measurements were noted as total length (TL) measured to the nearest 0.1 cm by using a digital Vernier caliper. A digital balance was used for weight measurements with an accuracy of 0.01 g. The study recorded a new maximum length of 48 cm for H. punctatus. The LWR data generated from the present study are significant for proper assessment of the stock status and their management, if collected together with other essential biological and physical parameters.     

Green biosynthesis of silver nanoparticles from Annona squamosa leaf extract and its in vitro cytotoxic effect on MCF-7 cells

The biological method for the synthesis of silver nanoparticles (AgNPs) using Annona squamosa leaf extract and its cytotoxicity against MCF-7 cells are reported. The synthesized AgNPs using A. squamosa leaf extract was determined by UV–visible spectroscopy and it was further characterized by FT-IR, X-ray diffraction (XRD), Transmission electron microscopy (TEM), Zeta potential and energy dispersive spectrometric (EDS) analysis. The UV–visible spectrum showed an absorption peak at 444 nm which reflects surface plasmon resonance (SPR) of AgNPs. TEM photography showed biosynthesized AgNPs were predominantly spherical in shape with an average size ranging from 20 to 100 nm. The Zeta potential value of ?37 mV revealed the stability of biosynthesized AgNPs. Furthermore, the green synthesized AgNPs exhibited a dose-dependent cytotoxicity against human breast cancer cell (MCF-7) and normal breast epithelial cells (HBL-100) and the inhibitory concentration (IC₅₀) were found to be 50 μg/mL, 30 μg/mL, and 80 μg/mL, 60 μg/ml for AgNPs against MCF-7 and normal HBL-100 cells at 24 h and 48 h incubation respectively. An induction of apoptosis was evidenced by (AO/EtBr) and DAPI staining. Application of such eco-friendly nanoparticles makes this method potentially exciting for the large scale synthesis of nanoparticles.     

Role of some riparian wetland plants in reducing erosion of organic carbon and selected cations

Five riparian herbaceous plants, Leonotis nepetaefolia, Cassia tora, Ageratum conyzoides, Parthenium hysterophorus and Sida acuta, dominant on the banks of the Rihand river at Renukoot (India), were selected to assess experimentally their quantitative role in conserving organic-C, Na, K and Ca. Young seedlings from the river bank were planted on sloping experimental plots made of alluvial soil. Simulated rainfall totalling 42.5 mm was applied at 300 mm h⁻¹ on five vegetated and one bare plots. Runoff water and eroded soil were collected from each experimental plot in artificial reservoirs and their quantities were measured. The soil conservation value of the five selected species ranged between 33 and 84% while the water conservation value varied between 19 and 50%. The overall nutrient conservation value, based on the losses in runoff water and eroded soil taken together, varied from 30 to 83% for organic-C, 19 to 78% for Na, 13 to 72% for K and 29 to 52% for Ca under different species. Loss of these four nutrients in response to 42.5 mm simulated rainfall was much higher than their input through rainfall. Loss value for the nutrients were in following order: organic-C > Ca > K > Na. The fraction of organic-C transported down the slope was higher in eroded soil (averaging 73%) and of exchangeable bases in runoff water (averaging 86% for Na, 82% for K and 90% for Ca). Flow-weighted concentrations of all the studied nutrients were consistently greater from bare stands. Number of fine roots was found to play greater role in the case of organic-C (92%; p < 0.01) and Na (70%; p < 0.05) runoff and their conservation by different plant species but canopy cover played greater role for K (58%; p < 0.08) and Ca (90%; p < 0.01).     

SNP discovery from next-generation transcriptome sequencing data and their validation using KASP assay in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Single nucleotide polymorphisms (SNPs) are becoming the most amenable form of DNA-based molecular markers for genetic analysis. In hexaploid bread wheat (Triticum aestivum L.), it is difficult to discern true polymorphic SNPs due to homoeologous and paralogous genes. Two serial analysis of gene expression (SAGE) libraries were developed utilizing leaves from resistant plants carrying leaf rust resistance gene Lr28; one library was derived from leaves that were mock inoculated and the other was derived from leaves inoculated with the urediniospores of the leaf rust pathogen Puccinia triticina. Next-generation sequencing reads, after quality trimming and removal of fungal sequences, were mapped to wheat reference sequences at Ensembl Plants. CLC Genomics Workbench and Freebayes softwares were employed for SNP calling. A total of 611 SNPs were predicted to be common by both softwares, of which 207 varietal SNPs were identified by ConservedPrimer software. A subset of 100 SNPs was used for validation across 47 wheat genotypes using Kompetitive Allele Specific PCR (KASP) assay; 83 SNPs could be successfully validated. These SNPs were positioned on wheat subgenomes and chromosome arms. When functionally annotated, many sequences harboring SNPs showed homology to resistance and resistance-like genes listed in Plant Resistance Gene database (PRGdb) as well as pathogenesis-related (PR) and stress-responsive genes. The results of the present study involving discovery of SNPs associated with resistance to leaf rust, a major threat to wheat production worldwide, will be valuable for molecular breeding for rust resistance.     

