(Epi)genetic and clinical characteristics in 84 patients with pseudohypoparathyroidism type 1B.

OBJECTIVE: Pseudohypoparathyroidism type 1B (PHP1B) caused by methylation defects of differentially methylated regions (DMRs) on the GNAS locus can be categorized into groups according to etiologies and methylation defect patterns of the DMRs. The aim of this study was to clarify the clinical characteristics of each group. DESIGN: Comprehensive molecular analyses consisting of methylation, copy number, and microsatellite analyses. METHODS: Eighty-four patients with PHP1B were included in this study. We classified them into 5 groups, namely, autosomal dominant inheritance-PHP1B (Group 1, G1), sporadic-PHP1B (G2), and atypical-PHP1B (G3-G5), based on the methylation defect patterns in 4 DMRs on the GNAS locus and etiologies and evaluated the clinical findings in each group and compared them among the groups. RESULTS: G2 had the youngest age and the highest serum intact parathyroid hormone levels among the 5 groups at the time of diagnosis. The most common symptoms at the time of diagnosis were tetany in G1, and seizures or loss of consciousness in G2. Albright's hereditary osteodystrophy and PHP-suggestive features were most frequently observed in the G2 proband. Nine patients had neurodevelopmental disorders (NDs) consisting of mild to borderline intellectual disability and/or developmental delay. There were no significant correlations between the average methylation ratios of 7 CpG sites in the GNAS-A/B:TSS-DMR and hormonal and biochemical findings. CONCLUSION: This study revealed the differences in some clinical characteristics, particularly clinical features, and ages at the time of diagnosis between G2 and other groups and detailed NDs observed in some patients with PHP1B.

The Effect of a Pre-consultation Tablet-Based Questionnaire on Changes in Consultation Time for First-Visit Patients With Diabetes: A Single-Case Design Preliminary Study.

Background and objective It has been reported that physicians spend about 50% of their time in the outpatient department doing non-face-to-face patient work, such as charting and desk activities. Work outside of patient consultations is often considered a burden. In this study, we aimed to examine whether the use of pre-consultation tablet-based questionnaires for first-visit diabetic patients had any impact on the time spent for consultation. Methods The sole participant was a diabetologist with more than 20 years of experience. The time spent in the clinic was compared via a single-case experimental design (ABAB) using paper- and tablet-based questionnaires for a total of 20 first-visit diabetic patients. Results The median pre-clinical time without patients was significantly shorter in the tablet group than in the paper group (two minutes and 45 seconds vs. five minutes and 39 seconds; p=0.003). The median clinical time with patients in the tablet group was significantly longer than that in the paper group (19 minutes and 37 seconds vs. 11 minutes and 25 seconds; p=0.026). The total clinical time was not significantly different between the two groups (p=0.25). Conclusions Our results suggest that tablet-based pre-consultation questionnaires may have an impact on the allocation of time for medical examinations and improve the quality of diabetes care.

Detection of Euglycemic Diabetic Ketoacidosis During Thoracic Surgery 75 Hours After Empagliflozin Discontinuation.

Euglycemic diabetic ketoacidosis (eDKA) has been increasingly reported as an adverse event of sodium-glucose cotransporter 2 inhibitors (SGLT2i), and the accompanying information on the drug recommends discontinuation three days prior to scheduled surgery. We present a case of a 50-year-old woman who developed eDKA during surgery for a metastatic lung tumor 75 hours after discontinuing SGLT2i. In this case, the onset of eDKA was detected using intraoperative blood gas analysis and urinary ketone measurements. Hence, perioperative eDKA can occur even after three or more days of SGLT2i withdrawal.

Recurrent hyperglycemic hyperosmolar state after re-administration of dose-reduced ceritinib, an anaplastic lymphoma kinase inhibitor.

Ceritinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor with clinical activity in crizotinib-resistant ALK-positive non-small cell lung cancer and in treatment-naïve ALK-positive disease. Hyperglycemia is a known adverse event, but the mechanism by which ceritinib causes hyperglycemia is unknown, and whether ceritinib causes hyperglycemic emergencies is unclear. Here, we report the case of a patient with a hyperglycemic hyperosmolar state (HHS) recurrence after the re-administration of dose-reduced ceritinib. A 78-year-old man with type 2 diabetes diagnosed as having advanced lung adenocarcinoma had been treated with alogliptin (25 mg/day) for the diabetes and with ceritinib for the lung cancer. After 28 days of ceritinib administration, he was admitted to our hospital due to HHS. His blood glucose level improved with insulin therapy after discontinuation of the ceritinib. He then received re-administration with a decreased ceritinib dose while maintaining the insulin treatment to control his blood glucose, but his HHS recurred. We discontinued the ceritinib for other side effects and noticed the HHS disappeared. Our findings suggest that ceritinib can cause HHS and that HHS may recur even after dose reductions.

Eomesodermin promotes interaction of RelA and NFATc2 with the Ifng promoter and multiple conserved noncoding sequences across the Ifng locus in mouse lymphoma BW5147 cells.

The T-box transcription factor Eomesodermin (Eomes) regulates the lineage-dependent expression of interferon γ (IFN-γ). We previously showed that Eomes promotes IFN-γ production and interacts with multiple conserved noncoding sequences (CNS) across the Ifng locus in mouse lymphoma BW5147 cells. In the present study, we investigated the transcriptional regulation of IFN-γ by the nuclear factor κB (NF-κB) subunit RelA and nuclear factor of activated T cells c2 (NFATc2, also known as NFAT1) in Eomes-transfected BW5147 cells. Eomes promoted the interaction of RelA and NFATc2 with the Ifng promoter and five CNS, including CNS-22 and CNS+30 upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). The dual NF-κB and STAT3 inhibitor TPCA-1 moderately reduced the PMA- and IM-induced IFN-γ transcription in Eomes-transfected BW5147 cells. TPCA-1 interfered with RelA binding to the Ifng promoter, CNS-22 and CNS+30. Moreover, TPCA-1 reduced the interaction of Eomes or NFATc2 with the Ifng promoter and CNS+30. The present results indicate that Eomes promotes the interaction of RelA and NFATc2 with the Ifng promoter and multiple CNS across the Ifng locus in BW5147 cells.

Different localization of lysosomal-associated membrane protein 1 (LAMP1) in mammalian cultured cell lines.

Lysosomal-associated membrane protein 1 (LAMP1) mainly localizes to lysosomes and late endosomes. We herein investigated the intracellular localization of lysosomal membrane proteins in five mammalian cultured cell lines. Rat LAMP1 fused to enhanced green fluorescent protein (EGFP) mostly accumulated at a particular cytoplasmic area and barely co-localized with LysoTracker® Red DND-99 in golden hamster kidney BHK-21 cells and Chinese hamster ovary CHO-K1 cells. Golden hamster, Chinese hamster, and human LAMP1-EGFP showed a similar intracellular distribution to rat LAMP1-EGFP in BHK-21 cells. Endogenous LAMP1 was also detected in a perinuclear area in BHK-21 cells and CHO-K1 cells, and co-localized with rat CD63-EGFP in BHK-21 cells. Moreover, rat LAMP1-DsRed-Monomer co-localized well with the human trans-Golgi network protein 2-EGFP in BHK-21 cells. These results reveal that LAMP1 predominantly localizes to the trans-Golgi network in BHK-21 cells.

Eomesodermin promotes interferon-γ expression and binds to multiple conserved noncoding sequences across the Ifng locus in mouse thymoma cell lines.

The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-γ (IFN-γ). However, in contrast to T-bet, the role of Eomes in the expression of IFN-γ remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-γ in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners.

Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins.

Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins. To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library [established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontermann, Jones, Low, Allison et al. (1994) EMBO J. 13, 3245-3260] to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI. By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence. By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins. Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites.