Whole genome sequencing and characteristics of extended-spectrum beta-lactamase producing Escherichia coli isolated from poultry farms in Banaskantha, India.

Worldwide dissemination of extended-spectrum -lactamase (ESBL)-producing Escherichia coli constitutes an emerging global health issue, with animal food products contributing as potential reservoirs. ESBL E. coli infection is associated with the high mortality and mobility rate in developing countries due to less susceptibility to antibiotics. The present study aimed to elucidate the molecular characteristics and sequence-based analysis of ESBL E. coli in the Gujarat state of India. This study included 108 E. coli strains were isolated from different poultry farms (broiler and layer) in the Banaskantha District. PCR was employed to identify genotypic ESBL-producing antimicrobial resistance genes. Overall, a high occurrence of ESBL genes was found in poultry farms due to the high usage of antimicrobials. The PCR analysis revealed that 79.62% of isolates were detected positive with one or more ESBL genes. Among them, bla TEM (63.88%) was found to be the predominant genotype, followed by bla SHV (30.55%) and bla OXA (28.70%). In the bla CTX-M group, a higher occurrence was observed in bla CTX-M-9 (23.14%), followed by bla CTX-M-2 (24.07%) and bla CTX-M-1 (22.22%). We used the whole-genome sequencing (WGS) method to evaluate the antimicrobial resistance genes, virulence factors, single nucleotide polymorphisms (SNPs), plasmid replicons, and plasmid-mediated AMR genes of one ESBL E. coli isolated. We examined the genetic relatedness of a human pathogenic E. coli strain by comparing its sequence with the broad geographical reference E. coli sequences. Escherichia coli ST 681 was determined using multi-locus sequence typing. We compared our findings to the reference sequence of Escherichia coli str. K- 12 substr. MG1655. We found 24,937 SNPs with 21,792 in the genic region, 3,145 in the intergenic region, and six InDels across the genome. The WGS analysis revealed 46 antimicrobial resistance genes and seven plasmid-mediated AMR genes viz., tetA, qnrS1, dfrA14, sul2, aph(3")-lb, aph(6)-ld, and Aph(3')-la. The ST 681 was found to have Cib, traT, and terC virulence factors and two plasmid replicons, IncFII(pHN7A8) and IncI1-I(Alpha). This study revealed a higher occurrence of ESBL E. coli detected in poultry.

Parthenogenetic activation of buffalo (Bubalus bubalis) oocytes: comparison of different activation reagents and different media on their developmental competence and quantitative expression of developmentally regulated genes.

This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8-71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6-46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus-oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.

Potential Risk by Disposal of Bottom Ash from Thermal Power Plants and Minimization by Addition of NaHCO3.

In the present study, the leachability of traced elements from the bottom ash of three different Indian power plants was investigated. Environmental impact of bottom ash was studied by varying the liquid-to-solid (L/S) ratio from 20:1 to 60:1. Leaching results show the presence of a major proportion of elements Mn, Mg, Cr, Zn and Cu and a minor proportion of Pb, Fe, Co, and Mo. The effect of the addition of sodium bicarbonate (NaHCO3) on leaching characteristics of bottom ash was also studied. Leaching concentration of bottom ash samples reduces with addition of additive from 0.2% to 0.6% and found to be optimum with 0.4% the addition of additive. This aspect of the investigation helps to design the ash disposal system for higher solid concentrations to minimize the environmental pollution.

Role of particle size in assessment of physico-chemical properties and trace elements of Indian fly ash.