Pollination Ecology and Endemic Trends in Pedicularis bracteosa var. atrosanguinea Pennell & Thompson (Scrophulariaceae) in North America

Abstract Pedicularis bracteosa var. atrosanguinea occurs locally in association with P. racemosa or P. groenlandica in the Olympic Mountains in Washington. Other plant species, e.g., Polygonum bistortoides, Lupinus argenteus var. parviflorus and Valeriana sitchensis compete for space and bumblebee pollinators within the study area. Pollinator sharing, resulting from such competition, may increase the frequency of unvisited flowers of P. bracteosa var. atrosanguinea. P. bracteosa var. atrosanguinea , with blood purple nectariferous flowers, is presumed to lack intense reflections of blue spectral components from its corollas in attracting bumblebees compared to those of P. racemosa and P. groenlandica . Six species of bumblebees ( Bombus ) and cuckoo bees ( Psithyrus ) pollinate P. bracteosa var. atrosanguinea . Of these, Bombus mixtus and B. occidentalis occur in higher frequencies and are the major pollinators of P. bracteosa var. atrosanguinea . Queen and larger worker bumblebees pollinate nototribically as they probe for nectar, while smaller worker bumblebees pollinate sternotribically while scraping pollen from anthers deeply hidden in the dome-shaped galea. Corbicular pollen loads of bumblebees collected in the study area contain Pedicularis pollen alone/in combination of Polygonum, Valeriana, Lupinus, Erigeron and Bidens , or exclusively of Polygonum or Valeriana. P. bracteosa var. atrosanguinea does not suffer seriously from deficient pollination but seedlings resulting from pollinated flowers may be subjected to natural selection pressure exerted by colonial plant species for space. P. bracteosa var. atrosanguinea does not propagate asexually but resumes aerial growth seasonally from the self-same underground root stocks. If seedlings are under continuous selection pressure for lack of space, P. bracteosa var. atrosanguinea is presumed to regenerate primarily by perennial root stocks. This behavior may favor endemism in P. bracteosa var. atrosanguinea .     

Evaluation of photosynthetic efficiency of yam bean (Pachyrhizus erosus L.) at saturating photon flux density under elevated carbon dioxide

Physiology and Molecular Biology of Plants - The future CO2 concentration is projected to reach 900–1000&nbsp;ppm levels by the end of twenty-first century, pertaining to global climatic...     

A Meta-Analysis of the Association between the CC Chemokine Ligand 5 (CCL5) -403 G>A Gene Polymorphism and Tuberculosis Susceptibility

Aim

Many case-control studies have been performed in the recent past to investigate the association between CCL5 -403 G>A (rs2107538) gene polymorphism and tuberculosis (TB) susceptibility in various ethnic groups. However, these studies have produced inconsistent and contradictory results. In the present study, meta-analysis was performed to assess the association between CCL5 -403 G>A polymorphism and TB risk.

Methodology

Quantitative synthesis was done for the published studies based upon association between CCL5 -403 G>A polymorphism and TB risk from PubMed (Medline), EMBASE web search. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for allele contrast, homozygous, heterozygous, dominant and recessive genetic models.

Results

A total of six studies comprising 1638 confirmed TB cases and 1519 healthy controls were included in this meta-analysis. Variant A allele (A vs. G: p = 0.035; OR = 1.301, 95% CI = 1.019 to 1.662) and variant homozygous (AA vs. GG; p = 0.001; OR = 1.520, 95% CI = 1.202 to 1.923) carriers were significantly associated with TB susceptibility. Similarly, recessive model (AA vs. GG+GA: p = 0.016; OR = 1.791, 95% CI = 1.117 to 2.873) also indicated increased TB risk. Whereas, heterozygous (GA vs. GG: p = 0.837; OR = 1.028, 95% CI = 0.791 to 1.335) and dominant (AA+GA vs. GG: p = 0.222; OR = 1.188, 95% CI = 0.901 to 1.567) models failed to show increased risk of developing TB.

Conclusions

This meta-analysis suggests that there is a significant association between the CCL5 -403 G>A polymorphism and increased risk of TB. However, larger well-designed epidemiological studies with stratified case control and biological characterization may be helpful to validate this association.     

Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.