Triterpenes from Ganoderma lucidum inhibit hepatocellular carcinoma by regulating enhancer-associated lncRNA in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (G. lucidum) has been widely used as adjuvant of anti-tumor therapy for variety tumors. The bioactive ingredients of G. lucidum mainly include triterpenes, such as Ganoderic acid A, Ganoderic acid B, Ganoderenic acid A, Ganoderenic acid B, Ganoderenic acid D, and Ganoderic acid X. However, the effects and underlying mechanisms of G. lucidum are often challenging in hepatocellular carcinoma (HCC) treatment. AIM OF THE STUDY: To explore the potential role and mechanism of enhancer-associated lncRNAs (en-lncRNAs) in G. lucidum treated HCC through the in vivo and in vitro experiments. MATERIALS AND METHODS: Hepa1-6-bearing C57 BL/6 mice model were established to evaluate the therapeutic efficacy of G. lucidum treated HCC. Ki67 and TUNEL staining were used to detect the tumor cell proliferation and apoptosis in vivo. The Mouse lncRNA 4*180K array was implemented to identify the differentially expressed (DE) lncRNAs and mRNAs of G. lucidum treated tumor mice. The constructed lncRNA-mRNA co-expression network and bioinformatics analysis were used to selected core en-lncRNAs and its neighboring genes. The UPLC-MS method was used to identify the triterpenes of G. lucidum, and the in vitro experiments were used to verify which triterpene monomers regulated en-lncRNAs in tumor cells. Finally, a stable knockdown/overexpression cell lines were used to confirm the relationship between en-lncRNA and neighboring gene. RESULTS: Ki67 and TUNEL staining demonstrated G. lucidum significantly inhibited tumor growth, suppressed cell proliferation and induced apoptosis in vivo. Transcriptomic analysis revealed the existence of 126 DE lncRNAs high correlated with 454 co-expressed mRNAs in G. lucidum treated tumor mice. Based on lncRNA-mRNA network and qRT-PCR validation, 6 core lncRNAs were selected and considered high correlated with G. lucidum treatment. Bioinformatics analysis revealed FR036820 and FR121302 might act as enhancers, and qRT-PCR results suggested FR121302 might enhance Popdc2 mRNA level in HCC. Furthermore, 6 main triterpene monomers of G. lucidum were identified by UPLC-MS method, and in vitro experiments showed FR121302 and Popdc2 were significantly suppressed by Ganoderenic acid A and Ganoderenic acid B, respectively. The knock/overexpression results demonstrated that FR121302 activating and enhancing Popdc2 expression levels, and Ganoderenic acid A and Ganoderenic acid B dramatically suppressed FR121302 and decreased Popdc2 level in Hepa1-6 cells. CONCLUSIONS: Enhancer-associated lncRNA plays a crucial role as an enhancer during hepatocarcinogenesis, and triterpenes of G. lucidum significantly inhibited tumor cell proliferation and induced apoptosis by regulating en-lncRNAs. Our study demonstrated Ganoderenic acid A and Ganoderenic acid B suppressed en-lncRNA FR121302 may be one of the critical strategies of G. lucidum inhibit hepatocellular carcinoma growth.

Chromatographic Purification and Polishing of AAV Particles.

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.

SCN1A intronic variants impact on Nav1.1 protein expression and sodium channel function, and associated with epilepsy phenotypic severity.

High-throughput sequencing has identified numerous intronic variants in the SCN1A gene in epilepsy patients. Abnormal mRNA splicing caused by these variants can lead to significant phenotypic differences, but the mechanisms of epileptogenicity and phenotypic differences remain unknown. Two variants, c.4853-1 G>C and c.4853-25 T>A, were identified in intron 25 of SCN1A, which were associated with severe Dravet syndrome (DS) and mild focal epilepsy with febrile seizures plus (FEFS+), respectively. The impact of these variants on protein expression, electrophysiological properties of sodium channels and their correlation with epilepsy severity was investigated through plasmid construction and transfection based on the aberrant spliced mRNA. We found that the expression of truncated mutant proteins was significantly reduced on the cell membrane, and retained in the cytoplasmic endoplasmic reticulum. The mutants caused a decrease in current density, voltage sensitivity, and an increased vulnerability of channel, leading to a partial impairment of sodium channel function. Notably, the expression of DS-related mutant protein on the cell membrane was higher compared to that of FEFS+-related mutant, whereas the sodium channel function impairment caused by DS-related mutant was comparatively milder than that caused by FEFS+-related mutant. Our study suggests that differences in protein expression levels and altered electrophysiological properties of sodium channels play important roles in the manifestation of diverse epileptic phenotypes. The presence of intronic splice site variants may result in severe phenotypes due to the dominant-negative effects, whereas non-canonical splice site variants leading to haploinsufficiency could potentially cause milder phenotypes.

