Serine protease Rv2569c facilitates transmission of Mycobacterium tuberculosis via disrupting the epithelial barrier by cleaving E-cadherin.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

Novel Enzyme-Assisted Recycle Amplification Strategy for Tetracycline Detection Based on Oxidized Single-Walled Carbon Nanohorns.

In this study, oxidized single-walled carbon nanohorns (oxSWCNHs) were prepared using nitric acid oxidation and subsequently combined with 3'6-carboxyfluorescein through charge transfer to prepare fluorescent probes. These oxSWCNHs were used to quench fluorogen signals at short distances and dissociate ssDNA using cryonase enzymes. We established a method for rapidly detecting tetracycline (TC) in complex samples based on the amplification of cryonase enzyme signals. After optimizing the experimental conditions, our method showed a detection limit of 5.05 ng/mL, with good specificity. This method was used to determine the TC content in complex samples, yielding a recovery rate of 90.0-103.3%. This result validated the efficacy of our method in detecting TC content within complex samples.

Expression profiling of ALOG family genes during inflorescence development and abiotic stress responses in rice (Oryza sativa L.).

The ALOG (Arabidopsis LSH1 and Oryza G1) family proteins, namely, DUF640 domain-containing proteins, have been reported to function as transcription factors in various plants. However, the understanding of the response and function of ALOG family genes during reproductive development and under abiotic stress is still largely limited. In this study, we comprehensively analyzed the structural characteristics of ALOG family proteins and their expression profiles during inflorescence development and under abiotic stress in rice. The results showed that OsG1/OsG1L1/2/3/4/5/6/7/8/9 all had four conserved helical structures and an inserted Zinc-Ribbon (ZnR), the other four proteins OsG1L10/11/12/13 lacked complete Helix-1 and Helix-2. In the ALOG gene promoters, there were abundant cis-acting elements, including ABA, MeJA, and drought-responsive elements. Most ALOG genes show a decrease in expression levels within 24 h under ABA and drought treatments, while OsG1L2 expression levels show an upregulated trend under ABA and drought treatments. The expression analysis at different stages of inflorescence development indicated that OsG1L1/2/3/8/11 were mainly expressed in the P1 stage; in the P4 stage, OsG1/OsG1L4/5/9/12 had a higher expression level. These results lay a good foundation for further studying the expression of rice ALOG family genes under abiotic stresses, and provide important experimental support for their functional research.

Anti-Inflammatory and α-Glucosidase Inhibitory Triterpenoid with Diverse Carbon Skeletons from the Fruits of Rosa roxburghii.

The fruits of Rosa roxburghii Tratt. are edible nutritional food with high medicinal value and have been traditionally used as Chinese folk medicine for a long time. In this study, 26 triterpenoids including four new pentacyclic triterpenoids, roxbuterpenes A-D (1, 4, 5, and 24), along with 22 known analogues (2, 3, 6-23, 25, and 26), were isolated from the fruits of R. roxburghii. Their chemical structures were determined on the basis of extensive spectroscopic analyses (including IR, HRESIMS and NMR spectroscopy). The absolute configuration of roxbuterpene A (1) was determined by an X-ray crystallographic analysis. This is the first report of the crystal structure of 5/6/6/6/6-fused system pentacyclic triterpenoid. Notably, roxbuterpenes A and B (1 and 4) possessed the A-ring contracted triterpenoid and nortriterpenoid skeletons with a rare 5/6/6/6/6-fused system, respectively. Compounds 1-7, 11, 13-15, 18-20, 24, and 25 exhibited moderate or potent inhibitory activities against α-glucosidase. Compounds 2, 4, 6, 11, and 14 showed strong activities against α-glucosidase with IC50 values of 8.4 ± 1.6, 7.3 ± 2.2, 13.6 ± 1.4, 0.9 ± 0.4, and 12.5 ± 2.4 µM, respectively (positive control acarbose, 10.1 ± 0.8 µM). Compounds 13, 14, and 16 moderately inhibited the release of NO (nitric oxide) with IC50 values ranging from 25.1 ± 2.0 to 51.4 ± 3.1 µM. Furthermore, the expressions of TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6) were detected by ELISA (enzyme-linked immunosorbent assay), and compounds 13, 14, and 16 exhibited moderate inhibitory effects on TNF-α and IL-6 release in a dose-dependent manner ranging from 12.5 to 50 µM.

Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept: a quantitative analysis based on 4D label-free technique.

AIM: To identify different metabolites, proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy (PDR) and resistance to anti-vascular endothelial growth factor (VEGF) drugs, and to provide biomarkers for the diagnosis and treatment of PDR. METHODS: Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analyses based on 4D label-free technology. Statistically differentially expressed proteins (DEPs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representation and protein interactions were analyzed. RESULTS: A total of 12 samples were analyzed. The proteomics results showed that a total of 58 proteins were identified as DEPs, of which 47 proteins were up-regulated and 11 proteins were down-regulated. We found that C1q and tumor necrosis factor related protein 5 (C1QTNF5), Clusterin (CLU), tissue inhibitor of metal protease 1 (TIMP1) and signal regulatory protein alpha (SIRPα) can all be specifically regulated after aflibercept treatment. GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR. In addition, protein-protein interaction (PPI) network evaluation revealed that TIMP1 plays a central role in neural regulation. In addition, CD47/SIRPα may become a key target to resolve anti-VEGF drug resistance in PDR. CONCLUSION: Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR. Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept, among which C1QTNF5, CLU, TIMP1 and SIRPα may become targets for future treatment of PDR and resolution of anti-VEGF resistance.

Characterization of FBA genes in potato (Solanum tuberosum L.) and expression patterns in response to light spectrum and abiotic stress.

Fructose-1, 6-bisphosphate aldolase (FBA) plays vital roles in plant growth, development, and response to abiotic stress. However, genome-wide identification and structural characterization of the potato (Solanum tuberosum L.) FBA gene family has not been systematically analyzed. In this study, we identified nine StFBA gene members in potato, with six StFBA genes localized in the chloroplast and three in the cytoplasm. The analysis of gene structures, protein structures, and phylogenetic relationships indicated that StFBA genes were divided into Class I and II, which exhibited significant differences in structure and function. Synteny analysis revealed that segmental duplication events promoted the expansion of the StFBA gene family. Promoter analysis showed that most StFBA genes contained cis-regulatory elements associated with light and stress responses. Expression analysis showed that StFBA3, StFBA8, and StFBA9 showing significantly higher expression levels in leaf, stolon, and tuber under blue light, indicating that these genes may improve photosynthesis and play an important function in regulating the induction and expansion of microtubers. Expression levels of the StFBA genes were influenced by drought and salt stress, indicating that they played important roles in abiotic stress. This work offers a theoretical foundation for in-depth understanding of the evolution and function of StFBA genes, as well as providing the basis for the genetic improvement of potatoes.

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

The ldp1 Mutation Affects the Expression of Auxin-Related Genes and Enhances SAM Size in Rice.

Panicle type is one of the important factors affecting rice (Oryza sativa L.) yield, and the identification of regulatory genes in panicle development can provide significant insights into the molecular network involved. This study identified a large and dense panicle 1 (ldp1) mutant produced from the Wuyunjing 7 (WYJ7) genotype, which displayed significant relative increases in panicle length, number of primary and secondary branches, number of grains per panicle, grain width, and grain yield per plant. Scanning electron microscopy results showed that the shoot apical meristem (SAM) of ldp1 was relatively larger at the bract stage (BM), with a significantly increased number of primary (PBM) and secondary branch (SBM) meristematic centers, indicating that the ldp1 mutation affects early stages in SAM development Comparative RNA-Seq analysis of meristem tissues from WYJ7 and ldp1 at the BM, PBM, and SBM developmental stages indicated that the number of differentially expressed genes (DEGs) were highest (1407) during the BM stage. Weighted gene coexpression network analysis (WGCNA) revealed that genes in one module (turquoise) are associated with the ldp1 phenotype and highly expressed during the BM stage, suggesting their roles in the identity transition and branch differentiation stages of rice inflorescences. Hub genes involved in auxin synthesis and transport pathways, such as OsAUX1, OsAUX4, and OsSAUR25, were identified. Moreover, GO and KEGG analysis of the DEGs in the turquoise module and the 1407 DEGs in the BM stage revealed that a majority of genes involved in tryptophan metabolism and auxin signaling pathway were differentially expressed between WYJ and ldp1. The genetic analysis indicated that the ldp1 phenotype is controlled by a recessive monogene (LDP1), which was mapped to a region between 16.9 and 18.1 Mb on chromosome seven. This study suggests that the ldp1 mutation may affect the expression of key genes in auxin synthesis and signal transduction, enhance the size of SAM, and thus affect panicle development. This study provides insights into the molecular regulatory network underlying rice panicle morphogenesis and lays an important foundation for further understanding the function and molecular mechanism of LDP1 during panicle development.

High expression of SULF1 is associated with adverse prognosis in breast cancer brain metastasis.

BACKGROUND: Breast cancer is the most common cancer in women, and in advanced stages, it often metastasizes to the brain. However, research on the biological mechanisms of breast cancer brain metastasis and potential therapeutic targets are limited. METHODS: Differential gene expression analysis (DEGs) for the datasets GSE43837 and GSE125989 from the GEO database was performed using online analysis tools such as GEO2R and Sangerbox. Further investigation related to SULF1 was conducted using online databases such as Kaplan-Meier Plotter and cBioPortal. Thus, expression levels, variations, associations with HER2, biological processes, and pathways involving SULF1 could be analyzed using UALCAN, cBioPortal, GEPIA2, and LinkedOmics databases. Moreover, the sensitivity of SULF1 to existing drugs was explored using drug databases such as RNAactDrug and CADSP. RESULTS: High expression of SULF1 was associated with poor prognosis in advanced breast cancer brain metastasis and was positively correlated with the expression of HER2. In the metastatic breast cancer population, SULF1 ranked top among the 16 DEGs with the highest mutation rate, reaching 11%, primarily due to amplification. KEGG and GSEA analyses revealed that the genes co-expressed with SULF1 were positively enriched in the 'ECM-receptor interaction' gene set and negatively enriched in the 'Ribosome' gene set. Currently, docetaxel and vinorelbine can act as treatment options if the expression of SULF1 is high. CONCLUSIONS: This study, through bioinformatics analysis, unveiled SULF1 as a potential target for treating breast cancer brain metastasis (BM).

Energy band modulation of Li2O-rGO core-shell as cathode sacrificial additive enables capacity enhancement of hard carbon anode in Li-ion batteries.

Lithium oxides (Li2O) possess a considerable theoretical capacity, rendering them highly promising as cathodic pre-lithiation additives. However, its decomposition voltage exceeds the charging cut-off voltage of most cathode materials, hindering its direct use as a cathode sacrificial additive. Herein, we design a facile and safe method to reduce the decomposition energy of Li2O at room temperature to offset the irreversible capacity loss by using a core-shell structured Li2O-reduced graphene oxide (rGO)-polyethylene glycol (PEG) composite (denoted as Li2O-rGO-PEG). The graphene oxide (GO) was heat-treated to remove oxygen functional groups to synthesize rGO, and then reacted with Li2O to form a Li2O-rGO composite. According to the DFT calculations, the density of states at the Fermi level of Li2O-rGO becomes continuous and features a metallic nature, which significantly improves the electrical conductivity of Li2O and facilitates electron conduction that modify the delithiation potential of Li2O. PEG was used to enhance the cohesive force between rGO and Li2O and to protect Li2O from atmospheric contamination. Moreover, in order to demonstrate the excellent pre-lithiation ability of Li2O-rGO-PEG composite, hard carbon (HC) with low initial coulombic efficiency (ICE) was used as the anode. In the application of LFP (Li2O)/HC full cell, Li2O was decomposed to Li+ to effectively improve the initial charge capacity from 149.7 to 200 mAh/g and discharge capacity from 104.2 to 147.5 mAh/g, which are 33.6 % and 41.6 % higher than those of the pristine LFP/HC full cell, respectively. The cathode pre-lithiation method proposed in this work is simple and environmentally friendly. The successful utilization of Li2O as a pre-lithiation additive effectively addressed the issue of low initial coulombic efficiency of the HC, indicating excellent prospects for practical applications.

A Sequentially Activated Probe for Imaging of Superoxide Anion and Peroxynitrite in PC12 Cells under Oxidative Stress.

Superoxide anion (O2·-) and peroxynitrite (ONOO-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O2·- and ONOO- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O2·- and ONOO-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O2·- and ONOO- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O2·- and ONOO- in PC12 cells under oxidative stress.

Identification of PDGFA as a Neutrophil-related Biomarker Linked to the Advancement of Diabetic Retinopathy through Integrated Bioinformatics Analysis.

BACKGROUND: The dysregulation of the innate immune system plays a crucial role in the development of Diabetic Retinopathy (DR). To gain an insight into the underlying mechanism of DR, it is essential to identify specific biomarkers associated with immune cell infiltration. METHODS: In this study, we retrieved the GSE94019 and GSE60436 datasets from the Gene Expression Omnibus (GEO) database. By utilizing CIBERSORT, MCPcounter, and xCell algorithms, we conducted a comprehensive analysis of the immune cell infiltration landscape in DR. The limma package was employed to identify Differentially Expressed Necroptosis-related Genes (DENRGs). Subsequently, enrichment analysis was performed to investigate the potential functions of the DENRGs. To identify the core DENRGs, the CytoHubba plug-in in Cytoscape software was utilized. The expression levels of these core DENRGs were verified in an independent dataset. RESULTS: Our analysis identified 213 DENRGs, and among them, Platelet-derived Growth Factor subunit A (PDGFA) was identified as a core DENRG. Notably, the expression of PDGFA was found to be upregulated in DR, and this finding was further validated in the GSE102485 dataset. Additionally, the results of GSVA and GSEA revealed that in the high PDGFA group, there was activation of pathways related to inflammation and the immune system. Moreover, analysis of immune infiltration demonstrated a significant association between PDGFA gene expression and the infiltration levels of specific immune cells, including basophils, macrophages M1, macrophages, neutrophils, monocytes, NK cells, and B cells. CONCLUSION: The involvement of neutrophils in the development and progression of DR is suggested. PDGFA has emerged as a potential marker and is linked to the infiltration of immune cells in DR. These findings shed new light on the underlying mechanisms of DR.

The role of HLA-DR on plasmacytoid dendritic cells in mediating the effects of Butyrivibrio gut microbiota on Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is viewed as a progressively deteriorating neurodegenerative disorder, the exact etiology of which remains not fully deciphered to this date. The gut microbiota could play a crucial role in PD development by modulating the human immune system. OBJECTIVE: This study aims to explore the relationship between gut microbiota and PD, focusing on how immune characteristics may both directly and indirectly influence their interaction. METHODS: Utilizing cumulative data from genome-wide association studies (GWAS), our research conducted a two-sample Mendelian randomization (MR) analysis to clarify the association between the gut microbiome and PD. Additionally, by employing a two-step MR approach, we assessed the impact of gut microbiota on PD development via immune characteristics and quantified HLA-DR mediation effect on plasmacytoid dendritic cells (pDCs). RESULTS: We discovered significant associations between PD and microbiota, comprising one class, one order, two families, and two genera. Furthermore, we explored the extent to which HLA-DR on pDCs mediates the effect of Butyrivibrio gut microbiota on PD. CONCLUSION: Our study emphasizes the complex interactions between the gut microbiota, immune characteristics, and PD. The relationships and intermediary roles identified in our research provide important insights for developing potential therapies that target the gut microbiome to alleviate symptoms in PD patients.

