Store operated calcium (Ca2+) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2+ store depletion causes Orai1-composed Ca2+ channels on the plasma membrane (PM) to open, mediating a ...rise in cytosolic Ca2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2+ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile α motif (SAM) domains (EF–SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF–SAM domains have been determined at high Ca2+ concentrations; however, no direct structural view of the Ca2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF–SAM at low Ca2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF–SAM binds a single Ca2+ ion with pentagonal bipyramidal geometry and an ancillary α-helix formed by the N-terminal region acts as a brace to stabilize EF–SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary α-helix and the canonical EF-hand in low Ca2+. Remarkably, we also detected an α-helix (+Ca2+) to β-strand (−Ca2+) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation.
Display omitted
•Caenorhabditiselegans and human STIMs activate SOCE after sensing decreases in ER Ca2+.•A crystal structure revealed pentagonal bipyramidal coordination of a single Ca2+.•An ancillary α-helix formed in the variable N-terminal region stabilizes EF–SAM.•Ca2+ depletion causes unfolding and conformational exchange of the EF-hand.•The terminal α-helix transitions to β-strand after Ca2+ depletion of EF–SAM.
Background: In recent years, various biomarkers of ulcerative colitis (UC) have emerged; however, few studies have simultaneously examined the utility of multiple biomarkers for monitoring disease ...activity. Additionally, serum leucine-rich alpha-2 glycoprotein (LRG), a new biomarker, may show a blunt response to anti-TNF antibody therapy. This prospective study explored effective biomarkers that could monitor disease activity changes in patients with UC. In addition, we examined the effect of anti-TNF antibody therapy on changes in LRG. Methods: Blood and stool samples were collected twice from patients with UC: at baseline and at least 8 weeks later. Changes in serum LRG, interleukin (IL)-6, prealbumin (pre-Alb), high-sensitivity C-reactive protein (hs-CRP), CRP, and fecal calprotectin (FC) were measured and correlated with changes in disease activity. The relationship between anti-TNF antibody therapy and LRG levels was also examined in patients with the same disease activity. Results: Forty-eight patients with UC (96 samples) were analyzed. ΔLRG and ΔIL-6 correlated strongly with the change in the partial Mayo (pMayo) score between the two time points (ΔpMayo) (r = 0.686, 0.635, respectively). In contrast, FC and IL-6 were particularly accurate predictors of clinical remission, and their area under the curves (AUCs) were significantly higher than that of CRP (AUC: 0.81, 0.76 vs. 0.50; p = 0.001, 0.005). No association was found between the administration of anti-TNF antibody preparations and the LRG values. Conclusions: Correlations were found between changes in UC disease activity and LRG, IL-6, pre-Alb, hs-CRP, CRP, and FC. LRG reflects disease activity during anti-TNF antibody therapy.
Cancer detection is a global research focus, and novel, rapid, and label-free techniques are being developed for routine clinical practice. This has led to the development of new tools and techniques ...from the bench side to routine clinical practice. In this study, we present a method that uses Raman spectroscopy (RS) to detect cancer in unstained formalin-fixed, resected specimens of the esophagus and stomach. Our method can record a clear Raman-scattered light spectrum in these specimens, confirming that the Raman-scattered light spectrum changes because of the histological differences in the mucosal tissue.
To evaluate the use of Raman-scattered light spectrum for detecting endoscop-ically resected specimens of esophageal squamous cell carcinoma (SCC) and gastric adenocarcinoma (AC).
We created a Raman device that is suitable for observing living tissues, and attempted to acquire Raman-scattered light spectra in endoscopically resected specimens of six esophageal tissues and 12 gastric tissues. We evaluated formalin-fixed tissues using this technique and captured shifts at multiple locations based on feasibility, ranging from six to 19 locations 200 microns apart in the vertical and horizontal directions. Furthermore, a correlation between the obtained Raman scattered light spectra and histopathological diagnosis was performed.
