Virus-like particles (VLPs) are nanostructures that possess diverse applications in therapeutics, immunization, and diagnostics. With the recent advancements in biomedical engineering technologies, commercially available VLP-based vaccines are being extensively used to combat infectious diseases, whereas many more are in different stages of development in clinical studies. Because of their desired characteristics in terms of efficacy, safety, and diversity, VLP-based approaches might become more recurrent in the years to come. However, some production and fabrication challenges must be addressed before VLP-based approaches can be widely used in therapeutics. This review offers insight into the recent VLP-based vaccines development, with an emphasis on their characteristics, expression systems, and potential applicability as ideal candidates to combat emerging virulent pathogens. Finally, the potential of VLP-based vaccine as viable and efficient immunizing agents to induce immunity against virulent infectious agents, including, SARS-CoV-2 and protein nanoparticle-based vaccines has been elaborated. Thus, VLP vaccines may serve as an effective alternative to conventional vaccine strategies in combating emerging infectious diseases.

Flavonoids are secondary metabolites that represent a heterogeneous family of plant polyphenolic compounds. Recent research has determined that the health benefits of fruits and vegetables, as well as the therapeutic potential of medicinal plants, are based on the presence of various bioactive natural products, including a high proportion of flavonoids. With current trends in plant metabolite research, flavonoids have become the center of attention due to their significant bioactivity associated with anti-cancer, antioxidant, anti-inflammatory, and anti-microbial activities. However, the use of traditional approaches, widely associated with the production of flavonoids, including plant extraction and chemical synthesis, has not been able to establish a scalable route for large-scale production on an industrial level. The renovation of biosynthetic pathways in plants and industrially significant microbes using advanced genetic engineering tools offers substantial promise for the exploration and scalable production of flavonoids. Recently, the co-culture engineering approach has emerged to prevail over the constraints and limitations of the conventional monoculture approach by harnessing the power of two or more strains of engineered microbes to reconstruct the target biosynthetic pathway. In this review, current perspectives on the biosynthesis and metabolic engineering of flavonoids in plants have been summarized. Special emphasis is placed on the most recent developments in the microbial production of major classes of flavonoids. Finally, we describe the recent achievements in genetic engineering for the combinatorial biosynthesis of flavonoids by reconstructing synthesis pathways in microorganisms via a co-culture strategy to obtain high amounts of specific bioactive compounds

Background: The invention of Polymerase Chain Reaction (PCR) marked a breakthrough in biomedical research. Its invention divided the timeline into an era before and after PCR. Because of its multiple applications, it has become a vital tool for clinical researchers and diagnosticians. A effective PCR experiment requires adequate knowledge of each reaction component and stepby- step procedure to attain the optimized results. This research aims to employ optimization strategies that are easy to perform, cost-effective, and do not require PCR kits for the generation of amplicons for TYR, MITF, and SOX10 genes and can be used in sequence analysis. Materials and Methods: Whole blood samples were used to extract genomic DNA with an inorganic method. DNA quantification was done by spectrophotometry analysis. Optimization strategies were adopted to generate PCR products of candidate genes and visualized by agarose gel electrophoresis. Sanger sequencing was performed to check the quality and specificity of generated amplicons via optimization strategies. Results: This study demonstrates a novel approach for troubleshooting failed reactions without the use of PCR kits. The result indicated that use of sterilized material and reagents along with optimum MgCl2 concentration (2.0-3.0 mM), DNA quantity (25 ng/μL), and annealing temperature (54-60°C) are necessary to achieve successful amplification. Sequence analysis revealed no background noise often associated with sequencing results. Conclusion: Optimizing MgCl2 concentration, DNA quantity, annealing temperature, along with the use of contamination-free material and reagents are essential steps in PCR optimization. Following this guide, anyone lacking proper supervision, and with little or no knowledge of the procedure should attain the desired results.

