Loss of 5-Hydroxymethylcytosine Is an Independent Unfavorable Prognostic Factor for Esophageal Squamous Cell Carcinoma

Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine and 5-carboxylcytosine, which result in genomic DNA demethylation. It was reported that 5-hmC levels were decreased in a variety of cancers and could be regarded as an epigenetic hallmark of cancer. In the present study, 5-hmC levels were detected by immunohistochemistry (IHC) in 173 esophageal squamous cell carcinoma (ESCC) tissues and 91 corresponding adjacent non-tumor tissues; DNA dot blot assays were used to detect the 5-hmC level in another 50 pairs of ESCC tissues and adjacent non-tumor tissues. In addition, the mRNA level of TET1, TET2 and TET3 in these 50 pairs of ESCC tissues was detected by real-time PCR. The IHC and DNA dot blot results showed that 5-hmC levels were significantly lower in ESCC tissues compared with corresponding adjacent non-tumor tissues (P = 0.029). TET2 and TET3 expression was also significantly decreased in tumor tissues compared with paired non-tumor tissues (TET2, P < 0.0001; TET3, P = 0.009), and the decrease in 5-hmC was significantly associated with the downregulation of TET2 expression (r = 0.405, P = 0.004). Moreover, the loss of 5-hmC in ESCC tissues was significantly associated with poor overall survival among patients with ESCC (P = 0.043); multivariate Cox regression analysis showed that the loss of 5-hmC in ESCC tissues was an independent unfavorable prognostic indicator for patients with ESCC (HR = 1.569, P = 0.029). In conclusion, 5-hmC levels were decreased in ESCC tissues, and the loss of 5-hmC in tumor tissues was an independent unfavorable prognostic factor for patients with ESCC.     

Characterization of Distinct T Cell Receptor Repertoires in Tumor and Distant Non-tumor Tissues from Lung Cancer Patients

T cells and T cell receptors(TCRs) play pivotal roles in adaptive immune responses against tumors.The development of next-generation sequencing technologies has enabled the analysis of the TCRb repertoire usage.Given the scarce investigations on the TCR repertoire in lung cancer tissues,in this study,we analyzed TCRb repertoires in lung cancer tissues and the matched distant non-tumor lung tissues(normal lung tissues) from 15 lung cancer patients.Based on our results,the general distribution of T cell clones was similar between cancer tissues and normal lung tissues;however,the proportion of highly expanded clones was significantly higher in normal lung tissues than in cancer tissues(0.021% ± 0.002% vs.0.016% ± 0.001%,P = 0.0054,Wilcoxon signed rank test).In addition,a significantly higher TCR diversity was observed in cancer tissues than in normal lung tissues(431.37 ± 305.96 vs.166.20 ± 101.58,P = 0.0075,Mann-Whitney U test).Moreover,younger patients had a significantly higher TCR diversity than older patients(640.7 ± 295.3 vs.291.8 ± 233.6,P = 0.036,Mann-Whitney U test),and the higher TCR diversity in tumors was significantly associated with worse cancer outcomes.Thus,we provided a comprehensive comparison of the TCR repertoires between cancer tissues and matched normal lung tissues and demonstrated the presence of distinct T cell immune microenvironments in lung cancer patients.     

Interactions between gastric microbiota and metabolites in gastric cancer

The development and progression of gastric cancer (GC) is greatly influenced by gastric microbiota and their metabolites. Here, we characterized the gastric microbiome and metabolome profiles of 37 GC tumor tissues and matched non-tumor tissues using 16s rRNA gene sequencing and ultrahigh performance liquid chromatography tandem mass spectrometry, respectively. Microbial diversity and richness were higher in GC tumor tissues than in non-tumor tissues. The abundance of Helicobacter was increased in non-tumor tissues, while the abundance of Lactobacillus, Streptococcus, Bacteroides, Prevotella, and 6 additional genera was increased in the tumor tissues. The untargeted metabolome analysis revealed 150 discriminative metabolites, among which the relative abundance of the amino acids, carbohydrates and carbohydrate conjugates, glycerophospholipids, and nucleosides was higher in tumor tissues compared to non-tumor tissues. The targeted metabolome analysis further demonstrated that the combination of 1-methylnicotinamide and N-acetyl-D-glucosamine-6-phosphate could serve as a robust biomarker for distinction between GC tumors and non-tumor tissues. Correlation analysis revealed that Helicobacter and Lactobacillus were negatively and positively correlated with the majority of differential metabolites in the classes of amino acids, carbohydrates, nucleosides, nucleotides, and glycerophospholipids, respectively, suggesting that Helicobacter and Lactobacillus might play a role in degradation and synthesis of the majority of differential metabolites in these classes, respectively. Acinetobacter, Comamonas, Faecalibacterium, Sphingomonas, and Streptococcus were also significantly correlated with many differential amino acids, carbohydrates, nucleosides, nucleotides, and glycerophospholipids. In conclusion, the differences in metabolome profiles between GC tumor and matched non-tumor tissues may be partly due to the collective activities of Helicobacter, Lactobacillus, and other bacteria, which eventually affects GC carcinogenesis and progression.Subject terms: Cancer metabolism, Gastrointestinal cancer     

