DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Usefulness of aMAP Risk Score for Predicting Recurrence after Curative Treatment for Hepatocellular Carcinoma within Milan Criteria.

INTRODUCTION: The aMAP score is a prediction model for hepatocellular carcinoma (HCC) risk in chronic hepatitis patients. This study was conducted to elucidate the utility of this model for predicting initial recurrence of HCC in patients within the Milan criteria after undergoing curative treatment. METHODS: Patients with naïve HCC within the Milan criteria (n = 1,020) and treated from January 2000 to August 2022 were enrolled. The cohort was divided into two groups according to the aMAP score (high ≥60, low <60) and then compared for recurrence-free survival (RFS) and overall survival (OS). RESULTS: Comparisons between the high and low groups showed that etiology (HBV:HCV:HBV+HCV:NBNC = 41:79:2:37 vs. 65:589:11:196, p < 0.001), AST (36 vs. 46 IU/L, p < 0.001), and multiple HCC occurrence (15% vs. 22%, p = 0.026) were significantly different. Additionally, median RFS (59.8 vs. 30.9 months; p < 0.001) and median OS (154.1 vs. 83.4 months, p < 0.01) were greater in the low group. As for patients with HCC due to chronic viral hepatitis, there was a significant difference in median RFS between the groups (59.8 vs. 30.6 months, p < 0.001), especially for HCV-positive patients (53.1 vs. 27.2 months, p = 0.002). In patients with HCC due to a nonviral cause, the difference in median RFS between the low (70.9 months) and high (32.0 months) groups was not significant. DISCUSSION: Findings of this retrospective study indicate a significant association of elevated aMAP with worse RFS in patients with HCC caused by chronic viral hepatitis, especially those with HCV. The aMAP score is considered useful to predict not only HCC-carcinogenesis risk but also risk of recurrence following curative treatment.

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.

Fatal Cerebral Venous Thrombosis in a Pregnant Woman with Inherited Antithrombin Deficiency after BNT162b2 mRNA COVID-19 Vaccination.

Antithrombin deficiency is a high-risk factor for venous thromboembolism during pregnancy, whereas cerebral venous thrombosis is rare. Cerebral venous thrombosis related to coronavirus disease 2019 (COVID-19) vaccines has been reported; however, there are a few reports of cerebral venous thrombosis after a messenger RNA (mRNA) vaccination. A 25-year-old female in her sixth week of pregnancy presented with headache 24 days after BNT162b2 mRNA COVID-19 vaccination. The following day, she presented with altered sensorium and was diagnosed with severe cerebral venous thrombosis. She demonstrated heparin resistance and was found to have an inherited antithrombin deficiency. A heterozygous missense variant in SERPINC1 (c.379T>C, p.Cys127Arg, 'AT Morioka') was detected by DNA analysis. Despite intensive care with unfractionated heparin, antithrombin concentrate, and repeated endovascular treatments, she died on the sixth day of hospitalization. Cerebral venous thrombosis in pregnant women with an antithrombin deficiency can follow a rapid and fatal course. Treatment with unfractionated heparin and antithrombin concentrate may be ineffective in severe cerebral venous thrombosis cases with antithrombin deficiency. Early recognition of antithrombin deficiency and an immediate switch to other anticoagulants may be required. Although the association between cerebral venous thrombosis and the vaccine is uncertain, COVID-19 vaccinations may require careful evaluation for patients with prothrombic factors.

Refractive changes with post-rotatory nystagmus in healthy individuals.

PURPOSE: Post-rotatory nystagmus has been used to detect autism spectrum disorders in clinical settings. Although previous studies have focused on eye movements, they did not evaluate the change in ocular refraction during post-rotatory nystagmus. This study aimed to evaluate the changes in ocular refraction during post-rotatory nystagmus in healthy individuals. METHODS: A total of 34 healthy volunteers (mean age ± standard deviation, 20.9 ± 0.6 years) participated in this study. The ocular refraction during post-rotatory nystagmus was measured using MR-6000 (Tomey Inc.) on quick mode with a sampling rate of 30 Hz under noncycloplegic and cycloplegic conditions. The amplitude of post-rotatory nystagmus was calculated on the basis of the anterior eye images, while the ocular refraction measurements were simultaneously recorded. The accommodative convergence per accommodation ratio was calculated using the heterophoria method. Video oculography was performed to measure the angle of convergence during post-rotatory nystagmus. RESULTS: The changes in ocular refraction during post-rotatory nystagmus were significantly greater under the noncycloplegic condition than under the cycloplegic condition. The changes in ocular refraction during the post-rotatory nystagmus were significantly and positively correlated with the amplitude of post-rotatory nystagmus under the noncycloplegic condition. The angle of convergence during post-rotatory nystagmus was significantly higher under the noncycloplegic condition than under the cycloplegic condition. The changes in the angle of convergence were significantly and positively correlated with the predicted accommodative convergence. CONCLUSIONS: These findings suggest that the accommodation was functional during the post-rotatory nystagmus to compensate for the retinal image slip, and the accommodative convergence can help weaken the nystagmus.

