In search of the best pollinators for canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) production in Pakistan

To discover the pollinator community of canola (Brassica napus L.) and the best pollinators for canola production, an experiment was performed at the research farm of Bahauddin Zakariya University, Multan, Pakistan. The insect pollinator community was composed of 35 species in 3 orders and 14 families. Most of the bees (Hymenoptera) and a butterfly species (Lepidoptera) foraged for nectar, whereas all the flies (Diptera) foraged either for pollen or both nectar and pollen. Eight major pollinators were tested for their pollination efficiency. The nectar-robbing behavior of many species made it difficult to judge the efficiency of an insect on the basis of visitation rate and stay time; therefore, the amounts of pollen deposited and pollen harvested per visit were also measured. The single visit efficiency in terms of the number of seeds per pod revealed that Apis dorsata, A. florea, and Halictus sp. were superior for canola pollination, having Spears’ values of 1.62, 1.55 and 1.73, respectively. With the increase in the number of seeds per pod, seed weight per pod also increased, confirming the importance of these three pollinator species in canola production.     

Effect of porcine placenta steroid extract on myogenic satellite cell proliferation, transdifferentiation, and lipid accumulation

Intramuscular long-chain fatty acids (LCFAs) play an important role in energy production and initiation of mitochondrial oxidation of lipids. Herein, we report a natural porcine placenta steroid extract (PPSE) that stimulates transdifferentiation and lipid accumulation in bovine myogenic satellite cells (MSCs). The steroids hormones in PPSE were analyzed using enzyme-linked immunosorbant assay and presence of LCFA was established using gas chromatography. At 70% confluent growth, cells were treated with PPSE, LCFAs, transdifferentiation cocktail and commercially available steroid hormones. The working concentrations of all chemicals were manipulated similar to PPSE. The cells were observed for morphological changes and subjected to quantitative analysis of lipid deposition on Days 2, 4, and 6 of treatment. PPSE-treated MSCs exclusively transformed into lipid-accumulated adipose-like cells (ALCs). However, myotubes or adipocytes were formed in cells treated with other chemicals. Expression of different genes was studied to ascertain the molecular mechanism involved in ALC formation. CD36, fatty acid binding protein 4, and peroxisome proliferator-activated receptor-gamma were up-regulated. The expression of CD36 was established through immunocyto-chemical analysis. A viability assay was used to confirm the effect of PPSE on proliferation of MSCs. Hence, a natural steroid extract from porcine was found as a nontoxic mixture, which induces lipid accumulation and transdifferentiation of MSCs to ALCs. From the gene expression studies, it was established that the extract works almost in homogenous manner with other lipid inducers.     

Comparative GC–FID and GC–MS analysis of the mono and sesquiterpene secondary metabolites produced by the field grown and micropropagated plants of <Emphasis Type="Italic">Artemisia amygdalina</Emphasis> Decne

The essential oil obtained by hydrodistillation from the leaves of micropropagated plants of Artemisia amygdalina was analyzed by capillary GC–FID and GC–MS and compared with that obtained from the leaves of field growing parent plants. The oil yield from the micropropagated plants was lower (0.05% v/w) than the oil yield obtained from field-grown plants (0.2% v/w). The major constituents of the field-grown plants were p-cymene (21.0%), 1,8-cineole (24.9%), α-terpineol (5.9%), β-caryophyllene (4.7%), germacrene D (4.0%), while as the major constituents from the micropropagated plants were p-cymene (11.3%),1,8-cineole (10.2%), borneol (7.9%), α-longipinene (5.5%), α-copaene (5.5%) and β-caryophyllene (17%). The essential oil from field-grown plant was dominated by the presence of oxygenated monoterpenes (41.5%), monoterpene hydrocarbons (35.9%) and sesquiterpene hydrocarbons (16.3%) while as the essential oil of micropropagated plants was characterized by sesquiterpene hydrocarbons (40.0%), oxygenated monoterpenes (25.2%) and monoterpene hydrocarbons (21.6%).     

Development and Clinical Evaluation of Clotrimazole–β-Cyclodextrin Eyedrops for the Treatment of Fungal Keratitis

Fungal keratitis is a serious corneal disease that may result in loss of vision. There are limited treatment options available in Iraqi eye hospitals which might be the main reason behind the poor prognosis of many cases. The purpose of this study was to prepare and pharmaceutically evaluate clotrimazole–β-cyclodextrin (CTZ–β-CD) eyedrops then clinically assess its therapeutic efficacy on fungal keratitis compared with extemporaneous amphotericin B eyedrops (0.5% w/v). A CTZ–β-CD ophthalmic solution was prepared and evaluated by various physicochemical, microbiological, and biological tests. The prepared formula was stable in 0.05 M phosphate buffer pH 7.0 at 40 ± 2°C and 75 ± 5% RH for a period of 6 months. Light has no significant effect on the formula’s stability. The CTZ–β-CD eyedrops efficiently complied with the isotonicity, sterility, and antimicrobiological preservative effectiveness tests. Results of the clinical study revealed that 20 (80%) patients showed a favorable response to the CTZ–β-CD eyedrops, while 16 patients (64%) exhibited a favorable response to amphotericin B (P > 0.05). The mean course of treatment was significantly (P < 0.05) less in the CTZ treatment group than in the amphotericin group (21.5 ± 5.2 vs. 28.3 ± 6.4 days, respectively). The CTZ formulation was significantly (P < 0.05) more effective in the management of severe cases and also against Candida sp. than amphotericin B. There was no significant difference (P < 0.05) between both therapies against filamentous fungi. The CTZ–β-CD formulation can be used alternatively to other ophthalmic antimycotic treatment options in developing countries where stability, cost, or efficacy is a limiting factor.Key words: clotrimazole, β-cyclodextrin, eyedrops, fungal keratitis, Iraq     

