Fine mapping and transcriptome profiling reveal CpAPRR2 to modulate immature fruit rind color formation in zucchini (Cucurbita pepo).

KEY MESSAGE: A large fragment deletion of CpAPRR2, encoding a two-component response regulator-like protein, which influences immature white rind color formation in zucchini (Cucurbita pepo). Fruit rind color is an important agronomic trait that affects commodity quality and consumer choice in zucchini (Cucurbita pepo). However, the molecular mechanism controlling rind color is unclear. We characterized two zucchini inbred lines: '19' (dark green rind) and '113' (white rind). Genetic analysis revealed white immature fruit rind color to be controlled by a dominant locus (CpW). Combining bulked segregant analysis sequencing (BSA-seq) and Kompetitive Allele-Specific PCR (KASP) markers, we mapped the CpW locus to a 100.4 kb region on chromosome 5 and then narrow down the candidate region to 37.5 kb using linkage analysis of 532 BC1 and 1613 F2 individuals, including 6 coding genes. Among them, Cp4.1LG05g02070 (CpAPRR2), encoding a two-component response regulator-like protein, was regarded to be a promising candidate gene. The expression level of CpAPRR2 in dark green rind was significantly higher than that in white rind and was induced by light. A deletion of 2227 bp at the 5' end of CpAPRR2 in '113' might explain the white phenotype. Further analysis of allelic diversity in zucchini germplasm resources revealed rind color to be associated with the deletion of CpAPRR2. Subcellular localization analysis indicated that CpAPRR2 was a nuclear protein. Transcriptome analysis using near-isogenic lines with dark green (DG) and white (W) rind indicated that genes involved in photosynthesis and porphyrin metabolism pathways were enriched in DG compared with W. Additionally, chlorophyll synthesis-related genes were upregulated in DG. These results identify mechanisms of zucchini rind color and provide genetic resources for breeding.

Comparing the differences in quality profiles and antioxidant activity in seven pumpkin cultivars (Cucurbita moschata and Cucurbita maxima) at harvest and during postharvest storage.

Pumpkin, nutritious vegetable, is renowned for its extended shelf life. In this study, seven pumpkin cultivars from Cucurbita moschata and Cucurbita maxima were comparatively characterized for 25 physiochemical quality factors, starch granule structures, antioxidant activity, and correlations at 0-60 days of postharvest (dop). The findings revealed that sucrose and carotenoid contents increased in C. moschata, while they initially increased and then decreased in C. maxima. Additionally, acidity, primarily driven by malic acid, decreased in C. maxima but increased in C. maxima. The starch content of C. moschata and C. maxima reached its maximum value at 30 dop and 20 dop, respectively. The DPPH radical scavenging activity correlated with the carotenoid content in both pumpkin species. Conclusively, C. moschata demonstrated improved nutritional and quality at 20-30 dop, while C. maxima exhibited higher commercial suitability at 10-20 dop. The findings suggested that pumpkin storage was crucial for quality improvement.

Morphological Analyses and QTL Mapping of Mottled Leaf in Zucchini (Cucurbita pepo L.).

The mottled leaf is one of the agronomic traits of zucchini and can be applied as a marker trait in aggregation breeding. However, the genetic mechanism responsible for mottled leaf has yet to be elucidated. In the present study, we used two inbred lines (line '19': silver mottled leaf; line '113': normal leaf) as parents for the physiological and genetic analysis of mottled leaf. The synthesis and net photosynthetic rate of chlorophyll were not significantly affected in the mottled areas of leaves. However, we detected a large space between the palisade parenchyma in the leaf mottle area of line '19', which may have caused the mottled leaf phenotype. Light also plays an important role in the formation of mottled leaf, and receiving light during the early stages of leaf development is a necessary factor. Genetic analysis has previously demonstrated that mottled leaf is a quantitative trait that is controlled by multiple genes. Based on the strategy of quantitative trait locus sequencing (QTL-seq), two QTLs were identified on chromosomes 1 and 17, named CpML1.1 and CpML17.1, respectively. Two major loci were identified using R/qtl software version 1.66 under greenhouse conditions in April 2019 (2019A) and April 2020 (2020A) and under open cultivation conditions in May 2020 (2020M). The major QTL, CpML1.1, was located in a 925.2-kb interval on chromosome 1 and explained 10.51%-24.15% of the phenotypic variation. The CpML17.1 was located in a 719.7-kb interval on chromosome 17 and explained 16.25%-38.68% of the phenotypic variation. Based on gene annotation, gene sequence alignment, and qRT-PCR analysis, the Cp4.1LG01g23790 at the CpML1.1 locus encoding a protein of the TPX2 family (target protein of Xklp2) may be a candidate gene for mottled leaf in zucchini. Our findings may provide a theoretical basis for the formation of mottled leaf and provide a foundation for the fine mapping of genes associated with mottled leaf. Molecular markers closely linked to mottled leaf can be used in molecular-assisted selection for the zucchini mottled leaf breeding.

Gut commensal Christensenella minuta modulates host metabolism via acylated secondary bile acids.

A strong correlation between gut microbes and host health has been observed in numerous gut metagenomic cohort studies. However, the underlying mechanisms governing host-microbe interactions in the gut remain largely unknown. Here we report that the gut commensal Christensenella minuta modulates host metabolism by generating a previously undescribed class of secondary bile acids with 3-O-acylation substitution that inhibit the intestinal farnesoid X receptor. Administration of C. minuta alleviated features of metabolic disease in high fat diet-induced obese mice associated with a significant increase in these acylated bile acids, which we refer to as 3-O-acyl-cholic acids. Specific knockout of intestinal farnesoid X receptor in mice counteracted the beneficial effects observed in their wild-type counterparts. Finally, we showed that 3-O-acyl-CAs were prevalent in healthy humans but significantly depleted in patients with type 2 diabetes. Our findings indicate a role for C. minuta and acylated bile acids in metabolic diseases.

GPNMB+ Gal-3+ hepatic parenchymal cells promote immunosuppression and hepatocellular carcinogenesis.

Hepatocellular carcinoma (HCC) formation is a multi-step pathological process that involves evolution of a heterogeneous immunosuppressive tumor microenvironment. However, the specific cell populations involved and their origins and contribution to HCC development remain largely unknown. Here, comprehensive single-cell transcriptome sequencing was applied to profile rat models of toxin-induced liver tumorigenesis and HCC patients. Specifically, we identified three populations of hepatic parenchymal cells emerging during HCC progression, termed metabolic hepatocytes (HCMeta ), Epcam+ population with differentiation potential (EP+Diff ) and immunosuppressive malignant transformation subset (MTImmu ). These distinct subpopulations form an oncogenic trajectory depicting a dynamic landscape of hepatocarcinogenesis, with signature genes reflecting the transition from EP+Diff to MTImmu . Importantly, GPNMB+ Gal-3+ MTImmu cells exhibit both malignant and immunosuppressive properties. Moreover, SOX18 is required for the generation and malignant transformation of GPNMB+ Gal-3+ MTImmu cells. Enrichment of the GPNMB+ Gal-3+ MTImmu subset was found to be associated with poor prognosis and a higher rate of recurrence in patients. Collectively, we unraveled the single-cell HCC progression atlas and uncovered GPNMB+ Gal-3+ parenchymal cells as a major subset contributing to the immunosuppressive microenvironment thus malignance in HCC.

Neutrophil-to-Lymphocyte Ratio and Early Tumor Shrinkage as Predictive Biomarkers in Unresectable Hepatocellular Carcinoma Patients Treated With Lenvatinib, PD-1 Inhibitors, in Combination With TACE.

Purpose: The purpose of this prospective observational study was to investigate the relationship between pretreatment neutrophil-to-lymphocyte ratio (NLR) and posttreatment early tumor shrinkage (ETS), and clinical outcomes in patients with unresectable hepatocellular carcinoma (uHCC) who received lenvatinib, programmed death-1 inhibitors plus transcatheter arterial chemoembolization. Patients and Methods: A total of 63 uHCC patients were treated with this triple combination. Multivariate analyses to determine the independent factors associated with overall survival (OS) were employed. The link between NLR and clinical results was further analyzed. Furthermore, the predictive value of combining NLR with ETS should be investigated to stratify patients receiving treatment for survival benefits. Results: Progression-free survival and OS were 9.8 and 23.0 months, respectively, with a median follow-up of 20.8 months. On a multivariate analysis of OS, NLR was the only independent prognostic factor. Patients with NLR low (NLR < 3.2) had longer progression-free survival (19.3 vs 7.3 months, P < 0.001) and OS (28.9 vs 16.9 months, P < 0.001), higher objective response rate (86.7% vs 39.4%, P < 0.001), and a higher chance of achieving ETS ≥ 10% (ETS high) (73.3% vs 21.1%, P < 0.001) compared with patients with NLR high (NLR ≥ 3.2). The Spearman correlation analysis also showed the strong consistency between NLR and ETS (R2 = 0.6751). In the subgroup analysis, greater OS benefit was found in the NLR low/ETS high group than the NLR high/ETS low group (χ2 = 31.258, P < 0.001), while there was no survival difference for patients in the NLR low/ETS low group compared with in the NLR high/ETS high group (χ2 = 0.046, P = 0.830). Conclusion: NLR has the potential to identify which patients would benefit from this triple therapy, and when combined with ETS, it has the potential to provide greater predictive power in selecting the appropriate candidates for this combination treatment.

