Epidural administration of large dose of opioid µ receptor agonist may impair cardiac functions and myocardial viability via desensitizing transient receptor potential vanilloid 1.

The incidence of postoperative myocardial injury remains high as the underlying pathogenesis is still unknown. The dorsal root ganglion (DRG) neurons express transient receptor potential vanilloid 1 (TRPV1) and its downstream effector, calcitonin gene-related peptide (CGRP) participating in transmitting pain signals and cardiac protection. Opioids remain a mainstay therapeutic option for moderate-to-severe pain relief clinically, as a critical component of multimodal postoperative analgesia via intravenous and epidural delivery. Evidence indicates the interaction of opioids and TRPV1 activities in DRG neurons. Here, we verify the potential impairment of myocardial viability by epidural usage of opioids in postoperative analgesia. We found that large dose of epidural morphine (50 µg) significantly worsened the cardiac performance (+dP/dtmax reduction by 11% and -dP/dtmax elevation by 24%, all P < 0.001), the myocardial infarct size (morphine vs Control, 0.54 ± 0.09 IS/AAR vs. 0.23 ± 0.06 IS/AAR, P < 0.001) and reduced CGRP in the myocardium (morphine vs. Control, 9.34 ± 2.24 pg/mg vs. 21.23 ± 4.32 pg/mg, P < 0.001), while induced definite suppression of nociception in the postoperative animals. It was demonstrated that activation of µ-opioid receptor (µ-OPR) induced desensitization of TRPV1 by attenuating phosphorylation of the channel in the dorsal root ganglion neurons, via inhibiting the accumulation of cAMP. CGRP may attenuated the buildup of ROS and the reduction of mitochondrial membrane potential in cardiomyocytes induced by hypoxia/reoxygenation. The findings of this study indicate that epidurally giving large dose of µ-OPR agonist may aggravate myocardial injury by inhibiting the activity of TRPV1/CGRP pathway.

Circ-sh3rf3/GATA-4/miR-29a regulatory axis in fibroblast-myofibroblast differentiation and myocardial fibrosis.

The transdifferentiation from cardiac fibroblasts to myofibroblasts is an important event in the initiation of cardiac fibrosis. However, the underlying mechanism is not fully understood. Circ-sh3rf3 (circular RNA SH3 domain containing Ring Finger 3) is a novel circular RNA which was induced in hypertrophied ventricles by isoproterenol hydrochloride, and our work has established that it is a potential regulator in cardiac hypertrophy, but whether circ-sh3rf3 plays a role in cardiac fibrosis remains unclear, especially in the conversion of cardiac fibroblasts into myofibroblasts. Here, we found that circ-sh3rf3 was down-regulated in isoproterenol-treated rat cardiac fibroblasts and cardiomyocytes as well as during fibroblast differentiation into myofibroblasts. We further confirmed that circ-sh3rf3 could interact with GATA-4 proteins and reduce the expression of GATA-4, which in turn abolishes GATA-4 repression of miR-29a expression and thus up-regulates miR-29a expression, thereby inhibiting fibroblast-myofibroblast differentiation and myocardial fibrosis. Our work has established a novel Circ-sh3rf3/GATA-4/miR-29a regulatory cascade in fibroblast-myofibroblast differentiation and myocardial fibrosis, which provides a new therapeutic target for myocardial fibrosis.

Effects of Postmortem Hemolysis and Ultrafiltration on Creatinine Detection Results.

OBJECTIVES: To investigate the interference of postmortem hemolysis on the detection of creatinine and whether ultrafiltration can reduce the interference. METHODS: A total of 33 non-hemolyzed whole blood samples from the left heart were collected. Hemolyzed samples with 4 hemoglobin mass concentration gradients H1-H4 were artificially prepared. Ultrafiltration was performed on each hemolyzed sample. Creatinine concentrations in non-hemolyzed serum (baseline serum), hemolyzed samples and ultrafiltrate were detected. Bias (B), Pearson correlation and receiver operator characteristic (ROC) of baseline creatinine concentration between before and after ultrafiltration were analyzed. RESULTS: As the hemoglobin mass concentration increased, B of the hemolyzed samples in the H1-H4 groups gradually increased, the |B| was 2.41(0.82, 8.25)-51.31(41.79, 188.25), reaching a maximum of 589.06%, and there was no statistically significant between the creatinine concentration and the baseline creatinine concentration (P=0.472 7, r=0.129 5). After ultrafiltration of hemolyzed samples, the interference of creatinine concentration in ultrafiltrate was significantly reduced, the |B| was 5.32(2.26, 9.22)-21.74(20.06, 25.58), reaching a maximum of 32.14%, and there was a positive correlation with baseline creatinine concentration (P<0.05, r=0.918 2). In the hemolyzed samples of H3 and H4 groups, there were 7 false-positive samples and 1 false-negative sample; in the ultrafiltrate samples, there were no false-positive sample and 1 false-negative sample. ROC analysis results showed the hemolyzed samples were lack of diagnostic value (P=0.117 5). CONCLUSIONS: The postmortem hemolysis significantly interferes creatinine detection results of blood samples, ultrafiltration can reduce hemolysis-induced interference in postmortem creatinine detection.

