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Tomato mosaic virus (ToMV) is one of the lethal tomato pathogens. In this study, we collected tomato leaves exhibiting interveinal chlorosis from farmer’s fields in district Lahore, Pakistan. The presence of tobamoviruses was confirmed through extracting RNA, cDNA synthesis, PCR amplification and subsequent sequencing of capsid protein (CP) gene of ToMV that contained 480 nucleotides (encoded for 160 amino acids making ~17 kDa protein) located at 5703rd to 6182nd nucleotide position in ToMV genome. In sequence analysis, Pakistani isolates shared 98.9-99.5% and 97.3-99.3% nucleotide and amino acid homologies with each other and 96-99% and 90-98.6% with other isolates respectively. In phylogenetic analysis the Pakistani isolates grouped with Iranian and Chines isolates, exhibiting higher nucleotides and amino acids identities as well. This research reveals a geographical association among Pakistani, Chines and Iranian ToMV isolates. The study suggests extensive surveys of tobamoviruses infecting field crops for their detailed genetic information.

Gene expression analyses to study the development of cotton fibers require high-quality nucleic acid. The conventional methods of nucleic acid extraction results in sub-quality nucleic acids with low yields. Young fibers are rich in polyphenols and sugars that react with nucleic acid to form phenols and insoluble substances. Furthermore, mature fibers contain more than 95% cellulose, hindering the nucleic acid isolation. Cytoplasm collapse and cellulose deposition also result in a very low yield of nucleic acid. Three different methods of RNA isolation from different cotton tissues were compared in this study to determine the best and most efficient one. The integrity and quality of RNA were analyzed using UV spectrum, agarose gel electrophoresis, RIN values, PCR, and Northern blot hybridization. RNA of functional quality was observed when using the high ion and pH method, with an A260/A280 ratio up to 1.87 and an average yield of 0.68 mg g-1 from fiber cells. From leaves, we found an A260/A280 ratio of 2.02 and an average yield of 6.35 mg g-1, which is suitable for molecular biology experiments. The extraction buffer with a high ion density and pH value include Tris-HCl, LiCl, EDTA, SDS, sodium deoxycholate, Nonident P-40, mercaptoethanol, and PVP. The addition of sodium deoxycholate and Nonider-40 (NP-40) enhances the density of other salt compounds and elevates the pH value. The results depicted that the high ion and pH method is a simple and effective way to extract a copious amount of high-quality RNA from polysaccharide-rich tissues. This method is also suitable for the extraction of cotton genomic DNA with high purity. Genomic DNA extracted from cotton using this method showed an A260/A230 ratio up to 2.09 and a yield of 1.44 mg g-1. This method is useful for isolating DNA and RNA from cotton fibers and produces high yields and quality at a comparatively low cost.

There has been an increasing demand for bioengineered blood vessels for utilization in both regenerative medicine and drug screening. However, the availability of a true bioengineered vascular graft remains limited. Three-dimensional (3D) bioprinting presents a potential approach for fabricating blood vessels or vascularized tissue constructs of various architectures and sizes for transplantation and regeneration. In this review, we summarize the basic biology of different blood vessels, as well as 3D bioprinting approaches and bioink designs that have been applied to fabricate vascular and vascularized tissue constructs, with a focus on small-diameter blood vessels.

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. The authors acknowledge Dr. Muhammad Ali, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA, for checking the manuscript for English language, grammar, style and syntax. The authors acknowledge Higher Education Commission Pakistan for providing funding for conduct of this work from TDF 02-163 (Dr. Azra Mehmood) and HEC NRPU Project number 8762 (Dr. Muhammad Yar). Scopus

Adipose-derived stem cells (ASCs) have been proven to have tumoricidal effects against hepatic cancer cell lines. However, it appears that exposure to oxidative microenvironment compromises the potential outcome of ASCs in real hepatoma. Herein, we aimed to examine the tumoricidal effects of ASCs under oxidative conditions and to investigate the impact of curcumin priming on ASCs' therapeutic potential.We used human hepatoma (HepG2) cells in a coculture system with unprimed or curcumin-primed ASCs (Cur-ASCs) under HCur-ASCs suppressed HepG2 proliferation, migration, and invasion as well as prompted apoptosis more significantly compared to unprimed ASCs under oxidative conditions. Expressional studies also revealed an obvious decline in the BCL-2/BAX ratio in HepG2 cocultured with Cur-ASCs. In addition, we noticed a marked elevation of apoptosis and senescence in unprimed ASCs compared to Cur-ASCs after coculture experiments, which demonstrated that curcumin priming preserved the survival and growth potential of ASCs; hence, Cur-ASCs performed better tumoricidal functions under oxidative conditions.Our findings suggest that ASCs have the intrinsic ability to induce cell death in HepG2 cells; however, their functions can be compromised under oxidative conditions. We believe that curcumin priming is an effective approach for improving the therapeutic effectiveness of ASCs in the cancerous microenvironment.

