Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel.     

A novel xylose isomerase from Neurospora crassa

Summary Highest production of xylose Isomerase by Neurospora crassa grown with different carbon sources was at 0.014 U mg^-1 with D-xylose. The enzyme exhibited maximum activity at pH 8.0 and 70°C and retained 100% activity at 45°C for 30 min at pH 8.0. It was activated by 8 mM Mg²⁺ whereas 2 mM Co²⁺ afforded protection against inactivation by heat. The K _m for xylose was 10 mM and 22 mM for xylose Isomerase and xylose reductase respectively at 28°C and pH 7.0. This is the first report on the presence of xylose isomerase in N. crassa and the existence of two different pathways for the utilization of D-xylose.     

A serine alkaline protease from the fungusConidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.     

Prospecting endophytes from different Fe or Zn accumulating wheat genotypes for their influence as inoculants on plant growth,yield, and micronutrient content

The plant microbiome is known to play a significant role in improving plant productivity and quality of produce, and the endophytic component was explored toward developing inoculants for enhancing micronutrient concentration in grains. A set of 213 endophytes (201 bacteria and 12 fungi) were isolated, from a set of 13 wheat genotypes, identified through preliminary screening as low and high iron and zinc accumulating genotypes. A pot experiment, with two low accumulator genotypes and eight selected endophytes, was undertaken, followed by field evaluation with both high and low Zn or Fe accumulator genotypes. Screening of these endophytes identified 11 bacteria and 2 fungi as being efficient for zinc solubilization, while 10 bacteria and 2 fungi were promising siderophore producers. Zinc and iron uptake were enhanced by one to several folds over the recommended dose of NPK (RDF) in the pot experiment. Two sets of promising endophytes, identified as Bacillus subtilis (DS-178) and Arthrobacter sp. (DS-179) for zinc accumulation, and Arthrobacter sulfonivorans (DS-68) and Enterococcus hirae (DS-163) for iron acquisition in grains, were selected. Significant increase of 14–20% in plant growth and yield was recorded in field experiment, with 75% increase, over RDF, in terms of Fe or Zn accumulation in wheat grains. Phytic acid, an anti-nutritional factor, was significantly lower in grains from endophyte inoculated treatments in the wheat genotypes evaluated. This illustrated the promise of these endophytes in improving both the translocation of micronutrients and enhancing the quality of wheat grains.     

12,13-diHOME: An Exercise-Induced Lipokine that Increases Skeletal Muscle Fatty Acid Uptake

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A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay

Background

Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available.

Methods

We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa.

Results

The assay’s analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6–0.9) and 84% (95% CI 0.6–0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77–1) and 94% (95% CI 0.7–0.99), respectively.

Conclusion

This novel approach may permit simple and rapid detection of TB using pediatric stool samples.     

Characterization of SNP and Structural Variations in the Mitochondrial Genomes of Tilletia indica and Its Closely Related Species Formed Basis for a Simple Diagnostic Assay

Tilletia indica causes the disease Karnal bunt in wheat. The disease is under international quarantine regulations. Comparative mitochondrial (mt) genome analysis of T. indica ({"type":"entrez-nucleotide","attrs":{"text":"KX394364","term_id":"1128620031","term_text":"KX394364"}}KX394364 and {"type":"entrez-nucleotide","attrs":{"text":"DQ993184","term_id":"115361453","term_text":"DQ993184"}}DQ993184) and T. walkeri ({"type":"entrez-nucleotide","attrs":{"text":"EF536375","term_id":"144925993","term_text":"EF536375"}}EF536375) has found 325 to 328 SNPs, 57 to 60 short InDels (from 1 to 13 nt), two InDels (30 and 61 nt) and five (>200 nt) presence/absence variations (PAVs) between the two species. The mt genomes of both species have identical gene order. The numbers of SNPs and InDels between the mt genomes of the two species are approximately nine times of the corresponding numbers between the two T. indica isolates. There are eight SNPs between T. indica and T. walkeri that resulted in amino acid substitutions in the mt genes of cob, nad2 and nad5. In contrast, there is no amino acid substitution in the mt genes of the T. indica isolates from the SNPs found. The five PAVs present in T. indica ({"type":"entrez-nucleotide","attrs":{"text":"DQ993184","term_id":"115361453","term_text":"DQ993184"}}DQ993184) are absent in T. walkeri. Four PAVs are more than 1 kb and are not present in every T. indica isolate. Analysis of their presence and absence separates a collection of T. indica isolates into 11 subgroups. Two PAVs have ORFs for the LAGLIDAG endonuclease and two have ORFs for the GIY-YIG endonuclease family, which are representatives of homing endonuclease genes (HEGs). These intron- encoded HEGs confer intron mobility and account for their fluid distribution in T. indica isolates. The small PAV of 221 bp, present in every T. indica isolate and unique to the species, was used as the genetic fingerprint for the successful development of a rapid, highly sensitive and specific loop mediated isothermal amplification (LAMP) assay. The simple procedure of the LAMP assay and the easy detection formats will enable the assay to be automated for high throughput diagnosis.     

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.