Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.Total internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K(+)-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca(2+) concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K(+) (K(ATP)) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.We provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, K(ATP) channel responsiveness and the subsequent activity of Ca(2+) channels through pathways other than CDK5-mediated regulation

P1 gene of Mycoplasma pneumoniae isolated from 2016 to 2019 and relationship between genotyping and macrolide resistance in Hokkaido, Japan

We characterized 515 Mycoplasma pneumoniae specimens in Hokkaido. In 2013 and 2014, the p1 gene type 1 strain, mostly macrolide-resistant, was dominant and the prevalence of macrolide resistance was over 50 %. After 2017, the p1 gene type 2 lineage, mostly macrolide-sensitive, increased and the prevalence of macrolide resistance became 31.0 % in 2017, 5.3 % in 2018 and 16.3 % in 2019

Therapeutic efficacy of azithromycin, clarithromycin, minocycline and tosufloxacin against macrolide-resistant and macrolide-sensitive Mycoplasma pneumoniae pneumonia in pediatric patients

Objective: To clarify therapeutic effects of azithromycin, clarithromycin, minocycline and tosufloxacin against macrolide-resistant Mycoplasma pneumoniae (MRMP) pneumonia and against macrolide-sensitive Mycoplasma pneumoniae (MSMP) pneumonia in pediatric patients. Methods: A prospective, multicenter observational study was conducted from July 2013 to August 2015. The therapeutic effects of azithromycin, clarithromycin, minocycline and tosufloxacin were evaluated in 59 patients with pneumonia caused by MRMP and in 50 patients with pneumonia caused by MSMP. In vitro activities of antimicrobial agents against isolates of Mycoplasma pneumoniae were also measured. Results: Mean durations of fever following commencement of treatment in patients infected with MRMP and MSMP were 5.2 and 1.9 days, respectively (log-rank test, P < 0.0001). Among patients infected with MRMP, mean durations of fever were 4.6, 5.5, 1.0 and 7.5 days for patients treated with azithromycin, clarithromycin, minocycline and tosufloxacin, respectively (log-rank test, P < 0.0001). Among patients infected with MSMP, mean durations of fever were 2.5, 1.7, 0.9 and 4.3 days for patients treated with azithromycin, clarithromycin, minocycline and tosufloxacin, respectively (log-rank test, P = 0.0162). The MIC90s of azithromycin and clarithromycin among the 27 isolates of MRMP were 64 and 256 μg/ml, respectively, and those among the 23 isolates of MSMP were <0.000125 and 0.001 μg/ml, respectively. The MIC90s of minocycline and tosufloxacin among the 27 isolates of MRMP were 1.0 and 0.25 μg/ml, respectively, and those among the 23 isolates of MSMP were 1.0 and 0.5 μg/ml, respectively. Conclusion: Both minocycline and tosufloxacin showed good in vitro activities against MRMP. Minocycline, but not tosufloxacin, shortened the duration of fever in pediatric patients infected with MRMP compared to the duration of fever in patients treated with macrolides

ELKS/Voltage-Dependent Ca²⁺ Channel-beta Subunit Module Regulates Polarized Ca²⁺ Influx in Pancreatic beta Cells

Pancreatic β cells secrete insulin by Ca²⁺-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in β cells to regulate Ca²⁺ influx for insulin secretion. β cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca²⁺ channel (VDCC) current density. In situ Ca²⁺ imaging of β cells within islets expressing a membrane-bound G-CaMP8b Ca²⁺ sensor demonstrated initial local Ca²⁺ signals at the ELKS-localized vascular side of the β cell plasma membrane, which were markedly decreased in ELKS-KO β cells. Mechanistically, ELKS directly interacted with the VDCC-β subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the β cell plasma membrane for polarized Ca²⁺ influx and first-phase insulin secretion from pancreatic islets

Clinical effectiveness of four neuraminidase inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir) for children with influenza A and B in the 2014-2015 to 2016-2017 influenza seasons in Japan

The clinical effectiveness of four neuraminidase inhibitors (NAIs) (oseltamivir, zanamivir, laninamivir, and peramivir) for children aged 0 months to 18 years with influenza A and B were investigated in the 2014-2015 to 2016-2017 influenza seasons in Japan. A total of 1207 patients (747 with influenza A and 460 with influenza B) were enrolled. The Cox proportional-hazards model using all of the patients showed that the duration of fever after administration of the first dose of the NAI was shorter in older patients (hazard ratio = 1.06 per 1 year of age, p < 0.001) and that the duration of fever after administration of the first dose of the NAI was shorter in patients with influenza A infection than in patients with influenza B infection (hazard ratio = 2.21, p < 0.001). A logistic regression model showed that the number of biphasic fever episodes was 2.99-times greater for influenza B-infected patients than for influenza A-infected patients (p < 0.001). The number of biphasic fever episodes in influenza A-or B-infected patients aged 0-4 years was 2.89-times greater than that in patients aged 10-18 years (p = 0.010), and the number of episodes in influenza A-or B-infected patients aged 5-9 years was 2.13times greater than that in patients aged 10-18 years (p = 0.012)

Effects of <i>CDKAL1</i> KO on glucose-induced biphasic insulin exocytosis.

<p><b>A</b>. Insulin release (for 30 min) in batch-incubated WT and <i>CDKAL1</i> KO β cells in the presence of 2.2 mM or 16.7 mM glucose (n = 8 per group). <b>B</b>. Histogram showing the number of fusion events from GFP-tagged granules in wild-type (WT) and <i>CDKAL1</i> KO β cells (per 200 µm²) at 1-min intervals after stimulation with 22 mM glucose and measured by TIRF microscopy. Data are mean±SEM (WT, n = 16 cells; KO, n = 14 cells). Time 0 indicates the addition of 22 mM glucose. The red column shows fusion events from previously docked granules, and the green column shows those from newcomers. <b>C</b>. Histogram showing the number of fusion events in WT and KO β cells (per 200 µm²) at 1-min intervals after 40 mM high K⁺ stimulation measured by TIRF microscopy (n = 8 cells per group).</p

<i>CDKAL1</i> KO did not affect the number of morphologically docked granules or granule fusion kinetics.

<p><b>A</b>. Total internal reflection fluorescence (TIRF) microscopy of insulin granules morphologically docked to the plasma membrane. (top) Typical TIRF images of docked insulin granules in WT and <i>CDKAL1</i> KO β cells. The surrounding lines represent the outline of cells attached to the cover glass. Scale bar: 5 µm. Pancreatic β cells were prepared from WT and KO mice, fixed, and immunostained for insulin. (bottom) Number of insulin granules morphologically docked to the plasma membrane. Individual fluorescent spots shown in the TIRF images were manually counted per 200 µm² in 15 cells per group. <b>B</b>. Electron micrograph of β cell sections. (top) Typical images of the plasma membrane area facing the blood capillary (C) of WT and KO β cells (B). Bar: 500 nm. (bottom) Number of morphologically docked insulin granules per 10 µm of the plasma membrane. Granules at their shortest distance of <10 nm from the plasma membrane were defined as morphologically docked granules (red arrowheads). Results are means±SEM. <b>C</b>. Expression of SNARE proteins in wild-type (WT) and KO islets by immunoblotting. Equal amounts of islet protein (30 µg) were separated by SDS-PAGE and immunoblotted. β-actin was used as a loading control.</p

Pancreatic histology and insulin content in <i>CDKAL1</i> KO mice.

<p><b>A</b>. <i>CDKAL1</i> KO mice have normal islet architecture. Pancreatic sections were peroxidase stained for insulin. Scale bar: 100 µm. <b>B</b>. Relative area occupied by β cells (percentage of total pancreatic area). Random sections of the entire pancreas from WT and <i>CDKAL1</i> KO mice were immunostained (as shown in <b>A</b>) and analyzed (60 sections from each of three mice per group). <b>C–F</b>. Electron micrographs of pancreatic tissue sections. <b>C</b> Representative sections (scale bar: 5 µm), <b>D</b> β cell size, <b>E</b> total number of granules per cell section, and <b>F</b> mean granule diameter in ultra-thin sections (100 nm) (n = 20 cells per group) of <i>CDKAL1</i> KO and WT β cells. <b>G–I</b>. Insulin content in <i>CDKAL1</i> KO mice. <b>G</b> Pancreatic insulin content measured in acid-ethanol extracts from WT and KO mice by ELISA (n = 6 per group). <b>H</b> DNA content per islet and <b>I</b> islet insulin content per DNA from WT and KO mice (n = 6 per group). Results are means±SEM.</p

Effects of <i>CDKAL1</i> KO on changes in intracellular free calcium ([Ca²⁺]_i).

<p><b>A</b> 22 mM glucose- and <b>B</b> 40 mM high K⁺-induced changes in [Ca²⁺]_i in WT and <i>CDKAL1</i> KO β cells. Changes in [Ca²⁺]_i were measured by Fura-2 acetoxymethyl (2 µM). Time 0 indicates when the stimulants were added. The fluorescence ratio (340/360) at time 0 was taken as 1. Results are means±SEM (n = 12 cells per group).</p