Rhnull phenotype in an Indian patient due to a novel c.1138 + 2 t > a mutation in the RHAG gene.

BACKGROUND: The Rh system is an extremely important RBC antigen system with over 50 antigens, 5 of which (D, C, E, c and e) are considered most clinically significant. The rare Rhnull phenotype can result from mutations in the RHD and RHCE genes or the RHAG gene that affects their expression. This is a case report of the second type. CASE REPORT: This case reports a multiparous lady who had to be evaluated for a panreactive antibody. The discrepancy was first identified at the centre she reported to. A thorough immunohematological workup was performed at a second reference laboratory. Suspecting Rhnull phenotype, a third referral (molecular typing) was requested at International Blood Group Reference Laboratory (IBGRL), Bristol. RESULTS: A novel RHAG null allele (c.1138+2t>a), causing a Rhnull phenotype was identified. The antibody was most likely an anti-Rh 29 antibody. CONCLUSION: The novel c.1138+2 t > a mutation in the RHAG gene causing the Rhnull phenotype and development of a pan reacting antibody(ies) made the patient's pregnancy challenging. Confirmation of the diagnosis, an important step in her management, required use of both serological immunohematology and molecular techniques.

Indirect Response of the Temperature, Humidity, and Rainfall on the Spread of COVID-19 over the Indian Monsoon Region.

This article examines the role of the meteorological variable in the spread of the ongoing pandemic coronavirus disease 2019 (COVID-19) across India. COVID-19 has created an unprecedented situation for public health and brought the world to a standstill. COVID-19 had caused more than 1,523,242 deaths out of 66,183,029 confirmed cases worldwide till the first week of December 2020. We have examined the surface temperature, relative humidity, and rainfall over five cities: Delhi, Mumbai, Kolkata, Bengaluru, and Chennai, which were severely affected by COVID-19. It is found that the prevailing southwest (SW) monsoon during the pandemic has acted as a natural sanitizer in limiting the spread of the virus. The mean rainfall is ~ 20-40 mm over the selected cities, resulting in an average decrease in COVID cases by ~ 18-26% for the next 3 days after the rainfall. The day-to-day variations of the meteorological parameters and COVID-19 cases clearly demonstrate that both surface temperature and relative humidity play a vital role in the indirect transport of the virus. Our analysis reveals that most COVID-19 cases fall within the surface temperature range from 24 to 30 °C and relative humidity range from 50% to 80%. At a given temperature, COVID-19 cases show a large dependency on the relative humidity; therefore, the coastal environments were more prone to infections. Wavelet transforms coherence analysis of the daily COVID-19 cases with temperature and relative humidity reveals a significant coherence within 8 days.

Identification of SNPs in crucial starch biosynthesis genes in rice.

Rice is the foremost crop catering to the major calorific requirement of the human population but has the disadvantage of having high glycaemic index (GI). The fine quality rice varieties, BPT and RNR have been recently identified as having low GI in nature and are grown mostly in southern parts of India. Starch (80%) is the major component of rice endosperm attributing to GI. The study aimed to unravel the molecular basis of low GI through targeted pooled amplicon sequencing of major starch biosynthetic genes. A total of 13 candidate genes involved in starch synthesis were amplified and pooled in equimolar proportion for sequencing. Single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) were detected in both coding and noncoding regions. Among the genes that are under study, the highest number of variations were identified in starch synthase I (SSI) followed by starch synthase IIIA (SSIIIA) genes. Nonsynonymous SNPs with high probability of effecting gene function were validated by Sanger sequencing and molecular docking. Identified causative SNPs were mapped on 3000 rice genome database and their allele frequencies were obtained. The outcome of this study has a potential to be applied in breeding programmes to obtain low GI rice varieties with added beneficial traits.

Heavy metal contamination in river water and sediments of the Swarnamukhi River Basin, India: risk assessment and environmental implications.

