RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

Expression of alcohol acyltransferase is a potential determinant of fruit volatile ester variations in Capsicum.

KEY MESSAGE: The transcript level of alcohol acyltransferase 1 (AAT1) may be the main factor influencing the variations in volatile esters that characterizing the fruity/exotic aroma of pepper fruit. Volatile esters are key components for characterizing the fruity/exotic aroma of pepper (Capsicum spp.) fruit. In general, the volatile ester content in the fruit is the consequence of a delicate balance between their synthesis by alcohol acyltransferases (AATs) and degradation by carboxylesterases (CXEs). However, the precise role of these families of enzymes with regard to volatile ester content remains unexplored in Capsicum. In this study, we found that the volatile ester content was relatively low in C. annuum and much higher in C. chinense, particularly in pungent varieties. Additionally, fruits collected from multiple non-pungent C. chinense varieties, which harbor loss-of-function mutations in capsaicinoid biosynthetic genes, acyltransferase (Pun1), putative aminotransferase (pAMT), or putative ketoacyl-ACP reductase (CaKR1) were analyzed. The volatile ester contents of non-pungent C. chinense varieties (pamt/pamt) were equivalent to those of pungent varieties, but their levels were significantly lower in non-pungent NMCA30036 (pun12/pun12) and C. chinense (Cakr1/Cakr1) varieties. Multiple AAT-like sequences were identified from the pepper genome sequences, whereas only one CXE-like sequence was identified. Among these, AAT1, AAT2, and CXE1 were isolated from fruits of C. chinense and C. annuum. Gene expression analysis revealed that the AAT1 transcript level is a potential determinant of fruit volatile ester variations in Capsicum. Furthermore, enzymatic assays demonstrated that AAT1 is responsible for the biosynthesis of volatile esters in pepper fruit. Identification of a key gene for aroma biosynthesis in pepper fruit will provide a theoretical basis for the development of molecular tools for flavor improvement.

TEG6s Platelet Mapping assay for the estimation of plasma fibrinogen concentration during cardiovascular surgery: a single-center prospective observational study.

PURPOSE: The Activator F (ActF) test on the TEG6s Platelet Mapping assay system is a means of quantifying blood viscoelasticity caused by fibrin network formation, triggered by reptilase and factor XIII, while platelets are inhibited. This unique methodology enables the measurement of blood viscoelasticity, even in highly heparinized blood. Here, we investigated whether fibrinogen concentration could be estimated using the ActF test in blood samples obtained during cardiopulmonary bypass (CPB) and after CPB in patients undergoing cardiovascular surgery. METHODS: We performed a single-center prospective observational study at a university hospital. Forty patients aged ≥ 18 years who underwent elective cardiovascular surgery with CPB were enrolled. Blood samples were drawn after the induction of anesthesia, after declamping of the aorta during CPB, and after the reversal of heparinization using protamine (after CPB). Coagulation profiles were evaluated using the Platelet Mapping assay and standard laboratory tests. RESULTS: There were strong correlations between the maximal amplitude of clot strength (MA) in the ActF test and fibrinogen concentration in samples drawn during CPB (R = 0.84, 95% confidence interval [CI] 0.72-0.91; P < 0.001) and after CPB (R = 0.83, 95% CI 0.70-0.91; P < 0.001). The areas under the receiver-operating characteristic curve for the ActF MA for fibrinogen concentrations < 150 mg/dL were 0.86 (95% CI 0.73-1.0) during CPB and 0.98 (95% CI 0.94-1.0) after CPB. CONCLUSION: TEG6s Platelet Mapping ActF MA values strongly correlated with plasma fibrinogen concentration in highly heparinized blood during CPB and yielded highly accurate measurements of low fibrinogen concentrations.

A prospective, randomized comparison between single- and multiple-injection techniques for ultrasound-guided subgluteal sciatic nerve block.

