The significance of detecting HBV pgRNA and HBcrAg in HBV patients treated with NAs.

The value of detecting hepatitis B virus (HBV), pregenomic RNA (pgRNA), and hepatitis B core-related antigen (HBcrAg), both separately and jointly, in the management of HBV patients undergoing treatment with Nucleotide Analog was investigated. A total of 149 HBV patients who were being treated with Nucleotide Analog were enrolled in this study. The quantitative levels of HBV pgRNA and HBcrAg in the sera of these patients were determined, aiming to comprehend their replication levels and expression during the course of antiviral therapy. The patients were separated into 3 groups based on treatment duration: treatment time ≤ 12 months, treatment time ranging from 12 months to <60 months, and treatment time ≥ 60 months. Significantly different levels of HBcrAg and HBV pgRNA were observed among 3 groups (P < .05). In the group of patients with positive hepatitis B e antigen, both HBcrAg and pgRNA levels were higher compared to the group with negative hepatitis B e antigen, and this difference between the 2 groups was found to be statistically significant. Stratified analysis based on levels of hepatitis B surface antigen (HBsAg) revealed that the group with HBsAg levels < 100 IU/mL had lower levels of both HBcrAg and pgRNA compared to the group with HBsAg levels ≥ 100 IU/mL (P < .001). Following antiviral therapy, various degrees of transcription of covalently closed circular DNA continue to exist within the liver of HBV patients. The levels of serum HBcrAg and HBV pgRNA vary among patients with different treatment durations, indicating their efficacy in evaluating disease conditions during antiviral therapy.

Genome-Wide Identification and Expression Analysis of YTH Gene Family for Abiotic Stress Regulation in Camellia chekiangoleosa.

N6-methyladenosine (m6A) is essential for RNA metabolism in cells. The YTH domain, conserved in the kingdom of Eukaryotes, acts as an m6A reader that binds m6A-containing RNA. In plants, the YTH domain is involved in plant hormone signaling, stress response regulation, RNA stability, translation, and differentiation. However, little is known about the YTH genes in tea-oil tree, which can produce edible oil with high nutritional value. This study aims to identify and characterize the YTH domains within the tea-oil tree (Camellia chekiangoleosa Hu) genome to predict their potential role in development and stress regulation. In this study, 10 members of the YTH family containing the YTH domain named CchYTH1-10 were identified from C. chekiangoleosa. Through analysis of their physical and chemical properties and prediction of subcellular localization, it is known that most family members are located in the nucleus and may have liquid-liquid phase separation. Analysis of cis-acting elements in the CchYTH promoter region revealed that these genes could be closely related to abiotic stress and hormones. The results of expression profiling show that the CchYTH genes were differentially expressed in different tissues, and their expression levels change under drought stress. Overall, these findings could provide a foundation for future research regarding CchYTHs in C. chekiangoleosa and enrich the world in terms of epigenetic mark m6A in forest trees.

Formulation development of a stable influenza recombinant neuraminidase vaccine candidate.

Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Analysis of global gene expression using RNA-sequencing reveals novel mechanism of Yanghe Pingchuan decoction in the treatment of asthma.

BACKGROUND: Yanghe Pingchuan decoction (YPD) has been used for asthma treatment for many years in China. We sought to understand the mechanism of YPD, and find more potential targets for YPD-based treatment of asthma. METHODS: An ovalbumin-induced asthma model in rats was created. Staining (hematoxylin and eosin, Masson) was used to evaluate the treatment effect of YPD. RNA-sequencing was carried out to analyze global gene expression, and differentially expressed genes (DEGs) were identified. Analysis of the functional enrichment of genes was done using the Gene Ontology database (GO). Analysis of signaling-pathway enrichment of genes was done using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Real-time reverse transcription-quantitative polymerase chain reaction was undertaken to measure expression of DEGs. RESULTS: Pathology showed that YPD had an improvement effect on rats with asthma. RNA-sequencing showed that YPD led to upregulated and downregulated expression of many genes. The YPD-based control of asthma pathogenesis may be related to calcium ion (Ca2+) binding, inorganic cation transmembrane transporter activity, microtubule motor activity, and control of canonical signaling (e.g., peroxisome proliferator-activated receptor, calcium, cyclic adenosine monophosphate). Enrichment analyses suggested that asthma pathogenesis may be related to Ca2 + binding and contraction of vascular smooth muscle. A validation experiment showed that YPD could reduce the Ca2 + concentration by inhibiting the Angiopoietin-II (Ang-II)/Phospholipase (PLA)/calmodulin (CaM0 signaling axis. CONCLUSION: Control of asthma pathogenesis by YPD may be related to inhibition of the Ang-II/PLA/CaM signaling axis, reduction of the Ca2+ concentration, and relaxation of airway smooth muscle (ASM).