The concentration of heavy metals was analyzed each of 20 river water, suspended sediments and bed sediments along the stretch of Swarnamukhi River Basin. River water is not contaminated with heavy metals except Fe and Mn. Contamination factor in sediments shows considerable to very high degree contamination with Cr, Cu, Pb and Zn. The sources of these metals could be residential wastes, sewer outfall, fertilizers, pesticides (M-45 + carbondine) and traffic activities apart from natural weathering of granitic rocks present in the basin area. Principal component analyses indicate the interaction between metals in different media. The comparison of metals (Cu, Pb and Zn) in bed sediments of Swarnamukhi River with the Indian and world averages indicates that the values obtained in the basin are above the Indian averages and far below to the world averages. Average shale values and sediment quality guidelines point toward the enrichment and contamination of Cu, Cr, Pb and Zn to several fold leading to eco-toxicological risks in basin.

Retrievability of calcium hydroxide intracanal medicament with three calcium chelators, ethylenediaminetetraacetic acid, citric acid, and chitosan from root canals: An in vitro cone beam computed tomography volumetric analysis.

AIM: This study compared the amount of aqueous-based and oil-based calcium hydroxide remaining in the canal, after removal with two different chelators 17% EDTA, 20% Citric acid and 0.2% Chitosan in combination with ultrasonic agitation. METHODS AND MATERIAL: Cleaning and shaping of root canals of 28 mandibular premolar was done and canals were filled either with Metapex or Ca(OH)2 mixed with distilled water. Volumetric analysis was performed utilizing cone beam-computed tomography (CBCT) after seven days of incubation. Ca(OH)2 was removed using either 17% EDTA, 20% Citric acid or 0.2% Chitosan in combination with ultrasonic agitation. STATISTICAL ANALYSIS USED: Volumetric analysis was repeated and percentage difference was calculated and statistically analyzed using Kruskal-Wallis and Mann-Whitney U test. RESULTS: All the three chelators failed to remove aqueous-based as well as oil-based Ca(OH)2 completely from the root canal. Aqueous-based Ca(OH)2 was easier to be removed than oil-based Ca(OH)2. 0.2% Chitosan in combination with ultrasonics performed better than 17% EDTA and 20% citric acid in removal of Ca(OH)2. CONCLUSION: Combination of 0.2% Chitosan and ultrasonic agitation results in lower amount of Ca(OH)2 remnants than 17% EDTA, 20% Citric acid irrespective of type of vehicle present in the mix.

Streptomyces hyderabadensis sp. nov., an actinomycete isolated from soil.

A novel actinomycete, designated strain OU-40(T), was isolated from farm soil collected from the Hyderabad region of Andhra Pradesh, southern India. The strain was found to have morphological and chemotaxonomic characteristics typical of species of the genus Streptomyces. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain OU-40(T) belonged to the genus Streptomyces, and was related most closely to Streptomyces pactum NBRC 13433(T) (99.0 % sequence similarity), Streptomyces olivaceus NBRC 12805(T) (99.0 %) and Streptomyces parvulus NBRC 13193(T) (98.8 %). Strain OU-40(T) could be distinguished from the type strains of its closest phylogenetic relatives based on levels of DNA-DNA relatedness and comparison of morphological and phenotypic data. It is therefore concluded that strain OU-40(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces hyderabadensis sp. nov. is proposed. The type strain is OU-40(T) (=CCTCC AA 209024(T) =PCM 2692(T)).

Streptomyces osmaniensis sp. nov., isolated from soil.

A novel actinomycete, designated strain OU-63T, was isolated from garden soil collected on the campus of Osmania University in Hyderabad, southern India. The strain was found to have morphological and chemotaxonomic characteristics typical of the genus Streptomyces. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belonged to the genus Streptomyces, and was related most closely to Streptomyces chartreusis NBRC 12753T (98.6% similarity). However, the mean level of DNA-DNA relatedness between the two strains was only 25+/-1.73%. Based on DNA-DNA relatedness, morphological and phenotypic data, strain OU-63T could be distinguished from the type strains of phylogenetically related species. It is therefore considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces osmaniensis sp. nov. is proposed. The type strain is OU-63T (=CCTCC AA209025T =PCM 2690T).

