Canonical Wnt and TGF-ß/BMP signaling enhance melanocyte regeneration but suppress invasiveness, migration, and proliferation of melanoma cells.

Melanoma is the deadliest form of skin cancer and develops from the melanocytes that are responsible for the pigmentation of the skin. The skin is also a highly regenerative organ, harboring a pool of undifferentiated melanocyte stem cells that proliferate and differentiate into mature melanocytes during regenerative processes in the adult. Melanoma and melanocyte regeneration share remarkable cellular features, including activation of cell proliferation and migration. Yet, melanoma considerably differs from the regenerating melanocytes with respect to abnormal proliferation, invasive growth, and metastasis. Thus, it is likely that at the cellular level, melanoma resembles early stages of melanocyte regeneration with increased proliferation but separates from the later melanocyte regeneration stages due to reduced proliferation and enhanced differentiation. Here, by exploiting the zebrafish melanocytes that can efficiently regenerate and be induced to undergo malignant melanoma, we unravel the transcriptome profiles of the regenerating melanocytes during early and late regeneration and the melanocytic nevi and malignant melanoma. Our global comparison of the gene expression profiles of melanocyte regeneration and nevi/melanoma uncovers the opposite regulation of a substantial number of genes related to Wnt signaling and transforming growth factor beta (TGF-ß)/(bone morphogenetic protein) BMP signaling pathways between regeneration and cancer. Functional activation of canonical Wnt or TGF-ß/BMP pathways during melanocyte regeneration promoted melanocyte regeneration but potently suppressed the invasiveness, migration, and proliferation of human melanoma cells in vitro and in vivo. Therefore, the opposite regulation of signaling mechanisms between melanocyte regeneration and melanoma can be exploited to stop tumor growth and develop new anti-cancer therapies.

Comparative membrane lipidomics of hepatocellular carcinoma cells reveals diacylglycerol and ceramide as key regulators of Wnt/ß-catenin signaling and tumor growth.

Hepatocellular carcinoma (HCC) is largely associated with aberrant activation of Wnt/ß-catenin signaling. Nevertheless, how membrane lipid composition is altered in HCC cells with abnormal Wnt signaling remains elusive. Here, by exploiting comprehensive lipidome profiling, we unravel the membrane lipid composition of six different HCC cell lines with mutations in components of Wnt/ß-catenin signaling, leading to differences in their endogenous signaling activity. Among the differentially regulated lipids are diacylglycerol (DAG) and ceramide, which were downregulated at the membrane of HCC cells after Wnt3a treatment. DAG and ceramide enhanced Wnt/ß-catenin signaling by inducing caveolin-mediated endocytosis of the canonical Wnt-receptor complex, while their depletion suppressed the signaling activity along with a reduction of caveolin-mediated endocytosis in SNU475 and HepG2 cells. Moreover, depletion of DAG and ceramide significantly impeded the proliferation, tumor growth, and in vivo migration capacity of SNU475 and HepG2 cells. This study, by pioneering plasma membrane lipidome profiling in HCC cells, exhibits the remarkable potential of lipids to correct dysregulated signaling pathways in cancer and stop abnormal tumor growth.

Brain Regeneration Resembles Brain Cancer at Its Early Wound Healing Stage and Diverges From Cancer Later at Its Proliferation and Differentiation Stages.

Gliomas are the most frequent type of brain cancers and characterized by continuous proliferation, inflammation, angiogenesis, invasion and dedifferentiation, which are also among the initiator and sustaining factors of brain regeneration during restoration of tissue integrity and function. Thus, brain regeneration and brain cancer should share more molecular mechanisms at early stages of regeneration where cell proliferation dominates. However, the mechanisms could diverge later when the regenerative response terminates, while cancer cells sustain proliferation. To test this hypothesis, we exploited the adult zebrafish that, in contrast to the mammals, can efficiently regenerate the brain in response to injury. By comparing transcriptome profiles of the regenerating zebrafish telencephalon at its three different stages, i.e., 1 day post-lesion (dpl)-early wound healing stage, 3 dpl-early proliferative stage and 14 dpl-differentiation stage, to those of two brain cancers, i.e., low-grade glioma (LGG) and glioblastoma (GBM), we reveal the common and distinct molecular mechanisms of brain regeneration and brain cancer. While the transcriptomes of 1 dpl and 3 dpl harbor unique gene modules and gene expression profiles that are more divergent from the control, the transcriptome of 14 dpl converges to that of the control. Next, by functional analysis of the transcriptomes of brain regeneration stages to LGG and GBM, we reveal the common and distinct molecular pathways in regeneration and cancer. 1 dpl and LGG and GBM resemble with regard to signaling pathways related to metabolism and neurogenesis, while 3 dpl and LGG and GBM share pathways that control cell proliferation and differentiation. On the other hand, 14 dpl and LGG and GBM converge with respect to developmental and morphogenetic processes. Finally, our global comparison of gene expression profiles of three brain regeneration stages, LGG and GBM exhibit that 1 dpl is the most similar stage to LGG and GBM while 14 dpl is the most distant stage to both brain cancers. Therefore, early convergence and later divergence of brain regeneration and brain cancer constitutes a key starting point in comparative understanding of cellular and molecular events between the two phenomena and development of relevant targeted therapies for brain cancers.

Comparative Transcriptome Analysis of the Regenerating Zebrafish Telencephalon Unravels a Resource With Key Pathways During Two Early Stages and Activation of Wnt/ß-Catenin Signaling at the Early Wound Healing Stage.

Owing to its pronounced regenerative capacity in many tissues and organs, the zebrafish brain represents an ideal platform to understand the endogenous regeneration mechanisms that restore tissue integrity and function upon injury or disease. Although radial glial and neuronal cell populations have been characterized with respect to specific marker genes, comprehensive transcriptomic profiling of the regenerating telencephalon has not been conducted so far. Here, by processing the lesioned and unlesioned hemispheres of the telencephalon separately, we reveal the differentially expressed genes (DEGs) at the early wound healing and early proliferative stages of regeneration, i.e., 20 h post-lesion (hpl) and 3 days post-lesion (dpl), respectively. At 20 hpl, we detect a far higher number of DEGs in the lesioned hemisphere than in the unlesioned half and only 7% of all DEGs in both halves. However, this difference disappears at 3 dpl, where the lesioned and unlesioned hemispheres share 40% of all DEGs. By performing an extensive comparison of the gene expression profiles in these stages, we unravel that the lesioned hemispheres at 20 hpl and 3 dpl exhibit distinct transcriptional profiles. We further unveil a prominent activation of Wnt/ß-catenin signaling at 20 hpl, returning to control level in the lesioned site at 3 dpl. Wnt/ß-catenin signaling indeed appears to control a large number of genes associated primarily with the p53, apoptosis, forkhead box O (FoxO), mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) signaling pathways specifically at 20 hpl. Based on these results, we propose that the lesioned and unlesioned hemispheres react to injury dynamically during telencephalon regeneration and that the activation of Wnt/ß-catenin signaling at the early wound healing stage plays a key role in the regulation of cellular and molecular events.

CRISPR/Cas9: An RNA-guided highly precise synthetic tool for plant genome editing.

CRISPR/Cas9 is a newly developed and naturally occurred genome editing tool, which is originally used by bacteria for immune defence. In the past years, it has been quickly employed and modified to precisely edit genome sequences in both plants and animals. Compared with the well-developed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 has lots of advantages, including easier to design and implement, higher targeting efficiency, and less expensive. Thus, it is becoming one of the most powerful tools for knockout of an individual gene as well as insertion of one gene and/or control of gene transcription. Studies have shown that CRISPR/Cas9 is a great tool to edit many genes in a variety of plant species, including the model plant species as well as agriculturally important crops, such as cotton, maize, wheat, and rice. CRISPR/Cas9-based genome editing can be used for plant functional studies and plant improvement to yield, quality, and tolerance to environmental stress.

The effect of rabeprazole on LES tone in experimental rat model.

INTRODUCTION: Despite adequate treatment with proton pump inhibitors (PPIs), symptoms of gastroesophageal reflux disease (GERD) may remain persistent as well as Barrett's esophagus may emerge. It may be proposed that the relaxant effect of PPIs on the smooth muscles may lead to resistance of symptoms. The aim of this study is to investigate effects of rabeprazole on the lower esophageal sphincter (LES) pressure with a rat model. MATERIALS AND METHODS: Sixteen rats were grouped as control and treatment groups. After obtaining LES tissues followed by a 60 min equilibration period for stabilization, contractile response to carbachol was obtained by application of single dose of carbachol to have a final concentration of 10(-6) M in the organ bath. After the contractions reached a plateau, concentration-response relationships for rabeprazole were obtained in a cumulative manner in the treatment group. RESULTS: In the carbachol contracted LES preparations; 1.5 × 10(-6) and 1.5×10(-5) M of rabeprazole caused 6.08% and 11.34% relaxations respectively which were not statistically significant. However, mean integral relaxation value for 4.5 × 10(-5) M of rabeprazole was 17.34% and this relaxation was significant compared with controls. CONCLUSIONS: In the present study, rabeprazole caused no direct significant change in LES tone in the therapeutic dose range applied to the organ bath. However, rabeprazole at the high dose caused a significant decrease in the LES tone.

Superiority of ceftriaxon to cefazolin in a rat model of obstructive jaundice: an experimental study.

OBJECTIVE: The objective of this study was to evaluate the serum and bile concentrations of cefazolin and ceftriaxone at the third and sixth hours in an experimental obstructive jaundice model and to identify the rate of excretion of these antibiotics into the bile. MATERIAL AND METHODS: Thirty-two Wistar albino rats were used in this study. The bile and serum levels of cefazolin were measured at the third hour in the A1 group and at the sixth hour in the A2 group, with cefazolin administered as 5 mg/rat; while the bile and serum levels of ceftriaxone were studied at the third hour in the B1 group and at the sixth hour in the B2 group, with ceftriaxone administered as 5 mg/rat. RESULTS: After 3 hr of cefazolin administration, the serum concentration in the A1 group reached a mean of 1.8 µg/ml, while the bile concentration was 90% of the serum concentration, with a mean of 1.6 µg/ml; whereas in the B1 group, the third-hour serum concentration of ceftriaxone was 18.6 µg/ml, while the bile concentration was found to be as high as 330% of this level, i.e., 56 µg/ml. The serum value of cefazolin decreased to 1.4 µg/ml in the A2 group and ceftriaxone decreased to 3.7 µg/ml in the B2 group at the sixth hour. CONCLUSIONS: Although the excretory level of cefazolin and ceftriaxone into the bile reaches therapeutic doses, the duration for which these levels are above those required for bactericidal activity is short. Ceftriaxone is better concentrated in the serum and bile than cefazolin.

Impact of genetic factors (CYP2C9, VKORC1 and CYP4F2) on warfarin dose requirement in the Turkish population.

