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Astroviruses (AstV) and adenoviruses (AdV) are associated with diarrhoea in young animals. However, the epidemiology and genetic diversity of AstVs and AdVs in animals is not well studied. Hence, the present study was conducted to detect and characterize AstVs and AdVs in calves, piglets and puppies from Western Maharashtra, India. Out of the processed porcine (48), canine (80), and bovine (65) faecal samples, the porcine AstV (PAstV), bovine AstV (BAstV), canine AstV (CAstV), and porcine AdV (PAdV) were detected in 12.5%, 7.69%, 3.75% and 4.1% of samples, respectively. In the RNA-dependent RNA polymerase region-based phylogenetic analysis, the detected BAstV strains grouped with MAstV-28, MAstV-33, and MAstV-35, CAstV strains belonged to MAstV-5; PAstV strains belonged to MAstV-24, MAstV-26, and MAstV-31. However, in hexon gene-based phylogeny, both the detected PAdV were of genotype 3, exhibiting 91.9-92.5% nucleotide identity with Ivoirian and Chinese strains. The study reports first-time BAstVs from calves and PAdV-3 from piglets in India. The study revealed diversity in the circulation of AstVs in tested animals and AdVs in pigs, and suggested that they alone might be associated with other diarrhoea or in combination with other enteric pathogens, thus highlighting the necessity of extensive epidemiological investigations to develop diagnostic tools and control measures.

The main route of the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are through respiratory pathways and close contact of human-to-human. While information about other modes of transmission is comparatively less, some published literature supporting the likelihood of a fecal-oral mode of transmission has been accumulating. The diagnosis of SARS-COV-2 infected cases is based on the real-time reverse transcription-PCR (RT-PCR). The fecal excretion of SARS-COV-2 has been reported frequently, however, the role of fecal viral load with the severity of disease is not yet clear. Our study focused on the investigation of SARS-CoV-2 shedding in the fecal samples of patients with coronavirus disease 2019 (COVID-19). A total of 280 RT-PCR-positive patients were enrolled, among them 15.4% had gastrointestinal (GI) symptoms. It was shown that 62% of the patients were positive for SARS-CoV-2 RNA in fecal specimens. This positivity was not related to the presence of GI symptoms and the severity of disease. The next generation sequencing [NGS] of SARS-CoV-2 from fecal samples of patients was performed to analyze mutational variations. Findings from this study not only emphasized the potential presence of SARS-CoV-2 in feces, but also its continuing mutational changes and its possible role in fecal-oral transmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lavania, Joshi, Ranshing, Potdar, Shinde, Chavan, Jadhav, Sarkale, Mohandas, Sawant, Tikute, Padbidri, Patwardhan and Kate.)

Four gastroenteritis viruses were responsible for 54% of the acute gastroenteritis (AGE) cases in children hospitalized between May 2017 and December 2019 in Pune city of Maharashtra state, Western India. The majority (79%) of the children were <2 years of age. The prevalence of Rotavirus A (RVA) was 30.5% followed by 14.3% for norovirus, 8.4% for adenovirus, and 5.5% for astrovirus. The severity of the disease was highest in patients with coinfections compared with the patients with a single infection or negative for all (p = 0.024). Genotyping analysis showed that the majority of the RVA-positive samples (66%) could be typed as G3P[8], 63.6% of the norovirus as GII.4 Sydney [P16], 44% of the adenovirus as type 41%, and 56.2% of the astrovirus as astrovirus type 1. The almost equivalent prevalence of rotavirus and nonrotaviruses and acute gastroenteritis (AGE) cases without known etiology in around 46% of the cases was noted in the present study. Our data highlight that after the recent inclusion of rotavirus vaccines as a part of the National Immunization schedule in India, conducting extensive AGE surveillance in children should include nonrotaviruses such as norovirus., (© 2021 Wiley Periodicals LLC.)

