MdTPR16, an apple tetratricopeptide repeat (TPR)-like superfamily gene, positively regulates drought stress in apple.

The Tetratricopeptide repeat (TPR)-like superfamily with TPR conserved domains is widely involved in the growth and abiotic stress in many plants. In this report, the gene MdTPR16 belongs to the TPR family in apple (Malus domestica). Promoter analysis reveal that MdTPR16 incorporated various stress response elements, including the drought stress response elements. And different abiotic stress treatments, drought especially, significantly induce the response of MdTPR16. Overexpression of MdTPR16 result in better drought tolerance in apple and Arabidopsis by up-regulating the expression levels of drought stress-related genes, achieving a higher chlorophyll content level, more material accumulation, and overall better growth compared to WT in the drought. Under drought stress, the overexpressed MdTPR16 also mitigate the oxidative damage in cells by reducing the electrolyte leakage, malondialdehyde content, and the H2O2 and O2- accumulation in apples and Arabidopsis. In conclusion, MdTPR16 act as a beneficial regulator of drought stress response by regulating the expression of related genes and the cumulation of reactive oxygen species (ROS).

Post-Harvest Application of Nanoparticles of Titanium Dioxide (NPs-TiO2) and Ethylene to Improve the Coloration of Detached Apple Fruit.

In this study, we analyzed the effects of treatments with titanium dioxide nanoparticles (NPs-TiO2) and ethylene on anthocyanin biosynthesis and reactive oxygen species (ROS) metabolism during light exposure in ripe 'red delicious' apples. Both treatments led to improved anthocyanins biosynthesis in detached mature apples, while the NPs-TiO2 had less impact on the fruit firmness, TSS, TA, and TSS/TA ratio. Furthermore, the effects of both treatments on the expression of anthocyanin-related enzymes and transcription factors in the apple peel were evaluated at the gene level. The differentially expressed genes induced by the two treatments were highly enriched in the photosynthesis and flavonoid biosynthesis pathways. The expression of structural genes involved in anthocyanin biosynthesis and ethylene biosynthesis was more significantly upregulated in the ethylene treatment group than in the NPs-TiO2 treatment group, and the opposite pattern was observed for the expression of genes encoding transcription factors involved in plant photomorphogenesis pathways. In addition, the ROS levels and antioxidant capacity were higher and the membrane lipid peroxidation level was lower in fruit in the NPs-TiO2 treatment group than in the ethylene treatment group. The results of this study reveal differences in the coloration mechanisms induced by NPs-TiO2 and ethylene in apples, providing new insights into improving the color and quality of fruits.

SLHSD: hybrid scaffolding method based on short and long reads.

In genome assembly, scaffolding can obtain more complete and continuous scaffolds. Current scaffolding methods usually adopt one type of read to construct a scaffold graph and then orient and order contigs. However, scaffolding with the strengths of two or more types of reads seems to be a better solution to some tricky problems. Combining the advantages of different types of data is significant for scaffolding. Here, a hybrid scaffolding method (SLHSD) is present that simultaneously leverages the precision of short reads and the length advantage of long reads. Building an optimal scaffold graph is an important foundation for getting scaffolds. SLHSD uses a new algorithm that combines long and short read alignment information to determine whether to add an edge and how to calculate the edge weight in a scaffold graph. In addition, SLHSD develops a strategy to ensure that edges with high confidence can be added to the graph with priority. Then, a linear programming model is used to detect and remove remaining false edges in the graph. We compared SLHSD with other scaffolding methods on five datasets. Experimental results show that SLHSD outperforms other methods. The open-source code of SLHSD is available at https://github.com/luojunwei/SLHSD.

Clinical features of 375 COVID-19 cases imported from Russia through the Suifenhe port and countermeasures.

BACKGROUND AND OBJECTIVES: At present, the focus of the fighting against COVID-19 in China is shifting to strictly prevent the entrance of cases from abroad and disease transmission. Therefore, it is extremely urgent to better understand the clinical features of imported cases from overseas countries, which is conductive to formulate the corresponding countermeasures. This study aimed to describe the clinical features of COVID-19 cases imported from Russia through the Suifenhe port, in order to identify baseline and clinical data associated with disease progression and present corresponding countermeasures. METHODS: All COVID-19 cases imported from Russia through the Suifenhe port were included in this retrospective study. According to the "Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (seventh edition)", imported COVID-19 cases were divided into asymptomatic infection, mild, moderate, severe, and critical groups. Baseline and clinical data, including age, gender, comorbidities, disease severity, symptoms at onset, body temperature, white blood cell (WBC) count, lymphocyte (LYMPH) count, lymphocyte percentage (LYM%), C-reactive protein (CRP), oxygenation index (OI), and the use therapeutic modalities were obtained on admission, and then compared between groups. RESULTS: A total of 375 COVID-19 cases imported from Russia through Suifenhe port were included, of whom the asymptomatic infection, mild, moderate, severe, and critical groups accounted for 4.0%, 13.9%, 75.5%, 5.3%, and 1.3%, respectively. The majority of the imported COVID-19 cases were men (61.9%) with a median age of 38.72 years who had no comorbidity (87.7%). Nearly one-third of them (33.1%) were asymptomatic at onset, and common initial symptoms included fever (36.5%), cough (36.0%), pharyngeal discomfort (12.3%), expectoration (8.0%), and chest tightness (5.3%). In total, 180 (48%) and 4 (1.1%) enrolled imported cases received nasal tube oxygen inhalation therapy and high-flow oxygen absorption, respectively; the remaining patients did not undergo oxygen therapy. The values of age, body temperature, WBC, LYMPH, LYM%, CRP, and OI were 38.72 ± 10.50, 35.10 ± 7.92, 5.59 ± 1.97, 1.67 ± 0.68, 31.05 ± 10.22, 8.00 ± 14.75, and 389.03 ± 74.07, respectively. Gender, age, LYMPH, LYM%, symptoms at onset, cough, fever, other rare symptoms, and oxygen therapy showed significant differences between groups (P = 0.036, < 0.001, < 0.001, < 0.001, < 0.001, < 0.001, < 0.001, = 0.045, < 0.001, respectively). CONCLUSIONS: Compared with domestic confirmed patients, COVID-19 patients who arrived at China from Russia through the Suifenhe port had significantly different clinical features, and the differences in gender, age, LYMPH, LYM%, symptoms at onset, cough, fever, other rare symptoms, and oxygen therapy between groups were statistically significant. Therefore, detailed and comprehensive countermeasures were developed to manage and prevent another outbreak based on these clinical features.

Identification of Key Regulators of Hepatitis C Virus-Induced Hepatocellular Carcinoma by Integrating Whole-Genome and Transcriptome Sequencing Data.

Background: Hepatitis C virus (HCV) infection is a major cause of cirrhosis and hepatocellular carcinoma (HCC). Despite recent advances in the understanding of the biological basis of HCC development, the molecular mechanisms underlying HCV-induced HCC (HCC-HCV) remain unclear. The carcinogenic potential of HCV varies according to the genotype and mutation in its viral sequence. Moreover, regulatory pathways play important roles in many pathogenic processes. Therefore, identifying the pathways by which HCV induces HCC may enable improved HCC diagnosis and treatment. Methods: We employed a systematic approach to identify an important regulatory module in the process of HCV-HCC development to find the important regulators. First, an HCV-related HCC subnetwork was constructed based on the gene expression in HCC-HCV patients and HCC patients. A priority algorithm was then used to extract the module from the subnetworks, and all the regulatory relationships of the core genes of the network were extracted. Integrating the significantly highly mutated genes involved in the HCC-HCV patients, core regulatory modules and key regulators related to disease prognosis and progression were identified. Result: The key regulatory genes including EXO1, VCAN, KIT, and hsa-miR-200c-5p were found to play vital roles in HCV-HCC development. Based on the statistics analysis, EXO1, VCAN, and KIT mutations are potential biomarkers for HCV-HCC prognosis at the genomic level, whereas has-miR-200c-5P is a potential biomarker for HCV-HCC prognosis at the expression level. Conclusion: We identified three significantly mutated genes and one differentially expressed miRNA, all related to HCC prognosis. As potential pathogenic factors of HCC, these genes and the miRNA could be new biomarkers for HCV-HCC diagnosis.

