Proteomic profiling and biomarker discovery for predicting the response to PD-1 inhibitor immunotherapy in gastric cancer patients.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment; however, a significant proportion of gastric cancer (GC) patients do not respond to this therapy. Consequently, there is an urgent need to elucidate the mechanisms underlying resistance to ICIs and identify robust biomarkers capable of predicting the response to ICIs at treatment initiation. Methods: In this study, we collected GC tissues from 28 patients prior to the administration of anti-programmed death 1 (PD-1) immunotherapy and conducted protein quantification using high-resolution mass spectrometry (MS). Subsequently, we analyzed differences in protein expression, pathways, and the tumor microenvironment (TME) between responders and non-responders. Furthermore, we explored the potential of these differences as predictive indicators. Finally, using machine learning algorithms, we screened for biomarkers and constructed a predictive model. Results: Our proteomics-based analysis revealed that low activity in the complement and coagulation cascades pathway (CCCP) and a high abundance of activated CD8 T cells are positive signals corresponding to ICIs. By using machine learning, we successfully identified a set of 10 protein biomarkers, and the constructed model demonstrated excellent performance in predicting the response in an independent validation set (N = 14; area under the curve [AUC] = 0.959). Conclusion: In summary, our proteomic analyses unveiled unique potential biomarkers for predicting the response to PD-1 inhibitor immunotherapy in GC patients, which may provide the impetus for precision immunotherapy.

Identification of the exosomal PD-L1 inhibitor to promote the PD-1 targeting therapy of gastric cancer.

Programmed death 1/programmed death-ligand 1 (PD-1/PD-L1) targeting therapy is widely applied in clinics for gastric cancer treatment. Nevertheless, the clinical response is not well acceptable due to the exosomal PD-L1. Hence, abrogation of the exosomal PD-L1 may be a strategy to sensitize the gastric cancer cell to PD-1 targeting therapy. With the aid of CD63 targeting antibody and PD-L1 targeting aptamer, HTRF based assay was established to quantify the exosomal PD-L1, and applied to our in-house compound library, resulting in the identification of moclobemide. Further optimization of moclobemide lead to EP16, which can inhibit the generation of exosomal PD-L1 with IC50 = 0.108 µM. By applying EP16 to gastric cancer cell line coupled with T-cell activity related experiment, it was validated to activate T-cell and can promote the response of PD-1 targeting therapy for gastric cancer treatment in vitro and in vivo. Collectively, our findings give a promising tool to promote the sensitivity of anti-PD-1 for gastric cancer treatment, and EP16 can serve as a leading compound for exosomal PD-L1 abrogation.

Genome-Wide Identification, Characterization, and Expression Analysis of Tubby-like Protein (TLP) Gene Family Members in Woodland Strawberry (Fragaria vesca).

Tubby-like proteins (TLPs) play important roles in plant growth and development and in responses to abiotic stress. However, TLPs in strawberry remain poorly studied. In this study, eight TLPs were identified in woodland strawberry (Fragaria vesca subspecies vesca 'Ruegen'). Protein structure analysis revealed that the structure of FvTLPs is highly conserved, but evolutionary and gene structure analyses revealed that the evolutionary pattern of FvTLP family members differs from that of their orthologous genes in Arabidopsis, poplar, and apple. Subcellular localization assays revealed that FvTLPs were localized to the nucleus and plasma membrane. FvTLPs showed no transcriptional activity. Yeast two-hybrid assays revealed that FvTLPs interact with specific FvSKP1s. The expression patterns of FvTLPs in different tissues and under various abiotic stresses (salt, drought, cold, and heat) and hormone treatments (ABA (abscisic acid) and MeJA (methyl jasmonate)) were determined. The expression patterns of FvTLPs indicated that they play a role in regulating growth and development and responses to abiotic stress in F. vesca. The GUS (beta-glucuronidase) activity of FvTLP1pro::GUS plants in GUS activity assays increased under salt and drought stress and abscisic acid treatment. The results of this study provide new insights into the molecular mechanisms underlying the functions of TLPs.

FaAKR23 Modulates Ascorbic Acid and Anthocyanin Accumulation in Strawberry (Fragaria × ananassa) Fruits.

