DSpace Collection:

In vitro propagation studies and partial biochemical characterization for drought stress in Jatropha curcas L.

2020-08-21T06:42:50Z

Title: In vitro propagation studies and partial biochemical characterization for drought stress in Jatropha curcas L. Authors: Basharat, Sadia Abstract: Development of rapid and efficient propagation methods for Jatropha curcas are highly desirable since its seed oil can be used as biofuel and hence of high economic value around the world. In this study, tissue culture techniques were employed to resolve conventional propagation issues. In vitro seed germination experiments in soil and on half or full strength MS medium, specifically during the dormant periods by using some pretreatments were performed. Pretreatments included presoaking of seeds in water overnight, scarification, stratification, removal of seed coats (before/after disinfection) and combination of these treatments. It was observed that the orientation of the seeds on the culture media also had significant effect on its germination rate. Disinfection of naked seeds could not support subsequent germination so the seeds were disinfected before removing the seed coats. It was observed that the removal of seed coats only could break the dormancy of seeds to get 100% in vitro germination on full strength MS medium kept in the dark at 25 ± 2˚C in the months of December to January. Such seedlings were shifted in light conditions (16 h photoperiod) after the root emergence at the same temperature to support chlorophyll development. Seedlings were successfully acclimatized by shifting to the soil containing a mixture of peat, clay and silt (1:1:1 v/v) in greenhouse. Efficient callus-mediated regeneration system was developed using various explants of Jatropha curcas like young/mature/cotyledonary leaf and hypocotyl. Different growth regulators including TDZ, Kin, BAP, NAA, IAA, 2, 4-D were supplemented in MS medium either singly or in combinations of different concentrations for callus induction and its proliferation. Cultures were kept in either darkness or 16/8 h photoperiod. It was observed that 22.17 µM BAP + 5.35 µM NAA supplemented in medium gave 100% embryogenic callus induction with all the explants used except mature leaf, when kept in 16/8 h photoperiod. Cultures kept in complete darkness also give good callus induction frequency (90%) but calluses were white friable and non-embryogenic. Developed calluses were shifted to shoot bud induction medium. Shoot bud induction medium was also MS medium supplemented with different plant growth regulators both auxins and cytokinins (BAP, NAA, GA3, TDZ, Kin, IBA) in combinations of two or three. Calluses developed on medium containing 22.17 µM BAP + 5.35 µM NAA, shifted to same combination of growth regulators have shown maximum number of shoot buds per culture vessel (17). However, frequency of shoot bud induction was low. Addition of GA3 or Kin in the medium having BAP and NAA have enhanced the frequency of shoot bud induction. However, when both GA3 and Kin were used together, they did not show any significant effect on shoot bud induction frequency. TDZ supplemented in the medium having BAP and NAA, have shown negative effect on regeneration potential. Maximum shoot bud induction frequency (37%) was achieved on MS medium with 6.65 µM BAP + 2.45 µM IBA added. Direct shoot regeneration from young leaf explant of Jatropha currcas was also achieved on MS medium supplemented with 6.65 µM BAP + 2.45 µM IBA. Developed and elongated shoots of average 2 cm length were shifted to another medium for root development. Maximum root induction frequency was achieved on MS medium supplemented with 4.9 µM IBA. Rooting was not very successful in recent experiments because of the callus formation at the base of shoots shifted to the rooting medium. Effect of water/osmotic stress (synonymously referred as drought stress in literature) on morphological and biochemical activities of Jatropha curcas plants were elucidated in the present experiments. The experiments were performed both under in vitro (seed germination, early growth of seedlings and callus cultures) and field conditions (pot-grown 5-month old plants). Different sorbitol treatment levels (0, 0.05, 0.1, 0.15, 0.20, 0.25, 0.3, 0.35, 0.40, 0.45 and 0.5 M) were supplemented to MS medium in order to increase the osmotic stress for in vitro studies. Five-month-old greenhouse plants were subjected to different field capacities of water (100, 75, 50, 25 and 0%). Results have shown that increased osmotic stress in the medium resulted in decreased germination along with its delayed onset. However above 0.3 M sorbitol concentration, germination process was stopped. Similarly fresh/dry weights and shoot lengths of germinating seedlings were also influenced significantly with increase in osmotic stress. Among the biochemical parameters of germinating seedlings studied, it was observed that there was trend towards significant increase in SOD and peroxidase activities with an increase in osmotic stress. However, the soluble protein contents were not affected significantly. Callus cultures were not influenced physiologically and biochemically with increased osmotic stress however, higher osmotic stress lead to reduction in fresh weight and water content and slight enhancement in soluble protein and peroxidase activity. Five-month-old plants subjected to different field capacities of water for 30 days have not shown any visual symptoms of stress like necrosis or chlorosis. However, minimum fresh weight per unit area of leaves was observed in lowest field capacity (0%). Similarly minimum SOD activity was observed in plants subjected to 50% field capacity and there was trend towards increase in SOD activity both in lower and higher field capacities. Peroxidase activities remained unaffected. However, slight increase in soluble protein contents was observed in 0% field capacity. Hence it can be concluded that germination and early seedling growth are influenced by drought stress to a great extent as compared to mature plants where no remarkable changes were observed in both physical and biochemical activities except in extreme stress condition. Same was the case with callus cultures derived from mature leaf explants. Hence Jatropha curcas plants may be planted in areas of low water availability if irrigated properly at seed germination stage.

Cloning Dof1 transcription factor in plant expression vector and its tranformation in wheat

2020-08-21T09:54:57Z

Title: Cloning Dof1 transcription factor in plant expression vector and its tranformation in wheat Authors: Hasnain, Ammarah Abstract: A single transcription factor modulates coordinated expression of a set of metabolites in a biochemical pathway, therefore, the use of transcription factors is an imperative strategy in generating plants with desirable traits. The earlier approaches of single-gene modulation were not highly effective because more than one enzymes are involved in a metabolic pathway and the expression of any single enzyme might be masked by concurrent mechanisms in order to maintain homoeostasis. The aim of the study was to develop transgenic wheat overexpressing Triticum aestivum Dof1 (TaDof1) transcription factor that affects the activity of multiple genes involved in nitrogen and carbon metabolisms that ultimately modulates nitrogen assimilation in plants. The TaDof1 was got synthesized, cloned under the control of CaMV35S promoter and terminated by Nos terminator in a binary expression vector pSB219 that carried phosphinothricin N-acetyltransferase cassette as a plant selection marker. The vector pSB219 with TaDof1 construct was transformed in AGL1 strain of Agrobacterium tumefaciens by electroporation method. The immature embryos of two elite wheat cultivars Galaxy and Faisalabd-2008 were excised and placed on callus induction medium (CIM) to initiate callus formation. One to two weeks old embryogenic calli were selected for infection with freshly grown culture of A. tumefaciens harboring TaDof1 cassette. The infected calli were shifted to co-cultivation medium containing 400 µM of acetosyringone. The calli were incubated on co-cultivatoin medium for 48 hours for TDNA delivery into plant genome. In order to suppress the overgrowth of bacterial cells, calli were placed on callus induction medium containing antibiotic timentin (160 mg/L) for 3-4 weeks. To induce shoot formation, the calli were shifted to regeneration (MSK) medium supplemented with kinetin hormone (1 mg/L). The plants were subjected to first round of selection in which they were shifted to regeneration (MSK) medium containing phosphinothricin (2 mg/L) for 2 weeks. The survived plants were transferred to MSO medium for 2 weeks for root formation. The second round of selection was done in which the plants were transferred to regeneration (MSK) medium containing 3 mg/ L of phosphinothricin for another 2 weeks. After selection, the plants showing resistance against phosphinothricin were transferred to MSO medium until long and healthy roots were developed. The plants having healthy shoots and roots were transferred to a mixture of peat moss, vermiculite and perlite (ratio 2:1:1 respectively) in plastic pots. To confirm the integration of complete TaDof1 cassette in plant genome, gene junction PCR was performed on the putative transgenic plants. Out of 31 positive events, only 8 plants possessed complete TaDof1 cassette while the rest of plants had truncated constructs. The seeds of T0 plants were sown in pots that were kept under controlled conditions to obtain T1 transgenic wheat lines. To induce the expression of TaDof1, six selected T1 wheat lines were subjected to nitrogen stress. Total RNA was isolated after 2 and 4 weeks of nitrogen stress in order to quantify the expression of TaDof1 and other related genes. The results of quantitative RT-PCR revealed that only a few transgenic lines showed upregulation of TaDof1 and related genes after 2 weeks of nitrogen stress. The increase in expression levels of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC) and pyruvate kinase (PK) genes ranged from 0.33-0.80 fold in transgenic Faisalabad-2008 and 0.33-17 fold in transgenic Galaxy. However, a substantial rise in the expression of TaDof1 and the genes affected by TaDof1 was observed after 4 weeks of nitrogen stress. The expression profiles of the genes CS, ICDH, PEPC and PK ranged from 0.4-88 fold in transgenic Faisalabad-2008 and 0.35464 fold in transgenic Galaxy. The maximum increase of 464 fold was recorded for ICDH gene expression after 4 weeks of stress. The effect of overexpression of TaDof1 on various agronomic, physiological and biochemical traits of T1 plants was also investigated. For morphological analysis, the data of plant height (cm), number of spikes plant-1, spike length (cm), number of grains spike-1 and seed weight (g) was recorded. Significantly increased values were observed in most of the selected T1 lines for agronomic traits. Under low-nitrogen conditions, all the selected T1 lines exhibited a remarkable increment in chlorophyll a, b and total chlorophyll contents as compared to wild type plants. For chlorophyll a, b and total chlorophyll, the maximum rise of 31 mg g-1FW, 40 mg g-1FW and 35 mg g-1FW respectively was observed in transgenic line of Galaxy (G4) as compared to control plants having 4 mg g-1FW, 6 mg g-1FW and 5 mg g-1FW of chlorophyll a, b and total chlorophyll contents respectively. For soluble protein content, 4 out of 6 T1 lines exhibited significantly increased values with transgenic line of Galaxy (G4) showing the highest value (12 mg g-1FW) in comparison with wild type plant (7 mg g-1FW). A considerable rise in soluble sugar content was recorded in all the T1 lines. The maximum rise of 9.9 mg g-1FW was recorded for transgenic line of Galaxy (G1) as compared to control plant (4.9 mg g-1FW). These findings clearly indicated that overexpression of Dof1 transcription factor not only enhances nitrogen assimilation in plants by modulating the pathway associated with nitrogen and carbon metabolisms but also improves plant growth. The results depict the potential of employing transcription factors in engineering plant metabolisms that pave the way for future characterization of Dof1 plants.

Prevelence of Fatty Liver Disease in Ageing Population and Effects of Nigella Sativa Tablets on Fatty Liver

2020-08-21T07:36:41Z

Title: Prevelence of Fatty Liver Disease in Ageing Population and Effects of Nigella Sativa Tablets on Fatty Liver Authors: Shah, Abdus Saboor Abstract: Non-alcoholic fatty liver disease (NAFLD) prevalence has not been well established. The aim of this study was to define prospectively non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease prevalence in hospitalized and ambulatory patients 20-65 years old during June 2013 to June 2014 were selected from Combined Military Hospital Peshawar Cantonment area. A base line questionnaire and right upper quadrant ultrasound was completed by all patients. On identifications of fatty liver among the selected cases further lab test data and liver biopsy reports were obtained. Mean BMI of female was 29.9 ± 5.65 while prevalence of hypertension and diabetes were 49.8% and 16.6% respectively. Among all patients 62% were Punjabis, 23% were Pathans while 12% were Sindhies. Overall NAFLD prevalence was 47% while NASH was confirmed in 20 patients (12.3% of total and 30%of ultrasound positive patients). Pathans had the highest prevalence of NAFLD (58.5%) as compared to Punjabi’s (44.5%) and Sindhies (35.3%). Pathans also had a higher prevalence of NASH compared with Punjabies (19.5% VS 10%: p-value= .03). In general NAFLD patients were more prevalent among male (59%), Diabetic (p-value <0.00005), hypertensive (p-value< 0.00005) and older (p-value< 0.005). They consumed more fast food (p-value = 0.049) had a higher BMI (p-value<0.0005) and had little or no exercise as compared to their normal or non NAFLD counter parts (p-value= 0.02). NAFLD was found in 75% and NASH in 22.5% among the 26 diabetic patients. ALT, AST, BMI, insulin, quantitative insulin sensitivity checks index and cytokeratin – 18 correlated with NASH. It was concluded that NAFLD and NASH prevalence is higher than estimated previously, Pathans and Patients with diabetes are at high risk.In this part of the study, the effects of tablets Nigella Sativa on fatty liver disease in rats were determined. Fatty liver disease are increasing worldwide due to physical inactivity and increased fructose intake in processed foods. The seeds and oil of the plant Nigella Sativa have been widely used in various countries of the world to promote health and cure disease. The study was conducted on 52 aged (16-18 months) male albino rats to investigate the effect of Nigella sativa tablets dietary supplementation on fatty liver disease. Rats were randomly divided in; Group-I (Control rats C) (n = 20) fed standard rat diet; Group-II (Fatty liver Group-F) (n=14) fed high fructose diet (diet having 60% fructose w/w) and Group-III (Fatty liver/ Nigella sativaGroup-F/NS) (n = 18) fed Group-F diet but mixed with crushed tablets (1.6 g/kg) in order to achieve Nigella sativa daily intake of 170mg/kg b.w. During this study rats were monitored for daily food intake and weekly body weight. The parameters including body weight final, BMI, liver weight, serum glucose,insulin, HOMA-R, TC, HDL-c, LDL-c, vLDL-c, Adiponectin, TNF-α, AST, ALT and bilirubin were monitored after six weeks in the selected group of rats. The rats livers, brains and kidneys histopathological examination were also carried out.The Real-Time PCR(RT- PCR) of superoxide dismutase (Sod1, Sod2) and DNA glycosylase (Ogg1, MutY) were used for the detection ofenzymatic antioxidant defense system. The western blot anlysis of OGG1 expression was used for the detection of DNA repaire. Our results show that that there was a significant increase in visceral fat weight in Group-F compared to Group-C and a significant decrease in Group-F/NS compared to Group-F. Both Group-F and Group-F/NS had significant increase in glucose fasting, insulin, HOMA R, TC, LDL-c, vLDL-c, TNF-α, AST, ALT and bilirubin. While a significant decrease in serum adiponectin compared to Group-C. However, Group-F/NS showed significant decrease in serum glucose fasting, insulin, HOMA-R, TC, LDL-c, TNF-α, AST, ALT and bilirubin as well adiponectin increased significantly compared to Group-F. Histopathological examination in Group-F showed vascular congestion in the liver, kidneys, necrosis of renal tubular cells as well as focal cerebral hemorrhage while Group-F/NS showed almost normal histology. Gene expression of antioxidant defense mechanism: Sod1, Sod2, Ogg1 and MutY decreased significantly in the Group-F but reversed by the Nigella Sativa tablets co-administeration in Group-F/NS. Nigella sativa crushed tablets co-feeding with higher fructose diet showed significant decrease in serum level of glucose fasting, insulin, total cholesterol(TC), LDL-c, HOMA-R, TNF-α, AST, ALT and bilirubin and a significant increase in serum adiponectin in fatty liver disease in aged rats. TheNigella Sativa tablets role in attenuation of hepatic oxidative DNA damage in Group-F/NS may be mediated by up regulation of the antioxidant defense mechanism and oxidative DNA repair activity. The clinical benefits of the Nigella Sativa treatment in the shape of diminution of oxidative damage may explain its role in patients with fatty liver disease. In this part of our study the ad-on effects of tablets Nigella Sativa on different clinical and biochemical parameters in human were determined. Metabolic syndrome and its associated complications have been reported to be extensively increased worldwide. The seeds and oil of the plant Nigella Sativa have been widely used globally to cure various disease and promote health since ancient times especially in the Southeast Asia and in the mejority of the Middle East countries. A lot of work has been done already on this plant. This prospective clinical study was designed to evaluate the ad-on effect of tablets Nigella Sativa on different parameters in fatty liver disease. This clinical study was conducted in medicine departement of a tertiary health care facility the Combined Military Hospital (CMH) Peshawar cantonment from June 2013 to May 2014. After considering inclusion and exclusion criteria and final diagnoses, 60 patients in this prospective study were enrolled. Upon approval from hospital ethical committee all the patients were properly consented in writing and were informed about study scope, Patients were equally distributed in two groups containing 30 each. In Group-1 (Control group) Patients were given Tab Metformine 500mg PO BD and Tab Rosuvastatine 10mg PO at night for a period of six weeks while in Group-II (Nigella Sativa treated group) in additions to standard treatment tablets Nigella sativa 600mg PO BD for a duration of six weeks were given. The parameters including waist circumference, body weight, body mass index, lipid profile, Blood sugar fasting and post parandial, Liver functions test (LFT’s) and Ultrasound right upper quadrant were recorded before and upon treatment completion.The above mentioned methodology was followed and the study findings were : Fasting blood sugar, low density Lipoprotein cholesterol (LDL-c)and total cholesterol (TC) percentage improvements in Group-II were statistically significant (p-value<0.05). In both the groups serum Alanintransaminase (ALT) declined significantly, in the Nigella sativa tablets treated Group-II the reduction was almost 2 fold (48% vs. 26.4%). On ultrsonography a statistically significant reversal of fatty liver was found only in the Nigella sativa tablets treated Group-II (p-value<0.05). As an ad-on therapy tablets Nigella sativa was found effective in fatty liver disease. Nigella sativa tablets has shown significant results especially in diabetic and dyslipidemic patients.

Preparation, Characterization, in Silico Modelling and Bioavailability of Nanosuspention for Poorly Water Soluble Drugs Dexibuprofen and Domperidone

2020-08-25T06:23:20Z

Title: Preparation, Characterization, in Silico Modelling and Bioavailability of Nanosuspention for Poorly Water Soluble Drugs Dexibuprofen and Domperidone Authors: , Naseem Ullah Abstract: The aim of this study was to prepare and evaluate the impact of polymers on fabricating stable dexibuprofen (Dexi) and domperidone nanocrystals with enhanced therapeutic potential, using a low energy, anti-solvent precipitation method (APSP). Combinations of Hydroxypropyl methyl cellulose-Polyvinyl pyrrolidone and Hydroxypropyl methyl cellulose-Eudragit were shown to be very effective in producing stable dexi-nanocrystals with particle sizes of 85.0±2.5 nm and 90±3.0 nm, and polydispersity of 0.179± 0.01, 0.182± 0.02 respectively. Produced nanocrystals of DOMP and Ethucel combination, the average particle size and polydespersity index were found to be approximately 130.00 ± 3.0 nm, 0.15 ± 0.01, moreover, the combination of hydroxyl propyl methyl cellulose and polyvinyl alcohol also found very affective to produce DOMP nanocrystals with small particle size (200.0 ±3.5nm) and PDI (0.2±0.02). The stability studies conducted for 90 days demonstrated that nanocrystals stored at 2-8°C and 25°C were more stable than those at 40°C. The maximum recovery of dexi-nanocrystals was observed from the formulations using the combination of Hydroxypropyl methylcellulose- Polyvinyl pyrrolidone and Hydroxy propyl methyl cellulose- Eudragit, which equated to 98% and 94% of the nominal active drug content respectively. In case of DOMP nanosuspensions stored at 2-8 ºC and room temperature (25 ºC) exhibited higher stability compared to the samples stored at 40 ºC. Crystallinity of the processed and unprocessed dexi and DOMP particles was confirmed using Differential Scanning Calorimetry, Powdered X-ray Diffraction and Transmission Electron Microscope. FTIR studies for both Dexi and DOMP nanocrystals confirmed that the process did not affect chemical integrity of DOMP. The DOMP nanoparticles exhibited significantly enhanced dissolution rate (P<0.05) compared to the raw counterpart. The saturation solubility of the ix dexinanocrystals was substantially increased to 270.0±3.5 µg/mL compared to the raw dexi in water (51.0± 2.0μg/mL) and stabilizer solution (92.0 ± 3.0 μg/mL). Enhanced dissolution rate (P<0.05) was observed for the dexi-nanocrystals compared to the unprocessed drug substance and marketed tablets. The saturation solubility of the DOMP nanocrystals was significantly increased to 2700.0± 3.5 µg/mL compared to the raw dexi in water (952.0± 2.0μg/mL) and stabiliser solutions which include HPMC (1253.0± 3.5) and HPMC-PVA (1133.0 ± 3.5 µg/ml). DOMP nanocrystals resulted in enhanced dissolution rate (P<0.05) compared to the unprocessed drug substance. Molecular Modeling studies underpinned and substantiated the experimental studies, including the impact of polymers on nanoparticle sizes, nanocrystals recovery and stability studies of the produced dexinanocrystals. The highest potential was shown by combination of HPMC-PVP- Dexi (-4.7) and HPMC EUD- DEXi (-4.6) . In case of DOMP, Molecular Modelling studies underpinned the molecular level understanding of the DOMP-Polymer nanocrystal interaction and substantiated the experimental studies, including the impact of polymers on nanocrystals sizes, and stability studies of the produced DOMP nanocrystals. Ethocel as single polymer showed highest binding potential (-27.26±0.24 ) for DOMP nanocrystals and HPMC-PVA was found the best dimer to effectively bind (-25.22 ± 0.79) with the nanocrystals with subsequent small particle size and high stability profile. The findings of the nociceptive assay showed that the dexinanocrystals exhibited significant analgesia, compared to the raw dexibuprofen and the control standard diclofenac sodium. The analgesic effect was, however, produced at much lower doses (5mg/kg) than that of control standard, diclofenac sodium (20mg/kg) and dexibuprofen counterparts (40 mg/kg). While the activity results in anti-emetic assay confirmed that the nano-formulation provided significant anti-emetic effect (P< 0.05) x at a dose of 0.5 mg/kg, comparable to that of the unprocessed DOMP and the control standard Metoclopramide administered at the doses of 1.0m/kg and 30mg/kg respectively. However, DOMP nanoparticles showed the anti-emetic effect at lower doses compared to that of control standard and unprocessed DOMP. Furthermore, there was observed a substantial % increase of antiemetic potential for DOMP nanoparticles compared to the unprocessed DOMP and the standard metoclopramide.