Dot Blot Research Articles

NSUN2 promotes lung adenocarcinoma progression through stabilizing PIK3R2 mRNA in an m5C-dependent manner.

It is well known that 5-methylcytosine (m5C) is involved in variety of crucial biological processes in cancers. However, its biological roles in lung adenocarcinoma (LAUD) remain to be determined. The LUAD samples were used to assess the clinical value of NOP2/Sun RNA Methyltransferase 2 (NSUN2). Dot blot was used to determine global m5C levels. ChIP and dual-luciferase assays were performed to investigate the MYC-associated zinc finger protein (MAZ)-binding sites in NSUN2 promoter. RNA-seq was used to explore the downstream molecular mechanisms of NSUN2. Dual luciferase reporter assay, m5C-RIP-qPCR, and mRNA stability assay were conducted to explore the effect of NSUN2-depletion on target genes. Cell viability, transwell, and xenograft mouse model were designed to demonstrate the characteristic of NSUN2 in promoting LUAD progression. The m5C methyltransferase NSUN2 was highly expressed and caused elevated m5C methylation in LUAD samples. Mechanistically,MAZ positively regulated the transcription of NSUN2 and was related to poor survival of LUAD patients. Silencing NSUN2 decreased the global m5C levels, suppressed proliferation, migration and invasion, and inhibited activation of PI3K-AKT signaling in A549 and SPAC-1 cells. Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2) was upregulated by NSUN2-mediated m5C methylation by enhancing its mRNA stabilization and activated the phosphorylation of the PI3K-AKT signaling. The present study explored the underlying mechanism and biological function of NSUN2-meditated m5C RNA methylation in LUAD. NSUN2 was discovered to facilitate the malignancy progression of LUAD through regulating m5C modifications to stabilize PIK3R2 activating the PI3K-AKT signaling, suggesting that NSUN2 could be a novel biomarker and promising therapeutic target for LUAD patients.

Serine protease inhibitor 3 (Serpin3) from Penaeus vannamei selectively interacts with Vibrio parahaemolyticus PirAvp.

Acute Hepatopancreatic Necrosis Disease (AHPND) represents a significant challenge in the field of shrimp aquaculture. This disease is primarily caused by Vibrio parahaemolyticus strains harbouring the pVA1 plasmid encoding the PirAvp and PirBvp toxins. To combat this epidemic and mitigate its devastating consequences, it is crucial to identify and characterize the receptors responsible for the binding of these pathogenic toxins. Our studied discovered that Penaeus vannamei's Serine protease inhibitor 3 (PvSerpin3) derived from shrimp hepatopancreatic tissues could bind to recombinant PirAvp , confirming its role as a novel PirAvp -binding protein (PA BP). Through comprehensive computational methods, we revealed two truncated PirAvp -binding proteins derived from PvSerpin3 called Serpin3(13) and Serpin3(22), which had higher affinity to PirAvp than the full-length PvSerpin3. The PA BP genes were amplified from a cDNA library that was reversed from total RNA extracted from shrimp, cloned and expressed in Escherichia coli. Three PA BP inclusion bodies were refolded to obtain the soluble form, and the recovery efficacy was found to be 100% for Serpin3 and Serpin3(13), while Serpin3(22) had a recovery efficacy of roundly 50%. Co-Immunoprecipitation (co-IP) and dot blot assays substantiated the interaction of these recombinant PA BPs with both recombinant PirAvp and VPAHPND (XN89)-producing natural toxins.

Laboratory Assessment of the Effects of AGA@4life Multidisciplinary Intervention on the Inflammatory Profile, MMPs, and TIMPs in a Geriatric Population.

In recent years, the world's aging population has increased, contributing to the development of age-related pathologies, which have been aggravated by physical inactivity and excessive fat intake. This study aimed to evaluate the effect of implementing a nutritional program (control group-CG) combined with exercise (intervention group-IG) on the inflammatory profile, MMPs, and TIMPs in a group of 34 elderly participants (IG, n = 18; CG, n = 16). Participants underwent a full multidisciplinary diagnostic evaluation (T0), with the gathering of clinical information and biochemical and hematological determinations being re-evaluated eight weeks later (T1). A diet manual was made, which provided a selection of different types of diets resulting from the nutritional needs of the different users at the center. The aerobic exercise consisted of two sessions per week with a total duration of 1 h. The laboratory evaluation was performed by slot blot. Statistical analysis included a paired sample t-test and Spearman's correlation coefficient. We observed that in the IG, there was a significant increase at T1 of TNF-α (p < 0.05) and MMP-2 (p < 0.05), without changes in IL-6 and MMP-9, showing that the intervention did not cause an exacerbated inflammatory response in exercised elderly people. The intervention program implemented showed potential to contribute to better active aging strategies, taking advantage of the known benefits of exercise without inducing a harmful inflammatory response in elderly participants.

Salvianolic Acid B Significantly Suppresses the Migration of Melanoma Cells via Direct Interaction with β-Actin.

Melanoma is the most aggressive and difficult to treat of all skin cancers. Despite advances in the treatment of melanoma, the prognosis for melanoma patients remains poor, and the recurrence rate remains high. There is substantial evidence that Chinese herbals effectively prevent and treat melanoma. The bioactive ingredient Salvianolic acid B (SAB) found in Salvia miltiorrhiza, a well-known Chinese herbal with various biological functions, exhibits inhibitory activity against various cancers. A375 and mouse B16 cell lines were used to evaluate the main targets and mechanisms of SAB in inhibiting melanoma migration. Online bioinformatics analysis, Western blotting, immunofluorescence, molecular fishing, dot blot, and molecular docking assays were carried out to clarify the potential molecular mechanism. We found that SAB prevents the migration and invasion of melanoma cells by inhibiting the epithelial-mesenchymal transition (EMT) process of melanoma cells. As well as interacting directly with the N-terminal domain of β-actin, SAB enhanced its compactness and stability, thereby inhibiting the migration of cells. Taken together, SAB could significantly suppress the migration of melanoma cells via direct binding with β-actin, suggesting that SAB could be a helpful supplement that may enhance chemotherapeutic outcomes and benefit melanoma patients.

Regional AT-8 reactive tau species correlate with intracellular Aβ levels in cases of low AD neuropathologic change

The amyloid cascade hypothesis states that Aβ aggregates induce pathological changes in tau, leading to neurofibrillary tangles (NFTs) and cell death. A caveat with this hypothesis is the spatio-temporal divide between plaques and NFTs. This has been addressed by the inclusion of soluble Aβ and tau species in the revised amyloid cascade hypothesis. Nevertheless, despite the potential for non-plaque Aβ to contribute to tau pathology, few studies have examined relative correlative strengths between total Aβ, plaque Aβ and intracellular Aβ with tau pathology within a single tissue cohort. Employing frozen and fixed frontal cortex grey and white matter tissue from non-AD controls (Con; n = 39) and Alzheimer’s disease (AD) cases (n = 21), biochemical and immunohistochemical (IHC) measures of Aβ and AT-8 phosphorylated tau were assessed. Biochemical native-state dot blots from crude tissue lysates demonstrated robust correlations between total Aβ and AT-8 tau, when considered as a combined cohort (Con and AD) and when as Con and AD cases, separately. In contrast, no associations between Aβ plaques and AT-8 were reported when using IHC measurements in either Con or AD cases. However, when intracellular Aβ was measured via the Aβ specific antibody MOAB-2, a correlative relationship with AT-8 tau was reported in non-AD controls but not in AD cases. Collectively the data suggests that accumulating intracellular Aβ may influence AT-8 pathology, early in AD-related neuropathological change. Despite the lower levels of phospho-tau and Aβ in controls, the robust correlative relationships observed suggest a physiological association of Aβ production and tau phosphorylation, which may be modified during disease. This study is supportive of a revised amyloid cascade hypothesis and demonstrates regional associative relationships between tau pathology and intracellular Aβ, but not extracellular Aβ plaques.

Design and development of multiepitope chimeric antigens by bioinformatic and bacterial based recombinant expression methods, with potential application for bovine tuberculosis serodiagnosis

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.

Investigating Protein Binding with the Isothermal Ligand-induced Resolubilization Assay.

Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand-induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate-linked moiety X motif 5 (NUDT5), poly [ADP-ribose] polymerase 1 (PARP1), and protein arginine N-methyltransferase 1 (PRMT1). Ligand-induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand-induced protein solubility were assessed by Coomassie blue staining for SDS-PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.

Detection of skin α-synuclein using RT-QuIC as a diagnostic biomarker for Parkinson’s disease in the Chinese population

BackgroundSeveral studies have indicated that skin holds promise as a potential sample for detecting pathological α-Syn and serving as a diagnostic biomarker for α-synucleinopathies. Despite reports in Chinese PD patients, comprehensive research on skin α-Syn detection using RT-QuIC is lacking.ObjectiveThis study aimed to evaluate the diagnostic performance of skin samples using RT-QuIC from PD patients in the Chinese population.MethodsPatients with sporadic PD and controls were included according to the British PD Association Brain Bank diagnostic criteria. The seeding activity of misfolded α-Syn in these skin samples was detected using the RT-QuIC assay after protein extraction. Biochemical and morphological analyses of RT-QuIC products were conducted by atomic force microscopy, transmission electron microscopy, Congo red staining, and dot blot analysis.Result30 patients clinically diagnosed with PD and 28 controls with non-α-synucleinopathies were included in this study. 28 of 30 PD patients demonstrated positive α-Syn seeding activity by RT-QuIC assay. In contrast, no α-Syn seeding activity was detected in the 28 control samples, with an overall sensitivity and specificity of 93.3% and 100%, respectively (P < 0.001). Biochemical characterization of the RT-QuIC product indicated fibrillary α-Syn species in PD-seeded reactions, while control samples failed in the conversion of recombinant α-Syn substrate.ConclusionThis study applied RT-QuIC technology to identify misfolded α-Syn seeding activity in skin samples from Chinese PD patients, demonstrating high specificity and sensitivity. Skin α-Syn RT-QuIC is expected to be a reliable approach for the diagnosis of PD.

Standardization of Semi-Quantitative Dot Blotting Assay-Application in the Diagnosis, Follow-Up, and Relapse of Paracoccidioidomycosis.

This study standardized a semi-quantitative dot blotting assay (DB) and a quantitative real-time polymerase chain reaction (qPCR) to detect specific antibodies for Paracoccidioides brasiliensis and its DNA in PCM patients. We evaluated 42 confirmed PCM patients upon admission using a serological double agar gel immunodiffusion test (DID), DB, and molecular tests (qPCR in total blood). The control groups included 42 healthy individuals and 37 patients with other infectious diseases. The serological progress during treatment was evaluated in eight patients, and there was a relapse diagnosis in ten patients using the Pb B.339 strain antigen. The cut-off points for the serological tests were determined by a receiver operator characteristic curve. The DB and DID tests showed similar accuracy, but the DB identified lower antibody concentrations. Cross-reactions were absent in the DB assay. In the relapse diagnoses, DB exhibited much higher sensitivity (90%) than DID (30%). A DB assay is easier and faster than a DID test to be performed; DB and DID tests show the same accuracy, while blood qPCR is not recommended in the diagnosis at the time of admission; cross-reactions were not observed with other systemic diseases; DB and DID tests are useful for treatment monitoring PCM patients; and a DB assay is the choice for diagnosing relapse. These findings support the introduction of semi-quantitative DB assays in clinical laboratories.

METTL14-mediated m6A epitranscriptomic modification contributes to chemotherapy-induced neuropathic pain by stabilizing GluN2A expression via IGF2BP2.

