Establishment of Star-edited Y1 cells as a novel in vitro functional assay for STAR.

Genetic variants involving steroidogenic acute regulatory protein cause lipoid congenital adrenal hyperplasia, which is characterized by impaired steroidogenesis in the adrenal glands and gonads. Functional assessment of variant STAR proteins is necessary for accurate genetic diagnosis. Ideally, steroidogenic cells should be used to assess the functionality of STAR proteins, but the presence of endogenous STARs in steroidogenic cells precludes such a method. Here, we generated Star-edited cells from steroidogenic Y1 mouse adrenocortical tumor cells by genome editing. Star-edited Y1 cells exhibited very low but measurable cAMP-dependent pregnenolone production. Furthermore, stimulation of the cAMP pathway for two weeks resulted in the formation of lipid droplets in the cytoplasm of Star-edited Y1 cells, which resembled the histology of the adrenal glands of patients with lipoid congenital adrenal hyperplasia. The steroidogenic defect of Star-edited Y1 cells can be restored by transient over-expression of mouse Star. We found that human STAR can also restore defective steroidogenesis of Star-edited Y1 cells, and were able to construct a novel in vitro system to evaluate human STAR variants. Collectively, we established Star-edited Y1 cells that retain the steroidogenic pathway downstream the Star protein. Star-edited Y1 cells recapitulate the functional and morphological changes of lipoid congenital adrenal hyperplasia, and can be used to evaluate the functionality of human STAR variants.

A Novel Characteristic Gastric Mucus Named "Web-like Mucus" Potentially Induced by Vonoprazan.

Background: In the absence of Helicobacter pylori (HP) infection, a characteristic gastric mucus adhesion may appear during the use of vonoprazan. We named this novel characteristic mucus "web-like mucus" (WLM). This study aimed to determine the incidence and risk factors for WLM. Methods: Between January 2017 and January 2022, 5665 patients were enrolled in this study. The patients were divided into a proton-pump inhibitor (PPI)-prescribed group (n = 2000), a vonoprazan-prescribed group (n = 268), and a no-PPI/vonoprazan-prescribed (n = 3397) group, and the presence of WLM was examined. After excluding four patients with autoimmune gastritis, the remaining 264 patients in the vonoprazan group were divided into WLM and non-WLM groups, and their clinical features were analyzed. Results: A total of 55 (21%) patients had WLM, all in the vonoprazan-prescribed group. There were no significant differences in factors such as, sex, age, chronic kidney disease, diabetes mellitus, HP eradication history, smoking, or alcohol consumption between the WLM and non-WLM groups. The median duration from the start of vonoprazan administration to the endoscopic detection of WLM was 2 (1-24) months. Conclusions: WLM appears to be a characteristic feature in patients treated with vonoprazan.

Activation of STING in pancreatic cancer-associated fibroblasts exerts an antitumor effect by enhancing tumor immunity.

Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate; therefore, the development of effective treatments is a priority. The stimulator of interferon genes (STING) pathway enhances tumor immunity by inducing the production of type 1 interferon (IFN) and proinflammatory cytokines and chemokines and promoting the infiltration of cytotoxic T cells. To assess the function of STING on pancreatic tumorigenesis, Ptf1aER-Cre/+ LSL-KrasG12D/+ p53loxP/loxP mice (KPC mice) and Ptf1aER-Cre/+ LSL-KrasG12D/+ p53loxP/loxP/STING-/- mice (KPCS mice) were generated. However, STING deletion did not affect pancreatic tumorigenesis in mice. Because STING is expressed not only in immune cells but also in cancer-associated fibroblasts (CAFs), we evaluated the STING function in PDAC CAFs. A mouse STING agonist 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA) was administered to KPC mice and CAFs from KPC mice and the resulting immune response was evaluated. DMXAA activated STING in PDAC CAFs in KPC mice, promoting cytotoxic T cell infiltration by secreting proinflammatory cytokines and enhancing tumor immunity. We next generated STING-deficient PDAC cells and subcutaneous tumors in which STING was expressed only in CAFs by performing bone marrow transplantation and assessed the antitumor effect of STING-activated CAFs. The administration of DMXAA to subcutaneous tumors expressing STING only in CAFs sustained the antitumor effect of DMXAA. About half of human PDACs lacked STING expression in the cancer stroma, suggesting that STING activation in PDAC CAFs exerts an antitumor effect, and STING agonists can be more effective in tumors with high than in those with low STING expression in the stroma.

Identification of amino acids in transmembrane domains of mutated cytokine receptor-like factor 2 and interleukin-7 receptor α required for constitutive signal transduction.

Cytokine receptor-like factor 2 (CRLF2) and interleukin-7 receptor α (IL-7Rα) form a receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation consisting of the substitution of five amino acids (SLLLL) in the transmembrane domain of CRLF2 with three amino acids, including glutamic acid, isoleucine, and methionine (insEIM), which has been identified in acute lymphocytic leukemia, causes the TSLP-independent dimerization with IL-7Rα and activation. However, the dimerization mechanism remains unclear. In this study, we examined the involvement of the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα in TSLP-independent activation. HEK293 cells were transfected with vectors encoding CRLF2 and IL-7Rα, or their mutants, in which the amino acid of the transmembrane domain was replaced with alanine. STAT5 phosphorylation was detected using western blotting, and receptor dimerization was analyzed using the NanoBiT assay. The substitution of glutamic acid within the insEIM mutation for alanine failed to cause the STAT5 phosphorylation in the absence of TSLP. Moreover, the alanine substation of the specific leucine residues in the transmembrane domains of both CRLF2 and IL-7Rα abrogated the TSLP-independent signal transduction and dimerization. The mutation of IL-7Rα W264 partially reduced the phosphorylation of STAT5 without affecting receptor dimerization. These results suggest that the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα play at least three possible functions: interaction through hydrogen bonds, hydrophobic interaction, and signal transduction. Our findings contribute to a better understanding of the function of the transmembrane domains of cytokine receptors in their dimerization and signal transduction.

Effects of Short-Term Lenvatinib Administration Prior to Transarterial Chemoembolization for Hepatocellular Carcinoma.

AIM: Transarterial chemoembolization (TACE) combined with lenvatinib, employing a 4-day lenvatinib administration followed by TACE without an interval (short-term LEN-TACE), was performed for hepatocellular carcinoma (HCC). The aim was to assess tumor hemodynamics following the 4-day lenvatinib and to evaluate the treatment outcomes after the short-term LEN-TACE. METHODS: 25 unresectable HCC patients received this combined therapy. Lenvatinib (4-12 mg) was administrated for 4 days prior to TACE. Perfusion CT scans were obtained before and after the lenvatinib administration. Either cTACE (76%) or DEB-TACE (24%) were performed. RESULTS: intra-tumor blood flow significantly decreased after the 4-day lenvatinib (p < 0.05). The TACE procedure was successful with no severe adverse events in all patients. The overall complete response (CR) rate was 75% (cTACE 84%, DEB-TACE 40%). The lipiodol-washout ratio between 1 week and 4 months after cTACE correlated with the arterial flow reduction ratio by lenvatinib prior to TACE (r = -0.55). The 12-month progression-free survival (PFS) rate was 75.0%. CONCLUSIONS: The short-term LEN-TACE is feasible and safe, demonstrating promising outcomes with a high CR ratio, contributing to lipiodol retention in the tumor after cTACE, and extended PFS. To confirm the advantages of this treatment protocol, a prospective clinical trial is mandatory.

RMRP-related short stature: A report of six additional Japanese individuals with cartilage hair hypoplasia and literature review.

Biallelic pathogenic variants in RMRP, the gene encoding the RNA component of RNase mitochondrial RNA processing enzyme complex, have been reported in individuals with cartilage hair hypoplasia (CHH). CHH is prevalent in Finnish and Amish populations due to a founder pathogenic variant, n.71A > G. Based on the manifestations in the Finnish and Amish individuals, the hallmarks of CHH are prenatal-onset growth failure, metaphyseal dysplasia, hair hypoplasia, immunodeficiency, and other extraskeletal manifestations. Herein, we report six Japanese individuals with CHH from four families. All probands presented with moderate short stature with mild metaphyseal dysplasia or brachydactyly. One of them had hair hypoplasia and the other immunodeficiency. By contrast, the affected siblings of two families showed only mild short stature. We also reviewed all previously reported 13 Japanese individuals. No n.71A > G allele was detected. The proportions of Japanese versus Finnish individuals were 0% versus 70% for birth length < -2.0 SD, 84% versus 100% for metaphyseal dysplasia and 26% versus 88% for hair hypoplasia. Milder manifestations in the Japanese individuals may be related to the difference of genotypes. The mildest form of CHH phenotypes is mild short stature without overt skeletal alteration or extraskeletal manifestation and can be termed "RMRP-related short stature".

Percutaneous Trans-jejunum Pancreatojejunostomy Reconstruction for Intractable Pancreatic Fistula after Pancreatoduodenectomy.

We present an interventional radiology technique for percutaneous trans-jejunal pancreatojejunostomy reconstruction for intractable pancreatic fistula. A 70-year-old man with pancreatic cancer who had undergone pancreatoduodenectomy underwent percutaneous drainage for leakage from the anastomosis of the pancreatic duct to the jejunum. The leakage continued and the hole at the anastomosis site in the jejunum closed completely after 5 months. We performed percutaneous jejunostomy; the previously placed drainage catheter was then replaced with a balloon catheter, which was punctured by a 19-gauge needle from inside the jejunum through the percutaneous jejunostomy tube. The seeking catheter was inserted into the pancreatic duct. Finally, a side-holed 6-Fr straight catheter was successfully placed in the pancreatic duct through the percutaneous jejunostomy route.

Long-term voice evaluation after arytenoid adduction surgery in patients with unilateral vocal fold paralysis.

PURPOSE: Laryngeal framework surgery, including medialization laryngoplasty and arytenoid adduction (AA), is expected to have a lasting or permanent effect in patients with unilateral vocal fold paralysis (UVFP); however, there are few reports about the long-term outcomes of AA. This study aimed to evaluate the long-term postoperative effects of AA surgery and examine its stability and reliability. METHODS: This study collected the voice handicap index (VHI) questionnaire from patients with UVFP who underwent AA more than 2 years previously. The VHI values preoperatively and 3 months postoperatively (early postoperative evaluation) were retrospectively calculated, and VHI values more than 2 years after surgery (late postoperative evaluation) were collected by mailing a sheet to the patients and asking to fill and return it. Possible influenced subscales such as age, sex, causes of UVFP, affected side, and surgeons were also analyzed. RESULTS: A total of 77 patients with UVFP who underwent AA had significantly lower early and late postoperative evaluations than preoperative evaluations. In 38 patients with no missing values, there were no significant differences between early and late postoperative evaluations, measured at a median of approximately 5 years. There were also no significant differences between early and late postoperative evaluations in any of the subscale groups. CONCLUSION: Patients with UVFP who underwent AA surgery achieved stable voice improvement in the long term after surgery.

Characterizing interaction between the juxtamembrane region of the single transmembrane protein and membrane using chemically synthesized peptides.

In membrane proteins, a transmembrane region and a juxtamembrane region play important roles in its function. Here, we present a protocol for characterizing membrane protein dynamics between the juxtamembrane region of the single transmembrane protein and acidic membrane. We describe steps for solid-phase peptide synthesis, peptide purification, and labeling. We then detail reconstitution of the transmembrane peptide into lipid bilayers and its evaluation and structural analysis. For complete details on the use and execution of this protocol, please refer to Prasada Rao et al.1.

Transarterial Chemoembolization with Irinotecan-loaded Beads Followed by Arterial Infusion of 5-Fluorouracil for Metastatic Liver Tumors Refractory to Standard Systemic Chemotherapy.

We report two cases of liver metastases from colorectal and anal cancers after the failure of systemic chemotherapies that were successfully treated with a combination therapy of transarterial chemoembolization using irinotecan-loaded drug-eluting beads and hepatic arterial infusion chemotherapy. In both cases, hepatic arterial infusion chemotherapy was performed as maintenance therapy after irinotecan-loaded drug-eluting beads. Irinotecan at a dose of 120 mg was loaded on drug delivery beads for irinotecan-loaded drug-eluting bead-transarterial chemoembolization. A weekly high-dose 5-fluorouracil regimen (1000 mg/m2/5 h) was used for hepatic arterial infusion chemotherapy. The liver metastases shrank remarkably in both cases, and progression-free survivals of 13 and 9 months, respectively, were obtained without any severe adverse events.

Predictive Factors of Complete Response to Transarterial Chemoembolization in Intermediate Stage Hepatocellular Carcinoma beyond Up-To-7 Criteria.

AIM: To clarify the prognosis and identify predictors for obtaining a complete response (CR) by transarterial chemoembolization (TACE) in intermediate stage HCC beyond up-to-7 criteria. METHODS: Of the 120 patients with intermediate stage HCC who were treated by TACE as the initial treatment from February 2007 to January 2016, 72 finally matched the following inclusion criteria: beyond up-to-7 criteria; Child-Pugh score under 7; and no combined therapy within 4 weeks after the initial TACE. The CR rate and overall survival (OS) were evaluated. Logistic regression analysis was performed to identify predictors of CR. The deterioration of liver function after TACE was also evaluated. RESULTS: The CR rate was 56.9%, and the overall median survival time (MST) was 37.7 months. The MST was 38.7 months in the CR group and 28.0 months in the non-CR group (p = 0.018). HCC within up-to-11 criteria was the only predictor of CR. The CR rate and MST were 70.7% and 37.7 months, respectively, in patients with HCC within up-to-11 criteria and 38.7% and 32.7 months, respectively, in the patients beyond up-to-11 criteria. Deterioration of the Child-Pugh score after the initial TACE and the 2nd TACE occurred in 24.2% and 12.0%, respectively, and deterioration of the modified albumin-bilirubin (mALBI) grade occurred in 17.6% and 7.4%, respectively. CONCLUSION: TACE can achieve high CR rates with prolonged overall survival for intermediate stage HCC beyond up-to-7 criteria. The predictor of CR was within up-to-11 criteria. Deterioration of liver function was not severe, but requires caution. Multidisciplinary approach as additional treatment after TACE is important.

Vascular Normalization Caused by Short-Term Lenvatinib Could Enhance Transarterial Chemoembolization in Hepatocellular Carcinoma.

We describe the clinical effects of short-term lenvatinib administration prior to conventional transarterial chemoembolization (cTACE) on tumor vasculature. Two patients with unresectable hepatocellular carcinoma underwent high-resolution digital subtraction angiography (DSA) and perfusion four-dimensional computed tomography during hepatic arteriography (4D-CTHA) before and after administration of lenvatinib treatment. The doses and periods of lenvatinib administration were, respectively, 12 mg/day for 7 days and 8 mg/day for 4 days. In both cases, high-resolution DSA revealed a decrease in dilatation and tortuosity of the tumor vessels. Furthermore, the tumor staining became more refined, and newly formed tiny tumor vessels were observed. Perfusion 4D-CTHA revealed a decrease in arterial blood flow to the tumor by 28.6% (from 487.9 to 139.5 mL/min/100 mg) and 42.5% (from 288.2 to 122.6 mL/min/100 mg) in the two cases, respectively. The cTACE procedure resulted in good lipiodol accumulation and complete response. Patients have remained recurrence-free for 12 and 11 months after the cTACE procedure, respectively. The administration of short-term lenvatinib in these two cases resulted in the normalization of tumor vessels, which likely led to improved lipiodol accumulation and a favorable antitumor effect.

Transcriptional Mechanism of the Mouse ß4-Galactosyltransferase 6 Gene in Mouse Neuroblastoma Cell Line Neuro-2a.

Lactosylceramide (Lac-Cer) constitutes the backbone structure of various gangliosides whose abnormal expression is associated with malignancy of neuroblastoma. The understanding of the regulatory mechanism of Lac-Cer contributes to the development of neuroblastoma therapy. In this study, the transcriptional mechanism of mouse ß4-galactosyltransferase (ß4GalT) 6, which is one of Lac-Cer synthase, was analyzed using mouse neuroblastoma cell line Neuro-2a. The -226 to -13 region relative to the most downstream transcriptional start site was determined to be the promoter region by luciferase assay using the 5'-deletion constructs. The mutation into the activating protein (AP) 4-binding site -110/-101 drastically decreased the promoter activity, indicating that this site is mainly implicated in the transcription. Furthermore, the mutation into the GATA-binding site -210/-201 or another AP4-binding site -202/-193 partially decreased the promoter activity. The study suggests that the mouse ß4GalT6 gene is transcriptionally regulated by AP4 in cooperation with GATA family transcription factor in neuroblastoma.

Quantitative Analysis of Signal Heterogeneity in the Hepatobiliary Phase of Pretreatment Gadoxetic Acid-Enhanced MRI as a Prognostic Imaging Biomarker in Transarterial Chemoembolization for Intermediate-Stage Hepatocellular Carcinoma.

BACKGROUND: In the era of local and systemic therapies for intermediate-stage hepatocellular carcinoma (HCC), personalized therapy has become available. The aim of our study was to evaluate the usefulness of quantitative analysis of pretreatment gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) to predict prognosis following transarterial chemoembolization (TACE). METHODS: This retrospective study included patients with treatment-naïve intermediate-stage HCC who underwent EOB-MRI before the initial TACE and were treated by initial TACE between February 2007 and January 2016. Signal heterogeneity in the hepatobiliary phase (HBP) of EOB-MRI was quantitatively evaluated by the coefficient of variation (CV). The cutoff CV value was determined using the Classification and Regression Tree algorithm. RESULTS: A total of 64 patients were enrolled. In multivariate analysis, High CV (≥0.16) was significantly associated with poor prognosis (p = 0.038). In a subgroup analysis of patients within up-to-7 criteria, MST was significantly shorter in the High CV group than in the Low CV group (37.7 vs. 82.9 months, p = 0.024). In patients beyond up-to-7 criteria, MST was 18.0 and 38.3 months in the High CV and Low CV groups, respectively (p = 0.182). In both groups scanned at 1.5 T or 3.0 T, High CV was significantly associated with poor prognosis (p = 0.001 and 0.003, respectively). CONCLUSION: CV of the tumor in the HBP of EOB-MRI is a valuable prognostic factor of TACE.