Large-Scale Transient Transfection of Suspension-Adapted Chinese Hamster Ovary Cells for the Production of the Trimeric SARS-CoV-2 Spike Protein.

The production and purification of the secreted ectodomain of SARS-CoV-2 spike protein (S protein) were performed by transiently transfecting suspension-adapted Chinese hamster ovary cells (ExpiCHO). The method involved the separate addition of plasmid DNA expressing the S protein and polyethyleneimine to a suspension culture at a density of 5 × 106 cells/mL; and the subsequent addition of dimethyl sulfoxide at 2% (v/v). The transfected ExpiCHO cells were cultivated at 31 °C with agitation by orbital shaking under 5% CO2. On day six post-transfection, the culture was centrifuged, and the supernatant was filtered to remove cells and cell debris. Finally, the secreted recombinant S protein was recovered from the supernatant by a single step of affinity chromatography to the Twin-Strep-Tag of the recombinant S protein.

Antiapoptosis Engineering for Improved Protein Production from CHO Cells.

Improving the time integral of viable cell concentration by overcoming cell death, namely apoptosis, is one of the most widely used strategies for the efficient production of therapeutic proteins. By establishing stable cell lines that overexpress antiapoptotic genes or downregulate proapoptotic genes, the final product yields can be enhanced as cells become more resistant to environmental stresses. From the selection of high-expressing clones to verification of antiapoptotic activity, the method to construct a stable antiapoptotic cell line is discussed in this chapter.

Relationship between pathogenic E.coli O78-induced intestinal epithelial barrier damage and Zonulin expression levels in yaks.

To explore whether the intestinal damage of yak colibacillosis resulted from the regulation of Zonulin expression by its pathogenic bacteria, the overexpression and interference plasmids of Zonulin were designed and cultured in Tranwell after cell transfection. Then qRT-PCR and Western blot were used to detect the results of cell transfection, 200 mL 1×105 CFU/mL E.coli O78 was added for 4 hours, transmembrane resistance was measured by transmembrane resistance meter, FD4 fluorescence concentration in the lower chamber was detected by enzyme labeling instrument, bacterial translocation was measured by CFU counting method, and epithelial mucin (MUC1, MUC2) and tight junction protein (FABP2, Occludin, ZO-1) were detected by qRT-PCR. Results: The Zonulin gene overexpression and knockout cell lines were successfully constructed, the TEER value of the barrier of Zonulin overexpression cell lines began to decrease at 1 h after the addition of E.coli O78 and reached the lowest value at 4 h, and the TEER value of Zonulin interference cell lines decreased within 1-4 h after the addition of E.coli O78. At 4 h, the FD4 passing capacity of Zonulin overexpression cell lines was significantly higher than that of interfering cell lines, reaching twice as much as siRNA-1. The amount of bacterial translocation in overexpressed cell lines increased rapidly within 1-4 h, and the concentration of E.coli in the lower chamber was significantly higher than that in the siRNA-1 group at 4 h, but there was no significant change in the siRNA-1 group in the 1-4 h. There was no significant change in the mRNA level of MUC1 in Zonulin overexpression and interference cell lines after the addition of E.coli O78. In the overexpression group, the mRNA levels of MUC2, Occludin, and ZO-1 were significantly decreased, and the mRNA level of FABP2 was increased considerably. These results suggest stimulate epithelial cells to secrete Zonulin protein. Many Zonulin proteins regulate the opening of tight junction structures, reduce the transmembrane resistance of the cell barrier, and improve the permeability of the cell barrier and the amount of bacterial translocation.

Determination of the role of miR-451a on Plasmodium falciparum red blood cell stages, oxidative stress, and proteomic profiling.