DDX20 positively regulates the interferon pathway to inhibit viral infection.

The DEAD-box (DDX) family comprises RNA helicases characterized by the conserved sequence D(Asp)-E(Glu)-A(Ala)-D(Asp), participating in various RNA metabolism processes. Some DDX family members have been identified for their crucial roles in viral infections. In this study, RNAi library screening of the DDX family unveiled the antiviral activity of DDX20. Knockdown of DDX20 enhanced the replication of viruses such as vesicular stomatitis virus (VSV) and herpes simplex virus type I (HSV-1), while overexpression of DDX20 significantly diminished the replication level of these viruses. Mechanistically, DDX20 elevated the phosphorylation level of IRF3 induced by external stimuli by facilitating the interaction between TBK1 and IRF3, thereby promoting the expression of IFN-ß. The increased IFN-ß production, in turn, upregulated the expression of interferon-stimulated genes (ISGs), including Cig5 and IFIT1, thereby exerting the antiviral effect. Finally, in an in vivo infection study, Ddx20 gene-deficient mice exhibited increased susceptibility to viral infection. This study provides new evidence that DDX20 positively modulates the interferon pathway and restricts viral infection.

Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1.

Tumor-resident microbiota in breast cancer promote cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increases the chemosensitivity of breast cancer by impairing BCSCs.

Identification and Characterization of the BZR Transcription Factor Genes Family in Potato (Solanum tuberosum L.) and Their Expression Profiles in Response to Abiotic Stresses.

Brassinazole resistant (BZR) genes act downstream of the brassinosteroid signaling pathway regulating plant growth and development and participating in plant stress responses. However, the BZR gene family has not systematically been characterized in potato. We identified eight BZR genes in Solanum tuberosum, which were distributed among seven chromosomes unequally and were classified into three subgroups. Potato and tomato BZR proteins were shown to be closely related with high levels of similarity. The BZR gene family members in each subgroup contained similar conserved motifs. StBZR genes exhibited tissue-specific expression patterns, suggesting their functional differentiation during evolution. StBZR4, StBZR7, and StBZR8 were highly expressed under white light in microtubers. StBZR1 showed a progressive up-regulation from 0 to 6 h and a progressive down-regulation from 6 to 24 h after drought and salt stress. StBZR1, StBZR2, StBZR4, StBZR5, StBZR6, StBZR7 and StBZR8 were significantly induced from 0 to 3 h under BR treatment. This implied StBZR genes are involved in phytohormone and stress response signaling pathways. Our results provide a theoretical basis for understanding the functional mechanisms of BZR genes in potato.

Analysis of the browning reaction in a sorbitol/glycine model: Formation and degradation of precursors, glucose and α-dicarbonyl compounds during heating.

Browning can occur in the matrices of alditol and amino acids due to heating or long-term storage, which poses challenges in achieving the desired appearance stability. To investigate the mechanism of browning in the sorbitol-glycine system, we evaluated the evolution of typical intermediates, including glucose and α-dicarbonyl compounds (α-DCs), during heating at 100 ˚C. The browning index and intermediate products of the sorbitol-glycine system increased more rapidly compared to those of the sorbitol system. After heating for 10 h, the browning index of the sorbitol-glycine system was eight times higher than that of sorbitol alone. In the presence of glycine, sorbitol underwent continuous conversion into glucose. After 10 h of heating, the concentration of glucose in the sorbitol-glycine system reanched 726.6 mg/L, which was approximately 63 times higher than that in the sorbitol system. Mass spectrometry analysis revealed the presence of α-DCs such as 3-deoxyglucosone (3-DG), glyoxal (GO), methylglyoxal (MGO), 2,3-butanedione (2,3-BD), in the sorbitol-glycine system. These compounds were precursors of melanoidins, indicating the occurrence of the Maillard reaction and resulting in the browning of the system. Therefore, the browning process in the sorbitol-glycine system involved two stages of reactions: the conversion of sorbitol to glucose and the Maillard reaction between glucose and glycine.

Gemcitabine Inhibits the Progression of Pancreatic Cancer by Restraining the WTAP/MYC Chain in an m6A-Dependent Manner.

PURPOSE: Pancreatic cancer (PC) is a common malignant tumor of the digestive system, and its 5-year survival rate is only 4%. N6-methyladenosine (m6A) RNA methylation is the most common post-transcriptional modification and dynamically regulates cancer development, while its role in PC treatment remains unclear. MATERIALS AND METHODS: We treated PC cells with gemcitabine and quantified the overall m6A level with m6A methylation quantification. Real-time quantitative reverse transcription polymerase chain reaction and Western blot analyses were used to detect expression changes of m6A regulators. We verified the m6A modification on the target genes through m6A-immunoprecipitation (IP), and further in vivo experiments and immunofluorescence (IF) assays were applied to verify regulation of gemcitabine on Wilms' tumor 1-associated protein (WTAP) and MYC. RESULTS: Gemcitabine inhibited the proliferation and migration of PC cells and reduced the overall level of m6A modification. Additionally, the expression of the "writer" WTAP was significantly downregulated after gemcitabine treatment. We knocked down WTAP in cells and found target gene MYC expression was significantly downregulated, m6A-IP also confirmed the m6A modification on MYC. Our experiments showed that m6A-MYC may be recognized by the "reader" IGF2BP1. In vivo experiments revealed gemcitabine inhibited the tumorigenic ability of PC cells. IF analysis also showed that gemcitabine inhibited the expression of WTAP and MYC, which displayed a significant trend of co-expression. CONCLUSION: Our study confirmed that gemcitabine interferes with WTAP protein expression in PC, reduces m6A modification on MYC and RNA stability, thereby inhibiting the downstream pathway of MYC, and inhibits the progression of PC.

Bacteria-loaded biochar for the immobilization of cadmium in an alkaline-polluted soil.

The combination of biochar and bacteria is a promising strategy for the remediation of Cd-polluted soils. However, the synergistic mechanisms of biochar and bacteria for Cd immobilization remain unclear. In this study, the experiments were conducted to evaluate the effects of the combination of biochar and Pseudomonas sp. AN-B15, on Cd immobilization, soil enzyme activity, and soil microbiome. The results showed that biochar could directly reduce the motility of Cd through adsorption and formation of CdCO3 precipitates, thereby protecting bacteria from Cd toxicity in the solution. In addition, bacterial growth further induces the formation of CdCO3 and CdS and enhances Cd adsorption by bacterial cells, resulting in a higher Cd removal rate. Thus, bacterial inoculation significantly enhances Cd removal in the presence of biochar in the solution. Moreover, soil incubation experiments showed that bacteria-loaded biochar significantly reduced soil exchangeable Cd in comparison with other treatments by impacting soil microbiome. In particular, bacteria-loaded biochar increased the relative abundance of Bacillus, Lysobacter, and Pontibacter, causing an increase in pH, urease, and arylsulfatase, thereby passivating soil exchangeable Cd and improving soil environmental quality in the natural alkaline Cd-contaminated soil. Overall, this study provides a systematic understanding of the synergistic mechanisms of biochar and bacteria for Cd immobilization in soil and new insights into the selection of functional strain for the efficient remediation of the contaminated environments by bacterial biochar composite.