Search

Expansion microscopy (ExM) is a promising technology that enables nanoscale imaging on conventional optical microscopes by physically magnifying the specimens. Here, we report the development of a strategy that enables i) on-demand labeling of subcellular organelles in live cells for ExM through transfection of fluorescent proteins that are well-retained during the expansion procedure; and ii) non-fluorescent chromogenic color-development towards efficient bright-field and photoacoustic imaging in both planar and volumetric formats, which is applicable to both cultured cells and biological tissues. Compared to the conventional ExM methods, our strategy provides an expanded toolkit, which we term as expansion fluorescence and photoacoustic microscopy (ExFLPAM), by allowing on-demand fluorescent protein labeling of cultured cells, as well as non-fluorescent absorption contrast-imaging of biological samples.

Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers.To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events.This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance.

Nucleic acid-based therapeutics are a common approach that is increasingly popular for a wide spectrum of diseases. Lipid nanoparticles (LNPs) are promising delivery carriers that provide RNA stability, with strong transfection efficiency, favorable and tailorable pharmacokinetics, limited toxicity, and established translatability. In this review article, we describe the lipid-based delivery systems, focusing on lipid nanoparticles, the need of their use, provide a comprehensive analysis of each component, and highlight the advantages and disadvantages of the existing manufacturing processes. We further summarize the ongoing and completed clinical trials utilizing LNPs, indicating important aspects/questions worth of investigation, and analyze the future perspectives of this significant and promising therapeutic approach.

Intradermal delivery of DNA vaccines via electroporation (ID-EP) has shown clinical promise, but the use of needle electrodes is typically required to achieve consistent results. Here, delivery of a DNA vaccine targeting the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is achieved using noninvasive intradermal vacuum-EP (ID-VEP), which functions by pulling a small volume of skin tissue into a vacuum chamber containing noninvasive electrodes to perform EP at the injection site. Gene expression and immunogenicity correlated with EP parameters and vacuum chamber geometry in guinea pigs. ID-VEP generated potent humoral and cellular immune responses across multiple studies, while vacuum (without EP) greatly enhanced localized transfection but did not improve immunogenicity. Because EP was performed noninvasively, the only treatment site reaction observed was transient redness, and ID-VEP immune responses were comparable to a clinical needle-based ID-EP device. The ID-VEP delivery procedure is straightforward and highly repeatable, without any dependence on operator technique. This work demonstrates a novel, reliable, and needle-free delivery method for DNA vaccines.

These days carbon dots have been developed for multiple biomedical applications. In the current study, the transfection potential of synthesized carbon dots from single biopolymers such as chitosan, PEI-2kDa, and PEI-25kDa (CS-CDs, PEI2-CDs, and PEI25-CDs) and by combining two biopolymers (CP2-CDs and CP25-CDs) through a bottom-up approach have been investigated. The characterization studies revealed successful synthesis of fluorescent, positively charged carbon dots

Mutations within the IL7-R-JAK-STAT signaling pathway are important drivers of T-cell acute lymphoblastic leukemia (T-ALL). Here we describe the important steps required to generate retroviral particles for the stable expression of mutant JAK3 constructs that induce constitutive JAK/STAT signaling. These are subsequently used for the viral transduction of the IL-3 cytokine-dependent Ba/F3 cell line or murine hematopoietic stem and progenitor cells (HSPCs) for in vitro and in vivo modelling of cytokine-independent growth or leukemia initiation respectively.

Ionizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their biological performance would extend the existing knowledge in the field. In the present study, we examined the effects of the three clinically-approved ICLs, Dlin-MC3-DMA, ALC-0315 and SM-102, as well as DODAP, on the in vitro and in vivo performance of LNPs for mRNA delivery and vaccine efficacy. mRNA-LNPs containing these lipids were successfully prepared, which were all found to be very similar in their physicochemical properties and mRNA encapsulation efficiencies. Furthermore, the results of the in vitro studies indicated that these mRNA-LNPs were efficiently taken up by immortalized and primary immune cells with comparable efficiency; however, SM-102-based LNPs were superior in inducing protein expression and antigen-specific T cell proliferation. In contrast, in vivo studies revealed that LNPs containing ALC-0315 and SM-102 yielded almost identical protein expression levels in zebrafish embryos, which were significantly higher than Dlin-MC3-DMA-based LNPs. Additionally, a mouse immunization study demonstrated that a single-dose subcutaneous administration of the mRNA-LNPs resulted in a high production of intracellular cytokines by antigen-specific T cells, but no significant differences among the three clinically-approved ICLs were observed, suggesting a weak correlation between in vitro and in vivo outcomes. This study provides strong evidence that ICLs modulate the performance of mRNA-LNPs and that in vitro data does not adequately predict their behavior in vivo.

Repairing genetic defects using exogenous DNA is a major challenge the science is currently facing. This requires the design of vectors that can effectively encapsulate, protect and target nucleic acids to specific cells safely and precisely. Here we have designed silica-based physiologically responsive particles to encapsulate, store, and transfer DNA. Unlike existing vectors (e.g., viral or lipidic particles), these DNA@SiO2 systems are very stable at room temperature. We also demonstrate how they protect the encapsulated DNA from exposure to different biological and physicochemical stresses, including DNase, denaturation temperatures (>100 °C), or reactive oxygen species (ROS). Remarkably, upon cellular uptake, these vectors dissolve safely unpacking the DNA and transfecting the cells.The versatility of the design is such that it can encapsulate genes without gene/size restrictions, in single or multiple layers of silica, so different genes can be expressed sequentially. This allows the time-controlled transcription of several genes, mimicking viral gene expression cascades, or even “fine-tuning” gene expression in transfected cells on demand. In addition, the method is easily scalable, reproducible, and inexpensive, enabling large-scale production and batch-quality testing, all of which are important for the personalized therapeutics industry. The high stability of these DNA vectors allows for easy and low-cost transport from the point of production to virtually any destination, making them unique as gene delivery tools.