BACKGROUND: This study examines the feasibility and effects of introducing microRNA mimic into red blood cells (RBCs) at the initial phases of Plasmodium falciparum 3D7 (Pf3D7) infection. The aim is to determine the correlation between increased expression of miR-451a and parasitaemia. METHODS: In this study miR-mimic-451a labelled with Cy3 and transfected into control and infected RBCs using lipofectamine and analysed using the fluorescence microscopy and flow cytometry. The study demonstrated the efficacy of miR-451a by treating pre-and post-transfected control RBCs and Pf3D7-infected RBCs with miR-mimic-451a. We also examined its impact on % growth inhibition of Pf3D7, oxidative stress markers (Luminometry, LPO, SOD, CAT, GSH and GPx). Additionally, determination of pH, haemoglobin (Hb), and proteomic profile performed using SDS-PAGE. RESULTS: Modified expression level of mir-451a has the potential to change the progression of the infection and yielded a 50% decrease in parasitaemia within 48 h. Moreover, transfected samples were shown to be efficacious in counteracting the oxidative stress-induced alterations during Pf3D7 infection and enable to return the cells towards the normalcy. Modified proteomic profile of transfected iRBCs demonstrates the correlation between overexpression of miRNA and protein expression. where, the major changes were observed in the heavy molecular weight proteins more than 57 kDa. CONCLUSION: The study reveals promising effects of miR-mimic-451a enrichment during RBC stages of Pf3D7, offering insights into potential malaria therapeutic strategies and potential biomedical research implications.

Hybrid fibrous architectures-mediated gene transfer by pDNA nanoparticles into macrophages.

Gene therapy is one of the most potential therapeutic approaches in direct and specific regulation of biological functions of macrophages at the gene level for efficient cell therapy. However, the delivery of genetic material to macrophages is extremely challenging, because of low stability, specificity and inability of therapeutic genes to efficiently enter the cells. Here, we present a method that uses the hybrid electrospun architectures based on gelatin-alginate decorated with carboxylated graphene oxide (HAG/G) as efficient substrate for loading and in vitro local and controlled delivery of plasmid DNA (pDNA) to macrophages as an alternative to systemic gene delivery carriers. Polyethyleneimine (PEI) is employed to assemble PEI/pDNA nanoparticles (Np) - used as model of carrier. The dispersion of GO-COOH sheets shifts the surface zeta potential of HAG/G to high negative value (SZP = -16.8 ± 2.21 mV) and further increases the encapsulation efficiency of PEI/pDNA Np onto hybrid HAG/G electrospun architectures to âˆ¼ 69 % (HAG/G-Np). The in vitro biological investigations show a good metabolic activity of macrophages seeded onto HAG/G-Np (MTT assay), while gene expression experiments (fluorescent microscopy) show a 30 % increase in transient gene transfection of cells cultured in the presence of HAG/G-Np as compared to those incubated with free PEI/pDNA Np.

MicroRNA-205-5p inhibits the growth and migration of breast cancer through targeting Wnt/ß-catenin co-receptor LRP6 and interacting with lncRNAs.

Breast cancer is the most prevalent type of cancer among women worldwide. Non-coding RNAs play a fundamental role in regulating the expression of different genes. MicroRNAs (miRNAs) are known to bind to mRNA and either induce its degradation or repress its translation. Also, miRNA can modulate the expression of long non-coding RNAs (lncRNA) through different mechanisms. This study aims to determine the role of miRNA-205-5p in breast cancer cell lines. miR-205-5p was bioinformatically predicted to interact with LRP6 mRNA and lncRNAs MALAT1, NEAT1, SNHG5, and SNHG16. Then, the levels of miR-205-5p and its target genes and lncRNAs in breast cancer cell lines MCF-7 and MDA-MB-231 were determined. In addition, MCF-7 and MDA-MB-231 breast cancer cells were transfected with miR-205-5p mimic or miRNA mimic negative control using lipofectamine 3000, and the effect of miR-205-5p overexpression on cellular proliferation and migration was assessed. Moreover, we probed the impact of miR-205-5p overexpression on the expression levels of LRP6, Wnt/ß-catenin pathway genes, lncRNAs, and apoptotic markers. miR-205-5p upregulation resulted in decreasing the growth and migration and induced apoptosis markers in the two tested breast cancer subtypes. Additionally, miR-205-5p overexpression resulted in decreasing the expression of LRP6 in MCF-7 and MDA-MB-231 cells leading to downregulation of Wnt/ß-catenin target genes, c-Myc, cyclin D1, and PPARδ and had various regulatory effects on the expression of lncRNAs MALAT1, NEAT1, SNHG5, and SNHG16. miR-205-5p inhibits the proliferation and migration of breast cancer through diverse mechanisms including targeting LRP6, Wnt/ß-catenin pathway, and its regulatory effects on lncRNAs.