Expression profiling and single nucleotide polymorphism of mitogen-activated protein kinase kinase kinase 8 MAP3K8 in white muscovy ducks (Cairina moschata).

A previous study on ovarian and hypothalami transcriptome analysis in white Muscovy duck revealed that MAP3K8 gene participated in MAPK signaling pathway that influence egg production. Additionally, MAP3K8 was predicted as a target gene of miRNA-509-3p that promotes the secretion of oestradiol which is an important hormone in egg ovulation. This suggested that MAP3K8 might have a functional role in the reproductive performance "egg production" of white Muscovy ducks. Herein, we focused on expression level of MAP3K8 in reproductive and non-reproductive tissues of highest (HP) and lowest (LP) egg producing white Muscovy ducks and identified the polymorphism in MAP3K8 and its association with three egg production traits; Age at first egg (AFE), number of eggs at 300 days (N300D) and 59 weeks (N59W). The results of expression level indicated that mRNA of MAP3K8 was significantly (p < 0.01) expressed in the oviduct than in the ovary and hypothalamus. Seven synonymous SNPs were detected, and association analysis showed that g.148303340 G>A and g.148290065 A>G were significantly (p < 0.05) associated with N300D and N59W. The results of this study might serve as molecular marker for marker-assisted selection of white Muscovy ducks for egg production.

Evaluation of Gene Expression Profiling by QuantiGene™ 2.0 RNA Assay.

QuantiGene™ 2.0 technique could be used to investigate the gene expression signature of the immune system senescence and thus to understand the molecular mechanism involved in the defects of the immune response during aging.QuantiGene™ 2.0 technique is a multiplex platform allowing the simultaneous analysis of several target RNA molecules (up to 80) present in a single sample. QuantiGene Assays use an accurate method for multiplexed or for single gene expression quantitation. QuantiGene 2.0 uses magnetic beads which are dyed internally with two fluorescence dyes, exhibiting a unique spectral signal and providing specificity and multiplexing capability of the technique. QuantiGene Assays incorporate branched-DNA technology for gene expression profiling.Branched-DNA system is responsible for the high sensitivity of the system. In fact, it permits to detect low levels of mRNA molecules. This branched-DNA system allows for the direct measurement of RNA transcripts by using signal amplification rather than target amplification. The assay protocol is spread over 2 days. First, immune cells are lysed to release the target RNA, which is incubated with oligonucleotide probe set targeted with beads capable to hybridize with the target RNA. Signal amplification is performed by sequential hybridization of the branched-DNA pre-amplifier, amplifier, and label probe molecules. The last step involves the incubation with Streptavidin-conjugated R-phycoerythrin. The fluorescent reporter generates a signal directly proportional to the levels of RNA molecules present in the cells. Luminex instrument evaluates the median intensity of fluorescence, which is proportional to the number of RNA target molecules present in the cells.

Analysis of Cytokine-Inducible SH2-Containing Protein (CISH) in the Study of Immunosenescence.

The aging immune system undergoes significant changes, leading to a state known as immunosenescence. Understanding the molecular mechanisms underlying immunosenescence is crucial for developing targeted interventions to enhance immune functions in older individuals. This bio-protocol review focuses on the application of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the mRNA quantification of cytokine-inducible SH2-containing protein (CISH), an immune regulator overexpressed in T-cell responses from older adults. We outline a comprehensive protocol for the quantitative assessment of CISH mRNA expression, providing a valuable tool for researchers investigating immunosenescence.

Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

Golden Gate Cloning of Expression Plasmids for Synthetic Small RNAs in Bacteria.

Bacterial small RNAs (sRNAs) are well known for their ability to modulate gene expression at the post-transcriptional level. Their rather simple and modular organization provides the user with defined building blocks for synthetic biology approaches. In this chapter, we introduce a plasmid series for Escherichia coli and describe protocols for fast and efficient construction of synthetic sRNA expression plasmids based on Golden Gate assembly. In addition, we present the G-GArden tool, which assists with the design of oligodeoxynucleotides and overhangs for scarless assembly strategies. We propose that the presented procedures are suitable for many applications in different bacteria, which are related to E. coli and beyond.

YeastFab Cloning of Toxic Genes and Protein Expression Optimization in Yeast.

YeastFab is a Golden Gate-based cloning standard and parts repository. It is designed for modular, hierarchical assembly of transcription units and multi-gene assemblies for expression in Saccharomyces cerevisiae. This makes it a suitable toolbox to optimize the expression strength of heterologous genes in yeast. When cloning heterologous coding sequences into YeastFab vectors, in several cases we have observed toxicity to the cloning host Escherichia coli. The provided protocol details how to clone such toxic genes from multiple synthetic DNA fragments while adhering to the YeastFab standard. The presented cloning strategy includes a C-terminal FLAG tag that allows screening for constructs with a desired protein expression in yeast by western blot. The design allows scarlessly removing the tag through a Golden Gate reaction to facilitate cloning of expression constructs with the native, untagged transgene.

Comparing Gene Expression Between Planktonic and Biofilm Cells of Foodborne Bacterial Pathogens Through RT-qPCR.

Like most microorganisms, important foodborne pathogenic bacteria, such as Salmonella enterica, Listeria monocytogenes, and several others as well, can attach to surfaces, of either abiotic or biotic nature, and create biofilms on them, provided the existence of supportive environmental conditions (e.g., permissive growth temperature, adequate humidity, and nutrient presence). Inside those sessile communities, the enclosed bacteria typically present a gene expression profile that differs from the one that would be displayed by the same cells growing planktonically in liquid media (free-swimming cells). This altered gene expression has important consequences on cellular physiology and behavior, including stress tolerance and induction of virulence. In this chapter, the methodology to use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to monitor and comparatively quantify expression changes in preselected genes of bacteria between planktonic and biofilm growth modes is presented.

From the cauldron of conflict: Endogenous gene regulation by piRNA and other modes of adaptation enabled by selfish transposable elements.

Transposable elements (TEs) provide a prime example of genetic conflict because they can proliferate in genomes and populations even if they harm the host. However, numerous studies have shown that TEs, though typically harmful, can also provide fuel for adaptation. This is because they code functional sequences that can be useful for the host in which they reside. In this review, I summarize the "how" and "why" of adaptation enabled by the genetic conflict between TEs and hosts. In addition, focusing on mechanisms of TE control by small piwi-interacting RNAs (piRNAs), I highlight an indirect form of adaptation enabled by conflict. In this case, mechanisms of host defense that regulate TEs have been redeployed for endogenous gene regulation. I propose that the genetic conflict released by meiosis in early eukaryotes may have been important because, among other reasons, it spurred evolutionary innovation on multiple interwoven trajectories - on the part of hosts and also embedded genetic parasites. This form of evolution may function as a complexity generating engine that was a critical player in eukaryotic evolution.

Learning Enhancer-Gene associations from Bulk Transcriptomic and Epigenetic Sequencing Data with STITCHIT.

To reveal gene regulation mechanisms, it is essential to understand the role of regulatory elements, which are possibly distant from gene promoters. Integrative analysis of epigenetic and transcriptomic data can be used to gain insights into gene-expression regulation in specific phenotypes. Here, we discuss STITCHIT, an approach to dissect epigenetic variation in a gene-specific manner across many samples for the identification of regulatory elements without relying on peak calling algorithms. The obtained genomic regions are then further refined using a regularized linear model approach, which can also be used to predict gene expression. We illustrate the use of STITCHIT using H3k27ac ChIP-seq and RNA-seq data from the International Human Epigenome Consortium (IHEC).

Identification and expression analysis of dmc1 in Chinese soft-shell turtle (Pelodiscus sinensis) during gametogenesis.

DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3' UTR, a 22 bp 5' UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86-88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles' Dmc1 and close to the reptiles', but far away from the teleost's. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells' meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells' development and differentiation in Chinese soft-shell turtles, even in the reptiles.

Deep learning detects subtle facial expressions in a multilevel society primate.

Facial expressions in nonhuman primates are complex processes involving psychological, emotional, and physiological factors, and may use subtle signals to communicate significant information. However, uncertainty surrounds the functional significance of subtle facial expressions in animals. Using artificial intelligence (AI), this study found that nonhuman primates exhibit subtle facial expressions that are undetectable by human observers. We focused on the golden snub-nosed monkeys (Rhinopithecus roxellana), a primate species with a multilevel society. We collected 3427 front-facing images of monkeys from 275 video clips captured in both wild and laboratory settings. Three deep learning models, EfficientNet, RepMLP, and Tokens-To-Token ViT, were utilized for AI recognition. To compare the accuracy of human performance, two groups were recruited: one with prior animal observation experience and one without any such experience. The results showed human observers to correctly detect facial expressions (32.1% for inexperienced humans and 45.0% for experienced humans on average with a chance level of 33%). In contrast, the AI deep learning models achieved significantly higher accuracy rates. The best-performing model achieved an accuracy of 94.5%. Our results provide evidence that golden snub-nosed monkeys exhibit subtle facial expressions. The results further our understanding of animal facial expressions and also how such modes of communication may contribute to the origin of complex primate social systems.

AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation.

Objective: Metabolic rewiring is a characteristic of cancer cells. Cancer cells require more nutrients for survival and proliferation. Although glutamine can be produced in cells via a series of enzymatic reactions, a group of cancer cells are dependent on extracellular glutamine for survival. TET2 plays a role in DNA demethylation and is a tumor suppressor gene. The TET2 gene is frequently mutated in various cancers, including acute myeloid leukemia (AML). Our study aimed to investigate the association between TET2-knockdown AML cell line HL-60 cells and glutamine metabolism. Methods: To evaluate the association between TET2 expression and glutamine limitation, TET2 was downregulated in HL-60 cells using shRNA plasmids. The proliferation of TET2-knockdown HL-60 cells was calculated in normal and glutamine-deficient medium. GLUL mRNA expression was investigated using quantitative reverse transcription polymerase chain reaction and protein levels were evaluated using immunoblotting. Results: The numbers and viability of TET2-knockdown HL-60 cells were decreased in low glutamine-containing medium, but the viability of TET2-knockdown HL-60 cells was higher than that of control cells. GLUL mRNA expressions were increased in TET2-knockdown cells in low glutamine. In addition, P-AMPKα protein expression was increased in TET2-knockdown HL-60 cells in low glutamine-containing medium. Conclusions: Our findings indicate that TET2-knockdown HL-60 cells may be more resistant to glutamine deprivation. In glutamine-deficient medium, the mRNA expression of glutamine synthetase is increased, which could be related to glutamine addiction in cells. In addition, low-glutamyl medium increased the P-AMPKα protein level in TET2-knockdown HL-60 cells.

The Role of MicroRNAs and Stem cells in Rheumatoid Arthritis: A Therapeutic and Diagnostic Approach.