BACKGROUND: It is believed that local anesthetic injected to obtain circumferential spread around nerves produces a more rapid onset and successful blockade after some ultrasound-guided peripheral nerve blocks. However, evidence demonstrating this point is limited only to the popliteal sciatic nerve block, which is relatively easy to perform by via a high-frequency linear transducer. In the present study, we tested the hypothesis that multiple injections of local anesthetic to make circumferential spread would improve the rate of sensory and motor blocks compared with a single-injection technique for ultrasound-guided subgluteal sciatic nerve block, which is considered a relatively difficult block conducted with a low-frequency, curved-array transducer. METHODS: Ninety patients undergoing knee surgery were divided randomly into 2 groups to receive the ultrasound-guided subgluteal approach to sciatic nerve block with 20 mL of 1.5% mepivacaine with epinephrine. For group M (the multiple-injection technique), the local anesthetic was injected to create circumferential spread around the sciatic nerve without limitation on the number of needle passes. For group S (the single-injection technique), the number of needle passes was limited to 1, and the local anesthetic was injected to create spread along the dorsal surface of the sciatic nerve, during which no adjustment of the needle tip was made. Sensory and motor blockade were assessed in double-blind fashion for 30 minutes after completion of the block. The primary outcome was sensory blockade of all sciatic components tested, including tibial, superficial peroneal, and sural nerves at 30 minutes after injection. RESULTS: Data from 86 patients (43 in each group) were analyzed. Block execution took more time for group M than group S. The proportion of patients with complete sensory blockade of all sciatic components at 30 minutes after injection was significantly larger for group M than group S (41.9% vs 16.3%, P = 0.018). Complete motor blockade of foot and toes extension also was observed more frequently in group M than in group S (67.4% vs 34.9%, P = 0.005 and 51.2% vs 25.6%, P = 0.027, respectively). CONCLUSIONS: When ultrasound-guided subgluteal sciatic nerve block is conducted, multiple injections of local anesthetic to make a circumferential spread around the sciatic nerve improve the rate of sensory and motor blocks compared with a single injection.

Elevated levels of angiopoietin-2 as a biomarker for respiratory failure after cardiac surgery.

OBJECTIVES: Angiopoietin-1 and angiopoietin-2 are important factors in regulating endothelial vascular permeability. This study evaluated perioperative changes in serum levels of angiopoietin-1 and -2 in patients undergoing cardiac surgery. DESIGN: Measurement of serum levels of angiopoietin-1 and angiopoietin-2 in samples collected during a previously conducted prospective, multicenter, observational study. SETTING: Three university hospitals. PARTICIPANTS: Eighty-four adult patients undergoing cardiac surgery. INTERVENTION: Serum levels of angiopoietins were measured at baseline, immediately after surgery, and the day after surgery (POD-1). MEASUREMENTS AND MAIN RESULTS: Serum levels of angiopoietin-2 were elevated by POD-1 (median 3.3 ng/mL, interquartile range [IQR] 2.5-4.6 ng/mL) compared with baseline (median 1.6 ng/mL, IQR 1.3-2.1 ng/mL, p < 0.0001), and angiopoietin-1 levels were decreased immediately after surgery (baseline median 23.2 ng/mL, IQR 10.2-32.8 ng/mL; postoperative median 8.0 ng/mL, IQR 1.5-13.2 ng/mL, p<0.0001). Angiopoietin-2 levels on POD-1 in patients undergoing off-pump coronary artery bypass grafting were significantly lower than those in patients undergoing aortic surgery (p = 0.0009) and valve surgery (p = 0.008). Angiopoietin-2 levels on POD-1 had a predictive performance of the area under the curve (AUC) of the receiver operating characteristic curve 0.74 for mechanical ventilation>3 days. Angiopoietin-1 levels and the angiopoietin-2/angiopoietin-1 ratio showed lower predictive performance (AUC values 0.58 and 0.68, respectively). CONCLUSIONS: Angiopoietin-2 serum levels were elevated after cardiac surgery. Elevated angiopoietin-2 had a good predictive performance for respiratory failure after cardiac surgery, perhaps reflecting the severity of lung dysfunction related to postoperative increases in vascular permeability.

Detection of invasive ductal carcinoma in quadrant breast areas by electrical impedance tomography implemented with gaussian relaxation-time distribution (EIT-GRTD).

Invasive ductal carcinoma (IDC) in breast specimens has been detected in the quadrant breast area: (I) upper outer, (II) upper inner, (III) lower inner, and (IV) lower outer areas by electrical impedance tomography implemented with Gaussian relaxation-time distribution (EIT-GRTD). The EIT-GRTD consists of two steps which are (1) the optimum frequencyfoptselection and (2) the time constant enhancement of breast imaging reconstruction.foptis characterized by a peak in the majority measurement pair of the relaxation-time distribution functionγ,which indicates the presence of IDC.γrepresents the inverse of conductivity and indicates the response of breast tissues to electrical currents across varying frequencies based on the Voigt circuit model. The EIT-GRTD is quantitatively evaluated by multi-physics simulations using a hemisphere container of mimic breast, consisting of IDC and adipose tissues as normal breast tissue under one condition with known IDC in quadrant breast area II. The simulation results show that EIT-GRTD is able to detect the IDC in four layers atfopt= 30, 170 Hz. EIT-GRTD is applied in the real breast by employed six mastectomy specimens from IDC patients. The placement of the mastectomy specimens in a hemisphere container is an important factor in the success of quadrant breast area reconstruction. In order to perform the evaluation, EIT-GRTD reconstruction images are compared to the CT scan images. The experimental results demonstrate that EIS-GRTD exhibits proficiency in the detection of the IDC in quadrant breast areas while compared qualitatively to CT scan images.

Imprint Cytology of Tall Cell Carcinoma with Reversed Polarity of the Breast: A Case Report.

INTRODUCTION: Tall cell carcinoma with reversed polarity (TCCRP) is a rare histologic subtype of breast cancer that was newly categorized in 2020. TCCRP is a relatively novel tumor, and there are no detailed reports about its cellular morphology. We were able to obtain imprint cytological specimens from fresh TCCRP tissue, and we provide our detailed observations. CASE PRESENTATION: The patient was a 73-year-old Japanese female with a 15-mm mass in her right breast. After invasive breast carcinoma was diagnosed based on a core needle biopsy, a lumpectomy was performed. The pathological examination revealed TCCRP, and Sanger sequencing detected IDH2 p.R172M hotspot mutation, which is characteristic of TCCRP. Soon after the surgery, the lumpectomy specimen was sliced before fixation for use in a clinical trial, and imprint cytological materials were obtained from the tumor's cut surface. Cytologically, the tumor showed papillary-like cell clusters and isolated cells with moderate cellularity. Neoplastic cell aggregates and clusters with thick vascular cores as the axis or with delicate fibrovascular stroma were observed. Most of the neoplastic cells were cuboidal-to-columnar in shape, with mildly to moderately irregularly shaped blunt nuclei. Some intranuclear cytoplasmic inclusions and nuclear grooves were present, resembling the nuclear findings of papillary thyroid carcinoma. The most characteristic finding was the columnar cell clusters with apically located nuclei, giving the impression of reversed polarity. CONCLUSION: We described cytological findings in TCCRP, a newly classified rare mammary tumor. Most of the characteristic histologic findings were also observed in imprint cytological specimens. Further studies on practical specimens such as fine-needle aspiration are needed for clinical application.

Precursor-derived versus de-novo carcinogenesis depends on lineage-specific mucin phenotypes of intramucosal gland-forming gastric neoplasms.

AIMS: To clarify the lineage-specific carcinogenesis of gland-forming gastric neoplasms, by characterizing mucin phenotypes and proliferation patterns immunohistochemically using monoclonal antibodies against MUC2, MUC5AC, MUC6, CD10 and Ki67. METHODS AND RESULTS: We analysed 49 gland-forming intramucosal neoplasms, including 15 non-invasive low-grade neoplasms (group A), 10 non-invasive high-grade neoplasms (group B) and 24 intramucosal adenocarcinomas (group C). The mode of gland-forming gastric carcinoma development was different between the intestinal and gastric lineages. The pure intestinal-type accounted for 93% of group A, 50% of group B and 4.2% of group C tumours. A zonal pattern of cell proliferation was well retained in group A tumours and was lost size-dependently in group B tumours. These findings suggest that non-invasive low-grade neoplasms of the intestinal lineage progress to non-invasive high-grade neoplasms, but rarely to intramucosal adenocarcinomas. In tumours of the gastric lineage, which exhibited pure gastric or mixed phenotypes, the polarity of cell proliferation and differentiation was well retained in small (≦5 mm) tumours but was lost in larger tumours in groups B and C. CONCLUSIONS: Intramucosal adenocarcinomas of the gastric lineage may often arise de novo, develop in the proper gastric mucosa, and are partially derived from non-invasive high-grade neoplasms.

Less necessity of adjuvant S-1 treatment in non-monarchE-eligible patients.

BACKGROUND: In monarchE and Postoperative Therapy with Endocrine and TS-1 (POTENT) trials, abemaciclib and S-1 have, respectively, shown to be effective as adjuvant therapies for luminal breast cancer (BC), although whether patients who meet the criteria are at high risk of recurrence compared to non-eligible patients is still unknown. Here, we investigated recurrence risk according to the criteria of each trial in Japanese patients. METHODS: We reviewed the records of 992 patients who received surgery at Chiba University Hospital for stage I-III BC from January 2017 to May 2022 and selected 553 analytic cohort patients and retrospectively analyzed the relapse-free survival of the patients as the primary endpoint. High-recurrence risk was defined according to monarchE trial and POTENT trial. RESULTS: The 5-year RFS for monarchE cohort 1 and cohort 2 eligible patients were 77.78% and 89.33%, respectively, which were significantly lower than monarchE non-eligible patients (98.31%; p < 0.0001). However, the 5-year RFS rate for POTENT eligible patients (90.51%) was lower than for POTENT non-eligible patients (98.75%; p = 0.0001); excluding those who met the monarchE criteria, the prognosis of POTENT eligible patients had no significant differences from the prognosis of patients with POTENT non-eligible BC (p = 0.3100). CONCLUSION: MonarchE criteria accurately identify patients who are prone to relapse. Moreover, although POTENT criteria also suggested a reasonable capacity for recurrence prediction, there was no significant difference in recurrence between POTENT non-eligible patients and the patients who were POTENT but not monarchE eligible. This might offer justification for reconsidering the use of S-1 in monarchE non-eligible patients.

Ex vivo dual gene therapy using human adipocytes secreting anti-HER2 antibody on HER2-positive xenograft tumor models.

