Sonoprocessing enhances the stabilization of fisetin by encapsulation in Saccharomyces cerevisiae cells.

The objective of this study was to investigate for the first time the role of S. cerevisiae natural barriers and endogenous cytoplasmatic bodies on the stabilization of fisetin encapsulated via sonoprocessing coupled to freeze-drying (FD) or spray drying (SD). Both protocols of encapsulation improved the resistance of fisetin against thermal treatments (between 60 and 150 °C) and photochemical-induced deterioration (light exposition for 60 days) compared to non-encapsulated fisetin (antioxidant activity retention of approximately 55% and 90%, respectively). When stored under constant relative humidity (from 32.8 to 90%) for 60 days, yeast carriers improved the half-life time of fisetin by up to 4-fold. Spray dried particles were smaller (4.9 µm) and showed higher fisetin release after simulated gastrointestinal digestion (55.7%) when compared to FD. Freeze-dried particles, in turn, tended to agglomerate more than SD (zeta potential -19.7 mV), resulting in reduced loading features (6.3 mg/g) and less efficient protection of fisetin to heat, photo, and moisture-induced deterioration. Overall, spray-dried sonoprocessed fisetin capsules are an efficient way to preserve fisetin against harsh conditions. Altogether, this report shows that sonoprocessing coupled to drying is an efficient, creative, and straightforward route to protect and deliver lipophilic fisetin using yeast capsules for food applications.

Synthesis and characterization of new acid-functionalized porphyrins displaying antimicrobial activity against gram positive bacteria, yeasts and filamentous fungi with or without ultra-high irradiance.

The antimicrobial activity of new acid-functionalized porphyrins, with or without ultra-high irradiance, was investigated. Antibacterial efficacy was evaluated against Staphylococcus aureus (methicillin-resistant or methicillin-sensitive strains) and antifungal efficacy was evaluated against the yeast Candida albicans and the filamentous fungi Aspergillus fumigatus. Overall, the porphyrins tested are more effective against S. aureus. The best results were obtained with zinc diacid porphyrins 4 and 5 after only 3 min of ultra-high irradiation (500 mW/cm2, 405 nm), demonstrating that acid-functionalized porphyrins are promising as novel antimicrobial drugs for surface disinfection.

Smooth Muscle Cell Relaxation Worsens Aortic Dilatation and Clinical Presentation in a BAPN/Angiotensin II-Induced Aortic Dissection Model in Rats.

INTRODUCTION: Beta-aminopropionitrile (BAPN) administration is a chemically induced model for preclinical aortic pathologies research. Angiotensin II (AngII) has been widely used to promotes aortic dissections in mice. Here, we provide insight on a modified aortic dissection model in rats. The effect of smooth muscle cell (SMC) relaxation with vasodilators is studied in this model. METHODS: Forty Sprague-Dawley rats were divided in 4 groups: control, isosorbide dinitrate (ISDN, 30 mg/kg/day) in the drinking water, BAPN (0.02%) in the food, BAPN + ISDN (same doses). Thoracic and abdominal aortic diameters were evaluated through transthoracic ultrasound echography. After 6 weeks, all rats were infused with AngII (1 mg/kg/day) subcutaneously. Survival and type of aortic events were numbered. Histological and histochemical analyses of aorta were performed. RESULTS: Initial telesystolic ascending aorta diameters were equal in all groups and became significantly larger in the BAPN + ISDN group compared to the BAPN group (control: 3.37 ± 0.17 mm, ISDN: 3.49 ± 0.16 mm, BAPN: 3.53 ± 0.13 mm, BAPN + ISDN: 3.61 ± 0.16 mm, analysis of variance p < 0.0001). BAPN followed by AngII infusion showed a significant lower survival rate (p = 0.029) and produced a large panel of aortic events. Association of ISDN and BAPN significantly reduces survival (p = 0.001) and provides more aortic events compared to BAPN alone (p = 0.031). In both BAPN-treated groups, orcein staining revealed split and dissected elastic fibers in the media, alcian blue staining showed mucoid degeneration of the aortic wall, and Perls-diaminobenzidine staining revealed an accumulation of Fe2+. CONCLUSION: SMC relaxation with ISDN increases aortic dilatation, worsens aortic prognosis, and reproduces human histological findings in a low-dose BAPN/AngII-induced aortic dissection model in rats.

