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Exosomes are natural membrane-enclosed nanovesicles (30–150 nm) involved in cell-cell communication. Recently, they have garnered considerable interest as nanocarriers for the controlled transfer of therapeutic agents to cells. Here, exosomes were derived from bone marrow mesenchymal stem cells using three different isolation methods. Relative to filtration and spin column condensation, the size exclusion chromatography led to the isolation of exosomes with the highest purity. These exosomes were then hybridized with liposomes using freeze-thaw cycles and direct mixing techniques to evaluate whether this combination enhances the transfection efficiency of large plasmids. The efficiency of these hybrids in transferring the Cas9-green fluorescent protein plasmid (pCas9-GFP) into the human embryonic kidney 293T (HEK293T) cells was evaluated compared to the pure exosomes. Both Cas9-GFP-loaded exosomes and exosome-liposome hybrids were taken up well by the HEK293T cells and were able to transfect them with their plasmid loads. Meanwhile, the treatment of the cells with plasmids alone, without any vesicles, resulted in no transfection, indicating that the exosome and exosome-liposome hybrids are essential for the transfer of the plasmids across the cell membrane. The pure exosomes and the hybrids incorporating liposomes obtained by the heating method transfected the cells more efficiently than those containing liposomes obtained by the thin film hydration technique. Interestingly, the method of combining exosomes with liposomes (freeze-thaw vs. direct mixing) proved to be more decisive in determining the size of the vesicular hybrid than their composition. In contrast, the liposome component in the hybrids proved to be decisive for determining the transfection efficiency. [ABSTRACT FROM AUTHOR]

Aim: The present study examined the protective potential of human adipose tissue-derived mesenchymal stem cells (hASCs) modified to overexpress alpha-1 antitrypsin (AAT), in a mouse model of the liver fibrosis. Background: For the treatment of end-stage liver diseases, cell therapy has emerged as a promising noninvasive alternative to liver transplantation. Mesenchymal stem cells (MSCs) are being evaluated due to their dual capabilities of promoting liver regeneration and modulating the pathogenic inflammation of the immune system. Methods: Liver fibrosis was induced in mice via the intraperitoneal injection of carbon tetrachloride (CCl4). MSCs were extracted from the human adipose tissue. After stemness confirmation, the cells were transduced with the lentiviruses containing the AAT gene, and then injected into the mice's tail vein. Fourteen days' post-transplantation, mice were sacrificed, and blood and tissue samples were collected for analysis. Important liver enzymes, including alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, and total bilirubin (TB), were measured. Histological studies were carried out using the hematoxylin and eosin (H&E), as well as Masson's trichrome (MT) staining. Results: Compared to hASCs, treatment with AAT-hASCs resulted in greater reductions in ALT, AST, ALP, and TB, as well as normalized albumin levels. AAT-hASCs promoted enhanced liver regeneration histologically, likely attributable to anti-inflammatory and anti-proteolytic properties of AAT. Conclusion: These findings indicate AAT-engineered hASCs as a promising cell-gene therapy candidate for further study in liver cirrhosis models. [ABSTRACT FROM AUTHOR]

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains' interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production. [ABSTRACT FROM AUTHOR]

Background and Objectives: The process of chronic diseases and aging is associated with reduced telomere length. The aim of this study was to investigate the effect of high-intensity interval training (HIIT) and low-intensity interval training (LIIT) on telomere repeat binding factor 1 and 2 (TRF1 and TRF2) in Soleus (SOL) muscle as a slow-twitch (ST) and Extensor Digitorum Longus (EDL) muscle as a fast-twitch (FT) of mice skeletal muscle. Materials and Methods: In this experimental study, the subjects were C57BL/6 mice (n=24) that were randomly divided into four groups of 6 mice: 1-Base control, 2-control, 3-high-intensity interval training (HIIT), and 4-lowintensity interval training (LIIT). The exercise included 5 days a week for 8 weeks. The gene expression of muscle tissues was measured using RT-PCR. The data were evaluated using two-way ANOVA with Tukey’s post hoc test. Results: By comparing ST and FT muscles (p=0.791) and HIIT and LIIT groups (p=0.164) and also the interaction between muscle type and training intensity (p=0.929), there was no significant difference in the expression of TRF1 gene. There was no significant interaction between TRF2 gene expression in ST and FT muscles at different intensities (p=0.868). But the results of Tukey’s post hoc test showed that in terms of TRF2 gene expression, HIIT (p=0.031) and LIIT (p=0.035) groups showed more increase compared to the control group. Conclusion: HIIT and LIIT similarly improved the telomere system in skeletal muscle of both muscle types, and this system was more affected by telomere elongation effect of TRF2. [ABSTRACT FROM AUTHOR]

Background and Objectives This study aims to investigate the effect of detraining on the telomere length in skeletal muscles of male mice. Subjects and Methods The samples were 24 C57BL/6 male mice that were randomly divided into four groups: Base control (n=6), control (n=6), low-intensity training (n=6), and high-intensity training (n=6). The training program was performed for 8 weeks, 5 days per week following 4 weeks of detraining. The factors were measured after DNA and RNA extraction using real time polymerase chain reaction method. The data were analyzed using two-way ANOVA. Results The main effects of muscle type (slow-twitch and fast-twitch) (P=0.825) and detraining type (P=0.062) and their interaction effect (P=0.408) on the expression of TRF1 gene was not significant. The main effects of muscle type (P=0.073) and detraining type (P=0.309) and their interaction effect (P=0.093) on the expression of TRF2 gene was not significant, either. The main effects of muscle type (P=0.763) and detraining type (P=0.053) and their interaction effect (P=0.651) on the telomere length was not significant, either. Conclusion Detraining after high-intensity or low-intensity training affects the telomere length of slowtwitch and fast-twitch muscles equally. It seems that detraining in people with a history of exercise with different intensities has no different effect on skeletal muscles. [ABSTRACT FROM AUTHOR]

This chapter reviews the different promoters used to control gene expression in the yeast Pichia pastoris, mainly for recombinant protein production. It covers natural inducible, derepressed, and constitutive promoters, as well as engineered synthetic/hybrid promoters, orthologous promoters from related yeasts, and emerging bidirectional promoters. Key examples, characteristics, and regulatory mechanisms are discussed for each promoter class. Recent efforts in promoter engineering through rational design, mutagenesis, and computational approaches are also highlighted. Looking ahead, we anticipate further developments that will enhance promoter design for Pichia pastoris. Overall, this comprehensive overview underscores the importance of promoter choice and engineering for fully harnessing Pichia pastoris biotechnological potential., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)