Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy.

Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.

Myxinidin-Derived Peptide against Biofilms Caused by Cystic Fibrosis Emerging Pathogens.

Chronic lung infections in cystic fibrosis (CF) patients are triggered by multidrug-resistant bacteria such as Pseudomonas aeruginosa, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia. The CF airways are considered ideal sites for the colonization and growth of bacteria and fungi that favor the formation of mixed biofilms that are difficult to treat. The inefficacy of traditional antibiotics reinforces the need to find novel molecules able to fight these chronic infections. Antimicrobial peptides (AMPs) represent a promising alternative for their antimicrobial, anti-inflammatory, and immunomodulatory activities. We developed a more serum-stable version of the peptide WMR (WMR-4) and investigated its ability to inhibit and eradicate C. albicans, S. maltophilia, and A. xylosoxidans biofilms in both in vitro and in vivo studies. Our results suggest that the peptide is able better to inhibit than to eradicate both mono and dual-species biofilms, which is further confirmed by the downregulation of some genes involved in biofilm formation or in quorum-sensing signaling. Biophysical data help to elucidate its mode of action, showing a strong interaction of WMR-4 with lipopolysaccharide (LPS) and its insertion in liposomes mimicking Gram-negative and Candida membranes. Our results support the promising therapeutic application of AMPs in the treatment of mono- and dual-species biofilms during chronic infections in CF patients.

A Re-Purposing Strategy: Sub-Lethal Concentrations of an Eicosanoid Derived from the Omega-3-Polyunsaturated Fatty Acid Resolvin D1 Affect Dual Species Biofilms.

The fungal species Candida parapsilosis and the bacterial species Staphylococcus aureus may be responsible for hospital-acquired infections in patients undergoing invasive medical interventions or surgical procedures and often coinfect critically ill patients in complicating polymicrobial biofilms. The efficacy of the re-purposing therapy has recently been reported as an alternative to be used. PUFAs (polyunsaturated fatty acids) may be used alone or in combination with currently available traditional antimicrobials to prevent and manage various infections overcoming antimicrobial resistance. The objectives of the study were to evaluate the effects of Resolvin D1 (RvD1) as an antimicrobial on S. aureus and C. parapsilosis, as well as the activity against the mixed biofilm of the same two species. Microdilution assays and time-kill growth curves revealed bacterial and fungal inhibition at minimum concentration values between 5 and 10 µg mL-1. In single-species structures, an inhibition of 55% and 42% was reported for S. aureus and C. parapsilosis, respectively. Moreover, RvD1 demonstrated an eradication capacity of 60% and 80% for single- and mixed-species biofilms, respectively. In association with the inhibition activity, a downregulation of genes involved in biofilm formation as well as ROS accumulation was observed. Eradication capability was confirmed also on mature mixed biofilm grown on silicone platelets as shown by scanning electron microscopy (SEM). In conclusion, RvD1 was efficient against mono and polymicrobial biofilms in vitro, being a promising alternative for the treatment of mixed bacterial/fungal infections.

Polystyrene Microplastics Exacerbate Candida albicans Infection Ability In Vitro and In Vivo.

Plastic pollution is an important environmental problem, and microplastics have been shown to have harmful effects on human and animal health, affecting immune and metabolic physiological functions. Further, microplastics can interfere with commensal microorganisms and exert deleterious effects on exposure to pathogens. Here, we compared the effects of 1 µm diameter polystyrene microplastic (PSMPs) on Candida albicans infection in both in vitro and in vivo models by using HT29 cells and Galleria mellonella larvae, respectively. The results demonstrated that PSMPs could promote Candida infection in HT29 cells and larvae of G. mellonella, which show immune responses similar to vertebrates. In this study, we provide new experimental evidence for the risk to human health posed by PSMPs in conjunction with Candida infections.

Omics Analyses of Stromal Cells from ACM Patients Reveal Alterations in Chromatin Organization and Mitochondrial Homeostasis.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by ventricular arrhythmias, contractile dysfunctions and fibro-adipose replacement of myocardium. Cardiac mesenchymal stromal cells (CMSCs) participate in disease pathogenesis by differentiating towards adipocytes and myofibroblasts. Some altered pathways in ACM are known, but many are yet to be discovered. We aimed to enrich the understanding of ACM pathogenesis by comparing epigenetic and gene expression profiles of ACM-CMSCs with healthy control (HC)-CMSCs. Methylome analysis identified 74 differentially methylated nucleotides, most of them located on the mitochondrial genome. Transcriptome analysis revealed 327 genes that were more expressed and 202 genes that were less expressed in ACM- vs. HC-CMSCs. Among these, genes implicated in mitochondrial respiration and in epithelial-to-mesenchymal transition were more expressed, and cell cycle genes were less expressed in ACM- vs. HC-CMSCs. Through enrichment and gene network analyses, we identified differentially regulated pathways, some of which never associated with ACM, including mitochondrial functioning and chromatin organization, both in line with methylome results. Functional validations confirmed that ACM-CMSCs exhibited higher amounts of active mitochondria and ROS production, a lower proliferation rate and a more pronounced epicardial-to-mesenchymal transition compared to the controls. In conclusion, ACM-CMSC-omics revealed some additional altered molecular pathways, relevant in disease pathogenesis, which may constitute novel targets for specific therapies.

Ca2+ dysregulation in cardiac stromal cells sustains fibro-adipose remodeling in Arrhythmogenic Cardiomyopathy and can be modulated by flecainide.

BACKGROUND: Cardiac mesenchymal stromal cells (C-MSC) were recently shown to differentiate into adipocytes and myofibroblasts to promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM). A calcium (Ca2+) signaling dysfunction, mainly demonstrated in mouse models, is recognized as a mechanism impacting arrhythmic risk in ACM cardiomyocytes. Whether similar mechanisms influence ACM C-MSC fate is still unknown. Thus, we aim to ascertain whether intracellular Ca2+ oscillations and the Ca2+ toolkit are altered in human C-MSC obtained from ACM patients, and to assess their link with C-MSC-specific ACM phenotypes. METHODS AND RESULTS: ACM C-MSC show enhanced spontaneous Ca2+ oscillations and concomitant increased Ca2+/Calmodulin dependent kinase II (CaMKII) activation compared to control cells. This is manly linked to a constitutive activation of Store-Operated Ca2+ Entry (SOCE), which leads to enhanced Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors. By targeting the Ca2+ handling machinery or CaMKII activity, we demonstrated a causative link between Ca2+ oscillations and fibro-adipogenic differentiation of ACM C-MSC. Genetic silencing of the desmosomal gene PKP2 mimics the remodelling of the Ca2+ signalling machinery occurring in ACM C-MSC. The anti-arrhythmic drug flecainide inhibits intracellular Ca2+ oscillations and fibro-adipogenic differentiation by selectively targeting SOCE. CONCLUSIONS: Altogether, our results extend the knowledge of Ca2+ dysregulation in ACM to the stromal compartment, as an etiologic mechanism of C-MSC-related ACM phenotypes. A new mode of action of flecainide on a novel mechanistic target is unveiled against the fibro-adipose accumulation in ACM.

The Arylamidine T-2307 as a Novel Treatment for the Prevention and Eradication of Candida tropicalis Biofilms.