The β-thalassemias and sickle cell disorders pose a considerable health burden in India. Of the more than 10,000 annual births of children with a severe hemoglobinopathy, only around 10.0% are managed optimally. Thus, genetic counseling and prenatal diagnosis (PND) is a valid option for a large and diverse country. Our center was one of the first to initiate PND and we present our experience over 30 years to evaluate the impact of awareness in changing the trends of PND of hemoglobinopathies. Both second and first-trimester diagnoses were undertaken by fetoscopy/cordocentesis and globin biosynthesis/high-performance liquid chromatography (HPLC) analysis of fetal blood and chorionic villus sampling (CVS) and DNA analysis. Over 30 years, 3478 couples (first trimester: 2475; second trimester: 1003) from all over India were offered PND. The number of couples coming in the first trimester increased significantly over each decade and couples coming prospectively increased from 2.5 to 18.4%. A cost-effective stepwise approach was used for molecular analysis. Eight hundred and one fetuses (23.0%) were affected and all except three couples opted for termination of these pregnancies. Genetic counseling and PND is the only way to reduce the burden of disease. With awareness, there was a shift from second trimester to first trimester PND over each decade, with an increasing number of couples coming during the first pregnancy. There are only 15 to 20 centers in India offering PND. We have compared our study with other reports on PND from different regions in India.

Group A rotaviruses (RVA) are a major cause of diarrhea in neonatal calves and children. The present study examined G/P combinations and genetic characteristics of RVAs in diarrheic bovine calves in Western India. RVAs were detected in 27 samples (17.64%) with a predominance of G10P[11] (51.85%), followed by previously unreported genomic constellations, G6P[14] (14.81%), and, G6P[4] (7.40%) and G10P[33] (3.70%). Sequencing and phylogenetic analysis revealed circulation of G10 (Lineage-5), G6 (Lineage-2), P[11] (Lineage-3), P[14] (proposed Lineage-8) and P[4] (Lineage-3) genotypes. The predominant G10P[11] strains were typical bovine strains and exhibited genotypic homogeneity. The rare, G10P[33] strain, had VP7 and VP4 genes of bovine origin but, a resemblance of the VP6 gene with simian strain indicated possible reassortment between bovine and simian (SA11-like) strains. The VP6 and VP7 genes of two rare strains, G6P[14] and G6P[4], were identical to those of bovine stains, but the VP4 was closely related to those of the human-bovine like and human strains, respectively. Additionally, in the VP4 gene phylogenetic tree, Indian P[14] strains constituted a closely related genetic cluster distinct from the other P[14] strains. Hence Lineage-8 was proposed for them. These findings indicated that bovines could serve as a source for anthropozoonotic transmission of G6P[14] strains while zooanthroponotic transmission followed by reassortment with human strain gave rise to G6P[4] strains. The observations of a present study reinforce the potential of rotaviruses to cross the host-species barrier and undergo reassortant to increase genetic diversity which, necessitates their continuous surveillance for development and optimization of prevention strategies against zoonotic RVAs., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Porcine enterovirus G (EV-G) and teschovirus (PTV) generally cause asymptomatic infections. Although both viruses have been reported from various countries, they are rarely detected from India. To detect these viruses in Western India, fecal samples (n = 26) of diarrheic piglets aged below three months from private pig farms near Pune (Maharashtra) were collected. The samples were screened by reverse transcription-polymerase chain reaction using conserved enterovirus specific primers from 5' untranslated region. For genetic characterization of detected EV-G strain, nearly complete genome, and for PTV, partial VP1 gene were sequenced. EV-G strain showed the highest identity in a VP1 gene at nucleotide (78.61%) and amino acid (88.65%) level with EV-G15, prototype strain. However, its complete genome was homologous with the nucleotide (78.38% identity) and amino acid (91.24% identity) level to Ishi-Ka2 strain (LC316832), unassigned EV-G genotype detected from Japan. The nearly complete genome of EV-G15 consisted of 7398 nucleotides excluding the poly(A) tail and has an open reading frame that encodes a 2170 amino acid polyprotein. Genetic analysis of the partial VP1 gene of teschovirus identified porcine teschovirus 4 (PTV-4) and putative PTV-17 genotype. To the best of our knowledge, this is the first report on nearly full genome characterization of EV-G15, and detection of PTV-4 and putative PTV-17 genotypes from India. Further, detection and characterization of porcine enteroviruses are needed for a comprehensive understanding of their genetic diversity and their association with symptomatic infections from other geographical regions of India., (© FEMS 2020.)

