Isolation of gene conferring salt tolerance from halophilic bacteria of Lunsu, Himachal Pradesh, India.

BACKGROUND: Halophiles offer an attractive source of genes conferring salt tolerance. Halobacillus trueperi SS1 strain of Lunsu, Himachal Pradesh, India, a strict halophile, was exploited to isolate and clone the genes for salt tolerance. The genomic library of BamH1 digest of H. trueperi SS1 was constructed in pUC19, and recombinants were screened for salt tolerance on an LB medium containing ampicillin (100 µg/ml) and NaCl (0 to 1.5 M). RESULTS: One recombinant clone named as salt-tolerant clone (STC) conferred salt tolerance to host Escherichia coli/DH5α, which showed growth in the LB medium supplemented with ampicillin and 1.2 M NaCl. Restriction digestion and PCR analysis revealed the presence of an insert of approximately 2000 bp in the STC. DNA sequencing of the 2-kb insert on both strands yielded a sequence of 2301 nucleotides. Protein BLAST analysis of 2301-bp sequence of H. trueperi SS1 present in STC showed 97% identity to multidrug transport ATP binding/permease protein of Halobacillus karajensis. The insert contained in STC was subcloned into pGEX4T2 vector, and the recombinant clone STC/pGEX4T2 conferred salt tolerance to the bacterial host E. coli. CONCLUSIONS: The present study led to the isolation of salt tolerance gene encoding a putative multidrug transport ATP binding/permease protein from H. trueperi SS1. The salt tolerance gene can be subcloned for transferring salt tolerance traits into agricultural crop plants for cultivation in saline and coastal lands.

Draft Genome Sequence of Halobacillus trueperi SS1, Isolated from Lunsu, a Saltwater Body in the Northwest Himalayas.

We report here the genome sequence of halophilic Halobacillus trueperi SS1, isolated from the Lunsu saltwater body in India. The bacteria are Gram positive and rod shaped. The genome of H. trueperi SS1 has 4.14 Mbp, with 4,329 coding sequences, 35 RNA genes (29 tRNAs, 2 rRNAs, and 4 noncoding RNAs), and 42.15% G+C content.

Draft Genome Sequence of Hyperthermophilic, Halotolerant Parageobacillus toebii PW12, Isolated from the Tattapani Hot Spring, Northwest Himalayas.

Here, we report the genome sequence of hyperthermophilic and halophilic Parageobacillus toebii PW12, isolated from the Tattapani hot spring in the northwest Himalayas. The genome size of Parageobacillus toebii PW12 is 3,210,377 bp. The G+C content is 42.05%, and 3,382 coding sequences (CDS), 80 tRNAs, 5 noncoding RNAs (ncRNAs), and 4 CRISPR arrays were predicted.

Halophilic Bacteria of Lunsu Produce an Array of Industrially Important Enzymes with Salt Tolerant Activity.

The halophilic bacterial isolates SS1, SS2, SS3, SS5, and SS8 were characterized for production of industrially important enzymes like amylase, protease, lipase, and glutaminase. Halophilic bacterial isolates SS1 and SS3 exhibited salt dependent extracellular amylase and protease activities. Both the halophilic isolates SS1 and SS3 exhibited maximum amylase and protease activities in the presence of 1.5 and 1.0 M NaCl, respectively, with the optimum pH 8 and temperature 40°C. SS2 showed maximum extracellular protease and lipase activities in the presence of 0.75 M NaCl, at optimum pH of 7, and temperature 37°C. The glutaminase activity of SS3 increased with increase in concentration of NaCl up to 2.5 M. The optimum pH and temperature for L-glutaminase activity of SS3 was 8 and 40°C, respectively. The combined hydrolytic activities of these halophilic bacterial isolates can be used for bioconversion of organic materials to useful products.

Secretome analysis of chickpea reveals dynamic extracellular remodeling and identifies a Bet v1-like protein, CaRRP1 that participates in stress response.

Secreted proteins maintain cell structure and biogenesis besides acting in signaling events crucial for cellular homeostasis during stress adaptation. To understand the underlying mechanism of stress-responsive secretion, the dehydration-responsive secretome was developed from suspension-cultured cells of chickpea. Cell viability of the suspension culture remained unaltered until 96 h, which gradually declined at later stages of dehydration. Proteomic analysis led to the identification of 215 differentially regulated proteins, involved in a variety of cellular functions that include metabolism, cell defence, and signal transduction suggesting their concerted role in stress adaptation. One-third of the secreted proteins were devoid of N-terminal secretion signals suggesting a non-classical secretory route. Screening of the secretome identified a leaderless Bet v 1-like protein, designated CaRRP1, the export of which was inhibited by brefeldin A. We investigated the gene structure and genomic organization and demonstrated that CaRRP1 may be involved in stress response. Its expression was positively associated with abiotic and biotic stresses. CaRRP1 could complement the aberrant growth phenotype of yeast mutant, deficient in vesicular transport, indicating a partial overlap of protein secretion and stress response. Our study provides the most comprehensive analysis of dehydration-responsive secretome and the complex metabolic network operating in plant extracellular space.

A diverse group of halophilic bacteria exist in Lunsu, a natural salt water body of Himachal Pradesh, India.

Five halophilic bacterial isolates namely SS1, SS2, SS3, SS5 and SS8 were isolated from soil sediments of Lunsu, a salty water body. All the bacterial isolates showed growth in LB medium containing up to 8.7% NaCl, pH 7-8 and at temperature range of 30-37°C. The bacterial isolates SS1 and SS3 require at least 3.8% NaCl for their growth, indicating their strict halophilic nature. Interestingly, bacterial isolates SS2, SS5 and SS8 but not SS1 and SS3 exhibited growth in medium supplemented with KCl. Accordingly, Na(+) and K(+) ions were detected at 1.39 and 0.0035%, respectively in Lunsu water. All the bacterial isolates were analyzed by random amplification of polymorphic DNA (RAPD) using four different random primers and produced PCR fragments ranging from 0.1 to 5 kb in size. Phylogenetic tree based on RAPD finger prints showed that SS1 and SS3 formed one group, while SS2 and SS5 formed the second group, whereas SS8 was out group. Sequence analysis of 16S rDNA identified SS1 and SS3 as Halobacillus trueperi, SS2 as Shewanella algae, SS5 as Halomonas venusta, and SS8 as Marinomonas sp. were deposited in GenBank with accession numbers of KM260166, KF751761, KF751760, KF751762 and KF751763, respectively. This is the first report on the presence of diverse halophilic bacteria in the foot hills of Himalayas.

Overexpression of CaTLP1, a putative transcription factor in chickpea (Cicer arietinum L.), promotes stress tolerance.

Dehydration is the most crucial environmental constraint on plant growth and development, and agricultural productivity. To understand the underlying mechanism of stress tolerance, and to identify proteins for improving such important trait, we screened the dehydration-responsive proteome of chickpea and identified a tubby-like protein, referred to as CaTLP1. The CaTLP1 was found to predominantly bind to double-stranded DNA but incapable of transcriptional activation. We investigated the gene structure and organization and demonstrated, for the first time, that CaTLP1 may be involved in osmotic stress response in plants. The transcripts are strongly expressed in vegetative tissues but weakly in reproductive tissues. CaTLP1 is upregulated by dehydration and high salinity, and by treatment with abscisic acid (ABA), suggesting that its stress-responsive function might be associated with ABA-dependent network. Overexpression of CaTLP1 in transgenic tobacco plants conferred dehydration, salinity and oxidative stress tolerance along with improved shoot and root architecture. Molecular genetic analysis showed differential expression of CaTLP1 under normal and stress condition, and its preferential expression in the nucleus might be associated with enhanced stress tolerance. Our work suggests important roles of CaTLP1 in stress response as well as in the regulation of plant development.

Resultados de un programa de pesquisa prenatal de la talasemia grave / Results of an antenatal screening programme for beta-thalessemia

