Polyphenols from hickory nut reduce the occurrence of atherosclerosis in mice by improving intestinal microbiota and inhibiting trimethylamine N-oxide production.

BACKGROUND: Trimethylamine N-oxide (TMAO), a metabolite produced by intestinal microbiota through metabolizing phosphatidylcholine, choline, l-carnitine and betaine in the diet, has been implicated in the pathogenesis of atherosclerosis (AS). Concurrently, dietary polyphenols have garnered attention for their potential to ameliorate obesity, diabetes and atherosclerosis primarily by modulating the intestinal microbial structure. Hickory (Carya cathayensis) nut, a polyphenol-rich food product favored for its palatability, emerges as a candidate for exploration. HYPOTHESIS/PURPOSE: The relationship between polyphenol of hickory nut and atherosclerosis prevention will be firstly clarified, providing theoretical basis for the discovery of natural products counteracting TMAO-induced AS process in hickory nut. STUDY DESIGN AND METHODS: Employing Enzyme-linked Immunosorbent Assay (ELISA) and histological examination of aortic samples, the effects of total polyphenol extract on obesity index, inflammatory index and pathological changes of atherosclerosis in C57BL/6 J mice fed with high-fat and high choline diet were evaluated. Further, the composition, abundance, and function of mouse gut microbiota were analyzed through 16srDNA sequencing. Concurrently, the levels of TMAO and the expression of key enzymes (CutC and FMO3) involved in its synthesis are quantified using ELISA, Western Blot and Real-Time Quantitative PCR (RT-qPCR). Additionally, targeted metabolomic profiling of the hickory nut polyphenol extract was conducted, accompanied by molecular docking simulations to predict interactions between candidate polyphenols and the CutC/FMO3 using Autodock Vina. Finally, the docking prediction were verified by microscale thermophoresis (MST) . RESULTS: Polyphenol extracts of hickory nut improved the index of obesity and inflammation, and alleviated the pathological changes of atherosclerosis in C57BL/6 J mice fed with high-fat and high-choline diet. Meanwhile, these polyphenol extracts also changed the composition and function of intestinal microbiota, and increased the abundance of microorganisms in mice. Notably, the abundance of intestinal microbiota endowed with CutC gene was significantly reduced, coherent with expression of CutC catalyzing TMA production. Moreover, polyphenol extracts also decreased the expression of FMO3 in the liver, contributing to the reduction of TMAO levels in serum. Furthermore, metabonomic profile analysis of these polyphenol extracts identified 647 kinds of polyphenols. Molecular docking predication further demonstrated that Casuariin and Cinnamtannin B2 had the most potential inhibition on the enzymatic activities of CutC or FMO3, respectively. Notably, MST analysis corroborated the potential for direct interaction between CutC enzyme and available polyphenols such as Corilagin, (-)-Gallocatechin gallate and Epigallocatechin gallate. CONCLUSION: Hickory polyphenol extract can mitigate HFD-induced AS by regulating intestinal microflora in murine models. In addition, TMA-FMO3-TMAO pathway may play a key role in this process. This research unveils, for the inaugural time, the complex interaction between hickory nut-derived polyphenols and gut microbial, providing novel insights into the role of dietary polyphenols in AS prevention.

Characteristic analysis of BZR genes family and their responses to hormone treatments and abiotic stresses in Carya illinoinensis.

As the core of Brassinosteroids (BR) signaling pathway, BR-resistant (BZR) transcription factor regulates thousands of targeted genes mediating photomophogenesis, pollen sterility, cell expansion and stress response. Pecan (Carya illinoinensis) is a famous trees species of Carya, and its nut has high nutritional and economic values. However, there has no report on BZR genes family in pecan yet. Herein, totals of seven CiBZR members were identified in pecan genome, which were predicted to be hydrophilic unstable proteins and located in the nucleus. CiBZR genes had close evolutionary relationships with CcBZRs and JrBZRs in both Carya cathayensis and Juglans regia. These seven CiBZR genes were located independently on 7 chromosomes without doubling or tandem duplication. Based on the analysis of conserved motifs and gene structures, CiBZR genes were divided into three categories. More than 40 cis-acting elements were found in the 2 kb promoter regions of CiBZRs, which were mainly involved in hormone, light, and stress response, and plant growth and development. Notably, some of these CiBZR proteins were mainly located in the nucleus, had the self-activation ability and interaction relationship with BIN2 kinase, and negatively regulated the expression of CiCPD and CiDWF4. Gene expressions analysis further showed that CiBZR genes could express in many tissues and shared similar expression trends during embryo development. Moreover, most CiBZR genes responded to BR, Gibberellin (GA), Strigolactone (SL), salt, acid and osmotic stress. This study provides theoretical basis for the subsequent study on the role of CiBZR family genes in plant growth, development and stress responses.

