Influence of cell-cell contact between L. thermotolerans and S. cerevisiae on yeast interactions and the exo-metabolome.

Sequential fermentation of grape must inoculated with L. thermotolerans and then S. cerevisiae 24 h later (typical wine-making practice) was conducted with or without cell-cell contact between the two yeast species. We monitored cell viability of the two species throughout fermentation by flow cytometry. The cell viability of S. cerevisiae decreased under both conditions, but the decrease was greater if there was cell-cell contact. An investigation of the nature of the interactions showed competition between the two species for nitrogen compounds, oxygen, and must sterols. Volatile-compound analysis showed differences between sequential and pure fermentation and that cell-cell contact modifies yeast metabolism, as the volatile-compound profile was significantly different from that of sequential fermentation without cell-cell contact. We further confirmed that cell-cell contact modifies yeast metabolism by analyzing the exo-metabolome of all fermentations by FT-ICR-MS analysis. These analyses show specific metabolite production and quantitative metabolite changes associated with each fermentation condition. This study shows that cell-cell contact not only affects cell viability, as already reported, but markedly affects yeast metabolism.

Clostridial Strain-Specific Characteristics Associated with Necrotizing Enterocolitis.

We aimed at identifying potential bacterial factors linking clostridia with necrotizing enterocolitis (NEC). We compared the phenotypic traits, stress responses, cellular cytotoxicity, and inflammatory capabilities of the largest collection of Clostridium butyricum and Clostridium neonatale strains isolated from fecal samples of NEC preterm neonates (PN) and control PNs. When strain characteristics were used as explanatory variables, a statistical discriminant analysis allowed the separation of NEC and control strains into separate groups. Strains isolated from NEC PN were characterized by a higher viability at 30°C (P = 0.03) and higher aerotolerance (P = 0.01), suggesting that NEC strains may have a competitive and/or survival advantage in the environmental gastrointestinal tract conditions of NEC PN. Heat-treated NEC bacteria induced higher production of interleukin-8 in Caco-2 cells (P = 0.03), suggesting proinflammatory activity. In vitro, bacteria, bacterial components, and fecal filtrates showed variable cytotoxic effects affecting the cellular network and/or cell viability, without specific association with NEC or control samples. Altogether, our data support the existence of a specific clostridial strain signature associated with NEC.IMPORTANCE Clostridia are part of the commensal microbiota in preterm neonates (PN). However, microbiota analyses by culture and metagenomics have linked necrotizing enterocolitis (NEC) and intestinal colonization with clostridial species. Nevertheless, little is known about the specific characteristics that may be shared by clostridia associated with NEC compared to commensal clostridia. Therefore, our goal was to identify specific bacterial factors linking clostridial strains with NEC. We report the existence of a specific bacterial signature associated with NEC and propose that activation of the innate immune response may be a unifying causative mechanism for the development of NEC independent of a specific pathogenic organism. The present study provides new insights into NEC pathophysiology that are needed for better diagnostics and strategies for implementing prevention of the disease.

Importance of Post-translational Modifications in the Interaction of Proteins with Mineral Surfaces: The Case of Arginine Methylation and Silica surfaces.

Understanding the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest for both basic research and practical applications involving nanotechnology. From the list of cellular proteins with the highest affinity for silica nanoparticles, we highlighted the group of proteins containing arginine-glycine-glycine (RGG) motifs. Biochemical experiments confirmed that RGG motifs interact strongly with the silica surfaces. The affinity of these motifs is further increased when the R residue is asymmetrically, but not symmetrically, dimethylated. Molecular dynamics simulations show that the asymmetrical dimethylation generates an electrostatic asymmetry in the guanidinium group of the R residue, orientating and stabilizing it on the silica surface. The RGG motifs (methylated or not) systematically target the siloxide groups on the silica surface through an ionic interaction, immediately strengthened by hydrogen bonds with proximal silanol and siloxane groups. Given that, in vivo, RGG motifs are often asymmetrically dimethylated by specific cellular methylases, our data add support to the idea that this type of methylation is a key mechanism for cells to regulate the interaction of the RGG proteins with their cellular partners.

Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

Competition for Nitrogen Resources: An Explanation of the Effects of a Bioprotective Strain Metschnikowia pulcherrima on the Growth of Hanseniaspora Genus in Oenology.

As a biological alternative to the antimicrobial action of SO2, bioprotection has been proposed to winemakers as a means to limit or prevent grape musts microbial alteration. Competition for nitrogenous nutrients and for oxygen are often cited as potential explanations for the effectiveness of bioprotection. This study analyses the effect of a bioprotective M. pulcherrima strain on the growth of one H. valbyensis strain and one H. uvarum strain. Bioprotection efficiency was observed only against H. valbyensis inoculated at the two lowest concentrations. These results indicate a potential species-dependent efficiency of the bioprotective strain and a strong impact of the initial ratio between bioprotective and apiculate yeasts. The analysis of the consumption of nitrogen compounds revealed that leucine, isoleucine, lysine and tryptophan were consumed preferentially by all three strains. The weaker assimilation percentages of these amino acids observed in H. valbyensis at 24 h growth suggest competition with M. pulcherrima that could negatively affects the growth of the apiculate yeast in co-cultures. The slowest rate of O2 consumption of H. valbyensis strain, in comparison with M. pulcherrima, was probably not involved in the bioprotective effect. Non-targeted metabolomic analyses of M. pulcherrima and H. valbyensis co-culture indicate that the interaction between both strains particularly impact lysin and tryptophan metabolisms.

Bio-protection in oenology by Metschnikowia pulcherrima: from field results to scientific inquiry.

Finding alternatives to the use of chemical inputs to preserve the sanitary and organoleptic quality of food and beverages is essential to meet public health requirements and consumer preferences. In oenology, numerous manufacturers already offer a diverse range of bio-protection yeasts to protect must against microbiological alterations and therefore limit or eliminate sulphites during winemaking. Bio-protection involves selecting non-Saccharomyces yeasts belonging to different genera and species to induce negative interactions with indigenous microorganisms, thereby limiting their development and their impact on the matrix. Although the effectiveness of bio-protection in the winemaking industry has been reported in numerous journals, the underlying mechanisms are not yet well understood. The aim of this review is to examine the current state of the art of field trials and laboratory studies that demonstrate the effects of using yeasts for bio-protection, as well as the interaction mechanisms that may be responsible for these effects. It focuses on the yeast Metschnikowia pulcherrima, particularly recommended for the bio-protection of grape musts.

Use of Oenological Tannins to Protect the Colour of Rosé Wine in a Bioprotection Strategy with Metschnikowia pulcherrima.

Although bioprotection is now recognised as an alternative to SO2 for limiting microbial spoilage, it does not guarantee protection against oxidation. This limits its application, more specifically for rosé winemaking. Oenological tannins present antioxidant properties, which could represent an interesting alternative to SO2 to protect must and wines against oxidation. A combination of the inoculation of a bioprotectant yeast strain and the addition of oenological tannins was tested to eliminate sulfites during the pre-fermentative step of rosé winemaking. In this experiment carried out in a winery, two oenological tannins were compared: quebracho and gall nut tannins. The antioxidant efficiency of tannins was compared to that of SO2. Colorimetric assays associated with chemical analyses of anthocyanins and phenolic compounds confirmed that the use of bioprotection alone did not protect the wine from oxidation. An addition of oenological tannins on musts stabilized the colour of bioprotected rosé wine in a similar way that SO2 addition did. Quebracho tannins appeared more efficient than gall nut tannins. The colour differences observed cannot be explained either by the concentration or forms of anthocyanins. However, the addition of tannins led to better protection of oxidation-sensitive phenolic compounds comparable to that obtained with the addition of sulfites.

