Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits.

Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneously screen 2,756 variants in strong linkage disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164. Functional follow-up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type-specific transcriptional regulatory pathways.

Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency.

Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.

Genome-wide chromatin state transitions associated with developmental and environmental cues.

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming.

Transcriptional and epigenetic dynamics during specification of human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.

The AGAMOUS-LIKE 16-GENERAL REGULATORY FACTOR 1 module regulates axillary bud outgrowth via catabolism of abscisic acid in cucumber.

Lateral branches are important components of shoot architecture and directly affect crop yield and production cost. Although sporadic studies have implicated abscisic acid (ABA) biosynthesis in axillary bud outgrowth, the function of ABA catabolism and its upstream regulators in shoot branching remain elusive. Here, we showed that the MADS-box transcription factor AGAMOUS-LIKE 16 (CsAGL16) is a positive regulator of axillary bud outgrowth in cucumber (Cucumis sativus). Functional disruption of CsAGL16 led to reduced bud outgrowth, whereas overexpression of CsAGL16 resulted in enhanced branching. CsAGL16 directly binds to the promoter of the ABA 8'-hydroxylase gene CsCYP707A4 and promotes its expression. Loss of CsCYP707A4 function inhibited axillary bud outgrowth and increased ABA levels. Elevated expression of CsCYP707A4 or treatment with an ABA biosynthesis inhibitor largely rescued the Csagl16 mutant phenotype. Moreover, cucumber General Regulatory Factor 1 (CsGRF1) interacts with CsAGL16 and antagonizes CsAGL16-mediated CsCYP707A4 activation. Disruption of CsGRF1 resulted in elongated branches and decreased ABA levels in the axillary buds. The Csagl16 Csgrf1 double mutant exhibited a branching phenotype resembling that of the Csagl16 single mutant. Therefore, our data suggest that the CsAGL16-CsGRF1 module regulates axillary bud outgrowth via CsCYP707A4-mediated ABA catabolism in cucumber. Our findings provide a strategy to manipulate ABA levels in axillary buds during crop breeding to produce desirable branching phenotypes.

Natural variation in CRABS CLAW contributes to fruit length divergence in cucumber.

Fruit length is a key domestication trait that affects crop yield and appearance. Cucumber (Cucumis sativus) fruits vary from 5 to 60 cm in length. Despite the identification of several regulators and multiple quantitative trait loci (QTLs) underlying fruit length, the natural variation, and molecular mechanisms underlying differences in fruit length are poorly understood. Through map-based cloning, we identified a nonsynonymous polymorphism (G to A) in CRABS CLAW (CsCRC) as underlying the major-effect fruit size/shape QTL FS5.2 in cucumber. The short-fruit allele CsCRCA is a rare allele that has only been found in round-fruited semi-wild Xishuangbanna cucumbers. A near-isogenic line (NIL) homozygous for CsCRCA exhibited a 34∼39% reduction in fruit length. Introducing CsCRCG into this NIL rescued the short-fruit phenotype, and knockdown of CsCRCG resulted in shorter fruit and smaller cells. In natural cucumber populations, CsCRCG expression was positively correlated with fruit length. Further, CsCRCG, but not CsCRCA, targets the downstream auxin-responsive protein gene CsARP1 to regulate its expression. Knockout of CsARP1 produced shorter fruit with smaller cells. Hence, our work suggests that CsCRCG positively regulates fruit elongation through transcriptional activation of CsARP1 and thus enhances cell expansion. Using different CsCRC alleles provides a strategy to manipulate fruit length in cucumber breeding.

Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells.

Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.

GLABRA 2 regulates ETHYLENE OVERPRODUCER 1 accumulation during nutrient deficiency-induced root hair growth.

Root hairs (RHs), extensive structures of root epidermal cells, are important for plant nutrient acquisition, soil anchorage, and environmental interactions. Excessive production of the phytohormone ethylene (ET) leads to substantial root hair growth, manifested as tolerance to plant nutrient deficiencies. However, the molecular basis of ET production during root hair growth in response to nutrient starvation remains unknown. Herein, we found that a critical transcription factor, GLABRA 2 (GL2), inhibits ET production during root hair growth in Arabidopsis (Arabidopsis thaliana). GL2 directly binds to the promoter of the gene encoding ET OVERPRODUCER 1 (ETO1), one of the most important ET-production-regulation factors, in vitro and in vivo, and then regulates the accumulation and function of ETO1 in root hair growth. The GL2-regulated-ETO1 module is required for promoting root hair growth under nitrogen, phosphorus, or potassium deficiency. Genome-wide analysis revealed numerous genes, such as ROOT HAIR DEFECTIVE 6-LIKE 4, ETHYLENE-INSENSITIVE 3-LIKE 2, ROOT HAIR SPECIFIC 13, are involved in the GL2-regulated-ETO1 module. Our work reveals a key transcription mechanism in the control of ET production during root hair growth under three major nutrient deficiencies.

Comparative epigenomic analysis of murine and human adipogenesis.

We report the generation and comparative analysis of genome-wide chromatin state maps, PPARγ and CTCF localization maps, and gene expression profiles from murine and human models of adipogenesis. The data provide high-resolution views of chromatin remodeling during cellular differentiation and allow identification of thousands of putative preadipocyte- and adipocyte-specific cis-regulatory elements based on dynamic chromatin signatures. We find that the specific locations of most such elements differ between the two models, including at orthologous loci with similar expression patterns. Based on sequence analysis and reporter assays, we show that these differences are determined, in part, by evolutionary turnover of transcription factor motifs in the genome sequences and that this turnover may be facilitated by the presence of multiple distal regulatory elements at adipogenesis-dependent loci. We also utilize the close relationship between open chromatin marks and transcription factor motifs to identify and validate PLZF and SRF as regulators of adipogenesis.

The CsHEC1-CsOVATE module contributes to fruit neck length variation via modulating auxin biosynthesis in cucumber.

Fruit neck is the proximal portion of the fruit with undesirable taste that has detrimental effects on fruit shape and commercial value in cucumber. Despite the dramatic variations in fruit neck length of cucumber germplasms, the genes and regulatory mechanisms underlying fruit neck elongation remain mysterious. In this study, we found that Cucumis sativus HECATE1 (CsHEC1) was highly expressed in fruit neck. Knockout of CsHEC1 resulted in shortened fruit neck and decreased auxin accumulation, whereas overexpression of CsHEC1 displayed the opposite effects, suggesting that CsHEC1 positively regulated fruit neck length by modulating local auxin level. Further analysis showed that CsHEC1 directly bound to the promoter of the auxin biosynthesis gene YUCCA4 (CsYUC4) and activated its expression. Enhanced expression of CsYUC4 resulted in elongated fruit neck and elevated auxin content. Moreover, knockout of CsOVATE resulted in longer fruit neck and higher auxin. Genetic and biochemical data showed that CsOVATE physically interacted with CsHEC1 to antagonize its function by attenuating the CsHEC1-mediated CsYUC4 transcriptional activation. In cucumber germplasms, the expression of CsHEC1 and CsYUC4 positively correlated with fruit neck length, while that of CsOVATE showed a negative correlation. Together, our results revealed a CsHEC1-CsOVATE regulatory module that confers fruit neck length variation via CsYUC4-mediated auxin biosynthesis in cucumber.

The exocyst subunit CsExo70B promotes both fruit length and disease resistance via regulating receptor kinase abundance at plasma membrane in cucumber.

Plant defence against pathogens generally occurs at the expense of growth and yield. Uncoupling the inverse relationship between growth and defence is of great importance for crop breeding, while the underlying genes and regulatory mechanisms remain largely elusive. The exocytosis complex was shown to play an important role in the trafficking of receptor kinases (RKs) to the plasma membrane (PM). Here, we found a Cucumis sativus exocytosis subunit Exo70B (CsExo70B) regulates the abundance of both development and defence RKs at the PM to promote fruit elongation and disease resistance in cucumber. Knockout of CsExo70B resulted in shorter fruit and susceptibility to pathogens. Mechanistically, CsExo70B associates with the developmental RK CsERECTA, which promotes fruit longitudinal growth in cucumber, and contributes to its accumulation at the PM. On the other side, CsExo70B confers to the spectrum resistance to pathogens in cucumber via a similar regulatory module of defence RKs. Moreover, CsExo70B overexpression lines showed an increased fruit yield as well as disease resistance. Collectively, our work reveals a regulatory mechanism that CsExo70B promotes both fruit elongation and disease resistance by maintaining appropriate RK levels at the PM and thus provides a possible strategy for superior cucumber breeding with high yield and robust pathogen resistance.

Evolutionary gain of oligosaccharide hydrolysis and sugar transport enhanced carbohydrate partitioning in sweet watermelon fruits.

How raffinose (Raf) family oligosaccharides, the major translocated sugars in the vascular bundle in cucurbits, are hydrolyzed and subsequently partitioned has not been fully elucidated. By performing reciprocal grafting of watermelon (Citrullus lanatus) fruits to branch stems, we observed that Raf was hydrolyzed in the fruit of cultivar watermelons but was backlogged in the fruit of wild ancestor species. Through a genome-wide association study, the alkaline alpha-galactosidase ClAGA2 was identified as the key factor controlling stachyose and Raf hydrolysis, and it was determined to be specifically expressed in the vascular bundle. Analysis of transgenic plants confirmed that ClAGA2 controls fruit Raf hydrolysis and reduces sugar content in fruits. Two single-nucleotide polymorphisms (SNPs) within the ClAGA2 promoter affect the recruitment of the transcription factor ClNF-YC2 (nuclear transcription factor Y subunit C) to regulate ClAGA2 expression. Moreover, this study demonstrates that C. lanatus Sugars Will Eventually Be Exported Transporter 3 (ClSWEET3) and Tonoplast Sugar Transporter (ClTST2) participate in plasma membrane sugar transport and sugar storage in fruit cell vacuoles, respectively. Knocking out ClAGA2, ClSWEET3, and ClTST2 affected fruit sugar accumulation. Genomic signatures indicate that the selection of ClAGA2, ClSWEET3, and ClTST2 for carbohydrate partitioning led to the derivation of modern sweet watermelon from non-sweet ancestors during domestication.

Deep learning-based 3D quantitative total tumor burden predicts early recurrence of BCLC A and B HCC after resection.

OBJECTIVES: This study aimed to evaluate the potential of deep learning (DL)-assisted automated three-dimensional quantitative tumor burden at MRI to predict postoperative early recurrence (ER) of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This was a single-center retrospective study enrolling patients who underwent resection for BCLC A and B HCC and preoperative contrast-enhanced MRI. Quantitative total tumor volume (cm3) and total tumor burden (TTB, %) were obtained using a DL automated segmentation tool. Radiologists' visual assessment was used to ensure the quality control of automated segmentation. The prognostic value of clinicopathological variables and tumor burden-related parameters for ER was determined by Cox regression analyses. RESULTS: A total of 592 patients were included, with 525 and 67 patients assigned to BCLC A and B, respectively (2-year ER rate: 30.0% vs. 45.3%; hazard ratio (HR) = 1.8; p = 0.007). TTB was the most important predictor of ER (HR = 2.2; p < 0.001). Using 6.84% as the threshold of TTB, two ER risk strata were obtained in overall (p < 0.001), BCLC A (p < 0.001), and BCLC B (p = 0.027) patients, respectively. The BCLC B low-TTB patients had a similar risk for ER to BCLC A patients and thus were reassigned to a BCLC An stage; whilst the BCLC B high-TTB patients remained in a BCLC Bn stage. The 2-year ER rate was 30.5% for BCLC An patients vs. 58.1% for BCLC Bn patients (HR = 2.8; p < 0.001). CONCLUSIONS: TTB determined by DL-based automated segmentation at MRI was a predictive biomarker for postoperative ER and facilitated refined subcategorization of patients within BCLC stages A and B. CLINICAL RELEVANCE STATEMENT: Total tumor burden derived by deep learning-based automated segmentation at MRI may serve as an imaging biomarker for predicting early recurrence, thereby improving subclassification of Barcelona Clinic Liver Cancer A and B hepatocellular carcinoma patients after hepatectomy. KEY POINTS: Total tumor burden (TTB) is important for Barcelona Clinic Liver Cancer (BCLC) staging, but is heterogenous. TTB derived by deep learning-based automated segmentation was predictive of postoperative early recurrence. Incorporating TTB into the BCLC algorithm resulted in successful subcategorization of BCLC A and B patients.