Warfarin has a narrow therapeutic index and displays marked person-to-person variation in dose requirement. Functional polymorphisms at candidate genes can therefore offer utility as biomarkers to individualize warfarin treatment. The main objective of this study was to determine whether and to what extent variability in warfarin dose requirements was determined by polymorphisms in CYP2C9, VKORC1, CYP4F2 (rs2108622) and EPHX1 (rs2292566) in the Turkish population. Patients (n = 107) who had stable doses and international normalized ratio (INRs) at their last three consecutive visits were registered. Their demographic factors, concurrent medications, warfarin-related bleedings or thromboembolisms, smoking, alcohol intake and weekly green vegetable consumption were recorded. From a blood sample, DNA was isolated and genotyped by real-time PCR for polymorphisms of CYP2C9, VKORC1, CYP4F2 and EPHX1. A regression analysis was used to determine the independent effects of genetic and non-genetic factors on warfarin dose optimization. In our study, in addition to age, genetic variants of CYP2C9, VKORC1 and CYP4F2 were found to be significant predictor variables for the maintenance dose for warfarin, explaining 39.3% of dose variability. VKORC1 and CYP2C9 genotypes remain predictor variables of the warfarin dose, and we first found that CYP4F2 (rs2108622) contributes to dose variability in the Turkish population as well. These observations may be of benefit to future translation research with a view to global personalized medicine in regions hitherto understudied such as the Turkish population so as to rationalize initial warfarin dose and reduce the burden of frequent INR measurements.

In vitro effects of famotidine and ranitidine on lower esophageal sphincter tone in rats.

BACKGROUND/AIMS: The aim of this study was to investigate the effects of the H2 receptor antagonists famotidine and ranitidine on lower esophageal sphincter pressure in the rat isolated lower esophageal sphincter preparation contracted with carbachol. MATERIALS AND METHODS: Lower esophageal sphincter tissues of eight rats for each group were placed in a standard organ bath. After contraction with carbachol, freshly prepared famotidine and ranitidine were added directly to the tissue bath in cumulatively increasing concentrations. Activities were recorded on an online computer using the software BSL PRO v 3.7, which also analyzed the data. RESULTS: Ranitidine caused a small statistically insignificant relaxation in the contracted lower esophageal sphincter at the two applied concentrations. Although 1.5 x 10⁻5 M famotidine did not cause a significant relaxation in lower esophageal sphincter tone, this value for 4.5 x 10⁻5 M famotidine was 9.33%, and the relaxation was significant when compared with controls (p<0.05). CONCLUSIONS: Neither famotidine nor ranitidine caused any direct significant change in lower esophageal sphincter tone in the therapeutic dose range applied to the organ bath. However, the higher dose of famotidine caused a significant relaxation in the lower esophageal sphincter tone. Further in vivo human studies may affect the usage of these drugs during gastroesophageal reflux disease treatment.

In vitro effect of pantoprazole on lower esophageal sphincter tone in rats.

AIM: To investigate the in vitro effects of pantoprazole on rat lower esophageal sphincter (LES) tone. METHODS: Rats weighing 250-300 g, provided by the Yeditepe University Experimental Research Center (YÜDETAM), were used throughout the study. They were anesthetized before decapitation. LES tissues whose mucosal lining were removed were placed in a standard 30-mL organ bath with a modified Krebs solution and continuously aerated with 95% oxygen-5% carbon dioxide gas mixture and kept at room temperature. The tissues were allowed to stabilize for 60 min. Subsequently, the contractile response to 10(-6) mol/L carbachol was obtained. Different concentrations of freshly prepared pantoprazole were added directly to the tissue bath to generate cumulative concentrations of 5 × 10(-6) mol/L, 5 × 10(-5) mol/L, and 1.5 × 10(-4) mol/L. Activities were recorded on an online computer via a 4-channel transducer data acquisition system using the software BSL PRO v 3.7, which also analyzed the data. RESULTS: Pantoprazole at 5 × 10(-6) mol/L caused a small, but statistically insignificant, relaxation in the carbachol-contracted LES (2.23% vs 3.95%). The 5 × 10(-5) mol/L concentration, however, caused a significant relaxation of 10.47% compared with the control. 1.5 × 10(-4) mol/L concentration of pantoprazol caused a 19.89% relaxation in the carbachol contracted LES (P < 0.001). CONCLUSION: This is the first study to demonstrate that pantoprazole has a relaxing effect in isolated LESs. These results might have significant clinical implications for the subset of patients using proton pump inhibitors who do not receive full symptomatic alleviation from gastroesophageal reflux disease.