Mn Doping and P Vacancy Induced Fast Phase Reconstruction of FeP for Enhanced Electrocatalytic Oxygen Evolution Reaction in Alkaline Seawater.

Due to the shortage of pure water resources, seawater electrolysis is a promising strategy to produce green hydrogen energy. To avoid chlorine oxidation reactions (ClOR) and the production of more corrosive hypochlorite, enhancing OER electrocatalyst activity is the key to solving the above problem. Considering that transition metal phosphides (TMPs) are promising OER eletrocatalysts for seawater splitting, a method to regulate the electronic structure of FeP by introducing Mn heteroatoms and phosphorus vacancy on it (Mn-FePV ) is developed. As an OER electrocatalyst in seawater solution, the synthesized Mn-FePV achieves extremely low overpotentials (η500  = 376, η1000  = 395 mV). In addition, the Pt/C||Mn-FePV couple only requires the voltage of 1.81 V to drive the current density of 1000 mA cm-2 for overall seawater splitting. The density functional theory (DFT) calculation shows that Mn-FePV (0.21 e- ) has more charge transfer number compared with FeP (0.17 e- ). In-situ Raman analysis shows that phosphorus vacancy and Mn doping can synergistically regulate the electronic structure of FeP to induce rapid phase reconstruction, further improving the OER performance of Mn-FePV . The new phase species of FeOOH is confirmed to can enhance the adsorption kinetics of OER intermediates.

Analysis of Delta(9) fatty acid desaturase gene family and their role in oleic acid accumulation in Carya cathayensis kernel.

Carya cathayensis, commonly referred to as Chinese hickory, produces nuts that contain high-quality edible oils, particularly oleic acid (18:1). It is known that stearoyl-ACP desaturase (SAD) is the first key step converting stearic acid (C18:0, SA) to oleic acid (C18:1, OA) in the aminolevulinic acid (ALA) biosynthetic pathway and play an important role in OA accumulation. Thus far, there is little information about SAD gene family in C. cathayensis and the role of individual members in OA accumulation. This study searched the Chinese Hickory Genome Database and identified five members of SAD genes, designated as CcSADs, at the whole genome level through the comparison with the homologous genes from Arabidopsis. RNA-Seq analysis showed that CcSSI2-1, CcSSI2-2, and CcSAD6 were highly expressed in kernels. The expression pattern of CcSADs was significantly correlated with fatty acid accumulation during the kernel development. In addition, five full-length cDNAs encoding SADs were isolated from the developing kernel of C. cathayensis. CcSADs-green fluorescent protein (GFP) fusion construct was infiltrated into tobacco epidermal cells, and results indicated their chloroplast localization. The catalytic function of these CcSADs was further analyzed by heterologous expression in Saccharomyces cerevisiae, Nicotiana benthamiana, and walnut. Functional analysis demonstrated that all CcSADs had fatty acid desaturase activity to catalyze oleic acid biosynthesis. Some members of CcSADs also have strong substrate specificity for 16:0-ACP to synthesize palmitoleic acid (C16:1, PA). Our study documented SAD gene family in C. cathayensis and the role of CcSSI2-1, CcSSI2-2, and CcSAD6 in OA accumulation, which could be important for future improvement of OA content in this species via genetic manipulation.

Tenogenic differentiation of human tendon-derived stem cells induced by long non-coding RNA LINCMD1 via miR-342-3p/EGR1 axis.

BACKGROUND: Tendon-derived stem cells (TDSCs) are proposed as a potential cell-seed for the treatment of tendon injury due to their tenogenic differentiation potential. In this work, we defined the action of long non-coding RNA (lncRNA) muscle differentiation 1 (LINCMD1) in tenogenic differentiation of human TDSCs (hTDSCs). METHODS: Quantitative real-time PCR (qRT-PCR) was used to assess the levels of LINCMD1, microRNA (miR)-342-3p, and early growth response-1 (EGR1) mRNA. Cell proliferation was detected by the XTT colorimetric assay. Protein expression was quantified by western blot. hTDSCs were grown in an osteogenic medium to induce osteogenic differentiation, and the extent of osteogenic differentiation was assessed by Alizarin Red Staining (ARS). The activity of alkaline phosphatase (ALP) was measured by the ALP Activity Assay Kit. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-342-3p and LINCMD1 or EGR1. RESULTS: Our results showed that enforced expression of LINCMD1 or suppression of miR-342-3p accelerated the proliferation and tenogenic differentiation and reduced osteogenic differentiation of hTDSCs. LINCMD1 regulated miR-342-3p expression by binding to miR-342-3p. EGR1 was identified as a direct and functional target of miR-342-3p, and knockdown of EGR1 reversed the effects of miR-342-3p suppression on cell proliferation and tenogenic and osteogenic differentiation. Furthermore, the miR-342-3p/EGR1 axis mediated the regulation of LINCMD1 on hTDSC proliferation and tenogenic and osteogenic differentiation. CONCLUSION: Our study suggests the induction of LINCMD1 in tenogenic differentiation of hTDSCs through miR-342-3p/EGR1 axis.

Platelet-rich plasma promotes skeletal muscle regeneration and neuromuscular functional reconstitution in a concentration-dependent manner in a rat laceration model.

Abnormal function of injured muscle with innervation loss is a challenge in sports medicine. The difficulty of rehabilitation is regenerating and reconstructing the skeletal muscle tissue and the neuromuscular junction (NMJ). Platelet-rich plasma (PRP) releases various growth factors that may provide an appropriate niche for tissue regeneration. However, the specific mechanism of the PRP's efficacy on muscle healing remains unknown. In this study, we injected PRP with different concentration gradients (800, 1200, 1600 × 109 pl/L) or saline into a rat gastrocnemius laceration model. The results of histopathology and neuromyography show that PRP improved myofibers regeneration, facilitated electrophysiological recovery, and reduced fibrosis in a concentration-dependent manner. Furthermore, we found that PRP promotes the activity of satellite cells by upregulating the expression of the myogenic regulatory factor (MyoD, myogenin). Meanwhile, PRP promotes the regeneration and maturation of acetylcholine receptor (AChR) clusters of the Neuromuscular junction (NMJ) on the regenerative myofibers. Finally, we found that the expression of the Agrin, LRP4, and MuSK was upregulated in the PRP-treated groups, which may contribute to AChR cluster regeneration and functional recovery. The conclusions proposed a hypothesis for PRP treatment's efficacy and mechanism in muscle injuries, indicating promising application prospects.

WRINKLED1 Positively Regulates Oil Biosynthesis in Carya cathayensis.

Hickory (Carya cathayensis Sarg.) is a kind of important woody oil tree species, and its nut has high nutritional value. Previous gene coexpression analysis showed that WRINKLED1 (WRI1) may be a core regulator during embryo oil accumulation in hickory. However, its specific regulatory mechanism on hickory oil biosynthesis has not been investigated. Herein, two hickory orthologs of WRI1 (CcWRI1A and CcWRI1B) containing two AP2 domains with AW-box binding sites and three intrinsically disordered regions (IDRs) but lacking the PEST motif in the C-terminus were characterized. They are nucleus-located and have self-activated ability. The expression of these two genes was tissue-specific and relatively high in the developing embryo. Notably, CcWRI1A and CcWRI1B can restore the low oil content, shrinkage phenotype, composition of fatty acid, and expression of oil biosynthesis pathway genes of Arabidopsis wri1-1 mutant seeds. Additionally, CcWRI1A/B were shown to modulate the expression of some fatty acid biosynthesis genes in the transient expression system of nonseed tissues. Transcriptional activation analysis further indicated that CcWRI1s directly activated the expression of SUCROSE SYNTHASE2 (SUS2), PYRUVATE KINASE ß SUBUNIT 1 (PKP-ß1), and BIOTIN CARBOXYL CARRIER PROTEIN2 (BCCP2) involved in oil biosynthesis. These results suggest that CcWRI1s can promote oil synthesis by upregulating some late glycolysis- and fatty acid biosynthesis-related genes. This work reveals the positive function of CcWRI1s in oil accumulation and provides a potential target for improving plant oil by bioengineering technology.

CcMYB12 Positively Regulates Flavonoid Accumulation during Fruit Development in Carya cathayensis and Has a Role in Abiotic Stress Responses.

Flavonoid, an important secondary metabolite in plants, is involved in many biological processes. Its synthesis originates from the phenylpropane metabolic pathway, and it is catalyzed by a series of enzymes. The flavonoid biosynthetic pathway is regulated by many transcription factors, among which MYB transcription factors are thought to be key regulators. Hickory (Carya cathayensis) is an economic forest tree species belonging to the Juglandaceae family, and its fruit is rich in flavonoids. The transcriptome of exocarp and seed of hickory has previously been sequenced and analyzed by our team, revealing that CcMYB12 (CCA0691S0036) may be an important regulator of flavonoid synthesis. However, the specific regulatory role of CcMYB12 in hickory has not been clarified. Through a genome-wide analysis, a total of 153 R2R3-MYB genes were identified in hickory, classified into 23 subclasses, of which CcMYB12 was located in Subclass 7. The R2R3-MYBs showed a differential expression with the development of hickory exocarp and seed, indicating that these genes may regulate fruit development and metabolite accumulation. The phylogenetic analysis showed that CcMYB12 is a flavonol regulator, and its expression trend is the same as or opposite to that of flavonol synthesis-related genes. Moreover, CcMYB12 was found to be localized in the nucleus and have self-activation ability. The dual-luciferase reporter assay demonstrated that CcMYB12 strongly bonded to and activated the promoters of CcC4H, CcCHS, CcCHI, and CcF3H, which are key genes of the flavonoid synthesis pathway. Overexpression of CcMYB12 in Arabidopsis thaliana could increase the content of total flavonoids and the expression of related genes, including PAL, C4H, CHS, F3H, F3'H, ANS, and DFR, in the flavonoid synthesis pathway. These results reveal that CcMYB12 may directly regulate the expression of flavonoid-related genes and promote flavonoid synthesis in hickory fruit. Notably, the expression level of CcMYB12 in hickory seedlings was significantly boosted under NaCl and PEG treatments, while it was significantly downregulated under acid stress, suggesting that CcMYB12 may participate in the response to abiotic stresses. The results could provide a basis for further elucidating the regulation network of flavonoid biosynthesis and lay a foundation for developing new varieties of hickory with high flavonoid content.

Comparative plastomes of Carya species provide new insights into the plastomes evolution and maternal phylogeny of the genus.

Carya, in the Juglandiodeae subfamily, is to a typical temperate-subtropical forest-tree genus for studying the phylogenetic evolution and intercontinental disjunction between eastern Asia (EA) and North America (NA). Species of the genus have high economic values worldwide for their high-quality wood and the rich healthy factors of their nuts. Although previous efforts based on multiple molecular markers or genome-wide SNPs supported the monophyly of Carya and its two EA and NA major subclades, the maternal phylogeny of Carya still need to be comprehensively evaluated. The variation of Carya plastome has never been thoroughly characterized. Here, we novelly present 19 newly generated plastomes of congeneric Carya species, including the recently rediscovered critically endangered C. poilanei. The overall assessment of plastomes revealed highly conservative in the general structures. Our results indicated that remarkable differences in several plastome features are highly consistent with the EA-NA disjunction and showed the relatively diverse matrilineal sources among EA Carya compared to NA Carya. The maternal phylogenies were conducted with different plastome regions and full-length plastome datasets from 30 plastomes, representing 26 species in six genera of Juglandoideae and Myrica rubra (as root). Six out of seven phylogenetic topologies strongly supported the previously reported relationships among genera of Juglandoideae and the two subclades of EA and NA Carya, but displayed significant incongruencies between species within the EA and NA subclades. The phylogenetic tree generated from full-length plastomes demonstrated the optimal topology and revealed significant geographical maternal relationships among Carya species, especially for EA Carya within overlapping distribution areas. The full-length plastome-based phylogenetic topology also strongly supported the taxonomic status of five controversial species as separate species of Carya. Historical and recent introgressive hybridization and plastid captures might contribute to plastome geographic patterns and inconsistencies between topologies built from different datasets, while incomplete lineage sorting could account for the discordance between maternal topology and the previous nuclear genome data-based phylogeny. Our findings highlight full-length plastomes as an ideal tool for exploring maternal relationships among the subclades of Carya, and potentially in other outcrossing perennial woody plants, for resolving plastome phylogenetic relationships.

Peri-tumoral infiltrate associates with postoperative prognosis of patients with OSCC: Stronger association in HPV negative patients.