Evaluation of decontamination process of heart valve and artery tissues in European Homograft Bank (EHB): a retrospective study of 1,055 cases

To evaluate the efficiency of decontamination practice in European Homograft Bank (EHB), the data of the cardiovascular tissues received during recent 2?years were retrospectively analysed in this study. After initial assessment, the tissues were incubated in a 3-antibiotics?? cocktail at 4°C for 20?C48?h. The states of contamination were evaluated before and after incubation with the focus on the differences in donor type, tissue type, germ type and incubation time. Amongst 1,055 eligible tissues, 77.2% were hearts and 22.8% were arteries. 82.2% of the tissues were retrieved from the multi-organ donors (MOD), 15.4% from the recipients of heart transplantation (RHT) and 2.4% from the non-heart beating donors (NHBD). The initial contamination rate was 27.4% with a significantly higher incidence in arteries. The RHT tissues had the lowest contamination rate comparing to that of MOD and NHBD. Staphylococcus species was the major source of contamination. After antibiotic incubation, 76.8% of the contaminated tissues were disinfected, which was significantly higher for the hearts than the arteries. The RHT tissues had the highest decontamination rate than that of MOD and NHBD tissues. Propionibacterium acnes was detected in 48.1% of the remaining contaminated cases. The average incubation time of the Propionibacterium-positive tissues was significantly shorter than that of decontaminated tissues. In conclusion, the current decontamination protocol of EHB is sufficient for most of the initially contaminated bacteria, whereas it is inadequate for Propionibacterium acnes. This may be related to the slow-growing nature of this bacterium and thereby the relative shorter antibiotic incubation time.     

Sensitive determination of bromazepam in human tissues using capillary gas chromatography-mass spectrometry

A reliable and sensitive gas chromatographic-mass spectrometric method was devised to determine the levels of bromazepam in human tissues. Bromazepam was extracted from body tissues using a three-step solvent extraction procedure. N-Desmethyldiazepam served as the internal standard. Selected ion monitoring withm/z 317 for bromazepam andm/z 270 for internal standard was used for quantitation. Calibration curves in all body tissues were linear over the concentration range from 50–500 ng/g. The lower detection limit in body tissues was 2–5 ng/g and the absolute recovery in body tissues was 27.8–68.0%. This method was used to determine the levels of bromazepam in tissues of an autopsied individual who had been prescribed psychotropic drugs and who was found dead in a car.     

Methylation of the DFNA5 increases risk of lymph node metastasis in human breast cancer

The pathogenesis of breast cancer involves multiple genetic and epigenetic events. In this study, we report an epigenetic alteration of DFNA5 in human breast cancer. DFNA5 gene was silenced in breast cancer cell lines that were methylated in the DFNA5 promoter, and restored by treatment with the demethylating agent, 5-aza-dC, and gene knock-down of DFNA5 increased cellular invasiveness in vitro. The mRNA expression of DFNA5 in breast cancer tissues was down-regulated as compared to normal tissues. Moreover, the DFNA5 promoter was found to be methylated in primary tumor tissues with high frequency (53%, 18/34). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary breast cancer tissues from normal breast tissues (15.3%, 2/13). Moreover, methylation status of DFNA5 was correlated with lymph node metastasis in breast cancer patients. Our data implicate DFNA5 promoter methylation as a novel molecular biomarker in human breast cancer.     