In this paper, the effect of particle size on the physico-chemical, mineralogical, and leaching behavior of Indian fly ash was studied. Experiments were carried out to study the leaching of different elements such as Mg, Cr, Zn, Pb, Mn, Fe, Cu, Co, Mo, and Ni from Indian fly ash. During the experiments, the liquid-to-solid (L/S) ratio of the fly ash was taken as 9/1, 8/2, 7/3, 3/2, 1/1, and 2/3. The effect of four different particle size ranges (below 53, 53-75, 75-106, and 106-150 µm) of fly ash was analyzed. The ASTMD-3987 method was used to analyze the presence of trace elements from fly ash. In the ASTM D-3987 method, distilled water was used for extraction of leachate. Fly ash slurry samples were agitated in a lubricating type temperature-controlled Remi orbital shaker for a time duration of 18 hours with speed of 100 revolutions per minute (rpm) at a temperature of 25ºC. Distilled water does not save cost as well as being easily available. The leaching test of trace elements from fly ash was investigated at different pH conditions in order to predict the environmental effect from the ash disposal on the groundwater quality. Results revealed that pH of slurry suspension increases with increase in particle size. The pH value of fly ash slurries was negligibly affected by the decrease in L/S ratio for all particle sizes. Fine particles of fly ash produce a more harmful effect as compared to the coarser range of fly ash particles.

Production of hand-made cloned buffalo (Bubalus bubalis) embryos from non-viable somatic cells.

Use of non-viable somatic cells for hand-made cloning (HMC) can enable production of cloned animals from tissues obtained from elite or endangered dead animals. Buffalo skin fibroblast cells were rendered non-viable by heat treatment and used for HMC. Although fusion (93.6 ± 1.72 vs 67.1 ± 2.83%) and cleavage (90.3 ± 1.79 vs 65.8 ± 1.56%) rate was lower (P < 0.001) than that for controls, blastocysts could be successfully produced. However, blastocyst rate (34.1 ± 2.43 vs 6.9 ± 2.18%, P < 0.001) and total cell number of blastocysts (TCN, 221.3 ± 25.14 vs 151.1 ± 21.69, P < 0.05) were lower and apoptotic index (4.8 ± 1.06 vs 10.9 ± 1.21) was higher (P < 0.001) than that of controls. In another experiment, ear tissue of slaughterhouse buffaloes was preserved in mustard oil at room temperature for 48 h following which somatic cells were harvested by enzymatic digestion and used for HMC. Although fusion (96.8 ± 1.48 vs 84.2 ± 3.19%), cleavage (89.6 ± 3.59 vs 77.2 ± 3.99%), and blastocyst rate (36.9 ± 7.45 vs 13.1 ± 6.87%) were lower (P < 0.01), TCN (223.0 ± 27.89 vs 213.3 ± 28.21) and apoptotic index (3.97 ± 0.67 vs 5.22 ± 0.51) of blastocysts were similar to those of controls. In conclusion, HMC can be successfully used for production of blastocysts from non-viable cells and from cells obtained from freshly slaughtered buffaloes. This can pave the way for the restoration of farm or wild animals by HMC if somatic cells could be obtained within a few hours after their death.

Performance and exhaust emission characteristics of variable compression ratio diesel engine fuelled with esters of crude rice bran oil.

As a substitute to petroleum-derived diesel, biodiesel has high potential as a renewable and environment friendly energy source. For petroleum importing countries the choice of feedstock for biodiesel production within the geographical region is a major influential factor. Crude rice bran oil is found to be good and viable feedstock for biodiesel production. A two step esterification is carried out for higher free fatty acid crude rice bran oil. Blends of 10, 20 and 40 % by vol. crude rice bran biodiesel are tested in a variable compression ratio diesel engine at compression ratio 15, 16, 17 and 18. Engine performance and exhaust emission parameters are examined. Cylinder pressure-crank angle variation is also plotted. The increase in compression ratio from 15 to 18 resulted in 18.6 % decrease in brake specific fuel consumption and 14.66 % increase in brake thermal efficiency on an average. Cylinder pressure increases by 15 % when compression ratio is increased. Carbon monoxide emission decreased by 22.27 %, hydrocarbon decreased by 38.4 %, carbon dioxide increased by 17.43 % and oxides of nitrogen as NOx emission increased by 22.76 % on an average when compression ratio is increased from 15 to 18. The blends of crude rice bran biodiesel show better results than diesel with increase in compression ratio.

Comparative expression profiling of insulin-like growth factor binding protein-5 in milk of Bos indicus and Bubalus bubalis during lactation.