Epigenetics is a biological process that modifies and regulates gene expression, affects neuronal function, and contributes to pain. However, the mechanism by which epigenetics facilitates and maintains chronic pain is poorly understood. We aimed to determine whether N6-methyladenosine (m6A) specifically modified by methyltransferase-like 14 (METTL14) alters neuronal activity and governs pain by sensitizing the GluN2A subunit of the N-methyl-d-aspartate receptor (NMDAR) in the dorsal root ganglion (DRG) neurons in a model of chemotherapy-induced neuropathic pain (CINP). Using dot blotting, immunofluorescence, gain/loss-of-function, and behavioral assays, we found that m6A levels were upregulated in L4-L6 DRG neurons in CINP in a DBP/METTL14-dependent manner, which was also confirmed in human DRGs. Blocking METTL14 reduced m6A methylation and attenuated pain hypersensitivity. Mechanistically, METTL14-mediated m6A modification facilitated the synaptic plasticity of DRG neurons by enhancing the GluN2A subunit of NMDAR, and inhibiting METTL14 blocked this effect. In contrast, overexpression of METTL14 upregulated m6A modifications, enhanced presynaptic NMDAR activity in DRG neurons, and facilitated pain sensation. Our findings reveal a previously unrecognized mechanism of METTL14-mediated m6A modification in DRG neurons to maintain neuropathic pain. Targeting these molecules may provide a new strategy for pain treatment.

Linc-smad7 is involved in the regulation of lipid synthesis in mouse mammary epithelial cells

Long intergenic non-coding RNA(lincRNA) is transcribed from the intermediate regions of coding genes and plays a pivotal role in the regulation of lipid synthesis. N6-methyladenosine (m6A) modification is widely prevalent in eukaryotic mRNAs and serves as a regulatory factor in diverse biological processes. This study aims to delineate the mechanism by which Linc-smad7 mediates m6A methylation to regulate milk fat synthesis. Tissue expression analysis in this study revealed a high expression of Linc-smad7 in breast tissue during pregnancy. Cell proliferation assays, including CCK8 and EdU assays, demonstrated that Linc-smad7 had no significant impact on the proliferation of mammary epithelial cells. However, during mammary epithelial cell differentiation, the overexpression of Linc-smad7 led to reduced lipid formation, whereas interference with Linc-smad7 promoted lipogenesis. Mechanistically, Linc-smad7 was found to modulate RNA m6A levels, as evidenced by dot blot assays and methylated RNA immunoprecipitation sequencing (MeRIP-Seq). Subsequent validation through RT-qPCR corroborated these findings, aligning with the m6A sequencing outcomes. Furthermore, co-transfection experiments elucidated that Linc-smad7 regulates lipid synthesis in mammary epithelial cells by influencing the expression of METTL14. In summary, these findings underscore the regulatory role of Linc-smad7 in controlling METTL14 gene expression, thereby mediating m6A modifications to regulate lipid synthesis in mammary epithelial cells.

Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9

BackgroundMucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917.ResultsThe HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) – rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/108 cfu.ConclusionsThe CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

LRPPRC promotes glycolysis by stabilising LDHA mRNA and its knockdown plus glutamine inhibitor induces synthetic lethality via m6 A modification in triple-negative breast cancer.

