Systematic elucidation of the multi-target pharmacological mechanism of Terminalia arjuna against congestive cardiac failure via network pharmacology and molecular modelling approaches.

Congestive cardiac failure (CCF) is a pathophysiologic state when the heart is not able to maintain its cardiac output to meet the demand of metabolising tissues. CCF is responsible for approximately 2.9 million deaths worldwide. The heterogeneous nature of CCF draws the attention of researchers to find more enthralling and promising diagnostic and treatment options. Terminalia arjuna (Arjuna) is an evergreen, deciduous tree exhibited various astringent, anti-bacterial, and anti-microbial properties. T. arjuna is being used in various regions for anginal pain, hypertension, congestive heart failure, and dyslipidemia. Although previous in vitro studies have demonstrated the therapeutic potential of T. arjuna, the exact molecular mechanism underlying its protective effect on the heart remains unclear. In this study, a network pharmacology technique was used to explore the active ingredients, potential targets in T. arjuna for the treatment of CCF. In the framework of this study, we explored the active ingredient-target-pathway network and figured out that oleanolic acid, arjunolic acid, luteolin, kaempferol, cholesterol, ellagic acid 4-O-xylopyranoside 3,3'-dimethyl ether, and cyclohexyl (2,4-dimethyl phenyl) methanone contributed significantly to the development of CCF by affecting AKT1, MAPK14, TNF, IL6, ESR1, and HSP90AA1 genes. Molecular docking analysis further validated the activities of these compounds against potential targets. To sum up, integrated network pharmacology and docking analysis revealed that T. arjuna exerts its cardioprotective effect by acting on various signalling pathways, including the thyroid hormone, VEGF signalling pathway, AGE-RAGE signalling pathway in diabetic complications, HIF signalling pathway, sphingolipid signalling pathway, and oestrogen signalling pathways. Overall, this study provides valuable insights into the molecular mechanism of T. arjuna in CCF and highlights its potential as a promising preventive treatment for this condition.

Development of transgenic algae strain expressing CTB-M2e fusion gene an approach towards the development of a universal edible vaccine in algae.

Avian Influenza, the most studied virus, is of high concern due to its zoonotic pandemic potential. In recent years, several influenza vaccines have been used with the broad goal of managing and in certain cases, eliminating the disease. The matrix 2 extracellular domain (M2e), is one of the key targets of the universal influenza vaccine, a liner peptide that is conserved throughout all influenza A subtypes virus. Many recombinant influenza proteins have been expressed in yeast and plants for vaccine development. A remarkable development has been made in the field of biotechnology to explore the potential of microalga as an expression host. In this study, we designed a fusion gene code for M2e peptide and CTB protein as M2e's natural form has a low level of immunogenicity. The fusion gene was cloned in the Chloroplast transformation vector pSRSapI and expressed in the TN72 mutant strain of Chlamydomonas reinhardii. The expression of the targeted protein was confirmed by ECL western blot analysis. A GM1-ELISA was carried out to detect the affinity of fusion protein for GM1 monosialoganglioside and the significant P-value is lower than 0.05. Immunogenicity assay on chicken detected the anti-M2e bodies in chicken serum. This study gives evidence of therapeutic protein production through algae chloroplast and a stable, selection free and low cost oral delivery for universal vaccine against influenza A virus.

Cloning and expression of an anti-cancerous cytokine: human IL-29 gene in Chlamydomonas reinhardtii.

Green algae, Chlamydomonas reinhardtii, with low cultivation cost, absence of endotoxins and insusceptibility to human pathogens is emerging as a potential system for the future production of recombinant proteins. The recent development of molecular tools enabling recombinant protein expression in algae chloroplast has provided new research and advance opportunities for developing low-cost therapeutic proteins. In the present study, algae chloroplast expression system was evaluated for the recombinant production of an anti-cancerous therapeutic protein, Interleukin 29 (IL29). The IL29 gene was cloned into algae chloroplast expression vector (pSRSapI). After the transformation, the positive clones were screened for homoplasmy and the presence of the IL29 gene by spot test and PCR analysis, respectively. The expressed SDS-PAGE and western blotting assay characterized IL-29. The algae expressed IL-29 was biologically active in an anti-proliferating bioassay using HepG2 cells. The results suggest that the Chlamydomonas reinhardtii expression system is convenient, low-cost, eco-friendly, and safe to express IL29.

Inorganic Nanoparticles: Toxic Effects, Mechanisms of Cytotoxicity and Phytochemical Interactions.