Insulin-like growth factor binding protein-5 (IGFBP-5) is a key molecule in mammary gland development, which facilitates the removal of mammary epithelial cells (MECs) by apoptosis that takes place during remodeling of the mammary gland during involution. IGFBP-5 binds with IGFs for their bioavailability. IGFBP-5 has been reported to perform pleiotropic roles such as cellular apoptosis, proliferation and differentiation. To understand the role of IGFBP-5 during lactation and clinical mastitis, expression profiling of IGFBP-5 at the protein level was performed in both indigenous cows (Bos indicus) and buffaloes (Bubalus bubalis) belonging to two different breeds - Sahiwal cows and Murrah buffaloes. Reverse-transcriptase PCR (RT-PCR) of IGFBP-5 mRNA confirmed its expression in milk somatic cells and MECs of Sahiwal cows. ELISA was performed for quantitative measurement of IGFBP-5 concentrations in milk during different days (0, 50, 100, 150, 200, 250 and 300) of lactation, during the involution period and in animals exhibiting short lactation and clinical mastitis. The highest concentration of IGFBP-5 in milk was observed during the involution period followed by colostrum, late and early lactation, respectively, in both cattle and buffaloes. No significant difference in the concentration of IGFBP-5 was observed during the first 150 days of lactation between cows and buffaloes. However, higher concentration of IGFBP-5 was observed in cows during late lactation (200 to 300 days) in comparison with buffaloes. To validate the ELISA data, quantitative real-time PCR was performed in MECs of Sahiwal cows. The relative mRNA abundance of IGFBP-5 was found to be significantly (P<0.05) higher on day 15 than between 50 and 150 days of lactation in case of Sahiwal cows. Highest mRNA expression of IGFBP-5 was observed around 300 days of lactation followed by 200 and 250 days (P<0.05), respectively. Murrah buffaloes showed low levels of IGFBP-5 protein in milk as compared with Sahiwal cows during lactation in ELISA. Animals having history of short lactation length (short lactating animals) showed higher levels of IGFBP-5 expression (at protein level) in comparison with normal lactating animals. We propose that higher level IGFBP-5 expression may have functional significance in lactation persistency. As a pro-apoptotic molecule, higher expression of IGFBP-5 was observed to be inversely related to lactation length and milk production.

Quantitative expression of pluripotency-related genes in parthenogenetically produced buffalo (Bubalus bubalis) embryos and in putative embryonic stem cells derived from them.

Parthenogenetically produced embryos and embryonic stem (ES) cells derived from them offer a unique model for investigating the role of transcription factors in embryonic genome activation (EGA), pluripotent lineage specification and in pluripotency and self-renewal of ES cells because of the unique nature of these embryos. There is little information on the quantitative expression of important genes in parthenogenetically produced embryos and in ES cells derived from them. The present study examined the quantitative expression of some important genes in parthenogenetically produced buffalo embryos and in putative parthenogenetic ES cells (pES) cells. The quantitative expression of OCT-4, SOX-2, NANOG, REX-1, FOXD-3 and NUCLEOSTEMIN, which is very low in immature and mature oocytes, and in embryos at 2-, 4- and 8- to 16-cell stage, increases significantly at morula and blastocyst stage. The expression level of TELOMERASE, c-MYC and STAT-3, which is high in immature oocytes decreases during embryonic development followed by either an increase at the morula stage (TELOMERASE) or a low expression level maintained throughout development till blastocyst stage (c-MYC and STAT-3). There is a progressive decline in the expression level of OCT-4, SOX-2, c-MYC, REX-1, NUCLEOSTEMIN, TELOMERASE and STAT-3 during long term culture of pES cells.

Comparative Studies on Performance Characteristics of CI Engine Fuelled with Neem Methyl Ester and Mahua Methyl Ester and Its Respective Blends with Diesel Fuel.

