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In our previous studies, expression of human interferon α-2b gene in Escherichia coli gave three types of polypeptide chains in nearly equimolar ratio. They include molecules with N-terminal methionine, molecules without N-terminal methionine, and molecules with acetylated N-terminal. This heterogeneity is not required in therapeutic applications. In order to improve the removal rate of N-terminus methionine, co-expression of human interferon α-2b and methionine aminopeptidase genes was performed in E. coli. The co-expression resulted in production of human interferon α-2b with dominance of molecules without N-terminal methionine and very few of them were acetylated at the N-terminus. Our findings contribute significantly to efforts for the production of homogenous therapeutic proteins which represent the exact recombinant replica of their native proteins.

Nano-food packaging, the emergence of latest technology based on nanomaterials, created enormous trust to the synthesis of novel packaging incorporated by zinc oxide nanoparticles (ZnO-NPs) in polylactic acid (PLA) polymer. The morphology and structure of biogenically fabricated ZnO-NPs by aloe barbadensis leaves extract were analyzed by X-ray diffraction, scanning electron microscopy and Fourier transforms infrared spectroscopy. The fabricated ZnO-NPs were hexagonal in shape and 36.5 nm in size. The zinc oxide nanoparticles at concentration of 0.4% (w/w) and 4% (w/w) are incorporated in polylactic acid (PLA) by solution casting method. Loading of zinc oxide nanoparticle in PLA improved stability, film thickness and elongation. The Vitis vinifera fruit packed in the biofilm was observed for appearance, taste and freshness during ambient storage. ZnO-NPs/PLA packed fruit samples retained their original taste and freshness for 15 days in comparison with the reference samples packed in ZnO-NPs/ PLA (4%) at 400C. The biofilm did not affect the fruit Vitis vinifera quality attributes. The decay content, color change, browning index, gases and metal accumulation observations for Vitis vinifera fruit packed in ZnO-NPs/ PLA (4%) exhibited marvellous results. There were no significant differences between the materials tested (p > 0.05). Intercalation of ZnO-NPs was involved in the form of fillers in PLA by solvent casting method for shelf life augmentation of the selected delicate fruit, Vitis vinifera.

The present study aims to enhance the morpho-phsyiological traits, effective utilization of phosphorus and potassium, nutritional quality, and maize productivity using combinations of inorganic and organic amendments in alkaline calcareous soil. The inorganic fertilizers were applied as control (PK0), recommended PK (PKR), and 75% of PKR (PKR75) either alone or in combination with different composts; university compost (UC), NIAB compost (NC) and vermicompost (VC), and organic acids; citric acid (CA) and oxalic acid (OA). The results indicated a significant effect of treatments on morpho-physiological and biochemical attributes of maize during crop growth. Stomatal conductance (gs) and transpiration rate (E) increased up to 93% and 139%, respectively, over control when PKR75 was applied with NC + CA. While, photosynthesis and chlorophyll contents were maximum at PKR75 along with UC + CA. The PKR along with UC + CA increased grain yield (108%) and biological yield (98%) over control. Moreover, the respective treatment increased P content in straw and grain up to 100% and 122% over control (P content: 0.08 and 0.18%), respectively, while 14.3 and 21.2% higher over PKR treatment (0.14 and 0.33%), respectively. The maximum K accumulation in maize grains was noted with PKR combined with NC + CA which was further confirmed with principle component analysis. The results clearly suggest integrated use of mineral PK fertilizers along with composts and organic acids could be an effective strategy to increase P and K supply, and enhance qualitative and quantitative parameters of maize grown on calcareous soil.

Melatonin is important due to its involvement in regulation of diverse mechanisms in plants. Its presence in plants is universal and provides primary defense against environmental stresses. In this study the effect of foliarly applied indole-3-Acetic Acid (IAA) and melatonin (control, 100, 150 µg/g each) on wheat seedling growth under water deficit condition was examined. The mitigation of stress was seen in melatonin treated wheat plants facing abiotic stress, with less accumulation of the H(2)O(2), MDA and anthocyanin. A marked decrease in chlorophyll, total soluble proteins, total soluble sugars, ascorbic acid, phenolic contents and yield- related attributes was noticed in stressed condition. Treatment with melatonin and IAA alleviated stress induced decrease in biochemical attributes, and growth of wheat plants in a dose-dependent manner. A significant increase in yield was achieved by melatonin treatments in Ujala-2016 under limited water supply. It is worthy to mention that melatonin spray at 150 µg/g followed by IAA proved to be the most pronounced treatment in the buildup of osmolytes and regulation of antioxidant defense system with increase in yield under water limited environment.

Nickel (Ni) element is strongly phytotoxic at high concentrations for several plants, but due to its dual behavior and complicated chemistry, it has received little attention in plant nutrition and relevant experimental data are limited. The current research was carried out to study the effect of Ni on maize (Zea mays L.) growth and phytoextraction potential with EDTA assistance, a process termed as chemical assisted phytoremediation. Treatments included two levels of EDTA (0 and 0.5 mM), two levels of Ni (0 and 40 µM) and their combination (EDTA+Ni) that were applied to maize plants grown in a pot experiment. Application of Ni alone or in combination with EDTA reduced maize root and shoot length by 7.8% to 13.3% and by 15.6% to 21.1%, respectively, compared with control, as well as root and shoot dry weight by 42.0% to 60.0% and by 29.8% to 46.6%, respectively. A similar declining trend was observed also for the content of photosynthetic pigments (chl-a, chl-b, total chlorophyll, and carotenoids) as well as total proteins. However, proline, total soluble sugars, and free amino acids showed an increasing trend with application of Ni and EDTA alone or in combination. These treatments significantly decreased P and Na content in maize roots, stems, leaves, and grains, while increased K content compared with control. Application of EDTA with Ni was the most effective treatment to enhance Ni accumulation in maize (50.23 mg per plant) compared with Ni alone (40.62 mg per plant), EDTA alone (27.75 mg per plant), and control (15.51 mg per plant). Application of EDTA in combination with Ni enhanced Ni accumulation by 4.9 folds in maize shoots and by 2.6 folds in roots over control. In conclusion, application of EDTA in suitable concentrations may enhance Ni uptake by maize providing an effective and economic phytoremediation method of Ni-contaminated soils.

Objective: To profile the secondary metabolites and to evaluate the antidiabetic potential of hydroethanolic leaf extracts of Conocarpus lancifolius. Methods: The various hydroethanolic extracts of Conocarpus lancifolius leaf were prepared by ultrasonication assisted freeze- drying. Total phenolic contents, flavonoid contents, antioxidant activity, α-glucosidase and α-amylase inhibitions of leaf extracts were determined. The metabolite profiling was accomplished by UHPLC-Q-TOF-MS/MS analysis. The antidiabetic assessment of the most potent extract was carried out by measuring the hypoglycemic and hypolipidemic effect in the high fat diet-fed diabetic albino mice. The blood glucose level, haemoglobin, total cholesterol, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were determined. Results: The 60% ethanolic extract exhibited the highest phenolic and flavonoid contents of (349.39 ± 2.13) mg GAE/g dry extract and (116.95 ± 2.34) mg RE/g dry extracts, respectively, and the highest DPPH scavenging activity with an IC50 value of (32.87 ± 1.11) μg/ mL. The IC50 values for α-glucosidase and α-amylase inhibitions were (38.64 ± 0.93) μg/mL and (44.80 ± 1.57) μg/mL, respectively. UHPLC-Q-TOF-MS/MS analysis confirmed the presence of gallic acid, ellagic acid, corilagin, kaempherol-3-O-rutinoside, caffeic acid derivative, isorhamnetin and galloyl derivatives in the 60% ethanolic extract. Plant extract at a dose of 450 mg/kg body weight reduced blood glucose level, total cholesterol, LDL and HDL, and increased haemoglobin in alloxan-induced diabetic mice, Conclusions: Conocarpus lancifolius leaves are proved as a good source of biologically functional metabolites and possess antidiabetic activity which may be further explored to treat diabetes.

Meat is a rich source of energy that provides high-value animal protein, fats, vitamins, minerals and trace amounts of carbohydrates. Globally, different types of meats are consumed to fulfill nutritional requirements. However, the increasing burden on the livestock industry has triggered the mixing of high-price meat species with low-quality/-price meat. This work aimed to differentiate different meat samples on the basis of metabolites. The metabolic difference between various meat samples was investigated through Nuclear Magnetic Resonance spectroscopy coupled with multivariate data analysis approaches like principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA). In total, 37 metabolites were identified in the gluteal muscle tissues of cow, goat, donkey and chicken using 1H-NMR spectroscopy. PCA was found unable to completely differentiate between meat types, whereas OPLS-DA showed an apparent separation and successfully differentiated samples from all four types of meat. Lactate, creatine, choline, acetate, leucine, isoleucine, valine, formate, carnitine, glutamate, 3-hydroxybutyrate and α-mannose were found as the major discriminating metabolites between white (chicken) and red meat (chevon, beef and donkey). However, inosine, lactate, uracil, carnosine, format, pyruvate, carnitine, creatine and acetate were found responsible for differentiating chevon, beef and donkey meat. The relative quantification of differentiating metabolites was performed using one-way ANOVA and Tukey test. Our results showed that NMR-based metabolomics is a powerful tool for the identification of novel signatures (potential biomarkers) to characterize meats from different sources and could potentially be used for quality control purposes in order to differentiate different meat types.

Mixing of phosphate fertilizer with farmyard manure (FYM) is a simple technique for optimizing phosphorus (P) availability and then improving the productivity of wheat (Triticum aestivum) grown in alkaline calcareous soils. Diammonium phosphate (DAP) and phosphoric acid (PA) were applied to soil at 36 mg P kg−1, either as sole or after amending 1-g P fertilizer with 2-g FYM (1:2, w/w basis). After 45-day incubation, concentration of P ions in the soil solution (Cp) and exchangeable P present in soil solid (E-value) were determined to evaluate the amount of total plant-available pool. The FYM-amended fertilizers, i.e., PA+FYM and DAP+FYM, showed higher E-values, i.e., 114 and 97 mg kg−1 soil, respectively. Similarly, PA+FYM exhibited the highest proportion of P derived from fertilizer (Pdff = 51.5%) and induced the highest P uptake by wheat seedlings (L-value = 72.1 mg kg−1). Consequently, PA+FYM and DAP+FYM treatments caused higher grain yield and P-use efficiency. The regression analysis revealed strong and positive correlation between L-value and grain yield (r = 0.86), biomass production (r = 0.84) and P-use efficiency (r = 0.87) by wheat crop. Results suggested that FYM-amended inorganic P fertilizer can be a promising technique to optimize supply of P from soil, improve efficiency of inorganic P fertilizers, and improve wheat yield in alkaline calcareous soils.

The supplementation of rumen bypass fat (RBF) has remained one of the preferred approaches used to decrease undesirable saturated fatty acids (FA) and increase beneficial unsaturated FA in the meat. This study was planned to evaluate the influences of rumen bypass fats on meat quality, fatty acid and metabolic profiles in male Dorper sheep (n = 36) with 24.66 ± 0.76 kg (mean ± standard error) initial body weight. Treatment comprised a basal diet (30:70 rice straw to concentrate) with no added RBF as a control (CON), basal diet with prilled fat (PF), basal diet with prilled fat plus lecithin (PFL) and basal diet with calcium soap of palm fatty acids (CaS). The findings revealed that cooking loss, drip loss and shear force in longissimus dorsi (LD) muscle were not affected by RBF supplementation, while meat pH was significantly higher in the CaS on aging day 1. However, the diet supplemented with prilled fat and lecithin modified the meat’s fatty acid profile significantly by increasing unsaturated fatty acids and decreasing saturated fats. The relative quantification of the major differentiating metabolites found in LD muscle of sheep showed that total cholesterol, esterified cholesterol, choline, glycerophosphocholine and glycerophospholipids were significantly lower in CaS and PFL diets, while glycerol and sphingomyelin were significantly higher in CaS and PFL diets. Most of the metabolites in the liver did not show any significant difference. Based on our results, the supplementation of protected fats did not have a negative influence on meat quality and the meat from Dorper sheep fed prilled fat with lecithin contained more healthy fatty acids compared to other diets.

Southern Lower Indus basin is an oil and gas prone basin, which is important for producing hydrocarbon in Pakistan. Due to limited Rock-Eval pyrolysis data availability in the prospective shale gas basin, the organic facies geochemical characteristics study is a challenging task for researchers. The primary objective of this study is an evaluation of the Lower Goru Formation organic facies hydrocarbon potential by geochemical data and its subsurface facies distribution study by well tops and seismic data. The study area is situated at the Southern Lower Indus basin of Pakistan that is considering the prospective shale gas basin with immense unconventional hydrocarbon potential. In the present study, Rock-Eval pyrolysis data analysis along with the integration of both seismic and well tops data were conducted for investigation of source rock generation potential of organic facies variability within the Lower Goru Formation. The geochemical approaches used in the current study show that the Lower Goru Formation encountered in, bears fair to excellent total organic carbon (TOC) content values of 0.50–3.60% in Well-A and 0.10–4.69% in Well-B. Additionally, the results of Rock-Eval pyrolysis data show that interbedded shales of the Lower Goru Formation contain different types of kerogen (III, II–III, and II) with variable generation potential of oil and gas.

Background Many studies shed light on the therapeutic importance of Azadirachta indica leaves extract as a source of drugs and neutracuticles for treatment of different diseases including diabetes mellitus. The extraction of these bioactive phytochemicals is optimized according to different pharmacological applications of extract. Study design The current study was performed to optimize the extraction from leaves of Azadirachta indica. The temperature, extraction time, solid to liquid ratio, ethanol concentration and ultrasonication frequency were optimized to improve extract yield with improved antioxidant and α-glucosidase inhibitory properties. Methods Utrasonication assisted extraction from Azadirachta indica leaves was optimized using Response Surface Methodology. Sets of extraction conditions that gave optimum yield, antioxidant activity and α-glucosidase inhibition were determined through statistical analysis. The phytochemicals present in extract were identified by 1H NMR and docked with homology modelled α-glucosidase structure to get and insight of enzyme-phytochemicals interaction. Results The maximum extract yield and α-glucosidase inhibition were observed when plant sample was extracted with 60% ethanol (15% solid-liquid ratio), and ultrasonication was done at 35 °C for 75 min having frequency of 30 KHz. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging observed the same extraction strategy except the time duration for ultasonication which was 52.5 min. The 1H NMR based metabolite profiling revealed the identification of 2α,4α,dihydroxy-pregn-5-en-(6-one-3α-O-D-glu-copyranoside, ferruginol, azadirachtin A, meliatetraolenone, odoratone, 7α‑hydroxy-15-β‑hydroxy-7′15,dioxonimbin, zeeshanol, nimbinene, epoxyazadiradione, azadirachtin B, 17-(5‑methoxy-2-oxo-furan-3-yl)-28-deoxonimbolide, epicatechin, 2-oxo-3-deacetyl salanin (nimbolide derivative), nimonol, nimbic acid B, 3‑methoxy-cumerine 6–0-(1,4) triglycoside, 17‑hydroxy-sandaracopimar-8,15-en-11-one(diterpene), and 23-O-methylnimocinolide. The molecular docking studies provided a deep insight on molecular interactions which might be useful for antidiabetic drug development. Conclusion Azadirachta indica leaves extract contain various bioactive compounds and can be considered as a source of α-glucosidase inhibitors hence, it may also be used for the development of functional foods.

Pepsin enzyme was used to pretreat the bovine skin at the rate of 5, 15, and 25 units of enzyme/g of skin to recover gelatin, and the recovered gelatins were referred to as Pe5, Pe15, and Pe25, respectively. The gelatin yield increased significantly (p &lt, 0.05) from 18.17% for Pe5 to 24.67% for Pe25 as the level of pepsin increased, but the corresponding gel strength and viscosity decreased significantly (p &lt, 0.05) from 215.49 to 56.06 g and 9.17 to 8.17 mPa.s for Pe5 and Pe25, respectively. β- and α1- and α2-chains were degraded entirely in all the gelatins samples as observed in protein pattern elaborated by gel electrophoresis. 1H nuclear magnetic resonance (1H NMR) analysis indicated the coiled structure of gelatin protein chains. The lowest amide III amplitude of Pe25 as found by Fourier transform infrared (FTIR) spectroscopy indicated that α-helix structure of protein chains were lost to more irregular coiled structure. Thus, it could be summarized that pepsin might be used at the lower level (5 units/g of wet skin) to extract gelatin from bovine skin with good functional properties and at higher level (15/25 units/g of wet skin) to obtain gelatin of industrial grade with high yield.

Propolis is a highly adhesive and resinous product of honey bee (Apis mellifera L.) which is produced from the exudations of plants. Bee propolis being a source of bioactive compounds like polyphenols and flavonoids imparts numerous biological properties including, antioxidant, anti-inflammatory, antimicrobial and anticancer activities. Present study was designed to elucidate the composition and antioxidant status of locally available propolis using in-vitro conditions. Propolis collected from locally found apiaries and its hydroalcoholic extract of propolis was prepared using different concentrations of ethanol and methanol. The results regarding proximate composition of propolis showed a higher proportion of ether extract (85.59±0.87%) and lowest contents of crude fiber (0.31±0.08%). Among the mineral's sodium, potassium and calcium was found in a concentration of 11.33±0.91, 52.10±2.9 and 10.53±0.83.59±0.23mg/Kg respectively whilst zinc was noticed as 3.59±0.23mg/Kg. HPLC characterization indicates a highest concentration of Chlorogenic acid 31.80±2.56mg/Kg whereas gallic acid (0.21±0.01mg/Kg) was found in lowest concentration among the polyphenols. Ethanol extract represents more phenolic contents, DPPH activity and antioxidant status as 327.30±14.89mg/gGAE, 73.18±4.43% and 60.59±4.38% accordingly in comparison to methanol and water extract. Bee propolis found an effective source of natural antioxidants which retards the production of free radicals and reactive oxygen species thus help to cope oxidative stress.

Human diets with functional ingredients showed promising role in management of diseases of modern age like hyperglycemia and hyperlipidemia and even cancer. The study designed to elucidate role of honeybee propolis for management of hyperglycemia and hyperlipidemia states through animal modeling system. Hydroalcoholic extract of propolis was used for development of functional drink with standard recipe and addition of specified dose of extracts (400mg/500mL). Animals were grouped into three studies including study-I fed on regular diet, study-II fed on sucrose enrich diet and study-III fed on diet enriched with cholesterol and monitored to evaluate the results. Various parameters like feed consumption, liquid intake of animals measured regularly whereas body weight recorded at the end of each week of study. At the end of the study animals were analyzed for different blood indicators like blood lipid indices (cholesterol, LDL, HDL concentration and triglyceride contents)), glucose concentration and insulin contents as well. The maximum feed and drink intake were examined in animals, fed with control diet whereas a non substantial mode of intake was recorded in rest of two groups of animals. The consumption of honeybee propolis based drink reduced cholesterol (6.63% to 10.25%) and LDL (9.96% to 11.23%), whilst a sharp increase in HDL level was ranged as 4.12 to 4.49% among animal groups fed with high cholesterol and high sucrose diet. Blood glucose level was decreased by 10.25% and 6.98% however 6.99% and 4.51% increase were observed in plasma insulin level in both studies, study-II and study-III correspondingly. The overall findings of the study showed that drinks prepared using propolis of propolis found effective for management of hyperglycemia and hypercholesterolemia in present animal modelling system.

Poly(lactic-co-glycolic acid) (PLGA) is the most commonly described biocompatible copolymer used in biomedical applications. In this work, a green synthetic approach based on the biocompatible zinc proline complex, as an initiator for PLGA synthesis, is reported for the first time for the synthesis of methoxy-poly(ethylene glycol)-block-poly(l-lactic-co-glycolic acid) (mPEG-PLGA). mPEG-PLGA with controlled molecular weight and narrow polydispersity was synthesised. Its potential for delivery of irinotecan (Ir), a poorly water-soluble chemotherapeutic drug used for the treatment of colon and pancreatic cancer, was studied. Nanoparticles of controlled size (140-160 nm), surface charge (∼-10 mV), release properties and cytotoxicity against CT-26 (colon) and BxPC-3 (pancreatic) cancer cells, were prepared. Tumor accumulation was confirmed by optical imaging of fluorescently labelled nanoparticles. Unlike Tween® 80 coated NP-Ir, the Pluronic® F-127 coated NP-Ir exhibits significant tumor growth delay compared to untreated and blank formulation treated groups in the CT-26 subcutaneous tumor model, after 4 treatments of 30 mg irinotecan per kg dose. Overall, this proof-of-concept study demonstrates that the newly synthesized copolymer, via a green route, is proven to be nontoxic, requires fewer purification steps and has potential applications in drug delivery.

Sodium aescinate (SA) is a vital salt of sodium escin from Aesculus wilsonii Rehd seeds. SA injection (SAI) has received great success in treating cerebral edema, venous reflux disease and other inflammatory conditions. Recently, high incidences of immediate hypersensitivity reactions were reported after SA infusion, which raised questions on safety and risk associated with its clinical application. This study was designed to check whether SAI and its four components induce degranulation using RBL-2H3 mast cells. For this purpose, we evaluated different treatment levels of SAI (20, 40, 60, 80 and 100 μg ml−1) and its four characteristic components, SA-A, SA-B, SA-C and SA-D, at 60 μg ml−1 in different tests including cell viability test, β-hexosaminidase and histamine assays, oxidative stress indices, apoptosis analysis and intracellular calcium ions in RBL-2H3 cells. Our results demonstrated that SAI at 80 μg ml−1 and 100 μg ml−1, and its two components (SA-B and SA-D) at 60 μg ml−1 were responsible for disturbing cell morphology and cell viability, elevated levels of β-hexosaminidase, histamine, modulation of oxidative stress indices, induced apoptosis and increase in intracellular calcium ions in RBL-2H3 cells, when compared with the control. Our results demonstrated for the first time that SAI was more likely to induce immediate hypersensitivity reactions attributable to degranulation via oxidative stress caused by SA-B and SA-D components. These results would not only be useful for the safety of end user but also for the industry to improve the quality of SA infusion.

Butea monosperma is one of the extensively used plants in traditional system of medicines for many therapeutic purposes. In this study, the antioxidant activity, α-glucosidase and α-amylase inhibition properties of freeze drying assisted ultrasonicated leaf extracts (hydro-ethanolic) of B. monosperma have been investigated. The findings revealed that 60% ethanolic fraction exhibited high phenolic contents, total flavonoid contents, highest antioxidant activity, and promising α-glucosidase and α-amylase inhibitions. The UHPLC-QTOF-MS/MS analysis indicated the presence of notable metabolites of significant medicinal potential including apigenin, apigenin C-hexoside C-pentoside, apigenin C-hexoside C-hexoside, apigenin-6,8-di-C-pentoside and genistin etc., in B. monosperma leave extract. Docking studies were carried out to determine the possible role of each phytochemical present in leaf extract. Binding affinity data and interaction pattern of all the possible phytochemicals in leaf extract of B. monosperma revealed that they can inhibit α-amylase and α-glucosidase synergistically to prevent hyperglycemia.

Acute lung injury (ALI) is considered as an uncontrolled inflammatory response that can leads to acute respiratory distress syndrome (ARDS), which limits the therapeutic strategies. Ginsenosides Rb1 (Rb1), an active ingredient obtained from Panax ginseng, possesses a broad range of pharmacological and medicinal properties, comprising the anti-inflammatory, anti-oxidant, and anti-tumor activities. Therefore, the purpose of the present study was to investigate the protective effects of Rb1 against S. aureus-induced (ALI) through regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) and mitochondrial-mediated apoptotic pathways in mice (in-vivo), and RAW264.7 cells (in-vitro). For that purpose, forty Kunming mice were randomly assigned into four treatment groups; (1) Control group (phosphate buffer saline (PBS); (2) S. aureus group; (3) S. aureus + Rb1 (20 mg/kg) group; and (4) Rb1 (20 mg/kg) group. The 20 μg/mL dose of Rb1 was used in RAW264.7 cells. In the present study, we found that Rb1 treatment reduced ALI-induced oxidative stress via suppressing the accumulation of malondialdehyde (MDA) and myeloperoxidase (MPO) and increase the antioxidant enzyme activities of superoxidase dismutase 1 (SOD1), Catalase (CAT), and glutathione peroxidase 1 (Gpx1). Similarly, Rb1 markedly increased messenger RNA (mRNA) expression of antioxidant genes (SOD1, CAT and Gpx1) in comparison with ALI group. The histopathological results showed that Rb1 treatment ameliorated ALI-induced hemorrhages, hyperemia, perivascular edema and neutrophilic infiltration in the lungs of mice. Furthermore, Rb1 enhanced the antioxidant defense system through activating the Nrf2 signaling pathway. Our findings showed that Rb1 treated group significantly up-regulated mRNA and protein expression of Nrf2 and its downstream associated genes down-regulated by ALI in vivo and in vitro. Moreover, ALI significantly increased the both mRNA and protein expression of mitochondrial-apoptosis-related genes (Bax, caspase-3, caspase-9, cytochrome c and p53), while decreased the Bcl-2. In addition, Rb1 therapy significantly reversed the mRNA and protein expression of these mitochondrial-apoptosis-related genes, as compared to the ALI group in vivo and in vitro. Taken together, Rb1 alleviates ALI-induced oxidative injury and apoptosis by modulating the Nrf2 and mitochondrial signaling pathways in the lungs of mice.

The prime objective of the current study was to investigate the occurrence of mycotoxin patulin (PAT) in apples, grapes and their value added products. PAT was determined by a validated method based on HPLC with UV detector. A total of 381 samples comprising apple and grape fruits (n = 133 each), apple-based products (n = 76, juice, puree, jam) and grape juice (n = 39) were analyzed. PAT was found in 58.9% samples of apple and apple-based products, with a mean of 49.8 &micro, g/kg (maximum 396 &micro, g/kg), while 27.3% samples contained PAT beyond the maximum regulatory limit of 50 &micro, g/kg. The average levels of PAT contamination in apple-derived products was higher in apple juice concentrate, followed by apple puree, apple juice and apple jam. The incidence of PAT in table grapes was 65.1%, with a mean of 53.9 &micro, g/kg (maximum 505 &micro, g/kg), whereas 23.8% exceeded the maximum level. Among the fruit samples, there were differences in PAT contents due to apple variety (6 types) or grape variety (8 types), as well as for sampling location. Our investigations showed the wide PAT occurrence in fruits and derived value-added products affecting consumer product safety, so that the population is chronically exposed to this toxin.

Our previous studies, using precursors for two classes of estrogens, estrone and estriol, have highlighted the following facets of aromatase. The overall reaction, converting androgens into estrogens, occurs in three steps, each requiring NADPH and O2. In Step 1, a 19-hydroxy intermediate is produced, which in Step 2, is converted into a 19-oxo derivative via a gem -diol intermediate with the stereospecific loss of HRe. In Step 3, a scission of the C-10-C-19 bond occurs releasing C-19 as formic acid (HCOOH) and incorporating an atom of oxygen from O2, The other oxygen atom of formic acid is derived from the hydroxyl group introduced in Step 1. These experiments were performed using the classical placental microsomal system. Our findings were confirmed and extended by (the late) Caspi’s group. However, incorporation of oxygen in Step 3, has been challenged in a subsequent study using a soluble reconstituted system. The latter authors have implied the superiority of their system over the microsomal preparation. However, several assumptions under pinning their own work were derived from the use of placental microsomes. Furthermore, the authors have not considered that when a previous work is challenged it needs to be repeated under the conditions described in the original publication.

The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100 °C and pH of 5.5. The recombinant enzyme followed Michaelis–Menten kinetics exhibiting apparent Vmax and Km values of 20 ± 0.5 μmol min−1 mg−1 and 140 ± 10 µM, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 ± 0.5 kJ mol−1. A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8 M urea or 5 M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200 min at 100 °C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date.

Hederacoside-C (HDC) is a biological active ingredient, extracted from the leaves of Hedera helix. It has been reported to have anti-inflammatory properties. However, the effects of HDC on Staphylococcus aureus (S. aureus)-induced mastitis have not been reported yet. Here, we evaluated the anti-inflammatory effects of HDC on S. aureus-induced mastitis both in vivo on mammary gland tissues and in vitro on RAW 264.7 cells. The ascertained histopathological changes and MPO activity revealed that HDC defended mammary glands from tissue destruction and inflammatory cell infiltration induced by S. aureus. The results of ELISA, western blot, and qRT-PCR indicated that HDC significantly inhibited the expressions IL-6, IL-1β, and TNF-α and enhanced the IL-10 by downregulating and upregulating their relevant genes, respectively. Furthermore, HDC markedly suppressed the TLR2 and TLR4 expressions by attenuating the MAPKs (p38, ERK, JNK) and NF-κB (p65 and IκBα) pathways followed by decreasing the phosphorylation of p38, ERK, JNK, p65, and IκBα. The above parameters enhanced the mammary gland defense and reduced inflammation. These findings suggested that HDC may have the potential to be an effective anti-inflammatory drug for the S. aureus-induced mice mastitis and in RAW 264.7 cells.

Biaryls are the potential source of synthetic drugs. The present study describes the synthesis of a series of functionalized biphenyl derivatives (3a-3g) using Pd-catalyzed Suzuki coupling reaction. The experimental results revealed the facile synthesis of biphenyl derivatives (3a-3g) with notably high yield (80-88%). Density functional theory (DFT) studies were performed by Gaussian 09 software in order to rationalize the selectivity of coupling at C-Br bond instead of C-Cl bond. In addition of synthesis, the biological activities (biofilm inhibition, hemolytic and anti-thrombolytic) of these novel compounds were investigated. These results exhibited good biofilm inhibition (5.86-65.8%), hemolytic (1.32-30.1%) and anti-thrombolytic activities (9.64-42.5%), indicating the potential use of these compounds for pharmaceutical applications

The antiphoto aging property of Zanthoxylum rhetsa obtained from Pangkor Island, Malaysia, was evaluated. Solvent fractions of different polarity obtained from the methanolic extract of the bark material were initially tested for anticollagenase and antielastase activities. The ethyl acetate fraction showed bioactivity against the protease enzymes. Hence, it was subjected to further purification via column chromatography, to yield a major constituent, hesperidin. Subsequently, the ethyl acetate fraction and hesperidin were tested for their effects against UVB-induced cytotoxicity and expressions of inflammatory cytokines (IL-6, IL-1β, and TNF-α), NF-κB, and MMPs (MMP1, 3, and 9) in human dermal fibroblasts (HDF). Both fraction and pure compound prevented UVB-induced cytotoxicity in HDF cells, in a dose dependent manner. Moreover, the ethyl acetate fraction inhibited the increase of pro-inflammatory cytokines induced by UVB to a level similar to the control (without UV treatment). Additionally, the fraction significantly inhibited the expressions of NF-κB, MMP 1, MMP 3, and MMP 9 in HDF cells treated with UVB. Similar effects were observed with hesperidin. The results obtained suggested that the ethyl acetate fraction of Z. rhetsa and its bioactive constituent, hesperidin, have the potential to be used as active ingredients in sunscreen and antiphoto aging formulations.

Clitorea ternatea has been used in Ayurvedic medicine as a brain stimulant to treat mental illnesses and mental functional disorders. In this study, the metabolite profiles of crude C. ternatea root extract (CTRE), ethyl acetate (EA), and 50% aqueous methanol (50% MeOH) fractions were investigated using ultrahigh-performance liquid chromatography–diode array detector–tandem mass spectrometry (UHPLC–DAD–MS/MS), while their effect on the stress-like behavior of zebrafish, pharmacologically induced with reserpine, was investigated. A total of 32 compounds were putatively identified, among which, a series of norneolignans, clitorienolactones, and various flavonoids (flavone, flavonol, isoflavone, and isoflavanone) was found to comprise the major constituents, particularly in the EA and 50% MeOH fractions. The clitorienolactones, presently unique to the species, were present in both the free and glycosylated forms in the roots. Both the EA and 50% MeOH fractions displayed moderate effects on the stress-induced zebrafish model, significantly decreasing freezing duration and elevating the total distance travelled and average velocity, 72 h post-treatment. The results of the present study provide further evidence that the basis for the use of C. ternatea roots in traditional medicine to alleviate brain-related conditions, such as stress and depression, is attributable to the presence of clitorienolactones and the isoflavonoidal constituents.

The present work was aimed at investigating hydroethanolic leaf extracts of Cassia fistula for their antioxidant and pancreatic lipase (PL) enzyme inhibitory properties. The most active extract was selected to profile the phytoconstituents by UHPLC-QTOF-MS/MS technique. Among the tested extracts, the 80% hydroethanolic extract exhibited the maximum levels of total phenolic and flavonoid contents (TPC and TFC) with a contribution of 201.3 ± 2.6 mg of gallic acid equivalent per gram of extract (GAE/g extract), and 116.3 ± 2.4 mg of rutin equivalent per gram of extract (RE/g extract), respectively. The same extract also showed promising 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and PL inhibitory activity with an IC50 (half maximal inhibitory concentration) of 30.5 ± 2.8 µg/mL and 17.31 ± 1.18 μg/mL, respectively. The phytochemical profiling of 80% hydroethanolic extract confirmed the presence of 23 metabolites of immense medicinal significance. Docking studies were conducted to investigate the potential interactions of compounds identified in the study. The docking study-based binding energy data and the interaction scheme both revealed the possible role of the identified compounds towards PL inhibitor. Moreover, energies of frontier molecular orbitals (FMOs), ionization potentials (IP), electron affinities (EA) and molecular electrostatic potentials (MEP) were also explored. The findings of the current work suggest that C. fistula is a promising natural source of antioxidant and antiobesity agents, which may be exploited to add pharmacological functionalities to food.

Pneumonia is the acute inflammation of lung tissue and is multi-factorial in etiology. Staphylococcus aureus (S. aureus) is a harmful pathogen present as a normal flora of skin and nares of dairy cattle. In bovine pneumonia, S. aureus triggers to activates Toll-Like Receptors (TLRs), that further elicits the activation of the inflammation via NF-κB pathway, oxidative stress and apoptotic pathways. In the current study, pathogen-associated gene expression of the pro-inflammatory cytokines, oxidative stress and apoptotic markers in the lung tissue of cattle was explored in bovine pneumonia. Fifty lung samples collected from abattoir located in Wuhan city, Hubei, China. Histopathologically, thickening of alveolar wall, accumulation of inflammatory cells and neutrophils in perivascular space, hyperemia, hemorrhages and edema were observed in infected lungs as compared to non-infected lung samples. Furthermore, molecular identification and characterization were carried by amplification of S. aureus-specific nuc gene (270 base pairs) from the infected and non-infected lung samples to identify the S. aureus. Moreover, qPCR results displayed that relative mRNA levels of TLR2, TLR4, pro-inflammatory gene (IL-1β, IL-6 and TNF-α) and apoptosis-associated genes (Bax, caspase-3 and caspase-9) were up-regulated except Bcl-2, which is antiapoptotic in nature, and oxidative stress related genes (Nrf2, NQO1, HO-1 and GCLC) which was down-regulated in infected pulmonary group. The relative protein expression of NF-κB, mitochondria-mediated apoptosis gene was up-regulated while Bcl-2 and Nrf2 pathway genes were downregulated in infected cattle lungs. Our findings revealed that genes expression levels of inflammatory mediators, oxidative stress and apoptosis were associated with host immunogenic regulatory mechanisms in the lung tissue during infection. Conclusively, the present study provides insights of active immune response via TLRs-mediated inflammatory, oxidative damage, and apoptotic paradox.

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

Eight proinsulin encoding genes were prepared and their translation products, when treated with a cocktail of trypsin and carboxypeptidase B, analyzed for the following features. One, their ability to undergo facile removal of the N-terminal linker, generating the phenylalanine residue destined to be the N-terminal of the B-chain of insulin, at a rate similar to that involved in the removal of the C-peptide. Two, processing of diarginyl insulin, produced in the latter process, by carboxypeptidase B then needed to be rapid to remove the two arginine residues, Three, both these operations were to be efficient whether the N-terminal methionine was acylated or not. Four, the proinsulin constructs needed to contain a minimum number of sites for acylation. The aforementioned features were monitored by mass spectrometry and the proinsulin derivative containing MRR at the N-terminal and K64 mutated to Q64, designated as MRR-(Q64) human proinsulin [MRR-(Q64) hpi] optimally fulfilled these requirements. The derivative was smoothly acylated with reagents of two chain lengths (acetyl and dodecanoyl) to give acetyl/dodecanoyl MRR-(Q64) hpi. Acetyl MRR-(Q64) hpi, using the cocktail of the two enzymes, was smoothly converted into, acetyl insulin. However, when dodecanoyl MRR-(Q64) hpi was processed with the above cocktail, carboxypeptidase B (whether from animal pancreas or recombinant) showed an unexpected specificity of acting on the K29-T30 bond of the insulin derivatives when K29 contained a large hydrophobic acyl group, generating dodecanoyl des-30 insulin.

Evaluation of shale gas hydrocarbon potential of Talhar Shale member of Lower Goru Formation, by using well logs data only, has been a challenging task in Southern Lower Indus Basin Pakistan. This paper is helpful to evaluate shale gas potential of source rock by using well log data. We have introduced and applied Empirical and Graphical method to resolve this task and have derived geochemical parameters from well logs data. The way mentioned is fastand the cheapest. Talhar Shale has kerogen typeIII and type II which has potential to produce, mainly, oil and gas. Talhar Shale is of Montmorillonite clay type. So, Talhar Shale has better sorption property. Empirical formulas are used to derive parameters, using well logs of Porosity, Density and Uranium. Porosity and Volume of Kerogen, calculated from density log, gives average values of 11.8% and 11.4%. Level of Maturity Index (LMI), derived by log, average value is 0.54 which indicates that it is on early stage of maturity. Vitrinite reflectance is between 0.5-0.55% as calculated by graphical method and empirical formula. Talhar Shale with main products of oil and gasis onset of oil generation,;could be a good potential source in the study area.

Genome search of Geobacillus thermopakistaniensis, formerly Geobacillus sp. SBS-4S, revealed the presence of an open reading frame (ESU71923) annotated as laccase. However, the gene product did not display any laccase-like activity against the substrates examined. The laccase activity was, therefore, purified from G. thermopakistaniensis cells and N-terminal amino acid residues of the enzyme were determined. These residues matched the N-terminal sequence of an open reading frame annotated as a copper oxidase (ESU72270). In order to characterize the enzyme, recombinant ESU72270 was prepared in Escherichia coli. The recombinant protein was found to exhibit a negligible amount of laccase activity when produced in the absence of copper in the growth medium. However, the recombinant protein exhibited significantly high laccase activity when produced in the presence of copper. The recombinant enzyme showed highest activity at 60 °C and a pH of 7-7.5. The purified enzyme was highly tolerant to various halides and organic solvents, thus having a potential for various industrial applications. To the best of our knowledge, this is the first characterization of a laccase from genus Geobacillus which identifies a gene responsible for functional laccase in this genus.

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

The Choice of new rice varieties by farmers and consumers at the expense of indigenous varieties have become a source of panic to scientists in Pakistan. In the present research the cooking quality and physico-chemical characteristics of 14 newly developed lines and two check varieties widely grown in Punjab, Pakistan were investigated. Significant variation (P

Sunburn, premature skin aging, skin cancers and suppression of the immune system are linked to exposure of the skin to UV light. In recent years, plant extracts are becoming a popular active ingredient in natural sunscreen formulations. In the present study, the ethyl acetate fraction of Zanthoxylum rhetsa bark (commonly called as Indian prickly Ash) was used as an active ingredient in two sunscreen cream formulations (F1 and F2). Primarily, the constituents present in the active fraction were identified using LC–MS/MS analysis. Coumaric acid, benzoic acid, p-hydroxybenzoic acid and its isomers, hesperitin, trihydroxyoctadecenoic acid and columbamine were identified in the ethyl acetate fraction of Z. rhetsa bark extract. The UV protection properties of the formulated creams were evaluated by assessment of parameters such as their SPF values (F1: 3.60 ± 0.28, F2: 6.90 ± 0.57), UVA effectiveness (moderate for both test formulations) and critical wavelengths (F1: 365.4, F2: 360.3). Moreover, the physicochemical and microbial count of the formulated creams was also assessed based on various parameters such as colour, pH, centrifugation, viscosity and microbial load over a storage period of 28 days. Both formulations showed pseudo plastic behaviour and were stable at all conditions except for samples kept at 40 °C. Altogether, these results suggested that the ethyl acetate fraction of Z. rhetsa bark has great potential to reduce exposure to harmful UVA/UVB radiations and may be utilized as an active ingredient in natural sunscreen formulation.

Background Diabetes mellitus type II (DMT-2) is a widely spread metabolic disorder both in developed and developing countries. The role of oxidative stress is well established in DMT-2 pathogenesis. The synthetic drugs for DMT-2 are associated with serious side complications. Antioxidant and α-glucosidase inhibitory actions of phytochemicals from various plant species are considered as an alternative to synthetic drugs for DMT-2 management. The present study aimed to evaluate the antioxidant activity, α-glucosidase inhibitory potential and phytochemical profiling of Hyophorbe lagenicaulis. Methods The total phenolic and flavonoid contents, in vitro antioxidant activity (α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging and phosphomolybdenum method) and α-glucosidase inhibition of ultrasonicated hydroethanolic H. lagenicaulis leaf extracts were determined spectrophotometrically. The results of DPPH assay and α-glucosidase inhibition were reported in terms of IC50 value. The phytochemical profiling was accomplished by UHPLC-Q-TOF/MS/MS technique. Results and Discussion Findings leaped 60% ethanolic extract as rich fraction regarding total phenolic and flavonoid contents. The 60% ethanolic fraction was a promising source of natural antioxidants and α-glucosidase inhibitory agents as indicated by anti-radical and enzyme inibitory activities. Kaempferol, rutin, hesperetin 5-O-glucoside, kaempferol-coumaroyl-glucoside, luteolin 3-glucoside, Isorhamnetin-3-O-rutinoside, trimethoxyflavone derivatives and citric acid were identified by UHPLC-Q-TOF-MS/MS. These compounds were believed to be responsible for the strong antioxidant and enzyme inhibitory activity of plant extracts. The extensive metabolite profiling of H. lagenicaulis was carried out the first time as never reported previously. The H. lagenicaulis might be an appropriate choice to manage diabetes mellitus in an alternate way. The findings may be further exploited extensively for toxicity evaluation to proceed with functional food development having antidiabetic attributes.

The present study deals with mass spectrometric investigation to characterize the nature of proinsulin in inclusion bodies. Various derivatives of human proinsulin were cloned, expressed in E. coli and inclusion bodies prepared under weak acidic conditions (pH 6.5), which protected the native thiols. Non-reductive PAGE showed that proinsulin migrated as monomer (approximately 10 kDa). MALDI-MS protocol was developed for the direct analysis of proinsulin derivatives in inclusion bodies. It was found that the masses of the derivatives corresponded to polypeptides containing six cysteines in reduced form. Iodoacetamide or iodoacetic acid treatment of proinsulin inclusion bodies, in suspension under non-reducing conditions and without any chaotropic agents, showed six alkylations, suggesting that these cytoplasmic aggregates were assembled from reduced monomers, with their –SH groups pointing towards hydrophilic surface. The MALDI analysis of inclusion bodies was extended to a proinsulin derivatives labelled with 13C and 15N giving an excellent agreement between experimental and theoretical masses. These mass spectrometric studies also provide early information about post-translational modification as evident in one of the derivatives MTRR-pi showing N-terminal cleavage of methionine. This shows the potential value of the protocol for the accurate analysis of polypeptides, expressed as inclusion bodies, prior to undertaking further purification.

Background: Low phosphorus (P) availability to wheat from commercial fertilizers is one of the reasons for lower grain yield and hence justifies search for more efficient P source under alkaline calcareous soils.; Results: Phosphoric acid (PA) and diammonium phosphate (DAP), applied through conventional and modified methods, were assessed for P supply and wheat yield in a calcareous soil. Under laboratory conditions, pre-incubated soil with 70 mg P kg-1 soil as PA and DAP was assessed for solution P (Cp ) for 4 weeks. Phosphorus sorption data were fitted using the Freundlich model for describing analyzed sorption in soil incubated with or without DAP and PA. The fitted model equations exhibited comparatively higher effluxes of P from the solution system in control treatment. Compared to DAP, lower quantities (19.6%) of P for PA-treated soil were required for producing optimum P concentration in soil solution, i.e. 0.2 mg P L-1 . The greenhouse study involved 32 P tracer technique to quantify the proportion of applied P derived by wheat from fertilizer or soil. The results showed that P derived from fertilizer was highest (47.5%) in PA placement, while the lowest (31.5%) was in DAP broadcast treatment. The field study also showed similar trends to that of the greenhouse study. The PA placement treatment resulted in highest (23.4%) phosphorus use efficiency, whereas the lowest one (17.1%) was recorded for DAP broadcast treatment.; Conclusion: PA proved to be a better P source than DAP for improving P content and achieving higher yield and recovery of applied P by wheat grown in alkaline calcareous soils. © 2016 Society of Chemical Industry.; © 2016 Society of Chemical Industry.

Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM-1 s-1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.

Genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis revealed the presence of an open reading frame, Pcal_0768, corresponding to a putative 4-α-glucanotranferase belonging to glycoside hydrolases (GH) family 77. We have produced, in Escherichia coli, and purified recombinant Pcal_0768 which exhibited high disproportionation (690 U mg(-1)) activity. To the best of our knowledge, this is the highest ever reported activity for any member of family GH77. Maltooligosaccharides, when used as sole substrates, were disproportionated into linear maltooligohomologues. The analysis of the reaction end products revealed no evidence for the production of cycloamyloses. Catalytic activity of the enzyme remained unchanged in the presence or the absence of ionic and nonionic detergents. γ-cyclodextrin, an inhibitor of 4-α-glucanotransferases, did not show any inhibitory effect on Pcal_0768 activity. These properties make Pcal_0768 a potential candidate for starch processing industry.

The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

BACKGROUND Low phosphorus (P) efficiency from existing granular fertilisers necessitates searching for efficient alternatives to improve wheat productivity in calcareous soil. RESULTS Multi-location trials have shown that phosphoric acid (PA) produced 16% higher wheat grain over commercial P fertilisers, i.e. diammonium phosphate (DAP) and triple superphosphate (TSP). Methods of P application significantly influenced grain yield and the efficiency of methods was observed in the order: PA placement below seed > PA, DAP or TSP fertigation > DAP or TSP broadcast. The sub-surface application of PA produced highest grain yields (mean of all rates), i.e. 4669, 4158 and 3910 kg ha−1 in Bagh, Bhalwal and Shahpur soil series, respectively. Phosphoric acid at 66 kg P2O5 ha−1 was found more effective in increasing gain yield over that of control. Trend in grain P uptake was found similar to that observed for grain yield. Maximum P uptake by grain was recorded at the highest P rate and the lowest at zero P. The significant increase in P uptake with P rates was generally related to the increase in yield rather than its concentration in grain. Phosphorus agronomic efficiency (PAE) and phosphorus recovery efficiency (PRE) were found higher at lower P rate (44 kg P2O5 ha−1) and decreased with P application. However, PA applied by the either method resulted in higher PAE and PRE compared to DAP and TSP. CONCLUSION Phosphoric acid is suggested as an efficient alternative to commercial granular P fertilisers for wheat production in alkaline calcareous soils. © 2015 Society of Chemical Industry

Aspartate aminotransferase catalyzes the transfer of an amino group from l -aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, ASTGt, from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant ASTGt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified ASTGt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that ASTGt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. ASTGt exhibited apparent Vmax and Km values of 120 μmol min−1 mg−1 and 1.5 mM, respectively, against l -aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus.

The naturopathic treatment of obesity is a matter of keen interest to develop efficient natural pharmacological routes for disease management with low or negligible toxicity and side effects. For this purpose, optimized ultrasonicated hydroethanolic extracts of Taraxacum officinale were evaluated for antiobesity attributes. The 2,2-diphenyl-1-picrylhydrazyl method was adopted to evaluate antioxidant potential. Porcine pancreatic lipase inhibitory assay was conducted to assess the in vitro antiobesity property. Ultra-high performance chromatography equipped with a mass spectrometer was utilized to profile the secondary metabolites in the most potent extract. The 60% ethanolic extract exhibited highest extract yield (25.05 ± 0.07%), total phenolic contents (123.42 ± 0.007 mg GAE/g DE), total flavonoid contents (55.81 ± 0.004 RE/g DE), DPPH-radical-scavenging activity (IC50 = 81.05 ± 0.96 µg/mL) and pancreatic lipase inhibitory properties (IC50 = 146.49 ± 4.24 µg/mL). The targeted metabolite fingerprinting highlighted the presence of high-value secondary metabolites. Molecular-binding energies computed by docking tool revealed the possible contribution towards pancreatic lipase inhibitory properties of secondary metabolites including myricetin, isomangiferin, icariside B4, kaempferol and luteolin derivatives when compared to the standard drug orlistat. In vivo investigations revealed a positive impact on the lipid profile and obesity biomarkers of obese mice. The study presents Taraxacum officinale as a potent source of functional bioactive ingredients to impart new insights into the existing pool of knowledge of naturopathic approaches towards obesity management.

Acute lung injury (ALI) is clinically characterized by excessive inflammation leading to acute respiratory distress syndrome (ARDS), having high morbidity and mortality both in human and animals. Ginsenoside Rb1 (Rb1) is a major primary bioactive component extracted by Panax ginseng, which has numerous pharmacological functions such as anti-cancer, anti-inflammatory, and antioxidant. However, the anti-inflammatory effects of Rb1 in Staphylococcus aureus (S. aureus)-induced ALI in mice have not been investigated. The aim of the current study was to determine the anti-inflammatory influence of Rb1 on S. aureus-induced ALI in mice, and to explore its possible underlying principle mechanisms in RAW 264.7 macrophage cells. The results of physical morphology, histopathological variation and wet-to-dry weight ratio of lungs revealed that Rb1 significantly attenuated S. aureus-induced lung injury. Furthermore, qPCR results displayed that Rb1 inhibited IL-1β, IL-6 and TNF-α production both in vivo and in vitro. The activation of Toll-like receptor 2 (TLR2) by S. aureus was inhibited by application of Rb1 as confirmed by results of immunofluorescence assay. The expression of NF-kB and MAPK signaling proteins revealed that Rb1 significantly attenuated the phosphorylation of p65, ERK, as well as JNK. Altogether, the results of this experiment presented that Rb1 has ability to protect S. aureus-induced ALI in mice by attenuating TLR-2-mediated NF-kB and MAPK signaling pathways. Consequently, Rb-1 might be a potential medicine in the treatment of S. aureus-induced lung inflammation.

Background Cosmos caudatus is an annual plant known for its medicinal value in treating several health conditions, such as high blood pressure, arthritis, and diabetes mellitus. The α-glucosidase inhibitory activity and total phenolic content of the leaf aqueous ethanolic extracts of the plant at different growth stages (6, 8. 10, 12 and 14 weeks) were determined in an effort to ascertain the best time to harvest the plant for maximum medicinal quality with respect to its glucose-lowering effects. Methods The aqueous ethanolic leaf extracts of C. caudatus were characterized by NMR and LC-MS/MS. The total phenolic content and α-glucosidase inhibitory activity were evaluated by the Folin-Ciocalteu method and α-glucosidase inhibitory assay, respectively. The statistical significance of the results was evaluated using one-way ANOVA with Duncan’s post hoc test, and correlation among the different activities was performed by Pearson’s correlation test. NMR spectroscopy along with multivariate data analysis was used to identify the metabolites correlated with total phenolic content and α-glucosidase inhibitory activity of the C. caudatus leaf extracts. Results It was found that the α-glucosidase inhibitory activity and total phenolic content of the optimized ethanol:water (80:20) leaf extract of the plant increased significantly as the plant matured, reaching a maximum at the 10th week. The IC50 value for α-glucosidase inhibitory activity (39.18 μg mL− 1) at the 10th week showed greater potency than the positive standard, quercetin (110.50 μg mL− 1). Through an 1H NMR-based metabolomics approach, the 10-week-old samples were shown to be correlated with a high total phenolic content and α-glucosidase inhibitory activity. From the partial least squares biplot, rutin and flavonoid glycosides, consisting of quercetin 3-O-arabinofuranoside, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and quercetin 3-O-xyloside, were identified as the major bioactive metabolites. The metabolites were identified by NMR spectroscopy (J-resolve, HSQC and HMBC experiments) and further supported by dereplication via LC-MS/MS. Conclusion For high phytomedicinal quality, the 10th week is recommended as the best time to harvest C. caudatus leaves with respect to its glucose lowering potential. Electronic supplementary material The online version of this article (10.1186/s12906-019-2655-9) contains supplementary material, which is available to authorized users.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km) toward R1P was 11.5 mM−1 · s−1. ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota. IMPORTANCE The discovery of the pentose bisphosphate pathway in Thermococcus kodakarensis has clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase in Pyrobaculum calidifontis. The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

Paddy soils have a heterogenous nature, with complex physico-chemical interactions and varying soil characteristics. Paddy soils remain flooded and are considered as rich sources of nutrients for plant growth. The nutrient levels mostly depend on different management practices, such as fertilizer application, irrigation, and tillage, and the movement of nutrients in the soils. These paddy soils normally show less movement of applied nutrients out of the medium than other soils, because of stagnant water that reduces the mobility rate. Paddy soils can become polluted by anthropogenic practices such as the use of sewage wastewater; industrial wastewater containing heavy metals; fertilizers; and pesticides, and the leakage of petrochemicals. Some natural pollutants can be oxidized by microbial activity, but most pollutants do not undergo biotic and chemical degradation. Inorganic (heavy metals) and organic pollutants (polychlorinated biphenyls, polychlorinated dibenzodioxins, and polychlorinated dibenzofurans) are the major types of pollutants in paddy soils. The numerous organic and inorganic pollutants resulting from anthropogenic activities can remain for long periods in nature and can be transported over long distances. In particular, organic pollutants can be bioaccumulated and biomagnified, thus reaching high levels that can be dangerous for human wellbeing and biological communities. Inorganic pollutants such as the heavy metals Pb, Cr, As, Zn, Cd, Cu, Hg, and Ni cause hazards for human health, for plants, for animals, and for the fertility status of the soil. These heavy metals are common pollutants in paddy soil and they bioaccumulate; in this way the concentrations of these pollutants increase in living systems, owing to their retention rates being higher than their discharge rates in these systems. The fate of these pollutants depends on their bioavailability, degradation by microorganisms, adsorption, desorption, leaching, and runoff. The transport and degradation of these pollutants in paddy soils and groundwater results in contamination. The physico-chemical characteristics of the paddy soil framework; for example, the water content, soil organic matter, presence of clay, and pH, influence the sorption or desorption and degradation of pollutants and also influence leaching to the groundwater and runoff to surface waters. The translocation of natural pesticides in paddy soils depends upon the ionic or neutral behavior of the soil constituents, on the pesticides’ solubility in water, extremity on the substance, and the colloidal nature of the paddy soils.