Ultra-high irradiance (UHI) blue light: highlighting the potential of a novel LED-based device for short antifungal treatments of food contact surfaces.

Microbial food spoilage is an important cause of health and economic issues and can occur via resilient contamination of food surfaces. Novel technologies, such as the use of visible light, have seen the light of day to overcome the drawbacks associated with surface disinfection treatments. However, most studies report that photo-inactivation of microorganisms with visible light requires long time treatments. In the present study, a novel light electroluminescent diode (LED)-based device was designed to generate irradiation at an ultra-high power density (901.1 mW/cm2). The efficacy of this technology was investigated with the inactivation of the yeast S. cerevisiae. Short-time treatments (below 10 min) at 405 nm induced a ~4.5 log reduction rate of the cultivable yeast population. The rate of inactivation was positively correlated to the overall energy received by the sample and, at a similar energy, to the power density dispatched by the lamp. A successful disinfection of several food contact surfaces (stainless steel, glass, polypropylene, polyethylene) was achieved as S. cerevisiae was completely inactivated within 5 min of treatments. The disinfection of stainless steel was particularly effective with a complete inactivation of the yeast after 2 min of treatment. This ultra-high irradiance technology could represent a novel cost- and time-effective candidate for microbial inactivation of food surfaces. These treatments could see applications beyond the food industry, in segments such as healthcare or public transport. KEY POINTS : • A novel LED-based device was designed to emit ultra-high irradiance blue light • Short time treatments induced high rate of inhibition of S. cerevisiae • Multiple food contact surfaces were entirely disinfected with 5-min treatments.

Osmoporation is a versatile technique to encapsulate fisetin using the probiotic bacteria Lactobacillus acidophilus.

The objective of this study was to evaluate the performance of Lactobacillus acidophilus cells as a novel encapsulating carrier for fisetin via osmoporation. Initially, the effects of osmotic pressure and initial fisetin concentration on the performance of the osmoporation process were evaluated. The best results were achieved when 15 MPa was applied, while the maximum loading capacity was reached when fisetin concentration of 2.0 mg·mL-1 was used. For these conditions, the cell viability, encapsulation efficiency (EE), and encapsulated fisetin content (EF) were 72%, 28%, and 0.990 mg, respectively. Further, the encapsulation was confirmed by Fourier transform-infrared (FT-IR), differential scanning calorimetry (DSC), and X-ray diffraction (XRD) analysis. DSC thermograms revealed an increase of 40 °C in the melting point of fisetin after encapsulation. In addition, the enhancement of fisetin bioaccessibility by osmoporated biocapsules is shown for the first time in the literature. When the fisetin biocapsules were subjected to in vitro gastrointestinal digestion, 99.6% of the encapsulated content were retained through the gastric stage and 45.5% were released during the intestinal stage, despite no active cells were detected during simulated digestion. These results suggest that alive cells are required for an effective osmoporation-assisted encapsulation process; however, osmoporated biocapsules can efficiently protect and preserve labile compounds, independently of their activity. Overall, this study demonstrated that osmoporation using probiotic L. acidophilus is a simple, versatile, and efficient technique to encapsulate and deliver lipophilic fisetin for food applications. KEY POINTS : •Fisetin is efficiently encapsulated into L. acidophilus via osmoporation. •Fisetin bioaccessibility is improved by osmoporation into L. acidophilus. •Release mechanisms of osmoporation carriers are independent of the cell activity.

Sonoprocessing is an effective strategy to encapsulate fisetin into Saccharomyces cerevisiae cells.

The encapsulation of fisetin into S. cerevisiae cells through sonoporation coupled with drying is reported for the first time in the literature. To establish the best conditions to maximize the amount of internalized fisetin, the cell density (5-10% w/v), fisetin concentration (1-3 mg/mL), acoustic energy density (0-333.3 W/L), and drying method (freeze-drying and spray drying) were analyzed through a Box-Behnken experimental design (BBD) coupled with response surface methodology (RSM). Higher encapsulation efficiency (EE) was achieved with a cell density of 10% w/v, while fisetin concentration of 3 mg/mL favored the encapsulation yield (EY) and antioxidant activity (AA). Higher EE (67.7%), EY (25.7 mg/g), and AA (90%) were registered when an acoustic density of 333.3 W/L was used. Furthermore, both drying protocols promoted fisetin encapsulation, but through spray drying, the EE, EY, and AA were 11.5%, 11.1%, and 26.6% higher than via freeze-drying, respectively. This work proved that fully filled biocapsules were produced through sonoprocessing, and their morphology was influenced by the acoustic energy and drying process. Overall, these results open new perspectives for the application of sonoprocessing-assisted encapsulation, paving the way for developing innovative yeast-based delivery systems for lipophilic compounds such as fisetin. KEY POINTS: • Sonoprocessing improves the encapsulation of fisetin into S. cerevisiae cells • Spray drying promotes fisetin loading into yeasts' intracellular space and cavities • Fisetin binding with yeast extracellular agents are favored by freeze-drying.

