Antimicrobial resistance (AMR) poses a significant threat to the health, social, environment, and economic sectors on a global scale and requires serious attention to addressing this issue. Acinetobacter baumannii was given top priority among infectious bacteria because of its extensive resistance to nearly all antibiotic classes and treatment options. Carbapenem-resistant A. baumannii is classified as one of the critical-priority pathogens on the World Health Organization (WHO) priority list of antibiotic-resistant bacteria for effective drug development. Although available genetic manipulation approaches are successful in A. baumannii laboratory strains, they are limited when employed on newly acquired clinical strains since such strains have higher levels of AMR than those used to select them for genetic manipulation. Recently, the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as one of the most effective, efficient, and precise methods of genome editing and offers target-specific gene editing of AMR genes in a specific bacterial strain. CRISPR-based genome editing has been successfully applied in various bacterial strains to combat AMR; however, this strategy has not yet been extensively explored in A. baumannii. This review provides detailed insight into the progress, current scenario, and future potential of CRISPR-Cas usage for AMR-related gene manipulation in A. baumannii.

Extracts of Plumeria obtusa are widely used in ethnomedicine and have been investigated for a variety of biological activities; however, the antimicrobial activity of P. obtusa flowers is poorly characterized. In this study, the antimicrobial activities of different solvents (petroleum ether, ethyl acetate, chloroform, isobutanol and ethanol) extracts from flowers of P. obtusa were investigated by a disc diffusion method against Gram-positive bacteria, Gram-negative bacteria and a fungus. All extracts exhibited growth inhibition of all microorganisms at variable degrees as measured by relative zones of inhibition, however, the petroleum ether extract was ineffective against Klebsiella pneumonia and ethyl acetate and isobutanol extracts were ineffective against Pseudomonas aeruginosa. The most susceptible Gram-positive bacterium was Bacillus subtilis while the most resistant Gram-positive bacterium was Staphylococcus aureus. Erwinia carotovora was the most susceptible Gram-negative bacterium while P. aeruginosa was highly resistant among the Gram-negative bacteria. In this study, for the first time, we investigated the antimicrobial activity of several different solvent extracts from flowers of P. obtusa against a broad spectrum of human-pathogenic microorganisms. These compounds warrant further investigation by isolation and structural elucidation with the aim to find novel and affordable bioactive compounds for the treatment of infectious diseases.

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K m, k cat, and k cat/K m for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K m, k cat, and k cat/K m for Ac-nKRR-amc substrate were 100 μM, 0.112 s−1, and 1120 M−1·s−1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.

The present research was conducted to study heavy metal accumulation and other physiological and biochemical parameters in three different field crops i.e. Alfalfa, Brassica juncea and Chickpea. The samples were collected from the field-grown crops at Malakandher Research Farm of Khyber Pakhtunkhwa Agricultural University Peshawar. Plant materials were analyzed for different physiological and biochemical parameters along with the determination of heavy metal accumulation by the collected samples of Medicago sativa L. Brassica juncea (L.) Czern. and Cicer arietinum L. . The data revealed that maximum plant fresh weight was recorded by Chickpea while minimum plant fresh weight was noted for Brassica juncea. Maximum plant dry weight was noted for Alfalfa and minimum plant dry weight was produced by Chickpea. Higher concentration of proline was noted in Brassica juncea while Chickpea produced lowest proline concentration. Maximum DNA concentration was noted in Chickpea while minimum DNA concentration was noted in Chickpea. Chickpea had the highest DNA purity. Minimum DNA purity was noted in Brassica juncea. Highest concentration protein was recorded by Brassica juncea while lowest protein concentration was noted for Chickpea. The data also showed that Chickpea recorded maximum Cd concentration. Minimum Cd concentration was noted in Alfalfa. Highest concentration of Cr was noted in Chickpea. Minimum Cr concentration was recorded by Alfalfa. Maximum levels of Pb was accumulated by Chickpea. Minimum levels of Pb were noted in Brassica juncea.

The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (kcat) and a lower Km value than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities.