Determining nitrogen topdressing rates in accordance with actual seedling density and crop nitrogen status in direct-seeded rice.

BACKGROUND: Adjusting nitrogen (N) input based on actual seedling density (ASD) and plant N status is a practical approach for improving the yield stability of direct-seeded rice. However, the adjustment of topdressing N rates has been empirical in the past. This study aimed to establish a quantitative approach for determining N topdressing rates during tillering (Ntil ) and panicle development (NPI ) based on ASD and crop N status in direct-seeded rice. Field experiments were conducted involving 12 treatments, consisting of four Ntil and three seeding rates in 2017, and eight treatments combining seeding rate, Ntil , and NPI in 2020. RESULTS: Linear regression analysis revealed that the tiller number at panicle initiation (TILPI ) was predominantly influenced by ASD and Ntil . The determination coefficients (R2 ) of the regression models ranged from 0.887 to 0.936 across the four-season experiments. The results indicated that Ntil could be determined accurately using ASD and the target maximum tiller number. Similarly, grain yield was influenced significantly by the N uptake at panicle initiation (NUPPI ) and NPI , with R2 of 0.814 and 0.783 in the early and late seasons of 2020, respectively. This suggested that NPI could be calculated based on NUPPI and the target grain yield. CONCLUSION: The findings offer a quantitative method for establishing N topdressing rates for tillering and panicle development, relying on the monitoring of actual seedling density and plant N status in direct-seeded rice production. © 2023 Society of Chemical Industry.

Improving grain yield and nitrogen use efficiency of direct-seeded rice with simplified and nitrogen-reduced practices under a double-cropping system in South China.

BACKGROUND: Enhancing grain yield and nitrogen use efficiency (NUE) of rice is of great importance for sustainable agricultural development. Little effort has been made to increase grain yield and NUE of direct-seeded rice under the double-cropping system in South China. Field trials were conducted during 2018-2020 with four treatments, including nitrogen-free, farmers' fertilization practice (FP), 'three controls' nutrient management (TC), and simplified and nitrogen-reduced practice (SNRP). RESULTS: Grain yield under SNRP averaged 6.46 t ha-1 during the three years and was 23.0% higher than that of FP but comparable to that of TC. Recovery efficiency (REN ), agronomic efficiency (AEN ), and partial factor productivity (PFPN ) of nitrogen under SNRP increased by 12.0-22.7%, 159.3-295.0% and 94.6-112.5% respectively compared with FP. Harvest index and sink capacity increased by 7.3-10.8% and 14.9-21.3% respectively. Percentage of productive tillers (PPT) and biomass after heading increased by 24.0% and 104.5% respectively. Leaf nitrogen concentration at heading and nitrogen accumulation after heading increased by 16.3% and 842.0% respectively. Grain yield was positively correlated with PPT, sink capacity, harvest index, biomass and nitrogen accumulation after heading, REN , AEN , and PFPN . CONCLUSION: Grain yield and NUE under SNRP were superior to those under FP and comparable to those under TC. Increase in sink capacity, higher PPT, more biomass and nitrogen accumulation after heading, and greater harvest index were responsible for high grain yield and NUE in SNRP with reduced nitrogen fertilizer and labor input. SNRP is a feasible approach for direct-seeded rice under a double-cropping system in South China. © 2023 Society of Chemical Industry.

Conditional deficiency of m6A methyltransferase Mettl14 in substantia nigra alters dopaminergic neuron function.

