Hybrid Warfare and its Impacts on Pakistan

In modern times, the conventional means of warfare are increasingly becoming less usable. However, the states are involved in waging hybrid warfare to the maximum to fulfill their foreign policy goals. In nuclearized South Asia, direct war between India and Pakistan seems unlikely given that both the states know that escalation could lead to nuclear catastrophe in the region. This compels both the states to find other means of warfare to undermine each other’s interests. India wants to weaken Pakistan so that it may abandon claim on Indian occupied Jammu and Kashmir. For that, India is using all tools of hybrid warfare against Pakistan. In this context, this paper aims at to unearth India’s hybrid warfare in the region and its implications for Pakistan. The main focus of the paper is to explain tools and methods of India hybrid warfare. At the same time the research also tries to unravel few other case studies. It also notes how Pakistan can counter hybrid threats posed by its arch rival.

Exploitation of Aspergillus and Cladosporium Species for Biologically Active Secondary Metabolites

Aspergillus carbonarius (NRRL–369) and Aspergillus oryzae from Aspergillus genus as well as Cladosporium carrionii and Cladosporium resinae (NRRL–6437) from Cladosporium genus were selected for the present study. Nutrient media were optimized for the growth and production of secondary metabolites. Out of five different media used, A. carbonarius and A. oryzae produced relatively more metabolites in Czapek–dox (Glucose and Starch) broth media (CGSB). Whereas; C. carrionii and C. resinae produced relatively more metabolites in Czapek yeast extracts broth (CYB). To further increase secondary metabolites productivity, two additional chemical compounds (suberoyl anilide hydroxamic acid; SAHA and 5–azacytidine; 5–AZA) were also used as chemical inducers for all fungi except C. carrionii. A dose of 10 μM/100 mL of SAHA resulted in higher secondary metabolites production from Aspergillus species and 15 μM/100 mL of SAHA resulted in higher secondary metabolites production from C. resinae. While a dose 15 μM/100 mL of 5–AZA resulted in higher secondary metabolites production from all the species. Secondary metabolites produced were then studied for its respective biological activities. In antibacterial assay a dose of 500 μg/mL of ethyl acetate extracted from A. carbonarius inhibited the growth of B. subtilis (64.5%), while for antifungal testing a dose of 1000 μg/mL ethyl acetate extract inhibited the linear growth of C. glabrata (58.5%). Whereas, in cytotoxic activities, dose of 1000 μg/mL of ethyl acetate extract showed 94% mortality against brine shrimps, while for phytotoxic activities, a dose 1000 μg/mL showed 90% mortality against Lemna. A dose of 500 μg/mL of ethyl acetate extracted from A. oryzae inhibited the growth of B. subtilis (94%), while for antifungal testing, a dose of 1000 μg/mL of ABSTRACT xxi ethyl acetate extract inhibited the linear growth of M. Canis (84%). Whereas, in cytotoxic activities a dose of 1000 μg/mL of ethyl acetate extract showed 52% mortality against brine shrimps, while for phytotoxic activities, a dose of 1000 μg/mL of ethyl acetate extract showed 67% mortality against Lemna. Furthermore, during the antibacterial assay a dose of 500 μg/mL of ethyl acetate extracted from C. carrionii inhibited the growth of B. subtilis (66%), while for antifungal testing a dose of 1000 μg/mL ethyl acetate extract inhibited the growth of C. albicans (60%). Whereas, in cytotoxic activities a dose of 1000 μg/mL of ethyl acetate extract showed 87% mortality against brine shrimps, while for phytotoxic activities, a dose of 1000 μg/mL ethyl acetate extract showed 80% mortality against Lemna. Finally during the antibacterial assay a dose of 500 μg/mL of ethyl acetate extracted from C. resinae inhibited the growth of S. aureus (81%), while for antifungal testing a dose of 1000 μg/mL of ethyl acetate extract inhibited the growth of A. flavus (15%), while in cytotoxic activities a dose of 1000 μg/mL of ethyl acetate showed 93% mortality against brine shrimps, while for phytotoxic activities, a dose of 1000 μg/mL of ethyl acetate showed 80% mortality against Lemna. The biological activities indicates that, the extracts from A. oryzae and C. carrionii inhibited the growth of experimental organisms with greater extent as compared to A. carbonarius and C. resinae; therefore, A. oryzae and C. carrionii were further selected for the isolation of pure metabolites. A total of three new and four known metabolites were isolated. Two new metabolites were isolated from A. oryzae while one new and four known metabolites were isolated from C. carrionii using preparative High Performance Liquid Chromatography (HPLC) and column chromatography techniques. The structures of all the compounds isolated were ABSTRACT xxii elucidated using (1D and 2D) NMR, IR and HR–MS techniques. The new metabolites were 6–butyl–3–methylene–2–oxotetrahydro–2H–pyran–4–carboxylic acid (A–41), 6–butyl–3–methylene–2–oxo–3,6–dihydro–2H–pyran–4–carboxylic acid (A–42) and (3S,6S)–3–allyl–6–benzylpiperazine–2,5–dione (D–44) whereas, the known metabolites were 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (C–43), 6–(3– methylbut–2–enyl)–1H–indole–3–carboxylic acid (45), 2-(4,6-dihydroxy-3-oxo-1,3- dihydroisobenzofuran-1-yl) acetic acid (46) and 2-(4-hydroxy-1,3- dihydroisobenzofuran-1-yl) acetic acid (47). The two new metabolites (A–41 and B–42) from A. oryzae were selected for the determination of their biosynthetic pathways using [1– 13C] labelled acetate. The [1– 13C] labelled acetate was added to the media on 4th, 5th and 6th days respectively. After the feeding of isotopic [1– 13C] labelled acetate as precursor, the labelled metabolites were isolated using HPLC and the pattern of their incorporation were determined using high field NMR. The basic idea of the present work was to isolate biologically active secondary metabolite(s) from fungi and to produce good quality of antibiotics for the welfare of the society.