Free energy component analysis for drug design: a case study of HIV-1 protease-inhibitor binding.

A theoretically rigorous and computationally tractable methodology for the prediction of the free energies of binding of protein-ligand complexes is presented. The method formulated involves developing molecular dynamics trajectories of the enzyme, the inhibitor, and the complex, followed by a free energy component analysis that conveys information on the physicochemical forces driving the protein-ligand complex formation and enables an elucidation of drug design principles for a given receptor from a thermodynamic perspective. The complexes of HIV-1 protease with two peptidomimetic inhibitors were taken as illustrative cases. Four-nanosecond-level all-atom molecular dynamics simulations using explicit solvent without any restraints were carried out on the protease-inhibitor complexes and the free proteases, and the trajectories were analyzed via a thermodynamic cycle to calculate the binding free energies. The computed free energies were seen to be in good accord with the reported data. It was noted that the net van der Waals and hydrophobic contributions were favorable to binding while the net electrostatics, entropies, and adaptation expense were unfavorable in these protease-inhibitor complexes. The hydrogen bond between the CH2OH group of the inhibitor at the scissile position and the catalytic aspartate was found to be favorable to binding. Various implicit solvent models were also considered and their shortcomings discussed. In addition, some plausible modifications to the inhibitor residues were attempted, which led to better binding affinities. The generality of the method and the transferability of the protocol with essentially no changes to any other protein-ligand system are emphasized.

Chronic toxicity of 1,3,5-trinitrobenzene in Fischer 344 rats.

The chronic toxicity of 1,3,5-trinitrobenzene (TNB) in male and female Fischer 344 (F344) rats was evaluated by feeding a diet containing 0, 5, 60, and 300 ppm of TNB for 2 years. The calculated average TNB intake over 2 years for males and females was 0.22, 2.64, 13.44 and 0.23, 2.68, 13.31 mg/kg body weight (BW)/day respectively. Terminal body weights were decreased and water intake was increased in both sexes (300 ppm), whereas food consumption was decreased in males (60 and 300 ppm groups) only. The relative spleen weights were significantly decreased in both sexes (300 ppm), whereas the relative brain weights were increased in females only (300 ppm). Hematological effects were not observed in animals killed at the 2-year time point, except significant decrease in the mean corpuscular hemoglobin (MCH) in males (300 ppm) and in females (60 and 300 ppm). Methemoglobin levels were increased in both sexes in the high dose group. Histopathological examination showed treatment-related changes in the kidney (hyaline droplets; 60 and 300 ppm) and the spleen (erythroid cell hyperplasia and pigment deposition; 300 ppm) of both sexes. Cytoplasmic hyaline droplets in the kidneys were characterized by immunohistochemistry as alpha-2mu-globulin. We propose a chronic, oral no-observable-adverse-effect level (NOAEL) of 2.68 mg/kg BW/day for TNB in the rat, based on the hematological and renal changes.

Assessment of environmental hazards of 1,3,5-trinitrobenzene.

The remedial investigation/feasibility studies conducted at certain Army installations showed a need to clean up contaminated sites, where high levels of ammunition chemicals such as 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 1,3-dinitrobenzene (DNB), and their degradation products/metabolites were detected in surface soil and groundwater. TNB is a photodegradation product of TNT; it is not easily degraded, and persists in the environment. The toxicity data on TNB are scanty. Hence the U.S. Environmental Protection Agency in 1988 (U.S. EPA, 1997) developed a reference dose (RfD) for TNB (0.00005 mg/kg/d for chronic toxicity) based on the toxicity of DNB, which is structurally similar to TNB. Since then we have completed acute, subacute, subchronic, chronic, reproductive, and developmental toxicity studies and toxicokinetics studies. We have reviewed the mammalian toxicity data for TNB and have determined the no observed adverse effect levels (NOAEL) and low observed adverse effect levels (LOAEL) for subchronic, chronic, reproductive, and developmental toxicity. Based on the newly determined NOAEL and LOAEL values, we have now developed a new RfD for TNB (0.03 mg/kg/d), based on the chronic toxic effects on hematology and histopathological changes in testes and kidney.