Candida tropicalis is an emerging pathogen with a high mortality rate due to its virulence factors, including biofilm formation, that has important repercussions on the public health system. The ability of C. tropicalis to form biofilms, which are potentially more resistant to antifungal drugs and the consequent increasing antimicrobial resistance, highlights an urgent need for the development of novel antifungal. The present study analyzed the antibiofilm capacity of the arylamidine T-2307 on two strains of Candida tropicalis. Antimicrobial activity and time-killing assays were performed to evaluate the anticandidal effects of T-2307, the antibiofilm ability on biomass inhibition and eradication was evaluated by the crystal violet (CV) method. Furthermore, in Galleria mellonella infected larvae an increased survival after pre-and post- treatment with T-2307 was observed. The MTT test was used to determine the viability of immortalized human prostate epithelial cells (PNT1A) after exposure to different concentrations of T-2307. Levels of interleukin IL-4, IL-8, IL-10 were quantified after Candida infection of PNT1A cells and treatment. Active doses of T-2307 did not affect the viability of PNT1A cells, and drug concentrations of 0.005 or 0.01 µg mL-1 inhibited the secretion of inflammatory cytokines. Taken together, these results provide new information on T-2307, indicating this drug as a new and promising alternative therapeutic option for the treatment of Candida infections.

WMR Peptide as Antifungal and Antibiofilm against Albicans and Non-Albicans Candida Species: Shreds of Evidence on the Mechanism of Action.

Candida species are the most common fungal pathogens infecting humans and can cause severe illnesses in immunocompromised individuals. The increased resistance of Candida to traditional antifungal drugs represents a great challenge in clinical settings. Therefore, novel approaches to overcome antifungal resistance are desired. Here, we investigated the use of an antimicrobial peptide WMR against Candida albicans and non-albicans Candida species in vitro and in vivo. Results showed a WMR antifungal activity on all Candida planktonic cells at concentrations between 25 µM to >50 µM and exhibited activity at sub-MIC concentrations to inhibit biofilm formation and eradicate mature biofilm. Furthermore, in vitro antifungal effects of WMR were confirmed in vivo as demonstrated by a prolonged survival rate of larvae infected by Candida species when the peptide was administered before or after infection. Additional experiments to unravel the antifungal mechanism were performed on C. albicans and C. parapsilosis. The time-killing curves showed their antifungal activity, which was further confirmed by the induced intracellular and mitochondrial reactive oxygen species accumulation; WMR significantly suppressed drug efflux, down-regulating the drug transporter encoding genes CDR1. Moreover, the ability of WMR to penetrate within the cells was demonstrated by confocal laser scanning microscopy. These findings provide novel insights for the antifungal mechanism of WMR against Candida albicans and non-albicans, providing fascinating scenarios for the identification of new potential antifungal targets.

GC-MS-Based Metabolomics Study of Single- and Dual-Species Biofilms of Candida albicans and Klebsiella pneumoniae.

Candida albicans and Klebsiella pneumoniae frequently co-exist within the human host as a complex biofilm community. These pathogens are of interest because their association is also related to significantly increased morbidity and mortality in hospitalized patients. With the aim of highlighting metabolic shifts occurring in the dual-species biofilm, an untargeted GC-MS-based metabolomics approach was applied to single and mixed biofilms of C. albicans and K. pneumoniae. Metabolomic results showed that among the extracellular metabolites identified, approximately 40 compounds had significantly changed relative abundance, mainly involving central carbon, amino acid, vitamin, and secondary metabolisms, such as serine, leucine, arabitol, phosphate, vitamin B6, cyclo-(Phe-Pro), trehalose, and nicotinic acid. The results were related to the strict interactions between the two species and the different microbial composition in the early and mature biofilms.

Cardiac Biomarkers and Autoantibodies in Endurance Athletes: Potential Similarities with Arrhythmogenic Cardiomyopathy Pathogenic Mechanisms.

