Malik Mobeen Ahmad

Chitinases: An update

Chitin, the second most abundant polysaccharide in nature after cellulose, is found in the exoskeleton of insects, fungi, yeast, and algae, and in the internal structures of other vertebrates. Chitinases are enzymes that degrade chitin. Chitinases contribute to the generation of carbon and nitrogen in the ecosystem. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications, especially the chitinases exploited in agriculture fields to control pathogens. Chitinases have a use in human health care, especially in human diseases like asthma. Chitinases have wide-ranging applications including the preparation of pharmaceutically important chitooligosaccharides and N-acetyl D glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, mosquito control and morphogenesis, etc. In this review, the various types of chitinases and the chitinases found in different organisms such as bacteria, plants, fungi, and mammals are discussed.

Advances in molecular detection of Aspergillus: an update

Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.

Fusarium solani, P1, a new endophytic podophyllotoxin-producing fungus from roots of Podophyllum hexandrum

Podophyllotoxin, a well-known naturally occurring aryl tetralin lignan produced by few plant species is used as precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophos phosphate. The availability of this lignan is limited due to the scarce occurrence of its natural sources. Further, synthetic approaches for its production are still commercially unacceptable. This paper reports the synthesis of podophyllotoxin by an endophytic fungus Fusarium solani isolated from the roots of Podophyllum hexandrum. The presence of podophyllotoxin in fungal biomass was confirmed and quantified by HPLC and mass spectrometry. The fungus is able to produce 29.0 µg/g podophyllotoxin on dry weight basis.

Efficient Method for Agrobacterium Mediated Transformation of Artemisia annua L.

Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.

Methods in Transgenic Technology

Genetic engineering had turned into a basic instrument reproducing of yields. In the course of the most recent decade, a significant advancement has been made in new and efficient transformation strategies in plants. In spite of an assortment of accessible DNA delivery techniques, Agrobacterium-mediated transformation remains a prevalently utilized methodology. Specifically, advance in Agrobacterium-mediated transformation of grains and other recalcitrant dicot species has been very noteworthy. Meanwhile, other transgenic-empowering advancements have developed, including gateway-based method, generation of marker-free transgenics, gene targeting, and chromosomal engineering. Despite the fact that alteration of some plant species or elite germplasm remains a challenge, further headway in transformation innovation is expected on the grounds that the components of overseeing the recovery and transformation procedures are currently better comprehended and are as a rule innovatively connected to outlining enhanced transformation techniques or to growing new empowering advancements.

RNAi-mediated modulation of squalene synthase gene expression in Artemisia annua L. and its impact on artemisinin biosynthesis

Artemisinin, an endoperoxide sesquiterpene lactone, is an antimalarial phytoconstituent isolated from Artemisia annua L. plants. Artemisinin-based combination therapies (ACTs) are recommended by WHO for treating malaria caused by multidrug-resistant Plasmodium falciparum sp. The main cause of instability in supply and cost escalation of ACTs in countries endemic to malaria is low content (0.01–1.1%) of artemisinin in these plants. In this study, the expression of SQS gene encoding squalene synthase (SQS), a key enzyme of the sterol biosynthetic pathway that competes for carbon precursor with that of artemisinin biosynthetic pathway was suppressed by hpRNAi-technology in A. annua to study its impact on artemisinin biosynthesis. Southern blot analyses of transgenic lines obtained have shown single (TR2, TR4 and TR10) and multiple copies (TR1, TR3, TR7, TR8 and TR9) of transgenes in their genomes. The GC/MS analysis showed decreased in squalene content by 65.7%, while HPLC result showed an increase in artemisinin content by 40.2% in transgenic lines, TR3. The plants from TR3 were also grown in environmentally controlled polyhouse and yielded 28.3% higher artemisinin as compared to the non-transgenic plants. The increased artemisinin levels coupled with reduced squalene contents in the transgenic lines of A. annua were well correlated with the up-regulation of artemisinin biosynthetic pathway genes and suppression of SQS in the qPCR analyses.

Development of standard operating procedure and standardization of Habb-e-Banafsha Qawi-A Unani polyherbal formulation

Introduction: Herbals drugs became a boon for mankind since ancient times and still are used worldwide for the treatment of various human ailments. The safety of alternative medicinal preparations has been questioned due to reports of unwanted side effects. To maintain the quality and efficacy of these drugs, it is essential to standardize them in order to avoid the use of substandard or adulterated medicines in the market. Unani system of medicine mainly focuses on the treatment by natural drugs. Habb-e-Banafsha Qawi is useful in a cough, catarrah, and coryza. Materials and Methods: Physiochemical constants of Habb-e-Banafsha Qawi were determined through organoleptic characters, macro- and micro-scopic characters, ash value, solubility, pH values. Chromatographic fingerprints were developed using thin layer chromatography method. Aflatoxin (AF) contamination, heavy metal, and pesticide residues were also evaluated by standard methods. Objectives: In the present study, an attempt has been made to develop standard operating procedure and physiochemical parameters to monitor the quality of a Unani poly-herbal formulation, Habb-e-Banafsha Qawi. Results: The tablets tasted sweetish bitter with a pleasant odor, water soluble and acidic in nature. Rf values were almost same in all the extracts. No AF, heavy metal, and pesticide residues were observed. Conclusions: It may be concluded that the values and chromatographic fingerprints obtained can be used for quality evaluation and to set new pharmacopoeial standards for the preparation of Habb-e-Banafsha Qawi.