Gastrointestinal transit tolerance, cell surface hydrophobicity, and functional attributes of Lactobacillus Acidophilus strains isolated from Indigenous Dahi.

Strains of Lactobacillus acidophilus WFA1 (KU877440), WFA2 (KU877441), and WFA3 (KU877442) were isolated from indigenous Dahi (yogurt), screened, and selected based on acid and bile tolerance along with the antimicrobial activity. These selected strains were further assessed for their probiotic and functional attributes. Results for simulated gastric and intestinal tolerance/ resistance revealed that all three strains can resist and survive under the following mentioned conditions. To access cell surface hydrophobicity, bacterial adhesion to hydrocarbons (BATH), cellular auto-aggregation, and salt aggregation were performed. In BATH, adhesion of strains against three hydrocarbons namely xylene, dichloromethane, and hexadecane was conducted. The results show that strains showed the least adhesion to xylene (54.25%) as compared to dichloromethane (55.25%) and hexadecane (56.65%). WFA1 showed maximum adherence percentage (55.48%) followed WFA2 (55.48%) and WFA3 (51.38%). Cellular auto-aggregation varied from 21.72% to 30.73% for WFA3 and WFA1, respectively. In the salt aggregation test (SAT), WFA1, WFA2, and WFA3 aggregated at 0.6, 1.0, and 2.0 molar concentrations of ammonium sulfate, respectively. PCR amplification of bile salt hydrolase gene (bsh) was performed and sequences were submitted to the public database of NCBI and Gene bank under accession numbers, KY689139, KY689140, and KY689141. Additionally, a cholesterol-lowering assay was conducted and up to 26% reduction in cholesterol was observed by the strains. Regarding functional properties, exopolysaccharide (EPS) production, and antioxidant potential, strain WFA1 showed promising results EPS (1.027mg/ml), DPPH (80.66%), ABTS (81.97%), and reducing power (1.787). It can be concluded from the present study that the mentioned strains of L. acidophilus (WFA1, WFA2, and WFA3) are strongly hydrophobic; thus having an ability to survive and colonize under the gastrointestinal tract which confirms their probiotic nature. Regarding their functional properties, L. acidophilus WFA1 (KU877440) showed excellent properties of antioxidants and EPS production.

N-glycosylation on Oryza sativa root germin-like protein 1 is conserved but not required for stability or activity.

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251 ± 70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

Formulation and evaluation of antioxidant and antityrosinase activity of Polygonum amplexicaule herbal gel.

Medicinal plants are long been used for pharmaceutical and cosmetic industry. Among medicinal plants, Polygonum amplexicaule of family polygonaceae has traditional use in medicines and skin care. P. amplexicaule belongs to genus Polygonum that contains several important phytochemicals and considered as a rich source of antioxidants. The present study was designed to formulate herbal gel containing P. amplexicaule extract and evaluate its different physical properties as well as antioxidants and antityrosinase activities. Chitosan gel base was used as gelling agent and different gel formulations were prepared by different concentrations of extracts and polymers. Physical properties like pH, colour, odour, appearance and homogeneity, spreadability, extrudability and stability were optimized and analysed. A stable gel formulation containing 1% chitosan gel base and 5% plant extract was prepared that showed good appearance and homogeneity, easily spread ability and excellent extrudability. This gel formulation was tested for antioxidant and skin whitening properties by DPPH free radical scavenging assay and tyrosinase inhibition assay respectively and ascorbic acid was used as reference standard. DPPH scavenging activity with an IC50 value of 0.446 mg/mL and tyrosinase inhibition activity with an IC50 value of 0.805 mg/mL was observed and results indicated that this herbal gel formulation has a good potential for cosmetic use.

Body Mass Index versus Other Adiposity Traits: Best Predictor of Cardiometabolic Risk.

BACKGROUND: A number of anthropometric indices have been used in different world populations as markers to estimate obesity and its related health risks. The present study is large population based study dealing with five anthropometric obesity scales; Body mass index (BMI), waist circumference (WC), waist to hip ratio (WHR), basal adiposity index (BAI), and Visceral adiposity index (VAI) to identify common adiposity trait(s) that best predict obesity and associated health complication(s). METHODS: A total of 4000 subjects including 1000 in each category of BMI from four provinces (Punjab, Sindh, Kahyber pakhtoonkha and Balochistan) of Pakistan from 2012-2017 were collected. Complete anthropometric measurementswere obtained and blood samples were collected and Biochemical profiling was performed. Descriptive statistics, linear regression, binary and multiple regression analysis was done. RESULTS: Our data analysis explored the relationships of obesity five indices; BMI, WC, WHR, BAI, and VAI with common metabolic health complications. Effect size analysis clearly indicates that a unit increase in BMI significant raised all anthropometric and clinical parameters. General and sex specific association analysis of adiposity traits with risk phenotypes (hypertension, hyperglycemia and dyslipidemia) indicated significant associations of WC with all three metabolic risks. Varying degrees of correlations of other adiposity traits with metabolic risks were observed. Frequency of different obesity classes among obese population group were as follows; 55.7% class I, 28.50% Class II and 15.80% Class III. CONCLUSION: WC is the strong predictor of obesity associated metabolic health issues in Pakistani populations. While BMI has significant increasing effect on other obesity indices like WHR, VAI and BAI.

