Regional differences of tachykinin effects on smooth muscle and pacemaker potentials of the stomach, duodenum, ileum and colon of an emetic model, the house musk shrews.

BACKGROUND AND AIMS: The contractile effects of tachykinins on the gastrointestinal tract are well-known, but how they modulate slow-waves, particularly in species capable of emesis, remains largely unknown. We aimed to elucidate the effects of tachykinins on myoelectric and contractile activity of isolated gastrointestinal tissues of the Suncus murinus. METHODS: The effects of substance P (SP), neurokinin (NK)A, NKB and selective NK1 (CP122,721, CP99,994), NK2 (SR48,968, GR159,897) and NK3 (SB218,795, SB222,200) receptor antagonists on isolated stomach, duodenum, ileum and colon segments were studied. Mechanical contractile activity was recorded using isometric force displacement transducers. Electrical pacemaker activity was recorded using a microelectrode array. RESULTS: Compared with NKA, SP induced larger contractions in stomach tissue and smaller contractions in intestinal segments, where oscillation magnitudes increased in intestinal segments, but not the stomach. CP122,721 and GR159,897 inhibited electrical field stimulation-induced contractions of the stomach, ileum and colon. NKB and NK3 had minor effects on contractile activity. The inhibitory potencies of SP and NKA on the peristaltic frequency of the colon and ileum, respectively, were correlated with those on electrical pacemaker frequency. SP, NKA and NKB inhibited pacemaker activity of the duodenum and ileum, but increased that of the stomach and colon. SP elicited a dose-dependent contradictive pacemaker frequency response in the colon. CONCLUSION: This study revealed distinct effects of tachykinins on the mechanical and electrical properties of the stomach and colon vs. the proximal intestine, providing a unique aspect on neuromuscular correlation in terms of the effects of tachykinin on peristaltic and pacemaker activity in gastrointestinal-related symptoms.

The Actions of Centrally Administered Nesfatin-1 on Emesis, Feeding, and Locomotor Activity in Suncus murinus (House Musk Shrew).

Nesfatin-1 is an anorectic peptide expressed in both peripheral tissues and brain areas involved in the regulation of feeding, emotion and emesis. The aim of the present study is to characterize the distribution of NUCB2/nesfatin-1 in Suncus murinus and to investigate the actions of nesfatin-1 to affect gastrointestinal contractility, emesis, food and water intake, and locomotor activity. The deduced amino acid sequence of S. murinus nesfatin-1 using in silico cloning showed high homology with humans and rodents. NUCB2 mRNA was detected throughout the entire brain and in the gastrointestinal tract, including the stomach and gut. Western blot analysis and immunohistochemistry confirmed the expression of nesfatin-1 protein in these regions. The NUCB2 mRNA levels in the hypothalamus, hippocampus and brainstem were significantly decreased, whereas that in the striatum were increased after 24 h starvation compared to ad libitum-fed animals (p < 0.05). In in vitro studies, nesfatin-1 (0.3-1,000 pM) failed to contract or relax the isolated gastric antrum and intestinal segments. In conscious, freely moving animals, intracerebroventricular administration of nesfatin-1 (1-50 pmol) induced emesis (p < 0.05) and suppressed 6-h cumulative food intake (p < 0.05), without affecting the latency to feeding. Nesfatin-1 (25 pmol, i.c.v.) decreased 24-h cumulative food and water intake by 28.3 and 35.4%, respectively (p < 0.01). No significant differences in locomotor activity were observed. In conclusion, NUCB2/nesfatin-1 might be a potent regulator of feeding and emesis in S. murinus. Further studies are required to elucidate the mechanism of actions of this peptide as a mediator linking the brainstem NUCB2/nesfatin-1 to forebrain system.

Whole Genome Transcriptomic Analysis of Ovary Granulosa Cells Revealed an Anti-Apoptosis Regulatory Gene DLGAP5 in Polycystic Ovary Syndrome.

