Development of a process for large scale production of PfRH5 in E. coli expression system.

The Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) has recently shown great promise to be developed as a vaccine candidate to prevent blood-stage malaria. However, because of its molecular complexity, most previous efforts were focused on expressing PfRH5 in its native and soluble form. Here, we describe the E. coli expression of full-length PfRH5 as inclusion bodies (IBs), followed by its high cell density fermentation at 1, 5 and 30 L scale. Denatured full-length PfRH5 was purified using a two-step chromatography process before being refolded using design of experiments (DoE). Refolded PfRH5 was further purified using size exclusion chromatography (SEC), recovering high purity antigen with an overall yield of 102 mg/L from fermentation cell harvest. Purified PfRH5 was further characterized using orthogonal analytical methods, and a short-term stability study revealed -80 °C as an optimum storage temperature. Moreover, refolded, and purified PfRH5, when formulated with adjuvant Glucopyranosyl A lipid stable emulsion (GLA-SE), elicited high antibody titers in BALB/c mice, proving its potential to neutralize the blood-stage malarial parasite. Here, we establish an E. coli-based process platform for the large-scale cGMP production of full-length PfRH5, enabling global malaria vaccine development efforts.

Insights into interplay of immunopathophysiological events and molecular mechanistic cascades in psoriasis and its associated comorbidities.

Psoriasis is an inflammatory skin disease with complex pathogenesis and multiple etiological factors. Besides the essential role of autoreactive T cells and constellation of cytokines, the discovery of IL-23/Th17 axis as a central signaling pathway has unraveled the mechanism of accelerated inflammation in psoriasis. This has provided insights into psoriasis pathogenesis and revolutionized the development of effective biological therapies. Moreover, genome-wide association studies have identified several candidate genes and susceptibility loci associated with this disease. Although involvement of cellular innate and adaptive immune responses and dysregulation of immune cells have been implicated in psoriasis initiation and maintenance, there is still a lack of unifying mechanism for understanding the pathogenesis of this disease. Emerging evidence suggests that psoriasis is a high-mortality disease with additional burden of comorbidities, which adversely affects the treatment response and overall quality of life of patients. Furthermore, changing trends of psoriasis-associated comorbidities and shared patterns of genetic susceptibility, risk factors and pathophysiological mechanisms manifest psoriasis as a multifactorial systemic disease. This review highlights the recent progress in understanding the crucial role of different immune cells, proinflammatory cytokines and microRNAs in psoriasis pathogenesis. In addition, we comprehensively discuss the involvement of various complex signaling pathways and their interplay with immune cell markers to comprehend the underlying pathophysiological mechanism, which may lead to exploration of new therapeutic targets and development of novel treatment strategies to reduce the disastrous nature of psoriasis and associated comorbidities.

Insight into Structure-Function Relationships of ß-Lactamase and BLIPs Interface Plasticity using Protein-Protein Interactions.

BACKGROUND: Mostly BLIPs are identified in soil bacteria Streptomyces and originally isolated from Streptomyces clavuligerus and can be utilized as a model system for biophysical, structural, mutagenic and computational studies. BLIP possess homology with two proteins viz., BLIP-I (Streptomyces exofoliatus) and BLP (beta-lactamase inhibitory protein like protein from S. clavuligerus). BLIP consists of 165 amino acid, possessing two homologues domains comprising helix-loop-helix motif packed against four stranded beta-sheet resulting into solvent exposed concave surface with extended four stranded beta-sheet. BLIP-I is a 157 amino acid long protein obtained from S. exofoliatus having 37% sequence identity to BLIP and inhibits beta-lactamase. METHODS: This review is intended to briefly illustrate the beta-lactamase inhibitory activity of BLIP via proteinprotein interaction and aims to open up a new avenue to combat antimicrobial resistance using peptide based inhibition. RESULTS: D49A mutation in BLIP-I results in a decrease in affinity for TEM-1 from 0.5 nM to 10 nM (Ki). It is capable of inhibiting TEM-1 and bactopenemase and differs from BLIP only in modulating cell wall synthesis enzyme. Whereas, BLP is a 154 amino acid long protein isolated from S. clavuligerus via DNA sequencing analysis of Cephamycin-Clavulanate gene bunch. It shares 32% sequence similarity with BLIP and 42% with BLIP-I. Its biological function is unclear and lacks beta-lactamase inhibitory activity. CONCLUSION: Protein-protein interactions mediate a significant role in regulation and modulation of cellular developments and processes. Specific biological markers and geometric characteristics are manifested by active site binding clefts of protein surfaces which determines the specificity and affinity for their targets. TEM1.BLIP is a classical model to study protein-protein interaction. ß-Lactamase inhibitory proteins (BLIPs) interacts and inhibits various ß-lactamases with extensive range of affinities.

