Valorization of agro-wastes for the biosynthesis and characterization of polyhydroxybutyrate by Bacillus sp. isolated from rice bran dumping yard.

Investigations have been made to determine the usage of inexpensive agro-waste products as an alternative carbon source for the production of degradable bacterial polyester. Among 33 bacterial isolates, a gram-positive bacterium PPECLRB-16 isolated from rice bran dumping yard was found to accumulate a relatively higher quantity of PHB and identified as Bacillus sp. through 16S rRNA gene sequence analysis. The higher PHB producing bacterial isolate was grown with different inexpensive agro-wastes to determine the suitable carbon source for its growth and PHB production. The one-factor-at-a-time approach comparatively enhanced PHB yield (5.64 g/L) when grown for 48 h with 1.5% (w/v) of defatted oil cake at a pH of 7.0. The bacterially accumulated PHB was isolated from the cells, purified, and characterized using solid-state 13C NMR, FT-IR, Powder XRD, TGA, GPC, Tensile and HR-SEM analyses. The hydrophobicity and printing accessibility of recovered PHB were demonstrated using contact angle measurement by coating on different surfaces. The results obtained in the present investigation have thrown light on the potential usage of agro-waste by-products, mainly oil cake, as an appropriate carbon source for the commercial production of PHB by Bacillus sp. in a cost-effective way.

Statistical augmentation of polyhydroxybutyrate production by Isoptericola variabilis: Characterization, moulding, in vitro cytocompatibility and biodegradability evaluation.

This study aimed to explore the production of polyhydroxybutyrate (PHB), a polyhydroxyalkanoate (PHA), which has been widely considered as a potential substitute for the synthetic polymers. Among 53 actinomycete isolates, 11 of them were found to be PHB positive and the quantity of PHB from the positive isolates varied from 10.5 to 29.82 wt% on a dry cell weight basis. A strain designated as PPLAT 012, accumulated relatively higher PHB and has been identified as Isoptericola variabilis by 16S rRNA gene sequence analysis. An effort has also been made to optimize the PHB production by the hyper-producing strain using the conventional, one-factor-at-a-time, and statistical response surface methodologies and the maximum PHB production (46.18%) in DSMZ medium, amended with 12% glucose and 9% potassium nitrate with a pH of 7.0. Further, the characteristic properties such as processability, cytocompatibility and biodegradability of the extracted PHB was also demonstrated. The physical properties of the recovered PHB was further improved by blending with PLA and the resultant blends were characterized. The present investigation has demonstrated that the isolate, Isoptericola variabilis, could be utilized as a potential source for the production of PHB with desirable characteristics, suitable for biomedical applications.

Enhanced method for High Spatial Resolution surface imaging and analysis of fungal spores using Scanning Electron Microscopy.

Efficient, fast and new micro-analytical methods for characterization of ultrastructures of fungal spores with electron microscopy are very much required and essential. SEM analysis of biological materials, especially fungi, requires optimal preparation of the specimen and often requires the usage of dried samples which demands a challenging sample preparation. In the present investigation, we described a fast and improved method for the preparation of fungal specimen for scanning electron microscopy (SEM). The fungus, Curvularia lunata was grown on the surface of sterile Whatman No.1 filter paper which was overlaid on Potato Dextrose Agar (PDA) medium, gold coated immediately after removal from the growth medium and subjected to imaging. Generally, SEM imaging is done with samples that were fixed with chemical fixatives, dehydrated and gold coated specimens, but here we describe an easy and more efficient sample preparation for SEM which enabled enhanced image quality and precision visualization of fungal cells, especially the spores. The developed method has enabled the analysis of even the robust samples like fungal spores that to eliminating special temperature requirement. The ultimate goal was to develop an improved protocol/method applied to analysis of fungal spores with greater coverage about fungal specimen preparation. This method permits the use of rapid sample preparation and will allow us to imaging of individual spore or conidia structures in the context of fungal cell architecture which clarifies our understanding in fungal taxonomy/biology.

Impact of geography on adaptation of Phyllanthus amarus seeds.

The present study was aimed to evaluate adaptive mechanism in terms of seed characters of Phyllanthus amarus collected from ten different locations of Tamil Nadu, India. The adaptive variations among the collected populations were assessed based on the sink and float percentages of the seeds in water, the percentage of seed germination, total protein, carbohydrates and their seedling's growth ability such as shoot and root lengths. From this, we observed that the population had a significantly higher germination percentage of sinking seeds that were attributed to its relatively higher carbohydrate and protein contents than the floating seeds. A comparison of the seed population by cluster analysis and principal coordinate analysis showed that the Chennai population constituted a single clade that was very distinct from the other nine populations, which were further grouped into two sub-clusters. They exhibited a trend consistent with their geographical proximity. Standardised Mantel's t tests had revealed that the adaptive diversity of the P. amarus population was significantly affected by the geographic distance (r = 0.78, t = 2.68, P > 0.001), altitude (r = 0.35, t = 21.53, P > 0.05), minimum temperature (r = 0.43, t = 1.49, P > 0.01) and maximum temperature (r = 0.49, t = 1.67, P > 0.001). Seed's characteristics and geographical conditions were correlated along with 19 bioclimatic variables. In dry season, the seedling's rooting ability showed positive correlation, while its protein content exhibited a negative correlation. It is clearly evident from this study that the geographical variables significantly influence the adaptive ability of the P. amarus.

