Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway

BACKGROUND Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved.Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process.AIM To assess the influence of interleukin-10(IL-10)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)following their interaction with macrophages in an inflammatory environment.METHODS IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment.In this study,we investigated its impact on the proliferation,migration,and osteogenesis of BMSCs.The expression levels of signal transducer and activator of transcription 3(STAT3)and its activated form,phos-phorylated-STAT3,were examined in IL-10-stimulated macrophages.Subsequently,a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling.RESULTS IL-10-stimulated macrophages underwent polarization to the M2 type through substitution,and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs.Mechanistically,STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages.Specifically,IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response,as evidenced by its diminished impact on the osteogenic differentiation of BMSCs.CONCLUSION Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs.The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs’osteogenic differentiation.

Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.

Heat-inducible SlWRKY3 confers thermotolerance by activating the SlGRXS1 gene cluster in tomato

High temperature stress is one of the major environmental factors that affect the growth and development of plants. Although WRKY transcription factors play a critical role in stress responses, there are few studies on the regulation of heat stress by WRKY transcription factors,especially in tomato. Here, we identified a group I WRKY transcription factor, SlWRKY3, involved in thermotolerance in tomato. First, SlWRKY3 was induced and upregulated under heat stress. Accordingly, overexpression of SlWRKY3 led to an increase, whereas knock-out of SlWRKY3 resulted in decreased tolerance to heat stress. Overexpression of SlWRKY3 accumulated less reactive oxygen species(ROS), whereas knock-out of SlWRKY3 accumulated more ROS under heat stress. This indicated that SlWRKY3 positively regulates heat stress in tomato. In addition,SlWRKY3 activated the expression of a range of abiotic stress-responsive genes involved in ROS scavenging, such as a SlGRXS1 gene cluster.Further analysis showed that SlWRKY3 can bind to the promoters of the SlGRXS1 gene cluster and activate their expression. Collectively, these results imply that SlWRKY3 is a positive regulator of thermotolerance through direct binding to the promoters of the SlGRXS1 gene cluster and activating their expression and ROS scavenging.

Regulation and function of signal transducer and activator of transcription 3

Signal transducer and activator of transcription 3(STAT3), a member of the STAT family, is a key regulator of many physiological and pathological processes. Significant progress has been made in understanding the transcriptional control, posttranslational modification, cellular localization and functional regulation of STAT3. STAT3 can translocate into the nucleus and bind to specific promoter sequences, thereby exerting transcriptional regulation. Recent studies have shown that STAT3 can also translocate into mitochondria, participating in aerobic respiration and apoptosis. In addition, STAT3 plays an important role in inflammation and tumorigenesis by regulating cell proliferation, differentiation and metabolism. Conditional knockout mouse models make it possible to study the physiological function of STAT3 in specific tissues and organs. This review summarizes the latest advances in the understanding of the expression, regulation and function of STAT3 in physiological and tumorigenic processes.

Inhibition of signal transducer and activator of transcription 3 expression by RNA interference suppresses invasion through inducing anoikis in human colon cancer cells

AIM:To investigate the roles and mechanism of signal transducer and activator of transcription 3 (STAT3) in invasion of human colon cancer cells by RNA interference. METHODS: Small interfering RNA (siRNA) targeting Signal transducer and activator of transcription 3 (STAT3) was transfected into HT29 colon cancer cells. STAT3 protein level and DNA-binding activity of STAT3 was evaluated by western blotting and electrophoretic mobility shift assay (EMSA), respectively. We studied the anchorage-independent growth using colony formation in soft agar, and invasion using the boyden chamber model, anoikis using DNA fragmentation assay and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL), respectively. Western blot assay was used to observe the protein expression of Bcl-xL and survivin in colon cancer HT29 cells. RESULTS: RNA interference (RNAi) mediated by siRNA leads to suppression of STAT3 expression in colon cancer cell lines. Suppression of STAT3 expression by siRNA could inhibit anchorage-independent growth, and invasion ability, and induces anoikis in the colon cancer cell line HT29. It has been shown that knockdown of STAT3 expression by siRNA results in a reduction in expression of Bcl-xL and survivin in HT29 cells. CONCLUSION: These results suggest that STAT3 siRNA can inhibit the invasion ability of colon cancer cells through inducing anoikis, which antiapoptotic genes survivin and Bcl-xL contribute to regulation of anoikis.These studies indicate STAT3 siRNA could be a useful therapeutic tool for the treatment of colon cancer.

Signal transducer and activator of transcription 3 promotes the Warburg effect possibly by inducing pyruvate kinase M2 phosphorylation in liver precancerous lesions

BACKGROUND Study shows that signal transducer and activator of transcription 3(STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2(PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.AIM To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.METHODS A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with Nmethyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen(PCNA), STAT3,and PKM2 were examined by Western blot and immunofluorescence.RESULTS We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liverprecancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression,PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells.Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.CONCLUSION The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

Basic transcription factor 3 is involved in gastric cancer development and progression

AIM: To further analyse cancer involvement of basic transcription factor 3 (BTF3) after detection of its upregulation in gastric tumor samples. METHODS: BTF3 transcription rates in human gastric tumor tissue samples (n = 20) and adjacent normal tissue (n = 18) specimens as well as in the gastric cancer cell lines AGS, SGC-7901, MKN-28, MKN-45 and MGC803 were analyzed via quantitative real-time polymerase chain reaction. The effect of stable BTF3 silencing via infection with a small interfering RNA (siRNA)-BTF3 expressing lentivirus on SGC-7901 cells was measured via Western blotting analysis, proliferation assays, cell cycle and apoptosis profiling by flow cytometry as well as colony forming assays with a Cellomic Assay System. RESULTS: A significant higher expression of BTF3 mRNA was detected in tumors compared to normal gastric tissues (P < 0.01), especially in section tissues from female patients compared to male patients, and all tested gastric cancer cell lines expressed high levels of BTF3. From days 1 to 5, the relative proliferation rates of stable BTF3-siRNA transfected SGC7901 cells were 82%, 70%, 57%, 49% and 44% compared to the control, while the percentage of cells arrested in the G 1 phase was significantly decreased (P = 0.000) and the percentages of cells in the S (P = 0.031) and G 2 /M (P = 0.027) phases were significantly increased. In addition, the colony forming tendency was significantly decreased (P = 0.014) and the apoptosis rate increased from 5.73% to 8.59% (P = 0.014) after BTF3 was silenced in SGC7901 cells. CONCLUSION: BTF3 expression is associated with enhanced cell proliferation, reduced cell cycle regulation and apoptosis and its silencing decreased colony forming and proliferation of gastric cancer cells.

Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

AIM： To explore the effect of silencing of signal transducer and activator of transcription 3 （STAT3） expression by RNA interference （RNAi） on growth of human hepatocellular carcinoma （HCC） in tumorbearing nude mice in vivo.METHODS： To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP （pSHI-siRNA- STAT3） and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSHI-siRNA- STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction （RT- PCR）. Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay was used to detect apoptosis of tumor cells.RESULTS： The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups （P 〈 0.01）. The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased （P 〈 0.01）. Most of the tumor tissue ceils in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION： Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

STAT3 Correlates with Stem Cell-related Transcription Factors in Cervical Cancer

Summary： Cancer stem cells （CSCs） are considered responsible for the high recurrence rate in cervical carcinoma. It has been demonstrated that the signal transducer and activator of transcription 3 （STAT3） is involved in the oncogenesis and takes part in mediating the effects of maintaining stem cell phenotype and pluripotency by regulating the expression of stem cell-related transcription factors. However, the correlation between STAT3 and stem cell-related transcription factors in cervical cancer has not been elucidated. In this study, we established overexpressing plasmid （GV316-STAT3） and siRNA-STAT3 for transfecting Siha cells. Cells negative or positive for Nanog, Oct4, or Sox2 were selected by flow cytometry. Proliferation and differentiation rate of Siha cells was determined by detecting the efficiency of tumor sphere formation. The expression of Nanog, Oct4 and Sox2 （cancer stem cell markers） and STAT3 was detected by quantitative real-time PCR and immunoblotting for Siha cells and by immuno- histochemistry （IHC） for cervical tissues, respectively. The results showed that Nanog＋, Oct4＋, and Sox2＋ Siha-STAT3 over-expressing cells displayed the typical non-adherent spheres. The sphere for- mation efficiency was significantly different between Siha-STAT3 overexpressing cells and siRNA-STAT3 cells （P〈0.05）. Meanwhile, the expression levels of Oct4, Nanog and Sox2 rrtRNA and protein were significantly higher in Siha-STAT3 overexprssing cells than in siRNA-STAT3 cells （P〈0.05）. In addition, the positive rate of STAT3, Nanog, Oct4 and Sox2 in cervical cancer tissues was higher than that in chronic cervicitis group （P〈0.05）. There was a significantly positive relationship between STAT3 and Nanog or Oct4 or Sox2 expression （all P〈0.001）. These results suggested that Oct4＋, Sox2＋, and Nanog＋ cell population possesses stem cell properties in cervical cancer, which may contribute to cervical carcinogenesis and be regulated by STAT3.

