Dot Blot Research Articles

Cathelicidin-HG Alleviates Sepsis-Induced Platelet Dysfunction by Inhibiting GPVI-Mediated Platelet Activation.

Platelet activation contributes to sepsis development, leading to microthrombosis and increased inflammation, which results in disseminated intravascular coagulation and multiple organ dysfunction. Although Cathelicidin can alleviate sepsis, its role in sepsis regulation remains largely unexplored. In this study, we identified Cath-HG, a novel Cathelicidin from Hylarana guentheri skin, and analyzed its structure using nuclear magnetic resonance spectroscopy. The modulatory effect of Cath-HG on the symptoms of mice with sepsis induced by cecal ligation and puncture was evaluated invivo, and the platelet count, degree of organ damage, and microthrombosis were measured. The antiplatelet aggregation activity of Cath-HG was studied invitro, and its target was verified. Finally, we further investigated whether Cath-HG could regulate thrombosis invivo in a FeCl3 injury-induced carotid artery model. The results showed that Cath-HG exhibited an α-helical structure in sodium dodecyl sulfate solution and effectively reduced organ inflammation and damage, improving survival in septic mice. It alleviated sepsis-induced thrombocytopenia and microthrombosis. Invitro, Cath-HG specifically inhibited collagen-induced platelet aggregation and modulated glycoprotein VI (GPVI) signaling pathways. Dot blotting, enzyme-linked immunosorbent assay, and pull-down experiments confirmed GPVI as the target of Cath-HG. Molecular docking and amino acid residue truncations/mutations identified crucial sites of Cath-HG. These findings suggest that GPVI represents a promising therapeutic target for sepsis, and Cath-HG may serve as a potential treatment for sepsis-related thrombocytopenia and thrombotic events. Additionally, identifying Cath-HG as a GPVI inhibitor provides insights for developing novel antithrombotic therapies targeting platelet activation mediated by GPVI.

Purification and structural characterization of three novel anti-adhesive peptides against Helicobacter pylori from corn gluten meal

More than 50% of the world population is infected with Helicobacter pylori (H. pylori). Potential anti-adhesive peptides from corn protein hydrolysate were developed using in vitro methodologies to prevent Helicobacter pylori infection. To obtain anti-adhesive peptides against Helicobacter pylori, corn gluten meal (CGM) was hydrolyzed by neutrase, and the hydrolysate (CPHN) was purified by using Superdex peptide gel filtration, Q anion exchange and Mono Q anion exchange. Then, sequences of peptides were identified by Nano-LC-MS/MS, and the structural and functional properties of the identified peptides were characterized. Three novel peptides Pro-Tyr-Ala-Glu-Tyr (PYAEY, PY5), Ile-Ile-Pro-Gln-Cys-Ser (IIPQCS, II6) and Thr-Ile-Ile-Pro-Gln (TIIPQ, TI5) were identified, respectively. PY5 exhibited the best anti-adhesive activity among the three novel peptides, and its anti-adhesive activity was 40.75 ± 0.72% at 4 mg/mL. Meanwhile, PY5 also possessed significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and excellent 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity, with IC50 values of 1.650 ± 0.184 and 0.007 ± 0.001 mg/mL, respectively, suggesting that PY5 could alleviate stomach damage induced by Helicobacter pylori infection. In addition, molecular docking and dot blot results showed that PY5 could also conjugate with the Helicobacter pylori adhesins, SabA and BabA, through hydrophobic interactions and hydrogen bond inhibiting Helicobacter pylori adhesion. These results indicate that three novel peptides from corn protein hydrolysate have potential anti-adhesive and antioxidant effects and could serve as functional foods or drugs to prevent Helicobacter pylori infection.

Antigenic epitopes and cross-reactivity analysis of tropomyosin from Oratosquilla oratorio.

Tropomyosin (TM) has been determined as the major allergen in Oratosquilla oratoria. However, little information is available about its antigenic epitopes and cross-reactivity. In this study, recombinant TM was obtained through genetic engineering and its IgG-/IgE-binding activity was similar to native TM. Subsequently, four T-cell epitopes were identified by using bioinformatics technology combined with CD4+ T-cell proliferation assay, and seven B-cell epitopes were identified by bioinformatics technology combined with serological assay. Moreover, dot blot analysis showed that TM had cross-reactivity among O. oratoria and other shellfish by using IgE polycolonal antibody from sera of shrimp-sensitive individuals or O. oratoria TM-immunized mice. Interestingly, three T-cell epitopes (AA4-24, AA194-212, and AA221-243) and four B-cell epitopes (AA85-100, AA121-138, AA209-233, and AA242-259) of TM in O. oratoria were conserved between O. oratoria and other shellfish, which may be the main reason for the cross-reactivity between them. In summary, these findings could serve for the diagnosis and prevention of shrimp allergy.

Mitochondria-encoded peptide MOTS-c participates in plasma membrane repair by facilitating the translocation of TRIM72 to membrane.

Rationale: An impairment of plasma membrane repair has been implicated in various diseases such as muscular dystrophy and ischemia/reperfusion injury. MOTS-c, a short peptide encoded by mitochondria, has been shown to pass through the plasma membrane into the bloodstream. This study determined whether this biological behavior was involved in membrane repair and its underlying mechanism. Methods and Results: In human participants, the level of MOTS-c was positively correlated with the abundance of mitochondria, and the membrane repair molecule TRIM72. In contrast to high-intensity eccentric exercise, moderate-intensity exercise improved sarcolemma integrity and physical performance, accompanied by an increase of mitochondria beneath the damaged sarcolemma and secretion of MOTS-c. Furthermore, moderate-intensity exercise increased the interaction between MOTS-c and TRIM72, and MOTS-c facilitated the trafficking of TRIM72 to the sarcolemma. In vitro studies demonstrated that MOTS-c attenuated membrane damage induced by hypotonic solution, which could be blocked by siRNA-TRIM72, but not AMPK inhibitor. Co-immunoprecipitation study showed that MOTS-c interacted with TRIM72 C-terminus, but not N-terminus. The dynamic membrane repair assay revealed that MOTS-c boosted the trafficking of TRIM72 to the injured membrane. However, MOTS-c itself had negligible effects on membrane repair, which was recapitulated in TRIM72-/- mice. Unexpectedly, MOTS-c still increased the fusion of vesicles with the membrane in TRIM72-/- mice, and dot blot analysis revealed an interaction between MOTS-c and phosphatidylinositol (4,5) bisphosphate [PtdIns (4,5) P2]. Finally, MOTS-c blunted ischemia/reperfusion-induced membrane disruption, and preserved heart function. Conclusions: MOTS-c/TRIM72-mediated membrane integrity improvement participates in mitochondria-triggered membrane repair. An interaction between MOTS-c and plasma lipid contributes to the fusion of vesicles with membrane. Our data provide a novel therapeutic strategy for rescuing organ function by facilitating membrane repair with MOTS-c.

Serum CCDC25 Levels as a Potential Marker for Metabolic Syndrome.

Metabolic syndrome (MetS) stands as a significant risk for developing various severe health problems. Therefore, the discovery of biomarkers capable of predicting the progression of metabolic conditions is crucial for improving overall health outcomes. Recently, we reported that coiled-coil domain containing 25 (CCDC25) might be associated with key proteins involved in metabolic pathways, by bioinformatics analysis. Thus, we assumed that serum CCDC25 levels might have an association with MetS status. In this study, based on the modified National Cholesterol Education Program-Adult Treatment Panel III (modified NCEP-ATP III) criteria, the participants who had three or more of abnormal criteria were defined as MetS, and those who had 1 or 2 abnormal criteria as pre-MetS groups; those who had no abnormal criteria were classified as the healthy control (HC) group. Serum CCDC25 levels were measured using the dot blot assay. The results showed that serum CCDC25 levels of the MetS group (0.072±0.026 ng/μl) were significantly higher (p<0.001) than that of pre-MetS (0.031±0.011 ng/μl) or HC groups (0.018±0.007 ng/μl). We can discern a consistent trend indicating that serum CCDC25 level is well correlated with the number of abnormal criteria of MetS of each participant. Although serum CCDC25 levels correlated with the distribution of all 5 MetS criteria, the highest correlation was seen in serum CCDC25 levels and triglyceride (TG) levels, with r=0.563, followed by systolic blood pressure (SBP) levels (r=0.557) and high-density lipoprotein-cholesterol (HDL-C) levels (r=-0.545). CCDC25 showed correlations with all MetS parameters, particularly with TG, SBP, and HDL-C. This prompts speculation that heightened CCDC25 levels may indicate the development and/or progression of those MetS-associated diseases.

Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice.

Intestinal stem cells (ISCs) play a pivotal role in maintaining intestinal homeostasis and facilitating the restoration of intestinal mucosal barrier integrity. Glutamine (Gln) is a crucial energy substrate in the intestine, promoting the proliferation of ISCs and mitigating damage to the intestinal mucosal barrier after burn injury. However, the underlying mechanism has not yet been fully elucidated. The objective of this study was to explore the mechanism by which Gln facilitates the proliferation of ISCs. A mouse burn model was established to investigate the impact of Gln on intestinal function. Subsequently, crypts were isolated, and changes in TP53-induced glycolysis and apoptosis regulator (TIGAR) expression were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, immunohistochemistry, and immunofluorescence. The effects of TIGAR on cell proliferation were validated through CCK-8, EdU, and clonogenicity assays. Furthermore, the effect of TIGAR on Yes-associated protein (YAP) nuclear translocation and ferroptosis was examined by western blotting and immunofluorescence staining. Finally, dot blot analysis and methylation-specific PCR were performed to evaluate the effect of Gln on TIGAR promoter methylation. The mRNA and protein levels of TIGAR decreased after burn injury, and supplementation with Gln increased the expression of TIGAR. TIGAR accelerates the nuclear translocation of YAP, thereby increasing the proliferation of ISCs. Concurrently, TIGAR promotes the synthesis of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione to suppress ferroptosis in ISCs. Subsequent investigations demonstrated that Gln inhibits TIGAR promoter methylation by increasing the expression of the demethylase ten-eleven translocation. This change increased TIGAR transcription, increased NADPH synthesis, and reduced oxidative stress, thereby facilitating the restoration of intestinal mucosal barrier integrity post-burn injury. Our data confirmed the inhibitory effect of Gln on TIGAR promoter methylation, which facilitates YAP translocation into the nucleus and suppresses ferroptosis, ultimately promoting the proliferation of ISCs.

METTL3 Inhibition Suppresses Cell Growth and Survival in Colorectal Cancer via ASNS Downregulation.

Background: Colorectal cancer (CRC) presents a significant global health burden, with high rates of incidence and mortality, and an urgent need to improve prognosis. STM2457, a novel small molecule inhibitor specific for N6-methyladenosine (m6A) catalytic enzyme Methyltransferase-like 3 (METTL3) has implicated significant treatment potentials in a few of types of cancer. However, its impact and underlying mechanism are still unclear in CRC cells. Methods: We used CCK-8 and colony formation assay to observe cell growth, flow cytometry and TUNEL approaches to detect cell apoptosis under the treatment of STM2457 on CRC cells in vitro or in vivo. RNA-sequencing, qRT-PCR and western blotting were performed to explore downstream effectors of STM2457. Messenger RNA stability was evaluated by qRT-PCR after treatment with actinomycin D. The methylated RNA immunoprecipitation (MeRIP) qPCR, dual-luciferase reporter analyses and m6A dot blotting were carried out to measure the m6A modification. Associated gene expression pattern and clinical relevance in CRC clinical tissue samples were analyzed using online database. Results: STM2457 exhibited a strong influence on cell growth suppression and apoptosis of CRC cells in vitro and subcutaneous xenograft growth in vivo. Asparagine synthetase (ASNS) was markedly downregulated upon STM2457 treatment or METTL3 knockdown and exogenous overexpression of ASNS could rescue the biological defects induced by STM2457. Mechanistically, the downregulation of ASNS by STM2457 may be due to the decrease of m6A modification level in ASNS mRNA mediated by METTL3. Conclusions: Our findings suggest that STM2457 may serve as a potential therapeutic agent and ASNS may be a new promising therapeutic target for CRC.

Bing–Neel Syndrome: An Unknown GCA Mimicker

Giant cell arteritis (GCA) is a chronic granulomatous vasculitis of medium and large arteries leading to cranial and extracranial manifestations. Temporal artery biopsy is considered the gold standard; however, its sensitivity is low at 47%. We report a unique case of Bing–Neel Syndrome (BNS) presenting as biopsy‐proven GCA. BNS is a rare complication (1%) of Waldenstrom Macroglobulinemia (WM), which results from infiltration of lymph plasmacytoid cells and plasma cells into the central nervous system. A 77‐year‐old female with a past medical history of glaucoma, hypertension, diabetes, and chronic ocular ischemic syndrome in her right eye presented with progressive left eye vision loss for 5 days. Fundoscopic examination was notable for pseudophakic pseudopallor but no optic disc edema. Intraocular pressure was &gt;40 and normalized after acetazolamide. The patient was started on pulse dose steroids by her neuro‐ophthalmologist. She was discharged home on 60 mg of prednisone. At follow up with her neuro‐ophthalmologist, new dot blot hemorrhages in the left eye were noted and she was readmitted for pulse dose of intravenous methylprednisolone. Temporal artery biopsy was consistent with GCA spectrum. Work up revealed paraproteinemia and subsequent bone marrow biopsy demonstrated WM. The patient was treated for her WM and her ophthalmic complications stabilized.

Comparison of multiplex PCR capillary electrophoresis assay and PCR-reverse dot blot assay for human papillomavirus DNA genotyping detection in cervical cancer tissue specimens.

The study aimed to evaluate the positivity rates and genotype distribution of the multiplex PCR capillary electrophoresis (MPCE) and PCR-Reverse Dot Blot (PCR-RDB) assays for human papillomavirus (HPV) detection in cervical cancer tissue specimens, and to explore their detection principles and applications in large-scale population screening. The MPCE and PCR-RDB assays were performed separately on 425 diagnosed cervical cancer tissue specimens. Subsequently, the results of both assays were compared based on the HPV infection positivity rates and genotype distribution. The overall positive rates of HPV genotypes for the MPCE and PCR-RDB assays were 97.9% and 92.9%, respectively. A p-value < 0.001 indicated a statistically significance difference in consistency between the two assays. The kappa value was 0.390, indicating that the consistency between both assays was fair. HPV16 was the most common single-genotype infection type, with infection rates detected via MPCE and PCR-RDB assays being 75.7% and 68.3%, respectively. In the age group >50 years, the HPV multiple-type infection rate detected via MPCE assay was significantly higher than that detected by the PCR-RDB assay, with a statistically significant difference (p = 0.002). To reduce the false-negative rate and improve screening efficiency, the MPCE assay, which targets the oncogenic gene E6/E7 segments, can be extended to the general female population for the early detection, diagnosis, and treatment of cervical cancer.

Isolation of Peroxidase enzyme from various vegetables and coupling to IgG

Coupling of IgG with peroxidase enzyme finds various applications in determining the antigen-antibody interactions, in diagnostics or for research purpose. In the present study, Peroxidase enzyme was isolated from various vegetables like tomato (Solanum lycopersicum), cabbage (Brassica oleracea) and Radish (Raphanus sativus) and partial purification of enzymes was done with ammonium sulphate fractionation to 80% saturation. The active peroxidase enzyme was coupled to Goat anti-human IgG and the efficiency of coupling procedure was tested against Protein A sepharose purified human IgG. The coupling procedure was done by using sodium periodate and coupling was confirmed by dot blot and ELISA procedures. The present study provides an indigenous method to isolate an active peroxidase enzyme and couple to various IgG and use in immuno detection methods at very cost effective manner.

No significant change of N6 -methyladenosine modification landscape in mouse brain after morphine exposure.

