Ultrasonographic evaluation of the caudal vena cava in dogs with right-sided heart disease.

INTRODUCTION/OBJECTIVES: In humans with impaired right-sided cardiac function, the caudal vena cava (CVC) diameter serves as a marker of venous congestion. This study aimed to investigate whether ultrasonographic CVC variables could identify the presence of right-sided congestive heart failure (R-CHF) in dogs with right-sided heart disease (RHD). ANIMALS: Fifty client-owned control dogs and 67 dogs with RHD were enrolled. The dogs with RHD were subdivided into the non-R-CHF (n = 43) and R-CHF (n = 24) groups. MATERIALS AND METHODS: We measured and compared the ultrasonographic CVC variables and echocardiographic variables among the groups. Receiver operating characteristic (ROC) curve analysis was performed to calculate the sensitivity and specificity of the variables at optimal cutoff values. RESULTS: We obtained the highest accuracies of the ratio of the shortest diameter (SD) of the minimal CVC area to the aorta diameter (Ao) during inspiration [SD(min)/Ao] and of the ratio of SD(min) to the longest diameter of the minimal CVC area during inspiration [LD(min),SD/LD(min)], with high sensitivities, specificities, and an area under the ROC curve greater than 0.925. CONCLUSIONS: In addition to the echocardiographic assessment of right-sided cardiac function, the CVC variables in this study, especially SD(min)/Ao and SD/LD(min), would be useful diagnostic indices for identifying R-CHF in dogs with RHD.

Novel lipoprotein density profiling in laminitic, obese, and healthy horses.

Lipoproteins are water-miscible macromolecules enabling the transport of lipids in blood. In humans, altered proportions of lipoproteins are used to detect and classify metabolic diseases. Obesity and obesity-related comorbidities are common in horses. The pathophysiology of obesity is poorly understood and likely multifactorial. Development of new diagnostic tests to identify horses at risk of developing obesity to implement preventative measures is critical; however, a necessary first step to accomplish this goal is to improve our understanding of the pathophysiology of disease. Thus, the objective of this study was to characterize and compare lipoprotein profiles of horses with normal and excess body conditions, with and without laminitis using a novel method of continuous lipoprotein density profiling (CLPDP). Comparisons were made between 4 groups of horses: (1) laminitic, obese horses (n = 66); (2) laminitic, nonobese horses (n = 35); (3) nonlaminitic, obese horses (n = 41); and (4) nonlaminitic, nonobese horses (n = 95). Lipoprotein profiling, including evaluation of triglyceride-rich lipoprotein (TRL), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs) was performed using CLPDP, and all 4 groups were compared. A significant difference was observed among groups for the subfractions TRL, LDL1, LDL2, HDL2b, HDL2a, HDL3a, HDL3b, HDL3c, and total HDL. This is the first known description of CLPDP to characterize equine lipid profiles and holds promise as a useful method for lipid characterization of horses.

MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species.

We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163-185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer.

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

TNF-α/TNFR1 signaling promotes gastric tumorigenesis through induction of Noxo1 and Gna14 in tumor cells.

Helicobacter pylori infection induces chronic inflammation that contributes to gastric tumorigenesis. Tumor necrosis factor (TNF-α) is a proinflammatory cytokine, and polymorphism in the TNF-α gene increases the risk of gastric cancer. We herein investigated the role of TNF-α in gastric tumorigenesis using Gan mouse model, which recapitulates human gastric cancer development. We crossed Gan mice with TNF-α (Tnf) or TNF-α receptor TNFR1 (Tnfrsf1a) knockout mice to generate Tnf-/- Gan and Tnfrsf1a-/- Gan mice, respectively, and examined their tumor phenotypes. Notably, both Tnf-/- Gan mice and Tnfrsf1a-/- Gan mice showed similar, significant suppression of gastric tumor growth compared with control Tnf+/+ or Tnfrsf1a+/+ Gan mice. These results indicate that TNF-α signaling through TNFR1 is important for gastric tumor development. Bone marrow (BM) transplantation experiments showed that TNF-α expressed by BM-derived cells (BMDCs) stimulates the TNFR1 on BMDCs by an autocrine or paracrine manner, which is important for gastric tumor promotion. Moreover, the microarray analysis and colony formation assay indicated that NADPH oxidase organizer 1 (Noxo1) and Gna14 are induced in tumor epithelial cells in a TNF-α-dependent manner, and have an important role in tumorigenicity and tumor-initiating cell property of gastric cancer cells. Accordingly, it is possible that the activation of TNF-α/TNFR1 signaling in the tumor microenvironment promotes gastric tumor development through induction of Noxo1 and Gna14, which contribute to maintaining the tumor cells in an undifferentiated state. The present results indicate that targeting the TNF-α/TNFR1 pathway may be an effective preventive or therapeutic strategy for gastric cancer.