Downregulation of signal transducer and activator of transcription 3 by sorafenib:A novel mechanism for hepatocellular carcinoma therapy

Hepatocellular carcinoma is one of the most common cancers worldwide,and a leading cause of cancer-related death.Owing to unsatisfactory clinical outcomes under the current standard of care,there is a need to search for and identify novel and potent therapeutic targets to improve patient outcomes.Sorafenib is the first and only approved targeted therapy for the treatment of hepatocellular carcinoma.Besides functioning as a multiple tyrosine kinase,sorafenib also acts via a kinase-independent mechanism to target signal transducer and activator of transcription 3(STAT3) signaling in hepatocellular carcinoma cells.STAT3 is a key regulator of inflammation,cell survival,and tumorigenesis of liver cells,and the high percentage of hepatocellular carcinoma cells with constitutively active STAT3 justifies targeting it for the development of novel therapeutics.Sorafenib inactivates STAT3 and STAT3-related signaling by inducing a conformational change in and releasing the autoinhibition of Src homology region 2 domaincontaining phosphatase-1.This phosphatase negatively regulates STAT3 activity,which leads to the subsequent apoptosis of cancer cells.The novel anti-cancer property of sorafenib will be discussed in this review,not only adding information regarding its mechanism of action but also providing an innovative approach for the development of cancer therapeutics in the future.

Diffusion-weighted magnetic resonance imaging reflects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/reperfusion

Signal transducer and activator of transcription（STAT）is a unique protein family that binds to DNA,coupled with tyrosine phosphorylation signaling pathways,acting as a transcriptional regulator to mediate a variety of biological effects.Cerebral ischemia and reperfusion can activate STATs signaling pathway,but no studies have confirmed whether STAT activation can be verified by diffusion-weighted magnetic resonance imaging（DWI）in rats after cerebral ischemia/reperfusion.Here,we established a rat model of focal cerebral ischemia injury using the modified Longa method.DWI revealed hyperintensity in parts of the left hemisphere before reperfusion and a low apparent diffusion coefficient.STAT3 protein expression showed no significant change after reperfusion,but phosphorylated STAT3 expression began to increase after 30 minutes of reperfusion and peaked at 24 hours.Pearson correlation analysis showed that STAT3 activation was correlated positively with the relative apparent diffusion coefficient and negatively with the DWI abnormal signal area.These results indicate that DWI is a reliable representation of the infarct area and reflects STAT phosphorylation in rat brain following focal cerebral ischemia/reperfusion.

Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean(Ricinus communis)

The B3 transcription factors(TFs)in plants play vital roles in numerous biological processes.Although B3 genes have been broadly identified in many plants,little is known about their potential functions in mediating seed development and material accumulation.Castor bean(Ricinus communis)is a non-edible oilseed crop considered an ideal model system for seed biology research.Here,we identified a total of 61 B3 genes in the castor bean genome,which can be classified into five subfamilies,including ABI3/VP1,HSI,ARF,RAV and REM.The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development,indicating that these genes may be associated with the accumulation of storage oils.Furthermore,through yeast one-hybrid and tobacco transient expression assays,we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2,which encodes an oil-body structural protein.This finding suggests that RcLEC2,as a seed-specific TF,may be involved in the regulation of storage materials accumulation.This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

Silencing the SLB3 transcription factor gene decreases drought stress tolerance in tomato

BRI1-EMS-SUPPRESSOR 1(BES1)transcription factor is closely associated with the brassinosteroid(BR)signaling pathway and plays an important role in plant growth and development.SLB3 is a member of BES1 transcription factor family and its expression was previously shown to increase significantly in tomato seedlings under drought stress.In the present study,we used virus-induced gene silencing(VIGS)technology to downregulate SLB3 expression to reveal the function of the SLB3 gene under drought stress further.The downregulated expression of SLB3 weakened the drought tolerance of the plants appeared earlier wilting and higher accumulation of H2 O2 and O2^–·,decreased superoxide dismutase(SOD)activity,and increased proline(PRO)and malondialdehyde(MDA)contents and peroxidase(POD)activity.Quantitative real-time PCR(qRT-PCR)analysis of BR-related genes revealed that the expression of SlCPD,SlDWARF and BIN2-related genes was significantly upregulated in SLB3-silenced seedlings under drought stress,but that the expression of TCH4-related genes was downregulated.These results showed that silencing the SLB3 gene reduced the drought resistance of tomato plants and had an impact on the BR signaling transduction which may be probably responsible for the variation in drought resistance of the tomato plants.

Ectopic expression of VvFUS3,B3-domain transcription factor,in tomato influences seed development via affecting endoreduplication and hormones

FUSCA3(FUS3)is a member of B3-domain transcription factor family and master regulator of seed development.It has potential roles in hormone biosynthesis and signaling pathways and therefore plays diverse roles in plant life cycle,especially in seed germination,dormancy,embryo formation,seed and fruit development,and maturation.However,there is limited information about its functions in seed and fruit development of grapevine.In this study,we expressed VvFUS3 in tomato for its functional characterization.Overexpression of VvFUS3 in tomato led to a reduction in seed number and seed weight without affecting the fruit size.Histological analysis found that both cell expansion and cell division in transgenic seed and fruit pericarp have been affected.However,there were no obvious differences in pollen size,shape,and viability,suggesting that VvFUS3 affects seed development but not the pollen grains.Moreover,the expression of several genes with presumed roles in seed development and hormone signaling pathways was also influenced by VvFUS3.These results suggest that VvFUS3 is involved in hormonal signaling pathways that regulate seed number and size.In conclusion,our study provides novel preliminary information about the pivotal roles of VvFUS3 in seed and fruit development and these findings can potentially serve as a reference for molecular breeding of seedless grapes.

18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3

BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GRA)has a variety of pharmacological effects.The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC,in order to provide effective ideas for the clinical prevention and treatment of GC.AIM To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.METHODS Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs(miRNAs)in GC cells after 18β-GRA intervention.Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability,cell colony formation ability was detected by the clone formation assay,and flow cytometry was used to detect the cell cycle and apoptosis.A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells.Tumor tissue morphology was observed by hematoxylin and eosin staining,and microtubule-associated protein light chain 3(LC3)expression was detected by immunohistochemistry.TransmiR,STRING,and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3(STAT3)-related information.Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction(qPCR)and the expression levels of STAT3,phosphorylated STAT3(p-STAT3),and LC3 were detected by western blot analysis.The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system.AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label.LC3 was labeled and autophagy flow was observed under a confocal laser microscope.RESULTS The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells(P=4.51E-06).Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability,arrested the cell cycle in the G0/G1 phase,promoted cell apoptosis,and inhibited the growth of subcutaneous tumors in BALB/c nude mice(P<0.01).No obvious necrosis was observed in the tumor tissue in the negative control group(no drug intervention or lentivirus transfection)and vector group(the blank vector for lentivirus transfection),and more cells were loose and necrotic in the miR-328-3p group.Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3,and STAT3 was closely related to autophagy markers such as p62.After overexpressing miR-328-3p,the expression level of STAT3 mRNA was significantly decreased(P<0.01)and p-STAT3 was downregulated(P<0.05).The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT33’untranslated regions of the wild-type reporter vector group was significantly decreased(P<0.001).Overexpressed miR-328-3p combined with bafilomycin A1(Baf A1)was used to detect the expression of LC3 II.Compared with the vector group,the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated(P<0.05),and compared with the Baf A1 group,the expression level of LC3 II in the overexpressed miR-328-3p+Baf A1 group was upregulated(P<0.01).The expression of LC3 II was detected after intervention of 18β-GRA in GC cells,and the results were consistent with the results of miR-328-3p overexpression(P<0.05).Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis(P<0.001).qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention(P<0.05).Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention(P<0.05).CONCLUSION 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

