A novel genetic circuitry governing hypoxic metabolic flexibility, commensalism and virulence in the fungal pathogen Candida albicans.

Inside the human host, the pathogenic yeast Candida albicans colonizes predominantly oxygen-poor niches such as the gastrointestinal and vaginal tracts, but also oxygen-rich environments such as cutaneous epithelial cells and oral mucosa. This suppleness requires an effective mechanism to reversibly reprogram the primary metabolism in response to oxygen variation. Here, we have uncovered that Snf5, a subunit of SWI/SNF chromatin remodeling complex, is a major transcriptional regulator that links oxygen status to the metabolic capacity of C. albicans. Snf5 and other subunits of SWI/SNF complex were required to activate genes of carbon utilization and other carbohydrates related process specifically under hypoxia. snf5 mutant exhibited an altered metabolome reflecting that SWI/SNF plays an essential role in maintaining metabolic homeostasis and carbon flux in C. albicans under hypoxia. Snf5 was necessary to activate the transcriptional program linked to both commensal and invasive growth. Accordingly, snf5 was unable to maintain its growth in the stomach, the cecum and the colon of mice. snf5 was also avirulent as it was unable to invade Galleria larvae or to cause damage to human enterocytes and murine macrophages. Among candidates of signaling pathways in which Snf5 might operate, phenotypic analysis revealed that mutants of Ras1-cAMP-PKA pathway, as well as mutants of Yak1 and Yck2 kinases exhibited a similar carbon flexibility phenotype as did snf5 under hypoxia. Genetic interaction analysis indicated that the adenylate cyclase Cyr1, a key component of the Ras1-cAMP pathway interacted genetically with Snf5. Our study yielded new insight into the oxygen-sensitive regulatory circuit that control metabolic flexibility, stress, commensalism and virulence in C. albicans.

Repurposing the anthelminthic salicylanilide oxyclozanide against susceptible and clinical resistant Candida albicans strains.

Current antifungal drugs suffer from limitations including toxicity, adverse interactions with other commonly prescribed drugs, and the emergence of resistant strains. Here, we repurposed the anthelmintic oxyclozanide as a potent antifungal agent against both sensitive and resistant clinical isolates of Candida albicans, as well as other human opportunistic fungi. Antifungal activity of oxyclozanide was enhanced when C. albicans grew in nonfermentable carbon sources. Our data support a mechanism of action where oxyclozanide uncoupled the mitochondrial electron transport from oxidative phosphorylation and perturbed the mitochondrial membrane potential.

Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress.

Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.

Fine-tuning of MEK signaling is pivotal for limiting B and T cell activation.

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development.

Antimicrobial Resistance in the Environment: Towards Elucidating the Roles of Bioaerosols in Transmission and Detection of Antibacterial Resistance Genes.

Antimicrobial resistance (AMR) is continuing to grow across the world. Though often thought of as a mostly public health issue, AMR is also a major agricultural and environmental problem. As such, many researchers refer to it as the preeminent One Health issue. Aerial transport of antimicrobial-resistant bacteria via bioaerosols is still poorly understood. Recent work has highlighted the presence of antibiotic resistance genes in bioaerosols. Emissions of AMR bacteria and genes have been detected from various sources, including wastewater treatment plants, hospitals, and agricultural practices; however, their impacts on the broader environment are poorly understood. Contextualizing the roles of bioaerosols in the dissemination of AMR necessitates a multidisciplinary approach. Environmental factors, industrial and medical practices, as well as ecological principles influence the aerial dissemination of resistant bacteria. This article introduces an ongoing project assessing the presence and fate of AMR in bioaerosols across Canada. Its various sub-studies include the assessment of the emissions of antibiotic resistance genes from many agricultural practices, their long-distance transport, new integrative methods of assessment, and the creation of dissemination models over short and long distances. Results from sub-studies are beginning to be published. Consequently, this paper explains the background behind the development of the various sub-studies and highlight their shared aspects.

Mass spectrometry-based functional proteomics of poly(ADP-ribose) polymerase-1.

