An oral toxicity assessment of a mosquito larvicidal transgenic algae (Chlamydomonas reinhardtii) using adult Zebrafish and its embryos.

Mosquito-borne diseases pose a global health threat, with pathogens like Malaria, Dengue fever, and others transmitted by mosquitoes. Our study focuses on evaluating the toxicity of genetically engineered mosquito larvicidal algae (Chlamydomonas reinhardtii) to non-target organisms, specifically Zebrafish. We conducted a 90-day experiment, feeding Zebrafish different combinations of larvicidal algae and commercial fish feed. Statistical analysis revealed no significant differences in mortality, allergenicity, or moribundity among groups. Hematology, molecular analysis, and necropsy showed no physiological differences. Our findings indicate that the transgenic algae (TN72.cry11Ba) had no adverse effects on adult Zebrafish or their larvae. This study confirmed the safety of algae on non-target organisms, such as zebrafish.

A Missense Variant in HACE1 Is Associated with Intellectual Disability, Epilepsy, Spasticity, and Psychomotor Impairment in a Pakistani Kindred.

Intellectual disability (ID), which affects around 2% to 3% of the population, accounts for 0.63% of the overall prevalence of neurodevelopmental disorders (NDD). ID is characterized by limitations in a person's intellectual and adaptive functioning, and is caused by pathogenic variants in more than 1000 genes. Here, we report a rare missense variant (c.350T>C; p.(Leu117Ser)) in HACE1 segregating with NDD syndrome with clinical features including ID, epilepsy, spasticity, global developmental delay, and psychomotor impairment in two siblings of a consanguineous Pakistani kindred. HACE1 encodes a HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1), which is involved in protein ubiquitination, localization, and cell division. HACE1 is also predicted to interact with several proteins that have been previously implicated in the ID phenotype in humans. The p.(Leu117Ser) variant replaces an evolutionarily conserved residue of HACE1 and is predicted to be deleterious by various in silico algorithms. Previously, eleven protein truncating variants of HACE1 have been reported in individuals with NDD. However, to our knowledge, p.(Leu117Ser) is the second missense variant in HACE1 found in an individual with NDD.

Revolutionizing personalized cancer treatment: the synergy of next-generation sequencing and CRISPR/Cas9.

In the context of cancer heterogeneity, the synergistic action of next-generation sequencing (NGS) and CRISPR/Cas9 plays a promising role in the personalized treatment of cancer. NGS enables high-throughput genomic profiling of tumors and pinpoints specific mutations that primarily lead to cancer. Oncologists use this information obtained from NGS in the form of DNA profiling or RNA analysis to tailor precision strategies based on an individual's unique molecular signature. Furthermore, the CRISPR technique enables precise editing of cancer-specific mutations, allowing targeted gene modifications. Harnessing the potential insights of NGS and CRISPR/Cas9 heralds a remarkable frontier in cancer therapeutics with unprecedented precision, effectiveness and minimal off-target effects.

Biosafety and toxicity assessment of transgenic cotton-harboring insecticide and herbicide tolerant genes on albino mice.

Introduction: Genetic engineering has revolutionized agriculture by transforming biotic and abiotic stress-resistance genes in plants. The biosafety of GM crops is a major concern for consumers and regulatory authorities. Methodology: A 14-week biosafety and toxicity analysis of transgenic cotton, containing 5 transgenes ((Cry1Ac, Cry2A, CP4 EPSPS, VIP3Aa, and ASAL)), was conducted on albino mice. Thirty mice were divided into three groups (Conventional, Non-transgenic, without Bt, and transgenic, containing targeted crop) according to the feed given, with 10 mice in each group, with 5 male and 5 female mice in each group. Results: During the study, no biologically significant changes were observed in the non-transgenic and transgenic groups compared to the control group in any of the study's parameters i.e. increase in weight of mice, physiological, pathological, and molecular analysis, irrespective of the gender of the mice. However, a statistically significant change was observed in the hematological parameters of the male mice, while no such change was observed in the female study group mice. The expression analysis, however, of the TNF gene increases many folds in the transgenic group as compared to the non-transgenic and conventional groups. Conclusion: Overall, no physiological, pathological, or molecular toxicity was observed in the mice fed with transgenic feed. Therefore, it can be speculated that the targeted transgenic crop is biologically safe. However, more study is required to confirm the biosafety of the product on the animal by expression profiling.

Cassia Angustifolia Primed ASCs Accelerate Burn Wound Healing by Modulation of Inflammatory Response.