The "Extreme Exercise Hypothesis" states that when individuals perform training beyond the ideal exercise dose, a decline in the beneficial effects of physical activity occurs. This is due to significant changes in myocardial structure and function, such as hemodynamic alterations, cardiac chamber enlargement and hypertrophy, myocardial inflammation, oxidative stress, fibrosis, and conduction changes. In addition, an increased amount of circulating biomarkers of exercise-induced damage has been reported. Although these changes are often reversible, long-lasting cardiac damage may develop after years of intense physical exercise. Since several features of the athlete's heart overlap with arrhythmogenic cardiomyopathy (ACM), the syndrome of "exercise-induced ACM" has been postulated. Thus, the distinction between ACM and the athlete's heart may be challenging. Recently, an autoimmune mechanism has been discovered in ACM patients linked to their characteristic junctional impairment. Since cardiac junctions are similarly impaired by intense physical activity due to the strong myocardial stretching, we propose in the present work the novel hypothesis of an autoimmune response in endurance athletes. This investigation may deepen the knowledge about the pathological remodeling and relative activated mechanisms induced by intense endurance exercise, potentially improving the early recognition of whom is actually at risk.

An Integrated Analysis of Intracellular Metabolites and Virulence Gene Expression during Biofilm Development of a Clinical Isolate of Candida tropicalis on Distinct Surfaces.

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidiasis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis consider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections.

Excess TGF-ß1 Drives Cardiac Mesenchymal Stromal Cells to a Pro-Fibrotic Commitment in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is characterized by the replacement of the myocardium with fibrotic or fibro-fatty tissue and inflammatory infiltrates in the heart. To date, while ACM adipogenesis is a well-investigated differentiation program, ACM-related fibrosis remains a scientific gap of knowledge. In this study, we analyze the fibrotic process occurring during ACM pathogenesis focusing on the role of cardiac mesenchymal stromal cells (C-MSC) as a source of myofibroblasts. We performed the ex vivo studies on plasma and right ventricular endomyocardial bioptic samples collected from ACM patients and healthy control donors (HC). In vitro studies were performed on C-MSC isolated from endomyocardial biopsies of both groups. Our results revealed that circulating TGF-ß1 levels are significantly higher in the ACM cohort than in HC. Accordingly, fibrotic markers are increased in ACM patient-derived cardiac biopsies compared to HC ones. This difference is not evident in isolated C-MSC. Nevertheless, ACM C-MSC are more responsive than HC ones to TGF-ß1 treatment, in terms of pro-fibrotic differentiation and higher activation of the SMAD2/3 signaling pathway. These results provide the novel evidence that C-MSC are a source of myofibroblasts and participate in ACM fibrotic remodeling, being highly responsive to ACM-characteristic excess TGF-ß1.

OctoPartenopin: Identification and Preliminary Characterization of a Novel Antimicrobial Peptide from the Suckers of Octopus vulgaris.

Microorganism resistance to conventional antibiotics represents one of the major global health concerns. This paper focuses on a peptide (OctoPartenopin) extracted from suckers of Octopus vulgaris; bioassay-guided chromatographic fractionation was used to identify this sequence, which holds significant antibacterial activity against Gram-positive and Gram-negative bacteria. OctoPartenopin is encrypted within the calponin sequence and was associated with the high levels of proteolytic activity already reported in octopus arm suckers. We synthesized the parent peptide and four analogues; all peptide were tested for their antibacterial and antibiofilm activities. Preliminary antibiofilm experiments showed that that one of the analogues had the best activity in both inhibition and eradication of biofilm of all three microorganisms tested. The occurrence of OctoPartenopin in arm suckers provided novel speculative information on animal behavior, as concerns maternal care of fertilized eggs. Our results highlight that suckers are a rich source of multifaceted peptides to develop alternative antimicrobial agents and food preservatives.