In the present investigation, neem and mahua methyl ester were prepared by transesterification using potassium hydroxide as a catalyst and tested in 4-stroke single cylinder water cooled diesel engine. Tests were carried out at constant speed of 1500 rev/min at different brake mean effective pressures. A series of tests were conducted which worked at different brake mean effective pressures, OkPa, 1kPa, 2kPa, 3kPa, 4kPa, 5kPa, 6kPa and 6.5kPa. The performance and exhaust emission characteristics of the diesel engine were analyzed and compared with diesel fuel. Results showed that BTE of NME was comparable with diesel and it was noted that the BTE of N0100 is 63.11% higher than that of diesel at part load whereas it reduces 11.2% with diesel fuel at full load. In case of full load, NME showed decreasing trend with diesel fuel. BTE of diesel was 15.37% and 36.89% at part load and full load respectively. The observation indicated that BTE for MME 100 was slightly higher than diesel at part loads. The specific fuel consumption (SFC) was more for almost all blends at all loads, compared to diesel. At part load, the EGT of MME and its blends were showing similar trend to diesel fuel and at full load, the exhaust gas temperature of MME and blends were higher than diesel. Based on this study, NME could be a substitute for diesel fuel in diesel engine.

Development of enzyme-controlled colonic drug delivery using amylose and hydroxypropyl methylcellulose: optimization by factorial design.

The aim of the present study is to develop colon-targeted drug delivery systems for diclofenac sodium which release the drug specifically and instantly at target site using amylose as a carrier. Coating formulations were designed based on the full factorial design. The evaluated responses were lag time prior to drug release and T90. Compression-coated tablets of diclofenac sodium containing various proportions of amylose and HPMC were prepared. In vitro drug release studies were done by changing pH method with enzyme. In vivo studies were done to confirm the potential of formulation to release the drug at target site. The dissolution data revealed that the ratio of polymers is very important to achieve optimum formulation. Results showed that the tablet prepared according to the above formulation released drug instantly at pH 6.8 (simulating colonic pH). An in vivo study shows that optimized formulation disintegrated in the target region. The results of this study revealed that factorial design is a suitable tool for optimization of coating formulations to achieve colon delivery. It was shown that coating formulation consisting of amylose 285 mg and HPMC 150 mg coating has the potential for colonic delivery of diclofenac sodium irrespective of change in pH in a patient with IBD.

Host-interactive genes in Amerindian Helicobacter pylori diverge from their Old World homologs and mediate inflammatory responses.

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.

Primary CNS lymphoma as second malignancy in a case of carcinoma larynx treated with chemoradiation.

A 66-year-old male was diagnosed as carcinoma larynx in the year April 2004. He was treated with concurrent chemoradiation and remained disease free for three consecutive years. After 3 years he suddenly complained of giddiness and seizures. Magnetic Resonance Imaging of brain showed features of primary CNS lymphoma (PCNSL) which was confirmed by histopathology test. To the best of our knowledge PCNSL as second malignancy in a case of carcinoma of head and neck has not been reported till date. The PCNSL in this patient may have resulted from depressed immunity due to previous radiotherapy. Whatever may be the predisposing cause, this case is most probably the first reported case of PCNSL in a patient of squamous cell carcinoma of larynx.

Growth of carbon nanotubes on nanoporous titania templates.

This work reports the synthesis of multiwalled carbon nanotubes (MWCNTs) on nanoporous titanium dioxide (TiO2) templates. TiO2 nanotubular templates for MWCNTs are produced by anodization of titanium followed by pulsed electrodeposition (PED) of cobalt inside the TiO2 nanotubes. Cobalt acts as a catalyst for the growth of MWCNTs using chemical vapor deposition (CVD) technique. The cobalt catalyst initiates the growth of well graphitized MWCNTs inside the titania pores as well as beyond the titania pores. These materials have been characterized by SEM, EDS, GXRD, XPS, TEM, and ED techniques. The MWCNTs were about 10 mu in length and 80-120 nm in diameter.

Synthesis, characterization and catalytic properties of trivalent iron substituted hexagonal mesoporous aluminophosphates.

Trivalent iron substituted hexagonal mesoporous aluminophosphate molecular sieve has been synthesized and characterized for the first time, which showed excellent catalytic activity and reusability for the cyclohexane oxidation reaction under mild conditions.

Artificial neural network and medicine.

