Optimization and Characterization of PHA (SCL-SCL) Copolymer by Indigenous Bacillus thuringiensis A102 Strain for Biomedical Applications.

PHA is one of the leading commercially important bio-polyesteric compounds piled up as an intracellular lipid-based energy storage compound by numerous microorganisms. An indigenous Gram-positive bacterium isolated from fire ant (Solenopsis invicta) has known to potentially accumulate PHA. Various nutritional elements like carbon, nitrogen, phosphate and C/N ratio were optimized. The indigenous B.t.A102 strain grown in optimized RC medium yielded PHA of about 3.25 g/L. The extracted polymer was characterized by NMR, GC-MS, X-ray diffraction and thermal analysis via TGA & DTA. The characterized PHA was used to prepare scaffold using the solvent casting method. The non-toxic nature of the composite material was evaluated on NIH/3T3 fibroblast cell lines using different staining (like Giemsa staining, AO/EB dual staining, neutral red staining) techniques and cell viability assay. This paper dealt with the optimization of the media components that increase PHA production and primary in vitro testing for its possible application as wound dressing materials.

Enhancement of Purified Human Colon Cancer-Specific Parasporal Toxin from Bacillus thuringiensis-LDC-501.

Parasporal inclusion protein of Bacillus thuringiensis-LDC-501 (Bt-LDC-501) exhibits selective cytocidal action towards human colon cancer cells. The yield of this parasporal protein was minimum in the normal culture. In order to increase the yield of protein from Bt-LDC-501 various agro-based cost-efficient nutrient sources such as corn steep liquor (CSL), sesame oil cake extract (SOC), groundnut oil cake extract (GOC), neem oil cake extract (NOC), rice bran extract (RB), wheat bran extract (WB), red gram hull extract (RGH), green gram hull extract (GGH), black gram hull extract (BGH), Mysore gram hull extract (MGH), and maize flour waste extract (MFW) were screened. Statistical experimental designs such as Plackett-Burman design (PBD) and response surface methodology (RSM) were the tools employed for the optimization of medium. Groundnut cake extract (GOC) served as a potential carbon and nitrogen source, as it induced twofold higher production of parasporal protein. Among the optimized seven media components KH2PO4, K2HPO4, GOC, NaCl, MgSO4, MnSO4, and FeSO4, the concentrations of GOC, NaCl, and MgSO4 have significant effect on parasporin production as well as cytotoxicity against colon cancer cell line, HCT-116. Bt-LDC-501 was found to produce 0.88 mg/ml of parasporal protein in optimized medium. In the un-optimized medium, the yield was 0.23 mg/ml only. This indicated that there was 382% of increase in the production of Parasporal protein. Parasporin protein with the molecular weight of 27 kDa has been purified with the purification fold of 27.1. It showed a LC50 value of 0.91 and 1.21 µg/ml against colorectal cancer cell lines such as HCT-116 and HCT-15, respectively. Purified parasporin exhibited stable cytocidal activity between pH 4.0 and 9.0 at room temperature. The present study revealed that the quantity and quality of media composition were necessary for eliciting cytocidal activity against human colon cancer and the importance of alternate cost-effective production of clinically significant parasporin. Moreover, this is the first report regarding optimization of media components for parasporal protein production from Bt.

Bioprospection of Basidiomycetes and molecular phylogenetic analysis using internal transcribed spacer (ITS) and 5.8S rRNA gene sequence.

Macrofungi belonging to the phylum Basidiomycota are mostly used as medicinal mushrooms in many countries. In the present study, hundred basidiocarp of macrofungi were collected from Tamilnadu during rainy season. The basidiocarp was found in association with root/trunk of living trees, wood log and decayed matter. Among the hundred basidiocarp, 49 were grown into axenic cultures. Notable variations in the macroscopic characteristics of the basidiome and culture morphology were observed. To study the genetic diversity, the molecular taxonomy of the isolates was carried out using internal transcribed spacer (ITS) and 5.8S rRNA gene sequence marker. Thirty-two strains belonging to the order Polyporales, Hymenochataeles and Russuales under the division Basidiomycota were classified based on phylogeny analysis. This study provides first evidence for the occurrence of species Fulvifomes fastuosus (LDCMY39 and LDCMY43) and Ganoderma wiiroense (LDCMY02, LDCMY08, LDCMY11, LDCMY17 and LDCMY19) from southern India. Molecular evidence for the existence of Phellinus badius was given for the first time as well. These data enhance our understanding on the diversity of macrofungi in India, which could be further exploited for biomedical applications.