Bioprotection Efficiency of Metschnikowia Strains in Synthetic Must: Comparative Study and Metabolomic Investigation of the Mechanisms Involved.

Three Metschnikowia strains marketed as bioprotection yeasts were studied to compare their antimicrobial effect on a mixture of two Hanseniaspora yeast strains in synthetic must at 12 °C, mimicking pre-fermentative maceration by combining different approaches. The growth of the different strains was monitored, their nitrogen and oxygen requirements were characterised, and their metabolomic footprint in single and co-cultures studied. Only the M. fructicola strain and one M. pulcherrima strains colonised the must and induced the rapid decline of Hanseniaspora. The efficiency of these two strains followed different inhibition kinetics. Furthermore, the initial ratio between Metschnikowia and Hanseniaspora was an important factor to ensure optimal bioprotection. Nutrient consumption kinetics showed that apiculate yeasts competed with Metschnikowia strains for nutrient accessibility. However, this competition did not explain the observed bioprotective effect, because of the considerable nitrogen content remaining on the single and co-cultures. The antagonistic effect of Metschnikowia on Hanseniaspora probably implied another form of amensalism. For the first time, metabolomic analyses of the interaction in a bioprotection context were performed after the pre-fermentative maceration step. A specific footprint of the interaction was observed, showing the strong impact of the interaction on the metabolic modulation of the yeasts, especially on the nitrogen and vitamin pathways.

eGFP Gene Integration in HO: A Metabolomic Impact?

Integrating fluorescent genes including eGFP in the yeast genome is common practice for various applications, including cell visualization and population monitoring. The transformation of a commercial S. cerevisiae strain by integrating a cassette including a gene encoding an EGFP protein in the HO gene was carried out using CRISPR-Cas9 technology. Although this type of integration is often used and described as neutral at the phenotypic level of the cell, we have highlighted that under alcoholic fermentation (in a Chardonnay must), it has an impact on the exometabolome. We observed 41 and 82 unique biomarkers for the S3 and S3GFP strains, respectively, as well as 28 biomarkers whose concentrations varied significantly between the wild-type and the modified strains. These biomarkers were mainly found to correspond to peptides. Despite similar phenotypic growth and fermentation parameters, high-resolution mass spectrometry allowed us to demonstrate, for the first time, that the peptidome is modified when integrating this cassette in the HO gene.

Shared decision-making in urology and female pelvic floor medicine and reconstructive surgery.

Shared decision-making (SDM) is a hallmark of patient-centred care that uses informed consent to help guide patients with making complex health-care decisions. In SDM, patients and providers work together to determine the best course of action based on both the current available evidence and the patient's values and preferences. SDM not only provides a framework for the legal and ethical obligations providers need to fulfil for informed consent, but also leads to improved knowledge of treatment options and satisfaction of decision-making for patients. Tools such as decision aids have been developed to support SDM for complex decisions. Several decision aids are available for use in the field of urology and female pelvic medicine and reconstructive surgery, but these decision aids are also associated with barriers to SDM implementation including patient, provider and systematic challenges. However, solutions to such barriers to SDM include continued development of SDM tools to improve patient engagement, expand training of providers in SDM communication models and a process to encourage implementation of SDM.

The anti-biofilm activity secreted by a marine Pseudoalteromonas strain.

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.

Living With Urinary Diversions: Patient Insights to Improve the Perioperative Experience.