The Gastrointestinal Tract as a Potential Infection Reservoir of Digital Dermatitis-Associated Treponemes in Beef Cattle and Sheep

Digital dermatitis (DD) is an important cause of lameness in dairy cattle worldwide. It has now been reported in beef cattle and also sheep (contagious ovine digital dermatitis [CODD]). Three Treponema phylogroups are consistently isolated from lesions, Treponema medium-like, Treponema phagedenis-like, and Treponema pedis. The gastrointestinal (GI) tract and feces are suggested sites of treponemal infection in dairy cattle; however, isolation of DD-associated treponemes from these areas has previously failed. This study surveyed gingival tissues, rectal tissues, and feces of beef cattle and sheep for the molecular presence (PCR) and isolation of the three cultivable DD-treponeme phylogroups. Of the sheep gingival (n = 40) and rectal (n = 40) tissues, 1/40 gingival tissues was positive for DD-associated treponemes (T. pedis), as were 3/40 rectal tissues (one containing T. medium-like and two containing T. pedis). No DD-associated treponeme DNA was amplified from beef cattle rectal tissues (n = 40); however, 4/40 beef gingival tissues were positive for DD-associated treponemes (all containing T. phagedenis-like). A T. phagedenis-like DD-associated treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle (n = 41) and sheep (n = 79) feces failed to amplify DD-associated Treponema DNA. Twenty-two treponemes were isolated from sheep feces; however, upon phylogenetic analysis, these clustered with the considered nonpathogenic treponemes. This study detected DD-associated treponemes in the GI tract tissues of sheep and beef cattle and successfully isolated a DD-associated treponeme from ruminant rectal tissue. This gives evidence that the GI tract is an important infection reservoir of DD-associated treponemes in multiple DD-infected species.     

Auxin binding activities in monocotyledonous and dicotyledonous tissue

A screening study was carried out in order to investigate the binding of NAA to subcellular fractions from various dark grown monocotyledonous and dicotyledonous tissues known to be physiologically responsive to NAA. Napthalene-1-acetic acid (NAA) binding results revealed differences between the monocots and dicots. Maize coleoptile tissue gave the highest concentration of saturable binding sites with a K_d-value of 1.56 μM and another in maize mesocotyl with a K_d-value of 0.83 M indicating a single class of binding site for NAA for both tissues. However, an inability to demonstrate saturable auxin binding in dicotyledonous tissues and maize root tissues was found. It is concluded that the results of this investigation do not negate the hypothesis that the high affinity saturable binding sites found in maize shoot tissues may function as auxin receptors.     

Dicer Is Required for Maintaining Adult Pancreas

Dicer1, an essential component of RNA interference and the microRNA pathway, has many important roles in the morphogenesis of developing tissues. Dicer1 null mice have been reported to die at E7.5; therefore it is impossible to study its function in adult tissues. We previously reported that Dicer1-hypomorphic mice, whose Dicer1 expression was reduced to 20% in all tissues, were unexpectedly viable. Here we analyzed these mice to ascertain whether the down-regulation of Dicer1 expression has any influence on adult tissues. Interestingly, all tissues of adult (8–10 week old) Dicer1-hypomorphic mice were histologically normal except for the pancreas, whose development was normal at the fetal and neonatal stages; however, morphologic abnormalities in Dicer1-hypomorphic mice were detected after 4 weeks of age. This suggested that Dicer1 is important for maintaining the adult pancreas.     

DNA-methylation by nitrosocimetidine and N-methyl-N′-nitro-N-nitrosoguanidine in the intact rat

The ability of the nitroso derivative of the drug cimetidine to interact with cellular macromolecules in the intact rat was investigated. Radiolabelled nitrosocimetidine (NC) was shown to methylate DNA in a variety of tissues in the rat after oral administration. Radioactivity was also detected in the RNA and protein extracted from these same tissues. Methylation of DNA by the parent compound, cimetidine, was not detected in any of the tissues studied. For comparison, the DNA methylation produced by the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) dosed orally was measured. DNA alkylation by MNNG was found to be approx. 2–36 times greater than that produced by NC, varying with the tissues studied. The highest yield of DNA alkylation was found in the stomach for MNNG and the small intestine for nitrosocimetidine suggesting pharmacokinetic differences.     

Water Relations in Pulvini from Samanea saman: II. Effects of Excision of Motor Tissues

Pulvinar motor tissues of Samanea saman are often excised for in vitro studies of ion transport. Because ion transport may be regulated in part by hydrostatic pressure (P), this study explores how P and water potential (Ψ) change when motor tissues are excised. Water potential (Ψ) of excised extensor and flexor tissues was measured by the Chardakov method and compared with Ψ measurements made on extensor and flexor tissues of intact pulvini (HL Gorton 1987 Plant Physiol 83: 945-950). Ψ values for excised extensor and flexor tissues were always substantially more negative than for the same tissues in intact pulvini. Extensor tissues excised from open pulvini had slightly more negative Ψ than excised flexor tissues, and the opposite was true for closed pulvini. Extensor and flexor tissues elongate immediately when excised from open or closed pulvini, suggesting that in intact pulvini they are constrained from elongating by the nonextensible vascular core. In addition, both tissues in both open and closed pulvini are under compression imposed by oppositely positioned motor tissue. Excision relieves constraint and compression, decreasing P, and thus decreasing Ψ. This finding may explain, at least in part, the difference between Ψ measurements on intact and excised motor tissues. Implications of these data for the planning and interpretation of in vitro experiments requiring excised strips of extensor and flexor tissues are discussed.     