Association of food folate with asthma in US children and adolescents: a cross-sectional study.

BACKGROUND: Asthma is a chronic inflammatory disease. Currently, contradictory findings exist regarding the association between food folate and asthma. Therefore, we hypothesize a positive correlation between food folate and asthma. PURPOSE: To investigate the possible relationship between food folate intake and the development of asthma in children and adolescents in the United States. METHODS: Data from the U.S. National Health and Nutrition Examination Survey (NHANES) from 2009 to 2018 were analyzed cross-sectionally by covariate adjustment using multivariate logistic regression, restricted triple spline curves, threshold effects, and stratified analyses. RESULTS: There were 8,821 participants, of whom 1,697 (19.2%) self-reported having received a diagnosis of asthma from a physician or other health professional. After accounting for potential confounders, the adjusted odds ratios (ORs) for asthma in the second (T2, 111-178 µg/day) and third (T3, >178 µg/day) groups were 1.15 (1-1.33) and 1.23 (1.04-1.46), respectively, compared with the group with the lowest food folate intake (T1, <111 µg/day). In addition, the association between food folate intake and asthma showed an inverse L-shaped curve (non-linear relationship, p = 0.003), and stratified analysis further validated the robustness of the results. The OR of asthma in subjects with food folate intake less than 263.9 µg/day was 1.002 (1.001-1.004). CONCLUSION: In children and adolescents in the United States, there is a non-linear association (inverted "L" shape) between food folate intake and asthma, with an inflection point at 263.9 micrograms per day.

Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation.

BACKGROUND: Immature cumulus-oocyte complexes are retrieved to obtain mature oocytes by in vitro maturation (IVM), a laboratory tool in reproductive medicine to obtain mature oocytes. Unfortunately, the efficiency of IVM is not satisfactory. To circumvent this problem, we therefore intended to commence with the composition of ovarian follicular fluid (FF), an important microenvironment influencing oocyte growth. It is well known that FF has a critical role in oocyte development and maturation. However, the components in human FF remain largely unknown, particularly with regard to small molecular peptides. RESULTS: In current study, the follicular fluid derived from human mature and immature follicles were harvested. The peptide profiles of FF were further investigated by using combined ultrafiltration and LC-MS/MS. The differential peptides were preliminary determined by performing differentially expressed analysis. Human and mouse oocyte culture were used to verify the influence of differential peptides on oocyte development. Constructing plasmids, cell transfecting, Co-IP, PLA etc. were used to reveal the detail molecular mechanism. The results from differentially expressed peptide as well as cultured human and mouse oocytes analyses showed that highly conserved C3a-peptide, a cleavage product of complement C3a, definitely affected oocytes development. Intriguingly, C3a-peptide possessed a novel function that promoted F-actin aggregation and spindle migration, raised the percentage of oocytes at the MII stage, without increasing the chromosome aneuploidy ratio, especially in poor-quality oocytes. These effects of C3a-peptide were attenuated by C3aR morpholino inhibition, suggesting that C3a-peptide affected oocytes development by collaborating with its classical receptor, C3aR. Specially, we found that C3aR co-localized to the spindle with ß-tubulin to recruit F-actin toward the spindle and subcortical region of the oocytes through specific binding to MYO10, a key regulator for actin organization, spindle morphogenesis and positioning in oocytes. CONCLUSIONS: Our results provide a new perspective for improving IVM culture systems by applying FF components and also provide molecular insights into the physiological function of C3a-peptide, its interaction with C3aR, and their roles in enabling meiotic division of oocytes.

The learning curve of the MS-TRAM/DIEP breast reconstruction by dual-trained breast surgeons.

BACKGROUND: Breast cancer surgeries involving MS-TRAM/DIEP breast reconstruction has traditionally been collaborative efforts between breast surgeons and plastic surgeons. However, in our institution, this procedure is performed by dual-trained breast surgeons who are proficient in both breast surgery and MS-TRAM/DIEP breast reconstruction. This study aims to provide insights into the learning curve associated with this surgical approach. MATERIALS AND METHODS: We included eligible breast cancer patients who underwent MS-TRAM/DIEP breast reconstruction by dual-trained breast surgeons between 2015 and 2020 at our institution. We present the learning curve of this surgical approach, with a focus on determining factors affecting flap harvesting time, surgery time, and ischemic time. Additionally, we assessed the surgical complication rates. RESULTS: A total of 147 eligible patients were enrolled in this study. Notably, after 30 cases, a statistically significant reduction of 1.7 h in surgery time and 21 min in ischemic time was achieved, signifying the attainment of a plateau in the learning curve. And the major and minor complications were comparable between the early and after 30 cases. CONCLUSION: This study explores the learning curve and feasibility experienced by dual-trained breast surgeons in performing MS-TRAM/DIEP breast reconstruction. TRIAL REGISTRATION: NCT05560633.

Integrative ATAC-seq and RNA-seq Analysis of the Longissimus Dorsi Muscle of Gannan Yak and Jeryak.

Jeryak is the F1 generation of the cross between Gannan yak and Jersey cattle, which has the advantages of fast growth and high adaptability. The growth and development of skeletal muscle is closely linked to meat production and the quality of meat. However, the molecular regulatory mechanisms of muscle growth differences between Gannan yak and Jeryak analyzed from the perspective of chromatin opening have not been reported. In this study, ATAC-seq was used to analyze the difference of chromatin openness in longissimus muscle of Gannan yak and Jeryak. It was found that chromatin accessibility was more enriched in Jeryak compared to Gannan yak, especially in the range of the transcription start site (TSS) ± 2 kb. GO and KEGG enrichment analysis indicate that differential peak-associated genes are involved in the negative regulation of muscle adaptation and the Hippo signaling pathway. Integration analysis of ATAC-seq and RNA-seq revealed overlapping genes were significantly enriched during skeletal muscle cell differentiation and muscle organ morphogenesis. At the same time, we screened FOXO1, ZBED6, CRY2 and CFL2 for possible involvement in skeletal muscle development, constructed a genes and transcription factors network map, and found that some transcription factors (TFs), including YY1, KLF4, KLF5 and Bach1, were involved in skeletal muscle development. Overall, we have gained a comprehensive understanding of the key factors that impact skeletal muscle development in various breeds of cattle, providing new insights for future analysis of the molecular regulatory mechanisms involved in muscle growth and development.

Unlocking the Transcriptional Control of NCAPG in Bovine Myoblasts: CREB1 and MYOD1 as Key Players.

Muscle formation directly determines meat production and quality. The non-SMC condensin I complex subunit G (NCAPG) is strongly linked to the growth features of domestic animals because it is essential in controlling muscle growth and development. This study aims to elucidate the tissue expression level of the bovine NCAPG gene, and determine the key transcription factors for regulating the bovine NCAPG gene. In this study, we observed that the bovine NCAPG gene exhibited high expression levels in longissimus dorsi and spleen tissues. Subsequently, we cloned and characterized the promoter region of the bovine NCAPG gene, consisting of a 2039 bp sequence, through constructing the deletion fragment double-luciferase reporter vector and site-directed mutation-identifying core promoter region with its key transcription factor binding site. In addition, the key transcription factors of the core promoter sequence of the bovine NCAPG gene were analyzed and predicted using online software. Furthermore, by integrating overexpression experiments and the electrophoretic mobility shift assay (EMSA), we have shown that cAMP response element binding protein 1 (CREB1) and myogenic differentiation 1 (MYOD1) bind to the core promoter region (-598/+87), activating transcription activity in the bovine NCAPG gene. In conclusion, these findings shed important light on the regulatory network mechanism that underlies the expression of the NCAPG gene throughout the development of the muscles in beef cattle.

CsRAXs negatively regulate leaf size and fruiting ability through auxin glycosylation in cucumber.