Immunosuppressive CD10+ALPL+ neutrophils promote resistance to anti-PD-1 therapy in HCC by mediating irreversible exhaustion of T cells.

BACKGROUND & AIMS: Remodeling the tumor microenvironment is a critical strategy for treating advanced hepatocellular carcinoma (HCC). Yet, how distinct cell populations in the microenvironment mediate tumor resistance to immunotherapies, such as anti-PD-1, remains poorly understood. METHODS: We analyzed the transcriptomic profile, at a single-cell resolution, of tumor tissues from patients with HCC scheduled to receive anti-PD-1-based immunotherapy. Our comparative analysis and experimental validation using flow cytometry and histopathological analysis uncovered a discrete subpopulation of cells associated with resistance to anti-PD-1 treatment in patients and a rat model. A TurboID-based proximity labeling approach was deployed to gain mechanistic insights into the reprogramming of the HCC microenvironment. RESULTS: We identified CD10+ALPL+ neutrophils as being associated with resistance to anti-PD-1 treatment. These neutrophils exhibited a strong immunosuppressive activity by inducing an apparent "irreversible" exhaustion of T cells in terms of cell number, frequency, and gene profile. Mechanistically, CD10+ALPL+ neutrophils were induced by tumor cells, i.e., tumor-secreted NAMPT reprogrammed CD10+ALPL+ neutrophils through NTRK1, maintaining them in an immature state and inhibiting their maturation and activation. CONCLUSIONS: Collectively, our results reveal a fundamental mechanism by which CD10+ALPL+ neutrophils contribute to tumor immune escape from durable anti-PD-1 treatment. These data also provide further insights into novel immunotherapy targets and possible synergistic treatment regimens. IMPACT AND IMPLICATIONS: Herein, we discovered that tumor cells reprogrammed CD10+ALPL+ neutrophils to induce the "irreversible" exhaustion of T cells and hence allow tumors to escape from the intended effects of anti-PD-1 treatment. Our data provided a new theoretical basis for the elucidation of special cell populations and revealed a molecular mechanism underpinning resistance to immunotherapy. Targeting these cells alongside existing immunotherapy could be looked at as a potentially more effective therapeutic approach.

QTL mapping and stability analysis of trichome density in zucchini (Cucurbita pepo L.).

Trichomes provide an excellent model for studying cell differentiation and proliferation. The aboveground tissues of plants with long dense trichomes (LDTs) can cause skin itching in people working in a zucchini field, in which management, pollination, and fruit harvesting are difficult. In this study, an F2 population was constructed with the LDT inbred line "16" and the sparse micro trichome (SMT) inbred line "63" for QTL analysis of type I and II trichome density. Two QTLs were identified on chromosomes 3 and 15 using the QTL-seq method. Additionally, 191 InDel markers were developed on 20 chromosomes, a genetic map was constructed for QTL mapping, and three QTLs were identified on chromosomes 3, 6, and 15. Two QTLs, CpTD3.1 and CpTD15.1, were identified in both QTL-seq and genetic map-based QTL analyses, and CpTD15.1 was the major-effect QTL. The stability of CpTD3.1 and CpTD15.1 was confirmed using data from F2 plants under different environmental conditions. The major-effect QTL CpTD15.1 was located between markers chr15-4991349 and chr15-5766791, with a physical distance of 775.44 kb, and explained 12.71%-29.37% of the phenotypic variation observed in the three environments. CpTD3.1 was located between markers chr3-218350 and chr3-2891236, in a region with a physical distance of 2,672.89 kb, and explained 5.00%-10.64% of the phenotypic variation observed in the three environments. The functional annotations of the genes within the CpTD15.1 region were predicted, and five genes encoding transcription factors regulating trichome development were selected. Cp4.1LG15g04400 encoded zinc finger protein (ZFP) and harbored nonsynonymous SNPs in the conserved ring finger domain between the two parental lines. There were significant differences in Cp4.1LG15g04400 expression between "16" and "63", and a similar pattern was found between germplasm resources of LDT lines and SMT lines. It was presumed that Cp4.1LG15g04400 might regulate trichome density in zucchini. These results lay a foundation for better understanding the density of multicellular nonglandular trichomes and the regulatory mechanism of trichome density in zucchini.

ACTB may serve as a predictive marker for the efficacy of lenvatinib in patients with HBV-related early-stage hepatocellular carcinoma following partial hepatectomy: a retrospective cohort study.

Background: The lack of effective biomarkers for the treatment of postoperative recurrence in hepatocellular carcinoma (HCC) persists despite lenvatinib therapy. This study aims to identify beta-actin (ACTB) as a predictive biomarker for lenvatinib that can facilitate individualized treatment for HCC. Methods: This retrospective study included a subset of patients with HCC who underwent partial hepatectomy, with some receiving postoperative lenvatinib treatment and others not receiving lenvatinib treatment. A propensity score matching (PSM) analysis of patients who underwent treatment with or without lenvatinib following HCC partial hepatectomy was performed. Immunohistochemistry was employed to determine the levels of ACTB expression in HCC samples obtained from matched patients (n=225) enrolled in this study. The X-Tile was employed to determine the optimal cut-off point of ACTB levels for predicting time to recurrence (TTR). To assess the correlation between ACTB levels and lenvatinib efficacy, a subgroup analysis of TTR was conducted. A Cox regression model with an interaction term was utilized to assess the predictive significance of the model. Subsequently, a nomogram was developed and its discriminative ability and predictive accuracy were assessed using the concordance index (C-index) and calibration curve. For the investigation of the ACTB expression, HCC and para-tumoral normal tissues were employed. The patient-derived xenograft (PDX) model was utilized to validate the correlation between ACTB levels and lenvatinib responsiveness. Results: After PSM, a total of 76 patients who underwent postoperative lenvatinib treatment were included in the analysis, with a median TTR of 24.35 months. Early-stage HCC patients with lower levels of ACTB exhibited a more favorable response to lenvatinib therapy compared to those with higher levels. The reduced expression of ACTB was indicative of the benefits of lenvatinib, as opposed to higher levels {hazard ratio (HR) =0.243 [95% confidence interval (CI): 0.096-0.619], P<0.001, P value for interaction =0.014}. In approximately 81.8% of cases involving HCC patients, there was an observed increase in the expression of ACTB. Multivariate analysis of the lenvatinib cohort revealed Child-Pugh [HR =5.416 (95% CI: 1.390-21.104), P=0.015], Barcelona Clinic Liver Cancer (BCLC) stage [HR =2.508 (95% CI: 1.116-5.639), P=0.026], and ACTB [HR =5.879 (95% CI: 2.424-14.259), P<0.001] score as independent factors for TTR, and all were included in the nomogram. The survival probability based on the calibration curve showed that the prediction of the nomogram was in good agreement with the actual observation. The C-index of the nomogram for predicting survival was 0.76 (95% CI: 0.71-0.84). Moreover, the PDXs derived from tumors exhibiting low levels of ACTB expression demonstrated a heightened sensitivity to lenvatinib treatment. Conclusions: In patients with tumors treated with lenvatinib, low ACTB expression can predict a lower risk of recurrence. The validation of this potential biomarker in independent cohorts is necessary prior to its implementation for precision treatment stratification in patients undergoing partial hepatectomy for early-stage HCC.

Genome-Wide Identification and Characterization of Members of the ACS Gene Family in Cucurbita maxima and Their Transcriptional Responses to the Specific Treatments.

Ethylene biosynthesis and signal transduction play critical roles in plant sex differentiation. ACS (1-aminocyclopropane-1-carboxylic acid synthase) is a rate-limiting enzyme in ethylene biosynthesis. However, the understanding of the ACS gene family in Cucurbita maxima is limited. Here, we identified and characterized 13 ACS genes in the C. maxima genome. All ACS genes could be divided into three groups according to a conserved serine residue at the C-terminus. Thirteen CmaACS genes were found to be randomly distributed on 10 of the 20 chromosomes of C. maxima. The ACS gene exhibits different tissue-specific expression patterns in pumpkin, and four ACS genes (CmaACS1, CmaACS4, CmaACS7, and CmaACS9) were expressed specifically in both the female and male flowers of C. maxima. In addition, the expression levels of CmaACS4 and CmaACS7 were upregulated after ethephon and IAA treatments, which ultimately increased the number of female flowers, decreased the position of the first female flower and decreased the number of bisexual flowers per plant. These results provide relevant information for determining the function of the ACS genes in C. maxima, especially for regulating the function of ethylene in sex determination.

Efficacy and Safety of TACE Combined With Lenvatinib Plus PD-1 Inhibitors Compared With TACE Alone for Unresectable Hepatocellular Carcinoma Patients: A Prospective Cohort Study.

Purpose: To compare the efficacy and safety of the combination of transcatheter arterial chemoembolization (TACE), Lenvatinib, and programmed cell death protein-1 (PD-1) inhibitors (combination group) with TACE (TACE group) in the treatment of patients with unresectable hepatocellular carcinoma (uHCC). Methods: We consecutively enrolled 110 patients with uHCC in this prospective cohort study, with 56 patients receiving combination treatment and 54 patients receiving TACE from November 2017 to September 2020. The differences in tumor response, survival benefit, and adverse events (AEs) were compared between the two groups. Factors affecting survival were identified via Cox regression analysis. Results: Compared with the TACE group, the combination group had a higher objective response rate (ORR) (67.9% vs. 29.6%, p < 0.001), longer median progression-free survival (mPFS) (11.9 vs. 6.9 months, P = 0.003) and overall survival (mOS) (23.9 vs. 15.3 months, p < 0.001). Multivariate analysis showed that the neutrophil-to-lymphocyte ratio (NLR) and the treatment option were independent factors associated with the PFS and OS. Further subgroup analysis showed that patients with low NLR (≤median 3.11) receiving combination therapy had better mPFS (20.1 vs. 6.2 months, P < 0.001) and mOS (28.9 vs. 15.2 months, P < 0.001) than those receiving TACE, while no obvious difference in PFS or OS was observed between the two groups in patients with high NLR (> 3.11). There were no unexpected toxicities in the combination group. Conclusion: Compared with TACE, the combination treatment demonstrated an improved clinical efficacy and manageable safety profile in patients with uHCC. Combination treatment showed better therapeutic efficacy in patients with low NLR; therefore, this ratio could be used to identify patients who will benefit from this treatment.

Single cell transcriptional diversity and intercellular crosstalk of human liver cancer.

Liver cancer arises from the evolutionary selection of the dynamic tumor microenvironment (TME), in which the tumor cell generally becomes more heterogeneous; however, the mechanisms of TME-mediated transcriptional diversity of liver cancer remain unclear. Here, we assess transcriptional diversity in 15 liver cancer patients by single-cell transcriptome analysis and observe transcriptional diversity of tumor cells is associated with stemness in liver cancer patients. Tumor-associated fibroblast (TAF), as a potential driving force behind the heterogeneity in tumor cells within and between tumors, was predicted to interact with high heterogeneous tumor cells via COL1A1-ITGA2. Moreover, COL1A1-mediated YAP-signaling activation might be the mechanistic link between TAF and tumor cells with increased transcriptional diversity. Strikingly, the levels of COL1A1, ITGA2, and YAP are associated with morphological heterogeneity and poor overall survival of liver cancer patients. Beyond providing a potential mechanistic link between the TME and heterogeneous tumor cells, this study establishes that collagen-stimulated YAP activation is associates with transcriptional diversity in tumor cells by upregulating stemness, providing a theoretical basis for individualized treatment targets.

Antioxidant and C5a-blocking strategy for hepatic ischemia-reperfusion injury repair.

BACKGROUND: Nonspecific liver uptake of nanomaterials after intravenous injection has hindered nanomedicine for clinical translation. However, nanomaterials' propensity for liver distribution might enable their use in hepatic ischemia-reperfusion injury (IRI) repair. During hepatic IRI, reactive oxygen species (ROS) are generated and the fifth component of complement (C5a) is activated. In addition, C5a is confirmed to exacerbate the vicious cycle of oxidative stress and inflammatory damage. For these reasons, we have investigated the development of nanomaterials with liver uptake to scavenge ROS and block C5a for hepatic IRI repair. RESULTS: To achieve this goal, a traditional nanoantioxidant of nanoceria was surface conjugated with the anti-C5a aptamers (Ceria@Apt) to scavenge the ROS and reduce C5a-mediated inflammation. High uptake of Ceria@Apt in the liver was confirmed by preclinical positron emission tomography (PET) imaging. The clinical symptoms of hepatic IRI were effectively alleviated by Ceria@Apt with ROS scavenging and C5a blocking in mice model. The released pro-inflammatory cytokines were significantly reduced, and subsequent inflammatory reaction involved in the liver was inhibited. CONCLUSIONS: The synthesized Ceria@Apt has great potential of medical application in hepatic IRI repair, which could also be applied for other ischemic-related diseases.

Identification and mapping of CpPM10.1, a major gene involved in powdery mildew (race 2 France of Podosphaera xanthii) resistance in zucchini (Cucurbita pepo L.).

KEY MESSAGE: Powdery mildew resistance in zucchini is controlled by one major dominant locus, CpPM10.1. CpPM10.1 was fine mapped. The expression of candidate gene Cp4.1LG10g02780 in resistant individuals was significantly upregulated after inoculation with the powdery mildew. Powdery mildew (PM) is one of the most destructive fungal diseases, reducing the productivity of Cucurbita crops globally. PM influences the photosynthesis, growth and development of infected zucchini and seriously reduces fruit yield and quality. In the present study, the zucchini inbred line 'X10' had highly stable PM resistance, and the inbred line 'Jin234' was highly susceptible to PM in the seedling stage and adult stages. Genetic analysis revealed that PM resistance in 'X10' is controlled by one major dominant locus. Based on the strategy of QTL-seq combined with linkage analysis and developed molecular markers, the major locus was found to be located in a 382.9-kb candidate region on chromosome 10; therefore, the major locus was named CpPM10.1. Using 1,400 F2 individuals derived from a cross between 'X10' and 'JIN234' and F2:3 offspring of the recombinants, the CpPM10.1 locus was defined in a region of approximately 20.9 kb that contained 5 coding genes. Among them, Cp4.1LG10g02780 contained a conserved domain (RPW8), which controls resistance to a broad range of PM pathogens. Cp4.1LG10g02780 also had nonsynonymous SNPs between the resistant 'X10' and susceptible 'Jin234.' Furthermore, the expression of Cp4.1LG10g02780 was strongly positively involved in PM resistance in the key period of inoculation. Further allelic diversity analysis in zucchini germplasm resources indicated that PM resistance was associated with two SNPs in the Cp4.1LG10g02780 RPW8 domain. This study not only provides highly stable PM resistance gene resources for cucurbit crops but also lays the foundation for the functional analysis of PM resistance and resistance breeding in zucchini.

Fine mapping identified the gibberellin 2-oxidase gene CpDw leading to a dwarf phenotype in squash (Cucurbita pepo L.).

Dwarfism is an important agronomic trait in pumpkin that can increase yield. In this study, the dwarf Cucurbita pepo L. line X10 exhibited significantly longitudinally shorter cell length in the stem than did the normal-vine line JIN234. The dwarf stature of X10 was recovered with exogenous gibberellin (GA3) application, suggesting that X10 might be sensitive to GA biosynthesis. Genetic analysis revealed that this dwarf trait is controlled by a single completely dominant locus: CpDw (Cucurbita pepo L. Dwarf). Using 1,300 F2 individuals derived from a cross between X10 and JIN234, we mapped the CpDw locus to a region of approximately 24.6 kb on chromosome 10 that contain 5 annotated genes. The high expression level of Cp4.1LG10g05910.1 and high GA2ox enzyme activity in X10 revealed that the GA 2-oxidase gene Cp4.1LG10g05910.1 is a candidate gene for CpDw. Alignment of the Cp4.1LG10g05910.1 gene revealed two nonsynonymous single nucleotide polymorphism (SNP) mutations in the two exons, as well as several SNPs and InDels in the important functional elements of promoter between parental lines. Further allelic diversity analysis of the Cucurbita spp. germplasm resources indicated that Cp4.1LG10g05910.1 may be involved in vine growth during the early developmental stage in C. pepo but not in C. maxima or C. moschata. This study provides an important theoretical basis for the genetic regulation of vine length and crop breeding in pumpkin.

miR-361-3p Regulates Liver Tumor-initiating Cells Expansion and Chemo-resistance.

