Longitudinal Study of Lactococcus Phages in a Canadian Cheese Factory.

The presence of virulent phages is closely monitored during cheese manufacturing, as these bacterial viruses can significantly slow down the milk fermentation process and lead to low-quality cheeses. From 2001 to 2020, whey samples from cheddar cheese production in a Canadian factory were monitored for the presence of virulent phages capable of infecting proprietary strains of Lactococcus cremoris and Lactococcus lactis used in starter cultures. Phages were successfully isolated from 932 whey samples using standard plaque assays and several industrial Lactococcus strains as hosts. A multiplex PCR assay assigned 97% of these phage isolates to the Skunavirus genus, 2% to the P335 group, and 1% to the Ceduovirus genus. DNA restriction profiles and a multilocus sequence typing (MLST) scheme distinguished at least 241 unique lactococcal phages from these isolates. While most phages were isolated only once, 93 of them (out of 241, 39%) were isolated multiple times. Phage GL7 was isolated 132 times from 2006 to 2020, demonstrating that phages can persist in a cheese factory for long periods of time. Phylogenetic analysis of MLST sequences showed that phages could be clustered based on their bacterial hosts rather than their year of isolation. Host range analysis showed that Skunavirus phages exhibited a very narrow host range, whereas some Ceduovirus and P335 phages had a broader host range. Overall, the host range information was useful in improving the starter culture rotation by identifying phage-unrelated strains and helped mitigating the risk of fermentation failure due to virulent phages. IMPORTANCE Although lactococcal phages have been observed in cheese production settings for almost a century, few longitudinal studies have been performed. This 20-year study describes the close monitoring of dairy lactococcal phages in a cheddar cheese factory. Routine monitoring was conducted by factory staff, and when whey samples were found to inhibit industrial starter cultures under laboratory conditions, they were sent to an academic research laboratory for phage isolation and characterization. This led to a collection of at least 241 unique lactococcal phages, which were characterized through PCR typing and MLST profiling. Phages of the Skunavirus genus were by far the most dominant. Most phages lysed a small subset of the Lactococcus strains. These findings guided the industrial partner in adapting the starter culture schedule by using phage-unrelated strains in starter cultures and removing some strains from the starter rotation. This phage control strategy could be adapted for other large-scale bacterial fermentation processes.

No evidence of tocilizumab treatment efficacy for severe to critical SARS-CoV2 infected patients: Results from a retrospective controlled multicenter study.

ABSTRACT: To assess tocilizumab (TCZ) efficacy associated to standard of care (SOC) compared to SOC alone in severe coronavirus associated disease 2019 (COVID-19) patients. In a matched case-control study from 3 French Hospital COVID-19 Departments, 27 patients with severe COVID-19 treated with TCZ and SOC were matched for baseline epidemiological and clinical features and compared to 27 severe COVID-19 patients treated with SOC alone. Baseline characteristics of the study population were comparable between groups. Eleven patients (20%) died. TCZ was not associated with clinical improvement as compared to SOC regarding oxygen-free status (44% vs 63%) and death (18.5% vs 22%), despite a higher decrease of the C-reactive protein at Day 7 (10.7 vs 52 mg/L; P < 10-3). Compared to the 43 patients alive at the end-of follow-up, patients who died were older (78 vs 64 years; P < 10-3), with 82% of them older than 72 years vs only 23% of live patients (P < 10-3). Age (OR = 1.15; 95%CI = 1.04-1.3; P = .008) and age over 72 years (OR) = 14.85; 95%CI = 2.7-80; P = .002) were independently associated with mortality. TCZ in addition to SOC for severe COVID-19 patients did not reduce mortality, subsequent need for invasive mechanical ventilation nor did it shorten the time of oxygen support, despite better control of the inflammatory response. More powerful and randomized controlled trials are warranted to determine if TCZ is effective in the management of COVID-19.

Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor.

OBJECTIVE: Obesity-associated metabolic dysfunctions are linked to dysregulated production of adipokines. Accumulating evidence suggests a role for fat-derived extracellular vesicles (EVs) in obesity-metabolic disturbances. Since EVs convey numerous proteins we aimed to evaluate their contribution in adipokine secretion. METHODS: Plasma collected from metabolic syndrome patients were used to isolate EV subtypes, namely microvesicles (MVs) and exosomes (EXOs). Numerous soluble factor concentrations were measured successively on total, MV- and EXO-depleted plasma by multiplexed immunoassays. RESULTS: Circulating MVs and EXOs were significantly increased with BMI, supporting a role of EVs as metabolic relays in obesity. Obesity was associated with dysregulated soluble factor production. Sequential depletion of plasma MVs and EXOs did not modify plasma levels of these molecules, with the exception of Macrophage Migration Inhibitory Factor (MIF). Half of plasma MIF circulated within MVs, and this MV secretory pathway was conserved over different MIF-producing cells. Although MV-associated MIF triggered rapid ERK1/2 activation in macrophages, these functional MV-MIF effects specifically relied on MIF tautomerase activity. CONCLUSION: Our results emphasize the importance of reconsidering MIF-metabolic actions with regard to its MV-associated form and opening new EV-based strategies for therapeutic MIF approaches.