Introduction: The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15 years., Materials and Methods: Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing., Results: The study includes 31 075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14 423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA 2 levels. We identified three δ gene mutations (HbA 2 Pellendri, HbA 2 St.George, HbA 2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA 2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ) 0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified., Conclusion: The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India., (© 2018 John Wiley & Sons Ltd.)

Genetic structure of the Indian population is influenced by waves of several immigrants from West Eurasia. Therefore, genetic information of various ethnic groups is valuable to understand their origins, the pattern of migration as well as the genetic relationship between them. No genetic data is available on Pathare Prabhu, which is a small indigenous Hindu community from Mumbai, Maharashtra State, India. The aim of this study was to screen the Pathare Prabhus for hemoglobinopathies, which is a major public health problem in India. Two hundred and fifty-seven unrelated Pathare Prabhus subjects were screened for various hemoglobinopathies. Complete blood counts (CBC) were done on an automated hematology counter. High performance liquid chromatography (HPLC) was used to identify β-thalassemia (β-thal) carriers. Molecular characterization of the β gene defects was done by reverse dot-blot hybridization, amplification refractory mutation system (ARMS) and DNA sequencing. Deletional α-thalassemia (α-thal) was detected by multiplex polymerase chain reaction (PCR). Hb A 2 -Saurashtra (HBD: c.301C>T) was identified by DNA sequencing; its modeling was also done. The prevalence of β-thal was 3.89%, while deletional α-thal was 5.4%. The initiation codon (ATG>ACG) (HBB: c.2T>C) was seen in eight individuals (80.0%), Hb D-Punjab (HBB: c.364G>C) and Hb A 2 -Saurashtra, was found in two and one individual, respectively. A community-specific β-thal mutation was found in Pathare Prabhus in significant proportions. This information is useful in developing an algorithm for a prenatal diagnosis (PND) program.

Introduction: In recent years, the Chandipura virus (CHPV) has emerged as an encephalitic pathogen and found associated with a number of outbreaks in different parts of India. Children under 15 years of age are most susceptible to natural infection. CHPV is emerging as a significant encephalitis, causing virus in the Indian subcontinent. Severe outbreaks caused by the virus have been reported from several parts of India., Expalanation: In the recent past, the noticeable association of CHPV with pediatric sporadic encephalitis cases as well as a number of outbreaks in Andhra Pradesh (2004, 2005, 2007 and 2008), Gujarat in (2005, 2009-12) and Vidarbha region of Maharashtra (2007, 2009-12) have been documented. Prevalence and seasonal activity of the virus in these regions are established by NIV through outbreak investigations, sero-survey and diagnosis of the referred clinical specimens. Recently CHPV has been isolated from pools of sand flies collected during outbreak investigations in Vidarbha region of Maharashtra. Since its discovery from India and above-mentioned activity of CHPV, it was suspected to be restricted only to India., Conclusion: However, CHPV has also been isolated from human cases during 1971-72 in Nigeria, and hedgehogs ( Atelerix spiculus ) during entomological surveillance in Senegal, Africa (1990-96) and recently referred samples from Bhutan and Nepal and from wild toque macaques ( Macaca sinica ) at Polonnaruwa, Sri Lanka during 1993 suggest its circulation in many tropical countries. Based on the limited study on vector related report, it appears that sandflies may be the principle vector.