Human mesenchymal stem cells (hMSCs) are primary cells with high clinical relevance that could be enhanced through genetic modification. However, gene delivery, particularly through nonviral routes, is inefficient. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological “priming” of hMSCs with clinically approved drugs can increase transfection in hMSCs by modulating transfection-induced cytotoxicity. However, even with priming, hMSC transfection remains inefficient for clinical applications. This work takes a complementary approach to addressing the challenges of transfecting hMSCs by systematically investigating key transfection parameters for their effect on transgene expression. Specifically, we investigated two promoters (cytomegalovirus [CMV] and elongation factor 1 alpha), four DNA vectors (plasmid, plasmid with no F1 origin, minicircle, and mini-intronic plasmid), two cationic carriers (Lipofectamine 3000 and Turbofect), and four donors of hMSCs from two tissues (adipose and bone marrow) for efficient hMSC transfection. Following systematic comparison of each variable, we identified adipose-derived hMSCs transfected with mini-intronic plasmids containing the CMV promoter delivered using Lipofectamine 3000 as the parameters that produced the highest transfection levels. The data presented in this work can guide the development of other hMSC transfection systems with the goal of producing clinically relevant, genetically modified hMSCs.

CD47 protects healthy cells from macrophage attack by binding to signal regulatory protein α (SIRPα), while its upregulation in cancer prevents immune clearance. Systemic treatment with CD47 antibodies requires a weakened Fc-mediated effector function or lower CD47-binding affinity to prevent side effects. Our approach combines “the best of both worlds,” i.e., maximized CD47 binding and full Fc-mediated immune activity, by exploiting gene therapy for paracrine release. We developed a plasmid vector encoding for the secreted fusion protein sCV1-hIgG1, comprising highly efficient CD47-blocking moiety CV1 and Fc domain of human immunoglobulin G1 (IgG1) with maximized immune activation. sCV1-hIgG1 exhibited a potent bystander effect, blocking CD47 on all cells via fusion protein secreted from only a fraction of cells or when transferring transfection supernatant to untransfected cells. The CpG-free plasmid ensured sustained secretion of sCV1-hIgG1. In orthotopic human triple-negative breast cancer in CB17-severe combined immunodeficiency (SCID) mice, ex vivo transfection significantly delayed tumor growth and eradicated one-third of tumors. In intratumoral transfection experiments, CD47 blockage and increased migration of macrophages into the tumor were observed within 17 h of a single injection. Natural killer (NK) cell-mediated lysis of sCV1-hIgG1-expressing cells was demonstrated in vitro. Taken together, this approach also opens the opportunity to block, in principle, any immune checkpoints.

The successful delivery of therapeutic nucleic acids (NAs) into eukaryotic cells is essential for numerous biomedical applications, including gene therapy, gene silencing, and genome editing. Cell-penetrating peptides (CPPs) have claimed significant attention as delivery vehicles due to their inherent ability to penetrate cellular membranes and efficiently transport cargo, including NAs, into the cells. However, further optimization and a deeper understanding of underlying mechanisms are necessary for such transfection methods. Previous studies have demonstrated that Ca 2+ ions can significantly enhance NA delivery efficiency when included in transfection media or CPP/NA nanoparticles during preparation. Similar effects have been observed for Mg 2+ , but the impact of other ions in this context has not been thoroughly investigated. In this study, we supplemented the CPP/NA formulations with various inorganic biocompatible ions by introducing solutions of the respective salts to colloidal nanoparticles at the preparation stage. Our results indicated that supplementing the CPP/NA formulations with certain salt solutions enhanced the biological effect achieved with NAs while also influencing nanoparticle size, surface charge, complexation stability, and, to some extent, the internalization route. Our findings offer valuable insights for optimizing the formation of CPP nanoparticles to improve NA delivery efficiency., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers. To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events. This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Ischemia-reperfusion injury (IRI) poses a significant challenge to liver transplantation (LT). The underlying mechanism primarily involves overactivation of the immune system. Heat shock protein 110 (HSP110) functions as a molecular chaperone that helps stabilize protein structures., Methods: An IRI model was established by performing LT on Sprague-Dawley rats, and HSP110 was silenced using siRNA. Hematoxylin-eosin staining, TUNEL, immunohistochemistry, ELISA and liver enzyme analysis were performed to assess IRI following LT. Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes., Results: Our findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT (P < 0.05). However, when rats were injected with siRNA-HSP110, IRI subsequent to LT was notably reduced (P < 0.05). Additionally, the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced (P < 0.05). Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver (P < 0.05)., Conclusions: HSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells. Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI., Competing Interests: Competing interest No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article., (Copyright © 2023 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

An important bottleneck for non-viral gene transfer commonly relates to translocation of nucleic acids into the nuclear compartment of target cells. So-called 3NFs are optimized short nucleotide sequences able to interact with the transcription factor nuclear factor κB (NF-κB), which can enhance the nuclear import of plasmid DNA (pDNA) carrying such motifs. In this work, we first designed a consistent set of six pDNAs featuring a common backbone and only varying in their 3NF sequences. These constructions were then transfected under various experimental settings. In vitro, cationic polymer-assisted pDNA delivery in five human-derived cell lines showed the potential advantage of 3NF carrying pDNA in diverse cellular contexts. In vivo, naked pDNAs were hydrodynamically delivered to muscle hindlimbs in healthy mice; this direct accurate comparative (in the absence of any gene carrier) revealed modest but consistent trends in favor of the pDNAs equipped with 3NF. In summary, the results reported emphasize the implications of various parameters on NF-κB-mediated pDNA nuclear import; under specific conditions, 3NF can provide modest to substantial advantages for pDNA gene transfer, in vitro as well as in vivo. This study thus further underscores the potential of optimized nuclear import for more efficient non-viral gene transfer applications.

Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.

Human mesenchymal stem cells (hMSCs) are under study for cell and gene therapeutics because of their immunomodulatory and regenerative properties. Safe and efficient gene delivery could increase hMSC clinical potential by enabling expression of transgenes for control over factor production, behavior, and differentiation. Viral delivery is efficient but suffers from safety issues, while nonviral methods are safe but highly inefficient, especially in hMSCs. We previously demonstrated that priming cells with glucocorticoids (Gcs) before delivery of DNA complexes significantly increases hMSC transfection, which correlates with a rescue of transfection-induced metabolic and protein synthesis decline, and apoptosis. In this work, we show that transgene expression enhancement is mediated by transcriptional activation of endogenous hMSC genes by the cytosolic glucocorticoid receptor (cGR) and that transfection enhancement can be potentiated with a GR transcription-activation synergist. We demonstrate that the Gc-activated cGR modulates endogenous hMSC gene expression to ameliorate transfection-induced endoplasmic reticulum (ER) and oxidative stresses, apoptosis, and inflammatory responses to prevent hMSC metabolic and protein synthesis decline, resulting in enhanced transgene expression after nonviral gene delivery to hMSCs. These results provide insights important for rational design of more efficient nonviral gene delivery and priming techniques that could be utilized for clinical hMSC applications.

Natural exosomes can express specific proteins and carbohydrate molecules on the surface and hence have demonstrated the great potentials for gene therapy of cancer. However, the use of natural exosomes is restricted by their low transfection efficiency. Here, we report a novel targeting tLyp-1 exosome by gene recombinant engineering for delivery of siRNA to cancer and cancer stem cells. To reach such a purpose, the engineered tLyp-1-lamp2b plasmids were constructed and amplified in Escherichia coli. The tLyp-1-lamp2b plasmids were further used to transfect HEK293T tool cells and the targeting tLyp-1 exosomes were isolated from secretion of the transfected HEK293T cells. Afterwards, the artificially synthesized siRNA was encapsulated into targeting tLyp-1 exosomes by electroporation technology. Finally, the targeting siRNA tLyp-1 exosomes were used to transfect cancer or cancer stem cells. Results showed that the engineered targeting tLyp-1 exosomes had a nanosized structure (approximately 100 nm) and high transfection efficiency into lung cancer and cancer stem cells. The function verifications demonstrated that the targeting siRNA tLyp-1 exosomes were able to knock-down the target gene of cancer cells and to reduce the stemness of cancer stem cells. In conclusion, the targeting tLyp-1 exosomes are successfully engineered, and can be used for gene therapy with a high transfection efficiency. Therefore, the engineered targeting tLyp-1 exosomes offer a promising gene delivery platform for future cancer therapy.

CRISPR genome editing is a widely used tool to perturb genes of interest within cells and tissues and can be used as a research tool to study the connection between genotypes and cellular phenotypes. Highly efficient genome editing is limited in certain cell types due to low transfection efficiency or single-cell survivability. This is true for BeWo cells, an in vitro model of placental syncytiotrophoblast cell-cell fusion and hormone secretion. Here we describe an optimized and easy-to-use protocol for knockout in BeWo cells using CRISPR Cas9 ribonucleoprotein (RNP) complexes delivered via electroporation. Further, we describe parameters for successful guide RNA design and how to assess genetic knockouts in BeWo cells so that users can apply this technique to their own genes of interest. We provide a positive control for inducing highly efficient knockout of the cell-cell fusion protein Syncytin-2 (ERVFRD-1) and assessing editing efficiency at this locus. We anticipate that efficient RNP-mediated genetic knockouts in BeWo cells will facilitate the study of new genes involved in cell-cell fusion and hormone secretion in this important cellular model system. Furthermore, this strategy of optimized nucleofection and RNP delivery may be of use in other difficult-to-edit trophoblast cells or could be applied to efficiently deliver transgenes to BeWo cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The understanding of the mechanisms involved in DNA electrotransfer in human skin remains modest and limits the clinical development of various biomedical applications, such as DNA vaccination. To elucidate some mechanisms of DNA transfer in the skin following electroporation, we created a model of the dermis using a tissue engineering approach. This model allowed us to study the electrotransfection of fibroblasts in a three-dimensional environment that included multiple layers of fibroblasts as well as the self-secreted collagen matrix. With the aim of improving transfection yield, we applied electrical pulses with electric field lines perpendicular to the reconstructed model tissue. Our results indicate that the fibroblasts of the reconstructed skin tissue can be efficiently permeabilized by applied millisecond electrical pulses. However, despite efficient permeabilization, the transfected cells remain localized only on the surface of the microtissue, to which the plasmid was deposited. Second harmonic generation microscopy revealed the extensive extracellular collagen matrix around the fibroblasts, which might have affected the mobility of the plasmid into deeper layers of the skin tissue model. Our results show that the used skin tissue model reproduces the structural barriers that might be responsible for the limited gene electrotransfer in the skin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

CD3ζ is part of the T cell receptor (TCR)/CD3 complex that plays a critical role in antigen recognition and subsequent T cell activation. Understanding the mechanisms that regulate CD3ζ can provide new insights into the T cell-mediated immune responses. However, it is challenging to deliver exogenous genes into T cells for functional and mechanistic analyses. To this end, we established a non-T cell transfection system based on HEK293FT cells to screen for candidate regulatory proteins. The transfection was optimized using relatively high confluent cultures and the transfection reagent PolyJet™. Pervanadate (PV) treatment sustained tyrosine phosphorylation of CD3ζ, and facilitated the subsequent activation-dependent ubiquitination by E3 ligase Cbl-b in the HEK293FT system. Lck and Zap70 kinases enhanced the levels of phosphorylated CD3ζ in the presence of PV. We compared the effects of E3 ligases and the corresponding adaptor proteins on activation-dependent ubiquitination of CD3ζ in the PV-stimulated cells, and found that Cbl-b was most effective. Taken together, we have demonstrated that a non-T cell transfection system based on PV-treated HEK293FT cells could effectively mimic CD3ζ phosphorylation and ubiquitination and is a promising model for studying the role of CD3ζ signaling in T cell activation., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Peripheral nerve injuries (PNIs) can cause neuropathies and significantly affect the patient's quality of life. Autograft transplantation is the gold standard for conventional treatment; however, its application is limited by nerve unavailability, size mismatch, and local tissue adhesion. Tissue engineering, such as nerve guidance conduits, is an alternative and promising strategy to guide nerve regeneration for peripheral nerve repair; however, only a few conduits could reach the high repair efficiency of autografts. The healing process of PNI is frequently accompanied by not only axonal and myelination regeneration but also angiogenesis, which initializes nerve regeneration through vascular endothelial growth factor A (VEGF-A). In this study, a composite nerve conduit with a poly (lactic-co-glycolic acid) (PLGA) hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with VEGF-A transfected Schwann cells (SCs) as the inner layer was established to evaluate its promising ability for peripheral nerve repair. A rat model of peripheral nerve defect was used to examine the efficiency of PLGA/GelMA-SC (VA) conduits, whereas autograft, PLGA, PLGA/GelMA, and PLGA/GelMA-SC (NC) were used as controls. VEGF-A-transfected SCs can provide a stable source for VEGF-A secretion. Furthermore, encapsulation in GelMA cannot only promote proliferation and tube formation of human umbilical vein endothelial cells but also enhance dorsal root ganglia and neuronal cell extension. Previous animal studies have demonstrated that the regenerative effects of PLGA/GelMA-SC (VA) nerve conduit were similar to those of autografts and much better than those of other conduits. These findings indicate that combination of VEGF-A-overexpressing SCs and PLGA/GelMA conduit-guided peripheral nerve repair provides a promising method that enhances angiogenesis and regeneration during nerve repair. STATEMENT OF SIGNIFICANCE: Nerve guidance conduits shows promise for peripheral nerve repair, while achieving the repair efficiency of autografts remains a challenge. In this study, a composite nerve conduit with a PLGA hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with vascular endothelial growth factor A (VEGF-A)-transfected Schwann cells (SCs) as the inner layer was established to evaluate its potential ability for peripheral nerve repair. This approach preserves growth factor bioactivity and enhances material properties. GelMA insertion promotes Schwann cell proliferation and morphology extension. Moreover, transfected SCs serve as a stable VEGF-A source and fostering angiogenesis. This study offers a method preserving growth factor efficacy and safeguarding SCs, providing a comprehensive solution for enhanced angiogenesis and nerve regeneration., Competing Interests: Declaration of competing interest The author(s) declared no potential conflicts of interest with respect to the research, author - ship, and/or publication of this article., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Sperm can be selected as a natural vector for the production of transgenic animals. Methyl-beta-cyclodextrin (MBCD) removes cholesterol from the phospholipid membrane of sperm and improves the efficiency of DNA uptake by sperm. In experiment 1, fresh sperm was treated with various concentrations of MBCD. The direct effects of MBCD on sperm parameters were monitored. In experiment 2, different concentrations of MBCD (0, 1, 2, and 4 mmol) were assessed for the transfection of genetically exogenous construction to rooster sperm. Washed semen was divided into 5 equal groups for the incubation and transfection with a pcDNA3.1+/hG-CSF vector (exogenous DNA) as follows; Treatment I—Control (washed semen without DNA); Treatment II—Control (washed semen with DNA); Treatment III—(washed semen incubated with DNA and 1 mmol MBCD); Treatment IV—(washed semen incubated with DNA and 2 mmol MBCD); and Treatment V—(washed semen incubated with DNA and 4 mmol MBCD). We demonstrated that rooster spermatozoa spontaneously can uptake exogenous DNA; this was assessed using exogenous DNA amplification (sperm genomic DNA used as a template for PCR reaction) after DNase I treatment. In addition, total motility (TM), progressive motility (PM), velocity parameters [curvilinear velocity (VCL), straight linear velocity (VSL), sperm track straightness (STR), linearity (LIN)], membrane integrity (MI), and membrane functionality were posttransfectionally evaluated. The concentrations of 1 and 2 mmol MBCD significantly (P < 0.05) improved the motion characteristics and membrane integrity of fresh sperm. The presence of hG-CSF in rooster sperm was detected by PCR and based on sperm analyses MBCD (1 mmol) improved the percentage of motility (98.9 ± 0.81), membrane functionality (64 ± 1.64), and MI (76.2 ± 1.65) after transfection when compared with the other groups (P < 0.05). For the production of transgenic chicken, hens were inseminated (AI) by transfected sperm treated with 1 and 0 mmol MBCD. A PCR analysis of the blood samples and dead embryo tissues of chicks did not reveal the transgene integration.

The prognosis of patients affected by malignant pleural mesothelioma (MPM) is presently poor and no therapeutic strategies have improved their survival yet. Introduction of miRNA mimics to restore their reduced or absent functionality in cancer cells is considered an important opportunity and a combination of miR's might be even more effective. In the present study, miR-16 and miR-34a were transfected, singularly and in combination, in MPM cell lines H2052 and H28, and their effects on cell proliferation and sensitivity to cisplatin are reported. Interestingly, the overexpression of both miRs, alone or combined, slows down the cell cycle progression, modulates the p53 and HMGB1 expression and increases the sensitivity of cells to cisplatin, producing a marked impairment of cell proliferation and strengthening the apoptotic effect of the drug. However, the co-overexpression of the two miRs results more effective only in the regulation of the cell cycle, but does not enhance the sensitivity of MPM cells to cisplatin. Consequently, although the potential of miR-16 and miR-34a is confirmed, we must conclude that their combination does not improve the response of MPM to chemotherapy.

Hepatocellular Carcinoma (HCC) is one of the most common malignant tumors, and high recurrence and metastasis are the major obstacles to successful treatment of HCC. Traditional Chinese medicine has little known and unique advantages in the treatment of HCC. Previous studies have confirmed that Chinese herbal formula Qingrejiedu (clears away heat and toxins), Huoxuehuayu (promotes blood flow to remove stasis) and Fuzhengguben (strengthens healthy qi and root) (QHF) has a significant effect on patients with advanced HCC, improves the quality of life and prolongs the survival time of patients significantly. In this study, we investigated the effect of QHF on proliferation, migration and invasion of human high metastatic hepatocellular carcinoma cell line HCCLM3 and its underlying mechanism. The results from our in vitro experiments showed that QHF has the ability to inhibit the proliferation by inducing G2/M phase cell cycle arrest and induce apoptosis. Moreover, QHF can also inhibit migration and invasion of HCCLM3 cells and the expression of the p-c-Met protein in HCCLM3 cells was down-regulated. c-Met is closely related to the metastasis of HCC, then we constructed a stable transfected cell line HepG2-met with high expression of c-Met by transfection. Further study in vivo revealed that c-Met gene will promote the growth of tumors and lung metastases in nude mice, and QHF intervention can reduce tumor lung metastases by inhibiting the HGF/c-Met signaling pathway. In conclusion, our study reveals that QHF can inhibit the proliferation, migration and invasion of HCCLM3, and this effect may be related to inhibiting HGF/c-Met signaling pathway.

The human MDR1 gene encodes the efflux transporter P-glycoprotein, which plays an important part of the blood-brain barrier function of brain microvascular endothelial cells (BMECs). Here, we report the generation of an iPSC line, where a construct of the human MDR1 gene was inserted into the safe-site locus AAVS1. This iPSC line (BIONi010-C-48) shows functional expression of P-gp and can further be differentiated and cultured into electrically tight BMEC-like monolayers exhibiting polarized expression of P-gp in the apical membrane., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cationic polymers have recently attracted considerable interest as research breakthroughs for various industrial and biomedical applications. They are particularly interesting due to their highly positive charges, acceptable physicochemical properties, and ability to undergo further modifications, making them attractive candidates for biomedical applications. Polyethyleneimines (PEIs), as the most extensively utilized polymers, are one of the valuable and prominent classes of polycations. Owing to their flexible polymeric chains, broad molecular weight (MW) distribution, and repetitive structural units, their customization for functional composites is more feasible. The specific beneficial attributes of PEIs could be introduced by purposeful functionalization or modification, long service life, biocompatibility, and distinct geometry. Therefore, PEIs have significant potential in biotechnology, medicine, and bioscience. In this review, we present the advances in PEI-based nanomaterials, their transfection efficiency, and their toxicity over the past few years. Furthermore, the potential and suitability of PEIs for various applications are highlighted and discussed in detail. This review aims to inspire readers to investigate innovative approaches for the design and development of next-generation PEI-based nanomaterials possessing cutting-edge functionalities and appealing characteristics., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lentiviral vectors (LVs) have provided an efficient way to integrate our gene of interest into eukaryote cells. Human immunodeficiency virus (HIV)-derived LVs have been vastly studied to become an invaluable asset in gene delivery. This abled LVs to be used in both research laboratories and gene therapy. Pseudotyping HIV-1 based LVs, abled it to transduce different types of cells, especially hematopoietic stem cells. A wide range of tropism, plus to the ability to integrate genes into target cells, made LVs an armamentarium in gene therapy. The third and fourth generations of self-inactivating LVs are being used to achieve safe gene therapy. Not only advanced methods enabled the clinical-grade LV production on a large scale, but also considerably heightened transduction efficiency. One of which is microfluidic systems that revolutionized gene delivery approaches. Since gene therapy using LVs attracted lots of attention to itself, we provided a brief review of LV structure and life-cycle along with methods for improving both LV production and transduction. Also, we mentioned some of their utilization in immunotherapy and gene therapy.

