Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein.

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (KD) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (KD = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.

Production and immunogenicity of Fubc subunit protein redesigned from DENV envelope protein.

Dengue virus (DENV) is a vector-borne human pathogen that usually causes dengue fever; however, sometime it leads to deadly complications such as dengue with warning signs (DWS+) and severe dengue (SD). Several studies have shown that fusion (Fu) and bc loop of DENV envelope domain II are highly conserved and consist some of the most dominant antigenic epitopes. Therefore, in this study, Fu and bc loops were joined together to develop a short recombinant protein as an alternative of whole DENV envelope protein, and its immunogenic potential as fusion peptide was estimated. For de novo designing of the antigen, Fu and bc peptides were linked with an optimised linker so that the three dimensional conformation was maintained as it is in DENV envelope protein. The redesigned Fubc protein was expressed in E. coli and purified. Subsequently, structural integrity of the purified protein was verified by CD spectroscopy. To characterise immune responses against recombinant Fubc protein, BALB/c mice were subcutaneously injected with emulsified antigen preparation. It was observed by ELISA that Fubc fusion protein elicited higher serum IgG antibody response either in the presence or in absence of Freund's adjuvant in comparison to the immune response of Fu and bc peptides separately. Furthermore, the binding of Fubc protein with mice antisera was validated by SPR analysis. These results suggest that Fu and bc epitope-based recombinant fusion protein could be a potential candidate towards the development of the effective subunit vaccine against DENV.

Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process.

BACKGROUND: The production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein. RESULTS: It was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation-refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer. CONCLUSIONS: Maltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.

Performances of Machine Learning Models for Diagnosis of Alzheimer's Disease.

In recent times, various machine learning approaches have been widely employed for effective diagnosis and prediction of diseases like cancer, thyroid, Covid-19, etc. Likewise, Alzheimer's (AD) is also one progressive malady that destroys memory and cognitive function over time. Unfortunately, there are no dedicated AI-based solutions for diagnoses of AD to go hand in hand with medical diagnosis, even though multiple factors contribute to the diagnosis, making AI a very viable supplementary diagnostic solution. This paper reports an endeavor to apply various machine learning algorithms like SGD, k-Nearest Neighbors, Logistic Regression, Decision tree, Random Forest, AdaBoost, Neural Network, SVM, and Naïve Bayes on the dataset of affected victims to diagnose Alzheimer's disease. Longitudinal collections of subjects from OASIS dataset have been used for prediction. Moreover, some feature selection and dimension reduction methods like Information Gain, Information Gain Ratio, Gini index, Chi-Squared, and PCA are applied to rank different factors and identify the optimum number of factors from the dataset for disease diagnosis. Furthermore, performance is evaluated of each classifier in terms of ROC-AUC, accuracy, F1 score, recall, and precision as well as included comparative analysis between algorithms. Our study suggests that approximately 90% classification accuracy is observed under top-rated four features CDR, SES, nWBV, and EDUC.

Spontaneous gallbladder perforation occurring at neck in an 8-year old boy.

Spontaneous gallbladder perforation (GBP) is a rare condition. Most of these perforations occur at the fundal region of the gallbladder. Perforation occurring at the neck of the gallbladder seems to be the rarest phenomenon. We herein describe a case of spontaneous GBP occurring at the neck of gallbladder in an 8-year-old boy, which was managed satisfactorily by surgical exploration and cholecystectomy.

Designing antibody against highly conserved region of dengue envelope protein by in silico screening of scFv mutant library.

Dengue being one of the deadliest diseases of tropical regions, enforces to put continuous efforts for the development of vaccine and effective therapeutics. Most of the antibodies generated during dengue infection are non-neutralizing and cause antibody dependent enhancement. Hence, making a potent neutralizing antibody against all four dengue serotypes could be very effective for the treatment. However, designing a single antibody for all serotypes is difficult due to variation in protein sequences. Therefore, the objective is to identify conserved region of dengue envelope protein and then develop an antibody against that conserved region. Before advancing to the development of such an antibody, it is desirable to validate the interactions between antibody and dengue envelope protein. In silico analysis of such interactions provides a good platform to find out a suitable region to design and construct an antibody against it by analyzing antigen-antibody interaction before synthesizing the antibody. In this study, two highly conserved regions of dengue envelope protein were identified and an scFv was constructed against it. Both scFv and FuBc proteins were expressed in bacterial expression system and binding efficiency was analyzed by SPR analysis with KD value 2.3 µM. In order to improve binding efficiency, an in silico scFv mutant library was created which was virtually screened for higher binding efficiency. Six mutants with high binding efficiency were selected for further analysis. The binding ability of these mutants were predicted using simulation analysis which shows these mutations were stabilizing scFv-FuBc complex.

Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells.

Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 µM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and ß-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 µM and 5-fold with 100 µM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells.

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock.

Paratuberculosis (pTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) in a wide variety of domestic and wild animals. Control of pTB is difficult due to the lack of sensitive, efficacious and cost-effective diagnostics and marker vaccines. Microscopy, culture, and PCR have been used for the screening of MAP infection in animals for quite a long time. Besides, giving variable sensitivity and specificity, these tests have not been considered ideal for large-scale screening of domestic livestock. Serological tests like ELISA easily detects anti-MAP antibodies. However, it cannot differentiate between the vaccinated and infected animals. Nanotechnology-based diagnostic tests are underway to improve the sensitivity and specificity. Newer generation diagnostic tests based on recombinant MAP secretory proteins would open new paradigm for the differentiation between infected and vaccinated animals and for early detection of the infection. Due to higher seroreactivity of secretory proteins vis-à-vis cellular proteins, the secretory proteins may be used as marker vaccine, which may aid in the control of pTB infection in animals. Secretory proteins can be potentially used to develop future diagnostics, surveillance and monitoring of the disease progression in animals and the marker vaccine for the control and eradication of pTB.

Chitinases from Bacteria to Human: Properties, Applications, and Future Perspectives.

Chitin is the second most plenteous polysaccharide in nature after cellulose, present in cell walls of several fungi, exoskeletons of insects, and crustacean shells. Chitin does not accumulate in the environment due to presence of bacterial chitinases, despite its abundance. These enzymes are able to degrade chitin present in the cell walls of fungi as well as the exoskeletons of insect. They have shown being the potential agents for biological control of the plant diseases caused by various pathogenic fungi and insect pests and thus can be used as an alternative to chemical pesticides. There has been steady increase in demand of chitin derivatives, obtained by action of chitinases on chitin polymer for various industrial, clinical, and pharmaceutical purposes. Hence, this review focuses on properties and applications of chitinases starting from bacteria, followed by fungi, insects, plants, and vertebrates. Designing of chitinase by applying directed laboratory evolution and rational approaches for improved catalytic activity for cost-effective field applications has also been explored.