Banana bunchy top virus genetic diversity in Pakistan and association of diversity with recombination in its genomes.

Banana Bunchy top virus (BBTV) is a multipartite circular single strand DNA virus that belongs to genus Babuvirus and family Nanoviridae. It causes significant crop losses worldwide and also in Pakistan. BBTV is present in Pakistan since 1988 however, till now only few (about twenty only) sequence of genomic components have been reported from the country. To have insights into current genetic diversity in Pakistan fifty-seven genomic components including five complete genomes (comprises of DNA-R, -U3, -S, -M, -C and -N components) were sequenced in this study. The genetic diversity analysis of populations from Pakistan showed that DNA-R is highly conserved followed by DNA-N, whereas DNA-U3 is highly diverse with the most diverse Common Region Stem-loop (CR-SL) in BBTV genome, a functional region, which previously been reported to have undergone recombination in Pakistani population. A Maximum Likelihood (ML) phylogenetic analysis of entire genomes of isolates by using sequence of all the components concatenated together with the reported genomes around the world revealed deeper insights about the origin of the disease in Pakistan. A comparison of the genetic diversity of Pakistani and entire BBTV populations around the world indicates that there exists a correlation between genetic diversity and recombination. Population genetics analysis indicated that the degree of selection pressure differs depending on the area and genomic component. A detailed analysis of recombination across various components and functional regions suggested that recombination is closely associated with the functional parts of BBTV genome showing high genetic diversity. Both genetic diversity and recombination analyses suggest that the CR-SL is a recombination hotspot in all BBTV genomes and among the six components DNA-U3 is the only recombined component that has extensively undergone inter and intragenomic recombination. Diversity analysis of recombinant regions results on average one and half fold increase and, in some cases up to four-fold increase due to recombination. These results suggest that recombination is significantly contributing to the genetic diversity of BBTV populations around the world.

Germin like protein genes exhibit modular expression during salt and drought stress in elite rice cultivars.

BACKGROUND: Germin-like proteins (GLPs) are ubiquitous plant proteins, which play significant role in plant responses against various abiotic stresses. However, the potential functions of GLPs in rice (Oryza sativa) against salt and drought stress are still unclear. METHODS AND RESULTS: In this study, transcriptional variation of eight OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-4, OsGLP8-7, OsGLP8-10, OsGLP8-11 and OsGLP8-12) was analyzed in leaves and roots of two economically important Indica rice cultivars, KS282 and Super Basmati, under salt and drought stress at early seedling stage. The relative expression analysis from qRT-PCR indicated the highest increase in expression of OsGLP3-6 in leaves and roots of both rice varieties with a significantly higher expression in KS282. Moreover, relative change in expression of OsGLP8-7, OsGLP8-10 and OsGLP8-11 under salt stress and OsGLP8-7 under drought stress was also commonly higher in leaves and roots of KS282 as compared to Super Basmati. Whereas, OsGLP3-7 and OsGLP8-12 after salt stress and OsGLP8-4 and OsGLP8-12 after drought stress were observed with higher relative expression in roots of Super Basmati than KS282. Importantly, the OsGLP3-6 and OsGLP4-1 from chromosome 3 and 4 respectively showed higher expression in leaves whereas most of the OsGLP genes from chromosome 8 exhibited higher expression in roots. CONCLUSION: Overall, as a result of this comparative analysis, OsGLP genes showed both general and specific expression profiles depending upon a specific rice variety, stress condition as well as tissue type. These results will increase our understanding of role of OsGLP genes in rice crop and provide useful information for the further in-depth research on their regulatory mechanisms in response to these stress conditions.

Overexpression of SlGSNOR impairs in vitro shoot proliferation and developmental architecture in tomato but confers enhanced disease resistance.