Can population differences in chemotherapy outcomes be inferred from differences in pharmacogenetic frequencies?

Inter-ethnic differences in drug handling and frequencies of pharmacogenetic variants are increasingly being characterized. In this study, we systematically assessed the feasibility of inferring ethnic trends in chemotherapy outcomes from inter-ethnic differences in pharmacogenetic variant frequencies. Frequencies of 51 variants and chemotherapy outcomes of East Asian and Caucasian colorectal cancer patients on standard chemotherapy regimens were summarized by meta-analyses, and variant frequencies were validated by MassARRAY analysis. Inferences of relative chemotherapy outcomes were made by considering minor allele function and population differences in their frequency. Significant population differences in genotype distributions were observed for 13/23 (60%) and 27/35 (77%) variants in the meta-analyses and validation series, respectively. Across chemotherapy regimens, East Asians had lower rates of grade 3/4 toxicity for diarrhea and stomatitis/mucositis than Caucasians, which was correctly inferred from 13/18 (72%, P=0.018) informative genetic variants. With appropriate variant selection, inferring relative population toxicity rates from population genotype differences may be relevant.

Inflammation-induced repression of tumor suppressor miR-7 in gastric tumor cells.

Inflammation has an important role in cancer development through various mechanisms. It has been shown that dysregulation of microRNAs (miRNAs) that function as oncogenes or tumor suppressors contributes to tumorigenesis. However, the relationship between inflammation and cancer-related miRNA expression in tumorigenesis has not yet been fully understood. Using K19-C2mE and Gan mouse models that develop gastritis and gastritis-associated tumors, respectively, we found that 21 miRNAs were upregulated, and that 29 miRNAs were downregulated in gastric tumors in an inflammation-dependent manner. Among these miRNAs, the expression of miR-7, a possible tumor suppressor, significantly decreased in both gastritis and gastric tumors. Moreover, the expression of miR-7 in human gastric cancer was inversely correlated with the levels of interleukin-1ß and tumor necrosis factor-α, suggesting that miR-7 downregulation is related to the severity of inflammatory responses. In the normal mouse stomach, miR-7 expression was at a basal level in undifferentiated gastric epithelial cells, and was induced during differentiation. Moreover, transfection of a miR-7 precursor into gastric cancer cells suppressed cell proliferation and soft agar colony formation. These results suggest that suppression of miR-7 expression is important for maintaining the undifferentiated status of gastric epithelial cells, and thus contributes to gastric tumorigenesis. Although epigenetic changes were not found in the CpG islands around miR-7-1 of gastritis and gastric tumor cells, we found that activated macrophage-derived small molecule(s) (<3 kDa) are responsible for miR-7 repression in gastric cancer cells. Furthermore, the miR-7 expression level significantly decreased in the inflamed gastric mucosa of Helicobacter-infected mice, whereas it increased in the stomach of germfree K19-C2mE and Gan mice wherein inflammatory responses were suppressed. Taken together, these results indicate that downregulation of tumor suppressor miR-7 is a novel mechanism by which the inflammatory response promotes gastric tumorigenesis.

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype.

The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin.

Endosonographic evaluation of c-kit-positive gastrointestinal stromal tumor.