Upregulation Mitochondrial Carrier 1 (MTCH1) Is Associated with Cell Proliferation, Invasion, and Migration of Liver Hepatocellular Carcinoma.

Among the primary causes of cancer-associated death in the world, liver hepatocellular carcinoma (LIHC) ranks the third. LIHC is defined as the sixth most frequently diagnosed carcinoma. The gene mitochondrial carrier 1 (MTCH1) is a protein-coding gene. Recent research suggests that MTCH1 may be associated with some diseases. Here, our study attempts to explore the role and implication of MTCH1 in LIHC. Kaplan Meier Plotter and GEPIA (Gene Expression Profiling Interactive Analysis) databases were employed to determine the expression of MTCH1 and its correlation with prognostic status in LIHC patients. For the first time, our results suggested that MTCH1 was aberrantly expressed in human pan-cancer and highly expressed in LIHC. Its high expression was closely associated with metastasis of tumor, stage of cancer, and poor survival of patients. Then, through enrichment analysis, MTCH1 was found to be closely related to RNA splicing in LIHC. Subsequently, we conducted a series of functional experiments. PCR data showed that LIHC cell lines and samples are highly expressed MTCH1. CCK-8 (Cell Counting Kit-8) assays and Transwell assays indicated that silencing MTCH1 certainly suppressed cell proliferation, migration, and invasion. These findings shed the clue that MTCH1 could be regarded as the potential prognosis biomarker of LIHC and a promising therapeutic target for LIHC.

Two Bariatric Surgical Procedures Differentially Alter the Intestinal Microbiota in Obesity Patients.

AIMS: To explore the intestinal microbiota composition affected by the two most widely used procedures of bariatric surgery, laparoscopic sleeve gastrectomy (LSG) and laparoscopic roux-en-Y gastric bypass (LRYGB), in Chinese obesity patients. METHODS: Stool samples were collected from the obese patients before (n = 87) and with follow-up after the surgery (n = 53). After DNA extraction, 16S rDNA (V3 + V4 regions) sequencing was completed on Illumina HiSeq 2500 sequencing platform. The samples were analyzed base on four groups, pre-LSG (n = 54), pre-LRYGB (n = 33), post-LSG (n = 33), and post-LRYGB (n = 20). The linear mixed models were used to analyze the alteration of intestinal microbiota before and after the surgeries of LSG or LRYGB. Student's t test and χ2 test were used for analysis of independent groups; Metastats analysis was used to compare the relative abundance of bacteria, and Pearson correlation and Spearman correlation analysis were used to test the correlation between indicated groups. RESULTS: 87 patients were included and 53 (60.92%) of them completed the follow-up (9.60 ± 3.92 months). Body mass index (BMI) decreased from 37.84 ± 6.16 kg/m2 to 26.22 ± 4.33 kg/m2 after LSG and from 45.75 ± 14.26 kg/m2 to 33.15 ± 10.99 kg/m2 after LRYGB. The relative abundance of 5 phyla and 42 genera were altered after the surgery in the cohort. Although no alteration of Firmicutes was observed at phylum level, 54.76% of the altered genera belong to phylum Firmicutes. Both LSG and LRYGB procedures increased the richness and evenness of intestinal microbiota in obese patients after the surgery. Particularly, 33 genera altered after LSG and 19 genera altered after LRYGB, in which 11 genera were common alterations in both procedures. CONCLUSION: Both LSG and LRYGB altered the composition of intestinal microbiota in Chinese obesity patients, and particularly increased the richness and evenness of microbiota. Genera belonging to phylum Firmicutes were the most altered bacteria by bariatric surgery. The procedure of LSG resulted in much more pronounced alteration of the intestinal microbiota abundance than that observed in LRYGB. While different genera were altered after LSG and LRYGB procedures, 10 genera were the common altered genera in both procedures. Bacteria altered after LSG and LRYGB were functionally associated with BMI, and with relieving of the metabolic syndromes.

An automatic detection method for 40-Hz auditory steady state response and its application in prognosis of comatose patients.