Targeted therapy for triple-negative breast cancer (TNBC) remains a challenge. N6-methyladenosine (m6 A) is the most abundant internal mRNA modification in eukaryotes, and it regulates the homeostasis and function of modified RNA transcripts in cancer. However, the role of leucine-rich pentatricopeptide repeat containing protein (LRPPRC) as an m6 A reader in TNBC remains poorly understood. Western blotting, reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry were used to investigate LRPPRC expression levels. Dot blotting and colorimetric enzyme linked immunosorbent assay (ELISA) were employed to detect m6 A levels. In vitro functional assays and in vivo xenograft mouse model were utilised to examine the role of LRPPRC in TNBC progression. Liquid chromatography-mass spectrometry/mass spectrometry and Seahorse assays were conducted to verify the effect of LRPPRC on glycolysis. MeRIP-sequencing, RNA-sequencing, MeRIP assays, RNA immunoprecipitation assays, RNA pull-down assays and RNA stability assays were used to identify the target genes of LRPPRC. Patient-derived xenografts and organoids were employed to substantiate the synthetic lethality induced by LRPPRC knockdown plus glutaminase inhibition. The expressions of LRPPRC and m6 A RNA were elevated in TNBC, and the m6 A modification site could be recognised by LRPPRC. LRPPRC promoted the proliferation, metastasis and glycolysis of TNBC cells both in vivo and in vitro. We identified lactate dehydrogenase A (LDHA) as a novel direct target of LRPPRC, which recognised the m6 A site of LDHA mRNA and enhanced the stability of LDHA mRNA to promote glycolysis. Furthermore, while LRPPRC knockdown reduced glycolysis, glutaminolysis was enhanced. Moreover, the effect of LRPPRC on WD40 repeat domain-containing protein 76 (WDR76) mRNA stability was impaired in an m6 A-dependent manner. Then, LRPPRC knockdown plus a glutaminase inhibition led to synthetic lethality. Our study demonstrated that LRPPRC promoted TNBC progression by regulating metabolic reprogramming via m6 A modification. These characteristics shed light on the novel combination targeted therapy strategies to combat TNBC.

Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboardtitrationtest was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

Establishment and application of a reverse dot blot assay for 13 mutations of hearing-loss genes in primary hospitals in China.

Hearing loss is a common sensorineural dysfunction with a high incidence in China. Although genetic factors are important causes of hearing loss, hearing-related gene detection has not been widely adopted in China. Establishing a rapid and efficient method to simultaneously detect hotspot hearing loss gene mutations. A reverse dot blot assay combined with a flow-through hybridization technique was developed for the simultaneous detection of 13 hotspot mutations of 4 hearing loss-related genes including GJB2, GJB3, SLC26A4, and the mitochondrial gene MT-RNR1. This method involved PCR amplification systems and a hybridization platform. The technique can detect 13 hotspot mutations of 4 hearing loss-related genes. And a total of 213 blood samples were used to evaluate the availability of this method. Our reverse dot blot assay was a simple, rapid, accurate, and cost-effective method to identify hotspot mutations of 4 hearing loss-related genes in a Chinese population.

N6-methyloxyadenine-mediated detoxification and ferroptosis confer a trade-off between multi-fungicide resistance and fitness.

Multi-fungicide resistance (MFR) is a serious environmental problem, which results in the excessive use of fungicides. Fitness penalty, as a common phenomenon in MFR, can partially counteract the issue of resistance due to the weakened vigor of MFR pathogens. Their underlying mechanism and relationship remain unexplained. By Oxford Nanopore Technologies sequencing and dot blot, we found that N6-methyloxyadenine (6mA) modification, the dominate epigenetic marker in Phytophthora capsici, was significantly altered after MFR emerged. Among the differently methylated genes, PcGSTZ1 could efficiently detoxify SYP-14288, a novel uncoupler, through complexing the fungicide with glutathione and induce MFR. Interestingly, PcGSTZ1 overexpression was induced by elevated 6mA levels and chromatin accessibility to its genomic loci. Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in P. capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici. In conclusion, the findings provide new insights into the biological role of 6mA as well as the mechanisms underlying the trade-off between MFR and fitness. These could also benefit disease control through the blockade of the epigenetic axis to resensitize resistant isolates.IMPORTANCEN6-methyloxyadenine (6mA) modification on DNA is correlated with tolerance under different stress in prokaryotes. However, the role of 6mA in eukaryotes remains poorly understood. Our current study reveals that DNA adenine methyltransferase 1 (DAMT1)-mediated 6mA modification at the upstream region of GST zeta 1 (GSTZ1) is elevated in the resistant strain. This elevation promotes the detoxification uncoupler and induces multifungicide resistance (MFR). Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in Phytophthora capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici. Overall, our findings uncover an innovative mechanism underlying 6mA modification in regulating PcGSTZ1 transcription and the ferroptosis pathway in P. capsici.