Chickpea (Cicer arietinum L.) is the second most important grain legume worldwide. Recent advances in the sequencing of the chickpea genome has provided a new and valuable resource to aid efforts in gene discovery and crop trait improvement. Technical difficulties in stable chickpea transgenics and the lack of a transient expression system for rapid analysis of gene expression and function; however, has limited the usefulness of this genomic resource. As a step toward alleviating this limitation, we report here the development of a simple and efficient transient gene expression protocol. Using leaves from chickpea seedlings, we have established a procedure that enables the generation of large quantities of vital chickpea protoplasts within only a few hours. In addition, we have optimized a PEG-calcium-mediated transfection method to efficiently deliver exogenous DNA into the chickpea protoplast. The current study is the first to present a detailed step-by-step procedures for protoplast isolation, evaluation, transfection, and application in chickpea. In addition, we optimize the transfection efficiency which has not been previously reported. Our protoplast transfection approach provides a platform that will allow rapid high-throughput screening and systematic characterization of gene expression and function. Knowledge gained through such studies will benefit current efforts to improve chickpea production and quality. • Modified enzymatic digestion solution for higher yield and viability. • Optimize transfection of chickpea protoplasts.

Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages.

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.

Background: CDC25 is a dual-specificity phosphatase that was first identified in the yeast Schizosaccharomyces pombe as a cell cycle-defective mutant. Although CDC25 is involved in the cell cycle of ovarian granulosa cells, the CDC25 signaling pathway has not been clarified fully. To explore the role of CDC25C in the cell cycle of goat ovarian granulosa cells, a CDC25C-overexpressing vector, pCMV-HA-CDC25C, was constructed and transfected into granulosa cells from adult and young white goats from Jiangsu Nantong. RT-PCR was used to measure CDC25C, CDK1, and WEE1 gene expression levels, and flow cytometry was used to distinguish ovarian granulosa cells in different phases of the cell cycle. Progesterone and estradiol levels in transfected ovarian granulosa cells were also measured. Results: In adult goat follicular granulosa cells transfected with pCMV-HA-CDC25C, CDC25C expression increased significantly, which greatly increased the relative gene expression levels of both CDK1 and WEE1. Additionally, progesterone and estradiol levels were increased in goat follicular granulosa cells overexpressing CDC25C. And the cell cycle results showed that transfection of pCMV-HA-CDC25C leads to a higher proportion of cells in S phase compared to the no vector-transfected groups. Conclusions: The results of this study indicated that the overexpression of CDC25C may increase the gene expression levels of both WEE1 and CDK1 in S phase and accelerate the transition of cells from G1 phase to S phase.

RNA interference (RNAi)-based strategies that mediate the specific knockdown of target genes by administration of small interfering RNAs (siRNAs) could be applied for treatment of presently incurable neurodegenerative diseases such as Parkinson’s disease. However, inefficient delivery of siRNA into neurons hampers in vivo application of RNAi. We have previously established the 4–12 kDa branched polyethylenimine (PEI) F25-LMW with superior transfection efficacy for delivery of siRNA in vivo. Here, we present that siRNA complexed with this PEI extensively distributes across the CNS down to the lumbar spinal cord after a single intracerebroventricular infusion. siRNA against α-synuclein (SNCA), a pre-synaptic protein that aggregates in Parkinson’s disease, was complexed with PEI F25-LMW and injected into the lateral ventricle of mice overexpressing human wild-type SNCA (Thy1-aSyn mice). Five days after the single injection of 0.75 μg PEI/siRNA, SNCA mRNA expression in the striatum was reduced by 65%, accompanied by reduction of SNCA protein by ∼50%. Mice did not show signs of toxicity or adverse effects. Moreover, ependymocytes and brain parenchyma were completely preserved and free of immune cell invasion, astrogliosis, or microglial activation. Our results support the efficacy and safety of PEI nanoparticle-mediated delivery of siRNA to the brain for therapeutic intervention.

Ketotifen has recently been reported to inhibit the growth of both asexual and sexual malaria parasites. A parasite transporter, PfgABCG2, has been implicated in its mechanism of action. Human dihydrofolate reductase (hDHFR) is the most commonly used selectable marker to create transgenic Plasmodium falciparum cell lines. Growth assays using transgenic P. falciparum parasites with different selectable markers revealed that the presence of hDHFR rather than the absence of PfgABCG2 is responsible for a shift in the parasite's sensitivity to ketotifen. Employing a range of in vitro assays and liquid chromatography-mass spectrometry we show that ketotifen influences hDHFR activity, but it is not metabolised by the enzyme. Our data also highlights potential pitfalls when functionally characterising transgenic parasites.