Structure-based drug designing and identification of Woodfordia fruticosa inhibitors targeted against heat shock protein (HSP70-1) as suppressor for Imiquimod-induced psoriasis like skin inflammation in mice model.

Heat shock proteins (HSPs) emerged as a therapeutic target and it was observed that inhibition of HSP70-1 plays a pivotal role in the management of psoriasis. In-silico investigation involving techniques like molecular docking and molecular dynamics (MD) simulation analysis was performed against HSP70-1. Further, anti-psoriatic activity of bioactive immunomodulatory compounds present in ethanolic extract of Woodfordia fruticosa flowers (Wffe) using combination of bioinformatics together with ethnopharmacological approach has been explored in this study. Myricetin (-8.024), Quercetin (-7.368) and Ellagic acid (-7.311) were the top three compounds with minimum energy levels as well as high therapeutic value/ADMET as compared to currently available marketed anti-psoriatic drug Tretinoin (-7.195). ADMET prediction was used to screen ligands for drug-likeness and efficacy. Further, biogenically Woodfordia fruticosa gold nanoparticles (WfAuNPs) were synthesized and characterized by UV-Visible Spectroscopy (UV-vis), Dynamic Light Scattering (DLS), Zeta Potential, X-Ray Diffraction (XRD) and High Resolution Transmission Electron Microscopy (HRTEM) techniques. Synthesized WfAuNPs observed in the size range of 10-20 nm and were used to develop WfAuNPs-Carbopol®934 ointment gel. Subsequently, the therapeutic efficacy of WfAuNPs-Carbopol® 934 was checked against 5% Imiquimod-induced psoriasis like skin inflammation. WfAuNPs-Carbopol® 934 was found to be exerting better therapeutic effect in reducing the mean DAI score (0.63 ±â€¯0.08), serum cytokines (TNF-α, IL-22 and IL-23) levels along with reduced epidermal thickness, parakeratosis and marked decrease in the hyperproliferation of keratinocytes. Results of the study revealed that the WfAuNPs-Carbopol® 934 could be an effective alternative treatment for psoriasis in near future.

Application of Computational Techniques to Unravel Structure-Function Relationship and their Role in Therapeutic Development.

Application of computational tools and techniques has emerged as an invincible instrument to unravel the structure-function relationship and offered better mechanistic insights in the designing and development of new drugs along with the treatment regime. The use of in silico tools equipped modern chemist with armamentarium of extensive methods to meticulously comprehend the structural tenacity of receptor-ligand interactions and their dynamics. In silico methods offers a striking property of being less resource intensive and economically viable as compared to experimental evaluation. These techniques have proved their mettle in the designing of potential lead compounds to combat life-threatening diseases such as AIDS, cancer, tuberculosis, malaria, etc. In the present scenario, computer-aided drug designing has ascertained an essential and indispensable gizmo in therapeutic development. This review will present a brief outline of computational methods used at different facets of drug designing and its latest advancements. The aim of this review article is to briefly highlight the methodologies and techniques used in structure-based/ ligand-based drug designing viz., molecular docking, pharmacophore modeling, density functional theory, protein-hydration and molecular dynamics simulation which helps in better understanding of macromolecular events and complexities.

In-vivo sustained release of nanoencapsulated ferulic acid and its impact in induced diabetes.

