Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5′ resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5′-overhanging DSBs (5′ DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3′ DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5′ DSBs were rejoined more efficiently than 3′ DSBs, consistent with a robust recruitment of NHEJ proteins to 5′ DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5′ DSBs and facilitated protection of 3′ DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5′ DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3′ and 5′ DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5′ DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5′ DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.     

Clusters in alpha/beta barrel proteins: implications for protein structure, function, and folding: a graph theoretical approach

The alpha/beta barrel fold is adopted by most enzymes performing a variety of catalytic reactions, but with very low sequence similarity. In order to understand the stabilizing interactions important in maintaining the alpha/beta barrel fold, we have identified residue clusters in a dataset of 36 alpha/beta barrel proteins that have less than 10% sequence identity within themselves. A graph theoretical algorithm is used to identify backbone clusters. This approach uses the global information of the nonbonded interaction in the alpha/beta barrel fold for the clustering procedure. The nonbonded interactions are represented mathematically in the form of an adjacency matrix. On diagonalizing the adjacency matrix, clusters and cluster centers are obtained from the highest eigenvalue and its corresponding vector components. Residue clusters are identified in the strand regions forming the beta barrel and are topologically conserved in all 36 proteins studied. The residues forming the cluster in each of the alpha/beta protein are also conserved among the sequences belonging to the same family. The cluster centers are found to occur in the middle of the strands or in the C-terminal of the strands. In most cases, the residues forming the clusters are part of the active site or are located close to the active site. The folding nucleus of the alpha/beta fold is predicted based on hydrophobicity index evaluation of residues and identification of cluster centers. The predicted nucleation sites are found to occur mostly in the middle of the strands. Proteins 2001;43:103-112.     

The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes

Glucuronoxylans with a backbone of 1,4-linked β-D-xylosyl residues are ubiquitous in the secondary walls of gymnosperms and angiosperms. Xylans have been reported to be present in hornwort cell walls, but their structures have not been determined. In contrast, the presence of xylans in the cell walls of mosses and liverworts remains a subject of debate. Here we present data that unequivocally establishes that the cell walls of leafy tissue and axillary hair cells of the moss Physcomitrella patens contain a glucuronoxylan that is structurally similar to glucuronoxylans in the secondary cell walls of vascular plants. Some of the 1,4-linked β-D-xylopyranosyl residues in the backbone of this glucuronoxylan bear an α-D-glucosyluronic acid (GlcpA) sidechain at O-2. In contrast, the lycopodiophyte Selaginella kraussiana synthesizes a glucuronoxylan substituted with 4-O-Me-α-D-GlcpA sidechains, as do many hardwood species. The monilophyte Equisetum hyemale produces a glucuronoxylan with both 4-O-Me-α-D-GlcpA and α-D-GlcpA sidechains, as does Arabidopsis. The seedless plant glucuronoxylans contain no discernible amounts of the reducing-end sequence that is characteristic of gymnosperm and eudicot xylans. Phylogenetic studies showed that the P. patens genome contains genes with high sequence similarity to Arabidopsis CAZy family GT8, GT43 and GT47 glycosyltransferases that are likely involved in xylan synthesis. We conclude that mosses synthesize glucuronoxylan that is structurally similar to the glucuronoxylans present in the secondary cell walls of lycopodiophytes, monilophytes, and many seed-bearing plants, and that several of the glycosyltransferases required for glucuronoxylan synthesis evolved before the evolution of tracheophytes.     

In vivo determination of mouse olfactory mucus cation concentrations in normal and inflammatory States

Objective

Olfaction is impaired in chronic rhinosinusitis (CRS). The study has two aims: (1) to determine whether changes in cation concentration occur in the olfactory mucus of mice with CRS, which may affect chemo-electrical transduction, (2) and to examine whether these alterations are physiologically significant in humans.

Study Design

Animal study in mice and translational study in humans.

Methods

Inflammation was induced by sensitization and chronic exposure of 16 C57BL/6 mice to Aspergillus fumigatus. The control group included 16 untreated mice. Ion-selective microelectrodes were used to measure free cation concentrations in the olfactory mucus of 8 mice from each treatment group, while the remaining mice were sacrificed for histology. To validate the findings in the animal model, olfactory threshold was measured in 11 healthy human participants using Sniffin’ Sticks before and after nasal irrigation with solutions that were composed of either of the cation concentrations.

Results

In 8 mice, olfactory mucus of chronically inflamed mice had lower [Na⁺] (84.8±4.45 mM versus 93.73±3.06 mM, p = 0.02), and higher [K⁺] (7.2±0.65 mM versus 5.7±0.20 mM, p = 0.04) than controls. No difference existed in [Ca²⁺] (0.50±0.12 mM versus 0.54±0.06 mM, p = 0.39). In humans, rinsing with solutions replicating ion concentrations of the mouse mucosa with chronic inflammation caused a significant elevation in the median olfactory threshold (9.0 to 4.8, p = 0.003) but not with the control solution (8.3 to 7.8, p = 0.75).

