Novel biotechnological approaches for monitoring and immunization against resistant to antibiotics Escherichia coli and other pathogenic bacteria.

The application of next-generation molecular, biochemical and immunological methods for developing new vaccines, antimicrobial compounds, probiotics and prebiotics for zoonotic infection control has been fundamental to the understanding and preservation of the symbiotic relationship between animals and humans. With increasing rates of antibiotic use, resistant bacterial infections have become more difficult to diagnose, treat, and eradicate, thereby elevating the importance of surveillance and prevention programs. Effective surveillance relies on the availability of rapid, cost-effective methods to monitor pathogenic bacterial isolates. In this opinion article, we summarize the results of some research program initiatives for the improvement of live vaccines against avian enterotoxigenic Escherichia coli using virulence factor gene deletion and engineered vaccine vectors based on probiotics. We also describe methods for the detection of pathogenic bacterial strains in eco-environmental headspace and aerosols, as well as samples of animal and human breath, based on the composition of volatile organic compounds and fatty acid methyl esters. We explain how the introduction of these low-cost biotechnologies and protocols will provide the opportunity to enhance co-operation between networks of resistance surveillance programs and integrated routine workflows of veterinary and clinical public health microbiology laboratories.

Breath Biopsy and Discovery of Exclusive Volatile Organic Compounds for Diagnosis of Infectious Diseases.

Exhaled breath contains thousand metabolites and volatile organic compounds (VOCs) that originated from both respiratory tract and internal organ systems and their microbiomes. Commensal and pathogenic bacteria and virus of microbiomes are capable of producing VOCs of different chemical classes, and some of them may serve as biomarkers for installation and progression of various common human diseases. Here we describe qualitative and quantitative methods for measuring VOC fingerprints generated by cellular and microbial metabolic and pathologic pathways. We describe different chemical classes of VOCs and their role in the host cell-microbial interactions and their impact on infection disease pathology. We also update on recent progress on VOC signatures emitted by isolated bacterial species and microbiomes, and VOCs identified in exhaled breath of patients with respiratory tract and gastrointestinal diseases, and inflammatory syndromes, including the acute respiratory distress syndrome and sepsis. The VOC curated databases and instrumentations have been developed through statistically robust breathomic research in large patient populations. Scientists have now the opportunity to find potential biomarkers for both triage and diagnosis of particular human disease.

Novel biotechnological approaches for monitoring and immunization against resistant to antibiotics Escherichia coli and other pathogenic bacteria

The application of next-generation molecular, biochemical and immunological methods for developing new vaccines, antimicrobial compounds, probiotics and prebiotics for zoonotic infection control has been fundamental to the understanding and preservation of the symbiotic relationship between animals and humans. With increasing rates of antibiotic use, resistant bacterial infections have become more difficult to diagnose, treat, and eradicate, thereby elevating the importance of surveillance and prevention programs. Effective surveillance relies on the availability of rapid, cost-effective methods to monitor pathogenic bacterial isolates. In this opinion article, we summarize the results of some research program initiatives for the improvement of live vaccines against avian enterotoxigenic Escherichia coli using virulence factor gene deletion and engineered vaccine vectors based on probiotics. We also describe methods for the detection of pathogenic bacterial strains in eco-environmental headspace and aerosols, as well as samples of animal and human breath, based on the composition of volatile organic compounds and fatty acid methyl esters. We explain how the introduction of these low-cost biotechnologies and protocols will provide the opportunity to enhance co-operation between networks of resistance surveillance programs and integrated routine workflows of veterinary and clinical public health microbiology laboratories.

Insights into breast cancer phenotying through molecular omics approaches and therapy response.

Breast cancer is the most common cancer in the world. Despite advances in early detection and understanding of the molecular bases of breast cancer biology, approximately 30% of all patients with early-stage breast cancer have metastatic disease. Breast cancers are comprised of molecularly distinct subtypes that respond differently to pathway-targeted therapies and neoadjuvant systemic therapy. However, no tumor response is observed in some cases and development of resistance is most commonly seen in patients with heterogeneous breast cancer subtype. To offer better treatment with increased efficacy and low toxicity of selecting therapies, new technologies that incorporate clinical and molecular characteristics of intratumoral heterogeneity have been investigated. This short review provides some examples of integrative omics approaches (genome, epigenome, transcriptome, immune profiling) and mathematical/computational analyses that provide mechanistic and clinically relevant insights into underlying differences in breast cancer subtypes and patients'responses to specific therapies.

Microbiome and Gut Dysbiosis.

