Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin(+)/CD45(+) cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis.     

Cortical rhythm of No-go processing in humans: An MEG study

ObjectiveWe investigated the characteristics of cortical rhythmic activity in No-go processing during somatosensory Go/No-go paradigms, by using magnetoencephalography (MEG).MethodsTwelve normal subjects performed a warning stimulus (S1) – imperative stimulus (S2) task with Go/No-go paradigms. The recordings were conducted in three conditions. In Condition 1, the Go stimulus was delivered to the second digit, and the No-go stimulus to the fifth digit. The participants responded by pushing a button with their right thumb for the Go stimulus. In Condition 2, the Go and No-go stimuli were reversed. Condition 3 was the resting control.ResultsA rebound in amplitude was recorded in the No-go trials for theta, alpha, and beta activity, peaking at 600–900 ms. A suppression of amplitude was recorded in Go and No-go trials for alpha activity, peaking at 300–600 ms, and in Go and No-go trials for beta activity, peaking at 200–300 ms.ConclusionThe cortical rhythmic activity clearly has several dissociated components relating to different motor functions, including response inhibition, execution, and decision-making.SignificanceThe present study revealed the characteristics of cortical rhythmic activity in No-go processing.     

Six-transmembrane epithelial antigen of prostate4 (STEAP4) is a tumor necrosis factor alpha-induced protein that regulates IL-6, IL-8, and cell proliferation in synovium from patients with rheumatoid arthritis

Human six-transmembrane epithelial antigen of prostate4 (STEAP4), an ortholog of mouse tumor necrosis factor-α-induced adipose-related protein (TIARP), plays a role in tumor necrosis factor (TNF)-dependent arthritis models. However, its role in rheumatoid arthritis (RA) is still obscure. This study explored such a role for STEAP4. The expressions of STEAP4, TNFα, and IL-6 were compared in synovia of RA and osteoarthritis patients. STEAP4 induction was examined in TNFα-stimulated fibroblast-like synoviocytes (FLS) in vitro. FLS (with/without TNFα stimulation) were also analyzed for IL-6 expression after STEAP4 knockdown, using siRNA or transfection with STEAP4-plasmid DNA. IL-8, cell proliferation, and apoptosis were also evaluated in STEAP4-overexpressing FLS. The expression of STEAP4 in joints correlated with TNFα expression, specifically in RA synovium. In the cultured FLS, STEAP4 protein expression was augmented by TNFα activation, and localized in endosomal/lysosomal compartments. STEAP4 downregulation by siRNA enhanced the expression of IL-6 mRNA, while STEAP4 overexpression suppressed IL-6 and IL-8 expression, inhibited cell proliferation, and induced apoptosis via caspase-3. The results indicated that human STEAP4 is regulated by TNFα in synovium, where it controls IL-6 secretion and proliferation of FLS, suggesting that STEAP4 might potentially suppress the pathogenesis of TNFα-induced arthritis such as RA.     

<Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> Mutations in 203 Esophageal Squamous Cell Carcinomas: Pyrosequencing Technology and Literature Review

Background

Epidermal growth factor receptor (EGFR) signaling is one of the most promising targets for molecular-targeted therapies in esophageal squamous cell carcinoma (ESCC). Thus, the molecular diagnosis of KRAS and BRAF mutations is clinically important in therapeutic decision making. However, the frequency of KRAS and BRAF mutations in ESCCs remains inconclusive because of the limited sample sizes of previous studies (all N ≤ 80). Pyrosequencing is a nonelectrophoretic nucleotide extension sequencing technology that can be used for mutation testing.

Methods

The frequency of KRAS and BRAF mutations was examined using a nonbiased database of 203 resected ESCCs and a high-throughput pyrosequencing assay.

Results

The validity of the KRAS pyrosequencing method was initially demonstrated by detection of all 4 types of KRAS mutations [c.35G>T (codon 12 GGT>GTT), c.35G>A (codon 12 GGT>GAT), c.34G>T (codon 12 GGT>TGT), c.38G>A mutation (codon 13 GGC>GAC)], which had been previously diagnosed using Scorpion-ARMS technology, in 9 colon cancer tissues (9 of 9; 100 %). Similar results were demonstrated for BRAF mutational status in 3 colon cancer cell lines (HCT116, Colo201, and HT29), which were validated by Sanger dideoxy sequencing. Subsequently, the KRAS mutation was found to be extremely rare (1 of 203; 0.5 %), and the BRAF mutation was absent (0 of 203; 0 %), in the dataset of 203 ESCCs.

Conclusions

These results suggest that KRAS and BRAF mutations play a limited role in the development of ESCC and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in ESCC.

     

The effect of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> MIYAIRI on the prevention of pouchitis and alteration of the microbiota profile in patients with ulcerative colitis

Purpose

Ulcerative colitis (UC) is a chronic, relapsing, and refractory disorder of the intestine. Total proctocolectomy with ileal pouch anal anastomosis (IPAA) is the preferred and standard surgical procedure for patients’ refractory to medical therapy. Pouchitis is one of the most common long-term complications after IPAA. In the present study, the safety and efficacy of Clostridium butyricum MIYAIRI (CBM) as a probiotic were examined.

Methods

A randomized and placebo-controlled study was performed. Seventeen patients were recruited from 2007 to 2013. Nine tablets of MIYA-BM^® or placebo were orally administered once daily. The cumulative pouchitis-free survival, pouch condition (using the modified pouch disease activity index), and blood parameters were evaluated. A fecal sample analysis was also performed.

Results

Subjects were randomly allocated to receive MIYA-BM or placebo (9 and 8 subjects, respectively). One subject in the MIYA-BM group and four subjects in the placebo group developed pouchitis. No side effects occurred in either group. Characteristic intestinal flora was observed in each group.

Conclusions

Our results suggest that probiotic therapy with CBM achieved favorable results with minimal side effects and might be a useful complementary therapy for the prevention of pouchitis in patients with UC who have undergone IPAA.

     

Perfusion with carbon monoxide does not affect extracellular glutamate in dialysates of the hippocampus of freely moving mice

Carbon monoxide (CO) produces several neurological effects, including cognitive, mood, and behavioral disturbance. Glutamate is thought to play a particularly important role in learning and memory. Thus, the present study was aimed at investigating the local effect of CO on the glutamate level in the hippocampus of mice using in vivo reverse microdialysis. Mice were perfused with Ringer’s solution (control) or CO (60–125?μM) in Ringer’s solution into the hippocampus via microdialysis probe. Dialysate samples were collected every 20?min, and then analyzed with high-performance liquid chromatography coupled to an electrochemical detector. The result revealed that the perfusion with CO had no significant effect on glutamate levels (p?=?0.316) as compared to the control group. This finding does not support a local CO rise as the cause of the increased glutamate level in the hippocampus of mice.     

PLXND1/SEMA3E Promotes Epithelial–Mesenchymal Transition Partly via the PI3K/AKT-Signaling Pathway and Induces Heterogenity in Colorectal Cancer

Annals of Surgical Oncology - Colorectal cancer (CRC) is a major cause of cancer-related deaths. Metastasis is enhanced through epithelial-mesenchymal transition (EMT), a process primarily induced...     

Morbidity of graft harvesting versus bone marrow aspiration in cell regenerative therapy

Purpose

Using bone marrow mesenchymal stem cells (MSCs) with aspiration from the iliac crest is commonly used in reconstructive orthopaedic surgery. Because bone marrow aspiration is a percutaneous technique, the morbidity as compared with the classical bone graft should be decreased.

Method

Therefore in a retrospective review of 523 consecutive cases of bone marrow aspiration performed at the Henri Mondor Hospital from 1990 to 2006 for the treatment of fractures, minor and major complications were identified and compared to the number of complications observed during the same period with 435 classical iliac crest bone graft procedures performed for the same indications of treatment of fractures. Minor complications included superficial infections, superficial seromas, and minor haematomas. Major complications included herniation of abdominal contents through massive bone graft donor sites, vascular injuries, deep infections at the donor site, neurologic injuries, deep haematoma formation requiring surgical intervention or transfusion, and iliac wing fractures.

Result

Bone marrow aspiration decreased significantly the number of complications as compared with harvesting classical iliac crest bone graft that was associated with significant morbidity. Adverse events were significantly lower (p?Conclusion In our series the number of complications with bone marrow aspiration was ten times less than the complications observed with the classical technique of bone piece harvesting from the iliac crest, and the complications were clearly less severe.     

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter‐like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high‐affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium‐3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na⁺‐independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H⁺ gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K⁺ concentration. In addition, the Na⁺‐independent uptake of choline was saturable, and the K_m value was estimated to be 108 µm . These findings suggest that the uptake of choline into LS180 cells is membrane potential‐dependent, but not outward H⁺ gradient‐dependent. Copyright © 2014 John Wiley & Sons, Ltd.