Design of a new lyoprotectant increasing freeze-dried Lactobacillus strain survival to long-term storage.

BACKGROUND: Stabilization of freeze-dried lactic acid bacteria during long-term storage is challenging for the food industry. Water activity of the lyophilizates is clearly related to the water availability and maintaining a low aw during storage allows to increase bacteria viability. The aim of this study was to achieve a low water activity after freeze-drying and subsequently during long-term storage through the design of a lyoprotectant. Indeed, for the same water content as sucrose (commonly used lyoprotectant), water activity is lower for some components such as whey, micellar casein or inulin. We hypothesized that the addition of these components in a lyoprotectant, with a higher bound water content than sucrose would improve lactobacilli strains survival to long-term storage. Therefore, in this study, 5% whey (w/v), 5% micellar casein (w/v) or 5% inulin (w/v) were added to a 5% sucrose solution (w/v) and compared with a lyoprotectant only composed of 5% sucrose (w/v). Protective effect of the four lyoprotectants was assessed measuring Lactiplantibacillus plantarum CNCM I-4459 survival and water activity after freeze-drying and during 9 months storage at 25 °C. RESULTS: The addition whey and inulin were not effective in increasing Lactiplantibacillus plantarum CNCM I-4459 survival to long-term-storage (4 log reduction at 9 months storage). However, the addition of micellar casein to sucrose increased drastically the protective effect of the lyoprotectant (3.6 log i.e. 0.4 log reduction at 9 months storage). Comparing to a lyoprotectant containing whey or inulin, a lyoprotectant containing micellar casein resulted in a lower water activity after freeze-drying and its maintenance during storage (0.13 ± 0.05). CONCLUSIONS: The addition of micellar casein to a sucrose solution, contrary to the addition of whey and inulin, resulted in a higher bacterial viability to long-term storage. Indeed, for the same water content as the others lyoprotectants, a significant lower water activity was obtained with micellar casein during storage. Probably due to high bound water content of micellar casein, less water could be available for chemical degradation reactions, responsible for bacterial damages during long-term storage. Therefore, the addition of this component to a sucrose solution could be an effective strategy for dried bacteria stabilization during long-term storage.

Pathogenic variants in THSD4, encoding the ADAMTS-like 6 protein, predispose to inherited thoracic aortic aneurysm.

PURPOSE: Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease with often unrecognized inherited forms. We sought to identify novel pathogenic variants associated with autosomal dominant inheritance of TAAD. METHODS: We analyzed exome sequencing data from 35 French TAAD families and performed next-generation sequencing capture panel of genes in 1114 unrelated TAAD patients. Functional effects of pathogenic variants identified were validated in cell, tissue, and mouse models. RESULTS: We identified five functional variants in THSD4 of which two heterozygous variants lead to a premature termination codon. THSD4 encodes ADAMTSL6 (member of the ADAMTS/L superfamily), a microfibril-associated protein that promotes fibrillin-1 matrix assembly. The THSD4 variants studied lead to haploinsufficiency or impaired assembly of fibrillin-1 microfibrils. Thsd4+/- mice showed progressive dilation of the thoracic aorta. Histologic examination of aortic samples from a patient carrying a THSD4 variant and from Thsd4+/- mice, revealed typical medial degeneration and diffuse disruption of extracellular matrix. CONCLUSION: These findings highlight the role of ADAMTSL6 in aortic physiology and TAAD pathogenesis. They will improve TAAD management and help develop new targeted therapies.

Glenzocimab does not impact glycoprotein VI-dependent inflammatory haemostasis.

Not available.

Effect of devitrification on the survival and resistance of dried Saccharomyces cerevisiae yeast.