N6-Methyladenosine (m6A) is the most prevalent internal modification in messenger RNAs (mRNAs) of eukaryotes and plays a vital role in post-transcriptional regulation. Recent studies demonstrated that m6A is essential for the normal function of the central nervous system (CNS), and the deregulation of m6A leads to a series of CNS diseases. However, the functional consequences of m6A deficiency within the dopaminergic neurons of adult brain are elusive. To evaluate the necessity of m6A in dopaminergic neuron functions, we conditionally deleted Mettl14, one of the most important part of m6A methyltransferase complexes, in the substantia nigra (SN) region enriched with dopaminergic neurons. By using rotarod test, pole test, open-field test and elevated plus maze, we found that the deletion of Mettl14 in the SN region induces impaired motor function and locomotor activity. Further molecular analysis revealed that Mettl14 deletion significantly reduced the total level of m6A in the mRNA isolated from SN region. Tyrosine hydroxylase (TH), an essential enzyme for dopamine synthesis, was also down-regulated upon Mettl14 deletion, while the activation of microglia and astrocyte was enhanced. Moreover, the expression of three essential transcription factors in the regulation of TH including Nurr1, Pitx3 and En1, with abundant m6A-binding sites on their RNA 3'-untranslated regions (UTR), was significantly decreased upon Mettl14 deletion in SN. Our finding first confirmed the significance of m6A in maintaining normal dopaminergic function in the SN of adult mouse.

Mutualistic Ophiostomatoid Fungi Equally Benefit from Both a Bark Beetle Pheromone and Host Tree Volatiles as Nutrient Sources.

Interactions between mutualistic bark beetles and ophiostomatoid fungi have received considerable attention in recent years. Studies have shown how volatile organic compounds emitted from mutualist fungi affect the behaviors of several bark beetle species. However, we currently lack sufficient knowledge regarding whether bark beetle pheromones can influence mutualist fungi. Here, we measured growth and biomass of two mutualistic fungi of the mountain pine beetle in response to headspace of a beetle pheromone (trans-verbenol), a blend of host tree volatiles, the combination of both, or control (no volatile source) in vitro experiments consisting of a nitrogen-based medium. The surface area and ergosterol content of the mycelia were used as surrogates for fungal growth and biomass respectively. We found that both growth and biomass of Grosmannia clavigera and Ophiostoma montium were greater in medium exposed to any type of volatile sources than the control. While growth and ergosterol content of G. clavigera were highest in the combination treatment, there were no differences in growth or biomass among the types of volatiles introduced for O. montium. These results suggest that both mutualistic fungi can utilize both bark beetle pheromone and host tree volatiles as nutrient sources. Overall, these results support the on-going studies on the role of volatile organic compounds mediating mutualistic bark beetle-fungi interactions.

High mobility group nucleosomal binding 2 reduces integrin α5/ß1-mediated adhesion of Klebsiella pneumoniae on human pulmonary epithelial cells via nuclear factor I.

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/ß1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/ß1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/ß1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/ß1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/ß1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.

Inhibition of COX-2 and EGFR by Melafolone Improves Anti-PD-1 Therapy through Vascular Normalization and PD-L1 Downregulation in Lung Cancer.

Checkpoint blockade therapy has been proven efficacious in lung cancer patients. However, primary/acquired resistance hampers its efficacy. Therefore, there is an urgent need to develop novel strategies to improve checkpoint blockade therapy. Here we tested whether dual inhibition of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) by flavonoid melafolone improves program death 1 (PD-1) checkpoint blockade therapy through normalizing tumor vasculature and PD-1 ligand (PD-L1) downregulation. Virtual screening assay, cellular thermal shift assay, and enzyme inhibition assay identified melafolone as a potential inhibitor of COX-2 and EGFR. In Lewis lung carcinoma (LLC) and CMT167 models, dual inhibition of COX-2 and EGFR by melafolone promoted survival, tumor growth inhibition, and vascular normalization, and ameliorated CD8+ T-cell suppression, accompanied by the downregulation of transforming growth factor-ß (TGF-ß), vascular endothelial growth factor (VEGF), and PD-L1 in the tumor cells. Mechanistically, dual inhibition of COX-2 and EGFR in lung cancer cells by melafolone increased the migration of pericyte, decreased the proliferation and migration of endothelial cells, and enhanced the proliferation and effector function of CD8+ T cells through VEGF, TGF-ß, or PD-L1 downregulation and PI3K/AKT inactivation. Notably, melafolone improved PD-1 immunotherapy against LLC and CMT167 tumors. Together, dual inhibition of COX-2 and EGFR by melafolone improves checkpoint blockade therapy through vascular normalization and PD-L1 downregulation and, by affecting vessels and immune cells, may be a promising combination strategy for the treatment of human lung cancer.

Upregulation of let-7f-2-3p by long noncoding RNA NEAT1 inhibits XPO1-mediated HAX-1 nuclear export in both in vitro and in vivo rodent models of doxorubicin-induced cardiotoxicity.

Clinical application of doxorubicin (Dox) is limited due to its undesirable side effects, especially cardiotoxicity. Several microRNAs (miRNAs) such as microRNA-140-5p and miR-23a aggravate Dox-induced cardiotoxicity. Here we demonstrate that upregulation of miRNA let-7f-2-3p by long noncoding RNA (lncRNA) NEAT1 inhibits exportin-1 (XPO1)-mediated nuclear export of hematopoietic-substrate-1 associated protein X-1 (HAX-1) in Dox-induced cardiotoxicity. Treatment of the H9c2 cells with the Dox (1 µM) for 6 h inhibited HAX-1 nuclear export and decreased XPO1 expression. Overexpression of XPO1 significantly attenuated the Dox-induced leakage of myocardial enzymes (creatine phosphokinase, creatine kinase-MB and lactate dehydrogenase) and cardiomyocyte apoptosis with the increased HAX-1 nuclear export. Differentially expressed miRNAs including let-7f-2-3p were selected from the Dox or vehicle-treated cardiomyocytes. TargetScan and luciferase assay showed that let-7f-2-3p targeted XPO1 3' UTR. Inhibition of let-7f-2-3p reduced Dox-induced cardiotoxicity and apoptosis by inhibiting XPO1-mediated HAX-1 nuclear export, whereas let-7f-2-3p overexpression aggravated these effects. In addition, lncRNA NEAT1 was identified as an endogenous sponge RNA to repress let-7f-2-3p expression. Overexpression of lncRNA NEAT1 abolished the increased let-7f-2-3p expression by Dox, and thereby attenuated cardiotoxicity. The loss function of let-7f-2-3p increased XPO1-mediated HAX-1 nuclear export and reduced myocardial injury in Dox (20 mg/kg)-treated rats. Importantly, let-7f-2-3p inhibition in mice alleviated Dox-induced cardiotoxicity and preserved the antitumor efficacy. Together, let-7f-2-3p regulated by lncRNA NEAT1 aggravates Dox-induced cardiotoxicity through inhibiting XPO1-mediated HAX-1 nuclear export, and may serve as a potential therapeutic target against Dox-induced cardiotoxicity.

CYP4X1 Inhibition by Flavonoid CH625 Normalizes Glioma Vasculature through Reprogramming TAMs via CB2 and EGFR-STAT3 Axis.

Tumor-associated macrophages (TAMs) are pivotal effector cells in angiogenesis. Here, we tested whether CYP4X1 inhibition in TAMs by flavonoid CH625 prolongs survival and normalizes glioma vasculature. CH625 was selected against the CYP4X1 3D model by virtual screening and showed inhibitory activity on the CYP4X1 catalytic production of 14,15-EET-EA in the M2-polarized human peripheral blood mononuclear cells (IC50 = 16.5 µM). CH625 improved survival and reduced tumor burden in the C6 and GL261 glioma intracranial and subcutaneous model. In addition, CH625 normalized vasculature (evidenced by a decrease in microvessel density and HIF-1α expression and an increase in tumor perfusion, pericyte coverage, and efficacy of temozolomide therapy) accompanied with the decreased secretion of 14,15-EET-EA, VEGF, and TGF-ß in the TAMs. Furthermore, CH625 attenuated vascular abnormalization and immunosuppression induced by coimplantation of GL261 cells with CYP4X1high macrophages. In vitro TAM polarization away from the M2 phenotype by CH625 inhibited proliferation and migration of endothelial cells, enhanced pericyte migration and T cell proliferation, and decreased VEGF and TGF-ß production accompanied with the downregulation of CB2 and EGFR-dependent downstream STAT3 expression. These effects were reversed by overexpression of CYP4X1 and STAT3 or exogenous addition of 14,15-EET-EA, VEGF, TGF-ß, EGF, and CB2 inhibitor AM630. These results suggest that CYP4X1 inhibition in TAMs by CH625 prolongs survival and normalizes tumor vasculature in glioma via CB2 and EGFR-STAT3 axis and may serve as a novel therapeutic strategy for human glioma.

Inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate crystal-induced inflammation.

Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E synthase-1 (mPGES-1)-derived eicosanoids play an essential role in human inflammatory disorders. Here, we investigated whether inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate (MSU) crystal-induced inflammation. Virtual screening assay and in vitro enzyme inhibition assay showed that leonurine was a potential inhibitor of COX-2, mPGES-1 and 5-LOX. Compared with COX-2 inhibitor celecoxib, leonurine (30 mg/kg) significantly decreased ankle perimeter, gait score and neutrophil number in synovial fluid in MSU crystal-treated rats, accompanied with the decreased expression of COX-2, mPGES-1 and 5-LOX and production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the synovial fluid macrophages. In addition, leonurine decreased representative M1 marker (iNOS and CD86) expression, NLRP3 inflammasome activation and M1 cytokine (TNF-α and IL-1ß) production. In the in vitro cultured RAW264.7 and human monocyte-derived macrophages (MDMs), blockade of COX-2/mPGES-1 and 5-LOX by leonurine inhibited macrophage M1 polarization and NLRP3 inflammasome activation in response to MSU crystals, and thus down-regulated IL-1ß and TNF-α with STAT1 and NF-κB inactivation. Conversely, these effects were partially abolished by overexpression of COX-2, mPGES-1, 5-LOX or STAT1. Furthermore, leonurine prevented a positive feedback loop between COX-2/mPGES-1/5-LOX and IL-1ß/TNF-α in MSU crystal-induced inflammation. Together, simultaneous down-regulation of COX-2/mPGES-1 and 5-LOX by leonurine ameliorates MSU crystal-induced inflammation through decreasing IL-1ß and TNF-α production. Our study may provide novel multi-target agents toward the arachidonic acid (AA) network for gouty arthritis therapy.

Droplet digital PCR-based circulating microRNA detection serve as a promising diagnostic method for gastric cancer.

BACKGROUND: Novel non-invasive biomarkers for gastric cancer (GC) are needed, because the present diagnostic methods for GC are either invasive or insensitive and non-specific in clinic. The presence of stable circulating microRNAs (miRNAs) in plasma suggested a promising role as GC biomarkers. METHODS: Based on the quantitative droplet digital PCR (ddPCR), four miRNAs (miR-21, miR-93, miR-106a and miR-106b) related to the presence of GC were identified in plasma from a training cohort of 147 participants and a validation cohort of 28 participants. RESULTS: All circulating miRNA levels were significantly higher in the plasma of GC patients compared to healthy controls (P < 0.05). Through a combination of four miRNAs by logistic regression model, receiver operating characteristic (ROC) analyses yielded the highest AUC value of 0.887 in discriminating GC patients from healthy volunteers. Furthermore, miR-21, miR-93 and miR-106b levels were significantly related to an advanced TNM stage in GC patients. ROC analyses of the combined miRNA panel also showed the highest AUC value of 0.809 in discriminating GC patients with TNM stage I and II from stage III and IV. Through combining four miRNAs and clinical parameters, a classical random forest model was established in the training stage. In the validation cohort, it correctly discriminated 23 out of 28 samples in the blinded phase (false rate, 17.8%). CONCLUSIONS: Using the ddPCR technique, circulating miR-21, miR-93, miR-106a and miR-106b could be used as diagnostic plasma biomarkers in gastric cancer patients.

Transareolar Video-Assisted Thoracoscopic Surgery in Females: A Novel Incision for Pulmonary Ground Glass Nodule Resection.

