Unravelling the Role of Candida albicans Prn1 in the Oxidative Stress Response through a Proteomics Approach.

Candida albicans Prn1 is a protein with an unknown function similar to mammalian Pirin. It also has orthologues in other pathogenic fungi, but not in Saccharomyces cerevisiae. Prn1 highly increases its abundance in response to H2O2 treatment; thus, to study its involvement in the oxidative stress response, a C. albicans prn1∆ mutant and the corresponding wild-type strain SN250 have been studied. Under H2O2 treatment, Prn1 absence led to a higher level of reactive oxygen species (ROS) and a lower survival rate, with a higher percentage of death by apoptosis, confirming its relevant role in oxidative detoxication. The quantitative differential proteomics studies of both strains in the presence and absence of H2O2 indicated a lower increase in proteins with oxidoreductase activity after the treatment in the prn1∆ strain, as well as an increase in proteasome-activating proteins, corroborated by in vivo measurements of proteasome activity, with respect to the wild type. In addition, remarkable differences in the abundance of some transcription factors were observed between mutant and wild-type strains, e.g., Mnl1 or Nrg1, an Mnl1 antagonist. orf19.4850, a protein orthologue to S. cerevisiae Cub1, has shown its involvement in the response to H2O2 and in proteasome function when Prn1 is highly expressed in the wild type.

Effect of miR-21 in mesenchymal stem cells-derived extracellular vesicles behavior.

BACKGROUND: A challenging new branch of research related to aging-associated diseases is the identification of miRNAs capable of modulating the senescence-associated secretory phenotype (SASP) which characterizes senescent cells and contributes to driving inflammation. METHODS: Mesenchymal stem cells (MSC) from human umbilical cord stroma were stable modified using lentivirus transduction to inhibit miR-21-5p and shotgun proteomic analysis was performed in the MSC-derived extracellular vesicles (EV) to check the effect of miR-21 inhibition in their protein cargo. Besides, we studied the paracrine effect of those modified extracellular vesicles and also their effect on SASP. RESULTS: Syndecan-1 (SDC1) was the most decreased protein in MSC-miR21--derived EV, and it was involved in inflammation and EV production. MSC-miR21--derived EV were found to produce a statistically significant inhibitory effect on SASP and inflammaging markers expression in receptor cells, and in the opposite way, these receptor cells increased their SASP and inflammaging expression statistically significantly when treated with MSC-miR-21+-derived EV. CONCLUSION: This work demonstrates the importance of miR-21 in inflammaging and its role in SASP through SDC1.

SILAC-Based Quantitative Phosphoproteomics in Yeast.

In this chapter, detailed procedures for stable isotope labeling with amino acids in cell culture, SILAC labeling of yeast auxotroph, optimization and evaluation of phosphopeptide enrichment, and sample preparation and analysis by high-resolution LC-MS/M, identification of phosphosites, and quantification methods are described.We report methods for the application of double SILAC to yeast using a combination of labeled lysine and labeled arginine.The combination of SILAC-based quantitation with phosphopeptides enrichment by TiO2 in a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways.

Candida albicans Hyphal Extracellular Vesicles Are Different from Yeast Ones, Carrying an Active Proteasome Complex and Showing a Different Role in Host Immune Response.

Candida albicans is the principal causative agent of lethal fungal infections, predominantly in immunocompromised hosts. Extracellular vesicles (EVs) have been described as crucial in the interaction of microorganisms with their host. Since the yeast-to-hypha transition is an important virulence trait with great impact in invasive candidiasis (IC), we have addressed the characterization of EVs secreted by hyphal cells (HEVs) from C. albicans, comparing them to yeast EVs (YEVs). YEVs comprised a larger population of bigger EVs with mainly cell wall proteins, while HEVs were smaller, in general, and had a much higher protein diversity. YEVs were able to rescue the sensitivity of a cell wall mutant against calcofluor white, presumably due to the larger amount of cell wall proteins they contained. On the other hand, HEVs also contained many cytoplasmic proteins related to protein metabolism and intracellular protein transport and the endosomal sorting complexes required for transport (ESCRT) pathway related to exosome biogenesis, pointing to an intracellular origin of HEVs. Interestingly, an active 20S proteasome complex was secreted exclusively in HEVs. Moreover, HEVs contained a greater number of virulence-related proteins. As for their immunogenic role, both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis; however, under our conditions, only HEVs showed a cytotoxic effect on human macrophages and could elicit the release of tumor necrosis factor alpha (TNF-α) by these macrophages. IMPORTANCE This first analysis of HEVs of C. albicans has shown clear differences between them and the YEVs of C. albicans, showing their relevance and possible use in the discovery of new diagnostic markers and treatment targets against C. albicans infections. The data obtained point to different mechanisms of biogenesis of YEVs and HEVs, as well as different involvements in cell biology and host interaction. YEVs played a more relevant role in cell wall maintenance, while HEVs were more closely related to virulence, as they had greater effects on human immune cells. Importantly, an active 20S proteosome complex was described as a fungal-EV cargo. A deeper study of its role and those of many other proteins exclusively detected in HEVs and involved in different relevant biological processes of this fungus could open up interesting new areas of research in the battle against C. albicans.

The Combination of Iron and Copper Increases Pathogenicity and Induces Proteins Related to the Main Virulence Factors in Clinical Isolates of Cryptococcus neoformans var. grubii.

In fungi, metals are associated with the expression of virulence factors. However, it is unclear whether the uptake of metals affects their pathogenicity. This study aimed to evaluate the effect of iron/copper in modulating pathogenicity and proteomic response in two clinical isolates of C. neoformans with high and low pathogenicity. METHODS: In both isolates, the effect of 50 µM iron and 500 µM copper on pathogenicity, capsule induction, and melanin production was evaluated. We then performed a quantitative proteomic analysis of cytoplasmic extracts exposed to that combination. Finally, the effect on pathogenicity by iron and copper was evaluated in eight additional isolates. RESULTS: In both isolates, the combination of iron and copper increased pathogenicity, capsule size, and melanin production. Regarding proteomic data, proteins with increased levels after iron and copper exposure were related to biological processes such as cell stress, vesicular traffic (Ap1, Vps35), cell wall structure (Och1, Ccr4, Gsk3), melanin biosynthesis (Hem15, Mln2), DNA repair (Chk1), protein transport (Mms2), SUMOylation (Uba2), and mitochondrial transport (Atm1). Increased pathogenicity by exposure to metal combination was also confirmed in 90% of the eight isolates. CONCLUSIONS: The combination of these metals enhances pathogenicity and increases the abundance of proteins related to the main virulence factors.

Sample Processing for Metaproteomic Analysis of Human Gut Microbiota.

Human gut microbiota can be studied through the characterization of microorganisms present in feces. Metaproteomics has arisen as a good approach to investigate this vast community. However, the processing of fecal samples in order to obtain the largest number of proteins from gut microbiota to be subsequently analyzed by means of metaproteomics is a challenge. Here we describe a protocol to approach this task. It includes two main steps: the first step of humectation and dispersion of the feces, followed by the separation of microorganisms from other fecal components such as roughage and food debris, and the second step in which microbial cells are broken up and microbiota proteins recovered for MS analysis. Detailed procedures for sample preparation, protein extraction, trypsin digestion, and mass spectrometry analysis for gut microbiota samples are provided.

Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistant Candida glabrata.

BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Extending the Proteomic Characterization of Candida albicans Exposed to Stress and Apoptotic Inducers through Data-Independent Acquisition Mass Spectrometry.

Candida albicans is a commensal fungus that causes systemic infections in immunosuppressed patients. In order to deal with the changing environment during commensalism or infection, C. albicans must reprogram its proteome. Characterizing the stress-induced changes in the proteome that C. albicans uses to survive should be very useful in the development of new antifungal drugs. We studied the C. albicans global proteome after exposure to hydrogen peroxide (H2O2) and acetic acid (AA), using a data-independent acquisition mass spectrometry (DIA-MS) strategy. More than 2,000 C. albicans proteins were quantified using an ion library previously constructed using data-dependent acquisition mass spectrometry (DDA-MS). C. albicans responded to treatment with H2O2 with an increase in the abundance of many proteins involved in the oxidative stress response, protein folding, and proteasome-dependent catabolism, which led to increased proteasome activity. The data revealed a previously unknown key role for Prn1, a protein similar to pirins, in the oxidative stress response. Treatment with AA resulted in a general decrease in the abundance of proteins involved in amino acid biosynthesis, protein folding, and rRNA processing. Almost all proteasome proteins declined, as did proteasome activity. Apoptosis was observed after treatment with H2O2 but not AA. A targeted proteomic study of 32 proteins related to apoptosis in yeast supported the results obtained by DIA-MS and allowed the creation of an efficient method to quantify relevant proteins after treatment with stressors (H2O2, AA, and amphotericin B). This approach also uncovered a main role for Oye32, an oxidoreductase, suggesting this protein as a possible apoptotic marker common to many stressors. IMPORTANCE Fungal infections are a worldwide health problem, especially in immunocompromised patients and patients with chronic disorders. Invasive candidiasis, caused mainly by C. albicans, is among the most common fungal diseases. Despite the existence of treatments to combat candidiasis, the spectrum of drugs available is limited. For the discovery of new drug targets, it is essential to know the pathogen response to different stress conditions. Our study provides a global vision of proteomic remodeling in C. albicans after exposure to different agents, such as hydrogen peroxide, acetic acid, and amphotericin B, that can cause apoptotic cell death. These results revealed the significance of many proteins related to oxidative stress response and proteasome activity, among others. Of note, the discovery of Prn1 as a key protein in the defense against oxidative stress as well the increase in the abundance of Oye32 protein when apoptotic process occurred point them out as possible drug targets.

Distinct Human Gut Microbial Taxonomic Signatures Uncovered With Different Sample Processing and Microbial Cell Disruption Methods for Metaproteomic Analysis.

The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspicion of being related to a specific taxon from the gut microbiota.

Mass Spectrometry-Based Proteomic and Immunoproteomic Analyses of the Candida albicans Hyphal Secretome Reveal Diagnostic Biomarker Candidates for Invasive Candidiasis.

Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.

Mesenchymal Stem Cell-Derived Extracellular Vesicle Isolation and Their Protein Cargo Characterization.

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.

Vultures from different trophic guilds show distinct oral pathogenic yeast signatures and co-occurrence networks.

Vultures have evolved adaptive mechanisms to prevent infections associated with their scavenging lifestyle. However, food-borne exposure to antimicrobial pharmaceuticals can promote opportunistic infections with adverse outcomes. Here, we used multivariate and network analyses to increase understanding of the behavior of the yeast communities causing oral mycosis outbreaks recently reported in wild nestling cinereous (Aegypius monachus), griffon (Gyps fulvus) and Egyptian (Neophron percnopterus) vultures (CV, GV and EV, respectively) exposed to antibiotics from livestock farming. Common and unique yeast signatures (of Candida, Debaromyces, Diutina, Meyerozyma, Naganishia, Pichia, Rhodotorula, Trichosporon and Yarrowia species) associated with oral mycoses were identified in the three vulture species. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) highlighted that oral lesions from CV and GV shared similar yeast signatures (of major causative pathogens of opportunistic mycoses, such as Candida albicans, Candida parapsilosis and Candida tropicalis), while EV had a distinct yeast signature (of uncommon pathogenic species, such as Candida dubliniensis, Candida zeylanoides, Pichia fermentans and Rhodotorula spp.). Synergistic interactions between yeast species from distinct fungal phyla were found in lesions from CV and GV, but not in EV. These formed co-occurrence subnetworks with partially or fully connected topology. This study reveals that the composition, assembly and co-occurrence patterns of the yeast communities causing oral mycoses differ between vulture species with distinct feeding habits and scavenging lifestyles. Yeast species widely pathogenic to humans and animals, and yeast co-occurrence relationships, are distinctive hallmarks of oral mycoses in CV and GV. These vulture species are more exposed to antibiotics from intensively medicated livestock carcasses provided in supplementary feeding stations and show higher incidence of thrush-like oral lesions than EV. These findings may be useful for development of new initiatives or changes in the conservation of these avian scavengers affected by anthropogenic activities.

Multiomics Substrates of Resistance to Emerging Pathogens? Transcriptome and Proteome Profile of a Vancomycin-Resistant Enterococcus faecalis Clinical Strain.

Antibiotic resistance and hospital acquired infections are on the rise worldwide. Vancomycin-resistant enterococci have been reported in clinical settings in recent decades. In this multiomics study, we provide comprehensive proteomic and transcriptomic analyses of a vancomycin-resistant Enterococcus faecalis clinical isolate from a patient with a urinary tract infection. The previous genotypic profile of the strain C2620 indicated the presence of antibiotic resistance genes characteristic of the vanB cluster. To further investigate the transcriptome of this pathogenic strain, we used whole genome sequencing and RNA-sequencing to detect and quantify the genes expressed. In parallel, we used two-dimensional gel electrophoresis followed by MALDI-TOF/MS (Matrix-assisted laser desorption/ionization-Time-of-flight/Mass spectrometry) to identify the proteins in the proteome. We studied the membrane and cytoplasm subproteomes separately. From a total of 207 analysis spots, we identified 118 proteins. The protein list was compared to the results obtained from the full transcriptome assay. Several genes and proteins related to stress and cellular response were identified, as well as some linked to antibiotic and drug responses, which is consistent with the known state of multiresistance. Even though the correlation between transcriptome and proteome data is not yet fully understood, the use of multiomics approaches has proven to be increasingly relevant to achieve deeper insights into the survival ability of pathogenic bacteria found in health care facilities.

Multiomics Assessment of Gene Expression in a Clinical Strain of CTX-M-15-Producing ST131 Escherichia coli.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments.

Trk1-mediated potassium uptake contributes to cell-surface properties and virulence of Candida glabrata.

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

Enrichment of ATP Binding Proteins Unveils Proteomic Alterations in Human Macrophage Cell Death, Inflammatory Response, and Protein Synthesis after Interaction with Candida albicans.

Macrophages are involved in the primary human response to Candida albicans. After pathogen recognition, signaling pathways are activated, leading to the production of cytokines, chemokines, and antimicrobial peptides. ATP binding proteins are crucial for this regulation. Here, a quantitative proteomic and phosphoproteomic approach was carried out for the study of human macrophage ATP-binding proteins after interaction with C. albicans. From a total of 547 nonredundant quantified proteins, 137 were ATP binding proteins and 59 were detected as differentially abundant. From the differentially abundant ATP-binding proteins, 6 were kinases (MAP2K2, SYK, STK3, MAP3K2, NDKA, and SRPK1), most of them involved in signaling pathways. Furthermore, 85 phosphopeptides were quantified. Macrophage proteomic alterations including an increase of protein synthesis with a consistent decrease in proteolysis were observed. Besides, macrophages showed changes in proteins of endosomal trafficking together with mitochondrial proteins, including some involved in the response to oxidative stress. Regarding cell death mechanisms, an increase of antiapoptotic over pro-apoptotic signals is suggested. Furthermore, a high pro-inflammatory response was detected, together with no upregulation of key mi-RNAs involved in the negative feedback of this response. These findings illustrate a strategy to deepen the knowledge of the complex interactions between the host and the clinically important pathogen C. albicans.

The development of a new parameter for tracking post-transcriptional regulation allows the detailed map of the Pseudomonas aeruginosa Crc regulon.

Bacterial physiology is regulated at different levels, from mRNA synthesis to translational regulation and protein modification. Herein, we propose a parameter, dubbed post-transcriptional variation (PTV), that allows extracting information on post-transcriptional regulation from the combined analysis of transcriptomic and proteomic data. We have applied this parameter for getting a deeper insight in the regulon of the Pseudomonas aeruginosa post-transcriptional regulator Crc. P. aeruginosa is a free-living microorganism, and part of its ecological success relies on its capability of using a large number of carbon sources. The hierarchical assimilation of these sources when present in combination is regulated by Crc that, together with Hfq (the RNA-binding chaperon in the complex), impedes their translation when catabolite repression is triggered. Most studies on Crc regulation are based either in transcriptomics or in proteomics data, which cannot provide information on post-transcriptional regulation when analysed independently. Using the PTV parameter, we present a comprehensive map of the Crc post-transcriptional regulon. In addition of controlling the use of primary and secondary carbon sources, Crc regulates as well cell respiration, c-di-GMP mediated signalling, and iron utilization. Thus, besides controlling the hyerarchical assimilation of carbon sources, Crc is an important element for keeping bacterial homeostasis and, consequently, metabolic robustness.

Diagnosis of Invasive Candidiasis: From Gold Standard Methods to Promising Leading-edge Technologies.

Invasive Candidiasis (IC) poses a major public health problem worldwide. Despite the introduction of new antifungal agents and changes in clinical practices, its morbidity and mortality rates and healthcare costs remain persistently high. This is mainly because of the serious underlying conditions of infected patients (critically ill or severely immunocompromised patients) and the difficulties encountered in early diagnosing this opportunistic mycosis and initiating prompt and appropriate antifungal therapy. In the light of this great clinical challenge, the past decades have witnessed the development of diverse early detection and therapeutic intervention strategies aimed at minimizing the clinical impact and economic burden of this healthcare-associated infection caused by Candida species. Here, we review the currently available methods for IC diagnosis. These encompass (i) gold standard methods (fungal culture and tissue histopathology), (ii) pathogen-derived biomarker detection tests (PCR, protein antigens, mannan, ß-D-glucan and D-arabinitol-based assays), (iii) host-derived biomarker detection tests (Candida albicans germ tube antibodies or CAGTA, anti-mannan antibodies, other infection-specific antibodies, procalcitonin, serum amyloid A, interleukin 17, interleukin 23 and transforming growth factor ß-based assays), (iv) clinical prediction algorithms (Candida score, Candida colonization index and other prediction rules), and (v) leading-edge molecular, proteomic and immunomic technologies (such as peptide nucleic acid-fluorescent in situ hybridization or PNA-FISH, T2 magnetic resonance or T2Candida assay, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or MALDI-TOF MS, among others). Their strengths, utility, limitations as well as combined use to assist in the diagnosis of this life-threatening and costly fungal infection (including candidemia and deepseated candidiasis) are also discussed.

Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord.

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.