Mutations in KARS,Encoding Lysyl-tRNA Synthetase,Cause Autosomal-Recessive Nonsyndromic Hearing Impairment DFNB89

Previously, DFNB89, a locus associated with autosomal-recessive nonsyndromic hearing impairment (ARNSHI), was mapped to chromosomal region 16q21–q23.2 in three unrelated, consanguineous Pakistani families. Through whole-exome sequencing of a hearing-impaired individual from each family, missense mutations were identified at highly conserved residues of lysyl-tRNA synthetase (KARS): the c.1129G>A (p.Asp377Asn) variant was found in one family, and the c.517T>C (p.Tyr173His) variant was found in the other two families. Both variants were predicted to be damaging by multiple bioinformatics tools. The two variants both segregated with the nonsyndromic-hearing-impairment phenotype within the three families, and neither mutation was identified in ethnically matched controls or within variant databases. Individuals homozygous for KARS mutations had symmetric, severe hearing impairment across all frequencies but did not show evidence of auditory or limb neuropathy. It has been demonstrated that KARS is expressed in hair cells of zebrafish, chickens, and mice. Moreover, KARS has strong localization to the spiral ligament region of the cochlea, as well as to Deiters’ cells, the sulcus epithelium, the basilar membrane, and the surface of the spiral limbus. It is hypothesized that KARS variants affect aminoacylation in inner-ear cells by interfering with binding activity to tRNA or p38 and with tetramer formation. The identification of rare KARS variants in ARNSHI-affected families defines a gene that is associated with ARNSHI.     

Linking oxidative and salinity stress tolerance in barley: can root antioxidant enzyme activity be used as a measure of stress tolerance?

Aims

A causal relationship between salinity and oxidative stress tolerance and a suitability of using root antioxidant activity as a biochemical marker for salinity tolerance in barley was investigated.

Methods

Net ion fluxes were measured from the mature zone of excised roots of two barley varieties contrasting in their salinity tolerance using non-invasive MIFE technique in response to acute and prolonged salinity treatment. These changes were correlated with activity of major antioxidant enzymes; ascorbate peroxidase, catalase, and superoxide dismutase.

Results

It was found that genotypic difference in salinity tolerance was largely independent of root integrity, and observed not only for short-term but also long-term NaCl exposures. Higher K⁺ retention ability (and, hence, salinity tolerance) positively correlated with oxidative stress tolerance. At the same time, antioxidant activities were constitutively higher in a sensitive but not tolerant variety, and no correlation was found between SOD activity and salinity tolerance index during large-scale screening.

Conclusion

Although salinity tolerance in barley correlates with its oxidative stress tolerance, higher antioxidant activity at one particular time does not correlate with salinity tolerance and, as such, cannot be used as a biochemical marker in barley screening programs.     

Anatomical characteristics and antioxidant ability of Centaurea sadleriana reveals an adaptation towards drought tolerance

The lamina, main vein and peduncle anatomical properties of Centaurea sadleriana Janka plants from two populations, were examined using light and scanning electron microscopy. The indumentum was comprised of glandular and non-glandular trichomes of two types. The leaves were amphistomatic, isolateral, with strongly developed palisade tissue. Secretory ducts were observed along the phloem or sclerenchyma of large vascular bundles. Collenchyma alternated with chlorenchyma in the main vein and peduncle. Large groups of strongly lignified sclerenchyma were present along the phloem of peduncle vascular bundles. These features, together with thickened walls of epidermal cells and cuticle, numerous trichomes and thick-walled parenchyma in the perimedullar zone, were perceived as a xeromorphic peduncle structural adaptation. Non-enzymatic antioxidant compounds of phenolic origin were detected in small amounts and their respective content was higher in leaves compared to inflorescences. Compounds of phenolic orgin showed positive correlation with total potenial of antioxidant activity indicated by the DPPH assay. Greater total quantity of polyphenols and tannins was detected in leaves of plants from Zobnatica locality, while leaves of plants from Rimski Sanac were characterized by higher content of total flavonoids and proantocyanidins. Phytochemical analysis showed that dominant secondary biomolecules in inflorescences were phenolic pigments including anthocyanins and leucoanthocyanins, and free quinones in leaves.     

Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System

In this study, we tested the NuGEN Ovation RNA-Seq System for library preparation followed by next-generation sequencing on an Illumina GAIIx. The cDNA product of the NuGEN kit may have significant amounts of ssDNA with hairpin structures that are generated during the amplification process. These structures interfere with efficient downstream end repair, A-tailing, and adapter ligation, all standard steps in post-amplification sequencing library construction. We were able to increase the efficiency of sequencing library yields 4- to 6-fold or greater by treatment of NuGEN-amplified cDNA product with the single-strand endonuclease S1. These results suggest that this treatment effectively cleaves hairpin structures generated during amplification that are resistant to the standard enzyme cocktails used for the end-repair step.     

Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLe(X)), and related lectin ligands on effector leukocytes. Based on anti-sLe(X) antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLe(X) formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLe(X) (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLe(X) structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLe(X) on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.     

The neuroprotective effect of Activin A and B: implication for neurodegenerative diseases

Activin is a member of the transforming growth factor-beta superfamily which comprises a growing list of multifunctional proteins that function as modulators of cell proliferation, differentiation, hormone secretion and neuronal survival. This study examined the neuroprotective effect of both Activin A and B in serum withdrawal and oxidative stress apoptotic cellular models and investigated the expression of pro- and anti-apoptotic proteins, which may account for the mechanism of Activin-induced neuroprotection. Here, we report that recombinant Activin A and B are neuroprotective against serum deprivation- and toxin- [either the parkinsonism-inducing neurotoxin, 6-hydroxydopamine (6-OHDA) or the peroxynitrite donor, 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1)] induced neuronal death in human SH-SY5Y neuroblastoma cells. Furthermore, we demonstrate for the first time that transient transfection with Activin betaA or betaB significantly protect SH-SY5Y and rat pheochromocytoma PC12 cells against serum withdrawal-induced apoptosis. This survival effect is mediated by the Bcl-2 family members and involves inhibition of caspase-3 activation; reduction of cleaved poly-ADP ribose polymerase and phosphorylated H2A.X protein levels and elevation of tyrosine hydroxylase expression. These results indicate that both Activin-A and -B share the potential to induce neuroprotective activity and thus may have positive impact on aging and neurodegenerative diseases to retard the accelerated rate of neuronal degeneration.     

What distinguishes the Olympic level heavyweight rowers from other internationally successful rowers?

The aim of the study was to determine which of the observed variables differentiate mostly the successful (Olympic Games contenders) from the less successful rowers (Internationally competitive rowers) and in order for that anthropometric and physiological profile of elite Croatian heavyweight rowers was assessed. Twenty nine national rowing champions were divided into two groups according to their international competitive achievements. Physiological characteristics were assessed during a continuous progressive rowing ergometer test. Differences were determined using the independent t-test with Bonferroni corrections. Successful rowers had somewhat larger values of almost all of the observed lengths, widths and girths (exceptions: chest and thigh girth). In addition, their maximal oxygen uptake was significantly higher (5.51 +/- 0.40 L/min vs. 5.16 +/- 0.39 L/min; p < 0.05) as was their power output at anaerobic threshold (346.3 +/-17.9 W vs. 319.2 +/- 20.1 W; p < 0.01). Less successful Croatian rowers should target their training in the upcoming years towards increasing, primarily, maximal oxygen uptake and power output at anaerobic threshold.