Generation of B6-Ddx4em1(CreERT2)Utr , a novel CreERT2 knock-in line, for germ cell lineage by CRISPR/Cas9.

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock-out mouse studies. In this study, we established a novel knock-in mouse line, B6-Ddx4 em1(CreERT2)Utr , which expresses CreERT2 recombinase under the control of the endogenous DEAD-box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen-treated B6-Ddx4 em1(CreERT2)Utr ::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6-Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.

Evaluation of the effects of dapagliflozin, a sodium-glucose co-transporter-2 inhibitor, on hepatic steatosis and fibrosis using transient elastography in patients with type 2 diabetes and non-alcoholic fatty liver disease.

AIMS: To investigate the effects of dapagliflozin on liver steatosis and fibrosis evaluated in patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: In a randomized, active-controlled, open-label trial, 57 patients with type 2 diabetes and NAFLD were randomized to a dapagliflozin group (5 mg/d; n = 33) or a control group (n = 24) and were treated for 24 weeks. Hepatic steatosis and fibrosis were assessed using transient elastography to measure controlled attenuation parameter (CAP) and liver stiffness, respectively. RESULTS: Baseline liver stiffness measurement (LSM) was positively correlated with several markers and scoring systems for liver fibrosis. In week 24, there was a significant decrease in CAP from 314 ± 61 to 290 ± 73 dB/m (P = 0.0424) in the dapagliflozin group, while there was no significant change in the control group. In addition, LSM tended to decrease from 9.49 ± 6.05 to 8.01 ± 5.78 kPa in the dapagliflozin group. In 14 patients from this group with LSM values ≥8.0 kPa, indicating significant liver fibrosis, LSM decreased significantly from 14.7 ± 5.7 to 11.0 ± 7.3 kPa (P = 0.0158). Furthermore, serum alanine aminotransferase and γ-glutamyltranspeptidase levels decreased in the dapagliflozin group, but not in the control group, and visceral fat mass was significantly reduced in the dapagliflozin group. CONCLUSIONS: Based on these findings, the sodium-glucose co-transporter-2 inhibitor dapagliflozin improves liver steatosis in patients with type 2 diabetes and NAFLD, and attenuates liver fibrosis only in patients with significant liver fibrosis, although the possibility cannot be excluded that a reduction in body weight or visceral adipose tissue by dapagliflozin may be associated with a decrease of liver steatosis or fibrosis.

Impact of dapagliflozin, an SGLT2 inhibitor, on serum levels of soluble dipeptidyl peptidase-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease.

AIMS: Soluble dipeptidyl peptidase-4 (sDPP-4) is secreted by hepatocytes and induces adipose tissue inflammation and insulin resistance. Sodium-glucose co-transporter-2 (SGLT2) inhibitors can improve hepatic steatosis by inhibiting hepatic de novo lipogenesis. We investigated the effects of dapagliflozin (an SGLT2 inhibitor) on serum levels of sDPP-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). METHODS: Fifty-seven patients with type 2 diabetes and NAFLD were randomized to a dapagliflozin group (5 mg/d for 24 weeks) (n = 33) or the control group (n = 24). Serum levels of sDPP-4 were measured with a commercial ELISA kit. Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas were measured by dual bioelectrical impedance analysis. RESULTS: In a total of 57 patients, baseline serum sDPP-4 was positively correlated with aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT) and HOMA-IR Both VAT and SAT areas decreased significantly in the dapagliflozin group alone. Liver enzymes were decreased at 24 weeks in the dapagliflozin group, but were unchanged in the control group. Although both groups showed significant reduction of serum sDPP-4 after 24 weeks of treatment, the magnitude of decrease was significantly larger in the dapagliflozin group. Changes in liver enzymes during treatment with dapagliflozin were positively correlated with the change in serum sDPP-4, but not with changes in VAT volume or HbA1c. CONCLUSIONS: Improvement of liver dysfunction after treatment with dapagliflozin was associated with a decrease in serum sDPP-4, suggesting that reduction of serum sDPP-4 by SGLT2 inhibitors may be a therapeutic strategy for NAFLD/NASH in patients with type 2 diabetes that is independent of glucose lowering or weight loss.

Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.

Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constructed a CRISPR/Cas9 expression vector for the Tyr gene (px330-Tyr-M). DNA cleavage activity of px330-Tyr-M at the target site of the Tyr gene was confirmed by the EGxxFP system. We also designed an ssDNA donor for homology-directed repair (HDR)-mediated gene modification. The px330-Tyr-M vector and ssDNA donor were co-microinjected into the pronuclei of 224 one-cell-stage embryos derived from C57BL/6J mice. We obtained 60 neonates, 28 of which showed the ocular albinism and absence of coat pigmentation. Genomic sequencing analysis of the albino mice revealed that the target of SNM, G291T in the Tyr gene, occurred in 11 mice and one founder was homozygously mutated. The remaining albino founders without Tyr G291T mutation also possessed biallelic deletion and insertion mutants adjacent to the target site in the Tyr locus. Simple production of albino C57BL/6J mice was provided by C57BL/6J zygote microinjection with px330-Tyr-M DNA vector and mutant ssDNA (G291T in Tyr) donor. A combination of CRISPR/Cas9 vector and optional mutant ssDNA could be expected to efficiently produce novel SNM-induced mouse models for investigating human diseases.

Diabetes with GLP-1R polymorphism (rs3765467) accompanied by myotonic dystrophy: A case of myotonic dystrophy with p.R131Q polymorphism at the glucagon-like peptide-1 receptor (rs3765467) resulting in marked effects of its agonist, dulaglutide.

A 47-year-old woman was diagnosed with myotonic dystrophy when admitted for traumatic subarachnoid hemorrhage. Her glycemic control was poor despite administration of pioglitazone, a PPARɤ agonist, and subcutaneous insulin infusion. However, adding a GLP-1 receptor (GLP-1R) agonist markedly improved blood glucose levels, resulting in eventual insulin withdrawal. Genetic testing revealed a heterozygous variant, p.R131Q, in the GLP1R (rs3765467), a common variant in Asia. This variant is known to be associated with increased endogenous insulin from beta cells in response to exogenous GLP-1 infusion. This is the first report and short review of a Japanese case of myotonic dystrophy accompanied by GLP-1R gene polymorphism.

Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions.

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.

Deletion of Exoc7, but not Exoc3, in male germ cells causes severe spermatogenesis failure with spermatocyte aggregation in mice.

Vesicular trafficking is essential for the transport of intracellularly produced functional molecules to the plasma membrane and extracellular space. The exocyst complex, composed of eight different proteins, is an important functional machinery for "tethering" in vesicular trafficking. Functional studies have been conducted in laboratory mice to identify the mechanisms by which the deletion of each exocyst factor affect various biological phenomena. Interestingly, each exocyst factor-deficient mutant exhibits a different phenotype. This discrepancy may be due to the function of the exocyst factor beyond its role as a component of the exocyst complex. Male germline-specific conditional knockout (cKO) mice of the Exoc1 gene, which encodes one of the exocyst factors EXOC1 (SEC3), exhibit severe spermatogenesis defects; however, whether this abnormality also occurs in mutants lacking other exocyst factors remains unknown. In this study, we found that exocyst factor EXOC3 (SEC6) was not required for spermatogenesis, but depletion of EXOC7 (EXO70) led to severe spermatogenesis defects. In addition to being a component of the exocyst complex, EXOC1 has other functions. Notably, male germ cell-specific Exoc7 cKO and Exoc1 cKO mice exhibited phenotypic similarities, suggesting the importance of the exocyst complex for spermatogenesis. The results of this study will contribute to further understanding of spermatogenesis from the aspect of vesicular trafficking.

[Behavior abnormality].

The aging rate of Japan is higher than 20% in 2005 and it is predominantly early and invites aged society in the developed countries. With increase in elderly person population, the emergency consultation of the elderly person increases, and the emergency medical treatment of the elderly person that complaining of a condition is vague must be careful to have possibilities to miss the severe condition of a patient. When an elderly person presents with sudden behavior abnormality and consults an emergency room, we must check presence of disturbance of consciousness. When there is disturbance of consciousness, we provide treatment for it. In this report, we describe an emergency disease with the behavior abnormality of the elderly person.

