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1.  Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India 
3 Biotech  2016;6(1):49.
In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg−1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0–14.0 pH and 30–80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.
PMCID: PMC4746201
Lipase; Bacillus licheniformis; Metal ions; Detergents; Inhibitors; Solvents
2.  Antibodies to Antigenic Site A of Influenza H7 Hemagglutinin Provide Protection against H7N9 Challenge 
PLoS ONE  2015;10(1):e0117108.
Identifying major antigenic and protective epitopes of the H7 hemagglutinin (HA) will be important for understanding the antibody response to vaccines developed against the novel influenza H7N9 viruses that emerged in China in 2013. To facilitate antigenic characterization of the H7N9 HA and to develop reagents for evaluation of H7N9 candidate vaccines, we generated a panel of murine monoclonal antibodies (mAbs) to the HA of A/Shanghai/2/2013 using mammalian cell-derived virus-like particles (VLP) containing the H7 HA. Neutralizing antibodies identified an HA epitope corresponding to antigenic site A on the structurally similar influenza H3 hemagglutinin. Importantly, the neutralizing antibodies protect against A/Shanghai/2/2013 challenge. This antigenic site is conserved among many H7 viruses, including strains of both Eurasian and North American lineage, and the isolated neutralizing antibodies are cross-reactive with older H7 vaccine strains. The results indicate that the identified antigenic site is a potentially important protective epitope and suggest the potential benefit of cross-reactive antibody responses to vaccination with H7 candidate vaccines.
PMCID: PMC4309539  PMID: 25629172
3.  Analysis of Equation of States for the Suitability at High Pressure: MgO as an Example 
The Scientific World Journal  2014;2014:289353.
A simple theoretical model is developed to study the high pressure behavior of solids and is applied to evaluate the pressure for MgO in case of large compression along with Shanker, Tait, Vinet, and Birch-Murnaghan equation of states (EOSs). These EOSs are also tested for the basic requirements revealed from the fundamental thermodynamics for solids in the limit of extreme compressions, as given by Stacey. It is found that for the high pressure compression behavior of MgO the present model, Tait, Vinet, and Birch-Murnaghan EOSs give the results compatible with the experimental data. It has also been found that in the regime of ultrahigh pressure the present model and Birch-Murnaghan EOS satisfy the Stacey criterion.
PMCID: PMC3914558  PMID: 24550701
4.  Oligomeric Recombinant H5 HA1 Vaccine Produced in Bacteria Protects Ferrets from Homologous and Heterologous Wild-Type H5N1 Influenza Challenge and Controls Viral Loads Better than Subunit H5N1 Vaccine by Eliciting High-Affinity Antibodies 
Journal of Virology  2012;86(22):12283-12293.
Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.
PMCID: PMC3486503  PMID: 22951833
5.  Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines 
Vaccine  2011;29(34):5657-5665.
Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009–2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently by using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially-expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.
PMCID: PMC3182405  PMID: 21704111
Pandemic influenza; Vaccine potency; Single-radial immunodiffusion assay; H5N1; H1N1; Vaccine
6.  H5N1 Virus-Like Particle Vaccine Elicits Cross-Reactive Neutralizing Antibodies That Preferentially Bind to the Oligomeric Form of Influenza Virus Hemagglutinin in Humans ▿ †  
Journal of Virology  2011;85(21):10945-10954.
Transmission of pathogenic avian influenza viruses (AIV) from wild birds to domestic poultry and humans is continuing in multiple countries around the world. In preparation for a potential AIV pandemic, multiple vaccine candidates are under development. In the case of H5N1 AIV, a clear shift in transmission from clade 1 to clade 2 viruses occurred in recent years. The virus-like particle (VLP) represents an economical approach to pandemic vaccine development. In the current study, we evaluated the humoral immune response in humans vaccinated with H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine manufactured in Sf9 insect cells. The VLPs were comprised of the influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins. In an FDA-approved phase I/II human clinical study, two doses of H5N1 VLPs at 15, 45, or 90 μg HA/dose resulted in seroconversion and production of functional antibodies. Moreover, cross-reactivity against other clade 2 subtypes was demonstrated using virus neutralization assays. H5N1 whole-genome fragment phage display libraries (GFPDL) were used to elucidate the antibody epitope repertoire in postvaccination human sera. Diverse epitopes in HA1/HA2 and NA were recognized by postvaccination sera from the two high-dose groups, including large segments spanning the HA1 receptor binding domain. Importantly, the vaccine elicited sera that preferentially bound to an oligomeric form of recombinant HA1 compared with monomeric HA1. The oligomeric/monomeric HA1 binding ratios of the sera correlated with the virus neutralizing titers. Additionally, the two high-dose VLP vaccine groups generated NA-inhibiting antibodies that were associated with binding to a C-terminal epitope close to the sialic acid binding site. These findings represent the first report describing the quality of the antibody responses in humans following AIV VLP immunization and support further development of such vaccines against emerging influenza virus strains.
PMCID: PMC3194932  PMID: 21865396
7.  Bacterial HA1 Vaccine against Pandemic H5N1 Influenza Virus: Evidence of Oligomerization, Hemagglutination, and Cross-Protective Immunity in Ferrets▿ †  
Journal of Virology  2010;85(3):1246-1256.
The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. We have developed bacterial systems for expression and purification of properly folded functional hemagglutinin as a rapid response to emerging pandemic strains. A recombinant H5N1 (A/Vietnam/1203/2004) hemagglutinin globular domain (HA1) was produced in Escherichia coli under controlled redox refolding conditions. Importantly, the properly folded HA1(1-320), i.e., HA1 lacking amino acids 321 to 330, contained ≥75% functional oligomers without addition of foreign oligomerization sequence. Site-directed mutagenesis mapped the oligomerization signal to the HA1 N-terminal Ile-Cys-Ile residues at positions 3 to 5. The purified HA1 oligomers (but not monomers) bound fetuin and agglutinated red blood cells. Upon immunization of rabbits, the oligomeric HA1(1-320) elicited potent neutralizing antibodies against homologous and heterologous H5N1 viruses more rapidly than HA1(28-320) containing only monomers. Ferrets vaccinated with oligomeric HA1 (but not monomeric HA1 with the N terminus deleted) at 15 and 3 μg/dose were fully protected from lethality and weight loss after challenge with homologous H5N1 (A/Vietnam/1203/2004, clade 1) virus, as well as heterologous clade 2.2 H5N1 (A/WooperSwan/Mongolia/244/2005) virus. Protection was associated with a significant reduction in viral loads in the nasal washes of homologous and heterologous virus challenged ferrets. This is the first study that describes the presence of an N-terminal oligomerization sequence in the globular domain of influenza virus hemagglutinin. Our findings suggest that functional oligomeric rHA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.
PMCID: PMC3020491  PMID: 21084473
8.  Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus 
PLoS ONE  2010;5(7):e11548.
In the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 “Spanish Flu”. The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.
Methodology/Principal Findings
Recombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1–330) and HA (1–480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1–330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1–330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1–480). Protein yield for the HA1 (1–330) ranged around 40 mg/Liter, while the HA (1–480) yield was 0.4–0.8 mg/Liter.
This is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.
PMCID: PMC2902520  PMID: 20634959
9.  Anesthetic management of a patient with prosthetic heart valve for non-cardiac surgery: A case report 
Cases Journal  2008;1:196.
Patients with prosthetic heart valves are a challenge to any anesthesiologist due to the risk of infective endocarditis, bleeding and thrombosis.
Case presentation
We present anesthetic management of a 58-year-old Indian lady with a prosthetic (mechanical) heart valve who underwent hemireplacement arthroplasty.
Patients with prosthetic heart valves, especially those with the mechanical valves are prone for thrombosis and resultant complications if anticoagulation is not maintained properly. However, when they are scheduled for major surgery, they can be best managed by normalising the coagulation profile immediately prior to surgery and restarting the anticoagulation as early as possible.
PMCID: PMC2569916  PMID: 18826632
10.  Phylogeography of mtDNA haplogroup R7 in the Indian peninsula 
Human genetic diversity observed in Indian subcontinent is second only to that of Africa. This implies an early settlement and demographic growth soon after the first 'Out-of-Africa' dispersal of anatomically modern humans in Late Pleistocene. In contrast to this perspective, linguistic diversity in India has been thought to derive from more recent population movements and episodes of contact. With the exception of Dravidian, which origin and relatedness to other language phyla is obscure, all the language families in India can be linked to language families spoken in different regions of Eurasia. Mitochondrial DNA and Y chromosome evidence has supported largely local evolution of the genetic lineages of the majority of Dravidian and Indo-European speaking populations, but there is no consensus yet on the question of whether the Munda (Austro-Asiatic) speaking populations originated in India or derive from a relatively recent migration from further East.
Here, we report the analysis of 35 novel complete mtDNA sequences from India which refine the structure of Indian-specific varieties of haplogroup R. Detailed analysis of haplogroup R7, coupled with a survey of ~12,000 mtDNAs from caste and tribal groups over the entire Indian subcontinent, reveals that one of its more recently derived branches (R7a1), is particularly frequent among Munda-speaking tribal groups. This branch is nested within diverse R7 lineages found among Dravidian and Indo-European speakers of India. We have inferred from this that a subset of Munda-speaking groups have acquired R7 relatively recently. Furthermore, we find that the distribution of R7a1 within the Munda-speakers is largely restricted to one of the sub-branches (Kherwari) of northern Munda languages. This evidence does not support the hypothesis that the Austro-Asiatic speakers are the primary source of the R7 variation. Statistical analyses suggest a significant correlation between genetic variation and geography, rather than between genes and languages.
Our high-resolution phylogeographic study, involving diverse linguistic groups in India, suggests that the high frequency of mtDNA haplogroup R7 among Munda speaking populations of India can be explained best by gene flow from linguistically different populations of Indian subcontinent. The conclusion is based on the observation that among Indo-Europeans, and particularly in Dravidians, the haplogroup is, despite its lower frequency, phylogenetically more divergent, while among the Munda speakers only one sub-clade of R7, i.e. R7a1, can be observed. It is noteworthy that though R7 is autochthonous to India, and arises from the root of hg R, its distribution and phylogeography in India is not uniform. This suggests the more ancient establishment of an autochthonous matrilineal genetic structure, and that isolation in the Pleistocene, lineage loss through drift, and endogamy of prehistoric and historic groups have greatly inhibited genetic homogenization and geographical uniformity.
PMCID: PMC2529308  PMID: 18680585

Results 1-10 (10)