Fourteen-day toxicity study of 1,3,5-trinitrobenzene in Fischer 344 rats.

Toxic effects of 1,3,5-trinitrobenzene (TNB) in male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 50, 200, 400, 800 and 1200 mg kg-1 diet) for 14 days. Food intake by female rats in 400, 800 and 1200 mg TNB diet groups was reduced and resulted in a significant decrease in absolute body weights (BW). Food and water consumption by male rats in high-dose groups (800 and 1200 mg TNB kg-1 diet) was also reduced and resulted in a significant decrease in body weight. The calculated average TNB intake (from 1200 mg TNB kg-1 diet) was 92 mg kg-1 BW day-1 for male rats and 80 mg kg-1 BW day-1 for females. A decrease in testicular weight in males and an increase in spleen weight of both sexes in high-dose groups was noted. In addition, histopathological examinations revealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hematopoiesis), brain (hemorrhage, malacia and gliosis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in Heinz bodies and increased methemoglobin concentration as compared to controls in both sexes. A lowest observed adverse effect level of 4.41 mg TNB kg-1 BW day-1 was established based on the findings of this study.

Carcinogenic evaluation of 2,3-dimethyl-2,3-dinitrobutane via the mouse skin bioassay.

Female SENCAR mice initiated with 2,3-dimethyl-2,3-dinitrobutane (DMDNB) and promoted with 12-O-tetradecanoylphorol-13-acetate (TPA) via the SENCAR mouse skin bioassay did not exhibit a significant increase in skin tumors. The mice received 20 mg kg-1 DMDNB divided into six intragastric doses over 2 weeks and were promoted three times per week for 20 weeks.

Chloroform inhibits the development of diethylnitrosamine-initiated, phenobarbital-promoted gamma-glutamyltranspeptidase and placental form glutathione S-transferase-positive foci in rat liver.

In this study we demonstrate that chloroform, a widely used industrial solvent, a medicinal chemical and a common drinking water contaminant, reduces the number of detectable preneoplastic enzyme-altered foci [gamma-glutamyltranspeptidase-positive (GGT+) and placental form glutathione S-transferase-positive (GST-P+)] in the liver of male Fischer 344 rats. The animals were given a partial hepatectomy and 18 h later received a single oral dose of either 0.5 mmol/kg diethylnitrosamine (DENA) or saline. Two weeks later, groups of 12 animals were started on drinking water containing phenobarbital with varying concentrations (200-1800 mg/l) of chloroform fro 12 weeks. Treated and control animals were killed and the number and the volume of GGT+ and GST-P+ expressing hepatic foci were tabulated. The numbers of foci per unit volume (and per unit area), the percent focal volume and the focal liver were reduced by chloroform in a dose-dependent manner. The mean focal volume was not influenced by chloroform. A plausible explanation for these results could be that chloroform exerts its focal inhibitory effect either by selectively killing the putative initiated cells, by retarding the inherent growth rate of enzyme-altered cells or by reducing the effectiveness of the promoter, phenobarbital. The available evidence suggests that the first hypothesis is the most likely explanation for these observations. These results are consistent with earlier studies showing that chloroform inhibits tumorigenesis in rodents.

Biological markers of exposure to benzene: S-phenylcysteine in albumin.