The Characterization and Cytotoxic Evaluation of Chondrosia reniformis Collagen Isolated from Different Body Parts (Ectosome and Choanosome) Envisaging the Development of Biomaterials.

Chondrosia reniformis is a collagen-rich marine sponge that is considered a sustainable and viable option for producing an alternative to mammalian-origin collagens. However, there is a lack of knowledge regarding the properties of collagen isolated from different sponge parts, namely the outer region, or cortex, (ectosome) and the inner region (choanosome), and how it affects the development of biomaterials. In this study, a brief histological analysis focusing on C. reniformis collagen spatial distribution and a comprehensive comparative analysis between collagen isolated from ectosome and choanosome are presented. The isolated collagen characterization was based on isolation yield, Fourier-transformed infrared spectroscopy (FTIR), circular dichroism (CD), SDS-PAGE, dot blot, and amino acid composition, as well as their cytocompatibility envisaging the development of future biomedical applications. An isolation yield of approximately 20% was similar for both sponge parts, as well as the FTIR, CD, and SDS-PAGE profiles, which demonstrated that both isolated collagens presented a high purity degree and preserved their triple helix and fibrillar conformation. Ectosome collagen had a higher OHpro content and possessed collagen type I and IV, while the choanosome was predominately constituted by collagen type IV. In vitro cytotoxicity assays using the L929 fibroblast cell line displayed a significant cytotoxic effect of choanosome collagen at 2 mg/mL, while ectosome collagen enhanced cell metabolism and proliferation, thus indicating the latter as being more suitable for the development of biomaterials. This research represents a unique comparative study of C. reniformis body parts, serving as a support for further establishing this marine sponge as a promising alternative collagen source for the future development of biomedical applications.

P054 R. intestinalis improves inflammatory bowel disease by preventing gut uric acid absorption through inhibiting METTL3 mediated m6A modification of SLC2A9

Abstract Background Inflammatory bowel disease (IBD) is a chronic inflammatory disease with persistent, recurrent, unsatisfactory response, and a poor prognosis. The incidence of Chinese IBD has increased rapidly, and Westernized diets such as purine-rich foods play an important role in its rapid growth. The alteration of purine-urate metabolism by microbiota may contribute to the development of IBD. Methods In this study, metagenomic and metabolomic analyses were performed on fecal samples from 30 untreated IBD patients and 30 healthy controls. R. intestinalis was used to observe the effect on serum uric acid levels in mice, respectively. Body weight, colon length, disease activity score, serum uric acid levels, and electron microscopy were all assessed. The RNAseq analysis of intestinal tissue from DSS-induced colitis mice intervened with R. intestinalis and co-cultured with R. intestinalis and the normal intestinal epithelial cell line NCM460, combined with m6A antibody dot blot assay, RM2Target database, and MeRIP sequencing data analysis, provided evidence that SLC2A9 was modified by METTL3, and SRAMP database predicted the potential for SLC2A9 mRNA to be modified by m6A after R. intestinalis intervention. Results Multi-omics analysis revealed that the abundance of R. intestinalis was significantly decreased in untreated IBD patients, and metabolomics revealed a correlation between high uric acid levels and disease severity, while the multi-omics integrated analysis suggested a significant negative correlation between R. intestinalis and uric acid. The research team confirmed that R. intestinalis can lower blood uric acid levels in mice and found that R. intestinalis intervention can alleviate the intestinal mucosal inflammation exacerbated by uric acid treatment in a DSS-induced mouse colitis model. Butyric acid has been reported to regulate m6A, and dot blot assays showed that R. intestinalis can reduce the m6A modification level of intestinal epithelial cells, METTL3, and SLC2A9. RM2Target database identified the protein-RNA interaction of the m6A regulator (WER) for SLC2A9 mRNA, and MeRIP sequencing data further indicated that METTL3 can bind to SLC2A9 mRNA through m6A modification. Meanwhile, based on sequence prediction, the research team identified 13 potential m6A modification sites on SLC2A9 mRNA, of which 4 sites had extremely high credibility . The highest credibility site had a score of 0.694, with a modification site sequence of GGACU located on an adenosine nucleotide. Conclusion R. intestinalis inhibit the METTL3 mediated m6A modification of intestinal epithelial SLC2A9, reducing the expression of SLC2A9, preventing the epithelial absorption of uric acid, and relieving IBD-related colitis