Diabetes mellitus (DM) is one of the most common lifestyle diseases, caused due to endocrine disorder. DM is commonly associated with hyperglycemia, a condition which is generally followed by an overproduction of free radicals leading to tissue oxidative stress. Currently, the focus of medical fraternity lies in developing therapeutic drugs based on natural origin in order to reduce the hyperglycemia associated toxicity. Ferulic acid (FA) is a ubiquitous hydroxycinnamic acid displaying an array of therapeutic properties, including anti-diabetic effect which could be attributed to its potent antioxidant capacity. However, due to low bioavailability and clinical efficacy of FA, its biomedical applications remained limited. In the present study, FA encapsulated chitosan nanoparticles (FANPs) were synthesized through ionotropic gelation process with an aim to enhance FA bioavailability. The plasma release and urinary excretion profiles of FANPs were compared with that of free FA using healthy Wistar albino rats as a model system. The encapsulated FA displayed extended plasma retention time and maximum plasma concentration was recorded at 60 min which implied four times enhancement of Tmax compared to free FA. The elimination of compound from the animal body also displayed a similar pattern where the peak urinary excretion of FA from nanoformulations. FANPs were also tested for their anti-hyperglycemic effects in streptozotocin (STZ) induced diabetes in Wistar albino rats and were found to attenuate the diabetes-associated symptoms. FANPs caused an enhancement in body weight, decrease in blood glucose level along with a regulatory effect on blood lipid profile of diabetic rats. Positive impact of FANPs in improving the hyperglycemic condition prevalent in diabetic rats might provide new avenues for the treatment of DM and help avoid secondary complications associated with the synthetic drugs.

Synergistic effects of Woodfordia fruticosa gold nanoparticles in preventing microbial adhesion and accelerating wound healing in Wistar albino rats in vivo.

Therapeutic effectiveness of biogenically synthesized Woodfordia fruticosa nano-gold particles (WfAuNPs) has been claimed in this study which prevents microbial adhesion and enhanced wound healing potential on Wistar albino rats. The synthesized WfAuNPs were characterized using several biophysical techniques such as UV-Visible Spectroscopy (UV-vis), X-Ray Diffraction (XRD), Dynamic Light Scattering (DLS), Zeta Potential, Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM) and High Resolution Transmission Electron Microscopy (HR-TEM) analysis. The synthesized WfAuNPs in the size range of 10-20nm were used to develop 1% Carbopol® 934 based nano gold formulation (WfAuNPs-Carbopol® 934). The WfAuNPs-Carbopol® 934 nanoformulation was evaluated using viscosity and spreadability measurements. The wound healing potential of WfAuNPs-Carbopol® 934 monitored up to 12days was confirmed by performing wound contraction (%), epithelialization, and histopathological studies done in vivo on Wistar albino rats. The hydroxyproline content was also measured in the re-epithelized skin for quantification of collagen content. The effects of WfAuNPs on microbial adhesion leading to biofilm formation were evaluated against Candida albicans and Cryptococcus neoformans fungal strains. The respective Minimum Inhibitory Concentration (MIC80), Biofilm Inhibitory Concentration (BIC80) and Biofilm Eradication Concentration (BEC80) values of C. albicans was found to be 16, 32, 256µg/ml respectively while for C. neoformans it was recorded to be 32, 64, 256µg/ml respectively. Data obtained, confirmed the effectiveness in preventing microbial adhesion and wound healing potential of the WfAuNPs as compared to current marketed formulations.

Accelerated in vivo wound healing evaluation of microbial glycolipid containing ointment as a transdermal substitute.

A potent biosurfactant (BS) producing Bacillus licheniformis SV1 (NCBI GenBank Accession No. KX130852) was isolated from oil contaminated soil sample. Physicochemical investigations (TLC, HPLC, FTIR, GC-MS and NMR) revealed it to be glycolipid in nature. Fibroblast culture assay showed cytocompatibility and increased cell proliferation of 3T3/NIH fibroblast cells treated with this biosurfactant when checked using MTT assay and DAPI fluorescent staining. To evaluate the wound healing potential, BS ointment was formulated and checked for its spreadability and viscosity consistency. In vivo wound healing examination of full thickness skin excision wound rat model demonstrated the prompt re-epithelialization and fibroblast cell proliferation in the early phase while quicker collagen deposition in later phases of wound healing when BS ointment was used. These results validated the potential usage of BS ointment as a transdermal substitute for faster healing of impaired skin wound. Biochemical evaluation also substantiated the highest concentration of hydroxyproline (32.18±0.46, p<0.001) in the BS ointment treated animal tissue samples compared to the control. Hematoxylin-Eosin (H&E) and Masson's Trichrome staining validated the presence of increased amount of collagen fibers and blood vessels in the test animals treated with BS ointment.

Ofloxacin loaded gellan/PVA nanofibers - Synthesis, characterization and evaluation of their gastroretentive/mucoadhesive drug delivery potential.