The pervasive presence of nitric oxide (NO) in cells and its role in modifying cystein residues through protein S-nitrosylation is a remarkable redox based signalling mechanism regulating a variety of cellular processes. S-NITROSOGLUTATHIONE REDUCTASE (GSNOR) governs NO bioavailability by the breakdown of S-nitrosoglutathione (GSNO), fine-tunes NO signalling and controls total cellular S-nitrosylated proteins. Most of the published data on GSNOR functional analysis is based on the model plant Arabidopsis with no previous report for its effect on in vitro regeneration of tissue cultured plants. Moreover, the effect of GSNOR overexpression (O.E) on tomato growth, development and disease resistance remains enigmatic. Here we show that SlGSNOR O.E in tomato alters multiple developmental programs from in vitro culture establishment to plant growth and fruit set. Moreover, constitutive SlGSNOR O.E in tomato showed enhanced resistance against early blight (EB) disease caused by Alternaria solani and reduction in hypersensitive response (HR)-mediated cell death after Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) infiltrations. High GSNOR transcript levels led to the inhibition of in vitro shoot proliferation in transformed explants as revealed by the fluorescence microscopy after YFP labelling. Transgenic tomato lines overexpressing SlGSNOR showed defective phenotypes exhibiting stunted plant growth and bushy-type plants due to loss of apical dominance, along with reduced seed germination and delayed flowering. Furthermore, SlGSNOR O.E plants exhibited altered leaf arrangement, fruit shape and modified locules number in tomato fruit. These findings give a novel insight into a multifaceted regulatory role of SlGSNOR in tomato plant development, reproduction and response to pathogens.

Elucidation of larvicidal potential of metallic and environment friendly food-grade nanostructures against Aedes albopictus.

To combat health challenges associated with mosquito-borne diseases, the larvicidal activity of metallic nanoparticles, food-grade polymeric nano-capsules and insecticides was investigated against larvae of Aedes albopictus as an effective alternate control approach. The Ae. albopictus was identified using sequencing and phylogenetic analyses of COXI, CYTB and ITS2 genes. The characterization of synthesized nanostructures was performed through Zetasizer, UV-VIS spectroscopy, atomic force microscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. The mosquito larvae were exposed to varying concentration of nanostructures and insecticides, and their percentage mortality was evaluated at different time intervals of 24 h and 48 h exposure. The highest efficacy was observed in zinc oxide nanoparticles (ZnO-NPs) and polymeric nanocapsules FG-Cur E-III (LC50 = 0.24 mg/L, LC90 = 0.6 mg/L) and (LC50 = 3.8 mg/L, LC90 = 9.33 mg/L), respectively, after 24 h; while (LC50 = 0.18 mg/L, LC90 = 0.43 mg/L) and (LC50 = 1.95 mg/L, LC90 = 6.46 mg/L), respectively, after 48 h against fourth instar larvae of Ae. albopictus. Ag, CuO, NiTiO3 and CoTiO3 nanoparticles evaluated in this study also showed promising larvicidal activity. Although ZnO-NPs proved to be effective larvicides, their possible toxicity (producing ROS species) can limit their use. The curcumin nanostructures (FG-Cur E-III) stabilized by food-grade materials are thought to exert their larvicidal activity by binding to sterol carrier protein-2, and depriving the larvae from the essential dietary cholesterol, and bears effective larvicidal potential as safe alternative for chemical larvicides, due to their environment friendly, food-grade and easy biodegradability.

Protein quantification and enzyme activity estimation of Pakistani wheat landraces.