BackgroundFollowing recent findings from our group that curcumin preconditioning augments the therapeutic efficacy of adipose-derived stem cells in the healing of diabetic wounds in rats, we aimed to investigate the regenerative effects of curcumin preconditioned adipose-derived mesenchymal stem cells (ASCs) for better recovery of acid inflicted burns in this study.MethodsASCs were preconditioned with 5 μM curcumin for 24 hours and assessed for proliferation, migration, paracrine release potential and gene expression comparative to naïve ASCs. Subsequently, the healing capacity of curcumin preconditioned ASCs (Cur-ASCs) versus naïve ASCs was examined using acidic wounds in rats. For this, acid inflicted burns of 20 mm in diameter were made on the back of male Wistar rats. Then, 2 × 106 cells of Cur-ASCs and naïve ASCs were intradermally injected in the wound periphery (n = 6) for comparison with an untreated saline control. Post-transplantation, wounds were macroscopically analysed and photographed to evaluate the percentage of wound closure and period of re-epithelization. Healed wound biopsies were excised and used for histological evaluation and expression analysis of wound healing markers at molecular level by quantitative PCR and western blotting.ResultsWe found that Cur-ASCs exhibited greater proliferation, migration and paracrine potential in vitro. Further, Cur-ASCs showed more effective recovery than naïve ASCs as exhibited by gross morphology, faster wound closure and earlier re-epithelialization. Masson’s trichrome and hematoxylin and eosin staining demonstrated the improved architecture of the healing burns, as evidenced by reduced infiltration of inflammatory cells, compact collagen and marked granulation in Cur-ASC treated rats. Corroborating these findings, molecular assessment showed significantly reduced expressions of pro-inflammatory factors (interleukin-1 beta, interleukin-6, tumor necrosis factor alpha) a with striking upsurge of an oxidative marker (superoxide dismutase 1), pro-angiogenic factors (vascular endothelial growth factor, hepatocyte growth factor, hypoxia-inducible factor-1 alpha) and collagen markers (transforming growth factor beta 1, fibroblast growth factor-2, collagen type 1 alpha 1), verifying that Cur-ASCs modulate the regulation of pro-inflammatory and healing markers at burn sites.ConclusionsTreatment with Cur-ASCs resulted in faster re-epithelization of acid inflicted burns compared to the treatment with naïve ASCs. Based on observed findings, we suggest the transplantation of Cur-ASCs is a valuable therapy for the potent clinical management of acidic burns.

Current in vitro anti-tumor drug screening strategies are insufficiently portrayed lacking true perfusion and draining microcirculation systems, which may post significant limitation in reproducing the transport kinetics of cancer therapeutics explicitly. Herein, we report the fabrication of an improved tumor model consisting of bioprinted hollow blood vessel and lymphatic vessel pair, hosted in a three-dimensional (3D) tumor microenvironment-mimetic hydrogel matrix, termed as the tumor-on-a-chip with bioprinted blood and lymphatic vessel pair (TOC-BBL). The bioprinted blood vessel was perfusable channel with opening on both ends while the bioprinted lymphatic vessel was blinded on one end, both of which were embedded in a hydrogel tumor mass, with vessel permeability individually tunable through optimization of the composition of the bioinks. We demonstrated that systems with different combinations of these bioprinted blood/lymphatic vessels exhibited varying levels of diffusion profiles for biomolecules and anti-cancer drugs. Our TOC-BBL platform mimicking the natural pathway of drug-tumor interactions would have the drug introduced through the perfusable blood vessel, cross the vascular wall into the tumor tissue via diffusion, and eventually drained into the lymphatic vessel along with the carrier flow. Our results suggested that this unique in vitro tumor model containing the bioprinted blood/lymphatic vessel pair may have the capacity of simulating the complex transport mechanisms of certain pharmaceutical compounds inside the tumor microenvironment, potentially providing improved accuracy in future cancer drug screening.

Aim: Pretreatment of stem cells with antioxidants accelerates their ability to counter oxidative stress and is associated with the overall therapeutic outcome of their transplantation. Material & methods: Wharton Jelly derived mesenchymal stem cells (WJMSCs) were cultured and pretreated with various doses of antioxidants; Vitamin C (Vit C), Vitamin E (Vit E), Vitamin D3 (Vit D3) and their Cocktail, followed by exposure to in vitro heat injury. Assessment of WJMSCs survival, paracrine release, in vitro wound healing and expression of angiogenic and survival markers was conducted. Results: The results displayed an enhanced survival of WJMSCs especially in the case of Cocktail priming. Conclusion: Our data suggest that antioxidant pretreatment of WJMSCs strengthens the endurance of the cells, within stress conditions.