BACKGROUND: Thermal traumas impose a huge burden on healthcare systems. This merits the need for advanced but cost-effective remedies with clinical prospects. In this context, we prepared a regenerative 3D-construct comprising of Cassia angustifolia extract (SM) primed adipose-derived stem cells (ASCs) laden amniotic membrane for faster burn wound repair. METHODS: ASCs were preconditioned with SM (30 µg/ml for 24 h), and subsequently exposed to in-vitro thermal injury (51 °C,10 min). In-vivo thermal injury was induced by placing pre-heated copper-disc (2 cm diameter) on dorsum of the Wistar rats. ASCs (2.0 × 105) pre-treated with SM (SM-ASCs), cultured on stromal side of amniotic membrane (AM) were transplanted in rat heat-injury model. Non-transplanted heat-injured rats and non-heat-injured rats were kept as controls. RESULTS: The significantly upregulated expression of IGF1, SDF1A, TGFß1, VEGF, GSS, GSR, IL4, BCL2 genes and downregulation of BAX, IL6, TNFα, and NFkB1 in SM-ASCs in in-vitro and in-vivo settings confirmed its potential in promoting cell-proliferation, migration, angiogenesis, antioxidant, cell-survival, anti-inflammatory, and wound healing activity. Moreover, SM-ASCs induced early wound closure, better architecture, normal epidermal thickness, orderly-arranged collagen fibers, and well-developed skin appendages in healed rat-skin transplanted with AM+SM-ASCs, additionally confirmed by increased expression of structural genes (Krt1, Krt8, Krt19, Desmin, Vimentin, α-Sma) in comparison to untreated-ASCs laden-AM transplanted in heat injured rats. CONCLUSION: SM priming effectively enabled ASCs to counter thermal injury by significantly enhancing cell survival and reducing inflammation upon transplantation. This study provides bases for development of effective combinational therapies (natural scaffold, medicine, and stem cells) with clinical prospects for treating burn wounds.

Analysis of Transgenic Cotton Plants Containing Universal Stress Protein (GaUSP-1, GaUSP-2) and Zinc Finger Transcriptional Factor (GaZnF) Genes under Drought Stress.

Water is the most limiting factor for plant growth and crop productivity. Drought stress adversely affects crop yield throughout the world. Up to 50% of crop yield in Pakistan is severely affected by the shortage of water. Cotton is an important cash crop for Pakistan known as "white gold." It accounts for 8.2% of the value added in agriculture and about 3.2% of GDP. Besides, being the world's fourth-largest cotton producer, our yield per acre ranks 13th in the world. If we look at the Pakistan scenario, water deficiency is one of the major yield-limiting factors. Limitations related to conventional breeding and the advancements in plant genomics and biotechnology applications have opened new horizons to plant improvements. Therefore, in the current study, we carry out a comparative analysis to evaluate the morphological, physiological biochemical and molecular parameters in transgenic plants containing GaUSP-1, GaUSP-2 and GaZinc Finger genes under different drought stress conditions. Data showed that transgenic plants showed more tolerance as compared to non-transgenic plants. Transgenic and non-transgenic assist us in our better understanding of the drought-responsive mechanism and its effect on different plant growth traits, so, in this way, we would be able to explore drought tolerance mechanism and this will open the doors for the identification of drought-related genes.

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.

Risk Loci For Chronic Obstructive Disease Reside On Chromosome 14: A Case-Control Study On The Pakistani Population.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD), the third leading cause of death worldwide, is characterized by airflow limitation that can be due to abnormalities in the airway and/or alveoli. Genetic diagnosis at an early stage can be a key factor in the provision of accurate and timely treatment. Single Nucleotide polymorphisms (SNPs) are an important tool to study genetic association/ predisposition of the disease and have great potential to be diagnostic markers for early diagnosis of disease. METHODS: This case-control COPD association study was designed for the five SNPs residing on potential candidate genes (SERPINA1, SERPINA3, RIN3), to check whether these genes are involved in the genetic predisposition for COPD in the Pakistani population or not. The SNAPshot method was used to find out the risk alleles and haplotypes using ABI Genetic analyzer 3130. GeneMapper, Haploview and PLINK 1.9 software were used for analyzing the genotypes and haplotypes taking smoking exposure and gender as covariates. RESULTS: Two of the SNPs, rs4934 and rs17473 were found to be independently and significantly associated with COPD in our studied population whereas haplotype H1 for two SNPs, rs754388 and rs17473 (that are in high linkage disequilibrium), was found to be a significant risk factor for developing COPD symptoms. CONCLUSIONS: SNP variants of SERPINA1 and SERPINA3 are significantly and independently associated with COPD in the local population of Pakistan.

Adeno-Associated Virus-Mediated Gene Therapy.