OBJECTIVE: To evaluate the perioperative decision-making process, post-operative decision regret and reflection on the peri-operative experience of patients undergoing radical cystectomy and urinary diversion through patient interviews. METHODS: Patients identified as having undergone radical cystectomy for malignancy were interviewed 6-24 months from the time of surgery and stratified by diversion type. Following written consent, interviews were conducted either in person or over the phone using a semi-structured script. Patients were asked 9 open-ended questions, with additional unscripted follow-up questions based on themes raised by the patient. The interviews were reviewed for common themes, preferences, and recommendations. RESULTS: A total of 13 interviews were conducted. No patient expressed decision regret about their choice of urinary diversion. Ten out of 13 interviewees specifically stated that they had adequate information about diversion options pre-operatively, none felt they did not have adequate pre-operative counseling. One area identified as improvable was postoperative counseling- specifically, for ostomy appliances, catheters, or irrigation. The most striking recurrent theme was the desire for a "buddy system" in which patients could contact and maintain discussion about their surgical experience with an experienced patient. The overall impression was that this system would be most useful in the recovery/maintenance phase rather than in the pre-operative decision-making process. CONCLUSION: This data furnishes a basis to develop more accessible and effective counseling and highlights the need to concentrate on post-surgical maintenance care, including management of urostomy appliances, catheters, and reinforcing irrigation technique.

Analyzing National Incidences and Predictors of Open Conversion During Minimally Invasive Partial Nephrectomy for cT1 Renal Masses.

Objectives: To analyze predictors of open conversion during minimally invasive partial nephrectomy (MIPN) for cT1 renal masses. Methods: The National Cancer Database (NCDB) was investigated for kidney cancer patients who underwent partial nephrectomy (PN) between 2010 and 2015. Patients who underwent MIPN were stratified into converted and nonconverted groups. Sociodemographics, facility characteristics, and surgical outcomes were compared between the two groups, and multivariate logistic regression model was fitted to identify independent predictors of open conversion. Results: In total, 54,246 patients underwent PN for kidney cancer during the 6-year period. Of those, 18,994 (35%) were open partial nephrectomies (OPNs) and 35,252 (64%) were MIPN. Overall, 1010 (2.87%) of MIPNs were converted to OPN. There was an increasing utilization of MIPN from 50.35% in 2010 to 74.73% in 2015. Patients who had open conversion had more 30-day readmissions (5.95% vs 3.31%, p < 0.01). On multivariate analysis; high-volume facility (>30 MIPNs/year), year of surgery (2015 vs 2010), and robotic approach predicted a lower likelihood of conversion (odds ratio [OR] 0.52, confidence interval [CI] 0.44-0.62; OR 0.59, CI 0.47-0.73; and OR 0.31, CI 0.27-0.35; respectively, p < 0.001 for all). Conversely, Medicaid (vs private insurance; OR 1.75, CI 1.39-2.19, p < 0.001) and male sex (OR 1.26, CI 1.11-1.44, p < 0.001) were independent predictors of conversion. Conclusions: Open conversion in MIPN occurred in 2.87% of cases. There was an increasing utilization of MIPN associated with decreased conversion rates. Higher volume hospitals and progressing year of surgery were associated with less likelihood of conversion.

Robotic Rectus Abdominis Muscle Flap following Robotic Extirpative Surgery.

SUMMARY: Vascularized tissue for obliteration of large pelvic dead spaces created by extirpative surgery has been shown to reduce complication rates. As more extensive resections are performed robotically, plastic surgeons have been challenged to reconstruct the resulting defects using a minimally invasive approach. The goal of this study was to report the authors' experience with robotic harvest of the rectus abdominis muscle for reconstruction of pelvic defects. The authors conducted a retrospective case series of patients who underwent robotic flap harvest following robotic extirpative surgery at their institution. Patient demographics, surgical characteristics, and postoperative outcomes were collected. These were compared to a retrospective cohort of patients who underwent open rectus abdominis muscle harvest. The authors identified seven male patients who underwent robotic flap harvest for pelvic reconstruction between 2013 and 2019. Their mean age was 66 ± 6 years and mean body mass index was 31 ± 5 kg/m2. Six patients (86 percent) had a history of radiation therapy and five patients (71 percent) received hormone therapy or chemotherapy. Surgical-site complications occurred in two patients. One patient developed ventral hernia. The donor-site complication rate was 19 percent (n = 18) in patients who underwent open rectus abdominis muscle harvest (n = 95). This study demonstrates the safety, efficacy, and reproducibility of robotic harvest of the rectus abdominis muscle in complex, multidisciplinary, minimally invasive pelvic surgery. The technique avoids violation of the anterior rectus sheath and wound complications related to open flap harvest, and early experience suggests reduced donor-site morbidity. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Antibiofilm activity of the marine bacterium Pseudoalteromonas sp. strain 3J6.

