MMP-9 gene ablation mitigates hyperhomocystenemia-induced cognition and hearing dysfunction

Hyperhomocysteinemia (HHcy) is associated with cognitive decline and hearing loss due to vascular dysfunction. Although we have shown that HHcy-induced increased expression of matrix metalloproteinase-9 (MMP-9) is associated with cochlear pathology in cystathionine-β-synthase heterozygous (CBS^+/?) mice, it is still unclear whether MMP-9 contributes to functional deficit in cognition and hearing. Therefore, we hypothesize that HHcy-induced MMP-9 activation causes vascular, cerebral and cochlear remodeling resulting in diminished cognition and hearing. Wildtype (WT), CBS^+/?, MMP-9^?/? and CBS^+/?/MMP-9^?/? double knock-out (DKO) mice were genotyped and used. Doppler flowmetry of internal carotid artery (ICA) was performed for peak systolic velocity [PSV], pulsatility index [PI] and resistive index [RI]. Cognitive functions were assessed by Novel Object Recognition Test (NORT) and for cochlear function Auditory brainstem response (ABR) was elicited. Peak systolic velocity, pulsatility and resistive indices of ICA were decreased in CBS^+/? mice, indicating reduced perfusion. ABR threshold was increased and maximum ABR amplitude and NORT indices (recognition, discrimination) were decreased in CBS^+/? mice compared to WT and MMP-9^?/?. All these parameters were attenuated in DKO mice suggesting a significant role of MMP-9 in HHcy-induced vascular, neural and cochlear pathophysiology. Regression analysis of PSV with ABR and cognitive parameters revealed significant correlation (0.44–0.58). For the first time, MMP-9 has been correlated directly to functional deficits of brain and cochlea, and found to have a significant role. Our data suggests a dual pathology of HHcy occurring due to a decrease in blood supply (vasculo-neural and vasculo-cochlear) and direct tissue remodeling.     

Induction of sex reversal in Oreochromis mossambicus by diethylstilbestrol

The efficacy of the synthetic estrogen, diethylstilbestrol (DES), for sex-reversal in the Java tilapia, Oreochromis mossambicus was investigated. Fry of 8–10 mm total length were fed diets containing 25, 50 and 100 ppm of DES for 30 days in plastic pools; this was followed by 45–65 days rearing in fertilized cement cisterns where hormone-free diet was given. DES at 50 and 100 ppm induced 100% sex-reversal; DES at 25 ppm resulted in only a slightly larger proportion of females to males. The untreated control group had a higher proportion of males than females. No intersex or sterile individuals were observed among the steroid-treated fish. The present investigation demonstrates that 50 ppm DES administered for 30 days is sufficient to induce a 100% sex-reversal in O. mossambicus.     

Amide H/D exchange in the thermal transition of bovine pancreatic ribonuclease A

The H/D exchange behavior of RNase A at pH 2.5 at a number of temperatures spanning the thermal transition region has been examined by NMR spectroscopy. The amide proton of V116 has a slow rate of H/D exchange even at temperatures above the midpoint of the thermal transition. The H/D exchange behavior of the peptide corresponding to residues 105-124 of RNase A and the peptide corresponding to residues 115-117 is compared with that of RNase A, showing that folding/unfolding cannot be described by a two-state model, and that both short- and long-range interactions are responsible for the slow rate of H/D exchange.     

A Synergistic Framework for Evaluating Business Process Improvements

The evaluation, selection, or justification of business process improvements, or business process reengineering efforts, is similar to many strategic initiatives and their justification methodologies. This similarity arises from the fact that there are multiple factors that need to be considered, many of which have long-term and broad implications for an organization. There are many intangible measures and qualitative concerns when evaluating business process improvements. These improvements necessarily have to link to the strategic objectives of the organization. The proposed methodological framework will involve the synthesis of the analytical network process and data envelopment analysis. These two techniques, when used together, can provide subjective and objective evaluations for managerial decision makers. An illustrative example provides some insights into the application of this methodology. Additional issues and research questions are also identified.     

Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support na?ve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.     

Identification of GR and TrxR systems in Setaria cervi: Purification and characterization of glutathione reductase

The glutathione reductase (GR) and thioredoxin reductase (TrxR) are important enzymes of the redox system that aid parasites to maintain an adequate intracellular redox environment. In the present study, the enzyme activity of GR and TrxR was investigated in Setaria cervi (S. cervi). Significant activity of both enzymes was detected in the somatic extract of adult and microfilariae stages of S. cervi. Both GR and TrxR were separated by partial purification using ammonium sulfate fractionation and DEAE ion exchange chromatography suggesting the presence of both glutathione and thioredoxin systems in S. cervi. The enzyme glutathione reductase (ScGR) was purified to homogeneity using affinity and ion exchange chromatography that resulted in 90 fold purification with a yield of 11.54%. The specific activity of the ScGR was 643 U/mg that migrated as a single band on SDS-PAGE. The subunit molecular mass was determined to be ~ 50 kDa while the optimum pH and temperature were found to be 7.0 and 35 °C respectively. The activation energy (Ea) was calculated from the slope of Arrhenius plot as 16.29 ± 1.40 kcal/mol. The Km and Vmax were determined to be 0.27 ± 0.045 mM; 30.30 ± 1.30 U/ml with NADPH and 0.59 ± 0.060 mM; 4.16 ± 0.095 U/ml with GSSG respectively. DHBA, a specific inhibitor for GR has completely inhibited the enzyme activity at 1 μM concentration. The inhibition of ScGR activity with NAI (IC₅₀ 0.71 mM), NEM (IC₅₀ 0.50 mM) and DEPC (IC₅₀ 0.27 mM) suggested the presence of tyrosine, cysteine and histidine residues at its active site. Further studies on characterization and understanding of these antioxidant enzymes may lead to designing of an effective drug against lymphatic filariasis.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

NOD2 gene mutations associate weakly with ulcerative colitis but not with Crohn's disease in Indian patients with inflammatory bowel disease

Background

Three mutations (two missense and one frameshift) in the NOD2 gene are associated with Crohn's disease (CD) in a proportion of patients with Crohn's disease in North America, Europe and Australia. These three mutations are not found in Indian patients with CD. We undertook new studies to identify polymorphisms in the NOD2 gene in the Indian population and to detect whether any of these were associated with inflammatory bowel disease (IBD) in this population.

Methods

Individual exons of the NOD2 gene were amplified by PCR and subjected to denaturing high performance liquid chromatography (DHPLC) to detect heteroduplex formation. All 12 exons of the NOD2 gene were amplified and Sanger-sequenced to detect polymorphisms in the NOD2 gene. 310 patients with CD, 318 patients with ulcerative colitis (UC) and 442 healthy controls (HC) were recruited for association studies. DNA from these participants was evaluated for the identified eight polymorphisms by Sequenom analysis.

Results

Heteroduplex formation was noted by DHPLC in exons 2 and 4 of the NOD2 gene. Sequencing of the entire NOD2 gene data revealed eight polymorphisms – rs2067085, rs2066842, rs2066843, rs1861759, rs2111235, rs5743266, rs2076753, and rs5743291 – of which the latter four were described for the first time in Indians. None of these polymorphisms was associated with CD. The SNPs rs2066842 and rs2066843 were in significant linkage disequilibrium. Both SNPs showed a significant association with UC (P = 0.03 and 0.04 respectively; odds ratio 1.44 and 1.41 respectively).

Conclusion

Four NOD2 polymorphisms were identified for the first time in the Indian population. Of 8 NOD2 polymorphisms, none were associated with CD but two were weakly associated with UC. NOD2 polymorphisms do not play a major role in CD genesis in India.     

Mutations in exons 3 and 7 resulting in truncated expression of human ATP6V1B1 gene showing structural variations contributing to poor substrate binding-causative reason for distal renal tubular acidosis with sensorineural deafness

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H⁺ secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding β1 subunit of H⁺-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A > G, c.308 C > A, c.310 C > G, c.704 T > C, c.705 G > T, c.709 A > G, c.710 A > G, c.714 G > A, c.716 C > A, c.717delC, c.722 C > G, c.728insG, c.741insT, c.753G > C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H⁺ ions contributing to dRTA with sensorineural deafness.