PARP-1 is an abundant nuclear protein that plays an essential role in the regulation of many genome integrity and chromatin-based processes, such as DNA repair, replication or transcriptional regulation. PARP-1 modulates the function of chromatin and nuclear proteins through several poly(ADP-ribose) (pADPr)-dependent pathways. Aside from the clearly established role of PARP-1 in the maintenance of genome stability, PARP-1 also emerged as an important regulator that links chromatin functions with extranuclear compartments. pADPr signaling has notably been found to be responsible for PARP-1-mediated mitochondrial dysfunction and cell death. Defining the mechanisms that govern the intrinsic functions of PARP-1 is fundamental to the understanding of signaling networks regulated by pADPr. The emergence of mass spectrometry-based proteomics and its broad applications in the study of biological systems represents an outstanding opportunity to widen our knowledge of the functional spectrum of PARP-1. In this article, we summarize various PARP-1 targeted proteomics studies and proteome-wide analyses that shed light on its protein interaction partners, expression levels and post-translational modifications.

Quantum dots in axillary lymph node mapping: biodistribution study in healthy mice.

BACKGROUND: Breast cancer is the first cause of cancer death among women and its incidence doubled in the last two decades. Several approaches for the treatment of these cancers have been developed. The axillary lymph node dissection (ALND) leads to numerous morbidity complications and is now advantageously replaced by the dissection and the biopsy of the sentinel lymph node. Although this approach has strong advantages, it has its own limitations which are manipulation of radioactive products and possible anaphylactic reactions to the dye. As recently proposed, these limitations could in principle be by-passed if semiconductor nanoparticles (quantum dots or QDs) were used as fluorescent contrast agents for the in vivo imaging of SLN. QDs are fluorescent nanoparticles with unique optical properties like strong resistance to photobleaching, size dependent emission wavelength, large molar extinction coefficient, and good quantum yield. METHODS: CdSe/ZnS core/shell QDs emitting around 655 nm were used in our studies. 20 microL of 1 microM (20 pmol) QDs solution were injected subcutaneously in the anterior paw of healthy nude mice and the axillary lymph node (ALN) was identified visually after injection of a blue dye. In vivo fluorescence spectroscopy was performed on ALN before the mice were sacrificed at 5, 15, 30, 60 min and 24 h after QDs injection. ALN and all other organs were removed, cryosectioned and observed in fluorescence microscopy. The organs were then chemically made soluble to extract QDs. Plasmatic, urinary and fecal fluorescence levels were measured. RESULTS: QDs were detected in ALN as soon as 5 min and up to 24 h after the injection. The maximum amount of QDs in the ALN was detected 60 min after the injection and corresponds to 2.42% of the injected dose. Most of the injected QDs remained at the injection site. No QDs were detected in other tissues, plasma, urine and feces. CONCLUSION: Effective and rapid (few minutes) detection of sentinel lymph node using fluorescent imaging of quantum dots was demonstrated. This work was done using very low doses of injected QDs and the detection was done using a minimally invasive method.

A phenotypic small-molecule screen identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell invasion.

A poorly exploited paradigm in the antimicrobial therapy field is to target virulence traits for drug development. In contrast to target-focused approaches, antivirulence phenotypic screens enable identification of bioactive molecules that induce a desirable biological readout without making a priori assumption about the cellular target. Here, we screened a chemical library of 678 small molecules against the invasive hyphal growth of the human opportunistic yeast Candida albicans. We found that a halogenated salicylanilide (N1-(3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide) and one of its analogs, Niclosamide, an FDA-approved anthelmintic in humans, exhibited both antifilamentation and antibiofilm activities against C. albicans and the multi-resistant yeast C. auris. The antivirulence activity of halogenated salicylanilides were also expanded to C. albicans resistant strains with different resistance mechanisms. We also found that Niclosamide protected the intestinal epithelial cells against invasion by C. albicans. Transcriptional profiling of C. albicans challenged with Niclosamide exhibited a signature that is characteristic of the mitochondria-to-nucleus retrograde response. Our chemogenomic analysis showed that halogenated salicylanilides compromise the potential-dependant mitochondrial protein translocon machinery. Given the fact that the safety of Niclosamide is well established in humans, this molecule could represent the first clinically approved antivirulence agent against a pathogenic fungus.

Influence of incubation time and sensitizer localization on meta-tetra(hydroxyphenyl)chlorin (mTHPC)-induced photoinactivation of cells.

The present study addresses the impact of different aggregation states of meta-tetra(hydroxyphenyl)chlorin (mTHPC) on the photoinactivation of cells. Measurements of the photophysical properties of mTHPC in MCF-7 cells showed progressive sensitizer aggregation with increasing incubation time. Reconstructed absorption spectra of intracellular mTHPC showed a significant decrease in the molar extinction coefficient and broadening of the Soret band at 24 h incubation compared to 3 h. Intracellular photobleaching of mTHPC slowed down, and the profile changed from mono- to bi-exponential upon incubation. Fluorescence lifetime imaging (FLIM) measurements revealed a substantial decrease in the lifetime of mTHPC fluorescence at 24 h compared to 3 h. In addition, the intracellular localization of mTHPC as observed by fluorescence microscopy changed from a diffuse homogeneous fluorescence pattern at short incubation times to a punctiform pattern at 24 h. The efficiency of photodynamic therapy (PDT) assessed by a clonogenic assay was three times greater at 24 h. However, when the survival curves were replotted as a function of the number of absorbed photons, the efficiency was 1.8 times greater at 3 h than at 24 h. The loss of photosensitizing efficiency at higher mTHPC concentrations was attributed to self-quenching of the triplet states of the sensitizers.

Reprogramming cellular events by poly(ADP-ribose)-binding proteins.

Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions.

Affinity-based assays for the identification and quantitative evaluation of noncovalent poly(ADP-ribose)-binding proteins.

Poly(ADP-ribose) polymerases have been linked to several cellular functions, most of which being mediated through the dynamics of poly(ADP-ribose) (pADPr). In several pathways, pADPr is the effector molecule that regulates cellular signaling and dictates biological outcomes. pAPDr is a central molecule that is capable of promoting both cell survival through the maintenance of genome integrity and cell death that occurs by way of a signal-mediated apoptotic-like process. Thus, interactions with pADPr are extremely important in bringing about the balanced regulation that controls cell fate. Further clues regarding these functions are emerging from a growing list of proteins with which pADPr interacts. Here, we describe the current approaches for investigating noncovalent protein interactions with pADPr.

Fluorescence imaging and whole-body biodistribution of near-infrared-emitting quantum dots after subcutaneous injection for regional lymph node mapping in mice.

PURPOSE: This study compares fluorescence imaging to mass spectroscopy (inductively coupled plasma-mass spectroscopy, ICP-MS) for detection of quantum dots (QDs) in sentinel lymph node (LN) mapping of breast cancer. PROCEDURES: We study the accumulation of near-infrared-emitting QDs into regional LNs and their whole-body biodistribution in mice after subcutaneous injection, using in vivo fluorescence imaging and ex vivo elemental analysis by ICP-MS. RESULTS: We show that the QD accumulation in regional LNs is detectable by fluorescence imaging as early as 5 min post-delivery. Their concentration reaches a maximum at 4 h then decreases over a 10-day observation period. These data are confirmed by ICP-MS. The QD uptake in other organs, assessed by ICP-MS, increases steadily over time; however, its overall level remains rather low. CONCLUSIONS: Fluorescence imaging can be used as a non-invasive alternative to ICP-MS to follow the QD accumulation kinetics into regional LNs.

Cadmium-free CuInS2/ZnS quantum dots for sentinel lymph node imaging with reduced toxicity.

Semiconductor quantum dots (QDs) could significantly impact the performance of biomedical near-infrared (NIR) imaging by providing fluorescent probes that are brighter and more photostable than conventional organic dyes. However, the toxicity of the components of NIR emitting II-VI and IV-VI QDs that have been made so far (Cd, Hg, Te, Pb, etc.) has remained a major obstacle to the clinical use of QDs. Here, we present the synthesis of CuInS(2)/ZnS core/shell QDs emitting in the NIR ( approximately 800 nm) with good quantum yield and stability even after transfer into water. We demonstrate the potential of these QDs by imaging two regional lymph nodes (LNs) in vivo in mice. We then compare the inflammatory response of the axillary LN induced by different doses of CuInS(2)/ZnS and CdTeSe/CdZnS QDs and show a clear difference in acute local toxicity, the onset of inflammation only occurring at a 10 times more concentrated dose for CuInS(2)/ZnS QDs than for their Cd-containing counterparts.

Quantification techniques and biodistribution of semiconductor quantum dots.

Quantum dots (QDs) are fluorescent inorganic nanocrystals with advantageous optical properties, which have been applied for biomedical purposes including imaging, diagnostic, drug delivery or therapy. Potential toxicity of QDs remains the major barrier to clinical translation, and as such the precise analysis of in vivo QDs distribution and pharmacokinetics is of major importance. Biodistribution studies in animal models are, however, sparse. The present review provides in a first lieu a summary of different techniques, which are currently used for relative quantification of QDs in vivo or their absolute quantification ex vivo. Fluorescence and radioactivity based techniques along with mass-spectrometry detection at the elementary level are addressed in this review. We further introduce biodistribution studies in animal models and discuss the possibilities to modify quantum dots biodistribution in function of different injection ways.