Collagen-derived dipeptide prolyl-hydroxyproline cooperates with Foxg1 to activate the PGC-1α promoter and induce brown adipocyte-like phenotype in rosiglitazone-treated C3H10T1/2 cells.

Background: The global obesity epidemic is a significant public health issue, often leading to metabolic disorders such as diabetes and cardiovascular diseases. Collagen peptides (CP) and their bioactive component, Prolyl-hydroxyproline (Pro-Hyp), have shown potential in reducing adipocyte size, with unclear mechanisms concerning brown adipocyte differentiation. Methods: We investigated the effects of Pro-Hyp on the differentiation of brown adipocytes in C3H10T1/2 mesenchymal stem cells, focusing on its impact on adipocyte size, gene expression related to brown fat function, and mitochondrial activity. Results: Pro-Hyp treatment decreased adipocyte size and upregulated brown fat-specific genes, including C/EBPα, PGC-1α, and UCP-1. Remarkably, it did not alter PPARγ expression. Pro-Hyp also elevated mitochondrial activity, suggesting enhanced brown adipocyte functionality. A Pro-Hyp responsive element was identified in the PGC-1α gene promoter, which facilitated the binding of the Foxg1 transcription factor, indicating a novel regulatory mechanism. Conclusion: Pro-Hyp promotes brown adipocyte differentiation, potentially offering a therapeutic strategy for obesity management. This study provides a molecular basis for the anti-obesity effects of CP, although further in vivo studies are needed to confirm these findings and to investigate the potential impact on beige adipocyte differentiation.

Medium-chain triglyceride-specific appetite is regulated by the ß-oxidation of medium-chain fatty acids in the liver.

Most studies on fat appetite have focused on long-chain triglycerides (LCTs) due to their obesogenic properties. Medium-chain triglycerides (MCTs), conversely, exhibit antiobesogenic effects; however, the regulation of MCT intake remains elusive. Here, we demonstrate that mice can distinguish between MCTs and LCTs, and the specific appetite for MCTs is governed by hepatic ß-oxidation. We generated liver-specific medium-chain acyl-CoA dehydrogenase (MCAD)-deficient (MCADL-/-) mice and analyzed their preference for MCT and LCT solutions using glyceryl trioctanoate (C8-TG), glyceryl tridecanoate (C10-TG), corn oil, and lard oil in two-bottle choice tests conducted over 8 days. In addition, we used lick microstructure analyses to evaluate the palatability and appetite for MCT and LCT solutions. Finally, we measured the expression levels of genes associated with fat ingestion (Galanin, Qrfp, and Nmu) in the hypothalamus 2 h after oral gavage of fat. Compared with control mice, MCADL-/- mice exhibited a significantly reduced preference for MCT solutions, with no alteration in the preference for LCTs. Lick analysis revealed that MCADL-/- mice displayed a significantly decreased appetite for MCT solutions only while the palatability of both MCT and LCT solutions remained unaffected. Hypothalamic Galanin expression in control mice was elevated by oral gavage of C8-TG but not by LCTs, and this response was abrogated in MCADL-/- mice. In summary, our data suggest that hepatic ß-oxidation is required for MCT-specific appetite but not for LCT-specific appetite. The induction of hypothalamic galanin upon MCT ingestion, dependent on hepatic ß-oxidation, could be involved in the regulation of MCT-specific appetite.NEW & NOTEWORTHY Whether and how medium-chain triglyceride (MCT) intake is regulated remains unknown. Here, we showed that mice can discriminate between MCTs and LCTs. Hepatic ß-oxidation participates in MCT-specific appetite, and hypothalamic galanin may be one of the factors that regulate MCT intake. Because of the antiobesity effects of MCTs, studying MCT-specific appetite may help combat obesity by promoting the intake of MCTs instead of LCTs.

Clinical course of small gastric subepithelial lesion less than 20 mm diagnosed by endoscopic ultrasound-guided fine-needle aspiration.