Co-inheritance of triplicated α-genes can alter the clinical and hematological phenotypes of β-thalassemias. We evaluated the phenotypic diversity and transfusion requirements in β-thalassemia heterozygotes, homozygotes, and normal individuals with associated α-gene triplication. Clinical and hematological evaluation was done and the β-thalassemia mutations characterized by a covalent reverse dot blot hybridization/amplification refractory mutation system. Alpha-globin gene triplication was assessed by multiplex PCR. During the last 2.5 years, 181 β-thalassemia patients and β-thalassemia carriers with an unusual clinical presentation were referred to us for screening for the presence of associated α-globin gene triplication. Twenty-nine of them had associated α-gene triplication (3 β-thalassemia homozygotes or compound heterozygotes and 26 β-thalassemia heterozygotes). One β-thalassemia compound heterozygote [IVS 1-5 (G → C) + CD 41/42 (-CTTT)] was anemic at birth and required blood transfusions unusually early by 6 weeks of age. The second patient (4.5 years) was also clinically severe and became transfusion dependent in spite of having one mild β-thalassemia mutation [Capsite +1 (A → C)]. The third case (3.5 years) who was homozygous for a mild β-gene mutation [-88 (C → T)] with α gene triplication was untransfused. The 26 β-thalassemia heterozygotes with associated triplicated α-genes presented variably, with a β-thalassemia intermedia-like presentation. While screening the family members of all these cases, we found another 10 β-thalassemia heterozygotes and 9 normal individuals with α-globin gene triplication; however, all of them were asymptomatic. Beta-thalassemia carriers, homozygotes, and compound heterozygotes with an unusual presentation should be screened for the possible presence of associated α-globin gene triplication which could influence the clinical and hematological presentation.

Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8(+) cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cortical spreading depression (CSD) occurs during various forms of brain injury such as stroke, subarachnoid hemorrhage, and brain trauma, but it is also thought to be the mechanism of the migraine aura. It is therefore expected to occur over a range of conditions including the awake behaving state. Yet it is unclear how such a massive depolarization could occur under relatively benign conditions. Using a microfluidic device with focal stimulation capability in a mouse brain slice model, we varied extracellular potassium concentration as well as the area exposed to increased extracellular potassium to determine the minimum conditions necessary to elicit CSD. Importantly, we focused on potassium levels that are physiologically plausible (≤145 mM; the intracellular potassium concentration). We found a strong correlation between the threshold concentration and the slice area exposed to increased extracellular potassium: minimum area of exposure was needed with the highest potassium concentration, while larger areas were needed at lower concentrations. We also found that moderate elevations of extracellular potassium were able to elicit CSD in relatively small estimated tissue volumes that might be activated under noninjury conditions. Our results thus show that CSD may be inducible under the conditions that expected in migraine aura as well as those related to brain trauma., (Copyright © 2014 the American Physiological Society.)

Background: Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk., Aim: To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma., Settings and Design: Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study., Materials and Methods: Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation., Results and Conclusions: There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable.

We report the problems in diagnosis faced by two families referred for prenatal diagnosis of thalassemia where cordocentesis and fetal blood analysis by high performance liquid chromatography (HPLC) had to be done. The Hb A levels of the fetal blood measured by HPLC on the VARIANT™ Hemoglobin Testing System were 1.2 and 6.7%, respectively, suggestive of a heterozygous β-thalassemia (β-thal) fetus in the first case and a normal fetus in the second case. In one family, one of the parents had a borderline Hb A(2) level and in the other, one parent had normal RBC indices. However, DNA sequencing, done later, showed that in the first case the fetus was a compound heterozygote for the IVS-I-5 (G>C) and the polyadenylation signal site [poly A (T>C)] mutation, while in the second case, the fetus was homozygous for the poly A mutation. This emphasizes that characterization of β-thal mutations must be done whenever one of the parents has a borderline Hb A(2) level or normal RBC indices, and one should not rely on fetal blood analysis by HPLC for prenatal diagnosis of β-thal so as to avoid misdiagnosis.