Studies on detection and analysis of proteases in leaf extract of medicinally important plants.

ETHNO-PHARMACOLOGICAL RELEVANCE: The whole plant or the extracts obtained from them have long been used as medicine to treat various human diseases and disorders. Notably, those plants endowed with protease activity have been traditionally used as the agents for treating tumors, digestion disorders, swelling, blood coagulation, fibrinolysis and also for immune-modulation. AIM OF THE STUDY: Proteases occupy a pivotal position in enzyme based industries. Plant proteases have been increasingly exploited for pharmaceutical, food, leather and textile processing industries. Earlier investigations have focused on the occurrence of proteases in medicinally unimportant plants. Therefore it has been aimed to study the occurrence of proteolytic enzymes from medicinally important plants establish any correlation exists between protease activity and medicinal use of individual plants. METHODS: Crude extract were obtained from the leaves of 80 different medicinal plants. Tris-HCl buffer was used as the extraction buffer and the supernatants obtained were used for determination of total protein and protease activity using spectrophotometric methods. Qualitative screening for the presence of protease was carried out with agar diffusion method by incorporating the substrate. SDS-PAGE was used to analyse the isoforms of protease and for determination of relative molecular mass. RESULTS: Relatively higher protease activities were observed in the extracts of leaves of Pongamia pinnata (Fabaceae), Wrightia tinctoria (Apocyanaceae) Acalypha indica (Euphorbiaceae), Adhatoda vasica (Acanthaceae) and Curcuma longa (Zingiberaceae). No correlation was found between the total protein content and protease activity in individual plant species. SDS-PAGE analysis indicated the presence of multiple forms of protease of higher molecular weight range in several plant species. We found a strong correlation between the protease activity and medicinal application of the plant CONCLUSION: The present study has unequivocally revealed that the leaves of medicinal plants could serve as excellent sources of proteases which could be exploited for various industrial, food and pharmaceutical applications.

Antibacterial Activity study of Musizin isolated from Rhamnus wightii Wight and Arn.

The crude extracts and the compounds isolated from traditional medicinal plants are used to treat infectious diseases caused by bacteria, fungi, and viruses. An attempt has been made in the present investigation to evaluate the antibacterial activity of musizin isolated from Rhamnus wightii, (Family: Rhamnaceae) against Gram-positive (Bacillus cereus, Staphylococcus aureus, Streptococcus faecalis), and Gramnegative (Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa) bacteria. The tested compound showed more pronounced antibacterial activity against the tested pathogens than the standard antibiotics like streptomycin and gentamycin with the lowest minimum inhibitory concentration (MIC). Molecular docking analysis was performed to study the effectiveness of musizin compared to the standard antibiotics; it showed a significant interaction with the target proteins such asalgR (P. arginosa), divIVA (E. faecalis), icaA (S. aureus), plcR(B. cereus), treC (K. pneumonia) and ftsl (E. coli) and found that musizin showed higher potential with least binding energy. It has also been found that musizin had better ADMET properties than the standard drugs. Thus,musizin acts as an inhibitor of bacterial growth for consideration as a drug to treat bacterial infections.

Polyhydroxybutyrate by Streptomyces sp.: Production and characterization.

A total number of 20 actinomycetes isolates were isolated from soil sediments obtained from Semmancheri coastal areas of Bay of Bengal, India and they were qualitatively screened for the production of polyhydroxybutyrate (PHB) on a medium containing Sudan black stain. Nine of the 20 isolates produced PHB and the quantity of PHB produced varied from 1.79 to 4.26g-L. Among the positive isolates an actinomycete isolate which was identified as Streptomyces sp. through 16S rRNA sequencing analysis (Accession No: KF667247) produced relatively higher PHB than other positive isolates. Subsequently, the growth conditions were optimized for the maximum PHB production by the chosen organism. Attempt was also made to utilize natural carbon sources such as paddy straw, wheat bran, rice bran, sugarcane molasses and oil cake for the production of PHB in an attempt to reduce the cost production of PHB. The purified PHB was analyzed by Solid-State 13C NMR, Fourier Transformed Infrared spectroscopy, Powder X-ray diffraction, Thermogravimetric Analysis, Scanning and Transmission Electron Microscopic analyses to determine the structure, crystallinity, purity and thermal stability. The present investigation has revealed that Streptomyces sp. could be a potential source for the production of PHB with desirable characteristics and could also be exploited for the industrial production.