Stable Polymer-Lipid Hybrid Nanoparticles Based on mcl-Polyhydroxyalkanoate and Cationic Liposomes for mRNA Delivery.

Background/Objectives: The development of polymer-lipid hybrid nanoparticles (PLNs) is a promising area of research, as it can help increase the stability of cationic lipid carriers. Hybrid PLNs are core-shell nanoparticle structures that combine the advantages of both polymer nanoparticles and liposomes, especially in terms of their physical stability and biocompatibility. Natural polymers such as polyhydroxyalkanoate (PHA) can be used as a matrix for the PLNs' preparation. Methods: In this study, we first obtained stable cationic hybrid PLNs using a cationic liposome (CL) composed of a polycationic lipid 2X3 (1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the hydrophobic polymer mcl-PHA, which was produced by the soil bacterium Pseudomonas helmantisensis P1. Results: The new polymer-lipid carriers effectively encapsulated and delivered model mRNA-eGFP (enhanced green fluorescent protein mRNA) to BHK-21 cells. We then evaluated the role of mcl-PHA in increasing the stability of cationic PLNs in ionic solutions using dynamic light scattering data, electrophoretic mobility, and transmission electron microscopy techniques. Conclusions: The results showed that increasing the concentration of PBS (phosphate buffered saline) led to a decrease in the stability of the CLs. At high concentrations of PBS, the CLs aggregate. In contrast, the presence of isotonic PBS did not result in the aggregation of PLNs, and the particles remained stable for 120 h when stored at +4 °C. The obtained results show that PLNs hold promise for further in vivo studies on nucleic acid delivery.

Machine Learning Elucidates Design Features of Plasmid Deoxyribonucleic Acid Lipid Nanoparticles for Cell Type-Preferential Transfection.

To broaden the accessibility of cell and gene therapies, it is essential to develop and optimize nonviral, cell type-preferential gene carriers such as lipid nanoparticles (LNPs). While high-throughput screening (HTS) approaches have proven effective in accelerating LNP discovery, they are often costly, labor-intensive, and do not consistently yield actionable design rules that direct screening efforts toward the most relevant chemical and formulation parameters. In this study, we employed a machine learning (ML) workflow, utilizing well-curated plasmid DNA LNP transfection data sets across six cell types, to extract compositional and chemical insights from HTS studies. Our approach achieved prediction errors averaging between 5 and 10%, depending on the cell type. By applying SHapley Additive exPlanations to our ML models, we uncovered key composition-function relationships that govern cell type-preferential LNP transfection efficiency. Notably, we identified consistent LNP composition parameters that enhance in vitro transfection efficiency across diverse cell types, including a helper lipid molar percentage of charged lipids between 9 and 50% and the inclusion of cationic/zwitterionic helper lipids. Additionally, several parameters were found to modulate cell type-preferentiality, such as the total molar percentage of ionizable and helper lipids, N/P ratio, PEGylated lipid molar percentage of uncharged lipids, and hydrophobicity of the helper lipid. This study leverages HTS of compositionally diverse LNP libraries combined with ML analysis to elucidate the interactions between lipid components in LNP formulations, providing insights that contribute to the design of LNP compositions tailored for cell type-preferential transfection.

