RNA Levels Research Articles

Structural variant allelic heterogeneity in MECP2 duplication syndrome provides insight into clinical severity and variability of disease expression

BackgroundMECP2 Duplication Syndrome, also known as X-linked intellectual developmental disorder Lubs type (MRXSL; MIM: 300260), is a neurodevelopmental disorder caused by copy number gains spanning MECP2. Despite varying genomic rearrangement structures, including duplications and triplications, and a wide range of duplication sizes, no clear correlation exists between DNA rearrangement and clinical features. We had previously demonstrated that up to 38% of MRXSL families are characterized by complex genomic rearrangements (CGRs) of intermediate complexity (2 ≤ copy number variant breakpoints < 5), yet the impact of these genomic structures on regulation of gene expression and phenotypic manifestations have not been investigated.MethodsTo study the role of the genomic rearrangement structures on an individual’s clinical phenotypic variability, we employed a comprehensive genomics, transcriptomics, and deep phenotyping analysis approach on 137 individuals affected by MRXSL. Genomic structural information was correlated with transcriptomic and quantitative phenotypic analysis using Human Phenotype Ontology (HPO) semantic similarity scores.ResultsDuplication sizes in the cohort ranging from 64.6 kb to 16.5 Mb were classified into four categories comprising of tandem duplications (48%), terminal duplications (22%), inverted triplications (20%), and other CGRs (10%). Most of the terminal duplication structures consist of translocations (65%) followed by recombinant chromosomes (23%). Notably, 65% of de novo events occurred in the Terminal duplication group in contrast with 17% observed in Tandem duplications. RNA-seq data from lymphoblastoid cell lines indicated that the MECP2 transcript quantity in MECP2 triplications is statistically different from all duplications, but not between other classes of genomic structures. We also observed a significant (p < 0.05) correlation (Pearson R = 0.6, Spearman p = 0.63) between the log-transformed MECP2 RNA levels and MECP2 protein levels, demonstrating that genomic aberrations spanning MECP2 lead to altered MECP2 RNA and MECP2 protein levels. Genotype–phenotype analyses indicated a gradual worsening of phenotypic features, including overall survival, developmental levels, microcephaly, epilepsy, and genitourinary/eye abnormalities in the following order: Tandem duplications, Other complex duplications, Terminal duplications/Translocations, and Triplications encompassing MECP2.ConclusionIn aggregate, this combined analysis uncovers an interplay between MECP2 dosage, genomic rearrangement structure and phenotypic traits. Whereas the level of MECP2 is a key determinant of the phenotype, the DNA rearrangement structure can contribute to clinical severity and disease expression variability. Employing this type of analytical approach will advance our understanding of the impact of genomic rearrangements on genomic disorders and may help guide more targeted therapeutic approaches.

RNA Stability: A Review of the Role of Structural Features and Environmental Conditions

The stability of RNA is a critical factor in determining its functionality and degradation in the cell. In recent years, it has been shown that the stability of RNA depends on a complex interaction of external and internal factors. External conditions, such as temperature fluctuations, the level of acidity of the environment, the presence of various substances and ions, as well as the effects of oxidative stress, can change the structure of RNA and affect its stability. Internal factors, including the specific structural features of RNA and its interactions with protein molecules, also have a significant impact on the regulation of the stability of these molecules. In this article, we review the main factors influencing RNA stability, since understanding the factors influencing this extremely complex process is important not only for understanding the regulation of expression at the RNA level but also for developing new methods for isolating and stabilizing RNA in preparation for creating biobanks of genetic material. We reviewed a modern solution to this problem and formulated basic recommendations for RNA storage aimed at minimizing degradation and damage to the molecule.