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field., Competing Interests: Declaration of competing interest All authors in this work declare competing financial interests. The novel image cytometry system and methods demonstrated in this manuscript were developed using the high-throughput image cytometer and fluorescent staining kits from Revvity Health Sciences, Inc., an indirect parent company of Nexcelom Biosciences, LLC., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Polyethylenimine (PEI) is a broadly exploited cationic polymer due to its remarkable gene-loading capacity. However, the high cytotoxicity caused by its high surface charge density has been reported in many cell lines, limiting its application significantly. In this study, two different molecular weights of PEI (PEI10k and PEI25k) were crosslinked with red blood cell membranes (RBCm) via disulfide bonds to form PEI derivatives (RMPs) with lower charge density. Furthermore, the targeting molecule folic acid (FA) molecules were further grafted onto the polymers to obtain FA-modified PEI-RBCm copolymers (FA-RMP25k) with tumor cell targeting and glutathione response. In vitro experiments showed that the FA-RMP25k/DNA complex had satisfactory uptake efficiency in both HeLa and 293T cells, and did not cause significant cytotoxicity. Furthermore, the uptake and transfection efficiency of the FA-RMP25k/DNA complex was significantly higher than that of the PEI25k/DNA complex, indicating that FA grafting can increase transfection efficiency by 15 %. These results suggest that FA-RMP25k may be a promising non-viral gene vector with potential applications in gene therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Efficient transfection remains a challenge for gene delivery in both cell biological scientific research and gene therapeutic fields. Existing transfection strategies rarely pay attention to altering the endocytosis pathway of nanocarriers for transfection efficiency improvement. In this work, we innovatively postulated that calcium phosphate nanoparticles coated with glycosaminoglycan could be internalized by cells mainly through caveolin-mediated endocytosis pathway allowing genes to bypass lysosome route, and hence enhance the transfection efficiency. To achieve this, we developed calcium phosphate nanoparticles (CP-ALN-CS) coated with chondroitin sulfate (CS) and alendronate (ALN) in a modular manner. The CP-ALN-CS had a hydrodynamic size of 131.0 ± 8.7 nm and exhibited favorable dispersity, stability, and resistance to nuclease degradation. Unlike conventional calcium phosphate and PEI-based transfection, CP-ALN-CS exhibited efficient cellular uptake with co-localization in Golgi apparatus and endoplasmic reticulum. Through bypassing the lysosome involved cellular uptake route, CP-ALN-CS can effectively protect genes from degradation and relieve cytotoxicity. After loading plasmid DNA, CP-ALN-CS showed extraordinary transfection efficiency in HEK 293T cells, outperforming the PEI which is considered as the gold standard. The current work provides a novel and facile approach to improve gene transfection efficiency and is valuable for the design of next-generation in vitro transfection reagents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nucleic acids provide a promising therapeutic platform by targeting various cell signaling pathways involved in cancer and genetic disorders. However, maintaining optimal stability during delivery limits their utility. Nucleic acid delivery vehicles are generally categorized into biological and synthetic carriers. Regardless of the efficiency of biological vectors, such as viral vectors, issues related to their immunogenicity and carcinogenesis are very important and vital for clinical applications. On the other hand, synthetic vectors such as lipids or polymers, have been widely used for nucleic acid delivery. Despite their transfection efficiency, low storage stability, targeting inefficiency, and tracking limitations are among the limitations of the clinical application of these vectors. In the past decades, gold nanoparticles with unique properties have been shown to be highly efficient mineral vectors for overcoming these obstacles. In this review, we focus on gold nanoparticle-nucleic acid combinations and highlight their use in the treatment of various types of cancers. Furthermore, by stating the biological applications of these structures, we will discuss their clinical applications., Competing Interests: Declaration of competing interest This paper has been read and approved by all the authors and all of them declared no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Calculations indicate that selectively heating the extracellular media induces membrane temperature gradients that combine with electric fields and a temperature-induced reduction in the electropermeabilization threshold to potentially facilitate exogenous molecular delivery. Experiments by a wide-field, pulsed femtosecond laser with peak power density far below typical single cell optical delivery systems confirmed this hypothesis. Operating this laser in continuous wave mode at the same average power permeabilized many fewer cells, suggesting that bulk heating alone is insufficient and temperature gradients are crucial for permeabilization. This work suggests promising opportunities for a high throughput, low cost, contactless method for laser mediated exogenous molecule delivery without the complex optics of typical single cell optoinjection, for potential integration into microscope imaging and microfluidic systems.

