A RecA-mediated exon profiling method

We have developed a RecA-mediated simple, rapid and scalable method for identifying novel alternatively spliced full-length cDNA candidates. This method is based on the principle that RecA proteins allow to carry radioisotope-labeled probe DNAs to their homologous sequences, resulting in forming triplexes. The resulting complex is easily detected by mobility difference on electrophoresis. We applied this exon profiling method to four selected mouse genes as a feasibility study. To design probes for detection, the information on known exonic regions was extracted from public database, RefSeq. Concerning the potentially transcribed novel exonic regions, RNA mapping experiment using Affymetrix tiling array was performed. As a result, we were able to identify alternative splice variants of Thioredoxin domain containing 5, Interleukin1β, Interleukin 1 family 6 and glutamine-rich hypothetical protein. In addition, full-length sequencing demonstrated that our method could profile exon structures with >90% accuracy. This reliable method can allow us to screen novel splice variants from a huge number of cDNA clone set effectively

Epithelial EP4 plays an essential role in maintaining homeostasis in colon

Colonic epithelial cells comprise the mucosal barrier, and their dysfunction promotes microbial invasion from the gut lumen and induces the development of intestinal inflammation. The EP4 receptor is known to mediate the protective effect of prostaglandin (PG) E2 in the gastrointestinal tract; however, the exact role of epithelial EP4 in intestinal pathophysiology remains unknown. In the present study, we aimed to investigate the role of epithelial EP4 in maintaining colonic homeostasis by characterizing the intestinal epithelial cell-specific EP4 knockout (EP4 cKO) mice. Mice harboring the epithelial EP4 deletion showed significantly lower colonic crypt depth and lower numbers of secretory cell lineages, as well as impaired epithelial cells in the colon. Interestingly, EP4-deficient colon epithelia showed a higher number of apoptotic cells. Consistent with the defect in mucosal barrier function of colonic epithelia and secretory cell lineages, EP4 cKO colon stroma showed enhanced immune cell infiltration, which was accompanied by increased production of inflammatory cytokines. Furthermore, EP4-deficient colons were susceptible to dextran sulfate sodium (DSS)-induced colitis. Our study is the first to demonstrate that epithelial EP4 loss resulted in potential "inflammatory" status under physiological conditions. These findings provided insights into the crucial role of epithelial PGE2/EP4 axis in maintaining intestinal homeostasis

JNK pathway plays a critical role for expansion of human colorectal cancer in the context of BRG1 suppression

Tumor stem cells (TSCs), capable of self-renewal and continuous production of progeny cells, could be potential therapeutic targets. We have recently reported that chromatin remodeling regulator Brg1 is required for maintenance of murine intestinal TSCs and stemness feature of human colorectal cancer (CRC) cells by inhibiting apoptosis. However, it is still unclear how BRG1 suppression changes the underlying intracellular mechanisms of human CRC cells. We found that Brg1 suppression resulted in upregulation of the JNK signaling pathway in human CRC cells and murine intestinal TSCs. Simultaneous suppression of BRG1 and the JNK pathway, either by pharmacological inhibition or silencing of c-JUN, resulted in even stronger inhibition of the expansion of human CRC cells compared to Brg1 suppression alone. Consistently, high c-JUN expression correlated with worse prognosis for survival in human CRC patients with low BRG1 expression. Therefore, the JNK pathway plays a critical role for expansion and stemness of human CRC cells in the context of BRG1 suppression, and thus a combined blockade of BRG1 and the JNK pathway could be a novel therapeutic approach against human CRC

Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis

膵癌悪性化の分子機構解明 --RECK発現の低下が膵癌の浸潤・転移を引き起こす--. 京都大学プレスリリース. 2023-09-19.RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast–like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC

Mechanisms underlying fatigue: a voxel-based morphometric study of chronic fatigue syndrome

BACKGROUND: Fatigue is a crucial sensation that triggers rest, yet its underlying neuronal mechanisms remain unclear. Intense long-term fatigue is a symptom of chronic fatigue syndrome, which is used as a model to study the mechanisms underlying fatigue. METHODS: Using magnetic resonance imaging, we conducted voxel-based morphometry of 16 patients and 49 age-matched healthy control subjects. RESULTS: We found that patients with chronic fatigue syndrome had reduced gray-matter volume in the bilateral prefrontal cortex. Within these areas, the volume reduction in the right prefrontal cortex paralleled the severity of the fatigue of the subjects. CONCLUSION: These results are consistent with previous reports of an abnormal distribution of acetyl-L-carnitine uptake, which is one of the biochemical markers of chronic fatigue syndrome, in the prefrontal cortex. Thus, the prefrontal cortex might be an important element of the neural system that regulates sensations of fatigue

Reduced responsiveness is an essential feature of chronic fatigue syndrome: A fMRI study

BACKGROUND: Although the neural mechanism of chronic fatigue syndrome has been investigated by a number of researchers, it remains poorly understood. METHODS: Using functional magnetic resonance imaging, we studied brain responsiveness in 6 male chronic fatigue syndrome patients and in 7 age-matched male healthy volunteers. Responsiveness of auditory cortices to transient, short-lived, noise reduction was measured while subjects performed a fatigue-inducing continual visual search task. RESULTS: Responsiveness of the task-dependent brain regions was decreased after the fatigue-inducing task in the normal and chronic fatigue syndrome subjects and the decrement of the responsiveness was equivalent between the 2 groups. In contrast, during the fatigue-inducing period, although responsiveness of auditory cortices remained constant in the normal subjects, it was attenuated in the chronic fatigue syndrome patients. In addition, the rate of this attenuation was positively correlated with the subjective sensation of fatigue as measured using a fatigue visual analogue scale, immediately before the magnetic resonance imaging session. CONCLUSION: Chronic fatigue syndrome may be characterised by attenuation of the responsiveness to stimuli not directly related to the fatigue-inducing task