Stable Gene Expression Research Articles

Lactate promotes neuronal differentiation of SH-SY5Y cells by lactate-responsive gene sets through NDRG3-dependent and -independent manners

Lactate serves as the major glucose alternative to an energy substrate in the brain. Lactate level is increased in the fetal brain from the middle stage of gestation, indicating the involvement of lactate in brain development and neuronal differentiation. Recent reports show that lactate functions as a signaling molecule to regulate gene expression and protein stability. However, the roles of lactate signaling in neuronal cells remain unknown. Here, we showed that lactate promotes the all stages of neuronal differentiation of SH-SY5Y and Neuro2A, human and mouse neuroblastoma cell lines, characterized by increased neuronal marker expression and the rates of neurites extension. Transcriptomics revealed many lactate-responsive genes sets such as SPARCL1 in SH-SY5Y, Neuro2A, and primary embryonic mouse neuronal cells. The effects of lactate on neuronal function were mainly mediated through monocarboxylate transporters 1 (MCT1). We found that NDRG family member 3 (NDRG3), a lactate-binding protein, was highly expressed and stabilized by lactate treatment during neuronal differentiation. Combinative RNA-seq of SH-SY5Y with lactate treatment and NDRG3 knockdown shows that the promotive effects of lactate on neural differentiation are regulated through NDRG3-dependent and independent manners. Moreover, we identified TEA domain family member 1 (TEAD1) and ETS-related transcription factor 4 (ELF4) are the specific transcription factors that are regulated by both lactate and NDRG3 in neuronal differentiation. TEAD1 and ELF4 differently affect the expression of neuronal marker genes in SH-SY5Y cells. These results highlight the biological roles of extracellular and intracellular lactate as a critical signaling molecule that modifies neuronal differentiation.

Stable Expression of FOXP3 and Core Tolerogenic Genes by Engineered Human Tregs

Abstract FOXP3 +Regulatory T cells (Tregs) play a critical role in the prevention of fatal autoimmunity and maintaining tissue homeostasis. The functional stability of Tregs remains critical in their contribution towards immune tolerance rather than runaway immunity, particularly for cell therapy whereby an inflammatory microenvironment may impact the expression of FOXP3 and associated tolerogenic genes. To address potential Treg instability, we generated human Engineered Tregs (EngTregs) through a gene editing approach of PBMC isolated CD4+ T cells, leading to stable FOXP3 expression and a rapamycin-activated signaling complex that provides tunable IL-2 signal, thereby effectively divorcing FOXP3 expression from existing regulatory elements known to promote Treg instability under inflammatory conditions. Using transcriptomic analysis, we evaluated the stability of cultured sorted Tregs (cTregs) compared to EngTregs. We found EngTregs expressed higher levels of key “Core Treg” and “FOXP3 Synergy” genes based on bulk and single cell RNA-seq analysis as confirmed by flow cytometry. Additionally, favorable expression pattern of key Treg stability markers, CD27, CD70 and IKZF4 (EOS) was observed in EngTregs compared to cTregs. Conversely, cTregs express higher levels of cytotoxic genes including GZMA and PERFORIN1 compared to EngTregs along with other inflammatory genes. Finally, we observed that these correlations have a functional implication as demonstrated by higher expression of key tolerogenic markers in EngTregs compared to cTregs. This work strongly supports EngTregs as stable, tolerogenic FOXP3+ T cells, thereby providing an invaluable asset for treating life-threatening autoimmune and autoinflammatory diseases.

Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes β-cell subpopulations with dynamic transcriptome profiles

BackgroundSingle-cell RNA sequencing (scRNA-seq) provides valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo. On the other hand, single-nucleus RNA sequencing (snRNA-seq) has compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and affords enhanced information from intronic sequences that can be leveraged to identify pre-mRNA transcripts.MethodsWe obtained nuclear preparations from fresh human islet cells and generated snRNA-seq datasets. We compared these datasets to scRNA-seq output obtained from human islet cells from the same donor. We employed snRNA-seq to obtain the transcriptomic profile of human islets engrafted in immunodeficient mice. In both analyses, we included the intronic reads in the snRNA-seq data with the GRCh38-2020-A library.ResultsFirst, snRNA-seq analysis shows that the top four differentially and selectively expressed genes in human islet endocrine cells in vitro and in vivo are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (β-cells); PTPRT, FAP, PDK4, LOXL4 (α-cells); LRFN5, ADARB2, ERBB4, KCNT2 (δ-cells); and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ-cells). Second, by integrating information from scRNA-seq and snRNA-seq of human islet cells, we distinguish three β-cell sub-clusters: an INS pre-mRNA cluster (β3), an intermediate INS mRNA cluster (β2), and an INS mRNA-rich cluster (β1). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA-rich cluster (β1) becomes the predominant sub-cluster in vivo.ConclusionsIn summary, snRNA-seq and pre-mRNA analysis of human islet cells can accurately identify human islet cell populations, subpopulations, and their dynamic transcriptome profile in vivo.

Partial erosion on under-methylated regions and chromatin reprogramming contribute to oncogene activation in IDH mutant gliomas

BackgroundIDH1/2 hotspot mutations are well known to drive oncogenic mutations in gliomas and are well-defined in the WHO 2021 classification of central nervous system tumors. Specifically, IDH mutations lead to aberrant hypermethylation of under-methylated regions (UMRs) in normal tissues through the disruption of TET enzymes. However, the chromatin reprogramming and transcriptional changes induced by IDH-related hypermethylation in gliomas remain unclear.ResultsHere, we have developed a precise computational framework based on Hidden Markov Model to identify altered methylation states of UMRs at single-base resolution. By applying this framework to whole-genome bisulfite sequencing data from 75 normal brain tissues and 15 IDH mutant glioma tissues, we identified two distinct types of hypermethylated UMRs in IDH mutant gliomas. We named them partially hypermethylated UMRs (phUMRs) and fully hypermethylated UMRs (fhUMRs), respectively. We found that the phUMRs and fhUMRs exhibit distinct genomic features and chromatin states. Genes related to fhUMRs were more likely to be repressed in IDH mutant gliomas. In contrast, genes related to phUMRs were prone to be up-regulated in IDH mutant gliomas. Such activation of phUMR genes is associated with the accumulation of active H3K4me3 and the loss of H3K27me3, as well as H3K36me3 accumulation in gene bodies to maintain gene expression stability. In summary, partial erosion on UMRs was accompanied by locus-specific changes in key chromatin marks, which may contribute to oncogene activation.ConclusionsOur study provides a computational strategy for precise decoding of methylation encroachment patterns in IDH mutant gliomas, revealing potential mechanistic insights into chromatin reprogramming that contribute to oncogenesis.

Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Megalurothrips usitatus (thysanoptera: thripidae).

Introduction: Gene expression analysis by reverse transcription quantitative polymerase chain reaction (qRT-PCR) has been widely used in research including insects. The selection of appropriate reference genes is the key to obtaining accurate and reliable results from qRT-PCR. However, studies on the expression stability of reference genes in Megalurothrips usitatus remain lacking. Methods: In this study, qRT-PCR was used to analyze the expression stability of candidate reference genes in M. usitatus. The expression levels of six candidate reference gene transcription of M. usitatus were analyzed. GeNorm, NormFinder, BestKeeper, and ΔCt were used to analyze the expression stability of M. usitatus treated with biological factors (developmental period treatment) and abiotic factors (light, temperature, insecticide treatment, respectively). Comprehensive stability ranking of candidate reference genes was recommended by RefFinder. Results and Discussion: Results showed that ribosomal protein S (RPS) was the most suitable expression in insecticide treatment. Ribosomal protein L (RPL) was the most suitable expression at developmental stage and light treatment, whereas elongation factor was the most suitable expression in temperature treatment. RefFinder was used to comprehensively analyze the above four treatments, and the results showed that RPL and actin (ACT) showed high stability in each treatment. Therefore, this study identified these two genes as reference genes in the qRT-PCR analysis of different treatment conditions of M. usitatus. Ourfindings will be beneficial for improving the accuracy of qRT-PCR analysis for future functional analysis of the target gene expression in M. usitatus.

Nanopore Cas9-targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells.

The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated, and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates, it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution.In this study, we present a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9-enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724× median on-target coverage and up to 153 kb long reads. The data generated by nCats provides sensitive, detailed, and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification-seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.

Low dose acetyl salicylic acid (LDA) mediates epigenetic changes in preeclampsia placental mesenchymal stem cells similar to cells from healthy pregnancy

IntroductionPreeclampsia (PE) affects 2–8% of all pregnancies, and is the leading cause of maternal and fetal morbidity and mortality. We reported on pathophysiological changes in placenta mesenchymal stem cells (P-MSCs) in PE. P-MSCs can be isolated from different layers of the placenta at the interface between the fetus and mother. The ability of MSCs from other sources to be immune licensed as immune suppressor cells indicated that P-MSCs could mitigate fetal rejection. Acetylsalicylic acid (aspirin) is indicated for treating PE. Indeed, low-dose aspirin is recommended to prevent PE in high risk patients. MethodsWe conducted robust computational analyses to study changes in gene expression in P-MSCs from PE and healthy term pregnancies as compared with PE-MSCs treated with low dose acetyl salicylic acid (LDA). Confocal microscopy studied phospho-H2AX levels in P-MSCs. ResultsWe identified changes in >400 genes with LDA, similar to levels of healthy pregnancy. The top canonical pathways that incorporate these genes were linked to DNA repair damage - Basic excision repair (BER), Nucleotide excision repair (NER) and DNA replication. A role for the sumoylation (SUMO) pathway, which could regulate gene expression and protein stabilization was significant although reduced as compared to BER and NER pathways. Labeling for phopho-H2AX indicated no evidence of double strand break in PE P-MSCs. DiscussionThe overlapping of key genes within each pathway suggested a major role for LDA in the epigenetic landscape of PE P-MSCs. Overall, this study showed a new insight into how LDA reset the P-MSCs in PE subjects around the DNA.

Evaluating and Validating Sunflower Reference Genes for Q-PCR Studies Under High Temperature Condition.

Q-PCR is the method of choice for PCR- based transcriptomics and validating microarray-based and RNA-seq results. Proper application of this technology requires proper normalization to correct as much as possible errors propagating during RNA extraction and cDNA synthesis. The investigation was performed to find stable reference genes in sunflower under shifting in ambient temperature. Sequences of five well-known reference genes of Arabidopsis (Actin, Ubiquitin, Elongation factor-1, GAPDH, and SAND) and one well-known reference gene inhuman, Importin, were subjected to BLASTX against sunflower databases and the relevant genes were subjected to primer designing for q-PCR. Two sunflower inbred lines were cultivated at two dates so that anthesis occurred at nearly 30 °C and 40 °C (heat stress). The experiment was repeated for two years. Q-PCR was run on samples taken for two planting date separately at the beginning of anthesis for each genotype from leaf, taproots, receptacle base, immature and mature disc flowers and on pooled samples comprising of the tissues for each genotype, planting dates and also all tissues for both genotypes and both planting dates. Basic statistical properties of each candidate gene across all the samples were calculated. Furthermore, gene expression stability analysis was done for six candidate reference genes on Cq mean of two years using three independent algorithms, geNorm, Bestkeeper, and Refinder. Designed primers for Actin2, SAND, GAPDH, Ubiquitin, EF-1a, and Importin yielded a single peak in melting curve analysis indicating specificity of the PCR reaction. Basic statistical analysis showed that Actin2 and EF-1a had the highest and lowest expression levels across all the samples, respectively. Actin2 appeared to be the most stable reference gene across all the samples based on the three used algorithms. Pairwise variation analysis revealed that for samples taken under ambient temperature of 30 °C, Actin2, EF-1a, SAND and for those taken under ambient temperature of 40 °C, Actin2, EF-1a, Importin and SAND have to be used for normalization in q-PCR studies. Moreover, it is suggested that normalization to be based on Actin2, SAND and EF-1a for vegetative tissues and Actin2, EF-1a, SAND and Importin for reproductive tissues. In the present research, proper reference genes for normalization of gene expression studies under heat stress conditions were introduced. Moreover, the presence of genotype-by- planting date interaction effects and tissue specific gene expression pattern on the behavior of the most three stable reference genes was indicated.

Immunoinformatic prediction of potential immunodominant epitopes from cagW in order to investigate protection against Helicobacter pylori infection based on experimental consequences.

Helicobacter pylori is a leading cause of stomach cancer and peptic ulcers. Thus, identifying epitopes in H. pylori antigens is important for disease etiology, immunological surveillance, enhancing early detection tests, and developing optimal epitope-based vaccines. We used immunoinformatic and computational methods to create a potential CagW epitope candidate for H. pylori protection. The cagW gene of H. pylori was amplified and cloned into pcDNA3.1 (+) for injection into the muscles of healthy BALB/c mice to assess the impact of the DNA vaccine on interleukin levels. The results will be compared to a control group of mice that received PBS or cagW-pcDNA3.1 (+) vaccinations. An analysis of CagW protein antigens revealed 8 CTL and 7 HTL epitopes linked with AYY and GPGPG, which were enhanced by adding B-defensins to the N-terminus. The vaccine's immunogenicity, allergenicity, and physiochemistry were validated, and its strong activation of TLRs (1, 2, 3, 4, and 10) suggests it is antigenic. An in-silico cloning and immune response model confirmed the vaccine's expression efficiency and predicted its impact on the immune system. An immunofluorescence experiment showed stable and bioactive cagW gene expression in HDF cells after cloning the whole genome into pcDNA3.1 (+). In vivo vaccination showed that pcDNA3.1 (+)-cagW-immunized mice had stronger immune responses and a longer plasmid DNA release window than control-plasmid-immunized mice. After that, bioinformatics methods predicted, developed, and validated the three-dimensional structure. Many online services docked it with Toll-like receptors. The vaccine was refined using allergenicity, antigenicity, solubility, physicochemical properties, and molecular docking scores. Virtual-reality immune system simulations showed an impressive reaction. Codon optimization and in-silico cloning produced E. coli-expressed vaccines. This study suggests a CagW epitopes-protected H. pylori infection. These studies show that the proposed immunization may elicit particular immune responses against H. pylori, but laboratory confirmation is needed to verify its safety and immunogenicity.

Quantitative evaluation of endogenous reference genes for ddPCR under salt stress using a moderate halophile.

Droplet digital PCR (ddPCR) is being increasingly adopted for gene detection and quantification because of its higher sensitivity and specificity. According to previous observations and our laboratory data, it is essential to use endogenous reference genes (RGs) when investigating gene expression at the mRNA level under salt stress. This study aimed to select and validate suitable RGs for gene expression under salt stress using ddPCR. Six candidate RGs were selected based on the tandem mass tag (TMT)-labeled quantitative proteomics of Alkalicoccus halolimnae at four salinities. The expression stability of these candidate genes was evaluated using statistical algorithms (geNorm, NormFinder, BestKeeper and RefFinder). There was a small fluctuation in the cycle threshold (Ct) value and copy number of the pdp gene. Its expression stability was ranked in the vanguard of all algorithms and was the most suitable RG for quantification of expression by both qPCR and ddPCR of A. halolimnae under salt stress. Single RG pdp and RG combinations were used to normalize the expression of ectA, ectB, ectC and ectD under four salinities. The present study constitutes the first systematic analysis of endogenous RG selection for halophiles responding to salt stress. This work provides a valuable theory and an approach reference of internal control identification for ddPCR-based stress response models.

Stability in gene expression and body-plan development leads to evolutionary conservation

BackgroundPhenotypic evolution is mainly explained by selection for phenotypic variation arising from factors including mutation and environmental noise. Recent theoretical and experimental studies have suggested that phenotypes with greater developmental stability tend to have a constant phenotype and gene expression level within a particular genetic and environmental condition, and this positively correlates with stronger evolutionary conservation, even after the accumulation of genetic changes. This could reflect a novel mechanism that contributes to evolutionary conservation; however, it remains unclear whether developmental stability is the cause, or whether at least it contributes to their evolutionary conservation. Here, using Japanese medaka lines, we tested experimentally whether developmental stages and gene expression levels with greater stability led to their evolutionary conservation.ResultsWe first measured the stability of each gene expression level and developmental stage (defined here as the whole embryonic transcriptome) in the inbred F0 medaka population. We then measured their evolutionary conservation in the F3 generation by crossing the F0 line with the distantly related Japanese medaka line (Teradomori), followed by two rounds of intra-generational crossings. The results indicated that the genes and developmental stages that had smaller variations in the F0 generation showed lower diversity in the hybrid F3 generation, which implies a causal relationship between stability and evolutionary conservation.ConclusionsThese findings suggest that the stability in phenotypes, including the developmental stages and gene expression levels, leads to their evolutionary conservation; this most likely occurs due to their low potential to generate phenotypic variation. In addition, since the highly stable developmental stages match with the body-plan-establishment stage, it also implies that the developmental stability potentially contributed to the strict conservation of animal body plan.

Housekeeping gene stability in Pesudomonas aeruginosa PAO1 under the pressure of commonly used antibiotics in molecular microbiology assays.

Pseudomonas aeruginosa is an opportunistic human pathogen notorious for its remarkable capacity of multi-drug resistance, and has become one of the most important model bacteria in clinical bacteriology research. Quantitative real-time PCR is a reliable method widely used in gene expression analysis, for which the selection of a set of appropriate housekeeping genes is a key prerequisite for the accuracy of the results. However, it is easy to overlook that the expression level of housekeeping gene may vary in different conditions, especially in the condition of molecular microbiology assays, where tested strains are generally cultured under the pre-set antibiotic selection pressures, and how this affects the stability of commonly used housekeeping genes remains unclear. In this study, the expression stability of ten classic housekeeping genes (algD, gyrA, anr, nadB, recA, fabD, proC, ampC, rpoS, and rpsL) under the pressure of eight laboratory commonly used antibiotics (kanamycin, gentamycin, tetracycline, chloramphenicol, hygromycin B, apramycin, tellurite, and zeocin) were tested. Results showed that the stability of housekeeping gene expression was indeed affected by the types of antibiotics added, and of course the best reference gene set varied for different antibiotics. This study provides a comprehensive summary of the effects of laboratory antibiotics on the stability of housekeeping genes in P. aeruginosa, highlighting the necessity to select housekeeping genes according to the type of antibiotics used in the initial stage of experiment.

Subchronic Toxicological Evaluation of Xiushui 134Bt Transgenic Insect-Resistant Rice in Rats

In this study, Xiushui 134Bt, a highly insect-resistant rice transgenic material with stable expression of CryIAc1 gene in rice plants and no significant changes in main agronomic traits, was obtained by means of Agrobacterium-mediated transformation method using japonica rice Xiushui 134 as receptor material. Biosecurity of the transgenic rice Xiushui 134Bt was assessed by rat feeding experiments. Wistar rats were fed a nutritionally balanced diet supplemented with 35% Genetically Modified (LGM) or 70% Genetically Modified (HGM) Xiushui 134Bt, respectively, for 90 days. Compared with the wild-type group (Xiushui 134), there were no significant differences in body weight, total food intake, food conversion rate, relative organ weight, blood routine, blood physiological or biochemical parameters, or histopathological examination between LGM and HGM Bt groups (p > 0.05). These results indicate that the transgenic rice Xiushui 134Bt is non-toxic in laboratory animals and provide guidance for the future commercial release of the transgenic rice Xiushui 134Bt in China.

The landscape of m1A modification and its posttranscriptional regulatory functions in primary neurons.

Cerebral ischaemia‒reperfusion injury (IRI), during which neurons undergo oxygen-glucose deprivation/reoxygenation (OGD/R), is a notable pathological process in many neurological diseases. N1-methyladenosine (m1A) is an RNA modification that can affect gene expression and RNA stability. The m1A landscape and potential functions of m1A modification in neurons remain poorly understood. We explored RNA (mRNA, lncRNA, and circRNA) m1A modification in normal and OGD/R-treated mouse neurons and the effect of m1A on diverse RNAs. We investigated the m1A landscape in primary neurons, identified m1A-modified RNAs, and found that OGD/R increased the number of m1A RNAs. m1A modification might also affect the regulatory mechanisms of noncoding RNAs, e.g., lncRNA-RNA binding proteins (RBPs) interactions and circRNA translation. We showed that m1A modification mediates the circRNA/lncRNA‒miRNA-mRNA competing endogenous RNA (ceRNA) mechanism and that 3' untranslated region (3'UTR) modification of mRNAs can hinder miRNA-mRNA binding. Three modification patterns were identified, and genes with different patterns had intrinsic mechanisms with potential m1A-regulatory specificity. Systematic analysis of the m1A landscape in normal and OGD/R neurons lays a critical foundation for understanding RNA modification and provides new perspectives and a theoretical basis for treating and developing drugs for OGD/R pathology-related diseases.

Re-analysis of publicly available methylomes using signal detection yields new information

Cytosine methylation is an epigenetic mark that participates in regulation of gene expression and chromatin stability in plants. Advancements in whole genome sequencing technologies have enabled investigation of methylome dynamics under different conditions. However, the computational methods for analyzing bisulfite sequence data have not been unified. Contention remains in the correlation of differentially methylated positions with the investigated treatment and exclusion of noise, inherent to these stochastic datasets. The prevalent approaches apply Fisher’s exact test, logistic, or beta regression, followed by an arbitrary cut-off for differences in methylation levels. A different strategy, the MethylIT pipeline, utilizes signal detection to determine cut-off based on a fitted generalized gamma probability distribution of methylation divergence. Re-analysis of publicly available BS-seq data from two epigenetic studies in Arabidopsis and applying MethylIT revealed additional, previously unreported results. Methylome repatterning in response to phosphate starvation was confirmed to be tissue-specific and included phosphate assimilation genes in addition to sulfate metabolism genes not implicated in the original study. During seed germination plants undergo major methylome reprogramming and use of MethylIT allowed us to identify stage-specific gene networks. We surmise from these comparative studies that robust methylome experiments must account for data stochasticity to achieve meaningful functional analyses.

Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human Tcells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) Tcells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR Tcells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

Plant-made pharmaceuticals: exploring studies for the production of recombinant protein in plants and assessing challenges ahead.

The production of pharmaceutical compounds in plants is attracting increasing attention, as plant-based systems can be less expensive, safer, and more scalable than mammalian, yeast, bacterial, and insect cell expression systems. Here, we review the history and current status of plant-made pharmaceuticals. Producing pharmaceuticals in plants requires pairing the appropriate plant species with suitable transformation technology. Pharmaceuticals have been produced in tobacco, cereals, legumes, fruits, and vegetables via nuclear transformation, chloroplast transformation, transient expression, and transformation of suspension cell cultures. Despite this wide range of species and methods used, most such efforts have involved the nuclear transformation of tobacco. Tobacco readily generates large amounts of biomass, easily accepts foreign genes, and is amenable to stable gene expression via nuclear transformation. Although vaccines, antibodies, and therapeutic proteins have been produced in plants, such pharmaceuticals are not readily utilized by humans due to differences in glycosylation, and few such compounds have been approved due to a lack of clinical data. In addition, achieving an adequate immune response using plant-made pharmaceuticals can be difficult due to low rates of production compared to other expression systems. Various technologies have recently been developed to help overcome these limitations; however, plant systems are expected to increasingly become widely used expression systems for recombinant protein production.

To determine the bioactivity of IL-1β monoclonal antibodies: Introduction of a reliable reporter gene assay.

IL-1β is a essential molecule in inflammatory signalling pathways and plays an essential role in inflammatory diseases. Accordingly, the development of monoclonal antibodies (mAbs) that target IL-1β has become the focus of developing new anti-inflammatory drugs. The successful clinical application of therapeutic antibodies is dependent on good quality control, which is based on accurate bioactivity determination. The aim of this work was to develop an elegant and accurate reporter gene assay to determine the bioactivity of anti-IL-1β antibody drugs. The D10-G4-1 cell line with a naturally high expression of IL-1 receptor was selected as the effector cell, and the plasmid containing luciferase reporter gene with NF-κB as a regulatory element was transfected into D10-G4-1 cells. After a period of pressure screening, a monoclonal cell line with good reactivity and stable expression of reporter gene was finally screened out. Stimulation of this cell line via IL-1β addition increased the expression of the luciferase gene by activating the NF-κB signalling pathway, with the addition of luciferase substrate, which can be quantified by relative luminescence units. When anti-IL-1β antibodies are present in the system, the expression of luciferase gene is inhibited, which demonstrates the bioactivity of anti-IL-1β antibodies. Detailed methodological optimization and comprehensive methodological validation were followed to establish a reporter gene assay for the bioactivity of anti-IL-1β antibodies.

Selection and Validation of Reference Genes for RT-qPCR Analysis of Gene Expression in Nicotiana benthamiana upon Single Infections by 11 Positive-Sense Single-Stranded RNA Viruses from Four Genera.

Quantitative real-time PCR (RT-qPCR) is a widely used method for studying alterations in gene expression upon infections caused by diverse pathogens such as viruses. Positive-sense single-stranded (ss(+)) RNA viruses form a major part of all known plant viruses, and some of them are damaging pathogens of agriculturally important crops. Analysis of gene expression following infection by ss(+) RNA viruses is crucial for the identification of potential anti-viral factors. However, viral infections are known to globally affect gene expression and therefore selection and validation of reference genes for RT-qPCR is particularly important. In this study, the expression of commonly used reference genes for RT-qPCR was studied in Nicotiana benthamiana following single infection by 11 ss(+) RNA viruses, including five tobamoviruses, four potyviruses, one potexvirus and one polerovirus. Stability of gene expression was analyzed in parallel by four commonly used algorithms: geNorm, NormFinder, BestKeeper, and Delta CT, and RefFinder was finally used to summarize all the data. The most stably expressed reference genes differed significantly among the viruses, even when those viruses were from the same genus. Our study highlights the importance of the selection and validation of reference genes upon different viral infections.

Prolonged Differentiation of Neuron-Astrocyte Co-Cultures Results in Emergence of Dopaminergic Neurons.

Dopamine is present in a subgroup of neurons that are vital for normal brain functioning. Disruption of the dopaminergic system, e.g., by chemical compounds, contributes to the development of Parkinson's disease and potentially some neurodevelopmental disorders. Current test guidelines for chemical safety assessment do not include specific endpoints for dopamine disruption. Therefore, there is a need for the human-relevant assessment of (developmental) neurotoxicity related to dopamine disruption. The aim of this study was to determine the biological domain related to dopaminergic neurons of a human stem cell-based in vitro test, the human neural progenitor test (hNPT). Neural progenitor cells were differentiated in a neuron-astrocyte co-culture for 70 days, and dopamine-related gene and protein expression was investigated. Expression of genes specific for dopaminergic differentiation and functioning, such as LMX1B, NURR1, TH, SLC6A3, and KCNJ6, were increasing by day 14. From day 42, a network of neurons expressing the catecholamine marker TH and the dopaminergic markers VMAT2 and DAT was present. These results confirm stable gene and protein expression of dopaminergic markers in hNPT. Further characterization and chemical testing are needed to investigate if the model might be relevant in a testing strategy to test the neurotoxicity of the dopaminergic system.