Strawberry (Fragaria × ananassa) fruits are rich in ascorbic acid (AsA) and anthocyanin, which are essential antioxidants for human health. However, the underlying regulatory mechanism of these antioxidant accumulation, especially AsA accumulation in strawberry fruits, remains largely unknown. In this study, we identified FaAKR23 was a regulator of AsA and anthocyanin accumulation. We transiently expressed FaAKR23 in strawberry fruits and conducted metabolic and molecular analyses to explore the role of FaAKR23 in AsA and anthocyanin accumulation. Transient silencing of FaAKR23 (FaAKR23-RNAi) in strawberry fruits significantly decreased the AsA and anthocyanin contents compared with control (empty vector-RNAi, EV-RNAi). Correspondingly, expression of some structural genes and regulatory factors involved in these two antioxidants' accumulation was dramatically repressed. In addition, transcriptome analysis of EV-RNAi and FaAKR23-RNAi fruits suggested that FaAKR23 was also involved in starch and sucrose metabolism as well as plant-pathogen interaction. Overall, these results not only provide the coordinated regulatory function of FaAKR23 on AsA and anthocyanin accumulation but also offer a promising candidate gene for strawberry breeding with high antioxidants.

Combined transcriptomic and metabolomic analysis reveals a role for adenosine triphosphate-binding cassette transporters and cell wall remodeling in response to salt stress in strawberry.

Strawberry (Fragaria × ananassa Duch) are sensitive to salt stress, and breeding salt-tolerant strawberry cultivars is the primary method to develop resistance to increased soil salinization. However, the underlying molecular mechanisms mediating the response of strawberry to salinity stress remain largely unknown. This study evaluated the salinity tolerance of 24 strawberry varieties, and transcriptomic and metabolomic analysis were performed of 'Sweet Charlie' (salt-tolerant) and 'Benihoppe' (salt-sensitive) to explore salt tolerance mechanisms in strawberry. Compared with the control, we identified 3412 differentially expressed genes (DEGs) and 209 differentially accumulated metabolites (DAMs) in 'Benihoppe,' and 5102 DEGs and 230 DAMs in 'Sweet Charlie.' DEGs Gene Ontology (GO) enrichment analyses indicated that the DEGs in 'Benihoppe' were enriched for ion homeostasis related terms, while in 'Sweet Charlie,' terms related to cell wall remodeling were over-represented. DEGs related to ion homeostasis and cell wall remodeling exhibited differential expression patterns in 'Benihoppe' and 'Sweet Charlie.' In 'Benihoppe,' 21 ion homeostasis-related DEGs and 32 cell wall remodeling-related DEGs were upregulated, while 23 ion homeostasis-related DEGs and 138 cell wall remodeling-related DEGs were downregulated. In 'Sweet Charlie,' 72 ion homeostasis-related DEGs and 275 cell wall remodeling-related DEGs were upregulated, while 11 ion homeostasis-related DEGs and 20 cell wall remodeling-related DEGs were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed only four KEGG enriched pathways were shared between 'Benihoppe' and 'Sweet Charlie,' including flavonoid biosynthesis, phenylalanine metabolism, phenylpropanoid biosynthesis and ubiquinone, and other terpenoid-quinone biosynthesis. Integrating the results of transcriptomic and metabolomics analyses showed that adenosine triphosphate-binding cassette (ABC) transporters and flavonoid pathway genes might play important roles in the salt stress response in strawberry, and DAMs and DEGs related to ABC transporter and flavonoid pathways were differentially expressed or accumulated. The results of this study reveal that cell wall remodeling and ABC transporters contribute to the response to salt stress in strawberry, and that related genes showed differential expression patterns in varieties with different salt tolerances. These findings provide new insights into the underlying molecular mechanism of strawberry response to salt stress and suggest potential targets for the breeding of salt-tolerant strawberry varieties.

Chromosome-level genome assembly of Fragaria pentaphylla using PacBio and Hi-C technologies.

Fragaria pentaphylla, a wild diploid quinquefoliolate species of Fragaria, is native to Southwest China. It has two morphs of red and white fruit color in nature and has characteristics of unique fragrance and resistance, which made it not only a valuable breeding material but also a potential model plant for molecular function researches. Here, we generate a high-quality chromosome-level genome assembly of a F. pentaphylla accession, BAAFS-FP039 employing a combination of PacBio Long-Read Sequencing, Illumina Short-Read Sequencing, and Hi-C Sequencing. The assembled genome contained 256.74 Mb and a contig N50 length of 32.38 Mb, accounting for 99.9% of the estimated genome (256.77 Mb). Based on Hi-C data, seven pseudo-chromosomes of F. pentaphylla-FP039 genome were assembled, covering 99.39% of the genome assembly. The genome was composed of 44.61% repetitive sequences and 29,623 protein-coding genes, 97.62% of protein-coding genes could be functionally annotated. Phylogenetic and chromosome syntenic analysis revealed that F. pentaphylla-FP039 was closely related to F. nubicola. This high-quality genome could provides fundamental molecular resources for evolutionary studies, breeding efforts, and exploring the unique biological characteristics of F. pentaphylla.

