Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration.

Synaptotagmin-1 and synaptotagmin-7 are two prominent calcium sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding calcium, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examine the biophysical properties of these two synaptotagmin isoforms and compare their interactions with phospholipid membranes. We discover that synaptotagmin-1-membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a super-penetrator. We exploit these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to calcium.

Development of an animal model for rosacea­like skin lesions caused by Demodex.

To develop an animal model of rosacea-like skin lesions caused by Demodex mites, a suspension of Demodex mites was injected into the skin of Japanese rabbits. The pathology of the skin lesion was assessed using H&E staining after 4 weeks of modeling. The skin lesions observed after 4 weeks were further treated with the recombinant bovine basic fibroblast growth factor (rbFGF) gel. Untreated lesions in the same rabbit were considered as the blank control. Erythema papules were observed in the model rabbit skin and could be observed most clearly in the 2nd week. Lumpy foreign bodies, telangiectasia and granuloma-like structure were observed in the model rabbit in the 1st, 2nd, and 3rd weeks, respectively. An organized granuloma-like structure was observed in the 4th week. The color of the skin lesions became lighter than that of the self-control after 4 weeks of rbFGF treatment. In conclusion, the model of Demodex-induced rosacea-like skin lesions can be developed through intradermal injection of suspension of Demodex mites into Japanese rabbits. The model can mimic the phenotype of skin lesions and histopathological manifestations in the Demodex mite-positive patient with rosacea.

Rapid and Gentle Immunopurification of Brain Synaptic Vesicles.

Current methods to isolate synaptic vesicles (SVs), the organellar quanta of synaptic transmission, require highly specialized materials and up to 24 h. These technical obstacles have thus far limited the study of SVs in models of synaptic function and pathophysiology. Here, we describe techniques for the rapid isolation of SVs by immunoprecipitation with widely available antibodies conjugated to magnetic beads. We report that the inexpensive rho1D4 monoclonal antibody binds SVs and show that elution with the 1D4 peptide yields native vesicles that are ≥ 10-fold purer than those obtained with classical techniques. These methods substantially widen the accessibility of SVs, enabling their purification in 60-90 min for downstream analyses including mass spectrometry and cryo-electron microscopy. Immunopurified SV preparations from mouse brain contained apolipoprotein E, the LDL receptor Lrp1, and enzymes involved in lipid metabolism, suggesting that SVs may play direct roles in lipid homeostasis and lipoprotein trafficking at the nerve terminal.SIGNIFICANCE STATEMENT SVs are small organelles that form and recycle at nerve terminals to enable synaptic transmission. Much remains unknown about the processes that enable the formation and function of SVs. Moreover, nerve terminals appear to be particularly vulnerable to pathophysiologic processes underlying neurodegenerative diseases and schizophrenia. Although techniques to purify synaptic vesicles thus have the potential to yield significant insights into physiology and pathophysiology of nerve terminals, current methods rely on either esoteric materials or expression of transgenes. This article addresses these problems by establishing robust, efficient methods for SV purification using widely available materials, and it highlights several promising areas of future study arising from proteomic analyses of immunopurified SVs.

Autophagy-Inflammation Interplay During Infection: Balancing Pathogen Clearance and Host Inflammation.

Inflammation is an essential immune response of the host against infections but is often over-activated, leading to a variety of disorders. Autophagy, a conserved degradation pathway, also protects cells by capturing intracellular pathogens that enter the cell and transporting them to the lysosome for clearance. Dysfunctional autophagy is often associated with uncontrolled inflammatory responses during infection. In recent years, more and more research has focused on the crosstalk between autophagy and inflammation. In this paper, we review the latest research advances in this field, hoping to gain insight into the mechanisms by which the body balances autophagy and inflammation in infections and how this mechanism can be used to fight infections better.

The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

Cholesterol stabilizes recombinant exocytic fusion pores by altering membrane bending rigidity.

SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.

Demodex-induced Lupus miliaris disseminatus faciei: A case report.

RATIONALE: Lupus miliaris disseminatus faciei (LMDF) is an inflammatory granulomatous skin disease without a clear etiology that frequently involves the middle area of the face and the upper eyelids. Pathological features of the disease include caseation necrosis and epithelioid granuloma. Consensus treatment for LMDF is currently unavailable. PATIENT CONCERNS: A 47-year-old Chinese female patient who presented with facial pruritic, erythematous papules 8 months before this study. She was diagnosed with skin tuberculosis at another hospital and given antituberculosis medication. However, the treatment was not efficacious. DIAGNOSES: In this study, the diagnosis of Demodex-induced LMDF was made by a dermatologist according to physical examination, skin biopsy pathology, and microscopic examination. INTERVENTIONS: The patient was given ornidazole tablets (500 mg twice a day) and recombinant bovine basic fibroblast growth factor gel (0.2 g/cm twice a day) for an 8-week period. OUTCOMES: Eight weeks after the treatment, the facial erythematous papules were improved, and no new skin lesions were observed. The patient showed no signs of recurrence during the 6-month follow-up. LESSONS SUBSECTIONS: This case showed that ornidazole combined with recombinant bovine basic fibroblast growth factor gel might be useful in treatment of Demodex-induced LMDF. In addition, the results suggested that pathological caseation necrosis was caused by a series of inflammatory and immune responses to Demodex infection.

Improved telangiectasia and reduced recurrence rate of rosacea after treatment with 540 nm-wavelength intense pulsed light: A prospective randomized controlled trial with a 2-year follow-up.

The aim of the present study was to evaluate the clinical efficacy and safety of 540 nm-wavelength intense pulsed light (IPL) for the treatment of telangiectasia in late-stage rosacea. Between July 2013 and January 2016, patients with rosacea who tested positive for Demodex folliculorum were recruited. Patients received anti-mite therapy and were then randomly apportioned to receive either three 540 nm-IPL treatments at 4-week intervals (IPL group), or no treatment (control group). Telangiectasia was assessed by the same clinician at baseline and at follow-up intervals over 2 years, where ≥90% clearance of telangiectasia was considered to indicate effective treatment. The rates of effective treatment, improvement (≥30% clearance) and recurrence (original or neo-location) were compared in both groups. After 33 patients were lost during follow-up, the IPL and control groups were comprised of 107 and 120 patients for the final analysis, respectively. The rates of effective treatment and total efficacy in the IPL group (66.36 and 95.33%, respectively) were found to be significantly higher compared with those of the control group (0 and 30.83%, respectively). By contrast, the rates of recurrence were found to be lower in the IPL group (8.41%) compared with the control group (48.33%). Redness-to-blisters associated with IPL treatment (9.7% of analyzed patients) subsided within one week and hyperpigmentation (1.9%) within 3 months. To conclude, treatment with 540 nm-IPL improved facial telangiectasia in late-stage rosacea that remained after sequential anti-mite therapy and effectively reduced the recurrence of rosacea. The present study was registered into the Chinese Clinical Trial Registry under the title 'Sequential therapy for mites folliculitis' (Trial registration number: ChiCTR-IPR-15006451; approved May 27, 2015).

Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores.

The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.