Involvement of ZEB1 and E-cadherin in the invasion of lung squamous cell carcinoma

This study intended to investigate the expression of the ZEB1 and E-cadherin proteins in lung squamous cell carcinoma (LSCC) tissues and to examine the clinicopathological correlation between protein levels and LSCC. RT-PCR and Western blot were used to examine the expression of ZEB1 and E-cadherin mRNAs and proteins in LSCC tissues as well as in adjacent normal tissues, and then analyze the relationship between the clinicopathological characteristics and the expression changes of ZEB1 and E-cadherin mRNAs in LSCC. In addition, RNAi was used to knockdown the expression of the ZEB1 gene in Human HCC827 cells; subsequently, changes in the invasive ability of the resultant cells were studied. The positive rates of ZEB1 and E-cadherin mRNAs in LSCC tissues were 69.2 and 38.5 %, respectively. They differed significantly from the corresponding positive rates in the adjacent normal lung tissues (15.4 and 80.8 %, p < 0.05). There was a negative correlation between the protein levels of ZEB1 and E-cadherin in LSCC tissues (r = -0.714, p < 0.001); in addition, it was found that ZEB1 protein expression in LSCC tissues was significantly higher than that in the neighboring normal lung tissues (p < 0.05), and its expression was also significantly higher in patients with lymph node metastases and distant metastases compared to those patients without metastatic disease (p < 0.05). On the contrary, E-cadherin expression was significantly lower in LSCC tissues than that in the neighboring normal tissue (p < 0.05). It was lower in patients with lymph node metastasis and distant metastasis compared to patients without metastatic disease (p < 0.05). However, the expression of ZEB1 and E-cadherin was independent of gender, age, tumor size, or tumor differentiation level (p > 0.05). Transfection of ZEB1 siRNA into HCC827 cells significantly reduced the ZEB1 protein level (p < 0.01) and significantly elevated E-cadherin levels (p < 0.01). Moreover, significantly less ZEB1 siRNA-transfected cells migrated through Transwell chambers in the LSCC tissue than that in the control groups (untransfected or transfected with control siRNA, p < 0.01). The expression of the ZEB1 gene in LSCC tissues is downregulated with the expression of E-cadherin. On the other hand, the expression of siRNA against ZEB1 promotes E-cadherin expression and suppresses the invasive ability conferred by E-cadherin. In conclusion, our data suggested that overexpression of the ZEB1 gene is possibly associated with the occurrence, development, invasion of LSCC.     

Decrease of PDSS2 expression,a novel tumor suppressor,in non-small cell lung cancer

Background: Prenyl diphosphate synthase subunit 2 (PDSS2) gene has recently been reported as a potential tumor suppressor. The association of PDSS2 and non-small cell lung cancer (NSCLC) has not been known. Methods: To investigate its association with NSCLC, we examined the expression level of PDSS2 in 28 paired clinical samples of non-small cell lung cancer tissues and surrounding normal tissues. Results: PDSS2 was constitutionally expressed in normal lung tissues regardless of sex, race and smoking history. An overall decreased PDSS2 expression was found in the tumor tissues compared to surrounding normal tissues. Decrease in PDSS2 expression was more severe in poorly and poor-to-moderately differentiated lung cancers, while the decrease was not significant in moderately to well-differentiated tumors. Moreover, the expression of PDSS2 decreased more in higher pathological stage, and in patients with lymph node metastasis. The decrease in PDSS2 expression in tumor tissues was not related to sex or histological type of NSCLC, but was related to smoking history. No correlation has been found between PDSS2 and the clinical factors of EGRF, Ki-67 and p53. Conclusion: Taken together, decreased expression of PDSS2 in NSCLC was evident. This is an initial report for the expression of PDSS2 in relation to different factors in lung cancer. Loss of PDSS2 could serve as a potential biomarker in NSCLC development. The role of PDSS2 as a tumor suppressor, and the mechanism of its potential anti-tumor action in NSCLC warrant further investigation.