Results of experiments in our laboratory have shown that benzene is metabolized by animals in part to an intermediate that binds to cysteine groups in hemoglobin to form the adduct S-phenylcysteine (SPC). These results suggested that SPC in hemoglobin may be an effective biological marker for exposure to benzene. However, we could not detect SPC in the globin of humans occupationally exposed to benzene concentrations as high as 28 p.p.m. for 8 h/day, 5 days/week. As another approach, we examined the binding of benzene to cysteine groups of a different blood protein, albumin. To facilitate the process, a new method for the precipitative isolation of albumin from plasma was also developed. The isolated albumin was analyzed for SPC by isotope dilution GC-MS. We used this approach to measure SPC in the albumin of F344/N rats exposed by gavage to 0-10,000 mumol/kg benzene. Amounts of albumin-associated SPC increased as a function of dose, followed by a leveling off in the amount of SPC seen at doses greater than 1000 mumol/kg. Levels of SPC were measured in humans occupationally exposed to average concentrations of 0, 4.4, 8.4 and 23 p.p.m. benzene 8 h/day, 5 days/week. Of nine controls, seven had levels of SPC below the limit of detection (0.1 pmol SPC/mg albumin). SPC increased in the exposed groups linearly, giving a statistically significant slope (P less than 0.001) of 0.044 +/- 0.008 pmol/mg albumin/p.p.m. with an intercept of 0.135 +/- 0.095 pmol/mg albumin. From this study, we conclude that SPC in albumin may prove useful as a biomarker for benzene exposure.

Macromolecular adduction by trichloroacetonitrile in the Fischer 344 rat following oral gavage.

Male Fisher 344 rats were administered 1- or 2-[14C]trichloroacetonitrile (TCAN) by oral gavage. DNA was isolated from the liver, kidneys and stomach and several protein fractions (globin, albumin and globulins) were isolated from blood. TCAN binds to both the DNA and the blood proteins in a dose-related manner. More radiolabel was associated with the DNA when the carbon at C2 position was labeled, than at C1 position. However, the position of the radiolabel did not influence the levels of radioactivity associated with the blood proteins. The stomach exhibited the highest level of DNA binding, followed in order by the liver and kidney. TCAN binding level was higher in DNA isolated from rats killed at 24 h than at 4 h after administration. In contrast, the three blood proteins showed similar binding levels, regardless of the exposure time. Radioactivity associated with DNA was not incorporated into the nitrogen bases (i.e. via de novo synthesis) and a covalent binding index (mumol chemical bound/mol nucleotide phosphate per mmol/kg body wt. of chemical administered) of 30-120 was observed for various tissues. Most of the radioactivity (60-80%) associated with globin could be released and separated from the protein by the treatment with concentrated ammonium hydroxide and precipitation of protein by organic solvent. Three peaks were observed in the HPLC elution profiles of the radioactivity released from the globin. Trichloroacetic acid co-eluted with one of these released products (peak II), however, the chemical identity of the material under the major peak (peak III) and peak I are still uncharacterized.

Induction of nuclear anomalies in the gastrointestinal tract by polycyclic aromatic hydrocarbons.

A selective list of polycyclic aromatic hydrocarbons (PAH) with varied carcinogenic and mutagenic potencies, which are identified as common contaminants at industrial sites and which often contaminate the neighboring ground water, are investigated for their ability to induce nuclear anomalies (NA) in the mouse gastrointestinal (G.I.) tract. These studies examined the hypothesis that a relationship between NA induction and carcinogenic potency of these PAH exists. Among the PAH tested, 7,12-dimethylbenzanthrene (DMBA) was most effective inducer of NA in all G.I. tract tissues examined, with the relative potency in duodenum of DMBA much much greater than benzo[a]pyrene (B[a]P) much greater than benzo[b]fluoranthene (B[b]F). The induction of NA by benzo[a]anthracene (B[a]A), pyrene (PY) and benzo[e]pyrene (B[e]P) was not different from that elicited by vehicle controls. MNU, a known potent inducer of NA in the mouse G.I. tract, yielded a high level of NA in duodenum and proximal colon but was less effective than DMBA in the forestomach. The data suggest that induction of NA by DMBA and B[a]P PAH are in approximate accordance with their relative carcinogenic potency in the gastrointestinal tract. When binary mixtures of some PAH were administered the yield of NA was less than that expected by simple additivity and closer to that expected by averaging the activities of the two PAH comprising the mixture. Thus, this short-term in vivo assay may be useful as a predictor of the genotoxic or carcinogenic strength of individual PAH and/or mixtures of these compounds.