Wheat is a major food grain in Pakistan having a prominent role in agriculture as well as the economic status of the country. In the current study, seeds of 99 wheat landraces were characterized for the quantification of seed storage proteins (Albumins, Globulin, Gliadins, and Glutenin), enzyme activities of antioxidant enzymes i.e. Ascorbate peroxidase (APX), Catalase (CAT), Superoxide dismutase (SOD), Peroxidase (POD), one hydrolytic enzyme Protease (PROT) and non-enzymatic antioxidant enzyme Ascorbic acid (AsA). The landraces were categorized into low, medium, and high based on protein concentration and enzymes activities/content. The majority of the landraces were placed in the medium category. However, for the AsA parameter majority of the landraces were placed in the low category. The highest concentration of total extracted protein (184.88±0.7 mg/g. wt.), globulins (21.35±0.43 mg/g. wt.) and glutenin (20±0.04 mg/g. wt.) as well as the high activity of SOD (303±16.80 Units/g. wt.), and Ascorbic acid (533±36.1 Units/g. wt.) was identified in the wheat landrace "11757" collected from district Panjgur (Balochistan). The wheat landrace "11760", collected from district Kech (Balochistan), contained the highest albumins concentration (65.42±0.02 mg/g. wt.) and highest activity for CAT (589.5±61.20 Units/g. wt.). The highest activity of POD (32341± 91.3) and PROT was observed in seeds of the wheat landrace "11618" collected from the Gilgit Baltistan region of Pakistan. The principal component analysis showed that the great variations existed for the tested parameters among the wheat landraces. The landraces with a high concentration of seed storage proteins and antioxidant enzyme activities can be used for breeding purposes to improve the nutrimental quality of wheat cultivars.

Isolation and characterization of culturable endophytic bacterial community of stripe rust-resistantand striperust-susceptible Pakistani wheat cultivars

In this study, endophytic bacteria isolated from root, stem, and leaf tissues of stripe rust-susceptible (Inqilab 91, Galaxy 2013, and 15BT023) and stripe rust-resistant (NARC 2011, Ujala 2015, TW1410) cultivars were identified and characterized. Abundance of endophytes was found in roots as compared with stems and leaves. Resistant and susceptible cultivars significantly differed in abundance of endophytic bacteria. Restriction analysis of 16S rRNA genes amplified from 100 bacterial isolates produced 17 unique patterns. Representatives of each of the 17 unique patterns were sequenced and identified. Among the sequenced bacteria, 8 belonged to Firmicutes, 7 were Proteobacteria, and 2 were Actinobacteria. Most of the isolates have plant growth-promoting properties and a few have the potential of producing hydrolytic enzymes. Two isolates showed significant inhibition of rust spore germination. These endophytic bacteria not only can be helpful in growth-promoting activities but also can assist in biocontrol of stripe rust disease

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Salt tolerance in indica rice cell cultures depends on a fine tuning of ROS signalling and homeostasis.

Among cereal crops, salinity tolerance is rare and complex. Multiple genes control numerous pathways, which constitute plant's response to salinity. Cell cultures act as model system and are useful to investigate the salinity response which can possibly mimic a plant's response to stress. In the present study two indica rice varieties, KS-282 and Super Basmati which exhibited contrasting sodium chloride (NaCl) stress response were used to establish cell cultures. The cell cultures showed a contrasting response to salt stress at 100 mM NaCl. High level of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) were observed in sensitive cell culture for prolonged period as compared to the tolerant cells in which an extracellular H2O2 burst along with controlled intracellular H2O2 and NO signal was seen. To evaluate the role of NO in inducing cell death under salt stress, cell death percentage (CDP) was measured after 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) pre-treatment. CDP was reduced significantly in both tolerant and sensitive cell cultures emphasizing NO's possible role in programmed cell death. Expression analysis of apoplastic NADPH oxidase, i.e. OsRbohA and recently characterised OSCA family members i.e. OsOSCA 1.2 and OsOSCA 3.1 was done. Intracellular H2O2/NO levels displayed an interplay between Ca2+ influx and ROS/RNS signal. Detoxifying enzyme (i.e. ascorbate peroxidase and catalase) activity was considerably higher in tolerant KS-282 while the activity of superoxide dismutase was significantly prominent in the sensitive cells triggering greater oxidative damage owing to the prolonged presence of intracellular H2O2. Salt stress and ROS responsive TFs i.e. OsSERF1 and OsDREB2A were expressed exclusively in the tolerant cells. Similarly, the expression of genes involved in maintaining high [K+]/[Na+] ratio was considerably higher and earlier in the tolerant variety. Overall, we suggest that a control over ROS production, and an increase in the expression of genes important for potassium homeostasis play a dynamic role in salinity tolerance in rice cell cultures.

Isolation and characterization of culturable endophytic bacterial community of stripe rust-resistant and stripe rust-susceptible Pakistani wheat cultivars.

In this study, endophytic bacteria isolated from root, stem, and leaf tissues of stripe rust-susceptible (Inqilab 91, Galaxy 2013, and 15BT023) and stripe rust-resistant (NARC 2011, Ujala 2015, TW1410) cultivars were identified and characterized. Abundance of endophytes was found in roots as compared with stems and leaves. Resistant and susceptible cultivars significantly differed in abundance of endophytic bacteria. Restriction analysis of 16S rRNA genes amplified from 100 bacterial isolates produced 17 unique patterns. Representatives of each of the 17 unique patterns were sequenced and identified. Among the sequenced bacteria, 8 belonged to Firmicutes, 7 were Proteobacteria, and 2 were Actinobacteria. Most of the isolates have plant growth-promoting properties and a few have the potential of producing hydrolytic enzymes. Two isolates showed significant inhibition of rust spore germination. These endophytic bacteria not only can be helpful in growth-promoting activities but also can assist in biocontrol of stripe rust disease.

Protein Profiling of Arabidopsis Roots Treated With Humic Substances: Insights Into the Metabolic and Interactome Networks.

Background and Aim: Humic substances (HSs) influence the chemical and physical properties of the soil, and are also known to affect plant physiology and nutrient uptake. This study aimed to elucidate plant metabolic pathways and physiological processes influenced by HS activity. Methods: Arabidopsis roots were treated with HS for 8 h. Quantitative mass spectrometry-based proteomics analysis of root proteins was performed using the iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. Out of 902 protein families identified and quantified for HS treated vs. untreated roots, 92 proteins had different relative content. Bioinformatic tools such as STRING, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of network amongst the identified proteins. Results: From this analysis it was possible to evaluate that all of the identified proteins were functionally classified into several categories, mainly redox homeostasis, response to inorganic substances, energy metabolism, protein synthesis, cell trafficking, and division. Conclusion: In the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Activation of enzymes of the glycolytic pathway and up regulation of ribosomal protein indicated a stimulation in energy metabolism and protein synthesis. Regulation of the enzymes involved in redox homeostasis suggest a pivotal role of reactive oxygen species in the signaling and modulation of HS-induced responses.

Antimicrobial activity, toxicity and anti-inflammatory potential of methanolic extracts of four ethnomedicinal plant species from Punjab, Pakistan.