Unraveling the significance of calcium as a biofilm promotion signal for Bacillus licheniformis strains isolated from dairy products.

Bacillus licheniformis, a quick and strong biofilm former, is served as a persistent microbial contamination in the dairy industry. Its biofilm formation process is usually regulated by environmental factors including the divalent cation Ca2+. This work aims to investigate how different concentrations of Ca2+ change biofilm-related phenotypes (bacterial motility, biofilm-forming capacity, biofilm structures, and EPS production) of dairy B. licheniformis strains. The Ca2+ ions dependent regulation mechanism for B. licheniformis biofilm formation was further investigated by RNA-sequencing analysis. Results revealed that supplementation of Ca2+ increased B. licheniformis biofilm formation in a dose-dependent way, and enhanced average coverage and thickness of biofilms with complex structures were observed by confocal laser scanning microscopy. Bacterial mobility of B. licheniformis was increased by the supplementation of Ca2+ except the swarming ability at 20 mM of Ca2+. The addition of Ca2+ decreased the contents of polysaccharides but promoted proteins production in EPS, and the ratio of proteins/polysaccharides content was significantly enhanced with increasing Ca2+ concentrations. RNA-sequencing results clearly indicated the variation in regulating biofilm formation under different Ca2+ concentrations, as 939 (671 upregulated and 268 downregulated) and 951 genes (581 upregulated and 370 downregulated) in B. licheniformis BL2-11 were induced by 10 and 20 mM of Ca2+, respectively. Differential genes were annotated in various KEGG pathways, including flagellar assembly, two-component system, quorum sensing, ABC transporters, and related carbohydrate and amino acid metabolism pathways. Collectively, the results unravel the significance of Ca2+ as a biofilm-promoting signal for B. licheniformis in the dairy industry.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Prolinyl Phosphoramidates of Nucleotides with Increased Reactivity.

Nucleoside monophosphates (NMPs) are the subunits of RNA. They are incorporated into growing complementary strands when sequences are copied in enzyme-free reactions using organic leaving groups at the phosphates. Amino acids are rarely considered as leaving groups, but proline can act as a leaving group when N-linked to NMPs, so that prolinyl NMPs hydrolyze in aqueous buffer at 37 °C, with half-life times as short as 2.4 h, and they act as monomers in enzyme-free primer extension. Still, their level of reactivity is insufficient for practical purposes, requiring months for some extensions. Herein we report the synthesis of eight substituted prolinyl AMPs together with seven related compounds and the results of a study of their reactivity. A δ-carboxy prolinyl NMP was found to be converted with a half-life time of just 11 min in magnesium-free buffer, and a δ-isopropyl prolinyl NMP was shown to react sevenfold faster than its prolinyl counterpart in enzyme-free genetic copying of RNA. Our results indicate that both anchimeric and steric effects can be employed to increase the reactivity of aminoacidyl nucleotides, i.e. compounds that combine two fundamental classes of biomolecules in one functional entity.

Pseudomonas aeruginosa transcriptome analysis of metal restriction in ex vivo cystic fibrosis sputum.

Chronic Pseudomonas aeruginosa lung infections are a feature of cystic fibrosis (CF) that many patients experience even with the advent of highly effective modulator therapies. Identifying factors that impact P. aeruginosa in the CF lung could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa studies using laboratory models or RNA isolated from sputum, we analyzed transcripts of strain PAO1 after incubation in sputum from different CF donors prior to RNA extraction. We compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in synthetic cystic fibrosis sputum medium to determine key genes, which are among the most differentially expressed or most highly expressed. Using the key genes, gene sets with correlated expression were determined using the gene expression analysis tool eADAGE. Gene sets were used to analyze the activity of specific pathways in P. aeruginosa grown in sputum from different individuals. Gene sets that we found to be more active in sputum showed similar activation in published data that included P. aeruginosa RNA isolated from sputum relative to corresponding in vitro reference cultures. In the ex vivo samples, P. aeruginosa had increased levels of genes related to zinc and iron acquisition which were suppressed by metal amendment of sputum. We also found a significant correlation between expression of the H1-type VI secretion system and CFTR corrector use by the sputum donor. An ex vivo sputum model or synthetic sputum medium formulation that imposes metal restriction may enhance future CF-related studies.IMPORTANCEIdentifying the gene expression programs used by Pseudomonas aeruginosa to colonize the lungs of people with cystic fibrosis (CF) will illuminate new therapeutic strategies. To capture these transcriptional programs, we cultured the common P. aeruginosa laboratory strain PAO1 in expectorated sputum from CF patient donors. Through bioinformatic analysis, we defined sets of genes that are more transcriptionally active in real CF sputum compared to a synthetic cystic fibrosis sputum medium. Many of the most differentially active gene sets contained genes related to metal acquisition, suggesting that these gene sets play an active role in scavenging for metals in the CF lung environment which may be inadequately represented in some models. Future studies of P. aeruginosa transcript abundance in CF may benefit from the use of an expectorated sputum model or media supplemented with factors that induce metal restriction.

Fingerprint analysis of dang-gui-Si-Ni decoction and its anticoagulant activity in vivo-in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Dang-Gui-Si-Ni (DGSN) decoction is a classic prescription in the clinical practice of traditional Chinese Medicine (TCM). DGSN decoction is often used to relieve symptoms of cold coagulation and blood stasis recorded by Treatise on Febrile Diseases (Shang Han Lun) and treat Raynaud's disease, dysmenorrhea, arthritis, migraine in TCM clinic. Accumulated evidences have suggested that this diseases are related to microcirculation disturbance. However, the anticoagulant activity and underlying mechanisms of DGSN decoction responsible for the therapeutic not well understood. AIM OF THE STUDY: The fingerprint and anticoagulant activity in vivo-in vitro of DGSN decoction were evaluated to strengthen the quality control and activity study of formulas. MATERIALS AND METHODS: The chemical components of DGSN decoction were analyzed by HPLC and its fingerprint similarity were evaluated by "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012 Edition)". The anticoagulant activity of DGSN decoction was assessed by measuring four coagulation factors (PT, TT, APTT, FIB) in vitro. Zebrafish thrombosis model induced by punatinib was established to evaluate the activity of improving microvascular hemodynamics in vivo. Quantitative real-time polymerase chain reaction (q-PCR) were adopted to compare the changes in the RNA expression levels of coagulation factor II (FII), VII (FVII), IX (FIX) and X (FX) in zebrafish thrombosis model. RESULTS: The fingerprint similarity evaluation method of DGSN decoction was established. The results showed that 18 samples had higher similarity (S1-S18 > 0.878). Pharmacodynamic results showed that DGSN decoction could extend PT, TT and APTT, and reduce FIB content in vitro. Meanwhile, it markedly enhanced the cardiac output and blood flow velocity at low dosage (500 µg mL-1) in vivo. q-PCR data demonstrated that DGSN decoction (500 µg mL-1) could downregulate the RNA expression of FII, FVII, FIX and FX. Interestingly, there were a bidirectional regulation of FII, FIX and FX in a certain concentration range. In general, DGSN decoction can significantly improve hemodynamics and downregulate coagulation factors, and the results were consistent both in vitro - in vivo. CONCLUSION: The fingerprint study provide a new perspective for improving the quality control of DGSN decoction. DGSN decoction possess anticoagulant activity by regulating multiple coagulation factors simultaneously. Thus, it has the potential to develop into the novel raw material of anticoagulant drugs.

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts.

The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy.

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1ß, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

Bioactive compounds and biological functions of medicinal plant-derived extracellular vesicles.

Extracellular vesicles (EVs) are tiny lipid bilayer-enclosed membrane particles released from a variety of cell types into the surrounding environment. These EVs have massive participated in cell-to-cell communication and interspecies communication. In recent years, plant-derived extracellular vesicles (PDEVs) and "exosome-like" EVs populations found in distinct plants have attracted widespread attention. Especially, research on medicinal plant-derived extracellular vesicles (MPDEVs) are increasing, which are considered a kind of promising natural compound. This review summarizes current knowledge on MPDEVs in terms of bioactive compounds, including small RNA, protein, lipid, and metabolite, have been found on the surface and/or in the lumen of MPDEVs. Moreover, both in vitro and in vivo experiments have shown that MPDEVs exert broad biomedical functions, such as anti-inflammatory, anticancer, antioxidant, modulate microbiota, etc. MPDEVs may be a better substitute than animal-derived extracellular vesicles (ADEVs) because of safety and biocompatibility in the future.

Pseudouridine-modified RNA probe for label-free electrochemical detection of nucleic acids on 2D MoS2 nanosheets.

RNA modification, particularly pseudouridine (Ψ), has played an important role in the development of the mRNA-based COVID-19 vaccine. This is because Ψ enhances RNA stability against nuclease activity and decreases the anti-RNA immune response. Ψ also provides structural flexibility to RNA by enhancing base stacking compared with canonical nucleobases. In this report, we demonstrate the first application of pseudouridine-modified RNA as a probe (Ψ-RNA) for label-free nucleic acid biosensing. It is known that MoS2 has a differential affinity for nucleic acids, which may be translated into a unique electronic signal. Herein, the Ψ-RNA probe interacts with the pristine MoS2 surface and causes a change in interfacial electrochemical charge transfer in the MoS2 nanosheets. Compared with an unmodified RNA probe, Ψ-RNA exhibited faster adsorption and higher affinity for MoS2. Moreover, Ψ-RNA could bind to complementary RNA and DNA targets with almost equal affinity when engaged with the MoS2 surface. Ψ-RNA maintained robust interactions with the MoS2 surface following the hybridization event, perhaps through its extra amino group. The detection sensitivity of the Ψ-RNA/MoS2 platform was as low as 500 attomoles, while the results also indicate that the probe can distinguish between complementary targets, single mismatches, and non-complementary nucleic acid sequences with statistical significance. This proof-of-concept study shows that the Ψ-RNA probe may solve numerous problems of adsorption-based biosensing platforms due to its stability and structural flexibility.

Hugan Qingzhi tablets attenuates endoplasmic reticulum stress in nonalcoholic fatty liver disease rats by regulating PERK and ATF6 pathways.

BACKGROUND: Endoplasmic reticulum (ER) stress, promoting lipid metabolism disorders and steatohepatitis, contributes significantly to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Hugan Qingzhi tablets (HQT) has a definite effect in the clinical treatment of NAFLD patients, but its mechanism is still unclear. This study aims to investigate the effects of HQT on ER stress in the liver tissues of NAFLD rats and explore the underlying mechanism. METHODS: The NAFLD rat model was managed with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, pro-inflammatory cytokines, liver histology were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the expression of ER stress-related signal molecules including glucose regulating protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic translation initiation factor 2α (EIF2α), p-EIF2α, activating transcription factor 4 (ATF4), acetyl-coenzyme A-carboxylase (ACC), activating transcription factor (ATF6), and nuclear factor-kappa B-p65 (NF-κB-p65) were detected by western blot and/or qRT-PCR. RESULTS: The histopathological characteristics and biochemical data indicated that HQT exhibited protective effects on HFD-induced NAFLD rats. Furthermore, it caused significant reduction in the expression of ERS markers, such as GRP78, PERK, p-PERK, and ATF6, and subsequently downregulated the expression of EIF2α, p-EIF2α ATF4, ACC, and NF-κB-p65. CONCLUSIONS: The results suggested that HQT has protective effect against hepatic steatosis and inflammation in NAFLD rats by attenuating ER stress, and the potential mechanism is through inhibition of PERK and ATF6 pathways.

Biomarker and transcriptomics profiles of serum selenium concentrations in patients with heart failure are associated with immunoregulatory processes.

BACKGROUND: Low selenium concentrations are associated with worse outcomes in heart failure (HF). However, the underlying pathophysiologic mechanisms remain incompletely understood. Therefore, we aimed to contrast serum selenium concentrations to blood biomarker and transcriptomic profiles in patients with HF. METHODS: Circulating biomarkers, whole blood transcriptomics and serum selenium measurements in a cohort of 2328 patients with HF were utilized. Penalized linear regression and gene expression analysis were used to assess biomarker and transcriptomics profiles, respectively. As a proof-of-principle, potential causal effects of selenium on excreted cytokines concentrations were investigated using human peripheral blood mononuclear cells (PBMCs). RESULTS: Mean selenium levels were 60.6 µg/L in Q1 and 122.0 µg/L in Q4. From 356 biomarkers and 20 clinical features, the penalized linear regression model yielded 44 variables with <5 % marginal false discovery rate as predictors of serum selenium. Biomarkers associated positively with selenium concentrations included: epidermal growth factor receptor (EGFR), IFN-gamma-R1, CD4, GDF15, and IL10. Biomarkers associated negatively with selenium concentrations included: PCSK9, TNFSF13, FGF21 and PAI. Additionally, 148 RNA transcripts were found differentially expressed between high and low selenium status (Padj.<0.05; log-fold-change<|0.25|). Enrichment analyses of the selected biomarkers and RNA transcripts identified similar enriched processes, including regulation processes of leukocyte differentiation and activation, as well as cytokines production. The mRNA expression of two selenoproteins (MSRB1 and GPX4) were strongly correlated with serum selenium, while GPX4, SELENOK, and SELENOS were associated with prognosis. In the in-vitro setting, PBMCs supplemented with selenium showed significantly lower abundance of several (pro-)inflammatory cytokines. CONCLUSION: These data suggest that immunoregulation is an important mechanism through which selenium might have beneficial roles in HF. The beneficial effects of higher serum selenium concentrations are likely because of global immunomodulatory effects on the abundance of cytokines. MSRB1 and GPX4 are potential modulators of and should be pursued in future research.

N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression.

N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology. As the m6A reader YTHDF1 has been reported to promote progression of hepatocellular carcinoma (HCC), it represents a potential therapeutic target. In this study, we evaluated the clinical significance of YTHDF1 using human HCC samples and found that YTHDF1 was significantly upregulated in HCCs with high stemness scores and was positively associated with recurrence and poor prognosis. Analysis of HCC spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSC). Stem cell-specific conditional Ythdf1 knockin (CKI) mice treated with diethylnitrosamine showed elevated tumor burden as compared with wild-type mice. YTHDF1 promoted CSCs renewal and resistance to the multiple tyrosine kinase inhibitors lenvatinib and sorafenib in patient-derived organoids and HCC cell lines, which could be abolished by catalytically inactive mutant YTHDF1. Multiomic analysis, including RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing identified NOTCH1 as a direct downstream of YTHDF1. YTHDF1 bound to m6A modified NOTCH1 mRNA to enhance its stability and translation, which led to increased NOTCH1 target genes expression. NOTCH1 overexpression rescued HCC stemness in YTHDF1-deficient cells in vitro and in vivo. Lipid nanoparticles targeting YTHDF1 significantly enhanced the efficacy of lenvatinib and sorafenib in HCC in vivo. Taken together, YTHDF1 drives HCC stemness and drug resistance through an YTHDF1-m6A-NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC. SIGNIFICANCE: Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.

Analysis of the relative frequencies of the multipartite BNYVV genomic RNAs in different plants and tissues.

Multipartite virus genomes are composed of two or more segments, each packaged into an independent viral particle. A potential advantage of multipartitism is the regulation of gene expression through changes in the segment copy number. Soil-borne beet necrotic yellow vein virus (BNYVV) is a typical example of multipartism, given its high number of genomic positive-sense RNAs (up to five). Here we analyse the relative frequencies of the four genomic RNAs of BNYVV type B during infection of different host plants (Chenopodium quinoa, Beta macrocarpa and Spinacia oleracea) and organs (leaves and roots). By successfully validating a two-step reverse-transcriptase digital droplet PCR protocol, we show that RNA1 and -2 genomic segments always replicate at low and comparable relative frequencies. In contrast, RNA3 and -4 accumulate with variable relative frequencies, resulting in distinct RNA1 : RNA2 : RNA3 : RNA4 ratios, depending on the infected host species and organ.

Calcium-mediated modulation of Pseudomonas fluorescens biofilm formation.

Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca2+) affects the biofilm formation of dairy Pseudomonas fluorescens isolates by investigating their growth, swarming motility, biofilm-forming capacity, extracellular polymeric substance production, and biofilm structures. Moreover, the regulation mechanism of Ca2+ involved in its biofilm formation was explored through RNA-sequencing analysis. This work revealed that supplementation of 5, 10, 15, and 20 mM Ca2+ significantly reduced the swarming motility of P. fluorescens strains (P.F2, P.F4, and P.F17), but the biofilm-forming ability and polysaccharide production were increased after the supplementation of 5 and 10 mM Ca2+. By the supplementation of Ca2+, complex structures with more cell clusters glued together in P. fluorescens P.F4 biofilms were confirmed by scanning electron microscopy, and increased biomass and coverage of P. fluorescens P.F4 biofilms were observed by confocal laser scanning microscopy. In addition, RNA-sequencing results showed that P. fluorescens P.F4 showed a transcriptional response to the supplementation of 10 mM Ca2+, and a total of 137 genes were significantly expressed. The differential genes were represented in 4 upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (nonribosomal peptide structures, quorum sensing, biosynthesis of siderophore group nonribosomal peptides, and phenylalanine metabolism), and 4 downregulated KEGG pathways (flagellar assembly, amino sugar and nucleotide sugar metabolism, nitrotoluene degradation, and cationic antimicrobial peptide resistance). The results indicate that Ca2+ might serve as an enhancer to substantially trigger the biofilm formation of dairy P. fluorescens isolates in the dairy industry.

A Fundamental Question for Complementary Medicine: Are There Other Forces in the Natural World Besides the Physical Forces?

BACKGROUND: The integration of conventional and complementary medicine reflects the pluralism in science. Still, a critical issue is the conception of the natural world. Many complementary therapy systems seem to contradict the reductionist-atomistic paradigm that all of natural reality is essentially based on the physical interactions of atoms and molecules. Thus, a fundamental question about the natural world is: Do other than the physical forces exist? SUMMARY: The assumption that no other than physical forces exist and work in the natural world is not tenable. For example, the formation and maintenance of the functional Gestalt of organisms cannot possibly be explained by molecular processes (e.g., from DNA to RNA and further to amino acids and proteins). The processes on each structural level - from molecules, organelles, cells, organs up to the whole organism - are regulated in regard to the formation of the next higher level. Specific Gestalt-forming forces exist and can be systematically investigated. Their existence implies an extended conception of matter. The Gestalt-forming forces and the extended concept of matter may be relevant for the scientific assessment of complementary therapies. KEY MESSAGES: (i) In the natural world, specific Gestalt-forming forces exist in addition to the physical forces, and can be systematically investigated. (ii) The existence of these forces implies an extended conception of matter. (iii) These forces and this extended concept of matter may be relevant for the scientific assessment of complementary therapies, e.g., homeopathy.In der Integration von konventioneller und komplementärer Medizin spiegelt sich der Methodenpluralismus der Wissenschaft. Die Ontologien vieler komplementärmedizinisches Systeme liegen allerdings außerhalb der Erklärbarkeit durch die Kräfte der Physik. Eine zentrale Frage ist deshalb: Gibt es Kräfte in der Natur, die eine materielle Wirkung haben, deren Ursprung aber nicht in Atomen oder Molekülen und in diesem Sinne nicht in der Materie liegt?Die Annahme, dass in der Natur keine anderen als die mit Atomen und Molekülen assoziierten physikalischen Kräfte existent und wirksam seien, ist wissenschaftlich nicht begründet. Beispielsweise ist die Bildung und Erhaltung der funktionsfähigen Gestalt von Organismen nicht durch molekulare Prozesse (z.B. von der DNA zur RNA und weiter zu Aminosäuren und Proteinen) erklärbar. Die Prozesse auf jeder strukturellen Ebene ­ von den Molekülen, Organellen, Zellen, Organen bis hinauf zum Gesamtorganismus ­ sind in Hinblick auf die Bildung der funktionsfähigen Gestalt der jeweils nächsthöheren Ebene gesteuert. Für diese Gestaltbildung gibt es spezifische Kräfte, die systematisch erforscht werden können. Ihre Existenz impliziert eine erweiterte Konzeption von Materie. Diese Gestalt-bildenden Kräfte und dieses erweiterte Konzept von Materie sind relevant für die wissenschaftliche Erfassung komplementärmedizinischer Systeme.(i) In der Natur sind außer den physikalischen Kräften noch weitere spezifische Kräfte wirksam, beispielsweise bei der Bildung und Erhaltung der funktionsfähigen Gestalt von Organismen. Diese Kräfte können systematisch erforscht werden. (ii) Die Existenz dieser Kräfte impliziert eine erweitere Konzeption von Materie. (iii) Diese Kräfte und das erweiterte Materiekonzept sind relevant für die wissenschaftliche Erfassung komplementärmedizinischer Systeme, beispielsweise der Homöopathie.

Tianhuang formula ameliorates liver fibrosis by inhibiting CCL2-CCR2 axis and MAPK/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In the progression of chronic liver diseases, liver fibrosis is a reversible pathophysiologic event for liver diseases prognosis and risk of cirrhosis. Liver injury factors of different etiologies mediate this process. There is still a lack of effective medications for treating liver fibrosis. Additionally, the ameliorative effects of traditional herbs on liver fibrosis have been commonly reported. Tianhuang formula (THF) is a drug combination consisting of 2 traditional Chinese herbs, which has been showing significant improvement in metabolic liver diseases. However, the hepatoprotective effect and mechanism of THF in ameliorating liver fibrosis are still unclear. AIM OF THE STUDY: This study aimed to investigate the effects of THF on carbon tetrachloride (CCl4)-induced and methionine-choline-deficient (MCD) diet-induced liver fibrosis model and to reveal the potential mechanisms. It can provide experimental evidence for THF as a therapeutic candidate for liver fibrosis. MATERIALS AND METHODS: In this study, CCl4-induced mice were treated with THF (80 mg/kg, 160 mg/kg) or Fuzheng Huayu (FZHY) capsules (4.8 g/kg) for 6 weeks. MCD-induced mice received the same doses of THF or FZHY for 4 weeks. FZHY is used as a comparative study in these two models. Following that, using kit reagents detected changes in relevant serum and liver biochemical indicators. Histological changes in mouse liver were measured by staining of H&E and Sirius Red. The markers expression of liver fibrosis and inflammation were detected using qRT-PCR, western blotting and immunohistochemical staining analysis. The potential regulatory mechanism of THF to ameliorate liver fibrosis was performed by RNA-sequencing analysis. Finally, the analysis results were verified by immunofluorescence co-staining, qRT-PCR and western blotting. RESULTS: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic triglyceride (TG) levels in CCl4 and MCD-induced liver fibrosis mice were significantly improved after THF treatment. Meanwhile, the expression of fibrosis and inflammation markers were significantly suppressed. Furthermore, THF downregulated the expression of the macrophage marker CD68. According to RNA-sequencing analysis, we found the CCL2-CCR2 axis and MAPK/NF-κB as the potential signaling pathway for THF against liver fibrosis. CONCLUSION: This study revealed that THF ameliorated liver injury, inflammation and fibrotic process by inhibiting CCL2-CCR2 axis and its downstream MAPK/NF-κB signaling pathway.