Antibacterial cysteine protease from Cissus quadrangularis L.

An antibacterial Cp was extracted from the stem of Cissus quadrangularis and purified with a 5.39 fold increase in specific activity and 8.67% recovery. The molecular weight of the purified enzyme was estimated to be 39kDa by SDS-PAGE. The purified enzyme appeared as a single band on Native-PAGE. The optimum pH and temperature for protease activity were around 6.0 and 50°C respectively. The Cp showed pH stability from 3 to 10 and retained more than 90% of its relative protease activity. The addition of metal ions such as Mg2+ and Ca2+ also exhibited relative protease activity. Cp showed a potent antibacterial activity against pathogenic bacteria. About 4.74Uml-1 of Cp from C. quadrangularis was tested for antibacterial activity against Bacillus cereus and Bacillus megaterium which subsequently showed zone of inhibition of 21 and 20mm respectively. Cp from C. quadrangularis degraded the peptidoglycan layer of bacteria by Cp was confirmed by transmission electron microscopic analysis.

An in vitro study on the burn wound healing activity of cotton fabrics incorporated with phytosynthesized silver nanoparticles in male Wistar albino rats.

In modern therapeutics, chemically synthesized drugs have been reported as causing adverse effects including allergies, rashes, itches, and swelling. For the past few decades, silver nanoparticles (AgNPs) have widely been applied in medical domains due to their antimicrobial and wound healing properties. In the present study, different concentrations of phytosynthesized AgNPs-saturated cotton dress fabrics - in comparison to cotton fabrics treated with commercial ointment - were tested for 18days to assess their ability to speed the healing of rats' burn wounds. No significant difference in body weight was observed during the course of treatment as compared to the normal rat group. The cotton fabrics observed under Scanning Electron Microscopy (SEM) confirmed the distribution of AgNPs in the cotton fibers. Energy-Dispersive X-ray analysis (EDX) spectrum also authenticated the AgNPs' distribution. At the end of the experimental period, the wound healing efficacy of dressing containing commercial ointment (Burn Heal) was slightly lower than that of treatment containing 100µg/kg of body weight (kg b.w.) of AgNPs. Additionally, it was also observed that the wound contraction area was higher than that of the positive drug 100µg/kg b.w. treated group, which indicates comparatively better-quality activity of ointments with AgNPs with regards to their burn healing properties. The histological and SEM observations showed better fibril alignments in repaired skin when compared with the negative and positive control groups. Perhaps due to the tensile strength of the comparatively higher concentration of nanoparticles, Groups IV and V (which contained the most nanoparticles out of all the groups) showed much better healing properties than did the positive drug treated group VI. Altogether, increased-concentration AgNPs show increased recovery action in comparison to the positive drug treated group. This study provides additional insight into the incorporation of AgNPs in wound dressings for speedy recovery of burn wounds, for improved human welfare.

Antibacterial serine protease from Wrightia tinctoria: Purification and characterization.

A serine protease was purified from the leaves of Wrightia tinctoria by sequential flow through method comprising screening, optimization, ammonium sulfate precipitation, gel filtration and ion exchange column chromatography. The yield and purification fold obtained were 11.58% and 9.56 respectively. A single band of serine protease was visualized on SDS-PAGE and 2-D gel electrophoretic analyses were revealed with the molecular mass of 38.5 kDa. Serine protease had an optimum pH of 8.0 and was stable at 45°C with high relative protease activity. The addition of metal ions such as Mg2+ and Mn2+ exhibits a high relative activity. Serine protease had a potent antibacterial activity against both Gram-positive and Gram-negative bacteria. A 10 µg/ml of serine protease was tested against S. aureus, M. luteus, P. aeruginosa and K. pneumoniae which had 21, 20, 18 and 17 mm of zone of inhibition respectively. Serine protease from W. tinctoria degrades the peptidoglycan layer of bacteria which was visualized by transmission electron microscopic analysis.

Qualitative and quantitative determination of R-phycoerythrin from Halymenia floresia (Clemente) C. Agardh by polyacrylamide gel using electrophoretic elution technique.

R-Phycoerythrin is one of the phycobiliproteins widely found in seaweeds. In this study, we have shown to extract and purify R-Phycoerythrin from the south east cost Indian red seaweed Halymenia floresia. R-Phycoerythrin was extracted in 50mM phosphate buffer (pH 7.0). The preparative native PAGE purification was employed alternative to the chromatography and therefore can be scaled up efficiently. Both the yield and the purity of R-Phycoerythrin are very effective. The purified R-Phycoerythrin showed a single band on the examination by native PAGE electrophrosis. SDS-PAGE analysis showed five bands at 16kDa, 21kDa, 30kDa, 39kDa and 47kDa which corresponds to the α, ß and γ', γ and αß subunits. This preparative method for R-Phycoerythrin purification can offer a reference for R-Phycoerythrin purification from other marine red macro algae.