The purpose of this investigation is to formulate a gastroretentive sustained drug release system for ofloxacin to improve its retention time, pharmacological activity, bioavailability and therapeutic efficacy in the stomach. Ofloxacin loaded gellan/poly vinyl alcohol (PVA) nanofibers were fabricated using a simple and versatile electrospinning technique. The fabricated nanofibers were evaluated for percent drug encapsulation efficiency and in vitro drug release in simulated gastric medium (pH1.2). The in vitro release profile and kinetic studies for drug indicated the sustained release of ofloxacin from the nanofibers through Fickian diffusion kinetics. The antimicrobial activity of the ofloxacin loaded nanofibers was assessed in comparison to the pure ofloxacin by means of minimal inhibitory concentrations (MIC) against microbial strains of Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The optimized ofloxacin loaded gellan/PVA nanofibers displayed biphasic drug release profile with considerable mucoadhesion and gastric retention in the rat's gastric mucosal membrane. Data obtained, suggested that the developed gastroretentive drug delivery can potentially enhance the pharmacological activity of ofloxacin and can also serve as a viable alternative for improving drug bioavailability via oral route.

Novel biogenic synthesis of silver nanoparticles and their therapeutic potential.

Here, we explored the medicinal uses of the novel biogenic silver nanoparticles of Pterospermum acerifolium (PaAgNPs) as a cost effective, eco-friendly, reducing and stabilizing compounds. The formation of PaAgNPs was confirmed by changing its color from colorless to yellowish brown, with maximum absorbance at 417 nm. FTIR spectrum of PaAgNPs suggested the presence of polycyclic compound similar to betulinic acid which plays as a capping agent and provided stability to PaAgNPs. FESEM and HRTEM images depicted the spherical shape of synthesized biogenic silver nanoparticles with an average particle size range of 10-20 nm. The EDX spectrum of the solution confirmed the presence of elemental silver signals. The crystalline nature of PaAgNPs was identified by XRD technique and its stability was recorded using Zeta potential analyzer. The antioxidant potential was assayed using diphenyl-beta-picrylhydrazyl (DPPH). Maximum free radical scavenging action of PaAgNPs was 69.52% as compared to 63.53% for PALE. Using a model of carrageenan-induced paw edema in rats, PaAgNPs showed two-fold enhanced anti-inflammatory activity in vivo.

Drug functionalized microbial polysaccharide based nanofibers as transdermal substitute.

In order to promote the natural healing process, drug-functionalized nanofibrous transdermal substitute was fabricated using gellan as chief polymer and polyvinyl alcohol (PVA) as supporting polymer via electrospinning technique. These fabricated nanofibers physiochemically mimic the extracellular matrix (ECM) which supports the cell growth. For neo-tissue regeneration in a sterilized environment, amoxicillin (Amx) was entrapped within these nanofibers. Entrapment of Amx in the nanofibers was confirmed by FESEM, FTIR, XRD and TG analysis. In vitro cell culture studies revealed that the fabricated non-cytotoxic nanofibers promoted enhance cell adherence and proliferation of human keratinocytes. A preliminary in vivo study performed on rat model for full thickness skin excision wound demonstrated the prompt re-epithelialization in early phase and quicker collagen deposition in later phases of wound healing in case of Amx-functionalized gellan/PVA nanofibers. Data collectively confirmed the potential usage of gellan based electrospun nanofibers as transdermal substitute for faster skin restoration.

Hydroxyproline: A Potential Biochemical Marker and Its Role in the Pathogenesis of Different Diseases.

Hydroxyproline is a non-essential amino acid found in collagen and few other extracellular animal proteins. It's two isomeric forms trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline play a crucial role in collagen synthesis and thermodynamic stability of the triple-helical conformation of collagen and associated tissues. Various abnormalities in hydroxyproline metabolism have been shown to play key roles in the pathophysiology and pathogenesis of different diseases. The elevated level of hydroxyproline is observed in several disorders, e.g., graft versus host disease, keloids, and vitiligo while its decreased level is a marker of poor wound-healing. This review explores the potential of using hydroxyproline as a biochemical marker to understand the pathogenesis, molecular pathophysiology and treatment of these diseases. The review concludes with an outlook on the scope and challenges in the clinical implementation of hydroxyproline as a biomarker.