Cost-effectiveness analysis of selpercatinib versus chemotherapy and pembrolizumab in the first-line treatment of rearranged during transfection fusion-positive non-small cell lung cancer in the United States.

BACKGROUND: Although selpercatinib has shown clinical benefits for rearranged during transfection (RET) fusion-positive non-small cell lung cancer (NSCLC), its cost-effectiveness requires further evaluation. AIM: This study aimed to evaluate the cost-effectiveness of selpercatinib versus chemotherapy and pembrolizumab in the first-line treatment of RET fusion-positive NSCLC from the perspective of the United States (US) payer. METHOD: A partitioned survival model was developed based on data from the LIBRETTO-431 trial. Cost and utility values for the health state were obtained from database data or published literature. The measured outcomes included quality-adjusted life-years (QALYs) and the incremental cost-effectiveness ratio (ICER). One-way sensitivity analysis and probabilistic sensitivity analyses (PSA) were conducted to assess the uncertainty of the model. RESULTS: Selpercatinib increased QALYs in patients with RET fusion-positive NSCLC by 0.90 compared to chemotherapy plus pembrolizumab, with an additional cost of $542,517.45, resulting in an ICER of $603,286.49/QALY, which exceeded the willingness-to-pay (WTP) threshold ($150,000) in the US. One-way sensitivity analysis suggested that the utility of progressed disease, the utility of progression-free survival, the price of selpercatinib, the discount, the price of pemetrexed, and the price of pembrolizumab had the greatest influence on the cost- effectiveness analysis process. In the PSA, 99.9% of the scatter points were distributed above the US WTP threshold of $150,000. CONCLUSION: From the perspective of the US payer, selpercatinib is not cost-effective compared to chemotherapy and pembrolizumab for first-line treatment in patients with RET fusion-positive NSCLC.

Single-Cell Meta-Analysis Uncovers the Pancreatic Endothelial Cell Transcriptomic Signature and Reveals a Key Role for NKX2-3 in PLVAP Expression.

BACKGROUND: The pancreatic vasculature displays tissue-specific physiological and functional adaptations that support rapid insulin response by ß-cells. However, the digestive enzymes have made it difficult to characterize pancreatic endothelial cells (ECs), resulting in the poor understanding of pancreatic EC specialization. METHODS: Available single-nuclei/single-cell RNA-sequencing data sets were mined to identify pancreatic EC-enriched signature genes and to develop an integrated atlas of human pancreatic ECs. We validated the findings using independent single-nuclei/single-cell RNA-sequencing data, bulk RNA-sequencing data of isolated ECs, spatial transcriptomics data, immunofluorescence, and RNAScope of selected markers. The TF (transcription factor) NKX2-3 was expressed in HUVECs via gene transfection, and the expression of pancreatic EC-enriched signature genes was assessed via RT-qPCR. RESULTS: We defined a pancreatic EC-enriched gene signature conserved across species and developmental stages that included genes involved in ECM (extracellular matrix) composition (COL15A1 and COL4A1), permeability and barrier function (PLVAP, EHD4, CAVIN3, HSPG2, ROBO4, HEG1, and CLEC14A), and key signaling pathways (S1P, TGF-ß [transforming growth factor-ß], RHO-RAC GTPase, PI3k-AKT, and PDGF [platelet-derived growth factor]). The integrated atlas revealed the vascular hierarchy within the pancreas. We identified and validated a specialized islet capillary subpopulation characterized by genes involved in permeability (PLVAP and EHD4), immune-modulation (FABP5, HLA-C, and B2M), ECM composition (SPARC and SPARCL1), IGF (insulin-like growth factor) signaling (IGFBP7), and membrane transport (SLCO2A1, SLC2A3, and CD320). Importantly, we identified NKX2-3 as a key TF enriched in pancreatic ECs. DNA-binding motif analysis found NKX2-3 motifs in ≈40% of the signature genes. Induction of NKX2-3 in HUVECs promoted the expression of the islet capillary EC-enriched genes PLVAP and SPARCL1. CONCLUSIONS: We defined a validated transcriptomic signature of pancreatic ECs and uncovered their intratissue transcriptomic heterogeneity. We showed that NKX2-3 acts upstream of PLVAP and provided a single-cell online resource that can be further explored by the community: https://vasconcelos.shinyapps.io/pancreatic_endothelial/.