The gastrointestinal (GI) tract is the residence of trillions of microorganisms that include bacteria, archaea, fungi and viruses. The collective genomes of whole microbial communities (microbiota) integrate the gut microbiome. Up to 100 genera and 1000 distinct bacterial species were identified in digestive tube niches. Gut microbiomes exert permanent pivotal functions by promoting food digestion, xenobiotic metabolism and regulation of innate and adaptive immunological processes. Proteins, peptides and metabolites released locally and at distant sites trigger many cell signalling and pathways. This intense crosstalk maintains the host-microbial homeostasis. Diet, age, diet, stress and diseases cause increases or decreases in relative abundance and diversity bacterial specie of GI and other body sites. Studies in animal models and humans have shown that a persistent imbalance of gut's microbial community, named dysbiosis, relates to inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), diabetes, obesity, cancer, cardiovascular and central nervous system disorders. Notably specific bacterial communities are promising clinical target to treat inflammatory and infectious diseases. In this context, intestinal microbiota transplantation (IMT) is one optional treatment for IBD, in particular to patients with recurrent Clostridium difficile-induced pseudo-membrane colitis. Here we discuss on recent discoveries linking whole gut microbiome dysbiosis to metabolic and inflammatory diseases and potential prophylactic and therapeutic applications of faecal and phage therapy, probiotic and prebiotic diets.

When and how NK cell-induced programmed cell death benefits immunological protection against intracellular pathogen infection.

NK cells are innate lymphoid cells that exert a key role in immune surveillance through the recognition and elimination of transformed cells and viral, bacterial, and protozoan pathogen-infected cells without prior sensitization. Elucidating when and how NK cell-induced intracellular microbial cell death functions in the resolution of infection and host inflammation has been an important topic of investigation. NK cell activation requires the engagement of specific activating, co-stimulatory, and inhibitory receptors which control positively and negatively their differentiation, memory, and exhaustion. NK cells secrete diverse cytokines, including IFN-γ, TNF-α/ß, CD95/FasL, and TRAIL, as well as cytoplasmic cytotoxic granules containing perforin, granulysin, and granzymes A and B. Paradoxically, NK cells also kill other immune cells like macrophages, dendritic cells, and hyper-activated T cells, thus turning off self-immune reactions. Here we first provide an overview of NK cell biology, and then we describe and discuss the life-death signals that connect the microbial pathogen sensors to the inflammasomes and finally to cell death signaling pathways. We focus on caspase-mediated cell death by apoptosis and pro-inflammatory and non-caspase-mediated cell death by necroptosis, as well as inflammasome- and caspase-mediated pyroptosis.

Gut Microbiome Dysbiosis and Immunometabolism: New Frontiers for Treatment of Metabolic Diseases.

Maintenance of healthy human metabolism depends on a symbiotic consortium among bacteria, archaea, viruses, fungi, and host eukaryotic cells throughout the human gastrointestinal tract. Microbial communities provide the enzymatic machinery and the metabolic pathways that contribute to food digestion, xenobiotic metabolism, and production of a variety of bioactive molecules. These include vitamins, amino acids, short-chain fatty acids (SCFAs), and metabolites, which are essential for the interconnected pathways of glycolysis, the tricarboxylic acid/Krebs cycle, oxidative phosphorylation (OXPHOS), and amino acid and fatty acid metabolism. Recent studies have been elucidating how nutrients that fuel the metabolic processes impact on the ways immune cells, in particular, macrophages, respond to different stimuli under physiological and pathological conditions and become activated and acquire a specialized function. The two major inflammatory phenotypes of macrophages are controlled through differential consumption of glucose, glutamine, and oxygen. M1 phenotype is triggered by polarization signal from bacterial lipopolysaccharide (LPS) and Th1 proinflammatory cytokines such as interferon-γ, TNF-α, and IL-1ß, or both, whereas M2 phenotype is triggered by Th2 cytokines such as interleukin-4 and interleukin-13 as well as anti-inflammatory cytokines, IL-10 and TGFß, or glucocorticoids. Glucose utilization and production of chemical mediators including ATP, reactive oxygen species (ROS), nitric oxide (NO), and NADPH support effector activities of M1 macrophages. Dysbiosis is an imbalance of commensal and pathogenic bacteria and the production of microbial antigens and metabolites. It is now known that the gut microbiota-derived products induce low-grade inflammatory activation of tissue-resident macrophages and contribute to metabolic and degenerative diseases, including diabetes, obesity, metabolic syndrome, and cancer. Here, we update the potential interplay of host gut microbiome dysbiosis and metabolic diseases. We also summarize on advances on fecal therapy, probiotics, prebiotics, symbiotics, and nutrients and small molecule inhibitors of metabolic pathway enzymes as prophylactic and therapeutic agents for metabolic diseases.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker.