Chemical characterization and evaluation of the neuroprotective potential of Indigofera sessiliflora through in-silico studies and behavioral tests in scopolamine-induced memory compromised rats

In the current study, we investigated the phytochemical and neuropharmacological potential of Indigofera sessiliflora, an indigenous least characterized plant widely distributed in deserted areas of Pakistan. The crude extract of the whole plant Indigofera sessiliflora (IS.CR) was preliminary tested in-vitro for the existence of polyphenol content, antioxidant and anticholinesterase potential followed by detailed chemical characterization through UHPLC-MS. Rats administered with different doses of IS.CR (100–300 mg/kg) for the duration of 4-weeks were behaviorally tested for anxiety and cognition followed by biochemical evaluation of dissected brain. The in-silico studies were employed to predict the blood–brain barrier crossing tendencies of secondary metabolites with the elucidation of the target binding site. The in-vitro assays revealed ample phenols and flavonoids content in IS.CR with adequate anti-oxidant and anticholinesterase potential. The dose-dependent anxiolytic potential of IS.CR was demonstrated in open field (OFT), light/dark (L/D) and elevated plus maze (EPM) tests as animals spent more time in open, illuminated and elevated zones (P < 0.05). In the behavioral tests for learning/memory, the IS.CR reversed the scopolamine-induced cognitive deficits, as animals showed better (P < 0.05) spontaneous alternation and discrimination index in y-maze and novel object recognition (NOR) tests. Similarly, as compared to amnesic rats, the step-through latencies were increased (P < 0.05) and escape latencies were decreased (P < 0.05) in passive avoidance (PAT) and Morris water maze (MWM) tests, respectively. Biochemical analysis of rat brains showed significant reduction in malondialdehyde and acetylcholinesterase levels, alongwith preservation of glutathione peroxidase and superoxide dismutase activity. The docking studies further portrayed a possible interaction of detected phytoconstituents with acetylcholinesterase target. The results of the study show valuable therapeutic potential of phytoconstituents present in IS.CR to correct the neurological disarrays which might be through antioxidant activity or via modulation of GABAergic and cholinergic systems by artocommunol, 1,9-dideoxyforskolin and 6E,9E-octadecadienoic acid.     

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5ʹ (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.     

Maintenance of photosynthesis and the antioxidant defence systems have key roles for survival of Halopeplis perfoliata (Amaranthaceae) in a saline environment

• Coastal salt marsh plants employ various combinations of morphological and physiological adaptations to survive under saline conditions. Little information is available on salinity tolerance mechanisms of Halopeplis perfoliata, a C3 stem succulent halophyte.
• We investigated the growth, photosynthesis and antioxidant defence mechanisms of H. perfoliata under saline conditions (0, 150, 300 and 600 mM NaCl) in an open greenhouse.
• Optimal shoot succulence, projected shoot area and relative growth rate were obtained in the low (150 mm NaCl) salinity treatment, while growth was inhibited at the highest salinity (600 mm NaCl). The CO₂ compensation point and carbon isotope composition of biomass confirmed C₃ photosynthesis. Increases in salinity did not affect the photosynthetic pigment content or maximum quantum efficiency of PSII of H. perfoliata. Assimilation of CO₂ (A) also remained unaffected by salinity. A modest effect on some gas exchange and photochemistry parameters was observed at 600 mm NaCl. With increasing salinity, there was a continual increase in respiration, suggesting utilisation of energy to cope with saline conditions. Under 300 and 600 mm NaCl, there was an increase in H₂O₂ and MDA with a concomitant rise in AsA, GR content and CAT activity.
• Hence, H. perfoliata appears to be an obligate halophyte that can grow up to seawater salinities by modulating photosynthetic gas exchange, photochemistry and the antioxidant defence systems.

     

Bio-pretreatment of municipal solid waste prior to landfilling and its kinetics

The conventional landfilling does not promote sustainable waste management due to uncontrolled emissions which potentially degrade the environment. Pretreatment of municipal solid waste prior to landfilling significantly enhances waste stabilization, reduces the emissions and provides many advantages. Therefore, pretreatment of municipal solid waste methods were investigated. The major objectives of biological pretreatment are to degrade most easily degradable organic matters of MSW in a short duration under controlled conditions so as to produce desired quality for landfill. To investigate the suitable pretreatment method prior to landfilling for developing countries four pretreatment simulators were developed in the laboratory: (i) anaerobic simulator (R₁), (ii) aerobic pretreatment simulator by natural convection of air (R₂), (iii) aerobic pretreatment simulator by natural convection of air with leachate recirculation (R₃) and (iv) forced aeration and leachate recirculation (R₄). During the pretreatment organic matter, elemental composition, i.e., carbon, hydrogen, nitrogen and settlement were determined for bench scale experiments. A two-component kinetic model is proposed for the biodegradation of organic matter. Biodegradation kinetic constants were determined for readily and slowly degradable organic matter. The biodegradation of organic matter efficiency in terms of kinetic rate constants for the pretreatment simulators was observed as R₄ > R₃ > R₂ > R₁. Biodegradation rate constants for readily degradable matter in simulators R₄ and R₃ were 0.225 and 0.222 per day. R₃ and R₄ simulators were more effective in reducing methane emissions about 45% and 55%, respectively, as compared to anaerobic simulator R₁. Pretreatment of MSW, by natural convection of air with leachate recirculation R₃ is sustainable method to reduce the emissions and to stabilize the waste prior to landfilling.