Formation Of Small Vesicles Research Articles

Phosphatidylserine-dependent structure of synaptogyrin remodels the synaptic vesicle membrane

Synaptic vesicles are small membrane-enclosed organelles that store neurotransmitters at presynaptic terminals. The uniform morphology of synaptic vesicles is important for brain function, because it enables the storage of well-defined amounts of neurotransmitters and thus reliable synaptic transmission. Here, we show that the synaptic vesicle membrane protein synaptogyrin cooperates with the lipid phosphatidylserine to remodel the synaptic vesicle membrane. Using NMR spectroscopy, we determine the high-resolution structure of synaptogyrin and identify specific binding sites for phosphatidylserine. We further show that phosphatidylserine binding changes the transmembrane structure of synaptogyrin and is critical for membrane bending and the formation of small vesicles. Cooperative binding of phosphatidylserine to both a cytoplasmic and intravesicular lysine-arginine cluster in synaptogyrin is required for the formation of small vesicles. Together with other synaptic vesicle proteins, synaptogyrin thus can sculpt the membrane of synaptic vesicles.

Combination of multifunctional ursolic acid with kinase inhibitors for anti-cancer drug carrier vesicles

A sterically stabilized unilamellar nanocarrier vesicle (SSV) system containing dipalmitoylphosphatidylcholine, cholesterol, ursolic acid and PEGylated phospholipid has been developed by exploiting the structural advantages of ursolic acid: by spontaneously attaching to the lipid head groups, it induces curvature at the outer side of the bilayers, allowing the preparation of size-limited vesicles without extrusion. Ursolic acid (UA) also interacts with the PEG chains, supporting steric stabilization even when the amount of PEGylated phospholipid is reduced. Using fluorescence immunohistochemistry, vesicles containing ursolic acid (UA-SSVs) were found to accumulate in the tumor in 3 h on xenografted mouse, suggesting the potential use of these vesicles for passive tumor targeting.Further on, mono- and combination therapy with UA and six different kinase inhibitors (crizotinib, erlotinib, foretinib, gefitinib, refametinib, trametinib) was tested on seven cancer cell-lines. In most combinations synergism was observed, in the case of trametinib even at very low concentration (0.001 μM), which targets the MAPK pathway most often activated in human cancers. The coupled intercalation of UA and trametinib (2:1 molar ratio) into vesicles causes further structural advantageous molecular interactions, promoting the formation of small vesicles. The high drug:lipid molar ratio (~0.5) in the novel type of co-delivery vesicles enables their direct medical application, possibly also overcoming the multidrug resistance effect.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Vaccinal prevention of chickenpox: relevance of the problem

Chickenpox is an acute viral infection which is a serious public health problem. According to the World Health Organization, there are 4,2 million severe forms of disease requiring hospitalization and 4,200 deaths annually. The CP cause is DNA-containing herpesvirus 3, Varicella-zoster. The first symptoms of the disease appear after the incubation period, which lasts from 10 to 21 days, and are manifested by fever, malaise and the appearance of a characteristic rash in the form of small vesicles with transparent content on the skin and mucous membranes. The vesicles are progressively covered with crust, which disappears within 7–10 days. Chicken pox is more frequent in the mild and moderate form, but it may develop severe forms, atypical course, complications (most often bacterial skin infections and lesions of the nervous system are registered in children (more often – chicken encephalitis), and in adults - pneumonia). Vaccinal prevention is the most effective and scientifically proven method of infection control, and chickenpox currently belongs to such infections. The currently used chickenpox vaccines were developed in the mid 1970s, used in some countries from the mid 1980s, and from the early 1990s began to be used in routine immunization programmes in leading countries. Since then, their safety and immunogenicity as well as high efficiency of routine chickenpox vaccine prevention in both healthy and high-risk individuals have been proved. Accumulated experience has shown that a vaccine can reduce the number of cases and the frequency of hospitalizations and deaths when mass immunization is carried out.

Treatment of herpes labialis by photodynamic therapy: Study protocol clinical trial (SPIRIT compliant).

Background:Lesions of herpes labialis are caused by the herpes simplex virus type 1 and cause pain and aesthetic compromise. It is characterized by the formation of small vesicles that coalesce and rupture forming extremely painful ulcers, that evolve to crusts, dry desquamations until their complete remission. Currently the treatment of these lesions is done with acyclovir. Although it diminishes the symptomatology, it causes viral resistance and does not prevent the recurrence of the lesions. It is known that antimicrobial photodynamic therapy (aPDT) has numerous advantages, among them: the reduction of the time of remission, and does not cause resistance. This protocol will determine the effectiveness of PDT in lesions of herpes labialis.Materials and methods:A total of 30 patients with herpes labialis in the prodromal stage of vesicles, ulcers, and crusts will be selected to participate in the study and randomized into 2 groups: G1 control and G2 experimental. After signing Research Ethics Committee and TA, patients in group G1 will undergo the standard gold treatment for herpes labialis with acyclovir and simulated PDT treatment. Patients in the experimental G2 group will be treated simulating the gold standard treatment of herpes labialis (placebo) and PDT. In all patients, saliva samples will be collected for analysis of cytokines, and will be performed exfoliative cytology in the lesions. The pain will be assessed through a pain scale and a questionnaire of quality of life related to oral health (OHIP-14) will be given to them. Patients will continue to be followed up after 7 days, 1 month, 3 months, and 6 months; if there is a recurrence of the lesion, they will contact the researchers.Clinical registration: clinicaltrials.gov - NCT 04037475. Registered on July 2019.

Freeze-substitution transmission electron microscopy of gentian shoot tips cryopreserved at ultra low temperatures.

Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells. In addition, the cells of PVS2-treated shoot tips exhibited considerable plasmolysis and formation of small vesicles in cytoplasm. These results clearly demonstrate that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot tips.

Evaluation of chromocystoscopy in the diagnosis of cystitis in female donkeys.

Early detection of cystitis in equine is essential to improve the prognosis and outcome of therapy. However, the conventional white light endoscopy is not sufficiently accurate for this purpose. Hence, this study evaluated chromoendoscopy as a recent diagnostic tool for cystitis in female donkeys. For this purpose, 5 apparently normal donkeys (control group) and 5 female donkeys with cystitis (diseased group) were used. Physical and rectal examinations, urine analysis, white light cystoscopy, methylene blue-based chromoendoscopy and histopathology were performed in all animals. Turbid urine exhibiting an alkaline pH and a significant (P = .02) increase in the numbers of RBCs and WBCs was observed in the diseased group compared to the control one. In the control group, white light cystoscopy showed a smooth pale pink glistening mucosa with two openings of the ureters and visible submucosal blood vessels. During chromocystoscopy, faint bluish discoloration of the mucosal surface with clearly visible submucosal blood vessels was detectable. These findings were correlated with the histopathological findings of the biopsies collected from the urinary bladder. In the diseased group, white-light cystoscopy showed clearly visible blood vessels, mildly hyperaemic mucosa in focal or diffuse forms and small vesicle formation. Chromocystoscopy revealed dark bluish oedematous and irregular mucosa either in a focal form or a diffuse form (marbled appearance) with deeply stained submucosal blood vessels. Histopathologically, the urothelium was hyperplastic with squamous metaplasia and the lamina propria was infiltrated with few leukocytes and congested blood vessels. Small bluish dots representing the absorbed methylene blue dye were seen in the inflamed areas against the lightly stained mucosa of the bladder. Severe diffuse necrotic cystitis was also seen with bacterial aggregations on the surface. Gram’s staining revealed both gram positive bacilli and Gram positive coccobacilli. In conclusion, chromoendoscopy is a helpful tool for early diagnosis of cystitis in female donkeys and enables targeted biopsies, which improves the prognosis and outcome of therapy.

Novel fluorochromes label tonoplast in living plant cells and reveal changes in vacuolar organization after treatment with protein phosphatase inhibitors.

The recently synthesized isocyanonaphtalene derivatives ACAIN and CACAIN are fluorochromes excitable at wavelengths of around 366nm and bind cysteine-rich proteins with hydrophobic motifs. We show that these compounds preferentially label tonoplasts in living Arabidopsis and tobacco (Nicotiana tabacum SR1) cells. ACAIN-labeled membranes co-localized with the GFP signal in plants expressing GFP-δ-TIP (TIP2;1) (a tonoplast aquaporin) fusion protein. ACAIN preserved the dynamics of vacuolar structures. tip2;1 and triple tip1;1-tip1;2-tip2;1 knockout mutants showed weaker ACAIN signal in tonoplasts. The fluorochrome is also suitable for the labeling and detection of specific (cysteine-rich, hydrophobic) proteins from crude cell protein extracts following SDS-PAGE and TIP mutants show altered labeling patterns; however, it appears that ACAIN labels a large variety of tonoplast proteins. ACAIN/CACAIN could be used for the detection of altered vacuolar organization induced by the heptapeptide natural toxin microcystin-LR (MCY-LR), a potent inhibitor of both type 1 and 2A protein phosphatases and a ROS inducer. As revealed both in plants with GFP-TIP2;1 fusions and in wild-type (Columbia) plants labeled with ACAIN/CACAIN, MCY-LR induces the formation of small vesicles, concomitantly with the absence of the large vegetative vacuoles characteristic for differentiated cells. TEM studies of MCY-LR-treated Arabidopsis cells proved the presence of multimembrane vesicles, with characteristics of lytic vacuoles or autophagosomes. Moreover, MCY-LR is a stronger inducer of small vesicle formation than okadaic acid (which inhibits preferentially PP2A) and tautomycin (which inhibits preferentially PP1). ACAIN and CACAIN emerge as useful novel tools to study plant vacuole biogenesis and programmed cell death.

Formation and dynamics of endoplasmic reticulum-like lipid nanotube networks.

We report on the self-organized formation and dynamics of artificial lipid nanotube networks, which, in terms of morphology and behavior, resemble the endoplasmic reticulum(ER) of biological cells. The networks, initially generated from a solid-supported planar phospholipid membrane, undergo a morphological transformation, triggered by the chelation and removal of Ca2+ from the environment surrounding the membrane. Calcium depletion gradually causes de-pinning, thus de-wetting, at the membrane-substrate interface. We observe dynamic re-arrangements very similar to the ones reported for the cellular ER, such as sliding of tubes and formation of new junctions, and quantify these transformations. We also show occurrences of the dynamic replacement of lipidic particles on nanotubes as indicators for the existence of a tension gradient throughout the network, as well as the spontaneous formation of small vesicles from semi-free floating tubes. We propose that these artificial networks are suitable to serve as a bottom-up-generated structural model for the cellular ER, whose fascinating characteristic morphology is suggested to be tied to its biological function, but with respect to formation, dynamics, and functional details still incompletely understood.

Benzo[ghi]perylene activates the AHR pathway to exert biological effects on the NL-20 human bronchial cell line.

Polycyclic aromatic hydrocarbons (PAH) are produced by incomplete combustion of organic material. In the Mexico City atmosphere, the most abundant PAH is benzo[ghi]perylene (BghiP), a gasoline combustion marker. At present, there are no reports of the effects of BghiP on human bronchial cells, so the aim of the study was to evaluate the effects in vitro of BghiP on the NL-20 cell line. Results showed that BghiP induced the formation of small vesicles throughout the cytoplasm, with absence of nuclear fragmentation. At 48h exposition, damage in cell membrane increased significantly at 1.24μg/mL of BghiP (p<0.05). Immunocytochemistry revealed that BghiP provokes nuclear translocation of AhR receptor, which indicates that this compound can induce transcription of genes via receptor binding (AhR pathway activation). BghiP induced a two-fold increase (p<0.05) in the expression of AhR and CYP4B1 (a lung-specific pathway effector). In the presence of the receptor antagonist CH-223191, the loss of viability, the nuclear translocation and the overexpression of genes decreased, though this did not prevent the formation of vesicles. BghiP induced oxidative stress and in presence of the receptor antagonist this increased significantly. In conclusion, BghiP can activate the overexpression of AhR and CYP4B1, and the effects are abated by the AhR receptor antagonist. This is the first report to prove that BghiP utilizes the AhR pathway to exert its toxic effects on the NL-20 human bronchial cell line .

Fluorescent sensibility of microarrays through functionalized adhesive polydiacetylene vesicles

We present a design of ‘smart’ gluing interfaces with combined adhesive properties and intrinsic fluorescent sensing properties. Therefore, polymer–peptide amphiphiles with adhesive functional head-groups were coupled to a diacetylene tail and polymerized after formation of small vesicles. The vesicles were immobilized and confined into micro-arrays created by a photo-patterned perfluorinated film onto the substrate. Fluorescent properties of the vesicles in aqueous suspension were quantified to build standard curves for fluorescent intensity as a function of vesicle concentration. The fluorescent properties of surface-confined vesicles were quantified for different vesicle concentration and external normal load, by using a micro-chip reader. A combination of fluorescent microscopy and image processing was applied to demonstrate the fluorescent sensitivity of the adhesive micro-arrays as a function of vesicle composition, concentration and thermal annealing. These observations allow estimating the quality of the adhesive interface. Finally, the fluorescent sensitivity of the adhesive micro-array to mechanical solicitations such as normal load, internal shear under parallel and rotational sliding was investigated. The observations serve as a proof-of-concept for the fluorescent sensitivity of the adhesive bond as a function of the operational conditions. In future, this design may serve as an assessment tool for optimization or detection of local defects and device failure, e.g. in integrated microsystem components.

Electron tomography and cryo-SEM characterization reveals novel ultrastructural features of host-parasite interaction during Chlamydia abortus infection

Chlamydia (C.) abortus is a widely spread pathogen among ruminants that can be transmitted to women during pregnancy leading to severe systemic infection with consecutive abortion. As a member of the Chlamydiaceae, C. abortus shares the characteristic feature of an obligate intracellular biphasic developmental cycle with two morphological forms including elementary bodies (EBs) and reticulate bodies (RBs). In contrast to other chlamydial species, C. abortus ultrastructure has not been investigated yet. To do so, samples were fixed by high-pressure freezing and processed by different electron microscopic methods. Freeze-substituted samples were analysed by transmission electron microscopy, scanning transmission electron microscopical tomography and immuno-electron microscopy, and freeze-fractured samples were analysed by cryo-scanning electron microscopy. Here, we present three ultrastructural features of C. abortus that have not been reported up to now. Firstly, the morphological evidence that C. abortus is equipped with the type three secretion system. Secondly, the accumulation and even coating of whole inclusion bodies by membrane complexes consisting of multiple closely adjacent membranes which seems to be a C. abortus specific feature. Thirdly, the formation of small vesicles in the periplasmic space of RBs in the second half of the developmental cycle. Concerning the time point of their formation and the fact that they harbour chlamydial components, these vesicles might be morphological correlates of an intermediate step during the process of redifferentiation of RBs into EBs. As this feature has also been shown for C. trachomatis and C. pneumoniae, it might be a common characteristic of the family of Chlamydiaceae.

Shear-Induced Structural Transition and Recovery in the Salt-Free Catanionic Surfactant Systems Containing Deoxycholic Acid

Shear-induced structural transition and recovery were studied by freeze-fracture transmission electron microscopy, cryogenic transmission electron microscopy (cryo-TEM), rheological measurements, and the variation of birefringent textures in the system of tetradecyltrimethylammonium hydroxide (TTAOH)/lauric acid (LA)/deoxycholic acid (DeCA)/H(2)O as a function of the molar fraction of DeCA [x = n(DeCA)/(n(DeCA) + n(LA))]. At x = 0.3, giant vesicles and planar lamellar structures were formed before exerting shearing. Shear thickening and large hysteresis loop are observed under shearing, indicating the occurrence of structural transition. Multilamellar and close-packed vesicles were determined by cryo-TEM observations. Exerting further shearing, the elastic properties of the sample were increased due to the strip off of the outer shell of the vesicles and formation of small vesicles. After shearing was stopped, the sample can slowly relax back to the original state. At x = 0.25, lamellar structures, giant vesicles, and small unilamellar vesicles were observed. Cryo-TEM observations show that the multilamellar vesicles can be formed after exerting shearing, and the sample cannot spontaneously recover to the original state. At x = 0.2, vesicles are dominant in the solution, and the aggregates structures are almost the same before and after shearing. Shearing can increase the elastic properties of the sample, which is ascribed to the strip off of the outer shells of the multilamellar vesicles, forming much smaller vesicles in the solution.

Further observation on histopathological alterations of adult Schistosoma japonicum harbored in mice following treatment with mefloquine at a smaller single dose

The purpose of the present study is to assess the histopathological alterations of adult schistosomes caused by a smaller dose of mefloquine. Mice were infected with Schistosoma japonicum cercariae for 35 days and then treated with a single 200 mg/kg oral dose of mefloquine. Groups of mice were killed between 12 h and 28 days posttreatment, and the livers were removed, fixed, and processed routinely and examined by light microscopy. Twelve hours to 48 h or 3 days posttreatment, 10.3% to 53.3% of male worms and 10% to 25% of female worms shifted to the liver revealed normal appearance. However, 46.7% to 69.2% of male worms and 45.5% to 75% of female worms showed signs of degeneration, including moderate or high swelling of tegument and/or muscles with roughing surface or formation of small vesicles beneath the tegument or collapse of damaged tegument, light swelling of parenchymal tissues, and light dilatation of gut. These histopathological changes aggravated either in extent or in severity along with time, especially the speed of emergence of dead worm granuloma that was faster in female worms than in male ones. In female worms, severe damage to the vitelline glands was universal, which was characterized by necrosis and karyopyknosis of vitelline cells and emergence of small vacuoles among the vitelline gland lobules. Seven days to 28 days posttreatment, almost all of the female worms shifted to the liver were classified as dead or dead worm granuloma, while the percentage of dead male worm and its granuloma examined in the liver was 52.1% to 53.3%. The results indicate that smaller dose of mefloquine (200 mg/kg) still shows strong histopathological response to kill female schistosomes, but its lethal effect against male worms is weakened.

Alternative Mechanisms for the Interaction of the Cell-Penetrating Peptides Penetratin and the TAT Peptide with Lipid Bilayers

Cell-penetrating peptides (CPPs) have recently attracted much interest due to their apparent ability to penetrate cell membranes in an energy-independent manner. Here molecular-dynamics simulation techniques were used to study the interaction of two CPPs: penetratin and the TAT peptide with 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospolipid bilayers shed light on alternative mechanisms by which these peptides might cross biological membranes. In contrast to previous simulation studies of charged peptides interacting with lipid bilayers, no spontaneous formation of transmembrane pores was observed. Instead, the simulations suggest that the peptides may enter the cell by micropinocytosis, whereby the peptides induce curvature in the membrane, ultimately leading to the formation of small vesicles within the cell that encapsulate the peptides. Specifically, multiple peptides were observed to induce large deformations in the lipid bilayer that persisted throughout the timescale of the simulations (hundreds of nanoseconds). Pore formation could be induced in simulations in which an external potential was used to pull a single penetratin or TAT peptide into the membrane. With the use of umbrella-sampling techniques, the free energy of inserting a single penetratin peptide into a DPPC bilayer was estimated to be ∼75 kJmol−1, which suggests that the spontaneous penetration of single peptides would require a timescale of at least seconds to minutes. This work also illustrates the extent to which the results of such simulations can depend on the initial conditions, the extent of equilibration, the size of the system, and the conditions under which the simulations are performed. The implications of this with respect to the current systems and to simulations of membrane-peptide interactions in general are discussed.

Surfactin-Triggered Small Vesicle Formation of Negatively Charged Membranes: A Novel Membrane-Lysis Mechanism

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri=200) and is complete for Ri=50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

Membrane interaction and perturbation mechanisms induced by two cationic cell penetrating peptides with distinct charge distribution

Independently from the cell penetrating peptide uptake mechanism (endocytic or not), the interaction of the peptide with the lipid bilayer remains a common issue that needs further investigation. The cell penetrating or antimicrobial properties of exogenous peptides require probably different preliminary interactions with the plasma membrane. Herein, we have employed 31P NMR, differential scanning calorimetry and CD to study the membrane interaction and perturbation mechanisms of two basic peptides with similar length but distinct charge distribution, penetratin (non-amphipathic) and RL16, a secondary amphipathic peptide. The peptide effects on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dipalmitoleoyl phosphatidylethanolamine (DiPoPE) were investigated. We have found that, even though both peptides are cationic, their interaction with zwitterionic versus anionic lipids is markedly distinct. Penetratin greatly affects the temperature, enthalpy and cooperativity of DMPG main phase transition but does not affect those of DMPC while RL16 presents opposite effects. Additionally, it was found that penetratin induces a negative curvature whereas RL16 induces a positive one, since a decrease in the fluid lamellar to inverted hexagonal phase transition temperature of DiPoPE ( T H) was observed for penetratin and an increase for RL16. Contrary to penetratin, 31P NMR of samples containing DMPC MLVs and RL16 shows an isotropic signal indicative of the formation of small vesicles, concomitant with a great decrease in sample turbidity both below and at the phase transition temperature. Opposite effects were also observed on DMPG where both peptides provoke strong aggregation and precipitation. Both CPPs adopt helical structures when contacting with anionic lipids, and possess a dual behavior by either presenting their cationic or hydrophobic domains towards the phospholipid face, depending on the lipid nature (anionic vs zwitterionic, respectively). Surprisingly, the increase of electrostatic interactions at the water membrane interface prevents the insertion of RL16 hydrophobic region in the bilayer, but is essential for the interaction of penetratin. Modulation of amphipathic profiles and charge distribution of CPPs can alter the balance of hydrophobic and electrostatic membrane interaction leading to translocation or and membrane permeabilisation. Penetratin has a relative pure CPP behavior whereas RL16 presents mixed CPP/AMP properties. A better understanding of those processes is essential to unveil their cell translocation mechanism.

A case of necrotizing fasciitis with septic shock in a cat caused byAcinetobacter baumannii

A 4-year-old, neutered female, domestic shorthair cat admitted to the animal hospital for recurrent constipation presumed to be due to post-traumatic injuries, went into shock with signs including fever and ataxia followed by stupor. On the fifth day of hospitalization, the cat developed severe, diffuse oedema of the ventral abdomen with multifocal to coalescing erythematous areas and small vesicle formation. The results of bacteriological cultures of liver, spleen and kidney specimens led to the diagnosis of Acinetobacter baumannii sepsis. Histopathological findings of skin samples taken during necropsy showed an extensive epidermal and dermal necrosis with septic vasculitis and numerous intralesional gram-negative bacteria. Detection of the bla(OXA-51-like) gene specific for A. baumannii by PCR, performed retrospectively on samples of the deep layers of the skin, confirmed the presence of A. baumannii also in the cutaneous lesions. To our knowledge this is the first report of a necrotizing fasciitis with septic shock in a cat caused by A. baumannii.

Myosin-1a powers the sliding of apical membrane along microvillar actin bundles

Microvilli are actin-rich membrane protrusions common to a variety of epithelial cell types. Within microvilli of the enterocyte brush border (BB), myosin-1a (Myo1a) forms an ordered ensemble of bridges that link the plasma membrane to the underlying polarized actin bundle. Despite decades of investigation, the function of this unique actomyosin array has remained unclear. Here, we show that addition of ATP to isolated BBs induces a plus end–directed translation of apical membrane along microvillar actin bundles. Upon reaching microvillar tips, membrane is “shed” into solution in the form of small vesicles. Because this movement demonstrates the polarity, velocity, and nucleotide dependence expected for a Myo1a-driven process, and BBs lacking Myo1a fail to undergo membrane translation, we conclude that Myo1a powers this novel form of motility. Thus, in addition to providing a means for amplifying apical surface area, we propose that microvilli function as actomyosin contractile arrays that power the release of BB membrane vesicles into the intestinal lumen.

Shape transitions in lipid membranes and protein mediated vesicle fusion and fission

In the cell, the plasma membrane is often densely decorated by transmembrane proteins. The morphology and dynamics of the membrane are strongly influenced by the presence of proteins. In this paper, we use a coarse-grained model to explore the composite membrane-protein system and develop a simulation methodology based on thermodynamic integration to examine free energy changes during membrane shape transitions. The authors show that a critical concentration of conical membrane proteins or proteins with nonzero spontaneous curvature can drive the formation of small vesicles. The driving force of vesicle budding stems from the preference of proteins to gather in regions of high curvature. A sufficiently high concentration of proteins therefore can influence the topology of the membrane. The biological significance of our results is discussed.