Increasing evidence shows that liver tumor-initiating cells (T-ICs) closely associated with the progression, metastasis, recurrence and chemo-resistance of hepatocellular carcinoma (HCC). However, the underlying mechanism for the propagation of liver T-ICs remains unclear. Here we show that miR-361-3p is upregulated in liver T-ICs. Knockdown of miR-361-3p impairs the self-renewal and tumorigenicity liver T-ICs. Conversely, forced miR-361-3p expression enhances the self-renewal and tumorigenicity liver T-ICs. Mechanistically, miR-361-3p directly targets SOX1 via binding its 3'-UTR in liver T-ICs. Moreover, miR-361-3p knockdown hepatoma cells are more sensitive to cisplatin or sorafenib treatment. Clinical cohort analysis demonstrates that miR-361-3p low HCC patients are benefited from TACE (transcatheter arterial chemoembolization) or sorafenib treatment. In conclusion, our findings revealed the crucial role of the miR-361-3p in liver T-IC expansion and TACE or sorafenib response, rendering miR-361-3p an optimal target for the prevention and intervention in HCC.

An 8 miRNA-Based Risk Score System for Predicting the Prognosis of Patients With Papillary Thyroid Cancer.

BACKGROUND: Dysregulation of microRNAs (miRNAs) in papillary thyroid cancer (PTC) might influence prognosis of PTC. This study is aimed to develop a risk score system for predicting prognosis of PTC. METHODS: The miRNA and gene expression profiles of PTC were obtained from The Cancer Genome Atlas database. PTC samples were randomly separated into training set (n = 248) and validation set (n = 248). The differentially expressed miRNAs (DE-miRNAs) in the training set were screened using limma package. The independent prognosis-associated DE-miRNAs were identified for building a risk score system. Risk score of PTC samples in the training set was calculated and samples were divided into high risk group and low risk group. Kaplan-Meier curves and receiver operating characteristic (ROC) curve were used to assess the accuracy of the risk score system in the training set, validation set and entire set. Finally, a miRNA-gene regulatory network was visualized by Cytoscape software, followed by enrichment analysis. RESULTS: Totally, 162 DE-miRNAs between tumor and control groups in the training set were identified. An 8 independent prognosis-associated DE-miRNAs, (including miR-1179, miR-133b, miR-3194, miR-3912, miR-548j, miR-6720, miR-6734, and miR-6843) based risk score system was developed. The area under ROC curve in the training set, validation set and entire set was all above 0.93. A miRNA-gene regulatory network involving the 8 DE-miRNAs were built and functional enrichment analysis suggested the genes in the network were significantly enriched into 13 pathways, including calcium signaling pathway and hedgehog signaling pathway. CONCLUSION: The risk score system developed this study might be used for predicting the prognosis of PTC. Besides, the 8 miRNAs might affect the prognosis of PTC via hedgehog signaling pathway and calcium signaling pathway.

Weighted Gene Coexpression Network Analysis Reveals the Dynamic Transcriptome Regulation and Prognostic Biomarkers of Hepatocellular Carcinoma.

This study was aimed at revealing the dynamic regulation of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in hepatocellular carcinoma (HCC) and to identify HCC biomarkers capable of predicting prognosis. Differentially expressed mRNAs (DEmRNAs), lncRNAs, and miRNAs were acquired by comparing expression profiles of HCC with normal samples, using an expression data set from The Cancer Genome Atlas. Altered biological functions and pathways in HCC were analyzed by subjecting DEmRNAs to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Gene modules significantly associated with disease status were identified by weighted gene coexpression network analysis. An lncRNA-mRNA and an miRNA-mRNA coexpression network were constructed for genes in disease-related modules, followed by the identification of prognostic biomarkers using Kaplan-Meier survival analysis. Differential expression and association with the prognosis of 4 miRNAs were verified in independent data sets. A total of 1220 differentially expressed genes were identified between HCC and normal samples. Differentially expressed mRNAs were significantly enriched in functions and pathways related to "plasma membrane structure," "sensory perception," "metabolism," and "cell proliferation." Two disease-associated gene modules were identified. Among genes in lncRNA-mRNA and miRNA-mRNA coexpression networks, 9 DEmRNAs and 7 DEmiRNAs were identified to be potential prognostic biomarkers. MIMAT0000102, MIMAT0003882, and MIMAT0004677 were successfully validated in independent data sets. Our results may advance our understanding of molecular mechanisms underlying HCC. The biomarkers may contribute to diagnosis in future clinical practice.