Immunostimulation by phospholipopeptide biosurfactant from Staphylococcus hominis in Oreochromis mossambicus.

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S. hominis at a dose of 2 mg, 20 mg and 200 mg kg(-1) body weight. Commercial surfactant surfactin (sigma) at 20 mg kg(-1) was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S. hominis stimulates the immunity of finfish thereby could enhance aquaculture production.

Immunostimulation by poly-ß hydroxybutyrate-hydroxyvalerate (PHB-HV) from Bacillus thuringiensis in Oreochromis mossambicus.

The present study was designed to test the immunostimulatory efficacy of poly-ß hydroxybutyrate-hydroxyvalerate (PHB-HV) extracted from Bacillus thuringiensis B.t.A102 on the immune system of Oreochromis mossambicus. Fish were fed with 0%, 1%, 3% or 5% PHB-HV supplemented feed and were bled at regular intervals of 5 days. The specific immune response was measured in terms of antibody response to sheep red blood cells, the nonspecific immune mechanisms were analysed in terms of serum lysozyme activity, total peroxidases activity and antiprotease activity. The overall functional immunity was tested by experimental challenge with live virulent Aeromonas hydrophila. The results revealed that all the doses of PHB-HV supplementation in feed were effective in stimulating both specific and nonspecific immune mechanisms. The bacterial challenge experiment showed that highest dose of 5% PHB-HV supplementation was more effective than 1% and 3% doses. The study concludes that PHB-HV can be used as a potential immunostimulant in finfish aquaculture.

Phenotypic and genotypic characterization of B.t.LDC-391 strain that produce cytocidal proteins against human cancer cells.

An indigenous Bacillus thuringiensis strain B.t.LDC-391 producing cytocidal proteins against human colon cancer cell line, HCT-116, was subjected to phenotypic and genotypic characterization to evaluate its relatedness to B.anthracis. The morphological features of this strain were meta-analyzed with data of other parasporin and insecticidal protein producing Bacillus thuringiensis strains. The conventional biochemical analysis and antibiotic sensitivity test proved it as an ampicillin resistant which is a salient feature, absent in B.anthracis Ames. PCR analysis showed the absence of cyt and parasporin related genes in the genome of B.t.LDC-391. But the strain was positive for cap gene. The sequencing and bio-informatic analysis of cap gene and 16S rDNA of B.t.LDC-391 placed it closer to B.thuringiensis and revealed significant divergence from that of any B.anthracis strain. However our strain lacked ß- hemolysis on human erythrocytes which is a common feature of B.anthracis strains and parasporin producers.

MEDDB: a medicinal plant database developed with the information gathered from tribal people in and around Madurai, Tamil Nadu.

Tribal peoples are endowed with enriched traditional wisdom to use available nature resources around them. They are well versed in the usage of plant for treating various diseases. They have used powder or extract or paste form of the plant parts such as root, shoot, whole plant, fruits and leaves etc. The recipe known by the tribal people was passed on only to their family members and community through mouth to mouth practice. Hence, the knowledge is confined to particular people alone. It is always expedient to store information in the database, so that it will be accessible by everyone from everywhere. To achieve this, MEDDB has been developed, which stores the details of 110 plant species that are commonly used by tribes for fever, asthma, cold, cough, diabetes, diarrhea, dysentery, eye infections, stomach ache, wounds and snake bite. The details of each plant were collected from the literature and through web search to give comprehensive and exhaustive information. Each plant entry is compiled under the subheadings viz., common name, classification, physical characteristics, medicinal uses, active constituents, and references.

Sequence analysis, structure prediction, and functional validation of phaC1/phaC2 genes of Pseudomonas sp. LDC-25 and its importance in polyhydroxyalkanoate accumulation.

Polyhydroxyalkanoates (PHAs) are attractive biomaterials in both conventional medical devices and tissue engineering. PHA synthase is responsible for catalyzing the formation of Polyhydroxyalkanoates (PHA), but its structural information is limited. Hence, this study focuses to predict 3D model for phaC1 and phaC2 genes of field-soil strain Pseudomonas sp. LDC-25 and to validate the functional properties through in vitro studies. The phaC1/phaC2 genes were amplified, cloned, and sequenced. The sequence analysis showed > 90% homology to phaC loci and presence of alpha/beta hydrolase fold, but phaC2 loci of LDC-25 exhibits variation in the conserved residue (Ser is replaced by Ala). Threading approach demonstrated that Carboxylesterase (d1tqha) can be used as the modeling template. The predicted models showed the presence of conserved residues at 122 (G), 205 (S), and 236 (S). In vitro studies also supported that PHA accumulation ability was less in Pseudomonas sp. LDC-25 compared to other field isolate, Pseudomonas sp. LDC-5. FT-IR spectrum showed PHA specific peaks at 1735.62 cm(-1). Results of this study would help to detect the functional domains of the protein in order to elucidate their structure/function characteristics with special emphasis on invariant conserved residues.

Serum zinc and albumin levels in pulmonary tuberculosis patients with and without HIV.

Limited data are available on the relationship between nutritional status and pulmonary tuberculosis (PTB). Zinc plays a vital role in the immune status of the individual. The present study was carried out to estimate serum zinc and albumin levels in newly detected adult active PTB patients with (n = 20) and without (n = 20) HIV, and to compare them with the levels in controls (healthy family members; n = 20) who satisfied rigid selection criteria. Standard methods were adopted to collect an early morning fasting blood sample for zinc (by Atomic Absorption Spectrometer) and albumin (estimated by the bromocresol green method). The mean +/- SD for BMI, zinc and albumin among the controls, HIV positive and HIV negative patients were 19.6 +/- 0.6, 18 +/- 0.4 and 18.5 +/- 0.6 kg/m(2); 117.13 +/- 4.2, 53.9 +/- 8 and 65.5 +/- 9.8 microg/dL; and 4.1 +/- 0.6, 2.9 +/- 0.4 and 3.6 +/- 0.7 g/dL, respectively. All three parameters were significantly low in active PTB patients irrespective of HIV status, but more so in HIV-positive individuals. These changes may be attributable to nutritional factors, enteropathy and acute phase reactant proteins. Hence, the National AIDS Control Organization (NACO) in India is providing nutritional supplements to those HIV-infected cases inducted for antiretroviral therapy and nutritional counseling for others as a part of a national policy.

Prevalence of intestinal parasitic infestation in HIV/AIDS patients with diarrhea in Madurai City, South India.

The prevalence and pattern of parasitic infestation among 80 HIV/AIDS patients with diarrhea in Madurai, south India, was studied by microscopy. Eighty HIV-negative patients were used as controls. Intestinal parasites were detected in 31 HIV/AIDS patients (38.7%) and in 14 (17.5%) HIV-negative patients, a difference that was statistically significant (P < 0.05). In HIV/AIDS patients with diarrhea, protozoa accounted for the majority of diarrhea cases (Entamoeba spp. 37.5%, Cryptosporidium parvum 28.7%). It is therefore suggested that enteric infections are more common in HIV-infected patients than in HIV-negative persons in south India, and this may be due to differences in immunological profile, susceptibility as well as factors related to sanitation and the environment.

Molecular characterization of Pseudomonas sp. LDC-5 involved in accumulation of poly 3-hydroxybutyrate and medium-chain-length poly 3-hydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are biological polyesters, of which, Short-Chain-Length-Medium-Chain-Length (SCL-MCL) PHA copolymers are important because of their wide range of applications. The present study focused on molecular characterization of Pseudomonas sp. LDC-5 that is identified as SCL-MCL producer. Phase contrast, fluorescent and electron microscopic observation confirmed the presence of PHA granules in Pseudomonas sp. LDC-5. PCR analysis indicated the presence of expected amplicon for SCL phaC gene ( approximately 500 bp), MCL phaC1 with phaZ ( approximately 1.3), and phaC2 with phaZ ( approximately 1.5 kb). Sequence analysis of the PHA synthase gene of Pseudomonas sp. LDC-5 revealed significant differences in phaC1 and phaC2 which were further confirmed by recombinant studies. Recombinant Escherichia coli harboring the partial phaC1 gene was able to accumulate PHA, whereas E. coli with phaC2 did not accumulate PHA as verified by fold analysis, immunoblotting, Gas Chromatography (GC), Differential scanning calorimetry (DSC), and FTIR studies. The predicted theoretical three-dimensional structure revealed that PhaC1 is consistent with alpha/beta hydrolase fold. Monomer composition showed the presence of monomer ranging from C4 to C12: 1 when glucose and sodium octanoate fed as the carbon source. DSC revealed melting temperature peak at 153.12 degrees C and glass transition (T(g)) peaks at -0.37 degrees C. Thermogravimetric analysis revealed that the polymer was stable up to 276 degrees C. Fourier Transform Infrared Spectroscopy (FT-IR) spectral analysis showed the PHA specific wave number at 1,739.67 and 1,161.07 cm(-1). The potential of Pseudomonas sp. LDC-5 and its properties are discussed.