Enhancing gene delivery efficiency with amphiphilic chitosan modified by myristic acid and tertiary amino groups.

The aim of this study is to synthesize new amphiphilic chitosan containing myristic acid as the hydrophobic tail and tertiary amine groups as the hydrophilic head and to evaluate the gene delivery efficiency. In this context, the primary amine groups of chitosan were first modified with myristic acid (Chi-M), followed by the modification of the methylol groups with 3-dimethylamino-1-propyl chloride hydrochloride. The chemical characterization of this chitosan formulation (Chi-MA) was determined using nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR) analysis and gel permeation chromatography-size exclusion chromatography. Chi-MA nanoparticles were prepared via ionic gelation, and particle size, polydispersity and zeta potential were determined. The nanoparticles were evaluated for their proton buffering capacity and gene complexing capacity. Additionally, the cytotoxicity of Chi-MA on HEK293 cells was determined via MTT assay, and the transfection efficiency of Chi-MA was analyzed by a flow cytometer. The results indicate a significant increase in gene complexing capacity (8-fold) and nanoparticle formation ability of Chi-MA compared to other chitosan formulations. Chi-MA nanoparticles showed no toxicity against HEK293 cells and exhibited the highest transfection efficiency with significantly lower nanoparticle: gene ratios compared to previous studies. These findings demonstrate the effective use of amphiphilic Chi-MA as a gene carrier.

Characterisation of the HLA-DQ alpha eplet 52SK targeting the DQA1*01 alleles family.

Eplet 52SK is unique in the HLA eplet registry as targeting the whole family of DQA1*01 alleles. It is proposed as an antibody-verified eplet but has not been validated enough to deserve this label. Especially, confusion can occur with reactivity targeting the 52PQ eplet which is present on the DQB1*05 and DQB1*06 alleles families, as DQ molecule stability imposes DQA1*01 to selectively associate with these DQ-ß families only. Using two Luminex single antigen (LSA) assays from two vendors, beads bearing DR-α/DQ6 heterodimers, a special build LSA panel of additional DQ beads, and an adsorption/elution strategy relying on cells from deceased donors or recombinant cells solely expressing one DQ antigen, we definitely established the antibody-verified status of eplet 52SK using patients' sera reacting only against the DQ5 and DQ6 beads of the One Lambda LSA panel in routine patients' follow up. We also show that reactivity against this eplet is not a rare event among anti-DQ1 immunisation. This study further strengthens the importance of considering the DQA1 locus in immunological studies of HLA and in organ allocation strategies.

Supplementation with ions enhances the efficiency of nucleic acid delivery with cell-penetrating peptides.

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 Ca2+ ions can significantly enhance NA delivery efficiency when included in transfection media or CPP/NA nanoparticles during preparation. Similar effects have been observed for Mg2+, 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.

Plate centrifugation enhances the efficiency of polyethylenimine-based transfection and lentiviral infection.

PURPOSE: To propose an efficient, reproducible, and consistent transgenic technology based on plate centrifugation, which is particularly useful for polyethylenimine (PEI) transfection and lentiviral infection. METHODS: We optimized multiple factors that could contribute to transfection efficiency, such as the dosage of the PEI or DNA, the working solution buffer used for diluting the PEI or DNA, the incubation time for the PEI/DNA complexes, and the transfection time. RESULTS: Plate centrifugation led to a 5.46-fold increase in the transfection efficiency of PEI-based transfection while maintaining the cell survival rate. Moreover, the average copy number of viral genes in each genome increased 4.96-fold with plate centrifugation. Plate centrifugation alters the spatial arrangement of the PEI/DNA complexes or lentiviruses, increasing the chances of these complexes or viruses coming into contact with target cells, ultimately resulting in improved transfection or infection efficiency. CONCLUSIONS: We present a protocol based on plate centrifugation for transfection or lentiviral infection that is suitable for genetic modification of primary cells or stem cells.

Cationized extracellular vesicles for gene delivery.

Last decade, extracellular vesicles (EVs) attracted a lot of attention as potent versatile drug delivery vehicles. We reported earlier the development of EV-based delivery systems for therapeutic proteins and small molecule chemotherapeutics. In this work, we first time engineered EVs with multivalent cationic lipids for the delivery of nucleic acids. Stable, small size cationized EVs were loaded with plasmid DNA (pDNA), or mRNA, or siRNA. Nucleic acid loaded EVs were efficiently taken up by target cells as demonstrated by confocal microscopy and delivered their cargo to the nuclei in triple negative breast cancer (TNBC) cells and macrophages. Efficient transfection was achieved by engineered cationized EVs formulations of pDNA- and mRNA in vitro. Furthermore, siRNA loaded into cationized EVs showed significant knockdown of the reporter gene in Luc-expressing cells. Overall, multivalent cationized EVs represent a promising strategy for gene delivery.

Sporadic and Familial Medullary Thyroid Carcinoma: A Retrospective Single Center Study on Presentation and Outcome.

BACKGROUND: Medullary Thyroid Carcinoma (MTC) is a neuroendocrine tumor that arises from the thyroid C-cells. Most cases are sporadic (sMTC) while, approximately 25%, are hereditary (hMTC) due to germline mutations of REarranged during Transfection (RET) gene mutations and manifest in the framework of multiple endocrine neoplasia (MEN) 2A or 2B, or as pure familial MTC syndrome (FMTC). OBJECTIVE: The aim of this study is to evaluate the clinical, histopathological, biochemical and outcome differences between sMTC and hMTC. METHODS: Retrospective analysis of a consecutive series of 102 patients with histologically proven MTC diagnosed in the period between 2000 and 2022. For the analysis patients with MTC diagnosed during screening through genetic test were excluded. RESULTS: Patients with hMTC had higher incidence of multifocal and bilateral MTC and younger age at diagnosis. We did not found differences on tumor stage at diagnosis between sMTC and hMTC, such as time to progression and rate of persistent and recurrent disease. At univariate analysis, factors associated with persistent and recurrent disease during follow-up in patients with sMTC were tumor size, extrathyroidal extension, presence of lymph node metastases at diagnosis, pre- and post-operative calcitonin, post-operative CEA; in patients with hMTC, features associated with persistent and recurrent disease were lymph node metastases, post-operative calcitonin and pre- and post-operative CEA values. CONCLUSION: Patients with hMTC and sMTC had similar histopathological characteristics and clinical outcome.

Cationic Carbosilane Dendrimers for Apmnkq2 Aptamer Transfection in Breast Cancer: An Alternative to Traditional Transfectants.

Transfection efficiency is a critical parameter in gene therapy and molecular biology, representing the success rate at which nucleic acids are introduced and expressed in target cells. The combination of aptamers with nanotechnology-based delivery systems has demonstrated remarkable improvements in the transfection efficiency of therapeutic agents and holds significant potential for advancing gene therapy and the development of targeted treatments for various diseases, including cancer. In this work, cationic carbosilane dendritic systems are presented as an alternative to commercial transfection agents, demonstrating an increase in transfection efficiency when used for the internalization of apMNKQ2, an aptamer selected against a target in cancer. Their potential therapeutic use has been evaluated in breast cancer cell lines, MDA-MB-468 and MDA-MB-231, studying the cytotoxicity of the nanoconjugate, the internalization process, and its effect on cellular migration processes.