A Randomized, Blinded, Vehicle-Controlled Dose-Ranging Study to Evaluate and Characterize Remdesivir Efficacy Against Ebola Virus in Rhesus Macaques

Ebola virus (EBOV) causes severe disease in humans, with mortality as high as 90%. The small-molecule antiviral drug remdesivir (RDV) has demonstrated a survival benefit in EBOV-exposed rhesus macaques. Here, we characterize the efficacy of multiple intravenous RDV dosing regimens on survival of rhesus macaques 42 days after intramuscular EBOV exposure. Thirty rhesus macaques underwent surgical implantation of telemetry devices for the fine-scale monitoring of body temperature and activity, as well as central venous catheters, to enable treatment administration and blood collection. Treatment, consisting of a loading dose of RDV followed by once-daily maintenance doses for 11 days, was initiated 4 days after virus exposure when all animals were exhibiting disease signs consistent with incipient EBOV disease as well as quantifiable levels of EBOV RNA in plasma. In the RDV treatment groups receiving loading/maintenance doses of 5/2.5 mg/kg, 10/5 mg/kg, and 20/10 mg/kg, a total of 6 of 8 (75%), 7 of 8 (87.5%), and 5 of 7 (71.4%) animals survived, respectively. In the vehicle control group, one of seven animals (14.3%) survived. The improved survival rate compared to the control group was statistically significant only for the 10/5 mg/kg RDV treatment group. This treatment regimen also resulted in a significantly lower systemic viral load compared to the vehicle control after a single RDV treatment. All three RDV regimens produced a significantly lower systemic viral load after two treatments. For most animals, RDV treatment, regardless of dose, resulted in the amelioration of many of the clinical–pathological changes associated with EBOV disease in this model.

IRX-related homeobox gene MKX is a novel oncogene in acute myeloid leukemia.

Homeobox genes encode transcription factors which organize differentiation processes in all tissue types including the hematopoietic compartment. Recently, we have reported physiological expression of TALE-class homeobox gene IRX1 in early myelopoiesis restricted to the megakaryocyte-erythroid-progenitor stage and in early B-cell development to the pro-B-cell stage. In contrast, sister homeobox genes IRX2, IRX3 and IRX5 are aberrantly activated in the corresponding malignancies acute myeloid leukemia (AML) and B-cell progenitor acute lymphoid leukemia. Here, we examined the role of IRX-related homeobox gene MKX (also termed IRXL1 or mohawk) in normal and malignant hematopoiesis. Screening of public datasets revealed silent MKX in normal myelopoiesis and B-cell differentiation, and aberrant expression in subsets of AML and multiple myeloma (MM) cell lines and patients. To investigate its dysregulation and oncogenic function we used AML cell line OCI-AML3 as model which strongly expressed MKX at both RNA and protein levels. We found that IRX5, JUNB and NFkB activated MKX in this cell line, while downregulated GATA2 and STAT5 inhibited its expression. MKX downstream analysis was conducted by siRNA-mediated knockdown and RNA-sequencing in OCI-AML3, and by comparative expression profiling analysis of a public dataset from MM patients. Analysis of these data revealed activation of CCL2 which in turn promoted proliferation. Furthermore, MKX upregulated SESN3 and downregulated BCL2L11, which may together underlie decreased etoposide-induced apoptosis. Finally, myeloid differentiation genes CEBPD and GATA2 were respectively up- and downregulated by MKX. Taken together, our study identified MKX as novel aberrantly expressed homeobox gene in AML and MM, highlighting the function of IRX1 in normal myelopoiesis and B-cell development, and of IRX-related genes in corresponding malignancies. Our data merit further investigation of MKX and its deregulated target genes to serve as novel markers and/or potential therapeutic targets in AML patient subsets.

LINC01089 governs the miR-1287-5p/HSPA4 axis to negatively regulate osteogenic differentiation of mesenchymal stem cells.

The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism.

Identifying the key role of mitochondrial respiration and lipid metabolism in regulating axillary osmidrosis through proteomics analysis.

Axillary osmidrosis (AO) affects a large number of young people in Asia, resulting from a combination of body and bacterial metabolism. This study aimed to explore the pathogenesis of AO through proteomics. Apocrine gland tissues from 3 mild and 3 severe AO patients were analyzed using 4D label-free proteomics, followed by bioinformatics analysis. The RNA and protein levels of the predicted key regulators were further validated by qPCR and immunohistochemistry in additional AO tissues. A total of 5066 proteins were identified, of which 323 were significantly upregulated and 412 were downregulated (by |log2FC|> 1 and p < 0.05). GO terms related to mitochondria, oxidation-reduction processes, and peroxisomes were significantly enriched among the upregulated DEPs, suggesting enhanced energy metabolism in severe AO patients. Downregulated DEPs were enriched in ribosome, phagosome, and platelet activation pathways according to KEGG, while upregulated DEPs were significantly enriched in metabolic pathways, valine, leucine, and isoleucine degradation, peroxisomes, and fatty acid degradation. The enriched pathways suggest that apocrine gland tissues develop AO by increasing blood flow to promote sweating and secreting excessive short-chain fatty acids by coupling mitochondrial respiration with incomplete metabolism of lipids and branched-chain amino acids. This metabolic coupling may have implications for studies on cardiovascular disease, metabolic disorders, and oxidative stress. Key proteins in the signaling network were further confirmed by qPCR and immunohistochemistry, including reduced FGA and ITGA2B, and increased EHHADH and ACOX1. Our proteomics analysis suggests a paradigm of lipid metabolism involving mitochondrial respiration and incomplete lipid and branched-chain amino acid metabolism as the pathogenesis of AO. We also suggest that EHHADH is a key regulator in promoting AO in this process.

Phenotypic Differences Between the Epidemic Strains of Vesicular Stomatitis Virus Serotype Indiana 98COE and IN0919WYB2 Using an In-Vivo Pig (Sus scrofa) Model

During the past 25 years, vesicular stomatitis virus (VSV) has produced multiple outbreaks in the US, resulting in the emergence of different viral lineages. Currently, very little is known about the pathogenesis of many of these lineages, thus limiting our understanding of the potential biological factors favoring each lineage in these outbreaks. In this study, we aimed to determine the potential phenotypic differences between two VSV Indiana (VSIV) serotype epidemic strains using a pig model. These strains are representative of the epidemic lineages that affected the US between 1997 and 1998 (IN98COE) and between 2019 and 2020 (IN0919WYB2), the latter responsible for one of the most extensive outbreaks in the US. Our initial genome analysis revealed the existence of 121 distinct mutations between both strains, including the presence of a 14-nucleotide insertion in the intergenic region between the G and L genes observed in IN0919WYB2. The levels of viral RNA in clinical samples between pigs infected with IN98COE or IN0919WYB2 were compared. Overall, higher and prolonged expression of viral RNA in pigs infected with IN98COE was observed. However, clinically, IN0919WYB2 was slightly more virulent than IN98COE, as well as more efficient at producing infection through contact transmission. Additionally, infectious virus was recovered from more samples when the pigs were infected with IN0919WYB2, as revealed by virus isolation in cell culture, indicating the increased ability of this virus to replicate in pigs. Sequence analyses conducted from isolates recovered from both experimental groups showed that IN0919WYB2 produced more variability during the infection, denoting the potential of this strain to evolve rapidly after a single infection–contact transmission event in pigs. Collectively, the results showed that epidemic strains of VSIV may represent disparate phenotypes in terms of virulence/transmissibility for livestock, a situation that may impact the intensity of an epidemic outbreak. This study also highlights the relevance of pathogenesis studies in pigs to characterize phenotypic differences in VSV strains affecting livestock in the field.

Mosquito-independent milk-associated transmission of zoonotic Wesselsbron virus in sheep.

Wesselsbron virus (WSLV) is a zoonotic, mosquito-borne orthoflavivirus endemic to sub-Saharan Africa, causing abortions and stillbirths in small ruminants. The life cycle of WSLV involves Aedes mosquitoes and various wildlife and domestic animals. Seminal studies in the 1950s have shown the zoonotic potential of WSLV, notably in accidental infections of laboratory workers exposed to infected material. More recent epidemiological studies suggest the emergence of clade I WSLV strains in peri-domestic and rural areas of western and eastern Africa. The pathobiology of recent clade I WSLV strains is unknown and no virus isolate is available. To address these gaps, we generated a recombinant clade I WSLV SA999 infectious clone (rSA999) by reverse genetics. Subsequently, lactating ewes were inoculated intravenously with the WSLV rSA999 strain or the clade II SAH177 strain in insect-free biocontainment stables. Inoculated ewes developed fever, viremia, and showed high levels of viral RNA at mucosal surfaces, and elevated viral titers in milk. Milk production was reduced, which directly affected the growth of the lambs, particularly within the rSA999 group. The ewes with higher WSLV titers in their milk in each group transmitted the infection to their lambs, which developed fever, prolonged viremia, and virus secretion. All infected animals produced high antibody titers with cross-neutralizing activity against both WSLV strains. Histopathology and blood biochemistry analysis indicated liver damage associated with necrotizing hepatitis lesions and active viral replication in some cases, which was more pronounced in the rSA999 group. Notably, only the SAH177-infected animals exhibited lesions consistent with meningoencephalitis, suggesting that WSLV clade II strains are neurotropic and that clade I strain are more hepatotropic. These findings demonstrate a previously unrecognized mode of vector-free transmission of WSLV that raises significant concerns for public and animal health.

A large-scale screening of hepatitis C among men who have sex with men in the community using saliva point-of-care testing

AimTo assess the feasibility and acceptability of massive hepatitis C virus (HCV) testing in point of care on the street using quick tests, determine the characteristics of the population included, and the prevalence of HCV infection in this population.MethodsCross-sectional community-based study including adult men who have sex with men (MSM) who attended the three most important LGTB+ events in Sitges (Catalonia, Spain) in 2022. Points of care were set up on tents on the street and attendees were offered voluntary anti-HCV antibody self-testing. Participants were informed of the study, provided consent, completed the test for identification of risk practices (TIRP), and took the test with the OraQuick® HCV test on a saliva sample (sensitivity: 97.8% [95% confidence interval (CI), 93.2–99.4%] and specificity: 100% [95% CI, 98.4–100%]; gold standard: IgG antibody test for HCV by immunoassay [serum]); participants with positive results were offered HCV virus testing with the Xpert HCV Fingerstick® on a blood drop.ResultsA total of 1249 adults participated in the large-scale screening, of which 1197 (95.8%) were identified as MSM. The screening time was 39 participants/h. Four (0.32%) participants had positive anti-HCV results, all with undetectable HCV RNA levels. Participants’ median (IQR) age was 44 (35, 54) years; most were Europeans, and 13% reported being unaware of their serological HCV status. The mean (SD) TIRP score was 1.40 (1.44) (n = 1062), with 67.41% reporting some risk, and the self-perceived sexually transmitted disease score was 3.0 (2.82) (n = 969).ConclusionThe point-of-care strategy on the street using a quick oral self-test at massive MSM events is feasible, well-accepted, and quick, and may be a useful strategy to reach other populations at risk of HCV infection.

Wastewater surveillance for SARS-CoV-2 during a mass sporting event in the City of Cape Town, Western Cape

BackgroundWastewater surveillance has become an important public health tool with numerous research studies indicating its potential for monitoring coronavirus disease 2019 (COVID-19) outbreaks. The aim of this study was to apply wastewater surveillance as an indicator for COVID-19 to monitor the impact of a mass sporting event in the City of Cape Town. The study compared the same event over 2 years (2022 and 2023).MethodsWeekly grab wastewater samples were collected from wastewater treatment plants in the City of Cape Town, and quantitative reverse transcription-polymerase chain reaction used to quantify severe acute respiratory syndrome 2 (SARS-CoV-2) RNA in wastewater.ResultsOur findings show a statistically significant correlation (rho = 0.68, p = 0.01) between clinical cases and concentrations of SARS-CoV-2 RNA in wastewater in the 2022 study period. During this specific period, a rise in clinical cases was observed 2 weeks after the event and the peaks in clinical cases coincided with the peaks in SARS-CoV-2 RNA level in wastewater. The study also found a statistically significant positive correlation (R2 = 0.03, F (1,208) = 6.56, p = 0.01) between the SARS-CoV-2 RNA in wastewater and the 2022 event of the marathon hosted in the city.ConclusionDue to the decrease in clinical testing and the country being a popular destination for mass gatherings such as sporting events, the results from this study indicate the potential of wastewater surveillance providing supplementary information to form part of public health risk evaluations for mass gatherings.

RRM2 Regulates Hepatocellular Carcinoma Progression Through Activation of TGF-β/Smad Signaling and Hepatitis B Virus Transcription

Background: Hepatocellular carcinoma (HCC) is a type of malignant tumor with high morbidity and mortality. Untimely treatment and high recurrence are currently the major challenges for HCC. The identification of potential targets of HCC progression is crucial for the development of new therapeutic strategies. Methods: Bioinformatics analyses have been employed to discover genes that are differentially expressed in clinical cases of HCC. A variety of pharmacological methods, such as MTT, colony formation, EdU, Western blotting, Q-PCR, wound healing, Transwell, cytoskeleton F-actin filaments, immunohistochemistry (IHC), hematoxylin–eosin (HE) staining, and dual-luciferase reporter assay analyses, were utilized to study the pharmacological effects and potential mechanisms of ribonucleotide reductase regulatory subunit M2 (RRM2) in HCC. Results: RRM2 expression is significantly elevated in HCC, which is well correlated with poor clinical outcomes. Both in vitro and in vivo experiments demonstrated that RRM2 promoted HCC cell growth and metastasis. Mechanistically, RRM2 modulates the EMT phenotype of HCC, and further studies have shown that RRM2 facilitates the activation of the TGF-β/Smad signaling pathway. SB431542, an inhibitor of TGF-β signaling, significantly inhibited RRM2-induced cell migration. Furthermore, RRM2 expression was correlated with diminished survival in HBV-associated HCC patients. RRM2 knockdown decreased the levels of HBV RNA, pgRNA, cccDNA, and HBV DNA in HepG2.2.15 cells exhibiting sustained HBV infection, while RRM2 knockdown inhibited the activity of the HBV Cp, Xp, and SpI promoters. Conclusion: RRM2 is involved in the progression of HCC by activating the TGF-β/Smad signaling pathway. RRM2 increases HBV transcription in HBV-expressing HCC cells. Targeting RRM2 may be of potential value in the treatment of HCC.

Aerobic exercise training attenuates ischemia-reperfusion injury in mice by decreasing the methylation level of METTL3-associated m6A RNA in cardiomyocytes.

Ischemic heart disease (IHD) presents a significant global health challenge, with myocardial ischemia-reperfusion injury (MIRI) being a major pathophysiological contributor and lacking effective interventions. While aerobic exercise training (AET) enhances cardiovascular health, its protective mechanism in MIRI remains elusive. This study aims to elucidate the protective effect of AET in MIRI and its underlying mechanism. A mouse model of AET and MIRI was established to evaluate basic indices, cardiac ultrasound, and myocardial injury markers. Dot Blot, qRT-PCR, and Western blot were employed to assess m6A RNA methylation levels and related protein expression in myocardial tissue. In vitro, primary cardiomyocyte culture was utilized to mimic MIRI, evaluating cell viability, mitochondrial membrane potential, etc. Finally, myocardial tissues of MIRI mice were immunoprecipitated for m6A RNA methylation and sequenced to analyze related signaling pathways. AET significantly improved cardiac function and mitigated myocardial injury and fibrosis. Moreover, AET protected myocardium from MIRI by reducing m6A RNA methylation levels and modulating METTL3 expression. In vitro experiments demonstrated that the decrease in m6A RNA methylation levels and METTL3 expression conferred resistance to hypoxia/reoxygenation-induced injury. Furthermore, sequencing results indicated elevated myocardial tissue m6A RNA methylation levels during MIRI, activation of the Nrf2-related signaling pathway, and AET-mediated regulation of the Nrf2/HO-1 signaling pathway, thereby attenuating MIRI through modulation of METTL3-related m6A methylation. AET attenuates MIRI by reducing the level of METTL3-related m6A RNA methylation in cardiomyocytes and activating the Nrf2/HO-1 antioxidant signaling pathway. This finding provides a novel insight and strategy for the prevention and treatment of IHD, holding significant clinical implications.

SIX transcription factors are necessary for the activation of DUX4 expression in facioscapulohumeral muscular dystrophy

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is a common and progressive muscle wasting disease that is characterized by muscle weakness often first noticed in the face, the shoulder girdle and upper arms before progressing to the lower limb muscles. FSHD is caused by the misexpression of the Double Homeobox 4 (DUX4) transcription factor in skeletal muscle. While epigenetic derepression of D4Z4 macrosatellite repeats underlies DUX4 misexpression, our understanding of the complex transcriptional activation of DUX4 is incomplete.MethodsTo identify potential DUX4-regulatory factors, we used small interfering RNAs (siRNAs) to knockdown SIX family transcription factors (SIX1, 2, 4, 5) in patient-derived FSHD1 and FSHD2 myoblasts that were differentiated to form multinucleated myotubes. Quantitative real-time polymerase chain reaction was used to measure changes in DUX4 mRNA, DUX4 target gene expression and myogenic markers. Staining for SIX1 and SIX2 with specific antibodies was performed in FSHD myoblasts and myotubes. To assess reciprocal effects of DUX4 on SIX1, 2, and 4 expression, we utilized a doxycycline-inducible DUX4 myoblast cell line.ResultWe show that SIX1, 2 and 4 transcription factors, regulators of embryonic development, muscle differentiation, regeneration and homeostasis, are necessary for myogenic differentiation-dependent DUX4 expression in FSHD muscle cells. Using siRNA, we demonstrate SIX1, SIX2, and SIX4 to be critical factors involved in the induction of DUX4 transcription in differentiating FSHD myotubes in vitro. siRNA dual knockdown of SIX1 and SIX2 resulted in a ~ 98% decrease of DUX4 and DUX4 target genes, suggesting that SIX1 and SIX2 are the most critical in promoting DUX4 expression. Importantly, we show that DUX4 downregulates SIX RNA levels, suggesting negative feedback regulation.ConclusionsIn this study, we identified a family of developmental regulators that promote aberrant DUX4 expression in FSHD1 and FSHD2 differentiating muscle cells. Our findings highlight the critical involvement of SIX transcription factors (SIX1, 2, 4) in the pathogenesis of FSHD by serving as necessary factors that function in the promotion of DUX4 expression following epigenetic derepression of the D4Z4 repeats.

HomoeOmicsDB: A global reference platform for accelerating basic and application research in homeopathy

Homeopathy, a globally practiced alternative medicine, faces criticism due to the lack of molecular mechanisms elucidating its drug actions. To address this, researchers are examining the mechanistic action of homeopathic drugs by assessing the molecular markers of various disorders in real-time patients and/or disease models. However, there is a gap: the lack of an assembled view of publicly available molecular-level studies hampers the research on homeopathic drugs. For example, the information on the mechanistic action of Arnica montana tinctures/dilutions - anti-inflammatory, apoptotic, anti-migratory, and healing properties through gene expression and cellular assays – are scattered over published scientific literature. To bridge this gap and accelerate research, we developed the HomoeOmicsDB database (https://ciods.in/homoeomicsdb/), the first reference and molecular analysis platform for multi-omic research in homeopathy. Currently, HomoeOmicsDB hosts information on 65 homeopathic drugs' effects on the expression of around 600 genes. This information is curated from over 100 peer-reviewed published research articles involving more than 50 human and animal cell lines. For each drug, the database integrates the studied host system (body fluid, tissue, host cell/line, cell type), and its regulated expression of genes at RNA, protein, and post-translational modification levels. The information assembled in HomoeOmicsDB, might galvanize research on molecular mechanisms of other homeopathic drugs with similar clinico-physiological outcomes. Integrated with the DisGeNet database, HomoeOmicsDB entails this comparative analysis of homeopathic drug efficacy for multiple disorders in practice. This would subsequently embrace translational/re-purposing options for diseases with limited or no therapeutic alternatives. Together, HomoeOmicsDB aims to serve as a platform for reference and integration of multi-omic data relevant to homeopathic research. The evaluation of the database by the practitioners and researchers would foster greater acceptance and integration of homeopathic medicine.

Berberine promotes K48-linked polyubiquitination of HNF4α, leading to the inhibition of HBV replication.

The current antiviral agents for the treatment of chronic infection with hepatitis B virus (HBV) do not completely remove covalently closed circular DNA (cccDNA) and integrated viral DNA fragments from patients. Berberine is an isoquinoline alkaloid extracted from various plants and has been reported to inhibit the replication of various types of DNA. In this study, we tested the effects of berberine and its derivatives on HBV infection. Berberine inhibited viral core promoter activity at the highest level among the compounds tested and suppressed HBV production and cccDNA synthesis in primary human hepatocytes and HBV-infected HepG2-NTCP cells at an EC50 value of 3.6 μM and a CC50 value of over 240.0 μM. Compared with other viral promoter activities, berberine treatment potently downregulated core promoter activity and reduced protein levels, but not RNA levels, of hepatic nuclear factor 4α (HNF4α), which primarily enhances enhancer II/core promoter activity. Furthermore, berberine treatment enhanced K48-linked, but not K63-linked, polyubiquitination and subsequent proteasome-dependent degradation of HNF4α. These results suggest that berberine enhances the polyubiquitination- and proteasome-dependent degradation of HNF4α and then inhibits HBV replication via the suppression of core promoter activity. The development of antiviral agents based on berberine may contribute to the amelioration of HBV-related disorders, regardless of the presence of residual cccDNA or integrated viral DNA fragments.

Biotin-Based Northern Blotting (BiNoB): A Cost-Efficient Alternative for Detection of Small RNAs.

Advances in sequencing technology have led to the discovery of diverse types of regulatory RNAs. Differential transcript levels regulate cellular processes and influence disease severity. Identifying these variations through reliable methods is crucial for understanding the regulatory roles and disease mechanisms of regulatory RNAs. Northern blotting, which is considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality and integrity, radioactivity exposure, and associated reagents and expenses. In this protocol, we employ a biotin-based northern blotting (BiNoB) approach that is both convenient and inexpensive, eliminating the need for specialized settings as required with radioactivity-based northern blotting. We comprehensively target various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conduct a comparison between 3'-end labeled probes that were labeled in-house and 5'-end labeled probes that were obtained commercially. Remarkably, our results reveal relatively higher sensitivity with 3'-end labeled probes. Furthermore, we demonstrated that the use of an in-house buffer offered comparable sensitivity to a commercially available buffer, providing another cost-effective alternative. We also aimed to determine the minimum quantity of total RNA required to detect small non-coding RNAs such as tRNA fragments. Whereas previous studies reported the use of 5-10 µg total RNA for tRNA fragment detection, our findings revealed that as little as 1 µg total RNA is sufficient to detect small RNAs like tRNAs and their fragments. This concentration may vary depending on the expression levels of the specific RNAs being detected. © 2024 Wiley Periodicals LLC. Basic Protocol: Biotin-based northern blotting.

Identification of Serum-Derived CricRNA Diagnostic Panel and Revealing Their Regulatory Mechanisms in HCV-HCC: A Cross-Sectional Study.

Hepatitis C virus (HCV) infection is a significant risk factor for the development of hepatocellular carcinoma (HCC). Serum-derived circular RNAs (circRNAs) play several crucial roles in HCV and HCC. They represent a promising area of research for improving the diagnosis and understanding the mechanisms of HCV-HCC. This study aims to identify a serum-derived circular RNA (circRNA) diagnostic panel for HCV-HCC and to elucidate the regulatory mechanisms underlying their role in cancer progression. In this study, data mining and in silico analysis were conducted to identify the role of circular RNAs (hsa_circ_0003288, circ-RNF13, hsa_circ_0004277, circANRIL, circUHRF1, hsa_circ_103047) and their associated biomarkers (IL-6 and NF-κB) in HCV-HCC pathogenesis. Additionally, RT-PCR was performed to assess their expression levels across different study groups (G0 = control, G1 = HCV, G2 = HCC, and G3 = HCV-induced HCC). The expression levels of circular RNAs, including hsa_circ_0003288, circ-RNF13, hsa_circ_0004277, circANRIL, circUHRF1, and hsa_circ_103047, as well as the biomarkers IL-6 and NF-κB, were significantly elevated in the G3 group compared to the G0 group. ROC analysis also revealed significantly different expression rates for G3 group and G0 group. The data revealed that cricRNAs panel (hsa_circ_0003288, circ-RNF13, circANRIL, circUHRF1, and hsa_circ_103047) could serve as a diagnostic biomarker and therapeutic target for HCV-induced HCC.

High cell throughput, programmable fixation reveals the RNA and protein co-regulation with spatially resolved NFκB pseudo-signaling.

RNA translation to protein is paramount to creating life, yet RNA and protein correlations vary widely across tissues, cells, and species. To investigate these perplexing results, we utilize a time-series fixation method that combines static stimulation and a programmable formaldehyde perfusion to map pseudo-Signaling with Omics signatures (pSigOmics) of single-cell data from hundreds of thousands of cells. Using the widely studied nuclear factor kappa B (NFκB) mammalian signaling pathway in mouse fibroblasts, we discovered a novel asynchronous pseudotime regulation (APR) between RNA and protein levels in the quintessential NFκB p65 protein using single molecule spatial imaging. Prototypical NFκB dynamics are successfully confirmed by the rise and fall of NFκB response as well as A20 negative inhibitor activity by 90 min. The observed p65 translational APR is evident in both statically sampled timepoints and dynamic response gradients from programmable formaldehyde fixation, which successfully creates continuous response measurements. Finally, we implement a graph neural network model capable of predicting APR cell subpopulations from GAPDH RNA spatial expression, which is strongly correlated with p65 RNA signatures. Successful decision tree classifiers on Potential of Heat-diffusion for Affinity-based Trajectory Embedding embeddings of our data, which illustrate partitions of APR cell subpopulations in latent space, further confirm the APR patterns. Together, our data suggest an RNA-protein regulatory framework in which translation adapts to signaling events and illuminates how immune signaling is timed across various cell subpopulations.

Long Non-Coding RNA LINC01116 Promotes the Proliferation of Lung Adenocarcinoma by Targeting miR-9-5p/CCNE1 Axis.

Long non-coding RNA (lncRNA) LINC01116 is crucial in promoting cell proliferation, invasion and migration in solid tumours, including lung adenocarcinoma (LUAD). LINC01116 acts as a competing endogenous RNAs (ceRNA) that binds competitively to microRNAs and plays a critical role in tumour migration and invasion. However, other mechanisms of action besides the ceRNA theory have been rarely reported and remain to be elucidated further. The differences in RNA and protein levels in cells and tissues were assessed through real-time quantitative PCR and Western blot analysis. Invitro functional assays and invivo xenograft models were used to analyse the function of LINC01116 in LUAD. Thus, the molecular correlation between miR-9-5p and CCNE1 was investigated through direct and indirect mechanism experiments. LINC01116, miR-9-5p and CCNE1 were upregulated in LUAD cell lines and tissues and were associated with a poor prognosis in patients. LINC01116 depletion inhibited proliferation but facilitated cell apoptosis. AGO2-RNA binding protein immunoprecipitation (AGO2-RIP) experiments confirmed that AGO2 binds to LINC01116 and miR-9-5p, indicating that LINC01116 interacts with miR-9-5p. The overexpression of miR-9-5p and CCNE1 effectively counteracts the biological effects of LINC01116 knockdown on reduced proliferation and cell cycle arrest in LUAD cells. The downregulation of miR-9-5p significantly reduces the CCNE1 level in A549 cells, and the upregulation of LINC01116 counteracts the downregulation of miR-9-5p effect, restoring the expression level of CCNE1. Our data demonstrated that LINC01116 regulates the expression of CCNE1 by positively regulating miR-9-5p, thereby affecting cell cycle, proliferation and participating in the development of LUAD.

Qufeng Tongluo Decoction May Alleviate Podocyte Injury Induced by High Glucose and Hydrogen Peroxide by Regulating Autophagy

Background: Diabetic kidney disease is a significant complication of diabetes. Previous studies have confirmed that Qufeng Tongluo (QFTL) decoction can alleviate podocyte injury in a diabetic rat model, but its mechanism remains unclear. The present in vitro study investigated QFTL’s mechanism in protecting podocytes. Methods: The mouse podocyte clone 5 (MPC-5) cell line stimulated by high glucose and hydrogen peroxide was used as a model for podocyte injury. The cells were treated with QFTL, QFTL + SC79 (activator of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), and C2 ceramide (inhibitor of the PI3K/Akt). Immunofluorescence, western blot and polymerase chain reaction (PCR) were employed to detect the expression of microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1/p62), PI3K, Akt, and phosphatase and tensin homolog (PTEN) proteins and their messenger RNA (mRNA) levels. Results: High glucose and hydrogen peroxide stimulation may disrupt podocytes’ cytoskeletal structure, while QFTL may mitigate these structural changes. After the SC79 intervention, QFTL’s alleviation of the cytoskeletal structure damage disappeared. High glucose and hydrogen peroxide stimulation decreased the expression of LC3 in MPC-5 cells, whereas QFTL upregulated its expression, but this effect was reversed by the SC79 intervention. Interestingly, P62 decreased after high glucose + hydrogen peroxide stimulation, and QFTL failed to upregulate P62. The expression levels of PI3K, Akt, and PTEN in the model group decreased, which were improved by QFTL. Conclusion: These results suggest that QFTL can maintain the stability of autophagic flux in injured podocytes, which may be related to its ability to improve P62 expression but not to regulating the PI3K/Akt signaling pathway.