PURPOSE: The current data on the relationship between local inflammatory infiltration and prognosis in oral squamous cell carcinoma (OSCC) are limited and controversial, especially in different HPV status. In this study, we analyzed the relationship between peri-tumoral inflammatory infiltrate (PTI) and HPV status and prognosis of patients with OSCC after surgery. METHODS: A retrospective cohort of 99 primary OSCC patients who underwent surgery was constructed. P16 immunohistochemistry was used to determine HPV status. PTI was determined by hematoxylin-eosin staining and quantified into four levels: none (Score 0), weak (Score 1), moderate (Score 2) and strong (Score 3). The associations of PTI with clinico-pathological characteristics, HPV status and survival were examined. RESULTS: Most OSCC patients had weak to moderate PTI. PTI was significantly associated with lymph node metastasis (P = 0.041), and patients with moderate PTI had significantly better OS (P = 0.009) than those with no PTI. In HPV negative OSCC, patients with moderate PTI also had significantly better OS (P = 0.019) than those with no PTI. However, PTI was not significantly associated with survival in HPV positive OSCC. CONCLUSIONS: In HPV negative OSCC, moderate PTI may suggest a better postoperative prognosis than no PTI.

Global Transcriptome Analysis Revealed the Molecular Regulation Mechanism of Pigment and Reactive Oxygen Species Metabolism During the Stigma Development of Carya cathayensis.

Hickory (Carya cathayensis Sarg.) is a monoecious plant of the genus Carya of the Juglandaceae family. Its nuts contain a number of nutritional compounds and are deeply loved by consumers. Interestingly, it was observed that the color of hickory stigma changed obviously from blooming to mature. However, the molecular mechanism underlying color formation during stigma development and the biological significance of this phenomenon was mostly unknown. In this work, pigment content, reactive oxygen species (ROS) removal capacity, and transcriptome analysis of developing stigma of hickory at 4 differential sampling time points (S1, S2, S3, and S4) were performed to reveal the dynamic changes of related pigment, antioxidant capacity, and its internal molecular regulatory mechanism. It was found that total chlorophyll content was decreased slightly from S1 to S4, while total carotenoids content was increased from S1 to S3 but decreased gradually from S3 to S4. Total anthocyanin content continued to increase during the four periods of stigma development, reaching the highest level at the S4. Similarly, the antioxidant capacity of stigma was also gradually improved from S1 to S4. Furthermore, transcriptome analysis of developing hickory stigma identified 31,027 genes. Time-series analysis of gene expressions showed that these genes were divided into 12 clusters. Cluster 5 was enriched with some genes responsible for porphyrin and chlorophyll metabolism, carotenoid metabolism, and photosynthesis. Meanwhile, cluster 10 was enriched with genes related to flavonoid metabolism, including anthocyanin involved in ROS scavenging, and its related genes were mainly distributed in cluster 12. Based on the selected threshold values, a total of 10432 differentially expressed genes were screened out and enriched in the chlorophyll, carotenoid, anthocyanin, and ROS metabolism. The expression trends of these genes provided plausible explanations for the dynamic change of color and ROS level of hickory stigma with development. qRT-PCR analyses were basically consistent with the results of RNA-seq. The gene co-regulatory networks of pigment and ROS metabolism were further constructed and MYB113 (CCA0887S0030) and WRKY75 (CCA0573S0068) were predicted to be two core transcriptional regulators. These results provided in-depth evidence for revealing the molecular mechanism of color formation in hickory stigma and its biological significance.

Genome-Wide Identification of TLP Gene Family and Their Roles in Carya cathayensis Sarg in Response to Botryosphaeria dothidea.

Hickory (Carya cathayensis) is a critical tree species of the genus Carya from the Juglandaceae family that contains nutrient-rich nuts. Due to large-scale soil degradation, the pests and diseases of hickory are becoming more and more serious. Thaumatin-like proteins (TLPs) are vital proteins involved in the complex defense process of plant pathogens. In this study, 40 CcTLP genes were identified genome-widely and phylogenetically grouped into three subfamilies. The sequence of CcTLPs had a conservative pattern, such as eight stable disulfide bonds, REDDD, and G-X-[GF]-X-C-X-T-[GA]-D-C-X(1,2)-G-X-(2,3)-C structure. In total, 57 cis-elements related to stress-responsive, light-responsive, phytohormone-responsive, and plant-responsive were discovered. Under salicylate (SA), methyl jasmonate (MeJA), and ethephon (ETH) treatments, the expressions of CcTLP28, CcTLP29, CcTLP30, CcTLP31, CcTLP32, CcTLP33, CcTLP37, CcTLP38, and CcTLP39 had different patterns. This is an indication that most of the TLP genes were upregulated by SA and downregulated by MeJA. Notably, seven TLP genes were significantly upregulated under the Botryosphaeria dothidea inoculation, especially CcTLP31, with an over 20-fold change. Nine genes were shown by subcellular localization analysis to be located at the plasma membrane and cytoplasm. The knowledge of the disease-resistant function of the CcTLP family in hickory is promoted by these results. A foundation reference for the molecular breeding of this plant in the future is provided by our findings.

Microcarriers in application for cartilage tissue engineering: Recent progress and challenges.

Successful regeneration of cartilage tissue at a clinical scale has been a tremendous challenge in the past decades. Microcarriers (MCs), usually used for cell and drug delivery, have been studied broadly across a wide range of medical fields, especially the cartilage tissue engineering (TE). Notably, microcarrier systems provide an attractive method for regulating cell phenotype and microtissue maturations, they also serve as powerful injectable carriers and are combined with new technologies for cartilage regeneration. In this review, we introduced the typical methods to fabricate various types of microcarriers and discussed the appropriate materials for microcarriers. Furthermore, we highlighted recent progress of applications and general design principle for microcarriers. Finally, we summarized the current challenges and promising prospects of microcarrier-based systems for medical applications. Overall, this review provides comprehensive and systematic guidelines for the rational design and applications of microcarriers in cartilage TE.

Minimally Invasive vs. Open Surgery for Hallux Valgus: A Meta-Analysis.

Purpose: In recent years, minimally invasive surgery (MIS) for hallux valgus has emerged and gained popularity. To date, evidence on the benefits of MIS for hallux valgus is still controversial. This updated meta-analysis aimed to comprehensively evaluate the efficiency of MIS vs. open surgery for hallux valgus. Methods: A systematic literature search of PubMed, Embase, and the Cochrane Library was performed. Two independent reviewers conducted data extraction and analyzed data with R software. Data were presented with risk ratio (RR) and standardized mean difference (SMD) along with 95% confidence interval (CI). Results: A total of 22 studies in which there were 790 ft treated with the MIS procedure and 838 ft treated with an open procedure were included. The correction of sesamoid position was better in the MIS group. The post-operative distal metatarsal articular angle (DMAA) of the MIS group was lower. There was less pain at the early phase in the MIS group. The MIS group had a shorter surgery time and shorter hospitalization time compared with the open group. Our meta-analysis revealed no statistically significant difference in hallux valgus angle (HVA), first intermetatarsal angle (IMA), the first metatarsal shortening, the American Orthopedic Foot and Ankle Society (AOFAS) score, visual analog scale (VAS) score at the final follow-up or complication rate (when all studies were considered). When taking into consideration only randomized controlled trial (RCT), the AOFAS score was higher in the MIS group while HVA, IMA, DMAA, and complication rate remained no significance. Post-operative IMA of the MIS group was significantly lower when only studies reporting the second-generation (2G) MIS were included. When just studies adopting the third-generation (3G) MIS were included, the HVA and DMAA were lower in the MIS group. Conclusion: The MIS procedures were more effective than open surgeries in the treatment of hallux valgus. Moreover, the MIS group achieved better radiologic and clinical outcomes compared with the open group.

CX3CL1 in the red bone marrow promotes renal cell carcinoma to metastasize to the spine by involving the Src-related pathway.