BACKGROUND: Endosonographic features of c-kit-positive gastrointestinal stromal tumors (GISTs) were compared with those of leiomyomas and schwannomas. METHODS: Twenty-four patients with gastric mesenchymal tumors who underwent endoscopic ultrasonography (EUS) and surgical treatment were enrolled. GISTs were defined as c-kit (CD117)-positive tumors, leiomyomas as desmin-positive and c-kit-negative tumors, and schwannomas as S-100-positive and c-kit-negative tumors. Invasion to adjacent organs or more than 20 mitotic counts per 50 high power fields indicated malignancy. RESULTS: There were 19 GISTs, three leiomyomas, and two schwannomas. All five malignant tumors were GISTs. A marginal halo was found in 12 of 19 GISTs and in both of the schwannomas, but not in any of the three leiomyomas. The echogenicities of GISTs were low but higher than that of the normal proper muscle layer, whereas those of leiomyomas and schwannomas were usually low. Lobulation of the tumor surface was documented only in GISTs, particularly in malignant ones. The tumor doubling time of a malignant GIST was 9.3 months, and that of six benign GISTs was 18.7 months (range = 10.7-28.0 months). CONCLUSION: Marginal halo and relatively higher echogenicity on EUS might suggest GIST. Marginal lobulation and a short doubling time may be signs of a malignant GIST.

Visualizing the gastric wall with a 30-MHz ultrasonic miniprobe: ex vivo imaging of normal gastric sites and sites of early gastric cancer.

BACKGROUND: This study was performed to determine the echo layer structures of the normal gastric wall and early gastric cancer when visualized with a 30-MHz ultrasonic miniprobe. METHODS: Twelve surgically resected gastric specimens were used for an ex vivo study. Eighteen normal sites and 12 early gastric cancer sites were scanned with an Olympus (XUM-S30-25R) probe with a frequency of 30-MHz. Endoscopic ultrasound images were compared with corresponding histopathologic sections stained with hematoxylin and eosin. RESULTS: The normal mucosa was visualized as at least four alternating echo layers; the muscularis mucosa was delineated at all normal sites. Lymphoid aggregates within the mucosa could be seen. The submucosa was clearly visualized in most cases, but the muscularis propria and subserosa were seldom depicted due to attenuation of ultrasound waves. At the sites of gastric cancer, the layered architecture of the mucosa was disturbed by an irregular hypoechoic lesion. Minimal submucosal infiltration (400 and 750 micrometer) was clearly depicted in two cases, without ulceration at or around the tumor site. However, attenuation at the site of a deep ulcer scar prevented adequate visualization of the tumor extent in two other cases with ulceration. CONCLUSION: A 30-MHz ultrasonic miniprobe may provide additional imaging information of the gastric wall and could play a role in the assessment of early cancer lesions.

Gene mutation as a target for early detection in cancer diagnosis.

The increasing number of genetic aberrations implicated in the development of human cancer has prompted a search to detect them at the earliest possible stage of their formation. Of the many such genetic changes identified thus far, relatively few meet the standard for markers in early diagnosis and prognosis, namely that the genetic modifications occur during the early onset phase of cancer development. Parallel to the increasing number of such genes is the growing availability of technologies using more powerful and cost-efficient methods that enable mass screening for genetic alterations. The purpose of this review is to summarize the currently available genes that can serve as markers for early detection of cancers and methods that allow their detection.

Distinct pattern of p53 phosphorylation in human tumors.

The protein product of the tumor suppressor gene p53 is phosphorylated on multiple residues by several protein kinases. Using a battery of 10 antibodies developed against different phosphorylated and acetylated residues of p53, we compared the pattern of p53 phosphorylation and acetylation in tumor-derived cell lines, tumor samples, and non-neoplastic cells. Irrespective of tumor types or the presence of p53 mutation, phosphorylation and acetylation of p53 was substantially higher in samples obtained from tumor tissues than those found in non-transformed samples. Among the 10 sites analysed, phosphorylation of residues 15, 81, 392, and acetylation were among the more frequent modifications. Analysis of two of the more abundant phosphorylation or acetylation sites on p53 is sufficient to detect 72% of tumor-derived p53 proteins. The distinct pattern of p53 phosphorylation and acetylation in human tumors may offer a new means to monitor the status and activity of p53 in the course of tumor development and progression.

Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

Presacral ganglioneuroma arising in an elderly man with persistent constipation.

Presacral ganglioneuroma in a 70-year-old man with persistent constipation and its features on computed tomography, magnetic resonance, and endoscopic ultrasonography are presented. Barium enema study and laparotomy showed that constipation was caused mainly by extrinsic compression from this tumor. Computed tomography, magnetic resonance, endoscopic ultrasonographic features such as well-defined solid tumor with a cystic component and punctate calcifications may facilitate early diagnosis of this rare tumor.

Altered expression of p53 and p27 proteins, alone or combined, as a predictor of metastatic potential in early invasive carcinoma of colon and rectum--a comparative clinicopathologic and molecular analysis.

To place the choice of therapy (endoscopic resection or radical surgery) in early invasive carcinoma (EIC) of colon and rectum on a more rational basis, this study sought to identify molecular predictors of metastasis. Several morphologic risk factors (histologic type, degree of tumor invasion, lymphatic and venous invasion) and expression of p53 and p27 proteins in the primary tumor were compared in 80 patients with EIC, including 12 (15%) with metastasis or recurrence (or both). Of the factors enumerated, deeper invasion of the submucosal layer, lymphatic-venous invasion, p53 overexpression, and decreased expression of p27 were correlated significantly with metastasis. The results also indicated that altered expression of p53 or p27 is independently relevant to metastasis of EIC. Analysis of these markers, together with determination of the morphologic risk factors, could complement the identification of patients with metastasis on the basis of known morphologic risk factors. Because the molecular factors can be assessed more objectively than can the morphologic parameters, they may strengthen the ability to identify EIC that has undergone, or will undergo, metastasis.

Wnt/beta-catenin signaling induces the expression and activity of betaTrCP ubiquitin ligase receptor.

Beta-transducing repeat-containing protein (betaTrCP) targets the ubiquitination and subsequent degradation of both beta-catenin and IkappaB, thereby playing an important role in beta-catenin/Tcf and NF-kappaB-dependent signaling. Here evidence is presented that beta-catenin/Tcf signaling elevates the expression of betaTrCP mRNA and protein in a Tcf-dependent manner, which does not require betaTrCP transcription. Induction of betaTrCP expression by the beta-catenin/Tcf pathway results in an accelerated degradation of the wild-type beta-catenin, suggesting that the negative feedback loop regulation may control the beta-catenin/Tcf pathway. This signaling also upregulated NF-kappaB transactivation without affecting the activity of IkappaB kinase, thereby establishing that the maintenance of the betaTrCP level is important for coordination between beta-catenin/Tcf and NF-kappaB signaling.

K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers--a review.

Multiple genomic alterations are involved in the development of most human cancers. They include alterations in oncogenes, tumor suppressor genes, DNA mismatch repair and excision repair genes. Genetic testing for susceptibility has been a part of the management of patients with well-defined but uncommon hereditary cancers in which certain susceptible gene mutations are determined in the germ line. However, a molecular diagnostic approach to sporadic cancers, which comprise the vast majority of malignant tumors in human beings, is still under development. One of the best characterized tumor-related genes is K-ras, which somatically mutates in several types of sporadic human cancers. Since mutations of this gene occur exclusively in three hot spots (codons 12, 13 and 61), and are frequently detected and well characterized in colorectal, pancreas and lung cancers, molecular diagnosis and susceptibility (risk) assessment targeting K-ras mutations are being developed. For this purpose, sample collection methods that reflect the state of the entire affected organ are important. Clinical samples used for molecular diagnosis and risk assessment include stool and lavage fluid, pancreatic and duodenal juices, and sputum and lavage fluids for colorectal, pancreas and lung cancers, respectively. The reported incidence of K-ras mutations detected in these samples ranges from 7% to 80% for colorectal cancers, 25% to 87% for pancreatic cancers, and 25% to 48% for lung cancers. Incidence of mutations clearly depends on the sensitivity of the method for detecting the mutant K-ras allele, as well as the nature and the quality of the clinical samples. Various methods including plaque hybridization, dot blot hybridization, combined PCR and RFLP or SSCP, and sensitive PCR have been used, and they exhibited high specificity (75 to 100%) in detecting mutations. Molecular analysis is demonstrating promise in assessing susceptibility to, or risk of developing, sporadic cancers.