N6 -methyladenosine (m6 A) plays a crucial role in regulating neuroplasticity and different brain functions at the posttranscriptional level. However, it remains unknown whether m6 A modification is involved in acute and chronic morphine exposure. In this study, we conducted a direct comparison of m6 A levels and mRNA expression of m6 A-associated factors between morphine-treated and nontreated C57BL/6 wild-type mice. We established animal models of both acute and chronic morphine treatment and confirmed the rewarding effects of chronic morphine treatment using the conditioned place preference (CPP) assay. The activation status of different brain regions in response to morphine was assessed by c-fos staining. To assess overall m6 A modification levels, we employed the m6 A dot blot assay, while mRNA levels of m6 A-associated proteins were measured using a quantitative polymerase chain reaction (qPCR) assay. These analyses were performed to investigate whether and how m6 A modification and m6 A-associated protein expression will change following morphine exposure. The overall m6 A methylation and mRNA levels of m6 A-associated proteins were not significantly altered in brain regions that were either activated or not activated during acute morphine stimulation. Similarly, the overall m6 A modification and mRNA levels of m6 A-associated proteins remained unaffected in several key brain regions associated with reward following chronic morphine exposure. This study showed that the overall m6 A modification level and mRNA expression levels of m6 A-associated factors were not affected after acute and chronic morphine exposure in different brain regions, indicating m6 A modification may not be involved in brain response to morphine exposure.

Rat hepatitis E virus (Rocahepevirus ratti) in people living with HIV

ABSTRACT Rat hepatitis E virus (ratHEV; species Rocahepevirus ratti) is considered a newly emerging cause of acute hepatitis of zoonotic origin. ratHEV infection of people living with HIV (PLWH) might portend a worse, as with hepatitis E virus (HEV; species Paslahepevirus balayani), and consequently this group may constitute a high-risk population. We aimed to evaluate the prevalence of ratHEV by measuring viral RNA and specific IgG antibodies in a large Spanish cohort of PLWH. Multicentre study conducted in Spain evaluating PLWHIV included in the Spanish AIDS Research Network (CoRIS). Patients were evaluated for ratHEV infection using PCR at baseline and anti-ratHEV IgG by dot blot analysis to evaluate exposure to ratHEV strains. Patients with detectable ratHEV RNA were followed-up to evaluate persistence of viremia and IgG seroconversion. Eight-hundred and forty-two individuals were tested. A total of 9 individuals showed specific IgG antibodies against ratHEV, supposing a prevalence of 1.1 (95% CI; 0.5%−2.1%). Of these, only one was reactive to HEV IgG antibodies by ELISA. One sample was positive for ratHEV RNA (prevalence of infection: 0.1%; 95% CI: 0.08%−0.7%). The case was a man who had sex with men exhibiting a slightly increased alanine transaminase level (49 IU/L) as only biochemical alteration. In the follow-up, the patients showed undetectable ratHEV RNA and seroconversion to specific ratHEV IgG antibodies. Our study shows that ratHEV is geographical broadly distributed in Spain, representing a potential zoonotic threat.

Analysis of Crude, Diverse, and Multiple Advanced Glycation End-Product Patterns May Be Important and Beneficial.

Lifestyle-related diseases (LSRDs), such as diabetes mellitus, cardiovascular disease, and nonalcoholic steatohepatitis, are a global crisis. Advanced glycation end-products (AGEs) have been extensively researched because they trigger or promote LSRDs. Recently, techniques such as fluorimetry, immunostaining, Western blotting, slot blotting, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, matrix-assisted laser desorption-mass spectrometry (MALDI-MS), and electrospray ionization-mass spectrometry (ESI-MS) have helped prove the existence of intra/extracellular AGEs and revealed novel AGE structures and their modifications against peptide sequences. Therefore, we propose modifications to the existing categorization of AGEs, which was based on the original compounds identified by researchers in the 20th century. In this investigation, we introduce the (i) crude, (ii) diverse, and (iii) multiple AGE patterns. The crude AGE pattern is based on the fact that one type of saccharide or its metabolites or derivatives can generate various AGEs. Diverse and multiple AGE patterns were introduced based on the possibility of combining various AGE structures and proteins and were proven through mass analysis technologies such as MALDI-MS and ESI-MS. Kampo medicines are typically used to treat LSRDs. Because various compounds are contained in Kampo medicines and metabolized to exert effects on various organs or tissues, they may be suitable against various AGEs.