Leaves are the main photosynthesis organ that directly determines crop yield and biomass. Dissecting the regulatory mechanism of leaf development is crucial for food security and ecosystem turn-over. Here, we identified the novel function of R2R3-MYB transcription factors CsRAXs in regulating cucumber leaf size and fruiting ability. Csrax5 single mutant exhibited enlarged leaf size and stem diameter, and Csrax1/2/5 triple mutant displayed further enlargement phenotype. Overexpression of CsRAX1 or CsRAX5 gave rise to smaller leaf and thinner stem. The fruiting ability of Csrax1/2/5 plants was significantly enhanced, while that of CsRAX5 overexpression lines was greatly weakened. Similarly, cell number and free auxin level were elevated in mutant plants while decreased in overexpression lines. Biochemical data indicated that CsRAX1/5 directly promoted the expression of auxin glucosyltransferase gene CsUGT74E2. Therefore, our data suggested that CsRAXs function as repressors for leaf size development by promoting auxin glycosylation to decrease free auxin level and cell division in cucumber. Our findings provide new gene targets for cucumber breeding with increased leaf size and crop yield.

[Trametenolic acid inhibits migration and invasion of hepatocellular carcinoma HepG2.2.15 cells via RhoC/ROCK1 pathway].

This study investigated the effect of trametenolic acid(TA) on the migration and invasion of human hepatocellular carcinoma HepG2.2.15 cells by using Ras homolog gene family member C(RhoC) as the target and probed into the mechanism, aiming to provide a basis for the utilization of TA. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the proliferation of HepG2.2.15 cells exposed to TA, and scratch and Transwell assays to examine the cell migration and invasion. The pull down assay was employed to determine the impact of TA on RhoC GTPase activity. Western blot was employed to measure the effect of TA on the transport of RhoC from cytoplasm to cell membrane and the expression of RhoC/Rho-associated kinase 1(ROCK1)/myosin light chain(MLC)/matrix metalloprotease 2(MMP2)/MMP9 pathway-related proteins. RhoC was over-expressed by transient transfection of pcDNA3.1-RhoC. The changes of F-actin in the cytoskeleton were detected by Laser confocal microscopy. In addition, the changes of cell migration and invasion, expression of proteins in the RhoC/ROCK1/MLC/MMP2/MMP9 pathway, and RhoC GTPase activity were detected. The subcutaneously transplanted tumor model of BALB/c nude mice and the low-, medium-, and high-dose(40, 80, and 120 mg·kg~(-1), respectively) TA groups were established and sorafenib(20 mg·kg~(-1)) was used as the positive control. The tumor volume and weight in each group were measured, and the expression of related proteins in the tumor tissue was determined by Western blot. The results showed that TA inhibited the proliferation of HepG2.2.15 cells in a concentration-dependent manner, with the IC_(50) of 66.65 and 23.09 µmol·L~(-1) at the time points of 24 and 48 h, respectively. The drug administration groups had small tumors with low mass. The tumor inhibition rates of sorafenib and low-, medium-and high-dose TA were 62.23%, 26.48%, 55.45%, and 62.36%, respectively. TA reduced migrating and invading cells and inhibited RhoC protein expression and RhoC GTPase activity in a concentration-dependent manner, dramatically reducing RhoC and membrane-bound RhoC GTPase. The expression of ROCK1, MLC, p-MLC, MMP2, and MMP9 downstream of RhoC can be significantly inhibited by TA, as confirmed in both in vitro and in vivo experiments. After HepG2.2.15 cells were transfected with pcDNA3.1-RhoC to overexpress RhoC, TA down-regulated the protein levels of RhoC, ROCK1, MLC, p-MLC, MMP2, and MMP9 and decreased the activity of RhoC GTPase, with the inhibition level comparable to that before overexpression. In summary, TA can inhibit the migration and invasion of HepG2.2.15 cells. It can inhibit the RhoC/ROCK1/MLC/MMP2/MMP9 signaling pathway by suppressing RhoC GTPase activity and down-regulating RhoC expression. This study provides a new idea for the development of autophagy modulators targeting HSP90α to block the proliferation and inhibit the invasion and migration of hepatocellular carcinoma cells via multiple targets of active components in traditional Chinese medicines.