An artificial intelligence model to predict survival and chemotherapy benefits for gastric cancer patients after gastrectomy development and validation in international multicenter cohorts.

BACKGROUND: Gastric cancer (GC) is a major health problem worldwide, with high prevalence and mortality. The present GC staging system provides inadequate prognostic information and does not reflect the chemotherapy benefit of GC. METHODS: Two hundred fifty-five patients who underwent surgical resection were enrolled in our study (training cohort = 212, internal validation cohort = 43). Nine clinicopathologic features were obtained to construct an support vector machine (SVM) model. The cohorts from 4 domestic centres and The Cancer Genome Atlas (TCGA) were used for external validation. RESULTS: In the training cohort, the AUCs were 0.773 (95% CI 0.708-0.838) for 5-year overall survival (OS) and 0.751 (95% CI 0.683-0.820) for 5-year disease-free survival (DFS); in the domestic validation cohort, the AUCs were 0.852 (95% CI 0.810-0.894) and 0.837 (95% CI 0.792-0.882), respectively. The model performed better than the TNM staging system according to the receiver operator characteristic(ROC) curve. GC patients were significantly divided into low, moderate and high risk based on the SVM. High-risk TNM stage Ⅱ and Ⅲ patients were more likely to benefit from adjuvant chemotherapy than low-risk patients. CONCLUSIONS: The SVM-based model may be used to predict OS and DFS in GC patients and the benefit of adjuvant chemotherapy in TNM stage Ⅱ and Ⅲ GC patients.

From Anti-HER-2 to Anti-HER-2-CAR-T Cells: An Evolutionary Immunotherapy Approach for Gastric Cancer.

Current Therapeutic modalities provide no survival advantage to gastric cancer (GC) patients. Targeting the human epidermal growth factor receptor-2 (HER-2) is a viable therapeutic strategy against advanced HER-2 positive GC. Antibody-drug conjugates, small-molecule tyrosine kinase inhibitors (TKIs), and bispecific antibodies are emerging as novel drug forms that may abrogate the resistance to HER-2-specific drugs and monoclonal antibodies. Chimeric antigen receptor-modified T cells (CAR-T) targeting HER-2 have shown considerable therapeutic potential in GC and other solid tumors. However, due to the high heterogeneity along with the complex tumor microenvironment (TME) of GC that often leads to immune escape, the immunological treatment of GC still faces many challenges. Here, we reviewed and discussed the current progress in the research of anti-HER-2-CAR-T cell immunotherapy against GC.

Genome-Wide Analysis of Ascorbic Acid Metabolism Related Genes in Fragaria × ananassa and Its Expression Pattern Analysis in Strawberry Fruits.

Ascorbic acid (AsA) is an important antioxidant for scavenging reactive oxygen species and it is essential for human health. Strawberry (Fragaria × ananassa) fruits are rich in AsA. In recent years, strawberry has been regarded as a model for non-climacteric fruit ripening. However, in contrast to climacteric fruits, such as tomato, the regulatory mechanism of AsA accumulation in strawberry fruits remains largely unknown. In this study, we first identified 125 AsA metabolism-related genes from the cultivated strawberry "Camarosa" genome. The expression pattern analysis using an available RNA-seq data showed that the AsA biosynthetic-related genes in the D-mannose/L-galactose pathway were downregulated remarkably during fruit ripening which was opposite to the increasing AsA content in fruits. The D-galacturonate reductase gene (GalUR) in the D-Galacturonic acid pathway was extremely upregulated in strawberry receptacles during fruit ripening. The FaGalUR gene above belongs to the aldo-keto reductases (AKR) superfamily and has been proposed to participate in AsA biosynthesis in strawberry fruits. To explore whether there are other genes in the AKR superfamily involved in regulating AsA accumulation during strawberry fruit ripening, we further implemented a genome-wide analysis of the AKR superfamily using the octoploid strawberry genome. A total of 80 FaAKR genes were identified from the genome and divided into 20 subgroups based on phylogenetic analysis. These FaAKR genes were unevenly distributed on 23 chromosomes. Among them, nine genes showed increased expression in receptacles as the fruit ripened, and notably, FaAKR23 was the most dramatically upregulated FaAKR gene in receptacles. Compared with fruits at green stage, its expression level increased by 142-fold at red stage. The qRT-PCR results supported that the expression of FaAKR23 was increased significantly during fruit ripening. In particular, the FaAKR23 was the only FaAKR gene that was significantly upregulated by abscisic acid (ABA) and suppressed by nordihydroguaiaretic acid (NDGA, an ABA biosynthesis blocker), indicating FaAKR23 might play important roles in ABA-mediated strawberry fruit ripening. In a word, our study provides useful information on the AsA metabolism during strawberry fruit ripening and will help understand the mechanism of AsA accumulation in strawberry fruits.

Clinical role of miR-421 as a novel biomarker in diagnosis of gastric cancer patients: A meta-analysis.

BACKGROUND: Gastric cancer (GC) has been identified as one of the most common malignancies. It was found that microRNAs can be used as potential biomarkers for GC diagnosis. The aim of this study was to estimate the diagnostic value of 4 potential microRNAs in GC. METHODS: PubMed, Embase, Cochrane Library, and Web of Science were used to search published studies. The quality of the studies was scored with the Quality Assessment of Diagnostic Accuracy Studies. The pooled sensitivity and specificity, diagnostic odds ratio (DOR) and area under the curve (AUC) were calculated. The heterogeneity was evaluated using Cochrane Q statistics and the inconsistency index. RESULTS: A total of 22 studies reporting the diagnostic value of miR-21 (n = 9), miR-106 (n = 10), miR-421 (n = 5) and miR-223 (n = 3) were included. Quality Assessment of Diagnostic Accuracy Studies scores showed the high quality of the selected 22 articles. The random effects model was adopted by evaluating the heterogeneity between articles. The DOR, AUC, and Q value of miRNA-21 were 12.37 (95% confidence interval [CI]: 5.36-28.54), 0.86 and 0.79, respectively. The DOR, AUC and Q value of miRNA-106 were 12.98 [95% CI: 7.14-23.61], 0.85 and 0.78, respectively. The DOR, AUC and Q value of miRNA-421 were 27.86 [95% CI: 6.04-128.48], 0.92 and 0.86, respectively. The DOR, AUC and Q value of miRNA-223 were 18.50 [95% CI: 7.80-43.86], 0.87 and 0.80, respectively. These results indicate that miRNA-421 has the highest diagnostic accuracy, followed by miR-223, miRNA-21, and miRNA-106 among the 4 microRNAs in GC. CONCLUSIONS: miR-21, miR-106, miR-421, and miR-223 have good diagnostic efficacy, especially miR-421, could be used as auxiliary diagnostic indicator for GC.

Pectoralis major muscle turnover flap reconstruction for treatment of deep sternal wound infection in infants and children.

OBJECTIVE: The aim of the study is to assess the therapeutic effect and applicability of pectoralis major muscle turnover flap (PMMTF) reconstruction for treatment of deep sternal wound infection (DSWI) after cardiac surgery in infants and children. METHODS: From March 2013 to October 2021, 23 patients with DSWI after cardiac surgery underwent PMMTF reconstruction. The data and outcomes of the patients were retrospectively analyzed. RESULTS: Twenty patients were treated with unilateral PMMTF reconstruction, and three patients were treated by bilateral PMMTF. All of the sternal wounds healed successfully. All patients survived and were discharged without evidence of infection. In a follow-up period, ranging from 15 to 83 months (mean 32.6 months), all patients demonstrated normal development with no limitations to limb movements. There were no signs of chronic sternal infection in all of them. CONCLUSION: PMMTF reconstruction is a simple, feasible, and effective treatment of DSWI after cardiac surgery in infants and children, with minimal developmental problems.

Gibberellin Induced Transcriptome Profiles Reveal Gene Regulation of Loquat Flowering.

Flowering is an integral part of the life cycle of flowering plants, which is essential for plant survival and crop production. Most woody fruit trees such as apples and pears bloom in spring, but loquat blooms in autumn and winter. Gibberellin (GA) plays a key role in the regulation of plant flower formation. In this study, we sprayed loquat plants with exogenous GA3, which resulted in vigorous vegetative growth rather than floral bud formation. We then performed a comprehensive RNA-seq analysis on GA3-treated and control-treated leaves and buds over three time periods to observe the effects of exogenous GA3 application on floral initiation and development. The results showed that 111 differentially expressed genes (DEGs) and 563 DEGs were down-regulated, and 151 DEGs and 506 DEGs were up-regulated in buds and leaves, respectively, upon treatment with GA3. Among those that are homologs of the DELLA-mediated GA signal pathway genes, some may be involved in the positive regulation of flower development, including EjWRKY75, EjFT, EjSOC1, EjAGL24, EjSPL, EjLFY, EjFUL, and EjAP1; while some may be involved in the negative regulation of flower development, including EjDELLA, EjMYC3, EjWRKY12, and EjWRKY13. Finally, by analyzing the co-expression of DEGs and key floral genes EjSOC1s, EjLFYs, EjFULs, EjAP1s, 330 candidate genes that may be involved in the regulation of loquat flowering were screened. These genes belong to 74 gene families, including Cyclin_C, Histone, Kinesin, Lipase_GDSL, MYB, P450, Pkinase, Tubulin, and ZF-HD_dimer gene families. These findings provide new insights into the regulation mechanism of loquat flowering.

Genome-Wide Identification of Aquaporin Gene Family in Pitaya Reveals an HuNIP6;1 Involved in Flowering Process.

Aquaporins (AQPs) are essential membrane proteins involved in seed maturation and germination, stomata movement, photosynthesis, and regulation of plant flowering processes. Pitaya flowers are open at night and wither at daybreak, which shows an obvious circadian rhythm. In this study, a comprehensive genome-wide analysis of AQPs in Hylocereus undantus was conducted to screen key genes associated with flowering processes. A total of 33 HuAQP genes were identified from the H. undantus genome. The 33 HuAQPs were grouped into four subfamilies: 10 PIPs, 13 TIPs, 8 NIPs, and 2 SIPs, which were distributed on 9 out of 11 pitaya chromosomes (Chr) (except for Chr7 and Chr10). Results from expression profiles showed that HuNIP6;1 may be involved in pitaya's floral opening. HuNIP6;1 was localized exclusively in the cell membrane. Overexpression of HuNIP6;1 in Arabidopsis thaliana significantly promoted early flowering through regulating negative flowering regulators of MJM30, COL9, and PRR5, suggesting that HuNIP6;1 plays key roles in regulating flowering time. The present study provides the first genome-wide analysis of the AQP gene family in pitaya and valuable information for utilization of HuAQPs.

The complete mitochondrial genome sequence of Fragaria orientalis (Rosaceae).

Fragaria orientalis Lozinsk. is valuable germplasm material for cross breeding in Fragaria. In this study, we assembled the complete mitochondrial genome of F. orientalis using a combination of Illumina data and Nanopore data. The mitochondrial genome was 275,143 bp in length, including 29 protein-coding genes, 20 tRNA genes, and three rRNA genes, with a total GC content 45.23%. Seven protein-coding genes contained introns, and three were trans-spliced. Phylogenetic analysis indicated that F. orientalis is making a sister clade to the Amygdaloideae species. The complete mitochondrial genome of F. orientalis reported in this study will improve our understanding of Fragaria evolution.

Polyploidy underlies co-option and diversification of biosynthetic triterpene pathways in the apple tribe.

Whole-genome duplication (WGD) plays important roles in plant evolution and function, yet little is known about how WGD underlies metabolic diversification of natural products that bear significant medicinal properties, especially in nonmodel trees. Here, we reveal how WGD laid the foundation for co-option and differentiation of medicinally important ursane triterpene pathway duplicates, generating distinct chemotypes between species and between developmental stages in the apple tribe. After generating chromosome-level assemblies of a widely cultivated loquat variety and Gillenia trifoliata, we define differentially evolved, duplicated gene pathways and date the WGD in the apple tribe at 13.5 to 27.1 Mya, much more recent than previously thought. We then functionally characterize contrasting metabolic pathways responsible for major triterpene biosynthesis in G. trifoliata and loquat, which pre- and postdate the Maleae WGD, respectively. Our work mechanistically details the metabolic diversity that arose post-WGD and provides insights into the genomic basis of medicinal properties of loquat, which has been used in both traditional and modern medicines.

Complete chloroplast genome sequencing of ten wild Fragaria species in China provides evidence for phylogenetic evolution of Fragaria.

Complete chloroplast genomes of ten wild Fragaria species native to China were sequenced. Phylogenetic analysis clustered Fragaria species into two clades: The south clade (F. iinumae, F. chinensis, F. pentaphylla, F. nilgerrensis, F. daltoniana, F. corymbosa, F. moupinensis, F. tibetica, F. nipponica, F. gracilis, and F. nubicola and north clade (F. viridis, F. orientalis, F. moschata, F. mandshurica, F. vesca, F. chiloensis, F. virginiana, and F. × ananassa), while F. iinumae is the oldest extant species. Molecular clock analysis suggested present Fragaria species share a common ancestor 3.57 million years ago (Ma), F. moschata and octoploid species evolve 0.89 and 0.97 Ma, respectively, but F. moschata be not directly involved in current octoploid species formation. Drastic global temperature change since the Palaeocene-Eocene, approx. 55 Ma, especially during uplifting of the Qinghai-Tibet plateau and quaternary glaciation may have driven the formation of Fragaria, separation of two groups and polyploidization.

EjRAV1/2 Delay Flowering Through Transcriptional Repression of EjFTs and EjSOC1s in Loquat.

Most species in Rosaceae usually need to undergo several years of juvenile phase before the initiation of flowering. After 4-6 years' juvenile phase, cultivated loquat (Eriobotrya japonica), a species in Rosaceae, enters the reproductive phase, blooms in the autumn and sets fruits during the winter. However, the mechanisms of the transition from a seedling to an adult tree remain obscure in loquat. The regulation networks controlling seasonal flowering are also largely unknown. Here, we report two RELATED TO ABI3 AND VP1 (RAV) homologs controlling juvenility and seasonal flowering in loquat. The expressions of EjRAV1/2 were relatively high during the juvenile or vegetative phase and low at the adult or reproductive phase. Overexpression of the two EjRAVs in Arabidopsis prolonged (about threefold) the juvenile period by repressing the expressions of flowering activator genes. Additionally, the transformed plants produced more lateral branches than the wild type plants. Molecular assays revealed that the nucleus localized EjRAVs could bind to the CAACA motif of the promoters of flower signal integrators, EjFT1/2, to repress their expression levels. These findings suggest that EjRAVs play critical roles in maintaining juvenility and repressing flower initiation in the early life cycle of loquat as well as in regulating seasonal flowering. Results from this study not only shed light on the control and maintenance of the juvenile phase, but also provided potential targets for manipulation of flowering time and accelerated breeding in loquat.

USP8 Inhibitor Suppresses HER-2 Positive Gastric Cancer Cell Proliferation and Metastasis via the PI3K/AKT Signaling Pathway.

PURPOSE: Referring to global cancer statistics, the incidence of gastric cancer (GC) was ranked sixth; however, detailed mechanisms underlying its development were not thoroughly investigated. Previous studies have reported that inhibition of ubiquitin-specific peptidase 8 (USP8) induced degradation of several receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), embryonic stem cells (ESCs), etc. Nevertheless, the regulation of HER-2 by USP8 and the molecular mechanisms controlling their role in the pathogenesis of GC remain unknown. PATIENTS AND METHODS: A total of 69 patients with histologically confirmed GC were recruited to satisfy the purpose of this study. Initially, tumor samples and GC cell lines were used to detect USP8 and HER-2 levels. Next, MTT and colony formation assays were applied to analyze cell proliferation capability. Cell migration and invasion ability were examined by transwell assays. To examine related mRNA and protein expressions, Western blot assays and quantitative real-time PCR (qRT-PCR) were performed. Immunofluorescence was used to detect the effect of USP8 inhibitor on GC cells. Finally, in vivo experiments were used to examine the effect of USP8 inhibitor. RESULTS: Patients with USP8 high-expression tumors have shown worse overall survival while opposite results found in patients with low USP8 expressions. Regarding disease prognosis, patients with low expression of USP8 and HER-2 were performed better prognosis, whereas those with overexpression of USP8 and HER-2 shown poor prognosis. USP8 inhibitor significantly inhibited HER-2 positive cell NCI-N87 proliferation and metastasis. In addition, USP8 stabilizes HER-2, preventing it from ubiquitin proteasome-mediated degradation. In vivo studies confirmed that the USP8 inhibitor inhibited HER-2 positive cell NCI-N87 tumor growth. However, it did not affect the HER2-negative cell MGC-803. Careful investigation unraveled that the USP8 inhibitor significantly inhibited NCI-N87 cell proliferation and metastasis via phosphatidylinositol-3-kinases/protein-serine-threonine kinase (PI3K/AKT) pathway. CONCLUSION: The USP8 inhibited HER-2 positive GC cell proliferation and migration in vivo and in vitro and probably served as a novel potential therapeutic biomarker for HER-2 positive GC.