Quantitative Trait Loci for Seed Size Variation in Cucurbits - A Review.

Cucurbits (Cucurbitaceae family) include many economically important fruit vegetable crops such as watermelon, pumpkin/squash, cucumber, and melon. Seed size (SS) is an important trait in cucurbits breeding, which is controlled by quantitative trait loci (QTL). Recent advances have deciphered several signaling pathways underlying seed size variation in model plants such as Arabidopsis and rice, but little is known on the genetic basis of SS variation in cucurbits. Here we conducted literature review on seed size QTL identified in watermelon, pumpkin/squash, cucumber and melon, and inferred 14, 9 and 13 consensus SS QTL based on their physical positions in respective draft genomes. Among them, four from watermelon (ClSS2.2, ClSS6.1, ClSS6.2, and ClSS8.2), two from cucumber (CsSS4.1 and CsSS5.1), and one from melon (CmSS11.1) were major-effect, stable QTL for seed size and weight. Whole genome sequence alignment revealed that these major-effect QTL were located in syntenic regions across different genomes suggesting possible structural and functional conservation of some important genes for seed size control in cucurbit crops. Annotation of genes in the four watermelon consensus SS QTL regions identified genes that are known to play important roles in seed size control including members of the zinc finger protein and the E3 ubiquitin-protein ligase families. The present work highlights the utility of comparative analysis in understanding the genetic basis of seed size variation, which may help future mapping and cloning of seed size QTL in cucurbits.

Morphological, Transcriptomic and Hormonal Characterization of Trimonoecious and Subandroecious Pumpkin (Cucurbita maxima) Suggests Important Roles of Ethylene in Sex Expression.

: Sex expression is a complex process, and in-depth knowledge of its mechanism in pumpkin is important. In this study, young shoot apices at the one-true-leaf stage and 10-leaf stage in Cucurbita maxima trimonoecious line '2013-12' and subandroecious line '9-6' were collected as materials, and transcriptome sequencing was performed using an Illumina HiSeqTM 2000 System. 496 up-regulated genes and 375 down-regulated genes were identified between shoot apices containing mostly male flower buds and only female flower buds. Based on gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the differentially expressed genes were mainly enriched in the ethylene and auxin synthesis and signal transduction pathways. In addition, shoot apices at the 4-leaf stage were treated with the ethylene-releasing agent 2-chloroethylphosphonic acid (Ethrel), aminoethoxyvinyl glycine (AVG), AgNO3 and indoleacetic acid (IAA). The number of female flowers up to node 20 on the main stem of '2013-12' increased significantly after Ethrel and IAA treatment and decreased significantly after AVG and AgNO3 treatment. The female flowers in '9-6' showed slight changes after treatment with the exogenous chemicals. The expression of key genes in ethylene synthesis and signal transduction (CmaACS7, CmaACO1, CmaETR1 and CmaEIN3) was determined using quantitative RT-PCR, and the expression of these four genes was positively correlated with the number of female flowers in '2013-12'. The variations in gene expression, especially that of CmaACS7, after chemical treatment were small in '9-6'. From stage 1 (S1) to stage 7 (S7) of flower development, the expression of CmaACS7 in the stamen was much lower than that in the ovary, stigma and style. These transcriptome data and chemical treatment results indicated that IAA might affect pumpkin sex expression by inducing CmaACS7 expression and indirectly affecting ethylene production, and the ethylene synthesis and signal transduction pathways play crucial roles in pumpkin flower sex expression. A possible reason for the differences in sex expression between pumpkin lines '2013-12' and '9-6' was proposed based on the key gene expression. Overall, these transcriptome data and chemical treatment results suggest important roles for ethylene in pumpkin sex expression.