Anaerophilus nitritogenes gen. nov., sp. nov., isolated from salt lake sediment in Xinjiang Province, China.

An obligately anaerobic, nitrate-reducing bacterial strain (MJB2T) was isolated from sediments of saline in Xinjiang province of China. Cells were Gram-stain-positive rods and motile by means of flagella and formed endospores. The novel strain MJB2T was able to grow at 15-37 °C (optimum 28-30 °C), pH 5.8-9.4 (optimum 7.8) and with 1.0-7.0% NaCl (optimum 5.0-6.0%, w/v). Sulfate, sulfite, thiosulfate, elemental sulfur, nitrite and Fe(III) were not used as terminal electron acceptors. Oxidase and catalase reactions were positive. H2S was producted from L-cystine. Complex substrates such as beef extract, peptone and yeast extract can be used as sole energy sources. The DNA G+C content was 29.4 mol%. The major cellular fatty acids (> 10%) were C14:0, C16:1 cis 7 and C16:1 cis 9. The main polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, three unidentified amino lipids, one unidentified amino glycolipid, two unidentified glycolipid, one unidentified aminophospholipid and two unidentified lipids. No respiratory quinones were detected. According to phylogenetic analysis based on 16S rRNA gene sequences, strain MJB2T was affiliated to the family Clostridiaceae (order Clostridiales) with highest 16S rRNA gene sequence similarity of 95.3% to Crassaminicella profunda Ra1766HT. Strain MJB2T exhibited 74.9% ANI values to C. profunda Ra1766HT. In silico DNA-DNA relatedness value between strain MJB2T and C. profunda Ra1766HT was 19.5%. The distinct biochemical, chemotaxonomic and phylogenetic differences from the previously described taxa supported that strain MJB2T represents a novel species of a new genus, for which the name Anaerophilus nitritogenes gen. nov., sp. nov. is proposed. The type strain is MJB2T (=KCTC 15800T=MCCC 1K03631T).

A boronic acid modified binary matrix consisting of boron nitride and α-cyano-4-hydroxycinnamic acid for determination of cis-diols by MALDI-TOF MS.

A MALDI-TOF mass spectrometric method is described for the determination of small molecule compounds with cis-diol. It is based on the use of a binary matrix consisting of boron nitride (BN) and α-cyano-4-hydroxycinnamic acid that was modified with the derivatization reagent of (3-(acridin-9-ylamino)phenyl)boronic acid which can recognize cis-diols. The binary matrix is used for desorption/ionization (DI) in the positive ion mode. The mechanism leading to DI enhancement was investigated. The results imply that BN is beneficial for the DI because it induces an enhancement in the positive ion mode. The boronic acid-functionalized binary matrix was successfully applied to capture the glucose, shikimic acid and quinic acid. The method was applied to the determination of 3-chloro-1,2-propanediol in plant oil. Graphical abstract Schematic representation of a method for detecting the cis-diol compounds on matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the binary matrix of boron nitride (BN)/α-cyano-4-hydroxycinnamic acid (CHCA) that was modified with (3-(acridin-9-ylamino)phenyl) boronic acid (AYPBA).

[Autism spectrum disorder-like symptoms in the population with intellectual disability aged 6 to 18 years].

OBJECTIVE: To investigate the incidence of autism spectrum disorder (ASD)-like symptoms in the population with intellectual disability (ID). METHODS: The students with ASD or ID, aged 6-18 years, who studied in a special school in Shanghai from January to June, 2017, as well as the typically developing (TD) population of the same age, who studied in a general school in Shanghai during the same period, were enrolled. Social Responsiveness Scale (SRS) was completed by their parents or other guardians, and the ASD-like symptoms were evaluated. RESULTS: A total of 69 subjects with ASD, 74 subjects with ID and 177 TD subjects were enrolled. The ID group had a significantly higher SRS-positive rate than the TD group (47.3% vs 1.7%; P<0.001) and a significantly lower SRS-positive rate than the ASD group (47.3% vs 87.0%; P<0.001). The total score of SRS was 114±26 in the ASD group, 80±24 in the ID group and 38±19 in the TD group. The ID group had a significantly higher total score of SRS than the TD group (P<0.05), and the score on the subscale of social cognition showed the most significant difference between the two groups (Cohen's d=2.00). There were no significant differences in the total score of SRS and the scores on each subscale of SRS between the mild-to-moderate ID and severe-to-extremely severe ID groups (P>0.05), and there was no significant correlation between SRS score and IQ (P>0.05). CONCLUSIONS: The ID population aged 6-18 years has more ASD-like symptoms than the general population, and ASD screening and intervention should be performed for the ID population as early as possible.

Effectiveness and feasibility of acupuncture for knee osteoarthritis: a pilot randomized controlled trial.

OBJECTIVE:: To evaluate the effectiveness of acupuncture for pain relief and function improvement in patients with knee osteoarthritis and to determine the feasibility of an eight-week acupuncture intervention. DESIGN:: Pilot randomized controlled trial. SETTING:: Three teaching hospitals in China. SUBJECTS:: Patients with knee osteoarthritis (Kellgren grade II or III). INTERVENTIONS:: Patients were randomly assigned to an eight-week (three sessions per week) intervention of either traditional Chinese acupuncture or sham acupuncture. MAIN MEASURES:: The primary outcome was response rate-the proportion of patients achieving score ⩾36% decrease in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and function at week 8 compared with baseline. Secondary outcomes included pain, function and quality of life. RESULTS:: Of 42 patients randomized, 36 (85.7%) completed the study. There was no significant difference in response rate between the traditional Chinese acupuncture and control groups: 61.9% (13 of 21) versus 42.9% (9 of 21) achieved score ⩾36% decrease in WOMAC pain and function at week 8 ( P = 0.217). The sum of WOMAC pain and function scores at week 8 was 11.6 (9.1) in the traditional Chinese acupuncture group compared with 16.3 (10.9) in the control group ( P = 0.183). There was no significant difference between groups. Three adverse events were recorded and were classified as mild. CONCLUSION:: It showed that three sessions per week acupuncture intervention of knee osteoarthritis was feasible and safe. No difference was observed between groups due to small sample size. Larger (sample size ⩾ 296) randomized controlled trials of this intervention appear justified.

Fast design of broadband terahertz diffusion metasurfaces.

A method for fast design of broadband terahertz diffusion metasurface is presented. The proposed metasurface is composed by three kinds of simply patterned elements with different resonant properties. To obtain the best broadband performance with the lowest backward reflections, a genetic algorithm is developed to manipulate the resonances for the fast determination of element geometries. An inverse discrete Fourier transform method is used to predict the scattering pattern of the metasurface with high accuracy and low time consumption, significantly enhancing the efficiency of the array-pattern design. The proposed fast design flow will benefit a broad range of terahertz applications, such as biological detection and imaging.

Discovery of pyrazolo[1,5-a]pyrimidine-3-carbonitrile derivatives as a new class of histone lysine demethylase 4D (KDM4D) inhibitors.

Herein we report the discovery of a series of new small molecule inhibitors of histone lysine demethylase 4D (KDM4D). Molecular docking was first performed to screen for new KDM4D inhibitors from various chemical databases. Two hit compounds were retrieved. Further structural optimization and structure-activity relationship (SAR) analysis were carried out to the more selective one, compound 2, which led to the discovery of several new KDM4D inhibitors. Among them, compound 10r is the most potent one with an IC50 value of 0.41±0.03µM against KDM4D. Overall, compound 10r could be taken as a good lead compound for further studies.

The synthesis and antistaphylococcal activity of dehydroabietic acid derivatives: Modifications at C-12.

A series of 12-oxime and O-oxime ether derivatives of dehydroabietic acid were synthesized and investigated for the antibacterial activity against Staphylococcus aureus Newman strain and five multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108, and NRS-271). The aromatic oximate derivative 11a showed the highest activity with MIC of 0.39-0.78µg/mL against S. aureus Newman. Of note, compounds 10b, 11 and 14 showed the most potent antibacterial activity against five multidrug-resistant S. aureus with MIC values of 1.25-3.13µg/mL. These results offered useful information for further strategic optimization in search of the antibacterial candidates against infection of multidrug-resistant Gram-positive bacteria.

Prevalence and genetic characterization of Toxoplasma gondii in pet dogs in Central China.

The prevalence and genotype of Toxoplasma gondii infection in dogs in Henan Province, Central China was investigated. A total of 125 blood samples were collected from pet dogs during April to June 2013, and all samples were examined by indirect hemagglutination antibody test (IHA) and nested PCR. The overall T. gondii prevalence in pet dogs was 24.0% (30/125), with 20.8% (26/125) in IHA and 10.4% (13/125) in PCR, respectively. No statistical associations were found between animal gender and age and the prevalence of T. gondii infection. Thirteen positive DNA samples were genotyped using 11 PCR-RFLP markers, including SAG1, (3'+5') SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Of these, only 2 samples were genotyped with complete data for all loci, and a novel genotype (type III at SAG3 and GRA6 loci, and type I at other loci) was identified. This is the first report of genetic characterization of T. gondii infection in dogs in China.

Di-(2-ethylhexyl) phthalate exacerbates abnormalities of testicular development in F1 males via inhibition the Wnt/ß-catenin signaling pathway.

The theory of "Developmental Origins of Health and Disease (DOHaD)" espouses that environmental exposures to toxicants during critical developmental stages can affect health outcomes in adulthood. Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer that can be transferred to developing organisms via the placenta and breast milk as an environmental endocrine disruptor. We herein implemented a cross-fostering model to decipher the contributions of prenatal vs. postnatal exposure to low or high dose DEHP (30 or 500 mg/kg-bw•d) on reproductive outcomes in male offspring and the underlying mechanism of action. Unexpectedly, we observed that postnatal DEHP exposure programmed weight gain in a dose-dependent manner, in-utero exposure to high dose DEHP appeared to constitute a significant factor in the weight loss of male offspring. Moreover, in the low dose group, offspring of control that were suckled by DEHP dams (CC-DE) generated a considerable number of adverse reproductive outcomes compared with the offspring of DEHP that were suckled by control dams (DE-CC), based on histopathologic alterations in the testis, blockage of sex hormone secretion, and transcriptional inhibition of steroid-hormone-related factors in the hypothalamic-pituitary-testicular (HPT) axis. However, DE-CC group affected reproductive dysfunction in male offspring more so than CC-DE in the high dose group. Mechanistically, DEHP contributed to the inhibition of steroidogenesis by perturbing the Wnt/ß-catenin-signaling pathway. These studies confirm the sensitivity window in which future reproductive outcomes in offspring are influenced following developmental exposure to DEHP at two different dosages, and reveals a critical role for the Wnt/ß-catenin signaling pathway in DEHP-induced male reproductive disorders.

Morphine timing-dependent modulation of TRPV1 phosphorylation correlates with differential morphine effects on myocardial ischemia/reperfusion injury.

Opioids are used for pain relief in patients suffering from acute myocardial ischemia or infarction. Clinical and laboratory studies demonstrate that morphine treated patients or the experimental animal model suffering acute myocardial ischemia and reperfusion, may worsen myocardial viability. As transient receptor potential vanilloid 1 (TRPV1) plays important roles in pain sensation and cardio-protection, we query whether opioids may exacerbate myocardial viability via interaction with TRPV1 activity in the pain relief. We found the co-expressions of TRPV1 and opioid µ, δ and κ receptors in adult rat cardiomyocytes. Intravenous injection of morphine (0.3 mg/kg) at 20 min after induction of myocardial ischemia, in the rat model of acute myocardial ischemia and reperfusion, induced significant reduction of phosphorylated TRPV1 (p-TRPV1) in the ventricular myocardium and increase in serum cardiac troponin I (cTnI), compared with the ischemia/reperfusion controls (all P < 0.05). The effects of morphine were completely reversed by selective opioid µ, δ and κ receptor antagonists. While significant upregulation of p-TRPV1 (P < 0.05) and improvement of ±dP/dt max (all P < 0.05) were detected in the animals giving the same dose of morphine before induction of myocardial ischemia. The changes in p-TRPV1 correlate with the alterations of cTnI (r = -0.5840, P = 0.0283) and ±dP/dt max (r = 0.8084, P = 0.0005 and r = -0.8133, P = 0.0004, respectively). The findings of this study may indicate that potentiation and attenuation of TRPV1 sensitivity correlate with the improvement of the cardiac performance and the aggravation of myocardial viability, respectively, by giving morphine before and during myocardial ischemia and reperfusion.

Insulin induces C2C12 cell proliferation and apoptosis through regulation of cyclin D1 and BAD expression.

Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis.

Sensitive determination of chloroanilines in water samples by hollow fiber-based liquid-phase microextraction prior to capillary electrophoresis with amperometric detection.

A hollow fiber-based liquid-phase microextraction method has been developed for enrichment of trace chloroanilines in water samples. Target analytes including aniline, three mono-chlorinated aniline isomers (o-chloroaniline, m-chloroaniline, and p-chloroaniline) and four mono-chlorinated methylaniline isomers (2-chloro-4-methylaniline, 3-chloro-4-methylaniline, 4-chloro-2-methylaniline, and 5-chloro-2-methylaniline) were determined by CE with amperometric detection after microextraction. Several factors that affect separation, detection, and extraction efficiency were investigated. Under the optimum conditions, eight aniline compounds could be well separated from other components coexisting in water samples within 25 min, exhibiting a linear calibration over three orders of magnitude (r > 0.998); the obtained enrichment factors were between 51 and 239, and the LODs were in the range of 0.01-0.1 ng/mL. The proposed method has been applied for the analyses of real environmental water and sewage samples with relative recoveries in the range of 83-108%.

[Analysis on chemical constituents of Gardenia jasminoides by UHPLC-Q-TOFMS].

OBJECTIVE: To analyze the chemical constituents of Gardenia jasminoides by UHPLC-Q-TOFMS. METHODS: A ACQUITY UPLC HSS T3 (100 mm x 2.1 mm, 1.8 microm) column was used. The mobile phase consisted of water containing 0.1% methane acid and acetonitrile was used in gradient elution. The flow rate was 0.4 mL/min. TOFMS was applied for qualitative analysis under negative ion mode. RESULTS: Under optimized condition, 18 major constituents in Gardenia jasminoides were identified by quadrupole-time of flight mass spectrometry and structure-relevant fragment ions. CONCLUSION: A simple and reliable method using UHPLC-Q-TOFMS is established and can be used to identify the chemical constituents of Gardenia jasminoides.

[Effect of Zinc Fertilizer Application on Cadmium Accumulation in Wheat Grain and Its Application Risk].

The interaction of zinc (Zn) and cadmium (Cd) is an important research direction in the prevention and control of Cd pollution of wheat in recent years. In this study, a typical wheat field in North China was selected as the object to explore the control effect and application risk of Zn fertilizer on Cd pollution in a soil-wheat system through field experiments. The results showed that under the treatment of a low dosage of Zn, the Cd concentrations in wheat grains in Jiyuan City and Kaifeng City decreased by 33.4% and 25.3% compared with those in the control, respectively. By contrast, Cd concentrations in wheat grains treated with a high dosage of Zn increased by 22.4% and 34.2% compared with that of the low-dosage Zn treatment. After the application of Zn, the total amount and available Zn concentrations increased significantly, and Cd was partially activated in these two locations. Canonical correlation analysis (CCA) showed that when the Zn concentrations in the soils were less than 200 mg·kg-1, soil Zn was the main factor affecting Cd accumulation in the soil-wheat system, whereas when Zn concentrations in soils were greater than 200 mg·kg-1, the activation of soil Cd was the main factor affecting Cd accumulation in wheat grains. Regression analysis showed that when the soil Cd/Zn ratio decreased to 0.0089 (low dosage of Zn), Zn and Cd showed an antagonistic effect, whereas when the soil Cd/Zn ratio decreased to 0.0078 (high dosage of Zn), Zn and Cd showed a synergistic effect. According to the characteristics of regional Cd pollution, adjusting the amount of Zn fertilizer can improve the efficiency of pollution control and avoid aggravating the harm of Cd pollution.

[Prediction of Cadmium Uptake Factor in Wheat Based on Machine Learning].

Applying machine learning methods to resolve the cadmium (Cd) uptake characteristics of regional soil-wheat systems can contribute to the accuracy and rationality of risk decisions. Based on a regional survey, we constructed a Freundlich-type transfer equation, random forest (RF) model, and neural network (BPNN) model to predict wheat Cd enrichment factor (BCF-Cd); verified the prediction accuracy; and assessed the uncertainty of different models. The results showed that both RF (R2=0.583) and BPNN (R2=0.490) were better than the Freundlich transfer equation (R2=0.410). The RF and BPNN were further trained repeatedly, and the results showed that the mean absolute error (MAE) and root mean square error (RMSE) of RF and BPNN were close to each other. Additionally, the accuracy and stability of RF (R2=0.527-0.601) was higher than that of BPNN (R2=0.432-0.661). Feature importance analysis showed that multiple factors led to the heterogeneity of wheat BCF-Cd, in which soil phosphorus (P) and zinc (Zn) were the key variables affecting the change in wheat BCF-Cd. Parameter optimization can further improve the accuracy, stability, and generalization ability of the model.

Localization and density of tertiary lymphoid structures associate with molecular subtype and clinical outcome in colorectal cancer liver metastases.

BACKGROUND: Tertiary lymphoid structures (TLSs) have been proposed to assess the prognosis of patients with cancer. Here, we investigated the prognostic value and relevant mechanisms of TLSs in colorectal cancer liver metastases (CRCLM). METHODS: 603 patients with CRCLM treated by surgical resection from three cancer centers were included. The TLSs were categorized according to their anatomic subregions and quantified, and a TLS scoring system was established for intratumor region (T score) and peritumor region (P score). Differences in relapse-free survival (RFS) and overall survival (OS) between groups were determined. Multiplex immunohistochemical staining (mIHC) was used to determine the cellular composition of TLSs in 40 CRCLM patients. RESULTS: T score positively correlated with superior prognosis, while P score negatively associated with poor survival (all p<0.05). Meanwhile, T score was positively associated with specific mutation subtype of KRAS. Furthermore, TLSs enrichment gene expression was significantly associated with survival and transcriptomic subtypes of CRCLM. Subsequently, mIHC showed that the densities of Treg cells, M2 macrophages and Tfh cells were significantly higher in intratumor TLSs than in peritumor TLSs (p=0.029, p=0.047 and p=0.041, respectively), and the frequencies of Treg cells and M2 macrophages were positively correlated with P score, while the frequencies of Tfh cells were positively associated with T scores in intratumor TLSs (all p<0.05). Next, based on the distribution and abundance of TLSs, an Immune Score combining T score and P score was established which categorized CRCLM patients into four immune classes with different prognosis (all p<0.05). Among them, patients with higher immune class have more favorable prognoses. The C-index of Immune Class for RFS and OS was higher than Clinical Risk Score statistically. These results were also confirmed by the other two validation cohorts. CONCLUSIONS: The distribution and abundance of TLSs is significantly associated with RFS and OS of CRCLM patients, and a novel immune class was proposed for predicting the prognosis of CRCLM patients.

[The application of a high resolution melting-based genotyping method in studying the association between FTO rs9930506 polymorphism and metabolic syndrome in Beijing population].

OBJECTIVE: To set up a new method, which is sensitive, low cost, rapid and suitable for clinical application for FTO gene rs9930506 variant genotyping basing on high resolution melting (HRM) platform, and to preliminarily put into practice in susceptibility analysis for metabolic syndrome (MS) in Beijing. METHODS: Unlabelled probe with C3-spacer block specific for rs9930506 variant has been designed according to the Refseq from GenBank. With LC-Green plus dye pre-mixed, we scanned the signal for the genotype analysis after PCR amplification and HRM reaction. Restriction fragment length polymorphism (RFLP) and PCR-sequencing methods were designed as 2 control genotyping methods for the evaluation of accuracy and convenience. Afterwards, the HRM-based method was put into practice in metabolic syndrome patients (n = 500) and control groups (n = 500) for rs9930506 genotyping, and primarily study the association between rs9930506 and MS. RESULTS: All the 3 methods could genotype rs9930506 appropriately, although the 2 control methods seemed to be a little time-inefficient. The call rate of HRM-method was 100% and sampling accuracy reached 99.3% according to sequencing results. In the MS group, AA, AG and GG genotypes were found in 290, 185 and 25 cases, respectively. And in the control group, those were found in 344, 138 and 18 cases. No genotype distribution difference was detected between control group and HapMap-CHB data (P = 0.520). The genotype distributions were all in Hardy-Weinberg equilibrium in each group. AA genotype of rs9930506 seemed to reduce the risk for MS (OR = 0.626, 95%CI = 0.483 - 0.812). CONCLUSIONS: The AA genotype of rs9930506 variant in FTO might be a protective factor for MS in Beijing population. The susceptibility related genotyping in clinical samples could be more rapid, precise and inexpensive with the development of HRM in genotyping.