Congenital idiopathic nystagmus (CIN) is an oculomotor disorder characterized by repetitive and rapid involuntary movement of the eye that usually develops in the first six months after birth. Unlike other forms of nystagmus, CIN is widely associated with mutations in the FRMD7 gene. This study involves the molecular genetic analysis of a consanguineous Pakistani family with individuals suffering from CIN to undermine any potential pathogenic mutations. Blood samples were taken from affected and normal individuals of the family. Genomic DNA was extracted using an in-organic method. Whole Exome Sequencing (WES) and analysis were performed to find any mutations in the causative gene. To validate the existence and co-segregation of the FRMD7 gene variant found using WES, sanger sequencing was also carried out using primers that targeted all of the FRMD7 coding exons. Additionally, the pathogenicity of the identified variant was assessed using different bioinformatic tools. The WES results identified a novel nonsense mutation in the FRMD7 (c.443T>A; p. Leu148 *) gene in affected individuals from the Pakistani family, with CIN resulting in a premature termination codon, further resulting in the formation of a destabilized protein structure that was incomplete. Co-segregation analysis revealed that affected males are hemizygous for the mutated allele c.443T>A; p. Leu148 * and the affected mother is heterozygous. Overall, such molecular genetic studies expand our current knowledge of the mutations associated with the FRMD7 gene in Pakistani families with CIN and significantly enhance our understanding of the molecular mechanisms involved in genetic disorders.

Oculocutaneous albinism (OCA) is a genetically heterogeneous condition that is associated with reduced or absent melanin pigment in the skin, hair, and eyes, resulting in reduced vision, high sensitivity to light, and rapid and uncontrolled eye movements. To date, seventeen genes have been associated with OCA including syndromic and non-syndromic forms of the condition. Whole exome sequencing (WES) was performed to identify pathogenic variants in nine Pakistani families with OCA, with validation and segregation of candidate variants performed using Sanger sequencing. Furthermore, the pathogenicity of the identified variants was assessed using various in-silico tools and 3D protein structural analysis software. WES identified biallelic variants in three genes explaining the OCA in these families, including four variants in TYR, three in OCA2, and two in HPS1, including two novel variants c.667C > T: p.(Gln223*) in TYR, and c.2009 T > C: p.(Leu670Pro) in HPS1. Overall, this study adds further knowledge of the genetic basis of OCA in Pakistani communities and facilitates improved management and counselling services for families suffering from severe genetic diseases in Pakistan.

Genes of folate-mediated 1 carbon metabolism are found to be highly upregulated in tumor cells and promote cancer cell proliferation. The current study aimed to determine the expression of the MTHFD2 gene in bladder and breast cancers. Furthermore, the determination of potential ligand-based inhibitors against MTHFD2 was performed in comparison with those of chemotherapeutic drugs and natural plant-based compounds. Semiquantitative expression analysis along with structure-based virtual ligand library screening was done to find plausible inhibitors. MTHFD2 expression was significantly increased with tumor stage progression both in low- and high-grade bladder cancer and especially in triple-negative breast cancer. Virtual ligand-based library screening against the three-dimensional MTHFD2 protein structure led to the identification of plausible inhibitors like MCULE-8109969891-0 and MCULE-9715677418-0-1 that displayed lower binding free energy as compared to that of already documented LY345899. Similar scaffold commercial drugs leucal (LEU), epirubicin (EPI), and lometrexol also displayed strong binding to the active site of MTHFD2. EPI and LEU in combinatorial therapy were also tested in vitro on MDA-MB-231 cells. The high doses of LEU in combination with EPI showed a significant reduction in cell viability at 2 and 3 μM concentrations. The interaction of breast cancer serum with high expression of MTHFD2 also showed an increase in binding of epirubicin in the presence of leucovorin. The decrease in the absorbance spectra of epirubicin at 37 and 53 °C displayed the stability induced by LEU on the interaction of EPI with the MTHFD2 binding pocket. Leucovorin tends to stabilize the interaction as the binding affinity is high even at 53 °C. Thus, MTHFD2 might be used as a cancer biomarker since its expression level changes drastically with tumor progression. Further experimental studies are required to establish the potential mode of inhibition of the novel small ligands. Future in vivo trials may validate the effectiveness of the combinatorial therapy.