Purpose: Uniportal video-assisted thoracoscopic surgery (VATS) is recognized for its minimally invasive nature, widely adopted globally. However, the evident scarring it leaves often triggers psychological apprehension and resistance to surgery. Transareolar incision, known for its superior cosmetic outcome with no visible scars, poses challenges in women due to the risk of mammary gland damage. In this report, we present successful pulmonary ground glass nodule (GGN) resection using transareolar VATS in female patients, aiming to address these concerns. Materials and Methods: We retrospectively analyzed the clinical data of 35 female patients who underwent GGN resection through transareolar VATS between August 2020 and March 2022. Results: There were no serious complications or perioperative deaths in this cohort of 35 female patients undergoing GGN resection through transareolar VATS. The operations, including local resection or segmentectomy, had an average duration of 70.1 ± 26.4 minutes, with a tube duration of 4.7 ± 2.1 days and a hospitalization time of 7.2 ± 2.3 days. The surgical approach varied, with 21 cases using transareolar uniport, 8 cases assisted by a 3-mm tiny port, and 6 cases converted to two-port VATS. Scar outcomes varied, with 21 cases showing no scar, 8 cases displaying a microscar, and 6 cases presenting a dominant scar of 1.7 ± 0.5 cm. Postoperative pain scores at 1 week and 1 month were 1.9 ± 0.9 and 1.0 ± 0.9, respectively, and the wound numbness occurred in 2.86% (1/35) of cases. Regarding breast complications, 2 patients suffered delayed healing of the incision. No damage and inflammation of glands were detected by breast B-mode ultrasonography. Conclusions: The transareolar incision emerges as a novel approach for VATS in female patients, offering advantages in terms of pain management and cosmetic outcomes.

Two-Centimeter Single-Port Incision Minimizing Intercostal Nerve Damage Applied in Video-Assisted Thoracoscopic Surgery Segmentectomy for Pulmonary Ground Glass Nodules.

Purposes: We introduced a novel modified 2-cm single-port incision made by blunt separation minimizing intercostal muscle and nerve damage applied in video-assisted thoracoscopic surgery (VATS) segmentectomy, and compared it with the traditional single-port incision or the novel incision plus a 3-mm tiny port, aiming to explore a more minimally invasive single-port technique for VATS segmentectomy. Materials and Methods: We retrospectively analyzed the clinical data of 174 pulmonary ground glass nodule patients who received single-port VATS segmentectomy (54 modified 2-cm single port, 67 modified single port plus tiny port, and 53 traditional single port, respectively) in our medical center from May 2020 to December 2022. Three kinds of approaches were compared retrospectively, concerning their safety, feasibility, and postoperative pain. Results: There were no serious complications and mortality in either group. The blood loss, tube duration, and hospitalization time were comparable among the three groups (P > .05). The 2-cm single-port and 2-cm single-port plus tiny-port group were obviously more advantageous in the visual analog scores of postoperative pain, the wound numbness, incision healing and appearance than that in the traditional group (P < .05), while they were comparable. Notably, the operation time of the 2-cm plus tiny-port group was shorter than that of the 2-cm group (P < .05) and similar to the traditional single-port group. Conclusions: The 2-cm modified single-port applied for VATS segmentectomy is feasible and safe, and has obviously advantages in postoperative pain, numbness, and appearance of incision. With addition of tiny port, the convenience of the operation can be significantly increased without increasing pain. Our finding could provide a promising new incision mode for VATS segmentectomy.

Growth, nutrient uptake and transcriptome profiling of rice seedlings in response to mixed provision of ammonium- and nitrate-nitrogen.

Nitrogen (N) is a principal macronutrient and plays a paramount role in mineral nutrition of rice plants. Mixed provision of ammonium- and nitrate-nitrogen (MPAN) at a moderate level could enhance N uptake and translocation and promote growth of rice, but current understanding of their molecular mechanisms is still insufficient. Two rice lines of W6827 and GH751, with contrasting ability of N uptake, were subjected to four levels of MPAN (NH4+/NO3- = 100:0, 75:25, 50:50, 25:75) in hydroponic experiments. In terms of plant height, growth rate and shoot biomass, growth of GH751 tended to increase firstly and then decrease with enhancement in NO3--N ratio. It attained maximal level under 75:25 MPAN, with an 8.3% increase in shoot biomass. In general, W6827 was comparatively less responsive to MPAN. For GH751, the uptake rate of N, phosphor (P) and potassium (K) under 75:25 MPAN was enhanced by 21.1%, 20.8% and 16.1% in comparison with that of control (100:0 MPAN). Meanwhile, the translocation coefficient and content in shoots of N, P and K were all increased significantly. In contrast to transcriptomic profile under control, 288 differentially expressed genes (DEGs) were detected to be up-regulated and 179 DEGs down-regulated in transcription under 75:25 MPAN. Gene Ontology analysis revealed that some DEGs were up-regulated under 75:25 MPAN and they code for proteins mainly located in membrane and integral component of membrane and involved in metal ion binding, oxidoreductase activity and other biological processes. KEGG pathway enrichment analysis indicated that DEGs related to nitrogen metabolism, carbon fixation in photosynthetic organisms, photosynthesis, starch and sucrose metabolism, and zeatin biosynthesis were up- or down-regulated in transcription under 75:25 MPAN, and they are responsible for improved nutrient uptake and translocation and enhanced growth of seedlings.

Mettl14 sustains FOXP3 expression to promote the differentiation and functions of induced-regulatory T cells via the mTOR signaling pathway.

Induced regulatory T cell (iTregs) can be generated in vitro. Thus, iTregs-based therapeutics are receiving increased attention for their potential to treat autoimmune diseases and prevent transplant rejection. However, iTregs fail to maintain FoxP3 expression and suppressive activity, which limits their clinical application. Increasing lines of evidence suggest that methyltransferase-like 14 (METTL14), a critical component of the m6A writer complex, regulates the stability and function of the Treg cells. However, beyond meeting the epigenetic modification of Treg cells, whether Mettl14 plays a role in the fate determination of iTregs is unclear. Here, we systemically investigated the potential function of METTL14 in iTregs differentiation and regulatory activity. In our study, iTregs were generated from CD4+ naïve T cells under iTreg-polarizing conditions, we found that the expression of METTL14 was increased in iTregs compared with CD4+naïve T cells. Subsequently, the expression of METTL14 was knocked down by siRNA-METTL14 interference in CD4+ naïve T cells and cultured under iTreg-polarizing conditions. According to the results, Mettl14 deficiency resulted in the disruption of iTregs differentiation evidenced by the limited FoxP3 expression. Meanwhile, inflammatory cytokines such as IFN-γ and IL-17a were upregulated in cultured iTregs. We next determined the functional change in METTL14-deficient iTregs. The results of the colitis development in Rag1-/- mice and CFSE assays revealed that loss of METTL14 significantly compromised the suppressive function of iTregs in vivo and in vitro. We further checked the altered signaling pathway in METTL14-deficient iTregs. We found that reduced METTL14 leads to activation of the mTOR pathway with increased p-mTOR and p-p70S6K, which are known to modulate the suppressive function of iTregs. In conclusion, our study revealed that Mettl14 plays a critical role in the development and suppressive function of iTregs in vitro and could thus serve as a regulatory element for stabilizing iTregs in cell-based therapy.

Functional diversity of subgroup 5 R2R3-MYBs promoting proanthocyanidin biosynthesis and their key residues and motifs in tea plant.

The tea plant (Camellia sinensis) is rich in polyphenolic compounds. Particularly, flavan-3-ols and proanthocyanidins (PAs) are essential for the flavor and disease-resistance property of tea leaves. The fifth subgroup of R2R3-MYB transcription factors comprises the primary activators of PA biosynthesis. This study showed that subgroup 5 R2R3-MYBs in tea plants contained at least nine genes belonging to the TT2, MYB5, and MYBPA types. Tannin-rich plants showed an expansion in the number of subgroup 5 R2R3-MYB genes compared with other dicotyledonous and monocot plants. The MYBPA-type genes of tea plant were slightly expanded. qRT-PCR analysis and GUS staining analysis of promoter activity under a series of treatments revealed the differential responses of CsMYB5s to biotic and abiotic stresses. In particular, CsMYB5a, CsMYB5b, and CsMYB5e responded to high-intensity light, high temperature, MeJA, and mechanical wounding, whereas CsMYB5f and CsMYB5g were only induced by wounding. Three genetic transformation systems (C. sinensis, Nicotiana tabacum, and Arabidopsis thaliana) were used to verify the biological function of CsMYB5s. The results show that CsMYB5a, CsMYB5b, and CsMYB5e could promote the gene expression of CsLAR and CsANR. However, CsMYB5f and CsMYB5g could only upregulate the gene expression of CsLAR but not CsANR. A series of site-directed mutation and domain-swapping experiments were used to verify functional domains and key amino acids of CsMYB5s responsible for the regulation of PA biosynthesis. This study aimed to provide insight into the induced expression and functional diversity model of PA biosynthesis regulation in tea plants.

Mettl14-mediated m6A modification enhances the function of Foxp3+ regulatory T cells and promotes allograft acceptance.

N6-methyladenosine (m6A), the most prevalent form of internal mRNA modification, is extensively involved in Treg cells differentiation and function. However, the involvement of m6A in functional Treg cells for transplantation tolerance remains to be elucidated. By using an experimental transplantation mouse model, we found that m6A levels in Treg cells were altered during the induction of transplant tolerance by performing a dot blotting assay. Subsequently, we used the heterogenic Treg-specific Mettl14 knockout mice (Foxp3-Mettl14f/+ cKO) to reduce METTL14 expression and performed islets allograft transplantation. Our result revealed that reduced expression of METTL14 prevented Treg cells expansion and promoted the infiltration of CD4+ and CD8+ T cells around the allograft, which led to rapid allograft rejection in Foxp3-Mettl14 f/+ cKO mice. The expression of regulatory cytokines including IL-10 and TGF-ß was significantly decreased in Foxp3-Mettl14 f/+ cKO mice, and the suppressive function of Treg cells was also abrogated. In addition, an analysis of RNA-seq data revealed that the SOCS family (SOCS1, SOCS2 and SOCS3) is the subsequent signaling pathway affected by the METTL14 mediated m6A modification in Treg cells to modulate the suppressive function after transplantation. Taken together, our study showed for the first time that the METTL14-mediated m6A modification is essential for the suppressive function of Treg cells in transplantation and may serve as a regulatory element of Treg cell-based therapy in transplant medicine.

Prevotellaceae produces butyrate to alleviate PD-1/PD-L1 inhibitor-related cardiotoxicity via PPARα-CYP4X1 axis in colonic macrophages.

BACKGROUND: Immune checkpoint inhibitor-related cardiotoxicity is one of the most lethal adverse effects, and thus, the identification of underlying mechanisms for developing strategies to overcome it has clinical importance. This study aimed to investigate whether microbiota-host interactions contribute to PD-1/PD-L1 inhibitor-related cardiotoxicity. METHODS: A mouse model of immune checkpoint inhibitor-related cardiotoxicity was constructed by PD-1/PD-L1 inhibitor BMS-1 (5 and 10 mg/kg), and cardiomyocyte apoptosis and cardiotoxicity were determined by hematoxylin and eosin, Masson's trichome and TUNEL assays. 16S rRNA sequencing was used to define the gut microbiota composition. Gut microbiota metabolites short-chain fatty acids (SCFAs) were determined by HPLC. The serum levels of myocardial enzymes (creatine kinase, aspartate transaminase, creatine kinase-MB and lactate dehydrogenase) and the production of M1 factors (TNF-α and IL-1ß) were measured by ELISA. The colonic macrophage phenotype was measured by mmunofluorescence and qPCR. The expression of Claudin-1, Occludin, ZO-1 and p-p65 was measured by western blot. The gene expression of peroxisome proliferator-activated receptor α (PPARα) and cytochrome P450 (CYP) 4X1 was determined using qPCR. Statistical analyses were performed using Student's t-test for two-group comparisons, and one-way ANOVA followed by Student-Newman-Keul test for multiple-group comparisons. RESULTS: We observed intestinal barrier injury and gut microbiota dysbiosis characterized by Prevotellaceae and Rikenellaceae genus depletion and Escherichia-Shigella and Ruminococcaceae genus enrichment, accompanied by low butyrate production and M1-like polarization of colonic macrophages in BMS-1 (5 and 10 mg/kg)-induced cardiotoxicity. Fecal microbiota transplantation mirrored the effect of BMS-1 on cardiomyocyte apoptosis and cardiotoxicity, while macrophage depletion and neutralization of TNF-α and IL-1ß greatly attenuated BMS-1-induced cardiotoxicity. Importantly, Prevotella loescheii recolonization and butyrate supplementation alleviated PD-1/PD-L1 inhibitor-related cardiotoxicity. Mechanistically, gut microbiota dysbiosis promoted M1-like polarization of colonic macrophages and the production of proinflammatory factors TNF-α and IL-1ß through downregulation of PPARα-CYP4X1 axis. CONCLUSIONS: Intestinal barrier dysfunction amplifies PD-1/PD-L1 inhibitor-related cardiotoxicity by upregulating proinflammatory factors TNF-α and IL-1ß in colonic macrophages via downregulation of butyrate-PPARα-CYP4X1 axis. Thus, targeting gut microbiota to polarize colonic macrophages away from the M1-like phenotype could provide a potential therapeutic strategy for PD-1/PD-L1 inhibitor-related cardiotoxicity.

Corrigendum: Mettl14-mediated m6A modification enhances the function of Foxp3+ regulatory T cells and promotes allograft acceptance.

[This corrects the article DOI: 10.3389/fimmu.2022.1022015.].

CYP4F2-Catalyzed Metabolism of Arachidonic Acid Promotes Stromal Cell-Mediated Immunosuppression in Non-Small Cell Lung Cancer.

SIGNIFICANCE: The identification of a role for CYP4F2-dependent metabolism in driving immune evasion in non-small cell lung cancer reveals a strategy to improve the efficacy of immunotherapy by inhibiting CYP4F2. See related article by Van Ginderachter, p. 3882.

Transplant Islets Into the Pinna of the Ear: A Mouse Islet Transplant Model.

BACKGROUND: Islets transplanted under the ear skin would allow easy observation of the graft response and survival in vivo. This research was designed to establish an efficient mouse islet transplant model to probe the dynamic cellular interplay in vivo. METHODS: Green fluorescent protein transgenic mice and BALB/c mice were used as donors and recipients. All recipients were divided into 6 groups of 6 mice each. First, we treated the transplant recipients, including diabetes induction, autologous epididymal fat pad, and MATRIGEL transplant to the ears. Then, 1. we transplanted isolated islets to the ear/ear with fat/ear with MATRIGEL; and 2. transplanted islets with collagen + basic fibroblast growth factor or islets with collagen + vascular endothelial growth factor. Mice in the control group received a sham transplantation with phosphate buffer saline. All recipients were then observed for 30 days with blood glucose (BG) monitoring. Finally, ears were removed with graft on day 28 for histologic examination. RESULTS: It was suggested that transplant of islets alone could not correct hyperglycemia. Fat, MATRIGEL, collagen, and growth factors have the similar function to form a microenvironment conducive to islet survival. The effect of islet transplantation for correcting hyperglycemia of the fat modification group was better than other groups (P < .05). BG could be normalized, and living islets were detected by anti-insulin immunohistochemistry. CONCLUSIONS: Transplant islets into the ear with transplanted autologous fat is the optimal way which can be used to analyze the allograft response in vivo and track cell population and migration using labels by confocal microscopy.