The introduction of human brain functions such as perception and cognition into the computer has been made possible by the use of Artificial Neural Network (ANN). ANN are computer models inspired by the structure and behavior of neurons. Like the brain, ANN can recognize patterns, manage data and most significantly, learn. This learning ability, not seen in other computer models simulating human intelligence, constantly improves its functional accuracy as it keeps on performing. Experience is as important for an ANN as it is for man. It is being increasingly used to supplement and even (may be) replace experts, in medicine. However, there is still scope for improvement in some areas. Its ability to classify and interpret various forms of medical data comes as a helping hand to clinical decision making in both diagnosis and treatment. Treatment planning in medicine, radiotherapy, rehabilitation, etc. is being done using ANN. Morbidity and mortality prediction by ANN in different medical situations can be very helpful for hospital management. ANN has a promising future in fundamental research, medical education and surgical robotics.

Screening for okadaic acid by immunoassay.

Increasing incidences of phytoplankton blooms with the potential danger of toxin release into the food chain have necessitated the search for new diagnostic methods that can detect toxins quickly and reliably. A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantitate okadaic acid in shellfish and phytoplankton extracts. To determine the specificity of the assay, a number of toxins, such as calyculin A, brevetoxin-1, and dinophysistoxins-1, -2, and -3 were analyzed. Both dinophysistoxins-2 and -1 could be detected by the assay but in concentration ranges 10- and 20-fold higher than that for okadaic acid, respectively. Dinophysistoxin-3, calyculin A, or brevetoxin-1 could not be detected with this assay. To validate the accuracy of the method, 18 mussel and 7 phytoplankton extracts were analyzed in parallel for okadaic acid content by ELISA and liquid chromatography combined with either fluorescence or mass spectrometric detection. Very high correlation between the results was found.

Purification and characterization of lutropin receptor from membranes of pig follicular fluid.

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).

Distribution of follitropin and deglycosylated follitropin in the rat: a quantitative and radioautographic study.

125I-labeled ovine follitropin (125I-oFSH) and deglycosylated follitropin (125I-DG-oFSH) were injected into rats and the tissue uptake was quantified and correlated with radioautographic data. Trichloroacetic acid (TCA)-precipitable radioactivity and gel filtration analysis of blood samples indicated no degradation of follitropin or analogue with time. Clearance of follitropin from the circulation was accelerated after its deglycosylation. Disappearance of both molecules from the blood was associated with uptake and/or loss of radioactivity from liver, kidney, ovary and spleen. The more rapid removal of deglycosylated follitropin from blood was associated with higher renal levels of accumulated radioactivity than native follitropin. This was associated with its localization within the cortex, specifically the proximal convoluted tubules of the nephron. Binding of 125I-labeled follitropin and analogue to granulosa cells was specific and time-dependent. 125-I-DG-oFSH demonstrated greater avidity of binding to rat granulosa cells with time than 125I-oFSH. This was associated with slower dissociation kinetics and/or metabolism for 125I-DG-oFSH. The absence of localization of either 125I-follitropin or analogue in hepatic tissue suggests that hepatic mechanisms may not significantly contribute to the clearance of these molecules. Implications of these findings in regard to the metabolism of oFSH and its antagonist are discussed.

On the identity of bovine seminal plasma inhibin.

In light of current discussions on multiple forms of inhibin, it was thought of interest to ascertain the identity of the postulated 'iso-hormones' of bull seminal plasma inhibin (Chari et al., 1978). By subjecting the biologically active fraction, obtained by Sephadex G-100 gel filtration of bull seminal plasma acetone powder, to extensive dialysis in distilled water adjusted to pH 5.8, it was possible to remove the bulk of inert protein as a precipitate. The resulting active preparation could be readily fractionated by preparative iso-electric focusing in the pH range 4.0-6.5 yielding 2 distinct homogeneous peptides, alpha and beta, capable of suppressing hCG-induced uterine weight increase in immature mice, in a 'reversed Steelman-Pohley' assay design. However, of these, alpha alone was able to suppress post-castrational serum gonadotropin rise in appropriate animal models. This peptide is highly acidic in nature (iso-electric point congruent to 2.2) and has a molecular weight (Mr) of 18200 and a Stokes radius of 1.90 nm. On the basis of currently available evidence, it is concluded that the molecule consists of a single peptide chain.