Zeaxanthin from Porphyridium purpureum induces apoptosis in human melanoma cells expressing the oncogenic BRAF V600E mutation and sensitizes them to the BRAF inhibitor vemurafenib

Abstract Zeaxanthin, an abundant carotenoid present in fruits, vegetables and algae was reported to exert antiproliferative activity and induce apoptosis in human uveal melanoma cells. It also inhibited uveal melanoma tumor growth and cell migration in nude mice xenograft models. Here we report that zeaxanthin purified from the rhodophyte Porphyridium purpureum (Bory) K.M.Drew & R.Ross, Porphyridiaceae, promotes apoptosis in the A2058 human melanoma cell line expressing the oncogenic BRAF V600E mutation. Zeaxanthin 40 µM (IC50) induced chromatin condensation, nuclear blebbing, hypodiploidy, accumulation of cells in sub-G1 phase, DNA internucleosomal fragmentation and activation of caspase-3. Western blot analysis revealed that zeaxanthin induced up-regulation of the pro-apoptotic factors Bim and Bid and inhibition of NF-κB transactivation. Additionally, zeaxanthin sensitized A2058 melanoma cells in vitro to the cytotoxic activity of vemurafenib, a BRAF inhibitor widely used for the clinical management of melanoma, suggesting its potential interest as dietary adjuvant increasing melanoma cells sensitivity to chemotherapy.

Breast cancer and ovarian tissue cryopreservation: Review of the literature.

INTRODUCTION: Ovarian tissue cryopreservation is a modern technique of fertility preservation, useful before using ovariotoxic therapies in the treatment of breast cancer. The aim of our literature review was to study ovarian cryopreservation experiences for women with breast cancer, to identify guidelines, constraints and results in the oncological and obstetrical fields. METHODS: We searched articles through the PubMed/Medline database, including all French and English references from January 2000 to October 2017. The combination of key words "breast cancer" and "ovarian tissue cryopreservation" allowed us to select 50 articles. We kept 18 publications which matched our subject. RESULTS: Sixteen cases of ovarian transplants among patients treated for breast cancer were published with 14 pregnancies, 11 births and 3 failures. Two cases of breast recurrences were published after ovarian grafting. However, the hindsight in this technique is limited, with a first transplant published in 2004 and only a low number of cases. PERSPECTIVES: A national census and comprehensive gathering of data among the patients treated for breast cancer using ovarian tissue cryopreservation would make it possible to better evaluate the occurrence of pregnancies and the carcinological risk of this technique.

Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles.

Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.

The amino acid substitution N136Y in Candida albicans sterol 14alpha-demethylase is involved in fluconazole resistance.

Resistance to fluconazole antifungal is an ongoing impediment to a successful treatment of Candida albicans infections. One of the most prevalent mechanisms leading to azole resistance is genetic alterations of the 14α-demethylase, the target of azole antifungals, through point mutations. Site-directed mutagenesis and molecular modeling of 14α-demethylase rationalize biological data about the role of protein substitutions in the azole treatment failure. In this work, we investigated the role of N136Y substitution by site-directed mutagenesis into Pichia pastoris guided by structural analysis. Single amino acid substitutions were created by site-directed mutagenesis into P. pastoris with C. albicans ERG11 gene as template. In vitro susceptibility of P. pastoris transformants expressing wild-type and mutants to azole compounds was determined by CLSI M27-A2 and spot agar methods. The fluconazole effect on ergosterol biosynthesis was analyzed by gas chromatography-mass spectrometry. By microdilution and spot tests, N136Y transformants showed a reduced in vitro susceptibility to fluconazole compared to wild-type controls. As expected, ergosterol/lanosterol ratios were higher in N136Y transformants compared to the wild-type controls after treatment with fluconazole. Molecular modeling suggests that residue Asn136 located within the first mutation hot spot, could play a role during heme and azole binding. These results provide new insights into the structural basis for 14α-demethylase-azole interaction and could guide the design of novel azole antifungals.

Hedgehog associated to microparticles inhibits adipocyte differentiation via a non-canonical pathway.

Hedgehog (Hh) is a critical regulator of adipogenesis. Extracellular vesicles are natural Hh carriers, as illustrated by activated/apoptotic lymphocytes specifically shedding microparticles (MP) bearing the morphogen (MP(Hh+)). We show that MP(Hh+) inhibit adipocyte differentiation and orientate mesenchymal stem cells towards a pro-osteogenic program. Despite a Smoothened (Smo)-dependency, MP(Hh+) anti-adipogenic effects do not activate a canonical Hh signalling pathway in contrast to those elicited either by the Smo agonist SAG or recombinant Sonic Hedgehog. The Smo agonist GSA-10 recapitulates many of the hallmarks of MP(Hh+) anti-adipogenic effects. The adipogenesis blockade induced by MP(Hh+) and GSA-10 was abolished by the Smo antagonist LDE225. We further elucidate a Smo/Lkb1/Ampk axis as the non-canonical Hh pathway used by MP(Hh+) and GSA-10 to inhibit adipocyte differentiation. Our results highlight for the first time the ability of Hh-enriched MP to signal via a non-canonical pathway opening new perspectives to modulate fat development.