The basic objective of this study was to enumerate whether co-administration of interferon-γ (IFN-γ) and/or interleukin-4 (IL-4) gene along with a bivalent Newcastle disease (ND) DNA vaccine construct could modulate the immune response to the DNA vaccine in chickens. pVIVO2 vector carrying Haemaglutinin-Neuraminidase (HN) and Fusion (F) genes of Newcastle disease virus (NDV) at its two cloning sites was used as a DNA vaccine. The same vector was used to clone the chicken IFN-γ and IL-4 genes at the multiple cloning site-1 separately. In vitro expression of IFN-γ and IL-4 gene constructs was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HN and F genes by indirect fluorescent antibody technique (IFAT) in addition to RT-PCR. The chickens were immunized thrice intramuscularly at 21, 36 and 46 days of age with the bivalent DNA vaccine alone, or in combination with IFN-γ/IL-4 or both cytokine gene constructs. The bivalent DNA vaccine led to increase in both NDV specific antibodies as assessed by enzyme linked immunosorbent assay (ELISA) and haemagglutination inhibition test (HI) and cell mediated immune (CMI) response as assessed by lymphocyte transformation test (LTT) employing MTT assay. Co-administration of the DNA vaccine with IL-4 gene resulted in highest IgY levels while IFN-γ produced highest CMI response. The DNA vaccine alone could afford only 10% protection against challenge infection by velogenic NDV. This protection was increased to 40% when IL-4 gene construct was co-administered with the DNA vaccine. Co-injection of IFN-γ as well as the combination of IFN-γ and IL-4 gene constructs with the DNA vaccine yielded 20% protection. Our study suggests that IL-4 may prove to be more appropriate as a genetic adjuvant than IFN-γ for ND DNA vaccine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Exposure to the excitotoxin domoic acid (DOM) has been shown to produce cardiac lesions in both clinical and animal studies. We have previously shown that DOM failed to directly affect cardiomyocyte viability and energetics, but the development of this cardiomyopathy has remained unexplained. The present study compared effects of high-level seizure induction obtained by intraperitoneal (2 mg/kg) or intrahippocampal (100 pmol) bolus administration of DOM on development of cardiac pathologies in a rat model. Assessment of cardiac pressure derivatives and coronary flow rates revealed a significant time-dependent decrease in combined left ventricular (LV) systolic and diastolic function at 1, 3, 7, and 14 days after intraperitoneal administration and at 7 and 14 days after intrahippocampal DOM administration. LV dysfunction was matched by a similar time-dependent decrease in mitochondrial respiratory control, associated with increased proton leakage, and in mitochondrial enzyme activities. Microscopic examination of the LV midplane revealed evidence of progressive multifocal ischemic damage within the subendocardial, septal, and papillary regions. Lesions ranged from reversible early damage (vacuolization) to hypercontracture and inflammatory necrosis progressing to fibrotic scarring. Plasma proinflammatory IL-1α, IL-1β, and TNF-α cytokine levels were also increased from 3 days after seizure induction. The observed cardiomyopathies did not differ between intraperitoneal and intrahippocampal groups, providing strong evidence that cardiac damage after DOM exposure is a consequence of a seizure-evoked autonomic response., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tuberculosis, a List B disease of World Organization for Animal Health, caused by M. avium or M. genavense predominantly affects poultry and pet or captive birds. Clinical manifestations in birds include emaciation, depression and diarrhea along with marked atrophy of breast muscle. Unlike tuberculosis in animals and man, lesions in lungs are rare. Tubercular nodules can be seen in liver, spleen, intestine and bone marrow. Granulomatous lesion without calcification is a prominent feature. The disease is a rarity in organized poultry sector due to improved farm practices, but occurs in zoo aviaries. Molecular techniques like polymerase chain reaction combined with restriction fragment length polymorphism and gene probes aid in rapid identification and characterization of mycobacteria subspecies, and overcome disadvantages of conventional methods which are slow, labour intensive and may at times fail to produce precise results. M. avium subsp. avium with genotype IS901+ and IS1245+ causes infections in animals and human beings too. The bacterium causes sensitivity in cattle to the tuberculin test. The paper discusses in brief the M. avium infection in birds, its importance in a zoonotic perspective, and outlines conventional and novel strategies for its diagnosis, prevention and eradication in domestic/pet birds and humans alike.

Domoic acid and its isomers are produced via algal blooms and are found in high concentrations in shellfish. Here, we assessed the acute seizurogenic potencies of isomers-D, -E and -F and their binding affinities at heterogeneous populations of KA receptors from rat cerebrum. In addition, binding affinities of all six isomers (Iso-A through -F) were assessed at AMPA receptors. Radioligand displacement studies indicated that the seizurogenic potency of Iso-F (E-configuration) closely correlates with its affinities at both KA and AMPA receptors, whereas isomers-D (Z) and -E (E), which exhibit distinctly lower seizurogenic potencies, are quite weak displacers. Previously observed functional potencies for isomers-A, -B and -C (Sawant et al., 2008) correlated with AMPA receptor affinities observed here. Taken together, these findings call into question previous structure-activity rules. Significantly, in our hands, Iso-D was ten-fold less potent than Iso-F. To further explain observed links between structural conformation and functional potency, molecular modeling was employed. Modeling results closely matched the rank order of potency and binding data observed. We further assessed the efficacy of isomers-D, -E and -F as pharmacological preconditioning agents. Acute preconditioning with low-dose Iso-D, -E or -F, before high-dose DA failed to impart behavioural tolerance. This study has shed new light on structural conformations affecting non-NMDA ionotropic glutamate receptor binding and functional potency, and provides a foundation for future work in areas of AMPA and KA receptor modeling., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Previously we have shown that low-dose domoic acid (DA) preconditioning produces tolerance to the behavioral effects of high-dose DA. In this study, we used electrocorticography (ECoG) to monitor subtle CNS changes during and after preconditioning. Young adult male Sprague-Dawley rats were implanted with a left cortical electrode, and acute recordings were obtained during preconditioning by contralateral intrahippocampal administration of either low-dose DA (15 pmoles) or saline, followed by a high-dose DA (100 pmoles) challenge. ECoG data were analyzed by fast Fourier transformation to obtain the percentage of baseline power spectral density (PSD) for delta to gamma frequencies (range: 1.25-100 Hz). Consistent with previous results, behavioral analysis confirmed that low-dose DA preconditioning 60 min before a high-dose DA challenge produced significant reductions in cumulative seizure scores and high level seizure behaviors. ECoG analysis revealed significant reductions in power spectral density across all frequency bands, and high-frequency/high-amplitude spiking in DA preconditioned animals, relative to saline controls. Significant correlations between seizure scores and ECoG power confirmed that behavioral analysis is a reliable marker for seizure analysis. The reduction of power in delta to gamma frequency bands in contralateral cortex does not allow a clear distinction between seizure initiation and seizure propagation, but does provide objective confirmation that pharmacological preconditioning by DA reduces network seizure activity., (Copyright 2009 Wiley-Liss, Inc.)

Background: Haemoglobin E (HbE)-beta-thalassaemia has a very variable clinical presentation. The management of severe cases that are often transfusion dependent is similar to that of cases of beta-thalassaemia major; however, this is often not possible in India because of its high cost and the lack of availability of safe blood at many places. Thus there was a need for a drug such as hydroxyurea, which is known to reduce the transfusion requirements of patients with thalassaemia intermedia. This study was undertaken to evaluate the response of Indian patients with HbE-beta-thalassaemia to hydroxyurea., Materials and Methods: 11 patients with HbE-beta-thalassaemia receiving regular transfusion plus two less frequently transfused patients were selected for hydroxyurea therapy. Clinical and haematological evaluation was performed before and after treatment for 2 years. Molecular studies included beta-globin genotype, beta-globin gene haplotype, Xmn I polymorphism and alpha-genotyping., Results: Four clinically severe patients became transfusion independent (responders) after hydroxyurea therapy, four patients showed a reduction in their transfusion requirements (partial responders), and three patients were non-responders. Responders showed a statistically significant increase in Hb, mean corpuscular volume, mean cell Hb, fetal Hb and F cells with a reduction in their transfusion requirements. A reduction in serum ferritin concentration was also seen in responders and non-responders., Conclusions: Genetic markers such as Xmn I polymorphism and alpha-gene deletions were not always beneficial for the response to hydroxyurea therapy. Thus many more markers and a larger cohort need to be studied to predict the response in these patients.