During the last few decades, nanotechnology has gained many applications in almost all fields of life because of the unique properties of nanoparticles (NPs). Nanotechnology has specially marked its name in the field of medicine. However, NPs toxicity is detrimental to human health and is a prime concern in applied medicine. They can cause insomnia, vertigo, madarosis, epistaxis, hypokalemia, lymphopenia, Alzheimer's and Parkinson's diseases, etc. There is a gap in knowledge regarding the study of the toxicological effects of NPs. Mechanisms that are responsible for this toxicity are not fully understood yet. Phytochemicals have natural therapeutic effects of reducing metal NPs' toxicity by acting as stabilizers and nontoxic reducing agents. However, the interaction between phytochemicals and NPs is remained to be elucidated. This review will provide in-depth knowledge about the various types of inorganic NPs and their associated toxicities, key parameters determining the toxic behaviour of NPs, and the mechanisms behind their cytotoxicity. It also emphasizes the need for further research to understand the interaction between various phytochemicals and NPs for therapeutic purposes.

Disease-associated variants of Gap Junction Beta 2 protein (GJB2) in the deaf population of Southern Punjab of Pakistan.

Hearing impairment (HI) is a highly heterogeneous genetic disorder and is classified into nonsyndromic (without any other clinical manifestations) and syndromic (if combined with other clinical presentations) forms. Variations in GJB2 gene are the leading cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) in several populations worldwide. This study was carried out to investigate the prevalence of GJB2 variations in severe-to-profound hearing impaired families of Southern Punjab of Pakistan. Ten families segregating ARNSHL were recruited from different areas of the region. Sanger sequencing of GJB2 coding region was carried out. In two out of ten families, NM_004004:c.*71G>A (p.(Trp24*)) and NM_004004:c.358_360del (p.(Glu120del)) homozygous variants were identified as the cause of hearing loss. Our study showed that GJB2-related hearing loss accounts for at least 20% of all cases with severe-to-profound hearing loss in the Southern Punjab population of Pakistan.

Rational design of chimeric Multiepitope Based Vaccine (MEBV) against human T-cell lymphotropic virus type 1: An integrated vaccine informatics and molecular docking based approach.

Human T-cell lymphotropic virus type 1 (HTLV-1) is an infectious virus that has been linked to adult T cell leukemia /lymphoma, aggressive CD4-T cell malignancy and many other immune-related medical illnesses. So far, no effective vaccine is known to combat HTLV-1, hence, the current research work was performed to design a potential multi-epitope-based subunit vaccine (MEBV) by adopting the latest methodology of reverse vaccinology. Briefly, three highly antigenic proteins (Glycoprotein, Accessory protein, and Tax protein) with no or minimal (<37%) similarity with human proteome were sorted out and potential B- and T-cell epitopes were forecasted from them. Highly antigenic, immunogenic, non-toxic, non-allergenic and overlapping epitopes were short-listed for vaccine development. The chosen T-cell epitopes displayed a strong binding affinity with their corresponding Human Leukocyte Antigen alleles and demonstrated 95.8% coverage of the world's population. Finally, nine Cytotoxic T Lymphocytes, six Helper T Lymphocytes and five Linear B Lymphocytes epitopes, joint through linkers and adjuvant, were exploited to design the final MEBV construct, comprising of 382 amino acids. The developed MEBV structure showed highly antigenic properties while being non-toxic, soluble, non-allergenic, and stable in nature. Moreover, disulphide engineering further enhanced the stability of the final vaccine protein. Additionally, Molecular docking analysis and Molecular Dynamics (MD) simulations confirmed the strong association between MEBV construct and human pathogenic immune receptor TLR-3. Repeated-exposure simulations and Immune simulations ensured the rapid antigen clearance and higher levels of cell-mediated immunity, respectively. Furthermore, MEBV codon optimization and in-silico cloning was carried out to confirm its augmented expression. Results of our experiments suggested that the proposed MEBV could be a potential immunogenic against HTLV-1; nevertheless, additional wet lab experiments are needed to elucidate our conclusion.

Rational design of multi epitope-based subunit vaccine by exploring MERS-COV proteome: Reverse vaccinology and molecular docking approach.

Middle East respiratory syndrome (MERS-COV), first identified in Saudi Arabia, was caused by a novel strain of coronavirus. Outbreaks were recorded from different regions of the world, especially South Korea and the Middle East, and were correlated with a 35% mortality rate. MERS-COV is a single-stranded, positive RNA virus that reaches the host by binding to the receptor of dipeptidyl-peptides. Because of the unavailability of the vaccine available for the protection from MERS-COV infection, the rapid case detection, isolation, infection prevention has been recommended to combat MERS-COV infection. So, vaccines for the treatment of MERS-COV infection need to be developed urgently. A possible antiviral mechanism for preventing MERS-CoV infection has been considered to be MERS-CoV vaccines that elicit unique T-cell responses. In the present study, we incorporated both molecular docking and immunoinformatic approach to introduce a multiepitope vaccine (MEP) against MERS-CoV by selecting 15 conserved epitopes from seven viral proteins such as three structural proteins (envelope, membrane, and nucleoprotein) and four non-structural proteins (ORF1a, ORF8, ORF3, ORF4a). The epitopes, which were examined for non-homologous to host and antigenicity, were selected on the basis of conservation between T-cell, B-cell, and IFN-γ epitopes. The selected epitopes were then connected to the adjuvant (ß-defensin) at the N-terminal through an AAY linker to increase the immunogenic potential. Structural modelling and physiochemical characteristic were applied to the vaccine construct developed. Afterwards the structure has been successfully docked with antigenic receptor, Toll-like receptor 3 (TLR-3) and in-silico cloning ensures that its expression efficiency is legitimate. Nonetheless the MEP presented needs tests to verify its safety and immunogenic profile.