Conclusion

Chronic inflammation elevates potassium and lowers sodium ion concentration in mice olfactory mucus. Nasal irrigation with a corresponding solution induced olfactory threshold shift in humans.     

Synthesis and photoluminescence properties of red emitting phosphor La2–xEuxLi0.5Al0.5O4 [x = 0.2–2] with K2NiF4 structure

Europium (Eu)³⁺‐substituted La₂Li_0.5Al_0.5O₄ red emitting phosphors were prepared by a conventional high‐temperature solid‐state reaction method. Powder X‐ray diffraction, diffuse reflectance spectra and spectrofluorometry were used as vital characterizing tools for the phosphors. The Eu concentration dependence luminescence properties and Judd–Ofelt intensity parameters were investigated and calculated, respectively. All compositions showed an orange red emission (due to the magnetic and electric dipole transitions of the Eu³⁺ ion) with the appropriate Commission Internationale de l'Eclairage (CIE) colour gamut under near ultraviolet or blue ray light excitation. The calculated critical distance showed that the energy transfer occured between Eu to Eu via an exchange mechanism. The Eu_1.4La_0.6Li_0.5Al_0.5O₄ composition showed the highest red emission intensity with CIE colour saturation compared with that of the commercial Eu‐activated yttrium oxysulfide red phosphor.     

Involvement of a glycerol-3-phosphate dehydrogenase in modulating the NADH/NAD+ ratio provides evidence of a mitochondrial glycerol-3-phosphate shuttle in Arabidopsis

A mitochondrial glycerol-3-phosphate (G-3-P) shuttle that channels cytosolic reducing equivalent to mitochondria for respiration through oxidoreduction of G-3-P has been extensively studied in yeast and animal systems. Here, we report evidence for the operation of such a shuttle in Arabidopsis thaliana. We studied Arabidopsis mutants defective in a cytosolic G-3-P dehydrogenase, GPDHc1, which, based on models described for other systems, functions as the cytosolic component of a G-3-P shuttle. We found that the gpdhc1 T-DNA insertional mutants exhibited increased NADH/NAD+ ratios compared with wild-type plants under standard growth conditions, as well as impaired adjustment of NADH/NAD+ ratios under stress simulated by abscisic acid treatment. The altered redox state of the NAD(H) pool was correlated with shifts in the profiles of metabolites concerning intracellular redox exchange. The impairment in maintaining cellular redox homeostasis was manifest by a higher steady state level of reactive oxygen species under standard growth conditions and by a significantly augmented hydrogen peroxide production under stress. Loss of GPDHc1 affected mitochondrial respiration, particularly through a diminished capacity of the alternative oxidase respiration pathway. We propose a model that outlines potential involvements of a mitochondrial G-3-P shuttle in plant cells for redox homeostasis.     

HPLC screening of natural vitamin E from mediterranean plant biofactories--a basic tool for pilot-scale bioreactors production of alpha-tocopherol

The study was performed in order to investigate a simple, efficient, reliable and rapid method of extracting and quantifying natural vitamin E for pressurized liquid extraction (PLE) as well as high-performance liquid chromatography (HPLC) analysis. Lyophilized Corylus avellana L. nut samples were powdered by high-speed milling with Waring blender for 40 s. alpha-Tocopherol was extracted from the nut tissue powder using dehydrated hexane fortified with 0.01% butylated hydroxytoluene (BHT, co-antioxidant). The rate of alpha-tocopherol accumulation showed differences among nut samples collected in different areas of Italy. Sarda Piccola nut biofactory contained higher amount (81.17 microg/g d.w) of alpha-tocopherol than other-local eleven Italian cultivar nuts. These results provide insight into the biofactory basis for alpha-tocopherol accumulation in hazelnut and give the suitable cultivar tissues to establish pilot-scale bioreactors production of natural bioactive vitamin E.     

Research on marine actinobacteria in India

Marine actinobacteriology is one of the major emerging areas of research in tropics. Marine actinobacteria occur on the sediments and in water and also other biomass (mangrove) and substrates (animal). These organisms are gaining importance not only for their taxonomic and ecological perspectives, but also for their unique metabolites and enzymes. Many earlier studies on these organisms were confined only to the temperate regions. In tropical environment, investigations on them have gained importance only in the last two decades. So far, from the Indian peninsula, 41 species of actinobacteria belonging to 8 genera have been recorded. The genus, Streptomyces of marine origin has been more frequently recorded. Of 9 maritime states of India, only 4 have been extensively covered for the study of marine actinobacteria. Most of the studies conducted pertain to isolation, identification and maintenance of these organisms in different culture media. Further, attention has been focused on studying their antagonistic properties against different pathogens. Their biotechnological potentials are yet to be fully explored.