Proximal gastrectomy with anti-reflux anastomosis for patients with adenocarcinoma of the esophagogastric junction: The simple and safe triangle-valve technique.

A simple and safe triangle-valve technique (TVT) was applied in proximal gastrectomy (PG) in order to prevent postoperative gastric reflux among patients with adenocarcinoma of the esophagogastric junction (AEG). The clinical outcomes were evaluated in comparison to those of canonical total gastrectomy (TG). This retrospective study of 74 AEG patients compared two surgical procedures, PG-TVT (n=44) and TG (n=30), in terms of surgical outcomes, postoperative complications and nutritional status. The Reflux Disease Questionnaire (RDQ) was used to evaluate reflux esophagitis, and patients with an RDQ score of ≥12 points were diagnosed with gastroesophageal reflux disease (GERD). The mean operative time was significantly shorter in the PG-TVT group (242.6 min) compared with that in the TG group (288.1 min). The overall postoperative complication rate did not differ significantly between the PG-TVT and TG groups. All the patients were followed up for 6 months, and none developed cancer recurrence in distant organs, gastric remnant, or lymph nodes. The GERD incidence was similar between the PG-TVT and TG groups. The mean levels of total protein and albumin within 6 months were significantly higher in the PG-TVT group compared with those in the TG group after adjustingtthe time effect and the interaction of time and surgical methods. The level of total protein significantly increased within 6 months in the PG-TVT group, but decreased in the TG group. Therefore, PG-TVT has several advantages over TG for patients with AEG, including a shorter operative time and better postoperative nutritional status, whereas the incidence of GERD was found to be similar between the two techniques.

Down-Regulation of USP8 Suppresses HER-3 Positive Gastric Cancer Cells Proliferation.

BACKGROUND: Ubiquitin specific peptidase 8 (USP8) has been reported to induce the degradation of several receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), among which human epidermal growth factor receptor-3 (HER-3) is one of them. However, the role and functional mechanisms of USP8 and HER-3 in gastric cancer (GC) remain unknown. OBJECTIVE: To explore the function and mechanism of USP8 and HER-3 in the progression of GC. MATERIALS AND METHODS: Eighty-eight patients with histologically confirmed GC were recruited for this study. Tumor samples and GC cell lines were used to detect USP8 and HER-3 levels. MGC803 (HER-3 negative GC cell) was selected as the control group and NCI-N87, MKN-45 and AGS (HER-3 positive GC cells) as the experimental group. USP8i and si-RNA were then used to down-regulate USP8 in each group. Apoptosis and cell-cycle experiments were performed to detect the effects of USP8 on GC cells. Cytotoxicity Assay Kit (MTT) and colony formation assays were used to analyze cell proliferation. Cell migration and invasion ability were examined by wound healing. The expression of related mRNA and protein was detected by Western blot and quantitative real-time PCR (qRT-PCR). In vivo experiments were used to examine the effect of USP8 and HER-3. RESULTS: Patients with high expression of USP8 or HER-3 tumors alone died earlier than those with low expression and the patients with both USP8 and HER-3 high expression had a shorter overall survival than those with the opposite pattern (both USP8 and HER-3 low expression). Down-regulation of USP8 inhibited cell proliferation and cell metastasis and also reduced the HER-3 expression. We also observed that down-regulation of USP8 inhibited the proliferation of GC cells which highly expressed HER-3. Moreover, down-regulation of USP8 could promote the apoptosis of HER3-positive GC cells and inhibit the proliferation of them by affecting the cell-cycle. In vivo studies demonstrated that down-regulation of USP8 inhibited HER-3 positive tumors growth. CONCLUSION: Down-regulation of USP8 inhibits HER-3 positive GC cells proliferation in vivo and in vitro, which indicate that USP8 represents a feasible choice as a therapeutic target for HER-3 positive GC cells.