AQP5 enriches for stem cells and cancer origins in the distal stomach.

LGR5 marks resident adult epithelial stem cells at the gland base in the mouse pyloric stomach1, but the identity of the equivalent human stem cell population remains unknown owing to a lack of surface markers that facilitate its prospective isolation and validation. In mouse models of intestinal cancer, LGR5+ intestinal stem cells are major sources of cancer following hyperactivation of the WNT pathway2. However, the contribution of pyloric LGR5+ stem cells to gastric cancer following dysregulation of the WNT pathway-a frequent event in gastric cancer in humans3-is unknown. Here we use comparative profiling of LGR5+ stem cell populations along the mouse gastrointestinal tract to identify, and then functionally validate, the membrane protein AQP5 as a marker that enriches for mouse and human adult pyloric stem cells. We show that stem cells within the AQP5+ compartment are a source of WNT-driven, invasive gastric cancer in vivo, using newly generated Aqp5-creERT2 mouse models. Additionally, tumour-resident AQP5+ cells can selectively initiate organoid growth in vitro, which indicates that this population contains potential cancer stem cells. In humans, AQP5 is frequently expressed in primary intestinal and diffuse subtypes of gastric cancer (and in metastases of these subtypes), and often displays altered cellular localization compared with healthy tissue. These newly identified markers and mouse models will be an invaluable resource for deciphering the early formation of gastric cancer, and for isolating and characterizing human-stomach stem cells as a prerequisite for harnessing the regenerative-medicine potential of these cells in the clinic.

Basal differentiation and expression status of SOX2 and KLF4 in basal layers are prognostic factors for disease-specific survival in oral squamous cell carcinoma.

BACKGROUND: Basal differentiation in oral squamous cell carcinoma is usually detected at invasive sites. However, its significance as a prognostic value has been poorly investigated. METHODS: COL17 was selected as a basal differentiation marker because of its stable expression in the basal-like cells of oral squamous cell carcinoma. Sixty-five cases of oral squamous cell carcinoma were subclassified into COL17-high (30 cases) and -low (35 cases) types, and the prognostic value was analyzed by Cox regression analysis. In addition, the stem cell markers such as SOX2, KLF4, MYC as well as the stem cell-related markers BMI1, EZH2, and YAP and its paralog TAZ, were immunohistochemically analyzed. Their prognostic values were investigated along with their COL17 status by Cox regression analysis. RESULTS: No significant difference was observed between the COL17-high and -low groups in the disease-specific survival and recurrence-free survival in oral squamous cell carcinoma. When the COL17-high and -low categories were combined with the SOX2, KLF4, EZH2, or YAP/TAZ status in the basal layers, together with gender and age as covariates, the hazard ratios reached 3.3, 3.7, 2.8, and 3.1, respectively. In addition, multivariate analysis, including COL17, SOX2, and KLF4, with gender and age as covariates, showed a significantly poor prognosis for disease-specific survival. CONCLUSION: Based on the relatively high hazard ratios, it is indicated that basal differentiation and the expression status of SOX2 and KLF4 in the basal layers are prognostic factors for oral squamous cell carcinoma.

A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells.

An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3ß, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.

Expression of Periostin in Benign Salivary Gland Tumors.

Parotid tumors present a wide range of histological features, from benign to malignant. Periostin, an extracellular matrix protein specifically expressed in the periosteum and periodontal ligament, is isolated from osteoblast cell lines. It regulates fibrosis and collagen deposition and plays an important role in myocardial repair after myocardial infarction. It is also known to be involved in otorhinolaryngological-diseases. This study included 36 patients [38 specimens; 16 men and 20 women, mean age 59.2 (range 26-82) years] who underwent parotid tumor resection at the Division of Otorhinolaryngology, Tohoku Medical and Pharmaceutical University, between April 2017 and March 2022 and were clinically and pathologically diagnosed as having benign parotid tumors. Formalin-fixed, paraffin-embedded sections from the surgical specimens were autoclaved and immunostained with anti-periostin antibodies to evaluate the expression and distribution of periostin. Histologically, the tumors were diagnosed as pleomorphic adenomas in 15 cases (15 specimens), Warthin's tumors in 13 cases (15 specimens), basal cell adenomas in 2 cases (2 specimens), oncocytomas in 4 cases (4 specimens), and myoepitheliomas in 2 cases (2 specimens). An increased expression of periostin was found in 32 of 38 samples (84.2%) in the stroma of benign parotid tumors. Four distinct patterns of periostin expression were observed in benign parotid gland tumors: negative, superficial, infiltrative, and diffuse. Statistically significant differences were found between periostin expression patterns and histological classification of the tumors. Our results suggest that periostin may be involved in the pathogenesis of benign parotid tumors and could serve as a new biomarker for these tumors.

