Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann–Pick C1-deficient cells

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.     

Synthesis of triazole- and tetrazole-xyloside analogues as potent hyaluronidase inhibitors

Hyaluronidase is one of the most important enzymes in the development of many diseases. In this study, a series of xyloside analogues bearing a triazole and tetrazole at the anomeric position were prepared from xylosylthioureas and evaluated their inhibitory effects on the hyaluronidase. Triazole and tetrazole skeletons were formed via the Hg(OAc)₂-mediated desulfurizative cyclization through carbodiimide intermediates. According to in vitro anti-hyaluronidase assay, tetrazole-xylosides having p-chloro- or p-nitro-substitution exhibited the high inhibition rates, whereas the compound having p-trifluoromethyl group on the structure did not show the potency. Our results demonstrated the importance of tetrazole-xylosides as hyaluronidase inhibitors.     

Long-term in vivo gene expression in mouse kidney using ϕC31 integrase and electroporation

Background: Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes.

Purpose: The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using ?C31 integrase.

Methods: Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5?µg of pORF-luc/attB as a donor plasmid and 20?µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression.

Results: Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, ?C31 integrase-transfected cells had less genomic DNA damage caused by integrase expression.

Discussion and conclusion: These results demonstrated that the ?C31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.     

Clinical significance of left atrial geometry in dilated cardiomyopathy patients: A cardiovascular magnetic resonance study

BackgroundClinical significance of left atrial (LA) function and geometry in patients with dilated cardiomyopathy (DCM) remains uncertain.HypothesisLA geometric parameters assessed by cardiac magnetic resonance (CMR) predict the prognosis in patients with DCM.MethodsThe present study included patients with DCM and sinus rhythm who underwent CMR between December 2007 and April 2018. LA volume was measured using CMR. LA sphericity index was computed as the ratio of the measured maximum LA volume by the volume of a sphere with maximum LA length diameter.ResultsWe included 255 patients in this study. During the mean follow‐up of 3.92 years, hospitalization for HF occurred in 37 patients. The LA sphericity index was significantly higher in patients with hospitalization for HF than in those without (0.78 ± 0.35 vs. 0.58 ± 0.18, p < .001). Multivariable Cox regression analysis identified a higher LA sphericity index as an independent predictor of hospitalization for HF. Patients were categorized based on the median of LA sphericity index. The Kaplan–Meier curve showed that patients with a high LA sphericity index (≥0.57) had a significantly higher risk of hospitalization for HF than those with a low LA sphericity index (<0.57).ConclusionLA sphericity index was an independent predictor of hospitalization for HF. Assessment of LA geometric parameters might be useful for risk stratification in patients with DCM.     

Molecular diagnosis and characterization of a culture-negative mycotic aneurysm due to ST54 Haemophilus influenzae type b with PBP 3 alterations

Mycotic aneurysm is a rare but life-threatening disease that warrants an integrated therapeutic approach involving surgical intervention and prolonged antibiotic use. However, the causative organisms are often unidentified because antibiotics started empirically render blood and tissue cultures negative. Molecular diagnosis has been reported to be useful in such culture-negative cases. We report a case of a culture-negative mycotic aortic aneurysm due to Haemophilus influenzae, diagnosed by direct 16S rRNA polymerase chain reaction (PCR) and sequencing of the resected aneurysm tissue. PCR for serotype revealed type b, and PCR and sequencing of the ftsI gene revealed alterations in penicillin-binding protein 3, suggesting resistance to ampicillin. Multilocus sequence typing demonstrated that the isolate belonged to sequence type 54.     

Resuscitative Endovascular Balloon Occlusion of the Aorta Using a Low-Profile Device is Easy and Safe for Emergency Physicians in Cases of Life-Threatening Hemorrhage

Background

Bleeding from hemorrhagic shock can be immediately controlled by blocking the proximal part of the hemorrhagic point using either resuscitative thoracotomy for aortic cross-clamping or insertion of a large-caliber (10–14Fr) resuscitative endovascular balloon occlusion of the aorta (REBOA) device via the femoral artery. However, such methods are very invasive and have various complications. With recent progress in endovascular treatment, a low-profile REBOA device (7Fr) has been developed.