Transfection - Science method

To screen potential differentially expressed genes related to immune function in nasopharyngeal carcinoma through an online database, and to verify their mechanism of action, so as to provide a reference for the diagnosis and treatment of nasopharyngeal carcinoma in the future. Differentially expressed genes were analyzed from the GSE227541 dataset...

Background The relevance of a plasma membrane Na⁺‐Ca²⁺ exchanger isoform‐3 protein, NCX3, is widely evidenced in neuronal physiology. However, the mechanisms leading to NCX3 expression deficits in Alzheimer’s disease (AD) pathology and its value as a target in AD pharmacological medicine remain incomplete. Methods Inhibition and rescue experiments...

Background TP53 variant classification benefits from the availability of large-scale functional data for missense variants generated using cDNA-based assays. However, absence of comprehensive splicing assay data for TP53 confounds the classification of the subset of predicted missense and synonymous variants that are also predicted to alter splicin...

Objectives SOX10 is crucially implicated in various cancer, yet the regulatory role in pancreatic cancer (PC) remains enigmatic. Underlying molecular mechanisms of SOX10 in PC were explored in our study. Methods Relationships between SOX10 and immune landscape were estimated using bioinformatic approaches. The expression of SOX10 and CMTM7 was ana...

Background Pancreatic cancer (PC) is a highly aggressive malignancy with a poor prognosis due to its late-stage diagnosis and limited treatment options. Objectives This study aimed to elucidate the molecular mechanisms underlying PC progression and identify potential molecular targets for its diagnosis and treatment. Methods DAZAP1 expression in...

A number of various human malignancies have been associated with abnormal microRNAs (miRNA) expression. There are evidence that miR-200 operates as both tumor suppressor and an onco-miR in a variety of tumors. In this study, we evaluated the effects of miR-200 on the proliferation and migration of pancreatic cancer cells, as well as the underlying...

Background Prion diseases are a group of neurodegenerative diseases associated with prion protein. The disease can be caused by mutations in the PRNP gene, the gene that encodes prion protein. An octapeptide repeat on the N‐terminus of prion protein plays an important role in normal intercellular function of prion protein. At present, numerous prio...

Messenger RNA (mRNA) therapeutics have garnered considerable attention due to their remarkable efficacy in the treatment of various diseases. The COVID‐19 mRNA vaccine and RSV mRNA vaccine have been approved on the market. Due to the inherent nuclease‐instability and negative charge of mRNA, delivery systems are developed to protect the mRNA from d...

Background The involvement of microRNA-668 (miR-668) in the onset and progression of renal fibrosis remains unclear. To this end, we aimed to explore the relevant mechanism of miR-668 in renal fibrosis. Methods C57BL/6 J male mice were randomly divided into sham-operated, unilateral ureteral obstruction (UUO), and UUO-fenofibrate groups. Based on...

The aim of our research was to explore the character of autophagy related 12 (ATG12) in the development of hepatocellular carcinoma (HCC). A total of 145 HCC tissues as well as paired adjacent normal tissues were collected, then immunohistochemistry was conducted to access the expression of ATG12. HCC cells were transfected with pcDNA ATG12 or si-A...

Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that binds MHC class Ilike molecule (MR1). It has garnered significant attention for its potential role in cancer immunity, although the immunological role of MAIT cells is still unclear in lung cancer. This study aim to investigate the impact of MR1 expressi...

To investigate the role of KLF6 in nasopharyngeal carcinoma (NPC) cells. NPC C666-1 cells were transfected with si-NC, si-KLF6, miR-181-5p mimics, and miR-NC. The transfection efficiency was evaluated using qRT-PCR to measure the expression of miR-181-5p and KLF6. The protein expression level of KLF6 was detected using WB assay. Cell proliferation...

Uncontrolled cell division and proliferation due to environmental and genetic factors are characteristics of cancer. Despite traditional treatments such as surgery, chemotherapy and radiation therapy, the incidence and mortality rate of cancer increases every year. Therefore, it is necessary to improve new treatment strategies such as microRNA (miR...

Simple Summary Previous studies have indicated that the presence of cancer stem cells may be a contributing factor to the development of metastasis in colorectal cancer patients. Cancer stem cells represent a small subpopulation within the tumor mass that exhibits heightened resistance to treatment and possesses the capacity for self-replication, e...

This study investigated the regulatory effect of LncRNA-GAS5 on FDX-1 in HEK293T cells by Acinetobacter baumannii . Transfected LncRNA-GAS5 overexpressing or knocking down plasmids into HEK293T cells, and the expression of FDX-1 was detected by Western blotting. A. baumannii inhibited the expression of FDX-1. Overexpression of LncRNA-GAS5 inhibited...

Viral and non-viral vectors for nucleic acids (DNA/RNA) therapeutics are important in development of pharmaceutical industries. Although viral vectors have shown significant impact in transfection of DNA/RNA, but numerus studies have shown that non-viral vectors are also effective for the safe and targeted delivery of DNA/RNA to exact location in t...

High-grade serous ovarian carcinoma (HGSOC) remains the most common and deadly form of ovarian cancer. However, available cell lines usually fail to appropriately represent its complex molecular and histological features. To overcome this drawback, we established OVAR79, a new cell line derived from the ascitic fluid of a patient with a diagnosis o...

Transfection of the mCherry tagged full length dystrophin plasmid (18.8 kb) using in-house synthesized R-HAp nanoparticles in mouse C2C12 cells, human skeletal muscle cells (hSKM) cells and DMD patient derived cells was accomplished.

Protease‐activated receptor 2 (PAR2) widely modulates various cytokine secretions and inflammatory responses, while excessive cytokine releases and immune hyperactivation may trigger cytokine storm. However, the potential participation of PAR2 in cytokine storm remains elusive. CRISPR‐Cas9 as an efficient gene editing tool can be used for investiga...

Our recently created RNA-sequence-based microRNA (miRNA) expression signature in breast cancer clinical specimens revealed that some miR-30 family members were significantly downregulated in cancer tissues. Based on TCGA database analyses, we observed that among the miR-30 family members, miR-30a-3p (the passenger strand derived from pre-miR-30a) w...

There are a number of new format antibody‐inspired molecules with multiple antigen binding capabilities in development and clinical evaluation. Here, we describe the impact of the sequence and configuration of a unique bispecific antibody format (termed BYbe) using a panel of four BYbe's and the three IgG1s from which they were derived on their pro...

GPCR-based fluorescent sensors are widely used to correlate neuromodulatory signaling with brain function. While experiments in transfected cells often reveal selectivity for individual neurotransmitters, sensor specificity in the brain frequently remains uncertain. Pursuing experiments in brain slices and in vivo, we find that norepinephrine and d...

Polyethylenimine (PEI) has been explored as an efficient non-viral system for delivering genes to cells; however, there were no protocols for its use in porcine fetal fibroblasts (PFF). Therefore, we compared different concentrations of FITC-PEI (0.625, 1.25, 2.5, 5, 10, 20, 40, or 80 µg/mL) and incubation times (30 min, 1 h, or 2 h). It was observ...

We have developed a cell-based relocalization (ReLo) assay for the detection of direct protein-protein interactions. In the assay, one protein is anchored to a membrane, causing the other protein to relocalize upon interaction. The assay can be used to study the influence of protein domains, post-translational modifications, protein conformations,...

Head and neck cancer (HNC) is the sixth most common cancer in the world, and its incidence is increasing year by year. Due to the late-stage diagnosis and poor prognosis of HNC, as well as the limitations of traditional treatment methods, it is urgent to improve early detection rates and explore alternative treatment approaches. Graphene-based nano...

Phase-separating peptides (PSPs) self-assembling into coacervate microdroplets (CMs) are a promising class of intracellular delivery vehicles that can release macromolecular modalities deployed in a wide range of therapeutic treatments. However, the molecular grammar governing intracellular uptake and release kinetics of CMs remains elusive. Here,...

To date, seven human coronaviruses (HCoVs) have been identified. Four of these viruses typically manifest as a mild respiratory disease, whereas the remaining three can cause severe conditions that often result in death. The reasons for these differences remain poorly understood, but may be related to the properties of individual viral proteins. Th...

Pralsetinib and selpercatinib have been approved as specific tyrosine kinase inhibitors (TKIs) for the treatment of patients with non-small cell lung cancer (NSCLC) harboring rearranged during transfection (RET) fusion and mutation. However, adverse events associated with pralsetinib and selpercatinib are not fully understood, especially in the rea...

The unprecedented success of mRNA-lipid nanoparticles (LNPs) has highlighted their power for protein expression, but the hours-long half-life of mRNA severely limits their use in chronic diseases. In contrast, DNA LNPs display months-long expression and genetically encode cell type specificity, but their use has been hindered by poor protein expres...

Objectives: This study described real-world patient characteristics and outcomes among selpercatinib-treated patients in the United States, using the Flatiron Health electronic health record-derived deidentified database (FHD) for advanced/metastatic non-small cell lung cancer (a/mNSCLC) and Optum’s de-identified Clinformatics® Data Mart Database (...

Background: SARS-CoV-2 enters cells primarily by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thereby blocking its physiological functions, affecting the apelinergic system, and inhibiting the cleavage of its peptides. The appropriate concentration of peptides in the apelinergic system influences the maintenance of homeostasis an...

Cationic lipids are widely used for gene delivery. Here, we report the transient formation of nuclear actin filaments in mammalian cells transfected with commercially available transfection reagents regardless of the proteins transfected. Readily detectable with phalloidin, nuclear actin ranges from short filaments to a fully developed network. Nuc...

Proton transfer is essential in virtually all biochemical processes, with enzymes facilitating this transfer by optimizing the proximity and orientation of reactants through site‐specific hydrogen bonds. Proton transfer is also crucial in the rate‐determining step for the ring‐opening polymerization of N‐carboxyanhydrides (NCAs), widely used to pre...

mRNA therapeutics are set to revolutionize disease prevention and treatment, inspiring the development of platforms for safe and effective mRNA delivery. However, current mRNA delivery platforms face some challenges, including limited organ tropism for nonvaccine applications and inflammation induced by cationic nanoparticle components. Herein, we...

This protocol describes the tissue preparation and immunohistochemistry protocol to verify the extent of dopamine denervation from 6-OHDA injections and check the transfection efficiency of GCamp6f-GFP vial construct that was injected into mouse brains.

Background Osteoporotic fractures (OPF) are fractures that occur with low-energy injuries or during daily activities, representing a serious consequence of osteoporosis (OP). With the worsening of population aging, the number of OPF patients continues to expand, causing a significant burden on families and society. Consequently, it is significant t...

Background Mitochondria-endoplasmic reticulum membrane contact (MERC) is an important mode of intercellular organelle communication and plays a crucial role in adipose tissue metabolism. Functionality of Hoxa5 is an important transcription factor involved in adipose tissue fate determination and metabolic regulation, but the relationship between Ho...

Recent advancements in oncology have led to the development of histology-agnostic therapies, which target genetic alterations irrespective of the tumor’s tissue of origin. This review aimed to provide a comprehensive update on the current state of histology-agnostic drug development, focusing on key therapies, including pembrolizumab, larotrectinib...

Background Selpercatinib is a selective RET inhibitor approved for treatment of RET-activated cancers. Adverse events (AEs) are manageable with dose modifications. This post hoc analysis characterized selpercatinib’s clinical safety profile after long-term follow-up in the safety population of LIBRETTO-001. Patients and Methods LIBRETTO-001 is an...

Background Transcription factor 7-like 2 (TCF7L2) variants seem to affect diabetes susceptibility through β-cell dysfunction, underlying basis of which has been considered to be β-cell dedifferentiation rather than apoptotic β-cell death. The Extracellular regulated protein kinases/Mitogen-activated protein kinase signaling pathway (ERK/MAPK signal...

GDP-fucose is synthesised via two pathways: de novo and salvage. The first uses GDP-mannose as a substrate, and the second uses free fucose. To date, these pathways have been considered to work separately and not to have an influence on each other. We report the mutual response of the de novo and salvage pathways to the lack of enzymes from a parti...

Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the endocrine system globally. Formononetin (FMNT), an isoflavonoid, exerts anti-tumorigenic effects and chromobox homolog 4 (CBX4) exerts tumor-promoting effect in specific types of tumors. Nevertheless, the predictive values and biological functions of FMNT and CBX4 in the pa...

Changes in intracellular concentrations of Na ⁺ and K ⁺ are shown to alter Fos gene expression. Here, we obtained a genetic construct encoding TurboGFP-dest1 gene under control of the human Fos promoter (-549; +155) and studied its expression in HEK293T. Amplification of the Fos promoter sequence from genomic DNA was only efficient in the presence...

An effective delivery platform is crucial for the development of mRNA vaccines and therapeutics. Here, a versatile platform utilizing cholesterol‐modified oligonucleotides (L‐oligo) that bind to the mRNA within lipid nanoparticles (LNP), and enables the effective delivery of the mRNA into target cells is introduced. mRNA incorporated into LNPs via...

This protocol covers the sorting of landing pad cells (HEK293F) for purity prior to starting MultiSTEP experiments. This avoids using cells which have undergone landing pad silencing, and increases the proportion of cells that are recombined during transfection experiments. Cells are sorted on blue fluorescence, as cells which do express the landin...

This protocol details the library transfection.

The incidence of thyroid cancer (THCA) has increased worldwide during the past 40 years. However, an understanding of the mechanisms and major transcription factors involved in THCA is insufficient to identify therapeutic targets against THCA. To reveal such mechanisms, we conducted bioinformatics analyses to assess the differential expression in h...

Gene therapy often fails due to enzyme degradation and low transfection efficiency, and single gene therapy usually cannot completely kill tumor cells. Several studies have reported that tripartite motif-containing protein 37 (TRIM37) plays a significant role in promoting the occurrence and development of triple negative breast cancer (TNBC). Herei...

Background High-risk human papillomaviruses are the causal agents of a subset of head and neck cancers. A previous transcriptomic analysis showed that cIAP2 protein, involved in cell survival and apoptosis, is upregulated in OKF6 oral cells that express HPV16 E6/E7. In addition, cIAP2 promotes radioresistance, a very important concern in HNC treatm...

Bioluminescence imaging (BLI) relies on the biochemical reaction between substrate and enzyme that triggers light emission upon convergence. Here, we present a protocol to study molecular oxygen dynamics in the in vivo mouse brain using the oxygen-dependent reaction between luciferase and its substrate. We describe steps for acute craniotomy, viral...

Genome-wide association study loci likely influence disease risk through cis-regulation of nearby genes. We present a protocol for measuring the cis-regulatory activity of risk variants on specific gene promoters in human primary astrocytes using luciferase reporter assays. We describe steps for transfection, conducting the assay, and data analysis...

Efficient cytosolic delivery is a significant hurdle when using short interfering RNA (siRNA) in therapeutic applications. Here we show that cholesterol-rich exosomes are prone to entering cancer cells through membrane fusion, achieving direct cytosolic delivery of siRNA. Molecular dynamics simulations suggest that deformation and increased contact...

Background The prognosis of breast cancer patients with visceral metastasis (VM) is significantly worse than that of patients without VM. We aimed to evaluate EZH2 (enhancer of zeste homolog 2) mutation as a biomarker associated with VM. Methods Data from forty-nine patients with metastatic breast cancer (MBC) pathologically confirmed at our hospi...

The Nox1-centered NADPH oxidase complex facilitates the transfer of electrons from intracellular NADPH across the cell membrane to extracellular molecular oxygen, resulting in the formation of superoxide. The complex is comprised of two membrane-bound subunits, namely Nox1 and p22phox, and the cytosolic subunits, namely NoxA1 and NoxO1. The presenc...

Aging is associated with decreased health span, and despite the recent advances made in understanding the mechanisms of aging, no antiaging drug has been approved for therapy. Therefore, strategies to promote a healthy life in aging are desirable. Previous work has shown that chronic treatment with extracellular vesicles (EVs) from young mice prolo...

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 a...

Background Reports have shown that several mutations of ring finger protein 123 ( RNF123 ) are related to ankylosing spondylitis (AS) and that the nuclear factor kappa B (NF‐κB) pathway may be involved in the etiology of AS. Herein, we sought to investigate the involvement of RNF123 and the NF‐κB pathway in AS. Methods Twenty AS patients and 20 he...

Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramu...

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral ge...

Lee and Broudy (2024) reported conspicuous microscopic objects in mRNA vaccines, which they interpreted as “nano-robots”. This is a misconception, because the wide range of different shapes can be readily explained in terms of self-assembling lipids (including cholesterol), as are used for transfection. Lipid nanostructures and their rearrangements...

The Pacific oyster Crassostrea gigas is known to have an exceptional ability to accumulate zinc, which endows it with robust resistance to pathogens and makes it an excellent source of dietary zinc. ZIP1 has been identified as an important zinc uptake protein in other species, but its role in oysters remains unclear. In the present study, a ZIP1 ho...

Dendritic cells (DCs) are critical regulators of T cell immunity, with immense therapeutic potential against tumors and autoimmune diseases. Efficient gene editing in DCs is crucial for understanding their regulatory mechanisms and maximizing their therapeutic efficacy. However, DCs are notoriously difficult to transfect, posing a major bottleneck...