The mechanisms underlining pathogenesis of polycystic ovary syndrome (PCOS) remain largely unknown. Dysfunction of ovarian granulosa cells plays an important role. The present study performed the lncRNA and mRNA profiling by whole genome transcriptomic sequencing of ovary granulosa cells from women with PCOS and investigated the potential role of differentially expressed gens (DEGs) in the pathomechanism of PCOS. In total, 1,936 DEGs (30 upregulated and 1,906 downregulated mRNAs and lncRNAs) were identified in the ovary granulosa cells between control and PCOS group. Functional enrichment analysis showed that DEGs were mainly associated with cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and olfactory transduction. qRT-PCR validated the upregulation of DLGAP5 mRNA in ovary from PCOS group when compared to control group. Immunostaining and TUNEL assays showed that DLGAP5 protein level was increased while apoptosis was decreased in follicles of ovary in PCOS group. In vitro functional assays showed that DLGPA5 knockdown repressed viability and proliferation, but enhanced apoptosis and disrupted cell cycle in granulosa cells; while DLGAP5 overexpression had the opposite effects in granulosa cells. In conclusion, the study showed differentially expressed lncRNA and mRNA profile in the granulosa cells in ovaries of PCOS. Functional results demonstrated that DLGAP5 is a dysregulated candidate gene in the pathogenesis of PCOS, especially granulosa cell apoptosis and proliferation.

Insights Into Acute and Delayed Cisplatin-Induced Emesis From a Microelectrode Array, Radiotelemetry and Whole-Body Plethysmography Study of Suncus murinus (House Musk Shrew).

Purpose: Cancer patients receiving cisplatin therapy often experience side-effects such as nausea and emesis, but current anti-emetic regimens are suboptimal. Thus, to enable the development of efficacious anti-emetic treatments, the mechanisms of cisplatin-induced emesis must be determined. We therefore investigated these mechanisms in Suncus murinus, an insectivore that is capable of vomiting. Methods: We used a microelectrode array system to examine the effect of cisplatin on the spatiotemporal properties of slow waves in stomach antrum, duodenum, ileum and colon tissues isolated from S. murinus. In addition, we used a multi-wire radiotelemetry system to record conscious animals' gastric myoelectric activity, core body temperature, blood pressure (BP) and heart rate viability over 96-h periods. Furthermore, we used whole-body plethysmography to simultaneously monitor animals' respiratory activity. At the end of in vivo experiments, the stomach antrum was collected and immunohistochemistry was performed to identify c-Kit and cluster of differentiation 45 (CD45)-positive cells. Results: Our acute in vitro studies revealed that cisplatin (1-10 µM) treatment had acute region-dependent effects on pacemaking activity along the gastrointestinal tract, such that the stomach and colon responded oppositely to the duodenum and ileum. S. murinus treated with cisplatin for 90 min had a significantly lower dominant frequency (DF) in the ileum and a longer waveform period in the ileum and colon. Our 96-h recordings showed that cisplatin inhibited food and water intake and caused weight loss during the early and delayed phases. Moreover, cisplatin decreased the DF, increased the percentage power of bradygastria, and evoked a hypothermic response during the acute and delayed phases. Reductions in BP and respiratory rate were also observed. Finally, we demonstrated that treatment with cisplatin caused inflammation in the antrum of the stomach and reduced the density of the interstitial cells of Cajal (ICC). Conclusion: These studies indicate that cisplatin treatment of S. murinus disrupted ICC networking and viability and also affected general homeostatic mechanisms of the cardiovascular system and gastrointestinal tract. The effect on the gastrointestinal tract appeared to be region-specific. Further investigations are required to comprehensively understand these mechanistic effects of cisplatin and their relationship to emesis.

Involvement of TRPV1 and TRPA1 in the modulation of pacemaker potentials in the mouse ileum.

BACKGROUND: The roles of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and subfamily A, member 1 (TRPA1) in mechanisms of gastrointestinal motility are complex. This study aimed to clarify the effects of several TRPV1 and TRPA1 ligands on the electrical potentials generated by pacemaker cells in the mouse-isolated ileum. METHOD: The pacemaker potentials of ileal segments of mice were recorded extracellularly using a 60-channel microelectrode array. The dominant frequencies, average waveform periods and propagation velocities were quantified. The effects of TRPV1 and TRPA1 agonist and antagonist were compared with the baseline recordings. RESULTS: The electrophysiological recordings showed that capsaicin (30 µM to 3 mM), resiniferatoxin (300 µM), capsazepine (100-300 µM), allyl isothiocyanate (300 µM), isovelleral (300 µM), icilin (300 µM), A-967,079 (10 µM), AP18 (20 µM) and HC-030,031 (50 µM) significantly reduced the pacemaker frequency and increased the waveform period relative to the baseline. Conversely, ruthenium red (300 µM) significantly increased the pacemaker frequency and reduced the waveform period. Capsaicin (3 mM) and AP18 (20 µM) also significantly reduced the propagation velocity. However, all tested antagonists failed to inhibit the effects of agonists. AMG9810 (300 µM), but not A-967,079 (300 µM), significantly inhibited the increases in pacemaker frequency caused by increased temperatures. CONCLUSION: Our findings suggest that TRPV1 and TRPA1 play a minor role in regulating pacemaker potentials and that at non-specific actions at other TRP and ion channels most likely contributed to the overall effects on the electrophysiological recordings that we observed.