Delivery of genetic material to cells is an integral tool to analyze and reveal the genetic interventions in normal cellular processes and differentiation, disease development and for gene therapy. It has profound applications in pharmaceutical, agricultural, environmental and biotechnological sectors. The major methods relied for gene delivery or transfection requires either viral vectors or xenogenic carrier molecules, which renders probabilistic carcinogenic, immunogenic and toxic effects. A newly evolved physical method, Cold atmospheric plasma induced transfection neither needs vector nor carriers. The 4th state of matter 'Plasma' is a quasineutral ionized gas-containing ions, neutral atoms, electrons and reactive radical molecules; and possess electric and magnetic field, along with emanating photons and UV radiations. Plasma produced at atmospheric pressure conditions, and having room temperature is conferred as Low temperature plasma or Cold atmospheric plasma. Selective and controlled application of cold atmospheric plasma on tissues creates temporary, restorable pores on cell membranes that could be diligently manipulated for gene delivery. Research in this regard attained pace since 2016. Cold atmospheric plasma induces transfection by lipid peroxidation, electroporation, and clathrin dependent endocytosis in cell membranes, by virtue of its reactive radicals and electric field. Plasma formed reactive radicals, especially 'OH' penetrates to the cell membrane and cleaves the phosphate head group of membrane lipids, peroxidize and detaches fatty acid tails. This decreases membrane thickness, increases membrane fluidity and permeability. Simultaneously plasma formed ions, electrons and reactive radicals accumulate over cells, generating local electric field and neutralize the negative charge of cell membrane. This induces stress on cell membrane and disrupts its structural integrity, by infringing the dynamic equilibrium between surface tension, spatial repulsion and linear tension between the head groups of phospholipids, generating minute pores. Neutralization of membrane charge promote foreign, external plasmid and gene movement towards cells and its enhanced binding with ligands and receptors on cell membrane, instigating clathrin dependent endocytosis. In vitro and in vivo studies have successfully delivered plasmids, linear DNA, siRNA and miRNA to several established cell lines like, HeLa, PC12, CHL, HUVEC, Jurkat, MCF, SH-SY5Y, HT, B16F10, HaCaT, LP-1, etc., and live C57BL/6 and BALB/c mice, using cold atmospheric plasma. This review delineates the cell surface mechanism of plasma-induced transfection, critically summarizes the research progress in this context, plasma devices used, and the inimitable features of this method. Metabolic activity, cell function, and viability are not adversely affected by this process; moreover, the cell permeating plasma-formed reactive radicals are effectively defended by cellular antioxidant mechanisms like superoxide dismutase, glutathione reductase and cytokines, alleviating its toxicity. A deeper understanding on mechanism of plasma action on cells, its aftermath, and the research status in this field would provide a better insight on future avenues of research., Competing Interests: Declaration of Competing Interest Authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fluorescent reporter genes are widely used to study the transfection of various types of primary cells and cell lines. The aim of our research was to investigate the expression dynamics of GFP and DsRed reporter genes individually and combined after gene electrotransfer of plasmids with two different electroporation protocols in B16F10 and CT26 cells in vitro. The cytotoxicity after gene electrotransfer of both plasmids was first determined. Second, the intensity of fluorescence and the percentage of cells transfected with both plasmids individually and in combination were monitored in real time. The results show that the percentage of viability after gene electrotransfer of plasmids using the EP2 pulses was significantly higher compared to the EP1 pulses. In contrast, the percentage of transfected cells and fluorescence intensity were higher after gene electrotransfer with the EP1 pulse protocol. Moreover, the percentage of transfected cells was higher and started earlier in the B16F10 cell line than in the CT26 cell line. However, fluorescence intensity was higher in CT26 cells. Co-expression of fluorescent proteins was achieved only in a small number of cells. In conclusion, this study elucidated some of the dynamics of reporter gene expression in cancer cell lines after gene electrotransfer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Electrotransfection is a non-viral method for delivery of nucleic acids into cells. In our previous study, we have determined the minimal copy number of plasmid DNA (pDNA) per cell required for transgene expression post electrotransfection, and developed a statistical framework to predict the pDNA copy number in the nucleus. To experimentally verify the prediction, the current study was designed to quantify the average copy number of pDNA per nucleus post electrotransfection. To achieve it, we developed a novel approach to effectively obtain isolated nuclei with minimal contamination by extranuclear pDNA. This sample preparation method enabled us to accurately measure intranuclear pDNA using quantitative real-time PCR. The data showed that the copy number of pDNA per nucleus was dependent on the period of cell culture post pulsing and the pDNA dose for electrotransfection. Additionally, the data were used to improve the statistical framework for understanding kinetics of pDNA transport in cells, and predicting how the kinetics depended on different factors. It is expected that the framework and the methodology developed in the current study will be useful for evaluating factors that may affect kinetics and mechanisms of pDNA transport in cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Aluminum (Al) is recognized as a neurotoxin. Studies have confirmed that the neurotoxicity induced by Al may be related to tau hyperphosphorylation. Phosphorylated tau is degraded through the ubiquitin-proteasome pathway (UPP), in which the carboxyl terminus of Hsc70-interacting protein (CHIP) plays an important role. However, whether the CHIP plays a role in regulating tau hyperphosphorylation induced by Al is yet to be determined. The purpose of this study was to explore the molecular mechanism of the CHIP in tau hyperphosphorylation induced by AlCl 3 in N2a cells. Mouse neuroblastoma cells (N2a) were exposed to different concentrations of AlCl 3 (0, 0.5, 1, and 2 mM) and treated with CHIP/CHIP shRNA/CHIP (ΔU-box)/CHIP (ΔTPR) plasmid transfection. The cell viability was determined by the CCK-8 kit. Protein expression was detected by Western blot. The interaction between CHIP and AlCl 3 exposure on the proteins was analyzed by factorial design ANOVA. The results showed that Al can cause tau hyperphosphorylation, mainly affecting the pThr231, pSer262, and pSer396 sites of tau in N2a cells. UPP is involved in the degradation of tau hyperphosphorylation induced by Al in N2a cells, of which CHIP may be the main regulatory target. Both the U-box and TPR domains of CHIP are indispensable and play an important role in the regulation of tau hyperphosphorylation induced by AlCl 3 in N2a cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)