Brassinosteroids regulate cotton fiber elongation by modulating very-long-chain fatty acid biosynthesis.

Brassinosteroid (BR), a growth-promoting phytohormone, regulates many plant growth processes including cell development. However, the mechanism by which BR regulates fiber growth is poorly understood. Cotton (Gossypium hirsutum) fibers are an ideal single-cell model in which to study cell elongation due to their length. Here we report that BR controls cotton fiber elongation by modulating very-long-chain fatty acid (VLCFA) biosynthesis. BR deficiency reduces the expression of 3-ketoacyl-CoA synthases (GhKCSs), the rate-limiting enzymes involved in VLCFA biosynthesis, leading to lower saturated VLCFA contents in pagoda1 (pag1) mutant fibers. In vitro ovule culture experiments show that BR acts upstream of VLCFAs. Silencing of BRI1-EMS-SUPPRESOR 1.4 (GhBES1.4), encoding a master transcription factor of the BR signaling pathway, significantly reduces fiber length, whereas GhBES1.4 overexpression produces longer fibers. GhBES1.4 regulates endogenous VLCFA contents and directly binds to BR RESPONSE ELEMENTS (BRREs) in the GhKCS10_At promoter region, which in turn regulates GhKCS10_At expression to increase endogenous VLCFA contents. GhKCS10_At overexpression promotes cotton fiber elongation, whereas GhKCS10_At silencing inhibits cotton fiber growth, supporting a positive regulatory role for GhKCS10_At in fiber elongation. Overall, these results uncover a mechanism of fiber elongation through crosstalk between BR and VLCFAs at the single-cell level.

An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony.

Stem strength is an important agronomic trait affecting plant lodging, and plays an essential role in the quality and yield of plants. Thickened secondary cell walls in stems provide mechanical strength that allows plants to stand upright, but the regulatory mechanism of secondary cell wall thickening and stem strength in cut flowers remains unclear. In this study, first, a total of 11 non-redundant Paeonia lactiflora R2R3-MYBs related to stem strength were identified and isolated from cut-flower herbaceous peony, among which PlMYB43, PlMYB83 and PlMYB103 were the most upregulated differentially expressed genes. Then, the expression characteristics revealed that these three R2R3-MYBs were specifically expressed in stems and acted as transcriptional activators. Next, biological function verification showed that these P. lactiflora R2R3-MYBs positively regulated stem strength, secondary cell wall thickness and lignin deposition. Furthermore, yeast-one-hybrid and dual luciferase reporter assays demonstrated that they could bind to the promoter of caffeic acid O-methyltransferase gene (PlCOMT2) and/or laccase gene (PlLAC4), two key genes involved in lignin biosynthesis. In addition, the function of PlLAC4 in increasing lignin deposition was confirmed by virus-induced gene silencing and overexpression. Moreover, PlMYB83 could also act as a transcriptional activator of PlMYB43. The findings of the study propose a regulatory network of R2R3-MYBs modulating lignin biosynthesis and secondary cell wall thickening for improving stem lodging resistance, and provide a resource for molecular genetic engineering breeding of cut flowers.

Cell cycle-dependent kinase inhibitor GhKRP6, a direct target of GhBES1.4, participates in BR regulation of cell expansion in cotton.

The steroidal hormone brassinosteroid (BR) has been shown to positively regulate cell expansion in plants. However, the specific mechanism by which BR controls this process has not been fully understood. In this study, RNA-seq and DAP-seq analysis of GhBES1.4 (a core transcription factor in BR signaling) were used to identify a cotton cell cycle-dependent kinase inhibitor called GhKRP6. The study found that GhKRP6 was significantly induced by the BR hormone and that GhBES1.4 directly promoted the expression of GhKRP6 by binding to the CACGTG motif in its promoter region. GhKRP6-silenced cotton plants had smaller leaves with more cells and reduced cell size. Furthermore, endoreduplication was inhibited, which affected cell expansion and ultimately decreased fiber length and seed size in GhKRP6-silenced plants compared with the control. The KEGG enrichment results of control and VIGS-GhKRP6 plants revealed differential expression of genes related to cell wall biosynthesis, MAPK, and plant hormone transduction pathways - all of which are related to cell expansion. Additionally, some cyclin-dependent kinase (CDK) genes were upregulated in the plants with silenced GhKRP6. Our study also found that GhKRP6 could interact directly with a cell cycle-dependent kinase called GhCDKG. Taken together, these results suggest that BR signaling influences cell expansion by directly modulating the expression of cell cycle-dependent kinase inhibitor GhKRP6 via GhBES1.4.

Salvia miltiorrhiza augments endothelial cell function for ischemic hindlimb recovery.

Salvia miltiorrhiza (Salvia miltiorrhiza) root, as a traditional herb, is widely applied to pharmacotherapy for vascular system disease. In this study, we elucidate the therapy mechanism of Salvia miltiorrhiza by using a model of hindlimb ischemia. Blood perfusion measurement showed that intravenous administration of the Water Extract of Salvia miltiorrhiza (WES) could facilitate damaged hindlimb blood flow recovery and blood vessel regeneration. In vitro mRNA screen assay in cultured human umbilical vein endothelial cells (HUVECs) show that WES induced increased NOS3, VEGFA, and PLAU mRNA levels. Endothelial NOS (eNOS) promotor reporter analysis revealed that WES and the major ingredients danshensu (DSS) could enhance eNOS promoter activity. Additionally, we found that WES and its ingredients, including DSS, protocatechuic aldehyde (PAI), and salvianolic acid A (SaA), promoted HUVECs growth by the endothelial cell viability assays. A mechanistic approach confirmed that WES augments HUVECs proliferation through the activation of extracellular signal-regulated kinase (ERK) signal pathway. This study reveals that WES promotes ischemic remodeling and angiogenesis through its multiple principal ingredients, which target and regulate multiple sites of the network of the blood vessel endothelial cell regenerating process.

The herbaceous peony transcription factor WRKY41a promotes secondary cell wall thickening to enhance stem strength.

Stem bending or lodging caused by insufficient stem strength is an important limiting factor for plant production. Secondary cell walls play a crucial role in plant stem strength, but whether WRKY transcription factors can positively modulate secondary cell wall thickness are remain unknown. Here, we characterized a WRKY transcription factor PlWRKY41a from herbaceous peony (Paeonia lactiflora), which was highly expressed in stems. PlWRKY41a functioned as a nucleus-localized transcriptional activator and enhanced stem strength by positively modulating secondary cell wall thickness. Moreover, PlWRKY41a bound to the promoter of the XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE4 (PlXTH4) and activated the expression of PlXTH4. PlXTH4-overexpressing tobacco (Nicotiana tabacum) had thicker secondary cell walls, resulting in enhanced stem strength, while PlXTH4-silenced P. lactiflora had thinner secondary cell walls, showing decreased stem strength. Additionally, PlWRKY41a directly interacted with PlMYB43 to form a protein complex, and their interaction induced the expression of PlXTH4. These data support that the PlMYB43-PlWRKY41a protein complex can directly activate the expression of PlXTH4 to enhance stem strength by modulating secondary cell wall thickness in P. lactiflora. The results will enhance our understanding of the formation mechanism of stem strength and provide a candidate gene to improve stem straightness in plants.

A brassinosteroid transcriptional regulatory network participates in regulating fiber elongation in cotton.

Brassinosteroids (BRs) participate in the regulation of plant growth and development through BRI1-EMS-SUPPRESSOR1 (BES1)/BRASSINAZOLE-RESISTANT1 (BZR1) family transcription factors. Cotton (Gossypium hirsutum) fibers are highly elongated single cells, and BRs play a vital role in the regulation of fiber elongation. However, the mode of action on how BR is involved in the regulation of cotton fiber elongation remains unexplored. Here, we generated GhBES1.4 over expression lines and found that overexpression of GhBES1.4 promoted fiber elongation, whereas silencing of GhBES1.4 reduced fiber length. DNA affinity purification and sequencing (DAP-seq) identified 1,531 target genes of GhBES1.4, and five recognition motifs of GhBES1.4 were identified by enrichment analysis. Combined analysis of DAP-seq and RNA-seq data of GhBES1.4-OE/RNAi provided mechanistic insights into GhBES1.4-mediated regulation of cotton fiber development. Further, with the integrated approach of GWAS, RNA-seq, and DAP-seq, we identified seven genes related to fiber elongation that were directly regulated by GhBES1.4. Of them, we showed Cytochrome P450 84A1 (GhCYP84A1) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (GhHMG1) promote cotton fiber elongation. Overall, the present study established the role of GhBES1.4-mediated gene regulation and laid the foundation for further understanding the mechanism of BR participation in regulating fiber development.

Lead exposure induces neurodysfunction through caspase-1-mediated neuronal pyroptosis.

Chronic lead (Pb) exposure causes neurodysfunction and contributes to the development of neurodegenerative disease. However, the mechanism of Pb-induced neurological dysfunction have yet to be fully elucidated. This study determined the role pyroptosis plays in Pb-induced neurodysfunction in neurons. We used both in vitro and in vivo approaches to explore whether Pb exposure induces caspase-1-mediated pyroptosis in neurons and its relationship to Pb-induced neurological disorders. Our findings showed that caspase-1-mediated pyroptosis in Pb-exposed neurons activated glycogen synthase kinase 3 protease activity by disrupting Ca2+/calmodulin-dependent protein kinase II/cAMP-response element binding protein pathway, leading to neurological disorders. Moreover, the caspase-1 inhibition VX-765 or the non-steroidal anti-inflammatory drug sodium para-aminosalicylic acid (PAS-Na) attenuated the Pb-induced neurological disorders by alleviating caspase-1 mediated neuronal pyroptosis. Our novel studies suggest that caspase-1-mediated pyroptosis in neurons represents a potential mechanism for Pb-induced neurodysfunction, identifying a putative target for attenuating the neurodegenerative effects induced by this metal.

miR-29b-3p regulates cardiomyocytes pyroptosis in CVB3-induced myocarditis through targeting DNMT3A.

BACKGROUND: Viral myocarditis (VMC) is a disease resulting from viral infection, which manifests as inflammation of myocardial cells. Until now, the treatment of VMC is still a great challenge for clinicians. Increasing studies indicate the participation of miR-29b-3p in various diseases. According to the transcriptome sequencing analysis, miR-29b-3p was markedly upregulated in the viral myocarditis model. The purpose of this study was to investigate the role of miR-29b-3p in the progression of VMC. METHODS: We used CVB3 to induce primary cardiomyocytes and mice to establish a model of viral myocarditis. The purity of primary cardiomyocytes was identified by immunofluorescence. The cardiac function of mice was detected by Vevo770 imaging system. The area of inflammatory infiltration in heart tissue was shown by hematoxylin and eosin (H&E) staining. The expression of miR-29b-3p and DNMT3A was detected by quantitative real time polymerase chain reaction (qRT-PCR). The expression of a series of pyroptosis-related proteins was detected by western blot. The role of miR-29b-3p/DNMT3A in CVB3-induced pyroptosis of cardiomyocytes was studied in this research. RESULTS: Our data showed that the expression of miR-29b-3p was upregulated in CVB3-induced cardiomyocytes and heart tissues in mice. To explore the function of miR-29b-3p in CVB3-induced VMC, we conducted in vivo experiments by knocking down the expression of miR-29b-3p using antagomir. We then assessed the effects on mice body weight, histopathology changes, myocardial function, and cell pyroptosis in heart tissues. Additionally, we performed gain/loss-of-function experiments in vitro to measure the levels of pyroptosis in primary cardiomyocytes. Through bioinformatic analysis, we identified DNA methyltransferases 3A (DNMT3A) as a potential target gene of miR-29b-3p. Furthermore, we found that the expression of DNMT3A can be modulated by miR-29b-3p during CVB3 infection. CONCLUSIONS: Our results demonstrate a correlation between the expression of DNMT3A and CVB3-induced pyroptosis in cardiomyocytes. These findings unveil a previously unidentified mechanism by which CVB3 induces cardiac injury through the regulation of miR-29b-3p/DNMT3A-mediated pyroptosis.

The application of blended teaching in medical practical course of clinical skills training.

BACKGROUND: Blended teaching is an effective approach that combines online and offline teaching methods, leading to improved outcomes in medical education compared to traditional offline teaching. In this study, we examined the impact of blended teaching in clinical skills training, a medical practice course. METHODS: This study involved forty-eight undergraduate students studying clinical medicine in the fifth semester at Wuhan University of Science and Technology. The students were divided into two groups: the control group, which received traditional offline teaching, and the experimental group, which received hybrid teaching. Following the completion of the 4-month course, both groups underwent the Objective Structured Clinical Examination (OSCE) to evaluate their proficiency in clinical skills. Furthermore, the experimental group was given a separate questionnaire to gauge their feedback on the Blended Teaching approach. RESULTS: Based on the OSCE scores, the experimental group outperformed the control group significantly (P<0.05). The questionnaire results indicated that a majority of students (54.2%, 3.71 ± 1.06) believed that blended teaching is superior to traditional offline teaching, and a significant number of students (58.3%, 3.79 ± 1.15) expressed their willingness to adopt blended teaching in other courses. Furthermore, students in the experimental group displayed varying levels of interest in different teaching contents, with emergency medicine (79.2%), internal medicine (70.8%), and surgery (66.7%) being the most popular among them. CONCLUSIONS: This research demonstrates for the first time that blended teaching can achieve a good pedagogical effectiveness in the medical practice course, clinical skills training and practice. Moreover, in different teaching contents, the teaching effects are different. In the content of Emergency Medicine and Surgery, which is more attractive to students, the application of blended teaching could result in a better pedagogical outcome than other contents.

GhBES1 mediates brassinosteroid regulation of leaf size by activating expression of GhEXO2 in cotton (Gossypium hirsutum).

KEY MESSAGE: We proposed a working model of BR to promote leaf size through cell expansion. In the BR signaling pathway, GhBES1 affects cotton leaf size by binding to and activating the expression of the E-box element in the GhEXO2 promoter region. Brassinosteroid (BR) is an essential phytohormone that controls plant growth. However, the mechanisms of BR regulation of leaf size remain to be determined. Here, we found that the BR deficient cotton mutant pagoda1 (pag1) had a smaller leaf size than wild-type CRI24. The expression of EXORDIUM (GhEXO2) gene, was significantly downregulated in pag1. Silencing of BRI1-EMS-SUPPRESSOR 1 (GhBES1), inhibited leaf cell expansion and reduced leaf size. Overexpression of GhBES1.4 promoted leaf cell expansion and enlarged leaf size. Expression analysis showed GhEXO2 expression positively correlated with GhBES1 expression. In plants, altered expression of GhEXO2 promoted leaf cell expansion affecting leaf size. Furthermore, GhBES1.4 specifically binds to the E-box elements in the GhEXO2 promoter, inducing its expression. RNA-seq data revealed many down-regulated genes related to cell expansion in GhEXO2 silenced plants. In summary, we discovered a novel mechanism of BR regulation of leaf size through GhBES1 directly activating the expression of GhEXO2.

Palaeobotanical evidence reveals the living conditions of Miocene Lufengpithecus in East Asia.

BACKGROUND: Understanding the relationship between human evolution and environmental changes is the key to lifting the veil on human origin. The hypothesis that environmental changes triggered the divergence of humans from apes (ca. 9.3-6.5 million years ago, Ma) has been poorly tested because of limited continuous environmental data from fossil localities. Lufengpithecus (12.5-6.0 Ma) found on the southeastern margin of the Tibetan Plateau (SEMTP) across the ape-human split provides a good chance for testing this hypothesis. RESULTS: Here, we reconstructed the habitats of L. keiyuanensis (12.5-11.6 Ma) with comprehensive vegetation, climate, and potential food web data by palaeobotanical evidence, together with other multidisciplinary data and partly tested the environment-driven hypothesis by revealing the living conditions of Lufengpithecus. CONCLUSION: A detailed comparison of hominoids on different continents reveals their behaviour and fate divergence across the ape-human split against the background of global climate change, i.e., the stable living conditions of SEMTP not only provided a so-called 'refuge' for arboreal Lufengpithecus but also acted as a 'double-edged sword', preventing their further evolution while vegetation shifts in East Africa probably stimulated the emergence of human bipedalism, and the intense climatic changes in Europe possibly prevented those hominoids from surviving that time interval. Our findings provide interesting insight into the environmental impacts on the behavioural evolution of hominoids.

PUS7 promotes the proliferation of colorectal cancer cells by directly stabilizing SIRT1 to activate the Wnt/ß-catenin pathway.

Pseudouridine synthase 7 (PUS7) may play key roles in cancer development. However, few studies have been conducted in this area. In the present study, we explored the function and potential mechanisms of PUS7 in colorectal cancer (CRC) progression. We found that PUS7 had higher expression in CRC tissues and cell lines. Clinically, high expression of PUS7 was associated with an unfavorable prognosis for CRC patients. Functionally, knockdown of PUS7 suppressed the proliferation of CRC cells in vitro and inhibited tumorigenicity in vivo. Mechanistically, RNA sequencing and coimmunoprecipitation (Co-IP) indicated that PUS7 exhibited oncogenic functions through the interaction of Sirtuin 1 (SIRT1) and activated the Wnt/ß-catenin signaling pathway. Thus, our findings suggest that PUS7 promotes the proliferation of CRC cells by directly stabilizing SIRT1 to activate the Wnt/ß-catenin pathway.

Galactooligosaccharides: Synthesis, metabolism, bioactivities and food applications.

Prebiotics are non-digestible ingredients that exert significant health-promoting effects on hosts. Galactooligosaccharides (GOS) have remarkable prebiotic effects and structural similarity to human milk oligosaccharides. They generally comprise two to eight sugar units, including galactose and glucose, which are synthesized from substrate lactose by microbial ß-galactosidase. Enzyme sources from probiotics have received particular interest because of their safety and potential to synthesize specific structures that are particularly metabolized by intestinal probiotics. Owing to advancements in modern analytical techniques, many GOS structures have been identified, which vary in degree of polymerization, glycosidic linkage, and branch location. After intake, GOS adjust gut microbiota which produce short chain fatty acids, and exhibit excellent biological activities. They selectively stimulate the proliferation of probiotics, inhibit the growth and adhesion of pathogenic bacteria, alleviate gastrointestinal, neurological, metabolic and allergic diseases, modulate metabolites production, and adjust ion storage and absorption. Additionally, GOS are safe and stable, with high solubility and clean taste, and thus are widely used as food additives. GOS can improve the appearance, flavor, taste, texture, viscosity, rheological properties, shelf life, and health benefits of food products. This review systemically covers GOS synthesis, structure identifications, metabolism mechanisms, prebiotic bioactivities and wide applications, focusing on recent advances.

Characterization of chromatin accessibility and gene expression reveal the key genes involved in cotton fiber elongation.

Cotton (Gossypium hirsutum L.) is an important economic crop, and cotton fiber is one of the longest plant cells, which provides an ideal model for the study of cell elongation and secondary cell wall synthesis. Cotton fiber length is regulated by a variety of transcription factors (TF) and their target genes; however, the mechanism of fiber elongation mediated by transcriptional regulatory networks is still unclear to a large extent. Here, we used a comparative assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) assay and RNA-seq analysis to identify fiber elongation transcription factors and genes using the short-fiber mutant ligon linless-2 (Li2 ) and wild type (WT). A total of 499 differential target genes were identified and GO analysis shows that differential genes are mainly involved in plant secondary wall synthesis and microtubule-binding processes. Analysis of the genomic regions preferentially accessible (Peak) has identified a number of overrepresented TF-binding motifs, highlighting sets of TFs that are important for cotton fiber development. Using ATAC-seq and RNA-seq data, we have constructed a functional regulatory network of each TF regulatory target gene and also the network pattern of TF regulating differential target genes. Further, to obtain the genes related to fiber length, the differential target genes were combined with FLGWAS data to identify the genes highly related to fiber length. Our work provides new insights into cotton fiber elongation.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

The cotton protein GhIQD21 interacts with GhCaM7 and modulates organ morphogenesis in Arabidopsis by influencing microtubule stability.

KEY MESSAGE: GhIQD21, a cotton IQ67-domain protein, interacts with GhCaM7 and alters organ shape in Arabidopsis by modulating microtubule stability. Calcium ion (Ca2+) and the calcium sensor calmodulin play crucial roles in the growth and development of plants. GhCaM7, a calmodulin in upland cotton (Gossypium hirsutum L.), is highly expressed in cotton fiber cells during the rapid elongation period and plays an important role in fiber cell development. In this study, we screened for GhCaM7-interacting proteins and identified GhIQD21, which contains a typical IQ67-domain. GhIQD21 was preferentially expressed at the fiber rapid elongation stage, and the protein localized to microtubules (MTs). Ectopic expression of GhIQD21 in Arabidopsis resulted in shorter leaves, petals, siliques, and plant height, thicker inflorescences, and more trichomes when compared with wild type (WT). Further investigation indicated that the morphogenesis of leaf epidermal cells and silique cells was altered. There was less consistency in the orientation of cortical microtubules in cotyledon and hypocotyl epidermal cells. Furthermore, compared with WT, transgenic seedling hypocotyls were more sensitive to oryzalin, a MT depolymerization drug. These results indicated that GhIQD21 is a GhCaM7-interacting protein located in MTs and that it plays a role in plant growth and potentially cotton fiber development. This study provides a foundation for further studies of the function and regulatory mechanism of GhIQD21 in fiber cell development.

Analysis of PAT1 subfamily members in the GRAS family of upland cotton and functional characterization of GhSCL13-2A in Verticillium dahliae resistance.

KEY MESSAGE: GhSCL13-2A, a member of the PAT1 subfamily in the GRAS family, positively regulates cotton resistance to Verticillium dahliae by mediating the jasmonic acid and salicylic acid signaling pathways and accumulation of reactive oxygen species. Verticillium wilt (VW) is a devastating disease of upland cotton (Gossypium hirsutum) that is primarily caused by the soil-borne fungus Verticillium dahliae. Scarecrow-like (SCL) proteins are known to be involved in plant abiotic and biotic stress responses, but their roles in cotton defense responses are still unclear. In this study, a total of 25 GhPAT1 subfamily members in the GRAS family were identified in upland cotton. Gene organization and protein domain analysis showed that GhPAT1 members were highly conserved. GhPAT1 genes were widely expressed in various tissues and at multiple developmental stages, and they were responsive to jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) signals. Furthermore, GhSCL13-2A was induced by V. dahliae infection. V. dahliae resistance was enhanced in Arabidopsis thaliana by ectopic overexpression of GhSCL13-2A, whereas cotton GhSCL13-2A knockdowns showed increased susceptibility. Levels of reactive oxygen species (ROS) and JA were also increased and SA content was decreased in GhSCL13-2A knockdowns. At the gene expression level, PR genes and SA signaling marker genes were down-regulated and JA signaling marker genes were upregulated in GhSCL13-2A knockdowns. GhSCL13-2A was shown to be localized to the cell membrane and the nucleus. Yeast two-hybrid and luciferase complementation assays indicated that GhSCL13-2A interacted with GhERF5. In Arabidopsis, V. dahliae resistance was enhanced by GhERF5 overexpression; in cotton, resistance was reduced in GhERF5 knockdowns. This study revealed a positive role of GhSCL13-2A in V. dahliae resistance, establishing it as a strong candidate gene for future breeding of V. dahliae-resistant cotton cultivars.

Preventive treatment with sodium para-aminosalicylic acid inhibits manganese-induced apoptosis and inflammation via the MAPK pathway in rat thalamus.

Excessive exposure to manganese (Mn) may lead to neurotoxicity, referred to as manganism. In several studies, sodium para-aminosalicylic acid (PAS-Na) has shown efficacy against Mn-induced neurodegeneration by attenuating the neuroinflammatory response. The present study investigated the effect of Mn on inflammation and apoptosis in the rat thalamus, as well as the underlying mechanism of the PAS-Na protective effect. The study consisted of sub-acute (Mn treatment for 4 weeks) and sub-chronic (Mn and PAS-Na treatment for 8 weeks) experiments. In the sub-chronic experiments, pro-inflammatory cytokines, namely tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and cyclooxygenase 2 (COX-2) were significantly increased in the Mn-exposed group compared to the control II. PAS-Na treatment led to a significant reduction in the Mn-induced neuroinflammation by inhibiting IL-1ß and COX-2 mRNA expression and reducing IL-1ß secretion and JNK/p38 MAPK pathway activity. Furthermore, immunohistochemical analysis showed that the expression of caspase-3 was significantly increased in both the sub-acute and sub-chronic experimental paradigms concomitant with a significant decrease in B-cell lymphoma 2 (Bcl-2) in the thalamus of Mn-treated rats. PAS-Na also decreased the expression levels of several apoptotic markers downstream of the MAPK pathway, including Bcl-2/Bax and caspase-3, while up-regulating anti-apoptotic Bcl-2 proteins. In conclusion, Mn exposure led to inflammation in the rat thalamus concomitant with apoptosis, which was mediated via the MAPK signaling pathway. PAS-Na treatment antagonized effectively Mn-induced neurotoxicity by inhibiting the MAPK activity in the same brain region.

Effects of sodium para-aminosalicylic acid on chelation treatment in Pb-exposed mice.

Lead (Pb) is a corrosion-resistant, heavy, non-ferrous metal. Several metal chelators have been used for the treatment of Pb poisoning. However, the efficacy of sodium para-aminosalicylic acid (PAS-Na) in enhancing Pb excretion has yet to be fully characterized. Healthy male mice (90) were divided into six groups, the normal control group was intraperitoneally (i.p.) injected with saline and the remaining group of mice i.p. 120 mg/kg Pb acetate. Four hour later, mice were subcutaneously (back) injected (s.c.) with (80, 160, 240 mg/kg) PAS-Na or 240 mg/kg edetate calcium disodium (CaNa2EDTA) or an equivalent amount of saline, once per day for 6 days. After 24-h urine sample collections, the animals were anesthetized with 5% chloral hydrate and sacrificed in batches on the 2nd, 4th, or 6th day. Levels of Pb [including manganese (Mn) and copper (Cu)] in the urine, whole blood, and brain tissues were analyzed by graphite furnace atomic absorption spectrometry. The results showed that Pb exposure increased its levels in urine and blood, and PAS-Na treatment may afford antagonistic effect on Pb poisoning, suggesting that PAS-Na is a potentially effective treatment to promote excretion of Pb.

Selection and verification of reliable internal reference genes in stem development of herbaceous peony (Paeonia lactiflora Pall.).

Herbaceous peony (Paeonia lactiflora Pall.) has emerged in the cut flower market due to its beautiful appearance. The bending flower stems caused by a lack of mechanical strength is the main problem restricting the development of the cut P. lactiflora industry. So it is of great worth to reveal the basis of stem development changes in P. lactiflora to improve its cut flower quality. Quantitative research on gene expression characteristics can provide clues for understanding their biological functions, and the screening of relatively stable expression genes is a prerequisite for the quantitative study of gene expression characteristics. Thus, it is necessary to find appropriate genes during stem development so as to analyze the qRT‒PCR results. In this study, 10 genes were screened, and these genes expressed stably in stems of different stem strengths at three different developmental stages. Then, their expressions were evaluated by RefFinder, BestKeeper, NormFinder, and GeNorm programs. The results demonstrated that γ-tubulin (γ-TUB) was the most suitable gene, followed by α-tubulin (α-TUB) and ß-D-glucosidase (ß-GUS), whereas histone H3 (His) was the least suitable gene. Besides, the temporal and spatial expression characteristics of PlCOMT1, the key gene concerned with the synthesis of cell wall fillers in P. lactiflora, were also used to evaluate the suitability of genes. Consequently, γ-TUB and α-TUB are the two best combinations during stem development, and their combination can be used for the stem development of P. lactiflora. These findings will provide a reference for the selection of genes related to stem development and the study of molecular mechanisms related to stem development in P. lactiflora. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01325-5.