OBJECTIVE: We proposed a 40-Hz auditory steady-state response (ASSR) automatic detection method, and studied the prognosis of comatose patients by combining the 40-Hz ASSR detection results of multiple paradigms of auditory stimulation. METHODS: The 40-Hz ASSR elicitation experiments were carried out on 32 comatose patients, with the detection results used as prognosis predictors. To achieve automatic detection, the detection was modeled as a binary hypothesis test for a sinusoidal waveform with unknown amplitude and phase, based on the generalized likelihood ratio test (GLRT). The patients were followed up for 6 months, and each patient's outcome was classified as either favorable outcome (severe disability, moderate disability or good recovery) or unfavorable outcome (vegetative state/unresponsive wakefulness syndrome or death) according to the Glasgow outcome scale (GOS). The performance of the prognosis predictors was assessed using the area under the receiver operating characteristic curve (AUC-ROC). RESULTS: The largest AUC in univariate analysis involving a single stimulation paradigm was 0.849, while the AUC obtained by combining multiple predictors was increased to 0.966. CONCLUSIONS: For comatose patients, the absence of 40-Hz ASSR in multiple stimulation paradigms may indicate an unfavorable prognosis. Furthermore, the combination of multiple auditory stimulation paradigms may increase the outcome prediction accuracy. SIGNIFICANCE: The combination of multi-paradigm 40-Hz ASSR automatic detection results may provide a feasible automatic outcome prediction method for comatose patients.

Role of depressive symptoms in subjective memory complaint in older adults with mild cognitive impairment.

AIMS: To explore the independent relationship between depressive symptoms and subjective memory complaint (SMC) amongst older adults with mild cognitive impairment (MCI) after adjusting for objective cognitive function and other important confounding factors. BACKGROUND: subjective memory complaint is a core symptom of MCI and is often the primary reason for older adults with MCI to seek for medical help. Improving subjective memory amongst older adults with MCI is important to enhance their quality of life and potentially delay further cognitive decline. Depressive symptoms, which are highly prevalent neuropsychiatric symptoms amongst older adults with MCI, may be one of the reasons that affect an individual's self-perception of memory function. However, there is a dearth of studies to provide a thorough evaluation of the independent relationship between depressive symptoms and SMC amongst older adults with MCI. DESIGN: A descriptive correlational study. METHODS: A consecutive sample (N = 154) of adults aged over 60 years was recruited from a community healthcare centre between June and September 2016. MCI was detected using the Montreal Cognitive Assessment. Depressive symptoms and subjective memory were measured using the Geriatric Depression Scale and Memory Inventory for the Chinese, respectively. Hierarchical regression was performed to explore the relationship between SMC and depressive symptoms, with control over objective cognitive function, socio-demographic and health-related confounding factors. RESULTS: After controlling objective cognitive function and other confounding factors, SMC was independently associated with depressive symptoms (standardised ß = 0.336, p < .001). This psychological status even explained for a greater variance (R2  = 8.8%) for SMC compared with objective cognitive function (R2  = 2.4%). CONCLUSION: subjective memory complaint was independently associated with depressive symptoms in older adults with MCI. Early detection and management of depressive symptoms are highly important amongst this clinical cohort. IMPLICATIONS FOR PRACTICE: Early detection and prompt treatment of depressive symptoms is a highly prioritised care agenda in managing SMC in older adults with MCI.

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice.

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2'-deoxyguanosine (to deplete donor's lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient's T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

Perturbations of Neuron-Restrictive Silencing Factor Modulate Corticotropin-Releasing Hormone Gene Expression in the Human Cell Line BeWo.

Stress exacerbates disease, and understanding its molecular mechanisms is crucial to the development of novel therapeutic interventions to combat stress-related disorders. The driver of the stress response in the hypothalamic-pituitary-adrenal axis (HPA) is corticotropin-releasing hormone (CRH), a neuropeptide synthesized in the paraventricular nucleus of the hypothalamus. Evidence supports that CRH expression is epigenetically modified at the molecular level by environmental stimuli, causing changes in the stress response. This effect is mediated by a concert of factors that translate environmental change into alterations in gene expression. An important regulator and epigenetic modulator of CRH expression is neuron-restrictive silencing factor (NRSF). Previously, our lab identified numerous splice variants of NRSF that are specific to humans and predictive of differential regulatory effects of NRSF variants on targeted gene expression. The human cell line BeWo has endogenous CRH and NRSF expression providing an in vitro model system. Here, we show that manipulation of NRSF expression through siRNA technology, overexpression by plasmid vectors, and direct cAMP induction that CRH expression is linked to changes in NRSF expression. Accordingly, this epigenetic regulatory pathway in humans might be a critical mechanism involved in the regulation of the stress response.

Modulation of nuclear REST by alternative splicing: a potential therapeutic target for Huntington's disease.

Huntington's disease (HD) is caused by a genetically mutated huntingtin (mHtt) protein with expanded polyQ stretch, which impairs cytosolic sequestration of the repressor element-1 silencing transcription factor (REST), resulting in excessive nuclear REST and subsequent repression of neuronal genes. We recently demonstrated that REST undergoes extensive, context-dependent alternative splicing, of which exon-3 skipping (∆E3 )-a common event in human and nonhuman primates-causes loss of a motif critical for REST nuclear targeting. This study aimed to determine whether ∆E3 can be targeted to reduce nuclear REST and rescue neuronal gene expression in mouse striatal-derived, mHtt-expressing STHdhQ111/Q111 cells-a well-established cellular model of HD. We designed two morpholino antisense oligos (ASOs) targeting the splice sites of Rest E3 and examined their effects on ∆E3 , nuclear Rest accumulation and Rest-controlled gene expression in STHdhQ111/Q111 cells. We found that (1) the ASOs treatment significantly induced ∆E3 , reduced nuclear Rest, and rescued transcription and/or mis-splicing of specific neuronal genes (e.g. Syn1 and Stmn2) in STHdhQ111/Q111 cells; and (2) the ASOs-induced transcriptional regulation was dependent on ∆E3 induction and mimicked by siRNA-mediated knock-down of Rest expression. Our findings demonstrate modulation of nuclear REST by ∆E3 and its potential as a new therapeutic target for HD and provide new insights into environmental regulation of genome function and pathogenesis of HD. As ∆E3 is modulated by cellular signalling and linked to various types of cancer, we anticipate that ∆E3 contributes to environmentally tuned REST function and may have a broad range of clinical implications.

Barbell-shaped stir bar sorptive extraction using dummy template molecularly imprinted polymer coatings for analysis of bisphenol A in water.

This study reports the development of a novel dummy template molecularly imprinted polymer (MIP)-coated barbell-shaped stir bar. The MIP stir bar coatings were prepared by using 2,2-bis(4-hydroxyphenyl)butane (BPB), 4,4'-dihydroxydiphenylmethane (BPF), 4-tert-butylphenol (PTBP), and tetrabromobisphenol A (TBBA) as dummy templates using a capillary in situ polymerization method. Uniform coatings can be prepared controllably. The method is simple, easy, and reproducible. The barbell-shaped stir bar was developed by using medical silicone tubes as wheels. The wheels could be removed and reinstalled when necessary; therefore, the barbell-shaped stir bar was easy to disassemble and reassemble. The novel MIP-coated stir bar showed good selectivity for the target analyte, bisphenol A (BPA). The established method is selective and sensitive with a lower detection limit for BPA of 0.003 µg/L. The dummy template MIP-coated stir bar is suitable for trace BPA analysis in real environmental water samples without template leakage. The novel stir bar can be used at least 100 times.

Gene-gene interactions among CX3CL1, LEPR and IL-6 related to coronary artery disease in Chinese Han population.

OBJECTIVE: The purpose of this study was to investigate the impact of the interactions among CX3CL1 (rs170364 and rs614230), LEPR (rs6700896), and IL-6 (rs2066992) polymorphisms on the risk of coronary artery disease (CAD) in Chinese Han population. METHODS: 120 CAD patients and 109 healthy controls were enrolled in the study. Polymerase chain reaction (PCR) and direct sequencing methods were used to analyze the genotypes of CX3CL1, LEPR, and IL-6 polymorphisms. Multifactor dimensionality reduction (MDR) software was utilized to analyze gene-gene interactions. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used for evaluating the association between gene polymorphisms or gene-gene interactions and CAD risk. RESULTS: In the study, TT genotype of rs170364 in CX3CL1 might decrease the CAD risk (OR=0.39, 95% CI=0.16-0.98). No significant correlation was found between T allele of rs170364 and CAD risk (P>0.05). CC genotype and C allele in rs614230 (CX3CL1) were significantly related with decreased risk of CAD (OR=0.38, 95% CI=0.17-0.86; OR=0.66, 95% CI=0.45-0.97). For IL-6 rs2066992 polymorphism. GG genotype could increase the risk of CAD (OR=2.32, 95% CI=1.04-5.17). Whereas, no significant correlation was observed between LEPR rs6700896 and CAD susceptibility. MDR analysis showed that CX3CL1, LEPR and IL-6 genes might jointly promote the occurrence of CAD. CONCLUSIONS: The interactions of CX3CL1, LEPR and IL-6 genes might increase the risk of CAD.

Depletion of neutrophils protects against L-arginine-induced acute pancreatitis in mice.

BACKGROUND/AIMS: Acute pancreatitis (AP) is an inflammatory disease characterized by acinar cell damage and inflammation of the pancreas with infiltration of leukocytes, predominantly neutrophils. We investigated whether neutrophil depletion protects against experimental AP induced by L-arginine. METHODS: AP was induced in C57BL/6 mice via two intraperitoneal L-arginine (4 g/kg) injections. Mice were pretreated with 250 and 100 µg anti-Gr-1 antibody via intraperitoneal injection at 24 and 4 h, respectively, before L-arginine challenge for neutrophil depletion. At 48 and 72 h after injection, the severity of AP was determined with the aid of biochemical and histological analyses. Amylase and MPO activity was detected using specific assay kits. The plasma cytokines levels were detected using ELISA. RESULTS: Neutrophil depletion resulted in significantly reduced plasma amylase levels in pancreas, myeloperoxidase (MPO) activity in pancreas and lung, reactive oxygen species (ROS) generation and cell apoptosis, and decreased circulating neutrophil, tissue damage as well as expression levels of nuclear factor NF-κB. CONCLUSION: Neutrophil depletion is capable of reducing tissue damage of pancreas and lung in mice with acute pancreatitis.

MicroRNA-377 suppresses cell proliferation and invasion by inhibiting TIAM1 expression in hepatocellular carcinoma.

Increasing evidence has suggested that microRNAs (miRNAs) play an important role in the initiation and progression of hepatocellular carcinoma (HCC). Here, we identified a novel tumor suppressive miRNA, miR-377, and investigated its role in HCC. The expression of miR-377 in HCC tissues and cell lines was detected by real-time reverse-transcription PCR. The effects of miR-377 on HCC cell proliferation and invasion were also investigated. Western blot and luciferase reporter assay were used to identify the direct and functional target of miR-377. The expression of miR-377 was markedly downregulated in human HCC tissues and cell lines. MiR-377 can dramatically inhibit cell growth and invasion in HCC cells. Subsequent investigation revealed that T lymphoma invasion and metastasis 1 (TIAM1) was a direct and functional target of miR-377 in HCC cells. Overexpression of miR-377 impaired TIAM1-induced promotion of proliferation and invasion in HCC cells. Finally, miR-377 is inversely correlated with TIAM1 expression in human HCC tissues. These findings reveal that miR-377 functions as a tumor suppressor and inhibits the proliferation and invasion of HCC cells by targeting TIAM1, which may consequently serve as a therapeutic target for HCC patients.

CCR7 expression and intratumoral FOXP3+ regulatory T cells are correlated with overall survival and lymph node metastasis in gastric cancer.

The aim of this study was to investigate the prognostic value of chemokine receptor CCR7 expression and intratumoral FOXP3(+) regulatory T cells (Tregs) in gastric cancer. CCR7(+) tumor cells and FOXP3(+) Tregs were assessed by immunohistochemistry in tissue microarrays containing gastric cancer from 133 patients. Prognostic effects of low or high CCR7 and FOXP3 expression were evaluated by Cox regression and Kaplan-Meier analysis, as well as the correlation between CCR7 positive score and intratumoral FOXP3(+) cell number in a longitudinal assessment. The analysis showed that the high expression levels of CCR7 and FOXP3 were detected in 69.9% and 65.4% of cases, respectively. High CCR7 expression in gastric cancer cells was significantly associated with poor overall survival (OS) (P = 0.010) and lymph node metastasis (P = 0.009), and was an independent factor for worse OS (P = 0.023) by multivariate analysis. High numbers of intratumoral FOXP3(+) Tregs significantly correlated with shorter OS (P = 0.021) and lymph node metastasis (P = 0.024), and was also an independent factor for adverse OS (P = 0.035). Furthermore, there was a significantly positive correlation between CCR7 positive score and intratumoral FOXP3(+) cell number (r = 0.949, P<0.001). These results revealed that CCR7 expression in gastric cancer cells and intratumoral FOXP3(+) Tregs could be considered as a co-indicator of clinical prognosis of gastric cancer.

Depletion of CD4+ CD25+ regulatory T cells promotes CCL21-mediated antitumor immunity.

CCL21 is known to attract dendritic cells (DCs) and T cells that may reverse tumor-mediated immune suppression. The massive infiltration of tumors by regulatory T cells (Tregs) prevents the development of a successful helper immune response. In this study, we investigated whether elimination of CD4(+) CD25(+) Tregs in the tumor microenvironment using anti-CD25 monoclonal antibodies (mAbs) was capable of enhancing CCL21-mediated antitumor immunity in a mouse hepatocellular carcinoma (HCC) model. We found that CCL21 in combination with anti-CD25 mAbs (PC61) resulted in improved antitumor efficacy and prolonged survival, not only inhibited tumor angiogenesis and cell proliferation, but also led to significant increases in the frequency of CD4(+), CD8(+) T cells and CD11c(+) DCs within the tumor, coincident with marked induction of tumor-specific CD8(+) cytotoxic T lymphocytes (CTLs) at the local tumor site. The intratumoral immune responses were accompanied by the enhanced elaboration of IL-12 and IFN-γ, but reduced release of the immunosuppressive mediators IL-10 and TGF-ß1. The results indicated that depletion of Tregs in the tumor microenvironment could enhance CCL21-mediated antitumor immunity, and CCL21 combined with anti-CD25 mAbs may be a more effective immunotherapy to promote tumor rejection.

Extensive alternative splicing of the repressor element silencing transcription factor linked to cancer.

The repressor element silencing transcription factor (REST) is a coordinate transcriptional and epigenetic regulator which functions as a tumor suppressor or an oncogene depending on cellular context, and a truncated splice variant REST4 has been linked to various types of cancer. We performed a comprehensive analysis of alternative splicing (AS) of REST by rapid amplification of cDNA ends and PCR amplification of cDNAs from various tissues and cell lines with specific primers. We identified 8 novel alternative exons including an alternate last exon which doubles the REST gene boundary, along with numerous 5'/3' splice sites and ends in the constitutive exons. With the combination of various splicing patterns (e.g. exon skipping and alternative usage of the first and last exons) that are predictive of altered REST activity, at least 45 alternatively spliced variants of coding and non-coding mRNA were expressed in a species- and cell-type/tissue-specific manner with individual differences. By examining the repertoire of REST pre-mRNA splicing in 27 patients with kidney, liver and lung cancer, we found that all patients without exception showed differential expression of various REST splice variants between paired tumor and adjacent normal tissues, with striking cell-type/tissue and individual differences. Moreover, we revealed that exon 3 skipping, which causes no frame shift but loss of a domain essential for nuclear translocation, was affected by pioglitazone, a highly selective activator of the peroxisome proliferator-activated receptor gamma (PPARγ) which contributes to cell differentiation and tumorigenesis besides its metabolic actions. Accordingly, this study demonstrates an extensive AS of REST pre-mRNA which redefines REST gene boundary and structure, along with a general but differential link between REST pre-mRNA splicing and various types of cancer. These findings advance our understanding of the complex, context-dependent regulation of REST gene expression and function, and provide potential biomarkers and therapeutic targets for cancer.