Celastrol suppresses human pancreatic cancer via m6A-YTHDF3-mediated downregulation of Claspin and Bcl-2

BackgroundCelastrol has been revealed to exhibit anticancer pharmacological activity, however, the molecular mechanisms of celastrol involved in pancreatic cancer remain to be further elucidated. The present study was to illustrate whether celastrol suppresses pancreatic cancer through modulating RNA m6A modification.MethodsEffect of celastrol treatment on the malignant phenotypes of pancreatic cancer cells was evaluated by CCK-8 assay, EdU assay, colony formation assay, flow cytometry analysis and subcutaneous xenograft experiments. RNA sequencing (RNA-seq) analysis was carried out to analyze the genes differentially expressed in celastrol-treated pancreatic cancer cells. RT-qPCR, Western blotting and immunohistochemistry were employed to evaluate the expression of the indicated genes. RNA dot blot and quantification of total RNA m6A modification assays, MeRIP-qPCR assay, RIP-qPCR assay, RNA stability and protein stability assays were applied to evaluate the regulatory mechanism of celastrol treatment in pancreatic cancer cells.ResultsWe demonstrated that celastrol suppressed cell proliferation and induced cell cycle arrest and apoptosis of pancreatic cancer cells in vitro, and decreased tumor growth in vivo. Specifically, Bcl-2, Claspin, METTL3 and YTHDF3 were identified as the potential targets of celastrol treatment in pancreatic cancer cells. Moreover, our results indicated that celastrol treatment downregulated METTL3 and decreased m6A levels of Claspin and Bcl-2 mRNA, leading to the degradation of Claspin and Bcl-2 mRNA in pancreatic cancer cells. Furthermore, we revealed that celastrol treatment downregulated Claspin and Bcl-2, at least in part, in an m6A-YTHDF3-mediated manner in pancreatic cancer cells.ConclusionOur study highlighted a novel mechanism underlying celastrol-induced cellular proliferation inhibition and apoptosis in pancreatic cancer cells via m6A-YTHDF3-mediated downregulation of Claspin and Bcl-2.

The identification of N6-methyladenosine-related miRNAs predictive of hepatocellular carcinoma prognosis and immunotherapy efficacy.

Hepatocellular carcinoma (HCC) has a high degree of malignancy and poor prognosis. N6-methyladenosine (m6A) modifications and microRNAs (miRNAs) play pivotal roles in tumorigenesis and development. However, the role of m6A-related miRNAs in HCC has not been clarified yet. This study aimed to identify the role of m6A-miRNAs in HCC prognosis through bioinformatics analysis. The clinicopathological information and RNA sequencing data of 369 HCC tumor tissues and 49 tumor-adjacent tissues were downloaded from the TCGA database. A total of 23 m6A regulators were extracted to evaluated the m6A-related miRNAs using Pearson's correlation analysis. Then, we selected prognosis-related m6A-miRNAs using a univariate Cox regression model and used the consensus cluster analysis to explore the characteristics of the m6A-miRNAs. The coefficient of the least absolute shrinkage and selection operator (LASSO) Cox regression was applied to construct a prognostic risk score model. The receiver operated characteristic (ROC) analysis was applied to evaluate the prognostic value of the signature. The biological functions of targeted genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Then, to validate the potential predictive value for prognosis, the miRNA expression profiles from the GSE76903 and GSE6857 were used. Single sample Gene Set Enrichment Analysis (ssGSEA) and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) were applied to assess the immune microenvironment of HCC. Additionally, a meta-analysis was used to verify the prognostic value of the m6A-microRNAs. RT-PCR was applied to validated the expression of miRNAs in HCC tissues. Cell viability, transwell assay and RNA m6A dot blot assays of HCC cells was applied to access the function of miR-17-5p. The expression of 48 m6A-related miRNAs was identified and 17 prognostic m6A-miRNAs was discovered. The expression profile of those 17 miRNAs was divided into three clusters, and these clusters were associated with the tumor microenvironment (TME) and prognosis. The nine m6A-related miRNA signature was associated with the prognosis of HCC, the AUC of the ROC was 0.771(TCGA dataset), 0.788(GSE76903) and 0.646(GSE6857). The TME and the expression of immune checkpoint molecules were associated with the risk score. The meta-analysis also validated the prognostic value of the m6A-related miRNAs (miR182-5p (HR:1.58, 95%CI:1.04-2.40) and miR-17-5p (HR:1.58, 95%CI: 1.04-2.40)). The expression of miR-17-5p was upregulated in HCC tissues and miR-17-5p showed an oncogenic role in HCC cells. The clinical innovation is the use of m6A-miRNAs as biomarkers for predicting prognosis regarding immunotherapy response in HCC patients.