Choice of vector is the most critical step in gene therapy. Adeno-associated viruses (AAV); third generation vectors, are getting much attention of scientists to be used as vehicles due to their non-pathogenicity, excellent safety profile, low immune responses, great efficiency to transduce non-dividing cells, large capacity to transfer genetic material and long-term expression of genetic payload. AAVs have multiple serotypes and each serotype shows tropism for a specific cell. Different serotypes are used to target liver, lungs, muscles, retina, heart, CNS, kidneys, etc. Furthermore, AAV based gene therapies have tremendous marketing applications that can be perfectly incorporated in the anticipated sites of the host target genome resulting in life long expression of transgenes. Some therapeutic products use AAV vectors that are used to treat lipoprotein lipase deficiency (LPLD) and it is injected intramuscularly, to treat mutated retinal pigment epithelium RPE65 (RPE65) that is introduced to subretinal space, an intravenous infusion to treat spinal muscular atrophy and rAAV2-CFTR vector is introduced into nasal epithelial cells to treat cystic fibrosis. AAV therapies and other such interdisciplinary methodologies can create the miracles for the generation of precision gene therapies for the treatment of most serious and sometimes fatal disorders.

Evaluation of exosomes encapsulated recombinant Interleukin-29 for its in vitro anticancer studies.

Exosomes have recently been considered ideal biotherapeutic nanocarriers that broaden the frontiers of current drug delivery systems to overcome the shortcomings associated with cytokine-based immunotherapy. Using this approach, the current study aimed to assess anti-proliferative activity of purified IL-29 and exosomes encapsulated IL-29. The IL-29+pET-28a construct was transformed into Rosetta 2(DE3) cells which was used for the large-scale production of IL-29. Exosomes isolated from H1HeLa, and SF-767 cells using Total Exosome Isolation reagent were loaded with IL-29 via sonication. Isolation of exosomes was validated using their core protein signature by western blotting and specific miRNA profiles by RT-PCR. The drug loading efficiency of exosomes derived from H1HeLa cells was higher than that of SF-767-derived exosomes. The drug release kinetics of IL-29 encapsulated exosomes exhibited stable release of the recombinant drug. Around 50% of all cancer cell lines survived when IL-29 was administered at a concentration of 20 µg/mL. A survival rate of less than 10% was observed when cells were treated with 20 µg/mL IL-29 loaded exosomes. It was concluded that IL-29 loaded exosomes had a more significant cytotoxic effect against cancer cells, which might be attributed to sustained drug release, improved half-life, superior targeting efficacy, capacity to harness endogenous intracellular trafficking pathways, and heightened biocompatibility of exosomes.

Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system.

Current research focuses on the soluble and high-level expression of biologically active recombinant human IL-29 protein in Escherichia coli. The codon-optimized IL-29 gene was cloned into the Champion™ pET SUMO expression system downstream of the SUMO tag under the influence of the T7 lac promoter. The expression of SUMO-fused IL-29 protein was compared in E. coli Rosetta 2(DE3), Rosetta 2(DE3) pLysS, and Rosetta-gami 2(DE3). The release of the SUMO fusion partner resulted in approximately 98 mg of native rhIL-29 protein with a purity of 99% from 1 l of fermentation culture. Purified rhIL-29 was found to be biologically active, as evaluated by its anti-proliferation assay. It was found that Champion™ pET SUMO expression system can be used to obtained high yield of biologically active soluble recombinant human protein compared to other expression vector.

Priming with caffeic acid enhances the potential and survival ability of human adipose-derived stem cells to counteract hypoxia.

The therapeutic effectiveness of stem cells after transplantation is hampered by the hypoxic milieu of chronic wounds. Prior research has established antioxidant priming as a thorough plan to improve stem cell performance. The purpose of this study was to ascertain how caffeic acid (CA) priming affected the ability of human adipose-derived stem cells (hASCs) to function under hypoxic stress. In order to study the cytoprotective properties of CA, hASCs were primed with CA in CoCl2 hypoxic conditions. Microscopy was used to assess cell morphology, while XTT, Trypan Blue, X-gal, LDH, Live Dead, scratch wound healing, and ROS assays were used to analyze viability, senescence, cell death, proliferation, and reactive oxygen species prevalence in the cells. According to our findings, CA priming enhances hASCs' ability to survive and regenerate in a hypoxic microenvironment more effectively than untreated hASCs. Our in-vitro research suggested that pre-treatment with CA of hASCs could be a unique way to enhance their therapeutic efficacy and ability to survive in hypoxic microenvironments.

Zinc oxide loaded chitosan-elastin-sodium alginate nanocomposite gel using freeze gelation for enhanced adipose stem cell proliferation and antibacterial properties.