Site-specific modulation of carcinogen-induced gastrointestinal tract nuclear anomalies in B6C3F1 mice by chloroform.

Chloroform (CHCl3) is an established rodent carcinogen and a prevalent contaminant of chlorine-disinfected drinking water. Thus in the United States CHCl3, along with other trihalomethanes, is regulated not to exceed 100 ppb in potable water. Recently, several studies have shown that CHCl3 also has anti-cancer properties as it inhibits tumor growth in mouse liver and in the gastrointestinal tract of the rat. In this paper we show that CHCl3 also inhibits the propensity for three gastrointestinal tract carcinogens, benzo(a)pyrene (BAP), 1,2-dimethylhydrazine (DMH) and methylnitrosourea (MNU), to induce nuclear anomalies in the proximal colon of the B6C3F1 mouse. For example, in mice pre-adapted to 1800 ppm CHCl3 for 30 days prior to the carcinogen administration the level of nuclear anomalies induced in the proximal colon by BAP was reduced by four-fold (0.9 +/- 0.7 v. 3.6 +/- 1.0 anomalies/10 crypts; p less than 0.001) and two-fold for MNU (2.4 +/- 1.0 v. 4.6 +/- 1.6; p less than 0.001) and DMH (0.9 +/- 0.9 v. 1.7 +/- 0.8; p = 0.03). In the duodenum CHCl3 was effective at inhibiting unclear anomalies only for MNU (45.3 +/- 4.6 v. 30.4 +/- 3.5; p = 0.02). The inhibitory effect of CHCl3 does not extend to nuclear anomalies of the forestomach. The anti-cancer properties of CHCl3 are discussed in light of its cancer causing potential and possible application to human risk assessment.

Effect of copper sulfate on hematology, blood chemistry, and hepato-somatic index of an Indian catfish, Heteropneustes fossilis (Bloch), and its recovery.

The present communication deals with the effect of copper sulfate (0.25 ppm) on hematology, blood chemistry, and hepato-somatic indices of an Indian catfish, Heteropneustes fossilis (Bloch) and its recovery from intoxication. Analysis of various parameters was made after 24 hr, and 5, 10, 20, and 30 days of CuSO4 exposure. Subsequently, the fish were allowed recovery periods of 24 hr and 5 days in normal water. Changes in various parameters from normal values during exposure and recovery were recorded and probable reasons for alterations have been discussed in detail.

DNA methylation and hepatocarcinogenesis in rats fed a choline-devoid diet.

Groups of male Fischer-344 rats were fed either a choline-supplemented or a choline-devoid (CD) diet, for up to 14 months. In rats fed the CD diet, hepatic lesions developed and progressed through two distinct stages, the first characterized by severe steatosis and an increase in cell turnover and the second by gradual clearance of the deposited fat, fibrosis and parenchymal nodularity. Large hepatocellular carcinomas were found in rats killed at 14 months. DNA was purified from the livers of all groups of rats and from the tumors, and its level of methylation was analyzed using the restriction endonucleases HpaII and MspI. DNA undermethylation was detected only in the livers of rats fed the CD diet for 14 months, whether bearing tumors or not, and in three of four hepatocellular carcinomas. Undermethylation of liver total DNA is therefore a late effect of dietary choline deficiency in the rat.