Yeasts are anhydrobiotes that accumulate large amounts of trehalose, which is involved in the vitrification of the cytoplasm during drastic desiccation. The effect of devitrification, which can be induced by the transient exposure of desiccated yeasts to increased humidity or elevated temperature, on the survival of yeast has been studied. A glass transition temperature (Tg)/water activity (aw) diagram of yeast was constructed based on differential scanning calorimetry analysis. The survival rate of yeasts that were equilibrated at different relative humidities (RHs) and temperature values over their Tg range was measured. The results revealed a long period of cell preservation at an intermediate RH (55%), with 100% survival observed after 3 months, a loss of 1.24 log colony-forming units/g recorded after 1 year at 25 °C and full preservation of viability at 75 °C for 60 min and at 100 °C and 12% RH for up to 10 min. These findings led us to conclude that dried yeast can resist low or intermediate RH values and elevated temperatures in the devitrified state. Considering the thermal and humidity fluctuations occurring in the yeast environments, we hypothesized that the supercooled state, which occurs immediately above the Tg after rehydration or heating, is a protective state that is involved in the persistence of yeasts at intermediate humidity levels. KEY POINTS: • Yeast survival for months in a supercooled state is observed at room temperature. • Dried yeasts survive a 10-min exposure to 100 °C in the supercooled state. • The supercooled state is suitable for yeast preservation.

Increased Survival of Lactococcus lactis Strains Subjected to Freeze-Drying after Cultivation in an Acid Medium: Involvement of Membrane Fluidity Cultivation in Acid Medium to Improve Bacterial Survival of Freeze-Drying.

RESEARCH BACKGROUND: Freeze-drying is the most widely used dehydration process in the food industry for the stabilization of bacteria. Studies have shown the effectiveness of an acid prestress in increasing the resistance of lactic acid bacteria to freeze-drying. Adaptation of bacteria to an acid stress is based on maintaining the properties of the plasma membrane. Indeed, the fatty acid composition of the membrane of lactic acid bacteria is often changed after an acid prestress. However, few studies have measured membrane fluidity after an acid stress during lactic acid bacterial strain cultivation. EXPERIMENTAL APPROACH: In order to use two pH profiles, the strains Lactococcus lactis NCDO 712 and NZ9000 were cultivated in two media, without any pH control. The two pH profiles obtained were representative of the initial medium composition, medium buffering properties and strain metabolism. Absorbance at 600 nm and pH were measured during bacterial cultivation. Then, the two strains were freeze-dried and their survival rates determined. Membrane fluidity was evaluated by fluorescence anisotropy measurements using a spectrofluorometer. RESULTS AND CONCLUSIONS: Cultivation under more acidic conditions significantly increased the survival during freeze-drying (p<0.05, ANOVA) of both strains. Moreover, in both strains of L. lactis, a more acidic condition during cultivation significantly increased membrane fluidity (p<0.05, ANOVA). Our results revealed that cultivation under such conditions, fluidifies the membrane and allows a better survival during freeze-drying of the two L. lactis strains. A more fluid membrane can facilitate membrane deformation and lateral reorganization of membrane components, critical for the maintenance of cellular integrity during dehydration and rehydration. NOVELTY AND SCIENTIFIC CONTRIBUTION: A better understanding of the involvement of membrane properties, especially of membrane fluidity, in bacterial resistance to dehydration is provided in this study.

DNA Content in Ischemic Stroke Thrombi Can Help Identify Cardioembolic Strokes Among Strokes of Undetermined Cause.

BACKGROUND AND PURPOSE: Identification of acute ischemic stroke (AIS) cause is crucial for guidance of secondary prevention. Previous studies have yielded inconsistent results regarding possible correlations between AIS cause and thrombus composition, as assessed by semiquantitative histological analysis. Here, we performed a correlation analysis between AIS cause and AIS thrombus cellular composition and content, as assessed using quantitative biochemical assays. METHODS: Homogenates of 250 patients with AIS thrombi were prepared by mechanical grinding. Platelet, red blood cell, and leukocyte content of AIS thrombi were estimated by quantification of GP (glycoprotein) VI, heme, and DNA in thrombus homogenates. AIS cause was defined as cardioembolic, noncardioembolic, or embolic stroke of undetermined source, according to the TOAST classification (Trial of ORG 10172 in Acute Stroke Treatment). RESULTS: Cardioembolic thrombi were richer in DNA (35.8 versus 13.8 ng/mg, P<0.001) and poorer in GPVI (0.104 versus 0.117 ng/mg, P=0.045) than noncardioembolic ones. The area under the receiver operating characteristic curve of DNA content to discriminate cardioembolic thrombi from noncardioembolic was 0.72 (95% CI, 0.63-0.81). With a threshold of 44.7 ng DNA/mg thrombus, 47% of thrombi from undetermined cause would be classified as cardioembolic with a specificity of 90%. CONCLUSIONS: Thrombus DNA content may provide an accurate biomarker for identification of cardioembolic thrombi in patients with AIS with embolic stroke of undetermined source. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03268668.

Persistence of Intraluminal Thrombus Makes Saccular Aneurysm More Biologically Active than Fusiform in an Experimental Rat Model.

INTRODUCTION: Saccular aneurysms are thought to have a worse prognosis than fusiform aneurysms in humans, due to hemodynamic reasons. However, data comparing hemodynamic and biology in saccular and fusiform aneurysms are lacking. The main objective was to evaluate the impact of aneurysm morphology on intra-luminal thrombus (ILT) formation and activity. METHODS: Forty Lewis rats were ran-domly divided into 2 groups of 20: "saccular" (Group A) and "fusiform" (Group B) aneurysms. Decellularized thoracic aortas from guinea pigs were xenografted to create saccular or fusiform aneurysms. Final imaging evaluation of the aneurysms was carried out during the third week, by quantitative Doppler ultrasound and magnetic resonance imaging. Assays of myeloperoxidase (MPO), platelet factor 4 (PF4), advanced oxidation protein products (AOPPs) iron and matrix metallopeptidase-9 (MMP-9) were performed as biological criteria. RESULTS: Quantitatively, saccular aneurysms are characterized by a more thicker ILT, lower inflow velocities and more important relative backflow velocities as compared to fusiform aneurysms. Compared to fusiform, saccular aneurysms released significantly more MPO (p = 0.004), PF4 (p = 0.02), AOPPs (p < 0.002), iron (p < 0.0001) and MMP-9 (p < 0.04). CONCLUSION: Experimental saccular and fusiform aneurysms show differential specific hemodynamics, which seem to impact the histology and the biology of the ILT in each type of aneurysm.

Haemodynamic stress-induced breaches of the arterial intima trigger inflammation and drive atherogenesis.

AIMS: Inflammatory mediators, including blood cells and their products, contribute critically to atherogenesis, but the igniting triggers of inflammation remain elusive. Atherosclerosis develops at sites of flow perturbation, where the enhanced haemodynamic stress could initiate the atherogenic inflammatory process due to the occurrence of mechanic injury. We investigated the role of haemodynamic stress-induced breaches, allowing the entry of blood cells in the arterial intima, in triggering inflammation-driven atherogenesis. METHODS AND RESULTS: Human coronary samples isolated from explanted hearts, (n = 47) displayed signs of blood entry (detected by the presence of iron, ferritin, and glycophorin A) in the subintimal space (54%) as assessed by histology, immunofluorescence, high resolution episcopic microscopy, and scanning electron microscopy. Computational flow dynamic analysis showed that intimal haemorrhagic events occurred at sites of flow disturbance. Experimental carotid arteries from Apoe deficient mice showed discrete endothelial breaches and intimal haemorrhagic events specifically occurring at the site of flow perturbation, within 3 days after the exacerbation of the local haemodynamic stress. Endothelial tearing was associated with increased VCAM-1 expression and, within 7 days, substantial Ly6G+ leucocytes accumulated at the sites of erythrocyte-derived iron and lipids droplets accumulation, pathological intimal thickening and positive oil red O staining. The formation of fatty streaks at the sites of intimal breaches was prevented by the depletion of Ly6G+ leucocytes, suggesting that the local injury driven by haemodynamic stress-induced breaches triggers atherogenic inflammation. CONCLUSION: Haemodynamic-driven breaches of the arterial intima drive atherogenic inflammation by triggering the recruitment of leucocyte at sites of disturbed arterial flow.

An Experimental Study of Paclitaxel Embolisation During Drug Coated Balloon Angioplasty.