Highly potential antifungal activity of quantum-sized silver nanoparticles against Candida albicans.

The antifungal activity of polyvinylpyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) against the growth of Candida albicans has been demonstrated in the present study. C. albicans is a known opportunistic human pathogen causing superficial and systemic infections. Research data carried out on C. albicans so far have shown unequivocally that it develops resistance against conventional antifungal drugs and that the infections it causes are difficult to cure with conventional antifungal agents. Hence, it is urgent to find newer materials for the treatment of infections caused by C. albicans that must be safe for the host. PVP-capped SNPs were synthesized, and its surface plasmon band was observed at 410 nm. The growth of C. albicans was markedly inhibited when the cells were incubated with SNP. The minimum inhibitory concentration (MIC) of SNP was determined as 70 ng/ml, and this value is relatively lower when compared with the conventionally used antifungal drugs such as amphotericin B (0.5 µg/ml), fluconazole (0.5 µg/ml), and ketoconazole (8 µg/ml). The viability of SNP-treated cells was checked by measuring the metabolic activity using XTT assay. Field emission scanning electron microscopic (FE-SEM) and transmission electron microscopic (TEM) analyses of the cells treated with SNP have lost the structural integrity to a greater extent.

Cell surface shaving of Candida albicans biofilms, hyphae, and yeast form cells.

We used a brief trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS to identify the surface proteins on live Candida albicans organisms growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty-one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.

Proteomic analysis of cytoplasmic and surface proteins from yeast cells, hyphae, and biofilms of Candida albicans.

Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non-covalently attached cell-surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (>or=1.5-fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi-organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.

Role for cell density in antifungal drug resistance in Candida albicans biofilms.

Biofilms of Candida albicans are less susceptible to many antifungal drugs than are planktonic yeast cells. We investigated the contribution of cell density to biofilm phenotypic resistance. Planktonic yeast cells in RPMI 1640 were susceptible to azole-class drugs, amphotericin B, and caspofungin at 1 x 10(3) cells/ml (standard conditions) using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay. As reported by others, as the cell concentration increased to 1 x 10(8) cells/ml, resistance was observed with 10- to 20-fold-greater MICs. Biofilms that formed in microtiter plate wells, like high-density planktonic organisms, were resistant to drugs. When biofilms were resuspended before testing, phenotypic resistance remained, but organisms, when diluted to 1 x 10(3) cells/ml, were susceptible. Drug-containing medium recovered from high-cell-density tests inhibited low-cell-density organisms. A fluconazole-resistant strain showed greater resistance at high planktonic cell density, in biofilm, and in resuspended biofilm than did low-density planktonic or biofilm organisms. A strain lacking drug efflux pumps CDR1, CDR2, and MDR1, while susceptible at a low azole concentration, was resistant at high cell density and in biofilm. A strain lacking CHK1 that fails to respond to the quorum-sensing molecule farnesol had the same response as did the wild type. FK506, reported to abrogate tolerance to azole drugs at low cell density, had no effect on tolerance at high cell density and in biofilm. These observations suggested that cell density has a role in the phenotypic resistance of biofilm, that neither the drug efflux pumps tested nor quorum sensing through Chk1p contributes to resistance, and that azole drug tolerance at high cell density differs mechanistically from tolerance at low cell density.

Analysis of RNA species of various sizes from stationary-phase planktonic yeast cells of Candida albicans.

We initiated a comparison of Candida albicans stationary-phase gene expression with other growth states. The widely used hot acid phenol method for RNA extraction did not extract rRNA from late stationary-phase cells. The RNA from growing yeast cells, hyphae and biofilm was biased towards small-sized RNA. The 2 : 1 ratio between the two large rRNA bands was rarely obtained. Real-time reverse transcriptase PCR was used to determine mRNA extraction by several methods for OXR1, IRA2, RAD50, PNC1 and CHS2, which have 300 bp-8 kb coding regions, and ACT1, EFB1 and TDH3, sometimes used as internal standards. Only smaller-sized cDNA species were amplified from some extracts. Crushing cells with glass beads in liquid nitrogen before RNA extraction by the hot phenol method (CGB) yielded an unbiased distribution for rRNA and mRNA as verified by real-time reverse transcriptase PCR. With the CGB method, the large mRNA species, RAD50, IRA2 and OXR1, were present throughout the stationary phase, whereas the CSH2 transcript increased. The ACT1, EFB1 and TDH3 transcripts decreased in the stationary phase, making them unsuitable for standardization. The CGB method yielded high-quality RNA with the various growth conditions and permitted the comparison of stationary-phase transcripts with those obtained under other conditions.