The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Sulprostone-Induced Gastric Dysrhythmia in the Ferret: Conventional and Advanced Analytical Approaches.

Nausea and emesis resulting from disease or drug treatment may be associated with disrupted gastric myoelectric activity (GMA). Conventional analytical techniques can determine the relative degrees of brady-, normo-, and tachygastric power, but lose information relative to the basic slow wave shape. The aim of the present study was to investigate the application of advanced analytical techniques in the analysis of disrupted GMA recorded after administration of sulprostone, a prostaglandin E3 / 1 agonist, in ferrets. Ferrets were implanted with radiotelemetry devices to record GMA, blood pressure, heart rate (HR) and core body temperature 1 week before the administration of sulprostone (30 µg/kg) or vehicle (saline, 0.5 mL/kg). GMA was initially analyzed using fast Fourier transformations (FFTs) and a conventional power partitioning. Detrended fluctuation analysis (DFA) was also applied to the GMA recordings to reveal information relative to the fluctuation of signals around local trends. Sample entropy (SampEn) analysis was used for examining the regularity of signals. Conventional signal processing techniques revealed that sulprostone increased the dominant frequency (DF) of slow waves, with an increase in the percentage power of the tachygastric range and a decrease in the percentage power of the normogastric range. DFA revealed that sulprostone decreased the fluctuation function, indicative of a loss of the variability of GMA fluctuations around local trends. Sulprostone increased SampEn values, indicating a loss of regularity in the GMA data. Behaviorally, sulprostone induced emesis and caused defecation. It also increased blood pressure and elevated HR, with an associated decrease in HR variability (HRV). Further analysis of HRV revealed a decrease in both low-frequency (LF) and high-frequency (HF) components, with an overall increase in the LF/HF ratio. Sulprostone did not affect core body temperature. In conclusion, DFA and SampEn permit a detailed analysis of GMA, which is necessary to understand the action of sulprostone to modulate gastric function. The action to decrease HRV and increase the LF/HF ratio may be consistent with a shift toward sympathetic nervous system dominance, commonly seen during nausea.

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.

Preliminary detection of the anti­tumour activity of indoline­2,3­dione derivative DH­12a targeting aminopeptidase N.

Aminopeptidase N (APN) is important in tumour processes. The present study detected the anti­tumour activity of the novel APN inhibitor DH­12a, which is an indoline­2,3­dione derivative. In the present study, Bestatin, a clinical APN inhibitor was used as a positive control. The expression of APN in the ES-2 and 3AO cell lines were assessed using flow cytometry and the drug inhibition constants of DH­12a (Ki=13.15 µM) and Bestatin (Ki=16.57 µM) were assessed using a double reciprocal method of competitive inhibition. The in vitro effects of DH­12a on cell proliferation were assessed using a 3­(4,5­dimethyl­thiazol­2­yl)­2,5­diphenyl tetrazolium bromide assay on human cell lines of ES­2 (IC50=43.8 µM), A549 (inhibition rate=41.5% at 160 µM DH­12a), HL60 (inhibition rate=47.83% at 160 µM DH­12a) and 3AO (IC50=70.2 µM). The inhibition rates were consistently higher than those of Bestatin. The effects of DH­12a on cell migration (inhibition rates in ES­2 cells and 3AO cells were 56.4 and 76.5%, respectively at 15 µM) and invasion (inhibition rates in ES­2 cells and 3AO cells were 75.6 and 66.5%, respectively at 15 µM) were assessed using transwell plates. The in vivo effects of DH­12a on tumour proliferation and lung tumour metastasis were determined using an H22 xenograft mice model, where DH­12a was administered in combination with genotoxic 5­fluorouracil. The anti­tumour activities of DH­12a in vivo were also greater than those of Bestatin. In conclusion, the in vitro effects of DH­12a on tumour proliferation, migration and invasion were consistent with the in vivo effects. In addition, DH­12a exhibited greater anti­tumour properties compared with Bestatin.