Biofilm formation results in medical threats or economic losses and is therefore a major concern in a variety of domains. In two-species biofilms of marine bacteria grown under dynamic conditions, Pseudoalteromonas sp. strain 3J6 formed mixed biofilms with Bacillus sp. strain 4J6 but was largely predominant over Paracoccus sp. strain 4M6 and Vibrio sp. strain D01. The supernatant of Pseudoalteromonas sp. 3J6 liquid culture (SN(3J6)) was devoid of antibacterial activity against free-living Paracoccus sp. 4M6 and Vibrio sp. D01 cells, but it impaired their ability to grow as single-species biofilms and led to higher percentages of nonviable cells in 48-h biofilms. Antibiofilm molecules of SN(3J6) were able to coat the glass surfaces used to grow biofilms and reduced bacterial attachment about 2-fold, which might partly explain the biofilm formation defect but not the loss of cell viability. SN(3J6) had a wide spectrum of activity since it affected all Gram-negative marine strains tested except other Pseudoalteromonas strains. Biofilm biovolumes of the sensitive strains were reduced 3- to 530-fold, and the percentages of nonviable cells were increased 3- to 225-fold. Interestingly, SN(3J6) also impaired biofilm formation by three strains belonging to the human-pathogenic species Pseudomonas aeruginosa, Salmonella enterica, and Escherichia coli. Such an antibiofilm activity is original and opens up a variety of applications for Pseudoalteromonas sp. 3J6 and/or its active exoproducts in biofilm prevention strategies.

Yeast-Yeast Interactions: Mechanisms, Methodologies and Impact on Composition.

During the winemaking process, alcoholic fermentation is carried out by a consortium of yeasts in which interactions occurs. The consequences of these interactions on the wine matrix have been widely described for several years with the aim of controlling the winemaking process as well as possible. In this review, we highlight the wide diversity of methodologies used to study these interactions, and their underlying mechanisms and consequences on the final wine composition and characteristics. The wide variety of matrix parameters, yeast couples, and culture conditions have led to contradictions between the results of the different studies considered. More recent aspects of modifications in the composition of the matrix are addressed through different approaches that have not been synthesized recently. Non-volatile and volatile metabolomics, as well as sensory analysis approaches are developed in this paper. The description of the matrix composition modification does not appear sufficient to explain interaction mechanisms, making it vital to take an integrated approach to draw definite conclusions on them.

Protein Corona Composition of Silica Nanoparticles in Complex Media: Nanoparticle Size does not Matter.

Biomolecules, and particularly proteins, bind on nanoparticle (NP) surfaces to form the so-called protein corona. It is accepted that the corona drives the biological distribution and toxicity of NPs. Here, the corona composition and structure were studied using silica nanoparticles (SiNPs) of different sizes interacting with soluble yeast protein extracts. Adsorption isotherms showed that the amount of adsorbed proteins varied greatly upon NP size with large NPs having more adsorbed proteins per surface unit. The protein corona composition was studied using a large-scale label-free proteomic approach, combined with statistical and regression analyses. Most of the proteins adsorbed on the NPs were the same, regardless of the size of the NPs. To go beyond, the protein physicochemical parameters relevant for the adsorption were studied: electrostatic interactions and disordered regions are the main driving forces for the adsorption on SiNPs but polypeptide sequence length seems to be an important factor as well. This article demonstrates that curvature effects exhibited using model proteins are not determining factors for the corona composition on SiNPs, when dealing with complex biological media.

National Trends and Impact of Regionalization of Radical Cystectomy on Survival Outcomes in Patients with Muscle Invasive Bladder Cancer.

OBJECTIVE: To evaluate national trends and the effect of surgical volume on perioperative mortality and overall survival (OS)in patients undergoing radical cystectomy (RC) for muscle invasive bladder cancer (MIBC). METHODS: We investigated the National Cancer Database to identify patients with localized MIBC (cT2a-T4, M0) who underwent RC from 2004 to 2014. Demographics, 30- and 90-day mortality rates, as well as OS were analyzed. Hospitals were stratified into low-, medium-, and high-volume centers according to median number of RCs performed per year. Multivariate logistic regression models were fitted to identify independent predictors of perioperative mortality. Kaplan-Meier survival curves were generated to evaluate OS. Cox proportional hazard modeling was performed to identify independent predictors of OS. RESULTS: A total of 24,763 patients with localized MIBC who underwent RC from 2004 to 2014 were included in the study. Overall, most (70.85%) RCs occurred at low-volume hospitals, whereas only 15.83% were performed at high-volume hospitals. Thirty-day mortality rates were 2.87%, 2.19%, and 1.83% (P < .01); and 90-day mortality rates were 8.25%, 6.9%, and 5.9% (P < .01) at low-, medium-, and high-volume hospitals, respectively. Multivariate analyses identified RC volume as an independent predictor of 30- and 90-day mortality. RC in high-volume hospitals was associated with a 35% risk reduction in 30-day mortality (odds ratio 0.65, 95% confidence interval [CI] 0.49-0.85; P < .01), and a 26% risk reduction in 90-day mortality (0.74, 95% CI, 0.63-0.87; P < .01). CONCLUSIONS: Treatment at high-volume centers offers improved outcomes and OS benefit. However, in the United States, only 16% of RCs are performed in high-volume hospitals.

RNA-binding proteins are a major target of silica nanoparticles in cell extracts.

Upon contact with biological fluids, nanoparticles (NPs) are readily coated by cellular compounds, particularly proteins, which are determining factors for the localization and toxicity of NPs in the organism. Here, we improved a methodological approach to identify proteins that adsorb on silica NPs with high affinity. Using large-scale proteomics and mixtures of soluble proteins prepared either from yeast cells or from alveolar human cells, we observed that proteins with large unstructured region(s) are more prone to bind on silica NPs. These disordered regions provide flexibility to proteins, a property that promotes their adsorption. The statistical analyses also pointed to a marked overrepresentation of RNA-binding proteins (RBPs) and of translation initiation factors among the adsorbed proteins. We propose that silica surfaces, which are mainly composed of Si-O- and Si-OH groups, mimic ribose-phosphate molecules (rich in -O- and -OH) and trap the proteins able to interact with ribose-phosphate containing molecules. Finally, using an in vitro assay, we showed that the sequestration of translation initiation factors by silica NPs results in an inhibition of the in vitro translational activity. This result demonstrates that characterizing the protein corona of various NPs would be a relevant approach to predict their potential toxicological effects.

Inverse Gas Chromatography with Film Cell Unit: An Attractive Alternative Method to Characterize Surface Properties of Thin Films.

Inverse gas chromatography (IGC) is widely used for the characterization of surfaces. The present work describes a novel IGC tool, the recently developed film cell module, which measures monolithic thin solid film surface properties, whereas only samples in powder or fiber state or polymer-coated supports can be studied by classic IGC. The surface energy of four different solid supports was measured using both classic IGC with columns packed with samples in the powder state, and IGC with the new film cell module or the sessile drop technique, using samples in the film state. The total surface energy and its dispersive and specific components were measured for glass, polyethylene, polyamide and polytetrafluoroethylene. Similar results were obtained for the four materials using the three different techniques. The main conclusion is that the new film cell module for IGC is an attractive alternative to the sessile drop technique as it gives very accurate and reproducible results for surface energy components, with significant savings in time and the possible control of sample humidity and temperature. This film cell module for IGC extends the application field of IGC to any thin solid film and can be used to study the effect of any surface treatment on surface energy.