Analysis of smooth pursuit eye movements in a clinical context by tracking the target and eyes.

In the evaluation of smooth pursuit eye movements (SPEMs), recording the stimulus onset time is mandatory. In the laboratory, the stimulus onset time is recorded by electrical signal or programming, and video-oculography (VOG) and the visual stimulus are synchronized. Nevertheless, because the examiner must manually move the fixation target, recording the stimulus onset time is challenging in daily clinical practice. Thus, this study aimed to develop an algorithm for evaluating SPEMs while testing the nine-direction eye movements without recording the stimulus onset time using VOG and deep learning-based object detection (single-shot multibox detector), which can predict the location and types of objects in a single image. The algorithm of peak fitting-based detection correctly classified the directions of target orientation and calculated the latencies and gains within the normal range while testing the nine-direction eye movements in healthy individuals. These findings suggest that the algorithm of peak fitting-based detection has sufficient accuracy for the automatic evaluation of SPEM in clinical settings.

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

Comparison of Surgical Resection and Percutaneous Ultrasonographic Guided Radiofrequency Ablation for Initial Recurrence of Hepatocellular Carcinoma in Early Stage following Curative Treatment.

Background/Aim: The SURF trial showed that surgical resection (SR) and percutaneous ultrasonographic guided radiofrequency ablation (RFA) had equal therapeutic effects for small hepatocellular carcinoma (HCC). However, consensus regarding which treatment is appropriate for initial recurrent early-stage HCC remains lacking. This study aimed to elucidate therapeutic efficacy differences between SR and RFA for initial recurrent early-stage HCC. Materials/Methods: From 2000 to 2021, 371 patients with recurrent early-stage HCC (≤3 cm, ≤3 nodules) after undergoing initial curative treatment with SR or RFA were enrolled (median age 72 years; males 269; Child−Pugh A:B, n = 328:43; SR:RFA, n = 36:335). Recurrence-free survival (RFS) and overall survival (OS) were retrospectively evaluated. Results: Although the median albumin−bilirubin (ALBI) score was better in the SR than the RFA group (−2.90 vs. −2.50, p < 0.01), there were no significant differences between them in regard to RFS (median 28.1 months, 95% CI 23.4−50.0 vs. 22.1 months, 95% CI 19.3−26.2; p = 0.34), OS (78.9 months, 95% CI 49.3­not applicable vs. 71.2 months 95% CI, 61.8−84.7; p = 0.337), or complications (8.3% vs. 9.3%; p = 1.0). In sub-analysis for RFS and OS according to ALBI grade revealed no significant differences between the SR and RFA groups (ALBI 1/2 = 28.2/17.5 vs. 24.0/23.4 months; p = 0.881/0684 and ALBI 1/2 = 78.9/58.9 vs. 115.3/52.6 months, p = 0.651/0.578, respectively). Conclusion: This retrospective study found no significant differences in regard to RFS or OS between patients in the SR and the RFA groups for initial recurrence of early-stage HCC after undergoing curative treatment. These results showing equal therapeutic efficacy of SR and RFA confirm the findings of the SURF trial.

Clinical Usefulness of Surgical Resection Including the Complementary Use of Radiofrequency Ablation for Intermediate-Stage Hepatocellular Carcinoma.

Background/Aim: For intermediate-stage hepatocellular carcinoma (HCC) (Barcelona Clinic Liver Cancer [BCLC]-B) cases, transarterial chemoembolization (TACE) is recognized as the standard treatment, while systemic therapy is recommended for TACE-unsuitable HCC. However, because the curative potential is not high, this study was conducted to elucidate the potential outcomes of surgical resection (SR) for BCLC-B HCC cases. Materials/Methods: From January 2000 to July 2022, 70 patients with BCLC-B HCC treated with surgery as the initial treatment were enrolled (median age 67.5 years, beyond up-to-7 criteria 44). Forty-five were treated with SR only (SR group), while twenty-five underwent that with complemental radiofrequency ablation (RFA) (Comb group). Recurrence-free survival (RFS) and overall survival (OS) were retrospectively evaluated in both groups. Results: The median albumin−bilirubin (ALBI) score was better in the SR as compared with the Comb group (−2.74 vs. −2.52, p = 0.02), while there were no significant differences between them for median RFS (17.7 vs. 13.1 months; p = 0.70) or median OS (66.6 vs. 72.0 months p = 0.54). As for those beyond up-to-7 criteria, there were no significant differences for median RFS (18.2 vs. 13.0 months; p = 0.36) or median OS (66.5 vs. 72.0 months; p = 0.57). An acceptable five-year cumulative survival rate (>50%) was obtained in both groups (54% vs. 64%). Conclusion: This retrospective study found no significant differences for RFS or OS between the present SR and Comb groups with BCLC-B HCC. When possible to perform, the outcome of SR for BCLC-B is favorable, with a five-year survival rate greater than 50%.

Serum high-molecular-weight adiponectin and response to dapagliflozin in patients with type 2 diabetes and non-alcoholic fatty liver disease.

A better baseline renal function is associated with a better response to sodium-glucose co-transporter-2 inhibitors in patients with type 2 diabetes. Low serum adiponectin is associated with visceral fat accumulation and hepatic steatosis. We investigated the relationship between baseline serum adiponectin and glycemic response to dapagliflozin in patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In a randomized, active-controlled, open-label trial, 57 patients with type 2 diabetes and NAFLD were randomized to either the dapagliflozin (5 mg/d) group or the control group. Both groups were treated for 24 weeks. Serum high-molecular-weight (HMW) adiponectin was measured with an ELISA kit. Visceral fat area (VFA) was measured by dual bioelectrical impedance analysis. Hepatic steatosis was assessed by the controlled attenuation parameter (CAP) measured by a transient elastography (FibroScan). Treatment with dapagliflozin significantly decreased HbA1c from 8.4%±1.5% at baseline to 7.4%±1.2% at 24 weeks. Both VFA and CAP decreased in the dapagliflozin group. Baseline serum HMW adiponectin was negatively correlated with changes in HbA1c from baseline to 24 weeks with dapagliflozin therapy. In the multivariate analysis, baseline HbA1c (ß=-0.559, p=0.002) and serum HMW adiponectin (ß=0.471, p=0.010) were independent determinants for the change (reduction) in HbA1c. In the dapagliflozin group, the change in HbA1c was positively correlated with the changes of CAP, but negatively correlated with the change in serum HMW adiponectin. In conclusion, a lower serum level of HMW adiponectin was associated with a better response to dapagliflozin in patients with type 2 diabetes and NAFLD.Trial registration numberUMIN000022155.

Serum levels of soluble dipeptidyl peptidase-4 in type 2 diabetes are associated with severity of liver fibrosis evaluated by transient elastography (FibroScan) and the FAST (FibroScan-AST) score, a novel index of non-alcoholic steatohepatitis with significant fibrosis.

AIM: To investigate the relationship in people with type 2 diabetes between serum soluble dipeptidyl peptidase-4 (sDDP-4) and degree of liver fibrosis assessed as the liver stiffness measurement (LSM) and FAST (FibroScan-AST) score, both of which were measured by transient elastography (FibroScan). SUBJECTS AND METHODS: In this cross-sectional study, we examined 115 patients with type 2 diabetes. With transient elastography (FibroScan), we assessed the controlled attenuation parameter (CAP) and liver stiffness measurement (LSM) as measures of hepatic steatosis and liver fibrosis, respectively. We calculated the FAST score, which identifies progressive non-alcoholic steatohepatitis (NASH), from CAP, LSM, and the serum aspartate aminotransferase level. Significant hepatic steatosis was defined as CAP ≥280 dB/m; and significant liver fibrosis, as LSM ≥ 8.0 kPa. LSM was divided into 3 severity levels: significant fibrosis (8.0 to <9.7 kPa); advanced fibrosis, (9.7 to <13.0 kPa); and liver cirrhosis (≥ 13.0 kPa). RESULTS: Serum sDPP-4 correlated positively with liver enzymes, CAP, LSM, and FAST score. Multivariate analysis showed that LSM remained to be an independent factor for serum sDDP-4. Serum sDPP-4 was significantly higher in patients with LSM ≥ 8.0 kPa than in those with LSM <8.0 kPa and was significantly elevated in patients who are at risk for non-alcoholic steatohepatitis (NASH) with fibrosis (FAST score ≥ 035 or 0.67). Patients with both hepatic steatosis and liver fibrosis had the highest serum sDPP-4. CONCLUSION: Serum sDPP-4 was strongly associated with severity of liver fibrosis evaluated by LSM and the FAST score and was markedly elevated in diabetic patients with LSM ≥ 13.0 kPa indicating probable cirrhosis.

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo.

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.