OBJECTIVE: Drug coated balloons (DCB) improve the patency of femoropopliteal angioplasty but their use in infrapopliteal lesions is debateable as paclitaxel (PTX) particle embolisation has been suspected in some trials. The aim of this study was to compare experimentally five DCBs in terms of distal embolism of PTX. METHODS: Twenty-five New Zealand rabbits were divided into five groups according to the DCB used: Lutonix (Bard), In.Pact (Medtronic), Passeo-18 Lux (Biotronik), Ranger (Boston Scientific), and Stellarex (Spectranetics) (n = 5 in each group). After ligation of the right common iliac artery, a 4 × 40 mm DCB was inflated in the infrarenal aorta for 180 seconds. Rabbits were euthanised two hours after inflation of the DCB. The infrarenal aorta, a blood sample and three left hind leg muscles (tensor fasciae latae [TFL], vastus lateralis [VL], and tibialis anterior [TA] muscles) were harvested for blind measurement of PTX concentrations and histological analysis (PTX emboli count). RESULTS: In the TA muscle (the most distal), concentrations of PTX were significantly lower for the Ranger (0.067 ng/mg) than for the Lutonix (0.342 ng/mg; p = .008), In.Pact (0.370 ng/mg; p = .012), and Passeo-18-Lux (0.160 ng/mg; p = .021) DCBs. Similarly, concentrations of PTX were significantly lower for the Passeo-18-Lux than for the In.Pact (p = .028). Concentrations of PTX were not significantly different between DCBs in the TFL and VL muscles. Concentrations of PTX were found to be significantly higher in the plasma and lower in the aorta and on the DCBs after use of Lutonix compared with the four other DCBs. Histological analysis revealed evidence of embolised PTX crystals in small arterioles of all muscle tissue samples without any significant difference between the DCBs. CONCLUSIONS: This study suggests some differences regarding distal embolisation profiles between the five assessed DCBs. Although clinical implications remain to be demonstrated, the present results may have implications when choosing a DCB, especially in a critical limb ischaemia setting.

Efficient stabilisation of curcumin microencapsulated into yeast cells via osmoporation.

This study proposes the investigation of curcumin encapsulation into Saccharomyces cerevisiae cells through osmoporation as an efficient way of increasing curcumin stability. The influence of three process parameters (cell, ethanol and curcumin concentrations) on the encapsulation process was evaluated, and the obtained biocapsules were characterised for physical and photochemical stabilisation. Results showed that encapsulation efficiency was favoured by the increase of cells/curcumin ratio and ethanol concentration up to 60%. Differential scanning calorimetry (DSC) curves revealed that yeast encapsulation delayed the curcumin melting point up to 207 °C. Encapsulated curcumin retained over 80% of antioxidant activity after thermal treatment (150 °C) and over 70% after a 50-day exposure to artificial light. Photochemical stability of yeast-encapsulated curcumin was increased by 5.7-fold, and half-life time reached 181 days under illumination conditions. Overall, osmoporation-produced yeast biocapsules confirmed the versatility of osmoporation as an encapsulation technique and successfully improved curcumin stability.

Exploring antibody-dependent adaptive immunity against aortic extracellular matrix components in experimental aortic aneurysms.

BACKGROUND: Recent evidence suggests that adaptive immunity develops during abdominal aortic aneurysm evolution. Uncertainties remain about the antigens implicated and their role in inducing rupture. Because antigens from the extracellular matrix (ECM) have been suspected, the aim of this experimental study was to characterize the role of adaptive immunity directed against antigens from the aortic ECM. METHODS: In a first step, an experimental model of abdominal aortic aneurysm rupture based on adaptive immunity against the ECM was developed and characterized. Forty 4-week-old male Lewis rats were divided into two groups. In the ECM group (n = 20), rats were presensitized against the guinea pig aortic ECM before implantation of a decellularized aortic xenograft (DAX). In the control group (n = 20), rats were not presensitized before DAX implantation. In each group, half the rats were sacrificed at day 3 to analyze early mechanisms involved after DAX implantation. In a second step, we aimed to assess which ECM component was most efficient in inducing rupture. For this purpose, the nonfibrillar and fibrillar ECM components were sequentially extracted from the guinea pig aortic wall. Forty Lewis rats were then divided into four groups. Each group was presensitized against one ECM component (structural glycoproteins and proteoglycans, collagen, elastin alone, and elastin-associated glycoproteins) before DAX implantation. Apart from those that experienced rupture, rats were sacrificed at day 21. Xenografts were harvested for histologic, immunofluorescence, and conditioned medium analyses. RESULTS: In total, early aortic rupture occurred in 80% of the ECM group vs 0% of the control group (P < .001). In the ECM group, major circumferential immunoglobulin deposits were observed in combination with the C3 complement fraction, without cell infiltration. Conditioned medium analysis revealed that matrix metalloproteinase 9 and myeloperoxidase levels and elastase activities were significantly increased in this group. Immunofluorescence analysis demonstrated that myeloperoxidase co-localized with tissue-free DNA and histone H4, highlighting local neutrophil activation and formation of neutrophil extracellular traps. Following differential presensitization, it appeared that rats presensitized against structural glycoproteins and proteoglycans were significantly more susceptible to rupture after DAX implantation. CONCLUSIONS: Stimulating adaptive immunity against the aortic ECM, especially structural glycoproteins and proteoglycans, triggers rupture after DAX implantation. Further studies are needed to assess the precise proteins involved.

Sng1 associates with Nce102 to regulate the yeast Pkh-Ypk signalling module in response to sphingolipid status.

All cells are delimited by biological membranes, which are consequently a primary target of stress-induced damage. Cold alters membrane functionality by decreasing lipid fluidity and the activity of membrane proteins. In Saccharomyces cerevisiae, evidence links sphingolipid homeostasis and membrane phospholipid asymmetry to the activity of the Ypk1/2 proteins, the yeast orthologous of the mammalian SGK1-3 kinases. Their regulation is mediated by different protein kinases, including the PDK1 orthologous Pkh1/2p, and requires the function of protein effectors, among them Nce102p, a component of the sphingolipid sensor machinery. Nevertheless, the mechanisms and the actors involved in Pkh/Ypk regulation remain poorly defined. Here, we demonstrate that Sng1, a transmembrane protein, is an effector of the Pkh/Ypk module and identify the phospholipid asymmetry as key for yeast cold adaptation. Overexpression of SNG1 impairs phospholipid flipping, reduces reactive oxygen species (ROS) and improves, in a Pkh-dependent manner, yeast growth in myriocin-treated cells, suggesting that excess Sng1p stimulates the Pkh/Ypk signalling. Furthermore, we link these effects to the association of Sng1p with Nce102p. Indeed, we found that Sng1p interacts with Nce102p both physically and genetically. Moreover, mutant nce102∆ sng1∆ cells show features of impaired Pkh/Ypk signalling, including increased ROS accumulation, reduced life span and defects in Pkh/Ypk-controlled regulatory pathways. Finally, myriocin-induced hyperphosphorylation of Ypk1p and Orm2p, which controls sphingolipid homeostasis, does not occur in nce102∆ sng1∆ cells. Hence, both Nce102p and Sng1p participate in a regulatory circuit that controls the activity of the Pkh/Ypk module and their function is required in response to sphingolipid status.

Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation.

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10 % and 5 h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

Fisetin yeast-based bio-capsules via osmoporation: effects of process variables on the encapsulation efficiency and internalized fisetin content.

Osmoporation is an innovative method that can be used with food-grade yeast cells of Saccharomyces cerevisiae as natural encapsulating matrices. This technique overcomes barriers that difficult encapsulation and enables the internalization of fragile bioactive molecules such as fisetin into yeasts. In the present study, we assessed the effects of concentration, osmotic pressure, and temperature on the encapsulation efficiency (EE) and internalized fisetin content (IF). Two different quantification strategies were investigated: direct extraction (DE) without cell washing or freeze-drying steps and indirect extraction (IE) performed after washings with ethanol and freeze-drying. Our results showed that osmoporation improved EE (33 %) and IF (1.199 mg). The best experimental conditions were found by using DE. High-resolution images showed that the yeast cell envelope was preserved during osmoporation at 30 MPa and 84 % of yeast cells remained viable after treatment. Washing cells with organic solvent led to decreased EE (0.65 %) and IF (0.023 mg). This was probably due to either damages caused to yeast cell envelope or fisetin dragged out of cell. Overall, the results demonstrated the adequacy and relevant biotechnological potential of yeasts as encapsulating matrices for hydrophobic compounds. This fresh biotechnological approach has proven to be a promising tool for the production of bioactive-rich food products.