Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy

Background

A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures.

Results

Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer’s paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6’s efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly.

Conclusions

Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

     

Selection of effective and highly thermostable <Emphasis Type="Italic">Bacillus subtilis</Emphasis> lipase A template as an industrial biocatalyst-A modern computational approach

Biocatalysts are intrinsically reactive and hence their operational stability is of vital significance for any bioprocess. The setback in biocatalyst stability has been tackled from diverse prospects. Inherently, stable biocatalysts are markedly realized and a regular attempt is being made to seek out new organisms that harbor them. Here, we analyzed the industrial biocatalyst lipase A (Native) of Bacillus subtilis and its six thermostable mutants (2M, 3M, 4M, 6M, 9M and 12M) computationally using conformational sampling technique. Consequently, the various structural events deciphering thermostability like root mean square deviation, root mean square fluctuation, radius of gyration and polar surface area showed mutant 12M to be highly stable with statistical validation. Besides, static model analysis involving intra-molecular interactions, secondary structure, solvent accessibility, hydrogen bond pattern, simulated thermal denaturation and desolvation energy also supported 12M comparatively. Of note, the presence of high secondary structural rigidity and hydrogen bonds increased thermostability and functionality of 12M, thus selecting it as a best template for designing thermostable lipases in future. Also, this study has a significant implication toward a better understanding of conformational sampling in enzyme catalysis and enzyme engineering.     

Evaluation of the cocktail recombinant antigens,LipL32 and LipL41 for serodiagnosis of canine leptospirosis

Leptospirosis is one of the diseases with economic impact, so its diagnosis and serosurveillance are very important for any control program. Many tests have been used in the field as screening tests of leptospirosis. The aim of this study was to evaluate the efficacy of combined recombinant antigens (rLipL41 + rLipL32) for serodiagnosis of canine leptospirosis and to compare the efficacy of IgG Enzyme linked immunosorbent assay and Latex agglutination test with standard MAT. A total of 533 canine serum samples were subjected to IgG-ELISA and LAT using recombinant LipL41 and LipL32 antigens in a single and in a combinations which were compared with standard MAT. The potential diagnostic cocktails of combined recombinant antigens (rLipL31 + rLipL41) developed in this study showed higher sensitivity and specificity in IgG ELISA and LAT (94.84, 88.50%; 98.21, 97.7%) when compared to the use of single recombinant antigen in this study. Results indicated that the both assays can be easily performed, and avoids the risk of infection in laboratory workers, and it seems to be a practical and suitable tool for serodiagnosis of leptospirosis.     

BIOCHEMICAL CHARACTERIZATION OF β-N-OXALYL-l-α,β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN AS AN EXCITANT AMINO ACID

Abstract— β- N -Oxalyl- l -α,β-diaminopropionic acid (ODAP), the toxin isolated from the seeds of Luthyrus sativus produces head retraction, tremors and convulsions when injected into a variety of experimental animals. In 12-day-old rats, it has been found that the convulsive behaviour is accompanied by profound biochemical changes in the brain. The brain homogenates prepared from ODAP injected animals show a higher rate of respiration. There is a decrease in the brain glucose, glycogen, ATP, phosphocreatine and acetylcholine levels of the convulsing animals. The inorganic phosphate, lactic acid and acetylcholineesterase levels increase. These results establish that ODAP is a typical convulsant.     

Hepatoprotective effect of Rhodiola imbricata rhizome against paracetamol-induced liver toxicity in rats

Rhodiola imbricata is a perennial herb of the family Crassulaceae, which has significant traditional usage as medicine and is also known to biosynthesize phytochemicals such as flavonoids, coumarins and phenyl glycosides. The present investigation was aimed to estimate the hepatoprotective activity of R. imbricata rhizome acetone extract against paracetamol (2 g/kg) induced liver toxicity. Paracetamol was administered to induce hepatic damage in Wistar rats. 200 and 400 mg/kg doses of rhizome acetone extract and silymarin (25 mg/kg) were used as treatment groups. The blood samples were analyzed for biochemical markers of hepatic injury and tissue samples were subjected for estimation of liver antioxidants and histopathological studies. Analysis of the extract treated rats (400 mg/kg) showed an elevation of superoxide dismutase (0.326 units/min/mg protein), catalase (185.03 μmole of H₂O₂ consumed/min/mg protein), glutothione peroxidase (19.26 mg GSH consumed/min/mg protein) and reduced glutathione (16.2 μmole of GSH/mg protein). Moreover, the biochemical parameters in serum like alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and lipid profiles were also improved in treated groups compared to the control. The oral administration of different doses of rhizome acetone extract significantly protected the hepatic cells from damage. The hematological and biochemical parameters were also normal in extract treated rats compared to the control and standard (silymarin) groups. The HPLC analysis revealed the presence of some important phenolic compounds which could be responsible for the hepatoprotective activity. This study proved that R. imbricata could be taken as a good natural source of the hepatoprotective agent.