Objetivos: Detectar el rasgo beta-talasémico durante el control prenatal de mujeres en la primera etapa del embarazo; reducir el nacimiento de niños homocigotos con beta-talasemia. Métodos: Se incluyeron embarazadas de hasta 18 semanas de gestación con antecedentes de no más de 3 embarazos, que asistían a una clínica de control prenatal. Se realizó una pesquisa mediante la estimación del volumen corpuscular medio, la hemoglobina corpuscular media y la prueba NESTROF. Se controló también a los esposos de las mujeres con pruebas positivas. Si ambos padres eran positivos, se confirmó el diagnóstico por medio de cromatografía líquida de alto rendimiento. Para la detección fetal de las parejas positivas se efectuó biopsia de vellosidades coriónicas o amniocentesis. Se ofreció la interrupción del embarazo a las mujeres con fetos con talasemia grave. Resultados: Se efectuó la pesquisa a 17339 madres en el período comprendido entre octubre de 1999 y marzo de 2010. Las pruebas de cribado fueron positivas para el volumen corpuscular medio, la hemoglobina corpuscular media y la pruebas de NESTROF en el 11.02%, 18.76% y 12.62% de las embarazadas, respectivamente. El 1.98% de las mujeres eran portadoras confirmadas. Un total de 54 parejas requirieron pruebas diagnósticas prenatales fetales. Se detectó talasemia grave en 19 fetos; todos esos embarazos fueron interrumpidos. Se encontró rasgo talasémico en 16 fetos. Conclusión: La pesquisa prenatal de la beta-talasemia es un abordaje rentable para evitar el nacimiento de niños afectados, en especial en países de alta prevalencia.

Characterization of the secretome of chickpea suspension culture reveals pathway abundance and the expected and unexpected secreted proteins.

The secretome of an organism is defined as a set of secreted proteins that encompasses all proteins exported to the extracellular space. To better understand the chickpea secretome, we used callus culture to isolate and identify secreted proteins as a step toward determining their functions. Proteins in the extracellular media of the suspension culture were examined using SDS-PAGE and mass spectrometry (LC-MS/MS). Proteomic analysis led to the identification of 773 proteins, presumably involved in a variety of functions including metabolism, signal transduction, transport, and cell defense, in addition to maintaining redox status of extracellular space. Bioinformatic analysis confirmed 724 proteins, accounting for 94% of the identified proteins, as constituents of the secretome. Analysis of the secretome revealed the presence of several proteins of unknown function and a large number of classical and nonclassical secreted proteins. This represents the first comprehensive secretome of a legume genome, which is yet to be sequenced. Comparative analysis of the chickpea secretome with those of Medicago, Arabidopsis, and rice revealed that the majority of identified proteins are seemingly species-specific. This study demonstrates that characterization of the chickpea secretome in vitro can be used to identify secreted proteins, which has implications for systems biology research.

Comparative proteomics of tuber induction, development and maturation reveal the complexity of tuberization process in potato (Solanum tuberosum L.).

Tuberization in potato ( Solanum tuberosum L.) is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multistep, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. To understand the molecular basis of tuberization in potato, a comparative proteomic approach has been applied to monitor differentially expressed proteins at different development stages using two-dimensional gel electrophoresis (2-DE). The differentially displayed proteomes revealed 219 protein spots that change their intensities more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of 97 differentially regulated proteins that include predicted and novel tuber-specific proteins. Nonhierarchical clustering revealed coexpression patterns of functionally similar proteins. The expression of reactive oxygen species catabolizing enzymes, viz., superoxide dismutase, ascorbate peroxidase and catalase, were induced by more than 2-fold indicating their possible role during the developmental transition from stolons into tubers. We demonstrate that nearly 100 proteins, some presumably associated with tuber cell differentiation, regulate diverse functions like protein biogenesis and storage, bioenergy and metabolism, and cell defense and rescue impinge on the complexity of tuber development in potato.

Enumeration of CD4 and CD8 T lymphocytes in healthy HIV seronegative adults of northwest India: a preliminary study.

CD4 T lymphocyte count is used to measure the progression of HIV infection and to monitor the response to antiretroviral therapy. Information on reference CD4 and CD8 T cell counts in healthy individuals is lacking in northwest India. Samples from 65 HIV-seronegative healthy volunteers (males, 37; females, 28) aged 18 through 59 years were analyzed using FACS (Fluorescent Antibody Cell Sorter) Count TM System. The values of mean and standard deviation of each lymphocyte subpopulation were estimated. The mean +/- SD of absolute numbers of CD4 and CD8 lymphocytes/microl was 743.4 +/- 307.8 and 541.7 +/- 176.4 in males and 790.7 +/- 280.4 and 497.03 +/- 203.6 in females respectively. The range of CD4 counts was 379 to 1800 in males and 321 to 1265 in females. The mean CD4:CD8 ratio was 1.43 +/- 0.56 in males and 1.78 +/- 0.76 in females. The results of this study show a wide variability in CD4 counts in the Indian population. A large multicentric study would define normal ranges of CD4, CD8, and CD4:CD8 ratios among the Indian population.