We successfully obtained Raman scattered light spectra from all six esophageal and 12 gastric specimens. After data capture, the tissue specimens were sent for histopathological analysis for further processing because RS is a label-free methodology that does not cause tissue destruction or alterations. Based on data analysis of molecular-level substrates, we established cut-off values for the diagnosis of esophageal SCC and gastric AC. By analyzing specific Raman shifts, we developed an algorithm to identify the range of esophageal SCC and gastric AC with an accuracy close to that of histopathological diagnoses.
Our technique provides qualitative information for real-time morphological diagnosis. However, further
evaluations require an excitation light source with low human toxicity and large amounts of data for validation.
Background: Tacrolimus (TAC) effectively induces remission in refractory ulcerative colitis (UC). However, TAC therapy usually lasts for 3 months. Although azathioprine (AZA) is often used in ...maintenance therapy, the relapse rate remains high. Herein, we evaluated the efficacy of adalimumab (ADA) for remission maintenance in patients with UC after induction therapy with TAC. Methods: We prospectively enrolled patients with moderate-to-severe UC who achieved clinical remission after 3 months of TAC therapy with endoscopic non-mucosal healing (Cohort A). After TAC discontinuation, the remission maintenance rate up to 1 year after starting ADA therapy was examined. We retrospectively enrolled patients with UC treated with TAC (Cohort B). Among patients in clinical remission after TAC treatment for 3 months, those who received AZA as remission maintenance therapy after TAC discontinuation constituted the AZA group. Patients in Cohort A who received ADA and AZA as remission maintenance therapy after TAC discontinuation constituted the ADA + AZA group. We compared the remission maintenance rates in the AZA and ADA + AZA groups for up to 5 years after TAC discontinuation. Results: In Cohort A, of the 46 patients with UC treated with TAC, 17 were eligible for analysis after receiving ADA as remission maintenance therapy. A notable 88.2% (15/17) were still in remission 1 year after starting ADA. The ADA + AZA group (n = 16) exhibited a significantly higher relapse-free rate than the AZA group (n = 26) (p < 0.05; log-rank test). Conclusion: switching to ADA for remission maintenance in patients with refractory UC who achieved clinical remission with TAC is clinically useful.
Vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Tumor cells are exposed to higher oxidative stress compared to normal cells. Numerous reports have demonstrated that ...the intracellular redox (oxidation/reduction) state is closely associated with the pattern of VEGF expression. Electrolyzed reduced water (ERW) produced near the cathode during the electrolysis of water scavenged intracellular H2O2 and decreased the release of H2O2 from a human lung adenocarcinoma cell line, A549, and down-regulated both VEGF transcription and protein secretion in a time-dependent manner. To investigate the signal transduction pathway involved in regulating VEGF expression, mitogen-activated kinase (MAPK) specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (ERK1/2 inhibitor) and JNKi (c-Jun N-terminal protein kinase inhibitor) were applied. The results showed that only PD98059 blocks VEGF expression, suggesting an important role for ERK1/2 in regulating VEGF expression in A549 cells. As well, ERW inhibited the activation of extracellular signal-regulated kinase (ERK) in a time-dependent manner. Co-culture experiments to analyze in vitro tubule formation assay revealed that A549 cell-derived conditioned medium significantly stimulated the formation of vascular tubules in all analyzed parameters; tubule total area, tubule junction, number of tubules, and total tubule length. ERW counteracted the effect of A549 cell-conditioned medium and decreased total tube length (p<0.01). The present study demonstrated that ERW down-regulated VEGF gene transcription and protein secretion through inactivation of ERK.
Genetic testing for inherited arrhythmias and discriminating pathogenic or benign variants from variants of unknown significance (VUS) is essential for gene-based medicine. KCNQ1 is a causative gene ...of type 1 long QT syndrome (LQTS), and approximately 30% of the variants found in type 1 LQTS are classified as VUS. We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. We generated homozygous kcnq1 deletion zebrafish (kcnq1del/del) using the CRISPR/Cas9 and expressed human Kv7.1/MinK channels in kcnq1del/del embryos. We dissected the hearts from the thorax at 48 h post-fertilization and measured the transmembrane potential of the ventricle in the zebrafish heart. Action potential duration was calculated as the time interval between peak maximum upstroke velocity and 90% repolarization (APD90). The APD90 of kcnq1del/del embryos was 280 ± 47 ms, which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (168 ± 26 ms, P < 0.01 vs. kcnq1del/del). A study of two pathogenic variants (S277L and T587M) and one VUS (R451Q) associated with clinically definite LQTS showed that the APD90 of kcnq1del/del embryos with these mutant Kv7.1/MinK channels was significantly longer than that of Kv7.1 WT/MinK channels. Given the functional results of the zebrafish model, R451Q could be reevaluated physiologically from VUS to likely pathogenic. In conclusion, functional analysis using in vivo zebrafish cardiac arrhythmia model can be useful for determining the pathogenicity of loss-of-function variants in patients with LQTS.
Display omitted
•Homozygous mutant zebrafish with deletion of the kcnq1 gene (kcnq1del/del) was created.•The ventricular APD90 of kcnq1del/del embryos was significantly longer.•It was shortened by co-injecting human KCNQ1 cRNA with human KCNE1 cRNA.•Injecting cRNA of pathogenic KCNQ1 variants did not rescue the prolonged APD90.•Injecting cRNA of KCNQ1 VUS, R451Q identified from a patient with LQTS did not rescue the prolonged APD90 either.
Abstract Background and study aims Mucosal defect closure after colorectal endoscopic submucosal dissection (ESD) has the potential to reduce the occurrence of delayed adverse events (AEs) such as ...bleeding and perforation. This study aimed to assess the feasibility and effectiveness of the Loop9 method for closing mucosal defects following colorectal ESD. Patients and methods A retrospective single-center study was conducted using prospectively collected data from May 2020 to March 2023. Loop9 was deployed through a single instrument channel and anchored with clips at the defect site. Closure was accomplished by tightening the loop and deploying additional conventional clips as needed for complete closure. The primary outcome was complete closure rate, with secondary outcomes including the sustained closure rate at 4 to 5 days post-ESD, closed defect size, closure time, number of additional clips, and incidence of delayed AEs. Results This study included 118 cases. Complete closure was achieved in 96.6% of cases (114/118) with a sustained closure rate of 93.9% (107/114). The median size of the closed mucosal defects was 30 mm (interquartile range IQR: 25–38, range: 15–74). The median closure time was 14 minutes (IQR: 11.25–17), and the median number of additional clips deployed was six (IQR: 4–7). Stenosis requiring balloon dilatation was observed in one patient; however, there were no instances of post-ESD bleeding or delayed perforation. Conclusions The Loop9 method proved feasible and effective for closing mucosal defects following colorectal ESD, achieving high rates of complete and sustained closure.
To examine the effects of 4-methylthio-3-butenyl isothiocyanate on esophageal carcinogenesis, male 6-week-old F344 rats were subcutaneously injected with 0.5 mg/kg body weight ...N-nitrosomethylbenzylamine three times per week for 5 weeks and fed a diet supplemented with 80 ppm 4-methylthio-3-butenyl isothiocyanate, equivalent to 6.05 mg/kg body weight/day for the initiation stage, 4.03 mg/kg body weight/day for the promotion stage, or 4.79 mg/kg body weight/day for all stages. Although the incidence of lesions was not affected by 4-methylthio-3-butenyl isothiocyanate treatment, the multiplicity of squamous cell papilloma in the esophagus was significantly decreased in rats in the 4-methylthio-3-butenyl isothiocyanate initiation stage group (1.13 ± 0.74), 4-methylthio-3-butenyl isothiocyanate promotion stage group (1.47 ± 0.99), and 4-methylthio-3-butenyl isothiocyanate all stage group (1.47 ± 1.13) as compared with rats treated with N-nitrosomethylbenzylamine alone (3.00 ± 1.46). Immunohistochemical analysis revealed that 4-methylthio-3-butenyl isothiocyanate induced apoptosis, suppressed cell proliferation, and increased p21 expression when administered in the promotion phase. These modifying effects were not observed in the rats treated with 4-methylthio-3-butenyl isothiocyanate alone. Our results indicated that 4-methylthio-3-butenyl isothiocyanate may exert chemopreventive effects against N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats.
PDF