The R2R3-MYB transcription factor GaPC controls petal coloration in cotton

Although a few cases of genetic epistasis in plants have been reported, the combined analysis of genetically phenotypic segregation and the related molecular mechanism remains rarely studied. Here, we have identified a gene(named GaPC) controlling petal coloration in Gossypium arboreum and following a heritable recessive epistatic genetic model. Petal coloration is controlled by a single dominant gene,GaPC. A loss-of-function mutation of GaPC leads to a recessive gene Gapc that masks the phenotype of other color genes and shows recessive epistatic interactions. Map-based cloning showed that GaPC encodes an R2R3-MYB transcription factor. A 4814-bp long terminal repeat retrotransposon insertion at the second exon led to GaPC loss of function and disabled petal coloration. GaPC controlled petal coloration by regulating the anthocyanin and flavone biosynthesis pathways. Expression of core genes in the phenylpropanoid and anthocyanin pathways was higher in colored than in white petals. Petal color was conferred by flavonoids and anthocyanins, with red and yellow petals rich in anthocyanin and flavonol glycosides, respectively. This study provides new insight on molecular mechanism of recessive epistasis,also has potential breeding value by engineering GaPC to develop colored petals or fibers for multifunctional utilization of cotton.

<i>Trapa japonica</i>Flerov Extract Attenuates Lipid Accumulation through Downregulation of Adipogenic Transcription Factors in 3T3-L1 Cells

Obesity is a major human health problem associated with various diseases, including cardiac injury and type 2 diabetes. Trapa japonica Flerov (TJF) has been used in traditional oriental medicine to treat diabetes. In this study, we evaluated the inhibitory effect of and the mechanism underlying the effect of TJF extract on adipogenesis in 3T3-L1 cells. The effects of TJF extract on cell viability were analyzed using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and the anti-adipogenic effect was measured by oil red O staining. The expression of peroxisomal proliferator activated receptor (PPAR)γ, CCAAT/enhancer-binding protein-α (C/EBP)α, adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), adiponectin, and fatty acid binding protein (FABP)4 involved in adipogenesis was determined by western blot analysis. TJF extract effectively inhibited lipid accumulation and the expression of PPARγ and C/EBPα in 3T3-L1 cells. TJF also increased the phosphorylation of AMPK and ACC, and decreased the expression of adiponectin and FABP4. These results indicate that TJF extract exerts its anti-obesity effect through the downregulation of adipogenic transcription factors and adipogenic marker genes.

Comparative transcriptomic analysis of Rosa sterilis inflorescence branches with different trichome types reveals an R3-MYB transcription factor that negatively regulates trichome formation

Rosa sterilis S.D.Shi is an important economic tree in China that produces fruits with high nutritional and medicinal value.Many of R.sterills’organs are covered with different types of trichomes or prickles that directly affect fruit appearance and plant management.This study used RNA sequencing technology to analyze the transcriptomes of two parts of the inflorescence branch,namely inflorescence stems with flagellated trichomes and pedicels with both flagellated and glandular trichomes.Comparative transcriptomic analysis showed that many transcription factors(TFs)are potentially involved in the formation and development of trichomes.The accumulation of RsETC1,a TF of the R3-MYB family,was significantly higher in inflorescence stems than in pedicels;quantitative reverse transcription PCR(qRTPCR)verified that its expression was significantly higher in inflorescence stems than in pedicels during the first three development stages,indicating its inhibitory action on the initiation of glandular trichomes in R.sterilis.The mRNA level of RsETC1 accumulated to significantly higher levels in trichomeless tissues than in tissues with trichromes,suggesting that this gene may inhibit the formation of trichomes in R.sterilis.Over-expression of RsETC1 in Arabidopsis resulted in glabrous phenotypes,and the expression of trichome-related endogenous genes,except for TTG1,was markedly reduced.In addition,the contents of the phytohormones jasmonic acid(JA),gibberellin A3(GA_(3)),and cytokinins(CKs)in pedicels were significantly higher than those in inflorescence stems,and the expression patterns of the genes related to hormone biosynthesis and signal transduction presented consistent responses,suggesting that the transduction of these hormones might be crucial for trichome initiation and development.These data provide a new perspective for revealing the molecular mechanism of trichome formation in R.sterilis.

特发性间质性肺炎病人血清STAT3和FOXM1水平与病情程度及预后的关系研究

目的探讨特发性间质性肺炎(IIP)病人血清信号转导及转录激活因子3(STAT3)、叉头框转录因子M1(FOXM1)表达水平与其病情程度和预后的关系。方法选取2017年1月至2022年10月四川省建筑医院收治的298例IIP病人为IIP组,选取同时期该院收治的298例普通型间质性肺炎(UIP)病人为UIP组,另选取同时期在该院体检的280例健康人员为对照组。采用酶联免疫吸附测定(ELISA)检测血清STAT3与FOXM1表达水平,并采用全肺纤维化高分辨率CT(HRCT)评分评价IIP病情严重程度。分析三组受试者血清STAT3与FOXM1表达水平差异。对IIP组病人随访3个月,根据其生存情况分为预后良好组与预后不良组,分析预后不良与预后良好组IIP病人血清STAT3与FOXM1表达水平差异,并采用Spearman法分析IIP病人血清STAT3与FOXM1表达水平与全肺纤维化HRCT评分之间的关系。采用logistic回归分析IIP病人预后不良的影响因素,绘制受试者操作特征曲线(ROC曲线)评估血清STAT3与FOXM1表达水平对IIP病人预后不良的预测效能。结果IIP组、UIP组、对照组血清STAT3[(1.50±0.39)ng/L、(1.32±0.31)ng/L、(1.01±0.27)ng/L]、FOXM1[(34.56±5.64)ng/L、(22.69±4.11)ng/L、(15.51±3.94)ng/L]水平均依次降低(P<0.05)。IIP病人随访3个月共有41例病人死亡,预后不良发生率为13.76%(41/298);预后不良组IIP病人血清STAT3、FOXM1水平均高于预后良好组(P<0.05)。Spearman相关性分析表明,IIP病人血清STAT3、FOXM1水平均与全肺纤维化HRCT评分正相关(rs=0.65,rs=0.57;P<0.001)。另预后不良组年龄、Ⅱ型肺泡细胞表面抗原(KL-6)、白细胞计数、红细胞沉降率(ESR)、C反应蛋白(CRP)水平、全肺纤维化HRCT评分均高于预后良好组(P<0.05);第一秒用力呼气量(FEV1)、最大自主通气量(MVV)均低于预后良好组(P<0.05)。多因素logistic回归分析显示,年龄、KL-6、白细胞、ESR、CRP、STAT3、FOXM1水平、全肺纤维化HRCT评分均是导致IIP病人预后不良的危险因素(P<0.05),FEV1、MVV为保护因素(P<0.05)。ROC分析结果表明,血清STAT3、FOXM1水平联合预测IIP病人预后的灵敏度高于STAT3、FOXM1单独预测的灵敏度(χ^(2)=8.10、6.12,P=0.002、0.008);联合预测的曲线下面积(AUC)高于STAT3、FOXM1单独预测的AUC(Z=3.15、2.54,P=0.002、0.011)。结论IIP病人血清STAT3、FOXM1表达水平均上升,二者与IIP病情程度正相关,均对IIP病人预后具有良好的预测价值,且联合预测效能更高。

基于超高通量筛选的D-阿洛酮糖3-差向异构酶定向进化

D-阿洛酮糖是一种低热量的甜味剂,具有多种生理功能,如维持血糖平衡和调节脂质代谢等。目前,D-阿洛酮糖工业生产主要通过D-阿洛酮糖3-差向异构酶(D-allulose-3-epimerase,DAEase)催化D-果糖的差向异构反应实现,但DAEase的应用性能仍需进一步提升以满足日益增长的市场需求。定向进化作为一种有效的分子改造策略,其筛选方法是限制效率的关键因素之一。通过构建并优化D-阿洛酮糖特异型生物传感器,结合微孔板筛选,对来自Clostridium cellulolyticum H10的DAEase(Cc DAEase)突变体文库进行超高通量筛选,最终获得了比活提高14.6%的优势突变体H209Q,其最适温度为60℃,最适pH值为7.5,并进一步解析了该突变体的构效关系。该研究证明了转录因子型生物传感器可以作为一种有效的定向进化筛选工具,为进一步优化DAEase的性能提供了可能性,亦为D-阿洛酮糖的工业制备提供了理论依据和技术支撑。