N6-methyladenosine demethylase FTO regulates synaptic and cognitive impairment by destabilizing PTEN mRNA in hypoxic-ischemic neonatal rats

Hypoxic-ischemic brain damage (HIBD) can result in significant global rates of neonatal death or permanent neurological disability. N6-methyladenosine (m6A) modification of RNA influences fundamental aspects of RNA metabolism, and m6A dysregulation is implicated in various neurological diseases. However, the biological roles and clinical significance of m6A in HIBD remain unclear. We currently evaluated the effect of HIBD on cerebral m6A methylation in RNAs in neonatal rats. The m6A dot blot assay showed a global augmentation in RNA m6A methylation post-HI. Herein, we also report on demethylase FTO, which is markedly downregulated in the hippocampus and is the main factor involved with aberrant m6A modification following HI. By conducting a comprehensive analysis of RNA-seq data and m6A microarray results, we found that transcripts with m6A modifications were more highly expressed overall than transcripts without m6A modifications. The overexpression of FTO resulted in the promotion of Akt/mTOR pathway hyperactivation, while simultaneously inhibiting autophagic function. This is carried out by the demethylation activity of FTO, which selectively demethylates transcripts of phosphatase and tensin homolog (PTEN), thus promoting its degradation and reduced protein expression after HI. Moreover, the synaptic and neurocognitive disorders induced by HI were effectively reversed through the overexpression of FTO in the hippocampus. Cumulatively, these findings demonstrate the functional importance of FTO-dependent hippocampal m6A methylome in cognitive function and provides novel mechanistic insights into the therapeutic potentials of FTO in neonatal HIBD.

HIF-1α/METTL1/m7G axis is involved in CRC response to hypoxia

BackgroundIn recent years, many studies have confirmed that hypoxia and hypoxia inducible factor (HIF)-1α drive the development of colorectal cancer (CRC). HIF-1α also modulates epitranscriptomic remodeling to regulate cancer development. However, the mechanism by which RNA methylation is altered under hypoxic conditions and the underlying regulatory mechanisms in CRC remain unclear. MethodsHere, seven common types of modifications of mRNA and tRNA were quantitated using liquid chromatography-tandem mass spectrometry. To validate the robustness of the profiling data, modifications that were consistently altered across the three CRC cell lines under hypoxia were validated via dot blot analysis. Then, 10 enzymes that could regulate the abundance of three RNA modifications in tRNA were measured in CRC cells after hypoxia treatment using quantitative real-time polymerase chain reaction. Furthermore, the regulatory role of HIF-1α in the expression of methyltransferase 1 (METTL1) under hypoxic conditions was confirmed using METTL1 promoter activity assays and HIF-1α small interfering RNA (siRNA). The binding capacity of HIF-1α to each hypoxia response element (HRE) in the promoter of METTL1 was investigated by performing Chromatin immunoprecipitation assay (ChIP). ResultsAbundance of RNA modifications was altered more consistently and significantly in tRNA than in mRNA under hypoxic conditions. In addition, the abundance of N7-methyleguanosine (m7G) modification in tRNA decreased significantly under hypoxic conditions. As a methyltransferase of the m7G modification in tRNA, the expression of METTL1 mRNA was drastically downregulated under hypoxic conditions. Mechanistically, suppression of HIF-1α by siRNA upregulated the METTL1 promoter activity. Furthermore, ChIP showed that HIF-1α could bind with an HRE in the promoter region of METTL1, indicating that METTL1 is a direct target of HIF-1α in CRC cells under hypoxic conditions. ConclusionsOur study revealed that the abundance of the m7G modification in tRNA was drastically reduced in CRC cells dependent on the HIF-1α-mediated inhibition of METTL1 transcription under hypoxic conditions.