Spinal metastasis (SM) frequently occurs in renal cell carcinoma (RCC) patients. Our preliminary work showed that CX3CL1 plays a positive role in SM. The objective of the present study was to verify whether CX3CL1 activates the downstream pathway by binding to CX3CR1 in RCC cells, ultimately promoting RCC to metastasize to the spine. The expression of CX3CL1 and CX3CR1 in tissue samples was detected by immunohistochemistry and western blotting. ELISA was used to quantify the concentration of CX3CL1 in the serum. The expression level of CX3CR1 in RCC cell lines was also detected. The CellTiter-Glo assay and flow cytometry were used to analyze cell viability and apoptosis of RCC cells. Transwell and wound healing assay were used to analyze the effect of CX3CL1 on the invasion and migration ability of RCC cells. Specific inhibitors were used to interfere with key molecules in the signaling pathway to further explore the signal transduction in RCC cells after CX3CL1 stimulation. The expression of CX3CR1 in SM from RCC was higher than that in limb bone metastases. Among the five RCC cell lines, 786O cells expressed the highest level of CX3CR1. CX3CL1 neither inhibited the proliferation of 786O cells nor promoted the apoptosis of 786O cells. However, it promoted the migration and invasion of RCC cells. After CX3CL1 stimulation, Src and Focal adhesion kinase (FAK) phosphorylation levels increased in RCC cells. Bosutinib and PF-00562271 inhibited Src/FAK phosphorylation and cell motility and invasion triggered by CX3CL1 stimulation. CX3CL1 in the red bone marrow of spinal cancellous bone enhances migration and invasion abilities of RCC cells, thereby promoting RCC metastasize to the spine. The migration and invasion of RCC cells activated by CX3CL1 are at least partially dependent on Src/FAK activation.

Identification and validation of a prognostic signature of autophagy, apoptosis and pyroptosis-related genes for head and neck squamous cell carcinoma: to imply therapeutic choices of HPV negative patients.

Introduction: An effective tool is needed to predict the prognosis of head and neck squamous cell carcinoma (HNSCC). Human papillomavirus (HPV) positive HNSCC patients generally have a favorable survival and a promising responsiveness to radiotherapy, chemoradiotherapy and checkpoint blockades. However, HPV negative patients, the majority of HNSCC patients, have been largely overlooked. Cell death has been involved in the therapeutic resistance of cancers. To this end, we aimed to identify the association of autophagy, apoptosis and pyroptosis-related genes with the prognosis of HNSCC, and construct a prognostic signature to predict the prognosis for HNSCC, especially for HPV negative HNSCC. Methods: Autophagy and apoptosis-related genes were obtained from Gene Set Enrichment Analysis (GSEA) website, and pyroptosis-related genes were obtained from GSEA and Gene Ontology (GO) database. We established the cell death index (CDI) based on RNA sequencing (RNA-seq) data and clinicopathological information from The Cancer Genome Atlas (TCGA) dataset. The prognostic value of CDI was verified by Kaplan-Meier, receiver operating characteristic (ROC) and univariate and multivariate Cox regression analyses in TCGA dataset, and validated with the datasets from Gene Expression Omnibus (GEO) and Qilu Hospital of Shandong University. We further assessed the immune microenvironment of patients with high and low CDI scores. Moreover, the expression of the signature genes in HNSCC cell lines were explored. Results: We found that CDI was an independent prognostic indicator for overall survival (hazard ratio 3.80, 95% confidential interval: 2.70-5.40, P < 0.001). Furthermore, HNSCC patients with high CDI scores obtained increased overall survival post radiation indicating benefits from radiotherapy of this subgroup. On the other hand, HPV negative HNSCC patients with low CDI exhibited increased checkpoint gene expressions, an inflamed tumor microenvironment and an enriched immune response-related functions, suggesting the potential benefits from checkpoint immunotherapies of this subgroup. Moreover, we validated the baseline and induced expressions of above 16 genes in two HPV negative HNSCC cell lines, CAL27 and SCC-15. Discussion: We established a prognostic signature and emphasized its implements in the therapeutic choices of HPV negative HNSCC patients, the majority and the poor outcome population of HNSCC.

Dysbiotic tumor microbiota associates with head and neck squamous cell carcinoma outcomes.

BACKGROUND: The need for an effective tool to predict prognosis of head and neck squamous cell carcinoma (HNSCC) patients is critical and unmet. Microbiota has recently been found involved in tumor progression and response to immunotherapy. However, the association of microbiota with the prognosis of HNSCC patients remains obscure. This study aims to investigate the association between tumor microbiota and outcomes of HNSCC patients. METHODS: A retrospective study including 129 primary tumors of HNSCC was conducted. Using 16S rRNA sequencing, the profile and the composition of tumor microbiota were measured and their associations with overall survival (OS) and disease free survival (DFS) were examined. RESULTS: We observed a reduced richness and enriched abundances of genera Schlegelella and Methyloversatilis in tumor microbiota of HNSCC patients with poor prognosis. However, a richer tumor microbiota with greater abundances of genera Bacillus, and Lactobacillus and Sphingomonas was characterized in the patients with favorable prognosis.The ratio of these differentially abundant taxa, microbial dysbiosis index (MDI), was significantly associated with OS (hazard ratio [HR], 4.67, 95% confidence interval [CI], 2.51 to 8.69,P < 0.001) and DFS (HR, 2.89; 95% CI, 1.74 to 4.80, P < 0.001) independently of age, tumor size, lymph node metastasis, differentiation and p16 status. The risk score of multivariate Cox regression exhibited an excellent performance for estimating three-year OS (AUC of 0.826). We also found a richer tumor microbiota was correlated with moderate peritumoral inflammatory infiltration. CONCLUSION: These results indicate that tumor microbiota associates with outcomes of HNSCC patients.