Evaluation of in situ and ex situ forage germplasm collections reveals the first occurrence and seed-transmission of alfalfa mosaic virus and southern bean mosaic virus in Urochloa species

Grasses of the genus Urochloa (Urochloa species syn. Brachiaria species) are among the most important tropical forage grasses of African origin. The International Livestock Research Institute (ILRI) Forage Genebank maintains 671 Urochloa grass accessions, belonging to 28 species, of which 261 (39%) accessions are Urochloa brizantha (A.Rich.) R.D.Webster. The susceptibility of Urochloa grasses to disease and insect pests is a key challenge to the sustainable production of these grasses in Africa. In this study, using a dot blot assay and RT-PCR, we report for the first time the association of Alfalfa Mosaic Virus (AMV) and Southern Bean Mosaic Virus (SBMV) with 92% of the tested accessions conserved in situ and ex situ in the ILRI forage genebank. In addition, virus transmission from seed to plant and from legume to grass was confirmed through the bioassay test. The detection of these two viruses in Urochloa species presents a new challenge to germplasm conservation, distribution, and their sustainable production in the region, and demands immediate attention towards developing an effective method of cleaning viruses from the conserved germplasm. Furthermore, there is an urgent need to assess the economic importance of diseases in the grasses, and for the selection of germplasm that shows resistance to diseases.

Comprehensive analysis of m6A modification in lipopolysaccharide-induced acute lung injury in mice

BackgroundN6-Methyladenosine (m6A) methylation is the most prevalent post-transcriptional modification in mRNA, and plays significant roles in various diseases. Nevertheless, the precise functions of m6A modification in the formation of ALI remain unclear. In this study we explore the transcriptome distribution of m6A methylation and its probable roles of in ALI.MethodsLipopolysaccharide (LPS) was utilized to establish an ALI mouse model. Real-time qPCR, Western blotting and m6A dot blot were utilized to assess m6A methylation level and the expression of m6A methylation enzymes. MeRIP-Seq and RNA-seq were utilized to explore differential m6A modifications and differentially expressed genes in ALI mice. The hub genes and enriched pathways were assessed by Real-time qPCR and Western blotting.ResultsOur findings showed that overall m6A methylation level was increased in ALI mice lung tissues, accompanied by lower levels of METTL3 and FTO. Notably, the protein expression of these methylases were different in various cells. There were 772 differently expressed m6A peaks in ALI as compared to the control group, with 316 being hypermethylated and 456 being hypomethylated. GO and KEGG analyses demonstrated these differentially methylated genes were associated with the calcium signaling pathway and cAMP signaling pathway. Furthermore, we identified 50 genes with distinct m6A peaks and mRNA expressions by combined analysis of MeRIP-Seq and RNA-Seq. KEGG analysis also demonstrated that these overlapped genes were closely associated with the calcium signaling pathway, cGMP-PKG signaling pathway, etc. Besides, Western blotting results demonstrated that the protein expression of Fibronectin leucine-rich transmembrane protein 3 (Flrt3) as well as the calcium signaling pathway and cGMP-PKG signaling pathway, increased significantly after ALI.Conclusionsm6A modification was paramount in the pathogenesis of ALI, and provided a foundation for the further investigation in the prevention and treatment of ALI.