Chemical denaturants induced folding unfolding pathway of the recombinant zebrafish dihydrofolate reductase

148-156Denaturation of proteins plays a crucial part in cellular activities. In this study, we have investigated the folding unfolding pathways of zebrafish dihydrofolate reductase (zDHFR) in presence of different chemical denaturants which were found to be an influential factor for the refolding yield by UV-visible spectrophotometric analysis. The activity change of zDHFR has been observed in presence of three different denaturants like Acetic Acid (AcOH), Sodium Dodecyl Sulphate (SDS), and Ethanol (C2H5OH). Spectrophotometric analysis reveals that protein unfolded completely at different concentrations and times by these denaturants. The spontaneous refolding experiments of chemically denatured zDHFR were also conducted to verify the spontaneous refolding yield. These investigations have helped us to decipher a picture about the denaturants contributing to achieving the refolding yield. We observed that acetic acid is a stronger denaturant among all, and the spontaneous refolding yields were higher from SDS denaturation. In the light of the above findings, higher spontaneous refolding yields were obtained from the low concentration of denaturants

Neurodevelopmental disorders in children aged 2-9 years: Population-based burden estimates across five regions in India.

BACKGROUND: Neurodevelopmental disorders (NDDs) compromise the development and attainment of full social and economic potential at individual, family, community, and country levels. Paucity of data on NDDs slows down policy and programmatic action in most developing countries despite perceived high burden. METHODS AND FINDINGS: We assessed 3,964 children (with almost equal number of boys and girls distributed in 2-<6 and 6-9 year age categories) identified from five geographically diverse populations in India using cluster sampling technique (probability proportionate to population size). These were from the North-Central, i.e., Palwal (N = 998; all rural, 16.4% non-Hindu, 25.3% from scheduled caste/tribe [SC-ST] [these are considered underserved communities who are eligible for affirmative action]); North, i.e., Kangra (N = 997; 91.6% rural, 3.7% non-Hindu, 25.3% SC-ST); East, i.e., Dhenkanal (N = 981; 89.8% rural, 1.2% non-Hindu, 38.0% SC-ST); South, i.e., Hyderabad (N = 495; all urban, 25.7% non-Hindu, 27.3% SC-ST) and West, i.e., North Goa (N = 493; 68.0% rural, 11.4% non-Hindu, 18.5% SC-ST). All children were assessed for vision impairment (VI), epilepsy (Epi), neuromotor impairments including cerebral palsy (NMI-CP), hearing impairment (HI), speech and language disorders, autism spectrum disorders (ASDs), and intellectual disability (ID). Furthermore, 6-9-year-old children were also assessed for attention deficit hyperactivity disorder (ADHD) and learning disorders (LDs). We standardized sample characteristics as per Census of India 2011 to arrive at district level and all-sites-pooled estimates. Site-specific prevalence of any of seven NDDs in 2-<6 year olds ranged from 2.9% (95% CI 1.6-5.5) to 18.7% (95% CI 14.7-23.6), and for any of nine NDDs in the 6-9-year-old children, from 6.5% (95% CI 4.6-9.1) to 18.5% (95% CI 15.3-22.3). Two or more NDDs were present in 0.4% (95% CI 0.1-1.7) to 4.3% (95% CI 2.2-8.2) in the younger age category and 0.7% (95% CI 0.2-2.0) to 5.3% (95% CI 3.3-8.2) in the older age category. All-site-pooled estimates for NDDs were 9.2% (95% CI 7.5-11.2) and 13.6% (95% CI 11.3-16.2) in children of 2-<6 and 6-9 year age categories, respectively, without significant difference according to gender, rural/urban residence, or religion; almost one-fifth of these children had more than one NDD. The pooled estimates for prevalence increased by up to three percentage points when these were adjusted for national rates of stunting or low birth weight (LBW). HI, ID, speech and language disorders, Epi, and LDs were the common NDDs across sites. Upon risk modelling, noninstitutional delivery, history of perinatal asphyxia, neonatal illness, postnatal neurological/brain infections, stunting, LBW/prematurity, and older age category (6-9 year) were significantly associated with NDDs. The study sample was underrepresentative of stunting and LBW and had a 15.6% refusal. These factors could be contributing to underestimation of the true NDD burden in our population. CONCLUSIONS: The study identifies NDDs in children aged 2-9 years as a significant public health burden for India. HI was higher than and ASD prevalence comparable to the published global literature. Most risk factors of NDDs were modifiable and amenable to public health interventions

Chemical denaturants induced folding unfolding pathway of the recombinant zebrafish dihydrofolate reductase

Denaturation of proteins plays a crucial part in cellular activities. In this study, we have investigated the folding unfolding pathways of zebrafish dihydrofolate reductase (zDHFR) in presence of different chemical denaturants which were found to be an influential factor for the refolding yield by UV-visible spectrophotometric analysis. The activity change of zDHFR has been observed in presence of three different denaturants like Acetic Acid (AcOH), Sodium Dodecyl Sulphate (SDS), and Ethanol (C2H5OH). Spectrophotometric analysis reveals that protein unfolded completely at different concentrations and times by these denaturants. The spontaneous refolding experiments of chemically denatured zDHFR were also conducted to verify the spontaneous refolding yield. These investigations have helped us to decipher a picture about the denaturants contributing to achieving the refolding yield. We observed that acetic acid is a stronger denaturant among all, and the spontaneous refolding yields were higher from SDS denaturation. In the light of the above findings, higher spontaneous refolding yields were obtained from the low concentration of denaturants

Evaluation of serologic screening of blood donors in India reveals a lack of correlation between anti-HBc titer and PCR-amplified HBV DNA

Background: Transfusion associated-HBV (TAHBV) is estimated at approximately 1.5 percent in postsurgical recipients and 50 percent or more in multiple-transfusion recipients in India. Not transfusing blood with high-titer anti-HBc, which reportedly correlates with the presence of HBV DNA, helped reduce TAHBV in Japan. This study tested anti-HBc-reactive donors for PCR-amplified HBV DNA and its correlation with anti-HBc titers. Study Design And Methods: In total, 30,853 donors from Cohort 1 (24,694 in 2001) and Cohort 2 (6159 in 2000) were screened for anti-HBc and anti-HBs. Amplification of HBV DNA was performed on an unselected subset of 147 out of 3304 anti-HBc-only sera from Cohort 1 and 230 out of 6159 from Cohort 2. Correlation of anti-HBc titers in DNA positive (n = 48), DNA negative (n = 40), anti-HBs reactive (n = 44), and HBsAg reactive (n = 45) donors was by Fisher's exact test. Results: In Cohort 1, 2673 (10.82%) donors were reactive for anti-HBc, of whom 1038 (4.20%) were anti-HBc only. HBV DNA was detected in 40 out of 147 (27.21%) and 48 out of 230 (20.87%) donors with anti-HBc only from the two cohorts. Anti-HBc titers detected no significant difference between the first three groups. Conclusion: Cryptic HBV infection was observed in approximately 25 percent of anti-HBc-only donors. No correlation was established between HBc titers and presence of HBV DNA

Hepatitis B virus surface (S) transactivator with DNA-binding properties

Chronic infection with hepatitis B virus (HBV) in humans is strongly linked to the development of hepatocellular carcinoma (HCC). Activation of growth-regulatory genes may play a crucial role in carcinogenesis. Proto-oncogene expression has been shown to be higher in HCC tissue with integrated HBV DNA than in the normal liver. Earlier, we showed that the 3' end of the HBV major surface gene (S) (426-855 nucleotides of the S region) is a transactivator of the X promoter-enhancer regulatory element in co-transfection experiments. This region expresses a truncated carboxy terminal S protein extending from amino acid residues 102 to 226. In this study, the truncated S protein (trc-S) was examined for its enhancing activity on several viral and cellular regulatory elements. The results indicate that trc-S activates rous sarcoma virus long terminal repeat (LTR), human T-lymphotropic virus 2 LTR, human immunodeficiency virus 1 LTR, and the c-jun and c-fos promoters. Electrophoretic mobility shift assays carried out to investigate its DNA-binding properties established that trc-S binds to HBV X promoter and oligonucleotides representing binding sites for the AP1 and TFIID transcription factors. The specificity of this interaction was confirmed by using competition experiments and supershift assays. These experiments suggest that trc-S is a transactivator of several cellular and viral promoters and that this activity is mediated by direct interaction with DNA

Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter

Background and Aims: Genome sequence of hepatitis B virus (HBV) from occult chronic infection is scarce. Fifty-six (9.4%) of 591 patients seronegative for hepatitis B surface antigen (HBsAg) with chronic liver disease were positive for HBV DNA. The complete HBV genome from 9 of these patients (S1-S9) and 5 controls positive for HBsAg (SWT.1-SWT.5) were analyzed. Methods: Overlapping genome fragment amplification, cloning, and sequencing was performed on these cases. Functional analysis of surface promoter was conducted using fusion construct. Results: All patients with occult infection except one (S8) had a low viral titer. Eight patients had infection with genotype A (S1-S5, SWT.1-2, SWT.5) and 6 had infection with genotype D (S6-S9, SWT.3-4). S4 and S5.1 of genotype A had the characteristic nucleotide deletions in core and pre-S1 region seen in genotype D. The major observations in patients with occult HBV infection were as follows: frequent quasispecies variation, deletions in pre-S2/S region affecting the surface promoters (nt 3025-54) and pre-S protein (S3, S5, S6, S8), truncated precore (S6, S8, S7.1) and core (S9) owing to stop signal, alternate start codon for the Polymerase gene (S3, S9), and YMDD mutation (S1, S4, S9) in patients not on antiviral therapy. HBsAg and core proteins could be shown immunohistochemically in 3 of 5 liver biopsy specimens available. The mutant surface promoters (pre-S2 and S) on functional analysis showed alterations in HBsAg expression. Conclusions: These changes in the regulatory region with possible alterations in the ratio of large and small surface proteins along with other mutations in the genome may decrease the circulating HBsAg level synergistically, making the immunodetection in serum negative

Conservation Prioritization in a Tiger Landscape: Is Umbrella Species Enough?

Conservation approaches in tiger landscapes have focused on single species and their habitat. Further, the limited extent of the existing protected area network in India lacks representativeness, habitat connectivity, and integration in the larger landscape. Our objective was to identify sites important for connected tiger habitat and biodiversity potential in the Greater Panna Landscape, central India. Further, we aimed to set targets at the landscape level for conservation and prioritize these sites within each district in the landscape as specific management/conservation zones. We used earth observation data to derive an index of biodiversity potential. Marxan was used to identify sites that met tiger and biodiversity conservation targets with minimum costs. We found that to protect 50% of the tiger habitat with connectivity, 20% of the landscape area must be conserved. To conserve 100% of high biodiversity potential, 50% moderate biodiversity potential, and 25% low biodiversity potential, 55% of the landscape area must be conserved. To represent both tiger habitat and biodiversity, 62% of the total landscape area requires conservation or restoration intervention. The prioritized zones can prove significant for hierarchical decision making, involving multiple stakeholders in the landscape, including other tiger range areas.Forestry, Faculty ofNon UBCReviewedFacultyResearche