Reduction of focal sweating by lipid nanoparticle-delivered myricetin.

Myricetin-a flavonoid capable of inhibiting the SNARE complex formation in neurons-reduces focal sweating after skin-application when delivers as encapsulated in lipid nanoparticles (M-LNPs). The stability of M-LNP enables efficient delivery of myricetin to sudomotor nerves located underneath sweat glands through transappendageal pathways while free myricetin just remained on the skin. Furthermore, release of myricetin from M-LNP is accelerated through lipase-/esterase-induced lipolysis in the skin-appendages, enabling uptake of myricetin by the surrounding cells. The amount of sweat is reduced by 55% after application of M-LNP (0.8 mg kg-1) on the mouse footpad. This is comparable to that of subcutaneously injected anticholinergic agents [0.25 mg kg-1 glycopyrrolate; 0.8 U kg-1 botulinum neurotoxin-A-type (BoNT/A)]. M-LNP neither shows a distal effect after skin-application nor induced cellular/ocular toxicity. In conclusion, M-LNP is an efficient skin-applicable antiperspirant. SNARE-inhibitory small molecules with suitable delivery systems have the potential to replace many BoNT/A interventions for which self-applications are preferred.

Characterization of Ginkgo biloba Leaf Flavonoids as Neuroexocytosis Regulators.

Ginkgo biloba leaf (GBL) is known as a potential source of bioactive flavonoids, such as quercetin, arresting the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-zippering. Here, the GBL flavonoids were isolated in two different manners and then examined for their bioactivity, physicochemical stability, and biocompatibility. The majority of flavonoids in the non-hydrolyzed and acidolyzed isolates, termed non-hydrolyzed isolate (NI) and acidolyzed isolate (AI) hereafter, were rich in flavonol glycosides and aglycones, respectively. Glycosidic/aglyconic quercetin and kaempferol were abundant in both NI and AI, whereas a little of apigenin, luteolin, and isorhamnetin were found in AI. NI was more thermostable in all pH ranges than quercetin, kaempferol, and AI. NI and AI both inhibited neurotransmitter release from differentiated neuronal PC-12 cells. NI and AI showed 1/2-1/3 lower EC50/CC50 values than quercetin and kaempferol. The NI and AI exhibited no toxicity assessed by the tests on chorioallantoic membranes of hen's eggs, removing toxicological concerns of irritation potential. Moreover, GBL isolates, particularly AI, showed antioxidant and anti-inflammatory activities in the use below the CC50 levels. Taken together, these results suggest that GBL isolates that are rich in antioxidant flavonoids are effective anti-neuroexocytotic agents with high stability and low toxicity.

Reliability of Korean Triage and Acuity Scale: Interrater Agreement between Two Experienced Nurses by Real-Time Triage and Analysis of Influencing Factors to Disagreement of Triage Levels.

BACKGROUND: All emergency centers in Korea use the Korean Triage and Acuity Scale (KTAS) as their initial triage tool. However, KTAS has been used without verification of its reliability. In this study, we assess the interrater agreement of KTAS by two independent nurses in real-time and analyse the factors which have an effect on the disagreement of KTAS levels. METHODS: This study was a prospective observational study conducted with patients who visited an emergency department (ED). Two teams, each composed of two nurses, triaged patients and recorded KTAS level and the main complaint from the list of 167 KTAS complaints, as well as modifiers. Interrater reliability between the two nurses in each team was assessed by weighted-kappa. Pearson's χ² test was conducted to determine if there were differences between each nurse's KTAS levels, depending on whether they chose the same complaints and the same modifiers or not. RESULTS: The two teams triaged a total of 1,998 patients who visited the ED. Weighted-kappa value was 0.772 (95% confidence interval [CI], 0.750-0.794). Patients triaged by different chosen complaints showed (38.0%) higher inconsistency rate in KTAS levels than those triaged by the same complaint (10.9%, P < 0.001). When nurses chose the same complaint and different modifiers, the ratio of different levels (50.5%) was higher than that of the same complaint and same modifier (8.1%, P < 0.001). CONCLUSION: This study showed that KTAS is a reliable tool. Selected complaints and modifiers are confirmed as important factors for reliability; therefore, selecting them properly should be emphasized during KTAS training courses.

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regarding the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening.

Visualization of SNARE-Mediated Hemifusion between Giant Unilamellar Vesicles Arrested by Myricetin.

Neurotransmitters are released within a millisecond after Ca2+ arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca2+-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca2+ triggering. It has been shown that the flavonoid myricetin arrests soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated vesicle fusion at hemifusion, but that the hemifused vesicles spontaneously convert to full fusion when the myricetin clamp is removed by the enzyme laccase. In the present study, we visualized SNARE-mediated hemifusion between two SNARE-reconstituted giant unilamellar vesicles (GUVs) arrested by myricetin. The large size of the GUVs enabled us to directly image the hemifusion between them. When two merging GUVs were labeled with different fluorescent dyes, GUV pairs showed asymmetric fluorescence intensities depending on the position on the GUV pair consistent with what is expected for hemifusion. The flow of lipids from one vesicle to the other was revealed with fluorescence recovery after photobleaching (FRAP), indicating that the two membranes had hemifused. These results support the hypothesis that hemifusion may be the molecular status that primes Ca2+-triggered millisecond exocytosis. This study represents the first imaging of SNARE-driven hemifusion between GUVs.

Co-expression of two heterologous lactate dehydrogenases genes in Kluyveromyces marxianus for l-lactic acid production.

Lactic acid (LA) is a versatile compound used in the food, pharmaceutical, textile, leather, and chemical industries. Biological production of LA is possible by yeast strains expressing a bacterial gene encoding l-lactate dehydrogenase (LDH). Kluyveromyces marxianus is an emerging non-conventional yeast with various phenotypes of industrial interest. However, it has not been extensively studied for LA production. In this study, K. marxianus was engineered to express and co-express various heterologous LDH enzymes that were reported to have different pH optimums. Specifically, three LDH enzymes originating from Staphylococcus epidermidis (SeLDH; optimal at pH 5.6), Lactobacillus acidophilus (LaLDH; optimal at pH 5.3), and Bos taurus (BtLDH; optimal at pH 9.8) were functionally expressed individually and in combination in K. marxianus, and the resulting strains were compared in terms of LA production. A strain co-expressing SeLDH and LaLDH (KM5 La+SeLDH) produced 16.0g/L LA, whereas the strains expressing those enzymes individually produced only 8.4 and 6.8g/L, respectively. This co-expressing strain produced 24.0g/L LA with a yield of 0.48g/g glucose in the presence of CaCO3. Our results suggest that co-expression of LDH enzymes with different pH optimums provides sufficient LDH activity under dynamic intracellular pH conditions, leading to enhanced production of LA compared to individual expression of the LDH enzymes.

A Feasibility Study of Real-Time Remote CT Reading for Suspected Acute Appendicitis Using an iPhone.

We aimed to evaluate the feasibility of an iPhone-based remote control system as a real-time remote computed tomography (CT) reading tool for suspected appendicitis using a third-generation (3G) network under suboptimal illumination. One hundred twenty abdominal CT scans were selected; 60 had no signs of appendicitis, whereas the remaining 60 had signs of appendicitis. The 16 raters reviewed the images using the liquid crystal display (LCD) monitor of a picture archiving and communication system (PACS) workstation, as well as using an iPhone connected to the PACS workstation via a remote control system. We graded the probability of the presence of acute appendicitis for each examination using a five-point Likert scale. The overall sensitivity and specificity for the diagnosis of suspected appendicitis using the iPhone and the LCD monitor were high, and they were not significantly different (sensitivity P = 1.00, specificity P = 0.14). The average areas under the receiver operating characteristic curves for all CT readings with the iPhone and LCD monitor were 0.978 (confidence interval 0.965-0.991) and 0.974 (0.960-0.988), respectively, and the two devices did not have significantly different diagnostic performances (P = 0.55). The inter-rater agreement for both devices was very good; the kappa value for the iPhone was 0.809 (0.793-0.826), and that for the LCD monitor was 0.817 (0.801-0.834). Each rater had moderate-to-very good intra-observer agreement between the two devices. We verified the feasibility of an iPhone-based remote control system as a real-time remote CT reading tool for identifying suspected appendicitis using a 3G network and suboptimal illumination.

Diagnostic accuracy of the inverted grayscale rib series for detection of rib fracture in minor chest trauma.

STUDY OBJECTIVE: To assess whether inverted grayscale rib series, used alone or as an additional imaging modality, improves diagnostic accuracy of rib fractures of emergency medicine (EM) residents in minor chest trauma. METHODS: Twenty readers, including 5 junior and 5 senior EM residents and 10 fourth-year medical students, independently reviewed 110 patients' radiographs during 3 sessions. Session 1 used conventional grayscale rib series, session 2 used inverted grayscale rib series, and session 3 used both conventional and inverted grayscale images. The McNemar test was used to compare the sensitivities and specificities of the diagnostic methods, and to compare their sensitivities and specificities for detecting more than 3 rib fractures. Interobserver agreement was assessed using Cohen κ analysis. RESULTS: For senior EM residents, there was no difference in sensitivity (P = .283) and accuracy (P = .888) between conventional rib series and the double-modality method. For junior EM residents and medical students, the double modality offered higher diagnostic sensitivity (P < .001, P = .001) and accuracy (P = .006, P = .002) than did conventional radiography. In cases with more than 3 rib fractures, who required specialist trauma care, the double modality provided greater sensitivity and accuracy among junior EM residents (P = .035 and P = .035, respectively) and medical students (P = .010, P = .010) than did conventional radiography. CONCLUSIONS: In the diagnosis of rib fractures, the combined use of conventional and inverted grayscale rib series increases the diagnostic accuracy of less biased readers by conventional grayscale image like junior EM residents and medical students.

An assessment of the iPad 2 as a CT teleradiology tool using brain CT with subtle intracranial hemorrhage under conventional illumination.

We examined the potential of the iPad 2 as a teleradiologic tool for evaluating brain computed tomography (CT) with subtle hemorrhage in the conventional lighting conditions which are common situations in the remote CT reading. The comparison of the clinician's performance was undertaken through detecting hemorrhage by the iPad 2 and the clinical liquid crystal display (LCD) monitor. We selected 100 brain CT exams performed for head trauma or headache. Fifty had subtle radiological signs of intracranial hemorrhage (ICH), while the other 50 showed no significant abnormality. Five emergency medicine physicians reviewed these brain CT scans using the iPad 2 and the LCD monitor, scoring the probability of ICH on each exam on a five-point scale. Result showed high sensitivities and specificities in both devices. We generated receiver operating characteristic curves and calculated the average area under the curve of the iPad 2 and the LCD (0.935 and 0.900). Using the iPad 2 and reliable internet connectivity, clinicians can provide remote evaluation of brain CT with subtle hemorrhage under suboptimal viewing condition. Considering the distinct advantages of the iPad 2, the popular out-of-hospital use of mobile CT teleradiology would be anticipated soon.