BACKGROUND: The plant species Aristolochia indica (AI), Melilotus indicus (MI), Tribulus terrestris (TT) and Cuscuta pedicellata (CP) are widely used in folk medicine in the villages around Chowk Azam, South Punjab, Pakistan. The aim of this study was to evaluate the antioxidant activity, phytochemical composition, and the antibacterial, antifungal, cytotoxic and anti-inflammatory potential of the four medicinal plants listed above. For CP stem, this study represents (to the best of our knowledge) the first time phytochemicals have been identified and the antioxidant and anti-inflammatory potential determined. METHODS: Phytochemicals were analyzed through chemical tests, thin layer chromatography (TLC) and spectrophotometric methods. Antioxidant activities (DPPH and H2O2) were also determined through spectrophotometric methods. Extracts were evaluated for antibacterial potential via the agar well diffusion method against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia and Acinetobacter baumannii. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the microdilution method. Antifungal activities were tested using the agar tube dilution method against three species: Aspergillus fumigatus, Aspergillus flavus and Rhizopus oryzae. The cytotoxic potential of the plant extracts was checked using the brine shrimp assay. In vitro anti-inflammatory activity of the selected plant extracts was evaluated using albumin denaturation, membrane stabilization and proteinase inhibitory assays. RESULTS: Of all the methanolic extracts tested, those from CP (stem) and TTF (T. terrestris fruit) had the highest phenolic, flavonoid and flavonol contents (497±4 mg GAE/g, 385±8 mg QE/g and 139±4 mg QE/g; 426±5 mg GAE/g, 371±8 mg QE/g and 138±6 mg QE/g, respectively) and also exhibited strong antioxidant potential in scavenging DPPH and hydrogen peroxide (IC50 values; 20±1 and 18±0.7 µg/mL; 92±2 and 26±2 µg/mL, respectively). CP, TTF and TTL (T. terrestris leaf) extracts substantially inhibited the growth of the bacteria A. baumannii, S. aureus, and K. pneumonia and also exhibited the highest antifungal potential. The ranking of the plant extracts for cytotoxicity was TTF > TTL > AI > CP > MI, while the ranking for in vitro anti-inflammatory potential at a concentration of 200 µg/mL of the selected plant extracts was CP > TTL, TTF > AI > MI. The lowest IC50 (28 µg/mL) observed in the albumin denaturation assay was for CP. Positive correlations were observed between total phenolics, antioxidants, antibacterial, antifungal and anti-inflammatory potential of the selected plant extracts, indicating a significant contribution of phenolic compounds in the plant extracts to these activities. CONCLUSIONS: This study revealed the strong antimicrobial, antioxidant, cytotoxic and anti-inflammatory potential of the plant species CP and TT used in folk medicine.

Purification and characterization of 2S albumin from Nelumbo nucifera.

The 2S albumins are a group of seed storage proteins that have recently attracted considerable attention in the field of allergen science due to their allergenic potential. A new 2S albumin from seeds of Nelumbo nucifera (Nn-2S alb) was purified to electrophoretic homogeneity by the combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The protein has a molecular mass of about 12 kDa estimated by SDS-PAGE, in good agreement with 12.5 ± 0.01 kDa determined by ESI-MS. Circular dichroism data showed that protein contained about 66% α-helices as estimated by K2D3, indicating that the protein was predominantly helical. The sedimentation coefficient (s°20,w) of the predicted model was 1.72 ± 0.21 S. The predicted 3-dimensional structure of the Nn-2S alb revealed that the protein has a region of 12 amino acids which largely corresponds to the conserved immuno-dominant epitope of 2S allergens.

Maternal and foetal outcome in gestational diabetes.

BACKGROUND: Gestational diabetes is impaired carbohydrate metabolism first diagnosed in pregnancy. Knowledge of diabetes dates back to centuries before Christ. Objective was to describe maternal complications during antenatal period and Maternal and neonatal outcome in gestational diabetes mellitus. METHODS: This Descriptive observational study was conducted at Department of Obstetrics and Gynaecology, Alhada Military Hospital, Taif, KSA. It was done during January-December 2009 to measure maternal and neonatal outcome. Hundred pregnant women diagnosed through glucose tolerance test as diabetic were enrolled as study subjects. All the subjects were enrolled and followed regularly at Obstetric and Gynaecology out-patient Department of Alhada Military Hospital, Taif. Blood glucose level was controlled either by diet or by diet and insulin. Study subjects were hospitalised for adjustment of dose of insulin and for management of complications. Foetal well being was assessed by kick count, cardiotocography and ultrasound. Time and mode of delivery was decided at 36 week of gestation. Intra-partum maternal blood glucose level was monitored and foetal monitoring was done by cardiotocography. RESULTS: Total numbers of women delivered were 2,858. Hundred cases of diabetes mellitus during pregnancy were studied. Eighty-eight patients were above 25 years of age, multiparous ladies with gestational diabetes in 76% of cases. Insulin was required in 64% of patients. Polyhydramnios was most common maternal complication. Two out of 100 had spontaneous miscarriage; 14 underwent preterm delivery while 84 reached term with two intrauterine death. Caesarean section was carried out in 58 patients. Total number of babies delivered alive were 92. There were 4 neonatal deaths. Hyperbilirubinemia was the most common neonatal complication. CONCLUSION: It was concluded that early detection, constant supervision, strict glycaemic control, delivery with intensive intrapartum monitoring and facilities of expert neonatologist can result in good maternal and foetal outcome, without much morbidity.