Common SYNE2 Genetic Variant Associated With Atrial Fibrillation Lowers Expression of Nesprin-2α1 With Downstream Effects on Nuclear and Electrophysiological Traits.

BACKGROUND: Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton. METHODS: Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2α1. After SYNE2 knockdown or SYNE2α1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity. RESULTS: The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2α1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2α1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2α1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity. CONCLUSIONS: rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2α1 expression. Downstream effects of SYNE2α1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF.

Spatially-Resolved Organoid Transfection by Porous Silicon-Mediated Optoporation.

Engineering the spatial organisation of organotypic cultures is pivotal for refining tissue models that are useful for gaining deeper insights into complex, non-cell autonomous processes. These advanced models are key to improving the understanding of fundamental biological mechanisms and therapeutic strategies. Controlling gene regulation through spatially-resolved delivery of nucleic acids provides an attractive approach to produce such tissue models. An emerging strategy for spatially-resolved transfection uses photosensitizing nanoparticles coupled with laser pulses to optoporate cells in culture and locally mediate gene delivery. However, localized optoporation in 3D systems remains challenging. Here we propose a solution to this longstanding hurdle, demonstrating that porous silicon nanoparticles are a safe and bioresorbable photosensitising nanomaterial capable of spatially-resolved transfection of mRNA in MCF-7 organoids by near-infrared two-photon optoporation. Functionalization with an azobenzene-lysine photo-switchable moiety enhances the transfection efficiency of the nanoparticles up to 84% in a 2D cell system. Moreover, the nanoparticles enable spatially selective mRNA transfection to MCF-7 spheroids, demonstrating targeted  gene delivery in complex 3D cellular environments. The approach for spatially-resolved 3D optoporation offers a way forward for the design of tailored spheroids and organoids by spatially selective nucleic acids delivery.

Molecular genetics, therapeutics and RET inhibitor resistance for medullary thyroid carcinoma and future perspectives.

Medullary thyroid carcinoma (MTC) is a rare type of thyroid malignancy that accounts for approximately 1-2% of all thyroid cancers (TCs). MTC include hereditary and sporadic cases, the former derived from a germline mutation of rearrangement during transfection (RET) proto-oncogene, whereas somatic RET mutations are frequently present in the latter. Surgery is the standard treatment for early stage MTC, and the 10-year survival rate of early MTC is over 80%. While for metastatic MTC, chemotherapy showing low response rate, and there was a lack of effective systemic therapies in the past. Due to the high risk (ca. 15-20%) of distant metastasis and limited systemic therapies, the 10-year survival rate of patients with advanced MTC was only 10-40% from the time of first metastasis. Over the past decade, targeted therapy for RET has developed rapidly, bringing hopes to patients with advanced and progressive MTC. Two multi-kinase inhibitors (MKIs) including Cabozantinib and Vandetanib have been shown to increase progression-free survival (PFS) for patients with metastatic MTC and have been approved as choices of first-line treatment. However, these MKIs have not prolonged overall survival (OS) and their utility is limited due to high rates of off-target toxicities. Recently, new generation TKIs, including Selpercatinib and Pralsetinib, have demonstrated highly selective efficacy against RET and more favorable side effect profiles, and gained approval as second-line treatment options. Despite the ongoing development of RET inhibitors, the management of advanced and progressive MTC remains challenging, drug resistance remains the main reason for treatment failure, and the mechanisms are still unclear. Besides, new promising therapeutic approaches, such as novel drug combinations and next generation RET inhibitors are under development. Herein, we overview the pathogenesis, molecular genetics and current management approaches of MTC, and focus on the recent advances of RET inhibitors, summarize the current situation and unmet needs of these RET inhibitors in MTC, and provide an overview of novel strategies for optimizing therapeutic effects.