Consanguinity: A blessing or menace at population level?

Consanguinity has highly complex and multifaceted aspects with sociocultural as well as biological debates on its pros and cons. The biological upshot of consanguinity includes the increased homozygosity, which results in manifold increased risk of genetic disorders at family and population levels. On the other hand, in addition to social, cultural, political, and economic benefits, consanguineous marriages have biological advantages at the population level. The consequence of consanguineous marriages is an upsurge in the number of homozygous diseased individuals with fewer chances of mating and reduced chances of survival, therefore evolutionarily confining the transmission of disease alleles to future generations and encouraging its elimination from a population. Protective effects of consanguinity have also been observed in a few diseases in different populations. Although attractive for many reasons, nonconsanguineous marriages will cause risk alleles to spread throughout the population, making most individuals carriers, and ultimately will resume the production of recessive diseases in subsequent generations. Although consanguinity, from an evolutionary point of view, is beneficial at the population level, it increases the risk of diseases in the very next generation. Presently, there is no treatment for most of the genetic disorders; we cannot opt for consanguinity for long-term benefits. Nonconsanguineous marriages are a better strategy by which we may delay disease manifestation for some generations until science offers a viable solution.

USH1K, a novel locus for type I Usher syndrome, maps to chromosome 10p11.21-q21.1.

We ascertained two large Pakistani consanguineous families (PKDF231 and PKDF608) segregating profound hearing loss, vestibular dysfunction, and retinitis pigmentosa; the defining features of Usher syndrome type 1 (USH1). To date, seven USH1 loci have been reported. Here, we map a novel locus, USH1K, on chromosome 10p11.21-q21.1. In family PKDF231, we performed a genome-wide linkage screen and found a region of homozygosity shared among the affected individuals at chromosome 10p11.21-q21.1. Meiotic recombination events in family PKDF231 define a critical interval of 11.74 cM (20.20 Mb) bounded by markers D10S1780 (63.83 cM) and D10S546 (75.57 cM). Affected individuals of family PKDF608 were also homozygous for chromosome 10p11.21-q21.1-linked STR markers. Of the 85 genes within the linkage interval, PCDH15, GJD4, FZD4, RET and LRRC18 were sequenced in both families, but no potential pathogenic mutation was identified. The USH1K locus overlaps the non-syndromic deafness locus DFNB33 raising the possibility that the two disorders may be caused by allelic mutations.

Allelic hierarchy of CDH23 mutations causing non-syndromic deafness DFNB12 or Usher syndrome USH1D in compound heterozygotes.

BACKGROUND: Recessive mutant alleles of MYO7A, USH1C, CDH23, and PCDH15 cause non-syndromic deafness or type 1 Usher syndrome (USH1) characterised by deafness, vestibular areflexia, and vision loss due to retinitis pigmentosa. For CDH23, encoding cadherin 23, non-syndromic DFNB12 deafness is associated primarily with missense mutations hypothesised to have residual function. In contrast, homozygous nonsense, frame shift, splice site, and some missense mutations of CDH23, all of which are presumably functional null alleles, cause USH1D. The phenotype of a CDH23 compound heterozygote for a DFNB12 allele in trans configuration to an USH1D allele is not known and cannot be predicted from current understanding of cadherin 23 function in the retina and vestibular labyrinth. METHODS AND RESULTS: To address this issue, this study sought CDH23 compound heterozygotes by sequencing this gene in USH1 probands, and families segregating USH1D or DFNB12. Five non-syndromic deaf individuals were identified with normal retinal and vestibular phenotypes that segregate compound heterozygous mutations of CDH23, where one mutation is a known or predicted USH1 allele. CONCLUSIONS: One DFNB12 allele in trans configuration to an USH1D allele of CDH23 preserves vision and balance in deaf individuals, indicating that the DFNB12 allele is phenotypically dominant to an USH1D allele. This finding has implications for genetic counselling and the development of therapies for retinitis pigmentosa in Usher syndrome. ACCESSION NUMBERS: The cDNA and protein Genbank accession numbers for CDH23 and cadherin 23 used in this paper are AY010111.2 and AAG27034.2, respectively.