Sequence Maneuverer: tool for sequence extraction from genomes.

UNLABELLED: The availability of genomic sequences of many organisms has opened new challenges in many aspects particularly in terms of genome analysis. Sequence extraction is a vital step and many tools have been developed to solve this issue. These tools are available publically but have limitations with reference to the sequence extraction, length of the sequence to be extracted, organism specificity and lack of user friendly interface. We have developed a java based software package having three modules which can be used independently or sequentially. The tool efficiently extracts sequences from large datasets with few simple steps. It can efficiently extract multiple sequences of any desired length from a genome of any organism. The results are crosschecked by published data. AVAILABILITY: URL 1: http://ww3.comsats.edu.pk/bio/ResearchProjects.aspx URL 2: http://ww3.comsats.edu.pk/bio/SequenceManeuverer.aspx.

Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism.

L-Ascorbate catabolism involves reversible oxidation to DHA (dehydroascorbic acid), then irreversible oxidation or hydrolysis. The precursor-product relationships and the identity of several major DHA breakdown products remained unclear. In the presence of added H2O2, DHA underwent little hydrolysis to DKG (2,3-dioxo-L-gulonate). Instead, it yielded OxT (oxalyl L-threonate), cOxT (cyclic oxalyl L-threonate) and free oxalate (~6:1:1), essentially simultaneously, suggesting that all three product classes independently arose from one reactive intermediate, proposed to be cyclic-2,3-O-oxalyl-L-threonolactone. Only with plant apoplastic esterases present were the esters significant precursors of free oxalate. Without added H2O2, DHA was slowly hydrolysed to DKG. Downstream of DKG was a singly ionized dicarboxy compound (suggested to be 2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone), which reversibly de-lactonized to a dianionic carboxypentonate. Formation of these lactones and acid was minimized by the presence of residual unreacted ascorbate. In vivo, the putative 2-carboxy-L-pentonolactones were relatively stable. We propose that DHA is a branch-point in ascorbate catabolism, being either oxidized to oxalate and its esters or hydrolysed to DKG and downstream carboxypentonates. The oxidation/hydrolysis ratio is governed by reactive oxygen species status. In vivo, oxalyl esters are enzymatically hydrolysed, but the carboxypentonates are stable. The biological roles of these ascorbate metabolites invite future exploration.

Pattern of thyroid autoimmunity in type 1 and type 2 diabetics.

OBJECTIVE: To determine the level of thyroid autoimmunity among clinically euthyroid patients of type 1 and type 2 diabetics and to correlate the levels with pattern of diabetes. DESIGN: Cross-sectional analytical study. PLACE AND DURATION OF STUDY: Diabetic Clinic of Shaikh Zayed Hospital, Lahore from August 2004 to April 2005. PATIENTS AND METHODS: Blood samples were collected from 163 subjects (51 of type 1, 61 of type 2 diabetics and 51 non- diabetic controls) and sera were separated. They were all clinically euthyroid and selected conveniently. Thyroid profile (FT4, TSH and anti-TPO) was assessed in the three groups and compared. RESULTS: It was observed that at a cut off value of less than 100 units of anti-TPO level was normal, raised anti-TPO antibodies were found in 12% of normal control group, 61% in type 1 and 42.3% in type 2 diabetics. In type 1 diabetics, the TSH was higher in the younger age patients with progressive rise in anti-TPO antibodies level by age. The TSH, anti-TPO antibodies and BSF were significantly higher in type 1 diabetics than in the controls. In type 2 diabetics, TSH was moderately low but anti-TPO antibodies and BSF were higher than in the controls. CONCLUSION: Thyroid autoimmune process seems to be correlated more with type 1 diabetic patients than with type 2 patients.