Hydrogels have been the material of choice for regenerative medicine applications due to their biocompatibility that can facilitate cellular attachment and proliferation. The present study aimed at constructing a porous hydrogel composite scaffold (chitosan, sodium alginate and elastin) for the repair of chronic skin wounds. Chitosan-based hydrogel incorporating varying concentrations of zinc oxide nanoparticles i.e. ZnO-NPs (0, 0.001, 0.01, 0.1 and 1 % w/w) as the antimicrobial agent tested against Escherichia coli (E.coli) and Staphylococcus aureus (S. aureus) exhibited good antibacterial activities. ZnO-NPs were characterized by UV visible spectroscopy, Scanning electron microscopy (SEM) analysis, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. Fabricated gels were characterized by SEM analysis, FTIR, XRD, swelling ratio, degradation behavior and controlled release kinetics of ZnO-NPs. In vitro cytocompatibility of the composite was investigated using human adipose stem cells (ADSCs) by MTT and lactate dehydrogenase (LDH) assay, further assessed by SEM analysis and PKH26 staining. The SEM and XRD analysis confirmed the successful loading of ZnO-NPs into these scaffolds. Fluorescence PKH26 stained images and SEM analysis of ADSCs seeded scaffolds revealed biocompatible nature. The findings suggested that the developed composite gels have potential clinically for tissue engineering and chronic wound treatment.

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.

An Insight into Modern Targeted Genome-Editing Technologies with a Special Focus on CRISPR/Cas9 and its Applications.

Genome-editing technology has enabled scientists to make changes in model organisms' DNA at the genomic level to get biotechnologically important products from them. Most commonly employed technologies for this purpose are transcription activator like effector nucleases (TALENs), homing-endonucleases or meganucleases, zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9). Among these tools, CRISPR/Cas9 is most preferred because it's easy to use, has a small mutation rate, has great effectiveness, low cost of development, and decreased rate of advancement. CRISPR/Cas9 has a lot of applications in plants, animals, humans, and microbes. It also has applications in many fields such as horticulture, cancer, food biotechnology, and targeted human genome treatments. CRISPR technology has shown great potential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic to provide early and easy detection methods, possible treatment, and vaccine development. In the present review, genome-editing tools with their basic assembly and features have been discussed. Exceptional notice has been paid to CRISPR technology on basis of its structure and significant applications in humans, plants, animals, and microbes such as bacteria, viruses, and fungi. The review has also shed a little light on current CRISPR challenges and future perspectives.

Priming of adipose-derived stem cells with curcumin prior to cryopreservation preserves their functional potency: Towards an 'Off-the-shelf' therapy for burns.

Stem cells-based treatment for burn wounds require frozen cells as an off-the-shelf therapy; however, cryopreservation-induced oxidative stress resulted in post-thaw cell death or loss of cell functions, thus arrested their clinical practicality. Although antioxidant priming to stem cells increase their resistant to oxidative stress, but this strategy is still unexplored on cryopreserved cells. Herein, we investigated whether curcumin priming before cryopreservation could preserve the therapeutic potency of thawed stem cells. For this, unprimed and curcumin-primed adipose-derived stem cells (ASCs) were cryopreserved for one month. Post-thawing, cells were assessed for viability by trypan blue assay; metabolic activity by MTT assay; senescence by senescence-associated (SA)-ß-galactosidase activity staining assay; migration by scratch healing assay and; mRNA expression by real-time PCR. Subsequently, the healing potential was examined by injecting cells around the wound periphery of acidic burn in rats. Post-healing, skin architecture was histologically examined. Results demonstrated that, curcumin-primed frozen cells (Cryo/Cur-ASCs) showed better post-thaw viability, metabolic activity, migration ability and lower percent of senescence comparative to unprimed frozen cells (Cryo/ASCs). Curcumin priming alleviated the oxidative damage by activating the ROS-reducing cellular antioxidant system as shown by the evident increase in GSH levels and upregulated mRNA expression of glutathione peroxidase (GPx), superoxide dismutases (SOD1, SOD2), and catalase (CAT). Further, invivo findings revealed that Cryo/Cur-ASCs-treated wounds exhibited earlier wound closure with an improved architecture comparative to Cryo/ASCs and depicted healing capacity almost similar to Fresh/ASCs. Our findings suggested that curcumin priming could be effective to alleviate the cryo-induced oxidative stress in post-thawed cells.

Adenovirus vector system: construction, history and therapeutic applications.

Since the isolation of adenovirus (AdV) in 1953, AdVs have been used as vectors for various therapeutic purposes, such as gene therapy in cancers and other malignancies, vaccine development and delivery of CRISPR-Cas9 machinery. Over the years, several AdV vector modifications have been introduced, including fiber switching, incorporation of ligands in the viral capsid and hexon modification of the fiber, to improve the efficiency of AdV as a vector. CRISPR-Cas9 has recently been used for these modifications and is also used in other adeno-associated viruses. These modifications further allow the production of AdV libraries that display random peptides for the production of cancer-targeting AdV vectors. This review focuses on the common methods of AdV construction, changes in AdV tropism for the improvement of therapeutic efficiency and the role of AdV vectors in gene therapy, vaccine development and CRISPR-Cas9 delivery.

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.