ZeOxaNMulti Trial: A Randomized, Double-Blinded, Placebo-Controlled Trial of Oral PMA-zeolite to prevent Chemotherapy-Induced Side Effects, in particular, Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is the most frequently reported adverse effect of oxaliplatin. In this study, we set out to evaluate the role of the panaceo-micro-activation (PMA) zeolite in the reduction of the incidence of CIPN and hematological and liver toxicity. The possible impact of the PMA-zeolite as an adjuvant therapeutic agent is based on its detoxification properties toward agents promoting the development of neuropathy (e.g., ammonium - recognized as a neurotoxic agent produced by tumors), as well as its positive impact on immunity and oxidative stress through its effects in the gastrointestinal tract. From April 2015 to October 2018, a total of 120 patients (pts) diagnosed with predominantly colorectal cancer requiring oxaliplatin-based chemotherapy were randomized to receive either the PMA-zeolite (Multizeo Med) or placebo while undergoing oxaliplatin-based chemotherapy. A nerve-conduction study (NCS) was planned at the baseline, after three and six months of chemotherapy, to evaluate CIPN. Furthermore, the evaluation of hematological and liver toxicity was performed during every cycle of chemotherapy. 70.6% and 64.3% of patients developed CIPN in the placebo and the PMA-zeolite group, respectively. Patients treated with the PMA-zeolite were able to undergo more cycles of chemotherapy (p = 0.03), which also indicates a significant improvement in tolerance to the therapy. The group treated with the PMA-zeolite showed a lower CIPN (although not statistically significant within the whole group of subjects) compared to patients receiving placebo. This advantage was, however, statistically significant in men (p = 0.047). In addition, supplementation with the PMA-zeolite resulted in a lower incidence of severe-grade hematological toxicity (trend toward statistical significance of p = 0.09 was observed). Cancer patients may benefit from the therapy with the appropriate certified zeolite-products (e.g., the PMA-zeolite) for human use in CIPN. The lower CIPN (statistically significant results in the male subgroup) was accompanied by a trend of lower incidence of severe-grade hematological toxicity. Furthermore, these benefits led to a better tolerance toward chemotherapy (increase in cycles) and allow an improved compliance with the oncological treatment protocol.

CaMKII Activity in the Inflammatory Response of Cardiac Diseases.

Inflammation is a physiological process by which the body responds to external insults and stress conditions, and it is characterized by the production of pro-inflammatory mediators such as cytokines. The acute inflammatory response is solved by removing the threat. Conversely, a chronic inflammatory state is established due to a prolonged inflammatory response and may lead to tissue damage. Based on the evidence of a reciprocal regulation between inflammation process and calcium unbalance, here we described the involvement of a calcium sensor in cardiac diseases with inflammatory drift. Indeed, the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in several diseases with an inflammatory component, such as myocardial infarction, ischemia/reperfusion injury, pressure overload/hypertrophy, and arrhythmic syndromes, in which it actively regulates pro-inflammatory signaling, among which includes nuclear factor kappa-B (NF-κB), thus contributing to pathological cardiac remodeling. Thus, CaMKII may represent a key target to modulate the severity of the inflammatory-driven degeneration.

Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration?

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.

The Amino-Terminal Domain of GRK5 Inhibits Cardiac Hypertrophy through the Regulation of Calcium-Calmodulin Dependent Transcription Factors.

We have recently demonstrated that the amino-terminal domain of G protein coupled receptor kinase (GRK) type 5, (GRK5-NT) inhibits NFκB activity in cardiac cells leading to a significant amelioration of LVH. Since GRK5-NT is known to bind calmodulin, this study aimed to evaluate the functional role of GRK5-NT in the regulation of calcium-calmodulin-dependent transcription factors. We found that the overexpression of GRK5-NT in cardiomyoblasts significantly reduced the activation and the nuclear translocation of NFAT and its cofactor GATA-4 in response to phenylephrine (PE). These results were confirmed in vivo in spontaneously hypertensive rats (SHR), in which intramyocardial adenovirus-mediated gene transfer of GRK5-NT reduced both wall thickness and ventricular mass by modulating NFAT and GATA-4 activity. To further verify in vitro the contribution of calmodulin in linking GRK5-NT to the NFAT/GATA-4 pathway, we examined the effects of a mutant of GRK5 (GRK5-NTPB), which is not able to bind calmodulin. When compared to GRK5-NT, GRK5-NTPB did not modify PE-induced NFAT and GATA-4 activation. In conclusion, this study identifies a double effect of GRK5-NT in the inhibition of LVH that is based on the regulation of multiple transcription factors through means of different mechanisms and proposes the amino-terminal sequence of GRK5 as a useful prototype for therapeutic purposes.