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

Using Pharmacogenomic Databases for Discovering Patient-Target Genes and Small Molecule Candidates to Cancer Therapy.

With multiple omics strategies being applied to several cancer genomics projects, researchers have the opportunity to develop a rational planning of targeted cancer therapy. The investigation of such numerous and diverse pharmacogenomic datasets is a complex task. It requires biological knowledge and skills on a set of tools to accurately predict signaling network and clinical outcomes. Herein, we describe Web-based in silico approaches user friendly for exploring integrative studies on cancer biology and pharmacogenomics. We briefly explain how to submit a query to cancer genome databases to predict which genes are significantly altered across several types of cancers using CBioPortal. Moreover, we describe how to identify clinically available drugs and potential small molecules for gene targeting using CellMiner. We also show how to generate a gene signature and compare gene expression profiles to investigate the complex biology behind drug response using Connectivity Map. Furthermore, we discuss on-going challenges, limitations and new directions to integrate molecular, biological and epidemiological information from oncogenomics platforms to create hypothesis-driven projects. Finally, we discuss the use of Patient-Derived Xenografts models (PDXs) for drug profiling in vivo assay. These platforms and approaches are a rational way to predict patient-targeted therapy response and to develop clinically relevant small molecules drugs.

Thymic and Postthymic Regulation of Naïve CD4(+) T-Cell Lineage Fates in Humans and Mice Models.

Our understanding of how thymocytes differentiate into many subtypes has been increased progressively in its complexity. At early life, the thymus provides a suitable microenvironment with specific combination of stromal cells, growth factors, cytokines, and chemokines to induce the bone marrow lymphoid progenitor T-cell precursors into single-positive CD4(+) and CD8(+) T effectors and CD4(+)CD25(+) T-regulatory cells (Tregs). At postthymic compartments, the CD4(+) T-cells acquire distinct phenotypes which include the classical T-helper 1 (Th1), T-helper 2 (Th2), T-helper 9 (Th9), T-helper 17 (Th17), follicular helper T-cell (Tfh), and induced T-regulatory cells (iTregs), such as the regulatory type 1 cells (Tr1) and transforming growth factor-ß- (TGF-ß-) producing CD4(+) T-cells (Th3). Tregs represent only a small fraction, 5-10% in mice and 1-2% in humans, of the overall CD4(+) T-cells in lymphoid tissues but are essential for immunoregulatory circuits mediating the inhibition and expansion of all lineages of T-cells. In this paper, we first provide an overview of the major cell-intrinsic developmental programs that regulate T-cell lineage fates in thymus and periphery. Next, we introduce the SV40 immortomouse as a relevant mice model for implementation of new approaches to investigate thymus organogenesis, CD4 and CD8 development, and thymus cells tumorogenesis.

Skeletal muscle wasting and renewal: a pivotal role of myokine IL-6.

Adult skeletal tissue is composed of heterogeneous population of cells that constantly self-renew by means of a controlled process of activation and proliferation of tissue-resident stem cells named satellite cells. Many growth factors, cytokines and myokines produced by skeletal muscle cells play critical roles in local regulation of the inflammatory process and skeletal muscle regeneration during different pathological conditions. IL-6 is a pleiotropic cytokine released in large amount during infection, autoimmunity and cancer. Low levels of IL-6 can promote activation of satellite cells and myotube regeneration while chronically elevated production promote skeletal muscle wasting. These distinct effects may be explained by a crosstalk of the IL-6/IL-6 receptor and gp130 trans-signaling pathway that oppose to regenerative and anti-inflammatory of the classical IL-6 receptor signaling pathway. Here we discuss on potential therapeutic strategies using monoclonal antibodies to IL-6R for the treatment of skeletal muscle wasting and cachexia. We also highlight on the IL-6/JAK/STAT and FGF/p38αß MAPK signaling pathways in satellite cell activation and the use of protein kinase inhibitors for tailoring and optimizing satellite cell proliferation during the skeletal muscle renewal. Future investigations on the roles of the IL-6 classical and trans-signaling pathways in both immune and non-immune cells in skeletal muscle tissue will provide new basis for therapeutic approaches to reverse atrophy and degeneration of skeletal muscles in cancer and inflammatory diseases.

Human microbiomes and their roles in dysbiosis, common diseases, and novel therapeutic approaches.