BACKGROUND AND AIM: Gastrointestinal stromal tumors (GISTs) are treated as malignant gastric subepithelial lesions (SELs), and resection is recommended. However, small gastric SELs < 20 mm with no malignant features are monitored without histopathological examination, and the frequency of malignancy is unknown. This study aimed to clarify the clinicopathological findings and clinical course of gastric SELs < 20 mm measured by endoscopic ultrasound (EUS). METHODS: This retrospective cohort study included consecutive patients with small gastric SELs < 20 mm diagnosed using EUS at a tertiary referral center between 2009 and 2021. The clinical course after diagnosis using EUS-guided fine-needle aspiration (EUS-FNA) was reviewed. RESULTS: Among 333 patients with small gastric SELs, 104 patients with 105 lesions underwent EUS-FNA. The pathological diagnosis was confirmed in 87 patients. GISTs were the most common pathology (47%). Among the 87 patients, 43 underwent therapeutic interventions, including tumor resection and chemotherapy. In groups of tumor resection, the pathological tumor size on the resected specimen was significantly larger than the size measured by EUS (19.5 mm vs 15.0 mm, P < 0.001), and 37% of resected SELs were 20 mm or over. No recurrence was observed after tumor resection during a mean follow-up period of 40 months. CONCLUSIONS: Approximately 40% of small gastric SELs were malignant tumors, such as GIST, with most of them requiring treatment. Additionally, considering that the EUS measurement is 5 mm smaller than the pathological tumor diameter, further examinations, such as systematic EUS-FNA, may be required for SEL, including those smaller than 20 mm.

Morphometric Analysis of the Eye by Magnetic Resonance Imaging in MGST2-Gene-Deficient Mice.

Strabismus, a neuro-ophthalmological condition characterized by misalignment of the eyes, is a common ophthalmic disorder affecting both children and adults. In our previous study, we identified the microsomal glutathione S-transferase 2 (MGST2) gene as one of the potential candidates for comitant strabismus susceptibility in a Japanese population. The MGST2 gene belongs to the membrane-associated protein involved in the generation of pro-inflammatory mediators, and it is also found in the protection against oxidative stress by decreasing the reactivity of oxidized lipids. To look for the roles of the MGST2 gene in the development, eye alignment, and overall morphology of the eye as the possible background of strabismus, MGST2 gene knockout (KO) mice were generated by CRISPR/Cas9-mediated gene editing with guide RNAs targeting the MGST2 exon 2. The ocular morphology of the KO mice was analyzed through high-resolution images obtained by a magnetic resonance imaging (MRI) machine for small animals. The morphometric analyses showed that the height, width, and volume of the eyeballs in MGST2 KO homozygous mice were significantly greater than those of wild-type mice, indicating that the eyes of MGST2 KO homozygous mice were significantly enlarged. There were no significant differences in the axis length and axis angle. These morphological changes may potentially contribute to the development of a subgroup of strabismus.

Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization.

The production of cell-type- and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)-mediated cell-type- and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier-penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target-flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.

Lobular distribution of enhanced expression levels of heat shock proteins using in-situ hybridization in the mouse liver treated with a single administration of CCl4.

This study was conducted to visualize the lobular distribution of enhanced mRNA expression levels of heat shock proteins (HSPs) in liver samples from carbon tetra chloride (CCl4)-treated mice using in-situ hybridization (ISH). Male BALB/c mice given a single oral administration of CCl4 were euthanized 6 hours or 1 day after the administration (6 h or 1 day). Paraffin-embedded liver samples were obtained, ISH for HSPs was conducted, as well as hematoxylin-eosin staining and immunohistochemistry (IHC). At 6 h, centrilobular hepatocellular vacuolization was observed, and increased signals for Hspa1a, Hspa1b, and Grp78, which are HSPs, were noted in the centrilobular area using ISH. At 1 day, zonal hepatocellular necrosis was observed in the centrilobular area, but mRNA signal increases for HSPs were no longer observed there. Some discrepancies between ISH and IHC for HSPs were observed, and they might be partly caused by post-transcriptional gene regulation, including the ribosome quality control mechanisms. It is known that CCl4 damages centrilobular hepatocytes through metabolization by cytochrome P450, mainly located in the centrilobular region, and HSPs are induced under cellular stress. Therefore, our ISH results visualized increased mRNA expression levels of HSPs in the centrilobular hepatocytes of mice 6 hours after a single administration of CCl4 as a response to cellular stress, and it disappeared 1 day after the treatment when remarkable necrosis was observed there.

Impact of endoscopy intervals on metachronous gastric cancer after endoscopic submucosal dissection: Comparison between 1 year and half-a-year.

OBJECTIVES: Japanese guidelines recommend posttreatment endoscopy once or twice a year after endoscopic submucosal dissection (ESD) for early gastric cancer. However, the impact of endoscopy intervals on metachronous gastric cancer (MGC) remains unclear, especially the difference between 1-year and half-a-year intervals. We aimed to investigate this difference. METHODS: This study retrospectively investigated 2429 patients who underwent gastric ESD between May 2001 and June 2019 at our hospital. Patients who developed MGC were classified based on those who underwent the previous endoscopy within at least 7 months (short-interval group) and within 8-13 months (regular-interval group). Propensity score matching (PSM) was used to adjust for possible confounders. The primary outcome was the proportion of MGC beyond curative ESD criteria established in the guidelines. RESULTS: A total of 216 eligible patients developed MGC. The short- and regular-interval groups included 43 and 173 patients, respectively. Overall, no patients in the short-interval group had MGC beyond curative ESD criteria, while 27 patients in the regular-interval group did. The proportion of MGC beyond curative ESD criteria was significantly lower in the short-interval group than in the regular-interval group before (P = 0.003) and after (P = 0.028) PSM. Although not significant, the short-interval group tended to have a higher stomach preservation rate than the regular-interval group (P = 0.093). CONCLUSION: Our study indicated a possible benefit of biannual surveillance endoscopy in the early post-ESD period.

Predictors of severe submucosal fibrosis during endoscopic submucosal dissection in patients with ulcerative colitis: Retrospective cohort study.

OBJECTIVES: Severe submucosal fibrosis is a crucial technical difficulty encountered during endoscopic submucosal dissection (ESD) in patients with ulcerative colitis (UC). We aimed to identify predictors of severe submucosal fibrosis in patients with UC. METHODS: We retrospectively included 55 tumors resected using ESD from 48 consecutive patients with UC. We analyzed the clinicopathological characteristics and treatment outcomes between the F0/1 (none to mild submucosal fibrosis) group (n = 28) and F2 (severe submucosal fibrosis) group (n = 27). RESULTS: No significant difference was found between the F0/1 and F2 groups in en bloc resection rate (100% vs. 96%, P = 0.49), the R0 resection rate (100% vs. 93%, P = 0.24), and the dissection speed (0.18 vs. 0.13 cm2 /min, P = 0.07). Intraoperative perforation was more common in the F2 group (30%) than in the F0/1 group (8%; P = 0.01). Multivariable analysis showed that a longer duration of UC (≥10 years; odds ratio [OR] 6.11; 95% confidence interval [CI] 1.20-31.03; P = 0.03) and scarring of background mucosa of the tumor (OR 39.61; 95% CI 3.91-400.78; P < 0.01) were independent predictors of severe submucosal fibrosis. CONCLUSION: Long UC duration and scarring background mucosa were predictors of severe submucosal fibrosis associated with perforation during ESD.

Determination of the Solid Content of Active Pharmaceutical Ingredient Powders in Suspension-Type Pharmaceutical Oral Jelly Using Time-Domain NMR.

This study determined the content of solid active pharmaceutical ingredient (API) powders dispersed in suspension-type pharmaceutical oral jellies using a low-field time-domain NMR (TD-NMR). The suspended jellies containing a designated API content were prepared and tested. Acetaminophen (APAP), indomethacin (IMC) and L-valine were used as test APIs. First, this study measured the T2 relaxation rate (the reciprocal of T2 relaxation time) by the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence, and then evaluated whether the API content could be determined by the acquired T2 relaxation rate. The T2 relaxation rate negatively correlated with API content to a certain extent, but their correlation was not sufficient for achieving a precise determination. Subsequently, the solid-echo pulse sequence measurement was adopted for this study. We found that NMR signals corresponding to solid components strongly correlated with API content. Thus, this method achieved a precise determination of API contents in suspended jellies. In addition, this study confirmed the effect of API particle size on the T2 relaxation rate by using an L-valine-containing jelly: the T2 relaxation rate became faster when a smaller API size was incorporated into the suspended jelly, while there was no difference in terms of the NMR signals measured by solid-echo pulse sequence. From these findings, TD-NMR could be a powerful tool for evaluating the API dispersion state in suspended oral jellies.

A Lipid Nanoparticle-Based Method for the Generation of Liver-Specific Knockout Mice.

Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.

Aberrant activation of estrogen receptor-α signaling in Mettl14-deficient uteri impairs embryo implantation.

The precise control of endometrial receptivity is crucial for successful embryo implantation, which is strictly regulated by the ovarian steroid hormones estrogen and progesterone. Despite our improved understanding of the genetic regulation of implantation downstream of the action of hormones, we do not know much about the epigenetic regulation that occurs during early pregnancy. To investigate the role of the N6-methyladenosine (m6A) RNA modification in embryo implantation, we generated mice with conditional deletion of Mettl14, a core component of the m6A writer complex, in the uterus. These mice were infertile due to implantation failure. We showed that Mettl14-deficient uteri had aberrant upregulation of estrogen receptor α (ERα) signaling and ERα phosphorylation, but progesterone receptor (PGR) signaling was largely unaffected. Additionally, Mettl14 deletion led to abnormal activation of the innate immune pathway in Mettl14-deficient uteri. This effect was accompanied by the infiltration of immune cells, such as macrophages and dendritic cells, into the basal region of the endometrial epithelium. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that genes involved in the innate immune response had decreased m6A peaks in Mettl14-deficient mice. These results suggest that Mettl14 plays a crucial role in successful implantation by precisely regulating both ERα signaling and innate immunity in the uterus.

Gene-Modified Blister Fluid-Derived Mesenchymal Stromal Cells for Treating Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis caused by variants in COL7A1-encoded type VII collagen, a major component of anchoring fibrils. In this study, we developed an ex vivo gene therapy for RDEB using autologous mesenchymal stromal cells (MSCs). On the basis of our previous studies, we first attempted to isolate MSCs from the blister fluid of patients with RDEB and succeeded in obtaining cells with a set of MSC characteristics from all 10 patients. We termed these cells blister fluid-derived MSCs. Blister fluid-derived MSCs were genetically modified and injected into skins of type VII collagen-deficient neonatal mice transplanted onto immunodeficient mice, resulting in continuous and widespread expression of type VII collagen at the dermal-epidermal junction, particularly when administered into blisters. When injected intradermally, the efforts were not successful. The gene-modified blister fluid-derived MSCs could be cultured as cell sheets and applied to the dermis with an efficacy equivalent to that of intrablister administration. In conclusion, we successfully developed a minimally invasive and highly efficient ex vivo gene therapy for RDEB. This study shows the successful application of gene therapy in the RDEB mouse model for both early blistering skin and advanced ulcerative lesions.

Generation of Floxed Mice by Sequential Electroporation.

Generation of conditional knockout mice using the Cre-loxP system is essential for the analysis of gene functions. The use of CRISPR-Cas9 in combination with two sets of guide RNAs and single-stranded oligonucleotides including loxP sites enables simultaneous insertion of two loxP sequences. Unfortunately, this method induces double-strand breaks at two sites in the same chromosome, which causes an undesirable large chromosomal deletion and reduces the flanked loxP (flox) rate. To overcome this problem, we have developed a method that sequentially introduces each loxP sequence by electroporation at the one- and two-cell embryonic stages, respectively. This sequential electroporation method improves the floxing efficiency compared with the conventional simultaneous method, leading to a high yield of offspring with floxed alleles.

Efficient Detection of Flox Mice Using In Vitro Cre Recombination.

Advances in CRISPR/Cas9 genome editing technologies have allowed for the rapid generation of Cre-loxP conditional knockout mice. However, current strategies for genotyping flox mice, typically based on Sanger sequencing following cloning of target sequences from dozens of pups, are time-consuming. Here, we describe a rapid screening method for flox mice, using in vitro Cre recombination that can be performed using simple enzymatic reactions and enables detection of functional flox mouse within 1 day. In addition, we introduce an efficient strategy for subsequent sequence analysis by cloning of floxed regions using the In-Fusion system. Our genotyping pipeline reduces laborious tasks and thus contributes to the rapid selection of accurately edited flox mice.

Endoscopic resection is feasible for high-grade dysplasia in patients with ulcerative colitis.

BACKGROUND: Endoscopic resection (ER) is feasible for treating well-circumscribed dysplasia in patients with ulcerative colitis (UC). However, long-term prognosis of ER for high-grade dysplasia (HGD) in patients with UC remains unclear. We aimed to evaluate the long-term prognoses of ER for HGD compared with low-grade dysplasia (LGD) and verify the feasibility of ER and follow-up with surveillance colonoscopy for HGD. METHODS: An observational, single-center retrospective study included 38 and 22 patients with LGD and HGD who were followed-up with surveillance colonoscopy after ER. We evaluated the cumulative incidence rate of metachronous HGD or colorectal cancer (CRC) and identified the characteristics of metachronous dysplasia. RESULTS: The median follow-up period was 56 months, and surveillance colonoscopies were performed 3.6 times (mean). The 5-year cumulative incidence rate of HGD/CRC was relatively high in HGD (24.6%) than in LGD (13.7%), but the difference was not significant (p = .16). In HGD cases, six metachronous dysplasia lesions (two LGD and four HGD) were detected 11.6-40.5 months after ER. However, these patients did not progress to CRC. All metachronous lesions were well-circumscribed and with no invisible dysplasia surrounding them; they were 'endoscopically resectable' lesions. Two of the four metachronous HGD lesions were treated endoscopically and two, by colectomy. No synchronous HGD or CRC was detected in the colectomy specimens. CONCLUSIONS: Our results suggest that ER and follow-up with surveillance colonoscopy is feasible in patients with HGD when histological complete resection is achieved.

Multiplexed genome editing by in vivo electroporation of Cas9 ribonucleoproteins effectively induces endometrial carcinoma in mice.

Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.

Outpatient flexible endoscopic diverticulotomy for the management of Zenker's diverticulum: a retrospective analysis of a large single-center cohort.

BACKGROUNDS AND AIMS: Flexible endoscopic Zenker's diverticulotomy (EZD) is well established as a safe and effective technique. Because of rare but concerning adverse events, most centers admit patients for observation and barium swallow study. Our center routinely performs EZD as a day procedure, discharging appropriate patients on the same day after clinical review. This study evaluates outcomes of this cohort compared with previously published studies where patients are admitted for observation. METHODS: A retrospective analysis was performed of EZD procedures done at our center using a flexible endoscope and, in most cases, a diverticulotomy overtube with patients under moderate sedation or general anesthesia. Patients were observed for 2 hours and discharged if no clinical concerns were found. Patient comorbidities, American Society of Anesthesiologists physical status, and endoscopic adverse events were recorded against the American Society for Gastrointestinal Endoscopy severity grading system. RESULTS: Two hundred forty EZD procedures were performed between January 2015 and February 2021. Eleven (4.6%) intraprocedural adverse events occurred: 4 perforations, 4 bleeds, and 1 each postprocedural pain, delirium, and vomiting, respectively. All were recognized within the 2-hour observation period and were managed conservatively, except 1 patient who required surgery. Six patients (2.5%) presented with delayed adverse events: 2 bleeds, 2 perforations, and 2 postprocedural pain. All patients recovered uneventfully with supportive care. CONCLUSIONS: All significant adverse events requiring endoscopic or surgical intervention were identified before discharge. Delayed adverse events occurred in 2.5% of cases, all of which were managed supportively. Our data are comparable with published cohorts of admitted patients, demonstrating that appropriately selected patients may be managed as outpatients while maintaining similar safety outcomes.

Electrosurgical knife with the water-jet function of tip-type during endoscopic treatment injection.

Objectives: This study aimed to objectively evaluate the water-jet-functioned electrosurgical knife injection performances in a desktop experiment. Methods: Five types of water-jet-functioned electrosurgical knives, including two injection styles of sheath-type (A: DualKnife J, KD-655L; B: FlushKnife, DK2620-J-B20S; C: Splash M-Knife, DN-D2718B; D: ISSEN, SN1650-20) and tip-type (E: ORISE ProKnife, M00519361) were evaluated. These knives were compared with an injection needle (Control: SuperGrip 25G) as a control. The injection speed under constant pressure and the injection efficiency for each knife against prepared porcine stomach mucosa were evaluated. The additional clear gel injections using an injection needle were observed using an indigo blue-colored gel to evaluate the difference between the locations of water-jet holes. Results: Four types of knives, except for A, showed significantly higher water-jet speeds (A: 0.79 ± 0.03 g/20 s, B: 2.56 ± 0.05 g/20 s, C: 3.09 ± 0.06 g/20 s, D: 2.86 ± 0.05 g/20 s, and E: 1.79 ± 0.03 g/20 s) compared to that of the control (1.21 ± 0.03 g/20 s). Meanwhile, significantly higher efficacy of injection was found in the tip-type water-jet function knife, second to the injection needle (Control: 37.2% ± 35.5%, A: 20.9% ± 20.2%, B: 1.1% ± 2.2%, C: 6.2% ± 12.6%, D: 12.5% ± 15.6%, and E: 33.3% ± 32.2%). An additional injection experiment revealed that the injection with a piercing tip into the gel could achieve sufficient additional injection inside the stacked clear gel. Conclusions: The tip-type water-jet function electrosurgical knife is preferable for effective submucosal injection during endoscopic treatments.