BACKGROUND: Although targeted treatments against human epidermal growth factor receptor 2 (HER2) have improved survival in patients with metastatic HER2-positive breast cancer, long and repeated treatment is time-consuming and costly for patients. To reduce these burdens, we developed ex vivo gene-transduced adipocytes that secrete anti-HER2 antibodies and evaluated their anti-tumor effects. METHODS: Ceiling culture-derived proliferative adipocytes (ccdPA) secreting anti-HER2 antibody against domain IV receptors: TRA-ccdPA, and domain II receptors: PER-ccdPA, were constructed using a plasmid lentivirus. Delivery of secreted antibody and its specific binding to HER2 breast cancer were evaluated in vitro and in vivo. To optimize antibody production from ccdPA, different conditions of ccdPA implantation were examined. Anti-tumor efficacy was evaluated in HER2-positive-cancer-inoculated nude mice. RESULTS: Anti-HER2 antibody against domain II was identified in supernatants from PER-ccdPAs. The optimal method to achieve the highest concentration of antibody in mouse sera was injecting differentiated ccdPA cells into the mammary fat pad. Antibody in supernatants from PER-ccdPAs bound to the surface of HER2-positive breast cancer cells similar to pertuzumab. Antibodies in mouse sera were delivered to HER2-positive breast cancer tumors and tumor necrosis was observed microscopically. One-time administration of combined TRA-ccdPAs and PER-ccdPAs produced antibody continuously in mouse sera, and anti-tumor effects were maintained for the duration of this study in xenograft models. Furthermore, combination therapy significantly suppressed tumor growth compared with a single administration. CONCLUSION: Ex vivo gene-transduced adipocytes might be useful for cell-based gene therapy. This system may be a platform for various antibody therapies.

Acalculous Ischemic Cholecystitis Caused by Spontaneous Celiac Artery Dissection.

We herein report a case of spontaneous isolated dissection of the celiac artery. A Japanese man in his 50s visited an emergency unit, complaining of sudden epigastralgia. Contrast-enhanced computed tomography indicated dissection of the celiac artery with patent false and true lumina, extending to the splenic and common hepatic arteries. On day 3 of hospitalization, the dissection progressed to the proper and right hepatic arteries. Progression of the dissection to the right hepatic artery provoked acalculous ischemic cholecystitis, and cholecystectomy followed. The resected gallbladder revealed extensive aseptic necrosis with little inflammatory reaction, and the gallbladder neck was spared from ischemia.

Acute psychiatric symptoms in a young woman with anti-N-methyl D-aspartate receptor encephalitis: A case of successful early diagnosis and therapeutic intervention.

This clinical image presents a report on the diagnosis and treatment of anti-NMDAR encephalitis, a rare disease. This report emphasizes the importance of a differential diagnosis for acute psychiatric symptoms. Accurate and timely diagnosis is critical for the selection and implementation of treatment and for optimal patient outcomes.

Down-regulation of FXYD3 is induced by transforming growth factor-ß signaling via ZEB1/δEF1 in human mammary epithelial cells.

FXYD3, a regulator of Na, K-ATPase, was identified as an mRNA overexpressed in murine breast cancers induced by neu oncogene, which had inactivated transforming growth factor (TGF)-ß signaling due to the defect of TGF-ß receptor I (TßRI) expression. To elucidate whether the expression of FXYD3 mRNA was regulated by TGF-ß signaling, we used a normal human mammary epithelial cell line, MCF-10A which responds to TGF-ß and tumor necrosis factor (TNF)-α, followed by induction of epithelial-to-mesenchymal transition (EMT). Here, we showed that FXYD3 at plasma membrane in epithelial state of MCF-10A cells was decreased by treatment of TGF-ß and TNF-α. The repression of FXYD3 mRNA induced by TGF-ß and TNF-α in MCF-10A cells was abolished by TßRI inhibitor or Smad3 inhibitor, but not by small interfering RNA (siRNA) for Smad2. In addition, expression level of FXYD3 mRNA was up-regulated by the silencing of ZEB1/δEF1 transcriptional repressor which was a down-stream target gene of TGF-ß and an inducer of EMT. On the other hand, expression level and cellular localization of E-cadherin and N-cadherin were not changed by siRNA for FXYD3 in MCF-10A and human breast cancer MCF-7 cells. These results suggest that FXYD3 is a target gene of TGF-ß signaling through ZEB1/δEF1, but is not directly involved in EMT.

Retrospective analysis of spontaneous recovery from neuromuscular blockade produced by empirical use of rocuronium.

PURPOSE: A train-of-four ratio (TOF ratio) of >0.9 should be the clinical cut-off to avoid residual paralysis. However, it is not rare to extubate patients without measurement of the TOF ratio, although the safe interval from the last administration of rocuronium assuring a TOF ratio of >0.9 has not been established in the daily clinical setting. In this study, to estimate the safe interval to avoid residual paralysis, we retrospectively selected patients in whom the TOF ratio was measured during remifentanil administration before extubation, and we studied the characteristics of recovery from the neuromuscular blockade produced by the empirical use of rocuronium. METHODS: Patients undergoing surgery under general anesthesia with sevoflurane and remifentanil were studied (n = 134). Rocuronium was administered at 0.7-1.0 mg/kg for tracheal intubation, and repeated bolus administration (10 mg) or continuous infusion (15-25 mg/h) was performed by the anesthesiologists in charge of the patient to maintain intraoperative paralysis. At the end of the surgery, the TOF ratio was measured, during remifentanil infusion and the contribution of clinical parameters to spontaneous recovery from the rocuronium-induced paralysis was studied by multivariate logistic regression analyses. RESULTS: Spontaneous recovery from rocuronium-induced paralysis within 2 h after the last administration of rocuronium varied among the patients. Multivariate logistic regression analyses showed that age (P = 0.002) and time elapsed from the last administration of rocuronium (P < 0.0001) significantly contributed to TOF recovery, and elderly patients demonstrated significantly slower recovery. CONCLUSION: Because of the large variation in the recovery from rocuronium-induced paralysis, TOF-based evaluation of residual paralysis is essential to determine the appropriate indication for reversal, especially for elderly patients.

Gastric perforation caused by secondary systemic amyloidosis.

Amyloid A amyloidosis secondary to chronic inflammation involves systemic organs and tissues, including the gastrointestinal tract. In the present case, massive amyloid deposit caused gastric perforation. IgM co-deposition in the glomeruli was another finding of note.

Initiation of malignancy by duodenal contents reflux and the role of ezrin in developing esophageal squamous cell carcinoma.

Gastroesophageal reflux has recently been implicated as a causative factor in upper aerodigestive tract carcinogenesis. Esophageal squamous cell carcinomas (ESCCs) have developed in duodenal-content reflux animals without any known carcinogen present. We established a cell line, designated ESCC-DR, from a thoracic metastatic tumor in a reflux animal. To gain insight into the genomic alterations associated with duodenal content reflux-induced carcinogenesis, we first performed comparative genomic hybridization using an Agilent rat 244K array in ESCC-DR and identified many chromosomal gains and losses. Of the many genes identified, we detected an interesting ezrin amplicon that has been recently reported in human ESCC. Ezrin, which cross-links the cytoskeleton and plasma membrane, is involved in the growth and metastatic potential of cancer cells. Overexpression of ezrin protein in ESCC-DR was confirmed by Western blotting. We also compared ezrin protein expression levels and patterns in hyperplastic, dysplastic, ESCC, and metastatic sites developed in two distinct reflux models using immunohistochemistry. Immunohistochemical staining of ezrin revealed overexpression in the nucleus, and the cytoplasm as well as plasma membrane of ESCC cells. Phosphorylated ERM (ezrin, radixin, moesin) was expressed at the leading edge, or invasive front, of larger metastatic sites. Taken together, duodenal reflux has a great potential for initiating malignancy, and thus likely plays a role in development of ESCC. Ezrin probably influences the growth and invasiveness of ESCC cells, and phosphorylation is only required in metastatic behavior of tumor cells at the leading edge and invasive front.

Severe COVID-19 Pneumonia in a 30-Year-Old Woman in the 36th Week of Pregnancy Treated with Postpartum Extracorporeal Membrane Oxygenation.

BACKGROUND There are few reports of coronavirus disease 2019 (COVID-19) in pregnant women. Although coagulation dysfunction was reported to affect the severity of COVID-19, the association between pregnancy, which is usually accompanied by changes in coagulation function, and the worsening of COVID-19 is unknown. We present a case of a 30-year-old woman in the 36th week of pregnancy who was diagnosed with severe COVID-19 pneumonia and required postpartum extracorporeal membrane oxygenation (ECMO) therapy. CASE REPORT A 30-year-old, 36-weeks pregnant woman presented to our hospital and was diagnosed with severe COVID-19 pneumonia soon after she had undergone a cesarean section. Her respiratory failure could not be managed by conventional therapeutic approaches. Therefore, ECMO was administered on day 7. Controlling coagulation function to maintain ECMO therapy was challenging. Nafamostat mesylate and cryoprecipitate were administered to treat the hypercoagulative status and severe hypofibrinogenemia, respectively. Since coagulopathy and her respiratory state improved, the ECMO therapy was terminated on day 15. CONCLUSIONS We report a case of severe COVID-19 pneumonia in a pregnant woman urgently treated with ECMO in the postpartum period. Thus, this case highlights the importance of close monitoring and appropriate medical care for pregnant women with severe COVID-19 pneumonia.

Transcriptome analysis of sevoflurane exposure effects at the different brain regions.

BACKGROUNDS: Sevoflurane is a most frequently used volatile anesthetics, but its molecular mechanisms of action remain unclear. We hypothesized that specific genes play regulatory roles in brain exposed to sevoflurane. Thus, we aimed to evaluate the effects of sevoflurane inhalation and identify potential regulatory genes by RNA-seq analysis. METHODS: Eight-week old mice were exposed to sevoflurane. RNA from medial prefrontal cortex, striatum, hypothalamus, and hippocampus were analysed using RNA-seq. Differently expressed genes were extracted and their gene ontology terms were analysed using Metascape. These our anesthetized mouse data and the transcriptome array data of the cerebral cortex of sleeping mice were compared. Finally, the activities of transcription factors were evaluated using a weighted parametric gene set analysis (wPGSA). JASPAR was used to confirm the existence of binding motifs in the upstream sequences of the differently expressed genes. RESULTS: The gene ontology term enrichment analysis result suggests that sevoflurane inhalation upregulated angiogenesis and downregulated neural differentiation in each region of brain. The comparison with the brains of sleeping mice showed that the gene expression changes were specific to anesthetized mice. Focusing on individual genes, sevoflurane induced Klf4 upregulation in all sampled parts of brain. wPGSA supported the function of KLF4 as a transcription factor, and KLF4-binding motifs were present in many regulatory regions of the differentially expressed genes. CONCLUSIONS: Klf4 was upregulated by sevoflurane inhalation in the mouse brain. The roles of KLF4 might be key to elucidating the mechanisms of sevoflurane induced functional modification in the brain.

Comparative analysis demonstrates cell type-specific conservation of SOX9 targets between mouse and chicken.

SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9's functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9's regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.

Effects of ezrin knockdown on the structure of gastric glandular epithelia.

Ezrin, an adaptor protein that cross-links plasma membrane-associated proteins with the actin cytoskeleton, is concentrated on apical surfaces of epithelial cells, especially in microvilli of the small intestine and stomach. In the stomach, ezrin is predominantly expressed on the apical canalicular membrane of parietal cells. Transgenic ezrin knockdown mice in which the expression level of ezrin was reduced to <7% compared with the wild-type suffered from achlorhydria because of impairment of membrane fusion between tubulovesicles and apical membranes. We observed, for the first time, hypergastrinemia and foveolar hyperplasia in the gastric fundic region of the knockdown mice. Dilation of fundic glands was observed, the percentage of parietal and chief cells was reduced, and that of mucous-secreting cells was increased. The parietal cells of knockdown mice contained dilated tubulovesicles and abnormal mitochondria, and subsets of these cells contained abnormal vacuoles and multilamellar structures. Therefore, lack of ezrin not only causes achlorhydria and hypergastrinemia but also changes the structure of gastric glands, with severe perturbation of the secretory membranes of parietal cells.