The human body is the residence of a large number of commensal (non-pathogenic) and pathogenic microbial species that have co-evolved with the human genome, adaptive immune system, and diet. With recent advances in DNA-based technologies, we initiated the exploration of bacterial gene functions and their role in human health. The main goal of the human microbiome project is to characterize the abundance, diversity and functionality of the genes present in all microorganisms that permanently live in different sites of the human body. The gut microbiota expresses over 3.3 million bacterial genes, while the human genome expresses only 20 thousand genes. Microbe gene-products exert pivotal functions via the regulation of food digestion and immune system development. Studies are confirming that manipulation of non-pathogenic bacterial strains in the host can stimulate the recovery of the immune response to pathogenic bacteria causing diseases. Different approaches, including the use of nutraceutics (prebiotics and probiotics) as well as phages engineered with CRISPR/Cas systems and quorum sensing systems have been developed as new therapies for controlling dysbiosis (alterations in microbial community) and common diseases (e.g., diabetes and obesity). The designing and production of pharmaceuticals based on our own body's microbiome is an emerging field and is rapidly growing to be fully explored in the near future. This review provides an outlook on recent findings on the human microbiomes, their impact on health and diseases, and on the development of targeted therapies.

Dermcidin exerts its oncogenic effects in breast cancer via modulation of ERBB signaling.

BACKGROUND: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines. METHODS: DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform. RESULTS: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands. CONCLUSIONS: These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.

Advances in the integration of optical and mass spectrometry molecular imaging technologies: from omics data to molecular signature discovery.

Many molecular mechanisms in complex biological processes of diseases cannot be fully understood without direct visualization. In the last decades, advances in imaging principles and technologies have expanded our ability to capture and analyze the morphology of tissues and cells' components on conventional widefield optical and fluorescence microscopies and their derivatives confocal and multiphoton fluorescence laser-scanning microscopes, as well as transmission electron microscopes. Innovative imaging technologies are now emerging for constructing fine structural features, precise localization and the dynamic interplay of single and macromolecular assemblies that drive cell growth, differentiation and cell death as well as stromal and chromatin remodeling within many cellular context. The newer super-resolution microscopies capture images with unprecedented sensitivity and clarity allowing the exploration of interactions between individual molecules with a distance resolution as low as 20 nm. But these techniques are not robust enough to quantitate molecules on a genome-wide scale. Mass spectrometry imaging is a high-throughput chemical imaging technique for the identification, quantitation and distribution of proteins, lipids and chemical metabolites at picomol level within a single-cell and complex multicellular tissue. Here we provide an overview on imaging instrumentations and computational platforms to store, data mining, analyze and retrieving genomic, proteomic and immunohistochemistry digital image information which are available for multilevel academic-private collaborations. The expansion of these data sets will lead to a merge picture from it we will retrieve knowledge for more rational-design systems to basic and clinical research in near future.

Cell death-associated molecular-pattern molecules: inflammatory signaling and control.

Apoptosis, necroptosis, and pyroptosis are different cellular death programs characterized in organs and tissues as consequence of microbes infection, cell stress, injury, and chemotherapeutics exposure. Dying and death cells release a variety of self-proteins and bioactive chemicals originated from cytosol, nucleus, endoplasmic reticulum, and mitochondria. These endogenous factors are named cell death-associated molecular-pattern (CDAMP), damage-associated molecular-pattern (DAMP) molecules, and alarmins. Some of them cooperate or act as important initial or delayed inflammatory mediators upon binding to diverse membrane and cytosolic receptors coupled to signaling pathways for the activation of the inflammasome platforms and NF-κB multiprotein complexes. Current studies show that the nonprotein thiols and thiol-regulating enzymes as well as highly diffusible prooxidant reactive oxygen and nitrogen species released together in extracellular inflammatory milieu play essential role in controlling pro- and anti-inflammatory activities of CDAMP/DAMP and alarmins. Here, we provide an overview of these emerging concepts and mechanisms of triggering and maintenance of tissue inflammation under massive death of cells.

Bortezomib (PS-341) treatment decreases inflammation and partially rescues the expression of the dystrophin-glycoprotein complex in GRMD dogs.

Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-ß1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and ß-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane.

The humankind genome: from genetic diversity to the origin of human diseases.

Genome-wide association studies have failed to establish common variant risk for the majority of common human diseases. The underlying reasons for this failure are explained by recent studies of resequencing and comparison of over 1200 human genomes and 10 000 exomes, together with the delineation of DNA methylation patterns (epigenome) and full characterization of coding and noncoding RNAs (transcriptome) being transcribed. These studies have provided the most comprehensive catalogues of functional elements and genetic variants that are now available for global integrative analysis and experimental validation in prospective cohort studies. With these datasets, researchers will have unparalleled opportunities for the alignment, mining, and testing of hypotheses for the roles of specific genetic variants, including copy number variations, single nucleotide polymorphisms, and indels as the cause of specific phenotypes and diseases. Through the use of next-generation sequencing technologies for genotyping and standardized ontological annotation to systematically analyze the effects of genomic variation on humans and model organism phenotypes, we will be able to find candidate genes and new clues for disease's etiology and treatment. This article describes essential concepts in genetics and genomic technologies as well as the emerging computational framework to comprehensively search websites and platforms available for the analysis and interpretation of genomic data.

New routes for transgenesis of the mouse.

Transgenesis refers to the molecular genetic techniques for directing specific insertions, deletions and point mutations in the genome of germ cells in order to create genetically modified organisms (GMO). Genetic modification is becoming more practicable, efficient and predictable with the development and use of a variety of cell and molecular biology tools and DNA sequencing technologies. A collection of plasmidial and viral vectors, cell-type specific promoters, positive and negative selectable markers, reporter genes, drug-inducible Cre-loxP and Flp/FRT recombinase systems are available which ensure efficient transgenesis in the mouse. The technologies for the insertion and removal of genes by homologous-directed recombination in embryonic stem cells (ES) and generation of targeted gain- and loss-of function alleles have allowed the creation of thousands of mouse models of a variety of diseases. The engineered zinc finger nucleases (ZFNs) and small hairpin RNA-expressing constructs are novel tools with useful properties for gene knockout free of ES manipulation. In this review we briefly outline the different approaches and technologies for transgenesis as well as their advantages and disadvantages. We also present an overview on how the novel integrative mouse and human genomic databases and bioinformatics approaches have been used to understand genotype-phenotype relationships of hundreds of mutated and candidate disease genes in mouse models. The updating and continued improvements of the genomic technologies will eventually help us to unraveling the biological and pathological processes in such a way that they can be translated more efficiently from mouse to human and vise-versa.

Identification of a metallopeptidase with TOP-like activity in Paracoccidioides brasiliensis, with increased expression in a virulent strain.

Paracoccidioidomycosis (PCM), caused by the pathogenic fungus Paracoccidioides brasiliensis, is a systemic mycosis with severe acute and chronic forms. The pathology of PCM is not completely understood, and the role of proteases in the infection is not clearly defined. In this report, we describe a metallopeptidase activity in P. brasiliensis total and cytosolic protein extracts similar to that of mammalian thimet oligopeptidase (TOP). The analogous enzyme was suggested by analysis of P. brasiliensis genome databank and by hydrolytic activity of the FRET peptide Abz-GFSPFRQ-EDDnp which was completely inhibited by o-phenanthrolin and significantly inhibited by the TOP inhibitor, JA-2. This activity was also partially inhibited by IgG purified from patients with PCM, but not from normal individuals. As shown by high-performance liquid chromatography (HPLC), the hydrolysis of bradykinin had the same pattern as that of mammalian TOP, and anti-mammalian TOP antibodies significantly inhibited fungal cytosolic peptidase activity. Moreover, anti-mammalian TOP antibodies recognized a component of 80 kDa on fungal cytosol. A P. brasiliensis virulent isolate showed higher gene expression and TOP-like peptidase activity than a non-virulent strain. The release of enzyme following fungal lysis would be consistent with host antibody production and may have a role in the pathogenesis, inflammation and further development of the mycosis.

TAF15 and the leukemia-associated fusion protein TAF15-CIZ/NMP4 are cleaved by caspases-3 and -7.

Caspases are central players in proteolytic pathways that regulate cellular processes such as apoptosis and differentiation. To accelerate the discovery of novel caspase substrates we developed a method combining in silico screening and in vitro validation. With this approach, we identified TAF15 as a novel caspase substrate in a trial study. We find that TAF15 was specifically cleaved by caspases-3 and -7. Site-directed mutagenesis revealed the consensus sequence 106DQPD/Y110 as the only site recognized by these caspases. Surprisingly, TAF15 was cleaved at more than one site in staurosporine-treated Jurkat cells. In addition, we generated two oncogenic TAF15-CIZ/NMP4-fused proteins which have been found in acute myeloid leukemia and demonstrate that caspases-3 and -7 cleave the fusion proteins at one single site. Broad application of this combination approach should expedite identification of novel caspase-interacting proteins and provide new insights into the regulation of caspase pathways leading to cell death in normal and cancer cells.