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•Myosin-1C is used for actomyosin-mediated expulsion of an essential blood clotting factor (VWF).•Myosin-1C links the exocytic actomyosin ring to PIP2 on the plasma membrane, forming anchor points that allow for maximal VWF secretion.

Membrane fusion triggered by Ca2+is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1–4. For neurotransmitter release, the Ca2+sensitivity is introduced by interactions between the Ca2+sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7–11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2+-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca2+-triggered fusion at physiological Ca2+concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca2+-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

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Blunt chest trauma causes pulmonary and systemic inflammation. It is still a matter of debate whether the long-term course of this inflammatory response is associated with persistent impairment of lung function. We hypothesized that an increase of inflammatory biomarkers may still be present at later time points after blunt chest trauma, eventually, despite normalized lung mechanics and gas exchange. Anesthetized spontaneously breathing male C57BL6J mice underwent a blast wave–induced blunt chest trauma or sham procedure. Twelve and 24 h later, blood gases and lung mechanics were measured, together with blood, bronchoalveolar lavage (BAL), and tissue cytokine concentrations (multiplex cytokine kit); heme oxygenase 1 (HO-1), activated caspase-3, Bcl-xL, and Bax expression (Western blotting); nuclear factor-B activation (electrophoretic mobility shift assay); nitrotyrosine formation; and purinergic (P2XR4 and P2XR7) receptor expression (immunohistochemistry). Histological damage was assessed by hematoxylin and eosin and periodic acid-Schiff staining. High-resolution respirometry allowed assessing mitochondrial respiration in diaphragm biopsies. Chest trauma significantly increased tissue and BAL cytokine levels, associated with a significant increase in HO-1, purinergic receptor expression, and tissue nitrotyrosine formation. In contrast, lung mechanics, gas exchange, and histological damage did not show any significant difference between sham and trauma groups. Activation of the immune response remains present at later time points after murine blunt chest trauma. Discordance of the increased local inflammatory response and preserved pulmonary function may be explained by a dissociation of the immune response and lung function, such as previously suggested after experimental sepsis.

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses.

AbstractRetinal pigment epithelial (RPE) cells provide crucial functions for the maintenance of the retinal environment. We investigated the phagocytotic mechanisms of RPE cells evaluating the question whether particle uptake underlies a diurnal rhythm. Additionally, a possible connection of volume regulation and the phagocytotic function of RPE cells was studied. As antiviral nucleoside analogues influence cell-volume-regulating mechanisms, we tested several antiviral drugs. Cultured primary RPE cells and a permanent cell line (ARPE-19) were tested for uptake of europium-labeled microspheres quantified by time-resolved fluorometry. Cells were also exposed to cyclic illumination or continuous light and dark culture conditions. Inhibitors of cytoskeleton (microtubuli, actin) and osmotic swelling were also tested. Ingested FITC-labeled microparticles were found in phagosomes strongly associated which the cytoskeleton as they could not be easily moved by laser tweezer microscopy. Phagocytosis was observed predominately during dark intervals and was reduced by continuous light exposure. The diurnal rhythm of unsynchronized RPE cultures was abolished by microtubule inhibitors although no inhibition of overall particle uptake by cytoskeletal blockers was observed. Hypoosmotic swelling of RPE also decreased phagocytosis. Acyclovir was found inhibitory in ARPE-19 cells, whereas azidothymidine showed a protracted inhibiting activity on primary RPE cells and ganciclovir was inactive in both cell types. The presence of a diurnal rhythm also in culture indicates genetic determination of light-regulated particle uptake. This mechanism appears to be influenced by the regulation of cell volume and microtubule function. Inhibition of RPE function by antiviral drugs is a novel finding and in accordance with interferences of the tested drugs with cellular chloride channels described earlier. It may give a hint towards possible ocular side effects in the long-term use of nucleoside-analogous substances.Copyright © 2006 S. Karger AG, Basel

The behavior of several simultaneously trapped, micrometer-sized particles in a fiber-optical trap consisting of two opposing single-mode fibers delivering counterpropagating, near-IR laser beams strongly depends on the size of the particles. Whereas beads that are considerably larger than the laser wavelength are pressed against each other in an axial line, smaller beads spontaneously arrange themselves into regular chains of equidistantly separated particles suspended in space with increasing separation for increasing bead diameter. A simple model based on self-organization by means of diffraction from the particles is capable of explaining the basic features of our experimental observations in the investigated range of bead diameters and refractive indices.

The release of surfactant from alveolar type II cells is essential to lower the surface tension in the lung and to facilitate inspiration. However, the factors controlling dispersal and diffusion of this hydrophobic material are still poorly understood. Here we report that release of surfactant from the fused vesicle, termed lamellar body (LB), resisted mechanical forces applied by optical tweezers: At constant trapping force, the probability to expand LB contents, i.e., to “pull” surfactant into the extracellular fluid, increased with time after LB fusion with the plasma membrane, consistent with slow fusion pore expansion in these cells. Elevations of the cytoplasmic Ca2+ concentration ([Ca2+]c) had a similar effect. Inasmuch as surfactant did not disintegrate in the extracellular space, this method permitted for the first time the determination of elastic and recoil properties of the macromolecular complex, yielding a spring constant of ∼12.5 pN/μm. This is the first functional evidence that release of hydrophobic material is mechanically impeded and occurs in an “all-or-none” fashion. This mode of release is most probably the result of cohesive forces of surfactant, combined with adhesive forces and/or retaining forces exerted by a constrictive fusion pore acting as a regulated mechanical barrier, withstanding forces up to 160 pN. In independent experiments equiaxial strain was exerted on cells without optical tweezers. Strain facilitated surfactant release from preexisting fused vesicles, consistent with the view of mechanical impediments during the release process, which can be overcome by cell strain.

Surfactant secretion must be regulated to maintain a low surface tension in the lung during various conditions such as exercise. In vitro studies reveal a slow, unique exocytotic process at the interface of stimulated and constitutive exocytosis. The exocytotic mechanisms and sites of regulation in vivo, however, are still poorly understood.

1Using conventional microelectrodes, the perforated patch clamp technique and fluorescence microscopy with fura‐2, we investigated the relationship between the cell membrane potential, whole‐cell currents and the free cytoplasmic Ca2+concentration ([Ca2+]i) in response to 10 nM endothelin‐1 (ET) in a rat respiratory epithelial cell line (L2).2Microelectrode experiments revealed that ET caused an immediate depolarization of the cell membrane potential (Vm) by 25 mV, which was unaffected by Na+replacement with N‐methyl‐D‐glucamine+(NMDG+) or by omission of bath Ca2+. In contrast, ET depolarized the cells by 61 mV in the presence of low Cl−(6 mM), resulting in a complete breakdown of Vm.3In perforated patch clamp experiments, the ET‐induced whole‐cell current (IET) exhibited a slight outward rectification with a reversal potential (Vrev) of ‐22·7 mV. IETwas reduced by 85 % in low Cl−(6 mM), but was unaffected by Ca2+removal, Na+replacement with NMDG+, pipette K+replacement with Cs+or 1 mM Ni2+in the bath.4IETwas unaffected by (+)‐isradipine (100 nM), a specific L‐type Ca2+channel (L‐VDCC) blocker. Transient inward Sr2+currents through L‐VDCCs were blocked by ET.5ET induced a biphasic Ca2+signal, consisting of a ‘peak’ and a ‘plateau’ elevation of [Ca2+]i. Simultaneous patch clamp and fura‐2 measurements revealed that IETcoincided with intracellular Ca2+release but clearly outlasted the elevation of [Ca2+]i. When the rise of [Ca2+]iwas prevented by pretreatment with thapsigargin in a Ca2+‐free bath, both activation time and amplitude of IETwere reduced. Under these conditions, ET caused a decrease of [Ca2+]i.6The Cl−channel blocker mefenamic acid (MFA) had a dual, concentration‐dependent effect on both IETand the ET‐induced ‘plateau’ elevation of [Ca2+]i: an increase at 10 μM, but an almost complete block at 100 μM. The effect of MFA on IETpreceded the effect on [Ca2+]i.7The ET‐induced ‘plateau’[Ca2+]ifell below control values in a low‐Cl−(6 mM) solution.8These data indicate an amplifying function of intracellular Ca2+release on an otherwise Ca2+‐independent, unique Cl−current by ET. Moreover, this Cl−current appears to be functionally coupled with dihydropyridine (DHP)‐insensitive Ca2+entry, suggesting a modulatory role for long‐lasting effects of ET.

Salivary antibodies were studied in 112 infants between 1 day and 8 yr of life. SIgA anticasein was present from birth in breast-fed and bottle-fed infants. Bottle-feeding resulted in significantly higher concentrations of SIgA anticasein at 3 wk to 3 months of life as compared to breast-feeding. Salivary anticasein declined toward the end of the 1st yr and was present in less than half of the children older than 1 yr. Salivary anti-lactoglobulin was also present at birth in some infants. Levels increased slightly over the following 3 months but remained low. Only a minority of older children had this antibody in their saliva.

ABSTRACT. Salivary antibodies were studied in 112 infants between 1 day and 8 yr of life. SIgA anticasein was present from birth in breast-fed and bottle-fed infants. Bottle-feeding resulted in significantly higher concentrations of SIgA anticasein at 3 wk to 3 months of life as compared to breast-feeding. Salivary anticasein declined toward the end of the 1st yr and was present in less than half of the children older than 1 yr. Salivary anti-lactoglobulin was also present at birth in some infants. Levels increased slightly over the following 3 months but remained low. Only a minority of older children had this antibody in their saliva.

The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho‐GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a “mix‐and‐match” approach containing the specific Rho‐inhibiting clostridial C3 enzyme and three PMN‐binding peptides using an avidin platform. Selective delivery of the C3 Rho‐inhibitor with these complexes into the cytosol of human neutrophil‐like NB‐4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type‐selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post‐traumatic injury. Supramolecular protein complexes comprising of neutrophil‐targeting peptides and the Rho‐inhibiting C3 enzymeare assembled by a “mix‐and‐match” strategy using avidin as a supramolecular “glue.” The complexes selectively address primary polymorphonuclear leukocytes (PMNs). The approach holds immense promise to decipher the role of PMNs in infection and inflammation and for diseases that are associated with hyperactivity and reactivity of PMNs.

Excessive recruitment of polymorphonuclear neutrophils (PMNs) into injured tissues after traumatic injuries can be highly detrimental to patients and remains a significant source of morbidity and mortality. In article number 1900665by Seah Ling Kuan, Tanja Weil, Holger Barth, and co‐workers, the authors develop supramolecular protein complexes for pharmacological modulation of PMNs. In this way, a novel therapeutic approach is formulated by fusing PMN‐binding peptides to a Rho‐inhibiting enzyme on a supramolecular platform.