Comparison analysis of widely-targeted metabolomics revealed the variation of potential astringent ingredients and their dynamic accumulation in the seed coats of both Carya cathayensis and Carya illinoinensis.

Pecan and hickory nuts are two of consumers' favorite ones. Pecan seeds can be eaten fresh, while hickory ones must remove astringency before eating. Here, we reported that total phenols, flavonoids and condensed tannins of hickory seeds were reduced after de-astringent treatments. They gradually increased with development, showing higher levels in hickory seed coat at mid-late periods than that in pecan's. Widely-targeted metabonomics analysis of developing testa identified 424 kinds of components, including 101, 38, 58, 27 classes of flavonoids, tannins, phenolic acids, organic acids and others, showing 16 different changing trends. Notably, most kinds of flavonoids, hydrolysable tannins and phenolic acids at maturity were more than that of pecan's, while oligomeric condensed tannins were opposite. Gene expression analysis provided further explanations for their dynamic accumulation. These results unraveled potential astringent components in hickory testa and preliminary molecular mechanisms of their dynamic changes, offering theoretical basis for the targeted de-astringency.

Analysis of lipidomics profile of Carya cathayensis nuts and lipid dynamic changes during embryonic development.

Hickory (Carya cathayensis) nuts contain higher amount of lipids, and possess high nutritional value and substantial health benefits. However, their lipid composition and dynamic changes during embryogenesis have not been thoroughly investigated. Therefore, lipidomics profile and lipid dynamic changes during embryonic development were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Totally, 544 kinds of lipids were identified in mature hickory nuts with higher proportions of glycerolipids (59.94%) and glycerophospholipids (38.66%). Notably, diacylglycerols showed gradual uptrends, which corresponded with total glycerolipid and glycerophospholipid at middle and late stage of embryogenesis, suggesting the pivotal role of diacylglycerols in the accumulation of glycerolipids and glycerophospholipids. Moreover, triacylglycerols, diacylglycerols, phosphatidylethanolamines and phosphatidylcholines had high relative content with abundance of unsaturated fatty acids, specifically oleic acid, linoleic acid and linolenic acid, localized mainly at sn-2 lipid position. Together, our study provides innovative perspectives for studying the nutritional benefits of hickory nut lipids.

Integrated transcriptome and proteome analysis of developing embryo reveals the mechanisms underlying the high levels of oil accumulation in Carya cathayensis Sarg.

Hickory (Carya cathayensis Sarg.) is an extraordinary nut-bearing deciduous arbor with high content of oil in its embryo. However, the molecular mechanism underlying high oil accumulation is mostly unknown. Here, we reported that the lipid droplets and oil accumulation gradually increased with the embryo development and the oil content was up to ~76% at maturity. Furthermore, transcriptome and proteome analysis of developing hickory embryo identified 32,907 genes and 9857 proteins. Time-series analysis of gene expressions showed that these genes were divided into 12 clusters and lipid metabolism-related genes were enriched in Cluster 3, with the highest expression levels at 95 days after pollination (S2). Differentially expressed genes and proteins indicated high correlation, and both were enriched in the lipid metabolism. Notably, the genes involved in biosynthesis, transport of fatty acid/lipid and lipid droplets formation had high expression levels at S2, while the expression levels of other genes required for suberin/wax/cutin biosynthesis and lipid degradation were very low at all the sampling time points, ultimately promoting the accumulation of oil. Quantitative reverse-transcription PCR analysis also verified the results of RNA-seq. The co-regulatory networks of lipid metabolism were further constructed and WRINKLED1 (WRI1) was a core transcriptional factor located in the nucleus. Of note, CcWRI1A/B could directly activate the expression of some genes (CcBCCP2A, CcBCCP2B, CcFATA and CcFAD3) required for fatty acid synthesis. These results provided in-depth evidence for revealing the molecular mechanism of high oil accumulation in hickory embryo.