Squamous cell carcinoma of cornea.

PURPOSE: To report a case of isolated squamous cell carcinoma of cornea. METHODS: It is an observational case report. RESULTS: An 80-year-old man presented with decreased vision and a white gelatinous mass on his left cornea. Ocular examination revealed sparing of the limbus. There was no lymphadenopathy and no evidence of metastasis. The mass was excised completely with superficial keratectomy. Histopathology of the mass revealed it to be an invasive well-differentiated squamous cell carcinoma of cornea. Postoperatively mitomycin C (0.002%) eye drops were prescribed for 4 weeks. There has been no recurrence of the lesion till date. CONCLUSIONS: Isolated squamous cell carcinoma of cornea, though a rare entity, should be kept in mind in patients with any corneal mass.

Spontaneous corneal melting in pregnancy: a case report.

BACKGROUND: To report a case of spontaneous corneal melting in pregnancy. We reviewed the literature on corneal melting and the effect of pregnancy on cornea and collagen containing tissues. CASE PRESENTATION: A 29-year-old woman who underwent radial keratotomy in both eyes followed by trabeculectomy in her left eye developed corneal melting in the same eye, in her seventh month of pregnancy. Despite screening, no infectious or immune mediated condition could be identified. She was managed conservatively with cyanoacrylate glue, bandage contact lens, lubricants and antibiotics. CONCLUSION: It may not always be possible to find the underlying cause of corneal melting but the more common underlying causes should be ruled out by proper investigations. Pregnancy with its host of hormonal changes could potentially have some effect on corneal collagen leading to corneal melting in compromised corneas.

Isolation and analysis of circulating tuberculous antigens in Mycobacterium tuberculosis.

BACKGROUND: Serological techniques like enzyme linked immunosorbent assay (ELISA) and immunoblotting are useful for detection of mycobacterial antigens of diagnostic importance in tuberculosis. AIM: To isolate and identify circulating tuberculous antigens reactive with sputum positive and sputum negative pulmonary tuberculosis (PTB) sera. METHODS: Circulating tuberculous antigen was isolated by ammonium sulphate fractionation from the sera of sputum positive and sputum negative (clinically and radiologically diagnosed) PTB cases. The circulating antigen fractions and individual patients' serum samples were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting was performed using anti M.tb sonicate IgG as a probe to detect antigens. RESULTS: Anti M.tb sonicate IgG was found to be reactive with mycobacterial proteins 170 kDa, 140 kDa, 85 kDa, 55 kDa, 43 kDa, 20 kDa and 16 kDa in the antigen fraction isolated from sputum positive tuberculosis sera by immunoblotting. However only 85 kDa, 55kDa, 43 kDa and 20 kDa antigenic proteins were found to be recognized by anti sonicate IgG in the antigen isolated from sputum negative sera. These observations were further confirmed by analysis of individual S+ and S- PTB serum by immunoblotting. CONCLUSION: Seroreactive studies of circulating tuberculous antigens showed the presence of 170 kDa, 140 kDa, 85 kDa, 55 kDa, 43 kDa, 20 kDa and 16 kDa protein antigens in sputum positive sera, while 85 kDa, 55 kDa, 43 kDa and 20 kDa antigens were found to be present in sputum negative PTB which need further evaluation for their use in serological diagnosis of tuberculosis.

Bilateral simultaneous central retinal artery occlusion following head injury.

PURPOSE: To report a case of bilateral simultaneous central retinal artery occlusion (CRAO) following head injury in a young 29-year-old man. METHODS: A 29-year-old man presented with head injury following road traffic accident. Posterior segment evaluation revealed CRAO in both eyes. RESULTS: The patient was treated for CRAO in the form of immediate ocular massage, paracentesis, intravenous mannitol and transdermal isosorbide dinitrate patch. Despite treatment the vision continued to be no perception of light. Systemic investigations were unremarkable. Color Doppler of carotid arteries showed plaque in left carotid bulb and thrombus in right internal carotid artery. CONCLUSION: Bilateral simultaneous CRAO following head trauma has not been reported earlier. Thorough ocular examination is recommended in all cases of head injury.