In autoimmune illnesses like Rheumatoid Arthritis (RA), the synovium is constantly inflamed, and joints are ruptured. It is becoming increasingly clear that stem cells, especially notably mesenchymal stem cells (MSCs) and microRNAs (miRNAs), play important roles in the onset and amelioration of RA. There is mounting evidence from both animal and human studies that suggests a potentially safe and effective method to treat RA and other intractable diseases by reducing chronic inflammation and spurring tissue regeneration through the transplantation of multipotent adult stem cells, such as mesenchymal stromal/stem cells. To control gene expression, which impacts immune cell differentiation and inflammatory pathways, microRNAs (miRNAs) are crucial, even though MSCs can self-renew and modify inflammatory reactions. The pathophysiology of RA is marked by the dysregulation of immunological responses and the proliferation of fibroblast-like synoviocytes, which are linked to the aberrant expression of specific microRNAs (miRNAs). This study mainly aims to examine the therapeutic implications of these microRNAs (miRNAs) in relation to RA, as they can function as diagnostic and prognostic indicators. This study explores the functions of microRNAs (miRNAs) and stem cells in rheumatoid arthritis (RA), drawing attention to the relevance of these biological processes and the potential for creating novel therapeutic strategies to improve patient outcomes.

Type III Collagen RNA Level Expression in Pelvic Organ Prolapse: A Systematic Review and Meta-Analysis.

INTRODUCTION AND HYPOTHESIS: Changes in the expression of type III collagen have been linked to women's predisposition to pelvic organ prolapse (POP); however, the findings of prior studies have been conflicting. This study was aimed at investigating whether changes in the type III collagen gene expression levels occur in POP development. METHODS: A systematic review and meta-analysis were conducted on research articles that evaluated type III collagen gene expression levels in patients with POP compared with those without the condition. The articles, published between January 2000 and February 2024, were obtained from PubMed, ScienceDirect, Semantic Scholar, and EBSCO databases. Data were analyzed using fixed-effect models, and the pooled standardized mean difference (SMD) was calculated. Cochrane's Review Manager 5.4 was used for the analysis. The aggregated SMD with 95% confidence interval (CI) regarding type III collagen gene expression levels relative to POP development was the main outcome measure. Results with p < 0.05 were considered statistically significant. RESULTS: Six studies were included in our analysis, comprising 229 POP cases and 139 non-POP cases. Our meta-analysis indicated that patients with POP had higher type III collagen gene expression levels than those without POP (SMD = 0.32; 95% CI: 0.07 to 0.56; p = 0.01). CONCLUSION: The results of this study provide evidence that a higher type III collagen gene expression levels is significantly associated with POP.

[Bipolar Action of Inhibitor of Vasculogenic Mimicry on Gene Expression in Melanoma Cells].

Multiple exogenous or endogenous factors alter gene expression patterns by different mechanisms that are poorly understood. We used RNA-Seq analysis in order to study changes in gene expression in melanoma cells that are capable of vasculogenic mimicry that is inhibited upon the action of an inhibitor of vasculogenic mimicry. Here, we show that the drug induces a strong upregulation of 50 genes that control the cell cycle and microtubule cytoskeleton coupled with a strong downregulation of 50 genes that control different cellular metabolic processes. We found that both groups of genes are simultaneously regulated by multiple sets of transcription factors. We conclude that one way for coordinated regulation of large groups of genes is regulation simultaneously by multiple transcription factors.

An anciently diverged family of RNA binding proteins maintain correct splicing of a class of ultra-long exons through cryptic splice site repression.

Previously, we showed that the germ cell-specific nuclear protein RBMXL2 represses cryptic splicing patterns during meiosis and is required for male fertility (Ehrmann et al., 2019). Here, we show that in somatic cells the similar yet ubiquitously expressed RBMX protein has similar functions. RBMX regulates a distinct class of exons that exceed the median human exon size. RBMX protein-RNA interactions are enriched within ultra-long exons, particularly within genes involved in genome stability, and repress the selection of cryptic splice sites that would compromise gene function. The RBMX gene is silenced during male meiosis due to sex chromosome inactivation. To test whether RBMXL2 might replace the function of RBMX during meiosis we induced expression of RBMXL2 and the more distantly related RBMY protein in somatic cells, finding each could rescue aberrant patterns of RNA processing caused by RBMX depletion. The C-terminal disordered domain of RBMXL2 is sufficient to rescue proper splicing control after RBMX depletion. Our data indicate that RBMX and RBMXL2 have parallel roles in somatic tissues and the germline that must have been conserved for at least 200 million years of mammalian evolution. We propose RBMX family proteins are particularly important for the splicing inclusion of some ultra-long exons with increased intrinsic susceptibility to cryptic splice site selection.