Extracellular vesicles as therapeutic tools in cardiovascular diseases.

Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, are small vesicles secreted from a wide variety of cells. Whereas MVs are particles released by the outward budding of the plasma membrane, exosomes are derived from endocytic compartments. Secretion of EVs can be enhanced by specific stimuli, and increased plasma circulating levels of EVs have been correlated with pathophysiological situations. MVs, already present in the blood of healthy individuals, are considerably elevated in several cardiovascular diseases associated with inflammation, suggesting that they can mediate deleterious effects such as endothelial dysfunction or thrombosis. Nonetheless, very recent studies also demonstrate that MVs may act as biological information vectors transferring proteins or genetic material to maintain cell homeostasis, favor cell repair, or even promote angiogenesis. Additionally, exosomes have also been shown to have pro-angiogenic and cardio-protective properties. These beneficial effects, therefore, reveal the potential therapeutical use of EVs in the field of cardiovascular medicine and regenerative therapy. In this review, we will provide an update of cellular processes modulated by EVs of specific interest in the treatment of cardiovascular pathologies. A special focus will be made on the morphogen sonic hedgehog (Shh) associated with EVs (EVs(Shh+)), which have been shown to mediate many pro-angiogenic effects. In addition to offer a potential source of cardiovascular markers, therapeutical potential of EVs reveal exciting opportunities to deliver specific agents by non-immunogenic means to cardiovascular system.

DNA microarray and miRNA analyses reinforce the classification of follicular thyroid tumors.

CONTEXT: Focusing on mitochondrial function and thyroid tumorigenesis, we used an integrative approach to identify relevant biomarkers for borderline thyroid lesions. DESIGN: Using cDNA and microRNA (miRNA) microarrays and quantitative RT-PCR analysis (qPCR), we explored samples of various types of thyroid tumors including 25 benign follicular adenomas represented by macrofollicular variants of thyroid adenomas, 38 oncocytic variants of follicular thyroid tumors, 19 papillary thyroid carcinomas, and 10 tumors of uncertain malignant potential, together with 53 normal thyroid tissue samples. RESULTS: Our transcriptomic analysis, which highlighted discrepancies between controls and tumor tissues, as well as between various tumor types, led to the identification of 13 genes, allowing discrimination between the thyroid adenomas, oncocytic variants of follicular thyroid tumors, and papillary thyroid carcinomas, whereas the tumors of uncertain malignant potential were found to overlap these classes. Five of these genes (TP53, HOXA9, RUNX1, MYD88, and CITED1), with a differential expression confirmed by qPCR analysis, are implicated in tumorigenesis, 4 in mitochondrial metabolism (MRPL14, MRPS2, MRPS28, and COX6A1), and 2 in thyroid metabolic pathways (CaMKIINalpha and TPO). The global miRNA analysis revealed 62 differential miRNAs, the expression level for 10 of these being confirmed by qPCR. The differential expression of the miRNAs was in accordance with the modulation of gene expression and the ontologies revealed by our transcriptomic analysis. CONCLUSIONS: These findings reinforce the classification of follicular thyroid tumors established by the World Health Organization, and our technique offers a novel molecular approach to refine the classification of thyroid tumors of uncertain malignant potential.

Amino acid substitutions at the major insertion loop of Candida albicans sterol 14alpha-demethylase are involved in fluconazole resistance.

BACKGROUND: In the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. The impact of amino acid substitutions on azole interaction has been postulated by homology modeling in comparison to the crystal structure of Mycobacterium tuberculosis (MT-CYP51). Modeling of amino acid residues situated between positions 428 to 459 remains difficult to explain to date, because they are in a major insertion loop specifically present in fungal species. METHODOLOGY/PRINCIPAL FINDING: Fluconazole resistance of clinical isolates displaying Y447H and V456I novel CaErg11p substitutions confirmed in vivo in a murine model of disseminated candidiasis. Y447H and V456I implication into fluconazole resistance was then studied by site-directed mutagenesis of wild-type CaErg11p and by heterogeneously expression into the Pichia pastoris model. CLSI modified tests showed that V447H and V456I are responsible for an 8-fold increase in fluconazole MICs of P. pastoris mutants compared to the wild-type controls. Moreover, mutants showed a sustained capacity for producing ergosterol, even in the presence of fluconazole. Based on these biological results, we are the first to propose a hybrid homology structure-function model of Ca-CYP51 using 3 different homology modeling programs. The variable position of the protein insertion loop, using different liganded or non-liganded templates of recently solved CYP51 structures, suggests its inherent flexibility. Mapping of recognized azole-resistant substitutions indicated that the flexibility of this region is probably enhanced by the relatively high glycine content of the consensus. CONCLUSIONS/SIGNIFICANCE: The results highlight the potential role of the insertion loop in azole resistance in the human pathogen C. albicans. This new data should be taken into consideration for future studies aimed at designing new antifungal agents, which circumvent azole resistance.