Isolation and antimicrobial susceptibility testing of Helicobacter pylori strains from gastric biopsies from Pakistani patients.

Helicobacter pylori is the etiological agent of gastritis and peptic ulcer. This importance had proposed antibiotics as a principle treatment of gastrointestinal pathologies. The focus of this research was to investigate the occurrence of H. pylori in patients having gastritis or gastric ulcer and also draw the susceptibility profile of isolates to several antibiotics. Blood and biopsy specimen from 96 acid peptic disease patients from both sexes were collected. Each sample was used for culture, gram staining, catalase, oxidase, urease and nitrate reduction test by conventional method. Serology using anti Helicobacter pylori IgG was done. The susceptibility profile to six common antibiotics was checked by E- test method. H. pylori was obtained from 40 patients (41.67%) with greater frequency in male (25%) than females (16.67%). With regards to age, H. pylori was recovered highest from the patients between 51-55 (75.86%) years of age. Tetracycline and rifampin were the most effective antibiotics in vitro, while metronidazole was less effective. Nine (22.5%) strains displayed resistance to at least one antimicrobial drug. Whereas, resistance to amoxicillin, ciprofloxacin, clarithromycin, metronidazole and combination of antibiotics like ciprofloxacin, clarithromycin and metronidazole, and ciprofloxin and metronidazole were 11.11, 55.56, 22.22, 33.33, 11.11 and 44.44% respectively. Lower susceptibility profile of H. pylori to antibiotics is because of frequent use of antibiotics to treat other infections.

Ectopic Expression of Plant RNA Chaperone Offering Multiple Stress Tolerance in E. coli.

Members of the plant glycine-rich RNA-binding proteins (GR-RBPs) family have been reported in flowering, development, circadian rhythms, biotic and abiotic stresses. Particularly, GR-RBPs are reported to function as RNA chaperones, promoting growth and acclimation during cold shock. It is indispensable to further question the efficacy and mechanism of GR-RBPs under various environmental strains. Monitoring the expression of stress-regulated proteins under stress conditions has been a beneficial strategy to study their functional roles. In an effort to elucidate the NtGR-RBP1 function, stress markers such as salinity, drought, low temperature and heat stresses were studied. The NtGR-RBP1 gene was expressed in E. coli followed by the exposure to stress conditions. Recombinant E. coli expressing NtGR-RBP1 were more tolerant to stresses, e.g., salinity, drought, cold and heat shock. Recombinants exhibited higher growth rates compared to control in spot assays. The tolerance was further confirmed by monitoring the growth in liquid culture assays. Cells expressing NtGR-RBP1 under salt (500 mM NaCl), drought (20% PEG), cold (4 and 20 °C) and heat stresses (50 °C) had enhanced growing ability and better endurance. Our study supports the notion that the protective role of NtGR-RBP1 may contribute to growth and survival during diverse environmental stresses.

Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen.

Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.

Cloning and sequence analysis of germin-like protein gene 2 promoter from Oryza sativa L. ssp. indica.

Germin and germin-like proteins (GLPs) are water soluble extracellular proteins reportedly expressed in response to some environmental and developmental signals. Some enzymatic activities have also been associated with germin/GLPs. However, their role in overall metabolism has not been fully understood. Significant insight into their function may also be gained by analysis of their promoter. During this study, about 1107 bp 5'region of OsRGLP2 gene was amplified, cloned and sequenced. The sequence analysis by BLAST showed that this promoter sequence has five common regions (CR1-CR5) of different sizes, which are repeated at 3-6 other locations in 30 kb region in which this gene driven by its promoter is located. Interestingly, all the genes driven by promoter harboring these common regions are GLPs/putative germins. Analysis of these common regions located on OsRGLP2 indicated presence of many elements including those for light responsiveness, dehydration and dark induced senescence, stresses (pathogen and salt), plant growth regulators, pollen specific expression and elements related to seed storage proteins. Analysis of the 30 kb germin/GLP clustered region by GenScan detected each gene to have a putative 40 bp promoter which contains TATA box and Dof factor which turned out to be a part of CR2.

Tobacco nectaries express a novel NADPH oxidase implicated in the defense of floral reproductive tissues against microorganisms.

Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary.

Nectarin IV, a potent endoglucanase inhibitor secreted into the nectar of ornamental tobacco plants. Isolation, cloning, and characterization.

We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii x Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5' and 3' rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a K(i) of 0.35 nm. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before.