Evaluation of antibacterial properties of nisin peptide expressed in carrots

Nisin, derived from Lactococcus lactis, is a well-known natural food preservative. In the present study, the gene of nisin was transformed to carrot by Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing neomycin phosphotransferase II (nptII) gene, peptide signal KDEL, and Kozak sequence. The integration of nisin and nptII transgenes into the plant genome was confirmed by polymerase chain reaction (PCR) and dot blot analysis. The gene expression was also performed by RT-PCR and Enzyme-Linked Immunosorbent Assay. The level of nisin expressed in one gram of transgenic plant ranged from 0.05 to 0.08 μg/ml. The stability of nisin varied in orange and peach juices depending on the temperature on the 70th day. The leaf protein extracted from the transgenic plant showed a significant preservative effect of nisin in peach and orange juice. A complete inhibition activity against Staphylococcus aureus and Escherichia coli in orange juice was observed within 24 h. After 24 h, log 1 and log 2 were obtained in a peach juice containing Staphylococcus aureus and Escherichia coli, respectively. Results of HPLC indicated that Chlorogenic and Chicoric acid compounds were increased in transgenic plants, but this increase was not significant. The study of determining the genetic stability of transgenic plants in comparison with non-transgenic plants showed high genetic stability between non-transgenic plants and transgenic plants. This study confirmed the significant inhibitory effect of nisin protein on gram-positive and gram-negative bacteria.

The inhibition of FTO attenuates the antifibrotic effect of leonurine in rat cardiac fibroblasts

BackgroundMyocardial fibrosis (MF) is a common pathological condition in cardiovascular diseases that often causes severe cardiac dysfunction. MF is characterized by changes in cardiomyocytes, cardiac fibroblasts (CFs), levels of collagen (Col) -1, -3, and overdeposition of the extracellular matrix. Our previous research showed that leonurine (LE) effectively inhibits collagen synthesis and differentiation of CFs, but the mechanism is not fully elucidated. Recent evidence indicates that fat mass and obesity-associated proteins (FTO) regulates the occurrence and development of MF. This study aimed to explore the role of FTO in the antifibrotic effects of LE. MethodsNeonatal rat CFs were isolated, and induced using angiotensin II (Ang II) to establish a cell model of MF. Cell viability, wound healing and transwell assays were used to detect cell activity and migration ability. The protein and mRNA levels of MF-related factors were measured following stimulation with Ang II and LE under normal conditions or after FTO knockdown. The RNA methylation level was measured by dot blot assay. ResultsThe results showed that LE (20, 40 μM) was not toxic to normal CFs. LE reduced the proliferation, migration and collagen synthesis of Ang II-induced CFs. Further investigation showed that FTO was downregulated by Ang II stimulation, whereas LE reversed this effect. FTO knockdown facilitated the migration of CFs, upregulated the protein levels of Col-3, α-SMA and Col-1 in Ang II and LE-stimulated CFs, and enhanced the fluorescence intensity of α-SMA. Furthermore, LE reduced N6-methyladenosine (m6A) RNA methylation, which was partially blocked by FTO knockdown. FTO knockdown also reduced the expression levels of p53 protein in Ang II and LE-stimulated CFs. ConclusionsOur findings suggest that the inhibition of FTO may attenuate the antifibrotic effect of LE in CFs, suggesting that FTO may serve as a key protein for anti-MF of LE.

A highly sensitive biotin-based probe for small RNA northern blot and its application in dissecting miRNA function in pepper.

Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope- and biotin- (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope-based probe is limited by strict environmental regulation and availability in many places in the world while biotin-based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase-based method for incorporation of a cluster of 33 biotin-labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P-labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low-abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus-based miRNA silencing in pepper, knocking down accumulation of Can-miR6027a and Can-miR6149L. Importantly, further analysis showed that knocking-down Can-miR6027a led to upregulation of a nucleotide binding-leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici.