High yield expression, characterization, and biological activity of IFNα2-Tα1 fusion protein.

The use of interferon α-2 in combination with thymosin α-1 shows higher anti-cancer effect in comparison when both are used individually because of their synergistic effects. In this study we produced an important human interferon α-2-thymosin α-1 (IFNα2-Tα1) fusion protein with probable pharmaceutical properties coupled to its high-level expression, characterization, and study of its biological activity. The IFNα2-Tα1 fusion gene was constructed by over-lap extension PCR and expressed in Escherichia coli expression system. The expression of IFNα2-Tα1 fusion protein was optimized to higher level and its maximum expression was obtained in modified terrific broth medium when lactose was used as inducer. The fusion protein was refolded into its native biologically active form with maximum yield of 83.14% followed by purification with ∼98% purity and 69% final yield. A band of purified IFNα2-Tα1 fusion protein equal to ∼23 kDa was observed on 12 % SDS-PAGE gel. The integrity of IFNα2-Tα1 fusion protein was confirmed by western blot analysis and secondary structure was assessed by CD spectroscopy. When IFNα2-Tα1 fusion protein was subjected to its biological activity analysis it was observed that it exhibits both IFNα2 & Tα1 activities as well as significantly higher anticancer activity as compared to IFNα-2 alone.

Hypoglycemic and hypolipidemic effects of Lactobacillus fermentum, fruit extracts of Syzygium cumini and Momordica charantia on diabetes induced mice.

A lot of treatment strategies available for diabetes but its complications are still a medical problem around the globe. It demands to find out some alternative therapeutic measures. In order to investigate the anti-diabetic potential of probiotics and natural extracts, this study was designed. Accordingly, a local source of yogurt probiotic strain Lactobacillus fermentum was isolated and characterized that showed its probiotic properties. Besides this, natural extracts of plants fruits like java plum (Syzygium cumini) and bitter gourd (M. charantia) were made. Lactobacillus fermentum and the extracts were administered individually as well as in combination to diabetes induced mice. Different parameters like body weight, blood glucose level and lipid profile including total cholesterol, HDL & LDL were analyzed before and after treatment. The results showed that Lactobacillus fermentum and natural extracts have hypoglycemic as well hypolipidemic activity against diabetic mice. This study can further investigated to screen potential compounds from these extracts to control the glucose and the lipid levels in diabetic patients.

Phytochemical, toxicological and antimicrobial evaluation of Lawsonia inermis extracts against clinical isolates of pathogenic bacteria.

BACKGROUND: The emerging resistance of pathogen against the currently available antimicrobial agents demands the search of new antimicrobial agents. The use of medicinal plants as natural substitute is the paramount area of research to overwhelm the drug resistance of infectious agents. Scientists have not made enough effort on the evaluation of safety of medicinal plant yet. METHODS: In the present study antimicrobial activity of Lawsonia inermis is investigated against clinical isolates of seven bacteria including four Gram negative (Escherichia coli, Salmonella typhi, Klebsiella spp., Shigella sonnei) and three Gram positive (Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis) using disc diffusion method. Four types of Lawsonia inermis extracts were prepared using methanol, chloroform, acetone and water as extraction solvents, while DMSO (Dimethyl sulfoxide) and water as dissolution solvents. The rate and extent of bacterial killing was estimated by time-kill kinetic assay at 1× MIC of each bacterial isolate. The overall safety of Lawsonia inermis extracts was assessed in mice. RESULTS: Lawsonia inermis displayed noteworthy antimicrobial activity against both gram positive and gram negative bacterial strains used in the study. The minimum value of MIC for different bacterial strains ranged from 2.31 mg/ml to 9.27 mg/ml. At 1x MIC of each bacterial isolate, 3log10 decrease in CFU was recorded after 6 hours of drug exposure and no growth was observed in almost all tested bacteria after 24 hours of exposure. No sign of toxidrome were observed during in vivo toxicity evaluation in mice at 300 mg/kg concentration. CONCLUSION: In conclusion, the present study provides the scientific rational for medicinal use of Lawsonia inermis. The use of Lawsonia inermis extracts is of great significance as substitute antimicrobial agent in therapeutics.

Anticancer medicines (Doxorubicin and methotrexate) conjugated with magnetic nanoparticles for targeting drug delivery through iron.

The uptake of iron is increased by cancer cells. Iron magnetic nanoparticles (MNP) can be used as a nanovehicle for immobilization of anticancer medicines and to integrate them at a target site. The anticancer medicines doxorubicin (DOX) and methotrexate (MTX) were immobilized separately and in combination onto MNP by a glutaraldehyde activation method and confirmed by magnetic nanoparticles linked immunosorbent assay (MagLISA) and Fourier-transform infrared (FTIR) spectroscopy. The phenol peaks of DOX and MTX at 2896.6 cm⁻¹ to 2912.5 cm⁻¹ in FTIR spectra of immobilized medicines indicated the conjugation. Affinity-purified anti-DOX and anti-MTX antibodies were used to evaluate the coupling of DOX and MTX onto MNP, and the binding was found 34.6% to 37.2% and 51.8% to 54.3% separately, respectively. The immobilization of DOX and MTX in combination onto MNP was 18% and 27%, respectively. HeLa and B cells were cultured with DOX-MNP, MTX-MNP, and DOX-MNP-MTX separately, and MagLISA indicated that the binding of DOX-MNP/MTX-MNP was 41.5% to 45% with HeLa cells and 20% to 26% with B cells. No significant difference was observed in binding of DOX-MNP-MTX with HeLa and B cells. Results also indicated that the release of medicines at pH 5.0 is more (39% to 44%) than at pH 7.4 (3.7% to 10.2%). Sixteen to 22% more killing effect was observed on HeLa cells than on B cells. In immunohistochemical staining, more deposition of brown color on HeLa cells than on B cells may be due to more expression of iron-binding sites on cancer cells. The dual property of MNP can be used for binding of medicines and for targeting drug delivery.

Identification of RdRp inhibitors against SARS-CoV-2 through E-pharmacophore-based virtual screening, molecular docking and MD simulations approaches.

The outbreak of novel Coronavirus, an enduring pandemic declared by WHO, has consequences to an alarming ongoing public health menace which has already claimed several million human lives. In addition to numerous vaccinations and medications for mild to moderate COVID-19 infection, lack of promising medication or therapeutic pharmaceuticals remains a serious concern to counter the ongoing coronavirus infections and to hinder its dreadful spread. Global health emergencies have called for urgency for potential drug discovery and time is the biggest constraint apart from the financial and human resources required for the high throughput drug screening. However, computational screening or in-silico approaches appeared to be an effective and faster approach to discover potential molecules without sacrificing the model animals. Accumulated shreds of evidence on computational studies against viral diseases have revealed significance of in-silico drug discovery approaches especially in the time of urgency. The central role of RdRp in SARS-CoV-2 replication makes it promising drug target to curtain on going infection and its spread. The present study aimed to employ E-pharmacophore-based virtual screening to reveal potent inhibitors of RdRp as potential leads to block the viral replication. An energy-optimised pharmacophore model was generated to screen the Enamine REAL DataBase (RDB). Then, ADME/T profiles were determined to validate the pharmacokinetics and pharmacodynamics properties of the hit compounds. Moreover, High Throughput Virtual Screening (HTVS) and molecular docking (SP & XP) were employed to screen the top hits from pharmacophore-based virtual screening and ADME/T screen. The binding free energies of the top hits were calculated by conducting MM-GBSA analysis followed by MD simulations to determine the stability of molecular interactions between top hits and RdRp protein. These virtual investigations revealed six compounds having binding free energies of -57.498, -45.776, -46.248, -35.67, -25.15 and -24.90 kcal/mol respectively as calculated by the MM-GBSA method. The MD simulation studies confirmed the stability of protein ligand complexes, hence, indicating as potent RdRp inhibitors and are promising candidate drugs to be further validated and translated into clinics in future.

Identification and purification of antigenic 34 kDa outer membrane protein of Salmonella typhi.

BACKGROUND: Salmonella Typhi is a pathogenic bacterium that causes a number of infectious diseases such as gastroenteritis and typhoid fever. In this study, an antigenic (34 kDa) protein was identified, purified, and characterized from outer membrane of Salmonella typhi. METHODS: Immunoblot analysis was used to screen antigenic proteins from outer membrane of Salmonella Typhi. Proteins from outer membrane were isolated and resolved on SDS-PAGE. In immunoblot analysis, four proteins with the following molecular weights of 60 kDa, 54 kDa, 34 kDa, and 26 kDa were identified as highly antigenic against the serum of patients suffering from typhoid fever. One of these outer membrane proteins, with a molecular mass of 34 kDa, was selected for this study. The 34 kDa protein was purified and characterized by a combination of anion exchange chromatography and gel permeation chromatography. The molecular weight of 34 kDa was determined using SDS-PAGE electrophoresis. The antigenic nature of the purified 34 kDa protein was determined by ELISA against serum proteins of patients suffering from typhoid fever and finally confirmed by immunoblot analysis. Antisera against the purified 34 kDa outer membrane protein of Salmonella typhi was produced and was used to recognize the epitope on the surface of an intact Salmonella typhi bacterium. RESULTS: The antigenic 34 kDa protein from the outer membrane of Salmonella typhi was identified, purified and characterized. The antigenecity of purified protein was confirmed by using antibodies present in serum of patient suffering from typhoid fever. It was also observed that antibody against 34 kDa outer membrane protein recognizes intact Salmonella typhi cells. CONCLUSIONS: It is established from the study that 34 kDa protein is antigenic in nature and antibody against this protein can also recognize epitopes on intact Salmonella typhi cells. Furthermore, this protein can be a good source material to produce vaccine against typhoid fever.

Hydrogel assisted synthesis of gold nanoparticles with enhanced microbicidal and in vivo wound healing potential.

The present study reports a hydrogel-based sunlight-assisted synthesis of gold nanoparticles (Au NPs) with enhanced antimicrobial and wound healing potential. The hydrogel extracted from the seeds of Cydonia oblonga was used as a reducing and capping agent to synthesize Au NPs for the first time. The as-synthesized Au NPs were characterized for an average size, shape, surface functionalization, antimicrobial, and wound healing capabilities. The cubic and rectangular-shaped Au NPs with an average edge length of 74 ± 4.57 nm depicted a characteristic surface plasmon resonance band at 560 nm. The hydrogel-based Au NPs inhibited the growth of microorganisms in zones with 12 mm diameter. In-vitro experiments showed that a minimum inhibitory concentration of Au NPs (16 µg/mL) was sufficient to mimic the 95% growth of pathogenic microorganisms in 24 h. In vivo treatment of wounds with Au NPs in murine models revealed a 99% wound closure within 5 days. Quantitative PCR analysis performed to decipher the role of Au NPs in enhanced wound healing showed an increase in the expression levels of NANOG and CD-34 proteins.

Interferon α2-Thymosin α1 Fusion Protein (IFNα2-Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect.

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2-thymosin α1 fusion protein (IFNα2-Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2-Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2-Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2-Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2-Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.

A Putative Prophylactic Solution for COVID-19: Development of Novel Multiepitope Vaccine Candidate against SARS-COV-2 by Comprehensive Immunoinformatic and Molecular Modelling Approach.

The outbreak of 2019-novel coronavirus (SARS-CoV-2) that causes severe respiratory infection (COVID-19) has spread in China, and the World Health Organization has declared it a pandemic. However, no approved drug or vaccines are available, and treatment is mainly supportive and through a few repurposed drugs. The urgency of the situation requires the development of SARS-CoV-2-based vaccines. Immunoinformatic and molecular modelling are time-efficient methods that are generally used to accelerate the discovery and design of the candidate peptides for vaccine development. In recent years, the use of multiepitope vaccines has proved to be a promising immunization strategy against viruses and pathogens, thus inducing more comprehensive protective immunity. The current study demonstrated a comprehensive in silico strategy to design stable multiepitope vaccine construct (MVC) from B-cell and T-cell epitopes of essential SARS-CoV-2 proteins with the help of adjuvants and linkers. The integrated molecular dynamics simulations analysis revealed the stability of MVC and its interaction with human Toll-like receptors (TLRs), which trigger an innate and adaptive immune response. Later, the in silico cloning in a known pET28a vector system also estimated the possibility of MVC expression in Escherichia coli. Despite that this study lacks validation of this vaccine construct in terms of its efficacy, the current integrated strategy encompasses the initial multiple epitope vaccine design concepts. After validation, this MVC can be present as a better prophylactic solution against COVID-19.

