A rapid novel strategy for screening of antibody phage libraries for production, purification, and functional characterization of amber stop codons containing single-chain antibody fragments.

Phage display antibody (PDA) libraries, allows the rapid isolation and characterization of high specificity monoclonal antibodies for therapeutic and diagnostic applications. However, selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias towards clones containing randomly generated amber stop codons, complicating the identification of high affinity binding antibodies. We screened Tomlinson I and J library against receptor binding domain (RBD) of SARS CoV2, eight clones which showed positive binding in phage ELISA, contained one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences. The presence of amber stop codons within the antibody sequence causes the premature termination of soluble form of scFv expression in nonsuppressor Escherichia coli strain. In the present study, we have used a novel strategy that allows soluble expression of scFvs having amber stop codon in their gene sequences (without phage PIII protein fusion), in the suppressor strain. This strategy of introduction of Ochre (TAA) codon at the junction of scFv and PIII gene, speeds up the initial screening process which is critical for selecting the right scFvs for further studies. Present strategy leads to the identification of a scFv, B8 that binds specifically with nanomolar affinity toward SARS CoV 2 RBD, which otherwise lost in terms of traditional methodology.

Gut microbiome: Microflora association with obesity and obesity-related comorbidities.

Obesity and obesity-related comorbidities have transformed into a global epidemic. The number of people suffering from obesity has increased dramatically within the past few decades. This rise in obesity cannot alone be explained by genetic factors; however, diet, environment, lifestyle, and presence of other diseases undoubtedly contribute towards obesity etiology. Nevertheless, evidence suggests that alterations in the gut microbial diversity and composition have a role to play in energy assimilation, storage, and expenditure. In this review, the impact of gut microbiota composition on metabolic functionalities, and potential therapeutics such as gut microbial modulation to manage obesity and its associated comorbidities are highlighted. Optimistically, an understanding of the gut microbiome could facilitate the innovative clinical strategies to restore the normal gut flora and improve lifestyle-related diseases in the future.

Prevention and Control Strategies to Counter Dengue Virus Infection.

Dengue is currently the highest and rapidly spreading vector-borne viral disease, which can lead to mortality in its severe form. The globally endemic dengue poses as a public health and economic challenge that has been attempted to suppress though application of various prevention and control techniques. Therefore, broad spectrum techniques, that are efficient, cost-effective, and environmentally sustainable, are proposed and practiced in dengue-endemic regions. The development of vaccines and immunotherapies have introduced a new dimension for effective dengue control and prevention. Thus, the present study focuses on the preventive and control strategies that are currently employed to counter dengue. While traditional control strategies bring temporary sustainability alone, implementation of novel biotechnological interventions, such as sterile insect technique, paratransgenesis, and production of genetically modified vectors, has improved the efficacy of the traditional strategies. Although a large-scale vector control strategy can be limited, innovative vaccine candidates have provided evidence for promising dengue prevention measures. The use of tetravalent dengue vaccine (CYD-TDV) has been the most effective so far in treating dengue infections. Nonetheless, challenges and limitation hinder the progress of developing integrated intervention methods and vaccines; while the improvement in the latest techniques and vaccine formulation continues, one can hope for a future without the threat of dengue virus.

Mutations in the reverse transcriptase and protease genes of human immunodeficiency virus-1 from antiretroviral naïve and treated pediatric patients.

The success of highly active antiretroviral therapy (HAART) is challenged by the emergence of resistance-associated mutations in human immunodeficiency virus-1 (HIV-1). In this study, resistance associated mutations in the reverse transcriptase (RT) and protease (PR) genes in antiretroviral therapy (ART) naïve and treated HIV-1 infected pediatric patients from North India were evaluated. Genotyping was successfully performed in 46 patients (30 ART naive and 16 treated) for the RT gene and in 53 patients (27 ART naive and 26 treated) for PR gene and mutations were identified using Stanford HIV Drug Resistance Database. A major drug resistant mutation in RT gene, L74I (NRTI), and two such mutations, K101E and G190A (NNRTI), were observed in two ART naïve patients, while M184V was detected in two ART treated patients. Overall, major resistance associated mutations in RT gene were observed in nine (30%) and seven (36%) of ART naïve and treated children respectively. Minor mutations were identified in PR gene in five children. Few non-clade C viral strains (≈30%) were detected, although subtype C was most predominant. The screening of ART naïve children for mutations in HIV-1 RT and protease genes, before and after initiation of ART is desirable for drug efficacy and good prognosis.