Accurate estimation of hemoglobin (Hbs) A, Hb A(2), Hb F and abnormal Hb is required for diagnosis of hemoglobinopathies and genetic counseling. High pressure liquid chromatography (HPLC) is the most suitable approach available. But for 70% of the rural Indian population, HPLC analysis facilities are not available and screening would require transportation of samples to laboratories in bigger cities. We thus evaluated the feasibility of using a kit designed for measuring Hb A(1c) using capillary blood for collection and preservation of samples over a period of 15 days at different temperatures for screening for hemoglobinopathies. Capillary blood (5 microl) of 90 individuals was collected in the capillary collection system and run on the Variant Hemoglobin Testing System on days 1, 3, 5, 8, 12 and 15 after incubation at 4, 22, 37, 42 and 50 degrees C. The stability of different Hbs varied at different temperatures. The stability was maintained for 12 to 15 days by most of the samples up to 37 degrees C. Hb E was stable for 3 days up to at 37 degrees C and Hb D and Hb Q for 3 days up to 42 degrees C. This capillary blood collection system would have tremendous potential for sample collection and transportation under adverse climatic conditions for screening of hemoglobinopathies in remote areas in different countries.

Background: The clinical and hematological response to hydroxyurea was evaluated in beta thalassemia patients in western India with variable clinical severity and correlated with genetic factors., Materials and Methods: Seventy-nine patients-[38-beta thalassemia intermedia-(group I), 41-beta thalassemia major-(group II)] on hydroxyurea therapy were followed-up for 20-24months., Results: Among the frequently transfused patients in group I, 58% became transfusion independent and 16% showed a 50% reduction in transfusions after therapy which correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. Forty-one percent of patients in group I had associated alpha-thalassemia and 72.7% were XmnI (+/+). beta thalassemia chromosomes among the responders of group I (41%) were linked to haplotype (- + + - + + - - +) as against haplotype (+ - - - - - - - +) being more common among the non-responders. Response was not linked to the beta thalassemia mutations. Thirty-two percent of group II patients showed a 50% reduction in their transfusion requirements after therapy which also correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. A significant decrease in serum ferritin was seen in both groups. 19% of patients could not tolerate the drug., Conclusions: In group I, clinical response to hydroxyurea was better in patients with alpha-thalassemia, XmnI (+/+) and a higher mean fold increase in gamma mRNA expression. In group II, only one-third of patients showed a partial response.

To date, nothing is known of the pharmacological properties of isomers of domoic acid (DA) in vivo in mammals. Here we assessed the acute seizurogenic and toxic properties of DA, isodomoic acids A, B and C (Iso-A, -B, -C), and the therapeutic potential of these compounds as pharmacological preconditioning agents. DA, Iso-A, Iso-B, and Iso-C all produced significant dose-dependent increases in seizure activity following intrahippocampal administration; doses producing half maximal cumulative seizure scores (ED50) were 137 pmol, 171 pmol, 13,000 pmol, and 3150 pmol, respectively. Pharmacological preconditioning with low-dose DA or Iso-A, 60 min before a high test dose of DA produced a significant reduction in seizure scores. In contrast, Iso-B and Iso-C each failed to induce any detectable tolerance to high-dose DA. Radioligand binding indicated a significant correlation between seizurogenic potency and kainate receptor affinity with KIs of 2.4 nM, 4.4 nM, 4990 nM and 170 nM for DA, Iso-A, Iso-B and Iso-C, respectively. Our in vivo results indicate that DA and Iso-A are functionally equipotent in acute seizure induction by direct intrahippocampal administration, while Iso-B and Iso-C are distinctly less potent.

Our purpose was to develop and evaluate isolation and enrichment of fetal erythroblasts and a nested polymerase chain reaction (PCR) approach using fetal erythroblasts for detecting the beta-globin gene mutations for a noninvasive prenatal diagnosis of hemoglobinopathies. Maternal blood at different periods of gestation was layered on a Percoll density gradient for enrichment of fetal nucleated RBCs (NRBCs). A combination of 3 monoclonal antibodies (CD45-peridinin chlorophyll protein, glycophorin A-phycoerythrin, and anti-hemoglobin F-fluorescein isothiocyanate) was used for flow cytometric sorting of fetal NRBCs from enriched cells. Different nested PCR-based approaches were used for identification of fetal mutations. Owing to heterogeneity of beta-thalassemia mutations in the population of India, we had to screen for 12 mutations and were able to give an accurate diagnosis in 84 (84.0%) of 100 cases when compared with chorionic villus sampling or cordocentesis and DNA analysis.This nested PCR approach enabled amplification of small quantities of DNA from fetal erythroblasts, providing a cost-effective method for noninvasive diagnosis.

Several natural isomers of the seizurogenic neurotoxin domoic acid (DA) have been found to occur at up to mg/kg levels in shellfish. The aim of the current study was to assess the neurotoxic potency of isodomoic acids A and C (Iso-A and Iso-C), recently isolated from commercial shellfish. Hippocampal slices were obtained from young adult rats and maintained in a tissue recording chamber. Synaptically evoked population spikes were recorded in region CA1 before and after exposure to DA or its isomers. Both Iso-A and Iso-C produced transient neuronal hyperexcitability followed by a dose-dependent suppression of population spikes, but were, respectively, 4- and 20-fold less potent than DA (spike area: EC50 DA=237 nM; Iso-A=939 nM; Iso-C=4.6 microM). In the hippocampus, DA preconditioning induces tolerance to subsequent DA toxicity. However, in the present study neither Iso-A nor Iso-C were effective as preconditioning agents. Competitive binding studies using homomeric GluR6 kainate (kainic acid, KA) receptors showed the affinity of Iso-A to be 40-fold lower than DA (Ki DA=3.35 nM; Iso-A=130 nM). Together with earlier work showing Iso-C affinity at GluR6 receptors to be 240-fold lower than DA, our results suggest that neuroexcitatory effects of Iso-A in CA1 may involve both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and KA receptors, while Iso-C likely involves the activation of AMPA receptors alone.

Clinical reports suggest that the elderly are hypersensitive to the neurological effects of domoic acid (DOM). In the present study we assessed DOM-induced seizures in young and aged rats, and seizure attenuation following low-dose DOM pretreatment (i.e. preconditioning). Seizure behaviours following saline or DOM administration (0.5-2mg/kg i.p.) were continuously monitored for 2.5h in naïve and DOM preconditioned rats. Competitive ELISA was used to determine serum and brain DOM concentrations. Dose- and age-dependent increases in seizure activity were evident in response to DOM. Lower doses of DOM in young and aged rats promoted low level seizure behaviours. Animals administered high doses (2mg/kg in young; 1mg/kg in aged) progressed through various stages of stereotypical behaviour (e.g., head tics, scratching, wet dog shakes) before ultimately exhibiting tonic-clonic convulsions. Serum and brain DOM analysis indicated impaired renal clearance as contributory to increased DOM sensitivity in aged animals, and this was supported by seizure analysis following direct intrahippocampal administration of DOM. Preconditioning young and aged animals with low-dose DOM 45-90 min before high-dose DOM significantly reduced seizure intensity. We conclude that age-related supersensitivity to DOM is related to reduced clearance rather than increased neuronal sensitivity, and that preconditioning mechanisms underlying an inducible tolerance to excitotoxins are robustly expressed in both young and aged CNS.