Development of latent Interferon alpha 2b as a safe therapeutic for treatment of Hepatitis C virus infection.

Interferon therapy for the treatment of hepatitis C virus infection has very limited clinical application due to short serum half-life and side effects of therapy in systemic route of administration. In the present study, we have focused to improve the interferon therapy by overcoming the limitation of side effects. We hypothesized that latent interferon alpha 2b (IFNα2b) produced by fusion of Latency associated protein (LAP) domain of TGFß and IFNα2b having HCV NS3 protease cleavage site as linker that will be activated only at target site (liver) by viral protease (HCV NS3 protease) present on the surface of infected cells. The fusion proteins were expressed in pichia pastoris as homodimer and cleaved by recombinant HCV NS3 protease in vitro into two fragments corresponding to the IFNα-2b and LAP respectively. The latency of chimeric proteins and biological activity after treatment with HCV NS3 protease was assessed by cytopathic effect inhibition assay in A594 cells infected with encephalomyocarditis virus (EMCV) and reduction in HCV viral load in Huh7 cells. The HCV NS3 protease was present on the surface of HCV replicating Huh7 cells in amount that activated half of the effective concentration (EC50) of latent IFNα2b fusion protein. As free circulating HCV NS3 protease was not detected in sera from chronic HCV patients and in vitro cleavage of intact latent IFNα2b fusion protein was not observed with peripheral blood mononuclear cells (PBMCs) isolated from chronic HCV patients, thus there are less likely chances of activation and off target binding of latent IFNα2b to show side effects during systemic route of administration. Therefore, most of the side effects of interferon can be overwhelmed at the cost of 50% reduced biological activity. Thus, the use of latent IFNα2b can be considered again as an option for treatment of HCV infection in combination with direct acting antivirals rather than alone with improved safety profile.

Use of Moringa oleifera Flower Pod Extract as Natural Preservative and Development of SCAR Marker for Its DNA Based Identification.

The use of Moringa oleifera as natural food preservative has been evaluated in the present study. In addition, for quality assurance, the study has also been focused on the shelf life of product to authenticate the identification of plant by development of DNA based marker. Among the different extracts prepared from flower pods of Moringa oleifera, methanol and aqueous extract exhibited high antibacterial and antioxidant activity, respectively. The high phenolic contents (53.5 ± 0.169 mg GAE/g) and flavonoid contents (10.9 ± 0.094 mg QE/g) were also recorded in methanol and aqueous extract, respectively. Due to instability of bioactive compounds in aqueous extract, methanol extract is considered as potent natural preservative. The shelf life of methanol extract was observed for two months at 4°C under dark conditions. The developed SCAR primers (MOF217/317/MOR317) specifically amplified a fragment of 317 bp from DNA of Moringa oleifera samples collected from different regions of Punjab province of Pakistan. The methanol extract of Moringa oleifera flower pods has great potential to be used as natural preservative and nutraceutical in food industry.

Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b.

Interferon alpha 2b (IFNα-2b) is an important cytokine and used for antiviral and anticancer treatment. The low cost production of IFNα-2b with high biological activity is necessary to provide the interferon therapy to the hepatitis patients in Pakistan. In the present study, human interferon alpha 2b (hIFNα-2b) gene from a healthy person was cloned and overexpressed in E. coli BL21(DE3). The molecular weight of the expressed hIFNα-2b is 19 kDa. The over expressed recombinant hIFNα-2b was checked by ELISA using antibodies raised against commercially available hIFNα-2b. The biocomputational analysis of recombinant hIFNα-2b gene showed the 99.9% nucleotide sequence and 100% deduced amino acid sequence homology with reported sequences of IFNα-2b. The predicted 3D-structure showed mainly five α-helices, one 310 helix and two disulfide bonds at Cys1-Cys98 and Cys129-Cys138. The amino acid sequence alignment indicated that the disulfide linkage position is conserved in all IFNα family members. On the basis of sequence homology among interferon alpha family, new potent variants of hIFNα-2b with enhance efficacy can be produced. Indigenous production of IFNα-2b from gene of local population will reduce the cost and increase tolerability of interferon therapy.