IgG subclass antibody response to mycobacterial serine protease at different stages of pulmonary tuberculosis.

BACKGROUND: Tuberculosis (TB) is a chronic bacterial infection caused by M. Tuberculosis. Studies of antibody response in TB have focussed mainly on their usefulness as a diagnostic serological tool, with little attention given to analysis of antibodies at the isotype and subclass level in relation to disease pathogenesis. Hence the present study was done to analyse IgG subclass response at different stages of tuberculosis, in order to understand the immunological events associated with disease development. MATERIAL/METHODS: Sera samples were collected from 104 subjects: 79 tuberculosis patients (fresh, relapse and chronic cases) and 25 healthy normals. IgG subclass antibody response was analysed by indirect plate peroxidase ELISA against previously reported mycobacterial serine protease (ES-31) antigen. RESULTS: Fresh cases of tuberculosis showed increased IgG1 and IgG3 antibodies, while a few cases showed moderately increased IgG2. IgG1 and IgG3 were found to be elevated with increased bacillary load. Relapse and chronic cases showed increased IgG1 and IgG3, while positivity to IgG2 was decreased. Chronic cases showed a moderate increase in IgG4 antibody. Thus IgG1 and IgG3 were predominant in all forms of tuberculosis. CONCLUSIONS: The elevated levels of IgG1 and IgG3 antibodies to mycobacterial serine protease in active tuberculosis observed in this study provide an additional marker for diagnosis of tuberculosis. Furthermore, the higher level of these antibodies with high bacillary load patients and in chronic cases of tuberculosis may provide valuable insight into their possible role in disease progression.

Serodiagnosis of childhood tuberculosis by ELISA.

OBJECTIVE: Diagnosis of childhood tuberculosis remains an enigma despite many recent technological developments. The present study has been taken up with the aim to assess the diagnostic potential of mycobacterium tuberculosis excretory-secretory ES-31 antigen and affinity purified anti ES-31 antibodies in the serodiagnosis of different spectrum of childhood tuberculosis. METHODS: Mycobacterium tuberculosis H37Ra excretory-secretory antigen (ES-31) and affinity purified goat anti ES-31 antibodies were used in stick penicillinase ELISA for IgG antibody detection and stick Sandwich penicillinase ELISA for detection of circulating free and immune complexed antigen in the sera of 230 children. RESULTS: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in both pulmonary and extrapulmonary form of childhood tuberculosis and overall sensitivity of 81.4% with a specificity of 93% was achieved for detection of antitubercular IgG antibodies. Of the five cases of pulmonary tuberculosis showing absence of IgG antibody, 3 showed the presence of CIC-Ag and one was found positive for both free and CIC-Ag. Similarly out of 8 cases of extrapulmonary childhood tuberculosis missed by IgG detection 5 were found to be positive for CIC-Ag and 1 showed the positive reaction for both free and immune complexed antigens. CONCLUSION: IgG antibody to excretory-secretory antigen ES-31 is found to be having good specificity with acceptable sensitivity in detecting different forms of childhood tuberculosis. Further detection of circulating free and/or immunecomplexed antigen can be used as an adjunct tool in the diagnosis of childhood tuberculosis.

Levels of antibody, free antigen and immune-complexed antigen by ELISA in different grades of sputum positive patients of pulmonary tuberculosis.

The ES-31 (31 kDa protein) antigen was isolated from culture filtrate of Mycobacterium tuberculosis H37Ra and was shown to have potential in immunodiagnosis of pulmonary tuberculosis. Serum samples from 38 confirmed sputum positive pulmonary tuberculosis patients were grouped into AFB+, AFB++, AFB+++ based on sputum bacillary load. Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in serum samples by Indirect and Sandwich ELISA using ES-31 antigen and affinity purified anti ES-31 antibody respectively. The analysis of Geometric mean titre (GMT) of all the three groups showed that GMT of tubercular antibody was considerably decreased compared to elevated levels of CIC-Ag (Antibody: 1360 to 816 and CIC-Ag: 534 to 1744) from low bacillary sample to high bacillary samples, whereas there is no significant change in the titre of circulating free antigen. Low levels of detectable antibody is observed possibly due to removal of antibody from circulation by immune complex formation as confirmed by its elevated levels in sputum AFB+++ positive patients.

Serodiagnosis of tuberculosis using two ELISA systems.

Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H(37)Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases.