Cellular and Enzymatic Determinants Impacting the Exolytic Action of an Anti-Staphylococcal Enzybiotic.

Bacteriophage endolysins are bacteriolytic enzymes that have been explored as potential weapons to fight antibiotic-resistant bacteria. Despite several studies support the application of endolysins as enzybiotics, detailed knowledge on cellular and enzymatic factors affecting their lytic activity is still missing. The bacterial membrane proton motive force (PMF) and certain cell wall glycopolymers of Gram-positive bacteria have been implicated in some tolerance to endolysins. Here, we studied how the anti-staphylococcal endolysin Lys11, a modular enzyme with two catalytic domains (peptidase and amidase) and a cell binding domain (CBD11), responded to changes in the chemical and/or electric gradients of the PMF (ΔpH and Δψ, respectively). We show that simultaneous dissipation of both gradients enhances endolysin binding to cells and lytic activity. The collapse of ΔpH is preponderant in the stimulation of Lys11 lytic action, while the dissipation of Δψ is mainly associated with higher endolysin binding. Interestingly, this binding depends on the amidase domain. The peptidase domain is responsible for most of the Lys11 bacteriolytic activity. Wall teichoic acids (WTAs) are confirmed as major determinants of endolysin tolerance, in part by severely hindering CBD11 binding activity. In conclusion, the PMF and WTA interfere differently with the endolysin functional domains, affecting both the binding and catalytic efficiencies.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes.

Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule-dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live-cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule-dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome-targeted MIRO1 fusion protein, we demonstrate that MIRO1-mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome-motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease.

The Use of Video-Head Impulse Test in Different Head Positions in Vertical Nystagmus and Ataxia Associated with Probable Thiamine Deficiency.

Upward and downward bias of eye movement signals in the semicircular canals (SCC)- and/or otolith-related central pathways have been proposed to explain the occurrence of vertical nystagmus (downbeat nystagmus [DBN] and upbeat nystagmus [UBN]) and its frequent modulation with head position. Video-head impulse test (VHIT), usually performed in upright position, is a recent development for measuring SCC function. We performed longitudinal nystagmus and VHIT assessments in different head positions in a patient with probable thiamine deficiency, in order to explore a possible relationship between the positional behavior of vertical nystagmus and SCC function. Initially, UBN in upright position changed to DBN in prone position and remained relatively unchanged in supine position. This was associated with both anterior and posterior SCC hyperactive responses in upright position, and a relative enhancement of the anterior SCC responses in prone position and the posterior SCC responses in supine position. Over 1 year, in prone position, change from UBN to DBN and the enhancement of anterior SCC responses remained, while in supine position, UBN either decreased or changed to DBN, when compared to upright position. This was associated with a relative enhancement of the anterior SCC responses in supine position, albeit inconsistently, and the presence of posterior SCC hypoactive responses in all positions, including prone. While not contradicting a primary otolithic dysfunction in the genesis of UBN change to DBN with head position, we provide evidence for positional modulation of SCC function in thiamine deficiency and a possible relationship with nystagmus positional behavior.

Telomere shortening and aortic plaque progression in Apoliprotein E knockout mice after pulmonary exposure to candle light combustion particles.

Particles from burning candles contribute to the overall indoor exposure to particulate matter (PM). However, little is known about the effects of indoor sources of particles on cardiovascular disease endpoints. This study investigated the effect of pulmonary exposure to particles from combustion of candles and progression of atherosclerosis. Telomere shortening was assessed in tissues due to its relationship to risk of cardiovascular diseases. The particles were collected from burning candles and used for toxicological studies in cultured endothelial cells and apolipoprotein E (ApoE) knockout mice. Three hours exposure to particles increased the production of reactive oxygen species in endothelial cells, whereas there was no effect on cytotoxicity. Intratracheal instillation of particles (0.5 or 5 mg/kg) once a week for 5 weeks in ApoE-/- mice was associated with an accelerated progression of atherosclerosis in aorta and telomere shortening in the lung and spleen, whereas there was no effect on inflammation in the lungs (i.e. cell numbers), cell damage (i.e. lactate dehydrogenase) and lung barrier damage (i.e. protein concentration) as measured in bronchoalveolar lavage fluid. The results indicate that particles from burning candles are hazardous and this indoor emission source is an important contribution to the health risk of exposure to PM.

Ferulic acid supresses Th2 immune response and prevents remodeling in ovalbumin-induced pulmonary allergy associated with inhibition of epithelial-derived cytokines.

Asthma is characterized by intermittent airway obstruction and chronic inflammation, orchestrated primarily by Th2 cytokines. There is a strong rationale for developing new asthma therapies, since current treatment protocols present side effects and may not be effective in cases of difficult-to-control asthma. The purpose of this study was to evaluate the effect of ferulic acid, a phenolic acid commonly present in plants, in the ovalbumin-induced pulmonary allergy murine model. METHODS: BALB/c mice were sensitized and challenged with ovalbumin, and treatments were provided by gavage. Six groups of mice (n = 6) were studied, labeled as: control, pulmonary allergy, dexamethasone, and 3 receiving ferulic acid (at 25, 50, and 100 mg/kg). Lung tissue, bronchoalveolar lavage fluid and serum were collected for analysis. RESULTS: Ferulic acid treatment inhibited an established allergic Th2-response by decreasing the key features of pulmonary allergy, including lung and airway inflammation, eosinophil infiltration, mucus production and serum levels of OVA-specific IgE. These results were associated with lower levels of CCL20, CCL11 and CCL5 chemokines and IL-4, IL-5, IL-13, TSLP, IL-25 and IL-33 cytokines in lung tissue homogenate. CONCLUSIONS: In this study it was demonstrated for the first time that ferulic acid treatment is able to suppress one of the main features of the airway remodeling, indicated by reduction of mucus production, besides the Th2 pathogenic response on ovalbumin-induced pulmonary allergy. Taken together, results shows that the immunopathological mechanism underlying these effects is linked to a reduction of the epithelial-derived chemokines and cytokines, suggesting that ferulic acid may be useful as a potential therapeutic agent for asthma.

Cerebrovascular Dissemination in Time and Space as a Predictor of Cardioembolism.

BACKGROUND: Cardioembolism has tendency to recur and cause lesions in distinct cerebrovascular territories. Using the imaging characteristics of cerebral lesions to determine dissemination in time and space (DTS) is a concept already used in other neurologic conditions; however, it has never been applied as a diagnostic tool in ischemic stroke etiology. AIM: This study aimed to assess DTS as a diagnostic marker of cardioembolism. METHODS: We enrolled consecutive patients with acute ischemic stroke of various etiologies admitted in a cerebrovascular disease nursery from a university hospital in a retrospective cohort study. We excluded patients with coexisting etiologies, incomplete study, or without an acute vascular lesion on computed tomography scan. Lacunar infarctions were not considered. Cerebrovascular territory was divided into right anterior, left anterior, and posterior. Localization of the acute vascular lesion(s), existence of previous vascular lesions, and their respective areas were analyzed. The presence of dissemination in time, space, or DTS was determined. RESULTS: We included 661 patients (mean age: 74.05 years (SD: 13.01)). Cardioembolism was the etiology with most DTS (30.47% of cardioembolic strokes); DT occurred more frequently within the atherosclerotic subtype (9.88%); DS was more prevalent within the arterial dissection group (3.33%). There was a statistically significant difference in stroke etiology between patients with DTS and patients without dissemination (P < .001). DTS had 81.67% specificity, 30.47% sensitivity, 66.67% positive predictive value, and 49.40% negative predictive value for the identification of cardioembolism. CONCLUSION: DTS is a specific diagnostic predictor of cardioembolic stroke and may be helpful in guiding etiologic investigation.

Hepatitis C virus NS3-4A inhibits the peroxisomal MAVS-dependent antiviral signalling response.

Hepatitis C virus (HCV) is the cause of one of the most prevalent viral infections worldwide. Upon infection, the HCV genome activates the RIG-I-MAVS signalling pathway leading to the production of direct antiviral effectors which prevent important steps in viral propagation. MAVS localizes at peroxisomes and mitochondria and coordinate the activation of an effective antiviral response: peroxisomal MAVS is responsible for a rapid but short-termed antiviral response, while the mitochondrial MAVS is associated with the activation of a stable response with delayed kinetics. The HCV NS3-4A protease was shown to specifically cleave the mitochondrial MAVS, inhibiting the downstream response. In this study, we have analysed whether HCV NS3-4A is also able to cleave the peroxisomal MAVS and whether this would have any effect on the cellular antiviral response. We show that NS3-4A is indeed able to specifically cleave this protein and release it into the cytosol, a mechanism that seems to occur at a similar kinetic rate as the cleavage of the mitochondrial MAVS. Under these conditions, RIG-I-like receptor (RLR) signalling from peroxisomes is blocked and antiviral gene expression is inhibited. Our results also show that NS3-4A is able to localize at peroxisomes in the absence of MAVS. However, mutation studies have shown that this localization pattern is preferred in the presence of a fully cleavable MAVS. These findings present evidence of a viral evasion strategy that disrupts RLR signalling on peroxisomes and provide an excellent example of how a single viral evasion strategy can block innate immune signalling from different organelles.

Atherosclerosis and vasomotor dysfunction in arteries of animals after exposure to combustion-derived particulate matter or nanomaterials.

Exposure to particulate matter (PM) from traffic vehicles is hazardous to the vascular system, leading to clinical manifestations and mortality due to ischemic heart disease. By analogy, nanomaterials may also be associated with the same outcomes. Here, the effects of exposure to PM from ambient air, diesel exhaust and certain nanomaterials on atherosclerosis and vasomotor function in animals have been assessed. The majority of studies have used pulmonary exposure by inhalation or instillation, although there are some studies on non-pulmonary routes such as the gastrointestinal tract. Airway exposure to air pollution particles and nanomaterials is associated with similar effects on atherosclerosis progression, augmented vasoconstriction and blunted vasorelaxation responses in arteries, whereas exposure to diesel exhaust is associated with lower responses. At present, there is no convincing evidence of dose-dependent effects across studies. Oxidative stress and inflammation have been observed in the arterial wall of PM-exposed animals with vasomotor dysfunction or plaque progression. From the data, it is evident that pulmonary and systemic inflammation does not seem to be necessary for these vascular effects to occur. Furthermore, there is inconsistent evidence with regard to altered plasma lipid profile and systemic inflammation as a key step in vasomotor dysfunction and progression of atherosclerosis in PM-exposed animals. In summary, the results show that certain nanomaterials, including TiO2, carbon black and carbon nanotubes, have similar hazards to the vascular system as combustion-derived PM.

Bullous hemorrhagic dermatosis induced by enoxaparin.

The bullous hemorrhagic dermatosis induced by enoxaparin is a rare adverse reaction, which may be under-reported given its favorable evolution. We report a 71-year-old man who developed hemorrhagic bullae at sites distant from subcutaneous enoxaparin injections. It is important that clinicians be aware of the different adverse reactions of these widely used drugs.