A metabolomics footprinting approach using GC-MS to study inhibitory effects of the fungal metabolite diplopyrone C against nosocomial pathogen biofilms.

Seen initially as wonder drugs, the widespread and often inappropriate use of antibiotics led to the development of microbial resistances. As a result, a true emergency has arisen, and a significant need has emerged to discover and develop new safe and valuable antibiotics. The captivating chemical structure of the fungal metabolite diplopyrone C has caught our attention as an excellent candidate for a circumstantial study aimed at revealing its antimicrobial and antibiofilm activities. In this work, we describe the full analytical strategy from the isolation/identification to the evaluation of the metabolomics effect on target microorganisms of this fungal metabolite. Our results show interesting antimicrobial and antibiofilm activities of diplopyrone C against two frequently isolated nosocomial pathogens (i.e., the fungus Candida albicans and the gram-negative bacterium Klebsiella pneumoniae). Moreover, a GC-MS based metabolomics footprinting approach gave an insight into the uptake and excretion of metabolites from and into the culture medium as a response to the presence of this active substance. The workflow employed in this study is suitable to exploit natural resources for the search of lead compounds for drug development.

A Preliminary Evaluation on the Antifungal Efficacy of VT-1161 against Persister Candida albicans Cells in Vulvovaginal Candidiasis.

Persister cells are a small fraction of the microbial population that survive lethal concentrations of antimicrobial agents. Candida albicans causes vaginal candidiasis, including recurrent vulvovaginal candidiasis, and may survive common antifungal treatments. The triazole VT-1161 is an antifungal agent that specifically targets fungal CYP51, as opposed to the human CYP enzyme. This work illustrates a new role of VT-1161 in eradicating the biofilm created from the persister cells of a primary biofilm of a clinical vaginal isolate of C. albicans. Antifungal activity was determined by the minimum inhibitory concentration (MIC), and the primary biofilm was treated with amphotericin B to obtain persister cells that were able to form a new biofilm. Results obtained using the new azole VT-1161 showed that VT-1161 not only eradicated a secondary biofilm formed from the persister-derived biofilm and counteracted the adhesion of C. albicans in vitro to human cells but also ameliorated C. albicans-induced infection in vivo in Galleria mellonella larvae, suggesting that it could be proposed as an alternative therapeutic strategy for the treatment of recurrent candidiasis.

Evaluation of Potential Probiotic Properties and In Vivo Safety of Lactic Acid Bacteria and Yeast Strains Isolated from Traditional Home-Made Kefir.

Probiotics are known for their health-promoting resources and are considered as beneficial microorganisms. The current study focuses on the isolation, and on a complete in vitro and in vivo characterization, of yeast and lactic acid bacteria acquired from traditional homemade kefir in order to assess their potentiality as probiotic candidates. In particular, the isolates Pichia kudriavzevii Y1, Lactococcus lactis subsp. hordniae LAB1 and Lactococcus lactis subsp. lactis LAB2 were subjected to in vitro characterization to evaluate their suitability as probiotics. Resistance to acid and bile salts, auto-aggregation, co-aggregation, hydrophobicity, and biofilm production capability were examined, as well as their antioxidant activity. A safety assessment was also conducted to confirm the non-pathogenic nature of the isolates, with hemolysis assay and antibiotic resistance assessment. Moreover, mortality in the invertebrate model Galleria mellonella was evaluated. Current findings showed that P. kudriavzevii exhibited estimable probiotic properties, placing them as promising candidates for functional foods. Both lactic acid bacteria isolated in this work could be classified as potential probiotics with advantageous traits, including antimicrobial activity against enteric pathogens and good adhesion ability on intestinal cells. This study revealed that homemade kefir could be a beneficial origin of different probiotic microorganisms that may enhance health and wellness.