Relationship between maximal isometric tongue pressure and limit of fracture force of gels in tongue squeezing.

BACKGROUND: There is no clear objective indicator for selecting soft foods that are required for food bolus formation in older people with impaired oral function. OBJECTIVE: This study aimed to investigate the relationship between maximal isometric tongue pressure (MITP) and the mechanical properties of gels that can be crushed by the tongue. METHODS: This study included 65 healthy participants aged 22-96 (young group; 15 males, 15 females; older dentate group; 7 males, 8 females; older edentulous group; 10 males, 10 females). MITP was measured by the balloon-probe device. Agar gel with 10 different kinds of fracture force from 10N to 100N was used. The limit of fracture force of gels (LFFG) that were crushed by the tongue was measured by the up-and-down method. In the older edentulous group, two items were measured with and without dentures. Spearman's rank correlation coefficient was used to evaluate the relationship between MITP and LFFG in each group (p < .05). RESULTS: There were positive correlations between MITP and LFFG in all groups (overall groups: rs = .66, young group: rs = .46, older dentate group: rs = .61, older edentulous group with dentures: rs = .60, older edentulous group without dentures: rs = .47). CONCLUSION: MITP and LFFG were positively correlated in young, older dentate and older edentulous groups, suggesting that MITP has the potential to be an objective indicator of the range of mechanical properties of soft food that can be crushed by the tongue.

[Cardiac Tamponade After Left Upper Lobectomy:Report of Two Cases].

We report two rare cases of cardiac tamponade after left upper lobectomy. Case 1:A 76-year-old man underwent thoracoscopic left upper lobectomy and lymph node dissection for lung cancer. The patient suddenly developed cardiac tamponade the day after surgery. Emergency surgery was performed to stop bleeding and confirm the source of bleeding, and dark red pericardial fluid and hematoma were observed in the pericardial sac. There was no postoperative recurrence of cardiac tamponade. He died 1 year and 2 months after the operation. Case 2:A 77-year-old woman underwent thoracoscopic left upper lobectomy and lymph node dissection for lung cancer. The patient did well until the 6th postoperative day. On the 7th postoperative day, she complained of sudden severe back pain, immediately after which she lost consciousness and went into cardiopulmonary arrest. The echocardiography revealed cardiac tamponade, and emergency pericardiocentesis was performed. The patient died without circulatory improvement despite drainage of approximately 200 ml of bloody pericardial fluid. The pathological findings of autopsy revealed penetrating atherosclerotic ulcer at the descending aorta. We speculated that severe back pain caused the afterload of left ventricle and the increase in left atrial pressure through mitral regurgitation, which might result in a bleeding from the staple-line of superior pulmonary vein in the pericardium.

Frequent loss of metastatic ability in subclones of Apc, Kras, Tgfbr2, and Trp53 mutant intestinal tumor organoids.

Cancer evolution is explained by the accumulation of driver mutations and subsequent positive selection by acquired growth advantages, like Darwin's evolution theory. However, whether the negative selection of cells that have lost malignant properties contributes to cancer progression has not yet been fully investigated. Using intestinal metastatic tumor-derived organoids carrying Apc, Kras, Tgfbr2, and Trp53 quadruple mutations, we demonstrate here that approximately 30% of subclones of the organoids show loss of metastatic ability to the liver while keeping the driver mutations and oncogenic pathways. Notably, highly metastatic subclones also showed a gradual loss of metastatic ability during further passages. Such non-metastatic subclones revealed significantly decreased survival and proliferation ability in Matrigel and collagen gel culture conditions, which may cause elimination from the tumor tissues in vivo. RNA sequencing indicated that stemness-related genes, including Lgr5 and Myb, were significantly downregulated in non-metastatic subclones as well as subclones that lost metastatic ability during additional passages. Furthermore, a CGH analysis showed that non-metastatic subclones were derived from a minor population of parental organoid cells. These results indicate that metastatic ability is continuously lost with decreased stem cell property in certain subpopulations of malignant tumors, and such subpopulations are eliminated by negative selection. Therefore, it is possible that cancer evolution is regulated not only by positive selection but also by negative selection. The mechanism underlying the loss of metastatic ability will be important for the future development of therapeutic strategies against metastasis.

Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.

Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/ß-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active ß-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the ß-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Immunohistochemical Localization of D-ß-Aspartic Acid and Periostin in Vocal Fold Polyps.

Long-term voice abuse or sudden vocal fold microvascular disruption may lead to injury and subsequent repair/remodeling in the vocal fold mucosa. Periostin is known to be involved in airway remodeling and also in various otolaryngological diseases. D-ß-aspartic acid is the major isomer of D-aspartic acid found in elderly tissue. In this study we investigated the expression and the role of D-ß-aspartic acid and periostin in the formation of vocal fold polyps. The expression patterns of D-ß-aspartic acid and periostin in 36 surgical specimens of vocal fold polyps from 36 patients were investigated immunohistochemically. In the epithelium of vocal polyps, D-ß-aspartic acid was expressed in all cases. Expression of D-ß-aspartic acid was detected in 25 samples obtained from patients with vocal fold polyps stroma. Expression of periostin was detected in 28 samples obtained from patients with vocal fold polyps. Two patterns of D-ß-aspartic acid expression were observed in vocal fold polyps stroma: positive type and negative type. The following four patterns of periostin expression were observed in vocal fold polyps: negative type, superficial type, infiltrative type, and diffuse type. An association was observed between D-ß-aspartic acid expression patterns and periostin expression patterns. From these findings we speculate that periostin and D-ß-aspartic acid participate in certain pathological changes in vocal fold polyps, such as extracellular matrix accumulation, local fibrosis, and the formation and development of vocal fold polyps.

Periostin is an aggravating factor and predictive biomarker of eosinophilic chronic rhinosinusitis.

BACKGROUND: Patients with eosinophilic chronic rhinosinusitis (ECRS) respond poorly to many treatment modalities. Overproduction of periostin in the nasal mucosa is reported to contribute to polyp formation. This study examined periostin levels in patients with ECRS in comparison with levels in patients with non-ECRS. METHODS: Fifty-nine patients with chronic rhinosinusitis were grouped into those with ECRS and those with non-ECRS. We compared the relationships between peripheral blood eosinophil level, serum periostin level, histopathological findings, clinical and laboratory findings, nose findings, diagnostic score of the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis Study, and postoperative recurrence of nasal polyps in each group. RESULTS: In the ECRS group, a positive correlation was found between peripheral blood eosinophil level and serum periostin level (rs = 0.49, P < 0.01: Spearman's rank correlation coefficient). ROC curve analysis was used to evaluate the serum periostin level that could predict postoperative recurrence of nasal polyps in the ECRS group: the area under the curve (AUC) was 0.95, sensitivity was 92%, and specificity was 100%; the serum periostin cutoff value for postoperative recurrence of nasal polyps was 130 ng/ml. In ROC curve analysis to evaluate peripheral blood eosinophil level, the AUC was 0.73, sensitivity was 69.2%, and specificity was 85.0%; the cutoff value was 8.8%. CONCLUSIONS: periostin was implicated in the pathophysiology of ECRS. Periostin shown to be a more useful biomarker than eosinophils in ECRS. Periostin was shown to likely be an important biomarker for pathological severity of ECRS and postoperative recurrence of nasal polyps.

NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers.

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.

Expression of Periostin in Vocal Fold Polyps.

Long-term voice abuse or sudden vocal fold microvascular disruption may lead to injury and subsequent repair/remodeling in the vocal fold mucosa. Periostin is known to be involved in airway remodeling and also in various otolaryngological diseases. The aim of this article was to investigate the expression and the role of periostin in the formation of vocal fold polyps. The expression patterns of periostin in 59 surgical specimens of vocal fold polyps from 54 patients were investigated immunohistochemically. Normal vocal fold mucosa specimens from 5 patients who had undergone total laryngectomy were used as the control group. Retrospective study with planned data collection was conducted at Tohoku Medical and Pharmaceutical University. Expression of periostin was detected in 43 (72.9%) samples and four patterns of periostin expression were observed in vocal fold polyps: negative type, superficial type, infiltrative type, and diffuse type. An association was observed between periostin expression patterns and the histological subtypes of vocal fold polyps. The infiltrative pattern of periostin expression was significantly dominant in vascular-hyaline types. Expression of transforming growth factor-ß (TGF-ß) was also detected in the vocal fold polyps. Our results confirmed that periostin might be involved in certain pathological changes in vocal fold polyps, such as extracellular matrix accumulation, local fibrosis, and formation and development of vocal fold polyps.

The effects of tongue elevation using a weighted plastic bottle on the tongue pressure and activity of suprahyoid muscles.

BACKGROUND: A rise in tongue pressure coincides with an increase in the suprahyoid muscle activity. OBJECTIVES: The aim was to investigate the effects of holding a weighted plastic bottle on tongue pressure and the suprahyoid muscle activity. METHODS: Eighteen participants (8 men and 10 women; mean age 42 ± 16 years) participated in this study. All participants had no history of speech, language, hearing or swallowing disorders and no tooth loss, and they did not require dentures. Healthy participants held gauzes connected with a plastic bottle with increasing resistive loads of 0 g, 250 g, 500 g and 750 g, between their palate and tongue. The maximum tongue pressure and average tongue pressure were measured during a 5 s hold. The average tongue pressure was defined as the mean tongue pressure data in each task. The suprahyoid muscle activity was measured using the electromyogram (EMG). The root mean square of the EMG signals measured while lifting different loads and while performing the head lifting exercises was compared. All variables were examined using the Friedman's test and Wilcoxon signed-rank test. RESULTS: The maximum tongue pressure (p < .05) and average tongue pressure values (p < .05) increased gradually in the anterior-median region with increasing resistive loads, and the root mean square amplitudes for 250 g, 500 g and 750 g were not significant compared with head lifting exercises. CONCLUSION: These results indicated that plastic bottle holding could be a potential strength training tool for the tongue and the suprahyoid muscles.

Compatibility of two types of gummy jelly tests for detecting decreased masticatory function.

OBJECTIVE: This study aimed to verify the compatibility of masticatory performance tests using different two types of gummy jellies. BACKGROUND DATA DISCUSSING THE PRESENT STATUS OF THE FIELD: Compatibility between the glucose concentration measuring method (GC) and 10-steps visual scoring method (VS), which are masticatory performance tests for diagnosing decreased masticatory function (DMF) in oral hypofunction and the use of different types of gummy jelly, has yet to be confirmed. MATERIALS AND METHODS: The participants consisted of 134 Japanese older people (51 men and 83 women). GC, VS and the increased surface area measuring method (ISA), as an alternative analysis method for VS, were conducted to evaluate masticatory performance. Cohen's Kappa coefficient was used to verify the consistency for detecting DMF between GC and VS. Pearson's correlation coefficient was used to analyse the relationship between GC and ISA. Each masticatory performance was compared among three different groups for the number of residual teeth. Cut-off values of VS and ISA for DMF that were consistent with GC were identified. RESULTS: Glucose concentration measuring method and VS showed an excellent consistency (Kappa coefficient = 0.86). There was a high positive correlation between GC and ISA (r = .70). However, the change in masticatory performance according to the number of teeth was different between GC and VS/ISA. The cut-off values of VS and ISA were a Score of 2 and 1687 mm2 , respectively. CONCLUSION: Glucose concentration measuring method and VS/ISA remained fairly consistent for detecting DMF, which might be useful information to interconnect the various studies on masticatory performance.

BEX2 is required for maintaining dormant cancer stem cell in hepatocellular carcinoma.

Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain-expressed X-linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67-negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC.

Analysis of implant stability changes in immediate loading using a laser displacement sensor in vivo and comparison of its sensitivity with that of resonance frequency analysis.

OBJECTIVE: The aim of this study was to analyze the stability changes in immediately loaded implants by using an in vivo quantitative measurement of micromotion under functional dynamic loading and to verify the sensitivity of Resonance Frequency Analysis (RFA) as compared to that of actual micromotion. MATERIALS AND METHODS: The micromotions of immediately loaded implants placed in the tibia of 11 rabbits were monitored using a laser displacement sensor. Functional dynamic loading forces were applied 5 days a week for 6 weeks. The implant stability quotient (ISQ) was monitored using RFA. RESULTS: The micromotion of the almost-loaded implants increased to peak values the day after loading was started and subsequently reached a plateau gradually. The ISQ changes in the loaded implants closely correlated with the alterations of the actual micromotion (r = -0.98, p < .01). Although the ISQ value itself correlated with the measured micromotion at the time of initial fixation (r = 0.73, p < .05), it did not correlate with the micromotion of the implant that acquired integration. No close correlation was observed between the ISQ and the histomorphometrical data. CONCLUSION: The immediately loaded implants showed the lowest stability immediately after the start of loading, which gradually increased thereafter. RFA is considered a useful method for examining stability changes and initial stability; however, it cannot determine the absolute magnitude of the stability after integration.

Effect of hard gummy candy chewing on masticatory function.

BACKGROUND: Although various training methods have been reported for improving oral function such as occlusal force, there are few reports that show the training effect of eating hard food on the oral functions. OBJECTIVE: To examine the training effects of habitually ating hard gummy candies on oral functions. METHODS: Participants of this cohort study were recruited into a training (six women, eight men; mean age 27.6 ± 3.5 years) and control group (two women, seven men; mean age 28.3 ± 4.9 years). The training group ate nine custom-developed hard gummy candies three times per week for 3 months. Training effects were evaluated by measuring the maximal occlusal force, masticatory performance, maximal tongue pressure, number of chewing cycles until swallowing the candy, duration of chewing and cycle time before training, after 1, 2 and 3 months of training, and 1 month after stopping training. The iEMG/force, which evaluates masseter muscle hypertrophy with electromyograms (EMG), was calculated. RESULTS: Masticatory performance after 1 month (p = 0.01), maximal occlusal force after 2 months (p < 0.01) and maximal tongue pressure after 3 months of training were significantly increased (p = 0.02), and the cycle time after 2 months of training (p = 0.02) was significantly decreased compared to before the intervention. Except for masticatory performance, the other effects were maintained for 1 month after stopping training. Changes in iEMG/force were not significant, but a tendency for muscle hypertrophy was observed in the training group. CONCLUSION: Habitual eating of hard gummy candies is a task-specific training that can improve overall masticatory function, including tongue pressure.

Interleukin 1 Up-regulates MicroRNA 135b to Promote Inflammation-Associated Gastric Carcinogenesis in Mice.

BACKGROUND & AIMS: Gastritis is associated with development of stomach cancer, but little is known about changes in microRNA expression patterns during gastric inflammation. Specific changes in gene expression in epithelial cells are difficult to monitor because of the heterogeneity of the tissue. We investigated epithelial cell-specific changes in microRNA expression during gastric inflammation and gastritis-associated carcinogenesis in mice. METHODS: We used laser microdissection to enrich epithelial cells from K19-C2mE transgenic mice, which spontaneously develop gastritis-associated hyperplasia, and Gan mice, which express activated prostaglandin E2 and Wnt in the gastric mucosa and develop gastric tumors. We measured expression of epithelial cell-enriched microRNAs and used bioinformatics analyses to integrate data from different systems to identify inflammation-associated microRNAs. We validated our findings in gastric tissues from mice and evaluated protein functions in gastric cell lines (SNU-719, SNU-601, SNU-638, AGS, and GIF-14) and knockout mice. Organoids were cultured from gastric corpus tissues of wild-type and miR-135b-knockout C57BL/6 mice. We measured levels of microRNAs in pairs of gastric tumors and nontumor mucosa from 28 patients in Japan. RESULTS: We found microRNA 135b (miR-135B) to be the most overexpressed microRNA in gastric tissues from K19-C2mE and Gan mice: levels increased during the early stages of gastritis-associated carcinogenesis. Levels of miR-135B were also increased in gastric tumor tissues from gp130F/F mice and patients compared with nontumor tissues. In gastric organoids and immortalized cell lines, expression of miR-135B was induced by interleukin 1 signaling. K19-C2mE mice with disruption of Mir-135b developed hyperplastic lesions that were 50% smaller than mice without Mir-135b disruption and had significant reductions in cell proliferation. Expression of miR-135B in gastric cancer cell lines increased their colony formation, migration, and sphere formation. We identified FOXN3 and RECK messenger RNAs (mRNAs) as targets of miR-135B; their knockdown reduced migration of gastric cancer cell lines. Levels of FOXN3 and RECK mRNAs correlated inversely with levels of miR-135B in human gastric tumors and in inflamed mucosa from K19-C2mE mice. CONCLUSIONS: We found expression of miR-135B to be up-regulated by interleukin L1 signaling in gastric cancer cells and organoids. miR-135B promotes invasiveness and stem-cell features of gastric cancer cells in culture by reducing FOXN3 and RECK messenger RNAs. Levels of these messenger RNA targets, which encode tumor suppressor, are reduced in human gastric tumors.

Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

Signal transducer and activator of transcription 3 (Stat3) has been shown to play a role in intestinal regeneration and colitis-associated colon carcinogenesis. However, the role of Stat3 in the Wnt-driven sporadic intestinal tumorigenesis remains poorly understood. We examined the roles of Stat3 in intestinal regeneration and tumorigenesis by organoid culture experiments using Stat3∆IEC mouse-derived intestinal epithelial cells in which Stat3 was disrupted. The regeneration of intestinal mucosa and organoid formation were significantly suppressed by Stat3 disruption, which was compensated by Wnt activation. Furthermore, once organoids were recovered, Stat3 was no longer required for organoid growth. These results indicate that Stat3 and Wnt signaling cooperatively protect epithelial cells at the early phase of intestinal regeneration. In contrast, intestinal tumorigenesis was not suppressed by Stat3 disruption in adenomatous polyposis coli ( Apc) Δ716 and Apc∆716 Tgfbr2∆IEC mice, thus indicating that Stat3 is not required for Wnt activation-driven intestinal tumorigenesis. Mechanistically, Itga5 and Itga6 were down-regulated by Stat3 disruption, and focal adhesion kinase (FAK) activation was also suppressed. Notably, FAK inhibitor suppressed the organoid formation of wild-type epithelial cells. These results indicate that Stat3 is indispensable for the survival of epithelial cells through the activation of integrin signaling and the downstream FAK pathway; however, it is not required for the Wnt signaling-activated normal or tumor epithelial cells.-Oshima, H., Kok, S.-Y., Nakayama, M., Murakami, K., Voon, D. C.-C., Kimura, T., Oshima, M. Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

Evaluation of hyoid movement during swallowing using a bend sensor.

BACKGROUND: Movement of the hyoid and laryngeal complex is critical for preventing aspiration, as well as smooth bolus passage through the pharynx. We have developed a non-invasive system for measuring laryngeal movement during swallowing with a bend sensor and have already reported the time coordination between the signal waveform from the sensor and hyoid movement. OBJECTIVE: The aim of this study was to clarify the quantitative association between the output value of the sensor and hyoid movement during swallowing. METHODS: A small bend sensor was fixed on the skin surface along the midline of the neck of 13 healthy men (mean age, 30.8 ± 4.5 years). Laryngeal and hyoid movements during swallowing of 5 mL of water were recorded synchronously by the bend sensor and videofluorography. The relationship between the bend sensor output value (LM value) and hyoid position (x- and y-axis displacements) by videofluorography from the onset to the offset of the signal waveform was analysed for all tasks and for each task using Pearson's correlation coefficients. RESULTS: There was a positive correlation between the LM value and hyoid position for all tasks (x-axis displacement: r = .647, y-axis displacement: r = .233). In particular, there was a moderate to high correlation between the LM value and x-axis displacement for each task (.453 ≤ r ≤ .934). CONCLUSION: The LM value can be a quantitative parameter of anterior hyoid movement during swallowing that might be associated with bolus flow and upper oesophageal sphincter opening.